Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[d-Lys

Jan 20, 2016 - receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their ... affinity 14-fold higher than the original hit but al...
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Potent μ‑Opioid Receptor Agonists from Cyclic Peptides Tyr‑c[D‑LysXxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation Yangmei Li,* Margret Cazares, Jinhua Wu, Richard A. Houghten, Laurence Toll, and Colette Dooley Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987, United States S Supporting Information *

ABSTRACT: To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe3 was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys2-Xxx3 is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

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the analogs lost affinity to the μ and δ opioid receptors. Cyclization of the linear analogs between the 4-amino of the cis4-amino-L-proline and the C-terminal Phe partly recovered the activity of endomorphin-2, suggesting the additional cationic center at the second position of endomorphin-2 may adversely affect the peptide backbone conformation and the receptor recognition.9 To identify potent peptide opioid ligands, we recently synthesized a cyclic peptide library consisting of over 24 624 anthraniloyl(Ant)-labeled peptides in a mixture-based format; a hit was identified exhibiting a binding affinity (Ki) of 14 nM to the MOR (Figure 1) through the screening-deconvolution of

INTRODUCTION It is widely accepted that new analgesics may be developed from peptides because opioid peptides are produced in the mammalian brain and spinal cord to alleviate pain.1 These endogenous opioid peptides, e.g., endomorphin-1 (EM-1, TyrPro-Trp-Phe-NH2) and endomorphin-2 (EM-2, Tyr-Pro-PhePhe-NH2), not only have high affinity and high selectivity toward the μ-opioid receptor (MOR) but also possess few tolerance and addiction side effects.2 It was reported that endomorphine-1 produces significant analgesia in rats without showing reward behavior,3 while N-terminus modified lipoendomorphine-1 show dose-dependent antinociceptive activity against neuropathic pain without producing constipation in rats.4 Though promising, endomorphins failed to meet the expectation of clinical analgesics due to their poor metabolic stability in vivo. Metabolic stability is a general hurdle for peptides in systemic therapeutic application. Therapeutic development of endomorphins has been focused on the discovery of the analogs that are metabolically stable while maintaining the potency of endomorphins to MOR. To increase the metabolic stability, residues of endomorphins were replaced with unnatural amino acids to generate analogs. For example, analogs having Phe4 replaced with (2S,3S)-β-MePhe4 were more potent toward MOR than endomorphins.5 A Pro2to-D-Ala2 replacement generally maintained the potency of endomorphins and was less prone to degradation than the parent peptides in vivo.6 Cyclization is a useful strategy to increase the metabolic stability and druggability of peptides. The head-to-tail cyclic analog of endormorphin-1 showed affinity 200 times weaker than the parent peptide, implying the N-amino group of Tyr1 is vital to the receptor binding and signal transduction.7 Cyclic endomorphin analogs synthesized by replacing Pro2 to D-Lys2 and then connecting the side chains of D-Lys2 to an additional Asp5 retained the potency of endomorphins.8 Endomorphin-2 analogs were synthesized by replacing Pro2 with a bivalent cis-4-amino-L-proline; however, © XXXX American Chemical Society

Figure 1. Structures of cyclic peptides: (a) structure of the hit; (b) scaffold of the mixture-based cyclic pentapeptide library.

the mixture-based library.10 This mixture-based library was designed to incorporate diverse residues/substituents at three positions, P5, P4, and P2; screening-deconvolution of such library reflects/involves multiple rounds of classic SAR optimization at these positions, even though the process of deconvolution of the mixture-based library may seem different from that of the classic SAR-employed hit-to-lead optimization. The cyclic peptide identified presents a sequence of H-Tyr-c[DReceived: December 8, 2015

A

DOI: 10.1021/acs.jmedchem.5b01899 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. (a) Amino Acids Employed and Sequences of the Resulting Cyclic Peptides and (b) Synthetic Approach to the Cyclic Peptides

Table 1. Binding Affinities (Ki) of the Tyr-c[D-Lys-Xxx-Tyr-Gly] to the Opioid Receptorsa Ki ± SD (nM) compd

Xxx3

MOR

EM-1 0 1 2 3 4 5 6 7 8 9

Dap(Ant) Phe Tyr Trp Tic 7-OH-Tic 3 Pal 4 Pal Arg Cit

0.7 ± 0.1 14 ± 0.54 0.99 ± 0.14 114 ± 27 1.99 ± 1.6 991 ± 326 1129 ± 65.8 1.95 ± 0.6 9.21 ± 1.2 17.4 ± 0.6 9.04 ± 2.0

