The discovery of non-benzimidazole and brain-penetrant prolylcarboxypeptidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.10.060

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC ₅₀ values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to

Full text of DOI:10.1016/j.bmcl.2011.10.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 658–665
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of non-benzimidazole and brain-penetrant
prolylcarboxypeptidase inhibitors
Thomas H. Graham a, , Hong C. Shen a, Wensheng Liu a, Yusheng Xiong a, Andreas Verras a, Kelly Bleasby c,
⇑
Urmi R. Bhatt d, Renee M. Chabin b, Dunlu Chen d, Qing Chen c, Margarita Garcia-Calvo d,
Wayne M. Geissler d, Huaibing He c, Michael E. Lassman d, Zhu Shen d, Xinchun Tong c, Elaine C. Tung c,
Dan Xie d, Suoyu Xu c, Steven L. Colletti a, James R. Tata a, Jeffrey J. Hale a, Shirly Pinto d, Dong-Ming Shen a
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Research Operations, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC50 values were prepared by replacing
the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substi-
tuted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition
shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The com-
pounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of com-
pound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 24 August 2011
Revised 17 October 2011
Accepted 18 October 2011
Available online 24 October 2011
Keywords:
Prolylcarboxypeptidase
PrCP
Angiotensinase C
P-glycoprotein
P-gP
Enzyme inhibitor
Obesity
Metabolic syndrome
Medicinal chemistry
Amide
Benzimidazole
Bioisostere
Melanocyte stimulating hormone
Prekallikrein
Angiotensin
Prolylcarboxypeptidase (PrCP) (angiotensinase C) is a widely
distributed serine protease which is expressed in many tissues
including liver, kidney, pancreas, heart, brain, adipose, hypothala-
mus and gut.1 In addition, extracellular forms of the enzyme are
present in plasma and urine.2 PrCP is known to cleave the amide
bond between a C-terminal amino acid and a proline residue (i.e.,
peptide-Pro-Xxx-OH), and degrades angiotensins II and III,3 plasma
mation induction,7 chemotherapy resistance8 and metabolism
regulation.9
Several recent studies focused on the metabolic role of PrCP in
body weight regulation. Genetic studies have suggested that inhi-
bition of PrCP expression leads to a lean phenotype in mice.10 In
addition, human genetic analysis has implicated PrCP mutations
in obesity and metabolic syndromes.11
The known biological activity and the genetic studies of PrCP
provided the evidence necessary to initiate a program focused on
identifying small molecule inhibitors of PrCP.12 The discovery of
potent inhibitors of PrCP would provide additional tools for under-
standing the biological role of PrCP and define the potential for
PrCP inhibition as a treatment for obesity. Initial efforts produced
two general classes of potent PrCP inhibitors (Fig. 1). The first class,
prekallikrein4 and
a-melanocyte stimulating hormone (
a
-MSH).5
The extensive tissue distribution and the ability to degrade numer-
ous bioactive peptides have led to several hypotheses concerning
the biological role of PrCP including cardiovascular effects,6 inflam-
⇑
Corresponding author. Tel.: +1 732 594 2918; fax: +1 732 594 3007.
E-mail address: thomas.graham@merck.com (T.H. Graham).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.060

T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
659
represented by 1, demonstrated statistically significant mecha-
nism-based weight loss in the established diet-induced obesity
(eDIO) mouse model.13 In addition, 1 was a substrate for P-glyco-
protein (P-gp) efflux transporters and the drug-level in the central
nervous system (CNS) was low. The observed weight decrease
caused by 1 is believed to be due to peripheral PrCP inhibition. A
second class, represented by 2, demonstrated potent in vitro PrCP
inhibition and is also believed to act peripherally.14 However,
in vivo studies of the second class using the PrCP knock-out eDIO
mouse indicated a weight decrease which was not mechanism-
based. The disparity in the results for the in vivo inhibition of PrCP
was the first indication of the potential complexity of the PrCP reg-
ulatory mechanism and prompted efforts to understand the effect
of inhibiting PrCP in the central and peripheral systems.
inhibition. Compound 3g, with the phenyl as a bioisostere of the
isopropyl or trifluoromethyl, showed similar activity to 3d and
3f.17 Exchanging an aryl group for a pyridyl group further im-
proved PrCP inhibition (3j and 3k) and was dependent on the posi-
tioning of the pyridyl nitrogen (3l and 3m).18 Introduction of
methyl groups to block potential sites of metabolism (3n) or shield
the pyridyl nitrogen (3o) decreased the inhibitory activity relative
to 3k.
