Scaffold decoration at positions 5 and 8 of 1,2,4-triazolo[1,5- c ]pyrimidines to explore the antagonist profiling on adenosine receptors: A preliminary structure-activity relationship study

Article

pubs.acs.org/jmc

Scaﬀold Decoration at Positions 5 and 8 of 1,2,4-

Triazolo[1,5‑c]Pyrimidines to Explore the Antagonist Proﬁling on

Adenosine Receptors: A Preliminary Structure−Activity Relationship

Study

Stephanie Federico,^†Antonella Ciancetta,^‡Nicola Porta,^‡Sara Redenti,^†Giorgia Pastorin,^§

Barbara Cacciari,^∥Karl Norbert Klotz,^⊥Stefano Moro,*^,‡and Giampiero Spalluto*^,†

^†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita

^‡Molecular Modeling Section (MMS), Dipartimento di Scienze del Farmaco, Universita

^§Department of Pharmacy, National University of Singapore, 3 Science Drive 2, Singapore 117543, Singapore

^∥Dipartimento di Scienze Farmaceutiche, Universita

degli Studi di Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy

^⊥Institut fur Pharmakologie, Universitat of Wurzburg, Versbacher Strasse 9, 97078 Wurzburg, Germany

̀

di Trieste, Piazzale Europa 1, 34127 Trieste, Italy

di Padova, via Marzolo 5, 35131 Padova, Italy

̀

̈

S

* Supporting Information

ABSTRACT: The structure−activity relationship (SAR) of new 5,8-disubstituted-

1,2,4-triazolo[1,5-c]pyrimidines as adenosine receptors (ARs) antagonists has been

explored. All the synthesized compounds show aﬃnity for the hA_2Aand hA₃ARs

depending on the substitution patterns at the 5 and 8 positions. In particular, a free

amino group at the 5 position with an ethoxycarbonyl group at the 8 position leads

to potent and quite selective hA_2Aantagonists (compound 12: hA_2AAR K_i= 3.32

nM; hA₁/hA_2A= 55.6; hA_2A/hA₃= 0.01), whereas the introduction of a methylamino

function at the 5 position yields a good binding proﬁle at the hA₃AR (compound 23:

hA₃AR K_i= 4.14 nM, hA₁/hA₃= 236; hA_2A/hA₃= 25). Through an in silico

receptor-driven approach, we have determined the most favorable orientation of the

substitutions at the 5 and 8 positions of the 1,2,4-triazolo[1,5-c]pyrimidine (TP)

scaﬀold and, accordingly, we have elucidated the observed SAR.

potent and selective ARs antagonists has been one of the most

highly investigated areas in medicinal chemistry in recent

years.¹⁷

Our research group and others have extensively investigated

the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus that,

after appropriate modiﬁcations and optimization of the N7, N8,

and 5 positions, led to very potent and selective human A_2AAR

antagonists such as compound 1 and preladenant¹⁸⁻²⁰or A₃

AR²¹⁻²⁶antagonists such as compound 2 (Chart 1).

Despite the achieved potency and selectivity, those

compounds suﬀered from poor water solubility and compli-

cated synthetic preparation. Taking into account these

drawbacks, in the last years we focused our attention on the

synthesis of more simpliﬁed heterocyclic derivatives, in

particular bicyclic systems such as 1,2,4-triazolo[1,5-a]-1,3,5-

triazine,²⁷⁻²⁹considering that ZM 241385 has been reported as

a potent A_2AAR antagonist.^30,31In addition, this compound

binds also with good aﬃnity to the human A_2BAR (28 nM),

and in its tritiated form, the compound is actually used in

radioligand binding studies for this subtype.³²In particular, at

INTRODUCTION

■

Activation of adenosine receptors (ARs) is responsible of

several eﬀects on diﬀerent organ systems. On the basis of the

widespread, and frequently beneﬁcial, eﬀects attributed to the

accumulation of endogenously released adenosine, it is

generally accepted that regulation of ARs has great therapeutic

potential. In particular, the cardioprotective^1,2and neuro-

protective^3,4eﬀects associated with AR activation during

periods of cardiac and cerebral ischemia, respectively, have

been widely investigated. Moreover, it has also been proposed

that antagonists for distinct AR subtypes may be used in the

treatment of asthma^5,6or neurological diseases such as

Parkinson’s disease.⁷

ARs are members of the superfamily of G protein-coupled

receptors (GPCRs), with four subtypes currently known, the A₁

AR, A_2AAR, A_2BAR, and A₃AR.⁸In the recent decades,

numerous medicinal chemistry groups have made intense

eﬀorts in developing ideal ligands for these receptor

subtypes.⁹⁻¹⁶In particular, the search for selective antagonists

held greater appeal than selective agonists, not only for their

potential therapeutic applications but also for their preferential

use as molecular probes for pharmacological characterization of

receptors. Considering all of these aspects, the development of

Received: May 15, 2014

Published: June 27, 2014

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With the aim to validate this hypothesis, we decided to

translate the substituent to the 4 position of the 1,2,4-triazolo-

[1,5-a]-1,3,5-triazine nucleus, but the presence of the nitrogen

at this position does not allow such a modiﬁcation (Chart 3).

We therefore designed a new class of compounds on the

basis of a 1,2,4-triazolo[1,5-c]pyrimidine nucleus which could

be considered a deaza analogue of the 1,2,4-triazolo-[1,5-a]-

1,3,5-triazine core, bearing diﬀerent substituents at the 5 and 8

positions, and we analyzed their binding proﬁle at the four

human ARs.

Chart 1. Pyrazolo-triazolo-pyrimidines as Human A_2Aand A₃

Adenosine Receptor Antagonists

RESULTS AND DISCUSSION

■

A. Chemistry. All the designed compounds 5−66 have

been synthesized as summarized in Schemes 1−4. Condensa-

tion between isothiourea 67 and diethyl ethoxymethylene

malonate 68 in basic condition led to a pyrimidine sodium salt

69, which after treatment with POCl₃under reﬂux aﬀorded

chloro derivative 70 that was immediately coupled with 2-furoyl

hydrazide in the presence of DBU leading to compound 71

(Scheme 1).^35,36

the N7 position of the 1,2,4-triazolo-[1,5-a]-1,3,5-triazine

nucleus, we introduced arylcarbamoyl (3) or arylacetyl (4)

moieties, which gave good results in terms of aﬃnity at the

21−25

33

A₃

and A_2BARs, respectively, when attached to the

pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine nucleus, to ob-

tain corresponding A_2Bor A₃AR antagonists with simpliﬁed

core (Chart 2).³⁴

By reacting the hydrazide 71 in the presence of P₂O₅/HMDS

in dry xylene at reﬂux, the corresponding triazolo-pyrimidine 5

was obtained. Nucleophilic substitution of methylthio group

with veratrylamine at 120 °C in a sealed tube yielded

benzylamino derivative 6, which under basic conditions

(LiOH H₂O/EtOH), led to the corresponding carboxylic acid

7.

Chart 2. N7 Phenylcarbamoyl and Phenylacetyl Triazolo-

triazines

By reacting acid derivative 7 with the appropriate amine in

the presence of pyridine and POCl₃, the corresponding

carboxamido derivatives 8,9 were obtained, which after amino

benzyl deprotection with TFA gave the ﬁnal compounds 10,11

(Scheme 2a). Aminoester derivative 12 could be easily obtained

by reacting intermediate 5 with ethanolic ammonia in a sealed

tube at 120 °C (Scheme 2b).

N5-Arylacetyl (15, 18, 21) or N5 urea (13, 14, 16, 17, 19,

20) derivatives were obtained by reacting the amino

compounds (10−12) with the appropriate acyl chloride or

isocyanate in dry THF at reﬂux overnight (Scheme 3a).

Treatment of compound 20 with LiOH in a mixture EtOH/

H₂O led to the corresponding acid derivative 22 (Scheme 3b).

5-Aminoalkyl derivatives (23−50) were obtained by reacting

methylthio intermediate 5 with the appropriate amine in

ethanol at 120 °C in a sealed tube (Scheme 4).

Carboxylic acid compounds 51−66 were obtained by

saponiﬁcation of the corresponding esters (23−38) in the

presence of LiOH/H₂O/EtOH.

B. Biology. Newly synthesized compounds (5−66) were

tested at the human A₁, A_2A, and A₃receptors expressed in

Unfortunately, both compounds showed poor aﬃnity and

selectivity for the hA₃and hA_2BARs. A possible explanation of

this lack of aﬃnity has been given comparing the 1,2,4-triazolo-

[1,5-a]-1,3,5-triazine derivatives 3,4 with the pyrazolo[4,3-e]-

1,2,4--triazolo[1,5-c]-pyrimidine 1,2. We hypothesized that

most probably the dimethylamino group in the 1,2,4-triazolo-

[1,5-a]-1,3,5-triazine nucleus is in a wrong position. In fact, the

dimethylamino group lacks correspondence with the N8-

methyl group of derivative 2 but is quite similar to its N7

pattern of substitution (e.g., compound 1), which has been

extensively demonstrated to be inactive at the hA₃ARs.^19,20,33

Chart 3. Rational Design and General Structures of Synthesized Compounds (5−66)

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a

Scheme 1. Synthesis Reagents and Conditions

a

(Upper left) i: NaOH, H₂O, EtOH, rt, 24 h; ii: POCl₃, reﬂux, 3 h; iii: 2-furoylhydrazide, DBU, THF, rt, 5 h; v: P₂O₅, HMDS, xylene, 90°C, 2 h,

reﬂux, 48 h; iv: 3,4-dimethoxybenzylamine, EtOH, 120 °C, sealed tube, 2 h; vii: LiOH·H₂O, H₂O/EtOH, 1:40, reﬂux, 2 h.

a

Scheme 2. Synthesis Reagents and Conditions

a

(Panel a) i: methylamine or β-phenylethyl amine, dry pyridine, POCl₃, rt, 2 h; ii: TFA, triﬂuoromethanesulfonic acid, anisole, rt, overnight. (Panel

b) i: NH₃7N in MeOH, 120 °C, 2 h.

a

Scheme 3. Synthesis Reagents and Conditions

Scheme 4. Synthesis Reagents and Conditions

a

(Left) i: R-NH₂or RR′NH, EtOH, sealed tube, 120 °C, 2 h; ii:

LiOH·H₂O, H₂O/EtOH 1:40, reﬂux, 2 h.

CHO cells: [³H]CCPA (A₁) and [³H]NECA (A_2A, A₃) were

used as radioligands in binding assays.³⁷Inhibition of NECA-

stimulated adenylyl cyclase activity was determined as an

aﬃnity measurement of the compounds at the A_2BAR (Table

1).

