Antioxidant properties of thio-caffeine derivatives: Identification of the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine as antioxidant and highly potent cytoprotective agent

Article abstract of DOI:10.1016/j.bmcl.2016.06.091

A series of nine thio-caffeine analogues were synthesized and characterised by NMR, FT-IR and MS spectroscopic methods. Molecular structures of four of them were determined using single crystal X-ray diffraction methods. The antioxidant properties of all

Full text of DOI:10.1016/j.bmcl.2016.06.091

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antioxidant properties of thio-caffeine derivatives: Identification
of the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]
caffeine as antioxidant and highly potent cytoprotective agent
a
b
a
Beata Jasiewicz a, , Arleta Sierakowska , Natalia Wandyszewska , Beata Warzajtis ,
⇑
_
a
a
b,
⇑
´
Urszula Rychlewska , Rafał Wawrzyniak , Lucyna Mrówczynska
a
´
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland
b Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan´, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nine thio-caffeine analogues were synthesized and characterised by NMR, FT-IR and MS spec-
troscopic methods. Molecular structures of four of them were determined using single crystal X-ray
diffraction methods. The antioxidant properties of all compounds, at concentration ranges from 0.025
to 0.1 mg/mL, were evaluated by various chemical- and cell-based antioxidant assays. Human erythro-
cytes were used to examine in vitro haemolytic activity of all compounds and their protective effect
against oxidative haemolysis induced by AAPH, one of the commonly used free radical generator. All
compounds studied showed no effect on the human erythrocytes membrane structure and permeability
with the exception of 8-(phenylsulfanyl)caffeine. Among the nine caffeine thio-analogues tested, the
newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine possessed exceptionally high
antioxidant properties. Moreover, it protects human erythrocytes against AAPH-induced oxidative dam-
age as efficiently as the standard antioxidant Trolox. Therefore, 8-[(pyrrolidin-1-ylcarbonothioyl)sul-
fanyl]caffeine may have a significant cytoprotective potential caused by its antioxidant activity.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 9 May 2016
Revised 28 June 2016
Accepted 29 June 2016
Available online xxxx
Keywords:
Caffeine thioderivatives
Antioxidant activity
Haemolysis
Human erythrocytes
Trolox
X-ray structure
Caffeine (1), the naturally occurring methylxanthine, is of an
unquestionable interest as a leading compound for the develop-
ment of new derivatives with enhanced activities and/or lower
toxicities. It modulates drugs used for curing lung, liver, uterine
cervix and breast cancer and enhances gastric secretion and urine
production, reduces the risk of developing gallstone disease and
also reduces asthma. The anticarcinogenic effect of caffeine has
been related to its effect on cell cycle and proliferation.1 Caffeine
easy penetrates through biological membrane2 and activates the
erythrocyte glutathione-S-transferase (GST) which is involved in
erythrocytes protection.3 It has been shown that caffeine inhibits
eryptosis (suicidal death) of human erythrocytes within the range
amyloid b-induced caspase-3 activity in neurons.7 According to
Kesavan and co-workers8–11 caffeine has abilities to scavenge
highly reactive free radicals and excited states of oxygen. Antioxi-
dant ability of caffeine is similar to that of the established biolog-
ical antioxidant glutathione and significantly much higher than
that of ascorbic acid. However, other studies demonstrate an
absence of antioxidant properties of caffeine using DPPH assay12–14
or even its pro-oxidant effects.15,16 Studies have shown that
C8-substituted caffeine derivatives are adenosine receptor antago-
nists,17–19 acetylcholinesterase inhibitors20 and monoamine
oxidase inhibitors, and that within this group the 8-thiocaffeine
analogues belong to a pharmacologically important subclass.21–24
In general, nearly all organosulfur compounds are considered as
antioxidants. Compounds such as allicin, methionine and methyl-
cysteine protect against metal-mediated oxidative DNA damage.
Moreover, the results of epidemiological, clinical, in vivo, and
in vitro studies have undoubtedly shown the protective effects of
sulfur compounds against cellular damage and disease.25 It there-
fore came as a surprise to us that although the antioxidant activity
of spent coffee extracts rich in caffeine26 and caffeine alone27,28 has
been extensively examined, similar studies devoted to the thio-
caffeine derivatives have not been so farreported. Notably, the
of its plasma concentration (100 l
M)4 and prevents the acceler-
ated clearance of erythrocytes from circulation and development
of anemia. On the other hand, the antiapoptotic effect of caffeine
on nucleated cells could be explained by inhibition of the phospho-
diesterase and c-AMP production,5 its inhibitory action on ATM
kinase and suppression of p53 phosforylation6 or suppression of
⇑
Corresponding authors. Tel.: +48 61 8291354; fax: +48 61 8291505.
