Tricyclic thiazolopyrazole derivatives as metabotropic glutamate receptor 4 positive allosteric modulators

Article abstract of DOI:10.1021/jm200290z

There is an increasing amount of evidence to support that activation of the metabotropic glutamate receptor 4 (mGlu4 receptor), either with an orthosteric agonist or a positive allosteric modulator (PAM), provides impactful interventions in diseases such as Parkinson's disease, anxiety, and pain. mGlu4 PAMs may have several advantages over mGlu4 agonists for a number of reasons. As part of our efforts in identifying therapeutics for central nervous system (CNS) diseases such as Parkinson's disease, we have been focusing on metabotropic glutamate receptors. Herein we report our studies with a series of tricyclic thiazolopyrazoles as mGlu4 PAMs.

Full text of DOI:10.1021/jm200290z

ARTICLE
pubs.acs.org/jmc
Tricyclic Thiazolopyrazole Derivatives as Metabotropic Glutamate
Receptor 4 Positive Allosteric Modulators
Sang-Phyo Hong,† Kevin G. Liu,† Gil Ma,† Michael Sabio,† Michelle A. Uberti,‡ Maria D. Bacolod,†
John Peterson,† Zack Z. Zou,† Albert J. Robichaud,† and Darío Doller†,*
†Chemical & Pharmacokinetic Sciences and ‡Synaptic Transmission, Lundbeck Research USA, 215 College Road, Paramus,
New Jersey 07652, United States
S Supporting Information
b
ABSTRACT: There is an increasing amount of evidence to support that activation of the metabotropic
glutamate receptor 4 (mGlu4 receptor), either with an orthosteric agonist or a positive allosteric modulator
(PAM), provides impactful interventions in diseases such as Parkinson’s disease, anxiety, and pain. mGlu4
PAMs may have several advantages over mGlu4 agonists for a number of reasons. As part of our efforts in
identifying therapeutics for central nervous system (CNS) diseases such as Parkinson’s disease, we have
been focusing on metabotropic glutamate receptors. Herein we report our studies with a series of tricyclic
thiazolopyrazoles as mGlu4 PAMs.
’ INTRODUCTION
(e.g., 10 and 11, Figure 2) as potential tool compounds.
Compound 9 displayed an EC50 of 410 nM when tested in a
calcium mobilization FLIPR assay of mGlu4 receptor modula-
tion, and it was inactive in the corresponding agonist, antagonist,
positive and negative modulation assays for the mGlu1, 2, 3, 5,
and 7 receptors. In a broad counterscreen of 70 CNS-relevant
GPCR receptors and ion channels, compound 9 showed some
level of cross-reactivity with the adenosine A2A and A3 receptors,
monoamine oxidase MAO-A, and norepinephrine transporter
(54.9, 77.4, 69.6, and 72.1% inhibition at 10 μM, respectively; see
Supporting Information). In terms of physicochemical proper-
ties, compound 9 is characterized by reasonable lipophilicity for
a CNS drug (LogD7.4 = 3.1), good kinetic solubility at pH 7.4
(120 μM), and good passive permeability in a PAMPA assay
(Papp = 32.9 ꢀ 10ꢁ6 cm/s). Human and rat plasma protein
binding are moderate (free fractions of 3.5% and 9.2%, re-
spectively), and nonspecific binding in a rat brain homogenate
dialysis study indicates a brain free fraction of 1.4%. hERG
channel inhibition potential is low (IC50 = 33 μM in an
electrophysiology screen). In vitro human and rat microsomal
stability studies yielded CLint of 11 L/min and 61 mL/min,
respectively. In an in vivo study of CNS partition in rat, sub-
cutaneous dosing of compound 9 (10 mg/kg, dosed as a solution
in 20% hydroxypropyl-β cyclodextrin) produced at 1 h brain,
plasma, and CSF concentrations of 744 ng/g, 766 ng/mL, and
52 ng/mL, respectively (brain/plasma ratio of 1).
Activation or inhibition of metabotropic glutamate receptor
function by small molecules is a strategy that has been utilized by
several research groups to identify novel therapeutic agents.1,2
Given the localization of the different metabotropic glutamate
receptors, many of the target indications for these small molecule
ligands are as CNS therapeutics. Recently, an increasing amount
of evidence has been accumulating to support the hypothesis that
activation of the metabotropic glutamate 4 receptor (mGlu4
receptor), either with an orthosteric agonist or a positive
allosteric modulator (PAM), may provide impactful pharmaco-
logical interventions in diseases such as Parkinson’s disease3 and
anxiety.4 Among these two types of ligands (orthosteric or
allosteric), the mGlu4 agonists reported to date are only sub-
type-selective, acting with some level of functional efficacy at all
Group 3 mGlu receptors, mGlu4, 6, 7, and 8 (based on in vitro
studies).5 Thus, the use of a PAM, targeting a unique site on the
mGlu4 receptor away from the native ligand binding site, would
have the potential to deliver selective receptor activation. Over
the past several years, a number of mGlu4 PAMs including
compounds 1,6 2,7 3,8 4,9 5,9 6,10 7,11 and 812 have been reported,
several of which are characterized by good brain exposure upon
peripheral administration (Figure 1). Herein we report our
efforts to identify a series of tricyclic pyrazoles as mGlu4 PAMs
to enable further testing of the biological hypothesis using in vivo
animal models.
In terms of structureꢁactivity relationships, substitution with
a methyl group on the thiazole ring increased the mGlu4 PAM
potency by over 30-fold (i.e., 10, EC50 = 13 nM), suggesting that
substitution at the 5-position of the thiazole ring enabled a
favorable interaction with the receptor. Likewise, substitution
with a methyl group on the pyrazole ring also significantly
Molecular Design. We have taken a multipronged approach
in an effort to identify lead compounds for our mGlu4 program.
These include high-throughput screening (HTS) of the Lund-
beck compound collection and rational design based on existing
knowledge of mGlu4 PAMs. Among the known mGlu4 ligands,
the thiazolopyrazole derivative 9 (Figure 2)13 attracted our
attention due to the low molecular weight and other favorable
physical properties such as cLogP (2.2) and tPSA (61 Å2).
Hence, we synthesized 9 together with several of its analogues
Received: March 11, 2011
Published: June 20, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Figure 1. Select known mGlu4 PAMs with reported activity at human receptors.
Figure 2. Design of mGlu4 PAMs.
Figure 3, portraying this “same-pocket” hypothesis. Methylation
in compound 10 resulted in a dihedral angle of only 13° between
the thiazole and pyrazole rings. These rings are essentially
coplanar in compound 11. Alternatively, in another low energy
conformation of 11, the pyrazole methyl group may point away
from the “upper pocket” and occupy another possible pocket
(“lower pocket”, highlighted by a blue dotted line for 11). To
further investigate this hypothesis, we synthesized dimethyl
derivative 12, only to find it to be inactive (EC50 > 10000 nM).
We surmised that due to the steric hindrance, the two methyl
groups in 12 cannot reside on the same side of the molecule but
will likely adopt a more favorable conformation with both methyl
groups pointing away from each other, as shown computationally
(Figure 4). The poor functional activity of 12 may further imply
that there is no room in the lower pocket to accommodate the
pyrazole methyl group when 12 adopts such conformation. To
further explore the “upper pocket” hypothesis and to identify
Figure 3. Overlay of 10 (with cyan carbon atoms) and 11 (with light-
pink carbon atoms) with each methyl group in the “top pocket”.
improved the potency as compared to that of 9 (i.e., 11, EC50
=
56 nM), which suggested to us that the methyl group on the
pyrazole 11 ring may occupy the same pocket (“upper pocket”,
Figure 2) as that of the methyl group from thiazole ring in
compound 10. Computational models14 of low energy confor-
mations of methyl derivatives 10 and 11 are superimposed in
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ARTICLE
mGlu4 positive allosteric modulators with potentially improved
properties, we constrained the two methyl groups by forming an
ethylene or a propylene bridge, therefore tethering the molecule
between the thiazole and pyrazole rings. This work yielded a class
of tricyclic thiazolopyrazole derivatives 13 with potent and
selective mGlu4 PAM affinity. It should be noted that a more
recent patent15 from Addex was published disclosing similar
compounds to those described herein after most of this work at
Lundbeck had been completed.16,17
Chemistry. The synthesis of the tricyclic thiazolopyrazole
derivatives isoutlined in Scheme 1. Heating 1,3-cyclohexanedione
14 to reflux in N,N-dimethylformamide dimethoxyacetal (DMF-
DMA) generated 2-dimethylamino-cyclohexane-1,3-dione 15,
and subsequent treatment of 15 with hydrazine in methanol
afforded the pyrazolo-cyclohexananone 16. The free nitrogen of
the pyrazole was then protected by either a trityl (Tr) or a
4-methoxybenzyl (PMB) group as regioisomeric mixtures (only
the 2-substituted regioisomer is shown), which were carried on
without further separation. Bromination of the isomeric mixtures
with CuBr2 in ethyl acetate afforded the common intermediate
17, which was then reacted with a variety of thioureas to provide
derivatives 18. The Tr or PMB protecting groups were then
removed, under standard conditions, to afford the final tricyclic
pyrazoles 20 and 21. An alternative synthesis was developed
when the aryl thioureas were not synthetically or commercially
available. In this case, unsubstituted thiourea and the correspond-
ing 17 were heated at reflux in EtOH overnight to afford
corresponding amino-thiazolo tricycles 18b. A typical Buchwald
coupling was then carried out between 18b and a variety of aryl
halides to afford derivatives 18a, which upon deprotection, as
before, provided final compounds 19 and 20.
