Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access to potent cyclin-dependent kinase inhibitor (R)-roscovitine analogue

Article abstract of DOI:10.1021/jm801340z

Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1- f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.

Full text of DOI:10.1021/jm801340z

J. Med. Chem. 2009, 52, 655–663
655
Pyrazolo[1,5-a]-1,3,5-triazine as a Purine Bioisostere: Access to Potent Cyclin-Dependent Kinase
Inhibitor (R)-Roscovitine Analogue
Florence Popowycz,† Guy Fournet,† Ce´dric Schneider,† Karima Bettayeb,‡ Yoan Ferandin,‡ Cyrile Lamigeon,| Oscar M. Tirado,§
Silvia Mateo-Lozano,§ Vicente Notario,§ Pierre Colas,‡,| Philippe Bernard,*, Laurent Meijer,*,‡ and Benoˆıt Joseph*,†
Institut de Chimie et Biochimie Mole´culaires et Supramole´culaires, UMR-CNRS 5246, Laboratoire de Chimie Organique 1, UniVersite´ de Lyon,
UniVersite´ Claude Bernard-Lyon 1, Baˆtiment Curien, 43 BouleVard du 11 NoVembre 1918, F-69622 Villeurbanne, France, CNRS, “Protein
Phosphorylation & Human Disease” Group, Station Biologique, B.P. 74, 26682 Roscoff, France, Laboratory of Experimental Carcinogenesis,
Lombardi ComprehensiVe Cancer Center, Georgetown UniVersity Medical Center, 3970 ReserVoir Road NW, Washington, D.C. 20057-1482,
Aptanomics SA, 181-203 AVenue Jean Jaure`s, 69007 Lyon, France, Greenpharma SA, 3 Alle´e du Titane, 45100 Orle´ans, France
ReceiVed October 23, 2008
Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among
the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays
rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure,
we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine
7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-
f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide
variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced
biomedical potential.
Introduction
roscovitine acts on multiple phases of the cell cycle and works
by inducing cell cycle arrest and apoptosis.6
Pharmacological inhibition of cyclin-dependent kinases (CDKs)
is anticipated to provide an effective approach to the control of
multiple human diseases in the clinic including cancers, chronic
neurodegenerative diseases (Alzheimer’s disease, Parkinson’s
disease), “acute” neuronal disorders (stroke, traumatic brain
injury, pain signaling), kidney diseases (glomerulonephritis,
lupus nephritis, collapsing glomerulopathy, polycystic kidney
disease, cisplatin-induced nephrotoxicity), pleural inflammation
and arthritis, diabetes type 2, viral infections (HSV, HCMV,
HPV, HIV), and unicellular parasites (Plasmodium, Leishmania,
etc.,...).1 Various CDK inhibitors have already been reported
to exhibit antiproliferative activities and are in clinical trials
against various cancers (Figure 1).2 Among them, flavopiridol
and (R)-roscovitine were the first CDK inhibitors to enter in
clinical phase. Second-generation CDK inhibitors such as SNS-
032 (former BMS-387032), AG-24322, R547, AT7519, AT7119,
AZD5438, and PD-0332991 are in advanced preclinical testing
or in clinical trials.3,4 The discovery of (R)-roscovitine has been
reported in detail previously.5 This compound is an orally
available 2,6,9-trisubstituted purine that specifically inhibits
CDK1, CDK2, CDK3, CDK5, CDK7, and CDK9 in the
submicromolar range. Preclinical studies demonstrate that (R)-
The 2,6,9-trisubstituted purine family I (Figure 2) has been
the subject of intensive structure-activity relationship studies
to identify more potent CDK inhibitory purine derivatives. Thus,
the modification of the nature of the substituents on positions
2, 6, and 9 led to the preparation of aminopurvalanol, purvalanol
A and B, displaying higher inhibitory CDK activities in vitro.7
Among the classical approaches to drug design, the bioisosterism
strategy8 is a powerful way to the rational design of new drugs.
Several features are favorable to this approach such as (1)
improvement of the metabolic stability of the drugs, (2)
reduction of certain adverse affects, (3) optimization of phar-
macokinetics and/or bioavailability, and (4) circumventing patent
issues. In this article, we report a bioisosterism study leading
to an original and promising new CDK inhibitor lead compound
based on (R)-roscovitine.
The pyrazolo[1,5-a]-1,3,5-triazine moiety II or the imi-
dazo[2,1-f]-1,2,4-triazine core III allows one to consider the
synthesis of a large number of “carbabioisosteres” of purine
drugs (Figure 2). In the case of the pyrazolo[1,5-a]-1,3,5-triazine
moiety, this scaffold perfectly mimicks the biophysicochemical
properties of the purine ring9 while being more stable in vivo
(no action of nucleosidases on C-8 position as encountered for
the purine ring).10 By this approach, a large number of
pyrazolo[1,5-a]-1,3,5-triazine derivatives have been designed
for the last several decades as remarkable purine bioisosteres.
Thus, this type of modification can lead to a large number of
derivatives showing potent biological activities.11
* To whom correspondence should be addressed. For P.B. (chemistry,
chemoinformatic): phone, +33 (0)2 38 25 99 80; fax, +33 (0)2 38 25 99
65; E-mail: philippe.bernard@greenpharma.com. For L.M. (biochemistry
and cell biology): phone, +33 (0)2 98 29 23 39; fax, +33 (0)2 98 29 25
26; E-mail: meijer@sb-roscoff.fr. For B.J. (chemistry): phone, +33
(0)4 72 44 81 35; fax, +33 (0)4 72 43 12 14; E-mail: benoit.joseph@
univ-lyon1.fr.
These data prompted us to synthesize novel pyrazolo[1,5-
a]-1,3,5-triazine and imidazo[2,1-f]-1,2,4-triazine roscovitine
bioisosteres in order to compare their CDK inhibitory and
antiproliferative activities to genuine (R)-roscovitine.
†
Institut de Chimie et Biochimie Mole´culaires et Supramole´culaires,
UMR-CNRS 5246, Laboratoire de Chimie Organique 1, Universite´ de Lyon,
Universite´ Claude Bernard-Lyon 1.
‡
CNRS, “Protein Phosphorylation & Human Disease” Group, Station
Results and Discussion
Biologique.
§
Laboratory of Experimental Carcinogenesis, Lombardi Comprehensive
Chemistry. Pyrazolo[1,5-a]-1,3,5-triazine derivatives 7a-c
were first prepared (Scheme 1). Regioselective iodination on
C-8 position of 112 was carried out in the presence of
Cancer Center, Georgetown University Medical Center.
|
Aptanomics SA.
