Synthesis and Analgesic Activity of Annelated Xanthine Derivatives in Experimental Models in Rodents

Article abstract of DOI:10.1002/ardp.201500169

A series of annelated derivatives of xanthine were synthesized and assayed as potential analgesic agents. All synthesized xanthine derivatives were tested in the writhing test and hot-plate test. The pharmacological assays demonstrated that all the compou

Full text of DOI:10.1002/ardp.201500169

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
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Full Paper
Synthesis and Analgesic Activity of Annelated Xanthine
Derivatives in Experimental Models in Rodents
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Małgorzata Zygmunt , Jacek Sapa , Anna Drabczynska , Tadeusz Karcz , Christa Muller , Meryem Kose ,
Gniewomir Latacz2, Jakub Schabikowski2, Marek Bednarski1,
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and Katarzyna Kiec-Kononowicz
1
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Department of Pharmacological Screening, Jagiellonian University Medical College, Krakow, Poland
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College,
Krakow, Poland
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3
PharmaCenter Bonn, Pharmaceutical Institute, University of Bonn, Bonn, Germany
A series of annelated derivatives of xanthine were synthesized and assayed as potential analgesic
agents. All synthesized xanthine derivatives were tested in the writhing test and hot-plate test. The
pharmacological assays demonstrated that all the compounds prepared, without exception, displayed
a significant activity in the mouse writhing assay. The analgesic action of the most active compounds,
expressed as ED50 was found to be 1.4–4.3 times more potent than that of acetylsalicylic acid used as
the reference compound. However, only some of the compounds demonstrated analgesic activity in
the hot-plate test. The analgesic effect of some compounds is probably related to their agonistic,
antagonistic, or partial agonistic activity at the adenosine receptors.
Keywords: Adenosine receptors / Analgesic activity / Pyrimido[2,1-f]purinediones / Xanthines
Received: May 19, 2015; Revised: July 2, 2015; Accepted: July 7, 2015
DOI 10.1002/ardp.201500169
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
antinociceptive effects in multiple preclinical models of acute
and chronic pain [4]. Spinal administration of adenosine
Introduction
Analgesic pharmacotherapy utilizes not only the classical
drugs (opioids and NSAIDs) but also agents belonging to other
pharmacological groups (analgesic adjuvants) used as an add-
on therapy in the treatment of intractable pain, especially
neuropathic pain syndromes [1].
Currently, the search for new drugs that interfere with
other mechanisms of the pain response is in progress.
Adenosine is one of the factors involved in the pain response
[2]. The role of adenosine receptors in nociception is complex
and may involve different mechanisms in the central nervous
system and in peripheral tissues [3]. A1AR agonists show
receptor agonists induces antinociception in a variety of
animal models of pain, presumably through the activation of
spinal A1 and to a lesser extent through A2 receptors [3].
Adenosine can produce analgesic or pronociceptive effects
through the activation of peripheral A1 and A2 receptors,
respectively [3].
Literature data on the effects of agonists and antagonists
of the A2AR in pain control are conflicting. For example,
studies with A2AR agonists have shown their antinociceptive
effects in the writhing test, a visceral pain model, but the
hypoalgesia observed in A2A
R knockout mice and the
antinociceptive effects of a specific A2AR antagonist in
inflammatory models suggest that this is the absence or
blockade of the A2AR which has therapeutic potential in pain
states [5]. In contrast, A2B receptor blockade may result in
analgesic effect [3]. Adenosine A3 receptor activation
produces pain behaviors due to the release of histamine
and 5-hydroxytryptamine from mast cells and their subse-
quent actions on the sensory nerve endings.
Correspondence: Dr. Małgorzata Zygmunt, Department of
Pharmacological Screening, Chair of Pharmacy, Jagiellonian
University Medical College, 9 Medyczna Street, 30-688 Krakow,
Poland.
E-mail: malgorzata.zygmunt@uj.edu.pl
Fax: þ48-12-6205552
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
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affinity. For selected compounds, antiproliferative effect and
metabolic stability were checked.
The endogenous adenosine systems contribute to antinoci-
ceptive properties of caffeine, opioids, noradrenaline, 5-
hydroxytryptamine, tricyclic antidepressants, and transcuta-
neous electrical nerve stimulation [6]. There are many reports
suggesting that some xanthine derivatives, for example,
caffeine, possess analgesic properties in laboratory animals.
Caffeine, an antagonist of adenosine A1, A2A, A2B, and A3
receptors, is known as an adjuvant analgesic in combination
with non-steroidal anti-inflammatory drugs (NSAIDs) and
acetaminophen in humans [3]. Caffeine potentiated the
analgesic effects of morphine and decreased the morphine-
induced hyperactivity in mice [7]. In preclinical studies,
caffeine produced intrinsic antinociceptive effects in several
rodent models [6]. Though it is thought that caffeine
analgesia is produced, at least in part, through adenosine
receptor antagonism, it is unclear which receptor subtypes are
involved [3].
Results and discussion
Chemistry
The tetrahydropyrimido[2,1-f]purinediones were synthesized
as depicted in Scheme 1. As starting materials, 7-(3-chlor-
opropyl)-8-bromotheophylline (2A) and 7-(3-chloropropyl)-8-
bromo-1,3-dipropylxanthine (2B) were used, which could be
obtained as previously described by us [8–10]. They were
cyclized with arylalkylamines (KD-56, KD-60, KD-114, and KD-
142) and alkylamines 4 and KD-179 under various reaction
conditions regarding the amount of amine, the reaction
medium, and the reaction time [11]. 4-Hydroxyphenylethyl-
amine (tyramine) was obtained from its hydrochloride by
alkalization to pH 9 of water solution of its hydrochloride by
10% aqueous sodium carbonate. The amine WZ-7 was
obtained as product of reaction of 5 (resulting from the
reaction of bromination of 4 with PBr3) with benzylamine
This paper reports the synthesis and results of a prelimi-
nary pharmacological screening of annelated xanthine
analogs. The synthesized compounds were tested for
analgesic activity as well as for adenosine receptor binding
Cl
O
O
O
H
N
R1
O
R1
O
R1
O
N
N
N
i
ii
N
N
R1 = CH3
N
Br
Br
R2
N
N
N
N
N
N
KD-56
R2 =
R1
R1
R1
2
3
N
1
KD-60
iii
OH
O
KD-114
H3C
O
N
N
N
N
R1 =
R2 =
R2 =
v
N
CH3
O
N
KD-142
KD-179
H3C
O
OH
iv
N
N
N
4
N
CH3
Br
5
O
N
R3 =
WZ-1
WZ-3
vi
H3C
O
N
N
N
N
O
N
Cl
F
H3C
O
O
N
N
CH3
N
WZ-4
WZ-5
O
R3
HN
N
6
Cl
CH3
NH
WZ-7
WZ-6
3
(i) Cl(CH2)3Br (ii) R2NH2 (iii) NH2(CH2)2OH (iv) R NCO
(v) PBr (vi) PhCH2NH2
3
Scheme 1. Chemical structures of the investigated compounds.
