Discovery of a novel potent GABAB receptor agonist

Article abstract of DOI:10.1016/j.bmcl.2011.08.006

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25-to 50-fold greater functional potency than R-baclofen at human and rodent GABA_B receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.

Full text of DOI:10.1016/j.bmcl.2011.08.006

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6582–6585
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel potent GABAB receptor agonist
Feng Xu, Ge Peng, Thu Phan, Usha Dilip, Jian Lu Chen, Tania Chernov-Rogan, Xuexiang Zhang,
⇑
Kent Grindstaff, Thamil Annamalai, Kerry Koller, Mark A. Gallop, David J. Wustrow
XenoPort Inc., 3410 Central Expressway, Santa Clara, CA 95051, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25-
to 50-fold greater functional potency than R-baclofen at human and rodent GABAB receptors in vitro.
Mouse hypothermia studies confirm that this compound crosses the blood–brain barrier and is approx-
imately 50-fold more potent after systemic administration.
Received 28 June 2011
Revised 29 July 2011
Accepted 1 August 2011
Available online 17 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
GABAB
Baclofen
SAR
Palladium
Boronate
Functional potency
Pyridylmethyl ether
Molecules that positively modulate GABA receptors continue to
be of great medicinal interest for a number of disease states.1 The
prototypical GABAB agonist baclofen 1 (Fig. 1) is widely used in the
treatment of spasticity,2 and has shown evidence for potential clin-
ical utility in treating other conditions including drug addiction
and alcoholism,3 gastroesophageal reflux disease (GERD),4 cancer
pain5 and overactive bladder.6 The GABAB activity of racemic bac-
lofen is known to reside primarily in the R-(À)-enantiomer 2. Cur-
rently R-(À)-baclofen is under development for the treatment of
behavioral symptoms of Fragile X Disorder. A prodrug of R-(À)-
baclofen, XP199867 3, is under clinical development as a potential
improved treatment for spasticity.
ture–activity study in which a broad range of substituents were
introduced to the aromatic ring of 2 in order to understand their
effects on GABAB receptor potency. In this paper, we report discov-
ery of a novel series of compounds that show improved GABAB
receptor potency in vitro and in vivo as compared to 2.
Synthesis began with the readily available lactam 4 which can
be prepared in high enantiomeric purity following a variety of lit-
erature methods.10–13 Nitration of lactam 4 followed by reduction
produced aniline 5 (Scheme 1). Conversion of the anilino group to
an iodide using Sandmeyer chemistry followed by Pd-catalyzed
substitution led to the dioxaborolane derivative 6. Oxidation of
the borolane with hydrogen peroxide afforded phenol 7, which
could be converted to the desired products by two alternate meth-
ods. Phenol ethers 8 could be prepared via a modified Mitsunobu
reaction procedure. The target baclofen derivatives were prepared
Since the initial synthesis of baclofen more than 40 years ago,8
relatively few studies of baclofen analogs exploring alternative aro-
matic substitution have been published.9 We undertook a struc-
Cl
Cl
Cl
O
O
O
H
O
O
N
H
2N
H
2N
OH
OH
OH
O
O
3
1
2
Figure 1.
⇑
Corresponding author.
E-mail address: Dave.Wustrow@XenoPort.com (D.J. Wustrow).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.006

F. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6582–6585
6583
Cl
Cl
Cl
O
B
NH2
O
c, d
a, b
e
N
N
H
N
H
Boc
O
O
O
4
5
6
Cl
Cl
OH
OR
Cl
OR
g
f
O
H
2N
N
N
OH
Boc
Boc
O
O
7
9a-n
8
Scheme 1. Reagents and conditions: (a) Guanidine nitrate, H2SO4 concd, 0 °C; (b) Fe, acetic acid, 50 °C; (c) NaNO2, KI, 12 N HCl, 0 °C 55% over three steps; (d) PdCl2(dppf)2,
octamethyl 2,2-bis-1,3,2-dioxaborolane, KOAc, DCM–dioxane 43%; (e) H2O2, DCM 82%; (f) alcohol, DIAD, PPh3 DCM; (g) (i) NaOH, (ii) 6 N HCl, 90 °C or TFA DCM.
Cl
Cl
OR
O
OR
O
Cl
N
N+O
Cl
b
a
7
O
O
O
O
a,b
H
2N
BOCNH
OEt
OH
BOCNH
H2N
OEt
OH
10a-n
9a-n
1Od
9k
Scheme 2. Reagents and conditions: (a) Cs2CO3, alkyl bromide, EtOH, 70 °C; (b) 6 N
HCl, 90 °C. Overall yield 23–50%.
Scheme 3. Reagents and conditions: (a) 35% H2O2; 6 N HCl, reflux 7 h, HPLC 8%
yield.
by hydrolysis and then isolated following HPLC purification in low to
moderate yields (Method A). Alternatively phenol 7 could be reacted
with alkyl halides in ethanol in the presence of Cs2CO3. In addition to
facilitating phenolic alkylation, the ethoxide formed under these
conditions also opened the lactam imide giving the BOC-protected
ester-ethers 10. BOC-esters 10 were converted to the target com-
pounds 9 via acidic hydrolysis (Method B, Scheme 2).
Cl
NH2
Cl
N
H
N
a,b
The pyridine N-oxide derivative 9k was prepared through reac-
tion of intermediate 10d with concentrated peroxide followed by
acid hydrolysis and HPLC purification (Scheme 3). The substituted
aniline derivative 9n could be prepared by reacting aniline 5 with
4-pyridinecarboxaldehyde under reductive amination conditions,
followed by hydrolysis in aqueous acid (Scheme 4). All target com-
pounds were isolated by semi-preparative reversed-phase HPLC
using a C18 stationary phase column and eluting with water–aceto-
nitrile gradients containing 0.5% formic acid. ES-MS was employed
during fraction collection and was used to guide isolation of the
appropriate sample-containing fractions. Purified compounds were
isolated by lyophilization from aqueous HCl and characterized using
mass spectrometry, 1H NMR and HPLC to assess structure and purity.
Compounds were assessed for their in vitro potency as agonists
at the human GABAB receptor. Human embryonic kidney (HEK)
cells constitutively expressing Gq/i proteins were transfected with
human GABAB1a and GABAB2 receptors under control of a tetracy-
cline-inducible promoter. Activation of the GABAB receptor com-
plex was quantified through measurement of the increased levels
of cytoplasmic Ca2+, released from intracellular stores, and de-
tected with a Ca2+ sensitive fluorescent probe. The concentration
of half-maximal stimulation was calculated and expressed as the
pEC50 (Table 1).
O
N
H
H2N
OH
O
5
9n
Scheme 4. Reagents and conditions: (a) 4-pyridinecarboxaldehyde, 2-picoline
borane, MeOH–AcOH 10:1; (b) 6 N HCl, acetonitrile 90 °C 3 h. HPLC 11% yield.
The active R isomer of baclofen 2 was shown to have a pEC50 of
7.1 at human GABAB receptors (with the Vmax defined as 100%).
pEC50 values for test compounds were calculated from duplicate
determinations. Substitution of the aromatic ring of R-baclofen
with a benzyl ether (9a) or 2-pyridylmethyl (9b) resulted in de-
creased agonist potency at the GABAB receptor. The loss in potency
was partially reversed by the replacement of the phenyl ring of 9a
with a 3-pyridyl ring (compound 9c). Introduction of the 4-pyridyl
substituent in 9d resulted in a 2.4 order of magnitude increase in
potency. Indeed compound 9d was more than one order of magni-
tude (25-fold) more potent than R-baclofen 2 as an agonist at the
human GABAB receptor in this assay.

