Synthesis and anticancer activity of multisubstituted purines and xanthines with one or two propynylthio and aminobutynylthio groups

Article abstract of DOI:10.1007/s00044-018-2155-3

A synthesis of new 2,6-disubstituted and 2,6,8-trisubstituted 7-methylpurines as well as 8-substituted 3,7-dimethylxanthines containing a triple bond chain have been worked out. Purinethiones and xanthinethiones were converted into propynylthio derivatives, which were then further transformed via a Mannich reaction into aminobutynylthio derivatives (amine = pyrrolidine, piperidine, morpholine, and diethylamine). The products thus obtained represent various types of the purine and xanthine structure: 8-mono-, 2,6- and 6,8-dipropynylthio, 6- and 8-monoaminobutynylthio, 2,6- and 6,8-diaminobutynylthio derivatives. All of these compounds were tested for their anticancer activity against human glioblastoma SNB-19, human adenocarcinoma MDA-MB-231, and melanoma C-32 cell lines. The anticancer activity depends on the nature of the substituent and its localization in the purine and xanthine framework. Generally, compounds possessing two alkynylthio groups (propynylthio or aminobutynylthio) were more active than those possessing only one group. Some compounds exhibited stronger or similar anticancer activity to cisplatin. All compounds were also tested for their cytotoxic activity against normal human fibroblasts (HFF-1). The most promising anticancer compounds were found to be 2,6-dipropynylthio-7-methylpurine 4, 2-chloro-6,8-dipropynylthio-7-methylpurine 14, and 2-chloro-6,8-di(N-morpholinylbutynylthio)-7-methylpurine 15c acting selectively on glioblastoma SNB-19, melanoma C-32, and adenocarcinoma MDA-MB-231 with the IC₅₀ = 0.07–4.08 μg/mL.

Full text of DOI:10.1007/s00044-018-2155-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2155-3
ORIGINAL RESEARCH
Synthesis and anticancer activity of multisubstituted purines and
xanthines with one or two propynylthio and aminobutynylthio
groups
Alicja Kowalska1 Krystian Pluta1 Małgorzata Latocha2
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Received: 3 November 2017 / Accepted: 10 February 2018
© The Author(s) 2018. This article is an open access publication
Abstract
A synthesis of new 2,6-disubstituted and 2,6,8-trisubstituted 7-methylpurines as well as 8-substituted 3,7-dimethylxanthines
containing a triple bond chain have been worked out. Purinethiones and xanthinethiones were converted into propynylthio
derivatives, which were then further transformed via a Mannich reaction into aminobutynylthio derivatives (amine =
pyrrolidine, piperidine, morpholine, and diethylamine). The products thus obtained represent various types of the purine and
xanthine structure: 8-mono-, 2,6- and 6,8-dipropynylthio, 6- and 8-monoaminobutynylthio, 2,6- and 6,8-diaminobutynylthio
derivatives. All of these compounds were tested for their anticancer activity against human glioblastoma SNB-19, human
adenocarcinoma MDA-MB-231, and melanoma C-32 cell lines. The anticancer activity depends on the nature of the
substituent and its localization in the purine and xanthine framework. Generally, compounds possessing two alkynylthio
groups (propynylthio or aminobutynylthio) were more active than those possessing only one group. Some compounds
exhibited stronger or similar anticancer activity to cisplatin. All compounds were also tested for their cytotoxic activity
against normal human fibroblasts (HFF-1). The most promising anticancer compounds were found to be 2,6-dipropynylthio-
7-methylpurine 4, 2-chloro-6,8-dipropynylthio-7-methylpurine 14, and 2-chloro-6,8-di(N-morpholinylbutynylthio)-7-
methylpurine 15c acting selectively on glioblastoma SNB-19, melanoma C-32, and adenocarcinoma MDA-MB-231 with
the IC50 = 0.07–4.08 μg/mL.
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Keywords Propynylthiopurines Aminobutynylthiopurines Aminobutynylthioxanthines Anticancer activity
Structure–activity relationship
Introduction
and function of nucleic acids and enzymes. Synthetically
modified purines are inhibitors of protein kinase, fructose
biphosphatase, adenylation enzyme, and adenosine receptor
modulators (Bliman et al. 2014; Sharma et al. 2016).
The physicochemical properties of many substituted
purine derivatives provide the potential for tight binding to
deep hydrophobic folds of physiologically disease relevant
proteins, such as thyrosine kinases, sulfatases, and phos-
phorylases. Purines could be the selectively target of these
protein hydrophobic folds, influencing the progression of a
disease (Riva-Toniolo et al. 2003).
Purine is a privileged heterocyclic nucleus existing in the
chemical architecture of various bioactive compounds. It is
an important pharmacophore interacting with the synthesis
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2155-3) contains supplementary
material, which is available to authorized users.
* Alicja Kowalska
kowalska@sum.edu.pl
Conventional thiopurines (6-mercaptopurine, 6-thiogua-
nine, and azathioprine) are incorporated into the structure of
natural biomolecules—they modify nucleic acid synthesis
and are commonly used as effective anticancer and immu-
nosuppressive drugs in the treatment of acute leukemia and
autoimmune hepatitis (Prima et al. 2013; Deswal and Sri-
vastava 2017). Thiopurines remain the first line of therapy
1
Department of Organic Chemistry, School of Pharmacy with the
Division of Laboratory Medicine The Medical University of
Silesia Jagiellońska 4, 41-200 Sosnowiec, Poland
2
Department of Cell Biology, School of Pharmacy with the
Division of Laboratory Medicine The Medical University of
Silesia Jedności 8, 41-200 Sosnowiec, Poland

Medicinal Chemistry Research
in inflammatory bowel disease, Crohn’s disease, ulcerative
colitis, and autoimmune hepatitis (Herreras and Iborra 2017;
Ward et al. 2017; Warner et al. 2016).
schizophrenia and epilepsy. They are also effective as
hypoglycemic agents (Baraldi et al. 1998; Endo et al. 2014).
Apadenoson (ATL-146e) has entered phase III clinical trials
as a pharmacologic stress agent for use in myocardial per-
fusion imaging (Foitzik et al. 2009).
6-Thiopurine derivatives containing the 1,2,3-triazole
moiety present higher values regarding the inhibition of
parasite multiplication than chloroquine and show anti-
malarial and antileishmanial activities (Corrales et al. 2011).
The pharmacologically active xanthines are commonly
used for their effects as stimulants, bronchodilators, phos-
phodiesterase inhibitors, CFTR chloride-channel activators,
and adenosine receptor antagonists. The selectivity of caf-
feine analogs for A2A adenosine receptors was increased by
the replacement of one or two methyl groups of caffeine
with a propyl or propynyl substituent. 3,7-Dimethyl-1-pro-
pynylxanthine has been used as a selective A2 antagonist. 1-
Propynyltheobromine and 1,3-dipropynyl-7-methylxanthine
are approximately fourfold more potent than caffeine at the
ryanodine sensitive calcium release channels, and fivefold
more potent than caffeine as GABAA receptor modulators.
The range of clinical applications for xanthines has con-
tinued to widen and currently includes their use as antic-
onvulsants, nootropics, and therapeutics for the treatment of
migraine (Daly et al. 2007; Thomas et al. 2013; Zagorska
et al. 2015).
