Identification of a novel orally bioavailable phosphodiesterase 10A (PDE10A) inhibitor with efficacy in animal models of schizophrenia.

Article abstract of DOI:10.1021/jm501651a

We report the continuation of a focused medicinal chemistry program aimed to further optimize a series of imidazo[1,2-a]pyrazines as a novel class of potent and selective phosphodiesterase 10A (PDE10A) inhibitors. In vitro and in vivo pharmacokinetic and pharmacodynamic evaluation allowed the selection of compound 25a for its assessment in preclinical models of psychosis. The evolution of our medicinal chemistry program, structure-activity relationship (SAR) analysis, as well as a detailed pharmacological profile for optimized lead 25a are described.

Full text of DOI:10.1021/jm501651a

Article
pubs.acs.org/jmc
Identification of a Novel Orally Bioavailable Phosphodiesterase 10A
(PDE10A) Inhibitor with Efficacy in Animal Models of Schizophrenia.
Jose Manuel Bartolome-Nebreda,† Sergio A. Alonso de Diego,† Marta Artola,† Francisca Delgado,†
́
́
†
†
†
†
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́
Oscar Delgado, María Luz Martín-Martín, Carlos M. Martínez-Viturro, Miguel Angel Pena,
Han Min Tong,† Michiel Van Gool,† Jose Manuel Alonso,‡ Alberto Fontana,‡ Gregor J. Macdonald,§
́
Anton Megens,∥ Xavier Langlois,∥ Marijke Somers,⊥ Greet Vanhoof,# and Susana Conde-Ceide*,†
‡
†Neuroscience Medicinal Chemistry and Discovery Sciences Analytical Chemistry, Janssen Research & Development,
Calle Jarama 75, Polígono Industrial, Toledo 45007, Spain
∥
⊥
#
§Neuroscience Medicinal Chemistry, Neuroscience Biology, Discovery Sciences ADME/Tox, and Discovery Sciences Cellular
Pharmacology, Janssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium
S
* Supporting Information
ABSTRACT: We report the continuation of a focused medicinal chemistry program aimed
to further optimize a series of imidazo[1,2-a]pyrazines as a novel class of potent and selective
phosphodiesterase 10A (PDE10A) inhibitors. In vitro and in vivo pharmacokinetic and
pharmacodynamic evaluation allowed the selection of compound 25a for its assessment in
preclinical models of psychosis. The evolution of our medicinal chemistry program, structure−
activity relationship (SAR) analysis, as well as a detailed pharmacological profile for optimized
lead 25a are described.
to five different molecules have progressed into clinical
evaluation to confirm this hypothesis.8
INTRODUCTION
■
Cyclic nucleotides, such as cAMP and cGMP, play a prominent
role as second messengers that activate several neuronal cellular
signaling pathways.1 Phosphodiesterases (PDEs) are enzymes
that metabolically inactivate these intracellular second mes-
sengers as such regulating and compartmentalizing the cyclic
nucleotide signaling cascades. The PDE family comprises 11
members (PDE1−11) encoded by distinct genes with each of
them encoding for several splice variants. Such diversity in
enzyme structure outside the conserved catalytic core suggests
that gene duplication and divergence have allowed for PDE
specialization in different tissues. Among the different PDEs,
PDE10A has one of the most restricted expression profiles
almost exclusively limited to the brain and testes in different
mammalian species.2 In the brain, PDE10A is highly
expressed in striatal medium spiny neurons (MSNs) which
are crucial for transmission and control of glutamatergic and
dopaminergic input within the basal ganglia, and hence it is
believed that inhibition of PDE10A contributes to the
regulation of cyclic nucleotide signaling in the cortico-
striato-thalamic circuit, which plays a key role in cognitive
and motor processes and in emotional behaviors.3 In light of
these features, during the past decade several pharmaceutical
companies have initiated research programs with the aim to
identify selective PDE10A inhibitors in order to evaluate their
potential as novel central nervous system (CNS) therapeutic
agents.4 These efforts have culminated in the identification
of several structurally different PDE10A inhibitors with
proved efficacy in a wide variety of preclinical models of
schizophrenia5 and other neurological disorders such as
Huntington’s disease (HD)6 and Lesch−Nyhan disease.7 Up
Our research group has recently reported the design,
synthesis, and preliminary SAR of a series of imidazo[1,2-a]-
pyrazine derivatives originated from a high-throughput screen-
ing campaign resulting in the identification of compound 1
(Figure 1) as a novel, potent, selective, and orally efficacious
Figure 1. Compound 1 (initial hit) and compound 2.
PDE10A inhibitor.9 Encouraged by its overall promising profile,
we considered to expand our SAR exploration by seeking for
alternatives to the N-(methoxyethyl)pyrazole moiety present
in 1, aiming to further improve its PDE10A inhibitory efficacy.
For this purpose, and based on our previous SAR findings,9 we
explored close analogues of the 8-morpholine-2-methyl-
imidazo[1,2-a]pyrazine. The most relevant findings of this
exploration are summarized herein.
Received: October 28, 2014
© XXXX American Chemical Society
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a
Scheme 1. Synthesis of Compound 9
a
Reagents and conditions: (a) ethyl 2-chloroacetoacetate, EtOH, 90 °C, 18 h, 82%; (b) isopropylmagnesium chloride, N,O-dimethylhydroxylamine
hydrochloride, DCM, THF, −20 °C to rt, 21 h, 68%; (c) methylmagnesium bromide, THF, −78 °C to rt, 16 h, 92%; (d) trimethylsilyl
trifluoromethanesulfonate, DIPEA, DCM, 0 °C to rt, 16 h, 97%; (e) NBS, NaHCO3, THF, −78 °C, 1 h, 67%; (f) isovaleramide, 1,4-dioxane, DMF,
90 °C, 18 h, 38%.
a
CHEMISTRY
Scheme 2. Synthesis of Compounds 11 and 13
■
As a first step, we targeted the synthesis of a small set of
analogues in which the pyrazole ring was replaced by other five-
membered heterocycles such as oxazole, thiazole, or pyrrole.
Prompted by synthetic accessibility, and supported by the in vitro
PDE10A inhibitory activity of the corresponding (isobutyl)-
pyrazole-holding prototype 2,9 an isobutyl side chain was
incorporated on the oxazole and thiazole containing analogues.
The synthesis of the oxazole derivative, outlined in Scheme 1,
commenced with the reaction of commercially available
2-amino-4-morpholinepyrazine 3 with ethyl 2-chloroacetoacetate
to give the corresponding 2-methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine-3-carboxylic acid ethyl ester 4. Activation of the
ester via the Weinreb amide 5, followed by treatment with
methylmagnesium bromide, gave ketone 6, which was then
selectively monobrominated at α position after its conversion
into the corresponding trimethylsilyl enol ether 7 and sub-
sequent reaction with N-bromosuccinimide (NBS). Finally, the
reaction of bromo ketone 8 with isovaleramide led to the
desired compound 9.
a
Reagents and conditions: (a) 2-isobutylthiazole, Pd(OAc)2,
K3PO4, tert-butyldicyclohexylphosphine, NMP, 125 °C, 18 h, 29%;
(b) 1-(triisopropylsilyl)pyrrole-3-boronic acid, PdCl2(PPh3)2,
Na2CO3, 1,4-dioxane, 100 °C, 16 h, 82%; (c) 2-bromoethyl methyl
ether, Cs2CO3, DMF, 160 °C, 30 min, microwave irradiation, 73%.
The thiazole-containing derivative 11 was prepared via C−H
activation and coupling of the 2-isobutylthiazole with precursor
bromoheteroarene 1010 catalyzed by palladium acetate and
in the presence of tert-butyldicyclohexylphosphine (Scheme 2).
The preparation of the corresponding pyrrole analogue 13 was
accomplished via Suzuki−Miyaura cross-coupling of 10 with
the 1-(triisopropylsilyl)pyrrole-3-boronic acid and subsequent
alkylation with 2-bromoethyl methyl ether to yield compound
13 (Scheme 2).
We next turned our attention to pyridines as possible re-
placements for the pyrazole ring. The key iodo intermediate 15
was prepared in two steps in a similar way as previously
described for the synthesis of bromo derivative 10,9 selective
iodination of 1411 at 3-position with N-iodosuccinimide
(NIS),12 followed by nucleophilic substitution with morpho-
line. From this common intermediate different synthetic
strategies were implemented depending upon the substitution
on the pyridine derivative targeted (see Schemes 3 and 5).
Thus, Suzuki−Miyaura cross-coupling of iodide 15 with
2-chloropyridyl-4-boronic acid yielded compound 16, and
subsequent Suzuki−Miyaura cross-coupling with vinylboronic
acid pinacolester followed by addition of sodium methoxide
afforded compound 17 (Scheme 3).
The synthetic route followed to prepare compound 22
was slightly different. In this case, the Wittig reaction of the
commercially available 6-bromonicotinaldehyde 18 with
(methoxymethyl)triphenylphosphonium chloride followed by
hydrogenation of the double bond formed in the presence of
rhodium as catalyst afforded 20. The transformation of
bromopyridine 20 into stannanyl derivative 21 followed by
Stille coupling yielded the desired analogue 22 (see Scheme 4).
To further expand the exploration of regioisomeric
C-alkylated pyridines, a similar approach as reported for the
synthesis of compound 17 was followed. Suzuki−Miyaura
cross-coupling of iodo derivative 15 with 2-chloropyridine-5-
boronic acid afforded intermediate 23, key in order to prepare a
variety of analogues as depicted in Scheme 5. Chloropyridine
23 was coupled with the vinylboronic acid pinacolester, afford-
ing compound 24 that was reacted with MeOH in the presence
of potassium hydrogen sulfate to yield 25a, was reacted with a
variety of different alcohols in basic media to obtain com-
pounds 25b−c, was treated with potassium hydrogen sulfate
and water to afford the hydroxy derivative 25d, or was hydro-
genated in the presence of Pd/C to yield the corresponding
ethyl analogue 25e. Analogues 25f−k were prepared via
B
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a
Scheme 3. Synthesis of 17
a
Reagents and conditions: (a) NIS, DCM, 0 °C, 2 h, 97%; (b) morpholine, DIPEA, ACN, 80 °C, 16 h, 80%; (c) 2-chloropyridine-4-boronic acid,
Pd(PPh3)4, Na2CO3, 1,4-dioxane, 90−110 °C, 10.5 h, 83%; (d) vinylboronic acid pinacolester, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 2 h, 78%;
(e) MeONa, MeOH, 100 °C, 18 h, 25%.
a
Scheme 4. Synthesis of 22
a
Reagents and conditions: (a) (methoxymethyl)triphenylphosphonium chloride, nBuLi, THF, 0 °C to rt, 16 h, 73%; (b) Rh/C 5% cartridge, EtOH,
full H2 mode, 70 °C, H-Cube, 21%; (c) Bu3SnCl, nBuLi, THF, −78 °C to rt, 1 h, 100%; (d) 21, Pd(PPh3)4, CuBr, 1,4-dioxane, 160 °C, 20 min,
microwave irradiation, 11%.
Suzuki−Miyaura cross-coupling of the corresponding bromo
intermediate 10 with the corresponding pyridinylboronic ester
derivatives.13 Alternatively, other side chains were introduced
on the pyridine by reaction with various Grignard derivatives
(compounds 25l,m), through Suzuki−Miyaura cross-coupling
reaction (compound 25n) or via nucleophilic substitution with
different amines (compounds 25o−p,r) or Buchwald type
reaction (compound 25q).
To further explore pyridine derivatives, we synthesized
analogues modified at 2- and 8-position of the central scaffold.
These analogues, 28a−g, were prepared via Suzuki−Miyaura
cross-coupling of the corresponding bromo derivatives 26a−g14
with the 2-(2-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)pyridine (27)15 (Scheme 6).
terms of their extraction ratio (ER) values and intrinsic clearance
(Clint), and those considered sufficiently metabolically stable
(ER < 0.5 and Clint < 40 μL/min/mg at 1 μM concentration)
were then tested in vivo for their ability to revert the
apomorphine-induced stereotypy in rats, a model that has been
extensively used to assess potential antipsychotic efficacy.16
Among the 5-member ring heterocycles evaluated as possible
replacements of the pyrazole ring, the oxazole and thiazole
containing analogues 9 and 11 maintained comparable in vitro
potency to prototype 2 while the pyrrole derivative 13 showed
decreased activity compared to lead 1. Unfortunately, neither 9
nor 11 were found metabolically stable enough to be con-
sidered for in vivo evaluation (see Table 1).
