Scholarly article on N-benzoyl-N-[8-methyl-2-(4-chlorophenyl)-8H-pyrazolo[4,3-e]-[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]benzamide 1221499-86-0 from Journal of Medicinal Chemistry p. 3361

J. Med. Chem. 2010, 53, 3361–3375 3361

DOI: 10.1021/jm100049f

The Significance of 2-Furyl Ring Substitution with a 2-(para-substituted) Aryl Group in a New Series of

Pyrazolo-triazolo-pyrimidines as Potent and Highly Selective hA₃Adenosine Receptors Antagonists:

New Insights into Structure-Affinity Relationship and Receptor-Antagonist Recognition

Siew Lee Cheong,^†Anna Dolzhenko,^†Sonja Kachler,^‡Silvia Paoletta,^§Stephanie Federico, Barbara Cacciari,^{^}

Anton Dolzhenko,^†Karl-Norbert Klotz,^‡Stefano Moro,*^,§Giampiero Spalluto,*^,and Giorgia Pastorin*^,†

^†Department of Pharmacy, National University of Singapore, 3 Science Drive 2, Block S15, no. 05-PI-03, Singapore 117543, ^‡Institut fu€r

Pharmakologie, Universita€t of Wu€rzburg, D-97078 Wu€rzburg, Germany, ^§Molecular Modeling Section (MMS), Dipartimento di Scienze

Farmaceutiche, Universitaꢀ di Padova, via Marzolo 5, I-35131 Padova, Italy, Dipartimento di Scienze Farmaceutiche, Universitaꢀ degli Studi di

Trieste, Piazzale Europa 1, I-34127 Trieste, Italy, and ^{^}Dipartimento di Scienze Farmaceutiche, Universitaꢀ degli Studi di Ferrara, via Fossato di

Mortara 17-19, I-44100 Ferrara, Italy

Received January 14, 2010

Among the heterocyclic structures identified as potent human A₃(hA₃) adenosine receptor’s antago-

nists, we have demonstrated that the new pyrazolo-triazolo-pyrimidines, bearing an aryl group in

replacement of the C²-furyl ring, not only confer a good pharmacological profile (with significantly

enhanced selectivity against other adenosine receptor subytpes) but also overcome the metabolic

transformation of the furan ring into toxic intermediates. All the synthesized [2-(para-substituted)

phenyl]-pyrazolo-triazolo-pyrimidines showed affinity at the hA₃receptor in the low nanomolar range.

The most potent derivative of the series presented better affinity and excellent selectivity (compound 31,

K_ihA₃=0.108 nM; hA₁/hA₃=5200; hA_2A/hA₃=7200), in comparison to the C²-furyl counterpart. A

receptor-driven molecular modeling investigation, based on a recently proposed model of A₃receptor

derived from the crystallographic structure of human A_2Areceptor, has been carried out in order to

support the experimental binding data and to justify the enhanced selectivity against the other receptor

subtypes.

Introduction

which render the discovery of new selective compounds more

unpredictable and complicated.

Human A₃adenosine receptor (hA₃AR), a member of the

seven transmembrane domains G-protein-coupled receptor

(GPCR^a) family, modulates several physiopathological con-

ditions.^1,2Its inhibition through selective hA₃antagonists is

found to be beneficial in inflammatory, asthmatic and is-

chemic conditions.³In addition, the A₃receptors have been

shown tobeoverexpressed in some tumor cell lines, suggesting

that this receptor could be a potential target in cancer

therapy.⁴However, different from the other adenosine recep-

tor subtypes (A₁, A_2A, and A_2B, respectively), there are

remarkable species differences in the case of A₃receptor,^5,6

Among the several diverse structures that have demon-

strated affinity at the hA₃adenosine receptor, our group

has particularly focused on the development of compounds

bearing a pyrazolo-triazolo-pyrimidine (PTP) nucleus (2)

(Chart 1).^7,8The PTP tricylic nucleus resembles the triazolo-

quinoline core of nonselective antagonist CGS15943 (1),⁹

except for the phenyl ring being replaced by a pyrazole, which

has resulted in an increase of selectivity against other receptor

subtypes.¹⁰Further exploitation of substituents, mainly at

positions N⁸and N⁵of such structure,^10,11has given rise to

highly potent and moderately selective hA₃antagonists. In

particular, the combination of a methyl group at N⁸and a 4-

pyridyl carbamoyl chain at N⁵has led to the most potent hA₃

antagonist ever synthesized.¹²Conversely, substitution at

position C²of the PTP tricyclic system has not been deeply

explored, being essentially limited to the introduction of a

furyl group. The furan ring has been considered as an essential

structural requirement for the binding of antagonists at all the

adenosine receptor subtypes, while its removal from the

tricyclic system has been associated with an irreversible loss

of affinity and selectivity, regardless of the receptor under

investigation.

*To whom correspondence should be addressed. For G. P.: phone,

þ65 6516 1876; fax, þ65 6779 1554; E-mail, phapg@nus.edu.sg. For S.

M.: phone, þ39-049-8275704; E-mail, stefano.moro@unipd.it. For G.

S.: phone, þ39-040-558-3726; E-mail, spalluto@univ.trieste.it.

^aAbbreviations: APCI, atmospheric pressure chemical ionization;

AR, adenosine receptor; ATP, adenosine triphosphate; cAMP, cyclic

adenosine monophosphate; CHO cells, Chinese hamster ovary cells;

DIPEA, diisopropylethylamine; EGTA, ethylene glycol tetraacetic acid;

EL2, extracellular loop 2; EtOAc, ethyl acetate; GPCR, G-protein-

coupled receptor; [³H]-CCPA, [³H]-2-chloro-6-cyclopentyl adenosine;

[³H]-NECA, [³H]-N-ethylcarboxamido adenosine; HPLC, high perfor-

mance liquid chromatography; MgCl₂, magnesium chloride; MOE,

Molecular Operating Environment software; NaCl, sodium chloride;

NaH, sodium hydride; Na₂SO₄, sodium sulfate; PTP, pyrazolo-triazolo-

pyrimidine; rmsd, root-mean-square deviation; R-PIA, R-N⁶-phenyli-

sopropyladenosine; SAR, structure-affinity relationship; TEA, triethy-

lamine; TLC, thin-layer chromatography; TM, transmembrane.

To reiterate, Baraldi and co-workers¹³have found that the

substitution of the furan ring in PTPs with phenyl or alkoxy-

phenyl rings led to a loss of affinity at A_2A, A_2B, and A₃

receptors, while the A₁subtype in some cases displayed a high

r

2010 American Chemical Society

Published on Web 03/22/2010

pubs.acs.org/jmc

3362 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

Chart 1. Rationale for the Design of 2-(para-substituted)phenyl Pyrazolo-triazolo-pyrimidine Derivatives

nanomolar binding profile. Similarly, the functionalization of

the furan ring with polar substituents resulted in completely

inactive derivatives, clearly indicating that an unsubstituted

furan ring at the C²position played a fundamental role in

ligand-receptor recognition. Notably, in most cases, substi-

tution at the pyrazole ring occurred at the N⁷rather than at

the N⁸position; hence, these compounds might not reflect the

same binding profile as their N⁸analogues. This observation

has triggered our interest to further investigate the effect of

concurrent substitution of alkyl groups at N⁸position and a

different moiety (other than furan ring) at the C²position of

PTPs on the affinity at hA₃receptor and selectivity over other

adenosine receptor subtypes. Interestingly, several reported

hA₃antagonists bearing tricyclic scaffolds (which are also

structural resemblance of CGS15943 (1), including tria-

zoloquinoxalinones (3),^14,15pyrazoloquinolines (4),¹⁶tria-

zolopyrazinones (5),¹⁷and triazolobenzotriazinones (6)¹⁸

(Chart 1)), comprise a substituted phenyl moiety at the

position equivalent to that of furan ring in PTP derivatives.

From the structure-affinity relationship (SAR) studies of

these derivatives, the presence of the phenyl ring seemed

crucial to maintain good affinity at hA₃receptor. In light of

the beneficial effect of this phenyl ring toward the hA₃affinity,

the substitution of the C²-furyl ring with an aryl group is

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3363

therefore deemed feasible for the design and synthesis of new

series of PTPs.

Some of the compounds bearing either a methyl group

(7-12) or a phenylethyl group (15, 19) at N⁸were dispersed in

toluene and treated with either benzoic anhydride or pheny-

lacetyl chloride (Scheme 2). The mixture was heated under

reflux and stirred for 6-12 h. The solvent was removed under

reduced pressure, and the residue was crystallized from an

appropriate solvent or subjected to chromatographic purifi-

cation, to afford the desired compounds (21-26, 29-36).

On top of that, two additional compounds, obtained from

the reaction of compounds 9 and 11 with benzoyl chloride,

were further isolated from the alcoholic mixtures of methanol

or ethanol: they included the bis-acylated products 27 and 28.

From the pharmacokinetic aspect, several studies reported

that drugs containing a furan in their chemical structure (e.g.,

prazosin and frusemide) were subjected to a metabolic cleavage

of the furyl ring by the cytochrome P450 enzymes in the liver,

^19-21resulting in remarkable hepatotoxicity in treated mice.²⁰

Some of these intermediate metabolites, for example epoxide

groups and γ-keto-R,β-unsaturated aldehydes, were shown to

be reactive and able to form adducts with cellular proteins or

DNA, therefore causing unpredictable effects. Although these

reactive metabolites can be detoxified by several enzymes in the

body, this still poses some threat of toxicity and should be

regarded as a safety issue. On the other hand, metabolites

derived from biotransformation of the phenyl ring through

oxidation by CYP450 enzymes are expected to be relatively less

reactive than those found in the metabolism of furan ring.

