Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design

Article abstract of DOI:10.1021/acs.jmedchem.5b02010

The N-methyl-d-aspartate receptor (NMDAR) is a Na⁺ and Ca²⁺ permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

Full text of DOI:10.1021/acs.jmedchem.5b02010

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Article
Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric
Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design
Matthew Volgraf, Benjamin D. Sellers, Yu Jiang, Guosheng Wu, Cuong Q. Ly, Elisia Villemure, Richard
M Pastor, Po-wai Yuen, Aijun Lu, Xifeng Luo, Mingcui Liu, Shun Zhang, Liang Sun, Yuhong Fu, Patrick
J Lupardus, Heidi J A Wallweber, Bianca M. Liederer, Gauri Deshmukh, Emile Plise, Suzanne Tay, Paul
Reynen, James Herrington, Amy Gustafson, Yichin Liu, Akim Dirksen, Matthias G A Dietz, Yanzhou
Liu, Tzu-Ming Wang, Jesse Hanson, David Hackos, Kimberly Scearce-Levie, and Jacob B. Schwarz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b02010 • Publication Date (Web): 26 Feb 2016
Downloaded from http://pubs.acs.org on March 5, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of GluN2AꢀSelective NMDA Receptor
Positive Allosteric Modulators (PAMs): Tuning
Deactivation Kinetics via StructureꢀBased Design
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,
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Matthew Volgraf,* Benjamin D. Sellers, Yu Jiang, Guosheng Wu, Cuong Q. Ly, Elisia
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†
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Villemure, Richard M. Pastor, Poꢀwai Yuen, Aijun Lu, Xifeng Luo, Mingcui Liu, Shun Zhang,
ø
ø
ß
ß
∆
Liang Sun, Yuhong Fu, Patrick J. Lupardus, Heidi J.A. Wallweber, Bianca M. Liederer, Gauri
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∆
∆
§
§
§
Deshmukh, Emile Plise, Suzanne Tay, Paul Reynen, James Herrington, Amy Gustafson, Yichin
§
#
#
†
∞
∞
Liu, Akim Dirksen, Matthias G. A. Dietz, Yanzhou Liu, TzuꢀMing Wang, Jesse Hanson, David
∞
∞
†
Hackos, Kimberly ScearceꢀLevie, Jacob B. Schwarz
†
∞
§
Department of Discovery Chemistry, Department of Neurosciences, Department of Biochemical
∆
ß
and Cellular Pharmacology, Department of Drug Metabolism and Pharmacokinetics, Department of
Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA
ø
PharmaronꢀBeijing Co. Ltd., 6 Taihe Road, BDA, Beijing 100176, PR China
#
Ion Channels Group, Evotec AG; Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg, Germany
*Correspondence: volgraf.matthew@gene.com
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
Abstract
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+
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The NꢀMethylꢀDꢀAspartate receptor (NMDAR) is a Na and Ca permeable ionotropic glutamate
1
2
receptor that is activated by the coꢀagonists glycine and glutamate. NMDARs are critical to synaptic
signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders,
including schizophrenia, depression, and Alzheimer’s disease. Herein we describe the discovery of
potent GluN2Aꢀselective NMDAR positive allosteric modulators (PAMs) starting from a highꢀ
throughput screening hit. Using structureꢀbased design we sought to increase potency at the GluN2A
subtype, while improving selectivity against related αꢀaminoꢀ3ꢀhydroxyꢀ5ꢀmethylꢀ4ꢀisoxazolepropionic
acid receptors (AMPARs). The structureꢀactivity relationship of channel deactivation kinetics was
studied using a combination of electrophysiology and protein crystallography. Effective incorporation of
these strategies resulted in the discovery of GNEꢀ0723 (46), a highly potent and brain penetrant
GluN2Aꢀselective NMDAR PAM suitable for in vivo characterization.
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Introduction
Glutamate is the primary excitatory neurotransmitter in the mammalian brian and acts on several
membraneꢀbound receptors, including both metabotropic receptors (mGluRs) and ionotropic receptors
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iGluRs). iGluRs are subdivided according to their function and pharmacology including αꢀaminoꢀ3ꢀ
hydroxyꢀ5ꢀmethylꢀ4ꢀisoxazolepropionic acid receptors (AMPARs), kainate receptors and NꢀmethylꢀDꢀ
aspartate receptors (NMDARs). NMDARs are tetrameric assemblies including two GluN1 and two
GluN2 subunits that bind the coꢀagonists glycine and glutamate, respectively. The four GluN2 subtypes
(GluN2A, GluN2B, GluN2C and GluN2D) largely determine the receptor’s functional and
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spatiotemporal expression heterogeneity. Functionally, NMDARs are blocked by Mg at resting
membrane potential and require both agonist binding and cell depolarization to open the transmembrane
channel. Within the nervous system NMDARs are implicated in controlling synaptic plasticity and are
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therefore thought to underlie both memory formation and other higher cognitive functions.
Due to their central role in neurotransmission, NMDARs have attracted significant interest as
pharmacological targets. Memantine, a nonꢀselective NMDAR antagonist, has been approved for
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treatment of moderateꢀtoꢀsevere Alzheimer’s disease, while GluN2Bꢀselective antagonists such as
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ifenprodil, have been studied as neuroprotective agents. NMDAR hypofunction has long been
implicated in the pathophysiology of schizophrenia beginning with the discovery that the dissociative
anaesthetics ketamine and phencyclidine (PCP) induce a psychotomimetic state resembling the disease
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while functioning as noncompetitive NMDAR antagonists. More recently, NMDARs have been shown
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to regulate gamma oscillations generated by fastꢀspiking (FS) parvalbumin (PV) interneurons, a cellular
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subtype that controls the inhibition of excitatory neurons. EEG studies have further demonstrated that
gamma oscillations are disrupted in schizophrenic patients, strengthening the link between NMDAR
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dysfuntion and schizophrenia.
Recently, a number of NMDAR positive allosteric modulators (PAMs) have been reported in the
literature targeting NMDAR hypofunction. Endogenous nonꢀselective NMDAR PAMs have been found,
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including the neurosteroid pregnenolone sulfate, along with the brain cholesterol metabolite 24(S)ꢀ
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hydroxycholesterol and closely related analogs. Subunit selective PAMs have also been
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described
, including the GluN2C/2D selective CIQ.
Our goal was to enhance physiological NMDAR activation while avoiding potentially toxic
overactivation. Therefore we focused on discovering PAMs that are selective for NMDARs containing
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the GluN2A subunit, which is broadly distributed in the mature brain and located at synapses . At the
same time selectivity against NMDARs containing only GluN2B subunits avoids the potential toxic
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consequences of overstimulating extrasynaptic GluN2Bꢀcontaining NMDARs
. We believed the
identification of GluN2A selective NMDAR PAMs would help dissect the physiological role of
NMDAR subtypes in disease and neuronal function while also providing a novel therapeutic approach in
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schizophrenia and other neurological disorders.
Here we report the discovery and optimization of a series of GluN2Aꢀselective PAMs starting from
the high throughput screening hit 1 (Table 1) which displayed low micromolar potency against GluN2A
and minimal activity against GluN2B. Compound 1 and related molecules from this series have been
previously confirmed to act as allosteric modulators rather than orthosteric ligands via
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electrophysiology, where compound alone resulted in little to no observable current, while significant
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potentiation was observed in the presence of glutamate. Additional pharmacological studies have also
been reported, including effects on GluN2A/GluN2B triheteromeric receptors as well as pyramidal
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neuron and interneuron NMDAR responses.
The GluN2A potency of 1 was quickly optimized by truncating the butyl chain to a methyl (compound
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) and adding a fluorine at the 4ꢀposition of the aniline (compound 3), while maintaining selectivity
against GluN2B. Compound 3 provided an excellent starting point for a focused lead optimization
campaign despite the poor observed selectivity against AMPARs (GluA2flip and GluA2flop isoforms).
Electrophysiology revealed that 3 and related PAMs slowed channel deactivation kinetics as the primary
means of potentiation, a characteristic we sought to minimize in order to preserve the timing and rhythm
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of neural firing patterns. The CNS drugꢀlike properties of 3 were also favorable; the measured LogD
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was moderate (<3), topographical polar surface area (TPSA) was low (<90 Å ), and there was no
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observable Pꢀglycoprotein (Pꢀgp) efflux in vitro (Efflux Ratio < 3). However, poor metabolic stability,
both in liver microsomes and in vivo (data not shown), prevented the use of 3 as an in vivo tool
compound. As metabolite identification studies revealed Nꢀdealkylation as the major metabolic pathway
for this series of PAMs (data not shown), efforts to replace the Nꢀethyl aniline and increase overall
potency while maintaining CNS drugꢀlike properties became a top priority.
Results and Discussion
A coꢀcrystal structure of 2 was obtained, using truncated GluN2A and GluN1 ligandꢀbinding domains
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(LBDs) (Figure 1A). The structure revealed a binding site at the dimer interface of the GluN1ꢀGluN2A
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LBD, the same site observed in the closely related AMPARs, where PAM binding stabilizes the LBDs
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in their closedꢀcleft, ligandꢀbound conformations. It was therefore unsurprising that 1 and related
analogs significantly potentiated AMPARs in addition to GluN2Aꢀcontaining NMDARs. We
hypothesized that the selectivity of this class of PAMs against other GluN2 subunits could be attributed
to van der Waals interactions with Val783 of GluN2A at the dimer interface. In the other subtypes, this
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residue is a phenylalanine (GluN2B), or leucine (GluN2C and GluN2D), while the GluA2 Flip and Flop
isoforms possess the polar residues serine and asparagine, respectively. After recognizing selectivity
against GluA2 receptors would be a concern for this series of PAMs, GluA2Flip and GluA2Flop calcium
imaging assays were established as the major counterꢀscreen assay, while GluN2B/C/D selectivity
panels were obtained much less frequently.