In the case of the replacement of the methoxyethyl pyrazole
moiety by pyridine, the three regioisomeric derivatives 17, 22,
and 25a showed comparable in vitro activity with the initial
lead 1 and displayed acceptable metabolic stability (Table 1) to
be considered for in vivo evaluation. The 3-pyridine analogue
25a was found the most potent within the set with an in vivo
activity ∼3.5 fold higher than 1 (ED50 = 1.02 mg/kg). With this
promising result in hand, the possibility to explore diversity on
the 3-pyridine was considered attractive and SAR development
continued along those lines. As in the case of the pyrazole
analogues previously reported,9 a wide range of variations of the
methoxyethyl side chain was allowed (Table 1).17 The
elongation of the methoxy group to an ethoxy afforded com-
pound 25b that showed a similar profile to 25a while the
incorporation of an isopropoxy group yielded compound 25c
that was not metabolically stable enough to be further profiled.
Compound 25d, where the methoxy group was replaced by a
hydroxy group while being much more stable metabolically lost
in vivo activity. The introduction of two gem-methyls on α
position to the methoxy moiety did not have a detrimental
impact on the in vitro profile and 25f presented similar in vivo
Finally, derivatives modified at C-6 of the scaffold were
prepared starting from the 3-chloro-5-iodo-pyrazin-2-amine
(29) as outlined in Scheme 7. Reaction with 2-chloroacetone
followed by bromination at the 3-position with NBS afforded
bicycle 30. Selective substitution of the chlorine atom by
morpholine yielded 31. The introduction of a methyl group at
the 6-position was accomplished by palladium catalyzed re-
action of the iodo derivative 31 with methylindium generated
in situ. The trifluoromethyl group was introduced via copper-
mediated cross coupling of 31 with methyl 2,2-difluoro-2-
(fluorosulfonyl)acetate (MDFA). Finally, Suzuki−Miyaura
cross-coupling of 26h and 26i with pyridine 27 yielded,
respectively, compounds 28h and 28i.
RESULTS AND DISCUSSION
■
The most relevant findings of this exploration are summarized
in Tables 1 and 2. Compounds were first evaluated for their
in vitro PDE10A inhibitory activity. Compounds with a pIC50 > 6.5
were selected to assess their in vitro metabolic stability in rat
liver microsomes preparations. Compounds were ranked in
C
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a
Scheme 5. Synthesis of 25a−r
a
Reagents and conditions: (a) 2-chloropyridine-5-boronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 23 h, 93%; (b) vinylboronic acid
pinacolester, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 1 h, 90%; (c) for 25a, KHSO4, MeOH, 80 °C, 3 days, 63%; for 25b, EtONa, EtOH, 100 °C,
16 h, 47%; for 25c, 2-propanol, NaH, 100 °C, 18 h, 25%; for 25d, KHSO4, water, 80 °C, 16 h, 18%; for 25e, Pd/C 10%, H2 atm, EtOH/EtOAc/
DCM, rt, 16 h, 91%; (d) heteroarylboronic derivative, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 140−150 °C, 15−20 min, microwave irradiation or 90 °C,
18 h, 11−94%; (e) For 25l, isopropylmagnesium chloride, Ni(dppf)2, THF, 0 °C to rt, 30 min, 56%; for 25m, isobutylmagnesium chloride,
Ni(dppf)2, THF, 0 °C to rt, 3 h, 56%; for 25n, cyclopropylboronic acid, 2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl, Pd(OAc)2, K3PO4,
toluene, 80 °C, 22 h, 29%; for 25o, pyrrolidine, 130 °C, 30 min, microwave irradiation, 60%; for 25p, (i) (R)-3-pyrrolidinol, 120 °C, 3 h, (ii) NaH,
MeI, THF, rt, 3 days, 19%; for 25q, isopropylamine, Pd(OAc)2, Binap, Cs2CO3, toluene, 50 °C, 16 h, 42%; for 25r, piperazine, 120 °C, 24 h, 22%.
potency to compound 25a. Elongation of the side chain by one
carbon atom (compound 25g) or introduction of an oxygen
linker on the β-position with regard to the pyridine ring
(compound 25h) had no significant impact on the measured in
vitro properties, but both compounds lacked in vivo activity
at the dose tested. Truncation of the side chain to a methyl
(compound 25k) or an ethyl (compound 25e) or the
incorporation of other small alkylic groups, such as an isoPr
(compound 25l), a cycloPr (compound 25n), or an isoBu
compound (25m), had little effect on in vitro primary activity.
These compounds were not further profiled due to lack of
adequate metabolic stability (25k−m) or in vitro potency
(25n). Additional SAR was carried out by the introduction of
an oxygen atom as linker. Ether 25i resulted in an attractive
compound with an in vivo ED50 of 0.79 mg/kg, while analogue
25j possessed much attenuated in vitro metabolic stability
under microsomal incubation and was not further profiled.
Likewise, the incorporation of nitrogen containing side chains
in pyrrolidine analogues 25o and 25p resulted in different
degrees of metabolic stability. The methoxy-substituted
pyrrolidine, 25p, is more stable than the unsubstituted
pyrrolidine, 25o. Unfortunately, 25p lacked relevant in vivo
efficacy. Isopropylamine derivative 25q was highly unstable,
while piperazine analogue 25r, sterically similar to the
pyrrolidine 25o, was highly stable in microsomes incubation
but did not show in vivo activity in the apomorphine model at
the dose tested. The relatively unresponsive in vitro pyridine
side chain SAR observed in the reported analogues could be
explained based on the fact that the R group of the pyridine, as
in the case of the pyrazole series reported in the previous paper,
gets oriented toward the solvent with no significant interactions
with the protein.
To determine levels of PDE10A occupancy, we evaluated the
most potent compounds from the induced stereotypy model
(25a, 25b, 25f, and 25i) in a central in vivo occupancy assay
that measures the displacement of the selective PDE10A ligand
[3H]MP-10.18 The specific binding was determined as the
difference between [3H]MP-10 binding quantified in the
striatum (a brain area showing a high density of PDE10A
enzyme) and in the cortex (a brain area where PDE10A is
virtually absent). Occupancy ED50 was calculated as the
inhibition of specific [3H]MP-10 binding in drug treated
animals relative to vehicle-treated animals. Gratifyingly,
occupancy data for compounds 25a, 25b, and 25f confirmed
target engagement in the brain at comparable ED50s to our
behavioral model. Unfortunately, and due to unknown reasons,
PDE10A occupancy could not be confirmed for the in vivo
most potent analogue, 25i, and therefore it was discarded for
additional profiling. Out of the remaining derivatives tested,
compound 25a proved to be the most promising analogue with
D
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a
increase of in vitro activity vs 25a (0.3 log units), an effect that
translated into a similar in vivo potency and occupancy
compared with 25a. Unfortunately, compound 28c showed
undesirable side effects in rats after administration of a single
dose of 40 mg/kg sc (passivity, hypotonia, flat body posture,
hypothermia, sedation) and was not further progressed. In a
similar fashion as previously described for our initial hit 1,9 we
also explored the possibility to replace the morpholine moiety
by other substituents. In this case, replacement by pyrrolidine
(28e), 4-pyridine (28f), or 3-pyridine (28g) was found
detrimental for the in vitro potency. Finally, introduction of
small groups, such as methyl (28h) or trifluoromethyl (28i) on
the 6-position of the central scaffold was not well-tolerated and
led to a decrease in in vitro potency. At this point in time and
because none of the modifications tried was successful,
compound 25a was selected to be further profiled.
Scheme 6. Synthesis of 28a−g
Further profiling of 25a confirmed a high degree of PDE10A
selectivity (79-fold toward PDE1B, more than 400-fold toward
others PDEs), high selectivity against a selection of GPCRs, ion
channels, and transporters (CEREP radioligand binding panel,
IC50 > 10 μM), and no relevant inhibition (>50% inhibition at
10 μM) of a selection of kinases in Millipore panel.19 In vitro
ADME evaluation revealed a clean CYP inhibition profile (IC50
all tested CYPs > 10 μM), a low binding to plasma proteins
(hPPB 58% and rPPB 60% bound) and to brain tissue (hBTB
69% and rBTB 85% bound) in the different species tested. We
also explored its pharmacokinetic properties in rats. Absorption
of 25a following oral administration of a single dose (10 mg/kg)
resulted in an absolute bioavailability of 45%. Compound 25a
showed moderate clearance (34 mL/min/kg), low volume of
distribution (1.9 L/kg), and a moderate short terminal half-life
(0.8 h). Additionally, 25a showed good brain exposure, reaching
total levels of 1212 ng/g in the brain after 1 h (10 mg/kg sc). On
the basis of these data, the compound was deemed an excellent
candidate for evaluation in acute behavioral studies in rats.20
a
Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, 1,4-dioxane,
CONCLUSION
150 °C, 15 min, microwave irradiation, 48−90%.
■
In conclusion, SAR expansion of our initial lead 1 focused on
the replacement of its pyrazole moiety by other heterocycles,
resulting in the identification of 25a. Compound 25a shows
improved efficacy in animal models believed to mimic the
positive symptoms of schizophrenia than the initial lead 1
and confirmed PDE10A engagement in the brain. Selectivity
profiling, in vitro ADME, and in vivo PK evaluation in rats
confirmed 25a as an excellent candidate for extensive efficacy
and side effect profiling evaluation in acute behavioral studies
in rats.20
improved in vivo potency and occupancy over compound 1
(see Table 1)
Table 2 summarizes some results of our subsequent efforts
aimed to further increase the potency of compound 25a.
Keeping the methoxyethylpyridine fixed at the 3-position, we
first evaluated the influence of the introduction of small groups
(CF3, isoPr, cycloPr, OMe) on the 2-position of the scaffold.
Among them, the best results were obtained by the introduc-
tion of a cyclopropyl substituent (28c), resulting in a moderate
a
Scheme 7. Synthesis of 28h,i
a
Reagents and conditions: (a) 2-chloroacetone, NaI, 90 °C, 24 h, 28%; (b) NBS, DCM, rt, 3 h, 83%; (c) morpholine, DIPEA, ACN, 160 °C, 30 min,
microwave irradiation, 83%; (d) For 26h, InCl3, MeLi, THF, −78 °C, 30 min then Pd(PPh3)4, 80 °C, 16 h, 78%; for 26i, MDFA, CuI, DMF, 90 °C,
16 h, 60%; (e) 27, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 150 °C, 15−30 min, microwave irradiation, 82%.
E
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Table 1. PDE10A Inhibitory Activity of the 5 and 6-Member Ring Heterocyclic Systems
F
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Table 1. continued
a
b
Values are mean SD of at least two experiments. Data refer to compound metabolized after incubation of tested compound with rat liver
c
microsomes, at 1 μM concentration across a time course (typically 0, 5, 10, 20, 40, and 60 min) (n = 1 per dose). Wistar rats (n = 3 per dose) were
pretreated with test compound (sc) or solvent and after 30 min, apomorphine (1.0 mg/kg, iv) induced stereotypy was scored every 5 min over the
d
e
first hour after injection of apomorphine. Compound 25i was administered oral (po). Dose−response experiments to measure PDE10 occupancy
were performed 1 h after sc administration (n = 3 per dose).
Labstation (Milestone, Inc.). Nuclear magnetic resonance (NMR)
spectra were recorded with either a Bruker DPX-400 or a Bruker
AV-500 spectrometer (Bruker AG) with standard pulse sequences
operating at 400 and 500 MHz, respectively, using CDCl3 and DMSO-
EXPERIMENTAL SECTION
General Procedures. Unless otherwise noted, all reagents and
solvents were obtained from commercial suppliers and used without
further purification. Thin layer chromatography (TLC) was carried out
■
d6 as solvents. Chemical shifts (δ) are reported in parts per million
(ppm) downfield from tetramethylsilane (δ = 0). Coupling constants
are reported in hertz. Splitting patterns are defined by s (singlet), d
(doublet), dd (double doublet), t (triplet), q (quartet), tt (triplet of
triplet), spt (septuplet), dquin (doublet of quintet), dt (doublet of triplet),
on silica gel 60 F254 plates (Merck). Flash column chromatography
was performed on silica gel, particle size 60 Å, mesh of 230−400
(Merck), under standard techniques. Microwave assisted reactions
were performed in a single-mode reactor, Biotage Initiator Sixty micro-
wave reactor (Biotage), or in a multimode reactor, MicroSYNTH
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Table 2. PDE10A Inhibitory Activity of Compounds 28a−i
a
b
Values are mean SD of at least two experiments. Data refer to compound metabolized after incubation of tested compound with rat liver
c
microsomes, at 1 μM concentration across a time course (typically 0, 5, 10, 20, 40, and 60 min) (n = 1 per dose). Wistar rats (n = 3 per dose) were
pretreated with test compound (sc) or solvent and after 30 min, apomorphine (1.0 mg/kg, iv) induced stereotypy was scored every 5 min over the
d
first hour after injection of apomorphine. Dose−response experiments were performed to measure PDE10A occupancy 1 h after sc administration
(n = 3 per dose)..
br (broad signal), or m (multiplet). Purities of all new compounds
were determined by analytical reverse phase RP (HPLC or UPLC)
coupled to a mass spectrometry detector, using the area percentage
method on the DAD trace, and were found to have ≥95% purity
unless otherwise specified. The HPLC or UPLC measurement was
performed using either an HP1100 (Agilent Technologies) system or
an Acquity UPLC system (Waters) comprising a pump (quaternary or
binary) with degasser, an autosampler, a column oven, a diode array
detector (DAD), and a column as specified in the respective methods.