Furthermore, the typical oxidation targeted at the para-posi-

tion of the phenyl ring can be bypassed through the synthesis of

para-substituted derivatives, leading to improvement of bioa-

vailability of the new bioactive compounds.

On the basis of potential structural and metabolic benefits

of the C²-aryl group in PTP derivatives, a novel structure-

-affinity relationship evaluation was conducted through the

rational design and synthesis of a new series of pyrazolo-

triazolo-pyrimidines bearing a (para-substituted)-phenyl ring

at C², while maintaining either methyl or phenyl-ethyl groups

at N⁸and a free amino, phenylacetamide or (bis-)benzamide

at the N⁵position (compounds 7-36).

Biological Assay

1. Binding Affinity at Human A₁, A_2A, and A₃Adenosine

Receptors. The affinity of an antagonist toward its receptor

(expressed in Chinese hamster ovary cells (CHO) for hA₁,hA_2A

,

hA₃receptors) was evaluated through the measurement of

displacement of selective radioligands,^24-27which were previ-

ously bound to the receptor expressed at the cell surface. In this

assay, we evaluated the displacement of: (i) specific [³H]CCPA

binding at hA₁receptors, (ii) specific [³H]NECA binding at

both the hA_2Aand hA₃receptors. The data were expressed as K_i

(dissociation constant), which was calculated from the Cheng

and Prusoff equation,²⁷with geometric means of at least three

experiments, including 95% confidence intervals.

2. Adenylyl Cyclase Activity. Because of the lack of a

suitable radioligand for hA_2Breceptor in binding assay, the

potency of antagonists at hA_2Breceptor (expressed on CHO

cells) was determined in adenylyl cyclase experiments in-

stead. The procedure was carried out as described previously

in Klotz et al. with minor modifications.^24,25In this assay, an

adenosine agonist (NECA) at known concentration was

subjected to inhibitory effect of tested antagonist. As a result,

the activation of adenylyl cyclase was inhibited with decrease

in the cAMP (cyclic adenosine monophosphate) production.

Moreover, in order to rationalize the observed structure-

-affinity relationship and the selectivity profile of this new

series of derivatives, a receptor-driven molecular modeling

investigation, based on a recently proposed model of hA₃

receptor derived from the crystallographic structure of human

A_2Areceptor,²²was also performed in this study.

Chemistry

Molecular Modeling

The synthetic pathways of new series of 2-(para-substitu-

ted)phenyl-pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines

derivatives 7-36 (Chart 1) are depicted in Schemes 1 and 2.

Compounds 7-20 were prepared following similar proce-

dures as already described elsewhere for the synthesis of the

pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines^10,23(Scheme 1).

The inseparablemixture of1-alkyl-5-amino-4-cyano-pyrazole

and 2-alkyl-5-amino-4-cyano-pyrazole regioisomers (39-42)

for both the ethyl and phenylethyl series were synthesized as

described in ref 10. For compounds bearing a methyl group,

an alternative procedure was followed to obtain only the

2-methyl-5-amino-4-cyano-pyrazole isomer (38) in good

yield.²³The obtained intermediates were directly refluxed in

triethyl orthoformate to give the corresponding imidates (43-

47), which were subsequently reacted with the appropriate

(para-substituted)benzoic hydrazide in refluxing 2-methoxy-

ethanol to afford the intermediates 48-61. Subsequently,

these compounds were subjected to a thermally induced

cyclization in diphenyl ether at 260 ꢀC. After the chromato-

graphic separation of N⁷(minor product) and N⁸(major

product) regioisomers, they provided the tricyclic compounds

(62-75) in rather good yield (about 60% for N⁸derivatives

and 30% for N⁷derivatives). Hydrolysis in 20% HCl gave rise

to the corresponding hydrolyzed intermediates (76-89),

which were subsequently converted into the 5-amino-7 or 8-

(substituted)-2-[(para-substituted)phenyl]pyrazolo-[4,3-e]1,2,4-

triazolo[1,5-c] pyrimidine derivatives (7-20).

The recently published crystallographic structure of human

A_2Aadenosine receptor, in complex with the high affinity

antagonist ZM241385 (PDB code: 3EML),²²provided a new

alternative template to perform homology modeling of other

GPCRs, particularly the adenosine receptors. Therefore, we

built up a homology model of the hA₃receptor based on the

crystal structure of the hA_2Areceptor.^28,29

In the process of selecting a reliable docking protocol to be

employed in the following docking studies of these new

derivatives, we have evaluated the ability of different docking

softwares to reproduce the crystallographic pose of ZM241385

inside the binding cavity of hA_2Areceptor. As reported in the

Experimental Section, among the four different types of pro-

grams used to calibrate our docking protocol, the GOLD

program was finally chosen because it showed the best perfor-

mance with regards to the calculated rmsd values relative to the

crystallographic pose of ZM241385.²⁸

Consequently, based on the selected docking protocol, we

performed the docking simulations to identify the hypothe-

tical binding mode of the new series of 2-(para-substituted)-

phenyl-PTPs inside the hA₃and hA_2Aadenosine receptors.

Results and Discussion

A. Structure-Affinity Relationship Studies. A new series of

2-(para-substituted)phenyl-pyrazolo[4,3-e]1,2,4-triazolo-[1,5-c]-

pyrimidines was successfully synthesized and characterized.

3364 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Scheme 1. Synthetic Scheme for Compounds 7-20^a

Cheong et al.

^aReagents: (i) 2-methylhydrazine, absolute EtOH; (ii) ethoxymethylene-malononitrile, benzene; (iii) conc HCl; (iv) NaH, R₂X, dimethylformamide;

(v) HC(OEt)₃, reflux; (vi) 2-(4-substituted)benzoic hydrazide, MeO(CH₂)₂OH; (vii) Ph₂O, 260 ꢀC, flash chromatography; (viii) HCl, reflux; (ix) NH₂CN,

1-methyl-2-pyrrolidinone, pTsOH.

Table 1 summarizes the receptor binding affinities of com-

pounds 7-36 determined at the human A₁, A_2A, and A₃

receptors and the corresponding adenylyl cyclase activity in

CHO cells which express the A_2Breceptors.

We introduced very little modifications in the pyrazole

ring in order to focus our investigation on the exploitation at

position C². In fact, only two compounds (compounds 13

and 17, bearing a substituent at N⁷instead of N⁸) were strate-

gically included in the library to confirm the necessity to have

a small alkyl group at N⁸for an optimal interaction with the

hA₃receptor subtype.¹⁰For the rest of the derivatives, we

fixed the methyl (compounds 7-12), ethyl (compound 14),

and phenylethyl (compounds 15, 16, 18-20) groups at N⁸,

with concurrent introduction of different moieties at posi-

tions C²and N⁵(compounds 21-36). The binding assay

results showed that the new series of 2-aryl-pyrazolo-triazo-

lo-pyrimidines presented good affinity at hA₃receptors,

as indicated by low nanomolar range of K_ivalues and

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3365

Scheme 2. Synthetic Scheme for Compounds 21-36^a

^aReagents: (i) benzoic anhydride, toluene (in synthesis of 21-26, 29, 30) or benzoyl chloride with DIPEA/TEA in toluene/dioxan (in synthesis of 27,

28), reflux; (ii) phenylacetyl chloride, DIPEA, toluene, reflux.

considerably improved selectivity toward the other AR sub-

types (Table 1).

affinity at hA₃AR to a certain extent. In particular, among

the substituents introduced at the para position of phenyl

ring, both the OCH₃and F groups have exerted relatively

more favorable effect on the affinity at hA₃receptor in all the

N⁵-unsubstituted (e.g., compound 11, with K_ihA₃=16.7 nM,

hA₁/hA₃= 583; hA_2A/hA₃= 71.3), N⁵-benzamide-substi-

tuted (e.g., compound 25, with K_ihA₃=2.1 nM, hA₁/hA₃=

>14300; hA_2A/hA₃= >47600) and N⁵-phenylacetamide-

substituted (e.g., compound 35, with K_ihA₃=0.241 nM; hA₁/

hA₃> 124,000; hA_2A/hA₃> 415000) derivatives.

Interestingly, although all the compounds with a 4-bromo

group in the N⁸-methyl series showed good affinity at the

hA₃receptor (e.g., compound 34, with K_ihA₃=0.345 nM;

hA₁/hA₃=70700; hA_2A/hA₃> 290000), its presence in the

N⁸-phenylethyl-substituted derivatives (e.g., compound 30,

with K_ihA₃=153 nM; hA₁/hA₃=1.76; hA_2A/hA₃> 654)

caused the opposite effect to the affinity at hA₃receptor. This

could be possibly due to the additional steric hindrance

caused by the phenylethyl group, which further limited the

C²Position. To examine the impact of the furan ring

substitution with a phenyl ring at C²position toward the

pharmacological profile, we compared the binding assay results

of compounds bearing the 2-furyl (e.g., compounds 90 and 91)

(Table 2)^10,30and the 2-aryl (e.g., compounds 7 and 31). It was

observed that the bioisosteric replacement of existing furan ring

with a phenyl ring resulted in a 3-7-fold increase in affinity

toward the hA₃receptor and significant improvement in

selectivity (of 2-3 order of magnitude) over other adenosine

receptor subtypes, namely hA₁, hA_2A, and hA_2Breceptors. In

other words, compounds with a phenyl ring at 2-position have

demonstrated better affinity and selectivity profiles toward hA₃

receptor as compared to the 2-furyl counterparts, indicating

that the aryl group at C²position played a more essential role

on the antagonistic activity at the hA₃receptor.

