Pyrazolopyridinones as functionally selective GABAA ligands

Article abstract of DOI:10.1016/j.bmcl.2005.08.006

2,5-Dihydro-3H-pyrazolo[4,3-c]pyridin-3-ones are GABA_A receptor benzodiazepine binding site ligands, which can exhibit functional selectivity for the α₃ subtype over the α₁ subtype. SAR studies to optimize this functional

Full text of DOI:10.1016/j.bmcl.2005.08.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4998–5002
Pyrazolopyridinones as functionally selective GABAA ligands
Wesley P. Blackaby,* John R. Atack, Frances Bromidge, Richard Lewis,
Michael G. N. Russell, Alison Smith, Keith Wafford, Ruth M. McKernan,
´
Leslie J. Street and Jose L. Castro
Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Terlings Park,
Eastwick Road, Harlow, Essex CM20 2QR, UK
Received 27 June 2005; revised 1 August 2005; accepted 2 August 2005
Available online 8 September 2005
Abstract—2,5-Dihydro-3H-pyrazolo[4,3-c]pyridin-3-ones are GABAA receptor benzodiazepine binding site ligands, which can
exhibit functional selectivity for the a3 subtype over the a1 subtype. SAR studies to optimize this functional selectivity are described.
ꢀ 2005 Elsevier Ltd. All rights reserved.
c-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the central nervous system. It acts
at the GABAA receptor on a pentameric supramolecular
complex that forms a ligand-gated chloride ion channel.
A functional receptor is formed by the co-assembly of
subunits selected from the family of 16 gene products
(a1–6, b1–3, c1–3, d, e, p and h), which are differentially
expressed throughout the brain.1,2 The most abundant
GABAA receptor subtypes contain at least one each of
the a, b and c subunits in a 2:2:1 stoichiometry. The ben-
zodiazepine class of drugs,3,4 widely used as a therapy
for anxiety and panic disorders, are generally unselective
agonists that allosterically modulate the GABA-medi-
ated chloride ion flux through the channel of GABAA
receptors containing b, c2 and either a1, a2, a3, or a5
subunits.1,2 However, the side-effect profile associated
with the classical benzodiazepines is far from ideal.5
Studies with transgenic mice and with subtype selective
compounds suggest that a1-containing receptors are
responsible for mediating the sedative/muscle relaxant
properties of benzodiazepines and that a3 and/or a2-con-
taining receptors are important for anxiety.6 The goal
of our research was to identify subtype selective ligands,
be they functionally selective or binding selective, with
the expectation of gaining an improved side-effect
profile over currently used benzodiazepines.
Tetrahydropyrazoloquinoline 1 (CGS-17867A)7 (Fig. 1)
was chosen as a starting lead, exhibiting high affinity for
the benzodiazepine binding site, anxiolytic activity in
several animal behavioral models,7 and with an im-
proved side-effect profile. We attributed this profile to
the functional selectivity of this compound, which, in
our hands, showed significant efficacy at a3- and a2-con-
taining receptors and a much reduced efficacy at the a1
subtype.8 This paper describes modification of the tetra-
hydro ring by substituting the pyrazolopyridine core at
the 6- and 7-positions to give a novel series (2) of high
affinity compounds. This modification allows access to
a similar region of space accessed by the 2,5-dihydro-
pyrazolo[4,3-c]pyridin-3-one scaffold described recently
by our laboratory.8
The 7-phenyl substituted pyrazolopyridinones were
synthesised by condensation of phenylacetones with
enamine 3, followed by thermal cyclisation of the
intermediate in Dowthermꢁ A to give the pyridones 5.
Cl
Cl
R
1
2
3
N
N
4
N
N
7
O
O
1
N
N
6
(CGS 17867A)
2
Keywords: Pyrazolopyridinones; GABAA.
