8-Benzylaminoxanthine scaffold variations for selective ligands acting on adenosine A2A receptors. Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.104033

A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position (benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving affinity and selectivity for the A_2A AR. The compounds were characterized by radioligand binding assays, and our study resulted in the development of the potent A_2A AR ligands including 8-((6-chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; K_i human A_2AAR: 68.5 nM) and 8-((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; K_i human A_2AAR: 71.1 nM). Moreover, dual A₁/A_2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine derivatives. Compound 14b displayed K_i values of 52.2 nM for the A₁AR and 167 nM for the A_2AAR. Selected A_2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the developed 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and selective A_2A ligands. Compound 12d with high selectivity for ARs, especially for the A_2AAR subtype, evaluated in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to display high selectivity and may therefore be of high interest for further development as drugs for treating cancer or neurodegenerative diseases.

Full text of DOI:10.1016/j.bioorg.2020.104033

BioorganicChemistry101(2020)104033
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
8-Benzylaminoxanthine scaffold variations for selective ligands acting on
adenosine A2A receptors. Design, synthesis and biological evaluation
Michał Załuskia, Jakub Schabikowskia, Piotr Jaśkob, Adrian Bryłab, Agnieszka Olejarz-Macieja,
Maria Kaletaa, Monika Głuch-Lutwinc, Andreas Brockmannd, Sonja Hinzd, Małgorzata Zygmuntb,
Kamil Kudera, Gniewomir Latacza, Christin Vielmuthd, Christa E. Müllerd,
⁎
Katarzyna Kieć-Kononowicza,
a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30688 Kraków, Poland
b Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30688 Kraków, Poland
c Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30688 Kraków, Poland
d PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction
with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position
(benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving
affinity and selectivity for the A2A AR. The compounds were characterized by radioligand binding assays, and
our study resulted in the development of the potent A2A AR ligands including 8-((6-chloro-2-fluoro-3-methox-
ybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; Ki human A2AAR: 68.5 nM) and 8-
((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; Ki human A2AAR:
71.1 nM). Moreover, dual A1/A2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine de-
rivatives. Compound 14b displayed Ki values of 52.2 nM for the A1AR and 167 nM for the A2AAR. Selected
Adenosine A2Areceptors
Anti-inflammatory activity
Metabolic stability
Molecular docking
Monoamine oxidase
Phosphodiesterase
Xanthine derivatives
A
2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of
phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the devel-
oped 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and
selective A2A ligands. Compound 12d with high selectivity for ARs, especially for the A2AAR subtype, evaluated
in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to
display high selectivity and may therefore be of high interest for further development as drugs for treating cancer
or neurodegenerative diseases.
1. Introduction
The A1- and A3ARs are coupled to Gi proteins resulting in inhibition of
adenylate cyclase, while A2A
- and A2BARs stimulate the enzyme
Adenosine is an endogenous extracellular signalling molecule in-
volved in the modulation of various physiological and pathological
processes. An important source of adenosine is its production by the
dephosphorylation of adenosine triphosphate (ATP), which is one of the
most abundant molecules in cells [1]. The extracellular functions of
adenosine are exerted through adenosine receptor (AR) activation. The
ARs belong to the G protein-coupled receptor (GPCR) family and are
subdivided into four subtypes: A1, A2A, A2B, A3, characterized by cou-
pling to various G protein isoforms, different distribution in tissues and
activation by different extracellular concentrations of adenosine [2].
through coupling to Gs proteins. Nevertheless, other second messenger
pathways can also be involved in intracellular signal transmission.
Their widespread distribution, as well as various cellular responses
mediated by modulation of ARs, and, in particular, their roles in dis-
eases, make them potential biological targets for therapeutic applica-
tion of (un)selective ligands for the different subtypes [3,4].
A
2AAR ligands have been investigated in inflammatory, neurolo-
gical and cardiovascular disorders, among others. High expression of
the A2AAR was detected in leukocytes, thymus, spleen, blood platelets
and olfactory bulb [5,6]. It is also enriched in heart, lungs, blood vessels
⁎ Corresponding author at: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków,
Poland.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kieć-Kononowicz).
https://doi.org/10.1016/j.bioorg.2020.104033
Received 24 March 2020; Received in revised form 1 June 2020; Accepted 15 June 2020
Availableonline19June2020
0045-2068/©2020PublishedbyElsevierInc.

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Fig. 1. Adenosine A2AAR antagonists with xanthine and non-xanthine scaffolds [12,15,44,45].
as well as some regions of the brain, especially in striatal nerves [6].
The activation of A2AARs mediates biological responses through Gs/Golf
proteins coupled with adenylate cyclase, and it may also modulate
extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal ki-
nase 1/2 (JNK1/2) [7,8].
inflammatory processes accompanied by oxidative stress, i.e. increased
production of free oxygen radicals [26–28]. According to this hypoth-
esis, a key role in the initiation of brain substantia nigra destruction
may be played by microglia [29–33]. The inflammation can cause an
activation of microglia, involving an increased production of numerous
inflammatory agents: cytokines (TNF-α, interleukin-1), free radicals,
and adhesion molecules, which lead to dopaminergic neuron apoptosis
[30,31]. Microglia activation is also associated with increased synthesis
of nitric oxide and prostaglandin PGJ2, whose formation is related to
cyclooxygenase-2 (COX-2) [34]. These changes promote dopamine
oxidation to its quinone derivative which interacts with protein amino
acid residues (cysteine, tyrosine, and lysine), leading to their irrever-
sible modification, and reduces cellular glutathione pool, which further
increases oxidative stress, causing the death of substantia nigra dopa-
minergic neurons [35]. Furthermore, activation of A2AARs decreases
dopaminergic transmission of D2 dopamine receptors, which is detri-
mental in PD [36,37]. Animal models indicated the potential utility of
A2A antagonists in decreasing the formation of β-amyloid plaques as
well as in recovering cognitive impairment in AD [38–40].
Predominantly, the response of peripheral tissues mediated by
A
2AARs is associated with protective effects in inflammation and is-
chemic processes. In those conditions, extracellular adenosine con-
centrations can rise from basic nanomolar to micromolar levels as a
response towards hypoxia or cells damage [4]. As those result, A2AARs
on different immune cells are activated modulating their activity, as
well as releasing of various pro- and anti- inflammatory molecules
[6,9]. The overall, modulation mediated by A2AARs results in a down-
regulation of immune responses [10,11]. Therefore, A2AAR agonists
show strong anti-inflammatory and immunosuppressive effects. Con-
versely, the blockade of A2AARs can result in immunostimulatory ef-
fects and has therefore been intensively explored with regard to im-
munotherapy of cancer. Preclinical studies of antitumor activity
mediated by a blockade of A2AARs provided promising outcomes sti-
mulating further development and investigation of A2AAR antagonists
[8,12,13].
Despite the above-mentioned effects mediated by A2AAR, there are
some hypotheses related to peripheral anti-inflammatory effect of A2A
AR antagonists [41,42]. Published papers provided ambiguous results
within A2AAR antagonists, that may exhibit anti-inflammatory and
antinociceptive effects in mice models [42]. However, those biological
effects require further evaluation. So, the functions of A2AARs in in-
flammatory processes and the protective effects of A2AAR agonists and
antagonists, in the brain and/or in the periphery, are still not fully
understood and need further investigations [11].
The intracellular response of A2AAR activation is mediated by an
increase in 3′,5′- cyclic adenosine monophosphate (cAMP) via adeny-
late cyclase activation. Therefore, modulation of intracellular level of
cAMP in immune cells can also have beneficial effects on antitumor
activity. Inhibition of adenylate cyclase isoform 7, as well as activation
of phosphodiesterase-4 (PDE-4), an enzyme that hydrolyzes in-
tracellular cAMP, are considered as further promising approaches to
protect immune effector cells from adenosine-mediated suppression of
antitumor functions [14]. The group of PDE isoforms include 11 fa-
milies of PDEs (PDE1 – PDE11) with various substrate preferences and
biological profiles, and, as mentioned above, modulation of selected
isoforms may display potential beneficial effects that enhance ther-
apeutic applications of ARs in inflammatory and neurodegenerative
processes [15,16].
Due to their immense therapeutic potential, novel molecules acting
through A2AARs are intensively explored in biological studies by med-
icinal chemists as selective antagonists or as multi target-directed li-
gands, which combine A2AAR blockade with activity on various biolo-
gical targets including enzymes and GPCRs to potentiate therapeutic
benefits [43,44]. (Fig. 1)
The chemical library of adenosine receptor antagonists consists of
xanthine derivatives and non-xanthine ligands. The xanthine-derived
antagonists comprise the naturally occurring methylxanthines theo-
phylline (1a), caffeine (1b) with weak subtype selectivity. They are
used as central stimulants, for the treatment of sleep apnea, for im-
proving lung functions in preterm babies, as antiasthmatics, and for the
treatment of pain in combination with analgesics [45,46].
Neurodegenerative diseases are another potential application for
A
2AAR antagonists. There is much evidence indicating their utility in
Parkinson disease (PD) as well as Alzheimer disease (AD) [17–20]. In
fact, the first A2AAR antagonist, istradefylline, has been approved in
Japan, and recently also in the USA for add-on therapy of PD [21,22].
The blockade of A2A can mediate neuroprotective effects, [23] and
appear to be beneficial for neuroinflammatory diseases [24,25]. One of
the hypotheses explaining the cause of PD is that degradation of do-
paminergic neurons of the substantia nigra are associated with
In the present study, our group developed small molecules con-
taining a xanthine scaffold as selective A2AAR antagonists with various
short alkyl substituents at N1, N3, and N7 position and substituted with
2

