Discovery of 4-amino-3-arylsulfoquinolines, a novel non-acetylenic chemotype of metabotropic glutamate 5 (mGlu5) receptor negative allosteric modulators

Article abstract of DOI:10.1016/j.ejmech.2017.03.071

Negative allosteric modulators of metabotropic glutamate receptor 5 (mGlu₅) showed efficacy in a number of animal models of different CNS diseases including anxiety and depression. Virtually all of the compounds which reached the clinic belong to the same chemotype having an acetylenic linker that connects (hetero)cyclic moieties. Searching for new chemotypes we identified a morpholino-sulfoquinoline derivative (1) by screening our corporate compound deck. The HTS hit showed reasonable affinity and selectivity towards mGlu₅ receptors, however, its inferior metabolic stability prevented its testing in?vivo. In a chemical program we aimed to improve the affinity, physicochemical properties and metabolic stability exploring three regions of the hit. Systematic variation of different amines at position 4 (region I) led to the identification of 4-methyl-piperidinyl analogues. Substituents of the quinoline core (region II) and the phenylsulfonyl moiety (region III) were mapped by parallel synthesis. Evaluation of both morpholino- and 4-methyl-piperidinyl-sulfoquinoline libraries of about 270 derivatives revealed beneficial substituent combinations in regions II and III. Blood levels of optimized 4-methyl-piperidinyl-sulfoquinolines, however, were still insufficient for robust in?vivo efficacy. Finally, introducing 4-hydoxymethyl-piperidinyl substituent to region I resulted in new sulfoquinolines with greatly improved solubility and reasonable affinity coupled with affordable metabolic stability. The most promising analogues (24 and 25) showed high blood levels and demonstrated significant efficacy in the experimental model of anxiety.

Full text of DOI:10.1016/j.ejmech.2017.03.071

Accepted Manuscript
Discovery of 4-amino-3-arylsulfoquinolines, a novel non-acetylenic chemotype of
metabotropic glutamate 5 (mGlu ) receptor negative allosteric modulators
5
János Galambos, Attila Bielik, Gábor Wágner, György Domány, János Kóti, Zoltán
Béni, Áron Szigetvári, Zsuzsanna Sánta, Zoltán Orgován, Amrita Bobok, Béla Kiss,
Mónika L. Mikó-Bakk, Mónika Vastag, Katalin Sághy, Mikhail Krasavin, Krisztina Gál,
István Greiner, Zsolt Szombathelyi, György M. Keserű
PII:
S0223-5234(17)30241-6
10.1016/j.ejmech.2017.03.071
EJMECH 9331
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2017
Revised Date: 9 March 2017
Accepted Date: 28 March 2017
Please cite this article as: J. Galambos, A. Bielik, G. Wágner, G. Domány, J. Kóti, Z. Béni, E. Szigetvári,
Z. Sánta, Z. Orgován, A. Bobok, B. Kiss, M.L. Mikó-Bakk, M. Vastag, K. Sághy, M. Krasavin, K. Gál,
I. Greiner, Z. Szombathelyi, G.M. Keserű, Discovery of 4-amino-3-arylsulfoquinolines, a novel non-
acetylenic chemotype of metabotropic glutamate 5 (mGlu ) receptor negative allosteric modulators,
5
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.071.
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ACCEPTED MANUSCRIPT
Graphical abstract
Library
rKi: 30.9 nM
met. stab. (FM): r35% h56%
Vogel (rat) MED: 10 mg/kg

ACCEPTED MANUSCRIPT
Discovery of 4-Amino-3-Arylsulfoquinolines, a Novel Non-acetylenic Chemotype
of Metabotropic Glutamate 5 (mGlu5) Receptor Negative Allosteric Modulators
János Galambos1, Attila Bielik1, Gábor Wágner2, György Domány1*, János Kóti1, Zoltán Béni1, Áron
Szigetvári1, Zsuzsanna Sánta1, Zoltán Orgován3, Amrita Bobok1, Béla Kiss1, Mónika L. Mikó-Bakk1,
Mónika Vastag1, Katalin Sághy1, Mikhail Krasavin4, Krisztina Gál1, István Greiner1, Zsolt Szombathelyi1,
György M. Keserű3*
1Gedeon Richter Plc, 19-21 Gyömrői út, Budapest, 1103 Hungary
2Division of Medicinal Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU
University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
3Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of
Sciences, 2 Magyar tudósok körútja, Budapest, 1117 Hungary.
4Institute of Chemistry, Saint Petersburg State University, 26 Universitetskii Prospekt, Peterhof
198504 Russia
*Corresponding authors e-mail address: gy.keseru@ttk.mta.hu; gy.domany@richter.hu
Abstract
Negative allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) showed efficacy in a
number of animal models of different CNS diseases including anxiety and depression. Virtually all of
the compounds which reached the clinic belong to the same chemotype having an acetylenic linker
that connects (hetero)cyclic moieties. Searching for new chemotypes we identified a morpholino-
sulfoquinoline derivative (1) by screening our corporate compound deck. The HTS hit showed
reasonable affinity and selectivity towards mGlu5 receptors, however, its inferior metabolic stability
prevented its testing in vivo. In a chemical program we aimed to improve the affinity,
physicochemical properties and metabolic stability exploring three regions of the hit. Systematic
variation of different amines at position 4 (region I) led to the identification of 4-methyl-piperidinyl
analogues. Substituents of the quinoline core (region II) and the phenylsulfonyl moiety (region III)
were mapped by parallel synthesis. Evaluation of both morpholino- and 4-methyl-piperidinyl-
sulfoquinoline libraries of about 270 derivatives revealed beneficial substituent combinations in
regions II and III. Blood levels of optimized 4-methyl-piperidinyl-sulfoquinolines, however, were still
insufficient for robust in vivo efficacy. Finally, introducing 4-hydoxymethyl-piperidinyl substituent to
region I resulted in new sulfoquinolines with greatly improved solubility and reasonable affinity
coupled with affordable metabolic stability. The most promising analogues (24 and 25) showed high
blood levels and demonstrated significant efficacy in the experimental model of anxiety.

ACCEPTED MANUSCRIPT
Keywords
mGlu5 receptor; negative allosteric modulator; 4-amino-3-arylsulfonyl-quinolines, parallel synthesis,
optimization
1. Introduction
Glutamate is an important neurotransmitter in the brain that is recognized by two main classes of
glutamatergic receptors, ionotropic glutamate (iGlu) receptors and metabotropic glutamate (mGlu)
receptors [1]. iGlu receptors (NMDA, AMPA and kainite receptors) are ligand gated ion channels
responsible for the fast glutamatergic transmission. The excitatory effect of glutamate is modulated
by the activated mGlu receptors impacting the synaptic transmission through ion channel activity and
neurotransmitter release [2]. The family of mGlu receptors consists of eight Class C G protein-
coupled receptors (GPCRs) that are grouped into three subclasses including groups I (mGlu1 and
mGlu5), II (mGlu2 and mGlu3), and III (mGlu4, mGlu6, mGlu7 and mGlu8). Group I mGlu receptors are Gq
coupled GPCRs located mainly postsynaptically. Their activation increases NMDA receptor activity
and neuronal excitability. Based on their functional role and also on their expression in the striatum,
hippocampus, amygdala, and frontal cortex mGlu5 receptors are connected to several disease states
and prompted the development of mGlu5 negative allosteric modulators in indications such as fragile
X syndrome [3], 'Parkinson's disease levodopa-induced dyskinesias (PD-LID) [4], anxiety [5],
gastroesophageal reflux disease (GERD) [6], and depression [7]. Although compounds did not show
promising therapeutic potential in either fragile X syndrome or L-DOPA-induced dyskinesia in
Parkinson's disease recent results with basimglurant in major depression [7] together with human
positron emission tomography (PET) data [8] reinforces the trends in the research and development
of mGlu5 negative allosteric modulators (NAMs).
Investigating the chemical structures of the mGlu5 NAMs reached the clinic (Figure 1) it seems that
virtually all of them contains a central acetylenic core that makes the compounds essentially rod-like
in shape.
Figure 1. Reference mGlu5 negative allosteric modulators entered to clinical trials
F
Cl
N
NH2
F
N
N
N
Raseglurant (ADX-10059)
Basimglurant (RG-7090)
O
O
H
H
N
N
N
N
F
OH
Dipraglurant (ADX-48621)
Mavoglurant (AFQ056)

