Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties

Article abstract of DOI:10.1021/acs.jmedchem.8b00372

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4-c]pyrazoles amides

Full text of DOI:10.1021/acs.jmedchem.8b00372

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Article
Design and Synthesis of Novel and Selective Glycine Transporter-1
(GlyT1) Inhibitors with Memory Enhancing Properties
Vincent J. Santora, Theresa A. Almos, Richard Barido, Jillian Basinger, Chris L. Bellows, Brett
Carder Bookser, J. Guy Breitenbucher, Nicola J. Broadbent, Clifford Cabebe, Chih-Kun Chai, Mi
Chen, Stephine Chow, De Michael Chung, Lindsay Crickard, Anne M. Danks, Graeme Freestone,
Dany Gitnick, Varsha Gupta, Christine Hoffmaster, Andrew R. Hudson, Alan P. Kaplan, Michael R.
Kennedy, Dong Lee, James Limberis, Kiev Ly, Chi Ching Mak, Brittany Masatsugu, Andrew C. Morse,
Jim Na, David Neul, John Nikpur, Marco Peters, Robert E. Petroski, Joel Renick, Kristen Sebring,
Samantha Sevidal, Ali Tabatabaei, Jenny Wen, Yingzhuo Yan, Zachary W Yoder, and Douglas Zook
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00372 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 11, 2018
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Yan, Yingzhuo; Dart NeuroScience LLC
Yoder, Zachary; Dart NeuroScience LLC, Chemistry
Zook, Douglas; Dart NeuroScience LLC
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Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with
Memory Enhancing Properties
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Vincent J. Santora,* Theresa A. Almos, Richard Barido, Jillian Basinger, Chris L. Bellows, Brett C.
Bookser, J. Guy Breitenbucher, Nicola J. Broadbent, Clifford Cabebe, Chih-Kun Chai, Mi Chen,
Stephine Chow, De Michael Chung, Lindsay Crickard, Anne M. Danks, Graeme C. Freestone,
Dany Gitnick, Varsha Gupta, Christine Hoffmaster, Andrew R. Hudson, Alan P. Kaplan, Michael
R. Kennedy, Dong Lee, James Limberis, Kiev Ly, Chi Ching Mak, Brittany Masatsugu, Andrew C.
Morse, Jim Na, David Neul, John Nikpur, Marco Peters, Robert E. Petroski, Joel Renick, Kristen
Sebring, Samantha Sevidal, Ali Tabatabaei, Jenny Wen, Yingzhuo Yan, Zachary W. Yoder, and
Douglas Zook
Abstract
We report here the identification and optimization of a novel series of potent GlyT1
inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led
to the identification of a novel series of pyrrolo[3,4-c]pyrazoles amides (21-50) with good in
vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties
produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of
GlyT1 was demonstrated for select compounds within this series by measuring the elevation of
glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately,
an optimized lead, compound 46, demonstrated in vivo efficacy in a rat Novel Object
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Recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.
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Introduction
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The amino acid glycine is both an inhibitory and excitatory neurotransmitter in the
mammalian central nervous system (CNS). Glycine acts as an inhibitory neurotransmitter at
strychnine sensitive glycine receptor ion channels which are predominantly found in the
cerebellum, brainstem and spinal cord (glycine A binding sites).1,2 Conversely, in areas such as
cortex, hippocampus and thalamus, glycine modulates excitatory neurotransmission as an
obligatory co-agonist, along with glutamate, of the N-methyl-D-aspartate (NMDA) receptor
(glycine B binding sites). Synaptic concentrations of glycine are regulated by two sodium-
dependent glycine transporters, GlyT1 and GlyT2, which maintain low extracellular glycine
concentrations by removing it from synapses via transport into glial cells and neurons. GlyT1 is
highly expressed in glial and neuronal cells in forebrain regions of the CNS including the cortex
and hippocampus where it acts as an indirect modulator of NMDA function. 1 In addition, GlyT1
is expressed in the retina,2 as well as outside the CNS in the dorsal root ganglia3 and in red
blood cells.4 By contrast, GlyT2 is found in the spinal cord, brainstem and cerebellum where it is
expressed on presynaptic glycinergic terminals and thought to regulate the function of glycine
receptors.
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Considerable evidence exists to implicate NMDA receptor-mediated signaling in learning
and memory. For example, NMDA receptor activation regulates new synapse formation and
long-term memories.5-8 Synaptic plasticity is dependent on NMDA receptor activation.