The MS detector (LCT, SQD, MSD) was configured with an electro-
spray ionization source. Nitrogen was used as the nebulizer gas. Data
acquisition was performed with MassLynx-Openlynx software or
Chemstation-Agilent Data Browser software. Compounds are
described by their experimental retention times (tR) and ions. If not
specified differently in the table of data, the reported molecular ion
corresponds to the [M + H]+ (protonated molecule). For molecules
with multiple isotopic patterns (Br, Cl, ...) the reported value is the
one obtained for the lowest isotope mass unless otherwise specified.
All results were obtained with experimental uncertainties that are
commonly associated with the method used. Low-resolution MS is
given for the compounds except for the ones analyzed on the LCT
detector which are validated by high-resolution MS. Detailed
H
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information about the different LCMS methods employed can be
found in the Supporting Information. Melting point (mp) values
are peak values and were obtained with experimental uncertainties that
are commonly associated with this analytical method. Melting points
were determined in open capillary tubes performing the experiment up
to a maximum temperature of 300 °C, measuring them either on a
Mettler apparatus (FP62 or FP 81HT/FP90 with a temperature
gradient of 10 °C/min) or on a Shanghai Precision/Scientific
Instrument Co. Ltd. apparatus (WRS-2A with a temperature gradient
of 0.2−5.0 °C/min).
hydrogen carbonate (0.192 g, 2.29 mmol) were added to a stirred
solution of intermediate 7 (0.38 g, 1.14 mmol) in THF (8 mL). The
mixture was stirred at −78 °C for 1 h and then diluted with Et2O and
extracted with a cold saturated solution of sodium hydrogen carbonate.
The organic layer was separated, dried (Na2SO4), filtered, and the
solvents evaporated in vacuo. The crude product was purified by flash
column chromatography (silica; EtOAc in DCM 0/100 to 10/90).
The desired fractions were collected and evaporated in vacuo to yield
intermediate 8 (0.26 g, 67%) as a pale-yellow solid. MS: m/z 339 [M + H]+.
tR =2.33 min (method 6). 1H NMR (500 MHz, CDCl3) δ ppm 2.82 (s,
3 H), 3.87 (t, J = 4.9 Hz, 4 H), 4.22 (br t, J = 4.6, 4.6 Hz, 4 H), 4.35 (s,
2 H), 7.63 (d, J = 4.6 Hz, 1 H), 8.91 (d, J = 4.3 Hz, 1 H).
2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine-3-carboxylic
Acid Ethyl Ester (4). A mixture of 3-morpholinopyrazin-2-amine 3
(2 g, 11.1 mmol) and ethyl 2-chloroacetoacetate (7.7 mL, 55.5 mmol)
in EtOH (8 mL) was stirred at 90 °C for 18 h. The mixture was cooled
down to rt and diluted with Et2O. The solid formed was filtered off
and dried in vacuo to yield intermediate 4 (2.98 g, 82%) as a white
solid (hydrochloride salt). MS: m/z 291 [M + H]+. tR = 1.25 min
(method 8). 1H NMR (500 MHz, CDCl3) δ ppm 1.46 (t, J = 7.2 Hz,
3 H), 2.69 (s, 3 H), 3.86−4.00 (m, 4 H), 4.47 (q, J = 7.1 Hz, 2 H),
4.50 (br s, 4 H), 7.56 (d, J = 4.9 Hz, 1 H), 8.65 (d, J = 4.9 Hz, 1 H).
2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine-3-carboxylic
Acid Methoxy-methyl-amide (5). A 2 M solution of isopropylmagne-
sium chloride in THF (10.33 mL, 20.67 mmol) was added over 15 min
to a stirred suspension of intermediate 4 (2 g, 6.89 mmol) and N,O-
dimethylhydroxylamine hydrochloride (1.0 g, 10.33 mmol) in a
mixture of THF (15 mL) and DCM (8 mL) at −20 °C under
nitrogen. The mixture was stirred at −5 °C for 1 h and then allowed to
warm to rt and stirred for a further 16 h. The mixture was cooled
to −20 °C and further N,O-dimethylhydroxylamine hydrochloride
(1 g, 10.33 mmol) and a 2 M solution of isopropylmagnesium chloride
in THF (10.33 mL, 20.67 mmol) were added. The mixture was stirred
at −20 °C for 5 min, allowed to warm to rt, and then stirred for a
further 5 h. The mixture was cooled to −10 °C, and a saturated
solution of ammonium chloride was added. The organic layer was
separated, dried (Na2SO4), filtered, and the solvents evaporated
in vacuo. The crude product was purified by flash column chromatog-
raphy (silica; EtOAc in DCM 0/100 to 50/50). The desired frac-
tions were collected and evaporated in vacuo to yield intermediate 5
(1.43 g, 68%) as a pink solid. MS: m/z 306 [M + H]+. tR = 1.99 min
3-(2-Isobutyl-oxazol-4-yl)-2-methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine (9). Isovaleramide (0.082 g, 0.81 mmol) was added to
a stirred solution of intermediate 8 (0.25 g, 0.74 mmol) in 1,4-dioxane
(5 mL). The mixture was stirred at 90 °C for 18 h under nitrogen, and
then the solvent was evaporated in vacuo and DMF (5 mL) and
further isovaleramide (0.082 g, 0.81 mmol) were added. The mixture
was stirred at 90 °C for a further 24 h, further isovaleramide (0.082 g,
0.81 mmol) was added and the mixture was stirred at 90 °C for a
further 24 h. The mixture was diluted with Et2O and extracted with
water. The organic layer was separated, dried (Na2SO4), filtered, and
the solvents evaporated in vacuo. The crude product was purified by
flash column chromatography (silica; EtOAc in DCM 0/100 to
50/50). The desired fractions were collected and evaporated in vacuo
to yield compound 9 (95 mg, 38%) as a white solid. MS: m/z 342
1
[M + H]+. tR = 3.85 min (method 5). H NMR (400 MHz, CDCl3)
δ ppm 1.04 (d, J = 6.7 Hz, 6 H), 2.24 (spt, J = 6.8 Hz, 1 H), 2.51 (s,
3 H), 2.75 (d, J = 7.2 Hz, 2 H), 3.88 (t, J = 4.9 Hz, 4 H), 4.22 (t, J =
4.6 Hz, 4 H), 7.40 (d, J = 4.6 Hz, 1 H), 7.76 (s, 1 H), 8.30 (d, J = 4.4
Hz, 1 H). Regioselectivity confirmed by NOE between methyl group
on imidazopyrazine core and CH of the oxazole ring; mp 121 °C.
3-(2-Isobutyl-thiazol-5-yl)-2-methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine (11). Palladium(II) acetate (0.01 g, 0.05 mmol) and
tert-butyldicyclohexylphosphine (0.027 mL, 0.1 mmol) were added
to a stirred solution of intermediate 10 (0.3 g, 1.01 mmol), 2-
isobutylthiazole (0.142 g, 1.01 mmol), and potassium phosphate
(0.428 g, 2.01 mmol) in NMP (4 mL). The mixture was stirred at rt
for 15 min under nitrogen and then at 125 °C for 18 h. The mixture
was diluted with Et2O and washed with a 1% solution of potassium
hydroxide. The organic layer was separated, dried (Na2SO4), filtered,
and the solvents evaporated in vacuo. The crude product was purified
by flash column chromatography (silica; EtOAc in DCM 0/100 to
40/60). The desired fractions were collected and the solvents evapo-
rated in vacuo. Then the crude product was purified by RP HPLC
(0.1% solution of ammonium bicarbonate/ammonium hydroxide
buffer pH 9 in ACN 80/20 to 0/100). The desired fractions were
collected and evaporated in vacuo to yield compound 11 (0.10 g, 29%)
as a yellow solid. ESI-HRMS: m/z for C18H24N5OS 358.1707
[M + H]+ calcd, 358.1701; found, 358.1707 (1.7 ppm). tR = 4.68 min
1
(method 5). H NMR (400 MHz, CDCl3) δ ppm 2.50 (s, 3 H), 3.41
(s, 3 H), 3.54 (s, 3 H), 3.88 (t, J = 4.9 Hz, 4 H), 4.21 (t, J = 4.9 Hz, 4
H), 7.42 (d, J = 4.6 Hz, 1 H), 7.83 (d, J = 4.4 Hz, 1 H).
1-(2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-etha-
none (6). A 1.4 M solution of methylmagnesium bromide in THF
(4.3 mL, 5.96 mmol) was added to a stirred solution of intermediate 5
(1.4 g, 4.59 mmol) in THF (30 mL) at −78 °C, under nitrogen. The
mixture was allowed to warm to rt and then stirred for 16 h. A
saturated solution of ammonium chloride was added, and the mixture
was extracted with EtOAc. The organic layer was separated, dried
(Na2SO4), filtered, and the solvents evaporated in vacuo. The crude
product was purified by flash column chromatography (silica; EtOAc
in DCM 0/100 to 40/60). The desired fractions were collected and
evaporated in vacuo to yield intermediate 6 (1.1 g, 92%, 71% pure) as
1
(method 2). H NMR (400 MHz, CDCl3) δ ppm 1.06 (d, J = 6.5 Hz,
6 H), 2.19 (spt, J = 6.7 Hz, 1 H), 2.46 (s, 3 H), 2.95 (d, J = 7.2 Hz, 2 H),
3.88 (br t, J = 4.9 Hz, 4 H), 4.26 (br t, J = 4.9 Hz, 4 H), 7.39 (d, 1 H), 7.48
(d, J = 4.4 Hz, 1 H), 7.71 (s, 1 H). Regioselectivity confirmed by HSQC,
CH of thiazole ring was detected at 143 ppm; mp 84.1 °C.
1
a white solid. MS: m/z 261 [M + H]+. tR = 1.98 min (method 5). H
NMR (400 MHz, CDCl3) δ ppm 2.62 (s, 3 H), 2.76 (s, 3 H), 3.88 (t,
J = 4.9 Hz, 4 H), 4.20 (dd, J = 5.1, 4.6 Hz, 4 H), 7.57 (d, J = 4.6 Hz,
1 H), 8.95 (d, J = 4.6 Hz, 1 H).
2-Methyl-8-morpholin-4-yl-3-(1H-pyrrol-3-yl)-imidazo[1,2-a]-
pyrazine (12). Dichlorobis(triphenylphosphine)palladium(II) (0.008 g,
0.012 mmol) was added to a stirred solution of intermediate 10 (0.071 g,
0.24 mmol) and (triisopropylsilyl)pyrrole-3-boronic acid (0.096 g,
0.36 mmol) in a mixture of 1,4-dioxane (2 mL) and a 1 M solution of
sodium carbonate (0.72 mL, 0.72 mmol). The mixture was stirred at
100 °C for 16 h, and then the solid formed was filtered off and the
filtrate was evaporated. The crude product was purified by flash column
chromatography (silica; EtOAc in heptane 40/60). The desired fractions
were collected and evaporated in vacuo to yield compound 12 (0.056 g,
82%) as a white solid. ESI-HRMS: m/z for C15H18N5O [M + H]+ calcd,
284.1511; found, 284.1483 (−9.9 ppm). tR = 2.74 min (method 1). 1H
NMR (500 MHz, DMSO-d6) δ ppm 2.38 (s, 3 H), 3.74 (t, J = 4.6 Hz,
4 H), 4.14 (t, J = 4.9 Hz, 4 H), 6.33 (q, J = 2.3 Hz, 1 H), 6.98 (q,
2-Methyl-8-morpholin-4-yl-3-(1-trimethylsilanyloxy-vinyl)-
imidazo[1,2-a]pyrazine (7). Trimethylsilyl trifluoromethanesulfonate
(2.23 mL, 12.3 mmol) and N,N-diisopropyl-ethylamine (2.84 mL,
16.3 mmol) were added to a stirred solution of intermediate 6 (0.8 g,
3.1 mmol) in DCM (12 mL). The mixture was stirred at 0 °C for
1.5 h, allowed to warm to rt, and then stirred for a further 16 h. The
mixture was partitioned between a cold saturated solution of sodium
hydrogen carbonate and DCM. The organic layer was separated, dried
(Na2SO4), filtered, and the solvents evaporated in vacuo to yield
intermediate 7 (0.99 g, 97%) as a colorless oil which was used in next
step without further purification.
