Design, synthesis, and SAR of N-((1-(4-(propylsulfonyl)piperazin-1-yl) cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1

Article abstract of DOI:10.1016/j.bmcl.2013.01.006

The design, synthesis, and structure-activity relationships (SAR) of a series of N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1) are described. Optimization of the benzamide and central ring

Full text of DOI:10.1016/j.bmcl.2013.01.006

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1257–1261
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and SAR of N-((1-(4-(propylsulfonyl)piperazin-1-
yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1
a
a
c
Christopher L. Cioffi a, , Mark A. Wolf , Peter R. Guzzo , Kashinath Sadalapure ,
⇑
Visweswaran Parthasarathy c, Dattatraya Dethe c, Jun-Ho Maeng c, Edmund Carulli c, David T. J. Loong c,
Xiao Fang c, Min Hu a, Priya Gupta c, Mark Chung c, Mei Bai c, Nick Moore a, Michele Luche b,
Yuri Khmelnitsky a, Patrick L. Love d, Megan A. Watson d, Andrew J. Mhyre b, Shuang Liu a
a AMRI, 26 Corporate Circle, PO Box 15098, Albany, NY 12212-5098, USA
b AMRI, Bothell Research Center, Inc., 22215 26th Ave., SE, Bothell, WA 98021-4425, USA
c AMRI, Singapore Research Center, 61 Science Park Road, Science Park III, Singapore 117525, Singapore
d Covance, Inc., 671 South Meridian Road, Greenfield, IN 46140, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and structure–activity relationships (SAR) of a series of N-((1-(4-(propylsulfo-
nyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1) are
described. Optimization of the benzamide and central ring components of the core scaffold led to the
identification of a GlyT-1 inhibitor that demonstrated in vivo activity in a rodent cerebral spinal fluid
(CSF) glycine model.
Received 30 October 2012
Revised 19 December 2012
Accepted 2 January 2013
Available online 11 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
GlyT-1
Schizophrenia
NMDA receptor
Transporter
Piperazine
Glycine
Schizophrenia is a chronic and devastating mental illness that
affects approximately 1% of the world’s population.1 The dopamine
(DA) D2 receptor antagonist activity for all currently approved typ-
ical and atypical antipsychotics led to the ‘dopamine hypothesis’ of
schizophrenia, which was the prevailing etiological hypothesis for
nearly 60 years.2 However, the lack of efficacy current antipsychot-
ics exhibit against negative and cognitive symptoms has called into
question whether the molecular basis for schizophrenia is due
exclusively to excessive dopaminergic neurotransmission and dys-
functional D2 signaling. Additionally, these agents generally exhibit
poor side effect profiles, resulting in patient compliance issues.
Therefore, safe and effective treatment for the entire spectrum of
symptoms remains a critical unmet need.3
critical role in the pathophysiology of schizophrenia.5–8 Therefore,
agents that enhance NMDA receptor signaling may potentially pro-
vide a means to effectively ameliorate the full symptomology asso-
ciated with the disease.
The NMDA receptor is a ligand and voltage gated ionotropic
receptor that requires the binding of both glutamate and the oblig-
atory co-agonist glycine to induce signaling.8 CNS glycine concen-
trations are regulated by two high affinity sodium chloride
dependent transporters: glycine transporter type-1 (GlyT-1) and
glycine transporter type-2 (GlyT-2).9–11
GlyT-1 is widely expressed throughout the CNS and is co-local-
ized with glutamatergic neurons within the forebrain, where it
tightly maintains synaptic glycine concentrations surrounding
the NMDA receptor at sub-saturation levels.9–11 GlyT-2 is largely
expressed on glycinergic neurons in the brain stem and spinal cord,
where glycine acts as an inhibitory neurotransmitter at strychnine-
sensitive glycine receptors.9,10 Thus, selective inhibition of GlyT-1
presents an attractive strategy to increase synaptic glycine levels
surrounding the NMDA receptor, thereby enhancing receptor sig-
naling. In fact, GlyT-1 inhibition has been validated in a variety
of pre-clinical models predictive of anti-psychotic and pro-cogni-
tive activity.7,12 Positive results have also been observed in clinical
The observation that the non-competitive N-methyl-D-aspartate
(NMDA) glutamate receptor antagonists ketamine and phencycli-
dine (PCP) induce positive, negative, and cognitive symptoms in
healthy individuals and in stable schizophrenic patients has led
to the emergence of the ‘glutamate hypothesis’.4 These observa-
tions suggest that NMDA receptor hypofunction may be playing a
⇑
Corresponding author.
E-mail address: christopher.cioffi@amriglobal.com (C.L. Cioffi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.006