The introduction of substituents in positions 2, 6, and 9
of the purine ring enhances both binding affinity and
selectivity regarding kinases. This enzyme family plays a
critical role in the proliferation of human tumor cells, as
well as in the survival and migration of neoplasia. There
was an observed selectivity of 2-acetylenyl-6,9-dis-
ubstituted purine analogs regarding the inhibition of cyclin-
dependent kinase (CDK1-cyclin B) (Legraverend et al.
2000; Sharma et al. 2016). The inhibitors of this enzyme
play an important role in cell cycle regulation in a number
of disease conditions, in particular cancer, and have been
characterized as tumor suppressors. It is highly notable that
the 2-nitrogen atom, which is characteristic of all other
purine-based cyclin-dependent kinase inhibitors (CKIs), can
be replaced by an sp-hybridized carbon atom, which
effectively occupies the ribose binding pocket (Sharma
et al. 2016).
Alkynyl chains in the 8 position of adenosine comprise a
very selective ligand for the A3 receptor subtype. Thus, it is
possible to modulate the activity at the adenosine receptor
subtype by introduction of alkynyl chains in either the 2 or
8 positions of 9-ethyladenine (Volpini et al. 2001, 2005). 8-
Substituted 2-alkynyl-N9-propynyladenine derivatives were
found to antagonize the receptor and may be effective in the
treatment of Parkinson’s disease (Endo et al. 2014;
Ivachtchenko et al. 2013). 2-Propynyladenosinyl phenyl
ether compositions as A2A receptor agonists stimulate
mammalian coronary vasodilatation and can be useful as
therapeutics for coronary artery disease (Zablocki et al.
2001).
The presence of the alkynyl chain in 2-substituted ade-
nosines is also very important for purinergic receptor
pharmacology. Purine ionotropic receptors could serve as
attractive targets for pharmacological interventions in var-
ious diseases. 2-Alkynyl derivatives of adenine, adenosine,
N-alkylcarboxamidoadenosine, and adenine nucleotide are
selective ligands for P2X ionotropic receptors (Koles et al.
2007; Dal Ben et al. 2011). 2-Hexynyl and 2-
phenylethynyladenosine mono-, di-, and triphosphates are
P2Y1 and P2Y12 receptor agonists and adenylyl cyclase
modulators. Through interaction with P2Y1 and P2Y12
receptors, they effectively inhibit platelet aggregation and
may be used in the prevention and treatment of arterial
thrombosis as highly potent antithrombotic agents (Cristalli
et al. 2005; Dal Ben et al. 2011).
Most 6-alkynyl-2-oxopurines and 6-phenylalkynylpurine
nucleoside analogs exhibit cytotoxicity against a human
chronic myelogenous leukemic cell line, K-562, that is
comparable to or better than the known anticancer drugs,
such as 6-mercaptopurine and fludarabine (Andersen et al.
2002; Brathe et al. 2003). The alkynyl-modified nucleosides
substituted specifically in the 8 position, such as 8-
alkynyladenosines and 2′-deoxyadenosines, proved to be
very selective ligands for the A3 adenosine receptor sub-
type, and showed moderate anti-HCV and anti HIV-1
activity in cell culture (Kuchar et al. 2008; Dimopoulou
et al. 2015). Oligonucleotides containing the alkynyl group
in the 1 position of 6-thiopurine or 6-thioguanine moiety
have demonstrated potent antiviral activity in several
assays, including the human immunodeficiency virus
(Broom et al. 1997). Some of the 2- or 6-alkynylpurine
derivatives were prepared for use in antibacterial prevention
and therapy (Atamanyuk et al. 2012). 6-Alkynylpurines also
show profound inhibitory activity against human 15-
lipoxygenase. The inhibitors of these enzymes have been
Some substituents in positions 2 and 6 of the purine
modulate both the affinity and selectivity of adenosine
agonists for the different receptor subtypes. The 2-alkynyl
adenosine and their N-ethylcarboxamide analogs, such as 2-
hexynyl (HENECA) and 2-(R,S)-phenylhydroxypropynyl
(R,S-PHPNECA), have proven to be highly potent A2A
adenosine receptor agonists (Volpini et al. 2002, 2005;
Endo et al. 2014). The type of alkynyl chain in the 2
position seems to be very important for the potency at the
A2A receptor. Selective A2A receptor subtype antagonists
have emerged as an attractive target for Parkinson’s disease
therapy as well as other neurodegenerative disorders such as

Medicinal Chemistry Research
implicated in the oxidation of low density lipoproteins, and
could have potential as drugs against artherosclerosis and
other major diseases, such as for instance cancer, Parkin-
son’s, Altzheimer’s disease, heart infraction, and rheuma-
toid arthritis, which are also linked to free radicals (Brathe
et al. 2002; Berg et al. 2005). 6-Alkynylpurines as cytokine
analogs also possess a profound plant growth stimulation
effect (Brathe et al. 2003).
2-Chloro-6-(4-N-piperidinylbut-2-ynylthio)-7-
methylpurine 2a
It was obtained as a pale yellow solid (0.14 g, 82%); mp
1
122–124 °C (EtOH); H NMR (CDCl3), δ: 1.44 (m, 2H,
CH2), 1.65 (m, 4H, 2CH2), 2.54 (m, 4H, 2NCH2), 3.33 (s,
2H, SCH2), 4.13 (s, 3H, NCH3), 4.24 (t, J = 2.1 Hz, 2H,
CCH2), 8.03 (s, 1H, H-8); (DMSO-d6), δ: 1.23 (m, 2H,
CH2), 1.41 (m, 4H, 2CH2), 2.28 (m, 4H, 2NCH2), 3.20 (s,
2H, SCH2), 4.05 (s, 3H, NCH3), 4.25 (t, J = 2.4 Hz, 2H,
CCH2), 8.60 (s, 1H, H-8), 13C NMR (DMSO-d6), δ: 18.86
(CH2, SCH2CC), 23.88 (CH2), 25.81 (2CH2), 34.87 (CH3,
NCH3), 47.44 (CH2, CCH2), 52.68 (2CH2, NCH2), 79.22
(C, SCH2CC), 80.34 (C, SCH2CC), 123.21 (C, C-5), 151.09
(CH, C-8), 152.38 (C, C-2), 154.27 (C, C-6), 160.55 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C15H19ClN5S
336.1050 found 336.1051.
Experimental
All commercially available organic solvents and reagents
were from Sigma-Aldrich and Chempur and were used
without further purification.
The melting points were determined in open capillary
tubes on a Boetius melting point apparatus and were
uncorrected. The 1H NMR and 13C NMR spectra were
recorded on a Bruker AscendTM 600 spectrometer operating
2-Chloro-6-(4-N-morpholinylbut-2-ynylthio)-7-
methylpurine 2b
1
at 600 MHz and 150 MHz for H and 13C nuclei, respec-
tively, in deuterochloroform and dimethyl sulphoxide-d6
with tetramethylsilane as internal standard. Shifts were
given in ppm, coupling constant (J) values was presented in
hertz (Hz), and the abbreviations were as follows: s (sing-
let), d (doublet), t (triplet), and m (multiplet). High resolu-
tion mass spectral analysis (HR ESIMS) was performed on
a Bruker Impact II spectrometer (in the acetonitrile: 1%
formic acid, 8:2 v/v). The reactions were monitored by thin
layer chromatography (TLC) using sheets coated with silica
gel 60F254 (Merck) and chloroform, ethanol (9:1 and 5:1) as
the solvents. Purity of the synthesized compounds was
confirmed by TLC on the same way. Spots were detected by
their absorption under UV light (λ = 254 nm) and the
chromatograms were further visualized by iodine vapor.
