Dihydrothiazolopyridone derivatives as a novel family of positive allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor

Article

pubs.acs.org/jmc

Dihydrothiazolopyridone Derivatives as a Novel Family of Positive

Allosteric Modulators of the Metabotropic Glutamate 5 (mGlu₅)

Receptor

Jose Manuel Bartolome-Nebreda,*^,†Susana Conde-Ceide,^†Francisca Delgado,^†Laura Iturrino,^¶

́

†

‡

́

Joaquín Pastor, Miguel Angel Pena, Andres A. Trabanco, Gary Tresadern, Carola M. Wassvik,

́

Shaun R. Stauﬀer,^§,∥,⊥,#Satyawan Jadhav,^§,∥Kiran Gogi,^§,∥Paige N. Vinson,^§,∥Meredith J. Noetzel,^§,∥

Emily Days,^§,△C. David Weaver,^§,△Craig W. Lindsley,^§,∥,⊥,#Colleen M. Niswender,^§,∥,⊥

Carrie K. Jones,^§,∥,⊥P. Jeﬀrey Conn,^§,∥,⊥Frederik Rombouts,^○Hilde Lavreysen,^●Gregor J. Macdonald,^○

Claire Mackie,^▲and Thomas Steckler^●

¶

‡

^†Neuroscience Medicinal Chemistry, CREATe Analytical Sciences, and CREATe Molecular Informatics, Janssen Research and

Development, Jarama 75, 45007 Toledo, Spain

∥

^§Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center,

Nashville, Tennessee 37232, United States

^⊥Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, Tennessee 37232, United States

^#Department of Chemistry and ^△Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232,

United States

^○Neuroscience Medicinal Chemistry, ^●Neuroscience Biology, and ^▲CREATe Discovery ADME/Tox, Janssen Research and

Development, Turnhoutseweg 30, B-2340, Beerse, Belgium

S

* Supporting Information

ABSTRACT: Starting from a singleton chromanone high throughput

screening (HTS) hit, we describe a focused medicinal chemistry

optimization eﬀort leading to the identiﬁcation of a novel series of

phenoxymethyl-dihydrothiazolopyridone derivatives as selective positive

allosteric modulators (PAMs) of the metabotropic glutamate 5 (mGlu₅)

receptor. These dihydrothiazolopyridones potentiate receptor responses in

recombinant systems. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) evaluation allowed us to select

compound 16a for its assessment in a preclinical animal screen of possible antipsychotic activity. 16a was able to reverse

amphetamine-induced hyperlocomotion in rats in a dose-dependent manner without showing any signiﬁcant motor impairment

or overt neurological side eﬀects at comparable doses. Evolution of our medicinal chemistry program, structure activity, and

properties relationships (SAR and SPR) analysis as well as a detailed proﬁle for optimized mGlu₅receptor PAM 16a are

described.

involve direct interaction with dopaminergic receptors have

been proposed and investigated.^2,3The ultimate hope is that

diﬀerent biological pathways may lead to eﬀective antipsychotic

medications with both increased eﬃcacy versus all three core

symptom domains and improved tolerability due to the lack of

direct D₂receptor blockade.

One of the proposed pathophysiological causes that may lead

to schizophrenia is a compromised function of N-methyl-^D-

aspartate (NMDA) receptors. This NMDA receptor hypo-

function triggers a cascade of events, i.e., reduced activation of

inhibitory γ-aminobutyric acidergic (GABAergic) interneurons

in thalamus and cortex with a consequent disinhibition of

excitatory glutamatergic pathways.⁴On the basis of these

INTRODUCTION

■

Schizophrenia is a mental disorder that aﬀects approximately

1% of the world population.¹This severely limiting illness is

characterized by a combination of so-called positive (e.g.,

hallucinations and delusions), negative (e.g., anhedonia and

poverty of speech), and cognitive symptoms.¹Currently

available therapies, “typical” and “atypical” antipsychotics, rely

on dopamine 2 (D₂) receptor antagonism to exert their action,

and, despite being highly eﬃcacious in addressing positive

symptoms, they are of limited value for the treatment of the

other core symptoms of the disease. Furthermore, their clinical

use is associated with a plethora of severe side-eﬀects (e.g.,

Parkinson-like extrapyramidal symptoms (EPS), prolactin

release, weight gain, or cardiac risk), and as such, patient

compliance is extremely poor. As a result, during the past 10−

15 years, several alternative mechanisms of action that do not

Received: May 2, 2013

Published: August 15, 2013

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Figure 1. mGlu₅receptor PAMs with reported eﬃcacy in preclinical models of schizophrenia and cognition.

observations, it has been proposed that compounds that

enhance NMDA receptor function may have beneﬁcial eﬀects

in schizophrenia. Unfortunately, direct agonists of the NMDA

receptor tend to be neurotoxic; hence, alternative strategies to

increase NMDA receptor function have been suggested. A

desired increase in NMDA receptor function may be obtained

through an indirect modulation of NMDA receptors via

metabotropic glutamate (mGlu) receptors, especially mGlu₅

receptors.²The mGlu₅receptor belongs to the Group I mGlu

receptor subfamily, together with the mGlu₁receptor.⁵They

activate phospholipase C (PLC) via coupling to G_qproteins

and are ubiquitously expressed on postsynaptic excitatory

terminals in limbic brain regions that are involved in

motivational, emotional, and cognitive processes.⁶At GABA-

ergic interneurons, mGlu₅and NMDA receptors are coex-

pressed⁷and are not only coupled via intracellular signaling

pathways but also physically through scaﬀolding proteins.⁸In

summary, mGlu₅receptor-mediated activation of NMDA

receptor functioning has recently emerged as a promising

non-dopamine based approach for potential therapeutic

intervention of schizophrenia.²

Taking into account the challenges associated with the

development of orthosteric agonists for mGlu receptors (e.g.,

poor druglike properties, elusive selectivity, or potential

tolerance development), current strategies have mainly focused

on the identiﬁcation of positive allosteric modulators (PAMs).⁹

This approach has already proven useful, and in vivo activity in

diﬀerent preclinical schizophrenia and cognition models has

been reported for early prototypical mGlu₅PAMs such as 3-

cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB,

1)^10,11or S-(4-ﬂuoro-phenyl)-{3-[3-(4-ﬂuoro-phenyl)-[1,2,4]-

oxadiazol-5-yl]-piperidin-1-yl}-methanone (ADX47273, 2)¹²

and more recently for novel structurally distinct chemical

series including N-cyclobutyl-6-((3-ﬂuorophenyl)ethynyl)-

nicotinamide (VU0360172, 3),¹³N-(1-methylethyl)-5-(pyri-

din-4-ylethynyl)pyridine-2-carboxamide (LSN2463359, 4),^14,15

4-butoxy-N-(2,4-diﬂuor-ophenyl)benzamide (VU0357121,

5),¹⁶2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile

(VU0364289, 6),¹⁷(1-(4-(2-chloro-4-ﬂuorophenyl)piperazin-

1-yl)-2-(pyridin-4-ylmethoxy)eth-anone (CPPZ, 7),¹⁸and

(7S)-3-tert-butyl-7-[3-(4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl]-

5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine (LSN2814617,

8).¹⁵These novel PAM classes represent a signiﬁcant evolution

from the ﬁrst generation as they oﬀer improved drug-likeliness

(e.g., improved solubility, unbound fraction in brain or oral

bioavailability and in vivo eﬃcacy). Although the preclinical

biological data clearly suggest the potential of mGlu₅receptor

PAMs as a novel class of antipsychotic agents, the identiﬁcation

of a compound suitable for clinical proof-of-concept studies still

remains a challenge and will be crucial to clarify the potential

role of mGlu₅receptor PAMs as improved antipsychotics.

RESULTS

■

Given the promise oﬀered by mGlu₅receptor PAMs as a

potential novel treatment for schizophrenia, we performed an

HTS campaign looking for for starting points for a medicinal

chemistry program. The in vitro mGlu₅PAM calcium

mobilization assay provides two measures of compound

activity: pEC₅₀, which is deﬁned as the negative logarithm of

the concentration at which half-maximal potentiation of

glutamate is reached, and the maximal increase in observed

glutamate response, the % Glu Max. Screening of the Janssen

corporate library in a cell line expressing the human mGlu₅

receptor with this functional calcium mobilization assay

resulted in the identiﬁcation of the subsequently reported

chromanone 9,¹⁹as a promising hit. Compound 9 possessed

potent in vitro mGlu₅PAM activity (EC₅₀= 128 nM, Glu Max

= 82%). As a ﬁrst step, and since molecule 9 contains labile

substructures such as the ester group, we veriﬁed that the

products of the ester hydrolysis, benzoic acid and 6-hydroxy-

chromanone, were not responsible for the mGlu₅PAM activity

of the HTS sample of 9. Both compounds were found to be

inactive under the same assay conditions (EC₅₀> 10 μM),

while quality control of the sample 9 was good (LCMS purity >

95%), providing conﬁdence in the HTS in vitro data.

Furthermore, 9 showed satisfactory selectivity²⁰versus other

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representative members of the mGlu family (mGlu_1,2,8EC₅₀

>

moieties have recently been reported for other mGlu₅PAM

chemotypes.^19,23

10 μM). Unfortunately, and as anticipated from the presence of

the carboxylic ester functionality, 9 was found to be completely

hydrolyzed in rat plasma, which precluded any further work for

its in vivo pharmacological characterization. Nevertheless,

compound 9 was considered as a good starting point for

optimization due to its low molecular weight, moderate

lipophilicity (clogP = 3.3), high ligand eﬃciency²¹(LE =

0.34), and promising selectivity.

For the second prong of our strategy, chromanone

replacements were sought using diﬀerent approaches. It is

now becoming clear in the literature that some of the most

potent in vitro mGlu₅receptor PAMs have a common

phenylacetylene moiety.⁹For comparative purposes, we

synthesized the corresponding phenylacetylene chromanone

analogue 12¹⁹which was also found to be an mGlu₅receptor

PAM (EC₅₀= 682 nM, Glu Max 84%) although with an

approximately 5-fold decrease in activity compared to the initial

HTS hit 9. Following this result, we decided to keep the

acetylenic spacer constant and combine it with diﬀerent bicyclic

systems with the underlying rationale that this could increase

the ability to sample and identify chromanone alternatives. To

prioritize among the multiple existing possibilities, our design

principle focused on bicyclic scaﬀolds possessing hydrogen

bond acceptors that could mimic those present in the

chromanone core. This strategy proved fruitful as among the

initial set of compounds synthesized, dihydrobenzothiazolone

derivative 13²⁴emerged as a highly potent (∼21 fold potency

increase vs 12), eﬃcacious and selective mGlu₅receptor PAM

(EC₅₀= 33 nM, 71% Glu Max, mGlu_1−4,6−8> 10 μM). Despite

its high potency and promising metabolic stability in human

liver microsomes (55% remaining after 15 min incubation), 13

was found to be highly unstable in rat liver microsomes (2%

remaining after 15 min incubation), likely due to the presence

of the exposed keto-group. Furthermore, 13 was poorly soluble

in aqueous buﬀer (30 μM, pH 7.4), and it could not be

formulated in our standard carrier for in vivo testing [<0.25

mg/mL in 20% hydroxypropyl-β-cyclodextrin (HP-β-CD)

formulation in water]. Therefore, we focused eﬀorts on the

identiﬁcation of metabolically stable analogues of 13 bearing a

carbonyl function less prone to metabolism. For this purpose,

we considered the conversion of the dihydrobenzothiazolone of

13 into a dihydrothiazolopyridone scaﬀold. Despite an

approximate 4-fold decrease in potency with respect to 13,

As seen in Figure 2, our strategy for the exploration of the

chromanone 9 followed two parallel approaches, ﬁrst ﬁxing the

compound 14a showed comparable mGlu₅potency (EC₅₀

=

123 nM, 79% Glu Max) to HTS hit 9. In addition, 14a was

selective versus the other mGlu receptors at the screening

concentration of 10 μM. Importantly, 14a showed a remarkably

reduced turnover in both human and rat liver microsomes

(85% and 63% respectively remaining after 15 min incubation

at 1 μM concentration). Furthermore, the amide functionality

present in the dihydrothiazolopyridone core of 14a represents

an attractive chemical handle amenable for further derivatiza-

tion for structure−activity relationship (SAR) expansion

purposes. A preliminary evaluation of tertiary amides showed

that the introduction of alkyl substituents could lead to a

comparable activity in the case of the methyl derivative 14b

(EC₅₀= 193 nM, 57% Glu Max) or the methoxyethyl

substituted example 14c (EC₅₀= 119 nM, 47% Glu Max)

although with a drop of Glu Max in both examples.