2-Bromo-1-(2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-
ethanone (8). N-Bromosuccinimide (0.224 g, 1.26 mmol) and sodium
I
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J = 2.5 Hz, 1 H), 7.12 (q, J = 1.7 Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H),
7.71 (d, J = 4.6 Hz, 1 H), 11.30 (br s, 1 H).
organic layer was separated, dried (Na2SO4), filtered, and the solvents
evaporated in vacuo. The crude product was purified by flash column
chromatography (silica; EtOAc in DCM 20/80 to 70/30). The desired
fractions were collected and evaporated in vacuo to yield 2-methyl-8-
morpholin-4-yl-3-(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine (0.23 g,
78%, 87% pure) as a white solid. MS: m/z 322 [M + H]+. tR =2.28 min
(method 5). Step 2: Synthesis of 17. Sodium methoxide (0.22 g, 4.04
mmol) was added to a stirred solution of 2-methyl-8-morpholin-4-yl-3-
(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine (0.23 g, 0.67 mmol) in
MeOH (8 mL). The mixture was stirred at 100 °C for 18 h in a sealed
tube and then poured into a saturated solution of sodium hydrogen
carbonate and extracted with DCM. The organic layer was separated,
dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The
crude product was purified by flash column chromatography (silica;
7 M solution of ammonia in MeOH and EtOAc in DCM 0/50/50 to
10/90/0). The desired fractions were collected and the solvents
evaporated in vacuo to yield compound 17 (60 mg, 25%) as a white
3-[1-(2-Methoxy-ethyl)-1H-pyrrol-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (13). 2-Bromoethyl methyl ether (0.024 mL,
0.254 mmol) and cesium carbonate (0.088 g, 0.271 mmol) were added
to a stirred solution of compound 12 (0.048 g, 0.169 mmol) in DMF
(3 mL). The mixture was stirred at 160 °C for 30 min under nitrogen
and under microwave irradiation, and then further 2-bromoethyl
methyl ether (0.072 mL, 0.762 mmol) was added. The mixture was
stirred at 160 °C for a further 30 min under microwave irradiation, and
then the solid formed was filtered off and the filtrate evaporated
in vacuo. The crude product was purified by flash column chromatog-
raphy (silica; EtOAc in heptane 40/60). The desired fractions were
collected and evaporated in vacuo to yield compound 13 (0.042 g,
73%) as an oil. ESI-HRMS: m/z for C18H24N5O2 [M + H]+ calcd,
342.1930; found, 342.1983 (15.5 ppm). tR = 3.48 min (method 1). 1H
NMR (500 MHz, DMSO-d6) δ ppm 2.38 (s, 3 H), 3.27 (s, 3 H), 3.66
(t, J = 5.3 Hz, 2 H), 3.74 (br t, J = 4.6 Hz, 4 H), 4.04−4.25 (m, 6 H),
6.30 (dd, J = 2.6, 1.7 Hz, 1 H), 6.99 (t, J = 2.3 Hz, 1 H), 7.16 (t, J = 1.7
Hz, 1 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.74 (d, J = 4.6 Hz, 1 H).
3-Iodo-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (15).
Step 1: Synthesis of 3-iodo-8-chloro-2-methyl-imidazo[1,2-a]pyrazine.
N-Iodosuccinimide (14.1 g, 62 mmol) was added to a stirred solution
of intermediate 14 (9.58 g, 57 mmol) in a mixture of DCM and acetic
acid at 0 °C. The mixture was allowed to warm to rt and then stirred
for 16 h. The mixture was diluted with further DCM and washed with
a saturated solution of sodium carbonate and sodium thiosulfite. The
organic layer was separated, dried (Na2SO4), filtered, and the solvents
evaporated in vacuo. The crude product was precipitated from
diisopropylether to yield intermediate 3-iodo-8-chloro-2-methyl-
imidazo[1,2-a]pyrazine (16 g, 97%) as a pale-brown solid which was
used in the next step without further purification. Step 2: Synthesis
of 15. A mixture 3-iodo-8-chloro-2-methyl-imidazo[1,2-a]pyrazine
(50 g, 170.36 mmol), morpholine (17.8 g, 204.43 mmol), and N,N-
diisopropylethylamine (44 g, 340.72 mmol) in ACN (500 mL) was
stirred at 80 °C for 16 h. The precipitate was filtered and washed with
petroleum ether. The resulting solid was dried to give intermediate 15
(46.8 g, 80%) as a white solid. MS: m/z 345 [M + H]+. tR = 2.43 min
(method 6). 1H NMR (500 MHz, CDCl3) δ ppm 7.46 (d, J = 4.6 Hz,
1 H), 7.44 (d, J = 4.6 Hz, 1 H), 4.20−4.25 (m, 4 H), 3.84−3.88 (m,
4 H), 2.45 (s, 3 H); mp 135.3−136.7 °C.
3-(2-Chloro-pyridin-4-yl)-2-methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine (16). Tetrakis(triphenylphosphine)palladium (0)
(0.165 g, 0.143 mmol) was added to a stirred solution of intermediate
15 (1.645 g, 4.780 mmol) and 2-chloropyridine-5-boronic acid (0.83 g,
5.26 mmol) in a mixture of 1,4-dioxane (44 mL) and a saturated
solution of sodium hydrogen carbonate (11 mL). The mixture was
stirred at 90 °C for 3 h, at 100 °C for 2.5 h and at rt for 48 h. Then
further 1,4-dioxane (10 mL) and a saturated solution of sodium
hydrogen carbonate (2.5 mL) were added. The mixture was stirred at
110 °C for a further 5 h and then diluted with EtOAc and water and
filtered over diatomaceous earth. The organic layer was separated,
dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The
crude product was purified by flash column chromatography (silica;
EtOAc in DCM 0/100 to 20/80). The desired fractions were collected
and evaporated in vacuo to yield intermediate 16 (1.3 g, 83%, 92%
pure) as a white solid. MS: m/z 330 [M + H]+. tR = 2.67 min (method 5).
1H NMR (500 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.86−3.91 (m, 4 H),
1
solid. MS: m/z 354 [M + H]+. tR = 1.92 min (method 7). H NMR
(400 MHz, CDCl3) δ ppm 2.49 (s, 3 H), 3.14 (t, J = 6.4 Hz, 2 H), 3.38
(s, 3 H), 3.83 (t, J = 6.4 Hz, 2 H), 3.86−3.93 (m, 4 H), 4.23−4.30 (m,
4 H), 7.22 (dd, J = 5.2, 1.5 Hz, 1 H), 7.31 (s, 1 H), 7.37 (d, J = 4.6 Hz,
1 H), 7.54 (d, J = 4.6 Hz, 1 H), 8.69 (d, J = 5.1 Hz, 1 H).
2-Bromo-5-(2-methoxy-vinyl)-pyridine (19). A 2.5 M solution of
n-butyllithium in hexanes (9.94 mL, 24.8 mmol) was added dropwise
to a stirred solution of (methoxymethyl)triphenylphosphonium
chloride (8.51 g, 24.8 mmol) in THF (150 mL) at 0 °C, and then
6-bromonicotinaldehyde (3.3 g, 17.7 mmol) was slowly added to the
red mixture. The mixture was stirred at rt for 16 h and then diluted
with Et2O and washed with water. The aqueous layer was extracted
with DCM, and the organic layer was dried (Na2SO4), filtered, and the
solvents evaporated in vacuo. The crude product was purified by flash
column chromatography (silica; DCM in heptane 0/100 to 50/50).
The desired fractions were collected and the solvents evaporated
in vacuo to yield intermediate 19 (2.8 g, 73%, 81% pure) as a mixture
57/43 of E and Z isomers. MS: m/z 214 [M + H]+. tR = 2.13/2.23 min
(sterochemistry not assigned) (method 5).
2-Bromo-5-(2-methoxy-ethyl)-pyridine (20). A solution of inter-
mediate 19 (2.3 g, 10.7 mmol) in EtOH (100 mL) was hydrogenated
in a H-cube reactor (1.5 mL/min, Rh/C 5% cartridge, full H2 mode,
70 °C, 3 cycles). The solvent was evaporated in vacuo and the crude
product purified by flash column chromatography (silica; EtOAc in
heptane and DCM 0/50/50 to 0/0/100 to 20/0/80). The desired
fractions were collected and evaporated in vacuo to yield intermediate
20 (0.48 g, 21%) as a colorless oil. MS: m/z 216 [M + H]+. tR = 1.83 min
(method 5).
5-(2-Methoxy-ethyl)-2-tributylstannanyl-pyridine (21). A 2.5 M
solution of n-butyllithium in hexanes (1.1 mL, 2.72 mmol) was added
dropwise to a solution of intermediate 20 (0.245 g, 1.13 mmol) in
THF (10 mL). The mixture was stirred at −78 °C for 1 h, and then
tributyltin chloride (0.74 mL, 2.72 mmol) was slowly added. The
mixture was allowed to warm to rt over 1 h, and then a saturated
solution of ammonium chloride was added. The mixture was ex-
tracted with Et2O and EtOAc. The combined organic layers were dried
(Na2SO4), filtered, and the solvents evaporated in vacuo to yield
intermediate 21 (0.72 g, quantitative, 67% pure), which was used in
the next step without any further purification. ESI-HRMS (120Sn):
m/z for C20H38NOSn [M + H]+ calcd, 428.1975; found, 428.1945
(7.0 ppm). tR = 3.91 min (method 2).
4.25−4.31 (m, 4 H), 7.33 (d, J = 4.3 Hz, 1 H), 7.37 (d, J = 4.6 Hz, 1 H),
7.52 (d, J = 8.1 Hz, 1 H), 7.74 (dd, J = 8.1, 2.3 Hz, 1 H), 8.49 (d, J =
1.7 Hz, 1 H).
3-[5-(2-Methoxy-ethyl)-pyridin-2-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (22). Intermediate 10 (0.392 g, 1.32 mmol),
tetrakis(triphenylphosphine)palladium (0) (0.038 g, 0.032 mmol), and
copper(I) bromide (0.010 g, 0.066 mmol) were added to a stirred
solution of 5-(2-methoxy-ethyl)-2-tributylstannanyl-pyridine (21)
(0.468 g, 1.1 mmol) in 1,4-dioxane (20 mL). The mixture was stirred
at 160 °C for 20 min in a sealed tube under nitrogen and under micro-
wave irradiation, and then the solvent was evaporated in vacuo. The
crude product was purified by flash column chromatography (silica;
EtOAc in DCM 40/60 to 90/10). The desired fractions were col-
lected, and the solvents evaporated in vacuo. The crude product was
purified again by flash column chromatography (silica; 7 M solution of
3-[2-(2-Methoxy-ethyl)-pyridin-4-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (17). Step 1: Synthesis of 2-methyl-8-
morpholin-4-yl-3-(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine.
Tetrakis(triphenylphosphine)palladium (0) (0.031 g, 0.027 mmol)
was added to a stirred solution of intermediate 16 (0.30 g, 0.909
mmol) and vinylboronic acid pinacolester (0.17 g, 1.09 mmol) in a
mixture of 1,4-dioxane (6 mL) and a saturated solution of sodium
carbonate (3 mL). The mixture was stirred at 80 °C for 3 h under
nitrogen and then diluted with DCM and extracted with water. The
J
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ammonia in MeOH in DCM 0/100 to 1/99). The desired fractions
were collected and the solvents evaporated in vacuo to yield com-
pound 22 (0.044 g, 11%) as a white solid. MS: m/z 354 [M + H]+.
chromatography (silica; 7 M solution of ammonia in MeOH in
DCM 2/98) and precipitation from heptane, yielded compound
25b as a white solid (47%). MS: m/z 368 [M + H]+. tR = 2.27 min
(method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.22 (t, J = 7.1 Hz,
3 H), 2.44 (s, 3 H), 3.16 (t, J = 6.7 Hz, 2 H), 3.56 (q, J = 6.9 Hz, 2 H),
3.84−3.92 (m, 6 H), 4.24−4.30 (m, 4 H), 7.32−7.39 (m, 2 H), 7.41
(d, J = 8.1 Hz, 1 H), 7.68 (dd, J = 7.9, 2.3 Hz, 1 H), 8.61 (d, J = 2.1
Hz, 1 H); mp 87.6 °C.
1
tR = 2.29 min (method 7). H NMR (500 MHz, CDCl3) δ ppm 2.60
(s, 3 H), 2.95 (t, J = 6.5 Hz, 2 H), 3.39 (s, 3 H), 3.68 (t, J = 6.5 Hz, 2
H), 3.87−3.92 (m, 4 H), 4.19−4.25 (m, 4 H), 7.40 (d, J = 4.6 Hz, 1
H), 7.47 (d, J = 7.8 Hz, 1 H), 7.71 (dd, J = 8.1, 2.0 Hz, 1 H), 8.45 (d,
J = 4.6 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H).
