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European Journal of Medicinal Chemistry 113 (2016) 11e27

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European Journal of Medicinal Chemistry


journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis and structure activity relationship investigation of triazolo


[1,5-a]pyrimidines as CB2 cannabinoid receptor inverse agonists
Mojgan Aghazadeh Tabrizi a, *, Pier Giovanni Baraldi a, **, Emanuela Ruggiero a,
Giulia Saponaro a, Stefania Baraldi a, Giulio Poli b, Tiziano Tuccinardi b, Annalisa Ravani c,
Fabrizio Vincenzi c, Pier Andrea Borea c, Katia Varani c
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
b  di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Dipartimento di Farmacia, Universita
c
Dipartimento di Scienze Mediche, Sezione di Farmacologia, Universita di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy

a r t i c l e i n f o a b s t r a c t

Article history: CB2 cannabinoid receptor ligands are known to be therapeutically important for the treatment of
Received 17 November 2015 numerous diseases. Recently, we have identified the heteroaryl-4-oxopyridine/7-oxopyrimidine de-
Received in revised form rivatives as highly potent and selective CB2 receptor ligands, showing that the pharmakodynamics of the
26 January 2016
new compounds was controlled by the nature of the heterocycle core. In this paper we describe the
Accepted 11 February 2016
Available online 16 February 2016
synthesis and biological evaluation of 7-oxo-4-pentyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide derivatives that led to the identification of novel CB2 receptor inverse agonists. Cyclic
AMP experiments on CB2 receptors expressed in CHO cells revealed that introduction of structural
Keywords:
Cannabinoid
modifications at position 2 of triazolopyrimidine template changes the functional activity from partial to
CB2 receptor inverse agonism. The molecular docking analysis of the novel structures is reported.
Inverse agonists © 2016 Elsevier Masson SAS. All rights reserved.
Triazolopyrimidine
Molecular docking

1. Introduction reported that these receptors are localized neuronally in different


species. They are expressed in hematopoietic cells, natural killer
The endocannabinoid system includes a group of neuro- cells, macrophages and neutrophils, and represent an attractive
modulatory lipids and their receptors that are involved in a di- target for the treatment of the inflammatory status [3]. These re-
versity of physiological and pathological conditions. The main ceptors also mediate a significant protection in brain microglia
endocannabinoids, or endogenous cannabis-like substances, are against neurotoxicity by modulating the release of anti- or pro-
small molecules resulting from arachidonic acid, N-arachidonoyl inflammatory cytokines [4]. CB2 receptors appear to be implicated
ethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2- in allergic dermatitis where their modulation mediates an inflam-
AG) [1]. Two diverse types of G protein-coupled receptors matory response obtained from antigen-specific effecter T cells
(GPCRs) have been discovered and named as cannabinoid CB1 and upon frequent allergen contact [5]. Contrasting preclinical studies
cannabinoid CB2 receptors. The CB1 and CB2 receptors are nega- are reported regarding the involvement of CB2 receptors and
tively coupled to adenylyl cyclase, exert an inhibitory effect on allergic dermatitis, supposing the potential use of both CB2 agonists
cyclic AMP (cAMP) production, alter the mitogen-activated protein or inverse agonists/antagonists in the pharmacological therapy [6].
kinase (MAPK) pathway, and regulate voltage-gated L-, N- and P- or In recent years, pharmaceutical companies and academic
Q-type Ca2þ channels [2]. research laboratories have challenged to identify novel CB2 agonists
CB2 receptors were originally supposed to be present in pe- without CB1 central effects by focusing on the discovery of high
riphery, primarily to immune cells, but at present it has been affinity and selective agonists for CB2 receptors with a therapeutic
potential in the treatment of different pathologies such as neuro-
degenerative diseases, pain transduction and perception, ischemic
* Corresponding author. stroke, severe inflammation, autoimmune diseases, osteoporosis
** Corresponding author. and cancers [4,7].
E-mail addresses: tbj@unife.it (M. Aghazadeh Tabrizi), baraldi@unife.it From the pharmacological point of view, CB2 agonists
(P.G. Baraldi).

http://dx.doi.org/10.1016/j.ejmech.2016.02.032
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
12 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

demonstrate various ways of interacting with the receptor site, heterocyclic scaffold. Within this series, the 8-methoxy derivative 6
suggesting different binding and functional properties well repre- (hCB2 Ki ¼ 0.6 nM, hCB1 Ki > 10,000 nM) (Chart 1) behaved as an
sented by pharmacodynamic concepts of full, partial and inverse inverse agonist and showed antihyperalgesic effects [16].
agonism [8e11]. Our group previously reported the synthesis and cannabinoid
The therapeutic potential of the cannabinoids has encouraged receptor binding of 4-oxopyrazolopyridines as CB2 partial agonists
the development of several CB2 receptor selective inverse agonists [10]. In this family, compound 7 (Chart 2) displayed high affinity
(Chart 1). In particular, the immunomodulatory activities associ- and selectivity for CB2 receptor. Changing the orientation of the
ated with cannabinoid CB2 receptor inverse agonists have been pyrazole ring led to the 7-oxopyrazolopyrimidines, such as com-
demonstrated by oral administration of the inverse agonists 1 (JTE- pound 8 that exhibited selective inverse agonist activity at CB2
907) and 2 (SR144528) that inhibited carrageenin-induced paw receptor [11]. Replacement of the pyrazolo ring of the parent nu-
edema in mice [12]. The efficacy of triaryl bissulphone 3 cleus with differently substituted 5-membered heterocycles
(Sch414319), a CB2 receptor inverse agonist, was investigated allowed to the isoxazolo/thienopyridines that were found to be CB2
in vivo in diverse disease models, showing its ability to modulate full agonists. In this context, the triazolopyrimidines 9, 10 (Chart 2)
immune cell mobility and bone damage in antigen-induced mono- were found to be potent CB2 receptor ligands and in functional
articular arthritis in the rat [13]. Additional reports suggest that assays compound 9 behaved as partial agonist.
compound 4 (MH), an inverse agonists of CB2 receptor, showed In light of these results, we sought to explore the influence of
anti-inflammatory and antiosteoclastogenic properties [14]. substituent arrangement at the C-2 position of triazolo heterocycle
Moreover, N,N0 -((4-(dimethylamino)phenyl)methylene)bis(2- on the activity versus CB2 receptor. In this attempt, we have syn-
phenylacetamide) derivatives such as 5 were found to be CB2 in- thesized a new series of 2-substituted-7-oxo-4-pentyl-[1,2,4]tri-
verse agonists and potential therapeutic agents for the treatment of azolo[1,5-a]pyrimidine-6-carboxamide derivatives based on the
antiosteoporosis [15]. previously well-known pharmacophores in the series of heteroaryl
The 4-oxo-1,8-naphthyridine-3-carboxamide and 4-oxo-1,4- pyridine/pyrimidine compounds. Consequently, the n-pentyl chain
dihydroquinoline-3-carboxamide cannabinoid based ligands are was retained at the N-4 position of heterocycle and the cyclohexyl,
reported with high affinity and selectivity toward CB2 receptors. cycloheptyl, and adamantyl moieties were introduced on the car-
Some of these analogues were demonstrated to act as agonists or boxamide moiety (Chart 2). All the newly synthesized compounds
inverse agonists in functional activity assays, depending on the were tested on membranes prepared from Chinese hamster ovary
nature of the substituents on the different positions of the (CHO) cells expressing human CB1 (hCB1) and CB2 (hCB2) receptors,

O
O
NH O
N
O H
O N O N
H N
O
Cl

1, JTE-907 2, SR144528

Cl
OCH3
NHSO2CF3
S O2S
O2
F OH

3, Sch414319 4, MH

N O O

NH

O O N
OCH3
N N
H H
5 6

Chart 1. Chemical structures of representative CB2 inverse agonists.


M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 13

O O O O
N N N N
N H H
N N N

7 8

O O O O
N N N N R2
N N
R1 H R1 H
N N N N

cyclohexyl,
9, R = H alkyl, phenyl, 4-substituted-phenyl, cycloheptyl,
10, R = phenyl furan-2-yl, pyridin-3-yl, amine adamant-1-yl,
3,5-dimethyl-adamant-1-yl

Chart 2. Structural investigations around the 4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide scaffold.

in order to estimate their affinity and selectivity for CB2 receptor. aminotriazoles 11aem with diethyl ethoxymethylenmalonate
The cyclic AMP assays on hCB2 CHO cells to test the functionality of (DEEM) in glacial acetic acid gave the ethyl 4,7-dihydro-2-
these ligands are reported. Moreover, a molecular docking analysis substituted-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylates
was carried out into the three-dimensional model of CB receptors 12aem in good yields. Alkylation of the bicyclic nucleus with 1-
recently constructed, in order to clarify the structure activity re- pentylbromide in the presence of K2CO3 led to the formation of
lationships (SARs) experimentally observed. 13aem. The subsequent hydrolysis with 10% aqueous NaOH yiel-
ded the related carboxylic acids 14aem, which were converted to
the corresponding carboxamide derivatives 15e41 by coupling re-
2. Chemistry action with appropriate amines using 1-ethyl-3-[3-(dimethyla-
mino)propyl]carbodiimide hydrochloride (EDCl) or N,N,N0 ,N0 -
Synthesis of derivatives 15e41 was carried out by the general tetramethyl-O-(1H-benzotriazol-1-yl)uronium
methodology shown in Scheme 1. Cyclocondensation of

O O O
A
N NH N N O
R1 NHNH2
i ii R1
R1 N NH2 N N
S H
N CN method A: 11a-h
S B 12a-m
method B: 11i-m

O O O O
N N N N R2
O N
iii R1 v R1 H
N N N N

13a-m
iv 15-41
14a-m

R1 = a: H; b: CH3; c: phenyl; d: 4-Cl-phenyl; e: 4-OCH3-phenyl;


f: 4-CH3-phenyl; g: furan-2-yl; h: pyridin-3-yl; i: S-CH3; j: morpholine;
k: N-methylpiperazine; l: N-benzyl-N-methyl; m: N,N-diallylamine.
Scheme 1. Reagents and conditions: i) A: S-methylisothiourea hemisulfate, water, 100  C, 6 h then NaOH 10%, 100  C, 6 h; B: R1NH2, hydrazine monohydrate, CH3CN, 70  C, 7 h; ii)
DEEM, CH3COOH, 3 h, 120  C; iii) K2CO3, DMF, 1-pentylbromide, 100  C, 6 h; iv) 10% NaOH, MeOH, rt, 3 h; v) EDC, HOBt, DMF, amine, rt, 16 h or HBTU, DIEA, DMF, amine, rt, 16 h.
14 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

hexafluorophosphate in dimethylformamide. The starting 1,2,4- WIN 55,212-2. According to our previous pharmacophore in-
triazol-3-amines 11aeb were commercially available, while de- vestigations, compounds bearing cyclohexyl, cycloheptyl, or ada-
rivatives 11cem were prepared following two different procedures. mantanyl moieties on the carboxamide function had shown the
According to method A via cyclocondensation of amidoguanidines, highest affinity for CB2 receptor. We had also confirmed that the
a suitable hydrazide was reacted with S-methylisothiourea hemi- presence of the 1-pentyl chain on the pyridine/pyrimidine nucleus
sulfate upon heating in water to give the amidoguanidine inter- was required for CB2 receptor affinity. Consequently, we introduced
mediate. The subsequent cyclization in 10% aqueous NaOH at structural modifications at position 2 of the 7-oxo-triazolo[1,5-a]
100  C yielded aminotriazoles 11ceh [17,18]. The aminotriazoles pyrimidine-6-carboxamide scaffold, in order to analyze the SARs of
11iem were prepared according to method B, by the reaction of N- the new series. Examination of the binding data for the novel
cyanocarbonimidodithioic acid dimethyl ester with one molar compounds reveals some potent and highly selective ligands for the
equivalent of an appropriate amine followed by the reaction with CB2 receptor, e.g. compounds 23, 26e28, 32 and 39 (Table 1). It is
hydrazine monohydrate in refluxing acetonitrile (as except for 11i worth noting that the new derivatives although showed decreased
that was prepared without adding hydrazine) [19]. in affinity for CB2 receptor in comparison to the reference com-
pound (10), highlighted a higher selectivity profile versus CB1 re-
3. Biological results and discussion ceptor. Introduction of cycloheptyl and 3,5-dimethyladamantan-1-
yl groups (15 and 16, respectively) produced drastic decrease in
All of the newly synthesized compounds were examined in [3H] affinity and selectivity at CB2 receptor compared with the parent
CP-55,940 competition binding experiments for their affinity and compound 9 bearing the adamantyl moiety. The same results were
selectivity toward the human recombinant CB1 and CB2 receptors. obtained by the 2-phenyl derivatives 19e21, when the adamantly
The results are reported in Table 1 together with Ki values of pre- carboxamide was replaced, showing lower affinity and selectivity
viously published triazolopyrimidines 9, 10 and the CB2 agonist in comparison with 10. The best affinity and selectivity profile was