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The crystal structure revealed a clear opportunity for increasing receptor interactions by building into a
waterꢀfilled pocket proximal to the thiadiazoleꢀcore nitrogen, in a region we termed the C’ꢀsubsite based
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on similarity to the AMPAR PAM pocket. We hypothesized that by displacing these crystallographic
waters we might gain significant GluN2A potency. To achieve this, we replaced the thiadiazoleꢀbased
core of 1 with a thiazoleꢀbased scaffold and sought to explore substitution at the newly created carbon
center with a variety of functional groups. We believed that substitution at this position might yield
more selective PAMs against AMPARs, as comparison of the binding sites from the two receptors
suggested that the C’ꢀsubsite of GluN2A was larger than the corresponding pocket in GluA2. Therefore
we hypothesized that larger groups at this position might afford both additional potency and selectivity.
Table 2 summarizes the results of the C’ꢀsubsite SAR, where overall potency was modestly improved
by replacing the thiadiazoleꢀbased core of 3 with the thiazoleꢀbased core of 4. Heteroaromatic rings such
as 3ꢀpyridyl (5) and pyrimidine (6) yielded an increase in potency with small improvements in
selectivity against GluA2 isoforms. Saturated heteroaromatics such as morpholine (7) were not as well
tolerated. Polar groups such as nitrile (8) and various aliphatic alcohols (9-13) all provided gains in
GluN2A potency via displacement and interaction with the C’ꢀsubsite water network. In contrast, nonꢀ
functionalized aliphatic groups such as isopropyl (14), cyclobutyl (15) and cyclopropyl (16) did not
improve GluN2A potency. However introduction of πꢀcharacter via isopropylene (17) and a methyl
ketone (18) brought double digit nanomolar EC50s, with selectivity against GluA2 receptors reaching
greater than 100ꢀfold.
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Throughout the SAR exploration at the C’ꢀsubsite, we sought to maintain favorable CNS drugꢀlike
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properties by maintaining Pꢀgp efflux ratios (ERs) less than 3. We were therefore pleased to find that Pꢀ
gp ERs for compounds 4-18 were all less than 2. In addition, we sought to improve selectivity against
GluA2 receptors as we believed even low levels of AMPAR potentiation might prove a liability in
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vivo. With this in mind, we explored polar groups not normally considered CNS drugꢀlike, such as
amides 19 and 20. The Pꢀgp ERs for these compounds exceeded our target ratio of 3 due to the increased
TPSA and the introduction of hydrogen bond donors, indicating a high likelihood of impaired bloodꢀ
brain barrier (BBB) penetration in vivo, but we were also encouraged by the improved balance of
GluN2A potency relative to GluA2 selectivity. In each case, only low levels of AMPAR potentiation
were observed at the highest concentrations studied in the assays (125 ꢀM).
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A crystal structure of the methyl amide 19 (Figure 1B) revealed that the amide NꢀH engages the
backbone carbonyl of Pro527 (GluN2A) in a hydrogen bond, enforcing a perpendicular orientation of
the amide relative to the core. We hypothesized that the forced perpendicular conformation of amides 19
and 20 was responsible for the observed selectivity against GluA2 receptors, as the amide carbonyl is
more likely to clash with the smaller AMPAR pocket. Other features of PAM binding remained the
same as previously described for the thiadiazoleꢀbased core.
Given the improved potency and selectivity of ethyl amide 20, we sought to explore SAR at the aniline
position, or “rightꢀhand side” (RHS), of the molecules using the crystal structure as a guide in attempts
to improve metabolic stability within the series (Table 3). The length of the Nꢀalkyl chain was critical to
potency; while the longer propyl chain (21) was roughly equipotent with 20, shorter permutations 22 and
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3 were less active. Meanwhile the aminopyridine 24 lost significant potency, without improving
metabolic stability relative to the anilines. The ether 25 also lost significant potency (8.5 ꢀM) but
possessed a notably reduced maximum potentiation (77%) relative to the previously studied Nꢀethyl
anilines (typically >140%) and significantly improved in vitro liver microsome stability in rodents.
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Metaꢀsubstituted phenyl groups 26 and 27 possessed micromolar potencies and reduced maximum
potentiation. Potency within the phenyl subseries was improved with addition of an orthoꢀfluorine (28
and 29), presumably through increased van der Waals interactions and maximizing the magnitude of the
aromatic dipole moment. We hypothesized that this dipole interacted favorably with the antiparallel
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dipole of the backbone amide of Thr759 (GluN2A) in a πꢀinteraction. Additional SAR within the
phenyl and aniline subseries supported this hypothesis (data not shown). Other substitutions on the
phenyl ring targeting regions of the binding site occupied by the ethyl group of the anilines (30 and 31),
as well as methylation of the methylene coreꢀlinker (32), proved unsuccessful at improving GluN2A
potency or dramatically impacting maximum potentiation.
We next explored substituted pyrazoles, a heterocycle previously reported in AMPAR PAMs to bind a
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homologous pocket. The 3ꢀtrifluoromethyl pyrazole 33 proved a reasonable starting point for our
investigations given the modest GluN2A potency and selectivity against AMPARs, while modeling
suggested that the 5ꢀposition of the pyrazoles provided a suitable vector to explore the pocket occupied
by the ethyl group of the anilines. Systematic investigation of functional groups at this site (34ꢀ38)
revealed that the chlorine at the 5ꢀposition (35) possessed a unique balance of GluN2A potency (0.439
ꢀM), moderate maximum potentiation (120%), and selectivity against GluA2 receptors (nearly 100ꢀfold
selective). Other larger groups, such as the cyclopropyl (36) and ethoxy (37), reduced AMPAR
selectivity and possessed larger maximum potentiation. Meanwhile, attempts to build further into the
small pocket off the 5ꢀposition, such as isobutoxy analog 38, proved unproductive.
To better study receptor deactivation kinetics, we developed an automated electrophysiology assay to
measure the fold slowing of deactivation in the presence of 100 ꢀM PAM; the fold deactivation was
defined as the the ratio between the inverse deactivation rate constants in the presence of a PAM versus
DMSO control. Figure 2A shows the relationship between maximum potentiation (%) and fold
deactivation, where various thiazolopyrmidinoneꢀcontaining PAMs have been grouped by the identity of
the aromatic RHS group, including anilines, pyrazoles, phenyls and ethers. The plot shows lower
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maximum potentiation values (40ꢀ100%) correlated with deactivation kinetics close to the DMSO
control (<3x), while higher maximum potentiation values (>150%) resulted in deactivation kinetics 10x
slower than control. Each aromatic group on the RHS possessed a characteristic deactivation profile;
anilines were the slowest deactivating, pyrazoles and phenyls possessed intermediate kinetics, while
ethers demonstrated very fast deactivation kinetics. This proved valuable during the SAR and design
stage of the program, allowing categorization of the RHS groups according to their deactivation kinetics
along with their DMPK properties.
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Individual PAMs were studied in greater detail using manual wholeꢀcell patch clamp
electrophysiology together with a fast perfusion system. Figure 2B compares the electrophysiology
profiles of five amide matched pairs with varying RHS groups including the Nꢀethyl aniline (20), ether
(25), substituted trifluorophenyl (29), and substituted trifluoropyrazole (35). Notably, the deactivation
trends observed for each aromatic group in the automated platform matched the trends in the fast
perfusion system (Figure 2A). Each RHS also possessed varying degrees of peak current potentiation,
following the same trend as the deactivation kinetics (ether 25 the smallest, aniline 20 the largest).
Crystallographic studies of representative compounds from each RHS subseries (ether 40, phenyl 29,
pyrazole 49 and aniline 20) did not reveal significant structural changes within the LBD that might
account for the observed differences in deactivation kinetics. The size and position of the functional
group occupying the “floor” of the RHS binding pocket appears to control the deactivation profile of the
receptor; ethers (Figure 2C) and phenyls (Figure 2D) without groups in this position possessed the
fastest deactivation kinetics, while Nꢀchloropyrazole (Figure 2E) and Nꢀethyl anilines (Figure 2F)
possessed slower kinetic profiles upon filling this site. Electron withdrawing substituents on each
aromatic ring enforce a dipole moment antiparallel to the backbone amide dipole of Thr759 in GluN2A,
supporting our design hypothesis that the direction and strength of the ring dipole contributes to
23
GluN2A potency (Figures 2CꢀF).
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Studies exploring the efflux properties of a broad set of amideꢀcontaining PAMs revealed the
difficulty in identifying molecules within this series that are not Pꢀgp substrates (Figure 3). Comparing
thiazolopyrimidinone PAMs containing amides in the C’subsite with those without amides demonstrated
the increased probability of Pꢀgp efflux for amides, even at equivalent TPSAs, due to the added Hꢀbond
donor. We recognized that unless the GluN2A potency of the amides could be greatly improved,
coverage of the in vivo EC50 would be limited due to the observed Pꢀgp efflux. Efforts shifted to
identifying GluN2A PAMs outside of the amide subseries that possessed both good selectivity and
predicted human pharmacokinetic (PK) properties without Pꢀgp efflux.
1
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Towards this goal, we recognized that the GluN2A potency of various alcohols (9ꢀ13) was improved
relative to unsubstituted aliphatic groups (14ꢀ16) and that the additional Hꢀbond donor of the alcohol did
not introduce Pꢀgp efflux. A key breakthrough came when the conformation of the propyl alcohol 13
was restricted through the addition of a cyclopropyl group to provide transꢀcyclopropyl methyl alcohol
3
9 (Table 4). Compound 39 was the most GluN2Aꢀpotent PAM on the program to date (24.4 nM) and
lacked Pꢀgp efflux, but had poor selectivity and metabolic stability. GluN2A potencies of aniline
replacements (40ꢀ43) were improved in each case relative to the amide matched pairs (25, 28, 29, and
3
5) although selectivities against AMPARs were poor, with the exception of ether 40. Compound 40
became a valuable in vitro tool compound to explore the properties of a fastꢀdeactivating GluN2A PAM
that did not possess any contaminating potentiation of AMPARs. It was later discovered that 40 behaves
as a silent allosteric modulator (SAM) at AMPARs, able to bind the receptor but unable to elicit a
1
7
positive or negative effect on the channel current. Replacement of the core methyl with a
trifluoromethyl (44) was found to improve selectivity against AMPARs, without greatly impacting
GluN2A potency.
