Development of Novel G-Protein-Coupled Receptor 54 Agonists with

belong to the RF-amide peptide family and share the amino acid sequence of ...... E. D.; Repine , J. T.; Roark , W. H.; Stier , M. A.; Tinney , F...
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J. Med. Chem. 2008, 51, 7645–7649

7645

Development of Novel G-Protein-Coupled Receptor 54 Agonists with Resistance to Degradation by Matrix Metalloproteinase Kenji Tomita,† Shinya Oishi,*,† Hiroaki Ohno,† Stephen C. Peiper,‡ and Nobutaka Fujii*,† Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501, Japan, and Anatomy and Cell Biology, Thomas Jefferson UniVersity, Philadelphia, PennsylVania 19017 ReceiVed July 24, 2008

Kisspeptin-GPR54 signaling is involved in the suppression of cancer metastasis and regulation of hormonal secretion. Recently, matrix metalloproteinase mediated deactivation of kisspeptins through hydrolysis of the Gly-Leu peptide bond has been reported. In the present report, GPR54 agonistic peptides having several nonhydrolyzable Gly-Leu dipeptide isosteres were designed and synthesized. (E)-Alkene- and hydroxyethylene-type isostere-containing analogues maintained the original activity with higher stability in murine serum and resistance to MMP-9-mediated cleavage. Introduction a

Table 1. Sequences of Kisspeptins and Pentapeptide GPR54 Agonist 1,2

GPR54 is a Gq-protein-coupled receptor, which was isolated as an orphan receptor sharing significant homology with galanin receptors.3 GPR54 is paired with endogenous kisspeptins (kisspeptin-54 has also been designated metastin),1,2,4 although galanin is not recognized by this receptor. Kisspeptins, which are generated by proteolytic cleavage of a precursor, belong to the RF-amide peptide family and share the amino acid sequence of the C-terminal decapeptide, which is highly conserved between human and mouse.5,6 High levels of kisspeptin expression have been observed in placenta, while GPR54 expression is high in the pancreas and placenta.4 Recent studies demonstrated that the kisspeptin-GPR54 system is involved in placentation and the onset of puberty.7Plasma concentration of kisspeptin is significantly increased in pregnancy, which returns to nearly the baseline level after delivery.8 GPR54 stimulation in the hypothalamus and pituitary prompts the release of gonadotropin-releasing hormone (GnRH),9 which is required for the onset of puberty.10 Furthermore, it is known that the mutation in GPR54 results in idiopathic hypogonadotrophic hypogonadism (IHH).11-13 These reports indicate the therapeutic potential of GPR54 ligands for disorders of endocrine secretion. Alternatively, the kisspeptin-GPR54 system was originally discovered in oncology research because a precursor protein of kisspeptins is encorded by the metastasis suppresser gene KiSS1.14 The motility of several cancer cell lines expressing GPR54 was found to be attenuated in the presence of kisspeptins.4,15,16 Although the mechanism by which kisspeptin-GPR54 signaling blocked cancer metastasis, a recent report indicated that GPR54 activation suppressed metastasis-related CXCL12 (SDF-1)CXCR4 signaling.17 Moreover, antimetastasis activity is also * To whom correspondence should be addressed. Phone: +81-75-7534551. Fax: +81-75-753-4570. E-mail: for S.O., [email protected]; for N.F., [email protected]. † Kyoto University. ‡ Thomas Jefferson University. a Abbreviations: GPR54, G-protein-coupled receptor 54; MMP, matrix metalloproteinase; Fmoc, 9-fluorenylmethoxycarbonyl; SPPS, solid-phase peptide synthesis; GnRH, gonadotropin-releasing hormone; IHH, idiopathic hypogonadotrophic hypogonadism; EADI, (E)-alkene dipeptide isostere; Mts, mesitylenesulfonyl; DIC, N,N′-diisopropylcarbodiimide; HOAt, N-hydroxy-7-azabenzotriazole.

