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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC Jan 13, 2012.
Published in final edited form as:
PMCID: PMC3071440
NIHMSID: NIHMS258688
Design and Synthesis of an Orally Active Metabotropic Glutamate Receptor Subtype-2 (mGluR2) Positive Allosteric Modulator (PAM) that Decreases Cocaine Self-administration in Rats
Raveendra-Panickar Dhanya,1 Shyama Sidique,1 Douglas J. Sheffler,2 Hilary Highfield Nickols,3 Ananda Herath,1 Li Yang,1 Russell Dahl,1 Robert Ardecky,1 Svetlana Semenova,4 Athina Markou,4 P. Jeffrey Conn,2 and Nicholas D. P. Cosford1*
1 Apoptosis and Cell Death Research Program and Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037
2 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232
3 Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232
4 Department of Psychiatry, School of Medicine, University of California San Diego, La Jolla, CA 92093
* Corresponding Author: To whom correspondence should be addressed. Tel: 858-646-3100. Fax: 858-795-5225. ncosford/at/sanfordburnham.org
These authors contributed equally to this work.
The modification of 3′-((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yloxy)methyl)biphenyl-4-carboxylic acid (BINA, 1) by incorporating heteroatoms into the structure and replacing the cyclopentyl moiety led to the development of new mGluR2 positive allosteric modulators (PAMs) with optimized potency and superior drug-like properties. These analogues are more potent than 1 in vitro, and are highly selective for mGluR2 vs. other mGluR subtypes. They have significantly improved pharmacokinetic (PK) properties, with excellent oral bioavailability and brain penetration. The benzisothiazol-3-one derivative 14 decreased cocaine self-administration in rats, providing proof-of-concept for the use of mGluR2 PAMs for the treatment of cocaine dependence.
Keywords: Metabotropic glutamate receptors, agonist, positive allosteric modulators, BINA, cocaine self-administration
Cocaine addiction is a chronic relapsing disorder affecting more than 1.6 million Americans.1 Relapse rates among cocaine users is in the range of 94–99%, the highest among all commonly abused drugs.2 Chronic cocaine abuse can lead to severe neurotoxicity, psychosis, lethargy, depression, or potentially death through a fatal overdose. Currently there is no effective treatment for cocaine dependence, and therefore cocaine addiction constitutes a major public health problem. Consequently, there is a significant need to identify new therapeutic agents for the treatment of cocaine and other psychomotor stimulant addictions. Recent findings suggest that neuroadaptations in glutamatergic transmission produced by repeated exposure to cocaine or other drugs of abuse are likely to contribute to the maintenance of addictive behaviors including drug use, craving and relapse to drug taking in humans.3 Specifically, it has been shown that repeated cocaine exposure alters the function of Group II metabotropic glutamate receptors (mGluRs).4a The Group II mGluRs include the mGluR2 and mGluR3 subtypes, which couple to Gi/o proteins to negatively regulate the activity of adenylyl cyclase.4b Brain regions implicated in different aspects of drug abuse and drug dependence, including the cerebral cortex, hippocampus, striatum, amygdala, frontal cortex and nucleus accumbens display high levels of mGluR2 and mGluR3 binding,5 suggesting a role for the mGluR2/3 subtypes in the development of cocaine dependence and as potential targets for therapeutic agents.3a,3e,3f,6
Orthosteric (glutamate site) mGluR2/3 agonists such as LY3792686g are constrained amino acid analogues that do not exhibit selectivity for mGluR2 versus mGluR3, presumably because of the high degree of sequence homology at the glutamate binding site for these two receptors.6h LY379268 has been shown to decrease glutamate levels7 and to attenuate cocaine self-administration both in rats6c,8 and in squirrel monkeys.6a However, LY379268 also inhibits responding for food and food-seeking behavior6c,6e,9, suggesting that mGluR2/3 agonists exhibit non-selective actions on responding for drug and non-drug reinforcers. We recently initiated investigations to determine the effect of selectively activating mGluR2 on cocaine dependence.8 Localization studies suggest that mGluR2 acts predominantly as a presynaptic autoreceptor to modulate the release of glutamate into the synaptic cleft.10 In recent years, there have been several reports on the use of high-throughput screening (HTS) for the identification of small molecule mGluR2 positive allosteric modulators (PAMs).11 The activity of these compounds is of interest because of their subtype-selectivity, ability to overcome receptor desensitization and potential for improved drug-like properties.12 The use of a selective mGluR2 PAM in an appropriate in vivo model therefore represents an attractive approach to elucidate the role of mGluR2 in cocaine dependence. We previously disclosed the structure activity relationship (SAR) around a series of pyrimidine derivatives with activity as mGluR2 PAMs.11b The synthesis and characterization of 3′-((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yloxy)methyl)biphenyl-4-carboxylic acid (BINA, 1) has been previously reported 13 and we have shown that this compound possesses antipsychotic and anxiolytic effects in mice.14 Most relevant to the present report, we recently showed that compound 1, unlike mGluR2/3 agonists, decreased cocaine self-administration in rats at doses that did not affect responding for food.8 A recent report by the Merck group on a series of oxazolobenzimidazole-based mGluR2 PAMs that are orally active in an in vivo model of schizophrenia prompts us to disclose our own efforts on the development of orally active mGluR2 PAMs.15b
While compound 1 is brain penetrant and selective, it lacks potency for mGluR2 both in vitro and in vivo and has sub-optimal pharmacokinetic (PK) properties. We therefore set out to design and synthesize new selective mGluR2 PAMs based on the compound 1 scaffold in order to improve both potency and PK properties. These new PAMs are suitable for in vivo proof-of-concept (POC) studies to evaluate the potential of mGluR2 as a therapeutic target for the treatment of cocaine dependence. We hypothesized that modifying the indanone ring of 1 by incorporating heteroatoms may lead to analogues with improved properties (Figure 1). Interestingly, to date there have been no reports on the synthesis and evaluation of analogues of compound 1 in which the indanone ring has been systematically modified in this way. In addition, as far as we are aware there have been no studies documenting the effects of modifying the cyclopentyl ring in this scaffold. Furthermore, despite several recent reports disclosing mGluR2 PAMs,11,15 none have been evaluated in an in vivo model of drug dependence.
Figure 1
Figure 1
Incorporation of heteroatoms into BINA (1).
Initially, we synthesized and tested compound 2, an analogue of compound 1 previously reported to be roughly 4-fold more potent than 1 at mGluR2 (Figure 1)13 The in vitro potency of the compounds was assessed using a thallium flux assay performed in HEK-GIRK cells expressing rat mGluR2.16 In this assay compound 1 exhibited an EC50 value of 380 nM, while compound 2 had an EC50 of 180 nM, confirming an improved potency of 2 compared with 1 (Table 1). We therefore incorporated the 4-chlorobiphenyl-3-carboxylic acid moiety from 2 into the design of the new analogues.
Table 1
Table 1
In vitro data for mGluR2 PAMs.
We next focused on the synthesis of isoindolinone analogues (8a-8k). As depicted in Scheme 1, the preparation of 8a-8k commenced with the conversion of commercially available 4-hydroxyphthalic acid 3 into the corresponding phthalic anhydride derivative 4 under thermal conditions, which was further converted to the phthalimide derivatives 5a-5k by treatment with the appropriate amine derivative. Regioselective reduction of the 3-carbonyl group with zinc in acetic acid afforded the desired 5-hydroxyisoindolinone derivatives 6a-6k. To verify the regioselectivity of the reduction, intermediate 6a (R1 = cyclopentyl) was also synthesized via an alternate procedure to confirm the structure unambiguously.17 Completion of the synthesis of compounds 8a-8k was achieved in two steps using an O-alkylation reaction with methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate to give the ester derivative 7a-7k which was saponified to the corresponding acid derivative 8a-8k using LiOH in THF.
Scheme 1
Scheme 1
Synthesis of 4-chloro-3′-((2-cyclopentyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid derivatives 8a-8k.a
The synthesis of an analogue of compound 1 based on the benzisothiazol-3-one scaffold is illustrated in Scheme 2. Methyl 4-methoxy thiosalicylate 918 was converted to the corresponding amide 10 by treatment with cyclopentylamine in the presence of trimethylaluminum. The cyclization to access the benzisothiazol-3-one derivative 11 was accomplished by a PIFA-meditated in situ formation of an N-acylnitrenium ion, and its intramolecular trapping by the thiol moiety.19 Demethylation of 11 was achieved with BBr3 in hot benzene, followed by O-alkylation with methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate to provide 13. Hydroxide-mediated demethylation led to the formation of byproducts, however clean saponification of 13 was accomplished with lithium iodide to complete the synthesis of 14.
Scheme 2
Scheme 2
Synthesis of 3-chloro-3′-((2-cyclopentyl-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yloxy)methyl)biphenyl-4-carboxylic acid 14a
We also designed and synthesized an analogue of compound 1 based on the isoxazol-3-one scaffold (21). Our approach to the synthesis of 21 began with the preparation of N-cyclopentylbenzohydroxamic acid derivative 18 as shown in Scheme 3. The synthesis began with 4-methoxysalicylic acid 15, which was transformed to 18 using a previously described procedure.20 Thus, 4-methoxysalicylic acid 15 was acetylated at the phenolic hydroxyl group prior to conversion to the acid chloride derivative 16. N-cyclopentylhydroxylamine 17 was reacted with 16 to provide hydroxamic acid 18 as a colorless solid. Cyclization of 18 under Mitsunobu conditions afforded N-cyclopentylbenzisoxazolinone 19, which was converted to the desired analogue 21 via intermediate 20 using the same conditions as those described for the preparation of compounds 8 and 14.
