Synthesis and biological evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents

Article abstract of DOI:10.1080/14756366.2016.1198902

A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4–21) were synthesized and evaluated for their serotonin (5-HT_1A/5-HT₇) receptor affinity and phosphodiesterase

Full text of DOI:10.1080/14756366.2016.1198902

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of 2-fluoro
and 3-trifluoromethyl-phenyl-piperazinylalkyl
derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-
dione as potential antidepressant agents
Agnieszka Zagórska, Adam Bucki, Marcin Kołaczkowski, Agata Siwek, Monika
Głuch-Lutwin, Gabriela Starowicz, Grzegorz Kazek, Anna Partyka, Anna
Wesołowska, Karolina Słoczyńska, Elżbieta Pękala & Maciej Pawłowski
To cite this article: Agnieszka Zagórska, Adam Bucki, Marcin Kołaczkowski, Agata Siwek,
Monika Głuch-Lutwin, Gabriela Starowicz, Grzegorz Kazek, Anna Partyka, Anna Wesołowska,
Karolina Słoczyńska, Elżbieta Pękala & Maciej Pawłowski (2016): Synthesis and biological
evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-
imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1198902
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1198902
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1198902
!
RESEARCH ARTICLE
Synthesis and biological evaluation of 2-fluoro and
3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of
1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential
antidepressant agents
1
1
1
2
Agnieszka Zagorska , Adam Bucki , Marcin Kołaczkowski , Agata Siwek , Monika Głuch-Lutwin , Gabriela
3
´
3
3
4
4
5
5
´
Starowicz , Grzegorz Kazek , Anna Partyka , Anna Wesołowska , Karolina Słoczynska , Elz_bieta Pe˛kala , and Maciej
Pawłowski1
2
3
4
1Department of Medicinal Chemistry, Department of Pharmacobiology, Department of Pharmacodynamics, Department of Clinical Pharmacy,
5
and Department of Pharmaceutical Biochemistry, Jagiellonian University Medical College, Krakow, Poland
´
Abstract
Keywords
A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-
f]purine-2,4(3H,8H)-dione (4–21) were synthesized and evaluated for their serotonin (5-HT1A
Depression, long-chain arylpiperazines, PDE,
phosphodiesterase inhibitors, serotonin
/
5-HT7) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The
study enabled the identification of potent 5-HT1A, 5-HT7 and mixed 5-HT1A/5-HT7 receptor
ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed
with the determination of lipophilicity and metabolic stability using micellar electrokinetic
chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary
pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-
yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential
antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects
evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam.
Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-
f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for
antidepressant and/or anxiolytic application.
History
Received 7 December 2015
Revised 27 May 2016
Accepted 1 June 2016
Published online 23 June 2016
Introduction
depression and schizophrenia1–6. A very important class of 5-
HT receptors ligands are derivatives of 1,4-disubstituted arylpi-
perazine (Figure 1). Such arylpiperazine derivative with long-
chain substituents incorporated on the basic nitrogen of the
phenylpiperazine ring – long-chain arylpiperazines (LCAPs) – are
commonly studied classes of bioactive compounds7–24. Despite
the enormous progress in central nervous system (CNS) drug
discovery, particularly in the areas of mood disorders and
schizophrenia, new drugs are still being sought. The main reasons
behind the search for novel therapeutics are poor patient
compliance, delayed onset of action and side effects, including
emesis, sexual dysfunction, weight gain, diabetes mellitus or
endocrinopathies. Roth et al. proposed that, for mood disorders
and schizophrenia, attempting to develop more effective treat-
ments by intentionally designing nonselective drugs that interact
with several molecular targets would lead to new and more
effective medications (‘‘magic shotguns’’)25.
Over the past decade, we have witnessed unparalleled advances in
our understanding of the basic biological processes that contribute
to many human disorders, although a detailed understanding of
the etiology of complex psychiatric disorders remains elusive.
Psychiatric diseases are chronic and recurrent and have a complex
etiology. Human genomics and biological studies have revealed
an unprecedented number of promising molecular targets for
neuropsychiatric disorders, including G-protein-coupled receptors
and transporters, intracellular and synaptic proteins and
microRNAs. Serotonin (5-HT) is one of the most attractive
targets for medicinal chemists and the discovery of ligands with
affinity for the family of 5-HT receptors (5-HTRs) is an area of
intense research, because of the potential to find new therapeutic
drugs, due to their involvement in numerous physiological and
pathophysiological processes. Among the 14 5-HTRs identified,
the 5-HT1A and 5-HT7 subtypes are the best studied due to effects
of their full or partial agonists or antagonists on anxiety,
Cyclic nucleotide phosphodiesterases (PDEs) are a super-
family of enzymes that are involved in the regulation of the
intracellular second messenger’s cyclic AMP (cAMP) and cyclic
GMP (cGMP) by controlling their rates of hydrolysis. There are
11 different PDE families and each family typically has multiple
isoforms and splice variants. The PDEs differ in their structures,
distribution, modes of regulation and sensitivity to inhibitors.
´
Address for correspondence: A. Zagorska, Department of Medicinal
Chemistry, Jagiellonian University Medical College, 9 Medyczna Street,
´
30-688 Krakow, Poland. Tel: +48 12 62 05 450. Fax: +48 12 62 05 458.
E-mail: agnieszka.zagorska@uj.edu.pl

´
A. Zagorska et al.
2
J Enzyme Inhib Med Chem, Early Online: 1–15
Figure 1. General structure of long-chain
arylpiperazine (LCAPs) and their represen-
tatives of 5-HT1A and 5-HT7 receptor ligands.
Amide/Imide
terminal
Aromaꢀc
fragment
N
N
Linker
O
O
N
N
F
N
N
N
NH2
O
O
N
CF3
comp 17 from [8]
comp 9 from [21]
Ki 5-HT1A = 141 nM
Ki 5-HT7 = 3134 nM
Ki 5-HT1A = 5.9 nM
Ki 5-HT7 = 134 nM
H
N
O
Cl
N
N
N
comp 5 from [20]
Ki 5-HT1A = 454 nM
Ki 5-HT7 = 7 nM
Since PDEs have been shown to play distinct roles in the
processes of emotion and related learning and memory processes,
selective PDE inhibitors, by preventing the breakdown of cAMP
and/or cGMP, modulate mood and related cognitive activity. It is
becoming increasingly clear that PDE inhibitors have therapeutic
potential for the treatment of neuropsychiatric disorders involving
disturbances of mood, emotion and cognition26. Moreover, PDE4
and PDE10 due to their presence in areas of the brain associated
with the site of action of psychotropic drugs and their inhibitors
have potential therapeutic relevance. In animal models, elevated
intracellular cAMP level achieved by PDE inhibition have been
shown to possess antidepressant-like effect. Moreover, antipsy-
chotic effects can be reproduce by inhibition of the enzymes that
degrade cAMP. Maxwell et al. used amphetamine-induced
abnormalities in auditory event-related potentials to study the
antipsychotic potential of nonreceptor based compounds such as
rolipram27,28. The therapeutic potentials of PDE inhibitors have
been explored in many areas; for instance, PDE4 inhibitors have
been evaluated for the treatment of cognitive disorders, depres-
sion, and anxiety and PDE10 inhibitors for the treatment of
schizophrenia and Huntington’s disease. Naturally occurring
methylxanthines (theophylline, caffeine) were the first inhibitors
of PDE to be discovered. To improve the potency and specificity
for the inhibition of various PDEs, thousands of compounds with
related structures have been synthesized. Today, selective
inhibitors of PDE4 and PDE10 show a large of chemical diversity
(Figure 2).
NO
2
O
O
N
O
O
N
N
N
O
N
O
RS 25344
Papaverine
PDE 10A inhibitor IC = 40 nM
PDE 4 inhibitor IC = 0.28 nM
50
50
O
O
N
O
O
N
NH
N
N
S
O
O
O
NH
Cl
O
D 28
Apremilast
PDE 4 inhibitor IC = 74 nM
PDE 4B selective inhibitor
50
Figure 2. Chemical structures and inhibitor activity (IC50) of inhibitors of
PDE4 and PDE10.
antidepressant and anxiolytic properties29–32. In view of this
and our earlier chemical and pharmacological investigation,
in the current study, we designed and synthesized a new series
of 2-fluoro and 3-trifluoromethylphenylpiperazinyalkyl deriva-
tives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-diones. The special
properties of fluorine, such as its high electronegativity, small size
and very low polarizability, impart a variety of properties
important in medicinal chemistry. Fluoro or trifluoromethyl
substitutions will generally have enhanced binding interactions,
metabolic stability, changes in physicochemical properties and
selective relativities toward of targeted molecules. Fluorinated
For years, our attention has been focused on the development
of LCAPs with the complex terminal part based on a purine
moiety. Annulated derivatives of theophylline with an LCAP
moiety demonstrate high affinity toward serotonin 5-HT1A and
moderate affinity to 5-HT2A and 5-HT7 and the dopaminergic D2
receptor site. Preclinical results indicate that monosubstituted
derivatives of LCAPs with an imidazo[2,1-f]purine-2,4-dione as
the terminal part act as antipsychotics with additional

