3,4-Diarylpiperidines as potent renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.044

The discovery and SAR of a series of potent renin inhibitors possessing a novel 3,4-diarylpiperidine scaffold are described herein. The resulting compound 38 exhibit low nanomolar plasma renin IC₅₀, had a clean CYP 3A4 profile and displayed mic

Full text of DOI:10.1016/j.bmcl.2012.01.044

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1953–1957
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3,4-Diarylpiperidines as potent renin inhibitors
⇑
Patrick Lacombe , Mélissa Arbour, Renée Aspiotis, Elizabeth Cauchon, Austin Chen, Daniel Dubé,
Jean-Pierre Falgueyret, Pierre-André Fournier, Michel Gallant, Erich Grimm, Yongxin Han, Hélène Juteau,
Suzanna Liu, Christophe Mellon, Yeeman Ramtohul, Daniel Simard, René St-Jacques, Gavin Chit Tsui
Merck Frosst Center for Therapeutic Research, PO Box 1005, Pointe Claire-Dorval, Québec, Canada H9R 4P8
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and SAR of a series of potent renin inhibitors possessing a novel 3,4-diarylpiperidine scaf-
fold are described herein. The resulting compound 38 exhibit low nanomolar plasma renin IC50, had a
clean CYP 3A4 profile and displayed micromolar affinity for the hERG channel. Furthermore, it was found
to be efficacious in the double transgenic rat hypertension model and show good to moderate oral bio-
availability in two animal species.
Received 4 November 2011
Revised 12 January 2012
Accepted 13 January 2012
Available online 26 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Renin
Inhibitor
Anti-hypertensive
Treatment of hypertension, one of the major risk factors for car-
diovascular diseases, has been an area of intense research for many
years. Despite this, most patients still cannot achieve the targeted
140/90 mmHg as recommended by the American Heart Associa-
tion (AHA) using the currently available therapies (i.e. diuretics,
b-blockers, calcium channel blockers, ACE inhibitors, AT1R antago-
nists, etc.).1 Disruption of the renin–angiotensin–aldosterone sys-
tem (RAAS) at its various points (Fig. 1) has proven to be an
effective strategy for the lowering of blood pressure.2 Inhibition
of renin, the enzyme responsible for the cleavage of angiotensino-
gen to angiotensin I, which is the first and rate limiting step of the
RAAS pathway, should then afford the maximum blood pressure
lowering effect.3 Furthermore, it is believed that the inhibition of
renin should have fewer mechanism-related side effects than the
current therapies targeting downstream events of the RAAS path-
way.4 Indeed, significant research effort and resources from the
pharmaceutical industry have been invested toward the discovery
of a renin inhibitor suitable for the treatment of hypertension.
Many compounds have entered clinical development,5 but so far,
only Aliskiren has received FDA approval (2007) for the treatment
of hypertension.6
channel encoded by the human ether-a-go-go gene (hERG) and
inhibited P450 CYP3A4 both reversibly and in a time-dependent
manner. Part of our efforts toward overcoming these two liabilities
Previously, we described the discovery of a new diaryl scaffold
generating potent renin inhibitors with compound 1 (Fig. 2) as a
representative example.7 Although 1 showed good potency against
human renin and displayed interesting pharmacokinetic proper-
ties, two major liabilities were revealed when it was further
profiled. Namely, 1 demonstrated a high affinity for the potassium
⇑
Corresponding author.
E-mail address: lacombepatrick@hotmail.com (P. Lacombe).
Figure 1. Renin–angiotensin–aldosterone system (RAAS).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.044

1954
P. Lacombe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1953–1957
the tertiary alcohol derivatives 34 and 35 (Scheme 4), can be ob-
tained using the synthetic routes described in Scheme 2. Starting
from commercially available alcohol 9, it can be converted to ben-
zyl ether 10 by treatment with 4-bromobenzyl bromide under base
promoted alkylation conditions. A two-step reduction–alkylation
sequence then afforded the desired intermediate 12. Starting from
known alcohol 13,10 it can be converted to methyl ether 14 by
treatment with MeI under base promoted alkylation conditions,
followed by TBAF-mediated removal of the silyl protecting group.