Figure 2. Parallel simultaneous evolution from HTS hit 9 to optimized

series 15 and 16.

chromanone scaﬀold while performing an extensive search for

replacements for the labile ester group (A) and, in addition,

seeking to replace and optimize the chromanone bicycle as it

also contains a ketone moiety, a potential metabolic hot spot

and biologically reactive group (B).

At this point, having accomplished our initial goal of

identifying separate replacements for the ester and keto

functions, we decided to combine both approaches and target

the synthesis of the corresponding series of benzyloxy- and

phenoxymethyl-dihydrothiazolopyridone combinations 15 and

16, respectively (Table 1).²⁵

Chemistry. Compound 13 was readily prepared in two

steps (39% overall yield), via Sandmeyer reaction and

subsequent Sonogashira cross-coupling, from 2-amino-benzo-

thiazolone 17²⁶(Scheme 1).

Approach A involved the synthesis and evaluation of several

libraries of diverse potential ester surrogates including, among

others, alkyls, amides, amines, heterocycles, and ethers as

alternate spacers between the chromanone core and the distal

phenyl ring.²²Among this set of linkers, only previously

published benzyloxy and phenoxymethyl derivatives 10 and

11¹⁹retained signiﬁcant mGlu₅receptor PAM activity (EC₅₀

=

950 nM, 76% Glu Max and EC₅₀= 2.14 μM, 94% Glu Max

respectively) and promising selectivity versus the other mGlu

receptor subtypes (mGlu_1−4,6−8EC₅₀> 10 μM). Similar

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Table 1. Structures and Activities of Substituted Benzyloxy- and Phenoxymethyl-dihydrothiazolopyridones 15 and 16

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Table 1. continued

a

Values were calculated from three independent experiments using a four-parameter logistic nonlinear regression model taking into account the

b

heterogeneity between experiments and concentration−response curves for each compound. HLM and RLM data refer to % of compound

metabolized after incubation of tested compound with human and rat microsomes respectively, for 15 min at 1 μM concentration. Calculated with

Biobyte software. Not tested. Data obtained from a single experiment not replicated.

c

d

e

a

following an analogous procedure to the one described for

Scheme 1

the synthesis of compound 13 in Scheme 1. Finally, reaction of

14a with the corresponding alkyl halides in the presence of

cesium carbonate as base, yielded derivatives 14b,c (19 and

20% yield, respectively).

The synthetic methods used for the preparation of the ﬁnal

compounds 15b,c and 16a−q are outlined in Schemes 3−9.

Preparation of most dihydrothiazolopyridones 15−16 followed

a cyclization step performed with a piperidine-2,4-dione, which

was commercially available or was prepared using a synthetic

sequence shown in Scheme 3. Thus, commercially available β-

amino esters 22a−e were reacted with ethyl malonyl chloride

to aﬀord the amides 23a−e in moderate to good yields (22−

70%). Intramolecular cyclization reaction of 23a−e, promoted

by sodium ethoxide, yielded the corresponding 3-carbethox-

ypiperidine-2,4-dione derivatives 24a−e. Acid mediated de-

carboxylation of 24a−e followed by regioselective bromination

aﬀorded the key intermediates 25a−e in good yields. These

intermediates were found to be highly unstable and had to be

used immediately after synthesis.

a

Reagents and conditions: (a) Isoamyl nitrite, CuBr₂, CH₃CN, 80 °C,

1 h, 75%; (b) phenyl acetylene, [1,1′-bis(diphenylphospino)-

ferrocene]dichloro-palladium(II), CuI, Et₃N, 1,4-dioxane, reﬂux, 2 h,

52%.

The synthesis of the targeted compounds 14a−c is shown in

Scheme 2. Starting from commercially available Boc-protected

2,4-dioxopiperidine 19, bromination and condensation with

thiourea in the presence of sodium hydrogen carbonate

followed by Boc deprotection aﬀorded key intermediate 20

(17% overall yield), which was transformed into the

dihydrothiazolopyridone 14a with moderate yield (55%)

a

Scheme 2

The benzyloxy-dihydrothiazolopiperidones 15b,c were pre-

pared as shown in Scheme 4. Nucleophilic substitution of the

bromothiazolopyridone 21 with benzyl alcohol yielded the

target intermediate 15a, which was subsequently N-alkylated

with methyl iodide aﬀording compound 15b in 30% yield

(equation (I), Scheme 4). Attempts to perform a copper

catalyzed N-arylation reaction on 15a (CuI, K₃PO₄, DMF,

microwave irradiation) were unsuccessful as compound 15c

was found to be unstable under these reaction conditions, and

the synthesis of compound 15c was carried out as shown in the

equation (II) of Scheme 4. Thus, intermolecular cyclization of

25d with thiourea followed by Sandmeyer reaction led with low

yield to the intermediate N-aryl bromothiazole derivative 26,

which was treated with benzyl alcohol in the presence of

sodium hydride to yield (63%) the target compound 15c.

In the case of the phenoxymethyl-dihydrothiazolopyridones

16, ﬁve main synthesis routes (Schemes 5−9) were used to

access the ﬁnal compounds 16a−q. Starting from the

bromothiazole derivative 21, palladium catalyzed carbonylation

a

Reagents and conditions: (a) (i) NBS, CCl₄, 10−15 °C, 2 h; (ii)

thiourea, NaHCO₃, EtOH, 80 °C, 2.5 h; (iii) HCl, 1,4-dioxane, rt, 30

min, 100%; (b) isoamyl nitrite, CuBr₂, CH₃CN, rt, 1.5 h, 55%; (c)

phenyl acetylene, [1,1′-bis(diphenylphosphino)ferrocene]dichloro-

palladium(II), CuI, Et₃N, 1,4-dioxane, reﬂux, 30%; (d) 14b, MeI or

14c, MeOCH₂CH₂Br, Cs₂CO₃, CH₃CN, 80 °C, 16 h, 19% for 14b and

20% for 14c.

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a

Scheme 3

a

Reagents and conditions: (a) Ethyl malonyl chloride, Et₃N or DIPEA, CH₂Cl₂, rt, 1 h, 22−70%; (b) NaOEt, EtOH, 85 °C, 16 h, 55−91%; (c) 24a-

c, CH₃CO₂H, H₂O, 90 °C, 16 h, 53−73%; or 24d,e, CH₃CN, H₂O, 90 °C, 2 h; (d) NBS, CH₂Cl₂, 0 °C, 30 min, 73−76%.

a

Scheme 4

a

Reagents and conditions: (a) BnOH, NaH, THF, 120 °C, 15 min, microwave, 54%; (b) MeI, NaH, THF, 120 °C, 15 min, microwave, 54%; (c) (i)

thiourea, NaHCO₃, EtOH, 80 °C, 1 h; (ii) isoamyl nitrite, CuBr₂, CH₃CN, rt, 45 min, 16%; (d) BnOH, NaH, THF, 120 °C, 15 min, microwave,

63%.

a

Scheme 5

a

Reagents and conditions: (a) CO, [1,1′-bis(diphenylphospino)ferrocene]dichloro-palladium(II), MeOH, Et₃N, 50 °C, 16 h, 21%; (b) MeI,

Cs₂CO₃, DMF, rt, 60 h, 42%; (c) NaBH₄, MeOH, 0 °C, 30 min, 99%; (d) PhOH, di-tert-butyl azodicarboxylate, PPh₃, THF, 120 °C, 20 min,

microwave, 20%.

a

Scheme 6

a

Reagents and conditions: (a) NaBH₄, MeOH, 0 °C, 30 min, 40%; (b) PhOH, di-tert-butyl azodicarboxylate, PPh₃, THF, 120 °C, 20 min,

microwave, 64%; (c) 32, Cs₂CO₃, DMF, rt, 16 h, 13−49%.

followed by alkylation with methyl iodide and reduction of the

intermediate ester 28 led to the alcohol 29 which after

Mitsunobu reaction with phenol aﬀorded 16a (Scheme 5).

Key intermediate 31 was obtained in 26% yield from

intermediate 27, in a similar way (Scheme 6). N-Alkylation of

31 with several primary alkyl halides 32 aﬀorded the

corresponding N-alkyl derivatives 16b,d,g in moderate yields

(13−49%) (Scheme 6).

Alternatively, several compounds (16c,e,f,h,i,k) were pre-

pared by intermolecular cyclization between the thioamides

33a,b and the corresponding 3-bromopiperidine-2,4-dione

25a−e (10−25%) (Scheme 7). Similarly, derivatives 16j,l

were accessed by nucleophilic substitution on the chloromethyl

thiazol 36, obtained via reaction of 25d with ethyl thiooxamate

followed by ester reduction and subsequent treatment with

thionyl chloride, with the corresponding phenols as shown in

Scheme 8.

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a

Scheme 7

Scheme 9

a

Reagents and conditions: (a) 25a−c, NaHCO₃, EtOH, 70 °C, 15

Reagents and conditions: (a) 16m-o,q, 33, CuI, K₂CO₃, N,N′-

dimethylethylenediamine, toluene, 120 °C, 16 h, 15−64% or 16p, 2-

bromo-4-ﬂuoropyridine, CuI, K₃PO₄, N,N′-dimethylethylenediamine,

toluene, 140 °C, 16 h, 64%.

min, 10−25% or 25d,e, NaHCO₃, DMF, 100 °C, 30 min, 10−23%.

Finally, intermediate 31 was also used to prepare the N-

pyridyl derivatives 16m-q by copper catalyzed coupling with

the corresponding halopyridines 36 as shown in Scheme 9, in

low to moderate yields (15−64%).

The introduction of a cyclopropyl group with pseudo aromatic

character in 16e resulted as well in a similar potency when

compared to 16a and its isopropyl congener 16c. Noteworthy,

the introduction of a methylene spacer between the cyclopropyl

group and the dihydrothiazolopyridone core (16f) delivered a

remarkable increase in potency (∼6 fold vs. 16a and 16e).

Finally, ring expansion to the more polar tetrahidropyrane ring

(16g) had almost negligible eﬀect on both potency and eﬃcacy

compared to 16a. As observed with the benzyloxy series, better

results were obtained with ﬂuorophenyl tethered amides such

as 16h or 16i with in vitro potencies below 100 nM (∼2−3 fold

more potent than reference compounds 1 and 2) and good

eﬃcacies. Taking 16h as a starting point, the introduction of

ﬂuorine substitution in the western aromatic ring was found to

be preferred at position 3, with 16k showing comparable

potency and minimally reduced eﬃcacy to 16h. A ∼7−10-fold

decrease in potency was observed for the 2- and 4-substituted

analogues 16j,l. In light of the promising in vitro results

obtained with the aryl derivatives 16h−l, our SAR exploration

concluded with the investigation of the eﬀects of replacing the

ﬂuorophenyl amide substituents by more polar and weakly

basic pyridine rings. From the data in Table 1, it can be

concluded that the 2-substituted pyridine analogue 16m was

preferred over the 3 and 4 regioisomers (16n and 16o). On the

other hand, although 3-ﬂuorine substitution (16q) did not

induce any increase on mGlu₅potency, a signiﬁcant improve-

ment was accomplished by the introduction of the ﬂuorine

atom in the 5-position of the 2-substituted pyridine ring, with

16p showing similar potency to 16m and increased eﬃcacy

(94% vs. 79%).

DISCUSSION

■

In the case of benzyloxy-dihydrothiazolopyridone derivatives

15, our SAR investigation started with the synthesis of the

methyl and 4-ﬂuorophenyl substituted analogues 15b (prepared

by analogy to 14b) and 15c (selected because ﬂuorine

containing aromatic rings are common motifs as distal aromatic

moieties for diﬀerent mGlu₅PAM chemotypes).^9,16As seen in

Table 1, the introduction of the 4-ﬂuorophenyl and the methyl

substituents resulted in compounds of similar potency.

Furthermore, 15c showed good metabolic stability in human

and rat fortiﬁed microsomes. Unfortunately, in the course of

the aqueous solubility assessment of derivatives 15, these were

found to be unstable in acidic media (20% HP-β-CD in water,

pH = 4) leading to the corresponding 2-hydroxy-dihydrothia-

zolopyridone fragments. In light of this ﬁnding, we decided to

stop the exploration of the benzyloxy-dihydrothiazolopyridone

series 15. Instead we focused eﬀorts on the phenoxymethyl

subseries 16, which proved to be stable under the same acidic

aqueous solubility conditions. As a ﬁrst step, the alkyl

substituent on the amide moiety was explored. Akin to the

benzyloxy series, introduction of a methyl group in 16a resulted

in a signiﬁcant reduction (∼6 fold) in mGlu₅PAM potency

compared to the corresponding acetylene analogue 14b, or

reference compounds 1 and 2, but still retained high eﬃcacy.