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
3
drugs constitute the first (spiperone, haloperidol, droperidol) General procedure for the deprotection of the
and second (risperidone, sertindole, paliperidone, iloperidone) phthalimide group (XII–XVI)
generation of antipsychotic drugs and antidepressants (fluvox-
A mixtures of 1.2mmol of appropriate compound VI–XI and 65%
hydrazine monohydrate aqueous solution (15 mmol) in 99.8%
ethanol were refluxed for 1 h. The reaction mixtures were cooled
down and treated with an additional amount of 99.8% ethanol and
concentrated HCl (1.3mL). Then, the reaction mixtures were
refluxed for 4 h and left overnight in a refrigerator. The precipitates
were filtered off, and the solvents were evaporated. The residues
were treated with n-hexane (20 mL) and NH3 (aqueous, 15mL).
The solutions were extracted with methylene chloride (3ꢀ 15mL),
the combined extracts were dried over anhydrous Na2SO4, and the
solvents were evaporated to give a products that were used without
further purification (yield: 70–80%).
amine, paroxetine, citalopram) from the selective serotonin
reuptake inhibitors (SSRIs) group with extremely famous
fluoxetine (ProzacÕ). Herein, we examined the effect of fluorine
substitution on receptor affinity, selectivity, intrinsic activity for
5-HT1A and 5-HT7 receptor, and lipophilicity. Moreover, studies
focused on the effect of the presence of annelated imidazole
moiety at the 7, 8 position of purine system on PDE4B/PDE10A
inhibition and metabolic stability. Finally, structure–activity
relationships were analyzed and explained by molecular
modeling.
Experimental protocols
Chemistry
General procedure for 7-ketonyl derivatives of 8-bromo-
1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (1–3)
Organic solvents (from Sigma-Aldrich and Chempur) were of
reagent grade and were used without purification. All other 2-(8-Bromo-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-
reagents were from Sigma-Aldrich and Alfa Aesar. Thin-layer yl)acetaldehyde (1) was obtained according to the previously
chromatography (TLC) was performed on Merck silica gel 60 described procedure29. 8-Bromo-1,3-dimethyl-7-(2-oxopropyl)-3,7-
F254 aluminum sheets (Merck, Darmstadt, Germany). Spots were dihydro-1H-purine-2,6-dione (2) and 8-bromo-1,3-dimethyl-
detected by their absorption under UV light (l ¼ 254 nm). 7-(2-oxo-2-phenylethyl)-3,7-dihydro-1H-purine-2,6-dione (3) were
Analytical high-performance liquid chromatography (HPLC) obtained as described below. 8-Bromo-1,3-dimethyl-3,7-dihydro-
was run on an Alliance Waters 2695 Separations Module 1H-purine-2,6-dione (10 mmol), 1-chloropropan-2-one or 2-chloro-
equipped with a Chromolith SpeedROD RP 18.5 mm column 1-phenylethan-1-one (15 mmol), potassium carbonate (30 mmol)
(4.6 ꢀ 50 mm). Standard conditions were eluent system A (water/ and acetonitrile (15 mL) were refluxed for 12h. After this time, the
0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of acetonitrile was removed in vacuo and the resulting residues were
5 mL/min and a gradient of (0–100)% B over 3 min were used. dissolved in dichloromethane (20 mL). The organic layers were
Detection was performed on a photodiode array (PDA) detector. washed with water (2 ꢀ 20 mL) and dried over anhydrous Na2SO4,
Retention times (tR) are given in minutes. Nuclear magnetic filtered and evaporated to dryness to give the crude products that
resonance (NMR) spectra were measured in CDCl3 on a Varian were recrystallized from 96% ethanol (yield: 85–91%).
Mercury-VX 300 spectrometer operating at 300.08 MHz (1H),
282 MHz (19F) and 75.46 MHz (13C). Sample concentrations were General procedure for the synthesis of 2-fluoro and 3-
in the range from 10 mg/1 mL (1H and 19F) to 50 mg/mL for 13C, trifluoromethylphenylpiperazinyalkyl derivatives of 1H-
all spectra were acquired at 26 ꢁC. The J values are in Hertz (Hz), imidazo[2,1-f]purine-2,4(3H,8H)-dione (4–21)
and splitting patterns are designated as follows: s (singlet), d
A mixture of 7-ketonyl derivatives of 8-bromo-1,3-dimethyl-3,7-
(doublet), t (triplet), m (multiplet). Mass spectrometry analyses:
dihydro-1H-purine-2,6-dione 1–3 (5mmol) with double amount of
Samples were prepared in acetonitrile/water (10/90 v/v) mixture.
appropriate 2-fluoro or 3-trifluoromethylphenylpiperazinyalkyla-
The LC/MS system consisted of a Waters Acquity UPLC, coupled
mine (10 mmol) was refluxed in 2-methoxyethanol (20 mL) for
to Waters TQD mass spectrometer (electrospray ionization mode
12h. The solvent was removed in vacuo, and the obtained residue
ESI-tandem quadrupole). All the analyses were carried out using
was purified by flash column chromatography on silica gel using
an Acquity UPLC BEH C18, 50 ꢀ 2.1 mm reversed-phase column.
mixture of dichloromethane/methanol (9/1) or (9/1.2) as an eluent.
A flow rate of 0.3 mL/min and a gradient of (5–5)% B over 5 min
was used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/
8-(3-(4-(2-Fluorophenyl)piperazin-1-yl)propyl)-1,3-dimethyl-1H-
0.1% HCO2H. Nitrogen was used for both nebulizing and drying
imidazo[2,1-f]purine-2,4(3H,8H)-dione (4)
gas. LC/MS data were obtained by scanning the first quadrupole
1
in 0.5 s in a mass range from 100 to 700 m/z; eight scans Yield: 62%, cream solid: m.p. 209–210 ꢁC, tR ¼ 0.831; H-NMR
were summed up to produce the final spectrum. Elemental (300 MHz, CDCl3): ꢀ (ppm): 2.07–2.16 (m, 2H, CH2CH2CH2),
analyses were found within 0.4% of the theoretical values. 2.46 (t, 2H, J ¼ 7.3 Hz, CH2CH2CH2), 2.64–2.65 (m, 4H,
Melting points (mp) were determined with a Bu¨chi apparatus and N(CH2)2), 3.09–3.12 (m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3),
are uncorrected. Column chromatography separations were 3.59 (s, 3H, N1–CH3), 4.17 (t, 2H, J ¼ 7.4 Hz, N8–CH2), 6.81–
carried out on column with Merck Kieselgel 60 (Darmstadt, 6.82 (d, 1H, J ¼ 2.3 Hz, arom), 6.96–7.05 (m, 4H, arom), 7.39–
Germany).
7.40 (d, 1H, J ¼ 2.3 Hz, C6H). 13C NMR (75 MHz, CDCl3) ꢀ
(ppm): 26.21, 27.77, 30.07, 44.16, 46.23, 50.45, 50.49, 53.10,
54.43, 105.00, 107.39, 115.98, 116.25, 118.62, 118.86, 122.51,
124.46, 139.98, 147.43, 151.98, 154.04, 157.34. 19F NMR
(282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19 (s, 1F). MS (EI) m/z:
440.38 [M +1]+. Anal. calcd for C22H26FN7O2: C, 60.12; H, 5.96;
N, 22.31; found: C, 60.2; H, 6.04; N, 22.37.
General procedure for the synthesis of phthalimide
derivatives (VI–XI)
A mixture of 10 mmol of 1-(2-fluorophenyl)- or 1-(3-trifluor-
omethyl)phenyl)piperazine (I–II) and 10 mmol of appropriate 2-
(bromoalkyl)-1H-isoindoline-1,3(2H)-dione (III–V), 30 mmol of
potassium carbonate, catalytic amount of potassium iodide and
20 mL of acetonitrile was stirred at 82 ꢁC temperature for 48 h.
The reaction mixture evaporated under reduced pressure to obtain
8-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-1,3-dimethyl-1H-
imidazo[2,1-f]purine-2,4(3H,8H)-dione (5)
1
a residue, which was purified by column chromatography using Yield: 69%, cream solid: m.p. 144–145 ꢁC, tR ¼ 1.011; H-NMR
dichloromethane/methanol (9/1) as an eluent.
(300 MHz, CDCl3): ꢀ (ppm): 1.61–1.66 (m, 2H, CH2CH2CH2CH2),

