Design, synthesis, and biological evaluation of novel pyridine-bridged analogues of combretastatin-A4 as anticancer agents

Article abstract of DOI:10.1021/jm500002k

A series of novel pyridine-bridged analogues of combretastatin-A4 (CA-4) were designed and synthesized. As expected, the 4-atom linker configuration retained little cytotoxicities in the compounds 2e, 3e, 3g, and 4i. Activities of the analogues with 3-atom linker varied widely depending on the phenyl ring substitutions, and the 3-atom linker containing nitrogen represents the more favorable linker structure. Among them, three analogues (4h, 4s, and 4t) potently inhibited cell survival and growth, arrested cell cycle, and blocked angiogenesis and vasculature formation in vivo in ways comparable to CA-4. The superposition of 4h and 4s in the colchicine-binding pocket of tubulin shows the binding posture of CA-4, 4h, and 4s are similar, as confirmed by the competitive binding assay where the ability of the ligands to replace tubulin-bound colchicine was measured. The binding data are consistent with the observed biological activities in antiproliferation and suppression of angiogenesis but are not predictive of their antitubulin polymerization activities.

Full text of DOI:10.1021/jm500002k

Article
pubs.acs.org/jmc
Open Access on 03/26/2015
Design, Synthesis, and Biological Evaluation of Novel Pyridine-
Bridged Analogues of Combretastatin-A4 as Anticancer Agents
Shilong Zheng,*,† Qiu Zhong,‡ Madhusoodanan Mottamal,† Qiang Zhang,† Changde Zhang,†
Elise LeMelle,§ Harris McFerrin,§ and Guangdi Wang*,†,‡
†RCMI Cancer Research Center, ‡Department of Chemistry, §Department of Biology, Xavier University of Louisiana, New Orleans,
Louisiana 70125, United States
ABSTRACT: A series of novel pyridine-bridged analogues of
combretastatin-A4 (CA-4) were designed and synthesized. As
expected, the 4-atom linker configuration retained little
cytotoxicities in the compounds 2e, 3e, 3g, and 4i. Activities
of the analogues with 3-atom linker varied widely depending
on the phenyl ring substitutions, and the 3-atom linker
containing nitrogen represents the more favorable linker
structure. Among them, three analogues (4h, 4s, and 4t)
potently inhibited cell survival and growth, arrested cell cycle,
and blocked angiogenesis and vasculature formation in vivo in
ways comparable to CA-4. The superposition of 4h and 4s in
the colchicine-binding pocket of tubulin shows the binding posture of CA-4, 4h, and 4s are similar, as confirmed by the
competitive binding assay where the ability of the ligands to replace tubulin-bound colchicine was measured. The binding data
are consistent with the observed biological activities in antiproliferation and suppression of angiogenesis but are not predictive of
their antitubulin polymerization activities.
These include various substituted phenyl rings12 and other
INTRODUCTION
■
aromatic rings.13 A few reports have attempted to modify the
Inhibition of tubulin polymerization disrupts the formation of
tumor vasculature, making the microtubule cytoskeleton an
trimethoxy ring with mixed outcomes. For example, the m-
methoxy group has been substituted by a fluoride to yield a
effective target for cancer chemotherapy.1−3 Combretastatin-A4
similarly potent compound.14 In another example, when the
(CA-4) is the prototype of a large group of vascular disrupting
trimethoxy ring was replaced by a trimethyl ring,15 the
agents that have been designed, synthesized, and tested in various
cytotoxicity of the compound was significantly reduced but the
antitubulin activity was largely retained. This suggests that it
biological models as potential therapeutic candidates for cancer
treatment.4,5 CA-4 binds to the colchicine binding site of tubulin
might be possible to achieve disruption of tumor vasculature with
to block microtubule assembly, causing rapid vascular shutdown
and cell death in the tumor.6 The water-soluble phosphate
prodrug form (CA-4P, also known as fosbretabulin) is in phase
fewer cytotoxic side effects.
Modifications of the double bond have also led to diverse
structural variations that remain viable as cytotoxic and
II/III clinical trials either alone or in combination with traditional
chemotherapeutic agents or with radiotherapy.7−10 Meanwhile,
antitubulin compounds. The olefinic bond is believed to be
critical in placing the two phenyl rings at an appropriate distance
over the past two decades, numerous novel derivatives of CA-4
and giving the molecule the right dihedral angle to maximize the
have been discovered to confer cytotoxic potency and antitubulin
activity that are comparable to CA-4, significantly expanding the
arsenal of vascular disrupting agents that could be further
explored for clinical applications. Despite the intense interest and
interaction with the target. As such, replacement of the double
bond by rings that facilitate a cis-locked configuration has proven
to be effective in retaining both cytotoxicity and antitubulin
activity.16−20 Indeed, this strategy has led to the discovery of
the large number of potent derivatives of CA-4 that have been
perhaps more active CA-4 analogues than any other types of
discovered that aimed at targeting the colchicine-binding site of
structural modifications. On the other hand, the observation that
tubulin, none of these inhibitors has reached the clinical stage.
two-carbon linkers are the optimal length of the bridge between
Thus, challenges remain in developing CA-4 analogues with
the two phenyl rings has somewhat limited exploration in this
improved pharmacological properties for eventual acceptance in
the clinic.
Modifications made on the two phenyl rings, for example, have
led to hundreds of active compounds that possess desirable
cytotoxicity while retaining varying degrees of antitubulin
activities.11 Most structural variations of the phenyl rings involve
different combinations of hydroxyl and methoxy substitutions.
strategy of modifications with some encouraging exceptions. For
example, when the methylene bridge is replaced by a carbonyl
group, the resulting analogue, phenstatin, actually retained much
Received: January 1, 2014
Published: March 26, 2014
© 2014 American Chemical Society
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of the antitubulin activity.21 Interestingly, increasing the bridge
length to three carbons such as a chalcone-like linker have been
reported to strongly inhibit tubulin polymerization as well as cell
survival.22 However, progress in this direction of structural
modifications has been limited.
The current study was undertaken to investigate the effect of a
novel variation of bridge length and structure on the anticancer
activities of resulting CA-4 analogues. Specifically we have
designed, synthesized, and evaluated a series of novel pyridine-
linked CA-4 analogues (Figure 1) in which the distance between
cytotoxicity as measured by the antiproliferative activity is largely
lost. Thus, analogues 3e, 3g, and 4i all showed high IC50 values,
suggesting that such stretched-out configuration of the whole
molecule does not bind well in the purported colchicine binding
site to be effective. When the two phenyl rings are 2,4
substitutions on the pyridine linker, making the bridge a 3-
carbon length where the pyridine nitrogen is not included (3f,
3h), results are mixed, with 3f showing modest cytotoxicity while
3h shows none. However, in the configuration where the 3-
carbon linker contains the pyridine nitrogen, i.e., the two phenyl
rings are now 2,6 meta to each other, significant improvement in
the antiproliferative activity is seen in some analogues with
further optimization of the phenyl ring substitutions. Indeed,
when separated by three carbons, ring substitutions appear to be
critical in conferring enhanced cytotoxicity. Trimethoxy
substitution on one phenyl ring generally affords few options
of substitutions on the other ring. For example, of the five
analogues (4a, 4c, 4f, 4g, 4t) containing the trimethoxy phenyl
group, 4a and 4f were inactive in all three cell lines, 4c was
inactive in A549 cells, and 4g was inactive in MDA-MB-231 cells.
The analogue 4t that differed from CA-4 in just the pyridine
linker was found to be the only one in this group that potently
inhibited growth of all three cancer cell lines that were tested.
The most significant enhancement of antiproliferative activity
was observed in the pyridine-linked analogues where one phenyl
ring accommodates the 2,4-dimethoxy substitutions. A sym-
metric analogue 3c with both phenyl ring adopting the 2,4-
dimethoxy configuration gave low nanomolar IC50 values in all
three cancer cell lines. However, when the dimethoxy
substitution on one phenyl ring is in any other position than
2,4 (4e, 3,4-dimethoxy; 4l, 3,5-dimethoxy; 4n, 2,3-dimethoxy;
4o, 2,6-dimethoxy), there is significant loss of the antiprolifer-
ative activity with the only exception of 4m, having a 2,5-
dimethoxyphenyl ring. Interestingly, 3,4,5-trimethoxy substitu-
tion (4g) on the second phenyl ring produced modest
antiproliferative activity in A549 and HeLa cells, but 2,3,5-
trimethoxy substitution (4q) abolished any such activity. When
only monomethoxy substitution is introduced into the second
ring while maintaining the 2,4-dimethoxy substitution on the first
ring, 3-methoxy (4p), 4-methoxy (4h), but not 2-methoxy (4r)
substitution affords nanomolar to micromolar cytotoxicity. In
fact, 4h is one of the most potent analogue in the series. Finally,
addition of a 3-hydroxy substitution to the second ring of 4h
leads to yet the most potent analogue, 4s, that shows low
nanomolar potency in all three cell lines. Thus, one phenyl ring of
4s is identical to that of CA-4 with 3-hydroxy-4-methoxy
substitutions and the other phenyl ring contains 2,4-dimethoxy
substitutions (Figure 1).
