Discovery and in vitro and in vivo profiles of 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as novel class of an orally active metabotropic glutamate receptor 1 (mGluR1) antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.07.097

We identified 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide 27 as a potent mGluR1 antagonist. The compound possessed excellent subtype selectivity and good PK profile in rats. It also demonstrated relatively potent a

Full text of DOI:10.1016/j.bmcl.2009.07.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5464–5468
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and in vitro and in vivo profiles of 4-fluoro-N-[4-[6-(isopropylamino)
pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as novel class of an orally
active metabotropic glutamate receptor 1 (mGluR1) antagonist
Atsushi Satoh, Yasushi Nagatomi, Yukari Hirata, Satoru Ito, Gentaroh Suzuki, Toshifumi Kimura,
Shunsuke Maehara, Hirohiko Hikichi, Akio Satow, Mikiko Hata, Hisashi Ohta, Hiroshi Kawamoto
*
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We identified 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide
27 as a potent mGluR1 antagonist. The compound possessed excellent subtype selectivity and good PK
profile in rats. It also demonstrated relatively potent antipsychotic-like effects in several animal models.
Suitable for development as a PET tracer, compound 27 would have great potential for elucidation of
mGluR1 functions in human.
Received 28 May 2009
Revised 17 July 2009
Accepted 18 July 2009
Available online 23 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
mGluR1 antagonist
Antipsychotic
Biarylamide
Glutamate is one of the major excitatory neurotransmitters in the
centralnervoussystem(CNS)andactsonionotropicglutamaterecep-
tors including NMDA and non-NMDA receptors and on G-protein
coupled metabotropic glutamate receptors (mGluRs). mGluRs are
classified into eight subtypes (three groups) based on sequence
homology, coupling mechanisms to G-protein and pharmacological
properties. mGluRs are considered tobe drug targets for the modulat-
ing glutamate transmission in the treatment of various neurological
and psychiatric diseases includingpain, epilepsy, Parkinson’s disease,
cognitive disorders, drug abuse, anxiety and schizophrenia.1–4
Since CPCCOEt5,6 was identified as the first noncompetitive
mGluR1 antagonist with moderate affinity, a number of rather po-
tent and selective ligands have been developed. In general, non-
competitive mGluR1 antagonists act at the seven transmembrane
domain, not at the glutamate binding N-terminal of the extracellu-
lar domain, and their structural features are very different from
glutamate. For example, thiazolo[3,2,a]benzimidazole amide deriv-
atives,7 2,4-dicarboxypyrrole esters,8 quinoline derivatives9 and
triazafluorenones10 were reported to be low nano-molar mGluR1
antagonists. These structurally diverse agents have facilitated the
elucidation of action mechanisms of noncompetitive mGluR1
antagonists in vitro and revealed their therapeutic potential by
using established animal models. In order to conduct clinical trail
to identify therapeutic utility of an mGluR1 antagonist, noncom-
petitive mGluR1 antagonists need to improve their oral bioavail-
ability. Previous attempts to improve oral bioavailability without
impairing potency have met with little success.
We have reported three kinds of potent and selective mGluR1
antagonist, such as FTIDC 1,11–13 isoindolinone analog 2,14 and quin-
oline analog 315 (Fig. 1). Our antagonists are structurally diverse but
have an aryl-triazole group in common. Consequently completely
structurally distinct classes were still needed to strengthen our ther-
apeutic concept. In order to obtain structurally novel hit compounds
suitable for development into an orally available mGluR1 antagonist,
we primarily screened Merck Research Laboratories (MRL) chemical
library using CHO cells expressing human mGluR1a, with antagonis-
tic activity measured by FLIPR assay. Among the hit compounds,
biarylamidecompound4possessingno triazolegroupshowedmod-
erate antagonistic activity on human mGluR1 receptors with IC50 va-
lue of 210 nM and good mGluR5/mGluR1 selectivity (Fig. 2).
In this Letter we describe the structure–activity relationship of
hit compound 4 and identify a potent, selective and orally active
mGluR1 antagonist in which potency and physicochemical proper-
ties are appropriately balanced.
