Optimization of orally bioavailable alkyl amine renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.11.066

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC₅₀ of 0.83 nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10 mg/kg

Full text of DOI:10.1016/j.bmcl.2009.11.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 694–699
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of orally bioavailable alkyl amine renin inhibitors
Zhenrong Xu a, Salvacion Cacatian a, Jing Yuan a, Robert D. Simpson a, Lanqi Jia a, Wei Zhao a, Colin M. Tice a,
,
*
Patrick T. Flaherty a, Joan Guo a, Alexey Ishchenko a, Suresh B. Singh a, Zhongren Wu a, Brian M. McKeever a,
Boyd B. Scott a, Yuri Bukhtiyarov a, Jennifer Berbaum a, Jennifer Mason a, Reshma Panemangalore a,
Maria Grazia Cappiello a, Ross Bentley b, Christopher P. Doe b, Richard K. Harrison a, Gerard M. McGeehan a,
Lawrence W. Dillard a, John J. Baldwin a, David A. Claremon a
a Vitae Pharmaceuticals, 502 West Office Center Drive, Fort Washington, PA 19034, USA
b GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo
potency. Lead compound 21a, has an IC50 of 0.83 nM for the inhibition of human renin in plasma (PRA).
Oral administration of 21a at 10 mg/kg resulted in >20 h reduction of blood pressure in a double trans-
genic rat model of hypertension.
Received 18 September 2009
Revised 11 November 2009
Accepted 16 November 2009
Available online 1 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Renin
Aspartyl protease
Structure-based drug design
X-ray crystallography
Hypertension
The renin angiotensin aldosterone system (RAAS) plays an
important role in regulating blood pressure.1 The highly selective
aspartyl protease, renin, is an important component of the RAAS
and has long been recognized as a desirable target for antihyper-
tensive drugs.2–8 In 2007, the first direct renin inhibitor, aliskiren
(1, Fig. 1), was approved for sale in the US.9,10
Recently, we reported the structure-based design of a new class
of potent, low MW renin inhibitors including 2 and 3.11 Benzyl
ether 2 had modest potency, especially in the presence of human
plasma, but good oral bioavailability in rat, while benzyl alcohol
3 had excellent intrinsic potency but lower oral bioavailability in
rat. Compound 3 was shown to cause a dose dependent reduction
of mean blood pressure in double transgenic rats expressing both
human renin and human angiotensinogen (dTGR). In the further
optimization of 3, our key objective was to improve activity in
the presence of plasma (PRA), while maintaining or improving oral
bioavailability.
hydrogen bonding opportunities. In the X-ray structure of 3 bound
to renin (PDB code: 3gw5), the ether oxygen of the 4-methoxybutyl
group accepts a hydrogen bond from the backbone NH of Tyr14.
Further inspection of the residues that form the Ss3p pocket sug-
gested that, in addition to this interaction, a suitably positioned do-
nor in the ligand could interact with the carbonyl of Gly218.
Workers at Roche had demonstrated that the terminus of the chain
occupying Ss3p pocket could be substituted with functional groups
bearing hydrogen bond donors and acceptors, for example, OH,
CH3CONH with retention of activity.15 Modeling of various chains
R from the benzylic carbon of scaffold 4 into this region indicated
that the 6–7 heavy atom chain seen in 2 and 3 would be optimal
and that both the Tyr14 backbone NH and the Gly218 carbonyl
could be engaged. Based on these studies, compounds of general
structure 4, wherein R was 4-hydroxybutyl (5, Scheme 1) and 3-
(acylamino)propyl (6, Scheme 3) were selected for synthesis.
4-Hydroxybutyl compound 5 was prepared as shown in Scheme 1.
Compound 7, an intermediate in the previously reported synthesis of 3,11
was O-demethylated with AlCl3 and n-Bu4NI with concomitant loss of
the Teoc group. The Teoc group was reintroduced to facilitate purification
and subsequently cleaved with Et4NF to afford 5.