The substituents (e.g., Cl, F, Br, OCH₃, NO₂) at the para

position of the C²-phenyl ring were found to modulate the

3366 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3367

Table 2. Binding Affinity (K_i) at hA₃Receptor and Selectivity against hA₁and hA_2AReceptors for Compounds 90, 91 and 7, 31

compd

R

R₁

R₂

hA₃(K_i, nM)

hA₁/hA₃

hA_2A/hA₃

90

7

H

CH₃

300^a

75

0.81^a

0.108

0.33^a

4.52

867^a

5204

0.009^a

1.61

522^a

7204

H

91

31

Ph-CH₂CO

^aData taken from refs 10 and 30.

accommodation of relatively bulky bromo group within the

binding site, resulting in detrimental effect on the hA₃affinity.

Besides that, it was observed that some N⁸-methyl-substituted

compounds bearing a 2-(para-nitro)phenyl ring (e.g., com-

pound 12, K_ihA₃=655 nM; hA₁/hA₃> 458; hA_2A/hA₃>

15.3) have acquired hA₃affinity in relatively high nanomolar

range. Similarly, compound 26 (with a 2-(para-nitro) phenyl

ring, a methyl at N⁸and a benzamide at N⁵) only demon-

stratedmoderate affinityathA₃receptor (K_ihA₃=56.4 nM) in

comparison to the other derivatives with different substituents

at C²position (compounds 22-25), which have shown a

better hA₃affinity profile (2-5 nM). These observations

could again be attributed to the steric constraint of the

relatively bigger NO₂group as compared to other substitu-

ents. This rendered the nitro group unable to bind firmly onto

the binding cavity, thus affecting the affinity at the hA₃

receptor. Therefore, because the nitro group might not be a

suitable substituent at this position, it was excluded from the

subsequent series of N⁸-phenylethyl derivatives.

N⁵Position. From the binding assay results, it was obser-

ved that the absence of any substituent at N⁵position (such

as in compounds 7-20) did not allow good discrimination

among the adenosine receptor subtypes, except for the hA_2B

receptor. In other words, the free amino group at N⁵not only

bound to the hA₃receptor but it also showed good interac-

tion at both hA₁and hA_2Areceptors. This observation was

consistent with previous SAR studies, which indicated high

affinity at both hA₁and hA_2Areceptors in N⁵-unsubstituted

derivatives.¹⁰In fact, further incorporation of substituents at

the N⁵position (as shown in compounds 21-36) enabled a

shift of affinity toward the hA₃subtype with a concomitant

decrease of affinity at hA₁and hA_2Areceptors, thus improv-

ing selectivity infavor of the hA₃subtype. Thisfinding seemed

to imply that the hA₃binding cavity around the N⁵position

was rather spacious to accommodate the extended chains, in

this case the benzoyl and phenyl acetyl groups. Between these

two substituents, the longer phenyl acetyl group (e.g., com-

crucial because its replacement with an additional benzoyl

chain (as in compounds 27, K_ihA₃=6.88 nM and 28, K_ihA₃=

6.94 nM), although less favorable than a single chain, still

maintained affinity at hA₃receptor and good selectivity

(>1400) against other receptor subtypes. This finding again

confirmed the postulation that the binding pocket of A₃

receptor around this N⁵position was roomy enough to

accommodate the bulky and branched bis-benzamidic sub-

stituent; at A₁and A_2Asubtypes, there was limited space

available for such bulky groups and resulted in decrease of

affinity with subsequent increase of selectivity against these

two receptors.

N⁸Position. It was found that when a small alkyl group

was present at N⁸, the compounds (7-12, 21-28, and

31-36) showed a preference for hA₃receptors, regardless

of substitution at N⁵and C²positions (e.g., compounds 7

[(with a phenyl at C²and a free amino at N⁵), K_ihA₃=75.0

nM] and 8 [(with a 4-fluorophenyl at C²and a free amino at

N⁵), K_ihA₃=31.4 nM] versus compounds 21 [(with a phenyl

at C²and a benzamide at N⁵), K_ihA₃=5.0 nM] and 22 [(with

a 4-fluorophenyl at C²and a benzamide at N⁵), K_ihA₃=3.43

nM]. This was further confirmed by the introduction of a

slightly longer alkyl chain (e.g., an ethyl group in compound

14, K_ihA₃=68.7 nM; hA₁/hA₃=22.9; hA_2A/hA₃=12.3),

which still showed hA₃antagonism and moderate selectivity.

Notably, when the same substituent was shifted from posi-

tion N⁸to N⁷(as in compounds 13, K_ihA₃=565 nM and 17,

K_ihA₃> 30000 nM), the affinity at hA₃receptors dropped to

at least 8 times, with the resulting binding affinity profile

inclined toward hA_2Areceptors. This observation was sub-

stantiated by the results previously reported on N⁷-substi-

tuted PTPs as potent hA_2Aantagonists.³¹

Conversely, when a group bigger than ethyl was intro-

duced at N⁸(e.g., phenylethyl group in compounds 15, 16,

18-20, and 29, 30), a decrease of affinity at hA₃receptor was

observed. In derivatives with free amino group at N⁵posi-

tion, the substitution of the methyl group (e.g., compound

11, K_ihA₃= 16.7 nM) to the long and bulky phenylethyl

group (e.g., compound 20, K_ihA₃=25.0 nM) only showed a

slight decrease of the hA₃affinity. When a substituent was

introduced at the N⁵position (e.g., compound 29, K_ihA₃=

23.9 nM), the presence of the phenylethyl group seemed to

pound 34, with K_ihA₃=0.345 nM, hA₁/hA₃=70700; hA_2A

/

hA₃> 290000) showed relatively better binding profile than

the shorter benzoyl chain (e.g., compound 24, with K_ihA₃=

5.24 nM, hA₁/hA₃> 5730; hA_2A/hA₃> 19100). Moreover,

the spare H on the nitrogen atom at N⁵did not seem to be

3368 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

Figure 1. Hypothetical binding modes of the N⁵-unsubstituted derivatives obtained after docking simulations: (A) compound 7 inside the

hA_2AAR binding site; (B) compound 7 inside the hA₃AR binding site; (C) compound 90 inside the hA_2AAR binding site; (D) compound 90

inside the hA₃AR binding site. Poses are viewed from the membrane side facing TM6, TM7, and TM1. The view of TM7 is voluntarily omitted.

Side chains of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not

displayed.

have resulted in a great decrease of hA₃affinity in compar-

ison to its N⁸-methyl counterpart (compound 21, K_ihA₃=

5.0 nM). This might be due to reason that in the N⁵-

unsubstituted derivatives, there was still enough space for

the bulky phenylethyl group to orientate itself inside the

binding cavity. Once an additional group was introduced at

the N⁵position, there was limited space remained to anchor

the phenylethyl, thus causing a considerable decrease of hA₃

affinity. Apparently, an inverse relationship appeared be-

tween the binding affinity values at hA₃receptor and the

molecular volume (MV) of the substituent at position N⁸: the

higher the MV, the lower the affinity toward the hA₃

receptor.¹⁰

On the whole, these results confirmed the importance of

the contemporary introduction, in the PTP system, of (a)

small substituents (e.g., CH₃) at the N⁸position to maintain

affinity and selectivity at hA₃AR, (b) a longer chain such as

a phenylacetyl group at the N⁵position to confer higher

affinity and a better selectivity especially toward hA₁and

hA_2Areceptors, (c) a 2-(para-substituted) phenyl ring at C²

to improve affinity and selectivity profile at hA₃receptors in

relative to the 2-furyl counterparts and protect from enzyme

deactivation with subsequent higher plasma bioavailability.

Among the newly synthesized PTP derivatives, compound

31, with a phenyl at C², a methyl group at N⁸, and a

phenylacetamidic chain at N⁵, showed the best hA₃affinity

profile (K_ihA₃=0.108 nM) and good selectivity against the

other adenosine receptors (hA₁/hA₃= 5200; hA_2A/hA₃=

7200).

docking simulations on both the crystallographic structure

22

of hA_2A

and the recently published hA₃receptor

model.^28,29All the newly synthesized 2-aryl-PTP derivatives

were docked into the orthosteric TMs binding cavities of

both adenosine receptors. In addition, docking studies were

also performed on the two previously reported PTPs bearing

a furan ring at C²(compounds 90 and 91 in Table 2),^10,30with

the aim of comparing the possible differences in binding

mode between the 2-aryl and 2-furyl derivatives. On top of

that, individual electrostatic contribution (ΔE^el_int) to the

interaction energy (ΔE_int) of each receptor residue involved

in binding interaction with the ligands (see Supporting

Information) was also calculated in order to analyze the

ligand-receptor recognition mechanism in a more quanti-

tative manner.

a. N⁵-Unsubstituted Derivatives. From the docking simu-

lation analysis, almost all the new derivatives with free amino

group at N⁵(compounds 7-20) were seen to share a similar

binding pose in the TM region of the hA₃AR. The ligand

recognition occurred in the upper region of the TM bundle,

and the PTP scaffold was surrounded by TMs 3, 5, 6, and 7

with the 2-aryl ring oriented toward TM2. Figure 1B showed

the hypothetical binding pose of compound 7. This com-

pound was anchored, inside the binding cleft, by two stabi-

lizing hydrogen-bonding interactions with the side chain of

Asn250 (6.55), which is highly conserved among all AR

subtypes and found to be important for ligand binding at

the hA₃AR.³²It also formed an aromatic π-π stacking

interaction with Phe168 (EL2), while the methyl group at

N⁸was in proximity with the highly conserved Trp243 (6.48),

an important residue in receptor activation and antagonist

recognition.³²It was also found to form hydrophobic inter-

actions with many residues of the binding site including

B. Molecular Modeling Studies. A receptor-driven mole-

cular modeling investigation has been performed in order to

rationalize the results obtained from the pharmacological

evaluation. For that purpose, we performed molecular

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3369

Ala69 (2.61), Val72 (2.64), Thr87 (3.29), Leu90 (3.32), Leu91

(3.33), Phe168 (EL2), Trp243 (6.48), Leu246 (6.51), Leu264

(7.35), Tyr265 (7.36), and Ile268 (7.39). On the other hand,

by comparing the binding mode of the 2-furyl counterpart

(compound 90 in Table 2) at the hA₃receptor (Figure 1D),

we observed a slight different binding orientation as com-

pared to compound 7. In fact, this compound was found to

be oriented almost parallel to the membrane plane, and it

formed only one H-bond with Asn250 (6.55) with concurrent

loss of the π-π stacking interaction with Phe168 (EL2).