Selected compounds were also tested in a whole serum assay
using mouse PrCP with angiotensin III (AngIII) as a substrate (Ta-
ble 1).19 The activity shifts for compounds (S)-3b, 3e and 3j in
the presence of whole mouse serum were minimal and compared
favorably to 1 and 2 which had 13-fold and 43-fold shifts in the
mouse AngIII assay, respectively (Fig. 1).
The in vivo results for the initial two classes of PrCP inhibitors
suggested that additional small molecule inhibitors were required
to understand the role of PrCP in body weight homeostasis. Efforts
were therefore directed towards diversifying the initial lead struc-
tures with a focus on controlling recognition by P-gp efflux trans-
porters and thus increasing PrCP inhibition in the CNS.15 The
obvious structural element common to both classes of PrCP inhib-
itors represented by 1 and 2 is the dichlorobenzimidazole-substi-
tuted pyrrolidine. The origin of this common motif resides in the
design logic which drove the development of 1 and 2; namely,
the scissile amide bond of the natural peptide-Pro-Xxx-OH sub-
strates was emulated with heterocyclic amide bioisosteres includ-
ing imidazoles and benzimidazoles.13 In a departure from the
original hypothesis, less obvious replacements for the scissile
amide bond were considered. Specifically, the removal of the het-
erocyclic hydrogen bond acceptor/donor pair was expected to in-
crease passive diffusion16 and decrease recognition by P-gp.15
The strategy was implemented by preparing and testing com-
pounds of the general structure 3 in an in vitro enzyme inhibition
assay according to previously described procedures (Table 1).13,14
Analogs 4 and 5 which contain replacements for the dichloro-
benzimidazole-substituted pyrrolidine in 1 were also prepared
(Table 2). Series 4, lacking the 2-aminoisobutyroyl group, demon-
strated diminished inhibitory activity relative to series 5. In con-
trast to 3c, which showed moderate inhibition relative to 2, 5a
demonstrated similar inhibitory activity to 1. Isopropyl substitu-
tion (5b) had decreased PrCP inhibition relative to 1. Fluorine sub-
stitution (5c and 5d) decreased the inhibition by 2- to 4-fold
relative to 1. Pyridine substitution (5e) demonstrated similar
inhibitory activity to 1. Compounds were also tested in the mouse
AngIII assay.19 Most compounds demonstrated large whole serum
activity shifts. Compound 5e is notable because it is the most po-
tent in the primary assay and has the lowest whole serum shift
of the tested compounds in Table 2.
A series of simplified analogs related to compound 3 were also
investigated (Table 3). Compound 6a, containing the dichlorobenz-
imidazole-substituted pyrrolidine was previously disclosed as a
potent inhibitor of human PrCP; however, it was significantly less
potent in the mouse AngIII assay.14 Most of the compounds in Ta-
ble 3 demonstrated inhibitory activities that were comparable to
6a and 2. Both (S)-6e and 6f had minimal serum shift in the mouse
AngIII assay.
The recognition of compounds 3 and 6 by P-gp efflux transport-
ers was assessed by using a standard assay (Table 4).20 All of the
compounds were cell permeable. Many of the compounds were
not substrates of human P-gp indicating that replacing the dichlo-
robenzimidazole-substituted pyrrolidine in 2 reduced recognition
by P-gp. In contrast, the pyridine-containing compounds 3k–n
were P-gp substrates. The results for 3k–m suggested that the
P-gp activity was dependent on the pyridyl nitrogen position.
Compounds 3f, (S)-6e and 6f were not in vitro substrates of mouse
P-gp, allowing for the study of PrCP inhibition in vivo with a
compound which was not a substrate of P-gp.
Substituted
able replacements for the dichlorobenzimidazole-substituted pyr-
rolidine in 2. The parent -methylbenzylamine (S)-3a was only
a-methylbenzylamines were rapidly identified as suit-
a
moderately active; however, para-substitution with fluorine ((S)-
3b) or chlorine ((S)-3c) afforded a 5-fold and 10-fold improvement,
respectively. The enantiomers, (R)-3a and (R)-3b, were less active.
