Octahydropyrrolo[3,4-c]pyrrole negative allosteric modulators of mGlu 1

Article abstract of DOI:10.1016/j.bmcl.2013.07.029

Development of SAR in an octahydropyrrolo[3,4-c]pyrrole series of negative allosteric modulators of mGlu₁ using a functional cell-based assay is described in this Letter. The octahydropyrrolo[3,4-c]pyrrole scaffold was chosen as an isosteric replacement for the piperazine ring found in the initial hit compound. Characterization of selected compounds in protein binding assays was used to identify the most promising analogs, which were then profiled in P450 inhibition assays in order to further assess the potential for drug-likeness within this series of compounds.

Full text of DOI:10.1016/j.bmcl.2013.07.029

Accepted Manuscript
Octahydropyrrolo[3,4-c]pyrrole negative allosteric modulators of mGlu
1
Jason T. Manka, Alice L. Rodriguez, Ryan D. Morrison, Daryl F. Venable,
Hyekyung P. Cho, Anna L. Blobaum, J. Scott Daniels, Colleen M. Niswender,
P. Jeffrey Conn, Craig W. Lindsley, Kyle A. Emmitte
PII:
S0960-894X(13)00874-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.07.029
BMCL 20694
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 May 2013
3 July 2013
16 July 2013
Please cite this article as: Manka, J.T., Rodriguez, A.L., Morrison, R.D., Venable, D.F., Cho, H.P., Blobaum, A.L.,
Scott Daniels, J., Niswender, C.M., Jeffrey Conn, P., Lindsley, C.W., Emmitte, K.A., Octahydropyrrolo[3,4-
c]pyrrole negative allosteric modulators of mGlu , Bioorganic & Medicinal Chemistry Letters (2013), doi: http://
1
dx.doi.org/10.1016/j.bmcl.2013.07.029
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Octahydropyrrolo[3,4-c]pyrrole negative allosteric modulators
of mGlu1
Jason T. Mankaa,b, Alice L. Rodrigueza,b, Ryan D. Morrisona,b, Daryl F. Venablea,b,
Hyekyung P. Choa,b, Anna L. Blobauma,b, J. Scott Danielsa,b,
Colleen M. Niswendera,b, P. Jeffrey Conna,b, Craig W. Lindsleya,b,c,
and Kyle A. Emmittea,b,c*
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232 USA
b
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232 USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232 USA
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract—Development of SAR in an octahydropyrrolo[3,4-c]pyrrole series of negative allosteric modulators of mGlu1 using a
functional cell-based assay is described in this Letter. The octahydropyrrolo[3,4-c]pyrrole scaffold was chosen as an isosteric replacement
for the piperazine ring found in the initial hit compound. Characterization of selected compounds in protein binding assays was used to
identify the most promising analogs, which were then profiled in P450 inhibition assays in order to further assess the potential for drug-
likeness within this series of compounds.
L-glutamic acid (glutamate) is the major excitatory
neurotransmitter in the mammalian central nervous
system (CNS). Activation of both ionotropic and
metabotropic glutamate receptors occurs following
binding to glutamate. The metabotropic glutamate
receptors (mGlus) are members of family C within the
broader G protein-coupled receptor (GPCR) family.