Structure−Activity Relationships. As clearly summarized in

Table 1, all the synthesized compounds (5−66) show aﬃnities

at the hA₃and/or hA_2AARs ranging from high nanomolar to

nanomolar concentrations, with diﬀerent degree of selectivity

versus the other subtypes. It is quite evident that a nitrogen at

the 5 position is fundamental for the aﬃnity, as compound (5)

bearing a thiomethyl group at the 5 position is inactive at all the

a

(Panel a) i: R-NCO, dry THF, reﬂux, overnight or RCOCl, Et₃N, dry

THF reﬂux, overnight. (Panel b) i: LiOH, H₂O/EtOH 1:40, reﬂux, 2

h.

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four AR subtypes. Also the presence of a carboxylic function at

the 8 position, which improves water solubility, leads to

compounds (7, 22, 51−66) exhibiting very poor aﬃnity at all

the ARs subtypes. Exceptions are observed for compounds

bearing a cyclopropylamino (56) or a dimethoxybenzylamino

(7) groups at the 5 position showing aﬃnity toward the hA₃AR

but poor selectivity versus the other receptor subtypes.

The introduction of a free amino group at the 5 position

leads to derivatives (10−12) with good aﬃnity versus the hA_2A

AR: In particular, the free amino function at the 5 position

combined with a ethoxycarbonyl moiety at the 8 position yields

a potent derivative (12) selective for the hA_2AAR (hA_2AAR K_i

= 3.32 nM; hA₁/hA_2A= 55.7; hA_2A/hA₃= 0.01). The

simultaneous introduction at the 8 position of a methyl

carboxamido (10) or β-phenethylcarboxamido (11) groups,

instead, produces a reduction of the aﬃnity (3−5-fold) at the

hA_2AAR with consequent drastic reduction of selectivity (e.g.,

compound 11: hA_2AAR K_i= 11.1 nM; hA_2A/hA₃= 0.08; hA₁/

hA_2A= 5.14).

In contrast with our expectations, the introduction at the 5

position of cycloalkylamino/arylureido or arylacetamido

moieties does not aﬀord an eﬀect at the hA₃or hA_2BARs. In

fact, the presence of a cyclohexylureido group at the 5 positon

(14, 17, 20) is detrimental for aﬃnity at all the AR subtypes,

and only when an ethoxycarbonyl moiety is present at the 8

position is a good aﬃnity for the hA_2AAR observed, albeit with

poor selectivity versus the other receptor subtypes (compound

20: hA_2AAR K_i= 183 nM; hA₁/hA_2A= 69.4; hA_2A/hA₃= 0.07).

On the contrary, the introduction of an arylureido moiety at the

5 position (13, 16, 19) gives diﬀerent results in terms of aﬃnity

and selectivity depending on the substitutions at the 8 position.

The simultaneous presence at the 8 position of an

ethoxycarbonyl (19) or a methyl carboxamido (13) function

leads to compounds with good aﬃnity and selectivity for the

hA_2AAR (e.g., compound 19: hA_2AAR K_i= 4.82 nM; hA_2A/hA₃

= 0.01; hA₁/hA_2A= 66.8), whereas the presence of a β-

phenethylcarboxamido group produces a reduction of the

aﬃnity (10-fold) at the hA_2AAR with a signiﬁcative loss of

selectivity (e.g., compound 16: hA_2AAR K_i= 51.4 nM; hA_2A/

hA₃= 0.04; hA₁/hA_2A= 7.1).

for the A_2AAR subtype (e.g., compound 37: hA₃AR K_i= 875

nM; hA_2AAR K_i= 187 nM). An improvement of hA_2AAR

aﬃnity with respect to hA₃AR, considering the 5-benzylamino

derivatives, is also observed when at the 8 position a substituted

carboxamido function (8,9) is present (e.g., compound 8: hA₃

AR K_i= 282 nM; hA_2AAR K_i= 25.8 nM). Also, introduction of

bulkier substituents at the 5 position such as benzhydrylamino

group (38) leads to a reduction of both aﬃnity and selectivity

at hA_2Aand hA₃ARs.

Concerning the aﬃnity at the hA₃AR, promising results are

observed when alkylamino substituents are introduced at the 5

position and an ethoxycarbonyl group is present at the 8

position (23−27, 30, 49). Small alkylamino groups such as

methylamino (23) or ethylamino (24) give good aﬃnity and

selectivity (e.g., compound 23: hA₃AR K_i= 4.14 nM, hA₁/hA₃

= 236; hA_2A/hA₃= 25.1), whereas bulkier and branched chains

(26, 27, 49) lead to a reduction of the aﬃnity with a signiﬁcant

reduction of selectivity versus the other receptor subtypes. This

reduction of the aﬃnity is more evident with bulkier

substituents such as 2-ethylhexylamino group (compound 27:

hA₃AR K_i= 39.3 nM; hA₁/hA₃= 29.3; hA_2A/hA₃= 5.47).

A similar behavior is observed when cycloalkylamino

substituents (28, 29, 39−41, 47) are introduced at the 5

position. Small cycloalkylamino groups, such as cyclopropyla-

mino (28) or cyclobutylamino (39) show good aﬃnity at the

hA₃AR (e.g., compound 28: hA₃AR K_i= 9.52 nM), although

bigger cycloalkylamino groups (29, 40, 41, 47) are not well-

tolerated (e.g., compound 41: hA₃AR K_i= 517 nM).

Nevertheless, independently from the cycloalkylamino group

dimension, the levels of selectivity versus the other receptor

subtypes are very low.

Introduction at the 5 position of polar groups such as

methoxyamino (50) or 2-methoxyethylamino (44) results in a

drastic reduction of the aﬃnity at the hA₃AR and low levels of

selectivity versus the other receptor subtypes (e.g., compound

44: hA₃AR K_i= 105 nM; hA₁/hA₃= 21.6; hA_2A/hA₃= 2.78).

Disubstitution of the nitrogen at the 5 position with methyl

groups (45) signiﬁcantly reduces both the aﬃnity and the

selectivity at the hA₃AR (hA₃AR K_i= 275 nM; hA₁/hA₃=

48.7; hA_2A/hA₃= 23.6). A more drastic reduction of aﬃnity and

selectivity at the hA₃AR is observed when cyclic amines are

introduced at the 5 position: Indeed, pyrrolidine (42) or

piperidine (43) derivatives show aﬃnity for the hA₃AR in the

high nanomolar range (836−1770 nM) and a total absence of

selectivity versus other receptor subtypes. More interestingly,

the introduction of heterocycles with a heteroatom at the 4

position such as morpholine (46) or insertion of other polar

functions as in the 4-aminomorpholin derivative (48) gives

compounds inactivity at all the four ARs, suggesting that polar

groups at the 5 position are not well-tolerated.

The introduction of a phenylacetamido group at the 5

position in the presence of a substituted carboxamido group at

the 8 position leads to compounds (15, 18) with good hA_2AAR

aﬃnity and quite good selectivity versus the other AR subtypes

(e.g., compound 15: hA_2AAR K_i= 12.4 nM; hA_2A/hA₃= 0.01;

hA₁/hA_2A= 23.9), although the presence of ethylester at the 8

(21) position results in this case detrimental for aﬃnity at all

the four AR subtypes (compound 21: hA₁AR K_i> 100 000

nM; hA_2AAR K_i= 42 700 nM; hA_2BAR K_i> 30 000 nM; hA₃

AR K_i= 2590 nM).

A signiﬁcantly diﬀerent binding proﬁle is observed when at

the 5 position an (ar)alkylamino moiety is introduced in the

presence of an ethoxycarbonyl function at the 8 position. In

fact, the introduction of a benzylamino group at the 5 position

(31) leads to an improvement of the aﬃnity at the hA₃AR, but

the selectivity is still low, in particular versus the hA_2AAR (hA₃

AR K_i= 10 nM; hA₁/hA₃= 94; hA_2A/hA₃= 3.2). Substitutions

on the para position of phenyl ring (6, 32−37) in general

reduces the aﬃnity (3−5-fold) at the hA₃AR with a signiﬁcant

reduction of selectivity (e.g., compound 32: hA₃AR K_i= 56.1

nM; hA₁/hA₃= 23.9; hA_2A/hA₃= 2.17). Substitution of the

phenyl ring with a 4-CF₃(36) or 4-Ph (37) is detrimental in

terms of hA₃AR aﬃnity and selectivity but shows better aﬃnity

C. Molecular Modeling. To explain from a molecular point

of view the observed binding data, we performed a receptor-

based molecular modeling study of the new derivatives by

running a previously reported computational protocol.³⁸The

crystallographic structure of the hA_2AAR and hA₃AR

homology model represent the macromolecular starting point

of our survey. Selected compounds were docked into the

putative TM binding site of the hA₃AR and the orthosteric

pocket of the hA_2AAR with the aim to identify their

hypothetical binding modes. The outcoming docking poses

were selected by taking into account optimal interaction

geometries with the residues surrounding the binding site. After

the selection of a representative docking pose for each

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Figure 1. Per residue IEele and IEhyd maps for the most energetically favorable docking poses of compounds with K_i< 100 nM. The maps have

been computed for selected poses of the considered compound inside the orthosteric binding site of hA₂AR (panels A and B) and the putative

binding site of hA_2AAR (panels C and D). Electrostatic energy contributions are expressed in kcal/mol, whereas hydrophobic contributions are in

arbitrary units. Ranges reported in the x-axes represent the length of protein sequences, truncated at 300 residues.

compound, we performed a general analysis of the binding

modes to identify the residues involved in the binding with the

ligands by computing per residue electrostatic and hydrophobic

contributions to the interaction energy (IEele and IEhyd,

respectively). This semiquantitative analysis was graphically

transferred into “Interaction Energy Fingerprints” (IEFs), that

is, maps that allow clear recognition of common features and

diﬀerences among the interaction proﬁles of the considered

compounds. We completed our analysis by the inspection of

individual docking poses to explain, from a molecular point of

view, the exhibited aﬃnity and selectivity proﬁles. A detailed

description of the methods used is reported in the Experimental

Section.

For our molecular modeling investigation we focused on

derivatives showing K_ivalues falling into the low nanomolar

range by applying an arbitrary cutoﬀ of 100 nM, whereas a

more detailed analysis has been performed for the ligands that

display higher activity toward the hA_2A(K_i< 15 nM) and hA₃

AR subtypes (K_i< 10 nM).

(Figure 1B) displays several residues involved in hydrophobic

contacts with the ligands, such as Phe168 (EL2) and Leu249

(6.51), for which shared electro-neutral surfaces are broadest.

Additionally, Ile66 (2.64), Val84 (3.32), Tyr271 (7.36), and

Ile274 (7.39) contribute to the stabilization of ligand−receptor

complexes. Similar interaction patterns are also observed for the

energetically more favorable poses of the ligands at the hA₃AR

(Figure 1C,D) In particular, the most prominent electrostatic

interactions are established with Asn250 (6.55), whereas

Gln261 (7.32), Tyr265 (7.36), and His272 (7.43) are engaged

in secondary interactions. Hydrophobic contacts include,

among others, Phe168 (EL2) and Leu246 (6.51), and to a

lesser extent also Val72 (2.64), Leu90 (3.32), Trp243 (6.48),

Leu264 (7.35), Tyr265 (7.36), and Ile268 (7.39).

