A novel class of trans-methylpyrazoline analogs of combretastatins: Synthesis and in-vitro biological testing

Article abstract of DOI:10.1016/j.ejmech.2011.03.064

Thirteen methylpyrazoline analogs (1a-m) of combretastatin A-4 (CA-4, 2) were synthesized. The trans-geometry of the two substituted phenyl moieties was ascertained by a single crystal X-ray diffraction study of compound 1d. The cytotoxicities of the analogs against the growth of murine B16 melanoma and L1210 lymphoma cells in culture were measured using the MTT assay. One of the derivatives, 1j, which has the same substituents as CA-4 was the most active in the series with IC₅₀ values of 3.3 μM and 6.8 μM against the growth of L1210 and B16 cells, respectively. The activity of this analog against human cancer cell lines was confirmed in the NCI 60 panel. The other active analogs against L1210 were 1b and 1f, which gave IC₅₀ values in the 6-8 μM range. Compound 1j caused microtubule depolymerization with an EC ₅₀ value of 4.1 μM. This compound has good water solubility of 372 μM. Molecular modeling studies using DFT showed that compound 1j adopts a "twisted" conformation mimicking CA-4 that is optimal for binding to the colchicine site of tubulin.

Full text of DOI:10.1016/j.ejmech.2011.03.064

European Journal of Medicinal Chemistry 46 (2011) 3099e3104
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A novel class of trans-methylpyrazoline analogs of combretastatins: Synthesis
and in-vitro biological testing
Megan Lee a, Olivia Brockway a, Armaan Dandavati a, Samuel Tzou a, Robert Sjoholm a, Vijay Satam a,
,
,
*
Cara Westbrook b, Susan L. Mooberry b c, Matthias Zeller d, Balaji Babu a, Moses Lee a
a Department of Chemistry, Division of Natural and Applied Sciences, Hope College, Holland, MI 49423, USA
b Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
c Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
d Department of Chemistry, Youngstown State University, Youngstown, OH 44555, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen methylpyrazoline analogs (1a-m) of combretastatin A-4 (CA-4, 2) were synthesized. The trans-
geometry of the two substituted phenyl moieties was ascertained by a single crystal X-ray diffraction
study of compound 1d. The cytotoxicities of the analogs against the growth of murine B16 melanoma and
L1210 lymphoma cells in culture were measured using the MTT assay. One of the derivatives, 1j, which
has the same substituents as CA-4 was the most active in the series with IC50 values of 3.3 mM and 6.8 mM
against the growth of L1210 and B16 cells, respectively. The activity of this analog against human cancer
Received 11 January 2011
Received in revised form
29 March 2011
Accepted 30 March 2011
Available online 6 April 2011
cell lines was confirmed in the NCI 60 panel. The other active analogs against L1210 were 1b and 1f,
Keywords:
Combretastatins
Tubulins
which gave IC50 values in the 6e8
m
M range. Compound 1j caused microtubule depolymerization with an
M. Molecular modeling studies
EC50 value of 4.1 M. This compound has good water solubility of 372
m
m
using DFT showed that compound 1j adopts a “twisted” conformation mimicking CA-4 that is optimal for
binding to the colchicine site of tubulin.
Microtubules
Pyrazolines
Cytotoxicity
Lymphoma
Melanoma
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
a potential antitumor drug, that is, its poor bioavailability and low
solubility in biological media [5]. This hindrance has led to the
There is an intense effort in cancer research to develop novel
compounds that are capable of rapid destruction of tumor vascu-
lature leading to tumor necrosis and antitumor efficacy. Such
vasculature disrupting agents (VTAs) are promising anticancer
drugs. One of the most prominent lead compounds is com-
bretastatin A-4 (CA-4, 2, see Fig. 1). CA-4 was originally isolated
from the african willow tree, combretum caffrum, by G.R. Pettit et al.