Through the analysis of electrostatic contributions per

residues to the whole interaction energy, we found that

Asn250 (6.55) showed a very negative electrostatic inter-

action energy in complex between compound 7 and hA₃

receptor due to the two stabilizing H-bonding interactions

with the ligand (see Supporting Information for details).

Conversely, for complex between compound 90 and hA₃

receptor, the same residue showed only a weak stabilizing

electrostatic interaction. Therefore, the loss of one H-bond

with Asn250 (6.55) and the π-π stacking interaction with

Phe168 (EL2) could be the reason to account for the ob-

servation that the 2-furyl derivative possessed lower affinity

toward the hA₃receptor as compared to the 2-aryl derivative

(compound 7, KihA₃= 75nM; compound 90, KihA₃=

300nM).

this region of the binding site. Moreover, the substituents at

the para position of the C²-phenyl ring could affect the

approaching of ligands into the hA_2Areceptor binding cavity

and subsequently mediating some interactions with residues

at the entrance of the binding site. This step could be crucial

for the recognition process and so for ligand affinity at

the receptor.³⁵Therefore, the presence of polar residues at

the entrance of the hA_2Areceptor binding cavity, such as

Glu169 (EL2) and His264 (EL3), might affect ligand orien-

tation while approaching the binding pocket, as already

proposed.²⁸

As for the N⁷-substituted derivative, it was found that

compound 13 showed similar binding mode at both the hA₃

and hA_2Areceptor subtypes to that of the N⁸-substituted

derivatives. Further details on the binding mode of this

compound are available in Supporting Information

(Figure S1).

b. N⁵-Substituted Derivatives. As shown in Figure 2, the

hypothetical binding modes at hA_2Aand hA₃receptors for

the N⁵-substituted derivatives with a 2-aryl ring (compounds

21-36) were the same as those obtained for 2-furyl counter-

part (compound 91). For these compounds, recognition at

hA₃receptor occurred in the upper region of the TM bundle,

and the PTP scaffold was surrounded by TMs 3, 5, 6, and 7

with the 2-aryl or 2-furyl ring oriented toward TM2 and the

substituent at N⁸located deep into the binding cavity. At the

hA₃receptor, compounds 31 and 91 formed two stabilizing

H-bonding interactions with Asn250 (6.55), a π-π stacking

interaction between the triazole ring and Phe168 (EL2) and

hydrophobic interactions with several residues of the binding

site including Ala69 (2.61), Val72 (2.64), Thr87 (3.29), Leu90

(3.32), Leu91 (3.33), Phe168 (EL2), Val169 (EL2), Trp243

(6.48), Leu246 (6.51), Ile249 (6.54), Ile253 (6.58), Val259

(EL3), Leu264 (7.35), Tyr265 (7.36), and Ile268 (7.39)

(Figure 2B,D). By analyzing the calculated individual elec-

trostatic contribution to the interaction energy of each recep-

tor residue, it was evident that the Asn250 (6.55) strongly

stabilized the ligand-hA₃receptor complex (negative elec-

trostatic interaction energy) due to the two hydrogen bond-

ing interactions (see Supporting Information).

Considering the hypothetical binding pose of these com-

pounds at the hA_2Areceptor (Figure 2A,C), it could be seen

that the PTP core was rotated of about 40ꢀ compared to the

binding pose of the same compounds at the hA₃subtypes.

Because of this different orientation of the molecules inside

the binding cleft at the hA_2Areceptor, both compounds 31

and 91 formed only one H-bond with Asn253 (6.55). Co-

herently, for these complexes, the electrostatic contributions

to the interaction energy of Asn253 (6.55) were shown to be

weakly stabilizing and no other amino acid with a note-

worthy negative electrostatic interaction energy was identi-

fied (see Supporting Information).

As regards the compounds with bulkier substituents at N⁸,

such as ethyl group (compound 14) and phenylethyl group

(compounds 15-16 and 18-20), they showed similar bind-

ing mode at hA₃receptor as compared to the N⁸-methyl

derivatives. There seemed to be enough space in the binding

cavity to accommodate such substituents at this position.

Moreover, the presence of different groups at the para

position of the phenyl ring at C²(e.g., Cl, F, Br, OCH₃)

was well tolerated at this receptor with the only exception for

the nitro group. Both steric and/or dipolar contributions of

nitro group can be responsible for the loss of activity of

compound 12.

In the context of hypothetical binding poses of compound

7 and compound 90 inside the cavity of hA_2Areceptor

(Figure 1, panels A and C, respectively), we noted that they

were very similar to each other. In fact, at the hA_2Areceptor,

both compounds formed two H-bonds with Asn253 (6.55)

and another H-bond with Glu169 (EL2), in which these two

residues have been indicated to play an important role in

ligand binding at the hA_2AAR.^33,34In addition, a π-π

stacking interaction with Phe168 (EL2) and hydrophobic

interactions with many residues of the binding site including

Val84 (3.32), Leu85 (3.33), Thr88 (3.36), Met177 (5.38),

Trp246 (6.48), Leu249 (6.51), His250 (6.52), Met270 (7.35),

and Ile274 (7.39), were also observed. Two of the strongly

negative electrostatic contributions to the interaction energy

were found to be related to the Glu169 (EL2) and Asn253

(6.55) for both complexes; however, the contribution due to

Glu169 (EL2) was of around -8 kcal/mol for the compound

7-hA_2AAR complex and -14 kcal/mol for the compound

90-hA_2AAR complex. This finding explained why com-

In fact, it has to be pointed out that at the position 169

(EL2) of the hA₃receptor subtype, a valine residue is present

while at the corresponding position of the hA_2Areceptor, this

valine is replaced by a glutamate residue (Glu169). It was

believed that at the hA_2Areceptor, the substituent at N⁵of

these derivatives were unable to occupy the same position as

that of the hA₃receptor and hence, the whole molecule was

shifted away from the Asn253 with a simultaneous loss of a

H-bond, as seen in compound 31 at the hA_2Abinding site.

This finding could be accounted for the low affinity profile at

the hA_2A, as observed in the majority of the N⁵-substituted

derivatives (compounds 22-36). In addition, due to the

pound 7 still possessed affinity for the hA_2Areceptor (KihA_2A

=

121 nM) but less as compared to the analogue with a 2-

furyl ring (compound 90, KihA_2A= 2.80 nM). Besides that,

the presence of substituents at the para position of the C²-

phenyl ring has imparted a detrimental effect toward the

hA_2Areceptor affinity. With the increase in size of the para-

substituents, a decrease in affinity at the hA_2Areceptor was

observed, which implied the existence of steric control within

3370 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

Figure 2. Hypothetical binding modes of the N⁵-substituted derivatives obtained after docking simulations: (A) compound 31 inside the

hA_2AAR binding site; (B) compound 31 inside the hA₃AR binding site; (C) compound 91 inside the hA_2AAR binding site; (D) compound

91 inside the hA₃AR binding site. Poses are viewed from the membrane side facing TM6, TM7, and TM1. The view of TM7 is voluntarily

omitted. Side chains of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are

not displayed.

presence of N⁵-substitutions, these derivatives could not

form the H-bonding interactions with Glu169 (EL2) as

observed for N⁵-unsubstituted analogues. Therefore, the

mutation of the valine at the position 169 with a glutamate

was hypothesized to be critical for the hA₃versus hA_2A

selectivity profile.²⁸Moreover, at the hA_2Areceptor, the

hydrophobic side cleft delimited by TM2 and TM3 could

well accommodate the 2-furyl ring of compound 91 but

hardly accommodated the 2-phenyl ring of compound 31.

This steric effect could be the reason of the difference in

affinity between the 2-aryl and the 2-furyl analogues at this

receptor subtype (compound 31, KihA_2A= 778 nM; com-

pound 91, KihA_2A= 432 nM). For the same reason, the

presence of para-substituents on the phenyl ring led to a

drastic decrease in affinity at hA_2Areceptor, up to complete

loss of affinity for bulkier substituents. Conversely, the 2-

phenyl and 2-furyl rings of compounds 31 and 91, respec-

tively, were located less deeply in the binding cavity of hA₃

receptor and hence, more space was available to accommo-

date them. Therefore, at the hA₃receptor subtype, the 2-

phenyl ring was preferred, and different para-substituents

were also well tolerated.

overcome the metabolic instability due to the C²-furan ring.

Through an additional molecular modeling investigation,

the experimental structure-affinity relationship (SAR) find-

ings have been rationalized by depicting the hypothetical

binding mode between these newly synthesized derivatives

and the specific amino acid residues within the binding site of

hA₃and hA_2Areceptors. In short, the rational design and

synthesis of this new series of 2-(para-substituted)phenyl-

pyrazolo-triazolo-pyrimidines has given rise to a class of

potent, highly selective, and metabolically stable hA₃AR

antagonists.

