Synthesis and structure activity relationship investigation of triazolo[1,5-a]pyrimidines as CB2 cannabinoid receptor inverse agonists

Article abstract of DOI:10.1016/j.ejmech.2016.02.032

CB₂ cannabinoid receptor ligands are known to be therapeutically important for the treatment of numerous diseases. Recently, we have identified the heteroaryl-4-oxopyridine/7-oxopyrimidine derivatives as highly potent and selective CB₂ receptor ligands, showing that the pharmakodynamics of the new compounds was controlled by the nature of the heterocycle core. In this paper we describe the 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 modifications at position 2 of triazolopyrimidine template changes the functional activity from partial to inverse agonism. The molecular docking analysis of the novel structures is reported.

Full text of DOI:10.1016/j.ejmech.2016.02.032

Accepted Manuscript
Synthesis and Structure Activity Relationship Investigation of
Triazolo[1,5-a]pyrimidines as CB Cannabinoid Receptor Inverse Agonists
2
Mojgan Aghazadeh Tabriz, Pier Giovanni Baraldi, Emanuela Ruggiero, Giulia
Saponaro, Stefania Baraldi, Giulio Poli, Tiziano Tuccinardi, Annalisa Ravani, Fabrizio
Vincenzi, Pier Andrea Borea, Katia Varani
PII:
S0223-5234(16)30112-X
10.1016/j.ejmech.2016.02.032
EJMECH 8383
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 November 2015
Revised Date: 26 January 2016
Accepted Date: 11 February 2016
Please cite this article as: M.A. Tabriz, P.G. Baraldi, E. Ruggiero, G. Saponaro, S. Baraldi, G. Poli, T.
Tuccinardi, A. Ravani, F. Vincenzi, P.A. Borea, K. Varani, Synthesis and Structure Activity Relationship
Investigation of Triazolo[1,5-a]pyrimidines as CB Cannabinoid Receptor Inverse Agonists, European
2
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.032.
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ACCEPTED MANUSCRIPT
Abstract graphic
Synthesis and Structure Activity Relationship Investigation of Triazolo[1,5-a]pyrimidines as CB2
Cannabinoid Receptor Inverse Agonists
O
N
O
O
N
O
N
N
N
N
N
N
N
N
H 2
R 2
H
H
C H
C H
5
11
5
11
Partial agonist
Inverse agonists

ACCEPTED MANUSCRIPT
Synthesis and Structure Activity Relationship Investigation of Triazolo[1,5-a]pyrimidines as
CB Cannabinoid Receptor Inverse Agonists
2
†
*
†*
†
†
Mojgan Aghazadeh Tabriz , Pier Giovanni Baraldi , Emanuela Ruggiero , Giulia Saponaro ,
†
‡
‡
⊥
⊥
Stefania Baraldi , Giulio Poli , Tiziano Tuccinardi , Annalisa Ravani , Fabrizio Vincenzi , Pier
⊥
⊥
Andrea Borea , Katia Varani
†
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Via Fossato di Mortara
17-19, 44121 Ferrara, Italy
‡
Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
⊥Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università di Ferrara, Via Fossato di
Mortara 17-19, 44121, Ferrara, Italy
*
Dr. Mojgan Aghazadeh Tabrizi, Dipartimento di Scienze Chimiche e Farmaceutiche, Università di
Ferrara, 44121, Ferrara, Italy, Via Fossato di Mortara 17-19, 44121, Ferrara Italy, Tel: +39-0532-
455926, Fax: +39-0532-455921, E-mail: tbj@unife.it
*
Prof. Pier Giovanni Baraldi, Dipartimento di Scienze Chimiche e Farmaceutiche, Università di
Ferrara, 44121, Ferrara, Italy, Via Fossato di Mortara 17-19, 44121, Ferrara Italy, Tel: +39-0532-
455921, Fax: +39-0532-455921, E-mail: baraldi@unife.it

ACCEPTED MANUSCRIPT
Abstract
CB cannabinoid receptor ligands are known to be therapeutically important for the treatment of
2
numerous diseases. Recently, we have identified the heteroaryl-4-oxopyridine/7-oxopyrimidine de-
rivatives as highly potent and selective CB receptor ligands, showing that the pharmakodynamics
2
of the new compounds was controlled by the nature of the heterocycle core. In this paper we de-
scribe the 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 CB receptor inverse
2
agonists. Cyclic AMP experiments on CB receptors expressed in CHO cells revealed that introduc-
2
tion of structural modifications at position 2 of triazolopyrimidine template changes the functional
activity from partial to inverse agonism. The molecular docking analysis of the novel structures is
reported.
Keywords
Cannabinoid; CB receptor; inverse agonists; triazolopyrimidine; molecular docking.
2
2

ACCEPTED MANUSCRIPT
Introduction
The endocannabinoid system includes a group of neuromodulatory lipids and their receptors that are
involved in a diversity of physiological and pathological conditions. The main endocannabinoids, or
endogenous cannabis-like substances, are small molecules resulting from arachidonic acid, N-
arachidonoyl ethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2-AG) [1]. Two di-
verse types of G protein-coupled receptors (GPCRs) have been discovered and named as canna-
binoid CB and cannabinoid CB receptors. The CB and CB receptors are negatively coupled to
1
2
1
2
adenylyl cyclase, exert an inhibitory effect on cyclic AMP (cAMP) production, alter the mitogen-
activated protein kinase (MAPK) pathway, and regulate voltage-gated L-, N- and P- or Q-type Ca2+
channels [2].
CB receptors were originally supposed to be present in periphery, primarily to immune cells, but at
2
present it has been reported that these receptors are localized neuronally in different species. They
are expressed in hematopoietic cells, natural killer cells, macrophages and neutrophils, and repre-
sent an attractive target for the treatment of the inflammatory status [3]. These receptors also medi-
ate a significant protection in brain microglia against neurotoxicity by modulating the release of an-
ti- or pro-inflammatory cytokines [4]. CB receptors appear to be implicated in allergic dermatitis
2
where their modulation mediates an inflammatory response obtained from antigen-specific effecter
T cells upon frequent allergen contact [5]. Contrasting preclinical studies are reported regarding the
involvement of CB receptors and allergic dermatitis, supposing the potential use of both CB ago-
2
2
nists or inverse agonists/antagonists in the pharmacological therapy [6].
In recent years, pharmaceutical companies and academic research laboratories have challenged to
identify novel CB agonists without CB central effects by focusing on the discovery of high affinity
2
1
and selective agonists for CB receptors with a therapeutic potential in the treatment of different pa-
2
thologies such as neurodegenerative diseases, pain transduction and perception, ischemic stroke, se-
vere inflammation, autoimmune diseases, osteoporosis and cancers [4,7].
3

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From the pharmacological point of view, CB agonists demonstrate various ways of interacting with
2
the receptor site, suggesting different binding and functional properties well represented by phar-
macodynamic concepts of full, partial and inverse agonism [8–11].
The therapeutic potential of the cannabinoids has encouraged the development of several CB re-
2
ceptor selective inverse agonists (Chart 1). In particular, the immunomodulatory activities associat-
ed with cannabinoid CB receptor inverse agonists have been demonstrated by oral administration
2
of the inverse agonists 1 (JTE-907) and 2 (SR144528) that inhibited carrageenin-induced paw oe-
dema in mice [12]. The efficacy of triaryl bissulphone 3 (Sch414319), a CB receptor inverse ago-
2
nist, was investigated in vivo in diverse disease models, showing its ability to modulate immune
cell mobility and bone damage in antigen-induced mono-articular arthritis in the rat [13]. Addition-
al reports suggest that compound 4 (MH), an inverse agonists of CB receptor, showed anti-
2
inflammatory
and
antiosteoclastogenic
properties
[14].
Moreover,
N,N′-((4-
(dimethylamino)phenyl)methylene)bis(2-phenylacetamide) derivatives such as 5 were found to be
CB inverse agonists and potential therapeutic agents for the treatment of antiosteoporosis [15].
2
The 4-oxo-1,8-naphthyridine-3-carboxamide and 4-oxo-1,4-dihydroquinoline-3-carboxamide can-
nabinoid based ligands are reported with high affinity and selectivity toward CB receptors. Some
2
of these analogues were demonstrated to act as agonists or inverse agonists in functional activity as-
says, depending on the nature of the substituents on the different positions of the heterocyclic scaf-
fold. Within this series, the 8-methoxy derivative 6 (hCB K = 0.6 nM, hCB K > 10000 nM)
2
i
1
i
(Chart 1) behaved as an inverse agonist and showed antihyperalgesic effects [16].
Chart 1 should be inserted here
Our group previously reported the synthesis and cannabinoid receptor binding of 4-
oxopyrazolopyridines as CB partial agonists [10]. In this family, compound 7 (Chart 2) displayed
2
high affinity and selectivity for CB receptor. Changing the orientation of the pyrazole ring led to
2
the 7-oxopyrazolopyrimidines, such as compound 8 that exhibited selective inverse agonist activity
at CB receptor [11]. Replacement of the pyrazolo ring of the parent nucleus with differently substi-
2
4