The IEFs analysis (Figure 2A) for the most active

compounds at the hA_2AAR identiﬁes Asn253 (6.55) and

Phe168 (EL2) as the residues mainly contributing to the

interaction energy for all the selected derivatives. The

interaction with Glu169 (EL2), instead, is a prerogative of

compounds bearing a free amino group at the 5 position (10,

12), whereas derivatives bearing arylcarbamoyl (19) and

phenylacetyl (15) groups establish additional hydrophobic

contacts with Ile66 (2.64), Val84 (3.32), and Tyr271 (7.36).

The corresponding hypothetical binding modes of 12 (Figure

2B) and 19 (Figure 2C) help in explaining these diﬀerences:

both ligands reside in the upper region of the transmembrane

The IEele map for the hA_2AAR subtype (Figure 1A) shows

three regions of negative potential energy corresponding to

residues mainly located in TM2, TM6, and EL2. Phe168 (EL2)

and Asn253 (6.55) take part to the most eﬀective polar

interactions, whereas Asp170 (EL2) and to a lesser extent

Asn181 (5.42) and the backbones of Ala63 (2.61) and Leu85

(3.33) are engaged in secondary interactions. The IEhyd map

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Figure 2. (A) Interaction Energy Fingerprints (IEFs) for the most active compound at the hA_2AAR (K_i< 15 nM). Hypothetical binding modes of

compounds 12 (B) and 19 (C) inside the orthosteric binding site of hA_2AAR. Poses are viewed from the membrane side facing TM6, TM7, and

TM1, hydrogen bonds are highlighted as dashed lines, and side chains of Asn253 (6.55), Phe168 (EL2), and Glu169 (EL2) are represented as sticks.

In panel C, residues interacting through hydrophobic contacts with compound 19 are displayed as surfaces.

(TM) bundle, with the triazole-pyrimidine (TP) core anchored

by an aromatic π−π stacking interaction with Phe168 (EL2).

Compound 12 (hA_2AAR K_i= 3.32 nM) is anchored in the cleft

by three hydrogen bonds established by the free amino group at

the 5 position and the N2 of the triazole ring. The ligand lies in

the pocket with the furan ring pointing toward TM6, by

adopting a conformation that resembles the crystallographic

binding mode of ZM 241385. For compound 19 (hA_2AAR K_i=

4.82 nM) the hydrogen bond acceptor role is played by the

ethoxycarbonyl group at the 8 position. Therefore, the scaﬀold

is rotated by about 180° with respect to the placement of 12,

and the furan ring is directed toward TM7. In this position the

furan ring establishes hydrophobic contacts with Val84 (3.32),

whereas the aromatic ring of the arylcarbamoyl moiety interacts

with Tyr271 (7.36) and Ile66 (2.64), represented as trans-

parent surfaces in Figure 2C.

The IEFs analysis (Figure 3A) for the most active

compounds at the hA₃AR shows a common interaction

pattern for all the considered derivatives, with the main

interactions established with Asn250 (6.55) and Phe168 (EL2).

A detailed analysis of the individual docking poses, however,

reveals that the scaﬀold is placed at least into two diﬀerent

orientations, as reported in Figure 3B,C, representing the

hypothetical binding mode of compounds 24 and 31,

respectively. Derivatives bearing small alkyl and cycloalkyl

groups like compound 24 interact with Asn250 (6.55) through

the ethoxycarbonyl group at the 8 position, by pointing the

furane ring toward TM2 and exposing the alkyl group to the

solvent. Conversely, compounds bearing branched chains and

benzyl groups like compound 31 interact with Ans250 (6.55)

with the N4 of the triazole ring, with the furane ring solvent-

exposed and the alkyl/benzyl group projected toward TM2.

A similar trend (e.g., diﬀerent binding modes depending on

the nature and the steric hindrance of the substituents) was also

previously observed for 5-alkylaminopyrazolo-[4,3-e]1,2,4-

triazolo[1,5-c]pyrimidines derivatives.³⁸

CONCLUSIONS

■

We have presented a novel series of 5,8-disubstituted-1,2,4-

triazolo[1,5-c]pyrimidines acting as antagonists of the hA_2Aand

hA₃ARs. We have analyzed the corresponding SAR and found

that the synthesized compounds show aﬃnity for the hA_2Aand

hA₃ARs depending on the substitution patterns at the 5 and 8

positions. In particular, a free amino group at the 5 position

combined with ethoxycarbonyl group at the 8 position aﬀords a

potent and quite selective hA_2Aantagonists (12, hA_2AAR K_i=

3.32 nM), while the introduction of a methylamino function at

the 5 position yields a good binding proﬁle at the hA₃AR (23,

hA₃AR K_i= 4.14 nM).

Using an in silico receptor-driven approach, we have

determined the most favorable orientation of the substitutions

at the 5 and 8 positions of the TP scaﬀold at both the

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Figure 3. (A) Interaction Energy Fingerprints (IEFs) for the most active compound at the hA₃AR (K_i< 10 nM). Hypothetical binding modes of

compounds 24 (B) and 31 (C) inside the putative binding site of hA₃AR. Poses are viewed from the membrane side facing TM6, TM7, and TM1,

hydrogen bonds are highlighted as dashed lines, and side chains of Asn250 (6.55) and Phe168 (EL2) are represented as sticks.

Chimica, University of Trieste, and were within 0.4% of the

theoretical values for C, H, and N. Complete elemental analyses are

given in the Supporting Information, and the chemical formula is

supplied in the section for each compound.

considered receptor subtypes. At the hA_2AAR compounds

bearing a free amino group at the 5 position are anchored

inside the binding pocket by a tight network of hydrogen

bonds, whereas derivatives bearing arylureido and phenyl-

acetamido groups establish several hydrophobic contacts. At the

hA₃AR, instead, although the most active derivatives show a

common interaction pattern, the corresponding docking poses

suggest at least two diﬀerent orientations of the TP scaﬀold

into the binding cleft, depending on the steric hindrance of the

substituent at the 5 position.

4-Hydroxy-2-methylthio-5-carbethoxypyrimidine Sodium Salt

(69). To a solution of 34.4 g (0.860 mol) of sodium hydroxide in

215 mL of water at 20 °C were added 59.8 g of 2-methyl-2-thio-

pseudourea sulfate (67, 0.215 mol). The mixture was stirred for 5 min,

by which time most of the pseudothiourea had dissolved. Diethyl

ethoxymethylenemalonate (68, 0.430 mol, 92.9 g), dissolved in 500

mL of ethanol, was added with stirring over a period of 1 h. Stirring

was continued for 3 h after the addition was completed. After it was

allowed to stand for 24 h, the mixture was ﬁltered to give a pink solid.

EXPERIMENTAL SECTION

1

■

Yield 60−70%; pale pink solid mp 310 °C. H NMR (D₂O) δ: 1.2

A. Chemistry. Reactions were routinely monitored by thin-layer

chromatography (TLC) on silica gel (precoated F₂₅₄Merck plates).

Infrared spectra (IR) were measured on a Jasco FT-IT instrument. ¹H

NMR were determined in CDCl₃or DMSO-d₆solutions with a Varian

Gemini 200 spectrometer, peaks positions are given in parts per

million (δ) downﬁeld relative to the central peak of the solvents, and J

values are given in Hz. Electrospray mass spectra were recorded on a

PerkinElmer PE SCIEX API 1 spectrometer, and compounds were

dissolved in methanol. Light petroleum ether refers to the fractions

boiling at 40−60 °C. Melting points were determined on a Buchi−

Tottoli instrument and are uncorrected. Flash chromatography was

performed using Merck 60−200 mesh silica gel. Elemental analyses

were performed by the microanalytical laboratory of Dipartimento di

(2H, t, J = 7); 2.29 (3H, s); 4.18 (2H, q, J = 7); 8.20 (1H, s); IR

(Nujol) cm⁻¹: 3545−3090, 1720, 1610, 1500, 1430. Anal.

(C₈H₉N₂NaOS) C, H, N.

4-[N′-(Furan-2-carbonyl)-hydrazino]-2-methylsulfanyl-pyrimi-

dine-5-carboxylic Acid Ethyl Ester (71). To 9 g (38 mmol) of dried

69 was added 27 mL of phosphoryl oxychloride with a rate at which

the temperature remained below 50 °C. After the addition was

complete, the mixture was reﬂuxed for 3 h. The solvent was then

removed under reduced pressure. Ice and diethyl ether were added to

the solid residue, and the mixture was shaken until all the solid had

dissolved. The organic layer was washed with water, dried over sodium

sulfate, and the solvent was removed by evaporation under reduced

pressure to give the 4-chloro-2-methylthio-5-carbethoxypyrimidine

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(70), which was weighted and readily used in the next step without

puriﬁcation due to stability problems. The 6 g obtained of 70 (25.8

mmol) were dissolved in THF and 5.78 g of 2-furoylhydrazide (45.4

mmol) were added while 6.8 mL of DBU (45.4 mmol) were added

dropwise. The mixture was stirred at room temperature for 5 h. The

solvent was then removed, the residue was dissolved in dichloro-

methane, and the resulting solution was washed with water. The

organic layer was concentrated, dried and puriﬁed by ﬂash

chromatography (diethyl ether 6: EtOAc 4). Yield 60−70%; yellow

solid (EtOAc-light petroleum) mp 125 °C. ¹H NMR (CDCl₃) δ: 1.36

(2H, t, J = 7); 2.55 (3H, s); 4.40 (2H, q, J = 7); 6.51 (1H, dd, J = 2, J =

4); 7.20 (1H, d, J = 4); 7.50 (1H, d, J = 2); 8.66 (1H, s); 9.15 (1H,

bs), 10.01 (1H, bs); IR (Nujol) cm⁻¹: 3345−3150, 1725, 1685, 1620,

1510, 1410. Anal. (C₁₃H₁₄N₄O₄S) C, H, N.

Ethyl 2-(Furan-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylate (5). To 73 mL of dry xylene were added

2 g of P₂O₅(14 mmol) and 6.59 mL of hexamethyldisiloxane (30

mmol), and the mixture was heated at 90 °C for 2 h. Three grams of

dry compound 71 was then added, and the reaction was reﬂuxed for 2

days. After that, the solvent was removed under reduced pressure, and

the residue was dissolved in EtOAc and washed with water. The

organic layer was then dried, concentrated and puriﬁed by ﬂash

chromatography using light petroleum 9: EtOAc 1. Yield 68%; white

solid (EtOAc-light petroleum) mp 165 °C. ¹H NMR (CDCl₃) δ: 1.41

(2H, t, J = 7); 2.82 (3H, s); 4.48 (2H, q, J = 7); 6.61 (1H, dd, J = 2, J =

4); 7.40 (1H, d, J = 4); 7.62 (1H, d, J = 2); 8.89 (1H, s); IR (Nujol)

cm⁻¹: 1720, 1620, 1515, 1425; Anal. (C₁₃H₁₂N₄O₃S) C, H, N.