Peptide

Sequence

Kisspeptin-54

H-Gly-Thr-Ser · · · Tyr-Asn-Trp-Asn-Ser-PheGly-Leu-Arg-Phe-NH2 H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-ArgPhe-NH2 4-fluorobenzoyl-Phe-Gly-Leu-Arg-Trp-NH2

Kisspeptin-10 1

mediated by suppressed expression of matrix metalloproteinases (MMPs), which contribute to cell invasion.18,19 Thus, kisspeptins may be useful as inhibitory peptides against cancer metastasis. We recently identified a pentapeptide 1 as a potent GPR54 agonist by structure-activity relationship studies on kisspeptin-10,20,21 which is the C-terminal decapeptide fragment of kisspeptins (Table 1). Kisspeptin-10 exerts the most potent receptor binding affinity among kisspeptins reported so far.4 It has been reported that kisspeptins are inactivated by the cleavage of their Gly-Leu peptide bond in the C-terminal region by MMPs.22 Since kisspeptins and peptide 1 share a common sequence (Phe-Gly-Leu-Arg) of the MMP-mediated cleavage site, peptide 1 would be also deactivated by MMP-mediated digestion. We envisioned that substitution of the Gly-Leu dipeptide moiety in peptide 1 with an appropriate dipeptide isostere would afford resistance to enzymatic degradation with maintenance of GPR54 agonistic activity, as well as the potential for additional inhibitory activity against MMPs. This could provide novel antimetastasis agents targeting two molecules, GPR54 and MMPs. Since it is possible that the introduction of a dipeptide isostere structure to bioactive peptides may lead to loss of the original bioactivity,23 several Gly-Leu dipeptide isosteres were analyzed for facile identification of appropriate amide equivalents. For efficient synthesis of a series of GlyLeu dipeptide isosteres, (E)-alkene dipeptide isostere (EADI) was converted into several isosteres by modification of the olefin moiety. In this manuscript, preparation and biological evaluation of peptidomimetics containing a Gly-Leu dipeptide isostere for GPR54 agonists are described. Results and Discussion Chemistry. Synthesis of Gly-Leu type EADI 5 was initiated from D-serine 2 as chiral educt (Scheme 1), which was converted into a chiral aziridine 3 in three steps.24 After DIBAL-H treatment of 3, (Z)-selective Horner-Wadsworth-Emmons reaction25 of the resulting aldehyde gave (Z)-enoate 4 with the

10.1021/jm800930w CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

7646 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23

Scheme 1. Synthesis of Hydrocarbon-Type Dipeptide Isosteresa

Brief Articles Table 2. Bioactivities and Stability of Peptide Analogues

a Reagents and conditions: (a) DIBAL-H, -78 °C, 30 min; (b) (oMePhO)2P(O)CH2CO2t-Bu, NaI, DBU, -78 °C, 1 h, then -40 °C, 11 h; (c) i-BuCu(CN)MgCl · 2LiCl, -78 °C, 30 min; (d) Pd/C, H2, room temp, 14 h; (e) DBU, room temp, 20 h; (f) 1 M TMSBr-thioanisole/TFA, room temp, 1 d; (g) Fmoc-OSu, Et3N, room temp, 3 h.

Scheme 2. Synthesis of Allyl Alcohol-Type Dipeptide Isosteresa

a IC50 values indicate the concentration needed for 50% inhibition of receptor binding of [125I]kisspeptin-15. b QIC values are calculated as QIC ) IC50(compound)/IC50(kisspeptin-10). c EC50 values mean the concentration needed for 50% of the full agonistic activity induced by 1 µM kisspeptin-10. d Half-life in murine serum. e Kisspeptin-10 was completely digested within 1 h.