E-mail addresses: beatakoz@amu.edu.pl (B. Jasiewicz), lumro@amu.edu.pl
(L. Mrówczyn´ ska).
http://dx.doi.org/10.1016/j.bmcl.2016.06.091
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jasiewicz, B.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.091

2
B. Jasiewicz et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
biochemistry of dithiocarbamates is of interest because of their
clinical use.29 Their biological properties include ability to influ-
ence oxidative stress, apoptosis and enzyme inhibition.30–33 More-
over, pyrrolidinedithiocarbamate is widely used as an inhibitor of
nuclear factor kappa B(NFkB) and this, or related compounds
may have therapeutic potential in inhibiting artheriosclerosis.34
Relying on the above reports, we expected that there will be some
advantage, in terms of biological activity, resulting from a syner-
gism between the biological actions of the caffeine and its sulfur
derivatives. We have therefore synthesized and characterised a
series of nine structurally diverse thio-caffeine analogues, that
included, 6-thiocaffeine, 2,6-dithiocaffeine, 8-thioalkyl derivatives,
8-(phenylsulfanyl)-caffeine and the newly synthesized 8-[(pyrro-
lidin-1-ylcarbonothioyl)sulfanyl]caffeine, and explored their
antioxidant activity as well as their effects on human erythrocytes
in vitro. Human red blood cells (RBCs) are widely used in the inves-
tigation of antioxidant activity of natural and newly obtained com-
pounds because there are the main targets for free radicals in the
circulatory system.35–38 Although RBCs contain enzymes that are
involved in defence against free radicals, namely catalase (CAT),
superoxide dismutase (SOD), glutathione reductase (GRd) and glu-
tathione peroxidase (GPx)39 they are not able to effectively elimi-
nate reactive oxygen species and as a result oxidative haemolysis
occurs. The water-soluble free radical generator 2,20-azobis(2-
methylpropionamidine) dihydrochloride (AAPH) is commonly
used for inducing RBCs membrane injury and oxidative haemolysis
in vitro. Thus, in the present study, AAPH was employed to exam-
ine the oxidative haemolysis in the presence or absence of the thio-
caffeine derivatives as well as the standard antioxidants, namely
Trolox and butylated hydroxytoluene (BHT).
The 6-thiocaffeine (2) and 2,6-dithiocaffeine (3) were synthe-
sized by reaction of caffeine with Lawesson’s reagent in toluene
(Scheme 1). Reaction of 8-bromocaffeine with an appropriate
sodium thiolate reagent in ethanol solution gives the C8 thiocaf-
feine analogues (4–10).40 The synthetic routes of these target com-
pounds are outlined in Scheme 2. All obtained compounds were
structurally (1H NMR, 13C NMR, FTIR, ESI-MS) characterised (see
Supplementary materials). For four of them the crystal structures
were determined.
The most noticeable differences in the NMR spectra were the
downfield shifts of signals corresponding to positions C5, C6 in
6-thio- or C2, C5 and C6 in 2,6-dithiocaffeine, as compared with
caffeine. The IR spectrum of 2 showed an absorption band near
O
O
CH3
CH3
H3C
O
H3C
O
N
N
6
6
N
2
N
2
8
8
+
R
Br
R-Na
N
N
N
N
CH3
CH3
(4-10)
R =
(4)
(5)
S
S
(8)
(9)
S
S
(6)
(7)
S
(10)
N
S
S
S
Scheme 2. Reaction and conditions: 1 equiv 8-bromocaffeine, 4 equiv sodium
thiolate, ethanol, reflux, 2–48 h. Time for completion of the reaction at reflux as
indicated by TLC.
1675 cmꢀ1 associated to the carbonyl group and absorption band
near 1110 cmꢀ1 associated to the thiocarbonyl group, whereas in
the spectrum of 3 we can observed absorption bands of two thio-
carbonyl groups at 1110 and 1070 cmꢀ1. The IR spectrum of com-
pound
5 as representative of the series (4–8), showed an
absorption band at 1319 and 1037 cmꢀ1 associated to the thioalkyl
group and absorption bands at 1702 and 1656 cmꢀ1 associated
to the carbonyl groups of the caffeine fragment. The IR spectrum
of compound
9 showed an absorption band at 3060 and
1440–1578 cmꢀ1 associated to the aromatic ring, whereas in the
IR spectrum of 10 the bands in 1400–1100 cmꢀ1 region are associ-
ated with NAC@SAS stretching vibrations. In the 1H NMR spectra
of compounds 4–10, three singlets in the range of 3.37–3.97 ppm
indicated the presence of the three methyl groups from caffeine
unit. Aromatic protons of compound 9 are present in the range of
7.60–7.30 ppm, while signals at 3.61 and 1.90 ppm present in the
spectra of 10 are connected with presence of pyrrolidine ring.