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Analgesic Activity of Xanthine Derivatives
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performed upon microwave irradiation under solvent free
conditions. Compound WZ-7 was isolated as hydrochloride.
The reaction of 4 and (un)substituted phenyl or cycloalkyl
isocyanates carried out in acetonitrile yielded the carbamates
WZ-1, WZ-3, WZ-4, WZ-5, and WZ-6.
ED50 value for WZ-7 is 31.4 mg/kg, for WZ-1 is 120.7 mg/kg,
whereasforWZ-6is150.7 mg/kg. ThecompoundsWZ-4andKD-
60 tested only at a dose of 100mg/kg statistically significantly
prolonged the nociceptive reaction time in mice. In comparison
to the reference compound which was morphine, none of the
xanthine derivatives had a stronger analgesic activity than the
reference compound (Tables 2 and 3).
Pharmacology
Radioligand receptor binding assays for adenosine
receptors
The writhing test
The affinity data for different adenosine receptor subtypes
are summarized in Table 1. The compound KD-179 (Ki ¼ 0.28
mM) showed the strongest affinity for A1 receptors, the
compounds KD-56 (Ki ¼ 0.32 mM), KD-114 (Ki ¼ 0.23 mM), and
KD-179 (Ki ¼ 0.30 mM) for A2A receptors, being not selective;
the compound KD-142 (Ki ¼ 0.59 mM) for A2B receptors, and
KD-179 (Ki ¼ 0.66 mM) for A3 receptors—both compounds
were not selective. Compounds WZ-1–WZ-7 with the excep-
tion of WZ-7 (showing weak affinity for A1 and A2ARs) were
deprived of adenosine receptors affinity.
The analgesic activity of the new compounds was also
evaluated in the writhing test. This test consists in the
intraperitoneal injection of a chemical irritant followed by
subsequent counting of “writhes” that is characteristic
contractions of abdominal muscles, accompanied by a hind
limb extensor motion [12]. Based on the available literature
data, this assay reveals significant activity of compounds
possessing anti-inflammatory properties, such as NSAIDs,
which would abolish the reflex stimulated by substances like
phenylbenzoquinone [12].
On the basis of the calculated ED50 values, it can be
established that the compound KD-142 (ED50 ¼ 9.0 mg/kg)
showed the strongest analgesic activity, and then in the
decreasing order of analgesic potency: WZ-5 (ED50 ¼ 10.2 mg/
kg), KD-60 (ED50 ¼ 11.3 mg/kg), WZ-4 (ED50 ¼ 12.1 mg/kg), WZ-
3 (ED50 ¼ 12.5 mg/kg), WZ-1 (ED50 ¼ 13.5 mg/kg), KD-179 (ED50
¼ 16.8 mg/kg), KD-56 (ED50 ¼ 21.4 mg/kg), KD-114 (ED50 ¼ 27.2
mg/kg), WZ-6 (ED50 ¼ 42.1 mg/kg), WZ-7 (ED50 ¼ 80.9 mg/kg).
In comparison to the reference compound in this assay which
was acetylsalicylic acid, except for WZ-7, the other compounds
showed a stronger or similar (compound WZ-6) analgesic
effect (Tables 4 and 5).
The hot-plate test
All annelated xanthine derivatives were examined in the hot
plate test, but only some of them showed analgesic activity
(Tables 2 and 3). The paws of mice are very sensitive to heat at
temperatures which are not damaging to the skin. The
reaction time is prolonged after administration of centrally
acting analgesics, whereas peripheral analgesics (COX-inhib-
itors) generally do not affect the responses [12].
The hot-plate test as a method of evaluating analgesic
activity of substances showed that five derivatives of xanthine
(WZ-1, WZ-4, WZ-6, WZ-7, and KD-60) in a dose-dependent
manner prolonged the nociceptive reaction time in mice. The
results were statistically significant. The highest analgesic
activity was observed for the compound WZ-7. The calculated
The results obtained from the analgesic tests showed that
new compounds significantly increased the pain threshold on
the hot plate and inhibited the writhing response induced by
Table 1. Adenosine receptor affinities of the investigated annelated xanthine derivatives.
Rat A2A Ki
[mM] ꢀ SEM (%
inhibition at
Human A2B Ki
[mM] ꢀ SEM (%
inhibition at
Human A3 Ki
[mM] ꢀ SEM (%
inhibition at
Rat A1 Ki [mM] ꢀ SEM
(% inhibition at
Compound
indicated conc.)
indicated conc.)
indicated conc.)
indicated conc.)
WZ-1
WZ-3
WZ-4
WZ-5
>10 (3%)
>10 (10%)
>10 (12%)
>10 (24%)
>10 (12)
7.55 ꢀ 2.35
>25 (32%)
>25 (22%)
ꢁ25 (46%)
0.620 ꢀ 0.220
0.28 ꢀ 0.02
41.0
>10 (35%)
>10 (33%)
ꢁ10 (42%)
>10 (32%)
ꢁ10 (48%)
20.0 ꢀ 2.0
>10 (6%)
>10 (4%)
>10 (15%)
>10 (6%)
>10 (19%)
>10 (8%)
>1 (0%)
>10 (3%)
>10 (0%)
>10 (13%)
>10 (16%)
>1 (0%)
>10 (20%)
>10 (0%)
>10 (9%)
3.66 ꢀ 0.77
0.66 ꢀ 0.02
13.3
WZ-6
WZ-7a)
KD-56
KD-60
KD-114
KD-142
KD-179b)
Caffeinec)
0.321 ꢀ 0.028 ca. 10 (57%)
>25 (37%)
13.4 ꢀ 4.7
7.20 ꢀ 0.60
0.590 ꢀ 0.060
1.32 ꢀ 0.23
10.4
0.230 ꢀ 0.080
0.860 ꢀ 0.090
0.30 ꢀ 0.09
43.0
a) Ref. [8].
b) Ref. [9].
c) Ref. [3].