KOR 5069 ± 3231 ± 4450 ± >10000 8160 ± >10000 >10000 5330 ± >10000 8530 ± 8430 ±

3003 863 1245 3271

3896 2070 2220

Ki ratio DOR

KOR/MOR

DOR/MOR

575 ± 175 865 ± 424 39 ± 4 171 ± 27 99 ± 2 600 ± 366 631 ± 163 1284 ± 362 2274 ± 1533 >10000 3320 ± 486

7241 231 4517 >324 2936 >91 >32 1316 1096 406 760

821 62 39 1.5 50 0.6 0.6 658 247 >805 367

a

Data represent the mean of two experiments, each experiment conducted in duplicate. The average Ki values for 2 assays obtained for standards: MOR, DAMGO, 2.2 nM; DOR, DPDPE, 2 nM; KOR, U50,488, 4 nM.

maintain at least one of the characters of the Dap(Ant)3 residue, aromatic ring, or basic nitrogen (the aniline N). Four types of amino acids, aromatic Phe, Tyr, and Trp, constrained aromatic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) and 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (7-OH-Tic), aromatic and basic β-3′-pyridyl alanine (3 Pal) and β-4′-pyridyl alanine (4 Pal), as well as linear and basic Arg and citrulline (Cit), were chosen to make these nine analogs (Scheme 1).

Lys-Dap(Ant)-Tyr-Gly], a free N-terminal tyrosine residue outside a heterodetic tetrapeptide ring. Three residues, Tyr1, DLys2, and Tyr4 of the cyclic peptide were identified through the deconvolution of P5, P4, and P2, which respectively constituted a mixture of 36, 19, and 36 amino acid residues from the original mixture-based library (Figure 1), indicating these three residues of the hit have been preliminarily optimized upon deconvolution. However, the Dap(Ant)3 of the hit resulted from a fixed Dap(Ant) residue at P3 in the original mixture library; therefore, it was not optimized during the deconvolution. Since a single residue replacement is able to dramatically improve biological activity in many cases of the hit-to-lead optimization in drug discovery,11 replacement of the Dap(Ant)3 with another residue is anticipated to generate leads with increased binding affinity toward the MOR. Moreover, the hit has a similar sequence to the endomorphins. Modification of the hit is very likely to yield new cyclic endomorphin analogs. Nine analogs were designed by replacing Dap(Ant)3 with other amino acid residues. These analogs were designed to

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RESULTS AND DISCUSSION Chemistry. These analogs were synthesized using our cyclization method: imidazole-promoted cyclization of thioesters.12 This method was employed to generate the original mixture-based cyclic peptide library and to synthesize the individual cyclic peptides including the hit. The peptide thioesters H-Tyr-D-Lys-Xxx-Tyr-Gly-SCH2Ph were synthesized by a solid-phase approach using mercaptomethylphenylfunctionalized silica gel as a “volatilizable” support.13 After B

DOI: 10.1021/acs.jmedchem.5b01899 J. Med. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Agonist potency of the compounds from a fluorescent membrane potential assay.

conformational alteration may affect its binding more than the residue’s aromaticity. The nine analogs were also tested for their binding affinities toward the κ-opioid receptor (KOR) and the δ-opioid receptor (DOR) in radioactive receptor binding assays using 3H-U69593 and 3H-DPDPE as competing radioligands, respectively. None of the analogs exhibited binding affinities to KOR higher than the original hit, indicating a single-residue replacement of Xxx3 did not improve the biological activity toward KOR. However, some analogs presented improved binding affinities to DOR; i.e., 1 increased over 20-fold from a binding affinity of 865 to 39 nM, and 3 increased over 8-fold to a binding affinity of 99 nM. The selectivity for MOR over KOR and over DOR was also calculated using the ratio of binding affinity to each receptor. These analogs are generally selective to MOR like the initial hit; but the analogs having Tic3 or 7-OH-Tic3 residues dramatically decreased their selectivity for MOR over DOR from a 62-fold of the original hit to less than 1. This is caused by the profoundly reduced affinities to the MOR, since their binding affinities to the DOR were slightly improved compared with the initial hit, suggesting the constrained aromatic Xxx3 residues may have advantage to the DOR. Other analogs also showed notable general structure−selectivity (MOR-over-DOR) relationships: aromatic and basic Xxx3 increased the selectivity to the MOR, and linear and basic Xxx3 increased the selectivity as well. Taken together, these data showed that a basic residue at Xxx3 may improve the selectivity for MOR over DOR. Though more studies may be needed to further confirm the structure− selectivity relationships, these data will be useful for the design of opioid ligand selective to MOR. Functional Activity toward MOR. Fluorescent membrane potential assay has been used to evaluate agonist and antagonist activity toward G-protein-coupled receptors and was reported highly sensitive in agonist assays.15 These analogs were therefore tested for their functional activity at the MOR in a fluorescent membrane potential assay using morphine, the known potent MOR agonist, as a positive control. All nine analogs activated the MOR to increase the membrane potential as does morphine, indicating that all of them were MOR agonists (Figure 2). The agonist potency (EC50) of each analog was calculated (Table 2). Among all analogs, 1 possessed the highest agonist potency of 2.3 nM, nearly 3-fold more potent than morphine (EC50 = 6.3 nM), suggesting this compound has agonist activity more potent than morphine in vitro. These compounds generally had the EC50 values in good correlation with their Ki values. NMR Structural Analysis. Peptide conformation plays a vital role in binding to the opioid receptors. It has been observed that endomorphins have both trans and cis isomers at Tyr1-Pro2 and the trans isomer is considered the active conformer to the MOR.16 The trans isomers take an extended conformation with the aromatic group of Trp3 away from that