Figure 1. Structures of CA-4 and three pyridine-linked analogues 4h, 4s,
and 4t.
the two phenyl rings is configured to be three or four atoms, i.e.,
meta- or para- to each other. Pyridine has been introduced to
replace the cis-double bond between the A ring and B ring.23
However, it was found that the antitubulin activities were largely
lost in these pyridine-containing analogues. We show that
cytotoxicity and antitubulin activities comparable to CA-4 can be
obtained when the bridge length is fixed at three atoms
(including the pyridine nitrogen) and substitutions on one or
both of the phenyl rings are optimized. Here we describe the
synthesis of 34 pyridine-bridged CA-4 analogues that were tested
for their ability to inhibit cancer cell growth and proliferation by
arresting cell cycles, their effect on tubulin polymerization as well
as their activities in blocking angiogenesis. Molecular modeling
and competitive binding assays were also performed to better
understand the structural requirements for the pyridine-linked
CA-4 analogues to retain antimitotic potency.
RESULTS AND DISCUSSION
■
Chemistry. As shown in Scheme 1, the designed analogues
were synthesized following the general procedures as detailed
below and in the Experimental Section. Starting from
commercially available dibromopyridines, palladium-catalyzed
monocoupling reactions gave mainly the products of 2 with
varying yields of the minor products of 3, some of which were
isolated and purified successfully. The products of 2 were further
transformed to symmetrically or unsymmetrically diaryl-
substituted pyridines (3 and 4) by the application of
aforementioned sequential Suzuki coupling. The reaction of 4s
with hydrogen chloride in ether afforded its chloride salt 5.
Antiproliferative Activities. To evaluate the cytotoxicities
of the pyridine-bridged combretastatin analogues, three human
cancer cell lines were cultured and treated with the pyridine-
bridged combretastatin analogues. Treatment at four different
concentrations was used in order to determine the IC50 values for
each compound along with the reference compound, CA-4. As
shown in Table 1, when the two phenyl rings are para to each
other on the pyridine linker, separated by a 4-carbon bridge, the
Antimicrotubule Effects in HeLa Cells. To determine the
microtubule disrupting effects of the pyridine-linked analogues,
we selected 4h as a representative compound in a cell-based
phenotypic screening. HeLa and MDA-MB-231 cells were used
to examine the effect of 4h on the reorganization of microtubules
during mitosis. Cells were treated with either 4h or CA-4 for 24 h,
and microtubules were visualized by indirect immunofluor-
escence techniques. An antibody for β-tubulin was used to
visualize interphase and mitotic microtubule structures. Vehicle-
treated cells exhibited a normal filamentous microtubule array,
with microtubules extending from the central regions of the cell
to the cell periphery (Figure 2A,D). Compound 4h caused
dramatic reduction of the interphase microtubule network, as
shown in Figure 2C in HeLa cells and in Figure 2F for MDA-MB-
231 cells. As demonstrated in parts B and E of Figure 2 for the
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a
Scheme 1. Synthesis of Novel Pyridine-Bridged Analogues of Combrestastatin-A4
a
Reagents and conditions: (a) PdCl2(dppf) (0.40 g, 0.5 mmol), RB(OH)2, toluene−EtOH (4:1), Na2CO3, 80 °C; (b) PdCl2(dppf) (0.40 g, 0.5
mmol), RB(OH)2, toluene−EtOH (4:1), Na2CO3, 120 °C; (c) HCl in ether, rt.
two cell lines, respectively, CA-4 induced very similar loss of the
microtubule network.
strong cell cycle inhibition. As indicated in Table 2, after
treatment with 4h and 4s, 73.4% and 70.6% of HeLa cells were
arrested in the G2/M phase, respectively, whereas treatment with
CA-4 resulted in 44.6% G2/M phase cells. Similarly, the
percentage of MDA-MB-231 cells in the G2/M phase increased
from 15.5% (control) to 57.8% (CA-4 treated), 70.5 (4h
treated), and 54.1 (4s treated).
Inhibition of Tubulin Polymerization in Vitro. The
inhibition of tubulin polymerization by the pyridine-linked CA-4
analogues 4h, 4s, and 4t were tested using bovine brain tubulin.
As shown in Figure 4, incubation with either vehicle (DMSO),
CA-4, 4h, 4s, or 4t resulted in various degrees of inhibition of
tubulin polymerization, depending on the compound and the
dose. At 1 μM, all pyridine-linked analogues (4h, 4s, and 4t)
failed to inhibit tubulin polymerization. Compared to vehicle, 4h
at this concentration appeared to be slightly stimulating tubulin
polymerization. In contrast, CA-4 at 1 μM inhibited tubulin
polymerization by 35%. When analogues concentrations were
Effect on Cell Cycle Arrest. To investigate the effect of the
pyridine-linked CA-4 analogues on cell cycle arrest, we used flow
cytometry to analyze the cell cycle distribution of HeLa cells
following treatment with 4h and 4s at 1 μM. Untreated cells were
used as a negative control, and cells treated with CA-4 were used
as a positive control. As shown in Figure 3, the two most potent
CA-4 analogues, 4h and 4s, were found to be as effective in
arresting the cell cycle at G2/M phase as CA-4. With the
untreated cells, the percentage of cells in the G0/G1 phase was at
61.6% with only 15.70% in the G2/M phase. After treatment with
4h or 4s, the percentage of cells in the G2/M phase increased to
73.4% and 70.6%, respectively. These results compare favorably
to 44.6% in the G2/M phase for cells treated with CA-4.
Similar results were obtained when another cancer cell line,
MDA-MB-231, was used to test the effect of these compounds on
cell cycle arrest. At 1 μM concentration, 4h and 4s again showed
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Table 1. Growth Inhibition IC50 Values of Synthetic Pyridine-Bridged CA-4 Analogues in Three Cancer Cell Lines: MDA-MB-231,
A549, and HeLa
growth inhibition IC50 (μM)
combretastatin analogues
MDA-MB-231
A549
9.0 0.1
HeLa
2a
2b
2c
2d
2e
2f
6.0 3.5
>25
12.0 0.2
>50
>25
>100
>50
>50
10.0 0.1
12.05 0.02
>100
2.0 0.2
>25
2.0
0
14.0 0.3
>25
>100
3a
3b
3c
3d
3e
3f
11.0 1.8
>50
>50
1.0 0.2
>25
>50
0.075 0.001
9.0 1.0
11.0 0.4
2.63 0.15
>100
0.079 0.019
>100
0.011 0.001
2.0 0.4
2.68 0.08
0.79 0.02
>50
>100
7.86 0.233
>100
3g
3h
4a
4b
4c
4d
4e
4f
>50
>50
>25
10.00 1.56
>50
>25
8.0 0.5
0.86 0.01
0.026 0.001
>25
>100
2.49 0.0022
>25
>50
>25
13.0 0.1
>25
>25
>50
>50
>50
4g
4h
4i
>100
0.69 0.05
0.089 0.008
>50
4.43 0.08
0.0038 0.0001
>25
0.0031 0.0003
>50
4j
>25
>25
>50
4k
4l
17.2 2.6
>25
4.22 0.58
>25
>100
>50
4m
4n
4o
4p
4q
4r
0.034 0.004
>50
0.33 0.01
>50
0.034 0.002
>50
>100
>100
>50
9.02 0.09
>50
0.56 0.01
>50
0.067 0.007
>25
>25
>50
>25
4s
0.0046 0.0001
0.069 0.0028
0.0054 0.0006
0.0028 0.0004
0.044 0.002
2.64 0.38
0.042 0.008
0.0038 0.0003
0.0014 0.0003
0.0047 0.0009
0.0026 0.0006
0.0009 0.0003
4t
5
CA-4
increased to 10 μM, 4h remained ineffective against tubulin
polymerization, whereas 4s and 4t were seen to inhibit tubulin
polymerization by 57% and 32%, respectively. In comparison,
CA-4 at 10 μM nearly completely blocked tubulin polymer-
ization (Figure 4). These data suggest that it is possible for the
pyridine-linked analogues to have potent cytotoxicity but very
little antitubulin polymerization activity, such as 4h.
Antiangiogenesis Assay Using Chick Embryo Chorioal-
lantoic Membrane (CAM). The three most potent pyridine-
linked combretastatin analogues, 4h, 4s, and 4t, were then tested
for antiangiogenic and vasculature disrupting properties using
the CAM assay. In this test, the vascular system of a fertilized
chicken embryo is used as a model. Figure 5 demonstrates the
effects of 4h, 4s, and 4t along with CA-4 on the development of
embryonal blood vessels compared to a negative control (PBS)
and a positive control (10 ng/plug basic fibroblast growth factor
(bFGF) and 25 ng/plug vascular endothelial growth factor
(VEGF)). The antiangiogenesis activities of CA-4 and the three
pyridine-bridged analogues were determined by the suppression
of angiogenic action of BV (bFGF + VEGF) when the compound
was added to a collagen plug containing BV and placed on the
chorioallantoic membrane of 10-day old embryos for 3 days. All
compounds, 4h, 4s, 4t, and CA-4, led to inhibition of new vessel
growth 4 days after treatment scored on a scale of 0−3 in a
blinded manner. The negative control (vehicle) and the test
compounds 4s and 4t scored significantly lower than the positive
control (BV). The CAM assay results suggest that the pyridine-
bridged analogues 4h, 4s, and 4t are potent inhibitors of
angiogenesis, with 4s being more effective than CA-4 in
suppressing new vessel growth.
Mouse Plasma Concentrations of 4h, 4s, and 4t vs CA-
4. To evaluate the possible effect of the pyridine bridge on the
analogues’s bioavailability, we measured the plasma concen-
tration levels of 4h, 4s, and 4t in comparison with CA-4 in mice
that were given a single dose of 5 mg/kg of the compound
(Figure 6). After oral administration, blood samples were
collected from the orbital sinus of the mice at 1, 3, 6, and 24 h.