General Procedure for Nucleophilic Substitution with Amines (6,

23−50). To 100 mg (0.328 mmol) of compound 5 dissolved in

ethanol, was added the appropriate amine (0.986 mmol). The mixture

was heated in a sealed tube at 120 °C for 2 h. The solvent was then

removed and the residue was puriﬁed by ﬂash chromatography (Light

petroleum-EtOAc 8:2 or 7:3).

5-((3,4-Dimethoxybenzyl)amino)-2-(furan-2-yl)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxylic Acid Ethyl Ester (6). Yield 72%; white

solid (EtOAc-light petroleum) mp 140 °C; ¹H NMR (CDCl₃) δ: 1.34

(2H, t, J = 7); 3.71 (3H, s); 3.72 (3H, s); 4.31 (2H, q, J = 7); 4.67

(2H, s); 6.74 (1H, dd, J = 2, J = 4); 6.82−6.98 (2H, m); 7.07 (1H, s);

7.24 (1H, d, J = 4); 7.98 (1H, d, J = 2); 8.57 (1H, s); 9.54 (1H, bs); IR

(Nujol) cm⁻¹: 3345−3170, 1720, 1615, 1520, 1430; Anal.

(C₂₁H₂₁N₅O₅) C, H, N.

2-Furan-2-yl-5-methylamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (23). Yield 83%; pale yellow solid (EtOAc-

light petroleum) mp 220 °C. ¹H NMR (CDCl₃) δ: 1.45 (3H, t, J = 7);

3.32 (3H, d, J = 4.4); 4.48 (2H, q, J = 7); 6.60 (1H, dd, J = 2, J = 4);

6.70 (1H, bs); 7.41 (1H, d, J = 4); 7.62 (1H, d, J = 2); 8.73 (1H, s); IR

(Nujol) cm⁻¹: 3355−31650, 1720, 1615, 1510, 1420. Anal.

(C₁₃H₁₃N₅O₃) C, H, N.

solid (EtOAc-light petroleum) mp 100 °C. ¹H NMR (CDCl₃) δ:

0.95−0.99 (6H, m); 1.35−1.48 (11H, m); 1.67−1.78 (1H, m); 3.67

(2H, t, J = 6); 4.47 (2H, q, J = 7); 6.56−6.63 (2H, m); 7.38 (1H, d, J =

4) 7.62 (1H, d, J = 2); 8.70 (1H, s); IR (Nujol) cm⁻¹: 3345−3160,

1720, 1650, 1520, 1425; ES-MS (methanol) m/z: 408.2 (M + 23).

Anal. (C₂₀H₂₇N₅O₃) C, H, N.

5-Cyclopropylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (28). Yield 75%; white solid

(EtOAc-light petroleum) mp 137 °C. ¹H NMR (CDCl₃) δ: 0.83 (2H,

bs); 1.05 (2H, d, J = 6); 1.44 (3H, t. J = 7); 3.11 (1H, bs); 4.46 (2H, q,

J = 7); 6.59 (1H, dd, J = 2, J = 4); 6.84 (1H, bs); 7.40 (1H, d, J = 4)

7.62 (1H, d, J = 2); 8.77 (1H, s); IR (Nujol) cm⁻¹: 3355−3180, 1715,

1630, 1510, 1410. Anal. (C₁₅H₁₅N₅O₃) C, H, N.

5-Cyclopentylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (29). Yield 64%; white solid

(EtOAc-light petroleum) mp 127 °C. ¹H NMR (CDCl₃) δ: 1.44 (3H,

t. J = 7); 1.64−1.85 (6H, m); 2.20 (2H, bs); 4.47 (2H, q, J = 7); 4.6

(1H, bs); 6.59 (2H, bs); 7.41 (1H, d, J = 4); 7.63 (1H, d, J = 2); 8.71

(1H, s); IR (Nujol) cm⁻¹: 3340−3165, 1720, 1630, 1515, 1420. Anal.

(C₁₇H₁₉N₅O₃) C, H, N.

5-(Cyclohexylmethylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (30). Yield 88%; white solid

(EtOAc-light petroleum) mp 127 °C. ¹H NMR (CDCl₃) δ: 1.02−1.33

(4H,m); 1.44 (3H, t. J = 7); 1.66−1.87 (7H, m); 3.59 (2H, t, J = 6);

4.47 (2H, q, J = 7); 6.60 (1H, dd, J = 2, J = 4); 6.73 (1H, bs); 7.41

(1H, d, J = 4); 7.63 (1H, d, J = 2); 8.70 (1H, s); IR (Nujol) cm⁻¹:

3350−3180, 1715, 1640, 1520, 1430. Anal. (C₁₉H₂₃N₅O₃) C, H, N.

5-Benzylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (31). Yield 76%; yellow solid (EtOAc-light

petroleum) mp 130 °C. ¹H NMR (CDCl₃) δ: 1.45 (3H, t. J = 7); 4.48

(2H, q, J = 7); 4.92 (2H, d, J = 5); 6.59 (1H, dd, J = 2, J = 4); 6.97

(1H, bs); 7.33−7.49 (6H, m); 7.60 (1H, d, J = 2); 8.74 (1H, s); IR

(Nujol) cm⁻¹: 3345−3185, 1725, 1650, 1540, 1430; ES-MS

(methanol) m/z: 364.1 (M + 1); 386.1 (M + 23). Anal.

(C₁₉H₁₇N₅O₃) C, H, N.

2-Furan-2-yl-5-(4-methyl-benzylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (32). Yield 85%; pale yellow

solid (EtOAc-light petroleum) mp 104 °C. ¹H NMR (CDCl₃) δ: 1.45

(3H, t, J = 7); 2.35 (3H, s); 4.48 (2H, q, J = 7); 4.87 (2H, d, J = 5);

6.58 (1H, dd, J = 2, J = 4); 6.93 (1H, bs); 7.24 (4H, dd, J = 8, J = 24);

7.38 (1H, d, J = 4); 7.60 (1H, d, J = 2); 8.74 (1H, s); IR (Nujol) cm⁻¹:

3340−3230, 1725, 1645, 1520, 1415. Anal. (C₂₀H₁₉N₅O₃) C, H, N.

2-Furan-2-yl-5-(4-methoxy-benzylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (33). Yield 76%; brown-

1

yellow solid (EtOAc-light petroleum) mp 149 °C. H NMR (CDCl₃)

δ: 1.44 (3H, t. J = 7); 3.81 (3H, s); 4.48 (2H, q, J = 7); 4.84 (2H, d, J =

5); 6.57 (1H, dd, J = 2, J = 4); 6.88−6.92 (3H, m); 7.32−7.36 (3H,

m); 7.60 (1H, d, J = 2); 8.74 (1H, s); IR (Nujol) cm⁻¹: 3335−3180,

1715, 1650, 1525, 1420. Anal. (C₂₀H₁₉N₅O₄) C, H, N.

5-(4-Fluoro-benzylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (34). Yield 84%; white solid

(EtOAc-light petroleum) mp 150 °C. ¹H NMR (CDCl₃) δ: 1.45 (3H,

t, J = 7); 4.48 (2H, q, J = 7); 4.88 (2H, d, J = 6); 6.59 (1H, dd, J = 2, J

= 4); 6.88 (1H, bs); 7.00−7.10 (2H, m); 7.37−7.43 (3H, m); 7.60

(1H, d, J = 2); 8.73 (1H, s); IR (Nujol) cm⁻¹: 3345−3255, 1720,

1645, 1530, 1415; ES-MS (methanol) m/z: 404.1 (M + 23). Anal.

(C₁₉H₁₆FN₅O₃) C, H, N.

5-Ethylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-car-

boxylic Acid Ethyl Ester (24). Yield 78%; pale yellow solid (EtOAc-

1

light petroleum) mp 157 °C. H NMR (CDCl₃) δ: 1.37−1.48 (6H,

m); 3.71−3.86 (2H, m); 4.48 (2H, q, J = 7); 6.60 (1H, dd, J = 2, J =

4); 6.68 (1H, bs); 7.40 (1H, d, J = 4); 7.63 (1H, d, J = 2); 8.71 (1H,

s); IR (Nujol) cm⁻¹: 3340−3130, 1720, 1650, 1530, 1410. Anal.

(C₁₄H₁₅N₅O₃) C, H, N.

2-Furan-2-yl-5-pentylamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (25). Yield 88%; pale yellow solid (EtOAc-

1

light petroleum) mp 100 °C. H NMR (CDCl₃) δ: 0.93−0.96 (3H,

5-(4-Chloro-benzylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (35). Yield 73%; yellow solid

(EtOAc-light petroleum) mp 135 °C. ¹H NMR (CDCl₃) δ: 1.45 (3H,

t, J = 7); 4.49 (2H, q, J = 7); 4.88 (2H, d, J = 6); 6.59 (1H, dd, J = 2, J

= 4); 6.96 (1H, bs); 7.30−7.35 (5H, m); 7.37−7.43 (3H, m); 7.60

(1H, d, J = 2); 8.72 (1H, s); IR (Nujol) cm⁻¹: 3350−3195, 1725,

1645, 1540, 1420; ES-MS (methanol) m/z: 398.1 (M + 1); 420.1 (M

+ 23). Anal. (C₁₉H₁₆ClN₅O₃) C, H, N.

2-Furan-2-yl-5-(4-triﬂuoromethyl-benzylamino)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxylic Acid Ethyl Ester (36). Yield 85%;

white solid (EtOAc-light petroleum) mp 163 °C. ¹H NMR (CDCl₃) δ:

1.44 (3H, t, J = 7); 4.48 (2H, q, J = 7); 4.94 (2H, d, J = 6); 6.59 (1H,

dd, J = 2, J = 4); 7.02 (1H, bs); 7.39−7.66 (6H, m); 8.74 (1H, s); IR

m); 1.41−1.48 (7H, m); 1.72−1.81 (2H, m); 3.67−3.82 (2H, m); 4.47

(2H, q, J = 7); 6.60−6.69 (2H, m); 7.41 (1H, d, J = 4) 7.63 (1H, d, J =

2); 8.71 (1H, s); IR (Nujol) cm⁻¹: 3355−3185, 1725, 1640, 1510,

1435. Anal. (C₁₇H₂₁N₅O₃) C, H, N.

2-Furan-2-yl-5-isopropylamino-[1,2,4]triazolo[1,5-c]pyrimidine-

8-carboxylic Acid Ethyl Ester (26). Yield 54%; white solid (EtOAc-

1

light petroleum) mp 125 °C. H NMR (CDCl₃) δ: 1.41−1.47 (6H,

m); 1.67−1.81 (3H, m); 3.65−3.69 (2H, m); 4.62 (2H, q, J = 7);

6.47−6.60 (2H, m); 7.41 (1H, d, J = 4) 7.63 (1H, d, J = 2); 8.70 (1H,

s); IR (Nujol) cm⁻¹: 3330−3140, 1725, 1635, 1520, 1415; ES-MS

(methanol) m/z: 316.2 (M+1). Anal. (C₁₅H17N₅O₃) C, H, N.