cleavage with anhydrous HF and removal of the HF with nitrogen stream, the fully unprotected peptide thioesters were released from the decomposed silica gel. The peptide thioesters were then dissolved in a mixture of 1.5 M aqueous imidazole solution and acetonitrile (1:7, v/v) at 1 mM and gently stirred at room temperature for 72 h to form the cyclic peptides (Scheme 1). Since the hit is a heterodetic peptide, we focused only on the biological activities of the heterodetic isomer in this study, though two cyclic isomers (the heterodetic peptide (sideto-tail cyclic) and the homodetic peptide (head-to-tail cyclic)) were obtained, as the cyclization has no regioselective preference when D-Lys is an internal residue in a linear peptide.12 The nine heterodetic cyclic peptides were then isolated by HPLC, and their structures were characterized and confirmed by NMR experiments. Next, 8 as a model cyclic peptide was tested for its stability against trypsin, as 8 has Arg3, a trypsin cleavable site between the Arg3-Tyr4. A linear nonapeptide, Trp-Arg-Arg-Trp-Trp-ArgIle-Arg-Arg-NH2, was used as control to monitor the activity of the trypsin. Stability toward trypsin degradation was tested by incubation of 1 mg of the peptide with freshly made trypsin solution containing 20 μg of the trypsin in 10 mL of 0.1 M NH4HCO3 buffer at pH 8.2.14 The control linear peptide was completely degraded after a 1 h incubation; however, no significant change was found for compound 8 after a 24 h incubation with trypsin, suggesting the 8 has increased stability against proteolysis as a result of cyclization. Binding Affinity toward Opioid Receptors. These nine analogs were tested for their binding affinities toward MOR in a radioactive receptor binding assay using 3H-DAMGO as a competing radioligand. Analogs having the same type of Xxx3 residues generally showed comparable binding affinity to each other (Table 1). Compound 1 containing a Phe3 had a binding affinity of 0.99 nM to MOR, while 3 containing a Trp3 had an affinity of 1.99 nM. Both compounds have an aromatic Xxx3 and showed a binding affinity 14-fold and 7-fold better than that of the original hit, individually. Compound 6 containing a 3 Pal3 and 7 a 4 Pal3, both having an aromatic and basic Xxx3 residue, had increased binding affinities compared with the original hit. Except 2, which has a Tyr3, compounds having an aromatic Xxx3 residue generally exhibited higher binding affinity than the original hit, implying aromatic residue at Xxx3 favored the ligand binding to the MOR. Compounds 8 (Arg3) and 9 (Cit3) having the linear and basic Xxx3 residues maintained a comparable binding affinity to the original hit. The other two compounds having the constrained aromatic Xxx3 residues (Tic3 of 4 and 7-OH-Tic3 of 5) dramatically decreased to an affinity of 1 μM to MOR, a 71-fold decrease from the original hit. Since these two constrained aromatic residues may take cis or trans conformation as does proline, the dramatically decreased binding affinities to the MOR indicate Xxx3 residue-induced C