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The acetylenic part of the molecules can be traced back to early mGlu5 NAM compounds from Sibia
Neurosciences (SIB-1893) [9] having a stilbene core that was subsequently replaced by an acetylene
to yield MPEP [10], M-MPEP [11] and MTEP [12] the compounds used in many pharmacology studies
dealing with the role of mGlu5 receptors in pathological states. Keeping this structural moiety for
more than a decade in virtually all published mGlu5 NAMs its role has first become evident from the
high resolution X-ray structure of the receptor complexed with mavoglurant [13]. The structure
solved by the Heptares team revealed that mavoglurant binds to an extremely narrow mostly
hydrophobic site between helices 2, 3, 5, 6 and 7 about 8 Å deep from the membrane surface. Since
most of the mGlu5 NAM programs lacked structural information the ligand based design strategy
resulted in pretty similar structures having structural diversity in acetylenic head groups rather than
the linker region. More recently there were major effort in pharmaceutical industry as well as
academic institutes to development of non-acetylenic containing mGlu5 NAMs that have yield a
number of leads [14, 15]. Although a number of structurally diverse non-acetylenic chemotypes were
reported, only two chemotypes were progressed to clinical development. An AstraZeneca team
disclosed AZD6538 and AZD9272 (Figure 2) containing an oxadiazole moiety instead of the acetylene
linker [16]. More recently Heptares disclosed another non-acetylenic pre-clinical candidate
(HTL14242) [17]. The pyridyl and phenyl substituted oxadiazole core of the AstraZeneca compounds
was picked up from an initial high throughput screening hit. The phenyl and heterocycle substituted
pyrimidine was a fragment screening hit at Heptares that was optimized by structure based design.
Despite of the very much different design paradigms there are striking similarities between the
overall structures of AZD9272 and HTL14242.
Figure 2. Representative non-acetylenic mGlu5 NAM clinical candidates
Looking for novel mGlu5 NAM chemotypes we identified a morpholino-sulfoquinoline derivative (1)
screening our corporate compound deck. Compound 1 (Figure 3) showed high binding affinity
towards both rat and human mGlu5 receptors, demonstrated selectivity towards mGlu1, but had
unacceptable metabolic stability as expressed by the estimated metabolic bioavailability (FM, see
5.2.2. in the Experimental section). In our optimization campaign we mapped three regions of the
sulfoquinoline core (Figure 1) with focused libraries synthesized by parallel synthesis approaches.
This methodology allowed us understanding structure-activity and structure-metabolism
relationships of the amino-sulfoquinoline family that finally led to an optimized compound having
improved metabolic stability associated with high affinity and acceptable selectivity towards mGlu5

ACCEPTED MANUSCRIPT
receptors. The advanced lead showed reasonable blood levels and demonstrated in vivo efficacy in
an experimental model of anxiety.
Figure 3. The amino-sulfoquinoline HTS hit and its regions selected for optimization
rKi: 68 nM; hKi: 52 nM
mGlu1: inactive
met.stab. (FM): r9%/h26%
1
2. Chemistry
4-Amino-3-arylsulfoquinolines were synthesized in 5 steps from substituted sodium arylsulfinates
(Scheme 1). The arylsulfinates were first converted to methyl 2-(arylsulfonyl)acetates with
ethylchloroacetate in DMF (reaction i). The reaction of arylsulfonyl acetates and triethyl
orthoformate gave methyl 3-ethoxy-2-(arylsulfonyl)-2-propenoates (reaction ii) that were cyclized to
corresponding quinolinones with substituted anilines (reaction iii). The resulting phenylsulfonyl-
4(1H)-quinolinones were chlorinated (reaction iv) by POCl3 in position 4 preparing the corresponding
4-chloro-3-phenylsulfonyl-quinolines that were coupled with different amines in DMF using DBU
(reaction v) to give the final products, 4-amino-3-aryl-sulfoquinolines.

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Scheme 1. Synthesis of 4-amino-3-aryl-sulfoquinolines
i – DMF, ClCH2COOEt, 2 h at 80°C, 80-95%; ii – HC(OEt)3, 2 h, reflux, 90-95%; iii – PhOPh, reflux 1 h,
35-55%; iv – POCl3, reflux 2-3 h, 90-95%; v – DMF, DBU, 1h at 80°C, 75-85%.
3. Results and discussion
Starting from the HTS hit (1) we first explored region I by investigating different amine substituents
on the sulfoquinoline core (Table 1). In addition to a variety of cyclic secondary amines including
piperidine, piperazine, azepane and pyrrolidine derivatives we tested aniline, benzyl and diethyl
amines. [3H]M-MPEP displacement studies revealed that the original morpholine substituent of 1 can
be replaced by piperidine and azepane rings as well as diethyl and benzyl amines. Interestingly we
found that piperazines are basically inactive and polar substituents at position 4 of the piperidines
are also not tolerated. Binding affinity (Ki) measurements (Table 1) suggested that out of the six
amines only the 4-methyl-piperidine derivative has comparable affinity to that of the HTS hit carrying
the 4-morpholino ring in region I. Therefore the exploration of regions II and III were focused to 4-
morpholino and 4-(4-methyl-piperidinyl) sulfoquinolines.
Having the two most tolerated substituents in region I identified we explored region II and III
synthesizing a large number of compounds by parallel synthesis. All of the library members
synthesized successfully were subjected to displacement studies and the evaluation of their
metabolic stability. Since human metabolic stability data run parallel to corresponding rat data we
restrict our structure-metabolism analysis to human data.