Efforts to reduce the hydrogen-bond accepting character of the
amide bond, and thus reduce P-gp recognition, focused on increas-
ing the steric shielding of the amide with an isopropyl group (3d)
or rendering the amide electron-deficient by fluorine substitution
(3e and 3f).15 Compounds 3d and 3f exhibited improved PrCP
The potential to attain significant drug levels of (S)-6e in the
brain was confirmed by in vivo studies with the CF1 MDR1A
wild-type (WT) and the MDR1A knock-out mouse (Table 5).
Administration of a 1 mpk IV dose of (S)-6e to the CF1 MDR1A
WT mouse (n = 3) afforded brain to plasma ratios of 4.7 and 2.5
at the 30 min and 4 h post-dose time points, respectively. The
CF1 MDR1A(ꢀ/ꢀ) mouse (n = 3) had brain to plasma ratios of 10
and 11 for the 30 min and 4 h post-dose time points, respectively.
Although the WT exhibited a lower brain to plasma ratio than the
MDR1A(ꢀ/ꢀ), the absolute concentrations indicated that a signifi-
cant brain level of (S)-6e was attained in the WT mouse. In con-
trast, when 1 was dosed PO at 30 mpk, the CF1 MDR1A WT
(n = 4), had brain to plasma ratios of 0.01 and 0.04 for the 2 h
and 6 h post-dose time points, respectively.21 The CF1 MDR1A(ꢀ/
ꢀ) mouse (n = 4) had brain to plasma ratios of 0.9 and 3.6 for the
2 h and 6 h post-dose time points, respectively. The results con-
firmed that the decreased recognition of (S)-6e by P-gp efflux
transporters afforded higher absolute brain levels of (S)-6e relative
to 1.
H
N
Me
N
Cl
Cl
N
O
O
HN
Me
1
PrCP IC50 (h,m): 1, 2 nM
NH2 Mouse AngIII IC5 0: 26 nM
Me
H
N
N
Cl
Cl
N
N
O
N
2
PrCP IC50 (h,m): 12, 4 nM
Mouse AngIII IC50: 173 nM
O
N
N
Figure 1. Previously disclosed structures of potent prolylcarboxypeptidase
inhibitors.

660
T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
Table 1
PrCP inhibition data for analogs of 2a
O
N
R
N
3
O
N
N
R=
Compound
PrCP (h, m)b IC50, nM
1520, —
Mouse AngIII IC50, nM
(S)-3a (X = H)
(R)-3a (X = H)
(S)-3b (X = F)
(R)-3b (X = F)
(S)-3c (X = Cl)
—
—
348
—
Me
32% (10
313, —
8700, —
151, —
lM)
N
H
X
—
Me
Me
3dc
3ec
21, —
—
N
H
Cl
Cl
Cl
CHF2
CF3
N
H
104, —
156
3fd
17, —
836, —
—
—
N
H
ent-3fd
3gc
15, —
—
N
H
Cl
Cl
Cl
3h
33, 10
305
N
H
N
N
3ic
26, —
—
N
H
3jc
7.6, —
15
N
H
F
N
3kc
2.7, —
—
—
N
H
Cl
Cl
N
3lc
37, —
N
H

T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
661
Table 1 (continued)
R=
Compound
PrCP (h, m)b IC50, nM
Mouse AngIII IC50, nM
N
3mc
64, —
—
—
N
H
Cl
Cl
Cl
N
3nc
11, —
Me
N
H
N
Me
3oc
26, —
—
N
H
a
b
c
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
Data are for the racemate.
d
Data are for the single enantiomer, absolute stereochemistry was not determined.
