Synthesis, structure–activity relationships and biological evaluation of 4,5,6,7-tetrahydropyrazolopyrazines as metabotropic glutamate receptor 5 negative allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2016.07.019

The design, synthesis and SAR studies of novel 4,5,6,7-tetrahydropyrazolopyrazines as metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) are presented in this letter. Starting from a HTS hit compound (1, IC₅₀?=?477

Full text of DOI:10.1016/j.bmcl.2016.07.019

Accepted Manuscript
Synthesis, Structure-Activity Relationships and Biological Evaluation of
4,5,6,7-Tetrahydropyrazolopyrazines as Metabotropic Glutamate Receptor 5
Negative Allosteric Modulators
Wataru Hirose, Yoshihiro Kato, Takayoshi Yamamoto, Momoe Kassai, Makoto
Takata, Shun Hayashi, Yukiyo Arai, Satoki Imai, Kohzo Yoshida
PII:
S0960-894X(16)30721-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.019
BMCL 24063
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
17 May 2016
5 July 2016
7 July 2016
Please cite this article as: Hirose, W., Kato, Y., Yamamoto, T., Kassai, M., Takata, M., Hayashi, S., Arai, Y., Imai,
S., Yoshida, K., Synthesis, Structure-Activity Relationships and Biological Evaluation of 4,5,6,7-
Tetrahydropyrazolopyrazines as Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.019
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Bioorganic & Medicinal Chemistry Letters
Synthesis, Structure-Activity Relationships and Biological Evaluation of 4,5,6,7-
Tetrahydropyrazolopyrazines as Metabotropic Glutamate Receptor 5 Negative
Allosteric Modulators
Wataru Hirosea,*, Yoshihiro Katoa, Takayoshi Yamamotoa, Momoe Kassaib, Makoto Takatab, Shun
Hayashib,Yukiyo Araia, Satoki Imaib and Kohzo Yoshidaa,
a Drug Research Division, Sumitomo Dainippon Pharma, 3-1-98 Kasugade-naka, Konohana-ku, Osaka, 554-0022, Japan
b Drug Research Division, Sumitomo Dainippon Pharma, 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The design, synthesis and SAR studies of novel 4,5,6,7-tetrahydropyrazolopyrazines as
metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) are
presented in this letter. Starting from a HTS hit compound (1, IC50 = 477 nM), optimization of
various groups led to the synthesis of a potent mGluR5 NAM (32, IC50 = 75 nM) with excellent
rat PK profile and good brain penetration. This compound produced oral antidepressant-like
effect in a mouse tale suspension model (MED: 30 mg/kg).
Keywords:
mGluR5
2009 Elsevier Ltd. All rights reserved.
Negative allosteric modulator
Hit to lead studies
CNS drug discovery
Metabotropic glutamate receptors (mGluRs) are members of
the G protein-coupled receptor (GPCR) class C and are divided
into eight subtypes based on their sequence homology,
downstream signal transduction and pharmacological output.1, 2
As the mGluR5 is post-synaptically expressed in the central
nervous system (CNS) and periphery,3 modulation of mGluR5
would be useful for the treatment of both central and peripheral
nervous systems disorders. Whereas mGluR5 orthosteric
antagonists often have poor selectivity for mGluR subtypes and
limited ability to penetrate biological membranes due to their
hydrophilicity,4 mGluR5 negative allosteric modulators (NAMs)
are favorable small molecule drug candidates due to their good
selectivity and appropriate pharmacokinetic properties.5 Several
preclinical and clinical evidence supports the therapeutic
potential of mGluR5 NAMs in the treatment of CNS disorders,
including anxiety, depression, Parkinson’s disease levodopa-
induced dyskinesia (PD-LID), and fragile X syndrome (FXS).6 In
addition, a number of phase II or III clinical trials with mGluR5
NAMs for different indications, including Mavoglurant (AFQ-
Most of the compounds advanced in clinical trials (Figure 1)
are characterized by a di-substituted alkyne as a common
structure that can potentially be influenced by metabolic
activation, leading to unacceptable toxicity.8 In addition, the
intellectual space around the disubstituted alkyne chemotypes is
quite crowded. These concerns continuously urged medicinal
056,
Novartis),
Dipraglurant
(ADX-48621,
Addex),
Basimglurant (RG-7090, Roche) and STX-107 (Seaside
Therapeutics, Roche) are completed or currently underway.7
However, no mGluR5 NAM drug has so far been launched.
