A novel H2S releasing-monastrol hybrid (MADTOH) inhibits L-type calcium channels

Article abstract of DOI:10.1039/d0nj04415f

A new alleged monastrol-H2S releasing hybrid, named MADTOH, was designed based on the structure of monastrol (M) and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) and synthesized in 7.8% overall yield. MADTOH was shown to be an H2S donor under physiological conditions. In addition, the hybrid causes a decrease in global calcium transient in cardiomyocytes similar to nifedipine (NIFE), taken as a positive control. Whole-cell voltage-clamp showed that MADTOH decreases L-type Ca2+ current in isolated ventricular cardiomyocytes.

Full text of DOI:10.1039/d0nj04415f

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A novel H2S releasing-monastrol hybrid
(MADTOH) inhibits L-type calcium channels†
Cite this: New J. Chem., 2021,
45, 671
Taniris Cafiero Braga,a Itamar Couto Guedes de Jesus,b Kathleen Viveiros Soares,a
Silvia Guatimosim,b Leonardo da Silva Neto,c Cristiane Jovelina da-Silva,d
Luzia Valentina Modolo,d Jose Evaldo Rodrigues Menezes Filho,e Paula Rhana,f
´
f
a
ˆ
Jader Santos Cruz and Angelo de Fatima
*
´
A new alleged monastrol-H2S releasing hybrid, named MADTOH, was designed based on the structure
of monastrol (M) and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) and synthesized in 7.8%
overall yield. MADTOH was shown to be an H2S donor under physiological conditions. In addition, the
hybrid causes a decrease in global calcium transient in cardiomyocytes similar to nifedipine (NIFE), taken
as a positive control. Whole-cell voltage-clamp showed that MADTOH decreases L-type Ca2+ current in
isolated ventricular cardiomyocytes.
Received 3rd September 2020,
Accepted 29th November 2020
DOI: 10.1039/d0nj04415f
rsc.li/njc
benzothiazepines and 1,4-dihydropyridines (1,4-DHPs; Fig. 1).12
The 1,4-DHP scaffold was the most investigated among the
1. Introduction
Voltage-dependent calcium channels are important regulators of CCBs, in which chemical modifications were carried out at
neurotransmission and membrane ion conductance in which almost every position (including the DHP moiety).13 The 4-aryl-
modulations of these channels influence a plethora of intracellular 3,4-dihydropyrimidin-2(1H)-ones(thiones) (DHPM) derivatives
events. L-Type Ca2+ channels (CaV1.2 isoform) are critical in the (Fig. 1) were determined to be the most potent and metabolically
excitation–contraction coupling in cardiomyocytes,1 comprising the stable CCBs.12,14 Monastrol (M; Fig. 1), the most studied DHPM,
main pathway of Ca2+ influx in myocardial cells. Thus, Ca2+ entry was reported in the 1990s as a potent anticancer agent.15
through these channels is the main trigger to SR Ca2+ release. The Compound M was later revealed as an inhibitor of centrin,16
a
control and maintenance of Ca2+ homeostasis plays a fundamental protein pivotal for the duplication of the centrosome. A role as a
role in myocardial physiology and pathology.2 Indeed, alterations in urease17,18 and L-type calcium channel inhibitor19,20 has also
the expression and function of L-type calcium channels have been been documented.
implicated in a variety of cardiovascular diseases such as atrial
Hydrogen sulfide (H2S), an endogenously generated gaseous
fibrillation,3 cardiac hypertrophy,4 heart failure5,6 and ischaemic transmitter, is a key player in the modulation of calcium
heart disease.7 Furthermore, administration of L-type calcium channels, besides regulating cardiac functions and preventing
channel blockers (CCBs) is recognized as an effective therapy for myocardial infarction, hypertrophy, arrhythmia, fibrosis and
hypertension,8 cardiac hypertrophy,9 arrhythmia,10 congestive heart
failure,7 and myocardial ischemia.11
Three main classes of CCBs are reported according to the
structure of the pharmacophore group namely arylalkylamines,
a
´
Departamento de Quımica, Universidade Federal de Minas Gerais, Belo Horizonte,
MG, 31270-901, Brazil. E-mail: adefatima@qui.ufmg.br; Tel: +55-31-3409-6373
b
´
ˆ
´
Departamento de Fisiologia e Biofısica, Instituto de Ciencias Biologicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901,
Brazil
c Instituto Federal Farroupilha, Alegrete, RS, 97555-000, Brazil
d
ˆ
ˆ
´
Departamento de Botanica, Instituto de Ciencias Biologicas, Universidade Federal
de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
e Faculdades Integradas de Sergipe, Tobias Barreto, SE, 49300-000, Brazil
f
´
ˆ
´
Fig. 1 Chemical structures of 1,4-dihydropyridines (1,4-DHPs) and
4-aryl-3,4-dihyropyrimidin-2(1H)-ones(thiones) (DHPM), monastrol (M),
5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) and the hybrid
(MADTOH).
Departamento de Bioquımica e Imunologia, Instituto de Ciencias Biologicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj04415f
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ischemia–reperfusion injury (I/R).21,22 In fact, exogenous H2S
Monastrol (M). M.p. 180–183 1C [lit. = 183–185 1C];40 IR
was shown to be cardioprotective.23–26 H2S was demonstrated (KBr, cmꢁ1): 3308, 3184, 3010, 1668, 1474, 1286, 1192.
to inhibit L-type calcium channels in cardiomyocytes,26 an 1H NMR (200 MHz, DMSO-d6) d 1.11 (t, 3H, J = 7.0 Hz), 2.28
effect that depends on the number of sulfhydryl groups.27
(s, 3H), 4.01 (q, 2H, J = 7.0 Hz), 5.09 (d, 1H, J = 2.8 Hz),
The use of hybrid molecules is a strategy that enhances 6.63–6.67(m, 3H), 7.11 (t, 1H, J = 7.0 Hz), 9.45 (s, 1H), 9.59
and improves biological proprieties based on two or more (brs, 1H), 10.29 (brs, 1H). 13C NMR (50 MHz, DMSO-d6): d 14.1,
pharmacophoric groups.28,29 The building block derivatives from 17.3, 54.1, 59.7, 100.9, 113.4, 114.7, 117.1, 129.6, 144.9, 157.6,
Lawesson’s reagent, 1,2-dithiole-3-thiones (DTT), and arylthio- 165.3, 174.3. HRMS calcd for C14H16N2O3S [M + 1] 293.096;
amides were successfully used as a H2S releasing agent.29,30 5-(4- found 293.1116 (error: ꢁ5.32 ppm), calcd for [M + Na] 315.078;
Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH, Fig. 1), a DTT found 315.0683 (error: 3.08 ppm).
