Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: Hit-to-lead optimization

Article abstract of DOI:10.1016/j.bmcl.2014.06.057

An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b] pyridines derivative, which fulfilled the lead criteria.

Full text of DOI:10.1016/j.bmcl.2014.06.057

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3845–3849
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thieno[2,3-b]pyridines as negative allosteric modulators
of metabotropic GluR5 receptors: Hit-to-lead optimization
⇑
Katalin Nógrádi, Gábor Wágner , György Domány, Amrita Bobok, Ildikó Magdó, Béla Kiss, Sándor Kolok,
}
Katalin Fónagy, István Gyertyán, Viktor Háda, János Kóti, Krisztina Gál, Sándor Farkas, György M. Keseru,
István Greiner, Zsolt Szombathelyi
Gedeon Richter Plc, Budapest 10, PO Box 27, H-1475, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits
with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective
was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different
modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled
the lead criteria.
Received 14 May 2014
Revised 18 June 2014
Accepted 19 June 2014
Available online 27 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
mGluR5
Negative allosteric modulator
Glutamate, the most prominent excitatory neurotransmitter in
human brain and the vertebrate central nervous system (CNS), acts
through both ionotropic and metabotropic glutamate receptors.1
Three types of ionotropic glutamate receptors (NMDA, AMPA,
and kainate receptors) are ligand-gated ion channels responsible
for fast excitatory transmission.
for the mGluR5 approach to the treatment of anxiety. Phase II or
III clinical trials with Dipraglurant (Addex), Mavoglurant (Novar-
tis), RG-7090 (Roche, Cugai) and STX-107 (Seaside, Merck, Roche)
(Fig. 1) are currently underway for different indications such as
fragile X syndrome, gastroesophageal reflux disease, Parkinson’s
disease levodopa induced dyskinesia, treatment-resistant depres-
sion and major depressive disorder.7
Metabotropic glutamate receptors (mGluRs) belong to family C
of G-protein-coupled receptors (GPCRs) and modulate ion channel
activity or influence neurotransmitter release.2 Eight metabotropic
glutamate receptor subtypes (mGluR1–mGluR8) are known to date
which have been clustered into three groups (I–III). Group I mGlu
receptors (mGluR1 and mGluR5) are positively coupled to phos-
pholipase C; group II mGlu receptors (mGluR2 and mGluR3) and
group III mGlu receptors (mGluR4, mGluR6, mGluR7 and mGluR8)
are negatively coupled to adenylate cyclase.3 In the last decade the
discovery of small molecules that selectively bind to both group I
and group II receptors has significantly facilitated the understand-
ing of their roles in brain physiology and pathophysiology.4
Several mGluR5 antagonists have now entered human clinical
trials. Before metabotropic glutamate receptor subtypes were
cloned, the non-GABAergic agent fenobam (Fig 1.) was investigated
in a double-blind, placebo-controlled clinical trial in which it
showed efficacy and onset of action comparable with that of diaz-
epam.5 In 2005 Roche reported that fenobam, a negative allosteric
modulator (NAM) of mGluR56 provided clinical proof of principle
According to patent reviews,8 most mGluR5 NAM clinical candi-
dates and in vivo tool compounds are alkyne chemotypes. This
crowded intellectual property space subsequently led scientists
to look for non-alkyne chemotypes for this molecular target.
High throughput screening (HTS) of our corporate compound
collection resulted in several non-alkyne chemotype hit clusters
which we optimized as reported in patent applications.9 Further
optimization of two hit clusters resulted in the lead compounds
(1, 2) (Fig. 2), as reported in our previous communications.10 This
Letter describes the optimization of another structural family, the
thieno[2,3-b]pyridines, represented by compounds 3a–c (Fig. 2,
Table 1). The hit cluster was supplemented by analogues 3d and
3e (Table 1). The preliminary SAR showed that halogen substitu-
tion of the phenyl rings improved both the binding and functional
activity, indicating the importance of this moiety for biological
activity. Compound 3e served as an advanced hit for the hit-to-lead
optimization which was monitored by calculating ligand efficiency
(LE)13 and lipophilic ligand efficiency metrics (LELP).14 LE values
were calculated using the formula: [LE[
D
g] = ÀRT log[Ki]/N non-
hydrogen atoms]. LELP was defined as the log P/LE ratio indicating
the price of ligand efficiency paid in log P. Consequently, the higher
the absolute value of LELP the less drug-like the lead compound.
