Preliminary finding on a new calcium channel entry blocker chemotype: 5,6-diamino-4-hydroxy-2-mercaptopyrimidine derivatives

Article abstract of DOI:10.1021/jm200414s

We report the preliminary in vitro characterization of a series of pyrimidines as a new chemotype that modulates cardiovascular parameters and relaxes ileum smooth muscle according to classical calcium entry blockers. Tested compounds showed an interestin

Full text of DOI:10.1021/jm200414s

BRIEF ARTICLE
pubs.acs.org/jmc
Preliminary Finding on a New Calcium Channel Entry Blocker
Chemotype: 5,6-Diamino-4-hydroxy-2-mercaptopyrimidine
Derivatives
||
Barbara Cosimelli,*,† Elda Severi,† Ettore Novellino,† Anna Cavaccini,‡ Mauro Cataldi,‡ Roberta Budriesi,*,§,
Matteo Micucci,§ Alberto Chiarini,§ and Pierfranco Ioan§,
||
†Dipartimento di Chimica Farmaceutica e Tossicologica, Universitꢀa degli Studi di Napoli “Federico II”, Via Montesano 49,
80131 Napoli, Italy
‡Divisione di Farmacologia, Dipartimento di Neuroscienze, Scuola di Medicina, Universitꢀa degli Studi di Napoli “Federico II”,
Via Pansini 5, 80131 Napoli, Italy
§Dipartimento di Scienze Farmaceutiche, Universitꢀa degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
S Supporting Information
b
ABSTRACT: We report the preliminary in vitro characterization of a series of pyrimidines as a new chemotype that modulates
cardiovascular parameters and relaxes ileum smooth muscle according to classical calcium entry blockers. Tested compounds
showed an interesting negative inotropic selectivity. In patch-clamp experiments they block L- over T-type calcium currents. Two
requisites seem essential for the activity: lipophilic substituents in positions 2 and 5 of the pyrimidine ring and the acetamidic
function in position 6.
’ INTRODUCTION
(Scheme 1), were synthesized. 2a was the starting compound,
whereas 2b and 2c were synthesized as its congeners. All other
compounds were derived from 2a, 2b, or 2c by further substitut-
ing the nitrogen atom in position 3 of the ring or the hydroxyl
function in position 4 and/or the amino group in position 6.
According to the substitutions made, compounds of the a, b, and
c series can be further classified into five additional subfamilies
designated as 3, 4, 5, 6, and 7. The synthesis of 2a has been
already reported elsewhere.11 Compounds 2b and 2c were
obtained by reaction of 5,6-diamino-4-hydroxy-2-mercaptopyr-
imidine (1) with 1-bromopropane and 1-bromo-2-methoxyethane,
respectively. Compounds 3aÀc and 4aÀc were obtained by
reaction of the respective compounds of subfamily 2 (2a, 2b,
and 2c) in acetonitrile with 1-bromoethane in the presence of
K2CO3. Compounds of the subfamilies 5, 6, and 7 are the N6-
acetylated derivatives of the members of the 2, 3 and 4
subfamilies, respectively. They were obtained by reaction of
the respective parent compounds (2aÀc, 3aÀc, and 4aÀc)
with acetic anhydride and concentrated H2SO4 as catalyst
(Scheme 1).
A substituted aminopyrimidine ring (Chart 1) represents the
common structural scaffold of drugs already used in clinical
practice and belonging to very different classes, including potas-
sium channel blockers (nifekalant1 and minoxidil2) imidazoline
receptor agonists (monoxidine3) and selective endotelin-1
receptor blockers (bosentan4). In addition, a pyrimidine ring with
substituents of moiety A (Chart 1) is also contained in a large
number of compounds with different biological activities such as
antimicotic, antihypertensive, and antithrombotic properties.5À10
Such a large representation of this heterocyclic nucleus in
drugs with diverse pharmacological properties suggests that this
heterocyclic moiety, if opportunely decorated with substituents
at a given distance, could lead to compounds with diverse
biological activities. Here we report the synthesis and the
pharmacological characterization of 18 pyrimidine derivatives
obtained by the chemical modification of the starting compound
2a (Scheme 1),11 obtained by reaction of 5,6-diamino-4-hydro-
xy-2-mercaptopyrimidine (1) with benzyl bromide. The new
compounds exhibited marked negative inotropic activity. Albeit
effective in blocking L-type voltage-gated Ca2+ channels, these
compounds differ from classical Ca2+ entry blockers (CEBs)
owing to the reduced negative chronotropic and vasorelaxant
activities. Because they potently block L-type with remarkable
selectivity over T-type voltage-gated Ca2+ channels, these com-
pounds could represent a novel class of L-type CEBs.