´
A. Zagorska et al.
4
J Enzyme Inhib Med Chem, Early Online: 1–15
1.92–2.02 (m, 2H, CH2CH2CH2CH2), 2.50 (t, 2H, J ¼ 7.1 Hz, 8-(5-(4-(2-Fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-
CH2CH2CH2CH2), 2.60–2.66 (m, 4H, N(CH2)2), 3.10–3.12 (m, 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9)
4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), 3.59 (s, 3H, N1–CH3), 4.19
1
Yield: 92%, Cream solid: m.p. 143–145 ꢁC, tR ¼ 1.197; H-NMR
(t, 2H, J ¼ 2.8 Hz, N8–CH2), 6.78–6.79 (d, 1H, J ¼ 2.3 Hz, arom),
(300 MHz, CDCl3): ꢀ (ppm): 1.37–1.45 (m, 2H, CH2CH2
6.92–7.04 (m, 4H, arom), 7.40–7.41 (d, 1H, J ¼ 3 Hz, C6H). 13C
CH2CH2CH2), 1.57–1.65 (m, 2H, CH2CH2CH2CH2CH2), 1.86–
NMR (75 MHz, CDCl3) ꢀ (ppm): 23.30, 26.96, 27.52, 30.09,
1.91 (m, 2H, CH2CH2CH2CH2CH2), 2.32 (s, 3H, CH3), 2.43 (t,
46.11, 46.23, 50.38, 53.24, 57.62, 100.05, 107.60, 115.96, 116.24,
2H, J ¼ 2.8 Hz, CH2CH2CH2CH2CH2), 2.60–2.66 (m, 4H,
117.96, 118.85, 122.49, 124.42, 135.33, 139.98, 147.46, 151.98,
N(CH2)2), 3.10–3.13 (m, 4H, (CH2)2N), 3.42 (s, 3H, N3–CH3),
157.32, 168.14. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19
3.59 (s, 3H, N1–CH3), 4.01 (t, 2H, J ¼ 7.1 Hz, N8–CH2), 6.91–
7.16 (m, 4H, arom), 7.26 (s, 1H, C6H). 13C NMR (75 MHz,
CDCl3) ꢀ (ppm): 5.67, 19.71, 21.40, 22.99, 24.52, 25.33, 38.50,
45.61, 48.51, 53.45, 95.12, 99.80, 111.19, 111.46, 114.13, 117.70,
111.81, 119.67, 122.07, 135.15, 143.01, 146.67, 147.25, 149.28,
152.57.19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19 (s, 1F).
MS (EI) m/z: 482.39 [M + 1]+. Anal. calcd for C25H32FN7O2: C,
62.35; H, 6.7; N, 20.36; found: C, 62.42; H, 6.78; N, 20.37.
(s, 1F). MS (EI) m/z: 454.34 [M+ 1]+. Anal. calcd for
C23H28FN7O2: C, 60.91; H, 6.22; N, 21.62; found: C, 61.2; H,
6.32; N, 21.37.
8-(5-(4-(2-Fluorophenyl)piperazin-1-yl)pentyl)-1,3-dimethyl-1H-
imidazo[2,1-f]purine-2,4(3H,8H)-dione (6)
1
Yield: 52%, Cream solid: m.p. 168–170 ꢁC, tR ¼ 1.145; H-NMR
(300 MHz, CDCl3): ꢀ (ppm): 1.40–1.43 (m, 2H, CH2CH2
CH2CH2CH2), 1.62–1.66 (m, 2H, CH2CH2CH2CH2CH2), 1.89–
1.97 (m, 2H, CH2CH2CH2CH2CH2), 2.45 (t, 2H, J ¼ 2.8 Hz,
CH2CH2CH2CH2CH2), 2.60–2.68 (m, 4H, N(CH2)2), 3.10–3.14
(m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), 3.60 (s, 3H, N1–CH3),
4.07 (t, 2H, J ¼ 7.1 Hz, N8–CH2), 6.78–6.79 (d, 1H, J ¼ 2.3 Hz,
arom), 6.90–7.05 (m, 4H, arom), 7.40–7.41 (d, 1H, J ¼ 3.1 Hz,
C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 24.32, 25.97, 27.75,
29.25, 30.07, 46.13, 50.23, 53.19, 58.11, 100.00, 107.52, 115.93,
116.21, 118.04, 118.89, 122.59, 124.42, 124.46, 139.94, 147.31,
151.95, 152.18, 154.01, 157.29. 19F NMR (282 MHz, CDCl3): ꢀ
(ppm): ꢂ123.19 (s, 1F). MS (EI) m/z: 468.43 [M + 1]+. Anal.
calcd for C24H30FN7O2: C, 61.65; H, 6.47; N, 20.97; found: C,
61.42; H, 6.66; N, 21.07.
8-(3-(4-(2-Fluorophenyl)piperazin-1-yl)propyl)-1,3-dimethyl-7-
phenyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (10)
1
Yield: 65%, Cream oil, tR ¼ 1.232; H-NMR (300 MHz, CDCl3):
ꢀ (ppm): 1.18–1.42 (m, 2H, CH2CH2CH2), 1.94–2.04 (m, 2H,
CH2CH2CH2), 2.53–2.73 (m, 4H, N(CH2)2), 2.96–3.12 (m, 4H,
(CH2)2N), 3.32 (s, 3H, N3–CH3), 3.55 (s, 3H, N1–CH3), 3.63 (t,
2H, J ¼ 4.1 Hz, N8–CH2), 6.50–6.55 (m, 2H, arom), 6.93–7.00 (m,
3H, arom), 7.35–7.58 (m, 4H, arom), 7.98–8.01 (d, J ¼ 3.4 Hz,
1H, C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 24.23, 25.92,
27.80, 29.69, 30.13, 42.36, 49.32, 50.37, 50.41, 52.99, 53.41,
54.78, 105.34, 115.90, 116.18, 118.80, 118.85, 122.40, 122.51,
124.40, 124.45, 128.26, 128.39, 151.74, 152.03, 154.06, 154.25,
157.32. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19 (s, 1F).
MS (EI) m/z: 516.36 [M + 1]+. Anal. calcd for C28H30FN7O2: C,
65.23; H, 5.86; N, 19.02; found: C, 65.38; H, 6.08; N, 18.96.
8-(3-(4-(2-Fluorophenyl)piperazin-1-yl)propyl)-1,3,7-trimethyl-
1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (7)
8-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-1,3-dimethyl-7-
phenyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (11)
1
Yield: 72%, Cream solid: m.p. 160–161 ꢁC, tR ¼ 1.005; H-NMR
(300 MHz, CDCl3): ꢀ (ppm): 2.09–2.16 (m, 2H, CH2CH2CH2),
2.35 (s, 3H, CH3), 2.45 (t, 2H, J ¼ 2.9 Hz, CH2CH2CH2), 2.63–
2.65 (m, 4H, N(CH2)2), 3.09–3.12 (m, 4H, (CH2)2N), 3.42 (s, 3H,
N3–CH3), 3.58 (s, 3H, N1–CH3), 4.10 (t, 2H, J ¼ 7.4 Hz, N8–
CH2), 6.92–7.05 (m, 4H, arom), 7.26 (s, 1H, C6H). 13C NMR
(75 MHz, CDCl3) ꢀ (ppm): 10.32, 26.01, 27.74, 30.04, 41.17,
50.43, 50.48, 53.16, 54.67, 104.43, 115.98, 116.25, 118.83,
118.88, 122.57, 124.46, 127.13, 139.88, 147.71, 151.43, 152.01,
154.02, 157.32. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19
(s, 1F). MS (EI) m/z: 454.34 [M + 1]+. Anal. calcd for
C23H28FN7O2: C, 60.91; H, 6.22; N, 21.62; found: C, 60.82; H,
6.41; N, 21.73.
1
Yield: 75%, Cream solid: m.p. 133–134 ꢁC, tR ¼ 1.363; H-NMR
(300 MHz, CDCl3):
ꢀ
(ppm): 1.21–1.42 (m, 2H,
CH2CH2CH2CH2), 1.70–1.92 (m, 2H, CH2CH2CH2CH2), 2.68–
2.74 (m, 2H, CH2CH2CH2CH2), 2.86–2.92 (m, 4H, N(CH2)2),
3.08–3.16 (m, 4H, (CH2)2N), 3.32–3.39 (m, 4H, N3–CH3 +N8–
CH2), 3.54–3.72 (m, 4H, N1–CH3 +N8–CH2), 6.80–7.05 (m, 5H,
arom), 7.45–7.63 (m, 4H, arom), 8.04–8.06 (d, J ¼ 8.0 Hz, 1H,
C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 23.30, 24.96, 27.52,
29.69, 30.90, 42.89, 49.26, 49.67, 57.68, 59.07, 60.90, 71.92,
115.98, 116.25, 118.92, 119.20, 122.78, 122.89, 124.45, 124.49,
127.38, 127.70, 128.08, 128.71, 128.87, 134.32, 149.19, 151.74,
154.19. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19 (s, 1F).
MS (EI) m/z: 530.60 [M +1]+. Anal. calcd for C29H32FN7O2: C,
65.75; H, 6.09; N, 18.51; found: C, 65.73; H, 6.02; N, 18.53.
8-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-1,3,7-trimethyl-
1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (8)
8-(5-(4-(2-Fluorophenyl)piperazin-1-yl)pentyl)-1,3-dimethyl-7-
phenyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (12)
Yield: 84%, Cream solid: m.p. 153–155 ꢁC, tR ¼ 1.155; 1H-
NMR (300 MHz, CDCl3):
ꢀ (ppm): 1.55–1.66 (m, 2H,
1
CH2CH2CH2CH2), 1.87–1.94 (m, 2H, CH2CH2CH2CH2), 2.33
(s, 3H, CH3), 2.53–2.68 (m, 6H, CH2CH2CH2CH2 + N(CH2)2),
3.10–3.12 (m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), 3.59 (s,
3H, N1–CH3), 4.05 (t, 2H, J ¼ 3.1 Hz, N8–CH2), 6.93–7.05 (m,
4H, arom), 7.26 (s, 1H, C6H). 13C NMR (75 MHz, CDCl3) ꢀ
(ppm): 10.41, 23.72, 24.53, 27.34, 27.74, 30.06, 43.17, 50.41,
53.25, 57.63, 58.31, 104.59, 115.96, 116.24, 118.88, 122.56,
124.42, 124.46, 126.98, 139.92, 147.77, 151.02, 151.43,
154.07. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ123.19 (s,
1F). MS (EI) m/z: 468.43 [M+ 1]+. Anal. calcd for
C24H30FN7O2: C, 61.65; H, 6.47; N, 20.97; found: C, 61.7;
H, 6.32; N, 21.03.
Yield: 77%, Cream oil, tR ¼ 1.422; H-NMR (300 MHz, CDCl3):
ꢀ (ppm): 1.19–1.28 (m, 2H, CH2CH2CH2CH2CH2), 1.40–1.45 (m,
2H, CH2CH2CH2CH2CH2), 1.75–1.80 (m, 2H, CH2CH2
CH2CH2CH2), 2.31 (t, 2H, J ¼ 2.2 Hz, CH2CH2CH2CH2CH2),
2.57–2.63 (m, 4H, N(CH2)2), 3.07–3.14 (m, 4H, (CH2)2N), 3.44
(s, 3H, N3–CH3), 3.63 (s, 3H, N1–CH3), 4.11 (t, 2H, J ¼ 3.4 Hz,
N8–CH2), 6.91–7.05 (m, 5H, arom), 7.41–7.50 (m, 4H, arom),
8.04–8.03 (d, J ¼ 5.0 Hz, 1H, C6H). 13C NMR (75 MHz, CDCl3) ꢀ
(ppm): 23.30, 24.96, 27.52, 29.69, 30.90, 42.89, 49.26, 49.67,
52.99, 57.68, 59.07, 60.90, 71.92, 115.98, 116.25, 118.92, 119.20,
122.78, 122.89, 124.45, 124.49, 127.38, 127.70, 128.08, 128.71,
128.87, 134.32, 149.19, 151.74, 154.19. 19F NMR (282 MHz,

