Discovery of 7-oxopyrazolo[1,5-a]pyrimidine-6-carboxamides as potent and selective CB2 cannabinoid receptor inverse agonists

Article abstract of DOI:10.1021/jm400182t

We recently described the medicinal chemistry of a new series of heteroaryl-4-oxopyridine/7-oxopyrimidines as CB₂ receptor partial agonists, showing that the functionality of these ligands is controlled by the nature of the heteroaryl function condensed with the pyridine ring. We describe herein the design and synthesis of the 7-oxopyrazolo[1,5-a]pyrimidine-6- carboxamides, structural isomers of our previously reported pyrazolo[3,4-b] pyridines. All of the new compounds showed high affinity and selectivity for the CB₂ receptor in the nanomolar range. In 3,5-cyclic adenosine monophosphate (cAMP) assays, the novel series shows stimulatory effects on forskolin-induced cAMP production acting as inverse agonists.

Full text of DOI:10.1021/jm400182t

Article
pubs.acs.org/jmc
Discovery of 7‑Oxopyrazolo[1,5‑a]pyrimidine-6-carboxamides as
Potent and Selective CB2 Cannabinoid Receptor Inverse Agonists
Mojgan Aghazadeh Tabrizi,*,† Pier Giovanni Baraldi,*,† Giulia Saponaro,† Allan R. Moorman,‡,#
Romeo Romagnoli,† Delia Preti,† Stefania Baraldi,† Emanuela Ruggiero,† Cristina Tintori,§
Tiziano Tuccinardi,∥ Fabrizio Vincenzi,⊥ Pier Andrea Borea,⊥ and Katia Varani⊥
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
‡Research and Development, King Pharmaceuticals, Inc., 4000 Centre Green Way, Suite 300, Cary, North Carolina 27707, United
States
§Dipartimento Farmaco Chimico Tecnologico, Universita
̀
di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
∥Dipartimento di Farmacia, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
⊥Dipartimento di Scienze Mediche e Sperimentale, Sezione di Farmacologia, Universita
̀
di Ferrara, 44121, Ferrara, Italy
S
* Supporting Information
ABSTRACT: We recently described the medicinal chemistry of a
new series of heteroaryl-4-oxopyridine/7-oxopyrimidines as CB2
receptor partial agonists, showing that the functionality of these
ligands is controlled by the nature of the heteroaryl function
condensed with the pyridine ring. We describe herein the design
and synthesis of the 7-oxopyrazolo[1,5-a]pyrimidine-6-carboxa-
mides, structural isomers of our previously reported pyrazolo[3,4-
b]pyridines. All of the new compounds showed high affinity and
selectivity for the CB2 receptor in the nanomolar range. In 3,5-
cyclic adenosine monophosphate (cAMP) assays, the novel series
shows stimulatory effects on forskolin-induced cAMP production
acting as inverse agonists.
antagonists may have utility in certain inflammatory and allergic
conditions.16 It has been suggested that inverse agonist may be
effective for the control of immune cell mobility in arthritis and
inflammatory states17 and for the treatment of osteoporosis via
the inhibition of osteoclast differentiation.18 This therapeutic
potential has prompted the development of several CB2
receptor selective ligands as agonists, partial agonists, or
antagonists/inverse agonists (Chart 1).
Among the selective agonists, the best known compound is
R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-
[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone me-
sylate 1 (R-(+)-WIN 55,212-2, Chart 1). This compound
displays activity at both CB1 and CB2 receptors, although it has
been found to possess slightly higher CB2 than CB1 affinity.8
(2-Methoxynaphthalen-1-yl)(1-pentyl-1H-indol-3-yl)-
methanone (JWH-267) is an N-alkylindole derivative with high
affinity at the CB2 receptor that exhibited partial activity in 35S-
labeled guanosine 5′-O-(3-thio)triphosphate ([35S]GTPγS)
binding assays.19
INTRODUCTION
■
Interest in the potential medicinal use of cannabinoids grew
recently with the discovery of two cannabinoid receptors, CB1
and CB2.1,2 The CB1 receptor is abundantly expressed in the
central nervous system (CNS) and is responsible for the
psychotropic side effects.3−5 The CB2 receptor is mainly found
in cells of the immune system, although it may be up-regulated
in the CNS under pathological conditions.6,7 The main signal
transduction pathway triggered is through Gi proteins, resulting
in an inhibition of adenylate cylase activity and a decrease in
cyclic AMP levels.8 Activation of CB2 receptors inhibits
adenylyl cyclase and activates mitogen-activated protein kinase
through binding of the α-subunit of the Gi/o protein but does
not modulate calcium or potassium conductances.9,10 Agonist
binding to CB1 receptors, by contrast, suppresses calcium and
activates inward-rectifying potassium conductances, effects
associated with depression of neuronal excitability and
transmitter release.11,12 Thus, differences in receptor distribu-
tion and signal transduction mechanisms are likely to account
for the relative absence of the CNS side effects induced by CB2
agonists. These considerations suggest that novel pharmaco-
therapies targeting CB2 receptors may have considerable
therapeutic potential. Agonists and partial agonists of the CB2
receptor have been investigated for their potential utility in the
treatment of inflammatory and neuropathic pain,13−15 while
Fewer classes of compounds have been reported as selective
antagonists or inverse agonists of the CB2 receptor.20 The
prototypical selective antagonist is SR1445289 (2, Chart 1),
Received: February 5, 2013
Published: May 22, 2013
© 2013 American Chemical Society
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Chart 1. Structures of Representative CB2 Selective Ligands
which is based upon a pyrazole scaffold. The 6-iodopravadoline
AM63021 (3, Chart 1) is an indole derived cannabinoid ligand
that acts as a potent and selective inverse agonist at the CB2
receptors. These compounds are thought to be CB2 receptor
inverse agonists rather than neutral antagonists because when
administered by themselves, they can produce inverse
cannabimimetic effects in CB2 receptor-expressing tissues.8 N-
(1,3-Benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-
(pentyloxy)-3-quinolinecarboxamide (JTE-907)22 is a potent
CB2 receptor ligand endowed with high selectivity for the CB2
receptor and inverse-agonistic properties. This compound
received attention because of its anti-inflammatory properties,
showing antipruritic activity in a model of dermatitis when
orally administered.22,23
controlled by the nature of the heteroaryl function condensed
with the pyridine ring. In 3,5-cyclic adenosine monophosphate
(cAMP) assays, they showed a dose-dependent effect in the
modulation of forskolin-induced cAMP production, revealing
different behaviors as full agonists, partial agonists, and inverse
agonists.
Here, we report a novel series of 7-oxopyrazolo[1,5-
a]pyrimidine-6-carboxamides that were found to be potent
and selective CB2 receptor ligands. Our goal was to design and
synthesize the structural isomers of our previously reported
pyrazolo[3,4-b]pyridine that allowed us to conduct a
pharmacophore exploration and optimization effort around
the heteroaryl central scaffold (Chart 2).
Recently, a new class of CB2 selective inverse agonists based
on a triaryl bis-sulfone scaffold has been described. This class is
represented by N-[1(S)-[4-[[4-methoxy-2-[(4-
methoxyphenyl)sulfonyl]phenyl]sulfonyl]phenyl]ethyl]-
methanesulfonamide (Sch225336),24 which was shown to
block the recruitment of leucocytes in vivo.25 Previously,
within a research program to identify novel CB2 agonists, our
group designed a hybrid chemical structure that incorporated
the structural features of known cannabinoid ligands. The new
series of oxazinoquinolone derivatives26 exemplified by N-
(adamant-1-yl)-3,7-dihydro-3-ethyl-7-oxo-10-(pyrrolidin-1-yl-
2H-[1,4]oxazino-[2,3,4-ij]quinoline-6-carboxamide (4, Chart
1) was synthesized and tested in binding assays, exhibiting
high affinity and selectivity for the CB2 receptor (hCB2 Ki =
8.12 nM, hCB1 Ki > 10000, selectivity index (SI) of >1231).
The potency of the new oxazinoquinoline-6-carboxamides was
measured in functional assays, revealing that the novel series
behaved as CB2 receptor full agonists.
In this context, very recently we have reported the medicinal
chemistry of a series of heteroaryl-4-oxopyridine/7-oxopyr-
imidine derivative27 as represented by trans-4,7-dihydro-1,3-
dimethyl-N-(4-methylcyclohexyl)-4-oxo-7-pentyl-1H-pyrazolo-
[3,4-b]pyridine-5-carboxamide 5 (Chart 1), which displayed
high affinity at the CB2 receptor (hCB2 Ki = 11.4 nM, hCB1 Ki
= 4568, SI = 401). In this study, additional CB2 ligands were
synthesized by replacing the pyrazolo ring with different
heterocycles that were found to be potent CB2 receptor ligands.
Moreover, it was shown that the functionality of these ligands is
Chart 2. Structure Variations around the 7-
Oxopyrazolo[1,5a]pyrimidine Scaffold
The newly synthesized pyrazolopyrimidines were tested in
competition binding assays toward both rat CB1 (rCB1) and rat
CB2 (rCB2) receptors expressed in native tissues (rat brain or
spleen) and human CB1 (hCB1) and human CB2 (hCB2)
receptors expressed in CHO cells. Affinity data (Ki, nM) were
used to calculate the selectivity of newly synthesized
compounds for the CB2 receptors. All of these ligands were
also examined in cyclic AMP assays on hCB2 CHO cells, with
the aim of evaluating their effect in the modulation of forskolin-
induced cAMP production.
Finally a molecular modeling study was performed in order
to explain the structure−activity relationships observed
experimentally. A series of compounds were docked into CB1
and CB2 receptor models, and the observed ligand−receptor
interactions were examined while taking into account the site-
directed mutagenesis data available for this receptor subtype.
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The preparation of the pyrazolopyrimidine derivatives 47−
49 was accomplished by the route shown in Scheme 2,
following synthetic procedures already established for the
previous compounds. Bromination of pyrazolo ester 7a in
position 3 led to compound 7p in high yields. Compound 7p
was alkylated with 1-pentyl bromide to give 8p. Hydrolysis
afforded the carboxylic acid 9p that was converted to the
carboxamide 47 by coupling with 1-adamantylamine. Suzuki
coupling with phenylboronic acid and 4-methoxyphenylboronic
acid yielded the final compounds 48 and 49, respectively.
CHEMISTRY
■
The synthetic routes to obtain the target 4,7-dihydro-7-oxo-4-
pentyl-2(3)-substituted[1,5-a]pyrimidine-6-carboxamide deriv-
atives 10−49 are outlined in Schemes 1 and 2. All substituents
are summarized in Table 1.
a
Scheme 1
RESULTS AND DISCUSSION
■
Structure−Activity Relationships (SARs). All of the
newly synthesized compounds were examined in [3H]CP-
55,940 competition binding experiments for their affinity and
selectivity toward the rat and human recombinant CB1 and CB2
receptors. The results, in terms of binding affinities for the two
receptors (Ki values), are reported in Table 1. While a
substantial degree of sequence homology between the human
and rat CB receptors is well-known, a significant divergence
between the two sequences still exists.31 In our previous
studies, we have observed significant differences in the binding
affinities of specific agonists to the receptors from the two
species.26 This may suggest that the ligands were binding to a
portion of the receptor where homology was not maintained
and could portend that efficacy in a rodent model may not
translate into efficacy in humans. As seen in Table 1, the
measured affinity values for all of the examined compounds do
not differ significantly between rat and human CB2 receptors.
For the first group of five synthesized 7-oxopyrazolo[1,5-
a]pyrimidine-6-carboxamides (10−14, Table 1), the structural
variations were focused on the moiety borne by the amide at
the C-6 position, maintaining the methyl group at the C-2
position and the n-pentyl chain at the N-4 constant. As was
seen previously, the compounds bearing cyclohexyl, cyclo-
heptyl, or adamantyl moieties on the amide showed high
affinity at the CB2 receptor. The best result in terms of both
activity and selectivity profiles was found with the N-
adamantan-1-ylcarboxamide chain (compound 12 hCB2 Ki =
12.7 nM, SI = 88). The benzylcarboxamide 13 displayed
modest affinity and slight selectivity for the cannabinoid
receptors (hCB2 Ki = 358 nM, hCB1 Ki = 3290 nM, SI =
9.2). The choice of the 4-methylcyclohexylcarboxamide group
in position 6 (compound 14) was based upon the binding
a
Reagents: (i) diethyl ethoxymethylenemalonate, AcOH, 120 °C, 2 h;
(ii) K2CO3, 1-pentyl bromide, DMF, 100 °C, 16 h; (iii) NaOH 10%,
MeOH, rt, 2 h; (iv) EDC, HOBt, amine, DMF, rt, 16 h or DIEA,
HBTU, adamantan-1-yl-amine, DMF, rt, 16 h. Information on
compound designations: a, R = Me; b, R = Et; c, R = t-But; d, R =
phenyl; e, R = 2-Me-phenyl; f, R = 4-tolyl-Me-phenyl; g, R = 2-Cl-
phenyl; h, R = 4-Cl-phenyl; i, R = 2,4-di-Cl-phenyl; j, R = 2,6-di-Cl-
phenyl; k, R = furan-2-yl; l, R = furan-3-yl; m, R = thiophen-2-yl; n, R
= 4-Me-thiophen-2-yl; o, R = 5-Me-thiophen-2-yl.
