Synthesis and antiproliferative evaluation of pyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives

Article abstract of DOI:10.1016/j.bmc.2009.03.007

Pyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives 1a-c were prepared from 4-(N-methyl-N-phenylamino)-2-methylsulfanylpyrazolo[1,5-a]-1,3,5-triazin e 2. Their cytotoxic activity, inhibition of tubulin polymerization, and cell cycle effects were evaluat

Full text of DOI:10.1016/j.bmc.2009.03.007

Bioorganic & Medicinal Chemistry 17 (2009) 3471–3478
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative evaluation of pyrazolo[1,5-a]-1,3,5-triazine
myoseverin derivatives
Florence Popowycz a, Cédric Schneider a, Salvatore DeBonis b, Dimitrios A. Skoufias b, Frank Kozielski c,
Carlos M. Galmarini d, Benoît Joseph a,
*
a Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR—CNRS 5246, Université de Lyon, Université Claude Bernard—Lyon 1, Bâtiment Curien, 43 Boulevard du 11
Novembre 1918, F-69622 Villeurbanne, France
b Institut de Biologie Structurale (CEA-CNRS-UJF), 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France
c The Beastson Institute for Cancer Research, Switchback Road, Bearsden, G61 1BD Glasgow, UK
d ENS-CNRS UMR 5239, UFR de Médecine Lyon-Sud, 165 chemin du Grand Revoyet, BP 12, 69921 Oullins, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives 1a–c were prepared from 4-(N-methyl-N-phenyla-
mino)-2-methylsulfanylpyrazolo[1,5-a]-1,3,5-triazine 2. Their cytotoxic activity, inhibition of tubulin
polymerization, and cell cycle effects were evaluated. Compounds 1a and 1c are potent tubulin inhibitors
and displayed specific antiproliferative activity in colorectal cancer cell lines at micromolar
concentrations.
Received 12 November 2008
Revised 2 March 2009
Accepted 3 March 2009
Available online 10 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Myoseverin
Pyrazolo[1,5-a]-1,3,5-triazine
Tubulin
Cancer
1. Introduction
P2Y1 receptor antagonist compared to the purine analogue.8 In
our group, we recently reported the design of pyrazolo[1,5-a]-
Myoseverin (Fig. 1) has been initially identified as a new micro-
tubule assembly inhibitor with moderate cytostatic activity from a
library of 2,6,9-trisubstituted purines.1 The same compound was
shown to convert multinucleated myotubes to proliferative myo-
blast-like cells by disrupting microtubule assembly.1a,2 Recently,
myoseverin was also described as a potential inhibitor of new ves-
sel growth by inhibiting endothelial cell function and differentia-
tion of progenitor cells.3 Further optimization of myoseverin led
to the preparation of two new myoseverin derivatives,1b,4 8-aza-
myoseverin and pyrazolo[4,3-d]pyrimidine (E2GG) analogues that
contain similar structural motifs (Fig. 1).5 E2GG displayed higher
antiproliferative activity compared to myoseverin in several hu-
man cancer cell lines. Trisubstituted 1,3,5-triazine derivatives6
such as tubulyzine were also designed with potent microtubule
disassembly properties.
1,3,5-triazine (R)-roscovitine bioisostere which displayed signifi-
cantly higher biological potency compared to purine (R)-
roscovitine.9
In this article, we investigated the synthesis of novel pyrazol-
o[1,5-a]-1,3,5-triazine myoseverin-like molecules 1a–c (Fig. 1) in
order to compare their antiproliferative and tubulin inhibitory
activities with those obtained for myoseverin.
2. Chemistry
The synthesis of 2,4,8-trisubstituted pyrazolo[1,5-a]-1,3,5-tria-
zines 1a–c is reported in Schemes 1 and 2. The starting material
4-(N-methyl-N-phenylamino)-2-(methylsulfanyl)pyrazolo[1,5-a]-
1,3,5-triazine 210 was oxidized by m-CPBA to afford the sulfonyl
derivative 3 in fair yield (78%). Nucleophilic aromatic substitution
was performed in the presence of 3 and 4-methoxybenzylamine in
excess in dioxane at 140 °C for 24 h (sealed tube). The final com-
pound 1a was obtained in 70% yield. The derivative 1b was also
prepared from 2. Bromination of 2 occurred regioselectively on
the C-8 position (NBS, CHCl3) to afford 4 in 82% yield. Oxidation
of 4 (84% yield), followed by a double addition–elimination reac-
tion on 5 in the presence of 4-methoxybenzylamine gave 1b in
70% yield. For the preparation of 1c, the alkyl chain was first
According to the literature,7 the purine nucleus and the pyraz-
olo[1,5-a]-1,3,5-triazine ring are structurally close and possess
strikingly similar biophysicochemical properties (electrostatic po-
tential surface, isopotential curve, charges). Improved metabolic
stability was also observed for the pyrazolo[1,5-a]-1,3,5-triazine
* Corresponding author. Tel.: +33 (0)4 72 44 81 35; fax: +33 (0)4 72 43 12 14.