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
5
CDCl3): ꢀ (ppm): ꢂ123.14 (s, 1F). MS (EI) m/z: 544.55 [M+ 1]+. 50.49, 52.99, 54.43, 105.00, 107.23, 112.07, 115.89, 118.32,
Anal. calcd for C30H34FN7O2: C, 66.28; H, 6.30; N, 18.04; found: 121.01, 123.36, 124.56, 129.53, 131.21, 151.28, 152.01, 154.04.
C, 66.42; H, 6.06; N, 18.07.
19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ60.34 (s, 3F). MS (EI)
m/z: 504.39 [M + 1]+. Anal. calcd for C24H28F3N7O2: C, 57.25; H,
5.61; N, 19.47; found: C, 57.20; H, 5.30; N, 19.37.
1,3-Dimethyl-8-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)propyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (13)
1,3,7-Trimethyl-8-(4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)butyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (17)
1
Yield: 78%, Cream solid: m.p. 110–112 ꢁC, tR ¼ 1.211; H-NMR
(300 MHz, CDCl3): ꢀ (ppm): 2.09–2.18 (m, 2H, CH2CH2CH2),
1
2.46 (t, 2H, J ¼ 7.3 Hz, CH2CH2CH2), 2.64–2.65 (m, 4H, Yield: 87%, Cream solid: m.p. 188–190 ꢁC, tR ¼ 1.306; H-NMR
N(CH2)2), 3.24–3.27 (m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), (300 MHz, CDCl3):
ꢀ
(ppm): 1.48–1.53 (m, 2H,
3.60 (s, 3H, N1–CH3), 4.18 (t, 2H, J ¼ 3.3 Hz, N8–CH2), 6.82– CH2CH2CH2CH2), 1.76–1.78 (m, 2H, CH2CH2CH2CH2), 2.24–
6.83 (m, 2H, arom), 7.04–7.10 (m, 3H, arom), 7.40–7.41 (d, 1H, 2.51 (m, 9H, CH3 +CH2CH2CH2CH2 +N(CH2)2), 3.10–3.13 (m,
J ¼ 2.3 Hz, C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 26.96, 4H, (CH2)2N), 3.30 (s, 3H, N3–CH3), 3.48 (s, 3H, N1–CH3),
27.52, 30.09, 42.16, 46.75, 50.45, 50.49, 52.99, 54.43, 105.00, 3.94–3.98 (m, 2H, N8–CH2), 6.95–6.98 (m, 3H, arom), 7.13–7.25
107.39, 111.37, 115.09, 116.25, 117.22, 121.01, 122.51, 124.16, (m, 2H, arom). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 13.30, 26.96,
129.53, 131.21, 151.28, 152.01, 154.04. 19F NMR (282 MHz, 27.52, 28.33, 29.69, 30.09, 42.89, 49.26, 49.67, 57.68, 59.07,
CDCl3): ꢀ (ppm): ꢂ60.34 (s, 3F). MS (EI) m/z: 490.36 [M +1]+. 71.92, 104.61, 107.33, 109.2, 111.77, 116.09, 118.12, 121.01,
Anal. calcd for C23H26F3N7O2: C, 56.44; H, 5.35; N, 20.03; 123.36, 129.53, 131.21, 151.28, 152.01, 154.04. 19F NMR
found: C, 56.20; H, 5.30; N, 20.37.
(282 MHz, CDCl3): ꢀ (ppm): ꢂ63.12 (s, 3F). MS (EI) m/z:
519.12 [M +1]+. Anal. calcd for C25H30F3N7O2: C, 58.02; H,
5.84; N, 18.94; found: C, 58.22; H, 5.58; N, 19.07.
1,3-Dimethyl-8-(4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)butyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (14)
1,3,7-Trimethyl-8-(5-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)pentyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (18)
1
Yield: 82%, Cream solid: m.p. 170–171 ꢁC, tR ¼ 1.254; H-NMR
(300 MHz, CDCl3):
ꢀ
(ppm): 1.48–1.53 (m, 2H,
1
CH2CH2CH2CH2), 1.82–1.92 (m, 2H, CH2CH2CH2CH2), 2.40– Yield: 80%, Cream solid: m.p. 168–169 ꢁC, tR ¼ 1.340, H-NMR
2.54 (m, 6H, CH2CH2CH2CH2 +N(CH2)2), 3.10–3.17 (m, 4H, (300 MHz, CDCl3): ꢀ (ppm): 1.35–1.37 (m, 2H, CH2CH2
(CH2)2N), 3.34 (s, 3H, N3–CH3), 3.51 (s, 3H, N1–CH3), 4.04 (t, CH2CH2CH2), 1.55–1.58 (m, 2H, CH2CH2CH2CH2CH2), 1.83–
2H, J ¼ 3.1 Hz, N8–CH2), 6.79–6.80 (d, 1H, J ¼ 2.3 Hz, arom), 1.85 (m, 2H, CH2CH2CH2CH2CH2), 2.28–2.93 (m, 9H,
6.91–7.01 (m, 2H, arom), 7.24–7.26 (m, 2H, arom), 7.40–7.41 (d, CH3 +CH2CH2CH2CH2CH2 +N(CH2)2), 3.20–3.24 (m, 4H,
1H, J ¼ 3 Hz, C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 26.96, (CH2)2N), 3.36 (s, 3H, N3–CH3), 3.54 (s, 3H, N1–CH3), 3.95–
27.52, 29.69, 30.09, 42.16, 46.75, 50.45, 50.49, 52.99, 54.43, 4.00 (m, 2H, N8–CH2), 6.99–7.07 (m, 3H, arom), 7.26–7.32 (m,
105.00, 107.39, 112.22, 115.75 115.98, 117.92, 118.62, 121.01, 2H, arom). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 10.20, 24.38,
123.56, 129.53, 129.91, 151.28, 152.01, 154.04. 19F NMR 25.91, 27.74, 29.25, 30.00, 43.21, 49.10, 52.84, 58.12, 99.83,
(282 MHz, CDCl3): ꢀ (ppm): ꢂ62.99 (s, 3F). MS (EI) m/z: 104.65, 112.13, 116.04, 118.76, 122.39, 126.00, 127.21, 129.51,
504.10 [M + 1]+. Anal. calcd for C24H28F3N7O2: C, 57.25; H, 131.10, 131.51, 147.63, 151.08, 151.38, 151.90, 154.04. 19F NMR
5.61; N, 19.47; found: C, 57.02; H, 5.32; N, 19.37.
(282 MHz, CDCl3): ꢀ (ppm): ꢂ63.14 (s, 3F). MS (EI) m/z: 532.15
[M + 1]+. Anal. calcd for C26H32F3N7O2: C, 58.75; H, 6.07; N,
18.44; found: C, 58.52; H, 6.10; N, 18.50.
1,3-Dimethyl-8-(5-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)pentyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (15)
1,3-Dimethyl-7-phenyl-8-(3-(4-(3-(trifluoromethyl)phenyl)pipera-
zin-1-yl)propyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (19)
Yield: 86%, Cream oil, tR ¼ 1.301, 1H-NMR (300 MHz, CDCl3): ꢀ
(ppm): 1.40–1.42 (m, 2H, CH2CH2CH2CH2CH2), 1.62–1.67 (m,
(m,
1
2H,
CH2CH2CH2CH2CH2),
CH2CH2CH2CH2CH2),
2.49
1.89–1.96
(t, 2H,
2H, Yield: 79%, Cream solid: m.p. 128–129 ꢁC, tR ¼ 1.501; H-NMR
J ¼ 2.8 Hz, (300 MHz, CDCl3): ꢀ (ppm): 1.82–2.02 (m, 2H, CH2CH2CH2),
CH2CH2CH2CH2CH2), 2.70–2.73 (m, 4H, N(CH2)2), 3.26–3.28 2.34 (t, 2H, J ¼ 7.3 Hz, CH2CH2CH2), 2.42–2.44 (m, 4H,
(m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), 3.59 (s, 3H, N1–CH3), N(CH2)2), 3.08–3.11 (m, 4H, (CH2)2N), 3.45 (s, 3H, N3–CH3),
4.06 (t, 2H, J ¼ 7.1 Hz, N8–CH2), 6.78–6.79 (d, 1H, J ¼ 2.3 Hz, 3.63 (s, 3H, N1–CH3), 4.21 (t, 2H, J ¼ 3.3 Hz, N8–CH2), 6.90–
arom), 7.05–7.09 (m, 4H, arom), 7.40–7.41 (d, 1H, J ¼ 3.1 Hz, 7.08 (m, 6H, arom), 7.33–7.49 (m, 4H, arom). 13C NMR (75 MHz,
C6H). 13C NMR (75 MHz, CDCl3) ꢀ (ppm): 26.96, 27.52, 28.33, CDCl3) ꢀ (ppm): 10.30, 25.92, 27.80, 29.69, 30.13, 42.36, 49.32,
29.69, 30.09, 42.16, 46.75, 50.45, 50.49, 52.99, 54.43, 105.00, 50.37, 50.41, 52.99, 53.41, 54.78, 105.34, 112.07, 115.90, 116.18,
107.39, 111.97, 114.89, 116.25, 118.02, 121.01, 122.96, 124.56, 118.32, 121.01, 122.40, 124.40, 125.22, 128.26, 129.53, 131.21,
129.53, 131.42, 151.28, 152.01, 154.04. 19F NMR (282 MHz, 151.28, 152.01, 154.04, 154.25, 157.32. 19F NMR (282 MHz,
CDCl3): ꢀ (ppm): ꢂ63.14 (s, 3F). MS (EI) m/z: 518.19 [M +1]+. CDCl3): ꢀ (ppm): ꢂ60.34 (s, 3F). MS (EI) m/z: 566.49 [M + 1]+.
Anal. calcd for C25H30F3N7O2: C, 58.02; H, 5.84; N, 18.94; Anal. calcd for C29H30F3N7O2: C, 61.58; H, 5.35; N, 17.34;
found: C, 58.22; H, 5.77; N, 18.97.
found: C, 61.60; H, 5.33; N, 17.43.
1,3,7-Trimethyl-8-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)propyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (16)
1,3-Dimethyl-7-phenyl-8-(4-(4-(3-(trifluoromethyl)phenyl)pipera-
zin-1-yl)butyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (20)
1
1
Yield: 95%, Cream oil, tR ¼ 1.172; H-NMR (300 MHz, CDCl3): Yield: 81%, Cream oil; tR ¼ 1.490; H-NMR (300 MHz, CDCl3):
ꢀ (ppm): 2.11–2.13 (m, 2H, CH2CH2CH2), 2.34 (s, 3H, CH3), ꢀ (ppm): 1.18–1.25 (m, 4H, CH2CH2CH2CH2), 1.42–1.52 (m, 6H,
2.47–2.49 (m, 2H, CH2CH2CH2), 2.64–2.65 (m, 4H, N(CH2)2), CH2CH2CH2CH2 +N(CH2)2), 3.39–3.64 (m, 10H, (CH2)2N +
3.23–3.26 (m, 4H, (CH2)2N), 3.43 (s, 3H, N3–CH3), 3.58 (s, 3H, N3–CH3 +N1–CH3), 4.16 (t, 2H, J ¼ 3.1 Hz, N8–CH2), 7.09–7.25
N1–CH3), 4.10 (t, 2H, J ¼ 3.3 Hz, N8–CH2), 7.03–7.16 (m, 4H, (m, 2H, arom), 7.43–7.52 (m, 8H, arom).
arom), 7.34–7.37 (m, 1H, C6H). 13C NMR (75 MHz, CDCl3) ꢀ
13C NMR (75 MHz, CDCl3) ꢀ (ppm): 22.97, 24.96, 27.52,
(ppm): 10.30, 26.96, 27.52, 28.33, 30.09, 42.16, 46.75, 50.45, 29.96, 30.89, 42.15, 49.26, 49.77, 57.68, 59.17, 60.94, 72.02,