Scheme 3
Scheme 3
Synthesis of 4-chloro-3′-((2-cyclopentyl-3-oxo-2,3-dihydrobenzo[d]isoxazol-6- yloxy)methyl)biphenyl-3-carboxylic acid 21a
In addition, we designed and synthesized an analogue of compound 1 (28) based on the tetrahydroisoquinolinone scaffold. Our approach to the synthesis of 28 is outlined in Scheme 4. Thus, commercially available 3-methoxyphenethylamine was alkylated with iodocyclopentane to provide N-(3-methoxyphenethyl)cyclopentanamine (23). Treatment of 23 with methylchloroformate in the presence of triethylamine gave methyl cyclopentyl(3-methoxyphenethyl)carbamate 24. Carbamate 24 was subjected to the Bischler-Napieralski cyclization conditions of Wang et al 21 (POCl3-P2O5) to afford the corresponding tetrahydroisoquinolone derivative 25. Compound 25 was then converted to the target BINA analogue 28 in three steps as described for the other analogues.
Scheme 4
Scheme 4
Synthesis of 4-Chloro-3′-((2-cyclopentyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6- yloxy)methyl)biphenyl-3-carboxylic acid 28a
The new analogues 8a-k, 14, 21 and 28 were tested, along with 1 and 2, to determine in vitro activity and the data are summarized in Table 1. For these in vitro experiments, a thallium flux assay was performed in HEK-GIRK cells16 expressing rat mGluR2. The concentration-response relationship that potentiates the effect of an EC20 concentration of glutamate was determined for each of the mGluR2 PAMs and this potency is expressed as an EC50 value. The efficacy for potentiation of an EC20 concentration of glutamate is presented as a percentage of the maximal glutamate response. Compounds 8a-j, 14, 21 and 28 were all more potent than 1 with compound 8a being especially potent (EC50 = 50 nM). Increase (8h) or decrease in ring size (8d, 8f) or the presence of bulky aryl substituents (8g, 8i) led to a decrease in potency compared with the N-cyclopentyl derivative 8a. On the other hand, branching (8b) or lengthening of the branched chain (8e, 8j) led to compounds with potency similar to that of 8a. Interestingly simple straight chain aliphatic substitution, as in the N-propyl derivative (8k), led to complete loss of activity. Enlargement of the lactam ring in the core scaffold from 5- (8a) to 6-membered (28) led to a 6-fold decrease in EC50. In addition, the new analogues are highly selective for mGluR2 vs. other mGluR subtypes with no activity observed up to 10 μM for 8a and 14 (data not shown). We also determined the membrane permeability, in vitro plasma stability, and microsomal stability of the analogues to predict their in vivo pharmacological profile. As shown in Table 1, several of the new analogues displayed superior drug-like properties compared with 1, suggesting that these improvements may translate to an in vivo test.
Based on their overall in vitro profile, an isoindolinone derivative (8a) and a benzothiazolone derivative (14) were therefore selected for an in vivo assessment of their PK properties in rats. The rat PK data for compounds 1, 8a and 14 are shown in Table 2. Gratifyingly, both 8a and 14 exhibited vastly superior PK profiles compared with 1, with high values for Cmax, AUC, and t1/2. Incorporation of sulfur into the isoindolinone ring (14) did lead to a three fold decrease in potency, but showed superior PK properties (Table 2) compared to 8a. Importantly, both compounds had excellent oral bioavailability and were highly brain penetrant. In particular, compound 14 showed exceptional oral bioavailability (86%) and brain levels (4.8 μM) following a 10 mg/kg oral dose, with a brain:plasma ratio of 4.8. In contrast, the brain levels for 1 were below the limit of quantitation (LOQ) following oral dosing (10 mg/kg).8 Based on these data compound 14 was selected for evaluation in the cocaine self-administration procedure in rats.
Table 2
Table 2
In vivo PK data for mGluR2 PAMs in rats following oral administration.a
The in vivo model was designed to investigate the effects of acute systemic (oral) administration of the mGluR2 PAM 14 on intravenous cocaine self-administration in rats that had 1 h access to cocaine (see Experimental for details). Additionally, we examined the effects of compound 14 on food-maintained responding to assess the selectivity of an orally active mGluR2 PAM on responding for cocaine compared to responding for the natural reinforcer food.
The results of the in vivo behavioral studies are shown in Figure 2. Oral administration of compound 14 significantly decreased cocaine self-administration in rats (Figure 2). Similarly, our previous work demonstrated that systemic (i.p.) administration of the mGluR2/3 agonist LY379268 significantly decreased cocaine self-administration in rats.8 However, compound 14 had a much less pronounced effect on food responding compared with the effects of LY379268.8 The effects of compound 14 on cocaine self-administration and the small effect of this compound on responding for food only at the highest dose tested (40 mg/kg) are consistent with our previous findings with compound 1.8 These data indicate that a selective, orally active mGluR2 PAM is as effective as an mGluR2/3 agonist at reversing the effects of cocaine self administration in rats, but with a diminished or no effect on food-maintained responding. This selectivity of the effect may be due to the selectivity of 14 for mGluR2 compared with the dual acting mGluR2/3 agonist, and/or because of the modulatory properties of a mGluR2 PAM compared to the direct actions of an agonist on the receptor(s).
Figure 2
Figure 2
Effects of compound 14 on cocaine self-administration and food-maintained responding in rats. Data are expressed as percent of baseline responding (mean ± SEM). The number of baseline cocaine infusions was 20 ± 4.0 (mean ± SEM) (more ...)
In conclusion, using the mGluR2 PAM 1 as a template, we have designed and synthesized a series of new, highly potent mGluR2 PAMs. These compounds are highly selective for mGluR2 vs. other mGluR subtypes and have significantly improved in vitro ADME and in vivo PK properties, providing compounds with excellent oral bioavailability and brain penetration. On the basis of these data compound 14 was selected for in vivo studies in a model of cocaine dependence in rats, providing proof-of-concept for the use of an orally active mGluR2 PAM for the treatment of cocaine addiction.
General Chemistry
All reactions were performed in oven-dried glassware under an atmosphere of argon with magnetic stirring. All solvents and chemicals used were purchased from Sigma-Aldrich or Acros, and were used as received without further purification. Purity and characterization of compounds were established by a combination of liquid chromatography-mass spectroscopy (LC-MS) and NMR analytical techniques and was >95% for all tested compounds. Silica gel column chromatography was carried out using prepacked silica cartridges from RediSep (ISCO Ltd.) and eluted using an Isco Companion system. Melting points were reordered on a MEL-TEMP® apparatus and are uncorrected. 1H- and 13C-NMR spectra were obtained using a Jeol 400 spectrometer at 400 MHz and 100 MHz, respectively, unless otherwise mentioned, in which case a Varian 300 MHz spectrometer was used. Chemical shifts are reported in δ (ppm) relative to residual solvent peaks or TMS as internal standards. Coupling constants are reported in Hz. High-resolution ESI-TOF mass spectra were acquired at the Center for Mass Spectrometry at The Scripps Research Institute (La Jolla, CA). HPLC-MS analyses were performed on a Shimadzu 2010EV LCMS using the following conditions: Kromisil C18 column (reverse phase, 4.6 mm × 50 mm); a linear gradient from 10% acetonitrile and 90% water to 95% acetonitrile and 5% water over 4.5 min; flow rate of 1 mL/min; UV photodiode array detection from 200 to 300 nm.
5-Hydroxyisobenzofuran-1,3-dione (4).23
4-Hydroxyphthalic acid (10 g, 54.9 mmol) was heated at 200 °C overnight in a round bottomed flask in the absence of solvent. The resulting material was cooled and washed with ethyl acetate to obtain 5-hydroxyisobenzofuran-1,3-dione 4 as a white solid in quantitative yield.
General procedure for the synthesis of thalimide derivatives (5a-5k)
4-Hydroxyphthalic anhydride 4 (5.4 g, 32.9 mmol), commercial amine (49.6 mmol) and 4Å molecular sieves in toluene (136 mL) were stirred at reflux overnight. The reaction mixture was filtered and the solvent was evaporated in vacuo to obtain 5a-5k as yellow solids in quantitative yield.
2-Cyclopentyl-5-hydroxyisoindoline-1,3-dione (5a)24
1H NMR (DMSO-d6): δ 10.8 (s,1H), 7.60 (d, J = 7.8 Hz, 1H), 7.05-7.01 (m, 2H), 4.50-4.40 (m, 1H), 1.97-1.52 (m, 8H). 13C NMR (DMSO-d6): δ 167.7, 163.1, 134.2, 124.9, 121.0, 120.2, 109.5, 50.0, 29.2, 24.7.