Figure 4. Preferred conformation of 12 (with light-gray carbon atoms)
to avoid steric hindrance of its two methyl groups, relative to the
conformation of 10 (with cyan carbon atoms).
Scheme 1a
a Reagents and conditions: (a) Me2NCH(OMe)2, reflux(15a, 80ꢁ100%;
15b, 69%); (b) hydrazine, MeOH, reflux (16a, 74ꢁ97%; 16b, 60%; (c)
for PG = Tr, (i) Ph3CCl, Et3N, DCM, (ii) CuBr2, EtOAc, reflux, for PG =
PMB, (i) PMBCl, K2CO3, CH3CN, 60 °C, (ii) CuBr2, EtOAc, reflux(17b,
21%; 17d, 36%, 2-step) (d) EtOH, reflux, overnight; (e) aryl halide, Pd2-
(dba)3, Xantphos, Cs2CO3, DMF/THF, microwave, 120 °C, 1 h; (f) TFA,
microwave, 150 °C, 30 min (PG = PMB) (5ꢁ50% overall from 17).
’ RESULTS AND DISCUSSION
To aid in the exploration of the structureꢁactivity relationship
(SAR), a number of 6-membered ring derivatives were prepared,
and the mGlu4 EC50 and Emax data, as well as some physico-
chemical and in vitro dmpk parameters, are summarized in
Table 1. The 2-pyridyl derivative 21a, which displayed an EC50
Table 1. SAR of 6-Membered Tricyclic Pyrazoles
EC50
(nM)
Emax
PSA
cLogP (Å2)
solubilityb permeability Papp
rCLint
hCLint
rPPBe
a
a
c
d
d
compd
X
Y
Z
W
R
(%)
(μM)
(10ꢁ6 cm/s)
(mL/min) (L/min) (%) hPPBe(%)
21a
21b
21c
N
CH CH CH
CH CH
CH
H
H
H
H
H
220
150
11
2.6
2.6
2.6
3.9
1.8
3.1
3.1
3.1
3.6
66.5
66.5
66.5
53.6
79.4
66.5
66.5
66.5
66.5
0.1
17
3.0
18.7
2.5
37
5.6
4.4
97.7
89.5
99.2
96.4
CH
N
>10000
>10000
2200
CH CH
N
10
180
<0.1
20
21d CH CH CH CH
160
180
11
<0.1
3.4
21e
21f
21g
21h
21i
N
N
N
N
N
CH CH
N
65
42
CH CH CH 4-Me
CH CH CH 5-Me
CH CH CH 6-Me
>10000
>10000
910
9
12.4
1.9
12
<0.1
2.9
120
2.0
400
48
CH CH CH 4,6-di-Me >10000
11
0.1
1.0
a EC50 was for the potentiation of an EC20 glutamate concentration; Emax (%) was the percent response compared with the maximum response of
glutamate alone. b Kinetic solubility from DMSO stock solutions at pH 7.4 and room temperature. c Passive permeability in a PAMPA assay. d Rat and
human microsomal intrinsic clearance values. Rat and human hepatic blood flows are 20 mL/min and 1.5 L/min, respectively. e Rat and human plasma
protein binding percentage.
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ARTICLE
of 220 nM, is twice as potent as its nontethered analogue 9 but is
∼15-fold less potent than the monomethylated analogue 10.
When computational models (Figure 5) of 21a and 10 were
superimposed, we noticed a change in the orientation of the
pyrazole NꢁH unit, a potential hydrogen-bond donor, in 21a
and 10 and that the thiazole and pyrazole rings in 21a are rotated
by only 11° from coplanarity. Thus, the lower potency of 21a
relative to that of 10 may be due to the change in the orientation
of the pyrazole NH moiety. The importance of the NꢁH group
of the pyrazole ring was further supported by the lack of activity
of the corresponding 1-methyl pyrazole analogue as well as its
2-methyl regioisomer (data not shown). Alternatively, it may
implicate that the relatively bigger cyclohexyl group cannot be
accommodated by the “top pocket”. The latter hypothesis was
quickly dismissed by the great potency of the 7-membered ring
analogues (vide infra). Compounds 21b and 21c were then
synthesized to investigate the effect of the pyridine nitrogen
position on the activity. As it can be seen from the Table 1, both
compounds were inactive, suggesting that the 2-pyridyl group
either participates in a very specific interaction with the receptor
or may be needed to stabilize a particular conformation. The
somewhat narrow SAR observed in this investigation is consis-
tent with what has been seen with other reported mGlu4 PAMs.
Phenyl derivative 21d was active although with a much reduced
potency as compared to that of 21a, confirming the favorable
interaction of the optimized pyridyl amine moiety. Introduction
of an additional nitrogen atom at the 3-position of the pyridine
ring (21e) increased the potency by 3-fold. One may hypothesize
that the improved potency of pyrimidine over pyridine deriva-
tives may be because the second nitrogen atom is involved in
additional interactions with the receptor or due to the reduced
Figure 5. An overlay of 21a (with green carbon atoms) and 10 (with
cyan carbon atoms) to show different orientations of the pyrazole ring.
Table 2. SAR of 7-Membered Tricyclic Pyrazoles (Part 1)
a EC50 was for the potentiation of an EC20 glutamate concentration; Emax (%) was the percent response compared with the maximum response of
glutamate alone. b Kinetic solubility from DMSO stock solutions at pH 7.4 and room temperature. c Passive permeability in a PAMPA assay. d Rat and
human microsomal intrinsic clearance values. Rat and human hepatic blood flows are 20 mL/min and 1.5 L/min, respectively. e Rat and human plasma
protein binding percentage.
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Table 3. SAR of 7-Membered Tricyclic Pyrazoles (Part 2)
permeability
solubilityb
Papp
(10ꢁ6 cm/s)
a
a
c
EC50
Emax
PSA
(Å2)
compd
R
X
(nM)
(%)
cLogP
(μM)
23a
23b
23c
23d
23e
23f
3-Me
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
3400
>10000
140
84
28
3.7
3.7
3.7
4.2
3.4
3.4
3.9
4.0
4.5
2.9
2.9
2.6
3.9
3.9
4.2
4.0
2.8
3.3
66.5
66.5
66.5
66.5
66.5
66.5
66.5
75.7
71.7
90.3
93.0
82.7
85.0
69.7
78.5
69.7
79.4
88.6
0.2
<0.1
110
0.4
8.0
2.7
5-Me
6-Me
200
4
4.9
4,6-di-Me
5-F
>10000
750
1.2
170
170
220
100
78
<0.1
0.2
0.3
6-F
105
0.1
23g
23h
23i
6-Cl
1100
650
38
15.4
0.1
6-OMe
6-OEt
6-CN
6-CO2Me
0.1
290
<0.1
<0.1
1.9
<0.1
<0.1
0.1
23j
4900
>10000
2700
390
50
23k
23l
5
6-(CH2OH)
6-(OCH2CH2OCH3)
6-N Me2
76
30
38.8
0.1
23m
23n
23o
23p
23q
23r
90
0.1
340
150
63
<0.1
0.9
<0.1
3.1
6-NHEt
600
6-(pyrrolidin-1-yl)
6-Me
>10000
51
14
<0.1
0.6
0.1
210
230
0.5
6-MeO
N
100
0.6
0.3
a EC50 was for the potentiation of an EC20 glutamate concentration; Emax (%) was the percent response compared with the maximum response of
glutamate alone. b Kinetic solubility from DMSO stock solutions at pH 7.4 and room temperature. c Passive permeability in a PAMPA assay.
basicity of the pyrimidine ring affording an optimized interaction
with the receptor. While the 4-methyl and 5-methyl derivatives,
21f and 20g, respectively, were totally inactive, the 6-methyl
derivative 21h had an EC50 of 910 nM, which represents less than
a 5-fold reduction from the potency of 21a. The 4,6-dimethyl
derivative 21i was totally inactive, which further underscores the
low tolerance for substitution on the pyridine ring of this series of
mGlu4 PAMs. Additionally, several heterocycles were explored
to replace the pyrazole ring, but all of them resulted in com-
pounds with significantly reduced potency and the respective
series were not followed up (data not shown). In terms of
physicochemical properties, cLogP values for these analogues
have increased over that for compound 9, as had been expected
from the ethylene tether. While pyrimidine 21e has reasonable
aqueous solubility and plasma free fraction, the values of these
important parameter for other compounds showing some level of
mGlu4 PAM activity (21a and 21h) are suboptimal. Passive
permeabilities for these three analogues are similar. The addition
of the methyl group in the 6-position in 21h impacts negatively
the microsomal stability, increasing human and rat intrinsic
clearances by 10-fold compared with unsubstituted 21a and 21e.
