Novel multi-target directed ligands based on annelated xanthine scaffold with aromatic substituents acting on adenosine receptor and monoamine oxidase B. Synthesis, in vitro and in silico studies

Article abstract of DOI:10.1016/j.bmc.2019.02.004

N9-Benzyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones were designed as dual-target-directed ligands combining A_2A adenosine receptor (AR) antagonistic activity with blockade of monoamine oxidase B (MAO-B). A library of 37 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. A systematic modification of the tricyclic structures based on a xanthine core by enlargement of the third heterocyclic ring or attachment of various substituted benzyl moieties resulted in the development of 9-(2-chloro-6-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (9u; K_i human A_2AAR: 189 nM and IC₅₀ human MAO-B: 570 nM) as the most potent dual acting ligand of the series displaying high selectivity versus related targets. Moreover, some potent, selective MAO-B inhibitors were identified in the group of pyrimido- and 1,3-diazepino[2,1-f]purinediones. Compound 10d (10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-2,4(3H,6H)-dione) displayed an IC₅₀ value at human MAO-B of 83 nM. Analysis of structure–activity relationships was complemented by molecular docking studies based on previously published X-ray structures of the protein targets. An extended biological profile was determined for selected compounds including in vitro evaluation of potential hepatotoxicity calculated in silico and antioxidant properties as an additional desirable activity. The new molecules acting as dual target drugs may provide symptomatic relief as well as disease-modifying effects for neurodegenerative diseases, in particular Parkinson's disease.

Full text of DOI:10.1016/j.bmc.2019.02.004

Accepted Manuscript
Novel multi-target directed ligands based on annelated xanthine scaffold with
aromatic substituents acting on adenosine receptor and monoamine oxidase B.
Synthesis, in vitro and in sillico studies
Michał Załuski, Jakub Schabikowski, Miriam Schlenk, Agnieszka Olejarz-
Maciej, Bartłomiej Kubas, Tadeusz Karcz, Kamil Kuder, Gniewomir Latacz,
Małgorzata Zygmunt, David Synak, Sonja Hinz, Christa E. Müller, Katarzyna
Kieć-Kononowicz
PII:
S0968-0896(18)32025-X
https://doi.org/10.1016/j.bmc.2019.02.004
BMC 14740
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 December 2018
25 January 2019
1 February 2019
Please cite this article as: Załuski, M., Schabikowski, J., Schlenk, M., Olejarz-Maciej, A., Kubas, B., Karcz, T.,
Kuder, K., Latacz, G., Zygmunt, M., Synak, D., Hinz, S., Müller, C.E., Kieć-Kononowicz, K., Novel multi-target
directed ligands based on annelated xanthine scaffold with aromatic substituents acting on adenosine receptor and
monoamine oxidase B. Synthesis, in vitro and in sillico studies, Bioorganic & Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.bmc.2019.02.004
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Novel multi-target directed ligands based on annelated xanthine scaffold with aromatic
substituents acting on adenosine receptor and monoamine oxidase B. Synthesis, in vitro
and in sillico studies.
1Michał Załuski, 1Jakub Schabikowski, 3Miriam Schlenk, 1Agnieszka Olejarz-Maciej, 1Bartłomiej
Kubas, 1Tadeusz Karcz, 1Kamil Kuder, 1Gniewomir Latacz, 2Małgorzata Zygmunt, 1David Synak,
3Sonja Hinz 3Christa E. Müller, 1Katarzyna Kieć-Kononowicz
1
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian
University Medical College, Kraków, Poland
2 Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College,
Kraków, Poland.
3PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An
der Immenburg 4, 53121 Bonn, Germany
*Correspondence: Katarzyna Kieć –Kononowicz, 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
Tel/fax: +48-12- 6205580/ +48-12- 6205596
Keywords: adenosine A2A receptors ligands, annelated xanthines, hepatotoxicity, molecular
docking, monoamine oxidase B inhibitors, neurodegenerative diseases, Parkinson’s disease,

Abstract
N9-benzyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones were
designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic
activity with blockade of monoamine oxidase B (MAO-B). A library of 37 novel compounds
was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and
for their ability to inhibit MAO-B. A systematic modification of the tricyclic structures based
on a xanthine core by enlargement of the third heterocyclic ring or attachment of various
substituted benzyl moieties resulted in the development of 9-(2-chloro-6-fluorobenzyl)-1,3-
dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (9u; Ki human A2AAR:
189 nM and IC50 human MAO-B: 570 nM) as the most potent dual acting ligand of the series
displaying high selectivity versus related targets. Moreover, some potent, selective MAO-B
inhibitors were identified in the group of pyrimido- and 1,3- diazepino[2,1-f]purinediones.
Compound
10d
(10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-
[1,3]diazepino[2,1-f]purine-2,4(3H,6H)-dione) displayed an IC50 value at human MAO-B of
83 nM. Analysis of structure-activity relationships was complemented by molecular docking
studies based on previously published X-ray structures of the protein targets. An extended
biological profile was determined for selected compounds including in vitro evaluation of
potential hepatotoxicity calculated in silico and antioxidant properties as an additional
desirable activity. The new molecules acting as dual target drugs may provide symptomatic
relief as well as disease-modifying effects for neurodegenerative diseases, in particular
Parkinson’s disease.

1. Introduction
Neurodegenerative diseases (NDs) such as Alzheimer’s, Parkinson’s, and
Huntington’s disease and amyotrophic lateral sclerosis (AD, PD, HD, ALS respectively) are
complex and multi-factorial disorders with unclear onset and continuous progression, caused
by genetic, environmental and endogenous pathogenic factors.[1–4] The characteristic
symptom of NDs is selective neuronal degeneration having various molecular mechanisms
and presenting different clinical manifestations. However, some general pathways leading to
neuronal death such as protein misfolding and aggregation, oxidative stress and free radical
formation, metal dyshomeostasis, mitochondrial dysfunction and phosphorylation impairment
occur concurrently in those disorders.[5–8]
Despite continuous progression in the understanding of the biochemical aspects of
NDs, the current therapeutic efficacy is still limited to palliative and/or symptomatic effects.
Moreover, molecules interacting with a single known biological target appear to be insufficient
to fully prevent, retard, halt or reverse NDs.[2,8,9] Therefore, alternative approaches such as
the development of multi target-directed ligands (MTDLs) have recently gained considerable
attention. The concept of MTDLs suggests one molecule interacting with two or more
precisely defined biological targets with the aim to increase therapeutic efficacy. This
approach presents a wide range of advantages related to pharmacokinetic and
pharmacodynamic profiles, reducing drug-drug interactions and improving patient compliance
as compared to polypharmacology by combination of drugs or single-pill drug
combinations.[8,10]
In the context of MTDLs designed for the treatment of NDs, molecules possessing
both neuroprotective and symptomatic effects seem to be especially desirable. Adenosine A2A
receptors and monoamine oxidase B are used as targets for the treatment of neurodegenerative
disorders, however recent studies indicate, as they provide not only symptomatic relief but
may also mediate neuroprotective effect.[11]
Monoamine oxidase B (MAO-B) is an isoform of the enzyme catalyzing the oxidative
deamination of endogenous and xenobiotic amines and thereby reducing levels of biogenic
amine neurotransmitters in the CNS.[12] Some studies suggest that elevated levels of MAO-B
in the aging brain accelerate the neurodegenerative process since potentially neurotoxic
substances such as dopaldehyde and hydrogen peroxide are formed as by-product of the
reaction catalyzed by this enzyme. These reactive compounds may lead to neuronal death by
oxidation of nucleic acids or proteins causing oxidative damage due to Fenton's reaction by
forming hydroxyl radicals.[8,13–15] Considering the abovementioned reasons, MAO-B
inhibitors, which are already well established therapeutics for PD, seem to be interesting
candidates for the development of MTDLs potentially exhibiting disease-modifying activity
in NDs. Actually, selective MAO-B inhibitors such as: selegiline (1a), rasagiline (1b) or
safinamide (1c) are already applied as adjunctive therapeutics for PD in order to elevate
dopamine concentration in the brain [16–18]. (Fig 1.)
Figure 1 Structures of potent MAO-B inhibitors
Adenosine A2A receptors (A2AARs) are one of the four subtypes of adenosine receptors
which are subdivided into A1, A2A, A2B, and A3ARs belonging to the G protein-coupled
receptor (GPCR) family. A2AARs are highly expressed in specific areas of the brain, such as

the caudate-putamen.[19,20] Blockade of A2AARs has resulted in neuroprotective effects in
preclinical studies by different cellular mechanism. A2AAR antagonists have beneficial impact
on the enhancement of D2-dopaminergic transmission related to PD and also reduce side
effects of L-DOPA therapy.[21–24] Moreover, there is evidence that blockade of the A2AAR
reduces -amyloid peptide aggregation [23,25].
Based on those considerations, structures with dual target activity as A2A AR
antagonists and MAO-B inhibitors may have potential application in PD or AD possibly
providing both symptomatic and neuroprotective effects. Therefore, the development of new
A2AAR/MAO-B dual targeted structures has been promoted, in particular among structures
based on a xanthine scaffold, however non-xanthine compounds have been also reported.[26–
28]
Caffeine (2a) and theophyline (2b) are naturally occurring unselective AR antagonists
from the group of methylxanthines. The structural modification of the xanthine scaffold,
especially in position 8, led to the discovery of the group of 8-styrylxanthines, among which
istradefylline (3a) and 8-chlorostyrylcaffeine (CSC, 3b) were obtained [24]. Istradefylline is a
potent and selective A2AAR antagonist, that has been approved in Japan for the treatment of
Parkinson’s disease as an adjunct therapy [29], while CSC is one of the first known
antagonists of A2AAR with ancillary MAO-B inhibitory activity [28]. (Fig 2.)
Figure 2 Structures of xanthine derivatives as adenosine receptors antagonists.
The class of tricyclic compounds containing a third ring fused to the f-bond of the 2,6-
purinedione system with a benzyl residue can be considered as bioisosteric analogues of (E)-
8-styrylxanthine. Therefore, the annelated xanthine derivatives with pyrimido[2,1-
f]purinediones scaffold were investigated as AR ligands by our group as well as by
others.[30–32] Koch et al. have reported structure 4 in this class of compounds as A1/A2AARs
antagonist with ancillary MAO-B inhibitory activity. They suggested the crucial role of
substituents in N1,N3-position of xanthine core for triple-target activity. 1,3-
dimethylpyrimido[2,1-f]purinedione derivatives 5 seem to be suitable for both A2A AR
antagonism and MAO-B inhibition, while structures with longer substituents in the N3
position such as propargyl presented higher affinity for ARs while the potency at MAO-B was
decreased[33]. (Fig. 3)
Figure 3 Pyrimido[2,1-f]purinediones derivatives as multiple target directed ligands.
Considering the abovementioned evidences, we made efforts to develop new dual
target structures as A2A antagonists/MAO-B inhibitors with enhanced and more balanced
potencies. As a suitable scaffold, annelated 1,3-dimethylxanthines with the imidazole,
pyrimidine or diazepine ring fused to the f-bond were selected. The investigation of structure-
activity relationships in the group of tricyclic compounds with an aromatic residue attached

by a short one/two carbon spacer was performed. Furthermore, we evaluated the impact of the
size of the heterocyclic ring fused to xanthine core on inhibition of MAO-B and affinity for
A2AARs. To rationalize the observed biological activities of the new compounds, molecular
docking studies using human A2A ARs and human MAO-B were performed. Moreover,
MAO-A inhibition potency was determined for some tricyclic xanthine derivatives, and their
MAO-B selectivity was evaluated. For all final products, selected molecular and ADMET
(absorption, distribution, metabolism, excretion, toxicity) properties were estimated by in
silico calculation. and the most active structures were tested in vitro for hepatotoxic and
antioxidant effects.
2. Results and discussion
2.1. Chemistry
The synthesis of tricyclic 1,3-dimethyl-imidazo-, pyrimido-, and diazepino[2,1-
f]purinediones with an aromatic residue in the 8-, 9-, or 10-position, respectively, of the
annelated xanthine was performed by a three-step procedure as shown in Scheme 1. The
diversity by different aromatic residues was introduced in the last step of the synthetic
pathway during the ring closure reaction with various aromatic amines.
Scheme 1 Synthesis of tricyclic xanthine derivatives) 40 % HBr, NaClO3, CH3COOH, 60 °C; ii) 1,2-dibromoethane/1-
bromo-3-chloropropane/1,4-dibromobutane, Benzyltriethylammonium chloride TEBA, K2CO3, acetone, reflux,
10h; iii) 1-propanol, NEt3, µM, 300W, 160 °C.
As a starting material, theophylline (2b) was oxidatively brominated according to a
previously described method [34]. Subsequently, 8-bromotheophylline (6) was subjected to
N7-alkylation with dihalogenoalkane such as 1,2-dibromoethane, 1-bromo-3-chloropropane or
1,4-dibromobutane. Phase transfer catalysis reaction conditions were applied providing 8-
bromo-7-(2-bromoethyl)theophylline (7a), 8-bromo-7-(3-chloropropyl)theophylline (7b) or 8-
bromo-7-(4-bromobutyl)theophylline (7c).[35] The last step of the synthetic pathway was the
condensation 7a-c with an appropriate amine providing the final tricyclic xanthine derivatives
in the series of 1,3-dimethyl-imidazo- (8a-f), pyrimido- (9a-v), and diazepino-[2,1-
f]purinediones (10a-i). Previously we had described a conventional approach for the ring-
closure reactions, which required long refluxing time in high boiling solvents such as
diethyleneglycol monomethyl ether or DMF [30]. Now we decided to use microwave
irradiation and sealed vessels to reduce the time of the reaction. This approach allowed us to
provide during 1 hour good reaction yields compared to 16h required upon conventional
heating.
The structures of all new synthesized compounds were confirmed by spectral analysis
1
including H NMR, 13C NMR, and MS. The melting points were determined for all new
compounds. The purity of all tested compounds was determined to be at least 95 % using
UPLC coupled to MS.

