Design and synthesis of potent, isoxazole-containing renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.014

The design and optimization of a novel isoxazole S₁ linker for renin inhibitor is described herein. This effort culminated in the identification of compound 18, an orally bioavailable, sub-nanomolar renin inhibitor even in the presence of human plasma. When compound 18 was found to inhibit CYP3A4 in a time dependent manner, two strategies were pursued that successfully delivered equipotent compounds with minimal TDI potential.

Full text of DOI:10.1016/j.bmcl.2012.03.014

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2670–2674
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of potent, isoxazole-containing renin inhibitors
⇑
Pierre-André Fournier , Mélissa Arbour, Elizabeth Cauchon, Austin Chen, Amandine Chefson,
Yves Ducharme, Jean-Pierre Falgueyret, Sébastien Gagné, Erich Grimm, Yongxin Han, Robert Houle,
Patrick Lacombe, Jean-François Lévesque, Dwight MacDonald, Bruce Mackay, Dan McKay,
M. David Percival, Yeeman Ramtohul, René St-Jacques, Sylvie Toulmond
Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Québec, Canada H9H 3L1
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and optimization of a novel isoxazole S1 linker for renin inhibitor is described herein. This
effort culminated in the identification of compound 18, an orally bioavailable, sub-nanomolar renin
inhibitor even in the presence of human plasma. When compound 18 was found to inhibit CYP3A4 in
a time dependent manner, two strategies were pursued that successfully delivered equipotent com-
pounds with minimal TDI potential.
Received 14 February 2012
Revised 2 March 2012
Accepted 5 March 2012
Available online 11 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Renin
Inhibitor
Anti-hypertensive
Hypertension is a major risk factor for a variety of cardiovascu-
lar diseases.1 Despite the fact that a wide variety of anti-hyperten-
sive medications are now available in the developed world, more
than 60% of hypertensive patients are still unable to reach the
blood pressure goal of 140/90 mm Hg as recommended by the
American Heart Association. As a result, hypertension is still an
area of active research in several pharmaceutical companies. In this
regard, the perturbation of the renin-angiotensin-aldosterone sys-
tem (RAAS) at its various stages continues to be an attractive strat-
egy for the development of novel anti-hypertensives (Fig. 1).2 Since
renin is involved in the first and rate-limiting step of this cascade,
the development of a suitable renin inhibitor should offer the best
potential for optimal blood pressure control and superior end or-
gan protection than therapies targeting other steps of the RAAS
pathway.3,4 However to date, only Aliskiren, marketed as Tekturna
in the U.S. and Rasilez in the EU and Japan, has received regulatory
approval for the treatment of moderate essential hypertension.5
We have previously reported that suitably functionalized 3,4-
biarylpiperidines can serve as highly potent renin inhibitors
(Fig. 2).6,7 In this scaffold, a 2-chlorophenyl ring at the S1 position
was employed as an equipotent replacement for the classic cyclo-
propyl amide linker.8 In the course of our investigations, we also
determined that piperidines substituted at the 4-position with a
pyridone ring were necessary to obtain compounds with a suffi-
ciently clean off-target profile (hERG and CYP 3A4 inhibition).6,9
However, the main issue associated with the use of such a
functionality is that pyridone’s inherent polarity renders the
resulting renin inhibitors poorly permeable to cellular membranes.
As a result, most of these compounds suffered from low and dose-
dependant oral exposure.10 For this reason, we hypothesized that
replacement of the lipophilic phenyl S1 linker in 1 with a more po-
lar heterocycle may enlarge the off-target/potency window suffi-
ciently such that the pyridone ‘band-aid’ would no longer be
necessary. In this regard, the suitability of several metabolically
stable heterocycles bearing the required substitution pattern were
evaluated and isoxazoles were rapidly identified as being optimal.
It is also worth highlighting that the isoxazole linker can be assem-
bled late in the synthesis to allow the convergent union of two
pieces of comparable synthetic complexity. This retrosynthetic dis-
section would in turn facilitate parallel SAR explorations at both
the northern and eastern aromatic rings as well as increase the
compound output.
