AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist: Identification, SAR and pharmacological characterization

Article abstract of DOI:10.1016/j.bmc.2014.09.033

Here we describe the identification, structure-activity relationship and the initial pharmacological characterization of AFQ056/mavoglurant, a structurally novel, non-competitive mGlu5 receptor antagonist. AFQ056/mavoglurant was identified by chemical der

Full text of DOI:10.1016/j.bmc.2014.09.033

Accepted Manuscript
AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist: Identi-
fication, SAR and pharmacological characterization
Ivo Vranesic, Silvio Ofner, Peter Josef Flor, Graeme Bilbe, Rochdi Bouhelal,
Albert Enz, Sandrine Desrayaud, Kevin McAllister, Rainer Kuhn, Fabrizio
Gasparini
PII:
S0968-0896(14)00679-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.033
BMC 11820
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 July 2014
11 September 2014
12 September 2014
Please cite this article as: Vranesic, I., Ofner, S., Flor, P.J., Bilbe, G., Bouhelal, R., Enz, A., Desrayaud, S.,
McAllister, K., Kuhn, R., Gasparini, F., AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist:
Identification, SAR and pharmacological characterization, Bioorganic & Medicinal Chemistry (2014), doi: http://
dx.doi.org/10.1016/j.bmc.2014.09.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist:
Identification, SAR and pharmacological characterization.
Ivo Vranesica,1, Silvio Ofnera,1, Peter Josef Flora,b, Graeme Bilbea,c, Rochdi Bouhelala, Albert Enza, Sandrine
Desrayauda, Kevin McAllistera, Rainer Kuhna,c and Fabrizio Gasparinia,*
aNovartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
ABSTRACT
Here we describe the identification, structure-activity relationship and the initial pharmacological
characterization of AFQ056/mavoglurant, a structurally novel, non-competitive mGlu5 receptor
antagonist. AFQ056/mavoglurant was identified by chemical derivatization of a lead compound
discovered in a HTS campaign. In vitro, AFQ056/mavoglurant had an IC50 of 30 nM in a
functional assay with human mGluR5 and was selective over the other mGluR subtypes, iGluRs
and a panel of 238 CNS relevant receptors, transporter or enzymes. In vivo,
AFQ056/mavoglurant showed an improved pharmacokinetic profile in rat and efficacy in the
stress-induced hyperthermia test in mice as compared to the prototypic mGluR5 antagonist
MPEP. The efficacy of AFQ056/mavoglurant in humans has been assessed in L-dopa induced
dyskinesia in Parkinson’s disease and Fragile X syndrome in proof of principle clinical studies.
Abbreviations
[Ca2+]i
Intracellular Ca2+ concentration
CDZ
chlordiazepoxide
95% CI
FLIPR
GABAB
L-glu
95-percent confidence interval (as obtained with best-curve fitting)
Fluorescence Imaging Plate Reader ™ (assay)
Gamma-amino butyric acid receptor, subtype B
L-glutamate
HTS
high-throughput screening
IC50 (EC50)
hmGluR5a
hmGluR1b
mGluR
P2Y2
Concentration at which half-maximal inhibition (stimulation) is observed
human metabotropic glutamate receptor, subtype 5a
human metabotropic glutamate receptor, subtype 1b
metabotropic glutamate receptor
purinoreceptor of the P2Y2 type
PI
phosphoinositol turnover (assay)
SIH
stress induced hyperthermia
s.d.
standard deviation
1Both authors contributed equally to this work
*Corresponding author. E-mail: fabrizio.gasparini@novartis.com
b PJF Current address: Faculty of Biology and Preclinical Medicine, University of Regensburg, D-93053 Regensburg, Germany.
c GB Current address: Drugs for Neglected Diseases initiative (DNDi, 15 Chemin Louis-Dunant, CH-1202 Geneva, Switzerland
d RK Current address: Evotec AG, Hamburg, Germany

1. Introduction
In the last two decades, pharmacological interventions to modulate the glutamate
neurotransmitter system have been the focus of intense drug discovery and development efforts
aimed at the identification of novel therapeutic solutions to a number of brain disorders. The first
attempts were aimed to inhibit the action of the ionotropic glutamate receptors such as the N-
Methyl-D-Aspartate receptor (NMDAR) with competitive antagonists for the therapeutic
indications stroke1 and epilepsy.2 The clinical development of antagonists acting at this
ionotropic glutamate receptor, however proved to be challenging due to either a limited efficacy
or the emergence of adverse effects that prevented their use as therapeutic agents.3 The
identification of the metabotropic glutamate receptor (mGluR) family and agents capable of
selectively modulating a sub-group or even individual subtypes gave rise to new possibilities for
pharmacological interventions aimed at treating dysfunctions of the glutamatergic
neurotransmission.4
NH2
N
N
N
N
N
1, MPEP
(Novartis)
2, SIB1757
(SIBIA)
3, SIB1893
(SIBIA)
N
N
N
F
Cl
N
N
F
N
4, ADX48621/Dipraglurant
(Addex Pharma)
5, R04917523/RG7090/Basimglurant
(Roche)
Figure 1. Selected mGluR5 antagonists
For the mGlu5 receptor subtype (mGluR5), significant progress in the understanding of its role
and potential therapeutic applications of ligands was made following the disclosure of the first
potent and selective non-competitive antagonist MPEP (1) and its precursor molecules SIB1757
(2) and SIB1893 (3).5,6 Subsequent investigations involving the use of mutated receptor
constructs revealed that MPEP and its derivatives interact with a novel binding pocket distal from
the glutamate binding site located in the transmembrane domain.7 More detailed investigations,
using site-directed mutagenesis revealed three amino acids, non-conserved between mGluR5 and
its sibling mGluR1, that are responsible for the binding and the selectivity of MPEP and its
derivatives at the mGluR5 subtype.7
Following the reporting of MPEP and its non-competitive mode of action, intense preclinical
discovery efforts involving industrial and academic research laboratories led to the identification
of a number of preclinical and clinical development candidates.8,9,10,11,12

Thanks to its overall favorable properties, MPEP proved to be a very useful tool to explore and
reveal the role of antagonizing the mGlu5 subtype in various pathologies such as inflammatory
pain, anxiety, Parkinson’s disease or Fragile X mental retardation syndrome.13
Here we describe the process which led to the identification, the initial preclinical
characterization and the selection of AFQ056/mavoglurant, a selective non-competitive
antagonist which showed efficacy in the treatment of L-dopa induced dyskinesias in Parkinson’s
disease and Fragile X mental retardation in proof of principle studies.14,15 Ongoing clinical
programs involving mGluR5 antagonists include ADX48621/Dipraglurant (4) (Addex Pharma)
(Fig. 1) for Parkinson’s disease16 and RG7090/Basimglurant (5) (Roche) for Fragile X and major
depressive disorders.17
2. Results and discussion
2.1.High throughput screening
With the aim to identify chemical scaffolds differing from the original MPEP structure, we
performed a high throughput functional screening using the human mGluR5 recombinantly
expressed in L(tk-) cells.18,19 Glutamate-evoked changes in intracellular calcium concentration
[Ca2+]i were measured using the FLIPR/Fluo-3 method and a total of 214,544 compounds were
tested in primary screening as mixtures of 10. 23 validated hits were identified to have good
selectivity (>10 fold) over the mGluR1 subtype and an endogenously expressed P2Y receptor.
Further filtering of the validated hits allowed the identification of (6) as lead compound (Figure
2).
OH
N
O
O
6
Figure 2. Chemical structure of ( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-phenylethynyl-octahydro-indole-1-carboxylic acid ethyl ester (5).
Although sharing part of the structural elements of the original lead MPEP, the newly identified
lead (6) was considered structurally sufficiently diverse owing to the presence of a tertiary
alcohol and a saturated octahydro-indole moiety, which was to be retained for a lead optimization
process.
2.2. Lead optimization/structure activity relationships
The previous work in the MPEP series did not allow us to identify a development candidate with
an appropriate combination of favorable drug-like properties. Particularly limiting were the poor
pharmacokinetic properties observed in rodents such as limited oral bioavailability and high
clearance. Therefore, the goal of the lead optimization process described here was to identify a
suitable preclinical development candidate with appropriate potency, selectivity, and physico-
chemical and drug-like properties. Three main directions for modification of the lead molecule

were defined 1) Explore the nature and the position of substituents on the aromatic ring 2)
Investigate the spacer between the aromatic ring and the octahydroindole moiety and 3)
Investigate the role of the nitrogen substituent as illustrated in Figure 3:
Triple/double/single bond
OH
Nature and position of
substituent
N
Type/size of substituent
O
O
Figure 3. Main modifications directions for exploration of SAR of compound 6
For the lead optimization phase, the in vitro pharmacological characterization was performed
with two functional assays (biochemical determination of PI levels20 and microfluorimetric
assessment of [Ca2+]i levels using a FLIPR™ device) with cells expressing recombinant human
mGluR5.18 Representatives of the new lead series displaced [3H]-M-MPEP binding to
preparations of rat brain,21 with binding affinities roughly in line with functional inhibition,
suggesting an allosteric interaction similar to MPEP. The results are reported in the Table 1.
The effect of the position of the substituent on the aromatic ring was investigated by introducing
a chlorine atom in ortho (7), meta (8) and para (9) positions. Clearly the meta position resulted in
the most active derivative with an IC50 of 0.11 µM for (8). Variation of the nature of the
substituent in the meta position showed that a methyl group (10) was more potent than the
fluorine (11) or methoxy (12) substituted derivatives. Double substitution in positions C-3 and C-
5 with fluorine (13) or methyl (14) significantly reduced the activity compared to the
corresponding monosubstituted derivatives.
To assess the role of the triple bond as spacer between the aromatic group and the octahydro-
indole moiety it was replaced with either a cis (15) or trans (16) double bond or a single bond
(17). This replacement resulted in a significant loss of potency. The exchange of the tertiary
alcohol by a methoxy group (18) led to a complete loss of activity.
The substituent R2 on the nitrogen was investigated by maintaining the 3-methyl substituent (R1)
on the aromatic ring and variation of the length and the nature of the substituent either as a
carbamate or an amide function. In the case of the carbamate derivatives introduction of a methyl
(10) instead of an ethyl substituent (19) had only a marginal effect on the potency: IC50 of 0.18
µM for (10) vs 0.25 µM for (19). The change of the carbamate function for an amide gave a
contrasting result with no activity for a methoxy-methyl (20), weak activity, 43% of inhibition at
1 µM, for an acetamide (21) and a higher activity, IC50 = 0.22 µM, with a 2-furanyl amide
substituent (22).