Replacement of the methyl group in 16a by other alkyl chains

with increased size and diverse polarity either had a limited

eﬀect or was detrimental to activity. Thus, comparable activity

was observed for the more lipophilic 16b (R¹= ethyl) and 16c

(R¹= isopropyl), although a modest reduction in eﬃcacy was

also noted for the bulkier 16c. In the case of the methoxyethyl

substituted 16d and in analogy with the acetylene containing

series 14, the potency was again in the same range as for 16a.

In light of their good mGlu₅PAM activity and eﬃcacy

compounds 16a, 16b, 16f, 16h, and 16p were selected as

representative examples for further characterization. Metabolic

stability evaluation showed signiﬁcant species diﬀerences, with

lower turnovers in human vs. rat liver microsomes in all cases.

a

Scheme 8

a

Reagents and conditions: (a) Ethyl thiooxamate, NaHCO₃, EtOH, 80 °C, 2 h, 15%; (b) (i) NaBH₄, MeOH, 0 °C, 2 h; (ii) SOCl₂, CH₂Cl₂, rt, 2 h,

27%; (c) Ar-OH, K₂CO₃, DMF, rt, 24 h, 8−20%.

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Figure 3. Glutamate CRC in the presence of increasing concentrations of 16a.

Thus, despite low (16a, 16h, and 16p, > 75% remaining) to

moderate-high (16b and 16f, 75−25% remaining) turnover in

HLM for the ﬁve compounds, metabolic instability in RLM

ranged from moderate (16p, ∼30% remaining) to very high

(16b and 16f, ∼100% metabolized). As high metabolic

instability in rodents would hamper further pharmacological

evaluation, only compounds 16a, 16h, and 16p were selected

for additional DMPK evaluation to conﬁrm their potential

utility for proof-of-concept in vivo studies for this series. Thus,

16a and 16b were evaluated in noncrossover in vivo

pharmacokinetic (PK) studies in rats. Because of its poor

solubility in aqueous buﬀer (<4 μM, pH 7.4) and in 20% HP-β-

CD in water formulation (<0.20 mg/mL), 16h could only be

dosed orally as a suspension (5 mg/mL) and was found to be

undetectable in the brain after 2.0 h of administration and

hence discarded for in vivo pharmacological evaluation. As a

consequence of these ﬁndings, and because the improvement in

kinetic solubility for 16p was minimal (10 μM, pH 7.4 and <1

mg/mL in 20% HP-β-CD in water) compared to 16h, 16p was

discarded for further investigation. PK studies (1 mg/kg iv, 10

mg/kg p.o.) with the more soluble 16a (97 μM, aqueous buﬀer

pH 7.4 and 4 mg/mL in 20% HP-β-CD in water), revealed a

glutamate shifted to the left with increasing concentration of

16a (55 nM to 30 μM) with no signiﬁcant eﬀect change of the

maximal response. A 5.4-fold leftward shift in the glutamate

EC₅₀was observed in the presence of 30 μM 16a, and a

predicted allosteric modulator aﬃnity of approximately 10 μM

(10069 nM) was calculated using the operational model of

allosterism described by Gregory et al. (Figure 3).²⁹These

results corroborate a positive allosteric interaction between

glutamate and 16a and were conﬁrmed in a cell line expressing

the human mGlu₅receptor,²⁷where 10 μM 16a produced a

4.4-fold leftward shift in the glutamate CRC.

Encouraged by its overall proﬁle, 16a (Figure 4) was

evaluated in a screen of potential antipsychotic eﬃcacy, the

reversal of amphetamine-induced hyperlocomotion in rats.^10,11

relatively high CL_pof 50 mL/min/kg and a short half-life (T_1/2

)

of 0.18 h. Examination of drug concentrations in portal vein,

plasma, and brain at diﬀerent time points (0.5, 1.0, 2.0, 4.0, and

7.0 h) showed a moderate ﬁrst-pass clearance (E_hep0.47) with a

relatively low volume of distribution (V_ss= 0.70 L/kg) and

moderate systemic exposure (plasma_AUC= 2.9 μM-h). The

Figure 4. Proﬁle summary of 16a.

As seen in Figure 5, when dosed orally in a 20% HP-β-CD in

water formulation, 16a showed robust dose-dependent eﬀects

in reversal of hyperlocomotion with a lowest active oral dose of

10 mg/kg. The maximal eﬀect (61%) was seen at the highest

tested dose of 100 mg/kg, corresponding with an estimated

average terminal unbound brain concentration of ∼3.2 μM,³⁰

which is well in line with the rat in vitro mGlu₅PAM EC₅₀of

16a and that, according with the progressive fold-shift

experiments (Figure 3), would correlate with a left-ward shift

in the glutamate CRC in the range of ∼1.5−2.9 fold.

The preliminary in vivo characterization of 16a concluded

with the evaluation of balance and motor coordination and

potential neurological side eﬀects by means of rotarod and

Irwin modiﬁed neurological battery tests respectively in rats. As

it can be seen in Figure 6, when dosed orally at 30, 56.6, and

100 mg/kg (20% HP-β-CD in water), 16a had no statistically

signiﬁcant eﬀect on general motor output as measured by

performance on the rotarod (120 s cutoﬀ) test in rats.³¹

Furthermore, 16a had no eﬀect on any autonomic or

somatomotor nervous system functions as measured using

the modiﬁed Irwin neurological test battery in rats when tested

at a single oral dose of 100 mg/kg (20% HP-β-CD in water).,³²

relative CNS exposure of 16a was moderate as well (brain_AUC

=

2.4 μM·h) with a good distribution to the brain (brain_AUC

/

plasma_AUC= 0.90). Additionally, a relatively high unbound

fraction in brain (rat f_ubrain = 18%) contributed favorably to

achieve good absolute brain levels (C_max= 522 nM, T_max= 0.5

h), which were considered adequate to warrant further

pharmacological evaluation.

Subsequent in vitro examination of 16a in a low expressing

rat receptor mGlu₅cell line²⁷resulted in a slightly reduced

potency (∼2 fold) and eﬃcacy (EC₅₀= 3408 nM, 62% Glu

Max) compared with the human receptor expressing cell line.

16a was found to be selective in an mGlu selectivity panel

(mGlu_1−4,6−8EC₅₀> 10 μM)²⁰and also when tested at 10 μM

concentration in a panel of 66 oﬀ-target class receptors, ion

channels and transporters.²⁸Finally, positive allosteric modu-

lator activity was further investigated by fold-shift experiments

to determine the degree of cooperativity with glutamate of the

compound. Thus, as shown in Figure 4, progressive fold-shift

experiments in our cell line expressing the rat mGlu₅receptor

revealed how the concentration response curve of (CRC)

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dose of 10 mg/kg p.o. and maximal eﬃcacy at a dose of 100

mg/kg p.o. without showing any signiﬁcant motor impairment

or overt neurological side eﬀects up to a dose of 100 mg/kg p.o.

These results underscore the potential of 16a as an interesting

tool compound suitable to unravel the in vivo pharmacology of

mGlu₅positive allosteric modulation. Further investigation and

evolution of this novel series of mGlu₅PAMs are underway and

will be reported in due course.

EXPERIMENTAL SECTION

■

Chemistry. Unless otherwise noted, all reagents and solvents were

obtained from commercial suppliers and used without further

puriﬁcation. Thin layer chromatography (TLC) was carried out 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 microwave

reactor (Biotage), or in a multimode reactor, MicroSYNTH Labstation

(Milestone, Inc.). Nuclear magnetic resonance (NMR) spectra were

recorded with a Bruker DPX-300 a Bruker DPX-400 or a Bruker AV-

500 spectrometer (Bruker AG) with standard pulse sequences,

operating at 300, 400, and 500 MHz, respectively, using CDCl₃and

DMSO-d₆as 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),

quin (quintet), sex (sextet), sep (septet), or m (multiplet). Liquid

chromatography combined with mass spectrometry (LCMS) was

performed on either a HP 1100 high-performance liquid chromatog-

raphy (HPLC) system (Agilent Technologies) or Advanced

Chromatography Technologies system composed of a quaternary or

binary pump with degasser, an autosampler, a column oven, a diode

array detector (DAD), and a column as speciﬁed in the respective

methods below. Flow from the column was split to a MS spectrometer.

The MS detector was conﬁgured with either an electrospray ionization

source or an electrospray combined with atmospheric pressure

chemical ionization (ESCI) dual ionization source. Nitrogen was

used as the nebulizer gas. Data acquisition was performed with

MassLynx- Openlynx software or with Chemstation-Agilent data

browser software. More detailed information about the diﬀerent

LCMS methods employed can be found in the Supporting

Information. Liquid chromatography combined with high resolution

mass spectrometry (LC-HRMS) was performed using a Micromass

(Waters) Q-Tof API-US calibrated and veriﬁed with sodium iodide.

The samples were diluted with a 50:50 0.1% formic acid (in Milli-Q)/

acetonitrile solution, directly infused using leucine-enkephalin ([M +

H]⁺= 556.2771) as a lockmass. Scan range was from 100 to 1000 Da,

with a scan time of 1 s. For a number of compounds, melting points

(Mp^a) were determined with a WRS-2A melting point apparatus

(Shanghai Precision and Scientiﬁc Instrument Co. Ltd.). Melting

points were measured with a linear heating up rate of 0.2−5.0 °C/min

(maximum temperature 300 °C), and the reported values are melt

ranges. For another number of compounds, melting points (Mp^b)

were determined in open capillary tubes on a FP62 or on a FP81HT-

FP90 apparatus (Mettler). Melting points were measured with a

temperature gradient of 10 °C/min (maximum temperature 300 °C),

and the melting point was read from a digital display. Melting point

values are peak values and were obtained with experimental

uncertainties that are commonly associated with this analytical

method.

Figure 5. Dose-dependent eﬀect of 16a on the reversal of

amphetamine-induced hyperlocomotion in rats.

Figure 6. Eﬀects of 16a on rotarod performance in rats.

These results highlight how 16a neither impacts motor

behavior nor shows any overt neurological side eﬀects in rats

up to a dose of 100 mg/kg p.o., a dose that has been previously

shown to produce maximal eﬃcacy in the inhibition of the

amphetamine-induced hyperlocomotion.

CONCLUSIONS

■

In summary, starting from a singleton phenyl ester chromanone

HTS hit, two parallel approaches allowed the identiﬁcation of

the benzyloxy and phenoxymethyl moieties and the dihy-

drothiazolopyridone core as optimal replacements for the ester

and the chromanone core respectively. While the benzyloxy-

dihydrothiazolopyridone subseries was found susceptible to

hydrolysis, the phenoxymethyl-dihydrothiazolopyridones 16

proved to be a promising mGlu₅PAM series. Preliminary

focused SAR exploration of amide substituents revealed

aromatic groups as preferred substituents for achieving high

in vitro mGlu₅potentiation although their poor DMPK

characteristics precluded further in vivo evaluation. On the

other hand, the N-methyl analogue 16a presented a more

balanced proﬁle in terms of in vitro potency, selectivity and

DMPK proﬁle, which made it an attractive candidate for in vivo

characterization in a model of antipsychotic activity. 16a

showed robust, dose-dependent eﬀects in the reversal of

amphetamine induced hyperlocomotion, with a lowest active

Purities of all new compounds were determined by analytical

reversed-phase high-performance liquid chromatography (RP-HPLC)

using the area percentage method on the UV trace recorded at a

wavelength of 254 nm, and compounds were found to have ≥95%

purity unless otherwise speciﬁed.

2-Bromo-5,6-dihydro-4H-benzothiazol-7-one (18). A mixture of

17²⁷(19 g, 112 mmol), CuBr₂(27 g, 120 mmol), and 3-methyl-1-

nitrosooxy-butane (18 g, 153 mmol) in CH₃CN (250 mL) was stirred

at 80 °C for 1 h. The mixture was then cooled to rt and poured into a

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10% solution of HCl. The mixture was extracted with CH₂Cl₂and the

organic layer was separated, dried (Na₂SO₄), and ﬁltered, and the

solvent was evaporated in vacuo to yield 19.6 g (75%) of 18 that was

used in the next step without any further puriﬁcation. H NMR (500

MHz, CDCl₃) δ ppm 2.47−2.60 (m, 2 H), 3.07−3.15 (m, 1 H), 3.18−

3.29 (m, 1 H), 4.64 (t, J = 3.9 Hz, 1 H).

used in the next step without any further puriﬁcation. LCMS: m/z 228

[M + H]⁺, t_R= 0.95 min.

1-Cyclopropyl-2,4-dioxo-piperidine-3-carboxylic Acid Ethyl Ester

(24b). Starting from 23b (40 g, 147 mmol) and following the

procedure described for 24a, 24b was obtained as an oil (20 g, 61%).

LCMS: m/z 226 [M + H]⁺, t_R= 0.91 min.

