Development of novel pyridazinone-based adenosine receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2018.03.086

With the aim of finding new adenosine receptor (AR) ligands, a preliminary investigation focusing on the thieno[2,3-d]pyridazin-5(4H)-one scaffold was undertaken. The synthesized compounds 1–11 were evaluated for their binding at hA₁, hA_2A

Full text of DOI:10.1016/j.bmcl.2018.03.086

Accepted Manuscript
Development of novel pyridazinone-based adenosine receptor ligands
Daniela Catarzi, Flavia Varano, Matteo Falsini, Katia Varani, Fabrizio Vincenzi,
Silvia Pasquini, Diego Dal Ben, Vittoria Colotta
PII:
S0960-894X(18)30291-9
https://doi.org/10.1016/j.bmcl.2018.03.086
BMCL 25740
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 February 2018
28 March 2018
29 March 2018
Please cite this article as: Catarzi, D., Varano, F., Falsini, M., Varani, K., Vincenzi, F., Pasquini, S., Dal Ben, D.,
Colotta, V., Development of novel pyridazinone-based adenosine receptor ligands, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.086
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Development of novel pyridazinone-based adenosine receptor
ligands
Daniela Catarzi,a* Flavia Varano,a Matteo Falsini,a Katia Varani,b Fabrizio Vincenzi,b Silvia
Pasquini,b Diego Dal Benc, Vittoria Colottaa
aDipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di
Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto
b
Fiorentino, Italy; Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli
c
Studi di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy; Scuola di Scienze del
Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, Via S. Agostino 1, 62032
Camerino (MC), Italy.
1

Abstract
With the aim of finding new adenosine receptor (AR) ligands, a preliminary investigation focusing
on the thieno[2,3-d]pyridazin-5(4H)-one scaffold was undertaken. The synthesized compounds 1-11
were evaluated for their binding at hA1, hA2A and hA3 ARs and efficacy at hA2B subtype in order to
determine the affinity at the human adenosine receptor subtypes. Small structural changes on this
scaffold highly influenced affinity; compound 5 (5-ethyl-7-(thiazol-2-yl)thieno[2,3-d]pyridazin-
4(5H)-one) emerged as the best of this series. The simplicity of the synthetic process, the capability
of the scaffold to be easily decorated, together with the predicted ADME properties confirm the role
of these compounds as promising hits. A molecular docking investigation at the hA1AR crystal
structure was performed to rationalize the SARs of the herein reported thienopyridazinones.
Key words: G protein-coupled receptors, adenosine receptor ligands, thieno[2,3-d]pyridazin-5(4H)-
ones, ADME properties.
Abbreviations: AB-MECA: (N6-(4-aminobenzyl)-N-methylcarboxamidoadenosine); AC, adenylate
cyclase; AR, Adenosine Receptor; CHO, Chinese Hamster Ovary; DPCPX, 8-cyclopentyl-1,3-
dipropylxanthine; MV, Molecular Volume; NECA, 5′-(N-ethylcarboxamido)adenosine; TPSA,
Topological Polar Surface Area.
2