Greenpharma SA.
10.1021/jm801340z CCC: $40.75
2009 American Chemical Society
Published on Web 01/07/2009

656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Popowycz et al.
Figure 1. CDK inhibitors in clinical trials: (R)-roscovitine, flavopiridol, SNS-032, AZD5438, PD-0332991, R547, AG-24322, and AT7519.
Figure 2. 2,6,9-Trisubstituted purine CDK inhibitor scaffold I and related bioisosteres II and III.
N-iodosuccinimide to give 8-iodopyrazolo[1,5-a]-1,3,5-triazine
213 in 88% yield. Introduction of the acetyl group on C-8
position was first performed through a Stille palladium-catalyzed
coupling reaction. Derivative 2 was treated with n-tributyl(1-
ethoxyvinyl)tin in DMF, in the presence of a catalytic amount
of freshly prepared tetrakis(triphenylphosphine)palladium (8
mol%) and LiCl to provide derivative 313 in good yield.
Similarly, compound 3 was prepared from 1 by a Friedel-Crafts
acylation.12 In a sealed tube, compound 1 was submitted to
acylation reaction with acetyl chloride in the presence of 1 M
SnCl4 in CH2Cl2 at 85 °C, affording compound 3 in 86% yield
(versus 74% for two steps). The same reaction was carried out
under various conditions (AcCl, AlCl3, CH2Cl2, room temper-
ature to reflux, or AcCl, SnCl4, CH2Cl2, reflux) leading to the
total recovery of the starting material. Nucleophilic addition of
1 M MeMgBr in Et2O on 3 followed by hydrolysis by a freshly
prepared NH4Cl solution gave tertiary alcohol, which was readily
treated by sodium borohydride/trifluoroacetic acid in CH2Cl2
at room temperature to yield isopropyl derivative 4. Direct
access to 4 from 1 via a Friedel-Crafts alkylation was
investigated. Attempts were performed using 1, isopropyl
chloride or iodide and Lewis acids (AlCl3, SnCl4, 1 M SnCl4 in
CH2Cl2) under different temperature conditions (room temper-
ature to 100 °C in a sealed tube). In each case, the starting
material was recovered. As already mentioned in the literature,10
the N-methyl-N-phenylamino group on C-4 position is a good
leaving group for nucleophilic addition-elimination reaction.
Nucleophilic aromatic substitution on C-4 position of 4 was
performed either in the presence of benzylamine or 3-chloroa-
niline, providing secondary amines 5a and 5b, respectively, in
84 and 40% yield. m-CPBA oxidation of methylsulfanyl group
of 5 furnished sulfones 6a and 6b in fair yields. (R)-roscovitine
bioisostere 7a (N-&-N1, GP0210)14 was obtained by nucleo-
philic aromatic substitution on C-2 position of 6a in the presence
of (R)-(-)-2-aminobutanol. The enantiomer 7b was prepared
from 6a and (S)-(+)-2-aminobutanol. Purvalanol A bioisostere
7c was obtained from 6b and (R)-(-)-2-amino-3-methylbutanol.
The synthesis of imidazo[2,1-f]-1,2,4-triazine bioisostere was
carried out in four steps from the known 6-amino-3,5-bis-
(methylsulfanyl)-1,2,4-triazine 815 as depicted in Scheme 2.
Treatment of 8 with 2-bromo-3-methylbutyraldehyde dimethyl
acetal 916 and molecular sieves 4 Å in the presence of a catalytic
amount of camphorsulfonic acid in acetonitrile at reflux led to
10 in 60% yield. Nucleophilic displacement of the methylsul-
fonyl substitutent on C-4 position of 10 by benzylamine in
excess gave compound 11 in good yield. It should be noted
that the first experiments were carried out in ethanol at 50 °C,
but even after a prolonged reaction time, the reactions were not
complete. Optimal condition reaction was finally obtained when
benzylamine was used as reagent and solvent. The 2-methyl-

Pyrazolo[1,5-a]-1,3,5-triazine as a Purine Bioisostere
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 657
Scheme 1a
Results show that compound 7a is on average more potent at
inhibiting cell proliferation than (R)-roscovitine (14-fold in terms
of GI50, 9-fold in terms of TGI) but only modestly more potent
at inducing net cell death (LC50). Nevertheless, the cytotoxic
effect of 7a is preferably observed on melanoma cancer cell
lines (see Table 3). Furthermore, like for (R)-roscovitine, no
specific tumor type displayed any particular sensitivity to
compound 7a. Finally, we performed a cell proliferation assay
on a small panel of 6 human cell lines derived from lung (A549),
prostate (PC3), cervix (Hela), kidney (293T) cancers, B-
lymphoma (Raji), and myeloma (U937) (see Supporting Infor-
mation, Figure S3). 7a inhibited the proliferation of all cell lines
in a dose-dependent fashion. Total growth inhibition was nearly
achieved at 5 µM for the other cell lines. In this assay as well,
7a proved significantly more potent than (R)-roscovitine (Sup-
porting Information, Figure S3), which only partially inhibited
tumor cell proliferation at 10 µM. All these data confirm that
7a is >5-fold more potent than its parent molecule, (R)-
roscovitine. A more detailed investigation on the intracellular
mechanism of action of 7a and 13, in comparison with (R)-
roscovitine, as well as the cocrystal structures of 7a and (R)-
roscovitine with CDK2/cyclin A, is presented elsewhere.14
In Vivo Antitumor Activity of Compound 7a and (R)-
Roscovitine. An Ewing’s sarcoma xenograft mouse model
system established as described in the Experimental Section was
used to compare the antitumor activity of 7a and (R)-roscovitine
in vivo. This model was selected on the basis of its clear-cut
sensitivity to roscovitine.18 Compound 7a was evaluated at 25
mg/kg, and (R)-roscovitine was tested at 50 mg/kg. Because
compound 7a was roughly 2 times more potent in vitro than
(R)-roscovitine (average on 5 kinases, Table 1), the dosing for
compound 7a was chosen as half of that of roscovitine, so that
both compounds could be compared on the basis of similar
activity. Results showed those 8 days after treatment initiation,
and both compounds had slowed tumor growth considerably
(Figure 3). At 25 mg/kg, compound 7a was as active as (R)-
roscovitine used at a concentration 2-fold higher (50 mg/kg).
