The synthesis and SAR of 2-arylsulfanyl-phenyl piperazinyl acetic acids as glyT-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.05.043

Elevation of glycine levels and activation of the NMDA receptor by inhibition of the glycine transporter 1 (GlyT-1) is a potential strategy for the treatment of schizophrenia. A novel series of GlyT-1 inhibitors have been identified containing the 2-arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)- phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC₅₀=150nM), which elevated glycine levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of 1.2-4.6mg/kg s.c.

Full text of DOI:10.1016/j.bmcl.2004.05.043

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4027–4030
The synthesis and SAR of 2-arylsulfanyl-phenyl
piperazinyl acetic acids as glyT-1 inhibitors
Garrick Smith,a,* Thomas Ruhland,a Gitte Mikkelsen,a Kim Andersen,a
Claus Tornby Christoffersen,b Lene Hjorth Alifrangis,b Arne Mørk,b Stephen P. Wren,c
Neil Harris,c Barry M. Wymanc and Guillaume Brandtc
aMedicinal Chemistry Research, H. Lundbeck A/S, 9 Ottiliavej, DK2500 Valby, Denmark
bBiological Research, H. Lundbeck A/S, 9 Ottiliavej, DK2500 Valby, Denmark
cArgenta Discovery Ltd, 8-9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, UK
Received 8 March 2004; revised 28 April 2004; accepted 18 May 2004
Available online 15 June 2004
Abstract—Elevation ofglycine levels and activation ofthe NMDA receptor by inhibition ofthe glycine transporter 1 (GlyT-1) is a
potential strategy of r the treatment ofschizophrenia. A novel series ofGlyT-1 inhibitors have been identified containing the 2-
arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)-
phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC50 ¼ 150 nM), which elevated glycine
levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of1.2–4.6 mg/kg s.c.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
sulfanyl)-phenyl-1-piperazine moiety. In this letter, we
describe novel GlyT-1 inhibitors ofthe general formula
Hypofunction of the glutamatergic system has been
suggested to be an underlying factor behind schizo-
phrenia.1 Thus, a potential therapeutic approach is to
enhance glutamatergic function by modulating the
function of the NMDA receptor.2 A strategy for
achieving this is to increase the amount ofthe co-agonist
glycine in the synapse by inhibiting the reuptake of
glycine into glial cells by the glycine transporter 1
(GlyT-1).3 This transporter has been shown to be co-
localised with NMDA receptors.4 The low potency
GlyT-1 inhibitor sarcosine displays clinical benefits as
add-on therapy to risperidone.5 It has been reported in
the literature that sarcosine derivatives such as Org-
245986 (1) and ALX54077 (2) are potent GlyT-1 inhib-
itors. Structural considerations in finding a replacement
for the 3-aryloxy-3-arylpropyl motif in these reference
structures lead us to the identification ofthe (2-aryl-
3.
2. Chemistry
Keywords: Glycine transporter inhibitors; Schizophrenia; NMDA dys-
function.
8
The solid phase synthesis ofRuhland et al. (method a)
was used to synthesise piperazine intermediates (e.g., 9)
* Corresponding author. Tel.: +45-3643-3226; fax: +45-3643-8237;
e-mail: gsm@lundbeck.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.043

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G. Smith et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4027–4030
as illustrated in Scheme 1. 4-[(4-Nitrophenoxy)carbonyl-
oxymethyl]phenoxymethyl polystyrene 4 was treated
with trans-2,5-dimethyl piperazine to afford resin-bound
piperazine 5. Reaction with 1,2-dichlorobenzene cyclo-
pentadienyl iron complex gave resin-bound 2,5-dimethyl
piperazinyl 2-chlorobenzene cyclopentadienyl iron
complex 6. Treatment with 4-methoxythiophenol under
basic conditions and subsequent decomplexation using
phenanthroline and light afforded resin-bound 2-(4-
methoxyphenylsulfanyl)phenyl-2,5-dimethylpiperazine (8).
Cleavage from the resin using trifluoroacetic acid
(TFA) yielded 1-[2-(4-methoxy-phenylsulfanyl)-phenyl]-
2,5-dimethyl-piperazine (9).
An alternative, solution phase, synthesis (method b) is
shown by the synthesis of 18 in Scheme 3. 4-Chloro-2-
fluoro-nitrobenzene was reacted with 2-methylpiper-
azine and then reduced to the aniline 16. Diazotisation
and reaction with 4-methoxythiophenol gave the 1-(2-
arylsulfanyl)-phenylpiperazine 17. Alkylation with ethyl
bromoacetate followed by aqueous hydrolysis yielded
the piperazine acetic acid 18.
3. Results and discussion
The prepared compounds were tested for inhibition
ofuptake of 3H glycine into cells transfected with the
human GlyT-1b transporter.9 The racemic forms of
compounds 1 (Org-24461) and ALX5407 (2) had IC50’s
of320 and 26 nM, respectively.
The (2-arylsulfanyl)-phenyl-1-piperazine intermediates
were then employed in a second solid phase synthesis as
illustrated in Scheme 2 for the preparation of the final
product 13. Chloroacetyl chloride was esterified onto
Wang resin 10, and the solid supported chloroacetate 11
was subsequently reacted with the piperazine 9 under
basic conditions. The resulting alkylated product 12 was
cleaved from the resin using trifluoroacetic acid to afford
the final piperazine acetic acid 13.
As can be seen in Table 1, introduction ofa methyl
group in the 2-position ofthe piperazine ring appears to
be important for activity as witnessed by the assay re-
sults ofcompounds 13, 24 and 26 compared to that of
compound 25. In contrast, introduction ofa methyl
Scheme 1. Reagents and conditions: (a) trans-2,5-dimethyl-piperazine, N-ethyldiisopropylamine, THF, DMF, 70 ꢁC, 16 h; (b) Cp(1,2-dichlorobenz-
ene)Fe, THF–DMF 1:1, 70 ꢁC, 16 h; (c) 4-methoxythiophenol, NaH, THF, 60 ꢁC, 6 h; (d) hm, phenanthroline, HCl, CH3CN, H2O, 12 h; (e) TFA,
DCM, 2.5 h.
Scheme 2. Reagents and conditions: (a) ClCH2COCl, DIEA, DCM, 16 h; (b) 1-[2-(4-methoxy-phenylsulfanyl)-phenyl]-2,5-dimethyl-piperazine,
DIEA, DMF, 70 ꢁC, 16 h; (c) TFA, DCM, 1 h, 14% over two steps.