ACCEPTED MANUSCRIPT
tuted 5-membered heterocycles allowed to the isoxazolo/thienopyridines that were found to be CB2
full agonists. In this context, the triazolopyrimidines 9, 10 (Chart 2) were found to be potent CB2
receptor ligands and in functional assays compound 9 behaved as partial agonist.
In light of these results, we sought to explore the influence of substituent arrangement at the C-2
position of triazolo heterocycle on the activity versus CB receptor. In this attempt, we have synthe-
2
sized a new series of 2-substituted-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide
derivatives based on the previously well-known pharmacophores in the series of heteroaryl pyri-
dine/pyrimidine compounds. Consequently, the n-pentyl chain was retained at the N-4 position of
heterocycle and the cyclohexyl, cycloheptyl, and adamantyl moieties were introduced on the car-
boxamide moiety (Chart 2). All the newly synthesized compounds were tested on membranes pre-
pared from Chinese hamster ovary (CHO) cells expressing human CB (hCB ) and CB (hCB ) re-
1
1
2
2
ceptors, in order to estimate their affinity and selectivity for CB receptor. The cyclic AMP assays
2
on hCB CHO cells to test the functionality of these ligands is reported. Moreover, a molecular
2
docking analysis was carried out into the three-dimensional model of CB receptors recently con-
structed, in order to clarify the structure activity relationships (SARs) experimentally observed.
Chart 2 should be inserted here
Chemistry
Synthesis of derivatives 15-41 was carried out by the general methodology shown in Scheme 1. Cy-
clocondensation of aminotriazoles 11a-m with diethyl ethoxymethylenmalonate (DEEM) in glacial
acetic acid gave the ethyl 4,7-dihydro-2-substituted-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylates 12a-m in good yields. Alkylation of the bicyclic nucleus with 1-pentylbromide in the
presence of K CO led to the formation of 13a-m. The subsequent hydrolysis with 10% aqueous
2
3
NaOH yielded the related carboxylic acids 14a-m, which were converted to the corresponding car-
boxamide derivatives 15-41 by coupling reaction with appropriate amines using 1-ethyl- 3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDCl) or N,N,N′,N′-tetramethyl-O-(1H-
5

ACCEPTED MANUSCRIPT
benzotriazol-1-yl)uronium hexafluorophosphate in dimethylformamide. The starting 1,2,4-triazol-3-
amines 11a-b were commercially available, while derivatives 11c-m were prepared following two
different procedures. According to method A via cyclocondensation of amidoguanidines, a suitable
hydrazide was reacted with S-methylisothiourea hemisulfate upon heating in water to give the ami-
doguanidine intermediate. The subsequent cyclization in 10% aqueous NaOH at 100 °C yielded
aminotriazoles 11c-h [17,18]. The aminotriazoles 11i-m were prepared according to method B, by
the reaction of N-cyanocarbonimidodithioic acid dimethyl ester with one molar equivalent of an ap-
propriate amine followed by the reaction with hydrazine monohydrate in refluxing acetonitrile (as
except for 11i that was prepared without adding hydrazine) [19].
Scheme 1 should be inserted here
Biological results and discussion
3
All of the newly synthesized compounds were examined in [ H]CP-55,940 competition binding ex-
periments for their affinity and selectivity toward the human recombinant CB and CB receptors.
1
2
The results are reported in Table 1 together with K values of previously published triazolopyrim-
i
idines 9, 10 and the CB agonist WIN55,212-2. According to our previous pharmacophore investi-
2
gations, compounds bearing cyclohexyl, cycloheptyl, or adamantanyl moieties on the carboxamide
function had shown the highest affinity for CB receptor. We had also confirmed that the presence
2
of the 1-pentyl chain on the pyridine/pyrimidine nucleus was required for CB receptor affinity.
2
Consequently, we introduced structural modifications at position 2 of the 7-oxo-triazolo[1,5-
a]pyrimidine-6-carboxamide scaffold, in order to analyze the SARs of the new series. Examination
of the binding data for the novel compounds reveals some potent and highly selective ligands for
the CB receptor, e.g. compounds 23, 26-28, 32 and 39 (Table 1). It is worth noting that the new de-
2
rivatives although showed decreased in affinity for CB receptor in comparison to the reference
2
compound (10), highlighted a higher selectivity profile versus CB receptor. Introduction of cyclo-
1
heptyl and 3,5-dimethyladamantan-1-yl groups ((15 and 16, respectively) produced drastic decrease
6

ACCEPTED MANUSCRIPT
in affinity and selectivity at CB receptor compared with the parent compound 9 bearing the ada-
2
mantyl moiety. The same results were obtained by the 2-phenyl derivatives 19-21, when the ada-
mantly carboxamide was replaced, showing lower affinity and selectivity in comparison with 10.
The best affinity and selectivity profile was observed by compound 21 (K CB = 8.4 nM, K CB =
i
2
i
1
6
53 nM, K CB / K CB = 77). Introduction of a methyl chain at position 2, such as in compounds 17
i 1 i 2
and 18, resulted in a substantial reduction of affinity and a total loss of selectivity for both deriva-
tives. Intriguingly, these results are in contrast with those of the previously pyrazolo[3,4-b]pyridone
series [10], where the most active compounds bore a methyl group or an hydrogen atom, indicating
a probable different binding mode of this new series to the receptor. Subsequently, analogues by in-
troduction of 4-substituted-phenyl at the C-2 of the triazolopyrimidine nucleus were prepared, while
the cyclohexyl, cycloheptyl, and adamantyl moieties on the carboxamide were maintained (com-
pounds 22-28, Table 1). The 4-chlorophenyl group at C-2 of the triazolopyrimidine nucleus (com-
pounds 22 and 23) resulted in a decreased of affinity and selectivity for the cyclohexylcarboxamide
22 compared to the parent phenyl analogue 19. The adamantlycarboxamide derivative 23 resulted in
a 10-fold decrease in affinity for the CB receptor (23: K CB = 5216 nM versus 10: K CB = 523
1
i
1
i
1
nM), showing higher selectivity and comparable affinity for CB receptor relative to that of ana-
2
logue compound 10 (23: K CB = 11.3 nM, 10: K CB = 3.5 nM).
i
2
i
2
Changing of the chlorine atom with an electron donating group, such as methoxy, resulted in de-
rivatives 24 and 25, which showed lower affinity for the CB receptors (K CB = 96 and 47 nM, re-
2
i
2
spectively) in comparison with the phenyl analogue (10). Introduction of a 4-methylphenyl group at
C-2, as with compounds 26-28, increased selectivity in comparison with 10, and the best results
were obtained with adamantylamide derivative 28 that showed high affinity and selectivity at CB2
receptor (K CB = 6.8 nM, K CB / K CB = 223). These results seem to indicate that electronic ef-
i
2
i
1
i
2
fects on the aromatic moiety do not significantly affect the binding for CB receptors confirming
2
our previously results found for the pyrazolopyrimidine series [11]. Replacement of the 2-phenyl
ring of 10 with a bioisosteric heterocycle such as 2-furyl (29) resulted in a decrease of both affinity
7

ACCEPTED MANUSCRIPT
and selectivity at the CB receptors (Table 1, K CB = 45 nM, K CB / K CB = 26). Substitution of
i
2
i
1
i
2
the 5-membered heterocycle with a 6-membered aromatic ring such as 3-pyridyl moiety (30) deter-
mined a dramatic loss of affinities at both CB receptors (K CB = 228 nM, K CB > 10000 nM).
i
2
i
1
Replacement of the aromatic ring of the C-2 position by additional substitution on the heterocyclic
nucleus had a profound effect on affinity versus CB receptor as shown by compounds 31-44 with a
1
K CB > 10000 nM (Table 1). The introduction of a thiomethyl at the C-2 position (compounds 31,
i
1
3
2) significantly alters affinity at the CB receptor relative to that of parent compounds 19 and 10,
2
respectively (K = 716 and 47 versus 43 and 3.5 nM), although the selectivity increased about 1.5-
i
fold for 32 (K CB /K CB > 213 versus K CB / K CB = 146). Among the 2-morpholino derivatives,
i
1
i
2
i
1
i
2
the highest affinity and selectivity was observed with the 6-adamantylcarboxamide 35 (K CB = 53
i
2
nM, K CB /K CB
2
> 188), while neither the 2-piperazine compounds (36, 37) nor the diallylamine
i
1
i
derivatives (40, 41) showed affinity against the CB receptor.
2
The enhancement in affinity and selectivity was obtained with the 2-N-methylbenzyl chain and ad-
amantyl moiety on the 6-carboxamide group (39, K CB = 21 nM, K CB /K CB = 476). This com-
i
2
i
1
i
2
pound was the most selective of whole series probably because of different binding mode into CB2
receptor due to the shape and volume of the C-2 substituent as shown by molecular modeling stud-
ies (Figure 3).
Human CB receptor functional assay
2
The most active compounds of this series and previously derivatives 9 and 10 were analyzed in
cAMP assays for evaluation of their effect on cAMP production. The new ligands, as well as com-
pound 10, revealed an inverse agonist behavior, as they were able to significantly increase cAMP
production, with the exception of compound 16 which acted as partial agonist (Figure 1). The best
values of Emax have been obtained by compounds 21, 23 and 28, that increased the second mes-
senger production by 243%, 223% and 256% at 10 ꢀM, respectively. It is noteworthy that the same
compounds were also identified as the most active derivatives in binding assays, revealing a good
8