in 1989 [1]. Combretastatin A-4 is an effective antitumor agent. This
compound is capable of rapid and selective disruption of the
abnormal tumor vasculature [2]. CA-4 binds to tubulin within the
colchicine binding site, thereby disrupting microtubules and
altering endothelial cell shape and disrupting the vascular barrier
causing rapid vasculature shutdown and tumor necrosis [3]. It had
been suggested that these anti-vasculature actions might be
mediated through the vascular endothelial-cadherin signaling
pathway [4]. Yet, CA-4 has a major drawback for development as
synthesis of many structural analogs of CA-4, including CA-4P (3),
a phosphate containing pro-drug, which is currently undergoing
clinical trials [6]. Another analog that has received significant
attention is A105972, 4, which has shown potent and promising
antitumor activity in mice [7].
A wide range of structural analogs of CA-4 have been synthe-
sized, evaluated, and reported from the author’s laboratory.
Examples include pyrazole [8], pyrazoline [9], cyclohexenone [10],
and oxadiazoline analogs [11]. Interestingly, the pyrazole-
containing compounds were not active at inhibiting the growth
of L1210 cancer cells in culture [8], but the pyrazoline analogs were
active [9]. This result was explained by the analysis of an X-ray
structure of a pyrazole compound [8] and molecular models of both
classes of compounds. The results revealed that the pyrazoles
essentially adopted a planar conformation but the pyrazolines
adopted a “twisted” geometry that was similar to the conformation
of CA-4 as determined by X-ray crystallography [12]. Several review
articles that catalog all combretastatin analogs have recently been
reported [6,13].
* Corresponding author. Tel.: þ1 616 395 7190; fax: þ1 616 395 7923.
E-mail address: lee@hope.edu (M. Lee).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.064

3100
M. Lee et al. / European Journal of Medicinal Chemistry 46 (2011) 3099e3104
Fig. 1. Structures of the methylpyrazolines 1a-m (the definition of R1 to R5 is given in Scheme 1 and drawn in Table 1), combretastatin A-4 (CA-4, 2), CA-4P (3), A-105972 (4), and the
4-bromomethylpyrazoline analog 1d.
The authors’ laboratory has also developed a series of acetylated
analogs of combretastatins [14]. Specifically, the acetylated analogs
of combretastatin were found to exhibit good cytotoxicity and
microtubule depolymerization potency, making them worthy of
further investigation [14]. One potential challenge for the ace-
tylcombretastatins is their susceptibility to detoxification by bio-
logical nucleophiles such as glutathione [15]. To overcome this
challenge while concomitantly enhancing the cytotoxic potency,
yield. The acetylcombretastatins were subsequently reacted with
hydrazine hydrate and acetic acid for 4 h in refluxing methanol. The
resulting methylpyrazoline analogs were collected and purified by
silica gel column chromatography using an ethyl acetate/hexane
solvent system. The chemical yields varied between 25 and 60%.
The structures of the methylpyrazoline analogs were confirmed
using mass spectrometry, infrared, and 400 MHz 1H NMR
measurements. The trans configuration of the two substituted
phenyl moieties on the pyrazoline unit was initially inferred from
the coupling constants of about 4 Hz between the two protons on
the ring, which is consistent with reported values [17]. A single
crystal X-ray crystallography study was performed on one of the
methylpyrazoline analogs, 1d, which contained a bromine atom in
the para-position. The X-ray results as shown in Fig. 2B provided
unambiguous evidence of the trans configuration for the phenyl
groups. The results also confirmed the “twisted” shape of 1d
determined from molecular modeling studies, which is similar to
the shape of combretastatin A-4 [12].
the acetylcombretastatins were modified by converting the
a,b-
unsaturated enone functionality into a methylpyrazoline unit. The
methylpyrazoline analogs were also designed because it was
proposed that they would adopt the necessary “twisted geometry”
similar to CA-4, and the substituted phenyl moieties would be fixed
in a trans configuration (see analog 1d in Fig. 1).
2. Results and discussion
2.1. Molecular modeling
Thirteen methylpyrazoline analogs were designed for this study.