2.2. Biological evaluation
The final compounds were tested in radioligand binding assays to evaluate their
affinity for all ARs subtypes. Rat brain cortical membranes were used for determining rat A1
AR affinity. Human A2A and A2B were recombinantly expressed in HEK293 cells. Human A1
and human A3ARs were recombinantly expressed in Chinese hamster ovary (CHO) cells.
[3H]2-Chloro-N6-cyclopentyladenosine ([3H]CCPA) [36], [3H]3-(3-hydroxypropyl)-1-
propargyl-7-methyl-8-(m-methoxystyryl)xanthine
([3H]MSX-2) [37],
[3H]8-(4-(4-(4-
chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine ([3H]PSB-603) [38] 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) [39] were used as radioligands for A1, A2A, A2B and A3 AR binding studies, respectively.
Several previous studies on pyrimido[2,1-f]purinediones had shown that compounds
belonging to this group displayed antagonistic properties for ARs.[30,33]
All new compounds were tested for inhibitory potency at human MAO-B. Selected
compounds were additionally tested for selectivity versus human MAO-A.
Table 1 Adenosine receptors affinity of tricyclic compounds and xanthine derivatives
A1 vs. [³H]CCPA
(h, human; r, rat)
A2A vs. [³H]MSX-2
(h, human; r, rat)
A2B vs. ³H]PSB-603 A3 vs. [³H]PSB-11
Compd
R1
Ki ± SEM (µM)
(or % inhibition ± SEM at 1 µM)
Caffeine
2a [40]
44.9 h
41.0 r
23.4 h
33.8 h
32.5 r
13.3 h
4.47 h
0.841 h
0.230 r
0.0912h
Istradefylline
>10.0 h
0.0044 r
3a [40]
>10.0 h (21%)
0.432 ± 0.027 r
1.42 ± 0.08 r
2.03 ± 0.16 h
KD240
0.802 ± 0.156 r
15.2 ± 5.5
>1.00 (10%)
5 [33,41]
1.12 ± 0.14 r
Imidazo[2,1-f]purinedione
>1.0
0.369 ± 0.115
>1.0
(22 ± 10)
0.396 ± 0.134
8a
8b
8c
8d
8e
(30 ± 4)
>1.0
(1 ± 1)
>1.0
>1.0
>1.0
(11 ± 7)
(29 ± 8)
(22 ± 11)
>1.0
(24 ± 3)
>1.0
(21 ± 0)
>1.0
(37 ± 2)
0.887 ± 0.214
>1.0
(6 ± 11)
>1.0
>1.0
>1.0
(1 ± 5)
(30 ± 7)
(2 ± 1)
>1.0
(23 ± 2)
>1.0
>1.0
>1.0
(30 ± 9)
(42 ± 4)
(21 ± 0)

>1.0
(30 ± 1)
>1.0
(3 ± 6)
>1.0
(15 ± 0)
0.784 ± 0.107
8f
Pyrimido[2,1-f]purinedione
1.780 ± 0.420 r
>1.0 r
1.210 ± 0.250
>1.0
>1.0
>1.0
9a
9b
9c
9d
9e
9f
>1.0
>1.0
>1.0
>1.0
>1.0 r
3.040 ± 0.440
>1.0
>1.0 r
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
4.360 ± 0.400 r
>1.0 r
8.270 ± 0.144
>1.0
2.650 ± 0.640 r
0.486 ± 0.022 r
>1.0 r
2.020 ± 0.290
>1.0
9g
9h
9i
>1.0
>1.0 r
8.000 ± 1.890
8.910 ± 2.310
>1.0
9j
4.900 ± 0.280 r
4.130 ± 1.340 r
0.965 ± 0.087 r
2.320 ± 0.130 r
>1.0 r
9k
9l
1.700 ± 0.090
1.610 ± 0.255
>1.0
9m
9n
9o
9p
9q
9r
0.927 ± 0.207 r
0.931 ± 0.242 r
>1.0 r
0.627 ± 0.078
>1.0
>1.0

1.410 ± 0.278 r
1.910 ± 0.648 r
1.550 ± 0.310 r
3.020 ± 0.067 r
1.020 ± 0.275
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
9s
9t
0.189 ± 0.047
>1.0
9u
9v
Diazepino[2,1-f]purinedione
>1.0
(10 ± 7)
>1.0
(41 ± 9)
>1.0
(15 ± 3)
0.881 ± 0.087
10a
10b
10c
10d
10e
10f
10g
10h
10i
>1.0
(11 ± 1)
1080 ± 350
(42 ± 14)
>1.0
(40 ± 1)
>1.0
(11 ± 1)
>1.0
(41 ± 6)
>1.0
(41 ± 2)
>1.0
(26 ± 2)
0.651 ± 0.057
>1.0
(21 ± 2)
>1.0
(33 ± 1)
>1.0
(40 ± 2)
>1.0
(26 ± 4)
>1.0
(21 ± 3)
>1.0
(43 ± 3)
>1.0
(42 ± 7)
0.767 ± 0.115
>1.0
(23 ± 4)
>1.0
(30 ± 5)
>1.0
(43 ± 3)
>1.0
(20 ± 7)
>1.0
(13 ± 4)
>1.0
(16 ± 4)
>1.0
(19 ± 10)
0.959 ± 0.154
>1.0
(16 ± 4)
1.580 ± 0.413
(52 ± 7)
>1.0
(5 ± 3)
>1.0
(31 ± 9)
>1.0
(13 ± 7)
>1.0
(9 ± 5)
>1.0
(13 ± 3)
>1.0
(10 ± 4)
2.3. Structure-activity relationships at adenosine receptors
From a structural point of view, the synthesized compounds can be subdivided into
three series according to the kind of the third heterocyclic ring fused to the xanthine core: 1)
N8-substituted
1,3-dimethylimidazo[2,1-f]purinediones,
2)
N9-substituted
1,3-
dimethylpyrimido[2,1-f]purinediones, 3) N10-substituted 1,3-dimethyldiazepino[2,1-
f]purinediones. Their affinities for ARs are presented in Table 1.
Within the three series, all investigated derivatives showed no affinity for the human
A2B and A3 ARs at the highest tested concentration. In general, most of the compounds
presented none or only negligible affinity for the A1 AR. Only the dual active structure 8a
possesses activity with a Ki value of approximately 0.4 µM for both human A1 and A2AAR.
Modification of this compound by the enlargement of the third heterocyclic ring (9m and 10c)

resulted in a decrease in affinity for both biological targets. Shifting of the bromine atom in
the aromatic ring (8c, 9n, 10c, 10e) led to a drop in affinity for A2AARs in all series, however
in contrast to the imidazo[2,1-f]purinediones, ortho-Br-substituted diazepino[2,1-
f]purinodione (10e) derivatives presented slightly higher affinity than meta-Br derivative
(10c). Within other mono-substituted structures para-Cl (8e, 10i), ortho-F (9b, 10b), ortho-
methyl (9s), meta-methyl (9t), and para-isopropyl (9r) were less well tolerated than bromine
containing derivatives. Exceptions were the ortho-methoxy (8f, 10g) and ortho-Cl (10a)
substituted compounds, which presented comparable affinity for the A2A AR with Ki values
slightly below 1 µM similar to the ortho-Br analogues.
In order to evaluate the structural determinants for interaction with the A2A AR in more
detail, di/tri-substituted benzyl derivatives of the tricyclic xanthines were investigated. Within
the group of di-substituted compounds, 2,4-dichloro (10h), 3,4-dichloro (8b, 10d) and 2,5-
dichloro (9v) displayed low affinity for the A2A AR with Ki values in the micromolar range .
However, replacement of one atom - chlorine or fluorine in the 2,6-disubstitued compound
(9u) increased the affinity remarkably (A2A: 0.185 µM). Further modification of the
substitution pattern of the aromatic ring containing two halogen atoms (8d, 9b, 9d, 9e, 9h, 9k,
9l, 10f) resulted in a reduction of affinity to micromolar values. The same effect was also
observed for 2/4-chloro-5-trifluoromethyl (9o, 9q) and 3-chloro-4-methoxy substituents.
Introduction of a 2-fluoro-3-methoxy-6-chlorobenzyl residue in the N9-position, as a
modification of the potent 2-chloro-6-fluoro derivative, decreased the affinity for A2A AR by
three-fold. Compared to compound 9u, structure 9p presented a Ki value three fold lower.
Another N9-tri-substituted benzyl-1,3-dimethylpyrimido[2,1-f]purinedione, 9f, displayed low
affinity for the A2A AR.
A further strategy was to replace the benzene ring by aromatic heterocyclic ones.
However, introduction of the bioisosteric 2-thienylmethyl moiety (9g) or 3/4-ethylpyridine
residues (9c, 9i) did not improve affinity for the A2A AR.
2.4. Structure-activity relationship at MAO
All compounds were evaluated for their human MAO-B inhibitory potencies.
Moreover, selectivity toward human MAO-A was additionally investigated for selected
structures. The results for MAO-A and MAO-B are presented in Table 2.
Within three series, none of the selected compounds was found to inhibit MAO-A by
50 % or higher at the highest tested concentration of 1 or 100 µM. Therefore, all of the
compounds that were found to inhibit MAO-B are selective versus MAO-A.
In the series of 1,3-dimethylpyrimido[2,1-f]purinediones, structures with IC50-values
in nanomolar range were found in the group N9-benzyl-substituted derivatives. Replacement
of the benzyl residue by a bioisosteric 2-thienylmethyl moiety (9g) or 3/4-ethylpyridine
resulted in
a
drop in inhibitory activity. Within N9-benzyl-substituted 1,3-
dimethylpyrimido[2,1-f]purinediones derivatives, the most potent compound was 9n bearing
an ortho-Br-benzyl moiety (IC50 human MAO-B = 150 nM). Other ortho-mono-substituted
derivatives (9f, 9s) were also relatively potent in the following rank order of potency: Br > Cl
> F > methyl. Generally, a shift of the substituent to the meta-position (9m) was slightly less
tolerated, however meta-methyl derivatives (9t) displayed higher inhibitory activity of MAO-

