Synthesis and Biological Activities of Conformationally Restricted

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Synthesis and Biological Activities of Conformationally Restricted Cyclopentenyl-Glutamate Analogues Alison T. Ung,† Karl Schafer,† Karl B. Lindsay,† Stephen G. Pyne,*,† Kitti Amornraksa,‡ Ria Wouters,§ Ilse Van der Linden,§ Ilse Biesmans,§ Anne S. J. Lesage,§ Brian W. Skelton,| and Allan H. White| Department of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia, Department of Chemistry, Thammasat University, Pathumthani, 12121, Thailand, CNS Discovery Research, Janssen Research Foundation, Beerse, B-2340, Belgium, and Department of Chemistry, University of Western Australia, Crawley, WA, 6009, Australia [email protected] Received August 23, 2001

An efficient method for preparing conformationally restricted cyclopentenyl-glutamate analogues in a regioselective and diastereoselective manner has been developed using a formal [3 + 2] cycloaddition reaction of dehydroamino acids. Methods for preparing optically active versions of these compounds have also been devised. Of these compounds, (S)-2 is an agonist at the mGlu5 (EC50 18 µM) and mGlu2 (EC50 45 µM) receptors. Introduction Considerable research efforts have been focused upon the development of selective agonists and antagonists for the ionotropic and metabotropic glutamate subtype receptors.1 Such selective compounds have potential applications as therapeutic agents for the treatment of a number of neurodegenerative diseases. A variety of cyclic, conformationally restrictive glutamate analogues have been prepared in laboratories around the world, but only a few are highly potent and subtype selective.1 One such compound is the conformationally restricted glutamate analogue, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) 1, which selectively activates metabotropic glutamate (mGlu) receptors over the ionotropic type. This compound, however, is not selective for the individual eight mGlu receptor subtypes that are currently known.2 In 1997,3 we reported the synthesis of the cyclopentenyl-glutamate analogue (R)-2 a novel dehydroanalogue of ACPD. This compound was prepared from the triphenylphosphine-catalyzed cycloaddition of ethyl buta-2,3-dienoate with the chiral dehydroamino acid derivative (R)-3.4 We now report here the full details of this synthesis and an extension of this study to the preparation of other optically active and racemic conformationally restricted glutamate analogues. The biological

activities of these compounds at specific glutamate receptors are also reported.

Results and Discussion Our general strategy for preparing the target glutamate analogues 7 is shown in Scheme 1. This method formally involves the [3 + 2] cycloaddition reaction between a dehydroamino acid derivative 6 with the ylide 5, generated in situ from the reaction from an appropriate alkyne or allene with a phosphine.5 In this study the dehydroamino acids (R)-3 and 4 were employed. Other dehydroamino acids that were tried (e.g. 6 [R ) Et, R1 ) H, R2 ) Boc])6 were not sufficiently reactive and resulted in low yields of cycloadduct and mainly recovered 6 and self-

†

University of Wollongong. Thammasat University. Janssen Research Foundation. | University of Western Australia. (1) For recent reviews see: Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. Ø, Madsen, U.; Krogsgaard-Larsen, P. J. Med. Chem. 2000, 43, 2609-2645. Trist, D. G. Pharm. Acta Helv. 2000, 74, 221-229. (2) For some recent papers, see: Jayaraman, V.; Keesey, R.; Madden, D. R. Biochemistry 2000, 39, 8693-8697. Bessis A.-S.; Jullian, N.; Coudert, E.; Pin, J.-P.; Acher, F. Neuropharmacology 1999, 38, 15431551. (3) Pyne, S. G.; Schafer, K.; Skelton, B. W.; White, A. H. Chem. Commun. 1997, 2267-8. (4) Pyne, S. G.; Dikic, B.; Gordon, P.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1993, 46, 73-93. ‡ §

(5) (a) Zhang, C.; Lu, X. J. Org. Chem. 1995, 60, 2906-2908. (b) Xu, Z.; Lu, X. Tetrahedron Lett. 1997, 38, 3461-3464. (c) Shu, L.-H.; Sun, W.-Q.; Zhang, D.-W.; Wu, S.-H.; Wu, S.-H.; Xu, J.-F.; Lao, X.-F. Chem. Commun. 1997, 79-81. (d) O’Donavan, B. F.; Hithcock, P. B.; Meidine, M. F.; Kroto, H. W.; Taylor, R.; Walton, D. R. M. Chem. Commun. 1997, 81-82. (e) Zhu, G.; Chen, Z.; Jiang, Q.; Xiao, D.; Cao, P.; Zhang, X. J. Am. Chem. Soc. 1997, 119, 3836-3837. (f) Xu, Z.; Lu, X. J. Org. Chem. 1998, 63, 5031-5041. (g) Kumar, K.; Kapur, A.; Ishar, M. P. S. Org. Lett. 2000, 2, 787-789. (h) Kumar, K.; Kapoor, R.; Kapur, A.; Ishar, M. P. S. Org. Lett. 2000, 2, 2023-2025. (i) Lu, X.; Zhang, C.; Xu, Z. Acc. Chem. Res. 2001, 34, 535-544. (6) Yokoyama, Y.; Takahashi, M.; Takashima, M.; Kohno, Y.; Kobayashi, H.; Katoaka, K.; Shidori, K.; Murakami, Y. Chem. Pharm. Bull. 1994, 42, 832-838.

10.1021/jo010864i CCC: $22.00 © 2002 American Chemical Society Published on Web 11/30/2001

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Ung et al. Scheme 3a

Scheme 1

a

Reagents: (i) (R)-methyl mandelate, Et3N, CH2Cl2, 7 d, 63%.

Figure 1. NOE cross-peaks for compounds 12 and 14.