5-Hydroxy-2-isopropylisoindoline-1,3-dione (5b)
1H NMR (300 MHz, DMSO-d6): δ 7.65 (d, J = 8.7 Hz, 1H), 7.22-7.09 (m, 2H), 4.38-4.29 (m, 1H), 1.37 (d, J = 6.9 Hz, 6H).
2-(Cyclopropylmethyl)-5-hydroxyisoindoline-1,3-dione (5c)
1H NMR (300 MHz, DMSO-d6): δ 7.70 (d, J = 8.1 Hz, 1H), 7.16-7.11 (m, 2H), 3.39 (d, J = 7.5 Hz, 2H), 1.11-1.06 (m, 1H), 0.47-0.39 (m, 2H), 0.32-0.28 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 168.5, 163.9, 135.1, 125.9, 122.4, 121.0, 110.5, 42.4, 11.0, 4.2.
2-Cyclobutyl-5-hydroxyisoindoline-1,3-dione (5d)
1H NMR (300 MHz, DMSO-d6): δ 7.61 (d, J = 8.4 Hz, 1H), 7.06-7.04 (m, 2H), 4.59-4.50 (m, 1H), 2.79-2.68 (m, 2H), 2.16-2.10 (m, 2H), 1.78-1.66 (m, 2H).
5-Hydroxy-2-isobutylisoindoline-1,3-dione (5e)
1H NMR (300 MHz, DMSO-d6): δ 7.65 (d, J = 8.4 Hz, 1H), 7.10-7.06 (m, 2H), 3.32 (d, J = 7.2 Hz, 2H), 2.00-1.91 (m, 1H), 0.84 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 168.7, 134.9, 125.9, 121.1, 110.6, 45.3, 28.1, 20.6.
2-Cyclopropyl-5-hydroxyisoindoline-1,3-dione (5f)
1H NMR (300 MHz, DMSO-d6): δ 7.70 (+d, J = 8.4 Hz, 1H), 7.20 – 7.10 (m, 2H), 2.70-2.60 (m, 1H), 0.91- 0.82 (m, 4H).
5-Hydroxy-2-phenylisoindoline-1,3-dione (5g)
1H NMR (DMSO-d6): δ 11.03 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.52-7.16 (m, 7H). 13C NMR (DMSO-d6): δ 166.7, 163.5, 134.3, 132.1, 128.8, 127.8, 127.3, 125.6, 120.8, 109.9.
2-Cyclohexyl-5-hydroxyisoindoline-1,3-dione (5h)
1H NMR (300 MHz, DMSO-d6): δ 7.48 (d, J = 7.8 Hz, 1H), 6.86-6.79 (m, 2H), 3.93-3.87 (m, 1H), 1.82-1.09 (m, 10H).
5-Hydroxy-2-(4-(trifluoromethoxy)benzyl)isoindoline-1,3-dione (5i)
1H NMR (300 MHz, DMSO-d6): δ 7.68-7.03 (m, 7H), 4.75 (s, 2H). 13C NMR (75 MHz, DMSO-d6): δ 168.5, 166.4, 137.2, 135.2, 130.1, 130.0, 126.2, 121.9, 121.6, 111.1, 44.7.
5-Hydroxy-2-isopentylisoindoline-1,3-dione (5j)
1H NMR (300 MHz, DMSO-d6): δ 7.62 (d, J = 7.8 Hz, 1H), 7.07-7.02 (m, 2H), 3.54-3.49 (m, 2H), 1.47-1.40 (m, 3H), 0.89 (d, J = 6.9 Hz, 6H).
5-Hydroxy-2-propylisoindoline-1,3-dione (5k)
1H NMR (DMSO-d6): δ 7.74 (d, J = 8.2 Hz, 1H), 7.19-7.16 (m, 2H), 3.56-3.52 (m, 2H), 1.66-1.61 (m, 2H), 0.92-0.89 (m, 3H). 13C NMR (DMSO-d6): δ167.8, 163.2, 134.3, 125.1, 121.7, 120.2, 109.7, 21.4, 11.2.
General procedure for the synthesis of isoindolinones (6a-6k)
To a solution of 1,3-dione 5a-5k (18.0 mmol), in CH3CO2H (129 mL) was added Zn dust (11.6 g, 180 mmol) and the mixture was heated at reflux for 5 min. The volatiles were removed in vacuo and the residue was dissolved in water and extracted with ethyl acetate. The crude residue was purified using automated preparative HPLC to yield the desired compounds (6a-6k) as amorphous solids.
2-Cyclopentyl-5-hydroxyisoindolin-1-one (6a)
(1.7 g, 44%) 1H NMR (DMSO-d6): δ 10.10 (bs, 1H), 7.44 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J = 7.9 Hz, 1H), 4.52 - 4.48 (m, 1H), 4.31 (s, 2H), 1.83–1.57 (m, 8H). 13C NMR (DMSO-d6): δ 167.2, 160.4, 144.1, 123.9, 123.7, 115.4, 109.6, 51.8, 45.4, 29.6, 23.8. LCMS calcd. for C13H15NO2 [M+H]+: 218; found: 218.
5-Hydroxy-2-isopropylisoindolin-1-one (6b)
(23 mg, 22%) 1H NMR (DMSO-d6): δ 10.04(s, 1H), 7.43 (d, J = 8.2 Hz, 1H), 6.89-6.81 (m, 2H), 4.38-4.31 (m, 1H), 4.28 (s, 2H), 1.16 (d, J = 6.4 Hz, 6H). 13C NMR (DMSO-d6): δ 166.7, 160.4, 144.1, 123.9, 115.4, 109.6, 44.4, 41.9, 20.4. LRMS (ESI) calcd. for C11H13NO2 [M+H]+: 192; found 191.95.
2-(Cyclopropylmethyl)-5-hydroxyisoindolin-1-one (6c)
(60 mg, 63%). LRMS (ESI) calcd. for C12H13NO2[M+H]+: 204; found 204.
2-Cyclobutyl-5-hydroxyisoindolin-1-one (6d)
(108 mg, 45%). 1H NMR (DMSO-d6): δ 7.43, (d, J = 8.2 Hz, 1H), 6.89-6.80 (m, 2H), 4.71-4.62 (m, 1H), 4.42 (s, 2H), 2.31-2.06 (m, 4H), 1.72-1.62 (m, 2H). 13C NMR (DMSO-d6): δ 166.7, 160.6, 144.1, 124.2, 123.6, 115.5, 109.6, 45.8, 45.6, 28.1, 14.5. LRMS (ESI) calcd. for C12H13NO2[M+H]+: 204; found 203.95.
5-Hydroxy-2-isobutylisoindolin-1-one (6e)
(86 mg, 34%). 1H NMR (DMSO-d6): δ 10.07 (s, 1H), 7.45 (d, J = 8.2 Hz, 1H), 6.88- 6.81 (m, 2H), 4.32 (s, 2H), 3.23 (d, J = 7.8 Hz, 2H), 2.00-1.90 (m, 1H), 0.83 (d, J = 6.8 Hz, 6H). 13C NMR (DMSO-d6): δ 167.6, 160.5, 144.1, 124.1, 123.5, 115.4, 109.5, 49.6, 49.1, 27.1, 20.0. LRMS (ESI) calcd. for C12H15NO2[M+H]+: 206; found 205.90.
2-Cyclopropyl-5-hydroxyisoindolin-1-one (6f)
(53 mg, 23%). 1H NMR (DMSO-d6):δ 7.36 (d, J = 8.2 Hz, 1H), 6.84-6.79 (m, 2H), 4.36 (s, 2H), 2.84-2.80 (m, 1H), 0.77-0.71 (m, 4H). 13C NMR (DMSO-d6): δ 168.9, 143.9, 123.8, 116.1, 109.7, 49.3, 24.9, 24.5, 5.1. LRMS (ESI) calcd. for C11H11NO2[M+H]+: 190; found 190.00.
5-Hydroxy-2-phenylisoindolin-1-one (6g)
(14 mg, 19%). 1H NMR (DMSO-d6): δ 7.83-7.81(m, 2H), 7.55-7.53 (m, 1H), 7.38-7.34 (m, 2H), 7.10-7.06 (m, 1H), 6.92-6.91 (m, 1H), 6.87-6.84 (m, 1H). LRMS (ESI) calcd. for C14H11NO2[M+H]+: 226; found 226.
2-Cyclohexyl-5-hydroxyisoindolin-1-one (6h)
(120 mg, 64%). 1H NMR (300 MHz, DMSOd6): δ10.08 (s, 1H), 7.47 (d, J = 8.7 Hz, 1H), 6.92-6.84 (m, 2H), 4.33 (s, 2H), 3.99-3.93 (m, 1H), 1.82-1.31 (m, 10H). 13C NMR (75 MHz, DMSO-d6): δ 167.4, 161.1, 144.9, 124.7, 116.1, 110.3, 50.7, 46.1, 31.4, 25.9, 25.8. LRMS (ESI) calcd. for C14H17NO2[M+H]+: 232; found 232.0.
5-Hydroxy-2-(4-(trifluoromethoxy)benzyl)isoindolin-1-one (6i)
(19 mg, 20%). LRMS (ESI) calcd. for C16H12 F3NO3[M+H]+: 324; found 323.95.