ACCEPTED MANUSCRIPT
correlation between affinity and efficacy of the developed series. The remaining compounds pro-
vided a comparable trend, since cAMP production increase was obtained within 53% and 135% at
1µM, and from 132% to 185% at 10 µM, respectively. Interestingly, the new 2-unsubstituted tria-
zolopyrimidine 16 acted as partial agonist as same as its parent compound 9 with values of effica-
cies (Emax) = 57 and 60%, respectively. Moreover, the potency value of 16 measured in functional
assays is in the nanomolar range (EC = 88 nM) and strictly correlated with the binding affinity
5
0
value. The difference in the functional activity between the 2-substituted derivatives and the com-
pounds lacking this substituent indicates that introduction of structural modifications at C-2 position
plays a crucial role in establishing the functionality of the novel cannabinoid ligands switching their
activity to inverse agonism. Furthermore, the antagonism of the new compounds was investigated,
evaluating their capability to counteract WIN55,212-2 inhibition of forskolin-stimulated cAMP
production. The IC values obtained with the concentration-response curves of the new canna-
5
0
binoids ligands were strictly correlated with their affinity values (Figure 2).
Table 1 should be inserted here
Figure 1 should be inserted here
Figure 2 should be inserted here
Molecular modeling studies
The synthesized compounds were docked into the inactive CB and CB receptor models by using
2
1
AUTODOCK4.2 [20], and for each ligand, the top-scored pose belonging to the cluster of solutions
with the best average of estimated free energy was considered. The resulting ligand-protein com-
plexes were then energy minimized with AMBER11 [21] in a lipid bilayer and water environment,
since molecular dynamic simulation studies carried out on the potent AMG3 CB ligand suggested
that the lipid bilayer environment can affect the binding of ligands to CB receptors [22,23]. Figure
3A shows the minimized structures of the receptor-ligand complexes obtained by docking com-
9

ACCEPTED MANUSCRIPT
pound 28, presenting the highest CB affinity, into both CB and CB receptor models. In the CB
2
2
2
1
receptor model the triazolopyrimidine core of the ligand is placed between the α-helixes belonging
to the third and the sixth transmembrane domains (TM3 and TM6), predominantly interacting with
M6.55(265) and L6.59(269) through hydrophobic interactions and forming an H-bond with
T3.33(114). The n-pentyl chain interacts into a sort of small lipophilic channel mainly delimited by
L3.26(107), L3.27(108) and L6.59(269), and is directed towards the TM4 α-helix, taking contacts
with P4.60(168) and L4.61(169). The adamantyl ring interacts with V4.56(164), P4.60(168),
Y5.39(190), W5.43(194) and feels the effects of a strong van der Waals interaction with F5.46(197),
which is a nonconserved residue (V5.46 in the CB receptor) and could thus contribute to the selec-
1
tivity of these ligands for CB versus CB receptor, consistently with site-directed mutagenesis stud-
2
1
ies. [24,25]. Finally, the 4-CH -phenyl moiety directed toward the α-helixes of the second and the
3
seventh transmembrane domains (TM2 and TM7) is located in a small lipophilic cavity mainly de-
limited by F2.57(87), I3.29(110), L6.54(264) and F7.35(281). In the CB receptor model, com-
1
pound 28 shows a binding pose in which it is orthogonally oriented with respect to its disposition
into the CB receptor (Figure 3B), thus interacting only with four transmembrane domains (TM3-
2
TM6). More in details, the 4-CH -phenyl moiety of the ligand is placed between TM3 and TM4 α-
3
helixes, surrounded by L3.26(190), F3.27(191) and L4.61(252) with which it establishes van der
Waals contacts, whereas the n-pentyl chain points toward TM6 interacting mainly with V6.59(367).
The central scaffold and the adamantyl ring of the ligand are placed in the hydrophobic pocket pri-
marily constituted by L3.29(193), T3.33(197), I4.56(247), A4.57(248), P4.60(251), and
W5.43(279), thus forming lipophilic interactions with these residues. Due to its disposition into CB1
receptor the ligand not only lacks the strong van der Waals interactions with residue 5.46 (which is
V282 instead of F197, as in CB ) but it is not even able to form any H-bond with T3.33(197)
2
through its triazolopyrimidine core. These two differences, together with the fewer hydrophobic
contacts established within the CB receptor, could be the main responsible for the selectivity for
1
CB versus CB receptor shown by compound 28 and, in general, for this series of ligands.
2
1
10

ACCEPTED MANUSCRIPT
Figure 3 should be inserted here
Figure 4 should be inserted here
Ligands such as compound 18, bearing no substituents or only a methyl group in position 2 of the
central scaffold, show a general decreased CB affinity with respect to ligands presenting phenyl or
2
aryl groups in the same positions. The energy minimized complex of compound 18 into CB recep-
2
tor (Figure 4A) shows a very similar binding mode for this ligand with respect to the one predicted
for compound 28. The two ligands share an almost identical disposition of their n-pentyl chains and
adamantyl ring, which thus take van der Waals contacts with the same residues. Anyway, com-
pound 18 is not able to fill the small lipophilic cavity mainly delimited by F2.57(87), I3.29(110),
L6.54(264) and F7.35(281), showing only limited contacts with I3.29(110) and L6.64(264). This
could be at the basis of the reduced affinity for CB receptor showed by this ligand and its ana-
2
logues lacking of larger substituents in C-2 position. Compound 18 shares with compound 28 also a
similar binding pose into CB receptor (Figure 4B). Anyway, the smaller C-2 substituent of this lig-
1
and allows it to adopt a slightly different orientation where the methyl group points toward the side
chain of L4.61(252) and the triazole ring of the central scaffold is closer to P4.60(251). In this dis-
position, the ligand is able to form an H-bond with T3.33(197) through the carbonyl oxygen of its
central scaffold; this interaction could constitute the reason of the highest affinity of this compound
for CB receptor with respect to what observed for compound 28 and generally for all those ligands
1
bearing bulkier C-2 substituents that wouldn’t allow such disposition due to steric bumps with
F3.27(191) and L4.61(252) side chains. Compound 39, bearing a N-benzyl-N-methyl group in 2 po-
sition of the central scaffold showed a strong affinity for CB receptor and the highest selectivity
2
versus CB receptor among this series of compounds. As shown in Figure 5A, the ligand adopts a
1
different binding mode into CB receptor with respect to the previously analyzed ligands, due to the
2
shape and volume of the C-2 substituent that cannot be placed into the small hydrophobic pocket
formed by F2.57(87), I3.29(110), L6.54(264) and F7.35(281). Nevertheless, the compound is still
11

ACCEPTED MANUSCRIPT
able to establish the strong van der Waals interactions with F5.46(197) through its adamantyl ring,
which shows the same location within the receptor as for compounds 18 and 28. Moreover, the lig-
and forms an H-bond with T3.33(114) through the carbonyl oxygen of its amide moiety, thus main-
taining both the interactions that are thought to be responsible of the CB versus CB selectivity of
2
1
these ligands. Finally, the C-2 substituent of the compound is efficiently positioned into the hydro-
phobic channel mainly delimited by L3.26(107), L3.27(108), L4.61(169) and Y5.39(190), forming
lipophilic interactions with all these residues and, in particular, a strong π-π stacking with
Y5.39(190). The energy minimized complex of compound 39 docked into CB receptor model
1
(
Figure 5B) shows that the ligand does not interact as in the CB receptor model. The compound
2
shows a binding mode similar to that observed for 28 and 18 into the CB receptor model, with a
1
slight rotation of the central core in order to place the bulky benzyl moiety between TM3 and TM4
α-helixes. Adopting this disposition the ligand is not able to form any H-bond with T3.33(197) and
although it maintains a good interaction with F3.27(191) through the benzyl group, it shows re-
duced contacts with L3.26(190) and V6.59(367), as well as weaker hydrophobic interactions with
T3.33(197), A3.34(198) and I4.65(247) through the adamantyl ring, with respect to compounds 28
and 18. This could explain the low CB affinity of compound 39 and thus its strong CB versus CB
1
2
1
selectivity.
Figure 5 should be inserted here
Conclusions
In order to progress the CB receptor affinity and selectivity and to develop structure activity rela-
2
tionship for the series of triazolo[1,5-a]pyrimidine-6-carboxamide, we synthesized and evaluated
twenty-seven new derivatives belonging to this chemical class. Structural modifications around the
central scaffold, especially at C-2 and C-6 positions, led us to achieve greater selectivity, with re-
spect to the parent compounds 9 and 10. The best affinity values associated with high selectivity
were obtained with compounds 23 (K CB = 11.3 nM; K CB /K CB = 462) and 39 (K CB = 21
i
2
i
1
i
2
i
2
12

ACCEPTED MANUSCRIPT
nM; K CB /K CB > 476) bearing an adamantan-1-yl moiety on the carboxamide function and a 4-
i
1
i
2
Cl-phenyl or N-benzyl-N-methylamine residues at C-2, respectively. cAMP experiments on CB re-
2
ceptors expressed in CHO cells surprisingly revealed a different behavior, basing on the substitu-
tions at C-2. Compound 16 as its parent 9 lacking the substituent in position C-2 of the triazolepy-
rimidine nucleus showed partial agonist activities. 2-Substituted derivatives provided an inverse ag-
onism behavior, with high efficacy levels from 152% to 246% of increase in cAMP production.
Thus, this novel series of compounds proposes an attractive starting point for more optimization,
representing novel pharmacological tools to evaluate the therapeutic potential of CB inverse ago-
2
nists in diverse disease settings.
Chemistry
Materials and methods
Positive ion electrospray ionization (ESI) mass spectra were performed with an Agilent 1100 Series
LC/MSD model. All melting points are uncorrected and were assigned by a Buchi-Tottoli appa-
1
ratus. The proton nuclear magnetic resonance ( H NMR) spectra were recorded on either a Bruker
AC 200 (200MHz) or a Varian Mercury Plus 400 (400MHz) spectrometer. Chemical shifts are re-
ported in δ values (parts per million), using appropriate deuterated solvents (DMSO-d and CDCl ).
6
3
The purity of tested compounds was determined by combustion elemental analyses performed by
the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a
Yanagimoto MT-5 CHN recorder elemental analyzer. All tested compounds obtained data con-
sistent with a purity of at least 95% as compared with the theoretical values.
Reactions were monitored by TLC (Thin Layer Chromatography) on silica gel (precoated Merck
F254 plates), and compounds visualization was performed by UV light. Flash chromatography was
accomplished using 230-400 mesh silica gel and a mixture of ethyl acetate and petroleum ether or
ethyl acetate and methanol in different ratios as eluting phase. Organic extracts were dried over an-
13