Specifically, pharmacological block or genetic disruption of NMDA receptor function can inhibit
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memory formation and long-term potentiation (LTP), a form of synaptic plasticity.9-12 In
addition, experimental manipulations that enhance NMDA receptor function enhance both
memory formation and LTP.13-16
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Given the obligatory role glycine plays in NMDA activation, it is not surprising that
pharmacological manipulation of synaptic glycine levels via GlyT1 inhibition has been an active
area of pharmaceutical research. Several GlyT1 antagonists have been described in the
literature.17-21 The first generation of GlyT1 inhibitors were predominantly sarcosine analogs
(e.g., 122 and 223) with initial in vitro profiles that appeared promising. However, further
investigation showed that many of these sarcosine derivatives produced a range of deleterious
side effects including ataxia, lateral recumbency, and respiratory depression.24,25 These side
effects are likely the result of sustained elevation of glycine in caudal regions of the CNS, where
GlyT1 and the glycine receptor (Gly A sites) are co-localized and play an important role in
control of motor function and respiration.26,27 More recent efforts to develop GlyT1 inhibitors
have focused on non-sarcosine chemotypes. A significant number of these second-generation
compounds, exemplified by compounds 3-7,28-33 have appeared in the literature recently and
some of these have demonstrated efficacy in preclinical animal models of cognition, pain,
addiction and anxiety.34,35 In addition, several compounds have advanced into clinical
development (including compounds 5-7), as potential treatments for the negative and cognitive
symptoms associated with schizophrenia and as treatments for obsessive compulsive disorder
(OCD), depression, addiction, and Parkinson’s disease.34, 36-41 To date no GlyT1 inhibitors have
received FDA approval.
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Figure 1. Representative GlyT1 inhibitors.
We initiated a program to identify a new series of GlyT1 inhibitors as potential
treatments for memory disorders using a ligand design approach based on known chemical
matter. In their SAR disclosures Hoffman La Roche identified several potent and selective GlyT1
inhibitors based on a benzoylpiperazine scaffold.31,42 We speculated that replacing the aryl-
piperazine moiety embedded within this chemotype with an aryl-pyrrolopyrazole group could
provide additional opportunities for optimization. In particular, based on Rapid Overlay of
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Chemical Structures (ROCS)43 overlays of these chemical structures the aryl ring of the
pyrrolopyrazole scaffold extends further, overlaying with the pyridyl CF3 group in compound 7
(Figure 2). We felt that this could allow us to investigate alternate aryl-groups in combination
with the pyrrolopyrazole linker.
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Figure 2. ROCS comparison of Roche compound 7 (blue) and aryl-pyrrolopyrazole 21
(grey).
Herein we describe the synthesis and optimization of this pyrrolopyrazole-containing
series (21-50) that led to the identification of several analogs with promising in vitro profiles. In
addition, we discuss the in vivo profiling of select examples from this new series in CNS
penetration and CSF glycine elevation studies, leading to the identification of an optimized lead,
compound 46, which had a favorable pharmacokinetic profile and showed in vivo efficacy in a
rat Novel Object Recognition (NOR) assay.
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Scheme 1. Synthetic Routes to Intermediates 10-17.a
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aReagents and conditions: (a) 8a or 9a, iPrOH, NaH, 70 °C; (b) 8a or 9a, R2OH, Cs2CO3,
Dioxane, 110 °C; (c) 8a or 9a, 4,4-difluoropiperidine, Cs2CO3, DMF, 120 °C; (d) 8b or 9b, 3-F-
phenyl boronic acid, Pd(PPh3)4 or Pd(dppf)Cl2, Na2CO3, Dioxane/H2O, 80 °C.
Chemistry
The new pyrrolopyrazole amides were synthesized using the methods outlined in
Schemes 1-3. Carboxylic acid precursors 10-17 were prepared from commercially available
acids 8 and 9 using methods similar to those described in the literature (Scheme 1).31,42
Specifically, 2-fluoro-5-sulfonyl or 2-fluoro-5-nitrile benzoic acids (8a or 9a) were reacted with
various alcohols or 4,4-difluoropiperidine under basic conditions to afford the 2-alkoxy
substituted derivatives 10-13 and the difluoropiperidine analogs 14 and 15. Alternatively,
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biphenyl carboxylic acids 16 and 17 were prepared from bromides 8b or 9b, respectively, by
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Suzuki coupling with 3-fluoro-phenyl boronic acid.
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The requisite pyrrolopyrazole intermediates 20a-f for this study were prepared in two
steps. The previously reported N-Boc-protected 4,6-dihydropyrrolo[3,4-c]pyrazole (18)44 was
first coupled with appropriate aryl halides using either Buchwald-Ullmann conditions45 or by
treating with an alkyl halide under basic conditions. The desired linear regiochemistry was
confirmed by NOESY-NMR, which showed the expected interaction between the ortho proton
of the aryl group (or CH2 of the cyclopentyl group) and the C-H proton of the pyrazole ring. The
intermediate 2-substituted pyrrolopyrazoles (19a-f) were then deprotected under acidic
conditions to provide pyrrolopyrazoles 20a-f. The final pyrrolopyrazole amides of this study (21-
50) were then prepared by amide coupling, either by conversion of acids 10-17 to acid chlorides
followed by addition of amines 20a-f, or by utilizing HATU as the coupling reagent in a standard,
one-step process.
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Scheme 2. Synthetic Routes to Intermediates 20a-f.a
aReagents and conditions: (a) ArI or ArBr, K3PO4, CuI, (1S, 2S)-(+)-1,2-diaminocyclohexane,
THF or Dioxane, 120 °C; (b) Alkyl halide, NaH, DMF; (c) HCl, Dioxane.
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Scheme 3. Synthetic Routes to Compounds 21-50.
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aReagents and conditions: (a) i. 10-17, SOCl2. ii. 20a-f, Et3N, DCM; (b) HATU, Et3N, DMF.