derivative, has already been used for the hybridization of several
2.1.2. 4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
modified drugs that are currently undergoing preclinical tetrahydropyrimidine-5-carboxylic acid (4)41. Monastrol (M; 1.7 mmol)
testing.31–36 The mechanism by which H2S is released from and KOH (5.0 mmol) were dissolved in 6 mL of water. The
ADTOH is not fully elucidated. Giustarini and coworkers (2010) mixture was stirred at room temperature for 3 days. After the
speculated that the mechanism of H2S release from ADTOH completion of the reaction, a small amount of 2.0 mol Lꢁ1 HCl
involves two possible pathways: (i) H2S could be formed from (ca. 0.5 mL each) was added to the mixture until it became
the hydrolysis of a thionyl group followed by the breakdown of the turbid. The mixture was then maintained under magnetic stir-
S–S bond to yield the dithiol derivative or (ii) the dithiol derivative ring for 20 min. The precipitate formed was filtered, washed with
is formed first following the hydrolysis of the thionyl group.37
The blocking action of monastrol (M) and H2S on calcium Compound 4 was obtained as a yellowish solid in 80% yield
channels prompted us to use the design and synthesis of a (359.4 mg–1.3 mmol).
monastrol-H2S releasing hybrid (MADTOH; Fig. 1) as a theoreti-
distilled water (3 ꢀ 10 mL) and dried under vacuum for 2 h.
cally dual CCB agent. The nucleus ADTOH was chosen as an H2S
4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
donor core to be attached to the M moiety. Hybrid MADTOH and 5-carboxylic acid (4). M.p. 195–198 1C; IR (KBr, cmꢁ1): 3308, 3192,
1
its precursors were tested as an H2S donor and on ventricular 3130, 1672, 1586, 1482, 1268, 1186. H NMR (200 MHz, DMSO-d6)
cardiomyocytes to investigate their effect as CCB inhibitors.
d 2.26 (s, 3H), 5.07 (brs, 1H), 6.63 (brs, 3H), 7.10 (t, 1H, J = 8Hz), 9.52
(brs, 2H), 10.19 (brs, 1H), 12.14 (brs, 1H). 13C NMR (50 MHz, DMSO-
d6): d 17.2, 54.1, 101.5, 113.4, 114.6, 117.2, 129.6, 144.4, 144.9, 157.5,
167.1, 174.3. HRMS calcd for C12H12N2O3S [M ꢁ 1] 263.0491; found
263.0446 (error: 1.71 ppm).
2. Materials and methods
2.1.3. 5-(4-Methoxyphenyl)-3H-1,2-dithiole-3-thione (9)42
.
All reagents were purchased from Sigma Aldrich. Tetrahydro-
furan (THF) and acetonitrile (ACN) were dried according to
Perrin and Amarengo.38 Chemical reactions were monitored
using thin layer chromatography (POLYGRAM-UV2540, 20 mm
MACHEREY – NAGEL; 20 ꢀ 20 cm). Silica gel (technical grade,
pore size 60 Å, 70–230 mesh, 63–200 mm) was used for flash
column chromatography. The melting point of the synthesized
compounds was determined on a GEHAKA-PF1500 device.
Infrared (IR) spectra were acquired in a Spectrometer One
PerkinElmer as KBr pellets at 400–4000 cmꢁ1. Mass spectra
were recorded on a SHIMADZU LC-ITTOF instrument. NMR
was recorded on Bruker AVANCE DPX 200 and Bruker AVANCE-
III 400 instruments. Chemical shifts (in ppm) are reported
relative to residual protic solvent resonances.
(E)-1-Methoxy-4-(prop-1-en-1-yl)benzene (8; 80 mmol) and sul-
fur (560 mmol) were dissolved in 70 mL of dimethylacetamide
(DMA). The mixture was heated until reflux and magnetically
stirred for 6 h. The excess of sulfur was removed and the
resulting solution was cooled to 0 1C for 5 h. The precipitate
formed was recrystallized from ethyl acetate furnishing the desir-
able product as an orange solid in 30% yield (5.8 g–24.0 mmol).
5-(4-Methoxyphenyl)-3H-1,2-dithiole-3-thione (9). M.p. 106–
108 1C [lit. 118 1C];42 IR (KBr, cmꢁ1): 3080, 1602, 1176;
1H NMR (200 MHz, DMSO-d6) d 3.84 (s, 3H), 7.05 (d, 2H, J = 3
Hz), 7.74 (s, 1H), 7.84 (d, 2H, J = 3 Hz). 13C NMR (50 MHz,
DMSO-d6): d 55.6, 115.0, 123.6, 128.9, 134.10, 162.6, 173.7,
214.7. HRMS calcd for C10H8OS3 [M + 1] 239.973; found
240.9889 (error: 3.07 ppm).
2.1. Synthesis of monastrol-H2S releasing hybrid MADTOH
2.1.1. Monastrol (M)39. Ethyl acetoacetate (1; 62.0 mmol),
2.1.4. 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH)43.