The eight known mGlus have been further classified
Figure 1. mGlu1 NAM initial hit 1 and tool compound VU0469650
according to their structure, preferred signal
transduction mechanisms, and pharmacology (Group I:
The design of selective small molecule negative
mGlu1 and mGlu5; Group II: mGlu2-3; Group III: mGlu4-
allosteric modulators (NAMs) of mGlu1 has been a
8).1 The majority of these receptors have attracted the
fruitful area of research within the mGlu allosteric
attention of researchers as potential therapeutic targets
modulator field.3 Multiple tool compounds have been
due to their association with a variety of CNS related
discovered during recent years, and their evaluation in
disorders. Initially, work toward the design of drug-like
behavioral models has further established a potential for
orthosteric ligands that selectively bind a specific mGlu
therapeutic benefit in a number of CNS-related
proved challenging. Perhaps this is not surprising,
disorders. Examples include addiction,4 anxiety,5
given that the orthosteric binding site across the mGlu
epilepsy,6 pain,5a,7 and psychotic disorders.5a,8 Recent
family is highly conserved. A more recent approach that
publications have also noted a potential role for mGlu1
yielded more selective compounds has been the design
inhibition in the treatment of melanoma9 and certain
and development of small molecules that modulate the
types of breast cancer.10 We recently reported our own
activity of the receptor, either positively or negatively,
initial efforts directed toward the discovery and
through binding to an allosteric site.2
optimization of structurally novel mGlu1 NAMs.11 In

that Letter we described the discovery and
characterization of VU0469650 through an optimization
program based on hit compound 1, which was identified
through internal cross screening (Fig. 1).12 VU0469650
is a potent mGlu1 NAM as measured in our functional
cell based assay, which measures the ability of the
compound to block the mobilization of calcium by an
EC80 concentration of glutamate in cells expressing
human mGlu1.13
conditions to afford 6. Acidic cleavage of the carbamate
protecting group provided amine intermediate 7, which
was readily converted to the target amide compounds 8-
19 using established methods. For evaluation of SAR
around the aryl portion of the chemotype (R2),
commercially available 20 was reacted with 1-
adamantoyl chloride to afford intermediate 21. Removal
of the benzyl protecting group was accomplished
through
a
palladium catalyzed hydrogenation.19
The SAR that was developed in the process leading
up to the discovery of VU0469650 was primarily
focused on evaluation of the amide and heteroaryl
portion of the scaffold. Concomitant to that work our
attention was also directed toward the design of
scaffolds that replaced the piperazine ring of 1
altogether. One piperazine isostere of interest was the
octahydropyrrolo[3,4-c]pyrrole. This particular ring
system has been successfully employed in drug
discovery research as an effective replacement for a
piperazine ring in the past.14 Furthermore, the N-
heteroaryl octahydropyrrolo[3,4-c]pyrrole chemotype
has proven to be useful for the design of drug-like small
molecules that interact with a number of CNS targets
(Fig. 2). Examples include the muscarinic acetylcholine
receptor M1 (2),14a the orexin receptor type 2 (3),15 the
α7 nicotinic acetylcholine receptor (A-582941),16 and
the cannabinoid receptor type 1 (4).17 The investigation
of the octahydropyrrolo[3,4-c]pyrrole scaffold as a
suitable chemotype for the development of novel mGlu1
NAMs is the subject of this Letter.
Conversion of amine 22 into target compounds 23-34
was achieved through nucleophilic aromatic substitution
reactions with aryl fluorides or Buchwald-Hartwig20
amination reactions with suitable aryl halides.
Scheme 1. Reagents and conditions: (a) R2F, DIEA, NMP, µwave, 180-
250 ºC; (b) HCl, MeOH, dioxanes; (c) R1COCl, DIEA, CH2Cl2; (d)
R1CO2H, HATU, DIEA, CH2Cl2; (e) 1-adamantoyl chloride, DIEA,
CH2Cl2; (f) H-cube®, Pd/C, MeOH, 80 bar, 80 ºC; (g) R2X (X = Cl, Br, or
I), Pd2(dba)3 or Pd(OAc)2, Xantphos, NaOtBu or Cs2CO3, dioxanes,
µwave, 120 ºC or thermal, 100 ºC.
Direct replacement of the piperazine ring of 1 with
the octahydropyrrolo[3,4-c]pyrrole group afforded 8,
which proved greater than seven fold more potent than 1
against human mGlu1 (Table 1). Furthermore, the
activity of 8 against rat mGlu5 was much reduced
relative to 1 (8 rmGlu5 IC50 = 3360 nM; % Glu Max =
1.1). Unfortunately, analogs with alternative amide
groups to the 1-adamantyl amide proved much less
potent, exhibiting only weak antagonism (10-13, 15, 17,
and 19) or were inactive (9 and 16) up to the top
concentration of 30 µM. Compounds 14 and 18 were
exceptions; however, these analogs were sixty and
seventeen fold less potent than 8, respectively. We had
Figure 2. Examples of small molecule octahydropyrrolo[3,4-c]pyrroles
that interact with CNS targets.