Both isoxazole isomers shown in Figure 2 can be readily ac-
cessed via a 1,3-dipolar cycloaddition between an alkyne and a ni-
trile oxide generated in situ from its corresponding oxime. In this
regard, we prepared oxime 4 and alkynes 5 and 6 from the known
carboxylic acid 2 (Scheme 1).7 Borane reduction of 2 and subse-
quent oxidation of the resulting alcohol with Dess-Martin’s period-
inane (DMP) yielded aldehyde 3 in excellent yield. Its condensation
with hydroxylamine then gave oxime 4 in quantitative yield. Alter-
natively, treatment of aldehyde 3 with Ohira-Bestmann’s reagent
delivered terminal alkyne 5.11 Finally, 2-bromoalkyne 6 was pre-
pared from aldehyde 3 via an initial Ramirez condensation with
CBr4 followed by potassium tert-butoxide promoted HBr
elimination.12
⇑
Corresponding author.
E-mail address: pierre-andre_fournier@merck.com (P.-A. Fournier).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.014

P.-A. Fournier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2670–2674
2671
Scheme 2. Reagents and conditions: Synthesis of 9–27: (a) 2 equiv 7, 2 equiv
Chloramine-T, MeOH, 20 min., then 1 equiv or 6, 60 °C, 3 h; (b) 1 equiv 4,
5
1.15 equiv Chloramine-T, MeOH, 20 min., then 3 equiv 8, 60 °C, 3 h; (c) 30 equiv 4 M
HCl in dioxane, RT, 1 h.
priate oxime 7 with Chloramine-T resulted in the in situ generation
of the corresponding nitrile oxide. Addition of alkyne 5 or 6 then
gave the desired isoxazole core. Deprotection of the Boc protecting
group using HCl gave renin inhibitors 9–17. Analogously, the same
steps were repeated using oxime 4 and alkynes 8 to deliver inhib-
itors 18–27.
Figure 1. The renin-angiotensin-aldosterone system.
The key data are summarized in Table 1. Firstly, compounds
lacking a 4-substituent at the isoxazole bridge (i.e., 9–11, 19, 21),
regardless of the substitution patterns of the terminal aromatic
group and which isoxazole isomer it contains, were all found to
be weaker inhibitors of renin. However, greater than 200Â boost
in renin potency could be realized following the installation of a
bromine at the 4-position of the isoxazole tether (i.e., 11 vs 12). In-
deed, the bromine substituent was found to occupy the previously
vacant renin S1 subpocket and effectively brought the potency of
the isoxazole series to the same range as that of the reference com-
pound 1.14
Figure 2. General topology of the renin active site and isoxazole as replacement for
phenyl linker.
With the 4-bromoisoxazole core in hand, we then set out to
optimize the terminal aromatic ring that sits in the highly lipo-
philic renin S3 pocket. On going from 2-chlorophenyl (i.e., 12) to
2,3-dichlorophenyl (i.e., 13), we were able to increase the intrinsic
renin potency further, although this gain was partially negated by
the concomitant increase in plasma shift. Replacement of the two
chlorine atoms by methyl groups (i.e., 14) unfortunately proved
to be detrimental for both renin potency and plasma shift. Simi-
larly, shuffling the meta-chlorine in compound 13 to the other
ortho-position of the phenyl ring (i.e., 15) was also not beneficial.