5-(2-Ethyl-hexylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (27). Yield 89%; pale yellow

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(Nujol) cm⁻¹: 3325−3150, 1715, 1645, 1530, 1425; ES-MS

(methanol) m/z: 454.1 (M + 23). Anal. (C₂₀H₁₆F₃N₅O₃) C, H, N.

5-[(Biphenyl-4-ylmethyl)-amino]-2-furan-2-yl-[1,2,4]triazolo[1,5-

c]pyrimidine-8-carboxylic Acid Ethyl Ester (37). Yield 87%; orange

solid (EtOAc-light petroleum) mp 110 °C. ¹H NMR (CDCl₃) δ: 1.45

(3H, t, J = 7); 4.48 (2H, q, J = 7); 4.95 (2H, d, J = 6); 6.59 (1H, dd, J

= 2, J = 4); 6.99 (1H, bs); 7.37−7.62 (11H, m); 8.75 (1H, s); IR

(Nujol) cm⁻¹: 3330−3190, 1720, 1655, 1550, 1420. Anal.

(C₂₅H₂₁N₅O₃) C, H, N.

5-(Benzhydryl-amino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (38). Yield 71%; pale yellow

solid (EtOAc-light petroleum) mp 160 °C. ¹H NMR (CDCl₃) δ: 1.42

(3H, t, J = 7); 4.45 (2H, q, J = 7); 6.59 (1H, dd, J = 2, J = 4); 6.71

(1H, d, J = 6); 7.23 (1H, bs); 7.28−7.48 (11H, m); 7.61 (1H, d, J =

2); 8.67 (1H, s); IR (Nujol) cm⁻¹: 3335−3200, 1715, 1650, 1540,

1430. Anal. (C₂₅H₂₁N₅O₃) C, H, N.

5-Cyclobutylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-

8-carboxylic Acid Ethyl Ester (39). Yield 76%; yellow solid (EtOAc-

light petroleum) mp 116 °C. ¹H NMR (CDCl₃) δ: 1.44 (3H, t, J = 7);

1.85−1.88 (2H, m); 2.04−2.28 (2H, m); 2.51 (2H, bs); 4.46 (2H, q, J

= 7); 4.72−4.90 (1H, m); 6.60 (1H, dd, J = 2, J = 4); 6.80 (1H, bs);

7.39 (1H, d, J = 4); 7.63 (1H, d, J = 2); 8.68 (1H, s); IR (Nujol) cm⁻¹:

3350−3220, 1725, 1630, 1520, 1430. Anal. (C₁₆H₁₇N₅O₃) C, H, N.

5-Cycloheptylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (40). Yield 86%; pale brown

solid (EtOAc-light petroleum) mp 123 °C. ¹H NMR (CDCl₃) δ: 1.43

(3H, t, J = 7); 1.54−1.80 (10H, m); 2.12 (2H, bs); 4.36−4.59 (3H,

m); 6.56−6.66 (2H, m); 7.38 (1H, d, J = 4); 7.62 (1H, d, J = 2); 8.70

(1H, s); IR (Nujol) cm⁻¹: 3335−3180, 1720, 1625, 1515, 1410. Anal.

(C₁₉H₂₃N₅O₃) C, H, N.

5-Cyclooctylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-

8-carboxylic Acid Ethyl Ester (41). Yield 95%; pale yellow solid

(EtOAc-light petroleum) mp 106 °C. ¹H NMR (CDCl₃) δ: 1.43 (3H,

t, J = 7); 1.63−1.84 (12H, m); 1.98−2.10 (2H, m); 4.41−4.51 (3H,

m); 6.56−6.59 (2H, m); 7.37 (1H, d, J = 4); 7.62 (1H, d, J = 2); 8.70

(1H, s); IR (Nujol) cm⁻¹: 3345−3210, 1715, 1625, 1515, 1420. Anal.

(C₂₀H₂₅N₅O₃) C, H, N.

2-Furan-2-yl-5-pyrrolidin-1-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (42). Yield 67%; white solid (EtOAc-light

petroleum) mp 170 °C. ¹H NMR (CDCl₃) δ: 1.43 (3H, t, J = 7); 2.07

(4H, bs); 4.10 (4H, bs); 4.44 (2H, q, J = 7); 6.59 (1H, dd, J = 2, J =

4); 7.29 (1H, d, J = 4); 7.60 (1H, d, J = 2); 8.61 (1H, s); IR (Nujol)

cm⁻¹: 1725, 1630, 1520, 1430. Anal. (C₁₆H₁₇N₅O₃) C, H, N.

2-Furan-2-yl-5-piperidin-1-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (43). Yield 77%; pale yellow solid (EtOAc-

light petroleum) mp 125 °C. ¹H NMR (CDCl₃) δ: 1.43 (3H, t, J = 7);

1.79 (6H, bs); 4.31 (4H, bs); 4.46 (2H, q, J = 7); 6.58 (1H, dd, J = 2, J

= 4); 7.32 (1H, d, J = 4); 7.61 (1H, d, J = 2); 8.64 (1H, s); IR (Nujol)

cm⁻¹: 1720, 1625, 1515, 1420. Anal. (C₁₇H₁₉N₅O₃) C, H, N.

2-Furan-2-yl-5-(2-methoxy-ethylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (44). Yield 92%; white solid

(EtOAc-light petroleum) mp 119 °C. ¹H NMR (CDCl₃) δ: 1.44 (3H,

t, J = 7); 3.42 (3H, s); 3.67 (2H, t, J = 5); 3.93 (2H, q, J = 5); 4.47

(2H, q, J = 7); 6.59 (1H, dd, J = 2, J = 4); 6.95 (1H, bs); 7.37 (1H, d, J

= 4); 7.63 (1H, d, J = 2); 8.69 (1H, s); IR (Nujol) cm⁻¹: 3340−3250,

1715, 1640, 1530, 1425. Anal. (C₁₅H₁₇N₅O₄) C, H, N.

(7H, m); 1.69−1.88 (4H, m); 2.12−2.17 (2H, m); 4.08−4.18 (1H,

m); 4.46 (2H, q, J = 7); 6.52 (1H, d, J = 8); 6.59 (2H, m); 7.38 (1H, d,

J = 4); 7.62 (1H, d, J = 2); 8.69 (1H, s); IR (Nujol) cm⁻¹: 3335−3190,

1720, 1625, 1510, 1415. Anal. (C₁₈H₂₁N₅O₃) C, H, N.

2-Furan-2-yl-5-(morpholin-4-ylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (48). Yield 75%; white solid

(EtOAc-light petroleum) mp 165 °C. ¹H NMR (CDCl₃) δ: 1.44 (3H,

t, J = 7); 3.91 (4H, t, J = 7); 4.38−4.52 (6H, m); 6.57 (1H, dd, J = 2, J

= 4); 7.35 (1H, d, J = 4); 7.61 (1H, d, J = 2); 8.64 (1H, s); IR (Nujol)

cm⁻¹: 3355−3230, 1715, 1640, 1520, 1420. Anal. (C₁₆H₁₈N₆O₄) C, H,

N.

2-Furan-2-yl-5-isobutylamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

1

carboxylic Acid Ethyl Ester (49). Yield 56%; sticky foam; H NMR

(CDCl₃) δ: 0.98 (3H, t, J = 7); 1.32−1.44 (6H, m); 1.70 (2H, m);

4.28−4.45 (3H, m); 6.56 (1H, dd, J = 2, J = 4); 7.35 (1H, d, J = 4);

7.60 (1H, d, J = 2); 8.67 (1H, s); IR (Nujol) cm⁻¹: 3350−3200, 1715,

1620, 1515, 1420. Anal. (C₁₆H₁₉N₅O₃) C, H, N.

2-Furan-2-yl-5-methoxyamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (50). Yield 83%; yellow solid (EtOAc-light

petroleum) mp 220 °C. ¹H NMR (CDCl₃) δ: 1.42 (3H, t, J = 7); 4.02

(3H, s); 4.44 (2H, q, J = 7); 6.55 (1H, dd, J = 2, J = 4); 7.22−7.24

(2H, m); 7.58 (1H, d, J = 2); 8.21 (1H, s); IR (Nujol) cm⁻¹: 3325−

3150, 1725, 1620, 1540, 1410; ES-MS (methanol) m/z: 326.1 (M +

23). Anal. (C₁₃H₁₃N₅O₄) C, H, N.

General Procedure for the Hydrolysis of Ethyl Ester Compounds

(7, 22, 51−66). To 0.167 mmol of ethyl ester compound (6, 20, 23−

38) dissolved in ethanol were added 1.67 mmol of LiOH·H₂O and 1.5

mmol of water. The mixture was reﬂuxed and stirred for 2 h. Then, a

small amount of water was added, followed by addition of HCl to

make pH 3. The carboxylic acid derivative precipitate and the solid was

ﬁltered oﬀ.

5-((3,4-Dimethoxybenzyl)amino)-2-(furan-2-yl)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxylic Acid (7). Yield 78%; white solid

(EtOAc-light petroleum) mp 135 °C. ¹H NMR (CDCl₃) δ: 3.70

(3H, s); 3.72 (3H, s); 4.68 (2H, d, J = 6); 6.74 (1H, dd, J = 2, J = 4);

6.88−6.98 (2H, m); 7.07 (1H, s); 7.34 (1H, d, J = 4); 7.97 (1H, d, J =

2); 8.49 (1H, s); 9.31 (1H, bs); 13.03 (1H, bs); IR (Nujol) cm⁻¹:

3545−3130, 1700, 1650, 1540, 1420. Anal. (C₁₉H₁₇N₅O₅) C, H, N.

5-(3-Cyclohexylureido)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (22). Yield 91%; pale yellow solid

1

(EtOAc-light petroleum) mp 260 °C. H NMR (d₆-DMSO) δ: 1.13−

1.38 (6H, m); 1.52−1.81 (4H, m); 3.73−3.79 (1H, m); 6.64 (1H, dd,

J = 2, J = 4); 7.41 (1H, d, J = 4); 7.66 (1H, d, J = 2); 8.50 (1H, bs);

8.78 (1H, s); 8.83 (1H, bs); 13.02 (1H, bs); IR (Nujol) cm⁻¹: 3545−

3090, 1700, 1685, 1630, 1540, 1430. Anal. (C₁₇H₁₈N₆O₄) C, H, N.

2-Furan-2-yl-5-methylamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid (51). Yield 70%; pale yellow solid (EtOAc-light

1

petroleum) mp 250 °C. H NMR (CDCl₃) δ: 3.34 (3H, d, J = 4.8);

6.64−6.51 (2H, m); 7.30 (1H, d, J = 4); 7.66 (1H, d, J = 2); 8.80 (1H,

s); IR (Nujol) cm⁻¹: 3555−3100, 1705, 1630, 1515, 1420. Anal.

(C₁₁H₉N₅O₃) C, H, N.