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Table 2. The influence of the test compounds on the pain reaction in the hot-plate test in mice.
Time of reaction to
Dose
pain stimulus
ED50
Compound
(mg/kg)
(s) ꢀ SEM
% Prolongation
(mg/kg)
Control
WZ-1
–
15.3 ꢀ 1.8
20.3 ꢀ 4.3
21.7 ꢀ 3.6
24.0 ꢀ 2.7a)
16.5 ꢀ 4.1
12.0 ꢀ 2.7
13.4 ꢀ 2.9
29.3 ꢀ 6.4b)
12.3 ꢀ 4.8
14.9 ꢀ 3.3
15.9 ꢀ 2.7
17.3 ꢀ 4.3
20.3 ꢀ 5.1
24.8 ꢀ 4.3a)
21.1 ꢀ 7.1
26.5 ꢀ 6.4b)
40.1 ꢀ 5.2c)
–
32.6
41.8
56.8
7.8
–
–
50
120.7
100
200
50
100
50
100
50
100
200
50
100
200
25
WZ-3
WZ-4
WZ-5
–
–
–
–
91.5
–
–
–
WZ-6
WZ-7
13.0
32.6
62.0
37.9
73.2
162.0
150.7
31.4
50
100
a) P < 0.05.
b) P < 0.01.
c) P < 0.001.
phenylbenzoquinone, which suggests that the new com-
pounds possess both centrally and peripherally mediated
analgesic properties [12]. The central analgesic action may be
mediated via the inhibition of central pain receptors, while
the peripheral analgesic effect may be mediated through the
inhibition of cyclooxygenase and/or lipoxygenase and other
mediators. This hypothesis is based on the previous reports
indicating that the hot-plate test and acetic acid writhing
method are useful techniques for the evaluation of centrally
and peripherally acting analgesic drugs, respectively [12].
The role of adenosine receptors in nociception is complex
and may involve different mechanisms in the central nervous
Table 3. The influence of the test compounds on the pain reaction in the hot-plate test in mice.
Time of reaction to
Dose
pain stimulus
ED50
Compound
(mg/kg)
(s) ꢀ SEM
% Prolongation
(mg/kg)
Control
KD-56
–
50
100
50
100
50
100
50
100
50
100
–
15.3 ꢀ 1.8
17.3 ꢀ 1.4
8.2 ꢀ 2.1
–
–
–
13.0
–
KD-60
11.4 ꢀ 2.6
30.7 ꢀ 3.5a)
13.2 ꢀ 2.4
8.8 ꢀ 1.7
–
–
–
–
–
100.6
–
KD-114
KD-142
KD-179
–
8.8 ꢀ 0.7
–
11.7 ꢀ 1.7
11.3 ꢀ 0.8
10.3 ꢀ 0.9
18.4 ꢀ 1.0
30.6 ꢀ 3.9b)
29.9 ꢀ 6.0c)
19.4 ꢀ 2.1
–
–
–
Control
Morphine
–
–
6
3
1
66.3
62.5
5.4
3.3
a) P < 0.001.
b) P < 0.01.
c) P < 0.05.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Analgesic Activity of Xanthine Derivatives
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Table 4. The influence of the test compounds on the pain reaction in the writhing test in mice.
Dose
(mg/kg)
Mean number of
writhes ꢀ SEM
ED50
(mg/kg)
Compound
% Inhibition
Control
WZ-1
–
26.0 ꢀ 1.8
14.2 ꢀ 2.4
4.0 ꢀ 1.3a)
0.0 ꢀ 0a)
–
–
12.5
25
50
100
12.5
25
45.3
84.6
100
13.5 (7.7–23.9)
0.0 ꢀ 0a)
100
WZ-3
WZ-4
WZ-5
12.3 ꢀ 2.5b)
9.7 ꢀ 1.6c)
4.2 ꢀ 1.8a)
2.2 ꢀ 0.9a)
13.1 ꢀ 2.7
6.0 ꢀ 2.6c)
2.7 ꢀ 0.9a)
1.7 ꢀ 0.3a)
12.6 ꢀ 3.1
7.5 ꢀ 2.8c)
6.2 ꢀ 1.6c)
2.2 ꢀ 0.5a)
18.7 ꢀ 3.4
12.5 ꢀ 2.7b)
3.4 ꢀ 0.8a)
21.5 ꢀ 3.9
9.0 ꢀ 2.8b)
4.8 ꢀ 2.1c)
52.6
62.6
83.8
91.5
49.6
76.9
89.6
93.4
51.5
71.1
76.1
91.5
28.0
51.9
86.9
17.3
65.3
81.5
12.5 (6.0–25.9)
12.1 (7.1–20.5)
10.2 (3.7–27.5)
50
100
12.5
25
50
100
12.5
25
50
100
25
50
100
50
100
WZ-6
WZ-7
42.1 (28.9–61.3)
80.9 (67.0–97.7)
200
a) P < 0.001.
b) P < 0.05.
c) P < 0.01.
system and in peripheral tissues [5]. In connection with
literature data about an important role of adenosine
receptors, especially A2B in the process of perception of
pain, we attempted to synthesize compounds with antago-
nistic action at these receptors [3]. The novel tricyclic
derivatives of xanthine, which were investigated for
analgesic activity in the present study, are an example of
these compounds.
receptors activation and blockade or stimulation of A2A
receptors.
However, elucidation of the mechanism of analgesic action
of other compounds that either did not bind to adenosine
receptors or did not have a marked affinity requires further
investigation.
Locomotor activity test
On the other hand, the xanthine derivatives (except for the
WZ-5, WZ-6, and WZ-7) showed a marked affinity for
adenosine receptors (Table 1). The compound KD-142
exhibiting the strongest analgesic effect in the writhing test,
showed an affinity for all types of adenosine A1 receptors
(Ki ¼ 0.62 mM), A2A (Ki ¼ 0.86 mM), A2B (Ki ¼ 0.59 mM), A3
(Ki ¼ 3.66 mM). Therefore, the analgesic effect of the test
compound may be due to effects on these receptors [3].
