Novel GlyT1 inhibitor chemotypes by scaffold hopping. Part 1: Development of a potent and CNS penetrant [3.1.0]-based lead

Article abstract of DOI:10.1016/j.bmcl.2014.01.013

This Letter describes the development and SAR of a novel series of GlyT1 inhibitors derived from a scaffold hopping approach that provided a robust intellectual property position, in lieu of a traditional, expensive HTS campaign. Members within this new [3.1.0]-based series displayed excellent GlyT1 potency, selectivity, free fraction, CNS penetration and efficacy in a preclinical model of schizophrenia (prepulse inhibition).

Full text of DOI:10.1016/j.bmcl.2014.01.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1067–1070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel GlyT1 inhibitor chemotypes by scaffold hopping.
Part 1: Development of a potent and CNS penetrant
[3.1.0]-based lead
Carrie K. Jones a,b, Douglas J. Sheffler a,b, , Richard Williams b, Sataya B. Jadhav b, Andrew S. Felts a,b,c
,
Ryan D. Morrison a,b,c, Colleen M. Niswender a,b,c, J. Scott Daniels a,b,c, P. Jeffrey Conn a,b,c
,
Craig W. Lindsley a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the development and SAR of a novel series of GlyT1 inhibitors derived from a scaf-
fold hopping approach that provided a robust intellectual property position, in lieu of a traditional,
expensive HTS campaign. Members within this new [3.1.0]-based series displayed excellent GlyT1
potency, selectivity, free fraction, CNS penetration and efficacy in a preclinical model of schizophrenia
(prepulse inhibition).
Received 21 November 2013
Revised 2 January 2014
Accepted 6 January 2014
Available online 13 January 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
GlyT1
Scaffold hopping
Transporter
Schizophrenia
Significant efforts are currently focused on non-dopaminergic
strategies to address the unmet medical needs in schizophrenia,
and targeting N-methyl-D-aspartate (NMDA) receptor hypofunc-
inhibitors,12 to accelerate a fast follower GlyT1 inhibitor program.
For this exercise, we were attracted to the GlyT1 inhibitors re-
ported from both Merck, represented by 1 and 2,13–15 and Pfizer’s
316 (Fig. 1), as they possessed potent GlyT1 inhibition with good
DMPK profiles and efficacy in preclinical models;13–16 moreover,
homology and overlap was noted between these otherwise dispa-
rate chemotypes.
Our initial approach was to replace the central piperidine core
of 1 and 213–15 with the [3.1.0] bicyclic ring system found in 3,16
to arrive at analogs such as 4 (Fig. 2). Synthetically, analogs 4 were
arrived at via a seven step route in low (ꢀ4%) overall yield. Starting
tion has garnered a great deal of attention.1–3 Elevation of synaptic
glycine levels near NMDA-containing synapses, by inhibition of the
glycine transporter type 1 (GlyT1), has proven to be a viable mech-
anism for achieving efficacy in multiple preclinical models of
schizophrenia with a diverse array of GlyT1 chemotypes.4–9 More
recently, Roche reported Phase II clinical efficacy with their GlyT1
inhibitor (RG6178) in improving the negative symptoms in pa-
tients with schizophrenia,10 a symptom cluster largely unmet with
available antipsychotic agents, that re-ignited the field. Further-
more, new data suggests roles for GlyT1 inhibition in alcohol
dependence, addiction and pain.11
N
N
O
Cl
O
Cl
Cl
F
O
N
H
N
H
N
Based on these data, the late-stage clinical status of GlyT1, and
the crowded intellectual property space,4–9 we elected to attempt
to develop novel chemical space by scaffold hopping, a strategy
we previously employed successfully for T-Type calcium channel
Cl
Cl
N
S
N
S
H
H
O
O
O
O
N
1, ACPPBI
GlyT1 IC50 = 2.4 nM
2, ACPPBII
GlyT1 IC50 = 26 nM
3, PF-0346275
GlyT1 IC50 = 11.6 nM
⇑
Corresponding author.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
Present address: Apoptosis and Cell Death Research Program and Conrad Prebys
Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901
N. Torrey Pines Rd., La Jolla, CA 92037, USA.
Figure 1. Structures and activities of GlyT1 inhibitors disclosed by Merck (1 and 2)
and Pfizer (3) utilized in subsequent scaffold-hopping to access new chemical
space.