Column chromatography separations were carried out with
Merck Kieselgel 60 or aluminum oxide 90 (Merck) using a
mixture of chloroform–ethanol (99:1, v/v) as an eluent.
2-Chloro-6-propynylthio-7-methylpurine 1, (Kowalska
et al. 2015), 7-methylpurine-2,6-dithione 3 (Kowalska
2007), and 2,6-dichloro-7-methylpurine 11 (Kowalska et al.
1985) were obtained according to the previously described
method. Theobromine was commercially available from
Sigma-Aldrich.
It was obtained as a pale yellow solid (0.16 g, 94%); mp
168–169 °C (EtOH); H NMR (CDCl3), δ: 2.62 (m, 4H,
1
2NCH2), 3.36 (s, 2H, SCH2), 3.78 (m, 4H, 2OCH2), 4.13 (s,
3H, NCH3), 4.25 (s, 2H, CCH2), 8.03 (s, 1H, H-8); (DMSO-
d6), δ: 2.36 (m, 4H, 2NCH2), 3.24 (t, J = 2.4 Hz, 2H,
SCH2), 3.52 (m, 4H, 2OCH2), 4.05 (s, 3H, NCH3), 4.27 (t,
J = 2.4 Hz, 2H, CCH2), 8.59 (s, 1H, H-8); 13C NMR
(DMSO-d6), δ: 18.74 (CH2, SCH2CC), 34.86 (CH3, NCH3),
46.96 (CH2, CCH2), 51.95 (2CH2, NCH2), 66.45 (2CH2,
OCH2), 78.82 (C, SCH2CC), 80.58 (C, SCH2CC), 123.12
(C, C-5), 151.09 (CH, C-8), 152.35 (C, C-2), 154.24 (C, C-
6), 160.53 (C, C-4); HR ESIMS m/z [M + H]+ calcd. for
C14H17ClN5OS 338.0842 found 338.0840.
2-Chloro-6-(4-diethylaminobut-2-ynylthio)-7-
methylpurine 2c
It was obtained as a pale yellow solid (0.14 g, 86%); mp
1
117–118 °C (EtOH); H NMR (CDCl3), δ: 1.06 (t, J =
7.2 Hz, 6H, 2CH3), 2.54 (q, J = 7.2 Hz, 4H, 2NCH2), 3.44
(s, 2H, SCH2), 4.13 (s, 3H, NCH3), 4.24 (t, J = 2.1 Hz, 2H,
CCH2), 8.02 (s, 1H, H-8); (DMSO-d6), δ: 0.87 (t, J =
7.2 Hz, 6H, 2CH3), 2.32 (q, J = 7.2 Hz, 4H, 2NCH2), 3.33
(s, 2H, SCH2), 4.05 (s, 3H, NCH3), 4.24 (s, 2H, CCH2),
8.59 (s, 1H, H-8), 13C NMR (DMSO-d6), δ: 12.84 (2CH3,
CH2CH3), 18.93 (CH2, SCH2CC), 34.86 (CH3, NCH3),
40.74 (CH2, NCH2), 46.85 (2CH2, CCH2), 78.66 (C,
SCH2CC), 80.10 (C, SCH2CC), 123.22 (C, C-5), 151.09
(CH, C-8), 152.38 (C, C-2), 154.26 (C, C-6), 160.55 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C14H19ClN5S
324.1050 found 324.1053.
Synthesis of 2-chloro-6-aminobut-2-ynyltio-7-
methylpurines 2a-c
2-Chloro-6-alkylaminobut-2-ynylthio-7-methylpurines 2a-c
were prepared from 6-propynylthio derivative 1 (0.12 g,
0.5 mmol) and amine (piperidine, morpholine, and diethy-
lamine, 0.7 mmol) according to the previously described
procedure (Kowalska et al. 2015).

Medicinal Chemistry Research
Synthesis of 2,6-di(prop-2-ynylthio)-7-methylpurine 4
4NCH2), 3.36 (s, 4H, SCH2), 4.01 (s, 3H, NCH3), 4.10 (t, J
= 1.8 Hz, 2H, CCH2), 4.30 (t, J = 1.8 Hz, 2H, CCH2), 8.46
(s, 1H, H-8); 13C NMR (DMSO-d6), δ: 18.41 (CH2,
SCH2CC), 19.97(CH2, SCH2CC), 23.72 (4CH2), 34.64
(CH3, NCH3), 42.70 (CH2, CCH2), 42.81 (CH2, CCH2),
52.01 (2CH2, NCH2), 52.07 (2CH2, NCH2), 78.36 (C,
SCH2CC), 79.30 (C, SCH2CC), 80.23 (C, SCH2CC),
81.23 (C, SCH2CC), 121.42 (C, C-5), 149.85 (CH, C-8),
152.28 (C, C-2), 159.86 (C, C-6), 161.88 (C, C-4); HR
ESIMS m/z [M + H]+ calcd. for C22H29N6S2 441.1895
found 441.1886.
The mixture of 7-methylpurine-2,6-dithione 3 (0.10 g,
0.5 mmol) and potassium tert-butoxide (0.16 g, 1.44 mmol)
in 10 mL of DMF was stirred at room temperature for 0.5 h.
Then, 80% solution of propargyl bromide (0.17 g,
1.44 mmol) in dry toluene (0.25 mL) was added. The
reaction mixture was stirred for an additional 24 h at room
temperature and then poured into 25 mL of water. The
resulted solid was filtered off and washed with water to give
compound 4.
It was obtained as a pale yellow solid (0.11 g, 79%); mp
1
162–164 °C (EtOH); H NMR (CDCl3), δ: 2.21 (t, J =
2,6-Di(4-N-piperidinylbut-2-ynylthio)-7-
methylpurine 5b
2.7 Hz, 1H, CH), 2.28 (t, J = 2.7 Hz, 1H, CH), 4.04 (d, J =
2.7 Hz, 2H, SCH2), 4.11 (s, 3H, NCH3), 4.22 (d, J = 2.7 Hz,
2H, SCH2), 8.09 (s, 1H, H-8); (DMSO-d6), δ: 3.14 (t, J =
2.4 Hz, 1H, CH), 3.26 (t, J = 2.4 Hz, 1H, CH), 4.02 (s, 3H,
NCH3), 4.06 (d, J = 2.4 Hz, 2H, SCH2), 4.27 (d, J = 2.4 Hz,
2H, SCH2), 8.47 (s, 1H, H-8); 13C NMR (DMSO-d6), δ:
17.82 (CH2, SCH2CCH), 19.50 (CH2, SCH2CCH), 34.62
(CH3, NCH3), 73.56 (CH, SCH2CCH), 74.52 (CH,
SCH2CCH), 80.09 (C, SCH2CCH), 81.24 (C, SCH2CCH),
121.42 (C, C-5), 149.92 (CH, C-8), 152.16 (C, C-2), 159.87
(C, C-6), 161.61 (C, C-4); HR ESIMS m/z [M + H]+ calcd.
for C12H11N4S2 275.0425 found 275.0417.