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Table 1. Structure-activity relationship in region I of the sulfoquinoline cores
R
O
O
S
N
[3H]M-MPEP
displacement (%) at 1
µM
R
mGlu5 Ki (nM)
N
N
79.9
7.1
310.2
ND
2
3
N
78.3
3.6
201.2
ND
4
N
5
6
7
N
73.5
7.7
198.0
ND
N
N CH3
N
N
N
CH2CH3
Ph
1.7
ND
8
N
13.5
71.4
89.2
24.8
33.0
5.6
ND
9
N
306.8
108.2
ND
10
N
N
N
N
CH3
11
12
13
CONH2
CO2CH2CH3
ND
O
O
ND
14

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Figure 5. Optimization of aromatic substituents in regions II and III of the sulfoquinoline core. Panel A shows the distribution of binding affinities (measured
as [3H]M-MPEP displacement (%) at 1 µM concentration) and human metabolic bioavailability (estimated from human intrinsic clearance data) in the 4-
morpholino-3-arylsulfonyl-quinoline library. The library contains 127 derivatives carrying a range of substituents in region II (rows) and region III (columns).
Panel B shows the distribution of binding affinities (measured as [3H]M-MPEP displacement (%) at 1 µM concentration) and metabolic bioavailability
(estimated from human intrinsic clearance data) in the 4-(4-methylpiperidinyl) -3-arylsulfonyl-quinoline library. The library contains 126 derivatives carrying
a range of substituents in region II (rows) and region III (columns). For binding affinity tables cells are color coded from red (no displacement) to green
(100% displacement). For metabolic bioavailability tables cells are color coded from red (not bioavailable) to green (100% bioavailability).
O
A. 4-morpholino-3-arylsulfonyl-quinoline library
N
N
O
O
S
Binding affinity ([3H]M-MPEP displacement, %)
Region II
Region III
Region III
6-OMe 65 -3 12 20 20 29
1
28
9
4
23
3
11 93
14 15
7
6
2
4
20.2
6-Cl
6-Me
6-F
18
0
24 20 28 63
2
90 -2 39 53 33 23 14
7
84 26.7
86 10 44 58 65 76 24
73 75 58 33 70 34 47 89 18 48 26 66 52.6
-5 58 69 26 28 17 43 78 -3 18 31.5
83 87 62 38 85 67 24 55 14
84 53 51 74 48 58 50 29
73 77 52 52 34
81
5
0
40 25 41 68 10
3
2
7-Cl
7-F
32 81 73 70 86 49 87
4
5
2
4
6
41 12 50.4
23 33 46.3
33 23 46.3
4
4
71 72 86 56 74
86 57
8-F
4
23
83
average 37.7 9.5 40.0 44.5 49.2 70.8 32.7 83.7 0.6 67.2 66.0 40.2 35.6 51.3 30.6 32.9 61.7 15.6 9.0 28.1 43.6

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Estimated metabolic bioavailability (FM, %)
Region III
6-OMe
6-Cl
6-Me
6-F
32 41 27 58 25 28
37
21 29 25 32 47 23 30 27 46
52 59 22 30 36 70
33.0
37 40 36 51 23 45 36 29 32 36 10 28
8
25 35.3
41 38 28 50 28 24 32
32 43 37 83 27 30 35
24 11 28 10 39 46 12 24 35 64 29 39 31.7
30 30 10 26 36 51 23 63 38 69 31 37.0
9
7-Cl
7-F
31 41 39 54 32 24 42 11 42 31 24 33 21 56 43 11 26 47 66 33 24 34.8
40 41
62 51
66 28 35 43 10 43
28 50
11 30 10 33 42 23 36 45 68 27 24 34.5
27 39 58 37 60 70 40 27 45.0
8-F
36
average 39.3 42.1 33.4 60.3 27.2 30.6 39.7 16.7 36.8 31.4 14.5 29.0 15.7 41.0 49.4 19.0 35.1 41.1 64.7 32.0 27.8

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B. 4-(4-methylpiperidinyl) -3-arylsulfonyl-quinoline library
CH3
N
O
O
S
N
Region III
Region II
Binding affinity ([3H]M-MPEP displacement, %)
Region III
6-OMe
6-Cl
6-Me
6-F
64 68
59 58 92 90 87 86 72 84 61
67 94 91 52 96 82 90
-3 90
0
87
-3
85 21 73 87 30 79 91 27
41 90 54.4
95 74.7
81 46 68 88 93 49 92 89 31
93 91 93 91 75 72 80 79 90 55 62 82 80.7
40 76 92 93 90 91 82 92 54 95 91 82 86 69
93 96 92 94 93 89 90 86 93 94 89 93 85 93 94
83 80 87 75 85 91 81.6
91 91 70 79 94 90.0
7-Cl
7-F
87
92 90 94 94 90 83 94 93
95 93 95
89 87 77 80 90 75 83 90 88 87.5
93 82 85 26 86 67 93 69.1
8-F
13 62
9
average 60.4 75.6 72.6 85.1 76.2 89.1 86.1 88.3 57.6 94.1 88.4 67.0 83.6 83.0 62.5 74.1 86.7 69.1 66.5 70.7 90.4

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Estimated metabolic bioavailability (FM, %)
Region III
6-OMe
6-Cl
6-Me
6-F
56 38 34 31 42 48
40 37 30 35 43 65 49 33 54
38 54 27 40 49 40 44
36 44 35 34 27 52 37 29 37 40 40 47 38 35 12 29 42 35 43 45 31 36.6
35 52 28 44 41 37 42 45 46 41 32 24 32 47 53 28 58 39 31 27 39.1
31 38 37 24 36 39 36 38 39 34.5
28 44 26 34 30 28 25 27.2
44
29 31 31 30 21 24 42 46
52 32 37.1
49 58 43 35 39 28 53 45 74
38 44.6
39 34 25 33 46 58 23 58 43 40 55 48 41.8
7-Cl
7-F
32
8
28 26 39 35 24 38 45 36
32 29 35
8-F
27
7
average 35.0 42.0 30.8 35.3 38.0 44.7 38.4 35.0 43.8 40.0 36.7 37.0 33.7 39.3 35.1 25.6 41.9 42.3 43.3 38.0 34.3

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Comparing the two sets of compounds we found that 4-methylpiperidinyl derivatives were generally
more active and metabolically more stable than corresponding morpholino analogues. In the case of
morpholine derivatives the most active compounds carry 6-Me, 7-Cl or 7-F substituents in region II
while halogens and methyl groups in positions 3 and 4 of region III contributed best to the binding
affinity. Unfortunately, metabolic stability data showed a different picture since compounds having
6-F and 8-F substituents in region II were the most stable and nitrile and fluoro substituents in the
positions 3 and 4 in region III were typically required for the acceptable metabolic stability. The
analysis of 4-methylpiperidine substituted sulfoquinolines revealed that similar to morpholine
derivatives positions 6 and 7 (6-Me, 6-F and 7-Cl, 7-F) are preferred for the affinity in region II.
Although this set of compounds showed similar preference for substituents in positions 3 and 4 of
region III interestingly a much wider group of substituents were found to be tolerated in this region
as compared to that found for morpholine derivatives. Since the affinity improvement achieved by
different substituents introduced to region II and III seems to be similar the higher affinity of the
unsubstituted 4-methylpiperidinyl sulfoquinoline might explain the higher affinity of this type of
compunds. Metabolic stability data obtained for 4-methylpiperidines suggest that compounds with
6-Me, 6- and 7-chloro substituents in region II and halogen or nitrile substituents in positions 3 and 4
of region III are generally more stable.
Biological evaluation of the two series revealed that 4-methylpiperidinyl derivatives are more active
than morpholines. Based on the comparative evaluation of metabolic stabilities we concluded that (i)
compounds should contain a chloro or fluoro substituted quinoline ring preferably in positions 6 or 7
in Region II, (ii) methyl or methoxy groups should be avoided in both region II and III, (iii) the
presence of one halogen substituent seems essential in positions 3 and/or 4 of region III, (iv) the
introduction of a fluoro or nitrile group to region III might increase the metabolic stability further.
Considering these observations, we selected the most promising 4-methylpiperidines from the library
(15-19) and tested them after resynthesis (Table 2). Based on their physicochemical properties,
mGlu5 affinity and metabolic stability data we prioritized compounds and three of them (16, 17 and
18) were selected for in vivo testing in the Vogel conflict model of anxiety in rats. Blood levels after
10 mg/kg doses were also determined as a surrogate for their pharmacokinetic profile. All of the
compounds had limited water solubility (less than 3 µM), high lipophilicity (clogP higher than 5) and
limited rat metabolic stability that resulted in generally low blood levels. Despite of that, however, at
least one of them (compound 17) showed statistically significant effect at 30 mg/kg i.p. that
strengthened our commitment with the further optimization of this chemotype.
The synthesis and testing about 250 different 4-amino-3-arylsulfoquinolines revealed that the
allosteric mGlu5 binding site prefers lipophilic compounds that are typically subjected to extensive
metabolism in both rat an human microsomes. Subsequent in vivo studies clarified that the
physicochemical and ADME parameters of 4-amino-3-arylsulfoquinolines should be improved by
simultaneously keeping their binding affinity lower than Ki=100 nM. Since both the solubility and the
metabolic stability correlate inversely to lipophilicity (shown by clogP) our objective was decreasing
clogP when keeping the binding affinity acceptable (Figure 6). In practical terms this means that we
should introduce polar groups to one of the regions we optimized, however, regions II and III did not
tolerate polar functionalities (see binding affinities depicted on Figure 5).

