Dihydrothiazolopyridone Derivatives as a Novel Family of Positive Allosteric Modulators of the Metabotropic Glutamate 5 (mGlu₅) Receptor

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 mGlu₅ receptor PAM 16a are described.

INTRODUCTION

Schizophrenia is a mental disorder that affects approximately 1% of the world population.¹ This 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.¹ Currently 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 involve direct interaction with dopaminergic receptors have been proposed and investigated.^2,3 The 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 D₂ receptor 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.⁴ On the basis of these 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.² The mGlu₅ receptor belongs to the Group I mGlu receptor subfamily, together with the mGlu₁ receptor.⁵ They activate phospholipase C (PLC) via coupling to G_q proteins and are ubiquitously expressed on postsynaptic excitatory terminals in limbic brain regions that are involved in motivational, emotional, and cognitive processes.⁶ At GABA-ergic interneurons, mGlu₅ and NMDA receptors are coex-pressed⁷ and are not only coupled via intracellular signaling pathways but also physically through scaffolding proteins.⁸ In summary, mGlu₅ receptor-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 mGlu₅ PAMs such as 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, 1)^10,11 or 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-(pyridin-4-ylethynyl)pyridine-2-carboxamide (LSN2463359, 4),^14,15 4-butoxy-N-(2,4-difluor-ophenyl)benzamide (VU0357121, 5),¹⁶ 2-(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),¹⁸ and (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).¹⁵ These 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 mGlu₅ receptor 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 mGlu₅ receptor PAMs as improved antipsychotics.

RESULTS

Given the promise offered by mGlu₅ receptor 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 mGlu₅ PAM 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 9,¹⁹ as a promising hit. Compound 9 possessed potent in vitro mGlu₅ PAM 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-hydroxychromanone, were not responsible for the mGlu₅ PAM 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 selectivity²⁰ versus other representative members of the mGlu family (mGlu^1,2,8 EC₅₀ > 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.

As seen in Figure 2, our strategy for the exploration of the chromanone 9 followed two parallel approaches, first fixing the 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).

Figure 2.

Parallel simultaneous evolution from HTS hit 9 to optimized series 15 and 16.

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.²² Among this set of linkers, only previously published benzyloxy and phenoxymethyl derivatives 10 and 11¹⁹ retained significant mGlu₅ receptor 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_1–4,6–8 EC₅₀ > 10 μM). Similar moieties have recently been reported for other mGlu₅ PAM chemotypes.^19,23

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 mGlu₅ receptor PAMs have a common phenylacetylene moiety.⁹ For comparative purposes, we synthesized the corresponding phenylacetylene chromanone analogue 12¹⁹ which was also found to be an mGlu₅ receptor 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 13²⁴ emerged as a highly potent (~21 fold potency increase vs 12), efficacious and selective mGlu₅ receptor 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, compound 14a showed comparable mGlu₅ potency (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 derivatization 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.

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

Table 1.

Structures and Activities of Substituted Benzyloxy- and Phenoxymethyl-dihydrothiazolopyridones 15 and 16