It was obtained as a dark yellow solid (0.18 g, 77%); mp
1
130–132 °C (EtOH); H NMR; (CDCl3), δ: 1.40 (m, 4H,
2CH2), 1.60 (m, 8H, 4CH2), 2.47 (m, 8H, 4NCH2), 3.28 (m,
4H, 2SCH2), 4.07 (s, 3H, NCH3), 4.07 (t, J = 2.1 Hz, 2H,
CCH2), 4.23 (t, J = 2.1 Hz, 2H, CCH2), 7.92 (s, 1H, H-8);
(DMSO-d6), δ: 1.23 (m, 4H, 2CH2), 1.43 (m, 8H, 4CH2),
2.30 (m, 8H, 4NCH2), 3.21 (m, 4H, 2SCH2), 4.01 (s, 3H,
NCH3), 4.10 (s, 2H, CCH2), 4.32 (s, 2H, CCH2), 8.46 (s,
1H, H-8); 13C NMR (DMSO-d6), δ: 18.49 (CH2, SCH2CC),
20.05 (CH2, SCH2CC), 23.89 (CH2), 23.90 (CH2), 25.78
(2CH2), 25.80 (2CH2), 34.62 (CH3, NCH3), 47.48 (CH2,
CCH2), 47.53 (CH2, CCH2), 52.80 (4CH2, NCH2), 78.22
(C, SCH2CC), 79.15 (C, SCH2CC), 80.72 (C, SCH2CC),
81.72 (C, SCH2CC), 121.42 (C, C-5), 149.82 (CH, C-8),
152.31 (C, C-2), 159.86 (C, C-6), 161.90 (C, C-4); HR
ESIMS m/z [M + H]+ calcd. for C24H33N6S2 469.2208
found 469.2205.
General procedure for synthesis of 2,6-di(aminobut-
2-ynylthio)-7-methylpurines 5a-d
To a mixture of propynylthio derivative 2 (0.137 g,
0.5 mmol) and paraformaldehyde (0.067 g, 1.4 mmol) in
10 mL of dry dioxane, appropriate amines (pyrrolidine,
piperidine, morpholine, or diethylamine, 1.4 mmol) and
CuCl (0.01 g) were added. The reaction mixture was stirred
at temperature 80 °C for 4 h. In the case of dimethylamine,
the reaction was performed at 50 °C for 24 h. After cooling,
the small amount of solid was filtered off and residue was
extracted with chloroform (3 × 5 mL), dried with anhydrous
Na2SO4, and evaporated in vacuo. The dried residue was
dissolved in CHCl3 and purified by thin layer preparative
chromatography (silica gel, CHCl3 – EtOH, 5:1 v/v) to give
compound 5a-d.
2,6-Di(4-N-morpholinylbut-2-ynylthio)-7-
methylpurine 5c
It was obtained as a dark yellow solid (0.175 g, 74%); mp
1
121–123 °C (EtOH); H NMR (CDCl3), δ: 2.55 (m, 8H,
4NCH2), 3.30 (m, 4H, 2SCH2), 3.73 (m, 8H, 4OCH2), 4.08
(s, 3H, NCH3), 4.08 (t, J = 2.1 Hz, 2H, CCH2), 4.23 (t, J =
2.1 Hz, 2H, CCH2), 7.93 (s, 1H, H-8); (DMSO-d6), δ: 2.35
(m, 8H, 4NCH2), 3.23 (s, 2H, SCH2), 3.25 (s, 2H, SCH2),
3.52 (m, 8H, 4OCH2), 4.01 (s, 3H, NCH3), 4.11 (s, 2H,
CCH2), 4.32 (s, 2H, CCH2), 8.46 (s, 1H, H-8); 13C NMR
(DMSO-d6), δ: 18.39 (CH2, SCH2CC), 19.99 (CH2,
SCH2CC), 34.63 (CH3, NCH3), 47.03 (CH2, CCH2), 47.06
(CH2, CCH2), 52.00 (2CH2, NCH2), 52.09 (2CH2, NCH2),
66.47 (4CH2, OCH2), 77.77 (C, SCH2CC), 78.76 (C,
SCH2CC), 80.95 (C, SCH2CC), 82.08 (C, SCH2CC),
121.43 (C, C-5), 149.83 (CH, C-8), 152.32 (C, C-2), 159.86
(C, C-6), 161.82 (C, C-4); HR ESIMS m/z [M + H]+ calcd.
for C22H29N6O2S2 473.1793 found 473.1795.
2,6-Di(4-N-pyrrolidinylbut-2-ynylthio)-7-
methylpurine 5a
It was obtained as a dark yellow solid (0.175 g, 79.5%); mp
97–99 °C (EtOH); 1H NMR (CDCl3), δ: 1.85 (m, 8H,
4CH2), 2.64 (m, 8H, 4NCH2), 3.44 (t, J = 2.1 Hz, 2H,
SCH2), 3.47 (t, J = 2.1 Hz, 2H, SCH2), 3.98 (s, 3H, NCH3),
4.06 (m, 2H, CCH2), 4.21 (m, 2H, CCH2), 7.92 (s, 1H, H-
8); (DMSO-d6), δ: 1.61 (m, 8H, 4CH2), 2.43 (m, 8H,

Medicinal Chemistry Research
2,6-Di(4-diethylaminobut-2-ynylthio)-7-
methylpurine 5d
NMR (DMSO-d6), δ: 30.39 (CH3, N3CH3), 32.29 (CH3,
N7CH3), 104.66 (C, C-5), 140.91 (CH, C-8), 150.12 (C, C-
2), 153.11 (C, C-6), 164.19 (C, C-4); HR ESIMS m/z [M +
H]+ calcd. for C7H9N4O2S 213.0446 found 213.0438.
It was obtained as a dark yellow solid (0.173 g, 78%); mp
77–79 °C (EtOH); 1H NMR (CDCl3), δ: 1.06 (m, 12H,
4CH3), 2.54 (m, 8H, 4NCH2), 3.45 (m, 4H, 2SCH2), 4.07 (t,
J = 2.1 Hz, 2H, CCH2), 4.08 (s, 3H, NCH3), 4.22 (t, J =
2.1 Hz, 2H, CCH2), 7.92 (s, 1H, H-8); (DMSO-d6), δ: 0.91
(m, 12H, 4CH3), 2.37 (m, 8H, 4NCH2), 3.35 (m, 4H,
SCH2), 4.01 (s, 3H, NCH3), 4.09 (m, 2H, CCH2), 4.29 (m,
2H, CCH2), 8.46 (s, 1H, H-8); 13C NMR (DMSO-d6), δ:
12.80 (4CH3, CH2CH3), 17.80 (CH2, SCH2CC), 20.01
(CH2, SCH2CC), 34.62 (CH3, NCH3), 40.84 (4CH2, NCH2),
46.92 (2CH2, CCH2), 74.52 (2C, SCH2CC), 80.03 (2C,
SCH2CC), 121.42 (C, C-5), 149.91 (CH, C-8), 152.02 (C,
C-2), 159.89 (C, C-6), 161.86 (C, C-4); HR ESIMS m/z [M
+ H]+ calcd. for C22H33N6S2 445.2208 found 445.2204.
Synthesis of 8-(prop-2-ynylthio)-3,7-
dimethylxanthine 9
8-(Prop-2-ynylthio)-3,7-dimethylxanthine 9 was obtained
from xanthine-8-thione 8 (0.11 g, 0.5 mmol) with propargyl
bromide in DMF according to the previously described
procedure (Kowalska et al. 2015).
It was obtained as a pale yellow solid (0.100 g, 80%); mp
1
252–253 °C (EtOH); H NMR (DMSO-d6), δ: 3.29 (t, J =
2.4 Hz, 1H, CH), 3.36 (s, 3H, N3CH3), 3.79 (s, 3H,
N7CH3), 4.07 (d, J = 2.4 Hz, 2H, SCH2), 11.17 (s, 1H,
NH); 13C NMR (DMSO-d6), δ: 21.64 (CH2, SCH2CCH),
28.99 (CH3, N3CH3), 32.79 (CH3, N7CH3), 75.26 (C,
SCH2CCH), 79.93 (C, SCH2CCH), 109.21 (C, C-5), 148.19
(CH, C-8), 149.80 (C, C-2), 151.12 (C, C-6), 154.69 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C10H11N4O2S
251.0603 found 251.0600.