Various biarylamide derivatives 4–17 and 23 were synthesized
as shown in Scheme 1. Aryl-methylkenone or aryl bromoketone
were cyclized with thiourea to afford 2-amino-5-aryl-thiazole.
Condensation of 2-aminothiazole with various acid chlorides un-
der basic condition provided compounds 4–17 and 23.
* Corresponding author. Current address: Medicinal Chemistry Laboratories,
Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho Kita-ku Saitama 331-9530,
Japan. Tel.: +81 48 669 3029; fax:+81 48 652 7254.
The terminal 2- or 5-pyrimidine derivatives were prepared from
commercially available 2,4-dibromothiazole as shown in Scheme
E-mail address: hiroshi.kawamoto@po.rd.taisho.co.jp (H. Kawamoto).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.097

A. Satoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5464–5468
5465
Figure 1. Structure of reported mGluR1 antagonists.
N-Alkylamide derivatives 20–22 were synthesized as shown in
Scheme 3. Non-alkylated amide 23 was prepared from non-substi-
tuted thiourea as shown in Scheme 1. Compound 23 was treated
with NaH, followed by appropriate alkyl iodide to yield N-alkyla-
mide derivatives 20–22.
6-Substituted pyrimidine derivatives 24–28 were synthesized
from 4,6-dichloropyrimidine as shown in Scheme 4. 4,6-Dichloro-
pyrimidine was reacted with 1-ethoxyvinyl-tri-N-butyltin under
Stille coupling condition to give mono-ethoxyvinylpyrimidine.
Ethoxyvinyl derivative was reacted with N-bromosuccinimide,
followed by treatment with N-methylthiourea to provide 4-pyri-
midinyl-2-methylaminothiazole. Aminothiazole derivative was
condensed with 4-fluorobenzoyl chloride, followed by substitution
of pyrimidine 6-position with several amines to give 6-substituted
pyrimidine derivatives 24–28.
Figure 2. Hit compound derived from HTS screening.
2. Dibromothiazole reacted with methylamine to give 5-bromo-2-
methylaminothiazole, followed by condensation with 4-fluor-
obenzoyl chloride to provide 2-benzoylamide-4-bromothiazole. Fi-
nally, Stille coupling with appropriate tri-butyltin compounds in
the presence of tetrakis (triphenylphosphine) palladium afforded
pyrimidine derivatives 18 and 19.
Scheme 1. Synthesis of biarylamide derivatives 4–17 and 23. Reagents and conditions: (a) I2, 100 °C, 3–60%; (b) Et3N, EtOH, reflux, 70% for R2: 4-pyridyl R3: Me, 95% for R2:
phenyl R3: Me; (c) Et3N, toluene, reflux, 8–77%.
Scheme 2. Synthesis of pyrimidine derivatives 18 and 19. Reagents and conditions: (d) MeNH2, MeOH, rt, 15%; (c) Et3N, toluene, reflux, 73%; (e) Pd(Ph3P)4, toluene, 100 °C, 8%
for R2: 4-pyrimidyl, 15% for R2: 2-pyrimidyl.
Scheme 3. Synthesis of N-alkyl derivatives 20–22. Reagents and conditions: (f) NaH, DMF, rt, 62% for R3: ethyl, 51% for R2: n-propyl, 5% for R2: iso-propyl.

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A. Satoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5464–5468
Scheme 4. Synthesis of 6-substituted pyrimidine derivatives 24–28. Reagents and conditions: (g) Pd(Ph3P)4, DMF, 120 °C, 75%; (h) THF–H2O, rt; (i) EtOH, rt, 73% for two
steps; (c) Et3N, toluene, reflux, 90%; (j) K2CO3, THF, 90 °C, 20–74%.
Our goals were to investigate the structure–activity relationship
of this lead class and to identify structurally diverse mGluR1 antag-
onists which have comparable profiles to our previously developed
antagonists 2 and 3.
