3-amido-3-aryl-piperidines: A novel class of potent, selective, and orally active GlyT1 inhibitors

Article abstract of DOI:10.1021/ml500005m

3-Amido-3-aryl-piperidines were discovered as a novel structural class of GlyT1 inhibitors. The structure-activity relationship, which was developed, led to the identification of highly potent compounds exhibiting excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo activity after oral administration.

Full text of DOI:10.1021/ml500005m

Letter
pubs.acs.org/acsmedchemlett
3‑Amido-3-aryl-piperidines: A Novel Class of Potent, Selective, and
Orally Active GlyT1 Inhibitors
Emmanuel Pinard,* Daniela Alberati, Ruben Alvarez-Sanchez, Virginie Brom, Serge Burner,
Holger Fischer, Nicole Hauser, Sabine Kolczewski, Judith Lengyel, Roland Mory, Christian Saladin,
Tanja Schulz-Gasch, and Henri Stalder
Pharmaceutical Research Basel, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
S
* Supporting Information
ABSTRACT: 3-Amido-3-aryl-piperidines were discovered as a novel structural
class of GlyT1 inhibitors. The structure−activity relationship, which was
developed, led to the identification of highly potent compounds exhibiting
excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo
activity after oral administration.
KEYWORDS: GlyT1 inhibitor, SAR, 3-amido-3-aryl-piperidines, Schizophrenia
chizophrenia is a devastating and chronic mental illness
that affects close to 1% of the population. Symptoms of
S
schizophrenia, which typically arise at young age (adolescence
or early adulthood), are categorized as positive, negative, or
cognitive. Currently approved drugs are efficacious in the
treatment of positive symptoms but do not address the negative
and cognitive symptoms. Numerous lines of evidence suggest
that hypofunction of glutamatergic transmission via N-methyl-
D-aspartate (NMDA) receptors may represent the final
common pathway leading to symptoms in schizophrenic
patients.1 One approach to normalize the reduced NMDA
receptor activity is to elevate the concentration of the coagonist
glycine at its modulatory site on the receptor through blockade
of the glycine transporter type 1 (GlyT1), which is colocalized
with the NMDA receptor.2 In the past decade, ample efforts
have been focused on the discovery and development of
selective GlyT1 inhibitors.3 The first examples reported were
sarcosine derivatives including 14 and 25 (Figure 1).
Figure 1. Selection of published GlyT1 inhibitors.
More recently, nonsarcosine-based compounds6 have been
described such as DCCCyB (3),7 GSK1018921 (4),8 and
bitopertin (5),9,10 which all progressed to clinical studies
(Figure 1). In a phase II proof of concept study, Bitopertin
demonstrated a beneficial effect in patients with schizophrenia
characterized with predominant negative symtoms.11 However,
recently, Hoffmann-La Roche announced that two phase III
studies of bitopertin did not meet their clinical end points.12
Four additional phase III studies with bitopertin are ongoing.
As part of our continued effort to discover and develop novel
and selective GlyT1 inhibitors, we considered the pyrrolidino-
ethyl-amide 4 as a potential starting point for further structural
modification. In the low energy conformation obtained for
compound 4 (Figure 2), the carbon (C1) adjacent to the
pyrrolidine nitrogen and the benzylic carbon (C2) bearing the
phenyl and amide substituents appeared as suitable attachment
points for forming, via a linker, a novel cyclic system (Figure 2).
We thus decided to investigate this cyclization approach
further. As connecting elements between C1 and C2, a bond, a
methylene, and an ethylene unit were evaluated (in red, Figure
2). To make our designed molecules chemically tractable, the
gem dimethyl group and the pyrrolidine ring present in 4 were
deleted. As a result, our approach led us to the generation of
the 3,3′-substituted-N-methyl-azetidine 6, pyrrolidine 7, and
piperidine 8 (Figure 2). In a glycine uptake inhibition assay
performed in CHO cells transfected with hGlyT1, azetine 6 and
pyrrolidine 7 were found inactive or poorly active. In contrast,
and to our delight, piperidine 8 displayed a promising GlyT1
potency with an IC50 of 140 nM. The X-ray crystal structure of
813 (Figure 2) showed that the amide and phenyl substituents
Received: January 6, 2014
Accepted: February 4, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
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Figure 2. Design principles leading to the identification of active 3-amido-3-aryl-piperidine 8 using pyrrolidino-ethyl-amide 4 as a seed structure.
at the position 3 on the piperidine ring adopted, respectively, an
axial and equatorial orientation. In addition, because of the
presence of the two ortho groups on the benzamide side, the
carbonyl function adopted a highly twisted orientation with a
torsion angle of 78°. Overall, as depicted in Figure 2, the crystal
structure of 8 aligned very well with the low energy
conformation of 4, a result perfectly in line with the good
GlyT1 potency measured for 8. A literature and patent search
revealed that, although quite structurally simple, 3-amido-3-
aryl-piperidines such as 8 had not been previously described.