R1
R3
(X)
O
H
N
H
R2
O
Table 1. Antagonistic activity of derivatives produced via Scheme 1
Cpd
R1
R2
R3
X
IC50[µM] (95% CI) or % inhibition at conc [µM]
Ca2+-FLIPR
IP3 turnover
M-MPEP
binding
displacementa
6
H
OEt
H
CꢀC
0.17 (0.14,
0.20)
0.13 (0.04-0.4)
0.37 (0.27,
0.50)
68% at 10 µM
-3% at 1 µM
2 % at 10 µM
7
8
2-Cl
3-Cl
OEt
OEt
H
H
CꢀC
CꢀC
0.11 (0.07-
0.17)
0.023
62% at 10 µM
10% at 1 µM
16 % at 10 µM
95% at 1 µM
9
4-Cl
OEt
H
H
CꢀC
CꢀC
10
3-Me
OMe
0.18 (0.13-
0.25)
0.052 (0.02-
0.13)
35 % at 0.11
µM
11
12
3-F
OEt
OEt
H
H
CꢀC
0.28 (0.27-
0.29)
0.31(0.16 –
0.64)
0.21 (0.14 –
0.31)
3-OMe
CꢀC
0.26 (0.21-
0.32)
0.13 (0.05 –
0.27)
0.26 (0.18 –
0.38)
13
14
15
16
3,5-F2
3,5-Me2
H
OEt
OEt
OEt
OEt
H
H
H
H
CꢀC
CꢀC
1.4 (0.92 - 2.0) 1.52 (1.1 – 2.2)
4 (1.0 - 16)
48% at 10 µM
-15% at 1 µM
54 % at 10 µM
(Z)-C=C
(E)-C=C
53% at 10 µM
81% at 10 µM
98% at 10 µM
54% at 1 µM
H
17
18
19
H
H
OMe
OEt
OEt
H
Me
H
C-C
CꢀC
CꢀC
36% at 10 µM
-6% at 10 µM
24% at 10 µM
-6% at 10 µM
3-Me
0.25 (0.13 -
0.35)
0.073 (0.04 –
0.12)
0.072 (0.044 -
0.12)
20
21
3-Me
3-Me
CH2OMe
Me
H
H
CꢀC
CꢀC
-4% at 1 µM
43% at 1 µM
20 % at 1 µM
2-
Furanyl
0.22 (0.16 -
0.31)
0.036 (0.026 -
0.05)
22
3-Me
H
CꢀC
a[3H]3-M-MPEP binding displacement in rat brain membranes[21]
While initially the optimization was performed using the racemic mixtures, the identification of
( )-10 as the most promising candidate prompted us to investigate whether the chirality played a
role for inhibitory activity. The choice of the 3-methyl substituted ( )-10 over the closely related

and more potent 3-chloro substituted ( )-8 was made based on the lower lipophilicity and higher
solubility of ( )-10, which showed a logP of 3.7 compared to 4.8 for ( )-8. For this purpose, the
two enantiomers of the derivative 10 were synthesized as described on scheme 1.
(-)-10/AFQ056/mavoglurant, the (-)-enantiomer showed IC50 of 0.11 µM and 0.03 µM (Ca2+ and
PI-turnover) whereas the (+)-enantiomer ((+)-10) showed only 37% and 18 % inhibition at 10
µM (Table 2 and supplementary data). The preference for the (-)-enantiomeric configuration was
confirmed with the two derivatives (-)-19 and (-)-22 which showed IC50s of 0.032 and 0.035 µM,
respectively (PI-turnover assay; Table 2).
Table 2. Antagonistic activity of AFQ056, its enantiomer and two close analogues
Cpd
R1
R2
R3
X
IC50[µM] (95% CI) or % inhibition at conc [µM]
Ca2+-FLIPR
IP3 turnover
M-MPEP
binding
displacement
(-)-10
AFQ056/mavoglurant
0.11 (0.9 –
0.15)
0.03 (0.02 –
0.05)
0.047 (0.03 –
0.07)
3-Me
OMe
H
CꢀC
(+)-10
(-)-19
3-Me
3-Me
OMe
OEt
H
H
CꢀC
CꢀC
37% at 10 µM 18% at 10 µM
n.d.
0.25 (0.13 –
0.5)
0.032 (0.015 –
0.07)
0.13 (0.13 –
0.2)
(-)-22
3-Me
2-
Furanyl
H
CꢀC
0.18 (0.15 –
0.27)
0.035 (0.016 – 0.073 (0.06 –
0.08) 0.09)
The absolute configuration was determined by resolving the crystal structure of a carbamate
derivative on the 4-hydroxy of the (+)-rotating enantiomer and was shown to be (3aS,4R,7aS) for
((+)-10) and (3aR,4S,7aR) for ((-)-10) (Supplementary data, Fig S1).
2.3. In vitro pharmacological characterization
The lead optimization process delivered the compound (-)-10 as the most promising candidate
based on the activity at the mGlu5 receptor, its reduced lipophilicity and increased solubility. We
therefore determined its pharmacological profile at the mGluR subtypes, the GABAB receptor
(which is structurally somewhat related to mGluRs) and its selectivity toward a large panel of
receptors, enzymes and transporters.
2.3.1. Activity at group I mGluRs
Figure 4 illustrates the in vitro functional activity of AFQ056/mavoglurant. AFQ056/mavoglurant
fully antagonized hmGluR5-mediated responses with IC50 values of 110 and 30 nM in Ca2+-and
PI-turnover assays but did neither inhibit mGluR1-mediated responses nor showed intrinsic
agonist-like activity up to 10 µM. Additionally, AFQ056/mavoglurant was assessed for inhibition
of ATP-stimulated PI formation mediated through endogenously expressed purinoreceptors (P2Y2
type) in the hmGluR5a-expressing cells; no effect was observed in the range between 0.1 to 30
µM.
A
B

100
50
0
100
50
0
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
log([AFQ056]/M)
log([AFQ056]/M)
IC50 = 110 (90, 150) nM
IC50 = 30 (20, 50) nM
Figure 4. A. Concentration-dependence of effects of AFQ056 on calcium mobilization in L(tk-) cells stably expressing mGluR5a as
determined with the FLIPR system. Data represent percentage of amplitude of maximal L-glu-(10 µM)-induced control response (means s.d.) of
pooled independent determinations (N = 2), one performed in duplicates, one in quadruplicates. Empty symbols (ꢁ): effect of test compounds (as
indicated in top row) applied alone; filled symbols (ꢂ): effect on L-glu-induced Ca2+-mobilization; solid curves: concentration-response curves
obtained by fitting the four-parametric logistic equation to the pooled data sets. The respective IC50 values (95% CI in brackets) are given below
each panel. B. Concentration-dependence of effects of AFQ056 on PI turnover in cells expressing hmGluR5a or hmGluR1b. Data (means s.d.)
represent percentage of amplitude with respect to maximal agonist-induced control response of N = 3 pooled independent determinations, each
performed in triplicates. Filled symbols (ꢂ): effect of test compound (as indicated in top row) on quisqualate (0.3 µM)-induced PI formation in
hmGluR5a-expressing cells; empty symbols (ꢁ): effect on quisqualate (30 µM)-induced PI formation in hmGluR1b-expressing cells, data of N = 2
independent determinations performed in triplicates. Triangles (ꢃ): Effect of AFQ056 on ATP (10 µM)-induced PI formation of N = 2 pooled
determinations performed in triplicates. IC50 values (95% CI in brackets) are given below each panel.
2.3.2. Site of action
To investigate whether the new series interacts with the previously described allosteric ligand
recognition site mediating the inhibition of MPEP and its structural derivatives,7 we measured
binding displacement of [3H]-AAE327/M-MPEP, a ligand interacting at the allosteric binding
site.21 AFQ056/mavoglurant fully displaced the radioligand in a concentration-dependent manner
with an IC50 of 47 nM, showing that AFQ056 indeed interacts with the same allosteric binding
site as MPEP (Figure 5).
100
50
0
-11
-10
-9
-8
-7
-6
-5
-4
log([AFQ056]/M)

IC50 = 47 (30,72) nM
ki = 26 (17, 40) nM
Figure 5. Concentration-dependence of radioligand displacement by AFQ056 determined in rat brain membranes (mixed hippocampus and cortex).
Data (means s.d.) represent percentage of specific binding of [3H]-AAE327 (2 nM) of N = 3 pooled independent determinations, each performed
in triplicates. IC50 and ki values (95% CI in brackets) are given below.
2.3.3. Mode of action
To assess the inhibitory mode of action of AFQ056/mavoglurant, we performed a Schild-like
analysis using the functional PI turnover assay, whereby the effects of the compound at different
fixed concentrations on the concentration-response relation of L-glu were determined. In two
independent experiments, AFQ056/mavoglurant clearly reduced the response amplitude to L-glu
in a concentration-dependent manner (Figure 6A). The EC50 of L-glu slightly increased in the
presence of increasing concentrations of AFQ056/mavoglurant (from approx. 3 µM to 12 µM),
while the Hill coefficient did not vary appreciably (Figure 6B). This result indicated that
AFQ056/mavoglurant mediates inhibition at hmGluR5 through a non-competitive mode of action.
A
B
fit parameters, 95% C.I.
L-glu EC50
+AFQ056
top
bottom
nHill
(% ctr)
(% ctr)
(uM)
(nM)
none
97
78
59
24
10
8
5
3
4
9
2
-1
0
10
3.3 (2.6, 4.2) 2.3 1.4
6.0 (4.9, 7.4) 1.7 0.5
8.6 (7.5, 9.9) 1.8 0.4
10
30
5
2
2
4
100
300
2
12.0 (8.3, 16)
8.6 (1.2, 63)
2.7 2.8
1.2 2.4
-2
Figure 6. A. Effects of AFQ056 on L-glu-stimulated PI turnover in L(tk-) cells expressing hmGluR5a. The graph
illustrates one of two independent experiments yielding equivalent results. B. Data (means s.d., normalized to control)
and concentration-response curves obtained by fitting the four-parametric logistic equation (determining parameters: top,
bottom, logEC50 and Hill coefficient) to the data sets obtained in the absence (L-glu ref) and presence of AFQ056 added at
10, 30, 100 and 300 nM.
2.3.4. Selectivity
AFQ056/mavoglurant was tested for agonistic/positive modulatory and antagonistic activity at
representative subtypes of group II/III mGlu and GABAB receptors, using a GTPγ35S based
assays and did not show agonist/positive modulator activity or antagonist action at 1-10 µM at
the hmGluR2, 3, 4, 6, 7, 8 or hGABABR1/2 (Table 3 and supplementary data).
Table 3. Selectivity profile of AFQ056/mavoglurant
Targets
Activity
Selectivity vs IC50 at
hmGluR5 (IP3)
mGluR1,2,4,7
> 300
NE at 10 µM

hmGluR3,6,8 (Tested at
Euroscreen)
> 300
NE at 10 µM
NE at 10 µM
hGABABR1/2
> 300
> 300
MDS Pharma screen (238
targets)
NE at 10 µM for all targets
except for the 5HT1A receptor
75 % Inh at 10 µM (IC50 =
13.2 µM)
NE: No effect
The selectivity profile of AFQ056/mavoglurant was further assessed in a panel of 238 targets
including receptors, enzymes, ion channels and transporters. AFQ056 was tested at a single
concentration of 10 µM in duplicates. Of the 238 targets tested, AFQ056 showed a weak affinity
only to the 5HT1A receptor (75% inhibition at 10 ꢄM). Further investigation in a concentration
response mode revealed an IC50 of 13.2 µM. To the other tested targets, AFQ056 did not exhibit
any significant binding to any of the receptor sites tested up to a concentration of 10 ꢄM. Taken
together, AFQ056 showed a > 300 fold selectivity for the mGluR5 over all targets tested (Table 3
and supplementary data for list of targets).
2.4. In vivo characterization
2.4.1. Pharmacokinetics
The pharmacokinetic properties of AFQ056/mavoglurant were determined in rats after a single
intravenous (3.1 mg/kg; 10 µmol/kg) or oral (9.4 mg/kg; 30 µmol/kg) administration (Figure 7A).
For comparison, the prototypic mGluR5 antagonist MPEP was tested under similar conditions
(2.3 mg/kg; 10 µmol/kg, i.v. and 6.9 mg/kg; 30 µmol/kg, p.o.) (Figure 7B).
A
B

Figure 7: A) Concentration of AFQ056 in rat plasma and brain following i.v. drug administration (3.1 mg/kg; 10
ꢄmol/kg) (left) or p.o. (9.4 mg/kg; 30 ꢄmol/kg) (right) Limit of quantification (LOQ): < 18 pmoles/ml plasma or < 90
pmoles/g brain. B) Concentration of MPEP•HCl in rat plasma and brain following i.v. drug administration (2.3 mg/kg; 10
ꢄmol/kg) (left) or p.o. (6.9 mg/kg; 30 ꢄmol/kg) (right). LOQ: 3 pmol/ml plasma and 15 pmol/g brain. Each point represents
the mean SD of n=6 rats.
The maximum concentration of AFQ056/mavoglurant after oral administration in plasma and
brain was reached at/or before 0.25 h, indicating that AFQ056/mavoglurant readily and
significantly penetrated into the brain with a brain/plasma concentration ratio between ~ 3 and 4.
An apparent terminal half-life of 2.9 hours was estimated for the removal of orally administered
AFQ056/mavoglurant from the plasma compartment. Following intravenous administration,
AFQ056/mavoglurant was quickly taken up in the brain, as it reached Tmax at/or before 0.08 h
(Figure 7; Table 4). The bioavailability of 32 % was clearly superior to that of MPEP, which
showed only 17 %. In a liver microsomal stability assay, both molecules demonstrated a fast
clearance in the rat tissue whereas AFQ056/mavoglurant showed a significantly lower clearance
in the human microsomes compared to MPEP (see Table 3). Overall, in rats
AFQ056/mavoglurant demonstrated a superior pharmacokinetic profile after oral administration
compared to MPEP.
Dose
Cmax
(plasma/brain)
Tmax
T1/2
Clearance
Oral
BAV
B/P
Ratio
Clearance in
microsomes
Human Rat
µmol/kg;
mg/kg
pmol/ml (p)
pmol/g (b)
h
h
ml•min-1•kg-1
%
32
17
µl•min-1•mg-1
AFQ056
Oral
113
578
277
254
30 / 9.4
10 / 3.1
950/3500
ꢅ 0.25
ꢅ 0.08
2.9
3.4-3.9
2.0-4.8
Intravenous
MPEP
3330/8400
0.69
65
Oral
30 / 6.8
10 / 2.3
290/930
1
1
1.6
1.1
2.4-3.3
2.3-3.4
Intravenous
410/1300
149