1

1-Cyclopropylmethyl-2,4-dioxo-piperidine-3-carboxylic Acid

Ethyl Ester (24c). Starting from 23c (20 g, 70.1 mmol) and following

the procedure described for 24a, 24c was obtained as an oil (10 g,

60%). LCMS: m/z 240 [M + H]⁺, t_R= 1.21 min.

2-Amino-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one hydro-

chloride salt (20). To a solution of 19 (40 g, 187.58 mmol) in

CCl₄(500 mL) was added NBS (33.38 g, 187.58 mmol) portionwise

keeping the reaction temperature in the range of 10−15 °C. The

mixture was further stirred at 10−15 °C for 2 h. The mixture was

allowed to warm to rt, and the solvent was evaporated in vacuo. The

residue thus obtained was dissolved in EtOAc and washed with H₂O.

The organic layer was separated, dried (Na₂SO₄), and ﬁltered and the

solvent was evaporated. The residue thus obtained was dissolved in

EtOH (400 mL), and then thiourea (6.5 g, 85.6 mmol) and NaHCO₃

(7.2 g, 85.6 mmol) were added. The resulting mixture was stirred at 80

°C for 2.5 h and then cooled to rt, and the solids were ﬁltered oﬀ. The

ﬁltrate was evaporated in vacuo to give a residue that was crystallized

in EtOH. The yellow crystals thus obtained were dissolved in a 4 M

solution of HCl in 1,4-dioxane (100 mL), and the resulting mixture

was stirred at rt for 30 min. The solvent was evaporated in vacuo to

yield 10 g (88% pure) of 20 as a yellow powder which was used in the

next step without any further puriﬁcation. LCMS: m/z 170 [M + H]⁺,

t_R= 0.38 min.

2-Bromo-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one (21). A

mixture of 20 (8 g, 39.8 mmol), CuBr₂(10.43 g, 46.68 mmol), and

3-methyl-1-nitrosooxy-butane (6.8 g, 58.35 mmol) in CH₃CN (100

mL) was stirred at rt for 1.5 h. The solvent was evaporated in vacuo.

The residue thus obtained was dissolved in EtOAc and washed with

H₂O. The organic layer was separated, dried (Na₂SO₄), and ﬁltered,

and the solvent was evaporated in vacuo to yield 5 g (55%) of 21 that

was used in the next step without any further puriﬁcation. LCMS: m/z

233 [M + H]⁺, t_R= 0.70 min.

N-(2-Ethoxycarbonyl-ethyl)-N-isopropyl-malonamic Acid Ethyl

Ester (23a). To a solution of 22a (13.5 g, 69 mmol) in CH₂Cl₂

(100 mL) was added Et₃N (26 g, 255 mmol). Then ethyl malonyl

chloride (14 g, 93 mmol) was added to the mixture at 0 °C, and the

mixture was stirred at rt for 5 h. Then H₂O was added and the organic

layer was separated, dried (Na₂SO₄), and ﬁltered and the solvent was

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica gel, EtOAc in petroleum ether, 10/90 to 33/

66). The desired fractions were collected and the solvents evaporated

in vacuo to yield 5.1 g (87% pure) of 23a as an oil. LCMS: m/z 274

[M + H]⁺, t_R= 1.25 min.

N-Cyclopropyl-N-(2-ethoxycarbonyl-ethyl)-malonamic Acid Ethyl

Ester (23b). Starting from 22b (40.3 g, 256 mmol) and following the

procedure described for 23a, 23b was obtained as an oil (40 g, 60%

pure). LCMS: m/z 272 [M + H]⁺, t_R= 0.91 min.

N-Cyclopropylmethyl-N-(2-ethoxycarbonyl-ethyl)-malonamic

Acid Ethyl Ester (23c). Starting from 22c (31.3 g, 183 mmol) and

following the procedure described for 23a, 23c was obtained as an oil

(33 g, 93% pure). LCMS: m/z 286 [M + H]⁺, t_R= 1.23 min.

N-(2-Ethoxycarbonyl-ethyl)-N-(4-ﬂuoro-phenyl)-malonamic Acid

Ethyl Ester (23d). Starting from 22d (10 g, 47.3 mmol) and following

the procedure described for 23a but using diisopropylethylamine as

base, 23d was obtained as an orange oil (11 g, 73% pure). LCMS: m/z

326 [M + H]⁺, t_R= 2.41 min.

1-(4-Fluoro-phenyl)-2,4-dioxo-piperidine-3-carboxylic Acid Ethyl

Ester (24d). A mixture of 23d (6.27 g, 19.27 mmol) in a 21% solution

of sodium ethoxide in EtOH (14.39 mL, 38.55 mmol) was stirred at 85

°C for 16 h. The solvent was evaporated in vacuo and the residue was

partitioned between EtOAc and H₂O. The aqueous layer was

separated, acidiﬁed by 1N HCl solution addition and extracted with

CH₂Cl₂. The organic layer was separated, dried (Na₂SO₄), ﬁltered and

the solvent evaporated in vacuo to yield 5 g (66% pure) of 24d which

was used in the next step without any further puriﬁcation. LCMS: m/z

280 [M + H]⁺, t_R= 0.73 min.

1-(2,4-Diﬂuoro-phenyl)-2,4-dioxo-piperidine-3-carboxylic acid

ethyl ester (24e). Starting from 23e (4.8 g, 13.98 mmol) and

following the procedure described for 24d, 24e was obtained as an oil

(4.16 g, 55% pure). LCMS: m/z 298 [M + H]⁺, t_R= 0.65 min.

3-Bromo-1-isopropyl-piperidine-2,4-dione (25a). A solution of

24a (3 g, 13.2 mmol) in a mixture of acetic acid (10 mL) and H₂O (90

mL) was stirred at 90 °C for 14 h. The mixture was cooled and

extracted with CH₂Cl₂. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvent was evaporated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography (silica gel;

EtOAc in petroleum ether 10/90 to 33/66). The desired fractions

were collected and the solvents evaporated in vacuo. The residue thus

obtained was dissolved in CH₂Cl₂(20 mL) and NBS (1.1 g, 6.44

mmol) was added. The mixture was stirred at rt for 4 h. Then H₂O

was added and the mixture was extracted with EtOAc. The organic

layer was separated, dried (Na₂SO₄), and ﬁltered, and the solvents

were evaporated in vacuo to yield 0.6 g (53% pure) of 26a which was

used in the next step without any further puriﬁcation. LCMS: m/z 234

[M + H]⁺, t_R= 0.35 min.

3-Bromo-1-cyclopropyl-piperidine-2,4-dione (25b). Starting from

24b (20 g, 89 mmol) and following the procedure described for 25a,

25b was obtained as an oil (11 g, 73% pure). LCMS: m/z 232 [M +

H]⁺, t_R= 0.29 min.

3-Bromo-1-cyclopropylmethyl-piperidine-2,4-dione (25c). Start-

ing from 24c (10 g, 41.8 mmol) and following the procedure described

for 25a, 25c was obtained as an oil (4 g, 66% pure). LCMS: m/z 246

[M + H]⁺, t_R= 1.053 min.

3-Bromo-1-(4-ﬂuoro-phenyl)-piperidine-2,4-dione (25d). Starting

from 24d (7.5 g, 26.86 mmol) and following the procedure described

for 25a, 25d was obtained as an oil (7.7 g, 76% pure). LCMS: m/z 285

[M + H]⁺, t_R= 0.42 min.

3-Bromo-1-(2,4-diﬂuoro-phenyl)-piperidine-2,4-dione (25e).

Starting from 24e (18 g, 60 mmol) and following the procedure

described for 25a, 25e was obtained as an oil (11 g, 38% pure). LCMS:

m/z 304 [M + H]⁺, t_R= 0.34 min.

2-Bromo-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (26). A mixture of 25d (4.14 g, 14.48 mmol), thiourea

(1.1 g, 14.48 mmol) and NaHCO₃(1.22 g, 14.48 mmol) in EtOH (60

mL) was stirred at 80 °C for 1 h. The mixture was then cooled to rt

and the solids were ﬁltered oﬀ. The ﬁltrate was evaporated in vacuo.

The residue thus obtained was dissolved in CH₃CN (80 mL) and then

CuBr₂(3.05 g, 13.67 mmol) and 3-methyl-1-nitrosooxy-butane (2.3

mL, 17.09 mmol) were added. The mixture was stirred at rt for 45 min

and then the solvent was evaporated in vacuo. The residue thus

obtained was partitioned between EtOAc and H₂O. The organic layer

was separated, dried (Na₂SO₄), ﬁltered and the solvent evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica gel, EtOAc in heptane, 0/100 to 30/70). The desired

fractions were collected and the solvents evaporated in vacuo to yield

N-(2-Ethoxycarbonyl-ethyl)-N-(2,4-diﬂuoro-phenyl)-malonamic

Acid Ethyl Ester (23e). Starting from 22e (29 g, 127 mmol) and

following the procedure described for 23a, 23e was obtained as an

orange oil (35 g, 69% pure). LCMS: m/z 344 [M + H]⁺, t_R= 1.21 min.

1-Isopropyl-2,4-dioxo-piperidine-3-carboxylic Acid Ethyl Ester

(24a). Sodium (0.86 g, 37.3 mmol) was added to EtOH (50 mL) at

0 °C. The mixture was stirred at rt for 1 h. Then 23a (5.1 g, 18.6

mmol) was added and the mixture was stirred at 85 °C for 16 h. The

mixture was poured into ice H₂O. The aqueous phase was neutralized

by addition of a 2 N HCl solution and extracted with EtOAc. The

organic layer was separated, dried (Na₂SO₄), and ﬁltered, and the

solvent was evaporated in vacuo to yield 3 g (91%) of 24a which was

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1.2 g (16%) of 26 as a white solid. LCMS: m/z 327 [M + H]⁺, t_R=

2.51 min.

H), 7.74 (br. s, 1 H), 7.97 (br. s, 1 H). LCMS: m/z 166 [M − H]⁻, t_R

= 1.18 min.

2-(3-Fluoro-phenoxy)-thioacetamide (33b). Starting from (3-

ﬂuoro-phenoxy)-acetonitrile (3.39 g, 22.41 mmol) and thioacetamide

(4.21 g, 56.02 mmol) and following the procedure described for 33a,

33b was obtained as a white solid (3.58 g, 86%). ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 4.78 (s, 2 H), 6.70−6.91 (m, 3 H), 7.25−7.42 (m, 1

H), 9.69 (m, 2 H). LCMS: m/z 184 [M − H]⁻, t_R= 1.19 min.

5-(4-Fluoro-phenyl)-4-oxo-4,5,6,7-tetrahydro-thiazolo[5,4-c]-

pyridine-2-carboxylic acid ethyl ester (34). A mixture of 25d (11.8 g,

41.3 mmol), ethyl thiooxamate (5.5 g, 41.3 mmol), and NaHCO₃(8.7

g, 103.5 mmol) in EtOH (400 mL) was stirred at 80 °C for 2 h. The

mixture was cooled and ﬁltered, and the ﬁltrate was evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica gel, EtOAc in petroleum ether, 10/90 to 33/66). The

desired fractions were collected and evaporated in vacuo to yield 2 g

(68% pure) of 34. LCMS: m/z 321 [M + H]⁺, t_R= 1.05 min.

2-Chloromethyl-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-thiazolo[5,4-

c]pyridin-4-one (35). Sodium borohydride (0.7 g, 18.7 mmol) was

added to a solution of 34 (2 g, 6.3 mmol) in MeOH (50 mL) at 0 °C.

The mixture was stirred at rt for 2 h, H₂O was added, and the mixture

was extracted with EtOAc. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvent was evaporated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography (silica gel,

EtOAc in petroleum ether, 20/80 to 66/33). The desired fractions

were collected, and the solvents were evaporated in vacuo. The residue

thus obtained was added to a mixture of thionyl chloride (10 mL) and

CH₂Cl₂(10 mL). The mixture was stirred at rt for 2 h. The solvents

were then evaporated in vacuo to yield 1 g (87% pure) of 35 as a solid

which was used in the next step without any further puriﬁcation.

LCMS: m/z 297 [M + H]⁺, t_R= 1.03 min.