B than an ortho-methyl analogue (9s). The analysis of structure-activity relationships in the
other two series was consistent with the results described above (8a, 8c, 8f, 10a-c, 10e, 10g).
Additionally, the para-mono-substituted compounds (8e, 10i) presented similar activity as the
meta-substituted analogues. Furthermore, the series of N8-substituted benzyl-1,3-
dimethylimidazo[2,1-f]purinediones seems to be less preferable for MAO-B inhibition
compared to the N9-substituted benzyl-1,3-dimethylpyrimido[2,1-f]purinediones and the N10-
substituted benzyl-1,3-dimethyldiazepino[2,1-f]purinediones, those displayed comparable
results.
Among the group of di-substituted compounds, 2,4- and3,4-disubstituted derivatives
with two halogen atoms or one halogen atom and a trifluoromethyl or methoxy group proved
to be suitable for MAO-B inhibition. The most potent compound was 10d bearing a 3,4-
dichloro-benzyl moiety and a seven-membered heterocyclic ring fused to the xanthine core.
Replacement of chlorine atoms by fluorine or bromine (10f) in 3,4-di-substituted structures
resulted in a slight decrease of activity. The same observation was true for compound 10h
with one chlorine atom in the benzene ring shifted from the 3- to the 2-position. Further
modification of 2,4- and 3,4-di-substituted benzyl derivatives (9b, 9d-e, 9j-l, 10f) did not
improve inhibitory activity, on the other hand they still maintained IC50-values for the enzyme
in the nanomolar range for both series: 1,3-dimethylpyrimido[2,1-f]purinedione and 1,3-
dimethyldiazepino[2,1-f]purinedione derivatives. Nevertheless, in the series of 1,3-
dimethylimidazo[2,1-f]purinediones, compound 8d with a 3-bromo-4-fluorobenzyl moiety
showed higher activity that the 3,4-dichlorobenzyl derivative (8b). A drop of activity was also
observed for 2,4-di- and tri-substituted benzyl compounds within all series.
Compound 9u, which was characterized as the most potent A2A AR ligand among all
investigated series, displayed also MAO-B inhibitory activity (IC50 human MAO-B = 570
nM). However, the 2-chloro-6-fluoro-substituted structure 9u and its derivative with a 2-
fluoro-3-methoxy-6-chlorobenzyl moiety (9p) were less active at MAO-B than 3,4-di-
substituted benzyl derivatives.
Table 2 MAO A and B inhibitory activity of tricyclic xanthine derivatives; nd - not determined
IC50 ± SEM (µM)
(or % inhibition ± SEM at 1 µM or 100 µM)
Compd
MAO-A
MAO-B
Rasagiline 1a
CSC 3b [33]
5 [33,41]
nd
15.4
>10.0
nd
0.0178
0.344
Imidazo[2,1-f]purinedione
> 1.0 (11 ± 1)
> 1.0 (-2 ± 1)
> 1.0 (13 ± 1)
> 1.0 (6 ± 1)
> 1.0 (16 ± 0)
> 1.0 (7 ± 0)
> 1.0 (39 ± 0)
0.515 ± 0.030
0.991 ± 0.090
0.177 ± 0.012
>1.0 (42 ± 0)
111.0 ± 2.9
8a
8b
8c
8d
8e
8f
Pyrimido[2,1-f]purinedione
> 1.0 (-1 ± 3)
> 100.0 (9 ± 3)
nd
0.583 ± 0.209
0.264 ± 0.036
>1.0 (1 ± 1) )
9a
9b
9c

nd
> 1.0 (1 ± 1)
nd
0.411 ± 0.050
0.346 ± 0.022
> 1.0 (24 ± 0)
>1.0 (20 ± 1)
1.18 ± 0.16
9d
9e
9f
9g
9h
9i
nd
nd
nd
>1.0 (4 ± 1)
> 100.0 (38 ± 1)
> 1.0 (2 ± 1.3)
> 1.0 (-2 ± 0.3)
> 1.0 (-1 ± 0.7)
> 1.0 (1 ± 1.2)
> 1.0 (2 ± 0.5)
> 100 (16 ± 5)
nd
0.407 ± 0.085
0.377 ± 0.071
0.426 ± 0.067
0.396 ± 0.011
0.150 ± 0.039
0.310 ± 0.134
0.840 ± 0.025
1.070 ± 0.010
1.350 ± 0.050
>1.0 (40 ± 0)
0.313 ± 0.021
0.570 ± 0.018
0.758 ± 0.009
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
nd
nd
> 100 (17 ± 2)
> 100 (10 ± 0)
nd
9t
9u
9v
Diazepino[2,1-f]purinedione
> 1.0 (4 ± 1)
> 1.0 (14 ± 0)
> 1.0 (6 ± 0)
> 1.0 (5 ± 1)
> 1.0 (3 ± 0)
> 1.0 (-6 ± 1)
> 1.0 (2 ± 1)
> 1.0 (-2 ± 1)
> 1.0 (6 ± 1)
0.343 ± 0.028
>1.0 (39 ±1)
0.371 ± 0.031
0.083 ± 0.016
0.275 ± 0.050
0.171 ± 0.009
1.917 ± 0.491
0.282 ± 0.053
0.364 ± 0.006
10a
10b
10c
10d
10e
10f
10g
10h
10i
2.5. Structure-activity relationships for both A2A AR and MAO-B
The detailed analysis of SAR for each biological target was described above.
Moreover, the global conclusion of structure-activity relationships within investigated library
of compounds based on tricyclic core is assembled in Scheme 2 for both A2A AR and MAO-
B.
Scheme 2 Structure-activity relationships of tricyclic structures
2.6. Molecular modelling studies
2.6.1. Adenosine A2A crystal structure

Among 34 X-ray structures of adenosine A2A receptor complexes deposited in the
PDB database, four are co-crystallized with xanthines: caffeine (PDB codes: 5MZP[42],
3RFM[43]), theophylline (PDB code: 5MZJ[42]) or XAC (PDB code: 3REY. The 3REY
crystal structure was used for docking studies due to the similar size and structure of the
described ligands.
In order to validate the methods used, XAC was redocked to its crystal structure. The
superposition of the phenylpurinedione core with the co-crystallized ligand was almost ideal,
although its flexible polar chain was bent towards the extracellular loop 2 (ECL2). As this
occurrence was in agreement with previous findings [44], the implemented docking protocol
was chosen for further docking studies. Detailed information on docking protocols can be
found in “Experimental protocols” section.

All of the docked ligands can be grouped in two clusters. The highest ranked cluster 1
includes ligand poses where the purinedione core is rotated, yet with still overlapping
carbonyl groups when compared to the XAC conformation in the crystal structure, in a way
that ASN2536.55 forms a hydrogen bond with the carbonyl group in position 2 of the
purinedione core. In this position, the annelated heterocyclic rings, independent of size, point
toward transmembrane helix 2 (TM2). Lower, but still highly ranked cluster 2 includes poses
Figure 4 Left panel: calculated binding pose of 9u (dark red) in the 3REY binding pocket; native ligand XAC shown as violet
wire. Right panel: top, surface view of superimposed calculated binding modes of 8a (green), 9u (dark red).
superimposing the whole phenylpurinedione core, as well as retaining the described ligand-
receptor interactions of XAC: the terminal amino group of ASN 2536.55 forms a hydrogen
bond with the carbonyl group in position 6 of the purinedione core, that is stabilized through
π- π stacking with the aromatic ring of PHE168 (ECL2). Paying attention to the poses and
ligand-receptor interactions of xanthine derivatives in their co-crystallized complexes, it
might be assumed that the binding mode of the ligands described herein in the adenosine A2A
receptor would reflect those of cluster 2 poses. Annelated heterocyclic rings, independent of
size, are located in a narrow pocket formed by PHE 168 and GLU 169 (ECL2) from one side
and TM7 amino acids MET 2707.35 and ILE 2747.39 from the other side, while substituted
benzyl moieties are located between TYR91.35, SER672.65 and TYR2717.36 (Fig. 4). No
additional interactions between halogen and/or alkyl(oxy) substituents of the benzyl moiety
were found for the most affine ligands from all of the subgroups. Therefore, no correlation
between in vitro / and in silico results could be observed, and no explanation for the A2A AR
affinity of particular ligands could be found. However, it can be stated that all of the ligands
fit well into the A2A AR binding pocket forming the expected receptor-ligand interactions.
2.6.2. Monoamine oxidase B crystal structure
As there is no available crystal structure of MAO-B with co-crystallized xanthine
derivatives, we used a structure with Safinamide in this studies (SAF; PDB ID: 2V5Z [45]),
which shows some structural similarities (methylated aminoformamide fragment) to the
purinedione core. An additional argument for using this as a model is documented by
molecular docking studies carried out on 8-styrylcaffeine structures. According to literature

[28], the position of SAF within the binding pocket is as follows: the halogen-substituted
aromatic ring lies within the entry cavity, while the polar fragment is housed in an aromatic
cage and is directed towards the cofactor flavin adenine dinucleotide (FAD). Due to the
structural analogy, a similar arrangement was expected for described herein ligands. Similar
to previous target, and in order to validate the methods used, SAF was redocked to its crystal
structure, and the resulting pose was perfectly superimposed with the native ligand, although
the terminal amide moiety was flipped by 180o horizontally.
All of the ligands fit well, and for most of the docked ligands, the substituted phenyl moiety is
pointing towards the entry cavity, while the purinedione core fits between the cage, formed by
TYR435 and GLN206 on one side, and TYR398 and LEU171 on the other side and is
stabilized by π- π stacking with TYR326 and/or PHE343. Two binding modes of the
purinedione core placement were found. One includes the N3-methyl group pointing toward
TYR326/PHE343, as well as an additional halogen bond formation with THR201 for several
compounds (e.g. 9n-9p), while the phenyl ring is pointing towards the entrance cavity. The
second binding mode differs by a deeper position of the ligands, in close proximity to the
FAD. In this mode, the purinedione core is flipped horizontally and stabilized by π- π stacking
with TYR 398, while the OH-group of TYR188 forms a hydrogen bond with the carbonyl
group in position 6 of the core, along with additional π- π stacking interactions between the
substituted phenyl ring and TYR326 (Fig. 5). This mode was observed for most of the highly
affine pyrimido- and diazepino- annelated derivatives. It might be therefore assumed, that this
binding mode will appear with higher probability, and the described interactions might
influence the ligands’ MAO-B affinity.

Figure 5 Calculated binding modes of 8d (green; upper panel) and 10d (violet; lower panel) superimposed
with the ligand safinamide (SAF, violet, thin wire)
2.7. Blood brain barrier permeability
The final compounds were evaluated in silico for their ability to exceed blood brain
barrier (BBB) as one of the most important parameter for structures acting in the CNS. The
results are assembled in Table S2 (see Supplementary data). Most of investigated structures
were estimated as potential able to penetrated across BBB. Only few compounds were found
with BBB parameter indicating on poor permeability, probably associated with additional
hydrogen bond acceptor group of these structures.
2.8. Hepatotoxicity of tricyclic structures
Prediction of selected drug-likeness parameters and ADME-Tox descriptors using
Molinspiration and pkCSM software provided promising results, with the only problematic
attribute that all compounds might be, according to the calculations, hepatotoxic. The details
of the in silico calculations are given in the Supplementary data. Therefore, the most active
compounds were also tested by in vitro studies to determine probable hepatotoxicity.
Structures 9n, 9u and 10d were incubated for 72 h with the hepatoma HepG2 cell line. For
compounds 9n and 9u similar, statistically significant, antiproliferative effect (p < 0.01, <
0.001) were observed, but only at the highest dose used of 100 µM (Fig. 6).

