Cyclic nucleotide phosphodiesterase type 4 inhibitors: Evaluation of pyrazolo[1,5-a]-1,3,5-triazine ring system as an adenine bioisostere

Article abstract of DOI:10.1016/j.ejmech.2007.05.016

A series of 8-substituted pyrazolo[1,5-a]-1,3,5-triazines were considered as a bioisosteric replacement for the 9-substituted adenine derivatives resulting in the discovery of 8-(2-methoxybenzyl)-4-(N-methylamino)-2-n-propylpyrazolo[1,5-a]-1,3,5-triazine (14d) and 2-trifluoromethyl-8-(2-methoxybenzyl)-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14e) as a new structural class of potent phosphodiesterase type 4 inhibitors (IC₅₀ = 13 nM and 11 nM, respectively) with high isoenzyme selectivity. An original tandem of reactions involving a palladium-mediated cross-coupling reaction (PMCCR) of the readily available 8-iodo-2-methyl-4-(N-methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (11a) and arylboronic acids or alkynes followed by the displacement of the N-methyl-N-phenylamino group constitute the key steps in a novel synthetic approach developed herein. The treatment of 11a-c with n-BuLi and selected aldehydes represents an interesting alternative to the PMCCR for the synthesis of benzylic derivatives 14a-i. Preliminary biological testing has shown that compounds 14d and 14e strongly inhibit LPS-induced TNFα release from human mononuclear cells from healthy subjects. These two compounds were selected for further biological evaluation.

Full text of DOI:10.1016/j.ejmech.2007.05.016

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European Journal of Medicinal Chemistry 43 (2008) 816e829
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Original article
Cyclic nucleotide phosphodiesterase type 4 inhibitors: Evaluation
of pyrazolo[1,5-a]-1,3,5-triazine ring system as an adenine bioisostere
Pierre Raboisson a,c, Dominique Schultz a, Christian Muller b, Jean-Marie Reimund b,
Guillaume Pinna b, Romain Mathieu a, Philippe Bernard d, Quoc-Tuan Do d,
c
Renee L. DesJarlais , Helene Justiano , Claire Lugnier , Jean-Jacques Bourguignon
b
b
a,
*
´ `
a Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7175 du CNRS, Universite´ Louis Pasteur,
Faculte´ de Pharmacie, 74, route du Rhin, 67401 Illkirch Cedex, France
b Laboratoire de Pharmacologie et de Physico-Chimie des Interactions Cellulaires et Mole´culaires,
UMR 7175 du CNRS, Universite´ Louis Pasteur, Faculte´ de Pharmacie, 74, route du Rhin, 67401 Illkirch Cedex, France
c Tibotec BVBA, Gen. De Wittelaan L11 B3, B-2800 Mechelen, Belgium
d GreenPharma S.A., 3, Alle´e du Titane, 45100 Orle´ans, France
Received 9 May 2007; accepted 31 May 2007
Available online 8 June 2007
Abstract
A series of 8-substituted pyrazolo[1,5-a]-1,3,5-triazines were considered as a bioisosteric replacement for the 9-substituted adenine deriva-
tives resulting in the discovery of 8-(2-methoxybenzyl)-4-(N-methylamino)-2-n-propylpyrazolo[1,5-a]-1,3,5-triazine (14d) and 2-trifluoro-
methyl-8-(2-methoxybenzyl)-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14e) as a new structural class of potent phosphodiesterase
type 4 inhibitors (IC50 ¼ 13 nM and 11 nM, respectively) with high isoenzyme selectivity. An original tandem of reactions involving a palla-
dium-mediated cross-coupling reaction (PMCCR) of the readily available 8-iodo-2-methyl-4-(N-methyl-N-phenylamino)pyrazolo[1,5-a]-
1,3,5-triazine (11a) and arylboronic acids or alkynes followed by the displacement of the N-methyl-N-phenylamino group constitute the key
steps in a novel synthetic approach developed herein. The treatment of 11aec with n-BuLi and selected aldehydes represents an interesting
alternative to the PMCCR for the synthesis of benzylic derivatives 14aei. Preliminary biological testing has shown that compounds 14d and
14e strongly inhibit LPS-induced TNFa release from human mononuclear cells from healthy subjects. These two compounds were selected
for further biological evaluation.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Bioisostere; Pyrazolo[1,5-a]-1,3,5-triazines; Phosphodiesterase; Type 4; TNFa; Inflammation
1. Introduction
findings have led to intense efforts in the last few years to
identify highly potent PDE4 inhibitors as promising treat-
ment for autoimmune and inflammatory diseases [1,3,4].
Moreover, several PDE4 inhibitors have demonstrated clini-
cal efficacy [4]. Unfortunately, the development of pioneer
PDE4 inhibitors, such as the archetypal rolipram (1, Fig. 1)
and structurally-related compounds (e.g. Ariflo, 2) has been
hampered by their propensity to induce various side effects,
such as nausea, emesis, gastric acid secretion, or central ner-
vous system activation [2,4e7]. As a result, these compounds
suffered from a limited therapeutic index [8]. Thus, the de-
sign of novel, potent and selective second-generation PDE4
Cyclic AMP phosphodiesterases (PDE4), responsible for the
hydrolysis of key second messenger cyclic AMP (cAMP), me-
diate a wide array of activities and are particularly abundant in
airway smooth muscle, and in inflammatory and immune cells,
where PDE4s regulate the production of inflammatory media-
tors, cytokines and reactive oxygen species [1,2]. These
* Corresponding author. Tel.: þ333 9024 4234; fax: þ333 9024 4310.
E-mail address: jacques.bourguignon@pharma.u-strasbg.fr (J.-J. Bourguignon).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.05.016

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
817
NH2
N
OCH3
O
CH3O
O
N
N
N
N
O
H
N
N
H2N
O
N
O
O
NC
N
H
O
P
OH
O
OH
O
COOH
cAMP
2
3
1
NH2
HNCH3
HNCH3
HNCH3
N
N
N
N
N
N
N
N
N
N
N
N
H3C
N
H3C
N
N
R2
O
N
R1
O
O
HOOC
(OH)2P(O)-O
(OH)2P(O)-O
(OH)2P(O)-O
5
O
OH
R1 = CH3, n-Pr, CF3, I
R2 = alkyl or aralkyl
HOOC
(OH)2P(O)-O
OH
4
7
6
Fig. 1.
inhibitors with reduced emetogenic properties represents
a critical need and is still a challenge in the pharmaceutical
industry [4].
improving the therapeutic interest over the parent adenines
[17,18].
We have recently reported that the pyrazolo[1,5-a]-1,3,5-
triazine C-nucleoside (7) as a potent purinergic P2Y1 receptor
antagonist with enhanced metabolic stability compared to the
parent purine derivative (6) [19]. In view of this promising re-
sult, we sought to apply the same strategy to the synthesis of
C-analogues of 9-substituted N6-methyladenines 5 as PDE4
inhibitors. In this article, we report the details of the synthetic
studies which have successfully led to the preparation of novel
pyrazolo[1,5-a]-1,3,5-triazines (19, 14aei, 16a,b, 18aek) as
potent and selective PDE4 inhibitors. Subsequently, the inhibi-
tion of TNFa release from mononuclear cells stimulated with
lipopolysaccharide (LPS) was also evaluated.
It was initially proposed that rolipram produced side effects
via the binding to a high affinity binding site (HARBS) dis-
tinct from the catalytic site of PDE4 [9]. It has now been clar-
ified that the HARBS corresponds to the holoenzyme
conformer of the PDE4 [10], and that the emetic response is
a consequence of inhibition of PDE4, especially the PDE4D,
in nontarget tissues [11]. Therefore, two ways to obtain potent
selective PDE4 inhibitors with an improvement in the thera-
peutic index are the following: (i) to develop new compounds
selective toward one subtype, especially the PDE4A and C
[12] and/or (ii) with original chemical structures, which are
completely unrelated to catechol ether derivatives (e.g. roli-
pram) [9]. The utility of the second approach has been
successfully demonstrated by the recently reported benzodiaz-
epine 3 (CI-1044) [13], which is well tolerated by ferrets re-
ceiving 3 at a dose of 40 mg kgꢀ1 i.v., whereas 2 was emetic
at 10 mg kgꢀ1 i.v. in the same model [4]. Another method to
obtain a potent and specific inhibitor that binds the PDE4 in
the catalytic site with improved margin of safety is to start
from the structure of the natural substrate (cAMP). The natural
product griseolic acid (4) was one of the first reported sub-
strate-related PDE4 inhibitors [14]. Moreover, several years
ago, we reported that adenine derivatives substituted at posi-
tion 9 (5, Fig. 1) strongly and selectively inhibited PDE4
and elicited anti-inflammatory properties [15,16]. The fact
that this series of PDE4 inhibitors did not stimulate the in
vivo gastric acid secretion in rats suggests that they may
produce fewer gastrointestinal side effects [16].
2. Semi-empirical calculations
In order to assess the similarity of the two scaffolds, com-
pounds 8 and 9 were first sketched in the Sybyl 6.8 package
[20] and minimized with the Tripos force field (Fig. 2) [21].
Then, a subsequent geometry optimization with the AM1 pa-
rameterization was performed [22] in Mopac [23] module of
Sybyl, with default parameters. Connolly surfaces were calcu-
lated with Molcad, and electrostatic potential properties were
mapped onto them. Fig. 2 shows the surface of each scaffold
colored by electrostatic potential values as determined from
the final minimized structures. The electrostatic potential sur-
faces of the two scaffolds (pyrazolotriazine 9 and adenine 8)
were found to be strikingly similar. Moreover, the amount of
surface area corresponding to the negative value is very close
2
2
˚
˚
(136 A for 8 and 137 A for 9).
However, since 9-substituted adenine derivatives have
been extensively studied, it is very difficult to patent such
compounds. One way to overcome this difficulty is to trans-
fer the structureeactivity relationships from the purine ring
to a novel bioisosteric heterocycle while maintaining or
In view of these promising preliminary results obtained by
comparing the two bicyclic compounds 8 and 9, we decided to
compare the charges (Fig. 3) and isopotential curve (Fig. 4)
properties [24e28] of the adenine 20d and its pyrazolo[1,5-
a]-1,3,5-triazine analogue 14d. Details of the calculations

