Structure-Activity Relationships for Negative Allosteric mGluR5 Modulators

Article abstract of DOI:10.1002/cmdc.201100578

A series of compounds based on the mGluR5-selective ligand 2-methyl-6-(phenylethynyl)pyridine (MPEP) were designed and synthesized. The compounds were found to be either structural analogues of MPEP, substituted monomers, or dimeric analogues. All compounds retained mGluR5 selectivity with only weak or no activity at other mGluRs or iGluRs. The substituted analogue, 1,3-bis(pyridin-2-ylethynyl)benzene (19), is a potent negative modulator at mGluR5, whereas all other compounds lost potency relative to MPEP and showed that activity is highly dependent on the position of the nitrogen atom in the pyridine moieties. A homology modeling and ligand docking study was used to understand the binding mode and the observed selectivity of compound 19.

Full text of DOI:10.1002/cmdc.201100578

MED
DOI: 10.1002/cmdc.201100578
Structure–Activity Relationships for Negative Allosteric
mGluR5 Modulators
Birgitte H. Kaae,[a, b] Kasper Harpsøe,[a] Trine Kvist,[a] Jesper M. Mathiesen,[a] Christina Mølck,[a]
David Gloriam,[a] Hermogenes N. Jimenez,[c] Michelle A. Uberti,[c] Søren M. Nielsen,[d]
Birgitte Nielsen,[a] Hans Brꢀuner-Osborne,[a] Per Sauerberg,[e] Rasmus P. Clausen,*[a] and
Ulf Madsen[a]
A series of compounds based on the mGluR5-selective ligand
2-methyl-6-(phenylethynyl)pyridine (MPEP) were designed and
synthesized. The compounds were found to be either structur-
al analogues of MPEP, substituted monomers, or dimeric ana-
logues. All compounds retained mGluR5 selectivity with only
weak or no activity at other mGluRs or iGluRs. The substituted
analogue, 1,3-bis(pyridin-2-ylethynyl)benzene (19), is a potent
negative modulator at mGluR5, whereas all other compounds
lost potency relative to MPEP and showed that activity is
highly dependent on the position of the nitrogen atom in the
pyridine moieties. A homology modeling and ligand docking
study was used to understand the binding mode and the
observed selectivity of compound 19.
Introduction
(S)-Glutamate ((S)-Glu, 1; Figure 1) is the major excitatory neu-
rotransmitter in the vertebrate central nervous system (CNS).
(S)-Glu nonselectively activates ionotropic and metabotropic
glutamate receptors (iGluRs and mGluRs).[1] The G-protein-cou-
pled mGluRs consist of eight cloned subtypes (mGluR1–8),
which are divided into three classes based on pharmacology
and sequence homology. Group I includes mGluR1 and 5,
Group II includes mGluR2 and 3, and Group III includes
mGluR4, 6, 7, and 8. Since the first evidence for mGluRs was re-
ported,[2] there has been an intense search for mGluR ligands
that can discriminate between the eight subtypes. Ligands de-
veloped include orthosteric agonists and antagonists such as
compounds 2 and 3, and more recently positive and negative
allosteric modulators such as compounds 4–7 (Figure 1).[1b]
Such ligands may help elucidate the physiological role of
mGluRs in the normal and diseased states of the mammalian
brain and they have the potential for therapeutic applica-
tions.[1a,b]
Group I mGluRs are important for CNS memory, learning,
and fear conditioning.[1b,3] In particular, the mGluR5 subtype
has been implicated in numerous psychiatric disorders such as
schizophrenia, depression, and anxiety as well as neurode-
generative diseases like Parkinson’s disease.[1b,4]
Interest is growing for allosteric ligands that bind the less
conserved seven-transmembrane (TM) domain of the mGluR5
protein, for the development of selective compounds. Com-
pound 5 is a negative allosteric modulator at mGluR1, whereas
compounds 6 and 7 are selective for mGluR5. Rational struc-
[a] Dr. B. H. Kaae, Dr. K. Harpsøe, T. Kvist, Dr. J. M. Mathiesen, C. Mølck,
Dr. D. Gloriam, B. Nielsen, Prof. H. Brꢀuner-Osborne, Dr. R. P. Clausen,
Dr. U. Madsen
Department of Molecular Drug Research
Faculty of Pharmaceutical Sciences, University of Copenhagen
Universitetsparken 2, 2100 Copenhagen (Denmark)
E-mail: rac@farma.ku.dk
[b] Dr. B. H. Kaae
Radiometer Innovation, Radiometer Medical Aps
ꢁkandevej 21, 2700 Brønshøj (Denmark)
[c] H. N. Jimenez, M. A. Uberti
ST-1 Glutamate Biology, Lundbeck Research USA
215 College Road, Paramus, NJ 07652-1431 (USA)
[d] S. M. Nielsen
Molecular Pharmacology, H. Lundbeck A/S
Ottiliavej 9, 2500 Valby (Denmark)
[e] P. Sauerberg
Diabetes Protein & Peptide Chemistry, Novo Nordisk A/S
Novo Nordisk Park 1, 2760 Mꢂløv (Denmark)
Figure 1. Examples of mGluR ligands: the endogenous ligand (S)-glutamate
(1); compounds 2 and 3 are competitive ligands, and compounds 4–7 are
allosteric modulators.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100578.
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Negative Allosteric mGluR5 Modulators
ture-based design of subtype-selective allosteric modulators is
hampered by the lack of crystal structures that contain the
membrane-spanning helices. However, published rhodopsin-
based homology models[5] of mGluR5 and pharmacophores for
the allosteric binding site[6] are available. Mutational studies
have shown that amino acid residues in the same areas of the
TM helical bundles of mGluR1 and mGluR5 are important for
the binding of certain negative allosteric modulators.[6]
To investigate potency and selectivity, we decided to pre-
pare potential mGluR ligands based on the concept of bivalent
ligands. The hypothesis that the affinity of a bivalent molecule
for its target is greater than the sum of its fragments was pro-
posed by Page and Jencks in the 1970s.[7] Assuming that the
translational and rotational entropy of each fragment in a sym-
metrical dimer equals the total translational and rotational en-
tropy for the bivalent molecule, the loss of entropy will de-
crease upon joining the two fragments, leading to increased
affinity. However, the dimerization of ligands has served other
purposes such as improving pharmacokinetic properties, selec-
tivity, potency, and solubility.[8]
Scheme 1. Synthesis of compounds 8, 11–12, and 16–21 using palladium-
catalyzed Sonogashira cross-couplings: a) PdCl2(PPh3)2, CuI, Et3N, CH2Cl2; 8
(96%), 11 (87%), 12 (89%), 16 (95%), 18 (87%), 19 (96%); b) PdCl2(PPh3)2,
CuI, Cs2CO3, DMF; 17 (97%), 20 (96%); c) PdCl2(PPh3)2, CuI, Et3N, THF; 21
(56%).
In the present study we investigated the dimerization of 2-
methyl-6-(phenylethynyl)pyridine (MPEP) analogues. Selective
inhibition of mGluR5 by MPEP has proven to be neuroprotec-
tive against N-methyl-d-aspartate (NMDA)-induced neuronal
damage,[9] and MPEP also displays anxiolytic properties.[4b]
Herein we present the synthesis and pharmacological charac-
terization of a variety of monomeric MPEP analogues, substi-
tuted monomers, and dimeric MPEP analogues. Furthermore,
we carried out homology modeling and a ligand docking
study to explain the observed selectivity toward mGluR5 over
mGluR1.
Scheme 2. Synthesis of monomeric compounds 9–10 and 13–15 using palla-
dium-catalyzed Suzuki cross-couplings: a) Diisopropylamine, nBuLi, Et2O,
B(OiPr)3; b) AcOH, 2,2-dimethyl-1,3-propanediol; 28 (68%), 29 (59%), 30
(58%); c) PdCl2(PPh3)2, Cs2CO3, DMF; 9 (92%), 13 (95%), 14 (92%), 15 (71%);
d) PdCl2(PPh3)2, Cs2CO3, DME, H2O; 10 (89%).
Results and Discussion
Chemistry
Compounds 8, 11, 12, and 16–21 (Figure 2) were synthesized
according to Scheme 1 using palladium(0)-catalyzed Sonoga-
shira cross-coupling reactions. The reactions were optimized
by using different bases and solvents. The electronic properties
of the electrophile, nucleophile, and palladium catalyst were
used to optimize and increase the yield of the reactions. In
general, PdCl2(PPh3)2-catalyzed reactions had fewer by-prod-
ucts and gave higher yields than any of the other tested cata-
lysts. Not surprisingly, the choice of nucleophile and electro-
phile greatly influenced the outcome too. The Sonogashira
cross-coupling reactions are very sensitive to atmosphere and
care should be taken to minimize the formation of homocou-
pled by-products, which severely complicates the purification
of compounds 16–21. Three by-products 43–45 (Figure 3)
were obtained in fairly large amounts and were isolated and
tested for pharmacological activity as well.
The dimeric analogues 22–27 of compounds 13–15 were
synthesized similarly by Suzuki cross-coupling reactions
(Scheme 3). The halogenated analogues 31–36 were purified
and metalated to generate the boronic esters 37–42, which
were used for the Suzuki cross-coupling reactions.
Pharmacology
All compounds depicted in Figures 2 and 3 were pharmacolog-
ically characterized on Group I mGluR1 and mGluR5, Group II
mGluR2, and Group III mGluR4 with respect to allosteric modu-
lation. Characterization at mGluR1 was performed by an inosi-
tol phosphate (IP) turnover assay with CHO cells expressing
mGluR1.[10] Compounds 19–21, however, were assayed by mea-
surement of intracellular Ca2+ concentrations essentially as de-
scribed for mGluR5 below. CHO cells expressing mGluR2 or
mGluR4[11] were employed for characterization on mGluR
Groups II and III, respectively. The activity was measured as in-
hibition of forskolin-stimulated cAMP production. We have pre-
Monomeric compounds 9, 10, and 13–15 (Figure 2) were
synthesized by palladium-catalyzed Suzuki cross-coupling reac-
tions according to Scheme 2. In general, crude compounds
28–30 could be used for the subsequent cross-coupling reac-
tions.
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Figure 3. Isolated and characterized homodimeric by-products 43–45.
mGluR4, and mGluR5. Furthermore, the compounds were char-
acterized pharmacologically at native iGluRs on rat cortical
membranes using [3H]AMPA, [3H]KA and [3H]CGP39653 binding
assays,[13] representing 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-
yl)propanoic acid (AMPA), kainic acid (KA), and NMDA recep-
tors, respectively. No significant affinity was observed for any
of the compounds in the assays representing iGluRs. Data
obtained at mGluRs are listed in Table 1.