’ BIOLOGICAL ASSAYS
The cardiovascular profile of all compounds was tested on
guinea pig left and right atria and on guinea pig aortic strips
(Table 1). The characterization of 2b, 3aÀc, 4a, and 6aÀc was
extended at guinea pig ileum longitudinal smooth muscle
(GPILSM) (Table 2) using nifedipine, verapamil, and diltiazem
as the reference drugs (for data see Supporting Information).
’ CHEMISTRY
Eighteen 5,6-diamino-4-hydroxy-2-mercaptopyrimidine deri-
vatives, classified in three main series, a, b, and c derivatives
Received: January 26, 2011
Published: June 29, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Some compounds were further examined for their effect on
L-type barium currents (IBa(L)) on CHO cells and T-type barium
currents (IBa(T)) on TT cells (Table 3). Additionally, adenosine
A1 receptor agonism of 3a was examined (see Supporting
Information for details of methods).
designed and synthesized (Scheme 1). The negative inotropic
and/or chronotropic activities were modulated by the transfor-
mation of the primary amino group in an acetamide moiety and/
or from the insertion of an ethyl chain. The transformation of 2a,
2b, and 2c in the corresponding acetamides 5a, 5b, and 5c had a
different result on the three moieties. In fact, on going from 2c to
5c, a decreased negative inotropic potency is observed; instead
the change 2a to 5a showed an increase in the negative inotropic
and chronotropic potencies. 5b increased the negative inotropic
potency but lost the vasorelaxant activity if compared to the close
relative 2b. Compounds 2aÀc were treated with bromoethane to
give the N3-ethylated 3aÀc together with the O-ethylated 4aÀc.
All these compounds showed a broad increased negative ino-
tropic activity in the a and b series but did not improve the
activity in the c series. In particular, in the a series, the
aromatization of cycle (4a) produced a loss of chronotropic
activity, while 3a possess a weak chronotropic potency with a
high selectivity versus the negative inotropic potency. In the b
series the derivatives 3b and 4b showed an increased negative
inotropic potency. The most potent compound in this selec-
tion is the derivative 4b. The acetamides 6aÀc and 7aÀc com-
pared with the amino moiety resulted in an equal or detrimental
negative inotropic activity in the b and c series (6b ∼ 3b;
7b , 4b; 6c < 3c; 7c ∼ 4c). Instead, in the a series, 6a and 7a
showed a good negative inotropic potency; in particular 7a was 5
times more potent as a negative inotropic agent with respect to
the parent 4a.
’ RESULTS AND DISCUSSION
The inotropic, chronotropic, and vasorelaxant activity of all
compounds is shown in Table 1 (for data of reference com-
pounds see Supporting Information). None of them, except 2b,
displayed vasorelaxant activity. Compound 2a showed inotropic
and chronotropic activity. When the steric hindrance is reduced,
changing the benzyl chains with the propyl ones as in 2b, the
negative chronotropic activity become negligible while slight
vasorelaxant potency occurred. Compound 2c in which the
propyl chains are substituted with the 2-methoxyethyl residues
showed the greatest and selective inotropic activity. Starting from
2aÀc, a number of chemical variations (3aÀ7c) have been
Chart 1
Since the L-type calcium channel (LTCC) modulators
endowed inhibitory effect in K+-depolarized GPILSM,12
some selected compounds have been tested in this biological
assay (Table 2) (for data of reference compounds see Support-
ing Information). N3-Alkylated 6a, 3a, 3b, and 6b were the
most potent. Passing from the ethyl to the methoxyethyl chain,
the effect on nonvascular smooth muscle is greatly reduced
(6b vs 6c).
