1,2,3,4-Tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4

Article abstract of DOI:10.1016/j.ejmech.2007.11.030

Eleven 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4 (CA-4) were synthesized and their cytotoxicity against the growth of two murine cancer cell lines (B16 melanoma and L1210 leukemia) in culture was determined using an MTT assay. Two 2-thioxopyrimidine analogs 8f and 9a exhibited significant activity (IC₅₀ < 1 μM for L1210 and <10 μM for B16 cells). Exposure of A-10 cells to 8f and 9a produced a significant reduction in cellular microtubules in interphase cells, with an EC₅₀ value of 4.4 and 2.9 μM, respectively, for microtubule loss. Molecular modeling studies using MacSpartan indicated that the two active 2-thioxopyrimidine analogs preferably adopt a twisted conformation, similar to CA-4, affirming that conformation and structure are connected to activity.

Full text of DOI:10.1016/j.ejmech.2007.11.030

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European Journal of Medicinal Chemistry 43 (2008) 2011e2015
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Short communication
1,2,3,4-Tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4
Lauren Lee a, Ryan Davis a, Jenna Vanderham a, Patrice Hills b,
Hilary Mackay a, Toni Brown a, Susan L. Mooberry b, Moses Lee a,
*
a Department of Chemistry and the Division of Natural and Applied Sciences, Hope College, 35E, 12th street, Holland, MI 49423, United States
b Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, TX 78227, United States
Received 23 September 2007; received in revised form 28 November 2007; accepted 29 November 2007
Available online 14 December 2007
Abstract
Eleven 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4 (CA-4) were synthesized and their cytotoxicity against
the growth of two murine cancer cell lines (B16 melanoma and L1210 leukemia) in culture was determined using an MTT assay. Two 2-
thioxopyrimidine analogs 8f and 9a exhibited significant activity (IC50 < 1 mM for L1210 and <10 mM for B16 cells). Exposure of A-10 cells
to 8f and 9a produced a significant reduction in cellular microtubules in interphase cells, with an EC50 value of 4.4 and 2.9 mM, respectively, for
microtubule loss. Molecular modeling studies using MacSpartan indicated that the two active 2-thioxopyrimidine analogs preferably adopt
a twisted conformation, similar to CA-4, affirming that conformation and structure are connected to activity.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Tubulin; Combretastatin; Chalcone; Thiourea; Cytotoxicity
Vascular targeting agents (VTAs), such as combretastatin-
A4 (CA-4, 1, Fig. 1), are effective antitumor agents. VTAs
rapidly and specifically disrupt the abnormal tumor vascula-
ture, resulting in vascular collapse and tumor necrosis [1].
There are data to suggest that the antivascular actions might
be mediated through the vascular endothelial-cadherin signal-
ing pathway [1b]. CA-4 (1) is a natural product extracted from
the African Willow Tree, Combretum caffrum and it inhibits
tubulin polymerization by interacting with the colchicine bind-
ing site on tubulin [2]. This alters the morphology of endothe-
lial cells and causes vascular shutdown and regression of
tumor vasculature. However, the use of CA-4 (1) as a clinical
antitumor agent is limited by its low bioavailability and poor
aqueous solubility [3]. These drawbacks have led to the devel-
opment of water-soluble derivatives and analogs as depicted in
Fig. 1, which include a phosphate-containing pro-drug (CA-
4P, 2), an amino analog 3 [5a] and an amino acid derivative
(AVE-8062, 4) [4]. These structural analogs have proven to
be effective VTAs and they have antitumor actions alone
and in combination with current cancer treatments such as
cytotoxic chemotherapy, radiation, radio-immunotherapy, and
anti-angiogenic agents [1].
Further examples of cytotoxic analogs of CA-4, 1, that have
been reported include furanones [5b,6a], isoxazoles [6b], im-
idazoles [6c], triazoles [6d], azetidinones [6e], pyrazoles
(e.g., 5) [6f], pyrazolines (e.g., 6) [7], and cyclohexenones
(e.g., 7) [8]. The latter three heterocyclic derivatives of CA-
4 (1) were synthesized in the author’s laboratory and their
structures are given in Fig. 1. The compounds showed varying
degrees of cytotoxic potency, but the pyrazole-compound 5
showed a significant loss in potency against murine cancer
cells growing in culture. The X-ray crystallography structure
of a close analog of pyrazole 5 (in which the eOH group is
replaced with an eOCH3 group) [6f] revealed that the com-
pound adopted a planar conformation, lacking the twisted ge-
ometry of CA-4 needed to bind optimally to tubulin [9]. The
planar conformation of 3,5-diarylpyrazoles was also predicted
from molecular modeling studies [7,8]. Interestingly, the pyr-
azoline 6 and cyclohexenone 7 compounds have significant cy-
totoxicity and they cause a major loss of cellular microtubules
* Corresponding author. Tel.: þ1 616 392 7190; fax: þ1 616 392 7923.
E-mail address: lee@hope.edu (M. Lee).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.030