Synthesis of 8-bromo-3,7-dimethylxanthine 7
To a stirred suspension of theobromine 6 (2.00 g, 10 mmol)
in 4 mL of carbon tetrachloride and 12 mL of nitrobenzene,
bromine (2.90 g, 0.9 mL, 18 mmol) dissolved in 1.5 mL of
nitrobenzene was added dropwise. When the addition was
completed, the reaction mixture was refluxed with stirring
for 5 h. The suspension was then carefully poured with
stirring into 40 mL of acetone. The resulting white pre-
cipitate was filtered, washed with acetone and ether, and
dried in vacuo (Koppel et al. 1962). The crude product was
purified by column chromatography (silica gel, CHCl3 -
EtOH, 99:1, v/v) to give compound 7.
It was obtained as a pale yellow solid (2.98 g, 69%), mp
298–300 °C, dec (EtOH), 298 °C dec (Koppel et al. 1962)
1H NMR (DMSO-d6), δ: 3.31 (s, 3H, N3CH3), 3.82 (s, 3H,
N7CH3), 11.28 (s, 1H, N1H); 13C NMR (DMSO-d6), δ:
28.96 (CH3, N3CH3), 34.10 (CH3, N7CH3), 109.58 (C, C-
5), 128.84 (CH, C-8), 149.48 (C, C-2), 151.04 (C, C-6),
154.58 (C, C-4); HR ESIMS m/z [M + H]+ calcd. for
C7H8BrN4O2 258.9831 found 258.9834.
Synthesis of 8-(aminobut-2-ynylthio)-3,7-
dimethylxanthine 10a-d
8-(Aminobut-2-ynylthio)-3,7-dimethylxanthines
10a-d
were obtained from 8-propynylthio derivative 9 (0.125 g,
0.5 mmol) and appropriate amines (pyrrolidine, piperidine,
morpholine, and diethylamine, 0.7 mmol) in dry dioxane
according to the described procedure (Kowalska et al.
2015).
8-(4-N-pyrrolidinylbut-2-ynylthio)-3,7-
dimethylxanthine 10a
It was obtained as a pale yellow solid (0.139 g, 84%); mp
195–197 °C (EtOH); 1H NMR (DMSO-d6), δ: 1.56 (m, 4H,
2CH2), 2.32 (m, 4H, 2NCH2), 3.33 (s, 2H, SCH2), 3.33 (s,
3H, N3CH3), 3.80 (s, 3H, N7CH3), 4.07 (t, J = 2.4 Hz, 2H,
CCH2), 11.19 (s, 1H, NH); 13C NMR (DMSO-d6), δ: 22.36
(CH2, SCH2CC), 23.65 (2CH2), 28.95 (CH3, N3CH3), 32.83
(CH3, N7CH3), 42.48 (CH2, CCH2), 51.73 (2CH2, NCH2),
80.10 (C, SCH2CC), 80.15 (C, SCH2CC), 109.11 (C, C-5),
148.28 (CH, C-8), 149.82 (C, C-2), 151.11 (C, C-6), 154.68
(C, C-4); HR ESIMS m/z [M + H]+ calcd. for
C15H20N5O2S 334.1338 found 334.1343.
Synthesis of 3,7-dimethylxanthine-8-thione 8
To the suspension of 8-bromo derivative 7 (0.26 g, 1 mmol)
in 40 mL of anhydrous ethanol, sodium hydrosulfide (NaSH
× 2 H2O, 0.92 g, 10 mmol) was added and the mixture was
stirred and refluxed for 2 h. After cooling the alcohol was
evaporated in vacuo and dry residue was dissolved in 5%
NaOH solution. The reaction product was precipitated with
15% hydrochloric acid to give compound 8.
8-(4-N-piperidinylbut-2-ynylthio)-3,7-
dimethylxanthine 10b
It was obtained as a dark yellow solid (0.20 g, 95%), mp
322–324 °C (EtOH), 319–320 °C (Carson et al. 1998); H
1
NMR (DMSO-d6), δ: 3.30 (s, 3H, N3CH3), 3.64 (s, 3H,
It was obtained as a pale yellow solid (0.120 g, 69%); mp
N7CH3), 11.34 (s, 1H, N1H), 13.64 (s, 1H, N9H); 13C
223–225 °C (EtOH); 1H NMR (DMSO-d6), δ: 1.23 (m, 2H,

Medicinal Chemistry Research
CH2), 1.39 (m, 4H, 2CH2), 2.19 (m, 4H, 2NCH2), 3.18 (t, J
= 1.8 Hz, 2H, SCH2), 3.32 (s, 3H, N3CH3), 3.81 (s, 3H,
N7CH3), 4.08 (t, J = 1.8 Hz, 2H, CCH2), 11.19 (s, 1H, NH);
13C NMR (DMSO-d6), δ: 22.42 (CH2, SCH2CC), 23.90
(CH2), 25.81 (2CH2), 28.95 (CH3, N3CH3), 32.87 (CH3,
N7CH3), 47.37 (CH2, CCH2), 52.57 (2CH2, NCH2), 79.92
(C, SCH2CC), 80.62 (C, SCH2CC), 109.12 (C, C-5), 148.25
(CH, C-8), 149.84 (C, C-2), 151.11 (C, C-6), 154.69 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C16H22N5O2S
348.1494 found 348.1489.
and ether, and dried in vacuo. Crude product was purified
by column chromatography (silica gel, CHCl3 - ETOH,
99:1, v/v) to give compound 12.
It was obtained as a pale yellow solid (2.15 g, 67%), mp
1
218–220 °C, (EtOH); H NMR (DMSO-d6), δ: 4.02 (s, 3H,
N7CH3), 13C NMR (DMSO-d6), δ: 35.22 (CH3, NCH3),
127.07 (C, C-5), 133.88 (CH, C-8), 142.90 (C, C-2), 151.46
(C, C-6), 161.03 (C, C-4); HR ESIMS m/z [M + H]+ calcd.
for C6H4Br2ClN4 324.8491 found 324.8489.
Synthesis of 2-chloro-7-methylpurine-6,8-dithione
13
8-(4-N-morpholinylbut-2-ynylthio)-3,7-
dimethylxanthine 10c
A solution of 6,8-dibromo derivative 12 (0.326 g, 1 mmol)
and sodium hydrosulfide (NaSH × 2H2O, 0.92 g, 10 mmol)
in 40 mL ethanol was refluxed for 2 h. The alcohol was
evaporated in vacuo and dry residue was dissolved in 5%
NaOH solution. The reaction product was precipitated with
15% hydrochloric acid to give compound 13.