E-mail address: benoit.joseph@univ-lyon1.fr (B. Joseph).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.007

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F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
OMe
OMe
HN
HN
N
H
N
N
N
N
Y
N
N
N
H
N
N
H
MeO
MeO
Y = CH myoseverin
Y = N 8-azamyoseverin
E2GG
OMe
OMe
HN
HN
N
N
N
N
N
N
N
H
N
N
H
NH
R
MeO
MeO
1a R = H
1b R = Br
1c R = CH(Me)2
tubulyzine
Figure 1. Chemical structures of myoseverin, 8-azamyoseverin, E2GG, tubulyzine and 1a–c.
built-up on the pyrazolo[1,5-a]-1,3,5-triazine moiety. The 8-acetyl-
4-(N-methyl-N-phenylamino)-2-(methylsulfanyl)pyrazolo[1,5-a]-
1,3,5-triazine 610 was treated with methylmagnesium bromide,
then the magnesium complex was smoothly hydrolyzed with
NH4Cl. Without further purification, the crude tertiary alcohol
was submitted to a dehydroxylation reaction in a NaBH4/TFA mix-
OMe
Ph
N
4-methoxybenzylamine
dioxane, 140 °C, 24 h
m-CPBA, CH2Cl2
0 °C to rt, 3 h
N
N
N
N
sealed tube
HN
N
N
N
78%
70%
S
N
N
N
N
O
O
S
N
3
N
N
MeO
2
H
1a
NBS, CHCl3
rflx, 2 h
82%
OMe
Ph
N
4-methoxybenzylamine
dioxane, 140 °C, 24 h
sealed tube
N
m-CPBA, CH2Cl2
0 °C to rt, 3 h
N
N
N
HN
N
N
N
70%
84%
N
S
N
N
N
S
N
Br
N
H
O
O
4
Br
N
MeO
5
Br
1b
Scheme 1.

F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
3473
1) MeMgBr, Et2O
2) NH4Cl
3) NaBH4, TFA
CH2Cl2, 0 °C to rt
N
N
N
N
N
N
m-CPBA, CH2Cl2
0 °C to rt, 3 h
N
N
N
N
N
N
N
N
N
65%
77%
S
S
S
O
O
O
6
7
8
4-methoxybenzylamine
dioxane, 140 °C, 24 h
sealed tube
79%
OMe
HN
N
N
N
N
N
MeO
H
1c
Scheme 2.
ture in CH2Cl2 to afford 7 in 77% two step yield. As reported for 1a
and 1b, oxidation of 7 (65% yield) followed by the SNAr reaction on
8 afforded 1c in good yield.
3.2. Cell cycle analysis
To characterize the effects of pyrazolo[1,5-a]-1,3,5-triazine
derivatives on the cell cycle, proliferating colorectal cancer cell
lines (HCT116, SW48 and SW480) were treated with 1a, 1b, 1c
3. Results and discussion
and myoseverin at 30 lM and analyzed by flow cytometry (Table
2). Myoseverin was found to arrest the cell cycle at the G2/M
phases in the three analyzed cell lines. Similarly, by 24 h of treat-
ment with 1c, a substantial number of cells were arrested at G2/
M phases at levels similar to those observed for myoseverin. In
contrast, 1a and 1b that presented a lower antiproliferative activity
than myoseverin in these cell lines, also showed a lower amount of
cells blocked at G2/M phases.
3.1. Growth inhibitory effects of myoseverin, 1a, 1b and 1c
Cell growth inhibition of myoseverin and pyrazolo[1,5-a]-1,3,5-
triazine derivatives 1a–c was evaluated using different cancer cell
lines. As shown in Table 1, myoseverin was active at low
lM ranges
(2.7–11.5 M) in all the tested cell lines being most active in the
l
colorectal cancer cell lines (HCT116, SW48 and SW480). Com-
pound 1c displayed a higher antiproliferative activity in colorectal
cancer cell lines and in CEM cells. The cell growth inhibition activ-
ity of 1c observed in these cell lines was similar to that observed
after myoseverin treatment. In contrast, the MCF-7 and the Mes-
sa cells responded only marginally to this compound at concentra-
3.3. Microtubule polymerization assays
The three pyrazolo[1,5-a]-1,3,5-triazine derivatives and myo-
severin as control were tested in microtubule polymerization as-
says using bovine brain purified tubulin (Table 3). The IC50 for
tions higher than 20 lM. A similar antiproliferative pattern was
myoseverin is 3.6
found an IC50 value of 7–8
equally potent effectors of microtubule polymerization than myo-
severin with IC50 values of 3.6 and 3.4 M, respectively. They both
l
M in good agreement with previous results that
observed after treatment of the cell lines with 1a. However, this
compound was less active than 1c and myoseverin in the CEM
cells. Finally, 1b displayed some antiproliferative activity only to-
wards the SW48 cells. In the other cell lines, the compound
l
M.1b,5 Both 1c (Fig. 2) and 1a are
l
reduced the rate and the final extent of microtubule assembly. Fifty
showed IC50 values higher than 20 lM.