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.013

1068
C. K. Jones et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1067–1070
N
Table 1
O
Cl
N
O
Cl
Cl
F
O
Structures and activities of [3.1.0] analogs 4
N
H
N
H
N
O
Cl
Cl
Cl
N
S
N
S
R
H
H
N
H
O
O
O
O
N
H
H
Cl
2
3
4
N
S
R
O
O
Figure 2. Initial approach to hybridize 2 and 3 to afford novel GlyT1 [3.1.0] scaffold 4.
4
a
a
Compound
R
GlyT1 IC50
(l
M)
GlyT2 IC50 (lM)
O
OEt
H
O
NH2
H
CN
4a
4b
1.3
>30
>30
CN
a,b
c
d
H
H
H
H
0.36
H
H
N
N
N
N
4c
3.4
>30
Boc
Boc
Boc
Boc
4d
4e
4f
0.23
5.5
>30
>30
>30
5
6
7
8
O
Cl
CN
2.9
N
H
e,f
g,h
H
H
H
H
Cl
N
4g
4h
4i
1.0
>30
>30
>30
N
S
R
N
O
O
O
S
R
O
N
0.89
>10
9
4
Scheme 1. Reagents and conditions: (a) LiOH, THF, MeOH, rt; (b) NH4Cl, EDC, HOBt,
DIEPA, DMF, 0 °C; (c) cyanuric chloride, DMF, 0 °C; (d) KHMDS, toluene, 0 °C,
cyclopropyl methylbromide; (e) TFA, CH2Cl2, 0 °C; (f) RSO2Cl, CH2Cl2, DIEPA, 0 °C;
(g) Raney Ni, H2 (45 psi), NH4OH, MeOH; (h) 2,4-dichlorobenzoyl chloride, CH2Cl2,
DIEPA, 0 °C.
4j
>10
>30
F
a
IC50s represent single determinations performed in duplicate.
from commercially available (1R,5S,6r)-3-tert-butyl 6-ethyl 3-aza-
bicyclo[3.1.0]hexane-3,6-dicarboxylate 5, two step conversion to
the primary carboxamide 6 proceeded smoothly, followed by treat-
ment with cyanuric chloride to afford the nitrile 7 (Scheme 1).
Deprotonation with KHMDS and alkylation with cyclopropyl
methylbromide in toluene at 0 °C provided 8 in 20% yield, as a sin-
gle diastereomer (stereochemical assignment based on literature
precedent17 and NOE studies). TFA-mediated removal of the Boc
group, followed by sulfonylation with various sulfonyl chlorides,
generated congeners 9. Finally, ‘Raney’ Ni reduction of the nitrile
and subsequent acylation with 2,4-dichlorobenzoyl chloride deliv-
ered analogs 4.
and with simple models could achieve an orientation in which the
N-methyl imidazole moiety could fill the same space as the alkyl
sulfonamides in 4b and 4d. Thus, we hypothesized that N-methyl-
imidazole sulfonamide congeners might enhance GlyT1 potency in
analogs 4.