4-Oxo-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-carboxylic

acid methyl ester (27). A mixture of Et₃N (17.2 g, 171 mmol) and

[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (2 g,

2.7 mmol) in THF (300 mL) was added to a solution of 21 (7.5 g,

23.6 mmol) in MeOH (300 mL). Then the mixture was stirred at 50

°C overnight under CO atmosphere (2.5 MPa). The mixture was

cooled and ﬁltered, and the solvents were evaporated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography (silica gel,

MeOH in CH₂Cl₂, 1/99). The desired fractions were collected and

evaporated in vacuo to yield 4.5 g (21%) of 27 that crystallized from

1

EtOAc as a yellow solid. Mp^a217.6−220.0 °C. H NMR (300 MHz,

CDCl₃) δ ppm 3.20 (t, J = 7.1 Hz, 2 H), 3.62−3.79 (m, 2 H), 4.02 (s,

3 H), 5.84 (br. s., 1 H). LCMS: m/z 213 [M + H]⁺, t_R= 2.94 min.

5-Methyl-4-oxo-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-car-

boxylic acid methyl ester (28). Iodomethane (4.4 mL, 70.7 mmol)

was added to a suspension of 27 (10 g, 47.1 mmol) and Cs₂CO₃(23 g,

70.68 mmol) in DMF (118 mL) under nitrogen. The mixture was

stirred at rt for 60 h and then diluted with H₂O and extracted with

EtOAc. The organic layer was separated, dried (MgSO₄), and ﬁltered,

and the solvent was evaporated in vacuo. The crude product was

puriﬁed by ﬂash column chromatography (silica gel, EtOAc in CH₂Cl₂,

0/100 to 50/50). The desired fractions were collected, and the

solvents were evaporated in vacuo to yield 4.46 g (42%) of 28 as a pale

1

brown oily solid. H NMR (400 MHz, CDCl₃) δ ppm 3.13 (s, 3 H),

3.23 (t, J = 7.2 Hz, 2 H), 3.71 (t, J = 7.2 Hz, 2 H), 4.03 (s, 3 H).

LCMS: m/z 227 [M + H]⁺, t_R= 0.60 min.

2-Hydroxymethyl-5-methyl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (29). Sodium borohydride (0.15 g, 4.0 mmol) was

added to a stirred solution of 28 (0.65 g, 2.87 mmol) in a mixture of

THF (8.8 mL) and MeOH (8.8 mL). The mixture was stirred at 0 °C

for 30 min in a sealed tube under nitrogen and then diluted with H₂O

and extracted with CH₂Cl₂. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered and the solvents were evaporated in vacuo. The

aqueous phase was acidiﬁed with 3 N HCl and extracted with CH₂Cl₂.

The combined organic layers were dried (Na₂SO₄) and ﬁltered, and

the solvents were evaporated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (silica gel, MeOH in EtOAc, 0/100

to 20/80). The desired fractions were collected and the solvents were

evaporated in vacuo to yield 0.59 g (99%) of 29 as a dark oil. ¹H NMR

(400 MHz, CDCl₃) δ ppm 2.81 (t, J = 6.1 Hz, 1H), 3.10 (t, J = 7.2 Hz,

2 H), 3.10 (s, 3 H), 3.66 (t, J = 7.2 Hz, 2 H), 4.93 (d, J = 6.2 Hz, 2 H).

LCMS: m/z 199 [M + H]⁺, t_R= 0.28 min.

2-Hydroxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one

(30). Starting from 27 (10 g, 47 mmol) and following the procedure

described for 29, 30 was obtained as an oil (3.5 g, 40%). Mp^a212.1−

230.9 °C. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.92 (t, J = 7.0 Hz,

2 H), 3.47 (td, J = 7.0, 2.5 Hz, 2 H), 4.72 (s, 2 H), 6.24 (br. s., 1 H),

7.79 (br. s., 1 H). LCMS: m/z 185 [M + H]⁺, t_R= 3.63 min.

2-Phenoxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one

(31). Di-tert-butyl azodicarboxylate (3.0 g, 13.03 mmol) was added to a

stirred solution of 30 (2 g, 10.86 mmol), phenol (1.23 g, 13.03 mmol),

and PPh₃(3.42 g, 13.03 mmol) in THF (31 mL) under nitrogen. The

mixture was stirred at 120 °C for 40 min under microwave irradiation.

The solvent was evaporated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (silica gel, EtOAc in CH₂Cl₂, 0/100

to 100/0). The desired fractions were collected and evaporated in

vacuo to yield 1.82 g (69% pure) of 31 as a white solid. LCMS: m/z

261 [M + H]⁺, t_R= 1.50 min.

2-Phenylethynyl-5,6-dihydro-4H-benzothiazol-7-one (13). To a

solution of 18 (2.3 g, 9.86 mmol), phenylacetylene (2.01 g, 19.7

mmol), CuI (0.2 g, 1.05 mmol) and Et₃N (2.98 g, 29.58 mmol) in 1,4-

dioxane (50 mL) at rt was added [1,1′-bis(diphenylphosphino)-

ferrocene]dichloro-palladium(II) (0.2 g, 0.25 mmol). The mixture was

stirred at reﬂux for 2 h under nitrogen. The solvent was evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica gel, EtOAc in petroleum ether, 66/33). The desired

fractions were collected, and the solvent was evaporated in vacuo to

1

yield 1.2 g (52%) of 13 as a solid. Mp^a117.1−119.9 °C. H NMR

(400 MHz, CDCl₃) δ ppm 2.28 (quin, J = 6.4 Hz, 2 H), 2.70 (t, J = 6.3

Hz, 2 H), 3.13 (t, J = 6.1 Hz, 2 H), 7.39 - 7.53 (m, 3 H), 7.60−7.70

(m, 2 H). LCMS: m/z 254 [M + H]⁺, t_R= 5.49 min.

2-Phenylethynyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one

(14a). To a solution of 21 (2.33 g, 10 mmol), phenylacetylene (2.0 g,

20 mmol) and Et₃N (4.5 g, 45 mmol) in 1,4-dioxane (50 mL) at rt

under nitrogen were added [1,1′-bis(diphenylphosphino)ferrocene]-

dichloro-palladium(II) (0.73 g, 1 mmol) and CuI (0.75 g, 4 mmol).

The mixture was stirred at 80 °C for 2 h and then cooled to rt, and the

solvent was evaporated in vacuo. The resulting residue was dissolved in

EtOAc and washed with H₂O. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvent was evaporated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography (silica gel,

EtOAc in petroleum ether, 10/90 to 50/50). The desired fractions

were collected, and the solvent was evaporated in vacuo to yield 0.7 g

1

(30%) of 14a as a brown solid. Mp^a232.1−235.1 °C. H NMR (400

MHz, DMSO-d₆) δ ppm 3.00 (t, J = 7.0 Hz, 2 H), 3.50 (td, J = 7.0, 2.5

Hz, 2 H), 7.44−7.57 (m, 3 H), 7.63−7.72 (m, 2 H), 8.07 (br. s, 1 H).

LCMS: m/z 255 [M + H]⁺, t_R= 5.00 min.

5-Methyl-2-phenylethynyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-

4-one (14b). A mixture of 14a (0.25 g, 0.98 mmol), methyl iodide

(0.84 g, 5.9 mmol), and Cs₂CO₃(1.9 g, 5.9 mmol) in CH₃CN (50

mL) was stirred at 80 °C for 16 h. The mixture was then cooled to rt

and concentrated in vacuo. The crude product was puriﬁed by reverse

phase HPLC (0.1% TFA in CH₃CN/0.1% TFA in H₂O). The desired

fractions were collected, washed with a saturated solution of NaHCO₃

and extracted with EtOAc. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvent was evaporated in vacuo to

yield 50 mg (19%) of 14b. Mp^a146.1−147.2 °C. ¹H NMR (400 MHz,

2-Phenoxy-thioacetamide (33a). To a solution of phenoxyacetoni-

trile (38 g, 285 mmol) in DMF (380 mL), thioacetamide (42.8 g, 450

mmol) and a solution of 4 M HCl in 1.4-dioxane (380 mL) were

added. The mixture was stirred at 100 °C for 16 h and then cooled to

rt and poured into a saturated solution of NaHCO₃. The solid formed

was ﬁltered oﬀ, washed with H₂O, and dried in vacuo to yield 45 g

(94%) of 33a as a beige solid which was used in the next step without

any further puriﬁcation. ¹H NMR (500 MHz, CDCl₃) δ ppm 4.88 (s, 2

H), 6.89−6.97 (m, 2 H), 7.04 (t, J = 7.4 Hz, 1 H), 7.29−7.36 (m, 2

7253

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DMSO-d₆) δ ppm 2.97 (s, 3 H), 3.09 (t, J = 7.2 Hz, 2 H), 3.67 (t, J =

7.2 Hz, 2 H), 7.43−7.58 (m, 3 H), 7.67 (d, J = 6.8 Hz, 2 H). LCMS:

m/z 269 [M + H]⁺, t_R= 5.29 min.

CDCl₃) δ ppm 25.64 (s, 1 C) 34.14 (s, 1 C) 48.56 (s, 1 C) 67.30 (s, 1

C) 114.85 (s, 2 C) 121.89 (s, 1 C) 127.04 (s, 1 C) 129.56 (s, 2 C)

157.41−157.65 (m, 1 C) 157.69−157.96 (m, 1 C) 160.76−161.07 (m,

5-(2-Methoxy-ethyl)-2-phenylethynyl-6,7-dihydro-5H-thiazolo-

[5,4-c]pyridin-4-one (14c). Starting from 14a (0.7 g, 2.9 mmol) and 2-

bromoethyl methyl ether and following the procedure described for

14b, 14c was obtained as a yellow solid (150 mg, 20%). Mp^a144.5−

1 C) 171.74 (s, 1 C). LCMS: m/z 275 [M + H] ̧

⁺t_R= 1.76 min. HRMS

(ES⁺, M + H) calcd. for C₁₄H₁₅N₂O₂S: 275.0854, found 275.0853.

5-Ethyl-2-phenoxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-

4-one (16b). Iodoethane (0.069 mL, 0.86 mmol) was added to a

suspension of 31 (150 mg, 0.058 mmol) and Cs₂CO₃(281 mg, 0.86

mmol) in anhydrous DMF (2.5 mL). The mixture was stirred at rt for

16 h under nitrogen then at 100 °C for 1 h. The mixture was diluted

with H₂O and extracted with EtOAc. The organic layer was separated,

washed with brine, dried (Na₂SO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica gel, EtOAc in CH₂Cl₂, 0/100 to 50/50). The

desired fractions were collected and the solvents evaporated in vacuo.

The impure product was triturated with diisopropyl ether and puriﬁed

by reverse phase HPLC (gradient elution: 80% 0.1% NH₄CO₃H/

NH₄OH pH 9 solution in H₂O, 20% CH₃CN to 0% 0.1% NH₄CO₃H/

NH₄OH pH 9 solution in H₂O, 100% CH₃CN). The desired fractions

were collected and evaporated in vacuo to yield 62 mg (37%) of 16b

as a yellow solid. Mp^b116.0 °C. ¹H NMR (400 MHz, CDCl₃) δ ppm

1.21 (t, J = 7.2 Hz, 3 H), 3.12 (t, J = 7.1 Hz, 2 H), 3.57 (q, J = 7.2 Hz,

2 H), 3.67 (t, J = 7.2 Hz, 2 H), 5.33 (s, 2 H), 6.95 - 7.07 (m, 3 H), 7.28

- 7.36 (m, 2 H). ¹³C NMR (101 MHz, CDCl₃) δ ppm 12.79 (s, 1 C)

25.90 (s, 1 C) 41.35 (s, 1 C) 45.97 (s, 1 C) 67.30 (s, 1 C) 114.87 (s, 2

C) 121.92 (s, 1 C) 127.46 (s, 1 C) 129.61 (s, 2 C) 157.54 (s, 1 C)

157.82 (s, 1 C) 160.31 (s, 1 C) 171.75 (s, 1 C). LCMS: m/z 289 [M +

H]⁺, t_R= 2.00 min. HRMS (ES⁺, M + H) calcd. for C₁₅H₁₇N₂O₂S:

289.1011, found 289.1013.

5-Isopropyl-2-phenoxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16c). A mixture of 25a (0.23 g, 1.29 mmol) and 33a

(0.24 g, 1.4 mmol) in EtOH (10 mL) was stirred at rt for 15 min

before NaHCO₃(0.4 g, 3.9 mmol) was added. Then the mixture was

stirred at 70 °C for 15 min, diluted with EtOAc, and washed with

H₂O. The organic layer was separated, washed with brine, dried

(Na₂SO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude product was puriﬁed by reverse phase HPLC (gradient

elution: 0.1% TFA in CH₃CN/0.1% TFA in H₂O). The desired

fractions were collected and washed with a saturated solution of

NaHCO₃and extracted with EtOAc (2 × 100 mL). The organic layer

was separated, washed with brine, dried (Na₂SO₄), ﬁltered and the

solvents evaporated in vacuo to yield 81 mg (21%) of 16c as a white

solid. Mp^a104.1−106.0 °C. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.13

(d, J = 6.8 Hz, 6 H), 3.00 (t, J = 7.0 Hz, 2 H), 3.49 (t, J = 7.0 Hz, 2 H),

4.87 (spt, J = 6.8 Hz, 1 H), 5.26 (s, 2 H), 6.86−7.01 (m, 3 H), 7.21−

7.31 (m, 2 H). LCMS: m/z 303 [M + H]⁺, t_R= 5.42 min.

