7-Amino-2-aryl/hetero-aryl-5-oxo-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles: Synthesis and adenosine receptor binding studies

Article abstract of DOI:10.1111/cbdd.13528

A series of novel 7-amino-5-oxo-2-substituted-aryl/hetero-aryl-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles (4a–4t) was synthesized, characterized and evaluated for their binding affinity and selectivity towards hA₁, hA_2A

Full text of DOI:10.1111/cbdd.13528

DR PRAN KISHORE DEB (Orcid ID : 0000-0002-8650-2874)
Article type : Research Letter
7-Amino-2-aryl/heteroaryl-5-oxo-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-
6-carbonitriles: Synthesis and Adenosine Receptor Binding Studies
Khasim Shaik,1 Pran Kishore Deb,2* Raghu Prasad Mailavaram,1* Balakumar
Chandrasekaran,2 Sonja Kachler,3 Karl-Norbert Klotz,3 Abdul muttaleb Yousef Jaber2
1
Pharmaceutical Chemistry Department, Shri Vishnu College of Pharmacy, Bhimavaram,
West Godavari, Andhra Pradesh, India;
2
Faculty of Pharmacy, Philadelphia University-Jordan, P.O BOX (1), Philadelphia
University (19392) – Jordan;
3 University of Würzburg Department of Pharmacology and Toxicology Versbacher Str. 9, D-
97078 Würzburg, Germany
*Corresponding authors
Professor Raghu Prasad Mailavaram
Dr Pran Kishore Deb
Head, Pharmaceutical Chemistry Department Assistant Professor, Pharmaceutical
Shri Vishnu College of Pharmacy,
Bhimavaram
West Godavari, Andhra Pradesh, India
E-mail: raghumrp@svcp.edu.in
ORCID: http://orcid.org/0000-0001-6126-
5860
Chemistry Department, Faculty of Pharmacy,
Philadelphia University-Jordan, P.O BOX (1),
Philadelphia University (19392) – Jordan
Email: prankishore1@gmail.com
pdeb@philadelphia.edu.jo
ORCID: http://orcid.org/0000-0002-8650-
2874
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13528
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ABSTRACT
A series of novel 7-amino-5-oxo-2-substituted-aryl/heteroaryl-5,8-dihydro[1,2,4]triazolo[1,5-
a]-pyridine-6-carbonitriles (4a-4t) was synthesized, characterized and evaluated for their
binding affinity and selectivity towards hA1, hA2A, hA2B and hA3 adenosine receptors (ARs).
Compound 4a with a phenyl ring at 2-position of the triazolo moiety of the scaffold showed
high affinity and selectivity for hA1 AR (Ki hA1 = 0.076 µM, hA2A = 25.6 µM and hA3 > 100
µM). Introduction of various electron donating and withdrawing groups at different positions
of the phenyl ring resulted in drastic reduction in affinity and selectivity towards all the ARs,
except compound 4b with a phenol group at 2-position. Interestingly, the replacement of the
phenyl ring with a smaller heterocyclic thiophene ring (π excessive system) resulted in
further improvement of affinity for hA1 AR of compound 4t (Ki hA1 = 0.051 µM, hA2A
=
9.01 µM and hA3 > 13.9 µM) while retaining the significant selectivity against all other AR
subtypes similar to compound 4a. The encouraging results for compounds 4a and 4t indicate
that substitution at 2-position of the scaffold with π-excessive systems other than thiophene
may lead to even more potent and selective hA1 AR antagonists.
Keywords: A1 adenosine receptors, adenosine receptor antagonists, cyanoacetic hydrazides,
triazolopyridines
1. INTRODUCTION
The physiological effects of endogenous adenosine on various organ systems are myriad and
complex which are generally elicited upon activation of any of the four G protein coupled
receptors (GPCRs) denoted as A1, A2A, A2B and A3 adenosine receptors (ARs) (Borea,
Varani, Gessi, Merighi, & Vincenzi, 2018; Fredholm, IJzerman, Jacobson, Klotz, & Linden,
2001). Receptor-mediated actions of A1 and A3 ARs are mainly linked to inhibition of
adenylyl cyclase (AC) activity and a consequent decrease in the intracellular level of cyclic
AMP (cAMP), whereas A2A and A2B ARs stimulate the AC activity leading to an increase of
cAMP (Merighi, Gessi, & Borea, 2018). The A1 AR is prominently expressed in the central
nervous system (CNS), spinal cord, heart and kidney. Agonists of A1 AR could be useful as
neuro- and cardio-protective agents as well as for the treatment of cardiac arrhythmias, pain,
diabetes type-2, reduction of lipolysis in adipose tissue and intraocular pressure in glaucoma
(Cosimelli et al., 2016; Gao et al., 2018). In contrast, A1 AR antagonists might be beneficial
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for the treatment of acute heart failure, asthma and chronic obstructive pulmonary disease
(COPD) (Brown, Spina, & Page, 2008; Gao et al., 2018; Kiesman, Elzein, & Zablocki, 2009).