However, compound KD-179, which had a stronger analgesic
effect than aspirin, showed a very strong affinity for A1
receptors (Ki ¼ 0.28 mM) and A2A receptors (Ki ¼ 0.30 mM). The
compounds KD-56 and KD-114, which were also stronger than
aspirin, tightly bound to the A2A receptors (Ki ¼ 0.32 mM,
Ki ¼ 0.23 mM, respectively). These results support a key role for
the adenosine A2A receptor (perhaps also A1) in peripheral
nociceptive pathways. There is a possibility that the analgesic
effect of the tested xanthine derivatives may result from A1
The effect of tested compounds on locomotor activity was
determined in order to exclude false positive results in the
assessment of analgesic activity. Investigations of the effects
of the compounds on the spontaneous motor activity in mice
showed that in comparison with the control group, the
compounds administered at the doses 12.5–100 mg/kg did not
influence on their analgesic effect in the writhing test (Tables
6 and 7). The exceptions are the two compounds, WZ-4 and
KD-60, which at an analgesic dose of 100 mg/kg, statistically
significantly decreased locomotor activity by 58.4 and by
55.4%, respectively. Taking this into account, it can be
concluded that the analgesic effect in part may be due to
reduced mobility. On the other hand, these compounds
showed significant analgesic effect in this test, at the doses 25
and 50 mg/kg, which did not influence in a statistically
significant way the spontaneous locomotor activity of mice.
We found that a high dose of the analgesic compound WZ-7
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Table 5. The influence of the test compounds on the pain reaction in the writhing test in mice.
Dose
(mg/kg)
Mean number of
writhes ꢀ SEM
ED50
(mg/kg)
Compound
% Inhibition
Control
KD-56
–
26.0 ꢀ 1.8
12.5 ꢀ 3.2a)
1.5 ꢀ 0.6b)
0.5 ꢀ 0.1b)
13.2 ꢀ 4.6
4.7 ꢀ 1.9c)
3.0 ꢀ 1.2b)
3.2 ꢀ 0.8b)
15.5 ꢀ 3.1
3.9 ꢀ 2.4b)
1.7 ꢀ 0.5b)
11.7þ4.5a)
7.0 ꢀ 2.6c)
5.2 ꢀ 1.0b)
2.5 ꢀ 0.6b)
18.5 ꢀ 2.7
5.6 ꢀ 2.1c)
1.2 ꢀ 0.7b)
0.5 ꢀ 0.4b)
11.2 ꢀ 2.1a)
8.5 ꢀ 1.3c)
3.2 ꢀ 1.2b)
24.3 ꢀ 1.9
20.5 ꢀ 2.5
1.5 ꢀ 0.1b)
–
–
25
50
100
12.5
25
50
100
25
51.9
94.2
98.0
49.2
81.9
88.4
87.6
40.3
85.0
93.4
55.0
73.0
80.0
90.3
28.8
78.4
95.3
98.0
56.9
67.3
87.6
6.5
21.4 (15.3–20.9)
KD-60
11.3 (6.5–19.5)
KD-114
KD-142
27.2 (18.6–39.9)
9.0 (3.5–22.9)
50
100
12.5
25
50
100
12.5
25
50
100
30
50
100
25
KD-179
16.8 (12.5–22.7)
Acetylsalicylic acid
Caffeine
39.1 (29.1–48.4)
63.5 (49.8–86.7)
50
100
23.9
94.2
a) P < 0.05.
b) P < 0.001.
c) P < 0.01.
(200 mg/kg) statistically significantly decreased the number of
light-beam crossings, as compared to the methylcellulose-
treated animals. Taking this into consideration, it seems likely
that its analgesic effect at this dose in the writhing test, in
part, may be due to the effect on the locomotor activity. We
did not observe any significant locomotor effects with the
antinociceptive compound WZ-7 at an analgesic dose of
100 mg/kg.
The compounds which showed significant analgesic activity
in the hot-plate test: WZ-1 (200 mg/kg), WZ-4 (100 mg/kg),
WZ-6 (200 mg/kg), WZ-7 (50, 100 mg/kg), and KD-60 (100 mg/
kg) at the same doses, statistically significantly decreased
locomotor activity. Taking this into account, probably the
analgesic effect of these compounds in the hot-plate test may
be wholly or partly due to the influence on locomotor activity.
Only compound WZ-7 did not alter locomotor activity at
100 mg/kg, although animals showed significantly decreased
hot-plate response latencies.
WZ-4 (100 mg/kg), WZ-6 (200 mg/kg), WZ-7 (200 mg/kg), and
KD-60 (100 mg/kg) cannot easily be accounted for their
locomotor effects.
Toxicity
Antiproliferative assay
In vitro toxicity screening of discovery compounds is an
important area of drug discovery and include hERG block
assays, mutagenicity/genotoxicity, teratogenicity, cytotoxici-
ty, reactivity screens, selectivity screens, and drug–drug
interactions which may cause toxic effects. Nowadays, the
in vitro evaluation of toxicity of the new compounds is
conducted at early discovery phases in parallel with the study
on their efficacy [13]. During this work, the potential toxicity
effect of the selected compounds was preliminary.
The antiproliferative effects of the selected compounds
(KD-60, KD-114, and KD-179; WZ-1, WZ-4, and WZ-7) as well as
the reference drug doxorubicin (DX) were determined against
HEK-293 cell line by using colorimetric EZ4U method [14]. As
seen in Fig. 1, the incubation of cells in the presence of
compounds WZ-1, WZ-4, and WZ-7 for 48 h showed no
antiproliferative effect, even in very high concentrations. No
effects were also observed for KD-60 and KD-114 (Fig. 2). The
In summary, no locomotor effects were observed with WZ-
3, WZ-5, KD-56, KD-114, KD-142, and KD-179, where strong
analgesia was observed. As for the other compounds, this
effect only at the highest dose. Thus, the analgesia observed
after administration of these compounds: WZ-1 (200 mg/kg),
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Table 6. The influence of the test compounds on the locomotor activity.