To test this concept, we took advantage of our large supply of
various 2-pyridyl containing [3.1.0] cores (inactive with alkyl or
aryl sulfonamides) and prepared the N-methylimidazole
sulfonamide analogs 10 and 11 (Fig. 3), as work form Merck
demonstrated that the 2-pyridyl moiety was superior to the origi-
nal 4-phenyl moiety in 1.14,15 These analogs all displayed
sub-micromolar potency at GlyT1 (IC50s of 247 nM for 10 and
185 nM for 11), clogPs of 2.3, large fraction unbound in plasma
The initial 10-membered library of analogs
4 displayed
somewhat unexpected SAR, with a significant diminution in
potency relative to the piperdine-based GlyT1 inhibitors 1 and 2
(Table 1).13–15 For example, 4a (GlyT1 IC50 = 1.3
l
M), the direct
analog of 2 (GlyT1 IC50 = 26 nM), lost 50-fold in potency, and 4b
(GlyT1 IC50 = 360 nM), the n-propyl congener of (GlyT1
IC50 = 2.4 nM), lost ꢀ150-fold. A trend towards increased activity
resulted with cyclopropyl methyl sulfonamide 4d (GlyT1
(fu 7–14%), clean CYP profiles (IC50 >30 lM) and in oral brain tissue
distribution studies, Kp ([brain]/[plasma]) ratios of 1.1. Interest-
ingly, replacement of the N-methylimidazole with either imidazole
or an N-methyl pyrazole led to a complete loss of GlyT1 activity. As
incorporation of the N-methyl imidazole sulfonamide increased
1
a
IC50 = 230 nM), but all other modifications, including aryl (4i and
4j) and heteroaryl sulfonamides (4g and 4h) led to significant loss
in GlyT1 potency. However, all analogs 4 retained high selectivity
O
CF3
O
Cl
versus GlyT2 (IC50 >30
ing indicated that this core retained the favorable disposition prop-
erties of 1–3 (fu 2–4%, IC50 >10 M vs CYP3A4, 2D6, 1A2 and 2C9).
Substitution of the 6-cyclopropyl methyl group in 4 with either
phenyl or 2-pyridyl moieties, also led to inactive analogs (GlyT1
lM) and preliminary in vitro DMPK profil-
N
N
H
N
N
H
H
H
H
H
Cl
N
S
N
S
l
O
O
O
O
N
N
N
N
IC50 >30 lM) and represented a divergence from the SAR of the
piperidine series 1 and 2.13–15 Analogs of 4 where the optimal
sulfonamides were maintained (4b and 4d), but the substitution
on the benzamide moiety was varied, once again led to a large
diminution in GlyT1 potency (data not shown).
10
11
GlyT1 IC50 = 247 + 29 nM
CYPs IC50 >30 µM
fu (human) = 14%
Kp = 1.1
GlyT1 IC50 = 185 + 33 nM
CYPs IC50 >30 µM
fu (human) = 7.6%
Kp = 1.1
As we began to assess and consider the data generated thus far,
Figure 3. Impact of incorporation of an N-methyl imidazole sulfonamide into the
[3.1.0] scaffold to afford 10 and 11.
we were attracted to the N-methyl imidazole moiety of Pfizer’s 3,16

C. K. Jones et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1067–1070
1069
potency in the 2-pyridyl [3.1.0] core from IC50s >30 lM to IC50s
<250 nM, we were excited to see the impact of this modification
to the already very potent cyclopropyl methyl [3.1.0] core of 4.
With a slight modification of the chemistry in Scheme 1, we
were able to readily prepare a library of analogs 12, where the
benzamide moiety was varied in the context of the cyclopropyl
methyl [3.1.0] core containing an N-methyl imidazole sulfonamide.
As shown in Table 2, this endeavor was very productive, affording
potent (IC50s from 4 to 119 nM) and selective GlyT1 inhibitors with
excellent DMPK profiles and CNS penetration in rat with oral dos-
ing (Kp of 0.3–0.8). While many analogs displayed excellent po-
tency at GlyT1, such as 12a, 12d and 12f, analog 12d stood out
as having the best balance of potency (GlyT1 IC50 = 5 nM), fraction
unbound in plasma (fu 7%), and in an oral brain tissue distribution
study, a Kp ([brain]/[plasma]) ratio of 0.8.