Experimental Section

A. Chemistry. Reactions were routinely monitored by thin-

layer chromatography (TLC) on silica gel plate (precoated 60

F

₂₅₄Merck plate). Column chromatographies were performed

using silica gel 60 (Merck, 70-230 mesh). Melting points were

determined on a Gallenkamp instrument and were uncorrected.

Compounds were dissolved in HPLC (high performance liquid

chromatography)-grade methanol for determination of mass to

charge ratio (m/z) via the LCQ Finnigan MAT mass spectro-

meter (source of ionization: atmospheric pressure chemical

ionization (APCI) probe). Purity of compounds was detected

by elemental analyses performed at the laboratory of micro-

analysis of the Department of Chemistry, University of Ferrara

(Italy), and all the compounds were confirmed to achieve g95%

Conclusions

In summary, the bioisosteric replacement of the furan ring

with a phenyl ring at the C²position has led to the identifica-

tion of new series of 2-(para-substituted)phenyl-pyrazolo-

triazolo-pyrimidine derivatives as hA₃antagonists with good

affinity and remarkably improved selectivity profile toward

the other adenosine receptor subtypes in comparison to the

2-furyl PTP derivatives. Moreover, the substitution of 2-furyl

with an aryl group in the new PTP derivatives is expected to

1

purity. H spectra were determined in deuterated chloroform

(CDCl₃) or deuterated dimethylsulfoxide (DMSO-d₆) through a

Bruker DPX 300 MHz spectrometer, with chemical shifts given

in parts per million (δ) downfield relative to the central peak of

the solvents, and J values (coupling constants) given in hertz.

The following abbreviations were used: s, singlet; bs, broad

singlet; d, doublet; dd, double doublet; bd, broad doublet; t,

triplet; m, multiplet.

Article

General Procedure for the preparation of 2-aryl-8-methyl-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3371

MS-APCI (methanol) m/z: 345.4 (M þ 1)^þ. Anal. (C₁₃H₁₀N₇Br)

C, H, N.

pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidin-5-amines (7-12),

2-(4-Chlorophenyl)-7- or -8-ethyl-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]

pyrimidin-5-amines (13, 14) and 2-aryl-7- or -8-phenylethyl-pyr-

azolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidin-5-amines (15-20). The

1- and 2-alkyl 5-amino-4-cyano-pyrazole regioisomers (38-42)

and corresponding imidates (43-47) for the methyl as well as the

ethyl and phenylethyl series were prepared as previously des-

cribed in refs 23 and 10.

2-(4-Methoxyphenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo-

[1,5-c]pyrimidin-5-amine (11). Yield 54%, mp 313-314 ꢀC

1

(DMF-EtOH). H NMR (300 MHz, DMSO-d₆) δ: 3.85 (3H,

s, OMe), 4.03 (3H, s, Me), 7.11 (2H, d, aromatic H, J=8.7), 7.57

(2H, s, NH₂), 8.16 (2H, d, aromatic H, J=8.7), 8.57 (1H, s,

pyrazolo-H). MS-APCI (methanol) m/z: 296.3 (M þ 1)^þ. Anal.

(C₁₄H₁₃N₇O) C, H, N.

The mixture of imidates (10 mmol) (43-47) were then dis-

solved in 2-methoxyethanol (50 mL), and (4-substituted)-ben-

zoic hydrazide (13 mmol) was added. The mixture was refluxed

for 12 h at 150 ꢀC to afford intermediates 48-61; after cooling,

the solvent was removed under reduced pressure and the dark

oily residue was cyclized further in diphenyl ether (50 mL) at

260 ꢀC. After 3 h, the mixture was poured onto cold hexane

(300 mL). The precipitate was filtered off and purified by

chromatography (AcOEt:Petroleum ether in different ratios).

The major product, N⁸-substituted compounds, was obtained in

good overall yield (60%).

The obtained regiosiomers (compounds 62-75) were dis-

solved in 20 mL of 1,4-dioxane, followed by addition of 5 mL

of concentrated hydrochloric acid, 37% w/w into the solution

(with final concentration of HCl ∼ 20%). The mixture was

refluxed for 2 h at 120 ꢀC, and after cooling, the solvent was

removed under reduced pressure. The mixture was further

basified with saturated sodium bicarbonate and extracted with

EtOAc (3 ꢀ 50 mL). The organic layers were dried over

anhydrous Na₂SO₄and evaporated under reduced pressure.

The residue obtained was crystallized from EtOAc to afford

the hydrolyzed compound as solid (76-89); this derivative

represented the hydrolyzed form of previous regioisomers, and

it was subjected to next step of reaction without further pur-

ification.

The hydrolyzed derivatives (76-89) (1 mmol) were subse-

quently dissolved in 10 mL of 1-methyl-2-pyrrolidinone and

cyanamide (12 mmol) was added into the solution, followed by

p-toluene sulfonic acid monohydrate (3 mmol). The mixture was

refluxed for 12 h at 160 ꢀC. The excess of cyanamide was pecipa-

tated by addition of 80 mL of EtOAc, followed by filtration and

solvent from the filtrate was removed under reduced pressure. A

brown-colored mixture consisting of pyrazolo-triazolo-pyri-

midines (compounds 7-20) was obtained. The mixture was

then purified by column chromatography (AcOEt:petroleum

ether in different ratios).

2-(4-Nitrophenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (12). Yield 53%, mp >300 dec ꢀC (DMF-

1

EtOH). H NMR (300 MHz, DMSO-d₆) δ: 4.04 (3H, s, Me),

7.72 (2H, s, NH₂), 8.43 (2H, d, aromatic H, J=9), 8.47 (2H,

d, aromatic H, J = 9), 8.63 (1H, s, pyrazolo-H). MS-APCI

(methanol) m/z: 311.4 (M þ 1)^þ. Anal. (C₁₃H₁₀N₈O₂) C,

H, N.

2-(4-Chlorophenyl)-7-ethylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (13). Yield 35%, pale-yellow solid, mp >300

dec ꢀC, (AcOEt-petroleum ether). ¹H NMR (300 MHz, CDCl₃)

δ: 1.59 (3H, t, CH₃, J=7), 4.61 (2H, q, N⁷-CH₂, J=7), 7.49 (2H,

d, aromatic H, J =8), 8.00 (2H, s, NH₂), 8.21-8.31 (3H, m,

aromatic 2H and pyrazolo-H). MS-APCI (methanol) m/z: 314.2

(Mþ)^þ. Anal. (C₁₄H₁₂N₇Cl) C, H, N.

2-(4-Chlorophenyl)-8-ethylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (14). Yield 18%, brown solid, mp >300 dec

ꢀC (AcOEt-petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

δ: 1.47 (3H, t, CH₃, J=7), 4.31 (2H, q, N⁸-CH₂, J=7), 7.61 (2H,

s, NH₂), 7.67 (2H, d, aromatic H, J=8), 8.22 (2H, d, aromatic H,

J = 8), 8.61 (1H, s, pyrazolo-H). MS-APCI (methanol) m/z:

314.4 (Mþ)^þ. Anal. (C₂₀H₁₆ClN₇) C, H, N.

2-(Phenyl)-8-phenylethylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (15). Yield 17%, yellow solid, mp 137-

139 ꢀC (AcOEt-petroleum ether). ¹H NMR (300 MHz,

DMSO-d₆) δ: 3.22 (2H, t, Ph-CH₂, J = 7), 4.54 (2H, t, N⁸-

CH₂, J=7), 7.16-7.28 (5H, m, aromatic H), 7.55-7.62 (5H, m,

NH₂and aromatic 3H), 8.23-8.26 (2H, d, aromatic H), 8.49

(1H, s, pyrazolo-H). MS-APCI (methanol) m/z: 356.5 (M þ 1)^þ.

Anal. (C₂₀H₁₇N₇) C, H, N.

2-(4-Fluorophenyl)-8-phenylethylpyrazolo[4,3-e]1,2,4-triazolo-

[1,5-c]pyrimidin-5-amine (16). Yyield 21%, pale-yellow solid,

mp 261-263 ꢀC (AcOEt-petroleum ether). ¹H NMR (300

MHz, DMSO-d₆) δ: 3.22 (2H, t, Ph-CH₂, J=7), 4.54 (2H, t,

N⁸-CH₂, J=7), 7.12-7.28 (5H, m, aromatic H), 7.39 (2H, dd,

aromatic H, J = 9, J = 9), 7.61 (2H, s, NH₂), 8.24 (2H, dd,

aromatic H, J=6, J=6), 8.47 (1H, s, pyrazolo-H). MS-APCI

(methanol) m/z: 374.8 (M þ 1)^þ. Anal. (C₂₀H₁₆FN₇) C, H, N.

2-(4-Chlorophenyl)-7-phenylethylpyrazolo[4,3-e]1,2,4-triazolo-

[1,5-c]pyrimidin-5-amine (17). Yield 25%, dark-yellow solid, mp

2-(Phenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrim-

idin-5-amine (7). Yield 52%, pale-yellow solid, mp 242-244 ꢀC

(AcOEt-petroleum ether). ¹H NMR (300 MHz, DMSO-d₆) δ:

4.03 (3H, s, Me), 7.56-7.63 (5H, m, NH₂and aromatic 3H), 8.23

(2H, d, aromatic H, J=8), 8.59 (1H, s, pyrazolo-H). MS-APCI

(methanol) m/z: 266.0 (M þ 1)^þ. Anal. (C₁₃H₁₁N₇) C, H, N.