Dose
(mg/kg)
Mean number of
movements ꢀ SEM
Locomotor activity decrease
(%)
Compound
Control
WZ-1
–
432.0 ꢀ 58.6
387.0 ꢀ 35.3
320.0 ꢀ 39.4
249.4 ꢀ 48.3
160.5 ꢀ 49.0a)
430.1 ꢀ 39.6
385.7 ꢀ 32.9
317.6 ꢀ 45.0
238.2 ꢀ 41.7
398.7 ꢀ 46.6
312.0 ꢀ 54.1
179.4 ꢀ 33.2b)
407.5 ꢀ 51.4
328.7 ꢀ 45.1
231.2 ꢀ 37.8
319.5 ꢀ 27.5
243.9 ꢀ 19.5
193.5 ꢀ 22.6b)
309.5 ꢀ 32.9
256.0 ꢀ 21.8
187.5 ꢀ 25.1b)
–
25
50
100
200
12.5
25
50
100
25
50
100
25
50
100
50
100
200
50
100
200
10.4
25.9
42.2
62.8
–
WZ-3
10.7
26.4
44.8
7.7
27.7
58.4
5.6
24.0
46.4
26.0
43.5
55.2
28.3
40.7
56.5
WZ-4
WZ-5
WZ-6
WZ-7
a) P < 0.01.
b) P < 0.05.
antiproliferative effect was observed only for compound KD-
179 with calculated IC50 value of 39.54 mM.
structures are responsible for the cognitive role of pain,
including its realization and affective reactions related to pain
(including anxiety, irritation, and aggression). Pain treatment
may involve both psychological and pharmacological
approaches. In our search for new more efficacious analgesics,
we identified several derivatives of xanthines that display
antinociceptive activity in two basic animal models of acute
pain, i.e., the hot-plate test and the writhing test.
The preliminary tests performed in the present study
indicated that new compounds demonstrated analgesic effect
first of all in the writhing test. Analgesic effect of some
compounds is probably related to their agonism/or antago-
nism or partial agonist at adenosine receptors. Preliminary
screening tests documented their potent central and/or
peripheral analgesic activity and affinity for different
adenosine receptor subtypes. Using the spontaneous locomo-
tor activity test, we showed that the results presented here
could not be falsely attributed to sedative influence of
xanthines. Explanation of the mechanism of action of some
derivatives which did not bind to adenosine receptors or did
not have a marked affinity, requires further investigations.
Drug–drug interactions
To predict the potential drug–drug interactions, influence of
chosen compounds on CYP3A4 cytochrome activity was also
examined. The effects of selected compounds (KD-60, KD-114,
and KD-179; WZ-1, WZ-4, and WZ-7) on CYP3A4 activity were
tested by using luminescent CYP3A4 P450-GloTM assay [15].
The results were compared to the reference compound,
strong CYP3A4 inhibitor, ketoconazole. The obtained data
showed that all examined compounds modulate cytochrome
activity. As seen in Fig. 3, the compounds possessed dose-
dependent and strong (KD-60 and KD-179) or weak (KD-114)
CYP3A4 inhibitor activity. Moreover, from compounds WZ-1,
WZ-4, and WZ-7, only WZ-4 did not inhibit significantly the
activity of CYP3A4 (Fig. 4). Relative to the calculated IC50 value
for ketoconazole (IC50 ¼ 0.14 mM), KD-60 showed similar, very
strong, and comparable inhibition activity with calculated
IC50 ¼ 0.21 mM. Additionally, KD-179 and WZ-7, with calculat-
ed IC50 values 1.79 and 1.01 mM, respectively, may be
considered as a strong CYP3A4 inhibitors.
Experimental
Conclusions
Chemistry
Pain perception, the endpoint of the whole nociception
process, occurs in the cortex and the limbic system. These
Melting points were measured in open capillaries on a Mel-
Temp. II apparatus (LD Inc., USA) and were not corrected. The
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M. Zygmunt et al.
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Table 7. The influence of the test compounds on locomotor activity.
Dose
(mg/kg)
Mean number of
movements ꢀ SEM
Locomotor activity decrease
(%)
Compound
Control
KD-56
–
432.0 ꢀ 58.6
389.1 ꢀ 51.3
357.5 ꢀ 42.8
271.8 ꢀ 39.8
364.5 ꢀ 24.8
294.2 ꢀ 39.4
192.4.2 ꢀ 36.2a)
345.9 ꢀ 43.5
264.7 ꢀ 51.8
435.2 ꢀ 41.9
375.0 ꢀ 39.7
308.2 ꢀ 47.1
254.5 ꢀ 42.0
396.6 ꢀ 31.0
337.2 ꢀ 42.9
248.5 ꢀ 37.9
–
25
50
100
25
50
100
50
100
12.5
25
50
100
25
9.9
17.2
37.0
15.7
31.8
55.4
19.9
38.7
–
KD-60
KD-114
KD-142
13.1
28.6
41.0
8.1
21.9
42.4
KD-179
50
100
a) P < 0.01.
purity of the synthesized compounds was confirmed by TLC
performed on Merck silica gel 60 GF254 aluminum sheets
(Merck, Darmstadt, Germany), using the following develop-
ing system: CHCl3/AcOEt (1:1, v/v). Spots were detected by
their absorption under UV light (l ¼ 254 nm). Column
chromatography was performed on Merck silica gel 60 (70–
230 mesh) using CH2Cl2/AcOEt 1:1, v/v as an eluent. Infrared
spectra were measured with an FT IR 410 spectrometer (Jasco)
in KBr pellets and are reported in cmꢂ1. 1H NMR spectra were
obtained on a Varian Mercury spectrometer (Varian Inc., Palo
Alto, CA, USA) in DMSO-d6, operating at 300 MHz. Chemical
shifts are reported in d values (ppm) relative to undeuterated
solvent signals. The J values are expressed in Hertz (Hz). Signal
multiplicities are represented by the following abbreviations:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
cycl (cyclohexyl). 13H NMR spectra were acquired on a Varian
Mercury spectrometer (Varian Inc.) in DMSO-d6, operating at
75 MHz. Mass spectrometry analyses: samples were prepared
in a mixture of acetonitrile/water (50:50, v/v). The LC/MS were
carried out on a system consisting of a Waters Acquity UPLC,
coupled to a Waters TQD mass spectrometer (electrospray
ionization mode ESI-tandem quadrupole). Retention times
(tR) are given in minutes. The UPLC/MS purity of all final
compounds was determined (%). Elemental analyses for C, H,
and N were carried out by a micro method using the
elemental Vario-EL III Elemental Analyser (Hanau, Germany)
and were found within ꢀ0.4% of the theoretical values. All
tested compounds possessed a purity of not less than 95%.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4,7,8-
hexahydropyrimido[1,2-a]purin-9(6H)-yl)ethyl
phenylcarbamate (WZ-1)
White powder, m.p: 234–236°C (ethanol); TLC Rf ¼ 0.63; yield
77.5%.; C19H22N6O4 (MW 398.38). 1H NMR (300 MHz, DMSO
d6) d: 2.05 (q, J ¼ 5.56 Hz, 2H, CH2–CH2–CH2), 3.10 (s, 3H,
N1–CH3), 3.17 (s, 3H, N3–CH3), 3.44 (t, J ¼ 5.50 Hz, 2H, 8–CH2),
3.74 (t, J ¼ 5.23 Hz, 2H, N9–CH2), 4.03 (t, J ¼ 5.91 Hz, 2H, 6–CH2),
4.29 (t, J ¼ 5.23 Hz, 2H, CH2–O), 6.93 (t, J ¼ 7,43 Hz,1H, Ar40–H),
Figure 1. The antiproliferative effect of WZ-1, WZ-4, WZ-7, and
DX (reference) against HEK-293 cell line.