ACCEPTED MANUSCRIPT
hydrous sodium sulfate (Na SO ). All starting materials, reagents and solvents were obtained from
2
4
Sigma-Aldrich or Alfa Aesar.
General procedure A for the synthesis of compounds 11c-h
A solution of S-methylisothiourea hemisulfate salt (22 mmol), appropriate hydrazide (22 mmol),
and water (35 mL) was refluxed for 6 h. The solvent was concentrated under reduced pressure and
the residue was washed with ethanol (3 x 10 mL). The resulting 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 adjusted to pH 5 using 1N HCl. The precipitate was filtered, washed with cold water and dried.
5-Phenyl-2H-1,2,4-triazol-3-amine (11c). Following general procedure A, compound 11c was iso-
1
lated as a white solid. Yield 82%, mp = 233-234 °C. H NMR (200 MHz, DMSO-d ) δ: 12.58 (br s,
6
1
5
1
1
5
1
1
3
5
H); 7.97-7.85 (m, 2H); 7.62-7.35 (m, 3H); 5.97 (br s, 2H).
-(4-Chlorophenyl)-2H-1,2,4-triazol-3-amine (11d). Following general procedure A, compound
1
1d was isolated as a white solid. Yield 90%, mp = 229-230 °C. H NMR (200 MHz, DMSO-d ) δ:
6
2.75 (br s, 1H); 7.95-7.89 (d, J = 8.6 Hz, 2H); 7.58-7.52 (d, J = 8.6 Hz, 2H); 5.11 (br s, 2H).
-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-amine (11e). Following general procedure A, compound
1
1e was isolated as pale yellow solid. Yield 87%, mp > 300 °C. H NMR (400 MHz, DMSO-d ) δ:
6
1.85 (br s, 1H); 7.80-7.78 (d, J = 9 Hz, 2H); 6.96-6.94 (d, J = 8.4 Hz, 2H); 5.98 (br s, 2H); 3.77 (s,
H).
-p-Tolyl-2H-1,2,4-triazol-3-amine (11f). Following general procedure A, compound 11f was iso-
1
lated as a white solid. Yield 78%, mp = 190-193 °C. H NMR (200 MHz, DMSO-d ) δ: 12.21 (br s,
6
1H); 7.78-7.73 (d, J = 8.2 Hz, 2H); 7.21-7.17 (d, J = 8 Hz , 2H); 5.91 (br s. 2H); 2.31 (s, 3H).
5-(Furan-2-yl)-2H-1,2,4-triazol-3-amine (11g). Following general procedure A, compound 11g was
1
isolated as a white solid. Yield 82%, mp = 225-226 °C. H NMR (400 MHz, DMSO-d ) δ: 12.11 (br
6
s, 1H); 7.60 (d, J = 1 Hz, 1H); 6.67 (d, J = 3.4 Hz, 1H); 6.53 (dd, J = 3.4 and 1.5 Hz, 1H); 6.07 (br
s, 2H).
14

ACCEPTED MANUSCRIPT
5-(Pyridin-3-yl)-2H-1,2,4-triazol-3-amine (11h). Following general procedure A, compound 11h
1
was isolated as a white solid. Yield 75%, mp = 203-204 °C. H NMR (200 MHz, DMSO-d ) δ:
6
12.23 (br s, 1H); 9.04-9.03 (d, J = 1.6 Hz, 1H); 8.54-8.52 (dd, J = 5.4 and 1.5 Hz, 1H); 8.18-8.15
(dt, J = 8 and 1.6 Hz, 1H); 7.44-7.41 (dd, J = 7.8 and 4.9 Hz, 1H); 6.17 (br s, 2H).
General procedure B for the synthesis of compounds 11i-m
A solution of N-cyanocarbonimidodithioic acid dimethyl ester (17.4 mmol), acetonitrile (10 mL),
and appropriate amine (17.4 mmol) was refluxed for 2 h. After cooling to room temperature, hydra-
zine monohydrate (25.6 mmol) was added and the reaction mixture was further refluxed for addi-
tional 5 h. The solvent was evaporated under reduced pressure and the resulting product recrystal-
lized from ethyl acetate.
5-(Methylthio)-2H-1,2,4-triazol-3-amine (11i). Following general procedure B, compound 11i was
1
isolated as a white solid. Yield 90%, mp = 134 °C. H NMR (200 MHz, DMSO-d ) δ: 11.78 (br s,
6
1H); 5.95 (br s, 2H); 2.39 (s, 3H).
5-Morpholino-2H-1,2,4-triazol-3-amine (11j). Following general procedure B, compound 11j was
1
isolated as a white solid. Yield 82%, mp = 161-162 °C. H NMR (200 MHz, DMSO-d ) δ: 10.98 (br
6
s, 1H); 5.71 (br s, 2H); 3.64-3.59 (m, 4H); 3.13-3.08 (m, 4H).
5-(4-Methylpiperazin-1-yl)-2H-1,2,4-triazol-3-amine (11k). Following general procedure B, com-
1
pound 11k was isolated as a pale yellow solid. Yield 80%, mp = 103-104 °C. H NMR (200 MHz,
DMSO-d ) δ: 10.95 (br s, 1H); 5.72 (br s, 2H); 3.16-3.11 (m, 4H); 2.34-2.29 (m, 4H); 2.17 (s, 3H).
6
N-benzyl-N-methyl-1H-1,2,4-triazole-3,5-diamine (11l). Following general procedure B, compound
11l was isolated as a white solid. Yield 86%, mp = 163-164 °C. 1H NMR (200 MHz, DMSO-d6) δ:
10.85 (br s, 1H); 7.27-7.23 (m, 5H); 5.75 (br s, 2H); 4.42 (s, 2H); 2.70 (s, 3H).
N,N-Diallyl-1H-1,2,4-triazole-3,5-diamine (11m). Following general procedure B, compound 11m
1
was isolated as a white solid. Yield 95%, mp = 244-245 °C. H NMR (400 MHz, DMSO-d ) δ:
6
15

ACCEPTED MANUSCRIPT
10.75 (br s, 1H); 5.82-5.69 (m, 2H); 5.54 (br s, 2H); 5.16-5.02 (m, 4H); 3.81-3.78 (d, J = 5.6 Hz,
4H).
General procedure C for the synthesis of ethyl 4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-
-carboxylates 12a-m
6
A solution of aminotriazole 11a-m (3.1 mmol), glacial acetic acid (5 mL), and diethyl ethoxymeth-
ylenemalonate (4.6 mmol) was refluxed for 3 h. After cooling to room temperature, the resulting
precipitate was collected by filtration, washed with cold water and dried. The residue was stirred
with cold ethyl ether (20 mL) and filtered to afford the desired product.
Ethyl 4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12a). Following general
1
procedure C, compound 12a was isolated as a white solid. Yield 76%, mp > 300 °C. H NMR (200
MHz, DMSO-d ) δ: 14.30 (br s, 1H); 8.95 (s, 1H); 8.84 (s, 1H); 4.20 (q, J = 6.8 Hz, 2H); 1.29 (t, J =
6
6.8 Hz, 3H).
Ethyl 4,7-dihydro-2-methyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12b). Following
1
general procedure C, compound 12b was isolated as a pale yellow solid 70%, mp = 237 °C dec. H
NMR (200 MHz, DMSO-d ) δ: 12.9 (br s, 1H); 8.58 (s, 1H); 4.28-4.17 (q, J = 7.4 Hz, 2H); 2.38 (s,
6
3H); 1.31-1.27 (t, J = 7 Hz, 3H).
Ethyl 4,7-dihydro-2-phenyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12c). Following
1
general procedure C, compound 12c was isolated as a white solid. Yield 67%, mp > 300 °C. H
NMR (200 MHz, DMSO-d ) δ: 14.00 (br s, 1H); 8.64 (s, 1H); 8.14–8.10 (m, 2H); 7.55 (m, 3H);
6
4.26 (q, J = 7.0 Hz, 2H); 1.28 (t, J = 6.8 Hz, 3H).
Ethyl 2-(4-chlorophenyl)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12d).
Following general procedure C, compound 12d was isolated as a pale yellow solid. Yield 60%, mp
1
>
300 °C. H NMR (200 MHz, DMSO-d ) δ: 13.41 (br s, 1H); 8.65 (s, 1H); 8.14-8.10 (d, J = 8.4
6
Hz, 2H); 7.63-7.59 (d, J = 8.6 Hz, 1H); 4.29-4.22 (q, J = 7 Hz, 2H); 1.33-1.25 (t, J = 7.4 Hz, 3H).
Ethyl 2-(4-methoxyphenyl)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
12e). Following general procedure C, compound 12e was isolated as a pale yellow solid. Yield
(
16