As was the case with the piperazine scaffold
exemplified by VU0469650, preparation of analogs
within this series was relatively straightforward
(Scheme 1).18 For evaluation of SAR around the amide
portion of the chemotype (R1), commercially available 5
was reacted with 2-fluoropyridine under SNAr

hoped that the cubyl amide of analog 19 might prove an
adequate amide replacement as that group has been
noted as a less lipophilic isosteric replacement for the
adamantyl group.21 Unfortunately, such a modification
resulted in a substantial loss of potency at mGlu1.
Table 2. Aryl/Heteroaryl SAR
% Glu
Max
Table 1. Amide SAR
mGlu1 pIC50
(± SEM)a
mGlu1 IC50
(nM)
cpd
8
R
(± SEM)a,b
7.07 ± 0.07
< 5.0c
85
> 10,000
> 10,000
> 10,000
669
2.2 ± 0.3
56.6 ± 8.6
10.3 ± 7.5
25.5 ± 5.7
-1.1 ± 1.8
2.3 ± 0.2
2.2 ± 1.2
2.1 ± 1.4
6.9 ± 1.5
12.2 ± 8.4
0.1 ± 0.5
36.8 ± 5.1
1.3 ± 1.1
23
24
25
26
27
28
29
30
31
32
33
34
% Glu
mGlu1 pIC50
(± SEM)a
mGlu1 IC50
(nM)
cpd
8
R
Max
(± SEM)a,b
< 5.0c
7.07 ± 0.07
< 4.5
85
2.2 ± 0.3
—
< 5.0c
> 30,000
> 10,000
> 10,000
> 10,000
> 10,000
5140
9
6.17 ± 0.21
7.03 ± 0.08
6.32 ± 0.10
6.00 ± 0.14
6.73 ± 0.07
5.25 ± 0.14
6.08 ± 0.17
< 5.0c
< 5.0c
57.7 ± 3.6
36.5 ± 13.2
14.9 ± 0.9
37.9 ± 4.9
9.3 ± 4.9
29.2 ± 2.6
—
10
11
12
13
14
15
16
17
18
19
93
< 5.0c
478
< 5.0c
1000
< 5.0c
185
5.29 ± 0.19
< 5.0c
5590
> 10,000
> 30,000
> 10,000
1430
833
< 4.5
> 10,000
553
< 5.0c
47.6 ± 3.7
0.0 ± 1.5
58.7 ± 4.0
6.26 ± 0.05
a Calcium mobilization mGlu1 assay; values are average of n ≥ 3
5.84 ± 0.18
< 5.0c
b Amplitude of response in the presence of 30 µM test compound as a
percentage of maximal response (100 µM glutamate); average of n ≥ 3
c CRC does not plateau
> 10,000
Exploration of SAR around the aryl ring of the
scaffold proved more fruitful (Table 2). While thiazole
23 and pyrimidines 24 and 25 were only weak
antagonists, pyrazine 26 proved more potent, albeit
eight-fold less than 8. Fluorination of 8 on the pyridine
a Calcium mobilization mGlu1 assay; values are average of n ≥ 3
b Amplitude of response in the presence of 30 µM test compound as a
percentage of maximal response (100 µM glutamate); average of n ≥ 3
c Concentration Response Curve (CRC) does not plateau

ring was tolerated with 6-fluoro analog 27 being
equipotent to 8 and 5-fluoro analog 28 and 3-fluoro
analog 29 being six-fold and twelve-fold less potent
than 8, respectively. The 2-cyanophenyl ring (30)
proved a reasonable alternative to the 2-pyridyl ring (8).