DOI: 10.1021/acs.jmedchem.5b01899 J. Med. Chem. XXXX, XXX, XXX−XXX

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66.9 ± 19.8a

of Tyr1, while the cis isomer takes a folded conformation with aromatic groups of Tyr1 and Trp3 packing against Pro2. To explore how conformation impacts the activity of these cyclic peptides, we employed NMR to probe the conformational preferences of the cyclic peptides. Among these peptides 4 and 5 was observed a NOE cross peak between Hα(D-Lys2)Hα(Tic3)/Hα(7-OH-Tic3), indicating both peptides were in cis conformation at the constrained amino acid residue. Since both peptides exhibited dramatically lower binding affinity and agonist potency compared with the rest of the analogs, their cis conformation at D-Lys2-Xxx3 should account for the weak binding affinity and functional activity to the MOR. While 1 shows its potent binding affinity to MOR, with its robust agonist potency consistent with the binding affinity, 2, having a Tyr3-to-Phe3 replacement of 1, had a much decreased binding affinity and agonist potency. Sequential HN NOE cross peaks and HN vicinal pairs in the two peptides were comparable, suggesting that the two peptides shared a similar backbone conformation. This was further investigated by temperature dependence of backbone HN chemical shifts by 1 H NMR experiments and confirmed by COSY experiments. The temperature shift coefficients of the HNs showed Gly5 and Tyr4 in 1 are in agreement with those in 2, both around −3.8 ppb/K; the temperature shift coefficients of the HN of D-Lys2Nα and Xxx3 in 1 are −2.7 and −2.5 ppb/K, while those in 2 are −3.3 and −3.3 ppb/K, suggesting both molecules had intramolecular hydrogen bonds at the HN of D-Lys2Nα and Xxx3,17 but the hydrogen bonds in 2 were weaker than those in 1. Hence, the backbone conformations of the two cyclic peptides were similar at Tyr4-Gly5-D-Lys2Nω and slightly differed at Tyr1-D-Lys2Nα-Xxx3. Notably, a NOE cross peak was observed between the aromatic protons at Tyr12,6 and Tyr32,6 in 2 but not between Tyr12,6 and Phe32,6 in 1. The NOE peak suggested the aromatic side chains of Tyr1 and Tyr 3 were in a close position in 2. This conformation is similar to the less active cis isomer of endomorphin-1, where the Tyr1 and Trp3 closely packed against Pro2.16 The adjacency of the aromatic groups of Tyr1 and Tyr3 in 2 suggests the peptide was in an undesirable conformation to the MOR binding. The side chain conformation was very likely responsible for the activity difference between these two peptides.

Data represent the mean of two experiments, each experiment conducted in triplicate. bData represent the mean of one experiment conducted in triplicate.

130.7 ± 95.6a

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CONCLUSION

A single residue of the initial hit was replaced to generate the cyclic peptide analogs in the hit-to-lead optimization for the identification of lead candidates targeting the MOR. Nine residues organized into four types of structures were designed to generate the analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly]. Half of the analogs showed binding affinity no lower than the original hit, with the analog containing a Phe3 showing the highest binding affinity of 0.99 nM toward MOR, 14-fold higher than the original hit. All the analogs activated the MOR to produce agonist activity, and their agonist potencies were in close agreement with the binding affinities. The most active analog exhibited an EC50 of 2.3 nM, nearly 3-fold more potent than morphine. The NMR study showed that a trans conformation at D-Lys2-Xxx3 is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the MOR. The study may provide insight into the design of endomorphin analogs for the development of potent analgesics with less adverse side effects.

a

8 7

32.0 ± 3.9a 11.1 ± 5.2a

6 5

1250.0 ± 316.1b

3

24 ± 9.2a

2 1 DAMGO

2.5 ± 0.6a 6.3 ± 3.0a EC50 ± SD (nM)

morphine

2.3 ± 1.0a

70.4 ± 52.0a

4

compd

Table 2. Agonist Potency of the Compounds to MOR

677.8 ± 313.6b

9

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DOI: 10.1021/acs.jmedchem.5b01899 J. Med. Chem. XXXX, XXX, XXX−XXX