The analogs were divided into two groups as shown in Table 1. The
first group I contains a single substituent on the B ring (1a-g). The
second group II contains two- or three substituents on the B ring
(1h-m). The conformation of analog 1d was assessed by molecular
modeling studies using MacSpartan to determine if it adopted the
“twisted geometry”. The molecular structures were optimized by
2.3. Cytotoxicity
Methylpyrazolines 1a-m were tested to determine their cyto-
toxicity against the growth of cancer cells in culture using an in-
vitro 72 h continuous exposure-MTT assay [8e11,14]. Two murine
cancer cell lines L1210 (lymphoma) and B16 (melanoma) were used
in this study. The IC50 values (mM) of each methylpyrazoline analog
molecular mechanics (MMFF) using
a molecular equilibrium
and CA-4 were determined and are provided in Table 1. It is
immediately apparent from the results that three compounds (1b,
1f, and 1j) display significant cytotoxicity. These compounds have
conformer procedure. The equilibrium geometry was subsequently
optimized using Hartree-Fock (3-21 G) calculations, followed by
another geometry optimization using a Density Functional calcu-
lation (B3LYP and 6-31G*). The results given in Fig. 2A provided
evidence that compound 1d does adopt a “twisted” conformation
similar to that of CA-4 [12].
IC50 values of less than 10
1j its IC50 value against B16 was also less than 10
values of 3.3 M and 6.8 M for L1210 and B16 cells, respectively,
compound 1j is the most potent. Compounds 1f and 1b have IC50
values of 8.4 M and 6 M for the L1210 cells, and 95 M and
45.5 M for the B16 cells, respectively. For comparison, CA-4 is
m
M against L1210 cells, and for compound
mM. With IC50
m
m
m
m
m
2.2. Chemistry
m
significantly more cytotoxic over the methylpyrazoline compounds
As described in Scheme 1, all thirteen derivatives were synthe-
sized in a two-step process. The first step included the reaction of
the appropriately substituted benzaldehyde with 3,4,5-trimethox-
yphenylacetone [16] in the presence of benzoic acid and piperidine
in refluxing toluene to yield acetylcombretastatins 5a-m in 15e95%
described in this paper. The reported IC50 values for CA-4 against
the growth L1210 and B16 cells in culture were 0.003 and 0.002
respectively [1b,7b].
mM,
With IC50 values of less than 50
mM, compounds 1a, 1d, 1h, 1i,
and 1k showed modest activity against L1210 cells. The other

M. Lee et al. / European Journal of Medicinal Chemistry 46 (2011) 3099e3104
Table 1 (continued )
3101
Table 1
IC50 values against the growth of L1210 and B16 cells. IC50 values are expressed in
M.
Compounds
L1210 B16
IC50 IC50
M) M)
m
Compounds
L1210 B16
IC50 IC50
M) M)
(
m
(m
Group I
(
m
(m
N
Group I
H3C
Ac
N
N
H C
Ac
H3CO
H3CO
Cl
Cl
N
1h
36
>100
H CO
H CO
1a
47
>100
OCH3
H3C
N
Ac
OCH
H3C
N
N
Ac
H3CO
H3CO
OCH3
OCH3
N
1i
34
>100
H3CO
H3CO
1b
1c
1d
6
45.5
OCH3
H3C
CH3
N
Ac
OCH3
H3C
N
H3CO
H3CO
OH
N
Ac
1j
3.3
6.8
N
H3CO
H3CO
OCH3
>100
>100
OCH3
H3C
Cl
N
Ac
N
OCH3
H3C
H3CO
H3CO
NO2
N
Ac
1k
32.5 >100
N
OCH3
H3CO
H3CO
33
>100
OCH3
N
H3
C
Ac
Br
OCH3
N
OCH3
H3CO
H3CO
1l
>100
>100
N
H3
C
Ac
N
H3CO
H3CO
OCH3
H3C
OCH3
1e
>100
>100
N
Ac
N
NO2
H3CO
H3CO
OCH3
OCH3
H3C
N
Ac
1m
>100
>100
N
OCH3
H3CO
H3CO
1f
8.4
95
OCH3
OCH3
CA-4 (2)
0.003
0.002
OCH3
[1b,7b]
OCH3
H3C
N
compounds described in this study did not show any cytotoxicity
against either cell line; their IC50 values were greater than 100 M.