ACCEPTED MANUSCRIPT
1
6
5%, mp > 300 °C. H NMR (400 MHz, DMSO-d ) δ: 13.25 (br s, 1H); 8.61 (s, 1H); 8.06-8.04 (d, J
6
=
8.8 Hz, 2H); 7.10-7.08 (d, J = 8.8 Hz, 1H); 4.25-4.23 (q, J = 7.2 Hz, 2H); 3.83 (s, 3H); 1.31-1.27
(t, J = 7.2 Hz, 3H).
Ethyl 4,7-dihydro-7-oxo-2-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12f). Following
general procedure C, compound 12f was isolated as a pale yellow solid. Yield 70%, mp = 268 °C
1
dec. H NMR (200 MHz, DMSO-d ) δ. 13.91 (br s, 1H); 8.63 (s, 1H); 8.03-7.99 (d, J = 8 Hz, 2H);
6
7
3
.37-7.32 (d, J = 8.2 Hz, 2H);4.27-4.23 (q, J = 7 Hz, 2H); 2.38 (s, 3H); 1.33-1.26 (t, J = 7.4 Hz,
H).
Ethyl 2-(furan-2-yl)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12g). Fol-
lowing general procedure C, compound 12g was isolated as a white solid. Yield 75%, mp > 300 °C.
1
H NMR (200 MHZ-DMSO-d ) δ: 13.24 (br s, 1H); 8.59 (s, 1H); 7.88-7.87 (d, J = 1 Hz, 1H); 7.11-
6
7.09 (d, J = 3.4 Hz, 1H); 6.67-6.65(d, J = 3.4 Hz, 1H); 4.23-4.20 (q, J = 7.2 Hz, 2H); 1.31-1.24 (t, J
=
7 Hz, 3H).
Ethyl 4,7-dihydro-7-oxo-2-(pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12h).
Following general procedure C, compound 12h was isolated as a white solid. Yield 65%, mp = 250
1
°
C. H NMR (400 MHz, DMSO-d ) δ: 11.98 (br s, 1H); 9.27 (d, J = 1.6 Hz, 1H); 8.74-8.72 (dd, J =
6
6.4 and 1.6 Hz, 1H); 8.67 (s, 1H); 8.48-8.46 (dt J = 6.9 and 1.7 Hz,1H); 7.62-7.60 (dd, J = 7 and 5.1
Hz, 1H); 4.28-4.23 (q, J = 7.2 Hz, 2H); 1.31-1.27 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-dihydro-2-(methylthio)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12i). Fol-
lowing general procedure C, compound 12i was isolated as a white solid. Yield 70%, mp = 277 °C
1
dec. H NMR (200 MHz, DMSO-d ) δ: 12.41 (br s, 1H); 8.56 (s, 1H); 4.27-4.20 (q, J = 7 Hz, 2H);
6
2.60 (s, 3H); 1.31-1.24 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-dihydro-2-morpholino-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12j). Fol-
lowing general procedure C, compound 12j was isolated as a white solid. Yield 75%, mp = 283-284
1
°
C dec. H NMR (200 MHz, DMSO-d ) δ: 12.5 (br s, 1H); 8.45 (s, 1H); 4.23-4.20 (q, J = 7.2 Hz,
6
2H); 3.70-3.66 (m, 4H); 3.41-3.37 (m, 4H); 1.30-1.23 (t, J = 7.2 Hz, 3H).
17

ACCEPTED MANUSCRIPT
Ethyl 4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
(12k). Following general procedure C, compound 12k was isolated as a white solid. Yield 78%, mp
1
=
220 °C dec. H NMR (200 MHz, DMSO-d ) δ: 9.95 (br s, 1H); 8.41 (s, 1H); 4.17-4.07 (q, J = 7
6
Hz, 2H); 3.78-3.45 (m, 4H); 2.76 (s, 3H); 2.52-2.49 (m, 4H); 1.26-1.19 (t, J = 7.2 Hz, 3H).
Ethyl 2-(N-benzyl-N-methylamino)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylates (12l). Following general procedure C, compound 12l was isolated as a white solid.
1
Yield 69%, mp = 284 °C. H NMR (200 MHz, DMSO-d6) δ: 13.37 (br s, 1H); 8.42 (s, 1H); 7.30-
7.28 (m, 5H); 4.64 (s, 2H); 4.25-4.17 (q, J = 7 Hz, 2H); 2.95 (s, 3H); 1.30-1.23 (t, J = 7 Hz, 3H).
Ethyl 2-(diallylamino)-4,7-dihydro-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12m).
Following general procedure C, compound 12m was isolated as a white solid. Yield 73%, mp =
1
2
5
40-241 °C. H NMR (200 MHz, DMSO-d ) δ: 13.91 (br s, 1H); 8.41 (s, 1H); 5.93-5.76 (m, 2H);
6
.21-5.13 (m, 4H); 4.26-4.16 (q, J = 7.2 Hz, 2H); 4.02-3.99 (d, J = 6.2 Hz, 4H); 1.30-1.23 (t, J = 7.2
Hz, 3H).
General procedure D for the synthesis of ethyl 4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxylates 13a-m
To a solution of appropriate carboxylic acid ethyl ester 12a-m (0.54 mmol) in anhydrous N,N-
dimethylformamide was added K CO (1.62 mmol), and the mixture was stirred at 70 °C for 1 h. 1-
2
3
Pentyl bromide (1.62 mmol) was added, and the mixture was heated at 100 °C for 16 h. The reac-
tion mixture was cooled to room temperature, the solvent was evaporated under reduced pressure
and the residue partitioned between water (20 mL) and ethyl acetate (60 mL). The organic phase
was dried was dried (Na SO ), and after removal of the solvent, the residue was purified by flash
2
4
column chromatography, eluting with ethyl acetate/petroleum ether or ethyl acetate/methanol.
Ethyl 4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13a). Following
general procedure D, compound 13a was isolated as a white solid. Eluent: petroleum ether-ethyl ac-
18

ACCEPTED MANUSCRIPT
1
etate 3/2. Yield 75%, mp = 85-86 °C. H NMR (200 MHz, DMSO-d ) δ: 8.88 (s, 1H), 8.33 (s, 1H),
6
4.32-4.22 (m, 4H), 1.95-1.79 (m, 2H), 1.33-1.26 (m, 7H), 0.89-0.82 (t, J = 6.8 Hz, 3H).
Ethyl 4,7-dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13b).
Following general procedure D, compound 13b was isolated as a white solid. Eluent: petroleum
1
ether-ethyl acetate 2/3. Yield 78%, mp = 151-152 °C. H NMR (200 MHz, DMSO-d ) δ: 8.82 (s,
6
1H), 4.30-4.21 (m, 4H), 2.39 (s, 3H), 1.91-1.77 (m, 2H), 1.32-1.25 (m, 7H), 0.89-0.84 (t, J = 6.8
Hz, 3H).
Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13c).
Following general procedure D, compound 13c was isolated as a pale yellow solid. Eluent: petrole-
1
um ether-ethyl acetate 2/3. Yield 75%, mp = 112-113 °C. H NMR (200 MHz, DMSO-d ) δ: 8.87
6
(
s, 1H), 8.16-8.11 (m, 2H), 7.56-7.53 (m, 3H), 4.37-4.23 (m, 4H), 1.93-1.81 (m, 2H), 1.37-1.26 (m,
H), 0.91-0.85 (t, J = 6.6 Hz, 3H).
Ethyl 2-(4-chlorophenyl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
7
carboxylate (13d). Following general procedure D, compound 13d was isolated as a white solid.
1
Eluent: petroleum ether-ethyl acetate 7/3. Yield 78%, mp = 155-156 °C. H NMR (200 MHz,
DMSO-d ) δ: 8.88 (s, 1H); 8.16-8.11 (d, J = 8.6 Hz, 2H); 7.64-7.60 (d, J = 8.4 Hz, 2H); 4.32-4.23
6
(m, 4H); 1.96-1.83 (m, 2H); 1.36-1.27 (m, 7H); 0.91-0.84 (t, J = 6.8 Hz, 3H).
Ethyl 4,7-dihydro-2-(4-methoxyphenyl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylate (13e). Following general procedure D, compound 13e was isolated as a white solid. El-
1
uent: petroleum ether-ethyl acetate 1/1. Yield 80%, mp = 147-148 °C. H NMR (400 MHz, DMSO-
d6) δ: 8.85 (s, 1H); 8.07-8.05 (d, J = 8.8 Hz, 2H); 7.10-7.08 (d, J = 8.8 Hz, 2H); 4.33-4.25 (m, 4H);
3.83 (s, 1H); 1.94-1.81 (m, 2H); 1.34-1.29 (m, 7H); 0.90-0.86 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (13f).
Following general procedure D, compound 13f was isolated as a pale yellow solid. Eluent: petrole-
1
um ether-ethyl acetate 1/4. Yield 82%, mp = 216-217 °C. H NMR (400 MHz, DMSO-d ) δ: 8.86
6
19