The A2A receptor antagonists are considered to be a potential non-dopaminergic therapeutic
approach for the treatment of neurodegenerative movement disorders such as Parkinson’s
disease (PD) and Huntington’s disease, which resulted in the discovery and development of
the xanthine derivative istradefylline as the first marketed selective A2A AR antagonist as an
adjunctive therapy of PD in Japan in 2013 (Pinna, 2014; Preti, Baraldi, Moorman, Borea, &
Varani, 2015). The A2B AR antagonists on the other hand are being investigated for the
treatment of type 2 diabetes, asthma and gastrointestinal disorders (Joseph et al., 2008; Kalla
& Zablocki, 2009; Sun & Huang, 2016). Similarly, agonists selective for A3 AR are currently
under investigation for the treatment of autoimmune inflammatory disorders, liver cancer,
rheumatoid arthritis, psoriasis, dry eye disease and cardioprotection, respectively, whereas A3
selective antagonists are developed for the treatment of asthma, inflammatory bowel disease,
glaucoma as well as cerebroprotective agent (Brown et al., 2008; Cruz Monteagudo,
Cordeiro, Teijeira, González, & Borges, 2010; Jacobson et al., 2018).
Theophylline (i) and caffeine (ii), the archetypal antagonists, have no selectivity for
the A1 AR. Thus, a lot of efforts have been made to develop xanthine and non-xanthine
analogues with higher selectivity and affinity towards specific AR subtypes. Despite the
continuous efforts made by various researchers only few analogues with promising A1
affinity have been identified so far (Borghini, Pietra, Leonardi, Giorgi, & Bianucci, 2013;
Cosimelli et al., 2016; Gao et al., 2018; Kiesman et al., 2009; Romagnoli, Baraldi, Moorman,
Borea, & Varani, 2015). In particular, 8-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-6-
carboxyl amides (ii) and isomeric 5-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-7-carboxyl
amide derivatives (iv) showed good affinity and selectivity towards hA2A versus hA1 ARs
(Guba, Nettekoven, Püllmann, Riemer, & Schmitt, 2004). Thus, it was our thought of interest
to further investigate any changes in affinity and selectivity towards ARs for a novel series of
triazolopyridine derivatives (4a-4t) with 7-amino (NH2) and 6-carbonitrile (CN) group, while
varying the substitution pattern at the 2-position of the scaffold with various aryl/heteroaryl
moieties (Figure 1). In continuation of our interest in the search of novel ligands for
adenosine receptors (Balakumar et al., 2012; Chandrasekaran et al., 2018; Deb et al., 2018;
Kaur et al., 2011; Kishore, Balakumar, Rao, Roy, & Roy, 2011; Prasad et al., 2008; Sirisha et
al., 2010; Veeraswamy et al., 2013) and in the light of the above mentioned literature reports,
some novel 5-oxo[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile derivatives (4a-4t) have been
synthesized as potential selective ligands for hA1 AR.
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2. RESULTS AND DISCUSSION
2.1. Chemistry
The synthetic route of the target compounds (4a-4t) is outlined in Figure 2. The 2-
cyanoacetohydrazide (1) was treated with various aryl/heteroaryl aldehydes (2) under mild
acidic conditions during which hydrazide reacts with the carbonyl group of the aldehyde and
undergoes dehydration to give rise to key intermediates, cyanoacetic acid substituted
benzylidene/hetero-arylidene hydrazides (3a-3t) (Gorobets, Yousefi, Belaj, & Kappe, 2004;
Metwally & Abdelrazek, 2005).