———
Figure 1. Structures of representative mGluR5 NAMs and the alkyne-
lacking HTS hit compound 1.
 Corresponding author. Tel.: +81-6-6466-5947; fax: +81-6-6466-5287; e-mail: wataru-hirose@ds-pharma.co.jp (W. Hirose), yoshida-
k@phs.osaka-u.ac.jp (K. Yoshida)

Scheme 1. Reagents and conditions: (a) ethyl diazoacetate, toluene, reflux; (b) chloroacetonitril, NaH, DMF, 0 °C to rt; (c) NaBH4, CoCl2, MeOH, rt, then
K2CO3; (d) LiALH4, THF, reflux; (e) tert-butyl (2-hydroxyethyl)carbamate, diisopropyl azodicarboxylate, Ph3P, THF, 0 °C to rt; (f) LiAlH4, THF, 0 °C; (g)
MsCl, iPr2NEt, CH2Cl2, 0 °C; (h) NBS, Ph3P, CH2Cl2, 0 °C; (i) NaH, DMF, 50 °C; (j) 4 M HCl in dioxane or AcOEt, rt; (k) R3COOH, EDCI, HOBt, DMF, rt; (l)
R3COCl, Et3N, CH2Cl2, 0 °C.
chemists to seek novel mGluR5 NAMs without the alkyne
structure.9 Our research program has also been directed toward
the development of novel non-alkyne mGluR5 NAMs. We
describe herein the design, synthesis, structure-activity
relationship (SAR) and biological profiles of a novel class of
mGluR5 NAMs possessing 4,5,6,7-tetrahydropyrazolopyrazine
as a core structure.
Table 1. Tetrahydropyrazolopyrazine analogues in vitro inhibitory activity
for human mGluR5.
compd
1
R1
R3
IC50 (nM)
477
In our drug discovery program for novel mGluR5 NAMs,
4,5,6,7-tetrahydropyrazolopyrazine derivative 1 (Figure 1) was
selected through a high-throughput screening using our corporate
library as a hit compound, which has a moderate inhibitory
activity for human mGluR5 (IC50: 477 nM). Our initial study was
an investigation of the SAR in order to identify a lead compound
and develop its optimization strategy. We first determined
functional IC50 values of the synthesized compounds for
inhibition of human mGluR5 using calcium mobilization assay
with HEK293 cells expressing recombinant human receptor and
EC90 concentration of L-glutamate.
tBu
2-naphthyl
11
Ph
Ph
Ph
Ph
Ph
127
12
4-pyridyl
3-pyridyl
2-pyridyl
1,293
>10,000
235
13
14
of 2-naphthyl group of 1 to a monocyclic substituent group, such
as phenyl, pyridyl, or pyrrolyl, also led to decrease of inhibitory
activity (data not shown). Various combinations of substitutions
were then explored as shown in Table 1, leading to compound 11
(R1 and R3 = Ph) with improved mGluR5 inhibitory activity
(IC50: 127 nM). However, this compound showed poor aqueous
solubility (2 g/mL at pH 7.4, 4 g/mL at 1.2). To overcome this
drawback, a nitrogen atom was introduced into the left phenyl
ring (12 - 14) affording only 14 (R1 = 2-pyridyl and R3 = Ph) as a
compound with moderate mGluR5 inhibitory activity (IC50: 235
nM) and sufficiently improved aqueous solubility (>100 g/mL
at both pH 7.4 and 1.2). Additionally, compound 14 showed good
liver microsomal stability (human: 92%, rat: 95%), and low IC50
value for CYP enzymes (IC50: >50 M for CYP1A2, 2A6, 2C8,
2C9, 2C19, 2D6 and 3A4 isoforms). These good
physicochemical and metabolic properties seemed to be
appropriate for selection of 14 as lead compound.