Scheme 3. Synthesis of dimeric compounds 22–27 using palladium-cata-
lyzed Suzuki cross-couplings: a) PdCl2(PPh3)2, CuI, Et3N, CH2Cl2; 31 (81%), 32
(82%), 33 (78%), 34 (57%), 35 (32%), 36 (63%); b) nBuLi, Et2O, B(OiPr)3
c) AcOH, 2,2-dimethyl-1,3-propanediol; 37 (91%), 38 (96%), 39 (81%), 40
(99%), 41 (98%), 42 (84%); d) PdCl2(PPh3)2, K2CO3, PPh3, 1,4-dioxane, H2O;
22 (62%), 23 (85%), 24 (78%), 25 (72%), 26 (66%), 27 (88%).
All compounds were devoid of agonist activity at mGluR1, 2,
4, and 5 at 100 mm, except for compound 19, which, interest-
ingly, is a weak agonist at mGluR4 (Table 1). All compounds
were also inactive as antagonists at 100 mm toward mGluR2
(data not listed). A few compounds displayed weak antagonist
activity at mGluR1. Some compounds displayed posi-
tive allosteric modulatory effects at mGluR4, which,
however, was no stronger than the weak effect of
MPEP[12a] and thus not characterized further. In gener-
al, the compounds maintained selectivity toward
mGluR5. However, the compounds vary significantly
and are very sensitive to the placements of hetero-
atoms. This is clearly demonstrated in comparing
compounds 19, 20, and 21, with activities spanning
almost two orders of magnitude at mGluR5. The thi-
azolyl MPEP analogues generally showed much
weaker potency than MPEP. However, compounds 11
and 12 were equipotent to corresponding MPEP ana-
logues. Among the pyridines, the 2-pyridyl analogues
showed the highest potency, with compounds 13,
16, 19, 22, and 25 being the most potent. Overall,
the bent structures, compounds 19–21 and 25–27,
had higher potency than their linear analogues, com-
pounds 16–18 and 22–24. In general, all potencies
were lower than that of MPEP, except for compound
19, which is equipotent.
The homocoupled pyridine-containing by-products
43–45 were also characterized pharmacologically and
showed only weak or no effects at mGluR5 and other
mGluRs. Interestingly, by-product 44 showed agonist
activity at mGluR5 as the only compound in the
series.
Figure 2. Synthesized and characterized compounds 8–27. Two series were investigated:
those containing 1) thiazoles and 2) pyridines. In general, series 2 displayed better solu-
bility than series 1. The pharmacological profiles are similar, and series 2 was chosen
based on solubility.
viously shown that MPEP is a weak positive allosteric modula-
tor at mGluR4,[12a] and therefore all compounds were also eval-
uated for positive allosteric modulatory effects. Activity at
Homology model of mGluR5 and putative binding mode of
compound 19
mGluR5 was evaluated by measuring changes in intracellular
Ca2+ concentrations in cells expressing human mGluR5, using
a Ca2+-sensitive fluorescent dye and a fluorescence image
plate reader (FLIPR). All compounds 8–27 and 43–45 depicted
in Figure 2 and Figure 3 were tested for agonist, antagonist,
negative, or positive modulator activity on mGluR1, mGluR2,
We constructed a homology model of the mGluR5 TM helical
bundle based on the human b2-adrenergic receptor co-crystal-
lized with an inverse agonist (PDB ID: 2RH1[14]) and the human
A2A adenosine receptor in complex with an antagonist (PDB ID:
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Negative Allosteric mGluR5 Modulators
Table 1. Pharmacological characterization of compounds 8–27 and 43–
45.
Compd
mGluR1
IC50 [mm]
(pIC50ÆSEM)
mGluR4
MPEP effect
[%][a]
mGluR5
IC50 [mm]
EC50 [mm]
(pIC50ÆSEM)
(pEC50ÆSEM)
MPEP
8
>100
>100
100Æ4.7
0.00099
0.25
(6.6Æ0.00)
1.37
(5.9Æ0.06)
2.33
(5.7Æ0.17)
0.026
(7.7Æ0.22)
0.017
(7.8Æ0.02)
0.021
(7.7Æ0.16)
0.13
(6.9Æ0.15)
0.68
(6.2Æ0.08)
0.026
(7.7Æ0.23)
1.06
(6.0Æ0.01)
0.99
(6.1Æ0.04)
0.00083
(9.0Æ0.05)
0.250
(6.6Æ0.1)
0.070
(7.2Æ0.2)
0.24
(6.6Æ0.05)
>100
1.04
>100
>100
77.0Æ12.5
9
>100
>100
>100
>100
>100
>100
À14Æ13.3
À17.8Æ11.8
À0.9Æ16.3
16.5Æ13.9
75.6Æ12.3
109Æ6.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
160
(3.8Æ0.03)
>100
134.2Æ3.3
73.9Æ14.5
69.6Æ12.4
21.6Æ15.4
61.0Æ12.0[b]
43.0Æ15.0
64.0Æ9.0
>100
>100
25
(4.6Æ0.09)
>100
>10
>100
>10
81
41.0Æ2.7
>100
(4.1Æ0.02)
>100
>100
Figure 4. Representations of the mGluR5 homology model with compound
19 docked into the putative binding site. a) The binding site is represented
as a grey transparent Connolly surface, and 19 is represented as spheres to
show the shape complementarity. b) Same view as above with truncated
helices and without the surface and spheres to more clearly show the rela-
23
24
41.6Æ2.7
>100
>100
46.8Æ10.1
(6.0Æ0.04)
0.029
(7.5Æ0.01)
1.35
(5.9Æ0.01)
0.10
(7.1Æ0.33)
0.16
25
26
27
43
44
45
6.4
(5.2Æ0.02)
28
(4.6Æ0.20)
>100
47.5Æ7.9
93.7Æ8.1
>100
>100
>100
>100
tive location of the residues discussed in the text. Residues Thr6322.61
,
Ile6513.32, Pro6553.36, Asn7475.46, Ser8097.39, and Ala8107.40 are shown as
sticks. The image was created in PyMOL.[18]
26.6Æ14.7
À9.0Æ10.4
À3.9Æ9.9
À14.2Æ10.2
78
mGluR5[5a] indicates that this is indeed the site of action. Thus,
to get an estimate of the binding site we docked compound
19 to the mGluR5 homology model and observed a pocket
with a Connolly surface that closely complements the shape of
19 (Figure 4a). The rigid nature of 19 and the shape comple-
mentarity render this a likely binding mode, and the lack of hy-
drogen bonds to the receptor may be solved by refinement of
(4.1Æ0.08)
>100
(6.8Æ0.16)
>100
0.46
(6.4Æ0.11)
>100
>100
>100
[a] Determined with MPEP at 100 mm; values represent the mean ÆSEM.
[b] EC50 [mm] (pEC50 ÆSEM): 25 (4.6Æ0.1).
the model, as potential interaction partners, Thr6322.61
,
Asn7475.46, and Ser8097.39 are found in the vicinity of the two
pyridine rings (Figure 4b).
3EML[15]). Both structures correspond to an inactive receptor
state,[16] and negative modulators are proposed to act by stabi-
lizing the inactive conformation, opposing activation.[5a] Like
many other GPCRs, mGluR5 was found to contain a cavity be-
tween the upper halves of the seven TM helices that also cor-
respond to the ligand binding sites of the two templates. Dis-
tant homology models must be interpreted with great care in
the generation of binding mode and SAR hypotheses. Map-
ping corresponding residues that have been previously shown
to be important for binding of a negative modulator in rat
mGluR5 selectivity of compound 19
To find a structural rationale that explains the selectivity of 19
for mGluR5 over mGluR1, we investigated the modulator bind-
ing site defined as all residues located within 5 ꢂ of the
docked 19 and with side chains pointing toward the modula-
tor. A comparison of the binding site residues at the sequence
level (Figure 5) shows that only four residues differ in mGluR5:
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Figure 5. Sequence alignment of the TM helices of mGluR1 and mGluR5. The red background highlights the residues of the modulator binding site defined
as residues located within 5 ꢂ of the suggested binding mode of compound 19 and side chains pointing toward the modulator.
Thr6322.61, Ile6513.32, Pro6553.36, and Ala 8107.40. In fact, the resi-
dues corresponding to Pro6553.36 and Ala8107.40 in rat mGluR5
have been shown to be important for binding and function of
the negative modulator, MPEP.[5a,17] In our binding model the
side chain of Ala8107.40 points toward TM1 (Figure 4b), but al-
lowing a small rotation of helix 7 would bring it into a position
where a valine, as in mGluR1, would sterically interfere with
the modulator. Ile6513.32 is in close van der Waals contact to 19
in our model, but the conservative change to the correspond-
ing valine in mGluR1 is not expected to significantly alter the
binding site to result in mGluR5 selectivity. Thr6322.61 and
Pro6553.36 both take part in shaping the binding site and corre-
spond respectively to proline and serine residues in mGluR1.
This represents a significant difference in the local environ-
ment of the binding site from hydrophilic to hydrophobic and
vice versa, which may likely impact modulator binding and
function.
pyridine moieties, with 2-pyridyl having the highest activity.
The bent analogues have greater activity than their linear
counterparts. All compounds retain mGluR5 selectivity with
only weak or no activity at other mGluRs or iGluRs.
In combination with the sequence alignment (Figure 5), our
mGluR5 homology model indicates that as few as three resi-
dues may be responsible for the differences in modulator IC50
values between mGluR1 and mGluR5. This has provided further
insight into the binding mode of mGluR5-modulatory agents
and gives directions for the future design of selective com-
pounds.
Experimental Section
Chemistry
All reactions involving air-sensitive reagents were performed under
an atmosphere of N2. Et2O was distilled and stored over Na. THF
was freshly distilled from Na/benzophenone under N2. Pd(PPh3)4
was freshly prepared.[19] ZnCl2 was flame-dried in vacuo. Commer-
cially available nBuLi and Grignard reagents were titrated prior to
reaction.[20] Column chromatography (CC) was performed using
Merck silica (0.063–0.200 mm), whereas Merck silica (0.035–
0.070 mm) or silica 60A (20–45 mm) were used for flash chromatog-
raphy (FC) and columns for CombiFlash purification (CF). Com-
Based on our binding model we predict that Thr6322.61
,
Pro6553.36, and/or Ala8107.40 could be responsible for the selec-
tivity of 19 for mGluR5 over mGluR1. In support of this,
Pagano et al. showed previously that Pro6553.36, Ser6583.39, and
Ala8107.40 are involved in MPEP selectivity between mGluR1
and mGluR5,[17] indicating an overlap of binding sites for MPEP
and 19. Due to differences in binding mode, Ser6583.39 is not
predicted to be involved in the binding and selectivity of 19,
but this may be the case for those compounds that are similar
in size to MPEP, such as compound 15. In addition, residues
from the second extracellular loop (ECL2) could contribute to
the observed selectivity for all compounds presented herein.
pounds were visualized on TLC plates (Merck silica gel 60 F254
)
using UV light, KMnO4, or CeMo spraying reagents. Melting points
were measured on a Bꢃchi melting point B-545 apparatus or on an
SRS Optimelt apparatus in open capillaries and are uncorrected.