Thus, the discovery of 8 offered us the unique opportunity to
develop a novel series of potent GlyT1 inhibitors having an
exquisite intellectual proprietary status. In this letter, we report
on our structure−activiy relationship (SAR) exploration effort
in this series14 that culminated in the identification of potent,
selective, and orally active GlyT1 inhibitors such as compound
29.
indicated in Scheme 2 were prepared from the 3-aryl-3-
piperidin-ol derivatives 46a−d under modified Ritter reaction
Scheme 2. Synthesis of N-Methyl-3-amino-3-aryl-piperidines
a
44a−d
a
Reagents and conditions: (a) NaN3, TFA, H2O, RT, 87−100%; (b)
LiAlH4, THF, RT, 32−63%.
conditions15 in the presence of sodium azide and trifluoroacetic
acid, followed by reduction of the obtained azido intermediates
47a−d with lithium aluminum hydride.
N-Methyl-3-amido-3-aryl-piperidine derivatives 8 and 18−43
were synthesized under standard amide coupling conditions
from N-methyl-3-amino-3-aryl-piperidines 44 (Scheme 1).
However, this synthetic approach turned out to be not
versatile enough. In particular, the azidation reaction performed
on 3-aryl-3-piperidinols carrying electron rich aryl R3 groups
such as 4-OMe-Ph failed. During the search of a more general
access route, we discovered that N-methyl-3-amino-3-aryl-
piperidine intermediates 44 could be efficiently prepared from
easily accessible 4-aryl-4-nitro-butyric acid methyl esters 51 via
a novel route involving as a key step a Mannich in situ
lactamization reaction as depicted in Scheme 3. When
performed in the presence of methylamine and formaldehyde,
this sequence provided with good to excellent yields N-methyl-
5-aryl-5-nitro-piperidin-2-one intermediates 52, which led to
the target building blocks 44 after the reduction of the nitro
and carbonyl functions. In addition, the synthetic access route
we have established for 44 was successfully applied to the
preparation of N-Boc-3-amino-3-phenyl-piperidine building
block 45 using ammonium acetate and formaldehyde as
reagents in the key Mannich in situ lactamization reaction
(Scheme 3).
Our first effort aimed at establishing the SAR at the
piperidine nitrogen (Table 1). Starting from 8, deletion of the
methyl group (compound 9) led to a 5-fold reduction in GlyT1
activity. A drop of activity was equally observed upon replacing
the methyl group with larger alkyl (10, 11) or cycloalkyl groups
(12, 13). Moreover, N-benzylation (14) as well as N-acylation
and sulfonylation (15−17) practically abolished the GlyT1
potency suggesting that a certain level of basicity of the
piperidine nitrogen is required for GlyT1 activity. Overall, our
a
Scheme 1. Synthesis of 3-Amido-3-aryl-piperidines 8−43
a
Reagents and conditions: (a) R2COCl, DIPEA, CH2Cl2, RT, 4−90%;
(b) R2CO2H, HATU, DIPEA, DMF, RT, 18−68%; (c) 2- OMe,2′,4-
(CF3)2-PhCOCl, DIPEA, CH2Cl2, RT, 86%; (d) HCl, dioxane, RT,
86%; (e) R1I, DIPEA, CH2Cl2, RT, 58%; (f) ketone or aldehyde,
NaCNBH3, AcOH, MeOH, RT, 63−89%; (g) acyl- or sulfonyl-
chloride, DIPEA, CH2Cl2, RT, 65−99%.
