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

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

²Department 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 A_2Areceptors 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 A_2Aadenosine 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_ihuman A_2AAR:

189 nM and IC₅₀human 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 IC₅₀value 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 A_2A

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 A_2Areceptors (A_2AARs) are one of the four subtypes of adenosine receptors

which are subdivided into A₁, A_2A, A_2B, and A₃ARs belonging to the G protein-coupled

receptor (GPCR) family. A_2AARs are highly expressed in specific areas of the brain, such as

the caudate-putamen.[19,20] Blockade of A_2AARs has resulted in neuroprotective effects in

preclinical studies by different cellular mechanism. A_2AAR 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 A_2AAR

reduces -amyloid peptide aggregation [23,25].

Based on those considerations, structures with dual target activity as A_2AAR

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

A_2AAR/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 A_2AAR 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 A_2AAR 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/A_2AARs

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 A_2AAR

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 A_2Aantagonists/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

A_2AARs. To rationalize the observed biological activities of the new compounds, molecular

docking studies using human A_2AARs 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 A_2Aand A_2Bwere 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, A_2A, A_2Band A₃AR 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

A₁vs. [³H]CCPA

(h, human; r, rat)

A_2Avs. [³H]MSX-2

(h, human; r, rat)

A_2Bvs. ³H]PSB-603 A₃vs. [³H]PSB-11

Compd

R¹

K_i± 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

0.0912^h

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

(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 ± 5)

(30 ± 7)

(2 ± 1)

>1.0

(23 ± 2)

>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

9a

9b

9c

9d

9e

9f

>1.0

>1.0 ^r

3.040 ± 0.440

>1.0

>1.0 ^r

>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

1.410 ± 0.278 ^r

1.910 ± 0.648 ^r

1.550 ± 0.310 ^r

3.020 ± 0.067 ^r

1.020 ± 0.275

>1.0

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

A_2Band A₃ARs at the highest tested concentration. In general, most of the compounds

presented none or only negligible affinity for the A₁AR. Only the dual active structure 8a

possesses activity with a K_ivalue of approximately 0.4 µM for both human A₁and A_2AAR.

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 A_2AARs 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 A_2AAR with K_ivalues

slightly below 1 µM similar to the ortho-Br analogues.

In order to evaluate the structural determinants for interaction with the A_2AAR 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 A_2AAR 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 (A_2A: 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 A_2AAR by

three-fold. Compared to compound 9u, structure 9p presented a K_ivalue three fold lower.

Another N9-tri-substituted benzyl-1,3-dimethylpyrimido[2,1-f]purinedione, 9f, displayed low

affinity for the A_2AAR.

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 A_2AAR.

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 (IC₅₀human 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 A_2AAR ligand among all

investigated series, displayed also MAO-B inhibitory activity (IC₅₀human 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

>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

> 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 A_2AAR 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 A_2AAR and MAO-

B.

Scheme 2 Structure-activity relationships of tricyclic structures

2.6. Molecular modelling studies

2.6.1. Adenosine A_2Acrystal structure

Among 34 X-ray structures of adenosine A_2Areceptor 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 ASN253^6.55forms 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 253^6.55forms 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 A_2A

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 270^7.35and ILE 274^7.39from the other side, while substituted

benzyl moieties are located between TYR9^1.35, SER67^2.65and TYR271^7.36(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 A_2AAR

affinity of particular ligands could be found. However, it can be stated that all of the ligands

fit well into the A_2AAR 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 180^ohorizontally.

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 A_2A

antagonism and MAO-B inhibition may provide synergistic neuroprotective effects in NDs.

