Solution-phase parallel synthesis and SAR of homopiperazinyl analogs as positive allosteric modulators of mGlu4

Article abstract of DOI:10.1021/co1000508

Using a functional high-throughput screening (HTS) and subsequent solution-phase parallel synthesis approach, we have discovered a novel series of positive allosteric modulators formGlu₄, a G-protein coupled receptor. This series is comprised of a homopiperazine central core. The solution-phase parallel synthesis and SAR of analogs derived from this series will be presented. This series of positive allosteric modulators of mGlu4 provide critical research tools to further probe the mGlu₄-mediated effects in Parkinson's disease.

Full text of DOI:10.1021/co1000508

RESEARCH ARTICLE
pubs.acs.org/acscombsci
Solution-Phase Parallel Synthesis and SAR of Homopiperazinyl
Analogs as Positive Allosteric Modulators of mGlu4
Yiu-Yin Cheung,†,‡,§ Rocio Zamorano,†,‡ Anna L. Blobaum,†,‡ C. David Weaver,†,‡ P. Jeffrey Conn,†,‡,¥
||
||
Craig W. Lindsley,†,‡,§, ,¥ Colleen M. Niswender,†,‡ and Corey R. Hopkins*,†,‡,§,
†Department of Pharmacology, ‡Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, Nashville,
Tennessee 37232, United States, §Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (MLPCN), Nashville,
Tennessee 37232-6600, United States
Department of Chemistry, ¥Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
S Supporting Information
b
ABSTRACT: Using a functional high-throughput screening (HTS) and subsequent
solution-phase parallel synthesis approach, we have discovered a novel series of positive
allosteric modulators for mGlu4, a G-protein coupled receptor. This series is comprised
of a homopiperazine central core. The solution-phase parallel synthesis and SAR of
analogs derived from this series will be presented. This series of positive allosteric
modulators of mGlu4 provide critical research tools to further probe the mGlu4-
mediated effects in Parkinson’s disease.
KEYWORDS: metabotropic glutamate receptor 4, mGlu4, structure-activity relationship, parallel synthesis
’ INTRODUCTION
scaffolds. In addition to known mGlu4 PAM, PHCCC, 1,7,10
we have reported a number of distinct structural classes of mGlu4
PAMs, such as VU0155041, 2,11,12 VU0080241, 3,13 VU0001171,
4,14 VU0092145, 5,14 VU0361737, 6,15 VU0364439, 7,16 and
VU0366037,18 8 (Figure 1); of which, two analogs (2 and 6) have
been shown to display efficacy in anti-Parkinsonian animal
models.11,15 However, all of the reported compounds bear striking
resemblance in that they all contain polyaromatic moieties, making
these compounds planar, and they do not contain any basic nitro-
gens. We now report a novel scaffold, represented by VU0105737,
9 (HTS hit, EC50 = 7.5 μM), as possessing mGlu4 PAM activity
(Figure 2). This new chemotype is comprised of a nonplanar
homopiperazine central core which contains a basic nitrogen
(which can presumably lead to more soluble compounds than
previously identified). Because of this structural novelty and the
encouraging calculated properties, this scaffold provided an ex-
cellent opportunity to explore the SAR around this initial HTS lead
as part of our hit-to-lead program.
The development of new strategies for the treatment of Parkin-
son’s disease (PD) continues to be a major focus of attention.1-4
PD is a neurodegenerative disease caused by the degeneration of
dopaminergic neurons in the substantia nigra pars compacta of the
basal ganglia, which leads to a debilitating movement disorder.