When tumors in control animals reached an average volume
about 2000 mm3, and animals had to be sacrificed to comply
with institutional animal care and use guidelines, tumors in drug-
treated animals had reached and average volume of only ∼550
mm3 (a near 73% inhibition of tumor growth). The inhibitory
effect of both drugs on tumor growth was also manifested by
the fact that tumors in treated animals took an additional period
of about 15 days to reach the maximum volume allowed by
institutional tumor burden standards (Figure 3).
a Conditions: (i) NIS, CHCl3, reflux, 90 min, 88%. (ii) AcCl, 1 M SnCl4,
CH2Cl2, 85 °C, 17 h, sealed tube, 86%. (iii) n-Tributyl(1-ethoxyvinyl)stan-
nane, Pd(PPh3)4, LiCl, DMF, 80-90 °C, 12 h, 84%. (iv) (a) 1 M MeMgBr,
THF, 0 °C to rt, 20 min; (b) NH4Cl. (v) NaBH4, TFA, CH2Cl2, 0 °C to rt,
1 h, 89% (two step yield). (vi) Benzylamine, EtOH, 90 °C, 12 h, 5b )
84%; 3-chloroaniline, dioxane, 120 °C, 12 h, 5a ) 40%. (vii) m-CPBA,
CH2Cl2, 0 °C to rt, 3 h, 6a ) 97%, 6b ) 60%. (viii) (R)-(-)-2-aminobutanol,
dioxane, 140 °C, 12 h, 7a ) 56%; (S)-(+)-2-aminobutanol, dioxane, 140
°C, 12 h, 7b ) 67%, (R)-(-)-2-amino-3-methylbutanol, dioxane, 140 °C,
12 h 7c ) 70%.
sulfanyl substituent was oxidized by m-CPBA to give the sulfone
12 in 81% yield. The ultimate nucleophilic aromatic substitution
on C-2 position of 12 was carried out at 170 °C in the presence
of (R)-(-)-2-aminobutanol in excess to afford (R)-roscovitine
analogue 13 (N-&-N2, GP0212)14 in fair yield.
Biological Evaluation. Inhibition of Cyclin-Dependent
Kinases. We first tested the synthesized compounds on a
selection of CDKs (CDK1, CDK2, CDK5, CDK7, and CDK9),
known to be inhibited by (R)-roscovitine.6 Results show that
all compounds were active on these kinases in the submicro-
molar range (Table 1). Compound 13 was essentially identical
to (R)-roscovitine in terms of its effects on the 5 CDKs. In
contrast, the roscovitine bioisostere 7a was significantly more
potent, by a factor of 3-5, than either 13 or (R)-roscovitine.
The purvalanol A analogue 7c was also more potent but,
surprisingly, totally inactive on CDK7/cyclin H and CDK9/
cyclin T. The enzymatic effects and selectivity of both 7a and
13 are described in more detail elsewhere.14
Inhibition of Tumor Cell Proliferation. We next tested the
synthesized compounds for their effects on cell survival using
the human neuroblastoma SH-SY5Y and human embryonic
kidney HEK293 cell lines (Table 2). Results show that while
compound 13 and (R)-roscovitine share a similar efficacy, 7a
and its (S)-isomer 7b are significantly more active (5-6 fold).
Interestingly, 7c was completely inactive, possibly accounting
for its lack of effects on CDK7 and CDK9. Compound 7a was
next evaluated against the full panel NCI of 60 tumor cell lines
(mean graphs data for 7a and (R)-roscovitine, see Supporting
Information, Figures S1 and S2).17 The biological data were
compared with those available for (R)-roscovitine (Table 3).
Conclusion
A rather straightforward bioisostere design approach, applied
to the clinical drug (R)-roscovitine, has allowed us to generate
one analogue that displays unexpected enhanced inhibitory
activity toward the CDK targets. This translates into enhanced
cell death inducing activity, a favorable property, which was
confirmed on more than 60 cell lines, and an improved in vitro
biological activity, which was confirmed in a first animal tumor
model. Altogether, this work shows that minor modification can
lead to better second generation analogues of (R)-roscovitine.
These findings support further clinical investigation to determine
the therapeutic potential of 7a.
Experimental Section
Chemistry. General Methods. Commercial reagents (Fluka,
Aldrich) were used without purification. Solvents were
distillated prior to use. (R)-Roscovitine was synthesized as

658 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Popowycz et al.
Scheme 2a
a Conditions: (i) CSA, Molecular sieves 4 Å, MeCN, 70 °C, 8 h, 60%; (ii) BnNH2, 50 °C, 16 h, 95%; (iii) m-CPBA, CH2Cl2, rt, 16 h, 81%; (iv) (R)-
(-)-2-aminobutanol, 170 °C, 4 h, 60%.
Table 1. Effects of (R)-Roscovitine, Purvalanol A, and Its Four
µ63 µM, Merck) using the indicated solvents. The light
petroleum ether (PE) refers to the fraction boiling at 40-
60 °C.
Analogues on the Catalytic Activity of Various CDKsa
IC50 (µM)b
CDK1/
cyclin B cyclin A
CDK2/
CDK5/
p25
CDK7/
cyclin H cyclin T
CDK9/
8-Iodo-4-(N-methyl-N-phenylamino)-2-(methylsulfanyl)pyra-
zolo[1,5-a]-1,3,5-triazine (2). A solution of 4-(N-methyl-N-
phenylamino)-2-(methylsulfanyl)pyrazolo[1,5-a]-1,3,5-triaz-
ine 112,13 (940 mg, 3.46 mmol) and NIS (1.09 g, 4.85 mmol) in
CHCl3 (35 mL) was stirred at reflux for 30 min. The solvent
was evaporated in vacuo. The residue was dissolved in CH2Cl2
(50 mL) and washed with a saturated aqueous Na2S2O3 solution
(3 × 20 mL). The organic layer was dried over MgSO4 and
concentrated in vacuo. The crude product was purified by flash
chromatography (PE/Et2O 9:1) to afford 2 (1.21 g, 88%) as a
solid; mp 162-163 °C (Et2O/PE). IR (KBr): ν 3085, 2915, 1605,
compd
(R)-roscovitine
0.33
0.004
0.073
ND
0.08
0.40
0.22
0.070
0.04
ND
0.06
0.22
0.27
0.075
0.07
0.13
0.06
0.32
0.80
NDc
0.50
ND
>100
0.60
0.23
ND
0.043
ND
>100
0.20
purvalanol A7a
7a
7b
7c
13
a (R)-Roscovitine and its analogues were tested at various concentrations
for their effects on five CDKs. b IC50 values were calculated from the
dose-response curves and are reported in µM. c ND, not determined.