Ac
N
m
It is interesting to note that the results are consistent with earlier
reports from our laboratory [8e11,14], the L1210 cells were more
sensitive to the methylpyrazoline analogs and other potential
vascular targeting agents than the B16 cells. This is presumably
because the L1210 cells are more aggressive than B16 cells, making
them more sensitive to tubulin inhibitors.
H3CO
H3CO
NO2
1g
>100
>100
Group II
OCH3
(continued on next page)

3102
M. Lee et al. / European Journal of Medicinal Chemistry 46 (2011) 3099e3104
Fig. 2. Structure and conformation of the 4-bromomethylpyrazoline analog 1d as determined by (A) molecular modeling and (B) single crystal X-ray diffraction. Complete X-ray
structural data for 1d were deposited with the Cambridge Structural Database.
CH3
H
O
O
R1
H3CO
H3CO
CH3
R2
R3
H3CO
H3CO
R5
R4
R1
R2
piperidine,
+
benzoic acid
O
toluene,
reflux, 4h
(15 - 95%)
R5
5a-m
OCH3
R4
OCH3
R3
a, R1=R2=R3=R4=R5=H
methanol, hydrazine
hydrate, acetic acid,
reflux, 4h
b, R1=R2=R4=R5=H; R3=CH3
c, R1=R2=R4=R5=H; R3=Cl
d, R1=R2=R4=R5=H; R3=Br
e, R1=R2=R4=R5=H; R3=NO2
f, R1=R2=R4=R5=H; R3=OCH3
g, R1=R3=R4=R5=H; R2=NO2
h, R1=R4=R5=H; R2=R3=Cl
i, R1=R4=R5=H; R2=R3=OCH3
(25 - 60%)
N
Ac
j, R1=R4=R5=H; R2=OH; R3=OCH3
k, R1=R4=R5=H; R2=NO2; R3=OCH3
l, R2=R3=R5=H; R1=R4=OCH3
H3C
R1
N
R2
H3CO
H3CO
m, R1=R5=H; R2=R3=R4=OCH3
R5
1a-m
R3
R4
OCH3
Scheme 1. Synthesis of the target methylpyrazoline analogs of CA-4 (1a-m).
From the cytotoxicity data obtained four other trends were also
noted. First, for the methylpyrazoline derivatives tested, there is no
correlation between the substitution pattern of group I or II with
cytotoxicity. Second, the data suggests that substituents in the 4-
position (para) contribute to optimal cytotoxicity. It can be seen
from compounds 1f and 1j that having a methoxy substitution at
the para-position makes the compounds more potent. Third, the
hydroxyl substitution in the 3-position of compound 1j seems to
have a significant effect on improving cytotoxicity against both the
L1210 and B16 cells. The fourth trend evident from the data is that
halides and nitro groups do not increase cytotoxicity. The most
cytotoxic compound 1j was selected for further evaluations to
Fig. 3. Effects of compound 1j (6.25 mM) on interphase microtubule depolymerization in A-10 aortic smooth muscle cells.

M. Lee et al. / European Journal of Medicinal Chemistry 46 (2011) 3099e3104
3103
determine its mechanism of action, cytotoxicity to other cancer cell
Appendix. Supplementary material
lines, and aqueous solubility.