5-Ethylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-car-

boxylic Acid (52). Yield 76%; pale yellow solid (EtOAc-light

1

petroleum) mp 240 °C. H NMR (CDCl₃) δ: 1.41 (3H, t, J = 7);

3.75−3.88 (2H, m); 6.64 (2H, bs); 7.31 (1H, d, J = 4); 7.66 (1H, d, J

= 2); 8.79 (1H, s); IR (Nujol) cm⁻¹: 3560−3120, 1700, 1625, 1515,

1430; ES-MS (methanol) m/z: 272.1 (M-1). Anal. (C₁₂H₁₁N₅O₃) C,

H, N.

5-Dimethylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (45). Yield 88%; pale yellow solid (EtOAc-

light petroleum) mp 139 °C. ¹H NMR (CDCl₃) δ: 1.43 (3H, t, J = 7);

3.67 (6H, s); 4.45 (2H, q, J = 7); 6.57 (1H, dd, J = 2, J = 4); 7.31 (1H,

d, J = 4); 7.61 (1H, d, J = 2); 8.63 (1H, s); IR (Nujol) cm⁻¹: 1725,

1620, 1530, 1420. Anal. (C₁₄H₁₅N₅O₃) C, H, N.

2-Furan-2-yl-5-pentylamino-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid (53). Yield 60%; pale orange solid (EtOAc-light

1

petroleum) mp 140 °C. H NMR (CDCl₃) δ: 0.93−0.96 (3H, m);

1.34−1.51 (4H, m); 1.65−1.88 (2H, m); 3.74−3.77 (2H, m); 6.63

(2H, bs); 7.30 (1H, d, J = 4); 7.66 (1H, d, J = 2); 8.78 (1H, s); IR

(Nujol) cm⁻¹: 3550−3070, 1695, 1635, 1525, 1400. Anal.

(C₁₅H₁₇N₅O₃) C, H, N.

2-Furan-2-yl-5-morpholin-4-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid Ethyl Ester (46). Yield 82%; white solid (EtOAc-light

petroleum) mp 164 °C. ¹H NMR (CDCl₃) δ: 1.44 (3H, t, J = 7); 3.94

(4H, bs); 4.38−4.56 (6H, m); 6.59 (1H, dd, J = 2, J = 4); 7.35 (1H, d,

J = 4); 7.62 (1H, d, J = 2); 8.65 (1H, s); IR (Nujol) cm⁻¹: 1715, 1630,

1515, 1420. Anal. (C₁₆H₁₇N₅O₄) C, H, N.

5-Cyclohexylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-

8-carboxylic Acid Ethyl Ester (47). Yield 86%; pale brown solid

(EtOAc-light petroleum) mp 123 °C. ¹H NMR (CDCl₃) δ: 1.37−1.51

2-Furan-2-yl-5-isopropylamino-[1,2,4]triazolo[1,5-c]pyrimidine-

8-carboxylic Acid (54). Yield 60%; pale yellow solid (EtOAc-light

1

petroleum) mp 210 °C. H NMR (CDCl₃) δ: 1.42 (3H, d, J = 6.6);

4.49−4.64 (1H, m); 6.48 (1H, d, J = 6.6); 6.64 (1H, dd, J = 2, J = 4);

7.30 (1H, d, J = 4); 7.67 (1H, d, J = 2); 8.78 (1H, s); IR (Nujol) cm⁻¹:

6220

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3545−3050, 1705, 1640, 1515, 1420; ES-MS (methanol) m/z: 286.1

(M-1). Anal. (C₁₃H₁₃N₅O₃) C, H, N.

5-[(Biphenyl-4-yl-methyl)-amino]-2-furan-2-yl-[1,2,4]triazolo[1,5-

c]pyrimidine-8-carboxylic Acid (65). Yield 94%; pale orange solid

(EtOAc-light petroleum) mp 300 °C. ¹H NMR (CDCl₃) δ: 4.98 (2H,

d, J = 6); 6.61 (1H, dd, J = 2, J = 4); 6.97 (1H, bs); 7.29−7.63 (11H,

m); 8.84 (1H, s); IR (Nujol) cm⁻¹: 3580−3090, 1700, 1655, 1550,

1420. Anal. (C₂₃H₁₇N₅O₃) C, H, N.

5-(Benzhydryl-amino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (66). Yield 88%; pale yellow solid

(EtOAc-light petroleum) mp 150 °C. ¹H NMR (CDCl₃) δ: 6.62 (1H,

dd, J = 2, J = 4); 6.72 (1H, d, J = 6); 7.19 (1H, bs); 7.30−7.43 (11H,

m); 7.65 (1H, d, J = 2); 8.75 (1H, s); IR (Nujol) cm⁻¹: 3610−3100,

1705, 1650, 1540, 1430. Anal. (C₂₃H₁₇N₅O₃) C, H, N.

General Procedure for the Synthesis of 5-((3,4-

Dimethoxybenzyl)amino)-2-(furan-2-yl)-N-substituted-[1,2,4]-

triazolo[1,5-c]pyrimidine-8-carboxamides (8, 9). One hundred

milligrams of 7 (0.253 mmol) was dissolved in 1 mL of dry pyridine.

The mixture was cooled to 0 °C, and 0.1 mL (1.1 mmol) of

phosphoryl oxychloride were added dropwise. The reaction was

further cooled to 10 °C, and the appropriate amine was added (1.3

mmol of phenethylamine for compound 8, and a large excess, 4 mmol,

of methylamine was used to obtain compound 9), and then the

mixture was left to reach room temperature and stirred for 2 h

monitoring by TLC (EtOAc). When the reaction was terminated, ice

and water were added to the mixture, and the product was extracted

with EtOAc. The organic layer was then washed with a NaHCO₃(aq),

dried, and the solvent was removed under reduced pressure. The crude

was puriﬁed by column chromatography.

5-((3,4-Dimethoxybenzyl)amino)-2-(furan-2-yl)-N-methyl-[1,2,4]-

triazolo[1,5-c]pyrimidine-8-carboxamide (8). Yield 68%; white solid

(EtOAc-light petroleum) mp 202 °C. ¹H NMR (CDCl₃) δ: 3.11 (3H,

d, J = 4); 3.66 (3H, s); 3.67 (3H, s); 4.83 (2H, d, J = 6); 6.64 (1H, dd,

J = 2, J = 4); 6.67 (1H, bt); 6.74−6.78 (2H, m); 7.01 (1H, s); 7.26

(1H, d, J = 4); 7.63 (1H, d, J = 2); 8.56 (1H, bs); 8.82 (1H, s); IR

(Nujol) cm⁻¹: 3350−3130, 1685, 1640, 1525, 1420. Anal.

(C₂₀H₂₀N₆O₄) C, H, N.

5-(2-Ethyl-hexylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (55). Yield 76%; pale yellow solid

(EtOAc-light petroleum) mp 100 °C. ¹H NMR (CDCl₃) δ: 0.66−1.11

(6H, m); 1.14−1.57 (8H, m); 1.65−1.82 (1H, m); 3.70 (2H, t, J = 6);

6.60 (2H, m); 7.28 (1H, d, J = 4); 7.65 (1H, d, J = 2); 8.77 (1H, s); IR

(Nujol) cm⁻¹: 3530−3030, 1700, 1625, 1530, 1420; ES-MS

(methanol) m/z: 304.3 (M-23). Anal. (C₁₈H₂₃N₅O₃) C, H, N.

5-Cyclopropylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (56). Yield 81%; white solid (EtOAc-

light petroleum) mp 135 °C. ¹H NMR (CDCl₃) δ: 0.84 (2H, bs); 1.06

(2H, d, J = 6); 3.13 (1H, bs); 6.63 (1H, dd, J = 2, J = 4); 6.78 (1H, bs)

7.27 (1H, d, J = 4); 7.66 (1H, d, J = 2); 8.85 (1H, s); IR (Nujol) cm⁻¹:

3545−3080, 1705, 1620, 1530, 1415. Anal. (C₁₃H₁₁N₅O₃) C, H, N.

5-Cyclopentylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (57). Yield 77%; white solid (EtOAc-

1

light petroleum) mp 250 °C. H NMR (CDCl₃) δ: 1.60−1.91 (6H,

m); 2.21 (2H, bs); 4.65 (1H, bs); 6.50−6.63 (2H, m); 7.31 (1H, d, J =

4); 7.66 (1H, d, J = 2); 8.78 (1H, s); ES-MS (methanol) m/z: 312.1

(M-1); IR (Nujol) cm⁻¹: 3560−3090, 1700, 1625, 1530, 1420. Anal.

(C₁₅H₁₅N₅O₃) C, H, N.

5-(Cyclohexylmethylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (58). Yield 70%; white solid (EtOAc-

light petroleum) mp 90 °C. ¹H NMR (CDCl₃) δ: 1.02−1.20 (4H, m);

1.43−1.87 (7H, m); 3.61 (2H, t, J = 6); 6.63 (1H, dd, J = 2, J = 4);

6.70 (1H, bs); 7.30 (1H, d, J = 4); 7.66 (1H, d, J = 2); 8.77 (1H, s); IR

(Nujol) cm⁻¹: 3550−3070, 1700, 1640, 1530, 1415. Anal.

(C₁₇H₁₉N₅O₃) C, H, N.

5-Benzylamino-2-furan-2-yl-[1,2,4]triazolo[1,5-c]pyrimidine-8-

carboxylic Acid (59). Yield 83%; pale yellow solid (EtOAc-light

petroleum) mp 205 °C. ¹H NMR (CDCl₃) δ: 4.94 (2H, d, J = 5); 6.61

(1H, dd, J = 2, J = 4); 6.95 (1H, bs); 7.29−7.40 (6H, m); 7.63 (1H, d,

J = 2); 8.82 (1H, s); IR (Nujol) cm⁻¹: 3610−3100, 1705, 1650, 1540,

1420. Anal. (C₁₇H₁₃N₅O₃) C, H, N.

5-((3,4-Dimethoxybenzyl)amino)-2-(furan-2-yl)-N-β-phenethyl-

[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxamide (9). Yield 69%; white

solid (EtOAc-light petroleum) mp 130 °C. ¹H NMR (CDCl₃) δ: 3.02

(2H, t, J = 6); 3.65−3.80 (2H, m); 3.89 (3H, s); 3.90 (3H, s); 4.83

(2H, d, J = 6); 6.63 (1H, dd, J = 2, J = 4); 6.68 (1H, bt); 6.78−6.82

(2H, m); 7.10 (1H, s); 7.21 (1H, d, J = 4); 7.25−7.55 (5H, m); 7.63

(1H, d, J = 2); 8.72 (1H, bs); 8.86 (1H, s); IR (Nujol) cm⁻¹: 3355−

3150, 1690, 1655, 1545, 1425. Anal. (C₂₇H₂₆N₆O₄) C, H, N.