The 13C NMR spectrum of compound
9 showed signals at
132–127 ppm corresponding to the phenyl ring. Signal at about
O
CH3
12
10
H3C
5
4
N
7
8
6
N
1
2
3
N
N
9
O
11
CH3
(1)
LR
LR
1. 5 eq
5 eq
S
S
S
CH3
CH3
CH3
H3C
O
H3C
S
H3C
O
N
N
N
N
6
6
N
N
N
6
N
N
+
8
8
8
2
2
2
N
N
N
CH3
CH3
CH3
(2)
(3)
Scheme 1. Reaction and conditions: Caffeine (1), 1.5 or 5 equiv LW (Lawesson’s reagent), toluene, reflux at 25 h. Time for completion of the reaction at reflux as indicated by
TLC.
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B. Jasiewicz et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 1. Sulfur analogue of caffeine 3 and C8 sulfur-substituted caffeines 4, 5 and 10 as present in crystals at 295 K (3, 4 and 5) and at 150 K (10). Atomic displacement
parameters are drawn at the 40% probability level. Hydrogen atoms are represented as spheres of arbitrary size. Only one of two alternative positions of the methyl hydrogen
atoms in 3 is displayed.
Figure 2. DPPH free radical scavenging activity of compounds tested and standard antioxidants Trolox and BHT at different concentrations. Results are presented as
average SD (n = 3). Different letters indicate samples that were significantly different (p <0.05). The numbers are respectively: caffeine (1); 6-thiocaffeine (2); 2,6-
dithiocaffeine (3); 8-(methylthio)caffeine (4); 8-(ethylthio)caffeine (5); 8-(propylthio)caffeine (6); 8-(isopropylthio)caffeine (7); 8-(tertbutylthio)caffeine (8); 8-(phenylsul-
fanyl)caffeine (9); 8-[(pyrrolidin-1-yl-carbonothioyl)sulfanyl]caffeine (10).
185 ppm are connected with thiocarbonyl group of 10. The signals
corresponding to caffeine carbonyl groups of 4–10 appearing near
150 and 154 ppm are assigned to C2 and C6 respectively. In the
ESI-MS spectra of compounds 2, 3 and 9 molecular ions [M+1]+
are observed, whereas for compounds 4–6, 8 and 10 sodium
adducts [M+Na]+ are present. The exception is compound 7 for
which the loss of (CH3)2 fragment from the [M+Na]+ ion gives
signals at m/z = 261.
The structural features of the molecules as present in crystals
are illustrated in Figure 1, prepared using the Mercury program.41
The xanthine ring and the CAS bonds in 4, 5 and 10 are coplanar,
while the terminal methyl group in 5 and the (pyrrolidin-1-ylcar-
bonothioyl)sulfanyl moiety in 10 are inclined at nearly right angles
with respect to the xanthine molecular plane. Unlike caffeine,
which crystallizes as a monohydrate, but also occurs in two
anhydrous, extensively disordered forms,42 all X-ray investigated
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B. Jasiewicz et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 3. Fe3+–Fe2+ reductive potential of compounds tested, and standard antioxidants Trolox and BHT at different concentrations. Results are presented as average SD
(n = 3). Different letters indicate samples that were significantly different (p <0.05). Other indications as in Figure 2.
Figure 4. In vitro protective effects of compounds tested and standard antioxidants Trolox and BHT against 60 mM AAPH-induced haemolysis after 20 min pre-incubation
with RBCs. Results are presented as means SD (n = 4). *—no haemolysis inhibition detected. Different letters indicate samples that were significantly different (p <0.05).
Other indications as in Figure 2.
compounds reported in this Letter form anhydrous crystals. A
hydrophilic centre at the imidazole nitrogen atom, utilised in the
hydrated caffeine crystals as H-bond acceptor from the included
water molecule, seems not to play a structure determining role
in the crystals of the dithioanalogue 3 as well as in the remaining
C8-substituted derivatives. More detailed description of X-ray
structures is provided in Supplementary material.
