Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.06.092

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1- yl]acetamide, a novel, selective T-type calcium channel (Ca_v3.2) inhibitor with in vivo antihypertensive effect in rats.

Full text of DOI:10.1016/j.bmcl.2011.06.092

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5557–5561
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives
as T-type calcium channel (Cav3.2) inhibitors
Fabrizio Giordanetto a, , Laurent Knerr a, Nidhal Selmi a, Antonio Llinàs b, Anders Lindqvist c,
⇑
Qing-Dong Wang c, Pernilla Ståhlberg a, Fredrik Thorstensson a, Victoria Ullah a, Kristina Nilsson a,
Gavin O’Mahony a, Ågot Högberg c, Emma Lindhardt b, Annika Åstrand c, Göran Duker c
a Medicinal Chemistry, AstraZeneca R&D CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b DMPK, AstraZeneca R&D CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
c Bioscience, AstraZeneca R&D CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-
tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Cav3.2)
inhibitor with in vivo antihypertensive effect in rats.
Received 18 May 2011
Revised 16 June 2011
Accepted 19 June 2011
Available online 30 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Calcium channel inhibition
Cav3.2
T-type calcium channels
Calcium influx across the cellular membrane is partly controlled
by a family of transmembrane proteins termed voltage-gated cal-
cium channels. These are activated by changes in electrical poten-
tial difference across the membrane and have been classified into
different subtypes according to various considerations: Cav1.x
(L-type), Cav2.x (N-, P/Q- and R-type), and Cav3.x (T-type).1
The T-type class is characterized by fast inactivation (transient)
and small conductance (tiny), and is composed of three members
Mibefradil (Fig. 1), the archetypal T-type channel inhibitor, was
launched in 1997 as an effective antihypertensive agent only to be
later withdrawn from the market due to the severity of observed
drug–drug interactions. As part of our efforts to discover novel
treatments for atrial fibrillation (AF), we were particularly intri-
gued by Mibefradil’s ability to protect against atrial remodeling,
a key contributing factor to AF’s own exacerbation, in a dog model
of AF.5,6 Although the ion channel selectivity of Mibefradil has been
questioned in vitro and in vivo, we hypothesized that a T-type
selective inhibitor could afford a similar cardioprotective effect to
Mibefradil. This is corroborated by the fact that Diltiazem (an
L-type calcium channel inhibitor) did not show any cardioprotec-
tive effect in the same AF dog model.6 Furthermore, as Cav3.2 is
the main T-type isoform expressed in the heart, we resolved to
identify Cav3.2 inhibitors to verify our initial hypothesis.
based on the different main pore-forming
a1 subunit: Cav3.1
(a
1G), Cav3.2 ( 1H) and Cav3.3 (
a
a
1I).1 While Cav3.1 and Cav3.3
are mainly expressed in the brain, Cav3.2 is found in brain and
peripheral tissues (e.g., heart, kidney, liver).2
In addition to their implications in a multitude of disease states,
T-type channels were proposed as therapeutic targets for a number
of cardiovascular afflictions including hypertension,3 angina pecto-
ris,3 heart failure,4 and atrial fibrillation.5,6 Specifically, in the car-
diovascular system, T-type calcium channels are mainly involved
in cardiac pacemaking7,8 and vascular smooth muscle contraction
regulation.9,10 Although several groups have reported T-type chan-
nels inhibitors (for a recent review see11), the cardiovascular phar-
macology of T-type blockers has remained elusive due to the
difficulties in characterizing the T-type current (ICaT) in the human
heart.12
High-throughput electrophysiology screening using a QPatchHTX
instrument (Sophion Bioscience A/S, Ballerup, Denmark) and hit-
triaging identified fragment 1 as a weak Cav3.2 inhibitor (IC50
:
70 M). Nevertheless, we considered it an attractive starting point
l
for further chemical evolution due its favorable size and lipophilicity
attributes (MW: 277; c log P: 1.7), and the readily availability of
structure–activity relationships (SAR) from nearest fragment neigh-
bors. We also realized the structural similarity between 1 and TTA-
P1, a T-type inhibitor previously reported by Merck,13 and decided
to verify whether potency improvements could be afforded through
series hybridization. As a result, compounds 2–4 were prepared
according to Scheme 1. Progressive growth of the alkyl group
⇑
Corresponding author. Tel.: +46 31 7065723; fax: +46 31 7763710.