At 1 and 3 h, CA-4 reached plasma concentration of 2.3 and 2.1
ng/mL, respectively. At 6 and 24 h, plasma concentration of CA-
4 was no longer detectable. Analogue 4h and 4t showed lower
peak concentrations than CA-4. Analogue 4s was detected at the
highest plasma concentration of 9.2 ng/mL at the first hour, with
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Figure 2. Effects of CA-4 and 4h on interphase microtubules. MDA-MB-231 and HeLa cells were treated with vehicle (A,D) or 1 μM compounds
(B,C,E,F) as indicated for 24 h. Cells were then fixed and microtubules visualized by indirect immunofluorescence techniques. Normal interphase
microtubules are visible in the control cells of HeLa and MDA-MB-231. The loss of interphase microtubules induced by CA-4 and 4h is shown in
respective treatments as labeled.
Figure 3. Flow cytometric analysis of cell cycle distributions of HeLa cells treated with pyridine-bridged CA-4 analogues 4h and 4s.
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Table 2. HeLa and MDA-MB-231 Cell Cycle Distribution for
after Treatment with CA-4, 4h, and 4s
G0/G1 (%)
S (%)
G2/M (%)
HeLa Cells
61.60
control (−)
22.10
16.70
15.40
17.10
15.70
44.60
73.40
70.60
treated with CA-4
treated with 4h
treated with 4s
37.00
10.30
12.10
MDA-MB-231 Cells
58.60
control (−)
25.70
11.80
16.40
17.10
15.50
57.80
70.50
54.10
treated with CA-4
treated with 4h
treated with 4s
27.50
13.10
18.60
Figure 5. Effect of 4h, 4s, 4t, and CA-4 on angiogenesis in chick embryo.
CA-4 and pyridine-bridged analogues 4h, 4s, and 4t potently inhibited
the induction of angiogenesis by bFGF and VEGF (BV). Angiogenesis
was scored by two scorers in a blinded fashion with similar results. *, p <
0.05; **, p < 0.01, ***, p < 0.001.
of CA-4 (Figure 7A) to the polar amino acids Asn258, Lys352,
Met259, and Cys241 suggests a likely stronger electrostatic
interaction with the protein. In addition, the hydrophobic moiety
of the CA-4 is well embedded in a pocket interacting with several
hydrophobic residues making CA-4 bind tightly to tubulin.
Despite the absence of one hydroxyl group in 4h, its binding pose
is essentially superimposable to 4s, and they both bind in a
fashion similar to CA-4. The close proximity of the functional
groups of 4h (Figure 7B) to the side chain polar residues Asn258,
Met259, Tyr202, Cys241, and Asn167 allows stronger
interactions with the protein. The additional hydroxyl group in
4s (Figure 7C) seems to be stabilized by its interaction with the
side chain of Lys352. Here again the hydrophobic moieties of
these compounds are well shielded by several hydrophobic
residues in the binding pocket. The binding posture of 4t,
however, is slightly different from CA-4, 4h, and 4s. The three
bulky methoxy groups on the para and meta positions of one of
the phenyl rings of 4t present steric hindrance to occupying the
exact colchicine binding site, causing it to bind to a better
position in the nearby colchicine binding site. Even though CA-4
also has three methoxy groups on one phenyl ring, the cis
conformation of CA-4 makes it more compact than 4t, thus it is
less impeded by steric hindrance to binding into the colchicine
binding site. The functional groups of 4t (Figure 7D) are in close
proximity to the side chains of Asn349 and Asn258, and the
hydrophobic moieties interact with the neighboring hydrophobic
side chains. The overall interactions between 4t and tubulin are
weaker than CA-4, 4h, and 4s.
Figure 4. Effects of pyridine-linked CA-4 analogues on microtubule
dynamics. Polymerization of tubulin at 37 °C in the presence of
paclitaxel (10 μM), CA-4 (1 and 10 μM), 4h (1 and 10 μM), 4s (1 and
10 μM), and 4t (1 and 10 μM) and were monitored continuously by
measuring the absorbance at 340 nm over 60 min. The reaction was
initiated by the addition of tubulin to have a final concentration of 3.0
mg of tubulin per mL of incubation solution.
its level dropping rapidly at 6 h and going below detection limit at
24 h. These preliminary results indicate that the pyridine-bridged
analogues varied widely in their bioavailability, as reflected in
their respective plasma concentrations. It appears that 4s
demonstrated the best bioavailability of the three analogues
tested.
Molecular Modeling. To elucidate the binding mode of
pyridine-linked combretastatin analogues, we postulated that the
analogues have the same binding site as colchicine and CA-4. We
have visually analyzed 20 top scoring postures for each
compound, and most of the top scoring poses of each compound
changed very little. The Surflex docking scores were 7.35 for CA-
4, 7.79 for 4h, 7.53 for 4s and 5.75 for 4t, where higher scores
indicate greater binding affinity. The order of the docking scores
appears to be consistent with the IC50 values of the compounds
for growth inhibition of MDA-MB-231 human breast cancer
cells. Here we examine the docking details of three most potent
pyridine-linked analogues, 4h, 4s, and 4t (IC50 values are 2.75
nM for CA-4, 3.13 nM for 4h, 4.56 nM for 4s, and 68.7 nM for 4t
in MDA-MB-231 cells) as compared to CA-4. The structures of
these compounds are shown in Figure 1.
The superposition of all four compounds in the binding pocket
is depicted in Figure 8. It clearly shows the binding posture of
CA-4, 4h, and 4s are similar, whereas the binding location of 4t is
slightly different. The free energy of binding of these compounds
with the receptor protein tubulin was calculated by employing
the MMGB/SA approach. The binding energies are −77.46,
−71.24, −66.72, and −49.54 kcal/mol for CA-4, 4h, 4s, and 4t,
The binding modes of these compounds in the colchicine-
binding site of tubulin are depicted in Figure 7. The binding
postures of CA-4, 4h, and 4s (Figure 7A−C) in the binding
pockets are similar. CA-4 formed a hydrogen bond with the
backbone carbonyl oxygen of Val238, which was absent when 4h
and 4s were bound. The close proximity of the functional groups
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Figure 6. Plasma concentrations of CA-4, 4h, 4s, and 4t in mice after the administration of a single oral dose of 5 mg/kg. Four blood samples were
collected, each at 1, 3, 6, and 24 h, respectively, after oral intake.
Figure 7. Molecular modeling of CA-4 and three pyridine-linked analogues in complex with tubulin. Shown is the proposed binding mode and
interaction between tubulin and selected compounds, (A) CA-4, (B) 4h, (C) 4s, and (D) 4t. The compounds and important amino acids in the binding
pockets are shown in stick model, whereas tubulin is depicted in the ribbon model.
respectively. The binding energies correlate with the exper-
imental IC50 values of these compounds in MDA-MB-231
human breast cancer cells. Both the docking scores and the free
energy of binding show weaker binding of 4t, which is in
agreement with their observed cytotoxic potencies (IC50: 4h < 4s
< 4t) but not the experimentally measured antitubulin
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differences in the order of binding scores where 4h and 4s scored
higher than 4t.
CONCLUSION
■
We have designed and synthesized a series of combretastatin
analogues with a pyridine linker that exceeds the standard two-
atom linker length. Structurally, these compounds represented
nonisomerizable analogues of CA-4 with three linker config-
urations: a 3-atom linker containing the pyridine nitrogen, a 3-
atom linker without the nitrogen, and a 4-atom linker with the
two phenyl rings para to each other on pyridine. As expected, the
4-atom linker configuration retained little cytotoxicities in the
compounds (2e, 3e, 3g, and 4i). Activities of the analogues with
3-atom linkers varied widely depending on the phenyl ring
substitutions, but the most potent compounds are exclusively
with the nitrogen-containing linker configuration. The pro-
gressive improvement of activity from 3e to 3f to 4c
demonstrates, at least qualitatively, that the 3-atom linker
containing nitrogen represents the more favorable linker
structure. More importantly, this novel 3-atom pyridine linker
led us to discover at three analogues (4h, 4s, and 4t) that potently
inhibited cell survival and growth, arrested cell cycle, and blocked
angiogenesis and vasculature formation in vivo in ways
comparable to CA-4. However, unlike CA-4, 4h was inactive in
inhibiting tubulin polymerization and 4s and 4t were significantly
weaker than CA-4 in blocking antitubulin polymerization.
Molecular modeling analysis indicates all three analogues bind
to the colchicine site of tubulin with 4h and 4s, showing greater
binding affinity. Competitive binding assay confirmed that the
pyridine-linked analogues bind to the colchicine site with
affinities similar to CA-4.
Figure 8. Superposition of CA-4, 4h, 4s, and 4t in the colchicine-binding
pocket of tubulin in surface representation. The compounds are shown
in stick model with carbon atoms in dark-green (CA-4), light-green
(4h), orange (4s), or purple (4t).
polymerization activities where 4h was not active in inhibiting
tubulin polymerization and 4s and 4t were minimally active. This
inconsistency suggests that cytotoxicity of the pyridine-linked
CA-4 analogues could be separated from their antitubulin
polymerization activities.