3-(6-Chloro-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine (23). Tetrakis(triphenylphosphine)palladium (0)
(1.5 g, 1.3 mmol) was added to a stirred solution of intermediate
15 (11.5 g, 33.42 mmol) and 2-chloropyridine-5-boronic acid (6.1 g,
3.87 mmol) in a mixture of 1,4-dioxane (200 mL) and a saturated
solution of sodium hydrogen carbonate (50 mL). The mixture was
stirred at 100 °C for 18 h under nitrogen, and then further
tetrakis(triphenylphosphine)palladium (0) (0.35 g, 0.3 mmol) and
2-chloropyridine-5-boronic acid (0.6 g, 2.8 mmol) were added. The
mixture was stirred at 100 °C for further 5 h and then concentrated
in vacuo and partitioned between DCM, water, and a saturated solution
of sodium carbonate. The organic layer was separated, dried (Na2SO4),
filtered, and the solvents evaporated in vacuo. The crude product was
precipitated from MeOH to yield intermediate 23 (10.3 g, 93%) as a
3-[6-(2-Isopropoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25c). 3-[6-(2-Isopropoxy-ethyl)-pyridin-3-yl]-
2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared ac-
cording to a protocol analogous to compound 17 (step 2) from
intermediate 24 and sodium isopropoxide. Flash column chromatog-
raphy (silica; EtOAc in DCM 0/100 to 100/0) and precipitation from
heptane yielded compound 25c as a white solid (25%). MS: m/z 382
[M + H]+. tR = 2.62 min (method 7). 1H NMR (400 MHz, CDCl3) δ
ppm 1.17 (d, J = 6.2 Hz, 6 H), 2.43 (s, 3 H), 3.14 (t, J = 6.7 Hz, 2 H),
3.63 (spt, J = 6.1 Hz, 1 H), 3.83−3.93 (m, 6 H), 4.24−4.31 (m, 4 H),
7.32−7.39 (m, 2 H), 7.42 (d, J = 7.9 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2
Hz, 1 H), 8.61 (d, J = 2.3 Hz, 1 H).
2-[5-(2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-pyr-
idin-2-yl]-ethanol (25d). 2-[5-(2-Methyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazin-3-yl)-pyridin-2-yl]-ethanol was prepared according to
a protocol analogous to compound 25a from intermediate 24,
potassium hydrogensulfate, and water. Flash column chromatography
(silica; 7 M solution of ammonia in MeOH in DCM 3/97) and flash
column chromatography (silica; MeOH in EtOAc 0/100 to 2/98)
yielded compound 25d as a white solid (18%). MS: m/z 340 [M + H]+.
tR = 1.36 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.44 (s,
3 H), 3.13 (t, J = 5.4 Hz, 2 H), 3.83−3.98 (m, 5 H), 4.11 (t, J = 5.4 Hz,
2 H), 4.25−4.30 (m, 4 H), 7.33−7.38 (m, 3 H), 7.70 (dd, J = 7.9, 2.3
Hz, 1 H), 8.59 (d, J = 2.1 Hz, 1 H).
1
white solid. MS: m/z 330 [M + H]+. tR = 1.78 min (method 7). H
NMR (500 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.86−3.91 (m, 4 H),
4.25−4.31 (m, 4 H), 7.33 (d, J = 4.3 Hz, 1 H), 7.37 (d, J = 4.6 Hz, 1
H), 7.52 (d, J = 8.1 Hz, 1 H), 7.74 (dd, J = 8.1, 2.3 Hz, 1 H), 8.49 (d,
J = 1.7 Hz, 1 H).
2-Methyl-8-morpholin-4-yl-3-(6-vinyl-pyridin-3-yl)-imidazo[1,2-
a]pyrazine (24). Tetrakis(triphenylphosphine)palladium (0) (0.623 g,
0.54 mmol) was added to a stirred solution of intermediate 23 (8.9 g,
26.99 mmol) and vinylboronic acid pinacolester (5.91 mL, 35.08
mmol) in a mixture of 1,4-dioxane (60 mL) and a saturated solution of
sodium carbonate (30 mL). The mixture was stirred at 100 °C for 1 h
under nitrogen and then diluted with DCM and extracted with water.
The organic layer was separated, dried (Na2SO4), filtered, and the
solvents evaporated in vacuo. The crude product was purified by flash
column chromatography (silica; 7 M solution of ammonia in MeOH in
DCM 0/100 to 2/98). The desired fractions were collected and the
solvents evaporated in vacuo to yield intermediate 24 (7.8 g, 90%, 91%
pure) as a white solid. MS: m/z 322 [M + H]+. tR = 1.72 min (method
7). 1H NMR (400 MHz, CDCl3) δ ppm 2.45 (s, 3 H), 3.86−3.92 (m,
4 H), 4.25−4.30 (m, 4 H), 5.59 (dd, J = 10.8, 1.0 Hz, 1 H), 6.32 (dd,
J = 17.5, 1.0 Hz, 1 H), 6.90 (dd, J = 17.5, 10.8 Hz, 1 H), 7.35 (d, J =
4.6 Hz, 1 H), 7.40 (d, J = 4.6 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.73
(dd, J = 8.1, 2.3 Hz, 1 H), 8.66 (d, J = 1.6 Hz, 1 H).
3-(6-Ethyl-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2-
a]pyrazine (25e). Palladium 10% on charcoal (0.042 g) was added to a
suspension of intermediate 24 (0.25 g, 0.78 mmol) in a mixture of
EtOH (3 mL), EtOAc (2 mL), and DCM (1 mL). The mixture was
hydrogenated (atmospheric pressure) at rt for 16 h and then filtered
through a pad of diatomaceous earth. The filtrate was evaporated
in vacuo and the crude product purified by flash column chromatography
(silica; 7 M solution of ammonia in MeOH in DCM 2/98). The
desired fractions were collected, and the solvents evaporated in vacuo
and triturated with Et2O to yield compound 25e (0.23 g, 91%) as a
1
white solid. MS: m/z 324 [M + H]+. tR = 2.32 min (method 7). H
NMR (500 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.7 Hz, 3 H), 2.36 (s,
3 H), 2.85 (q, J = 7.5 Hz, 2 H), 3.73−3.78 (m, 4 H), 4.14−4.20 (m,
4 H), 7.35 (d, J = 4.6 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H), 7.60 (d, J =
4.6 Hz, 1 H), 7.89 (dd, J = 7.9, 2.5 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H);
mp 80.3 °C.
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25a). Potassium hydrogensulfate (12 g,
88.13 mmol) was added to a stirred solution of intermediate 24
(6 g, 18.67 mmol) in MeOH (120 mL). The mixture was stirred at
80 °C for 3 days and then poured onto a saturated solution of sodium
carbonate and extracted with DCM. The organic layer was separated,
dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The
crude product was purified by open column chromatography (silica; 7
M solution of ammonia in MeOH in DCM 0/100 to 1.5/98.5). The
impure fractions were collected and evaporated in vacuo and the crude
product purified by flash column chromatography (silica; 7 M solution
of ammonia in MeOH in DCM 0/100 to 2/98). The combined
desired fractions were collected and the solvents evaporated in vacuo
and triturated with heptane to yield compound 25a (4.13 g, 63%) as a
3-[6-(2-Methoxy-2-methyl-propyl)-pyridin-3-yl]-2-methyl-8-mor-
pholin-4-yl-imidazo[1,2-a]pyrazine (25f). 3-[6-(2-Methoxy-2-methyl-
propyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]-
pyrazine was prepared according to a protocol analogous to inter-
mediate 16 from intermediate 10 and 2-(2-methoxy-2-methyl-propyl)-
pyridine-5-boronic acid pinacol ester at 140 °C for 15 min and under
microwave irradiation. Flash column chromatography (silica; EtOAc in
DCM 50/50 to 80/20), flash column chromatography (silica; 7 M
solution of ammonia in MeOH in DCM 0/100 to 2/98), and
precipitation from heptane yielded compound 25f as a white solid
(71%). MS: m/z 382 [M + H]+. tR = 2.40 min (method 7). 1H NMR
(400 MHz, CDCl3) δ ppm 1.26 (s, 6 H), 2.44 (s, 3 H), 3.07 (s, 2 H),
3.34 (s, 3 H), 3.86−3.93 (m, 4 H), 4.23−4.31 (m, 4 H), 7.32−7.41
(m, 2 H), 7.42 (d, J = 8.1 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2 Hz, 1 H),
8.61 (d, J = 2.1 Hz, 1 H) (regioselectivity confirmed by NOE between
gem-dimethyl group and CH2 attached to 6-position of pyridine ring);
mp 133.2 °C.
1
white solid. MS: m/z 354 [M + H]+. tR = 1.93 min (method 7). H
NMR (500 MHz, CDCl3) δ ppm 2.44 (s, 3 H), 3.16 (t, 2 H), 3.40 (s,
3 H), 3.86 (t, 2 H), 3.89 (br t, J = 4.9 Hz, 4 H), 4.27 (br t, J = 4.9 Hz,
4 H), 7.34 (d, 1 H), 7.39 (d, J = 4.6 Hz, 1 H), 7.40 (d, J = 8.1 Hz, 1 H),
7.68 (dd, J = 8.1, 2.3 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H); mp >300 °C.
3-[6-(2-Ethoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25b). 3-[6-(2-Ethoxy-ethyl)-pyridin-3-yl]-2-
methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared accord-
ing to a protocol analogous to compound 17 (step 2) from inter-
mediate 24 and sodium ethoxide. Flash column chromatography
(silica; EtOAc in DCM 50/50 to 100/0) then flash column
3-[6-(3-Methoxy-propyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25g). 3-[6-(3-Methoxy-propyl)-pyridin-3-yl]-
2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared ac-
cording to a protocol analogous to intermediate 16 from intermediate
10 and 2-(1-methoxy-propyl)pyridine-5-boronic acid pinacol ester at
K
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Journal of Medicinal Chemistry
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150 °C for 20 min and under microwave irradiation. Flash column
chromatography (silica; EtOAc in heptane 50/50 to 80/20) yielded
compound 25g as a white solid (11%). MS: m/z 368 [M + H]+. tR =
2.20 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.03−2.14
(m, 2 H), 2.44 (s, 3 H), 2.93−3.00 (m, 2 H), 3.38 (s, 3 H), 3.49 (t, J =
6.4 Hz, 2 H), 3.86−3.92 (m, 4 H), 4.25−4.30 (m, 4 H), 7.34 (d, J =
4.6 Hz, 1 H), 7.34 (d, J = 7.9 Hz, 1 H), 7.38 (d, J = 4.4 Hz, 1 H), 7.67
(dd, J = 7.9, 2.3 Hz, 1 H), 8.61 (d, J = 2.1 Hz, 1 H).
was quenched by addition of a saturated solution of ammonium
chloride. Then 1 M aqueous solution of hydrochloride acid and DCM
were added and the product extracted into the aqueous layer. The
organic layer was washed once more with diluted hydrochloride acid.
The combined aqueous layers were basified with saturated solution of
sodium carbonate and the product extracted with DCM. The organic
layer was separated, dried (Na2SO4), filtered, and the solvents
evaporated in vacuo. The crude product was purified by flash column
chromatography (silica; EtOAc in DCM 0/100 to 40/60). The desired
fractions were collected and the solvents evaporated in vacuo to yield
compound 25l (0.10 g, 56%) as a solid. MS: m/z 338 [M + H]+. tR =
3-(6-Ethoxymethyl-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25h). 3-(6-Ethoxymethyl-pyridin-3-yl)-2-
methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared accord-
ing to a protocol analogous to intermediate 16 from intermediate 10
and 2-ethoxymethylpyridine-5-boronic acid pinacol ester at 150 °C for
15 min and under microwave irradiation. Flash column chromatog-
raphy (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to
2/98) then RP HPLC (0.1% solution of ammonium bicarbonate/
ammonium hydroxide buffer pH 9 in EtOAc 80/20 to 0/100) yielded
compound 25h as a white solid (48%). MS: m/z 354 [M + H]+. tR =
2.23 min (method 7). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.23 (t,
J = 6.9 Hz, 3 H), 2.38 (s, 3 H), 3.63 (q, J = 7.1 Hz, 2 H), 3.76 (br t, J =
4.9 Hz, 4 H), 4.18 (br t, J = 4.9 Hz, 4 H), 4.64 (s, 2 H), 7.37 (d, J = 4.6
Hz, 1 H), 7.63 (d, J = 8.1 Hz, 1 H), 7.64 (d, J = 4.6 Hz, 1 H), 8.00 (dd,
J = 8.1, 2.3 Hz, 1 H), 8.68 (d, J = 1.7 Hz, 1 H); mp >300 °C (dec).