G. Smith et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4027–4030
4029
Table 2. Effect ofR phenyl ring substitution on inhibition of( 3H)-
2
glycine uptake at the GlyT-1b transporter (compounds were synthes-
ised by method b)
Com-
pound
R1
R2
GlyT-1b IC50
(nM)
18
27
28
29
30
31
32
2-Methyl
2-Methyl
2-Methyl
Cl
290
1600
CH3
MeO
MeO
Cl
4600
trans-2,5-Dimethyl
trans-2,5-Dimethyl
2R-Methyl
>50,000
1700
150
Scheme 3. Reagents and conditions: (a) 2-methylpiperazine, DIEA,
DMF, 80 ꢁC, 16 h, 88%; (b) H2, Pd/C, MeOH, 3 h, 98%; (c) 1. NaNO2,
H2SO4, H2O, 0.5 h; 2. Cu, NaOH, 4-methoxythiophenol, H2O, 60 ꢁC,
0.5 h, 11%; (d) BrCH2CO2Et, Et3N, CH3CN, 16 h, 48%; (e) NaOH,
H2O, EtOH, 16 h, 100%.
Cl
Cl
2S-Methyl
1400
unsubstituted (13). Stereochemistry at the 2-methyl
position ofthe piperazine ring is important of r affinity
with the R-isomer 31 displaying an order ofmagnitude
greater potency than the corresponding S-isomer 32.
This observation correlates with the preferred stereo-
chemistry ofthe 2-methyl group in compound 19.
Table 1. Effect ofR 1 piperazine ring substitution on inhibition of( 3H)-
glycine uptake at the GlyT-1b transporter
Variation ofR in Table 3 shows that substitution is
3
necessary for submicromolar potency as can be seen in
comparing the unsubstituted compound 33 with the
methyl-substituted analogue (34). This position appears
to be tolerant to small neutral groups as can be seen with
analogues 35, 36 and 13, but intolerant ofpolar groups
as seen with analogues 38 and 39 as well as phenyl
substitution (37).
Compound
R1
GlyT-1b IC50 (nM)
13a
19b
20b
21b
22b
23a
24a
25a
26b
trans-2,5-Dimethyl
2R,5S-Dimethyl
2S,5R-Dimethyl
2S,5S-Dimethyl
2R,5R-Dimethyl
3-Methyl
580
160
53,000
18,000
16,000
>10,000
1700
Table 3. Effect ofR 3 on inhibition of( 3H)-glycine uptake at the GlyT-
1b transporter
2-Methyl
––
2,2-Dimethyl
>10,000
1500
a Synthesised by method a.
b Synthesised by method b.10
group at the 3-position (compound 23) did not lead to
activity. However, incorporating a 5-methyl group onto
the 2-methylpiperazine unit appears to enhance potency
(13). The four stereoisomers of the 2,5-dimethyl substi-
tution demonstrate high stereoselectivity for the 2R,5S-
dimethyl analogue 19 with the other stereoisomers
showing much weaker potency.
Compound
R3
GlyT-1b IC50 (nM)
33a
34a
35a
36a
37b
38b
39b
––
CH3
2900
550
As can be seen in Table 2 where R1 is 2-methyl, varying
R2 indicates that the presence ofthe electron with-
drawing chlorine (18) confers better potency than
methyl (27) or methoxy (28) substitution. The preference
for R2 to be Cl (30) compared with methoxy (29) is not
reflected in the 2,5-dimethyl piperazine analogues where
neither compounds (29, 30) are more potent than the
Cl
360
1900
F
Ph
>10,000
3600
22,000
OH
NHCOCH3
a Synthesised by method a.
b Synthesised by method b.