Given that our new mercaptopyrimidines are related to
compounds described as ligand of the adenosine receptors14
and that the negative inotropic activity could be related to the
activation of the adenosine A1 receptors,15 we tested the negative
Scheme 1a
a Reagents and conditions: (i) CH3CH2Br, K2CO3, CH3CN, 80À85 °C, 4 h; (ii) (CH3CO)2O, H2SO4 cat., 50À55 °C, 7 h.
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BRIEF ARTICLE
Table 1. Cardiovascular Activity of Tested Compounds
chronotropic negative
activity
% decrease (M ( SEM)
inotropic negative activity
vasorelaxant activityd
c
c
c
negative inotropic
negative chronotropic
activityb
EC50
95% CL
EC30
activity
IC50
compd
activitya
(μM)
(Â10À6
)
(μM)
(mean ( SEM) (μM)
2a
3a
4a
5a
6a
7a
2b
3b
4b
5b
6b
7b
2c
3c
4c
5c
6c
7c
60 ( 2.1
56 ( 2.4
62 ( 3.4
90 ( 3.2
66 ( 3.1e
64 ( 2.7f
87 ( 0.3
56 ( 1.4f
68 ( 2.3f
62 ( 1.2g
60 ( 3.4e
71 ( 0.7e
93 ( 2.6f
76 ( 1.5f
79 ( 3.4e
90 ( 2.3
81 ( 3.6
80 ( 1.7f
80 ( 1.4
63 ( 3.6
33 ( 1.9
95 ( 4.3
72 ( 3.5e
44 ( 1.9
39 ( 2.4e
22 ( 1.5e
27 ( 2.2
7 ( 0.3f
13 ( 0.5e
2 ( 0.1f
13 ( 0.3f
11 ( 0.9
45 ( 2.3
6 ( 0.5e
2 ( 0.1
3.16
0.45
1.49
1.41
0.29
0.27
1.96
0.60
0.15
0.28
0.53
1.00
0.34
0.23
0.31
0.96
0.77
0.62
2.27À4.39
0.31À0.65
0.97À1.78
1.00À1.94
0.22À0.38
0.20À0.34
1.32À2.53
0.42À0.86
0.10À0.21
0.22À0.36
0.41À0.68
0.72À1.39
0.23À0.48
0.17À0.31
0.22À0.43
0.66À1.24
0.53À1.12
0.38À0.97
8.63
8.01À9.15
2 ( 0.1e
7 ( 0.5
2 ( 0.1e
31 ( 1.5
25 ( 1.6
20 ( 1.9e
18.12
17.58À18.83
6.79
6.32À7.03
13.85À14.52
14.06
54 ( 0.5
28 ( 2.5
16 ( 1.1
30 ( 1.5
13 ( 0.9e
23 ( 1.9
10 ( 0.7
33 ( 2.1
22 ( 1.9
2 ( 0.1
14.86
11.66À18.94
2 ( 0.1
3 ( 0.2
10 ( 0.7
a Activity: decrease on developed tension in isolated guinea pig left atrium at 10À4 M, expressed as percent changes from the control (n = 4À6). The left
atria were driven at 1 Hz. The 10À4 M concentration gave the maximum effect for most compounds. b Activity: decrease on atrial rate in guinea pig
spontaneously beating isolated right atria at 10À4 M, expressed as percent changes from the control (n = 6À8). Pretreatment heart rate ranged from 170
to 195 beats/min. The 10À4 M concentration gave the maximum effect for most compounds. c Calculated from log concentrationÀresponse curves
(Probit analysis according to Litchfield and Wilcoxon13 with n = 4À7). When the maximum effect was <50%, the EC50 inotropic, EC30 chronotropic, and
IC50 values were not calculated. d Activity: percent inhibition of calcium-induced contraction on K+-depolarized guinea pig aortic strip at 10À4 M. The
10À4 M concentration gave the maximum effect for most compounds. e At 5 Â 10À5 M. f At 10À5 M. g At 5 Â 10À6 M.