1

148.2 °C. H NMR (300 MHz, CDCl₃) δ ppm 3.06 (t, J = 7.0 Hz, 2

H), 3.30 (s, 3 H), 3.49−3.59 (m, 2 H), 3.59−3.68 (m, 2 H), 3.74 (t, J

= 7.0 Hz, 2 H), 7.26−7.43 (m, 3 H), 7.45−7.62 (m, 2 H). LCMS: m/z

313 [M + H]⁺, t_R= 6.01 min.

2-Benzyloxy-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-one (15a).

Benzyl alcohol (0.145 mL, 1.42 mmol) was added dropwise to a

suspension of NaH (0.067 g, 1.67 mmol, 60% in mineral oils) in THF

(6 mL) under nitrogen. The mixture was stirred at rt for 15 min and

then 21 (0.3 g, 1.29 mmol) was added. The mixture was stirred at 120

°C for 15 min under microwave irradiation in a sealed tube. The

solvent was removed with a nitrogen ﬂow to yield 0.340 g (70% pure)

of 15a. LCMS: m/z 261 [M + H]⁺, t_R= 2.15 min.

2-Benzyloxy-5-methyl-6,7-dihydro-5H-thiazolo[5,4-c]pyridin-4-

one (15b). 15a (0.34 g, 1.29 mmol) was added dropwise to a

suspension of NaH (0.067 g, 1.67 mmol, 60% in mineral oils) in THF

(2 mL) under nitrogen. The mixture was stirred for 15 min then

methyl iodide (0.12 mL, 1.68 mmol). The mixture was stirred at 120

°C for 15 min under microwave irradiation in a sealed tube. The

mixture was diluted with EtOAc and washed with a saturated solution

of NH₄Cl. The organic layer was separated, dried (Na₂SO₄), and

ﬁltered, and the solvent was evaporated in vacuo. The crude product

was puriﬁed by ﬂash column chromatography (silica gel, EtOAc in

CH₂Cl₂, 0/100 to 10/90). The desired fractions were collected and

the solvents evaporated in vacuo. The solid obtained was triturated

1

with heptane/Et₂O to yield 0.190 g (54%) of 15b. Mp^b106.4 °C. H

NMR (500 MHz, CDCl₃) δ ppm 2.96 (t, J = 7.2 Hz, 2 H), 3.05 (s, 3

H), 3.60 (t, J = 7.2 Hz, 2 H), 5.45 (s, 2 H), 7.34−7.50 (m, 5 H).

LCMS: m/z 275 [M + H]⁺, t_R= 1.68 min.

2-Benzyloxy-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (15c). Benzyl alcohol (0.38 mL, 3.67 mmol) was added

dropwise to a suspension of NaH (0.183 g, 4.58 mmol, 60% in mineral

oils) in THF (12 mL) under nitrogen. The mixture was stirred at rt for

15 min and then 26 (1 g, 3.06 mmol) was added. The mixture was

stirred at 120 °C for 25 min under microwave irradiation in a sealed

tube. The mixture was partitioned between CH₂Cl₂and H₂O. The

organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvent was evaporated in vacuo. The crude product was puriﬁed by

ﬂash column chromatography (silica gel, EtOAc in CH₂Cl₂in heptane,

0/0/100 to 10/10/80). The desired fractions were collected and

evaporated in vacuo to yield 0.68 g (63%) of 15c as a white solid. Mp^b

5-(2-Methoxy-ethyl)-2-phenoxymethyl-6,7-dihydro-5H-thiazolo-

[5,4-c]pyridin-4-one (16d). Starting from 31 (150 mg, 0.58 mmol)

and 2-bromoethyl methyl ether and following the procedure described

for 16b, 16d was obtained as a yellow solid (90 mg, 49%). Mp^b82.6

°C. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.10 (t, J = 7.1 Hz, 2 H), 3.36

(s, 3 H), 3.60 (t, J = 5.2 Hz, 2 H), 3.69 (t, J = 5.2 Hz, 2 H), 3.78 (t, J =

7.2 Hz, 2 H), 5.33 (s, 2 H), 6.97−7.04 (m, 3 H), 7.28−7.35 (m, 2 H).

LCMS: m/z 319 [M + H]⁺, t_R= 1.90 min.

5-Cyclopropyl-2-phenoxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16e). Starting from 25b (0.28 g, 1.2 mmol) and 33a

(0.23 g, 1.4 mmol) and following the procedure described for 16c, 16e

was obtained as a white solid (72 mg, 20%). Mp^a252.1−255.3 °C. ¹H

NMR (400 MHz, CDCl₃) δ ppm 0.62 - 0.69 (m, 2 H), 0.80 - 0.88 (m,

2 H), 2.65 (tt, J = 7.2, 3.7 Hz, 1 H), 2.99 (t, J = 7.0 Hz, 2 H), 3.63 (t, J

= 7.0 Hz, 2 H), 5.25 (s, 2 H), 6.89−6.99 (m, 3 H), 7.21−7.29 (m, 2

H). LCMS: m/z 301 [M + H]⁺, t_R= 4.86 min.

1

130.0 °C. H NMR (400 MHz, CDCl₃) δ ppm 3.08 (t, J = 6.9 Hz, 2

H), 4.01 (t, J = 6.9 Hz, 2 H), 5.49 (s, 2 H), 7.08 (t, J = 8.7 Hz, 2 H),

7.29 (dd, J = 9.0, 4.9 Hz, 2 H), 7.34−7.54 (m, 5 H). ¹³C NMR (126

MHz, CDCl₃) δ ppm 26.56 (s, 1 C) 49.57 (s, 1 C) 73.78 (s, 1 C)

115.68 (d, J = 22.00 Hz, 2 C) 119.25 (s, 1 C) 127.02 (d, J = 8.25 Hz, 2

C) 128.32 (s, 2 C) 128.64 (s, 2 C) 128.78 (s, 1 C) 134.65 (s, 1 C)

138.24 (d, J = 3.67 Hz, 1 C) 155.14 (s, 1 C) 160.62 (d, J = 245.61 Hz,

1 C) 160.68 (s, 1 C) 177.92 (s, 1 C). LCMS: m/z 355 [M+H]⁺, t_R=

2.44 min. HRMS (ES⁺, M + H) calcd. for C₁₉H₁₆N₂O₂SF: 355.0917,

found 355.0914.

5-Methyl-2-phenoxymethyl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16a). Di-tert-butyl azodicarboxylate (1.55 g, 5.90

mmol) was added to a stirred solution of 29 (0.90 g, 4.54 mmol),

phenol (0.534 g, 5.68 mmol) and PPh₃(1.55 g, 5.90 mmol) in THF

(15 mL) under nitrogen. The mixture was stirred at 120 °C for 20 min

under microwave irradiation. The solvent was evaporated in vacuo.

The crude product was puriﬁed by ﬂash column chromatography

(silica gel, EtOAc in CH₂Cl₂, 0/100 to 50/50). The desired fractions

were collected and evaporated in vacuo, and the residue was triturated

with diisopropyl ether to yield 0.253 g (20%) of 16a as a white solid.

Mp^a141.5−142.4 °C. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.10 (s, 3

H), 3.14 (t, J = 7.2 Hz, 2 H), 3.67 (t, J = 7.1 Hz, 2 H), 5.33 (s, 2 H),

6.96−7.05 (m, 3 H), 7.28−7.35 (m, 2 H). ¹³C NMR (126 MHz,

5-Cyclopropylmethyl-2-phenoxymethyl-6,7-dihydro-5H-thiazolo-

[5,4-c]pyridin-4-one (16f). Starting from 25c (250 mg, 1.01 mmol)

and 33a (0.2 g, 1.22 mmol) and following the procedure described for

16c, 16f was obtained as a white solid (78 mg, 25%). Mp^a292.6−

1

302.9 °C. H NMR (400 MHz, CDCl₃) δ ppm 0.23 (q, J = 4.9 Hz, 2

H), 0.40 - 0.56 (m, 2 H), 0.87 - 1.06 (m, 1 H), 3.08 (t, J = 7.2 Hz, 2

H), 3.35 (d, J = 7.0 Hz, 2 H), 3.72 (t, J = 7.2 Hz, 2 H), 5.28 (s, 2 H),

6.86 - 7.02 (m, 3 H), 7.21 - 7.33 (m, 2 H). ¹³C NMR (101 MHz,

7254

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CDCl₃) δ ppm 3.49 (s, 2 C) 9.53 (s, 1 C) 25.89 (s, 1 C) 46.52 (s, 1 C)

50.68 (s, 1 C) 67.32 (s, 1 C) 114.89 (s, 2 C) 121.93 (s, 1 C) 127.46 (s,

1 C) 129.62 (s, 2 C) 157.55 (s, 1 C) 157.85 (s, 1 C) 160.56 (s, 1 C)

171.80 (s, 1 C). LCMS: m/z 303 [M + H]⁺, t_R= 5.32 min. HRMS

(ES⁺, M + H) calcd. for C₁₇H₁₉N₂O₂S: 315.1167, found 315.1169.

2-Phenoxymethyl-5-(tetrahydro-pyran-4-ylmethyl)-6,7-dihydro-

5H-thiazolo[5,4-c]pyridin-4-one (16g). Starting from 31 (150 mg,

0.58 mmol) and 4-(bromomethyl)tetrahydropyran and following the

procedure described for 16a, 16g was obtained as a white solid (27

7.09 (br. t, J = 8.4, 8.4 Hz, 2 H), 7.26−7.35 (m, 2 H). LCMS: m/z 373

[M + H]⁺, t_R= 4.62 min.

2-Phenoxymethyl-5-pyridin-2-yl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16m). K₂CO₃(111 mg, 0.81 mmol) was added to a

stirred suspension of 31 (150 mg, 0.4 mmol), 2-iodopyridine (0.043

mL, 0.4 mmol), CuI (0.015 g, 0.081 mmol) and N,N′-dimethylethy-

lenediamine (0.026 mL, 0.24 mmol) in toluene (3 mL) in a sealed

tube and under nitrogen. The mixture was stirred at 120 °C for 16 h,

ﬁltered through a Celite pad, and washed with EtOAc and the ﬁltrate

was evaporated in vacuo. The crude product was puriﬁed by ﬂash

column chromatography (silica gel, EtOAc in CH₂Cl₂, 0/100 to 100/

0). The desired fractions were collected and the solvents were

evaporated in vacuo. The solid was triturated with diisopropyl ether to

1

mg, 13%). H NMR (400 MHz, CDCl₃) δ ppm 1.34−1.48 (m, 2 H),

1.59−1.68 (m, 2 H), 1.90−2.04 (m, 1 H), 3.11 (t, J = 7.0 Hz, 2 H),

3.32−3.42 (m, 2 H), 3.40 (d, J = 7.4 Hz, 2 H), 3.69 (t, J = 7.0 Hz, 2

H), 3.94−4.03 (m, 2 H), 5.33 (s, 2 H), 6.97−7.05 (m, 3 H), 7.28−7.35

(m, 2 H). LCMS: m/z 359 [M + H]⁺, t_R= 2.10 min.

1

yield 57 mg (57%) of 16m as a white solid. Mp^b143.9 °C. H NMR

(400 MHz, CDCl₃) δ ppm 3.23 (t, J = 6.8 Hz, 2 H), 4.47 (t, J = 6.8

Hz, 2 H), 5.37 (s, 2 H), 7.00−7.06 (m, 3 H), 7.07−7.13 (m, 1 H),

7.29−7.37 (m, 2 H), 7.71 (ddd, J = 8.6, 7.1, 1.8 Hz, 1 H), 7.89 (d, J =

8.3 Hz, 1 H), 8.44 (br. s, 1 H). LCMS: m/z 338 [M + H]⁺, t_R= 2.42

min.

5-(4-Fluoro-phenyl)-2-phenoxymethyl-6,7-dihydro-5H-thiazolo-

[5,4-c]pyridin-4-one (16h). A mixture of 25d (0.41 g, 1.45 mmol) and

33a (0.22 g, 1.3 mmol) in DMF (5 mL) was stirred at rt for 15 min

before NaHCO₃(0.19 g, 2.3 mmol) was added. Then the mixture was

stirred at 100 °C for 30 min, diluted with EtOAc and washed with

H₂O. The organic layer was separated, dried (MgSO₄), and ﬁltered,

and the solvents were evaporated in vacuo. The crude product was

puriﬁed by ﬂash column chromatography (silica gel, EtOAc in CH₂Cl₂,

0/100 to 2/98). The desired fractions were collected, and the solvents

were evaporated in vacuo to yield 0.084 g (16%) of 16h as a white

solid. Mp^b163.3 °C. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.26 (t, J =

6.9 Hz, 2 H), 4.08 (t, J = 6.9 Hz, 2 H), 5.36 (s, 2 H), 6.98−7.06 (m, 3

H), 7.07−7.13 (m, 2 H), 7.28−7.36 (m, 4 H). ¹³C NMR (126 MHz,

CDCl₃) δ ppm 26.32 (s, 1 C) 50.16 (s, 1 C) 67.37 (s, 1 C) 114.87 (s,

2 C) 115.82 (d, J = 22.91 Hz, 2 C) 122.01 (s, 1 C) 127.15 (d, J = 8.25

Hz, 2 C) 127.38 (s, 1 C) 129.64 (s, 2 C) 137.93 (d, J = 3.67 Hz, 1 C)

157.50 (s, 1 C) 158.79 (s, 1 C) 160.79 (d, J = 246.52 Hz, 1 C) 160.36

(s, 1 C) 172.95 (s, 1 C). LCMS: m/z 355 [M + H]⁺, t_R= 6.57 min.