The starting materials, 3-(substituted)-5-aminopyrazoles,
required for the synthesis of the title compounds were
synthesized by the condensation of appropriate β-ketonitriles28
with hydrazine monohydrate according to the literature
procedure.29 The aminopyrazoles (6a−o) were reacted
smoothly with diethyl ethoxymethylenemalonate (DEEM) in
glacial acetic acid to give the cyclized ethyl 2-substituted-4,7-
dihydro-7-oxopyrazolo[1,5-a]pyrimidine-6-carboxylates (7a−
o) in good yields. The subsequent N-alkylation with 1-pentyl
bromide in the presence of K2CO3 was performed to yield
compounds 8a−o. Hydrolysis in 10% aqueous NaOH yielded
the corresponding 4-pentylpyrazolo[1,5-a]pyrimidine-6-carbox-
ylic acids 9a−o30 that then underwent coupling reaction with
appropriate amines to yield the target compounds 10−46.
a
Scheme 2
a
Reagents: (i) Br2, AcOH, rt, 3 h; (ii) K2CO3, 1-pentyl bromide, DMF, 100 °C, 16 h; (iii) NaOH, 10%, MeOH, rt, 2 h; (iv) DIEA, HBTU,
adamantan-1-yl-amine, DMF, rt, 16 h; (v) (phenyl or 4-methoxyphenyl)boronic acid, Pd(PPh3)4, K2CO3, toluene, 100 °C, 16 h.
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Table 1. Affinity (Ki, nM) and Selectivity Index (SI) on Rat and Human CB1 and CB2 Receptors of the Novel CB Compounds
a
10−49
Ki (nM)
b
c
d
e
compd
R
R1
rCB1
rCB2
7.61 0.68
26
hCB1
hCB2
4.56 0.45
22
SI
WIN 55,212-2
10
15.3 1.3
12.2 1.4
2.68
58
Me
Me
Me
Me
Me
Et
cyclohexyl
1578 152
642 65
2
1278 110
527 48
2
11
cycloheptyl
adamant-1-yl
benzyl
12.4 1.1
15.3 1.6
428 17
10.3 1.2
12.7 1.3
358 32
51
12
1460 148
3688 386
2600 310
480 40
1118 96
88
13
3290 312
2432 272
420 35
9.2
14
4-Me-cyclohexyl
cyclohexyl
120 11
106
9
23
15
11.5 1.2
5.63 0.57
0.96 0.08
14.2 1.1
7.52 0.68
5.54 0.48
10.4 1.2
5.22 0.45
0.84 0.07
12.3 1.1
7.22 0.63
4.95 0.42
40
16
Et
cycloheptyl
adamantyl
220 20
200 15
38
17
Et
150 12
120 10
143
18
t-But
t-But
t-But
Ph
cyclohexyl
720 70
675 54
55
19
cycloheptyl
adamant-1-yl
cyclohexyl
650 60
605 50
84
20
675 65
612 57
124
21
750 80
50
35
5
4
702 68
42
31
4
3
17
22
Ph
cycloheptyl
adamant-1-yl
cyclohexyl
900 100
825 80
27
23
Ph
>10000 (40%)
>10000 (30%)
>10000 (45%)
>10000 (30%)
>10000 (25%)
>10000 (49%)
>10000 (45%)
>10000 (40%)
>10000 (42%)
>10000 (48%)
>10000 (8%)
>10000 (47%)
>10000 (10%)
>10000 (47%)
>10000 (49%)
>10000 (40%)
>10000 (18%)
>10000 (30%)
>10000 (20%)
>10000 (40%)
>10000 (17%)
>10000 (22%)
>10000 (15%)
>10000 (33%)
>10000 (1%)
>10000 (15%)
>10000 (5%)
2.74 0.28
48
>10000 (43%)
>10000 (16%)
>10000 (34%)
>10000 (22%)
>10000 (20%)
>10000 (39%)
>10000 (38%)
>10000 (34%)
>10000 (35%)
>10000 (41%)
>10000 (15%)
>10000 (40%)
>10000 (13%)
>10000 (38%)
>10000 (39%)
>10000 (37%)
>10000 (21%)
>10000 (21%)
>10000 (16%)
>10000 (34%)
>10000 (19%)
>10000 (26%)
>10000 (13%)
>10000 (31%)
>10000 (1%)
>10000 (15%)
>10000 (4%)
2.56 0.22
40
>3906
>250
>1119
>3496
>175
>970
>2932
>212
>970
>1610
>181
>1584
>2577
>192
>270
>333
>102
>1050
>1522
>2032
>990
>1602
>666
>862
>45
24
2-Me-phenyl
2-Me-phenyl
2-Me-phenyl
4-Me-phenyl
4-Me-phenyl
4-Me-phenyl
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
2,4-di-Cl-phenyl
2,4-di-Cl-phenyl
2,4-di-Cl-phenyl
2,6-di-Cl-phenyl
furan-2-yl
4
4
25
cycloheptyl
adamant-1-yl
cyclohexyl
9.53 0.92
3.21 0.30
8.93 0.86
2.86 0.25
26
27
62
6
57
5
28
cycloheptyl
adamant-1-yl
cyclohexyl
13.2 1.7
10.3 1.1
29
3.82 0.39
3.41 0.32
30
52
5
47
4
31
cycloheptyl
adamant-1-yl
cyclohexyl
12.3 1.6
10.3 1.1
32
6.32 0.63
6.21 0.55
33
58
6
55
5
34
cycloheptyl
adamant-1-yl
cyclohexyl
6.53 0.58
4.21 0.42
6.31 0.51
3.88 0.31
35
36
60
40
35
5
4
3
52
37
30
98
5
4
3
8
37
cycloheptyl
adamant-1-yl
adamant-1-yl
cyclohexyl
38
39
110 10
40
10.4 1.8
7.23 0.81
5.14 0.42
12.4 1.5
7.52 0.71
9.52 0.92
6.57 0.62
4.92 0.43
10.1 1.3
6.24 0.61
41
furan-2-yl
cycloheptyl
adamant-1-yl
adamant-1-yl
adamant-1-yl
adamant-1-yl
adamant-1-yl
42
furan-2-yl
43
furan-3-yl
44
thiophen-2-yl
4-Me-thiophen-2-yl
5-Me-thiophen-2-yl
Br
45
18
2
15
2
46
12.2 1.8
272 25
214 17
434 41
11.6 1.7
220 18
181 16
350 32
47
48
phenyl
>55
49
4-OCH3-Ph
>28
a
The data are espressed as the mean SEM of n = 4 independent experiments. The affinity values were calculated by using [3H]CP-55,940 as
b
c
d
e
radioligand on rat brain for CB1 receptors, rat spleen for CB2 receptors, human CB1 CHO membranes, and human CB2 CHO membranes.
results obtained from our previously reported work on
pyrazolo[3,4-b]pyridines, where this moiety gave the highest
affinity at the CB2 receptor. In the present series, the 4-
methylcyclohexylcarboxamide moiety did not enhance affinity
(14, hCB2 Ki = 106 nM, hCB1 Ki = 2432 nM, SI = 23) relative
to the cyclohexylcarboxamide (10), possibly because of a
different binding mode of the pyrazolopyrimidine derivatives.
Subsequently, the adopted strategy was to prepare analogues
by introduction of structural modifications at position 2 of the
pyrazolo[1,5-a]pyrimidine template while maintaining the
cyclohexyl, cycloheptyl, and adamantyl moieties on the
carboxamide to investigate their potential to bind to
cannabinoid CB1 and/or CB2 receptors and to study their
structure−affinity relationships.
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Extending the aliphatic chain at the C-2 position of
compounds 10−12 with ethyl or tert-butyl moieties was
investigated by preparation of compounds (15−20). These
compounds showed high affinity for the CB2 receptor (15−17,
0.84−10.4 nM; 18−20, 4.95−12.3 nM) with remarkable
selectivity over the CB1 receptor (15−17, 38 < SI < 143;
18−20, 55 < SI < 124). Replacing the cyclohexyl moiety with a
cycloheptyl or adamantly moiety led to an increase in affinity, as
shown by compounds 16, 17, 19, and 20. In addition the
highest selectivity over the CB1 receptor was observed with the
most lipophilic compounds 17 and 20 (SI of 143 and 124,
respectively). Compound 17 was found to have the highest CB2
receptor affinity among the series of 2-alkyl derivatives, with a
Ki value of 0.84 nM (Figure 1A).
(compounds 21−39) except for analogues 21 and 22 bearing
the cyclohexyl and cycloheptyl carboxamide groups, respec-
tively, that exhibited moderate affinities for the CB1 receptor.
More significantly, all of the 2-aryl derivatives displayed high
affinity for the CB2 receptor, in contrast with the results
obtained with the previous pyrazolo[3,4-b]pyridine series
where the phenyl or 4-Cl-phenyl moiety on the pyrazole led
to loss of affinity at cannabinoid receptors.
As seen with the 3-alkylpyrazolo[3,4-b]pyridine series,
introduction of an adamant-1-ylcarboxamide, as in compound
23, improved affinity and selectivity for the CB2 receptor in
comparison to the cyclohexylamide and cycloheptylamide (21
and 22, respectively). In contrast, the adamant-1-ylamide in this
group of compounds led to a loss of affinity for the CB1
receptor, showing a significant increase in selectivity and
suggesting a difference between the two receptor subtypes in
steric tolerance at this position. In particular, the adamant-1-
ylcarboxamide 23 is the most selective compound in this series
with high affinity for the target receptor (hCB2 Ki =2.56 nM, SI
> 3906).
Introduction of a 2-methylphenyl group at C-2 of the
pyrazolopyrimidine moiety, as with compounds 24−26,
resulted in a 15- and 41-fold increase of selectivity for the
cyclohexyl- and cycloheptylcarboxamide derivatives (SI > 250
and SI > 1119, respectively) relative to the analogous 2-phenyl
group (compounds 21 and 22, respectively). Affinity at the CB2
receptor was also increased by the 2-methylphenyl derivative
when the amide carried a cycloheptyl moiety (compound 25
hCB2 Ki = 8.93 nM). In contrast, the adamant-1-ylcarboxamide
(26) retained affinity and selectivity for the CB2 receptor
equivalent to that of the phenyl derivative 23. Moving the
methyl group from the ortho to the para position of the phenyl
moiety (compounds 27−29) did not induce a marked
alteration in affinity at the CB2 receptor. The most active
compound was the adamantyl carboxamide 29 (hCB2 Ki = 3.41,
SI > 2932), although the cycloheptylamide 28 displayed high
affinity with remarkable selectivity over the CB1 receptor (hCB2
Ki = 10.3 nM, SI > 970).
We therefore investigated the impact of other phenyl
substituents by introducing a chlorine atom at the ortho and
para positions of the 2-phenyl moiety (compounds 30−35,
Table 1). As shown by the biological assays, these new
compounds displayed affinity similar to that of the 2- and 4-
methyl compounds (24−29). The cycloheptyl- and adamant-1-
ylcarboxamide derivatives showed high affinity and selectivity
for the CB2 receptor (31, 32 hCB2 Ki = 10.3, 6.21 nM and 34,
35 hCB2 Ki = 6.31, 3.88 nM, respectively; hCB1 Ki > 10 000
nM). These results suggest that electronic effects on the
aromatic moiety do not significantly influence the affinity for
the cannabinoid CB2 receptor.
Disubstitution of the aryl moiety at C-2 was also examined,
specifically the 2,4-dichlorophenyl (36−38) and 2,6-dichlor-
ophenyl derivatives (39). These modifications resulted in a
significant loss of affinity for the CB2 receptors in comparison
with the corresponding unsubstituted or monosubstituted
phenyl compounds.
Figure 1. Competition curves on hCB2 receptors of WIN 55,212-2
and selected novel CB compounds (A). Effect of the same compounds
expressed as % of increase of forskolin-stimulated cAMP accumulation
in hCB2CHO cells (B). Concentration−response curves of the novel
compounds 17, 19, and 23 in cAMP assays (C). Results are the mean
SEM (n = 4 independent experiments).
Replacement of the phenyl ring of compounds 21−23 by the
bioisosteric 2- or 3-furyl groups (compounds 40−42 and 43,
respectively) mainly induced a marked improvement in affinity
at the CB2 receptor, as seen with compound 42, bearing a 6-
cyclohexylcarboxamide, in comparison to the parent 2-phenyl
analogue 21 (hCB2 Ki = 9.52 nM vs 42 nM, respectively). All of
the 2-furyl compounds showed excellent affinity at the CB2
These results clearly confirmed our previous studies. To
achieve good affinity and selectivity at the CB2 receptor, the
adamantyl group borne by the amide function should be
retained.
Interestingly, when the 2-alkyl group was replaced with an
aromatic phenyl or substituted phenyl moieties at the C-2
position, affinity for the CB1 receptors was completely lost
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Table 2. Effect of the Novel CB Compounds 10−49 in hCB2CHO Cells on Cyclic AMP Assays at 1 and 10 μM
a
a
increase in cAMP production (%)
at 1 μM
increase in cAMP production (%)
at 1 μM
compd
at 10 μM
compd
at 10 μM
WIN 55,212-2
−102
42
9
−104
54
8
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
37
4
6
55
69
5
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
4
5
6
3
5
5
6
8
4
5
53
44
63
114
79
9
136 12
94 10
56
75
9
38
52
121 13
144 13
162 14
183 15
48
67
121 11
137 13
173 14
146 12
159 16
243 22
115 10
213 18
189 16
123 11
134 12
195 17
45
61
87
38
73
42
4
5
8
4
7
4
65
77
6
8
112 10
85
9
62
6
151 12
114 11
103
88
9
9
92
8
107 11
124 13
103
9
56
89
71
38
32
34
36
6
8
6
4
4
4
4
92
102
97
68
48
51
60
8
163 15
9
73
6
94
8
8
5
4
6
6
107
8
125 10
184 17
155 15
63
81
7
7
93
9
107 11
168 16
138 12
a
The data are espressed as the mean SEM of n = 4 independent experiments and represent the % of increase of cAMP production in hCB2CHO
cells stimulated with forskolin (1 μM) obtained by novel CB compounds at 1 or 10 μM in comparison with the full agonist WIN 55,212-2 that
completely inhibited the forskolin-stimulated cAMP levels.
receptor ranging from 4.92 to 9.52 nM. The 3-furyl compound
43 is substantially equivalent to derivative 42 in in terms of
affinity, yet endowed with somewhat lower receptor selectivity.