Although replacement of the 2,3-dichlorobenzene in compound
13 with its well known 2-naphthalene isostere (i.e., 16) did im-
prove the intrinsic renin potency further, this again came at the ex-
pense of a concomitant increase in plasma protein binding. To get
around this, we decided instead to focus our attention on improv-
ing the interactions between our inhibitor and the less lipophilic
renin S3 subpocket. Indeed, following the replacement of the chlo-
rine in 12 with the same polar ethyl acetamide tail present in com-
pound 1 (i.e., 17), both the intrinsic renin potency and the plasma
shift were significantly improved. Furthermore on going from 12 to
17, there was a noticeable decrease in the compound’s affinity for
the hERG channel as well as its ability to reversibly inhibit CYP3A4,
although the extent of CYP3A4 time-dependent inhibition (TDI) re-
mained an issue.15
With the requisite alkynes and oximes in hand, we performed
the key 1,3-dipolar cycloaddition (Scheme 2).13 Treating the appro-
Scheme 1. Reagents and conditions: Synthesis of 6: (a) 3 equiv BH3DMS, THF, RT,
6 h, >99%; (b) 3 equiv DMP, 30 equiv pyridine DCM:t-BuOH (4:1), 18 h, 88% (c)
1.5 equiv hydroxylamine hydrochloride, 1.55 equiv Na2CO3, EtOH:H2O (9:1), >99%;
(d) 1.2 equiv Ohira-Bestmann’s reagent, 2 equiv K2CO3, MeOH, RT, 8 h, 72%; (e)
1 equiv Et3N, 2 equiv PPh3, 3.8 equiv CBr4, DCM, À78 °C to RT, 3 h, 88%; (f) 1.2 equiv
KOtBu, THF, RT, 2 h, 78%.
We then investigated which isoxazole isomer possessed the
best in vitro profile. Gratifyingly compound 18, which bears the
regioisomer of the isoxazole present in 17, exhibited superior renin
potency, reaching sub-nanomolar activity even in the presence of

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P.-A. Fournier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2670–2674
Table 1
SAR of select renin inhibitors
#
R1
R2
R3
R4
R5
R6
Renin potencya
Buffer (nM) Plasma (nM)
hERG Ki (
l
M)b
CYP 3A4 inhibition
l
M)c Time dependent (% loss)d
Reversible (
1
—
H
H
H
Br
Br
Br
Br
—
Br
Br
H
Br
H
Cl
C(O)Me
—
Br
Cl
Cl
Cl
Cl
Me
Cl
—
H
H
H
H
Cl
Me
H
—
H
H
H
H
H
H
H
H
H
H
H
—
—
F
H
F
—
H
Cl
H
H
H
H
Cl
—
H
H
Cl
H
H
H
H
H
H
H
H
—
—
—
—
—
—
—
—
—
0.5
402
7.5
1.1
1.7
2.8
3.4
6.2
—
6.0
3.9
20
3
8
49
22
59
36
76
60
77
82
67
74
68
60
78
21
74
32
48
75
60
86
18
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
8654
>9500
>9500
27
7.8
21
131
4.7
1.6
0.6
>9500
0.7
331
1.9
4.2
4.5
1.2
1.2
0.6
1.4
1213
2500
1.14
0.21
0.47
3.7
0.06
0.36
0.27
1333
0.14
166
0.73
2.82
4.30
0.26
0.17
0.22
0.37
7
H
H
H
H
—
H
H
H
F
H
H
H
H
H
H
H
—
18
19
20
15
>50
45
33
17
45
32
>50
8
>50
13
28
36
>50
—
—
—
4.2
C2H4NHAc
C2H4NHAc
Cl
10.5
10.7
9.8
23
3.3
15.3
13.7
33.6
19.5
21.2
30.0
>60
OH
OH
OH
OH
OH
OH
OH
H
C2H4NHAc
C2H4NHAc
C2H4NHAc
C2H4NHAc
C2H4NHAc
C2H4NHAc
C2H4NHAc
C2H4NHAc
—
CH(OH)Me
Br
Br
Br
—
OMe
C(O)NH2
—
a
b
c
Average of at least two replicates. See Ref. 16 for assay protocols.
Average of at least two replicates. Binding affinity.
Calculated as an IC50 for the inhibition of CYP3A4-mediated conversion of testosterone (50
l
M) to 6-b-hydroxytestosterone in the presence of compound.