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Brief Article

Tyr-c[D-Lys-Tic-Tyr-Gly] (4). 1H NMR (400 MHz, DMSO-d6) δ 1.09−1.35 (m, 5H), 1.73 (m, 1H), 2.39 (dd, 2H, J = 20, 12 Hz), 2.53 (dd, 1H, J = 16, 8 Hz), 2.67−2.90 (m, 3H), 3.09 (dd, 1H, J = 16, 4 Hz), 3.24−3.42 (m, 3H), 3.81 (dd, 1H, J = 20, 4 Hz), 3.96 (m, 1H), 4.25 (d, 1H, J = 16 Hz), 4.62 (m, 1H), 4.99 (d, 1H, J = 16 Hz), 5.50 (m, 1H), 6.33 (d, 2H, J = 8 Hz), 6.38 (d, 2H, J = 8 Hz), 6.60 (d, 2H, J = 8 Hz), 6.94 (d, 2H, J = 8 Hz), 7.13 (m, 2H), 7.23−7.25 (m, 3H), 8.11 (d, 1H, J = 8 Hz), 8.16 (d, 1H, J = 4 Hz), 8.32 (br s, 1H), 8.51 (t, 1H, J = 6.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ 22.8, 27.5, 31.4, 32.0, 35.4, 38.3, 40.7, 42.9, 43.6, 49.2, 56.1, 56.2, 115.3, 115.4, 126.9, 127.0, 127.1, 128.5, 128.7, 128.8, 130.1, 130.6, 132.7, 133.0, 156.0, 156.3, 169.1, 170.4, 170.8, 172.7, 174.1. [M + H]+ calcd 671.76; found 671.30. Tyr-c[D-Lys-7-OH-Tic-Tyr-Gly] (5). 1H NMR (400 MHz, DMSOd6) δ 1.06−1.34 (m, 5H), 1.70 (m, 1H), 2.45 (m, 1H), 2.56 (d, 1H, J = 20, 8 Hz), 2.68−2.72 (m, 1H), 2.86−2.92 (m, 3H), 3.2−3.31 (m, 3H), 3.45 (m, 1H), 3.78 (dd, 1H, J = 16, 4 Hz), 3.91 (m, 1H), 4.21 (d, 1H, J = 20 Hz), 4.58 (d, 1H, J = 4 Hz), 4.87 (d, 1H, J = 20 Hz), 5.43 (br s, 1H), 6.34 (d, 2H, J = 8 Hz), 6.46 (d, 2H, J = 4 Hz), 6.61 (d, 2H, J = 8 Hz), 6.66 (s, 2H), 6.93 (d, 1H, J = 8 Hz), 6.95 (d, 2H, J = 8 Hz), 7.02 (s, 1H), 7.16 (br s, 1H), 8.05 (d, 1H, J = 8 Hz), 8.16 (d, 1H, J = 8 Hz), 8.33 (s, 1H), 8.52 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 22.8, 27.6, 30.7, 32.1, 35.4, 38.4, 40.9, 43.2, 43.7, 49.2, 56.2, 56.4, 113.1, 114.7, 115.4, 115.5, 122.7, 128.5, 128.8, 129.7, 130.3, 130.6, 133.9, 156.1, 156.3, 156.6, 169.1, 170.6, 170.7, 172.6, 174.0. [M + H]+ calcd 687.76; found 687.25. Tyr-c[D-Lys-3 Pal-Tyr-Gly] (6). 1H NMR (400 MHz, DMSO-d6) δ 1.07−1.25 (m, 3H), 1.37 (m, 2H), 1.52 (m, 1H), 2.46−2.5 (m, 1H), 2.67−2.80 (m, 3H), 2.84−2.89 (m, 2H), 3.04 (m, 1H), 3.21−3.38 (m, 3H), 3.80 (dd, 1H, J = 17.6, 6 Hz), 3.97 (m, 1H), 4.14 (dd, 1H, J = 12.4, 8 Hz), 4.31 (t, 1H, J = 9.2 Hz), 6.67 (d, 2H, J = 7.2 Hz), 6.68 (d, 2H, J = 7.2 Hz), 6.96−7.01 (m, 5H), 7.23 (m, 1H), 7.32 (br s, 1H), 7.53 (d, 1H, J = 7.2 Hz), 7.94 (br s, 1H), 8.11 (d, 1H, J = 7.6 Hz), 8.32−8.36 (m, 5H), 8.5 (br s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 21.8, 28.5, 31.9, 34.9, 35.6, 38.0, 43.6, 54.2, 54.9, 55.9, 57.1, 115.5, 123.8, 128.4, 129.0, 130.5, 130.6, 133.8, 136.9, 148.0, 150.6, 156.3, 169.2, 171.3, 171.9, 172.3, 173.8. [M + H]+ calcd 660.74; found 330.75 ([M + 2H]2+), 660.25. Tyr-c[D-Lys-4 Pal-Tyr-Gly] (7). 