Synthesis and evaluation of [18F]FBNAF, a STAT3-targeting probe, for PET imaging of tumor microenvironment.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is a protein that regulates cell proliferation and differentiation, and it is attracting attention as a new index for evaluating cancer pathophysiology, as its activation has been highly correlated with the development and growth of tumors. With the development of STAT3 inhibitors, the demand for imaging probes will intensify. Noninvasive STAT3 imaging can help determine the cancer status and predict the efficacy of STAT3 inhibitors. In this study, we aimed to develop an imaging probe targeting STAT3 and synthesized [18F]FBNAF, which was derived from a STAT3-selective inhibitor as the lead compound, followed by in vitro and in vivo evaluations of [18F]FBNAF in positron emission tomography for STAT3. RESULTS: The results revealed that FBNAF concentration-dependently inhibited STAT3 phosphorylation, similar to the lead compound, thereby supporting radiosynthesis. [18F]FBNAF was easily synthesized from the pinacol boronate ester precursor with suitable radiochemical conversion (46%), radiochemical yield (6.0%), and radiochemical purity (> 97%). [18F]FBNAF exhibited high stability in vitro and in vivo, and radioactivity accumulated in tumor tissues expressing STAT3 with an increasing tumor/blood ratio over time, peaking at 2.6 ± 0.8 at 120 min after injection in tumor-bearing mice. Tumor radioactivity was significantly reduced by the coinjection of a STAT3-selective inhibitor. Furthermore, the localization of radioactivity was almost consistent with STAT3 expression based on ex vivo autoradiography and immunohistochemistry using adjacent tumor sections. CONCLUSIONS: Thus, [18F]FBNAF could be the first promising STAT3-targeting probe for PET imaging. A STAT3 imaging probe provides meaningful information on STAT3-associated cancer conditions and in tumor microenvironment.

Icing after skeletal muscle injury with necrosis in a small fraction of myofibers limits inducible nitric oxide synthase-expressing macrophage invasion and facilitates muscle regeneration.

Growing evidence from animal experiments suggests that icing after skeletal muscle injury is harmful to muscle regeneration. However, these previous experimental models yielded massive necrotic myofibers, whereas muscle injury with necrosis in a small myofiber fraction (<10%) frequently occurs in human sports activities. Although macrophages play a proreparative role during muscle regeneration, they exert a cytotoxic effect on muscle cells through an inducible nitric oxide synthase (iNOS)-mediated mechanism. In this study, we established an animal injury model with necrosis limited to a small myofiber fraction and investigated the effect of icing on muscle regeneration with a focus on macrophage-related events. Icing after muscle injury of this model resulted in an enlarged size of regenerating myofibers compared with those in untreated animals. During the regenerative process, icing attenuated the accumulation of iNOS-expressing macrophages, suppressed iNOS expression in the whole damaged muscle, and limited the expansion of the injured myofiber area. In addition, icing increased the ratio of M2 macrophages within the injured site at an earlier time point than that in untreated animals. Following these phenomena in icing-treated muscle regeneration, an early accumulation of activated satellite cells within the damaged/regenerating area occurred. The expression level of myogenic regulatory factors, such as MyoD and myogenin, was not affected by icing. Taken together, our results suggest that icing after muscle injury with necrosis limited to a small fraction of myofibers facilitates muscle regeneration by attenuating iNOS-expressing macrophage invasion, limiting muscle damage expansion, and accelerating the accumulation of myogenic cells which form regenerating myofibers.

Icing after skeletal muscle injury decreases M1 macrophage accumulation and TNF-α expression during the early phase of muscle regeneration in rats.