Competitive Binding to Colchicine Binding Site of
Tubulin. To determine if the CA-4 analogues indeed bind to the
colchicine-binding site of tubulin, we next performed a binding
experiment based on an HPLC-MS based method.26 In this
method, colchicine was incubated with tubulin in the absence or
presence of various concentrations of the test compounds, CA-4,
4h, 4s, and 4t in the incubation buffer (80 mM PIPES, 2.0 mM
MgCl2, 0.5 mM EGTA, pH 6.9) at 37 °C for 1 h. After
incubation, the concentration of the displaced colchicine was
measured by HPLC-MS/MS as previously described.26 The
ability of the analogue to inhibit the binding of colchicine was
expressed as a percentage of control binding in the absence of any
competitor. As shown in Figure 9, CA-4 demonstrated the
highest relative binding capacity, reaching 78% of colchicine
binding. All three analogues showed similar, but slightly weaker
binding affinity than CA-4. The binding results are largely
consistent with the molecular modeling predictions, with minor
EXPERIMENTAL SECTION
■
General Chemistry. All reagents and solvents were purchased from
CombiPhos Catalyst, Combi-Block, AK Scientific, Sigma-Aldrich
Chemical Co., Acros, and Pharmco-AAPER and were used as received.
1H and 13C NMR spectra were obtained on a Bruker-300 NMR
spectrometer. Chemical shifts are reported as parts per million (ppm)
relative to TMS. Mass spectral data were collected on an Agilent 2010
instrument, and HRMS spectra data were collected on a Thermo LTQ
Orbitrap-XL mass spectrometer in positive ion modes. Unless specified
otherwise, all tested compounds were confirmed to be >95% pure by
HPLC and GC-MS. Melting points were obtained on a Haake Buchler
melting point apparatus and are uncorrected.
General Procedure for Synthesis of Compounds 2 and 3. The
mixture of the dibromopyridines 1a−c (11.8 g, 0.05 mol), the phenyl
boronic acids (0.075 mol), sodium carbonate (15.9 g, 0.15 mol), and
PdCl2(dppf) (0.40 g, 0.5 mmol) in toluene−ethanol (4:1, 100 mL) was
stirred at 80 °C until completion of reaction (about 3−4 days). After
cooling down to room temperature, the reaction mixture was filtered
through Celite and the filtrate was concentrated. The residue crude was
purified to afford products 2 and 3 (minor) by flash column
chromatography on silica gel with hexane−ethyl acetate (9:1) as eluant.
2-Bromo-6-(3,4,5-trimethoxyphenyl)pyridine (2a). Yield 43%, 7.0
g. Solid, mp 100−102 °C. 1H NMR (CDCl3, 300 Hz): 7.64−7.55 (2H,
m), 7.39 (1H, d, J = 7.2 Hz), 7.21 (2H, s), 3.96 (6H, s), 3.90 (3H, s). 13C
NMR (CDCl3, 75 Hz): 158.3, 153.5, 142.0, 139.6, 139.0, 133.3, 126.2,
118.9, 104.3, 61.0, 56.3. MS-EI: 323, 325 (M+). HRMS (ESI(+)) calcd
for C14H15BrNO3 (M + H): 324.0235, 326.0215; found 324.0225,
326.0204.
2-Bromo-6-(3,4-dimethoxyphenyl)pyridine (2b). Yield 43%, 6.3 g.
Solid, mp 78−80 °C. 1H NMR (CDCl3, 300 Hz): 7.64−7.61 (2H, m),
7.56 (1H, d, J = 7.8 Hz), 7.51 (1H, dd, J = 2.1 and 8.4 Hz), 7.35 (1H, dd, J
= 0.9 and 7.8 Hz), 6.93 (1H, d, J = 8.4 Hz), 3.99 (3H, s), 3.93 (3H, s).
13C NMR (CDCl3, 75 Hz): 158.2, 150.4, 149.2, 142.0, 138.9, 130.5,
125.6, 119.6, 118.3, 110.9, 109.9, 56.0, 55.9. MS-EI: 293, 295 (M+).
Figure 9. Competitive mass spectrometry-based binding assay for
characterization of CA-4, 4h, 4s, and 4t binding to the colchicine binding
site of tubulin.
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HRMS (ESI(+)) calcd for C13H13BrNO2 (M + H): 294.0129, 296.0109;
found 294.0123, 296.0104.
Hz): 161.2, 161.1, 158.0, 157.8, 155.7, 148.8, 145.7, 132.0, 131.2, 125.1,
122.6, 121.8, 121.1, 104.9, 98.99, 98.87, 55.6, 55.5, 55.43, 55.41. MS-EI:
351 (M+). HRMS (ESI(+)) calcd for C21H22NO4 (M + H): 352.1549;
found 352.1537.
Procedure for Synthesis of Compounds 3d. The mixture of 2-
bromo-6-(4-methoxyphenyl)pyridine (2d, 1.3 g, 5 mmol), 4-methox-
yphenyl boronic acids (1.1 g, 7.5 mol), sodium carbonate (1.6 g, 0.015
mol), and PdCl2(dppf) (0.1 g, 0.1 mmol) in toluene−ethanol (4:1, 10
mL) was stirred overnight at 120 °C. After cooling down to room
temperature, the reaction mixture was filtered through Celite followed
by concentration of the filtrate. The residue crude was purified to afford
product 3d by flash column chromatography on silica gel with hexane−
ethyl acetate (9:1) as eluant.
2,6-Bis(4-methoxyphenyl)pyridine (3d). Yield 89%,1.3 g. Solid, mp
156−158 °C. 1H NMR (CDCl3, 300 Hz): 8.10 (4H, d, J = 9.0 Hz), 7.74
(1H, m), 7.57(2H, dd, J = 0.6 and 7.5 Hz), 7.01 (4H, d, J = 9.0 Hz), 3.87
(6H, s). 13C NMR (CDCl3, 75 Hz): 160.4, 156.3, 137.3, 132.3, 128.2,
117.2, 114.0, 55.4. MS-EI: 291 (M+). HRMS (ESI(+)) calcd for
C19H18NO2 (M + H): 292.1337; found 292.1327.
Procedure for Synthesis of Compounds 3c. The mixture of 2,6-
dibromopyridines 1a (5.9 g, 0.025 mol), 2,4-dimethoxyphenyl boronic
acids (13.7 g, 0.075 mol), sodium carbonate (15.9 g, 0.15 mol), and
PdCl2(dppf) (0.20 g, 0.25 mmol) in toluene−ethanol (4:1, 50 mL) was
stirred overnight at 120 °C. After cooling down to room temperature,
the reaction mixture was filtered through Celite and the filtrate was
concentrated. The residue crude was purified to afford product 3c by
flash column chromatography on silica gel with hexane−ethyl acetate
(9:1) as eluant.
2,6-Bis(2,4-dimethoxyphenyl)pyridine (3c). Yield 92%, 8.0 g. Solid,
mp 113−114 °C. 1H NMR (CDCl3, 300 Hz): 7.93 (2H, d, J = 8.4 Hz),
7.73−7.62 (3H, m), 6.62 (2H, dd, J = 2.1 and 8.4 Hz), 6.55 (2H, d, J =
2.1 Hz), 3.85 (12H, s). 13C NMR (CDCl3, 75 Hz): 161.1, 158.3, 155.0,
135.2, 132.3, 122.7, 122.2, 105.1, 98.8, 55.6, 55.5. MS-EI: 351 (M+).
HRMS (ESI(+)) calcd for C21H22NO4 (M + H) 352.1549; found
352.1537.
General Procedure for Synthesis of Compounds 4. The
mixture of the monobromopyridines 2a−f (3 mmol), the phenyl
boronic acids (4 mmol), sodium carbonate (0.5 g, 5 mmol), and
PdCl2(dppf) (0.1 g, 0.1 mmol) in toluene−ethanol (4:1, 100 mL) was
stirred overnight at 120 °C. After cooling down to room temperature,
the reaction was quenched with saturated ammonium chloride solution;
the organic layer was separated and extracted with ethyl acetate. The
combined organic solution was dried over MgSO4, filtered, and
concentrated. The residue crude was purified to afford products 4 by
flash column chromatography on silica gel with hexane−ethyl acetate as
eluant.
4-(6-(3,4,5-Trimethoxyphenyl)pyridin-2-yl)phenol (4a). Eluant:
hexane−ethyl acetate (7:3). Yield 41%, 0.42 g. Solid, mp 92−94 °C
(d). 1H NMR (DMSO-d6, 300 Hz): 9.79 (1H, s), 8.05 (2H, d, J = 8.1
Hz), 7.85−7.62 (3H, m), 7.47 (2H, s), 6.90 (2H, d, J = 8.1 Hz), 3.90
(6H, s), 3.72 (3H, s). 13C NMR (DMSO-d6, 75 Hz): 158.6, 155.5, 155.1,
153.1, 138.5, 137.9, 134.5, 129.6, 128.0, 117.7, 117.5, 115.5, 104.0, 60.1,
56.0. MS-EI: 337 (M+). HRMS (ESI(+)) calcd for C20H20NO4 (M +
H): 338.1392; found 338.1390.
4-(6-(3,4-Dimethoxyphenyl)pyridin-2-yl)phenol (4b). Eluant: hex-
ane−ethyl acetate (7:3). Yield 40%, 0.37 g. Solid, mp 70−72 °C (d). 1H
NMR (DMSO-d6, 300 Hz): 9.78 (1H, s), 8.05 (2H, d, J = 8.1 Hz), 7.85−
7.71 (5H, m), 7.06 (1H, m), 6.90 (2H, d, J = 8.1 Hz), 3.88 (3H, s), 3.82
(3H, s). 13C NMR (DMSO-d6, 75 Hz): 158.5, 155.5, 155.1, 149.8, 148.8,
137.8, 131.6, 129.7, 128.0, 119.2, 117.1, 116.9, 115.5, 111.7, 110.0, 55.5.
MS-EI: 307 (M+). HRMS (ESI(+)) calcd for C19H18NO3 (M + H):
308.1285; found 308.1278.
2-Bromo-6-(2,4-dimethoxyphenyl)pyridine (2c). Yield 75%, 11.0 g.