´
A. Zagorska et al.
6
J Enzyme Inhib Med Chem, Early Online: 1–15
111.87, 115.91, 116.27, 119.12, 120.99, 122.87, 122.89, 124.54, Inhibition of PDE4B1 and PDE10A was measured using PDElight
125.01, 128.08, 128.71, 128.87, 129.53, 131.01, 149.23, 151.18, HTS cAMP phosphodiesterase assay kit (Lonza) according to the
152.17, 154.01. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ62.82 (s, manufacturer’s recommendations. About 5 ng of human, recom-
3F). MS (EI) m/z: 580.14 [M +1]+. Anal. calcd for binant PDE4B1 (Sigma-Aldrich, St. Louis, MO) or 10 ng
C30H32F3N7O2: C, 62.17; H, 5.56; N, 16.92; found: C, 62.02; PDE10A (BPS Biosciences) in assay buffer was incubated with
H, 5.32; N, 16.73.
reference (papaverine for PDE10A, 3-isobutyl-1-methylxanthine
(IBMX) for PDE4B1) and tested compound for 20 min. After
1,3-Dimethyl-7-phenyl-8-(5-(4-(3-(trifluoromethyl)phenyl)pipera- incubation, the cAMP substrate (final concentration 1.25 mM for
zin-1-yl)pentyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (21)
PDE10A and 10 mM for PDE4B1) was added and incubated for
1 h. Then, PDELight AMP detection reagent was added and
incubated 10 min. All reactions were carried out at 37 ꢁC in
white-walled, 384-well plate. Luminescence was measured in a
multifunctional microplate reader (POLARstar Omega, BMG
Labtech, Germany). All the assays were carried out in duplicates
(n ¼ 2).
Yield: 85%, Cream oil, tR ¼ 1.540, 1H-NMR (300 MHz, CDCl3): ꢀ
(ppm): 1.18–1.25 (m, 4H, CH2CH2CH2CH2CH2), 1.56–1.81 (m,
8H, CH2CH2CH2CH2CH2 +N(CH2)2), 3.36–3.63 (m, 10H,
(CH2)2N + N3–CH3 +N1–CH3), 4.10–4.12 (m, 2H, N8–CH2),
7.10–7.25 (m, 2H, arom), 7.42–7.64 (m, 8H, arom). 13C NMR
(75 MHz, CDCl3) ꢀ (ppm): 23.33, 24.96, 27.52, 29.69, 30.90,
42.89, 49.26, 49.67, 52.99, 57.68, 59.07, 60.90, 71.92, 112.07,
115.89, 116.25, 118.32, 119.20, 121.01, 122.78, 122.89, 123.36,
Functional assays for 5-HT1A and 5-HT7 receptors
128.08, 128.71, 128.87, 129.53, 131.21, 149.19, 151.28, 152.01, Test and reference compounds were dissolved in dimethyl
154.04. 19F NMR (282 MHz, CDCl3): ꢀ (ppm): ꢂ62.81 (s, 3F). sulfoxide (DMSO) at a concentration of 1 mM. Serial dilutions
MS (EI) m/z: 594.27 [M + 1]+. Anal. calcd for C31H34F3N7O2: C, were prepared in 96-well microplate in assay buffers and eight
62.72; H, 5.77; N, 16.52; found: C, 62.61; H, 5.71; N, 16.67.
concentrations were tested. All the assays were carried out in
duplicates (n ¼ 2). The EC50 values (concentration producing a
half-maximal response) and IC50 values (concentration causing a
half-maximal inhibition of the control agonist response) were
determined by nonlinear regression analysis of the concentration
response curves generated with mean replicate values using Hill
equation curve fitting (GraphPad Prism 6.0 software; GraphPad
Software Inc., La Jolla, CA). The log IC50 was used to obtain the
antagonist equilibrium dissociation constant (Kb) by applying the
Cheng–Prusoff Equation (1):
Radioligand binding assays
All of the compounds were tested in radioligand binding assays
measuring their affinity for 5-HT1A and 5-HT7 receptors. The
inhibition constants Ki were determined based on previously
described protocols29,32. One millimolar stock solutions of the
compounds to be tested was prepared in DMSO. Serial dilutions
of compounds were prepared in 96-well microplate in assay
buffers using an automated pipetting system (epMotion 5070;
Eppendorf). Radioligand binding was performed using cryopre-
served membranes from cells stably expressing the relevant
human receptor. Reagents and condition: 50 mL working solution
of the tested compounds, 50 mL radioligand solution and 150 mL
diluted membranes prepared in assay buffer were transferred to
96-well microplates. These were covered with sealing tape, mixed
and incubated. The reaction was terminated by rapid filtration
through UniFilter 96 GF/B filter microplate and 10 rapid washes
with 200 mL 50 mM Tris buffer (4 ꢁC, pH 7.4) were performed
using vacuum manifold and 96-well pipettor. The UniFilter
microplates were dried overnight at 37 ꢁC in dry incubator. The
UniFilter bottoms were sealed and 30 mL of liquid scintillator
Betaplate Scint (PerkinElmer, Waltham, MA) was added to each
well. The plates were allowed to equilibrate for 1 h, and then,
radioactivity was counted in MicroBeta TriLux 1450 scintillation
counter (PerkinElmer, Waltham, MA) at approximately 30%
efficiency. Data were fitted to a one-site curve-fitting equation
with Prism 5 (GraphPad Software Inc., La Jolla, CA) and Ki
values were calculated using the Cheng–Prusoff equation33. Each
compound was tested in 10 concentrations from 1 ꢀ 10–5 M to
1 ꢀ 10–9 M (final concentration). All the assays were carried out
in duplicates (n ¼ 2). The inhibition constants Ki for D2 and 5-
HT2A,6 receptors were determined based on previously described
IC50
ꢀ
ꢁ
Kb ¼
ð1Þ
A
EC50A
1 þ
where Kb – antagonist equilibrium dissociation constant, A –
concentration of reference agonist in the assay, EC50A ¼EC50
value of reference agonist.
A cellular aeqorin-based functional assay was performed with
recombinant CHO-K1 cells expressing mitochondrially targeted
aequorin, human GPRC and the promiscuous G protein a16 for 5-
HT1A. Assay was executed according to previously described
protocol34. After thawing, cells were transferred to (DMEM/
HAM’s F12 with 0.1% protease-free BSA) and centrifuged. The
cell pellet was resuspended in assay buffer and coelenterazine h
was added at final concentration of 5 mM. The cell suspension was
incubated at 16 ꢁC, protected from light with constant agitation
for 16 h and diluted with assay buffer to the concentration of
100 000 cells/ml. after 1 h of incubation, 50 mM of the cells
suspension was dispensed using automatic injectors built into the
radiometric and luminescence plate counter MicroBeta2 LumiJET
(PerkinElmer, Waltham, MA) into white opaque 96-well micro-
plate preloaded with test compounds. Immediate light emission
generated following calcium mobilization was recordered for 30 s.
In antagonist mode, after 25 min of incubation, the reference
agonist was added to the above assay mix and light emission was
recordered again. Final concentration of the reference agonist was
to EC80 (100 nM for serotonin).
protocols29,32
.
The phosphodiesterase activity tests
For the 5-HT7 receptor, adenylyl cyclase activity was
monitored using cryopreserved CHO-K1 cells, expressing the
human serotonin 5-HT7 receptor. Thawed cells were resuspended
in stimulation buffer (HBSS, 5 mM HEPES, 0.5 IBMX and 0.1%
BSA at pH 7.4) at 200 000 cells/mL. Ten microliters of cell
suspension was added to 10 mL of tested compounds and loaded
onto a white opaque half area 96-well microplate. An antagonist
response experiment was performed with 10 nM serotonin as the
Inhibition of PDE4B1 and PDE10A was measured using PDElight
HTS cAMP phosphodiesterase assay kit (PDELightTM Lonza)
,
according to manufacturer’s recommendations. The percentage of
inhibition was calculated to vehicle control (DMSO). Test and
reference compounds were dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 1 mM and further diluted in
assay buffer (10 mM Tris-HCl, 10 mM magnesium chloride and
0.05% Tween-20; pH 7.4) and eight concentrations were tested.