Table 1
Affinity (Ki, nM) and selectivity on human CB1 and CB2 receptors.a

Compd R1 R2 Ki hCB1b (nM) Ki hCB2c (nM) CB1/CB2

WIN 55,212-2 12.5 ± 1.6 4.6 ± 0.4 2.7


9 H adamantan-1-yl 712 ± 64 20 ± 2 36
10 Ph adamantan-1-yl 523 ± 47 3.6 ± 0.3 146
15 H cycloheptyl 717 ± 66 514 ± 49 1
16 H 3,5-dimethyladamantan-1-yl 622 ± 53 25 ± 3 25
17 CH3 cyclohexyl 488 ± 46 674 ± 62 1
18 CH3 adamantan-1-yl 471 ± 42 383 ± 35 1
19 Ph cyclohexyl 3214 ± 28 43 ± 4 75
20 Ph cycloheptyl 2483 ± 237 48 ± 5 52
21 Ph 3,5-dimethyladamantan-1-yl 653 ± 61 8.4 ± 0.9 77
22 4-Cl-Ph cyclohexyl 4718 ± 413 117 ± 9 40
23 4-Cl-Ph adamantan-1-yl 5216 ± 483 11.3 ± 1.9 462
24 4-OCH3-Ph cyclohexyl 5849 ± 544 96 ± 8 61
25 4-OCH3-Ph adamantan-1-yl 3723 ± 318 47 ± 4 79
26 4-CH3-Ph cyclohexyl >10,000 31 ± 3 323
27 4-CH3-Ph cycloheptyl 6352 ± 594 22 ± 2 289
28 4-CH3-Ph adamantan-1-yl 1519 ± 141 6.8 ± 0.6 223
29 furan-2-yl adamantan-1-yl 1172 ± 106 45 ± 4 26
30 pyridin-3-yl adamantan-1-yl >10,000 228 ± 19 >44
31 S-CH3 cyclohexyl >10,000 716 ± 62 >14
32 S-CH3 adamantan-1-yl >10,000 47 ± 3 >213
33 morpholine cyclohexyl >10,000 >10,000
34 morpholine cycloheptyl >10,000 566 ± 52 >18
35 morpholine adamantan-1-yl >10,000 53 ± 5 140
36 N-CH3-piperazine cyclohexyl >10,000 >10,000 >188
37 N-CH3-piperazine cycloheptyl >10,000 982 ± 87 >10
38 N-benzyl-N-methyl cyclohexyl >10,000 1332 ± 128 >7
39 N-benzyl-N-methyl adamantan-1-yl >10,000 21 ± 2 >476
40 diallylamine cyclohexyl >10,000 1352 ± 124 >7
41 diallylamine adamantan-1-yl >10,000 158 ± 13 >63
a
The data are expressed as the mean ± SEM of n ¼ 4 independent experiments.
b
The affinity values were calculated by using [3H]CP-55,940 as radioligand on human CB1CHO membranes.
c
The affinity values were calculated by using [3H]CP-55,940 as radioligand on human CB2CHO membranes.
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 15

observed by compound 21 (KiCB2 ¼ 8.4 nM, KiCB1 ¼ 653 nM, KiCB1/ their effect on cAMP production. The new ligands, as well as
KiCB2 ¼ 77). Introduction of a methyl chain at position 2, such as in compound 10, revealed an inverse agonist behavior, as they were
compounds 17 and 18, resulted in a substantial reduction of affinity able to significantly increase cAMP production, with the exception
and a total loss of selectivity for both derivatives. Intriguingly, these of compound 16 which acted as partial agonist (Fig. 1). The best
results are in contrast with those of the previously pyrazolo[3,4-b] values of Emax have been obtained by compounds 21, 23 and 28,
pyridone series [10], where the most active compounds bore a that increased the second messenger production by 243%, 223% and
methyl group or an hydrogen atom, indicating a probable different 256% at 10 mM, respectively. It is noteworthy that the same com-
binding mode of this new series to the receptor. Subsequently, pounds were also identified as the most active derivatives in
analogues by introduction of 4-substituted-phenyl at the C-2 of the binding assays, revealing a good correlation between affinity and
triazolopyrimidine nucleus were prepared, while the cyclohexyl, efficacy of the developed series. The remaining compounds pro-
cycloheptyl, and adamantyl moieties on the carboxamide were vided a comparable trend, since cAMP production increase was
maintained (compounds 22e28, Table 1). The 4-chlorophenyl obtained within 53% and 135% at 1 mM, and from 132% to 185% at
group at C-2 of the triazolopyrimidine nucleus (compounds 22 10 mM, respectively. Interestingly, the new 2-unsubstituted tri-
and 23) resulted in a decreased of affinity and selectivity for the azolopyrimidine 16 acted as partial agonist as same as its parent
cyclohexylcarboxamide 22 compared to the parent phenyl compound 9 with values of efficacies (Emax) ¼ 57 and 60%,
analogue 19. The adamantlycarboxamide derivative 23 resulted in a respectively. Moreover, the potency value of 16 measured in func-
10-fold decrease in affinity for the CB1 receptor (23: tional assays is in the nanomolar range (EC50 ¼ 88 nM) and strictly
KiCB1 ¼ 5216 nM versus 10: KiCB1 ¼ 523 nM), showing higher correlated with the binding affinity value. The difference in the
selectivity and comparable affinity for CB2 receptor relative to that functional activity between the 2-substituted derivatives and the
of analogue compound 10 (23: KiCB2 ¼ 11.3 nM, 10: KiCB2 ¼ 3.5 nM). compounds lacking this substituent indicates that introduction of
Changing of the chlorine atom with an electron donating group, structural modifications at C-2 position plays a crucial role in
such as methoxy, resulted in derivatives 24 and 25, which showed establishing the functionality of the novel cannabinoid ligands
lower affinity for the CB2 receptors (KiCB2 ¼ 96 and 47 nM, switching their activity to inverse agonism. Furthermore, the
respectively) in comparison with the phenyl analogue (10). Intro- antagonism of the new compounds was investigated, evaluating
duction of a 4-methylphenyl group at C-2, as with compounds their capability to counteract WIN 55,212-2 inhibition of forskolin-
26e28, increased selectivity in comparison with 10, and the best stimulated cAMP production. The IC50 values obtained with the
results were obtained with adamantylamide derivative 28 that concentrationeresponse curves of the new cannabinoids ligands
showed high affinity and selectivity at CB2 receptor were strictly correlated with their affinity values (Fig. 2).
(KiCB2 ¼ 6.8 nM, KiCB1/KiCB2 ¼ 223). These results seem to indicate
that electronic effects on the aromatic moiety do not significantly 5. Molecular modeling studies
affect the binding for CB2 receptors confirming our previously
found results for the pyrazolopyrimidine series [11]. Replacement The synthesized compounds were docked into the inactive CB2
of the 2-phenyl ring of 10 with a bioisosteric heterocycle such as 2- and CB1 receptor models by using AUTODOCK4.2 [20], and for each
furyl (29) resulted in a decrease of both affinity and selectivity at ligand, the top-scored pose belonging to the cluster of solutions
the CB receptors (Table 1, KiCB2 ¼ 45 nM, KiCB1/KiCB2 ¼ 26). Sub- with the best average of estimated free energy was considered. The
stitution of the 5-membered heterocycle with a 6-membered aro- resulting ligandeprotein complexes were then energy minimized
matic ring such as 3-pyridyl moiety (30) determined a dramatic with AMBER11 [21] in a lipid bilayer and water environment, since
loss of affinities at both CB receptors (KiCB2 ¼ 228 nM, molecular dynamic simulation studies carried out on the potent
KiCB1 > 10 000 nM). AMG3 CB ligand suggested that the lipid bilayer environment can
Replacement of the aromatic ring of the C-2 position by addi- affect the binding of ligands to CB receptors [22,23]. Fig. 3A shows
tional substitution on the heterocyclic nucleus had a profound ef- the minimized structures of the receptoreligand complexes ob-
fect on affinity versus CB1 receptor as shown by compounds 31e44 tained by docking compound 28, presenting the highest CB2 af-
with a KiCB1 > 10,000 nM (Table 1). The introduction of a thio- finity, into both CB2 and CB1 receptor models. In the CB2 receptor
methyl at the C-2 position (compounds 31, 32) significantly alters model the triazolopyrimidine core of the ligand is placed between
affinity at the CB2 receptor relative to that of parent compounds 19 the a-helixes belonging to the third and the sixth transmembrane
and 10, respectively (Ki ¼ 716 and 47 versus 43 and 3.5 nM), domains (TM3 and TM6), predominantly interacting with
although the selectivity increased about 1.5-fold for 32 (KiCB1/ M6.55(265) and L6.59(269) through hydrophobic interactions and
KiCB2 > 213 versus KiCB1/KiCB2 ¼ 146). Among the 2-morpholino forming an H-bond with T3.33(114). The n-pentyl chain interacts
derivatives, the highest affinity and selectivity was observed with into a sort of small lipophilic channel mainly delimited by
the 6-adamantylcarboxamide 35 (KiCB2 ¼ 53 nM, KiCB1/ L3.26(107), L3.27(108) and L6.59(269), and is directed towards the
KiCB2 > 188), while neither the 2-piperazine compounds (36, 37) TM4 a-helix, taking contacts with P4.60(168) and L4.61(169). The
nor the diallylamine derivatives (40, 41) showed affinity against the adamantyl ring interacts with V4.56(164), P4.60(168), Y5.39(190),
CB2 receptor. W5.43(194) and feels the effects of a strong van der Waals inter-
The enhancement in affinity and selectivity was obtained with action with F5.46(197), which is a nonconserved residue (V5.46 in
the 2-N-methylbenzyl chain and adamantyl moiety on the 6- the CB1 receptor) and could thus contribute to the selectivity of
carboxamide group (39, KiCB2 ¼ 21 nM, KiCB1/KiCB2 ¼ 476). This these ligands for CB2 versus CB1 receptor, consistently with site-
compound was the most selective of whole series probably because directed mutagenesis studies [24,25]. Finally, the 4-CH3-phenyl
of different binding mode into CB2 receptor due to the shape and moiety directed toward the a-helixes of the second and the seventh
volume of the C-2 substituent as shown by molecular modeling transmembrane domains (TM2 and TM7) is located in a small
studies (Fig. 3). lipophilic cavity mainly delimited by F2.57(87), I3.29(110),
L6.54(264) and F7.35(281). In the CB1 receptor model, compound
4. Human CB2 receptor functional assay 28 shows a binding pose in which it is orthogonally oriented with
respect to its disposition into the CB2 receptor (Fig. 3B), thus
The most active compounds of this series and previously de- interacting only with four transmembrane domains (TM3eTM6).
rivatives 9 and 10 were analyzed in cAMP assays for evaluation of More in details, the 4-CH3-phenyl moiety of the ligand is placed
16 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

A
300
10 EC50 = 161 ± 15 nM
% increase of forskolin- stimulated 18 EC50 = 2078 ± 213 nM
cAMP production 200 19 EC50 = 756 ± 63 nM
20 EC50 = 849 ± 74 nM

100
21 EC50 = 489 ± 42 nM
23 EC50 = 637 ± 58 nM
25 EC50 = 858 ± 77 nM
0

9 EC50 = 87 ± 9 nM

-100 WIN 55,212-2 EC50 = 15.6 ± 1.3 nM

-10 -9 -8 -7 -6 -5 -4
log drug concentration (M)

B
300
26 EC50 = 464 ± 37 nM
% increase of forskolin- stimulated

27 EC50 = 393 ± 34 nM
200 28 EC50 = 652 ± 54 nM
cAMP production

29 EC50 = 771 ± 62 nM
100 32 EC50 = 494 ± 39 nM
35 EC50 = 670 ± 61 nM
39 EC50 = 453 ± 36 nM
0

16 EC50 = 88 ± 8 nM

-100

-10 -9 -8 -7 -6 -5 -4
log drug concentration (M)
Fig. 1. Effect of the new CB compounds on cAMP accumulation in comparison with the two previous derivatives 9 and 10, and the reference agonist WIN 55,212-2 in CB2-transfected
CHO cells. Values are reported as percentage of increase of forskolin-stimulated cAMP production and are expressed as mean ± SEM of three independent experiments performed in
duplicate.

between TM3 and TM4 a-helixes, surrounded by L3.26(190), ligands.