Among the 2-thienyl derivatives 44−46 bearing an adamant-
1-yl residue on the amide nitrogen, better affinity was seen with
the unsubstituted thiophene derivative 44 (hCB2 Ki = 6.24 nM,
SI > 1602) compared to the 4- or 5-methylthiophene analogues
(45 or 46, respectively) with slightly reduced the affinity at CB2
receptor.
An overall comparison between the binding assays of 2-
alkylpyrazolopyrimidine and 2-aryl derivatives indicates that C-
2 may be a site for enhancement in selectivity together with an
apparent improvement in affinity.
We also evaluated the effect on receptor affinity and
selectivity of structural modifications at the 3-position of the
pyrazolopyrimidone nucleus (46−48) within the 2-methyl
series. The introduction of a bromine (47, hCB2 Ki = 220 nM),
a phenyl (48, hCB2 Ki = 181 nM), or a 4-methoxyphenyl (49,
hCB2 Ki = 350 nM) resulted in a significant decrease in affinity
for CB2 receptor when compared to the 3-unsubstituted
analogue (12, hCB2 Ki = 12.7 nM), suggesting a severe steric
constraint at this position.
Functional Activity at CB2 Receptor and SAR. The
novel compounds were also evaluated in functional assays in
order to study their effects on forskolin-stimulated adenylate
cyclase production in hCB2 CHO cells at 1 and 10 μM
compared to the maximal effect (set at −100%) achieved with
the full CB2 agonist WIN 55,212-2. All of the tested
compounds show some level of efficacy, as evidenced by their
ability to increase forskolin-induced cAMP production, thus
characterizing these compounds as inverse agonists (Table 2).
Interestingly, the highest effect was achieved with the
compounds 17 and 19 that were able to increase forskolin-
induce cAMP production by 243% and 213% at 10 μM,
respectively (Figure 1B), yet the two compounds display
slightly different affinities at the CB2 receptor (Ki = 0.84 and
7.22 nM, respectively). When tested in the presence of WIN
55,212-2, the novel compound 17 was able to completely
abrogate the inhibitory effect of the agonist on forskolin-
stimulated cAMP production, confirming its opposite effect
with respect to WIN 55,212-2 (Figure 1B).
For the most effective compounds 17, 19, and 23, full dose−
response curves were measured and EC50 values were
determined. The obtained results that confirmed the inverse
agonism activity relative to the selected compounds are
presented in Figure 1C. The potencies of the novel compounds
17, 19, and 23 were 95 8, 351 31, and 195 17 nM,
respectively, confirming a good correlation with their affinity
for CB2 receptors.
Among the various compounds bearing an alkyl substituent
at C-2 (compounds 10−20), when the amide substituent is
held constant, there is a trend toward an ethyl substituent at C-
2 being more effective than tert-butyl, which is more effective
than methyl, suggesting that the size of the C-2 substituent may
influence the efficacy of these inverse agonists. For instance, in
the group of compounds having an adamantylamide, the C-2
ethyl compound (17) is one of the most effective compounds
evaluated, showing a 243% increase in cAMP levels at 10 μM,
while the tert-butyl (20) and methyl (12) analogues show 189%
and 75% increases, respectively, at the same concentration. A
similar trend is observed among the cyclohexylamides
(compounds 15, 18, and 10), while within the cyclo-
heptylamides (compounds 19, 16, and 11) the highest effect
is for the tert-butyl (19) analogue. Interestingly, within each
limited series of amides, there appears to be a correlation
between the observed efficacy as inverse agonists at 1 or 10 μM
and the affinity of the individual compounds for the CB2
receptor.
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Figure 2. Compound 17 docked into CB1 (A) and CB2 (B) receptors.
Figure 3. Docking of 13 (A), 23 (B), and 49 (C) into the CB2 receptor model.
We did not observe similar trends among the compounds
bearing a phenyl or substituted phenyl moiety at C-2 (21−39).
The adamantylamides (23, 26, 35, and 29) are the most
effective, showing 195%, 184%, 168%, and 183% increases in
cAMP levels at 10 μM, respectively. These compounds were
also found to bind to the CB2 receptor with very good affinities
(Ki < 4 nM). Similary, the cyclohexylamides (21, 24, 33, 27, 36,
and 30) show moderate effects at 10 μM with modest affinity
(40−57 nM). Among the cycloheptylamides (34, 22, 25, 28,
37, and 31) the increase in cAMP levels at 10 μM is correlated
with Ki values, while a different trend was observed only for the
2-Cl-phenyl derivative 31 that revealed a modest increase in
cAMP (69%) despite its good affinity at CB2 (Ki = 10.3 nM).
Molecular Modeling Studies. The synthesized ligands
were docked, using AUTODOCK 4.0,32 into the CB1 and CB2
receptor models. Figure 2A shows the docking of compound 17
into both receptors. In the CB1 receptor model, the
pyrazolopyrimidine central scaffold and the adamantane ring
interact in the lipohilic pocket mainly delimited by L3.29(193),
T3.33(197), I4.56(247), A4.57(248), P4.60(251), and
W5.43(279), while the n-pentyl chain is directed toward the
sixth transmembrane domain (TM6) and interacts with
L3.26(190) and V6.57(367).
I3.29(110), M6.59(465), and L6.59(269). Furthermore, the
adamantane ring interacts with V4.56(164), P4.60(168),
Y5.39(190), W5.43(194) and feels the effect of a strong
interaction with F5.46(197), which is a nonconserved residue
(V282 in the CB1 receptor). The interactions with T3.33(114)
and F5.46(197), together with the different disposition of the
pyrazolopyrimidine central scaffold, could be the reason for the
CB2 versus CB1 selectivity of these ligands. Site-directed
mutagenesis partially confirms our hypothesis, as mutations in
the CB2 subtype of F5.46(197) cause a substantial decrease of
WIN-55,212-2 affinity.33,34
The substitution of the adamantane ring with the benzylic
group (see 12 vs 13) causes an important decrease of the CB2
affinity. As shown in Figure 3A, the docking of compound 13
highlights that the benzyl group is not adapted to interact into
the lipophilic pocket delimited by V4.56(164), P4.60(168),
Y5.39(190), W5.43(194), and F5.46(197), causing the ligand
to assume a disposition very similar to that observed for 17 in
the CB1 receptor, with the loss of the H-bond interaction with
T3.33(114).
The addition of a phenyl ring in position 2 of the
pyrazolopyrimidine scaffold seems to be well tollerated, as
shown by the CB2 activity of compound 23. The docking of
this compound into the CB2 receptor model highlights that 23
and 17 interact in a very similar manner, showing the same
lipophilic interactions and the H-bond with T3.33(114) (see
Figure 3B). With regard to the phenyl group, it interacts with
F2.58(87) and is oriented toward an empty space delimited by
In the CB2 receptor model, the n-pentyl chain interacts with
L3.27(108) and L6.57(269) whereas the pyrazolopyrimidine
central scaffold is rotated about 90° with respect to the
disposition in the CB1 receptor model (clockwise sense from
the extracellular point of view) with the formation of an H-
bond with T3.33(114) and lipophilic interactions with
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at −80 °C until ready for use. The thawed rat brain tissue was
resuspended in 50 mM Tris-HCl buffer, pH 7.4, at 4 °C. The
suspension was homogenized with a Polytron, centrifuged for 10 min
at 2000g, and the supernatant was centrifuged again for 20 min at
40000g. The pellet was resuspended in a buffer containing 50 mM
Tris-HCl, 1 mM EDTA, 3 mM MgCl2, 0.5% fatty acid free BSA, pH
7.4, at 30 °C.
Competition binding experiments to rat CB1 receptors were
performed using [3H]CP-55,940 (1.0 nM), a membrane suspension
containing 40 μg of protein/100 μL, and different concentrations (1
nM to 10 μM) of the examined compounds.36
TM2 and TM7 that could correspond to the anandamide
binding pocket.35
Finally, the addition of a bulky group in position 3 of the
pyrazolopyrimidine scaffold determines a general decrease of
the CB2 affinity. As shown in Figure 3C, the docking of
compound 49 into the CB2 receptor model highlights that,
because of the steric hindrance produced by L3.26(107) and
I3.29(110), the presence of a 4-methoxyphenyl group in
position 3 determines a positional change of the pyrazolopyr-
imidine scaffold with the loss of the H-bond with T3.33(114)
and the lipophilic interactions with I3.29(110) and
M6.59(465).
To investigate CB2 receptors, [3H]CP-55,940 binding assay was
performed by using previously frozen rat spleen (male Sprague−
Dawley rats, Charles River) that was homogenized in 50 mM Tris-HCl
buffer, pH 7.4, at 4 °C with a Polytron and centrifuged for 10 min at
2000g, and the supernatant was centrifuged for 20 min at 40000g. The
pellet was resuspended in a buffer containing 50 mM Tris-HCl, 1 mM
EDTA, 3 mM MgCl2, 0.5% fatty acid free BSA, pH 7.4, at 30 °C.
Competition binding experiments to rat CB2 receptors were
performed using [3H]CP-55,940 (0.5 nM), a membrane suspension
containing 80 μg of protein/100 μL and different concentrations (1
nM to 10 μM) of examined compounds.36
CHO cells expressing human CB1 and CB2 receptors were grown
adherently and maintained in Ham’s F12 containing 10% fetal bovine
serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and
Geneticin (G418, 0.4 mg/ml) at 37 °C in 5% CO2/95% air.11,37,38 For
membrane preparations, the culture medium was removed and the
cells were washed with PBS, then scraped off T75 flasks in ice-cold
hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA, pH 7.4). The cell
suspension was homogenized with a Polytron and centrifuged for 10
min at 1000g, and the supernatant was then centrifuged for 30 min at
100000g. The membrane pellet was suspended in 50 mM Tris-HCl
buffer, 0.5% BSA (pH 7.4) containing 5 mM MgCl2, 2.5 mM EDTA or
1 mM EDTA for hCB1 or hCB2 receptor, respectively.
CONCLUSION
■
A series of 7-oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carbox-
amide derivatives was designed and synthesized as high affinity
CB2 receptor ligands, tested in radioligand binding studies, and
functionally characterized in cAMP accumulation assays.
A major focus of the optimization effort was to increase
selectivity of our previous series of heteroarylpyridine/
pyrimidine derivatives to avoid or reduce the potential CB1-
associated CNS adverse effects of this novel series. The best
affinity values associated with high selectivity were obtained
with compounds 23, 26, 29, 35, 42, and 44 (2.56 nM < hCB2
Ki < 6.24 nM, 1602 < SI < 3906) bearing an aryl group at the
C-2 position and an adamant-1-yl moiety on the C-6 amide
function.
The effect of novel compounds on forskolin induced
elevation of cyclic adenosine monophosphate (cAMP) levels
in Chinese hamster ovary (CHO) cells transfected with human
CB2 receptor was assessed. The potencies of the compounds
17, 19, and 23 were also measured in functional assays. These
results reveal that the novel series behaved as CB2 receptor
inverse agonists. A good correlation between receptor affinity
(expressed as Ki) and efficacy (represented by % increase in
cAMP levels at different concentrations of test compound) was
observed; thus, the wide range of relative efficacies seen with
this limited series of compounds offers the potential
opportunity to more closely examine the structural require-
ments for delineation between neutral antagonists and potent
inverse agonists. When compared to the triaryl bis-sulfones
described by the Schering group, the compounds herein display
similar levels of affinity and selectivity for the CB2 receptor17
while significantly exceeding the affinity and selectivity reported
recently for the related triarylsulfonamide derivatives.18 Thus,
this novel series of compounds offers an attractive starting point
for further optimization, representing novel pharmacological
tools to evaluate the therapeutic potential of CB2 inverse
agonists in various disease settings. To achieve these ambitious
objectives, an evaluation of the systemic bioavailability and
metabolic stability will be required, tasks beyond the scope of
this present study.
Competition binding experiments were performed using 0.5 nM
[3H]CP-55,940 and different concentrations (1 nM to 10 μM) of the
examined compounds or a reference agonist (WIN 55,212-2) for an
incubation time of 90 or 60 min at 30 °C for CB1 or CB2 receptors,
respectively.
Bound and free radioactivities were separated by filtering the assay
mixture through Whatman GF/C glass fiber filters using a Brandel cell
harvester (Brandel Instruments, Unterfohring, Germany). The filter
̈
bound radioactivity was counted on a Perkin-Elmer 2810 TR
scintillation counter (Perkin-Elmer Life and Analytical Sciences, U.S.).
Cyclic AMP Assay for Human CB2 Receptors. CHO cells
transfected with human CB2 receptors were washed with phosphate-
buffered saline, diluted trypsin and centrifuged for 10 min at 200g. The
pellet containing CHO cells (1 × 106 cells/assay) was suspended in
0.5 mL of incubation mixture: NaCl 150 mM, KCl 2.7 mM, NaH2PO4
0.37 mM, MgSO4 1 mM, CaCl2 1 mM, HEPES 5 mM, MgCl2 10 mM,
glucose 5 mM, pH 7.4 at 37 °C. Then 0.5 mM 4-(3-butoxy-4-
methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as a phosphodiester-
ase inhibitor was added and the mixture preincubated for 10 min in a
shaking bath at 37 °C.39 The effect of the novel CB compounds was
studied in the presence of forskolin, 1 μM, in comparison with the
well-known CB agonist WIN 55,212-2. The reaction was terminated
by the addition of cold 6% trichloroacetic acid (TCA). The TCA
suspension was centrifuged at 2000g for 10 min at 4 °C, and the
supernatant was extracted four times with water saturated diethyl
ether. The final aqueous solution was tested for cyclic AMP levels by a
competition protein binding assay. Samples of cyclic AMP standard
(0−10 pmol) were added to each test tube containing the incubation
buffer (Trizma base 0.1 M, aminophylline 8.0 mM, 2-mercaptoethanol
6.0 mM, pH 7.4) and [3H]cyclic AMP in a total volume of 0.5 mL.