Literature reports have indicated that decreasing log P often re-
duces plasma protein binding.12–14 Our working hypothesis was
that analogs of 3 incorporating additional polar groups would have
reduced plasma protein binding and thus improved PRA. The S3sp
pocket is a narrow, mostly hydrophobic channel, with some
The synthesis of piperidines 13, which comprise the left hand
half of the target compounds 6, is shown in Scheme 2. The cyclic
disilyl protected amine 8 was converted to the corresponding
Grignard reagent 9. (R)-Boc-nipecotic acid 10 was converted to
* Corresponding author. Tel.: +1 215 461 2042; fax: +1 215 461 2006.
E-mail address: ctice@vitaerx.com (C.M. Tice).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.066

Z. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 694–699
695
Figure 1. Nonpeptidic renin inhibitors.
Scheme 1. Reagents and conditions: (a) AlCl3, n-Bu4NI, MeCN, 0 °C, 4 h; (b) Teoc-OSu, K2CO3, H2O, CH2Cl2, rt, 2.5 h; (c) Et4NF, MeCN, 80 °C, 2 h.
Scheme 2. Yields are shown for 13j and are representative. Reagents and conditions: (a) Mg, THF, reflux, 2.5 h; (b) MeNHOMeÁHCl, EDC; i-Pr2NEt, CH2Cl2, rt, 16 h; (c) X-PhLi,
THF, À70 °C to rt, 2 h; (d) THF, À70 °C to À8 °C, 16 h; (e) (a) Ac2O, DMAP, Et3N, CH2Cl2; (b) (EtCO)2O, DMAP, Et3N, CH2Cl2; (c) (PrCO)2CO, DMAP, Et3N, CH2Cl2; (d) c-PrCO2H,
EDC, CH2Cl2; (e) MeSO2Cl, Et3N, CH2Cl2; (f) PhOC(@O)NH2, dioxane,
D; (g) (i) p-NO2C6H4OCOCl, i-Pr2NEt, CH2Cl2, THF; (ii) 33% MeNH2 in EtOH; (h) (i) p-NO2C6H4OCOCl,
i-Pr2NEt, CH2Cl2, THF; (ii) Me2NH; (i) H2NSO2NH2, dioxane, 110 °C; (j) MeOCOCl, DMAP, Et3N, CH2Cl2; (k) EtOCOCl, i-Pr2NEt, CH2Cl2; (f) 1:1 2 M aq HCl/MeCN, 16 h, rt.
its Weinreb amide and treated with various substituted phenyl-
lithiums to afford ketones 11. Addition of Grignard reagent 9 to
ketone 11 afforded, after aqueous work up during which the silyl
protecting group was lost, aminoalcohol 12 as the major product.
The stereochemistry of the newly formed carbinol center in 12
was initially assigned based on the results obtained in analogous
reactions reported previously.11 This assignment was subse-
quently confirmed by an X-ray structure of derived compound
6a (vide infra, Fig. 2). The primary amine at the terminus of the
side chain of 12 was reacted with various electrophiles and the

696
Z. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 694–699
Scheme 3. Yields are shown for 6j and are representative. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, À20 °C; (b) (a) 30% MeNH2 in EtOH, 55 °C, 16 h; (b) EtNH2, EtOH,
; (c) NaN3, DMF, 80 °C, 16 h followed by H2, Pd/C, MeOH; (c) Teoc-OSu, K2CO3, H2O, CH2Cl2; (d) TsOH, EtOH, Et2O, 60 °C; (e) p-NO2C6H4OCOCl, i-Pr2NEt, CH2Cl2, 0 °C; (f) 13, i-
Pr2NEt, 1:1 MeCN/CH2Cl2; (g) Et4N+FÀ, MeCN, microwave, 100 °C, 10 min.