⇑
Corresponding author. Tel.: +36 18898732; fax: +36 14326002.
E-mail address: g.wagner@richter.hu (G. Wágner).
http://dx.doi.org/10.1016/j.bmcl.2014.06.057
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3846
K. Nógrádi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3845–3849
Figure 1. mGluR5 NAMs in clinical trials.
Br
Cl
Y
O
S
O
O
Cl
N
O
N
N
H
O
N
S
N
N
N
N
X
3
1
2
Hit cluster
rmGluR5 Ki = 49 nM
rmGluR5 IC50 = 215 nM
rmGluR5 Ki = 9.1 nM
rmGluR5 IC50 = 109 nM
Figure 2. Non-alkyne chemotypes lead compounds (1, 2) and general structure of hit cluster (3).
Table 1
Hit cluster
The synthesis of thieno[2,3-b]pyridines (8) is shown in
Scheme 1. Reaction of 2-chloro-3-pyridinecarbonitrile (4) with
thiourea in aqueous ethanol under reflux first afforded
2-mercapto-nicotinonitrile (5) which was converted into the
Compd
X
Y
mGluR5
Ki (nM)11
IC50
LE13
LELP14
(l
M)12
amino-thieno[2,3-b]pyridines (6) by reaction with
a suitable
3a
3b
3c
3d
3e
—
4-MeO
4-F
3-F
4-F
—
—
—
4-F
4-F
730
2500
343
224
180
3.3
8.0
2.1
—
0.34
0.29
0.34
0.34
0.35
16.3
18.8
16.5
17.1
16.9
halomethyl-derivative in N,N-dimethylformamide. This amine
was then transformed into the bromide (7) by reaction with CuBr2
and tert-butyl nitrite in acetonitrile. Finally, derivatives of (8) were
prepared from the appropriate bromide (7) and boronic-acid using
the well known methods of Suzuki coupling reactions.9g (Scheme 1)
1.0
NH2
Br
N
N
R
a
b
d
c
Q
Q
Q
N
Cl
N
H
S
S
S
S
N
N
N
4
5
6
7
8
where
n = 0-2;
Ar = optionally substituted phenyl;
Har = optionally substituted heteroaryl
Q = CO(CH2)nAr, COOAr, CONHAr, CONMeAr, SO2Ar; Har
R = Ar;
Scheme 1. Reagents and conditions: (a) thiourea, 50% water/50% ethanol, reflux, 4 h, 90% yield; (b) Cl-CH2-Q, DMF, 70–100 °C, 1–2 h, 60–80% yield; (c) CuBr2, tert-butyl
nitrite, CH3CN, 60–80 °C, 1–2 h, 50–70% yield; (d) R-B(OH)2, Na2CO3, 40–50% toluene/50–60% ethanol, Pd(PPh3)4, 1–2 h, 70–110 °C, 50–70% yield.