3-hydroxybenzaldehyde (2; 41.0 mmol), thiourea (3; 62.0 mmol) A mixture of 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (9)
and p-toluenesulfonic acid (PTSA; 2.2 mmol) were dissolved in (4.2 mmol) and pyridine hydrochloride (PyꢂHCl) (24.0 mmol)
34 mL of ethanol. The mixture was stirred at room temperature was heated and magnetically stirred for 20 min in a microwave
for 8 h following the addition of aliquots of water (ca. 1 mL reactor (CEMs) with 4 min of ramp until 160 1C (300 W power
each) until the reaction became turbid. The reaction mixture rating). After complete consumption of 9, attested by TLC
was maintained under magnetic stirring for 20 min and analysis, hot water was slowly added to the reaction mixture.
the precipitate was filtered and dried under vacuum for 2 h. The solid formed was solubilized in 10% m/v K2CO3 solution
Monastrol (M) was obtained as a white solid in 57% yield (6.8 g– (80 mL) and extracted with dichloromethane (5 ꢀ 250 mL). The
23.3 mmol).
organic layer was concentrated, dried under vacuum for 4 h and
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ADTOH obtained as an orange solid in 60% yield (570.3 mg– water (3 ꢀ 15 mL) and dried under vacuum. Compound 11 was
2.5 mmol).
obtained as an orange solid in 86% yield (263.0 mg–0.86 mmol).
5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH). M.p.
5-(4-(2-Aminoethoxy)phenyl)-3H-1,2-dithiole-3-thione hydro-
chloride (11). M.p. 226–227 1C; IR (KBr, cmꢁ1): 3434, 3200,
3046, 1602, 1200; 1H NMR (200 MHz, DMSO-d6) d 3.22 (brs,
2H), 4.31 (brs, 2H), 7.11 (d, 2H, J = 8.0 Hz), 7.74 (s, 1H), 7.88 (d,
2H, J = 8.0 Hz), 8.46 (brs, 3H). 13C NMR (50 MHz, DMSO-d6): d
38.1, 64.7, 115.7, 124.2, 129.0, 134.4, 161.2, 173.6, 214.8.
HRMS calcd for C11H12NOS3+Clꢁ [M + 1]: 270.0076; found
[M + 1]: 270.0027 (error: 2.00 ppm).
178–180 1C [lit. 172–174 1C];43 IR (KBr, cmꢁ1): 3158, 3050,
1
1608, 1476, 1197; H NMR (200 MHz, DMSO-d6) d 6.90 (d, 2H,
J = 7 Hz), 7.70 (s, 1H), 7.78 (d, 2H, J = 7 Hz), 10.52 (s, 1H). 13C
NMR (50 MHz, DMSO-d6): d 116.4, 122.2, 129.2, 133.5, 161.7,
174.3, 214.4. HRMS calcd for C9H6OS3 [M ꢁ 1]: 224.9503; found
[M ꢁ 1]: 224.9477 (error: 1.16 ppm).
2.1.5. tert-Butyl(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)-
ethyl)carbamate (10)44,45. 2-Bromoethan-1-amine (6) (47.0 mmol)
was dissolved in 40 mL of dioxane and maintained at 0 1C under
magnetic stirring. Di-tert-butyl dicarbonate (5) (27.0 mmol) was
added to the reaction mixture followed by slow addition (during
5 min) of triethylamine (25.0 mmol). The reaction was kept
under magnetic stirring at 0 1C for 40 min. Afterwards, the
system was warmed up to room temperature and kept under
such conditions for 48 h. The liquid layer obtained after filtration
was concentrated under vacuum and the residue formed was
solubilized in dichloromethane (100 mL) and washed with water
(2 ꢀ 50 mL). The organic solvent was removed under vacuum to
furnish tert-butyl-(2-bromoethyl)carbamate (7) as a yellowish
viscous liquid in 73% yield (4.8 g–21.3 mmol). Carbamate
7 was used in the subsequent steps with no further chemical
characterization. Briefly, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-
thione (2.0 mmol) (ADTOH), KI (2.9 mmol) and Cs2CO3
(6.0 mmol) were dissolved in anhydrous ACN. The mixture was
heated to reflux for 20 min followed by addition of 6.0 mmol of
carbamate 7 prepared in anhydrous ACN. Forty-eight hours later,
the residue formed from the reaction concentration under
vacuum was solubilized in chloroform (40 mL) and extracted
with 0.4 mol Lꢁ1 NaOH (2 ꢀ 30 mL). The organic layer was dried
using MgSO4 and the solvent removed under vacuum. The
residue obtained was purified by column chromatography
using methanol/chloroform (1 : 100) as the eluent. The carba-
mate 10 was obtained as an orange solid in 79% yield
(583.8 mg–1.6 mmol).
2.1.7. 4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-N-(2-(4-(3-thioxo-
3H-1,2-dithiol-5-yl)phenoxy)ethyl)-1,2,3,4-tetrahydropyrimidine-
5-carboxamide (MADTOH). 4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (4) (0.7 mmol) and
N,N0-dicyclohexylcarbodiimide (DCC) (0.7 mmol) were dissolved in
anhydrous THF (10 mL). Five minutes later, a solution of 5-(4-
(2-aminoethoxy)phenyl)-3H-1,2-dithiole-3-thione
hydrochloride
(11) (0.3 mmol) prepared in N,N-disopropylethylamine (DIPEA)
(2.3 mmol) was added to this mixture. The reaction was magneti-
cally stirred for 24 h at room temperature and the residue formed
was purified by column chromatography using ethanol/chloroform
(1:10) as the eluent. The monastrol-H2S releasing (MADTOH)
hybrid was obtained as an orange solid in 64% yield (231.0 mg–
0.44 mmol).