Comp	X-Y	R1	R2	hmGlu5 PAM EC50a (nM)19	hmGlu5 PAM pEC50a19	Glu Maxa (%)19	HLMb (%)	RLMb (%)	clogPc
1	--	--	--	176	6.75 (86.73-6.78)	73 (70-76)	--	--	--
2	--	--	--	199	6.70 (6.66-6.74)	66 (63-70)	--	--	--
15b	CH2-O		H	1748	5.76 (5.43-6.08)	76 (55-97)	n.t.d	n.t.d	1.7
15c	CH2-O		H	1133	5.95 (5.76-6.13)	67 (55-79)	7	25	3.6
16a	O-CH2		H	1195	5.92 (5.84-6.01)	89 (79-99)	22	68	1.3
16b	O-CH2		H	696	6.16 (5.99-6.32)	76 (67-85)	72	99	1.8
16c	O-CH2		H	1211	5.92 (5.60-6.24)	42 (28-55)	n.t.d	n.t.d	2.1
16d	O-CH2		H	2314	5.64 (5.43-5.85)	66 (49-83)	n.t.d	n.t.d	1.6
16e	O-CH2		H	1459	5.84 (5.61-6.06)	66 (54-79)	n.t.d	n.t.d	1.8
16f	O-CH2		H	250	6.60 (6.34-6.86)	73 (55-91)	46	99	2.3
16g	O-CH2		H	851	6.07 (5.86-6.28)	71 (60-81)	n.t.d	n.t.d	1.5
16h	O-CH2		H	96	7.02 (6.78-7.25)	79 (63-95)	3	44	3.2
16i	O-CH2		H	66	7.18 (6.95-7.41)	69 (54-83)	14	43	3.2
16j	O-CH2		2-F	553	6.26 (6.09-6.43)	72 (64-80)	n.t.d	n.t.d	3.3
16k	O-CH2		3-F	77	7.12 (6.85-7.38)	61 (40-83)	18	48	3.5
16l	O-CH2		4-F	739	6.13 (6.02-6.24)	79 (73-85)	n.t.d	n.t.d	3.5
16m	O-CH2		H	1019	5.99 (5.82-6.16)	78 (69-88)	13	99	1.6
16n	O-CH2		H	1759	5.75 (5.59-5.91)	77 (64-90)	n.t.d	n.t.d	1.6
16o	O-CH2		H	2690e	5.57e	82e	n.t.d	n.t.d	1.6
16p	O-CH2		H	621	6.21 (6.12-6.29)	94 (89-99)	9	29	1.8
16q	O-CH2		H	791	6.10 (5.74-6.46)	56 (41-70)	33	97	1.6

^aValues were calculated from three independent experiments using a four-parameter logistic nonlinear regression model taking into account the heterogeneity between experiments and concentration—response curves for each compound.

^bHLM 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.

^cCalculated with Biobyte software.

^dNot tested.

^eData obtained from a single experiment not replicated.

Chemistry

Compound 13 was readily prepared in two steps (39% overall yield), via Sandmeyer reaction and subsequent Sonogashira cross-coupling, from 2-amino-benzothiazolone 17²⁶ (Scheme 1).

Scheme 1.

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%) following an analogous procedure to the one described for 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).

Scheme 2.

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-carbethoxypiperidine-2,4-dione derivatives 24a–e. Acid mediated decarboxylation 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.

Scheme 3.

Scheme 9.

The benzyloxy-dihydrothiazolopiperidones 15b,c were prepared 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.

Scheme 4.

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 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).

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).

Scheme 6.

Alternatively, several compounds (16c,e,f,h,i,k) were prepared 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.

Scheme 7.

Scheme 8.

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%).

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 mGlu₅ PAM chemotypes).^9,16 As 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-dihydrothiazolopyridone 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 mGlu₅ PAM 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. 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 mGlu₅ potency, a significant improvement 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%).

In light of their good mGlu₅ PAM 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. 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 relatively high CL_p of 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_hep 0.47) with a relatively low volume of distribution (V_ss = 0.70 L/kg) and moderate systemic exposure (plasma_AUC = 2.9 μM-h). The 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_u brain = 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 mGlu₅ cell line²⁷ resulted 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_1–4,6–8 EC₅₀ > 10 μM)²⁰ and also when tested at 10 μM concentration in a panel of 66 off-target class receptors, ion channels and transporters.²⁸ Finally, positive allosteric modulator 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 mGlu₅ receptor revealed how the concentration response curve of (CRC) 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 EC₅₀ was 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).²⁹ These results corroborate a positive allosteric interaction between glutamate and 16a and were confirmed in a cell line expressing the human mGlu₅ receptor,²⁷ where 10 μM 16a produced a 4.4-fold leftward shift in the glutamate CRC.

Figure 4.

Profile summary of 16a.

Figure 3.

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

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

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 mGlu₅ PAM EC₅₀ of 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.

Figure 5.

Dose-dependent effect of 16a on the reversal of amphetamine-induced hyperlocomotion in rats.

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 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.

Figure 6.

Effects of 16a on rotarod performance in rats.