2-(4-Fluorophenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (8).Yield54%, pale-yellowsolid, mp >300 dec

ꢀC, (AcOEt-petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

δ: 4.02 (3H, s, Me), 7.40 (2H, dd, aromatic H, J = 9,

J=9), 7.61 (2H, s, NH₂), 8.25 (2H, dd, aromatic H, J=6, J=

8), 8.59 (1H, s, pyrazolo-H). MS-APCI (methanol) m/z: 284.3

(M þ 1)^þ. Anal. (C₁₃H₁₀N₇F) C, H, N.

1

277-279 ꢀC (AcOEt-petroleum ether). H NMR (300 MHz,

DMSO-d₆) δ: 3.18 (2H, t, Ph-CH₂, J=7), 4.49 (2H, t, N⁸-CH₂,

J=7), 7.18-7.26 (5H, m, aromatic H), 7.60 (2H (d, aromatic H,

J=8), 8.01 (2H, s, NH₂), 8.14-8.24 (3H, m, aromatic 2H, and

pyrazolo-H). MS-APCI (methanol) m/z: 390.4 (Mþ)^þ. Anal.

(C₂₀H₁₆ClN₇) C, H, N.

2-(4-Chlorophenyl)-8-phenylethylpyrazolo[4,3-e]1,2,4-triazolo-

[1,5-c]pyrimidin-5-amine (18). Yield 17%, orange solid, mp

1

287-289 ꢀC, (AcOEt-petroleum ether). H NMR (300 MHz,

DMSO-d₆) δ: 3.22 (2H, t, Ph-CH₂, J=7), 4.54 (2H, t, N⁸-CH₂,

J=7), 7.18-7.29 (5H, m, aromatic H), 7.62-7.65 (4H, m, NH₂

and aromatic 2H), 8.21 (2H, d, aromatic H, J=8), 8.49 (1H, s,

pyrazolo-H). MS-APCI (methanol) m/z: 390.7 (Mþ)^þ. Anal.

(C₂₀H₁₆ClN₇) C, H, N.

2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (9). Yield 25%, brown solid, mp 177-179 ꢀC,

(AcOEt-petroleum ether). ¹H NMR (300 MHz, DMSO-d₆) δ:

4.19 (3H, s, Me), 7.62 (2H, s, NH₂), 7.66 (2H, d, aromatic H, J=

8), 8.23 (2H, d, aromatic H, J=8), 8.58 (1H, s, pyrazolo-H). MS-

APCI (methanol) m/z: 299.5 (Mþ)^þ. Anal. (C₁₃H₁₀N₇Cl) C,

H, N.

2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-

pyrimidin-5-amine (10). Yield 47%, brown solid, mp >300 dec

ꢀC, (AcOEt-petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

δ: 4.21 (3H, s, Me), 7.62 (2H, s,NH₂), 7.89 (2H, d, aromatic H,

J=8), 8.12 (2H, d, aromatic H, J=8), 8.29 (1H, s, pyrazolo-H).

2-(4-Bromophenyl)-8-phenylethylpyrazolo[4,3-e]1,2,4-triazolo-

[1,5-c]pyrimidin-5-amine (19). Yield 20%, beige solid, mp

1

295-297 ꢀC (AcOEt-petroleum ether). H NMR (300 MHz,

DMSO-d₆) δ: 3.22 (2H, t, Ph-CH₂, J=7), 4.54 (2H, t, N⁸-CH₂,

J=7), 7.18-7.29 (5H, m, aromatic H), 7.65 (2H, s, NH₂), 7.77

(2H, d, aromatic H, J=8); 8.14 (2H, d, aromatic H, J=8), 8.48

(1H, s, pyrazolo-H). MS-APCI (methanol) m/z: 434.6 (Mþ)^þ.

Anal. (C₂₀H₁₆BrN₇) C, H, N.

3372 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

2-(4-Methoxyphenyl)-8-phenylethylpyrazolo[4,3-e]1,2,4-triaz-

olo[1,5-c]pyrimidin-5-amine (20). Yield 15%, pale-yellow solid,

mp 217-219 ꢀC (AcOEt-petroleum ether). ¹H NMR (300

MHz, DMSO-d₆) δ: 3.22 (2H, t, Ph-CH₂, J=7), 3.84 (3H, s,

CH₃), 4.54 (2H, t, N⁸-CH₂, J=7), 7.10 (2H, d, aromatic H, J=

9), 7.13-7.26 (5H, m, aromatic H), 7.58 (2H, s, NH₂), 8.14 (2H,

d, aromatic H, J = 9), 8.46 (1H, s, pyrazolo-H). MS-APCI

(methanol) m/z: 386.8 (M þ 1)^þ. Anal. (C₂₁H₁₉N₇O) C, H, N.

General Procedure for N-(2-Aryl-8-methylpyrazolo[4,3-e]-

[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)benzamide (21-26). To a

suspension of 0.3 mmol of 2-aryl-8-methylpyrazolo[4,3-e]-

[1,2,4]triazolo[1,5-c]pyrimidin-5-amines (7-12) in 10 mL of

toluene, 0.27 g (1.2 mmol) of benzoic anhydride was added.

The mixture was heated under reflux and stirred for 6-12 h. The

solvent was removed under reduced pressure, and the residue

was crystallized from an appropriate solvent.

under reduced pressure, and the bis-acylated product, 27 was

later crystallized from EtOH. For the synthesis of 28, the same

procedure was followed, with the exceptions that 0.3 mmol of

2-(4-chloro)phenyl-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-

pyrimidin-5-amine (9) were suspended in 5 mL of dioxan in the

presence of TEA (0.42 mL, 3.0 mmol), and finallythe bis-acylated

product was crystallized from the MeOH.

N-Benzoyl-N-[8-methyl-2-(4-methoxyphenyl)-8H-pyrazolo-

[4,3-e][1,2,4]triazolo[1,5 c]pyrimidin-5-yl]benzamide (27). Yield

93%, mp 248-250 ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆)

δ: 3.81 (3H, s, OMe), 4.15 (3H, s, Me), 7.08 (2H, d, aromatic H,

J=9), 7.52 (4H, t, aromatic H, J=8), 7.64 (2H, t, aromatic H,

J=7), 7.88 (4H, d, aromatic H, J=8), 8.00 (2H, d, aromatic H,

J = 9), 8.96 (1H, s, pyrazolo-H). MS-APCI (methanol) m/z:

504.4 (Mþ)^þ. Anal. (C₂₈H₂₁N₇O₃) C, H, N.

N-Benzoyl-N-[8-methyl-2-(4-chlorophenyl)-8H-pyrazolo[4,3-e]-

[1,2,4]triazolo[1,5-c]pyrimidin-5-yl]benzamide (28). Yield 50%,

N-[2-(Phenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyri-

midin-5-yl]benzamide (21). Yield 53%, pale-yellow solid, mp

1

mp 278-280 ꢀC (MeOH). H NMR (300 MHz, DMSO-d₆) δ:

1

265-267 ꢀC (AcOEt-petroleum ether). H NMR (300 MHz,

4.15 (3H, s, Me), 7.51 (4H, t, aromatic H, J=7), 7.58-7.69 (4H,

m, aromatic H), 7.89 (4H, d, aromatic H, J=7), 8.08 (2H, d,

aromatic H, J=9), 8.99 (1H, s, pyrazolo-H). MS-APCI (metha-

nol) m/z: 508.2 (Mþ)^þ. Anal. (C₂₇H₁₈ClN₇O₂) C, H, N.

General Procedure for N-(2-Aryl-8-phenylethylpyrazolo[4,3-

e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)benzamide (29-30). To a

suspension of 1 mmol of 2-aryl-8-phenylethylpyrazolo[4,3-e]-

[1,2,4]triazolo[1,5-c]pyrimidin-5-amine (15, 19) in 10 mL of

toluene, 1.81 g (8 mmol) of benzoic anhydride was added. The

mixture was refluxed and stirred for 12 h at 120 ꢀC. Then the

solvent was removed under reduced pressure and the residue was

purified via column chromatography (AcOEt-petroleum ether

in different ratios) to obtain the pure N⁵substituted pyrazolo-

triazolo-pyrimidine derivatives (29 and 30).

CDCl₃) δ: 4.21 (3H, s, Me), 7.43-7.72 (6H, m, aromatic H),

8.08-8.15 (2H, bd, aromatic H), 8.25 (1H, s, pyrazolo-H),

8.27-8.38 (2H, bd, aromatic H), 9.84 (1H, s, NH). MS-APCI

(methanol) m/z: 370.2 (M þ 1)^þ. Anal. (C₂₀H₁₅N₇O) C, H, N.

N-[2-(4-Fluorophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (22). Yield 77%, mp 270-272

ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 4.20 (3H, s, Me),

7.37 (2H, dd, atomatic H, J=9, J=9), 7.62 (2H, t, aromatic H,

J=7), 7.71 (1H, t, aromatic H, J=7), 8.08 (2H, d, aromatic H,

J = 7), 8.22 (2H, dd, aromatic H, J = 6, J = 9), 8.93 (1H, s,

pyrazolo-H), 11.48 (1H, s, NH). MS-APCI (methanol) m/z:

388.3 (M þ 1)^þ. Anal. (C₂₀H₁₄FN₇O) C, H, N.

