Design, synthesis, and pharmacological properties of new heteroarylpyridine/heteroarylpyrimidine derivatives as CB2 cannabinoid receptor partial agonists

Article abstract of DOI:10.1021/jm301527r

Recent developments indicate that CB₂ receptor ligands have the potential to become therapeutically important. To explore this potential, it is necessary to develop compounds with high affinity for the CB₂ receptor. Very recently, we

Full text of DOI:10.1021/jm301527r

Article
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Properties of New
Heteroarylpyridine/Heteroarylpyrimidine Derivatives as CB2
Cannabinoid Receptor Partial Agonists
Mojgan Aghazadeh Tabrizi,*,† Pier Giovanni Baraldi,*,† Giulia Saponaro,† Allan R. Moorman,‡,∥
Romeo Romagnoli,† Delia Preti,† Stefania Baraldi,† Carmen Corciulo,§ Fabrizio Vincenzi,§
Pier Andrea Borea,§ and Katia Varani§
†Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, Universita
̀
di Ferrara, 44121, Ferrara, Italy
‡
King Pharmaceuticals Research and Development, Inc., 4000 Centre Green Way, Suite 300, Cary, North Carolina 27707, United
States
§Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita
̀
di Ferrara, 44121, Ferrara, Italy
ABSTRACT: Recent developments indicate that CB2 receptor
ligands have the potential to become therapeutically important. To
explore this potential, it is necessary to develop compounds with
high affinity for the CB2 receptor. Very recently, we have identified
the oxazinoquinoline carboxamides as a novel class of CB2 receptor
full agonists. In this paper we describe the medicinal chemistry of a
new series of heteroaryl-4-oxopyridine/7-oxopyrimidine derivatives.
Some of the reported compounds showed high affinity and potency
at the CB2 receptor while showing only modest affinity for the
centrally expressed CB1 cannabinoid receptor. Moreover, we found
that the functionality of these ligands is controlled by the nature of
the heteroaryl function condensed with the pyridine ring. In 3,5-cyclic adenosine monophosphate (cAMP) assays, the novel
series show dose-dependent effects on the modulation of forskolin-induced cAMP production, revealing different behaviors as
full agonists, partial agonists, and inverse agonists.
and mast cells, suggesting that a wide range of immune
functions can be regulated through CB2 modulators.5
INTRODUCTION
■
Cannabinoids are a group of distinct compounds found in
Cannabis sativa, with cannabinol, cannabidiol, and Δ9-
tetrahydrocannabinol (1, THC, Chart 1) being the most
representative molecules. The long history of Cannabis use has
led to the introduction into the clinic of 1 (dronabinol), for the
stimulation of appetite in AIDS patients), and of one of its
synthetic analogues, nabilone (for the suppression of nausea
and vomiting produced by chemotherapy).1,2 Despite the
clinical benefits, the therapeutic use of cannabis is limited by its
psychoactive effects including hallucination, addiction, and
dependence.3 The physiological effects of cannabinoids are
mediated by at least two G-protein-coupled receptors, CBl and
CB2. Autoradiographic studies have demonstrated that CBl
receptors are expressed primarily in the central nervous system,
specifically in the cerebral cortex, hippocampus, basal ganglia,
and cerebellum. They are also found to a lesser degree in the
reproductive system and other peripheral tissues, including
those of the immune system.4 CB2 receptors are almost
exclusively found in the immune system, with the greatest
density in the spleen. It is estimated that the expression level of
CB2 receptors in the immune cells is about 10−100 times
higher than that of CBl. Within the immune system, CB2 is
found in various cell types, including B cells, NK cells,
monocytes, microglial cells, neutrophils, T cells, dentritic cells,
CB2 receptors are also present in some central and peripheral
neurons,6,7and the role of neuronal CB2 receptors still has to be
established. Various strategies have been developed to increase
the benefit-to-risk ratio of cannabinoid therapies,8 one of which
is the development of CB2 selective agonists for circumventing
the unwanted consequences of cannabinoid CB1 receptor
activation.9 Selective agonists of CB2 receptors have been
shown to suppress inflammation in vivo as well as inhibiting
disease severity and spasticity in an animal model of multiple
sclerosis.10−12 Additionally, CB2 agonists have been shown to
inhibit inflammatory and neuropathic pain, as well as
emesis.13−16
A large number of CB2-selective agonists have been
discovered and classified according to their chemical structures
(Chart 1). The dibenzopyran derivatives include 1, the main
psychoactive constituent of Cannabis, and (6aR)-trans-3-(1,1-
dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimeth-
yl-6H-dibenzo[b,d]pyran-9-methanol 2 (HU-210), a synthetic
analogue of (−)-Δ8-tetrahydrocannabinol. Compound 2 dis-
plays high affinity for CB1 and CB2 receptors and also high
Received: October 19, 2012
Published: January 25, 2013
© 2013 American Chemical Society
1098
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
Chart 1. Structures of Representative CB2 Selective Ligands
potency and relative intrinsic activity as a cannabinoid receptor
agonist. A third member of this class is (6aR,10aR)-3-(1,1-
dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-
dibenzo[b,d]pyran (3, JWH-133), frequently used as a
pharmacological tool.17 Another well-known member of the
bicyclic and tricyclic analogues of 1 that lack a pyran ring is
(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-
hydroxypropyl)cyclohexanol 4 (CP-55,940, Chart 1). This has
been found to have slightly lower CB1 and CB2 affinities than 2
in some investigations but does seem to possess HU-210-like
CB1 and CB2 receptor relative intrinsic activity.17,18
Another member of the bicyclic analogues is {4-[4-
(1, 1dimethylheptyl)-2, 6-dimethoxyphenyl]-6, 6-
dimethylbicyclo[3.1.1]hept-2-en-2-yl}methanol 5 (HU-308)
that is selective for the CB2 receptor subtype, with a selectivity
of over 400× for CB2 vs CB1.17
The aminoalkylindole derivatives are represented by R-
(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-
[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanonemesy-
late 6 (R-(+)-WIN55212). This displays CP-55,940-like and
HU-210-like relative intrinsic activity at both CB1 and CB2
receptors. However, unlike 2 and 4, it has been found in some
investigations to possess slightly higher CB2 than CB1 affinity.
(2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone 7
(JWH-015), R-3-(2-iodo-5-nitrobenzoyl)-1-methyl-2-piperidi-
nylmethyl)-1H-indole 8 (AM1241), and methyl-1-[2-(4-
morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-
methanone 9 (AM630) are very important members of the
aminoalkylindoles, which are frequently used as pharmaco-
logical tools.4,19−21
Finally, the diarylpyrazole N-[(1S)-endo-1,3,3-
trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphen-
yl)-1-(4-methylbenzyl)pyrazole-3-carboxamide 10 (SR144528)
is one of the more potent compounds for blocking CB2 rather
than for CB1 receptor activation. It displays much higher
affinity for CB2 than CB1 receptors and blocks agonist-induced
CB2 receptor activation in a competitive manner.17−19
In recent years, structurally diverse CB2 selective agonists
exhibiting analgesic activity in various pain models have been
discovered and recently reviewed.22−24 These CB2 receptor
agonists include 4-oxo-1,4-dihydroquinoline-3-carboxamide
(general structure A, Chart 2) and 1,8-naphthyridine-4(1H)-
one-3-carboxamide derivatives (general structure B, Chart 2)
that have been described as structurally diverse ligands
endowed with high affinity and selectivity toward the CB2
receptor (Chart 2). Some of these analogues were demon-
strated to act as agonists or inverse agonists in functional
activity assays, depending on the nature of the substituents on
the different positions of the heterocyclic scaffold.25−27 In light
of these considerations and following these previous findings,
our group recently reported a series of oxazinoquinolone
derivatives (general structure C, Chart 2) with high affinity and
selectivity at the CB2 receptor.28 All of these new compounds
behaved as CB2 full agonists.
Taking into consideration these findings and as part of a new
research program to identify novel CB2 agonists, we designed a
1099
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
Chart 2. General Structures of 4-Oxo-1,4-dihydroquinoline-
3-carboxamide Derivatives (A), 1,8-Naphthyridine-4(1H)-
on-3-carboxamide (B), 4-Oxo-oxazinoquinoline (C), and the
New Synthesized Compounds 16−57
CHEMISTRY
■
The synthetic routes to obtain the target 4-oxo-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide derivatives 16−47 are
outlined in Scheme 1. All substituents are summarized in Table
1. The required aminopyrazoles 11a−f were prepared by
known procedures (see Experimental Section).
a
Scheme 1
new chemical structure to investigate the biological activity
toward cannabinoid receptors. Initially, our goal was to
synthesize a series of 4-oxo-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide derivatives to evaluate the effect of replacing the
phenyl ring of general structures A or B with different 1-
methyl-5-substituted pyrazolo nuclei (Chart 2). Indeed, the
condensed benzene ring of 4-oxo-1,4-dihydroquinoline-3-
carboxamide derivatives was replaced with the pyrazolo
nucleus, resulting in the 4,7-dihydro-4-oxo-1H-pyrazolo[3,4-
b]pyridine-5-carboxamides. Aliphatic or aromatic carboxamide
groups in position 5 have been selected on the basis of other
cannabinoid pharmacophores, such as those present in the
quinolone, naphthyridine, and oxazinoquinolone derivatives. A
hydrophobic substituent in position 7 was preferred, while
position 3 of the pyrazole ring was investigated with different
alkyl or aryl moieties.
a
Reagents: (a) diethyl ethoxymethylenemalonate, 120 °C, 2 h; (b)
diphenyl ether, 250 °C, 3 h; (c) NaOH 10%, 6 h, rt; (d) amine, EDC,
HOBt, DMF, 16 h, rt or amine, HBTU, DIEA, DMF, 16 h, rt; (e)
K2CO3, DMF, alkyl bromide, 16 h, 100 °C.
Subsequentely, we designed additional CB2 ligands by
replacing of the pyrazolo ring with different heterocycles via a
combination of structure−activity relationships (SARs). The 7-
pentylisoxazolo[5,4-b]pyridine, 7-pentylthieno[2,3-b]pyridine,
and 4-pentyl[1,2,4]triazolo[1,5-a]pyrimidine carboxamides (as
illustrated in Chart 2) were found to be potent CB2 receptor
ligands, and the consequences of such modifications deeply
affected the functionality of these compounds. These newly
synthesized compounds were tested in competition binding
assays toward both rat CB1 (rCB1) and rat CB2 (rCB2)
receptors expressed in native tissues as rat brain or spleen and
toward human CB1 (hCB1) and CB2 (hCB2) receptors
expressed in CHO cells. Affinity data (Ki, nM) were used to
calculate the selectivity of these novel compounds for the CB2
receptors. Most of these ligands were also examined in cyclic
AMP assays on hCB2 CHO cells, with the aim of evaluating the
potency values (EC50, nM) and efficacy (Emax, %) relative to the
efficacy of the reference compound WIN 55,212 (10 μM)
attributed as 100%.