D
17.16 Primary amine 17 was activated as its p-nitrophenyl carba-
mate, reacted with piperidines 13 and deprotected with Et4NF to
give the desired target compounds 6. Alternatively, carbonyl diim-
idazole was used to activate amine 17 for urea formation in some
cases.
The synthesis of ureas 21, in which the chain occupying Ss3p is
connected to the benzylic carbon by an ether linkage, is shown
in Scheme 4. Ketone 11 was reduced stereoselectively with (R)-
CBS borane to give a preponderance of the (R) alcohol 18.11,17
The side chain was elaborated as follows: alcohol 18 was alkylated
with ethyl bromoacetate and the resulting ester was reduced to the
corresponding primary alcohol with NaBH4. The alcohol was con-
verted to its mesylate which was displaced with sodium azide. Cat-
alytic hydrogenation of the azide afforded primary amine 19,
which was acylated with methyl chloroformate and deprotected
to afford piperidine 20. The urea linkage of 21 was constructed
as described for 6, using the p-nitrophenyl carbamate derivative
of 17a followed by removal of the Teoc group with Et4NF.
Replacement of the six heavy atom 4-methoxybutyl chain with
a five heavy atom 4-hydroxybutyl chain led to a 45 Â loss in activ-
ity (Table 1, 3 vs 5), presumably because the chain is shorter than
optimal and no acceptor is suitably positioned to interact with the
hydrogen of the OH group. Incorporation of an amide functionality
into a six or seven heavy atom chain (6a, 6b) restored excellent po-
tency. A 1.8-Å resolution X-ray structure of 6a bound to renin val-
idated the model we had developed (Fig. 2, PDB code: 3km4). The
amide NH of 6a donates a hydrogen bond to the carbonyl oxygen of
Gly218, while the amide carbonyl accepts a hydrogen bond from
the backbone NH of Tyr14. Other key interactions between 6a
and renin were similar to those seen in the previously reported
X-ray structure of 3 bound to renin (PDB code: 3gw5): the tertiary
alcohol donates a hydrogen bond to Ser219, the urea carbonyl
Figure 2. X-ray structure of 6a bound to renin.
Boc group was removed under mild aqueous conditions to afford
piperidines 13.
The synthesis of the right hand diamine portion and the final
assembly of target compounds 6 is depicted in Scheme 3. Commer-
cially available Boc protected aminoalcohol 14 was converted to its
mesylate 15 and the mesylate was displaced with methylamine or
ethylamine to give mono-Boc protected diamines 16a and 16b,
respectively. Alternatively, treatment of mesylate 15 with NaN3
afforded an azide which was subjected to catalytic hydrogenation
to give 16c. Amine 16 was protected as its Teoc derivative and
the Boc group was selectively removed with p-TsOH to afford
Scheme 4. Yields are shown for 21a and are representative. Reagents and conditions: (a) (R)-CBS borane, catecholborane, PhMe, À14 °C, 16 h; (b) BrCH2CO2Et, NaH, THF, 0 °C
to rt, 1 h; (c) NaBH4, MeOH, rt, 3 h; (d) MsCl, Et3N, CH2Cl2, 0 °C, 3 h; (e) NaN3, DMF, 80 °C, 16 h; (f) H2 (1 atm), 10% Pd/C, EtOAc; (g) MeOCOCl, Et3N, CH2Cl2; (h) 1:1 2 M aq HCl/
MeCN; (i) 17, p-NO2C6H4OCOCl, i-Pr2NEt, CH2Cl2; (j) Et4N+FÀ, MeCN, microwave, 100 °C, 10 min.