*
5
H
H
Corresponding author. Tel.: +44 1279 440404; fax: +44 1279
440187; e-mail: Wesley_Blackaby@Merck.com
Figure 1.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.006

W. P. Blackaby et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4998–5002
4999
R
R
O
EtO2C
i
EtO2C
H2N
CO2Et
ii
CO2Et
CO2Et
N
H
N
H
4
3
5
iii
Cl
R
H
Br
CN
CONH2
NO2
NH2
NHAc
a
b
c
d
e
f
g
h
i
2
R
iv
Cl
N
R
N
N
v
CO2Et
O
vi
2
6
vii
N
H
x,xi
viii,ix
2-oxopyrrolidin-1-yl
I
Scheme 1. Reagents and conditions: (i) ArCH2COMe, P2O5, THF, rt; (ii) Dowtherm A, reflux; (iii) POCl3, reflux; (iv) p-chlorophenylhydrazineÆHCl,
BuOH, reflux; (v) H2SO4 (85% v/v); (vi) Cu(acac)2, NaBH4, EtOH; (vii) AcCl, pyridine, DCM; (viii) isoamyl nitrite, tetrafluoroboric acid, DMF;
(ix) I2, KI, DMSO; (x) 4-chlorobutanoyl chloride, pyridine, DCM; (xi) NaH, DMF.
A range of dehydration conditions for the formation of
the enamine cyclisation precursor were examined; treat-
ment with phosphorus pentoxide9 in dichloromethane
or THF gave the best results. Treatment of 5 with phos-
phorus oxychloride gave the chloropyridines 6, which
were reacted with 4-chlorophenylhydrazine to give the
6-methyl-7-phenyl substituted pyrazolopyridinones 2a–
c, e (see Scheme 1).
substituents led to a loss in binding affinity (e.g., 2b)
compared to 1; however, polar substituents, such as
amides 2d and 2g, led to an increase in affinity. In our
high-throughput functional efficacy assay, based on
chloride ion flux,13 2d and 2g gave good efficacy at a3-
containing receptors. This was confirmed by patch-
clamp efficacy on L(tkÀ) cells containing a1b3c2 and
a3b3c2GABAA receptors, with both 2d and 2g imparting
functional selectivity for a3 over a1 similar to that of 1.
Interestingly, 2-oxopyrrolidin-1-yl 2h, lacking the amide
hydrogen bond donor (HBD), proved to be an unselec-
tive partial agonist. In a preliminary rat PK study, com-
pound 2a exhibited excellent oral bioavailability in rat
(F = 95%), a property not shared by 2g. It was hypoth-
esized that N-deacetylation of 2g could be responsible
for this lack of oral exposure and therefore we focused
on a series of heterocyclic replacements for the amide,
including heterocycles with a free NH group to probe
if this were critical for functional selectivity.
Copper-mediated borohydride reduction of the readily
available 4-nitro compound 2e gave aniline from which
iodide 2i was accessed by decomposition of the diazoni-
um tetrafluoroborate salt in the presence of iodine and
potassium iodide.10 Pyrrolidinone 2h was prepared by
acylation of the aniline with 4-chlorobutanoyl chloride,
followed by a base-promoted ring closure.11
Binding and efficacy data for the pyrazolopyridinones 2
are shown in Table 1. Introduction of small lipophilic
Table 1.