The binding protein previously prepared from beef adrenals was added
to the samples previously incubated at 4 °C for 150 min, and after the
addition of charcoal, they were centrifuged at 2000g for 10 min. The
clear supernatant was counted by using a Perkin-Elmer 2810 TR
scintillation counter (Perkin-Elmer Life and Analytical Sciences, U.S.).
EXPERIMENTAL SECTION
■
Pharmacology. Competition binding experiments were performed
by using [3H]CP-55,940 (specific activity, 180 Ci/mmol) that was
obtained from Perkin-Elmer Life and Analytical Sciences (U.S.).
Human CB1 and CB2 receptors expressed in CHO cells were
purchased from Perkin-Elmer Life and Analytical Sciences (U.S.). All
other reagents were of analytical grade and obtained from commercial
sources.
Competition Binding Experiments on CB1 and CB2
Receptors. To study CB1 receptors, rat brain (male Sprague−Dawley
rats, Charles River) was removed, frozen in liquid nitrogen, and stored
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1
Data Analysis. The protein concentration was determined
according to a Bio-Rad method (Bradford, 1976) with bovine albumin
as reference standard. Inhibitory binding constants, Ki, were calculated
from the IC50 values according to the Cheng and Prusoff equation:40
Ki = IC50/(1 + [C*]/KD*), where [C*] is the concentration of the
radioligand and KD* its dissociation constant. A weighted nonlinear
least-squares curve fitting program, LIGAND,41 was used for computer
analysis of the inhibition experiments. All the data are expressed as the
mean SEM of n = 4 independent experiments. Statistical analysis of
the data was performed using unpaired two-sided Student’s t test.
Docking Studies. The ligands were built by means of Maestro42
and were then minimized in a water environment (using the
generalized Born/surface area model) by means of Macromodel.43
They were minimized using the conjugate gradient, the MMFFs force
field, and a distance-dependent dielectric constant of 1.0 until they
reached a convergence value of 0.05 kcal Å−1 mol−1.
75%. MS: m/z 222.2 (M + H). H NMR (200 MHz, DMSO-d6): δ
12.98 (bs, 1H), 8.52 (s, 1H), 6.13 (s, 1H), 4.22 (q, J = 7 Hz, 2H), 2.23
(s, 3H), 1.27 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-Dihydro-2-ethyl-7-oxopyrazolo[1,5-a]pyrimidine-
6-carboxylate (7b). White solid; mp, 291 °C (dec); yield, 70%.
MS: m/z 235.8 (M + H). H NMR (200 MHz, DMSO-d6): δ 12.95
1
(bs, 1H), 8.52 (s, 1H), 6.16 (s, 1H), 4.22 (q, J = 7 Hz, 2H), 2.67 (q, J
= 7.6 Hz, 2H), 1.31−1.18 (m, 6H).
Ethyl 2-tert-Butyl-4,7-dihydro-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7c). White solid; mp, >300 °C; yield,
1
75%. MS: m/z 264.3 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.07 (bs, 1H), 8.47 (s, 1H), 6.15 (s, 1H), 4.17 (q, J = 7 Hz, 2H),
1.27−1.21 (m, 12H).
Ethyl 4,7-Dihydro-7-oxo-2-phenylpyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7d). White solid; mp, >300 °C; yield,
1
70%. MS: m/z 284.3 (M + H). H NMR (400 MHz, DMSO-d6): δ
The ligands were docked using AUTODOCK 4.032 in the
previously reported CB1 and CB2 receptor models44 optimized on
the basis of the recently deposited PDB structures of adenosine45 and
rhodopsine receptors.46 AUTODOCK TOOLS47 was used to identify
the torsion angles in the ligands, to add the solvent model, and to
assign partial atomic charges (Gasteiger for the ligands and Kollman
for the receptors). The regions of interest used by AUTODOCK were
defined by considering the previously published WIN 55,212-2 docked
into the CB1 and CB2 receptor44 as the central group of a grid of 54,
50, and 52 points in the x, y, and z directions. A grid spacing of 0.375
Å and a distance-dependent function of the dielectric constant were
used for the energetic map calculations. Since the compounds can
form an intramolecular H-bond and our previous study suggested that
the interaction was quite strong,44 this H-bond was considered to be
maintained during interaction in the binding site. For this reason,
during the AUTODOCK protocol, we blocked the torsions involved
in this intramolecular bond to prevent the loss of this interaction. By
use of the Lamarckian genetic algorithm, the compounds were
subjected to 100 runs of the AUTODOCK search using 500 000 steps
of energy evaluation and default values for the other parameters.
Cluster analysis was performed on the results using an rms tolerance of
1.0 Å. The cluster with the best average of estimated free energy was
chosen.
13.08 (bs, 1H), 8.56 (s, 1H), 8.00−7.98 (m, 2H), 7.50−7.42 (m, 3H),
6.77 (s, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 6.8 Hz, 3H).
Ethyl 4,7-Dihydro-2-(2-methylphenyl)-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7e). White solid; mp, >300 °C; yield,
1
65%. MS: m/z 298.2 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.12 (bs, 1H), 8.59 (s, 1H), 7.64−7.62 (m, 1H), 7.34−7.29 (m, 3H),
6.56 (s, 1H), 4.24 (q, J = 7 Hz, 2H), 2.52 (s, 3H), 1.29 (t, J = 7.4 Hz,
3H).
Ethyl 4,7-Dihydro-2-(4-methylphenyl)-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7f). White solid; mp, >300 °C; yield,
1
68%. MS: m/z 298.2 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.02 (bs, 1H), 8.56 (s, 1H), 7.88 (dd, J = 8 Hz, 2H), 7.29 (dd, J = 8
Hz, 2H), 6.58 (s, 1H), 4.24 (q, J = 7 Hz, 2H), 2.36 (s, 3H), 1.29 (t, J =
7 Hz, 3H).
Ethyl 2-(2-Chlorophenyl)-4,7-dihydro-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7g). White solid; mp, >300 °C; yield,
1
60%. MS: m/z 318.1 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.18 (bs, 1H), 8.64 (s, 1H), 7.87−7.45 (m, 4H), 6.55 (s, 1H), 4.25
(q, J = 7 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H).
Ethyl 2-(4-Chlorophenyl)-4,7-dihydro-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7h). Pale white solid; mp, >300 °C;
1
yield, 70%. MS: m/z 318.1 (M + H). H NMR (200 MHz, DMSO-
d6): δ 13.06 (bs, 1H), 8.57 (s, 1H), 8.03 (dd, J = 8.6 Hz, 2H), 7.54
(dd, J = 8.6 Hz, 2H), 6.81 (s, 1H), 4.24 (q, J = 7 Hz, 2H), 1.29 (t, J =
7.2 Hz, 3H).
1
Chemistry. H NMR spectra were recorded on a Bruker AC 200
spectrometer or a Varian Mercury Plus 400 spectrometer. Chemical
shifts (δ) are given in ppm, and the spectra were recorded in
appropriate deuterated solvents, as indicated. Positive ion electrospray
ionization (ESI) mass spectra were recorded on a double-focusing
Finnigan MAT 95 instrument with BE geometry. Melting points were
determined on a Buchi-Tottoli apparatus and are uncorrected. All
Ethyl 2-(2,4-Dichlorophenyl)-4,7-dihydro-7-oxopyrazolo-
[1,5-a]pyrimidine-6-carboxylate (7i). White solid; mp, >300 °C;
1
yield, 60%. MS: m/z 353.2 (M + H). H NMR (200 MHz, DMSO-
d6): δ 13.18 (bs, 1H), 8.65 (s, 1H), 7.93−7.80 (m, 2H), 7.60−7.54 (m,
1H), 6.75 (s, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7 Hz, 3H).
Ethyl 2-(2,6-Dichlorophenyl)-4,7-dihydro-7-oxopyrazolo-
[1,5-a]pyrimidine-6-carboxylate (7j). White solid; mp, 197 °C;
1
products reported showed H NMR spectra in agreement with the
assigned structures. The purity of the tested compounds was
determined by combustion elemental analyses conducted by the
Microanalytical Laboratory of the Department of Chemistry of the
University of Ferrara, Italy, with a Yanagimoto MT-5 CHN recorder
elemental analyzer. All tested compounds yielded data consistent with
a purity of at least 95% compared with the theoretical values. Reaction
progress and product mixtures were routinely monitored by TLC on
silica gel (precoated Merck F254 plates), and compounds were
visualized with aqueous KMnO4. Flash chromatography was
performed using 230−400 mesh silica gel and a mixture of ethyl
acetate/petroleum ether or ethyl acetate/methanol as eluent. Organic
solutions were dried over anhydrous Na2SO4. All chemicals and
reagents were purchased from Aldrich (Sigma-Aldrich) or Lancaster
(Alfa Aesar, Johnson Matthey Company).
Synthesis of Ethyl 4,7-Dihydro-7-oxo-2-(substituted)-
pyrazolo[1,5-a]pyrimidine-6-carboxylate (7a−o). General Pro-
cedure. A mixture of the appropriate 3-substituted-5-aminopyrazole
(6a−o, 10 mmol), diethyl ethoxymethylenemalonate (10 mmol), and
acetic acid (10 mL) was heated at reflux for 4 h. After the reaction was
complete (from TLC), the solvent was evaporated, water was added,
and the crude product was filtered and washed with cold ethanol.
Ethyl 4,7-Dihydro-2-methyl-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7a). White solid; mp, 295 °C; yield,
1
yield, 28%. MS: m/z 353.2 (M + H). H NMR (200 MHz, DMSO-
d6): δ 13.18 (bs, 1H), 8.64 (s, 1H), 7.60−7.56 (m, 2H), 7.21−7.16 (m,
1H), 6.41−6.37 (s, 1H), 4.25 (q, J = 7 Hz, 2H), 1.29 (t, J = 7 Hz, 3H).
Ethyl 4,7-Dihydro-2-(furan-2-yl)-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7k). White solid; mp, >300 °C; yield,
1
80%. MS: m/z 273.9 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.16 (bs, 1H), 8.59 (s, 1H), 7.84 (s, 1H), 7.06−7.01 (m, 1H), 6.66−
6.56 (m, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7 Hz, 3H).
Ethyl 4,7-Dihydro-2-(furan-3-yl)-7-oxopyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7l). White solid; mp, >300 °C; yield,
1
50%. MS: m/z 273.9 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.16 (bs, 1H), 8.57 (s, 1H), 8.32 (s, 1H), 7.88 (m, 1H), 6.97−6.89
(m, 1H), 6.56 (s, 1H), 4.24 (q, J = 7 Hz, 2H), 1.29 (t, J = 7 Hz, 3H).
Ethyl 4,7-Dihydro-7-oxo-2-(thiophen-2-yl)pyrazolo[1,5-a]-
pyrimidine-6-carboxylate (7m). White solid; mp, >300 °C; yield,
1
70%. MS: m/z 290.2 (M + H). H NMR (200 MHz, DMSO-d6): δ
13.16 (bs, 1H), 8.57 (s, 1H), 7.71−7.62 (m, 2H), 7.18−7.14 (m 1H),
6.70 (s, 1H), 4.24 (q, J = 7.Hz, 2H), 1.29 (t, J = 7 Hz, 3H).
Ethyl 4,7-Dihydro-2-(4-methylthiophen-2-yl)-7-
oxopyrazolo[1,5-a]pyrimidine-6-carboxylate (7n). White solid;
1
mp, >300 °C; yield, 59%. MS: m/z 304.3 (M + H). H NMR (200
MHz, DMSO-d6): δ 13.12 (bs, 1H), 8.56 (s, 1H), 7.52 (s, 1H), 7.20 (s,
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1
1H), 6.65 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), 2.25 (s, 3H), 1.29 (t, J = 7
Hz, 3H).
solid; mp, 196−197 °C; yield, 80%. MS: m/z 388.7 (M + H). H
NMR (200 MHz, DMSO-d6): δ 8.70 (s, 1H), 8.02 (dd, J = 8.4 Hz,
2H), 7.57 (dd, J = 8.4 Hz, 2H), 7.21 (s, 1H), 4.31−4.17 (m, 4H), 1.3
(m, 2H), 1.36−1.26 (m, 7H), 0.91−0.84 (t, J = 6.6 Hz, 3H).
Ethyl 2-(2,4-Dichlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8i). White
Ethyl 4,7-Dihydro-2-(5-methylthiophen-2-yl)-7-
oxopyrazolo[1,5-a]pyrimidine-6-carboxylate (7o). White solid;
1
mp, >300 °C; yield, 23%. MS: m/z 304.3 (M + H). H NMR (200
MHz, DMSO-d6): δ 13.16 (bs, 1H), 8.54 (s, 1H), 7.47 (d, J = 3.4 Hz,
1H), 6.85 (d, J = 2.8 Hz, 1H), 6.61 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H),
2.49 (s, 3 H), 1.28 (t, J = 7.2 Hz, 3H).
1
solid; mp, >300 °C; yield, 58%. MS: m/z 423.1 (M + H). H NMR
(200 MHz, DMSO-d6): δ 8.72 (s, 1H), 7.88−7.77 (m, 2H), 7.59−7.54
(m, 1H), 7.04 (s, 1H), 4.30−4.19 (m, 4H), 1.79 (m, 2H), 1.32−1.25
(m, 7H), 0.87−0.81 (t, J = 6.6 Hz, 3H).
Ethyl 2-(2,6-Dichlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8j). White
Synthesis of Ethyl 3-Bromo-4,7-dihydro-2-methyl-7-
oxopyrazolo[1,5-a]pyrimidine-6-carboxylate (7p). A solution of
bromine (38.5 mmol, 2.12 mL) in glacial acetic acid (5 mL) was added
dropwise and under vigorous stirring to a mixture of ethyl 4,7-dihydro-
2-methyl-7-oxopyrazolo[1,5-a]pyrimidine-6-carboxylate (7a, 11
mmol) in glacial acetic acid (40 mL). The reaction mixture was
stirred for 3 h at room temperature. The precipitate was removed by
filtration and washed with water. Yellow solid; mp, 163 °C; yield, 65%.