General Procedure for the Synthesis of 5-Amino-2-(furan-2-yl)-N-

substituted-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxamides (10,

11). One millimole of 8-carboxamido compound (8, 9) was dissolved

in triﬂuoroacetic acid (10 mL), and 5.5 mmol of triﬂuoromethane-

sulfonic acid (0.48 mL) and 3.7 mmol of anisole (0.40 mL) were

added. The reaction was stirred at room temperature overnight.

Dichloromethane was then added to the mixture, and the mixture was

washed three times with water. The organic layer was dried, and the

solvent was removed under reduced pressure. The crude was puriﬁed

by column chromatography (EtOAc-light petroleum 9:1).

5-Amino-2-(furan-2-yl)-N-methyl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxamide (10). Yield 71%; brown solid (EtOAc-

light petroleum) mp 220 °C. ¹H NMR (CDCl₃) δ: 2.95 (3H, d, J = 4);

6.64 (1H, dd, J = 2, J = 4); 6.77 (1H, bt); 7.40 (1H, d, J = 4); 7.99

(1H, d, J = 2); 8.49 (1H, s); 8.65 (2H, bs); IR (Nujol) cm⁻¹: 3350−

3090, 1685, 1635, 1525, 1415. Anal. (C₁₁H₁₀N₆O₂) C, H, N.

5-Amino-2-(furan-2-yl)-N-β-phenethyl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxamide (11). Yield 68%; pale yellow solid

(EtOAc-light petroleum) mp 130 °C.¹H NMR (CDCl₃) δ: 3.05

(2H, t, J = 6); 3.68−3.90 (2H, m); 6.22 (2H, bs); 6.65 (1H, dd, J = 2, J

= 4); 7.15 (1H, d, J = 4); 7.22−7.45 (5H, m); 7.66 (1H, d, J = 2); 8.76

(1H, bs); 8.80 (1H, s); IR (Nujol) cm⁻¹: 3360−3140, 1685, 1650,

1540, 1435. Anal. (C₁₈H₁₆N₆O₂) C, H, N.

2-Furan-2-yl-5-(4-methyl-benzylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (60). Yield 92%; pale yellow solid

(EtOAc-light petroleum) mp 105 °C. ¹H NMR (CDCl₃) δ: 2.36 (3H,

s); 4.88 (2H, bs); 6.61 (1H, dd, J = 2, J = 4); 6.95 (1H, bs); 7.17−7.38

(5H, m); 7.64 (1H, d, J = 2); 8.81 (1H, s); IR (Nujol) cm⁻¹: 3580−

3070, 1700, 1655, 1535, 1425; ES-MS (methanol) m/z: 350.1 (M+1).

Anal. (C₁₈H₁₅N₅O₃) C, H, N.

2-Furan-2-yl-5-(4-methoxy-benzylamino)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (61). Yield 88%; pale brown solid

1

(EtOAc-light petroleum) mp 80 °C. H NMR (CDCl₃) δ: 3.81 (3H,

s); 5.01 (2H, d, J = 5); 6.61 (1H, dd, J = 2, J = 4); 6.89−6.93 (3H, m);

7.27−7.36 (3H, m); 7.62 (1H, d, J = 2); 8.82 (1H, s); IR (Nujol)

cm⁻¹: 3575−3060, 1705, 1645, 1545, 1430. Anal. (C₁₈H₁₅N₅O₄) C, H,

N.

5-(4-Fluoro-benzylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (62). Yield 80%; pale yellow solid

(EtOAc-light petroleum) mp 120 °C. ¹H NMR (CDCl₃) δ: 4.91 (2H,

d, J = 6); 6.63 (1H, dd, J = 2, J = 4); 6.93 (1H, bs); 7.08 (2H, t, J = 8);

7.29 (1H, d, J = 4); 7.37−7.43 (2H, m); 7.64 (1H, d, J = 2); 8.82 (1H,

s); IR (Nujol) cm⁻¹: 3560−3070, 1700, 1640, 1530, 1420; ES-MS

(methanol) m/z: 352.1 (M-1). Anal. (C₁₇H₁₂FN₅O₃) C, H, N.

5-(4-Chloro-benzylamino)-2-furan-2-yl-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid (63). Yield 92%; pale yellow solid

1

(EtOAc-light petroleum) mp 280 °C. H NMR (d₆-DMSO) δ: 4.74

(2H, d, J = 6); 6.55 (1H, dd, J = 2, J = 4); 7.20 (2H, d, J = 8); 7.35

(2H, d, J = 8); 7.46 (1H, d, J = 4); 7.59 (1H, d, J = 2); 8.61 (1H, s);

8.65 (1H, bs); IR (Nujol) cm⁻¹: 3585−3100, 1700, 1645, 1540, 1435;

ES-MS (methanol) m/z: 368.0 (M-1). Anal. (C₁₇H₁₂ClN₅O₃) C, H,

N.

2-Furan-2-yl-5-(4-triﬂuoromethyl-benzylamino)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxylic Acid (64). Yield 80%; white solid

(EtOAc-light petroleum) mp 250 °C. ¹H NMR (CDCl₃) δ: 5.02 (2H,

d, J = 6); 6.63 (1H, dd, J = 2, J = 4); 7.02 (1H, bs); 7.28 (1H, d, J = 4);

7.52−7.66 (5H, m); 8.80 (1H, s); IR (Nujol) cm⁻¹: 3555−3090, 1695,

1650, 1525, 1430. Anal. (C₁₈H₁₂F₃N₅O₃) C, H, N.

5-Amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carbox-

ylic Acid Ethyl Ester (12). One hundred milligrams of 5 (0.33 mmol)

was dissolved in ethanol saturated with NH₃(10 mL), and then the

reaction was heated for 2 h. When the compound reacted completely,

the solvent was removed under reduced pressure, and the residue was

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recrystallized in EtOAc-light petroleum. Yield 95%; white solid mp 195

°C. ¹H NMR (CDCl₃) δ: 1.32 (3H, t, J = 7); 4.25 (2H, q, J = 7); 6.48

(2H, bs); 6.63 (1H, dd, J = 2, J = 4); 7.42 (1H, d, J = 4); 7.66 (1H, d, J

= 2); 8.69 (1H, s); IR (Nujol) cm⁻¹: 3350−3100, 1720, 1625, 1530,

1415. Anal. (C₁₂H₁₁N₅O₃) C, H, N.

bs); IR (Nujol) cm⁻¹: 3335−3120, 1695,1690, 1655, 1530, 1425. Anal.

(C₁₉H₁₆N₆O₃) C, H, N.

2-(Furan-2-yl)-N-phenethyl-5-(2-phenylacetamido)-[1,2,4]-

triazolo[1,5-c]pyrimidine-8-carboxamide (18). Yield 73%; white

solid (EtOAc-light petroleum) mp 83 °C. ¹H NMR (CDCl₃) δ:

3.01 (2H, t, J = 6); 3.85−3.8 (2H, m); 6.65 (1H, dd, J = 2, J = 4); 7.13

(1H, m, J = 4); 7.28−7.41 (5H, m); 7.70 (1H, d, J = 2); 8.79 (1H, bs);

8.97 (1H, s); 8.12 (1H, s); IR (Nujol) cm⁻¹: 3340−3160, 1690, 1685,

1645, 1540, 1435. Anal. (C₂₆H₂₂N₆O₃) C, H, N.

2-(Furan-2-yl)-5-(2-phenylacetamido)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (21). Yield 70%; white solid

(EtOAc-light petroleum) mp 243 °C. ¹H NMR (CDCl₃) δ: 1.32 (3H,

t, J = 7); 4.07 (2H, s); 4.54 (2H, q, J = 7); 6.63 (1H, dd, J = 2, J = 4);

7.08−7.44 (6H, m); 7.70 (1H, d, J = 2); 9.49 (1H, s); 9.75 (1H, bs);

IR (Nujol) cm⁻¹: 3340−3130, 1720, 1685, 1650, 1530, 1435. Anal.

(C₂₀H₁₇N₅O₄) C, H, N.

General Procedure for the Synthesis of 5-Ureido Derivatives (13,

14, 16, 17, 19, 20). To 0.3 mmol of 5-amino compound (10−12)

dissolved in dry THF was added 3 mmol of the appropriate isocyanate,

and the reaction was reﬂuxed overnight. To the mixture was then

added water, and it was extracted three times with EtOAc. The organic

layers were collected, dried, and the solvent was removed under

reduced pressure. The crude was puriﬁed by column chromatography.

2-(Furan-2-yl)-N-methyl-5-(3-phenylureido)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxamide (13). Yield 71%; white solid (EtOAc-light

petroleum) mp 150 °C. ¹H NMR (CDCl₃) δ: 2.95 (3H, d, J = 4); 6.66

(1H, dd, J = 2, J = 4); 7.11−7.52 (6H, bs); 7.87 (1H, d, J = 2); 8.63

(2H, bs); 8.95 (1H, s); 11.08 (1H, bs); IR (Nujol) cm⁻¹: 3350−3180,

1690, 1685, 1645, 1525, 1420. Anal. (C₁₈H₁₅N₇O₃) C, H, N.

5-(3-Cyclohexylureido)-2-(furan-2-yl)-N-methyl-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxamide (14). Yield 70%; white solid

B. Biology. Binding at Human A₁, A_2A, and A₃ARs. All

pharmacological methods followed the procedures as described

earlier.³⁷In brief, membranes for radioligand binding were prepared

from CHO cells stably transfected with human AR subtypes in a two-

step procedure. In a ﬁrst low-speed step (1000g), cell fragments and

nuclei were removed. The crude membrane fraction was sedimented

from the supernatant at 100 000g. The membrane pellet was

resuspended in the buﬀer used for the respective binding experiments,

frozen in liquid nitrogen and stored at 80 °C. For the measurement of

adenylyl cyclase activity, only one high speed centrifugation of the

homogenate was used. The resulting crude membrane pellet was

resuspended in 50 mM Tris/HCl, pH 7.4 and immediately used for

the cyclase assay.

1

(EtOAc-light petroleum) mp 199 °C. H NMR (CDCl₃) δ: 1.03−

1.42 (6H, m); 1.60−1.82 (4H, m); 3.02 (3H, d, J = 4); 3.31−3,35

(1H, m); 6.67 (1H, dd, J = 2, J = 4); 7.32 (1H, d, J = 4); 7.71 (1H, d, J

= 2); 8.46 (1H, bs); 8.61 (1H, bs); 8.86 (1H, s); 8.92 (1H, bs); IR

(Nujol) cm⁻¹: 3350−3170, 1685, 1680, 1625, 1515, 1410. Anal.

(C₁₈H₂₁N₇O₃) C, H, N.

2-(Furan-2-yl)-N-phenethyl-5-(3-phenylureido)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxamide (16). Yield 72%; yellow solid

(EtOAc-light petroleum) mp 165 °C. ¹H NMR (CDCl₃) δ: 3.05

(2H, t, J = 6); 3.78−3.92 (2H, m); 6.68 (1H, dd, J = 2, J = 4); 7.08−

7.37 (6H, m); 7.63 (1H, d, J = 2); 8.76 (1H, bs); 8.81 (1H, bs); 8.93

(1H, s); 11.08 (1H, bs); IR (Nujol) cm⁻¹: 3345−3130, 1695, 1687,

1650, 1535, 1415. Anal. (C₂₅H₂₁N₇O₃) C, H, N.