Figure 6 The effect of the cytostatic drug doxorubicin (DX), the mitochondrial toxin - carbonyl cyanide 3-
chlorophenylhydrazone (CCCP), 9n and 9u on HepG2 cell viability. Statistical significance was evaluated by one-way
ANOVA, followed by Bonferroni’s comparison test (∗∗p < 0.01, ∗∗∗p < 0.001, compared with the negative control).
Interestingly, N10-3,4-dicholorobenzyldiazepino[2,1-f]purinedione derivative 10d was
found as a very hepatotoxic agent. The statistically significant decrease of HepG2 cell
viability was observed at low doses 0.5 µM (p < 0.001) and 1 µM (p < 0.05), respectively
(Fig.7). Results from higher concentrations of 10 and 100 µM were not considered due to the
observed compound precipitation in the cell culture media.
Figure 7 The effect of the cytostatic drug doxorubicin (DX), the mitochondrial toxin - carbonyl cyanide 3-
chlorophenylhydrazone (CCCP) and 10d on HepG2 cell viability. Statistical significance was evaluated by one-way
ANOVA, followed by Bonferroni’s comparison test (∗p < 0.05, ∗∗∗p < 0.001, compared with the negative control).
The hepatotoxicity determined in the in vitro assay was not fully correlated with in
silico calculations. However, compound 10d showed a strong hepatotoxic effect in contrast to
structures 9n and 9u. Therefore, the tricyclic structures based on a xanthine scaffold may
present a certain risk of hepatotoxicity as in silico study predicted. Nevertheless, the toxic
effect seems to be dependent on structural modifications, especially in the attached substituted
aromatic residue rather than the tricyclic core of the molecule.
2.9. Antioxidant activity of selected tricyclic compounds
Antioxidants and free radical scavengers may have beneficial effects for ND treatment
as previously mentioned. Therefore, the selected compounds were tested in two assays to
determine their potential antioxidant properties and the utility in protection from lipid
peroxidation.
2.9.1. Determination of the antioxidant activity by the FRAP assay

Table 3. Influence of the test compounds on the total antioxidant activity in vitro
Compound
vitamin C
9n
Absorption (%)
100
3.40
0.59
10d
Compounds 9n, 10d were tested at a concentration 1000 µM. 9n increased the total
ferric-reducing antioxidant ability by 3.40%, while 10d was practically inactive in this test
(Table 3).
2.9.2. Influence of the test compounds on lipid peroxidation in rat brain homogenate
Table 4. Influence of the test compounds on lipid peroxidation
Compound
Carvedilol
9n
Absorption (%)
44.8
29.5
16.6
10d
Compounds 9n, 10d were tested at a concentration of 1000 µM. 9n and 10d inhibited
lipid peroxidation by 29.5% and 16.6% respectively, (Table 4). This effect is not superior to
the antioxidant activity of carvedilol, which has been used in this assays as reference
compound. However, the results showed additional biological activity that combined with A2A
antagonism and MAO-B inhibition may provide synergistic neuroprotective effects in NDs.
3. Conclusion
Looking for dual A2A AR/MAO-B targeted ligands as potential anti-neurodegenerative
agents, we have explored the xanthine scaffold with a third heterocyclic ring fused to the f-
bond and substituted with an (un)substituted aromatic residue. A library of 37 novel
compounds was synthesized and biologically evaluated providing an extensive structure-
activity relationship analysis for human A2A AR and human MAO-B. Within three
investigated series of tricyclic structures, we identified compounds 9p, 9u, 10a, 10e acting as
selective dual A2A/MAO-B ligands with sub-micromolar activity. These structures presenting
a dual pharmacological profile may have potential application in PD or AD providing both
symptomatic and neuroprotective effects.
Furthermore, the research has resulted in the discovery of selective MAO-B inhibitors
8d, 9n, 10d, 10f with IC50-values in nanomolar range. The 1,3-dimethylpyrimido[2,1-
f]purinedione and the 1,3-dimethyldiazepino[2,1-f]purinedione scaffolds were in particular
suitable for this activity, which was confirmed by molecular docking. The binding mode for
these tricyclic structures was slightly shifted as compared to that of the standard inhibitor
safinamide, being in close proximity to FAD, and the purinedione core was flipped
horizontally and stabilized by π- π stacking.
The in silico calculations of selected ADME-Tox parameters predicted hepatotoxicity
of all final compounds, however in vitro studies provided more insight into the structure-
activity relationships. The toxicity may potentially be elevated for the benzyl derivatives of

the tricyclic structures, nevertheless the most active compounds 9n and 9u presented low
hepatotoxicity when experimentally tested. Therefore, the increased risk of toxic effects
seems to be dependent on structural modifications in the aromatic substitutents of the
molecule.
Moreover, selected compounds were tested as antioxidants in two different assays.
Prevention of lipid peroxidation by tricyclic derivatives based on the xanthine scaffold may
indicate additional synergistic neuroprotective effects of the tested compounds, which makes
them promising candidates for the treatment of NDs.
4.
Experimental protocols
4.1. Chemistry
4.1.1. Material and methods
All commercially available reagents and solvents were used without further purification.
Melting points (mp.) were determined on a MEL-TEMP II (LD Inc., USA) melting point
1
apparatus and are uncorrected. H NMR spectra were performed with a Varian Mercury-VX
300 MHz PFG spectrometer or a Bruker AMX 300 (Bruker, Germany) spectrometer in
DMSO-d6 or CDCl3 with TMS as an internal standard. Chemical shifts were expressed in
parts per million (ppm). 13C NMR data were recorded on 75 MHz on Varian-Mercury-VX
300 MHz PFG or 400 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 final
compounds was determined (%) on an Waters TQD mass spectrometer coupled with an
Waters ACQUITY UPLC system. Retention times (tR) are given in minutes. Microwave
reactions were performed in CEM Discover System microwave oven. Silica gel 60 (0.063-
0.20 mm; Merck) was used for the column chromatography and the mixture of
dichloromethane with methanol was applied as a mobile phase. TLC data were obtained using
aluminium sheets coated with silica gel 60 F254 (Merck). Eluent system: DCM/MeOH 9:1,
DCM/MeOH 9.5:0.5.
The synthesis and physicochemical properties of the compounds 6 and 7a-c were reported
previously.[34,35]
4.1.2. General procedure for
the synthesis of imidazo[2,1-f]purinedione (8a-f),
pyrimido[2,1-f]purinedione (9a-v) and diazepino[2,1-f]purinedione (10a-i).
A mixture of 0.55 mmol of 8-bromo-7-(2-bromoethyl)theophylline 7a or 8-bromo-7-(3-
chloropropyl)theophylline 7b or 8-bromo-7-(4-bromobutyl)theophylline 7c, 1.1 mmol of
appropriate aromatic amine, 1,6 mmol of TEBA and 1.00 ml of propanol was heated in closed
vessels in microwave oven (300 Watt, Power Max Off, 160 °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). The precipitate was filtered off and dried.
8-(3-bromobenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-2,4(3H,6H)-dione
(8a)
1
Yield: 104 mg; 49 %; mp: 205-207°C; H NMR (300 MHz, DMSO-d6)  ppm 3.15 (s, 3 H,
N3CH3), 3.34 (s, 3 H, N1CH3), 3.73 - 3.81 (m, 2 H, C7H2), 4.05 - 4.13 (m, 2 H, C6H2), 4.48

(s, 2 H, N8CH2), 7.30 - 7.34 (m, 2 H, C5H, C6H, phe), 7.49 - 7.52 (m, 1 H, C2H), 7.72 - 7.77
(m, 1 H, C4H); UPLC/MS purity 98.3 %; tR = 5.53; C16H16BrN5O2; MW 390.23; [M]+ 390.04
8-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-2,4(3H,6H)-
dione (8b)
1
Yield: 124 mg; 59 %; mp: 202-204°C; H NMR (400 MHz, DMSO-d6)  ppm 3.19 (br. s., 3
H, N3CH3), 3.37 (br. s., 3 H, N1CH3), 3.76 - 3.87 (m, 2 H, C7H2), 4.06 - 4.19 (m, 2 H, C6H2),
4.52 (br. s., 2 H, N8CH2), 7.34 - 7.41 (m, 1 H, C6H, phe), 7.62 - 7.69 (m, 2 H, C2H, C5H,
phe); UPLC/MS purity 96.7%; tR = 5.99; C16H15Cl2N5O2; MW 380.23; [M]+ 380.07
8-(2-bromobenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-2,4(3H,6H)-dione
(8c)
1
Yield: 90 mg; 41 %; mp: 224-226°C; H NMR (300 MHz, DMSO-d6)  ppm 3.16 (s, 3 H,
N3CH3), 3.33 (s, 3 H, N1CH3), 3.78 - 3.90 (m, 2 H, C7H2), 4.08 - 4.18 (m, 2 H, C6H2), 4.55
(s, 2 H, N8CH2), 7.28 - 7.32 (m, 1 H, C6H, phe), 7.40 (td, J=7.62, 1.17 Hz, 1 H, C4H, phe),
7.45 - 7.50 (m, 1 H, C5H, phe), 7.65 (d, J=7.03 Hz, 1 H, C3H, phe); 13C NMR (DMSO-d6) δ
ppm 27.8 (N3CH3), 30.2 (N1CH3), 43.7 (C6), 50.7 (C7), 52.9 (N8CH2), 102.5 (C4a), 123.4
(C2, phe), 128.6 (C5, phe), 130.2 (C4, phe), 133.2 (C6, phe), 135.8 (C3, phe), 138.4 (C1,
phe), 149.7 (C10a), 151.4 (C2), 152.9 (C4), 160.8 (C9a); UPLC/MS purity 95.8%; tR = 5.47;
C16H16BrN5O2; MW 390.23; [M]+ 390.04
8-(3-bromo-4-fluorobenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-
2,4(3H,6H)-dione (8d)
Yield: 116 mg; 52 %; mp: 178-180°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm: 3.38
(s, 3 H, N3CH3), 3.55 (s, 3 H N1CH3), 3.77 - 3.84 (m, 2 H, C7H2), 4.21 (t, J=8.02 Hz, 2 H,
C6H2), 4.57 (s, 2 H, N8CH2), 7.17 - 7.24 (m, 1 H, C3H, phe), 7.31 - 7.34 (m, 2 H, C5H,C6H
13
phe); C NMR (CHLOROFORM-d) δ ppm: 27.8 (N3CH3), 30.1 (N1CH3), 43.4 (C6), 43.6
2
2
(C7), 52.0 (N8CH2), 103.0 (C4a), 119.4 (d, JC,F = 24.9 Hz, C3, phe), 121.7 (d, JC,F = 14.7
4
3
Hz, C5, phe), 122.6 (d, JC,F = 9.5 Hz, C6, phe), 128.0 (d, JC,F = 3.6 Hz, C2, phe), 131.5 (d,
3JC,F = 5.1 Hz, C1, phe), 151.7 (C9a), 153.2 (C8a), 153.8 (C2), 159.5 (C4) 161.2 (d, JC,F
1
=176.1 Hz, C4, phe),; UPLC/MS purity 100.0 %; tR = 5.19; C16H15BrFN5O2; MW 408.23;
[M+H]+ 409.04
8-(4-chlorobenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-2,4(3H,6H)-dione
(8e)
1
Yield: 126 mg, 66 %; mp: 216-218°C; H NMR (300 MHz, DMSO-d6)  ppm 3.16 (s, 3 H,
N3CH3), 3.34 (br. s., 3 H, N1CH3), 3.83 (t, J=8.21 Hz, 2 H, C7H2), 4.09 - 4.16 (m, 2 H,
C6H2), 4.58 (s, 2 H, N8CH2), 7.33 - 7.38 (m, 2 H, C2H, C6H, phe), 7.46 - 7.52 (m, 2 H, C3H,
13
C5H, phe); C NMR (DMSO-d6) δ ppm 27.8 (N3CH3), 30.2 (N1CH3), 43.7 (C6), 48.2 (C7),
52.8 (N8CH2), 102.5 (C4a), 128.0 (C3, C5, phe), 130.0 (C2, C6, phe), 133.1 (C4, phe), 134.3
(C1, phe), 151.4 (C10a), 153.0 (C2), 153.11 (C4), 160.9 (C9a); UPLC/MS purity 97.2%; tR =
4.79; C16H16ClN5O2; MW 345.78; [M+H]+ 346.11
8-(2-methoxybenzyl)-1,3-dimethyl-7,8-dihydro-1H-imidazo[2,1-f]purine-2,4(3H,6H)-
dione (8f)
1
Yield: 109 mg; 58 %; mp: 198-200°C; H NMR (300 MHz, DMSO-d6)  ppm 3.15 (s, 3 H,
N3CH3), 3.22 - 3.32 (m, 2 H, C7H2), 3.33 (s, 3 H, N1CH3), 3.78 (s, 3 H, OCH3), 3.96 - 4.15
(m, 2 H, C6H2), 4.45 (s, 2 H, N9CH2), 6.92 (t, J=7.03 Hz, 1 H, C4, phe), 7.02 (d, J=8.21 Hz,
1 H, C3, phe), 7.18 - 7.37 (m, 2 H, C5, C6, phe); 13C NMR (DMSO-d6) δ ppm: 27.8 (N3CH3),
30.2 (N1CH3), 43.7 (C6), 45.2 (C7), 52.5 (N9CH2), 55.9 (OCH3), 102.3 (C4a), 111.3 (C3,