Derivatives 10−17 having different R1 groups on the piperidine
nitrogen were obtained upon alkylation, reductive amination,
acylation, or sulfonylation of the unsubstituted piperidine 9,
itself prepared from N-Boc-protected-3-amino-3-phenyl-piper-
idine building block 45. N-Methyl-3-amino-3-aryl-piperidine
intermediates 44a−d carrying the aromatic R3 substituents
B
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 3. Synthesis of N-Methyl-3-amino-piperidines 44f−o and N-Boc-3-amino-3-phenyl-piperidine 45
a
Reagents and conditions: (a) methyl acrylate, amberlyst A-21, dioxane, RT, 41−85%; (b) cat. Pd2dba3, cat. di-tert-butyl-(2′-methyl-biphenyl-2-yl)-
phosphane, Cs2CO3, DME, reflux, 10−90%; (c) MeNH2 (41% in water), CH2O (36% in water), dioxane, RT then 65 °C, 49−94%; (d) zinc, HCl,
dioxane, RT, 13−86%; (e) LiAlH4, THF, RT, 71−86%; (f) Lawesson’s reagent, toluene, 80 °C, 77−100%; (g) NaBH4, MeOH, RT, 49−76%; (h)
Ra−Ni, H2 (1 atm.), THF, 0 °C, 87−100%; (i) NH4OAc, CH2O (36% in water), EtOH, reflux, 84%; (j) Boc2O, Et3N, CH2Cl2, RT, 70%.
a
a
Table 1. In Vitro Inhibitory Activity of 9−17 at GlyT1
Table 2. In Vitro Inhibitory Activity of 18−30 at GlyT1
b
compd
R1
GlyT1 EC50 (μM)
b
compd
R2
GlyT1 EC50 (μM)
9
H
0.66
0.23
0.23
0.27
0.38
6.7
18
19
20
21
22
23
24
25
26
27
28
29
30
2-OMe, 6-CF3−Ph
2-OMe, 4-CF3−Ph
2-CF3, 4-CF3−Ph
4-CF3−Ph
2-CN, 4-CF3−Ph
2-F, 4-CF3−Ph
2-Me, 4-CF3−Ph
2-Br, 4-CF3−Ph
2-SMe, 4-CF3−Ph
2-SMe, 2′-OMe, 4-CF3−Ph
S-enantiomer of 27
R-enantiomer of 27
2,4-Cl2−Ph
1.5
10
11
12
13
14
15
16
17
Et
4.8
i-Pr
0.54
>10
>10
9.9
c-C5H9
CH2-c-Pr
CH2−Ph
C(O)−Me
C(O)−Ph
SO2Me
>10
>10
>10
0.32
0.30
0.067
0.024
0.18
0.010
0.67
a
EC50 values are the average of at least two independent experiments.
[3H]-glycine uptake inhibition assay in cells transfected with
b
hGlyT1.10
a
EC50 values are the average of at least two independent experiments.
[3H]-glycine uptake inhibition assay in cells transfected with
exploration at this position revealed a highly restricted SAR
with the methyl group being the only well tolerated substituent.
Next, keeping the preferred N-methyl group in place, the
SAR evaluation at the benzamide region was conducted (Table
2). Starting from 8, selective deletion of the ortho, ortho′, and
para substituents (compounds 18 to 21) resulted in a
significant drop of GlyT1 potency suggesting that substitution
at all three positions on the benzamide moiety is important for
reaching a good level of potency. In particular, the lack of
activity observed for the p-CF3 monosubstituted compound 21,
devoid of any ortho groups suggested that for GlyT1 activity
the amide carbonyl group should adopt a twisted oriention as
seen in the X-ray structure of our initial compound 8. Next,
starting from inactive compound 21 and keeping the p-CF3
group in place, we explored various substituents at the ortho
b
hGlyT1.10
position (compounds 22−26). No improvement of activity was
observed with polar groups in place such as nitrile (22) as well
as with small substituents like fluorine (23). With larger
lipophilic groups such as methyl (24) or bromine (25), the
activity improved by more than 30-fold. Further exploration in
that direction resulted finally in the identification of the
thiomethyl group as one of the best substituents at the ortho
position providing a compound displaying a low nanomolar
activity (26, 67 nM). Our initial SAR studies (vide supra)
suggested that a further gain in activity could be predicted upon
introducing a second ortho group on 26. Pleasingly, by adding a
C
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Letter
methoxy group (27), a very potent compound was generated
demonstrating an EC50 of 24 nM. This compound existing as a
racemic mixture was subsequently separated by chiral HPLC to
provide the two enantiomers 28 ((S)-enantiomer) and 29
((R)-enantiomer). The R-configurated isomer 2916 showed the
best GlyT1 potency, reaching an EC50 as low as 10 nM at the
hGlyT1 (5 nM at the mouse GlyT1).