818
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
HNCH3
HNCH3
N
N
N
N
N
N
N
N
H3C
H3C
CH3
CH3
8
9
Fig. 2. Electrostatic potential: blue colors represent negative values while red colors positive values. Values close to zero are colored in green (for interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article).
are presented in the Section 6. Despite the existence of small
differences, these two heterocycles possess common features
and are structurally very similar. These computational studies
prompted the synthesis of novel pyrazolo[1,5-a]-1,3,5-triazines
substituted in position 8 (19, 14aei, 16a,b, 18aek), which were
tested as PDE4 inhibitors that were expected to act at the cata-
lytic site of the PDE4 with the same pharmacophoric contact
points as adenines 5.
under high pressure, followed by the regioselective iodination
at the 8-position by N-iodosuccinimide. Introduction of sub-
stituents and functions at the 8-position was then carried out
by two novel and versatile methods, both employing the suit-
able N-methyl-N-phenylamino (NMNPA) activating group,
which possesses a number of favorable characteristics includ-
ing stability, ease of handling, and reactivity [19,30]. The
first process (Fig. 5) utilizes the lithiation of key 8-iodo in-
termediates 11aec with n-BuLi and subsequent treatment
with selected aldehydes, followed by reduction of the
hydroxy group of intermediates 12bej leading to desired
8-substituted derivatives 13aei in reasonably good yields
(40e93%). Subsequent displacement of the NMNPA group
with methylamine provided the target pyrazolotriazines 19,
14aei (Table 1, 53e87% yield). The alternative route to 8-
substituted pyrazolotriazines 16aej utilizes an original tandem
of reactions involving a palladium-mediated cross-coupling
3. Chemistry
The synthetic strategies used for the preparation of the target
pyrazolo[1,5-a]-1,3,5-triazines are summarized in Figs. 5 and 6.
The starting materials, N-methyl-N-phenylamino derivatives
11aec, were prepared as previously reported [19,30] by treat-
ment of the corresponding pyrazolo[1,5-a]-1,3,5-triazin-4-ones
(10aec) with phosphorus oxychloride and dimethylaniline
Fig. 3. Mopac charges as calculated with AM1 and labelled on heavy atoms for 20d (right) and 14d (left).

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
819
Fig. 4. Isopotential curves for 20d (right) and 14d (left): red corresponds to positive values, blue to negative values, and yellow is neutral (for interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article).
reaction of the readily available 8-iodo-2-methyl-4-(N-
methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (11a)
and alkynes or boronic acids leading to intermediates 15a,b
and 17aej in 26e87% yield (Fig. 6). Subsequent displace-
ment of the NMNPA moiety with methylamine [19,30]
afforded final compounds 16a,b and 18aek (Table 1, 21e
95% yield).
structureeactivity relationships are very similar in both series.
Recently, we reported that the introduction of a n-propyl or
a trifluoromethyl group at position 2 of the adenine ring
strongly increased the potency of the resulting compounds
(compare 20a with 20b, 20c with 20d and 20e) [29]. In a similar
manner, when compared to 14a (IC50 ¼ 98 nM), the n-propyl
and trifluoromethyl derivatives (14b and 14c, respectively)
were found to be almost two fold more potent (IC50 ¼ 48 nM
and 77 nM, respectively). Moreover, the 2-methoxybenzyl
group at position 9 of the adenine (corresponding to the po-
sition 8 of the pyrazolotriazine) appeared to be especially
beneficial in both series, and increased by dfour to eight
fold the potency of the resulting compound, when compared
to the benzyl derivatives (compare 20a with 20c, 20b with
20d, 14b with 14d, and 14c with 14e). Although the 2-
furylmethyl (14g, IC50 ¼ 78 nM) and 2-thienylmethyl (14h,
IC50 ¼ 70 nM) compounds were found to be slightly more po-
tent than the benzyl derivative (14a, IC50 ¼ 98 nM), all other
substitutions at position 8 of the 2-methylpyrazolotriazine
4. Pharmacology, results and discussion
The 8-substituted pyrazolo[1,5-a]-1,3,5-triazines were
screened for their activity against PDE4 as described previ-
ously [15]. Results are summarized in Table 1. As reported
previously, a single substitution with a methyl group on the
exocyclic nitrogen atom at position 6 of the adenine ring
(corresponding to position 4 of the pyrazolotriazine ring)
was optimal [29]. Thus, the study was performed with N-
methyl derivatives. Comparison of adenines and their corre-
sponding pyrazolo[1,5-a]-1,3,5-triazines revealed that the
H3C
H3C
H3C
O
N
N
N
N
N
N
N
N
N
a, b, c
HN
N
N
N
N
N
d
N
N
e
(40%-93%)
(39%-86%)
N
N
R1
R1
R1
R1
I
R3
11a-c
R3
10a-c
12a-j
13a-i
HO
12a: R2 = CH2CH3
HNCH3
10a, 11a, 12a,b,g-j, 13a,f-i, 14a,f-i: R1 = CH3
10b, 11b, 12d,f, 13c,e, 14c,e: R1 = CF3
10c, 11c, 12c,e, 13b,d, 14b,d: R1 = n-Pr
12b-d, 13a-c,14a-c: R2 = Ph
12e,f, 13d,e,14d,e: R2 = 2-(MeO)Ph
12g, 13f,14f: R2 = 2-furyl
N
N
N
f
(53%-87%)
12h, 13g,14g: R2 = 3-furyl
12i, 13h,14h: R2 = 2-thienyl
12j, 13i,14i: R2 = 2-naphthyl
N
14a-i
R1
R3
Fig. 5. (a) POCl3, DMAP, CHCl3, 120 ꢁC, sealed tube, (b) N-methylaniline, CH2Cl2, 25 ꢁC, (c) NIS, CHCl3, reflux, (d) n-BuLi, R3CHO, THF, ꢀ78 ꢁC, (e) TMSCl,
NaI, CH3CN, (f) NH2CH3, EtOH, 100 ꢁC, sealed tube.

820
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
H3C
N
H3C
N
N
N
N
N
HNCH3
N
N
N
N
N
N
N
b
a
(71%-73%)
(62%-77%)
H3C
H3C
H3C
N
I
15a, R3 = Me
15b, R3 = n-Pr
16a, R3 = Me
16b, R3 = n-Pr
11a
R3
R3
H3C
N
HNCH3
N
c
b
N
N
N
N
N
(26%-87%)
(21%-95%)
H3C
N
N
H3C
R2
R2
R2
18a-j
17a-j
R2
17a, 18a
17b, 18b
17c, 18c
17d, 18d
Ph
2-ClPh
17f, 18f
2-furyl
17g, 18g
2-thienyl
1-naphthyl
3-acetylPh 17h, 18h
3-NO2Ph 17i, 18i
2-benzo[b]furyl
17j, 18j
2-benzo[b]thienyl
17e, 18e
3-CF3Ph
Fig. 6. (a) R3eC^CH, CuI, PdCl2, TEA, CH3CN, 25 ꢁC, (b) NH2CH3, EtOH, 100 ꢁC, sealed tube, (c) R2B(OH)2, Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, 90 ꢁC.
were found to be detrimental to the PDE4 inhibitory activity.
Finally, combining beneficial substituent effects in both posi-
tions R1 and R2 led to new compounds with increased
potencies. Thus, synergistic effects were observed when the
9-(2-methoxybenzyl) moiety was introduced in derivatives
with the most appropriate substituent in position 2. In partic-
ular, the 2-n-propylpyrazolotriazine (14d) exhibited almost
the same activity as that of the corresponding adenine 20d
(IC50 ¼ 13 nM and 7 nM, respectively). However, the benefi-
cial effect of the 2-trifluoromethyl substitution was only par-
tially recovered for the pyrazolotriazine 14e (IC50 ¼ 11 nM)
when compared to the parent adenine 20e (IC50 ¼ 1.4 nM).
Interestingly, this novel series of pyrazolotriazine derivatives
possess improved PDE4 selectivity profiles vs PDE1, -2, -3,
and -5 as demonstrated with compounds 14a, 14b, 14d, 14e,
18a, and 18k (Table 2).
With the above promising data, we selected compounds
14cee for further evaluation of their in vitro anti-inflammatory
activities by examining the suppression of TNFa release by li-
popolysaccharide (LPS)-activated peripheral blood mononu-
clear cells from healthy donors (Fig. 7). These compounds
elicited a strong concentration-dependent inhibition of the
TNFa release. However, we found no direct correlation be-
tween the activity of the compounds 14cee in PDE4 inhibition
and TNFa inhibition. These findings might be ascribed to the
difference of physicochemical properties (cell permeability,
water solubility) between these compounds or may suggest
that accumulation of cAMP is a major way to decrease
TNFa secretion but certainly not the only one.
5. Conclusion
The main objective of the present study was the bioisos-
teric replacement of the adenine ring by the pyrazolo[1,5-a]-
1,3,5-triazine in the field of PDE4 inhibitors. Thus, a series
of 8-substituted derivatives were synthesized using very effi-
cient and particularly attractive routes involving a palla-
dium-mediated cross-coupling reaction of the readily
available 8-iodo-2-methyl-4-(N-methyl-N-phenylamino)pyra-
zolo[1,5-a]-1,3,5-triazines (11a) and arylboronic acids or al-
kynes, or by the treatment of 11aec with n-BuLi and
selected aldehydes. The N-methyl-N-phenylamino group was
found to be particularly stable under these conditions and
was readily displaced at the end of the reaction sequence
with methylamine to give the target compounds 14aei,
16a,b, 18aek, and 19. As anticipated from computational
analysis, we have demonstrated that this series of pyrazolo-
triazine analogues 14aei, 16a,b, 18aek, and 19 are pharma-
cologically bioequivalent to the parent 9-substituted adenines
20aee and exhibit similar structureeactivity relationships.
Among these novel derivatives, 8-(2-methoxybenzyl)-4-meth-
ylamino-2-n-propylpyrazolo[1,5-a]-1,3,5-triazine (14d) and 2-
trifluoromethyl-8-(2-methoxybenzyl)-4-methylaminopyrazolo
[1,5-a]-1,3,5-triazine (14e) emerged as the most potent PDE4
inhibitors with IC50 values of 11 nM and 13 nM, respectively,
which was 100 fold more potent than rolipram (1.2 mM) and
2.5 fold when compared to Ariflo (30 nM). Additionally, pre-
liminary testing has shown that both 14d and 14e strongly in-
hibit cytokine production in LPS-induced human blood