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Table 2. Best 4-methylpiperidinyl-sulfoquinolines identified by library synthesis
Blood level
at 10 mg/kg
(rat, i.p. 40
min post
dose)
Vogel
(rat ip.)
MED
mGlu5
Ki nM
Structure
MW
clogP
rFM % hFM %
mg/kg
ng/mL
418.9
5.07 195.2
35
52
NT
NT
15
Me
N
N
O
O
S
F
436.9
5.22
68.2
37
53
29
>30
Cl
F
16
453.3
5.68
55.6
35
52
46
30
17
469.8
6.14 103.0
41
65
55
>30
18

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435.3
5.53 434,70
32
53
NT
NT
19
Figure 6. clogP-affinity diagram for 4-(4-methyl-piperazinyl)-3-arylsulfoquinolines
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
0
20
40
60
80
100
disp %
Although a couple of examples suggested that polar groups are not beneficial even in region I
(compounds 12-14 in Table 1) we selected a number of more polar cyclic secondary amines for
testing in this region (Figure 7). To achieve our goal we synthesized a small library of 7-F
sulfoquinolines equipped with thiomorpholine, 4-methoxymethyl-piperidine and N-sulfonamido-
piperazine moieties in region I and the metabolically most promising substituents in region III and
compared the lipophilicity and binding affinity of these compounds to corresponding morpholine and
4-methylpiperidinyl derivatives.

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Figure 7. Lipophilicity optimization in region I of 7-fluoro-3-arylsulfoquinolines. Panel A and B show
the distribution of calculated logP values (clogP) and binding affinities (measured as [3H]M-MPEP
displacement (%) at 1 µM concentration) in the 4-substituted-3-arylsulfonyl-7-fluoro-quinoline
library, respectively. The library contains 17 derivatives carrying a range of substituents in region I
(rows) and region III (columns). Cells are color coded from red to green indicating high to low
lipophilicity (Panel A) and low to high binding affinity (Panel B).
A. clogP distribution in the 4-substituted-7-fluoro-3-arylsulfoquinoline library
Region I
R
O
O
S
F
N
Region III
Region III
R
3F
3CN
4F
3,5F2
4.76
average
N
N
CH3
4.33
2.97
3.55
3.61
4.62
4.57
O
3.26
3.84
3.9
3.26
3.84
3.9
3.4
3.22
3.80
N
N
S
3.98
4.04
O
CH3
3.86
1.91
N
N SO2NH2
2.05
3.26
1.76
3.59
3.62
3.91
average

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B. Binding affinity ([3H]M-MPEP displacement, %) distribution in the 4-substituted-7-fluoro-3-
arylsulfoquinoline library
Region III
R
3F
3CN
92.4
4F
3,5F2
77.2
average
N
N
CH3
74.5
29
81.4
O
4.1
71.4
80.9
91
47.8
78.4
83
38.1
71.2
N
N
S
62.7
70.5
62.7
76.1
O
CH3
80.2
4.2
N
N SO2NH2
1.9
6.4
34.8
83.9
60.6
58.6
average
This analysis revealed that both the more polar thiomorpholine and 4-methoxymethyl-piperidine
derivatives have similar affinity to that of 4-methylpiperidines. It was also interesting to see that the
relatively most polar nitrile substituent in region III contributed significantly to the binding affinity.
When analyzing the metabolic stability data for this set of compounds we found, however, that
thiomorpholines are metabolically much more vulnerable than 4-methoxymethyl-piperidines.
Furthermore, combining the 4-methoxymethyl-piperidine moiety of region I with 7-chloro
substituent in region II gave more active compounds. These observations suggested to focus our
further efforts to 7-chloro-sulfoquinolines having halogen and/or nitrile groups in region III. As an
effort decreasing the lipophilicity of our best 4-methoxymethyl-piperidines further we removed the
methoxy group from region I and that yielded to the corresponding 4-hydroxymethyl analogues.
With these modifications we obtained 7-chloro-sulfoquinolines that are as polar as the
corresponding morpholine derivatives, have similar metabolic stability but much higher binding
affinity (Table 4).
Table 4. Comparison of morpholino- and 4-CH2OH-piperidinyl 7-chloro-sulfoquinolines
region III: 3-F
region III: 4-F
clogP disp. % hFM %
ID
20
22
clogP
disp. %
hFM % ID
region I:
morpholine
region I:
3.72
3.72
32
41
44
21
23
3.72
3.72
14
75
47
52
71
4-CH2OH-piperidine
The discovery of 4-hydroxymethyl-piperidine moiety for region I prompted us to combine our
knowledge accumulated during the optimization of 4-amino-3-arylsulfoquinolines and synthesized a

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couple of optimized compounds 24 and 25 with 7-chloro substituent in region II and halogens and
nitrile in position 3 of region III (Table 5).
Table 5. Optimized 4-hydroxymethyl-piperidinyl 7-chloro-sulfoquinolines
Blood level at 10
mg/kg (rat, i.p. 40
min post dose)
Vogel
(rat ip.)
MED
mGlu5 rFM hFM
Structure
MW clogP
Ki nM
%
%
ng/mL
mg/kg
459.92 3.58 24.1 47
55
608
>10
24
451.36 4.18 30.9 35
56
193
10
25
These compounds have much improved physicochemical parameters and consequently better
aqueous solubility (15-45 µM), high affinity and acceptable metabolic stability in both rats and
human microsomes that suggested two of them testing in vivo. Compounds 24 and 25 were
investigated in dose-response Vogel studies at 1, 3 and 10 mg/kg i.p. in rats (Figure 9). Compound 24
showed efficacy at 10 mg/kg, that is significant compared to the vehicle, however the p value of the
whole treated group was p=0.12 that resulted in MED>10 mg/kg. Compound 25 showed efficacy at
10 mg/kg, that is significant compared to the vehicle and the p value of the whole treated group was
p=0.000001 that gave the minimum effective dose of MED=10 mg/kg.