4-[2-Methyl-3-(6-methyl-3-pyridyl)imidazo[1,2-a]pyrazin-8-yl]-
morpholine (25i). 4-[2-Methyl-3-(6-methyl-3-pyridyl)imidazo[1,2-a]-
pyrazin-8-yl]morpholine was prepared according to a protocol
analogous to intermediate 16 from intermediate 15 and 2-picoline-5-
boronic acid pinacol ester at 90 °C for 18 h. Flash column chromato-
graphy (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to
2/98) yielded compound 25i as a white solid (48%, 92% pure). MS:
1
3.37 min (method 5). H NMR (500 MHz, CDCl3) δ ppm 1.38 (d,
J = 6.9 Hz, 6 H), 2.44 (s, 3 H), 3.16 (spt, J = 6.9 Hz, 1 H), 3.86−3.92
(m, 4 H), 4.24−4.31 (m, 4 H), 7.32−7.36 (m, 2 H), 7.39 (d, J = 4.3
Hz, 1 H), 7.68 (dd, J = 7.9, 2.2 Hz, 1 H), 8.62 (d, J = 1.7 Hz, 1 H).
4-[3-(6-Isobutyl-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]-
morpholine (25m). 4-[3-(6-Isobutyl-3-pyridyl)-2-methyl-imidazo[1,2-a]-
pyrazin-8-yl]morpholine was prepared according to a protocol analogous
to compound 25l from intermediate 23 and isobutylmagnesium bromide.
Flash column chromatography (silica; EtOAc in heptane 60/40) and
triturated with Et2O yielded compound 25m (56%). MS: m/z 352
[M + H]+. tR = 3.11 min (method 7). 1H NMR (400 MHz, DMSO-d6)
δ ppm 0.94 (d, J = 6.7 Hz, 6 H), 2.13 (dquin, J = 13.6, 6.8 Hz, 1 H),
2.36 (s, 3 H), 2.70 (d, J = 7.2 Hz, 2 H), 3.72−3.78 (m, 4 H), 4.14−
4.20 (m, 4 H), 7.35 (d, J = 4.4 Hz, 1 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.59
(d, J = 4.6 Hz, 1 H), 7.88 (dd, J = 8.1, 2.3 Hz, 1 H), 8.63 (d, J = 1.8
Hz, 1 H); mp >300 °C (dec).
3-(6-Cyclopropyl-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25n). Palladium(II) acetate (0.036 g, 0.16
mmol) and 2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl
(0.131 g, 0.32 mmol) were added to a stirred mixture of intermediate
23 (0.35 g, 1.06 mmol), cyclopropylboronic acid (0.137 g, 1.59 mmol),
and potassium phosphate (0.451 g, 2.12 mmol) in toluene (5 mL). The
mixture was stirred at 80 °C for 22 h under nitrogen and then diluted
with DCM and extracted with water. The organic layer was separated,
dried (MgSO4), filtered, and the solvents evaporated in vacuo. The
crude product was purified by flash column chromatography (silica; 7 M
solution of ammonia in MeOH in DCM 100/0 to 3/97). The desired
fractions were collected and the solvents evaporated in vacuo and the
crude product purified again by flash column chromatography (silica;
EtOAc in heptane 50/50 to 100/0). The desired fractions were
collected and the solvents evaporated in vacuo and triturated with
diisopropylether to yield compound 25n (0.103 g, 29%) as a pale-
1
m/z 310 [M + H]+. tR = 2.12 min (method 5). H NMR (500 MHz,
CDCl3) δ ppm 2.43 (s, 3 H), 2.66 (s, 3 H), 3.87−3.92 (m, 4 H),
4.24−4.30 (m, 4 H), 7.32−7.37 (m, 3 H), 7.65 (dd, J = 7.8, 2.3 Hz, 1
H), 8.58 (d, J = 2.0 Hz, 1 H); mp 87.3 °C.
3-[6-(2-Methoxy-ethoxy)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (25j). 3-[6-(2-Methoxy-ethoxy)-pyridin-3-yl]-
2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared ac-
cording to a protocol analogous to intermediate 16 from intermediate
10 and 2-picoline-5-boronic acid pinacol ester at 140 °C for 20 min
and under microwave irradiation. Flash column chromatography
(silica; MeOH in DCM 5/95) yielded compound 25j as a white solid
(43%). MS: m/z 370 [M + H]+. tR = 2.34 min (method 7). 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 3.32 (s, 3 H), 3.67−3.73
(m, 2 H), 3.75 (br t, J = 4.9 Hz, 4 H), 4.17 (br t, J = 4.9 Hz, 4 H),
4.40−4.53 (m, 2 H), 7.04 (d, J = 8.6 Hz, 1 H), 7.34 (d, J = 4.6 Hz, 1
H), 7.55 (d, J = 4.6 Hz, 1 H), 7.88 (dd, J = 8.7, 2.4 Hz, 1 H), 8.30 (d,
J = 2.3 Hz, 1 H).
1
brown solid. MS: m/z 336 [M + H]+. tR = 2.65 min (method 7). H
NMR (400 MHz, CDCl3) δ ppm 1.00−1.19 (m, 4 H), 2.05−2.19 (m,
1 H), 2.42 (s, 3 H), 3.89 (br t, J = 4.9 Hz, 4 H), 4.27 (br t, J = 4.6 Hz,
4 H), 7.31 (d, J = 8.3 Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H), 7.35 (d, J =
4.4 Hz, 1 H), 7.59 (dd, J = 8.1, 2.1 Hz, 1 H), 8.51 (d, J = 2.3 Hz, 1 H);
mp > 300 °C (dec).
4-[3-(6-Isopropoxy-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]-
morpholine (25k). 4-[3-(6-Isopropoxy-3-pyridyl)-2-methyl-imidazo-
[1,2-a]pyrazin-8-yl]morpholine was prepared according to a protocol
analogous to intermediate 16 from intermediate 10 and 2-
isopropoxypyridine-5-boronic acid pinacol ester at 150 °C for
15 min and under microwave irradiation. Flash column chromatog-
raphy (silica; 7 M solution of ammonia in MeOH in DCM 2/98) and
ion exchange chromatography using an ISOLUTE SCX2 cartridge
(eluting with MeOH then 7 M solution of ammonia in MeOH)
yielded compound 25k as a white solid (94%). ESI-HRMS: m/z for
C19H24N5O2 [M + H]+ calcd, 354.1930; found, 354.1922 (−2.3 ppm).
4-[2-Methyl-3-(6-pyrrolidin-1-yl-3-pyridyl)imidazo[1,2-a]pyrazin-
8-yl]morpholine (25o). A mixture of intermediate 23 (250 mg, 0.758
mmol) in pyrrolidine (0.314 g, 3.64 mmol) was stirred at 130 °C for
30 min under microwave irradiation. The solvent was evaporated in
vacuo. The crude product was purified by flash column chromatog-
raphy (silica; MeOH in DCM 0/100 to 5/95). The desired fractions
were collected and the solvents evaporated in vacuo. The product
triturated with diisopropylether to yield compound 25o (0.167 g,
60%) as a solid. MS: m/z 365 [M + H]+. tR = 2.88 min (method 7). 1H
NMR (400 MHz, CDCl3) δ ppm 2.00−2.12 (m, 4 H), 2.40 (s, 3 H),
3.50−3.57 (m, 4 H), 3.85−3.93 (m, 4 H), 4.22−4.29 (m, 4 H), 6.50 (d,
J = 8.8 Hz, 1 H), 7.30 (d, J = 4.4 Hz, 1 H), 7.35 (d, J = 4.6 Hz, 1 H), 7.46
(dd, J = 8.7, 2.4 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H); mp 132.2 °C.
(S)-3-[6-(3-Methoxy-pyrrolidin-1-yl)-pyridin-3-yl]-2-methyl-8-
morpholin-4-yl-imidazo[1,2-a]pyrazine (25p). A mixture of inter-
mediate 23 (0.15 g, 0.45 mmol) and (S)-3-hydroxypyrrolidine (0.159 g,
1.82 mmol) was stirred at 120 °C for 3 h, and then the mixture was
diluted with water and extracted with EtOAc. The organic layer was
separated, dried (Na2SO4), filtered, and the solvents evaporated
in vacuo. The crude product was dissolved in THF (3 mL), and a 60%
dispersion of sodium hydride in mineral oils (0.020 g, 0.5 mmol) was
1
tR = 3.85 min (method 2). H NMR (400 MHz, DMSO-d6) δ ppm
1.35 (d, J = 6.2 Hz, 6 H), 2.34 (s, 3 H), 3.71−3.79 (m, 4 H), 4.13−
4.21 (m, 4 H), 5.33 (spt, J = 6.2 Hz, 1 H), 6.94 (dd, J = 8.6, 0.7 Hz,
1 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.55 (d, J = 4.6 Hz, 1 H), 7.84 (dd, J =
8.6, 2.5 Hz, 1 H), 8.30 (dd, J = 2.5, 0.7 Hz, 1 H); mp 57.8 °C.
4-[3-(6-Isopropyl-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-
yl]morpholine (25l). [1,3-Bis(diphenylphosphino)propane]-
dichloronickel(II) (0.014 g, 0.027 mmol) was added to a solution of
intermediate 23 (0.18 g, 0.545 mmol) in THF (4 mL), and the
resulting mixture was cooled to 0 °C. A 2 M solution of
isopropylmagnesium chloride in Et2O (0.54 mL, 1.09 mmol) was
slowly added, and the stirring was continued for 30 min. The reaction
L
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Article
added. The mixture was stirred at rt for 5 min, and then iodomethane
(0.07 g, 0.49 mmol) was added. The mixture was stirred at rt for a
further 3 days and then extracted with a saturated solution of
ammonium chloride. The organic layer was separated, dried (Na2SO4),
filtered, and the solvents evaporated in vacuo. The crude product was
purified by flash column chromatography (silica; 7 M solution of
ammonia in MeOH in DCM 2/98 to 10/90). The desired fractions
were collected and the solvents evaporated in vacuo to yield com-
pound 25p (0.033 g, 19%) as a white solid. MS: m/z 395 [M + H]+.
tR = 2.44 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.08−
2.29 (m, 2 H), 2.40 (s, 3 H), 3.40 (s, 3 H), 3.56−3.74 (m, 4 H), 3.85−
3.92 (m, 4 H), 4.14 (tt, J = 4.8, 2.6 Hz, 1 H), 4.22−4.29 (m, 4 H), 6.51
(d, J = 8.8 Hz, 1 H), 7.30 (d, J = 4.6 Hz, 1 H), 7.33 (d, J = 4.6 Hz, 1
H), 7.47 (dd, J = 8.8, 2.3 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H).
N-Isopropyl-5-(2-methyl-8-morpholino-imidazo[1,2-a]pyrazin-3-yl)-
pyridin-2-amine (25q). Palladium(II) acetate (0.010 g, 0.045 mmol)
and racemic-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.042 g,
0.068 mmol) were added to a stirred solution of intermediate 23
(0.30 g, 0.91 mmol), isopropylamine (0.78 mL, 9.09 mmol), and
cesium carbonate (0.74 g, 2.27 mmol) in toluene (4 mL). The mixture
was stirred at 60 °C for 4 days and then diluted with DCM, washed
with water, and the organic layer was separated, dried (Na2SO4),
filtered, and the solvents evaporated in vacuo. The crude product was
purified by flash column chromatography (silica; EtOAc in heptane
40/60 to 100/0). The desired fractions were collected, the solvents
evaporated in vacuo and then triturated from diisopropylether to yield
a compound that was washed with a saturated solution of sodium car-
bonate and then diluted with DCM, the organic layer was separated,
dried (Na2SO4), and filtered and the solvents evaporated in vacuo. The
resulting residue was purified by flash column chromatography (silica;
EtOAc in heptane 30/70 to 70/30). The desired fractions were
collected, the solvents evaporated in vacuo to yield compound 25q
(0.19 g, 59%) as a white solid. MS: m/z 353 [M + H]+. tR = 2.57 min
(method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.30 (d, J = 6.2 Hz,
6 H), 2.40 (s, 3 H), 3.84−3.92 (m, 4 H), 3.92−4.02 (m, 1 H), 4.21−
4.30 (m, 4 H), 4.61 (br d, J = 7.9 Hz, 1 H), 6.50 (d, J = 8.6 Hz, 1 H),
7.30 (d, J = 4.6 Hz, 1 H), 7.35 (d, J = 4.4 Hz, 1 H), 7.43 (dd, J = 8.8,
2.3 Hz, 1 H), 8.12 (d, J = 2.3 Hz, 1 H); mp 105.2 °C.
2-Methyl-8-morpholin-4-yl-3-(6-piperazin-1-yl-pyridin-3-yl)-
imidazo[1,2-a]pyrazine (25r). A mixture of intermediate 23 (0.3 g,
0.91 mmol) and piperazine (0.314 g, 3.64 mmol) was stirred at 120 °C
for 24 h. The mixture was diluted with EtOAc and extracted with
water and a 1N solution of sodium hydroxide. The organic layer was
separated, dried (Na2SO4), filtered, and the solvents evaporated
in vacuo. The crude product was purified by flash column chromatog-
raphy (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to
10/90). The desired fractions were collected and evaporated in vacuo
and the crude product purified again by flash column chromatography
(silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 3/97) and
by RP-HPLC (0.1% solution of ammonium bicarbonate/ammonium
hydroxide buffer pH 9 in ACN 80/20 to 0/100). The desired fractions
were collected and the solvents evaporated and the crude product
triturated with diisopropylether to yield compound 25r (0.074 g, 22%)
as a white solid. MS: m/z 380 [M + H]+. tR = 1.19 min (method 7).