5-Hydroxy-2-isopentylisoindolin-1-one (6j)
(213 mg, 32%). 1H NMR (300 MHz, DMSO-d6): δ 7.46 (d, J = 8.1 Hz, 1H), 6.91 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.34 (s, 2H), 1.49-1.42 (m, 3H), 0.91 (d, J = 5.4 Hz, 6H). LRMS (ESI) calcd. for C13H17NO2[M+H]+: 220; found 219.85 (M+1)+.
5-Hydroxy-2-propylisoindolin-1-one (6k)
(12 mg, 22%). LRMS (ESI) calcd. for C11H13NO2[M+H]+: 192; found 191.95.
Methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate
Pd(OAc)2 (0.393 g, 1.75 mmol) was added to a solution of 2-chloro-5-iodobenzoic acid (9.9 g, 35 mmol), m-tolylboronic acid (5.71 g, 42 mmol) and Na2CO3 (11.45 g, 105 mmol) in water (45 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 50 °C for 2 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was acidified with 1M HCl. The precipitated 4-chloro-3′-methylbiphenyl-3-carboxylic acid was filtered, washed with water and dried to provide a tan solid that was used without further purification in the next step (quantitative yield). 1H NMR (DMSO-d6, 300 MHz): δ 8.03 (s, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.55-7.50 (m, 2H), 7.40 (t, J = 7.8 Hz,1H), 7.25 (d, J = 6.6 Hz, 1H), 2.41 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): δ 167.4, 139.9, 139.1, 138.6, 132.7, 131.9, 131.2, 129.7, 129.5, 129.3, 128.0, 124.5, 21.8.
To a solution of 4-chloro-3′-methylbiphenyl-3-carboxylic acid (7 g, 28.5 mmol) in acetone was added K2CO3 (27.5 g, 199 mmol) and MeI (12.5 mL, 28 mmol). The resulting solution was heated at 50 °C for 1 h. After cooling the excess solvent was removed in vacuo. The crude material was dissolved in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4, and the solvent was evaporated to afford methyl 4-chloro-3′-methylbiphenyl-3-carboxylate as a reddish yellow viscous liquid (7.3 g, 98%) which was used in the next step without further purification. 1H NMR (DMSO-d6): δ 8.04 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.52-7.47 (m, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 3.90 (s, 3H), 2.39 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): δ 166.2, 139.9, 139.1, 138.4, 131.9, 131.7, 131.4, 131.3, 129.7, 129.6, 129.5, 128.0, 124.5, 53.3, 21.7.
A catalytic amount of AIBN was added to a solution of methyl 4-chloro-3′-methylbiphenyl-3- carboxylate (7.3 g, 28 mmol) and NBS (5.5 g, 30.8 mmol) in CCl4. The reaction mixture was heated under reflux for 12 h. After cooling, the precipitated succinimide was removed by filtration. The filtrate was concentrated in vacuo to afford the crude product. Silica gel column chromatography using 4:1 hexanes/ethyl acetate afforded methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate (8.8 g, 91%) as a colorless solid. 1H NMR (CDCl3): δ 8.07(s, 1H), 7.68-7.45 (m, 6H), 4.58 (s, 2H), 4.00 (s, 3H). 13C NMR (CDCl3): δ 166.1, 139.5, 139.2, 138.7, 133.0, 131.6, 131.0, 130.4, 129.9, 129.6, 128.7, 127.7, 52.6, 33.2.
4-Chloro-3′-((2-cyclopentyl-1-oxoisoindolin-5-yloxy)-methyl)biphenyl-3-carboxylic acid (8a)
To a solution of 2-cyclopentyl-5-hydroxyisoindolin-1-one (50 mg, 0.23 mmol) in acetone (15 mL), K2CO3 (181 mg, 1.31mmol) was added methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate (92 mg, 0.27 mmol) and the mixture was heated at reflux for 1 h. The reaction mixture was filtered and the solvent was evaporated in vacuo to obtain methyl 4-chloro-3′-((2- cyclopentyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylate 7a as a yellow oil in quantitative yield.
Crude methyl 4-chloro-3′-((2-cyclopentyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylate 7a (109 mg, 0.23 mmol) was dissolved in tetrahydrofuran (17 mL), 2M LiOH (0.57 mL, 1.15 mmol) was added and the mixture was heated at reflux for 3 h. The solvent was evaporated and the residue was dissolved in water and neutralized using 2M HCl. The aqueous layer was extracted with ethyl acetate and the organic layer was dried over anhydrous Na2SO4. The solvent was evaporated in vacuo to obtain the crude acid as a yellow solid. The crude residue was purified using automated preparative HPLC to yield the desired compound 8a (48 mg, 46%) as an amorphous white solid. 1H NMR (CDCl3): δ 8.18 (s, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.65-7.41 (m, 6H), 7.05 (d, J = 8.5 Hz, 1H) 6.99 (s, 1H), 5.16 (s, 2H), 4.74 (m, 1H), 4.30 (s, 2H), 1.97 (m, 2H), 1.76 – 1.59 (m, 6H). 13C NMR (CDCl3): δ 168.7, 161.5, 143.3, 139.3, 137.2, 133.5, 131.7, 131.4, 130.7, 129.7, 129.4, 127.0, 126.8, 126.0, 125.9, 125.1, 115.4, 108.7, 70.1, 52.7, 46.1, 30.1, 24.1. HRMS: calcd. for C27H24ClNO4 [M+H]+: 462.1467; found: 462.1468.
Following the above mentioned procedure 8b-8k were synthesized using the appropriate starting materials.
4-Chloro-3′-((2-isopropyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8b)
Amorphous white solid (14 mg, 32%). 1H NMR (300 MHz, DMSO-d6): δ 8.06 (s, 1H), 7.85-7.82 (m, 2H), 7.68-7.51 (m, 5H), 7.24 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 5.26 (s, 2H), 4.45-4.40 (m, 1H), 4.34 (s, 2H), 1.19 (d, J = 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 166.8, 166.6, 161.1, 144.2, 138.8, 138.3, 137.7, 132.1, 131.4, 131.0, 130.6, 129.5, 128.8, 127.6, 126.4, 126.2, 125.7, 124.1, 115.4, 109.2, 69.5, 44.8, 42.3, 20.5. LRMS (ESI): 435.95 (M+1)+. HRMS: calcd. for C25H22ClNO4 436.131; found 436.1309.
4-Chloro-3′-((2-(cyclopropylmethyl)-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3- carboxylic acid (8c)
Amorphous white solid (49 mg, 39%). 1H NMR (300 MHz, DMSO-d6): δ 8.07 (s, 1H), 7.88 – 7.84 (m, 2H), 7.71 – 7.52 (m, 5H), 7.28 (s, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.28 (s, 2H), 4.50 (s, 2H), 3.33 (d, J = 7.5 Hz, 2H), 1.06 – 0.98 (m, 1H), 0.51 – 0.48 (m, 2H), 0.31 – 0.28 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 167.5, 167.3, 161.8, 144.8, 139.4, 138.9, 138.4, 132.8, 131.9, 131.6, 131.2, 130.1, 129.4, 128.3, 127.1, 126.9, 126.0, 124.7, 116.0, 109.8, 70.2, 50.0, 46.8, 10.6, 4.1. LRMS (ESI): 448.00 (M+1)+. HRMS: calcd. for C26H22ClNO4 448.131; found 448.1313.
4-Chloro-3′-((2-cyclobutyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8d)
Amorphous white solid (183 mg, 41%). 1H NMR (300 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.85-7.83 (m, 2H), 7.68-7.51 (m, 5H), 7.25 (s, 1H), 7.12 (d, J = 8.7 Hz, 1H), 5.27 (s, 2H), 4.78-4.62 (m, 1H), 4.49 (s, 2H), 2.38- 2.22 (m, 2H), 2.15-2.09 (m, 2H), 1.78-1.66 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 166.6, 166.4, 161.1, 144.0, 138.7, 138.2, 137.6, 132.0, 131.2, 130.9, 130.5, 129.4, 128.7, 127.5, 126.3, 126.2, 125.4, 124.1, 115.4, 109.1, 69.4, 46.0, 45.7, 28.1, 14.5. LRMS (ESI): 448.00 (M+1)+. HRMS: calcd. for C26H22ClNO4 448.131; found 448.131.
4-Chloro-3′-((2-isobutyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8e)
Amorphous white solid (75 mg, 34%). 1H NMR (CDCl3): δ 8.17 (s, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.66 – 7.62 (m, 2H), 7.55 – 7.41 (m, 4H), 7.07 – 7.05 (m, 1H), 7.00 (s, 1H), 5.17 (s, 2H), 4.33 (s, 2H), 3.39 (d, J = 7.3 Hz, 2H), 2.07 – 1.96 (m, 1H), 0.92 (d, J = 6.7 Hz, 6H). 13C NMR (CDCl3): δ 169.1, 168.2, 161.6, 143.4, 139.4, 137.2, 133.6, 131.7, 131.5, 130.7, 129.4, 127.1, 126.8, 125.9, 125.6, 125.3, 115.4, 108.7, 70.1, 50.6, 50.2, 27.8, 20.1. LRMS (ESI): 450.00 (M+1)+. HRMS: calcd. for C26H24ClNO4 450.1467; found 450.1472.