Z. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 694–699
697
Table 1
SAR of tertiary alcohols 6
Methyl carbamate 21a (Table 3) had superior PRA to the corre-
sponding tertiary alcohol analog 6j. The m-chloro substituent could
be replaced with F, Me or H with retention of subnanomolar intrin-
sic potency (21b–21d, respectively) but with inferior PRA. The ef-
fect of changing R2 from methyl to hydrogen (21e, 21f) and to
ethyl (21g, 21h) was investigated in both the m-chloro and m-flu-
oro phenyl analogs. The m-chloro primary amine analog 21e had
the best PRA of these four analogs but was inferior to methylamine
21a. Compound 21a had comparable enzyme potency and PRA to
aliskiren (1)
Following up on the favorable properties of 21a, a number of
analogs with disubstituted phenyl rings occupying S3 were pre-
pared. Earlier SAR and modeling had indicated that, in addition
to the favorable effect of chlorine at the 3-position, small lipophilic
substituents such as F and Cl could be accommodated at the 2-, 5-,
and 6-positions. The 3,5-difluorophenyl and 3-chloro-5-fluoro-
phenyl analogs 21n and 21o both had excellent intrinsic potency
although their PRA values were slightly poorer than 21a.
R1
X
IC50 (nM)
PRAb,c (nM)
a,b
Compd no.
3d
0.47
21
13
315
16
—
5e
—
6a
6b
6c
6d
6e
6f
6g
6h
6i
MeC(@O)–
EtC(=O)–
PrC(@O)–
c-PrC(@O)–
MeSO2–
H2NC(@O)–
MeNHC(@O)–
Me2NC(@O)–
H2NSO2–
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1.3
1.5
57
17
177
31
21
79
200
141
46
2260
46
0.18
14
297
3.7
170
6j
6k
MeOC(@O)–
EtOC(@O)–
a
b
c
See Ref. 18 for assay protocol.
Average of at least two replicates.
See Ref. 19 for assay protocol.
See Figure 1 for structure of 3.
See Scheme 1 for structure of 5.
d
e
Rat PK studies were performed on four benzyl ether compounds
of general structures 21 (Table 2). Gratifyingly, reintroduction of
the ether linkage into the chain occupying Ss3p restored oral bio-
availability (21a vs 6j). The IV clearance of 21a was comparable
to that of lead compound 3. m-Fluoro analog 21b had greater oral
bioavailability and AUC than 21a despite higher IV clearance, while
primary amine analog 21e had comparable oral bioavailability to
21a, lower AUC and higher IV clearance. 3-Chloro-5-fluoro analog
21o had comparable oral bioavailability and IV clearance to 21a
but a considerably lower AUC value. Oral bioavailabilities for com-
pounds 21 in rat uniformly exceeded that for aliskiren (1).
forms a water mediated hydrogen bond with the backbone NHs of
Ser77 and Thr78, the urea NH donates a hydrogen bond to the
backbone carbonyl of Gly217 and the terminal –N+H2Me group is
positioned between the catalytic carboxylates of Asp32 and
Asp215.11 The steric limitations on substituents filling the Ss3p pock-
et were further demonstrated by the diminished potency of the
eight heavy atom butanoamide chain in 6c and the branched cyclo-
propanecarboxamide 6d. Furthermore, methylation of the amide
NH of 6a caused a >3000Â loss in potency (data not shown).
Replacement of the carbonyl in 6a with a sulfonyl group to give
6e led to >100Â loss in activity. Ureas 6f and 6g, and sulfamide
6i demonstrated that additional hydrogen bond donors are not
well accommodated, while the dimethylurea 6h again showed
the lack of tolerance for branching. Finally, methyl and ethyl carba-
mates 6j and 6k had superior intrinsic potency to the isosteric
amides 6b and 6c. In addition, the PRA of methyl carbamate 6j
was threefold better than that of lead compound 3. Interestingly,
the SAR reported by Märki et al. differed somewhat from that of
our compounds. In our series, the analogs with chains terminating
with H2NCONH–, H2NSO2NH– and CH3SO2NH– groups (6e, 6f, and
6i) suffered >20Â losses in potency compared to acetamide 6a,
whereas the corresponding literature analogs differed in potency
only by 2Â.15
Table 3
SAR of benzyl ethers 21
a,b
Compd no.