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C. L. Cioffi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1257–1261
studies with sarcosine13–15 and in a recent proof-of-concept Phase
II study with the Roche inhibitor RG1678 (3, Fig. 1).16,17
Cl
H
N
A number of structurally diverse GlyT-1 inhibitors have been re-
ported and several comprehensive reviews have appeared in the
literature.18 These inhibitors are categorized as either sarcosine-
derived or non-sarcosine-derived and representative compounds
from each class are shown in Figure 1. Drawing from the ample
patent and journal literature accrued to date, we initiated a GlyT-
1 inhibitor program with the goal of rationally designing a novel
series of non-sarcosine-derived inhibitors that are highly potent,
demonstrate good selectivity against GlyT-2, and exhibit favorable
CNS drug-like properties.
We report herein the design of hit N-((1-(4-(propylsulfonyl)pip-
erazin-1-yl)cycloalkyl)methyl)benzamide 719 (Fig. 2) and the
structure–activity relationships (SAR) uncovered that led to im-
proved analogue 40, which was studied for in vivo activity in the
rodent cerebral spinal fluid (CSF) glycine biomarker model.
The design of 7 incorporated structural elements common to
several reported GlyT-1 inhibitors.18,20–22 Specifically, we sought
to tether a benzamide and propyl sulfonamide moiety within a rel-
ative proximity that shared a pharmacophoric similarity with
other reported inhibitors. The preparation of hit 7 and subsequent
analogues are shown in Schemes 1 and 2. In Scheme 1, following
sulfonylation and subsequent Boc-deprotection of commercially
available N-Boc-piperazine, a Strecker reaction with cyclohexa-
none was employed to furnish quaternary nitrile 11. Reduction of
11 yielded key aminomethyl intermediate 12, which allowed for
rapid SAR exploration of the benzamide portion of the scaffold
via iterative analogue synthesis using standard peptide coupling
procedures with various benzoic acids.19
N
O
N
O
NH2
S
O
7
Figure 2. Non-sarcosine-derived GlyT-1 inhibitor 7.
O
S
O
O
S
a
b
HN
NBoc
N
NBoc
N
NH
O
8
10
9
NH2
c
d
N
N
CN
O
O
N
N
S
S
O
O
11
12
Scheme 1. Reagents and conditions: (a) 1-propanesulfonyl chloride, Et3N, CH2Cl2,
0 °C to room temperature, 90%; (b) TFA, CH2Cl2, 0 °C to room temperature, 93%; (c)
cyclohexanone, Na2S2O5, KCN, CH3CN, H2O, room temperature, 67%; (d) LiAlH4, THF,
room temperature, 60%.
b
a
N
CN
HN
NBoc
BocN
8
13
Sarcosine-derived GlyT-1 Inhibitors
c
N
CN
N
CN
O
N
O
HN
S
14
O
11
N
CO2H
N
CO2H
Scheme 2. Reagents and conditions: (a) cyclohexanone, TMSCN, ZnI2, Et2O, CH3OH,
0 °C to reflux, 80%; (b) TFA, CH2Cl2, 0 °C to room temperature, 95%; (c) 1-
propanesulfonyl chloride, Et3N, CH2Cl2, 0 °C to room temperature, 87%.
H3CO
F
2
1
ORG-25935
NFPS
Nitrile intermediate 11 could also be furnished from a Strecker
reaction between N-Boc-piperazine and cyclohexanone, followed
by Boc-deprotection and subsequent sulfonylation (Scheme 2).19
GlyT-1 inhibition was assessed using a functional whole cell
scintillation proximity assay (SPA) that measured [14C]glycine up-
take by JAR cells that endogenously express hGlyT-1a.18 GlyT-2
inhibition was similarly measured using a transfected HEK293 cell
line stably expressing hGlyT-2.18 Compound 7 was identified as a
potent and selective inhibitor of GlyT-1 (GlyT-1 IC50 = 15 nM,
Non-sarcosine-derived GlyT-1 Inhibitors
Cl
O
CF3
O
O
S
N
H
Cl
N
N
N
F
GlyT-2 IC50 >75 lM) and the initial SAR strategy was to study the
O
S O
O
O
impact of substitution on the benzamide phenyl ring. Over 50 ana-
logues were synthesized and a wide range of substituents were tol-
erated (Table 1). In most cases, at least two substituents were
required for appreciable potency with optimal potency requiring
at least one substituent to be positioned ortho to the benzamide
carbonyl. Exquisite potency could be achieved with 2,4,6-trisubsti-
tution of the phenyl ring, whereas mono-substitution at the 3-po-
sition was disfavored. The analogues also exhibited excellent
selectivity for GlyT-1, as they were silent at GlyT-2 (IC50 values
>75 lM). However, the series suffered from poor human liver
microsomal (HLM) stability and further refinement of the scaffold
was required to address this issue.
We next evaluated changes to the central ring of the scaffold.
The size of the ring imparted a significant influence on potency
F3C
3
4
RG1678
DCCCyB
O
Cl
O
CF3
N
H
N
H
NH
N
5
6
SSR-504734
GSK931145
Figure 1. Reported sarcosine-derived and non-sarcosine-derived GlyT-1 inhibitors.