E-mail address: fabrizio.giordanetto@astrazeneca.com (F. Giordanetto).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.092

5558
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5557–5561
O
O
O
O
Cl
N
O
NH
N
H
N
N
N
H
N
F
H
N
Cl
F
Mibefradil
TTA-P1
1
Figure 1. Selected T-type calcium channel inhibitors (Mibefradil, TTA-P1) and initial fragment hit 1.
a
R1
Table 2
N
O
Cav3.2 potency and metabolic stability for compounds in the ‘adamantane replace-
ment’ set
1-4, 20-29
N
NH2
N
H
R1 = Me,
R1 = Boc
O
N
b
R1
*
N
N
H
R1 = H
c,d
R1 = CH2R2
a
b
Compound R1
Cav3.2 IC50
M)
HLM Clint
mg)
(lL/min/
Scheme 1. General synthesis of diverse N-alkylated piperidines. Reagents and
conditions: (a) (i) 2-chloroacetyl chloride, DIEA, DCM 0–20 °C, 16 h, 92%, (ii) tert-
butyl piperazine-1-carboxylate or 1-methylpiperazine, K2CO3, acetonitrile, reflux,
4 h, 85–90%; (b) TFA, DCM, 20 °C, 16 h, 90%; (c) R2CHO, NaBH(OAc)3, CH3COOH,
MeOH, 20 °C, 16 h or R2CH2Br/Cl, K2CO3, acetonitrile, reflux, 5 h (or 120 °C,
45 min) or (i) R2CH2OH, Dess–Martin periodinane, NaHCO3, DCM, 20 °C, 3 h , (ii)
NaBH(OAc)3, DCM, 20 °C, 16 h, 24–81%; (d) For 29: NaBH4, MeOH, 20 °C, 2 h, 82%.
(
l
4
0.8
>30
2
48
<5
36
lw,
HO
*
5
6
Table 1
*
Cav3.2 potency and ligand efficiencies for compounds 1–4
R1
*
N
O
7
8
14.6
8.8
<5
26
N
OH
N
H
*
Compound
R1
Cav3.2
LEb
LipEc
a
IC50 (lM)
9
7.3
12.5
3
*
*
1
2
H
Me
70
30
0.28
0.29
2.5
3.3
10
11
12
15.2
1.5
3
2
0.31
2.5
*
*
33
<5
*
*
4
0.8
0.32
2.5
N
>30
Mibefradil
TTA-P1
1
0.2
0.23
0.37
ꢀ0.4
1.1
Cl
a
Results are mean of at least two experiments. Experimental errors within 20% of
13
0.9
<5
value.22
b
Ligand efficiency (LE): calculated as ꢀRTln(IC50)/HAC.
Cl
*
c
Lipophilicity efficiency (LipE): calculated as pIC50ꢀc log P.
a
Results are mean of at least two experiments. Experimental errors within 20% of
value.22
b
Rate of disappearance in human liver microsomes, measured from 45 min
incubation with human hepatic liver microsomes (1 mg/mL at 37 °C).23
NH
N
O
a,b
N
c
O
N
N
O
HN
O
substituting the piperazine N4 atom of 1 resulted in incremental
Cav3.2 inhibition gains, as detailed in Table 1.
d
Having succeeded in improving Cav3.2 potency to a level com-
parable with Mibefradil (Table 1), we set out to systematically
modify the adamantane ring of 4 (Scheme 2) to verify the effect
on Cav3.2 potency and metabolic stability, as metabolite identifica-
tion analysis indicated it as the main site of oxidative metabolism.
The results from such exploration are summarized in Table 2.