K. Nógrádi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3845–3849
3847
Table 2
Modification of the central ring
Compd
Structure
X
Y
mGluR5 binding inhib. at 1 l
M11 (%)
9
—
—
0
10
—
—
17
11
12
—
—
—
0
0
4-F
13
14
4-F
4-F
4-F
4-F
8
4
Table 3
Modification of linker
Y
mGluR5
Ph
Ki (nM) or bind. inhib. at 1
l
M11
IC50 (nM)12
L
Ph
Compd
LE13
LELP14
X
S
N
X
L
Y
O
3e
23
4-F
4-F
4-F
180
3%
1000
—
0.35
—
16.9
—
OH
4-F
24
25
4-F
4-F
4-F
4-F
0%
—
—
—
—
—
—
12%
O
O
O
26
27
4-F
4-F
4-Cl
4-F
41
150
—
0.38
0.34
16.3
14.4
O
311
NH
28
29
4-F
4-F
4-Cl
4-Cl
13%
15%
—
—
—
—
—
—
N
CH3
O
O
(continued on next page)

3848
K. Nógrádi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3845–3849
Table 3 (continued)
Y
S
mGluR5
Ph
Ki (nM) or bind. inhib. at 1
l
M11
IC50 (nM)12
L
Ph
Compd
LE13
LELP14
X
N
X
L
Y
O
O
30
31
4-F
4-F
4-Cl
0%
—
—
—
—
—
—
NH
N
4-F
19%
CH3
O
O
32
33
34
4-F
4-F
4-Cl
4-F
4-Cl
4-F
140
83
—
0.37
0.38
-
14.8
15.5
-
350
-
O
30%
O
N
O
35
36
37
38
4-Cl
4-F
4-F
4-F
4-Cl
4-F
12.3
52%
0%
46
—
0.43
—
14.0
—
S
O
N
O
N
4-Cl
4-Cl
—
—
—
N
N
24%
—
—
—
O
70
60
50
40
30
20
10
0
The final products were purified by either crystallization or column
chromatography to >95% purity (HPLC, 1H, and 13C NMR).9c
Multiple objectives were set for our hit-to-lead optimization,
the most important of which were identification of the essential
moieties and modification of the central ring and propan-1-one lin-
ker. First, the need for the phenyl-propyl side chain was checked.
Binding results of the fragments (9) and (10) (Table 2) showed
the necessity of the cleaved moiety. The next steps were omission
of the pyridine ring from the central core (11, 12), and replacement
of the thieno[2,3-b]pyridines core by benzo(b)thiophene (13), or
indole (14). None of these modifications were tolerated, as shown
by their poor affinity for the mGluR5 allosteric binding site
(Table 2).
The next change was to modify the propanone spacer. (Table 3)
Changing oxo- to a hydroxyl-group (23), or omission of the oxo-
group from the linker (24, 25) were non-tolerated modifications.
Modification of linker’s component showed mixed results: thus,
the ester linker (26) improved activity whereas the simple amide
linker (27) maintained it and the methyl substituted amide linker
(28) decreased activity. Shortening the linker also produced mixed
results. Analogues with shorter ester or amide linkers (29–31) lost
their affinity to the mGluR5 allosteric binding site but compounds
with the ethanone spacer (32, 33) had slightly better activity than
that of the advanced hit (3e). In parallel with these activity
improvements, the LE and LELP data were also slightly better for
both compounds. The last step of linker shortening resulted in an
inactive compound (34).
+++
vehiculum
20 mg/kg
(dose, ip. -30 min)
40 mg/kg
Figure 3. Effect of (35) in the Vogel punished drinking test in rats. +++: p <0.001
versus control.
in an order of magnitude better binding and functional data, with
significantly improved LE and LELP values, than those of the
advanced hit (3e). As the improved affinity result showed, the sul-
fonyl group solves as a bioisostere of ethanone linker in case of
mGluR5 NAMs. Moreover, compound (35)15 showed significant,
dose-dependent activity in the Vogel punished drinking model16
(Fig. 3), which is a well established and widely accepted anxiolytic
method. mGluR5 antagonist reference compound (MPEP) showed
robust efficacy in this test.10b
In summary, our HTS campaign identified thieno[2,3-b]pyri-
dines derivatives (3) as a novel class of mGluR5 negative allosteric
modulator. During the hit-to-lead optimization process both
fragmentation of the phenyl-propyl side chain at position 2, and
As these modifications did not significantly improve the biolog-
ical activity our next step was to replace the ethanone linker with
the sulfonyl spacer, or either bioisosteric heterocycles. While com-
pounds with heterocyclic linkers showed only weak affinity (36),
or lacked activity (37–38) to the mGluR5 allosteric binding site,
the sulfonyl compound (35)15 brought a breakthrough. It resulted

K. Nógrádi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3845–3849
3849
Kolok, S.; Nagy, J.; Kurkó, D.; Bakk, M. L.; Vastag, M.; Sághy, K.; Gyertyán, I.; Gál,
K.; Greiner, I.; Szombathelyi, Z.; Keseru, G. M.; Domány, G. Bioorg. Med. Chem.
Lett. 2010, 20, 4371.
modification of central thieno[2,3-b]pyridines core were futile.
Focusing on the propanone linker, modification of its elements
resulted in more active compounds. Finally, replacing of ethanone
by the sulfonyl linker resulted in compound (35), which fulfilled
the lead criteria of the project.