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
821
Table 1
R1
R2
HNCH3
HNCH3
6
4
6
7
5
N
5
N
N
2
N
N
1
3
N
7
8
2
N
8
R1
4
N
R1
9
1
3
R2
R2
B
A
Compd
PDE4
Compd
PDE4
a
IC50 (mM)
a
IC50 (mM)
CH3
CH3
n-C3H7
CF3
CH(OH)C2H5
Bn
Bn
e
20a, [29]
20b, [29]
e
19
4.8
0.28
0.088
e
14a
14b
14c
e
0.098
0.048
0.077
e
Bn
e
20c, [29]
CH3
n-C3H7
CF3
2-(MeO)Bn
2-(MeO)Bn
2-(MeO)Bn
Furfuryl
0.061
0.007
0.0014
e
20d, [29]
20e, [29]
14d
14e
14f
14g
14h
14i
0.013
0.011
0.10
0.078
0.070
0.27
6.4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
a
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
3-Furylmethyl
2-Thienylmethyl
2-Naphthylmethyl
1-Propynyl
1-Pentynyl
Ph
e
e
e
e
16a
16b
18a
18b
18c
18d
18e
18f
18g
18h
18i
e
e
0.41
0.27
2.9
2-ClPh
e
3-Acetylph
3-NO2Ph
3-CF3Ph
e
0.54
1.2
e
e
0.13
3.1
2-Furyl
e
2-Thienyl
1-Naphthyl
2-Benzo[b]furyl
2-Benzo[b]thienyl
Bz
e
2.4
e
e
0.21
6.2
e
18j
18k, [30]
3.5
0.12
e
The IC50 was calculated by linear regression (correlation coefficient r ¼ 0.95) and represents the mean value of three determinations; the experimental error is
about 15%.
mononuclear cell preparations. Therefore, compounds 14d
and 14e were selected for further biological evaluation.
As demonstrated in our previous work on P2Y1 receptor an-
tagonists, the results presented in this study indicate that the
nitrogen in position 9 of adenine is unnecessary for activity
in both systems and appeared as a point of anchorage for
the N9 substituent. Moreover, the replacement of the purine
ring with the pyrazolotriazine would provide an increased in
Table 2
a
IC50 or % of inhibition at 10 mM
Compd PDE1
PDE2
PDE3 (%) PDE5
ꢀCaM (%) þCaM ꢀGMPc (%) þGMPc
14a
14b
14d
14e
18a
18k
a
37
24
27
20
21
16
14 mM 19
56 mM
20
31
8.6
10
33
16
38
74
11
8
25
35
e
20
19
16
16
24
e
11
20
29
3.6
15
2.7
11
17
The IC50 was calculated by linear regression (correlation coefficient
r ¼ 0.95) and represents the mean value of three determinations; the experi-
Fig. 7. Suppression of TNFa release by LPS-activated peripheral blood mono-
nuclear cells from healthy donors.
mental error is about 15%.

822
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
vivo stability when compared to the parent adenines [19].
Thus, pyrazolo[1,5-a]-1,3,5-triazine does appear to serve as
a valuable potent ‘‘carbabioisostere’’ for the purine nucleus
present in various biologically active molecules.
After the mixture was cooled to room temperature, the solvent
was evaporated in vacuo. The residue was diluted with ice-
cold water (7 mL) and the pH was adjusted to 7 with HCl
1 N. After 2 h at 0 ꢁC, the precipitate was collected by filtra-
tion and washed with water, ethanol and ether. Recrystalliza-
tion from ethanol/ether gave compound 10b (182 mg, 89%)
as a colorless solid: mp > 300 ꢁC. 1H NMR (200 MHz,
DMSO-d6) d 6.20 (d, J ¼ 1.8, 1H), 7.79 (d, J ¼ 1.8, 1H); m/z
205 (M þ H)þ.
6. Experimental section
6.1. Chemical synthesis
6.1.1. General
Reagents used for the synthesis were purchased from Sigma-
Aldrich (Isle d’Abeau Chesnes, France) and Lancaster (Bis-
chheim-Strasbourg, France). With the exception of THF and
Et2O, all solvents were obtained from commercial suppliers
and used without further purification. These two solvents were
freshly distilled from sodium benzophenone ketyl. Flash
chromatography was performed on GeduranÒ Silica gel Si 60
(40e63 mm, Merck). Thin-layer chromatography was carried
out using Silica gel 60 F254 plates (Merck). The spots
were visualized either under UV light (l ¼ 254 nm) or by
spraying with molybdate reagent (H2O/concentrated H2SO4/
(NH4)6Mo7O24$4H2O/(NH4)2-Ce(SO4)4$2H2O, 90:10:25:1, v/
v/w/w) and charring at 140 ꢁC for a few minutes. All chemical
yieldsareunoptimizedandgenerallyrepresenttheresultofasin-
gle experiment.
1H NMR spectra were recorded on a Bruker AC 200
(200 MHz) or a Bruker DPX 300 (300 MHz) spectrophotome-
ter at room temperature. Chemical shifts are given in ppm (d),
coupling constants (J ) are in Hertz (Hz) and signals are desig-
nated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet;
quint., quintuplet; m, multiplet; br s, broad singlet; etc.
The mass spectra were obtained on a Mariner API-TOF.
Melting points were determined with a Mettler FP62
apparatus and are uncorrected. Elemental analyses were per-
formed by the CNRS department of microanalysis (CNRS,
Vernaison, France) and are indicated only by the elemental
symbols within ꢂ0.4% of the theoretical values unless other-
wise noted.
6.1.3. 2-Trifluoromethyl-8-iodo-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (11b)
A solution of 10b (1.0 g, 4.87 mmol), 4-(N,N-dimethylami-
no)pyridine (2.90 g, 23.7 mmol), and phosphorus oxychloride
(8 mL, 107 mmol) was refluxed for 2 h. After the solution was
cooled to room temperature, the excess of phosphorus oxy-
chloride was removed under reduced pressure. Then, the resi-
due was redissolved in CH2Cl2 (40 mL), and N-methylaniline
(1.5 mL, 11.8 mmol) was added dropwise with stirring at 0 ꢁC
under argon. After 10 min, the reaction mixture was allowed to
warm to room temperature. After evaporation of the solvent,
the residue was chromatographed on silica (EtOAc/hexanes,
1:1) to give 2-trifluoromethyl-4-(N-methyl-N-phenylamino)-
pyrazolo[1,5-a]-1,3,5-triazine (1.27 g, 88%), as a white
1
powder: H NMR (300 MHz, CDCl3) d 3.82 (s, 3H, CH3),
6.54 (d, J ¼ 2.2, 1H, 8-H), 7.21e7.24 (m, 2H, ArH), 7.42e
7.44 (m, 3H, ArH), 7.81 (d, J ¼ 2.2, 1H, 7-H); m/z 296
(M þ H)þ.
To a solution of 2-trifluoromethyl-4-(N-methyl-N-phenyla-
mino)pyrazolo[1,5-a]-1,3,5-triazine (1.23 g, 4.18 mmol) in
dry chloroform (100 mL) was added N-iodosuccinimide
(1.33 g, 5.91 mmol), and the mixture was refluxed for 0.5 h.
After the mixture was cooled at room temperature, the solvent
was evaporated in vacuo. Then, the residue was partitioned be-
tween dichloromethane (100 mL) and water (70 mL). The
aqueous layer was extracted twice with additional dichlorome-
thane (2 ꢃ 50 mL). The combined organic phases was washed
with 10% sodium bisulfite (70 mL). The organic layer was
dried (Na2SO4) and concentrated under reduced pressure.
The crude material was purified by column chromatography
on silica (EtOAc/CH2Cl2/hexanes, 2:3:5). Recrystallization
from ethanol yielded compound 11b (1.42 g, 81%) as color-
less crystals: mp 155 ꢁC. 1H NMR (300 MHz, CDCl3)
d 3.81 (s, 3H, CH3), 7.19e7.24 (m, 2H, ArH), 7.40e7.45
(m, 3H, ArH), 7.79 (s, 1H, 7-H); m/z 420 (M þ H)þ.
6.1.2. 2-Trifluoromethylpyrazolo[1,5-a]-1,3,5-triazin-4-one
(10b)
To
14.2 mmol) in acetonitrile (15 mL) at room temperature was
added S-p-chlorophenyltrifluorothioacetimidate (3.4 g,
a stirred solution of 3-aminopyrazole (1.18 g,
14.2 mmol). After 5 min of stirring at 20 ꢁC, acetic acid
(812 mL, 14.2 mmol) was added dropwise under argon. After
8 h, the solvent was evaporated to dryness and the residue
was triturated with ether (5 mL) and hexane (30 mL), filtered
and washed successively with hexane and water to yield N-
(pyrazol-3-yl)trifluoroacetamidine (2.35 g, 93%) as a white
powder: 1H NMR (200 MHz, CDCl3) d 6.38 (d, J ¼ 2.4,
1H), 7.51 (d, J ¼ 2.4, 1H); m/z 201 (M þ Na)þ.
6.1.4. 8-Iodo-4-(N-methyl-N-phenylamino)-2-n-
propylpyrazolo[1,5-a]-1,3,5-triazine (11c)
Prepared from 4-(N-methyl-N-phenylamino)-2-n-propyl-
pyrazolo[1,5-a]-1,3,5-triazine (10c) [30] using the procedure
described for 11b. Compound 11c was obtained (76%) as
a white powder: 1H NMR (200 MHz, CDCl3) d 1.03 (t,
J ¼ 7.3, 3H, CH3), 1.85e1.89 (m, 2H, CH2), 2.80 (t, J ¼ 7.3,
2H, CH2), 3.73 (s, 3H, CH3), 7.13e7.44 (m, 5H, ArH), 7.68
(s, 1H, 7-H); m/z 394 (M þ H)þ.
Sodium ethoxide (1.0 M in EtOH, 5.45 mL) was slowly
added, at room temperature, to a stirred solution of N-(pyra-
zol-3-yl)trifluoroacetamidine (178 mg, 1.0 mmol) and diethyl
carbonate (605 mL, 5.45 mmol) in anhydrous ethanol (5 mL).
The resulting mixture was heated at reflux for 5 h under argon.