Scheme

2a

a Reagents: (i) Ph P (10 mol %), PhH, RT, 5-24 h [12 (17%) + 3 13 (49%); 14 (38%); 15 (78%)]; (ii) 6 N HCl, reflux, 14-16 h; (iii) ion-exchange; (iv) HCl (overall yields (72-91%).

condensation products of the alkyne or allene. The ylide 5 has been used to prepare a number of carbocylic and heterocyclic compounds from its [3 + 2] cycloaddition reactions with other electron deficient alkenes5 and imines.5b,f Furthermore, the cycloaddition reactions of chiral and achiral dehydroamino acid derivatives has been well documented.7 The ylide 5 (R1 ) H, R2 ) Ph, R ) Et) was generated in situ from the reaction of ethyl buta-2,3-dienoate 98 (5 equiv) and triphenylphosphine (0.1 equiv) in the presence of (R)-3 in benzene solution at RT. This reaction gave a mixture of the two regioisomers 12 and 13 that were readily separated by column chromatography (Scheme (7) (a) For chiral derivatives, see: Chinchilla, R.; Falvello, L. R.; Galindo, N.; Na´jera C. J. Org. Chem. 2000, 65, 3034-3041 and references therein. (b) For achiral derivatives, see: Stammer, C. H. Tetrahedron 1990, 46, 2231-2254. (8) Lang, R. W.; Hansen, H.-J. Org. Synth. 1984, 62, 202-209.

2). Pure 12 and 13 were obtained in yields of 17% and 49%, respectively, based on the moles of (R)-3. The known dimer of 9, diethyl 2-methylene-3-cyclopentene-1,3dicarboxylate5a was also isolated in 27% yield. The regioisomeric ratio of 12 and 13 was estimated as 77:23 from 1H NMR analysis of the crude reaction mixture. The structure of the major regioisomer 13 was secured by a single-crystal X-ray study.3 The enantiomeric excess of 13 was determined to be 88% from 1H NMR analysis of its (R)-methyl mandelate derivative 19, which was isolated as a 94:6 mixture of diastereomers [major: δH (CDCl3) 5.99 (s), minor 5.98 (s)] (Scheme 3). The stereochemistry of the minor regioisomer 12 was established from NOESY experiments that showed cross-peaks between the signal for H-2 and that for one H-3′ proton, as shown on structure 12 (Figure 1). Thus, compounds 12 and 13 have the 4S and 4R configurations, respectively, at the spiro carbon, consistent with the known tendency of (R)-3 in [4 + 2] and [3 + 2] cycloaddition reactions to give products that arise from attack of the four- or threeatom component anti to the C-2 phenyl group.4,9 Acid hydrolysis of compounds 12 and 13 with 6 N HCl at reflux followed by purification by ion-exchange chromatography gave the corresponding amino acids which were stored and characterized as their hydrochloride salts, (R)-2 and (S)-16, respectively (Scheme 2). The analogous triphenylphoshine-catalyzed reaction between 9 and the dihydroamino acid 4 produced diethyl 2-methylene-3-cyclopentene-1,3-dicarboxylate but no 21 could be detected in the crude reaction mixture from 1H NMR analysis. Using ethyl butynoate 20 and the more nucleophilic tributylphoshine, however, provided compound 21 as a single regioisomer in 98% yield after purification by column chromatography (Scheme 4). The structure of 21 was confirmed by a single-crystal X-ray study. Acid hydrolysis of 21 with 6 N HCl at reflux gave (9) (a) Pyne, S. G.; Dikic, B.; Gordon, P.; Skelton, B. W.; White, A. H. J. Chem. Soc., Chem Commun. 1991, 1505-1506. (b) Pyne, S. G.; J. Safaei-G., J.; Hockless, D. C. R.; Skelton, B. W.; Sobolev A. N.; White, A. H. Tetrahedron 1994, 50, 941-956. (c) Pyne, S. G.; Safaei-G.; J.; Koller, F. Tetrahedron Lett. 1995, 36, 2511-2514. (d) Pyne, S. G.; Javidan, A.; Skelton, B. W.; White, A. H. Tetrahedron 1995, 51, 51575168. (e) Pyne, S. G.; Safaei-G., J.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1995, 48, 1511-1533. (f) Pyne, S. G.; Safaei-G., J. J. Chem. Res. (S) 1996, 160-161. (g) Javidan, A.; Schafer, K.; Pyne, S. G.; Skelton, B. W.; White, A. H. Synlett 1997, 101-102. (h) Pyne, S. G.; Schafer, K.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1998, 51, 127136. (i) Pyne, S. G.; Safaei-G., J.; Schafer, K.; Javidan, A.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1998, 51, 137-158. (j) Pyne S. G.; Schafer, K. Tetrahedron 1998, 54, 5709-5720.

Cyclopentenyl-Glutamate Analogues Scheme 4a

J. Org. Chem., Vol. 67, No. 1, 2002 229 Scheme 5

Scheme 6

a Reagents: (i) For 20: Bu P (10 mol %), PhH, RT, 24 h (98%), 3 For 11:Ph3P (10 mol %), PhH, RT, 24 h (40%); (ii) 6 N HCl, reflux, 6 h (91-100%).