11. The mGluR5 receptor binding was determined according to Gasparini et al. (
Gasparini, F.; Andres, H.; Flor, P. J.; Heinrich, M.; Inderbitzin, W.; Lingenhöhl,
K.; Müller, H.; Munk, V. C.; Omilusik, K.; Stierlin, C.; Stoehr, N.; Vranesic, I.;
Kuhn, R. Bioorg. Med. Chem. Lett. 2002, 2, 407.
) with modifications. Rat
cerebrocortical membrane preparation was used to determine the binding
characteristics of reference compounds and novel compounds to the rat
mGluR5. As radioligand [3H]-M-MPEP (2 nM) was used. The non-specific
Acknowledgment
binding was determined in the presence of 10
displacement by the compounds was determined in duplicates or triplicates.
The ligand displacement at 1 M concentration of ligand were named as
binding inhibition at M. For IC50 (Ki) determinations concentration–
displacement curves were generated consisting of minimum of six
concentrations. Ki values (i.e., inhibition constants) were calculated using the
Cheng–Prusoff equation: Ki = IC50/[1 + (L/Kd)], where [L] is the radioligand
concentration and Kd the affinity of the labeled ligand for receptor. Kd was
lM M-MPEP. The ligand
K.N. is indebted to Katalin Koch for preparative assistance.
References and notes
l
1
l
1. Meldrum, B. S. J. Nutr. 2000, 130, 1007S.
2. Alagarsamy, S.; Sorensen, S. D.; Conn, P. J. Curr. Opin. Neurobiol. 2001, 11, 357.
3. (a) Schoepp, D. D.; Conn, P. J. Trends Pharmacol. Sci. 1993, 14, 13; (b) Schoepp, D.
D. Neurochem. Int. 1994, 24, 439; (c) Pin, J. P.; Duvoisin, R. Neuropharmacology
1995, 34, 1; (d) Bordi, F.; Ugolini, A. Prog. Neurobiol. 1999, 59, 55.
4. (a) Conn, P. J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205; (b) Schoepp,
D. D.; Jane, D. E.; Monn, J. A. Neuropharmacology 1999, 38, 1431; (c) Kew, J. N.
Pharmacol. Ther. 2004, 104, 233; (d) Niswender, C. M.; Jones, C. K.; Conn, P. J.
Curr. Top. Med. Chem. 2005, 5, 847; (e) Pitsikas, N. Eur. J. Pharmacol. 2014, 723,
181.
5. Pecknold, J. C.; McClure, D. J.; Appeltauer, L.; Wrzesinski, L.; Allan, T. J. Clin.
Psychopharmacol. 1982, 2, 129.
6. Porter, R. H.; Jaeschke, G.; Spooren, W.; Ballard, T. M.; Büttelmann, B.;
Kolczewski, S.; Peters, J. U.; Prinssen, E.; Wichmann, J.; Vieira, E.;
Mühlemann, A.; Gatti, S.; Mutel, V.; Malherbe, P. J. J. Pharmacol. Exp. Ther.
2005, 315, 711.
determined from the Scatchard plot. Binding inhibition at 1
displacement.
lM means the
12. Functional activity at mGluR5 was measured in primary rat neuronal cultures
of neocortical origin, taking advantage of the high expression level of mGluR5
in this brain area ( Romano, C.; Sesma, M. A.; McDonald, C. T.; O’Malley, K.; Van
den Pol, A. N.; Olney, J. W. J. Comp. Neurol. 1995, 355, 455. ) Functional activity
at mGluR5 was measured by Ca2+-fluorometry according to Nagy et al. with
modifications. Briefly, cells were isolated from E17 rat embryos, seeded in 96-
well plates and cultured at least for 5 days before being subjected to Ca2+
-
measurements. For the Ca2+-measurements cells were loaded with the Ca2+-
sensitive dye, fluo-4/AM. Baseline and agonist evoked signals were recorded
with a plate reader fluorometer. Agonist was (S)-3,5-dihydroxyphenylglycine.
For IC50 determination sigmoidal (4-parameter) concentration–inhibition
curves were fitted to the percent inhibition data derived from at least three
independent experiments using GraphPad Prism software.