It was obtained as a pale yellow solid (0.126 g, 72%); mp
174–176 °C (EtOH); 1H NMR (DMSO-d6), δ: 2.24 (m, 4H,
2NCH2), 3.24 (t, J = 1.8 Hz, 2H, SCH2), 3.33 (s, 3H,
N3CH3), 3.81 (s, 3H, N7CH3), 4.09 (t, J = 1.8 Hz, 2H,
CCH2), 4.47 (t, J = 1.8 Hz, 4H, 2OCH2), 11.15 (s, 1H, NH);
13C NMR (DMSO-d6), δ: 22.30 (CH2, SCH2CC), 28.96
(CH3, N3CH3), 32.86 (CH3, N7CH3), 46.80 (CH2, CCH2),
51.53 (2CH2, NCH2), 66.43 (2CH2, 2OCH2), 79.28 (C,
SCH2CC), 81.15 (C, SCH2CC), 109.18 (C, C-5), 148.13
(CH, C-8), 149.86 (C, C-2), 151.15 (C, C-6), 154.73 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C15H20N5O3S
350.1287 found 350.1279.
It was obtained as a dark yellow solid (0.225 g, 97%), mp
1
296–298 °C (EtOH); H NMR (DMSO-d6), δ: 4.03 (s, 3H,
N7CH3), 13.88 (s, 1H, N1H), 13.91 (s, 1H, N9H); 13C
NMR (DMSO-d6), δ: 34.62 (CH3, NCH3), 121.42 (C, C-5),
149.92 (CH, C-8), 152.16 (C, C-2), 159.87 (C, C-6), 161.61
(C, C-4); HR ESIMS m/z [M + H]+ calcd. for C6H6ClN4S2
232.9722 found 232.9720.
8-(4-diethylaminobut-2-ynylthio)-3,7-
dimethylxanthine 10d
Synthesis of 2-chloro-6,8-di(prop-2-ynylthio)-7-
methylpurine 14
It was obtained as a pale yellow solid (0.131 g, 78%); mp
186–188 °C (EtOH); H NMR (DMSO-d6), δ: 0.86 (t, J =
2-Chloro-6,8-di(prop-2-ynylthio)-7-methylpurine 14 was
obtained by alkylation of purine-6,8-dithione 13 (0.12 g,
0.5 mmol) with propargyl bromine in DMF according to the
procedure described for the synthesis compound 4.
1
7.2 Hz, 6H, 2CH3), 2.24 (q, J = 7.2 Hz, 4H, 2NCH2), 3.30
(s, 2H, SCH2), 3.30 (s, 3H, N3CH3), 3.80 (s, 3H, N7CH3),
4.07 (s, 2H, CCH2), 11.18 (s, 1H, NH); 13C NMR (DMSO-
d6), δ: 12.82 (2CH3,), 22.43 (SCH2CC), 28.94, (CH3,
N3CH3), 32.85 (CH3, N7CH3), 40.69 (CH2, CCH2), 46.71
(2CH2, NCH2), 79.33 (C, SCH2CC), 80.36 (C, SCH2CC),
109.10 (C, C-5), 148.26 (CH, C-8), 149.85 (C, C-2), 151.12
(C, C-6), 154.67 (C, C-4); HR ESIMS m/z [M + H]+ calcd.
for C15H22N5O2S 336.1494 found 336.1491.
It was obtained as a pale yellow solid (0.105 g, 68%); mp
1
161–163 °C (EtOH), H NMR (DMSO-d6), δ: 3.26 (t, J =
2.4 Hz, 1H, CH), 3.31 (t, J = 2.4 Hz, 1H, CH), 3.88 (s, 3H,
NCH3), 4.22 (d, J = 2.4 Hz, 2H, SCH2), 4.26 (d, J = 2.4 Hz,
2H, SCH2); 13C NMR (DMSO-d6), δ: 18.22 (CH2,
SCH2CCH), 21.02 (CH2, SCH2CCH), 33.90 (CH3, NCH3),
74.68 (CH, SCH2CCH), 75.05 (CH, SCH2CCH), 79.61 (C,
SCH2CCH), 79.68 (C, SCH2CCH), 125.50 (C, C-5), 150.98
(CH, C-8), 152.08 (C, C-2), 159.62 (C, C-6), 159.97 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C12H10ClN4S2
309.0035 found 309.0032.
Synthesis of 2-chloro-6,8-dibromo-7-methylpurine
12
To a stirred suspension of 2,6-dichloro-7-methylpurine 11
(2.00 g, 9.8 mmol) in 4 mL of carbon tetrachloride and
12 mL of nitrobenzene, bromine (4.90 g, 1.6 mL, 31 mmol)
dissolved in 3.0 mL of nitrobenzene was added dropwise.
When the addition was completed, the reaction mixture was
refluxed with stirring for 5 h. The suspension was then
carefully poured with stirring into 40 mL of acetone. The
resulting white precipitate was filtered, washed with acetone
Synthesis of 2-chloro-6,8-di(aminobut-2-ynylthio)-7-
methylpurines 15a-d
2-Chloro-6,8-di(aminobut-2-ynylthio)-7-methylpurines
15a-d were obtained from 6,8-dipropynylthio derivative 14
(0.15 g, 0.5 mmol) and appropriate amines ((pyrrolidine,
piperidine, morpholine, and diethylamine, 0.7 mmol) in dry

Medicinal Chemistry Research
dioxane according to the previously described procedure for
the synthesis of 2,6-di(aminobut-2-ynylthio)-7-methylpur-
ines 5a-d.
80.62 (C, SCH2CC), 80.74 (C, SCH2CC), 125.51 (C, C-5),
143.05 (CH, C-8), 151.23 (C, C-2), 152.18 (C, C-6), 159.95
(C, C-4); HR ESIMS m/z [M + H]+ calcd. for
C22H28ClN6O2S2 507.1404 found 507.1398.
2-Chloro-6,8-di(4-N-pyrrolidinylbut-2-ynylthio)-7-
methylpurine 15a
2-Chloro-6,8-di(diethylaminobut-2-ynylthio)-7-
methylpurine 15d
It was obtained as a pale yellow solid (0.183 g, 77%); mp
133–135 °C (EtOH); 1H NMR (DMSO-d6), δ: 1.58 (m, 8H,
4CH2), 2.38 (m, 8H, 4NCH2), 3.34 (t, J = 2.4 Hz, 2H,
SCH2), 3.36 (t, J = 2.4 Hz, 2H, SCH2), 3.88 (s, 3H, NCH3),
4.22 (t, J = 2.4 Hz, 2H, CCH2), 4.27 (t, J = 2.4 Hz, 2H,
CCH2); 13C NMR (DMSO-d6), δ: 18.93 (CH2, SCH2CC),
18.96 (CH2, SCH2CC), 21.70 (CH3, NCH3), 23.66 (2CH2),
23.71 (2CH2), 33.91 (CH2, CCH2), 33.92 (CH2, CCH2),
42.57 (CH2, NCH2), 42.63 (CH2, NCH2), 51.83 (CH2,
NCH2), 51.95 (CH2, NCH2), 79.32 (C, SCH2CC), 79.38 (C,
SCH2CC), 79.89 (C, SCH2CC), 80.03 (C, SCH2CC),
125.52 (C, C-5), 143.07 (CH, C-8), 151.20 (C, C-2), 152.19
(C, C-6), 159.84 (C, C-4); HR ESIMS m/z [M + H]+ calcd.
for C22H28ClN6S2 475.1505 found 475.1491.