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded
on a Varian Gemini spectrometer, a Varian Mercury spectrometer,
or on a Bruker DRX-300 instrument. 1H NMR (400 MHz), 13C NMR
(100 MHz), COSY and Fluoro-Eretic spectra were recorded on a
Bruker DRX-400 instrument. When CDCl3 was employed as solvent
TMS or CDCl3 were used as internal standards. For other solvents
the solvent residual peak was used as internal standard. Chemical
shifts (d) are expressed in ppm and coupling constants (J) in hertz.
Conclusions
We have designed and synthesized a series of ligands based
on the selective mGluR5 negative modulator, MPEP. The com-
pounds are either structural analogues of MPEP, substituted
monomers, or dimeric analogues. This led to the potent
mGluR5-selective compound 19. All other compounds are less
potent than MPEP and showed that the activity is highly de-
pendent on the position of the nitrogen heteroatom in the
GC–MS was performed on either a Shimadzu QP5050A instrument
using EI or CI, or on an Agilent 6890 GC system connected to an
Agilent 5973 network mass-selective detector using EI, API-ES+, or
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ChemMedChem 2012, 7, 440 – 451

Negative Allosteric mGluR5 Modulators
CI. GC was carried out by direct inlet on a Chrompack gas chroma-
tograph CP 9001 system.
Mikro Kemi AB, Seminariegatan, Uppsala, Sweden; or by J. Theiner,
Department of Physical Chemistry, University of Vienna, Austria.
Analytical HPLC was performed on one of the following systems:
1) An HPLC system consisting of a Jasco 880 pump, a Rheodyne
7125 injector equipped with a 100 mL Rheodyne loop, a TSP
AS3000 autosampler, and a TSP UV100 detector set at the desired
wavelength. The system was controlled by a computer using TSP
PC100 software.
2-(Phenylethynyl)thiazole (8): 2-Bromothiazole (1.78 mL, 20 mmol)
was dissolved in dry CH2Cl2 (40 mL). CuI (40 mg, 0.2 mmol), PdCl2-
(PPh3)2 (145 mg, 0.2 mmol), Et3N (20 mL), and phenylacetylene
(3.29 mL, 30 mmol) were added, and the reaction mixture was
heated at reflux for 5 h. The mixture was diluted with Et2O (60 mL),
filtered and evaporated. FC (CH2Cl2/MeOH gradient (0–5%)) afford-
ed 8 as a red–brown oil, which was further purified by Kugel Rohr
distillation (2.8 mbar, 1558C) to yield the target compound as a
yellow oil that crystallizes upon standing (3.56 g, 19.2 mmol, 96%);
2) An HPLC system consisting of a Dionex Ultimate 3000 pump, a
Rheodyne 7125 injector equipped with a 100 mL Rheodyne loop, a
TSP AS3000 autosampler, and a Dionex Ultimate 3000 photodiode
array detector set at the desired wavelength. The system was con-
trolled by a computer using Chromeleon version 6.80.
1
mp: <358C; H NMR (CDCl3): d=7.33–7.41 (m, 4H), 7.61 (m, 2H),
7.87 ppm (d, J=3.23 Hz, 1H); 13C NMR (CDCl3): d=82.70, 91.50,
121.44, 123.23, 130.00, 130.23, 131.85, 137.29, 144.10 ppm; Anal.
CHN for C11H7NS.
3) A Gilson HPLC system consisting of a 402 syringe pump
(500 mL), and a 234 autoinjector. The flow was maintained at 0.7–
1.5 mLminÀ1, depending on the column, by two 306 pumps con-
nected to an 811c dynamic mixer, of which one was via an 805
manometric module. Four valvemates secured injection of sample
onto the desired column. Signals were detected by a UV/Vis 155
detector. The system was controlled by a computer using Unipoint
version 3.30.
2-(Pyridin-2-ylethynyl)thiazole (9): 2-Bromothiazole (0.05 mL,
0.5 mmol) was dissolved in DMF (2 mL) and PdCl2(PPh3)2 (17 mg,
0.03 mmol) was added. The mixture was stirred for 15 min at RT
before the addition of 28 (161 mg, 0.75 mmol), Cs2CO3 (489 mg,
1.5 mmol) and DMF (2 mL). The reaction mixture was heated at
808C for 5 h. H2O (15 mL) and EtOAc (15 mL) were added and the
phases separated. The aqueous phase was further extracted with
EtOAc (2ꢄ10 mL). The combined organic phases were washed
with H2O (5 mL), 1m NaOH (2ꢄ10 mL) and H2O (10 mL), and after-
ward dried (Na2SO4). Evaporation afforded crude 9. FC (CH2Cl2/Et3N
(5%)/MeOH gradient (0–5%)) gave 9 as brown crystals, which were
dried in vacuo (86 mg, 92%); mp: 72.4–73.38C; 1H NMR (CDCl3/
TMS): d=7.32 (ddd, J=2.0, 4.5, 7.5 Hz, 1H), 7.45 (d, J=3.5 Hz, 1H),
7.62 (td, J=1.2, 6.0 Hz, 1H), 7.73 (dt, J=3.0, 7.5 Hz, 1H), 7.91 (d,
J=3.5 Hz, 1H), 8.66 ppm (d, J=7.5 Hz, 1H); 13C NMR (CDCl3/TMS):
d=81.5, 92.4, 121.7, 123.8, 127.8, 136.4, 142.0, 143.9, 149.9,
150.4 ppm; MS (EI) 186; Anal. CHN for C10H6N2S.
A number of columns were used: Waters Xterra MS C18 (50 mmꢄ
3 mm, 5 mm), Supelcosil Lichrosorb SI-60 (150 mmꢄ3.2 mm, 5 mm),
Kromasil SI-60 (250 mmꢄ4.6 mm, 10 mm), one of the following
Phenomenex columns: Luna C18 (150 mmꢄ4.6 mm, 5 mm), Gemini
C6-phenyl 110 (50 mmꢄ3 mm, 5 mm).
Preparative HPLC was carried out on a Gilson HPLC system consist-
ing of a 5 mL Rheodyne loop, a 215 liquid handler, and an 819 in-
jection valve actuator connected to the column via a prime/purge
valve and a injection valve. The flow was maintained between 7
and 50 mLminÀ1, depending on the column, by four 306 pumps
connected (one via an 806 manometric module) to an 811c dy-
namic mixer. A UV/Vis 155 detector and a fraction collector secured
collection of fractions. The system was controlled by a computer
using Unipoint version 3.30.
2-(Pyridin-3-ylethynyl)thiazole (10): 2-Bromothiazole (0.05 mL,
0.5 mmol) was dissolved in DME (2 mL) and PdCl2(PPh3)2 (17 mg,
0.03 mmol) was added. The mixture was stirred for 15 min at RT
before the addition of 29 (161 mg, 0.75 mmol), Cs2CO3 (489 mg,
1.5 mmol) and H2O (2 mL). The reaction mixture was heated at
808C for 14 h. H2O (15 mL) and EtOAc (15 mL) were added and the
phases separated. The aqueous phase was further extracted with
EtOAc (2ꢄ10 mL). The combined organic phases were washed
with H2O (5 mL), 1m NaOH (2ꢄ10 mL) and H2O (10 mL), and after-
ward dried (Na2SO4). Evaporation afforded crude 10. Preparative
HPLC (H2O/MeCN gradient/0.1% TFA) using a Luna column C18(2)
(250 mmꢄ21.2 mm, 5 mm) yielded 10. TFA residing from LC–MS
was removed by dissolving 10 in MeOH and run through a SPE-PL-
HCO3 (MP) tube. Compound 10 was obtained as red–brown crys-
tals, which were dried in vacuo (82 mg, 89%); mp>2008C (dec);
1H NMR (CDCl3/TMS): d=7.34 (dd, J=2.8, 6.1 Hz, 1H), 7.44 (d, J=
6.9 Hz, 1H), 7.85–7.92 (m, 2H), 8.62 (d, J=6.2 Hz, 1H), 8.84 ppm (d,
J=0.8 Hz, 1H); 13C NMR (CDCl3/TMS): d=76.6, 87.3, 121.7, 123.0,
126.0, 143.4, 143.8, 145.0, 146.6, 160.2 ppm; Anal. CHN for
C10H6N2S.
Analytical LC–MS was carried out on an Agilent 1100 apparatus
consisting of a gradient pump, an isocratic pump 3, a 1100 diode
array detector, and a mass spectrometer, a 1100 valve 1 G1160A
12 ps 13 pt, a 1100 valve 2 G1158 2 ps 6 pt, an analog digital con-
verter, and a 1100 dual-loop autosampler ps1. One of the following
columns was used: Supelcosil Lichrosorb SI-60 (150 mmꢄ3.2 mm,
5 mm), Waters Xterra MS C18 (50 mmꢄ3.0 mm, 5 mm), Phenomenex
Luna (150 mmꢄ4.6 mm, 5 mm), or Phenomenex Gemini C6-phenyl
110 (50 mmꢄ3 mm, 5 mm). Fractions were reported on the basis of
either UV or MS (M, M+1, or M+23).
Preparative LC–MS was performed on an Agilent 1100 apparatus
consisting of a gradient preparative pump, an isocratic pump 3, a
1100 diode array detector, and a mass spectrometer, a 1100 valve 1
G1160A 12 ps 13 pt, a 1100 valve 2 G1158 2 ps 6 pt, an analog digi-
tal converter, a 1100 dual-loop autosampler ps1, and four 1100
fraction collectors. Fractions were collected on the basis of either
UV or MS (M, M+1, or M+23).
1,3-Bis(thiazol-2-ylethynyl)benzene
(11):
2-Bromothiazole
(0.90 mL, 10 mmol) was dissolved in dry CH2Cl2 (20 mL). CuI
(20 mg, 0.1 mmol), PdCl2(PPh3)2 (75 mg, 0.1 mmol), Et3N (10 mL),
and 1,3-diethynylbenzene (0.90 mL, 6.8 mmol) were added, and
the reaction mixture was held at reflux for three days. The mixture
was diluted with Et2O (50 mL), filtered and evaporated. CC using
CH2Cl2/MeOH gradient (0–5%) afforded 11 as light-brown crystals,
which were recrystallized from EtOAc/heptane (1.34 g, 92%); mp:
110.2–114.68C (dec). Preparative HPLC (H2O/MeCN gradient/0.1%
HRMS was performed at the Mass Spectrometry Research Unit, De-
partment of Chemistry, University of Copenhagen, Denmark. Micro-
analyses of tested compounds agree with theoretical values
Æ0.4%, and were carried out at the Analytical Research Depart-
ment, H. Lundbeck A/S, Denmark; at Mikroanalytisk Afd. by Birgitta
Kegel, Department of Chemistry, University of Copenhagen; by
ChemMedChem 2012, 7, 440 – 451
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
445

R. P. Clausen et al.
MED
TFA) using a Luna C18(2) column (250ꢄ21.2 mm, 5 mm) gave 11.
