Synthesis and evaluation of 1,5-disubstituted tetrazoles as rigid analogues of combretastatin A-4 with potent antiproliferative and antitumor activity

Article abstract of DOI:10.1021/jm2013979

Tubulin, the major structural component of microtubules, is a target for the development of anticancer agents. Two series of 1,5-diaryl substituted 1,2,3,4-tetrazoles were concisely synthesized, using a palladium-catalyzed cross-coupling reaction, and identified as potent antiproliferative agents and novel tubulin polymerization inhibitors that act at the colchicine site. SAR analysis indicated that compounds with a 4-ethoxyphenyl group at the N-1 or C-5 position of the 1,2,3,4-tetrazole ring exhibited maximal activity. Several of these compounds also had potent activity in inhibiting the growth of multidrug resistant cells overexpressing P-glycoprotein. Active compounds induced apoptosis through the mitochondrial pathway with activation of caspase-9 and caspase-3. Furthermore, compound 4l significantly reduced in vivo the growth of the HT-29 xenograft in a nude mouse model, suggesting that 4l is a promising new antimitotic agent with clinical potential.

Full text of DOI:10.1021/jm2013979

Article
pubs.acs.org/jmc
Synthesis and Evaluation of 1,5-Disubstituted Tetrazoles as Rigid
Analogues of Combretastatin A-4 with Potent Antiproliferative and
Antitumor Activity
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Kimatrai Salvador,† Delia Preti,†
Mojgan Aghazadeh Tabrizi,† Andrea Brancale,‡ Xian-Hua Fu,§ Jun Li,§ Su-Zhan Zhang,§ Ernest Hamel,⊥
Roberta Bortolozzi,‡ Giuseppe Basso,∥ and Giampietro Viola*,∥
†Dipartimento di Scienze Farmaceutiche, Universita
̀
di Ferrara, 44100 Ferrara, Italy
‡The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, U.K.
§Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, The Second
Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province 310009, People’s Republic of China
∥Laboratorio di Oncoematologia, Dipartimento di Pediatria, Universita
̀
di Padova, 35131 Padova, Italy
⊥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National
Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
ABSTRACT: Tubulin, the major structural component of
microtubules, is a target for the development of anticancer
agents. Two series of 1,5-diaryl substituted 1,2,3,4-tetrazoles
were concisely synthesized, using a palladium-catalyzed cross-
coupling reaction, and identified as potent antiproliferative
agents and novel tubulin polymerization inhibitors that act at
the colchicine site. SAR analysis indicated that compounds
with a 4-ethoxyphenyl group at the N-1 or C-5 position of the
1,2,3,4-tetrazole ring exhibited maximal activity. Several of these compounds also had potent activity in inhibiting the growth of
multidrug resistant cells overexpressing P-glycoprotein. Active compounds induced apoptosis through the mitochondrial pathway
with activation of caspase-9 and caspase-3. Furthermore, compound 4l significantly reduced in vivo the growth of the HT-29
xenograft in a nude mouse model, suggesting that 4l is a promising new antimitotic agent with clinical potential.
clinical trials,6 thus stimulating significant interest in a variety of
INTRODUCTION
Microtubules are a key component of the cytoskeleton, and
they are involved in a wide range of cellular functions, including
■
combretastatin A-4 analogues.7
Previous SAR studies with analogues of 1 showed that both
the 3′,4′,5′-trimethoxy substitution pattern on the A-ring and
the cis-olefin configuration at the bridge were essential for
regulation of motility, cell division, organelle transport,
maintenance of cell morphology, and signal transduction.1
optimal activity, while B-ring structural modifications were
The essential role of microtubules in mitotic spindle formation
tolerated by the target.7 Despite the remarkable anticancer
and proper chromosomal separation makes them one of the
most attractive targets for the design and development of many
activity of 1, the cis-configured double bond is prone to
small natural and synthetic antitumor drugs.2 Many of them
isomerize to the chemically more stable trans form during
storage and metabolism, resulting in a dramatic loss in
exert their effects by inhibiting the noncovalent polymerization
antitumor activity. Thus, to retain the appropriate configuration
of the two adjacent aryl groups required for bioactivity,
heterocombretastatin derivatives with general structure 2 were
obtained by replacing the stilbene core of 1 with 1,2-diaryl
substituted five-member aromatic heterocyclic rings, such as
thiophene,8 furan,8 pyrazole,9 imidazole,9,10 thiazole,9a iso-
xazole,11 1,2,3-thiadiazole,12 and isomeric triazoles.9,13
Recently, we described a series of 1,5-diaryl-1,2,4-triazoles,
with general structure 3, as potent inhibitors of cell growth with
antimitotic activity.13c Among the synthesized compounds,
of tubulin into microtubules. Therefore, there has been great
interest in identifying and developing novel antimicrotubule
molecules. Among the naturally occurring compounds,
combretastatin A-4 (1, Chart 1) is one of the best characterized
antimitotic agents. Combretastatin A-4, isolated from the bark
of the South African tree Combretum caffrum,3 is a highly
effective natural tubulin-binding molecule affecting microtubule
dynamics by binding to the colchicine site.4 1 shows potent
cytotoxicity against a wide variety of human cancer cell lines,
including those that are multidrug resistant, based on
overexpressing P-glycoprotein (P-gp).5 A water-soluble diso-
dium phosphate derivative of 1 (named combretastatin A-4
phosphate) has shown promising results in human cancer
Received: October 18, 2011
Published: December 2, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
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Chart 1. Inhibitors and Potential Inhibitors of Tubulin Polymerization
derivatives 3a and 3b are the most active as inhibitors of the
growth of human tumor cells, with IC50 values of 5−100 and
3−20 nM for 3a and 3b, respectively. In addition, these
compounds were comparable to 1 in inhibiting tubulin
polymerization.
We examined the efficacy of the newly synthesized
compounds on a panel of human cancer cell lines, including
multidrug resistant lines overexpressing the 170 kDa P-gp drug
efflux pump, and we obtained initial in vivo data on a murine
xenograft nude mouse model that indicated high activity in
tumor growth suppression. In addition, the mechanism of
action of the most active compounds was investigated in detail.
These results led us to start a pharmacophore exploration
and optimization effort around the 1,2,4-triazole nucleus,
replacing the CH at its C-3 position with a fourth nitrogen,
to afford the bioisosteric 1,2,3,4-tetrazole derivatives with
general structure 4.14 A noteworthy point was that the
preparation of most derivatives in this series was carried out
via an efficient and flexible one-step procedure, starting from a
common N-1-(3′,4′,5′-trimethoxyphenyl)tetrazole intermediate.
For compounds 4a−p, modifications were focused on
varying the aryl moiety at the C-5 position of the tetrazole
skeleton, corresponding to the B-ring of combretastatin A-4, by
adding electron-withdrawing (COCH3) or electron-releasing
(alkyl and alkoxy) groups (EWG and ERG, respectively). In
addition, the B-ring was replaced with a thien-2-yl moiety. Since
the methoxy and ethoxy groups proved to be favorable for
bioactivity, we maintained one of these substituents at the para-
position and introduced an additional substituent (F, Cl, Me, or
MeO) at the meta-position of the phenyl ring.