1H NMR (400 MHz, DMSO-d6) δ 1.15−1.54 (m, 6H), 2.43 (dd, 1H, J = 24, 12 Hz), 2.67−2.74 (m, 3H), 2.84−2.90 (m, 2H), 3.04 (dd, 1H, J = 12, 4 Hz), 3.22−3.35 (m, 3H), 3.80 (dd, 1H, J = 16, 6 Hz), 3.98 (br s, 1H), 4.14 (dd, 1H, J = 16, 8 Hz), 4.37 (t, 1H, J = 8 Hz), 6.67 (d, 2H, J= 8 Hz), 6.69 (d, 2H, J = 8 Hz), 6.97 (d, 2H, J = 2 Hz), 6.99 (d, 2H, J = 8 Hz), 7.02 (s, 1H), 7.12 (s, 2H), 7.33 (s, 1H), 7.94 (br s, 1H), 8.11 (d, 1H, J = 4 Hz), 8.34− 8.36 (m, 5H), 8.52 (d, 1H, J = 4 Hz). 13C NMR (100 MHz, DMSOd6) δ 21.8, 28.5, 31.9, 35.7, 36.9, 38.1, 40.5, 43.7, 54.2, 54.3, 55.9, 57.1, 115.5, 124.8, 128.4, 129.0, 130.5, 130.6, 147.1, 149.7, 156.3, 156.4, 169.2, 171.3, 172.1, 172.2. [M + H]+ calcd 660.74; found 330.75 ([M + 2H]2+), 660.25. Tyr-c[D-Lys-Arg-Tyr-Gly] (8). 1H NMR (400 MHz, DMSO-d6) δ 1.09−1.52 (m, 10H), 2.57 (dd, 1H, J = 20, 8 Hz), 2.79−2.82 (m, 3H), 2.90−3.02 (m, 3H),3.24−3.34 (m, 2H), 3.46 (m, 1H), 3.81 (dd, 1H, J = 16.8, 6 Hz), 4.04−4.10 (m, 3H), 6.66 (d, 2H, J = 7.2 Hz), 6.68 (d, 2H, J = 7.2 Hz), 6.91 (d, 2H, J = 7.6 Hz), 6.98 (d, 2H, J = 7.6 Hz), 7.03 (s, 1H), 7.35 (s, 1H), 7.61 (m, 4H), 8.13 (m, 1H), 8.17 (br s, 1H), 8.32 (br s, 1H), 8.40 (br s, 1H), 8.48 (br s, 1H), 8.67 (br s, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 21.7, 25.4, 28.4, 29.0, 31.7, 35.8, 38.2, 40.3, 43.7, 53.6, 54.6, 55.8, 56.8, 115.5, 128.3, 128.7, 130.5, 156.4, 157.8, 169.1, 171.3, 172.2, 172.9. [M + H]+ calcd 668.76; found 334.8 ([M + 2H]2+), 668.3. Tyr-c[D-Lys-Cit-Tyr-Gly] (9). 1H NMR (400 MHz, DMSO-d6) δ 1.09−1.48 (m, 10H), 2.47 (m, 1H), 2.62 (m, 1H), 2.76−2.88 (m, 5H), 3.01 (dd, 1H, J = 12, 4 Hz), 3.26 (m, 2H), 3.46 (m, 1H), 3.80 (dd, 1H, J = 16.8, 6 Hz), 4.05 (m, 3H), 5.45 (s, 2H), 6.00 (s, 1H), 6.65 (d, 2H, J = 7.2 Hz), 6.67 (d, 2H, J = 7.2 Hz), 6.91 (d, 2H, J = 7.6 Hz), 6.98 (d, 2H, J = 7.6 Hz), 7.32 (s, 1H), 7.91 (d, 1H, J = 6.4 Hz), 8.04 (br s, 1H), 8.22 (d, 1H, J = 5.6 Hz), 8.32 (br s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 27.2, 28.4, 29.3, 31.8, 34.3, 35.2, 35.7, 38.2, 39.2, 43.7, 54.1, 54.4, 55.9, 56.8, 115.5, 128.4, 128.6, 130.6, 156.3, 156.4, 159.4,