4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-N-(2-(4-(3-thioxo-3H-1,2-
dithiol-5-yl)phenoxy)ethyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide
(MADTOH). M.p. 206–208 1C; IR (KBr, cmꢁ1): 3200, 2968, 2868,
2880, 1602, 1198, 1180; 1H NMR (400 MHz, DMSO-d6) d 2.01 (s, 3H),
3.37–3.54 (m, 2H), 4.05 (t, 2H, J = 6 Hz), 5.18 (d, 1H, J = 3.2 Hz),
6.61–6.64 (m, 3H), 7.03 (d, 2H, J = 8.8 Hz), 7.75 (brs, 1H), 7.86
(d, 2H, J = 8.8 Hz), 7.97 (t, 1H, J = 6 Hz), 9.28 (s, 1H), 9.41 (s, 1H),
9.81 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): d 16.3, 38.2, 54.8,
66.4, 106.8, 113.2, 114.5, 155.5, 166.9, 123.7, 129.0, 129.3, 134.2,
134.7, 144.5, 157.5, 161.8, 166.3, 173.8, 174.1, 214.8. HRMS calcd for
C23H21N3O3S4 [M ꢁ 1]: 515.0466; found [M ꢁ 1]: 516.0425 (error:
2.31 ppm).
tert-Butyl(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl)carbamate
(10). M.p. 116–117 1C; IR (KBr, cmꢁ1): 3044, 1694, 1602, 1078;
2.2. In vitro assay of H2S release
1H NMR (200 MHz, CDCl3) d 1.44 (s, 9H), 3.54 (q, 2H, J = 5.2 Hz), The ability of monastrol (M), 5-(4-hydroxyphenyl)-3H-1,2-
4.07 (t, 2H, J = 5.2), 5.02 (brs, 1H), 6.94 (d, 2H, J = 8.8 Hz), 7.35 dithiole-3-thione (ADTOH), 4-(3-hydroxyphenyl)-6-methyl-2-
(s, 1H), 7.58 (d, 2H, J = 8.8). 13C NMR (50MHz, CDCl3): d 28.5, 40.0, thioxo-N-(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl)-1,2,3,4-
67.6, 79.8, 115.5, 124.5, 128.7, 134.7, 155.9, 162.0, 172.9, 215.1. tetrahydropyrimidine-5-carboxamide (MADTOH) and nifedipine
HRMS calcd for C16H19NO3S3 [M + 1]: 370.0605; found [M + 1]: (NIFE) to release H2S was investigated using a TBR4100 Free
370.0600 (Error: 0.14 ppm); calcd for [M + Na] 392.0425; found [M + Radical Analyzer (WPI, USA) equipped with an ISO-H2S-2
Na]: 392.0445 (error: 0.51 ppm).
amperometric sensor (detection limit r5 nmol Lꢁ1). Each
2.1.6. 5-(4-(2-Aminoethoxy)phenyl)-3H-1,2-dithiole-3-thione compound (10 mmol Lꢁ1) was prepared in Tyrode solution and
hydrochloride (11). tert-Butyl(2-(4-(3-thioxo-3H-1,2-dithiol-5- analyzed at 0, 3, 6 and 12 min after preparation by individually
yl)phenoxy)ethyl)carbamate (10) (1.0 mmol) was solubilized adding 50 mL of each solution to 100 mmol Lꢁ1 PBS. The amount
in ethyl acetoacetate followed by addition of 4 mol Lꢁ1 of HCl of H2S released from the solutions at the specified times
(10 mL). The reaction mixture was maintained for 3 h under was determined using a standard curve of Na2S and the equation
magnetic stirring at room temperature and then at 4 1C for other H2S = [Na2S]/3.04173977, according to the manufacturer’s
5 h. The precipitate obtained was filtrated, washed in distilled instructions.
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2.3. Ex vivo effects of MADTOH on L-type calcium channels
applied to each measurement. Cardiomyocytes presenting Rs
higher than 8 MO were discarded. The composition of the
internal solution was 120 mmol Lꢁ1 CsCl, 20 mmol Lꢁ1 TEACl,
5 mmol Lꢁ1 NaCl, 10 mmol Lꢁ1 HEPES and 5 mmol Lꢁ1 EGTA
(pH 7.2) and the external solution was 140 mmol Lꢁ1 NaCl,
5.4 mmol Lꢁ1 KCl, 1 mmol Lꢁ1 MgCl2, 1.8 mmol Lꢁ1 CaCl2,
10 mmol Lꢁ1 HEPES and 10 mmol Lꢁ1 glucose (pH 7.4). The
time course of the ICa,L peak current was recorded in the
absence and presence of M, ADTOH and MADTOH (each at
10 mmol Lꢁ1) to evaluate the acute effect of these xenobiotics.
Pre-pulses from a holding potential (ꢁ80 mV to ꢁ40 mV), for 50
ms, were applied to inactivate Na+ and/or T-type Ca2+ channels.
A test pulse (0 mV) was then applied for 300 ms to measure ICa,
L, and time between sweeps was set at 10 s.
2.3.1. Animal model. This study was carried out with 10 to
12 week-old male C57BL/6 mice (n = 10 animals). The animals
were housed in the animal facility, kept at controlled tempera-
ture (22–24 1C) under 12 h photoperiod. Experiments were
performed according to protocols approved by the Institutional
Animal Care and Use Committee at the Federal University of
Minas Gerais (UFMG, Brazil) under protocol – CEUA183/2020.
All procedures were performed in accordance with the National
Institutes of Health (NIH) Guide for the Care and Use of
Laboratory Animals (NIH publication 86–23, revised 1985;
nih.gov/regs/index.htm).
2.3.2. Ventricular cardiomyocyte isolation. After appropri-
ate anesthesia (ketamine 100 mg kgꢁ1, xylazine 10 mg kgꢁ1
,
2.3.6. Statistical analysis. In intracellular measurement of
H2S and Ca2+ transients experiments, data are presented as
mean ꢃ SEM of at least 3 independent experiments, and for
statistical comparison, we used one-way ANOVA followed
by Newman–Keuls post hoc test. For the L-type Ca2+ current
experiments, data are presented as mean + SD of at least 3
independent experiments, and for statistical comparison, we
used a paired Student’s t-test. The level of significance was set
to values of p o 0.05.