Table 1
In vitro cell growth inhibition activities of myoseverin and 1a–c in human cancer cell lines
a,b
Compound
IC50
(lM)
MCF-7
Mes-sa
CEM
5.7 0.3
HCT116
3.3 0.1
SW48
SW480
Myoseverin
11.5 0.7
8.5 0.7
2.7 0.4
5.2 0.3
7.5 0.7
2.6 0.2
4.8 1.6
8.1 2.3
26.5 2.1
5.5 0.9
1a
1b
1c
34
8
45
7
13
38.5
7
9
6.2
25 4.2
4.6
1
35.5 4.9
51.5 4.9
43 1.4
23 2.4
5.4 0.5
3
a
Values are means of three different experiments SD.
IC50: Inhibitory concentration 50.
b

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F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
Table 2
Percentage of cells (%) in different phases of the cell cycle
Compounda
HCT116
S
SW48
SW480
b
G1
G2/M
G1
S
G2/M
G1
S
G2/M
Control
Myoseverin
1a
1b
1c
62.5 11.5
1.8 0.3
19 2.8
26.1 9.7
9.5 3.5
29 1.4
0.5 0.7
2.8 2.5
11.3 4.4
88 4.2
51 4.2
60.5 3.6
23.3 5.5
16.1 8.8
98 1.4
62 9.9
42.5 0.7
75.4 4.7
57.6 6.9
12.5 0.8
43 1.4
28.7
3
5
1.4
13.6 4.5
1
1.4
1
0
84.5
2
16 4.2
50 9.9
22.4 5.3
21 5.7
0.5 0.7
1.3 0.4
15 1.4
1.4
5.9 0.8
42 2.8
28 5.7
91.2 0.7
44 2.8
55.5 2.1
62 2.8
9
8
1.5
89.3
1
3
0.2
a
Compounds were tested at 30
Values are means of three different experiments SD.
l
M.
b
percentage of inhibition occurred at a mole ratio of about 0.12 mol
of compound per mole of tubulin indicating that the compounds
are stoichiometric or weakly substoichiometric inhibitors of micro-
tubule polymerization. Compound 1b was considerably less active
than the other three tested compounds showing only partial inhi-
bition of microtubule assembly.
Table 3
In vitro inhibition of bovine brain purified tubulin polymerization of myoseverin and
1a–c
Compound
IC50 (lM)
Myoseverin
3.6
3.4
1a
1b
1c
50% Baseline
3.6
3.4. Cell based assays
The observed G2/M arrest, coupled with the observed inhibition
of microtubule assemblyin in vitro assays indicated that myosever-
in, and its analogues 1a and 1c, may induce mitotic defects leading
to mitotic arrest. Indeed, live cell imaging of HeLa cells expressing
0.45
A
GFP-tubulin showed that addition of myoseverin (at 12 lM) in pre-
0.4
0.35
0.3
assembled bipolar metaphase spindles led to the complete collapse
of the spindle microtubules (Supplementary data, movie 1). Fur-
thermore prometaphase cells were not capable of forming a nor-
mal bipolar spindle (Supplementary data, movie 2). In contrast,
the interphase microtubule network was not visibly affected at
similar conditions. (Supplementary data, movie 3). HeLa cells, ex-
posed for 24 h at different concentrations of either myoseverin or
analogues 1a and 1c, exhibited also a pronounced block at G2/M
similar to that presented in Table 2 (Fig. 3A). HeLa cells treated
with myoseverin, 1a and 1c, were also observed by indirect immu-
0.25
0.2
nofluorescence microscopy. At 12 lM, a concentration that led to
strong G2/M arrest (Fig. 3A), almost 100% of the mitotic spindles
were aberrant, being monopolar or multipolar and in the rare occa-
sions that a bipolar spindle was present not all of the chromosomes
were aligned to the metaphase plate (Fig. 3B). A concentration
dependent increase in the percentage of aberrant spindles in HeLa
cells was also observed (Fig. 3C). Compound 1b did not induce sim-
0.15
0.1
0
500
1000
1500
Time (sec)
ilar mitotic defects at concentrations as high as 50 lM.
100
80
60
40
20
0
B
4. Conclusion
Contrary to our previous work on CDK kinase analogue inhib-
itors,9 no improvement of the biological activity was observed
when the purine nucleus was substituted by the pyrazolo[1,5-
a]-1,3,5-triazine scaffold. Nevertheless, myoseverin derivatives
1a and 1c constitute a new series of tubulin inhibitors and dis-
played micromolar antiproliferative activities towards colorectal
cancer cell lines. Compounds 1a and 1c can become new leads
in the ongoing search of novel specific and potent anticancer
drugs.