M. Załuski, et al.
BioorganicChemistry101(2020)104033
benzyl-amino or benzyloxy moieties in the 8-position. Based on our
previous results in the group of pyrimido[2,1f]purinedione derivatives,
we selected the most promising substitution patterns for high A2AAR
affinity creating a new chemical library of compounds derived from 8-
benzylaminoxanthine. A principal aim of our efforts was the evaluation
of various substitution patterns on the aromatic ring of the benzyl re-
sidue and also on the xanthine core with regard to affinity and se-
lectivity for the A2AAR subtype. The structure–activity relationship
analysis was additionally supported by molecular docking for human
hydroxymethylxanthine (16c). The selective alkylation in position N7
was performed using mild reaction conditions with diisopropylethyla-
mine (DIPEA) as a base, and the N1-position was alkylated by io-
doethane in the presence of K2CO3 and DMF at room temperature. 3,7-
Dimethyl-1-ethyl-8-hydroxymethylxanthine (16e) was treated with
PBr3 to replace the hydroxyl group by a bromine, which enabled the
direct conjugation with the appropriate benzylamine derivative in the
last synthetic step according to a previously described protocol with
some modifications [44].
A
2AAR. Selected products were tested for ancillary inhibitory activity
The structure of all new synthesized compounds was confirmed by
spectral analysis including 1H NMR, 13C NMR, and MS. The melting
points were determined for the new compounds. The purity of all tested
compounds was confirmed to be at least 95% using UPLC coupled to
UV/MS.
on the monoamineoxidases (MAO) MAO-A and MAO-B as well as some
isoforms of phosphodiesterase (PDE) as potential biological targets
since those targets share some mutual pharmacophoric features.
Moreover, the most active selective A2AAR ligand was firstly evaluated
in metabolic stability studies and then examined in in vivo biological
experiments including anti-inflammatory, anti–nociceptive and an-
algesic properties.
2.2. Biological evaluation
The final compounds were tested in radioligand binding assays to
evaluate their affinity for all four ARs subtypes. Human A1, A2A, A2B
and A3ARs were recombinantly expressed in Chinese hamster ovary
(CHO) or human embryonic kidney (HEK) cells. [3H]2-Chloro-N6-cy-
clopentyladenosine ([3H]CCPA) [49], [3H]3-(3-hydroxypropyl)-1-pro-
pargyl-7-methyl-8-(m-methoxystyryl)-xanthine ([3H]MSX-2) [6], [3H]
8-(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-pro-
2. Results
2.1. Chemistry
The synthetic strategy leading to the target structures is depicted in
Schemes 1, 2 and 3. For compounds 9a-h, 10a-g, 12a-j, 15a-e 8-bromo-
theophylline, -caffeine or –theobromine were used as starting material.
8-Br-1,3-diethylxanthine (13d) was obtained from 1,3-diethylurea
(13a) and cyanoacetic acid (13b) by a modified Traube synthesis ap-
proach.
pylxanthine ([3H]PSB-603) [50] and [3H]2-phenyl-8-ethyl-4-methyl-
(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purine-5-one ([3H]PSB-11)
[51], respectively, were used as radioligands in A1-, A2A-, A2B-, and
A3AR binding studies. Selected compounds were additionally evaluated
for their inhibitory potencies on human MAO-A, MAO-B, PDE-4B1 and
PDE-10A. In vivo tests for antinociceptive, anti-inflammatory and per-
ipheral analgesic properties were performed.
Oxidative bromination was applied for xanthine analogues ac-
cording to previously described procedures [47,48]. Subsequently, the
8-bromoxanthine derivatives (11, 13d) were alkylated in position N1 or
N7 by alkyl bromide (or chloride/iodide) in the presence of K2CO3 and
DMF as a solvent. The last step leading to the target compounds 9a-h,
10a-g, 11a-j, 14a-c was performed using microwave irradiation. The 8-
bromoxanthine derivatives were treated with the appropriate benzyla-
mine derivatives using triethylamine as a base and 1-propanol as a
solvent. Previously developed procedures of microwave irradiation
were applied in order to yield the final products which resulted in re-
duced solvent amount and reaction time compared to conventional
heating.
2.3. Structure-activity relationships at adenosine receptors
The presented library of xanthine derivatives combines the same
1,3,7- alkyl substituted xanthine core with a variety of aromatic re-
sidues in the 8-position. Therefore, the final compounds can be divided
into subgroups with common xanthine cores as depicted in Table 1
presenting their adenosine receptor affinities.
The most potent compounds included 12d (Ki, A2AAR = 68.5 nM)
and 12h (Ki, A2AAR = 71.1 nM) which belong to the series of 1-ethyl-
3,7-dimethylxanthine derivatives. Nevertheless, in each series, potent
molecules with affinity in the submicromolar range for the A2AAR were
The benzyloxy-substituted compounds 15a-e were synthesized in
analogy to the benzylamino-substituted derivatives but using benzyl
alcohols and different reaction conditions in the last step. As a base
sodium hydride was applied and DMF was used as a solvent with
conventional heating.
identified. Compounds 14b (Ki, A1AR
=
55.0 nM; Ki,
A
2AAR = 283 nM) and compound 14c (Ki, A1AR = 52.2 nM; Ki,
For compound 17 6-amino-1-methyluracil (16a) was used as
starting material, which was converted to 5,6-diamino-1-methyluracil
(16b) and subsequently conjugated with glycolic acid leading to 8-
A2AAR = 167 nM) showed dual affinity toward A1/A2A ARs with higher
affinity for the A1- than the A2AAR. Moreover, structure 12g displayed
moderate affinity for the A2AAR with ancillary potency at the A2BAR in
Scheme 1. Synthesis of 8-bromoxanthine derivatives; i, ii – K2CO3, DMF, alkyl bromide (or chloride/iodide).
3

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Scheme 2. Synthesis of final structures; i – benzylamine, Et3N, propanol, µW, ii – benzylalcohol, NaH, DMF.
the submicromolar range (Ki, A2AAR = 281 nM; Ki, A2BAR = 706 nM).
None of the final compounds displayed significant affinity for the A3AR.
The series of theophylline-based structures showed moderate affi-
nity for the A2AAR of approximately 1 µM (Ki value) with lower affinity
for the other AR subtypes. An exception was compound 9b with a meta-
bromobenzylamino residue in position 8 of the xanthine core displaying
additional moderate affinity for the A1AR (Ki, A1AR = 493 nM; Ki,
The best substitution pattern on the aromatic ring for high A2AAR af-
finity was identical to that observed for the caffeine analogues with the
following rank order of potencies: ortho > meta > para for mono-
substituted derivatives (halides, methoxy group). The crucial role of
ortho-substitution was also found in the group of di- and tri-substituted
benzyl derivatives. Structures containing an ortho-chlorine substituent
displayed high affinity for A2AAR even with other halogens or a
methoxy group in other positions. Interestingly, compound 12d con-
taining a 6-chloro-2-fluoro-3-methoxybenzylamino residue in the 8-
position showed similar affinity as its ortho-chlorobenzylamino-sub-
stituted analogue. Additionally, structures 12f and 12a with 2,6- or 2,4-
di-halogen substitutions of the aromatic ring displayed high affinity for
the A2AAR with Ki values of approximately 100 nM and good selectivity
versus the other AR subtypes.
A
2AAR = 718 nM). In this series, meta-substitution of the aromatic ring
appeared to be preferable for high A2AAR affinity. Compounds 9b, 9f
and 9g containing chlorine or bromine atoms in the meta-position dis-
played A2AAR affinity in the submicromolar range.
Higher potency and the highest selectivity for the A2AAR were found
within the caffeine-derived structures, while for almost all compounds
of this series A1- A2B-, and A3AR affinity was very low indicating high
A
2AAR selectivity. The preferred aromatic substitution was ortho-ha-
Replacement of the amine function in the benzylamino moiety by an
oxygen linker in the benzyloxy analogues belonging to the series of 1-
ethyl-3,7-dimethylxanthine derivatives resulted in a decrease in affinity
for the A2AAR while maintaining selectivity. The most potent com-
pound 15c (Ki, A2AAR = 185 nM) with an ortho-bromine substituent on
the aromatic ring was slightly (2-fold) more potent than the ortho-
chlorine analogue 15b (Ki, A2AAR = 445 nM). Nevertheless, comparing
structures 15b and 12 h, the exchange of the amine linker by an oxygen
atom decreased the affinity for the A2AAR significantly by about 6-fold.
A change of the AR subtype selectivity profile was observed for the
series 1,3-diethyl-7-methylxanthine derivatives. Within the series, a
crucial increase in A1AR affinity while maintaining high affinity the
logen- or -methoxy-benzyl moiety as in 10a, 10e and 10f. Compounds
10a and 10e containing a bromine atom or a methoxy group in the
ortho-position represented the structures with the highest A2AAR affi-
nity of the series with Ki values of 283 nM and 218 nM, respectively.
The A2AAR ligands with the highest affinity were found within the
series of 1-ethyl-3,7-dimethylxanthine derivatives. The compounds’
selectivity was slightly lower as compared to the caffeine derivatives,
nevertheless, it was still high with Ki values at A1-,A2B-, and A3ARs
of > 1 µM with one previously mentioned exception, compound 12g (1-
ethyl-8-((2-fluorobenzyl)amino)-3,7-dimethyl-3,7-dihydro-1H-purine-
2,6-dione) displaying dual A2A/A2BAR affinity in the nanomolor range.
Scheme 3. Synthesis of target compound 17; i-glycolic acid, NaOH; ii – DIPEA, DMF, iodomethane; iii –iodoethane, K2CO3, DMF; iv – PBr3, DCM, v – amine, DIPEA,
DCM.
4

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Table 1
Affinities of xanthine derivatives for adenosine receptors.
Compd
R1
A1 vs. [3H]CCPA
A2A vs. [3H]MSX-2
A2B vs. 3H]PSB-603
A3 vs. [3H]PSB-11
Ki
SEM (nM) (or % inhibition SEM at 1 µM)
1b caffeine
44 900
841
23 400
12
33 800
10 000
13 300
4700
2 istradefylline
N-7-Unsubstituted derivatives
9a
> 1000 (19
4)
1220
290
216
> 1000 (13
3)
> 1000 (27
4)
9b
9c
9d
9e
9f
493
157
718
> 1000 (37
> 1000 (34
> 1000 (15
> 1000 (4
9)
0)
3)
> 1000 (25
> 1000 (18
> 1000 (15
> 1000 (0
> 1000 (24
> 1000 (17
> 1000 (8
0)
2)
3)
> 1000 (40
> 1000 (23
> 1000 (38
> 1000 (31
> 1000 (39
> 1000 (24
2)
> 1000 (46
3)
3)
1380
1250
768
390
380
96
55
10)
9)
1)
2)
> 1000 (30
> 1000 (10
> 1000 (11
0)
2)
3)
4)
2)
9g
9h
4)
859
10)
> 1000 (31
6)
2)
N-7-Methylsubstituted derivatives
10a
10b
> 1000 (13
> 1000 (23
4)
5)
218
631
49 (79
2)
3)
> 1000 (-5
> 1000 (-2
3)
> 1000 (-7
> 1000 (-7
2)
45 (61
2)
2)
10c
10d
10e
10f
> 1000 (6
> 1000 (7
> 1000 (7
> 1000 (4
> 1000 (25
5)
682
789
401
283
552
70 (65
39 (58
8 (71
3)
5)
> 1000 (0
> 1000 (-3
> 1000 (0
> 1000 (10
> 1000 (12
2)
7)
4)
2)
> 1000 (3
> 1000 (3
> 1000 (7
> 1000 (-1
> 1000 (-1
3)
3)
1)
5)
12)
7)
5)
67 (77
68 (66
2)
2)
1)
1)
10g
7)
1)
1)
N-1-Ethylsubstituted derivatives
12a
> 1000 (31
10)
148
22 (91
1)
> 1000 (26
> 1000 (5
3)
12b
12c
12d
> 1000 (38
> 1000 (21
> 1000 (23
2)
571
419
68.5
271 (68
75 (77
5)
> 1000 (21
> 1000 (26
> 1000 (46
13)
1)
> 1000 (-14
> 1000 (9
21)
0)
6)
14)
10)
4)
15.5 (101
2)
3)
> 1000 (18
12e
12f
12g
12h
12i
> 1000 (15
> 1000 (36
> 1000 (15
> 1000 (22
> 1000 (39
11)
2)
1020
103
281
71.1
323
224
> 1000 (38
> 1000 (31
1)
0
> 1000 (22
> 1000 (10
> 1000 (35
> 1000 (14
> 1000 (8
1)
4)
5)
1)
32 (85
35 (76
5.8 (88
81 (73
4)
6)
7)
706
112 (44
5)
8)
5)
2)
> 1000 (44
> 1000 (31
10)
10)
6)
1)
(continued on next page)
5