the hydrochloride salt of the racemic amino acid rac-2 (Scheme 4) that has identical spectral properties to (R)2. The regiochemical outcome of the reaction of 4 with ylide 5 (R1 ) H, R2 ) Bu, R ) Et) occurred in the same regiochemical sense as the reaction of acrylate esters with 5 (R1 ) H, R2 ) Ph or Bu, R ) Et), that is, preference for 3-cyclopentene-1,3-dicarboxylate regioisomers (e.g., 7, Scheme 1) over the 2-cyclopentene-1,2-dicarboxylate regioisomers (e.g., 8, Scheme 1). However, unlike acrylate esters, the reaction of 4 with 5 (R1 ) H, R2 ) Bu, R ) Et), is completely (>98%) regioselective. In contrast, the reaction of (R)-3 with ylide 5 (R1 ) H, R2 ) Ph, R ) Et) favored the opposite 2-cyclopentene regioisomer (13). Semiempirical calculations (AM1, PC Spartan Pro) on these captodative alkenes suggest that the FMO coefficients at the alkene carbons of the LUMO of 4 are significantly larger than those in (R)-3 (that is the alkene group in 4 is more highly polarized). This is consistent with the expected stronger electron-withdrawing effect/ decreased electron donor effect of the imino group in 4 over the N-benzoyl group in (R)-3. Steric considerations aside, the enhanced diastereoselectivity in the case of 4 is readily understandable in terms of its larger FMO coefficients. The ylide 5 (R1 ) Ph, R2 ) Ph, R ) Et) that was generated in situ from the reaction of ethyl 4-phenylbuta-2,3-dienoate 11 (5 equiv) and triphenylphosphine (0.1 equiv) in the presence of (R)-3 or 4 in benzene solution at RT produced the adducts 15 and (1SR,5RS)23 respectively, as single regioisomers, in 78% and 40% yields, respectively (Schemes 2 and 4). The structure and relative stereochemistry of (1SR,5RS)-23 was unequivocally determined by a single-crystal X-ray study that showed the 1,2-cis relative configuration between the C-5 phenyl substituent and the C-1 diphenylmethylideneamino group. The same relative configuration was found in 15 from NOESY experiments on the alcohol 24 that was prepared from the sodium borohydride reduction of lactone moiety of 15 (Scheme 5). These NMR experiments showed significant cross-peaks between H-3 and the

diastereotopic protons of the C-4 hydroxymethyl group, which indicated their 3,4-cis relative configuration. Acid hydrolysis of compounds 15 and (1SR,5RS)-23 with 6 N HCl at reflux gave the corresponding amino acids (1S,5R)-18 and (1SR,5RS)-18, respectively, as their hydrochloride salts (Schemes 2 and 4). These compounds had identical 1H and 13C NMR spectra, consistent with the above configurational assignments of their precursors. Treatment of a benzene solution of (R)-3 and ethyl penta-2,3-dienoate 10 (5 equiv) with triphenylphosphine (0.1 equiv) at RT for 5 h gave a single cycloadduct 14 in 38% isolated yield after purification by column chromatography. No other regioisomeric product could be detected by 1H NMR analysis of, or was isolated from, the crude reaction mixture. The 3′S,4S stereochemistry of 14 was evident from NOESY experiments which showed cross-peaks between H-2 and the C-3′ methyl group and between H-3′ and the ortho protons of the benzamido group, as shown in Figure 1. Acid hydrolysis of 14 gave the amino acid 17 that was characterized as its hydrochloride salt (Scheme 2). The stereochemical outcomes of the phosphine-catalyzed reactions of (R)-3 with 10 and 11 are clearly different with respect to the newly created stereogenic center at C-3′ in the cyclopentenyl ring. This difference may be rationalized as arising from the ylides 25 and 26, respectively, in which the latter ylide has the 3,4-(E) geometry to avoid adverse steric interactions between the bulky 3-triphenylphosphonium group and the 4-phenyl group. The former ylide, with the less sterically demanding 4-methyl group would appear to react via 25 having the 3,4-(Z) geometry. The proposed transition state structures for these cycloaddition reactions are shown in Scheme 6. The relative orientation of the reacting partners shown is expected to minimize steric interactions between the sterically demanding 3-triphenylphosphonium group on the ylide and the bulky substituent on the nitrogen atom on the dipolarophile [(R)-3] and between the ylide and the C-2 phenyl group of the oxazolidinone ring in (R)-3. The (S)-enantiomer of 2 was also required for testing at glutamate receptors and was prepared according to Scheme 7. The known imino (1R,2S,5R)-menthyl ester

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J. Org. Chem., Vol. 67, No. 1, 2002 Scheme 7a

Ung et al. Table 1. Agonist Activity at the Metabotropic Glutamate Receptors MGlu1a, MGlu5a and MGlu2 EC50, µM (mean ( SD, n) (R)-2 (S)-2 rac-2 (1SR,5RS)-18 (1S,5R)-17 glutamate (1S,3R)-ACPD-1 LY-354740

a Reagents: (i) Bu P (10 mol %), PhH, RT, 16 h (87%); (ii) 1 N 3 HCl, ether, 16 h, RT (95%); (iii) BnOCOCl, Na2CO3, 2 h, 0 °C (70%), then HPLC (iv) 6 N HCl, 4 d, 85 °C (90-98%).

2710 was prepared using modifications to the literature procedures (Supporting Information). The tributylphosphine-catalyzed reaction between 20 and 27 gave the expected cycloadducts 28 and 29 as a 60:40 mixture of diastereomers in 87% yield. While these diastereomers could not be separated, their corresponding N-benzyloxycarbonyl (Cbz) derivatives, 32 and 33, could be separated by preparative HPLC to give pure samples. Acid hydrolysis of the individual diastereomers 32 and 33 gave (R)-2 ([R]D22 +5.9 c 0.6, 1 N HCl) and (S)-2 ([R]D22 -5.8 c 0.87, 1 N HCl), respectively, as their hydrochloride salts. These compounds had essentially equal and opposite optical rotations and identical NMR spectra to (R)-2 and rac-2 prepared according to Schemes 2 and 4, respectively.11 Biological Activity: Signal Transduction at Cloned Metabotropic Glutamate Receptors. The potency of (R)-2, (S)-2, rac-2, (1S,5R)-17, and (1SR,5RS)18 for signaling at metabotropic glutamate receptors was investigated. Signal transduction experiments were performed with CHO cells heterologously expressing rat mGlu1a, rat mGlu5a, or human mGlu2 receptors. Signaling at the mGlu1a and mGlu5a receptor was analyzed in a calcium-ion mobilization fluorimetric assay, signaling at the mGlu2 receptor was measured by means of a [35S]GTPγS binding assay on membranes from these cells. (10) Tarzia, G.; Balsamini, C.; Spadoni, G.; Duranti, E. Synthesis 1988, 514-517. (11) The optical rotations of (R)-2 and (S)-2 were lower in magnitude to that for (R)-2 that was reported by us in an earlier communication.3 The optical rotation of this sample was performed at a different concentration and acid concentration to that of the more recently prepared compounds (R)-2 and (S)-2. We assume that these differences are a result of the different concentrations, unfortunately a sample (R)-2, prepared according to Scheme 2 is no longer available for a direct comparison.