Table 4: Summary of PK parameters for AFQ056/mavoglurant and MPEP after i.v. and p.o. administration to rats.
Results were determined using nonlinear curve fitting procedures (software TOPFIT;22 Non-compartmental model).
Based on the in vivo properties in rat and the in vitro metabolic stability in human microsomes for
AFQ056 we extrapolated a potential favorable PK profile in human, these predicted favorable
properties were confirmed in human studies published recently.23
2.4.2. Activity in Stress induced hyperthermia in mice
The prototypic mGluR5 antagonist MPEP showed robust anxiolytic-like effects in rodents, and
anxiety was one of the earliest therapeutic uses proposed for mGlu5 antagonists.24,25 To assess
anxiolytic-like properties of AFQ056/mavoglurant we have chosen the stress induced
hyperthermia in rodents, a test which was shown to be sensitive to the effect of mGluR5
antagonists25 and to the genetic ablation of the mGlu5 receptor.26 The study was performed in
mice with oral administration of 10/1/0.1 mg/kg as described by Spooren et al.27 and using the
benzodiazepine chlordiazepoxide (CDZ) at a dose of 10 mg/kg p.o. as a positive control.
***
***
***
***
Figure 8: Stress induced hyperthermia in mice. Individually housed mice were treated with AFQ056 at the indicated
doses, CDZ (10 mg/kg p.o.) or with vehicle (n=24 per group) and one hour later rectal temperature was measured
(T1). In control subjects, stress caused by this procedure resulted in a significant hyperthermia as measured 15
minutes later (T2). Stress-induced hyperthermia (SIH) is defined as T2-T1. Bars represent mean ( s.e.m.). Levels of
statistical significance versus the vehicle group are shown with *** p< 0.001 (Dunnett's multiple comparison of
means).
The results, shown in Figure 8, demonstrate a dose-dependent effect of AFQ056/mavoglurant on
SIH at 0.1, 1 and 10 mg/kg p.o. The efficacy of AFQ056/mavoglurant in attenuating the SIH was
comparable to the positive control CDZ and was achieved without affecting motor performance
as measured in the rotarod assay (data not shown). We attempted to determine the PK/PD
relationships by measuring the brain levels of AFQ056 following the SIH assessment (90 minutes
post dose). We could only measure brain concentrations of 780 pmol/mg and 104 pmoles/mg in
mice dosed with 10 and 1 mg/kg respectively, whereas no drug could be detected in animals
dosed at 0.1 mg/kg. Only after improvement of the sensitivity of the detection, we were able to
determine that the total brain concentration achieved after administration of the 0.1 mg/kg dose
was 8 3 pmol/mg. The observed significant inhibition of the SIH achieved with such a low
brain level can be explained with the very short half-life of AFQ056 in mice (T1/2 = 0.2 h; data
not shown) and the effect on SIH results from an action of the compound in the brain between 60

and 75’ post dose whereas the PK was measured 90’ post dose. Additionally, as demonstrated by
other mGluR5 antagonists such as CTEP, the effect on SIH can be achieved at very low brain
concentrations far below the levels needed to achieve full receptor occupancy.28
2.5. Chemistry
The examples reported have been synthesized using a linear synthesis starting with the
commercially available 1,5,6,7-Tetrahydro-indol-4-one (23) as illustrated on scheme 1.
O
O
H
O
b, c
a
N
N
H
O
N
H
O
O
O
23
24
( )-25
O
H
H
N
O
(+)-25
O
d
O
H
Me
OH
H
N
H
O
e
N
H
O
O
(-)-25
(-)-26
O
OH
g
f
OH
H
H
N
N
H
HCl
H
H
(-)-27
O
(-)-10
O
Scheme 1. Reagents and conditions: (a) BOC2, tert-BuOK, THF, reflux, 2 h, 92 %; (b) H2, Pt/C, MeOH, 1 bar, 92 h, 67 %; (c) (COCl)2,
DMSO, TEA, THF, 65 %; (d) Chromatographical separation of enantiomers on Chiralpak AD; (e) 1) BuLi, THF, - 20°C; 2) (-)-25, 0°C, 2 h, 85 %;
(f) HCl, AcOEt, RT, 6 h, 93%; (g) MeOCOCl, TEA, THF, 2 h, 90 %.
1,5,6,7-Tetrahydro-indol-4-one (23) is treated with di-tert–butyldicarbonate in the presence of
KOtBu in THF to obtain the protected tetrahydro-indolone 24. 24 is exhaustively hydrogenated
with Pt (5%) on charcoal and the resulting alcohol is oxidized with oxalyl chloride and DMSO in
THF to afford racemic 4-oxo-octahydro-indole-1-carboxylic acid tert-butyl ester ( )-25. The
racemate ( )-25 is separated into its enantiomers by chromatography on a Chiralpak AD column.
The (+) enantiomer (+)-25 and the (-) enantiomer (-)-25 are obtained with an enantiomeric purity
of 99% and 99.9% respectively.
Lithiated 1-ethynyl-3-methyl-benzene is added to (-)-25 stereoselectively to give the tertiary
alcohol (-)-26 which is deprotected with dry hydrochloride in ethyl acetate. The resulting
hydrochloride salt of octahydro-indole (-)-27 is treated with methylchloroformate in the presence
of triethylamine to afford after crystallization pure (-)-(3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-
octahydro-indole-1-carboxylic acid methyl ester ((-)-10, AFQ056/mavoglurant).

3. Summary/Conclusions
At the time this work was initiated, MPEP was the only selective mGluR5 antagonist described.5
Using MPEP as a scaffold, we conducted extensive derivatization efforts with the aim to identify
and develop suitable candidates for various therapeutic indications.29 Unfortunately, these efforts
did not succeed due to various reasons, such as preclinical safety issues or poor pharmacokinetic
properties.
In order to identify new chemical matter differing from MPEP, we conducted a HTS campaign
yielding 23 validated hits. Further filtering using molecular weight limits, functional potency and
selectivity over the sibling mGlu1 receptor subtype and the P2Y receptor allowed the
identification of the novel lead compound 6. Despite sharing some of the structural elements with
MPEP, the presence of a saturated carbocyclic element, a tertiary alcohol group and the overall
tri-dimensional structure were considered significantly different and the hit molecule 6 was
retained for a hit-to-lead program. The derivatization of 6 was conducted along three main
directions and allowed the identification of the meta substitution, a triple bond and a
methylcarbamate as the optimal combination as illustrated with the derivative ( )-10 as the best
derivative of the series considering its activity at the receptor and lower lipophilicity compared to
the 3-chloro derivative 8. Separation of the enantiomers of ( )-10 and their characterization
revealed the (-)-(3aR,4S,7aR) stereochemical conformation of the octahydro-indole as the most
active conformation as illustrated with (-)-10/AFQ056/mavoglurant.
Further in vitro characterization demonstrated that AFQ056/mavoglurant is a functional
antagonist with a potency of 30 nM (IC50 in the PI3-turnover assay) with a non-competitive mode
of action. AFQ056/mavoglurant completely displaced the binding of the allosteric binding ligand
[3H]-AAE327 demonstrating that it exerts its antagonistic action by interacting with the site
located in the transmembrane domain characterized by Pagano et al.7 This finding was very
recently confirmed with the publication of a co-crystal structure of the TM domain of mGluR5
with AFQ056 by Doré et al.30 AFQ056/mavoglurant is selective for the mGlu5 subtype without
significant functional activity at any of the other mGluR subtypes or the GABAB receptor up to a
concentration of 10 µM. Additionally, the activity of AFQ056/mavoglurant was assessed at a
large panel of receptors, transporters and enzymes and demonstrated a highly selective profile.
With the exception of weak antagonistic activity at the 5HT1A receptor (IC50 = 13.2 µM), no
activity was detected up to 10 µM at any of the other targets.
In vivo, AFQ056/mavoglurant demonstrated superior pharmacokinetic elements, such improved
bioavailability as compared to MPEP in rats, and the significantly reduced in vitro clearance in
human liver microsomes. In the stress induced hyperthermia model in mice, AFQ056
demonstrated significant reduction of the SIH after single oral administrations of 0.1 and 1 mg/kg
with an almost complete inhibition at 10 mg/kg, which was comparable to the effects of the
prototypical anxiolytic chlordiazepoxide (CDZ).
Based on the in vitro and in vivo profiles described here, AFQ056/mavoglurant was selected as
preclinical candidate, further pre-clinically developed and successfully assessed clinically in
proof-of-principle studies.14,15

4. Experimental part
4.4.General methods
4.5. General procedure for the synthesis of AFQ056/mavoglurant derivatives
4.5.1.4-Oxo-4,5,6,7-tetrahydro-indole-1-carboxylic acid tert-butyl ester (24)
1,5,6,7-Tetrahydro-indol-4-one 23 (Aldrich # 35,783-9; 38.4 g, 28.1 mmol), di-tert-
butyldicarbonate (66 g; 302 mmol) and potassium tert-butylate (6 g; 62.5 mmol) in 1 l
tetrahydrofuran are heated under reflux for 2 h. After cooling at room temperature the reaction
mixture is poured on brine (1 l) and extracted with tert.-butylmethylether (4 X 500 ml). The
combined organic phases are dried over Na2SO4, filtered and evaporated in vacuo. 51 g of a
yellowish oil are isolated and purified by column chromatography on silica gel (600 g; eluent
hexane/ethyl acetate 8:2 v/v). 30.5 g (92 %) of 24 as white crystals are isolated (mp 84-86°C).
4.5.2.( )-(3aRS,7aRS)-4-Oxo-octahydro-indole-1-carboxylic acid tert-butyl ester (( )-25 )
Hydrogenation:
A mixture of 24 (60 g; 255 mmol) and 15 g of 5% Pt on charcoal (given in three portions of 5 g
each; 24h, 48, 72h) in 1 l of methanol are hydrogenated (1 bar ) at room temperature under
stirring for 92 h. The mixture is filtered and the solvent evaporated in vacuo. The residual
brownish oil is purified by chromatography on silica gel to yield ( )-(3aRS,4SR,7aRS)-4-
hydroxy-octahydro-indole-1-carboxylic acid tert-butyl ester as a yellowish oil (41.3 g; yield =
67 %). This mixture was used without purification for the next step.
Oxidation of alcohols:
To a solution of oxalylchloride (1.54 ml; 17.6 mmol) in THF (320 ml) cooled to –60°C a solution
of DMSO (2.28 ml; 32 mmol) in THF (32 ml) is added dropwise under stirring. After 5 min a
solution of ( )-(3aRS,4SR,7aRS)-4-hydroxy-octahydro-indole-1-carboxylic acid tert-butyl ester
(3.96 g; 16.4 mmol) in THF (64 ml) is added and the reaction mixture is stirred for 100 min at –
60°C. Triethylamine (11.2 ml; 80 mmol) is added and the cooling bath removed and the reaction
mixture stirred for further 60 min. The reaction mixture is diluted with ethyl acetate (1 l) and
washed with sat. NaHCO3(150 ml). The water phase is extracted with ethyl acetate (300 ml). The
combined organic phases are dried over Na2SO4, filtered and evaporated in vacuo. The residue is
purified by column chromatography on silica gel (150 g) and the fractions containing the desired
compound are collected and evaporated in vacuo to yield ( )-25/( )-(3aRS,7aRS)-4-Oxo-
octahydro-indole-1-carboxylic acid tert-butyl ester (2.51 g; yield = 65 %).
4.5.3.(-)-(3aR,7aR)-4-Oxo-octahydro-indole-1-carboxylic acid tert-butyl ester ((-)-25)
(Separation on chiral column)
4 g of ( )-25/( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic acid tert-butyl ester are
dissolved in 200 ml of hexane-ethanol 80:20 (v/v). This solution is injected via the pump on a 5
cm by 50 cm Chiralpak AD column (Daicel Chemical Industries). The chromatography is
achieved at room temperature at a flow-rate of 100 ml/min and UV detection is performed at 210
nm. The mobile phase consists of a mixture of hexane-ethanol 80:20 (v/v). Under the applied

