Benzyl ether structure-activity relationships in a series of ketopiperazine-based renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.07.063

Inhibition of renin enzymatic activity by a series of ketopiperazine-based compounds containing a C6 benzyloxymethyl substituent correlated with a +(π + σ) effect. A 3-pyridinyloxymethyl substituent was also found to be equipotent as higher molecular weig

Full text of DOI:10.1016/j.bmcl.2005.07.063

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4713–4716
Benzyl ether structure–activity relationships in a series
of ketopiperazine-based renin inhibitors
Noel A. Powell,* Emma H. Clay, Daniel D. Holsworth, John W. Bryant, Michael J. Ryan,
Mehran Jalaie and Jeremy J. Edmunds
Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 11 June 2005; revised 26 July 2005; accepted 26 July 2005
Available online 6 September 2005
Abstract—Inhibition of renin enzymatic activity by a series of ketopiperazine-based compounds containing a C6 benzyloxymethyl
substituent correlated with a +(p + r) effect. A 3-pyridinyloxymethyl substituent was also found to be equipotent as higher molec-
ular weight analogs, and exhibited decreased CYP3A4 inhibition levels and improved pharmacokinetic properties.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Hypertension is a leading risk factor for cardiovascular
disease, such as congestive heart failure, stroke, myocar-
dial infarction, and is a major cause of death in the
Western world.1 The renin angiotensin system (RAS) is
well-established as an endocrine system that is involved
in blood pressure regulation and fluid electrolyte balance
(Fig. 1).2 Activation of the RAS is stimulated by several
signals, including a drop in blood pressure, a decrease in
the circulating volume, or a reduction in plasma sodium
concentration. These signals stimulate the release of
renin, which cleaves angiotensinogen to angiotensin I
(AngI). Angiotensin converting enzyme (ACE) then
converts AngI into the vasopressor peptide angiotensin
II (AngII). The binding of AngII to the AT1 receptor
triggers a number of physiological effects, such as sodium
and water retention and vasoconstriction, ultimately
leading to an increase in blood pressure. Since renin is
the rate-limiting step in the RAS cascade, renin inhibition
is considered to be an attractive antihypertensive strategy.
Renin inhibitors have been predicted to be more
efficacious with fewer side effects than ACE
inhibitors and AT1 receptor antagonists, which target
downstream events.3
Angiotensinogen
Angiotensin I
Renin
Bradykinin
Inactives
Chymase
ACE
Angiotensin II
AT1 Receptor
Blood Volume
Peripheral Resistance
Vasoconstriction
Central pressor activation
Sympathetic tone
Aldosterone release
Sodium re-absorption
Thirst
Figure 1. The renin angiotensin system (RAS).
the programs were based on peptidic or peptidomimetic
scaffolds. Poor pharmacokinetic properties, including
low oral bioavailability and high clearance, and a high
cost of goods for large-scale synthesis made these agents
unattractive as clinical candidates.4
Several pharmaceutical companies have attempted to
advance renin inhibitors to the clinic. Although potent
in vitro renin inhibitory activity was obtained, most of
In 1999, the first renin inhibitors based on a trans-3-
alkoxymethyl-4-arylpiperidine scaffold were disclosed.5
We have recently disclosed a similar series of non-pep-
tidic renin inhibitors that is based on a 6-alkoxymethyl-
1-aryl-2-ketopiperazine scaffold, as exemplified by 1.6
Keywords: Renin inhibitor; Hypertension; Topliss tree.
*
Corresponding author. Tel.: +1 734 622 2151; fax: +1 734 622
3909; e-mail: noel.powell@pfizer.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.063