4-Chloro-3′-((2-cyclopropyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8f)
Amorphous white solid (78 mg, 23%). 1H NMR (300 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.86-7.81 (m, 2H), 7.67-7.51 (m, 5H), 7.21 (s, 1H), 7.12 (d, J = 7.8 Hz, 1H), 5.26 (s, 2H), 4.32 (s, 2H), 2.92-2.86 (m, 1H), 0.80-0.76 (m, 4H). 13C NMR (75 MHz, DMSO-d6): δ 168.3, 166.8, 161.2, 144.1, 138.8, 138.3, 137.7, 132.1, 131.4, 130.9, 130.6, 129.5, 128.7, 127.6, 126.4, 126.2, 125.7, 124.0, 115.5, 108.9, 69.5, 49.5, 25.1, 5.11. LRMS (ESI): 434.05 (M+1)+. HRMS: calcd. for C25H20ClNO4 434.1154; found 434.1150.
4-Chloro-3′-((1-oxo-2-phenylisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8g)
Amorphous white solid (28 mg, 36%). 1H NMR (CDCl3): δ 8.20 (s, 1H), 7.86 – 7.36 (m, 11H), 7.15 – 7.05 (m, 3H), 5.19 (s, 2H), 4.77 (s, 2H). 13C NMR (CDCl3): δ 169.2, 167.4, 162.2, 142.4, 139.5, 139.4, 139.3, 137.1, 133.7, 131.9, 131.7, 130.8, 129.5, 129.1, 127.1, 126.9, 126.1, 126.0, 125.8, 124.3, 119.4, 115.9, 108.4, 70.2, 50.6. LRMS (ESI): 470. (M+1)+. HRMS: calcd. for C28H20ClNO4: 470.1154; found 470.1157.
4-Chloro-3′-((2-cyclohexyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8h)
Amorphous white solid (87 mg, 36%). 1H NMR (300 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.83 (m, 2H), 7.68 – 7.45 (m, 5H), 7.24 (s, 1H), 7.12 (d, J = 8.4 Hz, 1H), 5.27 (s, 2H), 4.36 (s, 2H), 3.99 – 3.92 (m, 1H), 1.79 - 1.14 (m, 10H). 13C NMR (75 MHz, DMSO-d6): δ 167.3, 167.0, 161.7, 144.8, 139.4, 138.9, 138.4, 132.8, 131.9, 131.6, 131.2, 130.1, 129.4, 128.2, 127.0, 126.8, 126.3, 124.6, 116.0, 109.8, 70.1, 50.8, 46.3, 31.3, 25.9, 25.7. LRMS (ESI): 476.05 (M+1)+. HRMScalcd. for C28H26ClNO4: 476.1623; found 476.1621.
4-Chloro-3′-((1-oxo-2-(4-(trifluoromethoxy)benzyl)isoindolin-5-yloxy)methyl)biphenyl-3- carboxylic acid (8i)
Amorphous white solid (12 mg, 30%). 1H NMR (CDCl3): δ 8.13 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.62 – 7.28 (m, 8H),7.14 (d, J = 7.9 Hz, 2H), 7.07 – 7.05 (m, 1H), 6.93 (s, 1H), 5.14 (s, 2H), 4.74 (s, 2H), 4.20 (s, 2H). 13C NMR (CDCl3): δ 168.5, 161.8, 148.7, 143.4, 139.9, 139.4, 137.1, 135.8, 131.7, 131.5, 130.7, 129.5, 127.1, 126.8, 125.9, 125.4, 125.3, 121.3, 115.5, 108.9, 70.2, 49.3, 45.7. LRMS (ESI): 567.95 (M+1)+. HRMS: calcd. for C30H21ClF3NO5 568.1133; found 568.1136.
4-Chloro-3′-((2-isopentyl-1-oxoisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8j)
Amorphous white solid (82 mg, 41%). 1H NMR (CDCl3): δ 8.18 (s, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.66 – 7.62 (m, 2H), 7.55 – 7.42 (m, 4H), 7.06 (dd, J = 8.5, 1.8 Hz, 1H), 6.99 (s, 1H), 5.17 (s, 2H), 4.31 (s, 2H), 3.62 – 3.58 (m, 2H), 1.63- 1.49 (m, 3H), 0.93 (d, J = 6.1 Hz, 6H). 13C NMR (CDCl3): δ 168.6, 161.5, 143.3, 139.4, 137.2, 133.6, 131.8, 131.5, 130.7, 129.4, 127.1, 126.8, 125.9, 125.2, 115.3, 108.7, 70.1, 49.8, 40.8, 37.2, 25.8, 22.5. LRMS (ESI): 463.95 (M+1)+. HRMS: calcd. for C27H26ClNO4 464.1623; found 464.1626.
4-Chloro-3′-((1-oxo-2-propylisoindolin-5-yloxy)methyl)biphenyl-3-carboxylic acid (8k)
Amorphous white solid (30 mg, 34%). 1H NMR (CDCl3): δ 8.18 (s, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.65 – 7.62 (m, 2H), 7.53 – 7.43 (m, 4H), 7.06 (dd, J = 8.5 Hz, J = 2.4 Hz, 1H), 6.99 (s, 1H), 5.17 (s, 2H), 4.31 (s, 2H), 3.54 (t, J = 7.3 Hz, 2H), 1.70 – 1.61 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3): δ 168.7, 168.4, 161.5, 143.3, 139.4, 137.2, 133.5, 131.7, 131.4, 130.7, 129.5, 129.4, 127.0, 126.8, 125.9, 125.9, 125.2, 115.3, 108.7, 70.1, 49.9, 44.1, 21.7, 11.3. LRMS (ESI): 436.00 (M+1)+. HRMS: calcd. for C25H22ClNO4 436.131; found 436.1315.
N-Cyclopentyl-2-mercapto-4-methoxybenzamide (10)
The title compound was prepared using the literature procedure as follows. AlMe3 (10 mmol, 2.0 M in toluene) was added dropwise to a cooled (0 °C) suspension of cyclopentylamine (0.85 g, 10 mmol) in CH2Cl2 (30 mL). When the addition was complete, the reaction mixture was allowed to warm to room temperature and stirring was continued for 30 min until gas evolution had ceased. A solution of methyl 2-mercapto-4-methoxybenzoate 9 (0.990 g, 5 mmol) was then added. After stirring at 60 °C for12 h, the mixture was cooled, and carefully quenched with 5% aq HCl (20 mL). The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3 × 15 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (15 mL) and brine. The organic phase was dried using MgSO4 and evaporated, filtered and the solvent was evaporated in vacuo. The resulting residue was purified by crystallization from Et2O to afford amide 10 as a tan solid (1.18 g, 94%). 1H NMR (300 MHz, DMSO- d6): δ 8.26 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.01 (s, 1H), 6.72 (d, J = 8. 1 Hz, 1H), 5.62 (s, 1H), 4.16-4.14 (m, 1H), 3.78 (s, 3H), 1.99-1.80 (m, 2H), 1.79-1.68 (m, 2H), 1.66-1.53 (m, 4H).13C NMR: δ 164.6, 162.7, 142.7, 127.1, 118.4, 115.2, 105.0, 56.3, 54.8, 32.0, 23.9. LC-MS (ESI) calcd. for C13H17NO2S [M+H]+: 252.09; found: 251.95.
2-Cyclopentyl-6-methoxybenzo[d]isothiazol-3(2H)-one (11)
A solution of PIFA (645 mg, 1.5 mmol) in CH2Cl2 (20 mL) was added at 0 °C to a solution of N-cyclopentyl-2-mercapto-4-methoxybenzamide 10 (251mg, 1 mmol) and TFA (0.23 mL, 3 mmol) in CH2Cl2 (5 mL). The resulting mixture was gradually warmed to room temperature. After 1 h, the solvent was evaporated under reduced pressure and the resulting residue was purified by column chromatography using CH2Cl2 to afford the 2-cyclopentyl-6-methoxybenzo[d]isothiazol-3(2H)-one 11 as a white amorphous solid (84%, 0.210 g). 1H NMR (300 MHz, CDCl3): δ 7.87 (d, J = 8.5 Hz, 1H), 6.93 (s, 1H), 6.92 (d, J = 8.2 Hz, 1H), 5.50-5.01 (m, 1H), 3.86 (s, 1H), 2.13-2.12 (m, 2H), 1.83-1.67 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 165.3, 162.7, 142.2, 127.6, 118.8, 114.6, 103.1, 55.8, 55.0, 32.2, 24.4. LC-MS (ESI) calcd. for C13H15NO2S [M+H]+: 250.08; found: 249.95.