Table 2. Relaxant Activity of Some Compounds on K+-
Depolarized GPILSM
Table 3. Percent Decrease in L- and T-Type Ba2+ Current
Induced by Tested Compounds
% decrease (mean ( SEM)a
mean
activitya ( SEM
compd
IC50b(μM)
95% CL (Â10À6
)
compd
CHO cell
TT cell
2b
3a
3b
3c
4b
6a
6b
6c
87 ( 1.7c
94 ( 1.3
93 ( 1.5c
60 ( 1.6
83 ( 1.4
90 ( 1.4d
72 ( 1.6d
91 ( 1.5
13.89
2.91
10.65À18.13
2.23À3.81
3a
3b
3c
6a
6b
6c
60.66 ( 5.9 (n = 5)
54.00 ( 7.8 (n = 6)
52.05 ( 8.0 (n = 6)
57.97 ( 5.8 (n = 5)
56.02 ( 5.3 (n = 6)
37.04 ( 4.8 (n = 6)
11.9 ( 2.9 (n = 6)
35.3 ( 5.0 (n = 6)
24.7 ( 3.7 (n = 7)
15.2 ( 5.7 (n = 5)
22.3 ( 2.7 (n = 6)
12.3 ( 4.6 (n = 6)
3.39
2.24À4.54
21.51
10.72
1.48
15.52À29.81
7.41À13.87
1.13À1.94
3.68À6.02
19.10À25.31
4.86
a The values reported in the table represent the mean ( SEM of the
percent decrease in peak current amplitude induced by compounds
under examination in CHOCR9β3R2/δ4 cells for L-type current and
TT cells for T-type current. The percent decrease was calculated as the
percent ratio between the averages of peak current values recorded at
steady state conditions in five consecutive sweeps in control condition
and in the presence of the drug. The number of cells in each experi-
mental group is reported in parentheses.
23.77
a Percent inhibition of calcium-induced contraction on K+-depolarized
(80 mM) guinea-pig longitudinal smooth muscle at 10À4 M. The 10À4
M concentration gave the maximum effect for most compounds.
b Calculated from log concentrationÀresponse curves (Probit analysis
according to Litchfield and Wilcoxon13 with n = 6À8). c At 5 Â 10À5 M.
d At 10À5 M.
inotropic activity of 3a in the presence of the A1 antagonist
8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 μM). Since the
concentrationÀresponse curve to 3a in the presence of DPCPX
does not differ from that of the control (EC50 = 0.45, CL =
0.31À0.65 and EC50 = 0.50, CL 0.28À0.60, respectively; see
Supporting Information for details), no A1 adenosine receptor
pathways are involved in the negative inotropic effect of com-
pounds. To support the hypothesis that the strong negative
inotropic activity and the ability to relax nonvascular smooth
muscles of these compounds are related to inhibit LTCCs, we
evaluated the effect of 3aÀc, 6aÀc (10 μM) on IBa(L) current in
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BRIEF ARTICLE
CHO cells stably expressing the whole cardiac L-type Ca2+
channel.16 As shown in Table 3, all compounds tested caused a
decrease of ∼60% in IBa(L) amplitude except 6c, which was
remarkably less effective (for recording traces see Supporting
Information).
determine the purity of target compounds. All compounds showed
g95% purity. 6-Amino-2-(benzylthio)-5-dibenzylaminopyrimidin-
4(3H)-one 2a was obtained which used 5,6-diamino-4-hydroxy-2-mer-
captopyrimidine as starting material.11
General Procedure for the Synthesis of 2b and 2c. 1-
Bromopropane (62 mmol) (or 1-bromo-2-methoxyethane) was added
dropwise at 50À55 °C to a stirred solution of 5,6-diamino-4-hydroxy-2-
mercaptopyrimidine 1 (5.0 g, 31 mmol) in NaOH 1 M (62 mL). The
mixture was stirred at this temperature for 7 h and then at room
temperature overnight. The crude product, a solid mass, was collected by
filtration in vacuo and then purified by flash chromatography (CHCl3/
MeOH = 97:3 v/v) to give in order of elution, the following three pure
compounds: 2b (13%, mp 130.6À131.2 °C); 8b (36%, mp 177.7À
178.7 °C); 9b (10%, mp 127.9À129.2 °C dec). Yields, melting points,
and spectral data of 2c, 8b, 8c, 9b, and 9c are in the Supporting
Information.