Calculated as a percentage of the difference in the rate of CYP3A4-mediated conversion of testosterone (250 M) to 6-b-hydroxytestosterone before and after 30 min
M in DMSO). A 0% loss corresponds to no measurable time-dependent CYP 3A4 inhibition.
d
l
incubation period with the compound (10
l
human plasma. Unfortunately, the extent of TDI observed was not
improved with 18. To address this, we sought to decrease the lipo-
philicity of the molecule further by changing the bromine at the 4
position of the isoxazole core. To evaluate the viability of this ap-
proach, we first prepared compound 21 where the bromine was re-
placed by a hydrogen atom. Although this led to a major loss in
renin potency, the CYP3A4 TDI was indeed improved, going from
a 68% loss in CYP3A4 activity after a 30 min pre-incubation period
with 18 to a 21% loss with 21. Encouraged by this result, we then
prepared a variety of compounds with different R1 groups (i.e.,
compounds 22–24). Although improvements in their respective
off-target profile versus compound 20 were observed, the atten-
dant loss of potency against renin was unfortunately too large to
warrant further profiling of these compounds since the safety mar-
gin was not improved.
As an alternative approach to reduce the CYP3A4 TDI potential
of 18, we also tried to increase the polarity of the molecule. Previ-
ous in silico docking experiments involving scaffolds similar to that
found in compound 18 suggested that the tertiary alcohol residue
does not engage in any stabilizing interactions with the renin en-
zyme. Indeed, this was independently confirmed by the observa-
tion that both compounds 25 and 26, where the tertiary alcohol
in question was replaced by either a hydrogen or a methoxy group,
respectively, were equipotent to 18 in the absence of human plas-
ma. Furthermore, since the tertiary alcohol was suspected to be
solvent-exposed, we felt that this would be an ideal position to ap-
pend a polar residue. While carbamate 27 exhibited comparable
renin potency to that of 18, its CYP3A4 profile was not improved.
However, the introduction of an even more polar 2,3-dihydroxy-
propyl chain (i.e., 28) had the desired impact on both reversible
and time-dependent CYP3A4 inhibition, with only a small loss in
renin potency versus compound 18. Unfortunately, this compound
was not orally bioavailable in rats (F = 0%, Table 2), most likely a
consequence of its poor cellular permeability. This further exam-
plifies the delicate balance between lipophilicity, CYP3A3 time-
dependant inhibition and permeability present in multiple renin
inhibitor series.
Since it was suspected that the TDI observed with compound 18
was caused by the formation of an unknown reactive metabolite,
concurrent with the above effort, we were intrigued by whether
this liability could also be addressed through the introduction of
a distal metabolic soft spot that would shunt the metabolism to
a more innocuous position of the molecule. With this idea in mind,
we prepared compound 32 from the known piperidine 29 (Scheme
3). Removal of the trityl group with p-toluenesulfonic acid and a
subsequent protecting group switch under hydrogenolysis condi-
tion yielded compound 30. Following chiral resolution by HPLC,
the desired diastereomer was oxidized to aldehyde 31 using
DMP. The synthesis of 32 was then completed using the same syn-
thetic manipulations as described above for 18.
The introduction of a metabolic soft spot turned out to be a
highly promising approach. Compound 32 was found to be equipo-
tent to 18 both in the absence and presence of human plasma. Fur-
thermore, the off-target profile of 32 was significantly improved
over that of 18. Most importantly, compound 32 does not irrevers-
ibly inactivate CYP3A4 at a measurable rate and hence should ex-
hibit minimal potential to perpetrate drug-drug interactions (DDI)
if co-dosed with drugs cleared mainly by CYP3A4 (e.g., simvasta-

P.-A. Fournier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2670–2674
2673
Table 2
Key profiles of compounds 18, 28 and 32
Compound
18
28
32
a,b
Renin IC50 (nM)
Buffer
Plasma
F (%)
0.27
0.63
16
0.37
1.4
0
0.26
0.67
18
SD Rat (3 mpk PO, 0.5 mpk IV)
PO AUC (
Cl (mL/min/kg)
T1/2 (h)
Vdss (L/kg)
l
M h)
0.14
108
5.7
36
—
44
0
0.13
124
1.4
12
—
27
70
1.9
12
<1
—
c
Papp (Â10-6 cm sÀ1
)
Efficacy in dTGR (3 mpk PO)
max. BP ; (mm Hg)
duration (h)
18
—
8
hERG Ki (
CYP 3A4 IC50 (nM)
l
M)
11.7
33
68
>60
>50
18
—
31.2
29
10
Reversible (
Time dependent (% loss)d
l
M)d
CYP 3A4 kobs (minÀ1
)
0.093
<0.004
a
b
c
See Ref. 13 for assay protocols.