Replacement of the ethoxy group of diethyl ethoxymethy-
lenemalonate by the 5-amino function of the appropriate
pyrazoles 11a−f afforded the intermediate diethyl 5-pyrazoly-
laminomethylenemalonates 12a−f, which were cyclized in
excellent yields, giving the ethyl 3-substituted-4-oxo-1-methyl-
4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylates 13a−
f.29 In a first attempt, alkylation at N7 in anhydrous DMF in
the presence of K2CO3 with appropriate alkyl bromides was
performed on the esters 13a−f, yielding 1:1 mixtures of the N-/
O-alkylated compounds. To increase the regioselectivity of N7
alkylation, intermediates 13a−f were transformed to the
corresponding 4-oxo-4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-
5-carboxylic acids 14a−f30 by hydrolysis in 10% aqueous
NaOH. Subsequently, the amides 15a−w were obtained by a
coupling reaction mediated by an acid-activating agent between
selected amines and the 4-oxopyrazolo[3,4-b]pyridinecarboxylic
acids 14a−f. Alkylation of the resulting 4-oxo-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carboxamides 15a−w yielded the N-
1100
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
Table 1. Affinity (Ki, nM) and Selectivity Index (SI) on Rat and Human CB1 and CB2 Receptors of the Novel CB Compounds
a
16−57
Ki (nM)
hCB1/hCB2
b
c
d
e
compd
R
R1
R2
rCB1
rCB2
hCB1
hCB2
4.95 0.43
85
SI
6
R-(+)-WIN 55,212-2
14.4 1.3
7.73 0.68
100 12
11.3 1.2
2.3
5.9
9.1
1.8
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
pentyl
butyl
propyl
allyl
1420 138
1533 145
832 78
500 49
9
145 13
1256 127
758 72
138 12
593 18
412 38
>10000 (27%)
>10000 (10%)
>10000 (25%)
3220 310
4560 428
>10000 (38%)
>10000 (13%)
1040 95
>10000 (46%)
>10000 (1%)
>10000 (5%)
480 32
>10000 (34%)
>10000 (14%)
>10000 (28%)
3870 322
4248 415
>10000 (35%)
>10000 (16%)
1124 126
4568 422
723 63
>10000 (42%)
>10000 (1%)
>10000 (11%)
325 33
2-ethoxyethyl
4-cyanobutyl
hexyl
12
benzyl
1100 58
>10000 (29%)
>10000 (32%)
508 22
956 94
4.4
4-CH3-benzyl
2-(morpholin-4-yl)ethyl-
pentyl
>10000 (33%)
>10000 (36%)
372 35
cyclohexyl-CH2
trans-4-CH3-cyclohexyl
cis-4-CH3-cyclohexyl
cycloheptyl
3
pentyl
4800 570
750 73
13.3 1.5
11.8 1.3
11.4 1.2
401
68
pentyl
10.6 1.1
pentyl
1620 165
832 82
35
3
252 22
11.2 1.2
23
adamant-1-yl
3,5-di-Me-adamant-1-yl
1,4-di-Me-pentyl
phenyl
pentyl
11.4 1.7
16.7 1.8
262 25
800 84
9.51 0.93
9.83 1.01
212 20
84
pentyl
3260 352
2256 216
1020 95
1050 95
107
8.5
1.7
pentyl
1795 164
865 82
pentyl
740 38
524 53
4-OCH3-phenyl
4-F-phenyl
pentyl
>10000 (46%)
>10000 (48%)
>10000 (42%)
1120 126
2670 253
>10000 (19%)
3430 322
5650 525
5200 550
5342 493
>10000 (18%)
>10000 (1%)
>10000 (15%)
>10000 (12%)
187 15
>10000 (24%)
>10000 (27%)
4517 188
516 25
>10000 (38%)
>10000 (44%)
>10000 (38%)
957 92
>10000 (33%)
>10000 (37%)
3652 355
469 42
pentyl
benzyl
pentyl
>2.7
2
cyclohexyl
pentyl
Et
adamant-1-yl
cyclohexyl
pentyl
97 12
440 35
37
4
12
t-Bu
t-Bu
H
pentyl
4033 260
417 33
>10000 (23%)
3753 385
5150 480
4550 435
4752 381
>10000 (23%)
>10000 (1%)
>10000 (22%)
>10000 (17%)
155 16
3526 227
363 34
>2.8
10
adamant-1-yl
cyclohexyl
pentyl
pentyl
250 20
237 21
22
H
cycloheptyl
pentyl
130 14
103 12
44
H
adamant-1-yl
cyclohexyl
pentyl
13.3 1.2
>10000 (1%)
>10000 (1%)
5017 188
4050 132
11.6 1.1
>10000 (1%)
>10000(1%)
3620 326
2830 264
8.74 0.92
18.5 1.7
432
Ph
pentyl
Ph
cycloheptyl
pentyl
p-Cl-Ph
p-Cl-Ph
cyclohexyl
pentyl
>2.8
>3.5
17.7
7.8
cycloheptyl
pentyl
cyclohexyl
38
50
26
4
4
3
cycloheptyl
184 19
145 13
adamant-1-yl
cyclohexyl
1495 153
1468 138
21
2
69.9
7.2
H
98
67
9
5
14.8 1.4
8.23 0.81
1.25 0.13
4.26 0.42
86
52
7
4
12.3 1.1
6.95 0.71
1.12 0.09
3.27 0.29
H
cycloheptyl
7.5
H
adamant-1-yl
adamant-1-yl
cyclohexyl
16.5 1.5
13.7 1.2
12.5
15.9
Me
H
52
nd
5
43
nd
4
f
f
f
f
nd
nd
H
adamant-1-yl
850 70
21
2
712 64
20
2
35.6
146
phenyl
adamant-1-yl
560 52
4.83 0.46
523 47
3.58 0.31
a
b
The data are espressed as the mean SEM of n = 4 independent experiments. Affinity was calculated by using [3H]CP-55,940 as radioligand on
c
d
rat brain for CB1 receptors. Affinity was calculated by using [3H]CP-55,940 as radioligand calculated on rat spleen for CB2 receptors. Affinity was
e
calculated by using [3H]CP-55,940 as radioligand calculated on human CB1 CHO membranes. Affinity was calculated by using [3H]CP-55,940 as
f
radioligand calculated on human CB2 CHO membranes nd = not determined.
substituted regioisomers as the major products (80%). This was
probably favored because of the stabilization of the pyridine-4-
one tautomeric form by intramolecular hydrogen bonding
between the NH of the amide group and the 4-carbonyl
function of the 1-alkyl-4-oxopyrazolo[3,4-b]pyridines.29 Alky-
1
lation at N7 was confirmed by 2D H NMR (ROESY) as
1101
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
a
evidenced by the presence of cross-peaks between H6 and N-
CH2 protons.
Scheme 4
By use of the general procedures shown in Scheme 1, starting
from 3-methyl-5-aminoisoxazole31 11g as depicted in Scheme
2, the target isoxazolopyridone derivatives 48−50 were
prepared.
a
Scheme 2
a
Reagents: (a) diethyl ethoxymethylenemalonate, CH3COOH, 3 h,
reflux; (b) K2CO3, DMF, 1-pentyl bromide, 16 h, 100 °C; (c) NaOH
10%, 6 h, rt; (d) amine, EDC, HOBt, DMF, 16 h, rt or amine, HBTU,
DIEA, DMF, 16 h, rt.
malonate in glacial acetic acid afforded 7-oxo-4,7-dihydro-1,2,4-
triazolo[1,5-a]pyrimidine-6-carboxylic acid ethyl esters 12k,l.35
Alkylation of the resulting ester at the N4 position with 1-
bromopentane in dry DMF and K2CO3 and subsequent
hydrolysis of the ester function afforded compounds 14k,l,
which were transformed into the target compounds 55−57 in
accordance with the previously described synthetic approaches.
a
Reagents: (a) diethyl ethoxymethylenemalonate, 120 °C, 2 h; (b)
diphenyl ether, 250 °C, 3 h; (c) K2CO3, DMF, 1-pentyl bromide, 100
°C, 16 h; (d) NaOH 10%, 6 h, rt; (e) amine, EDC, HOBt, DMF, 16 h,
rt or amine, HBTU, DIEA, DMF, 16 h, rt.
The thieno[2,3-b]pyridine-4-one derivatives were synthe-
sized according to the reported procedure shown in Scheme 3.
RESULTS AND DISCUSSION
■
a
Scheme 3
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 (Table 1). These findings suggest a
high degree of homology between the human and rat CB2
receptors and their respective binding sites. While translation of
these binding results from rodent to man will require a
consideration of numerous other factors, including receptor
distribution, signaling pathways, and ADME parameters,
evaluation in rat models of inflammatory and/or neuropathic
pain can be considered without undue concern for differences
in receptor affinity. As noted previously, we hypothesized that
replacement of the benzene nucleus of the 4-oxo-1,4-
dihydroquinoline-3-carboxamide system with a 1-methyl-5-
substituted pyrazolo nucleus would afford novel and selective
CB2 receptor agonists. The initial choice of the cyclo-
hexylcarboxamide group in position 5 and the 1-pentyl chain
substituent in position 7 (compound 16) was based upon the
binding results obtained for other cannabinoid pharmaco-
phores. This compound displayed modest affinity and slight
selectivity for the cannabinoid CB2 receptor (hCB2 Ki = 85 nM;
hCB1 Ki = 500 nM, SI = 5.9). Subsequently, the adopted
strategy was to prepare analogues by stepwise introduction of
structural modifications on positions 3, 5, and 7 of the
pyrazolopyridine template of compound 16 to define the
correct structural requirements for binding to the CB2
cannabinoid receptor.
a
Reagents: (a) diethyl ethoxymethylenemalonate, 2 h, 120 °C; (b)
KOH−EtOH, dioxane, 10 min, 70 °C, then 30 min, rt; (c) PPA, 120
°C, 1 h; (d) NaOH 10%, 6 h, rt; (e) amine, EDC, HOBt, DMF, 16 h,
rt or amine, HBTU, DIEA, DMF, 16 h, rt; (f) K2CO3, DMF, 1-pentyl
bromide, 16 h, 100 °C.
Reaction of aminothiophenes 11i,j,32 prepared from the
corresponding ketones using Gewald’s methodology, with
diethyl ethoxymethylenemalonate followed by selective alkaline
hydrolysis of the 3-ethyl ester gave the intermediates 13i,j.33
The synthesis of target compounds 51−54 was accomplished
using a procedure similar to that utilized for preparing the
pyrazolopyridinones 16−47.
The synthesis of 1,2,4-triazolo[1,5-a]pyrimidine-6-carboxa-
mide analogues 55−57 is outlined in Scheme 4. The starting
4H-1,2,4-triazol-3-amine 11k was available commercially, while
5-phenyl-4H-1,2,4-triazol-3-amine 11l was prepared by a
reported procedure.34 Refluxing with diethyl ethoxymethylene-
The structural modifications in position 7 consisted of a set
of nine compounds (17−25, Table 1) synthesized on the basis
of previously reported work.27,36 The 7-allyl derivative (19)
showed a complete loss of affinity for both CB receptors. The
same activity profile was found by introduction of an
ethoxyethyl or a cyanobutyl substituent (20 and 21,
respectively), revealing that the affinity is quite sensitive to
1102
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
modifications at the 7-position of the pyrazolopyridine nucleus.
Compounds 23 and 24, bearing benzyl and 4-methylbenzyl
substituents, respectively, also exhibited a marked decrease or a
complete loss of affinity at both CB receptors. Introduction of a
2-(morpholin-4-yl)ethyl chain (25), mimicking the morpholi-
noethyl substituent found in WIN-55,212-2 (6), also resulted in
an inactive compound at the CB2 and CB1 receptors. It is
important to highlight that the benzyl and 2-(morpholin-4-
yl)ethyl moieties are widely found in the structures of
cannabinoid ligands with agonist properties.25,27 Because the
n-pentyl residue appears to be preferred, subsequent
modifications of the 4-oxopyrazolopyridine-5-carboxamide
nucleus were performed while keeping this N7-substituent
constant.
The SAR studies were extended by examining modifications
of the cyclohexyl ring on the carboxamide moiety at the 5-
position. Compound 26, bearing a methylene spacer, showed 4-
fold lower affinity toward the CB2 receptor and lower selectivity
when compared with compound 16. Substitution of the
cyclohexylamide group at position 5 of the pyrazolopyridine
nucleus with 4-methylcyclohexylamide (compounds 27, 28)
afforded compounds with higher affinity at the CB2 receptor
(hKi of 11.4 and 10.6 nM, respectively). Indeed, these
compounds proved to have some of the highest CB2 receptor
affinities in this series. A stereogenic preference is observed
with the trans-isomer 27 exhibiting better selectivity against the
CB1 receptor (SI = 401) compared with the cis-isomer 28 (SI =
68). These data are not in accordance with results previously
reported by Tuccinardi et al.,36 that the cis-isomer of 4-
methylcyclohexylcarboxamides synthesized by them showed
increases of affinity for the CB2 receptor when compared with
the corresponding trans-isomers.
Replacing the cyclohexyl moiety of compound 16 with
cycloheptyl, adamantyl, or 3,5-dimethyladamantyl led to a
marked increase in affinity, as shown by compounds 29, 30, and
31. In addition to compounds 27 and 28, these were among the
most potent analogues tested, with comparable potencies (hKi
values of 11.2, 9.5, and 9.8 nM respectively), although the
highest selectivity among the three against the CB1 receptor
was observed with the most lipophilic compound 31 (SI =
107). Replacing the cyclohexyl moiety with a noncyclic, yet
lipophilic chain such as the (1,4-dimethylpentyl)carboxamide
induced a dramatic reduction in affinity toward CB2 receptors
(32, hKi = 212 nM), with a concomitant reduction in
selectivity.
Replacement of the cyclohexyl group of derivative 16 by the
aromatic phenyl moiety (33) resulted in a 6-fold decreased
affinity for the CB2 receptor. Substitution on the aromatic
moiety in the para-position led to a complete loss of affinity for
cannabinoid receptors, as shown by the 4-methoxyphenyl and
4-fluorophenyl derivatives (34, 35, respectively, hKi > 10 000
nM). Introduction of a methylene spacer between the
carboxamide nitrogen and the phenyl ring (compound 36)
afforded a similar result.
to decreased affinity (hKi values of 3526 and 363 nM,
respectively).
Replacing the methyl group at position 3 by a hydrogen
yielded compounds 41−43, bearing the cyclohexyl, cycloheptyl,
and adamantyl carboxamide groups, respectively. Although
these compounds showed decreased affinity for the CB2
receptor relative to the corresponding 3-methyl compounds
(16, 29, and 30, respectively), they each showed increased
selectivity, suggesting an exploitable difference in the bindings
sites of the CB2 and CB1 receptors. In particular, the adamant-
1-ylamide 43 was one of the most potent compounds evaluated
and the most selective compound in this series (hKi =11.6 nM,
SI = 432).
Aromatic substituents at the 3-position of the pyrazole, as in
compounds 44−47, led to a significant loss of affinity at both
the CB2 and CB1 receptors (Ki > 10 000 nM for 44 and 45).
Interestingly, the 3-p-chlorophenyl derivatives 46 and 47
displayed a slight affinity for the CB2 receptor. Compared to
the 3-phenyl analogues (44 and 45), this may suggest that the
steric intolerance at the 3-position can be at least partially
overcome by electronic effects.
Replacement of the pyrazolo ring of the parent nucleus with
differently substituted 5-membered heterocycles allowed us to
further extend these SAR studies. In particular various
isoxazolo, thiophene, and triazolo derivatives were prepared.
The isoxazolo[5,4-b]pyridines 48−50 each included an N7-
pentyl chain, a 3-methyl group, and a cyclohexyl, cycloheptyl, or
adamantyl susbstituent on the 5-carboxamide group, respec-
tively. Optimal affinity within this group of compounds was
obtained with the cyclohexyl moiety on the carboxamide chain
(compound 48, hCB2 Ki = 8.74 nM, SI = 17.7), which showed
significantly greater affinity and selectivity than the correspond-
ing 4-oxopyrazolopyridine derivative (16). In contrast, neither
the N-cycloheptyl (49) nor the N-adamantyl (50) derivatives
showed the same relative enhancement in affinity. Although
there was an apparent increase in selectivity for the 5-adamantyl
derivative 50 (hCB2 Ki = 21 nM, SI = 69.9), the cycloheptyl
derivative (49) displayed lower affinity and selectivity relative
to analogue 29 (hCB2 Ki = 18.5 nM, SI = 7.8 vs hCB2 Ki = 11.2
nM, SI = 23).
All of the thieno[2,3-b]pyridine derivatives (51−54) showed
high affinity at the CB receptors (hKi values from 1.12 to 12.3
nM for the CB2 receptor). In this group, the highest affinities
were obtained with the adamant-1-ylamides (compounds 53,
54 hKi = 1.12, 3.27 nM respectively), in analogy to the
pyrazolopyridine series, although there was an apparent
decrease in selectivity.