6-Amino-5-(dipropylamino)-2-(propylthio)pyrimidin-4(3H)-one 2b.
1H NMR (400 MHz, DMSO-d6, δ ppm): 11.42 (bs, 1H, NH); 6.15 (bs,
2H, NH2); 3.02 (t, 2H, J = 7.1 Hz, SCH2); 2.95À2.93 (m, 2H, NCH2);
2.68À2.64 (m, 2H, NCH2); 1.68À1.60 (m, 2H, SCH2CH2); 1.34À1.21
(m, 4H, 2 Â NCH2CH2); 0.95 (t, 3H, J = 7.3 Hz, SCH2CH2CH3); 0.81
(t, 6H, J = 7.4Hz, 2 Â NCH2CH2CH3). 13C NMR (100 MHz, DMSO-d6,
δ ppm): 162.0; 160.4; 157.5; 104.8; 55.3 (2C); 31.4; 22.2; 21.4 (2C);
13.1; 11.9 (2C).
General Procedure for the Synthesis of O-Ethyl and N-
Ethyl Products. To a suspension of 2b (or 2a or 2c) (2.3 mmol) and
K2CO3 (0.32 g, 2.3 mmol) in acetonitrile (25 mL) was slowly added
0.35 mL (4.7 mmol) of bromoethane. The mixture was stirred at
80À85 °C for 4 h until disappearance of substrate (TLC, petroleum
ether/ethyl acetate 8:2 v/v) and then cooled at room temperature. The
white precipitate was filtered off and washed with ethyl acetate. The
organic solution was evaporated to dryness and then purified by flash
chromatography (petroleum ether/ethyl acetate = 85:15 v/v) to give N-
ethyl 3b (3a or 3c) and O-ethyl 4b (4a or 4c) compounds in ratio of 1:3.
Yields, melting points, and spectral data of 3aÀ4c are in the Supporting
Information.
Some of the classical CEBs also block low voltage activated
T-type channels,17 a class of voltage-gated calcium channels with a
well-established role in epilepsy18 that are now also regarded as
crucial in vascular and cardiac hypertrophy and remodeling.19,20
Therefore, we performed whole cell patch clamp experiments in
TT cells, whose only voltage-gated Ca2+ currents are of the
T-type21 to establish whether new compounds are also effective
on T-type channels. Different from what was observed with
IBa(L), the effect on IBa(T) was small or negligible (Table 3). In a
comparative analysis of the different compounds belonging to
the subfamilies 3 and 6, the activity on LTCC was predictive of
the effect on cardiac inotropism and on GPILSM tone: the higher
the activity was on the channels, the higher the effect on heart and
ileum (see Figure 4 in Supporting Information. This result
suggests that LTCC blockade could be responsible for the
above-mentioned pharmacological effects. In addition these
results demonstrate good selectivity of this new chemotype for
LTCC with respect to TTCCs; 3a was the most selective
compound, while 3b was the most effective. A notable exception
was 6c that showed appreciable negative inotropic potency
despite its low activity on LTCC.
’ CONCLUSIONS
In this work, we presented a preliminary characterization on a
series of pyrimidine structures as a new chemotype able to
modulate the activity of LTCCs. These compounds showed
good ability to potently and selectively modulate cardiovascular
parameters according to classical calcium antagonist. Patch-
clamp experiments showed their ability to block LTCC cur-
rents. TTCC or adenosine A1 receptors seem to be not
involved in their biological activity. Four of 18 compounds
kept the negative chronotropic activity while just one was
endowed with vasorelaxant proprieties. Despite the low num-
ber of compounds, it is possible to find some structureÀactivity
relationships (see Chart 1 in Supporting Information): the
aromatic core cancels the negative chronotropic activity in the
benzyl series, whereas the nature of substituents on pyrimidine
ring is important to modulate the potency and selectivity of
compounds. In conclusion this new chemotype might be
considered a new class of LTCCs modulators. Of course, more
in depth studies are required to understand which site they bind
at the LTCC level and if additional mechanisms are involved in
the biological activity we here showed. However, these pre-
liminary findings pave the way for the design, synthesis, and
biological evaluation of this new chemotype as calcium channel
blocker.