Adenosine is an endogenous nucleoside that plays pivotal roles in different physiological and
pathophysiological processes by triggering specific cell-surface receptors both in the central
nervous system (CNS) and in the periphery. The adenosine receptors (ARs) are four different
subtypes of G protein-coupled receptors (GPCRs) classified as A1, A2A, A2B and A3 on the basis of
their tissue localization, respective coupling to adenylate cyclase (AC), and specific
pharmacological criteria.1,2 ARs are widely distributed in the body and are expressed with different
density in the various tissues. The classical transduction intracellular pathways associated with AR
stimulation are inhibition, via Gi/o protein (A1 and A3 subtypes), or activation, via Gs protein (A2A
and A2B receptors), of adenylate cyclase (AC)1. More recently, other second messenger systems
have been described as relevant for AR signaling.1,3,4 Due to the widespread distribution of ARs1,5
and to the ubiquity of the natural ligand adenosine, selective modulation of each receptor subtype
could be exploited for therapeutic advantages. ARs have recently been recognized as novel and
potential drug targets for therapeutic intervention in many pathological conditions such as cardiac
and cerebral ischemia, asthma, renal failure, CNS disorders, inflammatory and neurodegenerative
diseases.6 Over the years, many ligands, agonists as well as antagonists, have been identified for the
ARs that, in turn, have been extensively characterized.6 In fact, a large number of selective AR
antagonists belonging to different chemical classes have been developed, leading to the
identification of several recurring structural features to define the affinity or efficacy of a ligand for
the target receptor subtype.6,7 Although the considerable number of AR ligands have been
discovered and clinically evaluated, only a few have been approved by the FDA. Moreover, some
phase I studies were interrupted due to low absorption, short half-life or other correlated
limitations.6,7 Thus, recent attention has focused on preliminary detection of pharmacokinetic
(ADME) properties whose optimization should be pursued together with the improvement of
potency.8,9 For all these reasons, there is a great deal of interest in the search for new chemical
entities (NCEs) using privileged structures as a support approach. In fact, the use of privileged
framework in drug discovery has proven to be an effective strategy for the generation of novel leads
3

and optimization processes.10 The pyridazin-3(2H)-one has been recognized as a privileged
structure for the design of novel compounds with potential pharmacological interest.11 In particular,
fruitful medicinal chemistry approaches using this core skeleton have been performed in the field of
AR ligands allowing discovery of some NCEs (series A-C, Figure 1A).12-14
Moreover, it is important to mention that the pyridazinone moiety is also present in both agonists
and antagonists of ARs as a versatile substituent in critical side chains (series D-F, Figure 1B).15-17
With this in mind, we have embarked on a project focused on the discovery of NCEs that
incorporate the pyridazin-3(2H)-one substructure. In the past, we identified the phthalazin-1(2H)-
one scaffold as a new decorable core skeleton for the design of potent and selective human (h)
A3AR antagonists.12 These studies were a part of a wider research program focused on the
development of new potent and selective adenosine receptor ligands belonging to different chemical
classes.12,18-27 Recently, our attention has been devoted to the thieno[2,3-d]pyridazin-5(4H)-one
scaffold that, to our knowledge, has never been functionalized for the design of AR ligands.
Moreover, this scaffold seems to be an attractive synthetic building block to perform a wide array of
structural modifications, allowing an easy synthesis of potential AR ligands characterized by good
yields. Thus, we have started a pilot study to investigate the versatility of the thieno[2,3-
d]pyridazin-5(4H)-one core as the basis of new compounds binding the ARs. The currently reported
thienopyridazinone derivatives (compounds 1-11, Figure 1C) possess, in addition to a bicyclic core
typical of AR ligands, structural similarities with a series of thieno[3,2-d]pyrimidines (series G,
Figure 1C) reported by Gillespie R. J. et al28 as A2A AR antagonists. A partial overlapping of the
critical features can be found also for the series of differently anellated thienopyridazinones
reported by Ferguson G. N. et al.14 (series C, Figure 1A) as A1AR antagonists able to recognize the
receptor allosteric site too. The R2, R5 and R7 positions of the thieno[2,3-d]pyridazin-5(4H)-one
framework were explored by designing compounds 1-11.
4