Table 2. Effects of (R)-Roscovitine and Its Four Analogues on the
Survival of Two Cell Linesa
1
1585, 745, 695 cm-1. H NMR (300 MHz, CDCl3): δ 7.61 (s,
IC50 (µM)b
1H, H7), 7.39-7.36 (m, 3H, Harom), 7.16 (broad d, 2H, J ) 7.1
Hz, Harom), 3.71 (s, 3H, CH3), 2.59 (s, 3H, CH3). MS (ESI):
m/z 398 (M + H+). Anal. (C13H12IN5S) C, H, N.
compd
SH-SY5Y
HEK293
(R)-roscovitine
16.1
2.7
2.0
>100
15.4
48.9
7.6
7.2
7a
7b
7c
13
8-Acetyl-4-(N-methyl-N-phenylamino)-2-(methylsulfa-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (3). Method A: A mixture
of 1 (871 mg, 3.21 mmol), acetyl chloride (458 mL, 6.42 mmol),
and 1 M SnCl4 in CH2Cl2 (16.1 mL) was stirred in a sealed
tube for 17 h at 85 °C. The reaction mixture was then poured
into crushed ice and extracted with EtOAc (3 × 15 mL). The
combined organic extracts were dried over MgSO4 and con-
centrated in vacuo. The crude residue was purified by flash
chromatography (CH2Cl2/PE/EtOAc 5:6:1) to provide 3 (868
mg, 86%) as a solid. Method B: A mixture of 2 (513 mg, 1.3
mmol), freshly prepared Pd(PPh3)4 (223 mg, 0.19 mmol), and
LiCl (137 mg, 3.23 mmol) in DMF (10 mL) was added to
n-tributyl(1-ethoxyvinyl)stannane (627 µL, 1.94 mmol). The
solution was stirred for 12 h at 90 °C. The solvent was
evaporated in vacuo. The crude residue was purified by flash
chromatography (CH2Cl2/PE/EtOAc 5:6:1) to give 3 (341 mg,
84%) as a solid; mp 223-225 °C (MeOH). IR (KBr): ν 3005,
2935, 1670, 1560, 755, 700 cm-1. 1H NMR (300 MHz, CDCl3):
δ 8.08 (s, 1H, H7), 7.45-7.38 (m, 3H, Harom), 7.19-7.16 (m,
2H, Harom), 3.75 (s, 3H, CH3), 2.70 (s, 3H, CH3), 2.59 (s, 3H,
CH3). MS (ESI): m/z 314 (M + H+). Anal. (C15H15N5OS) C,
H, N.
>100
26.8
a Compounds were tested at various concentrations for their effects on
the survival of the human neuroblastoma SH-SY5Y cells and human
embryonic kidney cells (HEK293). b Cell survival was estimated 48 h after
the addition of each compound using the MTS reduction assay. IC50 values
were calculated from the dose-response curves and are reported in µM.
described previously6 and kindly provided by Dr. Herve´
Galons and Nassima Oumata (Universite´ Rene´ Descartes,
Paris 5). Melting points were determined using a Bu¨chi
capillary instrument and are uncorrected. IR spectra were
1
recorded on a Perkin-Elmer Spectrum One. H spectra were
recorded on a Bruker Advance 300 MHz spectrometer.
Chemical shifts are reported in ppm (δ) relative to tetram-
ethylsilane as an internal standard. Mass spectra were
recorded with a Perkin-Elmer SCIEX API spectrometer.
Elemental analyses were performed on a Thermoquest Flash
1112 series EA analyzer. Thin layer chromatography (TLC)
analyses were conducted on aluminum sheets silica gel Merck
60F254. The spots were visualized using an ultraviolet light.
Flash chromatography was carried out on silica gel 60 (40

Pyrazolo[1,5-a]-1,3,5-triazine as a Purine Bioisostere
Table 3. Effects of (R)-Roscovitine and 7a on Cell Proliferation and Cell Death as Assayed in the NCI 60 Cell Line Screening Panela
GI50 (µM)b TGI (µM)c LC50 (µM)d
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 659
cell line
(R)-roscovitine (R)-roscovitine (R)-roscovitine
7a
7a
7a
Leukemia
CCRF-CEM
>100
19.9
50.1
25.1
25.1
5.01
0.65
0.41
2.44
2.18
2.23
0.25
>100
>100
>100
>100
>100
>100
9.26
2.68
7.38
9.45
6.93
1.59
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
NSCL Cancer
A549/ATCC
EKVX
12.6
19.9
25.1
50.1
19.9
12.6
31.6
25.1
15.8
1.58
2.41
2.10
1.67
1.84
1.71
1.86
1.05
1.46
>100
50.1
41.4
9.13
8.72
7.10
10.1
7.79
8.92
10.9
8.98
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
HOP-62
>100
>100
>100
50.1
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
>100
>100
63.1
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
12.6
31.6
5.01
19.9
19.9
5.01
79.4
1.12
1.20
0.51
0.87
2.00
0.71
1.38
31.6
3.15
3.27
15.0
10.5
15.5
2.79
5.56
>100
>100
>100
>100
>100
>100
>100
8.86
>100
>100
>100
>100
63.1
8.96
>100
>100
>100
9.30
KM12
SW-620
>100
>100
CNS Cancer
SF-268
SF-295
SF-539
SNB-19f
SNB-75
U251f
19.9
25.1
15.8
NDe
12.6
50.1
1.82
0.81
1.22
1.44
1.31
1.40
>100
>100
79.4
7.20
4.74
3.62
12.3
3.33
10.5
>100
>100
>100
ND
>100
>100
>100
>100
11.9
ND
>100
8.48
31.6
>100
>100
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
12.6
19.9
25.1
15.8
ND
10
1.07
2.60
2.00
1.47
1.16
1.78
3.56
1.37
>100
50.1
>100
39.8
ND
10.3
6.99
7.93
6.00
3.82
4.96
15.0
5.50
>100
>100
>100
>100
ND
34.4
43.5
34.3
>100
16.9
17.4
>100
31.1
31.6
ND
79.4
ND
7.9
ND
>100
31.6
Ovarian Cancer
IGROV1
6.3
0.78
1.15
2.57
1.85
2.15
5.55
>100
>100
>100
>100
ND
>100
4.60
37.0
15.0
54.8
40.2
>100
>100
>100
>100
ND
>100
42.4
>100
>100
>100
>100
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
12.6
31.6
39.8
ND
79.4
>100
>100
Renal Cancer
786-0
A498