In order to understand the positional effect of the 3′,4′,5′-
trimethoxyphenyl moiety on the tetrazole ring, by interchang-
ing the 4′-alkoxyaryl and 3′,4′,5′-trimethoxyphenyl moieties at
the C-5 and N-1 positions of compounds 4f and 4l, we
synthesized the corresponding regioisomeric analogues 5a and
5b, respectively. Compounds 4a−p, 5a, and 5b were
characterized by the presence of a 3′,4′,5′-trimethoxyphenyl
moiety, identical with the A-ring of 1, which was considered
essential for maximal tubulin binding activity.15
CHEMISTRY
■
1,5-Aryltetrazoles 4a−p and 5a,b were prepared following the
procedure reported in Scheme 1. The common intermediates
1-aryl substituted tetrazoles 7a−c16 were efficiently generated
in a one-step procedure by the condensation of the appropriate
aniline 6a−c with triethyl orthoformate and sodium azide in
refluxing acetic acid.17 The subsequent regioselective bromina-
tion of 7a−c with N-bromosuccinimide and a catalytic amount
of benzoyl peroxide in refluxing CCl4, furnished 5-bromote-
trazoles 8a−c with variable yields (70%, 50%, and 24% for 8a,
8b, and 8c, respectively). Starting from these latter compounds,
derivatives 4d, 4i, 4j, 4k, 4m, 5a, and 5b were synthesized by
Suzuki cross-coupling reaction with the appropriate arylboronic
acid under heterogeneous conditions [Pd(PPh3)4, aqueous
Na2CO3] in toluene at reflux.18
The low yield for the preparation of 5-bromotetrazole 8c
encouraged us to follow a concise and versatile synthetic
approach for the synthesis of 5-aryl-1-(3′,4′,5′-
trimethoxyphenyl)tetrazoles 4a−c, 4e−h, 4l, and 4n−p by
the direct arylation of 7c with the appropriate aryl iodide in the
presence of Pd(OAc)2, CuI, and CsCO3, with tri-(2-furyl)-
phosphine (TFP) as phosphine ligand in dry acetonitrile.19
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Journal of Medicinal Chemistry
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a
31-fold more active than its 1,2,3-triazole congener 3a, and the
regioisomeric derivative 5b was from 15- to 50-fold more active
than 3a.
Scheme 1
The unsubstituted C-5 phenyl derivative 4a was weakly
active (IC50 > 0.9 μM) against all the cell lines screened, with
the exception of the Jurkat cells, with which its IC50 was 3.8
nM. This activity against the Jurkat cells was completely lost
when the phenyl moiety was replaced with the bioisosteric 2-
thienyl ring (compound 4b).
Further structural optimization was conducted with variation
in the ERG or EWG on the phenyl group at the 5-position of
tetrazole ring. With the exception of the Jurkat, the A549, and
the MCF-7 cells, the introduction of a weak electron-
withdrawing acetyl group (4c) produced a slight increase of
activity with respect to 4a. Comparing the two p-alkylphenyl
derivatives 4d and 4e, replacing the methyl (4d) with an ethyl
(4e) moiety resulted in enhanced antiproliferative activities in
five of the cell lines. Both 4d and 4e were substantially more
potent in all cells, except the Jurkat cells, than the unsubstituted
4a. Replacement of the p-methyl group of 4d with a p-methoxy
moiety (4f) resulted in a 10- to 150-fold increase in
antiproliferative activity. Similarly, changing the p-ethyl moiety
of 4e to a p-ethoxy group in 4l resulted in enhanced
antiproliferative activity against all six cell lines. In addition,
4l was substantially more active than 4f in half the cell lines
(A549, Jurkat, and MCF-7 cells) and somewhat more active in
the others. In combination with data where still longer p-alkoxy
groups were examined (see below) and the data for 4e (p-ethyl
moiety), we conclude that the optimum length for the para-
substituent appears to be three non-hydrogen atoms.
Relative to the activity of 4f, the insertion of an additional
EWG or ERG on the 3′-position of the 4′-methoxyphenyl ring
had varying effects on antiproliferative activity, suggesting that
steric rather than electronic factors account for the differing
potencies of these compounds. A second, and especially a third,
strong electron-releasing methoxy group (4g and 4h, the
methoxy groups present at the meta position(s)) caused
substantial loss in antiproliferative activity relative to 4f. In most
of the cell lines, 4k with the weak electron-releasing m-methyl
group was more potent than compounds 4i and 4j, with the
electron-withdrawing fluorine and chlorine atoms, respectively.
By comparison of 4j with 4f, the addition of a m-chlorine atom
in 4j to the p-methoxy group in both compounds led to little
change in activity in four cell lines, while 4f was the more active
in the Jurkat and the HT-29 cells. The effect of a m-EWG (a
chlorine atom) in compound 4m was also examined in
combination with the p-ethoxy group of 4l. As with the 4f
and 4j pair, nearly identical activities were observed in most cell
lines, the exception being the A549 cells, in which 4l was the
more active.
Lengthening the p-alkyl moiety from ethoxy (4l) to propoxy
(4n) caused a significant reduction in antiproliferative activity
in all cell lines. A further increase in size of this moiety (4o and
4p) resulted in inactive compounds.
Inhibition of Tubulin Polymerization and Colchicine
Binding. To investigate whether the antiproliferative effects of
compounds 4e−f, 4i−n, and 5a,b were derived from
interactions with tubulin, these agents were evaluated for
their inhibition of tubulin polymerization and for effects on the
binding of [3H]colchicine to tubulin (Table 2).20 For
comparison, 1 and 3a were examined in contemporaneous
experiments. In the assembly assay, compound 4l was found to
be the most active (IC50 = 1.1 μM), with activity being in the
a
Reagents: (a) NaN3, HC(OEt)3, CH3CO2H, reflux; (b) N-
bromosuccinimide, benzoyl peroxide (cat.), CCl4, reflux; (c) ArB-
(OH)2, Pd(PPh3)4, Na2CO3, PhMe−H2O−EtOH, 120 °C; (d) ArI,
Pd(OAc)2, CuI, TFP, CsCO3, CH3CN, 45 °C.
RESULTS AND DISCUSSION
■
In Vitro Antiproliferative Activities. The 1-(3′,4′,5′-
trimethoxyphenyl)-5-aryltetrazoles 4a−p and the correspond-
ing regioisomeric analogues 5a and 5b were evaluated for their
inhibition of the growth of six different human cancer cell lines
in comparison with the 1,2,4-triazole derivative 3a and the
reference compound 1. As reported in Table 1, the
antiproliferative activities of the tested compounds were less
pronounced against A549 cells than the other cell lines.
Compound 4l, bearing a 4′-ethoxyphenyl at the C-5 position of
tetrazole ring, and its isomeric derivative 5b exhibited the
greatest antiproliferative activity among the tested compounds,
with IC50 values of 1.3−8.1 and 0.3−7.4 nM, respectively.
These values were similar to those obtained with 1 against HL-
60, while 4l and 5b were from 2- to 1000-fold more active than
1 against the other five cell lines. While there was little
difference in activity between 4l and 5b against each cancer cell
line, this similarity did not occur with the other isomeric pair,
the 4′-methoxyphenyl derivatives 4f and 5a. Compound 5a was
from 4- to 50-fold less active than 4f in five of the six cancer cell
lines, the exception being the MCF-7 cells, in which 5a was
somewhat more active.