5-(3-Cyclohexylureido)-2-(furan-2-yl)-N-β-phenethyl-[1,2,4]-

triazolo[1,5-c]pyrimidine-8-carboxamide (17). Yield 69%; white

solid (EtOAc-light petroleum) mp 186 °C. ¹H NMR (CDCl₃) δ:

1.02−1.42 (6H, m); 1.66−1.98 (4H, m); 3.03 (2H, t, J = 6); 3.40−

3,53 (1H, m); 3.86−3.91 (2H, m); 6.64 (1H, dd, J = 2, J = 4); 7.16

(1H, d, J = 4); 7,36 (1H, bs); 7.71 (1H, d, J = 2); 8.41 (1H, bs); 8.67

(1H, bs); 8.73 (1H, s); IR (Nujol) cm⁻¹: 3335−3120, 1690, 1685,

1635, 1525, 1410. Anal. (C₂₅H₂₇N₇O₃) C, H, N.

2-(Furan-2-yl)-5-(3-phenylureido)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (19). Yield 66%; white solid

(EtOAc-light petroleum) mp 170 °C. ¹H NMR (CDCl₃) δ: 1.47 (3H,

t, J = 7); 4.50 (2H, q, J = 7); 6.66 (1H, dd, J = 2, J = 4); 7.15 (1H, d, J

= 4); 7.28−7.42 (3H, m); 7.62−7.64 (2H, m); 7.68 (1H, d, J = 2);

8.74 (1H, bs); 8.83 (1H, s); 11.05 (1H, bs); IR (Nujol) cm⁻¹: 3330−

3140, 1685, 1680, 1650, 1540, 1415. Anal. (C₁₉H₁₆N₆O₄) C, H, N.

5-(3-Cyclohexylureido)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]-

pyrimidine-8-carboxylic Acid Ethyl Ester (20). Yield 65%; white solid

(EtOAc-light petroleum) mp 172 °C. ¹H NMR (CDCl₃) δ: 1.10−1.40

(6H, m); 1.47 (3H, t, J = 7); 1.55−1.78 (4H, m); 3.78−3.84 (1H, m);

4.50 (2H, q, J = 7); 6.63 (1H, dd, J = 2, J = 4); 7.42 (1H, d, J = 4);

7.69 (1H, d, J = 2); 8.57 (1H, bs); 8.75 (1H, s); 8.88 (1H, bs); IR

(Nujol) cm⁻¹: 3340−3130, 1690, 1685, 1635, 1520, 1415. Anal.

(C₁₉H₂₂N₆O₄) C, H, N.

General Procedure for the Synthesis of 5-(2-phenylacetamido)

derivatives (15, 18, 21). To 0.3 mmol of 5-amino compound (10−

12) dissolved in dry THF (10 mL), 1.8 mmol (0.24 mL) of

phenylacetyl chloride and 1.8 mmol (0.25 mL) of Et₃N were added.

The reaction was reﬂuxed overnight. The solvent was removed under

reduced pressure and the residue was dissolved in EtOAc and washed

with water. The organic layer was dried, and the solvent was removed

under reduced pressure and the residue was puriﬁed by column

chromatography (EtOAc-light petroleum).

For radioligand binding at A₁adenosine receptors 1 nM [³H]CCPA

was used, whereas 30 and 10 nM [³H]NECA were used for A_2Aand A₃

receptors, respectively. Nonspeciﬁc binding of [³H]CCPA was

determined in the presence of 1 mM theophylline, in the case when

[³H]NECA 100 μM R-PIA was used.³⁹

Adenylyl Cyclase Activity. The potency of antagonists at the A_2B

AR was determined in adenylyl cyclase experiments. The procedure

was carried out as described previously with minor modiﬁcations.^37,40

Membranes were incubated with about 150 000 cpm of [α-³²P]ATP

for 20 min in the incubation mixture as described without EGTA and

NaCl.^37,40For agonists, the EC₅₀values for the stimulation of adenylyl

cyclase were calculated with the Hill equation. Hill coeﬃcients in all

experiments were near unity. IC₅₀values for concentration-dependent

inhibition of NECA-stimulated adenylyl cyclase caused by antagonists

was calculated accordingly. Dissociation constants (K_i) for antagonist

were then calculated with the Cheng and Prusoﬀ equation.⁴¹

C. Molecular Modeling. Energy computation and analyses of

docking poses were performed using the MOE suite (Molecular

Operating Environment, version 2013.08).⁴²The software package

MOPAC,⁴³implemented in the MOE suite, was utilized for all

quantum mechanical calculations. GOLD (Genetic Optimization for

Ligand Docking, version 5.1)⁴⁴suite was used to carry out all the

docking simulations.

Three-Dimensional Structures of hARs. Among all the currently

available crystallographic structures of human A_2AAR, we selected for

our docking simulations a complex with the selective and high aﬃnity

inverse agonist ZM 241385. Among the structures cocrystallized with

ZM 241385, we selected the one with the highest resolution and

fewest missing atoms (PDB code: 4EIY, 1.80 Å resolution).⁴⁵As, to

date, no crystallographic information about the hA₃AR is available, we

used a previously build homology model deposited in our web

platform dedicated to ARs, Adenosiland.⁴⁶The numbering of the

amino acids follows the arbitrary scheme proposed by Ballesteros and

Weinstein:⁴⁷each amino acid identiﬁer starts with the helix number

(1−7), followed by a dot and the position relative to a reference

residue among the most conserved amino acids in that helix, to which

the number 50 is arbitrarily assigned.

2-(Furan-2-yl)-N-methyl-5-(2-phenylacetamido)-[1,2,4]triazolo-

[1,5-c]pyrimidine-8-carboxamide (15). Yield 67%; white solid

(EtOAc-light petroleum) mp 160 °C. ¹H NMR (CDCl₃) δ: 3.07

(3H, d, J = 4); 4.37 (2H, s); 6.67 (1H, dd, J = 2, J = 4); 7.22−7.42

(6H, m); 7.71 (1H, d, J = 2); 8.55 (2H, bs); 8.99 (1H, s); 9.11 (1H,

Molecular Docking. Ligand structures were built using the MOE-

builder tool, as part of the MOE suite,⁴²and were subjected to a

MMFF94x energy minimization until the rms conjugate gradient was

<0.05 kcal mol ⁻¹Å ⁻¹. We used the Protonate 3D methodology, part

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Journal of Medicinal Chemistry

Article

of the MOE suite, for protonation state assignment by selecting a

protonation state for each chemical group that minimizes the total free

energy of the system (taking titration into account). According to the

docking benchmark study recently completed by our research group,⁴⁸

we perform the simulations employing the docking tool of GOLD⁴⁴

suite as conformational search program and GoldScore as scoring

function. Indeed, the latter protocol resulted one of the most

successful at reproducing the crystallographic pose of ligand−hA_2AAR

complexes and the best for the considered crystal structure. For each

selected compound, 25 independent docking runs were performed and

searching was conducted within a user-speciﬁed docking sphere (20 Å

radius and centered on the barycenter of the Asn(6.55) residue) with

the Genetic Algorithm protocol and the GoldScore scoring function.

Prediction of antagonist−receptor complex stability (in terms of

corresponding pK_ivalue) and quantitative analysis for nonbonded

intermolecular interactions (H-bonds, transition metal, water bridges,

hydrophobic, electrostatic) were calculated and visualized using several

tools implemented in the MOE suite.⁴²Electrostatic and hydrophobic

contributions to the binding energy of individual amino acids have

been calculated using the MOE suite. To estimate the electrostatic

contributions, atomic partial charges for the ligands were calculated

using PM3/ESP methodology. Partial charges for protein amino acids

were calculated on the basis of the AMBER99 force ﬁeld.

Interaction Energy Fingerprints (IEFs). To analyze the ligand−

receptor recognition mechanism in a more quantitative manner, we

calculated the individual electrostatic and hydrophobic contributions

to the interaction energy (hereby denoted as IEele and IEhyd,

respectively) of each receptor residue involved in the binding with the

ligand. In particular, the IEele was computed on the basis of the

nonbonded electrostatic interaction energy term of the force ﬁeld,

whereas the IEhyd contributions was calculated by using the

directional hydrophobic interaction term based on contact surfaces

as implemented in the MOE scoring function.⁴²As a consequence, an

energy (expressed in kcal/mol) is associated with the IEele, instead an

adimensional score (the higher the better) is related to the IEhyd. The

analysis of these contributions have been reported as IEFs, that is,

interaction energy patterns (graphically displayed as histograms)

reporting the key residues involved in the binding with the considered

ligands along with a quantitative estimate of the occurring interactions.

ABBREVIATIONS

■

AR, adenosine receptor; ATP, adenosine triphosphate; CCPA,

2-chloro-N6-cyclopentyladenosine; CHO, chinese hamster

ovary; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMSO,

dimethyl sulfoxide; EGTA, ethylene glycol tetraacetic acid;

HMDS, hexamethyldisiloxane; IEele, per residue electrostatic

contribution to the interaction energy; IEF, interaction energy

ﬁngerprint; IEhyd, per residue hydrophobic contribution to the

interaction energy; NECA, 5′-N-ethylcarboxamidoadenosine;

R-PIA, R-(−)-N6-(2-phenylpropyl)adenosine; SAR, structure−

activity relationship; TFA, triﬂuoroacetic acid; THF, tetrahy-

drofuran; TLC, thin-layer chromatography; TP, 1,2,4-triazolo-

[1,5-c]pyrimidine; ZM 241385, 4-[2-[7-amino-2-(2-furyl)-

1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol; TM,

transmembrane; EL2, second extracellular loop

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ASSOCIATED CONTENT

■

S

* Supporting Information

Supporting Information contains elemental analyses for the

compounds used in this study. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Authors

*E-mail: (G.S.) spalluto@units.it. Fax: +39 040 52572Tel.: +39

040 5583726.

*E-mail: (S.M.) stefano.moro@unipd.it. Fax: +39 049 8275366.

Tel.: +39 049 8275704.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The work was carried out with ﬁnancial support Italian Ministry

for University and Research (MIUR), Rome, Italy. The

molecular modelling work coordinated by S.M. has been

carried out with ﬁnancial support from the University of

Padova, Italy, and the Italian Ministry for University and

Research, Rome, Italy. S.M. is also very grateful to Chemical

Computing Group for the scientiﬁc and technical partnership.

S.M. participates in the European COST Action CM1207

(GLISTEN)

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Article Doi

Scaffold decoration at positions 5 and 8 of 1,2,4-triazolo[1,5- c ]pyrimidines to explore the antagonist profiling on adenosine receptors: A preliminary structure-activity relationship study

DOI: 10.1021/jm500752h

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Full text of DOI:10.1021/jm500752h

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