mGlu1a

mGlu5a

mGlu2

>100 (1) >100 (5) >300 (1) >300 (2) >300 (2) 6.5 ( 2.1 (6) 76 (1) >10 (1)

>100 (2) 18 ( 6 (3) 30 ( 6 (2) >300 (2) >300 (1) 1.4 ( 0.66 (6) 30 ( 18 (3) >100 (2)

>300 (3) 45 ( 10 (3) 55 ( 14 (3) >300 (3) 158 ( 46 (5) 7.2 ( 2.2 (6) 15 ( 2 (4) 0.059 ( 0.015 (5)

The results for agonist activity are summarized in Table 1. These data show that (R)-2 is not an agonist for mGlu1a, mGlu5a, or mGlu2. Its enantiomer, however, (S)-2, is an agonist at the mGlu5 and mGlu2 receptors. This compound stimulates Ca2+ mobilization with a potency of 18 µM in CHO cells expressing mGlu5a, and stimulates [35S]GTPγS binding with a potency of 45 µM on membranes of CHO cells that express mGlu2. These potencies of (S)-2 for mGlu2 and mGlu5 are very similar to the potencies of (1S,3R)-ACPD 1. As expected, we find that the racemic mixture, rac-2, shows potencies at mGlu5 and mGlu2 that are slightly lower than that seen for the pure S-form. The concentration-response curves illustrated that (S)-2 and rac-2 are full agonists at the mGlu5 and mGlu2 receptors (Figure 2). Table 1 further illustrates that (1S,5R)-17 behaves as an agonist at the mGlu2 receptor with a potency of 158 µM while it does not show activity at mGlu1 or mGlu5. The concentration-response curve reveals that this compounds is able to maximally stimulate [35S]GTPγS binding far above the efficacy of glutamate (Figure 3). This could suggest that (1S,5R)-17 dramatically influences the coupling between receptor and G protein or the intrinsic properties of the G protein (e.g., affinity for GTP). Further experiments are needed to fully understand this remarkable finding. Table 1 further illustrates that (1SR,5RS)-18 did not affect signaling of mGlu1a, mGlu5a, or mGlu2, suggesting that steric hindrance in the 5 position destroys activity at the mGlu2 receptor. None of the compounds tested were antagonists at mGlu1a, mGlu5a, or mGlu2 (data not shown). Our preliminary data further suggests that (R)-2, (S)-2, rac2, (1S,5R)-17, and (1SR,5RS)-18 and did not bind to the ionotropic glutamate receptors such as the NMDA and AMPA receptors. There was no interaction with the channel pore site, the glutamate or the glycine site of the NMDA receptor, nor with the AMPA receptor as measured in rat cortical membranes by radioligand binding (data not shown). In conclusion, we have developed an efficient method for preparing conformationally restricted cyclopentenylglutamate analogues in a regioselective and diastereoselective manner. Methods for preparing optically active versions of these compounds have also been devised. Compound (S)-2 is a relatively potent agonist for mGlu5 and mGlu2 while compound (1S,5R)-17 behaves as a weak but very efficacious agonist for mGlu2. Both agents appear selective for metabotropic glutamate receptors as compared to ionotropic receptors. Interestingly, the 3-phosphono analogue of 2 has been recently reported to be a selective mGlu4a receptor agonist (EC50 17 µM) and ineffective at mGlu2 receptor subtypes.12

Cyclopentenyl-Glutamate Analogues

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Figure 2. Agonist activity of (S)-2, (R)-2 and rac-2 at the cloned mGlu5a and mGlu2 receptors in CHO cells: Ca2+ mobilization and [35S]GTPγS binding.

Figure 3. Comparison of (1S,5R)-17 to glutamate for agonist activity at mGlu2 in CHO cells.

Experimental Section General methods were as described previously.9 Unless specified, all NMR spectra were recorded at 300 MHz (1H NMR) or 75 MHz (13C NMR) in deuteriochloroform solution. 13C NMR assignments were based on DEPT experiments. Preparative HPLC was performed using a Waters Delta prep 4000 HPLC. Compounds were detected using a Waters 486 tunable UV absorbance detector. Ion exchange chromatography was performed using Dowex 50WX8-100 acidic cationexchange resin that was packed by the slurry method in a 5 mm diameter glass column. The material to be purified was added as a solution in 1 N HCl, and after washing with 120 mL of demineralized water, the amino acid product was eluted from the column with 1 M NH3 solution. [35S]GTPγS (specific activity 37 MBq/ml) was obtained from Amersham (Little Chalfort, UK). Dulbecco’s modified Eagle medium (DMEM) and dialyzed fetal calf serum were from Life technologies (Gaithersburg, MD). Scintillation fluid Ultimaflo AF as well as the Unifilter-96 GF/B plates were from Packard (Meriden, CT). Guanosine-5′-diphosphate dilithium salt (GDP) was from Boehringer Manheim (Basel, Switzerland), Fluo 3-AM was from Molecular Probes (Leiden, The Netherlands). Probenecid was from Sigma (St. Louis, MO). Black 96-well plates were from Costar (Merck, Overijse Belgium). (12) Amori, L.; Costanino, G.; Marinozzi, M.; Pellicciari, R.; Gasparini, F.; Flor, P. J.; Kuhn, R.; Vranesic, I. Bioorg. Med. Chem. Lett. 2000, 10, 1447-1450.