C. L. Cioffi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1257–1261
1259
Table 1
Benzamide SAR for representative cyclohexyl analogues
Cl
H
H
N
N
N
O
N
O
H
O
N
O
Cl
NH2
N
O
O
Cl
R
S
S
N
N
O
33
GlyT-1 IC50 = 661 nM
( )-34
N
O
GlyT-1 IC50 = 6.4 mM
S
O
H
H
H
H
N
N
N
O
O
N
O
O
a
Compound
R
GlyT-1 IC50 (nM) % Remainingb HLMc
N
O
F
F
N
O
F
F
S
S
7
2-NH2, 6-Cl
H
2-CF3
2-OCF3
4-Cl
4-OCF3
3-CF3
2-Cl, 3-CF3
2-F, 4-OCH3
2,4-diCl
2-CH3, 4-F
3,4-diCl
2,6-diF
2,6-diCl
15.1
782
48.5
22.0
175
118
523
29
NDd
5
2
4
11
NDd
1
4
11
2
5
16
3
10
1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
(1:1 mixture of cis:trans)-35
36
GlyT-1 IC50 = 621 nM
GlyT-1 IC50 = 162 nM
Figure 4. Central ring modifications.
that blockade of such sites might improve HLM stability. Indeed,
installation of a gem-difluoro cyclohexyl central ring provided
stand-out analogue 40 that possessed the best balance of potency,
selectivity, and favorable HLM stability for further evaluation
(Fig. 5).
In vitro pharmacological and ADME evaluation of 40 revealed a
desirable profile that included no CYP inhibition liabilities or sig-
nificant off-target activity at the hERG channel or within a standard
screening panel of seventy GPCRs, ion channels, enzymes, and
transporters (Table 2). Compound 40 also displayed favorable
pharmacokinetics (PK) in rat (Table 3). Encouraged by the
in vitro and PK profiles, we decided to evaluate the in vivo activity
of 40 in the CSF glycine model, in which CSF glycine levels are mea-
sured in response to oral administration of test compound.19,23
Drug concentration is also measured in the CSF, which is be-
lieved to be an approximate representation of unbound drug con-
centration in the brain.24
Drug naïve rats (n = 5) were administered vehicle or a 10, 30, or
75 mg/kg dose of compound 40 orally and sacrificed after 30 min,
at which point CSF was extracted and analyzed for glycine and
40 levels. It was observed that compound 40 induced a dose
dependent increase in CSF glycine relative to vehicle (set at
100%) and glycine elevation reached statistical significance for
the 30 and 75 mg/kg doses (Fig. 6). Additionally, a dose dependent
increase of 40 in the CSF (Fig. 7) and brain (mean brain/plasma ra-
tio for all doses was ꢀ0.13) was also observed. These positive re-
77.9
17.1
22.7
28.1
245
23.2
12.8
4.4
2,4-diCF3, 6-OCH3
2-OCH3, 4-F, 6-SCH3
6.1
a
b
c
Values are means of at least two determinations.
Compound concentration was 10
HLM = human liver microsomes.
ND = no data.
lM and incubation time was 60 min.
d
as contraction from cyclohexyl to cyclopentyl or cyclobutyl de-
creased potency significantly (Fig. 3). Conversely, expansion to a
cycloheptyl ring maintained or slightly increased potency for
GlyT-1 (Fig. 3). The benzamide SAR trends for the cyclopentyl
and cycloheptyl scaffolds were scouted and were found to be sim-
ilar to the cyclohexyl series (data not shown). The cycloheptyl ser-
ies provided analogues that were equipotent or slightly more
potent (threefold) than analogues of the cyclohexyl series, whereas
the cyclopentyl series was globally 10-fold less potent than the
cyclohexyl series. Regardless of potency trends, poor HLM stability
remained a common issue for both the cyclopentyl and cycloheptyl
series.
Additional exploration of the central ring was conducted utiliz-
ing the aforementioned synthetic routes (Fig. 4). However, many of
the modifications were not well tolerated by the transporter, sug-
gesting a narrow SAR for this region of the scaffold.
Further optimization efforts led to incorporation of heteroatoms
into the central ring, which did improve microsomal stability, yet
potency was significantly diminished for many of these analogues
(Fig. 5). These findings suggested that the central ring provided a
potential metabolic soft-spot(s) for CYP-mediated oxidation and
O
N
Cl
Cl
H
N
H
N
N
N
O
O
N
O
Cl
N
O
NH2
S
S
O
O
37
38
GlyT-1 IC50 = 500 nM
% remaining (HLM) = 100
GlyT-1 IC50 = 78.4 nM
% remaining (HLM) = 40
n
N
F
F
O
N
HN
O
O
S
F
F
H
N
H
N
O
Cl
NH2
N
N
O
O
N
O
F
N
O
F
S
S
30: n = 1; GlyT-1 IC50 = 2445 nM
31: n = 2; GlyT-1 IC50 = 183 nM
O
O
40
( )-39
GlyT-1 IC50 = 238 nM
7
32
: n = 3; GlyT-1 IC50 = 15.1 nM
GlyT-1 IC50 = 67.5 nM
% remaining (HLM) = 64
: n = 4; GlyT-1 IC50 = 6.3 nM
Figure 3. Impact of central ring size on GlyT-1 potency.
Figure 5. Additional central ring modifications.