intraperitoneal), the animals were euthanized by rapid cervical
dislocation. Briefly, the hearts were perfused via the Langen-
dorff method with Digestion Buffer containing 1.2 mg mLꢁ1 of
collagenase type 2 (Worthington Biochemical Corporation)
(DB: 130 mmol Lꢁ1 NaCl, 5.4 mmol Lꢁ1 KCl, 25 mmol Lꢁ1
HEPES, 0.5 mmol Lꢁ1 MgCl2, 0.33 mmol Lꢁ1 NaH2PO4, 22 mmol
Lꢁ1 glucose; 50 mmol Lꢁ1 CaCl2 and insulin 100 U mLꢁ1
,
pH = 7.4 adjust with NaOH). Ventricular myocytes were stored
in regular Tyrode solution at pH 7.4 (140 mmol Lꢁ1 NaCl, 4 mmol
L
ꢁ1 KCl, 1 mmol Lꢁ1 MgCl2, 1.8 mmol Lꢁ1 CaCl2, 10 mmol Lꢁ1
glucose and 5 mmol Lꢁ1 HEPES) until they were used.46
2.3.3. Intracellular measurement of H2S. Cardiomyocytes
were incubated at 37 1C with 25 mmol Lꢁ1 WSP-5 (Cayman
Chemical) prepared in Tyrode solution. After 30 min, the
cardiomyocytes were washed in Tyrode solution to remove the
3. Results and discussion
3.1. Synthesis of the monastrol-H2S releasing hybrid
MADTOH
The monastrol-H2S releasing hybrid MADTOH was constructed
excess of the H2S probe. Samples were analyzed by confocal by connecting the building blocks 4 and ADTOH using the
microscopy (Zeiss LSM 880 Meta) at the Center for Acquisition linker 7 (Panels A and C, Scheme 1). Initially, 4, the corresponding
carboxylic acid of monastrol (M) was prepared by a Biginelli
reaction of the ethyl acetoacetate (1), 3-hydroxybenzaldehyde (2)
and thiourea (3) catalyzed by p-toluenesulfonic acid (PTSA) (Panel
A, Scheme 1).39 Further treatment of M under basic conditions41
furnished the desirable carboxylic acid 4 with 45% overall yield
(2 steps). The preparation of ADTOH (the other coupling partner)
and Processing of Images (Institute of Biological Sciences,
UFMG) to verify changes in fluorescence. The software ImageJ
(NIH) was used for the analyses of micrographies.
2.3.4. Intracellular Ca2+ transients. Ventricular cardiomyo-
cytes were incubated at room temperature with 6 mmol Lꢁ1
Fluo-4/AM (Thermo Fisher Scientific) prepared in Tyrode
solution. After 30 min, the cells were washed in Tyrode solution
to remove the excess Ca2+ probe. Intracellular Ca2+ transients
were evoked by electrical stimulation of the cardiomyocytes
using field stimulating platinum electrodes with voltage pulses
of 5 ms, 40 V intensity and biphasic pulses at 1 Hz. The
scanning image was acquired by a confocal microscope Zeiss
LSM 880 Meta and 63ꢀ oil objective. Cardiomyocytes were
scanned with a 512 pixel line at 1.6 ms and sequential scans
were obtained to create two-dimensional images with time on
the x-axis. ImageJ software (NIH) was used for image analyses.
2.3.5. Measurement of L-type Ca2+ current in isolated left
ventricular cardiomyocytes. Electrophysiological recordings
were obtained by whole-cell voltage-clamp mode using an
EPC-10 patch-clamp amplifier (HEKA Electronics, Germany).
After achieving whole-cell configuration, equilibrium between
the pipette solution and the intracellular environment was
achieved in 3–5 min. Pipette resistances of 1–2 MO and
electronic correction for a series resistance (Rs) of 50–60% were
Scheme 1 Convergent synthesis of the monastrol-H2S releasing hybrid
MADTOH. Reagents and reactions conditions: (a) PTSA, ethanol, r.t., 8 h;
(b) KOH, H2O, r.t., 3 d; (c) dioxane, 0 1C, TEA, then r.t. 48 h; (d) S8, DMA,
reflux, 6 h; (e) PyꢂHCl, MWI, 160 1C, 20 min; (f) 7, KI, Cs2CO3, ACN, reflux,
48 h; (g) HCl (4 mol Lꢁ1), EtOAc, r.t., 3 h; (h) 4, DCC, THF, DIPEA, r.t., 24 h.
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was done by initially treating (E)-1-methoxy-4-(prop-1-en-1-
yl)benzene (8) and sulfur in dimethylacetamide to obtain 942
in 30% yield (Panel C, Scheme 1). The demethylation of
9 using pyridine hydrochloride (PyꢂHCl) under microwave
irradiation (MWI) at 160 1C for 20 min43 afforded ADTOH in
60% yield. The next step comprised the synthesis of the linker
7 (Panel B, Scheme 1), prepared by treating the amine 6 with
di-tert-butyl dicarbonate (5) in the presence of trimethylamine
(TEA) at room temperature for 48 h (73% of yield).47 Both
ADTOH and the linker 7 were treated with an excess of Cs2CO3
and KI under reflux in ACN for 48 h at room temperature to
provide the N-BOC-protected amine 10 with 79% yield (Panel
C, Scheme 1). An acid-mediated BOC-deprotection of 10 finally
led to the desired amine hydrochloride 11 (86% yield). Then,
11 was coupled to 4 to obtain the new monastrol-H2S releasing
hybrid MADTOH in 64% yield (Panel C, Scheme 1). The
convergent strategy herein used provided the target hybrid
MADTOH in an overall yield of 7.8% (Scheme 1). In conclu-
sion, from the point of view of our synthetic approach to
MADTOH, a new monastrol-H2S releasing molecule was suc-
cessfully synthesized and such compounds may present a dual
inhibitory effect on L-type calcium channels.
Fig. 2 Extent of H2S release from a hybrid constituted of monastrol (M)
and ADTOH, (MADTOH) under physiological conditions as a function
of time. ADTOH, MADTOH, M and nifedipine (NIFE) were prepared at
10 mmol Lꢁ1 in Tyrode solution and the release of H2S (if any) was
monitored in the interval 0–12 min after solution preparation. The values
are the means + SD of experiments carried out in triplicate.