Figure 2. The antiproliferative effect of KD-60, KD-114, KD-179,
and DX (reference) against HEK-293 cell line.
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Analgesic Activity of Xanthine Derivatives
Archiv der Pharmazie
NMR (75 MHz; DMSO d6): 21.22 (CH2); 27.57 (N3CH3); 29.55
(N1CH3); 41.82 (N9CH2); 44.82 (N9CH2); 48.59 (N5CH2); 62.03
0
(OCH2); 102.29 (C10 ); 105.00 (ArCH); 116.83 (ArCH); 117.78
(ArCH); 122.42 (ArCH); 130.75 (ArCH); 133.55 (ArCH); 141.09
–
–
0
0
(ArCH); 148.51(C ); 151.33 (C O); 151.91 (C O); 153.01 (C );
–
–
9
4
–
153.66 (OC O). IR n: 3289 (N–H), 3083 (C–H), 2943 (–CH –),
–
2
–
–
1733 (C O), 1696 (C O), 1224, 1064, 778 (C–H), 748 (–CH –);
–
–
2
UV (lmax) ¼ 300 nm (loge ¼ 4.6); LC/MSꢀ: purity 96.47%, tR
¼ 5.12; (ESI) m/z [MþH]þ: 433.33; Anal. calcd. for
C19H21N6O4Cl: C, 52.72; H, 4.89; N, 19.42. Found: C, 52.73;
H, 4.77; N, 19.40.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4,7,8-
hexahydropyrimido[1,2-a]purin-9(6H)-yl)ethyl 4-
fluorophenylcarbamate (WZ-4)
Figure 3. The effect of KD-60, KD-114, KD-179, and ketocona-
zole on CYP3A4 activity.
White crystals, m.p: 262–266°C (ethanol); TLC: Rf ¼ 0.60; yield
66.9%. C19H21N6O4F (MW ¼ 416.41). 1H NMR (ppm) d: 2.03–
2.06 (m, 2H, CH2–CH2–CH2), 3.11 (s, 3H, N1–CH3), 3.18 (s, 3H,
N3–CH3), 3.43 (t, J ¼ 5.37 Hz, 2H, 8–CH2), 3.73 (t, J ¼ 5.01 Hz, 2H,
N9–CH2), 4.03 (t, J ¼ 5.78 Hz, 2H, 6–CH2), 4.28 (t, J ¼ 5.09 Hz, 2H,
CH2–O), 7.05 (t, J ¼ 8.80 Hz, 2H, Ar20-H þ Ar60–H), 7.37 (br.s, 2H,
Ar30-H þ Ar50–H), 9.58 (s, 1H, NH). 13C NMR: 21.21 (CH2); 27.55
(N3CH3); 29.58 (N1CH3); 41.82 (N9CH2); 44.88 (N9CH2); 48.65
7.20 (t, J ¼ 7.98 Hz, 2H, Ar30–H þ Ar50–H), 7.37 (d, J ¼ 7.43 Hz,
2H, Ar20–H þ Ar60-H), 9.54 (s, 1H, NH); 13C NMR (75 MHz; DMSO
d6) d: 21.21 (CH2); 27.57 (N3CH3); 29.58 (N1CH3); 41.81 (N9CH2);
0
44.93 (N9CH2); 48.63 (N5CH2); 61.79 (OCH2); 102.29 (C10
)
(ArCH); 118.46 (ArCH); 122.75 (ArCH); 129.06 (ArCH); 139.47
–
–
0
0
(ArCH); 148.54 (C ); 151.34 (C O); 151.85(C O); 153.01 (C );
–
–
9
4
–
0
153.75 (OC O); IR n: 3289 (N–H), 3076 (C–H), 2949 (–CH –),
(N5CH2); 61.84 (OCH2); 102.29 (C10 ); 115.46 (ArCH); 115.76
–
2
–
–
–
0
1727 (C O), 1698 (C O), 1222, 1068, 849 (C–H), 759 (–CH –);
(ArCH); 119.95 (ArCH); 135.82 (ArCH); 148.53 (C ); 151.34 (C
–
–
–
2
9
UV (lmax) ¼ 300 nm (loge ¼ 5.2); LC/MSꢀ: purity 97.48%, tR
¼ 4.43; (ESI) m/z [MþH]þ: 399.36. Anal. calcd. for C19H22N6O4:
C, 57.27; H, 5.57; N, 21.10; Found: C, 57.17; H, 5.59; N, 21.00.
O); 151.88 (C O); 153.01 (C ); 153.84 (–OC O); 156.41; 159.56.