F3.27(191) and L4.61(252) with which it establishes van der Waals Ligands such as compound 18, bearing no substituents or only a
contacts, whereas the n-pentyl chain points toward TM6 interact- methyl group in position 2 of the central scaffold, show a general
ing mainly with V6.59(367). The central scaffold and the adamantyl decreased CB2 affinity with respect to ligands presenting phenyl or
ring of the ligand are placed in the hydrophobic pocket primarily aryl groups in the same positions. The energy minimized complex
constituted by L3.29(193), T3.33(197), I4.56(247), A4.57(248), of compound 18 into CB2 receptor (Fig. 4A) shows a very similar
P4.60(251), and W5.43(279), thus forming lipophilic interactions binding mode for this ligand with respect to the one predicted for
with these residues. Due to its disposition into CB1 receptor the compound 28. The two ligands share an almost identical disposi-
ligand not only lacks the strong van der Waals interactions with tion of their n-pentyl chains and adamantyl ring, which thus take
residue 5.46 (which is V282 instead of F197, as in CB2) but it is not van der Waals contacts with the same residues. Anyway, compound
even able to form any H-bond with T3.33(197) through its tri- 18 is not able to fill the small lipophilic cavity mainly delimited by
azolopyrimidine core. These two differences, together with the F2.57(87), I3.29(110), L6.54(264) and F7.35(281), showing only
fewer hydrophobic contacts established within the CB1 receptor, limited contacts with I3.29(110) and L6.64(264). This could be at
could be the main responsible for the selectivity for CB2 versus CB1 the basis of the reduced affinity for CB2 receptor showed by this
receptor shown by compound 28 and, in general, for this series of ligand and its analogues lacking of larger substituents in C-2
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 17

A
120 10 IC50 = 8.6 ± 0.7 nM
18 IC50 = 865 ± 72 nM
% of WIN 55,212-2 response 100 19 IC50 = 111 ± 9 nM
20 IC50 = 142 ± 13 nM
80
21 IC50 = 18.4 ± 1.5 nM
60 23 IC50 = 24.6 ± 1.9 nM
25 IC50 = 128 ± 11 nM
40

20

0
-12 -10 -8 -6 -4
log drug concentration (M)

B
120 26 IC50 = 79 ± 6 nM
27 IC50 = 64 ± 5 nM
% of WIN 55,212-2 response

100 28 IC50 = 12.6 ± 1.1 nM


29 IC50 = 124± 10 nM
80
32 IC50 = 139 ± 12 nM
60 35 IC50 = 162 ± 15 nM
39 IC50 = 29 ± 3 nM
40

20

0
-12 -10 -8 -6 -4
log drug concentration (M)
Fig. 2. Antagonism of the new CB ligands evaluating their capability to counteract the inhibitory effect of WIN 55,212-2 (20 nM) on forskolin-stimulated cAMP production in CB2-
transfected CHO cells. Values are expressed as mean ± SEM of three independent experiments performed in duplicate.

position. Compound 18 shares with compound 28 also a similar selectivity versus CB1 receptor among this series of compounds. As
binding pose into CB1 receptor (Fig. 4B). Anyway, the smaller C-2 shown in Fig. 5A, the ligand adopts a different binding mode into
substituent of this ligand allows it to adopt a slightly different CB2 receptor with respect to the previously analyzed ligands, due to
orientation where the methyl group points toward the side chain of the shape and volume of the C-2 substituent that cannot be placed
L4.61(252) and the triazole ring of the central scaffold is closer to into the small hydrophobic pocket formed by F2.57(87), I3.29(110),
P4.60(251). In this disposition, the ligand is able to form an H-bond L6.54(264) and F7.35(281). Nevertheless, the compound is still able
with T3.33(197) through the carbonyl oxygen of its central scaffold; to establish the strong van der Waals interactions with F5.46(197)
this interaction could constitute the reason of the highest affinity of through its adamantyl ring, which shows the same location within
this compound for CB1 receptor with respect to what observed for the receptor as for compounds 18 and 28. Moreover, the ligand
compound 28 and generally for all those ligands bearing bulkier C- forms an H-bond with T3.33(114) through the carbonyl oxygen of
2 substituents that wouldn't allow such disposition due to steric its amide moiety, thus maintaining both the interactions that are
bumps with F3.27(191) and L4.61(252) side chains. Compound 39, thought to be responsible of the CB2 versus CB1 selectivity of these
bearing a N-benzyl-N-methyl group in 2 position of the central ligands. Finally, the C-2 substituent of the compound is efficiently
scaffold showed a strong affinity for CB2 receptor and the highest positioned into the hydrophobic channel mainly delimited by
18 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

Fig. 3. Energy minimized complex of compound 28 docked into CB2 (A) and CB1 (B) receptors.

Fig. 4. Energy minimized complex of compound 18 docked into CB2 (A) and CB1 (B) receptors.

Fig. 5. Energy minimized complex of compound 39 docked into CB2 (A) and CB1 (B) receptors.

L3.26(107), L3.27(108), L4.61(169) and Y5.39(190), forming lipo- and 18 into the CB1 receptor model, with a slight rotation of the
philic interactions with all these residues and, in particular, a strong central core in order to place the bulky benzyl moiety between TM3
pep stacking with Y5.39(190). The energy minimized complex of and TM4 a-helixes. Adopting this disposition the ligand is not able
compound 39 docked into CB1 receptor model (Fig. 5B) shows that to form any H-bond with T3.33(197) and although it maintains a
the ligand does not interact as in the CB2 receptor model. The good interaction with F3.27(191) through the benzyl group, it
compound shows a binding mode similar to that observed for 28 shows reduced contacts with L3.26(190) and V6.59(367), as well as
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 19

weaker hydrophobic interactions with T3.33(197), A3.34(198) and with cold water and dried.
I4.65(247) through the adamantyl ring, with respect to compounds
28 and 18. This could explain the low CB1 affinity of compound 39 7.1.1.1. 5-Phenyl-2H-1,2,4-triazol-3-amine (11c). Following general
and thus its strong CB2 versus CB1 selectivity. procedure A, compound 11c was isolated as a white solid. Yield 82%,
mp ¼ 233e234  C. 1H NMR (200 MHz, DMSO-d6) d: 12.58 (br s, 1H);
6. Conclusions 7.97e7.85 (m, 2H); 7.62e7.35 (m, 3H); 5.97 (br s, 2H).

In order to progress the CB2 receptor affinity and selectivity and 7.1.1.2. 5-(4-Chlorophenyl)-2H-1,2,4-triazol-3-amine (11d).
to develop structure activity relationship for the series of triazolo Following general procedure A, compound 11d was isolated as a
[1,5-a]pyrimidine-6-carboxamide, we synthesized and evaluated white solid. Yield 90%, mp ¼ 229e230  C. 1H NMR (200 MHz,
twenty-seven new derivatives belonging to this chemical class. DMSO-d6) d: 12.75 (br s, 1H); 7.95e7.89 (d, J ¼ 8.6 Hz, 2H);
Structural modifications around the central scaffold, especially at C- 7.58e7.52 (d, J ¼ 8.6 Hz, 2H); 5.11 (br s, 2H).
2 and C-6 positions, led us to achieve greater selectivity, with
respect to the parent compounds 9 and 10. The best affinity values 7.1.1.3. 5-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-amine (11e).
associated with high selectivity were obtained with compounds 23 Following general procedure A, compound 11e was isolated as pale
(KiCB2 ¼ 11.3 nM; KiCB1/KiCB2 ¼ 462) and 39 (KiCB2 ¼ 21 nM; KiCB1/ yellow solid. Yield 87%, mp > 300  C. 1H NMR (400 MHz, DMSO-d6)
KiCB2 > 476) bearing an adamantan-1-yl moiety on the carbox- d: 11.85 (br s, 1H); 7.80e7.78 (d, J ¼ 9 Hz, 2H); 6.96e6.94 (d,
amide function and a 4-Cl-phenyl or N-benzyl-N-methylamine J ¼ 8.4 Hz, 2H); 5.98 (br s, 2H); 3.77 (s, 3H).
residues at C-2, respectively. cAMP experiments on CB2 receptors
expressed in CHO cells surprisingly revealed a different behavior, 7.1.1.4. 5-p-Tolyl-2H-1,2,4-triazol-3-amine (11f). Following general
basing on the substitutions at C-2. Compound 16 as its parent 9 procedure A, compound 11f was isolated as a white solid. Yield 78%,
lacking the substituent in position C-2 of the triazolepyrimidine mp ¼ 190e193  C. 1H NMR (200 MHz, DMSO-d6) d: 12.21 (br s, 1H);
nucleus showed partial agonist activities. 2-Substituted derivatives 7.78e7.73 (d, J ¼ 8.2 Hz, 2H); 7.21e7.17 (d, J ¼ 8 Hz, 2H); 5.91 (br s.
provided an inverse agonism behavior, with high efficacy levels 2H); 2.31 (s, 3H).
from 152% to 246% of increase in cAMP production. Thus, this novel
series of compounds proposes an attractive starting point for more 7.1.1.5. 5-(Furan-2-yl)-2H-1,2,4-triazol-3-amine (11g).
optimization, representing novel pharmacological tools to evaluate Following general procedure A, compound 11g was isolated as a
the therapeutic potential of CB2 inverse agonists in diverse disease white solid. Yield 82%, mp ¼ 225e226  C. 1H NMR (400 MHz,
settings. DMSO-d6) d: 12.11 (br s, 1H); 7.60 (d, J ¼ 1 Hz, 1H); 6.67 (d,
J ¼ 3.4 Hz, 1H); 6.53 (dd, J ¼ 3.4 and 1.5 Hz, 1H); 6.07 (br s, 2H).
7. Chemistry
7.1.1.6. 5-(Pyridin-3-yl)-2H-1,2,4-triazol-3-amine (11h).
7.1. Materials and methods Following general procedure A, compound 11h was isolated as a
white solid. Yield 75%, mp ¼ 203e204  C. 1H NMR (200 MHz,
Positive ion electrospray ionization (ESI) mass spectra were DMSO-d6) d: 12.23 (br s, 1H); 9.04e9.03 (d, J ¼ 1.6 Hz, 1H);
performed with an Agilent 1100 Series LC/MSD model. All melting 8.54e8.52 (dd, J ¼ 5.4 and 1.5 Hz, 1H); 8.18e8.15 (dt, J ¼ 8 and
points are uncorrected and were assigned by a Buchi-Tottoli 1.6 Hz, 1H); 7.44e7.41 (dd, J ¼ 7.8 and 4.9 Hz, 1H); 6.17 (br s, 2H).
apparatus. The proton nuclear magnetic resonance (1H NMR)
spectra were recorded on either a Bruker AC 200 (200 MHz) or a 7.1.2. General procedure B for the synthesis of compounds 11iem
Varian Mercury Plus 400 (400 MHz) spectrometer. Chemical shifts A solution of N-cyanocarbonimidodithioic acid dimethyl ester
are reported in d values (parts per million), using appropriate (17.4 mmol), acetonitrile (10 mL), and appropriate amine
deuterated solvents (DMSO-d6 and CDCl3). (17.4 mmol) was refluxed for 2 h. After cooling to room tempera-
The purity of tested compounds was determined by combustion ture, hydrazine monohydrate (25.6 mmol) was added and the re-
elemental analyses performed by the Microanalytical Laboratory of action mixture was further refluxed for additional 5 h. The solvent
the Chemistry Department of the University of Ferrara with a was evaporated under reduced pressure and the resulting product
Yanagimoto MT-5 CHN recorder elemental analyzer. All tested recrystallized from ethyl acetate.
compounds obtained data consistent with a purity of at least 95% as
compared with the theoretical values. 7.1.2.1. 5-(Methylthio)-2H-1,2,4-triazol-3-amine (11i).
Reactions were monitored by TLC (Thin Layer Chromatography) Following general procedure B, compound 11i was isolated as a
on silica gel (precoated Merck F254 plates), and compounds visu- white solid. Yield 90%, mp ¼ 134  C. 1H NMR (200 MHz, DMSO-d6)
alization was performed by UV light. Flash chromatography was d: 11.78 (br s, 1H); 5.95 (br s, 2H); 2.39 (s, 3H).
accomplished using 230e400 mesh silica gel and a mixture of ethyl
acetate and petroleum ether or ethyl acetate and methanol in 7.1.2.2. 5-Morpholino-2H-1,2,4-triazol-3-amine (11j). Following
different ratios as eluting phase. Organic extracts were dried over general procedure B, compound 11j was isolated as a white solid.
anhydrous sodium sulfate (Na2SO4). All starting materials, reagents Yield 82%, mp ¼ 161e162  C. 1H NMR (200 MHz, DMSO-d6) d: 10.98
and solvents were obtained from Sigma-Aldrich or Alfa Aesar. (br s, 1H); 5.71 (br s, 2H); 3.64e3.59 (m, 4H); 3.13e3.08 (m, 4H).