As can be seen in Figure 2, compound 10 showed the strongest
DPPH radical scavenging activity in the concentration-dependent
manner. The scavenging effect of 10 at the highest concentration
(0.1 mg/mL) was equal to 67% of the activity of standard Trolox
and was identical to standard BHT. Compound 7 exhibited 34%
activity of Trolox and 50% activity of BHT. Other thiocaffeines
showed weak DPPH scavenging activity (less than 10%), compara-
ble to that of caffeine.
The reducing capacity of a compound may act as an important
indicator of its potential antioxidant activity. The method is based
on the reduction of (Fe3+)-ferricyanide complex to ferrous form due
to the presence of antioxidant. As shown in Figure 3, compound 10
exhibits the dose-dependent reducing power. At the highest con-
centration activity of 10 was equal to 65% of Trolox activity and
was significantly higher than standard BHT. Similarly to results
obtained with DPPH assay, reducing activity of compound 7 (38%
of Trolox activity) was significantly higher than that of other
derivatives and identical to BHT.
Caffeine as well as its thioanalogues have no capacity to ferrous
ions chelate (data not shown).
The haemolytic activity of compounds 1–10 was estimated
against human erythrocytes in vitro. Neither caffeine nor its
thioanalogues were able to induce alterations in the erythrocyte
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B. Jasiewicz et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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materials). Exposure of human erythrocytes to compound
9
induced haemolysis equal to 57.17% 7.32 at 0.1 mg/mL and
26.91% 11.9 at 0.05 mg/mL, respectively. No significant haemoly-
sis (less than 5%) was observed at concentration of 9 equal
0.025 mg/mL. No significant RBCs shape changes were observed
after incubation (60 min) with caffeine and its thio-analogues up
to concentration 0.1 mg/mL. Compound 9 with confirmed mem-
brane-permeabilizing activity, was an exception again. Optical
microscopic evaluation of the erythrocytes morphology after
60 min incubation with 9 showed mostly the presence of echino-
cytes and spherocytes (Table S2, Supplementary materials), with
some RBCs-ghosts between them. It is known that echinocytic
transformation undergoes when molecules of compound incorpo-
rate into egzoplazmatic layer of the erythrocyte membrane,43
therefore it can be concluded that 9 incorporates into the outer
layer of a membrane and in consequence induce alterations in its
molecular structure. The statistically significant decrease
(p <0.05) of RBCs osmotic resistance (0.488% NaCl) under 9 treat-
ment (versus control cells resistance equal to 0.451% NaCl) confirm
its partitioning into the RBCs membrane (Table S2, Supplementary
materials).
As shown in Figure 4, analogue 10 was a potent agent in RBC
protecting against free radicals induced haemolysis, as effective
as Trolox. Interestingly, 6-thiocaffeine (2) and 2,6-dithiocaffeine
(3) did not exhibit an antioxidant potential in both DPPH and
reducing power assay, but significantly inhibited haemolysis
induced by AAPH. The protective activity of caffeine (1) and com-
pounds 4, 5 and 7 was similar and significantly lower (equal to
10%). Compounds 6, 8 and 9 were not active in RBCs protection
against free radicals damage.
To conclude, our results demonstrate that insertion of the
(pyrrolidin-1-ylcarbonothioyl)sulfanyl moiety into the caffeine
molecule leads to compound 10 that shows antioxidant potential
significantly high to explain its cytoprotective effect. This finding
substantiates classification of 8-[(pyrrolidin-1-ylcarbonothioyl)-
sulfanyl]caffeine for further studies aimed at the use of this com-
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Supplementary data
40. Representative synthesis: 8-(Methylthio)caffeine (4).
A mixture of 8-
bromocaffeine (1 mmol) and sodium methanethiolate (4 mmol) in ethanol
was heated under reflux for 2 h and when cooled to room temperature. The
solution was evaporated until the product started to precipitate. The resulting
precipitate was filtered off and recrystallized from ethanol. Yield 70%: mp 183–
185 0C. ESI-MS: m/z 263 [M+Na]+. 1H NMR d 3.84 (s, 3H), 3.57 (s, 3H), 3.40 (s,
3H), 2.72 (s, 3H); 13C NMR d154.52, 151.76, 151.24, 148.44, 108.65, 32.14,
29.71, 27.75, 14.85.
CCDC-1477518–1477521 contain the supplementary crystallo-
graphic data for 3, 4, 5 and 10, respectively. Supplementary data
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.06.091.
41. Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M.; Macrae, C. F.; McCabe, P.;
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Please cite this article in press as: Jasiewicz, B.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.091