The synthesized cyanoacetic acid substituted benzylidene/hetero-arylidene hydrazides (3a-3t)
were treated with malononitrile in the presence of an amount of a catalytic base (piperidine)
using ethanol as solvent to obtain the final products (4a-4t). The products thus obtained were
purified and characterized as 7-amino-5-oxo-2-phenyl/substitutedphenyl/hetero-aryl-5,8-
dihydro-[1,2,4]triazolo[1,5-α]pyridine-6-carbonitriles (4a-4t) based on their spectrometric
analysis. Compounds 4a, 4b, 4f, 4p preparation were reported earlier by one pot synthetic
procedure (El-Hamid Ismail, 1996; Ismail, 1996) and their reported melting points were well
corroborated with our melting points of these compounds. However, this method was not
applicable for the synthesis of other derivatives. Hence a two pot synthetic procedure was
developed. It is worth mentioning that LCMS data of all the target compounds indicated a
purity of > 95 %.
Details of materials and methods for the synthesis and characterization data (IR, NMR and
Mass spectra) of compounds along with physical properties of intermediate compounds (3a-
3t) are presented in Appendix S1 and Table S1 respectively, under supporting information.
2.2. Biological activity
All the synthesized target compounds (4a-4t) were evaluated for their affinity and selectivity
towards human A1, A2A, A2B and A3 ARs, expressed in CHO cells (Table 1) by following the
previously reported procedures. The radioligands [3H]-2-chloro-N6-cyclopentyl adenosine
([3H]CCPA), [3H]-5’-N-ethylcarboxamido adenosine ([3H]NECA) and [3H]-2-(1-hexynyl)-
N6-methyl adenosine ([3H]HEMADO) were used in binding assays for hA1, hA2A, and hA3
ARs, respectively. Due to the lack of a useful radioligand for A2B receptor a possible
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interaction of the new compounds with the A2B AR was tested in functional experiments. A
possible agonistic activity at hA2B AR was determined by stimulation of adenylyl cyclase
activity, while inhibition of NECA-stimulated adenylyl cyclase activity was used as a
measurement of an antagonist interaction. Data for hA2B AR are not shown as none of the
compounds showed measurable interaction with the hA2B AR in an agonistic or antagonistic
fashion (EC50 or IC50 values > 60 µM). Materials and methods required for the evaluation of
binding affinity and selectivity of compounds (4a-4t) towards human A1, A2A, A2B and A3
ARs are described in Appendix S1.
In this study, five-membered thiazole and six-membered benzene rings with diverse
functional groups were substituted at the 2-position of the triazolo ring to get the targeted
analogues (4a-4t), while retaining a nitrile and a free amino group at 6- and 7-position of the
scaffold. The AR binding data shows that the affinity and selectivity of the target compounds
(4a-4t) varied with a change in the aryl/heteroaryl substitution at the 2-position of the
scaffold. Among the 2-aryl analogues (4a-4r), compound 4a with an un-substituted phenyl
ring at the 2-position of the triazolo moiety of the scaffold was found to possess the highest
affinity and selectivity for hA1 AR (Ki hA1 = 0.076 µM, hA2A = 25.6 µM and hA3 > 100 µM).
Substitution of the phenyl ring with hydroxyl (OH) and methyl (CH3) group at the para
position decreased the affinity for hA1 AR of compounds 4b and 4c respectively (4a > 4b >
4c). Further introduction of a ring activating N,N-dimethyl amino group at para position of
the phenyl ring (compound 4d) and methoxy (OCH3) substitution at various positions of the
phenyl ring (compounds 4e-4i) drastically reduced the affinity for hA1 AR. Compounds with
a combination of both hydroxyl (OH) and methoxy (OCH3) substitutions at various positions
of the phenyl ring (4j and 4k) showed little improvement in affinity for hA1 AR as compared
to compounds 4e-4i, in particular, compound 4j with hydroxyl and methoxy group at ortho
and meta positions of the phenyl ring exhibited moderate affinity with high selectivity for
hA1 AR (Ki hA1 = 0.875 µM, hA2A = > 100 µM and hA3 > 47.60 µM).
Introduction of an electron withdrawing nitro group (NO2) at the ortho or para position of the
phenyl ring resulted in compounds 4l and 4m with insignificant affinity for all the ARs.