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N. A. Powell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4713–4716
Table 1. Renin inhibition activity for selected analogs
The ketopiperazine scaffold offers several advantages
over the trans-3-alkoxymethyl-4-arylpiperidine scaffold,
notably the ease of synthesis, removal of a chiral center,
and more amenability to modular SAR studies.
R3
H
N
R2
R4
R5
O
O
N
Although the ketopiperazine scaffold effected sub-
nanomolar renin inhibition and demonstrated in vivo
blood pressure reduction in transgenic mice,6 these
inhibitors suffered from high molecular weights and
suboptimal PK properties. Our SAR studies indicated
that the A, B, and D-ring portions were required for
good renin inhibition activity, whereas the C-ring por-
tion was amenable to variation. Thus, we chose to
investigate if the substituted tetrahydroquinoline C-
ring in analog 1 could be replaced with a substituted
benzyl ether (2, Fig. 2), which would reduce molecular
weight, increase ease of synthesis, and potentially pro-
vide opportunities for improving pharmacokinetic
properties.
O
O
O
R5 IC50
R2
H
R3
H
R4
pb
rc
p + r
(nM)a
7000
6800
5
6
7
8
9
H
H
F
0.00
0.88
0
0.54
0
1.42
CF3
OCH3
3900 À0.20 À0.27 À0.47
1600 0.29 0.4 0.688
1200 À0.57 0.56 À0.01
900 À0.02 À0.27 À0.29
F
CN
10 OCH3
11
12
OCH3
600 À0.20
0.12 À0.08
F
F
450
410
410
370
340
330
300
270
270
270
260
250
230
230
180
180
140
120
0.14
0.87
1.04
0.06
0.44
0.38
0.2
13 Cl
14
15
1.308
1.42
0.49
The analogs 5–29 described were synthesized by the
route exemplified with analog 5 (Scheme 1). The inter-
mediate alcohol 3 was prepared, as previously de-
scribed.6 Deprotonation of the alcohol with NaH,
followed by alkylation of the resulting alkoxide with
benzyl bromide in the presence of 15-crown-5, yielded
the desired benzyl ether 4. The N-Boc-protecting
group was removed by treatment of 4 with cold anhy-
drous HCl generated by the addition of AcCl to anhy-
drous MeOH to yield analog 5 in good overall yield.
The additional analogs, as given in Table 1, were pre-
pared in a similar fashion using the appropriately
OCF3
CH3
0.56 À0.07
0.56 À0.17
16
17
CH3
0.39
1.08
Cl
0.71
0.71
0.37
0.23
18
19
Cl
F
0.94
CH3
CF3
0.64 À0.01
0.634
1.518
1.628
1.514
0.618
1.31
20
21 CF3
F
1.03
1.03
1.21
0.22
0.88
0.15
1.25
1.43
1.03
1.59
0.49
0.6
22
23
24
Cl
CH3
F
0.3
0.4
F
CF3
0.43
0.06
0.52
0.75
0.49
0.66
25
26
27
28
29
F
0.205
1.77
Cl
Cl
Cl
Cl
2.178
1.518
2.25
CF3
CF3
F
Cl
O
a IC50 values obtained in duplicate using a fluorescent tGFP assay.6
b Non-literature p values were calculated by the formula p = (c logP of
the substituted benzene ring) À2.14.
HN
H
N
H
N
R
c r values for disubstituted rings are a sum of the r values of the
individual substituents.
A
O
N
O
O
N
O
N
C
B
substituted benzyl halide. The heterocyclic analogs giv-
en in Table 2 were prepared using the corresponding
heterocyclic methylene halide.
D
O
O
O
O
OMe
OMe
2
1
IC50 = 0.4 nM
Replacement of the tetrahydroquinolinyl C-ring in 1
with a benzyl ether resulted in a dramatic loss of renin
inhibition (Table 1, 5 IC50 = 7000 nM). However, sub-
stitution on the benzyl ether led to greatly improved
renin inhibition. Increases in potency were achieved by
following the Topliss tree for substituents that have a
net +(p + r) effect on the lipophilic and electronic nature
of the aromatic ring.7 For example, introduction of a 4-
Cl substituent led to a >20-fold improvement in renin
inhibition (18, IC50 = 300 nM). A further potency gain
was achieved by the addition of a second lipophilic elec-
tron-withdrawing group at the 3-position, such as the
3,4-dichlorobenzyl ether 26 (IC50 = 180 nM), which
was almost 1.6-fold more potent than 18. As predicted
by the Topliss tree, the most active compounds in
this series were the 3-CF3-4-fluorobenzyl ether 28
(IC50 = 140 nM) and the 3-CF3-4-chlorobenzyl ether
Figure 2. Ketopiperazine-based renin inhibitors.
Boc
N
R
N
OH
O
Br
O
N
O
N
NaH, 15-crown-5
DMF, 0 ˚C to r.t.
83%
O
O
O
O
OMe
3
OMe
AcCl, MeOH
0 ˚C
4 R = Boc
5 R = H
81%
Scheme 1.