Solid, mp 48−50 °C. 1H NMR (CDCl3, 300 Hz): 7.86 (2H, m), 7.50
(1H, m), 7.30 (1H, d, J = 7.8 Hz), 6.60 (1H, dd, J = 2.1 and 8.4 Hz), 6.52
(1H, d, J = 2.1 Hz), 3.85 (6H, s). 13C NMR (CDCl3, 75 Hz): 161.8,
158.3, 156.7, 141.2, 138.0, 132.3, 127.0, 125.1, 123.2, 105.2, 98.7, 55.5,
55.4. MS-EI: 293, 295 (M+). HRMS (ESI(+)) calcd for C13H13BrNO2
(M + H): 294.0129, 296.0109; found 294.0126, 296.0100.
2-Bromo-6-(4-methoxyphenyl)pyridine (2d). Yield: 62%, 8.1 g.
Solid, mp 99−100 °C. 1H NMR (CDCl3, 300 Hz): 7.95 (2H, d, J = 9.0
Hz), 7.61 (1H, dd, J = 0.9 and 7.8 Hz), 7.55 (1H, m), 7.34 (1H, dd, J =
1.9 and 7.5 Hz), 6.98 (2H, d, J = 9.0 Hz), 3.87 (3H, s). 13C NMR
(CDCl3, 75 Hz): 160.9, 158.2, 142.0, 138.9, 130.2, 128.3, 125.4, 118.1,
114.1, 55.3. MS-EI: 263, 265 (M+). HRMS (ESI(+)) calcd for
C12H11BrNO (M + H): 264.0024, 266.0004; found 264.0011, 266.0000.
5-Bromo-2-(3,4,5-trimethoxyphenyl)pyridine (2e). Yield 39%, 6.3 g.
Solid, mp 49−50 °C. 1H NMR (CDCl3, 300 Hz): 8.70 (1H, d, J = 2.4
Hz), 7.85 (1H, dd, J = 2.4 and 8.7 Hz), 7.58 (1H, d, J = 8.4 Hz), 7.21
(2H, s), 3.96 (6H, s), 3.91 (3H, s). 13C NMR (CDCl3, 75 Hz): 155.4,
153.5, 150.5, 139.3, 139.2, 133.8, 121.4, 119.1, 103.9, 60.9, 56.2. MS-EI:
323, 325 (M+). HRMS (ESI(+)) calcd for C14H15BrNO3 (M + H):
324.0235, 326.0215; found 324.0224, 326.0203.
5-Bromo-2-(2,4-dimethoxyphenyl)pyridine (2f). Yield 49%, 7.2 g.
Solid, mp 90−92 °C. 1H NMR (CDCl3, 300 Hz): 8.70 (1H, m), 7.79−
7.75 (3H, m), 6.61 (1H, dd, J = 2.4 and 8.7 Hz), 6.55 (1H, d, J = 2.4 Hz),
3.86 (3H, s), 3.85 (3H, s). 13C NMR (CDCl3, 75 Hz): 161.6, 158.1,
154.2, 150.1, 138.2, 131.8, 125.8, 120.7, 118.0, 105.1, 98.8, 55.6, 55.5.
MS-EI: 293, 295 (M+). HRMS (ESI(+)) calcd for C13H13BrNO2 (M +
H): 294.0129, 296.0109; found 294.0121, 296.0099.
2,6-Bis(3,4,5-trimethoxyphenyl)pyridine (3a). Yield 4%, 0.4 g. Solid,
mp 175−176 °C. 1H NMR (CDCl3, 300 Hz): 7.81 (1H, m), 7.64 (2H, d,
J = 7.8 Hz), 7.41 (4H, s), 3.98 (12H, s), 3.92 (6H, s). 13C NMR (CDCl3,
75 Hz): 156.3, 153.5, 139.1, 137.5, 135.0, 118.3, 104.2, 61.1, 56.2. MS-
EI: 411 (M+). HRMS (ESI(+)) calcd for C23H26NO6 (M + H):
412.1760; found 412.1745.
2,6-Bis(3,4-dimethoxyphenyl)pyridine (3b). Yield 14%, 2.4 g. Solid,
mp 140−141 °C. 1H NMR (CDCl3, 300 Hz): 7.84 (2H, d, J = 1.8 Hz),
7.76 (1H, m), 7.65 (2H, dd, J = 1.8 and 8.1 Hz), 7.60 (2H, d, J = 7.8 Hz),
6.98 (2H, d, J = 8.1 Hz), 4.02 (6H, s), 3.96 (6H, s). 13C NMR (CDCl3,
75 Hz): 156.2, 150.0, 149.1, 137.4, 132.5, 119.4, 117.5, 111.0, 110.0,
56.0, 55.9. MS-EI: 351 (M+). HRMS (ESI(+)) calcd for C21H22NO4 (M
+ H): 352.1549; found 352.1538.
2,5-Bis(3,4,5-trimethoxyphenyl)pyridine (3e). Yield: 7%, 1.5 g.
Solid, mp 176−177 °C. 1H NMR (CDCl3, 300 Hz): 8.88 (1H, dd, J =
0.9 and 2.4 Hz), 7.90 (1H, dd, J = 2.4 and 8.1 Hz), 7.76 (1H, dd, J = 0.9
and 8.4 Hz), 7.30 (2H, s), 6.81 (2H, s), 3.99 (6H, s), 3.95 (6H, s), 3.922
(3H, s), 3.915 (3H, s). 13C NMR (CDCl3, 75 Hz): 155.7, 153.7, 153.5,
147.8, 139.1, 138.2, 135.0, 134.5, 133.4, 120.0, 104.2, 103.9, 61.03, 61.00,
56.2. MS-EI: 411 (M+). HRMS (ESI(+)) calcd for C23H26NO6 (M +
H): 412.1760; found 412.1744.
2,4-Bis(3,4,5-trimethoxyphenyl)pyridine (3f). Yield 9%, 1.9 g. Solid,
mp 116−118 °C. 1H NMR (CDCl3, 300 Hz): 8.70 (1H, d, J = 5.1 Hz),
7.79 (1H, s), 7.41 (1H, d, J = 5.1 Hz), 7.26 (2H, s), 6.86 (2H, s), 3.98
(6H, s), 3.96 (6H, s), 3.93 (3H, s), 3.92 (3H, s). 13C NMR (CDCl3, 75
Hz): 157.7, 153.8, 153.6, 149.9, 149.7, 139.2, 139.1, 135.0, 134.4, 120.4,
118.8, 104.5, 104.4, 61.0, 60.9, 56.39, 56.35. MS-EI: 411 (M+). HRMS
(ESI(+)) calcd for C23H26NO6 (M + H): 412.1760; found 412.1746.
2,5-Bis(2,4-dimethoxyphenyl)pyridine (3g). Yield 36%, 6.2 g. Solid,
mp 124−125 °C. 1H NMR (CDCl3, 300 Hz): 8.79 (1H, m), 7.84−7.81
(3H, m), 7.30 (1H, d, J = 8.1 Hz), 6.65−6.57 (4H, m), 3.68 (9H, s), 3.83
(3H, s). 13C NMR (CDCl3, 75 Hz): 161.1, 160.8, 158.1, 157.7, 153.5,
149.5, 136.3, 131.9, 131.3, 131.0, 123.8, 122.0, 120.0, 105.0, 104.8, 99.0,
98.8, 55.59, 55.53, 55.48, 55.45. MS-EI: 351 (M+). HRMS (ESI(+))
calcd for C21H22NO4 (M + H): 352.1549; found 352.1538.
2-(3,4-Dimethoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine
(4c). Eluant: hexane−ethyl acetate (8:2). Yield 80%, 0.91 g. Solid, mp
107−108 °C. 1H NMR (CDCl3, 300 Hz): 7.83 (1H, d, J = 1.8 Hz), 7.78
(1H, m), 7.67−7.59 (3H, m), 7.40 (2H, s), 6.98 (1H, d, J = 8.4 Hz), 4.01
(3H, s), 3.98 (6H, s), 3.96 (3H, s), 3.92 (3H, s). 13C NMR (CDCl3, 75
Hz): 156.2, 153.4, 150.0, 149.1, 139.0, 137.4, 135.2, 132.3, 119.4, 117.8,
111.0, 109.9, 104.1, 61.0, 56.1, 56.0, 55.8. MS-EI: 381 (M+). HRMS
(ESI(+)) calcd for C22H24NO5 (M + H): 382.1654; found 382.1639.
2,4-Bis(2,4-dimethoxyphenyl)pyridine (3h). Yield 12%, 2.2 g. Solid,
mp 95−97 °C. 1H NMR (CDCl3, 300 Hz): 8.65 (d, J = 5.1 Hz, 1H), 7.92
(dd, J = 1.5 and 0.6 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 5.1
and 1.5 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 6.66−6.56 (m, 4H), 3.880 (s,
3H), 3.878 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H). 13C NMR (CDCl3, 75
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2-(3,4-Dimethoxyphenyl)-6-(4-methoxyphenyl)pyridine (4d).
Eluant: hexane−ethyl acetate (8:2). Yield 97%, 0.94 g. Solid, mp
114−115 °C. 1H NMR (CDCl3, 300 Hz): 8.10 (2H, d, J = 9.0 Hz), 7.81
(1H, d, J = 1.8 Hz), 7.75 (1H, m), 7.66 (1H, dd, J = 1.8 and 8.4 Hz), 7.58
(2H, d, J = 7.8 Hz), 7.02 (2H, d, J = 9.0 Hz), 6.97 (1H, d, J = 8.4 Hz),
4.02 (3H, s), 3.95 (3H, s), 3.88 (3H, s). 13C NMR (CDCl3, 75 Hz):
160.4, 156.3, 149.9, 149.1, 137.3, 132.6, 132.2, 128.2, 119.5, 117.4, 114.0,
111.0, 110.0, 55.9, 55.3. MS-EI: 321 (M+). HRMS (ESI(+)) calcd for
C20H20NO3 (M + H): 322.1443; found 322.1432.