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Table 1 (continued)
Compd
R1
A1 vs. [3H]CCPA
A2A vs. [3H]MSX-2
A2B vs. 3H]PSB-603
A3 vs. [3H]PSB-11
Ki
SEM (nM) (or % inhibition SEM at 1 µM)
12j
> 1000 (0
1)
> 1000 (39
5)
> 1000 (19
> 1000 (18
7)
4)
> 1000 (19
> 1000 (6
1)
N-1, N-3-Diethylsubstituted derivatives
14a
240
26 (64
3)
132
9 (99
9)
1)
3)
14b
14c
52.2
55.0
8.5 (86
2)
167
283
22 (83
18 (76
4)
> 1000 (33
> 1000 (22
12)
8)
> 1000 (8
12.3 (76
2)
4)
> 1000 (16
12)
8-Benzyloxy derivatives
15a
15b
> 1000 (9
1)
4)
> 1000 (8
3)
> 1000 (22
> 1000 (24
4)
> 1000 (-1
> 1000 (10
4)
3)
> 1000 (34
445
200
18)
15c
15d
> 1000 (31
> 1000 (37
5)
5)
185
14
> 1000 (23
> 1000 (9
1)
> 1000 (9
7)
1045
563
20)
> 1000 (14
1)
8-Benzylaminomethyl derivative
17
> 1000 (15
2)
> 1000 (28
3)
> 1000 (9
6)
> 1000 (-3
1)
A2AAR was discovered. Moreover, meta-substitution of the aromatic
responses, selected compounds were evaluated for their inhibitory po-
tency toward two isoforms of PDE (Table S2 and S3 of Supplementary
Data). PDE-4B1 and PDE-10A were examined in our biological studies
due to their potential role in inflammation and/or neurodegenerative
disorders being parallel with the application of A2AAR antagonists.
The selected compounds represented the most diverse structural
modifications within the synthesized chemical library. All tested
structures were only very weak PDE-10A and -4B1 inhibitors. The
percent inhibition was not higher than 12% for the most active struc-
ture in the highest tested concentration (10 µM).
ring by halogen had a slightly decreasing effect on the affinity for the
A
2AAR while remarkably improving A1AR affinity. Compounds 14b and
14c (Ki, A1AR
= 52.2 nM; Ki, A2AAR = 167 nM) and (Ki,
A1AR = 55 nM; Ki, A2AAR = 283 nM) were the most potent dual A1/
A2A AR ligands of the presented chemical library.
Structure 17 can be envisaged as an analogue of 12a with translo-
cated 2-fluoro-6-chlorobenzylamino residue more distant from the
xanthine core by an additional methylene linker in the 8-position. This
modification resulted in a significant drop of affinity for A2AAR. This
effect may suggest an important role of having a small methyl sub-
stituent in the N7-position, as well as some preferences in distance
between the aromatic ring in position 8 of the xanthine core with re-
gard to the compounds’ affinity for AR subtypes.
2.6. Docking to A2AAR crystal structure
Out of a number of available A2AAR crystal structures, paying at-
tention to well described proposed binding interactions of (annelated)
xanthines [48], for docking purposes we used the 3REY crystal struc-
ture in complex with XAC [53].
2.4. Inhibition of monoamine oxidase A and B
Selected final structures were tested for their inhibitory potency of
monoamine oxidase A and B. According to previously published lit-
erature, benzylamino-substituted derivatives of tricyclic xanthine de-
rivatives displayed inhibitory activity on MAO-B in the nanomolar
range, but were inactive on MAO-A [52]. Therefore, inhibition
screening on both, MAO-A and MAO-B, was performed to compare the
new library of 8-benzyl-(amino)/(oxy)-xanthine derivatives with the
tricyclic xanthines. The results showed very weak activity towards
MAO-A and MAO-B for the new xanthine derivatives with inhibition
percentage of significantly < 50% at the highest tested concentration
(1 µM). The results are collected in Table S1 of Supplementary Data.
They clearly indicate selectivity of the tested compounds for ARs versus
MAOs.
For all of the docked ligands, similar putative interaction patterns
can be found. The terminal amino group of ASN2536.55 is suggested to
form a key hydrogen bond with C6 carbonyl group of the purinedione
(superscript numbers denote the aminoacids according the Ballesteros-
Weinstein numbering system), that itself is located in a narrow pocket
formed by PHE168 (ECL2), that stabilizes the system through π-π
stacking and GLU169 (ECL2) from one side, and TM7 amino acids
MET2707.35 and ILE 2747.39 from the other side, likely displaying hy-
drophobic interactions with both of these amino acids. N1-alkyl sub-
stituents occupy a small hydrophobic sub-pocket formed by LEU2496.51
and MET1775.38 on sides, closed at the bottom by HIS2506.52. Halogen
substituted aminobenzyl fragment appear to be placed in a subpocket
between TM2 apex and EL2, stabilized by hydrogen bond between
amine group hydrogen and GLU169. However, in case of methoxy
substituted ligands (eg. 12e) a shift of the benzylamine fragment to-
wards hydrophobic cage of TYR91.35 and TYR2717.36 could be ob-
served. This “methoxy shift” in fact might be responsible for twice as
high A2AAR affinity of 12d, when compared to 12a – by allowing the
2.5. Inhibition of phosphodiesterases
According to the previously mentioned role of PDEs on intracellular
concentration of cAMP and thus on cAMP-mediated biological
6

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Fig. 2. Putative binding pose of 12a (blue) and 12d (golden) in orthosteric binding pocket of 3REY with corresponding ligand interaction diagram for 12d (left and
right panel respectively). ECL3 and partly TM7 residues were removed for better viewing clarity. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
halogen bond formation between chlorine atom and GLU169 (Fig. 2).
The stability of the calculated poses was evaluated by means of mole-
cular dynamics simulations. Receptor-ligand complexes appeared stable
through the whole 600 ps simulation, retaining the key interactions,
and the potential energy (U) of the atomic system at the level of
~1000 kcal/mol. Furthermore, during the simulation additional arene-
H interactions of the substituted benzene ring with SER672.65and/or
ILE2747.39 were observed.
the other evaluated biological targets (MAO-B, PDE-4B1 and -10A). The
compound was studied in selected animal models to estimate its anti-
inflammatory, antinociceptive and peripheral analgesic properties.
2.8.1. Antinociceptive activity in the formalin test
In the formalin test, which is a model of chronic pain induced by the
administration of 5% formalin solution, the evaluated 12d showed
antinociceptive activity (Table 2). 12d Shortened the licking/biting
time of the right hind paw of mice in response to the irritating chemical
stimulus.
2.7. Metabolic stability of compound 12d
The injection of formalin into the dorsal surface of the hind paw of a
mouse produces a biphasic nocifensive behavioral response, i.e. licking,
biting, flinching, or lifting of the injected paw. The acute (neurogenic)
nociceptive phase lasts for the first 5 min, and is followed by a period of
little activity during the next 10 min. The first phase of the test is di-
rectly associated with the stimulation of nociceptors and the develop-
ment of neurogenic inflammation. The second (late) phase occurs be-
tween 15 and 30 min after formalin injection. The second phase is
dependent on peripheral inflammation and central sensitization of pain.
Since this phase reflects the activation of inflammatory processes, the
compounds active in this phase of the experiment have also anti-in-
flammatory effect.
To determine the metabolic stability of 12d in rodents the com-
pound was incubated with mouse (MLMs) and rat liver microsomes
(RLMs) for 120 min at 37 °C. The UPLC analyses showed very high
metabolic stability of 12d, as 96.56 and 97.97% of the parent com-
pound remained in the reaction mixtures after incubation with MLMs or
RLMs, respectively (Fig. 3). Each performed reaction resulted in the
presence of only one metabolite, however depending on the used mi-
crosomes the different retention times were observed (5.02 min with
MLMs, and 4.80 min with RLMs) (Fig. 3). Indeed, the MS analyses
showed different molecular mass of obtained metabolites (m/
z = 382.20 (MLMs) and (m/z = 368.18 (RLMs) (Fig. S1 supplementary
material). The prediction of 12d metabolism performed by MetaSite
6.0.1 showed methoxy- substituent as the most probably site of bio-
transformation (Fig. 4).
12d shortened the licking/biting time of the right hind paw of mice
only in phase II of the test (15–30 min) (Table 2). The effect was con-
centration-dependent, and the calculated ED50 value for this compound
is 31.4 (21.3–42.6) mg/kg b.w. 12d showed about 4-fold higher activity
than reference compound acetyl salicylic acid (ASA) and similar as
xanthine derivative HC-030031 known TRAP1 antagonists [54]. ASA
(administered in doses of 50, 100 and 200 mg/kg), attenuated statis-
tically significant pain responses only in the late phases of the formalin
test. The calculated ED50 value is 126.3 (90.2 – 147.1) mg/kg. While
HC-030031 (administered in doses of 20, 40 and 100 mg/kg), atte-
nuated statistically significant pain responses in the late phases of the
formalin test. The calculated ED50 value is 33.8 mg/kg. In conclusion,
the tested compound demonstrated stronger antinociceptive and anti-
inflammatory effect than ASA.
This in silico result correlates with the molecular mass of demethy-
lated metabolite obtained by MLMs. The structure of this metabolite
was generated in silico with very high probability and was presented at
Fig. 5A. The molecular mass of the metabolite obtained by RLMs sug-
gests that N-deethylation at the xanthine moiety occurred. This meta-
bolite was also predicted in silico (Fig. 5B). In conclusion, 12d was
found as very metabolically stable compound in both used species with
different main metabolic pathways: demethylation in mice and N-dee-
thylation at xanthine moiety in rats.
2.8. Preliminary in vivo studies
Compound 12d was selected for further biological in vivo tests being
the most potent A2AAR ligand within the presented compound library
while showing high AR subtype selectivity as well as selectivity versus
2.8.2. Anti-inflammatory (antiedematous) effect in the carrageenan-
induced edema model
12d was administered at a dose of 20 mg/kg body weight.
7

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Fig. 3. UPLC spectra after 120 min incubation of compound 12d with MLMs (A) and RLMs (B).
Ketoprofen was used as a reference compound (Table 3).
of an irritating substance, like phenylbenzoquinone.
The carrageenan test was used to evaluate the anti-inflammatory
effect; injection of carrageenan into the hind paw of an animal induces
a long-lasting edema. The aim of the present study was to evaluate the
anti-inflammatory (antiedematous) activity of 12d. Ketoprofen ad-
ministered ip at a dose of 20 mg/kg b.w., inhibited edema formation by
23.3%, 54.2% and 66.0% in three consecutive hours of the experiment,
respectively.
Structure 12d showed analgesic activity (Table 4). Acetylsalicylic
acid (ASA) was used as a reference compound.
Statistically significant decrease in the number of writhings was
observed by 12d, showing an analgesic effect; at doses of 5 mg/kg,
10 mg/kg and 20 mg/kg. It reduced the number of writhes in response
to phenylbenzoquinone by 32.9%, 54.9% and 82.0%. The ED50 value
obtained for 12d was 8.1 (6.5–10.2) mg/kg b.w. being more potent
than that for the reference compound ASA with an ED50 = 39.1
(26.3–49.8) mg/kg b.w. Compound 12d demonstrated stronger anti-
nociceptive effect than ASA.
12d decreased statistically significantly the volume of edema in-
duced by carrageenan injection into the hind paw of rats. A statistically
significant effect was observed in the 2nd and 3rd hour of the experi-
ment, inhibiting edema development by 43.8% and 52.8%.
3. Discussion
2.8.3. Peripheral analgesic activity of 12d in the writhing test
The writhing test is a chemical method used to induce pain of
peripheral origin by injection of an irritant like phenylbenzoquinone or
acetic acid in mice. Analgesic activity of the test compound is inferred
from a decrease in the frequency of writhings. This test consists of in-
traperitoneal injection of the chemical irritant followed by subsequent
counting of “writhes”, i.e. characteristic contractions of abdominal
muscles accompanied by a hind limb extensor motion. This test detects
peripheral analgesic activity; however, some psychoactive agents (in-
cluding clonidine and haloperidol) also show activity in this test.
Compounds with anti-inflammatory properties, such as non-steroidal
anti-inflammatory drugs (NSAIDs) show a significant activity in this
assay, namely they abolish the reflex stimulated by the administration
In this studies, we investigated a novel library of xanthine deriva-
tives and evaluated their preliminary biological profile, looking for
potent and selective A2AAR ligands. The explored scaffold of 8-ben-
zylxanthine was chosen as closely related to N9-benzylpyrimido[2,1-f]
purinedione derivatives, which were originally designed as bioisosteric
analogues of 8-styrylxanthine to overcome some stability drawbacks
regarding to styryl fragment.[55,56] The previously reported tricyclic
compounds presented dual AR/MAO-B activity with emphasis on MAO-
B inhibitory potency.[52] Optimizing the chemical scaffold toward
A
2AAR activity, the third heterocyclic ring was removed from N9-ben-
zylpyrimido[2,1-f]purinedione core providing a novel library of xan-
thine derivatives. As consequence of structural modification, the
8