1
solid; mp, >300 °C; yield, 55%. MS: m/z 423.1 (M + H). H NMR
(200 MHz, DMSO-d6): δ 8.76 (s, 1H), 7.67−7.54 (m, 3H), 6.83 (s,
1H), 4.28−4.18 (m, 4H), 1.80−1.69 (m, 2H), 1.32−1.25 (m, 7H),
0.87−0.81 (t, J = 6.4 Hz, 3H).
Ethyl 4,7-Dihydro-2-(furan-2-yl)-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylate (8k). White solid; mp, 98−99
°C; yield, 75%. MS: m/z 344.4 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 8.69 (s, 1H), 7.86−7.78 (m, 1H), 7.04 (m, 1H), 6.92 (s,
1H), 6.68−6.67 (m, 1H), 4.27−4.20 (m, 4H), 1.89−1.78 (m, 2H),
1.34−1.26 (m, 7H), 0.86 (t, J = 6.6 Hz, 3H).
Ethyl 4,7-Dihydro-2-(furan-3-yl)-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylate (8l). White solid; mp, 127−129
°C; yield, 80%. MS: m/z 344.4 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 8.68 (s, 1H), 8.29−8.21 (m, 1H), 7.81 (m, 1H), 6.95 (s,
1H), 6.89 (s, 1H), 4.30−4.14 (m, 4H), 1.89−1.78 (m, 2H), 1.34−1.26
(m, 7H), 0.87 (t, J = 6.2 Hz, 3H).
1
MS: m/z 301.7 (M + H). H NMR (200 MHz, DMSO-d6): δ 13.38
(bs, 1H), 8.36 (s, 1H), 4.23 (q, J = 7.2 Hz, 2H), 2.29 (s, 3H), 1.28 (t, J
= 7.4 Hz, 3H).
Synthesis of Ethyl 4,7-Dihydro-7-oxo-4-pentyl-2-(substi-
tuted)-pyrazolo[1,5-a]pyrimidine-6-carboxylates (8a−p). Gen-
eral Procedure. A mixture of ethyl 4,7-dihydro-7-oxo-2-(substi-
tuted)-pyrazolo[1,5-a]pyrimidine-6-carboxylates (7a−o, 1.3 mmol)
and K2CO3 (4 mmol) in dry DMF (10 mL) was heated at 100 °C.
After 1 h, 1-pentyl bromide (2 mmol) was added and the mixture was
stirred at 80−100 °C for 10−18 h. The solution was evaporated to
dryness in vacuo and treated with water. The precipitate was collected
and was purified by crystallization (ethyl acetate/hexane).
Ethyl 4,7-Dihydro-2-methyl-7-oxo-4-pentylpyrazolo[1,5-a]-
Ethyl 4,7-Dihydro-7-oxo-4-pentyl-2-(thiophen-2-yl)-
pyrimidine-6-carboxylate (8a). White solid; mp, 93 °C; yield,
pyrazolo[1,5-a]pyrimidine-6-carboxylate (8m). Pale white solid;
1
1
65%. MS: m/z 292.4 (M + H). H NMR (400 MHz, DMSO-d6): δ
mp, 171 °C; yield, 46%. MS: m/z 360.2 (M + H). H NMR (200
8.63 (s, 1H), 6.41 (s, 1H), 4.23 (q, J = 7.2 Hz, 2H), 4.13 (t, J = 7.2 Hz,
2H), 2.32 (s, 3H), 1.75−1.63 (m, 2H), 1.30−1.26 (m, 7H), 0.86 (t, J =
6.8 Hz, 3H).
MHz, DMSO-d6): δ 8.69 (s, 1H), 7.69−7.64 (m, 2H), 7.21−7.17 (m,
1H), 7.06 (s, 1H), 4.31−4.16 (m, 4H), 1.82 (m, 2H), 1.36−1.26 (m,
7H), 0.87 (t, J = 6.6 Hz, 3H).
Ethyl 2-Ethyl-4,7-dihydro-7-oxo-4-pentylpyrazolo[1,5-a]-
Ethyl 4,7-Dihydro-2-(4-methylthiophen-2-yl)-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8n). Pale yel-
low solid; mp, 211−213 °C; yield, 90%. MS: m/z 374.1 (M + H). 1H
NMR (200 MHz, DMSO-d6): δ 8.68 (s, 1H), 7.50 (s, 1H), 7.23 (s,
1H), 7.02 (s, 1H), 4.27−4.15 (m, 4H), 2.26 (s, 3H), 1.81−1.75 (m,
2H), 1.35−1.24 (m, 7H), 0.87 (t, J = 6.4 Hz, 3H).
pyrimidine-6-carboxylate (8b). White solid; mp, 106 °C; yield,
1
74%. MS: m/z 306.4 (M + H). H NMR (200 MHz, DMSO-d6): δ
8.63 (s, 1H), 6.47 (s, 1H), 4.25−4.14 (m, 4H), 2.69 (q, J = 7.6 Hz,
2H), 1.81−1.72 (m, 2H), 1.32−1.20 (m, 10H), 0.86 (t, J = 6.6 Hz,
3H).
Ethyl 2-tert-Butyl-4,7-dihydro-7-oxo-4-pentylpyrazolo[1,5-
Ethyl 4,7-Dihydro-2-(5-methylthiophen-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8o). Yellow
a]pyrimidine-6-carboxylate (8c). White solid; mp, 87 °C; yield,
1
1
80%. MS: m/z 334.5 (M + H). H NMR (200 MHz, DMSO-d6): δ
solid; mp, 200 °C; yield, 65%. MS: m/z 374.1 (M + H). H NMR
8.62 (s, 1H), 6.56 (s, 1H), 4.25−4.11 (m, 4H), 1.79−1.68 (m, 2H),
(200 MHz, DMSO-d6): δ 8.67 (s, 1H), 7.47 (d, J = 3.4 Hz, 1H), 6.99
(s, 1H), 6.87−6.79 (m, 1H), 4.27−4.18 (m, 4H), 2.48 (s, 3H), 1.82−
1.64 (m, 2H), 1.34−1.26 (m, 7H), 0.87 (t, J = 6.6 Hz, 3H).
1.31−1.24 (m, 16H), 0.86 (t, J = 6.4 Hz, 3H).
Ethyl 4,7-Dihydro-7-oxo-4-pentyl-2-phenylpyrazolo[1,5-a]-
pyrimidine-6-carboxylate (8d). White solid; mp, 130 °C; yield,
Ethyl 3-Bromo-4,7-dihydro-2-methyl-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8p). White
1
60%. MS: m/z 354.6 (M + H). H NMR (200 MHz, DMSO-d6): δ
1
8.70 (s, 1H), 8.01−7.99 (m, 2H), 7.52−7.44 (m, 3H), 7.18 (s, 1H),
4.27−4.20 (m, 4H), 1.83−1.76 (m, 2H), 1.34−1.28 (m, 7H), 0.87 (t, J
= 3.4 Hz, 3H).
solid; mp, 163 °C; yield, 55%. MS: m/z 370.1−372.1 (M + H). H
NMR (200 MHz, DMSO-d6): δ 8.45 (s, 1H), 4.23 (q, J = 7.2 Hz, 2H),
4.15 (t, J = 7.2 Hz, 2H), 2.24 (s, 3H), 1.75−1.68 (m, 2H), 1.30−1.26
(m, 7H), 0.85 (t, J = 6.8 Hz, 3H).
Ethyl 4,7-Dihydro-2-(2-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8e). White
Synthesis of 4,7-Dihydro-2-(substituted)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylic Acids (9a−p).
General Procedure. The appropriate 4,7-dihydro-7-oxo-4-pentyl-2-
substituted-pyrazolo[1,5-a]pyrimidine-6-carboxylates (8a−p, 1.7
mmol) was stirred in a mixture of aqueous 10% sodium hydroxide
(5 mL) and methanol (5 mL) at room temperature for 3 h. The
solution was adjusted to pH 4 with aqueous 10% hydrochloric acid.
The resulting precipitate was collected by filtration and washed with
H2O and cold methanol.
1
solid; mp, 100 °C; yield, 74%. MS: m/z 368.4 (M + H). H NMR
(200 MHz, DMSO-d6): δ 8.71 (s, 1H), 7.67−7.64 (m, 1H), 7.34−7.31
(m, 3H), 6.93 (s, 1H), 4.31−4.20 (m, 4H), 2.52 (s, 3H), 1.82−1.76
(m, 2H), 1.33−1.26 (m, 7H), 0.87 (t, J = 6.4 Hz, 3H).
Ethyl 4,7-Dihydro-2-(4-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8f). White
1
solid; mp, 194 °C; yield, 71%. MS: m/z 368.4 (M + H). H NMR
(200 MHz, DMSO-d6): δ 8.68 (s, 1H), 7.89 (dd, J = 8 Hz, 2H), 7.31
(dd, J = 8 Hz, 2H), 7.12 (s, 1H), 4.27−4.20 (m, 4H), 2.36 (s, 3H),
1.85−1.77 (m, 2H), 1.33−1.26 (m, 7H), 0.87 (t, J = 6.4 Hz, 3H).
Ethyl 2-(2-Chlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8g). White
4,7-Dihydro-2-methyl-7-oxo-4-pentylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9a). White solid; mp, 134 °C; yield,
1
68%. MS: m/z 264.3 (M + H). H NMR (200 MHz, DMSO-d6): δ
12.89 (bs, 1H), 8.80 (s, 1H), 6.53 (s, 1H), 4.19 (t, J = 7.4 Hz, 2H),
2.35 (s, 3H), 1.77−1.68 (m, 2H), 1.29−1.26 (m, 4H), 0.85 (t, J = 6.6
Hz, 3H).
1
solid; mp, 91 °C; yield, 78%. MS: m/z 388.9 (M + H). H NMR
(200 MHz, DMSO-d6): δ 8.73 (s, 1H), 7.89−7.81 (m, 1H), 7.60−7.47
(m, 3H), 7.03 (s, 1H), 4.28−4.21 (m, 4H), 1.89−1.80 (m, 2H), 1.34−
1.25 (m, 7H), 0.89−0.83 (m, 3H).
Ethyl 2-(4-Chlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylate (8h). White
4,7-Dihydro-2-ethyl-7-oxo-4-pentylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9b). White solid; mp, 126 °C; yield,
1
60%. MS: m/z 278.3 (M + H). H NMR (200 MHz, DMSO-d6): δ
12.80 (bs, 1H), 8.77 (s, 1H), 6.59 (s, 1H), 4.20 (t, J = 7.4 Hz, 2H),
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2.69−2.58 (m, 2H), 1.77−1.69 (m, 2H), 1.32−1.22 (m, 7H), 0.89−
0.82 (t, J = 6.6 Hz, 3H).
1H), 7.26 (s, 1H), 7.12 (s, 1H), 4.23 (t, J = 7 Hz, 2H), 2.26 (s, 3H),
1.82−1.73 (m, 2H), 1.35−1.26 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H).
4,7-Dihydro-2-(5-Methylthiophen-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylic Acid (9o). White
2-tert-Butyl-4,7-dihydro-7-oxo-4-pentylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9c). White solid; mp, 110 °C; yield,
1
1
58%. MS: m/z 306.3 (M + H). H NMR (200 MHz, DMSO-d6): δ
solid; mp, 185 °C; yield, 76%. MS: m/z 345.6 (M + H). H NMR
12.78 (bs, 1H), 8.78 (s, 1H), 6.71 (s, 1H), 4.21 (t, J = 7.4 Hz, 2H),
1.78−1.66 (m, 2H), 1.33−1.29 (m, 13H), 0.89 (t, J = 7.4 Hz, 3H).
4,7-Dihydro-7-oxo-4-pentyl-2-phenylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9d). White solid; mp, 185 °C; yield,
(200 MHz, DMSO-d6): δ 12.73 (bs, 1H), 8.79 (s, 1H), 7.51 (d, J = 3.4
Hz, 1H), 7.09 (s, 1H), 6.89 (d, J = 2.8 Hz, 1H), 4.23 (t, J = 7.2 Hz,
2H), 2.49 (s, 3H), 1.82−1.76 (m, 2H), 1.34−1.21 (m, 4H), 0.87 (t, J =
6.2 Hz, 3H).
1
58%. MS: m/z 326.1 (M + H). H NMR (200 MHz, DMSO-d6): δ
3-Bromo-4,7-dihydro-2-methyl-7-oxo-4-pentylpyrazolo[1,5-
12.78 (bs, 1H), 8.28 (s, 1H), 8.03−8.01 (m, 2H), 7.51−7.46 (m, 3H),
7.28 (s, 1H), 4.26 (t, J = 7.2 Hz, 2H), 1.84−1.71 (m, 2H), 1.34−1.30
(m, 4H), 0.88−0.85 (t, J = 6.8 Hz, 3H).
a]pyrimidine-6-carboxylic Acid (9p). White solid; mp, 222 °C;
1
yield, 68%. MS: m/z 342.2−344.2 (M + H). H NMR (200 MHz,
DMSO-d6): δ 12.71 (bs, 1H), 8.73 (s, 1H), 4.39 (t, J = 7.8 Hz, 2H),
2.32 (s, 3H), 1.79−1.77 (m, 2H), 1.35−1.32 (m, 4H), 0.87 (t, J = 6.8
Hz, 3H).
4,7-Dihydro-2-(2-methylphenyl)-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9e). White solid; mp, 159
°C; yield, 56%. MS: m/z 340.5 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 12.72 (bs, 1H), 8.84 (s, 1H), 7.69−7.45 (m, 1H), 7.35−
7.30 (m, 3H), 7.05 (s, 1H), 4.28 (t, J = 7.2 Hz, 2H), 2.53 (s, 3H),
1.83−1.69 (m, 2H), 1.34−1.31 (m, 4H), 0.86 (t, J = 6.6 Hz, 3H).
4,7-Dihydro-2-(4-methylphenyl)-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9f). White solid; mp, 215 °C;
Synthesis of 4,7-Dihydro-7-oxo-4-pentyl-2-(substituted)-
pyrazolo[1,5-a]pyrimidine-6-carboxamides (10, 11, 13−16,
18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 40, 41).