We developed two versatile synthetic routes that allow easy
access to tetrahydropyrazolopyrazine derivatives with various
substituents (Scheme 1). The required pyrazole derivatives 3
were purchased or prepared via 1,3-dipolar cycloaddition
reaction with the commercially available alkynes 2 and ethyl
diazoacetate. In route A, alkylation of 3 afforded the nitrile
derivatives 4. Reduction of the cyano group of 4 with NaBH4 in
the presence of CoCl2 followed by cyclization generated the
tetrahydropyrazolopyrazinones 5 whose lactam moiety was
reduced by LiAlH4 to give the precursor amines 6. In route B,
Mitsunobu
reaction
of
3
and
tert-butyl
(2-
hydroxyethyl)carbamate afforded 7. Reduction of the ester of 7
with LiAlH4 produced the corresponding alcohols 8.
Halogenation of 8 with MsCl or N-bromosuccinimide generated
9,10 which were then cyclized using NaH to give 10. Deprotection
of the Boc group in 10 with hydrochloric acid gave 6. Finally,
amidation of 6 with appropriate carboxylic acids or acyl halides
We next turned our attention to the effects of various
substituents on the left-hand 2-pyridyl group and the right-hand
phenyl group of compound 14 (Table 2). Substitution of a
chloride at the ortho-, meta- or para-position of the phenyl group
(15-17) resulted in only the meta-substitution being tolerated. Di-
substitution of the chloride in the 3,5-position (18) was also
tolerated. Next, the effects of a substitution at the 3-, 4-, 5- or 6-
position of the left-hand 2-pyridyl group (19-24) were
investigated. The 3-Me (19), 4-Me (20) and 6-Me (22)
substituted compounds had weaker mGluR5 inhibitory activity
than the parent lead compound 14, whereas the 5-Me (21), 5-F
provided compounds 11-32. The precursor amines
6 of
compound 11-18 (R1 = Ph, 4-pyridyl, 3-pyridyl or 2-pyridyl)
were synthesized via route A, and the others (R1
monosubstituted 2-pyridyl) were synthesized via route B.
=
In order to identify the minimum pharmacophore, we initially
replaced the left-hand and the right-hand partial structures of
compound
1
with
a
smaller substituent, respectively.
t
Replacement of Bu group of 1 by methyl or a lower alkyl
resulted in severe decrease of the inhibitory activity. Conversion

Table 2. Tetrahydropyrazolopyrazine analogues in vitro inhibitory activity
for human mGluR5.
Table 3. Compound 32 physicochemical properties and in vitro ADMET
profile.
compound 32
CYP inhibition (IC50)
CYP induction
>40 M
Negative
37.0 x 10-6 cm/s
1.2
compd
14 (lead)
15
R4
R5
IC50 (nM)
235
PAMPA (pH 7.4)
P-gp efflux ratio
H
H
H
2-Cl
3-Cl
4-Cl
3,5-di-Cl
H
2,477
176
hERG inhibition (IC50)
15 M
16
H
17
H
9,469
270
Table 5. Compound 32 brain exposure profile in mice.
18
H
dose
B/P
1.38
Cp (M)
Cb (M)
Cb,u (M)
19
3-Me
4-Me
5-Me
6-Me
5-F
5-Cl
5-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
>10,000
451
30 mg/kg, po
21.4
29.5
1.38
20
H
21
H
244
In addition, compound 32 showed sufficient cell permeability
and low potential as P-glycoprotein substrate, which suggest
good brain penetration. Finally, compound 32 has low risk of
hERG channel inhibition. Considering all of these encouraging
results, we decided to evaluate the pharmacological effects of 32
in rodents.