M. Załuski, et al.
BioorganicChemistry101(2020)104033
ortho and meta position of benzyl moiety. The 10-fold increase of A2AAR
affinity was obtained for the most potent ligand 12d regarding to its
tricyclic analogue previously published [52]. The improvement of
A
2AAR selectivity for 12d included AR subtypes as well as MAO-B
presented classical target for xanthine derivatives.
Although, the A2AAR antagonists are mostly investigated as anti-
parkinsonian and antitumor agents as mentioned above, there are some
literature evidence that A2AAR antagonists included MSX-3 and un-
selective caffeine provided anti-inflammatory, antinociceptive and/or
analgesic effects in animals models. Moreover, the A2AAR knockout
mice show a hypoalgesic phenotype [42,57,58].
Therefore, compound 12d as the potent and selective A2A ligand was
investigated in animal models for anti-inflammatory, antinociceptive and
peripheral analgesic properties. Our in vivo studies provided outcomes,
which were compared to references NSAID such as ASA and ketoprofen.
The previous published data characterised those properties within the
group of potent and subtype-selective AR antagonists in inflammation and
hyperalgesia. The MSX-3, which was used as selective A2A antagonist
presented inhibition of formalin or carrageenan induced edema formation,
reduction of chemical irritant but not inflammatory pain, and a total block
of hyperalgesia in a limited dose range [57].
In the present study, the general effect of 12d was convergent with
effect of MSX-3. Moreover, 12d showed peripheral analgesic activity in
the writhing test. The antinociceptive activity was evaluated as de-
pendent on peripheral inflammation and central sensitization of pain
with 4-fold higher activity than reference compound ASA. Moreover,
anti-inflammatory effect was additionally detected for 12d in the car-
rageenan test. Considering the presented results and literature data, it
seems that A2AAR antagonists may display anti-inflammatory, anti-
nociceptive and/or analgesic effects. Moreover, presented biological
activities are probably mediated mostly by the selective blockade of
Fig. 4. The MetaSite 6.0.1. software prediction of the most probably sites of
compound 12d metabolism. The darker red color - the higher probability to be
involved in the metabolism pathway. The blue circle marked the site of com-
pound with the highest probability of metabolic bioconversion. (For inter-
pretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
designed compounds presented very low inhibitory potencies of MAO-B
and higher affinity for A2AAR comparing to the series of N9-benzyl-
pirymido[2,1-f]purinedione derivatives. These outcomes indicated im-
portant roles of the N7-substituent and the distance between the aro-
matic residue and the xanthine core on the ability to block MAO-B and
MAO-A. Moreover, the 8-benzylaminoxanthine scaffold seems to be
more tolerable for AR activity with preference of substitution pattern in
A
2AAR. The tested structure 12d was in vitro estimated as inactive for
another AR subtypes as well as other targets, the modulation of which
may produce similar anti-inflammatory response: MAO-B, PDE-4B1 and
PDE-10. The further investigation may provide more information and
potential utility of those effects related to therapeutically application of
A
2AAR antagonists.
Fig. 5. The in silico prediction of the most probable structure of confirmed in vitro demethylated 12d metabolite obtained by MLMs (A) and N-deethylated metabolite
obtained by RLMs (B).
9

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Table 2
The influence of 12d on the duration of pain reaction in the formalin test in mice.
Treatment
Dose [mg/kg]
Licking of the hind paw (sec)
Early phase (0–5 min)
Inhibition [%]
Late phase (15–30 min)
Inhibition [%]
Mean
46.2
44.8
48.8
46.5
63.8
62.0
58.5
59.8
97.0
71.0
73.8
82.8
SEM
Mean
181.2
45.0
SEM
5.6
Control
–
1.1
–
–
12d
40
30
20
–
0.5
3.1
3.4c
75.2
52.5
5.5
0.5
0.0
86.0
4.1c
3.3
0.6
0.0
171.3
120.8
40.6
Control
2.03
8.6
–
3.43
–
ASA
200
100
50
-
9.2
14.0b
9.5b
13.8
12.3
1.3c
66.3
50.0
10.0
–
4.5
14.3
12.4
–
60.3
6.3
108.7
158.3
13.9
Control
6.9
HC-030031[54]
100
40
20
4.1
26.8%
23.8%
14.6%
91.2%
51.7%
29.7%
5.31
4.82
76.5
9.0a
12.2
111.3
Data are presented as the means
SEM of 6–8 mice per group.
a
p < 0.05,
b
p < 0.01,
c
p < 0.001 vs. control.
Table 3
responsible for high A2AAR affinity. Interestingly, analogues containing
a 1,3-diethyl-7-methylxanthine scaffold in which the methyl substituent
in position N3 was replaced by an ethyl moiety showed high affinity for
the A1AR while maintaining A2AAR affinity. Compounds 14a-c dis-
played a dual A1/A2AAR affinity profile.
Anti-inflammatory (antiedematous) effect of 12d in the carrageenan-induced
paw edema test.
Treatment
Dose [mg/kg]
Change in edema volume [ml]
1 h
2 h
3 h
Selected compounds were tested for their inhibitory potency on
MAO-A and MAO-B, as well as isoforms 4B1 and 10A of PDE. The po-
tency to inhibit isoforms of PDE and MAO was evaluated for selected
compounds showing that the tested molecules all were virtually in-
active and therefore showing high selectivity for the ARs.
Control
12d
–
0.42
0.36
0.32
0.08
0.05
0.03
0.89
0.50
0.41
0.11
1.25
0.59
0.43
0.06
20
20
0.05a
0.04a
0.06b
0.03b
Ketoprofen
Data are presented as the means
SEM of six to eight animals per group,
a
b
p < 0.05,
Finally, compound 12d as the most potent A2AAR ligand, which was
evaluated as metabolic stable, was investigated in in vivo models for
anti-inflammatory, antinociceptive and peripheral analgesic properties.
Surprisingly the molecule was active in all three animal models. The
results of the in vivo tests as well as the outcomes of in vitro assays
confirm the hypothesis that in vivo activity could be mediated by the
p < 0.01,
Table 4
The effect of the investigated 12d in the writhing test.
Treatment
Dose
Number of writhings
Inhibition
[%]
[mg/kg]
Mean
SEM
A
2AAR. Nevertheless, further biological evaluation is required to con-
firm the biochemical mechanism of the presented in vivo properties.
5. Experimental protocols
Control
–
29.5
5.3
0.3
–
12d
20
10
5
1.1c
82.0
54.9
32.9
–
13.3
19.8
19.2
3.2
1.0b
1.3a
3.2
Control
–
5.1. Chemistry
Acetysalicylic acid
100
50
30
1.2c
83.3
55.7
41.6
8.5
1.3b
2.1
5.1.1. Material and methods
11.2
All commercially available reagents and solvents were purchased
and used without further purification. Melting points (mp.) were de-
termined on a MEL-TEMP II (LD Inc., USA) melting point apparatus and
are uncorrected. 1H NMR spectra were recorded on a Varian Mercury
300 MHz or JEOL FT-NMR 500 MHz apparatus in CDCl3 or in DMSO‑d6.
13C NMR data were recorded on 75 MHz on Varian-Mercury-VX
300 MHz PFG or JEOL FT-NMR 500 MHz spectrometer. The J values are
reported in Hertz (Hz), and the splitting patterns are designated as
follows: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), dt
(doublet of triplets), quin (quintet), m (multiplet). The purity of the
tested compounds was determined (%) on an Waters TQD mass spec-
trometer coupled with an Waters ACQUITY UPLC system. Retention
times (tR) are given in minutes. The reactions were monitored by thin
layer chromatography (TLC) using aluminium sheets coated with silica
gel 60F254 (Merck) using as developing system dichloromethane/me-
tanol9:1. Spots were detected under UV light.
Data are presented as the means
SEM of six to eight mice per group.
a
p < 0.05,
b
c
p < 0.01,
p < 0.001 vs. control.
4. Conclusion
The synthesis and preliminary biological evaluation of a new xan-
thine-based library of 8-benzyl-substituted derivatives were performed.
The compound library represented diversity in substitution patterns of
the xanthine core in the N1-, N3–, and N7-positions as well as on the
aromatic benzyl residue. The most potent A2AAR structures (12d and
12 h) were found in the group of 1-ethyl-3,7-dimethylxanthine deri-
vatives containing ortho-halogen substitution on aromatic residue. This
structural modification of the explored xanthine derivatives was found
to be preferable for high A2A potency as well as selectivity. To evaluate
interactions with A2A AR of most active structure 12d, the molecular
docking was performed. Comparing the poses of tested library, the
substitution pattern of 12d resulted in a slightly shift of the benzyla-
mine fragment towards the hydrophobic cage, probably being
The synthesis and physicochemical properties of the compounds 9,
10, 12, 14, and 16c were reported previously [48,59,60].
5.1.1.1. General procedure for the synthesis of 8-substitutedb-
enzylaminoxanthine derivatives. A mixture of 0.55 mmol of 8-
10