chromatographic conditions, the (+)-enantiomer is isolated from a first fraction collected between
11 and 18 min, and the (-)-enantiomer from a second fraction collected between 20 and 40 min.
After 6 injections of a total of 27 g of the racemate, the fractions containing the corresponding
enantiomers are combined to yield 12.6 g of the (+)-enantiomer and 12.2 g of the (-)-enantiomer,
with an enantiomeric purity of 99% and 99.9%, respectively. The enantiomeric purity is
determined on an analytical Chiralpak AD column (0.4 x 25 cm); mobile phase, hexane-ethanol
90:10 (v/v). (-)-25/(-)-(3aR,7aR)-4-oxo-octahydro-indole-1-carboxylic acid tert-butyl ester
([α]D= -111.6, ethanol, c = 0.96 %); (+)-25/(+)-(3aS,7aS)-4-oxo-octahydro-indole-1-carboxylic
acid tert-butyl ester ( [α]D= +105, ethanol, c = 1.0 %).
4.5.4.(-)-(3aR,4S,7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic acid tert-
butyl ester ((-)-26)
To a solution of 1-ethynyl-3-methyl-benzene (3.248 g; 28 mmol) in THF (168 ml) cooled to –
20°C, a solution of butyllithium (17.5 ml; 28 mmol; 1.6M in hexane) is added. The reaction
mixture is further stirred at –20°C for 2 h then a solution of (-)-25 (-)-4-oxo-octahydro-indole-1-
carboxylic acid tert-butyl ester (3.346 g; 14 mmol) in THF (70 ml) is added and the reaction
mixture further stirred at 0-5°C. After 2 h the reaction mixture is diluted with ethyl acetate (900
ml) and washed with sat. NaHCO3 (2 x 90 ml). The aqueous phase is extracted with ethyl acetate
(400 ml). The combined organic phases are dried over Na2SO4, filtered and evaporated in vacuo.
The residue is purified by column chromatography on silica gel (300 g) and the fractions
containing the desired compound are collected and evaporated in vacuo to yield (-)-26/(-)-
(3aR,4S,7aR)-4-Hydroxy-4-[2-(3-methylphenyl)ethynyl]-octahydro-1H-indole-1-carboxylic acid
tert-butyl ester (4.27 g; yield = 85 %). 1H-NMR (400 MHz; DMSO-d6): δ 7.3-7.1 (m, 4H), 5.5 (d,
J = 5 Hz, 1H), 3.85-3.65 (m, 1H), 3.35-3.25 (m, 1H), 3.25-3.1 (m, 1H), 2.6-2-45 (m, 1H), 2.28 (s,
3H), 1.9-1.4 (m, 7H), 1.36 (s, 9H), 1.13-0.98 (m, 1H). [α]D = - 21 (ethanol c = 1.15 %).
4.5.5.(-)-(3aR,4S,7aR)-4-m-Tolylethynyl-octahydro-indol-4-ol hydrochloride salt ((-)-27)
In a 1M solution of HCl in ethyl acetate (249 ml) (-)-26 (-)-(3aR,4S,7aR)-4-Hydroxy-4-m-
tolylethynyl-octahydro-indole-1-carboxylic acid tert-butyl ester (4.27 g; 12 mmol) is dissolved
and stirred at room temperature for 6 h. After completion of the hydrolysis (TLC) the solvent is
evaporated in vacuo to yield (-)-27/(-)-(3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-octahydro-
indole hydrochloride (3.39 g; yield = 93 %). m.p. = 181-183°C. 1H-NMR (400 MHz; DMSO-d6):
δ 7.28-7.14 (m, 4H), 5.94 (broad s), 3.6-3.53 (m, 1H), 3.37-3.25 (m, 1H), 3.21-3.11 (m, 1H),
2.56-2.45 (m, xH), 2.28 (s, 3H), 2.16-2.04 (m, 1H), 2.02-1.92 (m, 1H), 1.91-1.41 (m, 6H). ES-
MS (+): 256.2 [M-H2O +1]+. [α]D = -72.7 (ethanol c = 1 %).
4.5.6.(-)-(3aR,4S,7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic acid
methyl ester ((-)-10/AFQ056/mavoglurant)
Triethylamine (3.6 ml; 25.52 mmol) is added to a suspension of (-)-27/(-)-(3aR,4S,7aR)-4-
hydroxy-4-m-tolylethynyl-octahydro-indole hydrochloride (3.38 g; 11.6 mmol) in CH2Cl2 (174
ml) and the mixture is cooled to 5°C. Methylchloroformate (1.2 ml; 15.1 mmol) is added
dropwise. After completion of the addition, the cooling bath is removed and the solution stirred
for 2 h. The reaction mixture is diluted with CH2Cl2 (250 ml) and washed with brine (1 x 50 ml).
The aqueous phase is extracted with CH2Cl2 (50 ml), the combined organic phases are dried over
Na2SO4, filtered and the solvent evaporated in vacuo. The residue is column chromatographed on
silica gel (240 g), eluent toluene/acetone 9:1 v/v. The fractions containing the desired compound
are collected and evaporated in vacuo to yield 3.39 g of (-)-10/(-)-(3aR,4S,7aR)-4-hydroxy-4-m-
tolylethynyl-octahydro-indole-1-carboxylic acid methyl ester (yield = 90 %). m.p. = 110-112 °C.

1H-NMR (600 MHz; DMSO-D6): δ 7.29-7.23 (m, 2H), 7.23-7.15 (m, 2H), 5.76 (br. s., 1H), 3.82
(dddd, J = 23.7, 11.4, 6.1, 5.8 Hz, 1H), 3.45-3.37 (m, 1H), 3.55 (s, 3H), 3.31-3.2 (m, 1H), 2.63-
2.52 (m, 1H), 2.29 (s, 3H), 1.96-1.77 (m, 3H), 1.72 (d, J = 11.3 Hz, 1H), 1.66 – 1.45 (m, 3H),
1.18-1.03 (m, 1H). ES-MS (+): 314.1 [M+H]+. [α]D= -21.2 (methanol, c = 1%).
4.5.7.( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-phenylethynyl-octahydro-indole-1-carboxylic acid
ethyl ester (( )-6)
To a solution of phenyl acetylene (6.2 ml, 57 mmol) in THF (100 ml) is added a solution of n-
butyllithium in hexane (35 ml, 57 mmol) at 0 °C dropwise via a syringe. After stirring for 75 min
at 0°C a solution of 10 g of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic acid ethyl
ester31 (10 g, 47 mmol) in THF (50 ml) is added dropwise at 0 °C and the reaction mixture is
allowed to warm to room temperature. Stirring is continued for 90 minutes. The solvent is
removed in vacuo, the residue is dissolved in CH2Cl2 and washed two times with aqueous
ammonium chloride solution and with brine. The combined organic phases are dried over Na2SO4,
filtered and the solvent evaporated in vacuo. The residue is flash chromatographed on silica gel,
eluent hexane / CH2Cl2 / acetone 5:4:1 v/v. The fractions containing the desired compound are
collected and the product is crystallized from ether / hexane to afford ( )-6 ( )-
(3aRS,4SR,7aRS)-4-hydroxy-4-phenylethynyl-octahydro-indole-1-carboxylic acid ethyl ester
(yield = 56 %), m.p. = 109 – 110 °C. 1H-NMR (400 MHz, CDCl3): δ 7.42 (b, 2H), 7.33 (m, 3H),
4.15 (q, 2H), 4.0 (m, 1H), 3.55 (b, 1H), 3.40 (b, 1H), 2.63 (m, 1H), 2.05-1.1 (m, 9H), 1.25 (t, 3H).
4.5.8.( )-(3aRS,4SR,7aRS)-4-(2-Chloro-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester (( )-7)
To a solution of 1-chloro-2-ethynyl benzene (0.388 g, 2.84 mmol) in THF (5 ml) is added a
solution of n-butyllithium in hexane (1.78 ml, 2.84 mmol) at 0 °C dropwise via a syringe. After
stirring for 60 min at 0°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.5 g, 2.37 mmol) in THF (1 ml) is added dropwise at 0 °C and the reaction
mixture is allowed to warm to room temperature. Stirring is continued for 30 minutes. The
solvent is removed in vacuo, the residue is dissolved in CH2Cl2 and washed three times with
aqueous ammonium chloride solution and with brine. The combined organic phases are dried
over MgSO4, filtered and the solvent evaporated in vacuo. The residue is flash chromatographed
on silica gel, eluent hexane / ethyl acetate 2:1 v/v. The fractions containing the desired compound
are collected and the product is crystallized from hexane / ethyl acetate to afford ( )-7 ( )-
(3aRS,4SR,7aRS)-4-(2-Chloro-phenylethynyl)-4-hydroxy-octahydro-indole-1-carboxylic
acid
1
ethyl ester (yield = 47 %), m.p. = 134 – 135 °C. H-NMR (400 MHz, CDCl3): δ 7.46 (d, 1H),
7.41 (d, 1H), 7.30 – 7.18 (m, 2H), 4.12 (q, 2H), 4.10 (m, 1H), 3.55 (b, 1H), 3.40 (b, 1H), 2.65 (m,
1H), 2.1 - 1.1 (m, 9H), 1.25 (t, 3H). ES-MS (+): 348.2 [M+H]+.
4.5.9.( )-(3aRS,4SR,7aRS)-4-(3-Chloro-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester (( )-8)
To a solution of 1-chloro-3-ethynyl-benzene (0.5 g, 3.66 mmol) in THF (10 ml) is added a
solution of n-butyllithium in hexane (2.9 ml, 3.6 mmol) at -5 °C dropwise via a syringe. After
stirring for 90 min at -5°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.386 g, 1.83 mmol) in THF (1 ml) is added dropwise at -5 °C and the reaction
mixture is allowed to warm to room temperature and diluted with diethylether (150 ml). The
resulting solution is washed two times with aqueous sat NaHCO3, H2O and dried over MgSO4,
filtered and the solvent evaporated in vacuo. The residue is flash chromatographed on silica gel,

eluent hexane / Ethyl acetate 3:1 v/v. The fractions containing the desired compound are
collected and 8 is obtained as yellowish oil (Yield = 27%). The product is obtained in 27 % yield.
1H NMR (400MHz; CDCl3): δ 7.40 (m, 1H), 7.22-7.31 (m, 3H), 4.10 (m, 2H), 3.54 (m, 1H), 3.38
(m, 1H), 2.27 (m, 1H), 1.88-2.11 (m, 5H), 1.60-1.80 (m, 4H), 1.27 (t, 3H).
4.5.10. ( )-(3aRS,4SR,7aRS)-4-(4-Chloro-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester (( )-9)
To a solution of 4-chloro-phenyl acetylene (0.388 g, 2.84 mmol) in THF (5 ml) is added a
solution of n-butyllithium in hexane (1.78 ml, 2.84 mmol) at 0 °C dropwise via a syringe. After
stirring for 30 min at 0°C this solution is added dropwise to a solution of ( )-(3aRS,7aRS)-4-oxo-
octahydro-indole-1-carboxylic acid ethyl ester31 (0.5 g, 2.37 mmol) in THF (2 ml) cooled at -
78°C. After 15’ at -78°C, the reaction mixture is allowed to warm to room temperature. The
solvent is removed in vacuo, the residue is dissolved in CH2Cl2 and washed three times with
aqueous ammonium chloride solution and with brine. The combined organic phases are dried
over MgSO4, filtered and the solvent evaporated in vacuo. The residue is flash chromatographed
on silica gel, eluent hexane / ethyl acetate 1:1 v/v. The fractions containing the desired compound
are collected and the product is crystallized from hexane / ethyl acetate to afford ( )-9, mp. 108-
109 °C, yield 40 %. 1H-NMR (400 MHz, CDCl3): δ 7.35 (d, 2H), 7.30 (d, 2H), 4.12 (q, 2H), 3.97
(m, 1H), 3.55 (b, 1H), 3.40 (b, 1H), 2.62 (m, 1H), 2.1 - 1.1 (m, 9H), 1.25 (t, 3H). ES-MS (+):
348.2 [M+H]+.
4.5.11. ( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic
acid methyl ester (( )-10)
Triethylamine (0.44 ml; 3.18 mmol) is added to a suspension of ( )-(3aR,4S,7aR)-4-hydroxy-4-
m-tolylethynyl-octahydro-indole hydrochloride (0.54 g; 2.12 mmol) in CH2Cl2 (5 ml) and the
mixture is cooled at 0°C. Methylchloroformate (0.4 ml; 5.1 mmol) is added dropwise. After
completion of the addition, the cooling bath is removed and the reaction mixture allowed to warm
at room temperature. The reaction mixture was washed with a 10% solution of citric acid, brine
and dried over K2CO3. After filtration the solvent evaporated in vacuo. The residue is column
chromatographed on silica gel (240 g), eluent toluene/acetone 9:1 v/v. The fractions containing
the desired compound are collected, evaporated in vacuo and the product is crystallized from
ethyl ether / hexane to afford ( )-10, mp. 107.5- 108 °C, yield 63 %. 1H-NMR (400 MHz, CDCl3):
δ 7.26-7.07 (m, 4H), 5.26 (s, 1H), 4.09 (m, 1H), 3.94 (m, 1H), 3.65 (s, 3H), 3.52 (m, 1H), 3.36 (m,
1H), 2.68 (m, 1H), 2.3 (s, 3H), 2.14-1.61 (m, 6H), 1.07 (m, 1H). ES-MS (+): 336.2 [M+Na]+.
4.5.12. ( )-(3aRS,4SR,7aRS)-4-(3-Fluoro-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester (( )-11)
To a solution of 1-ethynyl-3-fluoro-benzene (0.18 g, 1.5 mmol) in THF (2.5 ml) is added a
solution of n-butyllithium in hexane (1.05 ml, 1.5 mmol) at -20 °C dropwise via a syringe. After
stirring for 60 min at -20°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.22 g, 1 mmol) in THF (0.5 ml) is added dropwise at -20 °C. The reaction
mixture is allowed to warm to room temperature and after 2 h diluted with diethylether. The
resulting solution is washed with aqueous sat NH4Cl, H2O and dried over MgSO4, filtered and the
solvent evaporated in vacuo. The residue is flash chromatographed on silica gel, eluent hexane /
ethyl acetate 7:1 v/v. The fractions containing the desired compound are collected and ( )-11 is