ACCEPTED MANUSCRIPT
(s, 1H), 8.03-8.01 (d, J = 8 Hz, 2H), 7.36-7.34 (d, J = 7.6 Hz, 2H), 4.32-4.25 (m, 4H), 2.38 (s, 3H),
1
.91-1.72 (m, 2H), 1.35-1.29 (m, 7H), 0.89-0.86 (t, J = 6.8 Hz, 3H).
Ethyl 2-(furan-2-yl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
13g). Following general procedure D, compound 13g was isolated as a white solid. Eluent: petro-
(
1
leum ether-ethyl acetate 2/3. Yield 77%, mp = 151-152 °C. H NMR (200 MHz, DMSO-d ) δ: 8.86
6
(s, 1H); 7.94-7.93 (d, J = 1.6 Hz, 1H); 7.20-7.18 (d, J = 3.4 Hz, 1H); 6.72-6.69 (dd, J = 3.4 and 1.6
Hz, 1H); 4.33-4.23 (m, 4H); 1.93-1.79 (m, 2H); 1.33-1.26 (m, 7H); 0.90-0.84 (t, J = 6.6 Hz, 3H).
Ethyl 4,7-dihydro-7-oxo-4-pentyl-2-(pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
(13h). Following general procedure D, compound 13h was isolated as a white solid. Eluent: petro-
1
leum ether-ethyl acetate 1/1. Yield 77%, mp = 104-105 °C. H NMR (400 MHz, DMSO-d ) δ: 9.46
6
(
d, J = 1.6 Hz, 1H); 8.72-8.70 (dd, J = 6.6 and 1.6 Hz, 1H); 8.57-8.55 (dt, J = 7.8 and 1.6 Hz, 1H);
.47 (s, 1H); 7.42-7.39 (dd, J = 8 and 5 Hz, 1H); 4.45-4.39 (q, J = 7.2 Hz, 2H); 4.34-4.30 (t, J = 7.6
Hz, 2H); 2.01-1.97 (m, 2H); 1.43-1.39 (m, 7H); 0.95-0.92 (t, J = 7.2 Hz, 3H).
Ethyl 4,7-dihydro-2-(methylthio)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
13i). Following general procedure D, compound 13i was isolated as a white solid. Eluent: petrole-
8
(
1
um ether-ethyl acetate 1/1. Yield 81%, mp = 120-121 °C. H NMR (200 MHz, DMSO-d ) δ: 8.79
6
(s, 1H); 4.30-4.20 (m, 4H); 2.61 (s, 3H); 1.91-1.72 (m, 2H); 1.32-1.25 (m, 7H); 0.89-0.84 (t, J = 6.8
Hz, 3H).
Ethyl
4,7-dihydro-2-morpholino-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
(13j). Following general procedure D, compound 13j was isolated as a white solid. Eluent: ethyl ac-
1
etate-methanol 9.5/0.5. Yield 82%, mp = 113-114 °C. H NMR (200 MHz, DMSO-d ) δ: 8.68 (s,
6
1
7
H); 4.30-4.13 (m, 4H); 3.71-3.66 (m, 4H); 3.43-3.38 (m, 4H); 1.95-1.72 (m, 2H); 1.31-1.24 (m,
H); 0.89-0.83 (t, J = 6.6 Hz, 3H).
Ethyl
4,7-dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylate (13k). Following general procedure D, compound 13k was isolated as a white solid.
1
Eluent: ethyl acetate-methanol 9/1. Yield 70%, mp = 183-184 °C. H NMR (200 MHz, DMSO-d )
6
20

ACCEPTED MANUSCRIPT
δ: 8.67 (s, 1H); 4.26-4.16 (m, 4H); 3.45-3.42 (m, 4H); 2.41-2.37 (m, 4H); 2.20 (s, 3H); 1.96-1.79
(m, 2H); 1.31-1.24 (m, 7H); 0.89-0.82 (t, J = 6.8 Hz, 3H).
Ethyl 2-(N-benzyl-N-methylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylate (13l). Following general procedure D, compound 13l was isolated as a white solid. pe-
1
troleum ether-ethyl acetate 1/4. Yield 82%, mp = 77-78 °C. H NMR (200 MHz, DMSO-d )
6
δ: 8.66 (s, 1H); 7.34-7.29 (m, 5H); 4.65 (s, 2H); 4.29-4.15 (m, 4H); 2.95 (s, 3H); 1.91-1.78 (m,
2H); 1.31-1.24 (m, 7H); 0.88-0.81 (t, J = 6.8 Hz, 3H).
Ethyl 2-(diallylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate
(13m). Following general procedure D, compound 13m was isolated as a white solid. Eluent: petro-
1
leum ether-ethyl acetate 1/4. Yield 81%, mp = 70-72 °C. H NMR (200 MHz, DMSO-d ) δ: 8.65 (s,
6
1
H); 5.87-5.76 (m, 2H); 5.23-5.14 (m, 4H); 4.25-4.16 (m, 4H); 4.03-4.00 (d, J = 5.8 Hz, 2H); 1.91-
.75 (m, 2H); 1.32-1.24 (m, 7H); 0.89-0.82 (t, J = 7 Hz, 3H).
1
General procedure E for the synthesis of carboxylic acid derivatives 14a-m
A solution of appropriate carboxylic acid ethyl ester 13a-m (1.27 mmol), methanol (20 mL), and
NaOH 10% (10 mL) was stirred at room temperature for 6 h. The suspension was acidified with
10% HCl, the precipitate was collected by filtration and washed with cold water to afford the de-
sired product.
4,7-Dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14a). Following
1
general procedure E, compound 14a was isolated as a white solid. Yield 72%, mp = 215 °C. H
NMR (200 MHz, DMSO-d ) δ: 12.12 (br s, 1H); 8.73 (s, 1H); 8.28 (s, 1H); 4.27-4.20 (t, J = 7 Hz,
6
2H); 1.84-1.77 (m, 2H); 1.32-1.25 (m, 4H); 0.88-0.81 (t, J = 6.6 Hz, 3H).
4,7-Dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14b).
Following general procedure E, compound 14b was isolated as a white solid. Yield 85%, mp = 164-
1
1
2
65 °C. H NMR (200 MHz, DMSO-d ) δ: 12.37 (br s, 1H); 8.68 (s, 1H); 4.23-4.16 (t, J = 7.2 Hz,
6
H); 2.38 (s, 3H); 1.81-1.73 (m, 2H); 1.30-1.25 (m, 4H); 0.88-0.81 (t, J = 6.8 Hz, 3H).
21

ACCEPTED MANUSCRIPT
4,7-Dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14c).
Following general procedure E, compound 14c was isolated as a white solid. Yield 91%, mp >300
1
°
C. H NMR (200 MHz, DMSO-d ) δ:12.24 (br s, 1H); 8.63 (s, 1H); 8.14-8.10 (m, 2H); 7.54-7.51
6
(m, 3H); 4.30-4.26 (t, J = 7 Hz, 2H); 1.98-1.81 (m, 2H); 1.39-1.27 (m, 4H); 0.89-0.82 (t, J = 6.6 Hz,
3H).
2-(4-Chlorophenyl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid
(14d). Following general procedure E, compound 14d was isolated as a white solid. Yield (87%),
1
mp = 204-205 °C. H NMR (200 MHz, DMSO-d ) δ: 12.81 (br s, 1H); 8.92 (s, 1H); 8.16-8.12 (d, J
6
=
8.6 Hz, 2H); 7.64-7.60 (d, J = 8.6 Hz, 2H); 4.37-4.30 (t, J = 7.2 Hz, 2H); 1.81-1.75 (m, 2H); 1.36-
1.28 (m, 4H); 0.90-0.84 (t, J = 6.6 Hz, 3H).
4,7-Dihydro-2-(4-methoxyphenyl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic ac-
id (14e). Following general procedure E, compound 14e was isolated as a white solid. Yield 86%,
1
mp = 200-201 °C. H NMR (400 MHz, DMSO-d ) δ: 12.79 (br s, 1H); 8.90 (s, 1H); 8.08-8.06 (d, J
6
=
8.8 Hz, 2H); 7.11-7.08 (d, J = 8.4 Hz, 2H); 4.35-4.31 (t, J = 7.2 Hz, 2H); 1.89-1.86 (m, 2H); 1.34-
1.32 (m, 4H); 0.89-0.86 (t, J = 6.4 Hz, 3H).
4,7-Dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (14f). Fol-
lowing general procedure E, compound 14f was isolated as a pale yellow solid. Yield 87%, mp =
1
2
07-208 °C. H NMR (200 MHz, DMSO-d ) δ: 12.65 (br s, 1H); 8.87 (s, 1H); 8.05-8.01 (d, J = 8.4
6
Hz, 2H); 7.37-7.33 (d, J = 8 Hz, 2H); 4.34-4.31 (t, J = 6.8 Hz, 2H); 2.38 (s, 3H); 1.96-1.79 (m, 2H);
1.35-1.30 (m, 4H); 0.90-0.84 (t, J = 6.6 Hz, 3H).
2-(Furan-2-yl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic
acid
(14g). Following general procedure E, compound 14g was isolated as a pale yellow solid. Yield
1
8
7%, mp = 187-188 °C. H NMR (200 MHz, DMSO-d ) δ: 12.81 (br s, 1H); 8.90 (s, 1H); 7.95-7.94
6
(
d, J = 1.8 Hz, 1H); 7.21-7.19 (d, J = 3.4 Hz, 1H); 6.72-6.70 (dd, J = 3.4 and 1.6 Hz, 1H); 4.32-4.26
t, J = 6.8 Hz, 2H); 1.87-1.81 (m, 2H); 1.34-1.27 (m, 4H); 0.90-0.83 (t, J = 6.6 Hz, 3H).
(
22