After assessing ancillary pharmacology in a Eurofin Lead Profil-
ing panel of radioligand binding assays against 68 GPCRs, ion chan-
nels and transporters, our focus narrowed on 12d, as it displayed
no inhibition >50%@10
was a potent and selective GlyT1 inhibitor (GlyT1 IC50 = 5 0.5 nM
(N = 3), GlyT2 IC50 >30 M) (Fig. 4). Eadie–Hoffstee plots, where the
lM against any target in the panel, yet
Figure 4. Concentration response curves (N = 3) for glycine and 12d in the [14C]-
glycine uptake assay.
l
reaction rate is plotted versus the ratio of the reaction rate and
substrate concentration, provide useful insight into the mechanism
of enzymatic inhibition, with competitive and noncompetitive
enzymatic inhibition demonstrating distinct patterns. An Eadie–
Hoffstee plot (Fig. 5) of the effect of this series, represented by
12c, on the enzyme kinetics of [14C]-glycine transport showed that
this series is competitive with respect to glycine, in accordance
with the known mechanism of action for 1 and 213–15, and distinct
from the non-competitive mechanism of action of the sarcosine-
derived GlyT1 inhibitors, such as NFPS.5–8 In addition, 12d pos-
a short half-life (t1/2 = 14.5 min). This is in-line with data reported
for Merck’s 2, which was moderate to high clearance in rat, but low
clearance in dog. We evaluated 12d in two separate rat brain tissue
distribution studies: one with subcutaneous (10 mg/kg sc in 10%
tween 80) dosing and one with oral (10 mg/kg po in 0.5% metho-
cellulose) dosing. Both dosing routes exhibited good exposure (sc
plasma AUC0–6h
: 976 nM h; sc brain, AUC0–6h: 431 nM h (or
Kp = 0.44); po plasma AUC0–6h: 1156 nM h, po brain, AUC0–6h
:
sessed a clean CYP profile (IC50 >30
lM against 1A2, 2C9 and
956 nM h (or Kp = 0.83)) with unbound concentrations above the
GlyT1 IC50 at 6 h. Due to the higher exposure, for an in vivo proof
of concept study in a preclinical model of schizophrenia, we pro-
ceeded with oral dosing.
2D6; 14.9 M against 3A4) and good unbound fraction in both hu-
l
man (fu = 6.8%) and rat (fu = 44.8%). We assessed stability in rat
plasma for 4 h at 37 °C, and 12d was stable, indicating the free frac-
tion in rat plasma is truly high. In vitro intrinsic clearance experi-
ments suggest 12d possesses moderate to high predicted clearance
for both human (ClHEP = 17.7 mL/min/kg) and rat (ClHEP = 43.0 mL/
min/kg). A rat IV PK study was conducted with 12d and it displayed
moderate in vivo clearance (0.5 mg/kg) (Clp = 31 mL/min/kg) with
Based on the precedent with other GlyT1 inhibitors such as 1,13
we evaluated both 215 and 12d for their ability to enhance prepulse
inhibition (PPI) of the rodent acoustic startle response, a measure
of sensorimotor gating known to be deficient in schizophrenic
patients.18,19 In this study (Fig. 6), both 2 and 12d were dosed
Table 2
Structures and activities of [3.1.0] analogs 12
O
N
H
Ar
H
O
H
N
S
O
N
N
12
a
b
Compound
Ar
GlyT1 IC50 (nM)
clogP
fu (hum) (%)
Kp (rat PO)
12a
12b
12c
12d
12e
12f
12g
12h
12i
2,4-diCIPh
2,4-diCIPh
2,6-diCIPh
2-CF3Ph
3-CF3Ph
2-CIPh
3,4-diCIPh
2-CIPh
3,5-diCIPh
4-CIPh
4
44
28
5
64
19
82
119
100
32
2.3
1.9
1.9
2.5
2.5
2.6
3.2
2.7
3.3
2.7
5
8
8
7
4
4
2
3
2
3
0.3
0.5
0.5
0.8
0.7
0.4
0.3
0.4
0.3
0.3
12j
a
IC50s represent single determinations performed in duplicate. All analogs were inactive on GlyT2 (IC50 >30 lM).