To further explore the SAR, a number of 7-membered
derivatives (22aꢁ22l) were then synthesized, with the data
summarized in Tables 2 and 3. The 7-membered ring 2-pyridyl
derivative 22a has an EC50 of 9 nM and is 24-fold more potent
than its 6-membered ring counterpart 21a, and equipotent to the
monomethylated analogue 10. In 22a, a dihedral angle of only 4°
is found between the pyrazole and thiazole rings (Figure 6).
Unlike its 6-membered ring derivative 21a, in which the pyrazole
Figure 6. An overlay of 22a (with orange carbon atoms) and 9 (with
cyan carbon atoms).
NH moiety shifts from that of noncyclic 9, low energy con-
formations of compounds 22a and 10 are almost completely
superimposed. As expected, the corresponding 3-pyridyl deriva-
tive 22b was inactive, consistent with the SAR from the 6-mem-
bered ring compounds. While the 4-pyrimidyl and the
2-pyrazinyl compounds 22c and 22d, respectively, had signifi-
cantly reduced or no activity, the 2-pyrimidyl (22e) and 2-pyridyl
were shown to be the optimal groups, again in concert with the
SAR of the 6-membered ring system. Other groups including
some 5-membered heterocycles, substituted phenyls and cyclo-
hexyl derivatives (22fꢁ22k, Table 2) have also been explored,
and all were significantly less active. Some of these compounds
displayed good levels of aqueous solubility and passive perme-
ability (22f, 22h), however those for active analogues 22a and
22e were suboptimal, as it was their rat and human plasma
protein binding.
Once the 2-pyridyl and 2-pyrimidyl derivatives indicated
these were the ring systems enabling mGlu4 PAM activity, the
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Table 4. Profile of Compounds 9, 21a, 22a, and 22e
compd
9
21a
22a
22e
a
EC50
410
72
220
150
9
7
a
Emax
120
160
selectivity @ mGlu 1,2,3,5,7
MW
>10 μM
243
2.2
>10 μM
>10 μM
283
>10 μM
269
284
cLogP
2.6
3.2
2.3
LogD7.4
3.1
4.0
5.0
3.8
solubility pH 1.5 (μM) b
220
33
220
240
100
hERG IC50 (μM)
17
28
30
permeability Papp c (10ꢁ6 cm/s)
MDCK/mdr1d (10ꢁ6 cm/s, AfB; BfA; ratio)
hCLint (L/min)e
32.9
n/a
11
3.0
9.4
1.5
22; 7.7; 0.36
24; 13; 0.54
10
33; 21; 0.64
5.6
37
9
rCLint (mL/min)e
61
36
92
1.6
0.6
rBrain free fraction, fu (%)
rPlasma free fraction, fu (%)
9.2
0.43
2.3
0.43
1.1
1.4
exposure (10 mg/kg, PO, rats)
plasma (ng/mL), 1 h
brain (ng/g), 1 h
766
744
1
987
517
0.5
259
200
0.8
0.7
3.1
f
brain/plasma ratio
a EC50 was for the potentiation of an EC20 glutamate concentration; Emax (%) was the percent response compared with the maximum response of
glutamate alone. b Kinetic solubility from DMSO stock solutions at pH 7.4 and room temperature. c Passive permeability in a PAMPA assay.
d Permeability in MadinꢁDarby canine kidney cells transfected with the human MDR1 gene; P-gp substrate assay. e Rat and human microsomal intrinsic
clearance values. Rat and human hepatic blood flows are 20 mL/min and 1.5 L/min, respectively. f Not calculated as absolute brain and plasma values
are low.
tolerance of substitution on these skeletons was extensively
investigated aiming to extract the most benefit from this chemo-
type. The data for select examples are summarized in Table 3.
Although EC50 values were used as the primary parameter to
drive SAR for this chemical series, Emax may also play a significant
role in determining the in vivo effects of mGlu4 PAMs, the exact
nature of which deserves further investigations. It became
immediately obvious that the 6-position is the only one that
allows substitution, even with a small group such as fluorine
(23aꢁ23f, Table 3). A variety of other moieties (23gꢁ23r) were
then explored, however, all resulted in compounds with signifi-
cantly reduced potency further confirming the limited SAR for
this chemotype around this allosteric site. Most of these analo-
gues are within the cLogP range for CNS drug candidates and are
characterized by borderline solubility and passive permeability.
Notably, in spite of being substituted with a lipophilic group on
position 6, compounds 23c and 23g are more soluble than their
unsubstituted counterpart 22a. This effect may arise from
disturbances to crystal packing caused by lack of coplanarity by
methyl or chloro C-6 substitution.18 Among polar C-6 substit-
uents, only the hydroxymethyl analogue 23l shows solubility in
an appropriate range.
Compounds 21a, 22a, and 22e were selected for further
characterization, and selected properties are summarized
in Table 4. All three compounds were found to be inactive
[EC50 >10 μM in agonist and PAM mode, IC50 >10 μM in
negative allosteric modulator (NAM) mode] at mGlu 1, 2, 3, 5,
and 7 receptors and showed low potential for hERG channel
inhibition. These compounds have excellent drug-like properties
with low molecular weight (<300) and favorable cLogP values for
CNS drugs (2.5ꢁ3.5). While kinetic solubilities at pH 7.4 are in
the low μM range, those at pH 1.5 are higher. Experimental
LogD7.4 values are significantly larger than cLogP, indicating the
lipophilic nature of these compounds and in agreement with the
relatively low free fractions observed both in plasma and brain
homogenate. High in vitro intrinsic clearance (CLint) in both
human and rat liver microsomes and high protein binding in a
brain homogenate and in plasma were observed for all these. The
pyridine derivatives 21a and 22a displayed good plasma (987 and
259 ng/mL, respectively) and brain exposure levels (517 and
200 ng/g, respectively) as well as good brain penetration (brain/
plasma ratios of 0.5 and 0.8, respectively) after 1 h following a
10 mg/kg oral administration in SpragueꢁDawley rats (com-
pounds dosed as suspensions in 20% aqueous hydroxypropyl-
β cyclodextrin). Asymmetry ratios in the MDCK-mdr1 assay
(BfA/AfB) were less than 1, indicating no evidence of these
compounds being a P-glycoprotein substrate. On the other hand,
the pyrimidine derivative 22e was characterized by very poor
in vivo exposure levels and was not profiled further. In a broad
counterscreen of 70 CNS-relevant GPCR receptors and ion
channels, compound 22a (10 μM concentration) maintained
the level of cross-reactivity previously seen with compound 9 for
the case of the adenosine A3 receptor (65% inhibition) and
monoamine oxidase MAO-A (71% inhibition). Notably, inter-
actions with the A2A receptor (13% inhibition) and the nor-
epinephrine transporter (28.9% inhibition) were significantly
reduced compared with those liabilities for compound 9 (see
Supporting Information). The improved selectivity against these
two receptors will eliminate caveats interpreting results while
testing the mGlu4 PAM biological hypothesis in animal models
of Parkinson’s disease.
’ CONCLUSION
In summary, we identified a series of tricyclic thiazolopyrazole
derivatives as mGlu4 PAMs through medicinal chemistry design
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aided by molecular modeling. The SAR and SPR of this series
were explored in detail. While several mGlu4 PAM chemotypes
reported in the literature show very shallow and narrow SAR, in
this chemotype some characteristics of mGlu4 PAM SAR and
SPR translated between the open chain (e.g., 9), the 5,7-dihydro-
4H-thiazolo[4,5-e]indazol-2-amine analogues (e.g., 21e), and
the 4,5,6,8-tetra-hydropyrazolo-[30,40:6,7]cyclohepta[1,2-d]thiazol-
2-amine (e.g., 22a) analogues. Potent and orally bioavailable
compounds were identified (21a and 22a) with excellent brain
penetration and good physicochemical properties. Among these,
compound 22a showed an improved selectivity profile over lead
compound 9, warranting further studies to elucidate the value of
mGlu4 PAMs as potential CNS therapeutics.
(30 μL/well) with Calcium 3 no wash dye (Molecular Devices,
Sunnyvale, CA) made in assay buffer (Hank’s Balanced Saline Solution
(HBSS)): 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, plus
20 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid
(HEPES), pH 7.4, 0.1% bovine serum albumin (BSA), and 2.5 mM
probenecid) for 1 h in 5% CO2 at 37 °C. Basal fluorescence is monitored
in a fluorometric imaging plate reader (FLIPR) (Molecular Devices,
Sunnyvale, CA). Cells were stimulated with an EC20 concentration of
glutamate in the presence of a compound to be tested, and relative
fluorescent units were measured at defined intervals. Concentra-
tionꢁresponse curves derived from the maximum change in fluores-
cence were analyzed by nonlinear regression (Hill equation). A positive
modulator can be identified from these concentrationꢁresponse curves
if a compound produces a concentration dependent increase in the EC20
glutamate response. EC50 and Emax values were measured at least in two
independent experiments, each one in duplicate, and the mean values are
reported.