Following skeletal muscle injury, both myogenic and immune cells interact closely during the regenerative process. Although icing is still a common acute treatment for sports-related skeletal muscle injuries, icing after muscle injury has been shown to disrupt macrophage accumulation and impair muscle regeneration in animal models. However, it remains unknown whether icing shortly after injury affects macrophage-related phenomena during the early stages of muscle regeneration. Therefore, we focused on the distribution of M1/M2 macrophages and cytokines expressed predominantly by macrophages during the early stages of muscle regeneration after muscle crush injury. Icing resulted in a decrease, not retardation, in the accumulation of M1 macrophages, but not M2 macrophages, in injured muscles. Consistent with the decrease in M1 macrophage accumulation, icing led to a reduction, instead of delay, in the level of tumor necrosis factor-α (TNF-α) expression. Additionally, at subsequent timepoints, icing decreased the number of myogenic precursor cells in the regenerating area and the size of centrally nucleated regenerating myofibers. Together, our findings suggest that icing after acute muscle damage by crushing disturbs muscle regeneration through hindering tM1 macrophage-related phenomena.

Icing after eccentric contraction-induced muscle damage perturbs the disappearance of necrotic muscle fibers and phenotypic dynamics of macrophages in mice.

Icing is still one of the most common treatments to acute skeletal muscle damage in sports medicine. However, previous studies using rodents reported the detrimental effect of icing on muscle regeneration following injury. This study aimed to elucidate the critical factors governing the impairment of muscle regeneration by icing with a murine model of eccentric contraction-induced muscle damage by electrical stimulation. Because of icing after muscle injury, the infiltration of polynuclear and mononuclear cells into necrotic muscle fibers was retarded and attenuated, leading to the persistent presence of necrotic cellular debris. These phenomena coincided with the delayed emergence and sustained accumulation of Pax7+ myogenic cells within the regenerating area. In addition, due to icing, delayed and/or sustained infiltration of M1 macrophages was noted in accordance with the perturbed expression patterns of inflammation-related factors, including tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). The key myogenic regulatory factors (i.e., MyoD and myogenin) involved in the activation/proliferation and differentiation of myogenic precursor cells were not altered by icing during the regenerative process. A detailed analysis of regenerating myofibers by size distribution at day 14 after muscle damage showed that the ratio of small regenerating fibers to total regenerating fibers was higher in icing-treated animals than in untreated animals. These findings suggest that icing following muscle damage blunts the efficiency of muscle regeneration by perturbing the removal of necrotic myofibers and phenotypic dynamics of macrophages rather than affecting myogenic factors.NEW & NOTEWORTHY Icing blunted the muscle regeneration by perturbing the infiltration of polynuclear and mononuclear cells into necrotic myofibers and the phenotypic dynamics of macrophages rather than affecting the myogenic regulatory factors. Because of icing, the disappearance of necrotic muscle debris was retarded, coinciding with the delayed emergence and sustained accumulation of Pax7+ cells within the regenerating area. The expression patterns of TNF-α and IL-10 were altered by icing consistent with the perturbation of the macrophage phenotype.

A solvent-dependent chirality-switchable thia-Michael addition to α,ß-unsaturated carboxylic acids using a chiral multifunctional thiourea catalyst.

An asymmetric thia-Michael addition of arylthiols to α,ß-unsaturated carboxylic acids using a thiourea catalyst that bears arylboronic acid and tertiary amine moieties is reported. Both enantiomers of the Michael adducts can be obtained in high enantioselectivity and good yield merely by changing the solvent. The origin of the chirality switch in the products was examined in each solvent via spectroscopic analyses.

PET probe detecting non-small cell lung cancer susceptible to epidermal growth factor receptor tyrosine kinase inhibitor therapy.

Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR-TKIs) are used as molecular targeted therapy for non-small cell lung cancer (NSCLC) patients. The therapy is applied to the patients having EGFR-primary L858R mutation, but drug tolerance caused by EGFR-secondary mutation is occurred within one and half years. For the non-invasive detection of the EGFR-TKIs treatment positive patients by positron emission tomograpy (PET) imagaing, fluorine-18 labeled thienopyrimidine derivative, [18F]FTP2 was newly synthesized. EGFR inhibition assay, cell uptake study, and blocking study indicated [18F]FTP2 binds with high and selective affinity for EGFR with L858R mutation, and not with L858R/T790M dual mutations. On animal PET study using tumor bearing mice, H3255 cells expressing L858R mutated EGFR was more clearly visualized than H1975 cells expressing L858R/T790M dual mutated EGFR. [18F]FTP2 has potential for detecting NSCLC which is susceptible to EGFR-TKI treatment.