The initial attempts to replace the adamantyl group by known,
less bulky and lipophilic surrogates,14–17 were met with limited suc-
cess (5–8, Table 2). These initial results also clearly indicated that
introduction of polarity in that region, while beneficial to metabolic
stability, was deleterious to Cav3.2 potency, as exemplified by the
N
O
e
N
O
N
HO
N
R1
N
H
4-13
Scheme 2. Synthesis of the ‘adamantane replacement’ set. Reagents and condi-
tions: (a) 3,3-dimethylbutyraldeyde, NaBH(OAc)3, MeOH, 20 °C, 16 h; (b) TFA, DCM,
20 °C, 95% over two steps; (c) benzyl bromoacetate, K2CO3, acetone, 60 °C, 40 h,
92%; (d) H2, 10% Pd/C, EtOH, 20 °C, 2 h, 95%; (e) R1NH2, EDAC, HOBt, DCM/water
(7:2), 20 °C, 16 h, 5–60%.

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5557–5561
5559
Table 3
Cav3.2 potency, metabolic stability, hERG and CYP450 2D6 inhibition for compounds in the ‘core variation’ set
O
R1
CORE
N
H
c
Compound
R1
CORE
Cav3.2
hERG
IC50
HLM Clint
(
lL/min/mg)
CYP450 2D6d
(lM)
a
b
IC50
0.8
0.9
0.5
0.3
0.8
0.2
0.9
(l
M)
(
lM)
4
1-Adamantyl
N
N
N
N
N
N
N
N
32
1.7
15
1.6
15
1.2
26
48
<5
59
13
38
<5
85
>20
1
N
*
*
*
*
*
*
*
*
*
13
14
15
16
17
18
3,5-Dichlorophenyl
1-Adamantyl
N
*
19
0.8
10
0.5
9
N
*
3,5-Dichlorophenyl
1-adamantyl
N
*
N
*
3,5-Dichlorophenyl
1-Adamantyl
N
*
N
*
N
*
19
1-Adamantyl
1.5
—
210
13
a
b
c
Results are mean of at least two experiments. Experimental errors within 20% of value.22
Patch clamp assay using IONWORKS™ technology in hERG-expressing CHO cells.
Rate of disappearance in human liver microsomes, measured from 45 min incubation with human hepatic liver microsomes (1 mg/mL at 37 °C).23
d
Inhibition of metabolic degradation of MAMC (7-methoxy-4-(aminomethyl)-coumarine) by human recombinant cytochrome P450 2D6 at 37 °C—The percent inhibition is
determined at five different concentrations and reported as IC50
.
O
matched pairs 4–5 and 6–7 (Table 2). The difficulty in maintaining
potency and improving metabolic stability was subsequently con-
firmed by attempting a more systematic simplification of the ada-
mantly ring with 1,4-dimethypentyl (8), 2-norbornyl (9) and
cyclohexyl (10) moieties. Substitution on the 4 position of cyclohex-
ane had again the opposite effect on the potency-metabolic stability
equation (cf. 11 and 12, Table 2). Aromatic replacement (13) of the
adamantly ring proved successful in combining metabolic stability
a
b
Br
N
NH2
Cl
H
A
Cl
O
Cl
Cl
Cl
N
NH2
H
B
c,d
e
O
N
13
HN
NH
NH
(HLM Clint: <5 lL/min/mg) with Cav3.2 potency (IC50: 0.9 lM).
N
O
We then turned our attention to the central core structure and
explored substituted piperazine rings in an effort to reduce metab-
olism originating from N-dealkylation mechanisms, as shown in
Table 3. Compounds 14–19 were prepared using standard methods
as described in Scheme 3.