ACCEPTED MANUSCRIPT
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 procedure E, compound 14h was isolated as a white solid. Yield 86%, mp
1
=
158-159 °C. H NMR (400 MHz, DMSO-d ) δ: 12.84 (br s, 1H); 9.28 (d, J = 1.6 Hz, 1H); 8.94 (s,
6
1
8
H); 8.74-8.73 (dd, J = 6.4 and 1.8 Hz, 1H); 8.48-8.45 (dt, J = 8 and 1,6 Hz, 1H); 7.61-7.58 (dd, J =
and 4.8 Hz, 1H); 4.37-4.33 (t, J = 7.2 Hz, 2H); 1.90-1.87 (m, 2H); 1.37-1.32 (m, 4H); 0.89-0.86 (t,
J = 6.8 Hz, 3H).
,7-Dihydro-2-(methylthio)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic
4
acid
(14i). Following general procedure E, compound 14i was isolated as a white solid. Yield 98%, mp =
1
1
65-166 °C. H NMR (200 MHz, DMSO-d ) δ: 12.51 (br s, 1H); 8.78 (s, 1H); 4.22-4.19 (t, J = 6.8
6
Hz, 2H); 2.61 (s, 3H); 1.84-1.77 (m, 2H); 1.35-1.26 (m, 4H); 0.88-0.83 (t, J = 6.4 Hz, 3H).
,7-Dihydro-2-morpholino-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic
4
acid
(14j). Following general procedure E, compound 14j was isolated as a white solid. Yield 98%, mp
1
=
276-277 °C. H NMR (200 MHz, DMSO-d ) δ: 12.69 (br s, 1H); 8.38 (s, 1H); 4.13-4.09 (t, J =
6
6
0
4
.8 Hz, 2H); 3.70-3.66 (m, 4H); 3.41-3.36 (m, 4H); 1.78-1.73 (m, 2H); 1.30-1.25 (m, 4H); 0.88-
.81 (t, J = 6.6 Hz, 3H).
,7-Dihydro-2-(4-methylpiperazin-1-yl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylic acid (14k). Following general procedure E, compound 14k was isolated as a white solid.
1
Yield 81%, mp = 225 °C dec. H NMR (200 MHz, DMSO-d ) δ: 12.72 (br s, 1H); 8.73 (s, 1H);
6
4
2
2
.21-4.17 (t, J = 6.6 Hz, 2H); 3.50-3.47 (m, 4H); 3.38-3.33 (m, 4H); 2.28 (s, 3H); 1.81-1.77 (m,
H); 1.29-1.26 (m, 4H); 0.88-0.82 (t, J = 6.6 Hz, 3H).
-(N-Benzyl-N-methylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxylic acid (14l). Following general procedure E, compound 14l was isolated as a white solid.
1
Yield 94%, mp = 130 °C. H NMR (400 MHz, DMSO-d ) δ: 12.71 (br s, 1H); 8.76 (s, 1H); 7.33-
6
7.24 (m, 5H); 4.64 (s, 2H); 4.22-4.19 (t, J = 7.2 Hz, 2H); 2.95 (s, 3H); 1.81-1.78 (m, 2H); 1.29-1.24
(m, 4H); 0.84-0.81 (t, J = 6.8 Hz, 3H).
23

ACCEPTED MANUSCRIPT
2-(Diallylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic
acid
(14m). Following general procedure E, compound 14m was isolated as a white solid. Yield 95%,
1
mp = 142-143 °C. H NMR (400 MHz, DMSO-d ) δ: 12.95 (br s, 1H); 8.77 (s, 1H); 5.92-5.78 (m,
6
2
H); 5.23-5.15 (m, 4H); 4.24-4.17 (t, J = 6.8 Hz, 2H); 4.05-4.02 (d, J = 5.6 Hz, 4H); 1.84-1.79 (m,
H); 1.30-1.26 (m, 4H); 0.88-0.82 (t, J = 6.6 Hz, 3H).
2
General procedure F for the synthesis of adamantan-1-yl-carboxamide derivatives 16, 18, 21, 23,
5, 28-30, 32, 35, 39, 41
2
To a stirred solution of the appropriate carboxylic acid (0.72 mmol) in anhydrous dimethylforma-
mide (10 mL) was added diisopropylethylamine (3.24 mmol). The resulting solution was stirred at
room temperature for 10 min before addition of O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU, 1.08 mmol). The solution was stirred for another 3 h at room temper-
ature. 1-Adamantylamine or 3,5-dimethyl-1-adamantylamine (1.08 mmol) was added and stirring
was continued for 16 h. The solvent was removed under reduce pressure, the residue was dissolved
in ethyl acetate (50 mL), then washed with saturated aqueous sodium bicarbonate (10 mL), water
(
10 mL), and brine (10 mL). The organic extract was dried (Na SO ), filtered, and evaporated to
2 4
dryness, and the residue was purified by flash chromatography on silica gel, eluting with ethyl ace-
tate-petroleum ether.
4,7-Dihydro-N-(3,5-dimethyladamantan-1-yl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (16). Following general procedure F, compound 16 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 7/3. Yield 60%, mp = 182 °C. MS (ESI): m/z 412.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.82 (s, 1H); 8.67 (br s, 1H); 8.38 (s, 1H); 4.33-4.30 (t, J = 7 Hz,
6
2H); 2.18-2.04 (m, 1H); 1.90-1.65 (m, 9H); 1.38-1.24 (m, 9H); 0.89-0.82 (m, 9H). Anal. for
C H N O . Calcd: C, 67.12; H, 8.08; N, 17.02. Found: C, 67.22; H, 8.18; N, 17.051.
23
33
5
2
N-Adamantan-yl-4,7-dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (18). Following general procedure F, compound 18 was isolated as a white solid. Elu-
ent: petroleum ether-ethyl acetate 3/2. Yield 56%, mp = 189-190 °C. MS (ESI): m/z 398.2 (M+H).
24

ACCEPTED MANUSCRIPT
1
H NMR (200 MHz, DMSO-d ) δ: 8.78 (s, 1H); 8.68 (br s, 1H); 4.30-4.22 (t, J = 7 Hz, 2H); 2.41 (s,
6
3H); 2.05 (s, 9H); 1.93-1.81 (m, 2H); 1.66 (s, 6H); 1.34-1.24 (m, 4H); 0.89-0.82 (t, J = 6.4 Hz, 3H).
Anal. for C H N O . Calcd: C, 66.47; H, 7.86; N, 17.62. Found: C, 66.22; H, 7.73; N, 17.37.
2
2
31
5
2
4,7-Dihydro-N-(3,5-dimethyladamantan-1-yl)-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxamide (21). Following general procedure F, compound 21 was isolated as a
white solid. Eluent: petroleum ether-ethyl acetate 4/1. Yield 55%, mp = 96 °C. MS (ESI): m/z 488.2
1
(
M+H). H NMR (200 MHz, DMSO-d ) δ: 8.81 (s, 1H); 8.72 (br s, 1H); 8.16-8.13 (m, 2H); 7.57-
6
7
0
7
.54 (m, 3H); 4.39-4.35 (t, J = 7 Hz, 2H); 2.19-2.07 (m, 1H); 1.93-1.68 (m, 9H); 1.39-1.18 (m, 9H);
.91-0.84 (m, 9H). Anal. for C H N O . Calcd: C, 71.43; H, 7.65; N, 14.36. Found: C, 71.34; H,
2
9
37
5
2
.60; N, 14.29.
N-Adamantan-1-yl-2-(4-chlorophenyl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxamide (23). Following general procedure F, compound 23 was isolated as a
white solid. Eluent: petroleum ether-ethyl acetate 4/1. Yield 53%, mp = 213 °C. MS (ESI): m/z
1
4
94.2 (M+H). H NMR (200 MHz, DMSO-d ) δ: 8.85 (s, 1H); 8.69 (br s, 1H); 8.16-8.12 (d, J = 8.6
6
Hz, 2H); 7.65-7.61 (d, J = 8.4 Hz, 2H); 4.38-4.34 (t, J = 7.2 Hz, 2H); 2.06 (s, 9H); 1.97-1.84 (m,
2
H); 1.68 (s, 6H); 1.36-1.31 (m, 4H); 0.90-0.84 (t, J = 6.2 Hz, 3H). Anal. for C H ClN O . Calcd:
27 32 5 2
C, 65.64; H, 6.53; N, 14.18. Found: C, 65.59; H, 6.42; N, 14.29.
N-Adamantan-1-yl-4,7-dihydro-2-(4-methoxyphenyl)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxamide (25). Following general procedure F, compound 25 was isolated as a
white solid. Eluent: petroleum ether-ethyl acetate 4/1. Yield 46%, mp = 176 °C. MS (ESI): m/z
1
4
90.2 (M+H). H NMR (200 MHz, DMSO-d ) δ: 8.81 (s, 1H); 8.73 (br s, 1H); 8.09-8.05 (d, J = 8.8
6
Hz, 2H); 7.12-7.08 (d, J = 9 Hz, 2H); 4.37-4.34 (t, J = 7.2 Hz, 2H); 3.83 (s, 3H); 2.06 (s, 9H); 1.96-
1
.81 (m, 2H); 1.67 (s, 6H); 1.36-1.32 (m, 4H); 0.91-0.85 (t, J = 6.6 Hz, 3H). Anal. for C H N O .
28 35 5 3
Calcd: C, 68.69; H, 7.21; N, 14.30. Found: C, 68.75; H, 7.28; N, 14.39.
N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (28). Following general procedure F, compound 28 was isolated as a white solid. Elu-
25