’ EXPERIMENTAL SECTION
General. Unless specifically stated otherwise, the experimental
procedures were performed under the following conditions. All opera-
tions were carried out at room temperature (about 18 °C to about
25 °C) under nitrogen atmosphere. Evaporation of solvent was carried
out using a rotary evaporator under reduced pressure or in the high
performance solvent evaporation system HT-4X (Genevac Inc., Valley
Cottage, NY, USA). The microwave oven used was a Biotage Initiator
synthesizer (Charlottesville, VA, USA). The course of the reaction was
followed by thin layer chromatography (TLC) or liquid chromatogra-
phyꢁmass spectrometry (LC-MS), and reaction times are given for
illustration only. Silica gel chromatography was carried out on a
CombiFlash system (Teledyne Isco, Inc., Lincoln, NE, USA) with
prepacked silica gel cartridges or performed on Merck Silica Gel 60
(230ꢁ400 mesh). The final compound structures were confirmed using
both nuclear magnetic resonance (NMR) and low and high resolution
mass spectrometry. Purity of all final products was determined to be
>95% based on LC-MS trace using UV detection in the range of
240ꢁ400 nm as well as at a single 254 nm wavelength. Purifications
were carried out on a reversed phase liquid chromatography/mass
spectrometry (RP-HPLC/MS) purification system. Flow rate:
100 mL/min. Mobile phase additive: 48 mM of ammonium formate.
Column: Inertsil C18, 30 mm ꢀ 50 mm, 5 μm particle size. Gradient
conditions were as follows:
4-(1H-Pyrazol-4-yl)-N-(pyridin-2-yl)thiazol-2-amine (9). The
title compound was prepared according the procedure reported in the
literature.13 1H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 8.30 (d,
J = 5.0 Hz, 1H), 7.94 (s, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 7.87 Hz,
1H), 7.01 (s, 1H), 6.92 (dd, J = 6.3, 5.2 Hz, 1H). ESMS m/e: 244.0 (M +
H)+. HRMS calcd for (M + H)+: C11H9N5S+ 244.06514; found
244.06450.
5-Methyl-4-(1H-pyrazol-4-yl)-N-(pyridin-2-yl)thiazol-2-amine
(10). The title compound was prepared according the procedure
reported in the literature.13 1H NMR (400 MHz, DMSO-d6) δ 12.94
(br, 1H), 11.15 (br, 1H), 8.26 (d, J = 4.8 Hz, 1H), 7.88 (br, 2H), 7.67 (t,
J = 7.8 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.88 (t, J = 5.8 Hz, 1H), 2.41 (s,
3H). ESMS m/e: 258.0 (M + H)+. HRMS calcd for (M + H)+:
C12H11N5S+ 258.08079; found 258.08017.
4-(3-Methyl-1H-pyrazol-4-yl)-N-(pyridin-2-yl)thiazol-2-amine
(11). The title compound was prepared according the procedure
reported in the literature.13 1H NMR (400 MHz, DMSO-d6) δ 12.59
(br, 1H), 11.28 (s, 1H), 8.30 (d, J = 4.9 Hz, 1H), 7.77 (br, 1H), 7.69 (t,
J = 7.8 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.91 (t, J = 6.3 Hz, 1H), 6.83 (s,
1H), 2.45 (s, 3H). ESMS m/e: 258.0 (M + H)+. HRMS calcd for (M +
H)+: C12H11N5S+ 258.08079; found 258.08029.
2-Dimethylaminomethylene-cyclohexane-1,3-dione (15a).
Into a round-bottom flask, 1,3-cyclohexanedione (2.00 g, 17.8 mmol)
and 1,1-dimethoxy-N,N-dimethylmethanamine (30 mL, 200 mmol)
were added. The reaction was heated to reflux for 3 h and was con-
centrated in vacuo to remove 1,1-dimethoxy-N,N-dimethylmethana-
mine. The desired title compound was obtained as a brown solid (2.89 g,
100% crude yield). 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 3.40
(s, 3H), 3.20 (s, 3H), 2.50 (m, 4H), 2.00 (m, 2H).
time (min)
acetonitrile % 18 18
0
0.50 0.65 3.00 3.50 4.95 5.00
22 45 95 95 18
High resolution mass spectra were recorded using an LTQ Orbitrap
XL, Thermo Electron Corp., Waltham, MA, USA. NMR spectra were
recorded on a Bruker Avance 300 spectrometer (Bruker BioSpin Corp.,
Billerica, MA, USA) or a Varian UNITY INOVA 400 (Varian, Inc., Palo
Alto, CA, USA) using the indicated solvent. Chemical shift (δ) is given
in parts per million (ppm) relative to tetramethylsilane (TMS) as an
internal standard. Coupling constants (J) are expressed in hertz (Hz),
and conventional abbreviations used for signal shape are: s = singlet; d =
doublet; t = triplet; m = multiplet; br = broad, etc. Unless stated
otherwise, mass spectra were obtained using electrospray ionization
(ESMS) via either a Micromass Platform II system or a Quattro Micro
system (both from Waters Corp., Milford, MA, USA) and (M + H)+ is
reported.
Calcium Mobilization Assay. The hmGlu4 and murine GR15
(G-protein) cDNAs were stably expressed in a BHK cell line and grown
in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad,
CA) with supplements (10% dialyzed fetal bovine serum, 1% glutamax,
1% sodium pyruvate, 1% Pen/strep, 1 mg/mL Geneticin, and 0.2 mg/
mL hygro B) at 37 °C, 5% CO2 . Twenty-four hours prior to assay, cells
were seeded into 384-well black-wall microtiter plates coated with poly-
D-lysine. Just prior to assay, media was aspirated and cells dye-loaded
2-Dimethylaminomethylene-cycloheptane-1,3-dione (15b).
A mixture of cycloheptane-1,3-dione (10 g, 79 mmol) and DMF-DMA
(29 g, 244 mmol) was stirred at refluxing for 1 h. The reaction mixture
was concentrated in vacuo. The resulting residue was triturated with
toluene and filtered to give the title compound (10 g, 69%). 1H NMR
(300 MHz, CD3OD) δ 7.84 (s, 1H), 3.37 (s, 3H), 2.82 (s, 3H),
2.61ꢁ2.50 (m, 4H), 1.90ꢁ1.72 (m, 4H).
2,5,6,7-Tetrahydro-indazol-4-one (16a). The crude material
of step 1 (2-dimethylaminomethylene-cyclohexane-1,3-dione, 2.89) was
dissolved in methanol (50 mL), followed by addition of hydrazine
(0.629 g, 19.6 mmol). The reaction was heated to reflux for 5 h.
The reaction was cooled to room temperature and filtered. A light-
brown solid was generated. The solid was washed with MeOH/hexanes
and dried to give the title compound (2.30 g, 95% crude yield). 1H NMR
(400 MHz, DMSO-d6) δ 8.4ꢁ7.7 (m, 1H), 2.8ꢁ2.3 (s, 4 h), 2.1ꢁ1.8
(m, 2H).
5,6,7,8-Tetrahydro-2H-cycloheptapyrazol-4-one (16b). To
a cold solution of 2-dimethylaminomethylene-cycloheptane-1,3-dione
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Journal of Medicinal Chemistry
ARTICLE
(20 g, 110 mmol) in MeOH (500 mL) was added dropwise a solution of
hydrazine in THF (112 mL, 112 mmol, 1 M THF) over 5 min. The
reaction was stirred for 1 h and concentrated in vacuo. The resulting
residue was recrystallized from EtOAc to afford the title compound
(10 g, 60%). 1H NMR (300 MHz, CDCl3) δ 8.00 (s, 1H), 3.04ꢁ2.95
(m, 2H), 2.78ꢁ2.65 (m, 2H), 2.04ꢁ1.85 (m, 4H).
(s,3H), 3.78 (s, 1.5H), 3.17ꢁ3.10 (m, 1H), 3.03ꢁ2.96 (m, 0.5H),
2.91ꢁ2.83 (m, 1H), 2.78ꢁ2.70 (m, 0.5H), 2.38ꢁ2.22 (m, 4.5H),
2.05ꢁ1.90 (m,1.5H). MS (ES+) m/e 351 (M + H)+.
8-(4-Methoxy-benzyl)-4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-
cyclopenta[e]azulen-2-ylamine (18b, n = 2). The reaction mixture
of 17d (2.00 g, 5.73 mmol) and thiourea (436 mg, 5.73 mmol) in EtOH
(120 mL) was refluxed overnight, cooled to room temperature, and
concentrated under the reduced pressure to afford the title compound
(1.82 g, 97%), which was used without further purification. MS (ES+) m/e
327 (M + H)+.
5-Bromo-2-trityl-2,5,6,7-tetrahydro-indazol-4-one (17a).