Overall, crowding of each of the piperazine nitrogen atoms did
not significantly decrease oxidative metabolism of the compounds
in the 1-adamantyl subseries, as exemplified by 14, 16, and 18 in
Table 3. As previously discussed, the 3,5-dichlorophenyl moiety
offered a substantial advantage in terms of metabolic stability over
c,d
e,d
e
O
O
14,15
N
HN
N
N
c
O
O
16,17
18
N
HN
R1
NH
N
H
O
the 1-adamantyl ring, while displaying
a tendency towards
e
c,d
f
O
N
HN
NH
increased Cav3.2 potency (cf. 13 and 4, Table 3). However, it also
yielded a consistent worsening of hERG and CYP450 2D6 inhibition
compared to the 1-adamantyl, as exemplified by the matched pairs
4–13, 14–15 and 16–17 in Table 3. These findings are in agreement
with earlier reports on the liabilities associated with aromaticity in
drug-like molecules.18,19
N
O
19
NH
.2HCl
HN
The original adamantyl-containing Cav3.2 inhibitor 4 offered
the best combination of Cav3.2 potency, selectivity over the hERG
ion channel and CYP450 enzymes, but displayed suboptimal meta-
bolic stability. Having failed to improve on the overall profile of the
ligands with chemical modifications of the adamantane and piper-
azine rings, we focused on the neohexyl side chain to reduce the
degree of oxidative metabolism. Compounds 20–29 were designed
to reduce the lipophilic character of the ligands and thus improve
metabolic stability, as summarized in Table 4.
Scheme 3. Synthesis of the ‘core variation’ set. Reagents and conditions: (a) 2-
bromoacetyl bromide, Na2CO3, DCM, ꢀ65 to 20 °C, 16 h, 77%; (b) 2-chloroacetyl
chloride, DIEA, DCM 20 °C, 16 h, 93%; (c) (i) 3,3-dimethylbutyraldehyde, NaB-
H(OAc)3, MeOH, 20 °C, 16 h, (ii) TFA, DCM, 20 °C, 2–16 h, 76–95% over two steps; (d)
(i) A or B, K2CO3, acetonitrile 110 °C, lw, 10 min or DIEA, DCM, 20 °C, 16 h or N,N-
(diisopropyl)aminomethylpolystyrene, DCM, 20 °C, 16 h, (ii) TFA, DCM, 20 °C,
2–16 h, 40–90% over two steps; (e) 3,3-dimethylbutyraldehyde, NaBH(OAc)3,
MeOH, 50 °C, 16 h, 5–20%; (f) A or B, K2CO3, acetonitrile 110 °C,
lw, 10 min,
25–90%; (g) (i) A, DIEA, DCM, 20 °C, 16 h, (ii) 3,3-dimethylbutyraldehyde,
NaBH(OAc)3, MeOH, 20 °C, 16 h, <5% over two steps.

5560
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5557–5561
Table 4
Table 5
Cav3.2 potency, metabolic stability and hERG inhibition for compounds in the
‘neohexyl variation’ set
In vitro/vivo comparison of 25 and Mibefradil
25
Mibefradil
R1
a
N
O
Cav3.2 IC50
Cav1.2 IC50
hERG IC50
(
(
l
l
l
M)
M)
M)
2
1
3
b
N
>31.6
20
>33
>20
<8
53
39
5/2
42.5
59
N
H
c
(
3.3
3.8, 22, 24
0.06 (3A4, 2D6)
57
0.5
2.5
1/3
59.3
50
c
Nav1.5, IKs, Kv4.3 IC50
‘Worst’ CYP450 IC50
(
M)
l
M)
d
(l
c
Compound R1
Cav3.2
IC50
hERG
IC50
HLM Clint
mg)
(lL/min/
e
HLM Clint
(
lL/min/mg)
a
b
f
Human PPB Fu (%)
(lM)
(lM)
f
Rat PPB Fu (%)
Rat dose iv/pog
(
l
mol/kg)
Rat CLg (mL/min/kg)
4
0.85
3.6
32
14
48
94
*
Rat Fg (%)
O
N
a
Results are mean of at least four experiments. Experimental errors within 20%
20
of value.22
*
b
Patch clamp assay using IONWORKS™ technology in CHO cells expressing
CACNA1C, CACNB2 and CACNA2D1 (ChanTest, Cleveland, OH).24
N
NDd
NDd
NDd
NDd
<5
Patch clamp assay using IONWORKS™ technology in CHO cells expressing the
c
21
5.0
30
12
N
*
relevant human ion channel.
d
Inhibition of metabolic degradation of the corresponding substrate by human
N
recombinant cytochrome P450s (3A4, 2C9, 2C19, 2D6, 1A2, 2C8) at 37 °C—The
percent inhibition is determined at five different concentrations and reported as
22
*
IC50
.