22
H
568
23
H
164
24
H
201
25
3-Cl
3-F
150
Pharmacokinetic (PK) studies were performed in rats at 1
mg/kg, iv and 10 mg/kg, p.o. Compound 32 PK parameters
(Table 4) show a favorable profile with long half-life (9.2 hr) and
low clearance (2.3 mL/min/kg), consistent with high liver
microsomal stability in rat. Oral administration of 32 to rats
resulted in sufficient plasma concentration (Cmax: 2930 ng/mL,
AUC: 68,744 hr·ng/mL) and excellent bioavailability (95%).
26
257
27
3-Me
3-OMe
3-CN
3-CF3
3-F, 5-Me
3-Cl, 5-F
150
28
482
29
>10,000
383
30
31
197
Brain exposure studies of compound 32 were performed in
ICR mice at 30 mg/kg, p.o. (Table 5). One hour after
administration, compound 32 showed good brain penetration
ratio (B/P = 1.38) based on plasma/brain concentration. Protein
binding in the mouse brain was 95%, and brain concentration for
unbound compound 32 was 1.38 M, which is sufficiently above
this compound in vitro mGluR5 IC50 value (75 nM).
32
75
(23) or 5-Cl (24) substituted compounds had an inhibitory
activity comparable to that of compound 14 (IC50: 244, 150 and
201 nM, respectively). Fixing the 5-F group as the most suitable
R4 substituent for good mGluR5 inhibitory activity, several R5
substituents at the meta-position of the right-hand phenyl group
were examined. As shown in Table 2, small substituents, such as
a chloro (25), fluoro (26), and methyl (27) groups were tolerable
(IC50: 150, 257 and 150 nM, respectively), while a methoxy (28),
cyano (29) and triflouromethyl (30) groups decreased mGluR5
inhibitory activity (IC50: 482, >10,000 and 383 nM, respectively).
A marked improvement in the activity was seen with compound
32 (R4 = 5-F and R5 = 3-Cl, 5-F), which showed 6-fold increased
in inhibitory activity (IC50: 75 nM) compared to the HTS hit 1.
Compound 32 also showed moderate aqueous solubility (7
g/mL at pH 7.4, 79 g/mL at pH 1.2) and appropriate liver
microsomal stability (human: >97%, rat: >97%). Other
physicochemical properties as well as in vitro ADMET profile of
32 are shown in Table 3. There seems to be no serious concern
regarding compound 32 CYP enzyme inhibition (IC50: >40 M
for CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4 isoforms) or
induction (human AhR and human PXR: negative).
Encouraged by good PK and brain exposure profiles of 32, we
evaluated the in vivo pharmacological effect of this compound in
the mouse tail suspension test as a behavioral model of
depression (Figure 2). Single oral administration of 32 at 30
mg/kg significantly decreased mice immobility time, suggesting
an antidepressant-like effect. This effect, which was predicted
from the results of brain exposure in mice, was comparable to
that of imipramine, an antidepressant drug used as a positive
control in the same model.
In conclusion, optimizing the HTS hit compound 1 led to a
series of tetrahydropyrazolopyrazine derivatives as novel
mGluR5 NAMs. In particular, compound 32 showed high in vitro
affinity for mGluR5, good in vivo oral bioavailability and brain
penetration, and clear antidepressant-like effect in a mouse tale
suspension model. Further studies of compound 32 aswell as
other mGluR5 NAMs are expected to generate novel therapeutic
Table 4. Compound 32 pharmacokinetic parameters in rat following single administrationa.
dose
t1/2 (hr)
CL (mL/min/kg)
2.3
Vdss (L/kg)
AUCb (hr·ng/mL)
7,220
Cmax (ng/mL)
BA (%)
N/A
1 mg/kg, iv
10 mg/kg, po
9.2
1.5
68,744
2,930
95
a Data are presented as the mean of 2 (iv) or 3 (po) rats per cohort.
b AUC from time 0 to infinity after test compound administration.