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Figure 9. Anxiolytic efficacy of 24 (A) and 25 (B) in the Vogel punished drinking test at 1, 3 and 10
mg/kg doses after i.p. administration. Mean±SEM values are given, group sizes were 8 at each dose,
*** indicates p<0.001 vs. vehicle-treated group (ANOVA followed by post-hoc Duncan-test).
A.
24
B.
25

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The availability of the high resolution X-ray structure of HTL14242 allowed us to predict and compare
the binding mode of 25 (Figure 10). Similar to HTL14242 the sulfoquinoline part of 25 is located in a
pocket formed by Pro6553.40, Ser8097.39, Gly6282.49, Ala8107.40, Ser6543.39, Ile6252.46 and Ser6583.43
(Ballesteros-Weinstein [18] numbering in superscript). In contrast to the Heptares compound that
forms a hydrogen bond to Ser8097.39 with its pyridyl nitrogen, the quinolone nitrogen of compound
25 is somewhat higher in the binding pocket and therefore could not form similar interaction.
Instead, we found that one of the sulfone oxygens of 25 makes a hydrogen bond with Ser8057.35
opposite to Ser8097.39. Ser8097.39 is in direct contact to the crystallographic water present in the
lower part of the binding pocket in both the mavoglurant and the HTL14242 structures. Although
compound 25 do not form hydrogen bond to Ser8097.39 the hydroxyl group of the 4-hydroxymethyl-
piperidine moiety makes direct hydrogen bond to Tyr6593.44 and Thr7816.46 from which the former is
in direct contact to the crystallographic water molecule proposed to be to crucial in receptor
activation. The allosteric pocket of mGlu5 involves a narrow channel between Tyr6593.44, Ser8097.39
,
Val8067.36, and Pro6553.40. In the mavoglurant structure this is filled by the acetylenic linker while in
the case of HTL14242 Val8067.36 is shifted outward from the binding site that allowed the central
pyrimidine fit to the pocket. Since compound 25 has a very different shape as compared to both
mavoglurant and HTL14242 we predict that the binding of our compound requires even more
significant change at Val8067.36 while it still contacts to Tyr6593.44, Ser8097.39, and Pro6553.40. In the
upper part of the binding pocket Trp7856.50 is rotated out toward helix 5 that was observed to form
aromatic interactions with HTL14242. The Heptares compound made similar contact with Phe7886.53
that was observed for compound 25 as well. Interestingly, however, there were no interactions with
Trp7856.50 found.
Figure 10. Predicted binding mode of compound 25

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4. Conclusions
A screening campaign on our corporate compound deck identified a new non-acetylenic chemotype
of mGlu5 receptor negative allosteric modulators. Optimization of the HTS hit (1) included systematic
variation of cyclic and acyclic amines at position 4 of the sulfoquinoline core and mapping the
aromatic substituents at both the quinoline ring and the phenylsulfonyl moiety by parallel synthesis.
Our efforts resulted in new sulfoquinoline derivatives with improved physicochemical properties and
reasonable metabolic stability showing high affinity towards mGlu5 receptors. The most promising
derivatives were tested in the Vogel’s punished drinking model of anxiety and showed significant
efficacy correlated with much improved blood levels. The advanced lead (25) identified in our
medicinal chemistry program represents a new chemotype of mGlu5 negative allosteric modulators
and is available for testing in further experimental models of CNS diseases.
5. Experimental section
5.1. Chemistry
All reactions were carried out under dry nitrogen. Commercially available reagents were used
without further purification. Solvents and gases were dried according to standard procedures.
Organic solvents were evaporated with reduced pressure using a rotary evaporator.

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Low-resolution (LRMS) electron impact (EI+) or fast ion bombardment (FIB+) mass spectra were
recorded either on a Finnigan MAT 95SQ or a Finnigan MAT 95XP mass spectrometer (Finnigan,
Bremen, Germany). High-resolution MS (HRMS) measurements were performed on a Thermo LTQ FT
Ultra spectrometer (Thermo Fisher Scientific, Bremen, Germany). The ionization method was
electrospray (ESI+). Nuclear magnetic resonance (NMR) measurements were performed on Varian
400 MHz (equipped with 5mm OneNMR 15N-31P/{1H-19F} PFG Probe), Varian 500 MHz (equipped with
1H{13C/15N} 5 mm PFG Triple Resonance 13C Enhanced Cold Probe) and Varian 800 MHz (equipped
with 1H{13C/15N} Triple Resonance 13C Enhanced Salt Tolerant Cold Probe) spectrometers. 1H chemical
shifts are given on the delta scale as parts per million (ppm) with tetramethylsilane (TMS) as the
internal standard (0.00 ppm). 13C chemical shifts are given on the delta scale as parts per million
(ppm) with tetramethylsilane (TMS) or dimethylsulfoxide-d6 as the internal standard (0.0 ppm and
39.5 ppm, respectively). The quinoline part, the benzenesulfonyl moiety, and the third ring of the
molecules are numbered as <1…8a>, <1’…6’> and <1’’…6’’ or 7’’ depending on the structure>,
respectively.
5.1.1. General procedure for the synthesis of 4-amino-3-arylsulfonyl-quinolines19
The equimolar mixture of ethylchloroacetate and sodium arylsulfinates in DMF (1 mL/mmol) was
stirred and heated at 80°C for 2 h. The solution was diluted with water (3 mL/mmol). The
separated oil was extracted with chloroform and washed with water. The organic phase was
evaporated in vacuo to yield the corresponding methyl 2-(arylsulfonyl)acetate in 80-95%. The
arylsulfonyl acetate was refluxed with equimolar triethyl orthoformate for 2 h with simultaneous
distillation of ethanol, and then evaporated to dryness to give methyl 3-ethoxy-2-arylsulfonyl-2-
propenoates (90–95%) that were used in the next step without purification. 25 mmol of
arylsulfonyl propenoates and 25 mmol of the corresponding aniline in 20 ml of diphenyl ether
was heated at near reflux for 1 h. After cooling the precipitate was filtered, washed with ether
(crystallized from e.g. pyridine if isolated) to give the corresponding 3-arylsulfonyl-4(1H)-
quinolinone in 35-55% yield. In the next step 10 mmol of the quinolinone was refluxed in 50
mmol of POCl3 for 2-3 h. Excess POCl3 was evaporated in vacuo and the residue was extracted
with chloroform and washed with water. The organic phase was evaporated in vacuo to yield the
corresponding 4-chloro-3-arylsulfonyl-quinoline in 90-95% yield. This intermediate (0.25 mmol)
was stirred with the suitable secondary amine (0.3 mmol) and 0.3 mmol of DBU in 3 ml of DMF
for 1 h at 80°C. After cooling, water was added; the corresponding 4-amino-3-arylsulfonyl-
quinolines were filtered out as solids and crystallized from methanol. The yield was 75-85%.
5.1.1.1. 3-[(3,4-dimethylphenyl)sulfonyl]-4-(morpholin-4-yl)quinoline (1)
1H NMR (500 MHz, DMSO-d6) δ 9.38 (s, 1H, H-2), 8.32–8.29 (m, 1H, H-5), 8.19–8.17 (m, 1H, H-8),
7.97–7.94 (m, 1H, H-7), 7.76–7.73 (m, 1H, H-6), 7.66–7.65 (m, 1H, H-2’), 7.64–7.61 (m, 1H, H-6’),
7.39–7.37 (m, 1H, H-5’), 3.49–3.46 (m, 4H, H2-2’’, H2-6’’), 3.09–3.05 (m, 4H, H2-3’’, H2-5’’), 2.28–
2.26 (2x s, 6H, C(3’)-Me, C(4’)-Me).
LRMS (FIB+), [M+H]+ = 383.
5.1.1.2. 4-(azepan-1-yl)-3-(phenylsulfonyl)quinoline (2)