(2′R,4S)-Ethyl 3′-Benzoyl-5′-oxo-2′-phenyl-spiro[cyclopent-1-ene-4,4′-oxazolidine]carboxylate (12) and (2′R,5S)Ethyl 3′-Benzoyl-5′-oxo-2′-phenyl-spiro[cyclopent-1-ene5,4′-oxazolidine]carboxylate (13). A solution of ethyl 2,3butadienoate 9 (0.200 g, 1.79 mmol), (R)-3 (0.100 g, 0.36 mmol), and triphenylphosphine (5 mg, 0.18 mmol) in dry benzene (4 mL) was stirred at RT for 5 h. The solvent was removed to give a dark thick oil which was purified by column chromatography (10% ether/hexane) to give 12 (0.024 g, 17%), 13 (0.068 g, 49%), and the dimer diethyl 2-methylene-3-cyclopentene-1,3-dicarboxylate (0.0216 g, 27%). 12: 1H NMR δ 7.357.20 (m, 6H), 7.12 (d, 2H, J 7.2 Hz), 6.91 (d, 2H, J 7.5 Hz), 6.71 (br s, 1H), 6.67 (br s, 1H), 4.23 (q, 2H), 3.66-3.60 (m, 1H), 3.39-3.32 (m, 2H), 3.26-3.20(m, 1H), 1.32 (t, 3H); 13C NMR δ 175.39 (CO), 168.58 (CO), 163.78 (CO), 138.92 (CH), 136.10 (C), 135.56 (C), 133.38 (C), 130.38 (CH), 130.05 (CH), 128.81 (CH), 128.57 (CH), 126.43 (CH), 126.33 (CH), 90.14 (CH), 64.70 (C), 60.55 (CH2), 44.65 (CH2), 43.72 (CH2), 14.29 (CH3); [R]D23 +202 (c 0.1 CHCl3); MS (ES +ve) m/z 392.0 (M + 1+, 100%); HRMS (ES +ve) Calcd for C23H21NO5 (MH+) 392.1498. Found: 392.1479. 13: 1H NMR δ 7.39-7.29 (m, 6H), 7.25 (d, 2H, J 6.9 Hz), 7.02 (d, 2H, J 6.9 Hz), 6.97 (br s, 1H), 6.77 (br s, 1H), 4.28 (q, 2H), 3.19-2.75 (m, 4H), 1.35 (t, 3H); 13C NMR δ 173.68 (CO), 168.72 (CO), 163.22 (CO), 151.78 (CH), 136.48 (C), 136.20 (C), 131.88 (C), 129.92 (CH), 129.71 (CH), 128.54 (CH), 128.31 (CH), 126.94 (CH), 126.05 (CH), 90.72 (CH), 72.38 (C), 61.06 (CH2), 35.74 (CH2), 32.32 (CH2), 14.16 (CH3); [R]D21 +155 (c 0.6 CHCl3); MS (ES +ve) m/z 392.0 (M + 1+, 30%). Anal. Calcd for C23H20NO5: C, 70.58; H, 5.41; N, 3.58. Found: C, 70.53; H, 5.43; N, 3.45%. (R)-1-Amino-3-cyclopentene 1,3-dicarboxylic Acid (hydrochloride salt) (2). Cycloadduct 12 (0.18 g, 0.45 mmol) was dissolved in 6 M HCl (10 mL). The solution was heated at reflux for 14 h. After cooling, the aqueous layer was extracted with ether (2×), and then the water was removed under reduced pressure. The product was purified by ion-exchange chromatography and the free amino acid that was isolated was treated with 6 N HCl, and the solution was evaporated to dryness. Drying under high vacuum gave a pale brown solid (0.067 g, 72%). 1H NMR (D2O) δ 6.66 (br s, 1H), 3.22-3.27 (br d, 2H, J 16.8 Hz), 2.84-2.78 (br d, 2H, J 16.2 Hz); 13C NMR (D2O) δ 172.80 (CO), 166.52 (CO), 140.39 (CH), 131.97 (C), 62.61 (C), 42.97 (CH2), 41.19 (CH2); [R]D22 +15 (c 0.2, 6N HCl); MS (ES +ve) m/z 171.7 (M + 1+. 100%); HRMS (ES +ve) Calcd for C7H10NO4 (MH+): 172.0610. Found: 172.0616. Anal. Calcd for C7H9NO4‚HCl: C, 40.50; H, 4.86; N, 6.74; Found: C, 40.48; H, 4.73; N, 6.70%. (S)-1-Amino-2-cyclopentene 1,2-dicarboxylic Acid (hydrochloride salt) (16). The titled compound was prepared from cycloadduct 13 (0.13 g, 0.33 mmol) as described above for the synthesis of 2. This compound was obtained as a pale