6-Amino-5-(dipropylamino)-3-ethyl-2-(propylthio)pyrimidin-4(3H)-
1
one 3b. Yield, 24%; pale yellow oil. H NMR (400 MHz, DMSO-d6,
δ ppm): 6.05 (bs, 2H, NH2); 3.86 (q, 2H, J = 7.0 Hz, NCH2CH3); 3.11
(t, 2H, J = 7.2 Hz, SCH2); 2.98À2.77 (m, 2H, NCH2); 2.71À2.61 (m,
2H, NCH2); 1.66 (sext, 2H, J = 7.2 Hz, SCH2CH2); 1.38À1.22 (m, 4H,
2 Â NCH2CH2); 1.13 (t, 3H, J = 7.0 Hz, NCH2CH3); 0.98 (t, 3H, J = 7.2
Hz, SCH2CH2CH3); 0.81 (t, 6H, J = 7.3 Hz, 2 Â NCH2CH2CH3). 13
C
NMR (100 MHz, DMSO-d6, δ ppm): 160.1; 158.6; 157.0; 104.2; 55.2
(2C); 37.9; 32.8; 21.8; 21.4 (2C); 13.3; 13.2; 12.0 (2C).
6-Ethoxy-N5,N5-dipropyl-2-(propylthio)pyrimidine-4,5-diamine 4b.
Yield, 63%; mp 49.8À50.3 °C (n-hexane). 1H NMR (400 MHz, DMSO-
d6, δ ppm): 6.01 (bs, 2H, NH2); 4.28 (q, 2H, J = 7.2 Hz, OCH2); 2.95 (t,
2H, J = 7.1 Hz, SCH2); 2.82À2.76 (m, 2H, NCH2); 2.74À2.68 (m, 2H,
NCH2); 1.64 (sext, 2H, J = 7.1 Hz, SCH2CH2); 1.33À1.22 (m, 7H, 2 Â
NCH2CH2 and OCH2CH3); 0.95 (t, 3H, J = 7.1 Hz, SCH2CH2CH3);
0.80 (t, 6H, J = 7.1 Hz, 2xNCH2CH2CH3). 13C NMR (100 MHz,
DMSO-d6, δ ppm): 164.4; 164.3; 162.2; 106.0; 61.0; 55.4; 55.4; 32.0;
22.7; 21.1; 21.1; 14.6; 13.3; 11.8; 11.8.
General Procedure for the Synthesis of Acetylated
Products 5aÀ7c. To 0.7 mmol of the appropriate amine were added
0.5 mL of acetic anhydride and two drops of sulfuric acid. The mixture
was stirred at 50À60 °C for 1 h. After cooling at room temperature, the
solution was poured into iceÀwater (20 mL) and then extracted with
chloroform (4 Â 15 mL). The combined organic phases were dried
(Na2SO4) and evaporated to dryness. The crude product was purified by
chromatography (see Supporting Information) to give the desired
products. Yields, melting points, and spectral data of 5aÀ7c are in the
Supporting Information.
’ EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a B€uchi appara-
tus B 540 and are uncorrected. Routine nuclear magnetic resonance
spectra were recorded on a Varian Mercuryplus400 spectrometer oper-
1
ating at 400 MHz for H nucleus and 100 MHz for 13C nucleus, re-
spectively. Evaporation was performed in vacuo (rotary evaporator).
Analytical TLC was carried out on Merck 0.2 mm precoated silica gel
aluminum sheets (60 F-254). Silica gel 60 (230À400 mesh) was used for
column flash chromatography. Combustion analyses were used to
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BRIEF ARTICLE
N-[5-(Dipropylamino)-1-ethyl-6-oxo-2-(propylthio)-1,6-dihydro-
pyrimidin-4-yl]acetamide 6b. Yield, 25%. Ordinary pressure chroma-
tography, eluant mixture: petroleum ether/ethyl acetate = 8:2 v/v.