Next, we investigated the SAR of R2 portion in order to remove
potentially toxic 4-pyridine structure of the left-hand substituent
which may cause CYP inhibition.16 Replacement of the 4-pyridine
group with various kinds of functional groups was tested as shown
in Table 2. Alkyl groups demonstrated poor antagonistic activity
(data not shown). Phenyl analogue 13 also did not show mGluR1
antagonistic activity. Nitrogen containing aromatic groups were
essential to show potent antagonistic activity. Various kinds of het-
ero-aromatic groups were examined. Among six-membered hetero
aryl analogues 14–19, 2-pyridyl analogue 15 and 4-pyrimidyl ana-
logue 17 showed potent antagonistic activity. The effects of N-alkyl
group substitution at R3 were checked using 2-pyridyl analogue 15
as a template (15 and 20–23). Only small alky groups such as
Modification of R1 moiety was initially performed since bromo-
benzene is not a preferable group for drug design, and derivatiza-
tion of R1 is relatively simple. Various kinds of carboxylic acids
were introduced and revealed that an aromatic ring is essential
to show mGluR1 antagonistic activity (data not shown). Therefore,
substituted benzene analogues were tested as a bromobenzene
replacement, Table 1 highlights the SAR of the R1 substitutent. Flu-
oro analogue 6 was the best among halide analogues 2–4. We con-
sidered that an electron-withdrawing group might be necessary.
However electron-donating methyl derivative 7 also showed po-
tent antagonistic activity. Relatively large substituents 8 and 9
were not tolerated in this lead class. Based on these results, only
small substituents were accepted in terms of mGluR1 antagonistic
activity. Among fluorobenzene analogues 6, 11 and 12, it became
clear that substitution at 4- (para) position was preferable.
Table 2
SAR of left-hand substituent
Table 1
SAR of right-hand substituent
No R2
R3
IC50 SEM
No R2
R3
IC50 SEM
a
a
(nM) hmGluR1
(nM) hmGluR1
6
Me 49 16
Me >10,000
Me 230 130
18
Me 1500 470
Me 5700 440
No R1
IC50 SEM
No R1
IC50 SEM
a
a
(nM)
(nM)
13
14
15
16
19
20
21
22
23
hmGluR1
hmGluR1
4
5
6
210 68
62 17
49 16
9
>10,000
80 9.7
100 10
Et
18 2.4
81 24
10
11
Me
Me
Me
6.0 1.0
49 17
10 2.9
nPr
iPr
H
2000 340
980 120
7
83 15
12
130 15
17
8
1500 15
a
a
The IC50 value is the mean of multiple results (at least three independent
determinations performed in duplicate) with standard error of the means.
The IC50 value is the mean of multiple results (at least three independent
determinations performed in duplicate) with standard error of the means.

A. Satoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5464–5468
5467
Table 3
Table 4
SAR of 5-substituted pyrimidine derivatives
Profiles of 27
In vitro profiles
Group 1
a
a
hmGluR1 (IC50
hmGluR5 (IC50
Group 2
)
)
5.1 nM
7000 nM
a
hmGluR2 (IC50
Group 3
)
>10,000 nM
a
hmGluR8 (IC50
)
>10,000 nM
Quisqualic acid binding site (IC50
>10,000 nM
No R4
IC50 SEM (nM)
Metabolic
stability in
human liver
microsomesb
(remaining%)
Solubility in
pH7.4
a
)
hmGluR1
hmGluR5
potassium
phosphate
In vivo profiles
Brain penetrabilityb
Mouse brain/plasma concn 0.17 nmol/g/
bufferc
(lM)
0.19 lM
18
24
25
26
10 2.9
2.4 0.32 >10,000
1.8 0.25 7900 2100 20
3.7 0.74 9200 770 35
6600 1400 37
11.5
1.6
Pharmacokineticsc
Rat F: 53%, T1/2: 2.3 h, , CLp: 28 mL/min/kg
64
In vivo efficacyd
4.5
Rat PPI disruption model MED 1.0 mg/kg,
PO
Mouse hyperlocomotion model MED
0.3 mg/kg, PO
1.3
27
28
a
5.1 2.0
77d
7000 1900 60
>10,000d
45
a
The IC50 value is the mean of multiple results (at least three independent
determinations performed in duplicate) with standard error of the means.
b
Plasma and brains of mice were collected at 0.5 h after drug administration
(3 mg/kg, po) and drug concentrations were measured (n = 3 mice/group).
c
Pharmacokinetic study was conducted in fasted Sprague-Dawley rats (n = 3)
dosed at 1 mg/kg for intravenous dosing and dosed at 3 mg/kg for oral dosing as a
solution in water.