1
obtained as colorless foam (Yield = 46 %). H-NMR (400 MHz; DMSO-D6): δ 7.44-7.31 (m,
1H), 7.29-7.18 (m, 3H), 5.79 (d, J = 5 Hz, 1H), 4.05-3.88 (m, 2H), 3.85-3.74 (m, 1H), 3.43-3.17
(m, 2H), 2.62-2.47 (m, 1H), 1.92-1.76 (m, 3H), 1.73-1.65 (m, 1H), 1.64-1.44 (m, 3H), 1.17-1.01
(m, 4H). HPLC-MS: 354 (M+Na)+.
4.5.13. ( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-(3-methoxy-phenylethynyl)-octahydro-indole-1-
carboxylic acid ethyl ester (( )-12)
To a solution of 1-ethynyl-3-methoxy-benzene (0.2 g, 2 mmol) in THF (3.5 ml) is added a
solution of n-butyllithium in hexane (1.3 ml, 2.1 mmol) at -20 °C dropwise via a syringe. After
stirring for 60 min at -20°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.22 g, 1 mmol) in THF (0.5 ml) is added dropwise at -20 °C. The reaction
mixture is allowed to warm to room temperature and after 2 h diluted with diethylether. The
resulting solution is washed with aqueous sat NH4Cl, H2O and dried over MgSO4, filtered and the
solvent evaporated in vacuo. The residue is flash chromatographed on silica gel, eluent hexane /
ethyl acetate 7:1 v/v. The fractions containing the desired compound are collected and ( )-12 is
obtained as colorless gum (Yield = 64 %). 1H-NMR (400 MHz; DMSO-d6): δ 7.33-7.24 (m, 1H),
7.03-6.89 (m, 3H), 5.75 (d, J = 5 Hz, 1H), 4.08-3.92 (m, 2H), 3.87-3.72 (m, 1H), 3.75 (s, 3H),
3.44-3.19 (m, 2H), 2.61-2.48 (m, 1H), 1.9-1.79 (m, 3H), 1.76-1.67 (m, 1H), 1.64-1.46 (m, 3H),
1.19-1.03 (m, 4H). HPLC-MS: 366 (M+Na)+.
4.5.14. ( )-(3aRS,4SR,7aRS)-4-(3,5-Difluoro-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester (( )-13)
To a solution of 1-ethynyl-3,5-difluoro-benzene (0.26 g, 1.9 mmol) in THF (2 ml) is added a
solution of n-butyllithium in hexane (1.3 ml, 2.1 mmol) at -20 °C dropwise via a syringe. After
stirring for 60 min at -20°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.22 g, 1 mmol) in THF (0.5 ml) is added dropwise at -20 °C. The reaction
mixture is allowed to warm to room temperature and after 2 h diluted with diethylether. The
resulting solution is washed with aqueous sat NH4Cl, H2O and dried over MgSO4, filtered and the
solvent evaporated in vacuo. The residue is flash chromatographed on silica gel, eluent hexane /
ethyl acetate 7:1 v/v. The fractions containing the desired compound are collected and ( )-13 is
1
obtained as colorless oil (Yield = 86 %). H-NMR (400 MHz; DMSO-D6): δ 7.33-7.26 (m, 1H),
7.21-7.14 (m, 2H), 5.82 (d, J = 5 Hz, 1H), 4.08-3.88 (m, 2H), 3.86-3.74 (m, 1H), 3.43-3.18 (m,
2H), 2.6-2.49 (m, 1H), 1.89-1.77 (m, 3H), 1.76-1.41 (m, 1H), 1.63-1.43 (m, 3H), 1.21-1.0 (m,
4H). HPLC-MS: 350 (M+H)+.
4.5.15. ( )-(3aRS,4SR,7aRS)-4-(3,5-Dimethyl-phenylethynyl)-4-hydroxy-octahydro-indole-
1-carboxylic acid ethyl ester (( )-14)
4.5.15.1.( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-trimethylsilyl-ethynyl-octahydro-indole-1-
carboxylic acid ethyl ester
Prepared from ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic acid ethyl ester and
trimethylsilyl acetylene according to the same procedure as in 4.5.7. The product is crystallized
from CH2Cl2 / hexane. ES-MS (+): 332.2 [M+Na]+.

4.5.15.2.( )-(3aRS,4SR,7aRS)-4-Ethynyl-4-hydroxy-octahydro-indole-1-carboxylic acid ethyl
ester
A mixture of 3.3 g (10.7 mmol) of ( )-(3aRS,4SR,7aRS)-4-hydroxy-4-trimethylsilyl-ethynyl-
octahydro-indole-1-carboxylic acid ethyl ester and 1.6 g (11.7 mmol) K2CO3 in 15 ml methanol
is stirred for 2 h at r.t. The solvent is removed in vacuo and the residue is partitioned between
CH2Cl2 and water. The organic phases are washed with brine, dried over Na2SO4, filtered and
evaporated in vacuo. The product is obtained as a white solid (2.5 g, 98 %), 1H-NMR (300 MHz,
CDCl3): δ 4.12 (q, 2H), 4.0 (b, 1H), 3.52 (bt, 1H), 3.38 (bq, 1H), 2.63 (m, 1H), 2.53 (s, 1H), 2.1 -
1.0 (m, 9H), 1.25 (t, 3H).
4.5.15.3.( )-(3aRS,4SR,7aRS)-4-(3,5-Dimethyl-phenylethynyl)-4-hydroxy-octahydro-indole-1-
carboxylic acid ethyl ester
A mixture of 100 mg (0.42 mmol) ( )-(3aRS,4SR,7aRS)-4-ethynyl-4-hydroxy-octahydro-indole-
1-carboxylic acid ethyl ester, 117 mg (0.63 mmol) 1-bromo-3,5-dimethyl-benzene, 15 mg (0.021
mmol) bis(triphenylphosphine)palladium(II) dichloride, 8 mg (0.042 mmol) copper(I) iodide and
of 64 mg (0.46 mmol) K2CO3 in 0.5 ml DME and 0.5 ml water are heated at 90 °C under argon
for 18 h. The reaction mixture is partitioned between diethyl ether and water. The organic phases
are washed with brine, dried over Na2SO4, filtered and evaporated in vacuo. The residue is flash
chromatographed on silica gel, eluent hexane / ethyl acetate 85:15 v/v. The fractions containing
the desired compound are collected to afford ( )-14 ( )-(3aRS,4SR,7aRS)-4-(3,5-dimethyl-
phenylethynyl)-4-hydroxy-octahydro-indole-1-carboxylic acid ethyl ester as a white solid (yield
1
= 21 %). H-NMR (400 MHz, CDCl3): δ 7.04 (s, 2H), 6.93 (s, 1H), 4.15 (q, 2H), 3.97 (b, 1H),
3.52 (b, 1H), 3.40 (b, 1H), 2.88 (b, 1H), 2.70 (m, 1H), 2.36 (t, 1H), 2.27 (s, 6H), 2.07-1.1 (m, 7H),
1.25 (t, 3H). ES-MS (+): 364.2 [M+Na]+.
4.5.16. ( )-(3aR,4R,7aR)-4-Hydroxy-4-((Z)-styryl)-octahydro-indole-1-carboxylic acid ethyl
ester (( )-15)
A solution of 150 mg ( )-6/( )-(3aRS,4SR,7aRS)-4-hydroxy-4-phenylethynyl-octahydro-indole-
1-carboxylic acid ethyl ester (4.5.7) in 6 ml of a mixture of THF and ethanol (3:1 v/v) is
hydrogenated in the presence of 30 mg of a Lindlar catalyst (1 bar/ r.t.) for 30 min. The mixture
of starting material and product is purified by flash chromatography with hexane / CH2Cl2 /
acetone (6:3:1) as the eluent. Pure fractions are collected to yield the product ( )-15/( )-
(3aR,4R,7aR)-4-Hydroxy-4-((Z)-styryl)-octahydro-indole-1-carboxylic acid ethyl ester (38 mg)
1
as an oil. H-NMR (400 MHz, CDCl3): δ 7.48 (d, 2H), 7.32 (t, 2H), 7.24 (t, 1H), 6.53 (d, 1H),
5.93 (d, 1H), 4.10 (q, 2H), 3.92 (m, 1H), 3.47 (m, 1H), 3.31 (m, 1H), 2.1 – 1.1 (m, 9H), 1.24 (t,
3H). ES-MS (+): 316.3 [M+H]+.
4.5.17. ( )-(3aR,4R,7aR)-4-Hydroxy-4-((E)-styryl)-octahydro-indole-1-carboxylic acid ethyl
ester (( )-16)
To a mixture of 0.145 ml (1.14 mmol) (E)- and (Z)-(2-bromovinyl)benzene (85:15) in 3 ml THF
is added a solution of 0.715 ml (1.14 mmol) butyllithium in hexane at -78 °C. To the red solution
is added dropwise a solution of 200 mg (0.95 mmol) of ( )-(3aRS,7aRS)-4-oxo-octahydro-
indole-1-carboxylic acid ethyl ester in 1 ml THF at -78 °C. The cooling is removed and stirring is