Despite the lack of observed Pꢀgp efflux, all cyclopropyl methyl alcohols remained metabolically
labile due to glucoronidation of the primary alcohol as predicted in hepatocytes. As a result, a
cyclopropyl methyl alcohol could not be dosed in rodents with sufficient exposures to enable biological
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Journal of Medicinal Chemistry
evaluation (data not shown). Among the many strategies employed to reduce the metabolic lability of
the cyclopropyl methyl alcohol, a cyclopropyl nitrile was explored to maintain polar interactions with
the C’ꢀsubsite waters, without the risk of glucoronidation. We were pleased to see that the GluN2A
potency of transꢀcyclopropyl nitrile 45 (Table 5) was very similar to the transꢀcyclopropyl methyl
alcohols (30 nM), and that addition of a core trifluoromethyl (GNEꢀ0723, 46) improved both
selectivities against AMPARs and metabolic stability without affecting potency (21 nM). Gratifyingly,
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6 possessed a rare combination of potency, metabolic stability and lack of Pꢀgp efflux in a single
GluN2Aꢀselective PAM.
A crystal structure of 46 bound to the LBD dimer interface confirmed our design hypothesis. The
unique conformation of the transꢀcyclopropyl allows the nitrile to reach into a distal waterꢀfilled pocket
off the C’ꢀsubsite (Figure 4). Similar to the methyl substituent, the trifluoromethyl group interacts with
Val783 of GluN2A in a van der Waals interaction, but the source of the improved selectivity against
AMPARs is less clear. We believe the selectivity of 46 and related analogs could be derived from the
clash of a larger, hydrophobic trifluoromethyl against the more polar serine and asparagine residues of
the Flip and Flop isoforms of GluA2, respectively. A separate small molecule Xꢀray crystal structure of
4
6 confirmed the relative and absolute stereochemistry of the 1,2ꢀdisubstituted cyclopropyl (see
Supporting Information).
Full receptor profiling of 46 against GluN2B/C/D subtypes revealed good selectivity (Figure 5A and
Table 6), with a greater than 250ꢀfold difference between GluN2A and the next most potent glutamate
receptor, GluA2Flop (Figure 5B and Table 6). Manual electrophysiology demonstrated moderate slowing
of deactivation kinetics relative to previously studied PAMs (Figure 5C).
A Mouse IV PK study with 46 revealed a good correlation between predicted clearance based on
mouse hepatocyte and microsomal stability studies (17 and 23 ml/min/kg, respectively) and in vivo
clearance (26 ml/min/kg) (Table 6). To support in vivo efficacy studies, a mouse PK study explored
brain exposure and BBB permeability at a 10 mg/kg PO dose where unbound brain concentrations
reached 14 nM at 1 h, compared with unbound plasma concentrations of 17 nM at the same timepoint.
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The brain/plasma ratio of these free concentrations at 1 h (0.82) revealed that 46 was BBB permeable, in
agreement with the in vitro MDCKꢀMDR1 ER (B:A/A:B = 1.4). This dose was suitable for in
vivo experiments exploring the bioactivity of 46 in mice, where the GluN2A selective PAM
demonstrates promising activity relative to NMDAR antagonists in both locomotor assays and direct
measurements of circuit activity using EEG. A detailed account of these studies will be reported in due
course.
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Chemistry
1
7
The syntheses of 1-3 were previously described. The majority of GluN2A PAMs described above
were all derived from a common thiazolopyrmidinone core. Depending on the SAR explored, we ideally
sought a lateꢀstage functionalization of the vector of interest. Towards this goal, previously described
1
7
core 47 could be treated with Nꢀethyl 4ꢀfluoroaniline to obtain the bromide 48 which was poised for a
variety of functionalizations at the C’ꢀsubsite (Scheme 1). Standard Suzuki crossꢀcouplings afforded
final targets 5, 6, 16, and 17, while copperꢀ and zincꢀmediated transformations afforded morpholine 7
and nitrile 8, respectively. Lithiation of 48, followed by quench with ethyl formate provided aldehyde
4
9, which could be reduced to provide primary alcohol 7 or treated with methylmagnesium bromide to
afford the secondary alcohol 10. A Suzuki crossꢀcoupling afforded enol ether 50, which then set the
stage for a hydrolysis/reduction sequence to yield ethyl alcohol 12. Heck coupling between ethyl acrylate
and bromide 48 provided enoate 51 which could be fully reduced to propyl alcohol 13.
Other C’ subsite analogs could not be easily obtained from bromide 48 and required preꢀinstallation of
various functional groups on a precursor aminothiazole (55-58), accessed via αꢀbromo ketones (52-54).
PPA cyclizations afforded alkyl chlorides (59-62) which could be treated with Nꢀethyl 4ꢀfluoroaniline
under basic conditions to afford final compounds 4, 14ꢀ15, and 18. Ketone 18 could in turn be
transformed into tertiary alcohol 20.
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Amide analogs 19ꢀ38 were most easily accessed by first forming the aminothiazole amides 64ꢀ65 and
cyclizing directly to the alkyl chlorides 66 and 67 (Scheme 2). Anilines 20ꢀ23, aminopyridine 24, and
ether 25 were all synthesized by alkylation of the corresponding nucleophiles under basic conditions,
while various substituted phenyls 26ꢀ31 were formed via palladiumꢀcatalyzed Suzuki reactions from the
boronic acids. To explore alkylation at the benzylic position adjacent to the phenyl ring, 29 could be
treated with excess nꢀbutyl lithium and quenched with methyl iodide to yield methylated 32.
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1
,5ꢀsubstituted pyrazoles were synthesized by two primary methods (Scheme 3). Access to the
chloropyrazole 71 was accomplished by first protecting 3ꢀtrifluoromethylpyrazole 68 as the N,Nꢀ
dimethyl sulfamoyl 69, and then selectively deprotonating at the Cꢀ5 position with nꢀbutyllithium,
followed by treatment with hexachloroethane. Chloropyrazole 71 was then obtained by TFA
deprotection of sulfamoyl 70. Other pyrazoles, such as cyclopropylpyrazole 75, were synthesized by
treatment of 1,3ꢀdiketones (74) with hydrazine hydrate. These pyrazoles could then be easily alkylated
by core alkyl chlorides (67) using the same methods as Nꢀalkyl anilines (Scheme 2) to provide targets
3
3ꢀ38.
Access to monoꢀsubstituted transꢀcyclopropyls required synthesis of the potassium trifluoroborate
building blocks which were found to perform better in Suzuki cross coupling reactions relative to the
parent pinacol boronate esters (Scheme 4). The transꢀcyclopropyl methyl alcohol intermediate 78 was
accessed from Eꢀsubstituted vinyl boronate ester 76 via SimmonsꢀSmith cyclopropanation using the Shiꢀ
2
5
modified conditions, providing clean transꢀcyclopropyl 77. The TBSꢀprotected pinacol boronate ester
could then be treated with potassium bifluoride in methanol and water to effect both formation of the
potassium trifluoroborate and TBS deprotection towards the final coupling partner 78. The nitrile 81
was also accessed in two steps (Scheme 4), first borylating cyclopropyl nitrile (79) using Hartwig’s
26
iridium catalysis protocol (ꢀ80), followed by the same potassium bifluoride conditions previously
described to access the final potassium trifluoroborate 81.
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Most cyclopropyl methyl alcohol analogs in Table 4 were synthesized by first installing the requisite
benzylic aromatic group on difunctionalized core 47 using previously described methods, followed by
Suzuki cross coupling with the trifluoroborate salt 78 (Scheme 5). Nitrile 45 could be accessed by an
analogous method using the cyclopropyl building block 81 (Scheme 4).
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The trifluoromethylated core of 44 and 46 required a de novo synthesis as the trifluoromethyl group
could not be installed on a lateꢀstage intermediate (Scheme 6). Key trifluoromethyl aminothiazole 90
was synthesized beginning from aminothiazole 86, which was chlorinated using Sandmeyer conditions
to provide 87, as the amine proved detrimental to the key trifluoromethylation event and required
protection/removal. Trihalogenated thiazole 88 was generated upon treatment with LDA and elemental
2
7
iodine after a “halogen dance reaction” at the 4ꢀ and 5ꢀpositions. The three halogens could then be
sequentially manipulated, first reacting the iodine under copperꢀmediated trifluoromethylation
conditions to provide trifluoromethyl 89, followed by regeneration of the aminoꢀfunctionality via
condensation with ammonium (ꢀ90). Core formation using previously described PPA conditions then
proceeded smoothly to provide alkyl chloride 91. Pyrazole installation (compound 92) and final Suzuki
crossꢀcoupling using intermediates 78 and 81 provided the final compounds 44 and 46.
Conclusions
Starting from an optimized HTS hit (1), 46 was identified as a potent, GluN2A selective, and BBB
penetrable NMDAR PAM suitable for in vivo characterization. During the course of this work we
identified the PAM binding site via Xꢀray crystallography at the dimer interface of the LBD and using
structureꢀbased design, we optimized both potency and kinetics of channel potentiation, characterizing a
variety of aromatic groups that can tune channel deavation kinetics. We also identified a subseries of
transꢀsubstituted cyclopropyls that provided superior potency within the thiazolopyrimidinone series of
PAMs. Upon adding a core trifluoromethyl, we successfully designed a GluN2Aꢀselective PAM suitable
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Journal of Medicinal Chemistry
for in vivo characterization. The results of the in vivo studies derived from 46 will be reported in due
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course.