Entry
R
Ki (nM)a
Efficacyb
a1
a3
a1
a3
1
—
0.29 0.04
1.5 0.2
0.14 0.06
[0.21 0.03]
—
0.38 0.05
[0.45 0.05]
—
2a
b
H
Br
6.8 2.2
—
—
9.9 3.0
57
2.7 0.3
7
—
—
—
c
CN
CONH2
0.13 0.03
0.38 0.06
[0.41 0.06]
0.48 0.03
[0.43 0.05]
0.32 0.05
[0.46 0.06]
d
0.30 0.07
0.67 0.11
0.22 0.08
[0.19 0.02]
—
g
h
NHC(O)Me
0.25 0.04
—
0.42 0.03
2.5 1.2
[0.23 0.03]
0.29 0.04
[0.41 0.04]
2-Oxopyrrolidin-1-yl
a Affinity was determined by the inhibition of [3H]Ro 15-1788 (flumazenil) binding to human recombinant GABAA receptors containing b3c2 plus
either a1 or a3 stably expressed in L(tkÀ) cells. Values are (means SD) of 2–10 separate determinations.12
b Modulation of chloride ion flux in cells expressing b3c2 plus either a1 or a3 produced by an EC20 equivalent concentration of GABA in the presence
of an approximate 1000 · Ki concentration of the test compound. Efficacy is expressed, relative to the full agonist chlorodiazepoxide (relative
efficacy = 1.0), from at least seven independent experiments.13
Efficacy data, given in square brackets [] were measured at GABAA receptors stably expressed in L(tkÀ) cells using whole cell patch-clamp recording
and represent the effect of the test compound on the current produced by an EC20-equivalent of GABA, relative to the full agonist chlorodiazepoxide
(relative efficacy = 1.0).14

5000
W. P. Blackaby et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4998–5002
Cl
Cl
Cl
Ar
Ar
I
N
N
i
N
N
iv
N
N
ii
O
O
O
8a,b
N
7a-e
N
N
2i
H
H
H
iii
Cl
Cl
Cl
R
N
N
HN
N
N
vii
N
N
N
N
N
N
O
O
O
N
a) R=OH
b) R=OMs
c) R=N3
10
v
N
N
11
N
9
H
H
vi
N
H
Scheme 2. Reagents and conditions: (i) ArZnCl, Pd(PPh3)4, THF, reflux; (ii) ArB(OH)2, K2CO3 (aq), Pd(PPh3)4, DME, reflux or ArSnBu3,
Pd(PPh3)4, DME, reflux; (iii) imidazole, copper bronze, K2CO3, NMP, reflux; (iv) propargyl alcohol, Pd(PPh3)4, CuI, piperidine 80 ꢂC; (v) MsCl,
LiCl, 2,6-lutidine, DMF; (vi) NaN3, DMSO; (vii) N-methylpiperazine, DMSO, 100 ꢂC.
The aryl iodide 2i proved to be an invaluable intermedi-
ate for the introduction of heterocycles via a range of
metal-catalysed chemistries, examples of which are
shown in Scheme 2. Compounds 7a–e were accessed
by Negishi cross couplings with the appropriate hetero-
cycle, NH heterocycles protected as the SEM deriva-
tives,15 prior to metallation, and deprotected after the
coupling reaction with HCl in ethanol. The N-linked
imidazole 9 was prepared by copper catalysed amination
of the aryl iodide. Suzuki and Stille couplings also pro-
ceeded smoothly to give 8a and 8b, respectively. Sono-
gashira coupling with propargyl alcohol gave acetylene
10a, which could be transformed to the mesylate 10b.
Displacement by azide (10c), followed by base-catalysed
rearrangement,16 gave amino substituted 1,2,3-triazoles
11 via an allenyl azide, which was trapped with nucleo-
philic amine bases. This type of group has been shown
to increase solubility in a series of NK1 antagonists.17
show excellent binding affinity at a3 containing receptors,
although the more lipophilic heterocycles 7e and 8b led to
a significant loss in affinity. None of the heterocycles
lacking a HBD exhibited any functional selectivity (7d,
8a and 8b). However, although NH triazoles 7a, 7b also
lack functional selectivity, imidazole 7c and triazolopip-
erazine 11 do exhibit functional selectivity for a3 contain-
ing receptors. Triazolopiperazine 11, having a higher
efficacy at a3, was chosen for further profiling but gave
no in vivo displacement of [3H]Ro 15-1788 binding when
dosed at 10 mg/kg po in 0.5% methocel to rats.18 The
poor physical properties (high crystallinity and poor sol-
ubility) of this series, which have also been observed in
the related pyrimidin-5(6H)-one series,19 precluded fur-
ther in vivo evaluation of any of the other derivatives.