Figure 2. Effects of compound 32 and imipramine in the mouse tail
suspension test. Each bar represents the mean ± S.E.M of immobility time
during a 6 min test session (n = 8 per each group). **P < 0.01, Differences
between imipramine-treated group and the vehicle-treated group were
assessed using Student's t-test. Differences between compound 32-treated
group and the vehicle-treated group were determined using parametric
Dunnett's multiple comparison test, following significant results of one-way
ANOVA. **P < 0.01 was considered as significant.
agents for depress ion and other CNS disorders.
Acknowledgments
We thank Ms. Maki Yamaguchi for chemical synthesis of
compounds, Ms. Keiko Yokogawa for HRMS, Mr. Osamu Odai
and Dr. Norio Fujiwara for useful discussions.
Supporting Information
Supporting information including compound data and
experimental procedure of biological tests can be found in
the online version, at
References and notes
1. For reviews on metabotropic glutamate receptors, see: (a) Conn, P.
J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205. (b)
Niswender, C. H.; Conn, P. J. Annu. Rev. Pharmacol. Toxicol.
2010, 50, 295.
2. Crawford, J. H.; Wainwright, A.; Heavens, R.; Pollock, J.; Martin,
D. J.; Scott, R. H.; Seabrook, G. R. Neuropharmacology 2000, 39,
621.
3. Chizh, B. A. Amino Acids 2002, 23, 169.
4. Schoepp, D. D.; Jane, D.E.; Monn, J. A. Neuropharmacology 1999,
38, 1431.
5. Ritzén1, A.; Mathiesen, J. M.; Thomsen, C. Basic Clin. Pharmacol.
Toxicol. 2005, 97, 202.
6. (a) Bach, P.; Isaac, M.; Slassi, A. Expert Opin. Ther. Pat. 2007,
17, 371. (b) Jacschke, G.; Wettstein, J. G.; Nordquist, R. E.;
Spooren, W. Expert Opin. Ther. Pat. 2008, 18, 123.
7. Conn, P. J.; Lindsley, C. W.; Meiler, J.; Niswender, C. M. Nat.
Rev. 2014, 13, 692.
8. Smith, G. F. Prog. Med. Chem. 2011, 50, 1.
9. (a) Emmitte K. A. ACS Chem. Neurosci. 2011, 2, 411. (b) Emmitte,
K. A. Expert Opin. Ther. Patents. 2013, 23, 393.
10. Direct chlorination was achieved by treatment with MsCl.
11. Compound 32; white solid; 1H NMR (300 MHz, CDCl3,  ppm):
8.47 (d, J = 2.8 MHz, 1H), 7.90 (dd, J = 8.8, 4.4 Hz, 1H), 7.44
(ddd, J = 8.8, 8.8, 2.9Hz, 1H), 7.27-7.22 (m, 2H), 7.10 (ddd, J =
7.9, 2.4, 1.3Hz, 1H), 6.64 (brs, 1H), 4.85 (brs, 2H), 4.33-3.80 (brm,
4H); 13C NMR (100 MHz, CDCl3, ppm):  168.1, 163.9, 161.4,
160.3, 157.7, 151.3, 148.3, 148.3, 137.8, 137.5, 136.2, 136.1,
123.7, 123.5, 121.0, 120.9, 118.6, 118.4; MS (ESI) m/z: 375.3
(M+H); HRMS (ESI) m/z calcd. for C18H13ClF2N4ONa
(M+Na)+: 397.0638, found: 397.0648.

Synthesis, Structure-Activity Relationships
and Biological Evaluation of 4,5,6,7-
Tetrahydropyrazolopyrazines as
Leave this area blank for abstract info.
Metabotropic Glutamate Receptor 5
Negative Allosteric Modulators
Wataru Hirose, Yoshihiro Kato, Takayoshi Yamamoto, Momoe Kassai, Makoto Takata, Shun Hayashi, Yukiyo
Arai, Satoki Imai, Kohzo Yoshida
mouse tale suspension test