5. Experimental
0
20
40
1c (µM)
60
80
100
5.1. General methods
Melting points were measured with a Büchi Tottoli SMP-20
heating unit and are uncorrected. IR spectra were recorded on a
Perkin–Elmer 681 infrared spectrophotometer. NMR spectra were
Figure 2. (A) In vitro inhibition of tubulin assembly for 1c measured at a protein
concentration of 29 M and increasing inhibitor concentrations up to 100 M. (B)
Concentration–response plot for compound 1c, derived from A.
l
l

F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
3475
Figure 3. Induction of a mitotic block with aberrant spindles in the presence of myoseverin, and its analogues 1a and 1c, in HeLa cells. (A) HeLa cells were exposed to
increasing concentrations of the inhibitors (myoseverin, 1a and 1c) for 24 h and the percentage of G2/M cells was determined by flow cytometry. Nocodazole (Noc) was used
as a control mitotic inhibitor. (B) Following addition of the drugs, cells were fixed and stained for indirect immunofluorescence microscopy (anti-tubulin: green; chromatin:
red). Representative spindles of treated cells at 12
lM inhibitor concentration are shown. Control (CNTRL) refers to absence of inhibitor. (C) The percentages of aberrant
spindles out of total spindles counted were determined for the different concentrations of each drug added.
recorded at 300 K on a Bruker Avance spectrometer at 300 MHz for
1H and 75 MHz for 13C. Tetramethylsilane (TMS) was used as inter-
nal reference. Chemical shifts are expressed in parts per million
(ppm) relative to TMS. It should be noted that quaternary carbon
peaks of pyrazolo[1,5-a]-1,3,5-triazine ring of final compounds
1a, 1b and 1c are not observed, these 13C NMR spectra are not re-
ported in the experimental part. Mass spectra were recorded with
a Perkin–Elmer SCIEX API spectrometer and Thermo Finnigan Mat
95 XL. Elemental analyses were performed on a Thermoquest Flash
1112 series EA analyser. TLC was conducted on precoated silica gel
plates (Merck 60F254) and the spots were visualized under UV light.
Flash chromatography was carried out on a column filled with
(petroleum ether (PE): bp 40–60 °C). All reactions requiring anhy-
drous conditions were conducted in flame-dried apparatus.
5.2. 4-(N-Methyl-N-phenylamino)-2-(methylsulfonyl)
pyrazolo[1,5-a]-1,3,5-triazine (3)
Under inert atmosphere, 50% m-CPBA (656 mg, 1.90 mmol) was
added to a solution of 4-(N-methyl-N-phenylamino)-2-(meth-
ylsulfanyl)pyrazolo[1,5-a]-1,3,5-triazine 210 (172 mg, 0.63 mmol)
in CH2Cl2 (10 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h and was allowed to warm up to room temperature
for 2 h. The reaction mixture was diluted by addition of CH2Cl2
(5 mL). The solution was washed with saturated aqueous solution
flash silica gel 60 (40–63 lm, Merck) using the indicated solvents

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F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
of NaHCO3 (10 mL), then water (10 mL). The organic phase was
dried over MgSO4, then evaporated. The crude residue was purified
by flash chromatography (EtOAc/PE 4:6) to afford compound 3 as a
0.29 mM) and 4-methoxybenzylamine (1.43 mmol) in dry dioxane
(1.5 mL). Chromatography eluent: EtOAc/PE 3:7. Mp = 136–138 °C
(MeOH washing); IR (KBr)
m 3245, 2925, 1640, 1580, 1430, 1245,
solid (150 mg, 78%). Mp = 174–175 °C (EtOH); IR (KBr)
m
3130,
1175, 765 cmÀ1 1H NMR (CDCl3) d 3.79 (s, 6H, 2 CH3), 4.60 (s,
;
2930, 2850, 1605, 1565, 1450, 1310, 930, 780 cmÀ1
;
1H NMR
2H, CH2), 4.62 (s, 2H, CH2), 5.60 (br s, 1H, NH), 6.62 (br s, 1H,
NH), 6.84–6.86 (m, 4H, Harom), 7.21–7.24 (m, 4H, Harom), 7.69 (s,
1H, H7); MS (ESI) m/z 469 (79Br, M+H)+, 471 (81Br, M+H)+; Anal.
Calcd for C21H21BrN6O2: C, 53.74; H, 4.51; N, 17.91. Found: C,
53.80; H, 4.44; N, 18.14.
(CDCl3) d 3.31 (s, 3H, CH3), 3.88 (s, 3H, CH3), 6.57 (d, 1H,
J = 2.0 Hz, H8), 7.22–7.25 (m, 2H, Harom), 7.42–7.45 (m, 3H, Harom),
7.83 (d, 1H, J = 2.0 Hz, H7); 13C NMR (CDCl3) d 39.0 (CH3), 43.1
(CH3), 98.4 (CH), 126.4 (2 CH), 128.2 (CH), 129.3 (2 CH), 143.7
(C), 146.1 (CH), 149.8 (C), 150.4 (C), 159.5 (C); MS (ESI) m/z 304
(M+H)+; Anal. Calcd for C13H13N5O2S: C, 51.47; H, 4.32; N, 23.09.