Evaluation of the 4-pentyl[1,2,4]triazolo[1,5-a]pyrimidine
carboxamides 55−57 showed further improvements in receptor
affinity and selectivity relative to the adamantyl derivatives 30,
38, 43, 50, 53, 54, although the compound with a
cyclohexylamide (55) lacked sufficient solubility in DMSO/
water mixtures to permit evaluation. In particular, the N-
adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-phenyl[1,2,4]-
triazolo[1,5-a]pyrimidine-6-carboxamide (compound 57)
proved to have the greatest affinity and selectivity in this
group for the CB2 receptor, with Ki = 3.58 nM and SI = 146.
We also evaluated the efficacy (Emax), expressed as a
percentage relative to that of WIN 55,212-2 representing the
full CB agonist with Emax of 100. The majority of the novel
compounds (from 17 to 43 and 56) showed moderate potency,
inhibiting adenylate cyclase activity with EC50 values between
41 and 3824 nM. Interestingly, most of these compounds reveal
Within this series of 4-oxopyrazolopyridine-5-carboxamides,
we also examined the effect of structural modifications at the 3-
position of the pyrazolopyridine nucleus on receptor affinity
and selectivity (compounds 37−47). The 3-ethyl analogues
bearing a cyclohexyl (37) or adamant-1-yl (38) carboxamide
displayed lower affinity and poor selectivity at the CB receptors
when compared with the parent 3-methyl compounds (16 and
30, respectively). Similarly, replacement of the 3-methyl group
with a bulky tert-butyl moiety, as in compounds 39 and 40, led
1103
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
Table 2. Potency (EC50, nM), Efficacies (Emax, %) in hCB2CHO Cells on Cyclic AMP Assays and CLogP of the Novel CB
Compounds 16−57
a
b
c
a
b
c
compd
hCB2 EC50 (nM)
Emax (%)
CLogP
compd
hCB2 EC50 (nM)
Emax (%)
CLogP
6
14.8 1.2
323 29
100
4.28
3.20
2.66
2.13
1.97
1.61
1.23
3.73
3.41
3.09
0.83
3.73
3.69
3.69
3.76
4.02
4.98
3.96
3.42
3.37
3.87
3.09
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
2348 227
162 14
32
2
4
3.73
4.55
4.42
5.25
2.74
3.30
3.56
4.34
4.91
4.91
5.48
2.69
3.25
3.51
4.50
5.07
5.33
5.79
2.12
2.94
5.38
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
52
38
5
3
51
1895 193
1764 167
872 79
404 42
43
48
56
67
3
5
5
6
587 52
58
4
41
4
1203 117
3824 392
27
36
2
3
1562 149
41
14
16
65
68
71
46
38
3
1
1
6
6
6
4
3
14.3 1.2
98
7
6
8
58
52
51
49
45
6
5
6
5
5
35
83
4
8
99
97
107 10
−64
−95
−167 15
−178 16
5
9
65
31
34
6
3
3
827 75
2159 248
87
9
60
5
a
b
EC50 values were calculated on cAMP experiments performed on human CB2 CHO cells. Efficacies (Emax) for CB2 of the examined compounds
are expressed as a percentage relative to the efficacy of the reference compound WIN 55,212-2 at the 10 μM. Emax of ∼100% indicated that the
compounds behave as full agonists. Emax between 0% and 100% indicated that the compounds behave as partial agonists. Negative Emax indicated that
c
the compounds behave as inverse agonists. CLogP (calculated logarithm of the partition coefficient between n-octanol and water) values were
obtained using ACDLabs/ChemSketch.
Table 3. Comparison between Direct Analogues of Four Core Series
a low Emax activity, suggesting they act as partial agonists (Table
1). As can be seen from Tables 2 and 3, the different structural
classes display different behaviors as full agonist, partial agonist,
and inverse agonist. The pyrazolopyridines 16−47 and
1104
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
protein/100 μL and different concentrations (1 nM to 10 μM) of
examined compounds.38
triazolopyrimidine 56 were found to act as partial agonists,
while the thienopyridines 51−54 act as inverse agonists.
Interestingly, the isoxazolopyridines 48−50 act as full agonists.
Thus, it appears that the core structure of these molecules
defines the type of activity to be seen, while the substituents
around the core structure can be used to modulate the potency
of that activity. This finding is unexpected in light of the recent
findings of Pasquini, et al., where substituted 6-arylquinolones
and 5-arylindoles were compared as ligands of the CB2
receptor. In this case, the authors suggested that the two
heterocyclic systems simply act as scaffolds to simply orient the
substituents in 3D space, with both classes of compounds
exerting the same functional effects.37 The calculation of log P
of the synthesized compounds (Table 2) has indicated the
absence of a significant correlation between the lipophilicity
and biological activities of this series.
Human CB1 and CB2 receptors expressed in CHO cells 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.39−41 For
membrane preparation, 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, 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 receptors, respectively.
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
CONCLUSION
■
The purpose of this study was to design and synthesize a novel
chemical class of compounds as selective ligands for CB2
receptor. In this effort, we have discovered a new series of
heteroarylpyridine/heteroarylpyrimidine derivatives that were
tested on CB1 and CB2 receptors. In light of the results
presented, this represents a new class of heterocyclic derivatives
acting as potent CB2 receptor partial agonists. The best affinity
values were obtained with compounds 30, 31, 48, 52−54 (1.12
nM < hCB2 Ki < 9.83 nM) bearing a lipophilic carboxamido
substituent. Compounds containing aromatic moieties on the
amide resulted in a decreased affinity for the CB2 cannabinoid
receptors, as shown by 33−36. The preferred N7 side chain
was the 1-pentyl moiety. Taken together, the present data could
be a suitable starting point for the optimization of novel CB2
receptor ligands as high quality leads.
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 to 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 phosphodiester-
ase inhibitor was added and the mixture preincubated for 10 min in a
shaking bath at 37 °C.42 The potency of the novel CB compounds was
studied in the presence of 1 μM forskolin 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. After the addition of
charcoal, the samples 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.).
Data Analysis. The protein concentration was determined
according to a Bio-Rad method with bovine albumin as reference
standard. Inhibitory binding constants, Ki, were calculated from the
EC50 values according to the Cheng and Prusoff equation:43 Ki =
EC50/(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,44 was used for computer
analysis of the inhibition experiments. All the data are expressed as the
mean SEM of n = 4 independent experiments for the in vitro assays.
Statistical analysis of the data was performed using unpaired two-sided
Student’s t test.
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
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.38
1
Chemistry. H NMR spectra were recorded on a Bruker AC 200
To investigate CB2 receptors, [3H]CP-55,940 binding assay was
performed by using 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, 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
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
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
1105
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
Microanalytical Laboratory of the Chemistry Department 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). The 5-aminopyrazoles were
prepared by known procedures: 1,3-dimethyl-1H-pyrazol-5-amine
(11a),45 3-ethyl-1-methyl-1H-pyrazol-5-amine (11b),46 3-tert-butyl-1-
methyl-1H-pyrazol-5-amine (11c),47 1-methyl-3-phenyl-1H-pyrazol-5-
amine (11d),48 3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-amine
(11e),49 1-methyl-1H-pyrazol-5-amine (11f).30
Synthesis of 2-[(Pyrazol-3-ylamino)methylene]malonic Acid
Diethyl Ester (12a−f) and 2-[(Isoxazol-5-ylamino)methylene]-
malonic Acid Diethyl Ester (12g). General Procedure. A mixture of
the appropriate pyrazol-5-amines 11a−f or isoxazol-5-amine 11g (10
g, 0.09 mol) and diethyl ethoxymethylenemalonate (19.4 g, 0.09 mol)
was heated at 120 °C for 2 h with stirring. The reaction mixture was
poured into n-hexane, and the precipitate was collected by filtration to
give the desired compounds 12a−g.
vacuo and the precipitate was collected by filtration and purified by
crystallization (ethanol/water) to yield 12i (93%) and 12j (95%) as
yellow solids.
1
12i. Mp 186−187 °C. H NMR (DMSO-d6): δ 13.38 (bs, 1H),
12.34 (bd, J = 13.4 Hz, 1H), 8.08 (d, J = 13.6 Hz, 1H), 6.72 (q, 4J = 1
Hz, 1H), 4.28−4.09 (m, 4H), 2.29 (d, 4J = 1.2 Hz, 3H), 1.30−1.20 (m,
6H).
1
12j. Mp 90 °C (dec). H NMR (DMSO-d6): δ 13.25 (bs, 1H),
12.25 (d, J = 13.5 Hz, 1H), 8.15 (d, J = 13.6 Hz, 1H), 4.34−4.25 (m,
4H), 2.28 (s, 6H), 1.39−1.29 (m, 6H).
Synthesis of Ethyl 4,7-Dihydro-7-oxo[1,2,4]triazolo[1,5-a]-
pyrimidine-6-carboxylate (12k) and Ethyl 4,7-Dihydro-7-oxo-
2-phenyl[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12l). A
mixture of 11k or 11l34 (3.1 mmol), glacial acetic acid (5 mL), and
diethyl ethoxymethylene malonate (1 g, 4.6 mmol) was heated at
reflux for 3 h. After the mixture was cooled, the white precipitate was
collected by filtration, washed with ice-cold water, and dried to give
the corresponding ester compounds 12k,l (75%) as a white crystalline
powder.
1
12k. Mp > 300 °C. H NMR (DMSO-d6): δ 14.30 (bs, 1H), 8.95
(s, 1H), 8.84 (s, 1H), 4.20 (q, J = 6.8 Hz, 2H), 1.29 (t, J = 6.8 Hz,
3H).
12l. Mp > 300 °C. 1H NMR (DMSO-d6): δ 14.00 (bs, 1H), 8.64 (s,
1H), 8.14−8.10 (m, 2H), 7.55 (m, 3H), 4.26 (q, J = 7.0 Hz, 2H), 1.28
(t, J = 6.8 Hz, 3H).
Diethyl 2-[(1,3-Dimethyl-1H-pyrazol-5-ylamino)methylene]-
malonate (12a). Yield: 86%. 1H NMR (DMSO-d6): δ 10.50 (bd, J =
13.4 Hz, 1H), 8.79 (d, J = 13.4 Hz, 1H), 6.06 (s, 1H), 4.23 (q, J = 7.2
Hz, 2H), 4.09 (q, J = 7.2 Hz, 2H), 3.63 (s, 3H), 2.10 (s, 3H), 1.30 (t, J
= 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H).
Ethyl 4-Oxo-4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-car-
boxylates (13a−f) and Ethyl 4,7-Dihydro-3-methyl-4-
oxoisoxazolo[5,4-b]pyridine-5-carboxylate (13g). General Pro-
cedure. A solution of the appropriate diethyl [(pyrazol-5-ylamino)-
methylene]malonate 12a−f or diethyl [(isoxazol-5-ylamino)-
methylene]malonate 12g (8.89 mmol) in diphenyl ether (4 mL)
was heated at 250 °C for 3 h. After the mixture was cooled to room
temperature, the ethanol formed was removed under reduced pressure,
leaving the crude product as an oily residue. The residue was purified
by column chromatography on silica using a mixture of ethyl acetate/
petroleum ether as eluent.
Diethyl 2-[(3-Ethyl-1-methyl-1H-pyrazol-5-ylamino)-
1
methylene]malonate (12b). Yield: 82%. H NMR (DMSO-d6): δ
11.00 (bd, J = 12.8 Hz, 1H), 8.15 (d, J = 12.8 Hz, 1H), 5.87 (s, 1H),
4.27 (m, 4H), 3.79 (s, 3H), 2.57 (q, J = 7.6 Hz, 2H), 1.36−1.17 (m,
9H).
Diethyl 2-[(3-tert-Butyl-1-methyl-1H-pyrazol-5-ylamino)-
1
methylene]malonate (12c). Yield: 50%. H NMR (DMSO-d6): δ
Ethyl 1,3-Dimethyl-4,7-dihydro-4-oxo-1H-pyrazolo[3,4-b]-
10.98 (bd, J = 12.6 Hz, 1H), 8.12 (d, J = 12.6 Hz, 1H), 5.90 (s, 1H),
4.27 (m, 4H), 3.79 (s, 3H), 1.43 (s, 9H), 1.39−1.22 (m, 6H).
Diethyl 2-[1-Methyl-1H-pyrazol-5-ylamino)methylene]-
pyridine-5-carboxylate (13a). White solid, mp 110 °C. Yield:
1
65%. H NMR (CDCl3): δ 12.20 (bs, 1H), 8.81 (s, 1H), 4.44 (q, J =
7.0 Hz, 2H), 4.01 (s, 3H), 2.65 (s, 3H), 1.43 (t, J = 7.0 Hz, 3H).
Ethyl 3-Ethyl-4,7-dihydro-1-methyl-4-oxo-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate (13b). White solid, mp 77 °C. Yield:
1
malonate (12d). Yield: 77%. H NMR (DMSO-d6): δ 10.49 (bd, J
= 12.6 Hz, 1H), 7.98 (bd, J = 12.6 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H),
6.28 (d, J = 2.0 Hz, 1H), 4.12 (m, 4H), 4.01 (s, 3H), 1.27−1.13 (m,
6H).
1
82%. H NMR (CDCl3): δ 12.24 (bs, 1H), 8.81 (s, 1H), 4.42 (q, J =
7.2 Hz, 2H), 4.01 (s, 3H), 3.04 (q, J = 7.6 Hz, 2H), 1.46−1.23 (m,
6H).
Diethyl 2-[(3-Phenyl-1-methyl-1H-pyrazol-5-ylamino)-
1
methylene]malonate (12e). Yield: 77%. H NMR (DMSO-d6): δ
Ethyl 3-tert-Butyl-4,7-dihydro-1-methyl-4-oxo-1H-pyrazolo-
[3,4-b]pyridine-5-carboxylate (13c). White solid, mp 156 °C.