Metabotropic glutamate receptor 4 (mGlu4) is expressed at a key
synapse in the indirect pathway of the basal ganglia circuitry.5,6
Several studies have shown that the activation of mGlu4 has anti-
Parkinsonian and neuroprotective effects in rodent PD models.6,7
These results have established this receptor as a viable target for the
symptomatic and disease modifying treatment of PD. We have
taken the approach of selectively activating mGlu4 using positive
allosteric modulators (PAMs), compounds which increase the
potency of the endogenous neurotransmitter glutamate at mGlu4.8,9
Compounds in Vanderbilt’s small molecule library were
screened in a triple-add assay format at 10 μM to identify agonist,
antagonist, or allosteric modulator activity in CHO cells stably
expressing human mGlu4 and the chimeric G protein Gqi5. Test
compounds were added to cells 140 s prior to addition of an EC20
concentration of glutamate; 90 s later an EC80 concentration of
glutamate was added. Response was measured in a fluorometric
calcium assay, and data were analyzed by finding the maximum
value for each fluorescence trace. This “triple add” approach to
HTS is utilized to identify compounds with different modes of
pharmacology (agonists prior to EC20 addition, antagonists and
negative allosteric modulators after EC80 addition, and positive
allosteric modulators after EC20 addition) in a single experiment.
Using this functional high-throughput screening protocol, we
have previously identified a number of novel mGlu4 PAM
’ RESULTS AND DISCUSSION
The SAR surrounding compound 9 centered around 4 struc-
tural motifs: 1, exploring the 2,4-dimethoxyphenylamide; 2, explor-
ing the linker chain length; 3, exploring the central homopiperazine
ring; and 4, exploring the 4-methoxyphenylsulfonamide (Figure 2).
The homopiperazine, 9, was readily modified using commercially
available acid chlorides, carboxylic acids, sulfonyl chlorides and
Received: October 29, 2010
Revised:
January 5, 2011
Published: February 21, 2011
r
2011 American Chemical Society
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RESEARCH ARTICLE
80 ꢀC, 45-50%) and saponification of the ester (LiOH, THF/
MeOH/H2O, 35%-quant.) afforded the penultimate acid. Finally,
HATU-mediated amide coupling with 2,4-dimethoxyaniline
(DMF, DIEA) provided the final compounds, 15a and 15b.
Results from Table 1 show that little modification of the core
portion of the molecule is tolerated. All modifications of the core
structure led to compounds that were inactive and showed no
potentiation of glutamate (as assessed by the %GluMax). We
utilized the Chinese Hamster Ovary cells expressing human
mGlu4 and the chimeric G protein Gqi5 to induce calcium
mobilization as our pharmacological assay to determine mGlu4
potency. This assay does show fluctuation in the day-to-day
maximal PAM response; because of this, all data have been
normalized to the control compound, 1, as a comparison of
relative efficacy (as noted in % PHCCC).10,16
Keeping the right-side of the molecule constant, investigation
of replacement of the amide aromatic ring with various hetero-
cycles and alkyl groups, and exploration of the linker was next
undertaken (Scheme 2, see Supporting Information for full
experimental procedures). To this end, commercially available
N-Boc homopiperazine, 16, was reacted with 4-methoxybenze-
nesulfonyl chloride (CH2Cl2, DIEA) to give 17 in high yield
(98%). Deprotection of the Boc group (4 M HCl, dioxane,
quant.) furnished the key intermediate 18 which was converted
to the urea, 20i, via reaction with 2,4-dimethoxyphenyl isocya-
nate (CH2Cl2, DIEA) in good yield (80%). Alternatively, 18
was N-alkylated with methyl bromoacetate (Cs2CO3, CH3CN,
80 ꢀC, 76%), followed by saponification of the ester (LiOH,
THF/H2O/MeOH, quant.), which gave the penultimate inter-
mediate, 19, in good overall yield. The carboxylic acid was then
coupled to a variety of anilines and amines utilizing HATU as the
coupling reagent (HATU, DMF, DIEA, 45-85%) to provide the
desired compounds (20a-j).
Figure 1. Structures of PHCCC, 1, and other recently reported mGlu4
PAMs, 2-9.
The results in Table 2 show that the original hit with the 2,4-
dimethoxyphenyl substituent was weakly active (9, >10 μM, 69.5%
PHCCC) as was the 2-methoxyphenyl substituent (20a, >10 μM,
63.9% PHCCC); however, both compounds showed good efficacy
(>100% PHCCC). Replacing the 2-methoxyphenyl with 4-meth-
oxyphenyl led to an inactive compound (20b, 14.8% PHCCC).