25.1
50.1
12.6
6.3
1.98
1.25
0.81
1.02
0.25
2.41
2.00
1.59
>100
>100
79.4
10.7
3.44
10.7
8.35
0.79
14.4
10.9
>100
>100
>100
>100
>100
>100
ND
>100
9.47
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
49.1
>100
>100
ND
>100
4.25
5.0
ND
63.1
12.6
58.1
>100
>100
>100
>100
>100
>100
Prostate Cancer
PC-3
DU-145
19.9
15.8
0.93
1.30
63.1
>100
12.3
9.52
>100
>100
>100
>100
Breast Cancer
MCF7
12.6
6.3
0.59
2.08
2.37
1.59
2.06
1.56
2.48
1.88
>100
63.1
>100
12.6
50.1
ND
7.99
15.7
9.47
6.16
10.1
5.29
13.3
5.19
>100
>100
>100
79.4
>100
>100
>100
>100
41.4
NCI/ADR-RESg
MDA-MB-231/ATCC
HS 578T
39.8
12.8
12.6
ND
MDA-MB-435h
BT-549
>100
ND
36.1
T-47D
16.8
ND
79.4
ND
>100
ND
>100
63.5
MDA-MB-468
MG_MIDi
19.3
1.41
78.3
8.71
99.4
61.6
a Data obtained from NCI’s in vitro-oriented tumor cells screen ((R)-roscovitine ) NSC 701554; 7a ) NSC 743927). b GI50 is the molar concentration
causing 50% growth inhibition of tumor cells. c TGI is the molar concentration giving total growth inhibition. d LC50 is the molar concentration leading to
50% net cell death. e ND ) not determined. f CNS cell lines SNB-19 and U251 are derived from the same individual. g NCI/ADR-RES is an ovarian tumor
cell line, not a breast tumor line. h MDA-MB-435 is a melanoma cell line, not a breast cancer cell line cell line. i MG_MID (mean graph midpoint) is the
arithmetical mean value for all tested cancer lines.
4-(N-Methyl-N-phenylamino)-8-(1-methylethyl)-2-(meth-
ylsulfanyl)pyrazolo[1,5-a]-1,3,5-triazine (4). At 0 °C, a solu-
tion of 3 M MeMgBr in Et2O (297 µL, 2.58 mmol) was added
dropwise to a solution of 3 (269 mg, 0.86 mmole) in THF (7
mL). After completion of the addition, the solution was stirred
at room temperature for an additional 20 min. The reaction

660 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Popowycz et al.
flash chromatography (PE/Et2O 20:1 then 9:1) to afford 5b as
a solid (51 mg, 40%); mp 85-87 °C (EtOAc/PE). IR (KBr): ν
3335, 2955, 1615, 1570 cm-1. 1H NMR (300 MHz, CDCl3): δ
8,.47 (s, 1H, NH), 7.93 (s, 1H, Harom), 7.82 (s, 1H, H7), 7.60 (d,
1H, J ) 7.5 Hz, Harom), 7.32 (t, 1H, J ) 8.1 Hz, Harom), 7.16 (d,
1H, J ) 7.4 Hz, Harom), 3.19 (hept, 1H, J ) 7.0 Hz, CH), 2.61
(s, 3H, CH3), 1.35 (d, 6H, J ) 7.0 Hz, 2 CH3). MS (ESI): m/z
334 (M + H+). Anal. (C15H16ClN5S) C, H, N.
4-(N-Benzylamino)-8-(1-methylethyl)-2-(methylsulfo-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (6a). Under an inert atmo-
sphere, 50% m-CPBA (249 mg, 0.72 mmol) was added to a
solution of 5a (74 mg, 0.24 mmol) in CH2Cl2 (5 mL) at 0 °C.
The reaction mixture was stirred for 1 h at 0 °C and was allowed
to warm up to room temperature for 3 h. The reaction mixture
was diluted with CH2Cl2 (10 mL), washed with a saturated
aqueous NaHCO3 solution (20 mL), and then H2O (20 mL).
The organic phase was dried over MgSO4 and concentrated in
vacuo. The crude product was purified by flash chromatography
(PE/EtOAc 4:1 then 2:1) to afford 6a as a solid (81 mg, 97%);
mp 123-125 °C (EtOAc/PE). IR (KBr): ν 3395, 2955, 2870,
Figure 3. In vivo antitumor activity of 7a. Compound 7a treatment
of Ewing’s sarcoma xenografts established in mice resulted in tumor
growth delay similar to that produced by (R)-roscovitine. Xenografts
established by sc inoculation of A4573 cells (4 × 106 per animal) in
nude mice were grown to a mean volume of about 195 mm3. Animals
carrying tumors were randomized into three groups. One group (n )
6) was treated with compound 7a in DMSO, administered as single
daily ip injections, at a dose of 25 mg/kg, for five days. Another group
(n ) 6) received (R)-roscovitine under the same conditions, except that
the dose was 50 mg/kg. The control group (n ) 6) received ip injections
of DMSO following identical schedules. Results shown correspond to
one of the experimental replicas. Tumor growths in compound 7a and
(R)-roscovitine-treated animals were significantly slower than the growth
of tumors in control animals.
1
1645, 1595, 1465, 1045, 750, 700 cm-1. H NMR (300 MHz,
CDCl3): δ 7.83 (s, 1H, H7), 7.43 (t, 1H, J ) 5.9 Hz, NH),
7.30-7.26 (m, 5H, Harom), 4.82 (d, 2H, J ) 5.9 Hz, CH2), 3.29
(s, 3H, CH3), 3.19 (hept, 1H, J ) 7.0 Hz, CH), 1.28 (d, 6H, J
) 7.0 Hz, 2 CH3). MS (ESI): m/z 346 (M + H+). Anal.
(C16H19N5O2S) C, H, N.
4-(3-Chloroanilino)-8-(1-methylethyl)-2-(methylsulfo-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (6b). Under an inert atmo-
sphere, 50% m-CPBA (159 mg, 0.93 mmol) was added to a
solution of 5b (51 mg, 0.15 mmol) in CH2Cl2 (3.5 mL) at 0 °C.
The reaction mixture was stirred for 1 h at 0 °C and was allowed
to warm up to room temperature for 3 h. The reaction mixture
was diluted with CH2Cl2 (10 mL), washed with a saturated
aqueous NaHCO3 solution (10 mL), and then H2O (10 mL).