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
823
6.1.5. 8-(1-Hydroxypropyl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12a)
d 1.02 (t, J ¼ 7.5, 3H, CH3), 1.88e1.91 (m, 2H, CH2), 2.83
(t, J ¼ 7.5, 2H, CH2), 3.73 (s, 3H, CH3), 3.74 (br s, 1H,
OH), 3.84 (s, 3H, CH3), 6.12 (d, J ¼ 1.7, 1H, CH), 7.11e
7.46 (m, 10H, ArH þ 7-H).
A solution of n-BuLi (15% in hexane, 220 mL, 0.52 mmol)
was slowly added under nitrogen at ꢀ78 ꢁC, to a stirred solu-
tion of 11a [19] (157 mg, 0.43 mmol) in anhydrous THF
(25 mL). After 5 min, propionaldehyde (93 mL, 1.29 mmol)
was added and the reaction mixture was allowed to warm up
to 0 ꢁC, then quenched with acetic acid (50 mL). The mixture
was diluted with ethyl acetate, washed with water, dried
(Na2SO4), and concentrated to dryness under reduced pres-
sure. Chromatography on silica (AcOEt/hexane, 1:1) afforded
6.1.10. 2-Trifluoromethyl-8-[(hydroxy)(2-methoxyphenyl)
methyl]-4-(N-methyl-N-phenyl amino)pyrazolo[1,5-a]-
1,3,5-triazine (12f)
Prepared from 11b and 2-methoxybenzaldehyde using the
procedure described for 12a. Compound 12f was obtained
(80%) as a white powder: 1H NMR (200 MHz, CDCl3)
d 3.78 (s, 3H, CH3), 3.82 (s, 3H, CH3), 4.70 (d, J ¼ 4.2, 1H,
CH), 6.38 (d, J ¼ 4.2, 1H, OH), 6.86e6.98 (m, 3H, ArH),
7.18e7.45 (m, 6H, ArH), 7.66 (s, 1H, 7-H); m/z 430
(M þ H)þ.
1
the desired product 12a (50 mg, 39%) as a white powder. H
NMR (200 MHz, CDCl3) d 0.96 (t, J ¼ 7.5, 3H, CH3),
1.82e1.94 (m, 2H, CH2), 2.53 (s, 3H, CH3), 3.08 (d,
J ¼ 3.3, 1H, OH), 3.72 (s, 3H, CH3), 4.89e4.95 (m, 1H,
CH), 7.16e7.20 (m, 2H, ArH), 7.27e7.42 (m, 3H, ArH),
7.65 (s, 1H, 7-H); 13C NMR (CDCl3, 75 MHz)
d
10.41, 26.01, 31.11, 42.42, 67.82, 111.06, 126.49, 127.43,
129.36, 143.10, 145.11, 148.74, 149.40, 162.37; m/z 298
(M þ H)þ.
6.1.11. 8-[(2-Furyl)(hydroxy)methyl]-2-methyl-4-(N-methyl-
N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12g)
Prepared from 11a [19] and 2-furaldehyde using the proce-
dure described for 12a. Compound 12g was obtained (81%) as
a white powder: 1H NMR (300 MHz, CDCl3/DMSO-d6)
d 2.35 (s, 3H, CH3), 3.54 (s, 3H, CH3), 4.66 (d, J ¼ 5.1, 1H,
OH), 5.92 (d, J ¼ 5.1, 1H, CH), 5.99e6.12 (m, 2H, ArH),
6.97e7.28 (m, 6H, ArH), 7.59 (s, 1H, 7-H); m/z 336
(M þ H)þ.
6.1.6. 8-[(Hydroxy)(phenyl)methyl]-2-methyl-4-(N-methyl-
N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12b)
Prepared from 11a [19] and benzaldehyde using the proce-
dure described for 12a. Compound 12b was obtained (82%) as
1
a white powder: H NMR (200 MHz, CDCl3) d 2.57 (s, 3H,
CH3), 3.72 (s, 3H, CH3), 3.74 (br s, 1H, OH), 6.14 (d,
J ¼ 1.7, 1H, CH), 7.13e7.45 (m, 11H, ArH þ 7-H); 13C
NMR (CDCl3, 75 MHz) d 25.95, 42.44, 68.30, 111.28,
126.61, 126.67, 127.52, 127.81, 128.73, 129.33, 143.79,
143.98, 144.99, 148.70, 149.33, 162.87; m/z 346 (M þ H)þ.
6.1.12. 8-[(3-Furyl)(hydroxy)methyl]-2-methyl-4-(N-methyl-
N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12h)
Prepared from 11a [19] and 3-furaldehyde using the proce-
dure described for 12a. Compound 12h was obtained (82%) as
a white powder: 1H NMR (200 MHz, DMSO-d6) d 2.46 (s, 3H,
CH3), 3.68 (s, 3H, CH3), 5.54 (d, J ¼ 4.2, 1H, OH), 5.89 (d,
J ¼ 4.2, 1H, CH), 6.49 (s, 1H, ArH), 7.26e7.56 (m, 7H,
ArH), 7.82 (s, 1H, 7-H); m/z 336 (M þ H)þ.
6.1.7. 8-[(Hydroxy)(phenyl)methyl]-4-(N-methyl-N-
phenylamino)-2-n-propylpyrazolo[1,5-a]-1,3,5-
triazine (12c)
Prepared from 11c and benzaldehyde using the procedure
described for 12a. Compound 12b was obtained (83%) as
1
a white powder: H NMR (200 MHz, DMSO-d6) d 1.03 (t,
6.1.13. 8-[(Hydroxy)(2-thienyl)methyl]-2-methyl-4-(N-
methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12i)
Prepared from 11a [19] and thiophene-2-carboxaldehyde
using the procedure described for 12a. Compound 12i was ob-
J ¼ 7.5, 3H, CH3), 1.88e1.90 (m, 2H, CH2), 2.81 (t, J ¼ 7.5,
2H, CH2), 3.73 (s, 3H, CH3), 3.74 (br s, 1H, OH), 6.12 (d,
J ¼ 1.7, 1H, CH), 7.13e7.45 (m, 11H, ArH þ 7-H).
1
tained (85%) as a white powder: H NMR (300 MHz, CDCl3)
6.1.8. 2-Trifluoromethyl-8-[(hydroxy)(phenyl)methyl]-4-(N-
methyl-N-phenylamino)pyrazolo [1,5-a]-1,3,5-triazine (12d)
Prepared from 11b and benzaldehyde using the procedure
described for 12a. Compound 12d was obtained (86%) as
d 2.53 (s, 3H, CH3), 3.74 (s, 3H, CH3), 4.15 (d, J ¼ 4.7, 1H,
OH), 6.38 (d, 1H, J ¼ 4.7, CH), 6.92e6.97 (m, 2H, ArH),
7.16e7.40 (m, 6H, ArH), 7.59 (s, 1H, 7-H); m/z 352
(M þ H)þ.
1
a white powder: H NMR (200 MHz, DMSO-d6) d 3.76 (s,
3H, CH3), 5.91 (d, J ¼ 4.4, 1H, CH), 5.99 (d, J ¼ 4.4, 1H,
OH), 7.21e7.45 (m, 10H, ArH), 7.94 (s, 1H, 7-H); m/z 400
(M þ H)þ.
6.1.14. 8-[(Hydroxy)(2-naphthyl)methyl]-2-methyl-4-(N-
methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (12j)
Prepared from 11a [19] and 2-naphthaldehyde using the
procedure described for 12a. Compound 12j was obtained
6.1.9. 8-[(Hydroxy)(2-methoxyphenyl)methyl]-4-(N-methyl-
N-phenylamino)-2-n-propyl pyrazolo[1,5-a]-1,3,5-triazine
(12e)
1
(80%) as a white powder: H NMR (200 MHz, DMSO-d6)
d 2.49 (s, 3H, CH3), 3.68 (s, 3H, CH3), 5.87 (d, J ¼ 4.6, 1H,
CH), 6.13 (d, J ¼ 4.6, 1H, OH), 7.24e7.57 (m, 8H, ArH),
7.75 (s, 1H, 7-H), 7.81e7.91 (m, 4H, ArH); m/z 396
(M þ H)þ.
Prepared from 11c and 2-methoxybenzaldehyde using the
procedure described for 12a. Compound 12e was obtained
1
(80%) as a white powder: H NMR (200 MHz, DMSO-d6)