It was obtained as a pale yellow solid (0.194 g, 81%); mp
88–90 °C (EtOH); H NMR (DMSO-d6), δ: 0.87 (m, 12H,
1
4CH3), 2.32 (m, 8H, 4NCH2), 3.33 (s, 4H, 2SCH2), 3.88 (s,
3H, NCH3), 4.22 (t, J = 2.4 Hz, 2H, CCH2), 4.27 (t, J =
2.4 Hz, 2H, CCH2); 13C NMR (DMSO-d6), δ: 12.76
(4CH3,), 19.06 (CH2, SCH2CC), 19.09 (CH2, SCH2CC),
21.82 (CH3, NCH3), 33.93 (CH2, CCH2), 33.94 (CH2,
CCH2), 46.83 (CH2, 2NCH2), 46.85 (CH2, 2NCH2), 78.41
(C, SCH2CC), 79.17 (C, SCH2CC), 80.19 (C, SCH2CC),
80.36 (C, SCH2CC), 125.97 (C, C-5), 143.09 (CH, C-8),
151.22 (C, C-2), 152.22 (C, C-6), 159.81 (C, C-4); HR
ESIMS m/z [M + H]+ calcd. for C22H32ClN6S2 479.1818
found 479.1813.
2-Chloro-6,8-di(4-N-piperidinylbut-2-ynylthio)-7-
methylpurine 15b
Antiproliferative assay in vitro
Cell culture
It was obtained as a pale yellow solid (0.165 g, 74%); mp
108–110 °C (EtOH); 1H NMR (DMSO-d6), δ: 1.23 (m, 4H,
2CH2), 1.40 (m, 8H, 4CH2), 2.26 (m, 8H, 4NCH2), 3.19 (s,
4H, 2SCH2), 3.89 (s, 3H, NCH3), 4.23 (t, J = 2.4 Hz, 2H,
CCH2), 4.28 (t, J = 2.4 Hz, 2H, CCH2); 13C NMR (DMSO-
d6), δ: 18.97 (2CH2, SCH2CC), 21.77 (CH3, NCH3), 23.85
(CH2), 23.88 (CH2), 25.76 (2CH2), 25.81 (2CH2), 33.94
(CH2, CCH2), 33.95 (CH2, CCH2), 47.41 (CH2, NCH2),
47.43 (CH2, NCH2), 52.67 (CH2, NCH2), 52.71 (CH2,
NCH2), 79.14 (C, SCH2CC), 79.75 (C, SCH2CC), 80.38 (C,
SCH2CC), 80.43 (C, SCH2CC), 125.54 (C, C-5), 143.10
(CH, C-8), 151.24 (C, C-2), 152.22 (C, C-6), 159.81 (C, C-
4); HR ESIMS m/z [M + H]+ calcd. for C24H32ClN6S2
503.1818 found 503.1814.
Compounds were evaluated for their anticancer activity
using three cultured cell lines: SNB-19 (human glio-
blastoma, DSMZ - German Collection of Microorganisms
and Cell Cultures, Braunschweig, Germany), C-32 (human
amelanotic melanoma, ATCC-American Type Culture
Collection, Manassas, VA, USA), MDA-MB-231 (human
adenocarcinoma mammary gland, ATCC, Manassas, VA,
USA), and HFF-1 (human fibroblast cell line, ATCC,
Manassas, VA, USA). The cultured cells were kept at 37 °C
and 5% CO2. The cells were seeded (1 × 104 cells/well/100
μL DMEM supplemented with 10% FCS and streptomycin
and penicillin) using 96-well plates (Corning). The cells
were counted in a hemocytometer (Burker’s chamber) using
a phase contrast Olympus IX50 microscope equipped with
Sony SSC-DC58 AP camera and Olympus DP10 digital
camera.
2-Chloro-6,8-di(4-N-morpholinylbut-2-ynylthio)-7-
methylpurine 15c
It was obtained as a pale yellow solid (0.172 g, 70%); mp
112–113 °C (EtOH); 1H NMR (DMSO-d6), δ: 2.30 (m, 4H,
2NCH2), 2.35 (m, 4H, 2NCH2), 3.23 (m, 4H, SCH2), 3.47
(m, 4H, 2OCH2), 3.51 (m, 4H, 2OCH2), 3.89 (s, 3H,
NCH3), 4.25 (t, J = 2.4 Hz, 2H, CCH2), 4.29 (t, J = 2.4 Hz,
2H, CCH2); 13C NMR (DMSO-d6), δ: 18.89 (CH2,
SCH2CC), 18.91 (CH2, SCH2CC), 21.73 (CH3, NCH3),
33.93 (CH2, CCH2), 33.94 (CH2, CCH2), 46.90 (CH2,
NCH2), 46.96 (CH2, NCH2), 51.85 (CH2, 2NCH2), 66.46
(CH2, 4OCH2), 78.83 (C, SCH2CC), 79.33 (C, SCH2CC),
Proliferation assay
The antiproliferative effect of the compounds obtained from
both the cancer and the normal cells was determined using
the Cell Proliferation Reagent WST-1 assay (Roche Diag-
nostics, Mannheim, Germany). This colorimetric assay is
based on the viable cell’s ability to cause the bright red-
colored stable tetrazolium salt (2-(4-iodophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono-
sodium salt) to cleave to the dark red soluble formazan by

Medicinal Chemistry Research
cellular enzymes. An expansion in the number of viable
cells results in an increase in the overall activity of mito-
chondrial dehydrogenases in the sample. An increase in the
amount of formazan dye formed correlates to the number of
metabolically active cells in the culture. The formazan dye
produced by metabolically active cells is quantified by a
scanning ELISA reader that measures the absorbance of the
dye solution at appropriate wavelengths. The examined
cells were exposed to the tested compounds for 72 h at
various concentrations between 0.1 and 100 μg/mL (pre-
pared initially at a concentration of 1 mg/mL in DMSO).
The control was performed in order to check that DMSO
has no effect on the cells at the concentration used. The cells
were incubated with WST-1 (10 μL) for 1 h and the
absorbance of the samples was measured against a back-
ground control at 450 nm using a microplate reader with a
reference wavelength at 600 nm. The results are expressed
as the means of at least two independent experiments per-
formed in triplicate. The antiproliferative activity of the
tested compound was compared to cisplatin. The IC50
values (a concentration of a compound that is required for
50% inhibition) were calculated from the dose–response
relationship with respect to control.
halogen atom in the 8 position requires halogenation with
bromine (Koppel et al. 1962). Compounds 6 and 11 were
brominated with bromine (as a solution in nitrobenzene)
followed by a refluxing of the reaction mixture for 5 h. In
the case of compound 11, the bromination proceeded not
only in the 8 position but also in ipso in the 6 position. 8-
Xanthinethione 8 and 8-purinethione 13 were obtained in
the sodium hydrosulfide reaction. In order to obtain 8-
aminobutynylthioxanthines 10a-d and 6,8-diaminobuty-
nylthiopurines 15a-d, the same sequence of propynylation
and a Mannich reaction was used.