Other substitution patterns, such as 2,4-difluorophenyl (20c),
2-fluorophenyl (20d), 4-pyridyl (20e), cycloalkyl (20f, 20g), and
cyclicdioxoaryl compound (20h) were all inactive. In addition,
changing the length of the carbon linker also produced inactive
compounds (20i, 20j). This rather “shallow” SAR is a common
occurrence with allosteric modulators.4,5
The last portion of the molecule for modification was the
right-hand sulfonamide moiety (Scheme 3, Table 3, see Support-
ing Information for full experimental details). Starting with 2,4-
dimethoxyaniline, 21, amide formation with bromoacetyl bro-
mide (CH2Cl2, DIEA, 81%) gave the desired R-bromo-amide,
22, which was N-alkylated with Boc-homopiperazine (Cs2CO3,
CH3CN, 80 ꢀC, 79%) to give 23 in 64% yield (2 steps). The Boc-
protecting group was removed (4 M HCl, dioxane, quant.) giving
the key homopiperazine intermediate, 24, which was coupled
with the appropriate sulfonyl chloride (CH2Cl2, DIEA, 45-
85%) to furnish the desired sulfonamides 25a-al.
Figure 2. Areas of SAR Exploration of VU0105737, 9.
other core starting materials. The parallel synthesis and biological
evaluation of this structure class is detailed below. All parallel
synthetic procedures were conducted utilizing either test tube
reaction blocks or stir-plate mounted vial racks.
Modification of the central homopiperazine core was per-
formed to address the basicity of the homopiperazine nitrogens
(piperazine, oxohomopiperazine, oxopiperazine) as well as to
change the shape by introduction of the bridged [3.3.0] scaffold
(Scheme 1. All experimental procedures are contained in the-
Supporting Information). The piperazine compound, 11, and
[3.3.0]-compound, 13, were prepared from N-Boc starting
materials, 10 and 12, via a three step protocol of sulfonylation,
Boc removal and alkylation. The oxo-containing compounds
started from piperazin-2-one (14a) or 1,4-diazepan-5-one (14b).
Thus, sulfonamide formation with 4-methoxybenzenesulfonyl
chloride (CH2Cl2, DIEA, 43%-quant.), followed by alkylation
of the amide with methyl bromoacetate (Cs2CO3, CH3CN,
The compounds evaluated in Table 3 cover a range of phenyl,
benzyl, alkyl, and heteroaryl groups. The phenyl group showed good
activity and efficacy (25a, 3.5 μM, 69.7% PHCCC). However,
substitution at the 2-position only tolerated F (25b, 3.5 μM, 36.6%
PHCCC) and Cl (25c, >10 μM, 25.8% PHCCC) with the latter
being only a weak potentiator. The 4-position was the most
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RESEARCH ARTICLE
Scheme 1a
a Reagent and conditions: (a) 4-methoxybenzenesulfonyl chloride, CH2C12, EtNiPr2 (43% -quant.); (b) 4 M HC1 dioxane (quant.); (c) 22 or methyl
bromoacetate, Cs2CO3, CH3CN 80 ꢀC (45-50%); (d) LiOH, THF, MeOH, H2O (35%-quant.); (e) NaH, DMF, methyl bromoacetate; (f) R1R2NH,
HATU, DMF, EtNiPr2 (19-60%).
Table 1. SAR Evaluation of Linker and Scaffold
a EC50 and GluMax, are the average of at least three independent determinations performed in triplicate (mean ( SEM shown in table). PHCCC is run
as a control compound each day, and the maximal response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs varies slightly in each
experiment. Therefore, efficacy data were further normalized to the relative PHCCC response obtained in each day’s run. b Inactive compounds are
c
defined as %GluMax did not surpass 2X the EC20 value for that day’s run. All yields were obtained by reverse phase preparative HPLC and were
optimized for purity (>95%) not yield.
tolerated with 4-CF3-phenyl (25g, 2.1 μM, 26.6% PHCCC) and
4-Me-phenyl (25i, 3.3 μM, 48.7% PHCCC) being the most potent.