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
7
reference agonist, and the agonist and antagonist were added stopped by the addition of perchloric acid (69–72%, by volume).
simultaneously. Cell stimulation was performed for 60 min at Proteins were sedimented by centrifugation. The supernatant was
room temperature. After incubation, cAMP measurements were analyzed using UPLC/MS (Waters Corporation, Milford, MA) for
taken with homogeneous time-resolved fluorescence energy qualitative analysis. The peak area ratios of analyte versus internal
transfer (TR-FRET) immunoassay using the LANCE Ultra standard were used to calculate the % remaining at the end of
cAMP kit (PerkinElmer, Waltham, MA). Ten microliters of 15 min of biotransformation. All the assays were carried out in
EucAMP tracer working solution and 10 mL of ULight-anti-cAMP duplicates (n ¼ 2).
tracer working solution were added, mixed and incubated for 1 h.
The TR-FRET signal was read on an EnVision microplate reader Pharmacology – general condition
(PerkinElmer, Waltham, MA).
The experiments were performed on male Albino Swiss mice (22–
28 g). The animals were kept at a room temperature of 20 1 ꢁC
and had free access to food (standard laboratory pellets) and tap
Determination of lipophilicity
Borate-phosphate
buffer
pH
7.0
with
SDS water before the experiment. All the experiments were conducted
(100 mM + 50 mM + 50 mM), SDS, 0.1 M of sodium hydroxide in the light phase between 9 a.m. and 2 p.m. All the experimental
´
were purchased from Sigma-Aldrich (Poznan, Poland). Methanol procedures were approved by the Local Ethics Commission for
(MeOH) HPLC purity. Reference drugs (purity499.5%) were Animal Experiments of Jagiellonian University in Cracow.
purchased from different sources: barbital, phenobarbital (Polfa- Citalopram (Sigma-Aldrich, St. Louis, MO) was dissolved in
Tarchomin, Poland), aminophenazone, caffeine (Polfa-Pabianice, distilled water. The investigated compound (9) and diazepam were
´
Poland), chloramphenicol (Polfa-Krakow, Poland), phenytoin suspended in a 1% aqueous solution of Tween 80 and was injected
(Polfa-Warszawa, Poland), phenacetin (Azienda Chimica intraperitoneally (ip) 60 min before the test (citalopram 30 min
e
Farmaceutica, Italy), benzocaine (Alfa Aesar, Germany), procaine before the test). Each experimental group consisted of six to ten
(Amara Co., Poland). Trazodone was extracted from Trittico CR animals, and all the animals were used only once.
tablets (Aziende Chimiche Riunite Angelini Francesco
(A.C.R.A.F.), Italy). The analyses were performed on Beckman Forced swim (porsolt) test in swiss albino mice
P/ACE MDQ instrument (Beckman, Brea, CA) with DAD with
The experiment was carried out according to the method of
UV detection at 220 nm. The electropherograms were recorded
Porsolt et al.37. Mice were individually placed in a glass cylinder
and analyzed with the 32 Karat Software version 8.0. Uncoated
(25 cm high; 10 cm in diameter) containing 10 cm of water
fused-silica capillaries (Beckman, Brea, CA; 39 cm distance to the
maintained at 23–25 ꢁC and were left there for 6 min. A mouse
detector, 50 mm internal diameter, 49 cm total length, 375 mm
was regarded as immobile when it remained floating on the water,
external diameter) were used. Background electrolyte, samples
making only small movements to keep its head above it. The total
preparation and separation conditions were prepared according to
duration of immobility was recorded during the last 4 min of a
methods of Bajda et.al.35.
6-min test session.
The logarithm of retention factor, log k, was calculated from
the MEKC migration times according to the Equation (2)35:
Four-plate test in Swiss albino mice
tR ꢂ tEOF
tMC
log k ¼ log
ð2Þ
The four-plate apparatus (BIOSEB) consists of a plastic cage
(25 ꢀ 18 ꢀ 16 cm) floored by four identical rectangular metal
plates (8 ꢀ 11 cm) separated from one another by a gap of 4 mm.
The top of the cage is covered by a transparent Perspex lid that
prevents escape behavior. The plates are connected to a device that
can generate electric shocks. Following a 15-s habituation period,
the animal’s motivation to explore a novel environment was
suppressed by an electric foot shock (0.8 mA, 0.5 s) every time it
moves from one plate to another during a 1-min test session. This
action is referred to as a ‘‘punished crossing’’ and was followed bya
3-s shock interval, during which the animal can move across plates
without receiving a shock38. The number of punished crossings
received by an animal was recorded during the 60-s period.
tR
tEOF
ꢀ
where tR, tEOF and tMC are the migration time (min) of the solute,
the EOF marker (MeOH) and the micelle marker (Sudan III),
respectively. Estimated log p (cLog p) coefficients for the tested
compounds were calculated from calibration curve obtained by
linear regression of the log k and log p values of reference
compounds according to Equation (3), where parameters b and a
represent the slope and intercept of the curve, respectively:
clog P ¼ blog k þ a
ð3Þ
Metabolism study
Spontaneous locomotor activity in mice
Human liver microsomes (HLMs, microsomes from liver, pooled,
from human adult male and female), NADPH-regenerating
system components and pentoxifylline were purchased from
Locomotor activity was recorded with an Opto M3 multichannel
activity monitor (MultiDevice Software v.1.3, Columbus
Instruments). Swiss albino mice were individually placed in
plastic cages (22 ꢀ 12 ꢀ 13 cm) for 30-min habituation period,
and then, the ambulation was counted from 2 to 6 min or for 1 min
15 s, that is the time equal to the observation period in the forced
swim test or the four-plate test, respectively. The cages were
cleaned up with 70% ethanol after each mouse.
´
Sigma Aldrich (Poznan, Poland). Stock solutions of test
compounds were prepared in ethanol. The final ethanol
concentration in the incubation mixture was 0.38%. The
incubation systems were composed of test compound (20 mM),
HLMs (0.4 mg/mL), NADPH-regenerating system and potassium
phosphate buffer (100 mM, pH 7.4). NADPH-regenerating system
contained NADP, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase and potassium phosphate buffer (100 mM, pH
7.4). Firstly, the mixtures containing HLM, the test compound and
a buffer were preincubated at 37 ꢁC for 15 min before the addition
of the NADPH-regenerating system. The resulting mixture was
incubated for 15 min at 37 ꢁC in a thermoblock. Next, pentoxifyl-
line (20 mM, internal standard) was added, and the reaction was
Statistics
All the data are presented as the mean SEM. The statistical
significance of results was evaluated by a one-way analysis of
variance (ANOVA) followed by Bonferroni’s comparison test.
p 50.05 was considered statistically significant.

´
A. Zagorska et al.
8
J Enzyme Inhib Med Chem, Early Online: 1–15
¨
(Schrodinger, Inc.), which was licensed for Jagiellonian
University Medical College.
Molecular modeling
The homology models of human 5-HT1A and 5-HT7 serotonin
receptors adopted for docking in the present study were built on
the template of b2 adrenergic receptor crystal structure (PDB ID:
2RH1). The homology models were generated using thoroughly
tested method29 and were described in previously published
papers34,39–41. Sequence alignments between target receptors
(UniProt database accession numbers P08908 and P34969,
respectively) and the template were performed by hhsearch
tool via GeneSilico Metaserver42. The crude receptor models
were obtained using SwissModel43 and were validated by
processing in Protein Preparation Wizard. For each receptor
type, a set of bioactive compounds was selected for ligand-
steered binding site optimization, which was performed using
induced fit docking (IFD) workflow44. The procedure resulted in
a variety of comparative models, mirroring conformational
plasticity of the protein. Ligand structures were optimized
using LigPrep tool and were docked to the target models using
Glide XP-docking procedure, setting flexible docking option,
retaining ring conformations and nitrogen inversions as were
selected in the previous step. H-bond constraints and centroid of
Results and discussion
Chemistry
Following our previously developed route, the final derivatives of
1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (4–21) were
achieved in via multistep synthesis. In order to carry out the
process, two series of synthons were necessary: 7-ketonyl
derivatives of 8-bromotheophylline (1–3) and N-(aminoalkyl)ar-
ylpiperazines (XII–XVI). Compound 1 was obtained from 8-
bromotheophylline according to described earlier method29.
Compounds 2 and 3 were obtained from 8-bromotheophylline in
reaction of alkylation with a-chloro ketones in biphasic system, in
the presence of potassium carbonate in acetonitrile. N-(aminoalk-
yl)arylpiperazines (XII–XVI) were obtained in two steps, accord-
ing to the Gabriel method45, with minor modifications. First, 1-(2-
fluorophenyl)- or 1-(3-trifluoromethyl)-phenyl)piperazine (I–II)
were alkylated in biphasic system, using catalytic amount of
potassium iodide with appropriate 2-(bromoalkyl)-1H-isoindoline-
1,3(2H)-dione (III–V), in the presence of potassium carbonate in
acetonitrile, to give the corresponding phthalimido derivatives. All
intermediates were purified by column chromatography using
dichloromethane/methanol (9/1) as an eluent. Next, phthalimido
derivatives were hydrolyzed with hydrazine monohydrate to afford
the N-(aminoalkyl)arylpiperazines (XII–XVI)45. Final ligands
˚
a grid box (22 ꢀ 22 ꢀ 22 A) for docking studies were located on
Asp3.32.
The
remaining
parameters
were
default.
Phisicochemical (PSA) and ADME (QPPMDCK) properties
were calculated with use of QikProp module. Glide, induced fit
docking, LigPrep, Protein Preparation Wizard and QikProp were
implemented in Small-Molecule Drug Discovery Suite
R
R
Scheme 1. General method for synthesis of
compounds 4–21a.
O
()n
N
N
N
O
O
()
+
Br nN
(a)
(b)
N
NH
O
I,II
III, IV, V
VI-XI
R1=-H, -CH3, -C6H5
R=-2F, -3CF3
n=3, 4, 5
R
R1
R
R1
()n
O
O
N
O
N
N
N
N
N
N
N
N
Br
N
+
(c)
N
N
O
N
O
()
n NH2
4-21
XII-XVI
1-3
4
2-F
H
5
2-F
H
6
7
8
9
10
11
12
2-F
R
R1
n
2-F
H
2-F
2-F
2-F
2-F
2-F
CH3 CH3 CH3 C6H5 C6H5 C6H5
3
4
5
3
4
5
3
4
5
13
14
15
16
17
18
19
20
21
R
R1
n
3-CF3 3-CF3 3-CF3 3-CF3 3-CF3 3-CF3 3-CF3 3-CF3 3-CF3
H
3
H
4
H
5
CH3
3
CH3
4
CH3
5
C6H5
3
C6H5
4
C6H5
5
areagents and conditions (a) K2CO3, KI, CH3CN, 82 oC, 48h; (b) hydrazine monohydrate 65%, EtOH, reflux;
(c) 2-methoxyethanol, reflux, 12h.