Figure 1. (A) Pyridazinone-based and (B) pyridazinone-containing adenosine receptor ligands. (C)
Currently reported thieno[2,3-d]pyridazin-4(5H)one derivatives and the structurally related
thieno[3,2-d]pyrimidine series.
O
O
O
R6
H2N
S
R2
N
N
N
N
N
N
N
N
N
R1
HN
O
C
B
R9
RO
O
R4
A
R1 = (hetero)aryl, (cyclo)alkyl
R6 = H, alkyl
R9 = H, (hetero)aryl
R4 = (hetero)aryl
R = H, CH3
R2 = aryl
(A)
R
N
N
N
O
H
R1
O
R
O
N
N
N
(CH2)n
(CH2)n
N
N
N
N
N
N
HN
R1
R3
R
O
N
N
D
O
E
F
N
N
(B)
Rib
O
R5
R5
N
N
N
R2
N
S
S
R7
R7
1-11
Series G
R2 = H, phenyl
R5 = alkyl, NH2
R7 = ethyl, (hetero)aryl
R5 = alkyl, NH2, NHEt,
N(CH3)2, OCH3, Cl,
NHCH2CH2OH
R7 = (hetero)aryl
(C)
Synthesis, pharmacological evaluation for the binding at hA1, hA2A and hA3 ARs, and efficacy at
hA2B subtype of the synthesized compounds, together with the assessment of their theoretical
ADME properties were the guidelines in this preliminary drug discovery investigation.
The synthetic pathways which yielded compounds 1-1129 and the relative intermediates are
illustrated in Schemes 1, 2. Litiation of commercially available thiophene-3-carboxylic acid
5

followed by (hetero)arylaldehyde addition provided the suitable substituted 2-
hydroxymethylthiophene-3-carboxylic acid which was not isolated (Scheme 1).
Scheme 1.
O
O
O
a
OH
OH
OH
OH
S
S
O
S
R7
R7
12-15
b
R5
cmpd
O
O
R5
C2H5
1, 2, 4, 5
N
N
NH
N
c
CH(CH3)2
3
S
S
R7
R7
NH2
6-9
1-9
16-19
R7
phenyl
cmpd
12, 16, 1, 6
2-pyridinyl
2-thienyl
13, 17, 2, 3, 7
14, 18, 4, 8
15, 19, 5, 9
2-thiazolyl
Reagent and conditions. a) i) R7CHO, LDA, anhydrous THF, rt, nitrogen atmosphere; ii) KMnO4;
b) NH2NH2•xH2O, EtOH, reflux; c) R5Br, NaH, anhydrous DMF, rt (compound 1-5) or
NH2OSO3H, NaH, anhydrous DMF, rt, nitrogen atmosphere.
Oxidation of the alcohol function with KMnO4 generated the ketoacids 12-15,30-32 which were
reacted with hydrazine hydrate in ethanol at reflux to yield the 7-substituted[2,3-d]pyridazin-4(5H)-
ones 16-19.29,32-33 The final targets 1-929 were synthesized following two different procedures as
follows. The 5-alkyl substituted derivatives 1-5 and the corresponding 5-N-amino-compounds 6-929
were obtained by reacting 16-1929,32-33 with NaH followed by addition respectively of the opportune
alkylbromide or hydroxylamine-O-sulfonic acid in DMF at room temperature. Both the 5,7-
6

diethylthieno[2,3-d]pyridazin-4(5H)-one 10 and 5,7-diethyl-2-phenylthieno[2,3-d]pyridazin-4(5H)-
one 11 were obtained following the procedure reported in Scheme 2.
Scheme 2.
O
O
O
O
a
OH
NH
N
O
b
OH
N
+
S
S
O
S
21
20
c
O
O
O
e
N
N
d
N
N
N
N
Br
S
S
S
10
22
11
Reagent and conditions. a) LDA, anhydrous THF, rt, nitrogen atmosphere; b) NH2NH2•xH2O,
EtOH, reflux; c) EtBr, NaH, anhydrous DMF, rt, nitrogen atmosphere; d) Br2, CH3COOH/H2O, 70
°C nitrogen atmosphere; e) C6H5B(OH)2, tetrakis, Na2CO3, DME/H2O, reflux, nitrogen atmosphere.
By reacting thiophene-3-carboxylic acid with LDA in anhydrous tetrahydrofuran followed by
addition of commercially available N-methoxy-N-methylpropanamide afforded the desired ketoacid
intermediate 20.34 The latter was cyclized with hydrazine hydrate to yield the pyridazinone 21
which was alkylated at 5-position with ethylbromide to give compound 10. Selective bromination
of the thienopyridazinone moiety34 of 10 at position 2 with bromine in aqueous acid acetic solution
at 70 °C produced the corresponding 2-bromo derivative 22 which was reacted with phenylboronic
acid in alkaline medium in the presence of tetrakis to afford the final compound 11.
All the reported compounds 1-11 were tested for their ability to displace specific [3H]DPCPX ([3H]-
8-cyclopentyl-1,3-dipropylxanthine),
[3H]ZM241385
( 4-(2-[7-Amino-2-(2-
furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) and [125I]AB-MECA ([125I]-N6-(4-
7