7.1.1. General procedure A for the synthesis of compounds 11ceh 7.1.2.3. 5-(4-Methylpiperazin-1-yl)-2H-1,2,4-triazol-3-amine (11k).
A solution of S-methylisothiourea hemisulfate salt (22 mmol), Following general procedure B, compound 11k was isolated as a
appropriate hydrazide (22 mmol), and water (35 mL) was refluxed pale yellow solid. Yield 80%, mp ¼ 103e104  C. 1H NMR (200 MHz,
for 6 h. The solvent was concentrated under reduced pressure and DMSO-d6) d: 10.95 (br s, 1H); 5.72 (br s, 2H); 3.16e3.11 (m, 4H);
the residue was washed with ethanol (3  10 mL). The resulting 2.34e2.29 (m, 4H); 2.17 (s, 3H).
solid was poured into 10 mL of 10% NaOH and heated to reflux for
additional 6 h. The reaction was cooled to room temperature and 7.1.2.4. N-benzyl-N-methyl-1H-1,2,4-triazole-3,5-diamine (11l).
adjusted to pH 5 using 1 N HCl. The precipitate was filtered, washed Following general procedure B, compound 11l was isolated as a
20 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

white solid. Yield 86%, mp ¼ 163e164  C. 1H NMR (200 MHz, (s, 1H); 7.88e7.87 (d, J ¼ 1 Hz, 1H); 7.11e7.09 (d, J ¼ 3.4 Hz, 1H);
DMSO-d6) d: 10.85 (br s, 1H); 7.27e7.23 (m, 5H); 5.75 (br s, 2H); 6.67e6.65 (d, J ¼ 3.4 Hz, 1H); 4.23e4.20 (q, J ¼ 7.2 Hz, 2H);
4.42 (s, 2H); 2.70 (s, 3H). 1.31e1.24 (t, J ¼ 7 Hz, 3H).

7.1.2.5. N,N-Diallyl-1H-1,2,4-triazole-3,5-diamine (11m). 7.1.3.8. Ethyl 4,7-dihydro-7-oxo-2-(pyridin-3-yl)-[1,2,4]triazolo[1,5-


Following general procedure B, compound 11m was isolated as a a]pyrimidine-6-carboxylate (12h). Following general procedure C,
white solid. Yield 95%, mp ¼ 244e245  C. 1H NMR (400 MHz, compound 12h was isolated as a white solid. Yield 65%,
DMSO-d6) d: 10.75 (br s, 1H); 5.82e5.69 (m, 2H); 5.54 (br s, 2H); mp ¼ 250  C. 1H NMR (400 MHz, DMSO-d6) d: 11.98 (br s, 1H); 9.27
5.16e5.02 (m, 4H); 3.81e3.78 (d, J ¼ 5.6 Hz, 4H). (d, J ¼ 1.6 Hz, 1H); 8.74e8.72 (dd, J ¼ 6.4 and 1.6 Hz, 1H); 8.67 (s,
1H); 8.48e8.46 (dt, J ¼ 6.9 and 1.7 Hz, 1H); 7.62e7.60 (dd, J ¼ 7 and
7.1.3. General procedure C for the synthesis of ethyl 4,7-dihydro-7- 5.1 Hz, 1H); 4.28e4.23 (q, J ¼ 7.2 Hz, 2H); 1.31e1.27 (t, J ¼ 7.2 Hz,
oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylates 12aem 3H).
A solution of aminotriazole 11aem (3.1 mmol), glacial acetic
acid (5 mL), and diethyl ethoxymethylenemalonate (4.6 mmol) was 7.1.3.9. Ethyl 4,7-dihydro-2-(methylthio)-7-oxo-[1,2,4]triazolo[1,5-a]
refluxed for 3 h. After cooling to room temperature, the resulting pyrimidine-6-carboxylate (12i). Following general procedure C,
precipitate was collected by filtration, washed with cold water and compound 12i was isolated as a white solid. Yield 70%, mp ¼ 277  C
dried. The residue was stirred with cold ethyl ether (20 mL) and dec. 1H NMR (200 MHz, DMSO-d6) d: 12.41 (br s, 1H); 8.56 (s, 1H);
filtered to afford the desired product. 4.27e4.20 (q, J ¼ 7 Hz, 2H); 2.60 (s, 3H); 1.31e1.24 (t, J ¼ 7.2 Hz, 3H).

7.1.3.1. Ethyl 4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6- 7.1.3.10. Ethyl 4,7-dihydro-2-morpholino-7-oxo-[1,2,4]triazolo[1,5-a]


carboxylate (12a). Following general procedure C, compound 12a pyrimidine-6-carboxylate (12j). Following general procedure C,
was isolated as a white solid. Yield 76%, mp > 300  C. 1H NMR compound 12j was isolated as a white solid. Yield 75%,
(200 MHz, DMSO-d6) d: 14.30 (br s, 1H); 8.95 (s, 1H); 8.84 (s, 1H); mp ¼ 283e284  C dec. 1H NMR (200 MHz, DMSO-d6) d: 12.5 (br s,
4.20 (q, J ¼ 6.8 Hz, 2H); 1.29 (t, J ¼ 6.8 Hz, 3H). 1H); 8.45 (s, 1H); 4.23e4.20 (q, J ¼ 7.2 Hz, 2H); 3.70e3.66 (m, 4H);
3.41e3.37 (m, 4H); 1.30e1.23 (t, J ¼ 7.2 Hz, 3H).
7.1.3.2. Ethyl 4,7-dihydro-2-methyl-7-oxo-[1,2,4]triazolo[1,5-a]py-
rimidine-6-carboxylate (12b). Following general procedure C, 7.1.3.11. Ethyl 4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-[1,2,4]
compound 12b was isolated as a pale yellow solid 70%, mp ¼ 237  C triazolo[1,5-a]pyrimidine-6-carboxylate (12k). Following general
dec. 1H NMR (200 MHz, DMSO-d6) d: 12.9 (br s, 1H); 8.58 (s, 1H); procedure C, compound 12k was isolated as a white solid. Yield
4.28e4.17 (q, J ¼ 7.4 Hz, 2H); 2.38 (s, 3H); 1.31e1.27 (t, J ¼ 7 Hz, 3H). 78%, mp ¼ 220  C dec. 1H NMR (200 MHz, DMSO-d6) d: 9.95 (br s,
1H); 8.41 (s, 1H); 4.17e4.07 (q, J ¼ 7 Hz, 2H); 3.78e3.45 (m, 4H);
7.1.3.3. Ethyl 4,7-dihydro-2-phenyl-7-oxo-[1,2,4]triazolo[1,5-a]py- 2.76 (s, 3H); 2.52e2.49 (m, 4H); 1.26e1.19 (t, J ¼ 7.2 Hz, 3H).
rimidine-6-carboxylate (12c). Following general procedure C,
compound 12c was isolated as a white solid. Yield 67%, 7.1.3.12. Ethyl 2-(N-benzyl-N-methylamino)-4,7-dihydro-7-oxo-
mp > 300  C. 1H NMR (200 MHz, DMSO-d6) d: 14.00 (br s, 1H); 8.64 [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylates (12l). Following
(s, 1H); 8.14e8.10 (m, 2H); 7.55 (m, 3H); 4.26 (q, J ¼ 7.0 Hz, 2H); 1.28 general procedure C, compound 12l was isolated as a white solid.
(t, J ¼ 6.8 Hz, 3H). Yield 69%, mp ¼ 284  C. 1H NMR (200 MHz, DMSO-d6) d: 13.37 (br s,
1H); 8.42 (s, 1H); 7.30e7.28 (m, 5H); 4.64 (s, 2H); 4.25e4.17 (q,
7.1.3.4. Ethyl 2-(4-chlorophenyl)-4,7-dihydro-7-oxo-[1,2,4]triazolo J ¼ 7 Hz, 2H); 2.95 (s, 3H); 1.30e1.23 (t, J ¼ 7 Hz, 3H).
[1,5-a]pyrimidine-6-carboxylate (12d). Following general proce-
dure C, compound 12d was isolated as a pale yellow solid. Yield 7.1.3.13. Ethyl 2-(diallylamino)-4,7-dihydro-7-oxo-[1,2,4]triazolo
60%, mp > 300  C. 1H NMR (200 MHz, DMSO-d6) d: 13.41 (br s, 1H); [1,5-a]pyrimidine-6-carboxylate (12m). Following general proce-
8.65 (s, 1H); 8.14e8.10 (d, J ¼ 8.4 Hz, 2H); 7.63e7.59 (d, J ¼ 8.6 Hz, dure C, compound 12m was isolated as a white solid. Yield 73%,
1H); 4.29e4.22 (q, J ¼ 7 Hz, 2H); 1.33e1.25 (t, J ¼ 7.4 Hz, 3H). mp ¼ 240e241  C. 1H NMR (200 MHz, DMSO-d6) d: 13.91 (br s, 1H);
8.41 (s, 1H); 5.93e5.76 (m, 2H); 5.21e5.13 (m, 4H); 4.26e4.16 (q,
7.1.3.5. Ethyl 2-(4-methoxyphenyl)-4,7-dihydro-7-oxo-[1,2,4]triazolo J ¼ 7.2 Hz, 2H); 4.02e3.99 (d, J ¼ 6.2 Hz, 4H); 1.30e1.23 (t, J ¼ 7.2 Hz,
[1,5-a]pyrimidine-6-carboxylate (12e). Following general procedure 3H).
C, compound 12e was isolated as a pale yellow solid. Yield 65%,
mp > 300  C. 1H NMR (400 MHz, DMSO-d6) d: 13.25 (br s, 1H); 8.61 7.1.4. General procedure D for the synthesis of ethyl 4,7-dihydro-7-
(s, 1H); 8.06e8.04 (d, J ¼ 8.8 Hz, 2H); 7.10e7.08 (d, J ¼ 8.8 Hz, 1H); oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylates
4.25e4.23 (q, J ¼ 7.2 Hz, 2H); 3.83 (s, 3H); 1.31e1.27 (t, J ¼ 7.2 Hz, 13aem
3H). To a solution of appropriate carboxylic acid ethyl ester 12aem
(0.54 mmol) in anhydrous N,N-dimethylformamide was added
7.1.3.6. Ethyl 4,7-dihydro-7-oxo-2-p-tolyl-[1,2,4]triazolo[1,5-a]py- K2CO3 (1.62 mmol), and the mixture was stirred at 70  C for 1 h. 1-
rimidine-6-carboxylate (12f). Following general procedure C, com- Pentyl bromide (1.62 mmol) was added, and the mixture was
pound 12f was isolated as a pale yellow solid. Yield 70%, heated at 100  C for 16 h. The reaction mixture was cooled to room
mp ¼ 268  C dec. 1H NMR (200 MHz, DMSO-d6) d. 13.91 (br s, 1H); temperature, the solvent was evaporated under reduced pressure
8.63 (s, 1H); 8.03e7.99 (d, J ¼ 8 Hz, 2H); 7.37e7.32 (d, J ¼ 8.2 Hz, and the residue partitioned between water (20 mL) and ethyl ac-
2H); 4.27e4.23 (q, J ¼ 7 Hz, 2H); 2.38 (s, 3H); 1.33e1.26 (t, J ¼ 7.4 Hz, etate (60 mL). The organic phase was dried was dried (Na2SO4), and
3H). after removal of the solvent, the residue was purified by flash col-
umn chromatography, eluting with ethyl acetate/petroleum ether
7.1.3.7. Ethyl 2-(furan-2-yl)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a] or ethyl acetate/methanol.
pyrimidine-6-carboxylate (12g). Following general procedure C,
compound 12g was isolated as a white solid. Yield 75%, 7.1.4.1. Ethyl 4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]py-
mp > 300  C. 1H NMR (200 MHz, DMSO-d6) d: 13.24 (br s, 1H); 8.59 rimidine-6-carboxylate (13a). Following general procedure D,
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 21