Experimental Section
Chemistry
0
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General Methods. Unless otherwise indicated, all commercial reagents and anhydrous solvents were
used without additional purification. 1HꢀNMR spectra were measured on Bruker Avance III 300, 400, or
5
00 MHz spectrometers. 13CꢀNMR spectra were measured on a Bruker Avance III 125.80 MHz
spectrometer. Chemical shifts (in ppm) were referenced to internal standard tetramethylsilane (δ = 0
ppm). The reported carbon multiplicities and coupling constants are from CꢀF coupling. Highꢀ
resolution mass spectrometry of final compounds were performed on a Thermo UHPLC/QE with a
ThermoꢀQ Exactive mass spectrometry detector using ESI ionization, after elution on a Acquity BEH
C18 (2.1 mm × 50 mm; 1.7 ꢁm particle size) stationary phase using a gradient of water/acetonitrile (3–
9
7% over 7 min; 0.1% formic acid in both phases). Reactions were monitored by walkup Shimadzu
LCMS/UV system with LCꢀ30AD solvent pump, 2020 MS, Silꢀ30AC autosampler, SPDꢀM30A UV
detector, CTOꢀ20A column oven, using 2ꢀ98% acetonitrile/0.1% formic acid (or 0.01% Ammonia) over
2
.5 min OR Waters Acquity LCMS system using 2ꢀ98% acetonitrile/0.1% formic acid (or 0.1%
Ammonia) over 2 min. Flash column chromatography purifications were done on a Teledyne Isco
Combiflash Rf utilizing Silicycle HP columns. Reverseꢀphase purification was carried out on a
Phenomenex GeminiꢀNX C18 (30 x 100mm,5um) with a gradient of 5−95% acetonitrile/water (with
0
.1% Formic Acid or 0.1% NH4OH) over 10 min at 60 mL/min. Preparative SFC separations were
performed on a PIC Solutions instrument, with conditions indicated in the Experimental Section.
Analytical purity was greater than 95% as determined by LCMS using UV 254 nM detection unless
stated otherwise. The melting point was determined by Differential Scanning Calorimetry (DSC) (TA
InstrumentsꢀWaters L.L.C.) by using 5mg of solid sample and measuring the onset melting temperature.
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1
2
The syntheses of compounds 1-3 and 47 have been described previously.
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7
-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (4). The
title compound was prepared in a manner analogous to 15 (29% yield, 2 steps). HRMS (ESI) Calc for
+
1
C H FN OS (M+H) : 318.1071. Found: 318.1079. H NMR (400 MHz, DMSOꢀd ) δ 7.84 (d, J = 1.8
1
6
17
3
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Hz, 1H), 7.03 – 6.92 (m, 2H), 6.66 – 6.56 (m, 2H), 5.93 (s, 1H), 4.35 (s, 2H), 3.47 (q, J = 7.0 Hz, 2H),
2.42 (d, J = 1.4 Hz, 3H), 1.13 (t, J = 7.0 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-3-(pyridin-3-yl)-5H-thiazolo[3,2-a]pyrimidin-
5
-one (5). The title compound was prepared from bromide 48 in a manner analogous to 6 (52% yield).
+
1
HRMS (ESI) Calc for C H FN OS (M+H) : 395.1336. Found: 396.1347. H NMR (400 MHz, DMSOꢀ
2
1
20
4
d6) δ 8.57 (d, J = 5.0 Hz, 1H), 8.54 (d, J = 2.1 Hz, 1H), 7.78 (dd, J = 7.9, 1.9 Hz, 1H), 7.42 (dd, J = 7.9,
.0 Hz, 1H), 6.98 (t, J = 8.8 Hz, 2H), 6.62 (dd, J = 9.1, 4.4 Hz, 2H), 5.81 (s, 1H), 4.35 (s, 2H), 3.47 (q, J
= 7.0 Hz, 2H), 2.16 (s, 3H), 1.13 (t, J = 7.0 Hz, 3H).
5
7
-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-3-(pyrimidin-5-yl)-5H-thiazolo[3,2-
a]pyrimidin-5-one (6). To a solution of bromide 48 (50 mg, 0.13 mmol) in acetonitrile/H O (1.3/0.5
2
mL) was added (pyrimidinꢀ5ꢀyl) boronic acid (20.3 mg, 0.16 mmol), sodium carbonate (40.5 mg, 0.379
mmol) and [1,1’ꢀbis(diphenylphosphino)ferrocene]palladium(II) dichloride (9.7 mg, 0.013 mmol). The
o
reaction mixture was heated under microwave irradiation for 20 min at 120 C. After cooling to room
temperature, the resulting mixture was concentrated in vacuo. The residue was purified by silica gel
chromatography with dichloromethane/methanol (95/5) to afford the title compound (14.8 mg, 30.0 %)
+
1
as an offꢀwhite solid. HRMS (ESI) Calc for C H FN OS (M+H) : 396.1289. Found: 396.1303. H
2
0
19
5
NMR (400 MHz, DMSOꢀd ) δ 9.18 (s, 1H), 8.83 (s, 2H), 6.98 (t, J = 8.8 Hz, 2H), 6.62 (dd, J = 9.2, 4.4
6
Hz, 2H), 5.85 (s, 1H), 4.36 (s, 2H), 3.47 (q, J = 7.0 Hz, 2H), 2.22 (s, 3H), 1.13 (t, J = 7.0 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-3-morpholino-5H-thiazolo[3,2-a]pyrimidin-5-
one (7). To a solution of bromide 48 (250 mg, 0.63 mmol) in dimethyl sulfoxide (3 mL) was added
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Journal of Medicinal Chemistry
potassium phosphate (268 mg, 1.26 mmol), Lꢀproline (22.0 mg, 0.19 mmol), morpholine (165 mg, 1.89
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mmol) and cuprous iodide (18.0 mg, 0.09 mmol) and the reaction mixture was stirred overnight at 90
o
C. The resulting mixture was then quenched with water (50 mL), extracted with ethyl acetate (30mL x
3
), washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was
purified by chromatography with ethyl acetate/petroleum ether (1/2) to afford the title compound (29.5
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
mg, 12%) as a white solid. HRMS (ESI) Calc for C H FN O S (M+H) : 403.1599. Found: 403.1608.
2
0
24
4
2
1
H NMR (300 MHz, CDCl ) δ 6.93ꢀ6.87 (m, 2H), 6.61ꢀ6.57 (m, 2H), 6.15 (s, 1H), 4.29 (s, 2H), 3.91ꢀ
3
3
.87 (m, 2H), 3.76ꢀ3.59 (m, 4H), 3.50ꢀ3.43 (m, 2H), 2.64ꢀ2.60 (m, 2H), 2.40 (s, 3H), 1.25ꢀ1.20 (m, 3H).
7-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carbonitrile (8). To a solution of bromide 48 (1 g, 2.523 mmol) in DMF (5 mL) was added zinc
cyanide (605 mg, 5.05 mmol), tris(dibenzylideneacetone)dipalladium(0) (118 mg, 0.126 mmol), and
dppf (143 mg., 0.2523 mmol). The reaction mixture was then heated at 160°C overnight. The resulting
mixture was then quenched with water (500 mL), extracted with ethyl acetate (300 mL x 3), washed
with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by
silica gel chromatography using ethyl acetate/heptane (0ꢀ100% ethyl acetate gradient) to afford the title
+
compound (284 mg, 33%) as a white solid. HRMS (ESI) Calc for C H FN OS (M+H) : 343.1023.
1
7
16
4
1
Found: 343.1031. H NMR (400 MHz, DMSOꢀd ) δ 6.98 (t, J = 8.7 Hz, 2H), 6.62 (dd, J = 9.1, 4.4 Hz,
6
2H), 6.01 (s, 1H), 4.36 (s, 2H), 3.47 (q, J = 7.0 Hz, 2H), 2.60 (s, 3H), 1.13 (t, J = 6.9 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-3-(hydroxymethyl)-2-methyl-5H-thiazolo[3,2-
a]pyrimidin-5-one (9). To a solution of aldehyde 49 (100 mg, 0.29 mmol) in tetrahydrofuran (10 mL)
was added water (2 mL) and sodium borohydride (33 mg, 0.87 mmol). The reaction mixture was stirred
overnight at room temperature and then quenched with water (50 mL). The resulting solution was
extracted with ethyl acetate (30 mL x 3), washed with brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was purified by chromatography with 50% ethyl acetate in
petroleum ether to afford the title compound (60 mg, 60%) as an offꢀwhite solid. HRMS (ESI) Calc for
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Journal of Medicinal Chemistry
Page 18 of 66
+
1
C H FN O S (M+H) : 348.1177. Found: 348.1188. H NMR (300 MHz, CDCl ) δ 6.93ꢀ6.86 (m, 2H),
1
7
19
3
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
.59 ꢀ 6.54 (m, 2H), 6.26 (s, 1H), 4.75 ꢀ 4.73 (m, 2H), 4.49 ꢀ 4.44 (m, 1H), 4.32 (s, 2H), 3.48 (q, J = 6.9
Hz,2H), 2.44 (s, 3H), 1.23 (t, J = 6.9 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-3-(1-hydroxyethyl)-2-methyl-5H-thiazolo[3,2-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a]pyrimidin-5-one (10). To a solution of aldehyde 49 (200 mg, 0.58 mmol) in tetrahydrofuran (15 mL)
was added methylmagnesium bromide (1.3 mL, 0.5 mol/L). The resulting solution was stirred overnight
at room temperature. The reaction was then quenched by saturated ammonium chloride (20 mL),
extracted with dichloromethane (3 x 30 mL), washed with brine, dried over sodium sulfate and
concentrated in vacuo. The residue was purified by chromatography with ethyl acetate/petroleum ether
(1/2) to afford the title compound (67.5 mg, 31%) as an offꢀwhite solid. HRMS (ESI) Calc for
+
1
C H FN O S (M+H) : 362.1333. Found: 362.1341. H NMR (300 MHz, CD OD) δ 6.95ꢀ6.88 (m,
1
8
21
3
2
3
2H), 6.71ꢀ6.65 (m, 2H), 6.20 (s, 1H), 5.43ꢀ5.36 (m, 1H), 4.39 (s, 2H), 3.53 (q, J = 7.2 Hz, 2H), 2.50 (s,
H), 1.52 (d, J = 6.6 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H).