Notably, by comparison of 3a and 4l, which shared common
3′,4′,5′-trimethoxyphenyl and 4′-ethoxyphenyl moieties at their
N-1 and C-5 positions, the 1,2,3,4-tetrazole 4l was from 8- to
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Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds 1, 3a, 4a−p, and 5a,b
a
IC50 (nM)
compd
HeLa
A549
HL-60
Jurkat
MCF-7
>10000
HT-29
>10000
>10000
4a
4b
4c
4d
4e
4f
4097 333
>10000
>10000
903 123
>10000
3.8 0.7
>10000
>10000
>10000
648 211
255 32.3
29.0 5.6
2.6 0.0
259 36.0
>10000
>10000
596 121
329 41
53.1 10
2.9 0.6
488 71
>10000
325 28
222 17
55.1 4.4
2.6 0.1
345 28
>10000
>10000
6033 1527
673 43
182 60
4.6 0.6
355 55
>10000
3420 127
3383 659
222 59
3574 280
>10000
1785 653
12.6 6.7
38.5 5.8
2716 625
>10000
4g
4h
4i
2.8 0.7
2.3 0.5
2.1 0.7
1.9 0.7
2.6 0.8
11.1 3.2
>10000
332 17
402 84
37.7 5.0
8.1 3.2
28.8 5.6
495 24
>10000
7.3 2.2
3.6 0.6
2.0 0.7
1.3 0.3
0.8 0.09
43.3 14.5
>10000
1.3 0.1
10.0 0.9
4.1 0.2
0.16 0.06
0.23 0.06
47.0 0.06
>10000
29.7 5.9
25.2 3.4
15.4 6.5
2.3 0.8
1.8 0.6
66.2 13.4
>10000
2.7 0.7
35.9 4.4
5.6 1.5
2.8 0.7
1.6 0.6
105 47
>10000
4j
4k
4l
4m
4n
4o
4p
5a
5b
3a
1
6404 1906
952 122
0.3 0.09
>10000
>10000
>10000
>10000
>10000
>10000
40.3 16.6
0.7 0.09
209 86
0.26 0.08
27.1 8.2
2.8 0.9
265 26
3.1 1.2
n.d.
7.4 2.2
100 20
180 50
15
4
4
20
1
3
5
5
0.2
0.6
50
9
1
0.2
370 100
3100 100
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean SE from the dose−response
curves of at least three experiments.
(70−78% inhibition), and they were slightly less potent than 1,
which in these experiments inhibited colchicine binding by
99%. For compounds 4e−f, 4j−k, and 4m,n, inhibition of
colchicine binding ranged from 46% to 58%, about half as
active as 1 in this assay. In this series of 10 compounds,
inhibition of [3H]colchicine binding correlated more closely
with inhibition of tubulin assembly than with antiproliferative
activity.
Effects of 4l, 4m, and 5b on Drug Resistant Cell
Lines. Drug resistance is an important therapeutic problem
caused by the emergence of tumor cells possessing different
mechanisms that confer resistance against a variety of
anticancer drugs.21,22 Among the more common mechanisms
are those related to the overexpression of a cellular membrane
protein called P-gp that mediates the efflux of various
structurally unrelated drugs.21,22 In this context, we evaluated
sensitivity to the most active compounds (4l, 4m, and 5b) of
two multidrug-resistant cell lines, one derived from a
lymphoblastic leukemia (CEMVbl‑100), the other derived from
a colon carcinoma (LovoDoxo). Both these lines express high
levels of the P-gp.23,24 As shown in Table 3, the three
compounds were equally potent toward parental cells and cells
resistant to vinblastine or doxorubicin showing a resistance
index, which is the ratio between IC50 values of resistant cells
and sensitive cells, of about 1.
Table 2. Inhibition of Tubulin Polymerization and
Colchicine Binding by Compounds 1, 3a, 4e,f, 4i−n, and
5a,b
a
b
tubulin assembly
colchicine binding
% inhibition SD
compd
IC50 SD (μM)
4e
4f
3.7 0.5
2.5 0.3
2.1 0.2
3.5 0.2
2.5 0.1
1.1 0.1
2.0 0.2
3.1 0.4
2.1 0.3
2.0 0.1
0.76 0.1
1.3 0.1
47
3
54 0.3
60 0.7
4i
4j
50
59
78
58
46
1
3
3
2
4
4k
4l
4m
4n
5a
5b
3a
1
70 0.1
71
86
2
2
99 0.4
a
Inhibition of tubulin polymerization. Tubulin was at 10 μM.
b
Inhibition of [3H]colchicine binding. Tubulin, colchicine, and tested
compound were at 1, 5, and 5 μM, respectively.
range of those of 1 and 3a (IC50 of 1.3 and 0.76 μM,
respectively). The remaining compounds were also good
inhibitors of tubulin polymerization, with IC50 values ranging
from 2 to 4 μM, so they were somewhat less active than 1 and
3a.
While this group of compounds was highly potent in
inhibition of cell growth and tubulin assembly, correlation
between these two assays was imperfect, a finding that is
frequently observed. Thus, compound 4l was the best inhibitor
of tubulin assembly, being 2-fold more potent than its isomeric
derivative 5b in this assay, but 4l and 5b were equipotent as
inhibitors of cell growth.
Resistance to microtubule inhibitors may also be mediated by
changes in the levels of expression of different β-tubulin
isotypes and by tubulin gene mutations that result in modified
tubulin with impaired polymerization properties. A-549-T12 is
a cell line with an α-tubulin mutation with increased resistance
to paclitaxel.25 Compounds 4l, 4m, and 5b had greater relative
activity than paclitaxel in this cell line, suggesting that these
compounds might be useful in the treatment of drug refractory
tumors resistant to other antitubulin drugs.
In the colchicine binding studies, derivatives 4l and 5a,b had
quantitatively similar effects, varying within a narrow range
Molecular Modeling. Molecular docking simulations were
performed on this series of compounds to understand the role
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Journal of Medicinal Chemistry
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larger groups, both in the meta and para positions (e.g., 4g−h
and 4o−p) did not dock successfully, probably because of a
steric clash with components of the binding pocket. These
results correlate well with the biological data observed for these
compounds.
Table 3. In Vitro Cell Growth Inhibitory Effects of
Compounds on Drug Resistant Cell Lines
a
IC50 (nM)
b
compd
LoVo
LoVoDoxo
resistance ratio
4l
1.1 0.9
0.7 0.06
15.7 6.5
96.9 42.6
13150 210
0.64
1.3
Analysis of Cell Cycle Effects. The effects of different
concentrations of compounds 4l, 4m, and 5b after 24 h of
treatment on cell cycle progression were examined in HeLa
cells (Figure 2A). The two isomers 4l and 5b caused a
significant G2/M arrest in a concentration-dependent manner,
with a rise in G2/M cells occurring at a concentration as low as
30 nM, while at higher concentrations more than 80% of the
cells were arrested in G2/M. This maximum was reached with
the compounds at 60 nM. There was a concomitant decrease of
cells in the other phases of the cell cycle (G1 and S). Treatment
with compound 4m produced similar G2/M arrest, but a
somewhat higher concentration was required.
4m
12.5 2.1
118.2 60.5
120 30
5b
0.8
c
doxorubicin
109.6
a
IC50 (nM)
CEMVbl100
b
CEM
resistance ratio
4l
3.1 1.0
0.7 0.1
3.8 1.4
4.1 0.2
0.7 0.09
0.7 0.2
0.5 0.1
230 32
0.2
4m
5b
1.0
0.1
c
vinblastine
56.1
a
IC50 (nM)
b
A549
A549-T12
resistance ratio
We next studied the association between 4l-induced G2/M
arrest and alterations in expression of proteins that regulate cell
division. Cell arrest at the prometaphase/metaphase to
anaphase transition is normally regulated by the mitotic
checkpoint.26 The major regulator of the G2 to M transition
is M phase promoting factor (MPF), a complex made up of the
catalytic subunit of cdc2 and the regulatory subunit of cyclin B.
This and other regulatory complexes are activated at different
points during the cell cycle and can also be regulated by several
exogenous factors. Cyclin B/cdc2 complexes are held in an
inactive state by phosphorylation of cdc2 at two negative
regulatory sites (Thr14 and Tyr15). Dephosphorylation of
these negative regulatory sites is needed to activate the cdc2/
cyclin B complex. cdc25c is a major phosphatase that
dephosphorylates both sites on cdc2 to activate its MPF
activity.27,28 As shown in Figure 2B in HeLa cells, 4l caused a
marked increase in cyclin B1 expression after both 24 and 48 h
treatments. In addition, slower migrating forms of phosphatase
cdc25c appeared at 24 h, indicating changes in the
phosphorylation state of this protein, followed by an overall
decrease in total cdc25c at 48 h. The phosphorylation of cdc25c
directly stimulates its phosphatase activity, and this is necessary
to activate cdc2/cyclin B on entry of cells into mitosis.27,28 In
addition, we observed dephosphorylation of cdc2 (Tyr15),
particularly at 24 h, which should result in inhibition of
formation of the cdc2/cyclinB complex.