2-(2,4-Dimethoxyphenyl)-6-(3,4-dimethoxyphenyl)pyridine (4e).
Eluant: hexane−ethyl acetate (8:2). Yield 95%, 1.0 g. Solid, mp 100−
101 °C. 1H NMR (CDCl3, 300 Hz): 8.02 (1H, d, J = 8.7 Hz), 7.80−7.78
(2H, m), 7.72 (1H, m), 7.63 (1H, dd, J = 2.1 and 8.4 Hz), 7.58 (1H, dd, J
= 1.2 and 7.5 Hz), 6.97 (1H, d, J = 8.4 Hz), 6.68 (1H, dd, J = 2.4 and 8.7
Hz), 6.59 (1H, d, J = 2.4 Hz), 4.01 (3H, s), 3.96 (3H, s), 3.89 (6H, s).
13C NMR (CDCl3, 75 Hz): 161.3, 158.4, 156.2, 155.0, 149.7, 149.1,
121.3, 116.6, 115.5, 105.7, 98.7. MS-EI: 307 (M+). HRMS (ESI(+))
calcd for C19H18NO3 (M + H): 308.1285; found 308.1276.
2-(2,4-Dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)pyridine (4l).
Eluant: hexane−ethyl acetate (9:1). Yield 63%, 0.66 g. Solid, mp 96−
98 °C. 1H NMR (CDCl3, 300 Hz): 8.02 (1H, d, J = 8.7 Hz), 7.83 (1H, d,
J = 7.5 Hz), 7.72 (1H, m), 7.57 (1H, d, J = 7.8 Hz), 7.26 (2H, s), 6.66
(1H, dd, J = 2.1 and 8.4 Hz), 6.57 (1H, d, J = 1.8 Hz), 6.53 (1H, m), 3.88
(12H, s). 13C NMR (CDCl3, 75 Hz): 161.3, 161.0, 158.4, 156.2, 155.0,
142.1, 136.3, 132.3, 123.3, 122.1, 117.9, 105.1, 100.8, 98.8, 55.6, 55.5.
MS-EI: 351 (M+). HRMS (ESI(+)) calcd for C21H22NO4 (M + H):
352.1549; found 352.1541.
2-(2,4-Dimethoxyphenyl)-6-(2,5-dimethoxyphenyl)pyridine (4m).
Eluant: hexane−ethyl acetate (7:3). Yield 71%, 0.75 g. Solid, mp 76−77
1
°C. H NMR (CDCl3, 300 Hz): 7.93 (1H, d, J = 8.4 Hz), 7.79−7.66
(3H, m), 7.53 (1H, d, J = 2.7 Hz), 6.96−6.87 (2H, m), 6.62 (1H, dd, J =
2.4 and 8.7 Hz), 6.56 (1H, d, J = 2.1 Hz), 3.86 (3H, s), 3.85 (3H, s), 3.83
(3H, s), 3.80 (3H, s). 13C NMR (CDCl3, 75 Hz): 161.2, 158.2, 155.1,
154.9, 153.9, 151.5, 153.3, 132.2, 130.5, 122.8, 122.5, 122.4, 116.5, 114.9,
113.1, 105.0, 98.8, 56.5, 55.8, 55.6, 55.4. MS-EI: 351 (M+). HRMS
(ESI(+)) calcd for C21H22NO4 (M + H): 352.1549; found 352.1560.
2-(2,3-Dimethoxyphenyl)-6-(2,4-dimethoxyphenyl)pyridine (4n).
Eluant: hexane−ethyl acetate (9:1). Yield 90%, 0.95 g. Solid, mp 79−
80 °C. 1H NMR (CDCl3, 300 Hz): 7.96 (1H, d, J = 8.4 Hz), 7.80 (1H,
dd, J = 2.1 and 7.2 Hz), 7.76−7.68 (2H, m), 7.50 (1H, dd, J = 1.5 and 7.8
Hz), 7.16 (1H, t, J = 8.1 Hz), 6.96 (1H, dd, J = 1.5 and 7.8 Hz), 6.63 (1H,
dd, J = 2.4 and 8.4 Hz), 6.56 (1H, d, J = 2.4 Hz), 3.91 (3H, s), 3.87 (3H,
s), 3.85 (3H, s), 3.71 (3H, s). 13C NMR (CDCl3, 75 Hz): 161.2, 158.2,
155.1, 155.0, 153.0, 147.2, 153.6, 134.9, 132.2, 124.1, 123.0, 122.9, 122.2,
112.3, 105.1, 98.8, 61.0, 55.9, 55.5, 55.4. MS-EI: 351 (M+). HRMS
(ESI(+)) calcd for C21H22NO4 (M + H): 352.1549; found 352.1557.
2-(2,4-Dimethoxyphenyl)-6-(2,6-dimethoxyphenyl)pyridine (4o).
Eluant: hexane−ethyl acetate (8:2). Yield 96%, 1.0 g. Solid, mp 155−
156 °C. 1H NMR (CDCl3, 300 Hz): 7.79 (1H, d, J = 8.4 Hz), 7.74−7.67
(2H, m), 7.29 (1H, t, J = 7.8 Hz), 7.18 (1H, dd, J = 2.1 and 6.9 Hz), 6.64
(2H, d, J = 8.4 Hz), 6.58 (1H, dd, J = 2.4 and 8.4 Hz), 6.54 (1H, d, J = 2.1
Hz), 3.85 (3H, s), 3.83 (3H, s), 3.74 (6H, s). 13C NMR (CDCl3, 75 Hz):
160.9, 158.3, 158.0, 155.2, 153.8, 135.2, 132.6, 129.4, 123.6, 122.8, 122.7,
119.8, 105.0, 104.4, 98.6, 56.0, 55.5, 55.4. MS-EI: 351 (M+). HRMS
(ESI(+)) calcd for C21H22NO4 (M + H): 352.1549; found 352.1536.
2-(2,4-Dimethoxyphenyl)-6-(3-methoxyphenyl)pyridine (4p).
Eluant: hexane−ethyl acetate (9:1). Yield 68%, 0.66 g. Solid, mp 75−
76 °C. 1H NMR (CDCl3, 300 Hz): 8.03 (1H, d, J = 8.7 Hz), 7.82 (1H,
dd, J = 0.6 and 7.8 Hz), 7.75−7.69 (2H, m), 7.65 (1H, d, J = 7.8 Hz), 7.59
(1H, dd, J = 0.6 and 7.5 Hz), 7.38 (1H, t, J = 7.8 Hz), 6.95 (1H, dd, J =
1.8 and 8.1 Hz), 6.66 (1H, dd, J = 2.4 and 8.7 Hz), 6.56 (1H, d, J = 2.1
Hz), 3.89 (3H, s), 3.87 (6H, s). 13C NMR (CDCl3, 75 Hz): 161.3, 159.9,
158.4, 156.3, 155.1, 141.4, 136.3, 132.3, 129.5, 123.2, 122.1, 119.4, 117.9,
114.3, 112.4, 105.1, 98.8, 55.6, 55.4, 55.3. MS-EI: 321 (M+). HRMS
(ESI(+)) calcd for C20H20NO3 (M + H): 322.1443; found 322.1452.
2-(2,4-Dimethoxyphenyl)-6-(2,4,6-trimethoxyphenyl)pyridine
(4q). Eluant: hexane−ethyl acetate (7:3). Yield 57%, 0.65 g. Solid, mp
136.2, 132.9, 132.2, 122.5, 122.3, 119.4, 117.2, 110.9, 110.1, 105.1, 98.8,
55.9, 55.6, 55.4. MS-EI: 351 (M+). HRMS (ESI(+)) calcd for
C21H22NO4 (M + H): 352.1549; found 352.1537.
2-(4-Methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine (4f).
Eluant: hexane−ethyl acetate (8:2). Yield 70%, 0.74 g. Solid, mp
112−114 °C. 1H NMR (CDCl3, 300 Hz): 8.09 (2H, d, J = 9.0 Hz), 7.77
(1H, m), 7.63−7.56 (2H, m), 7.37 (2H, s), 7.02 (2H, d, J = 9.0 Hz), 3.99
(6H, s), 3.92 (3H, s), 3.88 (3H, s). 13C NMR (CDCl3, 75 Hz): 160.5,
156.4, 153.4, 139.0, 137.4, 135.4, 132.0, 128.2, 117.8, 114.0, 104.2, 60.9,
56.2, 55.3. MS-EI: 351 (M+). HRMS (ESI(+)) calcd for C21H22NO4 (M
+ H): 352.1549; found 352.1538.
2-(2,4-Dimethoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine
(4g). Eluant: hexane−ethyl acetate (8:2). Yield 79%, 0.9 g. Solid, mp
115−117 °C. 1H NMR (CDCl3, 300 Hz): 7.99 (1H, d, J = 8.4 Hz), 7.80
(1H, dd, J = 0.9 and 7.8 Hz), 7.72 (1H, m), 7.55 (1H, dd, J = 0.9 and 7.5
Hz), 7.33 (2H, s), 6.66 (1H, dd, J = 2.1 and 8.4 Hz), 6.58 (1H, d, J = 2.1
Hz), 3.97 (6H, s), 3.90 (3H, s), 3.88 (6H, s). 13C NMR (CDCl3, 75 Hz):
161.3, 158.4, 156.3, 155.1, 153.4, 138.8, 136.3, 135.7, 132.2, 123.0, 122.1,
117.6, 105.1, 104.2, 98.8, 60.9, 56.2, 55.6, 55.4. MS-EI: 381 (M+). HRMS
(ESI(+)) calcd for C22H24NO5 (M + H): 382.1654; found 382.1644.