compound 13a was isolated as a white solid. Eluent: petroleum 7.1.4.9. Ethyl 4,7-dihydro-2-(methylthio)-7-oxo-4-pentyl-[1,2,4]tri-
ethereethyl acetate 3/2. Yield 75%, mp ¼ 85e86  C. 1H NMR azolo[1,5-a]pyrimidine-6-carboxylate (13i). Following general pro-
(200 MHz, DMSO-d6) d: 8.88 (s, 1H), 8.33 (s, 1H), 4.32e4.22 (m, 4H), cedure D, compound 13i was isolated as a white solid. Eluent:
1.95e1.79 (m, 2H), 1.33e1.26 (m, 7H), 0.89e0.82 (t, J ¼ 6.8 Hz, 3H). petroleum ethereethyl acetate 1/1. Yield 81%, mp ¼ 120e121  C. 1H
NMR (200 MHz, DMSO-d6) d: 8.79 (s, 1H); 4.30e4.20 (m, 4H); 2.61
7.1.4.2. Ethyl 4,7-dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo (s, 3H); 1.91e1.72 (m, 2H); 1.32e1.25 (m, 7H); 0.89e0.84 (t,
[1,5-a]pyrimidine-6-carboxylate (13b). Following general procedure J ¼ 6.8 Hz, 3H).
D, compound 13b was isolated as a white solid. Eluent: petroleum
ethereethyl acetate 2/3. Yield 78%, mp ¼ 151e152  C. 1H NMR 7.1.4.10. Ethyl 4,7-dihydro-2-morpholino-7-oxo-4-pentyl-[1,2,4]tri-
(200 MHz, DMSO-d6) d: 8.82 (s, 1H), 4.30e4.21 (m, 4H), 2.39 (s, 3H), azolo[1,5-a]pyrimidine-6-carboxylate (13j). Following general pro-
1.91e1.77 (m, 2H), 1.32e1.25 (m, 7H), 0.89e0.84 (t, J ¼ 6.8 Hz, 3H). cedure D, compound 13j was isolated as a white solid. Eluent: ethyl
acetateemethanol 9.5/0.5. Yield 82%, mp ¼ 113e114  C. 1H NMR
(200 MHz, DMSO-d6) d: 8.68 (s, 1H); 4.30e4.13 (m, 4H); 3.71e3.66
7.1.4.3. Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo
(m, 4H); 3.43e3.38 (m, 4H); 1.95e1.72 (m, 2H); 1.31e1.24 (m, 7H);
[1,5-a]pyrimidine-6-carboxylate (13c). Following general procedure
0.89e0.83 (t, J ¼ 6.6 Hz, 3H).
D, compound 13c was isolated as a pale yellow solid. Eluent: pe-
troleum ethereethyl acetate 2/3. Yield 75%, mp ¼ 112e113  C. 1H
7.1.4.11. Ethyl 4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-4-
NMR (200 MHz, DMSO-d6) d: 8.87 (s, 1H), 8.16e8.11 (m, 2H),
pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13k).
7.56e7.53 (m, 3H), 4.37e4.23 (m, 4H), 1.93e1.81 (m, 2H), 1.37e1.26
Following general procedure D, compound 13k was isolated as a
(m, 7H), 0.91e0.85 (t, J ¼ 6.6 Hz, 3H).
white solid. Eluent: ethyl acetateemethanol 9/1. Yield 70%,
mp ¼ 183e184  C. 1H NMR (200 MHz, DMSO-d6) d: 8.67 (s, 1H);
7.1.4.4. Ethyl 2-(4-chlorophenyl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4] 4.26e4.16 (m, 4H); 3.45e3.42 (m, 4H); 2.41e2.37 (m, 4H); 2.20 (s,
triazolo[1,5-a]pyrimidine-6-carboxylate (13d). Following general 3H); 1.96e1.79 (m, 2H); 1.31e1.24 (m, 7H); 0.89e0.82 (t, J ¼ 6.8 Hz,
procedure D, compound 13d was isolated as a white solid. Eluent: 3H).
petroleum ethereethyl acetate 7/3. Yield 78%, mp ¼ 155e156  C. 1H
NMR (200 MHz, DMSO-d6) d: 8.88 (s, 1H); 8.16e8.11 (d, J ¼ 8.6 Hz, 7.1.4.12. Ethyl 2-(N-benzyl-N-methylamino)-4,7-dihydro-7-oxo-4-
2H); 7.64e7.60 (d, J ¼ 8.4 Hz, 2H); 4.32e4.23 (m, 4H); 1.96e1.83 (m, pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13l).
2H); 1.36e1.27 (m, 7H); 0.91e0.84 (t, J ¼ 6.8 Hz, 3H). Following general procedure D, compound 13l was isolated as a
white solid. Eluent: petroleum ethereethyl acetate 1/4. Yield 82%,
7.1.4.5. Ethyl 4,7-dihydro-2-(4-methoxyphenyl)-7-oxo-4-pentyl- mp ¼ 77e78  C. 1H NMR (200 MHz, DMSO-d6) d: 8.66 (s, 1H);
[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13e). Following gen- 7.34e7.29 (m, 5H); 4.65 (s, 2H); 4.29e4.15 (m, 4H); 2.95 (s, 3H);
eral procedure D, compound 13e was isolated as a white solid. 1.91e1.78 (m, 2H); 1.31e1.24 (m, 7H); 0.88e0.81 (t, J ¼ 6.8 Hz, 3H).
Eluent: petroleum ethereethyl acetate 1/1. Yield 80%,
mp ¼ 147e148  C. 1H NMR (400 MHz, DMSO-d6) d: 8.85 (s, 1H); 7.1.4.13. Ethyl 2-(diallylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]
8.07e8.05 (d, J ¼ 8.8 Hz, 2H); 7.10e7.08 (d, J ¼ 8.8 Hz, 2H); triazolo[1,5-a]pyrimidine-6-carboxylate (13m). Following general
4.33e4.25 (m, 4H); 3.83 (s, 1H); 1.94e1.81 (m, 2H); 1.34e1.29 (m, procedure D, compound 13m was isolated as a white solid. Eluent:
7H); 0.90e0.86 (t, J ¼ 7.2 Hz, 3H). petroleum ethereethyl acetate 1/4. Yield 81%, mp ¼ 70e72  C. 1H
NMR (200 MHz, DMSO-d6) d: 8.65 (s, 1H); 5.87e5.76 (m, 2H);
5.23e5.14 (m, 4H); 4.25e4.16 (m, 4H); 4.03e4.00 (d, J ¼ 5.8 Hz, 2H);
7.1.4.6. Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]triazolo
1.91e1.75 (m, 2H); 1.32e1.24 (m, 7H); 0.89e0.82 (t, J ¼ 7 Hz, 3H).
[1,5-a]pyrimidine-6-carboxylate (13f). Following general procedure
D, compound 13f was isolated as a pale yellow solid. Eluent: pe-
7.1.5. General procedure E for the synthesis of carboxylic acid
troleum ethereethyl acetate 1/4. Yield 82%, mp ¼ 216e217  C. 1H
derivatives 14aem
NMR (400 MHz, DMSO-d6) d: 8.86 (s, 1H), 8.03e8.01 (d, J ¼ 8 Hz,
A solution of appropriate carboxylic acid ethyl ester 13aem
2H), 7.36e7.34 (d, J ¼ 7.6 Hz, 2H), 4.32e4.25 (m, 4H), 2.38 (s, 3H),
(1.27 mmol), methanol (20 mL), and NaOH 10% (10 mL) was stirred
1.91e1.72 (m, 2H), 1.35e1.29 (m, 7H), 0.89e0.86 (t, J ¼ 6.8 Hz, 3H).
at room temperature for 6 h. The suspension was acidified with 10%
HCl, the precipitate was collected by filtration and washed with
7.1.4.7. Ethyl 2-(furan-2-yl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]tri- cold water to afford the desired product.
azolo[1,5-a]pyrimidine-6-carboxylate (13g). Following general pro-
cedure D, compound 13g was isolated as a white solid. Eluent: 7.1.5.1. 4,7-Dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-
petroleum ethereethyl acetate 2/3. Yield 77%, mp ¼ 151e152  C. 1H 6-carboxylic acid (14a). Following general procedure E, compound
NMR (200 MHz, DMSO-d6) d: 8.86 (s, 1H); 7.94e7.93 (d, J ¼ 1.6 Hz, 14a was isolated as a white solid. Yield 72%, mp ¼ 215  C. 1H NMR
1H); 7.20e7.18 (d, J ¼ 3.4 Hz, 1H); 6.72e6.69 (dd, J ¼ 3.4 and 1.6 Hz, (200 MHz, DMSO-d6) d: 12.12 (br s, 1H); 8.73 (s, 1H); 8.28 (s, 1H);
1H); 4.33e4.23 (m, 4H); 1.93e1.79 (m, 2H); 1.33e1.26 (m, 7H); 4.27e4.20 (t, J ¼ 7 Hz, 2H); 1.84e1.77 (m, 2H); 1.32e1.25 (m, 4H);
0.90e0.84 (t, J ¼ 6.6 Hz, 3H). 0.88e0.81 (t, J ¼ 6.6 Hz, 3H).

7.1.4.8. Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-(pyridin-3-yl)-[1,2,4]tri- 7.1.5.2. 4,7-Dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]


azolo[1,5-a]pyrimidine-6-carboxylate (13h). Following general pro- pyrimidine-6-carboxylic acid (14b). Following general procedure E,
cedure D, compound 13h was isolated as a white solid. Eluent: compound 14b was isolated as a white solid. Yield 85%,
petroleum ethereethyl acetate 1/1. Yield 77%, mp ¼ 104e105  C. 1H mp ¼ 164e165  C. 1H NMR (200 MHz, DMSO-d6) d: 12.37 (br s, 1H);
NMR (400 MHz, DMSO-d6) d: 9.46 (d, J ¼ 1.6 Hz, 1H); 8.72e8.70 (dd, 8.68 (s, 1H); 4.23e4.16 (t, J ¼ 7.2 Hz, 2H); 2.38 (s, 3H); 1.81e1.73 (m,
J ¼ 6.6 and 1.6 Hz, 1H); 8.57e8.55 (dt, J ¼ 7.8 and 1.6 Hz, 1H); 8.47 (s, 2H); 1.30e1.25 (m, 4H); 0.88e0.81 (t, J ¼ 6.8 Hz, 3H).
1H); 7.42e7.39 (dd, J ¼ 8 and 5 Hz, 1H); 4.45e4.39 (q, J ¼ 7.2 Hz,
2H); 4.34e4.30 (t, J ¼ 7.6 Hz, 2H); 2.01e1.97 (m, 2H); 1.43e1.39 (m, 7.1.5.3. 4,7-Dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo[1,5-a]
7H); 0.95e0.92 (t, J ¼ 7.2 Hz, 3H). pyrimidine-6-carboxylic acid (14c). Following general procedure E,
22 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

compound 14c was isolated as a white solid. Yield 91%, Following general procedure E, compound 14k was isolated as a
mp > 300  C. 1H NMR (200 MHz, DMSO-d6) d:12.24 (br s, 1H); 8.63 white solid. Yield 81%, mp ¼ 225  C dec. 1H NMR (200 MHz, DMSO-
(s, 1H); 8.14e8.10 (m, 2H); 7.54e7.51 (m, 3H); 4.30e4.26 (t, J ¼ 7 Hz, d6) d: 12.72 (br s, 1H); 8.73 (s, 1H); 4.21e4.17 (t, J ¼ 6.6 Hz, 2H);
2H); 1.98e1.81 (m, 2H); 1.39e1.27 (m, 4H); 0.89e0.82 (t, J ¼ 6.6 Hz, 3.50e3.47 (m, 4H); 3.38e3.33 (m, 4H); 2.28 (s, 3H); 1.81e1.77 (m,
3H). 2H); 1.29e1.26 (m, 4H); 0.88e0.82 (t, J ¼ 6.6 Hz, 3H).

7.1.5.4. 2-(4-Chlorophenyl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]tri- 7.1.5.12. 2-(N-Benzyl-N-methylamino)-4,7-dihydro-7-oxo-4-pentyl-


azolo[1,5-a]pyrimidine-6-carboxylic acid (14d). Following general [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14l).
procedure E, compound 14d was isolated as a white solid. Yield Following general procedure E, compound 14l was isolated as a
(87%), mp ¼ 204e205  C. 1H NMR (200 MHz, DMSO-d6) d: 12.81 (br white solid. Yield 94%, mp ¼ 130  C. 1H NMR (400 MHz, DMSO-d6)
s, 1H); 8.92 (s, 1H); 8.16e8.12 (d, J ¼ 8.6 Hz, 2H); 7.64e7.60 (d, d: 12.71 (br s, 1H); 8.76 (s, 1H); 7.33e7.24 (m, 5H); 4.64 (s, 2H);
J ¼ 8.6 Hz, 2H); 4.37e4.30 (t, J ¼ 7.2 Hz, 2H); 1.81e1.75 (m, 2H); 4.22e4.19 (t, J ¼ 7.2 Hz, 2H); 2.95 (s, 3H); 1.81e1.78 (m, 2H);
1.36e1.28 (m, 4H); 0.90e0.84 (t, J ¼ 6.6 Hz, 3H). 1.29e1.24 (m, 4H); 0.84e0.81 (t, J ¼ 6.8 Hz, 3H).

7.1.5.5. 4,7-Dihydro-2-(4-methoxyphenyl)-7-oxo-4-pentyl-[1,2,4]tri- 7.1.5.13. 2-(Diallylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo


azolo[1,5-a]pyrimidine-6-carboxylic acid (14e). Following general [1,5-a]pyrimidine-6-carboxylic acid (14m). Following general pro-
procedure E, compound 14e was isolated as a white solid. Yield 86%, cedure E, compound 14m was isolated as a white solid. Yield 95%,
mp ¼ 200e201  C. 1H NMR (400 MHz, DMSO-d6) d: 12.79 (br s, 1H); mp ¼ 142e143  C. 1H NMR (400 MHz, DMSO-d6) d: 12.95 (br s, 1H);
8.90 (s, 1H); 8.08e8.06 (d, J ¼ 8.8 Hz, 2H); 7.11e7.08 (d, J ¼ 8.4 Hz, 8.77 (s, 1H); 5.92e5.78 (m, 2H); 5.23e5.15 (m, 4H); 4.24e4.17 (t,
2H); 4.35e4.31 (t, J ¼ 7.2 Hz, 2H); 1.89e1.86 (m, 2H); 1.34e1.32 (m, J ¼ 6.8 Hz, 2H); 4.05e4.02 (d, J ¼ 5.6 Hz, 4H); 1.84e1.79 (m, 2H);
4H); 0.89e0.86 (t, J ¼ 6.4 Hz, 3H). 1.30e1.26 (m, 4H); 0.88e0.82 (t, J ¼ 6.6 Hz, 3H).