Desiring to prepare less lipophilic compounds, various
pyridine derivatives of 30 were prepared (31-34) with
analogs 32 and 34 demonstrating moderate potency.
further profile these two compounds by measuring their
binding to rat brain homogenates. Gratifyingly, both
compounds exhibited a greater fraction unbound in rat
brain homogenates than VU0469650 (Fu = 0.016).
These two compounds were also tested at 10 µM in cell
based functional assays for their selectivity against the
other members of the mGlu family and both
demonstrated good overall selectivity.26,27 Compound 8
was chosen as representative of the series and submitted
to a commercially available radioligand binding assay
panel of 68 clinically relevant GPCRs, ion channels,
kinases, and transporters,28 and only four significant
responses were noted at a concentration of 10 µM.29
Table 3. Urea SAR
Scheme 2. Reagents and conditions: (a) 4-nitrophenyl chloroformate,
CH2Cl2; (b) HNR1R2, NMP, µwave, 200 ºC.
% Glu
mGlu1 pIC50
(± SEM)a
mGlu1 IC50
(nM)
cpd
36
R
Max
(± SEM)a,b
Having explored SAR around the amide and aryl
portions of the chemotype with limited success, we
turned our attention to a new strategy for replacement of
the 1-adamantyl amide with substituted ureas (Scheme
2).22 Reaction of intermediate 7 with 4-nitrophenyl
chloroformate afforded carbamate 35. Treatment of 35
with cyclic secondary amines under microwave
irradiation afforded analogs 36-42 (Table 3). This
strategy produced three new analogs 38-40 with mGlu1
IC50 values less than one micromolar. Interestingly,
each of these compounds contained similar spirocyclic
amine moieties. 2-Azaspiro[4.4]nonane analog 38 was
the most potent urea analog and only three fold less
potent than 8.
Having identified several interesting new compounds
with good to moderate mGlu1 activity, we examined
them for their propensity to non-specifically bind to rat
plasma proteins (Table 4).23 Since such binding can
limit the amount of drug available to interact with the
target, absolute functional potency and protein binding
are both important factors in the ultimate efficacy of a
compound in vivo. The measured fraction unbound in
rat plasma proteins generally tracked with the calculated
logP values. More lipophilic compounds were more
highly bound than less lipophilic compounds; however,
pyrazine 26 was an apparent exception to this trend.24
Taking into account both functional potency and
fraction unbound in rat plasma, 8 and 38 were deemed
two of our most interesting compounds. Examination of
the ligand-lipophilicity efficiency (LLE)25 values for
these same analogs also places these two analogs among
the most drug-like within this set. Given that we are
primarily interested in the application of mGlu1 NAMs
for the treatment of CNS disorders, we also chose to
4.93 ± 0.07
< 5.0c
11,900
> 10,000
297
-7.5 ± 14.6
23.2 ± 6.5
-0.1 ± 0.6
0.7 ± 0.5
1.5 ± 0.4
-5.8 ± 3.8
-1.5 ± 1.1
37
38
39
40
41
42
6.53 ± 0.12
6.11 ± 0.05
6.44 ± 0.08
5.29 ± 0.10
5.72 ± 0.04
769
364
5160
1910
a Calcium mobilization mGlu1 assay; values are average of n ≥ 3
b Amplitude of response in the presence of 30 µM test compound as a
percentage of maximal response (100 µM glutamate); average of n ≥ 3
c CRC does not plateau
Wanting to further understand the potential for this
chemotype to deliver molecules with drug-like
properties and specifically any potential liabilities
related to drug-drug interactions, we also profiled 8 and
38 in a cytochrome P450 inhibition assay (Table 5).30

The profile of VU0469650 is pictured alongside for
comparison. Both VU0469650 and 8 were moderate
inhibitors of CYP3A4. The extent to which a drug
this series include continued optimization of drug-like
properties and subsequent evaluation of
pharmacokinetics. Results and observations from such
studies will be the subject of future communications.
inhibits CYP3A4 is
a
particularly important
consideration since this isoform is responsible for the
metabolism of approximately half of the drugs in
clinical use.31 Fortunately, analog 38 proved to have a
superior P450 inhibition profile, demonstrating no
measurable inhibition up to the top concentration tested
(30 µM). Though the exact reason for this improved
profile with 38 has not been conclusively determined, it
may be related to the compounds reduced lipophilicity
relative to the other two compounds.32 Reducing
lipophilicity has been previously noted as a successful
strategy for mitigating P450 inhibition in other
chemotypes.33
Acknowledgements
We thank Seaside Therapeutics (VUMC36176) for their
support of our programs in the development of mGlu1
NAMs. We also thank Tammy S. Santomango for
technical contributions with the protein binding assays.