The resulting alcohol 14 was then converted to the desired phenyl
bromide 15 as described previously. Furthermore, phenyl bromide
derivative 18 could be accessed via a three-step sequence from
commercially available carboxylic acid 16. Indeed, borane reduc-
tion, Dess–Martin periodinane (DMP) oxidation and acetal forma-
tion under acidic conditions led to the desired aryl bromide 18.
The spirocyclic derivatives 27 and 44 were prepared according
to the synthetic route described in Scheme 3. Chemoselective
Suzuki cross-coupling reaction of 2-bromo-5-iodotoluene 19 with
commercially available 2-hydroxymethylbenzene boronic acid
afforded, after alcohol protection, the requisite diaryl intermediate
20. The latter was then converted to the desired keto-piperidine 21
Cl
Cl
O
O
O
truncation and
piperidine ring-
formation
Ar
Cl
O
NH
N
H
R
NH
H2N
1
Figure 2. 3,4-Diarylpiperidine as primary amine replacement.
was recently published.8 In an extension of this work, we report
herein our discovery of a novel series of 3,4-diarylpiperidine as po-
tent renin inhibitors.
Our initial SAR efforts were focused on the removal of the
northern lipophilic appendage of compound 1 (Fig. 1). However,
in order to minimize the magnitude of the anticipated loss in po-
tency associated with this truncation, the primary amine warhead
of 1 was replaced by a piperidine moiety. Indeed, as was demon-
strated in a previous publication,9 decreasing the number of freely
rotatable bonds has resulted in piperidine analogs with a tendency
to be more potent in vitro towards renin and more efficacious in
double transgenic rats than their acyclic counterparts. A series of
3,4-diarylpiperidines as represented in Tables 1 and 2 were then
prepared following the synthetic route described in Scheme 1.
Briefly, the Michael addition of 2-chloro-4-bromophenylacetonitri-
le 2 to a suitable ethyl aryl acrylate led to a diastereoisomeric mix-
ture of the desired intermediate 3. The latter was then cyclized
under acidic conditions to afford the racemic trans diastereomer
4. Subsequently, cyclic imide 4 was reduced with borane, protected
as the tert-butyl carbamate, and resolved to give the the enantio-
merically pure, trans-3,4-diarylpiperidine scaffold 5. Finally, Suzuki
cross-coupling of 5 with a suitable 2-substituted phenyl boronic
acid 6 afforded compound 7. Elaboration of derivative 7 could be
carried out by first unmasking the terminal primary amine func-
tionality via hydrogenation followed by acylation and Boc-depro-
tection with hydrochloric acid to give the desired amine 8.
In addition, the requisite 4-substituted phenyl bromides for the
preparation of the spirocycle derivative 27 (Scheme 3), as well as
using a Buchwald–Hartwig palladium catalyzed a-arylation proto-
col.11 Further elaboration of 21 was then accomplished with
Grignard reagents freshly prepared from either compound 16 to af-
fords the hydroxy-carboxylic acid derivative or 18 to afford com-
pound 22 which, following acidic treatment, gave the lactone
(not shown) or lactol 23, respectively. Reduction of lactol 23 with
lithium borohydride and removal of the silyl protecting group
afforded triol 24 which upon treatment with methanesulfonyl
chloride could be readily converted to spirocyclic ether 25. The
benzylic mesylate group of 25 was then displaced with cyanide, re-
duced with cobalt borohydride12 prepared in situ from cobalt(II)
chloride hexahydrate and sodium borohydride, and acetylated. Fi-
nally, resolution using preparative chiral HPLC afforded, following
Boc-deprotection with hydrochloric acid, the desired acetamide 27.