7. Thomson Reuters Cortellis™, www.cortellis.com.
8. (a) Emmitte, K. A. Expert Opin. Ther. Pat. 2013, 23, 393; (b) Lindsey, C. W.;
Emmitte, K. A. Curr. Opin. Drug Discov. Devel. 2009, 12, 446; (c) Jaeschke, G.;
Wettstein, J. G.; Nordquist, R. E.; Spooren, W. Expert Opin. Ther. Pat. 2008, 18,
123.
13. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
}
14. Keseru, G. M.; Makara, G. M. Nat. Rev. Drug Disc. 2009, 8, 203.
15. Structure characterization and synthesis of compound 35 is described in patent
application (compound 1 and example 3 in Ref. 9g).
9. a) (a) Gál, K.; Wéber, C.; Wágner, G. A.; Horváth, A.; Nyéki, G.; Vastag, M.;
16. The Vogel punished drinking test was according to Vogel et al. ( Vogel, J. R.;
Beer, B.; Clody, D. E. Psychopharmacology 1971, 100, 138. ) with modifications.
Rats are deprived of drinking water for 48 h prior to test. 24 h prior to test they
are placed into the test chambers equipped with a metal water spout mounted
on the wall of the chamber and a metal grid floor for delivering electric shocks.
260 during a 5-min adaptation period, they have free access to the drinking
spout. On the day of the measurement, the animals are treated with the test
compounds (intraperitoneally administration in solution of 5% Tween/distilled
water) then placed into the test chambers where they have free access to
drinking water for a 30-s unpunished period. After that, electric shocks (1 mA,
1 s) are applied through the drinking spout following every 10 licks during a 5-
min punished period. Number of licks and shocks delivered are recorded and
stored in a computer. Anxiolytic or anxiogenic activity is reflected by increased
or decreased number of accepted shocks, respectively. Test compounds are
studied minimally at three dose levels because of the occurrence of bell-
shaped dose–response curves.
}
Keseru, G. M. WO 2007039781, 2007.; a) Gál, K.; Wéber, C.; Wágner, G. A.;
}
Bobok, A. Á.; Nyéki, G.; Vastag, M.; Keseru, G. M.; Háda, V.; Kóti, J. WO
}
2007039782, 2007.; (c) Nógrádi, K.; Wágner, G. A.; Keseru, G. M.; Bielik, A.;
Gáti, T.; Háda, V.; Kóti, J.; Gál, K.; Vastag, M.; Bobok, A. Á. WO 2007072090,
}
2007.; (d) Nógrádi, K.; Keseru, G. M.; Bielik, A.; Gáti, T.; Gál, K.; Vastag, M.;
}
Bobok, A. Á. WO 2007072091, 2007.; (e) Keseru, G. M.; Wéber, C.; Bielik, A.;
Bobok, A. Á.; Gál, K.; Meszlényiné S. M.; Molnár, L.; Vastag, M. WO 2007072093,
}
2007.; (f) Nógrádi, K.; Wágner, G. A.; Keseru, G. M.; Bielik, A.; Gáti, T.; Háda, V.;
Kóti, J.; Gál, K.; Vastag, M.; Bobok, A. Á. WO 2007072094, 2007.; (g) Nógrádi, K.;
}
Wágner, G. A.; Keseru, G. M.; Bielik, A.; Gáti, T.; Háda, V.; Kóti, J.; Gál, K.; Vastag,
M.; Bobok, A. Á. WO 2007072095, 2007.
10. (a) Wágner, G.; Wéber, C.; Nyéki, O.; Nógrádi, K.; Bielik, A.; Molnár, L.; Bobok,
A.; Horváth, A.; Kiss, B.; Kolok, S.; Nagy, J.; Kurkó, D.; Gál, K.; Greiner, I.;
}
Szombathelyi, Z.; Keseru, G. M.; Domány, G. Bioorg. Med. Chem. Lett. 2010, 20,
3737. Erratum in: Bioorg. Med. Chem. Lett. 2011, 21, 3837; (b) Galambos, J.;
Wágner, G.; Nógrádi, K.; Bielik, A.; Molnár, L.; Bobok, A.; Horváth, A.; Kiss, B.;