10.56 (bs, 1H), 8.07 (s, 1H), 7.81−7.77 (m, 2H), 7.34−7.29 (m, 3H),
6.82 (s, 1H), 4.16 (m, 4H), 3.77 (s, 3H), 1.25 (m, 6H).
Diethyl 2-[(3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-
ylamino)methylene]malonate (12f). Yield: 87%. 1H NMR
(DMSO-d6): δ 10.60 (bs, 1H), 8.12 (bs, 1H), 7.80 (dd, J = 8.6 Hz,
2H), 7.45 (dd, J = 6.8 Hz, 2H), 6.85 (s, 1H), 4.12 (m, 4H), 3.76 (s,
3H), 1.24−1.19 (m, 6H).
1
Yield: 85%. H NMR (CDCl3): δ 12.24 (bs, 1H), 8.71 (s, 1H), 4.40
(q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 1.43 (s, 9H), 1.32 (m, 3H).
Ethyl 4,7-Dihydro-1-methyl-4-oxo-1H-pyrazolo[3,4-b]-
pyridine-5-carboxylate (13d). White solid, mp 139 °C. Yield:
1
48%. H NMR (DMSO-d6): δ 12.55 (bs, 1H), 8.73 (s, 1H), 8.25 (s,
1H), 4.37 (q, J = 7.2 Hz, 2H), 4.00 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-Dihydro-1-methyl-4-oxo-3-phenyl-1H-pyrazolo-
[3,4-b]pyridine-5-carboxylate (13e). Pale white solid, mp 110
°C. Yield: 65%. 1H NMR (DMSO-d6): δ 12.60 (bs, 1H), 8.76 (s, 1H),
8.05−8.00 (m, 2H), 7.48−7.42 (m, 3H), 4.44 (q, J = 7 Hz, 2H), 4.05
(s, 3H), 1.37 (t, J = 7.2 Hz, 3H).
Diethyl 2-[(3-Methylisoxazol-5-ylamino)methylene]-
1
malonate (12g). Yield: 90%. H NMR (DMSO-d6): δ 11.09 (bs,
1H), 8.10 (s, 1H), 5.95 (s, 1H), 4.23−4.12 (m, 4H), 2.17 (s, 3H),
1.28−1.20 (m, 6H).
Synthesis of (E)-2-((3-Ethoxy-2-(ethoxycarbonyl)-3-oxo-
prop-1-en-1-yl)amino)-4-methylthiophene-3-carboxylic Acid
(12i) and (E)-2-((3-Ethoxy-2-(ethoxycarbonyl)-3-oxoprop-1-
en-1-yl)amino)-4,5-dimethylthiophene-3-carboxylic Acid
(12j). A mixture of 11i or 11j32 (5.4 mmol) and diethyl
ethoxymethylenemalonate (1.4 g, 6 mmol) was stirred at 120 °C for
2 h. After being cooled, the mixture was triturated with Et2O. The
yellow precipitate was collected by filtration, washed with ice-cold
Et2O, and dried to give the corresponding triester compounds (85%)
as yellow crystalline powders. A solution of KOH (1.5 g, 26.7 mmol)
in EtOH (10 mL) was added to a solution of the triester compound in
dioxane (15 mL) at 70 °C. After being stirred at 70 °C for 10 min, the
mixture was stirred at room temperature for a further 30 min, then
acidified with 2 N HCl. The resulting suspension was concentrated in
Ethyl 3-(4-Chlorophenyl)-4,7-dihydro-1-methyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (13f). White solid, mp
1
172−173 °C. Yield: 72%. H NMR (CDCl3): δ 12.55 (bs, 1H), 8.75
(s, 1H), 8.05 (dd, J = 8.6 Hz, 2H), 7.48 (dd, J = 8.6 Hz, 2H), 4.35 (q, J
= 7.2 Hz, 2H), 3.99 (s, 3H), 1.30 (m, 3H).
Ethyl 4,7-Dihydro-3-methyl-4-oxoisoxazolo[5,4-b]pyridine-
1
5-carboxylate (13g). Pale yellow solid, mp 151 °C. Yield 45%. H
NMR (DMSO-d6): δ 12.40 (bs, 1H), 8.80 (s, 1H), 4.39 (q, J = 7.0 Hz,
2H), 2.58 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H).
Synthesis of Oxothienopyridine Carboxylate Derivatives
(13i−j). General Procedure. A mixture of the required diethyl ester
(12i or 12j, 6 mmol) and polyphosphoric acid (10 g) was heated at
1106
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
140 °C for 1 h. The mixture was poured into ice and water to form a
precipitate that was collected by filtration and washed with cold water.
Ethyl 4,7-Dihydro-3-methyl-4-oxothieno[2,3-b]pyridine-5-
carboxylate (13i). White solid, mp 189−190 °C (dec). Yield: 65%.
1H NMR (DMSO-d6): δ 12.04 (bs, 1H), 8.87 (s, 1H), 7.22 (q, 4J = 1.4
propyl]-3-ethylcarbodiimide (0.52 mmol), and 1-hydroxybenzotriazole
(0.52 mmol) in anhydrous dimethylformamide (5 mL) was stirred at
room temperature for 10 min. The appropriate amine (0.72 mmol)
was added, and stirring continued for 16 h at ambient temperature.
The reaction mixture was concentrated in vacuo, then purified by
column chromatography on silica gel, eluting with ethyl acetate/
methanol unless otherwise noted.
Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 2.52 (d, 4J = 1.4 Hz, 3H), 1.35 (t, J
= 7.0 Hz, 3H).
Ethyl 4,7-Dihydro-2,3-dimethyl-4-oxothieno[2,3-b]pyridine-
5-carboxylate (13j). White solid, mp 178−180 °C (dec). Yield: 60%.
1H NMR (CDCl3): δ 12.01 (bs, 1H), 8.77 (s, 1H), 4.46 (q, J = 7.2 Hz,
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-4-oxo-1H-pyrazolo-
[3,4-b]pyridine-5-carboxamide (15a). White solid, mp 105 °C.
Yield: 73%. 1H NMR (DMSO-d6): δ 12.90 (bs, 1H), 10.10 (bd, J = 7.8
Hz, 1H), 8.36 (s, 1H), 3.95 (m, 1H), 3.81 (s, 3H), 2.40 (s, 3H), 1.82−
1.46 (m, 10H).
2H), 2.51 (s, 3H), 2.43 (s, 3H), 1.44 (t, J = 7.0 Hz, 3H).
Synthesis of Carboxylic Acid Derivatives (14a−f, 14h−l).
General Procedure. A suspension of the appropriate carboxylic acid
ethyl ester 13a−f, 13i−1, 14g (1.27 mmol) in 20 mL of 10% aqueous
sodium hydroxide was stirred at room temperature for 6 h. After
cooling, the suspension was adjusted to pH 4 with 2 N HCl. The
resulting precipitate was collected by filtration, washed with H2O, and
recrystallized from ethanol/water.
N-(Cyclohexylmethyl)-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15b). White solid, mp
1
200 °C. Yield: 36%. H NMR (DMSO-d6): δ 12.30 (bs, 1H), 10.10
(bt, J = 7.8 Hz, 1H), 8.42 (s, 1H), 3.83 (s, 3H), 3.81 (m, 1H), 3.14 (t, J
= 6.6 Hz, 2H), 2.45 (s, 3H), 1.73−1.18 (m, 10H).
trans-4,7-Dihydro-1,3-dimethyl-N-(4-methylcyclohexyl)-4-
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15c) and cis-
4,7-Dihydro-1,3-dimethyl-N-(4-methylcyclohexyl)-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15d). The separation of
cis and trans isomers was obtained by flash chromatography on silica
gel using ethyl acetate/petroleum ether (9:1) as eluent.
4,7-Dihydro-1,3-dimethyl-4-oxo-1H-pyrazolo[3,4-b]-
pyridine-5-carboxylic Acid (14a). White solid, mp 237−238 °C.
1
Yield: 99%. H NMR (DMSO-d6): δ 15.30 (bs, 1H), 12.23 (bs, 1H),
8.54 (s, 1H), 3.90 (s, 3H), 2.48 (s, 3H).
4,7-Dihydro-3-ethyl-1-methyl-4-oxo-1H-pyrazolo[3,4-b]-
1
15c. Trans-isomer. White solid, mp 270 °C (dec). Yield: 22%. H
pyridine-5-carboxylic Acid (14b). White solid, mp 235−236 °C.
1
NMR (DMSO-d6): δ 13.02 (bs, 1H), 9.92 (bd, J = 7.8 Hz, 1H), 8.40
(s, 1H), 3.82 (s, 3H), 3.68 (m, 1H), 2.43 (s, 3H), 1.89−1.64 (m, 4H),
1.32−0.96 (m, 5H), 0.86 (d, J = 6.4 Hz, 3H).
Yield: 80%. H NMR (DMSO-d6): δ 15.80 (bs, 1H), 12.53 (bs, 1H),
8.51 (s, 1H), 3.94 (s, 3H), 2.89 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz,
3H).
3-tert-Butyl-4,7-dihydro-1-methyl-4-oxo-1H-pyrazolo[3,4-b]-
pyridine-5-carboxylic Acid (14c). White solid, mp 225 °C. Yield:
95%. 1H NMR (DMSO-d6): δ 15.90 (bs, 1H), 13.83 (bs, 1H), 8.59 (s,
1H), 3.91 (s, 3H), 1.41 (s, 9H).
4,7-Dihydro-1-methyl-4-oxo-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic Acid (14d). White solid, mp 236 °C (dec). Yield: 92%.
1H NMR (DMSO-d6): δ 15.10 (bs, 1H), 12.55 (bs, 1H), 8.65 (s, 1H),
1
15d. Cis-isomer. White solid, mp 252 °C (dec). Yield: 25% H
NMR (DMSO-d6): δ 13.02 (bs, 1H), 10.27 (bd, J = 7.8 Hz, 1H), 8.39
(s, 1H), 4.03 (m, 1H), 3.82 (s, 3H), 2.45 (s, 3H), 1.68−1.51 (m, 7H),
1.19−1.12 (m, 2H), 0.90 (d, J = 6.4 Hz, 3H).
N-Cycloheptyl-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15e). Pale white solid,
1
mp 249 °C (dec). Yield: 65%. H NMR (DMSO-d6): δ 12.90 (bs,
1H), 10.10 (bd, J = 7.8 Hz, 1H), 8.40 (s, 1H), 4.20 (m, 1H), 3.83 (s,
3H), 2.45 (s, 3H), 1.90−1.54 (m, 12H).
4,7-Dihydro-1,3-dimethyl-N-(5-methylhexan-2-yl)-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15h). White solid, mp
289 °C. Yield: 25%. 1H NMR (DMSO-d6): δ 13.20 (bs, 1H), 9.88 (bd,
J = 7.8 Hz, 1H), 8.43 (s, 1H), 3.90 (m, 1H), 3.82 (s, 3H), 2.51 (s, 3H),
1.51−1.41 (m, 3H), 1.11−1.20 (m, 5H), 0.87 (m, 6H).
4,7-Dihydro-1,3-dimethyl-4-oxo-N-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (15i). White solid, mp 273 °C. Yield:
32%. 1H NMR (DMSO-d6): δ 13.20 (bs, 1H), 12.40 (bs, 1H), 8.54 (s,
1H), 7.72−7.76 (m, 2H), 7.38−7.30 (m, 2H), 7.10−7.06 (m, 1H),
3.88 (s, 3H), 2.54 (s, 3H).
8.19 (s, 1H), 4.00 (s, 3H).
4,7-Dihydro-1-methyl-4-oxo-3-phenyl-1H-pyrazolo[3,4-b]-
pyridine-5-carboxylic Acid (14e). Pale yellow solid, mp 235 °C.
Yield: 78%. 1H NMR (DMSO-d6): δ 14.51 (bs, 1H), 12.60 (bs,
1H),8.67 (s, 1H), 8.22−8.18 (m, 2H), 7.48−7.41 (m, 3H), 4.04 (s,
3H).
3-(4-Chlorophenyl)-4,7-dihydro-1-methyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxylic Acid (14f). White solid, mp
> 300 °C. Yield: 88% . 1H NMR (DMSO-d6): δ 15.30 (bs, 1H), 12.50
(bs, 1H), 8.64 (s, 1H), 8.28 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz,
2H), 4.05 (s, 3H).
4,7-Dihydro-3-methyl-4-oxo-7-pentylisoxazolo[5,4-b]-
pyridine-5-carboxylic Acid (14h). White solid, mp 155 °C. Yield:
88%. 1H NMR (DMSO-d6): δ 14.78 (bs, 1H), 8.91 (s, 1H), 4.36 (t, J
= 7 Hz, 2H), 2.56 (s, 3H), 1.85−1.78 (m, 2H), 1.30−1.27 (m, 4H),
0.86 (t, J = 6.8 Hz, 3H).
4,7-Dihydro-1,3-dimethyl-N-(4-methoxyphenyl)-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15j). White solid, mp
1
171 °C. Yield: 40%. H NMR (DMSO-d6): δ 13.12 (bs, 1H), 12.35
(bs, 1 H), 8.54 (s, 1H), 7.63 (dd, J = 9 Hz, 2H), 6.92 (dd, J = 9 Hz,
2H), 3.87 (s, 3H), 3.74 (s, 3H), 2.50 (s, 3H).
4,7-Dihydro-3-methyl-4-oxothieno[2,3-b]pyridine-5-carbox-
1
ylic Acid (14i). White solid, mp 260−261 °C (dec). Yield: 89%. H
4,7-Dihydro-1,3-dimethyl-N-(4-fluorophenyl)-4-oxo-1H-
NMR (DMSO-d6): δ 15.98 (bs, 1H), 14.01 (bs, 1H), 8.76 (s, 1H),
pyrazolo[3,4-b]pyridine-5-carboxamide (15k). White solid, mp >
4
4
1
7.15 (d, J = 1.2 Hz, 1H), 2.55 (d, J = 1.2 Hz, 3H).
