Addressing time-dependent CYP 3A4 inhibition observed in a novel series of substituted amino propanamide renin inhibitors, a case study

Article abstract of DOI:10.1016/j.bmcl.2010.07.030

Time-dependent inhibitors of CYPs have the potential to perpetrate drug-drug interactions in the clinical setting. After finding that several leading compounds in a novel series of substituted amino propanamide renin inhibitors inactivated CYP3A4 in an NA

Full text of DOI:10.1016/j.bmcl.2010.07.030

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5074–5079
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Addressing time-dependent CYP 3A4 inhibition observed in a novel series
of substituted amino propanamide renin inhibitors, a case study
*
Austin Chen , Daniel Dubé, Laurence Dubé, Sébastien Gagné, Michel Gallant, Mireille Gaudreault,
Erich Grimm, Robert Houle, Patrick Lacombe, Sébastien Laliberté, Suzanna Liu, Dwight MacDonald,
*
Bruce Mackay, David Martin, Dan McKay, David Powell, Jean-François Lévesque
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:
Time-dependent inhibitors of CYPs have the potential to perpetrate drug–drug interactions in the clinical
setting. After finding that several leading compounds in a novel series of substituted amino propanamide
renin inhibitors inactivated CYP3A4 in an NADPH-dependent and time-dependent manner, a search to
identify the cause of this liability was initiated. Extensive SAR revealed that the amide bridge present
in compound 1 as a possible culprit. Through the installation of a metabolic soft spot distal to this moiety,
potent renin inhibitors with improved CYP profile were identified.
Received 9 June 2010
Revised 6 July 2010
Accepted 8 July 2010
Available online 11 July 2010
Keywords:
CYP3A4
Ó 2010 Elsevier Ltd. All rights reserved.
Time-dependent inhibition
Renin
Hypertension
Human cytochrome P450s (CYPs) are membrane-associated
heme-containing proteins localized in either the inner membrane
of mitochondria or endoplasmic reticulum of cells.1 Their main
function is to catalyze the oxidation of both endogenous (e.g., hor-
mones) as well as xenobiotic substrates. In the latter capacity, CYPs
often play a major role in the metabolism and subsequent excre-
tion of pharmaceuticals. Of the 18 families of P450 genes that have
been identified and characterized in humans, the CYP3A4 isoform
is known to be involved in the clearance of almost half of the drugs
currently on the market.2 The systemic exposure of these drugs can
therefore be altered in the presence of agents that can either in-
duce the biosynthesis of CYP3A4 or inhibit its enzyme activity,
either reversibly or in a time-dependent manner (TDI). Conse-
quently, it is important to evaluate and minimize the potential of
any promising clinical candidate to perpetrate such drug–drug
interactions (DDI) throughout the entire drug discovery process.
Hypertension affects more than a billion people worldwide and is
one of the major risk factors for strokes, heart attacks, heart failure
and arterial aneurysm, as well as the leading cause of chronic renal
failure.3 Despite the availability of numerous treatment options that
treat the disease from a variety of unique pathways,4 most of the pa-
tients still can not achieve their targeted blood pressure goals with-
out resorting to combination therapy.5 Furthermore, since
hypertension is often a consequence of other underlying metabolic
disorders such as diabetes or hypercholesterolemia,6 many of these
patients are treated with poly-pharmacy thereby making DDIs a ma-
jor concern for physicians. Recently, we reported the discovery of a
novel and potent series of amino propanamide renin inhibitors for
the treatment of essential hypertension (Fig. 1).7 We were therefore
quite alarmed by the observation that several of the most promising
compounds were all found to inhibit CYP 3A4 in a time-dependent
manner. Consequently, a study aimed at identifying the key cul-
prit(s) responsible for this liability was initiated.
Since the inactivation of CYP3A with compound 1 was found to
be an NADPH-dependent and time-dependent process, it was
hypothesized that the observed TDI was due to the formation of
an active metabolite capable of binding irreversibly to the CYP3A
enzyme. However, the search for the exact perpetrator was compli-
cated by two observations. Firstly, the addition of trapping agents
such as glutathione (GSH), cyanide and semicarbazide did not pre-
vent the in vitro inactivation of CYP 3A. Secondly, when compound
1 was incubated with either human liver microsomes or human
hepatocytes, only a few minor metabolites were observed while
the majority (>94%) of compound 1 remained intact (Fig. 2). Conse-
quently a classical SAR approach, where the impact of various
structural modifications on CYP3A4 activity was independently
evaluated, became necessary. The severity of the CYP3A4 TDI ob-
served for a given modification was gauged by the magnitude of
the IC50 fold shift observed upon 0 and 30 min pre-incubation.8
* Corresponding authors. Tel.: +1 514 428 2770 (A.C.).
E-mail address: austin_chen@merck.com (A. Chen).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.030

A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5074–5079
5075
Figure 1. Profile of a representative amino propanamide renin inhibitor.
Figure 2. In vitro metabolic profile of compound 1.
Metabolites (i.e., Ma and M13) derived from the de-methylation of
the methoxypropyl group found in compound 1 were observed
in vitro (Fig. 2) and this led us to focus the initial SAR study on
the P3 arene plate (Table 1). Although the extent of TDI observed
was slightly tempered by modifying the nature of the meta-substi-
tuent, TDI was not restricted to derivatives bearing the methoxy-
propyl tail. Indeed, analogues possessing either a cyano residue
(i.e., 3) or more polar capping groups such as amide 4 or sulfone
5 all suffered from TDI. Furthermore, replacement of the 2-chloro-
benzene plate found in compounds 1 with pyridine (i.e., 6), pyri-
dine N-oxide (i.e., 7) or quinoline (i.e., 8 and 9) also failed to
decrease the severity of time-dependent CYP3A4 inactivation.
Given the number of bio-transformations that can be envi-
sioned for primary amines,11 we then turned our attention to the
aminomethyl warhead. Unfortunately, its replacement by an ethyl
amine (i.e., 10), an amide (i.e., 11) or even simple hydrogen (i.e.,
12) still afforded compounds with significant potential to perpe-
trate DDI (Table 2). Furthermore, the small improvement in TDI

5076
A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5074–5079
Table 1
SAR of select renin compounds: P3 arene modifications
a,b
c
Compd
R1
R2
R3
X
Renin IC50 (nM)
CYP3A4 IC50 (lM)
Buffer
Plasma
30 min Pre-Inc
0 min Pre-Inc
Shift
1
2
3
4
5
6
7
8
9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
(CH2)3OMe
(CH2)2OMe
(CH2)3CN
CH2N(Me)Ac
(CH2)2SO2Me
(CH2)3OMe
(CH2)3OMe
(CH2)3OMe
(CH2)3CN
CH
CH
CH
CH
CH
N
0.09
0.04
0.10
2.9
3.1
0.19
0.3
2.4
2.9
4.6
250
720
2.2
1.8
4.4
1.5
0.12
0.12
0.10
0.21
0.10
0.14
0.17
0.10
0.08
14
14
12
7
110
110
120
35
50
90
70
120
140
5
12
12
12
10
N+–OÀ
N
–CH@CH–CH@CH–
–N@CH–CH@CH–
0.17
0.08
CH
a
b
c
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.
Table 2
SAR of select renin compounds: warhead modifications
a,b
c
Compd
R
Renin IC50 (nM)
CYP3A4 IC50
(lM)
Buffer
Plasma
30 min Pre-Inc
0 min Pre-Inc
Shift
2
10
11
12
CH2NH2
C(Me)NH2
C(@O)NH2
H
0.04
0.43
17
470
2.9
59
7000
—
0.12
0.20
0.41
0.30
14
12
22
12
110
60
55
d
d
40
a
b
c
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.
Compound was tested as a racemate.
d
realized with these analogues came at the expense of their renin
potency.
Incubation experiments with human hepatocytes and human li-
ver microsomes revealed the formation of phenol-bearing metabo-
lites (i.e., Ma and M2, Fig. 2) via the oxidative cleavage of the 2-
(2,6-dichloro-4-methyl-phenoxy)ethoxy northern terminus (vide
supra). Further oxidative processing of these phenol metabolites
could in theory deliver reactive intermediates capable of binding
irreversibly to CYPs.14 In this regard, compounds 17 and 18 were
prepared and tested (Table 4). Again, the TDI liability was not suc-
cessfully addressed with these modifications.
Although metabolites derived from the hydrolysis of compound
1’s amide bond were not detected in vitro, if formed, the observed
TDI may be ascribed to reactive intermediates generated from fur-
ther oxidation of the resulting cyclopropylamine metabolite.12,13
To test this hypothesis, several analogues lacking the cyclopropyl-
amine moiety were synthesized and tested. Replacement of the
cyclopropyl group by methyl (i.e., 13), cyclobutyl (i.e., 14), cyclo-
propylmethyl (i.e., 15) or cyclobutylmethyl (i.e., 16) again afforded
similar time-dependent inhibitors of CYP3A4 (Table 3). These ana-
logues also proved to be inferior inhibitors of renin.
Having sequentially ruled out some of the most likely suspects,
we then proceeded to evaluate the impact the amide functionality
has on TDI. Gratifyingly, removal of the carbonyl oxygen (i.e., 19)

A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5074–5079
5077
Table 3
Table 5
SAR of select renin compounds: P1 modifications
SAR of select renin compounds: P1 modifications
a,b
c
Compd Linker
Renin IC50
(nM)
CYP3A4 IC50 (lM)
a,b
c
Compd
R
Renin IC50 (nM)
CYP3A4 IC50
(lM)
Buffer
Plasma
2.9 0.12
30 min Pre-Inc 0 min Pre-Inc Shift
Buffer Plasma 30 min Pre-
Inc
0 min Pre-
Inc
Shift
2
0.04
0.47
14
12
110
40
O
13
26
0.30
0.10
Me
2
0.04
2.9
0.12
14
110
N
N
14
15
13
3100
18
9
180
100
19
29
40
190
3.5
16
50,000
1900
1.7
4
9.8
14
5.8
9.7
3700
0.09
0.10
3.5
1.7
16
210
26,000
13
130
a
b
c
1300
11
19
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.
a
b
c
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.
led to a significant improvement with respect to the extent of TDI
observed (Table 5). The renin enzyme unfortunately, did not toler-
ate this functional group deletion. In order to hopefully regain
some of the loss in renin potency, analogues where the nitrogen
in 19 was replaced by a carbon (i.e., 29 and 40) were synthesized
(Scheme 1). Briefly, palladium-catalyzed formylation of the known
aryl bromide 20 with sodium formate as the reducing agent15
afforded aldehyde 21 and subsequent olefination with ylide 22
lithium enolate generated from amide 26 with chiral iodide 39 oc-
curred with >95:5 diastereo-selectivity.17 Subsequent reductive re-
moval of the Myer’s auxiliary was best accomplished with lithiated
borane ammonia complex which delivered alcohol 27 with no loss
in stereochemical integrity.18 Finally, diphenyl phosphorazidate-
mediated conversion19 of 27 to azide 28 followed by Staudinger
reduction20 afforded compound 29. Synthesis of iodide 39 used
in the alkylation step described earlier began with the Suzuki-cou-
pling of aryl bromide 30 with vinylboronic acid pinacol ester.21
Iridium-catalyzed hydroboration of the resulting styrene 31 deliv-
ered, after oxidative workup with NaBO3, alcohol 32.22 Iodometh-
was carried out quantitatively.
a,b-Unsaturated ester 23 thus ob-
tained could then be hydrogenated in EtOAc with PtO2 as the cat-
alyst.16 Hydrolysis of the resulting ester 24 to carboxylic acid 25
with aq lithium hydroxide followed by coupling with (À)-pseudo-
ephedrine17 both proceeded without incidents. Alkylation of the
Table 4
SAR of select renin compounds: northern terminus
a,b
c
Compd
Renin IC50 (nM)
CYP3A4 IC50
(
lM)
Buffer
Plasma
30 min Pre-Inc
0 min Pre-Inc
Shift
2
17
18
0.04
2900
0.29
2.9
—
21
0.1
0.1
0.25
14
12
14
110
120
60
a
b
c
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.