Into a round-bottom flask, the 2,5,6,7-tetrahydro-indazol-4-one (1.00 g,
7.34 mmol), triethylamine (2.05 mL, 14.7 mmol), triphenylmethyl
chloride (2.25 g, 8.08 mmol), and methylene chloride (30 mL) were
added. The reaction was stirred for 3 h. The resulting mixture was
quenched with saturated NaHCO3 and extracted with DCM (3ꢀ). The
combined organic phase was washed with brine, dried with MgSO4, and
concentrated in vacuo. The crude material was purified over silica gel by
eluting with 0ꢁ60% EtOAc:hexanes to afford the intermediate as an
isomeric mixture (1.78, 64%). 1H NMR (400 MHz, CDCl3) δ 8.5ꢁ7.1
(m, 16H), 2.9ꢁ2.5 (m, 4H), 2.2ꢁ2.0 (m, 2H).
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-pyri-
din-2-yl-amine (21a). Crude 5-bromo-2-trityl-2,5,6,7-tetrahydro-in-
dazol-4-one (17a) (0.300 g, 0.657 mmol) and 2-pyridylthiourea (0.0486 g,
0.317 mmol) were dissolved in ethanol (5 mL), followed by refluxing
overnight. Using a rotary evaporator, the excess EtOH was removed
from the reaction mixture and the crude material was purified by reverse-
phase HPLC to afford the title compound (3 mg). 1H NMR (400 MHz,
CDCl3) δ 8.4 (m, 2H), 7.7 (s, 1H), 7.6 (m, 1H), 6.9 (m, 2H), 3.1 (m,
4H). MS (ES+) m/e 270 (M + H)+; HRMS calcd for (M + H)+:
C13H12N5S+ 270.08079; found 270.08066.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-pyri-
din-3-yl-amine (21b). Regio-mixture of 5-bromo-2-(4-methoxy-
benzyl)-2,5,6,7-tetrahydro-indazol-4-one (17b) (0.050 g, 0.179 mmol)
and (6-methyl-pyridin-2-yl)-thiourea (0.030 g, 0.18 mmol) were dis-
solved in ethanol (10 mL), followed by refluxing overnight. Using a
rotary evaporator, the excess EtOH was removed from the reaction
mixture and the crude was dissolved in TFA (3ꢁ5 mL), followed by
microwave irradiation at 150 °C for 30 min. The reaction mixture was
concentrated in vacuo, and the material was purified by reverse-phase
HPLC to afford the title compound (22 mg, 50%). 1H NMR (400 MHz,
DMSO-d6) δ 12.5 (s, 1H), 10.3 (s, 1H), 8.8 (d, J = 2.54 Hz, 1H), 8.2 (m,
1H), 8.1 (m, 1H), 7.7 (bs, 1H), 7.3 (dd, J = 4.6, 8.3 Hz, 1H) 2.9 (m, 4H).
MS (ES+) m/e 270 (M + H)+. HRMS calcd for (M + H)+: C13H12N5S+
270.08079; found 270.08108.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-pyri-
din-4-yl-amine. (21c). The title compound was prepared by the same
method as 21b (14 mg, 37%). 1H NMR (400 MHz, MeOD-d4) δ 8.3 (m,
2H), 7.7 (bs, 1H), 7.6 (m, 2H), 3.0 (m, 4H). MS (ES+) m/e 270 (M +
H)+. HRMS calcd for (M + H)+: C13H12N5S+ 270.08079; found
270.08051.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-phenyl-
amine (21d). The title compound was prepared by the same method
as 21b (6 mg, 10%). 1H NMR (400 MHz, MeOD-d4) δ 7.7 (s, 1H), 7.5
(d, J = 8.3 Hz, 2H), 7.3 (t, J = 8.3 Hz, 2H), 7.0 (t, J = 7.6 Hz, 1H), 3.0 (bs,
4H). MS (ES+) m/e 269 (M + H)+. HRMS calcd for (M + H)+:
C14H13N4S + 269.08554; found 269.08550.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-pyri-
midin-2-yl-amine (21e). The title compound was prepared by the
same method as 21b (15 mg, 37%). 1H NMR (400 MHz, DMSO-d6) δ
12.5 (s, 1H), 11.6 (s, 1H), 8.6 (d, J = 4.9 Hz, 2H), 7.6 (s, 1H), 7.0 (t, J =
4.9 Hz, 1H), 3.0ꢁ2.8 (m, 4H). MS (ES+) m/e 271 (M + H)+. HRMS
calcd for (M + H)+: C12H11N6S + 271.07604; found 271.07602.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-(4-
methyl-pyridin-2-yl)-amine (21f). The title compound was pre-
pared according to the method described for compound 21b (5 mg, 8%).
1H NMR (400 MHz, DMSO-d6) δ 12.3 (br, 1H), 11.1 (s, 1H), 8.00 (d,
J = 5.2 Hz, 2H), 7.46 (s, 1H), 6.67 (s, 1H), 6.62 (d, J = 5.3 Hz, 1H),
2.87ꢁ2.74 (m, 4H), 2.15 (s, 3H). MS (ES+) m/e 284 (M + H)+. HRMS
calcd for (M + H)+: C14H14N5S + 284.09644; found 282.08084.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-(5-
methyl-pyridin-2-yl)-amine (21g). The title compound was pre-
pared according to the method described for compound 21b (3 mg, 5%).
1H NMR (400 MHz, MeOD) δ 8.13 (s, 1H), 7.69 (s, 1H), 7.56ꢁ7.50
The 2-trityl-2,5,6,7-tetrahydro-indazol-4-one (0.5 g, 1.32 mmol) was
dissolved in ethyl acetate (20 mL), followed by addition of copper(II)
bromide (0.5 g, 2.24 mmol). The reaction was heated at 50 °C overnight,
filtered through Celite, and concentrated in vacuo. The crude title
compound was used without further purification.
5-Bromo-2-(4-methoxy-benzyl)-2,5,6,7-tetrahydro-indazol-
4-one (17b). A suspension of 2,5,6,7-tetrahydro-indazol-4-one (6 g,
0.044 mol), PMBCl (10 g, 0.064 mol), and K2CO3 (9.1 g, 0.66 mol) in
acetonitrile (300 mL) was stirred at 60 °C overnight. The reaction
mixture was cooled to room temperature and filtered. The filtrate was
concentrated to afford the crude product, which was purified by column
chromatography on silica gel (4:1 petroleum ether:ethyl acetate) to give
the intermediate as a mixture of regioisomers (8 g, 71%).
To a solution of 2-(4-methoxy-benzyl)-2,5,6,7-tetrahydro-indazol-4-
one (4 g, 16 mmol) in EtOAc (400 mL) was added CuBr2 (7 g, 31
mmol). The reaction mixture was stirred at refluxing for 4 h. The
resulting mixture was cooled to room temperature and filtered. The
filtrate was concentrated to give the crude compound, which was
purified by column chromatography on silica gel (3:1 petroleum
ether:ethyl acetate) to give the title compound as a mixture of regio-
isomers in a ratio of 1:2. (1.5 g, 29%). 1H NMR (400 MHz, CD3OD) δ
8.49 (s, 2H),7.93 (s, 1H), 7.31 (d, J = 8.4 Hz, 4H), 7.22 (d, J = 8.4 Hz,
2H), 6.99ꢁ6.87 (m, 6H), 5.31 (s, 2H), 5.26 (s, 4H), 4.85ꢁ4.75 (m, 3H),
3.80ꢁ3.70 (m, 9H), 3.07ꢁ3.02 (m, 1H), 2.89ꢁ2.75 (m, 5H),
2.48ꢁ2.29 (m, 6H). ESMS m/e: 337 (M + H)+.
5-Bromo-2-(4-methoxy-benzyl)-5,6,7,8-tetrahydro-2H-
cycloheptapyrazol-4-one (17d). To a solution of 5,6,7,8-tetra-
hydro-2H-cycloheptapyrazol-4-one (10 g, 67 mmol) in CH3CN
(200 mL) was added PMBCl (12.5 g, 80 mmol) and K2CO3 (13.8
g, 100 mmol). The reaction mixture was stirred at 60 °C for 2 h. The
resulting mixture was filtered, and the filtrate was concentrated in
vacuo. The resulting residue was purified by column chromatogra-
phy on silica gel (10:1ꢁ1:1 petroleum ether:EtOAc) to afford the
1
intermediate (15 g, 83%). H NMR (CDCl3 400 MHz): δ 8.01 (s,
0.5H), 7.78 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.8 Hz, 1H),
6.92ꢁ6.85 (m, 3H), 5.25 (s, 1H), 5.17 (s, 2H), 3.83 (s, 3H), 3.81
(s,1.5H), 2.97 (t, J = 6.0 Hz, 2H), 2.85 (t, J = 6.0 Hz, 1H), 2.77ꢁ2.65
(m, 3H), 2.01ꢁ1.85 (m, 6H).