Scheme 3. Synthesis of Peptidomimetics 24a,b and 25a,b Containing Hydroxyethylene-Type Isosterea a Reagents and conditions: (a) m-CPBA, room temp, 20 h; (b) NaH, room temp, 2 h; (c) K2CO3, CH3OH, 50 °C, 5.5 h; (d) 1 M TMSBr-thioanisol/ TFA, room temp, 1 d; (e) Fmoc-OSu, Et3N, room temp, 3 h; (f) TESCl, imidazole, 4 °C, 4 h.

concomitant formation of small amount of (E)-isomer (E/Z ) 1:4). Employment of 4 in the reaction with organocuprate afforded desired Gly-Leu type EADI 5 as a single product in 90% yield.26 The absolute configuration at the R-position in 5 can be determined by measurement of circular dichroism.27 A negative n f π* Cotton effect (∆ε ) -2.77 at 225.4 nm in isooctane) indicated the (R)-configuration (L-configuration) at the R-isobutyl group of 5. Reduction or isomerization of double bond in 5 gave other dipeptide isosteres 6 and 7. The simultaneous removal of N-mesitylenesulfonyl (Mts) and tertiary butyl groups of dipeptide isosteres 5-7 with 1 M TMSBrthioanisole/TFA followed by protection of the resulting amino groups afforded the Fmoc-protected dipeptide isosteres 8-10, respectively. Next, dipeptide isosteres containing a γ-hydroxy group were prepared from EADI 5 (Scheme 2). Epoxidation of 5 with m-CPBA gave a mixture of the diastereomers of desired epoxides 11 and 12 (11/12 ) 3:1). After separation of these

a

Reagents and conditions: (a) H2, Pd(OAc)2, room temp, 3 h.

diastereomers by column chromatography, each epoxide was treated with base to afford allyl alcohols 13 and 14.28-30 Stereochemistry of the γ-position of 13 and 14 was determined by transformation into the β-aziridinyl-R,β-enoates (see Supporting Information). Deprotection of 13 and 14 followed by sequential protections of the resulting amino and hydroxy groups gave the Fmoc-protected isosteres 17 and 18.

Brief Articles

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7647

Figure 1. Stability evaluation of GPR54 agonists 1, 19, and 25b by treatment with (a) MMP-9 and (b) murine serum: 1 (b), 19 (0), 25b (]), kisspeptin-10 (2). Kisspeptin-10 was completely digested in murine serum within 1 h.

Five Fmoc-protected Gly-Leu dipeptide isosteres 8-10, 17, and 18 in hand were applied to Fmoc-based solid-phase peptide synthesis (SPPS) for the mimetics of GPR54 agonist 1. All isosteres were smoothly introduced into the peptide on Rinkamide resin with N,N′-diisopropylcarbodiimide (DIC) and N-hydroxy-7-azabenzotriazole (HOAt). After completion of the peptide elongation, the desired peptides 19-23 were yielded (Table 2) as major products by release from the solid support followed by HPLC purification. Further hydrogenation of the olefin in 22 and 23 using H2/Pd(OAc)2 provided hydroxyethylene-type isostere-containing peptides 24a,b and 25a,b as mixtures of diastereomers,30,31 which were separated by HPLC (Scheme 3). Configuration of the R-carbon of 24a,b and 25a,b bearing an isobutyl group was determined using reference peptides, which were prepared through a separate approach using an Evans chiral auxiliary (see Supporting Information). Biological Evaluation and Structure-Activity Relationship for GPR54 Ligands. The bioactivity of peptides 19-25 is summarized in Table 2. Initially, we evaluated GPR54 binding affinity of all peptide analogues by competitive binding assay using [125I]kisspeptin-15.4 We also assessed GPR54 agonistic activity (EC50) of the compounds, which exerted high binding affinity, by FLIPR assay.32 Since biological values of the reference kisspeptin-10 varied among assay plates, relative bioactivities were calculated [QIC ) IC50(compound)/IC50(kisspeptin-10)]. Among a series of peptidomimetics 19-25, EADI-containing analogue 19 showed the highest binding affinity [QIC(19) ) 1.0], which is slightly more potent than the parent, peptide 1 [QIC(1) ) 7.3]. The constrained structure resulting from the double bond within EADI contributes to the high binding affinity, which is in contrast to the lower binding affinity of a flexible analogue 20 [QIC(20) ) 10]. On the other hand, R,β-unsaturated analogues (21-23) are not potent ligands for GPR54, which may be caused by loss of the chirality at the isostere R-position. This is consistent with the lower binding affinity of the D-Leucontaining analogue 26 [QIC(26) ) 240].20,32 The binding affinities of the analogues containing a hydroxyethylene-type isostere was dependent on the stereochemistry at the R- and γ-positions of the isostere. A higher binding affinity was also observed in 25b [QIC(25b) ) 2.5] with an (2R,4S)-configuration. Similarly, peptide 24b, which contains an L-Leu isosteric unit, was more potent than the D-Leu-containing congener 24a. These findings suggest that the L-configuration of Leu is needed for the bioactivity. Moreover, the ability of the peptides showing high binding affinities to activate GPR54 was also evaluated. All peptides functioned as full agonists, and the bioactivity showed a positive correlation with the binding affinity.33 Stability of Isostere-Containing GPR54 Ligands in MMPs and Serum and the Inhibitory Activity against MMPs. The stability of GPR54 agonists against digestion by MMPs was evaluated. Cleavage of the Gly-Leu peptide bond in 1 by