General Procedure. A mixture of 4,7-dihydro-7-oxo-4-pentyl-2-
(substituted)-pyrazolo[1,5-a]pyrimidine-6-carboxylic acids (9a−k, 9p,
0.48 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC,
1.44 mmol), and 1-hydroxybenzotriazole (HOBt, 1.44 mmol) in dry
DMF (5 mL), was stirred for 10 min at room temperature. The
appropriate amine was added, and the mixture was stirred for 12 h at
room temperature.The reaction mixture was concentrated and purified
by column chromatography on silica gel.
N - C y c l o h e x y l - 4 , 7 - d i h y d r o - 2 - m e t h y l - 7 - o x o - 4 -
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (10). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 1:1, as eluent); mp, 148 °C; yield, 45%. MS: m/z 345.2 (M +
H). 1H NMR (200 MHz, CDCl3): δ 9.11 (bd, J = 8 Hz, 1H), 8.48 (s,
1H), 5.96 (s, 1H), 4.02−3.94 (m, 3H), 2.45 (s, 3H), 1.91−1.32 (m,
16H), 0.91 (t, J = 6.6 Hz, 3H). Anal. (C19H28N4O2) C, H, N.
N-Cyclohep tyl-4, 7-di hyd ro-2-methyl-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (11). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 1:1, as eluent); mp, 149 °C; yield, 42%. MS: m/z 359.3 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.00 (bd, J = 8 Hz, 1H), 8.66
(s, 1H), 6.44 (s, 1H), 4.18 (t, J = 7 Hz, 2H), 4.02−3.90 (m, 1H), 2.34
(s, 3H), 1.84−1.41 (m, 14H), 1.39−1.28 (m, 4H), 0.85 (t, J = 6.6 Hz,
3H). Anal. (C20H30N4O2) C, H, N .
1
yield, 60%. MS: m/z 340.5 (M + H). H NMR (200 MHz, DMSO-
d6): δ 12.76 (bs, 1H), 8.82 (s, 1H), 7.92 (dd, J = 8.2 Hz, 2H), 7.33
(dd, J = 7.8 Hz, 2H), 7.24 (s, 1H), 4.26 (t, J = 7.4 Hz, 2H), 2.37 (s,
3H), 1.84−1.77 (m, 2H), 1.37−1.32 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H).
2-(2-Chlorophenyl)-4,7-dihydro-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9g). White solid; mp, 167
°C; yield, 64%. MS: m/z 360.7 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 12.71 (bs, 1H), 8.84 (s, 1H), 7.85−7.83 (m, 1H), 7.61−
7.48 (m, 3H), 7.12 (s, 1H), 4.30 (t, J = 7.4 Hz, 2H), 1.82−1.75 (m,
2H), 1.34−1.30 (m, 4H), 0.86 (t, J = 6.4 Hz, 3H).
2-(4-Chlorophenyl)-4,7-dihydro-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9h). White solid; mp, 199
°C; yield, 63%. MS: m/z 360.7 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 12.70 (bs, 1H), 8.82 (s, 1H), 8.04 (dd, J = 8.8 Hz, 2H),
7.59 (dd, J = 8.4 Hz, 2H), 7.30 (s, 1H), 4.26 (t, J = 7.4 Hz, 2H), 1.82−
1.73 (m, 2H), 1.35−1.32 (m, 4H), 0.86 (t, J = 6.4 Hz, 3H).
2-(2,4-Dichlorophenyl)-4,7-dihydro-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9i). White solid; mp, 175 °C;
1
yield, 47%. MS: m/z 395.1 (M + H). H NMR (200 MHz, DMSO-
d6): δ 12.65 (bs, 1H), 8.84 (s, 1H), 7.92−7.57 (m, 3H), 7.13 (s, 1H),
4.29 (t, J = 6.6 Hz, 2H), 1.81−1.71 (m, 2H), 1.33−1.30 (m, 4H), 0.86
(t, J = 6.4 Hz, 3H).
N-Benzyl-4,7-dihydro-2-methyl-7-oxo-4-pentylpyrazolo[1,5-
a]pyrimidine-6-carboxamide (13). White solid (purified by flash
chromatography, using ethyl acetate/petroleum ether, 3:7, as eluent);
2-(2,6-Dichlorophenyl)-4,7-dihydro-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxylic Acid (9j). White solid; mp, 178−
1
mp, 144 °C; yield, 50%. MS: m/z 353.19 (M + H). H NMR (200
1
179 °C; yield, 45%. MS: m/z 395.1 (M + H). H NMR (200 MHz,
MHz, DMSO-d6): δ 9.34 (bt, J = 6 Hz, 1H), 8.72 (s, 1H), 7.35−7.29
(m, 5H), 6.46 (s, 1H), 4.55 (d, J = 6 Hz, 2H), 4.19 (t, J = 7.4 Hz, 2H),
2.34 (s, 3H), 1.82−1.68 (m, 2H), 1.31−1.26 (m, 4H), 0.85 (t, J = 6.6
Hz, 3H). Anal. (C20H24N4O2) C, H, N.
DMSO-d6): δ 12.65 (bs, 1H), 8.86 (s, 1H), 7.68−7.52 (m, 3H), 6.91
(s, 1H), 4.24 (t, J = 7.6 Hz, 2H), 1.81−1.74 (m, 2H), 1.33−1.30 (m,
4H), 0.86 (t, J = 6.6 Hz, 3H).
4,7-Dihydro-2-(furan-2-yl)-7-oxo-4-pentylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9k). Pale white solid; mp, 164−166
°C; yield, 40%. MS: m/z 316.4 (M + H). 1H NMR (200 MHz,
DMSO-d6): δ 12.71 (bs, 1H), 8.81 (s, 1H), 7.88−7.73 (m, 1H), 7.10−
7.08 (m, 1H), 7.02 (s, 1H), 6.69 (t, J = 7.2 Hz, 1H), 4.26−4.13 (m,
2H), 1.77−1.68 (m, 2H), 1.36−1.25 (m, 4H), 0.86 (t, J = 6.4 Hz, 3H).
4,7-Dihydro-2-(furan-3-yl)-7-oxo-4-pentylpyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (9l). White solid; mp, 191 °C (dec);
4,7-Dihydro-2-methyl-N-(4-methyl-cyclohexyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (14). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 3:7, as eluent); mp, 149 °C; yield, 62%. MS: m/z 359.2 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.11 (bd, J = 8 Hz, 1H), 8.68
(s, 1H), 6.45 (s, 1H), 4.22−4.08 (m, 3H), 3.81−3.64 (m, 1H), 2.34 (s,
3H), 1.99−0.81 (m, 20H). Anal. (C20H30N4O2) C, H, N.
1
N-Cyclohexyl-4,7-dihydro-2-ethyl-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxamide (15). White solid (purified by
flash chromatography, using ethyl acetate/petroleum ether, 1:1, as
yield, 71%. MS: m/z 316.4 (M + H). H NMR (200 MHz, DMSO-
d6): δ 12.71 (bs, 1H), 8.81 (s, 1H), 8.34−8.33 (m, 1H), 7.83−7.75 (m,
1H), 6.99−6.89 (m, 2H), 4.23 (t, J = 7 Hz, 2H), 1.83−1.74 (m, 2H),
1.35−1.27 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H).
1
eluent); mp, 157 °C; yield, 40%. MS: m/z 359.2 (M + H). H NMR
(200 MHz, DMSO-d6): δ 9.01 (bd, J = 7.8 Hz, 1H), 8.67 (s, 1H), 6.50
(s, 1H), 4.19 (t, J = 7.4 Hz, 2H), 3.89−3.72 (m, 1H), 2.71 (q, J = 7.6
Hz, 2H), 1.99−1.21 (m, 19H), 0.85 (t, J = 6.6 Hz, 3H). Anal.
(C20H30N4O2) C, H, N.
4,7-Dihydro-7-oxo-4-pentyl-2-(thiophen-2-yl)pyrazolo[1,5-
a]pyrimidine-6-carboxylic Acid (9m). White solid; mp, 157 °C;
1
yield, 94%. MS: m/z 332.1 (M + H). H NMR (200 MHz, DMSO-
d6): δ 12.73 (bs, 1H), 8.81 (s, 1H), 7.72−7.68 (m, 2H), 7.22−7.16 (m,
2H), 4.24 (t, J = 7 Hz, 2H), 1.83−1.69 (m, 2H), 1.34−1.27 (m, 4H),
0.87 (t, J = 6.4 Hz, 3H).
4,7-Dihydro-2-(4-Methylthiophen-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxylic Acid (9n). Pale
white solid; mp, 162−164 °C; yield, 40%. MS: m/z 345.6 (M + H). 1H
NMR (200 MHz, DMSO-d6): δ 12.71 (bs, 1H), 8.80 (s, 1H), 7.54 (s,
N-Cycloheptyl-4,7-dihydro-2-ethyl-7-oxo-4-pentylpyrazolo-
[1,5-a]pyrimidine-6-carboxamide (16). White solid (purified by
flash chromatography, using ethyl acetate/petroleum ether, 4:6, as
1
eluent); mp, 116 °C; yield, 40%. MS: m/z 373.3 (M + H). H NMR
(200 MHz, DMSO-d6): δ 9.01 (bd, J = 7.6 Hz, 1H), 8.65 (s, 1H), 6.49
(s, 1H), 4.18 (t, J = 7.2 Hz, 2H), 4.03−3.81 (m, 1H), 2.67 (q, J = 7.6
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Hz, 2H), 1.82−1.20 (m, 21H), 0.84 (t, J = 6.6 Hz, 3H). Anal.
(C21H32N4O2) C, H, N.
d6): δ 8.98 (bd, J = 7.8 Hz, 1H), 8.77 (s, 1H), 7.96−7.82 (m, 1H),
7.65−7.61 (m, 1H), 7.52−7.48 (m, 2H), 7.07 (s, 1H), 4.30 (t, J = 7
Hz, 2H), 3.99−3.78 (m, 1H), 1.81−1.21 (m, 16H), 0.89 (t, J = 6.6 Hz,
3H). Anal. (C24H29ClN4O2) C, H, N.
2-tert-Butyl-N-cyclohexyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (18). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 3:7, as eluent); mp, 191 °C; yield, 40%. MS: m/z 387.2 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.01 (bd, J = 7.8 Hz, 1H), 8.66
(s, 1H), 6.60 (s, 1H), 4.20 (t, J = 7 Hz, 2H), 3.97−3.84 (m, 1H),
1.97−1.29 (m, 25H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C22H34N4O2) C,
H, N.
2-tert-Butyl-N-cycloheptyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (19). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 3:7, as eluent); mp, 186 °C; yield, 40%. MS: m/z 401.3 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.05 (bd, J = 8.2 Hz, 1H), 8.65
(s, 1H), 6.60 (s, 1H), 4.20 (t, J = 7.6 Hz, 2H), 4.11−3.91 (m, 1H),
1.83−1.76 (m, 4H), 1.55−1.28 (m, 23H), 0.86 (t, J = 6.4 Hz, 3H).
Anal. (C23H36N4O2) C, H, N.
N - C y c l o h e x y l - 4 , 7 - d i h y d r o - 7 - o x o - 4 - p e n t y l - 2 -
phenylpyrazolo[1,5-a]pyrimidine-6-carboxamide (21). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 2:8, as eluent); mp, 156 °C; yield, 59%. MS: m/z 407.1 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 8.99 (bd, J = 7.2 Hz, 1H), 8.73
(s, 1H), 8.04−8.00 (m, 2H), 7.57−7.45 (m, 3H), 7.21 (s, 1H), 4.27 (t,
J = 7.2 Hz, 2H), 3.93−3.77 (m, 1H), 1.87−1.31 (m, 16H), 0.87 (t, J =
6.4 Hz, 3H). Anal. (C24H30N4O2) C, H, N.
N - C y c l o h e p t y l - 4 , 7 - d i h y d r o - 7 - o x o - 4- p e n t y l - 2 -
phenylpyrazolo[1,5-a]pyrimidine-6-carboxamide (22). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 2:8, as eluent); mp, 190 °C; yield, 55%. MS: m/z 421.4 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.04 (bd, J = 7.8 Hz, 1H), 8.73
(s, 1H), 8.04−7.99 (m, 2H), 7.57−7.45 (m, 3H), 7.21 (s, 1H), 4.27 (t,
J = 6.6 Hz, 2H), 4.02−3.89 (m, 1H), 1.87−1.83 (m, 4H), 1.63−1.57
(m, 10H), 1.37−1.28 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H). Anal.
(C25H32N4O2) C, H, N.
2-(2-Chlorophenyl)-N-cycloheptyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (31). White
solid (purified by crystallization using methanol); mp, 117 °C; yield,
1
41%. MS: m/z 455.7 (M + H). H NMR (200 MHz, DMSO-d6): δ
8.99 (bd, J = 7.8 Hz, 1H), 8.76 (s, 1H), 7.87−7.83 (m, 1H), 7.64−7.61
(m, 1H), 7.52−7.48 (m, 2H), 7.07 (s, 1H), 4.30 (t, J = 7 Hz, 2H),
4.11−4.03 (m, 1H), 1.85−1.30 (m, 18H), 0.86 (t, J = 6.6 Hz, 3H).
Anal. (C25H31ClN4O2).
2-(4-Chlorophenyl)-N-cyclohexyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (33). White
solid (purified by crystallization using methanol); mp, >300 °C;
1
yield, 44%. MS: m/z 441.2 (M + H). H NMR (200 MHz, DMSO-
d6): δ 8.97 (bd, J = 7.6 Hz, 1H), 8.74 (s, 1H), 8.03 (dd, J = 8.6 Hz,
2H), 7.59 (dd, J = 8.6 Hz, 2H), 7.24 (s, 1H), 4.26 (t, J = 6.8 Hz, 2H),
3.96−3.84 (m, 1H), 1.87−1.33 (m, 16H), 0.87 (t, J = 6.4 Hz, 3H).