3.2. In vitro H2S releasing studies
were incubated with the Ca2+ sensitive fluorescent dye (Fluo-4/AM),
paced at 1 Hz and visualized by confocal microscopy. The peak
Ca2+ transient magnitude on ventricular myocytes was also mon-
itored after treatment with NIFE, as the structure of this repre-
sentative member of 1,4-dihydropyridines (1,4-DHPs) is closely
related to that of MADTOH. The peak Ca2+ amplitude decreased
Solutions of ADTOH, MADTOH, M and NIFE were prepared in
Tyrode solution to test the hypothesis that the allegedly
monastrol-H2S releasing hybrid herein synthesized (MADTOH)
is able to release H2S under physiological conditions. The
use of a highly sensitive H2S electrode and an amperometric
approach allowed it to be unequivocally demonstrated that
MADTOH, during the analyzed time, is an H2S donor in an
isotonic solution with interstitial fluid similar to that observed
for ADTOH, the well-known H2S donor used for the synthesis of
the monastrol hybrid (Fig. 2). Monastrol (M) does not release
H2S under the same experimental conditions tested for MAD-
TOH. As expected, no detectable H2S was released from Tyrode
solution or nifedipine (Fig. 2). Therefore, the strategy used to
link a known H2S donor (ADTOH) to M effectively provided a
new molecule eligible to release H2S donor under physiological
conditions.
3.3. Ex vivo effects of MADTOH on calcium channels
The H2S production was also evaluated ex vivo by incubating
left ventricular cardiomyocytes exposed to test molecules (MAD-
TOH, M or ADTOH) with WSP-5 for 10–20 min. WSP-5 is a
substance that in the presence of H2S emits fluorescence.48
As expected, ADTOH induced a significant increase in H2S
(25%; p o 0.05; Fig. 3). Likewise, MADTOH enhanced H2S
production in left ventricular myocytes by 17% (p o 0.05; Fig. 3)
while M had no significant effect on the H2S production (p o 0.05;
Fig. 3).
Hydrogen sulfide (H2S) is described as an important mod-
ulator of cardiovascular function.22,49,50 Ca2+ is the major ion
that orchestrates excitation–contraction coupling (E–C cou-
pling) in cardiomyocytes. To investigate the effect of MADTOH,
ADTOH and M on global Ca2+ transients, ventricular myocytes
Fig. 3 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) and the
monastrol-H2S releasing (MADTOH) hybrid increase H2S production in
ventricular cardiomyocytes. (A) Representative confocal images showing
WSP-5-loaded cardiomyocytes treated with ADTOH (10 mmol Lꢁ1), MAD-
TOH (10 mmol Lꢁ1) or M (10 mmol Lꢁ1). (B) Averaged-WSP-5 fluorescence
increase in ventricular myocytes after ADTOH or MADTOH treatment
(10 mmol Lꢁ1) for 10–20 min. J, number of cardiomyocytes isolated from
3 hearts. Bar = 20 mm. *p o 0.05 vs. Control and M. #p o 0.05 vs. Control.
Data are mean ꢃ SEM and were analyzed by one-way ANOVA followed by
Newman–Keuls post hoc test.
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Fig. 4 MADTOH negatively affects the peak Ca2+ magnitude in ventricular
cardiomyocytes. (A) Representative images of Ca2+ transients in control samples
and cardiomyocytes treated with MADTOH (100 nmol Lꢁ1–10 mmol Lꢁ1).
(B) Quantitative data for Ca2+ transient amplitude in cardiomyocytes treated
with MADTOH. (C) Representative images of Ca2+ transients in control samples
and cardiomyocytes treated with nifedipine (NIFE; 100 nmol Lꢁ1–10 mmol Lꢁ1).
(D) Quantitative data for Ca2+ transient amplitude in cardiomyocytes treated
with NIFE. J, number of cardiomyocytes isolated from 2–3 hearts. Bar =
20 mm. *p o 0.05 vs. the other groups. Data are mean ꢃ SEM and were
analyzed by one-way ANOVA followed by Newman–Keuls post hoc test.
Fig. 5 Increment of peak Ca2+ in ventricular cardiomyocytes caused by
monastrol (M). (A) Representative images of Ca2+ transients in control
samples and cardiomyocytes treated with ADTOH. (B) Quantitative data
for peak Ca2+ transient amplitude in cardiomyocytes treated with ADTOH.
(C) Representative images of Ca2+ transients in control samples and
cardiomyocytes treated with M. (D) Quantitative data for Ca2+ transient
amplitude in cardiomyocytes treated with M. J, number of cardiomyo-
cytes isolated from 3 hearts. Bar = 20 mm. *p o 0.05 vs. control. Data are
mean ꢃ SEM and were analyzed by one-way ANOVA followed by the
Newman–Keuls post hoc test.
in a concentration-dependent manner in ventricular cardiomyo- was previously described as an inhibitor of a calcium channel
cytes exposed to MADTOH (ꢁ22% at 100 nmol Lꢁ1; ꢁ28% at in HEK293 cells (100 mM)19 and aortic rings (10–100 mM).20 This
1 mmol Lꢁ1; ꢁ43% at 10 mmol Lꢁ1; p o 0.05; Fig. 4A and B). distinct effect observed for M on L-type calcium channels was
Likewise, NIFE provoked a decrease in Ca2+ transient magnitude also detected for nifedipine (NIFE)51 and H2S52 when they were
(ꢁ23% at 100 nmol Lꢁ1; ꢁ29% at 1 mmol Lꢁ1 and ꢁ38% at evaluated in different concentrations and/or cell lineages. Thus,
10 mmol Lꢁ1 p o 0.05; Fig. 4C and D).
we suggested that the inhibitory effect of M (0.1–100 mM) on
Key intermediate molecules used in the synthesis of MAD- L-type calcium channels from cardiomyocytes could be attributed
TOH were also tested for the effect on peak Ca2+ transient. Peak for such compounds. In addition, MADTOH effects could be due
Ca2+ was not altered in cardiomyocytes upon treatment with to a synergism between M and ADTOH – the hybrid subunits.