Structure-Activity Relationships
Peripheral functionality (R1-R3) for the new series was selected on the basis of
knowledge gained from SAR studies with an earlier, in-house series and literature
precedent.31,42,46 In vitro profiling of the pyrrolopyrazole amides consisted of extensive testing
across a diverse panel of assays. These included in vitro GlyT1 and GlyT2 functional potency in
HEK cells, as well as rat GlyT1 functional activity in rat cortical cultures. In vitro ADMET
properties for the new compounds were also assessed for intrinsic clearance in rat and human
liver microsomes (RLM and HLM, respectively), inhibition of CYP450 (1A2, 2C9, 2C19, 2D6 and
3A4 isozymes) and hERG channel inhibition as well as MDCK efflux and PAMPA-BBB
permeability.47
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The initial compound 21, incorporating an unsubstituted ring at the R3-aryl group,
exhibited high in vitro potency against both rat and human GlyT1 (IC50 = 4 and 15 nM,
respectively). The compound was also stable in rat and human LMs and showed no hERG
channel inhibition at concentrations < 10 μM. Based on these data, the SAR was expanded to
include several alkyl sulfones exemplified by compounds 22-39 (Table 1). Initial data were
promising, with similar IC50’s observed for all compounds against both rat and human forms of
GlyT1 and significant in vitro potency (hGlyT1 IC50 values < 38 nM) observed for all but one
compound in the sulfone series (39, hGlyT1 IC50 = 406 nM). In addition, the new compounds
exhibited high selectivity for GlyT1 over GlyT2 (all GlyT2 IC50’s > 10 μM) and minimal inhibition
of all CYP isozymes tested (all IC50’s > 10 μM). Finally, high in vitro permeability was observed
for most compounds in this series (17 of 19 compounds had PAMPA-BBB permeability values >
5 x 10-6 cm/s).
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Methyl sulfone analogs with S-trifluoromethyl-isopropoxy substituents at R2 served as
starting points for scanning the effects of various pyrazole N-substituents (R3) on in vitro
profiles. Several compounds within this group exhibited unacceptably high efflux ratios in the
MDCK assay, which can be indicative of being a substrate of P-glycoprotein (P-gp) or other CNS
efflux transport, an undesirable characteristic for CNS drugs.48 This was particularly true for N-
phenyl and N-cyclopentyl analogs 21 and 26 and pyridyl analogs 24 and 25. Interestingly, the
high efflux observed for N-phenyl substituted analogs such as 21 (efflux ratio = 5.9) was
significantly improved by installing an o-fluorine substituent on the phenyl ring (e.g., compound
22, efflux ratio = 2.1). Similar in vitro profiles were observed for methyl sulfones with other
functionality in the R2 position including alternate alkoxy groups (27-29), as well as
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difluoropiperidines (30-32) and 3-fluorophenyl substituents (33-35). Here again, high efflux
ratios were observed for compounds with N-pyridyl substituents at R3. The efflux issue
notwithstanding, the methyl sulfone analogs generally exhibited minimal hERG channel
inhibition and modest intrinsic clearance in rat liver microsomes, regardless of the structure of
R2 and R3 groups. The only exceptions were the neopentyloxy analog 28 and the m-
fluorophenyl derivatives 33 and 35.
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Attempts to reduce the MDCK efflux observed for certain methyl sulfones, by increasing
the size and lipophilicity of sulfone alkyl groups, were only marginally successful. Ethyl sulfones
36-38 (hGlyT1 IC50 = 12-29 nM) largely retained the potency observed with methyl sulfone
analogs and their efflux ratios were approximately equal to (36 cf. 22) or significantly lower
than their corresponding methyl sulfone analogs (37 cf. 24 and 38 cf. 25). However, despite
these improvements, none of these compounds demonstrated efflux ratios low enough to
warrant further testing (vide infra). Increasing the steric bulk of the sulfone alkyl group by
incorporating an isopropyl sulfone group (compound 39) considerably lowered the efflux (efflux
ratio = 2.2 cf. 5.9 for compound 21), but, unfortunately, this decrease in efflux was
accompanied by substantially reduced potency (hGlyT1 IC50 = 406 nM).
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Table 1. Properties of Sulfones Analogs 21-39.