2-(2,4-Dimethoxyphenyl)-6-(4-methoxyphenyl)pyridine (4h).
Eluant: hexane−ethyl acetate (8:2). Yield 90%, 0.87 g. Solid, mp 97−
98 °C. 1H NMR (CDCl3, 300 Hz): 8.04 (2H, d, J = 8.7 Hz), 8.00 (1H, d,
J = 8.4 Hz), 7.75 (1H, dd, J = 1.2 and 8.1 Hz), 7.69 (1H, m), 7.54 (1H,
dd, J = 1.5 and 7.5 Hz), 6.99 (1H, d, J = 9.0 Hz), 6.65 (1H, dd, J = 2.4 and
8.7 Hz), 6.56 (1H, d, J = 2.4 Hz), 3.864 (3H, s), 3.861 (3H, s), 3.856
(3H, s). 13C NMR (CDCl3, 75 Hz): 161.3, 160.2, 158.4, 156.3, 155.1,
136.3, 132.5, 132.3, 128.2, 12.4, 122.3, 117.1, 116.1, 114.8, 114.0, 105.1,
98.9, 55.6, 55.4, 55.3. MS-EI: 321 (M+). HRMS (ESI(+)) calcd for
C20H20NO3 (M + H): 322.1443; found 322.1434.
5-(3,4-Dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)pyridine (4i).
Eluant: hexane−ethyl acetate (7:3). Yield 88%, 1.0 g. Solid, mp 131−
132 °C. 1H NMR (CDCl3, 300 Hz): 8.88 (1H, d, J = 2.4 Hz), 7.91 (1H,
dd, J = 2.4 and 8.1 Hz), 7.75 (1H, d, J = 8.4 Hz), 7.29 (2H, s), 7.20 (1H,
dd, J = 2.4 and 8.1 Hz), 7.13 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 8.4 Hz),
3.99 (6H, s), 3.98 (3H, s), 3.95 (3H, s), 3.92 (3H, s). 13C NMR (CDCl3,
75 Hz): 155.2, 153.5, 149.4, 149.2, 147.6, 139.0, 134.7, 134.6, 130.4,
120.0, 119.4, 111.7, 110.0, 103.9, 60.9, 56.2, 56.0. MS-EI: 381 (M+).
HRMS (ESI(+)) calcd for C22H24NO5 (M + H): 382.1654; found
382.1638.
4-(6-(4-Methoxyphenyl)pyridine-2-yl)phenol (4j). Eluant: hexane−
ethyl acetate (7:3). Yield 73%, 0.60 g. Solid, mp 133−134 °C. 1H NMR
(DMSO-d6, 300 Hz): 9.78 (1H, s), 8.15 (2H, d, J = 8.7 Hz), 8.05 (2H, d,
J = 8.4 Hz), 7.81 (1H, m), 7.74−7.70 (2H, m), 7.05 (2H, d, J = 8.7 Hz),
6.90 (2H, d, J = 8.4 Hz), 3.82 (3H, s). 13C NMR (DMSO-d6, 75 Hz):
160.2, 158.6, 155.6, 155.1, 138.0, 131.4, 129.8, 128.04, 127.95, 116.9,
115.6, 114.1, 55.3. MS-EI: 277 (M+). HRMS (ESI(+)) calcd for
C18H16NO2 (M + H): 278.1181; found 278.1171.
1
144−145 °C. H NMR (CDCl3, 300 Hz): 7.79 (1H, d, J = 8.7 Hz),
7.72−7.65 (2H, m), 7.16 (1H, dd, J = 2.1 and 6.6 Hz), 6.58 (1H, dd, J =
2.1 and 8.4 Hz), 6.53 (1H, d, J = 2.1 Hz), 6.22 (2H, s), 3.85 (6H, s), 3.83
(3H, s), 3.73 (6H, s). 13C NMR (CDCl3, 75 Hz): 161.1, 160.9, 159.0,
158.0, 155.1, 153.8, 135.1, 132.6, 123.9, 122.8, 122.5, 113.1, 105.0, 98.6,
91.1, 56.0, 55.5, 55.4. MS-EI: 381 (M+). HRMS (ESI(+)) calcd for
C22H24NO5 (M + H): 382.1654; found 382.1662.
2-(2,4-Dimethoxyphenyl)-6-(2-methoxyphenyl)pyridine (4r).
Eluant: hexane−ethyl acetate (9:1). Yield 89%, 0.86 g. Solid, mp 95−
96 °C. 1H NMR (CDCl3, 300 Hz): 7.94 (1H, d, J = 8.7 Hz), 7.92 (1H,
dd, J = 1.8 and 7.8 Hz), 7.77−7.65 (3H, m), 7.36 (1H, m), 7.08 (1H, td, J
= 1.2 and 7.5 Hz), 7.00 (1H, d, J = 8.1 Hz), 6.62 (1H, dd, J = 2.4 and 8.7
Hz), 6.56 (1H, d, J = 2.4 Hz), 3.87 (3H, s), 3.86 (3H, s), 3.85 (3H, s).
13C NMR (CDCl3, 75 Hz): 161.1, 158.2, 157.1, 155.2, 155.1, 135.2,
132.3, 131.5, 129.7, 129.6, 122.7, 122.6, 122.5, 121.0, 111.4, 105.1, 98.8,
55.64, 55.61, 55.4. MS-EI: 321 (M+). HRMS (ESI(+)) calcd for
C20H20NO3 (M + H): 322.1443; found 322.1435.
4-(6-(2,4-Dimethoxyphenyl)pyridine-2-yl)phenol (4k). Eluant: hex-
ane−ethyl acetate (7:3). Yield 48%, 0.44 g. Solid, mp 112−114 °C (d).
1H NMR (DMSO-d6, 300 Hz): 9.74 (1H, s), 7.98 (2H, d, J = 8.7 Hz),
7.90 (1H, d, J = 9.0 Hz), 7.77−7.66 (3H, m), 6.88 (2H, d, J = 8.7 Hz),
6.70−6.68 (2H, m), 3.85 (3H, s), 3.83 (3H, s). 13C NMR (DMSO-d6, 75
Hz): 161.1, 158.5, 158.2, 155.6, 154.3, 136.8, 131.7, 130.0, 128.0, 121.9,
5-(6-(2,4-Dimethoxyphenyl)pyridine-2-yl)-2-methoxyphenol (4s).
Eluant: hexane−ethyl acetate (7:3). Yield 83%, 0.84 g. Solid, mp 142−
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144 °C. 1H NMR (DMSO-d6, 300 Hz): 9.16 (1H, s), 7.89 (1H, d, J = 9.3
Hz), 7.78−7.66 (4H, m), 7.53 (1H, dd, J = 1.8 and 8.4 Hz), 7.01 (1H, d, J
= 8.4 Hz), 6.70−6.69 (2H, m), 3.86 (3H, s), 3.83 (3H, s), 3.82 (3H, s).
13C NMR (DMSO-d6, 75 Hz): 161.1, 158.2, 155.4, 154.3, 148.7, 146.7,
136.8, 132.0, 131.7, 122.2, 121.2, 117.7, 116.9, 113.7, 112.1, 105.7, 98.7,
55.72, 55.66, 55.4. MS-EI: 337 (M+). HRMS (ESI(+)) calcd for
C20H20NO4 (M + H): 338.1392; found 338.1386.
compounds were preincubated with 10 μM bovine brain tubulin in
glutamate buffer at 30 °C and then cooled to 0 °C. After the addition of
0.4 mM GTP, the mixtures were transferred to 0 °C cuvettes in a
recording spectrophotometer and warmed to 30 °C. Tubulin assembly
was monitored by measuring the optical density at 340 nm using a
BioTek Synergy 4 multifunction microplate spectrophotometer.
Cytofluorimetric Analysis of Cell Cycle Distribution. Cells
treated with tested compounds for 24 h were washed once in PBS and
resuspended in 1 mL of 70% ice-cold ethanol and stored at −20 °C.
Fixed cells were washed twice in PBS and then treated with 1 mL of 0.1
mg/mL of RNase A solution at 37 °C for 1 h. DNA was then stained with
a PBS solution containing 0.1 mg/mL propidium iodide for 30 min at
room temperature in the dark. Cell cycle analysis was determined with
Accuri C6 (BD Biosciences, Mountain View, CA).
Chick Embryo Chorioallantoic Membrane (CAM) Assay.
Fertilized embryos were obtained from Charles River Laboratories
and incubated at 37.5 °C for 3 days, removed from their shell using a
Dremel tool and placed into a covered weighboat for 10 further days of
incubation. Solidified 30 μL onplants containing 2.1 mg/mL rat tail
collagen (BD Biosciences, Bedford, MA) and 10 ng/plug bFGF and 30
ng/plug VEGF in the presence or absence of Ca-4 and 4s were placed on
the embryo chorioallantoic membrane (CAM) over two pieces of nylon
mesh approximately 0.5 cm2. Four collagen onplants were added per egg
on at least three separate eggs. After 4 days of incubation, images were
taken of each plug on surviving embryos using a mini-Vid camera (LW
Scientific, Lawrenceville, GA) and quantified in a masked fashion on a
scale from 0 to 3 with 0 representing no angiogenesis and 3 representing
extreme angiogenesis. Data from one scorer (confirmed by a second
scorer) are presented as the means standard errors of the mean.