TFA residing from HPLC was removed by dissolving 11 in MeOH
and run through a SPE-PL-HCO3 (MP) tube. Compound 11 was ob-
tained as off-white powder-like crystals, (1.27 g, 87%); mp: 113.8–
4-(Phenylethynyl)pyridyl (15): Iodobenzene (0.22 mL, 2.0 mmol)
was dissolved in DMF (5 mL) and PdCl2(PPh3)2 (27 mg, 0.04 mmol)
was added. The mixture was stirred for 15 min before the addition
of 30 (633 mg, 2.9 mmol), Cs2CO3 (2.24 g, 6.9 mmol) and DMF
(5 mL). The reaction mixture was heated at 508C for 72 h. H2O
(50 mL) and EtOAc (50 mL) were added and the phases separated.
The aqueous phase was further extracted with EtOAc (2ꢄ30 mL).
The combined organic phases were washed with H2O (25 mL), 1m
NaOH (20 mL), and H2O (20 mL). Drying (Na2SO4) and evaporation
afforded crude 15. Preparative LC–MS (H2O (0.1% TFA)/MeCN gra-
dient) using a Waters Xterra Prep RP18 column (150 mmꢄ30 mm,
10 mm) afforded 15 when fractions were collected on the basis of
m/z=179. TFA residing from LC–MS was removed by dissolving 15
in MeOH and run through a SPE-PL-HCO3 (MP) tube. Compound
1
115.28C (dec); H NMR (CDCl3/TMS): d=7.48 (t, J=7.0 Hz, 1H), 7.55
(d, J=3.2 Hz, 2H), 7.68 (d, J=7.0 Hz, 2H), 7.87 (s, 1H), 8.05 ppm (d,
J=3.2 Hz, 2H); 13C NMR (CDCl3/TMS): d=83.1, 92.5, 121.1, 122.3,
128.9, 132.8, 135.0, 143.8, 148.4; Anal. CHN for C16H8N2S2.
1,4-Bis(thiazol-2-ylethynyl)benzene
(12):
2-Bromothiazole
(0.90 mL, 10 mmol) was dissolved in dry CH2Cl2 (20 mL). CuI
(20 mg, 0.1 mmol), PdCl2(PPh3)2 (75 mg, 0.1 mmol), Et3N (10 mL),
and 1,4-diethynylbenzene (0.90 mL, 7.0 mmol) were added, and
the reaction mixture was held at reflux for three days. The mixture
was diluted with Et2O (65 mL), filtered and evaporated. FC using
CH2Cl2/MeOH gradient (0–5%) afforded 12. Compound 12 was
dried in vacuo and obtained as brown crystals (1.33 g, 91%); mp:
189–1938C (dec). HPLC (heptane/EtOH gradient) using a Supelcosil
LC-SI column (250 mmꢄ21.2 mm, 5 mm) yielded 12, as yellow crys-
tals (1.29 g, 89%); mp: 191–1938C; 1H NMR (CDCl3/TMS): d=7.44
(d, J=3.9 Hz, 2H), 7.59 (s, 4H), 7.89 ppm (d, J=3.9 Hz, 2H);
13C NMR (CDCl3/TMS): d=84.5, 93.1, 121.2, 122.5, 132.0, 143.8,
1
15 was dried in vacuo (249 mg, 71%); H NMR (CD3CN): d=7.48–
7.57 (m, 3H), 7.69 (td, J=0.9, 4.4 Hz, 2H), 7.83 (d, J=7.4 Hz, 2H),
8.72 ppm (d, J=7.3 Hz, 2H); 13C NMR (CDCl3/TMS): d=77.7, 88.3,
124.0, 130.9, 131.8, 133.5, 135.2, 141.8, 147.3 ppm; HRMS (ESP+)
calcd: 180.0813 (C13H10N M+1), found: 180.0817; Anal. CHN for
C13H9N.
1,4-Bis(pyridin-2-ylethynyl)benzene (16): PdCl2(PPh3)2 (319 mg,
0.46 mmol) and CuI (87 mg, 0.46 mmol) were suspended in dry
CH2Cl2 (75 mL) and 1,4-diiodobenzene (3.0 g, 9.1 mmol) was added.
The mixture was stirred for 5 min at RT before the addition of 2-
ethynylpyridine (2.39 mL, 23.6 mmol) and Et3N (75 mL). The reac-
tion mixture was stirred overnight at RT and terminated by evapo-
ration to dryness. CF with a heptane/EtOAc gradient afforded 16
as dark-red crystals that turn dark brown overnight (2.52 g, 99%).
Preparative HPLC (H2O/MeCN gradient/0.1% TFA) using a Waters
Xterra Prep RP18 (150 mmꢄ30 mm, 10 mm) gave 16 as light-brown
crystals, which were dried in vacuo (2.42 g, 95%); mp>2008C
148.4; MS (EI) 292.0 ppm [M]+; Anal. CHN for C16H8N2S2·1= H2O.
3
2-(Phenylethynyl)pyridyl (13): Iodobenzene (0.06 mL, 102 mg,
0.6 mmol) was dissolved in DMF (2 mL) and PdCl2(PPh3)2 (17 mg,
0.025 mmol) was added. The mixture was stirred for 15 min at RT
before the addition of 28 (161 mg, 0.75 mmol) Cs2CO3 (489 mg,
1.5 mmol) and DMF (2 mL). The reaction mixture was heated at
808C for 3 h. H2O (15 mL) and EtOAc (15 mL) were added and the
phases separated. The aqueous phase was further extracted with
EtOAc (2ꢄ10 mL). The combined organic phases were washed
with H2O (5 mL), 1m NaOH (2ꢄ10 mL) and H2O (10 mL) and after-
ward dried (Na2SO4). Evaporation afforded crude 13. Preparative
LC–MS (H2O (0.1% TFA)/MeCN gradient) using a Waters Xterra Prep
RP18 column (150 mmꢄ30 mm, 10 mm) afforded 13 when fractions
were collected on the basis of m/z=179. TFA residing from LC–MS
was removed by dissolving 13 in MeOH and run through a SPE-PL-
HCO3 (MP) tube. Compound 13 was dried in vacuo, (85 mg, 95%);
1H NMR (CDCl3/TMS): d=7.20 (t, J=7.0 Hz, 1H), 7.33–7.36 (m, 3H),
7.51 (d, J=7.5 Hz, 1H), 7.59–7.67 (m, 3H), 8.61 ppm (d, J=7.0 Hz,
1H); 13C NMR (CDCl3/TMS): d=88.6, 89.2, 122.2, 122.8, 127.1, 128.4,
128.8, 132.0, 136.2, 143.4, 150.0 ppm; MS (API-ES+) 180.1 [M+1]+;
Anal. CHN for C13H9N.
1
(dec); H NMR (CD3CN): d=7.76 (s, 4H), 7.79 (t, J=7.1 Hz, 2H), 7.96
(d, J=7.5 Hz, 2H), 8.30 (dt, J=1.7, 8.1 Hz, 2H), 8.77 ppm (d, J=
4.1 Hz, 2H); 13C NMR ([D6]DMSO): d=72.5, 73.5, 120.7, 124.3, 130.6,
135.6, 139.8, 144.9, 148.7 ppm; MS (CI) 280; HRMS (ESP+) calcd:
281.1079 (C20H13N2 M+1), found: 281.1070; Anal. CHN for
C20H12N2·3TFA·1.8H2O.
1,4-Bis(pyridin-3-ylethynyl)benzene (17): 1,4-Diethynylbenzene
(300 mg, 2.4 mmol), 3-iodopyridine (1.71 g, 8.3 mmol), PdCl2(PPh3)2
(33 mg, 0.05 mmol), Cs2CO3 (2.71 g, 8.35 mmol), and CuI (23 mg,
0.12 mmol) were suspended in dry DMF (6 mL) and the reaction
mixture was stirred at 508C for two days. H2O (30 mL) and EtOAc
(50 mL) were added and the phases separated. The aqueous phase
was further extracted with EtOAc (2ꢄ30 mL). The combined organ-
ic phases were washed with H2O (30 mL), 1m NaOH (2ꢄ30 mL),
and H2O (30 mL). Drying (Na2SO4) and evaporation afforded crude
17. Preparative HPLC (H2O/MeCN gradient (0–18%)/0.1% TFA)
using a Luna C18(2) column (250 mmꢄ21.2 mm, 5 mm) gave 17.
TFA residing from HPLC was removed by dissolving 17 in MeOH
and run through a SPE-PL-HCO3 (MP) tube. Compound 17 was ob-
tained as brown crystals (647 mg, 97%); mp: 191.3–191.78C;
1H NMR (CDCl3/TMS): d=7.28–7.32 (dd, J=5.1, 7.9 Hz, 2H), 7.57 (s,
4H), 7.84 (d, J=8.0 Hz, 2H), 8.58 (d, J=5.2 Hz, 2H), 8.79 ppm (s,
2H); 13C NMR (CDCl3/TMS): d=88.0, 92.1, 120.2, 122.9, 123.1, 131.7,
138.5, 148.8, 152.3 ppm; MS (EI) 280; Anal. CHN for C20H12N2.
3-(Phenylethynyl)pyridyl (14): Iodobenzene (0.06 mL, 0.6 mmol)
was dissolved in DMF (2 mL) and PdCl2(PPh3)2 (17 mg, 0.03 mmol)
was added. The mixture was stirred for 15 min at RT before the ad-
dition of 29 (161 mg, 0.75 mmol), Cs2CO3 (489 mg, 1.5 mmol) and
DMF (2 mL). The reaction mixture was heated at 808C for 5 h. H2O
(15 mL) and Et2O (15 mL) were added and the phases separated.
The aqueous phase was further extracted with Et2O (2ꢄ10 mL).
The combined organic phases were washed with H2O (5 mL), 1m
NaOH (10 mL), and H2O (10 mL) and afterward dried (Na2SO4).
Evaporation afforded crude 14 Preparative HPLC (H2O/MeCN gradi-
ent (0–10%)/0.1% TFA) using a Phenomenex Gemini C6-phenyl
110A (250 mmꢄ30 mm, 5 mm) afforded 14 as light-brown crystals.
TFA residing from HPLC was removed by dissolving 14 in MeOH
and run through a SPE-PL-HCO3 (MP) tube. Evaporation afforded
14 which was dried in vacuo (82 mg, 92%); 1H NMR (CDCl3/TMS):
d=7.25 (t, J=8 Hz, 1H), 7.34–7.38 (m, 3H), 7.51–7.57 (m, 2H), 7.76
(d, J=8 Hz, 1H), 8.54 (d, J=5 Hz, 1H), 8.75 ppm (s, 1H); 13C NMR
(CDCl3): d=84.9, 88.8, 121.0 122.9, 127.1, 128.4, 128.7, 133.5, 139.3,
148.0, 149.7 ppm; MS (API-ES+) 180.1 [M+1]+; Anal. CHN for
C13H9N.