3. Conclusion

Looking for dual A_2AAR/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 A_2AAR and human MAO-B. Within three

investigated series of tricyclic structures, we identified compounds 9p, 9u, 10a, 10e acting as

selective dual A_2A/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-d₆or CDCl₃with 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 (t_R) 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 %; t_R= 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%; t_R= 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%; t_R= 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

(C7), 52.0 (N8CH₂), 103.0 (C4a), 119.4 (d, J_C,F= 24.9 Hz, C3, phe), 121.7 (d, J_C,F= 14.7

4

3

Hz, C5, phe), 122.6 (d, J_C,F= 9.5 Hz, C6, phe), 128.0 (d, J_C,F= 3.6 Hz, C2, phe), 131.5 (d,

³J_C,F= 5.1 Hz, C1, phe), 151.7 (C9a), 153.2 (C8a), 153.8 (C2), 159.5 (C4) 161.2 (d, J_C,F

1

=176.1 Hz, C4, phe),; UPLC/MS purity 100.0 %; t_R= 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%; t_R=

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 %; t_R= 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, N3CH_3,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, J_C,F= 4.6 Hz,

2

N9CH₂), 103.0 (C4a), 115.5 (d, J_C,F= 21.8 Hz, C3, phe), 123.6 (d, J_C,F= 15 Hz, C1, phe),

124.4 (d, ⁴J_C,F= 3.5 Hz, C5, phe), 129.6 (d, ³J_C,F= 8.1 Hz, C4, phe), 130.6 (d, ³J_C,F= 4.6 Hz,

1

C6, phe), 148.7 (C10a), 151.6 (C9a), 151.8 (C2), 153.9 (C4), 161.1 (d, J_C,F=246.4 Hz, C2,

phe). UPLC/MS purity 97.01 %, t_R= 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, J_C,F= 21.9 Hz, C2, phe), 120.2 (C4, phe), 124.4

(d, ⁴J_C,F= 3.4 Hz, C6, phe), 130.9 (C5, phe), 137.7 (d, ³J_C,F= 5.8 Hz, C1, phe), 148.5 (C10a),

1

151.3 (C9a), 151.8 (C2), 153.9 (C4), 158.2 (d, J_C,F=249.9 Hz, C3, phe). UPLC/MS purity

97.01 %, t_R= 5.45. C₁₇H₁₈FN₅O₂, MW 343.36, [M+H]⁺344.32. UPLC/MS purity 100 %, t_R=

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 %, t_R= 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, J_C,F= 20.5 Hz, C5, phe), 117.2 (d,

²J_C,F= 25 Hz, C3, phe), 130.1 (d, ⁴J_C,F= 3.7 Hz, C1, phe) , 131.1 (d, ³J_C,F= 8.8 Hz, C6, phe),

134.5 (d, ³J_C,F= 10.3 Hz, C2, phe), 148.1 (C10a), 151.1 (C9a), 151.8 (C2), 153.9 (C4), 162.0

1

(d, J_C,F=250.1 Hz, C4, phe). UPLC/MS purity 98.18 %, t_R= 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, ²J_C,F= 21.2 Hz, C3, phe), 116.7 (d, ²J_C,F= 22 Hz,

3

4

C5, phe), 128.7 (d, J_C,F= 7.4 Hz, C6, phe), 133.2 (C2, phe), 134.1 (d, J_C,F= 3.7 Hz, C1,

1

phe), 148.5 (C10a), 151.3 (C9a), 151.8 (C2), 153.9 (C4), 158.7 (d, J_C,F= 248 Hz, C4, phe).

UPLC/MS purity 100 %, t_R= 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, J_C,F= 10.3, J_C,F

=

3.7 Hz, C3/C5, phe), 154.0 (C4). UPLC/MS purity 100 %, t_R= 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 %, t_R= 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, J_H,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, ²J_C,F= 23.5 Hz, C3, phe), 125.9 (d, ²J_C,F= 16.9 Hz, C1, phe),

3

132.5 (d, J_C,F= 8.07 Hz, C4, phe), 133.6 (d, J_C,F= 4.4 Hz, C6, phe), 148.2 (C10a), 151.0

1

(C9a), 151.8 (C2), 154.0 (C4), 161.2 (d, J_C,F= 247.3 Hz, C2, phe). UPLC/MS purity 98.40