3
7
-((Ethyl(4-fluorophenyl)amino)methyl)-3-(2-hydroxypropan-2-yl)-2-methyl-5H-thiazolo[3,2-
a]pyrimidin-5-one (11). To a solution of ketone 18 (70 mg, 0.19 mmol) in tetrahydrofuran (15 mL) was
added methylmagnesium bromide in tetrahydrofuran (1 mol/L, 0.42 mL). The reaction was stirred for
4
8 h at room temperature and was then quenched by a saturated aqueous ammonium chloride solution
(20 mL). The resulting mixture was then extracted with dichloromethane (3 x 30 mL), washed with
brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by
chromatography with dichloromethane/methanol (50/1) to afford the title compound (40 mg, 52%) as a
+
1
yellow solid. HRMS (ESI) Calc for C H FN O S (M+H) : 376.1490. Found: 376.1501. H NMR (300
1
9
23
3
2
MHz, CD OD) δ6.95ꢀ6.89 (m, 2H), 6.70ꢀ 6.65 (m, 2H), 6.24 (s, 1H), 4.39 (s, 2H), 3.53 (q, J = 6.9 Hz,
3
2
H), 2.58 (s, 3H), 1.73 (s, 6H), 1.23 (t, J = 6.9 Hz, 3H).
7-((Ethyl(4-fluorophenyl)amino)methyl)-3-(2-hydroxyethyl)-2-methyl-5H-thiazolo[3,2-
a]pyrimidin-5-one (12). To a solution of enol ether 50 (250 mg, 0.65 mmol) in acetone was added 3 N
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Journal of Medicinal Chemistry
hydrogen chloride (15 mL). The resulting solution was refluxed for 3 h in an oil bath. The pH value of
the solution was then adjusted to pH 8 with a saturated aqueous sodium bicarbonate solution. The
mixture was extracted with dichloromethane (3 x 50 mL), washed with brine, dried over anhydrous
sodium sulfate and concentrated in vacuo to afford the title compound as a light yellow oil (180 mg).
The crude aldehyde product was used in the next step without further purification.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To a solution of the crude aldehyde (180 mg, 0.50 mmol) in methanol (10 mL) was added sodium
o
borohydride (40 mg, 1.06 mmol) at 0 C. After stirring overnight at room temperature, the reaction was
quenched by saturated aqueous ammonium chloride (20 mL). The resulting solution was extracted with
dichloromethane (3 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by chromatography with dichloromethane/methanol (30/1) to afford
the title compound (90 mg, 38% yield, 2 steps) as a white solid. HRMS (ESI) Calc for C H FN O S
1
8
21
3
2
+
1
(M+H) : 362.1333. Found: 362.1342. H NMR (300 MHz, CDCl ) δ 6.90ꢀ6.84 (m, 2H), 6.55ꢀ6.50 (m,
3
2
1
H), 6.14 (s, 1H), 4.26 (s, 2H), 3.89ꢀ3.85 (m, 2H), 3.47ꢀ3.39 (m, 4H), 2.95ꢀ2.91 (m, 1H), 2.32 (s, 3H),
.19 (t, J = 6.9 Hz, 3H).
7-((Ethyl(4-fluorophenyl)amino)methyl)-3-(3-hydroxypropyl)-2-methyl-5H-thiazolo[3,2-
a]pyrimidin-5-one (13). To a solution of enoate 51 (200 mg, 0.5 mmol) in methanol (10 mL) was added
0% palladium on carbon and the reaction solution was stirred 12 h at room temperature under a
1
hydrogen atmosphere (1.5 atm). After filtration the resulting solution was concentrated in vacuo to
afford the title compound (180 mg, 90%) as a light yellow solid. HRMS (ESI) Calc for C H FN O S
1
9
23
3
2
+
1
(
M+H) : 376.1490. Found: 376.1500. H NMR (300 MHz, CDCl ) δ 6.94ꢀ6.88 (m, 2H), 6.62ꢀ6.58 (m,
3
2H), 6.12 (s, 1H), 4.28 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 3.51ꢀ3.41 (m, 4H), 2.73 (t, J = 6.9 Hz, 2H),
2
.37 (s, 3H), 1.26ꢀ1.20 (m, 6H).
To a solution of ethyl ester from the previous step (180 mg, 0.43 mmol) in methanol (10 mL) was
o
added lithiumborohydride (20 mg, 0.91 mmol) at 0 C. The resulting solution was stirred for 5 h at
room temperature. The reaction was then quenched by a saturated aqueous ammonium chloride solution
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Page 20 of 66
(
20 mL), extracted with dichloromethane (3 x 50 mL), washed with brine, dried over anhydrous sodium
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sulfate, and then concentrated in vacuo. The residue was purified by silica gel chromatography with
dichloromethane/methanol (30/1) to afford the title compound (100 mg, 62%) as a light yellow solid.
+
1
LCMS, m/z = 376.0 [M+H] . HNMR (400 MHz, CDCl ) δ 6.94ꢀ6.90 (m, 2H), 6.63ꢀ6.59 (m, 2H), 6.15
3
(s, 1H), 4.30 (s, 2H), 3.67 (t, J = 5.6 Hz, 2H), 3.48 (q, J = 7.2 Hz, 2H), 3.33 (t, J = 7.2 Hz, 2H), 2.36 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3H), 1.90ꢀ1.95 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-3-isopropyl-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-
one (14). The title compound was prepared in a manner analogous to 15 (6% yield, 2 steps). LCMS, m/z
+
1
= 360.0 [M+H] . HNMR (300 MHz, CD OD) δ 6.95ꢀ6.86 (m, 2H), 6.19ꢀ6.69 (m, 2H), 6.05 (s, 1H),
3
4
6
.33 (s, 2H), 3.53 (q, J = 6.9 Hz, 2H), 2.44 (s, 3H), 2.10 (m, 1H), 1.40 (d, J = 7.2 Hz, 6H), 1.21 (t, J =
.9 Hz, 3H).
3-Cyclobutyl-7-((ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-
one (15). To a solution of alkyl chloride 60 (300 mg, 1.12 mmol) in acetonitrile (20 mL) was added
potassium iodide (93 mg, 0.56 mmol), potassium carbonate (309 mg, 2.24 mmol), and Nꢀethylꢀ4ꢀ
o
fluoroaniline (311 mg, 2.23 mmol). The resulting solution was stirred for 5 h at 70 C. After
concentrating in vacuo, the crude product was purified by PrepꢀHPLC with the following conditions
(Agilent 1200: Column, XꢀBrigde C18; mobile phase, 0.05% NH HCO in water and CH CN (CH CN
4 3 3 3
20% up to 60% in 15 min); Detector, UV254) to afford the title compound (36.1 mg, 8%) as a yellow
+
1
solid. HRMS (ESI) Calc for C H FN OS (M+H) : 372.1540. Found: 3372.1553. H NMR (300 MHz,
2
0
23
3
CD OD) δ6.93ꢀ6.86 (m, 2H), 6.69ꢀ6.62 (m, 2H), 6.01 (s, 1H), 4.44 ꢀ 4.35 (m, 1H), 4.31 (s, 2H), 3.51 (q,
3
J = 6.9 Hz, 2H), 2.48 (s, 3H), 2.45ꢀ2.35 (m, 3H), 1.89ꢀ1.890 (m, 1H), 2.06 ꢀ 1.94 (m, 1H), 1.21 (t, J =
6
.9 Hz, 3H).
3
-Cyclopropyl-7-((ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-
one (16). The title compound was prepared from bromide 48 in a manner analogous to 6 (31% yield).
+
1
HRMS (ESI) Calc for C H FN OS (M+H) : 358.1384. Found: 358.1393. H NMR (400 MHz, DMSOꢀ
1
9
21
3
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Journal of Medicinal Chemistry
d6) δ 6.98 (t, J = 8.7 Hz, 2H), 6.61 (dd, J = 9.2, 4.3 Hz, 2H), 5.82 (s, 1H), 4.29 (s, 2H), 3.46 (q, J = 7.0
Hz, 2H), 2.34 (s, 3H), 2.14 (p, J = 7.6 Hz, 1H), 1.12 (t, J = 7.0 Hz, 3H), 0.92 (t, J = 7.1 Hz, 2H), 0.66 (t,
J = 5.6 Hz, 2H).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
-((Ethyl(4-fluorophenyl)amino)methyl)-2-methyl-3-(prop-1-en-2-yl)-5H-thiazolo[3,2-
a]pyrimidin-5-one) (17). The title compound was prepared from bromide 48 in a manner analogous to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
6
6
2
(62% yield). LCMS, m/z = 358.1 [M+H] . H NMR (400 MHz, DMSOꢀd6) δ 6.98 (t, J = 8.6 Hz, 2H),
.61 (dd, J = 9.0, 4.3 Hz, 2H), 5.86 (s, 1H), 5.35 (s, 1H), 5.00 (s, 1H), 4.33 (s, 2H), 3.47 (q, J = 7.0 Hz,
H), 2.28 (s, 3H), 1.94 (s, 3H), 1.13 (t, J = 7.0 Hz, 3H).
3
-Acetyl-7-((ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one
(18). The title compound was prepared in a manner analogous to 15 (3% yield, 3 steps). HRMS (ESI)
+
1
Calc for C H FN O S (M+H) : 360.1177. Found: 360.1186. H NMR (300 MHz, CD OD) δ 6.95ꢀ6.89
1
8
19
3
2
3
(m, 2H), 6.70ꢀ6.65 (m, 2H), 6.16 (s, 1H), 4.40 (s, 2H), 3.53 (q, J = 7.4 Hz, 2H), 2.42 (s, 3H), 2.39 (s,
3
H), 1.23 (t, J = 7.4 Hz, 3H).