300 °C. Yield: 40%. H NMR (DMSO-d6): δ ppm 13.12 (bs, 1H),
4,7-Dihydro-2,3-dimethyl-4-oxothieno[2,3-b]pyridine-5-car-
boxylic Acid (14j). White solid, mp 254−255 °C (dec). Yield: 80%.
1H NMR (DMSO-d6): δ 16.01 (bs, 1H), 13.95 (bs, 1H), 8.71 (s, 1H),
12.53 (bs, 1 H), 8.55 (s, 1H), 7.77−7.70 (m, 2H), 7.22−7.14 (m, 2H),
3.87 (s, 3H), 2.50 (s, 3H).
N-Benzyl-4,7-dihydro-1,3-dimethyl-4-oxo-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (15l). White solid, mp 185−186 °C.
2.46 (s, 3H), 2.37 (s, 3H).
1
Yield: 60%. H NMR (DMSO-d6): δ 13.20 (bs, 1H), 10.40 (bt, J = 6
4,7-Dihydro-7-oxo-4-pentyl[1,2,4]triazolo[1,5-a]pyrimidine-
6-carboxylic Acid (14k). White solid, mp 215 °C (dec). Yield: 75%.
1H NMR (DMSO-d6): δ 12.10 (bs, 1H), 8.74 (s, 1H), 8.24 (s, 1H),
Hz, 1H), 8.42 (s, 1H), 7.34−7.32 (m, 5H), 4.51 (d, J = 6.0 Hz, 2H),
3.84 (s, 3H), 2.45 (s, 3H).
N-Cyclohexyl-4,7-dihydro-3-ethyl-1-methyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15m). White solid, mp
297 °C. Yield: 38%. H NMR (DMSO-d6): δ 13.20 (bs, 1H), 10.10
4.43 (q, J = 7.0 Hz, 2H), 1.82 (m, 2H), 1.36−1.29 (m, 4H), 0.88 (t, J =
6.6 Hz, 3H).
1
4,7-Dihydro-7-oxo-4-pentyl-2-phenyl[1,2,4]triazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (14l). White solid, mp > 300 °C
(bd, J = 7.8 Hz, 1H), 8.42 (s, 1H), 3.85 (s, 3H), 3.80 (m, 1H), 2.83 (q,
J = 7.6 Hz, 2H), 1.65−1.29 (m, 10H), 1.22−1.18 (m, 3H).
1
(dec). Yield: 75%. H NMR (DMSO-d6): δ 12.90 (bs, 1H), 8.93 (s,
3-tert-Butyl-N-cyclohexyl-4,7-dihydro-1-methyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15o). White solid, mp
1H), 8.17 (m, 2H), 7.55 (m, 3H), 4.34 (q, J = 7.0 Hz, 2H), 1.89 (m,
2H), 1.36−1.29 (m, 4H), 0.88 (t, J = 6.6 Hz, 3H).
Synthesis of Carboxamide Derivatives (15a−e, 15h−m, 15o,
15q,r, 15t−w, 15aa,ab, 48, 49, 55). General Procedure. A solution
of the appropriate carboxylic acid (0.48 mmol), 1-[3-(dimethylamino)-
1
134−135 °C. Yield: 48%. H NMR (DMSO-d6): δ 13.00 (bs, 1H),
10.10 (bd, J = 7.8 Hz, 1H), 8.45 (s, 1H), 3.98 (m, 1H), 3.84 (s, 3H),
1.82−1.29 (m, 19H).
1107
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
N-Cyclohexyl-4,7-dihydro-1-methyl-4-oxo-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (15q). White solid, mp 97−99 °C.
Yield: 28%. 1H NMR (DMSO-d6): δ 13.30 (bs, 1H), 10.03 (bd, J = 7.6
Hz, 1H), 8.50 (s, 1H), 7.96 (s, 1H), 4.04 (m, 1H), 3.94 (s, 3H), 1.65−
1.24 (m, 10H).
N-Cycloheptyl-4,7-dihydro-1-methyl-4-oxo-1H-pyrazolo-
[3,4-b]pyridine-5-carboxamide (15r). White solid, mp 214 °C.
Yield: 40%. 1H NMR (DMSO-d6): δ 13.30 (bs, 1H), 10.03 (bd, J = 7.6
Hz, 1H), 8.50 (s, 1H), 7.96 (s, 1H), 3.95 (s, 3H), 3.92 (m, 1H), 1.74−
1.23 (m, 12H).
After removal of the dimethylformamide under reduce pressure, the
residue was dissolved in ethyl acetate (100 mL), then washed with
saturated aqueous sodium bicarbonate (20 mL), water (10 mL), and
brine (10 mL). The organic extract was dried (Na2SO4), filtered, and
evaporated to dryness, and the residue was purified by flash
chromatography on silica gel, eluting with ethyl acetate/petroleum
ether.
N-(Adamantan-1-yl)-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15f). White solid, mp
284−285 °C. Yield: 45%.1H NMR (DMSO-d6): δ 13.00 (bs, 1H),
10.23 (bs, 1H), 8.37 (s, 1H), 3.78 (s, 3H), 2.42 (s, 3H), 2.03 (s, 9H),
1.65 (s, 6H).
4,7-Dihydro-1,3-dimethyl-N-(3,5-dimethyladamantan-1-yl)-
4-oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15g). White
solid, mp 241−242 °C. Yield: 42%. 1H NMR (DMSO-d6): δ 13.10 (bs,
1H), 10.26 (bs, 1H), 8.25 (s, 1H), 3.97 (s, 3H), 2.63 (s, 3H), 1.98−
1.19 (m, 19H).
N-Cyclohexyl-4,7-dihydro-1-methyl-4-oxo-3-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15t). White solid, mp
1
235 °C. Yield: 64%. H NMR (DMSO-d6): δ 13.09 (bs, 1H), 10.10
(bd, J = 7.4 Hz, 1H), 8.53 (s, 1H), 8.21−8.17 (m, 2H), 7.50−7.38 (m,
3H), 4.00 (s, 3H), 3.81 (m, 1H), 1.83−1.30 (m, 10H).
N-Cycloheptyl-4,7-dihydro-1-methyl-4-oxo-3-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15u). White solid, mp
1
228 °C (dec). Yield: 50%. H NMR (DMSO-d6): δ 13.10 (bs, 1H),
N-(Adamantan-1-yl)-4,7-dihydro-3-ethyl-1-methyl-4-oxo-
10.15 (bd, J = 7.6 Hz, 1H), 8.49 (s, 1H), 8.21−8.18 (m, 2H), 7.45−
1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15n). White solid,
1
7.40 (m, 3H), 4.05−3.99 (m, 4H), 1.85−1.55 (m, 12H).
mp 279 °C. Yield: 37%. H NMR (DMSO-d6): δ 13.00 (bs, 1H),
3-(4-Chlorophenyl)-N-cyclohexyl-4,7-dihydro-1-methyl-4-
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15v). White
10.00 (s, 1H), 8.36 (s, 1H), 3.84 (s, 3H), 2.85 (q, J = 7.6 Hz, 2H), 2.04
(s, 9H), 1.65 (s, 6H), 1.21 (t, J = 7.6 Hz, 3H).
1
solid, mp149−150 °C. Yield: 45%. H NMR (DMSO-d6): δ 13.20
N-(Adamantan-1-yl)-3-tert-butyl-4,7-dihydro-1-methyl-4-
(bs, 1H), 10.06 (bd, J = 7.6 Hz, 1H), 8.50 (s, 1H), 8.24 (dd, J = 8.0
Hz, 2H), 7.52 (dd, J = 8.6 Hz, 2H), 3.95 (s, 3H), 3.79 (m, 1H), 1.87−
1.29 (m, 10H).
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15p). White
1
solid, mp 256−257 °C. Yield: 54%. H NMR (DMSO-d6): δ 13.00
(bs, 1H), 10.10 (bs, 1H), 8.40 (s, 1H), 3.83 (s, 3H), 2.05 (s, 9H), 1.66
(s, 6H), 1.40 (s, 9H).
3-(4-Chlorophenyl)-N-cycloheptyl-4,7-dihydro-1-methyl-4-
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (15w). White
N-(Adamantan-1-yl)-4,7-dihydro-1-methyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (15s). White solid, mp >
300 °C. Yield: 45%. 1H NMR (DMSO-d6): δ 12.97 (bs, 1H), 8.70 (s,
1H), 8.47 (bs, 1H), 7.58 (bs, 1H), 4.01 (s, 3H), 2.06 (s, 9H), 1.67 (s,
6H).
N-(Adamantan-1-yl)-4,7-dihydro-3-methyl-4-oxothieno[2,3-
b]pyridine-5-carboxamide (15ac). White solid, mp 243 °C (dec).
Yield: 57%. 1H NMR (400 MHz, DMSO-d6): δ 13.02 (bs, 1H), 10.15
(bs, 1H), 8.51 (s, 1H), 6.92 (q, J = 1.2 Hz, 1H), 2.52 (d, J = 1.2 Hz,
3H), 2.03 (m, 9H), 1.65 (s, 6H).
1
solid, mp 154−155 °C. Yield: 40%. H NMR (DMSO-d6): δ 13.22
(bs, 1H), 10.03 (bd, J = 7.8 Hz, 1H), 8.52 (s, 1H), 8.24 (dd, J = 8.4
Hz, 2H), 7.52 (dd, J = 8.4 Hz, 2H), 4.02 (s, 3H), 3.98−3.96 (m,1 H),
1.85−1.54 (m, 12H).
N-Cyclohexyl-4,7-dihydro-3-methyl-4-oxothieno[2,3-b]-
pyridine-5-carboxamide (15aa). White solid, mp 247 °C (dec).
1
Yield: 29%. H NMR (400 MHz, DMSO-d6) δ 13.12 (bs, 1H), 10.35
(bd, J = 7.8 Hz, 1H), 8.56 (s, 1H), 6.88 (q, J = 1.2 Hz, 1H), 3.91−3.78
(m, 1H), 2.52 (d, J = 1.2 Hz, 3H), 1.95−1.19 (m, 10H).
N-Cycloheptyl-4,7-dihydro-3-methyl-4-oxothieno[2,3-b]-
pyridine-5-carboxamide (15ab). White solid, mp 252 °C (dec).
Yield: 25%. 1H NMR (400 MHz, DMSO-d6): δ 13.17 (bs, 1H), 10.36
(bd, J = 8 Hz, 1H), 8.56 (s, 1H), 6.89 (q, J = 1.2 Hz, 1H), 4.01−3.97
(m, 1H), 2.52 (d, J = 1.2 Hz, 3H), 1.86−1.48 (m, 12H).
N-(Adamantan-1-yl)-4,7-dihydro-2,3-dimethyl-4-oxothieno-
[2,3-b]pyridine-5-carboxamide (15ad). White solid, mp 218 °C
(dec). Yield: 55%.1H NMR (DMSO-d6): δ 13.03 (bs, 1H), 10.25 (bs,
1H), 8.47 (s, 1H), 2.50 (s, 3H), 2.32 (s, 3H), 2.04 (m, 9H), 1.66 (s,
6H).
N - C y c l o h e x y l - 4 , 7 - d i h y d r o - 3 - m e t h y l - 4 - o x o - 7 -
pentylisoxazolo[5,4-b]pyridine-5-carboxamide (48). White
N-(Adamantan-1-yl)-4,7-dihydro-3-methyl-4-oxo-7-
pentylisoxazolo[5,4-b]pyridine-5-carboxamide (50). White
1
1
solid, mp 187 °C. Yield: 71%. MS: m/z 346.6 (M + H). H NMR
solid, mp 170 °C. MS: m/z 398.3 (M + H). Yield: 59%. H NMR
(DMSO-d6): δ 9.83 (bd, J = 8.2 Hz, 1H), 8.63 (s, 1H), 4.28 (t, J = 7.2
Hz, 2H), 3.82 (m, 1H), 2.52 (s, 3H), 1.85−1.22 (m, 16H), 0.85 (t, J =
6.8 Hz, 3H). Anal. (C19H27N3O3) C, H, N.
(DMSO-d6): δ 8.36 (s, 1H), 8.22 (bs, 1H), 4.38 (t, J = 6.4 Hz, 2H),
2.58 (s, 3H), 2.05 (m, 9H), 1.81 (m, 2H), 1.65 (m, 6H), 1.40−1.36
(m, 4H), 0.89 (t, J = 7 Hz, 3H). Anal. (C23H31N3O3) C, H, N.
N-(Adamantan-1-yl)-4,7-dihydro-7-oxo-4-pentyl[1,2,4]-
triazolo[1,5-a]pyrimidine-6-carboxamide (56). White solid, mp
163 °C. Yield: 62%. MS: m/z 384.2 (M + H). 1H NMR (DMSO-d6): δ
8.85 (s, 1H), 8.65 (s, 1H), 8.38 (s, 1H), 4.31 (q, J = 7.4 Hz, 2H), 2.08
(m, 9H), 1.82 (m, 2H), 1.87 (m, 6H), 1.32−1.28 (m, 4H), 0.85 (t, J =
6.2 Hz, 3H). Anal. (C21H29N5O2) C, H, N.
N- Cycl oheptyl-4, 7-dihydro-3-methyl-4-oxo-7-
pentylisoxazolo[5,4-b]pyridine-5-carboxamide (49). White
1
solid, mp 180 °C. Yield: 75%. MS: m/z 360.6 (M + H). H NMR
(DMSO-d6): δ 9.83 (bd, J = 8.2 Hz, 1H), 8.62 (s, 1H), 4.28 (t, J = 7.2
Hz, 2H), 4.03 (m, 1H), 2.49 (s, 3H), 1.82−1.78 (m, 4H), 1.54 (m,
10H), 1.28−17 (m, 4H), 0.85 (t, J = 6.8 Hz, 3H). Anal. (C20H29N3O3)
C, H, N.