Average of at least two replicates.
See Ref. 9 for assay protocol.
d
Calculated as a percentage of the difference in the rate of CYP3A4-mediated conversion of testosterone (250
lM) to 6-b-hydroxy-testosterone before and after 30 min
incubation period with the compound (10
lM in DMSO). A 0% loss corresponds to no measurable time-dependent CYP 3A4 inhibition.
In conclusion, an isoxazole S1 linker was found to be a superior
replacement for the classic amide linker as well as the more recent
phenyl linker. Indeed, changing the 2-chlorophenyl linker in 1 for a
4-bromoisoxazole linker in 18 increased the plasma renin potency
by more than 10-fold and improved the attendant off-target pro-
file. Although compound 18 was found to inactivate CYP3A4 in a
time dependent manner, the judicious incorporation of either
polarity or a metabolic soft spot both proved to be viable strategies
for addressing this liability. Further optimization of the pharmaco-
kinetic properties of this series of inhibitors is currently underway
and will be reported in due course.
References and notes
1. http://ucatlas.ucsc.edu/cause.php.
2. Weber, M. A. Am. J. Hypertens. 1992, 5, 247S.
3. (a) Cooper, M. E. J. Hypertens. 2004, 17, 16S; (b) Norris, K.; Vaughn, C. Expert Rev.
Cardiovasc. Ther. 2003, 1, 5; (c) Cheng, H.; Harris, R. C. Expert Opin. Drug Saf.
2006, 5, 631; (d) Azizi, M.; Gradman, A. H.; Sever, P. S. J.R.A.A. Syst. 2009, 10, 65.
4. Zaman, M. A.; Oparil, S.; Calhoun, D. A. Nat. Rev. Drug Disc. 2002, 1, 621.
5. Hollenberg, N. K. Nat. Rev. Nephrol. 2010, 6, 49.
6. Lacombe, P.; Arbour, M.; Aspiotis, R.; Cauchon, E.; Chen, A.; Dubé, D.;
Falgueyret, J.-P.; Fournier, P.-A.; Gallant, M.; Grimm, E.; Han, Y.; Juteau, H.;
Liu, S.; Mellon, C.; Ramtohul, Y.; Simard, D.; St-Jacques, R.; Tsui, G. C. Bioorg.
Med. Chem. Lett. 2012, 22, 1953.
7. For a detailed discussion of the binding mode of renin inhibitors and of the
active site topology, see: Webb, R. L.; Schiering, N.; Sedrani, R.; Maibaum, J. J.
Med. Chem. 2010, 53, 7490.
8. 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.; Hughes, G.; Juteau, H.; Lacombe, P.; Lauzon, S.;
Lévesque, J. F.; Liu, S.; MacDinald, D.; Mackay, B.; Mackay, D.; Percival, D.; St-
Jacques, R.; Toulmond, S. Bioorg. Med. Chem. Lett. 2011, 21, 3976.
9. 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, M. D.; Regan, C.; Regan, H.; St-Jacques, R.; Stump, G.;
Soisson, S.; Toulmond, S. Bioorg. Med. Chem. Lett. 2011, 21, 3970.