Interestingly, the compounds 4n-4r with a halide (F/Cl/Br) substituted phenyl ring at the 2-
position of the scaffold showed moderate to good affinity for hA1 AR with significant
selectivity against the other AR subtypes. In particular, compound 4o with a chloro (Cl)
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substitution at 2-position of the phenyl ring showed significant affinity and selectivity
towards hA1 AR (Ki hA1 = 0.294 µM, hA2A > 100 µM and hA3 > 100 µM).
The replacement of the phenyl/substituted phenyl group (compounds 4a-4r) with a more
reactive smaller heterocyclic thiophene moiety (π excessive system) resulted in further
improvement for compound 4t displaying the highest affinity and significant selectivity
towards hA1 AR (Ki hA1 = 0.051 µM, hA2A = 9.01 µM and hA3 > 13.9 µM), whereas
replacement of the phenyl group with a pyridine ring (π-deficient system) showed detrimental
effect with reduced affinity for hA1 AR in case of compound 4s (Ki hA1 = 0.859 µM, hA2A =
6.43 µM and hA3 > 60 µM). This wide variation in affinity might be attributed to the higher
electron density of thiophene over pyridine and phenyl rings. The electron pairs on sulfur are
delocalized in the π-electron system to a significant extent and are readily available for bond
formation which might facilitate crucial interactions with the amino acid residues at the
binding site of the hA1 AR leading to greater binding affinity. Moreover, a six-membered
ring as a substituent could be too big in size to fit into the receptor binding site for proper
interaction with target amino acid residues. However, it is well known that pyridine because
of the negative inductive effect of nitrogen suffers from weaker resonance stabilization as
compared to a phenyl group and this further explains the differences in affinities of the
thiophene, phenyl and pyridyl derivatives.
3. CONCLUSIONS
In conclusion, a series of novel 7-amino-5-oxo-2-substituted phenyl/heteroaryl-5,8-dihydro-
[1,2,4]triazolo[1,5-α]pyridine-6-carbonitrile
derivatives
(4a-4t)
was
synthesized,
characterized and evaluated for A1 affinity and selectivity. Structure activity relationship
(SAR) studies revealed that among the 2-aryl analogues (4a-4r), the compound 4a with
unsubstituted phenyl ring at the 2-position of the scaffold possesses the highest affinity and
selectivity for hA1 AR (Ki hA1 = 0.076 µM, hA2A = 25.6 µM and hA3 > 100 µM).
Introduction of various electron donating and withdrawing group/s at different positions of
the phenyl ring showed detrimental effect in affinity and selectivity towards all the ARs. For
example, compound 4i with 3,4,5-trimethoxyphenyl group did not show any affinity for ARs
(Ki hA1 > 60 µM, hA2A > 60 µM and hA3 > 100 µM), whereas compound 4l with 3-
nitrophenyl group showed insignificant affinity and selectivity for ARs. Interestingly, the
compounds (4n-4r) with a halide (F/Cl/Br) substituted phenyl ring at the 2-position of the
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scaffold showed good affinity with significant selectivity for hA1 AR. In particular,
compound 4o with 2-chlorophenyl group showed maximum affinity and selectivity for hA1
AR (Ki hA1 = 0.294 µM, hA2A > 100 µM and hA3 > 340 µM) as compared to other halide
substituted compounds (4n, 4p-4r). The replacement of the phenyl group with a more
reactive smaller heterocyclic thiophene moiety (π excessive system) resulted in further
improvement of affinity as seen in the case of compound 4t displaying the highest affinity
and significant selectivity for hA1 AR (Ki hA1 = 0.051 µM, hA2A = 9.01 µM and hA3 > 13.9
µM). Thus, further optimization of this scaffold with various substituted five-membered
aromatic ring systems like thiophene may lead to the discovery and development of highly
potent A1 AR ligands with improved affinity and selectivity.
ACKNOWLEDGEMENTS
Authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi,
India for providing the research grant (Project Number: 01/2346/09/EMR-II) to financially
support this project. Author are also thankful to the Faculty of Pharmacy, Philadelphia
University, Jordan for providing the research grant (Project number: 46/34/100PU) and
necessary research facilities.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Pran Kishore Deb http://orcid.org/0000-0002-8650-2874
Karl-Norbert Klotz http://orcid.org/0000-0003-3553-3205
M. Raghu Prasad http://orcid.org/0000-0001-6126-5860
SUPPORTING INFORMATION
Appendix S1: Methods and materials (details) for synthesis and characterization data of
compounds, and biological activity studies.