Statistical significances were determined by one-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test (GraphPad
Prism, La Jolla, CA).
Molecular Modeling. All the docking studies were carried out using
Sybyl-X 1.3 on a linux workstation. The initial coordinates for tubulin
was taken from the crystal structure of tubulin in complex with
colchicine (PDB: 1SA0.pdb). After removing all the ions and
substructures, such as GDP and GTP present in the crystal structure,
hydrogen atoms were added to the protein system using Amber99 force
field. Four representative CA-4 analogues, including the parent
compound CA-4, were selected for the docking studies. The 3D
structures of these selected compounds were first built using Sybyl-X 1.3
sketch followed by energy minimization using the MMFF94 force field
and Gasteiger−Marsili charges. We employed Powell’s method for
optimizing the geometry with a distance dependent dielectric constant
and a termination energy gradient of 0.05 kcal/mol. Structural
resemblance of combretastatin to colchicine indicates that combretas-
tatin binds to tubulin at the same site as colchicine and presumably has a
similar mode of action. Hence we used colchicine binding site for
docking our selected compounds to tubulin. We used the Surflex
docking program to automatically dock all the selected compounds into
the binding pocket of tubulin. Surflex is a fully automatic flexible
molecular docking algorithm that combines Hammerhead’s empirical
scoring function with a molecular similarity method to generate putative
poses of ligand fragments. Docking process was guided by the protomol,
which is a computational representation of the intended binding site. We
used the position of the native ligand, colchicine, in the crystal structure
to generate the protomol. In all the cases, we used the default screening
protocol of Surflex that employs Surflex’s ligand preminimization and
postdocking all-atom minimization. Standard parameters were used to
estimate the binding affinity characterized by the Surflex-Dock scores. In
each docking experiment, 20 poses were saved for each compound based
on their total docking score values. A higher score represents stronger
binding affinity. The optimal binding pose of the docked compounds
was selected based on the Surflex scores and visual inspection of the
docked complexes. The binding free energies of the complexes were
calculated using the MM/GBSA method. OPLS-AA force field and GB/
SA continuum solvent model were used to calculate the necessary
energies of the complex.
2-Methoxy-5-(6-(3,4,5-trimethoxyphenyl)pyridine-2-yl)phenol
(4t). Eluant: hexane−ethyl acetate (7:3). Yield 66%, 0.72 g. Solid, mp
129−130 °C. 1H NMR (DMSO-d6, 300 Hz): 9.24 (1H, s), 7.87 (2H, d, J
= 4.2 Hz), 7.76 (1H, m), 7.71 (1H, d, J = 2.1 Hz), 7.60 (1H, dd, J = 2.1
and 8.4 Hz), 7.48 (2H, s), 7.03 (1H, d, J = 8.4 Hz), 3.91 (6H, s), 3.83
(3H, s), 3.73 (3H, s). 13C NMR (DMSO-d6, 75 Hz): 155.4, 155.1, 153.2,
148.9, 146.7, 138.6, 138.0, 134.5, 131.5, 118.1, 117.9, 113.7, 112.2, 104.1,
60.2, 56.1, 55.7. MS-EI: 367 (M+). HRMS (ESI(+)) calcd for
C21H22NO5 (M + H): 368.1498; found 368.1490.
2-(2,4-Dimethoxyphenyl)-6-(3-hydroxy-4-methoxyphenyl)-
pyridin-1-ium Chloride (5). To the solution of 4s (0.3 g, 0.88 mmol) in
THF (5 mL) was added HCl solution in ether (0.44 mL, 0.88 mmol, 2
M). The resultant mixture was stirred at rt overnight. The precipitate
was filtered and washed with dichloromethane to afford 5 (0.32 g) in
yield of 97%. Solid, mp 140 °C (d). 1H NMR (DMSO-d6, 300 Hz): 8.28
(1H, t, J = 7.8 Hz), 7.96 (1H, d, J = 7.8 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.75
(1H, d, J = 8.4 Hz), 7.45−7.42 (2H, m), 7.13 (1H, d, J = 8.7 Hz), 3.92
(3H, s), 3.861 (3H, s), 3.855 (3H, s). 13C NMR (DMSO-d6, 75 Hz):
163.1, 158.6, 152.8, 151.6, 150.6, 147.1, 143.1, 132.5, 126.4, 124.1, 121.0,
119.9, 115.7, 114.8, 112.5, 106.4, 99.0, 56.3, 56.0, 55.9. MS-ESI: 338
(M+-Cl). HRMS (ESI(+)) calcd for C20H20NO4 (M − Cl): 338.1392;
found 338.1387.
Antiproliferative Assays. Human breast adenocarcinoma (MDA-
MB-231), human nonsmall cell lung carcinoma (A549), and human
cervical carcinoma (HeLa) cells were grown in DMEM medium
supplemented with 115 units/mL of penicillin G, 115 μg/mL of
streptomycin, and 10% fetal bovine serum (all from Life Technologies,
Grand Island, NY). Cells were seeded in 96-well plates (5 × 103 cells/
well) containing 50 μL of growth medium for 24 h. After medium
removal, 100 μL of fresh medium containing individual analogue
compounds at different concentrations was added to each well and
incubated at 37 °C for 72 h. After 24 h of culture, the cells were
supplemented with 50 μL of analogue compounds dissolved in DMSO
(less than 0.25% in each preparation). After 72 h of incubation, 20 μL of
resazurin was added for 2 h before recording fluorescence at 560 nm
(excitation) and 590 nm (emission) using a Victor microtiter plate
fluorimeter (PerkinElmer, USA). The IC50 was defined as the
compound concentration required to inhibit cell proliferation by 50%
in comparison with cells treated with the maximum amount of DMSO
(0.25%) and considered as 100% viability.
Immunofluorescence. MDA-MB-231 and HeLa cells were grown
on a Laboratory-Tek chamber slide (VWR International, Radnor, PA)
and treated with vehicle (DMSO) or 1 μM CA-4 or 4h for 24 h to 4 days.
At the completion of treatment, cells were fixed with 3.7% formaldehyde
and permeabilized with 0.1% Triton X-100 in PBS for 4 min. The cells
were first incubated for 1 h in a solution of PBS containing 1% BSA and
calf serum to block nonspecific antibody binding. The cells were then
incubated with the mouse antitubulin antibody (1:200) (Life
Technologies, Grand Island, NY), washed three times in PBS containing
1% BSA, and incubated for 2 h at room temperature with secondary goat
antimouse antibody Alexa 488 labeled (1:200) for tubulin staining. The
chamber slides were examined and photographed using a Nikon ES800
fluorescence microscope with a digital camera.
Tubulin Polymerization Assays. A tubulin polymerization kit
(Cytoskeleton, Denver, CO) was used to evaluate effect of the pyridine-
linked CA-4 analogues on tubulin assembly in vitro.24,25 It is based on
the principal that light is scattered by microtubules to an extent that is
proportional to the concentration of the microtubule polymer.
Compounds that interact with tubulin will alter the polymerization of
tubulin, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37 °C is monitored. The experimental
procedure of the assay was performed as described in version 8.2 of the
tubulin polymerization assay kit manual. Varying concentrations of
Competitive Binding Assay Using LC-MS/MS. A Thermo TSQ
Advantage instrument was used to determine the competitive binding
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affinity of the pyridine-bridged CA-4 analogues by measuring the
concentration of colchicine displaced by the analogues. Briefly,
colchicine (1.2 μM) was incubated with tubulin (1.3 mg/mL) in the
incubation buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH
6.9) at 37 °C for 1 h. Varying concentrations (0.1−125 μM) of 4h, 4s,
and 4t were used to compete with colchicine originally bound to tubulin.
After incubation, the filtrate was obtained as previously described.26 The
ability of the analogue to inhibit the binding of colchicine was expressed
as a percentage of control binding in the absence of any competitor.
Pharmacokinetic Study in Mice. Female C57BL/6 mice were
used for the pharmacokinetic study of selected CA-4 analogues. Mice
were given oral gavage containing PBS and ethanol-dissolved CA-4, 4h,
4s, and 4t, at a single dose of 5 mg/kg/mouse. After oral administration,
blood samples were collected from the orbital sinus of the mice at 1, 3, 6,
and 24 h, with each group of mice subjected to only one sampling. All
procedures involving these animals were conducted in compliance with
State and Federal laws, standards of the U.S. Department of Health and
Human Services, and guidelines established by Xavier University Animal
Care and Use Committee. The facilities and laboratory animals program
of Xavier University are accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care.
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AUTHOR INFORMATION
Corresponding Authors
*For. S.Z.: phone, 504-520-7824; E-mail, szheng@xula.edu.
*For G.W.: phone, 504-520-5076; E-mail, gwang@xula.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH-NIMHD through grant
8G12MD007595 and NIH-NIGMS grant 8P20GM103424, and
in part by Louisiana Cancer Research Consortium. We thank
Thomas Vu, Bria Carmichael, Breanna Jarrett, Eric Stewart, and
Sydnie Turner for technical assistance in the CAM assays.
ABBREVIATIONS USED
■
CA-4, combretastatin-A4; G0/G1, Gap0/Gap1 phase; G2/M,
Gap2/mitosis phase; DMSO, dimethyl sulfoxide; CAM,
chorioallantoic membrane; PBS, phosphate buffered saline;
bFGF, basic fibroblast growth factor; VEGF, vascular endothelial
growth factor; BV, bFGF + VEGF
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