aminobenzyl)-N-methylcarboxamidoadenosine) binding from hA1, hA2A and hA3 receptors,
respectively. Potency of the compounds at the hA2B subtype was investigated by measuring their
inhibitory effect on 200 nM NECA (5’-N-ethylcarboxamidoadenosine)-stimulated cAMP levels in
Chinese Hamster Ovary (CHO) cells transfected with the hA2B receptor.27 The affinity data
(expressed as Ki) reported in Table 1 indicate that we have produced some new AR ligands
endowed with modest affinity for the hA1 subtype. None of the herein reported compounds 1-11
shows any activity at the hA2BAR since, in the cAMP experiments, the percentage of inhibition
(I%) of each is equal only to 1%. To a lesser extent, this can also be applied to the results obtained
in hA2A and hA3 AR binding experiments even though some compounds (1, 2, 5, 11 and 19) exhibit
Ki values in the high micromolar range. Affinity values in this range have also been found for the
A1 subtype, compounds 1, 7-9 and 19 showing 1153 < Ki (µM) < 3721. In contrast, compounds 2-5
present the highest affinity for the hA1AR, the Ki values ranging from 912 to 113 nM. Despite the
low or moderate affinity of the majority of the reported derivatives these data are encouraging and
suggest that the thieno[3,2-d]pyridazin-5(4H)-one ring system can be considered as a new scaffold
for the development of new AR ligands. Starting from the 7-phenyl-substituted derivative 1, better
results were obtained when 7-position was functionalized with a heteroaromatic substituent (see
compounds 2-5 for comparison) that exerts the maximum profitable effect for the binding at A1AR
when it is a thiazol-2-yl moiety (derivative 5). Other than position 7 of the thienopyridazinone
scaffold, also R5 substituent was modified by replacement of the ethyl group of 2 with the more
hindering isopropyl substituent leading to derivative 3 that maintains the hA1AR affinity in the
same range. No influence was observed on the other ARs. In contrast, introduction at position 5 of a
primary amino group produced a reduction of the hA1 binding affinity (compounds 6-9).
8

Table 1. Binding affinity (Ki) at hA1, hA2A and hA3 ARs.
O
R5
R5
N
R2
N
R2
N
N
S
S
R7
R7
1-11,19
G1,G2
Binding experimentsa
Ki(nM) or I%
b
c
d
R2
H
R5
C2H5
C2H5
CH(CH3)2
C2H5
C2H5
H
R7
hA1
hA2A
hA3
phenyl
2665 ± 214
782 ± 67
912 ± 82
724 ± 64
113 ± 12
1153 ± 95
16%
17%
2869 ± 238
2%
2590 ± 191
1%
1
2
H
2-pyridyl
2-pyridyl
2-thienyl
H
2%
3
H
1%
1%
4
H
2-thiazolyl
2-thiazolyl
phenyl
2487 ± 187
3392 ± 247
12%
2928 ± 211
1%
5
H
19
6e
7
H
NH2
1%
H
NH2
2-pyridyl
2-thienyl
2-thiazolyl
C2H5
3721 ± 238
3535 ± 305
2874 ± 244
3%
1%
1%
H
NH2
1%
1%
8
H
NH2
15%
5%
9
H
C2H5
C2H5
C2H5
NH2
7%
14%
10%
1813
N.Tf
10
11
G1f
G2f
C6H5
-
C2H5
4%
3141 ± 269
1.6
2-thiazolyl
2-thiazolyl
388
-
675
35
a Ki values are means ± SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) is
determined at 1µM concentration of the tested compounds.
b Displacement of specific [3H]DPCPX competition binding to hA1CHO cells.
c Displacement of specific [3H]ZM241385 competition binding to hA2ACHO cells.
d Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
eReference 29.
fReference 28.
9