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
9
4–21 (Scheme 1) were synthesized as a result of cyclocondesation belong to this group. This optimal length of spacer leads to an
of 7-ketonyl derivatives of 8-bromotheophylline (1–3) with absence of influence of the substituent at 7 position of the purine
corresponding N-(aminoalkyl)arylpiperazines (XII–XVI), occurs core for 5-HT1A affinities. In a group of 2-F analogs, it is easier to
by refluxing the in 2-methoxyethanol (Scheme 1). Note, in this prove the unfavorable impact of the propyl (C3) alkyl chain than
condition, final compounds were obtained in good to moderate C4 preference. Moreover, the weaker influence of the 2-F
yields, depending on 7-ketonyl derivatives of 8-bromotheophylline substituent on 5-HT1AR affinity exposes the impact of a methyl
used. All final compounds 4–21 in order to obtain analytic samples moiety at the 7-position of imidazo[2,1-f]purine-2,4-dione (5 vs.
and further biological evaluation, were purified by column 8, 8 vs. 11, 6 vs. 9, and 9 vs. 12). A spectrum of activities was
chromatography using a mixture of dichloromethane and methanol observed for 5-HT7R, and tested compounds exhibited either a
in various proportion as an eluent. The chemical structure of lack of affinity (4, 5, 13, 16), or high (comp. 12 Ki ¼ 9 nM, comp.
1
compounds 4–21 was verified by H, 13C and 19F NMR tests.
21 Ki ¼ 1 nM, comp. 18 and 20 Ki ¼ 10 nM) or moderate affinity
(comp. 6–11, 14, 17, Ki from 20–152 nM) (Table 1). The
structure–activity relationship revealed that, in the case of this
receptor, 3-CF3 derivatives in general bound with the highest
affinity, and compound 21 with Ki ¼ 1 nM belongs to this group.
Due to the structural similarity between 5-HT1A and 5-HT7
receptors, many of the LCAPs described in the literature as 5-
HT7R ligands derive from a structural modification of the
terminal fragment present in 5-HT1AR ligands. Moreover, studies
have revealed the critical role of the arylpiperazine substitution
pattern in determining the receptor affinity. Volk et al.46 found out
that electron-donating substituents (–OCH3) were superior to
electron-withdrawing (–Cl, –F) for binding to the 5-HT1A and 5-
HT7 receptors. Our study confirmed that 2-F and 3-CF3 substituents
at the phenyl ring of arylpiperazine were highly preferential for the
5-HT1AR over the 5-HT7R. The influence of substituents 2-F and 3-
CF3 on 5-HT7 receptor affinity in the group of tested compounds
appears to be equivocal, and a spectrum of activities was observed.
It appears that, for LCAPs with a terminal fragment based on
imidazo[2,1-f]purine-2,4-dione, the length of the aliphatic spacer
(C5) is decisive for 5-HT7R affinity. This is in accordance with
Leopoldo et al.47, who found that, in a group of N-(1,2,3,4-
tetrahydronaphthalen-1-yl)-4-arylpiperazinealkylamide derivatives,
Biological evaluation
In vitro examination of the newly synthesized compounds
occurred in three steps. First, all of the compounds were tested
in assays against cloned 5-HT1A and 5-HT7 receptors with
standard competitive displacement assays and the inhibition
constants Ki were determined. For structure–activity relationship
studies, we examined the impact of three chemical elements:
substitution at phenyl ring of LCAPs, the length of the linker
between the purine core and LCAPs and substitution at the 7-
position of the purine core.
All tested compounds possessed high affinity at 5-HT1ARs
with some binding in the same range as serotonine (Ki from 0.1 to
33 nM) (Table 1), but the 3-CF3-phenyl-piperazinylalkyl deriva-
tives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-diones were more
active than their analogs with a 2-F moiety. For example,
compound 7 exhibited the lowest affinity (Ki ¼ 33 nM), and the
affinity of compound 13 was more than six times higher than that
of compound 4. The tetramethylene spacer (C4) between the
purine core and LCAPs proved to be optimal for 5-HT1A sites and
compounds with the highest affinity (one exception comp. 11)
Table 1. The binding data and cLog p of compounds 4–21.
R
R1
O
N
N
N
N
N
()n
N
O
N
4-21
Ki [nM]*
Compound
R
n
R1
5-HT1A
5-HT7
Sy
cLog p
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
3
4
5
3
4
5
3
4
5
3
4
5
3
4
5
3
4
5
–H
–H
–H
–CH3
–CH3
–CH3
–C6H5
–C6H5
–C6H5
–H
11.7 0.8
0.6 0.2
4.8 0.5
33 2.0
NA
–
5.67
5.34
4.68
5.14
3.81
4.06
5.75
4.06
5.39
4.12
5.75
5.69
5.23
5.99
5.73
5.08
4.72
5.72
508 12.5
119.7 7.3
152 3.5
86.5 5.0
30.8 3.5
88 4.7
137 4.5
9
NA
136 5.2
16 1.2
NA
20 2.2
10 0.5
59.6 4.0
10 0.9
0.001
0.04
0.21
0.01
0.08
0.05
0.04
0.33
–
0.001
0.125
–
0.01
0.06
0.005
0.01
0.2
1.1 0.1
2.5 0.2
4.9 0.5
6.3 0.2
3.0 0.2
1.8 0.1
0.2 0.05
0.2 0.04
1.1 0.1
0.2 0.05
0.6 0.04
0.3 0.03
0.1 0.02
0.2 0.04
2-F
1.0
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
–H
–H
–CH3
–CH3
–CH3
–C6H5
–C6H5
–C6H5
1
0.2
*Data expressed as the mean SD of two independent experiments in duplicate.
y5-HT1A/5-HT7 selectivity.

´
10 A. Zagorska et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
Table 2. Inhibition of PDE (%) of compounds 4–21.
INHIBITION OF PDE10A ENZYME
100
80
60
40
20
0
PDE4B
PDE10A
10ꢂ5.5
[-5] PAPAVERINE
[-5.5] PAPAVERINE
[-5] THEOPHYLLINE
[-5.5] THEOPHYLLINE
[-5] 9
Compound
10ꢂ5
10ꢂ5.5
10ꢂ5
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2
3
1
3
2
6
7
2
3
5
1
0
1
0
1
3
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
–
75
6
6
5
5
4
2
3
9
6
5
0
0
4
3
1
2
3
3
2
78
3
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
[-5.5] 9
4
3
13
84
0
log (compounds) [M]
Theophylline
Papaverine
Rolipram
ꢂ5
57
8
Figure 3. Inhibitor activity (%) against PDE10A for compound 9,
theophylline and papaverine in concentration of 10–5 M and 10–5.5 M.
Data are expressed as the mean of two independent experiments in
duplicate, vehicle control (DMSO).
affinity and lack of stimulation of particular signaling pathway is
not equal to effective functional antagonism of the receptor. In
fact, the functional tests provide additional level of characteristics
for the tested compound and are more stringent than binding
experiments and therefore constituting good tools to select
compounds that are significantly active at 5-HT7 receptors.
For 5-HT1A receptors, Paluchowska et al.21 evaluated a series
of 1-substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives
with different cyclic imide/amide termini, and their flexible
tetramethylene analogs. Functional examination revealed that
rigid ligands with 2-OCH3 groups in the aryl moiety and a cyclic
imide system in the opposite terminal behaved like 5-HT1A
receptor antagonists. On the other hand, unsubstituted, 3-Cl or 3-
CF3 substituted derivatives, as well as those with a cyclic amide
group in the terminal fragment, exhibited agonistic or partial
agonistic activity. Our study revealed that for LCAPs with
terminal fragments based on imidazo[2,1-f]purine-2,4-dione and a
tetramethylene spacer, ligands with 3-CF3 group in the aryl
moiety (14, 17, 20) showed 5-HT1A antagonist properties.
Interestingly, in the group of ligands with a 2-F moiety and
tetramethylene spacer, only compound 5 exhibited the ability to
inhibit the 5-HT1A receptor. Elongation of the spacer from C4 to
C5 for 2-F (9 and 12) and 3-CF3 (14 vs. 15, 17 vs. 18, 20 vs. 21)
derivatives caused decreased in a value of Kb. The above
observation indicates that not only the combination of structural
features of arylpiperazinealkyl moiety found to be connected with
the antagonistic properties of the ligands, but their overall effect
also depends on the substitution in 7-position of terminal
imidazo[2,1-f]purine-2,4-dione fragments.
five methylene units separating the amide moiety and the
piperazine ring were preferred for the affinity to 5-HT7Rs.
The second step of the in vitro study was the PDE inhibitory
activity of targeted compounds using a bioluminescent detection
system, based on the activity of PDEs which utilized second
messenger cAMP. The AMP produced from the hydrolysis of
cAMP was quantified using an AMP detection reagent that
converts AMP directly to ATP. The therapeutic effect of
theophylline (bronchodilatory, positive inotropic effect on heart,
psychostimulant) has not been attributed to any single mechanism
of action, but the inhibition of PDE was the most important. In
order to identify which of the mechanisms involved in the central
nervous system action of the LCAPs with imidazo[2,1-f]purine-
2,4-dione as terminal fragment, PDE4B and PDE10A inhibitory
activity was measured. It is noteworthy that, to assess the relative
importance of receptor-dependent and/or receptor-independent
mechanisms of action, the same range of concentrations as for
receptor affinity were taken as a measure of PDE inhibitory
activity (starting from 10ꢂ5 M). The investigated compounds had
weak inhibitory potencies for PDE4B and PDE10A only at an
initial concentration of 10ꢂ5 M, and no inhibition was detected at
lower concentrations (Table 2, Figure 3). Moreover, the inhibitory
effect of the tested compounds was compared with theophylline.
On the basis of the binding affinity results, a series of
compounds possessing affinity below 3 nM for 5-HT1AR (5, 8, 9,
12–21) and below 50 nM for 5-HT7 receptors (9, 12, 15, 17, 18,
20, 21) was selected for functional profile characterization.
Intrinsic activity studies were performed using in vitro measures
of receptor activation. No activation of any of the receptors was
observed, and the compounds showed antagonist properties,
especially the 5-HT1A (5, 9, 12, 14, 15, 17–21) (Figures 4 and 5).
In case of 5-HT7R, there are significant differences between the
binding and functional tests results for selected compounds. The
reason for such a discrepancy is not clear, but one have to bear in
mind, that the binding experiments are reflecting simply the
competition at the level of a binding site which do not require
intracellular signaling mechanisms (e.g. G-proteins or cAMP),
whereas the functional tests require appropriate cellular machin-
ery in addition to 5-HT7 receptor expression. In other words,
Lipophilicity
In medicinal chemistry, numerous measured or calculated
parameters have been used for the characterization of lipophili-
city, but the logarithm of the n-octanol/water partition coefficient
(log p) is still the generally accepted and primarily applied
descriptor of lipophilicity in quantitative structure-activity
relationship (QSAR) studies. The lipophilicity of a drug candidate
can affect its both pharmacokinetic and pharmacodynamic
properties. In particular, the ability of a molecule to cross the
cell membrane depends on its partition coefficient36. The MEKC
system applied for estimation of lipophilicity uses micelles and
closely mimics biological membranes; therefore, it is more

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione 11
Figure 4. Antagonist mode (Kb) of the
selected compounds for 5-HT1A receptor.
a
Comp.
K
SEM [nM]
b
5
2.7 0.38
9
4
0.63
ANTAGONIST MODE 5HT1A
12
110 64
1.4 0.86
9.7 0.03
1.4 0.02
6.5 3.01
55 7.05
28 22.5
67 4.50
0.87 0.26
150
125
100
75
14
NAN-190
5
9
15
17
12
14
15
17
18
19
20
21
18
19
50
20
25
21
0
NAN-190
a
−12 −11 −10 −9 −8 −7 −6 −5 −4
data are expressed as the
mean SEM of three
independent experiments
performed in duplicate
NAN-190-1-(2-
log [compounds] (M)
metoxyphenyl)4-4-
phthalimidobutyl)piperazine
selective 5-HT antagonist
1A
a
Compd.
K
SEM [nM]
b
9
5257 68
15
nc
1040 30
nc
ANTAGONIST MODE 5-HT7
17
50
40
30
20
10
0
SB 269970
18
20
9
nc
15
17
18
20
21
21
nc
SB269970
2
0.2
a
data are expressed as the mean
SEM of three independent
experiments performed in
duplicate
nc - not calculable
−12 −11 −10 −9 −8 −7 −6 −5 −4
SB269970 – (2R)-1-[(3-
hydroxyphenyl)sulfonyl]-2-(2-
(4-methyl-1-
log [compounds] (M)
piperidinyl)ethyl)pyrrolidine
selective 5-HT antagonist
7
Figure 5. Antagonist mode (Kb) of selected compounds for the 5-HT7 receptor.
suitable for physicochemical characterization of bioactive mole- against the incubation time. Currently, commercially available
cules. The ‘‘rule of five’’ defines the range of log p for good human liver microsomes (HLM) have been the most widely used
intestinal permeability and penetration to the brain. The estimated in vitro models for metabolic stability studies49. Compounds 9
log p values for compounds 6, 8, 9, 11, 13 and 20 were in ranges and 20 were selected for in vitro metabolic stability assays. It is
defined by the ‘‘rule of five’’ (log p 55), which indicates good noteworthy that this is the first report of the metabolic stability of
intestinal permeability (Table 1).
LCAPs with terminal fragments based on imidazo[2,1-f]purine-
2,4(3H,8H-dione). In terms of the conditions of our study,
compounds 9 and 20 after 15 min of biotransformation, showed
acceptable metabolic stability but 20 was slightly more suscep-
tible to metabolism (Table 3).
Metabolic stability
Metabolic stability is a critical property for drug candidates,
because it contributes to oral bioavailability and overall
pharmacokinetic performance48. Metabolic stability is usually
quantified by monitoring the disappearance over time of the
parent compound in liver preparations. The disappearance of the
Pharmacological in vivo evaluation
parent compound was monitored by calculating the percentage of Finally, compound 9 was selected for further in vivo examination
each compound remaining relative to 0 min and this was plotted on the basis of the following. First, clinical and preclinical studies