Found: C, 51.23; H, 4.18; N, 22.89.
5.7. 8-(1-Methylethyl)-4-(N-methyl-N-phenylamino)-2-(meth-
ylsulfanyl)pyrazolo[1,5-a]-1,3,5-triazine (7)
5.3. N2,N4-Bis-(4-methoxybenzyl)pyrazolo[1,5-a]-1,3,5-
A solution of 3 M methylmagnesium bromide in Et2O (297 lL,
triazine-2,4-diamine (1a)
2.58 mmol) was added dropwise to a solution of 8-acetyl-4-(N-
methyl-N-phenylamino)-2-(methylsulfanyl)pyrazolo[1,5-a]-1,3,5-
triazine 610 (269 mg, 0.86 mmol) in dry Et2O (7 mL) cooled to 0 °C.
After addition, the solution was stirred at room temperature for an
additional 20 min. The reaction mixture was neutralized by a
freshly prepared aqueous solution of NH4Cl (5 mL). The aqueous
layer was extracted with CH2Cl2 (5 mL) then EtOAc (2 Â 5 mL).
The combined organic extracts were dried over MgSO4 and concen-
trated. The oily residue was used without further purification. Un-
In a sealed tube, a solution of compound 3 (120 mg, 0.38 mmol)
and 4-methoxybenzylamine (209 lL, 1.91 mmol) in dry dioxane
(1 mL) was heated at 140 °C for 20 h. After cooling, the solvent
was evaporated. The crude product was purified by flash chroma-
tography (EtOAc/PE 3:7 then 4:6) to afford compound 1a as a solid
(101 mg, 70%). Mp = 144–146 °C (MeOH); IR (KBr)
m 3245, 2995,
1640, 1570, 1430, 1250, 1175, 765 cmÀ1 1H NMR (CDCl3) d 3.80
;
(s, 6H, CH3), 4.58 (d, 2H, J = 5.6 Hz, CH2), 4.64 (d, 2H, J = 6.0 Hz,
CH2), 5.20 (br s, 1H, NH), 5.94 (br s, 1H, H8), 6.62 (br s, 1H, NH),
6.85–6.88 (m, 4H, Harom), 7.25–7.30 (m, 4H, Harom), 7.73 (br s, 1H,
H7); MS (ESI) m/z 391 (M+H)+; Anal. Calcd for C21H22N6O2: C,
64.60; H, 5.68; N, 21.52. Found: C, 64.55; H, 5.79; N, 21.46.
der nitrogen atmosphere, a solution of the tertiary alcohol
intermediate in CH2Cl2 (9 mL) was added at 0 °C to a solution of
NaBH4 (113.5 mg, 3.00 mmol) in TFA (9 mL). The reaction mixture
was stirred at room temperature for 1 h and then neutralized by a
solution of 1 M NaOH (15 mL). The aqueous layer was extracted
with CH2Cl2 (3 Â 10 mL). The combined organic extracts were
dried over MgSO4 and evaporated. The residue was purified by
flash chromatography (Et2O/PE 1:9) to afford 7 (241 mg, 89%) as
5.4. 8-Bromo-4-(N-methyl-N-phenylamino)-2-(methylsulfa-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (4)
a solid. Mp = 85–87 °C (Et2O/PE); IR (KBr)
m 3055, 2960, 2865,
A solution of 2 (200 mg, 0.74 mmol) and N-bromosuccinimide
(184 mg, 1.04 mmol) in dry CHCl3 (20 mL) was stirred at reflux for
3 h. After evaporation of the solvent, the crude product was purified
by flash chromatography (EtOAc/PE 1:9) to afford derivative 2
1615, 1535, 1460, 750, 695 cmÀ1 1H NMR (CDCl3) d 1.27 (d, 6H,
;
J = 6.8 Hz, CH3), 2.55 (s, 3H, CH3), 3.14 (hept, 1H, J = 6.8 Hz, CH),
3.70 (s, 3H, CH3), 7.15–7.19 (m, 2H, Harom), 7.31–7.41 (m, 3H, Har-
om), 7.54 (s, 1H, H7); 13C NMR (CDCl3) d 14.3 (CH3), 23.2 (2 CH3),
23.5 (CH), 42.2 (CH3), 113.9 (C), 126.2 (2 CH), 127.1 (CH), 129.1
(2 CH), 143.4 (CH), 144.9 (C), 147.4 (C), 148.4 (C), 164.9 (C); MS
(ESI) m/z 314 (M+H)+; Anal. Calcd for C16H19N5S: C, 61.32; H,
6.11; N, 22.34. Found: C, 60.99; H, 5.95; N, 22.36.