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brown solid (0.059 g, 86%). 1H NMR (D2O) δ 6.82 (br s, 1H, H-3), 2.68-2.57 (m, 2H), 2.48-2.39 (m, 1H), 2.18-2.09 (m, 1H); 13 C NMR (D2O) δ 175.98 (CO), 169.91 (CO), 146.42 (CH), 137.72 (C), 70.63 (C), 33.55 (CH2), 30.08 (CH2); [R]D22 +11 (c 0.4 H2O); MS (ES +ve) m/z 171.8 (M + 1, 20%); HRMS (ES +ve) Calcd for C7H10NO4 (MH+): 172.0610. Found: 172.0618. Methyl 1-[Diphenylmethylideneamino]-3-cyclopentene 1,3-dicarboxylate (21). A solution of ethyl butynoate 20 (0.196 g, 2 mmol), methyl 2-[N-(diphenylmethylideneamino)propenoate 4 (0.26 g, 1 mmol), and tributylphosphine (40 mg, 0.2 mmol) in benzene (5 mL) was stirred at RT for 24 h. The solvent was removed to give a black thick oil which was purified by column chromatography (25% ethyl acetate/ hexane) to give a bright yellow thick oil (0.372 g, 98%) which upon triteration in cold petroleum spirit gave a white crystalline solid. 1H NMR δ 7.58-7.11 (m, 10 H), 6.65 (m, 1H), 4.18 (q, 2H), 3.38-3.32 (m, 1H), 3.29 (s, 3H), 3.28-3.18 (m, 1H), 3.15-3.03 (m, 2H), 1.28 (t, 3H); 13C NMR δ 173.97 (CO), 167.93 (CO), 164.15 (CN), 140.13 (C), 139.56 (CH), 136.67 (C), 133.58 (C), 128.47 (CH), 128.24 (2 CH), 128.29 (2 CH), 127.76 (2 CH), 127.66 (2 CH), 72.38 (C), 59.94 (CH2), 51.36 (CH3), 47.92 (CH2), 46.30 (CH2), 13.99 (CH3); MS (ES +ve) m/z 378.2 (M + 1+). Anal. Calcd for C23H23NO4: C, 73.19; H, 6.14; N, 3.71. Found: C, 73.40; H, 6.34; N, 3.48%. 1-Amino-3-cyclopentene 1,3-dicarboxylic Acid (hydrochloride salt) (rac-2). Cycloadduct 21 (0.50 g, 1.32 mmol) was dissolved in 6 N HCl (3 mL). The solution was heated at under reflux for 6 h. After cooling, the aqueous layer was extracted with ether (2×), and the water was removed under reduced pressure to give a white solid (0.235 g, 91%). 1H NMR (D2O) δ 6.63 (s, 1H), 3.20 (dd, 2H, J 3.0, 19.8 Hz), 2.78 (dd, 2H, J 2.4, 19.4 Hz); 13C NMR (D2O) δ 172.13 (CO), 166.23 (CO), 140.69 (CH), 131.71 (C), 62.96 (C), 43.06 (CH2), 41.19 (CH2); MS (ES +ve) m/z 172.0 (M + 1, 100%). HRMS: Calcd for C7H10NO4 (MH+): 172.0616. 1-(1R,2S,5R)-Menthyl 3-Ethyl (1R)-1-Diphenylmethyleneamino-3-cyclopentene dicarboxylate (28) and 1-(1R,2S, 5R)-Menthyl 2-Ethyl (1S)-1-Diphenylmethyleneamino-3cyclopentene dicarboxylate (29). The acrylate 27 (305 mg, 0.783 mmol) was dissolved in dry benzene (8.0 mL), and then ethyl butynoate (0.22 mL, 2.20 mmol) and tributylphosphine (0.10 mL, 0.40 mmol) were added. The mixture was stirred at RT overnight, and then all volatiles were removed in vacuo to give a dark brown syrup. The pure product was obtained by column chromatography (9:1 petroleum spirit:ethyl acetate as eluent) which gave the title compound (340 mg, 0.678 mmol, 86.6%) as an amber syrup. A diastereoisomeric ratio of 60:40 was estimated from the 1H NMR spectrum. 1Η ΝΜR (400 MHz) δ 0.65 (3H, d, J 7.2 Hz, major), 0.66 (3H, d, J 7.2 Hz minor), 0.78 (3H, d, J 7.2 Hz, major), 0.80 (3H, d, J 7.2 Hz, minor), 0.86 (3H, d, J 6.4 Hz, minor), 0.87 (3H, d, J 6.4 Hz, major), 0.7-1.0 (4H, m), 1.28 (3H, t, J 7.2 Hz), 1.2-1.8 (5H, m), 2.85-3.30 (4H, m), 4.17 (2H, q, J 7.2 Hz), 4.40-4.50 (1H, m), 6.60 (1H, br s), 7.15-7.60 (10H, m); 13C ΝΜR δ major isomer: 14.2 (CH3), 15.9 (CH3), 20.8 (CH3), 22.0 (CH3) 22.9 (CH2), 25.7 (CH) 31.2 (CH), 34.1 (CH2), 40.1 (CH2), 46.7 (CH), 47.0 (CH2), 47.2 (CH2), 60.2 (CH2), 72.9 (C), 75.2 (CH), 127.8, 127.9, 127.9, 128.0, 128.6, 128.6, 128.7, 128.7, 130.0, 130.1 (d, Ar CH’s), 134.0 (C), 133.7 (C), 140.1 (CH), 140.8 (C), 164.4 (C), 168.8 (C), 173.6 (C); MS (ES +ve) m/z 502 (M + 1); HRMS Calcd for C32H40NO4 (MH+) 502.2957. Found 502.2955. 1-(1R,2S,5R)-Menthyl 3-Ethyl (1R)-1-Amino-3-cyclopentenedicarboxylate Hydrochloride Salt (30) and 1-(1R,2S,5R)-Menthyl 3-Ethyl (1S)-1-Amino-3-cyclopentenedicarboxylate Hydrochloride Salt (31). The above mixture of cycloadducts 28 and 29 (340 mg, 0.678 mmol) was dissolved in diethyl ether (10 mL), and then 1 N HCl (10 mL) was added. The mixture was stirred at RT for 16 h and then diluted with diethyl ether and 1 N HCl, and the two layers were separated. The organic portion was extracted with 1 N HCl, and the combined aqueous layers were evaporated in vacuo to give the title compound (250 mg, 0.669 mmol, 94.7%), as a clear gum. 1Η ΝΜR (CDCl3) δ 0.74 (3H, d, J 6.3 Hz), 0.801.20 (9H, m), 1.29 (3H, t, J 6.9 Hz), 1.46 (2H, m), 1.60-1.90 (3H, m), 2.02 (1H, br d), 3.26 (4H, m), 4.20 (2H, q, J 6.9 Hz),