Kp = Partitioning coefficient = [brain]/[plasma].
b

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C. K. Jones et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1067–1070
Figure 6. The effect of vehicle, 2 and 12d on PPI in rat at 30 mg/kg oral (po) dosing
in 0.5% methylcellulose. ⁄p < 0.05 in comparison to vehicle by Dunnett’s test (n = 6–
7).
on Schizophrenia and Depression (NARSAD)–Dylan Tauber Young
Investigator Award. Vanderbilt is a member of the MLPCN and
houses the Vanderbilt Specialized Chemistry Center for Acceler-
ated Probe Development supported by U54 MH084659. The
support of William K. Warren, Jr. who funded the William K. War-
ren, Jr. Chair in Medicine (to C.W.L.) is gratefully acknowledged.
References and notes
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Figure 5. (A) Saturation [14C]-glycine transport in the presence of vehicle (red
squares) or 40 nM 12c (blue triangles). (B) An Eadie–Hoffstee diagram for 12c and
[
14C]-glycine.
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orally at 30 mg/kg (a dose known to engender >90% occupancy for
2),15,20,21 and evaluated against four increasing prepulse intensities
(70–88 dB). Both 2 and 12d showed a statistically significant
enhancement in prepulse inhibition at the 82 and 88 dB prepulse
intensities, with no effect on basal startle amplitude during no-
stimulus trials. Thus, 12d (VU0240391), derived from a scaffold-
hopping exercise employing 2 and 3, led to a novel [3.1.0]-based
GlyT1 inhibitor with in vitro and in vivo properties comparable
to other advanced GlyT1 inhibitors in short order, and for which
a U.S. patent was issued.22
In conclusion, we were able to scaffold hop and merge elements
from both the Merck piperidine-based series of GlyT1 inhibitors,
represented by 1 and 2,13–15 and Pfizer’s 3,16 into a novel, patented
series of [3.1.0]-based N-methylimidazole sulfonamides 12. Mem-
bers of this series displayed exceptional GlyT1 potency, DMPK pro-
files, CNS penetration and comparable in vivo efficacy to advanced
GlyT1 inhibitors without the need for an HTS to enable a fast-fol-
lower program. Additional scaffolds developed during the course
of this scaffold-hopping program will be reported in due course.
17. Klapars, A.; Waldman, J. H.; Campos, K. R.; Jensen, M. S.; McLaughlin, M.;
Chung, J. Y. L.; Cvetovich, R. J.; Chen, C.-Y. J. Org. Chem. 2005, 70, 10186–10189.
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Wallace, M. A.; Raab, C.; Lindsley, C. W.; Sur, C.; Williams, D. L., Jr. Nucl. Med.
Biol. 2008, 35, 315.
Acknowledgments
21. Hamill, T. G.; Eng, W.; Jennings, A. S. R.; Lewis, R. T.; Thomas, S.; Wood, S.;
Street, L. J.; Wisnoski, D. D.; Wolkenberg, S. W.; Lindsley, C. W.; Sanabria-
Bohorquez, S. M.; Patel, S.; Ryan, C.; Riffel, K.; Cook, J.; Sur, C.; Burns, H. D.;
Hargreaves, R. Synapse 2011, 65, 261.
22. Lindsley, C. W.; Conn, P. J.; Williams, R.; Jones, C. K.; Sheffler, D. J. US 8,497,289
2013.
This work was supported by the NIH/NIMH under a National
Cooperative Drug Discovery and Development Grant U01
MH08795. D.J.S. is a recipient of a National Alliance for Research