phe), 112.6 (C5, phe), 115.4 (C1, phe), 120.9 (C4, phe), 124.4 (C6, phe), 129.6 (C10a), 151.4
(C9a), 153.1 (C2), 157.6 (C4), 161.2 (C2, phe); UPLC/MS purity 98.68 %; tR = 5.21;
C17H19N5O3; MW 341.36; [M+H]+ 342.04
9-(2-Fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-
dione (9a)
Yield: 136 mg, 72 %; mp: 219 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.13 (dt,
J=11.73, 5.80 Hz, 2 H, C7H2), 3.32 - 3.38 (m, 5 H, N3CH3, C8H2), 3.55 (s, 3 H, N1CH3), 4.22
(t, J=6.03 Hz, 2 H, C6H2), 4.83 (s, 2 H, N9CH2), 7.03 - 7.15 (m, 2 H, C5H/C6H, phe), 7.27 -
13
7.34 (m, 1 H, C3H, phe), 7.39 - 7.46 (m, 1 H, C4H, phe). C NMR (CHLOROFORM-d) δ
3
ppm: 21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.7 (C6), 44.2 (C8), 46.4 (d, JC,F = 4.6 Hz,
2
2
N9CH2), 103.0 (C4a), 115.5 (d, JC,F = 21.8 Hz, C3, phe), 123.6 (d, JC,F = 15 Hz, C1, phe),
124.4 (d, 4JC,F = 3.5 Hz, C5, phe), 129.6 (d, 3JC,F = 8.1 Hz, C4, phe), 130.6 (d, 3JC,F = 4.6 Hz,
1
C6, phe), 148.7 (C10a), 151.6 (C9a), 151.8 (C2), 153.9 (C4), 161.1 (d, JC,F =246.4 Hz, C2,
phe). UPLC/MS purity 97.01 %, tR = 5.45. C17H18FN5O2, MW 343.36, [M+H]+ 344.32.
9-(4-Chloro-3-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9b)
Yield: 100 mg, 53 %; mp: 196 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.15 (dt,
J=11.80, 5.90 Hz, 2 H, C7H2), 3.27 (t, J=5.60 Hz, 2 H, C8H2), 3.37 (s, 3 H, N3CH3), 3.52 (s, 3
H, N1CH3), 4.24 (t, J=6.03 Hz, 2 H, C6H2), 4.72 (s, 2 H, N9CH2), 7.02 - 7.08 (m, 1 H, C2H,
13
phe), 7.13 (dd, J=9.62, 1.92 Hz, 1 H, C6H, phe), 7.37 (m, J=15.64 Hz, 1 H, C5H, phe). C
NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.8 (N1CH3), 41.7 (C6), 44.0
2
(C8), 52.2 (N9CH2), 103.1 (C4a), 116.2 (d, JC,F = 21.9 Hz, C2, phe), 120.2 (C4, phe), 124.4
(d, 4JC,F = 3.4 Hz, C6, phe), 130.9 (C5, phe), 137.7 (d, 3JC,F = 5.8 Hz, C1, phe), 148.5 (C10a),
1
151.3 (C9a), 151.8 (C2), 153.9 (C4), 158.2 (d, JC,F =249.9 Hz, C3, phe). UPLC/MS purity
97.01 %, tR = 5.45. C17H18FN5O2, MW 343.36, [M+H]+ 344.32. UPLC/MS purity 100 %, tR =
6.13. C17H17ClFN5O2, MW 377.11, [M+H]+ 378.75.
1,3-Dimethyl-9-(2-(pyridin-3-yl)ethyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9c)
1
Yield: 90 mg, 48 %; mp: 206 °C; H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.07 -
2.14 (m, 2 H, C7H2), 3.02 (t, J=7.24 Hz, 2 H, N9CH2CH2), 3.29 (m, J=5.48 Hz, 2 H, C8H2),
3.38 (s, 3 H, N3CH3), 3.52 (s, 3 H, N1CH3), 3.76 - 3.80 (m, 2 H, N9CH2), 4.21 (t, J=5.87 Hz,
2 H, C6H2), 7.31 (dd, J=7.63, 4.89 Hz, 1 H, C5H, pyr), 7.65 (dt, J=7.83, 1.76 Hz, 1 H, C6H,
13
pyr), 8.51 (dd, J=4.69, 1.57 Hz, 1 H, C4H, pyr), 8.54 - 8.56 (m, 1 H, C2H, pyr). C NMR
(CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 31.2, (N9CH2CH2),
41.6 (C6), 45.4 (C8), 51.0 (N9CH2CH2), 102.9 (C4a), 123.7 (C5, pyr), 134.8 (C6, pyr), 137.2
(C1, pyr), 147.2 (C4, pyr), 148.9 (C10a), 149.3 (C2, pyr), 151.9 (C2), 153.9 (C4). UPLC/MS
purity 95.07 %, tR = 2.34. C17H20N6O2, MW 340.39, [M+H]+ 341.13.
9-(2-Chloro-4-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9d)
1
Yield: 75 mg, 36 %; mp: 190 °C; H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.14 -
2.21 (m, 2 H, C7H2), 3.35 (t, J=5.90 Hz, 2 H, C8H2), 3.39 (s, 3 H, N3CH3), 3.56 (s, 3 H,
N1CH3), 4.27 (t, J=6.06 Hz, 2 H, C6H2), 4.90 (s, 2 H, N9CH2), 7.00 (td, J=8.31, 2.54 Hz, 1
H, C5H, phe), 7.18 (m, J=2.74 Hz, 1 H, C6H, phe), 7.43 (dd, J=8.61, 5.87 Hz, 1 H, C3H,
13
phe). C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.7 (N3CH3), 30.0 (N1CH3), 41.7
2
(C6), 44.4 (C8), 49.9 (N9CH2), 103.0 (C4a), 114.5 (d, JC,F = 20.5 Hz, C5, phe), 117.2 (d,

2JC,F = 25 Hz, C3, phe), 130.1 (d, 4JC,F = 3.7 Hz, C1, phe) , 131.1 (d, 3JC,F = 8.8 Hz, C6, phe),
134.5 (d, 3JC,F = 10.3 Hz, C2, phe), 148.1 (C10a), 151.1 (C9a), 151.8 (C2), 153.9 (C4), 162.0
1
(d, JC,F =250.1 Hz, C4, phe). UPLC/MS purity 98.18 %, tR = 6.18. C17H17ClFN5O2, MW
377.80, [M+H]+ 378.29.
9-(3-Bromo-4-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9e)
1
Yield: 70 mg, 30 %; mp: 199 °C; H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.16
(quin, J=5.87 Hz, 2 H, C7H2), 3.28 (t, J=5.50 Hz, 2 H, C8H2), 3.39 (s, 3 H, N3CH3), 3.55 (s, 3
H, N1CH3), 4.25 (t, J=6.06 Hz, 2 H, C6H2), 4.71 (s, 2 H, N9CH2), 7.11 (t, J=8.41 Hz, 1 H,
13
C5H, phe), 7.25 - 7.28 (m, 1 H, C6H, phe), 7.55 (dd, J=6.46, 2.15 Hz, 1 H, C2H, phe). C
NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.7 (N3CH3), 29.8 (N1CH3), 41.7 (C6), 44.0
(C8), 52.0 (N9CH2), 103.1 (C4a), 109.4 (d, 2JC,F = 21.2 Hz, C3, phe), 116.7 (d, 2JC,F = 22 Hz,
3
4
C5, phe), 128.7 (d, JC,F = 7.4 Hz, C6, phe), 133.2 (C2, phe), 134.1 (d, JC,F = 3.7 Hz, C1,
1
phe), 148.5 (C10a), 151.3 (C9a), 151.8 (C2), 153.9 (C4), 158.7 (d, JC,F = 248 Hz, C4, phe).
UPLC/MS purity 100 %, tR = 6.23. C17H17BrFN5O2, MW 422.26, [M+H]+ 422.01.
1,3-Dimethyl-9-(3,4,5-trifluorobenzyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9f)
Yield: 95 mg, 46 %; mp: 236 °C; 1H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.16 - 2.23
(m, 2 H, C7H2), 3.31 (t, J=5.90 Hz, 2 H, C8H2), 3.39 (s, 3 H, N3CH3), 3.54 (s, 3 H, N1CH3),
4.27 (t, J=6.06 Hz, 2 H, C6H2), 4.71 (s, 2 H, N9CH2), 6.96 - 7.02 (m, 2 H, C2H/C6H, phe).
13C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.7 (N3CH3), 29.9 (N1CH3), 41.7 (C6),
44.2 (C8), 52.3 (N9CH2), 103.2 (C4a), 111.8-112.1 (m, C2/C6, phe), 133.0-133.2 (m, C1,
3
4
phe), 140.6 (C4, phe), 148.2 (C10a), 151.0 (C9a), 151.8 (C2), 152.6 (dd, JC,F = 10.3, JC,F
=
3.7 Hz, C3/C5, phe), 154.0 (C4). UPLC/MS purity 100 %, tR = 5.93. C17H16F3N5O2, MW
379.34, [M+H]+ 380.35.
1,3-Dimethyl-9-(thiophen-2-ylmethyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9g)
1
Yield: 75 g, 41 %; mp: 203 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.13 (dt,
J=11.72, 5.86 Hz, 2 H, C7H2), 3.28 - 3.33 (m, 2 H, C8H2), 3.35 (s, 3 H, N3CH3), 3.53 (s, 3 H,
N1CH3), 4.19 (t, J=6.15 Hz, 2 H, C6H2), 4.87 (s, 2 H, N9CH2), 6.95 (dd, J=4.69, 3.52 Hz, 1
H, C4H, thiophen-2-yl), 7.00 - 7.03 (m, 1 H, C5H, thiophen-2-yl), 7.23 (dd, J=4.69, 1.17 Hz,
1 H, C3H, thiophen-2-yl).13C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3),
29.7 (N1CH3), 41.7 (C6), 43.7 (C8), 47.7 (N9CH2), 103.1 (C4a), 125.7 (C5, thiophen-2-yl),
126.77 (C3/C4, thiophen-2-yl), 138.8 (C2, thiophen-2-yl), 148.8 (C10a), 151.3 (C9a), 151.9
(C2), 153.9 (C4). UPLC/MS purity 94.72 %, tR = 5.08. C15H17N5O2S, MW 331.39, [M+H]+
332.28.
9-(5-Bromo-2-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9h)
1
Yield: 88 mg, 38 %; mp: 194 °C; H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.18 (dt,
J=11.74, 5.87 Hz, 2 H, C7H2), 3.35 - 3.40 (m, 5 H, C8H2, N3CH3), 3.57 (s, 3 H, N1CH3), 4.25
(t, J=6.06 Hz, 2 H, C6H2), 4.79 (s, 2 H, N9CH2), 6.96 - 7.02 (m, 1 H, C3H, phe), 7.39 - 7.44
(m, 1 H, C6H, phe), 7.60 (dd, JH,F=6.46, 2.54 Hz, 1 H, C4, phe). 13C NMR (CHLOROFORM-
d) δ ppm: 21.4 (C7), 27.7 (N3CH3), 29.9 (N1CH3), 41.7 (C6), 44.6 (C8), 46.5 (N9CH2), 103.1
(C4a), 116.9 (C5, phe), 117.3 (d, 2JC,F = 23.5 Hz, C3, phe), 125.9 (d, 2JC,F = 16.9 Hz, C1, phe),
3
3
132.5 (d, JC,F = 8.07 Hz, C4, phe), 133.6 (d, JC,F = 4.4 Hz, C6, phe), 148.2 (C10a), 151.0