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Rat
GlyT1
Human
GlyT1
Rat
CLint
Human
CLint
Compound
R1
R2
R3
hERG
MDCK Permeabilitye
Papp
A-B
(x10-6
cm/s)
Papp
B-A
(x10-6
cm/s)
IC50
(nM)a
IC50
(nM)a
(μL/min (μL/min
/mg)c
IC50
(μM)d
Efflux
Ratio
/mg)c
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29
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4
5b
15
8b
2.0
0.0
>10
>10
>10
>10
>10
>10
>10
8.1
10.3
24.6
13.6
2.7
61.1
51.7
23.5
74.4
69.5
84.0
51.6
91.3
58.9
5.9
2.1
1.6
0.0
0.9
0.3
6.7
1.5
76.4
5.3
0.0
0.1
0.0
1.4
0.0
3.3
1.1
0.3
12
9
23
1.7
23
27.3
32.5
7.7
3
12
2.1
18
18b
3b
17
10.9
28.4
46.5
24.6
23b
10b
38b
1.8
2.0
28b
9.9
2.4
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CH3
CH3
CH3
CH3
CH3
CH3
Et
12
10
4
16
10
7
22.2
11.3
3.2
0.6
1.7
4.3
6.7
24.5
3.8
68.7
67.1
63.7
72.6
85.5
97.4
51.9
99.0
101.0
123.0
2.8
17.8
23.1
2.8
5
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8
31
9
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32
2.5
>10
4.5
2.8
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33
12
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2
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6
56.6
13.1
36.4
0.0
21.5
1.4
26.3
10.1
6.2
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34
21
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27
>10
>10
>10
>10
>10
>10
8.6
4
4.3
15.9
2.9
36
9b
8
29b
27
12
406b
0.9
18.0
5.8
28
29
30
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32
37
Et
5.4
1.3
17.2
16.8
2.2
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38
Et
4
2.3
5.3
6.1
37
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39
i-Pr
79b
1.8
10.0
56.6
aIn vitro GlyT1 inhibition determined using a scintillation proximity assay (SPA). Potency was assessed in inhibiting the
uptake of radiolabeled glycine (11-concentration dose response, 2 points per concentration, single determination) in
primary rat cortical neurons (rat IC50) or HEK cells expressing the human GlyT1c (human IC50).
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bIC50 determined in duplicate or higher (n≥2). Standard deviations (SD) values were typically less than 40%.
cIntrinsic in vitro clearance determined in rat and human liver microsomes (LM) in the presence of NADPH.
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stably expressing the hERG potassium channel.
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eBi-directional transport determined across MDCK-MDR1 cell monolayers.
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We next examined the in vitro profiles of several nitrile analogs (40-50) with continued
interest in lowering the efflux ratios in this series. As observed for the sulfone analogs,
compounds in the nitrile series consistently displayed low activity against GlyT2 (all GlyT2 IC50’s
> 10 μM), minimal inhibition of various CYP enzymes (all IC50’s > 10 µM) and high permeability
(PAMPA-BBB > 10 x 10-6 cm/s for all compounds). However, GlyT1 potency across the nitrile
series was generally lower compared to the sulfones, particularly for N-2-fluorophenyl and N-
cycloalkyl analogs (e.g., 40 and 43), which showed approximately 10-fold lower potency
compared to similarly substituted methyl sulfone analogs (22 and 26). In addition, a wider
range of potencies was observed within subsets of the nitrile series compared to those of the
sulfone series. For example, 3-fluoro-5-pyridyl analogs (41, 45 and 49) had hGlyT1 IC50 values
that ranged from 69 to 383 nM and 3-methyl-4-pyridyl analogs (42, 46-48 and 50) had hGlyT1
IC50 values that ranged from 18 to 441 nM, whereas the analogous sulfones had IC50 values that
were consistently below 12 nM. The loss of potency for some nitrile analogs notwithstanding,
several analogs from this class had IC50 values in the sub-100 nM range.
Analogs in the nitrile series generally exhibited modest intrinsic clearance in rat liver
microsomes, with the exception of compounds with non-polar pyrazole substituents, such as
40, 43 and 44, which were substantially less stable than their corresponding sulfone analogs.
These observations, coupled with the significant improvement in MDCK efflux ratios and the
lack of any significant hERG channel inhibition observed within the nitrile series, resulted in a
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number of analogs with promising in vitro profiles (e.g., 41, 42, 46, 48 and 50).
Table 2. In vitro Properties of Nitrile Analogs 40-50.
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10
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
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46
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Compou
nd
Rat
GlyT1
Human
GlyT1
Human
CLint
R1
R2
Rat CLint
hERG
MDCK Permeabilitye
Papp A-B
Papp B-A
(x10-6
cm/s)
IC50
(nM)a
IC50
(nM)a
(μL/min/ (μL/min/
IC50
(μM)d
Efflux
Ratio
(x10-6
cm/s)
mg)c
mg)c
40
41
42
43
44
45
46
47
48
108
82b
27
78
69b
18
451.3
13.5
>10
>10
10
57.4
33.8
33.6
67.8
42.9
42.2
30.8
41.0
50.4
61.7
62.9
70.9
80.8
48.3
59.6
66.7
51.6
93.0
1.1
1.9
2.1
1.2
1.1
1.4
2.2
1.3
1.8
7.3
5.7
0.4
12.5
10.0
6.3
331
161
183
38b
690b
4
210
105
113
21b
441b
20
162
375.8
9.0
>10
>10
>10
10
1.9
7.2
3.7
7.0
22.2
36.9
>10
6.9
19.4
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49
50
619
78b
383
55b
17.2
16.9
4.7
8.8
10
51.4
43.0
79.9
40.4
1.6
0.9
10.1
10
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19
aIn vitro GlyT1 inhibition determined using a scintillation proximity assay (SPA). Potency was assessed in inhibiting the uptake
of radiolabeled glycine (11-concentration dose response, 2 points per concentration, single determination) in primary rat
cortical neurons (rat IC50) or HEK cells expressing the human GlyT1c (human IC50).
bIC50 determined in duplicate or higher (n≥2). Standard deviations (SD) values were typically less than 40%.
20 cIntrinsic in vitro clearance determined in rat and human liver microsomes (LM) in the presence of NADPH.