The IC50 value is the mean of multiple results (at least three independent
determinations performed in duplicate) with standard error of the means.
d
b
Method is described in detail in Refs. 12,20.
1 lM of compound was incubated in NADPH fortified liver microsomes
(0.25 mg-protein/mL) for 30 min at 37 °C.
c
Method is described in detail in Ref. 17.
The values are the means of two experiments.
d
displacement of [3H]quisqualate (>10,000 nM) binding indicated
that compound 27 bound at a site different from the glutamate
binding site. Therefore, compound 27 is a potent and noncompet-
itive mGluR1 antagonist (allosteric antagonist) with excellent sub-
type selectivity.
Compound 27 also demonstrated antipsychotic-like activities in
several kinds of in vivo animal models. In mice, 27 antagonized
methamphetamine-induced hyperlocomotion at 0.3 mg/kg po.
Furthermore, 27 (1.0 mg/kg po) reversed disruption of prepulse
inhibition (PPI) caused by methamphetamine or ketamine in
rats.12,20 As a final point, 27 had acceptable DMPK profiles, im-
proved microsomal stabilities, and good oral bioavailability
(F = 53%) in rats as well as good brain penetrability.
methyl 15 and ethyl 20 were accepted and amide NH proton
should be blocked (23) in terms of mGluR1 antagonistic activity.
Since the pyridine at the end of the molecule still had little con-
cern for CYP inhibition, pyrimidine analogue 17 was used as a tem-
plate for further modification.16
Finally, the effects of substituents at pyrimidine ring on intrinsic
potency and solubility were investigated. Introduction of methyl
group at 2-, 5- or 6-position of the pyrimidine ring were tested and
revealed that position 6 was the most effective in terms of improve-
ment of intrinsic potency (data not shown). Characterization of
pyrimidine analogues revealed that they showed insufficient micro-
somal stability and poor solubility in aqueous media.17 Therefore, in
order to improve metabolic stability and water solubility, introduc-
tion of hydrophilic substituents was examined. Among them, amine
analogue 24 had good microsomal stability as well as good antago-
nistic activity. However, mGluR1 antagonists require good brain
penetration showing in vivo efficacy; therefore alkyl groups should
be introduced on the nitrogen at 5-position since H-bond donors
are known to impair brain penetration.18,19
The primary amine of 24 was replaced with alkylamines. Unfor-
tunately, metabolic stability and water solubility of 6-amino
pyrimidine derivatives 25 and 26 decreased compared with non-
amino analogue 18. Only isopropylaminopyrimidine analogue 27
showed good microsomal stability and excellent water solubility
in addition to good mGluR1 antagonistic activity (see Table 3).
Detailed profiles of isopropylaminopyrimidine analogue 27 are
shown in Table 4.
In summary, compound 2721 showed quite potent mGluR1
antagonistic activity, excellent subtype selectivity and also good
PK profile in rats. Moreover it also demonstrated antipsychotic-like
effects in several animal models. Furthermore development of 27
with an N-methyl amide moiety in which the methyl group can
be introduced in the molecule in the last step of preparation would
be suitable for development of a PET tracer to examine in vivo
pharmacodynamics of mGluR1 antagonist. Therefore, compound
27 has great potential for elucidating the functions of mGluR1 in
humans.
Acknowledgments
The authors would like to thank Atsushi Hirano (for solubility
measurement) and Noriko Akaogi (for in vivo technical assistance).
Compound 27 had potent antagonistic activity against human
mGluR1 receptor with an IC50 value of 5.1 nM. Excellent selectivity
over other subtypes was exhibited; IC50 values were 7000 nM (hu-
man mGluR5), >10,000 nM (human mGluR2) and >10,000 nM (hu-
man mGluR8).
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