The organic phase was dried over MgSO4 and concentrated in
vacuo. The crude product was purified by flash chromatography
(PE/EtOAc 9:1 then 2:1) to afford 6b as a solid (34 mg, 60%);
mp 172-174 °C (EtOAc/PE). IR (KBr): ν 3295, 2960, 2860,
1625, 1570, 1030, 755, 675 cm-1. 1H NMR (300 MHz, CDCl3):
δ 8.71 (s, 1H, NH), 8.02 (s, 1H, H7), 7.78-7.76 (m, 2H, Harom),
7.35 (t, 1H, J ) 8.3 Hz, Harom), 7.18 (d, 1H, J ) 8.3 Hz, Harom),
3.38 (s, 3H, CH3), 3.29 (hept, 1H, J ) 7.0 Hz, CH), 1.37 (d,
6H, J ) 7.0 Hz, 2 CH3). MS (ESI): m/z 366 (M + H+). Anal.
(C15H16ClN5O2S) C, H, N.
mixture was neutralized by a freshly prepared aqueous NH4Cl
solution (5 mL). The aqueous layer was extracted with
CH2Cl2 (5 mL) and EtOAc (2 × 5 mL). The combined
organic extracts were dried over MgSO4 and concentrated.
The oily residue was used without further purification. A
solution of the intermediate in CH2Cl2 (9 mL) was added at
0 °C under nitrogen atmosphere to a solution of NaBH4 (113
mg, 3.00 mmol) in trifluoroacetic acid (9 mL). The reaction
mixture was stirred for 1 h at room temperature and then
neutralized by a 1 M NaOH solution (15 mL). The aqueous
layer was extracted with CH2Cl2 (3 × 10 mL). The combined
organic extracts were dried over MgSO4 and concentrated
in vacuo. The residue was purified by flash chromatography
(PE/Et2O 9:1) to afford 4 as a solid (241 mg, 89%); mp
85-87 °C (EtOAc/PE); IR (KBr): ν 3055, 2960, 2865, 1615,
1
1535, 1460, 750, 695 cm-1. H NMR (300 MHz, CDCl3): δ
7.54 (s, 1H, H7), 7.41-7.31 (m, 3H, Harom), 7.19-7.15 (m,
2H, Harom), 3.70 (s, 3H, CH3), 3.14 (hept, 1H, J ) 6.8 Hz,
CH), 2.55 (s, 3H, CH3), 1.27 (d, 6H, J ) 6.8 Hz, 2 CH3).
MS (ESI): m/z 314 (M + H+). Anal. (C16H19N5S) C, H, N.
4-(N-Benzylamino)-8-(1-methylethyl)-2-(methylsulfa-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (5a). In a sealed tube, a
solution of 4 (120 mg, 0.38 mmol) and benzylamine (209 µL,
1.91 mmol) in ethanol (1 mL) was heated for 12 h at 90 °C.
Concentration of the reaction mixture provided the crude product
that was purified by flash chromatography (PE/Et2O 4:1) to
afford 5a as a solid (101 mg, 84%); mp 139-141 °C (MeOH).
IR (KBr): ν 3280, 2960, 1630, 1605 cm-1. 1H NMR (300 MHz,
CDCl3): δ 7.69 (s, 1H, H7), 7.33-7.28 (m, 5H, Harom), 6.93 (t,
1H, J ) 6.0 Hz, NH), 4.78 (d, 2H, J ) 6.0 Hz, CH2), 3.15
(hept, 1H, J ) 7.0 Hz, CH), 2.57 (s, 3H, CH3), 1.32 (d, 6H, J
) 7.0 Hz, 2 CH3). MS (ESI): m/z 314 (M + H+). Anal.
(C16H19N5S) C, H, N.
2-((R)-1-Amino-2-butanol)-4-(N-benzylamino)-8-(1-meth-
ylethyl)pyrazolo[1,5-a]-1,3,5-triazine (7a). In a sealed tube, a
solution of 6a (104 mg, 0.30 mmol) and (R)-(-)-2-aminobutanol
(142 µL, 1.50 mmol) in dry dioxane (1 mL) was heated for
12 h at 140 °C. After evaporation of the solvent, the residue
was purified by flash chromatography (PE/EtOAc 2:1) to afford
25
7a as a solid (60 mg, 56%); mp 37-39 °C. [R] ) +40.6 (c
589
0.5, CH2Cl2). IR (film): ν 3275, 2960, 2875, 1650, 1600, 1560,
1
1435, 765, 695 cm-1. H NMR (300 MHz, CDCl3): δ 7.56 (s,
1H, H7), 7.33-7.24 (m, 5H, Harom), 7.10 (s, 1H, NH), 5.23 (s,
1H, OH), 4.73-4.65 (m, 2H, CH2), 4.00-3.88 (m, 1H, CH),
3.83 (d, 1H, J ) 10.8 Hz, CH2O), 3.66 (dd, 1H, J ) 7.3, 10.8
Hz, CH2O), 3.02 (hept, 1H, J ) 6.6 Hz, CH), 1.70-1.52 (m,
2H, CH2), 1.28 (d, 6H, J ) 6.6 Hz, 2 CH3), 1.03 (t, 3H, J )
7.4 Hz, CH3). MS (EI + VE): m/z 354 (M+). Anal. (C19H26N6O)
C, H, N.
4-(3-Chloroanilino)-8-(1-methylethyl)-2-(methylsulfa-
nyl)pyrazolo[1,5-a]-1,3,5-triazine (5b). In a sealed tube, a
solution of 4 (120 mg, 0.38 mmol) and 3-chloroaniline (204
µL, 1.91 mmol) in dioxane (1 mL) was heated for 12 h at 120
°C. After concentration in vacuo, the residue was purified by
2-((S)-1-Amino-2-butanol)-4-(N-benzylamino)-8-(1-meth-
ylethyl)pyrazolo[1,5-a]-1,3,5-triazine (7b). In a sealed tube,
a solution of 6a (84 mg, 0.24 mmol) and (S)-(-)-2-aminobutanol
(115 µL, 1.22 mmol) in dry dioxane (1 mL) was heated for

Pyrazolo[1,5-a]-1,3,5-triazine as a Purine Bioisostere
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 661
12 h at 140 °C. After evaporation of the solvent, the residue
132-133 °C (EtOH). IR (KBr): ν 3250, 2970, 1620, 1380, 1330,
1
was purified by flash chromatography (PE/EtOAc 1:2) to afford
1145, 970, 755 cm-1. H NMR (300 MHz, CDCl3): δ 7.40
25
7b as a solid (58 mg, 67%); mp 37-39 °C. [R] ) -40.6 (c
(broad s, 1H, NH), 7.31-7.39 (m, 6H, Harom + H6), 4.88 (d,
2H, J ) 5.8 Hz, CH2), 3.43 (hept, 1H, J ) 7.0 Hz, CH), 3.32
(s, 3H, CH3), 1.39 (d, 6H, J ) 7.0 Hz, 2 CH3). MS (ESI): m/z
346 (M + H+). Anal. (C16H19N5O2S2) C, H, N.