ACCEPTED MANUSCRIPT
1
ent: petroleum ether-ethyl acetate 4/1. Yield 44%, mp = 229 °C. MS (ESI): m/z 474.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.82 (s, 1H); 8.72 (br s, 1H); 8.05-8.01 (d, J = 8 Hz, 2H); 7.38-7.34
6
(d, J = 8.2 Hz, 2H); 4.38-4.33 (t, J = 7 Hz, 2H); 2.38 (s, 3H); 2.06 (s, 9H); 1.90-1.83 (m, 2H); 1.68
(
s, 6H); 1.36-1.30 (m, 4H); 0.90-0.84(t, J = 6.4 Hz, 3H). Anal. for C H N O . Calcd: C, 71.01; H,
2
8
35
5
2
7.45; N, 14.79. Found: C, 71.15; H, 7.52; N, 15.75.
N-Adamantan-1-yl-2-(furan-2-yl)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (29). Following general procedure F, compound 29 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 1/1. Yield 54%, mp = 175 °C. MS (ESI): m/z 450.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.82 (s, 1H); 8.67 (br s, 1H); 7.96-7.94 (d, J = 1.6 Hz, 1H); 7.22-
6
7
.19 (d, J = 3.4 Hz, 1H); 6.73-6.70 (dd, J = 3.4 and 1.8 Hz, 1H); 4.37-4.29 (t, J = 6.8 Hz, 2H); 2.06
s, 9H); 1.96-1.82 (m, 2H); 1.67 (s, 6H); 1.37-1.30 (m, 4H); 0.90-0.84 (t, J = 6.2 Hz, 3H). Anal. for
C H N O . Calcd: C, 66.79; H, 6.95; N, 15.58. Found: C, 66.75; H, 6.96; N, 15.61.
(
25
31
5
3
N-Adamantan-1-yl-4,7-dihydro-7-oxo-4-pentyl-2-(pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (30). Following general procedure F, compound 30 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 1/1. Yield 45%, mp = 160 °C. MS (ESI): m/z 461.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 9.29 (d, J = 1.6 Hz, 1H); 8.87 (s, 1H); 8.76-8.72 (dd, J = 7.4 and 1.8
6
Hz, 1H); 8.68 (br s, 1H); 8.49-8.43 (dt, J = 7.8 and 1.6 Hz, 1H); 7.63-7.57 (dd, J = 7.8 and 4.2 Hz,
1H); 4.41-4.36 (t, J = 6.8 Hz, 2H); 2.07 (s, 9H); 1.99-1.85 (m, 2H); 1.74-1.59 (m, 8H); 1.36-1.32
(
m, 2H); 0.91-0.84 (t, J = 6 Hz, 3H). Anal. for C H N O . Calcd: C, 67.80; H, 7.00; N, 18.25.
26
32
6
2
Found: C, 67.74; H, 7.23; N, 18.33.
N-Adamantan-1-yl-4,7-dihydro-2-(methylthio)-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (32). Following general procedure F, compound 32 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 4/1. Yield 55%, mp = 161 °C. MS (ESI): m/z 430.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.75 (s, 1H); 8.67 (br s, 1H); 4.27-4.22 (t, J = 7 Hz, 2H); 2.61 (s,
6
3H); 2.05 (s, 9H); 1.91-1.74 (m, 2H); 1.67 (s, 6H); 1.33-1.25 (m, 4H); 0.89-0.83 (t, J = 6.4 Hz, 3H).
Anal. for C H N O S. Calcd: C, 61.51; H, 7.27; N, 16.30. Found: C, 61.34; H, 7.29; N, 16.45.
2
2
31
5
2
26

ACCEPTED MANUSCRIPT
N-Adamantan-1-yl-4,7-dihydro-2-morpholino-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (35). Following general procedure F, compound 35 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 2/3. Yield 40%, mp = 148 °C. MS (ESI): m/z 469.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.81 (br s, 1H); 8.64 (s, 1H); 4.08-3.99 (t, J = 6.8 Hz, 2H); 3.71-
6
3
0
6
.67 (m, 4H); 3.44-3.40 (m, 4H); 2.03 (s, 9H); 1.85-1.70 (m, 2H); 1.66 (s, 6H); 1.33-1.26 (m, 4H);
.89-0.83 (t, J = 6.6 Hz, 3H). Anal. for C H N O . Calcd: C, 64.08; H, 7.74; N, 17.93. Found: C,
2
5
36
6
3
4.19; H, 7.79; N, 17.90.
N-Adamantan-1-yl-2-(N-benzyl-N-methyl-amino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-
a]pyrimidine-6-carboxamide (39). Following general procedure F, compound 39 was isolated as a
white solid. Eluent: petroleum ether-ethyl acetate 2/3. Yield 60%, mp = 139 °C. MS (ESI): m/z
1
5
4
1
1
19.2 (M+H). H NMR (200 MHz, DMSO-d ) δ: 8.85 (br s, 1H); 8.61 (s, 1H); 7.34-7.29 (m, 5H);
6
.65 (s, 2H); 4.24-4.19 (t, J = 7 Hz, 2H); 2.97 (s, 3H); 2.03 (s, 9H); 1.87-1.75 (m, 2H); 1.66 (s, 6H);
.30-1.26 (m, 4H); 0.88-0.82 (t, J = 6.2 Hz, 3H). Anal. for C H N O . Calcd: C, 69.29; H, 7.62; N,
2
9
38
6
2
6.72. Found: C, 69.32; H, 7.77; N, 16.68.
N-Adamantan-1-yl-2-(diallylamino)-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-
-carboxamide (41). Following general procedure F, compound 41 was isolated as a white solid.
6
Eluent: petroleum ether-ethyl acetate 4/1. Yield 45%, mp = 110 °C. MS (ESI): m/z 479.2 (M+H).
1
H NMR (200 MHz, DMSO-d ) δ: 8.84 (br s, 1H); 8.60 (s, 1H); 5.87-5.78 (m, 2H); 5.24-5.14 (m,
6
4
1
6
H); 4.22-4.16 (t, J = 6.8 Hz, 2H); 4.04-4.01 (d, J = 5.6 Hz, 4H); 2.03 (s, 9H); 1.90-1.72 (m, 2H);
.66 (s, 6H); 1.32-1.25 (m, 4H); 0.88-0.82 (t, J = 6.6 Hz, 3H). Anal. for C H N O . Calcd: C,
2
7
38
6
2
7.75; H, 8.00; N, 17.56. Found: C, 67.71; H, 7.98; N, 17.49.
General procedure G for the synthesis of N-cyclohexyl/cycloheptyl carboxamide derivatives 15, 17,
9, 20, 22, 24, 26, 27, 31, 33, 34, 36-38, 40
1
To a solution of the appropriate carboxylic acid (0.57 mmol) in DMF, 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide (EDCI, 0.85 mmol) and 1-hydroxybenzotriazole (HOBt, 0.85 mmol)
were added and the reaction was stirred at room temperature for 10 min before adding the suitable
27

ACCEPTED MANUSCRIPT
amine (0.85 mmol) and the stirring continued at the same temperature for 16h. The solvent was
evaporated under reduced pressure and the residue was dissolved in ethyl acetate (30 mL), washed
with aqueous bicarbonate (10 mL), water (10 mL) and brine (10 mL). The organic extract was
dried, filtered and concentrated and resulting products were purified by flash chromatography on
silica gel, eluting with ethyl acetate and petroleum ether or ethyl acetate and methanol.
N-Cycloheptyl-4,7-dihydro-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxamide (15).
Following general procedure G, compound 15 was isolated as a white solid. Eluent: petroleum
1
ether-ethyl acetate 2/3. Yield 46%, mp = 149 °C. MS (ESI): m/z 346.2 (M+H). H NMR (200 MHz,
DMSO-d ) δ: 8.89 (s, 1H); 8.85-8.83 (br d, J = 7.8 Hz, 1H); 8.39 (s, 1H); 4.34-4.30 (t, J = 7 Hz,
6
2H); 4.17-4.01 (m, 1H); 1.96-1.75 (m, 4H); 1.70-1.42 (m, 10H); 1.35-1.24 (m, 4H); 0.88-0.83 (t, J =
6
.4 Hz, 3H). Anal. for C H N O . Calcd: C, 62.58; H, 7.88; N, 20.27. Found: C, 62.51; H, 7.82;
1
8
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5
2
N, 20.39.
N-Cyclohexyl-4,7-dihydro-2-methyl-7-oxo-4-pentyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (17). Following general procedure G, compound 17 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 2/3. Yield 41%, mp = 120 °C. MS (ESI): m/z 346.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.83 (s, 1H); 8.81-8.77 (br d, J = 7.8 Hz, 1H); 4.31-4.24 (t, J = 7
6
Hz, 2H); 3.91-3.78 (m, 1H); 2.41 (s, 3H); 1.94-1.54 (m, 6H); 1.43-1.24 (m, 10H); 0.89-0.82 (t, J =
6
.6 Hz, 3H). Anal. for C H N O . Calcd: C, 62.58; H, 7.88; N, 20.27. Found: C, 62.61; H, 7.59;
18 27 5 2
N, 20.32.
N-Cyclohexyl-4,7-dihydro-7-oxo-4-pentyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-
carboxamide (19). Following general procedure G, compound 19 was isolated as a white solid. Elu-
1
ent: petroleum ether-ethyl acetate 4/1. Yield 55%, mp = 161 °C. MS (ESI): m/z 408.2 (M+H). H
NMR (200 MHz, DMSO-d ) δ: 8.90 (s, 1H); 8.85-8.81 (br d, J = 8 Hz, 1H); 8.17-8.12 (m, 2H);
6
7.57-7.54 (m, 3H); 4.40-4.35 (t, J = 7 Hz, 2H); 3.94-3.81 (m, 1H); 1.91-1.58 (m, 6H); 1.39-1.28 (m,
1
0H); 0.90-0.84 (t, J = 6.4 Hz, 3H). Anal. for C H N O . Calcd: C, 67.79; H, 7.17; N, 17.19.
23
29
5
2
Found: C, 67.68; H, 6.94; N, 17.25.
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