N-Cyclohexyl-4,7-dihydro-7-oxo-4-pentyl[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxamide (55). White solid, mp 161 °C. Yield:
30%. MS: m/z 332.2 (M + H). 1H NMR (DMSO-d6): δ 8.91 (s, 1H),
8.78 (bd, J = 7.8 Hz, 1H), 8.39 (s, 1H), 4.33 (t, J = 7.2 Hz, 2H), 3.97−
3.79 (m, 1H), 1.86−1.25 (m, 16H), 0.86 (t, J = 6.6 Hz, 3H). Anal.
(C17H25N5O2) C, H, N.
N-(Adamantan-1-yl)-7-oxo-4-pentyl-2-phenyl-4,7-dihydro-
[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (57). White
1
solid, mp 158 °C. Yield: 38%. MS: m/z 460.3 (M + H). H NMR
(DMSO-d6): δ 8.83 (s, 1H), 8.71 (s, 1H), 8.16−8.11 (m, 2H), 7.57−
7.53 (m, 3H), 4.37 (q, J = 7.2 Hz, 2H), 2.06 (m, 9H), 1.85 (m, 2H),
1.68 (m, 6H), 1.36−1.33 (m, 4H), 0.87 (t, J = 6.2 Hz, 3H). Anal.
(C27H33N5O2) C, H, N.
Synthesis of Adamantan-1-ylcarboxamides (15f, 15g, 15n,
15p, 15s, 15ac, 15ad, 50, 56, 57). General Procedure. To a stirred
solution of the appropriate carboxylic acid (0.97 mmol) in anhydrous
dimethylformamide (15 mL) was added diisopropylethylamine (5.42
mmol). The resulting solution was stirred at room temperature for 10
min before adding o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (1.45 mmol). The solution was stirred for an
additional 3 h at room temperature, at which time 1-adamantylamine
or 3,5-dimethyl-1-adamantylamine (1.45 mmol) was added. The
mixture was then stirred at ambient temperature for an additional 16 h.
Synthesis of N7-Substituted Pyrazolo[3,4-b]pyridine-5-car-
boxamides 16−47, 7-Pentylthieno[2,3-b]pyridine-5-carboxa-
mides 51−54, 13k, l, and 14g. General Procedure. To a solution
of the suitable carboxamide or carboxylic acid ethyl ester (0.27 mmol)
in anhydrous N,N-dimethylformamide (5 mL) was added K2CO3
(0.81 mmol), and the mixture was heated at 80 °C for 1 h. Alkyl
bromide (0.81 mmol) was added, and the mixture was stirred at 100
°C for 16 h. After the mixture was cooled to room temperature, the
solvent was concentrated in vacuo and the residue partitioned between
ethyl acetate (30 mL) and water (5 mL). The organic phase was
1108
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
evaporated and purified by flash chromatography on silica gel, eluting
with ethyl acetate/petroleum ether.
Hz, 2H), 4.10 (s, 3H), 3.27 (t, J = 6.6 Hz, 2H), 2.61 (s, 3H), 1.85−1.
37 (m, 17H), 0.92 (t, J = 6.4 Hz, 3H). Anal. (C21H32N4O2) C, H, N.
trans-4,7-Dihydro-1,3-dimethyl-N-(4-methylcyclohexyl)-4-
oxo-7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (27).
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-4-oxo-7-pentyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (16). White solid, mp 69
1
1
White solid, mp 153 °C. Yield: 57%. MS: m/z 373.2 (M + H). H
°C. Yield: 42%. MS: m/z 359.2 (M + H). H NMR (DMSO-d6): δ
NMR (DMSO-d6): δ 9.95 (bd, J = 7.2 Hz, 1H), 8.43 (s, 1H), 4.42 (t, J
= 7 Hz, 2H), 4.07 (s, 3H), 3.73 (bm, 1H), 2.44 (s, 3H), 2.00−1.66 (m,
6H), 1.31−0.83 (m, 15H). Anal. (C21H32N4O2) C, H, N.
10.00 (bd, J = 7.6 Hz, 1H), 8.37 (s, 1H), 4.22 (t, J = 7.6 Hz, 2H), 4.10
(s, 3H), 3.99 (m, 1H), 2.61 (s, 3H), 2.00−1.27 (m, 16H), 0.88 (t, J =
7.2 Hz, 3H). Anal. (C20H30N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-7-propyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (17). White solid, mp
158 °C. Yield: 40%. MS: m/z 331.1 (M + H). 1H NMR (DMSO-d6): δ
10.06 (bd, J = 8.0 Hz, 1H), 8.44 (s, 1H), 4.39 (t, J = 7.0 Hz, 2H), 4.06
(s, 3H), 3.90 (m, 1H), 2.44 (s, 3H), 1.82−1.29 (m, 12 H), 0.89 (t, J =
7.2 Hz, 3H). Anal. (C18H26N4O2) C, H, N.
7-Allyl-N-(cyclohexyl)-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (18). White solid, mp
212 °C. Yield: 55%. MS: m/z 329.3 (M + H). 1H NMR (DMSO-d6): δ
10.04 (bd, J = 7.8 Hz, 1H), 8.41 (s, 1H), 6.23−6.20 (m, 1H), 5.27−
5.24 (m, 1H), 5.16−5.14 (m, 2H), 4.88−4.79 (m, 1H), 4.00 (s, 3H),
3.97 (m, 1H), 2.45 (s, 3H), 1.64−1.20 (m, 10H). Anal. (C18H24N4O2)
C, H, N.
7-Butyl-N-(cyclohexyl)-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (19). White solid. mp
160 °C. Yield: 35% MS: m/z 345.2 (M + H). 1H NMR (DMSO-d6): δ
10.05 (bd, J = 7.8 Hz, 1H), 8.43 (s, 1H), 4.43 (t, J = 7.0 Hz, 2H), 4.07
(s, 3H), 3.80 (m, 1H), 2.50 (s, 3H), 1.84−1.23 (m, 14H), 0.94 (t, J =
7.0 Hz, 3H). Anal. (C19H28N4O2) C, H, N.
cis-4,7-Dihydro-1,3-dimethyl-N-(4-methylcyclohexyl)-4-oxo-
7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (28).
1
White solid, mp 116 °C. Yield: 57%. MS: m/z 373.2 (M + H). H
NMR (DMSO-d6): δ 10.28 (bd, J = 7.8 Hz, 1H), 8.44 (s, 1H), 4.42 (t,
J = 7.6 Hz, 2H), 4.07 (m, 4H), 2.46 (s, 3H), 1.75−1.09 (m, 15H),
0.93−0.83 (m, 6H). Anal. (C21H32N4O2) C, H, N.
N-Cycloheptyl-4,7-dihydro-1,3-dimethyl-7-pentyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (29). White solid, mp 80
1
°C. Yield: 40%. MS: m/z 373.1 (M + H). H NMR (DMSO-d6): δ
10.10 (bd, J = 7.8 Hz, 1H), 8.42 (s, 1H), 4.39 (t, J = 7.8 Hz, 2H), 4.06
(s, 3H), 3.99 (m, 1H), 2.44 (s, 3H), 1.82−1.27 (m, 18H), 0.89 (t, J =
6.8 Hz, 3H). Anal. (C21H32N4O2) C, H, N.
N-(Adamantan-1-yl)-4,7-dihydro-1,3-dimethyl-7-pentyl-4-
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (30). White
1
solid, mp 143 °C. Yield: 25%. MS: m/z 411.2 (M + H). H NMR
(DMSO-d6): δ 9.90 (bs, 1H), 8.38 (s, 1H), 4.39 (t, J = 7.8 Hz, 2H),
4.06 (s, 3H), 2.43 (s, 3H), 2.03 (s, 9H), 1.65 (m, 8H), 1.32 (m, 4H),
0.86 (t, J = 6.4 Hz, 3H). Anal. (C24H34N4O2) C, H, N.
4,7-Dihydro-1,3-dimethyl-N-(3,5-dimethyladamantan-1-yl)-
4-oxo-7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
(31). White solid, mp 201 °C. Yield: 25%. MS: m/z 439.2 (M + H).
1H NMR (DMSO-d6): δ 9.97 (bd, J = 7.6 Hz, 1H), 8.35 (s, 1H), 4.41
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-7-(2-ethoxyethyl)-4-
oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (20). White
1
solid, mp 98 °C. Yield: 40%. MS: m/z 361.4 (M + H). H NMR
(t, J = 7.2 Hz, 2H), 4.06 (s, 3H), 2.43 (s, 3H), 1.92−1.14 (m, 25H),
0.84 (m, 3H). Anal. (C26H38N4O2) C, H, N.
(DMSO-d6): δ 10.02 (bd, J = 7.8 Hz, 1H), 8.41 (s, 1H), 4.35−4.31
(m, 2H), 3.93 (s, 3H), 3.73−3.69 (m, 3H), 3.47 (q, J = 7 Hz, 2H),
2.57 (s, 3H), 1.97−1.42 (m, 5H), 1.32−1.27 (m, 5H), 1.09 (t, J = 7.2
Hz, 3H). Anal. (C19H28N4O3) C, H, N.
4,7-Dihydro-1,3-dimethyl-7-pentyl-N-(5-methylhexan-2-yl)-
4-oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (32). White
1
solid, mp 195 °C. Yield: 25%. MS: m/z 375.4 (M + H). H NMR
7-(4-Cyanobutyl)-N-(cyclohexyl)-4,7-dihydro-1,3-dimethyl-
(CDCl3): δ 9.88 (bd, J = 7.8 Hz, 1H), 8.37 (s, 1H), 4.16 (m, 3H), 4.10
(s, 3H), 2.61 (s, 3H), 1.68−1.54 (m, 14H), 0.89 (m, 9H). Anal.
(C21H34N4O2) C, H, N.
4-oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (21). White
1
solid, mp 209 °C. Yield: 22%. MS: m/z 370.3 (M + H). H NMR
(DMSO-d6): δ 10.05 (bd, J = 7.8 Hz, 1H), 8.46 (s, 1H), 4.47 (t, J = 7.2
Hz, 4H), 4.07(s, 3H), 3.90 (m, 1H), 2.48 (s, 3H), 1.88−1.29 (m,
16H). Anal. (C20H27N5O2) C, H, N.
4,7-Dihydro-1,3-dimethyl-4-oxo-7-pentyl-N-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (33). White solid, mp
138 °C. Yield: 40%. MS: m/z 353.3 (M + H). 1H NMR (DMSO-d6): δ
12.45 (bs, 1H), 8.61 (s, 1H), 7.70 (m, 2H), 7.35 (m, 2H), 7.07 (m,
1H), 4.48 (t, J = 7.2 Hz, 2H), 4.01 (s, 3H), 2.48 (s, 3H), 1.85 (m, 2H),
1.31 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H). Anal. (C20H24N4O2) C, H, N.
4,7-Dihydro-1,3-dimethyl-N-(4-methoxyphenyl)-4-oxo-7-
pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (34). White
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-7-hexyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (22). White solid, mp 81
1
°C. Yield: 40%. MS: m/z 373.1 (M + H). H NMR (DMSO-d6): δ
10.20 (bd, J = 7.6 Hz, 1H), 8.43 (s, 1H), 4.42 (t, J = 7.6 Hz, 2H), 4.06
(s, 3H), 3.85 (bm, 1H), 2.41 (s, 3H), 1.95−1.27 (m, 18H), 0.88 (t, J =
7.2 Hz, 3H). Anal. (C21H32N4O2) C, H, N.
1
solid, mp 167 °C. Yield: 40%. MS: m/z 384.7 (M + H). H NMR
7-Benzyl-N-cyclohexyl-4,7-dihydro-1,3-dimethyl-4-oxo-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (23). White solid, mp
240 °C. Yield: 40%. MS: m/z 379.2 (M + H). 1H NMR (DMSO-d6): δ
10.05 (bd, J = 7.6 Hz, 1H), 8.55 (s, 1H), 7.40−7.06 (m, 5H), 5.81 (s,
2H), 3.81 (s, 3H), 3.80 (bm, 1H), 2.45 (s, 3H), 1.84−1.27 (m, 10H).
Anal. (C22H26N4O2) C, H, N.
(DMSO-d6): δ 12.35 (bs, 1 H), 8.62 (s, 1H), 7.63 (dd, J = 9 Hz, 2H),
6.92 (dd, J = 9 Hz, 2H), 4.84 (t, J = 7.4 Hz, 2H), 3.89 (s, 3H), 3.76 (s,
3H), 2.49 (s, 3H), 1.81 (m, 2H), 1.32−1.27 (m, 4H), 0.87 (t, J = 6.4
Hz, 3H). Anal. (C21H26N4O3) C, H, N.
4,7-Dihydro-1,3-dimethyl-N-(4-fluorophenyl)-4-oxo-7-pen-
tyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (35). White solid,
mp 176 °C. Yield: 40%. MS: m/z 371.6 (M + H). 1H NMR (DMSO-
d6): δ 12.46 (bs, 1 H), 8.61 (s, 1H), 7.77−7.70 (m, 2H), 7.22−7.14
(m, 2H), 4.84 (t, J = 7.4 Hz, 2H), 4.09 (s, 3H), 2.49 (s, 3H), 1.79 (m,
2H), 1.35−1.28 (m, 4H), 0.87 (t, J = 6.4 Hz, 3H). Anal.
(C20H23FN4O2) C, H, N.
N-Benzyl-4,7-dihydro-1,3-dimethyl-4-oxo-7-pentyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (36). White solid, mp
137 °C. Yield: 40%. MS: m/z 367.2 (M + H). 1H NMR (DMSO-d6): δ
10.39 (bt, J = 6.0 Hz, 1H), 8.49 (s, 1H), 7.34−7.32 (m, 5H), 4.51 (d, J
= 6.0 Hz, 2H), 4.21 (t, J = 7.2 Hz, 2H), 4.07 (s, 3H), 2.43 (s, 3H), 1.75
(m, 2H), 1.39−1.28 (m, 4H), 0.88 (t, J = 6.8 Hz, 3H). Anal.