(104 mg, 82%) as a solid. Mp = 154–156 °C (EtOAc) IR (KBr)
m 3095,
2920, 1610, 1540, 760, 695 cmÀ1 1H NMR (CDCl3) d 2.58 (s, 3H,
;
CH3), 3.71 (s, 3H, CH3), 7.16 (d, 2H, J = 7.1 Hz, Harom), 7.37–7.39 (m,
3H, Harom), 7.56 (s, 1H, H7); 13C NMR (CDCl3) d 14.4 (CH3), 42.6
(CH3), 81.1 (C), 126.2 (2 CH), 127.6 (CH), 129.2 (2 CH), 144.3 (CH),
145.0 (C), 147.8 (C), 147.9 (C), 168.3 (C); MS (ESI) m/z 350 (79Br,
M+H)+, 352(81Br, M+H)+; Anal. Calcd for C13H12BrN5S: C, 44.58; H,
3.45; N, 20.00. Found: C, 44.83; H, 3.62; N, 19.97.
5.8. 8-(1-Methylethyl)-4-(N-methyl-N-phenylamino)-2-(meth-
ylsulfonyl)pyrazolo[1,5-a]-1,3,5-triazine (8)
Following the procedure described for the preparation of 3,
5.5. 8-Bromo-4-(N-methyl-N-phenylamino)-2-(methylsulfo-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (5)
compound 8 was obtained in 65% yield from 7 (140 mg,
0.45 mmol) and m-CPBA (1.34 mmol) in CH2Cl2 (8 mL). Chroma-
tography eluent: EtOAc/PE 1:2. Mp = 144–146 °C (EtOAc/PE); IR
Following the procedure described for the preparation of 3,
(KBr) m ;
2960, 2870, 1600, 1555, 1460, 1045, 755, 705 cmÀ1 1H
compound
5
was obtained in 84% yield from
4
(132 mg,
NMR (CDCl3) d 1.27 (d, 6H, J = 6.8 Hz, CH3), 3.21 (hept, 1H,
J = 6.8 Hz,CH), 3.33 (s, 3H, CH3), 3.83 (s, 3H, CH3), 7.19–7.22 (m,
2H, Harom), 7.39–7.42 (m, 3H, Harom), 7.69 (s, 1H, H7); 13C NMR
(CDCl3) d 23.2 (2 CH3), 23.5 (CH), 38.8 (CH3), 42.9 (CH3), 119.2
(C), 126.5 (2 CH), 128.0 (CH), 129.1 (2 CH), 143.9 (C), 144.5 (CH),
145.5 (C), 150.1 (C), 158.2 (C); MS (ESI) m/z 346 (M+H)+; Anal.
Calcd for C16H19N5O2S: C, 55.64; H, 5.54; N, 20.27. Found: C,
55.88; H, 5.71; N, 20.39.
0.38 mmol) and m-CPBA (1.13 mmol) in CH2Cl2 (6 mL). Chroma-
tography eluent: EtOAc/PE 2:3. Mp = 195–197 °C (CH2Cl2/PE); IR
(KBr)
m ;
3030, 2930, 2850, 1610, 1565, 1140, 745, 695 cmÀ1 1H
NMR (CDCl3) d 3.37 (s, 3H, CH3), 3.82 (s, 3H, CH3), 7.19–7.22 (m,
2H, Harom), 7.40–7.45 (m, 3H, Harom), 7.76 (s, 1H, H7); 13C NMR
(CDCl3) d 39.1 (CH3), 43.6 (CH3), 87.3 (C), 126.7 (2 CH), 128.6
(CH), 129.6 (2 CH), 143.6 (C), 146.4 (C), 146.9 (CH), 150.3 (C),
160.7 (C); MS (ESI) m/z 382 (79Br, M+H)+, 384 (81Br, M+H)+; Anal.
Calcd for C13H12BrN5O2S: C, 40.85; H, 3.16; N, 18.32. Found: C,
40.87; H, 3.08; N, 18.44.
5.9. N2,N4-Bis-(4-methoxybenzylamine)-8-(1-methylethyl)-
pyrazolo[1,5-a]-1,3,5-triazine (1c)
5.6. 8-Bromo-N2,N4-bis-(4-methoxybenzylamino)pyrazolo[1,5-
a]-1,3,5-triazine (1b)
Following the procedure described for the preparation of 1a,
compound 1c was obtained in 79% yield from 7 (70 mg, 0.20 mmol)
and 4-methoxybenzylamine (1.01 mmol) in dry dioxane (1 mL).
Chromatography eluent: EtOAc/PE 1:4 then 1:2. Mp = 96–98 °C
Following the procedure described for the preparation of 1a,
compound 1b was obtained in 70% yield from
5
(110 mg,
(EtOAc/PE); IR (KBr) m 3285, 2955, 2865, 1635, 1555, 1445, 1250,

F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
3477
1175, 810 cmÀ1; 1H NMR (CDCl3) d 1.30 (d, 6H, J = 6.8 Hz, CH3), 3.06
5.14. Live cell imaging and video microscopy
(hept, 1H, J = 6.8 Hz, CH), 3.78 (s, 3H, CH3), 3.79 (s, 3H, CH3), 4.47 (s,
2H, CH2), 4.49 (s, 2H, CH2), 5.49 (br s, 1H, NH), 6.81 (d, 2H, J = 8.3 Hz,
Harom), 6.85 (d, 2H, J = 8.5 Hz, Harom), 6.95 (br s, 1H, NH), 7.17 (d, 2H,
J = 8.3 Hz, Harom), 7.29 (d, 2H, J = 8.5 Hz, Harom), 7.58 (br s, 1H, H7); MS
(ESI) m/z 433 (M+H)+; Anal. Calcd for C24H28N6O2: C, 66.65; H, 6.53;
N, 19.43. Found: C, 66.88; H, 6.44; N, 19.49.