A solution of compound 2-(4-methoxy-benzyl)-5,6,7,8-tetrahydro-
2H-cycloheptapyrazol-4-one (8 g, 30 mmol) and CuBr2 (11.2 g, 50.2 mmol)
in EtOAc (150 mL) was stirred at reflux for 1 h. The reaction mixture
was filtered, and the filter cake was washed with EtOH several times. The
combined filtrate was concentrated in vacuo. The resulting residue was
purified by reverse-phase HPLC to afford the title compound (5.4 g,
52%). 1H NMR (CDCl3 400 MHz): δ 8.02 (s, 0.5H), 7.81 (s, 1H), 7.22
(dd, J = 6.4 Hz, 2.0 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.91ꢁ6.83 (m,
3H), 5.31ꢁ5.17 (m, 1H), 5.14 (s, 2H), 4.88ꢁ4.76 (m, 1.5H), 3.80
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Journal of Medicinal Chemistry
ARTICLE
(m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 3.07ꢁ2.97 (m, 4H), 2.29 (s, 3H). MS
(ES+) m/e 284 (M + H)+.
4H), 2.19 (s, 3H), 2.01ꢁ1.92 (m, 2H). MS (ES+) m/e 287 (M + H)+.
HRMS calcd for (M + H)+: C13H15N6S+ 287.10734; found 287.10762.
(1-Methyl-1H-pyrazol-3-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (22h). The title com-
pound was prepared according to the method described for compound
23c (30 mg, 40%). 1H NMR (400 MHz, DMSO-d6) δ 12.4 (br, 1H),
10.3 (s, 1H), 7.66 (s, 1H), 7.53 (d, J = 2.2 Hz, 1H), 5.96 (d, J = 2.0 Hz,
1H), 3.74 (s, 3H), 2.98ꢁ2.89 (m, 4H), 2.01ꢁ1.93 (m, 2H). MS (ES+)
m/e 287 (M + H)+. HRMS calcd for (M + H)+: C13H15N6S+ 287.10734;
found 287.10779.
(4,5,6,8-Tetrahydro-3-thia-1,7,8-triaza-cyclopenta[e]azulen-
2-yl)-thiazol-2-yl-amine (22i). To a solution of 18a (60 mg, 0.18
mmol) in THF (3.0 mL) was added NaH (60% in mineral oil, 15 mg,
0.37 mmol) at 0 °C, and the mixture was stirred at room temperature for
10 min, followed by the addition of 2-bromothiazole (30 mg, 8.18 mmol).
The reaction mixture was refluxed overnight, cooled to room tempera-
ture, and quenched with ice. The aqueous layer was extracted with DCM
(2 ꢀ 10 mL). The combined organic layers were concentrated, and the
residue was purified by CombiFlash system (gradient: 10ꢁ90% ethyl
acetate in hexanes) to afford PMB-protected intermediate, which was
dissolved in TFA (2.5 mL). The resulting mixture was microwaved at
150 °C for 30 min and concentrated. The residue was quenched with
saturated aqueous NaHCO3, and the aqueous layer was extracted with i-
PrOH/CHCl3 (1:3, 3 ꢀ 15 mL). The combined organic layers were
concentrated and the resulting residue was purified by reverse-phase
HPLC to afford the title compound (2 mg, 4% over 2 steps). MS (ES+)
m/e 290 (M + H)+.
(6-Methyl-pyridazin-3-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (22j). The title com-
pound was prepared according to the method described for compound
23q (16 mg,c 29%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br s, 1H),
11.2 (s, 1H), 7.70 (s, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.26 (d, J = 9.0 Hz,
1H), 3.03ꢁ2.93 (m, 4H), 2.51 (s, 3H), 2.05ꢁ1.95 (m, 2H). MS (ES+)
m/e 299 (M + H)+. HRMS calcd for (M + H)+: C14H15N6S+ 299.10734;
found 299.10769.
Cyclohexyl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclop-
enta[e]azulen-2-yl)-amine (22k). The title compound was pre-
pared according to the method described for compound 23c (14 mg,
17%). 1H NMR (400 MHz, DMSO-d6) δ 12.4 (s, 1H), 7.58 (s, 1H), 7.19
(d, J = 7.7 Hz, 1H), 2.99ꢁ2.79 (m, 4H), 2.53ꢁ2.51 (m, 1H), 1.98ꢁ1.89
(m, 4H), 1.75ꢁ1.53 (m, 3H), 1.37ꢁ1.11 (m, 5H). MS (ES+) m/e 289
(M + H)+. HRMS calcd for (M + H)+: C15H21N4S+ 289.14814; found
288.28970.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-(6-
methyl-pyridin-2-yl)-amine (21h). The title compound was pre-
pared according to the method described for compound 21a (3 mg, 6%).
1H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 1H), 11.2 (s, 1H), 7.6 (s,
1H), 7.5 (t, J = 8.0 Hz, 1H), 6.8 (dd, J = 8.0, 16.8 Hz, 2H), 3.0ꢁ2.9 (m,
4H), 2.4 (s, 3H). MS (ES+) m/e 284 (M + H)+.
(5,7-Dihydro-4H-3-thia-1,6,7-triaza-as-indacen-2-yl)-(4,6-
dimethyl-pyridin-2-yl)-amine (21i). The title compound was pre-
pared according to the method described for compound 21b (3 mg, 4%).
1H NMR (400 MHz, DMSO-d6) δ 12.3 (br, 1H), 11.1 (s, 1H), 7.38
(s, 1H), 6.60 (s, 1H), 6.54 (s, 1H), 2.99ꢁ2.86 (m, 4H), 2.40 (s, 3H),
2.22 (s, 3H). MS (ES+) m/e 298 (M + H)+. HRMS calcd for (M + H)+:
C15H16N5S+ 298.11209; found 298.11240.
Pyridin-2-yl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclopenta-
[e]azulen-2-yl)-amine (22a). The title compound was prepared
according to the method described for compound 23c (0.16 g, 39%). 1H
NMR (400 MHz, DMSO-d6) δ 12.4 (s, 1H), 11.1 (s, 1H), 8.2 (d, J =
8.2 Hz, 1H), 7.7 (m, 2H), 7.0 (d, J = 8.2 Hz, 1H), 6.8 (dd, J = 5.7, 6.8 Hz,
1H), 3.0 (m, 4H), 2.0 (m, 2H). MS (ES+) m/e 284 (M + H)+. HRMS
calcd for (M + H)+: C14H14N5S + 284.09644; found 284.09650.
Pyridin-3-yl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclop-
enta[e]azulen-2-yl)-amine (22b). The title compound was pre-
pared according to the method described for compound 23c (9 mg,
1
10%). H NMR (400 MHz, MeOD) δ 8.73 (s, 1H), 8.45 (s, 1H),
8.21ꢁ8.14 (m, 1H), 8.05ꢁ7.95 (m, 1H), 7.77 (s, 1H), 7.28 (dd, J = 8.4,
4.8 Hz, 1H), 2.97ꢁ2.86 (m, 4H), 2.05ꢁ1.96 (m, 2H). MS (ES+) m/e
284 (M + H)+. HRMS calcd for (M + H)+: C14H14N5S+ 284.09644;
found 284.09646.
Pyrimidin-4-yl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclo-
penta[e]azulen-2-yl)-amine (22c). The title compound was pre-
pared according to the method described for compound 23q (16 mg,
23%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.6 (s, 1H),
8.70 (s, 1H), 8.34 (d, J = 5.9 Hz, 1H), 7.63 (s, 1H), 6.97 (d, J = 5.7 Hz,
1H), 2.97ꢁ2.87 (m, 4H), 1.96ꢁ1.88 (m, 2H). MS (ES+) m/e 285 (M +
+
H)+. HRMS calcd for (M + H)+: C13H13N6S 285.09169; found
285.09175.
Pyrazin-2-yl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclo-
penta[e]azulen-2-yl)-amine (22d). The title compound was pre-
pared according to the method described for compound 23q (2 mg,
4%). 1H NMR (400 MHz, DMSO-d6) δ 8.70ꢁ7.66 (m, 4H), 3.07ꢁ2.93
(m, 4H), 2.04ꢁ1.95 (m, 2H). MS (ES+) m/e 285 (M + H)+. HRMS
calcd for (M + H)+: C13H13N6S+ 285.09190; found 285.09208.
Pyrimidin-2-yl-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclo-
penta[e]azulen-2-yl)-amine (22e). The title compound was pre-
pared according to the method described for compound 23c (60 mg,
10%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 1H), 11.5 (s, 1H), 8.6
(d, J = 4.9 Hz, 2H), 7.7 (s, 1H), 7.0 (d, J = 4.9 Hz, 1H), 3.0 (m, 4H), 2.0
(m, 2H). MS (ES+) m/e 285 (M + H)+; HRMS Calcd for (M + H)+:
C13H13N6S+ 285.09169; found 285.09177.
(1H-Pyrazol-3-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-
cyclopenta[e]azulen-2-yl)-amine (22f). The title compound was
prepared according to the method described for compound 23c (45 mg,
38%). 1H NMR (400 MHz, DMSO-d6) δ 12.4 (br s, 1H), 12.1 (s, 1H),
10.3 (s, 1H), 7.66 (s, 1H), 7.59 (s, 1H), 5.98 (s, 1H), 3.00ꢁ2.88 (m,
4H), 2.04ꢁ1.91 (m, 2H). MS (ES+) m/e 273 (M + H)+. HRMS calcd for
(M + H)+: C12H13N6S + 273.09169; found 273.09164.