21
22
23
dhERG channel inhibition was measured in an electrophysiology assay (QPatch) using Chinese hamster ovary (CHO) cells stably
24
25
expressing the hERG potassium channel.
26
27
eBi-directional transport determined across MDCK-MDR1 cell monolayers.
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32
DMPK Profiling and Glycine Elevation Studies
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34
35
In order to identify promising compounds for in vivo behavioral studies, the CNS
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penetration properties of four representative compounds (two from the sulfone series and two
from the nitrile series) were measured in Sprague−Dawley rats 90 minutes aꢀer administraꢁon
of a single 10 mg/kg oral dose of each compound (Table 3). Sulfone analogs 22 and 29 had
similar exposure profiles in terms of plasma concentrations (Cplasma = 3093 and 2138 nM,
respectively), total brain concentrations (Cbrain = 687 and 605 nM, respectively) and CSF levels
(CCSF = 44 and 26 nM, respectively). In contrast, plasma exposure for nitrile analogs 41 and 46
were higher (Cplasma = 4684 and 3237 nM, respectively) and CNS penetration was considerably
higher (Cbrain = 2862 and 1700 nM, and CCSF = 319 and 304 nM, respectively). These results were
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somewhat unexpected considering the calculated properties and in vitro profiles of the test
compounds (Table 3). Specifically, sulfones 22 and 29 showed higher permeability (PAMPA-BBB
= 43.1 and 77.9 x 10-6 cm/s, respectively) compared to nitriles 41 and 46 (PAMPA-BBB = 25.5
and 16.3 x 10-6 cm/s, respectively) and the calculated topological polar surface area (tPSA)
values for sulfones 22 and 29 (tPSA = 81.5 for both compounds) were slightly lower than the
nitriles 41 and 46 (tPSA = 84.0 and 85.3, respectively).
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In additional experiments, in vivo GlyT1 target engagement for these compounds was
assessed by measuring changes in glycine concentrations in the CSF of rats in an acute, single
dose-response study (Table 3). Specifically, CSF samples from rats were collected 90 minutes
after oral administration of 10 mg/kg of test compound, a time point close to Tmax (data not
shown). The CSF glycine concentrations were determined by derivation of glycine with Marfey’s
reagent49 and subsequent LC-MS/MS analysis. Interestingly, statistically significant increases in
CSF levels of glycine were only observed for one compound in each series (sulfone 22 and nitrile
46) when compared to vehicle treated animals. These discrepancies are not easily explained
given the in vitro potency, high structural similarity and similar in vitro and CNS penetration
profiles shared by each pair of compounds. However, examination of the relationship between
free brain concentrations (Cu, Brain) and Rat GlyT1 potency for each compound suggests that
significant glycine elevation occurs only when the ratio of Cu, Brain/Rat IC50 is greater than 2
(Table 3). In addition, it is possible that these compounds have different binding kinetics
profiles (i.e., kon and koff) with GlyT1, which could result in different in vivo target engagement
profiles.50 To date, the binding kinetics of these analogs have not been explored to confirm if
such differences correlate with the levels of glycine elevation observed experimentally.
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Of the two compounds that showed in vivo elevation of CSF glycine, nitrile 46 emerged
as the preferred lead compound by virtue of its superior CNS penetration profile. Subsequent
evaluation of its pharmacokinetic profile in rat after a single 1 mg/kg IV or PO dose (Figure 3
and Table 4) revealed that 46 had low systemic clearance (CL = 14.6 min/mL/kg; 23% of liver
blood flow) with an elimination half-life of 2.4 h. Compound 46 was rapidly absorbed after oral
administration (Tmax = 0.67 h) with good oral bioavailability (F = 81%). In addition, compound 46
was profiled in a general off-target selectivity panel consisting of 95 targets, including enzymes
and receptors, and was found to be highly selective for GlyT1 with < 50% activity when
screened at 10 μM against all targets.
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Table 3. Summary of CNS Penetration, Glycine Elevation, In vitro ADME, and Calculated Properties for Compounds 22, 29, 41 and 46a
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6
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8
9
Rat
IC50
CSF Glycine
Glycine Elevation
PAMPA
-BBB
Cu,
Cu, brain/
Rat IC50
Compound
CPlasma CBrain CCSF
tPSAf cLogPf
Plasma Fu
Brain Fu
brain
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35
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37
38
39
40
41
42
43
44
45
46
47
48
10-6
cm/s
nM
4
nMb
3093
2138
nMb nMb
µMc
9.5
7.6
7.2
11
% controld
169 ± 29e
110 ± 26
109 ± 20
167 ± 18e
% unboundg % unboundg nMh
22
29
41
46
687
605
44
26
81.5
81.5
84
2.98
2.85
2.8
43.1
77.9
25.5
16.3
5.6
4.7
1.3
1.7
3.2
6.3
8.9
10.3
91.6
107.1
2.23
0.37
1.12
2.98
28
82
36
4684 2862 319
10.4
3237 1700 304
85.3
2.87
11.3
aMale SD rats (n=5) 90 min after administration of a single 10 mg/kg dose formulated in a mixture of NMP:PEG400:Water (10:50:40)
bStandard deviation typically < 35%
cStandard deviation typically < 15%
dCSF glycine mean % of treatment group vs. vehicle control ± propagated absolute uncertainty of the ratio (test compound glycine conc./vehicle
glycine conc.).
eStatistical significance, p < 0.05 vs vehicle control.
fCalculated using Chemdraw 12.