1H NMR (400 MHz, DMSO-d6) δ ppm 2.32 (s, 3 H), 2.81 (br t,
The organic layer was separated, dried (Na2SO4), filtered, and the
solvents evaporated in vacuo. The crude product was purified by flash
column chromatography (silica; MeOH in DCM 0/100 to 2/98). The
desired fractions were collected and the solvents evaporated in vacuo
to yield intermediate 26h (0.86 g, 78%) as a white solid. MS: m/z 311
[M + H]+. tR = 2.62 min (method 5). 1H NMR (400 MHz, CDCl3) δ
ppm 2.32 (d, J = 0.9 Hz, 3 H), 2.39 (s, 3 H), 3.83−3.88 (m, 4 H),
4.19−4.26 (m, 4 H), 7.26−7.28 (m, 1 H).
3-Bromo-2-methyl-6-trifluoromethyl-8-morpholin-4-yl-imidazo-
[1,2-a]pyrazine (26i). Copper(I) iodide (0.18 g, 0.95 mmol) and
MDFA (0.12 mL, 0.95 mmol) were added to a stirred solution of
intermediate 31 (0.20 g, 0.47 mmol) in DMF (2 mL). The mixture
was stirred at 90 °C for 16 h in a sealed tube under nitrogen and then
diluted with Et2O and washed with a saturated solution of ammonium
hydroxide. The organic layer was separated, dried (Na2SO4), filtered,
and the solvents evaporated in vacuo. The crude product was purified
by flash column chromatography (silica; EtOAc in heptane 0/100 to
50/50). The desired fractions were collected and the solvents
evaporated in vacuo to yield intermediate 26i (0.15 g, 60%, 69%
pure) as a pale-brown solid. MS: m/z 365 [M + H]+. tR = 2.94 min
1
(method 6). H NMR (400 MHz, DMSO-d6) δ ppm 2.36 (s, 3 H),
3.72−3.77 (m, 4 H), 4.17−4.27 (m, 4 H), 8.01 (s, 1 H).
2-(2-Methoxyethyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-
2-yl)-pyridine (27). Step 1: Synthesis of 2-(5-bromo-pyridin-2-yl)-
ethanol. A 2.5 M solution of n-butyllithium in pentane (6.97 mL, 17.44
mmol) was added dropwise to a solution of N,N-diisopropylamine
(3.29 mL, 23.25 mmol) in THF (50 mL). The mixture was stirred at
0 °C for 30 min, cooled down to −78 °C, and then a solution of
5-bromo-2-methylpyridine (2.0 g, 11.63 mmol) in THF (50 mL) was
added. The mixture was stirred at −78 °C for a further 2 h, and then
DMF (8.5 g, 116.26 mmol) was added dropwise. The mixture was
stirred at −78 °C for 2 h, at 0 °C for 30 min, and finally allowed to
warm to rt. MeOH (25 mL) and sodium borohydride (0.439 g, 11.6
mmol) were added, and the mixture was stirred at rt for further
30 min. A saturated solution of ammonium chloride was added, and
the organic layer was separated. The aqueous layer was extracted with
EtOAc, and the combined organic extracts were dried (Na2SO4) and
filtered, and the solvents evaporated in vacuo to yield 2-(5-bromo-
pyridin-2-yl)-ethanol (2.8 g, 87%, 73% pure) as a colorless oil. MS:
m/z 202 [M + H]+. tR = 1.07 min (method 5). Step 2: Synthesis of
5-bromo-2-(2-methoxy-ethyl)-pyridine. A 60% dispersion of sodium
hydride in mineral oils, (0.43 g, 11.1 mmol) was added portionwise to
a stirred solution of 2-(5-bromo-pyridin-2-yl)-ethanol (2.8 g, 10.1
mmol) in THF (50 mL). The mixture was stirred at 0 °C for 30 min
and at rt for 16 h. A saturated solution of ammonium chloride was
added, and the organic layer was separated. The aqueous layer was
extracted with DCM, and the combined organic extracts were dried
(Na2SO4), filtered, and the solvents evaporated in vacuo to yield
5-bromo-2-(2-methoxy-ethyl)-pyridine (0.9 g, 41%) as a colorless oil.
MS: m/z 216 [M + H]+. tR = 2.25 min (method 5). Step 3: Synthesis
of 27. [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
(0.061 g, 0.083 mmol) was added to a stirred suspension of 5-bromo-
2-(2-methoxy-ethyl)-pyridine (0.6 g, 2.77 mmol), 4,4,4′,4′,5,5,5′,5′-
octamethyl-2,2′-bi-1,3,2-dioxaborolane (0.846 g, 3.33 mmol), and
potassium acetate (0.817 g, 8.33 mmol) in a mixture of 1,4-dioxane
(9 mL) and DMF (1.2 mL). The mixture was stirred at 150 °C for 40
min in a sealed tube under nitrogen and under microwave irradiation.
The mixture was filtered through a pad of diatomaceous earth and the
filtrate diluted with DCM and washed with water. The organic layer
was separated, dried (Na2SO4), filtered, and the solvents evaporated
in vacuo to yield intermediate 27 (1.1 g, 64%, 43% pure) used in the
next step without further purification. ESI-HRMS (11B): m/z for
C14H23BNO3 [M + H]+ calcd, 264.1771; found, 264.1819 (18.2 ppm).
tR = 2.37 min (method 2).
J = 5.1 Hz, 4 H), 3.32 (br s, 1 H), 3.51 (dd, J = 5.3, 4.9 Hz, 4 H), 3.74
(br t, J = 4.9 Hz, 4 H), 4.16 (dd, J = 4.9, 4.4 Hz, 4 H), 6.96 (d, J = 8.8
Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 4.6 Hz, 1 H), 7.64 (dd,
J = 8.8, 2.3 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H).
3-Bromo-2,6-dimethyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine
(26h). A 1.6 M solution of methyllithium in THF (2.66 mL,
4.25 mmol) was added dropwise to a solution of indium(III) chloride
(0.35 g, 1.59 mmol) in THF (35 mL) at −78 °C. The mixture was
stirred at −78 °C for 30 min and then allowed to warm to rt. The
trimethylindium pale-white solution obtained was transferred via
cannula to a stirred solution of intermediate 31 (1.5 g, 3.55 mmol) and
tetrakistriphenylphosphine palladium (0) (0.21 g, 0.18 mmol) in THF
(20 mL). The mixture was stirred at 80 °C for 16 h, and then the
solvent was evaporated in vacuo. The crude product was dissolved in
DCM and washed with a saturated solution of ammonium chloride.
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-yl-2-trifluoro-
methyl-imidazo[1,2-a]pyrazine (28a). 28a was prepared according to
a protocol analogous to intermediate 16 from intermediate 26a and
intermediate 27 at 150 °C for 15 min and under microwave irradia-
tion. Flash column chromatography (silica; 7 M solution of ammonia
in MeOH and EtOAc in DCM 3/0.3/96.7) then flash column
M
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Journal of Medicinal Chemistry
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chromatography (silica; 7 M solution of ammonia in MeOH in DCM
0/100 to 0.5/99.5) and freeze-drying yielded compound 28a as a
white solid (50%). MS: m/z 408 [M + H]+. tR = 2.80 min (method 7).
1H NMR (400 MHz, CDCl3) δ ppm 3.17 (t, J = 6.5 Hz, 2 H), 3.40 (s,
3 H), 3.83−3.91 (m, 6 H), 4.30−4.37 (m, 4 H), 7.24 (d, J = 4.6 Hz, 1
H), 7.40−7.44 (m, 2 H), 7.70 (dd, J = 8.0, 2.2 Hz, 1 H), 8.63 (d, J =
2.1 Hz, 1 H); mp 118.4 °C.
2.53 (s, 3 H), 3.10 (t, J = 6.6 Hz, 2 H), 3.29 (s, 3 H), 3.79 (t, J = 6.6
Hz, 2 H), 7.56 (d, J = 7.9 Hz, 1 H), 8.03 (dd, J = 8.1, 2.3 Hz, 1 H),
8.06 (d, J = 4.6 Hz, 1 H), 8.44 (d, J = 4.6 Hz, 1 H), 8.68−8.72 (m, 2
H), 8.76 (d, J = 2.1 Hz, 1 H), 8.80−8.84 (m, 2 H); mp 279.0 °C.
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-pyridin-3-yl-
imidazo[1,2-a]pyrazine (28g). 28g was prepared according to a
protocol analogous to intermediate 16 from intermediate 26g and
intermediate 27 at 150 °C for 15 min and under microwave irradia-
tion. Flash column chromatography (silica; 7 M solution of ammonia
in MeOH in DCM 1/99 then EtOAc in DCM 0/100 to 100/0) and
precipitation from Et2O yielded compound 28g as a white solid (48%).
2-Isopropyl-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (28b). 28b was prepared according to a proto-
col analogous to intermediate 16 from intermediate 26b and inter-
mediate 27 at 150 °C for 15 min and under microwave irradiation.
Flash column chromatography (silica; 7 M solution of ammonia in
MeOH in DCM 2/98) and ion exchange chromatography using an
ISOLUTE SCX2 cartridge (eluting with MeOH then 7 M solution of
ammonia in MeOH) yielded compound 28b as a clear syrup (52%).
1
MS: m/z 346 [M + H]+. tR = 1.68 min (method 7). H NMR (400
MHz, DMSO-d6) δ ppm 2.52 (s, 3 H), 3.09 (t, J = 6.6 Hz, 2 H), 3.29
(s, 3 H), 3.79 (t, J = 6.6 Hz, 2 H), 7.56 (d, J = 8.1 Hz, 1 H), 7.63 (dd,
J = 8.1, 4.9 Hz, 1 H), 8.02 (dd, J = 5.8, 2.3 Hz, 1 H), 8.03 (d, J = 4.4
Hz, 1 H), 8.38 (d, J = 4.6 Hz, 1 H), 8.74 (dd, J = 4.6, 1.6 Hz, 1 H),
8.76 (d, J = 2.3 Hz, 1 H), 9.04 (dt, J = 8.0, 1.9 Hz, 1 H), 9.85 (d, J =
2.1 Hz, 1 H); mp > 300 °C (dec).
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2,6-dimethyl-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (28h). 28h was prepared according to a pro-
tocol analogous to intermediate 16 from intermediate 26h and inter-
mediate 27 at 150 °C for 30 min and under microwave irradiation.
Flash column chromatography (silica; MeOH in DCM 4/96) yielded
compound 28h as a white solid (82%). MS: m/z 368 [M + H]+. tR =
2.39 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.22−2.25
(m, 3 H), 2.41 (s, 3 H), 3.15 (t, J = 6.5 Hz, 2 H), 3.41 (s, 3 H), 3.81−
3.92 (m, 6 H), 4.23−4.32 (m, 4 H), 7.18−7.23 (m, 1 H), 7.39 (d, J =
7.9 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2 Hz, 1 H), 8.59−8.64 (m, 1 H); mp
112.2 °C.
1
MS: m/z 382 [M + H]+. tR = 2.90 min (method 7). H NMR (400
MHz, DMSO-d6) δ ppm 1.24 (d, J = 6.7 Hz, 6 H), 2.95−3.05 (m, 1
H), 3.07 (t, J = 6.6 Hz, 2 H), 3.28 (s, 3 H), 3.74−3.80 (m, 6 H), 4.16−
4.23 (m, 4 H), 7.33 (d, J = 4.6 Hz, 1 H), 7.49 (d, J = 4.6 Hz, 1 H), 7.51
(d, J = 8.1 Hz, 1 H), 7.86 (dd, J = 7.9, 2.3 Hz, 1 H), 8.58 (d, J = 2.3
Hz, 1 H).