HRMS (ES⁺, M + H) calcd. for C₁₉H₁₆N₂O₂SF: 355.0917, found

355.0916.

2-Phenoxymethyl-5-pyridin-3-yl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16n). Starting from 31 (150 mg, 0.58 mmol) and 3-

bromopyridine and following the procedure described for 16m, 16n

1

was obtained as a white solid (29 mg, 15%). H NMR (400 MHz,

CDCl₃) δ ppm 3.30 (t, J = 6.8 Hz, 2 H), 4.17 (t, J = 6.8 Hz, 2 H), 5.37

(s, 2 H), 6.96−7.09 (m, 3 H), 7.29−7.41 (m, 3 H), 7.71−7.80 (m, 1

H), 8.50 (dd, J = 4.7, 1.3 Hz, 1 H), 8.65 (d, J = 2.3 Hz, 1 H). LCMS:

m/z 338 [M + H]⁺, t_R= 2.00 min.

2-Phenoxymethyl-5-pyridin-4-yl-6,7-dihydro-5H-thiazolo[5,4-c]-

pyridin-4-one (16o). Starting from 31 (150 mg, 0.58 mmol) and 4-

iodopyridine and following the procedure described for 16m, 16o was

obtained as a white solid (5 mg, 2.5%). ¹H NMR (500 MHz, CDCl₃) δ

ppm 3.29 (t, J = 6.8 Hz, 2 H), 4.19 (t, J = 6.8 Hz, 2 H), 5.37 (s, 2 H),

6.90−7.12 (m, 3 H), 7.29 - 7.41 (m, 4 H), 8.62 (d, J = 6.4 Hz, 2 H).

LCMS: m/z 338 [M + H]⁺, t_R= 2.09 min.

5-(5-Fluoro-pyridin-2-yl)-2-phenoxymethyl-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16p). K₃PO₄(1.6 g, 7.49 mmol) was

added to a stirred suspension of 31 (650 mg, 2.5 mmol), 2-bromo-5-

ﬂuoropyridine (879 mg, 4.99 mmol), CuI (475 mg, 2.5 mmol) and

N,N′-dimethylethylenediamine (0.81 mL, 7.49 mmol) in toluene (12

mL) in a sealed tube and under nitrogen. The mixture was stirred at

140 °C for 16 h and then ﬁltered through a Celite pad. The ﬁltrate was

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica gel, EtOAc in CH₂Cl₂, 0/100 to 30/70). The

desired fractions were collected and the solvents evaporated in vacuo.

The solid was triturated with diisopropyl ether to yield 570 mg (64%)

5-(2,4-Diﬂuoro-phenyl)-2-phenoxymethyl-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16i). Starting from 25e (300 mg, 0.99

mmol) and 33a (148 m_g, 0.89 mmol) and following the procedure

described for 16h, 16i was obtained as a yellow solid (37 mg, 10%).

Mp^a268.9−284.1 °C. ¹H NMR (400 MHz, CDCl₃) δ ppm 3.28 (t, J =

6.9 Hz, 2 H), 4.01 (t, J = 6.8 Hz, 2 H), 5.37 (s, 2 H), 6.89−6.99 (m, 2

H), 6.99−7.07 (m, 3 H), 7.29−7.39 (m, 3 H). LCMS: m/z 373 [M +

H]⁺, t_R= 5.63 min.

2-(2-Fluoro-phenoxymethyl)-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16j). A mixture of 35 (0.3 g, 1.01

mmol), 2-ﬂuorophenol (0.15 g, 1.3 mmol) and K₂CO₃(0.4 g, 3

mmol) in DMF (40 mL) was stirred at rt for 1 h. The mixture was

ﬁltered and the solvent evaporated in vacuo. The crude product was

puriﬁed by reverse phase HPLC (gradient elution: 0.1% TFA in

CH₃CN/0.1% TFA in H₂O). The desired fractions were collected and

washed with a saturated solution of NaHCO₃and extracted with

EtOAc (2 × 100 mL). The organic layer was separated, dried

(Na₂SO₄), ﬁltered and the solvent evaporated in vacuo to yield 31 mg

(8%) of 16j as a solid. Mp^a> 280 °C. ¹H NMR (300 MHz, CDCl₃) δ

ppm 3.25 (t, J = 6.9 Hz, 2 H), 4.07 (t, J = 6.8 Hz, 2 H), 5.41 (s, 2 H),

6.92−7.19 (m, 6 H), 7.19−7.42 (m, 2 H). LCMS: m/z 373 [M + H]⁺,

t_R= 4.58 min.

1

of 16p as a white solid. Mp^a131.7−132.8 °C. H NMR (500 MHz,

CDCl₃) δ ppm 3.23 (t, J = 6.8 Hz, 2 H), 4.42 (t, J = 6.9 Hz, 2 H), 5.37

(s, 2 H), 6.86−7.12 (m, 3 H), 7.29−7.38 (m, 2 H), 7.39 - 7.50 (m, 1

H), 7.89 (dd, J = 9.2, 4.0 Hz, 1 H), 8.27 (d, J = 3.2 Hz, 1 H). ¹³C

NMR (101 MHz, CDCl₃) δ ppm 26.15 (s, 1 C) 46.39 (s, 1 C) 67.41

(s, 1 C) 114.83 (s, 2 C) 120.72 (d, J = 4.24 Hz, 1 C) 122.02 (s, 2 C)

124.21 (d, J = 19.78 Hz, 1 C) 127.41 (s, 1 C) 129.65 (s, 2 C) 134.92

(d, J = 25.43 Hz, 1 C) 149.26 (d, J = 2.12 Hz, 1 C) 158.76 (d, J =

259.96 Hz, 1 C) 157.89 (s, 1 C) 160.67 (s, 1 C) 173.65 (s, 1 C).

LCMS: m/z 356 [M + H]⁺, t_R= 5.99 min. HRMS (ES⁺, M + H) calcd.

for C₁₈H₁₅N₃O₂SF: 356.0869, found 356.0870.

5-(3-Fluoro-pyridin-2-yl)-2-phenoxymethyl-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16q). Starting from 31 (150 mg, 0.58

mmol) and 2-chloro-3-ﬂuoropyridine and following the procedure

described for 16m, 16q was obtained as a white solid (22 mg, 15%).

¹H NMR (400 MHz, CDCl₃) δ ppm 3.29 (t, J = 6.8 Hz, 2 H), 4.25 (t,

J = 6.8 Hz, 2 H), 5.38 (s, 2 H), 6.98−7.06 (m, 3 H), 7.23−7.29 (m, 1

H), 7.30 - 7.40 (m, 2 H), 7.51 (ddd, J = 9.7, 8.1, 1.6 Hz, 1 H), 8.31 (dt,

J = 4.8, 1.2 Hz, 1 H). LCMS: m/z 356 [M + H]⁺, t_R= 2.37 min.

Biology. Cell Culture. Human embryonic kidney (HEK) 293 cells

were stably transfected with human mGluR_5acDNA in expression

vector pcDNA4/TO. hmGlu₅receptor-expressing HEK293 cells were

maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM)

supplemented with 10% heat inactivated dialyzed fetal bovine serum

and antibiotics (88 IU/mL penicillin G and 88 μg/mL streptomycin

2-(3-Fluoro-phenoxymethyl)-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16k). Starting from 25d (0.39 g, 1.38

mmol) and 33b (230 mg, 1.24 mmol) and following the procedure

described for 16h, 16k was obtained as a white solid (120 mg, 23%).

Mp^b152.8 °C. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.27 (t, J = 6.9 Hz,

2 H), 4.09 (t, J = 6.9 Hz, 2 H), 5.35 (s, 2 H), 6.69−6.84 (m, 2 H),

7.07−7.16 (m, 2 H), 7.23−7.35 (m, 4 H). LCMS: m/z 372 [M + H]⁺,

t_R= 2.88 min.

2-(4-Fluoro-phenoxymethyl)-5-(4-ﬂuoro-phenyl)-6,7-dihydro-5H-

thiazolo[5,4-c]pyridin-4-one (16l). Starting from 35 (0.3 g, 1.01

mmol) and 4-ﬂuorophenol (0.15 g, 1.3 mmol) and following the

procedure described for 16j, 16l was obtained as a solid (74 mg, 20%).

1

Mp^a> 280 °C. H NMR (300 MHz, CDCl₃) δ ppm 3.25 (t, J = 6.7

Hz, 2 H), 4.07 (t, J = 6.8 Hz, 2 H), 5.31 (s, 2 H), 6.85−7.05 (m, 4 H),

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sulfate). Cells were detached with PBS w/o Ca²⁺/Mg²⁺containing

0.04% EDTA and 0.005% trypsin. The cells were grown in 175 cm²

culture ﬂasks at 37 °C in a 5% CO₂environment and hold under

permanent selection with 200 μg/mL zeocin and 5 μg/mL blasticidin.

Cells were cultured at 40 000 cells/cm²or 20 000 cells/cm²for

respectively 3 or 4 days of growth in a F175 ﬂask.

dose of 16a p.o., followed by a subcutaneous injection of 1 mg/kg

amphetamine or vehicle and monitored for an additional 60 min.

Treatment groups. Dose group 1: VAMP = 20% β-CD (16a vehicle),

p.o. +1 mg/kg AMP, sc (n = 8). Dose group 2: 3AMP = 3 mg/kg 16a,

p.o. +1 mg/kg AMP, sc (n = 6). Dose group 3: 10AMP = 10 mg/kg

16a, po +1 mg/kg AMP, sc (n = 7). Dose group 4: 30AMP = 30 mg/

kg 16a, p.o. +1 mg/kg AMP, sc (n = 8). Dose group 5: 56.6AMP =

56.6 mg/kg 16a, p.o. + 1 mg/kg AMP, sc (n = 8). Dose group 6:

100AMP = 100 mg/kg 16a, p.o. +1 mg/kg AMP, sc (n = 8). Dose

group 7: 100 V = 100 mg/kg 16a, p.o. + sterile water (AMP vehicle),

sc (n = 8). Changes in locomotor activity were recorded for a total of

120 min. Data were expressed as changes in ambulation deﬁned as the

total number of photobeam breaks per 5 min interval. At the end of

this behavioral study, each animal was euthanized, then decapitated,

and the plasma and brain tissues were collected for the evaluation of

exposure levels of 16a. Data Analysis. Behavioral data were analyzed

using a one-way ANOVA with main eﬀects of treatment and time. Post

hoc analyses were performed using a Dunnett’s t-test with all treatment

groups compared to the VAMP group using JMP 8.0 (SAS Institute,

Cary, NC) statistical software. Data were graphed using SigmaPlot for

Windows Version 11.0 (Saugua, MA). A probability of p ≤ 0.05 was

taken as the level of statistical signiﬁcance. Percent Eﬀect and Reversal

calculations for the 3AMP, 10AMP, 30AMP, 56.6AMP, and 100AMP

treatment groups were performed with the following formula relative

to the VAMP treatment group: (1) Total number of photobeam

breaks in the time interval from t = 60 to t = 120 was calculated for

each rat in each treatment group; (2) Mean total number of

photobeam breaks in the time interval from t = 60 to t = 120 was

calculated for the VAMP group; (3) Percent eﬀect = ratio of the total

number of photobeam breaks for each rat in each treatment group

divided by the mean total number of photobeam breaks in the time

interval from t = 60 to t = 120 of the VAMP group multiplied by 100;

(4) Percent reversal = 100-the percent eﬀect for each rat in each

treatment group; (5) ﬁnally the mean SE percent reversal for each

treatment group was calculated from the individual percent reversal

values. Percent eﬀect and change calculations for the VAMP and 100 V

treatment groups relative to the VV treatment group were also

performed with the following formula: (1) Total number of

photobeam breaks in the time interval from t = 60 to t = 120 was

calculated for each rat in each treatment group; (2) Mean total

number of photobeam breaks in the time interval from t = 60 to t =

120 was calculated for the VV group; (3) Percent Eﬀect = Ratio of the

total number of photobeam breaks for each rat in each treatment

group divided by the mean total number of photobeam breaks in the

time interval from t = 60 to t = 120 of the VV group multiplied by 100;

(4) percent change = the percent eﬀect for each rat in each treatment

Calcium Mobilization Assay. Cells were seeded at 40 000 cells/well

in PDL-coated MW384, and a day later, medium was removed before

addition of a ﬂuo-4-AM solution (HBSS, 2.5 mM probenecid and ∼2

μM ﬂuo-4-AM, pH 7.4). Measurements were performed 90 min later

(while incubation at 37 °C and 5% CO₂) using the FDSS. Two

additions were made: ﬁrst, compound alone was added at 6 s to detect

any agonist activity. Next, a suboptimal concentration (nominally 20%

of the maximum response, EC₂₀) of glutamate was added at 157 s to

detect any potentiation of the glutamate response. For each interval,

Ca²⁺ﬂuorescence of the Ca²⁺-bound dye was monitored every second,

at an excitation wavelength of 480 nm and emission at 540 nm. The

ratio of peak versus basal ﬂuorescent signals was used for data analysis.