Disubstituted phenyl groups in general showed good activity
especially with dimethylated phenyl groups where the 2,4-dimethyl-
phenyl was the most potent compound of this series (25n, 1.3 μM,
42.8% PHCCC), along with 2,5-dimethyl phenyl (25o, 2.7 μM,
32.5% PHCCC). 2,4,5-Trisubstituted phenyl groups also showed
potencies as good or better than the original HTS hit (25x, 3.1 μM,
43.6% PHCCC and 25y, 2.7 μM, 36.9% PHCCC).
The limited SAR around the benzyl group showed contrasting
activity compared to the phenyl group. For example, the benzyl
group was a weak potentiator (25z, >10 μM, 46.5% PHCCC).
Furthermore the 2,4-dichlorobenzyl group showed good
potency (25aa, 2.8 μM, 34.4% PHCCC), which in the case of
the dihalogenated phenyl groups were all inactive. Compounds
25ab and 25ac showed that alkyl groups were not tolerated.
Substitution with heteroaryl groups led to 3 compounds with
acceptable potency. The most potent was the 2-thienyl (25ae, 1.8
μM, 52.9% PHCCC), followed by the 2-methyl-4-trifluoro-
methylthiazol-5-yl (25al, 2.5 μM, 28.1% PHCCC) and the
2-furyl (25af, 3.3 μM, 36.2% PHCCC).
Compounds 9, 20a, 25a, 25i, and 25ae were selected based on
potency and efficacy for further in vitro pharmacokinetic (PK)
studies, which included CYP450 inhibition, metabolic stability
(CLINT),19 and rat plasma protein binding (rPPB) (Table 4,
see Supporting Information for assay details). Although these
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RESEARCH ARTICLE
Scheme 2. Synthetic Procedure for the Initial Homopiperazine Analogs 20a-ja
a Reagent and conditions: (a) 4-methoxybenzenesulfonyl chloride, CH2Cl2, EtNiPr2 (98%); (b) 4 M HCl dioxane (quant.); (c) Br(CH2)nCO2Me,
Cs2CO3, CH3CN 80 ꢀC (40-76%); (d) LiOH, THF, MeOH, H2O (quant.); (e) R1R2NH, HATU, DMF, EtNiPr2 (2-80%); (f) 2,4-dimethoxyphenyl
isocyanate, CH2Cl2, EtNiPr2 (80%)
Table 2. SAR Evaluation of Amide Aromatic Ring
cmpd
n
R
hEC50 (μM)a
%PHCCCa
yieldc (%)
9
1
1
1
1
1
1
1
1
1
0
3
2,4-dimethoxyphenyl
2-methoxyphenyl
4-methoxyphenyl
2,4-difluorophenyl
2-fluorophenyl
>10
69.5 ( 7.7
63.9 ( 3.6
14.8 ( 0.8
17.7 ( 1.6
13.2 ( 0.4
13.9 ( 1.2
15.5 ( 1.3
15.0 ( 0.8
17.6 ( 1.0
13.5 ( 0.7
14.8 ( 1.3
61
64
20d
3d
2d
2d
12d
19d
12d
80
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
>10
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
3.1 ( 0.3
4-pyridyl
cyclohexyl
isopropyl
2,3-dihydrobenzo[b][1,4]dioxin-6-yl
2,4-dimethoxyphenyl
2,4-dimethoxyphenyl
(()-PHCCC
70
a EC50 and GluMax are the average of at least three independent determinations performed in triplicate (mean ( SEM shown in table). PHCCC is run as
a control compound each day, and the maximal response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs varies slightly in each
experiment. Therefore, efficacy data were further normalized to the relative PHCCC response obtained in each day’s run. b Inactive compounds are
defined as %GluMax did not surpass 2Â the EC20 value for that day’s run. c All yields were obtained by reverse phase preparative HPLC unless otherwise
stated and were optimized for purity (>95%) not yield. d Yields obtained by mass directed HPLC.17
aScheme 3
a Reagent and conditions: (a) bromoacetyl bromide, CH2Cl2, EtNiPr2(81%); (b) Boc-homopiperazine, CH3CN, Cs2CO3, 80 ꢀC (79%); (c) 4 M HCl
dioxane (quant.); (d) RSO2Cl, EtNiPr2, CH2Cl2 (5-98%).