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 dihydrothiazolopyridone core as optimal replacements for the ester and the chromanone core respectively. While the benzyloxydihydrothiazolopyridone subseries was found susceptible to hydrolysis, the phenoxymethyl-dihydrothiazolopyridones 16 proved to be a promising mGlu₅ PAM series. Preliminary focused SAR exploration of amide substituents revealed aromatic groups as preferred substituents for achieving high in vitro mGlu₅ potentiation 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 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 mGlu₅ positive allosteric modulation. Further investigation and evolution of this novel series of mGlu₅ PAMs 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 CDCl₃ and DMSO-d₆ as solvents. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane (δ = 0). Coupling constants are reported in Hertz. Splitting patterns are defined by s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), 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 chromatography (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 FP81HTFP90 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.

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 10% solution of HCl. The mixture was extracted with CH₂Cl₂ and 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. ¹H 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).

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

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 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-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.

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 24e (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)

Starting 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 chromatography (silica gel, EtOAc in heptane, 0/100 to 30/70). The desired fractions were collected and the solvents evaporated in vacuo to yield 1.2 g (16%) of 26 as a white solid. LCMS: m/z 327 [M + H]⁺, t_R = 2.51 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 EtOAc as a yellow solid. Mp^a 217.6–220.0 °C. ¹H 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-carboxylic 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 brown oily solid. ¹H 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^a 212.1–230.9 °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-Phenoxy-thioacetamide (33a)

To a solution of phenoxyacetonitrile (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 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 chromatography (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.

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 chromatography (silica gel, EtOAc in petroleum ether, 66/33). The desired fractions were collected, and the solvent was evaporated in vacuo to yield 1.2 g (52%) of 13 as a solid. Mp^a 117.1–119.9 °C. ¹H 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 (30%) of 14a as a brown solid. Mp^a 232.1–235.1 °C. ¹H 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^a 146.1–147.2 °C. ¹H NMR (400 MHz, 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.

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^a 144.5–148.2 °C. ¹H 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 with heptane/Et₂O to yield 0.190 g (54%) of 15b. Mp^b 106.4 °C. ¹H 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 CH₂Cl₂ and 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 CH₂Cl₂ in 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 130.0 °C. ¹H 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^a 141.5–142.4 °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, 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, 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^b 116.0 °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 NaHCO₃ and 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^a 104.1–106.0 °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.

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^b 82.6 °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^a 252.1–255.3 °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.

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^a 292.6–302.9 °C. ¹H 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, 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 mg, 13%). ¹H 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.

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^b 163.3 °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.

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^a 268.9–284.1 °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 NaHCO₃ and 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.

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^b 152.8 °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%). Mp^a > 280 °C. ¹H 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), 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′-dimethylethylenediamine (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 yield 57 mg (57%) of 16m as a white solid. Mp^b 143.9 °C. ¹H 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.

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 was obtained as a white solid (29 mg, 15%). ¹H 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%) of 16p as a white solid. Mp^a 131.7–132.8 °C. ¹H 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_5a cDNA in expression vector pcDNA4/TO. hmGlu₅ receptor-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 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% CO₂ environment and hold under permanent selection with 200 μg/mL zeocin and 5 μg/mL blasticidin. Cells were cultured at 40 000 cells/cm² or 20 000 cells/cm² for respectively 3 or 4 days of growth in a F175 flask.

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 EC₅₀ and 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_max of 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 administration 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 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 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 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_u plasma and f_u brain, 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.

Figure 1.

mGlu₅ receptor PAMs with reported efficacy in preclinical models of schizophrenia and cognition.

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

concentration response curve

D₂

dopamine 2

DAD

diode array detector

dihydrobenzothiazolone

5,6-dihydro-7(4H)-benzothiazolone

dihydrothiazolopyridone

6,7-dihydro-thiazolo[5,4-c]pyridin-4(5H)-one

DMPK

drug metabolism and pharmacokinetic

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 chromatography

HTS

high throughput screening

iv

intravenous

LCHRMS

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

RPHPLC

reversed-phase high-performance liquid chromatography

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 Neuroscience Drug Discovery