124
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 119–124
21. stable cell line was constructed by transfecting T-Rex 293 cells (Life
A
methyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide (21), a no-
vel, selective Cav3.2 calcium channel blocker. Based on its favour-
able in vitro profile as well as its demonstrated in vivo efficacy,
21 was selected for a proof-of-concept study in dogs to verify the
cardioprotective role of Cav3.2 blockade in AF. The results of a
head-to-head comparison of 21 and mibefradil in a dog model of
AF will be published soon. Based on the favourable selectivity
and distribution profile, 21 could represent a useful chemical probe
to verify the pathophysiological effects of peripheral T-type cal-
cium channel inhibition.
Technology Corp., Carlsbad, CA) with a pcDNA4TO vector encoding human
CACNA1H. Patch-clamp measurements were performed in the standard whole
cell configuration using a QPatchHTX instrument (Sophion Bioscience A/S,
Ballerup, Denmark). The extracellular solution contained (mM): NaCl 145, KCl
4, CaCl2 2, MgCl2 1, HEPES 10, glucose 10 (pH 7.4) and the intracellular (mM):
KCl 120, MgCl21.75, CaCl2 5.374, EGTA 10, HEPES 10, Na2ATP 4 (pH 7.2). All
experiments were performed at room temperature. The cells were depolarized
from a holding potential (Vhold) of ꢀ100 or ꢀ80 mV to ꢀ20 mV.
22. The extent of a compound entering the brain is given as the ratio between the
unbound fraction of this compound in the brain and the unbound fraction in
the blood using the following equation: Cubr/Cupl = Cbr x fubr/Cpl x fupl , where
Cubr and Cupl are the unbound concentrations in brain and plasma, Cbr and Cpl
are the total concentrations in brain and plasma and fubr and fupl are the free
unbound fractions in brain and plasma respectively. Two separate experiments
are run in order to calculate the Cubr/Cupl ratio: (i) The in vitro measurement of
the unbound volume of distribution (Vubr) and the free fraction (fubr) in the
brain using a Brain Slice Assay. The in vitro Brain Slice assay employs freshly
prepared slices from rat brain. The slices are incubated in buffer solution
spiked with a cassette of compounds for 5 h at 37 °C. The concentrations are
measured (slice and buffer) by LC/MS/MS and the unbound volume of
distribution and the free fraction of the test compound are calculated. (ii)
The compound concentrations in blood and brain are obtained from the
terminal sampling of blood and brain collected after iv infusing female
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
Sprague-Dawley rats for 4 h. The infusion doses are 2 lmol/kg/h. The brain is
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the brain) when calculating the Cbr/Cpl ratio.
23. Test compounds (6 lL of 50 lM DMSO/MeCN) are incubated with liver
microsomes⁄ (1 mg/mL in 0.1 M phosphate buffer pH 7.4) and NADPH
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T1/2 = Ln2/–slope
CLint = Ln2ꢁ1000/T1/2ꢁ[protein]
24. Ratio obtained by dividing the total integrated peak area of glutathione
adducts of the test compound by the integrated peak area of the major
glutathione adduct of the control compound clozapine (average value, N = 3).
RM formation is ranked as follows: ratio 1 = ‘High’; Ratio between 0.25–
1 = ‘Medium’; Ratio <0.25 = ‘Low’.
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27. NMR data for 21: 1H NMR (400 MHz, DMSO-d6) d 8.52 (t, 1H), 7.86 (d, 2H), 7.82
(t, 1H), 5.80 (s, 1H), 5.52 (t, 1H), 4.51 (t, 1H), 3.31–3.24 (m, 4H), 3.08–3.01 (m,
2H), 2.42–2.25 (m, 6H), 1.48–1.33 (m, 4H), 1.20 (s, 9H).
28. A large scale synthesis used for making 50 g of compound 21 is depicted in
Scheme 4. This route is with the exception for the last step, a chromatography
free process. Instead, purifications of intermediates are done through
crystallisations or alternatively, intermediates are used as such in the next
step without further purification. The reduction of the nitrile 24 to the
corresponding amine 25 turned out to be a major challenge as dimerisation
easily occurs between the amine 25 and the starting material 24. The solution
to this problem was to perform the reaction in the presence of NH3 which
efficiently suppressed this background reaction and gave a clean reaction when
performed under continous flow conditions in an H-cube apparatus. In the last
step in the transformation of the salt 27 to the urea 21 we also found that one
major byproduct turned out to be the result of a dimerisation of the starting
material 27 with the product 21. This byproduct along with various other
minor unidentified byproducts could not be removed to an acceptable level
through crystallisation only. Instead, we had to employ chromatography
followed by a final crystallisation from EtOH which gave the desired compound
21 in 99% purity by HPLC. Full description of methods and experimental data
available as Supplementary data.
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20. To avoid the confounding effect of baroreflex changes in HR, the dogs were
subjected to autonomic blockade by pretreatment with Metoprolol (0.5 mg/kg
iv) and Methylscopolamine (0.018 mg/kg iv) 5 min prior to start of 21 or
vehicle (3% HPbCD) infusion. 21 or vehicle (0.1 mL/kg/min) was infused over
20 min followed by 60 min of washout. One week later, the animals were
crossed-over to vehicle or 21 infusion (i.e., the animal that received 21 was
given vehicle, and vice versa). Blood was taken for analysis of plasma levels of
21 before start of infusion, at 10 and 19 min of infusion as well as at 10, 30, and
60 min of washout. Lead II ECG was registered and heart rate was analysed
using PharmLab (AstraZeneca) software. Effect of 21 on HR was normalized
based on the effect recorded for the vehicle in each animal.