EXPERIMENT SECTION

Synthesis of Cyclic Peptides H-Tyr-c[D-Lys-Xxx-Tyr-Gly]. All cyclic peptide were prepared by imidazole-promoted cyclization of peptide thioesters as we described earlier.12 Peptide α-thioesters were synthesized by SPPS using mercaptomethylphenyl-functional silica gel as a “volatilizable” support as we published.13 Parallel solid-phase synthesis of peptide α-thioesters were carried out using the “teabag” approach. Stepwise peptide synthesis was carried out using a standard PyBOP/DIEA coupling protocol. After peptide chain elongation, the resin bound peptide was treated with anhydrous HF for 2 h at 0 °C. Following evaporation of the anhydrous HF with a gaseous nitrogen stream, the unprotected peptide α-thioester was obtained. Linear peptide α-thioesters were dissolved in a mixture of acetonitrile and 1.5 M aqueous imidazole solution with a volume ratio of 7:1 at 1 mM. The reaction was allowed to proceed at room temperature for 72 h. An aliquot of 0.1 mL was withdrawn and quenched with 15% TFA in water. The mixture was analyzed by LC− MS. The reaction was quenched with 15% TFA in water after no linear peptide α-thioester was detected by LC−MS. The solvents were then removed with lyophilization. The cyclic peptide was isolated by preparative HPLC. The molecular weight of each cyclic peptide was analyzed by ESI-MS.12 NMR Spectroscopy and Determination of Temperature Shift Coefficients. All NMR spectra were recorded using a Bruker Avance III 400 high performance digital NMR spectrometer and processed using Bruker TopSpin3.2 software. All samples were run in DMSO-d6. The NH peaks were assigned by TOCSY spectra and 1H NMR spectra. After determination of the NH, temperature coefficients have been measured by 1D and 2D NMR methods for the amide chemical shifts. 1H NMR and COSY spectra were recorded at 5° intervals from 297 to 327 K for each cyclic peptide. A linear equation was fitted for the chemical shift versus temperature data, and the NH temperature shift coefficients were obtained from the gradient of the best-fit line. Tyr-c[D-Lys-Phe-Tyr-Gly] (1). 1H NMR (400 MHz, DMSO-d6) δ 1.09−1.53 (m, 6H), 2.44 (dd, 1H, J = 10 Hz), 2.70−2.89 (m, 5H), 3.02 (dd, 1H, J = 12 Hz), 3.19 (dd, 1H, J = 13.6 Hz), 3.30−3.34 (m, 2H), 3.78 (dd, 1H, J = 17.6, 5.6 Hz), 4.02 (m, 1H), 4.14 (m, 1H), 4.25 (t, 1H, J = 8.8 Hz), 6.66 (d, 2H, J = 8.8 Hz), 6.68 (d, 2H, J = 8.8 Hz), 6.96 (d, 2H, J = 10 Hz), 6.98 (d, 2H, J = 10 Hz), 7.15−7.18 (m, 5H), 7.33 (br s, 1H), 7.92 (d, 1H, J = 5.6 Hz), 7.99 (d, 1H, J = 6.8 Hz), 8.30 (s, 1H), 8.34 (m, 1H), 8.39 (d, 1H, J = 4.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ 21.8, 28.4, 32.1, 35.8, 37.5, 38.2, 40.6, 43.7, 54.0, 55.8, 55.9, 57.2, 115.4, 115.5, 126.7, 128.3, 128.6, 128.9, 129.4, 130.7, 130.6, 138.3, 156.3, 169.2, 171.4, 172.1, 172.6, 173.9. [M + H]+ calcd 659.75; found 659.30. Tyr-c[D-Lys-Tyr-Tyr-Gly] (2). 1H NMR (400 MHz, DMSO-d6) δ 1.04−1.15 (m, 2H), 1.27−1.49 (m, 4H), 2.64−2.71 (m, 2H), 2.75 (m, 1H), 2.86 (m, 2H), 3.01 (m,1H), 3.18 (m, 1H), 3.29−3.38 (m, 2H), 3.77 (dd, 1H, J = 15.6, 4 Hz), 4.05 (m, 2H), 4.15 (m, 2H), 6.6 (d, 2H, J = 7.6 Hz), 6.66 (d, 2H, J = 7.6 Hz), 6.70 (d, 2 H, J = 8 Hz), 6.94 (m, 4H), 7.01 (d, 2H, J = 7.2 Hz), 7.32 (br s, 1H), 7.92 (d, 1H, J = 6.4 Hz), 7.94 (d, 1H, J = 10 Hz), 8.33 (br s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 21.8, 28.4, 32.1, 35.9, 36.8, 38.2, 43.8, 53.9, 55.9, 56.2, 57.2, 115.41, 155.43, 115.5, 128.2, 128.3, 129.0, 130.4, 130.5, 130.6, 156.3, 156.4, 169.1, 171.4, 172.0, 172.6, 173.9. [M + H]+ calcd 675.75; found 675.30. Tyr-c[D-Lys-Trp-Tyr-Gly] (3). 1H NMR (400 MHz, DMSO-d6) δ 1.12−1.47 (m, 6H), 2.45 (dd, 1H, J = 20, 12 Hz), 2.72 (dd, 1H, J = 16, 4 Hz), 2.87−2.98 (m, 6H), 3.0 (m, 1H), 3.33 (dd, 1H, J = 17.6, 6 Hz), 3.78 (dd, 1H, J = 20, 4 Hz), 4.05 (br s, 1H), 4.17 (d, 1H, J = 4 Hz), 4.31 (t, 1H, J = 9.2 Hz), 6.67 (d, 2H, J = 7.2 Hz), 6.68 (d, 4H, J = 7.2 Hz), 6.96 (m, 4H), 7.04 (d, 2H, J = 12 Hz), 7.30 (d, 1H, J = 8 Hz), 7.34 (m, 1H), 7.49 (d, 1H, J = 4 Hz), 7.94 (d, 1H, J = 4 Hz), 7.99 (d, 1H, J = 7.6 Hz), 8.35−8.40 (m, 3H), 10.8 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 21.7, 22.8, 28.3, 32.0, 35.9, 38.2, 40.3, 43.8, 47.5, 54.0, 55.2, 55.9, 57.2, 110.4, 111.8, 115.4, 118.6, 121.3, 124.0, 127.5, 128.4, 130.5, 136.5, 156.3, 156.4, 169.1, 171.4, 172.1, 173.0. [M + H]+ calcd 698.79; found 698.30. E