2012
L. Lee et al. / European Journal of Medicinal Chemistry 43 (2008) 2011e2015
S
N
NH
OH
HN
NH
R1
H3CO
H3CO
H3CO
OR
H3CO
H3CO
R2
R3
OCH3
A
B
H3CO
OCH3
OCH3
R5
5
OCH3
1, R=H (CA-4)
R4
OCH3
2, R=PO3-2 (CA-4P)
Type I
8a, R1=R5=H; R2=R3=R4=OCH3
8b, R2=R4=H; R1=R3=R5=OCH3
8c, R1=R4=R5=H; R2=R3=OCH3
8d, R2=R3=R5=H; R1=R4=OCH3
8e, R1=R3=R4=R5=H; R2=OCH3
8f, R1=R4=R5=H; R3=OCH3; R2=OH
8g, R1=R4=R5=H; R2=NO2; R3=OCH3
8h, R1=R2=R4=R5=H; R3=NO2
8i, R1=R2=R4=R5=H; R3=Cl
N
NH
OH
H3CO
H3CO
H3CO
NHR
OCH3
H3CO
OCH3
OCH3
OCH3
6
3, R=H (AC-7739)
4, R=amino acid (AVE-8062)
S
O
R1
OCH3
HN
NH
R2
R3
H3CO
H3CO
OH
R5
OCH3
OCH3
R4
OCH3
Type II
7
9a, R2=R4=H; R1=R3=R5=OCH3
9b, R1=R4=R5=H; R2=NO2; R3=OCH3
Fig. 1. Structures of combretastatin-A4 (CA-4, 1) and its water-soluble derivatives 2e4 and analogs 5e7, including pyrazole, pyrazoline and cyclohexenone de-
rivatives of chalcones, 5e7, respectively. Two general types of 1,2,3,4-tetrahydro-2-thioxopyrimidine, I for compounds 8aei and II for 9a,b.
in A-10 cells [7,8]. Molecular modeling studies confirmed that
both compounds 6 and 7 preferred twisted geometries [2].
Despite past successes in designing analogs of CA-4, 1, that
are more soluble in biological media, the task of creating mol-
ecules that are equally potent as an anticancer agent as CA-4
has been far more challenging. As part of a program to develop
novel heterocyclic analogs of CA-4, a series of novel 1,2,3,4-
tetrahydro-2-thioxopyrimidine analogs was designed with the
purpose of examining their cytotoxic properties and to corre-
late the results with the conformational ‘‘twist’’ of the mole-
cules. An additional objective for synthesizing this class of
compounds is to search for analogs of CA-4 that have good wa-
ter solubility and are biologically active. 1,2,3,4-Tetrahydro-2-
thioxopyrimidine analogs are attractive for our studies because
only a small handful of 1,2,3,4-tetrahydro-2-thioxopyrimidine
have been made and reported [10a], and none has been
Fig. 2. Energy optimized structures of two 1,2,3,4-tetrahydro-2-thioxopyrmidine analogs: 9a (left) and 8f (right), type II and I, respectively.