N-[2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (23). Yield 54%, mp 296-297

N-[2-(Phenyl)-8-phenylethyl-pyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (29). Yield 27%, light-yellow

solid, mp 122-124 ꢀC (AcOEt-petroleum ether). ¹H NMR

(300 MHz, CDCl₃) δ: 3.34 (2H, t, Ph-CH₂, J=7), 4.62 (2H, t, N⁸-

CH₂, J=7), 7.06-7.22 (5H, m, aromatic H), 7.52-7.68 (6H, m,

aromatic H), 7.96 (1H, s, pyrazolo-H), 8.08 (2H, d, aromatic H,

J=7), 8.26-8.27 (2H, bd, aromatic H), 9.85 (1H, s, NH). MS-

APCI (methanol) m/z: 457.7 (M - 1)^þ. Anal. (C₂₇H₂₁N₇O) C,

H, N.

N-[2-(4-Bromophenyl)-8-phenylethyl-pyrazolo[4,3-e][1,2,4]-

triazolo[1,5-c]pyrimidin-5-yl]benzamide (30). Yield 62%, light-

yellow solid, mp 237-239 ꢀC (AcOEt-petroleum ether). ¹H

NMR (300 MHz, CDCl₃) δ: 3.34 (2H, t, Ph-CH₂, J=7), 4.62

(2H, t, N⁸-CH₂, J=7), 7.06-7.25 (5H, m, aromatic H), 7.58-

7.68 (5H, m, aromatic H)), 7.95 (1H, s, pyrazolo-H), 8.08 (2H, d,

aromatic H, J=6), 8.14 (2H, d, aromatic H, J=8), 9.80 (1H, bs,

NH). MS-APCI (methanol) m/z: 539.0 (M þ 2)^þ. Anal. (C₂₇H₂₀-

BrN₇O) C, H, N.

General Procedure for N-(2-Aryl-8-methylpyrazolo[4,3-e][1,2,4]-

triazolo[1,5-c]pyrimidin-5-yl)phenylacetamide (31-36). To a sus-

pension of 0.3 mmol of 2-aryl-8-methylpyrazolo[4,3-e][1,2,4]-

triazolo[1,5-c]pyrimidin-5-amines (7-12) in 15 mL of toluene,

0.16 mL (1.2 mmol) of phenylacetyl chloride was added follo-

wed by 0.2-0.5 mL (1.2-3 mmol) of DIPEA. The mixture was

heated under reflux with stirring for 6-12 h. The solvent was

removed under reduced pressure, and the residue was crystal-

lized from an appropriate solvent.

N-[2-(Phenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyri-

midin-5-yl]phenylacetamide (31). Yield 50%, pale-yellow solid,

mp 283-285 ꢀC (AcOEt-petroleum ether). ¹H NMR (300

MHz, DMSO-d₆) δ: 4.03 (2H, s, CH₂), 4.15 (3H, s, Me), 7.28-

7.46 (5H, m, aromatic H), 7.57-7.59 (3H, m, aromatic H),

8.20-8.23 (2H, m, aromatic H), 8.83 (1H, s, pyrazolo-H), 10.90

(1H, bs, NH). MS-APCI (methanol) m/z: 384.3 (M þ 1)^þ. Anal.

(C₂₁H₁₇N₇O) C, H, N.

1

ꢀC (MeOH). H NMR (300 MHz, DMSO-d₆) δ: 4.20 (3H, s,

Me), 7.61 (2H, d, aromatic H, J=9), 7.62 (2H, t, aromatic H,

J=7), 7.71 (1H, t, aromatic H, J=7), 8.08 (2H, d, aromatic H,

J=7), 8.18 (2H, d, aromatic H, J=9), 8.93 (1H, s, pyrazolo-H),

11.48 (1H, s, NH). MS-APCI (methanol) m/z: 404.2 (Mþ)^þ.

Anal. (C₂₀H₁₄ClN₇O) C, H, N.

N-[2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (24). Yield 73%, mp 292-294

ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 4.20 (3H, s, Me),

7.62 (2H, t, aromatic H, J=8), 7.71 (1H, t, aromatic H, J=7),

7.75 (2H, d, aromatic H, J=8), 8.08 (2H, d, aromatic H, J=7),

8.11 (2H, d, aromatic H, J=8), 8.93 (1H, s, pyrazolo-9), 11.50

(1H, s, NH). MS-APCI (methanol) m/z: 449.4 (M þ 1)^þ. Anal.

(C₂₀H₁₄BrN₇O) C, H, N.

N-[2-(4-Methoxyphenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (25). Yield 74%, mp 295-296

ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 3.83 (3H, s,

OMe), 4.20 (3H, s, Me), 7.09 (2H, d, aromatic H, J=9), 7.62

(2H, t, aromatic H, J=7), 7.71 (1H, t, aromatic H, J=8), 8.08

(2H, d, aromatic H, J=7), 8.11 (2H, d, aromatic H, J=9), 8.91

(1H, s, pyrazolo-H), 11.42 (1H, s, NH). MS-APCI (methanol)

m/z: 400.5 (M þ 1)^þ. Anal. (C₂₁H₁₇N₇O₂) C, H, N.

N-[2-(4-Nitrophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]benzamide (26). Yield 47%, mp 295-296

ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 4.20 (3H, s, Me),

7.61 (2H, t, aromatic H, J=8), 7.70 (1H, t, aromatic H, J=7),

8.10 (2H, d, aromatic H, J=8), 8.39 (2H, d, aromatic H, J=9),

8.43 (2H, d, aromatic H, J=9), 8.94 (1H, s, pyrazolo-H), 11.57

(1H, bs, NH₂). MS-APCI (methanol) m/z: 414.3 (Mþ)^þ. Anal.

(C₂₀H₁₄N₈O₃) C, H, N.

General Procedure for N-Benzoyl-N-(2-aryl-8-methylpyrazolo-

[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)benzamide (27-28). To

a suspension of 0.3 mmol of 2-(4-methoxyphenyl)-8-methylpyra-

zolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (11) in 5 mL of

toluene, 0.35 mL (3.0 mmol) of benzoyl chloride was added

followed by DIPEA (0.52 mL, 3.0 mmol). The mixture was

heated under refluxwith stirringfor 8 h. The solvent was removed

N-[2-(4-Fluorophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]phenylacetamide (32). Yield 38%, mp 229-

231 ꢀC (MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 4.03 (2H, s,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3373

CH₂), 4.16 (3H, s, Me), 7.03 (1H, t, aromatic H, J = 7.0),

7.35-7.49 (6H, m, aromatic H), 8.25 (2H, dd, aromatic H, J=

6, J=9), 8.84 (1H, s, pyrazolo-H), 10.90 (1H, s, NH). MS-APCI

(methanol) m/z: 402.4 (M þ 1)^þ. Anal. (C₂₁H₁₆FN₇O) C, H, N.

N-[2-(4-Chlorophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]phenylacetamide (33). Yield 36%, mp

3h at 25 ꢀC. Nonspecific binding was determined in the presence

of 100 μM R-PIA (R-N⁶-phenylisopropyladenosine).²⁶

Binding of [³H]-NECA to CHO cells transfected with the

human recombinant A₃adenosine receptors was carried out as

previously described.^24,25The displacement experiments were

performed for 3h at 25 ꢀC in buffer solution containing 10 nM

[³H]-NECA, 20 μg membrane protein in 50 mM Tris-HCl, 1 mM

EDTA (ethylenediaminotetraacetate), 10 mM MgCl₂, pH 8.25

and tested compound in different concentrations. Nonspecific

binding was determined in the presence of 100 μM R-PIA.²⁶

Bound and free radioactivity was separated by filtering the

assay mixture through Whatman GF/B glass-fiber filters using a

Micro-Mate 196-cell harvester (Packard Instrument Company).

The filter bound radioactivity was counted on Top Count

(efficiency of 57%) with Micro-Scint 20. The protein concentra-

tion was determined according to the Bio-Rad method³⁶with

bovine albumin as a reference standard.

2. Adenylyl Cyclase Activity. Because of the lack of a suitable

radioligand for hA_2Breceptor in binding assay, the potency of

antagonists at A_2Breceptor (expressed on CHO cells) was

determined in adenylyl cyclase experiments instead. The proce-

dure was carried out as described previously with minor mod-

ifications.^24,25Membranes were incubated with about 150000

cpm of [R-³²P]ATP for 20 min in the incubation mixture as

described^24,25without EGTA and NaCl. For agonists, the EC₅₀

values 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

were calculated accordingly. Dissociation constants (K_i) for

antagonist were then calculated from the Cheng and Prusoff

equation.²⁷

1

223-225 ꢀC (EtOH). H NMR (300 MHz, DMSO-d₆) δ: 4.03

(2H, s, CH₂), 4.16 (3H, s, Me), 7.31 (1H, t, aromatic H, J=7),

7.38 (2H, t, aromatic H, J=7), 7.45 (2H, d, aromatic H, J=8),

7.66 (2H, d, aromatic H, J=9), 8.21 (2H, d, aromatic H, J=9),

8.84 (1H, s, pyrazolo-H), 10.93 (1H, s, NH). MS-APCI

(methanol) m/z: 418.4 (Mþ)^þ. Anal. (C₂₁H₁₆ClN₇O) C, H, N.

N-[2-(4-Bromophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]phenylacetamide (34). Yield 63%, mp

217-218 ꢀC (MeOH), ¹H NMR (300 MHz, DMSO-d₆) δ: 4.03

(2H, s, CH₂), 4.16 (3H, s, Me), 7.31 (1H, t, aromatic H, J=7),

7.38 (2H, t, aromatic H, J=7), 7.45 (2H, d, aromatic H, J=8),

7.80 (2H, d, aromatic H, J=9), 8.14 (2H, d, aromatic H, J=9),

8.84 (1H, s, pyrazolo-H), 10.93 (1H, s, NH). MS-APCI

(methanol) m/z: 463.3 (M þ 1)^þ. Anal. (C₂₁H₁₆BrN₇O) C, H, N.