18F-Labeled Pyrido[3,4-d]pyrimidine as an Effective Probe for Imaging of L858R Mutant Epidermal Growth Factor Receptor.

In nonsmall-cell lung carcinoma patients, L858R mutation of epidermal growth factor receptor (EGFR) is often found, and molecular target therapy using EGFR tyrosine kinase inhibitors is effective for the patients. However, the treatment frequently develops drug resistance by secondary mutation, of which approximately 50% is T790M mutation. Therefore, the ability to predict whether EGFR will undergo secondary mutation is extremely important. We synthesized a novel radiofluorinated 4-(anilino)pyrido[3,4-d]pyrimidine derivative ([18F]APP-1) and evaluated its potential as a positron emission tomography (PET) imaging probe to discriminate the difference in mutations of tumors. EGFR inhibition assay, cell uptake, and biodistribution study showed that [18F]APP-1 binds specifically to the L858R mutant EGFR but not to the L858R/T790M mutant. Finally, on PET imaging study using [18F]APP-1 with tumor-bearing mice, the H3255 tumor (L858R mutant) was more clearly visualized than the H1975 tumor (L858R/T790M mutant).

Synthesis and preliminary characterization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase-3ß (GSK-3ß) in the brain.

We report on the synthesis and preliminary characterization of two radioiodinated benzofuran-3-yl-(indol-3-yl)maleimides, 3-(benzofuran-3-yl)-4-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione ([(125) I]5), and 3-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-4-(6-methoxybenzofuran-3-yl)-1H-pyrrole-2,5-dione ([(125) I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase-3ß (GSK-3ß). In this study, we used (125) I as a surrogate of (123) I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10-30%. In vitro binding experiments suggested that both compounds show high affinity for GSK-3ß at a level similar to a known GSK-3ß inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK-3ß.

Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase ß.

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1-9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol ß, which is a DNA repair-related pol. Compounds 3-6 strongly inhibited the growth of human colon carcinoma HCT116 p53(+/+) cells. The influence of compounds 1-9 on HCT116 p53(-/-) cell growth was similar to that observed for HCT116 p53(+/+) cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol ß and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol ß activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53(-/-) cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol ß inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.

Inhibitory effect of somatostatin Peptide analogues on DNA polymerase activity and human cancer cell proliferation.

BACKGROUND AND OBJECTIVES: It was previously reported that ten small peptides derived from TT-232, somatostatin structural analogue (compounds 1-10), synthesised by a solution-phase method, exhibited potent antitumour activity on human epithelial tumour (A431) cells. MATERIALS AND METHODS: The present study investigated the inhibitory activity of these peptide compounds against DNA polymerase (pol) and human cancer cell growth. RESULTS: Among the compounds tested, compounds 1-5, which contain a t-butyloxycarbonyl (Boc) group, inhibited the activity of mammalian pols. Compounds 2 (Boc-Tyr-D-Trp-1-adamantylamide) and 3 (Boc-Tyr-D-Trp-2-adamantylamide) strongly suppressed the growth of a human colon carcinoma (HCT116) cell line and also arrested HCT116 cells in S phase, suggesting that these phenomena observed in cancer cells may be due to the selective inhibition of mammalian pols, especially DNA replicative pol α, by these compounds. Compound 2 induced apoptosis of the cells, although compound 3 did not. CONCLUSION: Compounds 2 and 3 had an enhanced anticancer effect based on pol inhibition.

New cyclic somatostatin analogues containing a pyrazinone ring: importance of Tyr for antiproliferative activity.

Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antiproliferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-D-Trp-Lys, with more hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicating that Tyr residue was an essential residue.

Synthesis of somatostatin analogues containing C-terminal adamantane and their antiproliferative properties.

On the basis of the structure of somatostatin analogue TT-232 (1), which exhibited a highly potent antitumor activity, we synthesized small linear peptide derivatives and evaluated their antitumor and apoptotic activity. Of them, Boc-Tyr-D-Trp-1-adamantylamide (5) had the most potent cell antiproliferative activity in SW480 and A431 cell lines, which was supported in A431 cell lines by FACS analysis that demonstrated a major increase in DNA fragmentation in the subG1 fraction.

Synthesis of opioidmimetics, 3-[H-Dmt-NH(CH(2))(m)]-6-[H-Dmt-NH(CH(2))(n)]-2(1H)-pyrazinones, and studies on structure-activity relationships.

Opioidmimetics containing 3-[H-Dmt-NH-(CH(2))(m)]-6-[H-Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinone symmetric (m = n, 1-4) (1 - 4) and asymmetric (m, n = 1 - 4) aliphatic chains (5 - 16) were synthesized using dipeptidyl chloromethylketone intermediates. They had high mu-affinity (K(i)mu = 0.021 - 2.94 nM), delta-affinity (K(i)delta = 1.06 - 152.6 nM), and mu selectivity (K(i)delta/K(i)mu = 14 - 3,126). The opioidmimetics (1 - 16) exhibited mu agonism in proportion to their mu-receptor affinity. delta-Agonism was essentially lacking in the compounds except (4) and (16), and (1) and (2) indicated weak delta antagonism (pA(2) = 6.47 and 6.56, respectively). The data verify that a specific length of aliphatic linker is required between the Dmt pharmacophore and the pyrazinone ring to produce unique mu-opioid receptor ligands.

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform.

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.9 nM) and weak delta-antagonism (MVD pA(2)=6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.

Bifunctional [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed mu-agonist/delta-antagonist and dual mu-agonist/delta-agonist opioid ligands.

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, containing alkylated Phe3 derivatives, 2'-monomethyl (2, 2'), 3',5'- and 2',6'-dimethyl (3, 3', and 4', respectively), 2',4',6'-trimethyl (6, 6'), 2'-ethyl-6'-methyl (7, 7'), and 2'-isopropyl-6'-methyl (8, 8') groups or Dmt (5, 5'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).

Transformation of mu-opioid receptor agonists into biologically potent mu-opioid receptor antagonists.

N-Allylation (-CH(2)-CHCH(2)) of [Dmt(1)]endomorphins yielded the following: (i) [N-allyl-Dmt(1)]endomorphin-2 (Dmt=2',6'-dimethyl-l-tyrosine) (12) and [N-allyl-Dmt(1)]endomorphin-1 (15) (K(i)mu=0.45 and 0.26nM, respectively) became mu-antagonists (pA(2)=8.59 and 8.18, respectively) with weak delta-antagonism (pA(2)=6.32 and 7.32, respectively); (ii) intracerebroventricularly administered 12 inhibited morphine-induced CNS-mediated antinociception in mice [AD(50) (0.148ng/mouse) was 16-fold more potent than naloxone], but not spinal antinociception, and (iii) 15 reversed the alcohol-elevated frequency in spontaneous inhibitory post-synaptic currents (IPSC) in hippocampal CA1 pyramidal cells in rat brain slices (P=0.0055). Similarly, N-allylation of the potent mu-opioidmimetic agonists, 1,6-bis-[H-Dmt-NH]-hexane and 3,6-bis-[Dmt-NH-propyl]-2(1H)-pyrazinone, converted them into mu-antagonists (pA(2)=7.23 and 7.17 for the N-allyl-derivatives 17 and 19, respectively), and exhibited weak delta-antagonism. Thus, N-allylation of Dmt containing opioid peptides or opioidmimetics continues to provide a facile means to convert selective mu-opioid agonists into potent mu-opioid antagonists.

Synthesis of 3,6-bis[H-Tyr/H-Dmt-NH(CH2)m,n]-2(1H)pyrazinone derivatives: function of alkyl chain length on opioid activity.

Dimeric opioid analogues linked to a pyrazinone platform, 3-[Tyr/Dmt-NH(CH2)m]-6-[Tyr/Dmt-NH(CH2)n]-2(1H)-pyrazinone (m, n=3 or 4), were synthesized. The Tyr-containing compound (m=4, n=3) exhibited mu-receptor affinity (K(i)mu; 7.58 nM) comparable to that of morphine, while the Dmt derivatives exhibited considerably higher affinity (K(i)mu; 0.021-0.051 nM) with corresponding agonism (IC50=1.79-4.93 nM). Interestingly one compound (m=4, n=3) revealed modest delta-opioid agonism; the converse analogue (m=3, n=4), however, was inactive in MVD assay.

Potent in vivo antinociception and opioid receptor preference of the novel analogue [Dmt1]endomorphin-1.

[Dmt1]Endomorphin-1 is a novel analogue of the potent mu-opioid agonist endomorphin-1. Given the physiological role of endomorphin-1 in vivo, this compound was investigated to determine if the antinociception occurred through systemic, supraspinal or in a combination of both neuronal pathways. This compound exhibited a potent dose-dependent effect intracerebroventricularly in both spinal and supraspinal regions, and was blocked by opioid antagonist naloxone, which verified the involvement of opioid receptors. Specific opioid antagonists characterized the apparent receptor type: beta-funaltrexamine (mu1/mu2-irreversible antagonist) equally inhibited spinal- and central-mediated antinociception; on the other hand, naloxonazine (mu1-subtype) was ineffective in both neural pathways and naltrindole (delta-selective antagonist) partially (26%), though not significantly, blocked only the spinal-mediated antinociception. Therefore, spinal antinociception was primarily triggered by mu2-subtypes without involvement of mu1-opioid receptors; however, although a slight enhancement of antinociception by delta-receptors cannot be completely ruled out since functional bioactivity indicated mixed mu-agonism/delta-antagonism. In terms of the CNS action, [Dmt1]endomorphin-1 appears to act through mu2-opioid receptor subtypes.

Potent Dmt-Tic pharmacophoric delta- and mu-opioid receptor antagonists.

A series of dimeric Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues (8-14, 18-22) were covalently linked through diaminoalkane and symmetric or asymmetric 3,6-diaminoalkyl-2(1H)-pyrazinone moieties. All the compounds exhibited high affinity for both delta-opioid receptors [Ki(delta) = 0.06-1.53 nM] and mu-opioid receptors [Ki(mu) = 1.37-5.72 nM], resulting in moderate delta-receptor selectivity [Ki(mu)/Ki(delta) = 3-46]. Regardless of the type of linker between the Dmt-Tic pharmacophores, delta-opioid-mediated antagonism was extraordinarily high in all analogues (pA2 = 10.42-11.28), while in vitro agonism (MVD and GPI bioassays) was essentially absent (ca. 3 to >10 microM). While an unmodified N-terminus (9, 13, 18) revealed weak mu-opioid antagonism (pA2 = 6.78-6.99), N,N'-dimethylation (21, 22), which negatively impacts on mu-opioid-associated agonism (Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435-5441), markedly enhanced mu-opioid antagonism (pA2 = 8.34 and 7.71 for 21 and 22, respectively) without affecting delta-opioid activity. These data are the first evidence that a single dimeric opioid ligand containing the Dmt-Tic pharmacophore exhibits highly potent delta- and mu-opioid antagonist activities.

New series of potent delta-opioid antagonists containing the H-Dmt-Tic-NH-hexyl-NH-R motif.

Heterodimeric compounds H-Dmt-Tic-NH-hexyl-NH-R (R=Dmt, Tic, and Phe) exhibited high affinity to delta- (K(i)delta=0.13-0.89nM) and mu-opioid receptors (K(i)mu=0.38-2.81nM) with extraordinary potent delta antagonism (pA(2)=10.2-10.4). These compounds represent the prototype for a new class of structural homologues lacking mu-opioid receptor-associated agonism (IC(50)=1.6-5.8muM) based on the framework of bis-[H-Dmt-NH]-alkyl (Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem.2003, 46, 3201), which exhibited both high mu affinity and bioactivity.