O
e
N
Rate of disappearance in human liver microsomes, measured from 45 min
23
<5
incubation with human hepatic liver microsomes (1 mg/mL at 37 °C).23
*
f
% Fraction unbound in plasma measured by equilibrium dialysis (18 h at 37 °C).
Pharmacokinetic parameters calculated from noncompartmental analysis con-
g
O
24
9
30
30
centrations in fasted Sprague–Dawley female rats (iv n = 12; po n = 2).
*
O
25
2
20
<5
72
profile in rats (Table 5). Additionally, a selectivity screening includ-
ing 98 biological targets20 did not reveal any significant hits (>50%
*
O
26
1.7
>33
*
binding) other than the
centration of 10 M.
r1 receptor, when 25 was tested at a con-
l
O
27
1.9
5.8
33
33
21
69
As Cav3.2 calcium channels are highly expressed in the sinoatri-
al node and are known to contribute to cardiac pacemaking in the
adult heart,8 in vivo Cav3.2 inhibition would result in heart rate
(HR) reduction. This therefore provides a practical surrogate bio-
marker to evaluate target engagement in vivo, and to derive phar-
macokinetic–pharmadynamic (PK–PD) relationships for Cav3.2
inhibitors. We thus hypothesized that free plasma concentrations
of a Cav3.2 inhibitor equivalent to the IC50 measured in vitro
(assuming no considerable species differences wrt potency) would
be sufficient to elicit a significant HR reduction in vivo. Accord-
*
N
28
*
29
0.9
33
18
*
OH
a
Results are mean of at least two experiments. Experimental errors within 20% of
value.22
ingly, based on in vitro Cav3.2 potency (IC50: 2
tein binding (Fu: 39%) and the preliminary rat PK profile (Table 5), a
50
mol/kg dose of 25 was infused to rats,21 as summarized in
Figure 2.
Thirty minutes infusion of 25 significantly reduced HR com-
lM), rat plasma pro-
b
Patch clamp assay using IONWORKS™ technology in hERG-expressing CHO
cells.
l
c
Rate of disappearance in human liver microsomes, measured from 45 min
incubation with human hepatic liver microsomes (1 mg/mL at 37 °C).23
pared to vehicle (p <0.05). After termination of compound infusion,
HR tended to recover toward baseline (Fig. 2). According to the
expected physiology of the rat system employed, the HR reduction
Incorporation of heteroaryl groups in the side chain (20 and 21)
had a negative effect on Cav3.2 potency, and the most potent deriv-
ative (20) still displayed very high intrinsic clearance, as shown in
Table 4. On the other hand, compounds containing saturated het-
erocycles (22–25) showed increased metabolic stability, whereas
the impact on Cav3.2 affinity was variable. Replacement of a mor-
pholine group with a tetrahydropyran (23 and 25, respectively)
led to
a 8–26% blood pressure reduction during compound
infusion. The HR reduction caused by 25 occurred in a concentra-
tion-dependent fashion with an effective unbound concentration
causing 10% HR reduction estimated to be 1.9 lmol/L ( Fig. 2).
While there might be differences between in vitro and in vivo
proved successful in combining acceptable potency (Cav3.2 IC50
:
potencies as well as potency differences across species, the free
2
l
M) and metabolic stability (HLM Clint: <5 L/min/mg). Hetero-
l
plasma concentrations of 25 did not exceed 4 lmol/L in this exper-
aliphatic side chains displayed improved Cav3.2 potency and selec-
tivity over the hERG channel but, unfortunately, moderate to high
intrinsic clearance (26–29, Table 4). The two enantiomers of com-
pound 29 were separated by chiral HPLC but displayed similar po-
tency and clearance values (data not shown).
Compound 25 emerged as an adequate Cav3.2 inhibitor for fur-
ther profiling, as outlined in Table 5. Compared to Mibefradil, it
showed enhanced selectivity over a range of ion channels of car-
diovascular importance (notably the L-type calcium channel—
Cav1.2) and CYP450 enzymes, and a comparable pharmacokinetic
iment, thus minimizing the risk for off-target-mediated effects (cf.