N-[2-(4-Methoxyphenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]phenylacetamide (35). Yield 52%, mp 225-

226 ꢀC (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 3.86 (3H, s,

OMe), 4.03 (2H, s, CH₂), 4.15 (3H, s, Me), 7.13 (2H, d, aromatic

H, J=9), 7.31 (1H, t, aromatic H, J=7), 7.39 (2H, t, aromatic H,

J=7), 7.45 (2H, d, aromatic H, J=7), 8.15 (2H, d, aromatic H,

J=9), 8.82 (1H, s, pyrazolo-H), 10.83 (1H, s, NH). MS-APCI

(methanol) m/z: 413.2 (Mþ)^þ. Anal. (C₂₂H₁₉N₇O₂) C, H, N.

N-[2-(4-Nitrophenyl)-8-methylpyrazolo[4,3-e][1,2,4]triazolo-

[1,5-c]pyrimidin-5-yl]phenylacetamide (36). yield 57%, mp 248-

249 ꢀC (MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ: 4.04 (2H, s,

CH₂), 4.17 (3H, s, Me), 7.32 (1H, t, aromatic H, J=7), 7.40 (2H,

t, aromatic H, J=7), 7.45 (2H, d, aromatic H, J=7), 8.45 (4H, s,

aromatic H), 8.88 (1H, s, pyrazolo-H), 11.02 (1H, s, NH). MS-

APCI (methanol) m/z: 429.4 (M þ 1)^þ. Anal. (C₂₁H₁₆N₈O₃) C,

H, N.

C. Molecular Modeling. All modeling studies were carried out

on a 20 CPU (Intel Core2 Quad CPU 2.40 GHz) Linux cluster.

Homology modeling, energy calculation, and analyses of dock-

ing poses were performed using the Molecular Operating En-

vironment (MOE, version 2008.10) suite.³⁷The software

package MOPAC (version 7),³⁸implemented in MOE suite,

was utilized for all quantum mechanical calculations. Docking

simulations were performed using GOLD suite.³⁹

B. Biology. CHO Membrane Preparation. All the pharmaco-

logical methods involving in membrane preparation for radi-

oligand binding experiments followed the procedures as

described earlier.²⁴

Membranes for radioligand binding were prepared from cells

stably transfected with the human adenosine receptor subtypes

(A₁, A_2A, and A₃expressed on CHO cells) in a two-step

procedure. In the first low-speed step (1000g for 4 min), the cell

fragments and nuclei were removed. After that, the crude

membrane fraction was sedimented from the supernatant at

100000g for 30 min. The membrane pellet was then resuspended

in the specific buffer used for the respective binding experiments,

frozen in liquid nitrogen, and stored at -80 ꢀC. For the

measurement of the adenylyl cyclase activity in A_2Breceptor

expressed on CHO cells, only one step of centrifugation was

used in which the homogenate was sedimented for 30 min at

54000g. The resulting crude membrane pellet was resuspended

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

adenylyl cyclase assay.

1. Human Cloned A₁, A_2A, A₃Adenosine Receptor Binding

Assay. Binding of [³H]-CCPA to CHO cells transfected with the

human recombinant A₁adenosine receptor was performed as

previously described.^24,25Displacement experiments were per-

formed for 3h at 25 ꢀC in 200 μL of buffer containing 1 nM [³H]

CCPA, 0.2 U/mL adenosine deaminase, 20 μg of membrane

protein in 50 mM Tris/HCl, pH 7.4 and tested compound in

different concentrations. Nonspecific binding was determined in

the presence of 1 mM theophylline.²⁶

1. Homology Models of hA₃AR. On the basis of the assump-

tion that GPCRs share similar TM boundaries and overall

topology, a homology model of the hA₃adenosine receptor

was constructed, as previously reported,^28,29based on a tem-

plate of the recently published crystal structure of hA_2Areceptor

(PDB code: 3EML).²²

The numbering of the amino acids follows the arbitrary

scheme by Ballesteros and Weinstein. According to this scheme,

each amino acid identifier starts with the helix number, followed

by the position relative to a reference residue among the most

conserved amino acid in that helix. The number 50 is arbitrarily

assigned to the reference residue.⁴⁰

First, the amino acid sequences of TM helices of the hA₃

receptor were aligned with those of the template, guided by the

highly conserved amino acid residues, including the DRY motif

(Asp3.49, Arg3.50, and Tyr3.51) and three proline residues

(Pro4.60, Pro6.50, and Pro7.50) in the TM segments of GPCRs.

The same boundaries were applied for the TM helices of hA₃

receptor as they were identified from the 3D structure for the

corresponding sequences of the template, the coordinates of

which were used to construct the seven TM helices for hA₃

receptor. Then, the loop domains were constructed by the loop

search method implemented in MOE on the basis of the

structure of compatible fragments found in the Protein Data

Bank. In particular, loops were modeled first in random order.

For each loop, a contact energy function analyzed the list of

candidates collected in the segment searching stage, taking into

account all atoms already modeled and any atoms specified by

the user as belonging to the model environment. These energies

were then used to make a Boltzmann-weighted choice from the

Binding of [³H]-NECA to CHO cells transfected with the

human recombinant A_2Aadenosine receptors was performed

following the conditions as that described for the A₁receptor

binding.^24,25In the displacement experiments, samples contain-

ing a protein concentration of 50 μg, 30 nM [³H]-NECA and

tested compound in different concentrations were incubated for

3374 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Cheong et al.

candidates, the coordinates of which were then copied to the

model. Subsequently, the side chains were modeled using a

library of rotamers generated by systematic clustering of the

Protein Data Bank data, using the same procedure. Side chains

belonging to residues whose backbone coordinates were copied

from a template were modeled first, followed by side chains of

modeled loops. Outgaps and their side chains were modeled last.

Special caution has to be given to EL2 because amino acids of

this loop could be involved in direct interactions with the

ligands. A driving force to the peculiar fold of the EL2 loop

might be the presence of a disulfide bridge between cysteines in

TM3 and EL2. Because this covalent link is conserved in both

hA_2Aand hA₃receptors, the EL2 loop was modeled using a

constrained geometry around the EL2-TM3 disulfide bridge.

The constraints were applied before the construction of the

homology model, in particular during the sequences alignment,

selecting the cysteine residues involved in the disulfide bridge in

hA_2Ato be constrained with the corresponding cysteine residues

in hA₃sequence. In particular, Cys166 (EL2) and Cys77 (3.25)

of the hA_2Areceptor were constrained, respectively, with

Cys166 (EL2) and Cys83 (3.25) of the hA₃receptor. During

the alignment, MOE-Align attempted to minimize the number

of constraint violations. Then, after running the homology

modeling, the presence of the conserved disulfide bridge in the

model was manually checked. After the heavy atoms were

modeled, all hydrogen atoms were added using the Protonate

3D methodology,⁴¹part of the MOE suite. This application

assigned a protonation state for each chemical groups that

minimized the total free energy of the system (taking titration

into account).⁴¹

individual amino acids have been calculated as implemented

in MOE suite.³⁷To estimate the electrostatic contributions,

atomic 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.

Acknowledgment. This work has been supported by the

National Medical Research Council, Singapore (NMRC/

NIG/0020/2008), and the National University of Singapore

(R-148-050-091-101/133).

Supporting Information Available: Electrostatic interaction

energy between the ligand and individual amino acid involved in

ligand recognition of compounds 7, 31, 90, 91; binding mode of

compound 13 inside the hA₃and hA_2Areceptors and combus-

tion analysis data of the synthesized compounds. This material

is available free of charge via the Internet at http://pubs.acs.

org.

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Protein stereochemistry evaluation was then performed by

several tools (Ramachandran plot; backbone bond lengths,

angles and dihedral plots; clash contacts report; rotamers strain

energy report) implemented in MOE suite.³⁷

2. Molecular Docking of hA₃AR Antagonists. Ligand struc-

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suite,³⁷and were subjected to MMFF94x energy minimization

-1

until the rms of conjugate gradient was <0.05 kcal mol^-1

.

˚

A

Partial charges for the ligands were calculated using PM3/ESP

methodology.

Four different programs have been used to calibrate our

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crystal structure of the hA_2Aadenosine receptor (PDB code:

3EML) with different docking algorithms and scoring func-

tions, as already described.^28,29Then, rmsd values between

predicted and crystallographic positions of ZM-241385 were

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˚

highest number of poses with rmsd value <2.5 A.

On the basis of the best docking performance, all antagonist

structures were docked into the hypothetical TM binding site of

the hA₃AR model and that of the hA_2AAR crystal structure by

using the docking tool of the GOLD suite.³⁹Searching was

conducted within a user-specified docking sphere, using the

Genetic Algorithm protocol and the GoldScore scoring func-

tion. GOLD performed a user-specified number of independent

docking runs (25 in our specific case) and wrote the resulting

conformations and their energies in a molecular database file.

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energy minimization until the rms of conjugate gradient was

˚

<0.1 kcal mol^-1

A

^-1. Charges for the ligands were imported

from the MOPAC output files using PM3/ESP methodology.

Prediction of antagonist-receptor complex stability (in terms

of corresponding pK_ivalue) and the quantitative analysis for

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suite.³⁷Electrostatic contributions to the binding energy of

Article

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Article Doi

The significance of 2-furyl ring substitution with a 2-(para -substituted) aryl group in a new series of pyrazolo-triazolo-pyrimidines as potent and highly selective hA3 adenosine receptors antagonists: New insights into structure-affinity relationship and receptor-antagonist recognition

DOI: 10.1021/jm100049f

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