N. A. Powell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4713–4716
Table 2. CYP3A4 inhibition and PK profile of selected analogsa
4715
CYP3A4 % inhibition at 3 lMb
Solubilityc (lg/mL)
Caco-2 permeability A to B (·10À6 cm/s)
Log D at pH 7.4
28
29
32
87
97
44
7
3
NTd
NTd
13
3.7
3.7
2.9
100
a Reported data are an average of results obtained in duplicate.
b 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) was used as a fluorogenic substrate for the CYP3A4 isozyme.9
c Apparent solubility at pH 6.5 measured by laser nepholometric method.
d Not tested due to low solubility.
introduction of a N atom at the 2- and 4-positions led
to inactive compounds (30 and 31), the 3-pyridinyl ether
32 inhibited renin with an IC50 = 120 nM. This finding
was of significant interest since the 3-pyridinyl ether 32
achieved the same renin inhibitory potency as the 3-
CF3-4-chlorobenzyl ether 29, but with a substantially
lower molecular weight (32 MW = 491 vs 29
MW = 593). Even more interesting was the observation
that 32 displayed a significantly less cytochrome P450
3A4 inhibition than either 28 or 29 in our primary
screens (Table 2). We believe that the decrease in
CYP3A4 inhibition is primarily a result of the lower
lipophilicity of 32, as measured by the logD at pH
7.4.8 In addition, 32 displayed improved solubility com-
pared to either 28 or 29, as well as measurable Caco-2
cell membrane permeability. To understand the basis
of this remarkable selectivity for the position of the N
atom in the benzene ring, we initiated docking studies
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
-1
2
2.2
2.4
2.6
2.8
3
log IC50
Figure 3. Graph showing the correlation between the log IC50 of the
analogs in Table 1 and p + r. Line is an orthogonal straight line fit
with r2 = 0.58.
29 (IC50 = 120 nM). A graph of the log IC50 of the ana-
logs in Table 1 (X axis) against p + r of the substituents
(Y axis) indicates a reasonable correlation between
potency and a +(p + r) effect (Fig. 3).7 In this series, in-
creased renin inhibition was driven by aryl substituents
that led to a net increase in the lipophilic and electron
deficient nature of the aromatic ring. This result is not
surprising, as previous crystal structures of the ketopip-
erazine series bound to the renin active site indicate that
the benzyl ether occupies a large lipophilic S3 binding
pocket.6
of 32 in the active site of renin in the flap-open confor-
6,10
mation (Fig. 5).
These studies indicated that the 3-
pyridinyl ether extended into the S3 subpocket, where
it could potentially make hydrogen bond contacts with
the Ser219 hydroxyl and amide NH along the rim of
the S3 subpocket. Analogs 30 and 31 are less active be-
cause they are unable to make these potential hydrogen
bonds.
We recently reported that both enantiomers of the keto-
piperazine scaffold with bicyclic C-ring portions exhibit-
ed equipotent renin inhibition and followed similar SAR
trends.10 To examine if this trend held good for benzyl
ethers, we prepared several compounds of the S config-
uration (Table 3). Surprisingly, the benzyl ether analogs
of S configuration were found to be much less potent
In conjunction with the benzyl ether SAR, a series of
pyridinyl ethers was also examined to explore the effect
of basic groups in the S3 pocket (Fig. 4). While
H
N
H
N
N
O
N
O
O
O
N
O
N
O
O
O
O
O
30
31
IC50 = >1000 nM
IC50 = >1000 nM
H
N
N
O
O
N
O
O
O
Figure 5. View of analog 32 (blue atom coloring) docked in the flap-
open conformation of the renin active site.6,10 Protein surface is
colored by atom (red, oxygen; blue, nitrogen). Residue Ser219 is
indicated by the green stick atom coloring.
32
IC50 = 120 nM
Figure 4. Pyridinyl ether structure–activity relationships.

4716
N. A. Powell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4713–4716
Table 3. Comparison of the activity of ketopiperazine benzyl ether
enantiomers
and electronic nature of the S3 pocket. It was also dis-
covered that equivalent renin inhibition potency could
be obtained through the use of a 3-pyridinyl ether,
resulting in a substantial reduction in molecular weight,
decreased CYP3A4 inhibition, and improved cell mem-
brane permeability and aqueous solubility.
H
N
H
N
O
Ar
O
O
Ar
O
O
N
O
O
N
(R)
(S)
Acknowledgments
O
O
O
We thank the Pfizer Global Research & Development
Discovery Technologies Department in Ann Arbor,
MI, for generation of the ADME data for analogs 28,
29, and 32, and Dr. Bruce D. Roth for stimulating
discussions.
Ar group
R analog IC50 (nM) S analog IC50 (nM)
F
12
29
32
450
120
12
33
34
35
5590
594
Cl
CF3
References and notes
N
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against renin. Although the 4-Cl-3-CF3-benzyl ether 34
(S configuration) was only 5-fold less active than the
corresponding analog with R configuration 29, both
the 4-fluorobenzyl ether 33 and 3-pyridinyl ether 35
showed a >10-fold difference in renin inhibition activity
compared to the corresponding R configuration analogs
12 and 32. A vector change in the orientation of the C6
hydroxymethyl linker is required to orient the bicyclic
C-ring in the S3 pocket in the analogs with S configura-
tion.10 We theorize that a similar vector change cannot
be induced in our C6 benzyloxymethyl analogs because
the benzyl ether does not occupy enough steric space in
the S3 pocket and the hydroxymethyl linker between the
ketopiperazine and benzyl ether rings contains too many
degrees of rotational freedom.
6. Holsworth, D. D.; Powell, N. A.; Downing, D. M.; Cai,
C.; Cody, W. L.; Ryan, M.; Ostroski, R.; Jalaie, M.;
Bryant, J. W.; Edmunds, J. J. Bioorg. Med. Chem. 2005,
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Topliss, J. G. J. Med. Chem. 1977, 20, 463.
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In summary, we have reported our efforts to replace the
bicyclic C-ring portion of the ketopiperazine scaffold
with benzyl ethers in an effort to achieve renin inhibition
potency, while reducing molecular weight and for ease
of synthesis. Increased renin potency was guided by fol-
lowing the Topliss tree for substituents that give a net in-
crease in lipophilicity and electron deficiency (p + r) of
the C6 benzyloxymethyl moiety to match the lipophilic