1,4-Bis(pyridin-4-ylethynyl)benzene (18): PdCl2(PPh3)2 (319 mg,
0.46 mmol) and CuI (87 mg, 0.46 mmol) were suspended in dry
CH2Cl2 (75 mL) and 1,4-diiodobenzene (3.0 g, 9.1 mmol) was added.
The mixture was stirred for 5 min at RT before the addition of 4-
ethynylpyridine hydrochloride (2.63 g, 18.8 mmol) and Et3N
(75 mL). The reaction mixture was stirred for 5 h at RT and termi-
nated by evaporation to dryness to yellow–green crystals. H2O
446
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 440 – 451

Negative Allosteric mGluR5 Modulators
(50 mL) and EtOAc (50 mL) were added and the phases separated.
The aqueous phase was further extracted with EtOAc (2ꢄ30 mL).
The combined organic phases were washed with H2O (15 mL), 1m
NaOH (2ꢄ15 mL), and H2O (15 mL). Drying (Na2SO4) and evapora-
tion afforded crude 18. Preparative HPLC (H2O/MeCN gradient/
0.1% TFA) using a Luna C18(2) column (250 mmꢄ21.2 mm, 5 mm)
afforded 18. Compound 18 was dried in vacuo and obtained as
brown crystals (2.22 g, 87%); mp: 185–1878C (dec); 1H NMR
([D6]DMSO): d=7.65 (d, J=8.7 Hz, 4H), 7.71 (s, 4H), 8.74 ppm (bs,
4H); 13C NMR (CDCl3/[D6]DMSO 1:1): d=88.6, 94.3, 122.3, 126.0,
128.5, 132.1, 148.3 ppm; HRMS (ESP+) calcd: 281.1079 (C20H13N2
M+1), found: 281.1071; Anal. CHN for C20H12N2·2 TFA·1H2O.
7.5 Hz, 1H), 7.55 (d, J=7.4 Hz, 2H), 7.76 (s, 1H), 7.83 (d, J=7.6 Hz,
2H), 8.58 (d, J=5.1 Hz, 2H), 8.77 ppm (s, 2H); 13C NMR (CDCl3/
TMS): d=86.7, 91.6, 120.1, 123.0, 123.1, 128.7, 131.8, 134.7, 138.5,
148.8, 152.3 ppm; MS (EI) 280; Anal. CHN for C20H12N2.
1,3-Bis(pyridin-3-ylethynyl)benzene (20); method b: Into a vial
containing
a
degassed suspension of 1,3-diethynylbenzene
(100 mg, 1 mmol), bromobenzene (200 mg, 1 mmol), 3-iodopyri-
dine (205 mg, 1 mmol) and Et3N (5 mL, 40 mmol), CuI (20 mg,
0.1 mmol) and PdCl2(PPh3)2 (40 mg, 0.05 mmol) were added. After
stirring at RT for 16 h the mixture was filtered through Celite, and
the Celite was washed with MeOH (5 mL). Concentration in vacuo
followed by purification on an RP HPLC–MS system afforded 20
(30 mg, 10%); 1H NMR (400 MHz, CDCl3): d=7.27–7.40 (m, 3H),
7.51–7.56 (m, 2H), 7.74–7.75 (m, 1H), 7.79–7.83 (m, 2H), 8.58 (bs,
2H), 8.78 ppm (bs, 2H); MS m/z: 280.9 [M+1]+.
1,3-Bis(pyridin-2-ylethynyl)benzene (19); method a: PdCl2(PPh3)2
(80 mg, 0.11 mmol) and CuI (22 mg, 0.11 mmol) were suspended in
dry CH2Cl2 (20 mL) and 1,3-diiodobenzene (750 mg, 2.3 mmol) was
added. The mixture was stirred for 5 min at RT before the addition
of 2-ethynylpyridine (0.58 mL, 5.7 mmol) and Et3N (20 mL). The re-
action mixture was held at reflux overnight, and terminated by
evaporation to dryness. H2O (50 mL) and EtOAc (50 mL) were
added and the phases separated. The aqueous phase was further
extracted with EtOAc (2ꢄ30 mL). The combined organic phases
were washed with H2O (30 mL), 1m NaOH (2ꢄ30 mL), and H2O
(30 mL), and afterward dried (Na2SO4). CF using heptane/EtOAc
gradient (Rf(heptane/EtOAc 3:2) =0.17) afforded 19. Preparative LC–MS
(H2O (0.1% TFA)/MeCN gradient) using a Luna C18(2) column
(250 mmꢄ21.2 mm, 5 mm) afforded 19 when fractions were collect-
ed on the basis of m/z=280. Compound 19 was obtained as dark-
brown crystals (610 mg, 96%); mp: 187.6–188.98C; 1H NMR
(CD3OD): d=7.28 (t, J=6.5 Hz, 2H), 7.39 (t, J=7.5 Hz, 1H), 7.55 (d,
J=6.0 Hz, 2H), 7.59 (d, J=7.5 Hz, 2H), 7.69 (t, J=7.1 Hz, 2H), 7.80
(s, 1H), 8.69 ppm (d, J=4.9 Hz, 2H); 13C NMR (CD3OD): d=87.1,
90.6, 122.7, 124.7, 128.7, 129.6, 133.3, 135.3, 138.3, 141.2,
148.5 ppm; GC–MS (CI) 280; HRMS (ESP+) calcd: 281.1079
1,3-Bis(pyridin-4-ylethynyl)benzene (21): PdCl2(PPh3)2 (557 mg,
0.73 mmol), CuI (151 mg, 0.73 mmol) and Et3N (8.3 mL) were sus-
pended in dry THF (20 mL). 1,3-Diethynylbenzene (1.05 mL,
7.9 mmol) and 4-bromopyridine hydrochloride (3.85 g, 19.8 mmol)
were suspended in dry THF (10 mL), and added to the former sus-
pension. The reaction mixture was stirred at RT for 1.5 h and then
heated at 508C for 4.5 h with monitoring by GC–MS. The reaction
mixture was filtered through Celite, and the Celite was washed
with EtOAc (5 mL). Et2O (15 mL) was added to the filtrate and the
HCl salt was precipitated by addition of saturated HCl in MeOH.
The HCl salt of 21 was filtered off and dried carefully under
vacuum (2.1 g, 56%); mp: >2008C (dec). Compound 21 is volatile
and should be followed by MS (API-ES+ or EI) or NMR when sub-
1
jected to heat or vacuum; H NMR (CD3OD): d=7.64 (t, J=6.8 Hz,
1H), 7.86 (dd, J=2.4, 9.2 Hz, 2H), 8.04 (s, 1H), 8.21 (d, J=9.6 Hz,
4H), 8.87 ppm (d, J=9.8 Hz, 4H); 13C NMR (CDCl3/TMS): d=87.5,
92.6, 122.7, 128.5, 128.8, 131.9, 133.0, 135.1, 149.8 ppm; HRMS
(ESP+) calcd: 281.1079 (C20H13N2 M+1), found: 281.1085 (using
MeCN proved vital for obtaining HRMS); Anal. CHN
C20H12N2·0.05PPh3O·5.5HCl.
(C20H13N2 M+1), found: 281.1070; Anal. CHN for C20H12N2·1= TFA.
2
1,3-Bis(pyridin-2-ylethynyl)benzene (19); method b: Into a vial
containing
a
degassed suspension of 1,3-diethynylbenzene
4,4’-Bis(pyridine-2-ylethynyl)biphenyl (22): K2CO3 (750 mg,
5.4 mmol) and PPh3 (33 mg, 0.11 mmol), were added to a solution
of 34 (331 mg, 1.1 mmol) in 1,4-dioxane (30 mL) and stirred for
15 min. PdCl2(PPh3)2 (76 mg, 0.11 mmol), 40 (394 mg, 1.36 mmol)
and H2O (30 mL) were added, and the reaction mixture was heated
at 808C for 14 h. H2O (100 mL) was added and the mixture was ex-
tracted with EtOAc (2ꢄ75 mL) and CH2Cl2 (2ꢄ75 mL). The com-
bined organic phases were washed with H2O (15 mL), 1m NaOH
(15 mL), and H2O (15 mL). Drying (Na2SO4) and evaporation afford-
ed crude 22. Preparative HPLC (H2O/MeCN gradient/0.1% TFA)
using a Phenomenex Gemini C6-phenyl 110A column (250 mmꢄ
30 mm, 5 mm) gave 22 as sticky brown crystals, which were dried
in vacuo (240 mg, 62%); 1H NMR (CDCl3/TMS): d=7.47 (d, J=
13.1 Hz, 2H), 7.51 (dt, J=3.6, 9.8 Hz, 2H), 7.59–7.67 (m, 2H), 7.72 (t,
J=11.9 Hz, 2H), 7.78 (d, J=13.1 Hz, 2H), 7.82–7.86 (m, 2H), 8.20 (t,
J=10.8 Hz, 2H), 8.84 ppm (t, J=5.0 Hz, 2H); 13C NMR (CDCl3/TMS):
d=83.2, 97.8, 126.6, 128.8, 132.3, 132.7, 133.8, 138.0, 142.6, 144.6,
160.1 ppm; Anal. CHN for C26H16N2·3.7TFA.
(100 mg, 1 mmol), bromobenzene (200 mg, 1 mmol), 2-iodopyri-
dine (205 mg, 1 mmol) and Et3N (5 mL, 40 mmol), CuI (20 mg,
0.1 mmol) and PdCl2(PPh3)2 (40 mg, 0.05 mmol) were added. After
stirring at RT for 16 h, the mixture was filtered through Celite, and
the Celite was washed with MeOH (5 mL). Concentration in vacuo
followed by purification on a reversed-phase liquid chromatogra-
phy/mass spectrometry (RP HPLC–MS) system afforded 19 (8 mg,
3%); 1H NMR (400 MHz, CDCl3): d=7.24–7.28 (m, 2H), 7.37 (t, J=
7.8 Hz, 1H), 7.53–7.55 (m, 2H), 7.58–7.62 (m, 2H), 7.70 (td, J=7.8,
1.8 Hz, 2H), 7.81–7.84 (m, 1H), 8.62–8.65 ppm (m, 2H); MS m/z:
280.9 [M+1]+.