6584
F. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6582–6585
Table 1
withdrawing and aromatic ring substituents. Similarly pyridine
N-oxide 9k also possessed reduced agonist potency at the GABAB
receptor. The saturated piperidines 9l and 9m had little or no ago-
nist activity at the human GABAB receptor. Replacement of the
ether oxygen linkage of 9d with nitrogen functionality resulted
in aniline 9n, which had decreased agonist potency.
Synthesis method and in vitro functional potency of compounds at human GABAB
receptor
Cl
R
O
To further characterize the GABAB agonist activity of 9d, it was
compared to R-baclofen 2 for its ability to induce GABAB receptor-
mediated G-protein activated inwardly rectifying K+ (GIRK) cur-
rents. Patch clamp electrophysiological experiments were per-
formed in rat hippocampal slices containing pyramidal cell CA1
neurons as previously described.14,15 Currents were measured in
the presence of varying concentrations of R-baclofen 2 and 9d. As
shown in Figure 2 compound 9d stimulated GIRK currents at
approximately 60-fold lower concentrations than 2. The magni-
tude of the maximal stimulation was the same as that observed
with R-baclofen (2).
H
2N
OH
2, 9a-n
No.
Method
—
R
h-GABAB agonist pEC50
7.1
2
H
9a
9b
A
B
6.1
4.0
O
O
N
The stimulation of GIRK currents caused by 9d could be com-
pletely reversed by the specific GABAB receptor antagonist
CGP55845 as shown in Figure 3. Taken together these studies dem-
onstrate that compound 9d also has a more potent interaction than
2 with the rodent isoform of the GABAB receptor. This is further
consistent with radioligand binding experiments using rat whole
brain tissue which showed that compound 9d displaced the GABAB
antagonist [3H]CGP54626 more potently than did 2, with pIC50 val-
ues of 6.7 and 5.5, respectively.
N
9c
B
B
6.3
8.5
O
O
O
N
N
9d
9e
9f
A
A
7.3
5.3
The in vivo pharmacology of GABAB agonist 9d was next com-
Me
pared with R-baclofen 2. The mouse hypothermia model16 was
H
N
O
O
110
100
90
H
Me
9g
9h
9i
A
B
B
7.4
7.2
6.6
N
80
9d
70
N
N
EC50 0.079 µM
R-baclofen (2)
O
O
60
50
40
30
20
10
0
EC50 5.0 µM
Cl
9j
B
3.9
6.7
N
N
O
-8
-7
-6
-5
-4
-3
+ O
Log compound concentration (mol/L)
9k
—
O
O
O
Figure 2. GABAB receptor evoked GIRK currents in rat pyramidal cell neurons.
NH
N
9l
B
<3
CGP55845 2 µM
9d 10 µM
9m
9n
A
—
3.6
6.8
N
NH
A series of analogs of 9d were prepared and evaluated. The
a-
methyl-substituted derivatives of 9d show a significant difference
in potency between the stereoisomers 9e and 9f. The introduction
of an ethyl spacer between the pyridine and oxygen functionality
in 9g resulted in a compound slightly more potent than baclofen,
but reduced in potency compared to 9d. Substitution of the pyri-
dine ring as exemplified by 9h–j led to decreases in potency com-
pared to 9d. The potency decrease was accentuated for electron
50
2
Figure 3. Reversal of GIRK current stimulation by 9d with CGP54626.

F. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6582–6585
6585
Table 2
Mouse hypothermia data
compound has been studied clinically as a possible treatment for
GERD but was recently dropped from development, in part, due
to liver toxicity issues. The potency of 9d was compared to 11,
12 and 13 in the functional GABAB agonist assay. The pEC50 values
were in the range of 7.4 and 8.1, respectively (Table 3). While the
phosphinic analogues were more potent than R-baclofen (2), com-
pound 9d with a pEC50 of 8.5 is more potent than any of the phos-
phinic acid compounds.
Compound
Dose (mol/kg)
MaxTemp decrease (°C)
Vehicle
2
9d
—
24
0.125
0.25
0.4
0
4.2
0
1
2.4
4.0
0.5
In summary a series of R-baclofen analogs have been prepared
that investigated the effect of substitution at the 3-position of
the phenyl ring. Analogs containing a 4-pyridyl substituent ap-
pended to this position had improved in vitro potency compared
to R-baclofen. The most potent of these compounds, 9d, demon-
strated in vitro GABAB agonist activity in functional assays with
cloned human receptors and in a rat neuronal electrophysiological
assay, appears to cross the blood–brain barrier and exhibits potent
activity in vivo.
Table 3
Aminophosphinic GABAB agonists
Compound
Structure
h-GABAB agonist pEC50
7.4
O
H
2N
P
11
OH
CH3
References and notes
O
P
H
H
2N
12
13
7.6
8.1
OH
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Compound 9d was shown to be more than 10-fold more potent
than R-baclofen. Previously aminophosphinic acids 11 and 12 were
shown to be more potent GABAB agonists than baclofen.17,18 More
recently the 3-fluoro analogue 13 (AZD-3355) was identified as a
potent GABAB agonist with restricted access to the CNS.19 This
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