To improve the physical properties, replacements for the
phenyl spacer were investigated. To facilitate this, the
synthetic route was modified to give access to a pyrazol-
opyridine core with a halogen at the 7-position (Scheme
3). Treatment of 4-hydroxy-6-methyl-2-pyrone 12 with
From the data shown in Table 2, it can be seen that a
range of polar heterocyclic replacements for the amide
Table 2.
Entry
R
Ki (nM)a
Efficacyb
a1
a3
a1
a3
2d
CONH2
0.30 0.07
0.67 0.11
0.22 0.08
0.38 0.06
7a
b
1,2,4-Triazol-3-yl
1,2,3-Triazol-5-yl
Imidazol-2-yl
—
—
—
—
—
0.85 0.22
0.79 0.18
1.2 0.2
0.01 0.04
0.23 0.06
0.03 0.04
0.34 0.03
—
0.03 0.04
0.26 0.04
0.22 0.04
0.4 0.04
—
c
d
1-Methylimidazol-2-yl
Thiazol-2-yl
1.5 0.1
e
51
2.4 0.3
14
4
8a
b
Pyridin-3-yl
Oxazol-2-yl
—
—
0.15 0.03
0.22 0.02
0.18 0.03
0.25 0.02
1
9
Imidazol-1-yl
—
—
1.8 0.7
0.1 0.05
0.08 0.04
0.43 0.04
11
4-[(4-Methylpiperazin-1-yl) methyl]-1,2,3-triazol-5-yl
0.44 0.02
0.23 0.04
a,bAs for Table 1.

W. P. Blackaby et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4998–5002
5001
O
OH
O
OH
N
O
i
ii, iii, iv
NMe2
OH
O
O
O
12
13
14
v
Cl
Cl
O
OH
O
N
N
viii
vi, vii
I
I
I
O
OMe
OH
N
N
15
N
17
16
H
Scheme 3. Reagents and conditions: (i) DMF/DMA, dioxan; (ii) NH4OH; (iii) NHMe2, 50 ꢂC; (iv) H2SO4; (v) ICl, CaCO3, DMF, 50 ꢂC; (vi) POCl3,
95 ꢂC; (vii) MeOH, DCM, 0 ꢂC, (viii) p-chlorophenylhydrazineÆHCl, BuOH, reflux.
Cl
Cl
N
N
N
N
O
ii
N
i
iii
MeO2C(CH2)3
O
MeO2C
I
O
N
N
H
Cl
N
18
H
Cl
N
N
O
N
v
N
N
iv
ii
i
NH
NC(CH2)3
O
NC
I
N
H
Cl
N
19
H
N
N
vi
O
N
N
N
N
O
N
N
N
H
Scheme 4. Reagents and conditions: (i) activated zinc, THF, 50 ꢂC; (ii) 17, Pd2(dba)3, tris(2-furylphosphine), DMF, 50 ꢂC; (iii) acetamide oxime,
NaOEt, EtOH, 50 ꢂC; (iv) aminoacetaldehyde dimethylacetal, CuCl, DMSO, 80 ꢂC; (v) formic acid, 70 ꢂC; (vi) 17, Pd2(dba)3, tris(2-furylphosphine),
CuI, piperidine, 80 ꢂC.