Table 5).
In summary, starting from a HTS-based fragment hit, a novel ser-
ies of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide-con-
taining Cav3.2 inhibitors was designed by systematic chemical
variation of its structural elements. Simultaneous optimization of
Cav3.2 potency, ion channel and CYP450 selectivity, as well as
metabolic stability identified N-(1-adamantyl)-2-[4-(2-tetrahydro-
pyran-4-ylethyl)piperazin-1-yl]acetamide (25) as an in vivo effica-
cious Cav3.2 inhibitor. Further medicinal chemistry optimization

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5557–5561
5561
Figure 2. HR effect in anaesthetized rats, following infusion of 50
relationship: arrows denote time course of infusion. Error bars indicate standard deviation of measurements.
l
mol/kg of 25 (solid squares). (left) Comparison to vehicle (squares): (⁄) p <0.05 (#) p <0.02; (right) PK–PD
ventilated with a Ugo ventilator at a frequency of 60 cycles/min and 2 mL/
breath. A PE50 catheter was put into the jugular vein for substance infusion
to validate the hypothesis of a Cav3.2-mediated cardioprotective ef-
fect in atrial fibrillation will be published in due course.
and another PE50 catheter into
a carotid artery for blood pressure
measurements and blood sampling. To achieve autonomic blockade, both left
and right vagal nerves were cut and Metoprolol (5 mg/kg iv) was given 20 min
prior to start of substance infusion. Test compound or vehicle (0.1 mL/kg/min)
was infused over 31 min followed by 75 min of washout. Blood was taken, for
analysis of plasma level of test compound, before start of infusion and at 5, 15,
25, and 30 min of infusion as well as 5, 1 0, 30, 50, and 75 min of washout. Lead
II ECG was registered and heart rate was analyzed using PharmLab
(AstraZeneca) software.
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A stable cell line was constructed by transfecting T-Rex 293 cells (Life
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20. Ricerca Biosciences, LLC 7528 Auburn Road Concord, OH 44077 US A.
21. Adult Sprague–Dawley rats (BW 300–350 g, N = 3) were anaesthetized with
ꢀ20 mV. Compound 25 blocked the t-type current in
dependent manner with an IC50 of 2.0 0.4
M (n = 4) at Vhold = ꢀ100 mV
and 3.7 0.6
a concentration
l
lM
(n = 5) at ꢀ80 mV (p <0.05 vs Vhold = ꢀ100 mV). The
compound had no effect on the current voltage relationship, steady-state
activation (V½ = 45 3 vs 46 1 mV, n = 5, p = n.s.), steady-state inactivation
(V½ 60 3 vs 59 2, n = 5, p = n.s.) or recovery after inactivation ( = 299 27
vs 303 26 ms, respectively, n.s.).
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
(0.5 mM in phosphate buffer pH 7.4) in a 96-well plate at 37 °C. An aliquot
of the incubation mixture is taken after 0, 5, 15, and 45 min and the reaction is
stopped with cold acetonitrile containing volume marker (1 mM 5,5-diethyl-1,
3-diphenyl-2-iminobarbituric acid). The samples are centrifuged at 4000 rpm
and supernatant is transferred and diluted with water in 96 well plates. The
intrinsic metabolic capacity, measured as intrinsic clearance (CLint) gives an
indication of the contribution of hepatic oxidative metabolism to the overall
clearance of the test compound. Calculation of CLint is based on the substrate
disappearance rate. The chromatographic peaks are integrated and a linear plot
of Ln areas vs. time is created. The slope is calculated for each data set. T1/2 and
CLint are calculated according to the following equations:
T1=2 ¼ Ln2=—slope
CLint ¼ Ln2 ꢁ 1000=T1=2 ꢁ ½proteinꢂ
24. Dilbaghi, S.; Abi-Gerges, N.; Morton, M. J.; Bridgland-Taylor, M. H.; Pollard, C.
E.; Valentin, J.-P. J. Pharmacol. Toxicol. Methods 2010, 62, e1.
pentobarbital sodium and placed on
a heating pad to keep the body
temperature at 38 °C throughout the experiment. The animals were