In addition, the tertiary alcohols 34 and 35 were prepared via
the route described in Scheme 4. Briefly, Buchwald–Hartwig a-ary-
lation of Boc-piperid-4-one with 2-bromo-4-hydroxytoluene (28)
readily delivered ketopiperidine 29. Subsequent triflate formation
and Suzuki cross-coupling reaction with aryl boronate ester 6 gave
diaryl 30. Hydrogenation in presence of acetic anhydride then
afforded the requisite derivative 31. Finally, addition of a suitable
Grignard reagent prepared from either aryl bromide 12 or 15 affor-
Table 1
In vitro data for compounds 36–42
Y
Cl
HN
O
N
H
R
Compounds
Y
R
Renin buffer
IC50 (nM)
Renin plasma
IC50 (nM)
hERG
IC50 (lM)
CYP 3A4
Time-dependent inhibitionc
(% activity loss @ 10 M)
a,b
a,b
a
Reversible inhibitionc
(% activity @ 10
lM)
l
36
37
38
39
40
41
42
3-4-Difluorophenyl
Pyridin-3-yl
Me
Me
Me
Et
OMe
OEt
CH2OMe
0.5
1.4
2.1
7.5
3.6
3.4
8.0
6.1
1.1
4.8
5.8
2.8
10.3
3.2
—
98
98
97
92
92
91
—
49
55
0
21
46
58
—
1-Methyl-4-pyridinyl-2-one
1-Methyl-4-pyridinyl-2-one
1-Methyl-4-pyridinyl-2-one
1-Methyl-4-pyridinyl-2-one
1-Methyl-4-pyridinyl-2-one
4.8
3.52
11.0
73.8
28.2
97.4
a
b
c
Values are means of at least two experiments.
See Ref. 16 for assay protocol.
See Ref. 17 for assay protocol.

P. Lacombe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1953–1957
1955
Table 2
In vitro data for tertiary alcohol and ether derivatives
F
R1
O
R1
F
HO
HN
O
O
NH
N
H
N
34, 35, 43
H
27, 44
Compounds R1
Renin buffer IC50 (nM) Renin plasma IC50 (nM) hERG IC50
(l
M)
CYP 3A4
Reversible inhibitionc Time-dependent inhibitionc
a,b
a,b
a
(% activity @ 10
lM)
(% activity loss @ 10 lM)
43
44
27
3,4-Difluorophenyl
CO
CH2
0.50
0.14
0.17
4.95
1.6
1.3
4.2
12.9
5.1
87
80
88
40
88
68
F
F
O
O
O
O
34
35
0.08
0.05
0.32
0.44
9.8
9.0
76
88
37
72
a
b
c
Values are means of at least two experiments.
See Ref. 10 for assay protocol.
See Ref. 11 for assay protocol.
Br
cally less potent against renin than 1 (for data, see Table 3), they
were however less shifted in the presence of human plasma.16 Con-
sequently, the measured renin plasma IC50s were found to be com-
parable across all four compounds. More importantly, these three
truncated derivatives were found to demonstrate much lower
affinity for the hERG channel as well as a decreased tendency to
reversibly inhibit CYP3A4 activity versus their non-truncated
counterpart 1 (Tables 1 and 2). Furthermore, the pyridone-bearing
analog 38 did not suffer from time-dependent CYP3A4 inhibition
and offered the optimum balance between renin potency and off-
target profile. Consequently, further fine-tuning of compound 38
ethyl acetamide side chain was carried out. Unfortunately, replace-
ment of the acetamide cap by either a propionamide (39), a methyl
carbamate (40), an ethyl carbamate (41), or 2-methoxyacetamide
(42) all failed to afford any tangible improvement. In fact, these
modifications led to a loss in renin potency, as well as an increase
in time-dependent CYP 3A4 inhibition. From the results in Table 1,
it is also apparent that the compound 36 bearing the 3,4-difluoro-
phenyl residue was the intrinsically most potent renin inhibitor in
this series. Although a high level of protein binding blunted its po-
tency in the presence of human plasma by 15-fold, we hypothe-
sized that the judicious introduction of polarity into the scaffold
of 36 would offer the best opportunity to decrease this plasma
shift. Indeed, the incorporation of a tertiary alcohol at the 4-posi-
tion of the piperidine ring led to a 33% drop in plasma shift without
adversely impacting the intrinsic renin potency (i.e. 43, Table 2).