M. Załuski, et al.
BioorganicChemistry101(2020)104033
bromotheophylline (9) or 8-bromocaffeine (10) or 8-bromo-1-ethyl-3,7-
dimethyl-3,7-dihydro-1H-purine-2,6-dione (12) or 8-bromo-1,3-
diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione (14), 1.1 mmol of
appropriate benzylamine, 1,6 mmol of TEA and 1.00 mL of propanol
was heated in closed vessels in microwave oven (300 Watt, Power Max
Off, 150 °C, 10 bar) for 1 h. The solvent was removed and the residue
was treated with ethanol. The products were purified by crystallization
from ethanol or flash column chromatography over silica gel with
CH2Cl2 : MeOH (100 : 0 to 80 : 20) as eluent.
dione (9h)
Yield: 79 mg; 48%; mp: 242–243 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.41 (d, J = 6.45 Hz,
2H, NHCH2), 7.31–7.39 (m, 4H, C2H, C3H, C5H, C6H, phe), 7.69 (t,
J = 6.45 Hz, 1H, NHCH2), 11.59 (s, 1H, N7H); UPLC/MS purity
97.70%, tR = 4.85, C14H14ClN5O2, MW 319.74, [M + H]+ 320.32.
5.1.1.1.2. N-7-Methylsubstituted derivatives. 8-((2-Methoxybenzyl)
amino)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (10a)
Yield: 100 mg; 55%; mp: 251–252 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 3.37 (s, 3H, N1CH3) 3.56 (s, 3H, N3CH3) 3.65 (s, 3H, N7CH3) 3.91
(s, 3H, OCH3) 4.66 (d, J = 5.87 Hz, 2H, NHCH2) 4.90 (br. s., 1H,
NHCH2) 6.91–6.98 (m, 2H, C4H, C5H, phe) 7.29–7.34 (m, 1H, C3H,
phe) 7.38 (dd, J = 7.43, 1.96 Hz, 1H, C6H, phe); 13C NMR (CHLOR-
OFORM-d) δ ppm: 27.6 (N1CH3), 29.5 (N7CH3), 29.7 (N3CH3), 43.6
(NHCH2), 55.4 (OCH3), 103.2 (C5), 110.5 (C3, phe), 120.7 (C5, phe),
126.1 (C1, phe), 129.3 (C4, phe), 130.1 (C6, phe), 148.5 (C4), 151.8
(C2), 153.4 (C6), 154.2 (C8), 157.6 (C2, phe). UPLC/MS purity 100%;
tR = 5.02; C16H19N5O3, MW 329.36; [M]+ 330.16.
5.1.1.1.1. N-7-Unsubstituted derivatives. 8-((2-Chlorobenzyl)amino)-
1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (9a)
Yield: 113 mg; 61%; mp: 243–244 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.33 (s, 3H, N3CH3), 4.49 (d, J = 6.45 Hz,
2H, NHCH2), 7.11–7.20 (m, 2H, C5H, C6H, phe), 7.25–7.37 (m, 2H,
C3H, C4H, phe), 7.59 (t, J = 6.15 Hz, 1H, NHCH2), 11.62 (s, 1H, N7H);
UPLC/MS purity 96.16%, tR = 4.80, C14H14ClN5O2, MW 319.74,
[M + H]+ 320.06.
8-((3-Bromobenzyl)amino)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione (9b)
8-((3-Methoxybenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-
purine-2,6-dione (10b)
Yield: 110 mg; 53%; mp: 206–207 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.42 (d, J = 6.45 Hz,
2H, NHCH2), 7.25–7.35 (m, 2H, C5H, C6H, phe), 7.42 (dt, J = 7.03,
1.76 Hz, 1H,C2H, phe),7.52 (t, J = 1.76 Hz, 1H,C4H, phe), 7.73 (t,
J = 6.45 Hz, 1H,NHCH2), 11.61 (s, 1H,N7H); UPLC/MS purity
100.00%, tR = 4.99, C14H14BrN5O2, MW 364.20, [M + H]+ 364.12.
8-((3,4-Dichlorobenzyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione (9c)
Yield: 93 mg; 51%; mp: 197–199 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm: 3.13 (s, 3H, N1CH3) 3.29 (s, 3H, N3CH3) 3.57 (s, 3H, N7CH3)
3.71 (s, 3H, OCH3) 4.49 (d, J = 6.45 Hz, 2H, NHCH2) 6.76–6.82 (m,
1H, C4H, phe) 6.90–6.95 (m, 2H, C2H, C6H, phe) 7.18–7.26 (m, 1H,
C5H, phe) 7.55 (t, J = 6.15 Hz, 1H, NHCH2);13C NMR (DMSO‑d6) δ
ppm: 27.6 (N1CH3), 29.7 (N7CH3), 30.2 (N3CH3), 46.1 (NHCH2), 55.4
(OCH3), 102.4 (C5), 112.7 (C2, phe), 113.5 (C4, phe), 120.0 (C6, phe),
129.8 (C5, phe), 141.6 (C1, phe), 148.6 (C4), 151.6 (C2), 153.3 (C6),
154.4 (C8), 159.7 (C3, phe). UPLC/MS purity 99.4%; tR = 4.82;
Yield: 115 mg;56%; mp: 202–203 °C;1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.41 (d, J = 7.03 Hz,
2H, NHCH2), 7.28–7.34 (m, 1H, C2H, phe), 7.55–7.61 (m, 2H, C5H,
C6H, phe), 7.73 (t, J = 6.74 Hz, 1H, NHCH2), 11.64 (s, 1H, N7H);
UPLC/MS purity 100.00%, tR = 5.52, C14H13Cl2N5O2, MW 354.19,
[M + H]+ 354.02.
C
16H19N5O3; MW 329.36; [M]+ 330.16.
8-((4-Methoxybenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-
purine-2,6-dione (10c)
Yield: 112 mg; 61%; mp: 241–242 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm: 3.13 (s, 3H, N1CH3) 3.30 (s, 3H, N3CH3) 3.55 (s, 3H, N7CH3)
3.70 (s, 3H, OCH3) 4.44 (d, J = 5.86 Hz, 2H, NHCH2) 6.83–6.89 (m,
2H, C3H, C5H, phe) 7.25–7.31 (m, 2H, C2H, C6H, phe) 7.48 (t,
J = 5.86 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm: 27.6 (N1CH3),
29.7 (N7CH3), 30.2 (N3CH3), 46.1 (NHCH2), 55.5 (OCH3), 102.40 (C5),
114.1 (C3/C5, phe), 129.3 (C1/C6, phe), 131.9 (C1, phe), 148.6 (C4),
151.4 (C2), 153.3 (C6), 154.4 (C8), 158.8 (C3, phe). UPLC/MS purity
100%; tR = 4.77; C16H19N5O3; MW 329.36; [M]+ 330.16.
8-((3,4-Dimethoxybenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-
purine-2,6-dione (10d)
8-((2-Bromobenzyl)amino)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione (9d)
Yield: 111 mg; 53%; mp: 206–207 °C; 1H NMR (500 MHz, DMSO‑d6)
δ ppm 3.14 (s, 3H, N1CH3), 3.30 (s, 3H, N3CH3), 4.40 (d, J = 6.30 Hz,
2H, NHCH2), 7.23–7.28 (m, 1H, C5H, phe), 7.29–7.32 (m, 1H, C4H,
phe), 7.40 (d, J = 8.02 Hz, 1H, C3H, phe), 7.48–7.52 (m, 1H, C6H,
phe), 7.72 (t, J = 6.59 Hz, 1H, NHCH2), 11.60 (s, 1H, N7H); UPLC/MS
purity 100.00%, tR = 4.91, C14H14BrN5O2, MW 364.20, [M + H]+
364.12.
8-((2-Bromo-4-fluorobenzyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione (9e)
Yield: 120 mg; 60%; mp: 222–224 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm: 3.13 (s, 3H, N1CH3) 3.31 (s, 3H, N3CH3) 3.56 (s, 3H, N7CH3)
3.70 (s, 3H, OCH3) 3.72 (s, 3H, OCH3) 4.43 (d, J = 5.86 Hz, 2H,
NHCH2) 6.85–6.88 (m, 2H, C5H, C6H, phe)7.01 (s, 1H, C2H, phe) 7.48
(t, J = 5.86 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm: 27.6
(N1CH3), 29.7 (N7CH3), 30.2 (N3CH3), 46.1 (NHCH2), 55.8 (OCH3),
56.0 (OCH3), 102.4 (C5), 112.0 (C2, phe), 112.0 (C5, phe), 120.1 (C6,
phe), 132.3 (C1, phe), 148.3 (C4, phe), 148.6 (C4), 148.9 (C3, phe),
151.4 (C2), 153.3 (C6), 154.4 (C8). UPLC/MS purity 98.6%; tR = 4.30;
C17H21N5O4; MW 359.39; [M]+ 360.14.
Yield: 93 mg; 42%; mp: 224–226 °C;1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.44 (d, J = 5.86 Hz,
2H, NHCH2), 7.25–7.32 (m, 1H, C5H, phe), 7.35–7.41 (m, 1H, C6H,
phe), 7.51 (m, 1H, C3H, phe), 7.62 (t, J = 6.15 Hz, 1H, NHCH2), 11.65
(s, 1H, N7H); UPLC/MS purity 100.00%, tR = 5.19, C14H13BrFN5O2,
MW 382.19, [M + H]+ 382.00.
8-((3-Bromo-4-fluorobenzyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione (9f)
Yield: 121 mg; 55%; mp: 196–197 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.40 (d, J = 7.03 Hz,
2H, NHCH2), 7.27–7.41 (m, 2H, C5H, C6H, phe), 7.65 (m, 1H, C2H,
phe), 7.71 (t, J = 6.45 Hz, 1H, NHCH2), 11.60 (br. s., 1H, N7H); UPLC/
MS purity 100.00%, tR = 5.11, C14H13BrFN5O2, MW 382.19, [M + H]+
382.07.
8-((2-Chlorobenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-purine-
2,6-dione (10e)
Yield: 83 mg; 45%; mp: 249–251 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm: 3.14 (s, 3H, N1CH3) 3.26 (s, 3H, N3CH3) 3.62 (s, 3H, N7CH3)
4.60 (d, J = 5.27 Hz, 2H, NHCH2) 7.24–7.34 (m, 2H, C4H, C5H, phe)
7.41–7.47 (m, 2H, C3H, C6H, phe) 7.60 (t, J = 5.86 Hz, 1H, NHCH2);
13C NMR (DMSO‑d6) δ ppm: 27.6 (N1CH3), 29.7 (N7CH3), 30.3
(N3CH3), 43.9 (NHCH2), 102.6 (C5), 127.6 (C4, phe), 129.2 (C6, phe),
129.6 (C3, phe), 132.5 (C2, phe), 136.9 (C1, phe), 148.5 (C4), 151.3
(C2), 153.4 (C6), 154.2 (C8). UPLC/MS purity 98.1%; tR = 5.43;
8-((3-Chlorobenzyl)amino)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione (9g)
Yield: 99 mg; 51%; mp: 213–214 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 3.16 (s, 3H, N1CH3), 3.32 (s, 3H, N3CH3), 4.43 (d, J = 6.45 Hz,
2H, NHCH2), 7.26–7.39 (m, 4H, C2H, C4H, C5H, C6H, phe), 7.72 (t,
J = 6.74 Hz, 1H, NHCH2), 11.58 (br. s., 1H, N7H); UPLC/MS purity
96.32%, tR = 4.92, C14H14ClN5O2, MW 319.74, [M + H]+ 320.25.
8-((4-Chlorobenzyl)amino)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
C
15H16ClN5O2; MW 333.78; [M]+ 334.15.
8-((2-Bromobenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-purine-
2,6-dione (10f)
11