2-((R)-1-Amino-2-butanol)-4-(N-benzylamino)-7-(1-meth-
ylethyl)imidazo[2,1-f]-1,2,4-triazine (13). A mixture of 12 (253
mg, 0.73 mmol) and (R)-(-)-2-aminobutanol (0.35 mL, 3.7
mmol) was heated for 4 h at 170 °C. Excess of amine was
removed by Kugelrohr distillation at 10-2 mmHg, and the crude
residue was purified by flash chromatography (PE/EtOAc 6:4
589
0.5, CH2Cl2). Anal. (C19H26N6O) C, H, N.
2-((1R)-(1-Methylethyl)-2-hydroxyethylamino)-4-(3-chlo-
roanilino)-8-(1-methylethyl)pyrazolo[1,5-a]-1,3,5-triazine (7c).
In a sealed tube, a solution of 6b (34 mg, 0.09 mmol) and (R)-
(-)-2-amino-3-methylbutanol (47.5 mg, 0.46 mmol) in dry
dioxane (1 mL) was heated for 12 h at 140 °C. After evaporation
of the solvent, the residue was purified by flash chromatography
(PE/EtOAc 4:1) to afford 7c as a solid (25 mg, 70%); mp
25
161-163 °C (EtOAc). [R] ) +32.5 (c 0.27, CH2Cl2). IR
589
(KBr): ν 3360, 3310, 2955, 1635, 1615, 1575, 1420, 1070, 760,
to 1:1) to give 13 (155 mg, 60%) as a solid; mp 105-106 °C
1
25
680 cm-1. H NMR (300 MHz, DMSO-d6 + D2O, 75 °C): δ
(PE/EtOAc 8:2). [R] ) +50.8 (c 0.5, CHCl3). IR (KBr): ν
589
8.05 (s, 1H, Harom), 7.88 (d, 1H, J ) 8.0 Hz, Harom), 7.77 (s,
1H, H7), 7.38 (t, 1H, J ) 8.0 Hz, Harom), 7.16 (d, 1H, J ) 8.0
Hz, Harom), 3.83 (q, 1H, J ) 5.9 Hz, CH), 3.60-3.50 (m, 2H,
CH2O), 2.96 (hept, 1H, J ) 7.0 Hz, CH), 1.92-2.02 (m, 1H,
CH), 1.27 (d, 6H, J ) 7.0 Hz, 2 CH3), 0.94 (d, 3H, J ) 6.8 Hz,
CH3), 0.93 (d, 3H, J ) 6.8 Hz, 3 CH3). MS (ESI): m/z 389 (M
+ H+). Anal. (C19H25ClN6O) C, H, N.
3330, 3220, 3100, 1615, 1570, 1540, 1450, 1370, 1055, 755,
1
720 cm-1. H NMR (300 MHz, CDCl3): δ 7.34-7.27 (m, 5H,
Harom), 7.08 (s, 1H, H6), 6.89 (broad s, 1H, NH), 4.73 (d, 2H, J
) 5.5 Hz, CH2), 4.57 (d, 1H, J ) 6.4 Hz, OH), 3.89-3.78 (m,
2H, CH2O + CH), 3.70-3.60 (m, 1H, CH2O), 3.37 (broad s,
1H, NH), 3.24 (hept, 1H, J ) 7.0 Hz, CH), 1.73-1.51 (m, 2H,
CH2), 1.35 (d, 3H, J ) 7.0 Hz, CH3), 1.34 (d, 3H, J ) 7.0 Hz,
CH3), 1.02 (t, 1H, J ) 7.4 Hz, CH3). MS (EI + VE): m/z 354
(M+). Anal. (C19H26N6O) C, H, N.
7-(1-Methylethyl)-2,4-bis(methylsulfanyl)imidazo[2,1-f]-
1,2,4-triazine (10). A solution of triazine 815 (558 mg, 2.96
mmol), camphorsulphonic acid (50 mg, 0.21 mmol), 2-bromo-
3-methylbutyraldehyde dimethyl acetal 916 (0.7 mL), and MS
4 Å (50 mg) in dry MeCN (30 mL) were heated at reflux for
48 h. The reaction was completed by addition of 9 (0.5 mL) to
the medium at 24 h. The solids were removed by filtration and
washed with CH2Cl2. After evaporation in vacuo, the residue
was taken up in CH2Cl2 (40 mL) and washed with saturated
aqueous NaHCO3 solution (10 mL), then brine (10 mL). The
organic phase was dried over MgSO4 and concentrated in vacuo.
The crude product was purified by flash chromatography (PE/
EtOAc 95:5) to give 10 (452 mg, 60%) as a solid; mp 85-86
°C. IR (KBr): ν 2960, 1570, 1510, 1435, 1355, 1150 cm-1. 1H
NMR (300 MHz, CDCl3): δ 7.40 (s, 1H, H6), 3.40 (hept, 1H, J
) 7.0 Hz, CH), 2.66 (s, 3H, CH3), 2.60 (s, 3H, CH3), 1.39 (d,
6H, J ) 7.0 Hz, 2 CH3). MS (ESI): m/z 255 (M + H+). Anal.
(C10H14N4S2) C, H, N.
Biology. Protein Kinase Assays. CDK1/cyclin B (native
affinity purified from starfish oocytes), CDK2/cyclin A, CDK7/
cyclin H, CDK9/cyclin T (human recombinant, expressed in
baculovirus-infected insect cells), and CDK5/p25 (human
recombinant, expressed in E. coli) were assayed in the presence
of 15 µM ATP as previously described.16 IC50 values were
determined from dose-response curves and are expressed in
µM.