Table 2
PrCP inhibition data for analogs of 1a
4, R2 = H
Me
O
R1
O
5, R2
=
HN
NH2
R2
R1=
Compound
PrCP (h, m)b IC50, nM
Mouse AngIII IC50, nM
Me
4a
5a
15, 11
0.9, 2.3
570
198
N
H
Cl
Me
Me
F
4bc
5bc
53, —
17, —
—
—
N
H
Cl
F
4cc
5cc
21, —
3.8, 6.6
—
458
N
H
Cl
Cl
CF3
4dc
5dc
32, —
1.7, —
—
—
N
H
N
N
4ec
5ec
2.5, 1.4
0.6, 0.4
108
6
N
H
Cl
Cl
Me
4fd
45, —
248, —
—
—
epi-4fd
N
H
(continued on next page)

662
T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
Table 2 (continued)
R1=
Compound
PrCP (h, m)b IC50, nM
Mouse AngIII IC50, nM
Me
N
Me
4gd
1250, —
5600, —
—
—
epi-4gd
N
H
Cl
a
b
c
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
Data are for the mixture of epimers.
d
Data are for the single epimer, absolute stereochemistry was not determined.
Table 3
PrCP inhibition data for simplified analogs of 3a
O
R
N
6
R=
Compound
PrCP (h, m)b IC50, nM
Mouse AngIII IC50, nM
>10000
H
N
N
Cl
Cl
6a
10, —
N
Me
6b (X = H)
6c (X = Cl)
80, —
10, 54
—
240
N
H
6d (X = Br)
8.4, 20
—
X
Me
Me
F
(S)-6e
(R)-6e
1.5, 5.2
199, —
12.6
—
N
H
Cl
F
6fd
3.2, 17
160, —
17
—
ent-6fd
N
H
Cl
Cl
CF3
6gd
1.4, 14
70, —
—
—
N
H
ent-6gd
Cl
6h
5.2, 4.0
58
N
H
Cl
F
N
N
6ic
3.8, —
—
—
N
H
6jc
0.3, 2.1
N
H
Cl

T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
663
Table 3 (continued)
R=
Compound
PrCP (h, m)b IC50, nM
Mouse AngIII IC50, nM
N
6kc
1.2, —
—
—
—
Me
N
H
Cl
Cl
N
Me
6lc
2.1, —
N
H
Me
N
Me
6mc
79, —
N
H
Cl
Me
N N
6nc
11, 25
—
—
N
H
Cl
Me
6od
2.8, 5.7
N
H
a
b
c
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
Data are for the racemate.
d
Data are for the single enantiomer, absolute stereochemistry was not determined.
Table 4
Table 5
P-gp efflux data for selected PrCP inhibitorsa
In vivo brain levels of (S)-6e in the WT and MDR1A deficient mousea
Compound
BA/AB ratio (h, m)b
Pappc (10ꢀ6 cm/s)
0.5 h (nM)
4 h (nM)
33
83 18
2.5
34 15
383 191
11
2
13, 25
23
31
35
24
30
32
33
27
21
28
21
18
MDR1A WT plasma
MDR1A WT brain
260 49
1197 23
4.7
336 44
3449 336
10
6
(S)-3c
3f
3g
3k
3l
3m
3n
(S)-6e
6f
6n
6o
1.4, 9.8
0.8, 2.2
1.0, 5.2
15, 21
14, 16
2.5, 8.2
11, 36
0.4, 1.6
0.3, 0.7
2.8, 13
0.4, 1.3
MDR1A WT brain/plasma
MDR1A (ꢀ/ꢀ) plasma
MDR1A (ꢀ/ꢀ) brain
MDR1A (ꢀ/ꢀ) brain/ plasma
a
CF1 MDR1A WT mouse (n = 3) or CF1 MDR1A(ꢀ/ꢀ) mouse (n = 3), 1 mpk IV
dose; matrix concentrations were measured at 0.5 and 4 h post-dose.
subcutaneous infusion pump dosing of (S)-6e at 10 mpk (n = 8)
failed to demonstrate statistically significant weight loss relative
to the vehicle dose (Fig. 2a). Daily food intake was similar to the
vehicle-dosed cohort. Terminal plasma concentration was
a
b
c
Compounds tested as 1 lM HBSS solutions.
Human LLC-MDR1 or mouse LLC-MDR1a cell lines.
LLC-PK1 cell line (control).
0.14 0.07
(p <0.0001) (Fig. 2b). The terminal brain exposure was
0.29 0.12 M and confirmed that substantial CNS exposure was
achieved. The results indicated that increased inhibition of PrCP
may be required for statistically significant weight loss to be
observed.