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G. Smith et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4027–4030
with in vitro potencies for 13, 18 and 31 comparable to
Org-24598, 1. It has been demonstrated that extracell-
ular glycine levels are increased in the ventral hippo-
campus after subcutaneous administration with
compound 31 and thus provides further tool compounds
for examining the potential use of GlyT-1 inhibitors
as novel antipsychotics. The identification ofthe novel
2-phenyl-sulfanyl-phenyl piperazinyl motif provides a
novel template for further lead optimisation.
References and notes
1. Coyle, J. T.; Goff, J. T. Am. J. Psychiat. 2001, 158, 1367–
1377.
2. Heresco-Levy, U. Int. J. Neurospsychopharm. 2000, 3,
243–258.
3. Slassi, A.; Egle, I. Expert Opin. Ther. Patents 2004, 14,
201–214.
Figure 1. Study ofthe effects ofcompound 31 on extracellular glycine
levels in the rat ventral hippocampus. Mean basal pre-injection values
normalised to 100%; n: number ofanimals.
4. Bergeron, R.; Meyer, T. M.; Coyle, J. T.; Greene, R. W.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15730–15734.
5. Tsai, G.; Lane, H.-Y.; Yang, P.; Chong, M.-Y.; Lange, N.
Biol. Psychiat. 2004, 55, 452–456.
6. Brown, A.; Carlyle, I.; Clark, J.; Hamilton, W.; Gibson,
S.; McGarry, G.; McEachen, S.; Rae, D.; Thorn, S.;
Walker, G. Bioorg. Med. Chem. Lett. 2001, 11, 2007–2009.
7. Atkinson, B. N.; Bell, S. C.; De Vivo, M.; Kowalski, L. R.;
Lechner, S. M.; Ognyanov, V. I.; Tham, C.-S.; Tsai, C.;
Jia, J.; Ashton, D.; Klitenick, M. A. Mol. Pharm. 2001, 60,
1414–1420.
Compound 31 was profiled further in vitro against the
GlyT-2 transporter and a representative set of75 targets
including dopaminergic and serotonergic receptors with
no appreciable inhibition at a concentration of10 lM
but compound 31 did show some affinity towards
GABA receptors as it increased 35S-TBPS binding to rat
brain membranes. From in vitro assessment in Caco-2
cells, penetration through the blood–brain barrier was
estimated to be good. The permeability coefficient was
51 · 10ꢀ6 cm/s, comparable to clozapine (34 · 10ꢀ6 cm/s)
and risperidone (31 · 10ꢀ6 cm/s) and in the bi-directional
Caco-2 set-up the permeability coefficients were equal in
both directions, indicating that compound 31 is proba-
bly not a substrate for efflux transporters such as p-
glycoprotein. Assessment ofpharmacokinetics in the rat
for compound 31 revealed an excellent oral bioavail-
ability of100% when administered as a solution with a
low plasma clearance of0.4 L/h/kg and a terminal half-
life of 4 h.
8. Ruhland, T.; Bang, K. S.; Andersen, K. J. Org. Chem.
2002, 67, 5257–5268.
9. Cells transfected with the human GlyT-1b transporter
were seeded in 96-well plates. The cells were pre-washed
twice with HBS (10 mM Hepes-Tris (pH 7.4), 2.5 mM KCl,
1 mM CaCl2, 2.5 mM MgSO4, and preincubated with the
test compound for 6 min. Afterwards, 10 nM of 3H-glycine
was added, and the cells were incubated for 15 min. The
cells were washed twice in HBS. Scintillation fluid was
added, and the plates were counted on a Trilux (Wallac)
scintillation counter.
10. Compounds 19, 20, 21 and 22 were prepared by a
modification ofmethod b. N-Benzyl-2R,5S- and 2S,5R-
dimethyl piperazine were prepared as described in patent
application WO/0071525 and Aicher, T. D. et al. J. Med.
Chem. 2000, 43, 236–249. These intermediates were then
employed in the SNAR reaction with 2-fluoronitrobenzene
(method b), and the resulting product was subjected to
catalytic hydrogenation at 1.5 bar using Pd/C in methanol
for removal of the benzyl group. The 2R,5R- and 2S,5S-
dimethyl piperazines were prepared by borane mediate
reduction ofthe commercially available diketopiperazines,
In order to assess the effect ofcompound 31 in vivo, a
microdialysis study on freely moving rats was under-
taken. After subcutaneous administration, a dose-
dependent increase in extracellular levels ofglycine in
the ventral hippocampus was observed, giving an in-
crease to 140% ofbaseline levels atfer 60 min with a
4.6 mg/kg dose (Fig. 1).
4. Conclusions
cyclo-L-ala-L-ala and cyclo-D-ala-D-ala as described by:
Jung, M. E.; Rohloff, J. C. J. Org. Chem. 1995, 50, 4909–
4913, followed by monobenzylation and then used as
described for the trans stereoisomers.
Novel 2-phenylsulfanyl-phenylpiperazinyl acetic acids
have been identified as centrally acting GlyT-1 inhibitors