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gUnbound fraction of compound in rat plasma or brain homogenate determined by rapid equilibrium dialysis.
hCalculated according to the formula Cu, brain = CBrain x Brain Fu.
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Figure 3. Rat plasma concentrations of 46 following either a single 1 mg/kg PO or
1 mg/kg IV administration in 10:50:40 NMP/PEG400/H20.
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Table 4. Rat Pharmacokinetic Parameters of 46 After 1 mg/kg Administration.
Route of Administrationa
Pharmacokinetic Parameter
i.v. (1 mg/kg)
p.o. (1 mg/kg)
Tmaxb (hr)
Cmaxc (ng/mL)
AUCinfd (ng.hr/mL)
t1/2e (hr)
ND
ND
0.67
290
958
2.6
ND
ND
81
1176
2.4
Vssf (L/kg)
1.83
14.6
-
CLg (mL/min/kg)
Fh (%)
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aMale SD rats (n = 3) following administration of a single dose formulated in a
mixture of NMP:PEG400:Water (10:50:40)
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bTime of maximum observed concentration of compound in plasma.
cMaximum observed concentration of compound in plasma.
dArea under the plasma concentration versus time curve from 0 to the last time
point compound was quantifiable in plasma.
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eApparent half-life of the terminal phase of elimination of compound from plasma.
fVolume of distribution at steady state.
gClearance
hBioavailability
Memory Study
Given its promising profile, nitrile 46 was evaluated for long term memory enhancement
in a NOR test in rat (Figure 4). The NOR task is a widely-used behavioral task to assess visual
recognition memory, and is based on an animal’s innate preference for novelty.51 This task
consists of a training phase, where rats are presented with two identical objects to explore.
Following a delay interval, memory is assessed by presenting rats with a trained object and a
novel object. Rats with memory of the previously presented object will preferentially explore
the novel object. Rats that receive a memory enhancing drug in conjunction with submaximal
training are expected to exhibit improved memory performance similar to that of untreated
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animals that have received stronger training (i.e., more exposure to objects during the training
phase).
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Figure 4. Effect of 46 in a rat model of recognition memory. A) Study design, n = 18-24
male, Long-Evans rats/treatment group. B) Effect on 24 h memory retention. Rats treated
with 0.1, 1 and 3 mg/kg 46 exhibited significantly higher memory scores than rats treated
with vehicle and trained for 3 min. Asterisk = p < 0.05 vs. 3 min training + vehicle. C)
Exploration of the novel and familiar object during 24 h memory testing. D) Compound 46
did not affect object exploration during training. E) Compound 46 did not affect
locomotion during training. The mean ± SEM. are shown.
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Rats were dosed orally with compound 46 or vehicle 90 minutes prior to training and
then given 3 mins (submaximal memory training) or 20 mins (strong memory training) to
explore the arena and objects (Figure 4). Rats treated with compound 46 (0.1, 1 or 3 mg/kg)
exhibited enhanced long-term object recognition memory compared to sub-maximally trained
vehicle-treated rats at the 24 h retention test (p < 0.05, Fig. 4B). Further, the memory scores for
the 46-treated rats were no different from that of vehicle-treated rats that had received strong
20 min training (p > 0.07) and these groups also exhibited a significant preference for the object
at test (one sample t-test, p < 0.05, Fig. 4 B/C). By contrast, sub-maximally trained vehicle-
treated rats did not exhibit a preference for the novel object at test (p = 0.91). Importantly,
analog 46 did not affect object exploration time or the distance traveled during training,
indicating that the drug did not alter locomotor activity or motivation to explore the objects
(Fig. 4 D/E).
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Conclusions
Activation of NMDA receptor-mediated signaling plays an important role in learning and
memory. Because glycine acts as an obligatory co-agonist of the NMDA receptor,
pharmacological enhancement of synaptic glycine concentrations via inhibition of GlyT1 may
provide the means to enhance NMDA signaling. We identified and optimized a novel series of
GlyT1 inhibitors using a ligand design/scaffold modification strategy starting from known GlyT1
inhibitors. Several compounds from this new series showed promising in vitro profiles and
selected examples demonstrated good CNS penetration. Further evaluation of these advanced
compounds revealed a disparity in their ability to elevate CSF glycine concentrations in rats
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following oral dosing at 10 mg/kg.
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Analog 46, which possessed a promising in vitro and in vivo profile, showed efficacy in a
rat NOR test of long-term recognition memory after oral dosing at 0.1, 1 and 3 mg/kg.
Unfortunately, subsequent testing of this compound at higher doses revealed a side effect
profile that was similar to those of previously reported GlyT1 inhibitors24,25 and a safety margin
we considered insufficient for further development. Taken together, these results suggest that
compounds such as 46 can serve as tools to examine GlyT1 inhibitors as potential treatments of
cognitive disorders where NMDA receptor hypofunction may play a role. The difficulty in
identifying GlyT1 inhibitors with sufficiently wide therapeutic indexes is also highlighted.