1
(C9a), 151.8 (C2), 154.0 (C4), 161.2 (d, JC,F = 247.3 Hz, C2, phe). UPLC/MS purity 98.40
%, tR = 6.25. C17H17BrFN5O2, MW 422.26, [M+H]+ 424.01.
1,3-Dimethyl-9-(2-(pyridin-4-yl)ethyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9i)
1
Yield: 40 mg, 21 %; mp: 205 °C; H NMR (400 MHz, CHLOROFORM-d)  ppm: 2.10 (dt,
J=11.74, 5.87 Hz, 2 H, C7H2), 3.03 (t, J=7.24 Hz, 2 H, N9CH2CH2), 3.28 (t, J=5.90 Hz, 2 H,
C8H2), 3.38 (s, 3 H, N3CH3), 3.53 (s, 3 H, N1CH3), 3.80 (t, J=7.40 Hz, 2 H, N9CH2), 4.21 (t,
J=6.06 Hz, 2 H, C6H2), 7.25 - 7.28 (m, 2 H, C2H/ C6H, pyr), 8.55 - 8.58 (m, 2 H, C3H/ C5H,
13
pyr). C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 33.5
(N9CH2CH2), 41.6 (C6), 45.5 (C8), 50.4 (N9CH2CH2), 103.0 (C4a), 124.5 (C2/C6, pyr),
148.7 (C10a), 148.9 (C1, pyr), 149.3 (C3/5, pyr), 151.1 (C9a), 151,9 (C2), 153.9 (C4).
UPLC/MS purity 96.37 %, tR = 2.30, C17H20N6O2, MW 340.39, [M+H]+ 341.13.
9-(3-Chloro-4-methoxybenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9j)
Yield: 88 mg, 41 %; mp: 213 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.11 (quin,
J=5.86 Hz, 2 H, C7H2), 3.23 (t, J=5.30 Hz, 2 H, C8H2), 3.36 (s, 3 H, N3CH3), 3.52 (s, 3 H,
N1CH3), 3.89 (s, 3 H, OCH3), 4.21 (t, J=5.86 Hz, 2 H, C6H2), 4.64 (s, 2 H, N9CH2), 6.89 (d,
J=8.21 Hz, 1 H, C5H, phe), 7.18 (dd, J=8.50, 2.05 Hz, 1 H, C6H, phe), 7.33 (d, J=2.34 Hz, 1
H, C2H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.7
(N1CH3), 41.6 (C6), 43.6 (C8), 52.0 (N9CH2), 56.2 (OCH3), 103.0 (C4a), 112.1 (C5, phe),
122.6 (C3, phe), 127.6 (C2, phe), 129.7 (C6, phe), 130.1 (C1, phe), 148.8 (C10a), 151.6
(C9a), 151,9 (C2), 153.9 (C4), 154.6 (C4, phe). UPLC/MS purity 95.94 %, tR = 5.77,
C18H20ClN5O3, MW 389.84, [M+H]+ 390.04.
9-(4-Chloro-2-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9k)
1
Yield: 72 mg, 35 %; mp: 192 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.14 (dt,
J=11.72, 5.86 Hz, 2 H, C7H2), 3.30 - 3.37 (m, 5 H, C8H2, N3CH3), 3.51 (s, 3 H, N1CH3), 4.21
(t, J=6.15 Hz, 2 H, C6H2), 4.74 (s, 2 H, N9CH2), 7.11 (d, J=8.79 Hz, 2 H, C5H/C6H, phe),
7.37 (m, J=15.82 Hz, 1 H, C3H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6
2
(N3CH3), 29.7 (N1CH3), 41.7 (C6), 44.4 (C8), 46.1 (N9CH2), 103.1 (C4a), 116.3 (d, JC,F
=
25.5 Hz, C3, phe), 122.5 (C1, phe), 124.9 (d, 4JC,F = 3.5 Hz, C5, phe), 131.5 (d, 3JC,F = 4.6 Hz,
3
C6, phe), 134.5 (d, JC,F = 10.4 Hz, C4, phe), 148.6 (C10a), 151.4 (C9a), 151.8 (C2), 153.9
(C4), 159.2 (C2, phe). UPLC/MS purity 100 %, tR = 6.30, C17H17ClFN5O2, MW 377.80,
[M+H]+ 378.01.
9-(4-Bromo-2-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9l)
Yield: 94 mg, 40 %; mp: 203 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.09 - 2.18
(m, 2 H, C7H2), 3.30 - 3.37 (m, 5 H, C8H2, N3CH3), 3.51 (s, 3 H, N1CH3), 4.21 (t, J=5.86 Hz,
2 H, C6H2), 4.72 (s, 2 H, N9CH2), 7.23 - 7.35 (m, 3 H, C3H/C5H/C6H, phe). 13C NMR
(CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.7 (C6), 44.4 (C8),
46.4 (N9CH2), 103.1 (C4a), 119.2 (d, 2JC,F = 25.3 Hz, C3, phe), 122.0 (d, 3JC,F = 10.8 Hz, C4,
2
4
3
phe), 122.9 (d, JC,F = 16.1 Hz, C1, phe), 127.8 (d, JC,F = 4.6 Hz, C5, phe), 131.8 (d, JC,F
=
4.6 Hz, C6, phe), 148.7 (C10a), 151.4 (C9a), 151,8 (C2), 153.9 (C4), 159.9 (d, 1JC,F = 251 Hz,
C2, phe). UPLC/MS purity 100 %, tR = 6.47, C17H17BrFN5O2, MW 422.26, [M+H]+ 423.94.

9-(3-Bromobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-
dione (9m)
1
Yield: 96 mg, 43 %; mp: 162 °C; H NMR (300 MHz, DMSO-d6)  ppm: 2.05 (quin, J=5.71
Hz, 2 H, C7H2), 3.15 (s, 3 H, N3CH3), 3.25 - 3.30 (m, 2 H, C8H2), 3.32 (s, 3 H, N1CH3), 4.06
(t, J=5.86 Hz, 2 H, C6H2), 4.67 (s, 2 H, N9CH2), 7.26 - 7.35 (m, 2 H, C5H/C6H, phe), 7.44 -
7.49 (m, 1 H, C2H, phe), 7.52 - 7.54 (m, 1 H, C4H, phe). 13C NMR (DMSO-d6) δ ppm: 21.2
(C7), 27.6 (N3CH3), 29.8 (N1CH3), 41.8 (C6), 44.2 (C8), 51.9 (N9CH2), 102.6 (C4a), 122.3
(C3, phe), 127.2 (C6, phe), 130.7 (C5, phe), 130.9 (C4, phe), 131.2 (C2, phe), 140.5 (C1,
phe), 148.5 (C10a), 151.4 (C9a), 151.8 (C2), 153.1 (C4). UPLC/MS purity 98.96 %, tR = 6.19,
C17H18BrN5O2, MW 404.27, [M+H]+ 406.99.
9-(2-Bromobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-
dione (9n)
1
Yield: 99 mg, 45 %; mp: 194 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.16 (dt,
J=11.72, 5.86 Hz, 2 H, C7H2), 3.30 - 3.37 (m, 5 H, N3CH3, C8H2), 3.49 (s, 3 H, N1CH3),
4.25 (t, J=6.15 Hz, 2 H, C6H2), 4.86 (s, 2 H, N9CH2), 7.13 - 7.20 (m, 1 H, C4H, phe), 7.28 -
7.36 (m, 2 H, C5H/C6H, phe), 7.55 - 7.60 (m, 1 H, C3H, phe). 13C NMR (CHLOROFORM-
d) δ ppm: 21.5 (C7), 27.6 (N3CH3), 29.8 (N1CH3), 41.7 (C6), 44.3 (C8), 53.0 (N9CH2), 103.1
(C4a), 123.7 (C2, phe), 127.8 (C5, phe), 129.2 (C4, phe), 129.3 (C6, phe), 133.1 (C3, phe),
135.8 (C1, phe), 148.8 (C10a), 151.7 (C9a), 151,9 (C2), 153.8 (C4). UPLC/MS purity 98.21
%, tR = 6.19, C17H18BrN5O2, MW 404.27, [M+H]+ 406.00.
9-(4-Chloro-3-(trifluoromethyl)benzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-
f]purine-2,4(1H,3H)-dione (9o)
1
Yield: 69 mg, 29 %; mp: 185 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.16 (dt,
J=11.72, 5.86 Hz, 2 H, C7H2), 3.28 (t, J=5.90 Hz, 2 H, C8H2), 3.36 (s, 3 H, N3CH3), 3.50 (s,
3 H, N1CH3), 4.24 (t, J=6.15 Hz, 2 H, C6H2), 4.73 (s, 2 H, N9CH2), 7.42 - 7.51 (m, 2 H,
C5H/C6H, phe), 7.66 - 7.69 (m, 1 H, C2H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 21.4
(C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.7 (C6), 44.2 (C8), 52.2 (N9CH2), 103.2 (C4a), 120.8
(CF3), 127.3-127.4 (m, C2, phe), 128.5 (C3, phe), 131.9 (C5, phe), 132.4 (C4/6, phe), 136.0
(C1, phe), 148.6 (C10a), 151.3 (C9a), 151,8 (C2), 154.0 (C4). UPLC/MS purity 96.35 %, tR =
6.80, C18H17ClF3N5O2, MW 427.81, [M+H]+ 428.06.
9-(6-Chloro-2-fluoro-3-methoxybenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-
f]purine-2,4(1H,3H)-dione (9p)
Yield: 78 mg, 35 %; mp: 193 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.04 - 2.12
(m, 2 H, C7H2), 3.23 (t, J=5.90 Hz, 2 H, C8H2), 3.35 (s, 3 H, N3CH3), 3.52 (s, 3 H, N1CH3),
3.88 (s, 3 H OCH3), 4.19 (t, J=6.15 Hz, 2 H, C6H2), 4.92 (d, J=1.76 Hz, 2 H, N9CH2), 6.89 (t,
J=9.08 Hz, 1 H, C4H, phe), 7.15 (dd, J=8.79, 1.76 Hz, 1 H, C5H, phe). 13C NMR
(CHLOROFORM-d) δ ppm: 21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.6 (C6), 43.4
3
(OCH3), 44.3 (C8), 56.4 (N9CH2), 103.0 (C4a), 113.5 (d, JC,F = 2.3 Hz, C4, phe), 122.3 (d,
2JC,F = 13.8 Hz, C1, phe), 124.8 (d, 3JC,F = 4.6 Hz, C6, phe), 126.5 (d, 4JC,F = 4.6 Hz, C5, phe),
2
146.8 (d, JC,F = 11.5 Hz, C3, phe), 148.6 (C10a), 151.3 (C9a), 151,8 (C2), 153.6 (C2, phe),
154.0 (C4). UPLC/MS purity 97.77 %, tR = 5.87, C18H19ClFN5O3, MW 407.83, [M+H]+
407.99.
9-(2-Chloro-5-(trifluoromethyl)benzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-
f]purine-2,4(1H,3H)-dione (9q)
Yield: 85 mg, 36 %; mp: 215 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.19 (quin,
J=5.86 Hz, 2 H, C7H2), 3.36 (s, 3 H, N3CH3), 3.41 (t, J=5.90 Hz, 2 H, C8H2), 3.49 (s, 3 H,