Furthermore, we were gratified to observe that a fourfold decrease
in hERG affinity was also achieved with this addition.
Br
Br
Ar
Ar
a
b
Cl
Cl
Cl
O
N
H
O
O
O
N
N
2
3
4(rac)
c,d,e
O
B
Br
6
O
Ar
Ar
N
CBz
H
N
H
N
CBz
f
Cl
Cl
N
Boc
7
5
Boc
g,h,i
Ar
H
N
R
Cl
O
N
H
8
Scheme 1. Synthesis of 8. Reagents and conditions: (a) NaOEt, EtOH, ethyl aryl
acrylate; (b) H2SO4, AcOH; (c) BH3ÁDMS, THF; (d) (BOC)2O, Hünig’s base, CH2Cl2; (e)
Chiralpak AD separation; (f) Pd(OAc)2, Ph3P, Na2CO3, n-PrOH; (g) H2, Pd/C, MeOH;
(h) RCOCl, Hünig’s base, CH2Cl2; (i) HCl, dioxane/CH2Cl2.
ded, after chiral resolution and acidic protecting group removal,
the desired 3,4-diarylpiperidine 34 and 35, respectively.
In agreement with what was previously observed, replacing the
lipophilic 2-(2,6-dichloro-4-methylphenoxy)ethoxy residue pres-
ent in 1 with either the 3,4-difluorophenyl (36),13 pyrid-3-yl
(37),14 or the 1-methyl-4-pyridinyl-2-one moiety (38)15 furnished
potent, low nanomolar inhibitors of renin (Table 1). Indeed, these
three privileged motifs are known to form both a stabilizing
Previously, we have demonstrated that the tertiary alcohol also
offered a handle to freeze the adjacent 3,4-difluorophenyl moiety
into its bioactive conformation via spirocyclization which led to a
marked improvement in both renin potency and off-target pro-
file.15 Indeed, the same benefits were realized here as both spirocy-
clic ether 27 and spirocyclic lactone 44 were both more effective
inhibitors of renin than their non-spirocyclic congener 43. In addi-
p
-stack and a hydrogen bond with Tyr83 and Trp45 of the renin
enzyme, respectively. Although all three compounds were intrinsi-

1956
P. Lacombe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1953–1957
O
OMe
b
O
OH
c
O
O
O
O
Me
O
OMe
a
OH
O
Br
Br
11
9
10
12
Br
F
F
F
F
F
F
a
g
c, d
HO
O
HO
O
Si
14
13
15
Br
F
Br
Br
F
O
Br
O
O
e, f
O
OH
16
F
F
17
18
F
F
Scheme 2. Synthesis of aryl bromide intermediates. Reagents and conditions: (a) 4-Bromobenzyl bromide, NaH, DMF; (b) DIBAL-H, CH2Cl2; (c) MeI, NaH, THF; (d) TBAF, THF;
(e) BH3ÁDMS, THF; (f) DMP, CH2Cl2; (g) ethylene glycol, TsOHÁH2O, benzene.