1,3-Bis(pyridin-3-ylethynyl)benzene (20); method a: 1,3-Diethyn-
ylbenzene (0.32 mL 2.4 mmol), 3-iodopyridine (1.46 g, 7.1 mmol),
PdCl2(PPh3)2 (33 mg, 0.05 mmol), Cs2CO3 (2.71 g, 8.3 mmol), and CuI
(23 mg, 0.12 mmol) were suspended in dry DMF (6 mL), and the re-
action mixture was stirred at 508C for two days. H2O (50 mL) and
EtOAc (50 mL) were added and the phases separated. The aqueous
phase was further extracted with EtOAc (2ꢄ30 mL). The combined
organic phases were washed with H2O (30 mL), 1m NaOH (2ꢄ
30 mL), and H2O (30 mL). Drying (Na2SO4) and evaporation afforded
crude 20. Preparative HPLC (H2O/MeCN gradient (0–18%)/0.1%
TFA) using a Luna C18(2) column (250 mmꢄ21.20 mm, 5 mm) gave
20. TFA residing from HPLC was removed by dissolving 20 in
MeOH and run through a SPE-PL-HCO3 (MP) tube. Compound 20
was obtained as brown crystals (640 mg, 96%); mp: 66.8–67.48C;
1H NMR (CDCl3/TMS): d=7.31 (dd, J=4.9, 7.5 Hz, 2H), 7.39 (t, J=
4,4’-Bis(pyridine-3-ylethynyl)biphenyl (23): Compound 23 was
prepared as light-green crystals (358 mg, 85%); mp: 179.8–
181.08C, in analogy to the procedure described for 22. As starting
materials, 35 (361 mg, 1.18 mmol) and 41 (431 mg, 1.48 mmol)
1
were used; H NMR ([D6]DMSO): d=7.53–7.64 (m, 10H), 8.08 (d, J=
6.8 Hz, 2H), 8.54 (d, J=7.1 Hz, 2H), 8.83 ppm (s, 2H). 13C NMR
([D6]DMSO): d=69.1, 74.3, 115.7, 116.8, 120.9, 126.1, 127.4, 129.2,
131.9, 132.6, 152.0 ppm; MS (CI) 356; HRMS (ESP+) calcd: 357.1392
ChemMedChem 2012, 7, 440 – 451
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447

R. P. Clausen et al.
MED
(C26H17N2
C26H16N2·3.9TFA·2H2O.
M+1),
found:
357.1356;
Anal.
CHN
for
2H), 7.28 (dd, J=5.3, 6.7 Hz, 1H), 7.49 (d, J=6.5 Hz, 1H), 7.68 (t,
J=6.4 Hz, 1H), 8.60 ppm (d, J=4.8 Hz, 1H); 13C NMR (CDCl3): d=
21.8, 35.2, 36.7, 71.8, 77.7, 82.9, 123.9, 127.9, 136.7, 142.6,
150.2 ppm.
4,4’-Bis(pyridine-4-ylethynyl)biphenyl (24): Compound 24 was
prepared as green crystals (328 mg, 78%); mp: 184–1878C (dec), in
analogy to the procedure described for 22. As starting materials,
36 (361 mg, 1.18 mmol) and 42 (431 mg, 1.479 mmol) were used;
1H NMR ([D6]DMSO/CDCl3): d=7.64 (d, J=5.7 Hz, 4H), 7.83 (d, J=
8.1 Hz, 4H), 7.92 (d, J=8.1 Hz, 4H), 8.77 ppm (d, J=5.7 Hz, 4H);
13C NMR ([D6]DMSO/CDCl3): d=79.9, 90.5, 124.9, 126.1, 127.8, 132.3,
133.1, 142.8, 150.3 ppm; MS (EI) 356; Anal. CHN for
C26H16N2·5TFA·2H2O.
3-((5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)ethynyl)pyridyl
(29):
Compound 29 was prepared as a yellow (semi)crystalline substance
(3.20 g, 59%) in analogy to the procedure described for 28 using
3-ethynylpyridine (2.60 g, 25.2 mmol). No extra Et2O was needed
for proper stirring; 1H NMR (CDCl3): d=0.93 (s, 6H), 3.25 (s, 2H),
3.52 (s, 2H), 7.25–7.28 (m, 1H), 7.77 (d, J=7.1 Hz, 1H), 8.55 (d, J=
4.8 Hz, 1H), 8.72 ppm (s, 1H); 13C NMR (CDCl3): d=21.7, 72.3, 77.6,
81.3, 123.9, 139.9, 149.1, 152.7 ppm.
3,3’-Bis(pyridine-2-ylethynyl)biphenyl (25): Compound 25 was
prepared as green sticky crystals (257 mg, 72%); mp: 159.8–
162.38C (dec), in analogy to the procedure described for 22. As
starting materials, 31 (215 mg, 0.71 mmol) and 37 (256 mg,
0.88 mmol) were used. The crystals were hygroscopic and turned
dark brown upon standing; 1H NMR (CDCl3/TMS): d=7.47 (t, J=
7 Hz, 2H), 7.54–7.62 (m, 6H), 7.67–7.73 (m, 4H), 7.88 (s, 2H),
8.63 ppm (d, J=5 Hz, 2H); 13C NMR ([D6]DMSO): d=82.6, 98.8,
120.9, 124.5, 128.9, 129.2, 129.4, 131.2, 131.8, 138.2, 140.5, 142.5,
144.6 ppm; HRMS (ESP+) calcd: 357.1392 (C26H17N2 M+1), found:
357.1384; Anal. CHN for C26H16N2·3TFA·2H2O·0.05PPh3O.
4-((5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)ethynyl)pyridyl
(30):
Compound 30 was prepared as a dark oil (4.02 g, 58%) in analogy
to the procedure described for 28 using 4-ethynylpyridine hydro-
1
chloride (4.50 g, 32.2 mmol); H NMR (CDCl3): d=0.91 (s, 6H), 3.34
(s, 2H), 3.48 (s, 2H), 7.37 (d, J=6.2 Hz, 2H), 8.59 ppm (d, J=6.1 Hz,
2H); 13C NMR (CDCl3): d=21.7, 21.9, 32.8, 72.4, 75.1, 82.3, 86.3,
122.0, 123.8, 150.6 ppm.
2-((3-Iodophenyl)ethynyl)pyridine (31): Compound 31 (1.12 g,
81%); mp: 74.9–75.48C, was prepared as brown crystals, in analogy
to the procedure described for 34 using 1,3-diiodobenzene (1.5 g,
4.5 mmol) and 2-ethynylpyridine (0.56 mL, 5.5 mmol). The reaction
mixture was stirred at 358C overnight; 1H NMR (CDCl3/TMS): d=
7.10 (t, J=7.0 Hz, 1H), 7.31–7.38 (m, 1H), 7.51–7.59 (m, 2H), 7.66–
7.72 (m, 2H), 7.97 (s, 1H), 8.65 ppm (d, J=4.9 Hz, 1H); 13C NMR
(CDCl3/TMS): d=90.0, 95.9, 100.1, 121.8, 123.3, 129.5, 130.1, 130.9,
138.0, 138.6, 140.4, 149.0, 152.4 ppm; MS (EI) 305.
3,3’-Bis(pyridine-3-ylethynyl)biphenyl (26): Compound 26 was
prepared as light-brown crystals (225 mg, 66%); mp: 145.6–
146.38C, in analogy to the procedure described for 22. As starting
materials, 32 (293 mg, 0.96 mmol) and 38 (349 mg, 1.2 mmol) were
used. Compound 26 was purified on preparative LC–MS (H2O(0.1%
TFA)/MeCN gradient) using a Luna C18(2) (250 mmꢄ21.20 mm,
1
5 mm). Fractions were collected on the basis of m/z=356; H NMR
(CDCl3/TMS): d=7.46 (d, J=7.5 Hz, 2H), 7.53 (td, J=1.5, 7.8 Hz,
2H), 7.58–7.61 (m, 2H), 7.61–7.62 (m, 2H), 7.76 (t, J=1.2 Hz, 2H),
8.13 (td, J=1.5, 8.4 Hz, 2H), 8.61 (dd, J=1.5, 5.1 Hz, 2H), 8.89 ppm
(d, J=1.5 Hz, 2H); 13C NMR (CD3OD): d=88.2, 95.8, 124.3, 126.1,
127.2, 130.8, 132.3, 133.1, 133.9, 143.1, 143.7, 150.5, 153.8 ppm;
HRMS (ESP+) calcd: 357.1392 [M+1]+ found: 357.1375; Anal. CHN
for C26H16N2·2TFA·1H2O.
3-((3-Iodophenyl)ethynyl)pyridine (32): Compound 32 (1.52 g,
82%); mp: 84.0–84.48C, was prepared as yellow–brown crystals in
analogy to the procedure described for 34 using 1,3-diiodoben-
zene (2.0 g, 6.1 mmol) and 3-ethynylpyridine (750 mg, 7.3 mmol).
The reaction mixture was stirred at 358C overnight; 1H NMR
(CDCl3/TMS): d=7.11 (t, J=7.4 Hz, 1H), 7.27–7.30 (m, 1H), 7.49 (d,
J=7.5 Hz, 1H), 7.70 (d, J=7.6 Hz, 1H), 7.79 (d, J=7.5 Hz, 1H), 7.91
(s, 1H), 8.56 (d, J=5.3 Hz, 1H), 8.75 ppm (s, 1H); 13C NMR (CDCl3/
TMS): d=87.1, 90.7, 93.6, 119.9, 123.0, 124.5, 129.9, 130.7, 137.7,
138.4, 140.1, 148.8, 152.2 ppm; MS (EI) 305.
3,3’-Bis(pyridine-4-ylethynyl)biphenyl (27): Compound 27 was
prepared as light-green crystals (221 mg, 88%); mp: 124.0–
125.18C, in analogy to the procedure described for 27. As starting
materials, 33 (215 mg, 0.71 mmol) and 39 (256 mg, 0.881 mmol)
1
4-((3-Iodophenyl)ethynyl)pyridine (33): Compound 33 (2.15 g,
78%) was prepared as a semicrystalline substance in analogy to
the procedure described for 34 using 1,3-diiodobenzene (3.0 g,
9.1 mmol) and 4-ethynylpyridine hydrochloride (1.33 g, 9.5 mmol);
1H NMR (CDCl3/TMS): d=7.12 (t, J=7.8 Hz, 1H), 7.38 (dd, J=1.1,
5.1 Hz, 2H), 7.52 (d, J=7.2 Hz, 1H), 7.73 (td, J=0.9, 7.8 Hz, 1H),
7.92 (t, J=1.1 Hz, 1H), 8.63 ppm (dd, J=1.1, 5.1 Hz, 2H); 13C NMR
(CDCl3/TMS): d=79.4, 87.8, 116.9, 125.5, 126.2, 130.0, 131.0, 136.7,
138.2, 140.4, 149.8 ppm; MS (EI) 305.
were used; H NMR (CD3OD): d=7.42 (t, J=7.8 Hz, 2H), 7.82–7.87
(m, 4H), 8.06 (d, J=7.5 Hz, 2H), 8.22–8.28 ppm (m, 4H), 8.99 (d, J=
6 Hz, 4H); 13C NMR (CDCl3): d=86.1, 93.6, 112.0, 122.8, 128.7, 128.8,
130.5, 131.6, 139.9, 140.9, 142.9 ppm; HRMS (ESP+) calcd:
357.1392
C26H16N2·5.5TFA·3.3H2O.
[M+1]+
found:
357.1381;
Anal.