Table 4. In Vitro Properties of 29
parameter
value
a
log D
3.12
147
7.4
b
solubility (μg/mL)
c
pampa pe (10−6 cm/s)
d
pKa
7.72
human, mouse CLint (μL/min/mg protein)
19, 54
5.4
Finally, the SAR around the aryl group at position 3 on the
piperidine ring was explored (Table 3).
e
hERG IC20 (μM)
CYP450 3A4, 2D6, 2C9, 2C19 IC50 (μM)
>50, 16, >50, 46
f
human, mouse P-gp ER
0.9, 1.5
a
Table 3. In Vitro Inhibitory Activity of 31−43 at GlyT1
a
b
Determined in 1-octanol/phosphate buffer, pH = 7.4. Measured in a
lyophilization solubility assay (LYSA) at pH = 6.5.18 Pe: PAMPA
c
permeation constant through artificial membranes.19 Determined by
d
e
f
capillary electrophoresis. Patch clamp assay. ER = efflux ratio in LLC-
PK1 cells stably expressing human MDR1 and mouse MDR1A.
b
compd
R3
4-F−Ph
4-Cl−Ph
4-Me−Ph
4-OMe−Ph
3-F−Ph
GlyT1 EC50 (μM)
metabolite after incubation in human hepatocytes. In agree-
ment with our SAR results (vide supra), this compound showed
only weak GlyT1 activity (EC50 = 0.45 μM). Gratifyingly, 29
showed low inhibitory activity at the hERG potassium channel
(IC20 = 5.4 μM), a result that may be attributed to the relatively
low basicity of its piperidine nitrogen (pKa = 7.72). For
comparison, pyrrolidino-ethyl-amide seed structure 4, which
displayed a higher basicity (pKa = 9.12), showed a significantly
higher hERG activity (IC20 = 0.7 μM). Moreover, 29
demonstrated a low level of inhibition at the major drug
metabolizing CYP450 enzymes (IC50 > 16 μM) and was
inactive in the genotoxicity assays (Ames and MNT tests).
Finally, 29 was not a substrate for the human and mouse P-gp
efflux systems.
31
32
33
34
35
36
37
38
39
40
41
42
43
0.026
0.028
0.23
0.17
0.048
0.025
0.110
0.054
0.060
0.180
1.670
>10
3-Cl−Ph
3-OMe−Ph
pyridin-3-yl
pyridin-4-yl
5-F-pyridin-2-yl
pyrazinyl-2-yl
pyrimidin-2-yl
pyrimidin-4-yl
1.7
a
EC50 values are the average of at least two independent experiments.
[3H]-glycine uptake inhibition assay in cells transfected with
In vivo pharmacokinetic studies in mouse (Table 5) revealed,
in addition, that 29 had an attractive profile, with, in particular,
b
hGlyT1.10
Table 5. Pharmacokinetics Properties of 29 in Mouse
This study was performed keeping the optimized 2-
a
parameter
value
thiomethyl-2-methoxy-4-trifluoromethyl-phenyl group in place
at the benzamide moiety. A very similar SAR was seen at the
para and meta position on the aryl moiety. Indeed, at both
positions, introduction of EWGs like fluorine or chlorine
iv dose (1.7 mg/kg)
CL (mL/min/kg)
Vss (L/kg)
54
8
(compounds 31−32 and 35−36) was well tolerated (EC50
=
po dose (7.7 mg/kg)
bioavailability F (%)
t1/2 (h)
23−48 nM), whereas with EDGs such as methoxy group (34,
37) the potency dropped significantly (EC50 = 110−170 nM).
Moreover, a 6-membered-ring-heteroaryl scan revealed that the
meta and para positions were the best centers to insert a
nitrogen atom as exemplified with compounds 38 (54 nM) and
39 (60 nM). All the synthetized heteroaryl derivatives having a
nitrogen atom in ortho (40−43) displayed much reduced
potencies.
100
4.2
4.3
1.7
brain/plasma (at 1.5 h)
PPB (% unbound)
a
Values are the average of two independent experiments.
a complete oral bioavailability despite a medium systemic
clearance and an excellent brain penetration (brain/plasma =
4.3), a result in agreement with its low P-gp efflux ratio (1.5)
and large volume of distribution. Interestingly, 29 demon-
strated a much superior brain penetration profile over seed
compound 4 for which a P-gp efflux ratio of 3.9 and a brain/
plasma ratio of only 0.4 was measured in the mouse.