5078
A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5074–5079
Scheme 1. Synthesis of 29: (a) Pd(PPh3)2Cl2, CO, NaCO2H, DMF, 110 °C, 8 h, 77%; (b) 22, THF, 24 h, >99%; (c) PtO2, H2, EtOAc, 14 h, 92%; (d) 2 N aq LiOH, MeOH, THF, 16 h,
>99%; (e) (ClCO)2, CH2Cl2, cat. DMF, 1 h, then (À)-pseudoephedrine, NEt3, THF, 0 °C, 91%; (f) 2.5 equiv LDA, 8 equiv LiCl, THF, À78 °C, 1 h, then 0 °C, 30 min, then 39, 0 °C to rt,
20 h; (g) 4 equiv LDA, 4.2 equiv BH3ÁNH3, THF, 0 °C to rt, 20 h, 36% yield over two steps; (h) (PhO)2P@O(N3), DBU, DMSO, rt to 80 °C, 4 h, 82%; (i) PPh3, H2O, THF, 50 °C, 2 h, 94%;
(j) Pd(OAc)2, PPh3, vinylboronic acid pinacol ester, 2 N aq Na2CO3, EtOH, DMF, 90 °C, 8 h, 86%; (k) [IrCl(COD)]2, DPPP, pinacolborane, THF, 24 h, then NaBO3, H2O, 2 h, 69%; (l)
NaH, MeI, THF, 30 min; (m) 1.5 M DIBAl-H in toluene, CH2Cl2, À78 °C to rt, 16 h, 98% over two steps; (n) I2, imidazole, PPh3, CH2Cl2, 0 °C to rt, 1.5 h, 77%; (o) (ClCO)2, CH2Cl2,
cat. DMF, 1 h, then (+)-pseudoephedrine, NEt3, THF, 0 °C, 60%; (p) 2.5 equiv LDA, 10 equiv LiCl, THF, À78 °C, 1 h, then 0 °C, 30 min, then 35, 0 °C to rt, 20 h, 88%; (q) 4 equiv
LDA, 4.2 equiv BH3ÁNH3, THF, 0 °C to rt, 20 h, 85% yield; (r) I2, imidazole, PPh3, CH2Cl2, 0 °C to rt, 1.5 h, 90%. Abbreviations: COD = 1,5-cyclooctadiene; DBU = 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIBAl-H = diisobutylaluminum hydride; DPPP = 1,3-bis(diphenylphosphino)propane; LDA = lithium diisopropyl-amide.
Table 6
ane methylation of alcohol 32, DIBAl-H reduction of ester 33 and
Arbusov iodination of alcohol 34 then furnished iodide 35. Treat-
SAR of select renin compounds: second appendage
ment of lithiated amide 37, itself synthesized from cyclopropyl-
acetic acid 36 and (+)-pseudoephedrine, with iodide 35 also pro-
ceeded with complete stereochemical control. Again, removal of
the Myer’s auxiliary from amide 38 was best accomplished with
lithiated borane ammonia complex. Finally, Arbusov iodination
cleanly delivered iodide 39. Unfortunately, although compound
29 and its diastereomer 40 were both much cleaner than com-
pound 2 in terms of TDI, neither compound was sufficiently potent
against renin to warrant further profiling.
The exact mechanism by which the amide moiety promotes the
observed time-dependent inactivation of CYP3A remains unclear. If
the amide itself was indeed the culprit, one could, as was observed
a,b
c
Compd
R
Renin IC50 (nM)
CYP3A4 IC50 (lM)
with compounds 29 and 40, dial out the TDI liability through the
installation of an appropriate amide isostere. Furthermore, since
independent docking studies have revealed that the amide itself
does not participate in any key stabilizing interaction with the re-
nin enzyme, it should be possible to design an amide-free, potent
renin inhibitor as long as the proper orientation of the flanking
substituents are preserved.23 Indeed, our successful realization of
this strategy will be disseminated in a separate communication.
An alternative explanation can also be envisioned, wherein the re-
Buffer
Plasma
30 min
Pre-Inc
0 min
Pre-Inc
Shift
1
41
—
OMe
0.09
0.02
2.4
3.5
0.12
1.3
14
2
110
1.5
42
0.03
2.3
3.2
3.0
<1
N
O
a
b
c
See Ref. 9 for assay protocols.
Average of at least two replicates.
See Ref. 10 for assay protocols.