continued for 1 h. The solvent is removed in vacuo and the mixture is quenched with aqueous
ammonium chloride solution and extracted with CH2Cl2. The organic phases are washed with
brine, dried over Na2SO4, filtered and evaporated in vacuo. The residue is flash chromatographed
on silica gel, eluent hexane / ethyl acetate 2:1 v/v to afford the product ( )-16 as an oil (21 mg),
1H-NMR (300 MHz, CDCl3): δ 7.40 (d, 2H), 7.32 (t, 2H), 7.26 (t, 1H), 6.73 (d, 1H), 6.47 8d, 1H),
4.12 (q, 2H), 3.96 (m, 1H), 3.53 (m, 1H), 3.38 (q, 1H), 2.5-1.1 (m, 9H), 1.26 (t, 3H). ES-MS (+):
338.2 [M+Na]+.
4.5.18. ( )-(3aR,4R,7aR)-4-Hydroxy-4-phenethyl-octahydro-indole-1-carboxylic acid methyl
ester (( )-17)
Prepared by hydrogenation of ( )-6/( )-(3aRS,4SR,7aRS)-4-hydroxy-4-phenylethynyl-
octahydro-indole-1-carboxylic acid ethyl ester (4.5.7.) using Pd/C 5% as the catalyst to give ( )-
(3aR,4R,7aR)-4-hydroxy-4-phenethyl-octahydro-indole-1-carboxylic acid ethyl ester, m.p. 91-
92 °C. 1H-NMR (400 MHz, CDCl3): δ 7.29 (t, 2H), 7.26 (d, 1H), 7.20 (d, 2H), 4.10 (q, 2H), 3.85
(b, 1H), 3.52 (m, 1H), 3.35 (q, 1H), 2.70 (m, 2H), 2.33 - 1.1 (m, 11H), 1.15 (t, 3H). ES-MS (+):
340.2 [M+Na]+. The ethyl carbamate (500 mg, 1.58 mmol) is treated with 5 ml of a solution of
NaOMe in methanol (5.4 M, 27 mmol) at 100 °C in an autoclave for 4 h. The solvent is removed
in vacuo. The residue is partitioned between CH2Cl2 and 1 M aqueous citric acid solution. The
organic phases are washed with brine, dried over Na2SO4, filtered and evaporated in vacuo. The
residue is flash chromatographed on silica gel, eluent hexane / ethyl acetate 1:1 v/v to afford the
( )-17 as a white foam after drying (345 mg, 72 %), 1H-NMR (300 MHz, CDCl3): δ 7.27 (t, 2H),
7.24 (d, 1H, 7.19 (d, 2H), 3.97 (b, 1H), 3.68 (s, 3H), 3.52 (m, 1H), 3.35 (m, 1H), 2.70 (m, 2H),
2.25 (q, 1H), 2.15 - 1.05 (m, 11H). ES-MS (+): 326.2 [M+Na]+.
4.5.19. ( )-(3aRS,4SR,7aRS)-4-Methoxy-4-phenylethynyl-octahydro-indole-1-carboxylic
acid ethyl ester (( )-18)
To a solution of 100 mg (0.32 mmol) ( )-(3aRS,4SR,7aRS)-4-hydroxy-4-phenylethynyl-
octahydro-indole-1-carboxylic acid ethyl ester in 3 ml DMF is added a suspension of 15 mg (0.38
mmol) NaH in 1.5 ml DMF under argon at 0 °C. After stirring for 30 minutes at 0°C 40 µl methyl
iodide (0.64 mmol) is added dropwise via a syringe. After 20 minutes stirring is continued at
room temperature for 30 min. The reaction mixture is quenched with water and extracted with
CH2Cl2. The organic phases are washed with brine, dried over Na2SO4, filtered and evaporated in
1
vacuo to afford ( )-18 as an oil (yield = 96 %), H-NMR (400 MHz, CDCl3): δ 7.46 (m, 2H),
7.33 (m, 3H), 4.12 (q, 2H), 3.98 (m, 1H), 3.57-3.33 (m, 3H), 3.46 (s, 3H), 2.77 (m, 1H), 2.15-1.1
(m, 7H), 1.25 (t, 3H). ES-MS (+): 328.3 [M+H]+.
4.5.20. ( )-(3aRS,4SR,7aRS)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic
acid ethyl ester (( )-19)
To a solution of 1-ethynyl-3-methyl benzene (0.33 g, 2.84 mmol) in THF (5 ml) is added a
solution of n-butyllithium in hexane (1.78 ml, 2.84 mmol) at 0 °C dropwise via a syringe. After
stirring for 60 min at 0°C a solution of ( )-(3aRS,7aRS)-4-oxo-octahydro-indole-1-carboxylic
acid ethyl ester31 (0.5 g, 2.37 mmol) in THF (1 ml) is added dropwise at 0 °C and the reaction
mixture is allowed to warm to room temperature. Stirring is continued for 60 minutes. The

solvent is removed in vacuo, the residue is dissolved in CH2Cl2 and washed three times with
aqueous ammonium chloride solution and with brine. The combined organic phases are dried
over MgSO4, filtered and the solvent evaporated in vacuo. The residue is flash chromatographed
on silica gel, eluent hexane / ethyl acetate 2:1 v/v. The fractions containing the desired compound
are collected and the product is crystallized from hexane / ethyl acetate to afford ( )-19, 123-
124°C. 1H-NMR (400 MHz; CDCl3): δ 7.29-7.11 (m, 4H), 4.17-4.04 (m, 3H), 3.97 (m, 1H), 3.55
(m, 1H), 3.39 (m, 1H), 2.71 (m, 1H), 2.33 (s, 3H), 2.08-1.84 (m, 4H), 1.76-1.65 (m, 3H), 1.25 (m,
3H), 1.1 (m, 1H). ES-MS (+): 350.2 [M+Na]+.
4.5.21. ( )-((3aRS,4SR,7aRS)-4-Hydroxy-4-m-tolylethynyl-octahydro-indol-1-yl)-2-
methoxy-ethanone (( )-20)
Triethylamine (0.053 ml; 0.376 mmol) is added to a suspension of ( )-(3aR,4S,7aR)-4-hydroxy-
4-m-tolylethynyl-octahydro-indole hydrochloride (0.08 g; 0.31 mmol) in CH2Cl2 (1.5 ml) and the
mixture is cooled at 0°C. 2-methoxy-acetyl chloride (0.03 ml; 0.33 mmol) is added dropwise.
After completion of the addition, the cooling bath is removed and the reaction mixture allowed to
warm at room temperature. The reaction mixture was washed with a 10% solution of citric acid,
brine and dried over MgSO4. After filtration the solvent evaporated in vacuo and ( )-20 is
1
obtained as an oil in 91 % yield. H-NMR (400 MHz, CDCl3, rotamers 1:1): δ 7. 25 – 7.10 (m,
4H), 4.45 (m, 0.5H), 4.01, 4.0 (2s, 2H), 4.0 (m, 0.5H), 3.62 (m, 1H), 3.43 (m, 1H), 3.45 (s, 3H),
2.75, 2.67 (2m, 1H), 2.35, 2.32 (2s, 3H), 2.3 – 1.25 (m, 9H). ES-MS (+): 328.3 [M+H]+.
4.5.22. ( )-1-((3aRS,4SR,7aRS)-4-Hydroxy-4-m-tolylethynyl-octahydro-indol-1-yl)-ethanone
(( )-21)
Triethylamine (0.053 ml; 0.376 mmol) is added to a suspension of ( )-(3aR,4S,7aR)-4-hydroxy-
4-m-tolylethynyl-octahydro-indole hydrochloride (0.08 g; 0.31 mmol) in CH2Cl2 (1.5 ml) and the
mixture is cooled at 0°C. Methylchloroformate (0.03 ml; 0.33 mmol) is added dropwise. After
completion of the addition, the cooling bath is removed and the reaction mixture allowed to warm
at room temperature. The reaction mixture was washed with a 10% solution of citric acid, brine
and dried over MgSO4. After filtration the solvent evaporated in vacuo. The residue is column
chromatographed on silica gel eluent hexane / ethyl acetate 1:3 v/v. The fractions containing the
desired compound are collected, evaporated in vacuo to afford ( )-21 as an oil in 86 % yield.1H-
NMR (400 MHz, CDCl3, rotamers): δ 7.25 – 7.10 (m, 4H), 4.40 (m, 0.5H), 3.92 (m, 0.5H), 3.65 –
3.40 (2m, 2H), 2.78, 2.70 (2m, 1H), 2.35, 2.33 (2s, 3H), 2.08, 2.01 (2s, 3H), 2.5 – 1.0 (m, 9H).
ES-MS (+): 298.1 [M+H]+.
4.5.23. ( )-Furan-2-yl-((3aRS,4SR,7aRS)-4-hydroxy-4-m-tolylethynyl-octahydro-indol-1-yl)-
methanone (( )-22)
Triethylamine (0.053 ml; 0.376 mmol) is added to a suspension of ( )-(3aR,4S,7aR)-4-hydroxy-
4-m-tolylethynyl-octahydro-indole hydrochloride (0.08 g; 0.31 mmol) in CH2Cl2 (1.5 ml) and the
mixture is cooled at 0°C. Furan-2 carbonyl chloride (0.07 ml; 0.07 mmol) is added dropwise.
After completion of the addition, the cooling bath is removed and the reaction mixture allowed to
warm at room temperature. The reaction mixture was washed with a 10% solution of citric acid,
brine and dried over MgSO4. After filtration the solvent evaporated in vacuo, the residue is

1
crystallized from CH2Cl2 / ethyl ether to afford ( )-22, mp 195-196 °C. H-NMR (400 MHz,
CDCl3): δ 7.5 (s, 1H), 7.31 – 7.04 (m, 5H), 4.40 (m, 0.54H), 3.92 (m, 0.46H), 3.65 – 3.40 (2m,
2H), 2.78, 2.70 (2m, 1H), 2.35, 2.33 (2s, 3H), 2.08, 2.01 (2s, 3H), 2.5 – 1.0 (m, 9H). ES-MS (+):
298.1 [M+H]+.
4.5.24. (+)-(3aS,4R,7aS)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic acid
methyl ester ((+)-10)
( )-10/( )-(3aRS,4RS,7aRS)-4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic
acid
methyl ester (1.12 g) was separated into its enantiomers by chromatography on a Chiralpak AD
column (Daicel Chemical Industries, 5 x 50 cm). The chromatography is achieved at room
temperature at a flow-rate of 150 ml/min and UV detection is performed at 210 nm. The mobile
phase consists of a mixture of hexane-ethanol 95:5 (v/v) + 0.1 % TFA. After iterative recycling
of fractions, the (+)-enantiomer is isolated first and the (-)-enantiomer second.
Crystallization of the (+)-enantiomer from diethyl ether / n-hexane gave 365 mg of (+)-
(3aS,4R,7aS)-4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic acid methyl ester ((+)-
10), m.p. 114.9-115.2 °C, [α]D= +21.3 (methanol 1%, T=21 °C, 589 nm). Optical purity was
determined on an analytical Chiralpak AD column, 250x 4.6 mm with n-hexane / EtOH (95:5 +
0.1 % TFA, flow 1 ml/min) as the mobile phase: 100 % ee (RT = 13.4 min, UV detection 210
nm). 1H-NMR (400 MHz, CDCl3): δ 7.25-7.10 (m, 4H), 4.17-3.90 (b, 1H), 3.68 (s, 3H), 3.53 (b,
1H), 3.40 (b, 1H), 2.80 (m, 1H), 2.33 (s, 3H), 2.20-1.50 (m, 9H). ES-MS (+): 336.2 [M+Na]+.
4.5.25. (-)-(3aR,4S,7aR)-4-Hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylic acid
ethyl ester ((-)-19)
Triethylamine (0.9 ml; 0.6.49 mmol) is added to a suspension of (-)-(3aR,4S,7aR)-4-hydroxy-4-
m-tolylethynyl-octahydro-indole hydrochloride (0.86 g; 2.95 mmol) in CH2Cl2 (44 ml) and the
mixture is cooled at 5°C. Ethylchloroformate (0.366 ml; 3.835 mmol) is added dropwise. After
completion of the addition, the cooling bath is removed and the reaction mixture stirred for 2h30’
at RT. The reaction mixture is diluted with CH2Cl2, washed brine and dried over Na2SO4. After
filtration the solvent evaporated in vacuo, the residue is column chromatographed on silica gel
eluent toluene / acetone 9:1 v/v. The fractions containing the desired compound are collected,
evaporated in vacuo to afford (-)-19, white crystals: mp 118-121°C. [α]D = -21.6 (Ethanol
1.09 %). 1H-NMR (400 MHz; DMSO-d6): δ 7.67-7.14 (m, 4H), 5.73(d, J = 5 Hz, 1H), 4.05-3.93
(m, 2H), 3.85-3.73 (m, 1H), 3.41-3.32 (m, 1H), 3.28-3.17 (m, 1H), 2.6-2-48 (m, 1H), 2.27 (s, 3H),
1.9-1.77 (m, 3H), 1.74-1.66 (m, 1H), 1.64-1.45 (m, 3H), 1.17-1.03 (m, 4H). ES-MS (+): 328.2
[M+H]+
4.5.26. (-)-Furan-2-yl-((3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-octahydro-indol-1-yl)-
methanone ((-)-22)
Triethylamine (0.53 ml; 3.8 mmol) is added to a suspension of (-)-(3aR,4S,7aR)-4-hydroxy-4-m-
tolylethynyl-octahydro-indole hydrochloride (0.5 g; 1.7 mmol) in CH2Cl2 (5 ml) and the mixture
is cooled at 0°C. Furan-2 carbonyl chloride (0.19 ml; 1.9 mmol) is added dropwise. After
completion of the addition, the cooling bath is removed and the reaction mixture allowed to warm