3-Methyl-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23a). The title com-
pound was prepared according to the method described for compound
23q (25 mg, 46%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br s, 1H),
10.1 (s, 1H), 8.13 (dd, J = 4.9, 1.2 Hz, 1H), 7.76 (s, 1H), 7.52 (dd, J = 7.2,
0.8 Hz, 1H), (dd, J = 7.2, 5.0 Hz, 1H), 3.02ꢁ2.94 (m, 4H), 2.33 (s, 3H),
2.04ꢁ1.95 (m, 2H). MS (ES+) m/e 298 (M + H)+. HRMS calcd for
(M + H)+: C15H16N5S+ 299.10734; found 299.10769.
(5-Methyl-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23b). The title com-
pound was prepared according to the method described for compound
23c (3 mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.0
(s, 1H), 8.10ꢁ8.06 (m, 1H), 7.68 (s, 1H), 7.50 (dd, J = 8.6, 2.2 Hz, 1H),
6.96 (d, J = 8.4 Hz, 1H), 3.02ꢁ2.93 (m, 4H), 2.21 (s, 3H), 2.03ꢁ1.93
(m, 2H). MS (ES+) m/e 298 (M + H)+. HRMS calcd for (M + H)+:
C15H16N5S+ 298.11209; found 298.11209.
(5-Methyl-1H-pyrazol-3-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (22g). The title com-
pound was prepared according to the method described for compound
23c (50 mg, 40%). 1H NMR (400 MHz, DMSO-d6) δ 12.4 (br s, 1H),
11.8 (s, 1H), 10.2 (s, 1H), 7.65 (s, 1H), 5.73 (s, 1H), 2.98ꢁ2.87 (m,
(6-Methyl-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23c). Into a vial was
added 5-bromo-2-(4-methoxy-benzyl)-5,6,7,8-tetrahydro-2H-cyclohep-
tapyrazol-4-one (17d) (0.100 g, 0.286 mmol), (6-methyl-pyridin-2-yl)-
thiourea (0.0479 g, 0.286 mmol), and ethanol (5 mL, 80 mmol). The
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Journal of Medicinal Chemistry
ARTICLE
reaction mixture was heated to reflux overnight, and then, using a rotary
evaporator, the excess EtOH was removed. The crude material was
dissolved in trifluoroacetic acid (3 mL, 40 mmol) and irradiated with
microwave at 150 °C for 30 min. The excess TFA was removed in vacuo.
The crude material was purified over silica gel eluting with 0ꢁ10%
MeOH (2 M NH3) in DCM to afford the title compound 23c (25 mg,
29%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 1H), 11.1 (s, 1H), 7.7
(s, 1H), 7.5 (t, J = 7.5 Hz, 1H), 6.8 (d, J = 8.3 Hz, 1H), 6.7 (d, J = 7.5 Hz,
1H), 3.0 (m, 4H), 2.4 (s, 3H), 2.0 (m, 2H). MS (ES+) m/e 298 (M + H)+.
HRMS calcd for (M + H)+: C15H16N5S+ 298.11209; found 278.10360.
(4,6-Dimethyl-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23d). The title com-
pound was prepared according to the method described for compound
23c (3 mg, 4%). 1H NMR (400 MHz, DMSO-d6) δ 11.0 (s, 1H), 7.68 (s,
1H), 6.63 (s, 1H), 6.60 (s, 1H), 3.01ꢁ2.92 (m, 4H), 2.39 (s, 3H), 2.22
(s, 3H), 2.03ꢁ1.94 (m, 2H). MS (ES+) m/e 312 (M + H)+. HRMS calcd
for (M + H)+: C15H16N5S + 298.11209; found 278.10360.
[6-(4,5,6,8-Tetrahydro-3-thia-1,7,8-triaza-cyclopenta[e]-
azulen-2-ylamino)-pyridin-2-yl]-methanol (23l). The title com-
pound was prepared according to the method described for compound
23q (8 mg, 10%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.1
(s, 1H), 7.71ꢁ7.63 (m, 1H), 6.97 (d, J = 7.0 Hz, 1H), 6.88 (d, J = 8.1 Hz,
1H), 5.34 (s, 1H), 4.55 (s, 1H), 3.02ꢁ2.92 (m, 4H), 2.04ꢁ1.94 (m, 2H).
MS (ES+) m/e 314 (M + H)+. HRMS calcd for (M + H)+: C15H16N5OS+
342.10192; found 342.10217.
[6-(2-Methoxy-ethoxy)-pyridin-2-yl]-(4,5,6,8-tetrahydro-3-
thia-1,7,8-triaza-cyclopenta[e]azulen-2-yl)-amine (23m). The
mixture of (6-fluoro-pyridin-2-yl)-[8-(4-methoxy-benzyl)-4,5,6,8-tetrahydro-
3-thia-1,7,8-triaza-cyclopenta[e]azulen-2-yl]-amine (50 mg, 0.052 mmol)
and NaOH (14 mg, 0.36 mmol) in 2-methoxyethanol (0.5 mL) was
microwaved at 150 °C for 30 min. The mixture was diluted with DCM
(20 mL). The organic layer was washed with brine and concentrated to
give a crude intermediate, which was dissolved in TFA (2 mL). The
resulting reaction mixture was microwaved at 130 °C for 30 min and
concentrated. The residue was dissolved in i-PrOH/CHCl3 (1:3. 30 mL)
and quenched with saturated aqueous NaHCO3. The combined organic
layers were concentrated, and the resulting residue was purified on a
reversed-phase liquid chromatography/mass spectrometry (RP-HPLC/
MS) purification system (Gradient: acetonitrile in water, 20ꢁ95% in 3.3
min with a cycle time of 5 min. A shallow gradient between 25 and 50% of
acetonitrile was used between 0.6 andd 3.0 min to separate close-eluting
impurities. Flow rate: 100 mL/min. Mobile phase additive: 39 mM of
ammonium acetate. Column: Inertsil C8, 30 mm ꢀ 50 mm, 5 μm particle
size) to afford the title compound (5 mg, 9% over 2 steps). The 1H NMR
(400 MHz, DMSO-d6) δ 12.4 (br, 1H), 11.0 (s, 1H), 7.61 (S, 1H), 7.49
(T, J = 7.9 Hz, 1H), 6.50 (d, J = 7.8 Hz, 1H), 6.20 (d, J = 7.9 Hz, 1H),
4.49ꢁ4.44 (m, 2H), 3.67ꢁ3.62 (m, 2H), 3.25 (s, 3H), 2.96ꢁ2.86 (m,
4H), 1.96ꢁ1.87 (m, 2H). MS (ES+) m/e 358 (M + H)+. HRMS calcd for
(M + H)+: C17H20N5O2S+ 358.13322; found 358.13348.
(6-Fluoro-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-
cyclopenta[e]azulen-2-yl)-amine (23f). The title compound was
prepared according to the method described for compound 23c (5 mg,
6%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (s, 1H), 11.4 (s, 1H), 7.8
(q, J = 8.0 Hz, 1H), 7.7 (s, 1H), 7.0 (d, J = 8.0 Hz, 1H), 6.6 (dd, J = 8.0,
2.1 Hz, 1H), 3.0 (m, 4H), 2.0 (m, 2H). MS (ES+) m/e 302 (M + H)+.
HRMS calcd for (M + H)+: C16H18N5S + 312.12774; found 312.12770.
(6-Chloro-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23g). The title com-
pound was prepared according to the method described for compound
23c (5 mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ 12.6 (s, 1H), 11.4 (s,
1H), 7.7 (m, 2H), 7.7 (s, 1H), 7.0 (d, J = 7.8 Hz, 1H), 6.9 (d, J = 7.4 Hz,
1H), 3.0 (m, 4H), 2.0 (m, 2H). MS (ES+) m/e 302 (M + H)+. HRMS
calcd for (M + H)+: C14H13FN5S + 302.08702; found 302.08736.
(5-Fluoro-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23e). The title com-
pound was prepared according to the method described for compound
23q (6 mg, 10%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.2
(s, 1H), 8.24 (d, J = 3.0 Hz, 1H), 7.72ꢁ7.64 (m, 2H), 7.13ꢁ7.07 (m, 1H),
3.02ꢁ2.91 (m, 4H), 2.04ꢁ1.93 (m, 2H). MS (ES+) m/e 302 (M + H)+.
HRMS calcd for (M + H)+: C14H13FN5S + 302.08702; found 302.08736.
(6-Methoxy-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23h). The title com-
pound was prepared according to the method described for compound
N,N-Dimethyl-N0-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-
cyclopenta[e]azulen-2-yl)-pyridine-2,6-diamine (23n). The
title compound was prepared according to the method described for
compound 23q (23 mg, 29%). 1H NMR (400 MHz, DMSO-d6) δ 12.4
(br, 1H), 10.7 (s, 1H), 7.59 (s, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.13 (d, J =
7.7 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H), 3.03 (s, 6H), 2.93ꢁ2.85 (m, 4H),
1.95ꢁ1.86 (m, 2H). MS (ES+) m/e 327 (M + H)+. HRMS calcd for
(M + H)+: C16H19N6S+ 327.13864; found 327.13912.