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1H NMR (800 MHz, DMSO-d6) δ 9.38 (s, 1H, H-2), 8.19–8.17 (m, 2H, H-5, H-8), 7.96–7.93 (m, 1H,
H-7), 7.89–7.87 (m, 2H, H-2’, H-6’), 7.75–7.70 (m, 2H, H-6, H-4’), 7.64–7.62 (m, 2H, H-3’, H-5’),
3.12–3.10 (m, 4H, H2-2’’, H2-7’’), 1.60–1.58 (m, 4H, H2-4’’, H2-5’’), 1.48–1.44 (m, 4H, H2-3’’, H2-6’’).
HRMS (ESI+), calcd for C21H23O2N2S [M+H]+: 367.14748; found: 367.14720. delta=-0.75 ppm.
5.1.1.3. N,N-diethyl-3-[(4-methylphenyl)sulfonyl]quinolin-4-amine (4)
1H NMR (800 MHz, DMSO-d6) δ 9.39 (s, 1H, H-2), 8.13–8.11 (m, 2H, H-5, H-8), 7.92–7.89 (m, 1H,
H-7), 7.82–7.80 (m, 2H, H-2’, H-6’), 7.69–7.66 (m, 1H, H-6), 7.45–7.43 (m, 2H, H-3’, H-5’), 3.31 (q,
J = 7.2 Hz, 4H, NCH2), 2.39 (s, 3H, C(4’)-CH3), 0.59 (t, J = 7.2 Hz, 6H, NCH2-CH3).
HRMS (ESI+), calcd for C20H23O2N2S [M+H]+: 355.14748; found: 355.14710. delta=-1.06 ppm.
5.1.1.4. N-benzyl-3-[(4-methylphenyl)sulfonyl]quinolin-4-amine (6)
1H NMR (800 MHz, DMSO-d6) δ 8.90 (s, 1H, H-2), 8.31–8.29 (m, 1H, H-5), 7.86–7.85 (m, 1H, H-8),
7.78–7.76 (m, 1H, H-7), 7.73–7.71 (m, 2H, H-2’, H-6’), 7.65 (t, J = 5.6 Hz, 1H, C(4)-NH), 7.45–7.42
(m, 1H, H-6), 7.38–7.32 (m, 5H, H-3’, H-5’, H-3’’, H-4’’, H-5’’), 7.24–7.22 (m, 2H, H-2’, H-6’), 5.00
(d, J = 5.6 Hz, 2H, C(4)-NH-CH2), 2.37 (s, 3H, C(4’)-CH3).
HRMS (ESI+), calcd for C23H21O2N2S [M+H]+: 389.13183; found: 389.13145. delta=-0.96 ppm.
5.1.1.5. 3-(phenylsulfonyl)-4-(piperidin-1-yl)quinoline (10)
1H NMR (800 MHz, DMSO-d6) δ 9.40 (s, 1H, H-2), 8.33–8.31 (m, 1H, H-5), 8.19–8.18 (m, 1H, H-8),
7.96–7.94 (m, 1H, H-7), 7.87–7.85 (m, 2H, H-2’, H-6’), 7.73–7.69 (m, 2H, H-6, H-4’), 7.63–7.60 (m,
2H, H-3’, H-5’), 3.07–3.05 (m, 4H, H2-2’’, H2-6’’), 1.53–1.49 (m, 2H, H2-4’’), 1.32–1.28 (m, 4H, H2-
3’’, H2-5’’).
HRMS (ESI+), calcd for C20H21O2N2S [M+H]+: 353.13183; found: 353.13129. delta=-1.52 ppm
5.1.1.6. 4-(4-methylpiperidin-1-yl)-3-(phenylsulfonyl)quinoline (11)
1H NMR (800 MHz, DMSO-d6) δ 9.40 (s, 1H, H-2), 8.30–8.28 (m, 1H, H-5), 8.19–8.18 (m, 1H, H-8),
7.96–7.94 (m, 1H, H-7), 7.86–7.84 (m, 2H, H-2’, H-6’), 7.73–7.69 (m, 2H, H-6, H-4’), 7.63–7.60 (m,
2H, H-3’, H-5’), 3.32–3.28 (m, 2H, Hax-2’’, Hax-6’’), 2.86–2.83 (m, 2H, Heq-2’’, Heq-6’’), 1.54–1.48 (m,
1H, H-4’’), 1.39–1.36 (m, 2H, Heq-3’’, Heq-5’’), 0.88 (d, J = 6.5 Hz, 3H, C(4’’)-Me), 0.88–0.83 (m, 2H,
Hax-3’’, Hax-5’’).
HRMS (ESI+), calcd for C21H23O2N2S [M+H]+: 367.14748; found: 367.14718. delta=-0.80 ppm.
5.1.1.7. 7-chloro-3-[(3-fluorophenyl)sulfonyl]-4-(4-methylpiperidin-1-yl)quinoline (14)
1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H, H-2), 8.16 (d, J = 2.0 Hz, 1H, H-8), 8.11 (d, J = 9.2 Hz, 1H,
H-5), 7.64–7.59 (m, 2H, H-6, H-2’), 7.53–7.47 (m, 2H, H-4’, H-6’), 7.34–7.28 (m, 1H, H-5’), 3.38–