%, t_R= 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 %, t_R= 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 %, t_R= 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, J_C,F

=

25.5 Hz, C3, phe), 122.5 (C1, phe), 124.9 (d, ⁴J_C,F= 3.5 Hz, C5, phe), 131.5 (d, ³J_C,F= 4.6 Hz,

3

C6, phe), 134.5 (d, J_C,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 %, t_R= 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, ²J_C,F= 25.3 Hz, C3, phe), 122.0 (d, ³J_C,F= 10.8 Hz, C4,

2

4

3

phe), 122.9 (d, J_C,F= 16.1 Hz, C1, phe), 127.8 (d, J_C,F= 4.6 Hz, C5, phe), 131.8 (d, J_C,F

=

4.6 Hz, C6, phe), 148.7 (C10a), 151.4 (C9a), 151,8 (C2), 153.9 (C4), 159.9 (d, ¹J_C,F= 251 Hz,

C2, phe). UPLC/MS purity 100 %, t_R= 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 %, t_R= 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

%, t_R= 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 %, t_R=

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, J_C,F= 2.3 Hz, C4, phe), 122.3 (d,

²J_C,F= 13.8 Hz, C1, phe), 124.8 (d, ³J_C,F= 4.6 Hz, C6, phe), 126.5 (d, ⁴J_C,F= 4.6 Hz, C5, phe),

2

146.8 (d, J_C,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 %, t_R= 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

(d, J_C,F= 3.5 Hz, C4, phe), 127.1 (d, J_C,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 %, t_R= 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 %, t_R= 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 %, t_R=

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 %, t_R= 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, J_C,F= 3.4 Hz,

N9CH₂), 103.0 (C4a), 114.3 (d, ²J_C,F= 21.9 Hz, C5, phe), 121.9 (d, ²J_C,F= 17.2 Hz, C1, phe),

125.7 (d, ⁴J_C,F= 3.5 Hz, C3, phe), 130.0 (d, ³J_C,F= 10.4 Hz, C4, phe), 136.2 (d, ³J_C,F= 5.8 Hz,

1

C2, phe), 148.8 (C10a), 151.4 (C9a), 151,9 (C2), 154.0 (C4), 162.3 (d, J_C,F= 245.0 Hz, C6,

phe). UPLC/MS purity 98.32 %, t_R= 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 %, t_R= 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 %; t_R= 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

51.3 (N10CH₂), 103.8 (C4a),115.8 (d, J_C,F= 21.3 Hz, C3, phe), 124.9 (d, J_C,F= 3 Hz, C5,

2

3

phe), 125.2 (d, J_C,F= 13,9 Hz, C1, phe), 129.8 (d, J_C,F= 8.1 Hz, C4, phe), 130.8 (d, J_C,F

=

4.5 Hz, C6, phe), 147.8 (C11a), 151.4 (C2), 153.9 (C4), 158.8 (C10a) ), 161.1 (d, ¹J_C,F=245.1

Hz, C2, phe); UPLC/MS purity 100.00%; t_R= 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 %; t_R= 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 %; t_R= 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 %; t_R= 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, ²J_C,F= 21.3 Hz, C3, phe), 116.5 (d, ²J_C,F= 22.8 Hz, C5, phe), 128.9 (d, ³J_C,F= 7.3 Hz, C6,

4

phe), 133.5 (C1, phe), 135.2 (d, J_C,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 %; t_R= 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

%; t_R= 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 %; t_R= 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 %; t_R= 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 A₁AR 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];

A_2A:

[³H]3-(3-hydroxypropyl)-1-propargyl-7-methyl-8-(m-

methoxystyryl)xanthine ([³H]MSX-2) [37]; A_2B: [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 × 10⁴cells/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 37^oC 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 FeCl₂and 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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Article Doi

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

DOI: 10.1016/j.bmc.2019.02.004

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