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Table 3. SAR Evaluation of Sulfonamide Aromatic Ringa
cmpd
R
hEC50 (μM)
%PHCCC
yieldc (%)
25a
25b
25c
phenyl
3.5 ( 0.7
3.5 ( 0.5
>10
Inactiveb
Inactiveb
Inactiveb
2.5 ( 0.5
>10
69.7 ( 5.1
36.6 ( 1.8
25.8 ( 2.5
21.8 ( 1.0
20.1 ( 0.2
18.2 ( 1.9
26.6 ( 2.3
48.2 ( 4.6
48.7 ( 1.1
27.6 ( 1.3
43.6 ( 4.4
32.6 ( 3.1
42.9 ( 2.7
42.8 ( 4.1
32.5 ( 1.2
22.7 ( 0.7
18.1 ( 0.9
22.3 ( 1.7
42.4 ( 5.0
39.2 ( 1.2
24.3 ( 1.5
27.0 ( 1.8
25.5 ( 1.2
43.6 ( 4.7
36.9 ( 2.7
46.5 ( 4.8
34.4 ( 3.5
17.5 ( 0.7
21.3 ( 0.9
20.6 ( 0.3
52.9 ( 5.0
36.2 ( 2.2
25.6 ( 4.6
34.1 ( 3.6
35.0 ( 3.3
46.4 ( 5.5
47.7 ( 2.1
28.1 ( 4.8
80
3d
2-fluorophenyl
2-chlorophenyl
2-methoxyphenyl
58
87
24d
6d
25d
25e
25f
2-(trifluoromethyl)phenyl
3-(trifluoromethyl)phenyl
4-(trifluoromethyl)phenyl
4-(trifluoromethoxy)phenyl
4-methylphenyl
25g
25h
25i
36
5d
3.3 ( 0.2
>10
77
33d
41
32
65
58
67
67
75
23
67
43
31
64
53
44
36
12d
44
22d
27d
36
83
98
20
70
79
60
73
49
25j
4-fluorophenyl
25k
25l
4-chlorophenyl
4.1 ( 0.6
>10
4-tertbutylphenyl
4-acetylphenyl
25m
25n
25o
25p
25q
25r
>10
2,4-dimethylphenyl
2,5-dimethylphenyl
2,5-dichlorophenyl
2-chloro-6-methylphenyl
2,6-dichlorophenyl
3,4-dimethylphenyl
3-chloro-4-methylphenyl
3,4-dichlorophenyl
3,4-difluorophenyl
4-chloro-3-fluorophenyl
2,4-dichloro-5-methylphenyl
4-chloro-2-fluoro-5-methylphenyl
benzyl
1.3 ( 0.5
2.7 ( 0.6
inactiveb
inactiveb
inactiveb
3.2 ( 0.2
3.4 ( 0.2
>10
25s
25t
25u
25v
25w
25x
>10
4.5 ( 0.7
3.1 ( 0.9
2.7 ( 0.2
>10
25y
25z
25aa
25ab
25ac
25ad
25ae
25af
25ag
25ah
25ai
25aj
25ak
25al
2,4-dichlorobenzyl
isopropyl
2.8 ( 0.01
inctiveb
inactiveb
inactiveb
1.8 ( 0.3
3.3 ( 0.3
>10
isobutyl
2-pyridyl
2-thiophene
2-furyl
3-pyridyl
3-furyl
>10
3-thiophene
>10
2-acetamidothiazol-5-yl
2,4-dimethylthiazol-5-yl
2-methyl-5-(trifluoromethyl)thiazol-5-yl
(()-PHCCC
>10
>10
2.5 ( 0.4
3.1 ( 0.3
a EC50 and GluMax, are the average of at least three independent determinations performed in triplicate (Mean ( SEM shown in table). PHCCC is run
as a control compound each day, and the maximal response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs varies slightly in each
experiment. Therefore, data were further normalized to the relative PHCCC response obtained in each day’s run. b Inactive compounds are defined as %
GluMax did not surpass 2Â the EC20 value for that day’s run. c All yields were obtained by reverse phase preparative HPLC unless otherwise stated and
were optimized for purity (>95%) not yield. d Yields obtained by mass directed HPLC17
compounds possessed favorable free fraction when tested for
plasma protein binding in rats (>3% free), this class of com-
pounds was very unstable in liver microsomes (CLINT) and were
equipotent with inhibiting CYP activity (2C9 and 3A4 < 10 μM).