at room temperature. The reaction mixture was washed with a 10% solution of citric acid, brine
and dried over MgSO4. After filtration the solvent evaporated in vacuo, the residue is crystallized
from CH2Cl2 / hexane to afford (-)-22, mp 195-196 °C. [α]D= -9.6 (methanol 0.5%, T=20 °C, 589
nm). Optical purity was determined on an analytical Chiralpak AD column, 250x 4.6 mm with n-
hexane / EtOH (9:1 + 0.1 % TFA, flow 1 ml/min) as the mobile phase: 100 % ee (RT = 11.1 min,
UV detection 210 nm). 1H-NMR (500 MHz, DMSO-d6, rotamers 2:1): δ 7.85 (d, 1H), 7.30 – 7.15
(m, 4H), 7.06 (d, 1H), 6.62 (m, 1H), 5.8 (b, 1H), 4.49 (m, 0.33 H), 4.30 (m, 0.67 H), 3.95 (t, 0.67
H), 3.25 (q, 0.67 H), 3.58 (m, 0.66 H), 2.70, 2.58 (2m, 1H), 2.30, 2.29 (2s, 3H), 2.10 – 1.10 (m,
8H).
4.6. Assessment of inhibitory activity at the human mGluR5
Generation of stable cell line and cell culture:
The generation and the culturing of the cell lines stably expressing hmGluR1b, hmGluR2,
hmGluR4, hmGluR5a and hmGluR7 were described in detail previously.18,32,33,34
Cell culture was performed essentially as described by Flor et al.32 Briefly, cells were cultured in
glutamate free medium composed of DMEM lacking L-glutamate (without phenol red, Gibco,
#11880-028) containing a reduced concentration of 2 mM L-glutamine (Gibco, #25030-024),
supplemented with 0.046 mg/ml proline (Sigma, #P0380), 10% dialyzed fetal calf serum (Gibco,
#26300-061) and 50 mg/ml geneticin (G-418 sulphate, Gibco, #11811-031).
High throughput screening:
Functional antagonists of human mGluR5 were identified by their ability to block glutamate-
induced intracellular calcium transients in L(tk-)/hmGluR5 cells utilizing a 96-well FLIPR assay
platform in combination with the fluorescent calcium indicator Fluo-3. For a description of the
methodology see e.g. Widler et al.19 In brief, L(tk-) cells were seeded at 60,000 cells/well in clear
bottom black plates for 24 h in the presence of 2 ꢄM dexamethasone to up-regulate mGluR5.
Cells were loaded with Fluo-3 and washed twice to remove excess of the dye. Cells were then
kept at room temperature for 2 hours until prior to testing in the FLIPR device.
Primary assay:
Assessment of hmGluR5 antagonism during the lead optimization phase was also assessed with a
FLIPR-based assay using a slightly modified protocol and the fluorescent calcium indicator Fluo-
4. Prior to testing, L(tk-)/hmGluR5 cells were cultured and treated essentially as described above,
then seeded at 40’000 cells/well, left to incubate with fluo-4 for 75 minutes, washed twice, left to
recover for 60 minutes at room temperature, washed once more and left to recover for another 30
minutes. In the FLIPR device, test compounds were dispensed to the cells and left to incubate for
10 minutes, following which cells were challenged by injecting L-glutamate (final assay
concentration 10 ꢄM). Concentration-response relations of test compounds were typically derived
from 8-point serial dilutions utilizing 2-4 replicates per concentration. IC50 values were obtained
by fitting the four-parametric logistic equation to the data sets utilizing GraphPad Prism software.
Secondary assay:
The functional phosphoinositol (PI) turnover assays using hmGluR5a- and hmGluR1b-expressing
cell lines were performed as described previously by Litschig et al.20

Radioligand displacement:
The radioligand displacement assay using a membrane preparation of rat brain (mixture of
hippocampus and cortex) was performed as described previously Gasparini et al.21
Data analysis and curve fitting
If not stated otherwise, results are given as percentages (mean s.d.) normalized to positive and
negative controls (i.e. maximal agonist-stimulated effect = 100% and unstimulated [basal] effect
= 0%); the percentages represent data pooled replicatewise from at least two independent
determinations. To quantify concentration-response relations (regardless of assay), the four-
parametric logistic equation was fitted to the pooled means. Where appropriate, the best-fit
parameters are quoted with their corresponding 95% confidence intervals (95% CI) as obtained
with the fitting procedure (GraphPad Prism 2.0).
4.7. Assessment of in vivo properties
Pharmacokinetic
Animals: Male Sprague-Dawley rats (175-250g) were obtained from Iffa Credo, France. They
were housed in groups of three. The animals had access to water and food (Ecosan, Eberle Nafag
AG, Gossau, Switzerland), ad libitum.
Drug treatment:
AFQ056/mavoglurant was administered as a microemulsion (made from a mixture of corn oil-
mono-di-triglycerides, polyoxyl 40 hydrogenated castor oil NF, DL-ꢆ-tocopherol USP, propylene
glycol USP and 12 % ethyl alcohol) at a dose of 3.1 mg/kg (10 µmol/kg; application volume 1
ml/kg) intravenously and 9.4 mg/kg (30 µmol/kg; application volume 2 ml/kg) orally. After 0.08,
0.5, 1, 2, 4, 8 or 24 hours (i.v.) and 0.25, 0.5, 1, 2, 4, 8 or 24 (p.o.) (N=6 rats per time-point per
injection procedure) the animals were decapitated and trunk-blood was collected in EDTA-
containing tubes and the brain was removed and immediately frozen on dry ice. Thereafter,
plasma samples and brains were stored at -80°C until analysis.
MPEP was administered to rats intravenously (suspension in 50 % PEG200/50 % H2O; volume 1
ml/kg) at the dose of 10 µmol/kg (~2.3 mg/kg salt) or per os (suspension in 0.5% methylcellulose;
volume 2 ml/kg) at the dose of 30 µmol/kg (~6.9 mg/kg salt). After 0.5, 1, 2, 4, 6 or 24 hours
(N=4 rats per time-point per injection procedure) the animals were decapitated and trunk-blood
was collected in EDTA-containing tubes and the brain was removed and immediately frozen on
dry ice. Thereafter, blood samples and brains were stored at -80°C until analysis.
Determination of brain/blood levels of AFQ056 in mice following SIH
Ninety minutes post dose (10/1/0.1 mg/kg) (N=6 mice) the animals were decapitated and trunk-
blood was collected in EDTA-containing tubes and the brain was removed and immediately
frozen on dry ice. Thereafter, blood samples and brains were stored at -80°C until analysis.
Analytical methodology, preparation of samples:
Frozen plasma and brain samples were thawed out. To 100 µL plasma 10 µL internal standard
(AAG561)35 (0.1 pmol) was added and extracted three times with 500 µL ethylacetate. The
combined extracts were then dried under a stream of nitrogen and redissolved in 100 µL
acetonitrile. Brains were weighed and then homogenized in water (1:5 w/v) using an Ultra-Turax

Mod T8 for 30 sec. Two 100 µL aliquots of each homogenate + 10 µL internal standard (0.1
pmol) were extracted three times with 500 µL ethylacetate and further processed as the plasma
samples.
Data processing:
The disposition and absorption parameters of AFQ056/mavoglurant and MPEP in rats were
analyzed by non-compartmental approach using the software TOPFIT (Tanswell et al 1993)22 and
using the plasma concentration versus time profiles after intravenous or oral administration. The
area under the curve (AUC) was obtained from the measured plasma concentrations versus time
using the linear trapezoidal rule and extrapolated to infinite time. The terminal half-life and total
clearance were calculated by the program from the plasma concentrations after i.v. or p.o.
administration versus time.
Stress induced hyperthermia
Animals:
Male OF1/IC mice (Iffa Credo, France) were housed in groups of twelve, in macrolon cages
(Type IV, 33 x 55 x 19 cm) in the experimental room of the laboratory, which was temperature
controlled and equipped with artificial illumination (6:00 hr - 18:00 hr, lights on). Animals had
always free access to food (Ecosan, Eberle Nafag AG, Gossau, Switzerland) and water.
Experimental protocol:
The test procedure for the modified stress-induced hyperthermia was adapted from Van der
Heyden et al. (1997)36 as described by Spooren et al (2002).27 Approximately 18 hours before
testing (i.e. on the afternoon before the test day) animals were individually housed in smaller
macrolon cages (26 x 21 x 14 cm) and transferred to the experimental room. The next morning,
test-compounds (or vehicle) were administered orally and one hour later rectal temperature was
measured (T1). Core temperatures were measured to the nearest 0.1 °C by an ELLAB
instruments thermometer (Copenhagen, Denmark) via a lubricated thermistor probe (2 mm
diameter) inserted 20 mm into the rectum while the mouse was hand held near the base of the tail.
The probe was left in place until steady readings were obtained ( 15 seconds). Immediately after
the measurement was completed the probe was removed and the mouse released in his cage.
Hyperthermia induced by this procedure was assessed 15 minutes later by a second temperature
measurement (T2). The dependent variable, i.e. stress-induced hyperthermia (SIH), was defined
as the difference T2 - T1. In addition, T1 was used to evaluate whether the test-compound by
itself would have a potential effect on basal body temperature.
Drug treatment:
AFQ056 was administered to mice orally as a suspension in 0.5% methylcellulose (volume 2
ml/kg) at the doses of 0.1/1/10 mg/kg.
Acknowledgments
We thank M. Heinrich, A. Dattler, C. Hunn, W. Inderbitzin, T.-M. Komla, P. Merklen, HP.
Mueller, N. Reymann and C. Stierlin for expert technical assistance. We also thank Ms G. Rihs
for the crystal structure determination of compound 28 (Supplementary data) and Dr. Terance W.
Hart for the synthesis of 8 (( )-(3aRS,4SR,7aRS)-4-(3-Chloro-phenylethynyl)-4-hydroxy-
octahydro-indole-1-carboxylic acid ethyl ester).