824
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
6.1.15. 8-Benzyl-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (13a)
(m, 1H, ArH), 7.26e7.50 (m, 6H, ArH), 7.75 (s, 1H, 7-H);
m/z 320 (M þ H)þ.
To a solution of NaI (260 mg, 1.74 mmol) in acetonitrile
(5 mL) was added chlorotrimethylsilane (220 mL, 1.74 mmol)
under argon atmosphere. After 10 min of stirring at 20 ꢁC,
a solution of 12b (100 mg, 0.29 mmol) in acetonitrile
(5 mL) was added. The mixture was stirred at room temper-
ature under argon for 15 min, then diluted with ether (60 mL)
and washed with water (30 mL). The organic layer was dried
(Na2SO4) and concentrated under reduced pressure. Chroma-
tography on silica (AcOEt/hexane, 1:1) afforded the desired
product 13a (79 mg, 83%) as colorless oil: 1H NMR
(300 MHz, CDCl3) d 2.60 (s, 3H, CH3), 3.74 (s, 3H, CH3),
4.02 (s, 2H, CH2), 7.16e7.43 (m, 10H, ArH), 7.50 (s, 1H,
7-H); m/z 330 (M þ H)þ.
6.1.21. 8-[(3-Furfuryl)methyl]-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (13g)
Prepared from 12h using the procedure described for 13a.
1
Compound 13g was obtained (93%) as a white powder: H
NMR (200 MHz, CDCl3) d 2.61 (s, 3H, CH3), 3.76 (s, 3H,
CH3), 3.84 (s, 2H, CH2), 6.34 (s, 1H, ArH), 7.18e7.43 (m,
7H, ArH), 7.58 (s, 1H, 7-H); m/z 320 (M þ H)þ.
6.1.22. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(2-
thienylmethyl)pyrazolo[1,5-a]-1,3,5-triazine (13h)
Prepared from 12i using the procedure described for 13a.
1
Compound 13h was obtained (86%) as a white powder: H
NMR (200 MHz, CDCl3) d 2.59 (s, 3H, CH3), 3.72 (s, 3H,
CH3), 4.01 (s, 2H, CH2), 7.14e7.42 (m, 8H, ArH), 7.47 (s,
1H, 7-H); m/z 336 (M þ H)þ.
6.1.16. 8-Benzyl-4-(N-methyl-N-phenylamino)-2-n-
propylpyrazolo[1,5-a]-1,3,5-triazine (13b)
Prepared from 12c using the procedures described for 13a.
Compound 13b was obtained (54%) as colorless oil: 1H NMR
(300 MHz, CDCl3) d 1.05 (t, J ¼ 7.5, 3H, CH3), 1.88e1.90 (m,
2H, CH2), 2.79 (t, J ¼ 7.5, 2H, CH2), 3.73 (s, 3H, CH3), 4.02
(s, 2H, CH2), 7.06e7.43 (m, 10H, ArH), 7.50 (s, 1H, 7-H); m/z
358 (M þ H)þ.
6.1.23. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(2-
naphthylmethyl)pyrazolo[1,5-a]-1,3,5-triazine (13i)
Prepared from 12j using the procedure described for 13a.
1
Compound 13i was obtained (81%) as a white powder: H
NMR (200 MHz, CDCl3) d 2.64 (s, 3H, CH3), 3.77 (s, 3H,
CH3), 4.22 (s, 2H, CH2), 7.19e7.90 (m, 13H, ArH þ 7-H);
m/z 380 (M þ H)þ.
6.1.17. 8-(Benzyl)-2-trifluoromethyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (13c)
Prepared from 12d using the procedure described for 13a.
6.1.24. 8-Benzyl-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-
1,3,5-triazine (14a)
1
Compound 13c was obtained (84%) as a white powder: H
NMR (300 MHz, CDCl3) d 3.80 (s, 3H, CH3), 4.06 (s, 2H,
CH2), 7.22e7.40 (m, 10H, ArH), 7.59 (s, 1H, 7-H); m/z 384
(M þ H)þ.
A solution of 13a (1.01 mmol) and methylamine (2 M,
2.0 mL, 4.0 mmol) in ethanol (10 mL) was stirred at 100 ꢁC in
a sealed tube for 12 h. After the mixture was cooled the solvent
was evaporated under reduced pressure. The crude reaction
product was purified by column chromatography on silica gel
(EtOAc/CH2Cl2/EtOH, 4:5:1). Recrystallization from ethanol
and diethyl ether yielded compound 14a (53%) as colorless
prisms: 1H NMR (300 MHz, CDCl3) d 2.98 (s, 3H, CH3), 3.41
(d, J ¼ 5.1, 3H, CH3), 4.42 (s, 2H, CH2), 7.18e7.39 (m, 5H,
ArH), 7.79 (s, 1H, 7-H), 7.90 (q, J ¼ 5.1, 1H, NH); m/z 254
(M þ H)þ. Anal. Found: C14H15N5 (C, H, N).
6.1.18. 8-(2-Methoxybenzyl)-4-(N-methyl-N-phenylamino)-
2-n-propylpyrazolo[1,5-a]-1,3,5-triazine (13d)
Prepared from 12e using the procedures described for 13a.
Compound 13d was obtained (40%) as colorless oil: 1H NMR
(200 MHz, CDCl3) d 1.07 (t, 3H, J ¼ 7.4, CH3), 1.89e1.93 (m,
2H, CH2), 2.81 (t, J ¼ 7.4, 2H, CH2), 3.74 (s, 3H, CH3), 3.85
(s, 3H, CH3), 4.03 (s, 2H, CH2), 6.80e7.45 (m, 9H, ArH), 7.58
(s, 1H, 7-H); m/z 388 (M þ H)þ.
6.1.25. 8-Benzyl-4-(N-methylamino)-2-n-propylpyrazolo
[1,5-a]-1,3,5-triazine hydrochloride (14b)
Prepared from 13b using the procedure described for 14a.
6.1.19. 2-Trifluoromethyl-8-(2-methoxybenzyl)-4-(N-methyl-
N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (13e)
Prepared from 12f using the procedure described for 13a.
1
Compound 14b was obtained (68%) as colorless prisms: H
1
Compound 13e was obtained (88%) as a white powder: H
NMR (300 MHz, CDCl3) d 1.11 (t, J ¼ 7.3, 3H, CH3), 1.95e
1.98 (m, 2H, CH2), 3.26 (t, J ¼ 7.6, 2H, CH2), 3.42 (d, J ¼ 5.0,
3H, CH3), 4.42 (s, 2H, CH2), 7.19e7.36 (m, 5H, ArH), 7.74
(s, 1H, 7-H), 8.08 (q, J ¼ 5.0, 1H, NH); 13C NMR (CDCl3,
75 MHz) d 14.32, 21.35, 28.31, 28.86, 37.32, 107.32, 126.93,
129.22, 129.33, 138.46, 141.27, 147.45, 149.67, 165.44; m/z
282 (M þ H)þ. Anal. Found: C16H19N5.HCl (C, H, N).
NMR (300 MHz, CDCl3) d 3.78 (s, 3H, CH3), 3.82 (s, 3H,
CH3), 4.04 (s, 2H, CH2), 6.83e6.88 (m, 2H, ArH), 7.16e
7.22 (m, 4H, ArH), 7.36e7.42 (m, 3H, ArH), 7.65 (s, 1H, 7-
H); m/z 414 (M þ H)þ.
6.1.20. 8-(Furfuryl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (13f)
Prepared from 12g using the procedure described for 13a.
1
Compound 13f was obtained (53%) as a white powder: H
NMR (200 MHz, CDCl3) d 2.46 (s, 3H, CH3), 3.67 (s, 3H,
CH3), 3.96 (s, 2H, CH2), 6.02e6.04 (m, 1H, ArH), 6.31e6.34
6.1.26. 8-Benzyl-2-trifluoromethyl-4-(N-methylamino)
pyrazolo[1,5-a]-1,3,5-triazine (14c)
Prepared from 13c using the procedure described for 14a.
1
Compound 12c was obtained (79%) as colorless prisms: H