2-Cyclopentyl-6-hydroxybenzo[d]isothiazol-3(2H)-one (12)
A solution of 2-cyclopentyl-6-methoxybenzo[d]isothiazol-3(2H)-one 11 (0.250 g, 1 mmol) in anhydrous C6H6 (20 mL) was cooled 0 °C before BBr3 (0.54 g, 2 mmol) was added dropwise. The mixture was gradually warmed to room temperature and then heated at 80 °C for 30 min. After that, the reaction mixture was cooled to room temperature and carefully quenched with H2O (20 mL). The resulting mixture was then heated at reflux for 1 h and filtered; the precipitate was washed with H2O and dried in vacuo to give the crude product. This material was purified by flash chromatography (silica gel, CHCl3:MeOH, 9:1) to afford 2-cyclopentyl-6-hydroxybenzo[d]isothiazol-3(2H)-one 12 (0.200 g, 85%) as a colorless amorphous solid. 1H NMR (300 MHz, DMSO-d6): δ 10.36 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.20 (s, 1H), 6.80 (d, J = 8.6 Hz, 1H), 4.84-4.81 (m, 1H), 2.12-1.98 (m, 2H), 1.75-1.60 (m, 6H). 13C NMR (75 MHz, DMSO- d6): δ 164.8, 161.3, 142.5, 127.4, 117.2, 115.6, 106.9, 54.6, 32.1, 24.4. LC-MS (ESI) calcd. for C12H13NO2S [M+H]+: 236.0667; found: 235.95.
3-Chloro-3′-((2-cyclopentyl-3-oxo-2,3-dihydrobenzo[d]isothiazol-6- yloxy)methyl)biphenyl-4-carboxylic acid (14)
Potassium carbonate (0.052 g, 0.38 mmol) was added to a solution of 2-cyclopentyl-6- hydroxybenzo[d]isothiazol-3(2H)-one 12 (0.075g, 0.32 mmol) and methyl 3′-(bromomethyl)-3-chlorobiphenyl-4-carboxylate (0.129 g, 0.38 mmol) in CH3CN (5 mL). After stirring for 12 h at 80 °C, the organic phase was evaporated under reduced pressure and the crude material was partioned between water and CH2Cl2. The aqueous layer was extracted with CH2Cl2 (3 × 15 mL). The organic layer was dried using Na2SO4 and evaporated to give methyl 3-chloro-3′-((2-cyclopentyl-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yloxy)methyl)biphenyl-4-carboxylate 13 (0.158 g, quantitative yield). The crude product was used in the next step without further purification. LC-MS (ESI) calcd. for C27H24NO2S [M+H]+: 494.11; found: 494.00.
LiI (0.042 g, 0.32 mmol) was added to a solution of the crude product (0.158 g, 0.32 mmol) in pyridine (5 mL). The reaction mixture was heated under reflux for 12 h and then cooled to room temperature. The excess solvent was removed in vacuo. The product was purified by preparative HPLC using isopropanol:water as the solvent system to afford the title compound 14 as a colorless solid (70 mg) in 46% yield. mp: 189-191 °C. 1H NMR (DMSO-d6): δ 8.01 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.71-7.76 (m, 4H), 7.49-7.47 (m, 2H), 7.05 (d, J = 8.5 Hz, 1H.), 5.23 (s, 2H), 4.83-4.81 (m, 1H), 2.02-1.99 (m, 2H), 1.62-1.60 (m, 6H). 13C NMR (DMSO-d6): δ 167.2, 164.0, 140.2, 138.7, 137.8, 131.7, 130.6, 131.0, 129.9, 129.2, 128.2, 127.2, 127.0, 126.9, 119.0, 115.73, 106.6, 70.2, 54.8, 32.2, 24.4. LC-MS (ESI) calcd. for C26H22ClNO4S [M+H]+: 480.09; found: 479.95. HRMS (ESI) calcd. for C26H22ClNO4S [M+H]+: 480.1031; found: 480.1031.
N-Cyclopentyl-2-dihydroxy-4-methoxybenzamide (18)
To a solution of 4-methoxysalicylic acid (0.84 g, 5 mmol) in CH2Cl2 was added pyridine (2.05 mL, 25 mmol) and acetyl chloride (0.89 mL, 12.5 mmol) at 0 °C. The reaction mixture was gradually warmed to room temperature over 30 min and then poured into water. The resulting biphasic mixture was stirred for 1h and then acidified with 1 M HCl and extracted with EtOAc. The crude acetoxy derivative was then converted to the corresponding acid choride by heating with oxalyl chloride (1.3 mL, 15 mmol) in CH2Cl2 for 1h at reflux. After removal of the volatile materials, the acid chloride (1.05 g, 4.38 mmol) in CH2Cl2 was added to a stirred mixture of sodium carbonate (1M, 17.5 mL) and C5H9NH. OH (1.32 g, 13.2 mmol) in THF at 0 °C. The resulting mixture was stirred at room temperature for 1 h, acidified with 0.5 M HCl and then extracted with EtOAc. The crude product was purified by column chromatography on silica gel using hexanes:ethyl acetate afforded hydroxamic acid 18 (1 g, 79%) as a colorless solid. 1H NMR (DMSO- d6): δ 7.65 (d, J = 9.1 Hz, 1H), 6.37-6.36 (overlapping doublet and singlet, 2 H), 4.10-3.98 (m, 1H), 3.70 (s, 1H), 1.69-1.63 (m, 2H), 1.55-1.52 (m, 6H). 13C NMR (DMSO- d6): δ 167.5, 162.5, 160.0, 131.4, 113.3, 105.5, 101.6, 56.6, 55.7, 29.5, 25.5. LC-MS (ESI) calcd. for C13H17NO4 [M+H]+: 251.12; found: 252.00.
2-Cyclopentyl-6-methoxybenzo[d]isoxazol-3(2H)-one (19)
Diethylazodicarboxylate was added dropwise to a cooled solution of hydroxamic acid 18 (0.71 g, 2.8 mmol) and triphenyl phosphine (0.89 g, 3.4 mmol) in THF. The reaction mixture was warmed to room temperature and quenched with 1:1 MeOH:AcOH (0.2 mL). Concentration of the solvent followed by chromatographic separation on silica gel afforded 19 as a pale yellow solid (0.3 g, 46%). 1H NMR (CDCl3): δ 7.67 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.32 (s, 1H), 4.35-4.33 (m, 1H), 3.84 (s, 3H), 1.89-1.68 (m, 8H). 13C NMR (CDCl3): δ 164.5, 162.8, 162.3, 124.9, 112.9, 109.8, 103.1, 55.8, 55.0, 32.2, 24.4. LC-MS (ESI) calcd. for C13H15NO3 [M+H]+: 233.10; found: 232.90.
4-Chloro-3′-((2-cyclopentyl-3-oxo-2,3-dihydrobenzo[d]isoxazol-6-yloxy)methyl)biphenyl- 3-carboxylic acid (21)
BBr3 (0.54 g, 2 mmol) was added dropwise to a solution of 19 (0.232 g, 1 mmol) in anhydrous benzene (20 mL) at 0 °C. The mixture was gradually warmed to room temperature and then heated at 80 °C for 30 min. After that the reaction mixture was cooled to room temperature and carefully quenched with H2O (20 mL). The resulting mixture was then heated at reflux for 1 h and then cooled. Extraction with EtOAc and removal of the solvent in vacuo afforded the crude demethylated product (0.170 g, 78%) which was used in the next step without further purification. A solution of 2-cyclopentyl-6-hydroxyybenzo[d]isoxazol-3(2H)-one (0.109 g, 0.5 mmol) was treated with methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate under the same conditions as described for compound 14 to give the corresponding ester in 76% yield (0.180 g). LC-MS (ESI) calcd. for C27H24ClNO5 [M+H]+: 477.13. Found: 478.05. The crude product obtained above was treated with LiI in pyridine to give the final compound 21 (0.063 g, after HPLC purification) as a colorless solid in 36% yield. mp: 185–187 °C. 1H NMR (DMSO- d6): δ 8.21 (s, 1H), 7.70-7.56 (m, 7H), 6.92 (d, J = 6.1 Hz, 1H), 5.18 (s, 2H), 4.98-4.95 (m, 1H), 2.06-2.02 (m, 2H), 1.92-1.88 (m, 4H), 1.64-1.63 (m, 2H). 13C NMR (DMSO- d6): δ 164.0, 163.4, 162.7, 139.4, 137.6, 131.7, 130.9, 129.9, 129.2, 127.1, 126.9, 125.2, 114.3, 109.4, 70.5, 95.5, 56.6, 30.2, 25.4. LC-MS (ESI) calcd. for C26H22ClNO5 [M+H]+: 464.13; found: 463.95. HRMS (ESI) calcd. for C26H22ClNO5 [M+H]+: 464.1259; found: 464.1261.
N-(3-Methoxyphenethyl)cyclopentanamine (23)
3-Methoxyphenethylamine (4.5 mL, 30 mmol), and iodocyclopentane (1.15 mL, 10 mmol) in CH3CN were irradiated for 10 min in a microwave reactor at 110 °C. After cooling to room temperature the mixture was diluted with CH2Cl2 and washed with NaHCO3 solution. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. The crude product was separated by silica gel chromatography using hexane as eluent and the pure product N-(3-methoxyphenethyl)cyclopentanamine (1.45 g, 66%) was obtained as a colorless liquid which solidified upon standing. 1H NMR (CDCl3): δ 8.48 (s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.79-6.73 (overlapping doublets and singlet, 3H), 3.74 (s, 3H), 3.44-3.39 (m, 1H), 3.38-3.11 (m, 4H), 1.84-1.83 (m, 2H), 1.82-1.81 (m, 4H), 1.66-1.56 (m, 2H). 13C NMR (CDCl3): δ 159.63, 140.93, 129.38, 121.07, 114.49, 111.45, 59.26, 55.18, 48.08, 32.46, 29.65, 23.84. LC-MS (ESI) Calcd for: C14H21NO [M+H]+: 220.16. Found: 220.00.