4l
8.1 3.27
28.8 5.6
7.4 2.2
7.2 0.1
8.2 5.0
1.0
4m
5b
31.5 11.4
4.2 1.8
1.1
0.6
c
paclitaxel
75.2 12.5
10.4
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%. Data are expressed as the mean SE from the
dose−response curves of at least three independent experiments. The
values express the ratio between IC50 values determined in resistant
b
c
and nonresistant cell lines. Data from ref 13c.
of the tetrazole ring in the binding interaction with tubulin,
using the same procedures reported previously.13c Figure 1
Compounds 4l and 5b Induce Apoptosis. To charac-
terize the mode of cell death induced by 4l and 5b, a
biparametric cytofluorimetric analysis was performed using
propidium iodide (PI), which stains DNA and enters only dead
cells, and fluorescent immunolabeling of the protein annexin-V,
which binds to phosphatidyl serine (PS) in a highly selective
manner.29 Dual staining for annexin-V and with PI permits
discrimination between live cells (annexin-V−/PI−), early
apoptotic cells (annexin-V+/PI−), late apoptotic cells (annex-
in-V+/PI+), and necrotic cells (annexin-V−/PI+) . As depicted in
Figure 3, both compounds induced an accumulation of
annexin-V positive cells in comparison with the control, in a
concentration and time-dependent manner.
Effect of 4l and 5b on Mitochondrial Depolarization.
Mitochondria play an essential role in the propagation of
apoptosis.30 It is well established that at an early stage,
apoptotic stimuli alter the mitochondrial transmembrane
potential (Δψmt). Δψmt was monitored by the fluorescence of
the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcar-
bocyanine (JC-1). With normal cells (high Δψmt), JC-1 displays
Figure 1. Proposed binding mode of 4l (purple) and 5b (orange) with
DAMA-colchicine (green) in the β-tubulin binding site.
shows that the proposed binding of these compounds is very
similar to the pose of the cocrystallized DAMA-colchicine, with
the trimethoxyphenyl rings placed in proximity to βCys241
(amino acid residue numbers follow the modeling convention).
Furthermore, the tetrazole is placed toward the external part of
the pocket, and it does not seem to establish any specific
interaction with tubulin. Indeed, the binding poses of the two
isomers 4l and 5b are virtually identical. Another important
consideration is that the binding modes of the tetrazole
analogues were very similar to those observed for the triazole
compounds 3a,b.13c With the current compounds, only
relatively small substituents were tolerated in the meta position
of the second phenyl ring (e.g., 4i−k), while analogues with
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Figure 2. (A) Effect of compounds 4l, 4m, and 5b on cell cycle distribution of HeLa cells. Cells were treated with different concentrations of the
indicated compounds, ranging from 15 to 250 nM for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry.
Data are presented as the mean SEM of three independent experiments. (B) Effect of 4l on G2/M regulatory proteins. HeLa cells were treated for
24 or 48 h with the indicated concentration of 4l. The cells were harvested and lysed for the detection of cyclin B, p-cdc2Y15, and cdc25c expression
by Western blot analysis. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.
a red fluorescence (590 nm). This is caused by spontaneous
and local formation of aggregates that are associated with a
large shift in the emission. In contrast, when the mitochondrial
membrane is depolarized (low Δψmt), JC-1 forms monomers
that emit at 530 nm. As shown in Figure 4, both 4l and 5b
induced a time and concentration-dependent increase in the
proportion of cells with depolarized mitochondria.
Mitochondrial membrane depolarization is associated with
mitochondrial production of ROS.31 Therefore, we investigated
whether ROS production increased after treatment with 4l and
5b. We utilized two fluorescence indicators: hydroethydine
(HE), whose fluorescence appears if ROS are generated,32 and
the dye 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA),
which is oxidized to the fluorescent compound dichlorofluor-
escein (DCF) by a variety of peroxides, including hydrogen
peroxide.32
through the mitochondrial pathway. In this context, triazole
derivative 3a also induces apoptosis through the intrinsic
pathway.13c
Compound 4l Induces Caspases Activation. Caspases,
which are proteolytic enzymes, are the central executioners of
apoptosis, and their activation is mediated by various
inducers.33 Synthesized as proenzymes, caspases are themselves
activated by specific proteolytic cleavage reactions. Caspase-2,
-8, -9, and -10 are termed apical caspases and are usually the
first to be activated in the apoptotic process. Following their
activation, they in turn activate effector caspases, in particular
caspase-3.34 Following treatment of HeLa cells with compound
4l for 24 or 48 h, we observed activation of caspase-3 and of the
caspase-3 substrate poly ADP-ribose polymerase (PARP)
(Figure 6A). In addition, the expression of X-linked inhibitor
of apoptosis protein (XIAP), a member of the apoptosis
inhibitors protein family, was strongly reduced concomitant
with caspase activation (Figure 6B). Consistent with the Δψmt
results described above, 4l treatment induced activation of
caspase-9, the major initiator caspase of the intrinsic
(mitochondrial) apoptosis pathway (Figure 6A).
The results presented in Figure 5 (panels B and C) show that
both 4l and 5b induced the production of significant amounts
of ROS in comparison with control cells, which agrees with the
previously described dissipation of Δψmt. Altogether, these
results indicate that these compounds induced apoptosis
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Figure 3. (A) Representative histograms of HeLa cells treated with 4l (100 nM) for 48 h and analyzed by flow cytometry after double staining of the
cells with annexin-V-FITC and PI. (B, C) Percentage of cells found in the different regions of the biparametric histograms obtained from
cytofluorimetric analysis, after incubation with 4l (B) or 5b (C) for 24 or 48 h, as indicated.
We also examined whether the induction of apoptosis by 4l
was associated with changes in the expression of two proteins
of the Bcl-2 family, since there is increasing evidence that they
share the signaling pathways induced by antimicrotubule
compounds.35 However, our results showed (Figure 6B) that
the antiapoptotic protein Bcl-2 was only slightly affected after
48 h of treatment, whereas 4l treatment had no effect on the
expression of the proapoptotic protein Bax.
reduction in tumor growth was statistically significant as early as
the day after the third dose of 4l, suggesting a rapid, effective
delivery of compound to the tumor mass. It is interesting to
note that the effect on tumor volume reduction by 4l was better
than that of 1, which caused a 43.7% of reduction at day 24.
During the whole treatment period, no significant weight
changes occurred in the treated animals (Figure 7B), suggesting
that 4l has minimal toxicity.
In Vivo Antitumor Activity of Compound 4l. To
evaluate the in vivo antitumor activity of 4l, human colon
adenocarcinoma xenografts were established by subcutaneous
injection of HT29 cells in the backs of nude mice. Once the
HT-29 xenografts reached a size of ∼300 mm3, 18 mice were
randomly assigned to one of the three groups. In two of the
groups, compound 4l or reference compound 1, dissolved in
DMSO, was injected intraperitoneally at a dose of 100 mg/kg.