M. Załuski, et al.
BioorganicChemistry101(2020)104033
Yield: 74 mg; 35%; mp: 251–253 °C; 1H NMR (300 MHz, DMSO‑d6)
[M]+ 396.16.
δ ppm: 3.14 (s, 3H, N1CH3) 3.27 (s, 3H, N3CH3) 3.63 (s, 3H, N7CH3)
4.56 (d, J = 5.86 Hz, 2H, NHCH2) 7.16–7.25 (m, 1H, C4H, phe)
7.32–7.45 (m, 2H, C5H, C6H, phe) 7.58–7.66 (m, 2H, C3H phe,
NHCH2); 13C NMR (DMSO‑d6) δ ppm: 27.6 (N1CH3), 29.8 (N7CH3),
30.4 (N3CH3), 46.4 (NHCH2), 102.7 (C5), 122.8 (C2, phe), 128.2 (C5,
phe), 129.4 (C4, phe), 129.5 (C6, phe), 132.8 (C3, phe), 138.4 (C1,
phe), 148.6 (C4), 151.4 (C2), 153.4 (C6), 154.2 (C8). UPLC/MS purity
98.6%; tR = 5.58; C15H16BrN5O2; MW 378.23; [M]+ 378,15.
8-((3-Chlorobenzyl)amino)-1,3,7-trimethyl-3,7-dihydro-1H-purine-
2,6-dione (10g)
8-((3,4-Dimethoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-
1H-purine-2,6-dione (12e)
Yield: 96 mg; 46%; mp: 242–245 °C; 1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.03 (t, J = 6.59 Hz, 3H, N1CH2CH3) 3.30 (br. s., 3H, N3CH3)
3.54 (s, 3H, N7CH3) 3.68 (br. s., 3H, OCH3) 3.70 (br. s., 3H, OCH3)
3.78–3.83 (m, 2H, N1CH2) 4.39–4.43 (m, 2H, NHCH2) 6.85 (s, 2H,
C5H, C6H, phe) 6.99 (br. s., 1H, C2H, phe) 7.45 (br. s., 1H, NHCH2);
13C NMR (DMSO‑d6) δ ppm: 13,9 (N1CH2CH3), 29.7 (N7CH3), 30.3
(N3CH3), 35.6 (N1CH2), 46.1 (NHCH2), 55.9 (OCH3), 56.1 (OCH3),
102.6 (C5), 112.2 (C2, phe), 112.2 (C5, phe), 120.2 (C6, phe), 132.5
(C1, phe), 148.4 (C4, phe), 148.8 (C4), 149.1 (C3, phe), 151.1 (C2),
153.1 (C6), 154.6 (C8). UPLC/MS purity 100%; tR = 4.75; C18H23N5O4;
MW 373.41; [M]+ 374.23.
Yield: 88 mg; 48%; mp: 223–224 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm: 3.13 (s, 3H, N1CH3) 3.28 (s, 3H, N3CH3) 3.58 (s, 3H, N7CH3)
4.52 (d, J = 5.86 Hz, 2H, NHCH2) 7.26–7.36 (m, 3H, C4H, C5H, C6H,
phe) 7.42 (d, J = 1.76 Hz, 1H, C2H, phe) 7.60 (t, J = 6.15 Hz, 1H,
NHCH2); 13C NMR (DMSO‑d6) δ ppm: 27.6 (N1CH3), 29.7 (N7CH3),
30.3 (N3CH3), 45.6 (NHCH2), 102.6 (C5), 126.5 (C6, phe), 127.3 (C5,
phe), 127.7 (C4, phe), 130.6 (C2, phe), 133.4 (C1, phe), 142.7 (C3,
phe), 148.5 (C4), 151.4 (C2), 153.4 (C6), 154.2 (C8). UPLC/MS purity
98.9%; tR = 5.51; C15H16ClN5O2; MW 333.78; [M]+ 334.15.
5.1.1.1.3. N-1-Ethylsubstituted derivatives. 8-((2-Chloro-6-fluorobe-
nzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione
(12a)
8-((2,4-Dichlorobenzyl)amino)-1-ethyl-3,7-dimethyl-1H-purine-
2,6(3H,7H)-dione (12f)
Yield: 94 mg; 47%; mp: 226–227 °C;1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.03 (t, J = 7.16 Hz, 3H, N1CH2CH3), 3.23 (s, 3H, N3CH3), 3.59
(s, 3H, N7CH3), 3.80 (q,
J = 6.87 Hz, 2H, N1CH2), 4.53 (d,
J = 5.73 Hz, 2H, NHCH2), 7.34–7.37 (m, 1H, C6H, phe), 7.41–7.44 (m,
1H, C5H, phe), 7.56 (d, J = 1.72 Hz, 1H, C3H, phe), 7.60 (t,
J = 6.01 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm 13.83 (CH3CH2),
29.71 (N7CH3), 30.41(N3CH3), 35.57 (CH3CH2), 43.59 (NHCH2),
102.83(C5), 127.81(C5, phe),129.08 (C6, phe), 130.87(C3, phe),
132.81 (C2, phe), 133.54(C4, phe), 136.28(C1, phe), 148.60 (C2),
151.01 (C4), 153.17 (C6), 154.08 (C8); UPLC/MS purity 96.99%,
tR = 6.75, C16H17Cl2N5O2, MW 382.24, [M + H]+ 382.07.
1-Ethyl-8-((2-fluorobenzyl)amino)-3,7-dimethyl-1H-purine-
2,6(3H,7H)-dione (12g)
Yield: 97 mg; 48%; mp: 256–257 °C;1H NMR (500 MHz, DMSO‑d6) δ
ppm 1.00–1.06 (m, 3H, N1CH2CH3) 3.32 (br. s., 3H, N3CH3) 3.47–3.52
(m, 3H, N7CH3) 3.78–3.84 (m, 2H, N1CH2) 4.58 (br. s., 2H, NHCH2)
7.21 (t, J = 8.02 Hz, 1H, C4H, phe) 7.27–7.39 (m, 3H, C3H, C5H, phe;
NHCH2); UPLC/MS purity 96.8%; tR = 5.87; C16H17ClFN5O2; MW
365.79; [M]+ 366.18.
8-((3-Chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (12b)
Yield: 55 mg; 32%; mp: 194–195 °C; 1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.03 (t, J = 6.87 Hz, 3H, N1CH2CH3), 3.25 (d, J = 14.89 Hz, 3H,
N3CH3), 3.58 (d, J = 12.60 Hz, 3H, N7CH3), 3.77–3.83 (m, 2H,
N1CH2), 4.53 (d, J = 5.16 Hz, 2H, NHCH2), 7.07–7.18 (m, 1H, C5H,
phe), 7.34–7.46 (m, 2H, C3H, C4H, phe), 7.50–7.64 (m, 2H, C6H, phe,
NHCH2); 13C NMR (DMSO‑d6) δ ppm 13.83 (CH3CH2), 29.71(N7CH3),
30.41(N3CH3), 35.57 (CH3CH2), 43.59 (NHCH2), 102.83(C5), 115.66
(C3, phe), 127.81 (C5, phe), 129.08 (C4, phe), 130.88 (C6, phe), 132.81
(C1, phe), 136.29 (C2, phe), 148.60 (C2), 151.01 (C4), 153.17 (C6),
154.08 (C8); UPLC/MS purity 100.00%, tR = 5.40, C16H18FN5O2, MW
331.34, [M + H]+ 332,16.
Yield: 104 mg; 54%; mp: 209–211 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 1.05 (t, J = 7.03 Hz, 3H, N1CH2CH3) 3.29 (s, 3H, N3CH3) 3.59
(s, 3H, N7CH3) 3.83 (q, J = 7.03 Hz, 2H, N1CH2) 4.52 (d, J = 5.86 Hz,
2H, NHCH2) 7.26–7.38 (m, 3H, C4H, C5H, C6H, phe) 7.41 (s, 1H, C2H,
phe) 7.61 (t, J = 6.15 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm:
13.8 (N1CH2CH3), 29.6 (N7CH3), 30.3 (N3CH3), 35.5 (N1CH2), 45.6
(NHCH2), 102.6 (C5), 126.5 (C6, phe), 127.3 (C5, phe),127.6 (C4, phe),
130.6 (C2, phe), 133.4 (C1, phe), 142.7 (C3, phe), 148.6(C4), 150.9
(C2), 153.0 (C6), 154.2 (C8).UPLC/MS purity 96.8%; tR = 5.96;
C16H18ClN5O2; MW 347.80; [M]+ 348.17.
8-((2-Chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-1H-purine-
2,6(3H,7H)-dione (12h)
8-((3-Bromobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (12c)
Yield: 101 mg; 56%; mp: 216–217 °C; 1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.03 (t, J = 7.16 Hz, 3H, N1CH2CH3), 3.24 (s, 3H, N3CH3), 3.60
(s, 3H, N7CH3), 3.77–3.85 (m, 2H, N1CH2), 4.58 (d, J = 5.73 Hz, 2H,
NHCH2), 7.22–7.31 (m, 2H, C4H, C5H, phe), 7.38–7.43 (m, 2H, C3H,
C6H, phe), 7.57 (t, J = 6.01 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6)
δ ppm13.85(CH3CH2), 29.72(N7CH3), 30.40 (N3CH3), 35.57 (CH3CH2),
43.97(NHCH2), 102.78(C5), 127.69(C5, phe), 129.22(C4, phe),
129.42(C6, phe), 129.65(C3, phe), 132.60(C2, phe), 137.01(C1, phe),
148.67(C2), 151.03(C4), 153.15(C6), 154.27(C8); UPLC/MS purity
94.96%, tR = 5.92, C16H18ClN5O2, MW 347.8, [M + H]+ 348.11.
1-Ethyl-8-((2-methoxybenzyl)amino)-3,7-dimethyl-1H-purine-
2,6(3H,7H)-dione (12i)
Yield: 116 mg; 53%; mp: 216–217 °C; 1H NMR (300 MHz, CDCl3) δ
ppm 1.21 (t, J = 6.74 Hz, 3H, N1CH2CH3) 3.52 (s, 3H, N3CH3) 3.68 (s,
3H, N7CH3) 4.02 (q, J = 6.64 Hz, 2H, N1CH2) 4.74 (d, J = 5.28 Hz,
2H, NHCH2) 4.94 (br. s., 1H, NHCH2) 7.12–7.21 (m, 1H, C6H, phe)
7.27–7.33 (m, 1H, C5H, phe) 7.47–7.59 (m, 2H, C2H,C4H, phe); 13C
NMR (CHLOROFORM-d) δ ppm: 13.4 (N1CH2CH3), 29.7 (N7CH3), 29.7
(N3CH3), 36.1 (N1CH2), 47.5 (NHCH2), 103.4 (C5), 123.8 (C3, phe),
127.7 (C6, phe),129.6 (C5, phe), 130.9 (C4, phe), 132.9 (C2, phe),137.1
(C1, phe), 148.1 (C4), 151.3 (C2), 152.6 (C6), 154.0 (C8).UPLC/MS
purity 97.6%; tR = 6.04; C16H18BrN5O2; MW 392.26; [M]+ 392.10.
8-((6-Chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-di-
methyl-3,7-dihydro-1H-purine-2,6-dione (12d)
Yield: 74 mg; 41%; mp: 218–219 °C;1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.04 (t, J = 6.87 Hz, 3H, N1CH2CH3), 3.25 (s, 3H, N3CH3), 3.59
(s, 3H, N7CH3), 3.79 (s, 3H, OCH3), 3.81 (q, J = 7.45 Hz, 2H, N1CH2),
4.48 (d, J = 5.73 Hz, 2H, NHCH2), 6.87 (t, J = 7.45 Hz, 1H, C5H, phe),
6.95 (d, J = 8.02 Hz, 1H, C3H, phe), 7.18–7.24 (m, 2H, C4H, C6H,
phe), 7.35 (t, J = 6.01 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm
Yield: 92 mg; 41%; mp: 254–255 °C; 1H NMR (300 MHz, DMSO‑d6)
δ ppm 1.05 (t, J = 6.74 Hz, 3H, N1CH2CH3) 3.33 (s, 3H, N3CH3) 3.52
(s, 3H, N7CH3) 3.81–3.85 (m, 5H, N1CH2, OCH3) 4.58 (dd, J = 4.69,
1.76 Hz, 2H, NHCH2) 7.09–7.17 (m, 1H, C4H, phe) 7.21–7.27 (m, 1H,
C5H, phe) 7.32 (s, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm: 13,8
(N1CH2CH3), 29.6 (N7CH3/N3CH3), 30.4 (N1CH3), 38.8 (d,
13.87(CH3CH2),
29.75(N7CH3),
30.38(N3CH3),35.56(CH3CH2),
3JC,F
=
4.6 Hz, NHCH2), 56.8 (OCH3), 102.7 (C5), 114.2 (d,
41.23(NHCH2), 55.86 (OCH3), 102.64(C5), 110.98(C3, phe),
120.64(C5, phe), 127.42 (C1, phe), 128.05(C4, phe), 128.61(C6, phe),
148.85(C2), 151.07(C4), 153.09(C6), 154.71(C8), 157.14(C2, phe);
4JC,F = 2.3 Hz, C5, phe), 124.5 (d, 2JC,F = 15.0 Hz, C1, phe), 125.1 (d,
3JC,F = 4.6 Hz, C6, phe), 125.6 (C4, phe), 146.8 (C3, phe), 148.6 (C4),
1
151.0 (C2), 151.5 (d, JC,F = 252.2 Hz, C6, phe), 153.1 (C6), 153.9
UPLC/MS purity 98.57%, tR = 5.51, C17H21N5O3, MW 343.38,
(C8). UPLC/MS purity 95.7%; tR = 5.85; C17H19ClFN5O3; MW 395.82;
[M + H]+ 344.19.
12