Further details on compounds from this and related series will be reported in future
publications from our laboratories.
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Experimental Section
General Methods. Solvents and reagents were purchased and used without further purification
unless otherwise noted. Flash column chromatography (FCC) was performed on Teledyne Isco
CombiFlash instruments equipped with silica gel columns. Preparative HPLC purification was
performed on a Waters Preparative LCMS system equipped with a Waters XBridge Prep C18 5
μm OBD 19 × 50 mm column. Elution was achieved with increasing concentration of CH3CN in
water and 0.05% TFA added as a modifier. Analytical HPLC was performed on an Acquity UPLC
system equipped with a BEH C18 1.7 μM 2.1 × 50 mm column. Elution was achieved with
increasing concentrations of CH3CN in water and 0.1% formic acid added as modifier. NMR was
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performed on a Bruker Ultrashield 400 Plus spectrometer. The purity of all compounds tested
was determined by LCMS to be > 95%.
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(S)-5-(methylsulfonyl)-2-((1,1,1-trifluoropropan-2-yl)oxy)benzoic acid (10a). General
Method A. To a 100 mL pressure vessel was added 2-fluoro-5-(methylsulfonyl)benzoic acid
(4.00 g; 18.33 mmol; 1.00 eq.), (S)-1,1,1-trifluoropropan-2-ol (3.27 mL; 36.66 mmol; 2.00
eq.) and cesium carbonate (11.95 g; 36.66 mmol; 2.00 eq.) in 65 mL of dioxane. The vessel
◦
was flushed with N2, sealed and the mixture was heated and stirred at 115 C for 23 h then
cooled to room temperature. The mixture was diluted with 100 mL H2O, acidified with conc.
HCl to pH 1 and extracted with EtOAc (2 x 75 mL). The combined organics were dried with
Na2SO4, and concentrated under vacuum to give the title compound as a white solid (5.50 g,
96%) 1H NMR (400 MHz, DMSO-d6) δ 13.26 (br. s., 1H), 8.13 (d, J = 2.3 Hz, 1H), 8.03 (dd, J = 2.5,
8.8 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 5.49 (td, J = 6.4, 12.6 Hz, 1H), 3.55 (s, 3H), 3.22 (s, 3H), 1.44
(d, J = 6.3 Hz, 3H) ppm. LCMS (ESI) [M + H]+ m/z 313.1.
2-Isopropoxy-5-(methylsulfonyl)benzoic acid (10b). General Method B. To a 500 mL round-
bottomed flask was added sodium hydride (60% dispersion in mineral oil, 2.75 g, 68.7
◦
mmol) and isopropanol (100 mL). The mixture was heated to 110 C and stirred for 10
◦
minutes. Then, the mixture was cooled to 70 C and 2-fluoro-5-(methyl-sulfonyl)benzoic
acid (3.0 g, 13.75 mmol) was added in one portion. Heating and stirring were continued for
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7 h. Solvents were removed under reduced pressure and the resulting solids were taken up
in water (150 mL). The resulting aqueous mixture was acidified to pH 1 using conc. HCl and
extracted with EtOAc (2 X 100 mL). The combined organic layers were dried over sodium
sulfate, filtered and evaporated to afford the title compound (2.9 g, 82%). 1H NMR (400
MHz, DMSO-d6) δ 13.00 (br s, 1H), 8.06 (d, J = 2.7 Hz, 1H), 7.95 (dd, J = 2.3, 9.0 Hz, 1H), 7.36
(d, J = 9.0 Hz, 1H), 4.81 (td, J = 6.1, 12.1 Hz, 1H), 3.18 (s, 3H), 1.29 (d, J = 6.3 Hz, 6H) ppm.
LCMS (ESI) [M + H]+ m/z 259.0.
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5-(Methylsulfonyl)-2-(neopentyloxy)benzoic acid (10c). The title compound was prepared
1
using General Method A (0.53 g, 20%). H NMR (400 MHz, DMSO-d6) δ 13.17 - 12.98 (m, 1H),
8.11 (d, J = 2.7 Hz, 1H), 7.98 (dd, J = 2.3, 8.6 Hz, 1H), 7.31 (d, J = 9.0 Hz, 1H), 3.79 (s, 2H), 1.00 (s,
9H) ppm. LCMS (ESI) [M + H]+ m/z 287.3.
2-(Cyclopropylmethoxy)-5-(methylsulfonyl)benzoic acid (10d). The title compound was
prepared using General Method A (1.7 g, 68%). 1H NMR (400 MHz, DMSO-d6) δ13.07 (br. s.,
1H), 8.10 (d, J = 2.6 Hz, 1H), 7.99 (dd, J = 2.6, 8.8 Hz, 1H), 7.34 (d, J =8.9 Hz, 1H), 4.05 (d, J = 6.7
Hz, 2H), 3.20 (s, 3H), 1.32 - 1.19 (m, 1H), 0.61 - 0.55 (m, 2H), 0.42 - 0.35 (m, 2H) ppm. LCMS
(ESI) [M + H]+ m/z 271.0.