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C. L. Cioffi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1257–1261
Table 2
In vitro profile of 40
a
b
CYP inhibition IC50 (3A4, 2D6, 2C19, 2C9)
hERG IC50
>100
Selectivity panel IC50 (1
All <50% inhibition
lM)
CLint
(l
L/min/mg)
% PPBc
Mini-amesd
Negative
All >10
l
M
l
M
Human = 13, rat = 5.5
Human = 87, rat = 84
a
b
c
‘Patch-Xpress’ patch-clamp assay; compound was tested (n = 3) in a 5-point concentration–response on HEK293 cells stably expressing the hERG channel.
Compound concentration at 1
Plasma protein binding.
lM.
d
TA98 and TA100 strains tested in the presence and absence of rat S9 fraction.
Table 3
PK properties of 40 in rat (2 mg/kg IV and 10 mg/kg POa)
sults indicated both CNS penetration of inhibitor 40 and in vivo
engagement with the target, which justified further optimization
and development of our series.
CLb (mL/h/kg)
Vss (mL/kg)
1321
3258
486
1995
2.9
In summary, we have rationally designed and developed a ser-
ies of potent and selective non-sarcosine-derived N-((1-(4-(propyl-
sulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of
GlyT-1. Poor HLM stability was identified as a key deficiency
requiring improvement for the series. Installation of a gem-difluoro
cyclohexyl central ring provided a competent strategy for greatly
improving this issue while maintaining an excellent in vitro profile
and favorable CNS drug-like characteristics. This modification pro-
vided compound 40, which demonstrated in vivo activity in the rat
CSF glycine biomarker model and provided proof-of-concept that
our series could generate a GlyT-1 inhibitor capable of in vivo
engagement with the target. Additional refinements to the series
that have led to advanced analogues demonstrating exquisite
in vitro potency and in vivo activity will be reported in due course.
Cmax (ng/mL)
AUC0 ? 1 (h ng/mL)c
Terminal T1/2 (h)
Tmax (h)
1.5
25.7
%Fd
a
IV and PO formulation: 10% EtOH, 40% PEG400, 50% H2O.
Total plasma clearance.
Area under the curve extrapolated to infinity.
Oral bioavailability.
b
c
d
250
225
*
200
*
Acknowledgments
175
150
125
100
75
The authors would like to thank Drs. Robert E. Davis, Jeffery S.
Sprouse, and Bryan L. Roth for helpful discussions.
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