I
c
O
a,b
Br
OTIPS
Br
OTIPS
N
19
20
21
Boc
d
O
N
F
F
F
OH
OH
OH
F
F
O
OH
f,g
e
F
F
F
O
OH
OTIPS
OTIPS
N
N
24
23
26
22
27
Boc
Boc
Boc
O
h
F
F
F
F
O
O
j,k,l,m
i
OMs
N
O
NH
N
N
N
25
Boc
Boc
H
Scheme 3. Synthesis of spirocyclic compound 27. Reagents and conditions: (a) 2-Hydroxymethylbenzene boronic acid, Pd(PPh3)2Br2, Na2CO3, toluene/ethanol; (b) TIPSiCl,
imidazole, DMF; (c) Boc-piperid-4-one, Pd2(dba)3, DTBPF, t-BuONa, THF; (d) aryl bromide 18, Mg, LiCl, THF; (e) PPTS, acetone/H2O; (f) LiBH4, THF; (g) TBAF, THF; (h) MsCl,
Et3N, CH2Cl2; (i) KCN, TBAI, DMF; (j) CoCl2Á6H2O, NaBH4, MeOH; (k) AcCl, Hunig’s base, CH2Cl2; (l) Chiralpak AD separation; (m) HCl, dioxane/CH2Cl2.
tion, lactone 44 exhibited the lowest affinity for the hERG channel
of all the diaryl compounds presented here. Although all three
compounds showed little affinity for CYP3A4, they unfortunately
did inhibit CYP3A4 in a time-dependent manner. To address this,
it was felt that further introduction of polarity may serve to de-
crease the oxidative stress on the molecule. Indeed, we hypothe-
sized that this was the main reason why pyridone 38 possessed
such a clean CYP profile. In this regard, the 3,4-difluorobenzene
of 43 was replaced by two representative phenyl derivatives bear-
ing polyether side-chain (i.e. 34, 35). While these modifications did
deliver sub-nanomolar renin inhibitors even in the presence of
plasma, they unfortunately failed to completely dial out the
CYP3A4 time-dependent inhibition liability.
potent against renin than 1, it was less protein shifted and found to
be 3Â more potent in the presence of plasma. Furthermore, analog
38 showed a 1000-fold improvement over 1 with regards to its
hERG binding affinity and, unlike 1, possessed a clean CYP3A4 pro-
file. When dosed orally as the hydrochloride salt to Sprague–Dawly
rats at 30 mpk, or to dogs at 3 mpk (0.5% methocel solution, 5 mL/
kg), compound 38 was found to have good to moderate bioavail-
ability. Finally, it was found to be efficacious in a hypertensive dou-
ble transgenic rat model (dTGR) harboring both human renin and
human angiotensinogen genes. At a dose of 10 mpk, robust blood
pressure lowering over a period of 24 h was achieved with com-
pound 38 (Table 3).
In conclusion, we were able to design a novel series of 3,4-
diarylpiperidine renin inhibitors with good in vitro potency and
lower affinity for the hERG channel. Furthermore within this series,
it was demonstrated that potent compounds devoid of CYP3A4-
driven drug–drug interaction liabilities can be identified. One such
Having established that 3,4-diarylpiperidine 38 was still the
best representative of this novel series of renin inhibitors, the com-
pound was profiled further (Table 3). As previously mentioned,
even though compound 38 was found to be intrinsically ꢀ50Â less

P. Lacombe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1953–1957
1957
OH
O
OH
O
a
b,c
Br
N
Boc
NHCbz
N
Boc
29
28
30
d
R1 R2
R1 R2
O
O
O
O
O
O
N
g
e,f
HO
HO
O
NH
O
NH
NH
Boc
31
N
N
H
Boc
34 R1=R2=F
32 R1=R2=F
33 R1=Me R2=H
35 R1=Me R2=H
Scheme 4. Synthesis of tertiary alcohol derivatives 34 and 35. Reagents and conditions: (a) Boc-piperid-4-one, Pd2(dba)3, Q-Phos, t-BuONa, THF; (b) Tf2O, pyridine, CH2Cl2; (c)
boronate ester, Pd(OAc)2, S-Phos, K3PO4, THF/H2O; (d) H2, Pd/C, Ac2O, EtOH; (e) aryl bromide 12 or 15, Mg, LiCl, THF; (f) Chiralpak AD separation; (g) HCl, dioxane/CH2Cl2.