Cell Cultures and Reagents. A549, PC3, Hela, and 293T
cells were cultured in complete medium (DMEM without phenol
red, supplemented with 10% heat-inactivated fetal calf serum,
2 mM Glutamax-I, 100 IU/mL penicillin, 100 µg/mL strepto-
mycin). Raji and U937 cells were cultured in complete medium
(RPMI 1640 without phenol red supplemented with 10% heat-
inactivated fetal calf serum, 2 mM Glutamax-I, 100 IU/mL
penicillin, 100 µg/mL streptomycin). All cells were kept at 37
°C in a humidified atmosphere (5% CO2 in air). Exponentially
growing cells were used for all experiments. Cell culture
reagents were purchased from Invitrogen. SH-SY5Y human
neuroblastoma cell line was grown in DMEM supplemented
with 2 mM L-glutamine (Invitrogen, Cergy Pontoise, France)
plus antibiotics and a 10% volume of FCS (Invitrogen). Cell
viability was determined by means of the MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium) method as previously described in
detail.19 Roscovitine was purchased from Sigma-Aldrich and
dissolved in DMSO to make a 10 mM stock, which was then
diluted as desired into complete medium. The DMSO concen-
tration was kept equal to 0.1% in all cell culture wells.
4-Benzylamino-7-(1-methylethyl)-2-(methylsulfanyl)imid-
azo[2,1-f]-1,2,4-triazine (11). A mixture of 10 (228 mg, 0.90
mmol) and benzylamine (1 mL) was stirred at 50 °C for 4 h.
Amine in excess was removed at 10-2 mm Hg using Kugelrohr
apparatus. The solid residue was recrystallized from EtOH to
yield 11 (232 mg, 83%). Mother liquors were concentrated and
the crude residue was purified by flash chromatography (PE/
EtOAc 7:3) gel to give an additional quantity (35 mg) of 11
(overall yield: 95%); mp 161-162 °C (EtOH). IR (KBr): ν
3245, 2960, 1620, 1340, 1270, 1195 cm-1. 1H NMR (300 MHz,
CDCl3): δ 7.30-7.34 (m, 5H, Harom), 7.13 (s, 1H, H6), 6.94
(broad s, 1H, NH), 4.79 (d, 2H, J ) 5.6 Hz, CH2), 3.35 (hept,
1H, J ) 7.0 Hz, CH), 2.55 (s, 3H, CH3), 1.37 (d, 6H, J ) 7.0
Hz, 2 CH3). MS (ESI): m/z 314 (M + H+). Anal. (C16H19N5S)
C, H, N.
Cell Proliferation Assays. Five thousand cells of each cell
line were seeded in 90 µL of culture medium/well in 96-well
plates (Greiner, no. 655098), and we incubated the plates for
6 h at 37 °C. We then added different concentrations of (R)-
roscovitine or compound 7a in triplicate, and plates were
incubated for 72 h. To quantify viable cells at the beginning of
the experiment, a Vialight assay (Cambrex, no. LT07-121) was
used in a control plate where cells were treated with 0.1%
DMSO. Briefly, 50 µL of cell lysis buffer were added, followed
by a 10 min incubation at room temperature. Then 100 µL of
Vialight assay buffer was added, and after a 2 min incubation,
luminescence was recorded in each well with an Envision plate
4-Benzylamino-7-(1-methylethyl)-2-(methylsulfonyl)imid-
azo[2,1-f]-1,2,4-triazine (12). To an ice-cold solution of 11 (331
mg, 1.06 mmol) in CH2Cl2 (10 mL) was added 70% m-CPBA
(781 mg, 3.17 mmol) in small portions. The reaction was stirred
for 30 min at 0 °C then 3.5 h at room temperature. The mixture
was diluted in CH2Cl2 (10 mL), washed with saturated aqueous
NaHCO3 solution (10 mL), and then brine (10 mL). The organic
phase was dried over MgSO4 and concentrated in vacuo. The
crude residue was purified by flash chromatography (PE/EtOAc
85:15 to 1:1) to provide 12 (295 mg, 81%) as a solid; mp

662 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Popowycz et al.
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reader (Perkin-Elmer). The average luminescence value was
calculated on 6 wells for each cell line. After 72 h, a Vialight
assay was performed on all test plates and on the control plate.
In Vivo Antitumor Activity. Experiments to evaluate the
antitumor activity of compound 7a were carried out essentially
as previously described14,19 under protocols approved by the
Georgetown University Animal Care and Use Committee, using
immunodeficient, male (5-6 weeks old) athymic nude (BALB/c
nu/nu) mice purchased from the National Cancer Institute.20
Mice were injected sc into the right posterior flank with 4 ×
106 A4573 Ewing’s sarcoma cells in 100 µL of Matrigel
basement membrane matrix (BD Biosciences, San Jose, Cali-
fornia). Once tumors reached a mean volume of about 195 mm3,
mice were randomized into three groups (six animals per group)
and treatment was initiated. Experimental groups were treated
with compound 7a or (R)-roscovitine, dissolved in DMSO, and
administered as a single daily ip injection, at a dose of 25 or
50 mg/kg, respectively, for 5 days. The control group received
ip injections of DMSO following an identical schedule. Tumor
growth was followed for up to 4 weeks after the first injection.
Tumor volumes were calculated by the formula V ) (1/2)a ×
b2, where a is the longest tumor axis, and b is the shortest tumor
axis. Whenever tumors reached the maximum volume allowed
by institutional tumor burden guidelines, animals were sacrificed
by asphyxiation with CO2. Tumors were immediately excised
from euthanized animals and measured. Data are given as mean
values ( SD. Statistical analysis of differences between groups
was performed by a one-way ANOVA, followed by an unpaired
Student’s t test.
Acknowledgment. This research was supported by grants
from the EEC (FP6-2002-Life Sciences & Health, PRO-
KINASE Research Project) (L.M.), the “Cance´ropole Grand-
Ouest” grant (L.M.), from the “Institut National du Cancer”
(INCa), Cancer De´tection d’Innovations 2006 (L.M.), from the
“Ligue Nationale contre le Cancer” (L.M.), and by U.S. NIH
grant PO1-CA74175 (V.N.). K.B. was supported by a fellowship
from the “Ministe`re de la Recherche” and from the “Association
pour la Recherche sur le Cancer”. We are grateful to J. Boix
for the SH-SY5Y cell line. We thank the National Cancer
Institute (Bethesda, MD) for the antitumor tests reported in this
paper.
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Supporting Information Available: Elemental analyses; NCI
60 mean graphs data for 7a; NCI 60 mean graphs data for (R)-
roscovitine; the effects of (R)-roscovitine (A) and 7a (B) on growth
of six human tumor cell lines (A549, PC3, Hela, 293T, Raji, and
U937). This material is available free of charge via the Internet at
http://pubs.acs.org.
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