Methyl cyclopentyl(3-methoxyphenethyl)carbamate (24)
To a solution of N-(3-methoxyphenethyl)cyclopentanamine 23 (1.31 g, 6 mmol) in diethyl ether was added triethylamine (1.3 mL, 9 mmol) and methyl chloroformate (0.7 mL, 9 mmol) at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 30 min. Filtration and concentration of the solvent under reduced pressure preceded silica gel column chromatography using hexanes/ethyl acetate to afford methyl cyclopentyl(3- methoxyphenethyl)carbamate as a pale yellow liquid (1.41g, 84%). 1H NMR (CDCl3): δ 7.19 (t, J = 7.6 Hz, 1H), 6.78-6.73 (overlapping doublets and singlet, 3H), 4.27-4.24 (m, 1H), 3.78 (s, 3H), 3.70 (s, 3H), 3.28 (t, J = 7.9 Hz, 2H), 2.80 (t, J = 7.9 Hz, 2H), 1.68-1.67 (m, 2H), 1.53-1.50 (m, 6H). 13C NMR (CDCl3): δ 159.63, 156.74, 140.93, 129.38, 121.07, 114.49, 111.45, 57.94, 55.09, 52.30, 45.75, 36.50, 29.37, 23.50. LC-MS (ESI) Calcd for C16H23NO3 [M+H]+: 278.17. Found: 278.00.
2-Cyclopentyl-6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (25)
To a solution of methyl cyclopentyl(3-methoxyphenethyl)carbamate 24 (1.39 g, 5 mmol) in POCl3 (20 mL) was added P2O5 (1.4 g, 10 mmol). The reaction mixture was heated at reflux for 2 h. Excess POCl3 was removed under reduced pressure and quenched with iced water. The mixture was neutralized with Na2CO3, extracted with EtOAc, and the organic phase was dried over Na2SO4. Concentration of the solvent followed by silica gel column chromatography using hexanes/ethyl acetate afforded 2-cyclopentyl-6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (1.30 g, 94%). 1H NMR (CDCl3): δ 8.00 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 8.6 Hz,1H), 6.63 (s, 1H), 5.18-5.14 (m, 1H), 3.85 (t, J = 6.7 Hz, 1H), 2.88 (t, J = 6.7 Hz, 1H), 1.88-1.70 (m, 2H), 1.64-1.63 (m, 4H), 1.62-1.61 (m, 2H). 13C NMR (CDCl3): δ 164.44, 162.08, 139.97, 130.56, 123.05, 112.43, 111.79, 55.43, 53.70, 40.32, 28.96, 28.81, 24.57. LC-MS (ESI) Calcd for C15H19NO2 [M+H]+: 246.14. Found: 246.00.
2-Cyclopentyl-6-hydroxy-3,4-dihydroisoquinolin-1(2H)-one (26)
BBr3 (3.7 g, 14.7 mmol) was added dropwise to a solution of 2-cyclopentyl-7-methoxy-3,4-dihydroisoquinolin-1(2H)-one 25 (1.2 g, 4.9 mmol) in benzene at 0 °C. The mixture was gradually warmed to room temperature and then heated at 80 °C for 30 min. The reaction mixture was then cooled to room temperature and carefully quenched with H2O. The resulting mixture was then heated at reflux for 1 h and then cooled. Extraction with EtOAc and removal of the solvent in vacuo, followed by chromatographic separation using MeOH-CHCl3 provided 2-cyclopentyl-6-hydroxy-3,4-dihydroisoquinolin-1(2H)-one 26 as a colorless solid (0.735 g, 65%). 1H NMR (DMSO-D6): δ 9.96 (s, 1H), 7.65 (d, J = 8.6 Hz, 1H), 6.63 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 4.95-4.93 (m, 1H), 3.32 (t, 2H, J = 6.1 Hz), 2.73 (t, 2 H, J = 6.7 Hz), 1.73-1.64 (m, 4H), 1.53-1.45 (m, 4H). 13C NMR (DMSO-d6): 163.82, 160.69, 141.02, 130.29, 121.51, 114.28, 113.62, 53.44, 42.20, 28.79, 28.57, 24.52. LC-MS (ESI) Calcd for C14H17NO2 [M+H]+: 232.13. Found: 232.00.
4-Chloro-3′-((2-cyclopentyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yloxy)methyl)biphenyl-3- carboxylic acid (28)
2-Cyclopentyl-6-hydroxy-3,4-dihydroisoquinolin-1(2H)-one 26 (0.106 g, 0.5 mmol) and methyl 3′-(bromomethyl)-4-chlorobiphenyl-3-carboxylate (0.208 g, 0.6 mmol) were processed according to the general procedure to provide methyl 4-chloro-3′-((2-cyclopentyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yloxy)methyl)biphenyl-3-carboxylate 27. NaOH (50% aqueous) was added to a solution of the crude product (0.150 g, 0.3 mmol) in dioxane (5 mL). The reaction mixture was heated at reflux for 1 h, and then cooled to room temperature. Excess solvent was removed in vacuo, the residue was ddiluted with water and then acidified using 1M HCl. Extraction using EtOAc, followed by purification using preparative HPLC afforded 28 (0.162 g, 69% over 2 steps) as a colorless solid. mp: 147–149 °C. 1H NMR (DMSO-d6): δ 8.00 (s, 1H), 7.87-7.75 (m, 3H), 7.65-7.57 (m, 2H), 7.49-7.41 (m, 2H), 6.95-6.90 (overlapping doublets and singlet, 2H), 5.20 (s, 2H), 4.97-4.93 (m, 1H), 3.34 (t, J = 6.1 Hz, 2H), 2.84 (t, J = 6.1 Hz, 2H), 1.69-1.46 (m, 8 H). 13C NMR (DMSO-d6): 163.32, 161.79, 141.60, 138.58, 137.55, 131.75, 130.99, 129.84, 129.16, 128.86, 126.45,113.78, 112.80, 69.70, 53.54, 42.20, 28.81, 28.63, 23.83. LC-MS (ESI) Calcd for C28H26ClNO4 [M+H]+: 476.16. Found: 476.00. HRMS (ESI) calcd for C28H26ClNO4 [M+H]+: 476.1623. Found: 476.1623.
mGluR2 in vitro assay
Human Embryonic Kidney (HEK-293) cell lines co-expressing rat mGluR 2, 3, 4, 6, 7 or 8 and G protein inwardly rectifying potassium (GIRK) channels16 were grown in Growth Media containing 45% DMEM, 45% F-12, 10% FBS, 20 mM HEPES, 2 mM L-glutamine, antibiotic/antimycotic, non-essential amino acids, 700 μg/ml G418, and 0.6 μg/ml puromycin at 37°C in the presence of 5% CO2. Rat mGluR1 and 5 cells were cultured as described in Hemstapat et al.25 All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted. Calcium assays were used to assess activity of compounds at mGluRs 1 and 5 as previously described in Engers et al.26 Compound activity at the rat group II (mGluRs 2 and 3) and group III (mGluRs 4, 6, 7, and 8) mGluRs was assessed using thallium flux through GIRK channels, a method that has been described in detail.16 Briefly, cells were plated into 384-well, black-walled, clear-bottomed poly-D-lysine-coated plates at a density of 15,000 cells/20 μl/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (assay media). Plated cells were incubated overnight at 37 °C in the presence of 5% CO2. The following day, the medium was removed from the cells, and 20 μL/well of 1.7 μM BTC-AM, an indicator dye (Invitrogen; prepared as a stock in DMSO and mixed in a 1:1 ratio with Pluronic acid F-127) in assay buffer [Hanks’ balanced salt solution (Invitrogen) containing 20 mM HEPES, pH 7.3] was added to the plated cells. Cells were incubated for 1 h at room temperature, and the dye was replaced with 20 μl assay buffer. Test compounds were diluted to 2 times their final desired concentration in assay buffer. Agonists were diluted in thallium buffer [125 mM sodium bicarbonate (added fresh the morning of the experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, and 10 mM HEPES, pH 7.3] at 5 times the final concentration to be assayed. Cell plates and compound plates were loaded onto a kinetic imaging plate reader (FDSS 6000; Hamamatsu Corporation, Bridgewater, NJ). Appropriate baseline readings were taken (10 images at 1 Hz; excitation, 470 ± 20 nm; emission, 540 ± 30 nm) and test compounds were added in a 20 μl volume and incubated for 2.5 min before the addition of 10 μl of thallium buffer with or without agonist. After the addition of agonist, data were collected for an additional 2.5 min. Data were analyzed using Excel (Microsoft Corp, Redmond, WA). The slope of the fluorescence increase beginning 5 s after thallium/agonist addition and ending 15 s after thallium/agonist addition was calculated, corrected to vehicle and maximal agonist control slope values, and plotted in Prism software (GraphPad Software, San Diego, CA) to generate concentration-response curves. Potencies were calculated from fits using a four-point parameter logistic equation. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves and were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA). Test compounds were applied and followed by EC20 concentrations of glutamate. For selectivity experiments, full concentration-response curves of glutamate or L-AP4 (for mGluR7) were performed in the presence of a 10 μM concentration of compound and compounds that affected the concentration-response by less than 2 fold in terms of potency or efficacy were designated as inactive.