S.; McKay, D.; Roy, P.; Toulmond, S.; Wu, T. Bioorg. Med. Chem. Lett. 2010, 20,
5822.
8. Chen, A.; Dubé, D.; Dubé, L.; Gagné, S.; Gallant, M.; Gaudreault, M.; Grimm, E.;
Table 3
Comparative profiles for compounds 1 and 38
Houle, R.; Lacombe, P.; Laliberté, S.; Liu, S.; MacDonald, D.; Mackay, B.; Martin,
D.; McKay, D.; Powell, Lévesque, J. F. Bioorg. Med. Chem. Lett. 2010, 20, 5074.
9. (a) Corminboeuf, O.; Bezençon, O.; Grisostomi, C.; Remen, L.; Richard-Bildstein,
S.; Bur, D.; Prade, L.; Hess, P.; Strickner, P.; Fischli, W.; Steiner, B.; Treiber, A.
Bioorg. Med. Chem. Lett. 2010, 20, 6286; (b) Corminboeuf, O.; Bezençon, O.;
Compounds
1
38
Renin
Renin
hERG
Buffer IC50, nMa,b
Plasma IC50, nMa,b
0.04
9.6
0.006
2.1
3.4
5.8
IC50
(l
M)a
ˇ
Remen, L.; Grisostomi, C.; Richard-Bildstein, S.; Bur, D.; Prade, L.; Strickner, P.;
CYP3A4
Reversible inhibitionc
53
74
97
0
Hess, P.; Fischli, W.; Steiner, B.; Treiber, A. Bioorg. Med. Chem. Lett. 2010, 20,
6291.
Time-dependent inhibitionc
10. Ozaki, F.; Ishida, T.; Soejima, M.; Norimine, Y.; Yamamoto, N.; Kobayashi, K.;
Hasegawa, D.; Kaneko, T.; Doi, E.; Kurusu, N. US2009/270369 A1, 2009.
11. Colacot, T. J.; Grasa, G. A. Org. Lett. 2007, 9, 5489.
F (%)
23
0.44
34
7
12
SD Rat
(30 mpk po)
(5 mpk iv)
Cmax
Cl (mL/min/kg)
(h)
(
l
M)
0.45
115
4
12. Lu, Y.; Miet, C.; Kunesh, N.; Poisson, J. E. Tetrahedron Asymmetry 1993, 4, 893.
13. Chen, A.; Cauchon, E.; Chefson, A.; Dolman, S.; Ducharme, Y.; Dubé, D.;
Falgueyret, J.-P.; Fournier, P.-A.; Gagné, S.; Gallant, M.; Grimm, E.; Han, Y.;
Houle, R.; Huang, J.-Q.; Hugues, G.; Juteau, H.; Lacombe, P.; Lauzon, S.;
Lévesque, J.-F.; Liu, S.; MacDonald, D.; Mackay, B.; McKay, D.; Percival, D.; St-
Jacques, R.; Toulmond, S. Bioorg. Med. Chem. Lett. 2011, 21, 3970.
14. Chen, A.; Campeau, L.-C.; Cauchon, E.; Chefson, A.; Ducharme, Y.; Dubé, D.;
Falgueyret, J.-P.; Fournier, P.-A.; Gagné, S.; Grimm, E.; Han, Y.; Houle, R.; Huang,
J.-Q.; Lacombe, P.; Laliberté, S.; Lévesque, J.-F.; Liu, S.; MacDonald, D.; Mackay,
B.; McKay, D.; Percival, D.; Regan, C.; Regan, H.; Stump, S.; Toulmond, S. Bioorg.
Med. Chem. Lett. 2011, 21, 3976.