Data Analysis. Data were normalized to the control agonist

response and sigmoid concentration−response curves plotting these

percentages versus the log concentration of the test compound were

analyzed using a 4 parameter logistic nonlinear regression model

programmed in R (R Development Core Team, 2010. R: A language

and environment for statistical computing. R Foundation for Statistical

Computing,Vienna, Austria. ISBN 3−900051−07−0, URL http://

www.R-project.org/.). The model was determined by the maximal

eﬀect of each compound (E_max), its EC₅₀and the response in the

absence of compound. The fourth parameter determining the slope

was ﬁxed to 1. In addition, the heterogeneity between experiments and

concentration−response curves for each compound was taken into

account by the inclusion of a random eﬀect in both ‘E₀’ (response in

the absence of compound) and E_maxof the model

In Vivo Pharmacology. (a) Animal Husbandry. Animals were

housed in the animal care facility certiﬁed by the American Association

for the Accreditation of Laboratory Animal Care (AAALAC) under a

12-h light/dark cycle (lights on: 7 a.m.; lights oﬀ: 7 p.m.) and had free

access to food and water. The animals used in these experiments were

food-deprived the evening before experimentation for oral admin-

istration of test compound. The experimental protocols performed

during the light cycle were approved by the Institutional Animals Care

and Use Committee of Vanderbilt University and conformed to the

guidelines established by the National Research Council Guide for the

Care and Use of Laboratory Animals.

(b) Preparation of Test Article. 16a was formulated in volumes

speciﬁc to the number of animals dosed each day. The solutions were

formulated so that animals were injected with a maximal dosing

volume of 10 mL/kg. The appropriate amount according to the dosage

was mixed into a 20% 2-(hydroxypropyl)-β-cyclodextrin in sterile

water (HP-β-CD; Sigma, Cat. No. C0926-10G) solution. Each mixture

was ultrahomogenized on ice for 2−3 min using a hand-held tissue

homogenizer. Next, the pH of all solutions was checked using 0−14

EMD pH strips and adjusted to a pH of 6−7 if necessary with 1N

NaOH. The mixtures were then vortexed, and stored in a sonication

bath at 40 °C until time of injection. d-Amphetamine hemisulfate

(AMP) was obtained from Sigma (Cat. No. A5880-1G; St. Louis,

MO). Salt-correction was used to determine the correct amount of the

d-amphetamine hemisulfate form in mg to add to sterile water in order

to yield a 1 mg/mL solution; injected with a maximal dosing volume of

10 mL/kg.

Reversal of Amphetamine-Induced Hyperlocomotion. Studies

were conducted using Smart Open Field activity chambers (27 × 27 ×

20 cm) (Kinder Scientiﬁc, Poway, CA) equipped with 16 horizontal

(x- and y-axes) infrared photobeams. Changes in locomotor activity

were measured as the number of photobeam breaks over time and

were recorded with a Pentium I computer equipped with rat activity

monitoring system software (Motor Monitor, Kinder Scientiﬁc,

Poway, CA). Male Harlan Sprague−Dawley rats (Harlan Laboratories,

Indianapolis, IN) weighing 250−375 g were used. Animals were

habituated in the locomotor activity test chambers for 30 min. Animals

were next pretreated for an additional 30 min with either vehicle or a

group −100; (5) ﬁnally the Mean

SE percent change for each

treatment group was calculated from the individual percent change

values. LC-MS/MS Sample Preparation and Analysis Methodology. Brain

samples were homogenized in 3 mL of 70:30 isopropanol/water and

then centrifuged at 3500 rpm for 5 min. Resulting brain sample

supernatant and plasma samples were transferred into a 96-well plate

containing plasma blanks, plasma double blank, a standard curve of the

test article, and quality control samples for subsequent LC-MS/MS

analysis. Each sample was diluted with acetonitrile containing 50 nM

carbamazepine (internal standard), centrifuged at 3500 rpm, and the

resulting supernatant was transferred to a new 96-well plate. After an

equal volume of water was added to each sample (to provide 50:50

acetonitrile/water solution), the plate was analyzed via ESI on a triple-

quadrupole mass spectrometer (AB Sciex API-4000) coupled with an

autosampling liquid chromatography system (Shimadzu LC-10AD

pumps, Leap Technologies CTC PAL autosampler). Analyte (test

article) was separated by gradient elution using a C18 3.0 × 50 mm, 3

μm column (Fortis Technologies) thermostatted at 40 °C. HPLC

mobile phase A was 0.1% formic acid in water (pH unadjusted),

mobile phase B was 0.1% formic acid in acetonitrile (pH unadjusted),

and the gradient used was 30−90% mobile phase B with 0.2 min hold

at 30% B, linear increase to 90% B over 0.8 min, hold at 90% B for 0.6

min, and a return to 30% B over 0.1 min followed by a re-equilibration

(0.5 min) to provide a 2.0 min total run time per sample. HPLC ﬂow

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Article

rate was 0.5 mL/min, source temperature was 500 °C, and mass

spectral analyses were performed using the following MRM

transitions: 353.5 → 259.2 and 353.5 → 123.2 m/z. ESI was achieved

by a Turbo-Ionspray source in positive ionization mode (5.0 kV spray

voltage). The raw plasma and brain concentration data obtained by

LC-MS/MS were analyzed by Analyst software (AB Sciex, version

1.5.1), which used the ratio of peak area responses of drug relative to

internal standard to construct a standard curve with a dynamic range

covering the concentrations found in the samples. Free plasma and

brain concentrations were calculated by multiplication of the total

concentration values by f_uplasma and f_ubrain, respectively, based on

data from in vitro rat plasma protein and rat brain homogenate binding

experiments.

Rotarod. The eﬀects of 16a on motor performance were evaluated

using an automated rotarod setup with a rotarod (7.0 cm in diameter)

rotating at a constant speed of 20 rotations/min (MedAssociates, Inc.,

St Albans, CA). Male Harlan Sprague−Dawley rats (Harlan

Laboratories, Indianapolis, IN) weighing 250−300 g were used.

Animals were given two training trials of 120 s on the rotarod with a

10 min interval between trials, followed by baseline assessment of

performance with a 120 s trial, any animals that did not reach a

performance criteria of 85 s were excluded from the study. Animals

were next pretreated for 30 min with vehicle (20% HP-β-CD) or a

dose of 16a (n = 6 each group) and then tested on the rotarod using a

120 s trial. The amount of time in s that each animal remained on the

rotarod was recorded; animals not falling oﬀ of the rotarod were given

a maximal score of 120 s. Data were expressed as the mean latency to

fall oﬀ the rotarod in seconds for each treatment group. Data Analysis.

Behavioral data were analyzed using a one-way ANOVA with main

eﬀects of treatment. Post hoc analyses were performed using a

Dunnett’s t test with all treatment groups compared to the vehicle

group using JMP 8.0 (SAS Institute, Cary, NC) statistical software.

Data were graphed using SigmaPlot for Windows Version 11.0

(Saugua, MA). A probability of p ≤ 0.05 was taken as the level of

statistical signiﬁcance.

Modiﬁed Irwin Neurological Test Battery. Male Harlan Sprague−

Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 250−

300 g were used. Animals were evaluated in the modiﬁed Irwin

neurological test battery at t = 0 to provide a baseline measurement

across each functional end point. Animals were next administered

either vehicle (20% HP-β-CD) or a 100 mg/kg p.o. dose of 16a (n = 6

each group) and then assessed after a 30 min, 1 h, and 4 h

pretreatment interval in the modiﬁed Irwin neurological test battery.

Changes in the diﬀerent functional end points of the Irwin test battery

were given a score of 0, 1 or 2; with 0 = no eﬀect, 1 = moderate eﬀect,

and 2 = robust full eﬀect. All data were collected blinded to treatment

for each animal. Following functional end points were scored.

Autonomic nervous system functions: ptosis, exophthalmos, miosis,

mydriasis, corneal reﬂex, pinna reﬂex, piloerection, respiratory rate,

writhing, tail erection, lacrimation, salivation, vasodilation, skin color,

irritability and rectal temperature; Somatomotor nervous system

functions: motor activity, ataxia, arch/roll, tremors, leg weakness, rigid

stance, spraddle, placing loss, grasping loss, righting loss, catalepsy, tail

pinch reaction, escape loss and physical appearance. Data Analysis.

Mean change values for each functional end point in the Irwin test

battery of each treatment group were calculated using Microsoft Excel.

Behavioral data were then analyzed using a one-way ANOVA with

main eﬀect of treatment. Post hoc analyses were performed using a

Dunnett’s t test with all treatment groups compared to the vehicle

group using JMP 8.0 (SAS Institute, Cary, NC) statistical software.

Data were graphed using SigmaPlot for Windows Version 11.0

(Saugua, MA). A probability of p ≤ 0.05 was taken as the level of

statistical signiﬁcance.

rats. This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: +34 925 245778. Fax: +34 925 245771. E-mail:

jbartolo@its.jnj.com.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Vanderbilt Center for Neuroscience Drug Discovery

(VCNDD) research was supported by grants from Janssen

Pharmaceutical Companies of Johnson & Johnson and in part

by the NIH (NS031373, MH062646). The authors would like

to thank Dr. Lieve Heylen and Dr. Tom Jacobs for their help

with data management and interpretation, Dr. Han Min Tong

for her assistance in the preparation of this manuscript, and the

members of the puriﬁcation and analysis group and the biology

and ADME/Tox teams from Janssen R&D for their

experimental contribution.

ABBREVIATIONS USED

■

AMP, d-amphetamine hemisulfate; AUC, area under the curve;

b/p, brain to plasma ratio; chromanone, 2,3-dihydro-4H-1-

benzopyran-4-one; CL_p, plasma clearance; C_max, peak plasma

concentration; CNS, central nervous system; CRC, concen-

tration response curve; D₂, dopamine 2; DAD, diode array

detector; dihydrobenzothiazolone, 5,6-dihydro-7(4H)-benzo-

thiazolone; dihydrothiazolopyridone, 6,7-dihydro-thiazolo[5,4-

c]pyridin-4(5H)-one; DMPK, drug metabolism and pharmaco-

kinetic; E_hep, hepatic extraction ratio; EPS, extrapyramidal

symptoms; ESCI, electrospray combined with atmospheric

pressure chemical ionization; f_u, fraction unbound; GABAergic,

γ-aminobutiric acidergic; HEK, human embryonic kidney;

HLM, human liver microsome; HP-β-CD, 2-hydroxylpropyl-

β-cyclodextrin; HPLC, high-performance liquid chromatogra-

phy; HTS, high throughput screening; iv, intravenous; LC-

HRMS, liquid chromatography combined with high resolution

mass spectrometry; LCMS, liquid chromatography combined

with mass spectrometry; LE, ligand eﬃciency; Mp, melting

point; NMDA, N-methyl-^D-aspartate; mGlu, metabotropic

glutamate; NMR, nuclear magnetic resonance (NMR);

mGlu₅, metabotropic glutamate 5; PAM, positive allosteric

modulator; PK, pharmacokinetic; PKC, protein kinase C; ppm,

parts per million; p.o., oral; RLM, rat liver microsome; RP-

HPLC, reversed-phase high-performance liquid chromatogra-

phy; SAR, structure activity relationship; sc, subcutaneous;

SPR, structure properties relationship; T_1/2, half-life; T_max, t_R,

retention time; time to reach peak concentration; TLC, thin

layer chromatography; VCNDD, Vanderbilt Center for Neuro-

science Drug Discovery

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ASSOCIATED CONTENT

■

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dx.doi.org/10.1021/jm400650w | J. Med. Chem. 2013, 56, 7243−7259

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Dihydrothiazolopyridone derivatives as a novel family of positive allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor

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