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
825
NMR (200 MHz, CDCl3) d3.28(d, J ¼ 5.1, 3H, CH3),4.11(s,2H,
CH2), 6.60 (br s, 1H, NH), 7.13e7.42(m, 5H, ArH), 7.83(s, 1H, 7-
H); m/z 308 (M þ H)þ. Anal. Found: C14H12F3N5 (C, H, N).
J ¼ 5.1, 3H, CH3), 4.25 (s, 2H, CH2), 6.47 (br s, 1H, NH),
7.43e7.82 (m, 7H, ArH), 7.86 (s, 1 H, 7-H); m/z 304
(M þ H)þ. Anal. Found: C18H17N5$0.3H2O (C, H, N).
6.1.27. 8-(2-Methoxybenzyl)-4-(N-methylamino)-2-n-
propylpyrazolo[1,5-a]-1,3,5-triazine (14d)
Prepared from 13d using the procedure described for 14a.
6.1.33. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(prop-1-
ynyl)pyrazolo[1,5-a]-1,3,5-triazine (15a)
Methylacetylene (2 mL) was condensed in a sealed tube at
ꢀ78 ꢁC. Then, 11a [19] (157 mg, 0.43 mmol), CuI (12 mg,
0.063 mg), PdCl2 (7 mg, 0.039 mmol), triphenylphosphine
(23 mg, 0.088 mmol), triethylamine (2 mL, 14 mmol) and ace-
tonitrile (3 mL) were added. The reaction mixture was stirred
at room temperature for 24 h. After the solution was cooled to
ꢀ78 ꢁC, the sealed tube was opened and slowly heated at room
temperature. The solvent was evaporated under reduced pres-
sure and the crude reaction product was purified by column
chromatography on silica gel (EtOAc/hexane, 1:1) as a yellow
solid (73%): 1H NMR (200 MHz, CDCl3) d 2.07 (s, 3H, CH3),
2.64 (s, 3H, CH3), 3.78 (s, 3H, CH3), 7.14e7.20 (m, 2H, ArH),
7.36e7.48 (m, 3H, ArH), 7.74 (s, 1H, 7-H).
1
Compound 14d was obtained (76%) as colorless prisms: H
NMR (300 MHz, CDCl3) d 1.04 (t, J ¼ 7.4, 3H, CH3),
1.86e1.89 (m, 2H, CH2), 2.77 (t, J ¼ 7.7, 2H, CH2), 3.21 (d,
J ¼ 5.3, 3H, CH3), 3.87 (s, 3H, CH3), 4.04 (s, 2H, CH2),
6.41 (br s, 1H, NH), 6.85e6.90 (m, 2H, ArH), 7.18e7.22
(m, 2H, ArH), 7.58 (s, 1H, 7-H); m/z 312 (M þ H)þ. Anal.
Found: C17H21N5O$0.1H2O (C, H, N).
6.1.28. 2-Trifluoromethyl-8-(2-methoxybenzyl)-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14e)
Prepared from 13e using the procedure described for 14a.
1
Compound 14e was obtained (76%) as colorless prisms: H
NMR (300 MHz, CDCl3) d 3.30 (d, J ¼ 4.9, 3H, CH3), 3.87
(s, 3H, CH3), 4.09 (s, 2H, CH2), 6.72 (br s, 1H, NH), 6.87e
6.92 (m, 2H, ArH), 7.19e7.28 (m, 2H, ArH), 7.89 (s, 1H, 7-
H); m/z 338 (M þ H)þ. Anal. Found: C15H14F3N5O (C, H, N).
6.1.34. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(pent-1-
ynyl)pyrazolo[1,5-a]-1,3,5-triazine (15b)
A solution of 11a [19] (200 mg, 0.55 mmol), 1-pentyne
(980 mL, 10 mmol), CuI (12 mg, 0.063 mmol), PdCl2 (7 mg,
0.039 mmol), triphenylphosphine (23 mg, 0.088 mmol), trie-
thylamine (2 mL, 14 mmol) and acetonitrile (3 mL) was
stirred under nitrogen at 25 ꢁC for 24 h. The solvent was evap-
orated under reduced pressure and the crude reaction product
was purified by column chromatography on silica gel (EtOAc/
hexane, 1:1). Recrystallization from dichloromethane and hex-
anes yielded compound 15b (119 mg, 71%) as colorless solid:
1H NMR (200 MHz, CDCl3) d 1.07 (t, J ¼ 7.3, 3H, CH3),
1.65e1.69 (m, 2H, CH2), 2.47 (t, J ¼ 7.3, 2H, CH2), 2.63 (s,
3H, CH3), 3.76 (s, 3H, CH3), 7.15e7.20 (m, 2H, ArH),
7.35e7.45 (m, 3H, ArH), 7.76 (s, 1H, 7-H); 13C NMR
(CDCl3, 75 MHz) d 14.07, 22.32, 22.68, 26.21, 42.54, 70.03,
92.42, 94.24, 126.35, 127.49, 129.40, 144.96, 147.19,
149.54, 152.02, 164.12; m/z 306 (M þ H)þ.
6.1.29. 8-(Furfuryl)-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14f)
Prepared from 13f using the procedure described for 14a.
1
Compound 14f was obtained (62%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.57 (s, 3H, CH3), 3.21 (d,
J ¼ 4.9, 3H, CH3), 4.06 (s, 2H, CH2), 6.01e6.04 (m, 1H,
ArH), 6.27e6.30 (m, 1H, ArH), 6.45 (br s, 1H, NH), 7.25e
7.34 (m, 1H, ArH), 7.84 (s, 1H, 7-H); m/z 244 (M þ H)þ.
Anal. Found: C12H13N5O (C, H, N).
6.1.30. 8-[(3-Furyl)methyl]-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14g)
Prepared from 13g using the procedure described for 14a.
1
Compound 14g was obtained (64%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.57 (s, 3H, CH3), 3.22 (d, J ¼ 4.9,
3H, CH3), 3.84 (s, 2H, CH2), 6.30e6.34 (m, 1H, ArH), 6.48
(br s, 1H, NH), 7.25e7.37 (m, 2H, ArH), 7.56 (s, 1H, 7-H); m/
z 244 (M þ H)þ. Anal. Found: C12H13N5O (C, H, N).
6.1.35. 2-Methyl-4-(N-methylamino)-8-(prop-1-
ynyl)pyrazolo[1,5-a]-1,3,5-triazine hydrochloride (16a)
Prepared from 15a using the procedure described for 14a.
Compound 16a was obtained (62%) as a white solid: 1H
NMR (200 MHz, CDCl3) d 2.05 (s, 3H, CH3), 2.75 (s, 3H,
CH3), 3.20 (d, J ¼ 4.2, 3H, CH3), 7.91 (s, 1H, 7-H), 9.50 (br
s, 1H, NH); m/z 202 (M þ H)þ. Anal. Found:
C10H11N5$HCl$0.2H2O (C, H, N).
6.1.31. 2-Methyl-4-(N-methylamino)-8-[(2-
thienyl)methyl]pyrazolo[1,5-a]-1,3,5-triazine (14h)
Prepared from 13h using the procedure described for 14a.
1
Compound 14h was obtained (87%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.57 (s, 3H, CH3), 3.22 (d, J ¼ 4.9,
3H, CH3), 4.25 (s, 2H, CH2), 6.45 (br s, 1H, NH), 7.86e7.94
(m, 2H, ArH), 7.11e7.25 (m, 1H, ArH), 7.81 (s, 1H, 7-H); m/z
260 (M þ H)þ. Anal. Found: C12H13N5S (C, H, N).
6.1.36. 2-Methyl-4-(N-methylamino)-8-(pent-1-
ynyl)pyrazolo[1,5-a]-1,3,5-triazine (16b)
Prepared from 15b using the procedure described for 14a.
1
Compound 16b was obtained (77%) as colorless prisms: H
6.1.32. 2-Methyl-4-(N-methylamino)-8-[(2-
NMR (300 MHz, CDCl3) d 1.06 (t, J ¼ 7.3, 3H, CH3),
1.62e1.72 (m, 2H, CH2), 2.46 (t, J ¼ 7.2, 2H, CH2), 2.60 (s,
3H, CH3), 3.23 (d, J ¼ 4.9, 3H, CH3), 6.49 (br s, 1H, NH),
7.9 (s, 1H, 7-H); 13C NMR (CDCl3, 75 MHz) d 14.07,
22.28, 22.65, 26.47, 27.67, 69.84, 85.81, 93.56, 94.18,
naphthyl)methyl]pyrazolo[1,5-a]-1,3,5-triazine (14i)
Prepared from 13i using the procedure described for 14a.
1
Compound 14i was obtained (86%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.63 (s, 3H, CH3), 3.26 (d,