DMF/DMA20 gave the enamine 13 which on treatment
with aqueous ammonia, followed by acidification with
sulfuric acid,21 gave the hydroxy acid 14. The acid was
halogenated with iodine monochloride and transformed
to pyrazolopyridine 17, as outlined in Scheme 3.
pling of 17 using the propargylic triazole (Scheme 4)
gave concomitant hydration of the acetylene to afford
ketone 20 as the sole product. This facile hydrolysis
(presumably via an allene intermediate) is probably
due to enhanced acidity of the propargylic methylene.ꢀ
From iodide 17, it was possible to gain access to com-
pounds with a flexible alkyl spacer via palladium-catal-
ysed coupling with an alkyl zinc reagent prepared by
the method of Knochel et al.22 Thus, methyl 4-iodobut-
anoate was converted to the alkyl zinc reagent, coupled
to 17, and reacted with acetamide oxime23 to give
oxadiazole 18. Similarly, coupling with the organozinc
derived from 4-iodobutyronitrile, followed by copper
catalysed amidine formation24 and acid-mediated ring
closure, gave imidazole 19. Attempted Sonogashira cou-
As shown in Table 3, replacement of the phenyl spacer
with alkyl spacers gave compounds with high affinity at
a3 receptors. In addition, compounds 18, 19 and 20
demonstrated a range of efficacy profiles. Compounds
18 and 19 have the efficacy profile of an antagonist and
ꢀ
The hydrolysis may be catalysed by trace amounts of the hydroxide
present in the triethylamine, which was dried over potassium
hydroxide before use.

5002
W. P. Blackaby et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4998–5002
Table 3.
Entry
R
Ki (nM)a
Efficacyb
a1
a3
a1
a3
18
4-(3-Methyl-1,2,4-oxadiazol-5-yl)
0.30 0.02
0.80 0.14
À0.2 0.05
[1.9%]
0.08 0.03
[À6.3%]
19
20
3-(Imidazol-2-yl)-propyl
2-Oxo-3-(1,2,4-triazol-1-yl)-propyl
0.30 0.04
0.45 0.04
0.75 0.04
0.75 0.18
0.23 0.04
À0.06 0.03
0.28 0.04
0.23 0.05
a,bAs for Table 1.
Carling, R. W.; Street, L. J.; Castro, J. L.; Ragan, C. I.;
Dawson, G. R.; Whiting, P. J. Nat. Neurosci. 2000, 3,
587.
unselective partial agonist, respectively. However, com-
pound 20 exhibited the desired efficacy profile, being a
partial agonist at a3 containing receptors and an antago-
nist at a1 containing receptors. The occupancy of com-
pound 20 was determined in vivo by displacement of
[3H]Ro 15-1788 binding in the mouse. When dosed at
3 mg/kg iv in 70% PEG, 20 gave a 44% receptor
occupancy.18
7. Bennett, D. A.; Amrick, C. L.; Wilson, D. E.; Boast, C.
A.; Loo, P.; Bernard, P. S.; Schmutz, M.; Gerhardt, S. C.;
Braunwalder, A.; Klebs, K.; Yokoyama, N.; Liebman, J.
M. Drug Dev. Res. 1987, 11, 219.
8. Mitchinson, A.; Atack, J. R.; Blurton, P.; Carling, R. W.;
Castro, J. L.; Curley, K. S.; Russell, M. G. N.; Marshall,
G.; McKernan, R. M.; Moore, K. W.; Narquizian, R.;
Smith, A.; Street, L. J.; Thompson, S.; Wafford, K.
Bioorg. Med. Chem. Lett. 2004, 14, 3441.
In conclusion, replacement of the fused tetrahydro ring
of CGS-17867A gave a novel series of high affinity GA-
BAA ligands by pendantly substituting the pyrazolopyri-
dine ring at the 6- and 7-positions. Although binding
selectivity for a3b3c2 over a1b3c2 receptors was rarely
greater than about 5-fold, modification of the 7-position
of the pyrazolopyridinone ring enabled modulation of
the efficacy profile to introduce functional selectivity.
Compounds, such as 2d and 11, achieved functional
selectivity comparable to that of the original lead.
Replacement of the 7-phenyl with a ketotriazole moiety
gave a compound (20) with subnanomolar affinity at
a3b3c2 receptors and an attractive efficacy profile, being
an antagonist at a1b3c2 receptors and a partial agonist at
a3b3c2 receptors.