T
½
Vdss (L/kg)
9
25
F (%)
—
—
—
—
—
30
0.35
24
6.2
4.6
BeagleDog
(3 mpk po)
(1 mpk iv)
Cmax
Cl (mL/min/kg)
(h)
(l
M)
T
½
Vdss (L/kg)
Efficacy in dTGR (10 mpk po)
Max. BP; (mmHg)
Duration (h)
—
—
32
24
15. Chen, A.; Aspiotis, R.; Campeau, L.-C.; Cauchon, E.; Chefson, A.; Ducharme, Y.;
Falgueyret, J.-P.; Gagné, S.; Han, Y.; Houle, R.; Laliberté, S.; Larouche, G.;
Lévesque, J.-F.; McKay, D.; Percival, D. Bioorg. Med. Chem. Lett. 2011, 21.
doi:10.1016/j.bmcl.2011.10.013.
a
b
c
Values are means of at least two experiments.
See Ref. 16 for assay protocol.
See Ref. 17 for assay protocol.
16. Buffer assay: Human recombinant renin (Proteos) at 100 pM was incubated in
the presence or absence of renin inhibitors and 6
lM of Q-FRET substrate 9
DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His- -Amp in 50 mM MOPS, 100 mM
D,L
compound, renin inhibitor 38, was found to exhibit good to mod-
erate bioavailability in both rodents and dogs and to be efficacious
in dTGR. Other biaryl-based renin inhibitors, as well as their asso-
ciated properties, will be disclosed in due course.
NaCl, pH 7.4, 0.002% Tween. The reactions were performed in Costar 384
well black plates at 37 °C for 3 h. Fluorescence was measured at times 0 and 3 h
in a SpectraMax Gemini EM reader with excitation and emission filters at
328 nm and 388 nm, respectively. Plasma assay: Frozen human EDTA-plasma
was rapidly thawed in warm water and centrifuged at 2900 g for 15 min at
40 °C. The supernatant was collected and recombinant human renin (Proteos)
were added at 1 nM nominal concentration. The plasma was transferred to
Costar black 384 well plates, renin inhibitors added and the mixture was pre-
incubated at 37 °C for 10 min. The renin Q-FRET substrate QXL520-Lys-His-Pro-
Phe-His-Leu-Val-Ile-His-Lys-(5-FAM) (Proteos), diluted in 3 M Tris/200 mM
EDTA, pH 7.2 was added to the plasma with final concentrations of 342 mM
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Chem. 1995, 32, 37; (c) Tice, C. M. Annu. Rep. Med. Chem. 2006, 41, 155.
6. Feldman, D. L.; Maibaum, J. Annu. Rep. Med. Chem. 2009, 44, 105.
7. Lacombe, P.; Aspiotis, R.; Bayly, C.; Chen, A.; Dubé, D.; Dubé, L.; Gagné, S.;
Gallant, M.; Gaudreault, M.; Grimm, E.; Houle, R.; Juteau, H.; Lévesque, J. F.; Liu,
Tris, 23 mM EDTA and 6.8 lM substrate. The plate was incubated at 37 °C for
1 h and the plates were analyzed in a SpectraMax Gemini EM reader with
excitation and emission filters at 490 nm and 520 nm, respectively, at time 0
and 1 h.
17. (a) Calculated as percentage of the difference in the rate of CYP3A4-mediated
conversion of testosterone (250
presence of compound (10 M in DMSO) versus blank DMSO. A 50% change
corresponds to a reversible CYP3A4 inhibition IC50 of 10 M.; (b) Calculated as
percentage of the difference in the rate of CYP3A4-mediated conversion of
testosterone (250 M) to 6-b-hydroxytestosterone before and after 30 min
incubation period with the compound (10 in DMSO). 0% change
corresponds to no measurable time-dependent CYP3A4 inhibition.
lM) to 6-b-hydroxytestosterone in the
l
l
l
lM
A