7.1.5.6. 4,7-Dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]triazolo[1,5-a] 7.1.6. General procedure F for the synthesis of adamantan-1-yl-


pyrimidine-6-carboxylic acid (14f). Following general procedure E, carboxamide derivatives 16, 18, 21, 23, 25, 28e30, 32, 35, 39, 41
compound 14f was isolated as a pale yellow solid. Yield 87%, To a stirred solution of the appropriate carboxylic acid
mp ¼ 207e208  C. 1H NMR (200 MHz, DMSO-d6) d: 12.65 (br s, 1H); (0.72 mmol) in anhydrous dimethylformamide (10 mL) was added
8.87 (s, 1H); 8.05e8.01 (d, J ¼ 8.4 Hz, 2H); 7.37e7.33 (d, J ¼ 8 Hz, diisopropylethylamine (3.24 mmol). The resulting solution was
2H); 4.34e4.31 (t, J ¼ 6.8 Hz, 2H); 2.38 (s, 3H); 1.96e1.79 (m, 2H); stirred at room temperature for 10 min before addition of O-ben-
1.35e1.30 (m, 4H); 0.90e0.84 (t, J ¼ 6.6 Hz, 3H). zotriazol-1-yl-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate
(HBTU, 1.08 mmol). The solution was stirred for another 3 h at room
7.1.5.7. 2-(Furan-2-yl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo temperature. 1-Adamantylamine or 3,5-dimethyl-1-
[1,5-a]pyrimidine-6-carboxylic acid (14g). Following general pro- adamantylamine (1.08 mmol) was added and stirring was
cedure E, compound 14g was isolated as a pale yellow solid. Yield continued for 16 h. The solvent was removed under reduce pres-
87%, mp ¼ 187e188  C. 1H NMR (200 MHz, DMSO-d6) d: 12.81 (br s, sure, the residue was dissolved in ethyl acetate (50 mL), then
1H); 8.90 (s, 1H); 7.95e7.94 (d, J ¼ 1.8 Hz, 1H); 7.21e7.19 (d, washed with saturated aqueous sodium bicarbonate (10 mL), water
J ¼ 3.4 Hz, 1H); 6.72e6.70 (dd, J ¼ 3.4 and 1.6 Hz, 1H); 4.32e4.26 (t, (10 mL), and brine (10 mL). The organic extract was dried (Na2SO4),
J ¼ 6.8 Hz, 2H); 1.87e1.81 (m, 2H); 1.34e1.27 (m, 4H); 0.90e0.83 (t, filtered, and evaporated to dryness, and the residue was purified by
J ¼ 6.6 Hz, 3H). flash chromatography on silica gel, eluting with ethyl aceta-
teepetroleum ether.
7.1.5.8. 4,7-Dihydro-7-oxo-4-pentyl-2-(pyridin-3-yl)-[1,2,4]triazolo
[1,5-a]pyrimidine-6-carboxylic acid (14h). Following general pro- 7.1.6.1. 4,7-Dihydro-N-(3,5-dimethyladamantan-1-yl)-7-oxo-4-
cedure E, compound 14h was isolated as a white solid. Yield 86%, pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (16).
mp ¼ 158e159  C. 1H NMR (400 MHz, DMSO-d6) d: 12.84 (br s, 1H); Following general procedure F, compound 16 was isolated as a
9.28 (d, J ¼ 1.6 Hz, 1H); 8.94 (s, 1H); 8.74e8.73 (dd, J ¼ 6.4 and white solid. Eluent: petroleum ethereethyl acetate 7/3. Yield 60%,
1.8 Hz, 1H); 8.48e8.45 (dt, J ¼ 8 and 1.6 Hz, 1H); 7.61e7.58 (dd, J ¼ 8 mp ¼ 182  C. MS (ESI): m/z 412.2 (MþH). 1H NMR (200 MHz, DMSO-
and 4.8 Hz, 1H); 4.37e4.33 (t, J ¼ 7.2 Hz, 2H); 1.90e1.87 (m, 2H); d6) d: 8.82 (s, 1H); 8.67 (br s, 1H); 8.38 (s, 1H); 4.33e4.30 (t, J ¼ 7 Hz,
1.37e1.32 (m, 4H); 0.89e0.86 (t, J ¼ 6.8 Hz, 3H). 2H); 2.18e2.04 (m, 1H); 1.90e1.65 (m, 9H); 1.38e1.24 (m, 9H);
0.89e0.82 (m, 9H). Anal. for C23H33N5O2. Calcd: C, 67.12; H, 8.08; N,
7.1.5.9. 4,7-Dihydro-2-(methylthio)-7-oxo-4-pentyl-[1,2,4]triazolo 17.02. Found: C, 67.22; H, 8.18; N, 17.051.
[1,5-a]pyrimidine-6-carboxylic acid (14i). Following general proce-
dure E, compound 14i was isolated as a white solid. Yield 98%, 7.1.6.2. N-Adamantan-yl-4,7-dihydro-2-methyl-7-oxo-4-pentyl-
mp ¼ 165e166  C. 1H NMR (200 MHz, DMSO-d6) d: 12.51 (br s, 1H); [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (18). Following
8.78 (s, 1H); 4.22e4.19 (t, J ¼ 6.8 Hz, 2H); 2.61 (s, 3H); 1.84e1.77 (m, general procedure F, compound 18 was isolated as a white solid.
2H); 1.35e1.26 (m, 4H); 0.88e0.83 (t, J ¼ 6.4 Hz, 3H). Eluent: petroleum ethereethyl acetate 3/2. Yield 56%,
mp ¼ 189e190  C. MS (ESI): m/z 398.2 (MþH). 1H NMR (200 MHz,
7.1.5.10. 4,7-Dihydro-2-morpholino-7-oxo-4-pentyl-[1,2,4]triazolo DMSO-d6) d: 8.78 (s, 1H); 8.68 (br s, 1H); 4.30e4.22 (t, J ¼ 7 Hz, 2H);
[1,5-a]pyrimidine-6-carboxylic acid (14j). Following general proce- 2.41 (s, 3H); 2.05 (s, 9H); 1.93e1.81 (m, 2H); 1.66 (s, 6H); 1.34e1.24
dure E, compound 14j was isolated as a white solid. Yield 98%, (m, 4H); 0.89e0.82 (t, J ¼ 6.4 Hz, 3H). Anal. for C22H31N5O2. Calcd: C,
mp ¼ 276e277  C. 1H NMR (200 MHz, DMSO-d6) d: 12.69 (br s, 1H); 66.47; H, 7.86; N, 17.62. Found: C, 66.22; H, 7.73; N, 17.37.
8.38 (s, 1H); 4.13e4.09 (t, J ¼ 6.8 Hz, 2H); 3.70e3.66 (m, 4H);
3.41e3.36 (m, 4H); 1.78e1.73 (m, 2H); 1.30e1.25 (m, 4H); 7.1.6.3. 4,7-Dihydro-N-(3,5-dimethyladamantan-1-yl)-7-oxo-4-
0.88e0.81 (t, J ¼ 6.6 Hz, 3H). pentyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide
(21). Following general procedure F, compound 21 was isolated as
7.1.5.11. 4,7-Dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-4-pentyl- a white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 55%,
[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14k). mp ¼ 96  C. MS (ESI): m/z 488.2 (MþH). 1H NMR (200 MHz, DMSO-
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 23