The same applies also to intermediate 19, formally obtained by elimination of the R5 substituent
from compound 5. If compared to the thienopyrimidine derivatives (series G, Figure 1C), the
thieno[3,2-d]pyridazin-5(4H)-ones 1-9 explored herein seem to maintain similar size and shape and
also analogue structural characteristics that could be important for the binding at ARs. In particular,
in addition to nature and position of the R5 and R7 substituents on both the scaffolds, also the
hydrogen bond acceptor atoms have been conserved at similar positions. In fact, the 4-oxo function
of the thienopyridazinone compounds could be considered to replace the N1 atom of the lead series
G.28 While the 5N-ethyl substituted derivatives 2, 4-5 maintain comparable hA1 binding affinity
with respect to G1, the analogue 5N-amino compounds 6-9 do not recognize this AR subtype or fit
it little (compare to G2). However, considering the close structural analogy between the two series,
the most unexpected result was the great difference in binding data for the hA2AAR since only
thienopyridazinones 2, 5 and 19 bind with low affinity at this subtype, most of them showing a
percentage of inhibition below 50%. To further explore the SAR of this new scaffold, the
heteroaromatic ring at position 7 was then replaced with an ethyl group in order to evaluate the
requirements at this position. Compound 10 was prepared but it was ineffective at all the ARs. An
important goal was synthesis of the 2-phenyl-substituted compound 11 in which similarities with
the well-known AR antagonists, i.e. 3,5-disubstituted-8-arylxanthine derivatives, can be
recognized.6,7 This modification was performed with the aim of exploring the versatility of the
thienopyridazinone as building block for future modifications at the level of the condensed
thiophene ring. Despite the low hA2AAR affinity of derivative 11, the result opened the door to
rational future modification at position 2 of the present scaffold.
A molecular docking investigation was performed to analyze the binding affinity data of the herein
reported thienopyridazinone derivatives. For this task, the recently reported crystal structure of the
hA1 receptor covalently bound to the xanthine-based ligand DU172(hA1 pKi = 7.4; pdb code:
5UEN; 3.2-Å resolution)35 was refined within MOE software36 and employed as protein target. For
this purpose, the covalently bound ligand DU172 and the engineered segment bRILwere removed.
10

Moreover, the IL3 missing loop was restored and the wild type receptor sequence changed by the
presence of the N159A mutation. Docking analyses were performed with CCDC Gold software.37
Figure 2. Docking studies of the synthesized derivatives. (A). Compound 5 is shown, together with
the key A1 residues for the ligand interaction. (B). Surface representation of the binding cavity,
where light-to-dark regions indicate increasing polarity of the cavity.
The lowest score and most populated kind of docking conformations presents the
thienopyridazinone scaffold located in the binding cavity between the Phe171 (EL2) and the
11