MMP-9 was significantly lower than the rapid digestion of kisspeptin-10. MMP-2-mediated digestion gradually cleaved the Gly-Leu peptide bond in kisspeptin-10 as reported,22 while parent peptide 1 was resistant to the treatment with MMP-2 (see Supporting Information). Isostere-containing peptides were quite stable in the presence of both enzymes (Figure 1a and Supporting Information). These results indicate that introduction of Gly-Leu isosteres and downsizing from kisspeptin-10 were beneficial to the resistance against MMPs-mediated digestion. Furthermore, the stability of peptide analogues in murine serum was investigated. Kisspeptin-10 was completely digested within 1 h. A small amount of residual peptide 1 was observed after treatment for 12 h. On the other hand, half-lives (t1/2) of isostere-containing peptides were significantly longer than that of the parent peptide 1 by at least 4-fold (Table 2, Figure 1b, and Supporting Information), indicating that introduction of the Gly-Leu dipeptide isostere successfully contributed to the improved stability of GPR54 agonist peptides. However, slow degradation of isostere-containing peptides in murine serum was still observed, implying the possible presence of alternative cleavage sites within GPR54 agonists by some enzymes. Next, their inhibitory activities for MMP-2 and -9 were evaluated. None of the peptides exhibited significant inhibition of either enzyme at 10 µM. These findings suggest that a series of C-terminal short peptides and peptidomimetics of kisspeptins are not recognized by the catalytic domain of MMPs. Conclusions In this study, several Gly-Leu dipeptide isosteres were prepared using EADI 5 as a common key intermediate. The resulting isosteres were utilized for improvement of the stability of a GPR54 agonist, peptide 1, against inactivation by peptidases such as MMP-9. Two isostere-containing peptides 19 and 25b showed resistance to degradation by MMP-2 and -9, with maintenance of the bioactivity for GPR54. Furthermore, these peptides are more stable in murine serum than the parent peptide 1. Information from this research is useful for development of novel GPR54 ligands having in vivo stability. Experimental Section tert-Butyl (2R,3E)-2-Isobutyl-5-[N-(2,4,6-trimethylphenylsulfonyl)amino]pent-3-enoate (5). To a stirred solution of CuCN (108 mg, 1.20 mmol) and LiCl (102 mg, 2.40 mmol) in dry THF (2.4 mL) at -78 °C was added by syringe i-BuMgCl (2.0 M THF solution, 600 µL, 1.20 mmol). The mixture was allowed to warm to 4 °C and was stirred at this temperature for 30 min. A solution of ester 4 (105 mg, 0.300 mmol) in dry THF (3 mL) was added dropwise to the above reagent at -78 °C with stirring, and the stirring was continued for 30 min followed by quenching at -78 °C with 6 mL of a mixture of saturated NH4Cl solution and 28% NH4OH solution (1:1). After concentration of the mixture, the residue was extracted with Et2O, and the extract was washed with brine and dried over MgSO4. The solvent was removed under