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3.32 (m, 2H, Hax-2’’, Hax-6’’), 3.08–3.03 (m, 2H, Heq-2’’, Heq-6’’), 1.63–1.57 (m, 3H, Heq-3’’, H-4’’,
Heq-5’’), 1.18–1.08 (m, 2H, Hax-3’’, Hax-5’’), 0.99 (d, J = 6.6 Hz, 3H, C(4’’)-Me).
LRMS (EI+), M+ = 418.
5.1.1.8. 7-chloro-3-[(3,5-difluorophenyl)sulfonyl]-4-(4-methylpiperidin-1-yl)quinoline (15)
1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H, H-2), 8.34 (d, J = 9.1 Hz, 1H, H-5), 8.26 (d, J = 2.2 Hz,
1H, H-8), 7.76–7.69 (m, 4H, H-6, H-2’, H-4’, H-6’), 3.35–3.27 (m, 2H, Hax-2’’, Hax-6’’), 2.93–2.87 (m,
2H, Heq-2’’, Heq-6’’), 1.62–1.51 (m, 1H, H-4’’), 1.49–1.43 (m, 2H, Heq-3’’, Heq-5’’), 0.90 (d, J = 6.5 Hz,
3H, C(4’’)-Me), 0.89–0.79 (m, 2H, Hax-3’’, Hax-5’’).
LRMS (EI+), M+ = 436.
5.1.1.9. 3-[(3,4-dichlorophenyl)sulfonyl]-6-fluoro-4-(4-methylpiperidin-1-yl)quinoline (16)
3
4
1H NMR: (400 MHz, DMSO-d6) δ 9.37 (s, 1H, H-2), 8.28 (dd, JHH = 9.3 Hz, JHF = 5.7 Hz, 1H, H-8),
8.21 (d, J = 2.2 Hz, 1H, H-2’), 8.00 (dd, 3JHF = 10.3 Hz, 4JHH = 2.7 Hz, 1H, H-5), 7.94–7.89 (m, 1H, H-
7), 7.88 (d, J = 8.4 Hz, 1H, H-5’), 7.81 (dd, J = 8.4, 2.2 Hz, 1H, H-6’), 3.39–3.30 (m, 2H, Hax-2’’, Hax-
6’’), 2.85–2.79 (m, 2H, Heq-2’’, Heq-6’’), 1.65–1.54 (m, 1H, H-4’’), 1.48–1.42 (m, 2H, Heq-3’’, Heq-5’’),
0.91 (d, J = 6.6 Hz, 3H, C(4’’)-Me), 0.85–0.73 (m, 2H, Hax-3’’, Hax-5’’).
LRMS (EI+), M+ = 452.
5.1.1.10. 6-chloro-3-[(3,4-dichlorophenyl)sulfonyl]-4-(4-methylpiperidin-1-yl)quinoline (17)
1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H, H-2), 8.24–8.19 (m, 3H, H-5, H-8, H-2’), 7.99 (dd, J =
9.0, 2.0 Hz, 1H, H-7), 7.88 (d, J = 8.5 Hz, 1H, H-5’), 7.82 (dd, J = 8.5, 2.2 Hz, 1H, H-6’), 3.35–3.28
(m, 2H, Hax-2’’, Hax-6’’), 2.92–2.86 (m, 2H, Heq-2’’, Heq-6’’), 1.65–1.52 (m, 1H, H-4’’), 1.50–1.44 (m,
2H, Heq-3’’, Heq-5’’), 0.91 (d, J = 6.7 Hz, 3H, C(4’’)-Me), 0.88–0.76 (m, 2H, Hax-3’’, Hax-5’’).
LRMS (EI+), M+ = 468.
5.1.1.11. 3-[(3-fluorophenyl)sulfonyl]-6-methyl-4-(4-methylpiperidin-1-yl)quinoline (18)
1H NMR (800 MHz, DMSO-d6) δ 9.33 (s, 1H, H-2), 8.10 (d, J = 8.6 Hz, 1H, H-8), 8.04–8.03 (m, 1H,
H-5), 7.80 (dd, J = 8.6, 1.7 Hz, 1H, H-7), 7.75–7.73 (m, 1H, H-2’), 7.69–7.64 (m, 2H, H-5’, H-6’),
7.60–7.57 (m, 1H, H-4’), 3.38–3.34 (m, 2H, Hax-2’’, Hax-6’’), 2.81–2.78 (m, 2H, Heq-2’’, Heq-6’’), 2.55
(s, 3H, C(6)-Me), 1.59–1.53 (m, 1H, H-4’’), 1.41–1.38 (m, 2H, Heq-3’’, Heq-5’’), 0.88 (d, J = 6.5 Hz,
3H, C(4’’)-Me), 0.82–0.75 (m, 2H, Hax-3’’, Hax-5’’).
HRMS (ESI+), calcd for C22H24O2N2FS [M+H]+: 399.15370; found: 399.15344. delta=-0.66 ppm.
5.1.1.12. 6-chloro-3-[(4-chlorophenyl)sulfonyl]-4-(4-methylpiperidin-1-yl)quinoline (19)

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1H NMR (500 MHz, DMSO-d6) δ 9.36 (s, 1H, H-2), 8.21 (d, J = 2.3 Hz, 1H, H-5), 8.20 (d, J = 9.0 Hz,
1H, H-8), 7.98 (dd, J = 9.0, 2.3 Hz, H-7), 7.92–7.88 (m, 2H, H-2’, H-6’), 7.71–7.67 (m, 2H, H-3’, H-
5’), 3.32–3.26 (m, 2H, Hax-2’’, Hax-6’’), 2.94–2.89 (m, 2H, Heq-2’’, Heq-6’’), 1.64–1.53 (m, 1H, H-4’’),
1.49–1.43 (m, 2H, Heq-3’’, Heq-5’’), 0.94–0.85 (m, 2H, Hax-3’’, Hax-5’’), 0.91 (d, J = 6.6 Hz, 3H, C(4’’)-
Me).
HRMS (ESI+), calcd for C21H21O2N2Cl2S [M+H]+: 435.06953; found: 435.06931. delta=-0.51 ppm.
5.1.1.13. 7-chloro-3-[(3-fluorophenyl)sulfonyl]-4-(morpholin-4-yl)quinoline (20)
1H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H, H-2), 8.38 (d, J = 9.1 Hz, 1H, H-5), 8.30 (d, J = 2.3 Hz,
1H, H-8), 7.83–7.79 (m, 2H, H-2’, H-6’), 7.77 (dd, J = 9.1, 2.3 Hz, 1H, H-6),7.73–7.67 (m, 1H, H-5’),
7.62–7.57 (m, 1H, H-4’), 3.46–3.43 (m, 4H, H2-2’’, H2-6’’), 3.11–3.07 (m, 4H, H2-3’’, H2-5’’).
HRMS (ESI+), calcd for C19H17O3N2ClFS [M+H]+: 407.06270; found: 407.06228. delta=-1.02 ppm.
5.1.1.14. 7-chloro-3-[(4-fluorophenyl)sulfonyl]-4-(morpholin-4-yl)quinoline (21)
1H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H, H-2), 8.37 (d, J = 9.1 Hz, 1H, H-5), 8.29 (d, J = 2.2 Hz,
1H, H-8), 8.05–8.00 (m, 2H, H-2’, H-6’), 7.76 (dd, J = 9.1, 2.2 Hz, 1H, H-6), 7.49–7.45 (m, 2H, H-3’,
H-5’), 3.52–3.48 (m, 4H, H2-2’’, H2-6’’), 3.11–3.06 (m, 4H, H2-3’’, H2-5’’).
HRMS (ESI+), calcd for C19H17O3N2ClFS [M+H]+: 407.06270; found: 407.06224. delta=-1.12 ppm.
5.1.1.15. (1-{7-chloro-3-[(3-fluorophenyl)sulfonyl]quinolin-4-yl}piperidin-4-yl)methanol (22)
1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H, H-2), 8.32 (d, J = 9.2 Hz, 1H, H-5), 8.25 (d, J = 2.2 Hz,
1H, H-8), 7.78–7.71 (m, 3H, H-6, H-2’, H-6’), 7.70–7.63 (m, 1H, H-5’), 7.61–7.56 (m, 1H, H-4’), 4.50
(t, J = 5.2 Hz, 1H, C(4’’)-CH2-OH), 3.33–3.25 (m, 2H, Hax-2’’, Hax-6’’), 3.25–3.21 (m, 2H, C(4’’)-CH2-
OH), 2.95–2.90 (m, 2H, Heq-2’’, Heq-6’’), 1.61–1.46 (m, 3H, Heq-3’’, H-4’’, Heq-5’’), 0.93–0.82 (m, 2H,
Hax-3’’, Hax-5’’).
LRMS (FIB+), [M+H]+ = 435.
5.1.1.16. (1-{7-chloro-3-[(4-fluorophenyl)sulfonyl]quinolin-4-yl}piperidin-4-yl)methanol (23)
1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H, H-2), 8.31 (d, J = 9.1 Hz, 1H, H-5), 8.24 (d, J = 2.1 Hz,
1H, H-8), 7.99–7.94 (m, 2H, H-2’, H-6’), 7.73 (dd, J = 9.1, 2.1 Hz, 1H, H-6), 7.48–7.42 (m, 2H, H-3’,
H-5’), 4.50 (t, J = 5.2 Hz, C(4’’)-CH2-OH), 3.36–3.24 (m, 4H, Hax-2’’, Hax-6’’, C(4’’)-CH2-OH), 2.96–
2.90 (m, 2H, Heq-2’’, Heq-6’’), 1.62–1.49 (m, 3H, Heq-3’’, H-4’’, Heq-5’’), 1.02–0.90 (m, 2H, Hax-3’’,
Hax-5’’).
LRMS (FIB+), [M+H]+ = 435.
5.1.1.17. 3-({7-chloro-4-[4-(hydroxymethyl)piperidin-1-yl]quinolin-3-yl}sulfonyl)-5-
fluorobenzonitrile (24)