ADTOH, regardless of the concentration tested, in comparison
to the control (Fig. 5; p 4 0.05). Notably, the treatment with
ADTOH at 100 mmol Lꢁ1 impaired the viability of cardiomyo-
cytes which, in turn, resulted in failure to record a Ca2+
transient. Surprisingly, M induced an increment in the peak
Ca2+ (31% at 100 nmol Lꢁ1; 37% at 1 mmol Lꢁ1; 34% at 10 mmol
L
ꢁ1; p o 0.05). Strikingly, the hybrid MADTOH shows an effect
very distinctive of that of ADTOH or M, whose carbon skeletons
are embedded in an MADTOH chemical structure.
Next, it was investigated the effects of MADTOH on L-type Ca2+
channels. Accordingly, L-type Ca2+ current (ICa,L) was measured
in isolated left ventricular cardiomyocytes using the whole-cell
voltage-clamp technique. MADTOH (10 mmol Lꢁ1) led to a higher
decrease in ICa,L peak when compared to ADTOH or M (Fig. 6). In
fact, the ICa,L peak was decreased by 36% (from ꢁ1.1 ꢃ 0.7 nA to
ꢁ0.4 ꢃ 0.2 nA; p o 0.05) 2 min after-incubation of cardiomyocytes
with MADTOH. Overall, these results indicate that the decrease of
Ca2+ influx in ventricular cardiomyocytes triggered by MADTOH
was due to the inhibition of L-type Ca2+ channels.
Fig. 6 Effects of MADTOH on L-type Ca2+ current. (A) Representative
recording showing the L-type calcium current (ICa,L) in the control and
after 2 or 4 min incubation with 10 mmol Lꢁ1 MADTOH, ADTOH or M.
(B) Summarized data of MADTOH (left chart), ADTOH (middle chart) and
M (right chart) on the ICa,L (n = 6, *p o 0.05). Data are mean + SD and
were analyzed by a paired Student’s t-test.
Monastrol (M), at 0.1–100 mM, triggered an increase in Ca2+
transient amplitude in ventricular cardiomyocytes, although it
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14 K. S. Atwal, G. C. Rovnyak, S. D. Kimball, D. M. Floyd,
S. Moreland, B. N. Swanson, J. Z. Gougoutas, J. Schwartz,
K. M. Smillie and M. F. Malley, J. Med. Chem., 1990, 33,
2629–2635.
15 T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King,
S. L. Schreiber and T. J. Mitchison, Science, 1999, 286, 971–974.
16 L. Duan, T.-Q. Wang, W. Bian, W. Liu, Y. Sun and B.-S. Yang,
Spectrochim. Acta, Part A, 2015, 137, 1086–1091.
17 U. Rashid, I. Batool, A. Wadood, A. Khan, Z. ul-Haq,
M. I. Chaudhary and F. L. Ansari, J. Mol. Graphics Modell.,
2013, 43, 47–57.
18 T. C. Braga, T. F. Silva, T. M. S. Maciel, E. C. D. da Silva,
E. F. da Silva-Ju´nior, L. V. Modolo, I. M. Figueiredo,
4. Conclusions
A new monastrol-H2S-releasing hybrid (MADTOH) was success-
fully synthesized via a convergent strategy which provided the
desired product in 7.8% overall yield. Opposed to monastrol
(M), MADTOH efficiently decreased global calcium transient
amplitude in cardiomyocytes by inhibiting L-type calcium
channels. Notably, the intermediates ADTOH and M are less
effective than the hybrid MADTOH in the control of Ca2+
homeostasis. Overall, the strategy of hybridization using the
ADTOH moiety has proven to be promising for obtaining
substances eligible to modulate calcium channels.
ˆ
´
J. C. C. Santos, T. M. de Aquino and A. de Fatima, New
J. Chem., 2019, 43, 15187–15200.
Conflicts of interest
19 Y. A. Abassi, B. Xi, W. Zhang, P. Ye, S. L. Kirstein,
M. R. Gaylord, S. C. Feinstein, X. Wang and X. Xu, Chem.
Biol., 2009, 16, 712–723.
There are no conflicts to declare.
Acknowledgements
´
´
20 E. Gonzalez-Hernandez, R. Aparicio, M. Garayoa,
´
M. J. Montero, M. A. Sevilla and C. Perez-Melero, Med.
Chem. Commun., 2019, 10, 1589–1598.
˜
`
This study was financed in part by Fundaçao de Amparo a
Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho
Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)
21 R. Wang, Physiol. Rev., 2012, 92, 791–896.
22 Y. Shen, Z. Shen, S. Luo, W. Guo and Y. Z. Zhu, Med. Cell
Longev., 2015, 925167.
23 J.-S. Bian, Q. C. Yong, T.-T. Pan, Z.-N. Feng, M. Y. Ali,
S. Zhou and P. K. Moore, J. Pharmacol. Exp. Ther., 2006,
316, 670–678.
´
´
˜
´
and Coordenaçao de Aperfeiçoamento de Pessoal de Nıvel
Superior (CAPES; Finance Code 001). ADF, LVM, SG and JSC
are supported by a CNPq Research Fellowship.
24 Y.-X. Shi, Y. Chen, Y.-Z. Zhu, G.-Y. Huang, P. K. Moore,
S.-H. Huang, T. Yao and Y.-C. Zhu, Am. J. Physiol., 2007, 293,
H2093–H2100.
25 Y. Z. Zhu, Z. J. Wang, P. Ho, Y. Y. Loke, Y. C. Zhu,
S. H. Huang, C. S. Tan, M. Whiteman, J. Lu and
P. K. Moore, J. Appl. Physiol., 2007, 102, 261–268.
26 Y.-G. Sun, Y.-X. Cao, W.-W. Wang, S.-F. Ma, T. Yao and
Y.-C. Zhu, Cardiovasc. Res., 2008, 79, 632–641.
27 R. Zhang, Y. Sun, H. Tsai, C. Tang, H. Jin and J. Du, PLoS
One, 2012, 7, e37073.
28 B. Meunier, Acc. Chem. Res., 2008, 41, 69–77.
29 S. Sestito, G. Nesi, R. Pj, M. Macchia and S. Rapposelli,
Front. Chem., 2017, 5, 72.
30 Y. Zheng, X. Ji, K. Ji and B. Wang, Acta Pharm. Sin. B, 2015, 5,
367–377.