Both drugs, as well as the vehicle control, were administered 3
times a week for 1 week. As shown in Figure 7 A, compound 4l
immediately caused a remarkable reduction in tumor growth,
and this reached a 66% reduction by the end of the observation
period compared with administration of vehicle only. The
CONCLUSIONS
■
We demonstrated that the 1,2,3,4-tetrazole ring is a suitable
mimic for the cis-olefinic configuration present in 1. N-1-
(3′,4′,5′-Trimethoxyphenyl)tetrazole was the key intermediate
for the preparation of many active compounds via a parallel
synthesis. The observed antiproliferative activities depended on
the substitution pattern on the phenyl at the 5-position of the
tetrazole ring. The methoxy and ethoxy groups at the para-
position, especially the latter, enhanced biological activity. The
introduction of an additional EWG, such as F or Cl, or weak
ERG (Me) group at the meta-position of this 4′-methoxy
phenyl ring led to little change or further increased
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Figure 4. Assessment of Δψmt after treatment of HeLa cells with compounds 4l and 5b. (A) Representative histograms of both control cells and cells
incubated 48 h in the presence of 4l, as indicated, and stained with the fluorescent probe JC-1 after treatment. The horizontal axis shows fluorescence
intensity of the JC-1 monomer, and the vertical axis shows fluorescence of JC-1 aggregates. (B, C). Induction of loss of mitochondrial membrane
potential after 24 or 48 h incubations with compound 4l (B) or 5b (C). Data are expressed as the mean SEM of three independent experiments.
antiproliferative activity, but a m-methoxy group caused
significant decrease in potency. The p-ethoxyphenyl derivative
4l and its isomeric derivative 5b had antiproliferative activities
with IC50 values below 10 nM (0.3−8.1 nM). Replacement of
the p-ethoxy group with larger alkoxy groups was detrimental
for antiproliferative activity. These results imply that the 3′-
hydroxy-4′-methoxyphenyl moiety of 1 (B-ring) can be
replaced by a 4′-ethoxyphenyl group. Compound 4l was a
strong inhibitor of tubulin polymerization (IC50 = 1.1 μM) and
strongly inhibited the binding of colchicine to tubulin (78%
inhibition), with activities not greatly different from those of 1.
We also showed that the tetrazole system that characterizes
compound 4l retained, or even improved on, the biological
activity of the 1,2,4-triazole analogue 3a previously reported.
The 3′-chloro-4′-ethoxyphenyl derivative 4m and the two
isomeric 4′-ethoxyphenyl derivatives 4l and 5b were shown to
arrest the cell cycle at the G2/M phase and induce apoptosis
through the mitochondrial (intrinsic) pathway. Importantly, the
antitumor efficacy of 4l was demonstrated in a xenograft-
bearing mouse. In this model, there was significant inhibition of
tumor growth with only three doses of 4l, and the treated
animals had no significant weight loss. Our results demon-
strated that 4l is a promising new tubulin binding agent and is
worthy of further evaluation as potential chemotherapeutic
agent.
EXPERIMENTAL SECTION
■
Chemistry. Materials and Methods. 1H NMR spectra were
recorded on a Bruker AC 200 spectrometer. Chemical shifts (δ) are
given in ppm upfield from tetramethylsilane as internal standard, and
the spectra were recorded in appropriate deuterated solvents, as
indicated. Positive-ion electrospray ionization (ESI) mass spectra were
recorded on a double-focusing Finnigan MAT 95 instrument with BE
geometry. Melting points (mp) were determined on a Buchi-Tottoli
apparatus and are uncorrected. All products reported showed 1H
NMR spectra in agreement with the assigned structures. The purity of
tested compounds was determined by combustion elemental analyses
conducted by the Microanalytical Laboratory of the Department of
Chemistry of the University of Ferrara, Italy, with a Yanagimoto MT-5
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Figure 5. Mitochondrial production of ROS in HeLa cells following treatment with compound 4l (A, B) or compound 5b (C, D). After the 24 or 48
h incubations, cells were stained with HE (A, C) or H2-DCFDA (B, D) and analyzed by flow cytometry. Data are expressed as the mean SEM of
three independent experiments.
Figure 6. (A) Western blot analysis of caspase-3, cleaved caspase-9,
and cleaved PARP after treatment of HeLa cells with 4l at the
indicated concentration and for the indicated times. (B) Western blot
analysis of Bcl-2, Bax, and XIAP after treatment of HeLa cells with 4l
at the indicated concentration and for the indicated times. To confirm
equal protein loading, each membrane was stripped and reprobed with
anti-β-actin antibody.
Figure 7. Inhibition of human xenograft growth in vivo by compound
4l. (A) HT29 tumor-bearing nude mice were administered the vehicle,
as control, or 100 mg/kg 4l or 1. Injections were given intra-
peritoneally on days 0, 2, and 4 (indicated by arrows). The figure
shows the average measured tumor volumes (A) and body weights of
the mice (B) recorded on the indicated days after treatments. Data are
expressed as the mean SEM of tumor volume and body weight at
each time point for six animals per group: ∗, p < 0.01 vs control.
CHN recorder elemental analyzer. All tested compounds yielded data
consistent with a purity of at least 95% compared with the theoretical
values. All reactions were carried out under an inert atmosphere of dry
nitrogen, unless otherwise indicated. Standard syringe techniques were
used for transferring dry solvents. Reaction courses and product
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mixtures were routinely monitored by TLC on silica gel (precoated
F254 Merck plates), and compounds were visualized with aqueous
KMnO4. Flash chromatography was performed using 230−400 mesh
silica gel and the indicated solvent system. Organic solutions were
dried over anhydrous Na2SO4. Anilines 6a−c, aryl iodides, and
arylboronic acids are commercially available and used as received. The
synthesis of 4-pentyloxyiodobenzene was performed as previously
described.:36
General Procedure A for the Synthesis of 1-Aryl Substituted
1H-Tetrazoles 7a−c. A stirred suspension of the appropriate aniline
6a−c (14 mmol), triethyl orthoformate (3.7 mL, 22.5 mmol), and
sodium azide (1.36 g., 21 mmol) in acetic acid (15 mL) was refluxed
for 4 h. The mixture was cooled, the solvent was removed in vacuo and
the residue dissolved in a mixture of dichloromethane (50 mL) and 0.1
N aqueous HCl (20 mL). The organic phase was washed with water
(20 mL), brine (10 mL), dried over Na2SO4, and concentrated. The
residue was suspended in ethyl ether (15 mL), stirred for 30 min, and
filtered to afford the desired product.
General Procedure D for the Direct Arylation of 1-(3,4,5-
Trimethoxyphenyl)-1H-tetrazole (7c). A suspension of 1-(3,4,5-
trimethoxyphenyl)-1H-tetrazole 7c (236 mg, 1.00 mmol), the
appropriate aryl iodide (1.00 mmol), cesium carbonate (358 mg, 1.1
mmol), copper(I) iodide (190 mg, 1 mmol), palladium(II) acetate
(11.2 mg, 0.05 mmol), and TFP (23.2 mg, 0.1 mmol) in dry
acetonitrile (5 mL) was heated under argon for 6 h at 45 °C. The
resulting mixture was diluted with dichloromethane (10 mL) and
filtered through Celite, and the solvents were removed under reduced
pressure. The residue was dissolved in a mixture of dichloromethane
(15 mL) and 0.1 N aqueous HCl (5 mL). The organic phase was
washed with water (5 mL) and brine (5 mL), dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (mixture of petroleum ether−
ethyl acetate as eluent) to afford the title compound.
1-(3,4,5-Trimethoxyphenyl)-5-phenyl-1H-tetrazole (4a). Fol-
lowing general procedure D, the crude residue was purified by flash
chromatography, using ethyl acetate−petroleum ether, 2:8 (v/v), as
eluent, to furnish 4a as a brown solid (48% yield), mp 148−149 °C.