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1H NMR (500 MHz, DMSO-d6) δ 9.41 (s, 1H, H-2), 8.35 (d, J = 9.2 Hz, 1H, H-5), 8.32–8.31 (m, 1H,
H-2’), 8.29–8.19 (m, 3H, H-8, H-4’, H-6’), 7.74 (dd, J = 9.2, 2.2 Hz, 1H, H-6), 4.51 (t, J = 5.2 Hz,
C(4’’)-CH2-OH), 3.36–3.26 (m, 2H, Hax-2’’, Hax-6’’), 3.24–3.21 (m, 2H, C(4’’)-CH2-OH), 2.94–2.89 (m,
2H, Heq-2’’, Heq-6’’), 1.62–1.53 (m, 1H, H-4’’), 1.53–1.48 (m, 2H, Heq-3’’, Heq-5’’), 0.80–0.72 (m, 2H,
Hax-3’’, Hax-5’’).
LRMS (EI+), M+ = 459.
5.1.1.18. (1-{7-chloro-3-[(3-chlorophenyl)sulfonyl]quinolin-4-yl}piperidin-4-yl)methanol (25)
1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H, H-2), 8.33 (d, J = 9.1 Hz, 1H, H-5), 8.26 (d, J = 2.2 Hz,
1H, H-8), 7.97–7.96 (m, 1H, H-2’), 7.81–7.78 (m, 2H, H-4’, H-6’), 7.74 (dd, J = 9.1, 2.2 Hz, 1H, H-6),
7.66–7.58 (m, 1H, H-5’), 4.51 (t, J = 5.2 Hz, 1H, C(4’’)-CH2-OH), 3.33–3.26 (m, 2H, Hax-2’’, Hax-6’’),
3.26–3.22 (m, 2H, C(4’’)-CH2-OH), 2.92–2.86 (m, 2H, Heq-2’’, Heq-6’’), 1.62–1.47 (m, 3H, Heq-3’’, H-
4’’, Heq-5’’), 0.88–0.78 (m, 2H, Hax-3’’, Hax-5’’); 13C NMR (100 MHz, DMSO-d6) δ 158.7 (C-8a), 152.0
(C-4), 150.7 (C-2), 143.6 (C-1’), 136.8 (C-7), 133.7 (C-3’), 133.0 (C-4’), 131.2 (C-5’), 129.2 (C-3),
128.5 (C-8), 127.8 (C-5, C-6), 125.6 (C-2’), 125.3 (C-4a), 124.7 (C-6’), 65.8 (C(4’’)-CH2-OH), 51.3 (C-
2’’, C-6’’), 36.9 (C-4’’), 27.7 (C-3’’, C-5’’).
LRMS (FIB+), [M+H]+ = 451.
5.2. Docking calculations
The binding mode of 25 was investigated by Induced Fit Docking. A receptor grid box with the
side length of 20 Å and centered on the ligand was generated. The initial Glide docking was
carried out with side chains trimmed automatically based on B-factors, with receptor and ligand
van der Waals scaling of 0.70 and 0.50, respectively generating 50 poses in total. Prime residue
refinement and side chain minimization were performed within 5.0 Å of ligand poses. Glide SP
was used for redocking into the top 30 receptor structures generated within 30 kcal/mol of the
best structure as obtained from the Prime refinement. The binding mode found in the best
scored mGlu 5 receptor-25 complex was analyzed.
5.3. Biological assays
5.2.1. mGlu5 binding assay
The mGlu5 receptor binding was determined according to Gasparini et al. [11] with modifications.
Rat cerebrocortical membrane preparation was used to determine the binding characteristics of
reference compounds and novel compounds to the rat mGlu5 receptor. The A18 cell line
recombinantly expressing human mGlu5a (purchased from Euroscreen) was used to determine
binding characteristics of the chemical compounds to the human mGlu5 receptor. The non-
specific binding was determined in the presence of 10 mM M-MPEP. Assays were carried out in
96-well format, the rat cortical membrane homogenates or membrane homogenates of A18
human mGlu5 receptor cells were incubated in binding buffer (50 mM Tris buffer (pH: 7.6)
supplemented with 2 mM MgCl2, 2 mM CaCl2) in the presence of radioligand [3H]M-MPEP and
drugs under investigation in a total volume of 0.3 ml for 60 minutes at 25°C. The radioligand

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displacement by the tested compounds was determined in duplicates or triplicates. For IC50 (Ki)
determinations concentration–displacement curves were generated consisting of generally 7
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 determined from the Scatchard plot.
5.2.2. Rat and human liver microsomal stability assay
In vitro metabolic stability was assessed using human (Xenotech, USA) and Wistar rat (In Vitro
Metabolism Research, Gedeon Richter Plc, Hungary) liver microsomes. Test compounds at 2.5
µM initial test concentration were incubated for various lengths of time with the liver
microsomes (0.5 mg/mL). In vitro intrinsic clearance (Clint mL/min*g liver) was calculated using
the basic concept of clearance prediction [20] according to following equations: Clint=Vmax/KM or if
S<<KM Clint=V/S; Vmax= maximal rate of enzyme reaction; KM= affinity constant of substrate
concentration; V=actual rate of enzyme reaction under first order conditions.
Estimated metabolic bioavailability (FM) was calculated according to the following equations: FM =
(1- EH) x 100; EH = CLint / (CLint + HBF); CLint = Vmax / KM or if [S] << KM CLint = V / S; EH = hepatic
extraction ratio; HBF = hepatic blood flow. As for hepatic blood flow (HBF) literature values of
1.18 mL/min/g liver for humans and 1.8 mL/min/g liver for rats have been used.
5.2.3. Vogel punished drinking conflict test in rats
The Vogel punished drinking test was performed according to Vogel et al. [21] 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. 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.
Acknowledgement
The authors are grateful to Aaron Keeley (RCNS, Hungary) for carefully reading the manuscript.
The support of the National Brain Research Program (KTIA_NAP_13-1-2013-0001) is acknowledged.
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HIGHLIGHTS
-
A novel non-acetylenic mGluR5 chemotype was discovered by high throughput
screening.
-
-
Efficient parallel synthesis methodology has been developed for library synthesis
Three regions of 4-amino-3-arylsulfoquinolines were optimized against potency and
metabolic stability
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Compounds with improved physicochemical properties and reasonable metabolic
stability showing high affinity towards mGlu5 receptors were evaluated in vivo
Further optimization around the scaffold yielded the advanced lead (25) that is
available for testing in the experimental models of CNS diseases.