For time-lapse microscopy, HeLa cells expressing GFP-tubulin
were plated in glass-bottom dishes then placed inside a video
microscopy platform equipped with an incubator enabling the reg-
ulation of the temperature. The cell growth medium was replaced
with the CO2 independent growth medium DMEM/F12 with
15 mM HEPES (GIBCO) supplemented with 10% serum. Time-lapse
Z series images (Z = 3) were collected with an inverted motorized
microscope. Images were acquired with an inverted Olympus
5.10. Cell lines
All cell lines were grown on 25 cm2 flasks and were placed at
37 °C in a humidified atmosphere containing 5% CO2. The MCF-7
breast cancer cell line and the HCT116, SW48 and SW480 colorec-
tal cancer cell lines were cultured in D-MEM culture medium con-
taining 10% fetal calf serum, 1% L-glutamine and 2% penicillin–
streptomycin. The Mes-sa sarcoma cell line and the CEM lym-
phoma cell line were cultured in RPMI1640 medium containing
IX81 epifluorescence motorized microscope equipped with a
motorized piezo stage (Ludl Electronic Products, USA) and a Reti-
ga-SRV CCD camera (QImaging) driven by VOLOCITY software
(Improvision) with a binning of 1, using a PlanApo 60xNA 1.42
objective (Olympus). Images were acquired every 5 min and the
acquisition time was 53 ms. For each Z series, the best focus images
was chosen before the reconstitution of the movie.
10% fetal calf serum, 1%
streptomycin.
L-glutamine and 1% penicillin–
5.15. Immunofluorescence microscopy
5.11. Cell growth inhibition assay
HeLa cells were grown on Dulbecco’s modified Eagle’s medium
(GIBCO, BRL) supplemented with 10% fetal bovine serum (Hyclone)
and maintained in a humid incubator at 37 °C in 5% CO2. Cells were
In vitro antiproliferative activities were determined in three sep-
arate experiments, each of which was performed in triplicate as pre-
viously described.11 Briefly, asynchronously growing cells were
transferred into 96-well culture plates (CostarÒ, Corning Inc., New
seeded and left to adhere for at least 36 h on poly-D-lysine-coated
glass coverslips in 24 well plates. Drugs were diluted appropriately
in medium from 50 mM stocks in 100% DMSO, and then added to
the cells. Following 6 h incubation with drugs, cells were fixed with
1% paraformaldehyde–PBS at 37 °C for 3 min followed by 5 min
incubation in 100% methanol at -20 °C. Coverslips were then
washed with PBS and cells were stained with an anti-b-tubulin
monoclonal antibody (Sigma) at 300-fold dilution for 1 h, then
with an FITC-conjugated goat anti-mouse secondary antibody
(Jackson ImmunoResearch Laboratories, West Grove, PA USA) at
250-fold dilution for 30 min and counterstained with propidium
iodide. Images were acquired with an Olympus BX61 epifluores-
cence microscope equipped with a Retiga-SRV CCD camera driven
by VOLOCITY software (Improvision).
York) in 100
l
L of medium at a final concentration of 5 Â 103 cells/
well and incubated for 24 h. Corresponding drug concentrations
were then added to each plate. After 72 h of drug exposure, 20 lL
of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) reagent (5 mg/mL) was added to each well. Cell growth was
expressed as the percent of absorbance of treated wells relative to
untreated control wells. IC50 values were defined as drug doses
resulting in 50% cell growth inhibition relative to untreated cells.
5.12. Flow cytometric detection of cell cycle (or cell cycle assay)
For analysis of DNA content and cell cycle distribution, colorec-
tal cancer cell lines were treated with compounds 1a, 1b, 1c, and
myoseverin for 24 h. Based on cytotoxicity assay, a concentration
Acknowledgments
of 30
drug exposure experiments. After drug-exposure, 106 cells/mL
were resuspended in 2 mL of propidium iodide solution (50 L/
l
M (approx. IC80 values for these cell lines) was chosen for
We thank Rose-Laure Indorato for excellent technical assis-
tance. We are grateful for financial support from the Association
pour la Recherche sur le Cancer (ARC), grant number 3973.
l
mL), incubated at 4 °C overnight and then analyzed by flow cytom-
etry. Flow cytometry was performed on a FACScalibur (Becton
Dickinson, San Jose, California). Cell cycle distribution and DNA
ploidy status were calculated after exclusion of cell doublets and
aggregates on a FL2-area/FL2-width dot plot using Modfit LT 2.0
software (Verity Software Inc. Topsham. ME).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.03.007.