7
-((Ethyl(4-fluorophenyl)amino)methyl)-N,2-dimethyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (19). The title compound was prepared from chloride 66 in a manner analogous to 48
+
1
(
64% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 375.1286. Found: 375.1298. H NMR (300
18 20 4 2
MHz, DMSOꢀd ) δ 8.36ꢀ8.37 (m, 1H), 6.95ꢀ7.14 (m, 2H), 6.59ꢀ6.68 (m, 2H), 5.90 (s, 1H), 4.42 (s, 2H),
6
3
.35ꢀ3.48 (q, J = 6.9 Hz, 2H), 2.72 (d, J = 4.8 Hz, 3H), 2.30 (s, 3H), 1.12 (t, J = 6.9 Hz, 3H).
N-Ethyl-7-((ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-
-carboxamide (20). The title compound was prepared from chloride 67 in a manner analogous to 48
3
+
1
(80% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 389.1442. Found: 389.1451. HꢀNMR
1
9
22
4
2
(
400MHz, CDCl ) δ 6.93ꢀ6.85 (m, 2 H), 6.61ꢀ6.56 (m, 2 H), 6.18 (s, 1 H), 6.00ꢀ5.97 (m, 1H), 4.30 (s,
3
2
H), 3.55ꢀ3.42 (m, 4 H), 2.39 (s, 3H), 1.29ꢀ1.18 (m, 6 H).
N-Ethyl-7-(((4-fluorophenyl)(propyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-
a]pyrimidine-3-carboxamide (21). The title compound was prepared from chloride 67 in a manner
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Journal of Medicinal Chemistry
Page 22 of 66
+
analogous to 48 (71% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 403.1599. Found:
2
0
24
4
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
4
03.1609. H NMR (400 MHz, DMSOꢀd ) δ 8.42 (t, J = 5.6 Hz, 1H), 6.96 – 6.85 (m, 2H), 6.60 – 6.49
6
(m, 2H), 6.27 (t, J = 6.3 Hz, 1H), 6.10 (s, 1H), 4.14 (d, J = 6.3 Hz, 2H), 3.22 (qd, J = 7.2, 5.5 Hz, 2H),
2
.31 (s, 3H), 1.09 (t, J = 7.2 Hz, 3H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-Ethyl-7-(((4-fluorophenyl)(methyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-
a]pyrimidine-3-carboxamide (22). The title compound was prepared from chloride 67 in a manner
+
analogous to 48 (30% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 375.1286. Found:
1
8
20
4
2
1
3
75.1296. H NMR (400 MHz, DMSOꢀd ) δ 8.41 (t, J = 5.6 Hz, 1H), 7.06 – 6.94 (m, 2H), 6.73 – 6.61
6
(m, 2H), 5.87 (s, 1H), 4.41 (s, 2H), 3.21 (qd, J = 7.2, 5.5 Hz, 2H), 3.05 (s, 3H), 2.31 (s, 3H), 1.08 (t, J =
7
.2 Hz, 3H).
N-Ethyl-7-((4-fluorophenylamino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (23). The title compound was prepared from chloride 67 in a manner analogous to 48
+
1
(
38% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 361.1129. Found: 361.1139. H NMR (400
17 18 4 2
MHz, DMSOꢀd ) δ 8.42 (t, J = 5.6 Hz, 1H), 6.96 – 6.85 (m, 2H), 6.60 – 6.49 (m, 2H), 6.27 (t, J = 6.3
6
Hz, 1H), 6.10 (s, 1H), 4.14 (d, J = 6.3 Hz, 2H), 3.22 (qd, J = 7.2, 5.5 Hz, 2H), 2.31 (s, 3H), 1.09 (t, J =
7
.2 Hz, 3H).
N-Ethyl-7-((ethyl(5-fluoropyridin-2-yl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-
a]pyrimidine-3-carboxamide (24). The title compound was prepared from chloride 67 in a manner
+
analogous to 48 (24% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 390.1395. Found:
1
8
21
5
2
1
3
90.1402. H NMR (400 MHz, DMSOꢀd ) δ 8.40 (t, J = 5.5 Hz, 1H), 8.02 (d, J = 3.2 Hz, 1H), 6.69 –
6
6.61 (m, 1H), 5.85 (s, 1H), 4.54 (s, 2H), 3.58 (q, J = 6.9 Hz, 2H), 3.22 (qd, J = 7.3, 5.5 Hz, 2H), 2.31 (s,
3
H), 1.11 (dt, J = 20.4, 7.1 Hz, 6H).
N-Ethyl-7-((4-fluorophenoxy)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (25). The title compound was prepared from chloride 67 in a manner analogous to 48
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Journal of Medicinal Chemistry
+
1
(
22% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 362.0969. Found: 362.0980. H NMR (300
1
7
17
3
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MHz, CDCl ) δ 7.00ꢀ6.96 (m, 2H), 6.90ꢀ6.86 (m, 2H), 6.45 (s, 1H), 5.89 (s, 1H), 4.91 (s, 2H), 3.57ꢀ3.51
3
(m, 2H), 2.42 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H).
7
-(3-Cyanobenzyl)-N-ethyl-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-carboxamide
(26).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The title compound was prepared from chloride 67 in a manner analogous to 28 (31% yield). HRMS
+
1
(ESI) Calc for C H N O S (M+H) : 353.1067. Found: 353.1078. H NMR (400 MHz, CDCl ) δ 7.56ꢀ
1
8
17
4
2
3
7
3
.48 (m, 3H), 7.44ꢀ7.40 (m, 1H), 6.07 (s, 1H), 5.83 (s, 1H), 3.90 (s, 2H), 3.56ꢀ3.49 (m, 2H), 2.42 (s,
H), 1.28 (t, J = 7 Hz, 3H).
N-Ethyl-2-methyl-5-oxo-7-(3-(trifluoromethyl)benzyl)-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (27). The title compound was prepared from chloride 67 in a manner analogous to 28
+
1
(
20% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 396.0988. Found: 396.0996. H NMR (300
18 17 3 3 2
MHz, CDCl ) δ7.51ꢀ7.50 (m, 2H), 7.45ꢀ7.43 (m, 2H), 6.06 (s, 1H), 5.81ꢀ5.77 (m, 1H), 3.93 (s, 2H),
3
3
.57ꢀ3.48 (m, 2H), 2.41 (s, 3H), 1.28 (m, 3H).
7
-(3-Cyano-2-fluorobenzyl)-N-ethyl-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (28). To a solution of alkyl chloride 67 (500 mg, 1.75 mmol) in 1,4ꢀdioxane/H O (10/3
2
ml) was added sodium carbonate (370 mg, 3.49 mmol), Pd(dppf)Cl (127 mg, 0.170 mmol) and 3ꢀ
2
cyanoꢀ2ꢀfluorophenylboronic acid (578 mg, 3.50 mmol). The reaction mixture was then stirred for 30
o
min at 120 C. After concentration under vacuum, the residue was purified on silica gel column with
dichloromethane/methanol (50/1) to afford the title compound (0.56 g, 75%) as an offꢀwhite solid. mp
o
+
1
91 C. HRMS (ESI) Calc for C H O N FS (M+H) : 371.0973. Found: 371.0986. 1H NMR (500
18 16 2 4
MHz, DMSOꢀd6) δ 8.41 (t, J = 5.5 Hz, 1H), 7.85 (ddd, J = 1.8, 6.1, 7.7 Hz, 1H), 7.76 (ddd, J = 1.8, 7.7,
7
.7 Hz, 1H), 7.39 (dd, J = 7.7, 7.7 Hz, 1H), 6.14 (s, 1H), 4.01 (s, 2H), 3.23 (qd, J = 5.6, 7.2 Hz, 2H),
.30 (s, 3H), 1.10 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 162.23, 161.67, 160.65 (d, JCF =
2
254.8 Hz), 158.37, 157.04, 137.38 (d, JCF = 5.3 Hz), 132.48, 128.52, 126.20 (d, JCF = 14.3 Hz) 125.43
(d, JCF = 3.8 Hz), 122.58, 113.99, 104.50, 100.12 (d, JCF = 14.8 Hz), 35.13, 33.92, 14.11, 11.42.
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Journal of Medicinal Chemistry
Page 24 of 66
N-Ethyl-7-(2-fluoro-3-(trifluoromethyl)benzyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (29). The title compound was prepared from chloride 67 in a manner analogous to 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
+
(68% yield). mp 193 C. HRMS (ESI) Calc for C H O N F S (M+H) : 414.0894. Found: 414.0897.
1
8
16
2
3 4
1
H NMR (500 MHz, DMSOꢀd6) δ 8.41 (t, J = 5.6 Hz, 1H), 7.76 – 7.66 (m, 2H), 7.39 (dd, J = 7.7, 7.7
Hz, 1H), 6.12 (s, 1H), 4.02 (s, 2H), 3.23 (qd, J = 5.5, 7.2 Hz, 2H), 2.30 (s, 3H), 1.09 (t, J = 7.2 Hz, 3H).
3C NMR (126 MHz, DMSO) δ 162.49, 161.65, 158.39, 157.09 (d, JCF = 254.0 Hz), 157.05, 136.58
d, JCF = 4.5 Hz), 128.53, 126.65 (d, JCF = 14.6 Hz), 125.82 (q, JCF = 4.7 Hz), 124.80 (d, JCF = 4.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
Hz), 122.62 (q, JCF = 272.2 Hz), 122.53, 116.54 (dq, JCF = 12.5, 32.4 Hz), 104.44, 35.12 (d, JCF = 3.1
Hz), 33.92, 14.09, 11.41.