L. Lee et al. / European Journal of Medicinal Chemistry 43 (2008) 2011e2015
2013
Table 1
Cytotoxicity of compounds 8aei (type I) and 9a,b (type II) against the growth
investigated as potential analogs of CA-4 [10b]. Eleven specific
analogs were synthesized and they are divided into two types: I,
which contains a 3,4,5-trimethoxyphenyl moiety in the A-ring
(8aei), and II, containing a 2,5-dimethoxyphenyl group in the
A-ring (9a,b). The conformation of analogs, 8f and 9a, were as-
sessed by molecular modeling studies using MacSpartan,
a strategy that was reported earlier [7,8]. The molecular struc-
tures were optimized by molecular mechanics (MMFF) using
a molecular equilibrium conformer procedure. The equilibrium
geometry was subsequently optimized using Hatree-Fock (3-
21 G) calculations, followed by a density-functional calcula-
tion (B3LYP and 6-31 G). The conformations of compounds
8f and 9a are shown in Fig. 2, and it is evident that the mole-
cules adopt a twisted geometry similar to that of CA-4 [9].
The 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs 8aei
and 9a,b were synthesized by reaction of the appropriate chal-
cone [11] with 2.5 mol equivalents of thiourea and potassium
carbonate in refluxing ethanol (overnight), Scheme 1. Upon
completion, the cooled reaction mixture was poured into ice-
water and the precipitate was collected. When needed, the
precipitate was recrystallized from methanol. The chemical
yields were between 40e90% and the structures were ascer-
tained by 400 MHz 1H NMR, infrared, and mass spectrometry
measurements.
of murine cancer cells grown in culturea
Compound
IC50 (mM) (type I)
B16
L1210
Ar¼
8a
>100
>100
8b
6.3
>100
36
5.7
8c
56.3
32
8d
8e
59.5
6.0
>100
8f
0.5
The cytotoxicity of the newly synthesized compounds was
tested in an in vitro 72-h MTT assay [12]. The analogs were
tested against L1210 and B16 cell lines (murine leukemia
and melanoma, respectively) and the compound concentration
(mM) required to reduce the cell population by 50% relative to
an untreated control (IC50 value) was determined. The results
are shown in Table 1. It is worthy to note that the thioxopyr-
imidine derivatives have good solubility in cell culture media,
ca. >17.5 mM.
8g
8h
>100
>100
55.5
>100
>100
8i
48.3
Compound
IC50 (mM) (type II)
B16
L1210
The cytotoxicity results showed three trends. First, three an-
alogs (8b,f and 9a) displayed significant activity against both
cell lines, comparable to the pyrazoline [7] and cyclohexenone
[8] analogs reported earlier; however, L1210 cells were gener-
ally more sensitive to the agents. It is reasonable to expect com-
pound 8f of the type I design to be active, since analogs 5
[5b,6], 6 [7], and 7 [8], and CA-4, (1), which have a similar sub-
stitution pattern are highly active. Second, the activity of the
analogs that contain a 2,4,6-trimethoxy substituted B-ring is in-
teresting; particularly as compound 9a, a type II molecule,
which has a completely different substitution pattern from
CA-4 (1), is as potent as 8f. Analysis of the molecular models
Ar¼
9a
6.1
42
0.4
2.2
9b
a
IC50 of CA-4 (1) against L1210 and B16 are 0.002 and 0.003 mM, respec-
tively [13,14].
S
O
HN
NH
Thiourea
K2CO3
H3CO
H3CO
OH
H3CO
OH
EtOH, reflux,
overnight
OCH3
H3CO
OCH3
OCH3
OCH3
8f
Scheme 1. A representative synthesis of 1,2,3,4-tetrahydro-2-thioxopyrimidine 8f.

2014
L. Lee et al. / European Journal of Medicinal Chemistry 43 (2008) 2011e2015
given in Fig. 2 provided a plausible explanation, in which the
conformation of 9a is twisted due to steric hindrance between
ortho-methoxy groups on both phenyl groups. This explanation
is consistent with the lack of activity for compound 8a, a type I
molecule that contains a 3,4,5-trimethoxy substituted pattern in
the B-ring. Finally, as with many of the analogs of CA-4 (1) that
have been synthesized and tested, the nitro-group containing
compounds were generally inactive even at 100 mM, except
compound 9b which gave IC50 values of 2.2 and 42 mM for
L1210 and B16, respectively.
In summary, the novel 1,2,3,4-tetrahydro-2-thioxopyrimi-
dine compounds 8aei and 9a,b, analogs of CA-4 (1), have
been found to have significant cytotoxic activity against cancer
cells grown in culture. Even though the IC50 values of the ‘‘ac-
tive’’ compounds are larger (or less potent) than CA-4 itself, 1,
the new compounds have significant advantages: enhanced
solubility in aqueous biological media and ease of synthesis.
Acknowledgements
To gain insight into the mechanism of action for the 2-
thioxopyrimidine analogs, the effects of compounds 8f and
9a on interphase cellular microtubules were evaluated in A-
10 aortic smooth muscle cells [8,]. The results given in Fig. 3
provided clear evidence that compound 8f was very active,
with an EC50 value of 4.4 mM (effective concentration to cause
50% loss of cellular microtubules). Compound 9a was also
very potent (data not shown) and it gave an EC50 value of
2.9 mM. With the EC50 values being comparable to the cytotox-
icity IC50 values, the data suggest that microtubule disruption is
a likely mechanism for both compounds to exert their biologi-
cal activity. For comparison, CA-4 is still significantly more
potent in disrupting microtubules under similar conditions,
with an EC50 value of 0.007 mM [9a]. These results provide ad-
ditional evidence to support our earlier suggestions [7,8] that
the conformational ‘‘twist’’ of CA-4 analogs as well as the
composition and positioning of substituents are important pa-
rameters for biological activity.
The authors are grateful to Taiho Pharmaceutical Co. of
Japan, Hope College and the William Randolph Hearst Foun-
dation (SLM) for support.
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