N1CH3), 4.26 (t, J=5.86 Hz, 2 H, C6H2), 4.86 (s, 2 H, N9CH2), 7.50 - 7.53 (m, 2 H,
C3H/C4H, phe), 7.73 - 7.76 (m, 1 H, C6H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 21.5
(C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.7 (C6), 45.2 (C8), 50.6 (N9CH2), 103.2 (C4a), 125.8
3
3
(d, JC,F = 3.5 Hz, C4, phe), 127.1 (d, JC,F = 2.5 Hz, C6, phe), 129.7 (C5, phe), 130.3 (C3,
phe), 135.6 (C1, phe), 137.5 (C2, phe), 148.8 (C10a), 151.7 (C9a), 151,9 (C2), 154.0 (C4).
UPLC/MS purity 100 %, tR = 6.80, C18H17ClF3N5O2, MW 427.81, [M+H]+ 428.06.
9-(4-(tert-Butyl)benzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9r)
Yield: 93 mg, 40 %; mp: 178 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 1.31 (s, 9
H, C(CH3)2), 2.11 (dt, J=11.72, 5.86 Hz, 2 H, C7H2), 3.26 (t, J=5.30 Hz, 2 H, C8H2), 3.37 (s,
3 H, N3CH3), 3.52 (s, 3 H, N1CH3), 4.21 (t, J=5.86 Hz, 2 H, C6H2), 4.71 (s, 2 H, N9CH2),
7.20 - 7.25 (m, 2 H, C2H/C6H, phe), 7.33 - 7.38 (m, 2 H, C3H/C5H, phe). 13C NMR
(CHLOROFORM-d) δ ppm: 21.3 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 31.3 (C(CH3)3), 34.5
(C(CH3)3), 41.7 (C6), 43.7 (C8), 52.6 (N9CH2), 103.0 (C4a), 125.6 (C3/5, phe), 126.0 (C2/6,
phe), 127.7 (C1, phe), 133.5 (C4, phe), 148.9 (C10a), 150.8 (C9a), 151,9 (C2), 153.9 (C4).
UPLC/MS purity 100 %, tR = 7.38, C21H27N5O2, MW 427.81, [M+H]+ 382.20.
1,3-Dimethyl-9-(2-methylbenzyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-
dione (9s)
1
Yield: 101 mg, 54 %; mp: 222 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.06 -
2.17 (m, 2 H, C7H2), 2.33 (s, 3 H, CH3), 3.20 (t, J=5.90 Hz, 2 H, C8H2), 3.37 (s, 3 H,
N3CH3), 3.50 (s, 3 H, N1CH3), 4.24 (t, J=6.15 Hz, 2 H, C6H2), 4.75 (s, 2 H, N9CH2), 7.16 -
7.24 (m, 4 H, C3H/C4H/C5H/C6H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 19.2 (CH3),
21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.7 (C6), 43.4 (C8), 50.8 (N9CH2), 103.0 (C4a),
126.1 (C5, phe), 127.8 (C4, phe), 128.5 (C6, phe), 130.7 (C2, phe), 134.2 (C1, phe), 136.8
(C4, phe), 148.9 (C10a), 151.9 (C9a), 151,9 (C2), 153.9 (C4). UPLC/MS purity 98.46 %, tR =
5.86, C18H21N5O2, MW 339.40, [M+H]+ 340.20.
1,3-Dimethyl-9-(3-methylbenzyl)-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-
dione (9t)
1
Yield: 97 mg, 52 %; mp: 171 °C; H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.11 (dt,
J=11.72, 5.86 Hz, 2 H, C7H2), 2.34 (s, 3 H, CH3), 3.24 (t, J=5.90 Hz, 2 H, C8H2), 3.37 (s, 3
H, N3CH3), 3.53 (s, 3 H, N1CH3), 4.22 (t, J=6.15 Hz, 2 H, C6H2), 4.71 (s, 2 H, N9CH2), 7.07
- 7.14 (m, 3 H, C2H/C4H/C6H, phe), 7.19 - 7.24 (m, 1 H, C5H, phe). 13C NMR
(CHLOROFORM-d) δ ppm: 21.3 (C7), 21.4 (CH3), 27.6 (N3CH3), 29.8 (N1CH3), 41.7 (C6),
43.6 (C8), 52.9 (N9CH2), 102.9 (C4a), 125.1 (C6, phe), 128.6 (C4, phe), 128.6 (C5, phe),
128.8 (C2, phe), 136.5 (C1, phe), 138.4 (C3, phe), 148.8 (C10a), 151.8 (C9a), 151,9 (C2),
153.9 (C4). UPLC/MS purity 99.11 %, tR = 5.94, C18H21N5O2, MW 339.40, [M+H]+ 340.20.
9-(2-Chloro-6-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9u)
Yield: 88 mg, 42 %; mp: 236 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.08 (quin,
J=5.86 Hz, 2 H, C7H2), 3.23 (t, J=5.90 Hz, 2 H, C8H2), 3.35 (s, 3 H, N3CH3), 3.51 (s, 3 H,
N1CH3), 4.19 (t, J=5.86 Hz, 2 H, C6H2), 4.92 (d, J=1.76 Hz, 2 H, N9CH2), 6.98 - 7.06 (m, 1
13
H, C4H, phe), 7.22 - 7.28 (m, 2 H, C3H/C5H, phe). C NMR (CHLOROFORM-d) δ ppm:
3
21.4 (C7), 27.6 (N3CH3), 29.7 (N1CH3), 41.6 (C6), 43.4 (C8), 44.0 (d, JC,F = 3.4 Hz,
N9CH2), 103.0 (C4a), 114.3 (d, 2JC,F = 21.9 Hz, C5, phe), 121.9 (d, 2JC,F = 17.2 Hz, C1, phe),
125.7 (d, 4JC,F = 3.5 Hz, C3, phe), 130.0 (d, 3JC,F = 10.4 Hz, C4, phe), 136.2 (d, 3JC,F = 5.8 Hz,

1
C2, phe), 148.8 (C10a), 151.4 (C9a), 151,9 (C2), 154.0 (C4), 162.3 (d, JC,F = 245.0 Hz, C6,
phe). UPLC/MS purity 98.32 %, tR = 5.92, C17H17ClFN5O2, MW 377.80, [M+H]+ 378.08.
9-(2,5-Dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-
2,4(1H,3H)-dione (9v)
Yield: 79 mg, 36 %; mp: 174 °C; 1H NMR (300 MHz, CHLOROFORM-d)  ppm: 2.14 - 2.23
(m, 2 H, C7H2), 3.33 - 3.38 (m, 5 H, C8H2, N3CH3), 3.49 (s, 3 H, N1CH3), 4.26 (t, J=6.15
Hz, 2 H, C6H2), 4.81 (s, 2 H, N9CH2), 7.19 - 7.24 (m, 1 H, C3H, phe), 7.30 - 7.38 (m, 2 H,
C4H/C6H, phe). 13C NMR (CHLOROFORM-d) δ ppm: 21.5 (C7), 27.6 (N3CH3), 29.7
(N1CH3), 41.7 (C6), 44.7 (C8), 50.3 (N9CH2), 103.2 (C4a), 129.0 (C4, phe), 129.4 (C6, phe),
130.8 (C3, phe), 131.8 (C2, phe), 133.0 (C5, phe), 136.2 (C1, phe), 148.7 (C10a), 151.5
(C9a), 151,8 (C2), 154.0 (C4). UPLC/MS purity 99.31 %, tR = 6.59, C17H17Cl2N5O2, MW
394.26, [M+H]+ 394.03.
10-(2-chlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10a)
Yield: 156 mg; 76 %; mp: 153-154°C; 1H NMR (300 MHz, DMSO-d6)  ppm 1.63 - 1.74 (m,
2 H, C7H2), 1.76 - 1.86 (m, 2 H, C8H2), 3.15 (s, 3 H, N3CH3), 3.24 - 3.28 (m, 2 H, C9H2),
3.31 (s, 3 H, N1CH3), 4.23 - 4.29 (m, 2 H, C6H2), 4.73 (s, 2 H, N10CH2), 7.29 - 7.36 (m, 2
H,C4H, C5H, phe), 7.41 - 7.47 (m, 1 H, C6H, phe), 7.49 - 7.52 (m, 1 H, C3H, phe); 13C NMR
(DMSO-d6) δ ppm: 26.2 (C7), 27.8 (N3CH3), 28.7 (C8), 29.8 (N1CH3), 45.7 (C6), 51.5 (C9),
53.8 (N10CH2), 103.9 (C4a), 127.7 (C3, phe), 129.5 (C6, phe), 129.9 (C4, phe), 130.5 (C5,
phe), 133.3 (C2, phe), 135.6 (C1, phe), 147.8 (C11a), 151.4 (C2), 157.8 (C4), 158.7 (C10a);
UPLC/MS purity 96.89 %; tR = 6.38; C18H20ClN5O2; MW 373.83; [M+H]+ 374.21
10-(2-fluorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10b)
Yield: 132 mg; 68 %; mp: 120-121°C; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.69 - 1.74 (m,
2 H, C7H2), 1.79 - 1.86 (m, 2 H, C8H2), 3.17 (s, 3 H, N3CH3), 3.26 - 3.31 (m, 2 H, C9H2),
3.34 (s, 3 H, N1CH3), 4.24 - 4.29 (m, 2 H, C6H), 4.72 (s, 2 H, N10CH2), 7.16 - 7.25 (m, 2 H,
13
C4H, C5H, phe), 7.31 - 7.38 (m, 1 H, C6H, phe), 7.47 - 7.54 (m, 1 H, C3H, phe); C NMR
(DMSO-d6) δ ppm: 26.2 (C7), 27.8 (N3CH3), 28.7 (C8), 29.8 (N1CH3), 45.7 (C6), 49.8 (C9),
2
2
51.3 (N10CH2), 103.8 (C4a),115.8 (d, JC,F = 21.3 Hz, C3, phe), 124.9 (d, JC,F = 3 Hz, C5,
2
3
3
phe), 125.2 (d, JC,F = 13,9 Hz, C1, phe), 129.8 (d, JC,F = 8.1 Hz, C4, phe), 130.8 (d, JC,F
=
4.5 Hz, C6, phe), 147.8 (C11a), 151.4 (C2), 153.9 (C4), 158.8 (C10a) ), 161.1 (d, 1JC,F =245.1
Hz, C2, phe); UPLC/MS purity 100.00%; tR = 6.35, C18H20FN5O2, MW 357.38, [M+H]+
358.1.
10-(3-bromobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10c)
Yield: 141 mg; 62 %; mp: 125-126°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.78 -
1.85 (m, 2 H, C7H2), 1.88 - 1.94 (m, 2 H, C8H2), 3.14 - 3.18 (m, 2 H, C9H2), 3.40 (s, 3 H,
N3CH3), 3.54 (s, 3 H, N1CH3), 4.39 - 4.44 (m, 2 H, C6H2), 4.67 (s, 2 H, N10CH2), 7.20 - 7.26
(m, 1 H, C6H, phe), 7.33 (d, J=7.82 Hz, 1 H, C5H, phe), 7.44 (d, J=7.82 Hz, 1 H, C4H, phe),
7.57 (s, 1 H, C2H, phe); 13C NMR (CHLOROFORM-d) δ ppm: 26.5 (C7), 27.7 (N3CH3),
29.2 (C8), 29.7 (N1CH3), 45.9 (C6), 51.1 (C9), 55.9 (N10CH2), 104.5 (C4a), 122.7 (C3, phe),
126.9 (C6, phe), 130.1 (C4, phe), 130.7 (C5, phe), 131.4 (C2, phe), 140.1 (C1, phe), 147.7
(C11a), 151.8 (C2), 154.6 (C4), 158.9 (C10a); UPLC/MS purity 100.0 %; tR = 7.13;
C18H20BrN5O2; MW 418.29; [M+H]+ 418.3