CHN
for
2-((5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)ethynyl)pyridyl (28): Di-
isopropylamine (7.71 mL, 54.9 mmol) and nBuLi (34 mL, 54.5 mmol,
1.6m in hexane) were added to dry Et2O (200 mL) and cooled to
À788C. The mixture was stirred for 20 min before B(OiPr)3
(16.5 mL, 72.0 mmol) was added. After another 5 min 2-ethynylpyri-
dine (2.74 mL, 27.2 mmol) dissolved in dry Et2O (14 mL) was added.
Extra Et2O (12 mL) was added to secure stirring. The resulting
yellow suspension was left stirring at À788C for 4 h and the cool-
ing bath was removed. After 15 min AcOH (2.50 mL) and then 2,2-
dimethyl-1,3-propanediol (7.49 g, 72.0 mmol) were added. The re-
action mixture was left stirring at RT for 3 h. CH2Cl2 (1 L) was
added, and the mixture washed with saturated NH4Cl(aq) (300 mL),
saturated NaHCO3(aq) (300 mL), and H2O (300 mL). Drying (Na2SO4),
filtration and evaporation of solvent afforded 28 as a brown oil
2-((4-Iodophenyl)ethynyl)pyridine (34): PdCl2(PPh3)2 (0319 mg,
0.46 mmol) and CuI (87 mg, 0.46 mmol) were suspended in dry
CH2Cl2 (75 mL) and 1,4-diiodobenzene (3.0 g, 9.1 mmol) was added.
The mixture was stirred for 5 min at RT before the addition of 2-
ethynylpyridine (1.10 mL, 11 mmol) and Et3N (75 mL). The reaction
mixture was stirred overnight at RT and terminated by evaporation
to dryness. CF (heptane/EtOAc gradient) afforded pure 34 as light-
brown crystals, which were dried in vacuo (1.55 g, 57%); mp: 82.5–
83.58C, 1H NMR (CDCl3/TMS): d=7.30–7.35 (m, 3H), 7.53 (d, J=
7.5 Hz, 1H), 7.67–7.74 (m, 3H), 8.63 ppm (d, J=6.3 Hz, 1H);
13C NMR (CDCl3/TMS): d=88.4, 89.8, 95.2, 121.7, 123.0, 127.2, 133.5,
136.3, 137.6, 143.1, 150.1 ppm; MS (EI) 305.
1
(3.97 g, 68%); H NMR (CDCl3): d=0.94 (s, 6H), 3.22 (s, 2H), 3.54 (s,
448
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ChemMedChem 2012, 7, 440 – 451

Negative Allosteric mGluR5 Modulators
3-((4-Iodophenyl)ethynyl)pyridine (35): Compound 35 (887 mg,
32%); mp: 134.0–134.58C, was prepared as yellow crystals, in anal-
ogy to the procedure described for 34 using 1,4-diiodobenzene
(3.0 g, 9.1 mmol) and 3-ethynylpyridine (1.13 g, 10.9 mmol);
1H NMR (CDCl3/TMS): d=7.23–7.32 (m, 3H), 7.72 (d, J=6.4 Hz, 2H),
7.82 (d, J=8.0 Hz, 1H), 8.58 (d, J=4.9 Hz, 1H), 8.78 ppm (s, 1H);
13C NMR (CDCl3/TMS): d=87.2, 91.8, 94.9, 116.9, 122.0, 123.1, 133.1,
137.7, 138.6, 148.6, 152.0 ppm; MS (EI) 305.
4-((4-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (42): Compound 42 (257 mg, 84%) was prepared as a dark-
brown semicrystalline substance in analogy to the procedure de-
1
scribed for 40 using 36 (320 mg, 1.05 mmol); H NMR (CDCl3/TMS):
d=1.03 (s, 6H), 3.78 (s, 4H), 7.37–7.39 (m, 2H), 7.53 (d, J=8.1 Hz,
2H), 7.81 (d, J=8.6 Hz, 2H), 8.61 ppm (bs, 2H); 13C NMR (CDCl3/
TMS): d=21.8, 35.5, 72.0, 72.4, 77.2, 124.0, 125.6, 130.9, 133.3,
133.8, 137.7, 149.7 ppm.
4-((4-Iodophenyl)ethynyl)pyridine (36): Compound 36 (1.75 g,
63%) was prepared as sticky yellow–brown crystals in analogy to
the procedure described for 34 using 1,4-diiodobenzene (3.0 g,
9.1 mmol) and 4-ethynylpyridine hydrochloride (1.33 g, 9.5 mmol);
1H NMR (CDCl3): d=7.31 (d, J=6.4 Hz, 2H), 7.62 (d, J=4.8 Hz, 2H),
7.69 (d, J=6.5 Hz, 2H), 8.25 ppm (d, J=4.8 Hz, 2H), 13C NMR
(CDCl3/TMS): d=87.9, 93.0, 95.5, 121.6, 125.5, 126.2, 133.3, 137.7,
149.8 ppm; MS (EI) 305.
1,4-Di(pyridine-2-yl)buta-1,3-diyne (43): Purified as a by-product
of other palladium-catalyzed cross-couplings. 2-Bromothiazole
(0.9 mL, 10 mmol), was dissolved in dry CH2Cl2 (20 mL). CuI (20 mg,
0.1 mmol), PdCl2(PPh3)2 (72 mg, 0.1 mmol), Et3N (10 mL), and 2-
ethynylpyridine (1.51 mL, 15 mmol) were added, and the reaction
mixture was held at reflux for 16 h. The mixture was diluted with
EtOAc (60 mL). The mixture was washed with H2O (100 mL). The
EtOAc phase was filtered and evaporated. CF (CH2Cl2/Et3N/MeOH
gradient 95:5:0 to 95:5:5) afforded 43 in the first fraction, and 9 in
the second fraction obtained. The fractions were evaporated and
dried in vacuo to afford 43 (597 mg, 39%) as light-brown crystals.
Compound 9 was obtained in 42% with data in agreement with
earlier described. 43: 1H NMR (CDCl3/TMS): d=7.25–7.29 (m, 2H),
7.49 (d, J=7.5 Hz, 2H), 7.67 (dt, J=2.9, 7.5 Hz, 2H), 8.6 ppm (d, J=
8.0 Hz, 2H) 13C NMR (CDCl3/TMS): d=77.2, 82.7, 123.4, 127.5, 136.2,
142.4, 150.1 ppm; MS (EI) 204 [M]+; Anal. CHN for C14H8N2.
2-((3-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (37): Compound 37 (352 mg, 91%) was prepared as a red–
brown semicrystalline substance in analogy to the procedure de-
1
scribed for 40 using 31 (300 mg, 0.98 mmol); H NMR (CDCl3/TMS):
d=1.02 (s, 6H), 3.79 (s, 4H), 7.28–733 (m, 1H), 7.37 (d, J=6.2 Hz,
1H), 7.54 (d, J=8.1 Hz, 1H), 7.65–7.75 (m, 2H), 7.80 (d, J=7.9 Hz,
1H), 8.07 (s, 1H), 8.63 ppm (d, J=5.0 Hz, 1H).
3-((3-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (38): Compound 38 (352 mg, 96%) was prepared as a red
semicrystalline substance in analogy to the procedure described
1,4-Di(pyridine-3-yl)buta-1,3-diyne (44): Purified as a by-product
of other palladium-catalyzed cross-couplings. 4,4’-Dibromobiphenyl
(700 mg, 2.2 mmol) was dissolved in dry CH2Cl2 (20 mL). CuI
(21 mg, 0.1 mmol), PdCl2(PPh3)2 (79 mg, 0.1 mmol), Et3N (20 mL),
and 3-ethynylpyridine (810 mg, 7.9 mmol) were added, and the re-
action mixture was held at reflux for 14 h. The mixture was diluted
with EtOAc (60 mL), Et2O (15 mL) was added to precipitate PPh3O
and filtered. The mixture was washed with H2O (100 mL). The or-
ganic phase was dried (MgSO4) and evaporated. CF (heptane/Et3N/
EtOAc 95:5:0 to 95:5:5) afforded 44 as well as unreacted 4,4’-di-
bromobiphenyl. The fractions holding 44 were evaporated and
dried in vacuo to afford 44 as light-grey crystals (589 mg, 73%);
mp: 157.0–157.98C; 1H NMR (CDCl3/TMS): d=7.30 (dd, J=4.4,
8.0 Hz, 2H), 7.83 (td, J=1.8, 7.4 Hz, 2H), 8.61 (dd, J=2.1, 7.1 Hz,
2H), 8.78 ppm (d, J=1.8 Hz, 2H); 13C NMR (CDCl3/TMS): d=76.6,
79.2, 118.9, 123.2, 139.4, 149.7, 153.2 ppm; MS (EI) 204; HRMS
(ESP+) calcd: 205.0766 (C14H9N2 M+1), found: 205.0765; Anal.
CHN for C14H8N2·3TFA·2.5H2O.
1
for 40 using a solution of 32 (386 mg, 1.27 mmol); H NMR (CDCl3/
TMS): d=0.98 (s, 3H), 104 (s, 3H), 3.59 (s, 2H), 3.78 (s, 2H), 7.39 (t,
J=7.0 Hz, 1H), 7.60–7.67 (m, 2H), 7.86 (d, J=7.9 Hz, 1H), 8.02 (s,
1H), 8.15 (d, J=7.2 Hz, 1H), 8.60 (d, J=4.8 Hz, 1H), 8.77 ppm (s,
1H).
4-((3-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (39): Compound 39 (295 mg, 81%) was prepared in analogy
to the procedure described for 40 using 33 (383 mg, 1.26 mmol);
1H NMR (CDCl3/TMS): d=1.04 (s, 6H), 3.79 (s, 4H), 7.35–7.40 (m,
3H), 7.60 (d, J=7.6 Hz, 1H), 7.81 (d, J=7.6 Hz, 1H), 8.01 (s, 1H),
8.59 ppm (d, J=6.1 Hz, 2H); 13C NMR (CDCl3/TMS): d=21.9, 31.9,
72.0, 72.4, 77.7, 121.4, 125.6, 127.7, 128.5, 131.8, 133.8, 134.5, 137.6,
149.7 ppm.