Anal. (C24H29ClN4O2) C, H, N.
2-(4-Chlorophenyl)-N-cycloheptyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (34). White
solid (purified by crystallization using methanol); mp, 184 °C; yield,
1
41%. MS: m/z 455.6 (M + H). H NMR (200 MHz, DMSO-d6): δ
9.01 (bd, J = 7.6 Hz, 1H), 8.73 (s, 1H), 8.03 (dd, J = 8.2 Hz, 2H), 7.59
(dd, J = 8.4 Hz, 2H), 7.24 (s, 1H), 4.25 (t, J = 6.8 Hz, 2H), 4.13−4.05
(m, 1H), 1.84−1.31 (m, 18H), 0.87 (t, J = 6.4 Hz, 3H). Anal.
(C25H31ClN4O2) C, H, N.
N-Cyclohexyl-2-(2,4-dichlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (36). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 4:6, as eluent); mp, 149 °C; yield, 41%. MS: m/z 476.1 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 8.92 (bd, J = 8 Hz, 1H), 8.76
(s, 1H), 7.91−7.80 (m, 2H), 7.61−7.57 (m, 1H), 7.09 (s, 1H), 4.30 (t,
J = 7.2 Hz, 2H), 3.96−3.84 (m, 1H), 1.87−1.30 (m, 16H), 0.85 (t, J =
6.6 Hz, 3H). Anal. (C24H28Cl2N4O2) C, H, N.
N-Cycloheptyl-2-(2,4-dichlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (37). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 4:6, as eluent); mp, 134 °C; yield, 41%. MS: m/z 490.2 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 8.97 (bd, J = 7.8 Hz, 1H), 8.76
(s, 1H), 7.91−7.80 (m, 2H), 7.63−7.57 (m, 1H), 7.09 (s, 1H), 4.30 (t,
J = 7.2 Hz, 2H), 4.12−4.06 (m, 1H), 1.84−1.30 (m, 18H), 0.85 (t, J =
6.6 Hz, 3H). Anal. (C25H30Cl2N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-2-(2-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (24). White
solid (purified by crystallization using methanol); mp, 122 °C; yield,
1
52%. MS: m/z 421.5 (M + H). H NMR (200 MHz, DMSO-d6): δ
9.02 (bd, J = 7.8 Hz, 1H), 8.74 (s, 1H), 7.76−7.61 (m, 1H), 7.35−7.34
(m, 3H), 6.98 (s, 1H), 4.28 (t, J = 7 Hz, 2H), 3.92−3.85 (m, 1H), 2.53
(s, 3H), 1.82−1.30 (m, 16H), 0.86 (t, J = 6.6 Hz, 3H). Anal.
(C25H32N4O2) C, H, N.
N-Cycloheptyl-4,7-dihydro-2-(2-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (25). White
solid (purified by crystallization using methanol); mp, 147 °C; yield,
N-Cyclohexyl-4,7-dihydro-2-(furan-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (40). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 6:4, as eluent); mp, 169 °C; yield, 44%. MS: m/z 397.2 (M +
1
45%. MS: m/z 435.2 (M + H). H NMR (200 MHz, DMSO-d6): δ
9.03 (bd, J = 7.8 Hz, 1H), 8.73 (s, 1H), 7.67 (m, 1H), 7.35−7.32 (m,
3H), 6.97 (s, 1H), 4.28 (t, J = 7 Hz, 2H), 4.03 (m, 1H), 2.54 (s, 3H),
1.85−1.27 (m, 18H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C26H34N4O2) C,
H, N.
1
H). H NMR (200 MHz, DMSO-d6): δ 8.94 (bd, J = 8 Hz, 1H) 8.72
(s, 1H), 7.88−7.87 (m, 1H), 7.06 (d, J = 3.4 Hz, 1H), 6.96 (s, 1H),
6.69−6.67 (m, 1H), 4.26 (t, J = 7.2 Hz, 2H), 3.92−3.79 (m, 1H),
1.87−1.30 (m, 16H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C22H28N4O2) C,
H, N.
N-Cyclohexyl-4,7-dihydro-2-(4-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (27). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 2:8, as eluent); mp, 190 °C; yield, 55%. MS: m/z 421.1 (M +
H). 1H NMR (200 MHz, DMSO-d6): δ 9.00 (bd, J = 7.8 Hz, 1H), 8.72
(s, 1H), 7.90 (dd, J = 7.8 Hz, 2H), 7.32 (dd, J = 7.8 Hz, 2H), 7.16 (s,
1H), 4.26 (t, J = 6.6 Hz, 2H), 3.97−3.83 (m, 1H), 2.37 (s, 3H), 1.86−
1.33 (m, 16H), 0.37 (t, J = 6 Hz, 3H). Anal. (C25H32N4O2) C, H, N.
N-Cycloheptyl-4,7-dihydro-2-(4-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (28). White
solid (purified by crystallization using methanol); mp, 208 °C; yield,
N-Cycloheptyl-4,7-dihydro-2-(furan-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (41). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 1:1, as eluent); mp, 168 °C; yield, 44%. MS: m/z 411.7 (M +
1
H). H NMR (200 MHz, DMSO-d6): δ 8.99 (bd, J = 8 Hz, 1H) 8.72
(s, 1H), 7.88−7.87 (m, 1H), 7.07 (d, J = 3.4 Hz, 1H), 6.96 (s, 1H),
6.68−6.67 (m, 1H), 4.26 (t, J = 7.2 Hz, 2H), 4.15−4.02 (m, 1H),
1.84−1.30 (m, 18H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C23H30N4O2) C,
H, N.
Synthesis of N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-
2-(substituted)-pyrazolo[1,5-a]pyrimidine-6-carboxamides
(12, 17, 20, 23, 26, 29, 32, 35, 38, 39, 42−47). General
Procedure. To a stirred solution of the desired 4,7-dihydro-7-oxo-4-
pentyl-2-(substituted)-pyrazolo[1,5-a]pyrimidine-6-carboxylic acid
(9a−p, 1.00 mmol) in dry DMF (15 mL) was added diisopropylethyl-
amine (5.42 mmol). The resulting solution was stirred at room
temperature for 10 min before adding HBTU (o-benzotriazol-1-yl-
N,N,N′,N′-tetramethyluronium hexafluorophosphate) (1.45 mmol)
1
43%. MS: m/z 435.5 (M + H). H NMR (200 MHz, DMSO-d6): δ
9.03 (bd, J = 7.8 Hz, 1H), 8.71 (s, 1H), 7.90 (dd, J = 7.8 Hz, 2H), 7.32
(dd, J = 7.8 Hz, 2H), 7.16 (s, 1H), 4.25 (t, J = 6.6 Hz, 2H), 4.15−4.07
(m, 1H), 2.37 (s, 3H), 1.85−1.31 (m, 18H), 0.87 (t, J = 6.6 Hz, 3H).
Anal. (C26H34N4O2) C, H, N.
2-(2-Chlorophenyl)-N-cyclohexyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (30). White
solid (purified by crystallization using methanol); mp, >300 °C;
1
yield, 43%. MS: m/z 441.2 (M + H). H NMR (200 MHz, DMSO-
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and stirring for an additional 3 h at room temperature. 1-
Adamantylamine (1.45 mmol) was then added, and the solution was
stirred for 24 h. The solvent was evaporated under reduced pressure,
and the residue was dissolved in ethyl acetate and successively washed
with aqueous saturated sodium bicarbonate, water, and brine. The
combined organic layers were dried over sodium sulfate and
concentrated under vacuum. The residue was purified by column
chromatography or crystallization.
N-Adamantan-1-yl-2-(2,4-dichlorophenyl)-4,7-dihydro-7-
oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (38).
White solid (purified by flash chromatography, using ethyl acetate/
petroleum ether, 3:7, as eluent); mp, 230 °C; yield, 41%. MS: m/z
1
527.2 (M + H). H NMR (200 MHz, DMSO-d6): δ 8.80 (bs, 1H),
8.72 (s, 1H), 7.91−7.80 (m, 2H), 7.62−7.58 (m, 1H), 7.09 (s, 1H),
4.28 (t, J = 6.6 Hz, 2H), 2.06 (s, 9H), 1.93−1.68 (m, 8H), 1.34−1.30
(m, 4H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C28H32Cl2N4O2) C, H, N.
N-Adamantan-1-yl-2-(2,6-dichlorophenyl)-4,7-dihydro-7-
oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (39).
White solid (purified by flash chromatography, using ethyl acetate/
petroleum ether, 3:7, as eluent); mp, 209 °C; yield, 57%. MS: m/z
N-Adamantan-1-yl-4,7-dihydro-2-methyl-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (12). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 1:1, as eluent); mp, 168 °C; yield, 44%. MS: m/z 397.4 (M +
1
1
527.2 (M + H). H NMR (200 MHz, DMSO-d6): δ 8.76 (bs, 1H),
H). H NMR (200 MHz, DMSO-d6): δ 8.85 (bd, 1H), 8.61 (s, 1H),
8.73 (s, 1H), 7.66−7.52 (m, 3H), 6.84 (s, 1H), 4.24 (t, J = 7.2 Hz,
2H), 2.04 (s, 9H), 1.95−1.78 (m, 2H), 1.66 (s, 6H), 1.35−1.23 (m,
4H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C28H32Cl2N4O2).
6.43 (s, 1H), 4.16 (t, J = 6.6 Hz, 2H), 2.34 (s, 3H), 2.05 (s, 9H), 1.82−
1.67 (m, 8H), 1.29−1.27 (m, 4H), 0.85 (t, J = 6.2 Hz, 3H). Anal.
(C23H32N4O2) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-2-(furan-2-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (42). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 4:6, as eluent); mp, 199 °C; yield, 44%. MS: m/z 449.3 (M +
N-Adamantan-1-yl-2-ethyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (17). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 1:1, as eluent); mp, 149 °C; yield, 45%. MS: m/z 411.2 (M +
1
1
H). H NMR (200 MHz, DMSO-d6): δ 8.83 (bs, 1H), 8.67 (s, 1H),
H). H NMR (200 MHz, DMSO-d6): δ 8.86 (bs, 1H), 8.61 (s, 1H),
7.88−7.87 (m, 1H), 7.05 (d, J = 3 Hz, 1H), 6.95 (s, 1H), 6.69−6.67
(m, 1H), 4.24 (t, J = 7.2 Hz, 2H), 2.06 (s, 9H), 1.67 (m, 8H), 1.33−
1.30 (m, 4H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C26H32N4O2) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-2-(furan-3-yl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (43). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 4:6, as eluent); mp, 241 °C; yield, 53%. MS: m/z 449.2 (M +
6.49 (s, 1H), 4.17 (t, J = 7 Hz, 2H), 2.71 (q, J = 7.6 Hz, 2H), 2.05 (s,
9H), 1.81−1.66 (m, 8H), 1.28−1.21 (m, 7H), 0.86 (t, J = 6.6 Hz, 3H).
Anal. (C24H34N4O2) C, H, N.
N-Adamantan-1-yl-2-tert-butyl-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (20). White
solid (purified by crystallization using methanol); mp, 168 °C; yield,
1
40%. MS: m/z 439.3 (M + H). H NMR (200 MHz, DMSO-d6): δ
1
H). H NMR (200 MHz, DMSO-d6): δ 8.86 (bs, 1H) 8.66 (s, 1H),
8.80 (bs, 1H), 8.60 (s, 1H), 6.59 (s, 1H), 4.17 (t, J = 7.6 Hz, 2H), 2.04
(s, 9H), 1.82−1.67 (m, 8H), 1.32−1.29 (m, 13H), 0.86 (t, J = 7.4 Hz,
3H). Anal. (C26H38N4O2) C, H, N.
8.30 (s, 1H), 7.84−7.81 (m, 1H), 6.96−6.94 (m, 1H), 6.91 (s, 1H),
4.20 (t, J = 7.2 Hz, 2H), 2.06 (s, 9H), 1.67 (m, 8H), 1.34−1.28 (m,
4H), 0.86 (t, J = 6.6 Hz, 3H). Anal. (C26H32N4O2) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-
phenylpyrazolo[1,5-a]pyrimidine-6-carboxamide (23). White
solid (purified by flash chromatography, using ethyl acetate as eluent);
N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-(thiophen-
2-yl)pyrazolo[1,5-a]pyrimidine-6-carboxamide (44). White solid
(purified by flash chromatography, using ethyl acetate/petroleum
ether, 4:6, as eluent); mp, >300 °C; yield, 64%. MS: m/z 465.4 (M +
1
mp, 206 °C; yield, 48%. MS: m/z 459.2 (M + H). H NMR (200
MHz, DMSO-d6): δ 8.87 (bs, 1H), 8.68 (s, 1H), 8.03−7.98 (m, 2H),
7.52−7.45 (m, 3H), 7.20 (s, 1H), 4.25 (t, J = 7.2 Hz, 2H), 2.06 (m,
9H), 1.83−1.80 (m, 2H), 1.68 (s, 6H), 1.35−1.28 (m, 4H), 0.87 (t, J =
6.4 Hz, 3H). Anal. (C28H34N4O2) C, H, N.
1
H). H NMR (200 MHz, DMSO-d6): δ 8.84 (bs, 1H) 8.66 (s, 1H),
7.71−7.66 (m, 2H), 7.22−7.17 (m, 1H), 7.09 (s, 1H), 4.22 (t, J = 7
Hz, 2H), 2.06 (s, 9H), 1.93−1.68 (m, 8H), 1.36−1.31 (m, 4H), 0.87
(t, J = 6.4 Hz, 3H). Anal. (C26H32N4O2S) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-2-(2-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (26). White
solid (purified by crystallization using methanol); mp, 240 °C (dec);
N-Adamantan-1-yl-4,7-dihydro-2-(4-methylthiophen-2-yl)-
7-oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (45).