IR n: 3300 (N–H), 3071 (C–H), 2939 (–CH –), 1731 (C O), 1695
–
–
–
–
0
4
–
–
2
–
–
–
–
(C O), 1646, 1218, 1074, 841 (C H), 749 (–CH –); UV (l
)
2
max
¼ 300 nm (loge ¼ 4.8); LC/MSꢀ: purity 97.21%. tR ¼ 4.61; (ESI)
m/z [MþH]þ: 417.31; Anal. calcd. for C19H21N6O4F: C, 54.80; H,
5.08; N, 20.18. Found: C, 54.74; H, 4.96; N, 20.18.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4,7,8-
hexahydropyrimido[1,2-a]purin-9(6H)-yl)ethyl 3-
chlorophenylcarbamate (WZ-3)
White solid, m.p: 244–252°C (ethanol); TLC: Rf ¼ 0.64; yield
69.6%; C19H21N6O4Cl (MW 432.87). 1H NMR (ppm) d: 2.03–2.07
(m, 2H, CH2–CH2–CH2), 3.11 (s, 3H, N1–CH3), 3.17 (s, 6H,
N3–CH3), 3.43 (t, J ¼ 5.36 Hz, 2H, 8–CH2), 3.75 (t, J ¼ 4.96 Hz, 2H,
N9–CH2), 4.03 (t, J ¼ 5.78 Hz, 2H, 6–CH2), 4.31 (t, J ¼ 5.01 Hz, 2H,
CH2–O), 6.97–6.99 (m, 1H, Ar50–H), 7.20–7.25 (m, 2H,
Ar40–H þ Ar60–H), 7.49 (s, 1H, Ar20–H), 9.74 (s, 1H, NH). 13C
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4,7,8-
hexahydropyrimido[1,2-a]purin-9(6H)-yl)ethyl 4-
chlorophenylcarbamate (WZ-5)
White crystals, m.p: 262–266°C; (ethanol); TLC: Rf ¼ 0.62; yield
66.9%. C19H21N6O4Cl MW ¼ 432.87. 1H NMR (ppm) d: 2.03–2.07
(m, 2H, CH2–CH2–CH2), 3.11 (s, 3H, N1–CH3), 3.17 (s, 3H, N3–CH3),
3.42–3.45(m,2H,8–CH2),3.74(t,J ¼ 4.98 Hz,2H,N9–CH2),4.03(t,
J ¼ 5.78Hz, 2H, 6–CH2), 4.29 (t, J ¼ 4.95Hz, 2H, CH2–O), 7.25 (t,
J ¼ 8.80Hz, 2H, Ar30–H þ Ar50–H), 7.38 (d, J ¼ 7.43Hz, 2H,
Ar20–H þ Ar60–H), 9.68 (s, 1H, NH). 13C NMR: 21.21 (CH2); 27.56
(N3CH3); 29.58 (N1CH3); 41.82 (N9CH2); 44.83 (N9CH2); 48.61
0
(N5CH2); 61.92 (OCH2); 102.29 (C10 ); 119.84 (ArCH); 126.36
–
0
(ArCH);128.94(ArCH);138.49(ArCH);148.51(C );151.33(C O);
–
9
–
–
0
151.90(C O);153.01(C );153.72(–OC O).IRn:3299(N–H),2939
–
–
4
–
–
(–CH –), 1735 (C O), 1698 (C O), 1638, 1219, 1088, 750 (–CH –);
–
–
2
2
UV (lmax) ¼ 300 nm (loge ¼ 4.6); LC/MSꢀ: purity 100.0%; tR
¼ 5.16; (ESI) m/z [MþH]þ: 433.33; Anal. calcd. for C19H21N6O4Cl:
C, 52.72; H, 4.89; N, 19.42. Found: C, 52.71; H, 4.75; N, 19.38.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4,7,8-
hexahydropyrimido[1,2-a]purin-9(6H)-yl)ethyl
cyclohexylcarbamate (WZ-6)
White crystals, m.p: 262–266°C; (ethanol); TLC: Rf ¼ 0.62; yield
Figure 4. The effect of WZ-1, WZ-4, WZ-7, and ketoconazole on
CYP3A4 activity.
12.36%. C19H28N6O4: MW ¼ 404.46. 1H NMR (ppm) d: 0.96–
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1.24 (m, 5H, cyclohexyl), 1.48–1.99 (m, 6H, cyclohexyl), 2.00–
2.06 (m, 2H, CH2–CH2–CH2), 3.13 (s, 3H, N1–CH3), 3.31 (s, 3H,
N3–CH3), 3.39 (t, J ¼ 5.39 Hz, 2H, 8–CH2), 3.63 (t, J ¼ 5.23 Hz, 2H,
N9–CH2), 4.02 (t, J ¼ 5.91 Hz, 2H, 6–CH2), 4.12 (t, J ¼ 5.28 Hz, 2H,
CH2–O), 7.03 (d, J ¼ 7.70 Hz, 1H, NH). 13C NMR: 21.18 (CH2);
25.03 (cyclC); 25.58 (cyclC); 27.60 (N3CH3); 29.76 (N1CH3); 33.03
(cyclC); 33.80 (cyclC); 41.79 (N9CH2); 45.15 (N9CH2); 48.80
the investigated compound vs. the control group) from the
writhing and hot-plate tests. The difference between the
means was statistically significant if P < 0.05.
Adenosine radioligand binding assay
The affinity of the examined compounds for adenosine
receptors was determined in cooperation with the University
of Bonn.
0
0
(N5CH2); 49.86 (cyclC); 61.17 (OCH2); 102.27 (C10 ); 148.64 (C9 );
–
–
–
0
151.43 (C O); 151.72 (–C O); 153.01 (C ); 155.59 (–OC O). IR
–
–
–
4
–
–
n: 3313 (N–H), 2933 (–CH –), 1708 (C O), 1689 (C O), 1652,
The hot-plate test
–
–
2
1225, 1044, 749 (–CH2–); UV (lmax) ¼ 300 nm (loge ¼ 4.6); LC/
MSꢀ: purity 98.62%; tR ¼ 4.86; (ESI) m/z [MþH]þ: 405.41; Anal.
calcd. for C19H28N6O4: C, 56.42; H, 6.98; N, 20.78. Found: C,
57.35; H, 7.09; N, 20.53.
In the hot-plate test, mice are treated ip either with the test
compound or the vehicle 30 min before placing the animal on
a hot-plate apparatus (Hot Plate 2A Type Omega) with the
temperature set at 55–56°C. The time until the animal licks its
hind paws or jumps is recorded by means of a stopwatch [16].