M. Załuski, et al.
BioorganicChemistry101(2020)104033
8-((4-Chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-1H-purine-
Yield: 200 mg; 28%;mp: 148–151 °C; 1H NMR (400 MHz, DMSO‑d6)
δ ppm 1.11 (t, J = 6.85 Hz, 3H, N1CH2CH3) 3.34 (s, 3H, N3CH3) 3.49
(s, 3H, N7CH3) 3.88 (q, J = 7.04 Hz, 2H, N1CH2) 5.26 (s, 2H, OCH2)
7.25 (d, J = 8.61 Hz, 2H, C2H, C6H, phe) 7.44 (d, J = 8.61 Hz, 2H,
C3H,C5H, phe); 13C NMR (DMSO‑d6) δ ppm: 13.4 (N1CH2CH3), 29.5
(N7CH3), 31.3 (N3CH3), 36.4 (N1CH2), 45.4 (OCH2), 99.2 (C5), 128.3
(C2/C6, phe), 129.4 (C3/C5, phe), 132.6 (C4, phe), 136.3 (C1, phe),
136.6 (C4), 150.3 (C2), 152.1 (C6), 153.1 (C8). UPLC/MS purity
99.35%; tR = 5.51; C16H17ClN4O3; MW 348.79; [M]+ 349.
2,6(3H,7H)-dione (12j)
Yield: 88 mg; 48%; mp: 238–239 °C; 1H NMR (500 MHz, DMSO‑d6)
δ ppm 1.03 (t, J = 6.87 Hz, 3H, N1CH2CH3), 3.26 (s, 3H, N3CH3), 3.56
(s, 3H, N7CH3), 3.80 (q, J = 6.87 Hz, 2H, CH3CH2), 4.48 (d,
J = 6.30 Hz, 2H, NHCH2), 7.29–7.38 (m, 4H, C2H, C6H, C3H, C5H,
phe), 7.58 (t, J = 6.01 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6) δ ppm
13.85(CH3CH2), 29.71(N7CH3), 30.33(N3CH3), 35.56(CH3CH2),
45.54(NHCH2), 102.69(C5), 128.72(C3, C5, phe), 129.77(C2, C6, phe),
131.98(C4, phe), 139.22(C1, phe), 148.70(C2), 151.04(C4),
8-((2-Chlorobenzyl)oxy)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (15b)
153.11(C6), 154.38(C2); UPLC/MS purity 100.00%, tR
= 6.01,
C16H18ClN5O2, MW 347.8, [M + H]+ 348.11.
Yield: 90 mg; 13%; mp: 126–129 °C; 1H NMR (400 MHz, DMSO‑d6)
δ ppm 1.10 (t, J = 7.04 Hz, 3H, N1CH2CH3) 3.38 (s, 3H, N3CH3) 3.61
(s, 3H, N7CH3) 3.88 (q, J = 6.91 Hz, 2H, N1CH2) 5.57 (s, 2H, OCH2)
7.39–7.47 (m, 2H, C5H, C6H, phe) 7.53–7.56 (m, 1H, C4H, phe) 7.67
(dd, J = 7.04, 2.35 Hz, 1H, C3H, phe); 13C NMR (DMSO‑d6) δ ppm:
13.6 (N1CH2CH3), 29.9 (N7CH3), 30.1 (N3CH3), 35.8 (N1CH2), 69.9
(OCH2), 103.3 (C5), 127.9 (C5, phe), 130.0 (C6, phe), 131.1 (C4, phe),
131.2 (C3, phe), 133.1 (C2, phe), 133.4 (C1, phe), 146.0 (C4), 150.9
(C2), 154.0 (C6), 155.2 (C8). UPLC/MS purity 100%; tR = 6.90;
5.1.1.1.4. N-1, N-3-Diethylsubstituted derivatives. 1,3-Diethyl-8-((2-
methoxybenzyl)amino)-7-methyl-3,7-dihydro-1H-purine-2,6-dione
(14a)
Yield: 87 mg; 44%; mp: 180–181 °C; 1H NMR (300 MHz, CDCl3) δ
ppm 1.16–1.25 (m, 3H, N1CH2CH3) 1.32 (t, J = 7.33 Hz, 3H,
N3CH2CH3) 3.62 (s, 3H, N7CH3) 3.89 (s, 3H, OCH3) 4.02 (q,
J = 7.03 Hz, 2H, N1CH2) 4.13 (q, J = 7.03 Hz, 2H, N3CH2) 4.63 (d,
J = 5.86 Hz, 2H, NHCH2) 4.79–4.86 (m, 1H, NHCH2) 6.88–6.96 (m,
2H, C4H, C5H, phe) 7.27–7.32 (m, 1H, C3H, phe) 7.37 (dd, J = 7.62,
1.76 Hz, 1H, C6H, phe); 13C NMR (CHLOROFORM-d) δ ppm: 13.4
(N1CH2CH3), 13.5 (N3CH2CH3), 29.4 (N7CH3), 36.0 (N1CH2), 38.3
(N3CH2), 43.6 (NHCH2), 55.4 (OCH3), 103.4 (C5), 110.4 (C3, phe),
120.6 (C5, phe), 126.3 (C1, phe), 129.2 (C4, phe), 130.3 (C6, phe),
148.1 (C4), 150.8 (C2), 153.4 (C6), 154.0 (C8), 157.6 (C2, phe). UPLC/
MS purity 100%; tR = 6.18; C18H23N5O3; MW 357.41; [M]+ 358.21.
8-((3-Bromobenzyl)amino)-1,3-diethyl-7-methyl-3,7-dihydro-1H-
purine-2,6-dione (14b)
C
16H17ClN4O3; MW 348.79; [M]+ 349.
8-((2-Bromobenzyl)oxy)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (15c)
Yield: 145 mg; 18%; mp: 150–152 °C; 1H NMR (400 MHz, DMSO‑d6)
δ ppm 1.10 (t, J = 6.85 Hz, 3H, N1CH2CH3) 3.34 (s, 3H, N3CH3) 3.62
(s, 3H, N7CH3) 3.88 (q, J = 7.04 Hz, 2H, N1CH2) 5.54 (s, 2H, OCH2)
7.33–7.39 (m, 1H, C4H, phe) 7.46 (t, J = 7.04 Hz, 1H, C6H, phe)
7.64–7.73 (m, 2H, C3H, C5H, phe); 13C NMR (DMSO‑d6) δ ppm: 13.6
(N1CH2CH3), 29.9 (N7CH3), 30.1 (N3CH3), 35.8 (N1CH2), 72.0
(OCH2), 103.3 (C5), 123.5 (C2, phe), 128.5 (C5, phe), 131.3 (C3, phe),
133.2 (C4, phe), 134.7 (C6, phe), 146.0 (C4), 150.9 (C2), 150.9 (C1,
phe), 154.0 (C6), 155.2 (C8). UPLC/MS purity 100%; tR = 7.05;
Yield: 103 mg; 46%; mp: 191–192 °C; 1H NMR (300 MHz, CDCl3) δ
ppm 1.22 (t, J = 7.03 Hz, 3H, N1CH2CH3) 1.31 (t, J = 7.03 Hz, 3H,
N3CH2CH3) 3.67 (s, 3H, N7CH3) 3.98–4.15 (m, 4H, N1CH2, N3CH2)
4.62 (d, J = 5.27 Hz, 2H, NHCH2) 4.75–4.82 (m, 1H, NHCH2)
7.16–7.23 (m, 1H, C6H, phe) 7.28–7.33 (m, 1H, C5H, phe) 7.38–7.44
(m, 1H, C4H, phe) 7.56 (s, 1H, C2H, phe); 13C NMR (CHLOROFORM-d)
δ ppm: 13.5 (N1CH2CH3/N3CH2CH3), 29.6 (N7CH3), 36.1 (N1CH2),
38.4 (N3CH2), 46.7 (NHCH2), 103.6 (C5), 122.7 (C3, phe), 126.6 (C6,
phe), 130.2 (C5, phe), 130.9 (C4, phe), 131.1 (C2, phe), 140.7 (C1,
phe), 147.8 (C4), 150.7 (C2), 152.8 (C6), 154.1 (C8). UPLC/MS purity
100%; tR = 6.81; C17H20BrN5O2; MW 406.28; [M]+ 408.12.
8-((3-Chlorobenzyl)amino)-1,3-diethyl-7-methyl-3,7-dihydro-1H-
purine-2,6-dione (14c)
C
16H17BrN4O3; MW 393,24; [M]+ 395.
8-((3-Bromobenzyl)oxy)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (15d)
Yield: 70 mg; 9%; mp: 147–150 °C; 1H NMR (400 MHz, DMSO‑d6) δ
ppm 1.11 (t, J = 7.04 Hz, 3H, N1CH2CH3) 3.39 (s, 3H, N3CH3) 3.62 (s,
3H, N7CH3) 3.89 (q, J = 7.04 Hz, 2H, N1CH2) 5.50 (s, 2H, OCH2)
7.37–7.42 (m, 1H, C5H, phe) 7.57 (m, 2H, C4H, C6H, phe) 7.76 (s, 1H,
C2H, phe); 13C NMR (DMSO‑d6) δ ppm: 13.6 (N1CH2CH3), 29.9
(N7CH3), 30.1 (N3CH3), 35.8 (N1CH2), 71.4 (OCH2), 103.3 (C5), 122.2
(C3, phe), 127.7 (C6, phe), 131.2 (C5, phe), 131.4 (C4, phe), 131.8 (C2,
phe), 138.5 (C1, phe), 146.0 (C4), 150.9 (C2), 154.0 (C6), 155.4 (C8).
UPLC/MS purity 100%; tR = 7.09; C16H17BrN4O3; MW 393,24; [M]+
395.
Yield: 99 mg; 49%; mp: 177–178 °C;1H NMR (500 MHz, DMSO‑d6) δ
ppm 1.03 (t, J = 6.87 Hz, 3H, N1CH2CH3) 1.11 (t, J = 7.16 Hz, 3H,
N3CH2CH3) 3.56 (s, 3H, N7CH3) 3.79–3.84 (m, 2H, N1CH2) 3.88 (q,
J = 6.87 Hz, 2H, N3CH2) 4.47 (d, J = 5.73 Hz, 2H, NHCH2) 7.25–7.33
(m, 3H, C4H, C5H, C6H, phe) 7.42 (s, 1H, C2H, phe) 7.61 (t,
5.1.1.3. Procedure for the synthesis of 8-(((2,6-dichlorobenzyl)amino)
methyl)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dion (17)
5.1.1.3.1. Synthesis of 1-ethyl-8-(hydroxymethyl)-3,7-dimethyl-3,7-
dihydro-1H-purine-2,6-dione (16e). A mixture of 10 mmol of 8-
(hydroxymethyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione (16c) and
12 mmol of iodomethane, 25 mmol of DIPEA and 10 mL of DMF was
heated over night at 40 °C under TLC control. To yield the crude
product, water was added to the mixture and the precipitate was
filtered off and used to the next reaction. The mixture of ca. 1 g of crude
8-(hydroxymethyl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione
(16d), 1.5 equiv. iodoethane, 2.5 equiv. K2CO3 and 10 mL DMF was
heated at 70° C for 4 h. To precipitate the product, water was added.
The product was purified by flash column chromatography over silica
gel with CH2Cl2 : MeOH (100 : 0 to 80 : 20).
J
=
5.73 Hz, 1H, NHCH2); 13C NMR (DMSO‑d6)
δ ppm: 13,7
(N1CH2CH3), 13,9 (N3CH2CH3), 30.3 (N7CH3), 35.5 (N1CH2), 38.0
(N3CH2), 45.8 (NHCH2), 102.9 (C5), 126.8 (C6, phe), 127.4 (C5, phe),
128.0 (C4, phe), 130.6 (C2, phe), 133.4 (C1, phe), 142.8 (C3, phe),
148.1 (C4), 150.5 (C2), 153.2 (C6), 154.4 (C8). UPLC/MS purity 100%;
tR = 6.64; C17H20ClN5O2; MW 361.83; [M]+ 362.20.
5.1.1.2. General procedure for the synthesis of 8-(substituted)benzyloxy-1-
ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione derivatives (15a-
15d). A mixture of
2
mmol 8-bromo-1-ethyl-3,7-dimethyl-3,7-
dihydro-1H-purine-2,6-dione (12),
2 mmol of appropriate benzyl
alcohol, 1,5 mmol of 60% NaH and 14 mL of DMF was heated at
80 °C for 4 h. The mixture was stirred for the next day at room
temperature and the product precipitated upon addition of water. For
purification, the compounds were crystallized from ethanol or
Yield: 1,02 g; 42%;mp: 249–250 °C; 1H NMR (300 MHz, DMSO‑d6) δ
ppm 1.04–1.12 (m, 3H, N1CH2CH3) 3.38 (s, 3H, N3CH3) 3.84–3.92 (m,
5H, N7CH3, N1CH2) 4.55 (s, 2H, CH2OH) 5.59–5.68 (m, 1H,CH2OH).
UPLC/MS purity 93.6%; tR = 2.64; C10H14N4O3; MW 238.25; [M]+
238.92
subjected column chromatography over silica gel with CH2Cl2
MeOH (100 : 0 to 80 : 20).
:
8-((4-Chlorobenzyl)oxy)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (15a)
5.1.1.3.2. Synthesis of 8-(((2,6-dichlorobenzyl)amino)methyl)-1-ethyl-
13