(C21H26N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-3-ethyl-1-methyl-4-oxo-7-pentyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide (37). White solid, mp
146 °C. Yield: 25%. MS: m/z 373.1 (M + H). 1H NMR (DMSO-d6): δ
10.10 (bd, J = 7.8 Hz, 1H), 8.44 (s, 1H), 4.40 (t, J = 7.6 Hz, 2H), 4.08
(s, 3H), 3.98 (m, 1H), 2.85 (q, J = 7.6 Hz, 2H), 1.79−1.30 (m, 16H),
1.22 (m, 3H), 0.83 (t, J = 6.6 Hz, 3H). Anal. (C21H32N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-7-(4-methylbenzyl)-
4-oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (24). White
1
solid, mp 234 °C. Yield: 40%. MS: m/z 393.3 (M + H). H NMR
(DMSO-d6): δ 10.05 (bd, J = 7.6 Hz, 1H), 8.52 (s, 1H), 7.20 (d, J =
8.0 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 5.75 (s, 2H), 3.83 (s, 3H), 3.80
(br m, 1H), 2.45 (s, 3H), 2.28 (s, 3H), 1.83-1. 26 (m, 10H). Anal.
(C23H28N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-1,3-dimethyl-7-[2-(morpholin-4-
yl)ethyl]-4-oxo-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
(25). White solid, mp 128 °C. Yield: 42%. MS: m/z 402.1 (M + H).
1H NMR (DMSO-d6): δ 10.06 (bd, J = 7.6 Hz, 1H), 8.41 (s, 1H), 4.52
(t, J = 7.0 Hz, 2H), 4.08 (s, 3H), 3.90 (br m, 1H), 3.49 (m, 4H), 2.59
(t, J = 7.0 Hz, 2H), 2.44 (s, 3H), 1.99−1.22 (m, 14H). Anal.
(C21H31N5O3) C, H, N.
N-(Cyclohexylmethyl)-4,7-dihydro-1,3-dimethyl-4-oxo-7-
pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (26). White
1
solid, mp 87 °C. Yield: 40%. MS: m/z 373.1 (M + H). H NMR
(DMSO-d6): δ 10.10 (bt, J = 7.8 Hz, 1H), 8.37 (s, 1H), 4.21 (t, J = 7.6
1109
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
N-(Adamantan-1-yl)-4,7-dihydro-3-ethyl-1-methyl-4-oxo-7-
pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (38). White
solid, mp 130−131 °C. Yield: 20%. MS: m/z 425.4 (M + H). 1H NMR
(DMSO-d6): δ 9.95 (s, 1H), 8.39 (s, 1H), 4.41 (t, J = 6.8 Hz, 2H),
4.08 (s, 3H), 2.86 (q, J = 6.0 Hz, 2H), 2.03 (s, 9H), 1.76 (m, 2H), 1.65
(s, 6H), 1.31 (m, 4H), 1.20 (t, J = 6.8 Hz, 3H), 0.88 (t, J = 6.4 Hz,
3H). Anal. (C25H36N4O2) C, H, N.
3H), 1.99−1.17 (m, 16H), 0.84 (t, J = 6.6 Hz, 3H). Anal.
(C20H28N2O2S) C, H, N.
N-Cycloheptyl-4,7-dihydro-3-methyl-4-oxo-7-pentylthieno-
[2,3-b]pyridine-5-carboxamide (52). White solid, mp 113 °C.
1
Yield: 28%. MS: m/z 375.2 (M + H). H NMR (400 MHz, DMSO-
d6): δ 10.26 (bd, J = 7.6 Hz, 1H), 8.63 (bs, 1H), 7.05 (q, 4J = 1.2 Hz,
1H), 4.24 (t, J = 7.4 Hz, 2H), 4.05−3.98 (m, 1H), 2.54 (d, 4J = 1.2 Hz,
3H), 1.86−1.79 (m, 4H), 1.62−1.28 (m, 14 H), 0.85 (t, J = 6.6 Hz,
3H). Anal. (C21H30N2O2S) C, H, N.
3-tert-Butyl-N-cyclohexyl-4,7-dihydro-1-methyl-4-oxo-7-
pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (39). White
1
solid, mp 177 °C. Yield: 30%. MS: m/z 401.3 (M + H). H NMR
N-(Adamantan-1-yl)-4,7-dihydro-3-methyl-4-oxo-7-
pentylthieno[2,3-b]pyridine-5-carboxamide (53). White solid,
(DMSO-d6): δ 10.20 (bd, J = 7.8 Hz, 1H), 8.47 (s, 1H), 4.43 (t, J = 7.6
Hz, 2H), 4.09 (s, 3H), 3.97 (m, 1H), 1.99−1.58 (m, 12H), 1.41 (s,
9H), 1.33−1.29 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H). Anal.
(C23H36N4O2) C, H, N.
1
mp 159 °C. Yield: 37%. MS: m/z 413.2 (M + H). H NMR (400
4
MHz, CDCl3): δ 10.08 (bs, 1H), 8.54 (s, 1H), 6.61 (q, J = 1.2 Hz,
1H), 4.03 (t, J = 7.6 Hz, 2H), 2.66 (d, 4J = 1.2 Hz, 1H), 2.09 (s, 9H),
1.97−1.82 (m, 2H), 1.66 (s, 6H), 1.30−1.28 (m, 4H), 0.86 (t, J = 6.6
Hz, 3H). Anal. (C24H32N2O2S) C, H, N.
N-(Adamantan-1-yl)-3-tert-butyl-4,7-dihydro-1-methyl-4-
oxo-7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (40).
1
White solid, mp 192 °C. Yield: 32%. MS: m/z 453.2 (M + H). H
N-(Adamantan-1-yl)-4,7-dihydro-2,3-dimethyl-4-oxo-7-
pentylthieno[2,3-b]pyridine-5-carboxamide (54). White solid,
mp 215 °C. Yield: 35%. MS: m/z 427.2 (M + H). 1H NMR (CDCl3):
δ 10.18 (bs, 1H), 8.49 (s, 1H), 3.99 (t, J = 7.6 Hz, 2H), 2.56 (s, 3H),
2.37 (s, 3H), 2.18 (s, 9H), 2.09−1.92 (m, 2H), 1.71 (s, 6H), 1.37−
1.30 (m, 4H), 0.90 (t, J = 6.6 Hz, 3H). Anal. (C25H34N2O2S) C, H, N.
Ethyl 4,7-Dihydro-7-oxo-4-pentyl[1,2,4]triazolo[1,5-a]-
pyrimidine-6-carboxylate (13k). White solid, mp 85−86 °C.
NMR (CDCl3): δ 10.07 (bs, 1H), 8.34 (s, 1H), 4.19 (t, J = 7.6 Hz,
2H), 4.10 (s, 3H), 2.17−2.09 (m, 9H), 1.72−1.70 (m, 8H), 1.47 (s,
9H), 1.36 (m, 4H), 0.91 (t, J = 7.2 Hz, 3H). Anal. (C27H40N4O2) C,
H, N.
N-Cyclohexyl-4,7-dihydro-1-methyl-4-oxo-7-pentyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (41). White solid, mp
146 °C. Yield: 33%. MS: m/z 345.3 (M + H). 1H NMR (DMSO-d6): δ
8.69 (bd, J = 7.6 Hz, 1H), 8.48 (s, 1H), 7.98 (d, J = 6.0 Hz, 1H), 4.72
(t, J = 6.2 Hz, 2H), 4.01 (s, 3H), 3.99 (m, 1H), 1.90−1.27 (m, 16H),
0.91 (t, J = 7.4 Hz, 3H). Anal. (C19H28N4O2) C, H, N.
N-Cycloheptyl-4,7-dihydro-1-methyl-4-oxo-7-pentyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide (42). White solid, mp
131 °C. Yield: 28%. MS: m/z 359.2 (M + H). 1H NMR (DMSO-d6): δ
8.69 (bd, J = 7.8 Hz, 1H), 8.47 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 4.72
(t, J = 6.0 Hz, 2H), 4.00 (s, 3H), 3.99 (m, 1H), 1.7−1.36 (m, 18H),
0.88 (t, J = 7.2 Hz, 3H). Anal. (C20H30N4O2) C, H, N.
1
Yield: 85%. H NMR (CDCl3): δ 8.47 (s, 1H), 8.09 (s, 1H), 4.42
(q, J = 7.0 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 1.93−1.85 (m, 2H),
1.43−1.35 (m, 7H), 0.91 (t, J = 6.6 Hz, 3H).
Ethyl 4,7-Dihydro-7-oxo-4-pentyl-2-phenyl[1,2,4]triazolo-
[1,5-a]pyrimidine-6-carboxylate (13l). White solid, mp 112−113
1
°C. Yield: 80%. H NMR (DMSO-d6): δ 8.87 (s, 1H), 8.16−8.11 (m,
2H), 7.58−7.53 (m, 3H), 4.36−4.3 (m, 4H), 1.92−1.85 (m, 2H);
1.37−1.27 (m, 7H); 0.91 (t, J = 6.4 Hz, 3H).
Ethyl 4,7-Dihydro-3-methyl-4-oxo-7-pentylisoxazolo[5,4-b]-
pyridine-5-carboxylate (14g). White solid, mp 91 °C. Yield: 42%.
1H NMR (DMSO-d6): δ ppm 8.46 (s, 1H), 4.24−4.17 (m, 4H), 2.48
N-(Adamantan-1-yl)-4,7-dihydro-1-methyl-4-oxo-7-pentyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide (43). White solid, mp
79 °C. Yield: 35%. MS: m/z 397.6 (M + H). 1H NMR (DMSO-d6): δ
8.70 (s, 1H), 8.47 (s, 1H), 7.58 (bs, 1H), 4.73 (t, J = 7.2 Hz, 2H), 4.01
(s, 3H), 2.06 (s, 9H), 1.98−1.82 (m, 2H), 1.67 (s, 6H), 1.59−1.35 (m,
4H), 0.92 (t, J = 7 Hz, 3H). Anal. (C23H32N4O2) C, H, N.
N-Cyclohexyl-4,7-dihydro-1-methyl-4-oxo-7-pentyl-3-phe-
nyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (44). White
(s, 3H), 1.77 (m, 2H), 1.32−1.25 (m, 7H), 0.86 (t, J = 7.2 Hz, 3H).
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.
1
solid, mp 176 °C. Yield: 45%. MS: m/z 421.1 (M + H). H NMR
(DMSO-d6): δ 10.01 (bd, J = 7.4 Hz, 1H), 8.54 (s, 1H), 8.04−8.00
(m, 2H), 7.45−7.41 (m, 3H), 4.51 (m, 2H), 4.23 (s, 3H), 3.83 (m,
1H), 1.85−1.28 (m, 16H), 0.88 (t, J = 6.6 Hz, 3H). Anal.
(C25H32N4O2) C, H, N.
Present Address
∥Alta Vetta Pharmaceutical Consulting, LLC, 3 Skipwith Court,
Durham, North Carolina 27707.
N-Cycloheptyl-4,7-dihydro-1-methyl-4-oxo-7-pentyl-3-phe-
nyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (45). White
Notes
1
solid, mp 208 °C. Yield: 33%. MS: m/z 435.2 (M + H). H NMR
The authors declare no competing financial interest.
(CDCl3) 10.06 (bd, J = 7.6 Hz, 1H), 8.44 (s, 1H), 8.04−7.98 (m, 2H),
7.46−7.43 (m, 3H), 4.24 (m, 6H), 1.95−1.27 (m, 18H), 0.94 (m, 3H).
Anal. (C26H34N4O2) C, H, N.
ACKNOWLEDGMENTS
■
We thank King Pharmaceuticals Research and Development,
Inc. for financial support.
3-(4-Chlorophenyl)-N-cyclohexyl-4,7-dihydro-1-methyl-4-
oxo-7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (46).
1
White solid, mp 203 °C. Yield: 25%. MS: m/z 455.3 (M + H). H
NMR (DMSO-d6): δ 9.94 (bd, J = 7.6 Hz, 1H), 8.53 (s, 1H), 8.08 (dd,
J = 6.8 Hz, 2H), 7.50 (dd, J = 7.0 Hz, 2H), 4.48 (t, J = 7.2 Hz, 2H),
4.21 (s, 3H), 3.89 (m, 1H), 1.82−1.25 (m, 16H), 0.85 (t, J = 6.8 Hz,
3H). Anal. (C25H31ClN4O2) C, H, N.
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
3-(4-Chlorophenyl)-N-cycloheptyl-4,7-dihydro-1-methyl-4-
oxo-7-pentyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (47).
1
White solid, mp 219 °C. Yield: 25%. MS: m/z 470.2 (M + H). H
NMR (DMSO-d6): δ 10.00 (bd, J = 7.8 Hz, 1H), 8.54 (s, 1H), 8.09
(dd, J = 8.2 Hz, 2H), 7.51 (dd, J = 8.2 Hz, 2H), 4.50 (t, J = 7.0, 2H),
4.23 (s, 3H), 3.99 (m, 1H), 1.56−1.23 (m, 18H), 0.85 (t, J = 6.8 Hz,
3H). Anal. (C26H33ClN4O2) C, H, N.
REFERENCES
■
(1) Robson, P. Human studies of cannabinoids and medicinal
cannabis. Handb. Exp. Pharmacol 2005, 168, 719−756.
(2) Beal, J. E.; Olson, R.; Laubenstein, L.; Morales, J. O.; Bellman, P.;
Yangco, B.; Lefkowitz, L.; Plasse, T. F.; Shepard, K. V. Dronabinol as a
treatment for anorexia associated with weight loss in patients with
AIDS. J. Pain Symptom Manage. 1995, 10, 89−97.
N-Cyclohexyl-4,7-dihydro-3-methyl-4-oxo-7-pentylthieno-
[2,3-b]pyridine-5-carboxamide (51). White solid, mp 130 °C.