Supplementary Material
Crystal structure determination of a derivative of (+)-10, selectivity profile at the mGluR
subtypes, binding battery, 1H-NMR spectra.
5. References and notes
1. Grotta, J.; Clark, W.; Coull, B.; Pettigrew, L. C.; Mackay, B.; Goldstein, L. B.; Meissner,
I.; Murphy, D.; LaRue, L. Safety and tolerability of the glutamate antagonist CGS 19755
(Selfotel) in patients with acute ischemic stroke. Results of a phase IIa randomized trial.
Stroke 1995, 26, 602-5.
2. Sveinbjornsdottir, S.; Sander, J. W.; Upton, D,; Thompson, P. J.; Patsalos, P. N.; Hirt, D.;
Emre, M.; Lowe, D.; Duncan, J. S.; The excitatory amino acid antagonist D-CPP-ene
(SDZ EAA-494) in patients with epilepsy. Epilepsy Res. 1993, 16, 165-74.
3. Davis, S. M.; Lees, K. R.; Albers, G. W.; Diener, H. C.; Markabi, S.; Karlsson, G.; Norris
J. Selfotel in Acute Ischemic Stroke: Possible Neurotoxic Effects of an NMDA
Antagonist. Stroke 2000, 31, 347 - 354.
4. Nicoletti, F.; Bockaert, J.; Collingridge, G. L.; Conn, P. J.; Ferraguti, F.; Schoepp, D. D.;
Wroblewski, J. T.; Pin, J. P.; Metabotropic glutamate receptors: from the workbench to
the bedside. Neuropharmacology 2011, 60, 1017-41.
5. Gasparini, F.; Lingenhöhl, K.; Stoehr, N., Flor, P. J.; Heinrich, M., Vranesic, I.; Biollaz,
M.; Allgeier, H.; Heckendorn, R.; Urwyler, S.; Varney, M. A.; Johnson, E. C.; Hess, S. D.;
Rao, S. P.; Sacaan, A. I.; Santori, E. M.; Veliçelebi, G.; Kuhn, R. 2-Methyl-6-
(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5
receptor antagonist. Neuropharmacology 1999, 38, 1493-503.
6. Varney, M. A.; Cosford, N. D.; Jachec, C.; Rao, S. P.; Sacaan, A.; Lin, F. F.; Bleicher, L.;
Santori, E. M.; Flor, P. J.; Allgeier, H.; Gasparini, F.; Kuhn, R.; Hess, S.D.; Veliçelebi, G.;
Johnson, E. C. SIB-1757 and SIB-1893: selective, noncompetitive antagonists of
metabotropic glutamate receptor type 5. J. Pharmacol. Exp. Ther. 1999, 290, 170-81.
7. Pagano, A.; Ruegg, D.; Litschig, S.; Stoehr, N.; Stierlin, C.; Heinrich, M.; Floersheim, P.;
Prezèau, L.; Carroll, F.; Pin, J. P.; Cambria, A.; Vranesic, I.; Flor, P. J.; Gasparini, F.;
Kuhn, R. The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-
hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with
overlapping binding pockets in the transmembrane region of group I metabotropic
glutamate receptors. J. Biol. Chem. 2000, 275, 33750-8.
8. Rocher, J, P.; Bonnet, B.; Boléa, C.; Lütjens, R.; Le Poul, E.; Poli, S.; Epping-Jordan, M.;
Bessis, A. S.; Ludwig, B.; Mutel, V. mGluR5 negative allosteric modulators overview: a
medicinal chemistry approach towards a series of novel therapeutic agents. Curr Top Med
Chem. 2011, 11, 680-95.
9. Cosford, N. D. P.; Tehrani, L.; Roppe, J.; Schweiger, E.; Smith, N. D.; Anderson, J.;
Bristow, L.; Brodkin, J.; Jiang, X. H.; McDonald, I.; Rao, S.; Washburn, M.; Varney, M.
A. 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-pyridine: a potent and highly selective
metabotropic glutamate subtype 5 receptor antagonist with anxiolytic activity. J. Med.
Chem. 2003, 46, 204–206.
10. Kulkarni, S. S.; Zou, M.-F.; Cao, J.; Deschamps, J. R.; Rodriguez, A. L.; Conn, P. J.;
Newman, A. H. Structure-Activity Relationships Comparing N-(6-Methylpyridin-yl)-
SubstitutedAryl Amides to 2-Methyl-6-(substituted-arylethynyl)pyridines or 2-Methyl-4

(substituted-arylethynyl)thiazoles as Novel Metabotropic Glutamate Receptor Subtype 5
Antagonists, J. Med. Chem. 2009, 52, 3563-3575.
11. Milbank, J.B.J.; Knauer, C.S.; Augelli-Szafran, C.E.; Sakkab-Tan, A.T.; Lin, K.K.;
Yamagata, K.; Hoffman, J.K.; Zhuang, N.; Thomas, J.; Galatsis, P.; Wendt, J. A.;
Mickelson, J. W.; Schwarz, R. D.; Kinsora, J. J.; Lotarski, S. M.; Stakich, K.; Gillespie, K.
K.; Lam, W. W.; Mutlib, A. E. Rational design of 7-arylquinolines as non-competitive
metabotropic glutamate receptor subtype 5 antagonists, Bioorg. Med. Chem. Lett. 2007,
17, 4415-4418.
12. Jaeschke, G.; Porter, R.; Büttelmann, B.; Ceccarelli, S. M.; Guba, W.; Kuhn, B.;
Kolczewski, S.; Huwyler, J.; Mutel, V.; Peters, J.-U.; Ballard, T.; Prinssen, E.; Vieira, E.;
Wichmann, J.; W. Spooren, Synthesis and biological evaluation of fenobam analogs as
mGlu5 receptor antagonists Bioorg. Med. Chem. Lett. 2007, 17, 1307-1311.
13. Gasparini, F.; Bilbe, G.; Gomez-Mancilla, B.; Spooren, W. mGluR5 antagonists:
discovery, characterization and drug development. Curr. Opin. Drug. Discov. Devel. 2008
11, 655-65.
14. Berg, D.; Godau, J.; Trenkwalder, C.; Eggert, K.; Csoti, I.; Storch, A.; Huber, H.; Morelli-
Canelo, M.; Stamelou, M.; Ries, V.; Wolz, M.; Schneider, C.; Di Paolo, T.; Gasparini, F.;
Hariry, S.; Vandemeulebroecke, M.; Abi-Saab, W.; Cooke, K.; Johns, D.; Gomez-
Mancilla, B. AFQ056 treatment of levodopa-induced dyskinesias: results of 2 randomized
controlled trials. Mov Disord. 2011, 26, 1243-50.
15. Jacquemont, S.; Curie, A.; des Portes, V.; Torrioli, M. G.; Berry-Kravis, E.; Hagerman, R.
J.; Ramos, F. J.; Cornish, K.; He, Y.; Paulding, C.; Neri, G.; Chen, F.; Hadjikhani, N.;
Martinet, D.; Meyer, J.; Beckmann, J. S.; Delange, K.; Brun, A.; Bussy, G.; Gasparini, F.;
Hilse, T.; Floesser, A.; Branson, J.; Bilbe, G.; Johns, D.; Gomez-Mancilla, B. Epigenetic
modification of the FMR1 gene in fragile X syndrome is associated with differential
response to the mGluR5 antagonist AFQ056 Sci. Transl. Med. 2011, 3, 1-9.
16. ADX48621 for the Treatment of Levodopa Induced Dyskinesia in Patients with
Parkinson's Disease, Addex Pharmaceuticals, Clinicaltrials.gov: A service of the US
National Institute of Health. http://clinicaltrials.gov/ct2/results?term=adx48621. Accessed
on June 2nd, 2014.
17. RG7090, Roche, Pipeline update April 15th, 2014.
http://www.roche.com/research_and_development/pipeline/roche_pharma_pipeline.htm.
Accessed on June 2nd, 2014.
18. Daggett, L. P.; Sacaan, A. I.; Akong, M.; Rao, S.P.; Hess, S.D.; Liaw, C.; Urrutia, A.;
Jachec, C.; Ellis, S. B.; Dreessen, J.; Knoepfel, T.; Landwehrmeyer, G. B.; Testa, C. M.;
Young, A. B.; Varney, M.; Johnson E. C.; Velicelebi, G. Neuropharmacology 1995, 34,
871.
19. Widler, L.; Altmann, E.; Beerli, R.; Breitenstein, W.; Bouhelal, R.; Buhl, T.; Gamse, R.;
Gerspacher, M.; Halleux, C.; John. M. R.; Lehmann, H.; Kalb, O.; Kneissel, M.;
Missbach, M.; Müller, I. R.; Reidemeister, S.; Renaud, J.; Taillardat, A.; Tommasi, R.;
Weiler, S.; Wolf, R. M.; Seuwen, K. 1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a
novel series of potent calcium-sensing receptor antagonists. J. Med. Chem. 2010, 53,
2250-63.
20. Litschig, S.; Gasparini, F.; Rueegg, D.; Stoehr, N.; Flor, P. J.; Vranesic, I.; Prezeau, L.;
Pin, J. P.; Thomsen, C.; Kuhn, R. CPCCOEt, a noncompetitive metabotropic glutamate
receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. Mol.
Pharmacol. 1999, 55, 453-461.

21. Gasparini, F.; Andres, H.; Flor, P. J.; Heinrich, M.; Inderbitzin, W.; Lingenhöhl, K.;
Müller, H.; Munk, V. C.; Omilusik, K.; Stierlin, C.; Stoehr, N.; Vranesic, I.; Kuhn R.
[3H]-M-MPEP, a potent, subtype-selective radioligand for the metabotropic glutamate
receptor subtype 5. Bioorg Med. Chem. Lett. 2002, 12, 407-9.
22. Tanswell, P.; Koup, J.; TopFit: a PC-based pharmacokinetic/pharmacodynamic data
analysis program Int. J. Clin. Pharmacol. Ther. Toxicol. 1993, 31, 514-20
23. Walles, M.; Wolf, T.; Jin, Y.; Ritzau, M.; Leuthold, L. A.; Krauser, J.; Gschwind, H. P.;
Carcache, D.; Kittelmann, M.; Ocwieja, M.; Ufer, M.; Woessner, R.; Chakraborty, A.;
Swart, P. Metabolism and disposition of the metabotropic glutamate receptor 5 antagonist
(mGluR5) mavoglurant (AFQ056) in healthy subjects. Drug Metab. Dispos. 2013, 41,
1626-41.
24. Spooren, W. P.; Vassout, A.; Neijt, H. C.; Kuhn, R.; Gasparini, F.; Roux, S.; Porsolt, R.
D.; Gentsch, C. Anxiolytic-like effects of the prototypical metabotropic glutamate
receptor 5 antagonist 2-methyl-6-(phenylethynyl)pyridine in rodents. J. Pharmacol. Exp.
Ther. 2000, 295, 1267-75.
25. Pilc, A.; Kłodziꢇska, A.; Braꢇski, P.; Nowak, G.; Pałucha, A.; Szewczyk, B.;
Tatarczyꢇska, E.; Chojnacka-Wójcik, E.; Wieroꢇska, J. M. Multiple MPEP
administrations evoke anxiolytic- and antidepressant-like effects in rats.
Neuropharmacology 2002, 43, 181-7.
26. Brodkin, J.; Bradbury, M.; Busse, C.; Warren, N.; Bristow, L. J.; Varney, M.A. Reduced
stress-induced hyperthermia in mGluR5 knockout mice. Eur. J. Neurosci. 2002, 16, 2241-
4.
27. Spooren, W. P.; Schoeffter, P.; Gasparini, F.; Kuhn, R.; Gentsch, C. Pharmacological and
endocrinological characterisation of stress-induced hyperthermia in singly housed mice
using classical and candidate anxiolytics (LY314582, MPEP and NKP608). Eur. J.
Pharmacol. 2002, 435, 161-70.
28. Lindemann, L.; Jaeschke, G.; Michalon, A.; Vieira, E.; Honer, M.; Spooren, W.; Porter,
R.; Hartung, T.; Kolczewski, S.; Büttelmann, B.; Flament, C.; Diener, C.; Fischer, C.;
Gatti, S.; Prinssen, E.P.; Parrott, N.; Hoffmann, G.; Wettstein, J.G. CTEP: a novel, potent,
long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor. J
Pharmacol Exp Ther. 2011, 339, 474-86.
29. Allgeier, H.; Auberson, Y.; Biollaz, M.; Cosford, N. D.; Gasparini, F.; Heckendorn, R.;
Johnson, E. C.; Kuhn, R.; Varney, M. A.; Velicelebi, G. From PCT Int. Appl. WO
9902497 A2 19990121; Chem. Abstr. 1999, 130, 153580.
30. Doré, A. S.; Okrasa, K.; Patel, J. C.; Serrano-Vega, M.; Bennett, K.; Cooke, R. M.; Errey,
J. C.; Jazayeri, A.; Khan, S.; Tehan, B.; Weir, M.; Wiggin, G. R.; Marshall, F. H.
Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain
Nature, 511, 557-62..
31. Dolfini, J. E.; Dolfini, D. M. Aziridinyl compounds as intermediates in pyrrolidine
synthesis Tetrahedron Letters 1965, 25, 2053-8.
32. Flor, P. J.; Lindauer, K.; Puettner, I.; Rueegg, D.; Lukic, S.; Knoepfel, T.; Kuhn, R.
Molecular cloning, functional expression and pharmacological characterization of the
human metabotropic glutamate receptor type 2. Eur. J. Neurosci. 1995a, 7, 622–629.
33. Flor, P. J.; Lukic, S.; Rueegg, D.; Leonhardt, T.; Knoepfel, T.; Kuhn, R. Molecular
cloning, functional expression and pharmacological characterization of the human
metabotropic glutamate receptor type 4. Neuropharmacology 1995b, 34, 149–155.

34. Flor, P. J.; van der Putten, H.; Rueegg, D.; Lukic, S.; Leonhardt, T.; Bence, M.; Sansig, G.;
Knoepfel, T.; Kuhn, R. A novel splice variant of a metabotropic glutamate receptor,
human mGluR7b. Neuropharmacology 1997, 36, 1532–1539.
35. Mickel, S.; Fischer, R.; Marterer, W. Broad Spectrum Chemistry as Practised by Novartis
Process Research Chimia 2004, 58, 640-648.
36. Van der Heyden, J. A.; Zethof, T. J.; Olivier, B. Stress-induced hyperthermia in singly
housed mice. Physiol. Behav. 1997, 62, 463-70.