M. Załuski, et al.
BioorganicChemistry101(2020)104033
3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dion (17). The synthesis was
performed according to previously described procedures [60]. 1-
Ethyl-8-(hydroxymethyl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-
dione (16d) 400 mg was dissolved in dry CH2Cl2 and PBr3 (0.4 mL) was
added dropwise at 0 °C. The solution was allowed to warm to rt and
stirred for 1 h. Then it was cooled to 0 °C and the excess of PBr3 was
hydrolyzed by addition of saturated aq. NaHCO3-solution to the pH
7–8. Then, the aqueous layer was extracted with CH2Cl2. The organic
extracts were combined, dried over Na2SO4 and the solvent was
removed by rotary evaporation. The residue was dissolved in a
mixture of dimethoxyethane (10 mL), DIPEA (0.5 mL) and 2-chloro-6-
fluorobenzylamine was added. The solution was stirred overnight at rt.
The product was precipitated upon addition of H2O. For purification,
the compound was subjected to flash-chromatography over silica gel
with CH2Cl2:MeOH (100 : 0 to 80 : 20).
percentage of inhibition and IC50s were computed using GraphPad
Prism Version 6.0 software.
5.2.4. Metabolic stability
In silico experiments of metabolic stability prediction was performed
by MetaSite 6.0.1 provided by Molecular Discovery Ltd. The most
probable sites of metabolism were predicted by liver computational
model and evaluated by in vitro studies using mouse or rat liver mi-
crosomes. All experiments were performed according to previously
described procedures [62,63].
5.3. A2A AR molecular docking studies
Crystal structure of A2Areceptor in complex with XAC (PDB ID:
3REY) [53] respectively were imported into MOE v. 2019.01 (ff used -
AMBER10:EHT) and prepared using implemented QuikPrep tool: pro-
tonation states were generated using Protonate-3D tool, receptor tether
strength – 5000, remove water molecules farther than 4.5 Å from ligand
[69]. Ligand library was prepared using Conformational Search tool
using default settings. 5 lowest energy conformers were then docked to
the rigid form of receptor, with site centred on ligand atoms using Wall
Constraint (Placement method: Triangle Matcher, 30 poses to retain
before refinement, 10 final poses scored GBVI/WSA dG scoring
method). Possible binding pocket adaptation in presence of certain li-
gands was examined using Induced fit refinement protocol. In order to
validate the methods used, co-crystallized ligands were redocked to
crystal structures. The superposition of the phenylpurinedione cores
with the co-crystallized ligands were of high confidence. Dynamics si-
mulations (in time of 600 ps, T = 300 K) were performed using the
Nosé-Poincaré-Andersen equations of motion, force field AM-
BER10:EHT; R-Field 1:80, Cutoff (8,10).
Yield: 300 mg; 47%;mp: 170–171 °C; 1H NMR (500 MHz, DMSO‑d6)
δ
ppm 1.06 (t, J = 6.87 Hz, 3H, N1CH2CH3) 2.56 (br. s.,
1H,CH2NHCH2) 3.34 (s, 3H, N3CH3) 3.79–3.87 (m, 9H, N7CH3, N1CH2,
CH2NHCH2, CH2NHCH2) 7.14 (t, J = 8.59 Hz, 1H, C4H, phe) 7.23–7.31
(m, 2H, C3H, C5H, phe); UPLC/MS purity 94.94%; tR
= 3.52;
C
17H19ClFN5O2; MW 379.82; [M]+ 380.01.
5.2. Biological experiments
5.2.1. Radioligand binding assays at adenosine receptors
Radioligand binding assays were performed as previously described
[50]. For assays at all four human AR subtypes, cell membranes of
Chinese hamster ovary (CHO) or human embryonic kidney cells (HEK)
recombinantly expressing the respective receptors were purchased from
PerkinElmer or prepared in our laboratory. The following compounds
were employed as radioligands: A1: [3H]2-chloro-N6-cyclopentylade-
nosine ([3H]CCPA) [49]; A2A: [3H]3-(3-hydroxypropyl)-1-propargyl-7-
methyl-8-(m-methoxystyryl)xanthine ([3H]MSX-2) [6]; A2B: [3H]8-(4-
(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine
([3H]PSB-603) [50]; A3: [3H]2-phenyl-8-ethyl-4-methyl-(8R)-4,5,7,8-
tetrahydro-1H-imidazo[2,1-i]purine-5-one ([3H]PSB-11) [51]. Initially,
a single high concentration of compound was tested (typically 1 µM).
For potent compounds, full concentration-inhibition curves were de-
termined using different concentrations of test compounds spanning 3
orders of magnitude. At least three independent experiments were
performed. Data were analyzed using the PRISM program version 4.0 or
higher (Graph Pad, San Diego, CA, USA).
Ligand interaction diagrams were generated using Schrödinger
Maestro [70] and MOE, ligand-receptor visualizations were generated
using UCSF Chimera [71]
5.4. Pharmacology
5.4.1. Animals
The in vivo experiments were carried out on male albino Swiss mice
weighing 18–26 g and male rats Wistar weighing 150–180 g. The ani-
mals were housed in constant temperature facilities exposed to 12:12
light–dark cycle and maintained on a standard pellet diet and tap water
given ad libitum. Control and experimental groups consisted of 6–8
animals each. The investigated compounds were administered in-
traperitoneally (ip) in the form of a suspension in 0.5% methylcellulose.
Control animals received the equivalent volume of solvent.
All procedures were conducted according to the guidelines of ICLAS
(International Council on Laboratory Animals Science) and were ap-
proved by The Local Ethics Committee of the Jagiellonian University in
Krakow (agreement nr 47/2014).
5.2.2. Monoamine oxidase assays
Inhibition activity of compounds was measured by a fluorometric
method for detecting monoamine oxidase activity using the Amplex™
Red Monoamine Oxidase Assay (ThermoFisher Scientific A12214) in a
96-well plate. Human recombinant MAO-B and MAO-A enzymes (Sigma
Aldrich M7441 and M7316) were used. The assays were conducted as
previously described.[52,61]
5.2.3. Phosphodiesterase inhibition assays
5.4.2. Statistical analysis
Tested and reference compounds were dissolved in dimethyl sulf-
oxide (DMSO) at a concentration of 10 mM and further diluted in assay
buffer (10 mM Tris-HCl, 10 mM magnesium chloride and 0,05% Tween-
20; pH 7,4). Inhibition of PDE-10A and -4B1 was measured using
PDElight HTS cAMP phosphodiesterase assay kit (Lonza) according to
manufacturer’s recommendations. 10 ng of PDE-10A and 5 ng of PDE-
4B1(BPS Biosciences) in appropriate buffer was incubated with re-
ference and tested compound for 20 min. After incubation the cAMP
substrate (final concentration 1,25 µM for PDE-10A and 5 µM for PDE-
4B1) was added and incubated for 1 h. Then PDELight AMP Detection
Reagent was added and incubated 10 min. All reactions were carried
out at 37 °C in white-walled, 96 half area-well plates which were ob-
tained from Perkin Elmer. Luminescence was measured in a multi-
function plate reader (POLARstar Omega, BMG Labtech, Germany). The
The data are expressed as the mean
SEM (standard error of the
mean). Differences between vehicle control and treatment groups were
tested using one-and two-way ANOVA followed by Duncan’s multiple
comparison test. The difference of means was statistically significant if
p < 0.05.
5.4.3. The formalin test
The mice were pretreated with the test compound or the vehicle and
were allowed to acclimate in Plexiglas observation chambers
(20 × 30 × 15 cm) for 30 min before the test. Then, 20 μL of a 5%
formalin solution was injected intraplantarly into the right hind paw
using a 26-gauge needle. Immediately after formalin injection, the an-
imals were placed individually into glass beakers and were observed
during the next 30 min. Time (in seconds) spent on licking or biting of
14

M. Załuski, et al.
BioorganicChemistry101(2020)104033
the injected paw in selected intervals, 0–5, 15–20, 20–25, and
25–30 min, was measured in each experimental group and was an in-
dicator of nociceptive behavior [64]. The ED50 values and their con-
fidence limits were estimated by the method of Litchfield and Wilcoxon
[65].
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Rats were divided into four groups, one of them being the control. In
order to produce inflammation, 0.1 mL of 1% carrageenan solution in
water was injected into the hind paw subplantar tissue of rats, ac-
cording to the modified method of C. A. Winter [66] and P. Lence [67].
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substances, paw diameters were measured by dividers and recorded.
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(as a suspension in methylcellulose), prior to carrageenan injection.
Methylcellulose was administered by the same route, to the control
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Edema inhibition % = (N N × 100)/N
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Mice were treated with 0.25 mL of 0.02% phenylbenzoquinone
solution 30 min after ip administration of the investigated compound or
the vehicle. Then the mice were placed individually in glass beakers
and 5 min were allowed to elapse. After that period of time each animal
was observed for 10 min and the number of characteristic writhes was
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
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Financial support by the National Science Center, Poland [grant
based on decision No DEC-2016/23/N/NZ7/00475] and the
Jagiellonian University Medical College in Kraków, Poland [grant no.
N42/DBS/000039, N42/DBS/000041], are gratefully acknowledged.
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