7648 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23

reduced pressure, and the residue was purified by flash chromatography over silica gel with n-hexane-EtOAc (5:1) to give the β,γenoate 5 (110 mg, 90% yield) as a colorless solid: mp 51-53 °C; [R]D23 -22.0 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.84 (d, J ) 6.3 Hz, 3H), 0.87 (d, J ) 6.3 Hz, 3H), 1.17-1.26 (m, 1H), 1.42 (s, 9H), 1.45-1.54 (m, 2H), 2.30 (s, 3H), 2.63 (s, 6H), 2.88 (ddd, J ) 8.5, 7.3, 7.3 Hz, 1H), 3.51 (dd, J ) 6.1, 6.1 Hz, 2H), 4.43 (t, J ) 6.1 Hz, 1H), 5.41 (dt, J ) 15.4, 6.1 Hz, 1H), 5.55 (dd, J ) 15.4, 8.5 Hz, 1H), 6.96 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 20.9, 22.3, 22.4, 23.0, 25.6, 28.0, 41.4, 44.5, 47.7, 80.6, 126.7, 132.0, 132.7, 133.6, 139.0, 142.2, 173.3. Anal. Calcd for C22H35NO4S: C, 64.51; H, 8.61; N, 3.42. Found: C, 64.39; H, 8.33; N, 3.48. (2R,3E)-5-[N-(9-Fluorenylmethoxycarbonyl)amino]-2-isobutylpent-3-enoic Acid (8). The ester 5 (819 mg, 2.00 mmol) was dissolved in 1 M TMSBr-thioanisole/TFA (40 mL) at room temperature, and the mixture was stirred for 1 day at this temperature. Concentration under reduced pressure gave an oily residue, which was poured into ice-cold dry diethyl ether. The resulting pellet was collected by centrifugation and washed with ice-cold dry diethyl ether, which was dissolved in water (10 mL). Et3N (1.65 mL, 12.0 mmol) and Fmoc-OSu (670 mg, 2.00 mmol) in CH3CN (10 mL) were successively added to the above solution at 4 °C. After being stirred for 3 h at room temperature, the reaction was quenched with 1 N HCl at 4 °C. After concentration under reduced pressure, the resulting residue was extracted with EtOAc. The extract was washed with 1 N HCl and brine and dried over MgSO4. Concentration under reduced pressure was followed by flash chromatography over silica gel with n-hexane/EtOAc (2:1) containing 1% acetic acid. The eluent was wash with brine and dried over MgSO4 followed by concentration to give the Fmocprotected δ-amino acid 8 (400 mg, 51% yield in two steps) as a colorless solid: mp 146-148 °C; [R]D24 -30.8 (c 1.01, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (d, J ) 6.3 Hz, 3H), 0.91 (d, J ) 6.3 Hz, 3H), 1.35-1.47 (m, 1H), 1.52-1.70 (m, 2H), 3.04-3.17 (m, 1H), 3.67-3.88 (m, 2H), 4.21 (t, J ) 6.6 Hz, 1H), 4.41 (d, J ) 6.6 Hz, 2H), 4.79-4.91 (m, 1H), 5.48-5.68 (m, 2H), 7.30 (dd, J ) 7.6, 7.3 Hz, 2H), 7.39 (dd, J ) 7.3, 7.3 Hz, 2H), 7.58 (d, J ) 7.3 Hz, 2H), 7.75 (d, J ) 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 22.1, 22.5, 25.5, 41.1, 42.6, 46.7, 47.2, 66.7, 120.0, 125.0, 127.0, 127.7, 129.2, 130.0, 141.3, 143.9, 156.2, 179.7. HRMS (FAB), m/z calcd for C24H28NO4 (MH+) 394.2013, found 394.2014. Peptide 19. Isostere 8 (120 mg, 0.3 mmol) was employed in Fmoc-based SPPS (see Supporting Information). The peptide 19 was yielded as a TFA salt (33 mg, 36% yield from Rink amide resin): [R]D23 -17.5 (c 0.22, CH3OH); HRMS (FAB), m/z calcd for C42H53FN9O5 (M + H+) 782.4148, found 782.4163. Peptide 23. Isostere 18 (160 mg, 0.3 mmol) was employed in Fmoc-based SPPS. The peptide 23 was yielded as a TFA salt (48 mg, 53% yield from Rink amide resin): [R]D21 -22.3 (c 0.15, CH3OH); HRMS (FAB), m/z calcd for C42H53FN9O6 (M + H+) 798.4097, found 798.4109. Peptides 25a and 25b. To a solution of the peptide 23 (18 mg, 0.020 mmol) in CH3OH (2 mL) was added Pd(OAc)2 (4.5 mg, 0.020 mmol), and the mixture was stirred for 3 h under H2 at room temperature. The mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC to afford the expected peptides 25a (5.4 mg, 30% yield) and 25b (2.9 mg, 16% yield). 25a: [R]D22 +3.6 (c 0.08, CH3OH); HRMS (FAB), m/z calcd for C42H55N9O6F (M + H+) 800.4254, found 800.4271. 25b: [R]D23 +10.7 (c 0.11, CH3OH); HRMS (FAB), m/z calcd for C42H55FN9O6 (M + H+) 800.4254, found 800.4250. Measurement of Binding Affinity. Assasys were performed as previously described.4 Membrane fraction was prepared using homogenizing buffer (10 mM NaHCO3, 2 mM EGTA, 0.2 mM MgCl2, protease inhibitors, pH 7.4) and stored in 50% glycerol-50% homogenizing buffer at -20 °C. Kisspeptin-15 was labeled with 125I-Na using lactoperoxidase and purified to a carrierfree single peak.