X
R2
IC50 (nM)
PRAb,c (nM)
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
21m
21n
21o
1
3-Cl
3-F
3-Me
H
3-Cl
3-F
3-Cl
3-F
2,3-diF
2,5-diF
2-F-3-Cl
2-F-5-Cl
3,4-diF
3,5-diF
3-F-5-Cl
Me
Me
Me
Me
H
H
Et
Et
Me
Me
Me
Me
Me
Me
Me
0.48
0.59
0.47
1.17
0.48
0.58
0.87
2.10
1.7
0.62
0.61
0.62
2.5
0.82
1.8
4.0
2.9
2.6
3.3
7.7
18.0
6.2
2.7
3.4
2.1
16.6
1.2
1.5
Acetamide 6a and methyl carbamate 6j were selected for rat PK
(Table 2) but, disappointingly, both had negligible oral bioavail-
ability. Noting that benzyl ether 2 had better oral bioavailability
than tertiary alcohol 3, we prepared compounds of general struc-
ture 21 (Scheme 4), in which the chain occupying Ss3p is attached
through an ether oxygen and the methyl carbamate feature at
the terminus of the chain is maintained.
0.23
0.52
0.53
0.65
a
b
c
See Ref. 18 for assay protocol.
Average of at least two replicates.
See Ref. 19 for assay protocol.
Table 2
Rat PK parametersa
Compd
Oral Cmax (ng/mL)
Oral tmax (h)
Oral AUC(0–t) (ng h/mL)
Oral t1/2 (h)
IV CL (mL/min kg)
Vss (L/kg)
F (%)
2
3
6a
6j
21a
21b
21e
21o
1
91
73
ndb
5
94
123
63
54
4
3.0
3.3
nd
0.9
3.0
2.0
4.0
4.0
0.1
832
472
nd
5.9
nd
nd
1.8
9.7
12.7
7.8
7.2
13.5
59
33
23
34
36
58
53
41
55
22
nd
117
29
19
61
51
38
62
35.8
12.9
nd
0.3
9.4
22.1
7.2
13.1
1.2
10
301
380
201
166
21
a
Compounds were administered as fumarate salts. PO dose 10 mg/kg. IV dose 2 mg/kg.
nd = not determined.
b

698
Z. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 694–699
Table 4
Monkey PK of 21a, 3, and 1a
Compd
Oral Cmax (ng/mL)
Oral tmax (h)
Oral AUC(0–t) (ng h/mL)
Oral t1/2 (h)
IVCL (mL/min kg)
Vss (L/kg)
F (%)
21ab,c
3b,d
33.3
116
29.5
1.0
2.7
1.3
129.7
615e
154
29.2
15
9.8
11.7
5.2
2.5
9.8
6.1
1.5
9.9
16
1.4
1b,d
a
b
c
Compounds were administered as fumarate salts.
IV dose = 1 mg/kg.
PO dose 1 mg/kg.
PO dose 2 mg/kg. IV dose 1 mg/kg.
t = 32 h.
d
e
Vehicle
(n=4)
Aliskiren (1) 10mg/kg PO (n=4)
21a 10mg/kg PO (n=6)
30
20
10
0
-10
-20
-30
-40
-50
-60
0
10
20
30
Time (hrs)
40
50
60
Figure 3. Effect of 21a and aliskiren (1) on mean arterial pressure in double transgenic rats.
8. Tice, C. M. Ann. Rep. Med. Chem. 2006, 155.
Based on its good PRA and rat PK parameters, compound 21a
9. Maibaum, J.; Stutz, S.; Goeschke, R.; Rigollier, P.; Yamaguchi, Y.; Cumin, F.;
Rahuel, J.; Baum, H.-P.; Cohen, N.-C.; Schnell, C. R.; Fuhrer, W.; Gruetter, M. G.;
Schilling, W.; Wood, J. M. J. Med. Chem. 2007, 50, 4832.