1H NMR (CDCl3) δ: 3.76 (s, 6H), 3.91 (s, 3H), 6.59 (s, 2H), 7.46 (m,
1-(3,4,5-Trimethoxyphenyl)-1H-tetrazole (7c). Following gen-
eral procedure A, compound 6c was isolated as a gray solid (yield
3H), 7.64 (m, 2H). MS (ESI): [M]+ = 312.6. Anal. (C16H16N4O3) C,
H, N.
1
>95%), mp 172−173 °C. H NMR (CDCl3) δ: 3.89 (s, 6H), 3.93 (s,
3H), 6.89 (s, 2H), 8.96 (s, 1H).
1-(3,4,5-Trimethoxyphenyl)-5-(thiophen-2-yl)-1H-tetrazole
(4b). Following general procedure D, the crude residue was purified
by flash chromatography, using ethyl acetate−petroleum ether, 3:7
(v.v), as eluent to furnish 4b as a pale brown solid (54% yield), mp
197−199 °C. 1H NMR (CDCl3) δ: 3.84 (s, 6H), 3.95 (s, 3H), 6.67 (s,
2H), 7.08 (dd, J = 5.2 and 4.0 Hz, 1H), 7.39 (dd, J = 3.6 and 1.0 Hz,
1H), 7.54 (dd, J = 5.2 and 1.6 Hz, 1H). MS (ESI): [M]+ = 318.6. Anal.
(C14H14N4SO3) C, H, N.
General Procedure B for the Synthesis of 5-Bromo-1-aryl
Substituted 1H-Tetrazoles 8a−c. To a suspension of the
appropriate 1-aryl substituted tetrazole 7a−c (10 mmol) in carbon
tetrachloride (100 mL) heated at reflux were added N-bromosucci-
nimide (3.56 g., 20 mmol) and benzoyl peroxide (120 mg, 5 mmol).
The reflux continued for 4 h, and another portion of benzoyl peroxide
was added to the mixture (120 mg, 5 mmol). After 18 h, the solution
was diluted with dichloromethane (100 mL) and subsequently washed
with 5% aqueous Na2S2O3 (50 mL), water (50 mL), brine (50 mL),
dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel using the indicated eluent
mixture to afford the desired product.
1-(4-(1-(3,4,5-Trimethoxyphenyl)-1H-tetrazol-5-yl)phenyl)-
ethanone (4c). Following general procedure D, the crude residue
was purified by flash chromatography, using ethyl acetate−petroleum
ether, 4:6 (v/v), as eluent, to furnish 4c as a pale brown solid (62%
1
yield), mp 146−147 °C. H NMR (CDCl3) δ: 2.63 (s, 3H), 3.77 (s,
5-Bromo-1-(4-methoxyphenyl)-1H-tetrazole (8a). Following
general procedure B, starting from 1-(4-methoxyphenyl)-1H-tetrazole
7a, the crude residue was purified by column chromatography on silica
gel (petroleum ether−ethyl acetate, 2:8) to afford 8a as a yellow solid.
6H), 3.92 (s, 3H), 6.58 (s, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.98 (d, J =
8.8 Hz, 2H). MS (ESI): [M]+ = 354.8. Anal. (C18H18N4O4) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-p-tolyl-1H-tetrazole (4d). Fol-
lowing general procedure C, the crude residue was purified by flash
chromatography, using ethyl acetate−petroleum ether, 3:7 (v/v), as
eluent, to furnish 4d as a pale brown solid (56% yield), mp 98−100
°C. 1H NMR (CDCl3) δ: 2.39 (s, 3H), 3.77 (s, 6H), 3.92 (s, 3H), 6.59
(s, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H). MS (ESI):
[M]+ = 326.8. Anal. (C17H18N4O3) C, H, N.
1
Yield 49%, mp 110−111 °C. H NMR (CDCl3) δ: 3.90 (s, 3H), 7.06
(d, J = 9.0 Hz, 2H), 7.45 (d, J = 9.0 Hz, 2H).
5-Bromo-1-(4-ethoxyphenyl)-1H-tetrazole (8b). Following
general procedure B, starting from 1-(4-ethoxyphenyl)-1H-tetrazole
7b, the crude residue was purified by column chromatography on silica
gel (petroleum ether−ethyl acetate, 2:8) to afford 8b as a gray solid.
Yield 70%, mp 116−117 °C. 1H NMR (CDCl3) δ: 1.47 (t, J = 6.8 Hz,
3H), 4.13 (q, J = 6.8 Hz, 2H), 7.04 (dd, J = 8.6 and 2.2 Hz, 2H), 7.44
(dd, J = 8.6 and 2.2 Hz, 2H).
1-(3,4,5-Trimethoxyphenyl)-5-(4-ethylphenyl)-1H-tetrazole
(4e). Following general procedure D, the crude residue was purified
by flash chromatography, using ethyl acetate−petroleum ether, 3:7 (v/
v), as eluent, to furnish 4e as a pale brown solid (52% yield), mp 156−
157 °C. 1H NMR (CDCl3) δ: 1.24 (t, J = 7.6 Hz, 3H), 2.71 (q, J = 7.6
Hz, 2H), 3.76 (s, 6H), 3.91 (s, 3H), 6.59 (s, 2H), 7.23 (d, J = 8.6 Hz,
2H), 7.55 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ = 340.7. Anal.
(C18H20N4O3) C, H, N.
5-Bromo-1-(3,4,5-trimethoxyphenyl)-1H-tetrazole (8c). Fol-
lowing general procedure B, starting from 1-(3,4,5-trimethoxyphenyl)-
1H-tetrazole 7c, the crude residue was purified by column
chromatography on silica gel (petroleum ether−ethyl acetate, 3:7)
1
to afford 8c as a brown solid. Yield 24%, mp 130−131 °C. H NMR
1-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-1H-tetra-
zole (4f). Following general procedure D, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4f as a yellow solid (49% yield),
(CDCl3) δ: 3.91 (s, 6H), 3.92 (s, 3H), 6.74 (s, 2H).
General Procedure C for the Arylation of 5-Bromo-1-aryl
Substituted 1H-Tetrazoles 8a−c. A solution of 5-bromo-1-aryl
substituted 1H-tetrazole 8a−c (0.5 mmol), the appropriate arylboronic
acid (1 mmol, 2 equiv), and 2 M aqueous Na2CO3 (0.5 mL) in a
mixture of toluene (5 mL) and ethanol (0.5 mL) was purged with
nitrogen for 5 min. Tetrakis(triphenylphosphine)palladium (54 mg,
0.05 mmol) was added and the mixture heated with stirring at 120 °C
under argon for 18 h. After this time, the suspension was cooled to
ambient temperature, diluted with dichloromethane (10 mL), and
filtered through a pad of Celite, and the filtrate was evaporated in
vacuo. The residue was dissolved with dichloromethane (20 mL), and
the resultant solution was washed sequentially with 2 M aqueous
Na2CO3 (10 mL), water (10 mL), and brine (10 mL). The organic
layer was dried, filtered, and evaporated, and the residue was purified
by flash chromatography on silica gel, using a mixture of ethyl acetate−
petroleum ether as eluent.
1
mp 144−145 °C. H NMR (CDCl3) δ: 3.78 (s, 3H), 3.84 (s, 3H),
3.86 (s, 3H), 3.92 (s, 3H), 6.60 (s, 2H), 6.91 (d, J = 9.0 Hz, 2H), 7.56
(d, J = 9.0 Hz, 2H). MS (ESI): [M]+ = 342.9. Anal. (C17H18N4O4) C,
H, N.