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Anticancer activity
For the biological tests, 18 propynylthio- and aminobuty-
nylthiopurines and xanthines of 4 types were selected:
a. 2-chloro-6-aminobutynylthio derivatives containing
the piperidinyl, morpholinyl, and diethylamino groups (2a-
c),
b. 2,6-dipropynylthio (4) and 2,6-diaminobutynylthio-
purines containing the pyrrolidinyl, piperidinyl, morpholi-
nyl, and diethylamino groups (5a-d),
Result and discussion
c. 8-propynylthio (9) and 8-aminobutynylthioxanthines
derivatives containing the pyrrolidinyl, piperidinyl, mor-
pholinyl, and diethylamino groups (10a-d),
Chemistry
2-Chloro-6-(prop-2-ynylthio)-7-methylpurine
1
was
d. the 2-chloro-6,8-dipropynylthio (14) and 2-chloro-6,8-
diaminobutynylthiopurines containing pyrrolidinyl, piper-
idinyl, morpholinyl, and diethylamino groups (15a-d),
The anticancer activity of the compounds obtained was
investigated in vitro using cultured human glioblastoma
SNB-19, human adenocarcinoma MDA-MB-231, and
melanoma C-32 cell lines. Normal human fibroblasts (HFF-
1) were used as a control and cisplatin was used as a
reference drug. Table 1 shows the activity of alkynylthio-
purines and xanthines 2a-c, 4, 5a-d, 9, 10a-d, 14, and 15a-d
as the IC50 values. The tested compounds exhibited differ-
ent activities against three cancer cell lines. The SNB-19
and C-32 lines were more sensitive than the MDA-MB-231
line for most compounds.
obtained via S-alkylation of the appropriate 6-purinethione
with propargyl bromide (as solution in toluene) in DMF at
room temperature in the presence of potassium tert-but-
oxide (Kowalska et al. 2015). Using this procedure, 2,6-
dipropynylthio-7-methylpurine 4 was obtained from 2,6-
purinethione 3. 2-Chloro-6-propynylthio-7-methylpurine 1
and 2,6-dipropynylthio-7-methylpurine 4, which were
transformed via a Mannich reaction with paraformaldehyde
and secondary amine (pyrrolidine, piperidine, morpholine,
and diethylamine) in dry dioxane in the presence of cata-
lytic amounts of CuCl into 2-chloro-6-aminobut-2-ynylthio-
7-methylpurines 2a-c, as well as 2,6-di(aminobut-2-ynyl-
thio)-7-methylpurines 5a-d in yields of 82–94% and
74–79.5%, respectively. Whereas 2- and 6-chloropurines
are easily obtained from oxopurines, the introduction of the
As shown in Table 1, 6,8-disubstituted derivatives 14
and 15a-d were the most active group of the tested
Scheme 1 Synthesis of 2-
chloro-6-aminobutynylthio-7-
methylpurines 2a-c

Medicinal Chemistry Research
Scheme 2 Synthesis of 2,6-
dipropynylthio- and 2,6-
diaminobutynylthio-7-
methylpurines 4 and 5a-d
Scheme 3 Synthesis of 8-
propynylthio- and 8-
aminobutynylthio-3,7-
dimethylxanthines 9 and 10a-d
compounds and in some cases were more or at least as
active as cisplatin. 2-Chloro-6,8-dipropynylthiopurine 14
and its pyrrolidinylmethyl derivative 15a exhibited strong
antiproliferative activity (IC50 values of <1 μg/mL) against
the SBN-19 line. The last compound was also active against
two other lines (IC50 = 3.8–4.5 μg/mL). Compounds 15c
and 15d (containing two morpholinylbutynylthio and die-
thylaminobutynylthio groups) showed selective activity
against the SNB-19 and C-32 lines (IC50 = 2.9–5.3 μg/mL).
The activity of 2,6-disubstituted compounds (4 and its
aminomethyl derivatives 5a-c) were also selective. 2,6-
Dipropynylthiopurine 4 was more active than cisplatin
against the MDA-MB-231 and C-32 cell lines (IC50 ≤ 1.77
μg/mL). The introduction of the pyrrolidinylmethyl and
piperidinylmethyl moieties to the propynylthio chain
(compounds 5a and 5b) slightly decreased activity against
selected lines. On the other hand, derivative 5c (the mor-
pholinylmethyl moiety that was introduced) was very active
(more so than cisplatin) with the IC50 value of 0.35 μg/mL
against the C-32 cell line.
2-Chloro-6-aminobutynyltiopurines 2a-c were less active
against all cell lines with IC50 values over 11.88 μg/mL.
The other monoalkynylthiosubstituted derivatives 9 and
10a-d containing the 3,7-dimethylxanthine framework showed

Medicinal Chemistry Research
Scheme 4 Synthesis of 2-
chloro-6,8-dipropynylthio- and
2-chloro-6,8-
diaminobutynylthio-7-
methylpurines 14 and 15a-d
Table 1 Antiproliferative activity of alkynylthiopurines and xanthines
its derivatives 10d and 10c (containing the diethylaminobu-
tynylthio and morpholinylbutynylthio groups) exhibited strong
activity against the SBN-19 line (IC50 < 1.7 μg/mL). Two
other derivatives (10a and 10b) were found rather inactive.
All the compounds were tested for their cytotoxicity on
the normal human fibroblasts (HFF-1). Some compounds
(5b-d, 10c-d, 15a-b, and 15d) as well as cisplatin also
turned out to be cytotoxic for fibroblasts. Very active
compounds 4 (against adenocarcinoma MDA-MB-231 and
melanoma C-32), 14 (against glioblastoma SNB-19), and
15c (against melanoma C-32) were found to have relatively
weak cytotoxicity.
Compounds
Antiproliferative activity IC50 (μg/mL)
SNB-19
C-32
MDA-MB-231
HFF-1
2a
28.80
35.40
17.40
15.01
19.41
4.64
21.01
125.00
11.88
1.77
33.40
66.28
48.06
1.00
35.42
20.09
8.65
2b
2c
4
10.00
10.00
3.98
5a
6.81
19.95
7.35
5b
6.39
5c
21.54
48.93
191.10
176.62
166.17
1.69
0.35
50.80
38.49
169.34
471.50
363.50
6.54
0.37
5d
23.86
103.46
51.08
355.00
222.72
26.36
270.35
4.55
5.95
9
153.65
34.26
30.39
5.87
10a
10b
10c
10d
14
Conclusion
While searching for alkynylthiopurines and aminoalk-
ynylthiopurines and xanthines with the anticancer activity,
we worked out a synthesis of 18 new multisubstituted 7-
methylpurines and 3,7-dimethylxanthines containing one or
two propynylthio groups (in 2, 6, and 8 positions), which
were further transformed via a Mannich reaction with cyclic
and open chain amines and formaldehyde into aminobuty-
nylthio derivatives. The products obtained represent various
types of the purine and xanthine structure containing the
triple bond: 8-mono-, 2,6-, and 6,8-dipropynylthio deriva-
tives 4, 9, and 14, 6- and 8-monoaminobutynylthio deri-
vatives 2a-c and 10a-d, 2,6- and 6,8-diaminobutynylthio
1.48
68.12
10.00
3.83
3.68
0,07
20.09
3.63
15a
15b
15c
15d
Cisplatin
0.52
11.62
26.10
2.91
19.08
4.08
38.07
74.05
10.00
11.85
5.50
36.38
1.00
5.33
1.46
5.71
1.21
very different activity. In contrast to the very active 2,6- and
6,8-dipropynylthio derivatives 4 and 14, 8-propynylthio deri-
vative 9 was found inactive (IC50 > 100 μg/mL). Surprisingly,

Medicinal Chemistry Research
derivatives 5a-d and 15a-d. All tested compounds exhibited
different anticancer activity against human glioblastoma
SNB-19, human adenocarcinoma MDA-MB-231, and
melanoma C-32 cell lines depending on the nature of the
substituent and its localization in the purine framework.
Some compounds exhibited stronger or similar anticancer
activity to cisplatin. A small number of compounds were
also cytotoxic against the normal human fibroblasts (HFF-
1). The most promising compounds with strong anticancer
activity and relatively low cytotoxicity turned out to be 2,6-
dipropynylthio-7-methylpurine 4, 2-chloro-6,8-dipropy-
nylthio-7-methylpurine 14, and 2-chloro-6,8-di(N-morpho-
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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