2-((4-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (40): A solution of 34 (350 mg, 1.15 mmol) in dry Et2O (10 mL)
was cooled to À788C. nBuLi (1.54 mL, 2.3 mmol, 1.49m in hexane)
was added dropwise and the mixture was left stirring for 10 min
before addition of B(OiPr)3 (0.70 mL, 3.0 mmol) dissolved in dry
Et2O (6 mL). After 3.5 h at À788C the cooling bath was removed
and after 15 min AcOH (0.011 mL) and then 2,2-dimethyl-1,3-pro-
panediol (299 mg, 2.9 mmol) were added. The reaction mixture
was stirred overnight at RT. CH2Cl2 (50 mL) was added and the mix-
ture washed with saturated NH4Cl(aq) (15 mL), saturated NaHCO3(aq)
(15 mL) and H2O (15 mL). Drying (Na2SO4), filtration and evapora-
1,4-Di(pyridine-4-yl)buta-1,3-diyne (45): Purified as a by-product
of other palladium-catalyzed cross-couplings. 2,5-Dibromothiazole
(250 mg, 1.0 mmol), 4-ethynylpyridine hydrochloride (359 mg,
2.6 mmol), Pd(PtBu3)2 (21 mg, 0.04 mmol), Pd2(dba)3 (38 mg,
0.04 mmol), and CuI (18 mg, 0.09 mmol) were dissolved in 1,4-diox-
ane (5 mL). Cy2NMe (2.2 mL, 10.3 mmol) was added and the reac-
tion mixture was heated at 608C for two days. The mixture was di-
luted with Et2O (3 mL), and filtered through Decalite. The Decalite
was washed with cold EtOAc (10 mL). The combined organic fil-
trates were concentrated in vacuo to afford crude product. CF
(heptane/Et3N/EtOAc 95:5:0 to 95:5:10) afforded 45 as well as
number of unidentified products in smaller amounts. The fractions
holding 45 were evaporated, and dried in vacuo to afford 45 as
brown crystals (64 mg, 24%); mp: >2008C (dec); 1H NMR
([D6]DMSO): d=7.60–7.68 (m, 4H), 8.75 ppm (bs, 4H); 13C NMR
([D6]DMSO): d=77.2, 80.2, 126.2, 150.0 ppm; MS (EI) 204; HRMS
(ESP+) calcd: 205.0766 (C14H9N2 M+1), found: 205.0765; Anal.
CHN for C14H8N2·0.5HCl.
1
tion afforded 40 (331 mg, 99%); H NMR (CDCl3/TMS): d=1.03 (s,
6H), 3.79 (s, 4H), 7.24–7.29 (m, 1H), 7.35–7.39 (m, 1H), 7.52 (d, J=
6.9 Hz, 1H), 7.58 (d, J=7.0 Hz, 1H), 7.63–7.71 (m, 1H), 7.78 (d, J=
7.0 Hz, 2H), 8.63 ppm (d, J=5.5 Hz, 1H).
3-((4-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)ethynyl)pyri-
dine (41): Compound 41 (361 mg, 98%) was prepared in analogy
to the procedure described for 40 using 35 (386 mg, 1.27 mmol);
1H NMR (CDCl3/TMS): d=1.05 (s, 6H), 3.79 (s, 4H), 7.27–7.31 (m,
1H), 7.54 (d, J=7.4, 2H), 7.72 (d, J=7.9 Hz, 1H), 7.76–7.83 (m, 2H),
8.53 (d, J=5.3 Hz, 1H), 8.78 ppm (m, 1H).
ChemMedChem 2012, 7, 440 – 451
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449

R. P. Clausen et al.
MED
30 min at RT followed by measurement on a NOVOstar instrument
(BMG Labtech). Test compounds were diluted in HEPES buffer and
added automatically after baseline measurements. For antagonist
testing cells were incubated with the test compound diluted in
HEPES buffer supplemented with 2.5 mm probenecid for 30 min at
RT before measurement by NOVOstar. Cells were stimulated using
15 mm Glu, corresponding to EC80, diluted in HEPES buffer. Glu was
added automatically after baseline measurements. For the fluores-
cent measurements on the NOVOstar instrument an excitation
filter at 485 nm and an emission filter at 520 nm were used. Data
originate from three independent experiments (duplicate measure-
ments).
Pharmacology
Materials: All cell culture reagents were obtained from Gibco
unless otherwise stated.
Inhibition of forskolin-stimulated cAMP production: CHO cells
expressing mGluR2 or mGluR4 (previously described by Tanabe
et al.)[11] were cultured in DMEM with GlutaMAX-I with 10% dia-
lyzed FBS, 1% penicillin–streptomycin, and l-proline (2.5 gLÀ1) at
5% CO2 at 378C. Cells were seeded at 26000 cells per well (96-well
plate) 24 h before assaying. Cells were washed with DPBS prior to
compound addition. For agonist testing cells were first incubated
with cAMP-ground buffer (DPBS with 1 mm CaCl2, 1 mm MgCl2 and
1 mm IBMX) for 20 min at 378C. The buffer was then replaced with
test compounds diluted in cAMP-ground buffer supplemented
with 25 mm forskolin and incubated at 378C for 10 min. For antago-
nist testing cells were first incubated with test compounds diluted
in cAMP-ground buffer for 20 min at 378C, and then replaced with
cAMP-ground buffer containing test compounds, 25 mm forskolin,
and l-glutamate corresponding to the EC80 (20 mm for mGluR2,
30 mm for mGluR4) for 10 min at 378C. All reactions were terminat-
ed by aspiration and addition of ice-cold sodium acetate buffer
pH 6.2 supplemented with 0.1% Triton X-100 and 0.1 mm IBMX.
cAMP levels were quantified using the Adenylyl Cyclase Activation
FlashPlate Assay (PerkinElmer) and interpolated from a cAMP stan-
dard curve.
Modeling
Protein sequence alignment of the TM helices of Family A–C
GPCRs and homology model of mGluR5
mGluR5 belongs to Family C of GPCRs, and because there are no
crystal structures available of the TM region of this family, we de-
cided to use two Family A GPCR crystal structures as templates for
construction of our homology model. Mutational studies have indi-
cated that the binding site for negative allosteric mGluR5 modula-
tors is located between the upper halves of the seven TM helices[5a]
and thus an alignment of only the helices was used for model
building. In both templates the second extracellular loop (ECL2)
between TM4 and 5 is, to some extent, involved in ligand binding,
but significantly different in structure and length. However, ECL2
of mGluR5 does not match any of the templates in length or se-
quence, making modeling of this loop extremely difficult. There-
fore, we decided to exclude ECL2 from homology modeling. The
two other extracellular loops (between TM2 and 3 and between
TM6 and 7) are shorter than in the templates and were included to
constrain the distances between the helices in the mGluR5 model.
To obtain a reliable sequence alignment between the TM helices of
mGluR5 and the two templates, an alignment was created of all
Family A, B, and C GPCRs as described below.
Inositol phosphate (IP) turnover assay: CHO cells expressing the
mGluR1 (previously described by Aramori and Nakanishi)[10] were
cultured as described in the cAMP assay above. Cells were seeded
at 26000 cells per well (96-well plate) and labeled with myo-[2-
3H]inositol (4 mCimLÀ1, TRK911, GE Healthcare) 24 h before assay-
ing. Prior to compound addition cells were washed with DPBS. For
agonist testing cells were first incubated with IP-ground buffer
(HBSS containing 20 mm HEPES, 1 mm CaCl2, 1 mm MgCl2 and
0.85 mgmLÀ1 LiCl, pH 7.4) for 30 min at 378C. The buffer was then
replaced with test compounds diluted in IP-ground buffer and in-
cubated at 378C for 30 min. For antagonist testing cells were first
incubated with test compounds diluted in IP-ground buffer for
30 min at 378C, and then replaced with IP-ground buffer contain-
ing test compounds and 30 mm l-glutamate corresponding to the
EC80 for 30 min at 378C. All reactions were terminated by aspira-
tion and addition of ice-cold 10 mm formic acid and incubation for
30 min at 48C. Yttrium silicate scintillation proximity assay beads
(RPNQ0010, GE Healthcare) were used for measuring radioactivity
from generated [3H]IP as previously described (Brandish et al.).[21]
Radioactivity was quantified in a Packard TopCount microplate
scintillation counter and responses were read as counts per minute
(CPM). All experiments were performed in triplicate and repeated
in at least two independent experiments.
Sequences for Family A, B, and C GPCRs were retrieved from previ-
ous studies,[22] and the entry numbers given below refer hereto.
First, a joint ungapped alignment was produced for the seven TM
helices of all human Family A GPCRs in MEGA4.[23] The alignment
was anchored on the conserved Family A residues used in the Bal-
lesteros–Weinstein indexing system.[24] Second, human Family B
and C GPCRs were aligned to the Family A receptor alignment,
again by anchoring on conserved residues, but to a lesser extent
those from the Ballesteros–Weinstein indexing and to a larger
extent depending on sequence conservation in a large number or
receptor homologues and orthologues.[25] For example, the TM3
alignment was anchored on the cysteine that forms the disulfide
bond to ECL2 because this residue is more conserved than R3.50
in Families B and C. Third, the alignment of the human mGluR5
(entry: P41594) to the human b2-adrenergic and adenosine A2A re-
ceptors (entries: P07550 and P29274, respectively) was extracted
from the overall alignment, and the TM2–3 and TM6–7 loops were
added in the mGluR5 sequence.
Intracellular Ca2+ measurements (test of 19–21 on mGluR1):
CHO cells expressing mGluR1 were cultured as described for the
cAMP assay. Cells were seeded at 30000 cells per well in flat clear-
bottom black microplates (96-well format) the day before the
assay. Cells were washed with DPBS before loading with dye. The
dye loading solution (Fluo-4 NW Calcium Assay Kit, F36206, Molec-
ular Probes) was prepared according to the manufacturer’s instruc-
tions by dissolving it in HEPES buffer (1ꢄ HBSS supplemented with
1 mm MgCl2, 1 mm CaCl2, 20 mm HEPES, pH 7.4) supplemented
with 2.5 mm probenecid; 50 mL dye loading solution was added to
each well. Cells were incubated with the dye for 60 min at 378C,
after which they were washed with HEPES buffer supplemented
with 2.5 mm probenecid. For agonist testing, cells were incubated
with HEPES buffer supplemented with 2.5 mm probenecid for
The human mGluR5 homology model was constructed with MOD-
ELLER,[26] version 9v8 using the human b2-adrenergic and adeno-
sine A2A receptor crystal structures as templates and the alignment
presented in the Supporting Information. The template structures
(PDB IDs: 2RH1[14] and 3EML[15]) were retrieved from the RCSB Pro-
tein Data Bank[27] (http://www.rcsb.org/), and all residues that are
not part of the TM helices were deleted. a-Helical restraints were
450
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ChemMedChem 2012, 7, 440 – 451

Negative Allosteric mGluR5 Modulators
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Acknowledgements
B.H.K. was supported by the Drug Research Academy, Faculty of
Pharmaceutical Sciences, University of Copenhagen. C.M. and
H.B.-O. were supported by the Drug Research Academy (Faculty
of Pharmaceutical Sciences, University of Copenhagen, Denmark),
the A. P. Møller Foundation for the Advancement of Medical Sci-
ences, and the Brødrene Hartmanns Fond. K.H. was supported
the Carlsberg Foundation and the Drug Research Academy. D.G.
was supported by the Alfred Benzon Foundation. J.M.M. was sup-
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Keywords: allosteric modulators · cross-coupling · mGluR5 ·
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Received: December 7, 2011
Revised: December 21, 2011
Published online on January 20, 2012
ChemMedChem 2012, 7, 440 – 451
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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