31 G. Caliendo, G. Cirino, V. Santagada and J. L. Wallace,
J. Med. Chem., 2010, 53, 6275–6286.
32 Z. J. Song, M. Y. Ng, Z.-W. Lee, W. Dai, T. Hagen,
P. K. Moore, D. Huang, L.-W. Deng and C.-H. Tan, Med.
Chem. Commun., 2014, 5, 557–570.
33 Y. Zhao, T. D. Biggs and M. Xian, Chem. Commun., 2014, 50,
11788–11805.
34 B. Gemici, W. Elsheikh, K. B. Feitosa, S. K. P. Costa,
M. N. Muscara and J. L. Wallace, Nitric oxide, 2015, 46,
25–31.
Notes and references
1 D. M. Bers, Nature, 2002, 415, 198–205.
2 P. Szentesi, C. Pignier, M. Egger, E. G. Kranias and E. Niggli,
Circ. Res., 2004, 95, 807–813.
¨
3 T. Christ, P. Boknik, S. Wohrl, E. Wettwer, E. M. Graf,
R. F. Bosch, M. Knaut, W. Schmitz, U. Ravens and
D. Dobrev, Circulation, 2004, 110, 2651–2657.
4 J. A. Wagner, F. L. Sax, H. F. Weisman, J. Porterfield,
C. McIntosh, M. L. Weisfeldt, S. H. Snyder and
S. E. Epstein, N. Engl. J. Med., 1989, 320, 755–761.
5 J. A. Wagner, I. J. Reynolds, H. F. Weisman, P. Dudeck,
M. L. Weisfeldt and S. H. Snyder, Science, 1986, 232,
515–518.
6 C. Boixel, W. Gonzalez, L. Louedec and S. N. Hatem, Circ.
Res., 2001, 89, 607–613.
7 A. M. Katz, J. Am. Coll. Cardiol., 1996, 28, 522–529.
8 M. Sabbatini, A. Leonardi, R. Testa, L. Vitaioli and
F. Amenta, Hypertension, 2000, 35, 775–779.
9 G. M. London, B. Pannier, A. P. Guerin, S. J. Marchais,
M. E. Safar and J. L. Cuche, Circulation, 1994, 90, 2786–2796.
10 M. A. Vos, A. P. M. Gorgels, J. D. Leunissen and
H. J. Wellens, Circulation, 1990, 81, 343–349.
11 P. A. Majid and J. de Jong, Circulation, 1982, 65, 1114–1118.
12 K. Singh, D. Arora, K. Singh and S. Singh, Mini. Rev. Med.
35 L. Zhang, Y. Wang, Y. Li, L. Li, S. Xu, X. Feng and S. Liu,
Front. Pharmacol., 2018, 9, 1066.
Chem., 2009, 9, 95–106.
ˆ
´
13 A. de Fatima, T. C. Braga, L. S. Neto, B. S. Terra,
36 C. R. Powell, K. M. Dillon and J. B. Matson, Biochem.
Pharmacol., 2018, 149, 110–123.
B. G. F. Oliveira, D. L. da-Silva and L. V. Modolo, J. Adv.
Res., 2015, 6, 363–373.
This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 671--678 | 677

View Article Online
Paper
NJC
37 D. Giustarini, P. D. Soldato, A. Sparatore and R. Rossi, Free 46 S. Guatimosim, E. A. Sobie, J. S. Cruz, L. A. Martin and
Radical Biol. Med., 2019, 48, 1263–1272.
W. J. Lederer, Am. J. Physiol. Cell. Physiol., 2001, 280,
38 D. D. Perrin and W. L. F. Amarengo, in Purification of
C1327–C1339.
´
Laboratory Chemicals, ed. A. P. G. Kieboom, Oxford, London, 47 F. A. Servın, J. A. Romero, G. Aguirre, D. Grotjahn,
´
3rd edn, 1988.
39 D. L. da Silva, S. A. Fernandes and A. A. Sabino, A. de
R. Somanathan and D. Chavez, J. Mex. Chem. Soc., 2017,
61, 23–27.
ˆ
´
Fatima, Tetrahedron Lett., 2011, 52, 6328–6330.
48 B. Peng, W. Chen, C. Liu, E. W. Rosser, A. Pacheco, Y. Zhao,
H. C. Aguilar and M. Xian, Chem. – Eur. J., 2014, 20,
1010–1016.
40 T. N. Glasnov, H. Tye and C. O. Kappe, Tetrahedron, 2008,
64, 2035–2041.
41 E. Klein, S. DeBonis, B. Thiede, D. A. Skoufias, F. Kozielski 49 L.-L. Pan, M. Qin, X.-H. Liu and Y.-Z. Zhu, Front. Pharmacol.,
and L. Lebeau, Bioorg. Med. Chem., 2007, 15, 6474–6488. 2017, 8, 1–13.
42 A. Sparatore, P. del Soldato and G. Santus, New anticancer 50 D. Polhemus, K. Kondo, S. Bhushan, S. C. Bir, C. G. Kevil,
compounds, WO2009/065926 A2, 2009, 1–46.
43 X.-C. Li, W. Fan, L. Hai, S. Qian, Q.-Q. Xiao and M. Guan,
Lett. Drug Des. Discovery, 2010, 7, 747–753.
44 H. Li, M. Hao, L. Wang, W. Liang and K. Chen, Org. Prep.
Proced. Int., 2009, 41, 301–307.
T. Murohara, D. J. Lefer and J. W. Calvert, Circ.: Heart
Failure, 2013, 6, 1077–1086.
51 J. Hensley, G. E. Billman, J. D. Johnson, C. M. Hohl and
R. A. Altschuld, J. Mol. Cell. Cardiol., 1997, 29, 1037–1043.
52 G. F. Sitdikova, N. N. Khaertdinov and A. L. Zefirov, Bull.
Exp. Biol. Med., 2011, 151, 163–166.
45 S. N. Georgiades and J. Clardy, Org. Lett., 2006, 8, 4251–4254.
678 | New J. Chem., 2021, 45, 671--678
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