Brief Articles

Measurement of [Ca2+]i Using FLIPR Technology.32 GPR54/ CHO cells (3.0 × 104 cells per 200 µL/well) were inoculated in 10% dFBS/DMEM onto a 96-well plate for FLIPR analysis (Black Plate Clear Bottom, Coster, Inc.), followed by incubation at 37 °C overnight in 5% CO2. After the medium was removed, 100 µL of the pigment mixture was dispensed into each well of the plate, followed by incubation at 37 °C for an hour in 5% CO2. Then 1 mM peptide in DMSO was diluted with HANKS/HBSS containing 2.5 mM probenecid, 0.2% BSA, and 0.1% CHAPS. The dilution was transferred to a 96-well plate for FLIPR analysis (V-Bottom plate, Coster, Inc.; hereafter referred to as a sample plate). After completion of the pigment loading onto the cell plate, the cell plate was washed 4 times with wash buffer (2.5 mM probenecid in HANKS/HBSS) using a plate washer. After the washing, 100 µL of wash buffer was left. The cell plate and the sample plate were set in FLIPR (Molecular Devices, Inc.), and 0.05 mL of a sample from the sample plate was automatically transferred to the cell plate.

Acknowledgment. We thank Takeda Pharmaceutical Co. Ltd. for the evaluation of biological activity for GPR54 of a series of compounds. This work is supported by a Grant-inAid for Scientific Research and Molecular Imaging Research Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. K.T. is grateful for Research Fellowships from the JSPS for Young Scientists. Supporting Information Available: Experimental procedures, characterization, and HPLC and bioassay data. This material is available free of charge via the Internet at http://pubs.acs.org.

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