1-(3,4,5-Trimethoxyphenyl)-5-(3,4-dimethoxyphenyl)-1H-
tetrazole (4g). Following general procedure D, the crude residue
was purified by flash chromatography, using ethyl acetate−petroleum
ether, 4:6 (v/v), as eluent, to furnish 4g as a pale brown solid (54%
1
yield), mp 158−159 °C. H NMR (CDCl3) δ: 3.78 (s, 6H), 3.82 (s,
3H), 3.84 (s, 3H), 3.91 (s, 3H), 6.63 (s, 2H), 6.83 (d, J = 8.6 Hz, 1H),
7.02 (dd, J = 8.6 and 2.0 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H). MS (ESI):
[M]+ = 372.9. Anal. (C18H20N4O5) C, H, N.
1,5-Bis(3,4,5-trimethoxyphenyl)-1H-tetrazole (4h). Following
general procedure D, the crude residue was purified by flash
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chromatography, using ethyl acetate−petroleum ether, 4:6 (v/v), as
eluent, to furnish 4h as a white solid (56% yield), mp 135−136 °C. 1H
NMR (CDCl3) δ: 3.71 (s, 6H), 3.81 (s, 3H), 3.85 (s, 3H), 3.88 (s,
3H), 3.90 (s, 3H), 6.65 (s, 2H), 6.87 (s, 2H). MS (ESI): [M]+ = 402.9.
Anal. (C19H22N4O6) C, H, N.
ether, 4:6 (v/v), as eluent, to furnish 5a as a white solid (64% yield),
mp 120−121 °C. H NMR (CDCl3) δ: 3.69 (s, 6H), 3.88 (s, 3H),
1
3.89 (s, 3H), 6.81 (s, 2H), 7.02 (d, J = 9.0 Hz, 2H), 7.34 (d, J = 9.0
Hz, 2H). MS (ESI): [M]+ = 342.8. Anal. (C17H18N4O4) C, H, N.
5-(3,4,5-Trimethoxyphenyl)-1-(4-ethoxyphenyl)-1H-tetra-
zole (5b). Following general procedure C, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 5b as a pale brown solid (54%
5-(3-Fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
1H-tetrazole (4i). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate−petroleum
ether, 4:6 (v/v), as eluent, to furnish 4i as a pale brown solid (49%
1
yield), mp 94−95 °C. H NMR (CDCl3) δ: 1.41 (t, J = 6.8 Hz, 3H),
1
yield), mp 171−172 °C. H NMR (CDCl3) δ: 3.79 (s, 6H), 3.91 (s,
3.68 (s, 6H), 3.87 (s, 3H), 4.10 (q, J = 6.8 Hz, 2H), 6.81 (s, 2H), 7.00
(d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H). MS (ESI): [M]+ = 356.9.
Anal. (C18H20N4O4) C, H, N.
3H), 3.92 (s, 3H), 6.58 (s, 2H), 6.96 (t, J = 8.0 Hz, 1H), 7.37 (m, 2H).
MS (ESI): [M]+ = 360.4. Anal. (C17H17FN4O4) C, H, N.
5-(3-Chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
1H-tetrazole (4j). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4j as a yellow solid (52% yield),
Biology. Materials and Methods. Antiproliferative Assays.
Human T-leukemia (Jurkat) and human promyelocytic leukemia (HL-
60) cells were grown in RPMI-1640 medium (Gibco, Milano, Italy).
Breast adenocarcinoma (MCF7), human non-small-cell lung carcino-
ma (A549), human cervix carcinoma (HeLa), and human colon
adenocarcinoma (HT-29) cells were grown in DMEM medium
(Gibco, Milano, Italy), all supplemented with 115 units/mL penicillin
G (Gibco, Milano, Italy), 115 μg/mL streptomycin (Invitrogen,
Milano, Italy), and 10% fetal bovine serum (Invitrogen, Milano, Italy).
LoVoDoxo cells are a doxorubicin resistant subclone of LoVo cells22 and
were grown in complete Ham’s F12 medium supplemented with
doxorubicin (0.1 μg/mL). CEMVbl‑100 are a multidrug-resistant line
selected against vinblastine.23 A549-T12 are non-small-cell lung
carcinoma cells exhibiting resistance to paclitaxel.24 They were
grown in complete DMEM medium supplemented with paclitaxel
(12 nM). Stock solutions (10 mM) of the different compounds were
obtained by dissolving them in DMSO. Individual wells of a 96-well
tissue culture microtiter plate were inoculated with 100 μL of complete
medium containing 8 × 103 cells. The plates were incubated at 37 °C
in a humidified 5% CO2 incubator for 18 h prior to the experiments.
After medium removal, 100 μL of fresh medium containing the test
compound at different concentrations was added to each well and
incubated at 37 °C for 72 h. The percentage of DMSO in the medium
never exceeded 0.25%. This was also the maximum DMSO
concentration in all cell-based assays described below. Cell viability
was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide test as previously described.37 The IC50 was defined as
the compound concentration required to inhibit cell proliferation by
50%, in comparison to cells treated with the maximum amount of
DMSO (0.25%) and considered as 100% viability.
Effects on Tubulin Polymerization and on Colchicine
Binding to Tubulin. To evaluate the effect of the compounds on
tubulin assembly in vitro,20a varying concentrations of compounds
were preincubated with 10 μM bovine brain tubulin in glutamate
buffer at 30 °C and then cooled to 0 °C. After 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
followed turbidimetrically at 350 nm. The IC50 was defined as the
compound concentration that inhibited the extent of assembly by 50%
after a 20 min incubation. The capacity of the test compounds to
inhibit colchicine binding to tubulin was measured as described20b
except that the reaction mixtures contained 1 μM tubulin, 5 μM
[3H]colchicine, and 5 μM test compound.
1
mp 175−177 °C. H NMR (CDCl3) δ: 3.80 (s, 6H), 3.92 (s, 3H),
3.94 (s, 3H), 6.61 (s, 2H), 6.91 (d, J = 8.6 Hz, 1H), 7.46 (dd, J = 8.4
and 2.2 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H). MS (ESI): [M]+ = 376.8.
Anal. (C17H17ClN4O4) C, H, N.
5-(3-Methyl-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
1H-tetrazole (4k). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4k as a pale brown solid (51%
1
yield), mp 152−153 °C. H NMR (CDCl3) δ: 2.19 (s, 3H), 3.78 (s,
6H), 3.84 (s, 3H), 3.92 (s, 3H), 6.62 (s, 2H), 6.79 (d, J = 8.6 Hz, 1H),
7.34 (dd, J = 8.6 and 1.4 Hz, 1H), 7.54 (d, J = 1.4 Hz, 1H). MS (ESI):
[M]+ = 356.8. Anal. (C18H20N4O4) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetra-
zole (4l). Following general procedure D, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4l as a pale brown solid (53%
yield), mp 170−171 °C. 1H NMR (CDCl3) δ: 1.43 (t, J = 7.0 Hz, 3H),
3.78 (s, 6H), 3.92 (s, 3H), 4.07 (q, J = 7.0 Hz, 2H), 6.61 (s, 2H), 6.91
(d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H). MS (ESI): [M]+ = 356.9.
Anal. (C18H20N4O4) C, H, N.
5-(3-Chloro-4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
1H-tetrazole (4m). Following general procedure C, the crude
residue was purified by flash chromatography, using ethyl acetate−
petroleum ether, 3:7 (v/v), as eluent, to furnish 4m as a red solid (52%
1
yield), mp 75−77 °C. H NMR (CDCl3) δ: 1.49 (t, J = 7.0 Hz, 3H),
3.80 (s, 6H), 3.92 (s, 3H), 4.13 (q, J = 7.0 Hz, 2H), 6.61 (s, 2H), 6.90
(d, J = 8.6 Hz, 1H), 7.39 (dd, J = 8.6 and 2.2 Hz, 1H), 7.76 (d, J = 2.2
Hz, 1H). MS (ESI): [M]+ = 390.9. Anal. (C18H19ClN4O4) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-(4-propoxyphenyl)-1H-tetra-
zole (4n). Following general procedure D, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4n as a yellow solid (49% yield),
1
mp 152−154 °C. H NMR (CDCl3) δ: 1.04 (t, J = 7.2 Hz, 3H), 1.84
(m, 2H), 3.79 (s, 6H), 3.92 (s, 3H), 4.03 (q, J = 7.2 Hz, 2H), 6.61 (s,
2H), 6.92 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H). MS (ESI):
[M]+ = 370.8. Anal. (C19H22N4O4) C, H, N.