9. Ohtsuka, Y. J. Org. Chem. 1976, 41, 629.
10. Citterio, A.; Arnoldi, A. Synth. Commun. 1981, 11, 639.
11. Michael, J. P.; Cirillo, P. F.; Denner, L.; Hosken, G. D.;
Howard, A. S.; Tinkler, O. S. Tetrahedron 1990, 46, 7923.
12. Hadingham, K. L.; Wingrove, P.; Le Bourdelles, B.;
Palmer, K. J.; Ragan, C. I.; Whiting, P. J. Mol. Pharma-
col. 1993, 43, 970.
13. Smith, A. J.; Alder, L.; Silk, J.; Adkins, C.; Fletcher, A. E.;
Scales, T.; Kerby, J.; Marshall, G.; Wafford, K. A.;
Mckernan, R. M.; Atack, J. R. Mol. Pharmacol. 2001, 59,
1108.
14. Brown, N.; Kerby, J.; Bonnert, T. P.; Whiting, P. J.;
Wafford, K. A. Br. J. Pharmacol. 2002, 136, 965.
15. Holzer, W.; Ruso, K. J. Heterocycl. Chem. 1992, 29, 1203.
16. Banert, K. Chem. Ber. 1989, 122, 1963.
17. Harrison, T.; Owens, A. P.; Williams, B. J.; Swain, C. J.;
Williams, A.; Carlson, E. J.; Rycroft, W.; Tattersall, F. D.;
Cascieri, M. A.; Chicchi, G. G.; Sadowski, S.; Rupniak,
N. M. J.; Hargreaves, R. J. J. Med. Chem. 2001, 44,
4296.
Acknowledgments
We thank Dr. Andrew Pike for generation of the phar-
macokinetic data.
18. Atack, J. R.; Smith, A. J.; Emms, F.; McKernan, R. M.
Neuropsychopharmacology 1999, 20, 255.
19. Albaugh, P. A.; Marshall, L.; Gregory, J.; White, G.;
Hutchison, A.; Ross, P. C.; Gallagher, D. W.; Tallman, J.
F.; Crago, M.; Cassella, J. V. J. Med. Chem. 2002, 45, 5043.
20. Kilbourn, E. E.; Seidel, M. C. J. Org. Chem. 1972, 37,
1145.
21. Yamada, H.; Tobiki, H.; Jimpo, K.; Gooda, H.; Takeuchi,
Y.; Ueda, S.; Komatsu, T.; Okuda, T.; Noguchi, H.
J. Antibiot. 1983, 36, 532.
References and notes
1. Simon, J.; Wakimoto, H.; Fujita, N.; Lalande, M.;
Barnard, E. A. J. Biol. Chem. 2004, 279, 41422.
2. Sieghart, W. Pharmacol. Rev. 1995, 47, 181.
3. The GABA Receptors; Bowery, G., Enna, S. J., Eds., 2nd
ed., 1997, p 332.
4. Gupta, S. P. Prog. Drug Res. 1995, 45, 67.
5. Atack, J. R. Curr. Drug Targets CNS Neurol. Disord.
2003, 2, 213.
6. McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.; Sur,
C.; Wafford, K. A.; Atack, J. R.; Farrar, S.; Myers, J.;
Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall,
G.; Macaulay, A.; Brown, N.; Howell, O.; Moore, K. W.;
22. Knochel, P.; Perea, J. J. A.; Jones, P. Tetrahedron 1998,
54, 8275.
23. Street, L. J.; Baker, R.; Book, T.; Kneen, C. O.; MacLeod,
A. M.; Merchant, K. J.; Showell, G. A.; Saunders, J.;
Herbert, R. H. J. Med. Chem 1990, 33, 2690.
24. Rousselet, G.; Capdevielle, P.; Maumy, M. Tetrahedron
Lett. 1993, 34, 6395.