Analog 1j was sent to the NCI (National Cancer Institute) for
further testing against a panel of 60 different human cancer cell
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.03.064.
lines [18]. At a fixed concentration of 10 mM, compound 1j was
found to be cytotoxic and selective against certain human cancer
cell lines. It was most cytotoxic against SF-539 CNS cancer, MDA-
MB-435 melanoma, OVCAR-3 ovarian cancer, and HS-578T breast
cancer cells. The results are summarized in a figure given in the
supplementary materials section. In a separate study conducted in
our laboratory, the cytotoxicity of compound 1j against human
melanoma MDA-MB-435 was found using an SRB assay (48 h
References
[1] (a) G.R. Pettit, S.B. Singh, M.L. Niven, E. Hamel, J.M. Schmidt, Isolation, struc-
ture, and synthesis of combretastatins A-1 and B-1, potent new inhibitors of
microtubule assembly, derived from Combretum caffrum, J. Nat. Prod. 50
(1987) 119e131;
(b) R.T. Dorr, K. Dvorakova, K. Snead, D.S. Alberts, S.E. Salmon, G.R. Pettit,
Antitumor activity of combretastatin-A4 phosphate, a natural product tubulin
inhibitor, invest, New Drugs 14 (1996) 131e137.
exposure) to have an IC50 value of 0.90 mM [11], which is consistent
with NCI’s GI50 value for the same cells of 0.2 mM.
[2] C.M. Lin, S.B. Singh, P.S. Chu, R.O. Dempcy, J.M. Schmidt, G.R. Pettit, E. Hamel,
Interactions of tubulin with potent natural and synthetic analogs of the
antimitotic agent combretastatin: a structureeactivity study, Mol. Pharmacol.
34 (1988) 200e208.
[3] D.J. Chaplin, G.R. Pettit, S.A. Hill, Anti-vascular approaches to solid tumour
therapy: evaluation of combretastatin A4 phosphate, Anticancer Res. 19
(1999) 189e195.
[4] L. Vincent, P. Kermani, L.M. Young, J. Cheng, F. Zhang, K. Shido, G. Lam,
H. Bompais-Vincent, Z. Zhu, D.J. Hicklin, P. Bohlen, D.J. Chaplin, C. May, S. Rafii,
Combretastatin A-4 phosphate induces rapid regression of tumor neovessels
and growth through interference with vascular endothelial-cadherin
signaling, J. Clin. Invest. 115 (2005) 2992e3006.
[5] (a) G.R. Pettit, B.E. Toki, D.L. Herald, M.R. Boyd, E. Hamel, R.K. Pettit,
J.C. Chapuis, Antineoplastic agents. 410. Asymmetric hydroxylation of trans-
combretastatin A-41, J. Med. Chem. 42 (1999) 1459e1465;
(b) G.R. Pettit, M.R. Rhodes, D.L. Herald, D.J. Chaplin, M.R.L. Stratford, E. Hamel,
R.K. Pettit, J.C. Chapuis, D. Oliva, Antineoplastic agents 393. Synthesis of the trans-
isomer of combretastatin A-4 prodrug, Anticancer Drug Des. 13 (1998) 981e993.
[6] D.J. Chaplin, G.R. Pettit, C.S. Parkins, S.A. Hill, Antivascular approaches to solid
tumour therapy: evaluation of tubulin binding agents, Br. J. Cancer Suppl. 27
(1996) S86eS88.
2.4. Microtubule depolymerization
Further studies were conducted to determine the mechanism of
action for compound 1j and its methylpyrazoline derivatives. The
effect of compound 1j on interphase microtubules was examined
on A-10 aortic cells (See Fig. 3). The EC50 value, the concentration
that is required to cause 50% loss of cellular microtubules in
A-10 cells, was found to be 4.1
mM. The results were further
analyzed by calculating the ratio of EC50/IC50 (MDA-MB-435) for
analog 1j. Compounds that display a low ratio of EC50/IC50, such as
compound 4.5 for 1j, are expected to derive their cytotoxic activity
from causing microtubule depolymerization. For comparison, the
ratio of EC50/IC50 (MDA-MB-435) for CA-4 is 2.3 [11]. This suggests
that microtubule depolymerization is most likely the mechanism of
action for the methylpyrazoline analogs.