N-Ethyl-N0-(4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclopenta-
[e]azulen-2-yl)-pyridine-2,6-diamine (23o). The title compound
was prepared according to the method described for compound 23q (21
1
23q (5 mg, 9%). H NMR (400 MHz, DMSO-d6) δ 11.1 (s, 1H),
7.74ꢁ7.52 (m, 2H), 6.57 (d, J = 7.8 Hz, 1H), 6.27 (d, J = 7.8 Hz, 1H),
4.00 (s, 3H), 3.01ꢁ2.94 (m, 4H), 2.03ꢁ1.95 (m, 2H). MS (ES+) m/e
314 (M + H)+. HRMS calcd for (M + H)+: C15H16N5OS+ 314.10701;
found 314.10631.
(6-Ethoxy-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23i). The title com-
pound was prepared according to the method described for compound
23q (8 mg, 10%). 1H NMR (400 MHz, DMSO-d6) δ 12.6 (br, 1H), 11.1
(s, 1H), 7.76 (s, 1H), 7.63 (t, J = 7.9 Hz, 1H), 6.64 (d, J = 7.8 Hz, 1H),
6.32 (d, J = 7.8 Hz, 1H), 4.55 (q, J = 7.0 Hz, 2H), 3.10ꢁ3.01 (m, 4H),
2.12ꢁ2.02 (m, 2H), 1.45 (t, J = 7.0 Hz, 3H). MS (ES+) m/e 328 (M +
H)+. HRMS calcd for (M + H)+: C16H18N5OS+ 328.12266; found
328.12323.
1
mg, 26%). H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 10.7 (s,
1H), 7.66 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.43 (d, J = 5.5 Hz, 1H), 6.08
(d, J = 7.6 Hz, 1H), 5.93 (d, J = 7.9 Hz, 1H), 3.48ꢁ3.38 (m, 2H),
3.02ꢁ2.89 (m, 4H), 2.03ꢁ1.94 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). MS
(ES+) m/e 326 (M + H)+; HRMS calcd for (M + H)+: C16H19N6S+
327.13864; found 327.13873.
(6-Pyrrolidin-1-yl-pyridin-2-yl)-(4,5,6,8-tetrahydro-3-thia-
1,7,8-triazacyclopenta[e]azulen-2-yl)-amine (23p). The title
compound was prepared according to the method described for
1
compound 23q (5 mg, 8%). H NMR (400 MHz, DMSO-d6) δ 12.4
(br, 1H), 10.7 (s, 1H), 7.59 (s, 1H), 7.30ꢁ7.23 (m, 1H), 6.07 (d, J = 7.7
Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 3.46ꢁ3.37 (m, 4H), 2.93ꢁ2.84 (m,
4H), 1.96ꢁ1.84 (m, 6H). MS (ES+) m/e 353 (M + H)+. HRMS calcd for
(M + H)+: C18H21N6S+ 353.15429; found 353.15444.
(4-Methyl-pyrimidin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23q). The mixture of
18b (60 mg, 18 mmol), 2-chloro-4-methylpyrimidine (23 mg, 18
mmol), Cs2CO3 (120 mg, 0.363 mmol), Pd2(dba)3 (2 mg, 0.002 mmol),
and Xantphos (2 mg, 0.004 mmol) in DMF/THF (1.5 mL/1.5 mL) was
microwaved at 125 °C for 1 h, filtered through the Celite pad, and
concentrated to afford the crude intermediate, [8-(4-methoxy-benzyl)-
6-(4,5,6,8-Tetrahydro-3-thia-1,7,8-triaza-cyclopenta[e]-
azulen-2-ylamino)-pyridine-2-carboxylic Acid Methyl Ester
(23k). The title compound was prepared according to the method
described for compound 23q (5 mg, 8%). 1H NMR (400 MHz, DMSO-d6)
δ 12.4 (br, 1H), 11.3 (s, 1H), 7.77 (dd, J = 8.3, 7.4 Hz, 1H), 7.63 (s, 1H),
7.50 (dd, J = 7.3, 0.6 Hz, 1H), 7.21(d, J = 8.3 Hz, 1H), 3.84 (s, 3H),
2.95ꢁ2.88 (m, 4H), 1.97ꢁ1.89 (m, 2H). MS (ES+) m/e 342 (M + H)+.
+
HRMS calcd for (M + H)+: C16H16N5O2S
342.10217.
342.10192; found
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Journal of Medicinal Chemistry
ARTICLE
4,5,6,8-tetrahydro-3-thia-1,7,8-triaza-cyclopenta[e]azulen-2-yl]-(4-methyl-
pyrimidin-2-yl)-amine, which was dissolved in TFA (3.0 mL). The
resulting reaction mixture was microwaved at 150 °C for 30 min and
concentrated. The residue was dissolved in i-PrOH/CHCl3 (1:3.
30 mL) and quenched with saturated aqueous NaHCO3. The combined
organic layers were concentrated, and the resulting residue was purified
on a reverse-phase HPLC to afford the title compound (5 mg, 9% over 2
steps). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.4 (s, 1H),
8.43 (d, J = 5.0 Hz, 1H), 7.70 (s, 1H), 6.88 (d, J = 5.0 Hz, 1H), 3.01ꢁ2.95
(m, 4H), 2.42 (s, 3H), 2.02ꢁ1.95 (m, 2H). MS (ES+) m/e 299 (M + H)+.
HRMS calcd for (M + H)+: C14H15N6S + 299.10734; found 299.10760.
(4-Methoxy-pyrimidin-2-yl)-(4,5,6,8-tetrahydro-3-thia-1,7,8-
triaza-cyclopenta[e]azulen-2-yl)-amine (23r). The title com-
pound was prepared according to the method described for compound
23q (5 mg, 9%). 1H NMR (400 MHz, DMSO-d6) δ 12.5 (br, 1H), 11.4
(s, 1H), 8.29 (d, J = 5.7 Hz, 1H), 7.70 (s, 1H), 6.42 (d, J = 5.7 Hz, 1H),
4.01 (s, 3H), 3.03ꢁ2.94 (m, 4H), 2.03ꢁ1.95 (m, 2H). MS (ES+) m/e
315 (M + H)+; HRMS calcd for (M + H)+: C14H15N6OS+ 315.10226;
found 315.10278.
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(14) Geometry optimizations were conducted with the DFT-
(B3LYP)/6-31G** method with the SM8 water solvation model as
implemented in the Spartan’08 software package, which is distributed by
Wave function, Inc. of 18401 Von Karman Avenue, Suite 370, Irvine, CA
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’ ASSOCIATED CONTENT
S
Supporting Information. Cross reactivity panel for com-
b
pounds 9 and 22a, reference compounds used in cross reactivity
panel and controls used in the MDCK-mdr1 in vitro assay for
P-gp substrate activity. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 201-350-0341. Fax: 201-261-0623. E-mail: dado@
lundbeck.com.
’ ACKNOWLEDGMENT
We thank Drs. Gamini Chandrasena, Robbin Brodbeck, and
Andrew White for their helpful advice and support. We also
thank Drs. Mark Hayward, Xu Zhang, Qing Ping Han, Chi
Zhang, Rajinder Bhardwaj, Mr. Manuel Cajina, and Mrs. Megan
Nattini for their analytical and DMPK support.
’ ABBREVIATIONS USED
DMPK, drug metabolism and pharmacokinetics; Tr, trityl; PMB,
4-methoxybenzyl; PAM, positive allosteric modulator; mGlu4,
metabotropic glutamate 4 receptor; HTS, high-throughput
screening; MTS, medium-throughput screening; DMF-DMA, N,
N-dimethylformamide dimethoxyacetal; CNS, central nervous
system; TLC, thin layer chromatography; LC-MS, liquid
chromatographyꢁmass spectrometry; NMR, nuclear magnetic
resonance; FLIPR, fluorescent imaging plate reader; tPSA, topo-
logical polar surface area; CLint, intrinsic clearance; CSF, cere-
brospinal fluid; TMS, tetramethylsilane; BHK, baby hamster
kidney; DMEM, Dulbecco's Modified Eagle Medium; HBSS,
Hank’s Balanced Saline Solution; HEPES, N-2-hydroxyethylpi-
perazine-N0-2-ethanesulfonic acid; BSA, bovine serum albumin;
NAM, negative allosteric modulator; SAR, structureꢁactivity
relationship; SPR, structureꢁproperty relationship; P-gp,
P-glycoprotein; GPCR, G protein coupled receptor; hERG,
human ether-ꢀa-go-go related gene
(15) Bolea, C.; Celanire, S. Preparation of heteroaryldihydrothiazo-
loindazoleamine derivatives and analogs as metabotropic glutamate
receptors modulators. Patent WO 2010079238, 2010.
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ARTICLE
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