10. Jensen, C.; Herold, P.; Brunner, H. R. Nat. Rev. Drug Disc. 2008, 7, 399.
11. Tice, C. M.; Xu, Z.; Yuan, J.; Simpson, R. D.; Cacatian, S.; Flaherty, P. T.; Zhao, W.;
Guo, J.; Ishchenko, A.; Singh, S.; Wu, Z.; Scott, B. B.; Bukhtiyarov, Y.; Berbaum,
J.; Mason, J.; Panemangalore, R.; Cappiello, M. G.; Harrison, R. K.; McGeehan, G.;
Dillard, L. W.; Baldwin, J. J.; Claremon, D. A. Bioorg. Med. Chem. Lett. 2009, 19,
3541–3545.
was selected for advanced in vitro and in vivo evaluation. The
compound showed >1000Â selectivity over the aspartic proteases
b-secretase, cathepsin D and cathepsin E. Its half-life in human
liver microsomes was >30 min. The cynomolgus monkey PK
parameters of 21a, 3, and aliskiren (1) are compared in Table 4.
While the oral bioavailability of 21a is lower than that of 3, it is
substantially better than that of aliskiren (1). Notably, 21a has a
half-life of 29 h in cynomolgus monkey.
12. Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure Design and Methods,
Academic Press, 2008. pp 193–194.
Compound 21a was studied in an animal model of hyperten-
sion. In the dTGR,20 a 10 mg/kg oral dose gave a statistically signif-
icant reduction in mean blood pressure compared to vehicle lasting
for >20 h (Fig. 3). The magnitude and duration of blood pressure
lowering were greater than that observed with the same dose of
aliskiren (1) and achieved a statistically significant difference over
the 24 h interval.
In conclusion, we have described the structure-based discovery
of renin inhibitors with improved plasma renin activity. Oral
administration of 21a elicited sustained reductions in mean blood
pressure in an animal model of hypertension.
13. Gleeson, M. P. J. Med. Chem. 2008, 51, 817.
14. Leach, A. G.; Jones, H. D.; Cosgrove, D. A.; Kenny, P. W.; Ruston, L.; MacFaul, P.;
Wood, J. M.; Colclough, N.; Law, B. J. Med. Chem. 2006, 49, 6672.
15. Märki, H. P.; Binggeli, A.; Bittner, B.; Bohner-Lang, V.; Breu, V.; Bur, D.;
Coassolo, P.; Clozel, J. P.; D’Arcy, A.; Doebeli, H.; Fischli, W.; Funk, C.; Foricher,
J.; Giller, T.; Gruninger, F.; Guenzi, A.; Guller, T.; Hartung, R.; Hirth, G.; Jenny,
C.; Kansy, M.; Klinkhammer, U.; Lave, T.; Lohri, B.; Luft, F. C.; Mervaala, E. M.;
Muller, D. N.; Muller, M.; Montavon, F.; Oefner, C.; Qiu, C.; Reichel, A.; Sanwald-
Ducray, P.; Scalone, M.; Schleimer, M.; Schmid, R.; Stadler, H.; Treiber, A.;
Valdenaire, O.; Vieira, E.; Waldmeier, P.; Wiegand-Chou, R.; Wilhelm, M.;
Wostl, W.; Zell, M.; Zell, R. Il Farmaco 2001, 56, 21.
16. Rosowsky, A.; Wright, J. E. J. Org. Chem. 1983, 48, 1539.
17. Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C. P.; Singh, V. K. J. Am. Chem. Soc.
1987, 109, 7925.