A sub-chronic weight loss study was performed in wild-type
(WT) and PrCP knockout (KO) mice to assess the in vivo PrCP target
engagement. Compound (S)-6e was dosed subcutaneously by con-
tinuous infusion pump over 7 days at 10 and 30 mpk (n = 8).23
Body weight and food intake were measured daily. For the
lM and the terminal plasma PrCP inhibition was 65%
Mouse pharmacokinetic (PK) profiles for several compounds re-
l
vealed a universally high clearance, high volume of distribution
and low oral bioavailability (Table 6). Despite the PK profile of
compound (S)-6e,22 the in vitro potency, low serum shift and cell
permeability resulted in its selection for in vivo studies. Continu-
ous subcutaneous dosing via infusion pump was required to com-
pensate for the poor PK profile and allowed for the evaluation of
(S)-6e as the first potent brain-penetrant PrCP inhibitor.
A study was conducted in the WT eDIO mouse model to mea-
sure the in vivo inhibition of PrCP. A 7-day study with continuous

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T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
Table 6
pyrrolidine, common to the previous PrCP inhibitors 1 and 2, was
Mouse PK data for selected PrCP inhibitorsa
replaced by a series of substituted benzylamines. Many of the com-
pounds demonstrated PrCP inhibition in whole serum, decreased
recognition by P-gp efflux transporters and were cell permeable.
Compound (S)-6e exhibited significant brain exposures during
in vivo studies; however, the dosing of (S)-6e at 30 mpk caused
non-mechanism-based weight loss and 10 mpk dosing did not
achieve statistically significant mechanism-based weight loss.
The in vivo results suggested that very high levels of PrCP inhibi-
tion may be required for efficacy in the eDIO model. Therefore,
compounds with improved oral pharmacokinetics and enhanced
in vivo efficacy will be required to fully understand the role of PrCP
in body weight homeostasis. The results from studies directed to-
wards this goal will be reported in due course.
Compound
Cl
Vd
(L/kg)
T
(h)
%F
AUCNpo
(M h kg/mg)
½
(mL/min/kg)
3h
5d
5e
6c
(S)-6e
6f
105
78
85
185
217
389
5.6
24
21
27
31
59
0.7
4.4
3.6
2.3
1.7
1.8
1.3
40
<0.01
0.19
ndb
<0.01
0.04
0.04
ndb
<1
22
35
a
Formulations: 1 mg/mL ethanol–PEG–water (20:40:40), IV dose: 1 mg/kg
(n = 2), PO dose: 2 mg/kg (n = 3), blood concentration was determined by LC/MS/MS
following protein precipitation with acetonitrile.
b
Not determined.
References and notes
(S)-6e (10 mpk)
Vehicle
1.0
0.5
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during a 7-day study; (b) Plasma PrCP inhibition at the termination of the study.
30 mpk dosing, both the KO and the WT cohorts lost weight and
this result indicated non-mechanism-based weight loss. In addi-
tion, significant skin-related adverse events (AEs) occurred with
compound precipitation at the infusion pump injection site. Termi-
nal plasma concentrations were 0.22 0.14 and 0.25 0.08 lM for
the WT and KO cohorts, respectively. The WT PrCP inhibition was
calculated to be 85% based on the heat-inactivated plasma shifted
Ki.24 The 10 mpk cohorts failed to demonstrate statistically signif-
icant weight loss relative to the vehicle dose and there were negli-
gible AEs at the injection site. Terminal plasma concentrations
were 0.27 0.17 and 0.16 0.06 lM for the WT and KO cohorts,
respectively. The WT PrCP inhibition was calculated to be 87%
based on a heat-inactivated plasma shifted Ki.24 The results dem-
onstrated that the required level of inhibition cannot simply be
achieved with higher doses. Increasing the dose above 10 mpk
did not afford dose proportional in vivo inhibition of PrCP and
led to skin-related AEs and precipitation of the compound at the
injection site.25
16. Pardridge, W. M. Adv. Drug Delivery Rev. 1995, 15, 5.
17. (a) Zanda, M. New J. Chem. 2004, 28, 1401; (b) Mikami, K.; Itoh, Y.; Yamanaka,
M. Chem. Rev. 2004, 104, 1.
18. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.
19. The mouse AngIII assay involved treating the whole mouse plasma with
protease inhibitors to suppress the conversion of AngIII to Ang (2–7) by non-
PrCP proteases. The PrCP inhibitor was then added to study the rate of
conversion of AngIII to Ang (2–7) as compared to a vehicle treated control.