These favorable data led us to evaluate the oral effect of 29 in
the mouse L-687,414-induced hyperlocomotion assay, a novel
and straightforward functional screening model,20 that allows
the detection of the in vivo activity of GlyT1 inhibitors (Figure
3). Pleasingly, 29 displayed a robust pharmacodynamic activity
after oral administration and reached an ID50 (dose producing
50% inhibition of L-687,414-induced hyperlocomotion) of 1.5
mg/kg po. Noteworthy, the in vivo efficacy of 29 was reached
at a very low plasma concentration (8 ng/mL), a result that can
From our SAR and optimization studies, compound 29
emerged as the most potent congener from our newly
discovered piperidine series. Further profiling revealed that
29 had excellent selectivity against GlyT2 (15 μM) as well as a
fairly good selectivity profile in a CEREP screen performed
against a panel of 80 targets including transmembrane and
soluble receptors, ion channels, and monoamine transporters.17
Moreover, as indicated in Table 4, 29 displayed good
physicochemical properties with a moderate lipophilicity, a
good aqueous solubility and membrane permeability. The
metabolic stability of 29 in mouse and human liver microsomes
was found to be intermediate with a measured intrinsic
clearance of 54 and 19 μL/min/mg protein, respectively. Not
surprisingly, in vitro metabolism studies on 29 revealed that the
corresponding N-des-methyl piperidine derivative was a major
D
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ACS Medicinal Chemistry Letters
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safety profile and in vivo efficacy in rodents after oral
administration.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the synthesis of compounds 8−43 and
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(E.P.) E-mail: emmanuel.pinard@roche.com.
■
Figure 3. Dose-dependent inhibition of L-687,414-induced hyper-
locomotion by 29 in mice. The data represent mean horizontal activity
counts per group recorded over a 60 min time period; error bars
indicate SEM (n = 8 per group). ##, p < 0.01 versus vehicle (Veh)
alone; ***, p < 0.001 versus L-687,414 alone.
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
be explained by its high GlyT1 potency and its excellent brain
penetration in the mouse.
In addition, the effect of 29 on the extracellular level of
glycine in rat striatum in a microdialysis study was evaluated
(Table 6). We were pleased to observe that, at an oral dose of
10 mg/kg, 29 produced a 1.7-fold glycine increase over basal
levels.
ACKNOWLEDGMENTS
We would like to thank Andre
Schnider, and Daniel Zimmerli for their dedicated technical
assistance.
■
́
Alker, Theo Stoll, Christian
ABBREVIATIONS
■
CYP, cytochrome P; dba, dibenzylideneacetone; DIPEA, N,N-
diisopropylethylamine; EWG, electron withdrawing group;
EDG, electron donating group; HATU, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri-di-
nium 3-oxid hexafluorophosphate; hERG, human ether-a-go-
̀
go-related gene; MDR, multidrug resistance protein; TFA,
Table 6. Effect of 29 on the Extracellular Glycine Levels in
a
Rat Striatum at 10 mg/kg po and Its Plasma and Brain
Exposures
b
b
b
max. fold incr.
of glycine
fold incr. of
glycine at 3 h
plasma
brain
brain
(ng/mL)
(ng/mL) GlyT1 EC50
trifluoro acetic acid
1.7
1.5
713
60 13
a
b
n = 6. Measured at 3 h postdosing.
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(16) The absolute configuration was determined by X-ray
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(17) The panel consisted in targets included in the standard CEREP
high throughput profile screen. The targets that were inhibited by
more than 50% at the concentration of 10 μM: H2R, MC4R, NK1,
NK2, BZDR (peripheral), Na Ch (site 2), and SSTR were followed up
in dose−response experiments. In these studies, a selectivity of at least
110-fold vs GlyT1 was measured.
(18) Solubility was measured in a lyophilization solubility assay:
Compound initially in DMSO solution is lyophilized then dissolved in
0.05 M phosphate buffer (pH 6.5), stirred for one hour, and shaken for
two hours. After one night, the solution is filtered and the filtrate
analyzed by direct UV measurement or by HPLC-UV.
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(21) Similar PD effects were observed at similar brain/GlyT1 EC50
multiples with our previously published structurally distinct series of
GlyT1 inhibitors: Pinard, E.; Alberati, D.; Bender, M.; Borroni, E.;
Brom, V.; Burner, S.; Fischer, H.; Hainzl, D.; Halm, R.; Hauser, N.
Discovery of benzoylisoindolines as a novel class of potent, selective
and orally active GlyT1 inhibitors. Bioorg. Med. Chem. Lett. 2010, 20,
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dx.doi.org/10.1021/ml500005m | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX