Phenylalanine Stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 Exhibit Distinctly Different Opioid Activity Profiles

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues’ stereochemistry on the peptides’ opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.


Introduction
The endogenous opioid system is a valuable therapeutic target for the treatment of pain as it is extensively involved in pain perception and experience [1]. The majority of opioid ligands used clinically for the treatment of pain are mu-opioid receptor (MOR) agonists, although agonists of kappa (KOR) and delta (DOR) receptors also produce analgesia. However, opioid-selective agonists also l-Trp-containing peptide exhibited mixed, multifunctional activity, with robust antinociception mediated by both KOR and MOR, followed by KOR-selective antagonism lasting several hours after the dissipation of antinociception. Both of these macrocyclic tetrapeptides are active after oral administration [20,21], increasing their potential as leads for drug discovery.
Therefore, we explored the influence of the stereochemistry of the two phenylalanine residues in CJ-15,208 and [d-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) on opioid activity. All of the stereoisomers retained significant antinociception with reduced liabilities, while the different stereochemistries of the aromatic residues in the five analogs resulted in significant variation in their multifunctional opioid activity.

Synthesis
The stereoisomers of CJ-15,208 and its d-Trp isomer ( Figure 1) were synthesized by a combination of solid phase synthesis of the linear precursors followed by cyclization in solution using modifications to our original strategy [20,22] to improve the yields of the macrocyclic peptides.
The linear sequences chosen contained the turn inducing D-Pro residue in the middle of the peptide to facilitate cyclization [17], and the use of the 2-chlorotrityl chloride resin minimized the potential for diketopiperazine formation. The peptides were purified by silica gel flash chromatography, which permitted the facile purification of larger quantities of the macrocyclic peptides for in vivo pharmacological evaluation following oral administration. The purified peptides were analyzed by electrospray ionization mass spectrometry, thin layer chromatography, and in two analytical HPLC systems. All of the stereoisomers were obtained in high purity and reasonable yields (34-50% from the linear precursors) after purification.

Metabolic Stability
We evaluated the metabolic stability of the stereoisomers in mouse liver microsomes. While macrocyclic peptides are stable to proteases, they can be metabolized by cytochrome P450 enzymes [23,24]. In all cases the peptides were stable in incubations lacking NADPH, but disappeared from the incubations containing NADPH, consistent with cytochrome P450 enzyme metabolism. The halflives of the stereoisomers in the mouse liver microsomes were ≤ 30 min for all of the stereoisomers except for 1, which displayed a half-life more than twice that of most of the other stereoisomers (

Metabolic Stability
We evaluated the metabolic stability of the stereoisomers in mouse liver microsomes. While macrocyclic peptides are stable to proteases, they can be metabolized by cytochrome P450 enzymes [23,24]. In all cases the peptides were stable in incubations lacking NADPH, but disappeared from the incubations containing NADPH, consistent with cytochrome P450 enzyme metabolism. The half-lives of the stereoisomers in the mouse liver microsomes were ≤30 min for all of the stereoisomers except for 1, which displayed a half-life more than twice that of most of the other stereoisomers ( Figure 2). The short half-lives of most of the stereoisomers are consistent with that of

Metabolic Stability
We evaluated the metabolic stability of the stereoisomers in mouse liver microsomes. While macrocyclic peptides are stable to proteases, they can be metabolized by cytochrome P450 enzymes [23,24]. In all cases the peptides were stable in incubations lacking NADPH, but disappeared from the incubations containing NADPH, consistent with cytochrome P450 enzyme metabolism. The halflives of the stereoisomers in the mouse liver microsomes were ≤ 30 min for all of the stereoisomers except for 1, which displayed a half-life more than twice that of most of the other stereoisomers (

In Vitro Pharmacological Evaluation
In radioligand equilibrium competition binding assays the D-Phe stereoisomers of CJ-15 [D-Trp]CJ-15,208 generally exhibited greatly reduced affinity for KOR and MOR compared to parent peptides (Table 1). Only the D-Phe 3 analogs (see Figure 1 for residue numbering) e sub-micromolar affinity for any of the opioid receptors (Ki = ~350 nM for KOR). Similar to C and [D-Trp]CJ-15,208 [19], the stereoisomers all exhibited negligible affinity for DOR. Also co with the results for CJ-15,208 and [D-Trp]CJ-15,208, none of the analogs exhibited appreciable at either KOR or MOR at 10 μM in [ 35 S]GTPγS assays.

In Vivo Pharmacological Evaluation
The stereoisomers were initially evaluated for their antinociceptive activity in the 55 °C water tail-withdrawal assay in C57BL/6J mice following i.c.v. administration (Figures 3A and of the D-Phe stereoisomers produced significant time-and dose-dependent antinociception two-way RM ANOVA). Peak antinociception was produced 20 min after i.c.v administr stereoisomers 1, 3, and 4 and at 30 min for isomers 2 and 5 ( Figure S1). The duration of si antinociception (p < 0.05; Dunnett's post hoc test) varied from 45 min (isomers 3 and 4) to (isomers 2 and 5), with isomer 1 exhibiting an intermediate duration. All of the isomers exhib and potent antinociception except for 2 ( Figure 3A), which produced approximat antinociception at the highest dose tested (100 nmol); the potencies of stereoisomers 1 and 3 comparable to that of CJ-15,208 ( Table 2). The maximal antinociception found for the stereoi

In Vitro Pharmacological Evaluation
In radioligand equilibrium competition binding assays the d-Phe stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 generally exhibited greatly reduced affinity for KOR and MOR compared to the two parent peptides (Table 1). Only the d-Phe 3 analogs (see Figure 1 for residue numbering) exhibited sub-micromolar affinity for any of the opioid receptors (K i =~350 nM for KOR

In Vivo Pharmacological Evaluation
The stereoisomers were initially evaluated for their antinociceptive activity in the 55 • C warm-water tail-withdrawal assay in C57BL/6J mice following i.c.v. administration ( Figure 3A and Figure S1). All of the d-Phe stereoisomers produced significant time-and dose-dependent antinociception (p < 0.05, two-way RM ANOVA). Peak antinociception was produced 20 min after i.c.v administration for stereoisomers 1, 3, and 4 and at 30 min for isomers 2 and 5 ( Figure S1). The duration of significant antinociception (p < 0.05; Dunnett's post hoc test) varied from 45 min (isomers 3 and 4) to 90 min (isomers 2 and 5), with isomer 1 exhibiting an intermediate duration. All of the isomers exhibited full and potent antinociception except for 2 ( Figure 3A), which produced approximately 70% antinociception at the highest dose tested (100 nmol); the potencies of stereoisomers 1 and 3-5 were comparable to that of CJ-15,208 ( Table 2). The maximal antinociception found for the stereoisomers 4 and 5 of [d-Trp]CJ-15,208 is in contrast to the parent peptide, which exhibits minimal antinociceptive activity [19]. and 5 of [D-Trp]CJ-15,208 is in contrast to the parent peptide, which exhibits minimal antinociceptive activity [19].  Table 2) that peaked between 20-30 min, while isomers 2 and 4 produced only 45-60% antinociception at the highest oral dose tested (30 mg/kg, p.o.). The duration of significant antinociception (p < 0.05, Dunnett's post hoc test) was 50-60 min for all stereoisomers except 4 (30-40 min). While isomer 2 exhibited low antinociceptive efficacy by both routes of administration, isomer 4 exhibited decreased efficacy only following oral administration.

In Vivo Assessment of Opioid Related Liabilities
The five stereoisomers were then assessed for several potential liabilities produced by opioid agonists, specifically impairment of locomotor coordination, respiratory depression, hyperlocomotion and analgesic tolerance.

Figure 5.
Opioid antagonist activity of the stereoisomers in the 55 • C warm-water tail-withdrawal assay. Mice were pretreated with a stereoisomer (30 or 100 nmol., i.c.v.) 3 h prior to the administration of (A) the KOR selective agonist U50,488 (10 mg/kg., i.p.), (B) the MOR preferring agonist morphine (10 mg/kg., i.p.), or (C) the DOR selective agonist SNC-30 (100 nmol, i.c.v.) to assess their ability to significantly reduce the antinociceptive effect of the opioid agonist. Mean % antinociception ± SEM from 8 mice for each bar. * significantly different from response of agonist alone (p < 0.05); one-way ANOVA with Sidak's multiple comparison post hoc test.

In Vivo Assessment of Opioid Related Liabilities
The five stereoisomers were then assessed for several potential liabilities produced by opioid agonists, specifically impairment of locomotor coordination, respiratory depression, hyperlocomotion and analgesic tolerance.

Evaluation of Respiratory and Spontaneous Locomotor Effects
The five stereoisomers (10 mg/kg, p.o.) were then assessed for their effect on spontaneous respiration rates and locomotor activity over a 1 h period using the Comprehensive Laboratory Animal Monitoring System (CLAMS) ( Figure 7). As expected, the positive control morphine produced significant, time-dependent respiratory depression compared to vehicle (10-30 min; F(10,165) = 2.48, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure S2), while treatment with U50,488 resulted in significant, time-dependent increases in respiration rates (20-40 and 50-60 min; p < 0.05, Dunnett's post hoc test; Figure S2). Stereoisomers 1-3 of CJ-15,208 did not have any significant effect on respiration compared to vehicle ( Figure 7A). Conversely, [D-Trp]CJ-15,208's isomers 4 and 5 produced significant decreases in respiration rates for the duration of the 60 min testing period (F(5,50) = 12.9, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure 7A). Among the D-Phe stereoisomers, only 3 had a significant effect on ambulation compared to vehicle, demonstrating elevated ambulations at several time-points (20-40 and 50-60 min; F(5,47) = 6.20, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure 7B). The positive control morphine produced robust, significant increases in ambulation over the last 40 min of testing (F(10,215) = 27.2, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure S2).

Evaluation of Respiratory and Spontaneous Locomotor Effects
The five stereoisomers (10 mg/kg, p.o.) were then assessed for their effect on spontaneous respiration rates and locomotor activity over a 1 h period using the Comprehensive Laboratory Animal Monitoring System (CLAMS) (Figure 7). As expected, the positive control morphine produced significant, time-dependent respiratory depression compared to vehicle (10-30 min; F (10,165) = 2.48, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure S2), while treatment with U50,488 resulted in significant, time-dependent increases in respiration rates (20-40 and 50-60 min; p < 0.05, Dunnett's post hoc test; Figure S2). Stereoisomers 1-3 of CJ-15,208 did not have any significant effect on respiration compared to vehicle ( Figure 7A). Conversely, [d-Trp]CJ-15,208's isomers 4 and 5 produced significant decreases in respiration rates for the duration of the 60 min testing period (F (5,50) = 12.9, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure 7A). Among the d-Phe stereoisomers, only 3 had a significant effect on ambulation compared to vehicle, demonstrating elevated ambulations at several time-points (20-40 and 50-60 min; F (5,47) = 6.20, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure 7B). The positive control morphine produced robust, significant increases in ambulation over the last 40 min of testing (F (10,215) = 27.2, p < 0.05, two-way RM ANOVA with Dunnett's multiple comparison post hoc test; Figure S2).

Assessment of Acute Antinociceptive Tolerance Development
The stereoisomers were also tested in a model of acute antinociceptive tolerance [25] with repeated dosing (at 0 and 8 h, 0.1-300 nmol, i.c.v.) of morphine, CJ-15,208 or one of the five analogs. The development of acute antinociceptive tolerance was assessed by pretreating with the ED50 i.c.v. dose of the test compound, followed 8 h later by treatment with one of a range of graded doses; antinociceptive tolerance was indicated by a significant increase in the ED50 value compared to the value observed in naïve animals. As expected, morphine demonstrated acute antinociceptive tolerance, with a significant 7.63-fold rightward shift in the dose-response curve of the second dose administered (F(1,4) = 26.2, p < 0.05; non-linear regression analysis; Table 3; see also Figure S3). Neither stereoisomer 4 or 5 demonstrated any significant changes in the ED50 values collected at 8 h vs. 0 h ( Table 3). Both stereoisomers 1 and 3 demonstrated greater acute antinociceptive tolerance than CJ-15,208, with significant rightward shifts in their second dose-response curves (31.2-fold (F(1,5) = 39.9 p < 0.05) and 5.19-fold (F(1,5) = 27.3, p < 0.05), respectively; non-linear regression analysis). While isomer 2 demonstrated a 6.13-fold increase in its ED50 value after pretreatment, the increase was not statistically significant (F(1,4) = 2.99, p = 0.16.)

Assessment of Acute Antinociceptive Tolerance Development
The stereoisomers were also tested in a model of acute antinociceptive tolerance [25] with repeated dosing (at 0 and 8 h, 0.1-300 nmol, i.c.v.) of morphine, CJ-15,208 or one of the five analogs. The development of acute antinociceptive tolerance was assessed by pretreating with the ED 50 i.c.v. dose of the test compound, followed 8 h later by treatment with one of a range of graded doses; antinociceptive tolerance was indicated by a significant increase in the ED 50 value compared to the value observed in naïve animals. As expected, morphine demonstrated acute antinociceptive tolerance, with a significant 7.63-fold rightward shift in the dose-response curve of the second dose administered (F (1,4) = 26.2, p < 0.05; non-linear regression analysis; Table 3; see also Figure S3). Neither stereoisomer 4 or 5 demonstrated any significant changes in the ED 50 values collected at 8 h vs. 0 h (Table 3). Both stereoisomers 1 and 3 demonstrated greater acute antinociceptive tolerance than CJ-15,208, with significant rightward shifts in their second dose-response curves (31.2-fold (F (1,5) = 39.9 p < 0.05) and 5.19-fold (F (1,5) = 27.3, p < 0.05), respectively; non-linear regression analysis). While isomer 2 demonstrated a 6.13-fold increase in its ED 50 value after pretreatment, the increase was not statistically significant (F (1,4) = 2.99, p = 0.16.)

Evaluation of Potential Reinforcing or Aversive Properties
Analogs 3 and 5 were further evaluated in a conditioned place-preference assay (Figure 8). Following a two-day place conditioning paradigm, mice conditioned with morphine (10 mg/kg, i.p.) demonstrated a significant place-preference for the morphine-paired chamber, whereas mice conditioned with U50,488 (10 mg/kg, i.p.) demonstrated a significant conditioned place avoidance (F (3,53) = 5.38, p < 0.05; two-way ANOVA with Sidak's multiple comparison post hoc test; Figure 8).

Evaluation of Potential Reinforcing or Aversive Properties
Analogs 3 and 5 were further evaluated in a conditioned place-preference assay (Figure 8). Following a two-day place conditioning paradigm, mice conditioned with morphine (10 mg/kg, i.p.) demonstrated a significant place-preference for the morphine-paired chamber, whereas mice conditioned with U50,488 (10 mg/kg, i.p.) demonstrated a significant conditioned place avoidance (F(3,53) = 5.38, p < 0.05; two-way ANOVA with Sidak's multiple comparison post hoc test; Figure 8). Figure 8. Evaluation of potential rewarding or aversive properties of isomer 3 and 5. After determination of initial preconditioning preferences, C57BL/6J mice were place conditioned daily for two days with morphine (10 mg/kg, i.p.), U50,488 (10 mg/kg, i.p.), or stereoisomer (10 mg/kg, p.o.) using a counterbalanced design. Data is presented as mean difference in time spent on the drugpaired side ± SEM, with positive and negative values indicating a preference for and avoidance of the drug-paired chamber, respectively. * significantly different from matching preconditioning preference (p < 0.05), two-way ANOVA. n = 14-28 mice/compound. However, mice place conditioned with either analog 3 or 5 (10 mg/kg, p.o.) demonstrated no significant preference or aversion for their respective drug-paired chamber.

Discussion
The macrocyclic tetrapeptide CJ-15,208 is structurally distinct from the endogenous opioid peptides, representing a novel lead compound for the development of new ligands for KOR. Its structure is particularly appealing for modification because its small macrocyclic structure imparts stability to degradation by proteases and facilitates penetration of biological barriers, including both the intestinal and blood-brain barriers [20,21]  Changes in stereochemistry had differential effects on the contributions of the three opioid receptors to antinociception in vivo. While all three aromatic residues of CJ,15-208 were previously found to be important for KOR agonist activity [26], all of the stereoisomers, including the isomers of [D-Trp]CJ-15,208, exhibited KOR agonist activity in vivo regardless of residue stereochemistry. In contrast, only stereoisomer 1 retained KOR antagonist activity. Unlike the parent compounds, all of the stereoisomers except 2 demonstrated antinociception also mediated in part by DOR. This is in contrast to the in vitro results, where the stereoisomers all lacked affinity for DOR in radioligand binding assays; such discrepancies between in vitro and in vivo activity have been found for other CJ-15,208 analogs [26][27][28]. D-Phenylalanine in position 1 appears to favor DOR agonist activity; of the four isomers where DOR contributes to the observed antinociception only 5 does not contain D-Phe 1 , and DOR appears to contribute less to the antinociception of this isomer than for the other isomers ( Figure 4B). MOR contributes to the antinociception of three of the stereoisomers, but not to Figure 8. Evaluation of potential rewarding or aversive properties of isomer 3 and 5. After determination of initial preconditioning preferences, C57BL/6J mice were place conditioned daily for two days with morphine (10 mg/kg, i.p.), U50,488 (10 mg/kg, i.p.), or stereoisomer (10 mg/kg, p.o.) using a counterbalanced design. Data is presented as mean difference in time spent on the drug-paired side ± SEM, with positive and negative values indicating a preference for and avoidance of the drug-paired chamber, respectively. * significantly different from matching preconditioning preference (p < 0.05), two-way ANOVA. n = 14-28 mice/compound. However, mice place conditioned with either analog 3 or 5 (10 mg/kg, p.o.) demonstrated no significant preference or aversion for their respective drug-paired chamber.

Discussion
The macrocyclic tetrapeptide CJ-15,208 is structurally distinct from the endogenous opioid peptides, representing a novel lead compound for the development of new ligands for KOR. Its structure is particularly appealing for modification because its small macrocyclic structure imparts stability to degradation by proteases and facilitates penetration of biological barriers, including both the intestinal and blood-brain barriers [20,21]  Changes in stereochemistry had differential effects on the contributions of the three opioid receptors to antinociception in vivo. While all three aromatic residues of CJ,15-208 were previously found to be important for KOR agonist activity [26], all of the stereoisomers, including the isomers of [d-Trp]CJ-15,208, exhibited KOR agonist activity in vivo regardless of residue stereochemistry. In contrast, only stereoisomer 1 retained KOR antagonist activity. Unlike the parent compounds, all of the stereoisomers except 2 demonstrated antinociception also mediated in part by DOR. This is in contrast to the in vitro results, where the stereoisomers all lacked affinity for DOR in radioligand binding assays; such discrepancies between in vitro and in vivo activity have been found for other CJ-15,208 analogs [26][27][28]. d-Phenylalanine in position 1 appears to favor DOR agonist activity; of the four isomers where DOR contributes to the observed antinociception only 5 does not contain d-Phe 1 , and DOR appears to contribute less to the antinociception of this isomer than for the other isomers ( Figure 4B). MOR contributes to the antinociception of three of the stereoisomers, but not to antinociception produced by isomers 3 and 4. The lack of MOR contribution in these peptides cannot be attributed to the change in stereochemistry of any specific residue; it may instead be related to differences in peptide backbone conformation and the resulting effect on the orientation of one or more of the aromatic residues. Thus only isomer 2 retained the mixed MOR/KOR agonism of CJ-15,208, but this isomer did not exhibit maximum antinociception nor KOR antagonist activity.
All of the stereoisomers retained antinociceptive activity following oral administration, despite the rapid metabolism of most of these peptides by liver microsomes. Such hydrophobic peptides, including the parent peptides (Khaliq et al., manuscript in preparation), generally have high plasma protein binding which can protect compounds from metabolism and clearance, and thereby extend the duration of their activity in vivo. Only in the case of isomer 4 was there a difference in the maximum antinociception following oral vs. central administration. The decreased maximum response and shorter duration following oral administration for this isomer are likely due to its pharmacokinetic properties (metabolism, clearance and/or intestinal absorption).
The stereoisomers exhibited different potential liabilities of use. While KOR agonism contributed to the antinociception of all of the stereoisomers, in the rotarod assay only isomer 5 exhibited significantly decreased locomotor coordination at multiple time points, suggesting that activity at multiple opioid receptors mitigated this known side effect of KOR agonists. The lack of significant decreases in respiratory rates for all three CJ-15,208 isomers was very promising. We have previously shown that the multifunctional macrocyclic tetrapeptide cyclo[Pro-Sar-Phe-d-Phe] exhibits reduced liabilities, particularly respiratory effects, and that the peptide's KOR agonist activity appears to offset respiratory depression mediated by MOR [9]. In contrast, treatment with [d-Trp]CJ-15,208 isomers 4 and 5 decreased respiratory rates; this effect of 4 was surprising, given the lack of MOR agonist activity by this isomer. While not significant, there was a trend towards decreased ambulation following treatment with 4 and 5, so it is possible that the decreased respiration rates could be due in part to decreased movement by these mice. In contrast to their effects on respiration rates, neither of the [d-Trp]CJ-15,208 stereoisomers exhibited evidence of acute tolerance, while, unlike the parent peptide, the CJ-15,208 isomers exhibited rightward shifts of the dose-response curves to varying degrees. Thus there was a dichotomy between the liabilities observed for the CJ-15,208 stereoisomers, which lacked respiratory depression but exhibited variable acute tolerance, and the [d-Trp]CJ-15,208 isomers, that exhibited the opposite pattern of decreased respiratory rates, but without significant acute tolerance. Two isomers, 3 and 5, were selected for testing in the conditioned place-preference assay for reinforcing (conditioned place preference, CPP) or aversive (conditioned place aversion, CPA) effects. Among the CJ-15,208 isomers, 3 demonstrated the most promising activity, producing full antinociception without locomotor impairment or respiratory effects, while the [d-Trp]CJ-15,208 isomer 5 produced full antinociception without acute antinociceptive tolerance. Mice place conditioned with either isomer demonstrated no significant preference or aversion for their respective drug-paired chamber. These results are consistent with earlier tests of multifunctional macrocyclic tetrapeptides [9,19] and could reflect the counteracting effects of agonism at multiple opioid receptors such as MOR and KOR [9,10]. In contrast to 5, isomer 3 did not demonstrate significant MOR-mediated agonism, but rather KOR-and DOR-mediated antinociception. Mixed action DOR/MOR agonists such as MMP-2200 reportedly do not produce CPP and exhibit limited reinforcing effects [29]. Isomer 5 also displayed DOR antagonism which has been shown to prevent the conditioned place preference of MOR agonists in studies of bivalent ligands [30] and peptidomimetics [31]. To the best of our knowledge, no one has examined the effect of combined KOR agonists/DOR agonists or antagonists. Additional testing of higher doses of 3 and 5 are required to confirm the absence of the place conditioning demonstrated here.
The liabilities of the different stereoisomers did not correlate with the receptor involvement determined in the antinociceptive assay. The antinociception of both the CJ-15,208 stereoisomer 3 and the [d-Trp]CJ-15,208 isomer 4 were mediated by DOR and KOR, without significant contribution from MOR, but these peptides had opposite liability profiles. The same was true for stereoisomers 1 and 5, where agonist activity mediated by all three opioid receptors contribute to the observed antinociception.
Additional studies will be necessary to explore potential mechanisms for the observed agonist (and in two cases antagonist) activity of these stereoisomers and to better understand receptor contributions to the observed side effects. Such studies are currently ongoing in our laboratories.
The stereoisomer [d-Phe 1,3 ]CJ-15,208 (3) is a very promising new lead compound for further exploration. Its potent antinociception after oral administration and lack of respiratory depression or locomotor impairment holds significant promise for the identification of safer analgesics.

Chemicals
The sources of the reagents, amino acids, solid phase resin and solvents for peptide synthesis are the same as reported previously [17,26,27]. Amino acids are the l-isomer unless otherwise specified, and abbreviations for amino acids follow the IUPAC-IUB joint commission of biochemical nomenclature (Eur. J. Biochem. 1984, 138, 9 -37). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Thin layer chromatography was performed on glass backed precoated silica gel plates (Sorbent Technologies, Atlanta, GA, USA, or Whatman, aluminum backed, 250 µm layer, Fisher Scientific, Pittsburg, PA, USA), and flash chromatography was performed on standard grade (32-63 µm) silica gel (Sorbent Technologies). HPLC analysis was performed on a Vydac 218TP C18 reversed phase column (Grace Davison, Columbia, MD, USA, 4.6 × 50 mm, 5 µm).

Instruments
Electrospray ionization mass spectra were acquired on a LCT Premier time of flight mass spectrometer (Waters Corp., Milford MA, USA) at the University of Kansas. HPLC analysis was performed using an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA).

Peptide Synthesis and Purification
The linear peptide precursors (based on the parent sequences l-/d-Trp-Phe-d-Pro-Phe-OH) were synthesized on a 2-chlorotrityl chloride resin by Fmoc (fluorenylmethoxycarbonyl) peptide synthesis, and the peptides cleaved from the resin with 1% trifluoroacetic acid (TFA) in dichloromethane as described previously [17,26,27]. The crude linear peptides were cyclized using the following general procedure [20,22,27]: The crude linear peptide (0.5 equiv, 21 mM in N,N-dimethylformamide, DMF) was added dropwise at a rate of 1.0 mL/h (using a KD Scientific single infusion syringe pump) to a dilute solution of HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 0.75 equiv, 1.2 mM) and N,N-diisopropylethylamine (DIEA, 4 equiv, 5 mM) in DMF. After 15 h additional HATU (0.75 equiv) was then added to the reaction in one portion, and additional linear peptide (0.5 equiv, 21 mM in DMF) was added dropwise at a rate of 1.0 mL/h as described above to give a final concentration of the linear tetrapeptide of 2.5 mM. The reaction was then stirred for 6 h at room temperature, followed by an additional 24 h at 37 • C. The solvent was evaporated under reduced pressure, and the crude macrocyclic tetrapeptides isolated as previously described [17,20,26].
The crude peptides were purified by silica gel chromatography using a step gradient of 60-90% EtOAc in hexane (with EtOAc increased in 10% increments), followed by 0-10% MeOH in EtOAc (with MeOH increased in 1% increments). The purified peptides were dissolved in aqueous acetonitrile (water:MeCN, 4:1) and then lyophilized to give the pure peptides as white solids. The purified peptides were analyzed by electrospray ionization mass spectrometry, thin layer chromatography, and in two analytical HPLC systems (see Table 4). The peptides were all >98% pure in both HPLC systems except for 4 which exhibited slightly lower purity. HPLC chromatograms are included in the Supplementary Materials.  (2) The linear peptide (400 mg) was cyclized according to the general procedure, except that the second addition of peptide was added at a rate of 0.8 mL/h and the final concentration of the linear peptide in the reaction mixture was 4.5 mM. The peptide was purified starting at 60% EtOAc in hexane as described above, followed by 0-30% MeOH in EtOAc (with MeOH increased in 3% increments) to yield stereoisomer 2 as a white solid (167 mg, 43% yield).

[d-Phe 1 ,d-Trp 4 ]CJ-15,208 (4)
The linear peptide (400 mg) was cyclized according to the general procedure above, except that the first addition of peptide was added at a rate of 1.2 mL/h and the final concentration of the linear peptide in the reaction mixture was 1.5 mM. The purification was performed starting at 30% EtOAc in hexane (with EtOAc increased in 10% increments), followed by 0-3% MeOH in EtOAc (with MeOH increased in 1% increments) to yield 4 as a white solid (174 mg, 45% yield).

[d-Phe 3 ,d-Trp 4 ]CJ-15,208 (5)
The linear peptide (430 mg) was cyclized according to the general procedure, except that the second addition of peptide was added at a rate of 0.8 mL/h and the final concentration of the linear peptide in the reaction mixture was 3.6 mM. The peptide was purified starting at 60% EtOAc in hexane as described above, followed by 0-5% MeOH in EtOAc (with MeOH increased in 5% increments) to yield stereoisomer 5 as a white solid (200 mg, 48% yield).

In Vitro Pharmacological Evaluation
Opioid receptor affinities were determined by equilibrium radioligand binding assays as previously described [19,26,33] [34]. The results are presented as the mean ± SEM from at least three separate experiments each performed in triplicate.
Agonist stimulation of [ 35 S]GTPγS binding to membranes from CHO cells stably expressing KOR or MOR was assayed as described previously [19,26,35]. The macrocyclic tetrapeptides were screened at 10 µM for efficacy compared to the reference full agonists dynorphin A-(1-13) amide for KOR and DAMGO for MOR. The stereoisomers all exhibited negligible stimulation of GTPγS binding at both KOR and MOR.

Animals and Drug Administration
Adult male wild-type C57BL/6J mice weighing 20-25 g were obtained from Jackson Labs (Bar Harbor, ME, USA). Food pellets and distilled water were available ad libitum. All mice were kept on a 12 h light-dark cycle and were housed and cared for in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All results of animal testing are reported in accordance with ARRIVE guidelines [36].
For intracerebroventricular (i.c.v.) administration the macrocyclic tetrapeptides were dissolved in dimethyl sulfoxide (DMSO), followed by addition of sterile saline (0.9%) so that the final vehicle was 50% DMSO and 50% saline, and the i.c.v. injections performed as described previously [26]. This concentration of DMSO was not observed to have antinociceptive or behavioral effects in previous use [9,19,21]. For per os (p.o.) administration the macrocyclic tetrapeptides were administered in 10% Solutol in 0.9% saline. All solutions for animal administration were prepared fresh daily.

Antinociceptive Testing
The 55 • C warm-water tail-withdrawal assay was performed in mice as previously described [37], with the latency of the mouse to withdraw its tail from the water taken as the endpoint (a cut-off time of 15 sec was used in this assay). Antinociception was calculated according to the following formula: % antinociception = 100 × (test latency − control latency)/(15 − control latency). Tail-withdrawal data points are the means of 8-16 mice, unless otherwise indicated, with SEM shown by error bars.
The opioid receptor involvement in the agonist activity of the macrocyclic peptides was determined by pretreating mice with a single dose of β-funaltrexamine (β-FNA, 10 mg/kg, i.p.) or nor-BNI (10 mg/kg, i.p.) 24 h in advance of administration of a dose of a macrocyclic tetrapeptide. Additional mice were pretreated with a single dose of naloxone (30 mg/kg, s.c.) or naltrindole (20 mg/kg, i.p.) 20 min in advance of administration of the macrocyclic tetrapeptide.
To determine antagonist activity, mice were pretreated with the macrocyclic tetrapeptide 140 min prior to the administration of the MOR-preferring agonist morphine (10 mg/kg, i.p.), KOR-selective agonist U50,488 (10 mg/kg, i.p.) or DOR-selective agonist SNC-80 (100 nmol, i.c.v.); at this time the antinociceptive activity of the stereoisomers had dissipated. Antinociception produced by these established agonists was then measured 40 min after their administration.

Acute Antinociceptive Tolerance Determination
A standardized state of tolerance was induced by administration of morphine or test compound at times 0 and 8 h [25,38,39] to quantitatively evaluate development of acute opioid tolerance. This assay was used to efficiently measure the potential of compounds to cause tolerance using a minimum amount of compound while yielding reliable results. Mice were administered an ED 50 dose (i.c.v.) of test compound in the morning (time = 0) and a second dose (varying between 0.1-300 nmol, i.c.v.) 8 h later. The degree of tolerance was calculated from the shift in ED 50 value from the singly-to repeatedly-treated condition [40]. All compounds were administered i.c.v., with antinociception assessed 30 min after injection of morphine or at the time of peak antinociceptive effect of the macrocyclic tetrapeptides, as determined in their initial antinociceptive characterization.

Coordinated Locomotor Activity
The stereoisomers were tested for their possible impairment of locomotor coordination in the rotarod assay as described previously [9,21]. Locomotor activity was recorded using an automated, computer-controlled rotarod apparatus (San Diego Instruments, San Diego, CA, USA). Mice were first habituated to the rotarod over seven trials, with the last trial serving as the baseline response. Mice so habituated were then administered a 10 mg/kg dose of a stereoisomer (p.o.), U50,488 (i.p.), or vehicle 15 min prior to assessment in accelerated speed trials (180 s max latency at 0-20 rpm) performed every 10 min over a 60 min period. Mice were thus tested a total of 14 trials (seven habituation trials prior to treatment + seven drug trials). Decreased latencies to fall in the rotarod test indicate impaired motor coordination/sedation.

Respiration and Ambulation
Respiration rates (in breaths per minute) and animal locomotive activity (as ambulations) were assessed using the Oxymax/CLAMS system (Columbus Instruments, Columbus, OH, USA) as described previously [9,25]. Mice were habituated to their individual sealed housing chambers for 60 min before testing. Mice were administered stereoisomer (10 mg/kg, p.o.), morphine (10 mg/kg, i.p.), U50,488 (10 mg/kg, i.p.), or vehicle, as indicated, and five min later confined to the CLAMS testing chambers. Pressure monitoring within the sealed chambers measured frequency of respiration. Infrared beams located in the floor measured locomotion as number of beam breaks. Respiration and locomotive data were averaged over 10 min periods for 60 min post-injection of the test compound. Data is presentenced as % vehicle response ± SEM, ambulation or breaths per minute.

Evaluation of Potential Conditioned Place Preference and Conditioned Place Aversion
An automated, balanced three-compartment place conditioning apparatus (San Diego Instruments, San Diego, CA, USA) and a 2-day counterbalanced place conditioning design was used similar to methods previously described [21]. The amount of time subjects spent in each of the three compartments was measured over a 30 min testing period. Prior to place conditioning an initial preference test was performed in which the animals could freely explore all open compartments; the animals did not demonstrate significant differences in their time spent exploring the outer left versus right compartments (p > 0.05, Student's t-test). For place conditioning mice were administered 0.9% saline (i.p.) and consistently confined in a randomly assigned outer compartment: half of each group in the right chamber, and half in the left chamber. Four hours later, mice were administered test compound and confined to the opposite compartment for 40 min. To determine if 3 or 5 (10 mg/kg, p.o.) produced CPP or CPA, mice were place conditioned in this way for two days, with a final preference test taken on the fourth day, as this has been shown to produce dependable morphine CPP and U50,488-induced CPA [41]. Additional groups of mice were placed conditions with morphine or U50,488 (10 mg/kg, i.p.) as positive controls.

Statistical Analysis
All dose-response lines were analyzed by regression, and ED 50 (effective dose producing 50% antinociception) values and 95% confidence intervals (C.I.) determined using individual data points from graded dose-response curves with Prism 8.0 software (GraphPad, La Jolla, CA, USA). Percent antinociception was used to determine within group effects and to allow comparison to baseline latency in tail-withdrawal experiments. The statistical significance of differences between ED 50 values was determined by evaluation of the ED 50 value shift via nonlinear regression modeling with Prism software. Significant differences in behavioral data were analyzed by ANOVA (one-way or two-way with repeated measures (RM), as appropriate). Significant results were further analyzed with Sidak's, Tukey's, or Dunnett's multiple comparison post hoc tests, as appropriate. Data for conditioned place preference experiments were analyzed by two-way RM ANOVA, with analyses examining the main effect of conditioned place preference phase (e.g., pre-or post-conditioning) and the interaction of drug pretreatment. Significant effects were further analyzed using Sidak's HSD post hoc testing. All data are presented as mean ± SEM, with significance set at p < 0.05.
Supplementary Materials: The following are available online, Figure S1: Time-course of antinociceptive activity in the 55 • C warm-water tail-withdrawal assay following (A) i.c.v. administration and (B) oral administration in C57Bl/6J mice of a maximally efficacious dose. Points represent average % antinociception ± SEM from 4-16 mice for each set presented, Figure S2. Effects of the U50,488 or morphine on (A) respiration and (B) ambulation in C57BL76J mice. Respiration and ambulation were monitored after administration of U50,488 or morphine (10 mg/kg, i.p.) using the CLAMS/Oxymax system. Data from 9-18 mice presented as % vehicle response ± SEM; breaths per minute, BPM (A) or ambulation, XAMB (B). * significantly different from response of saline alone (p < 0.05); two-way RM ANOVA with Dunnett's multiple comparison post hoc test., Figure S3. Evaluation of acute antinociceptive tolerance in the 55 • C warm-water tail-withdrawal assay following i.c.v. administration of morphine, (A) CJ-15,208, (B) 1, (C) 2, (D) 3, (E) 4 or (F) 5. All points represent antinociception at peak response in naïve mice (Time 0 h) and mice that were previously administered an ED 50 dose of test compound (as listed) prior to additional administration of a graded dose of test compound eight hours later (Time 8 h). Points represent average % antinociception ± SEM from 8-16 mice for each set presented, Figure S4.