826
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
147.67, 149.53, 165.27; m/z 230 (M þ H)þ. Anal. Found:
6.1.42. 8-(2-Furyl)-2-methyl-4-(N-methyl-N-
C12H15N5 (C, H, N).
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17f)
Prepared from 11a [19] and furan-2-boronic acid using the
procedure described for 17a. Compound 17f was obtained
(51%) as a white powder: 1H NMR (200 MHz, CDCl3)
d 2.63 (s, 3H, CH3), 3.74 (s, 3H, CH3), 6.47e6.50 (m, 1H,
ArH), 6.77e6.80 (m, 1H, ArH), 7.15e7.20 (m, 2H, ArH),
7.33e7.44 (m, 4H, ArH), 7.99 (s, 1H, 7-H); m/z 306
(M þ H)þ.
6.1.37. 2-Methyl-4-(N-methyl-N-phenylamino)-8-
phenylpyrazolo[1,5-a]-1,3,5-triazine (17a)
A solution of 11a [19] (157 mg, 0.43 mmol), tetrakis(tri-
phenylphosphine)palladium(0) (50 mg, 0.043 mmol), sodium
carbonate (2 M in H2O, 430 mL, 0.86 mmol), benzeneboronic
acid (58 mg, 0.47 mmol), and ethanol (500 mL) in toluene
(10 mL) was stirred under nitrogen at 90 ꢁC for 16 h. After
the solution was cooled to room temperature, the solvent
was evaporated under reduced pressure. The crude reaction
product was purified by column chromatography on silica
gel (EtOAc/hexanes, 1:1) to give 17a (94 mg, 69%) as a color-
6.1.43. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(2-
thienyl)pyrazolo[1,5-a]-1,3,5-triazine (17g)
Prepared from 11a [19] and thiphene-2-boronic acid using
the procedure described for 17a. Compound 17g was obtained
(54%) as a white powder: 1H NMR (200 MHz, CDCl3) d 2.67
(s, 3H, CH3), 3.78 (s, 3H, CH3), 7.08e7.48 (m, 8H, ArH), 7.93
(s, 1H, 7-H); m/z 322 (M þ H)þ.
1
less solid: mp 195 ꢁC. H NMR (200 MHz, CDCl3) d 2.67 (s,
3H, CH3), 3.78 (s, 3H, CH3), 7.20e7.50 (m, 8H, ArH), 7.95e
8.00 (m, 2H, ArH), 8.05 (s, 1H, 7-H); 13C NMR (CDCl3,
75 MHz) d 26.34, 31.33, 108.39, 126.38, 126.44, 126.60,
127.33, 129.09, 129.39, 132.31, 143.34, 145.29, 147.99,
149.71, 163.35; m/z 316 (M þ H)þ.
6.1.44. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(1-
naphthyl)pyrazolo[1,5-a]-1,3,5-triazine (17h)
Prepared from 11a [19] and 1-naphthaleneboronic acid us-
ing the procedure described for 17a. Compound 17h was ob-
6.1.38. 8-(2-Chlorophenyl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17b)
1
tained (57%) as a white powder: H NMR (300 MHz, CDCl3)
Prepared from 11a [19] and 2-chlorophenylboronic acid using
the procedure described for 17a. Compound 17b was obtained
d 2.57 (s, 3H, CH3), 3.80 (s, 3H, CH3), 7.26e7.98 (m, 12H,
ArH), 7.95 (s, 1H, 7-H); m/z 366 (M þ H)þ.
1
(79%) as a white powder: H NMR (200 MHz, CDCl3) d 2.62
(s, 3H, CH3), 3.80 (s, 3H, CH3), 7.19e7.51 (m, 8H, ArH),
7.86e7.91 (m, 1H, ArH), 8.19 (s, 1H, 7-H); m/z 350 (M þ H)þ.
6.1.45. 8-(2-Benzo[b]furyl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17i)
Prepared from 11a [19] and benzo[b]furan-2-boronic acid
using the procedure described for 17a. Compound 17i was ob-
6.1.39. 8-(3-Acetylphenyl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17c)
Prepared from 11a [19] and 3-acetylphenylboronic acid us-
ing the procedure described for 17a. Compound 17c was ob-
1
tained (58%) as a white powder: H NMR (200 MHz, CDCl3)
1
tained (26%) as a white powder: H NMR (200 MHz, CDCl3)
d 2.69 (s, 3H, CH3), 3.79 (s, 3H, CH3), 7.22e7.63 (m, 10H,
ArH), 8.17 (s, 1H, 7-H); m/z 356 (M þ H)þ.
d 2.67 (s, 3H, CH3), 2.68 (s, 3H, CH3), 3.78 (s, 3H, CH3),
7.24e7.86 (m, 7H, ArH), 8.08 (s, 1H, 7-H), 8.18e8.24 (m,
1H, ArH), 8.56e8.58 (m, 1H, ArH); m/z 358 (M þ H)þ.
6.1.46. 8-(2-Benzo[b]thienyl)-2-methyl-4-(N-methyl-N-
phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17j)
Prepared from 11a [19] and benzo[b]thiophene-2-boronic
acid using the procedure described for 17a. Compound 17j
6.1.40. 2-Methyl-4-(N-methyl-N-phenylamino)-8-(3-
nitrophenyl)pyrazolo[1,5-a]-1,3,5-triazine (17d)
1
was obtained (59%) as a white powder: H NMR (200 MHz,
CDCl3) d 2.69 (s, 3H, CH3), 3.76 (s, 3H, CH3), 7.18e7.43
(m, 7H, ArH), 7.73e7.80 (m, 3H, ArH), 8.06 (s, 1H, 7-H);
m/z 372 (M þ H)þ.
Prepared from 11a [19] and 3-nitrophenylboronic acid using
the procedure described for 17a. Compound 17d was obtained
(77%) as a white powder: 1H NMR (200 MHz, CDCl3) d 2.66
(s, 3H, CH3), 3.77 (s, 3H, CH3), 7.18e7.59 (m, 6H, ArH),
8.02e8.04 (m, 1H, ArH), 8.06 (s, 1H, 7-H), 8.30e8.35 (m,
1H, ArH), 8.80e8.82 (m, 1H, ArH); m/z 361 (M þ H)þ.
6.1.47. 2-Methyl-4-(N-methylamino)-8-phenylpyrazolo[1,5-a]-
1,3,5-triazine (18a)
Prepared from 17a using the procedure described for 14a.
1
6.1.41. 8-[3-(Trifluoromethylphenyl)]-2-methyl-4-(N-
methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (17e)
Prepared from 11a [19] and 3-trifluoromethylphenylbor-
onic acid using the procedure described for 17a. Compound
17e was obtained (87%) as a white powder: 1H NMR
(200 MHz, CDCl3) d 2.64 (s, 3H, CH3), 3.76 (s, 3H, CH3),
7.18e7.52 (m, 7H, ArH), 8.03 (s, 1H, 7-H), 8.14e8.21 (m,
2H, ArH); m/z 384 (M þ H)þ.
Compound 18a was obtained (83%) as colorless prisms: H
NMR (300 MHz, CDCl3) d 2.63 (s, 3H, CH3), 3.25
(d, J ¼ 5.0, 3H, CH3), 6.51 (br s, 1H, NH), 7.23e7.28 (m,
1H, ArH), 7.41e7.46 (m, 2H, ArH), 7.98e8.01 (m, 2H,
ArH), 8.24 (s, 1H, 7-H); 13C NMR (CDCl3, 75 MHz)
d 26.64, 27.64, 109.83, 126.50, 129.16, 132.27, 143.36,
145.72, 149.59, 164.47; m/z 240 (M þ H)þ. Anal. Found:
C13H13N5 (C, H, N).