9-(2-Benzylamino)ethyl)-1,3-dimethyl-6,7,8,9-
tetrahydropyrimido[1,2-a]purine-2,4(1H,3H)-dione
hydrochloride (WZ-7)
The writhing syndrome test
Mice are treated with 0.25 mL of 0.02% phenylbenzoquinone
solution 30 min after ip administration of the investigated
compound or the vehicle. Then the mice are placed
individually in glass beakers and 5 min are allowed to elapse.
After that period of time, a 10-min observation is conducted
for each animal and the number of characteristic writhes is
counted. The analgesic effect of the tested substances was
determined by a decrease in the number of the observed
writhes [17]. The analgesic effect of individual doses was
expressed in percent. The ED50 values and their confidence
limits were estimated by the method of Litchfield and
Wilcoxon [18].
White crystals, m.p: 280–285°C; (ethanol); TLC: Rf ¼ 0.30; yield
22.45%; C19H24N6O2: MW ¼ 368.43. 1H NMR (ppm) d: 2.06–
2.10 (m, 2H, CH2–CH2–CH2), 3.14 (s, 3H, N1–CH3), 3.29 (s, 3H,
N3–CH3), 3.14–3.21 (m, 2H, CH2–NH–), 3.34 (t, 2H, 8–CH2), 3.73
(t, J ¼ 6.05 Hz, 2H, N9–CH2), 4.03 (t, J ¼ 5.91 Hz, 2H, –NH–CH2),
4.19 (t, J ¼ 5.64 Hz, 2H, 8–CH2), 7.38–7.43 (m, 3H,
Ar30–H þ Ar40–H þ Ar50–H), 7.54–7.56 (m, 2H, Ar20–H þ Ar60–
H), 9.48 (s, 2H, NH2þ). 13C NMR: 21.02 (CH2); 27.67 (N3CH3);
29.80 (N1CH3); 41.72 (N9CH2); 44.41 (N9CH2); 45.05 (NCH2);
0
45.80 (NCH2); 50.17 (N5CH2); 102.46 (C10 ); 129.07 (ArCH);
0
129.31 (ArCH); 130.40 (ArCH); 132.52 (ArCH); 148.34 (C9 );
–
–
0
151.39 (C O); 151.76 (C O); 153.13 (C ). IR n: 2950 (–CH –),
–
–
4
2
–
1699 (C O), 1654, 881 (C–H), 748 (–CH –); UV (lmax) ¼ 300 nm
Locomotor activity test
–
2
(loge ¼ 4.7); LC/MSꢀ: purity 98.03%. tR ¼ 2.89; (ESI) m/z
[MþH]þ: 369.31; Anal. calcd. for C19H24N6O2 ꢃ HCl ꢃ 0.5 H2O
(MW 413.91): C, 55.13; H, 6.33; N, 20.30. Found: C, 55.12; H,
6.17; N, 20.37.
Tested compounds and the vehicle (methylcellulose) were
administered ip 30 min before the assay. The effects of
various doses of the investigated compounds administered
by the ip route were recorded in the photoresistor actimeters
(Multi-Serv, Lublin, Poland, 30 cm in diameter, illuminated by
two light beams) connected to the counter for the recording
of light-beam interruptions. The mice were placed individu-
ally in the actimeters and the number of light-beam crossings
was counted during the 30-min session. Each experimental
group consisted of six mice. The data obtained in the 30th
minute of the observation period were used for the
statistical analysis.
Pharmacology
Animals
The experiment was carried out on male Albino Swiss mice
weighing 18–30 g. The animals were housed in constant
temperature facilities exposed to 12:12 light-dark cycle and
maintained on a standard pellet diet; tap water was given ad
libitum. Control and experimental groups consisted of six
animals each. The investigated compounds were adminis-
tered intraperitoneally (ip) in the form of a suspension in 0.5%
methylcellulose. Control animals received the equivalent
volume of solvent.
All procedures were conducted according to guidelines of
ICLAS (International Council on Laboratory Animals Science)
and were approved by The Local Ethics Committee for Animal
Experimentation.
Antiproliferative assay
Cell line: Human embryonic kidney HEK-293 cell line was
cultured in Dulbecco’s modified Eagle medium (DMEM)
(Gibco) containing 10% fetal bovine serum, 100 mg/mL
streptomycin, and 100 U/mL penicillin. Cells were cultured
at 37°C in an atmosphere 5% CO2. HEK-293 cell line (ATCC
€
CRL1573) was kindly donated by Prof. Dr. Christa Muller
(Pharmaceutical Institute, Pharmaceutical Chemistry I, Uni-
versity of Bonn).
Statistical analysis
The data are expressed as the mean ꢀ SEM (standard error of
the mean). Student’s t-test was used to compare the results
between two different groups of animals (the group given
In vitro antiproliferative assay: EZ4U non-radioactive cell
proliferation and cytotoxicity assay (Biomedica) was used to
define the antiproliferative activity of examined compounds.
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Analgesic Activity of Xanthine Derivatives
Archiv der Pharmazie
First, the cells were seeded in 96-well plate at a concentration
of 1.5 ꢃ 104 cells/well in 200 mL culture medium and cultured
for 24 h to reach 60% of confluence. Next, the stock solutions
of examined compounds in DMSO were diluted into fresh
growth medium (the maximal DMSO concentration did not
exceed 1%) and added into the microplates at the final
concentrations from 0.01 to 250 mM. After 48 h of incubation,
20 mL of EZ4U labeling mixture was added to each well and
the cells were incubated under the same conditions for 5 h.
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The luminescent CYP3A4 P450-GloTM assay was purchased
from Promega. The CYP3A4 inhibitor ketoconazole was
purchased from Sigma–Aldrich. The enzymatic reactions were
performed in white polystyrene, flat-bottom NuncTM Micro-
WellTM 96-well microplates (Thermo Scientific). The CYP3A4
P450-GloTM assays were performed according to the manu-
facturer’s procedure. The final concentrations of all examined
compounds were 0.025–25 mM. The effect of ketoconazole
was estimated as we described previously [19]. The lumines-
cence signal was measured with a microplate reader in
luminescence mode (PerkinElmer). For calculation, the total
luminescence, the average luminescence of the control
reaction containing inactive membranes was subtracted from
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luminescence of the reactions containing luciferin-free water
instead of the tested compound indicated the total (100%)
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This study was partly supported by National Science Center
granted on the basis of decision no. DEC-2012/04/M/NZ4/
00219.
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The authors have declared no conflicts of interest.
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