Microsomal stability in vitro assay
Pooled rat liver microsomes (BD Biosciences, # 452701) were preincubated with test compounds at 37.5 °C for 5 min in the absence of NADPH. The reaction was initiated by addition of NADPH and incubated under the same conditions. The final incubation concentrations were 4 μM test compound, 2 mM NADPH, and 1 mg/mL (total protein) liver microsomes in phosphate-buffered saline (PBS) at pH 7.4. One aliquot (100 μL) of the incubation mixture was withdrawn at 15 min time points and combined immediately with 100 μL of ACN/MeOH. After mixing, the sample was centrifuged at approximately 13000 rpm for 12 min. The supernatant was filtered and transferred into an autosampler vial and the amount of test compound was quantified using a Shimadzu LCMS 2010EV mass spectrometer. The change of the AUC (area under the curve) of the parent compound as a function of time was used as a measure of microsomal stability. Test compounds were run in duplicate with a positive control.
Plasma stability in vitro assay
A 20 μL aliquot of a 10 mM solution in DMSO of the test compound was added to 2.0 mL of heparinized rat plasma (Lampire, P1-150N) to obtain a 100 μM final solution. The mixture was incubated for 1 h at 37.5 °C. Aliquots of 100 μL were taken at 15 min intervals and diluted with 100 μL of MeOH/ACN. After mixing, the sample was centrifuged at approximately 13000 rpm for 12 min. The supernatant was filtered and transferred into an autosampler vial and the amount of test compound was quantified using the Shimadzu LCMS-2010EV system. The change of the AUC of the parent compound in function of time was used as a measure of plasma stability.
Parallel artificial membrane permeation assay (PAMPA)
A 96-well microtiter plate (Millipore, # MSSACCEPTOR) was completely filled with aqueous buffer solution (pH 7.4) and covered with a microtiter filterplate (Millipore, # MAPBMN310) to create a sort of sandwich construction. The hydrophobic filter material was impregnated with a 10% solution of polar brain lipid extract in chloroform (Avanti) as the artificial membrane, and the organic solvent was allowed to completely evaporate. Permeation studies were started by the transfer of 200 μL of a 100 μM test compound solution on top of the filter plate. In general phosphate pH 7.2 buffer was used. The maximum DMSO content of the stock solutions was <1.5%. In parallel, an equilibrium solution lacking a membrane was prepared using the exact concentrations and specifications but lacking the membrane. The concentrations of the acceptor and equilibrium solutions were determined using the Shimadzu LCMS-2010EV and AUC methods. The Acceptor plate and equilibrium plate concentrations were used to calculate the permeability rate (Log Pe) of the compounds. The log Pe values were calculated using the following equation:
equation M1
Behavioral procedures
Apparati
Intravenous cocaine self-administration and food-maintained responding took place in 24 Plexiglas experimental chambers (25 × 31 × 24 cm; MED Associates, St. Albans, VT), each housed in a sound-attenuating box (San Diego Instruments, San Diego, CA). One wall of the chamber contained two levers, measuring approximately 3 cm in width and located approximately 3 cm above the metal grid floor of the chamber. There was also an opaque disk (2.5 cm diameter) mounted 10 cm above each lever which could be illuminated by a 2.5 W, 24 V white light bulb and served as a cue light paired with cocaine or food delivery. All data collection and test session functions were controlled by computers and MED-PC IV software (MED Associates).
Food training and food-maintained responding
Methodological details of food training and food-maintained responding procedures have been described previously. 2730, Rats were placed under food restriction (20 g/day/rat) and trained to lever press for 45 mg food pellets (Research Diets, New Brunswick, NJ). Training started on a fixed-ratio 1 time-out 1 s (FR1 TO1 s) schedule of reinforcement, which then changed to FR1 TO 20 s. During food training, only the active lever was used and there was no cue light presentation. Successful acquisition of the food response was defined as earning 100 pellets during a 60-min session. The training period lasted approximately 5 days. After successful acquisition of food-maintained responding, these rats were allowed to continue responding for food on an FR1 TO20 s schedule during 1-h test sessions 5–7 days per week. During these sessions, two levers (active and inactive) were present in the box. Responses on an active lever resulted in a food pellet delivery paired with a 20 s cue light presentation. Responses on inactive lever had no programmed consequences. An identical training procedure was used for both the subjects that responded for food and the rats that later were allowed to selfadminister cocaine.
Cocaine self-administration procedure
Methodological details of catheter construction and surgery and self-administration procedure have been described previously.2629 Briefly, rats were prepared with intravenous catheters inserted into the right jugular vein under isoflurane anesthesia (1–1.5% isoflurane/oxygen mixture). Cocaine self-administration training began after 7 days of recovery from surgery during daily 1-h self-administration testing sessions. These sessions were initiated by extension of both the active and inactive levers. Responses at the active lever were reinforced under an FR1 TO 20 s schedule of reinforcement by an intravenous injection of cocaine (0.5 mg/kg/infusion salt) delivered over a 2-s period in a volume of 0.05 ml. Upon completion of the operant response for cocaine delivery, a cue light above the lever was turned on for 20 s and signaled the time-out period, during which responses had no consequences. Responses at the inactive lever were recorded but had no scheduled consequences. All rats were trained to selfadminister cocaine for 21 days. The criteria for exhibiting stable performance (more than 10 infusions per session; less than 20% variation over three consecutive sessions) were reached by all rats during this 10-day period.
Experimental design for investigating the effects of the mGluR2 PAM on cocaine self-administration and food-maintained responding
After completion of the cocaine self-administration training, drug treatments were initiated. Compound 14 was administered to rats (n=9) according to a within-subjects Latin-square design. The compound was administered orally at doses of 0, 5, 10 and 20 mg/kg with 60 min pretreatment time. The highest dose (40 mg/kg) was administered to all rats after completion of the Latin-square. At least three days elapsed between drug/vehicle injections to re-establish stable self-administration behavior (less than 20% variation over three days).
To assess possible nonspecific effects, the effects of compound 14 at doses identical to those used for cocaine self-administration were assessed in a separate group of rats (n=8) trained to lever-press for food during 1-h sessions. The FR1 TO20 s schedule of reinforcement used for food-maintained responding was identical in all parameters to the schedule under which cocaine was self-administered.
Statistical analyses
Intravenous self-administration and food-maintained responding data were expressed as a percentage of the baseline number of rewards earned, with baseline defined as the mean number of rewards earned during the three days before each drug testing session. Percent values are presented to allow for direct comparisons for data derived from the two different reinforcers (i.e., cocaine and food). Acute effects of compound 14 on cocaine self-administration and food-maintained responding were analyzed using two-way repeated-measures ANOVA, with Dose as the within-subjects factor, and Reinforcer (food or cocaine) as the between-subject factor. Post hoc comparisons were conducted with Newman-Keuls and Bonferroni tests. The criterion for significance was set at 0.05.
Results
The number of baseline cocaine infusions was 20 ± 4.0 (mean ± SEM) and the number of baseline food pellets earned was 100 ± 0.0 (mean ± SEM). A two-way ANOVA on the number of baseline cocaine/food infusions earned during the three baseline days prior to the drug treatment days indicated no significant differences across days, demonstrating the stability of baseline food and cocaine self-administration in rats during the experiment (data not shown).
Systemic administration of compound 14 significantly decreased both cocaine and food self-administration in rats (Fig. 2 in main text). A two-way ANOVA revealed significant main effects for the factor compound 14 Dose (F4,60 = 14,88, p < 0.0001), the factor Reinforcer (F1,60 = 35.51, p < 0.0001) and their interaction (F4,60 = 8.75, p < 0.0001). Post hoc tests showed a significant decrease in cocaine intake after administration of 20 mg/kg (p<0.01) and 40 mg/kg (p<0.001) of compound 14 compared with the vehicle treatment condition. Compound 14 had no effect on inactive lever presses at any of the doses tested (data not shown). Compound 14 also decreased food-maintained responding and this effect was significant only at the highest dose used (40 mg/kg). Additionally, post-hoc tests comparing the effects of the same dose of compound 14 on cocaine- and food-maintained responding indicated that compound 14 had more pronounced effect in decreasing cocaine compared to food self-administration (Figure 2 in main text).
Acknowledgments
This work was supported by National Institutes of Health grant R01 DA023926 to NC. The authors would like to thank Dr. Xinchun Jin for collection of the cocaine and food self-administration data, and Dr. Anthony Pinkerton for helpful comments during the preparation of the manuscript.
Abbreviations
mGluR2metabotropic glutamate receptor subtype 2
PAMpositive allosteric modulator
BINA3′-((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yloxy)methyl)biphenyl-4-carboxylic acid
HTShigh-throughput screening
PKpharmacokinetic
POCproof-of-concept
AUCarea under curve
LOQlimit of quantitation
AIBN2,2′-azo bisisobutyronitrile
NBSN-bromosuccinimide
HEKHuman Embryonic Kidney
GIRKG protein inwardly rectifying potassium
PBSphosphate-buffered saline
DMEMDulbecco’s modified eagle medium
HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
FBSfetal bovine serum

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