10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-
f]purine-2,4(3H,6H)-dione (10d)
1
Yield: 143 mg; 64 %; mp: 147-149°C; H NMR (300 MHz, DMSO-d6)  ppm 1.74 (br. s., 2
H, C7H2), 1.77 - 1.83 (m, 2 H, C8H2), 3.16 (s, 3 H, N3CH3), 3.17 - 3.21 (m, 2 H, C9H2), 3.33
(s, 3 H, N1CH3), 4.25 - 4.29 (m, 2 H, C6H2), 4.63 (s, 2 H, N10CH2), 7.43 (dd, J=8.21, 2.34
Hz, 1 H, C6H, phe), 7.59 (d, J=8.79 Hz, 1 H, C2H, phe), 7.70 (d, J=1.76 Hz, 1 H, C2H, phe);
13C NMR (CHLOROFORM-d) δ ppm: 26.2 (C7), 27.8 (N3CH3), 28.7 (C8), 29.8 (N1CH3),
45.8 (C6), 51.1 (C9), 54.8 (N10CH2), 103.9 (C4a), 129.0 (C3, phe), 130.3 (C6, phe), 130.7
(C4, phe), 131.0 (C5, phe), 131.4 (C2, phe), 139.8 (C1, phe), 147.7 (C11a), 151.4 (C2), 153.9
(C4), 158.8 (C10a); UPLC/MS purity 97.69 %; tR = 7.62; C18H19Cl2N5O2; MW 408.28; [M]+
408.12
10-(2-bromobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10e)
Yield: 146 mg; 64 %; mp: 154-155°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.75 -
1.81 (m, 2 H, C7H2), 1.88 - 1.93 (m, 2 H, C8H2), 3.22 - 3.26 (m, 2 H, C9H2), 3.39 (s, 3 H,
N3CH3), 3.54 (s, 3 H, N1CH3), 4.36 - 4.43 (m, 2 H, C6H2), 4.79 (s, 2 H, N10CH2), 7.15 - 7.21
(m, 1 H, C6H, phe), 7.31 (td, J=7.53, 1.37 Hz, 1 H, C4H, phe), 7.45 (dd, J=7.43, 1.56 Hz, 1
13
H, C5H, phe), 7.60 (dd, J=7.82, 1.17 Hz, 1 H, C3H, phe); C NMR (CHLOROFORM-d) δ
ppm: 26.5 (C7), 27.7 (N3CH3), 29.0 (C8), 29.7 (N1CH3), 45.9 (C6), 51.5 (C9), 56.4
(N10CH2), 104.5 (C4a), 124.3 (C2, phe), 127.4 (C5, phe), 129.2 (C4, phe), 130.5 (C6, phe),
133.2 (C3, phe), 136.7 (C1, phe), 147.8 (C11a), 151.8 (C2), 154.6 (C4), 158.8 (C10a);
UPLC/MS purity 99.5 %; tR = 6.99; C18H20BrN5O2; MW 418.29; [M+H]+ 420.09
10-(3-bromo-4-fluorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-
f]purine-2,4(3H,6H)-dione (10f)
Yield: 179 mg; 75 %; mp: 152-153°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.79 -
1.85 (m, 2 H, C7H2), 1.89 - 1.94 (m, 2 H, C8H2), 3.13 - 3.17 (m, 2 H, C9H2), 3.40 (s, 3 H,
N3CH3), 3.54 (s, 3 H, N1CH3), 4.39 - 4.43 (m, 2 H, C6H2), 4.64 (s, 2 H, N10CH2), 7.11 (t,
J=8.41 Hz, 1 H, C6H, phe), 7.34 (ddd, J=8.31, 4.79, 2.15 Hz, 1 H, C2H, phe), 7.64 (dd,
J=6.65, 2.35 Hz, 1 H, C5H, phe); 13C NMR (CHLOROFORM-d) δ ppm: 26.4 (C7), 27.73
(N3CH3), 29.2 (C8), 29.7 (N1CH3), 45.9 (C6), 51.1 (C9), 55.3 (N10CH2), 104.5 (C4a), 109.1
(d, 2JC,F = 21.3 Hz, C3, phe), 116.5 (d, 2JC,F = 22.8 Hz, C5, phe), 128.9 (d, 3JC,F = 7.3 Hz, C6,
4
phe), 133.5 (C1, phe), 135.2 (d, JC,F = 3.6 Hz, C1, phe), 147.7 (C11a), 151.76 (C2), 154.6
(C4), 158.7 (C10a), 159.7 (C4, phe); UPLC/MS purity 95.2 %; tR = 7.16; C18H19BrFN5O2;
MW 436.27; [M]+ 436.38
10-(2-methoxybenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10g)
Yield: 128 mg; 63 %; mp: 157-159°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.73 -
1.78 (m, 2 H, C7H2), 1.87 - 1.92 (m, 2 H, C8H2), 3.24 - 3.27 (m, 2 H, C9H2), 3.40 (s, 3 H,
N3CH3), 3.55 (s, 3 H, N3CH3), 3.85 (s, 3 H, OCH3), 4.34 - 4.38 (m, 2 H, C6H2), 4.75 (s, 2 H,
N10CH2), 6.90 - 6.96 (m, 2 H, CC4H, C5H, phe), 7.24 - 7.33 (m, 1 H, C6H, phe), 7.38 (dd,
J=7.43, 1.56 Hz, 1 H, C3H, phe); 13C NMR (CHLOROFORM-d) δ ppm: 26.5 (C7), 27.7
(N3CH3), 28.9 (C8), 29.7 (N1CH3), 45.9 (C6), 50.9 (C9), 51.2 (N10CH2), 55.3 (OCH3), 104.3
(C4a), 110.4 (C3, phe), 120.4 (C5, phe), 125.9 (C1, phe), 128.6 (C4, phe), 129.5 (C6, phe),
148.2 (C11a), 151.9 (C2), 154.4 (C4), 157.7 (C2, phe), 159.5 (C10a); UPLC/MS purity 97.5
%; tR = 6.36; C19H23N5O3; MW 369.41; [M+H]+ 370.17

10-(2,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-
f]purine-2,4(3H,6H)-dione (10h)
Yield: 131 mg; 59 %; mp: 204-205°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.78 -
1.83 (m, 2 H, C7H2), 1.88 - 1.93 (m, 2 H, C8H2), 3.20 - 3.25 (m, 2 H, C9H2), 3.39 (s, 3 H,
N3CH3), 3.53 (s, 3 H, N1CH3), 4.37 - 4.41 (m, 2 H, C6H2), 4.77 (s, 2 H, N10CH2), 7.24 (dd,
J=8.22, 1.96 Hz, 1 H, C3H, phe), 7.40 - 7.44 (m, 2 H, C5H, C6H, phe); 13C NMR
(CHLOROFORM-d) δ ppm: 26.4 (C7), 27.7 (N3CH3), 29.1 (C8), 29.6 (N1CH3), 45.9 (C6),
51.9 (C9), 53.6 (N10CH2), 104.5 (C4a), 127.1 (C5, phe), 129.6 (C3, phe), 131.2 (C6, phe),
133.9 (C4, phe), 134.0 (C2, phe), 134.8 (C1, phe), 147.7 (C11a), 151.8 (C2), 154.6 (C4),
158.6 (C10a); UPLC/MS purity 97.62 %; tR = 7.75; C18H19Cl2N5O2; MW 408.28; [M]+ 408.3
10-(4-chlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-
2,4(3H,6H)-dione (10i)
Yield: 121 mg; 59 %; mp: 182-183°C; 1H NMR (300 MHz, DMSO-d6)  ppm 1.67 - 1.75 (m,
2 H, C7H2), 1.77 - 1.83 (m, 2 H, C8H2), 3.16 (s, 3 H, N3CH3), 3.17 - 3.21 (m, 2 H, C9H2),
3.33 (s, 3 H, N1CH3), 4.24 - 4.29 (m, 2 H, C6H2), 4.63 (s, 2 H, N10CH2), 7.35 - 7.41 (m, 2 H,
C2H, C6H, phe), 7.42 - 7.47 (m, 2 H, C3H, C5H, phe); 13C NMR (CHLOROFORM-d) δ
ppm: 26.2 (C7), 27.8 (N3CH3), 28.6 (C8), 29.8 (N1CH3), 45.7 (C6), 50.9 (C9), 55.1
(N10CH2), 103.2 (C4a), 125.58 (C2, phe), 128.8 (C5, phe), 130.4 (C4, phe), 132.3 (C6, phe),
137.5 (C3, phe), 140.3 (C1, phe), 145.1 (C11a), 147.8 (C2), 153.9 (C4), 158.9 (C10a);
UPLC/MS purity 98.7 %; tR = 7.02; C18H20ClN5O2; MW 373.83; [M+H]+ 374.36
4.2. Biological experiments
4.2.1.
Radioligand binding assays at adenosine receptors
AR radioligand binding assays were performed as previously described [38]
using rat brain cortical membrane preparations for rat A1 AR assays. Frozen rat brains
(unstripped) were obtained from Pel-Freez, Rogers, Arkansas, USA. For assays at all
four human AR subtypes, cell membranes of HEK or CHO cells recombinantly
expressing the respective receptors were purchased from PerkinElmer.The following
compounds were employed as radioligands: A1: [3H]2-Chloro-N6-cyclopentyladenosine
([3H]CCPA)
[36];
A2A:
[3H]3-(3-hydroxypropyl)-1-propargyl-7-methyl-8-(m-
methoxystyryl)xanthine ([3H]MSX-2) [37]; A2B: [3H]8-(4-(4-(4-chlorophenyl)-
piperazine-1-sulfonyl)phenyl)-1-propylxanthine ([3H]PSB-603) [38]; 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) [39]. Initially, a single high concentration of compound was tested. For
potent compounds, full concentration-inhibition curves were determined 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).
4.2.2. 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 [26].
4.2.3.Hepatotoxicity

Hepatoma HepG2 (ATCC HB-8065) cell line was kindly donated by the Department
of Pharmacological Screening, Jagiellonian University Medical College. To determine the
antiproliferative effect the HepG2 cells were seeded first in 96-well cell culture plate at
concentration of 1.5 × 104 cells/well in glucose (4.5 g/L) and FBS (10%) supplemented
Minimum Essential Medium Eagle (MEM) media (Gibco, Carlsbad, CA, USA) and incubated
at 37°C for 24 hours at 5% CO2. Next, the reference toxins and tested compounds were
diluted in the same media and added to the cells. After 72h of incubation CellTiter 96®
AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) was
added to the each well, and the cells were incubated under the same conditions for 2 h. The
absorbance of the samples was measured using a microplate reader EnSpire (PerkinElmer,
Waltham, MA USA) at 490 nm. The statistical significance was analyzed by GraphPad
Prism™ software (version 5.01, San Diego, CA, USA) using One-way ANOVA and
Bonferroni's Multiple Comparison Post Test.
4.2.4.
Determination of the antioxidant activity by FRAP assay
The FRAP assay was done according to Benzie and Strain [54] with some
modifications. The stock solutions included 300mM acetate buffer (3.1 g C2H3NaO2·3H2O
and 16 ml C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM
HCl, and 20 mM FeCl3·6H2O solution was prepared by mixing 10 parts of acetate buffer, 1
part of TPTZ solution, and 1 part of FeCl3·6H2O solution. 300 ul of the FRAP solution was
mixed with 10 ul of tested compound solution and incubated in room temperature for 10 min
in the dark condition. Readings of the colored product [ferrous tripyridyltriazine complex]
were then taken at 593 nm according to ethanol. Results for tested compounds are expressed
as an increase in absorbance of the test sample compared to a sample containing the solvent.
Trolox was used as reference compound, which the ability to inhibit lipid peroxidation was
determined in the concentration range of 1 to 300 mM.
In the FRAP assay, the antioxidant potential of sample was determined from a
standard curve plotted using FeSO4·7H2O at a concentration range between 37.5 and 1200
μM.
4.2.5.
Antioxidant effect - measurement of lipid peroxidation in rat brain
homogenate
The rat brain homogenate was made in 0.9% saline containing 10 mg tissue/ml. The
rates of membrane lipid peroxidation were measured by the formation of TBARS. Rat brain
homogenates (1 ml) were incubated at 37oC for 5 min with 10 µl of a test compound or
vehicle. Lipid peroxidation was initiated by the addition of 50 µl of 0.5 mM FeCl2 and 50 µl
of 2 mM ascorbic acid. After 30 min of incubation, the reaction was stopped by adding 0.1 ml
of 0.2% BHT. Thiobarbituric acid reagent was then added and the mixture was heated for 15
min in a boiling water bath. The TBARS was extracted by n-butanol and measured at 532 nm.
The amount of TBARS was quantified using a standard curve of MDA as described
previously.[55]
4.3.
Molecular docking
For docking purposes, Schrödinger Maestro Suite was used [46]. Ligands were built in
their 3D forms and their bioactive conformations were generated using ConfGen module
[47,48](water environment, target number of conformers – 20). Crystal structures: 3REY and
2V5Z were prepared for docking using Protein Preparation Wizard [49], ligand binding sites
were centered on bound ligands. Docking to rigid form of receptor was performed using
Glide module [50–52] (precision standard, flexible ligand sampling, max 5 poses per
conformer). Ligands were rated according their position in binding pocket, interactions with

binding pocket amino acids as well as the docking score value. Ligand interaction diagrams
were generated using Schrödinger Maestro, ligand-receptor visualizations were generated
using UCSF Chimera [53].
Acknowledgements
Financial support by the Jagiellonian University Medical College [grant no. K/DSC/003511,
K/ZDS/007121], Polish National Science Center grant based on decision No DEC-
2016/23/N/NZ7/00475 are gratefully acknowledged. We thank Anika Püsche, Angelika
Fischer, Alexandra Jünger-Leif, and Christin Vielmuth for expert technical assistance in
compound testing. Thanks are due to the European Commission for funding our collaboration
within the COST Action MuTaLig.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at ....
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