In conclusion, potent brain-penetrant inhibitors of PrCP
have been described. The dichlorobenzimidazole-substituted

T. H. Graham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 658–665
665
20. He, H.; Lyons, K. A.; Shen, X.; Yao, Z.; Bleasby, K.; Chan, G.; Hafey, M.; Li, X.; Xu,
S.; Salituro, G. M.; Cohen, L. H.; Tang, W. Xenobiotica 2009, 39, 687.
21. Data for 1, following 30 mpk PO dosing:
afforded (S)-6e (384.1 mg, 85%) as a clear, colorless wax which crystallized
after aging overnight at ambient temperature. 1H NMR (500 MHz, CDCl3) d 9.25
(d, J = 8.6 Hz, 1H); 7.36–7.18 (m, 9H); 4.82 (dd, J = 8.6, 6.7 Hz, 1H); 3.20 (d,
J = 11.5 Hz, 1H); 3.15 (d, J = 11.5 Hz, 1H); 2.75–2.56 (m, 3H); 2.52–2.39 (m, 2H);
2.22–2.12 (m, 2H); 2.07–1.93 (m, 3H); 1.86–1.70 (m, 2H); 0.96 (d, J = 6.8 Hz,
3H); 0.90 (d, J = 6.7 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 172.1, 145.7, 140.9,
132.7, 128.8, 128.6, 128.4, 126.8, 126.6, 58.0, 54.6, 54.0, 53.7, 42.4, 33.9, 33.6,
33.5, 32.1, 19.9, 18.6; Exact mass calcd for C24H31ClN2O: 398.2 (100.0%), 399.2
2 h (nM)
306 41
6 h (nM)
91 53
MDR1A WT plasma
(26%), 400.2 (32%); found [M+H+]: 399.3 (100%), 400.3 (25%), 401.3 (33%); [
a]
D
MDR1A WT brain
4
1
3
1
+25.0 (CHCl3, c 1.00, 20 °C).
MDR1A WT brain/plasma
MDR1A (ꢀ/ꢀ) plasma
MDR1A (ꢀ/ꢀ) brain
0.01
0.04
23. The vehicle for subcutaneous dosing was DMSO–1,2-propanediol (1:1 v/v).
24. The heat-inactivated plasma shift assay was developed to eliminate non-PrCP
protease activity while measuring PrCP inhibition in the plasma from
186 52
166 68
0.9
51 21
185 46
3.6
compound treated animals. Plasma (35
50% with de-ionized water (70 L total volume) and heat-inactivated at 65 °C
for 45 min. The heat-inactivated plasma was diluted to 25% final volume and
treated with PrCP FRET substrate (unpublished, 15 final), 100 mM
lL) from dosed mice was diluted to
MDR1A (ꢀ/ꢀ) brain/plasma
l
a
lM
sodium acetate (pH 5.5) and recombinant mouse PrCP (1 nM final). The
conversion of the FRET substrate to the product was measured kinetically by
fluorescence. Enzyme inhibition was calculated relative to vehicle and
inhibition in 100% plasma was calculated based on measured Kiobs. Control
experiments were performed for heat stability and plasma shift of the
22. The method for the preparation of (S)-6e: A mixture of 3-(4-phenylpiperidin-1-
yl)propanoic acid (265 mg, 1.14 mmol, 1.0 equiv) and (S)-1-(4-chlorophenyl)-
2-methylpropan-1-amine hydrochloride ([
a]
D
+5.0 (CH3OH, c 1.00, 20 °C))
(250 mg, 1.14 mmol, 1.0 equiv) in dichloromethane (4.0 ml) at 0 °C was treated
compound. The details of the assay will be disclosed in
communication.
a separate
with 1-hydroxy-benzotriazole hydrate (34.8 mg, 0.23 mmol, 0.2 equiv), N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(261 mg,
25. The brain exposure of (S)-6e was not measured for the WT/KO study, but CNS
1.36 mmol, 1.2 equiv) and N-methylmorpholine (0.150 mL, 1.36 mmol,
1.2 equiv). The mixture was stirred at rt for 12 h and then directly applied to
exposure was inferred from the previous in vivo studies.
a
SiO2 column (25 g). Gradient elution (0–10% methanol/ethyl acetate)