White solid (purified by flash chromatography, using ethyl acetate/
petroleum ether, 4:6, as eluent); mp, 217 °C; yield, 58%. MS: m/z
479.3 (M + H). 1H NMR (200 MHz, DMSO-d6): δ 8.84 (bs, 1H) 8.65
(s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 7.03 (s, 1H), 4.21 (t, J = 6.6 Hz,
2H), 2.26 (s, 3H), 2.06 (s, 9H), 1.92−1.67 (m, 8H), 1.34−1.31 (m,
4H), 0.87 (t, J = 6.2 Hz, 3H). Anal. (C27H34N4O2S) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-2-(5-methylthiophen-2-yl)-
7-oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (46).
White solid (purified by flash chromatography, using ethyl acetate/
petroleum ether, 4:6, as eluent); mp, 234 °C (dec); yield, 48%. MS:
m/z 479.4 (M + H). 1H NMR (200 MHz, DMSO-d6): δ 8.85 (bs, 1H)
8.64 (s, 1H), 7.48 (d, J = 3.8 Hz, 1H), 7.01 (s, 1H), 6.95 (d, J = 3.6
Hz, 1H), 4.21 (t, J = 6.6 Hz, 2H), 2.49 (s, 3H), 2.06 (s, 9H), 1.92−
1.67 (m, 8H), 1.34−1.30 (m, 4H), 0.87 (t, J = 6.4 Hz, 3H). Anal.
(C27H34N4O2S) C, H, N.
1
yield, 43%. MS: m/z 473.8 (M + H). H NMR (200 MHz, DMSO-
d6): δ 8.67 (bs, 1H), 8.69 (s, 1H), 7.73−7.62 (m, 1H), 7.35−7.28 (m,
3H), 6.96 (s, 1H), 4.26 (t, J = 7.2 Hz, 2H), 2.53 (s, 3H), 2.06 (s, 9H),
1.93−1.68 (m, 8H), 1.34−1.31 (m, 4H), 0.86 (t, J = 6.6 Hz, 3H). Anal.
(C29H36N4O2) C, H, N.
N-Adamantan-1-yl-4,7-dihydro-2-(4-methylphenyl)-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (29). White
solid (purified by crystallization using methanol); mp, 258 °C; yield,
1
50%. MS: m/z 473.8 (M + H). H NMR (200 MHz, DMSO-d6): δ
8.86 (bs, 1H), 8.64 (s, 1H), 7.88 (dd, J = 7.8 Hz, 2H), 7.30 (dd, J = 7.8
Hz, 2H), 7.13 (s, 1H), 4.22 (t, J = 7.2 Hz, 2H), 2.35 (s, 3H), 2.05 (s,
9H), 1.91−1.66 (m, 8H), 1.30 (m, 4H), 0.85 (t, J = 6 Hz, 3H). Anal.
(C29H36N4O2) C, H, N.
N-Adamantan-1-yl-2-(2-chlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (32). White
solid (purified by crystallization using methanol); mp, 300 °C; yield,
N-Adamantan-1-yl-3-bromo-4,7-dihydro-2-methyl-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (47). White
solid (purified by flash chromatography, using ethyl acetate/petroleum
ether, 3:7, as eluent); mp, 222 °C; yield, 45%. MS: m/z 474.2−476.2
1
42%. MS: m/z 493.9 (M + H). H NMR (200 MHz, DMSO-d6): δ
8.82 (bs, 1H), 8.71 (s, 1H), 7.85−7.83 (m, 1H), 7.63−7.60 (m, 1H),
7.52−7.47 (m, 2H), 7.06 (s, 1H), 4.28 (t, J = 7.2 Hz, 2H), 2.07 (s,
9H), 1.92−1.68 (m, 8H), 1.34−1.30 (m, 4H), 0.86 (t, J = 6.6 Hz, 3H).
Anal. (C28H33ClN4O2) C, H, N.
1
(M + H). H NMR (200 MHz, DMSO-d6): δ 8.70 (bs, 1H) 8.62 (s,
1H), 4.44 (t, J = 7.8 Hz, 2H), 2.32 (s, 3H), 2.05 (s, 9H), 1.85−1.71
(m, 2H), 1.67 (s, 6H), 1.36−1.30 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).
Anal. (C23H31BrN4O2) C, H, N.
Synthesis of N-Adamantan-1-yl-4,7-dihydro-2-methyl-7-
oxo-4-pentyl-[3-phenyl or 3-(4-methoxyphenyl)]pyrazolo[1,5-
a]pyrimidine-6-carboxamide (48 or 49). A suspension of N-
adamantyl-3-bromo-4,7-dihydro-2-methyl-7-oxo-4-pentylpyrazolo[1,5-
a]pyrimidine-6-carboxamide (47, 0.3 mmol), arylboronic acid (0.95
mmol), and K2CO3 (0.45 mmol) in toluene (5.5 mL) was thoroughly
N-Adamantan-1-yl-2-(4-chlorophenyl)-4,7-dihydro-7-oxo-4-
pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (35). White
solid (purified by crystallization using methanol); mp, 226 °C; yield,
1
42%. MS: m/z 493.9 (M + H). H NMR (200 MHz, DMSO-d6): δ
8.85 (bs, 1H), 8.68 (s, 1H), 8.02 (dd, J = 8.6 Hz, 2H), 7.59 (dd, J = 8.4
Hz, 2H), 7.23 (s, 1H), 4.23 (t, J = 6.6 Hz, 2H), 2.06 (s, 9H), 1.92−
1.68 (m, 8H), 1.31−1.25 (m, 4H), 0.87 (t, J = 6 Hz, 3H). Anal.
(C28H33ClN4O2) C, H, N.
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Journal of Medicinal Chemistry
Article
deaerated and stirred under argon. Tetrakis(triphenylphosphine)-
palladium(0) (0.04 mmol) was then added, and the mixture was
heated at 100 °C for 16 h. The solvent was removed under reduced
pressure, and the residue was dissolved in ethyl acetate and
successively washed with aqueous saturated sodium bicarbonate,
water, and brine. The organic layer was dried over sodium sulfate,
filtered, and concentrated under vacuum. The residue was purified by
column chromatography on silica gel, eluting with ethyl acetate/
petroleum ether, 3:7.
Mackie, K.; Mechoulam, R.; Ross, R. A. International Union of Basic
and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their
ligands: beyond CB1 and CB2. Pharmacol. Rev. 2010, 62, 588−631.
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cannabinoids in synaptic signaling. Physiol. Rev. 2003, 83, 1017−1066.
(6) Patel, K. D.; Davison, J. S.; Pittman, Q. J.; Sharkey, K. A.
Cannabinoid CB(2) receptors in health and disease. Curr. Med. Chem.
2010, 17, 1393−1410.
́
́
(7) Benito, C.; Tolon, R. M.; Pazos, M. R.; Nunez, E.; Castillo, A. I.;
̃
N-Adamantyl-4,7-dihydro-2-methyl-7-oxo-4-pentyl-3-
Romero, J. Cannabinoid CB2 receptors in human brain inflammation.
Br. J. Pharmacol. 2008, 153, 277−285.
phenylpyrazolo[1,5-a]pyrimidine-6-carboxamide (48). White
1
solid; mp, 236 °C; yield, 35%. MS: m/z 473.4 (M + H). H NMR
(8) Howlett, A. C.; Barth, F. I.; Cabral, G.; Casellas, P.; Devane, W.
A.; Felder, C. C.; Herkenham, M.; Mackie, K.; Martin, B. R.
International Union of Pharmacology. XXVII. Classification of
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(9) Rinaldi-Carmona, M.; Barth, F.; Millan, J.; Derocq, J. M.;
Casellas, P.; Congy, C.; Oustric, D.; Sarran, M.; Bouaboula, M.;
Calandra, B.; Portier, M.; Shire, D.; Breliere, J. C.; Le Fur, G. L.
SR144528, the first potent and selective antagonist of the CB2
cannabinoid receptor. J. Pharmacol. Exp. Ther. 1998, 284, 644−650.
(10) Bouaboula, M.; Poinot-Chazel, C.; Marchand, J.; Canat, X.;
Bourrie, B.; Rinaldi-Carmona, M.; Calandra, B.; Le Fur, G.; Casellas, P.
Signaling pathway associated with stimulation of CB2 peripheral
cannabinoid receptor. Involvement of both mitogen-activated protein
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(200 MHz, DMSO-d6): δ 8.84 (bs, 1H) 8.58 (s, 1H), 7.47−7.43 (m,
5H), 3.87 (t, J = 7.8 Hz, 2H), 2.12 (s, 3H), 2.06 (s, 9H), 1.67 (s, 6H),
1.41−1.23 (m, 2H), 1.12−0.92 (m, 2H), 0.71−0.63 (m, 5H). Anal.
(C29H36N4O2) C, H, N.
N-Adamantyl-4,7-dihydro-3-(4-methoxyphenyl)-2-methyl-7-
oxo-4-pentylpyrazolo[1,5-a]pyrimidine-6-carboxamide (49).
White solid; mp, 222 °C (dec); yield, 38%. MS: m/z 503.5 (M +
1
H). H NMR (200 MHz, DMSO-d6): δ 8.82 (bs, 1H), 8.55 (s, 1H),
7.33 (dd, J = 8.6 Hz, 2H), 7.02 (dd, J = 8.8 Hz, 2H), 3.99−3.82 (m,
2H), 3.79 (s, 3H), 2.10 (s, 3H), 2.04 (s, 9H), 1.66 (s, 6H), 1.39−1.25
(m, 2H), 1.19−1.04 (m, 2H), 0.70−0.63 (m, 5H). Anal.
(C30H38N4O3) C, H, N.
ASSOCIATED CONTENT
* Supporting Information
■
S
(11) Felder, C. C.; Joyce, K. E.; Briley, E. M.; Mansouri, J.; Mackie,
K.; Blond, O.; Lai, Y.; Ma, A. L.; Mitchell, R. L. Comparison of the
pharmacology and signal transduction of the human cannabinoid CB1
and CB2 receptors. Mol. Pharmacol. 1995, 48, 443−450.
Table 1 organized in ascending order by selectivity index (SI)
of the novel CB compounds 10−49. This material is available
free of charge via the Internet at http://pubs.acs.org.
(12) Guindon, J.; Hohmann, A. G. Cannabinoid CB2 receptors: a
therapeutic target for the treatment of inflammatory and neuropathic
pain. Br. J. Pharmacol. 2008, 153, 319−334.
(13) Quartilho, A.; Mata, H. P.; Ibrahim, M. M.; Vanderah, T. W.;
Porreca, F.; Makriyannis, A.; Malan, T. P. J. Inhibition of inflammatory
hyperalgesia by activation of peripheral CB2 cannabinoid receptors.
Anesthesiology 2003, 99, 955−960.
(14) Ibrahim, M. M.; Deng, H.; Zvonok, A.; Cockayne, D. A.; Kwan,
J.; Mata, H. P.; Vanderah, T. W.; Lai, J.; Porreca, F.; Makriyannis, A.;
Malan, T. P., Jr. Activation of the CB2 cannabinoid receptors by
AM1241 inhibits experimental neuropathic pain: pain inhibition by
receptors not present in the CNS. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 10529−10533.
(15) Vincenzi, F.; Targa, M.; Corciulo, C.; Aghazadeh Tabriz, M.;
Merighi, S.; Gessi, S.; Saponaro, G.; Baraldi, P. G.; Borea, P. A.; Varani,
K. Antinociceptive effects of the selective CB2 agonist MT178 in
inflammatory and chronic rodent pain models. Pain 2013, 154, 864−
873.
AUTHOR INFORMATION
Corresponding Author
*For M.A.T.: phone, 39-(0)532455926; fax, 39-(0)53245593;
e-mail, tbj@unife.it. For P.G.B.: phone, 39-(0)532455921; fax,
39-(0)532455953; e-mail, Baraldi@unife.it.
■
Present Address
#A.R.M.: Alta Vetta Pharmaceutical Consulting, LLC, 3
Skipwith Court, Durham, North Carolina 27707.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank King Pharmaceuticals, Inc., Research and Develop-
ment (4000 Centre Green Way, Suite 300, Cary, North
Carolina 27513), for financial support.
(16) Ashton, J. C.; Glass, M. The cannabinoid CB2 receptor as a
target for inflammation-dependent neurodegeneration. Curr. Neuro-
pharmacol. 2007, 5, 73−80.
(17) Lunn, C. A.; Reich, E. P.; Fine, J. S.; Lavey, B.; Kozlowski, J. A.;
Hipkin, R. W.; Lundell, D. J.; Bober, L. Biology and therapeutic
potential of cannabinoid CB2 receptor inverse agonists. Br. J.
Pharmacol. 2008, 153, 226−239.
ABBREVIATIONS USED
■
CHO, Chinese hamster ovary; DEEM, diethylethoxymethylene
malonate; SI, selectivity index; EDC, 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride; HOBt,
hydroxybenzotriazole; HBTU, O-benzotriazole-N,N,N′,N′-tet-
ramethyluronium hexafluorophosphate
(18) Yang, P.; Wang, L.; Feng, R.; Almehizia, A. A.; Tong, Q.; Myint,
K.-Z.; Ouyang, Q.; Alqarni, M. A.; Wang, L.; Xie, X.-Q. Novel triaryl
sulfonamide derivatives as selective cannabinoid receptor 2 inverse
agonists and osteoclast inhibitors: discovery, optimization, and
biological evaluation. J. Med. Chem. 2013, 56, 2045−2058.
(19) Huffman, J. W.; Zengin, G.; Wu, M. J.; Lu, J.; Hynd, G.; Bushell,
K.; Thompson, A. L.; Bushell, S.; Tartal, C.; Hurst, D. P.; Reggio, P.
H.; Selley, D. E.; Cassidy, M. P.; Wiley, J. L.; Martin, B. R. Structure−
activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the
cannabinoid CB(1) and CB(2) receptors: steric and electronic effects
of naphthoyl substituents. New highly selective CB(2) receptor
agonists. Bioorg. Med. Chem. 2005, 13, 89−112.
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