7
-(2-Chloro-5-cyanobenzyl)-N-ethyl-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (30). The title compound was prepared from chloride 67 in a manner analogous to 28 (9%
+
1
yield). LCMS, m/z = 386.7 [M+H] . H NMR (300 MHz, CDCl ) δ 7.59 (s, 1H), 7.50 (s, 2H), 6.05 (s,
3
1
H), 5.82 (m, 1H), 4.03 (s, 2H), 3.55ꢀ3.51 (m, 2H), 2.44 (s, 3H), 1.28 (t, J = 7.2Hz, 3H).
7
-(5-Cyano-2-cyclopropylbenzyl)-N-ethyl-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidine-3-
carboxamide (31). The title compound was prepared from chloride 67 in a manner analogous to 28
+
1
(
14% yield). HRMS (ESI) Calc for C H N O S (M+H) : 393.1380. Found: 393.1390. H NMR (300
21 21 4 2
MHz, CDCl ) δ 7.47ꢀ7.44 (m, 1H), 7.32ꢀ7.19 (m, 2H), 5.94 (s, 1H), 5.83ꢀ5.81 (m, 1H), 4.16 (s, 2H),
3
3
0
.56ꢀ3.52 (m, 2H), 2.41 (s, 3H), 1.92ꢀ1.82 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 1.02ꢀ0.96 (m, 2H), 0.70ꢀ
.65 (m, 2H).
N-Ethyl-7-(1-(2-fluoro-3-(trifluoromethyl)phenyl)ethyl)-2-methyl-5-oxo-5H-thiazolo[3,2-
a]pyrimidine-3-carboxamide (32). To a ꢀ78 °C solution of ethyl amide 29 (200 mg, 0.48 mmol) in
tetrahydrofuran (20 mL) was added nꢀbutyllithium (2.5 M in hexanes; 1 mL). After 30 min at ꢀ78 °C,
iodomethane (235 mg, 1.66 mmol) was added. The resulting solution was stirred for 3 h at room
temperature. The reaction was then quenched by water (20 mL), extracted with dichloromethane (3x30
mL), washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified
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Page 25 of 66
Journal of Medicinal Chemistry
by chromatography with dichloromethane/methanol (50:1) to afford the title compound (52.7 mg, 25%)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
as a white solid. HRMS (ESI) Calc for C H F N O S (M+H) : 428.1050. Found: 428.1059. HNMR
1
9
18
4
3
2
(300 MHz, CDCl ) δ 7.60ꢀ7.48 (m, 2H), 7.20ꢀ7.19 (m, 1H), 6.13 (s, 1H), 5.92 (bs, 1H), 4.38 (q, J = 7.2
3
Hz, 1H), 3.56ꢀ3.48 (m, 2H), 2.40 (s, 3H), 1.63 (d, J = 6.9 Hz, 3H), 1.28 (t, J = 7.2 Hz, 3H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-Ethyl-2-methyl-5-oxo-7-((3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-5H-thiazolo[3,2-
a]pyrimidine-3-carboxamide (33). The title compound was prepared from chloride 67 in a manner
+
analogous to 35 (63% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 386.0893. Found:
1
5
15
3
5
2
1
3
96.0904. H NMR (400 MHz, DMSOꢀd ) δ 8.39 (t, J = 5.6 Hz, 1H), 8.11 (dd, J = 2.3, 1.2 Hz, 1H),
6
6.80 (d, J = 2.4 Hz, 1H), 5.89 (s, 1H), 5.38 (s, 2H), 3.22 (qd, J = 7.2, 5.5 Hz, 2H), 2.31 (s, 3H), 1.18 –
1
.05 (m, 3H).
N-Ethyl-2-methyl-7-((5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-5-oxo-5H-
thiazolo[3,2-a]pyrimidine-3-carboxamide (34). The title compound was prepared from chloride 67 in
+
a manner analogous to 35 (15% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 400.1050.
1
6
17
3
5
2
1
Found: 400.1060. HNMR (300 MHz, CDCl ) δ 6.37 (s, 1H), 5.97 (bs, 1H), 5.93 (s, 1H), 5.18 (s, 2H),
3
3
.56ꢀ3.47 (m, 2H), 2.42 (s, 3H), 2.29 (s, 3H), 1.27 (t, J = 7 Hz, 3H).
7
-((5-Chloro-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-N-ethyl-2-methyl-5-oxo-5H-
thiazolo[3,2-a]pyrimidine-3-carboxamide (35). To a solution of chloride 67 (500 mg, 1.75 mmol) in
acetonitrile (50 mL) was added 5ꢀchloroꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole 71 (596 mg, 3.50 mmol),
potassium iodide (146 mg, 0.88 mmol) and potassium carbonate (484 mg, 3.50 mmol). The resulting
o
mixture was stirred for 4 h at 80 C, and then cooled to room temperature. The resulting mixture was
washed with brine (30 mL) and the solution was extracted with dichloromethane (3 x 50 mL) and
concentrated in vacuo. The residue was applied onto a prepꢀTLC with ethyl acetate/petroleum ether (2:1)
o
to afford the title compound (205 mg, 28%) as a white solid. mp 214 C. HRMS (ESI) Calc for
+
C H O N ClF S (M+H) : 420.0503. Found: 420.0521. 1H NMR (500 MHz, DMSOꢀd6) δ 8.44 (t, J =
1
5
14
2
5
3
5
.6 Hz, 1H), 7.16 (s, 1H), 5.91 (s, 1H), 5.43 (s, 2H), 3.23 (dq, J = 7.2, 5.6 Hz, 2H), 2.31 (s, 3H), 1.09 (t,
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Journal of Medicinal Chemistry
Page 26 of 66
J = 7.2 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 162.14, 158.53, 158.26, 156.83, 140.91 (q, JCF = 38.2
Hz), 129.53, 128.61, 123.18, 120.56 (q, JCF = 268.7 Hz), 104.15, 103.17, 52.93, 33.93, 14.08, 11.46.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7-((5-Cyclopropyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-N-ethyl-2-methyl-5-oxo-5H-
thiazolo[3,2-a]pyrimidine-3-carboxamide (36). The title compound was prepared from chloride 67 in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
a manner analogous to 35 (32% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 426.1206.
1
8
19
3
5
2
1
Found: 426.1217. H NMR (300 MHz, CDCl ) δ 6.17 (s, 1H), 5.84ꢀ5.81 (m, 1H), 5.66 (s, 1H), 5.32 (s,
3
2
0
H), 3.54ꢀ3.49 (m, 2H), 2.43 (s, 3H), 1.68ꢀ1.63 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 1.02ꢀ1.00 (m, 2H),
.75ꢀ0.73 (m, 2H).
7-((5-Ethoxy-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-N-ethyl-2-methyl-5-oxo-5H-
thiazolo[3,2-a]pyrimidine-3-carboxamide (37). The title compound was prepared from chloride 67 in
+
a manner analogous to 35 (15% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 430.1155.
1
7
19
3
5
3
1
Found: 430.1167. H NMR (400 MHz, DMSOꢀd ) δ 8.41 (t, J = 5.6 Hz, 1H), 6.32 (s, 1H), 5.70 (s, 1H),
6
5
.12 (s, 2H), 4.24 (q, J = 7.0 Hz, 2H), 3.22 (qd, J = 7.3, 5.4 Hz, 2H), 2.32 (s, 3H), 1.31 (t, J = 7 Hz, 3H),
.09 (t, J = 7 Hz, 3H).
1
N-Ethyl-7-((5-isobutoxy-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-2-methyl-5-oxo-5H-
thiazolo[3,2-a]pyrimidine-3-carboxamide (38). The title compound was prepared from chloride 67 in
+
a manner analogous to 35 (26% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 458.1468.
1
9
23
3
5
2
1
Found: 458.1483. H NMR (400 MHz, DMSOꢀd ) δ 8.41 (t, J = 6 Hz, 1H), 6.31 (s, 1H), 5.76 (s, 1H),
6
5
.13 (s, 2H), 3.96 (d, J = 7 Hz, 2H), 3.22 (qd, J = 8, 6 Hz, 2H), 2.31 (s, 3H), 1.99 (dq, J = 13.3, 7 Hz,
H), 1.09 (t, J = 7 Hz, 3H), 0.91 (d, J = 7 Hz, 6H).
1
7-((Ethyl(4-fluorophenyl)amino)methyl)-3-((1R,2R)-2-(hydroxymethyl)cyclopropyl)-2-methyl-
5
H-thiazolo[3,2-a]pyrimidin-5-one (39). The title compound was prepared from bromide 48 in a
+
manner analogous to 40 (35% yield). HRMS (ESI) Calc for C H FN O S (M+H) : 388.1490. Found:
20 23
3
2
1
3
88.1500. H NMR (300 MHz, CDCl ) δ 7.00 (d, J = 6.9 Hz, 4H), 6.30 (s, 1H), 4.35 (s, 2H), 4.06 (dd,
3
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Journal of Medicinal Chemistry
J1 = 6.6 Hz, J2 = 11.4 Hz, 1H), 3.57 (q, J = 7.2 Hz, 2H), 3.13 (t, J = 10.6 Hz, 1H), 2.41 (s, 3H), 2.22ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.30 (m, 1H), 1.27ꢀ1.31 (m, 4H), 0.99ꢀ1.09 (m, 2H).
7-((4-Fluorophenoxy)methyl)-3-((1R,2R)-2-(hydroxymethyl)cyclopropyl)-2-methyl-5H-
thiazolo[3,2-a]pyrimidin-5-one (40). To a mixture of bromide 82 (500 mg, 1.35 mmol) in 1,4ꢀ
dioxane/water (9mL/3mL) was added trifluoroborate salt 78 (480 mg, 2.70 mmol), [1,1'ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
bis(diphenylphosphino)ferrocene]palladium(II) dichloride (200 mg, 0.27 mmol) and sodium carbonate
o
(
290 mg, 2.70 mmol). The reaction mixture was irradiated with microwave radiation for 1.5 h at 120 C.