1
Yield: 32%. MS: m/z 361.2 (M + H). H NMR (400 MHz, DMSO-
d6): δ 10.23 (bd, J = 7.8 Hz, 1H), 8.64 (bs, 1H), 7.05 (q, 4J = 1.2 Hz,
1H), 4.25 (t, J = 7.2 Hz, 2H), 3.97−3.78 (m, 1H), 2.52 (d, 4J = 1.8 Hz,
1110
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
(3) Ranganathan, M.; D’Souza, D. C. The acute effects of
cannabinoids on memory in humans: a review. Psychopharmacology
2006, 188, 425−444.
(4) Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.;
Devane, W. A.; Felder, C. C.; Herkenham, M.; Mackie, K.; Martin, B.
R.; Mechoulam, R.; Pertwee, R. G. International Union of
Pharmacology. XXVII. Classification of cannabinoid receptors.
Pharmacol. Rev. 2002, 54, 161−202.
development of CB2-selective agonists. Curr. Med. Chem. 2008, 15,
1428−1443.
(23) Worm, K.; Dolle, R. E. Simultaneous optimization of potency,
selectivity and physicochemical properties for cannabinoid CB2
ligands. Curr. Pharm. Des. 2009, 15, 3345−3366.
(24) 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.
(25) Manera, C.; Benetti, V.; Castelli, M. P.; Cavallini, T.; Lazzarotti,
S.; Pibiri, F.; Saccomanni, G.; Tuccinardi, T.; Vannacci, A.; Martinelli,
A.; Ferrarini, P. L. Design, synthesis, and biological evaluation of new
1,8-naphthyridin-4(1H)-on-3-carboxamide and quinolin-4(1H)-on-3-
carboxamide derivatives as CB2 selective agonists. J. Med. Chem. 2006,
49, 5947−5957.
(26) Manera, C.; Cascio, M. G.; Benetti, V.; Allara, M.; Tuccinardi,
T.; Martinelli, A.; Saccomanni, G.; Vivoli, E.; Ghelardini, C.; Di Marzo,
V.; Ferrarini, P. L. New 1,8-naphthyridine and quinoline derivatives as
CB2 selective agonists. Bioorg. Med. Chem. Lett. 2007, 17, 6505−6510.
(27) Stern, E.; Muccioli, G.; Millet, R.; Goossens, J. F.; Farce, A.;
Chavatte, P.; Poupaert, J. H.; Lambert, D. M.; Depreux, P.; Henichart,
J. P. Novel 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives as
new CB2 cannabinoid receptors agonists: synthesis, pharmacological
properties and molecular modelling. J. Med. Chem. 2006, 49, 70−79.
(28) Baraldi, P. G.; Saponaro, G.; Moorman, A. R.; Romagnoli, R.;
Preti, D.; Baraldi, S.; Ruggiero, E.; Varani, K.; Targa, M.; Vincenzi, F.;
Borea, P. A.; Aghazadeh Tabrizi, M. 7-Oxo-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxamides as selective CB(2) cannabinoid receptor
ligands: structural investigations around a novel class of full agonists. J.
Med. Chem. 2012, 55, 6608−23.
(29) Hohn, H.; Denzel, T.; Jansen, W. 1H-Pyrazole[3,4-b]pyridines.
J. Heterocycl. Chem. 1972, 9, 235−253.
(30) Chu, I.; Lynch, B. M. Synthesis and biological evaluation of
xanthine oxidase inhibitors. Pyrazolo[3,4-d]pyrimidines and pyrazolo-
[3,4-b]pyridines. J. Med. Chem. 1975, 18, 161−165.
(31) Baraldi, P. G.; Simoni, D.; Moroder, F.; Manfredini, S.; Mucchi,
L.; Dalla Vecchia, F. Synthesis of 2-(5′-substituted isoxazol-3′-yl)-4-
oxo-3-thiazolidinylalkanoic acid. J. Heterocycl. Chem. 1982, 19, 557−
560.
(32) Golub, A. G.; Bdzhola, V. G.; Briukhovetska, N. V.; Balanda, A.
O.; Kukharenko, O. P.; Kotey, I. M.; Ostrynska, O. V.; Yarmoluk, S. M.
Synthesis and biological evaluation of substituted (thieno[2,3-
d]pyrimidin-4-ylthio)carboxylic acids as inhibitors of human protein
kinase CK2. Eur. J. Med. Chem. 2011, 46, 870−876.
(5) Klein, T. W.; Newton, C.; Larsen, K.; Lu, L.; Perkins, I.; Nong, L.;
Friedman, H. The cannabinoid system and immune modulation. J.
Leukocyte Biol. 2003, 74, 486−496.
(6) Mackie, K. Distribution of cannabinoid receptors in the central
and peripheral nervous system. Handb. Exp. Pharmacol. 2005, 168,
299−325.
(7) Ross, R. A.; Coutts, A. A.; McFarlane, S. M.; Anavi-Goffer, S.;
Irving, A. J.; Pertwee, R. G.; MacEwan, D. J.; Scott, R. H. Actions of
cannabinoid receptor ligands on rat cultured sensory neurones:
implications for antinociception. Neuropharmacology 2001, 40, 221−
232.
(8) Pertwee, R. G. Emerging strategies for exploiting cannabinoid
receptor agonists as medicines. Br. J. Pharmacol. 2009, 156, 397−411.
́
(9) Elphick, M. R.; Egertova, M. The neurobiology and evolution of
cannabinoid signalling. Philos. Trans. R. Soc. London, Ser. B 2001, 356,
381−408.
(10) Ashton, J. C. Cannabinoids for the treatment of inflammation.
Curr. Opin. Invest. Drugs 2007, 8, 373−384.
(11) Croxford, J. L.; Miller, S. D. Immunoregulation of a viral model
of multiple sclerosis using the synthetic cannabinoid R+WIN55,212. J.
Clin. Invest. 2003, 111, 1231−1240.
(12) Fernandez-Ruiz, J.; Romero, J.; Velasco, G.; Tolon, R. M.;
Ramos, J. A.; Guzman, M. Cannabinoid CB2 receptor: a new target for
controlling neural cell survival? Trends Pharmacol. Sci. 2007, 28, 39−
45.
(13) Whiteside, G. T.; Lee, G. P.; Valenzano, K. J. The role of the
cannabinoid CB2 receptor in pain transmission and therapeutic
potential of small molecule CB2 receptor agonists. Curr. Med. Chem.
2007, 14, 917−36.
(14) Cheng, Y.; Hitchcock, S. A. Targeting cannabinoid agonists for
inflammatory and neuropathic pain. Expert Opin. Invest. Drugs 2007,
16, 951−965.
(15) 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.
(16) Yao, B. B.; Hsieh, G. C.; Frost, J. M.; Fan, Y.; Garrison, T. R.;
Daza, A. V.; Grayson, G. K.; Zhu, C. Z.; Pai, M.; Chandran, P.; Salyers,
A. K.; Wensink, E. J.; Honore, P.; Sullivan, J. P.; Dart, M. J.; Meyer, M.
D. In vitro and in vivo characterization of A-796260: a selective
cannabinoid CB2 receptor agonist exhibiting analgesic activity in
rodent pain models. Br. J. Pharmacol. 2008, 153, 390−401.
(17) Pertwee, R. G.; Howlett, A. C.; Abood, M. E.; Alexander, S. P.
H.; Di Marzo, V.; Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos,
G.; Mackie, K.; Mechoulam, R.; Ross, R. A. International Union of
Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and
their ligands: beyond CB(1) and CB(2). Pharmacol. Rev. 2010, 62,
588−631.
(33) Imada, T.; Cho, N.; Imaeda, T.; Hayase, Y.; Sasaki, S.; Kasai, S.;
Harada, M.; Matsumoto, H.; Endo, S.; Suzuki, N.; Furuya, S. Design,
synthesis, and structure−activity relationships of thieno[2,3-b]pyridin-
4-one derivatives as a novel class of potent, orally active, non-peptide
luteinizing hormone-releasing hormone receptor antagonists. J. Med.
Chem. 2006, 49, 3809−3825.
(34) Dolzhenko, A. V.; Pastorin, G.; Dolzhenko, A. V.; Keung Chui,
W. An aqueous medium synthesis and tautomerism study of 3(5)-
amino-1,2,4-triazoles. Tetrahedron Lett. 2009, 50, 2124−2128.
(35) Abdel-Rahman, H. M.; El-Koussi, N. A.; Hassan, H. Y.
Fluorinated 1,2,4-triazolo[1,5-a]pyrimidine-6-carboxylic acid deriva-
tives as antimycobacterial agents. Arch. Pharm. 2009, 342, 94−99.
(36) Manera, C.; Saccomanni, G.; Adinolfi, B.; Benetti, V.; Ligresti,
A.; Cascio, M. G.; Tuccinardi, T.; Lucchesi, V.; Martinelli, A.; Nieri, P.;
Masini, E.; Di Marzo, V.; Ferrarini, P. L. Rational design, synthesis, and
pharmacological properties of new 1,8-naphthyridin-2(1H)-on-3-
carboxamide derivatives as highly selective cannabinoid-2 receptor
agonists. J. Med. Chem. 2009, 52, 3644−3651.
(18) Pertwee, R. G.; Cascio, M. G.; Gauson, L. A.; Stevenson, L. A.;
Ross, R. A. Evidence that the plant cannabinoid cannabigerol is a
highly potent alpha(2)-adrenoceptor agonist and moderately potent
5HT(1A) receptor antagonist. Br. J. Pharmacol. 2010, 159, 129−141.
(19) Pertwee, R. G. Pharmacology of cannabinoid receptor ligands.
Curr. Med. Chem. 1999, 6, 635−664.
(37) Pasquini, S.; Mugnaini, C.; Ligresti, A.; Tafi, A.; Brogi, S.;
Falciani, C.; Pedani, V.; Pesco, N.; Guida, F.; Luongo, L.; Varani, K.;
Borea, P. A.; Maione, S.; Di Marzo, V.; Corelli, F. Design, synthesis,
and pharmacological characterization of indol-3-ylacetamides, indol-3-
yloxoacetamides, and indol-3-ylcarboxamides: potent and selective
CB2 cannabinoid receptor inverse agonists. J. Med. Chem. 2012, 55,
5391−5402.
(20) Pertwee, R. G. Pharmacological actions of cannabinoids. Handb.
Exp. Pharmacol. 2005, 168, 1−51.
(21) Pertwee, R. G. Ligands that target cannabinoid receptors in the
brain: from THC to anandamide and beyond. Addict. Biol. 2008, 13,
147−159.
(22) Ashton, J. C.; Wright, J. L.; McPartland, J. M.; Tyndall, J. D.
Cannabinoid CB1 and CB2 receptor ligand specificity and the
1111
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112

Journal of Medicinal Chemistry
Article
(38) Rinaldi-Carmona, M.; Barth, F.; Congy, C.; Martinez, S.;
Oustric, D.; Per
́
io, A.; Poncelet, M.; Maruani, J.; Arnone, M.; Finance,
O.; Soubrie, P.; Le Fur, G. SR147778 [5-(4-bromophenyl)-1 (2,4-
́
dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxa-
mide], a new potent and selective antagonist of the CB1 cannabinoid
receptor: biochemical and pharmacological characterization. J.
Pharmacol. Exp. Ther. 2004, 310, 905−914.
(39) 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.
(40) MacLennan, S. J.; Reynen, P. H.; Kwan, J.; Bonhaus, D. W.
Evidence for inverse agonism of SR141716A at human recombinant
cannabinoid CB1 and CB2 receptors. Br. J. Pharmacol. 1998, 124, 619−
622.
(41) Merighi, S.; Simioni, C.; Gessi, S.; Varani, K.; Borea, P. A.
Binding thermodynamics at the human cannabinoid CB1 and CB2
receptors. Biochem. Pharmacol. 2010, 79, 471−477.
(42) Varani, K.; Merighi, S.; Gessi, S.; Klotz, K. N.; Leung, E.; Baraldi,
P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, P.; Borea, P. A. [3H]-
MRE3008F20: a novel antagonist radioligand for the pharmacological
and biochemical characterization of human A3 adenosine receptors.
Mol. Pharmacol. 2000, 57, 968−975.
(43) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes 50 per
cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol.
1973, 22, 3099−108.
(44) Munson, P. J.; Rodbard, D. Ligand: a versatile computerized
approach for characterization of ligand-binding systems. Anal. Biochem.
1980, 107, 220−239.
(45) Taylor, E. C.; Hartke, K. S. Reaction of malononitrile with
substituted hydrazines: new routes to 4-aminopyrazolo[3,4-d]-
pyrimidines. J. Am. Chem. Soc. 1959, 81, 2456−2464.
(46) Ochiai, H.; Ishida, A.; Ohtani, T.; Kusumi, K.; Kishikawa, K.;
Yamamoto, S.; Takeda, H.; Obata, T.; Nakai, H.; Toda, M. Discovery
of new orally active phosphodiesterase (PDE4) inhibitors. Chem.
Pharm. Bull. 2004, 52, 1098−1104.
(47) Adachi, I.; Yamamori, T.; Hiramatsu, Y.; Sakai, K.; Sato, H.;
Kawakami, M.; Uno, O.; Ueda, M. Studies on dihydropyridines. II.
Synthesis of 4,7-dihydropyrazolo[3,4-b]pyridines with vasodilating and
antihypertensive activities. Chem. Pharm. Bull. 1987, 35, 3235−3252.
(48) Nam, L. N.; Grandberg, I. I.; Sorokin, V. I. Condensation of 1-
substituted-5-aminopyrazoles with β-dicarbonyl compounds. Chem.
Heterocycl. Compd. 2003, 39, 937−941.
(49) Bernard, M. K.; Wrzeciono, U.; Koehler, T.; Nuhn, P. Azoles.
Part 25. Isomeric 3- and 5-aminopyrazole derivatives. Pharmazie 1989,
44, 535−539.
1112
dx.doi.org/10.1021/jm301527r | J. Med. Chem. 2013, 56, 1098−1112