2-Cyclopropyl-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin-
4-yl-imidazo[1,2-a]pyrazine (28c). 28c was prepared according to a
protocol analogous to intermediate 16 from intermediate 26c and
intermediate 27 at 150 °C for 15 min and under microwave irradia-
tion. Flash column chromatography (silica; 7 M solution of ammonia
in MeOH in DCM 4/96) and precipitation with Et2O yielded
compound 28c as a brown solid (43%). MS: m/z 380 [M + H]+. tR =
1
2.67 min (method 7). H NMR (400 MHz, DMSO-d6) δ ppm 0.86−
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-
6-trifluoromethyl-imidazo[1,2-a]pyrazine (28i). 28i was prepared
according to a protocol analogous to intermediate 16 from inter-
mediate 26i and intermediate 27 at 150 °C for 15 min and under
microwave irradiation. Flash column chromatography (silica; EtOAc in
DCM 0/100 to 100/0) and trituration with diisopropylether/heptane
yielded compound 28i as a solid (41%, 93% pure by RP-LCMS, 98%
0.97 (m, 4 H), 1.90−2.01 (m, 1 H), 3.07 (t, J = 6.6 Hz, 2 H), 3.27 (s,
3 H), 3.70−3.75 (m, 4 H), 3.77 (t, J = 6.7 Hz, 2 H), 4.07−4.18 (m,
4 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 7.9 Hz, 1 H), 7.59 (d, J =
4.6 Hz, 1 H), 7.94 (dd, J = 8.1, 2.3 Hz, 1 H), 8.69 (d, J = 1.6 Hz,
1 H); mp 103.9 °C.
2-Methoxy-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-yl-
imidazo[1,2-a]pyrazine (28d). 28d was prepared according to a pro-
tocol analogous to intermediate 16 from intermediate 26d and inter-
mediate 27 at 150 °C for 15 min and under microwave irradiation.
Flash column chromatography (silica; 7 M solution of ammonia in
MeOH in DCM 2/98), flash column chromatography (silica; EtOAc
in heptane 30/70 to 100/0), and freeze-drying yielded compound 28d
as a brown solid (50%). MS: m/z 370 [M + H]+. tR = 2.35 min
1
by NMR). MS: m/z 422 [M + H]+. tR = 3.24 min (method 7). H
NMR (400 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.17 (t, J = 6.5 Hz,
2 H), 3.41 (s, 3 H), 3.82−3.93 (m, 6 H), 4.30−4.46 (m, 4 H), 7.43 (d,
J = 7.9 Hz, 1 H), 7.67 (dd, J = 8.1, 2.3 Hz, 1 H), 7.71 (s, 1 H), 8.61 (d,
J = 2.3 Hz, 1 H); mp 118.4 °C.
3-Bromo-8-chloro-6-iodo-2-methyl-imidazo[1,2-a]pyrazine (30).
Step 1: Synthesis of 8-chloro-6-iodo-2-methyl-imidazo[1,2-a]pyrazine.
A mixture of intermediate 29 (2.5 g, 9.78 mmol), sodium iodide
(2.93 g, 19.57 mmol), and 2-chloroacetone (4.67 mL, 58.72 mmol)
was stirred at 90 °C for 24 h in a sealed tube protected from light.
After cooling to rt, Et2O was added and the solid formed was
suspended in a saturated solution of sodium hydrogen carbonate and
extracted with DCM. The organic layer was dried (Na2SO4), filtered,
and the solvents evaporated in vacuo. The crude product was purified
by open column chromatography (silica; DCM). The desired fractions
were collected and the solvents evaporated in vacuo to yield 8-chloro-
6-iodo-2-methyl-imidazo[1,2-a]pyrazine (0.85 g, 28%) as a white solid
(hydroiodide). MS: m/z 294 [M + H]+. tR = 2.01 min (method 4).
Step 2: Synthesis of 30. N-Bromosuccinimide (1.091 g, 6.133 mmol)
was added to a stirred solution of 8-chloro-6-iodo-2-methyl-imidazo-
[1,2-a]pyrazine (1.9 g, 6.47 mmol) in DCM (50 mL). The mixture
was stirred at rt for 3 h and then diluted with further DCM and
washed with a saturated solution of sodium carbonate. The organic
layer was separated, dried (Na2SO4), filtered, and the solvent
evaporated in vacuo. The crude product was purified by flash column
chromatography (silica; EtOAc in heptane 0/100 to 40/60). The
desired fractions were collected and the solvents evaporated in vacuo
to yield intermediate 30 (2 g, 83%) as a white solid. MS: m/z 372
1
(method 7). H NMR (400 MHz, DMSO-d6) δ ppm 3.02 (t, J = 6.6
Hz, 2 H), 3.26 (s, 3 H), 3.73 (t, J = 6.5 Hz, 2 H), 3.74−3.78 (m, 4 H),
4.00 (s, 3 H), 4.07−4.12 (m, 4 H), 7.45 (d, J = 8.3 Hz, 1 H), 7.46 (d,
J = 4.6 Hz, 1 H), 7.85 (d, J = 4.6 Hz, 1 H), 7.92 (dd, J = 8.1, 2.3 Hz, 1
H), 8.70 (d, J = 2.1 Hz, 1 H); mp > 300 °C (dec).
3-[6-(2-Methoxyethyl)-3-pyridyl]-2-methyl-8-pyrrolidin-1-yl-
imidazo[1,2-a]pyrazine (28e). 28e was prepared according to a
protocol analogous to intermediate 16 from intermediate 26e and
intermediate 27 at 150 °C for 15 min and under microwave irradia-
tion. Flash column chromatography (silica; 7 M solution of ammonia
in MeOH in DCM 1/99), flash column chromatography (silica;
EtOAc in heptane 0/100 to 100/0), and precipitation from
diisopropylether yielded compound 28e as a solid (56%). MS: m/z
1
338 [M + H]+. tR = 2.35 min (method 7). H NMR (400 MHz,
DMSO-d6) δ ppm 2.53 (s, 3 H), 3.10 (t, J = 6.6 Hz, 2 H), 3.29 (s, 3
H), 3.79 (t, J = 6.6 Hz, 2 H), 7.56 (d, J = 7.9 Hz, 1 H), 8.03 (dd, J =
8.1, 2.3 Hz, 1 H), 8.06 (d, J = 4.6 Hz, 1 H), 8.44 (d, J = 4.6 Hz, 1 H),
8.70 (dd, J = 4.6, 1.6 Hz, 2 H), 8.76 (d, J = 2.1 Hz, 1 H), 8.82 (dd, J =
4.4, 1.6 Hz, 2 H); mp 111.0 °C.
3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-pyridin-4-yl-
imidazo[1,2-a]pyrazine (28f). 28f was prepared according to a
protocol analogous to intermediate 16 from intermediate 26f and
intermediate 27 at 150 °C for 15 min and under microwave irradia-
tion. Flash column chromatography (silica; 7 M solution of ammonia
in MeOH in DCM 0/100 to 2/98) and precipitation from Et2O
yielded compound 28f as a white solid (90%). ESI-HRMS: m/z for
C20H20N5O [M + H]+ calcd, 346.1668; found, 346.1663 (−1.4 ppm).
1
[M + H]+. tR = 2.40 min (method 6). H NMR (500 MHz, CDCl3)
δ ppm 2.55 (s, 3 H), 8.25 (s, 1 H).
3-Bromo-6-iodo-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]-
pyrazine (31). Morpholine (0.241 mL, 2.75 mmol) was added to a
stirred solution of intermediate 30 (790 mg, 2.12 mmol) and N,N-
diisopropylethylamine (0.554 mL, 3.18 mmol) in ACN (10 mL).
1
tR = 2.44 min (method 2). H NMR (400 MHz, DMSO-d6) δ ppm
N
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The mixture was stirred at 160 °C for 30 min under microwave
irradiation. The mixture was diluted with DCM and washed with a
saturated solution of ammonium chloride. The organic layer was
separated, dried (Na2SO4), filtered, and the solvents evaporated
in vacuo. The crude product was purified by flash column chromatog-
raphy (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to
1/99). The desired fractions were collected and the solvents
evaporated in vacuo and the crude product precipitated from Et2O
to yield intermediate 31 (750 mg, 83%, 90% pure) as a white solid.
to the modified Spearman−Kaerber estimate, using theoretical
probabilities instead of empirical ones.21 This modification allows
the determination of the ED50 and its confidence interval as a function
of the slope of the log dose−response curve.22
ASSOCIATED CONTENT
* Supporting Information
■
S
Pharmacokinetics experimental details, and analytical methods.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
MS: m/z 423 [M + H]+. tR =3.49 min (method 3). H NMR (400
MHz, CDCl3) δ ppm 2.39 (s, 3 H), 3.78−3.87 (m, 4 H), 4.22−4.32
(m, 4 H), 7.68 (s, 1 H); mp 181.2−182.1 °C.
Biology Experimental. The Institutional Ethical Committee on
Animal Experimentation approved the experimental protocols, in
compliance with the Belgian law (Royal Decree on the Protection of
Laboratory Animals, April 6, 2010).
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 925232331. Fax: +34 925245771. E-mail:
scondec@its.jnj.com.
■
In Vitro Assay PDE10A. Rat recombinant PDE10A (rPDE10A) was
expressed in Sf9 cells using a recombinant rPDE10A baculovirus
construct. Cells were harvested after 48 h of infection, and the
rPDE10A protein was purified by metal chelate chromatography on
Ni-sepharose 6FF. Tested compounds were dissolved and diluted in
100% DMSO to a concentration 100-fold of the final concentration in
the assay. Compound dilutions (0.4 μL) were added in 384-well plates
to 20 μL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2,
1.7 mM EGTA). Then 10 μL of rPDE10A enzyme in incubation buffer
was added, and the reaction was started by addition of 10 μL substrate
to a final concentration of 60 nM cAMP and 0.008 μCi [3H]cAMP.
The reaction was incubated for 60 min at rt. After incubation, the
reaction was stopped with 20 μL of 17.8 mg/mL PDE SPA beads.
After sedimentation of the beads during 30 min, the luminescence was
measured in a PerkinElmer Topcount scintillation counter and results
were expressed as cpm. For blank values the enzyme was omitted from
the reaction and replaced by incubation buffer. Control values were
obtained by addition of a final concentration of 1% DMSO instead of
compound. A best fit curve was fitted by a minimum sum of squares
method to the plot of % of control value subtracted with blank value
versus compound concentration and the half-maximal inhibitory
concentration (IC50) value was derived from this curve.
PDE10A Occupancy. Dose−response or single dose experiments
were performed to measure PDE10A occupancy 1 h after oral (sc or
po) administration. Male Wistar rats (200 g) were treated by sc or po
administration of various PDE10 inhibitors. The PDE10 radioligand
[3H]MP-10 (10 μCi/animal) was injected intravenously (iv) 30 min
before sacrifice. Brains were immediately removed from the skull and
rapidly frozen. Then 20 μm thick brain sections were cut using a
cryostat-microtome, thaw-mounted on microscope slides and loaded
in a β-imager (Biospace Laboratory, Paris, France) for 8 h to quantify
PDE10A occupancy in the striatum. Digital autoradiograms were
quantified using the Beta Vision Program (Biospace Laboratory). The
specific binding was determined as the difference between [3H]MP-10
binding quantified in the striatum (a brain area showing a high density
of PDE10A) and in the cortex (a brain area where PDE10A is virtually
absent). Occupancy was calculated as the inhibition of specific [3H]-
MP-10 binding in drug-treated animals relative to vehicle-treated
animals.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank our co-worker Herman Hendrickx from the
Neuroscience Discovery team.
■
ABBREVIATIONS USED
■
ACN, acetonitrile; cAMP, cyclic adenosine monophosphate;
APO, apomorphine; BTB, brain tissue binding; calcd, calculated;
Clint, intrinsic clearance; CNS, central nervous system; CYPs,
cytochromes; D2, dopamine 2; DCM, dichloromethane; DAD,
diode array; dec, decomposition; DIPEA, N,N-diisopropylethyl-
amine; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH,
ethanol; ER, extraction ratio; cGMP, cyclic guanosine mono-
phosphate; h, hours; HPLC, high-performance liquid chroma-
tography; iv, intravenous; MSD, mass selective detector; MSNs,
medium spiny neurons; QTOF, quadrupole time-of-flight
detector; [M + H]+, the protonated mass of the free base of
the compound; MDFA, methyl 2,2-difluoro-2-(fluorosulfonyl)-
acetate; MeOH, methanol; min, minutes; mp, melting point; MS,
mass spectrum; NBS, N-bromosuccinimide; NIS, N-iodosucci-
nimide; NMP, N-methyl pyrrolidine; NMR, nuclear magnetic
resonance; PCP, phencyclidine; PDE, phosphodiesterase; PET,
positron emission tomography; rPPB, rat plasma protein
binding; po, per oral; rt, room temperature; RP-HPLC, reverse
phase high performance liquid chromatography; sc, subcuta-
neous; SAR, structure−activity relationship; SQD, single quadru-
pole detector; THF, tetrahydrofuran; TLC, thin layer
chromatography; TOF, time-of-flight detector; tR, retention
time; UPLC, ultraperformance liquid chromatography
REFERENCES
■
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molecular regulation to clinical use. Pharmacol. Rev. 2006, 58, 488−
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Apomorphine-Induced Stereotypy in Rats. Apomorphine
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stereotypy: fewer than 6 scores of 3 (0.16% false positives), fewer than
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ED50 for Functional Effects. All-or-none criteria for drug-induced
effects were defined by analyzing a frequency distribution of a series
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