d6) d: 8.81 (s, 1H); 8.72 (br s, 1H); 8.16e8.13 (m, 2H); 7.57e7.54 (m, white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 55%,
3H); 4.39e4.35 (t, J ¼ 7 Hz, 2H); 2.19e2.07 (m, 1H); 1.93e1.68 (m, mp ¼ 161  C. MS (ESI): m/z 430.2 (MþH). 1H NMR (200 MHz,
9H); 1.39e1.18 (m, 9H); 0.91e0.84 (m, 9H). Anal. for C29H37N5O2. DMSO-d6) d: 8.75 (s, 1H); 8.67 (br s, 1H); 4.27e4.22 (t, J ¼ 7 Hz, 2H);
Calcd: C, 71.43; H, 7.65; N, 14.36. Found: C, 71.34; H, 7.60; N, 14.29. 2.61 (s, 3H); 2.05 (s, 9H); 1.91e1.74 (m, 2H); 1.67 (s, 6H); 1.33e1.25
(m, 4H); 0.89e0.83 (t, J ¼ 6.4 Hz, 3H). Anal. for C22H31N5O2S. Calcd:
7.1.6.4. N-Adamantan-1-yl-2-(4-chlorophenyl)-4,7-dihydro-7-oxo- C, 61.51; H, 7.27; N, 16.30. Found: C, 61.34; H, 7.29; N, 16.45.
4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (23).
Following general procedure F, compound 23 was isolated as a 7.1.6.10. N-Adamantan-1-yl-4,7-dihydro-2-morpholino-7-oxo-4-
white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 53%, pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (35).
mp ¼ 213  C. MS (ESI): m/z 494.2 (MþH). 1H NMR (200 MHz, Following general procedure F, compound 35 was isolated as a
DMSO-d6) d: 8.85 (s, 1H); 8.69 (br s, 1H); 8.16e8.12 (d, J ¼ 8.6 Hz, white solid. Eluent: petroleum ethereethyl acetate 2/3. Yield 40%,
2H); 7.65e7.61 (d, J ¼ 8.4 Hz, 2H); 4.38e4.34 (t, J ¼ 7.2 Hz, 2H); 2.06 mp ¼ 148  C. MS (ESI): m/z 469.2 (MþH). 1H NMR (200 MHz,
(s, 9H); 1.97e1.84 (m, 2H); 1.68 (s, 6H); 1.36e1.31 (m, 4H); DMSO-d6) d: 8.81 (br s, 1H); 8.64 (s, 1H); 4.08e3.99 (t, J ¼ 6.8 Hz,
0.90e0.84 (t, J ¼ 6.2 Hz, 3H). Anal. for C27H32ClN5O2. Calcd: C, 2H); 3.71e3.67 (m, 4H); 3.44e3.40 (m, 4H); 2.03 (s, 9H); 1.85e1.70
65.64; H, 6.53; N, 14.18. Found: C, 65.59; H, 6.42; N, 14.29. (m, 2H); 1.66 (s, 6H); 1.33e1.26 (m, 4H); 0.89e0.83 (t, J ¼ 6.6 Hz,
3H). Anal. for C25H36N6O3. Calcd: C, 64.08; H, 7.74; N, 17.93. Found:
7.1.6.5. N-Adamantan-1-yl-4,7-dihydro-2-(4-methoxyphenyl)-7- C, 64.19; H, 7.79; N, 17.90.
oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (25).
Following general procedure F, compound 25 was isolated as a 7.1.6.11. N-Adamantan-1-yl-2-(N-benzyl-N-methyl-amino)-4,7-
white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 46%, dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
mp ¼ 176  C. MS (ESI): m/z 490.2 (MþH). 1H NMR (200 MHz, carboxamide (39). Following general procedure F, compound 39
DMSO-d6) d: 8.81 (s, 1H); 8.73 (br s, 1H); 8.09e8.05 (d, J ¼ 8.8 Hz, was isolated as a white solid. Eluent: petroleum ethereethyl ace-
2H); 7.12e7.08 (d, J ¼ 9 Hz, 2H); 4.37e4.34 (t, J ¼ 7.2 Hz, 2H); 3.83 (s, tate 2/3. Yield 60%, mp ¼ 139  C. MS (ESI): m/z 519.2 (MþH). 1H
3H); 2.06 (s, 9H); 1.96e1.81 (m, 2H); 1.67 (s, 6H); 1.36e1.32 (m, 4H); NMR (200 MHz, DMSO-d6) d: 8.85 (br s, 1H); 8.61 (s, 1H); 7.34e7.29
0.91e0.85 (t, J ¼ 6.6 Hz, 3H). Anal. for C28H35N5O3. Calcd: C, 68.69; (m, 5H); 4.65 (s, 2H); 4.24e4.19 (t, J ¼ 7 Hz, 2H); 2.97 (s, 3H); 2.03 (s,
H, 7.21; N, 14.30. Found: C, 68.75; H, 7.28; N, 14.39. 9H); 1.87e1.75 (m, 2H); 1.66 (s, 6H); 1.30e1.26 (m, 4H); 0.88e0.82
(t, J ¼ 6.2 Hz, 3H). Anal. for C29H38N6O2. Calcd: C, 69.29; H, 7.62; N,
7.1.6.6. N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl- 16.72. Found: C, 69.32; H, 7.77; N, 16.68.
[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (28). Following
general procedure F, compound 28 was isolated as a white solid. 7.1.6.12. N-Adamantan-1-yl-2-(diallylamino)-4,7-dihydro-7-oxo-4-
Eluent: petroleum ethereethyl acetate 4/1. Yield 44%, mp ¼ 229  C. pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (41).
MS (ESI): m/z 474.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.82 (s, Following general procedure F, compound 41 was isolated as a
1H); 8.72 (br s, 1H); 8.05e8.01 (d, J ¼ 8 Hz, 2H); 7.38e7.34 (d, white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 45%,
J ¼ 8.2 Hz, 2H); 4.38e4.33 (t, J ¼ 7 Hz, 2H); 2.38 (s, 3H); 2.06 (s, 9H); mp ¼ 110  C. MS (ESI): m/z 479.2 (MþH). 1H NMR (200 MHz, DMSO-
1.90e1.83 (m, 2H); 1.68 (s, 6H); 1.36e1.30 (m, 4H); 0.90e0.84 (t, d6) d: 8.84 (br s, 1H); 8.60 (s, 1H); 5.87e5.78 (m, 2H); 5.24e5.14 (m,
J ¼ 6.4 Hz, 3H). Anal. for C28H35N5O2. Calcd: C, 71.01; H, 7.45; N, 4H); 4.22e4.16 (t, J ¼ 6.8 Hz, 2H); 4.04e4.01 (d, J ¼ 5.6 Hz, 4H); 2.03
14.79. Found: C, 71.15; H, 7.52; N, 15.75. (s, 9H); 1.90e1.72 (m, 2H); 1.66 (s, 6H); 1.32e1.25 (m, 4H);
0.88e0.82 (t, J ¼ 6.6 Hz, 3H). Anal. for C27H38N6O2. Calcd: C, 67.75;
7.1.6.7. N-Adamantan-1-yl-2-(furan-2-yl)-4,7-dihydro-7-oxo-4- H, 8.00; N, 17.56. Found: C, 67.71; H, 7.98; N, 17.49.
pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (29).
Following general procedure F, compound 29 was isolated as a 7.1.7. General procedure G for the synthesis of N-cyclohexyl/
white solid. Eluent: petroleum ethereethyl acetate 1/1. Yield 54%, cycloheptyl carboxamide derivatives 15, 17, 19, 20, 22, 24, 26, 27,
mp ¼ 175  C. MS (ESI): m/z 450.2 (MþH). 1H NMR (200 MHz, 31, 33, 34, 36e38, 40
DMSO-d6) d: 8.82 (s, 1H); 8.67 (br s, 1H); 7.96e7.94 (d, J ¼ 1.6 Hz, To a solution of the appropriate carboxylic acid (0.57 mmol) in
1H); 7.22e7.19 (d, J ¼ 3.4 Hz, 1H); 6.73e6.70 (dd, J ¼ 3.4 and 1.8 Hz, DMF, 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide (EDCI,
1H); 4.37e4.29 (t, J ¼ 6.8 Hz, 2H); 2.06 (s, 9H); 1.96e1.82 (m, 2H); 0.85 mmol) and 1-hydroxybenzotriazole (HOBt, 0.85 mmol) were
1.67 (s, 6H); 1.37e1.30 (m, 4H); 0.90e0.84 (t, J ¼ 6.2 Hz, 3H). Anal. added and the reaction was stirred at room temperature for 10 min
for C25H31N5O3. Calcd: C, 66.79; H, 6.95; N, 15.58. Found: C, 66.75; before adding the suitable amine (0.85 mmol) and the stirring
H, 6.96; N, 15.61. continued at the same temperature for 16 h. The solvent was
evaporated under reduced pressure and the residue was dissolved
7.1.6.8. N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-(pyridin- in ethyl acetate (30 mL), washed with aqueous bicarbonate (10 mL),
3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (30). water (10 mL) and brine (10 mL). The organic extract was dried,
Following general procedure F, compound 30 was isolated as a filtered and concentrated and resulting products were purified by
white solid. Eluent: petroleum ethereethyl acetate 1/1. Yield 45%, flash chromatography on silica gel, eluting with ethyl acetate and
mp ¼ 160  C. MS (ESI): m/z 461.2 (MþH). 1H NMR (200 MHz, DMSO- petroleum ether or ethyl acetate and methanol.
d6) d: 9.29 (d, J ¼ 1.6 Hz, 1H); 8.87 (s, 1H); 8.76e8.72 (dd, J ¼ 7.4 and
1.8 Hz, 1H); 8.68 (br s, 1H); 8.49e8.43 (dt, J ¼ 7.8 and 1.6 Hz, 1H); 7.1.7.1. N-Cycloheptyl-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo
7.63e7.57 (dd, J ¼ 7.8 and 4.2 Hz, 1H); 4.41e4.36 (t, J ¼ 6.8 Hz, 2H); [1,5-a]pyrimidine-6-carboxamide (15). Following general procedure
2.07 (s, 9H); 1.99e1.85 (m, 2H); 1.74e1.59 (m, 8H); 1.36e1.32 (m, G, compound 15 was isolated as a white solid. Eluent: petroleum
2H); 0.91e0.84 (t, J ¼ 6 Hz, 3H). Anal. for C26H32N6O2. Calcd: C, ethereethyl acetate 2/3. Yield 46%, mp ¼ 149  C. MS (ESI): m/z
67.80; H, 7.00; N, 18.25. Found: C, 67.74; H, 7.23; N, 18.33. 346.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.89 (s, 1H);
8.85e8.83 (br d, J ¼ 7.8 Hz, 1H); 8.39 (s, 1H); 4.34e4.30 (t, J ¼ 7 Hz,
7.1.6.9. N-Adamantan-1-yl-4,7-dihydro-2-(methylthio)-7-oxo-4- 2H); 4.17e4.01 (m, 1H); 1.96e1.75 (m, 4H); 1.70e1.42 (m, 10H);
pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (32). 1.35e1.24 (m, 4H); 0.88e0.83 (t, J ¼ 6.4 Hz, 3H). Anal. for
Following general procedure F, compound 32 was isolated as a C18H27N5O2. Calcd: C, 62.58; H, 7.88; N, 20.27. Found: C, 62.51; H,
24 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

7.82; N, 20.39. 0.90e0.84 (t, J ¼ 6.4 Hz, 3H). Anal. for C24H31N5O2. Calcd: C, 68.38;
H, 7.41; N, 16.61. Found: C, 68.42; H, 7.45; N, 16.69.
7.1.7.2. N-Cyclohexyl-4,7-dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]
triazolo[1,5-a]pyrimidine-6-carboxamide (17). Following general 7.1.7.8. N-Cycloheptyl-4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]
procedure G, compound 17 was isolated as a white solid. Eluent: triazolo[1,5-a]pyrimidine-6-carboxamide (27). Following general
petroleum ethereethyl acetate 2/3. Yield 41%, mp ¼ 120  C. MS procedure G, compound 27 was isolated as a white solid. Eluent:
(ESI): m/z 346.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.83 (s, petroleum ethereethyl acetate 7/3. Yield 48%, mp ¼ 195  C. MS
1H); 8.81e8.77 (br d, J ¼ 7.8 Hz, 1H); 4.31e4.24 (t, J ¼ 7 Hz, 2H); (ESI): m/z 436.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.91e8.90
3.91e3.78 (m, 1H); 2.41 (s, 3H); 1.94e1.54 (m, 6H); 1.43e1.24 (m, (br d, J ¼ 7.8 Hz, 1H); 8.87 (s, 1H); 8.05e8.01 (d, J ¼ 8 Hz, 2H);
10H); 0.89e0.82 (t, J ¼ 6.6 Hz, 3H). Anal. for C18H27N5O2. Calcd: C, 7.38e7.34 (d, J ¼ 7.8 Hz, 2H); 4.39e4.36 (t, J ¼ 7 Hz, 2H); 4.17e4.02
62.58; H, 7.88; N, 20.27. Found: C, 62.61; H, 7.59; N, 20.32. (m, 1H); 2.38 (s, 3H); 1.97e1.81 (m, 4H); 1.74e1.49 (m, 10H);
1.36e1.28 (m, 4H); 0.90e0.84 (t, J ¼ 6.4 Hz, 3H). Anal. for
7.1.7.3. N-Cyclohexyl-4,7-dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4] C25H33N5O2. Calcd: C, 68.94; H, 7.64; N, 16.08. Found: C, 68.74; H,
triazolo[1,5-a]pyrimidine-6-carboxamide (19). Following general 7.68; N, 16.12.
procedure G, compound 19 was isolated as a white solid. Eluent:
petroleum ethereethyl acetate 4/1. Yield 55%, mp ¼ 161  C. MS 7.1.7.9. N-Cyclohexyl-4,7-dihydro-2-(methylthio)-7-oxo-4-pentyl-
(ESI): m/z 408.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.90 (s, [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (31). Following
1H); 8.85e8.81 (br d, J ¼ 8 Hz, 1H); 8.17e8.12 (m, 2H); 7.57e7.54 (m, general procedure G, compound 31 was isolated as a white solid.
3H); 4.40e4.35 (t, J ¼ 7 Hz, 2H); 3.94e3.81 (m, 1H); 1.91e1.58 (m, Eluent: petroleum ethereethyl acetate 7/3. Yield 50%, mp ¼ 192  C.
6H); 1.39e1.28 (m, 10H); 0.90e0.84 (t, J ¼ 6.4 Hz, 3H). Anal. for MS (ESI): m/z 378.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d:
C23H29N5O2. Calcd: C, 67.79; H, 7.17; N, 17.19. Found: C, 67.68; H, 8.80e8.77 (m, 2H); 4.27e4.21 (t, J ¼ 6.8 Hz, 2H); 3.87e3.75 (m, 1H);
6.94; N, 17.25. 2.60 (s, 3H); 1.91e1.59 (m, 6H); 1.42e1.21 (m, 10H); 0.88e0.81 (t,
J ¼ 6.4 Hz, 3H). Anal. for C18H27N5O2S. Calcd: C, 57.27; H, 7.21; N,
7.1.7.4. N-Cycloheptyl-4,7-dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4] 18.55. Found: C, 57.21; H, 7.25; N, 18.61.
triazolo[1,5-a]pyrimidine-6-carboxamide (20). Following general
procedure G, compound 20 was isolated as a white solid. Eluent: 7.1.7.10. N-Cyclohexyl-4,7-dihydro-2-morpholino-7-oxo-4-pentyl-
petroleum ethereethyl acetate 7/3. Yield 48%, mp ¼ 155  C. MS [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (33). Following
(ESI): m/z 422.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d; 8.90e8.98 general procedure G, compound 33 was isolated as a white solid.
(m, 2H); 8.15e8.12 (m, 2H); 7.57e7.54 (m, 3H); 4.40e4.36 (t, Eluent: petroleum ethereethyl acetate 1/1. Yield 44%, mp ¼ 143  C.
J ¼ 7 Hz, 2H); 4.15e4.03 (m, 1H); 1.93e1.82 (m, 4H); 1.71e1.44 (m, MS (ESI): m/z 417.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d:
10H); 1.36e1.30 (m, 4H); 0.91e0.85 (t, J ¼ 6.4 Hz, 3H). Anal. for 8.95e8.91 (d, J ¼ 7.8 Hz, 1H); 8.70 (s, 1H); 4.24e4.20 (t, J ¼ 7 Hz,
C24H31N5O2. Calcd: C, 68.38; H, 7.41; N, 16.61. Found: C, 68.22; H, 2H); 3.89e3.72 (m, 1H); 3.71e3.67 (m, 4H); 3.45e3.40 (m, 4H);
7.55; N, 16.55. 1.81e1.47 (m, 6H); 1.39e1.20 (m, 10H); 0.88e0.82 (t, J ¼ 6.4 Hz, 3H).
Anal. for C21H32N6O3. Calcd: C, 60.56; H, 7.74; N, 20.18. Found: C,
7.1.7.5. 2-(4-Chlorophenyl)-N-cyclohexyl-4,7-dihydro-7-oxo-4- 60.61; H, 7.69; N, 20.59.
pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (22).
Following general procedure G, compound 22 was isolated as a 7.1.7.11. N-Cycloheptyl-4,7-dihydro-2-morpholino-7-oxo-4-pentyl-
white solid. Eluent: petroleum ethereethyl acetate 4/1. Yield 53%, [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (34). Following
mp ¼ 212  C. MS (ESI): m/z 442.2 (MþH). 1H NMR (200 MHz, general procedure G, compound 34 was isolated as a white solid.
DMSO-d6) d: 8.90 (s, 1H); 8.83e8.79 (br d, J ¼ 8.2 Hz, 1H); 8.16e8.12 Eluent: petroleum ethereethyl acetate 1/1. Yield 50%, mp ¼ 172  C.
(d, J ¼ 8.4 Hz, 2H); 7.65e7.61 (d, J ¼ 8.4 Hz, 2H); 4.41e4.35 (t, MS (ESI): m/z 431.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d:
J ¼ 7.2 Hz, 2H); 3.97e3.82 (m, 1H); 2.41 (s, 3H); 1.90e1.57 (m, 6H); 9.00e8.96 (br d, J ¼ 7.8 Hz, 1H); 8.69 (s, 1H); 4.25e4.18 (t, J ¼ 6.8 Hz,
1.39e1.23 (m, 10H); 0.90e0.84 (t, J ¼ 6.4 Hz, 3H). Anal. for 2H); 4.15e4.02 (m, 1H); 3.71e3.67 (m, 4H); 3.45e3.40 (m, 4H);
C23H28ClN5O2. Calcd: C, 62.51; H, 6.39; N, 15.85. Found: C, 62.59; H, 1.89e1.73 (m, 4H); 1.66e1.48 (m, 10H); 1.32e1.24 (m, 4H);
6.20; N, 15.72. 0.88e0.82 (t, J ¼ 6.6 Hz, 3H). Anal. for C22H34N6O3. Calcd: C, 61.37;
H, 7.96; N, 19.52. Found: C, 61.46; H, 7.86; N, 19.58.
7.1.7.6. N-Cyclohexyl-4,7-dihydro-2-(4-methoxyphenyl)-7-oxo-4-
pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (24). 7.1.7.12. N-Cyclohexyl-4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-
Following general procedure G, compound 24 was isolated as a oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (36).
white solid. Eluent: petroleum ethereethyl acetate 7/3. Yield 53%, Following general procedure G, compound 36 was isolated as a
mp ¼ 190  C. MS (ESI): m/z 438.2 (MþH). 1H NMR (200 MHz, white solid. Eluent: ethyl acetateemethanol 4/1. Yield 40%,
DMSO-d6) d: 8.88 (s, 1H); 8.86 (br s, 1H); 8.10e8.05 (d, J ¼ 9.2 Hz, mp ¼ 168  C. MS (ESI): m/z 430.2 (MþH). 1H NMR (200 MHz,
2H); 7.13e7.08 (d, J ¼ 8.6 Hz, 2H); 4.39e4.35 (t, J ¼ 7.2 Hz, 2H); DMSO-d6) d: 8.96e8.92 (br d, J ¼ 8 Hz, 1H); 8.68 (s, 1H); 4.24e4.18
3.42e3.84 (m, 1H); 3.83 (s, 3H); 1.98e1.61 (m, 6H); 1.38e1.30 (m, (t, J ¼ 6.6 Hz, 2H); 3.89e3.78 (m, 1H); 3.47e3.42 (m, 4H); 2.40e2.36
10H); 0.90e0.85 (t, J ¼ 6.6 Hz, 3H). Anal. for C24H31N5O3. Calcd: C, (m, 4H); 2.21 (s, 3H); 1.81e1.49 (m, 6H); 1.41e1.22 (m, 10H);
65.88; H, 7.14; N, 16.01. Found: C, 65.68; H, 7.22; N, 16.12. 0.89e0.82 (t, J ¼ 6.8 Hz, 3H). Anal. for C22H35N7O2. Calcd: C, 61.51;
H, 8.21; N, 22.83. Found: C, 61.54; H, 8.24; N, 22.69.
7.1.7.7. N-Cyclohexyl-4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]
triazolo[1,5-a]pyrimidine-6-carboxamide (26). Following general 7.1.7.13. N-Cycloheptyl-4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-
procedure G, compound 26 was isolated as a white solid. Eluent: oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (37).
petroleum ethereethyl acetate 7/3. Yield 50%, mp ¼ 200  C. MS Following general procedure G, compound 37 was isolated as a
(ESI): m/z 422.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: 8.88 (s, white solid. Eluent: ethyl acetateemethanol 4/1. Yield 40%,
1H); 8.87e8.86 (br d, J ¼ 7.8 Hz, 1H); 8.05e8.01 (d, J ¼ 8.4 Hz, 2H); mp ¼ 183  C. MS (ESI): m/z 444.2 (MþH). 1H NMR (200 MHz,
7.38e7.34 (d, J ¼ 8 Hz, 2H); 4.39e4.36 (t, J ¼ 7 Hz, 2H); 3.93e3.81 DMSO-d6) d: 9.00e8.97 (br d, J ¼ 7.8 Hz, 1H); 8.67 (s, 1H); 4.24e4.17
(m, 1H); 2.38 (s, 3H); 1.89e1.58 (m, 6H); 1.36e1.25 (m, 10H); (t, J ¼ 7 Hz, 2H); 4.12e3.99 (m, 1H); 3.47e3.42 (m, 4H); 2.40e2.36
M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27 25