TM
References and notes
5.13. Microtubule polymerization assays
1. (a) Rosania, G. R.; Chang, Y.-T.; Perez, O.; Sutherlin, D.; Dong, H.; Lockhart, D.;
Schultz, P. G. Nat. Biotechnol. 2000, 18, 304; (b) Chang, Y.-T.; Wignall, S. M.;
Rosania, G. R.; Gray, N. S.; Hanson, S. R.; Su, A. I.; Merlie, J., Jr.; Moon, H.-S.;
Sangankar, S. B.; Perez, O.; Heald, R.; Schultz, P. J. Med. Chem. 2001, 44, 4497; (c)
Microtubule polymerization assays in the absence or presence
of inhibitors were performed in 96-well half-area
lclear plates
using a 96-well photometer (TECAN) measured at a wavelength
of 340 nm. Tubulin was prepared as previously described.12 Exper-
iments were performed at the same time using the same buffers
and tubulin from the same preparation to minimize variations
originating from different experimental conditions. The final test
volume was 50 lL prepared from solutions kept at 4 °C. To 33 lL
PEM buffer (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA), supple-
mented with increasing inhibitor concentrations in DMSO, tubulin
at 12 mg/mL (29 lM) in PEM buffer was added, and carefully
´
´
Dvorák, Z.; Modriansky, M.; Vrba, J.; Ulrichová, J.; Krystof, V.; Styskala, J.; Pávek,
P. Gen. Physiol. Biophys. 2007, 26, 173.
2. (a) Perez, O. M.; Chang, Y.-T.; Rosania, G.; Sutherlin, D.; Schultz, P. G. Chem. Biol.
2002, 9, 475; (b) Duckmanton, A.; Kumar, A.; Chang, Y.-T.; Brockes, J. P. A. Chem.
Biol. 2005, 12, 1117; (c) Ng, D. C. H.; Gebski, B. L.; Grounds, M. D.; Bogoyevitch,
M. A. Cell. Motil. Cytoskel. 2008, 65, 40.
3. Park, H.-E.; Baek, S. H.; Min, J.; Chang, Y.-T.; Kang, D.-K.; Chang, S.-I.; Joe, Y. A.
DNA Cell Biol. 2006, 25, 514.
4. Hocek, M.; Votruba, I.; Dvorakova, H. Tetrahedron 2003, 59, 607.
5. Krystof, V.; Moravcova, D.; Paprskarova, M.; Barbier, P.; Peyrot, V.; Hlobilkova,
A.; Havlicek, L.; Strnad, M. Eur. J. Med. Chem. 2006, 41, 1405.
6. Moon, H.-S.; Jacobson, E. M.; Khersonsky, S. M.; Luzung, M. R.; Walsh,
D. P.; Xiong, W.; Lee, J. W.; Parikh, P. B.; Lam, J. C.; Kang, T.-W.;
Rosania, G. R.; Schier, A. F.; Chang, Y.-T. J. Am. Chem. Soc. 2002, 124,
11608.
mixed. The polymerization into microtubules was followed at
340 nm over at least 1 h at 37 °C. The DMSO concentration was ad-
justed to be identical in all wells.

3478
F. Popowycz et al. / Bioorg. Med. Chem. 17 (2009) 3471–3478
7. Raboisson, P.; Schultz, D.; Muller, C.; Reimund, J.-M.; Pinna, G.; Mathieu, R.;
Bernard, P.; Do, Q.-T.; DesJarlais, R. L.; Justiano, H.; Lugnier, C.; Bourguignon, J.-
J. Eur. J. Med. Chem. 2008, 43, 816.
7, 2713; (b) Popowycz, F.; Fournet, G.; Schneider, C.; Bettayeb, K.; Ferandin, Y.;
Lamigeon, C.; Tirado, O. M.; Mateo-Lozano, S.; Notario, V.; Colas, P.; Bernard, P.;
Meijer, L.; Joseph, B. J. Med. Chem. 2009, 52, 655.
8. Raboisson, P.; Baurand, A.; Cazenave, J.-P.; Gachet, C.; Schultz, D.; Spiess, B.;
Bourguignon, J.-J. J. Org. Chem. 2002, 67, 8063.
9. (a) Bettayeb, K.; Sallam, H.; Ferandin, Y.; Popowycz, F.; Fournet, G.; Hassan, M.;
Echalier, A.; Bernard, P.; Endicott, J.; Joseph, B.; Meijer, L. Mol. Cancer Ther. 2008,
10. Popowycz, F.; Bernard, P.; Raboisson, P.; Joseph, B. Synthesis 2007, 367.
11. Galmarini, C. M.; Clarke, M. L.; Falette, N.; Puisieux, A.; Mackey, J. R.; Dumontet,
C. Int. J. Cancer 2002, 97, 439.
12. Williams, R. C., Jr.; Lee, J. C. Methods Enzymol. 1982, 85, 376.