(S)-5-(Ethylsulfonyl)-2-((1,1,1-trifluoropropan-2-yl)oxy)benzoic acid (11a). The title
compound was prepared using General Method A (1.4 g 83%). 1H NMR (400 MHz, DMSO-d6) δ
13.29 (br. s., 1H), 8.11 (d, J = 2.4 Hz, 1H), 8.01 (dd, J = 2.5, 8.9 Hz, 1H), 7.60 (d, J =8.9 Hz, 1H),
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5.52 (quin, J = 6.4 Hz, 1H), 3.36 - 3.32 (m, 2H), 1.47 (d, J = 6.2 Hz, 3H), 1.11 (t, J = 7.3 Hz, 3H)
ppm. LCMS (ESI) [M + H]+ m/z 327.0.
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(S)-5-(Isopropylsulfonyl)-2-((1,1,1-trifluoropropan-2-yl)oxy)benzoic acid (12a). The title
compound was prepared using General Method A (0.4 g, 61%). 1H NMR (400 MHz, DMSO-d6) δ
= 13.30 (br. s., 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.98 (dd, J = 2.4, 8.8 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H),
5.52 (spt, J = 6.4 Hz, 1H), 3.46 (spt, J = 6.8 Hz, 1H), 1.48 (d, J = 6.4 Hz, 3H), 1.17 (d, J = 6.7 Hz, 6H)
ppm. LCMS (ESI) [M + H]+ m/z 341.0.
(S)-5-Cyano-2-((1,1,1-trifluoropropan-2-yl)oxy)benzoic acid (13a). The title compound was
prepared using General Method A (8.0 g, 84%). 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 2.2 Hz,
1H), 7.84 (dd, J = 8.7, 2.2 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 4.93 – 4.83 (m, 1H), 1.63 (d, J = 6.5 Hz,
3H) ppm. LCMS (ESI) [M + H]+ m/z 260.2.
5-Cyano-2-isopropoxybenzoic acid (13b). The title compound was prepared using General
Method B (14.2 g, 76%). 1H NMR (400 MHz, DMSO-d6) δ 13.00 (br. s., 1H), 7.96 (d, J = 2.3 Hz,
1H), 7.89 (dd, J = 2.3, 8.6 Hz, 1H), 7.30 (d, J = 9.0 Hz, 1H), 4.80 (td, J = 5.9, 12.0 Hz, 1H), 1.40 -
1.14 (m, 6H) ppm. LCMS (ESI) [M + H]+ m/z 206.2.
5-Cyano-2-(cyclopropylmethoxy)benzoic acid (13d). The title compound was prepared using
General Method A, using DMF as solvent and heating at 120 °C for 30 min in a sealed vial (20
mg, 15%). 1H NMR (400 MHz, CD3OD) δ 8.09 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 2.2, 8.8 Hz, 1H),
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Page 29 of 66
Journal of Medicinal Chemistry
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7.27 (d, J =8.8 Hz, 1H), 4.07 (d, J = 6.7 Hz, 2H), 1.34 (tt, J = 1.5, 6.6 Hz, 1H), 0.77 - 0.57 (m, 2H),
0.50 - 0.34 (m, 2H) ppm. LCMS (ESI) [M + H]+ m/z 218.1.
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2-(4,4-Difluoropiperidin-1-yl)-5-(methylsulfonyl)benzoic acid (14). The title compound was
prepared using General Method A using 4,4-difluoropiperidine (1.7 g, 62%). 1H NMR (400
MHz, DMSO-d6) δ 13.40 (br. s., 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.90 (dd, J = 2.4, 8.8 Hz, 1H), 7.32 (d,
J = 8.7 Hz, 1H), 3.32 - 3.28 (m, 4H), 3.19 (s, 3H), 2.19 - 2.06 (m, 4H) ppm. LCMS (ESI) [M + H]+
m/z 320.1
5-Cyano-2-(4,4-difluoropiperidin-1-yl)benzoic acid (15). The title compound was prepared
using General Method A using 4,4-difluoropiperidine (0.1 g, 43%). 1H NMR (400 MHz, DMSO-
d6) δ 7.98 (d, J = 2.1 Hz, 1H), 7.81 (dd, J = 2.2, 8.7 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 3.29 (d, J = 5.3
Hz, 4H), 2.19 -2.03 (m, 4H) ppm. LCMS (ESI) [M + H]+ m/z 267.1.
3'-Fluoro-4-(methylsulfonyl)-[1,1'-biphenyl]-2-carboxylic acid (16). General Method C. A 100
mL round-bottomed flask, equipped with an argon inlet adaptor, was charged with 2.8 g (10
mmol) of 8b, 3-fluorophenyl boronic acid (1.5 g, 10.4 mmol), Pd(PPh3)4 (0.54 g, 0.5 mmol), and
Na2CO3 (2.1 g, 19.8 mmol) followed by 40 mL dioxane and 20 mL water. The reaction mixture
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was flushed with argon and stirred at 80 C for 2 days. The mixture was filtered and the filtrate
was washed with EtOAc. To the water phase, was added 1N HCl until the solution reached pH 3.
Then the mixture was stirred at room temperature for 2 days. The desired compound was
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filtered as a white solid (0.9 g, 31%). H NMR (400 MHz, DMSO-d6) δ 13.41 (br. s., 1H), 8.31 -
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