18. The activity of renin inhibitors in vitro was measured using the following test
protocol: All reactions were carried out in
microtiter plate. A 4- L aliquot of 400 M renin substrate (DABCYL-
His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS) in 192 L assay buffer (50 mM
BES, 150 mM NaCl, 0.25 mg/mL bovine serum albumin, pH 7.0) was added to
L of test compound in DMSO at various concentrations ranging from 10
to 1 nM final concentrations. Next, 100 L of trypsin-activated recombinant
human renin (final enzyme concentration of 0.2–2 nM) in assay buffer was
added, and the solution was mixed by pipetting. The increase in fluorescence at
495 nm (excitation at 340 nm) was measured for 60–360 min at rt using a
Perkin–Elmer Fusion microplate reader. The slope of a linear portion of the plot
of fluorescence increase as a function of time was then determined, and the
rate was used to calculate% inhibition in relation to uninhibited control. The%
a
flat bottom white opaque
References and notes
l
l
c
-Abu-Ile-
l
1. Zaman, M. A.; Oparil, S.; Calhoun, D. A. Nat. Rev. Drug Disc. 2002, 1, 621.
2. Greenlee, W. J. Med. Res. Rev. 1990, 10, 173.
3. Rosenburg, S. H. Prog. Med. Chem. 1995, 32, 37.
4. Rosenburg, S. H.; Boyd, S. A. Antihypertensive Drugs; Van Zweiten, P. A.;
Greenlee, W. J., Ed.; Harwood: Amsterdam, The Netherlands, 1997; pp 77–111.
5. Stanton, A. J. Renin Angiotensin Aldosterone Syst. 2003, 4, 6–10.
6. Fisher, N. D. L.; Hollenburg, N. K. J. Am. Soc. Nephrol. 2005, 16, 592.
7. Scott, B. B.; McGeehan, G.; Harrison, R. K. Curr. Protein Pept. Sci. 2006, 7, 241–
254.
4
l
lM
l

Z. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 694–699
699
inhibition values were plotted as a function of inhibitor concentration, and
the IC50 was determined from a fit of this data to a four parameter equation.
The IC50 is defined as the concentration of a particular inhibitor that reduces
the formation of product by 50% relative to a control sample containing no
inhibitor. (Wang, G. T. et al. Anal. Biochem. 1993, 210, 351; Nakamura, N. et al. J.
Biochem. (Tokyo) 1991, 109, 741; Murakami, K. et al. Anal Biochem. 1981, 110,
232).
mL/h) ꢀ2 pM of recombinant human renin was added to achieve PRA of 3–
4 ng/mL/h. The cleavage of endogenous angiotensinogen in plasma was carried
out at 37 °C for 90 min and the product angiotensin I was measured by
competitive radioimmunoassay using DiaSorin PRA kit. Uninhibited
incubations containing 2% DMSO and fully inhibited controls with 2 lM of
isovaleryl-Phe-Nle-Sta-Ala-Sta-OH were used to derive the% inhibition for each
concentration of inhibitors. The% inhibition values were plotted as a function of
inhibitor concentration, and the IC50 was determined from a fit of this data to a
four parameter equation. The IC50 is defined as the concentration of a particular
inhibitor that reduces the formation of product by 50% relative to a control
sample containing no inhibitor.
19. The activity of renin inhibitors in vitro in human plasma was measured by the
decrease in plasma renin activity (PRA) levels observed in the presence of the
compounds. Incubation mixtures contained in the final volume of 250 lL
95.5 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7.0, 8 mM
EDTA, 0.1 mM neomycin sulfate, 1 mg/mL sodium azide, 1 mM phenylme-
thanesulfonyl fluoride, 2% DMSO and 87.3% of pooled mixed-gender human
plasma stabilized with EDTA. For plasma batches with low PRA (less than 1 ng/
20. Ganten, D.; Wagner, J.; Zeb, K.; Bader, M.; Michel, J. B.; Paul, M.; Zimmermann,
F.; Ruf, P.; Hilgenfeldt, U.; Ganten, U.; Kaling, M.; Bachmann, S.; Fukamizu, A.;
Mullins, J. J.; Murakami, K. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7806.