Table S1: Physical properties of intermediate compounds 3a-3t.
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FIGURE LEGENDS
Figure 1: Chemical structures of xanthine analogues theophylline (i), caffeine (ii), isomeric
triazolopyridine-7-carboxylic acid derivatives (iii, iv) and newly designed compounds (4a-
4t).
Figure 2: Synthesis of 7-amino-2-aryl/heteroaryl-5-oxo-5,8-dihydro[1,2,4]triazolo[1,5-
a]pyridine-6-carbonitriles (4a-4t). Reagents and conditions: (i) acetic acid, EtOH, reflux; (ii)
CNCH2CN, piperidine, EtOH, reflux.
TABLE
Table 1: Binding affinity (Ki) of target compounds (4a-4t) at hA1, hA2A and hA3 ARs and
selectivity against hA2A and hA3 ARs.
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Table 1 Binding affinity (Ki) of target compounds (4a-4t) at hA1, hA2A and hA3 ARs and
selectivity against hA2A and hA3 ARs.
O
NC
N
N
N
R
H2N
(4a-4t)
Comp.
No.
R
Ki, µM*
hA1
Selectivity
a
b
c
hA2A
hA3
hA2A
hA1
/
hA3 / hA1
4a
4b
4c
4d
4e
4f
phenyl
0.076
(0.0694-0.0831)
0.115
(0.0826-0.160)
0.525
(0.499-0.552)
5.50
(4.99-6.05)
5.73
(4.81-6.83)
4.25
(4.12-4.39)
8.13
(7.13-9.28)
1.00
(0.859-11.8)
> 60
0.875
(0.83-0.922)
25.6
(17.7-36.9)
3.64
> 100
> 30
340
32
1300
> 260
> 110
> 11
> 17
> 24
> 7
4-hydroxy-phenyl
p-tolyl
(3.47-3.83)
> 100
> 100
> 100
> 100
> 60
> 60
> 190
> 18
> 17
> 24
> 7
4-N,N-dimethyl-phenyl
2-methoxy-phenyl
4-methoxy-phenyl
3,4-dimethoxy-phenyl
2,4-dimethoxy-phenyl
> 60
> 100
> 100
> 60
4g
4h
19.5
(11.5-32.9)
> 100
47.6
(42.4-53.5)
> 30
> 60
> 100
> 30
-
20
-
4i
4j
3,4,5-trimethoxy-phenyl
2-hydroxy-3-methoxy-phenyl
> 110
54
4k
4l
3,5-dimethoxy-4-hydroxy-phenyl 1.76
(1.54-2.01)
2.45
1.37
(1.01-1.86)
52.2
> 100
0.8
> 57
2
3-nitro-phenyl
4-nitro-phenyl
4-fluoro-phenyl
2-chloro-phenyl
4-chloro-phenyl
2,4-dichloro-phenyl
4-bromo-phenyl
2-pyridyl
5.49
(4.86-6.2)
21
(2.28-2.62)
7.29
(5.9-9.0)
0.413
(0.326-0.523)
0.294
(0.273-0.315)
0.832
(0.723-0.958)
0.732
(0.663-0.808)
(40.9-66.7)
4m
4n
4o
4p
4q
4r
4s
> 100
> 100
> 100
> 100
> 30
> 14
59
> 14
> 240
> 340
> 36
> 82
42
24.4
(15.9-37.4)
> 100
> 340
> 43
> 140
> 100
7
35.4
(30.5-40.9)
> 100
> 60
> 60
0.589
(0.481-0.721)
0.859
24.6
(21.6-27.9)
6.43
(3.25-12.7)
9.01
> 60
> 70
> 270
(0.755-0.976)
0.0510
4t
thiophene
13.9
(12.6-15.3)
176
(0.0451-0.0577)
(6.37-12.7)
* Ki values are given as geometric means with 95% confidence intervals in parentheses.
a Displacement of specific [3H]CCPA binding at human A1 AR expressed in CHO cells (n = 3).
b Displacement of specific [3H]NECA binding at human A2A AR expressed in CHO cells (n = 3).
c Displacement of specific [3H]HEMADO binding at human A3 AR expressed in CHO cells (n = 3).
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.