´
12 A. Zagorska et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
Table 3. Metabolic stability screen of compounds 9 and 20 in human liver microsomes (HLM) and major metabolites characteristics.
Metabolites
Comp [M + H] Retention time [min] % of parent comp. ID [M + H]+ Retention time [min] % of metabolites Metabolic pathway
9
482
580
3.93
5.28
43
39
M1
M2
M1
M2
498
320
596
352
3.35
3.40
4.33
4.64
90
10
82
18
Hydroxylation
Dealkylation
Hydroxylation
Dealkylation
20
9
Citalopram
100
80
60
40
20
0
*
*
*
****
****
5
5
10
2.5
2.5
1.25
dose [mg/kg]
The investigated compound was administered ip 60 min, while
citalopram 30 min before the test. n = 6-9 mice per group.
* p < 0.05, **** p < 0.0001 vs. vehicle
The investigated compound 9 and diazepam were administered
ip 60 min before the test. n = 8-10 mice per group.
* p < 0.05, ** p<0.01, *** p < 0.001 vs. vehicle
Figure 6. The effect of the tested compound and 9 citalopram in the
forced swim test (FST) in mice.
Figure 7. The effect of the tested compound 9 and diazepam in the
four-plate test in mice.
of dose–response curve. Compound 9 shows U-shaped dose–
response relationship in the forced swim test. Some anxiolytics
and antidepressants and newly synthesized compound (9) with
different mechanisms of action can produce such U-shaped dose–
response effects in animal models, for example Millan et al.2,
Wesołowska et al.52.
Compound 9 given at a dose of 2.5 mg/kg showed anxiolytic-
like activity in the four-plate test, increasing by 90%, in a
statistically significant manner, the number of punished crossings
accepted by animals during the observation sessions. The
reference anxiolytic drug, diazepam, administered at doses of
1.25, 2.5 and 5 mg/kg, significantly increased (by 38%, 52% and
57%, respectively) the number of punished crossings. It is worth
noting that the potency of antianxiety effects evoked by 9 (2.5 mg/
kg) is greater than that of the reference anxiolytic drug, diazepam,
given at the same dose of 2.5 mg/kg. Compound 9 shows inverted
U-shaped dose–response relationship in four-plate test. The
effects of compound 9 and diazepam in the four-plate test are
shown in Figure 7.
have shown that antidepressant-like and/or anxiolytic-like effects
are mainly produced by serotonin receptor ligands50. Compound 9
(8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-
1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione) showed very high 5-
HT1A receptor affinity (Ki ¼ 2.5 nM) and high 5-HT7 receptor
affinity (Ki ¼ 30.8 nM). In our previous studies, a spectrum of
receptor activities was observed for compounds with a substituent
at the 7th position of the imidazo[2,1-f]purine-2,4-dione system.
Some compounds were potent 5-HT1A and/or 5-HT7 receptor
ligands with additional affinity for dopamine D2 receptors.
Therefore, to test the feasibility of multireceptor activity,
compound 9 was evaluated for affinity for D2 (Ki ¼ 437 nM)
and other serotonin subtype (5-HT2A,6 Ki41000 nM) according to
the procedures described earlier32,34. Next, behavioral effects
(antidepressant like) similar to those of selective serotonin
reuptake inhibitors (SSRIs, e.g. fluoxetine) have been produced
by antagonists of 5-HT1A and 5-HT7 receptors51. Moreover, an
antagonist of 5-HT1A and/or 5-HT7 receptors induces an
anxiolytic-like effect. Compound 9 showed 5-HT1A antagonistic
properties exemplified by Kb ¼ 4 nM. Our cumulative results
prompted us to evaluate the activity of compound 9 in FST and
four-plate tests in mice.
Moreover, the antidepressant-and/or anxiolytic-like properties
of the investigated agents, citalopram and diazepam, seem to be
specific, since when administered at effective doses they did not
stimulate spontaneous locomotor activity in mice during the 4 min
and 1 min 15 s sessions (i.e. at a time identical to the observation
period in the FST and four-plate test, respectively) (data not
shown).
Compound 9 (1.25–5 mg/kg), as well as citalopram (1.25–
5 mg/kg) used as a reference compound, revealed antidepressant-
like properties. Compound 9 administered at two higher doses
(2.5 and 5 mg/kg) significantly reduced the immobility time of
mice in that test by 21%. The reference antidepressant citalopram
shortened (by 28%, 51% and 61%, respectively) the immobility
time of mice at all doses (Figure 6). The additional dose (10 mg/
kg) of compound 9 have been studied in FST and to check the type
Molecular modeling
Docking experiments were performed for compound 9. The
molecule was situated across the two cavities of the binding

DOI: 10.1080/14756366.2016.1198902
Derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione 13
Figure 8. Binding modes of compound 9 in
the site of 5-HT1A (A) and 5-HT7 (B)
receptors. Amino acid residues engaged in
˚
ligand binding (within 4 A from the ligand
atoms) are displayed as sticks, whereas those
forming H-bonds (dotted yellow lines) or
ꢁ–ꢁ/CH-ꢁ stacking (dotted cyan lines) are
represented as thick sticks. Van der Waals
molecular surface of the receptor binding site
is shown as grid, colored due to electrostatic
potential. For the sake of clarity, ECL2
residues were hidden. TMH – transmembrane
helix; ECL – extracellular loop.
site: the first one constituted by the transmembrane helices
(TMHs) 3–6 in both the receptor models, and the second one
between TMHs 2 and 7 (5-HT1A receptor) or TMHs 3 and 7 (5-
Conclusion
In conclusion, the paper presents the synthesis of arylpiperaziny-
HT7 receptor). The anchoring interaction in both sites was a lalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
charge-reinforced hydrogen bond between the protonated with 2-fluoro and 3-trifluoromethyl moieties. The study allowed
us to identify some potent 5-HT1A (5, 14, 17–21), 5-HT7 (17, 18,
20) and mixed 5-HT1A/5-HT7 (12, 15, 21) receptor ligands with
and aromatic amino acid cluster in the deeper cavity, mainly weak inhibitory potencies for PDE4B and PDE10A. The study
nitrogen atom of the ligand and the carboxyl group of
Asp3.32, as well as the CH-ꢁ interactions of the arylpiperazine
reveals that 5-HT1A and 5-HT7 receptor affinity benefits from the
introduction of a fluorine moiety while introduction of imidazole
turned toward the electropositive receptor surface (blue color of ring into 7, 8 position of theophylline had no significant effect on
Phe6.52 (Figure 8). In the case of the 5-HT1A receptor, the
electronegative 2-F group of the phenylpiperazine fragment was
inhibitory potencies for selected PDE. The tested compounds
were in the ranges defined by the ‘‘rule of five’’ (log p 55),
the electrostatic potential grid), finding beneficial fluorine-
bonding interaction with the side chain residues of Lys191 (the
e-amino group) from the second extracellular loop (ECL 2) which indicates good intestinal permeability and showed good
(Figure 8A). The 5-HT7 receptor-binding site accepted the 2- metabolic stability. In preliminary pharmacological in vivo
studies, selected 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-
fluorophenyl moiety in an analogous manner, exposing an
electropositive surface of Gln235 (Figure 8B). The weak 1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) in
fluorine bonding interactions are considered to enrich the the FST in mice behaved as a potential antidepressant. Moreover,
potency of antianxiety effects evoked by 9 in the four-plate test
(2.5 mg/kg) is greater than that of the reference anxiolytic drug,
of the molecule occupied the second cavity and established diazepam. The binding mode in the 5-HT1A and 5-HT7 receptors
ligand–receptor contacts and therefore favorably adjust the
ligand binding specificity. The imidazo[2,1-f]purine fragment
has been elucidated for the representative molecule by means of
molecular docking, pointing out favorable interactions, character-
stacking with phenyl ring of Tyr2.64 (Figure 8A). In the 5-HT7 istic for each receptor type. Studies might be beneficial to further
favorable, characteristic for each receptor type, aromatic
interactions there. In the 5-HT1A receptor, it interacted by ꢁ-ꢁ
investigation of the mechanism of CNS action of imidazo[2,1-
f]purine-2,4(3H,8H)-dione derivatives.
receptor-binding site, the heterocyclic system formed essential
ꢁ–ꢁ stacking with Phe3.28 (Figure 8B).

´
14 A. Zagorska et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
19. Medina RA, Sallander J, Benhamu B, et al. Synthesis of new
serotonin 5-HT7 receptor ligands. Determinants of 5-HT7/5-HT1A
receptor selectivity. J Med Chem 2009;52:2384–92.
Declaration of interest
This study was financially supported by the National Science
Center Poland (NSC) funded grant based on the decision DEC- 20. Siracusa MA, Salerno L, Modica MN, et al. Synthesis of new
arylpiperazinylalkylthiobenzimidazole, benzothiazole, or benzoxa-
zole derivatives as potent and selective 5-HT1A serotonin receptor
ligands. J Med Chem 2008;51:4529–38.
2012/07/B/NZ7/01173 and Funds of Statutory Activity UJ CM
(K/ZDS/005533). The authors declare no conflict of interest.
´
21. Paluchowska MH, Bugno R, Tatarczynska E, et al. The influence of
modifications in imide fragment structure on 5-HT(1A) and 5-HT(7)
receptor affinity and in vivo pharmacological properties of some
new 1-(m-trifluoromethylphenyl)piperazines. Bioorg Med Chem
2007;15:7116–25.
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Supplementary material available online