A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5074–5079
5079
diluted in DMSO and 25
3 h. At the end of the incubation period, 5 lL of the renin reaction (or standards
l
L added to the pre-mix, then incubated at 37 °C for
moval of the amide moiety instead served to reduce the formation
of non-amide derived reactive intermediate(s).24 Consequently, it
should be possible to keep the amide but to also introduce a distal
metabolic soft spot to divert the metabolism to a more innocuous
area in the molecule. In this regard, analogues possessing a second
appendage on the P3 aromatic plate were prepared and tested
(Table 6).
We were gratified to observe that these modifications not only
led to a significant improvement in TDI but did so without jeopar-
dizing their respective potency against renin. Furthermore when
these compounds were incubated with human liver microsomes
so that their respective in vitro clearance can be determined,25
compound 1 (Clint = 15 mL/min/kg) was indeed found to be more
metabolically stable than both compounds 41 (Clint = 32 mL/min/
kg) and 42 (Clint = 45 mL/min/kg).
in the assay buffer) were measured for AngI accumulation. The percentage of
renin inhibition (AngI decrease) was calculated for each concentration of
compound and an IC50 was determined by curve fitting software. Plasma assay:
citrate-plasma from human volunteers was pooled, aliquoted and stored frozen
at À20 °C. Renin activity in pooled plasma was supplemented with
recombinant human renin (150 pg/mL final concentration) in order to
increase the readout of the assay.
concentrations in DMSO, was added to 80
plasma and a fast trapping primary AngI antibody (anti-AngI: AS1, bleed 6, pre-
diluted 1:10 in horse serum) diluted initially 1:26.5 in assay buffer (PBS1X,
1 mM EDTA, 0.1% BSA, pH 7.4) and then diluted 1:3.3 in 3 M Tris, 200 mM
EDTA, pH 7.2 (final anti-serum dilution 1:50,000) and was incubated at 37 °C
5
l
L
of renin inhibitors, at various
L of a mixture (7:1) of human
l
for 2 h. As was done in the buffer assay, 12 lL of the renin reaction (or
standards in the assay buffer) were measured for AngI accumulation by
immunoassay.
10. CYP3A4 IC50 shift assay: 0.25 mg/mL of human liver microsomes, 0.03–200 lM
of test compound, and 1 mM NADPH in 125 mM phosphate buffer pre-
incubated for 0 or 30 min at 37 °C. Then, 500uM of testosterone (CYP3A
substrate) added and incubation carried-out for additional 15 min at 37 °C.
Samples are then quenched with acetonitrile, centrifuged and formation of 6b-
hydroxy-testosterone monitored by HPLC-MS/MS.
In conclusion, we have described our effort to identify the key
functionality responsible for the time-dependent CYP3A4 inhibi-
tion observed with the leading compounds in our recently dis-
closed novel series of renin inhibitors. Two unique strategies to
overcome this liability were also successfully implemented.
11. Brown, C. H.; Reisfeld, B.; Mayeno, A. N. Drug Metab. Rev. 2008, 40, 1.
12. MacDonald, T. L.; Eirri, K.; Burka, L. T.; Peyman, P.; Guengerich, F. P. J. Am. Chem.
Soc. 1982, 104, 2050.
13. Cerny, M. A.; Hanzlik, R. P. J. Am. Chem. Soc. 1982, 104, 3346.
14. Borheim, L. M.; Everhart, E. T.; Li, J.; Correia, M. A. Biochem. Pharmacol. 1993, 45,
1323.
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Am. Chem. Soc. 1997, 119, 6496.
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scenario.
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25. Metabolic stability assay: Incubations performed at 37 °C in 125 mM phosphate
buffer in presence of 1 mg/mL human liver microsomes, NADPH regenerating
system and 1uM of test compound. Incubates quenched at t = 2, 5, 10, 20, 45
and 75 min with acetonitrile, centrifuged, and supernatant analyzed by HPLC-
MS/MS to monitor disappearance of test compound over-time. Clint calculated
based on initial Ke.
9. Buffer assay: recombinant human renin (3 pg/
EDTA, 0.1% BSA, pH 7.4), human tetradecapeptide (1–14) substrate (5
10 mM HCl), hydroxyquinoline sulfate (30 mM in H2O) and assay buffer were
pre-mixed at 4 °C at a ratio of 100:30:10:145. 47.5 L per well of this pre-mix
was transferred into polypropylene plates. Test compounds were dissolved and
l
L) in assay buffer (PBS1X, 1 mM
l
M in
l