(m, 4H); 2.21 (s, 3H); 1.87e1.72 (m, 4H); 1.64e1.46 (m, 10H); Analytical Sciences, U.S.).
1.35e1.24 (m, 4H); 0.88e0.82 (t, J ¼ 6.6 Hz, 3H). Anal. for
C23H37N7O2. Calcd: C, 62.28; H, 8.41; N, 22.10. Found: C, 62.39; H, 8.2. Cyclic AMP assay for human CB2 receptors
8.49; N, 22.35.
CHO cells transfected with human CB2 receptors were washed
7.1.7.14. 2-(N-Benzyl-N-methylamino)-N-cyclohexyl-4,7-dihydro-7- with phosphate-buffered saline, diluted trypsin and centrifuged at
oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (38). 200g for 10 min. The pellet containing CHO cells (1  106 cells/
Following general procedure G, compound 38 was isolated as a assay) was suspended in 0.5 mL of incubation mixture: KCl 2.7 mM,
white solid. Eluent: petroleum ethereethyl acetate 1/1. Yield 62%, NaCl 150 mM, MgSO4 1 mM, NaH2PO4 0.37 mM, CaCl2 1 mM, MgCl2
mp ¼ 157  C. MS (ESI): m/z 451.2 (MþH). 1H NMR (400 MHz, CDCl3) 10 mM, HEPES 5 mM, glucose 5 mM, pH 7.4 at 37  C. Then 0.5 mM
d: 9.17e9.15 (br d, J ¼ 7.6 Hz, 1H); 8.47 (s, 1H); 7.33e7.24 (m, 5H); 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as
4.73 (s, 2H); 4.18e4.14 (t, J ¼ 7.2 Hz, 2H); 3.06 (s, 3H); 1.98e1.73 (m, a phosphodiesterase inhibitor was added and the mixture pre-
5H); 1.56e1.30 (m, 11H); 0.91e0.87 (t, J ¼ 6.8 Hz, 3H). Anal. for incubated for 10 min in a shaking bath at 37  C [11]. The effect of the
C25H34N6O2. Calcd: C, 66.64; H, 7.61; N, 18.65. Found: C, 66.51; H, novel CB compounds was studied in the presence of forskolin, 1 mM,
7.68; N, 18.76. in comparison with the well-known CB agonist WIN 55,212-2. The
antagonism of the new CB ligands was also investigated evaluating
7.1.7.15. N-Cyclohexyl-2-(diallylamino)-4,7-dihydro-7-oxo-4-pentyl- their inhibition of the agonist WIN 55,212-2 (20 nM) response. The
[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (40). Following reaction was terminated by the addition of cold 6% trichloroacetic
general procedure G, compound 40 was isolated as a white solid. acid (TCA). The TCA suspension was centrifuged for 10 min at 2000g
Eluent: petroleum ethereethyl acetate 7/3. Yield 42%, mp ¼ 78  C. at 4  C, and the supernatant was extracted four times with water
MS (ESI): m/z 427.2 (MþH). 1H NMR (200 MHz, DMSO-d6) d: saturated diethyl ether. The final aqueous solution was tested for
8.99e8.95 (br d, J ¼ 7.8 Hz, 1H); 8.66 (s, 1H); 5.87e5.79 (m, 2H); cyclic AMP levels by a competition protein binding assay. Samples
5.23e5.14 (m, 4H); 4.25e4.18 (t, J ¼ 7.2 Hz, 2H); 4.04e4.01 (d, of cyclic AMP standard (0e10 pmol) were added to each test tube
J ¼ 5.6 Hz, 4H); 3.96e3.82 (m, 1H); 1.89e1.55 (m, 6H); 1.43e1.24 containing the incubation buffer (Trizma base 0.1 M, aminophylline
(m, 10H); 0.88e0.81 (t, J ¼ 6.2 Hz, 3H). Anal. for C23H34N6O2. Calcd: 8.0 mM, 2-mercaptoethanol 6.0 mM, pH 7.4) and [3H]cyclic AMP in
C, 64.76; H, 8.03; N, 19.70. Found: C, 64.49; H, 7.96; N, 19.44. a total volume of 0.5 mL. The binding protein previously prepared
from beef adrenals was added to the samples previously incubated
8. Pharmacology at 4  C for 150 min, and after the addition of charcoal, they were
centrifuged for 10 min at 2000g. The clear supernatant was counted
The affinity and the functionality of the novel compounds were by using a Perkin-Elmer 2810 TR scintillation counter (Perkin-
studied by using human CB1 and CB2 receptors expressed in CHO Elmer Life and Analytical Sciences, U.S.).
cells (Perkin-Elmer Life and Analytical Sciences, U.S.). To calculate
the affinity values, competition binding assays were performed by 8.3. Data analysis
using [3H]CP-55,940 as radioligand (specific activity, 180 Ci/mmol;
Perkin-Elmer Life and Analytical Sciences, U.S.). The effect of these The protein concentration was determined following a Bio-Rad
compounds was also evaluated in cyclic AMP experiments that method (Bradford, 1976) with bovine albumin as reference stan-
used [3H]-cAMP as radioligand (specific activity, 58 Ci/mmol; dard [26]. Inhibitory binding constants, Ki, were calculated from the
Perkin-Elmer Life and Analytical Sciences U.S.). All other reagents IC50 values according to the Cheng and Prusoff equation [27]:
were of analytical grade and obtained from commercial sources. Ki ¼ IC50/(1 þ [C*]/KD*), where [C*] is the concentration of the
radioligand and KD* its dissociation constant. A weighted nonlinear
8.1. Competition binding experiments on CB1 and CB2 receptors least-squares curve fitting program, LIGAND [28], was used for
computer analysis of the inhibition experiments. All the data are
CHO cell lines expressing human CB1 and CB2 receptors were expressed as the mean ± SEM of n ¼ 4 independent experiments.
grown adherently and maintained in Ham's F12 containing 10% Statistical analysis of the data was performed using unpaired two-
fetal bovine serum, streptomycin (100 mg/mL), penicillin (100 U/ sided Student's t test.
mL), and Geneticin (G418, 0.4 mg/mL) in 5% CO2/95% air at 37  C
[8,10]. For membrane preparations, the culture medium was 9. Docking studies
removed and the cells were washed with PBS, then scraped off T75
flasks in ice-cold hypotonic buffer (5 mM TriseHCl, 2 mM EDTA, pH The ligands were built with Maestro [29] and subjected to
7.4). The cell suspension was homogenized with a Polytron and minimization into a water environment (employing the general-
centrifuged at 1000g for 10 min, and the supernatant was then ized Born/surface area model) by using Macromodel [30]. The
centrifuged at 1,00,000g for 30 min. The membrane pellet was minimizations were carried out by means of the conjugate
suspended in 50 mM TriseHCl buffer, 0.5% BSA (pH 7.4) containing gradient, the MMFFs force field and a distance-dependent dielectric
5 mM MgCl2, 1 mM EDTA or 2.5 mM EDTA for hCB1 or hCB2 receptor, constant of 1.0, until a convergence value of 0.05 kcal/(Å mol) was
respectively. attained. The inactive CB1 and CB2 receptor models employed for
Competition binding experiments were performed using 0.5 nM docking studies were generated by applying a previously reported
[3H]CP-55,940 along with different concentrations (1 nMe10 mM) procedure [31] and then further optimized based on the recently
of the examined compounds or a reference agonist (WIN 55,212-2) deposited PDB structures of adenosine (PDB code 3EML) [31] and
for an incubation time of 90 or 60 min at 30  C for CB1 or CB2 re- rhodopsine (PDB code 1U19) [32] receptors. The ligands were
ceptors, respectively. docked using AUTODOCK4.2 [20] AUTODOCK TOOLS [33] was
Bound and free radioactivity were separated by filtering the employed to define the torsion angles in the ligands, to add the
assay mixture through Whatman GF/C glass fiber filters using a solvent model and to assign partial atomic charges (Gasteiger for
Brandel cell harvester (Brandel Instruments, Unterfo € hring, Ger- the ligands and Kollman for the receptors). The docking sites were
many). The filter bound radioactivity was counted on a Perkin- defined according to the previously published WIN 55,212-2
Elmer 2810 TR scintillation counter (Perkin-Elmer Life and docked into the CB1 and CB2 receptor models [34]. The new CB1 and
26 M. Aghazadeh Tabrizi et al. / European Journal of Medicinal Chemistry 113 (2016) 11e27

CB2 models were aligned to the corresponding protein structures do not know about the cannabinoid receptor 2 (CB2), Semin. Immunol. 26
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complexed with WIN 55,212-2 and the ligand was considered as
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pose within the receptor sites and the torsions involved in this al., 7-Oxo-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamides as selective CB(2)
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