[7] (a) J.R. Wu-Wong, J.D. Alder, L. Alder, D.J. Burns, E.K.-H. Han, B. Credo, S.K. Tahir,
B.D. Dayton, P.J. Ewing, W.J. Chiou, Identification and characterization of A-
105972, an antineoplastic agent, Cancer Res. 61 (2001) 1486e1492;
(b) G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,
Isolation and structure of the Strong cell growth and tubulin inhibitor com-
bretastatin A-4, Experientia 45 (1989) 209e211;
2.5. Water solubility
(c) N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents,
Curr. Med. Chem. 10 (2003) 1697e1922;
(d) H.N. Pati, B.K. Mishra, B. Babu, S.G. Hiriyanna, Chemistry of combretastatin
and its analogs: an overview, Biomed 4 (2009) 1e22;
(e) A. Chaudhary, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni,
K.K. Verma, Combretastatin A-4 analogs as anticancer agents, Mini Rev. Med.
Chem. 7 (2007) 1186e1205;
(f) T. Brown, H. Holt Jr., M. Lee, Novel heterocyclic analogs of combretastatins,
Top. Heterocyl. Chem. 2 (2006) 1e52;
One important goal of this project was to design CA-4 analogs
that possess an increased solubility in biological media. Thus, the
lead compound 1j was subjected to a test in which the aqueous
solubility was measured. This assay utilized a modified version of
the Multi Screen Solubility Filter Plate protocol developed by Mil-
lipore [11]. The maximum aqueous solubility for the assay is
500
372
m
m
M, and the results showed that compound 1j had solubility of
M, which is slightly higher than that for CA-4, which was
(g) G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal
chemistry of combretastatin A4: present and future directions, J. Med. Chem. 49
(2006) 3033e3044.
recorded as 350 mM [11].
[8] R. LeBlanc, J. Dickson, T. Brown, M. Stewart, H.N. Pati, D. VanDerveer,
H. Arman, J. Harris, W. Pennington, H. Holt Jr., M. Lee, Synthesis and cyto-
toxicity of Epoxide and pyrazole analogs of the combretastatins, Bioorg. Med.
Chem. 13 (2005) 6025e6034.
3. Conclusion
[9] M. Johnson, B. Younglove, L. Lee, R. LeBlanc, H. Holt Jr., H. Patrice, H. Mackay,
T. Brown, S.L. Mooberry, M. Lee, Design, synthesis, and biological testing of
pyrazoline derivatives of Combretastatin-A4, Bioorg. Med. Chem. Lett. 17
(2007) 5897e5901.
[10] J. Ruprich, A. Prout, J. Dickson, B. Younglove, L. Nolan, K. Baxi, R. LeBlanc,
L. Forrest, P. Hills, H. Holt Jr., H. Mackay, T. Brown, S.L. Mooberry, M. Lee,
Design, synthesis, and biological testing of cyclohexenone derivatives of the
combretastatins, Lett. Drug Des. Disc. 4 (2007) 144e148.
[11] L. Lee, M.R. Lyda, M. Lee, R. Davis, H. Mackay, S. Chavda, B. Babu, E.L. O’Brien,
A.L. Risinger, S.L. Mooberry, M. Lee, Design, synthesis, and biological evalua-
tions of 2,5-Diaryl-2,3-Dihydro-1,3,4-Oxadiazoline analogs of Combretastatin-
A4, J. Med. Chem. 53 (2010) 325e334.
[12] (a) F. Lara-Ochoa, G. Espinosa-Perez, A new synthesis of combretastatins A-
4 and AVE-8062A, Tetrahedron Lett. 48 (2007) 7007e7010;
In conclusion, methylpyrazoline compounds 1a-m are effec-
tive analogs of combretastatin A-4. Several derivatives are
cytotoxic against a wide variety of cancer cell lines grown in
culture. The mechanism of action of the most cytotoxic analog 1j
was found to be microtubule depolymerization, consistent with
the actions of CA-4. In addition, methylpyrazoline compounds
were found to have noteworthy advantages in comparison to CA-
4, which include increased water solubility and an easier
synthetic scheme.