10. Lévesque, J.-F.; Bleasby, K.; Chefson, A.; Chen, A.; Dubé, D.; Ducharme, Y.;
Fournier, P.-A.; Gagné, S.; Gallant, M.; Grimm, E.; Hafey, M.; Han, Y.; Houle, R.;
Lacombe, P.; Laliberté, S.; MacDonald, D.; Mackay, B.; Papp, R.; Tschirret-Guth,
R. Bioorg. Med. Chem. Lett. 2011, 21, 5547.
Scheme 3. Reagents and conditions: Synthesis of 32: (a) 1.2 equiv TsOH,
THF:MeOH (1:4), 4 h, >99%; (b) 1.05 equiv BOC2O, 0.1 equiv Pd/C, MeOH, 18 h,
73%; (c) chiral resolution, AD column, 90:5:5 hexanes: i-PrOH: MeOH; (d) 3 equiv
DMP, 30 equiv pyridine DCM:t-BuOH (4:1), 18 h, 69%; (e) 1.5 equiv hydroxylamine
hydrochloride, 1.55 equiv Na2CO3, EtOH:H2O (9:1), >99%; (f) 1.15 equiv Chlora-
mine-T, MeOH, 20 min, then 3 equiv 8, 60 °C, 3 h; (g) 30 equiv 4 M HCl in dioxane,
RT, 1 h, 32% over 2 steps.
tin). Unlike compound 28, compound 32 is bioavailable (F = 18%) in
rats when dosed orally as a 3 mpk solution in 0.5% methyl cellu-
lose. As expected, the presence of a metabolic softspot in 32 was
manifested in a higher plasma clearance (Cl = 124 mL/min/kg)
and a shorter half life (T1/2 = 1.4 h) than compound 18.
When compounds 18 and 32 were given to hypertensive double
transgenic rats (dTGR) harboring both human renin and angioten-
sinogen, robust blood pressure lowering was observed with both
compounds.17 As is consistent with the higher volume of distribu-
tion and lower rate of plasma clearance in rat of compound 18, its
blood pressure lowering effect was both more extensive and pro-
longed than compound 32 in this hypertension efficacy model.
Unfortunately when compound 32 was profiled in higher order
species, namely beagle dogs and cynomolgus monkeys, the ob-
served systemic exposure following oral dosing was not sufficient
to warrant further characterization (data not shown).
11. Roth, G. J.; Liepold, B.; Müller, G. S.; Bestmann, H. J. Synthesis 2004, 59.
12. Meijere, A.; Kozhushkov, S.; Puls, C.; Haumann, T.; Boese, R.; Cooney, M. J.;
Scott, L. T. Angew. Chem. 1994, 106, 394.
13. Yedav, P. P.; Ahmad, G.; Maurya, R. Tetrahedron Lett. 2005, 46, 5621.
14. For a discussion on the binding mode of biaryl renin inhibitors, see: 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, S.; McKay,
D.; Roy, P.; Toulmond, S.; Wu, T. Bioorg. Med. Chem. Lett. 2010, 20, 5822.
15. For an in depth analysis of CYP3A4 time-dependent inhibition observed in
series of renin inhibitors, see: Chen, A.; Dubé, D.; Dubé, L.; Gagné, S.; Gallant,
M.; Gaudreault, M.; Grimm, E.; Houle, R.; Lacombe, P.; Laliberté, S.; Liu, S.;

2674
P.-A. Fournier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2670–2674
MacDonald, D.; Mackay, B.; Martin, D.; McKay, D.; Powell, D.; Lévesque, J.-F.
Bioorg. Med. Chem. Lett. 2010, 20, 5074.
16. Buffer assay: Human recombinant renin (Proteos) at 100 pM was incubated in
supernatant was collected and recombinant human renin (Proteos) added at
1 nM nominal concentration. The plasma was transferred to Costar black 384
well plates, renin inhibitors added and the mixture 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) (Anaspec), diluted in 3 M Tris/200 mM EDTA, pH 7.2 was
added to the plasma with final concentrations of 342 mM Tris, 23 mM EDTA
and 6.8 lM substrate. The plate was incubated at 37 °C for 1 h and the plate
read in a SpectraMax Gemini EM reader with excitation and emission filters at
490 and 520 nm, respectively, at time 0 and 1 h.
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-D,L-Amp in 50 mM MOPS, 100 mM
NaCl, pH 7.4, 0.002% Tween. The reactions take place in a Costar 384 well black
plate 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 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
17. Hess, P.; Clozel, M.; Clozel, J.-P. J. Appl. Physiol. 1996, 81, 1027.