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169.1, 171.3, 172.0, 172.6, 173.1, 173.6. [M + H]+ calcd 669.75; found 669.35. Radioligand Competition Binding Assays. MOR Binding Assay. Rat cortices were homogenized using 50 mM Tris, pH 7.4, and centrifuged at 16 500 rpm for 10 min. The pellets were resuspended in fresh buffer and incubated at 37 °C for 30 min. Following incubation, the suspensions were centrifuged as before, the resulting pellets resuspended in 100 volumes of 50 mM Tris, pH 7.4, plus 2 mg/mL bovine serum and the suspensions combined. Each assay tube contained 0.5 mL of membrane suspension, 2 nM [3H]DAMGO, and a tested compound at varying concentrations in a total volume of 0.65 mL. KOR Binding Assay. Guinea pig cortices and cerebella were homogenized using 50 mM Tris, pH 7.4, 10 mM MgCl2·6H2O, 200 μM PMSF and centrifuged and incubated as above. Each assay tube contained 0.5 mL of membrane suspension, 2 nM [3H]U69,593, and a tested compound in a total volume of 0.65 mL. Assay tubes were incubated for 2 h at 25 °C. Unlabeled U50,488 was used as a competitor to generate a standard curve and determine nonspecific binding. DOR Binding Assay. Rat cortices were homogenized using 50 mM Tris, pH 7.4, 10 mM MgCl2·6H2O, 200 μM PMSF, centrifuged, and incubated as above. Each assay tube contained 0.5 mL of membrane suspension, 2 nM [3H]DPDPE, and a tested compound in a total volume of 0.65 mL. Assay tubes were incubated for 2.5 h at 25 °C. Unlabeled DPDPE was used as a competitor to generate a standard curve and determine nonspecific binding. All three binding assays were terminated by filtration through GF/B filters, soaked in 5 mg/mL bovine serum albumin, 50 mM Tris, pH 7.4, on a Tomtec Mach II Harvester 96. The filters were subsequently washed with 6 mL of assay buffer. Bound radioactivity was counted on a Wallac Betaplate liquid scintillation counter. Experiments were conducted using 2 replicates and repeated twice. μ-Opioid Receptor Functional Assay. μ-Opioid receptor functional activity was determined by measuring μ-opioid receptorinduced membrane potential change, which can be directly read by Molecular Devices membrane potential assay kit (blue dye) (Molecular Devices), using the FlexStation 3 microplate reader (Molecular Devices). The CHO cells expressing human μ-opioid receptor are seeded in a 96-well plate (20 000 cells per well) 1 day prior to the experiments. For agonist assays, after brief washing, the cells are loaded with 225 μL of HBSS assay buffer (Hanks’ balanced salt solution with 20 mM of HEPES, pH 7.4), containing the blue dye, and incubated at 37 °C. After 45 min, an amount of 25 μL of the appropriate compounds is automatically dispensed into the wells by the FlexStation and μ-opioid receptor stimulation-mediated membrane potential change is recorded every 3 s for 60 s by reading 550−565 nm fluorescence excited at 530 nm wavelength. The relative change in fluorescence represents the maximum response minus the minimum response in each well. GraphPad Prism was used to determine the EC50 and MPE values.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the State of Florida, Drug Discovery and Development Acceleration Program, National Institute on Drug Abuse Grants DA023281 (to L.T.), DA031370 (to R.A.H.), DA03696801-01A1 and DA03696802 (to J.W.), and DA040995 (to C.D.).

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ABBREVIATIONS USED MOR, μ-opioid receptor; DOR, δ-opioid receptor; KOR, κopioid receptor; DAMGO, H-Tyr-D-Ala-Gly-NMe-Phe-Gly-ol; DPDPE, c[D-Pen2,D-Pen5]encephalin; SD, standard deviation; LC−MS, liquid chromatography−mass spectrometry; ESI−MS, electron spray ionization mass spectrometry; NOE, nuclear Overhauser effect; COSY, correlation spectroscopy; TOCSY, total correlation spectroscopy; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; DIEA, N,Ndiisopropylethylamine; TFA, trifluoroacetic acid; PMSF, phenylmethanesulfonyl fluoride

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01899.

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REFERENCES

Synthetic details, stability toward tryptic degradation, data of temperature shift coefficient, and profiles of LC− MS and NMR (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 772-345-4725. Fax: 772-3253636. F

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DOI: 10.1021/acs.jmedchem.5b01899 J. Med. Chem. XXXX, XXX, XXX−XXX