’ CONCLUSIONS
In summary, through a functional HTS campaign, we identi-
fied a novel chemotype as an mGlu4 PAM and SAR was explored
around 4-structural motifs. These compounds represent a series
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RESEARCH ARTICLE
Table 4. In Vitro Pharmacokinetic Data for Selected Compounds
CYP
b
cmpd
1A2
2C9
2D6
3A4
rPPB, %fua
human CLINT (CLHEP)
9
>30 μM
>30 μM
>20 μM
16.29 μM
8.46 μM
1.8 μM
2.0 μM
2.9 μM
2.3 μM
2.6 μM
>20 μM
>20 μM
>20 μM
13.9 μM
10.1 μM
4.2 μM
4.6 μM
7.0 μM
3.0 μM
7.4 μM
3.9
3.5
908.58 (20.53)
893.34 (20.52)
559.86 (20.24)
731.45 (20.41)
517.92 (20.18)
20a
25a
25i
25ae
11.5
3.1
27.3
a rat protein binding, % free unbound. b Intrinsic clearance, human (CLINT). Predicted hepatic clearance (CLHEP).
of homopiperazine sulfonamides. Limited SAR around this
scaffold suggests a possible alternate allosteric binding site
compared to previously disclosed mGlu4 PAMs, where more
robust SAR was identified. Unfortunately, these compounds
possess less than ideal PK properties (poor metabolic stability,
CYP 2C9, and CYP 2D6 inhibition) preventing their further
study. However, as this class of compounds represent a novel
chemotype in the mGlu4 PAM area, we anticipate these com-
pounds will inform the community for future scaffold design.
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’ ASSOCIATED CONTENT
S
Supporting Information. Details of experimental proce-
b
dures and spectroscopic data for synthesized compounds and
biological procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 615-936-6892. Fax: 615-936-4381. E-mail: corey.r.hopkins@
vanderbilt.edu.
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Funding Sources
This work was supported by the National Institute of Mental
Health, National Institute of Neurological Disorders and Stroke,
NIH/MLPCN (5U54MH084659-02), the Michael J. Fox Foun-
dation, the Vanderbilt Department of Pharmacology, and the
Vanderbilt Institute of Chemical Biology. Vanderbilt is a member
of the MLPCN and houses the Vanderbilt Specialized Chemistry
Center for Accelerated Probe Development (NIH/MLPCN;
5U54MH084659-02).
’ ACKNOWLEDGMENT
The authors thank Emily L. Days, Tasha Nalywajko, Cheryl A.
Austin, and Michael Baxter Williams for their critical contribu-
tions to the HTS portion of the project. In addition, we would
like to thank Katrina Brewer, Ryan Morrison, and Matt Mulder
for technical assistance with the PK assays, and Chris Denicola,
Nathan Kett, and Sichen Chang for the purification of com-
pounds utilizing the mass-directed HPLC system.
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