Leu2506.51 side chains (see compound 5, Figure 2). The carbonyl group makes a polar interaction
with the Asn2546.55 side chain, and this appears as the key ligand-target interaction for this series of
compounds. As indicated above, the 4-oxo function is considered to replace the N1 atom of the lead
series G. Docking results confirm these data as the G compounds present a binding arrangement
similar to that of the the newly synthesized derivatives, with the N1 atom providing a polar contact
with the Asn2546.55 side chain.
The 5-alkyl group is located between residues of TM3 (Val873.32 and Leu883.33), TM5 (Met1805.38),
and TM6 (Trp2476.48) helices. Docking results suggest that the hydrophobic TM3-TM5-TM6 sub-
pocket, in which the 5-ethyl group of 5 is inserted, was unsuitable for receiving the polar 5-amino
of derivative 9. The 7-substituent gets oriented toward residues of TM2 (Ala662.61), TM3
(Val873.32), and TM7 (Thr2787.43) domains. The slightly lower volume of the 7-heteroaryl moiety of
compounds 4 and 5 with respect to that of the 7-phenyl (derivative 1) and the 7-(2-pyridinyl) group
(compound 2) appears to provide a better interaction with the receptor cavity. Also the polarity of
the 7-heteroaryl group seems to play a positive effect in the receptor-ligand interaction (compare
compounds 2 and 5 to 1 and 5, respectively).
The potential use of derivatives belonging to this series as therapeutic agents mostly depends on
their pharmacokinetics and pharmacodynamics. The pharmacokinetic phase includes absorption,
distribution, metabolism and excretion (ADME) of the studied compounds. Therefore, preliminary
data for theoretical ADME profiles of the newly synthesized compounds were determined (Table 2)
with properties calculated using the methodology developed by Molinspiration property program.
Log P, expressed as the octanol-water partition coefficient, was calculated as a sum of fragment-
based contributions and correction factors.38 Molecular Volume (MV) and Topological Surface
Area (TPSA) were calculated as a sum of fragment contributions, the latter following the
methodology by Ertl et al.39 From the data obtained, it can be observed that no violation of
Lipinski’s rule (molecular weight (MW) < 500; Log P < 5; number of H-bond donors (HBD) < 5,
and acceptors (HBA) < 10) was found, making derivatives 1-5, 9, 11, 19 promising leads.39 Thus,
12

one can notice that all the herein reported compounds possess log P values compatible with those
required to cross membranes. Moreover, TPSA is also found to be as good for potential
therapeutics40 and also MV values are in agreement.
Table 2. Prediction of the molecular properties for selected thieno[2,3-d]pyridazin-5(4H)-one
derivatives.
MWa
(g/mol)
256.33
HBAb
HBDc
VMd
(Å3)
223.71
TPSAe
(Å2)
34.9
LogP
3.06
1.91
2.27
2.84
2.11
1.67
1.12
4.10
3
4
4
3
4
4
5
3
0
0
0
0
0
1
2
0
1
2
257.32
271.35
262.36
263.35
235.29
250.31
284.38
219.56
236.14
214.42
210.27
176.52
188.19
257.07
47.79
47.79
34.90
47.79
58.65
73.81
34.90
3
4
5
19
9
11
aMolecula Weight; bHBA: number of hydrogen-bond acceptors; cHBD: number of hydrogen-bond
donors; dMV: Molecular Volume; eTPSA: Topological Polar Surface Area.
These parameters have been shown to be very good descriptors for characterizing drug absorption,
including intestinal absorption and blood brain barrier (BBB) penetration.
Thus, the thieno[3,2-d]pyridazin-5(4H)-one scaffold proved to have potential for the design of
novel AR ligands. In fact, compound 5 emerged as the best of this series and can be considered a
lead. Also the simplicity of the synthetic process and the capability of the scaffold to be decorated
confirm this heteroaromatic core as a privileged structure. The theoretical evaluation of ADME
properties confirms the role of these compounds as promising hits. The optimization process is
currently in progress, having chosen as the most important targets i) insertion of a suitable spacer
between the heteroaryl substituent at R7 and the scaffold; ii) introduction of substituents with
13

different steric and electronic properties at position 5; iii) acylation of the amine group at R5; and
not less important, iv) introduction of aryl and aralkyl substituents at position 2 and/or 3.
ASSOCIATED CONTENT
Supporting information
Chemical procedures, characterization and combustion analysis data of the newly synthesized
compounds. Pharmacological assay methods.
Acknowledgments.
The work was financially supported by the Italian Ministry for University and Research (MIUR,
PRIN 2010-2011, 20103W4779_004 project) and the University of Florence.
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Graphical abstract
20

Highlights
 the pyridazin-3(2H)-one scaffold was evaluated as a privileged scaffold
 thieno[2,3-d]pyridazin-5(4H)-one derivatives were developed as novel adenosine receptor ligands
 some compounds showed micromolar affinity for the hA1AR
 preliminary data for theoretical ADME profiles of the synthesized compounds were determined
 molecular modelling studies were made to rationalize the SARs of the thienopyridazinones
21