5-(4-Butoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-tetra-
zole (4o). Following general procedure D, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 2:8 (v/v), as eluent, to furnish 4o as a white solid (51% yield),
1
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 8.
The tubulin structure was downloaded from the PDB (http://www.
rcsb.org/, PDB code 1SA0).38 Hydrogen atoms were added to the
protein, using Molecular Operating Environment (MOE),39 and
minimized keeping all the heavy atoms fixed until a rmsd gradient of
0.05 kcal mol−1 Å−1 was reached. Ligand structures were built with
MOE and minimized using the MMFF94x force field until a rmsd
gradient of 0.05 kcal mol−1 Å−1 was reached. The docking simulations
were performed using PLANTS.40
Flow Cytometric Analysis of Cell Cycle Distribution. For flow
cytometric analysis of DNA content, 5 × 105 HeLa cells in exponential
growth were treated with different concentrations of the test
compounds for 24 and 48 h. After the incubation period, the cells
were collected, centrifuged, and fixed with ice-cold ethanol (70%). The
mp 133−134 °C. H NMR (CDCl3) δ: 0.97 (t, J = 7.0 Hz, 3H), 1.52
(m, 2H), 1.84 (m, 2H), 3.78 (s, 6H), 3.92 (s, 3H), 3.98 (q, J = 7.0 Hz,
2H), 6.61 (s, 2H), 6.90 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H).
MS (ESI): [M]+ = 384.8. Anal. (C20H24N4O4) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-(4-(pentyloxy)phenyl)-1H-tet-
razole (4p). Following general procedure D, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
ether, 3:7 (v/v), as eluent, to furnish 4p as a pale brown solid (54%
yield), mp 143−144 °C. 1H NMR (CDCl3) δ: 0.93 (t, J = 7.0 Hz, 3H),
1.44 (m, 4H), 1.82 (m, 2H), 3.78 (s, 6H), 3.92 (s, 3H), 3.97 (q, J = 7.0
Hz, 2H), 6.61 (s, 2H), 6.90 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 9.0 Hz,
2H). [M]+ = 398.9. Anal. (C21H26N4O4) C, H, N.
5-(3,4,5-Trimethoxyphenyl)-1-(4-methoxyphenyl)-1H-tetra-
zole (5a). Following general procedure C, the crude residue was
purified by flash chromatography, using ethyl acetate−petroleum
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Journal of Medicinal Chemistry
Article
cells were then treated with lysis buffer containing RNase A and 0.1%
Triton X-100 and then stained with PI. Samples were analyzed on a
Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms
were analyzed using MultiCycle for Windows (Phoenix Flow
Systems).
Annexin-V Assay. Surface exposure of PS on apoptotic cells was
measured by flow cytometry with a Coulter Cytomics FC500
(Beckman Coulter) by adding annexin-V-FITC to cells according to
the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostics).
Simultaneously, the cells were stained with PI. Excitation was set at
488 nm, and the emission filters were at 525 and 585 nm, respectively,
for FTIC and PI.
Assessment of Mitochondrial Changes. The mitochondrial
membrane potential was measured with the lipophilic cationic dye JC-
1 (Molecular Probes), as described.13c The production of ROS was
measured by flow cytometry using either HE (Molecular Probes) or
H2DCFDA (Molecular Probes), as previously described.13c
39-(0)49-8211451; fax, 39-(0)49-8211462; e-mail, giampietro.
viola1@unipd.it.
ABBREVIATIONS USED
■
EWG, electron-withdrawing group; ERG, electron-releasing
group; P-gp, P-glycoprotein; TFP, tri-(2-furyl)phosphine;
MPF, M phase promoting factor; PI, propidium iodide; PS,
phosphatidylserine; Δψmt, mitochondrial transmembrane po-
tential; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimida-
zolcarbocyanine; ROS, reactive oxygen species; HE, hydro-
ethidine; H2-DCFDA, 2,7 dichlorodihydrofluorescein diacetate;
DCF, dichlorofluorescein; PARP, poly ADP-ribose polymerase;
XIAP, X-linked inhibitor of apoptosis protein; ESI, electrospray
ionization; PBS, phosphate buffered saline; SDS−PAGE,
sodium dodecyl sulfate−polyacrylamide gel electrophoresis
Western Blot Analysis. HeLa cells were incubated in the
presence of test compounds and, after different times, were collected,
centrifuged, and washed two times with ice cold phosphate buffered
saline (PBS). The pellet was then resuspended in lysis buffer. After the
cells were lysed on ice for 30 min, lysates were centrifuged at 15000g at
4 °C for 10 min. The protein concentration in the supernatant was
determined using the BCA protein assay reagents (Pierce, Italy). Equal
amounts of protein (20 μg) were resolved using sodium dodecyl
sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) (7.5−15%
acrylamide gels) and transferred to PVDF Hybond-P membrane (GE
Healthcare). Membranes were blocked with I-Block (Tropix), the
membrane being gently rotated overnight at 4 °C. Membranes were
then incubated with primary antibodies against Bcl-2, Bax, cleaved
PARP, cleaved caspase-9, p-cdc2Tyr15, cdc25c (Cell Signaling), caspase-
3 (Alexis), cyclin B (Upstate), or β-actin (Sigma-Aldrich) for 2 h at
room temperature. Membranes were next incubated with peroxidase-
labeled secondary antibodies for 60 min. All membranes were
visualized using ECL Advance (GE Healthcare) and exposed to
Hyperfilm MP (GE Healthcare). To ensure equal protein loading,
each membrane was stripped and reprobed with anti-β-actin antibody.
Antitumor Activity in Vivo. Four-week-old female BALB/c-nu
nude mice (15−18 g) were obtained from Shanghai SLAC Laboratory
Animal Co., Ltd. (Shanghai, China). The animals were maintained
under specific pathogen-free conditions with food and water supplied
ad libitum at Zhejiang University, Traditional Chinese Medicine
Laboratory Animal Center. Human colon adenocarcinoma HT-29 cells
in logarithmic growth phase were resuspended in RPMI 1640 without
fetal bovine serum at 1 × 107cells/mL and inoculated (0.2 mL) in the
hypodermis of the pars dorsalis of each mouse. Once the HT-29
xenografts reached a size of ∼300 mm3, 18 mice were randomly
assigned to either the control group or one of two treatment groups.
Compounds 4l and 1 were prepared in DMSO and injected
intraperitoneally at 0.01 mL/g body weight to give a dose of 100
mg/kg. The drugs or vehicle was administered 3 times a week for 1
week. After completing the treatment schedule and the evaluation
period, tumor-bearing mice were euthanized. Tumor volume was
calculated by the formula V = (LW2)/2 where L is the length and W is
the width of the tumor nodules measured by vernier caliper. The study
was approved by the Institutional Animal Ethical Committee of the
Second Affiliated Hospital, School of Medicine, Zhejiang University
(PRC).
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AUTHOR INFORMATION
■
Corresponding Author
*For R.R.: phone, 39-(0)532-455303; fax, 39-(0)532-455953;
e-mail, rmr@unife.it. For P.G.B.: phone, 39-(0)532-455293;
fax, 39-(0)532-455953; e-mail, pgb@unife.it. For G.V.: phone,
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