Ung et al. 4.79 (1H, m), 4.98 (1H, br s), 6.70 (1H, br s), 9.15 (2H, br s); 13C ΝΜR (D O) δ major isomer: 13.8 (CH ), 15.7 (CH ), 20.6 2 3 3 (CH3), 21.7 (CH3), 23.0 (CH2), 26.1 (CH), 31.2 (CH), 34.2 (CH2), 40.0 (CH2), 41.8 (CH2), 43.4 (CH2), 46.9 (CH), 61.9 (CH2), 62.9 (C), 77.6 (CH), 132.6 (C), 140.5 (CH), 165.0 (C), 170.8 (C) ppm. MS (CI +ve) m/z 338 (M + 1); HRMS Calcd for C19H32NO4 (MH+) 338.2331, Found 338.2330. 1-(1R,2S,5R)-Menthyl 3-Ethyl (1R)-1-Benzyloxycarbonylamino-3-cyclopentenedicarboxylate (32) and 1-(1R,2S, 5R)-Menthyl 3-Ethyl (1S)-1-Benzyloxycarbonylamino-3cyclopentenedicarboxylate (33). The above mixture of the salts 30 and 31 (250 mg, 0.669 mmo1) was dissolved in THF (10 mL), and the solution was cooled to 0 °C. Saturated sodium carbonate solution (10 mL), benzyl chloroformate (0.2 mL, 1.40 mmol) was added, and the mixture was stirred at 0 °C for 2 h. The volatiles were removed in vacuo, and the residue was diluted with water and extracted with chloroform (2×). The combined organic extracts were dried (MgSO4), filtered, and evaporated in vacuo to give a clear syrup. The pure product was obtained by column chromatography (9:1 petroleum spirit: ethyl acetate as eluent) which gave the title compounds (220 mg, 0.467 mmol, 69.7%) as a clear syrup. The two isomers could be partially separated by HPLC (6.5% ethyl acetate in petroleum spirit as eluent, 20 mL min-1, 10 mg injections), which afforded 30 mg and 15 mg of 33 and 32, respectively. 32: [R]D23 -10 (c 0.5, CHCl3); 1Η ΝΜR δ 0.74 (3H, d, J 7.2 Hz), 0.86 (3H, d, J 7.2 Hz), 0.89 (3H, d, J 7.2 Hz), 1.28 (3H, t, J 6.9 Hz), 0.70-2.10 (9H, m), 2.85-3.00 (2H, m), 3.10-3.30 (2H, m), 4.20 (2H, q, J 6.9 Hz), 4.71 (1H, td, J 10.5, 4.2 Hz), 5.09 (2H, AB system, J 12.3 Hz), 5.56 (1H, br. s), 6.69 (1H, br. s), 7.30-7.40 (5H, m); 13C ΝΜR δ 14.2 (CH3), 15.9 (CH3), 20.8 (CH3), 22.0 (CH3), 23.1 (CH2), 26.1 (CH), 31.3 (CH), 34.1 (CH2), 40.2 (CH2), 43.8 (CH2), 44.6 (CH2), 46.8 (CH), 60.5 (CH2), 64.2 (C), 66.7 (CH2), 76.1 (CH), 128.0, 128.0, 128.2, 128.5, 128.5 (Ar CH’s), 133.3 (C), 136.0 (C), 139.9 (CH), 155.0 (C), 164.0 (C), 172.8 (C). 33:[R]D23 -43 (c 1.0, CHCl3); 1Η ΝΜR δ 0.74 (3H, d, J 7.2 Hz), 0.86 (3H, d, J 7.2 Hz), 0.89 (3H, d, J 7.2 Hz), 1.28 (3H, t, J 6.9 Hz), 0.70-2.10 (9H, m), 2.85-3.00 (2H, m), 3.10-3.30 (2H, m), 4.19 (2H, q, J 6.9 Hz), 4.70 (1H, td, J 10.5, 4.2 Hz), 5.08 (2H, AB system, J 12.0 Hz), 5.55 (1H, br.s), 6.70 (1H, br. s), 7.30-7.40 (5H); 13C ΝΜR δ 14.2 (CH3), 15.9 (CH3), 20.7 (CH3), 21.9 (CH3), 23.0 (CH2), 26.0 (CH), 31.3 (CH), 34.1 (CH2), 40.3 (CH2), 43.5 (CH2), 44.3 (CH), 46.8 (CH), 60.4 (CH2), 64.2 (C), 66.7 (CH2), 76.0 (CH), 128.0, 128.0, 128.1, 128.5, 128.5 (Ar CH’s) 133.3 (C), 135.2 (C), 140.2 (CH), 155.0 (C), 164.1 (C), 172.7 (C); MS (CI +ve) m/z 472 (M + 1); HRMS Calcd for C27H38NO6 (MH+) 472.2699, Found 472.2700. (R)-1-Amino-3-cyclopentene 1,3-dicarboxylic Acid (hydrochloride salt) (2) and (S)-1-Amino-3-cyclopentene 1,3dicarboxylic Acid (hydrochloride salt) (2). Compound 33 (30 mg, 0.064 mmol) was dissolved in diethyl ether (5 mL) and transferred to a sealed tube, and the diethyl ether removed in vacuo. Hydrochloric acid solution (6N, 8 mL) was added and the tube sealed and shaken. The tube was warmed to 85 °C, with frequent shaking until all material was in solution, and then the mixture stirred at that temperature for 4 d. The mixture was cooled to RT and then diluted with water (40 mL) before it was washed with diethyl ether (2×) and evaporated in vacuo to give a pale yellow solid. The pure product was obtained by ion exchange chromatography which afforded (R)-2 (13 mg, 0.062 mmol, 98%) as the hydrochloride salt. (S)-2 was produced from compound 32 (15 mg, 0.032 mmol), in the same way giving 6 mg (0.029 mmol, 90.3%). NMR spectral data were identical to (R)-2 and rac-2 prepared according to Schemes 2 and 4, respectively. (R)-2: [R]D22 +5.9 (c 0.6, 1 N HCl). (S)-2: [R]D22 -5.8 (c 0.87, 1 N HCl). Signal Transduction at Cloned Rat mGlu1a, mGlu5a, and mGlu2 Receptors in CHO Cells. CHO cells expressing the cloned rat mGlu1a and mGlu5a receptors (a kind gift from S. Nakanishi, Kyoto University, Japan) and the human mGlu2 receptor (cloned and expressed in house) were grown in DMEM/Glutamax-I to which 2 mM glutamine, 46 mg/L proline, and 10% dialyzed fetal calf serum were added. Ca2+ Fluorimetric Assay for Rat mGlu1a and mGlu5a. CHO cells expressing the mGlu1a or mGlu5a were plated at