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Table 4. Protein Binding Results
mGlu1 IC50
(nM)
Rat PPB
(Fu)c
Rat BHB
(Fu)c
cLogPa
cpd
LLEb
4.28
4.96
4.53
4.44
4.55
3.39
2.41
3.73
85
2.79
1.21
2.50
1.88
2.18
2.87
4.12
2.71
0.029
0.103
0.012
0.015
0.014
0.045
0.090
0.015
0.023
—
8
669
93
26
27
28
30
34
38
40
—
478
185
553
297
364
—
—
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—
a Calculated using ADRIANA.Code (www.molecular-networks.com)
b LLE (ligand-lipophilicity efficiency) = pIC50 – cLogP
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8
38
7.0
6.2
> 30
CYP3A4
> 30
12.4
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> 30
24.3
> 30
> 30
> 30
> 30
CYP2C9
CYP2D6
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selective mGlu1 NAM compounds can be prepared
within a series of octahydropyrrolo[3,4-c]pyrroles that
were developed from a piperazine cross screening hit.
Key SAR gleaned from this effort included mechanisms
for increasing the fraction of unbound compound in the
presence of plasma proteins and brain homogenates as
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13. HEK 293A TREx cells stably expressing human
mGlu1 were plated in black-walled, clear-bottomed,
poly-D-lysine coated 384-well plates in 20 μL of assay
medium (DMEM containing 10% dialyzed FBS, 20
mM HEPES, and 1 mM sodium pyruvate) at a density
of 20K cells/well. The cells were induced with 50
ng/mL tetracycline and grown overnight at 37 °C in
the presence of 5% CO2. The next day, medium was
exchanged to assay buffer (Hank’s balanced salt
solution, 20 mM HEPES, and 2.5 mM probenecid)
using an ELX405 microplate washer, leaving 20
μL/well, followed by addition of 20 μL/well 2X Fluo-
4 AM (2.3 µM final) indicator dye (prepared as a
DMSO stock and mixed in a 1:1 ratio with pluronic
acid F-127) in assay buffer, and incubation for 45 m at
37 °C. The dye was then exchanged to assay buffer
using an ELX405, leaving 20 μL/well. Ca2+ flux was
measured using the Functional Drug Screening
System (FDSS7000, Hamamatsu, Japan). Compounds
were serially diluted 1:3 into 10 point concentration
response curves and transferred to daughter plates
using the Echo acoustic plate reformatter. Compounds
were diluted into assay buffer to a 2x stock using a
Thermo Fisher Combi which was applied to cells at t
= 3 seconds. Cells were incubated with the test
compounds for 2.3 minutes and then stimulated with
an EC20 concentration of glutamate; 1.9 minutes later
an EC80 concentration of glutamate was added and
readings taken for an additional 1.7 minutes. Data
were collected at 1 Hz. Concentration response curves
were generated using a four point logistical equation
with XLfit curve fitting software for Excel.
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noted for mGlu6-8 while no activity was observed at
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= 3.36 µM). In the case of 38, very weak and
marginally significant activity was noted for mGlu6
and mGlu8 while no activity was observed at mGlu2-4
and mGlu7. A full CRC for 38 against mGlu5 was run
(IC50 > 10 µM).
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inhibited more than 50% of radioligand binding. In the
case of 8, the following significant responses were
noted: human opiate κ (60%), human serotonin 5-HT3
(90%), human sigma σ1 (95%), and rat Na+ channel
site 2 (52%).
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