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1
Transparent oil. H NMR (400 MHz, DMSO-d6, δ ppm): 8.92 (bs,
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1H, NHCO); 3.95 (q, 2H, J = 7.0 Hz, NCH2CH3); 3.18 (t, 2H, J = 7.1
Hz, SCH2); 2.86 (t, 4H, J = 7.2 Hz, 2 Â NCH2); 2.35 (s, 3H, CH3); 1.70
(sext, 2H, J = 7.1 Hz, SCH2CH2); 1.29 (sext, 4H, J = 7.2 Hz, 2 Â
NCH2CH2); 1.19 (t, 3H, J = 7.0 Hz, NCH2CH3); 0.97 (t, 3H, J = 7.1 Hz,
SCH2CH2CH3); 0.83 (t, 6H, J = 7.2 Hz, 2 Â NCH2CH2CH3). 13C
NMR (100 MHz, DMSO-d6, δ ppm): 169.0; 158.8 (2C); 152.7; 111.3;
54.7 (2C); 39.0; 33.1; 24.8; 21.8; 21.2 (2C); 13.1; 12.6; 11.7 (2C).
’ ASSOCIATED CONTENT
S
Supporting Information. Physicochemical property, analy-
b
tical data, concentrationÀresponse curve for 3a in presence of DPCPX,
L- and T-type Ca2+ currents traces for tested compounds, functional
tissue assays, electrophysiological experiments, and Chart 1. This
material is available free of charge via the Internet at http://pubs.acs.
org.
(11) Cosimelli, B.; Iadanza, M.; Spisani, R.; Novellino, E. New
results on the reactivity of 5,6-diamino-4-hydroxy-2-mercaptopyrimi-
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’ AUTHOR INFORMATION
(12) Bolger, G. T.; Genco, P.; Klockowski, R.; Luchowski, E.; Siegel,
H.; Janis, R. A.; Triggle, A. M.; Triggle, D. J. Characterization of binding
of the Ca++ channel antagonist, [3H]nitrendipine, to guinea-pig ileal
smooth muscle. J. Pharmacol. Exp. Ther. 1983, 225, 291–309.
(13) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calcula-
tions with Computer Programs, 2nd ed.; Spinger-Verlag: New York, 1987.
(14) Cosimelli, B.; Greco, G.; Ehlardo, M.; Novellino, E.; Da
Settimo, F.; Taliani, S.; La Motta, C.; Bellandi, M.; Tuccinardi, T.;
Martinelli, A.; Ciampi, O.; Trincavelli, M. L.; Martini, C. Derivatives of
4-amino-6-hydroxy-2-mercapropyrimidine as novel, potent and selec-
tive A3 adenosine receptor antagonists. J. Med. Chem. 2008, 51, 1764–
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guinea-pig atrial and ventricular preparations: evidence against a role of
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Hofmann, F.; Klugbauer, N.; Di Renzo, G.; Annunziato, L. Studies on
maitotoxin-induced intracellular Ca2+ elevation in Chinese hamster
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Corresponding Author
*For B.C.: phone, +39-081-678614; fax, +39-081-678630; e-mail,
barbara.cosimelli@unina.it. For R.B.: phone, +39-051-2099737;
fax, +39-051-2099721; e-mail, roberta.budriesi@unibo.it.
Author Contributions
These authors contributed equally.
’ ACKNOWLEDGMENT
The authors thank Prof. F. Hofmann for his generous gift of
CHOCR9β3R2/δ4 cells, Dr. R. Pivonello for his generous gift of
TT cells, and Alessandro Casoni for animal care. This work was
supported by grants from University of Napoli and from Uni-
versity of Bologna.
’ ABBREVIATIONS USED
CEB, calcium entry blocker; LTCC, L-type calcium channel;
TTCC, T-type calcium channel; SAR, structureÀactivity rela-
tionship; GPILSM, guinea pig ileum longitudinal smooth
muscle;SEM, standard error of the mean; CHO, Chinese hamster
ovary
(18) Cataldi, M.; Lariccia, V.; Marzaioli, V.; Cavaccini, A.; Curia, G.;
Viggiano, D.; Canzoniero, L. M.; di Renzo, G.; Avoli, M.; Annunziato, L.
Zn(2+) slows down Ca(V)3.3 gating kinetics: implications for thala-
mocortical activity. J. Neurophysiol. 2007, 98, 2274–2284.
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the diseased heart. Channels (Austin) 2010, 4, 447–452.
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