The resulting mixture was concentrated in vacuo. The residue was purified by flash chromatography on
silica gel eluting with ethyl acetate/petroleum ether (1/2) to afford the racemic product (150 mg, 30%).
The racemic product was then separated with SFC with the following conditions (Column: Lux 5u
Celluloseꢀ3, 2*25 cm; Mobile Phase: CO : EtOH (0.2% DEA) =65:35; Flow rate: 45 g /min; detector:
2
220 nm) to afford two enantiomers.
Enantiomer 1 (40) (Peak 1, 68 mg, 14%, white solid) Retention time: 9.93 min. [α] −96.31 (c. 0.3, in
D
+
CH Cl ); HRMS (ESI) Calc for C H FN O S (M+H) : 361.1017. Found: 361.1029. 1H NMR (500
2
2
18 18
2
3
MHz, DMSOꢀd6) δ 7.18 – 7.11 (m, 2H), 7.08 – 7.02 (m, 2H), 6.21 (s, 1H), 4.94 (s, 2H), 4.59 (t, J = 5.6
Hz, 1H), 3.47 (t, J = 5.8 Hz, 2H), 2.38 (d, J = 1.5 Hz, 3H), 2.06 – 1.98 (m, 1H), 1.33 – 1.23 (m, 1H),
0
.92 – 0.80 (m, 2H). 13C NMR (126 MHz, DMSO) δ 162.10, 160.29, 159.48, 156.67 (d, JCF = 236.9
Hz), 153.90, 133.61, 120.35, 115.96 (d, JCF = 5.4 Hz), 115.83 (d, JCF = 19.3 Hz), 103.23, 68.51, 63.27,
2
2.96, 14.77, 12.72, 12.11.
Enantiomer 2 (Peak 2, 60 mg, 13%, white solid) Retention time: 11.06 min. LCMS, m/z = 361.0
+
1
[M+H] . H NMR (300 MHz, CDCl ) δ 7.02ꢀ6.89 (m, 4H), 6.47 (s, 1H), 4.91 (s, 2H), 4.08ꢀ4.03 (m,
3
1
H), 3.12 (t, J = 10, 1 Hz, 1H), 2.40 (s, 3H), 2.30ꢀ2.28 (m, 1H), 1.30ꢀ1.25 (m, 1H), 1.07ꢀ1.01 (m, 2H).
2
-Fluoro-3-((3-(2-(hydroxymethyl)cyclopropyl)-2-methyl-5-oxo-5H-thiazolo[3,2-a]pyrimidin-7-
yl)methyl)benzonitrile (41). The title compound was prepared from bromide 83 in a manner analogous
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 66
+
1
to 39 (10% yield). LCMS, m/z = 370.0 [M+H] . H NMR (300 MHz, CDCl ) δ 7.64ꢀ7.57 (m, 2H), 7.29ꢀ
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.24 (m, 1H), 6.07 (s, 1H), 4.07 (dd, J = 3.6, 11.4 Hz, 1H), 3.99 (s, 2H), 3.15 (dd, J = 9.6, 11.4 Hz, 1H),
2.41 (s, 3H), 2.32ꢀ2.26 (m, 1H), 1.29ꢀ1.24 (m, 1H), 1.06ꢀ0.99 (m, 2H).
7
-(2-Fluoro-3-(trifluoromethyl)benzyl)-3-(2-(hydroxymethyl)cyclopropyl)-2-methyl-5H-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
thiazolo[3,2-a]pyrimidin-5-one (42). The title compound was prepared from bromide 84 in a manner
+
analogous to 39 (11% yield). HRMS (ESI) Calc for C H F N O S (M+H) : 413.0941. Found:
1
9
17
4
2
2
1
4
13.0950. H NMR(400MHz, DMSOꢀd ) δ 7.76 – 7.64 (m, 2H), 7.38 (t,J = 7.8Hz, 1H), 6.02 (s, 1H),
6
4
.58 –4.51 (m, 1H), 3.96 (s, 2H), 3.50 – 3.40 (m, 5H), 2.35 (s, 3H), 2.00 (dtd,J = 8.6, 5.5, 1.8Hz, 1H),
1.33– 1.20 (m, 1H), 0.93 – 0.78 (m, 2H).
7
-((5-Chloro-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-3-((1R,2R)-2-
hydroxymethyl)cyclopropyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (43). The title compound
was prepared from bromide 85 in a manner analogous to 39 (7% yield). HRMS (ESI) Calc for
(
+
1
C H F N ClO S (M+H) : 419.0551. Found: 419.0565. HNMR (300 MHz, CDCl ) δ 6.59 (s, 1H),
1
6
15
3
4
2
3
5
.70 (s, 1H), 5.27 (s, 2H), 4.03 (dd, J = 3.6, 11.7 Hz, 1H), 3.11 (t, J = 9.9 Hz, 2H), 2.39 (s, 3H) 2.30ꢀ
2.22 (m, 1H) 1.28ꢀ1.24 (m, 1H), 1.03ꢀ0.97 (m, 2H).
7
-((5-Chloro-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-3-((1R,2R)-2-
hydroxymethyl)cyclopropyl)-2-(trifluoromethyl)-5H-thiazolo[3,2-a]pyrimidin-5-one (44). The title
compound was prepared from bromide 92 in a manner analogous to 39 (7% yield). HRMS (ESI) Calc
(
+
1
for C H F N ClO S (M+H) : 473.0268. Found: 473.0284. HNMR (300 MHz, CDCl ) δ 6.60 (s, 1H),
1
6
12
6
4
2
3
5
.81 (s, 1H), 5.28 (s, 1H), 3.97 (dd, J = 3.3, 12 Hz, 1H), 3.23 (dd, J = 8.4, 11.4 Hz, 1H), 2.41ꢀ2.39 (m,
1H), 1.42ꢀ1.33 (m, 2 H), 1.10ꢀ1.06 (m, 1H).
(1R,2R)-2-(7-((5-Chloro-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-2-methyl-5-oxo-5H-
thiazolo[3,2-a]pyrimidin-3-yl)cyclopropanecarbonitrile (45). The title compound was prepared from
+
1
bromide 85 in a manner analogous to 46 (38% yield). LCMS, m/z = 422 [M+H] . HNMR (400 MHz,
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Page 29 of 66
Journal of Medicinal Chemistry
CD3OD) δ 6.82 (s, 1H), 5.89 (s, 1H), 5.37 (s, 2H), 2.96ꢀ2.91 (m, 1H), 2.45 (s, 3H), 2.00ꢀ1.95 (m, 1H)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.81ꢀ1.76 (m, 1H), 1.60ꢀ1.55 (m, 1H).
(1R,2R)-2-(7-((5-Chloro-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)-5-oxo-2-(trifluoromethyl)-
5
H-thiazolo[3,2-a]pyrimidin-3-yl)cyclopropanecarbonitrile (46). To a mixture of bromide 92 (350
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mg, 0.73 mmol) in 1,4ꢀdioxane/water (15 mL/1.5 mL) was added trifluoroborate salt 81 (350 mg, 2.02
mmol), Pd(dppf)Cl (120 mg, 0.16 mmol) and sodium carbonate (350 mg, 3.30 mmol), and the resulting
2
o
mixture was stirred overnight at 85 C. The resulting mixture was filtered and concentrated in vacuo. The
residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1/2). Then the
racemic product was separated by PrepꢀChiral HPLC with the following conditions (Column:
CHIRALPAK ICꢀ3, 0.45*5 cm, 3 um; Mobile Phase: Hex:EtOH=50:50; Flow rate: 1.0 mL/min;
detector: 254 nm) to afford two enantiomers.
Enantiomer 1 (46) (Peak 1, 34.5 mg, 10%, white solid) Retention time: 1.461 min; [α] −586.46 (c.
D
o
+
0
4
1
.3, in CH Cl ); mp 137 C. HRMS (ESI) Calc for C H F N ClOS (M+H) : 468.0115. Found:
2 2 16 9 6 5
68.0133. 1H NMR (500 MHz, DMSOꢀd6) δ 7.16 (s, 1H), 6.05 (s, 1H), 5.43 (s, 2H), 3.08 – 3.01 (m,
H), 2.33 (dt, J = 5.8, 9.1 Hz, 1H), 1.85 (dt, J = 5.7, 8.9 Hz, 1H), 1.49 (dt, J = 5.9, 8.8 Hz, 1H). 13C
NMR (126 MHz, DMSO) δ 161.38, 158.98, 158.87, 141.02 (q, JCF = 38.2 Hz), 139.08 (q, JCF = 4.5
Hz), 129.65, 121.01 (q, JCF = 270.6 Hz), 120.54 (q, JCF = 268.8 Hz), 120.44, 113.14 (q, JCF = 39.2
Hz), 105.30, 104.15, 52.53, 18.26, 15.00 (q, JCF = 2.6 Hz), 5.97.
Enantiomer 2 (Peak 2, 34.5 mg, 10%, white solid) Retention time: 2.383 min; HRMS (ESI) Calc for
+
1
C H F N ClOS (M+H) : 468.0115. Found: 468.0134. HNMR (400 MHz, CDCl ) δ 6.60 (s, 1H), 5.81
1
6
9
6
5
3
(s, 1H), 5.27 (s, 2H), 3.11ꢀ3.00 (m, 1H), 1.91ꢀ1.80 (m, 2H), 1.65ꢀ1.62 (m, 1H).
-Bromo-7-((ethyl(4-fluorophenyl)amino)methyl)-2-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one
3
1
2
(
48). A mixture of alkyl chloride 47 (30 g, 0.1 mol), Nꢀethylꢀ4ꢀfluoroaniline (18.5 g, 0.13 mol),
potassium carbonate (28.2 g, 0.2 mol) and sodium iodide (7.66 g, 0.05 mol) in acetonitrile was heated
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