(b) G.R. Pettit, M.R. Rhodes, D.L. Herald, E. Hamel, J.M. Schmidt, R.K. Pettit,
Antineoplastic agents. 445. Synthesis and evaluation of structural modifica-
tions of (Z)- and (E)-combretastatin A-41, J. Med. Chem. 48 (2005) 4087e4099.
[13] (a) N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents,
Curr. Med. Chem. 10 (2003) 1697e1722;
Acknowledgements
Support from Conjura Pharmaceuticals, LLC, (ML) and Hope
College-Arnold & Mabel Beckman Scholars Program (RS) is greatly
appreciated. Support by the President’s Council.
(b) H.N. Pati, B.K. Mishra, B. Babu, S.G. Hiriyanna, Chemistry of combretastatin
and its analogs: an overview, Biomed 4 (2009) 1e22;
(c) A. Chaudhary, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni,
K.K. Verma, Combretastatin A-4 analogs as anticancer agents, Mini-Reviews
Med. Chem. 7 (2007) 1186e1205;
(d) T. Brown, H. Holt Jr., M. Lee, Synthesis of biologically active heterocyclic stilbene
and chalcone analogs of combretastatin, Top. Heterocyl. Chem. 2 (2006) 1e52;
Research Excellence award (SLM) is gratefully acknowledged.
The X-ray diffractometer at Youngstown State University was fun-
ded by NSF Grant 0087210, Ohio Board of Regents Grant CAP-491,
and by Youngstown State University.

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M. Lee et al. / European Journal of Medicinal Chemistry 46 (2011) 3099e3104
(e) G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal
chemistry of combretastatin A4: present and future directions, J. Med. Chem. 49
(2006) 3033e3044.
[17] (a) P.A. Wade, N.V. Amin, H.K. Yen, D.T. Price, G.F. Huhn, Acid-catalyzed
nitronate cycloaddition reactions. Useful syntheses and simple trans-
formations of 3-acyl- and 3-alkenylisoxazolines, J. Org. Chem. 49 (1984)
4595e4601;
[14] B. Babu, M. Lee, L. Lee, R. Strobel, O. Brockway, A. Nickols, R. Sjoholm, S. Tzou,
S. Chavda, D. Desta, G. Fraley, W. Pennington, C. Westbrook, S. Mooberry, K.
Kiakos, J.A. Hartley, M. Lee, Acetyl Analogs of Combretastatin A-4: Synthesis
and Biological Studies, Bioorg. Med. Chem, submitted.
[15] J.R. Dimmock, N.M. Kandepu, M. Hetherington, J.W. Quail, U. Pugazhenthi,
A.M. Sudom, M. Chamankhah, P. Rose, E. Pass, T.M. Allen, S. Halleran,
J. Szydlowski, B. Mutus, M. Tannous, E.K. Manavathu, T.G. Myers, E. De Clercq,
J. Balzarini, Cytotoxic activities of mannich bases of chalcones and related
compounds, J. Med. Chem. 41 (1998) 1014e1026.
(b) D. Simoni, G. Grisolia, G. Giannini, M. Roberti, R. Rondanin, L. Piccagli,
R. Baruchello, M. Rossi, R. Romagnoli, F.P. Invidiata, S. Grimaudo,
M.K. Jung, E. Hamel, N. Gebbia, L. Crosta, V. Abbadessa, A. Di Cristina,
L. Dusonchet, M. Meli, M. Tolomeo, Heterocyclic and phenyl double-
bond-locked combretastatin analogues possessing potent apoptosis-
inducing activity in HL60 and in MDR cell lines, J. Med. Chem. 48
(2005) 723e736.
[18] R.B. Boyd, The NCI in vitro anticancer drug discovery screen. in: B. Teicher
(Ed.), Anticancer Drug Development Guide: Preclinical Screening, Clinical
Trials, and Approval. Humana Press Inc., Totowa, NJ, 1997, pp. 23e42.
[16] R. Singh, H. Kaur, Advances in synthetic approaches for the preparation of
combretastatin-based anti-cancer agents, Synthesis 15 (2009) 2471e2491.