P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
827
6.1.48. 8-(2-Chlorophenyl)-2-methyl-4-(N-
6.1.54. 2-Methyl-4-(N-methylamino)-8-(1-naphthyl)
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (18b)
Prepared from 17b using the procedure described for 14a.
pyrazolo[1,5-a]-1,3,5-triazine hydrochloride (18h)
Prepared from 17h using the procedure described for 14a,
then converted to the HCl salt. Compound 18h was obtained
1
Compound 18b was obtained (64%) as colorless prisms: H
1
NMR (200 MHz, DMSO-d6) d 2.44 (s, 3H, CH3), 3.06 (d,
J ¼ 4.6, 3H, CH3), 7.31e7.86 (m, 4H, ArH), 8.46 (s, 1H, 7-
H), 8.82 (q, J ¼ 4.6, 1H, NH); m/z 274 (M þ H)þ. Anal.
Found: C13H12ClN5$0.5H2O (C, H, N).
(40%) as colorless prisms: H NMR (200 MHz, DMSO-d6)
d 2.43 (s, 3H, CH3), 3.15 (d, J ¼ 4.8, 3H, CH3), 7.48e7.65
(m, 4H, ArH), 7.86e8.30 (m, 3H, ArH), 8.46 (s, 1H, 7-H),
9.48 (q, J ¼ 4.8, 1H, CH3); m/z 290 (M þ H)þ. Anal. Found:
C17H15N5$HCl$0.7H2O (C, H, N).
6.1.49. 8-(3-Acetylphenyl)-2-methyl-4-(N-
6.1.55. 8-(2-Benzo[b]furyl)-2-methyl-4-(N-methylamino)
pyrazolo[1,5-a]-1,3,5-triazine (18i)
Prepared from 17i using the procedure described for 14a.
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (18c)
Prepared from 17c using the procedure described for 14a.
1
Compound 18c was obtained (43%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.55 (s, 3H, CH3), 2.68 (s, 3H,
CH3), 3.26 (d, J ¼ 5.0, 3H, CH3), 6.56 (br s, 1H, NH),
7.51e7.56 (m, 1H, ArH), 7.81e7.84 (m, 1H, ArH), 8.26e
8.30 (m, 2H, ArH), 8.56 (s, 1H, 7-H); m/z 282 (M þ H)þ.
Anal. Found: C15H15N5O (C, H, N).
1
Compound 18i was obtained (95%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.69 (s, 3H, CH3), 3.30 (d,
J ¼ 4.9, 3H, CH3), 6.57 (q, J ¼ 4.9, 1H, NH), 7.24e7.33 (m,
3H, ArH), 7.52e7.63 (m, 2H, ArH), 8.40 (s, 1H, 7-H); m/z
280 (M þ H)þ. Anal. Found: C15H13N5O (C, H, N).
6.1.56. 8-(2-Benzo[b]thienyl)-2-methyl-4-(N-methylamino)
pyrazolo[1,5-a]-1,3,5-triazine (18j)
Prepared from 17j using the procedure described for 14a.
6.1.50. 2-Methyl-4-(N-methylamino)-8-(3-nitrophenyl)
pyrazolo[1,5-a]-1,3,5-triazine hydrochloride (18d)
Prepared from 17d using the procedure described for 14a.
1
Compound 18j was obtained (58%) as colorless prisms: H
1
Compound 18d was obtained (32%) as colorless prisms: H
NMR (200 MHz, CDCl3) d 2.70 (s, 3H, CH3), 3.29 (d,
J ¼ 5.1, 3H, CH3), 6.55 (q, J ¼ 5.1, 1H, NH), 7.26e7.44 (m,
2H, ArH), 7.78e7.89 (m, 3H, ArH), 8.22 (s, 1H, 7-H); m/z
296 (M þ H)þ. Anal. Found: C15H13N5S (C, H, N).
NMR (200 MHz, DMSO-d6) d 2.52 (s, 3H, CH3), 3.07 (d,
J ¼ 2.3, 3H, CH3), 7.66e7.71 (m, 1H, ArH), 8.01e8.04 (m,
1H, ArH), 8.46e8.49 (m, 1H, ArH), 8.63 (br s, 1H, NH),
8.73 (s, 1H, 7-H), 8.96 (s, 1H, ArH); m/z 285 (M þ H)þ.
Anal. Found: C13H12N6O2$1H2O (C, H, N).
6.1.57. 8-(1-Hydroxypropyl)-2-methyl-4-(N-methylamino)
pyrazolo[1,5-a]-1,3,5-triazine (19)
Prepared from 12a using the procedure described for 14a.
6.1.51. 8-(3-Trifluoromethylphenyl)-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (18e)
Prepared from 17e using the procedure described for 14a.
1
Compound 19 was obtained (81%) as colorless crystals: H
NMR (300 MHz, CDCl3) d 1.02 (t, J ¼ 7.5, 3H, CH3),
1.90e2.04 (m, 2H, CH2), 2.47 (s, 3H, CH3), 2.94 (d,
J ¼ 4.4, 1H, OH), 3.25 (q, J ¼ 5.1, 3H, CH3), 4.94e5.03 (m,
1H, CH), 6.52 (br s, 1H, NH), 7.91 (s, 1H, 7-H); 13C NMR
(CDCl3, 75 MHz) d 10.50, 26.30, 27.60, 31.16, 67.57,
112.58, 143.37, 146.37, 149.38, 163.61; m/z 222 (M þ H)þ.
Anal. Found: C10H15N5O (C, H, N).
1
Compound 18e was obtained (29%) as colorless prisms: H
NMR (200 MHz, DMSO-d6) d 2.56 (s, 3H, CH3), 3.12 (d,
J ¼ 4.2, 3H, CH3), 7.32e7.45 (m, 2H, ArH), 7.97 (s, 1H, 7-
H), 8.04e8.14 (m, 2H, ArH), 8.29 (br s, 1H, NH); m/z 308
(M þ H)þ. Anal. Found: C14H12F3N5 (C, H, N).
6.1.52. 8-(2-Furyl)-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (18f)
Prepared from 17f using the procedure described for 14a.
6.2. Molecular modeling
1
Compound 18f was obtained (21%) as colorless prisms: H
Molecules were sketched in Sybyl 6.8 sketcher [20], and
Concord 4.05 [24] was used to generate 3D structures. They
were minimized in the Tripos force field [21] with 20 Simplex
[25] iterations as initial optimization step. Powell method [26]
NMR (200 MHz, CDCl3) d 2.62 (s, 3H, CH3), 3.25 (d,
J ¼ 5.0, 3H, CH3), 6.49e6.51 (m, 2H, ArH þ NH), 6.79 (d,
J ¼ 3.1, 1H, ArH), 7.45 (d, J ¼ 1.9, 1H, ArH), 8.20 (s, 1H,
7-H); m/z 230 (M þ H)þ. Anal. Found: C11H11N5O (C, H, N).
was appliedwithaterminationgradientof 0.05 kcal molꢀ1
.
ꢀ1
˚
A
The maximum iterations were set to 1000. No charge was
used. The minimized structures were submitted to AM1
[22] optimization in singlet state with zero net charge and
with a time limit of 3600 s as found in Mopac [23] imple-
mented in Sybyl. Normal convergence and full optimization
were selected. The keywords were DENSITY LOCAL
VECT MULLIK AM1 PI BONDS GRAPH T¼3600. Molcad
[27] surfaces were calculated using the Fast Connolly
6.1.53. 2-Methyl-4-(N-methylamino)-8-(2-
thienyl)pyrazolo[1,5-a]-1,3,5-triazine hydrochloride (18g)
Prepared from 17g using the procedure described for 14a,
then converted to the HCl salt. Compound 18g was obtained
1
(42%) as colorless prisms: H NMR (200 MHz, DMSO-d6)
d 2.50 (s, 3H, CH3), 3.05 (d, J ¼ 4.6, 3H, CH3), 7.09e7.14
(m, 1H, ArH), 7.43e7.55 (m, 2H, ArH), 8.51 (s, 1H, 7-H),
8.91 (q, J ¼ 4.6, 1H, NH); m/z 246 (M þ H)þ. Anal. Found:
C11H11N5S$0.5H2O (C, H, N).
˚
method with a probe radius of 1.4 A as found in Sybyl
6.8, and the electrostatic potential [28] was derived from

828
P. Raboisson et al. / European Journal of Medicinal Chemistry 43 (2008) 816e829
the AM1 charges calculated previously with Mopac and map-
ped onto the Connoly surfaces. Surface area was estimated
by the Molcad Surface Property Area utilities. Isopotential
curves are drawn in the contour surface tools available in
Sybyl.
supernatants were removed and stored at ꢀ80 ꢁC until ELISA.
Cell viability was assessed by the Trypan blue exclusion test.
6.3.4. Immunoassays for TNFa
Culture supernatants were assayed with two-site ELISAs
specific for human interleukin: TNFa antibodies (Antibody So-
lutions, Half Moon Bay, CA, USA). Quantitative evaluation of
TNFa secreted by PBMC’s was achieved by ELISA using con-
ditions as previously described [35]. Polyvinyl chloride plates
(Costar, #2596) were coated with 50 mL per well of antibodies
(15 mg mLꢀ1) and incubated overnight at 4 ꢁC. After the usual
wash and non specific saturation steps, 25 mL of standard or
sample was added to 25 mL of biotinylated monoclonal anti-
body (2 mg mLꢀ1) for 2 h at room temperature. Following
washing steps, 50 mL of peroxidase streptavidin dilution
(1:3000 in PBS) were added (1 h at room temperature). A
colorimetric reaction (O.D. at 450 nm) using O-phenyl-
enediamine as peroxidase substrate was performed ensuing
four washing steps. Concentrations (pg mLꢀ1) of unknown
samples were computed by interpolation with a standard curve
run on each plate using four parameters logistics analysis. Stan-
dard human recombinant protein, hr-TNFa, was purchased
from R&D Systems Europe (Abingdon, UK).
6.3. Pharmacology
6.3.1. PDE inhibition
PDE1, -3, -4 and -5 were isolated from the media layer of bo-
vine aorta according to a modification of the previously re-
ported method [31]. PDE2 was isolated from cultured bovine
aortic endothelial cells [32]. PDE activities were measured by
the two-step assay previously described [33] at a [3H]-cAMP
or [3H]-cGMP concentration of 1 mM as substrate in a buffer so-
lution of 50 mM of TriseHCl, at pH 7.5, containing 2 mM of
magnesium acetate, 1 mg mLꢀ1 of BSA. PDE1 was assayed
at 1 mM cGMP in calmodulin activated state (18 nM calmodu-
lin with 10 mM CaCl2). PDE2 was evaluated at 1 mM
cAMP þ 1 mM EGTA in activated state (in presence of 5 mM
cGMP). PDE3 and PDE4 were assayed at 1 mM cAMP þ 1 mM
EGTA. To prevent the influence of reciprocal cross-contamina-
tion between PDE3 and PDE4, the studies were always carried
out in the presence of 50 mM rolipram for PDE3 and in the pres-
ence of 50 mM cGMP for PDE4. PDE5 activity was measured at
1 mM cGMP in the presence of 1 mM of EGTA. PDE inhibitors
were dissolved in dimethyl sulfoxide (DMSO) at a final concen-
tration of 1%. At this concentration, DMSO had no significant
effect on PDE activity. The concentration of drugs that pro-
duced 50% inhibition of substrate hydrolysis (IC50) was calcu-
lated by nonlinear regression analysis from concentratione
response curves and included different concentrations of inhib-
itors. The results represent the mean of three determinations ob-
tained for three different enzymatic preparations. The
experimental error is about 15%.
Appendix. Supplementary data
Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.ejmech.
2007.05.016.
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