Cyclopentenyl-Glutamate Analogues a density of 30 000 cells/well in black 96-well plates. The effect of drugs on basal or glutamate-induced intracellular Ca2+ levels was evaluated 24 h later for mGlu1a cells and 48 h later for mGlu5a cells in a fluorescent based assay. Cells were incubated with 2 µM fluo-3 AM in culture medium supplemented with 5 mM probenecid for 1 h at 37 °C. Cells were washed, and to evaluate antagonism, test drug was added to the cells in assay buffer (phosphate buffered saline, containing 1.25 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 10 mM glucose, 5 mM Hepes, and 5 mM probenicid, pH 7.4) for 20 min at room temperature. Glutamate was added to the cell culture plates in the Fluorometric Imaging Plate Reader (FLIPR, Molecular Devises Inc.), and relative fluorescence units were recorded for each well in function of time. For the study of agonist properties of test compounds, cell culture plates were first incubated with assay buffer, and glutamate was replaced by test compound. Average data graphs of duplo wells were obtained, and the peak fluorescence (maximum signal between 1 and 120 s) was calculated. Concentration response curves were constructed based on peak fluorescence for each concentration of test compound. [35S]GTPγS Radioligand Binding Assay for Human mGlu2. Membrane Preparation. Cells were grown to confluence. Cells were washed twice with ice-cold PBS without Ca2+ and Mg2+, scraped off and homogenized in buffer (EDTA 10 mM, Hepes 20 mM). After centrifugation (18 000 rpm, 15 min, 4 °C), the pellet was washed with 0.1 mM EDTA, 20 mM Hepes, and resuspended in the same buffer for protein determination with the Biorad assay. Membrane aliquots were stored at -70 °C. [35S]GTPγS Radioligand Binding. Each incubate contained 10 µg of membrane protein in 250 µL of binding buffer (HEPES 20 mM, NaCl 100 mM, MgCl2 3 mM, GDP 3 µM, pH 7.4). The incubation was started by addition of an appropriate concentration of agonist and/or antagonist. Compounds were incubated with the membranes at 37 °C for 30 min. Subsequently, 0.1 nM [35S]GTPγS (approximately 2 × 105 DPM) was added in the presence or absence of 30 µM glutamate. The mixture was further incubated for 30 min at 37 °C. The reactions were terminated by separating free and

J. Org. Chem., Vol. 67, No. 1, 2002 233 bound radioactivity by rapid vacuum filtration using a Packard filtration manifold through GF/B prewetted glass fiber filters. Filters were rapidly washed two times with cold 10 mM NaH2PO4/10 mM Na2HPO4 buffer, pH 7.4. Filters were transferred to vials for subsequent counting in a scintillation counter. Results are expressed as % of glutamate-induced response, the latter being defined as 100%. Glutamate and amino acids were dissolved and diluted in water. Concentration-response curves were drawn on a logarithmic scale. Sigmoidal curves of best fit were calculated by nonlinear regression analysis using GraphPad software (San Diego, CA). The pIC50-value referred to the concentration of a compound producing half the maximum response.

Acknowledgment. We thank Johnson and Johnson Research Pty. Limited (Sydney, Australia) for financial support and Prof. Susan Pond (J&J), Dr. Vic Sipido, and Dr. John Stuart Andrews (Jannsen Pharmaceutica, Belgium) for their encouragement and support. Note Added after ASAP Posting. The version of this paper posted on November 30, 2001, had incorrect compound numbering in Scheme 7 and corresponding text. The corrected version was posted on December 12, 2001. Supporting Information Available: Copies of the ORTEP diagrams of the crystal structures 21 and 23 and their crystal/ refinement data (crystallographic data files have been deposited for 21 and 23 at the Cambridge Crystallographic Data Base, deposition numbers 168800 and 168801) and copies of the 1H NMR spectra of compounds rac-2, 12, 14-19, 24, 27, 28 + 29, 30 + 31, and 33 and full experimental details for the synthesis of compounds not mentioned in the Experimental Section. This material is available free of charge via the Internet at http://pubs.acs.org. JO010864I