Synthesis and pharmacological activity of dermorphin and its N-terminal sequences

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂ (demorphin), an opiate-like peptide, and tri-, tetra-, penta- and hexapeptide-amide analogs, were synthesized by conventional methods in solution, to determine the minimum peptide chain-length, required for analgesic activity.     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

Synthesis and preliminary biological investigations of O-sulphated dermorphin

Synthesis by a classical solution method of a sulphated analogue of dermorphin is reported. The product has proved to be devoid of any peripheral and central opiate- or CCK-like activity in the tests assayed.     

Synthesis and pharmacological activity of the N-terminal dermorphin tetrapeptide analogs with CH2-NH peptide bond isosteres

The synthesis of pseudotetrapeptides H-Tyr-D-Ala-Phe-NH-(CH₂)₂-NH₂ (1a), H-Tyr-D-Ala-Phe-ψ(CH₂-NH)-Gly-NH₂ (2a), H-Tyr-D-Ala-ψ(CH₂-NH)-Phe-Gly-NH₂ (3a), and H-Tyr-ψ(CH₂-NH)-D-Ala-Phe-Gly-NH₂ (4a), representing the N-terminal tetrapeptide sequence of dermorphin, in which amide bonds are replaced by CH₂-NH bond, is described. N-acetyl-Tyr and desamino-Tyr pseudopeptide analogs (1-4b), (1-3c) are also described. The analogs were assayed in binding studies based on displacement of μ and δ-receptor selective radiolabels from rat brain membrane and in a bioassay using guinea pig ileum (GPI). Pseudopeptides in which the C-terminal (1a) or D-Ala-Phe (3a) amide bond are substituted, exhibit higher μ-affinities and μ-receptor selectivity than the corresponding Phe-Gly or Tyr-D-Ala analogs (2a, 4a). Acetyl-and desamino-Tyr pseudopeptide analogs (1-4b) and (1-3c) did not exhibit μ and δ-opioid receptor affinity at nM concentration. The relevance of the single peptide replacement and of its association to acetylation or amino group elimination of Tyr, is discussed on the basis of a receptor model for μ and δ opioids.     

Synthesis and biological activity of carboxyl terminally extended dermorphins

Dermorphinoyl(DMR)-glycine, DMR-sarcosine and DMR-glycyl-arginine have been prepared in order to examine the effect of C-terminal extension of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) on opioid activity. On GPI preparation the addition of Gly, Sar, or Gly-Arg to the carboxyl terminus of dermorphinoic acid was detrimental to μ activity: dermorphinoyl-derivatives, in fact, retain only 5–20% of dermorphin potency. Following intracerebroventricular administration (tail-flick test), whereas the analgesic activities of compounds showed the trend dermorphin >DMR-Sar> DMR-Gly-Arg>DMR-Gly>morphine, the nonapeptide displayed highest activity after subcutaneous injection in mice: DMR-Gly-Arg was 2.5 and 10 times more potent than dermorphin and morphine, respectively.     

Opioid peptides Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) were replaced by N^α-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of μ, Δ, and κ opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the κ binding site. The introduction of N^α-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed μ/δ specificity.     

Direct electrophilic fluorination of tyrosine in dermorphin analogues and its effect on biological activity,receptor affinity and selectivity

In a preliminary communication we reported [(Tetrahedron Lett. 31, 619 (1990)] that acetyl hypofluorite can be used efficiently to introduce fluorine regiospecifically (ortho to OH) into the phenolic ring of tyrosine-containing peptides. This procedure has been applied to the fluorination of a number of μ-selective opioid peptides derived from dermorphin. While the procedure can be used even when the side chains of Arg, Lys, and Tyr are left unprotected, the sulfoxide of a Met(O)-containing analogue was oxidized to sulfone faster than fluorination of the phenolic ring. This method can also be used when the peptide is attached to Merrifield resin. Thus, Tyr(3-F)-d -Ala-Phe-Gly-NH₂ and Tyr(3-F)-d -Arg-Phe-Lys-NH₂ (F-DALDA) have been prepared, purified, and characterized. Affinities of these fluorinated peptides for both μ-and δ-opioid receptors are reduced (two- to nine-fold) relative to their nonfluorinated analogues, but their selectivity for μ-opioid receptors is not significantly altered. Similarly, the in vitro biological potencies (GPI and MVD assays) of the fluorinated analogues are reduced (two- to seven-fold) relative to their nonfluorinated parent peptides. Thus, F-DALDA, which has high affinity (K δ= 15.2nM) and selectivity (K δ/K δ= 5390) for μ-opioid receptors, has potential use in biochemical studies which utilize ¹⁹F or ¹⁸F- labeled compounds.     

Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor

The neuropeptide galanin (1–29) binds with high affinity to hypothalamic receptors (K_D～ 0.9 nM) and regulates feeding behavior. The N-terminal fragments (1–16), (1–16)NH₂ are high affinity (K_D～ 6nM) full agonists in vivo and in vitro.l -Ala substitutions show that amino acid residues Gly¹, Trp², Asn⁵, Tyr⁹, and Gly¹² are important for the high affinity binding of galanin (1–16). Shortening the fragment (1–16) to galanin (1–7) causes a gradual drop of affinity: galanin (1–15), (1–14), and (1–13) have submicromolar K_D values and galanin (1–12) has K_D～ 3 μm . Cyclic analogs of galanin (1–12) of different ring size were synthesized by condensing Gly¹ and Gly¹² without or with spacer groups. These analogs, independent of ring size, had a lower affinity than the linear galanin (1–12). Derivatization of the N-terminus of galanin (1–29), (1–16), and (1–12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N-terminal Gly.     

Synthesis,activity on NK‐3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16

Abstract: Two analogs of a tachykinin family peptides – scyliorhinin II (ScyII): [Aib¹⁶]ScyII and [Sar¹⁶]ScyII were synthesized by the solid‐phase method using Fmoc chemistry. Conformational studies in water and DMSO‐d₆ on these peptides were performed using a combination of two‐dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar¹⁶]ScyII in water appeared to be more flexible, especially in the C‐terminal fragment. A better defined structure for this analog was obtained in DMSO‐d₆, in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3₁₀‐helix in the N‐terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib¹⁶]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3₁₀‐helix in the 13–18 fragment. A common structural motif was found in conformationally constrained Cys⁷?Cys¹³ fragment, which resembles the Greek letter ‘ω’. The differences in the solution structure of the C‐terminal fragment of the peptides studied are responsible for their specificity. [Aib¹⁶]ScyII showed 25% the agonistic activity of selective NK‐3 agonist – senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar¹⁶]ScyII appeared to be a full agonist of NK‐3 tachykinin receptor.     

Methylation in positions 1 and 7 of angiotensin II A structure-activity relationship study

Six analogues of angiotensin II (Ang) were synthesized with modifications in positions 1 and 7. The study was undertaken in order to learn more about the influence of alkylations in positions 1 and 7 and their interdependence. Previous studies have shown that x,x-dimethylation of Gly (aminoisobutyric acid, Aib) in position 1 produces quite potent analogues, as does N-methylation of Gly (sarcosine). Combination of both C^x- and N^x-methylations to N-Me-Aib¹, however, did not produce an affinity increase. Decyclisation of the Pro⁷-residue produced moderately active analogues with position 7 N-methylation and inactive analogues if the N-alkylation was suppressed. In order to investigate a possible stereochemical interdependence of positions 1 and 7, a group of peptides with combinations of position 1 and 7 alkylations were investigated. The following analogues were prepared: [Sar¹, Aib⁷]Ang, [Sar¹,Aib⁷,Leu⁸]Ang, [Aib^1,7]Ang, [Aib^1,7, Leu⁸]Ang, [N-Me-Aib¹,Aib⁷]Ang, [N-Me-Aib¹,Aib⁷,Leu⁸]Ang. They were synthesized by classical solid phase synthesis using the BOC-TFA-HF scheme. The biological properties of these peptides were assessed on the rabbit aorta preparation and their binding potencies were measured on bovine adrenal membranes. Both on agonistic and antagonistic [Leu⁸]Ang analogues single Aib substitutions in position 1 or 7 induced affinity reduction in both bioassays. Simultaneous Aib modifications in positions 1 and 7 induced more important affinity loss in a synergic manner in both bioassays and as well for agonists and antagonists. The N-Me-Aib¹ modifications induce similar affinity loss with or without concomitant Aib⁷ modification. Agonistic (Phe⁸) analogues containing Aib in position 7 all have reduced intrinsic activity, indicating for the first time an influence of this position on the activation mechanism of the Ang receptor of the Type AT₁. © Munksgaard 1994.     

Synthesis of a potent enkephalin analog,Tyr-D-Thr-Gly-Phe-Δ3Pro-NH2, and some of its biological activities

The enkephalin analog, H-Tyr-D-Thr-Gly-Phe-Δ³Pro-NH₂ x HCl (VI)([D-Thr², Δ³Pro⁵]-enkephalinamide), has been synthesized by conventional methods in solution and purified to homogeneity by reversed phase HPLC. The analog is a potent analgesic agent. For evaluation of some of its biological activity a related compound [D-Thr², Thz⁵]-enkephalinamide (XI) was also synthesized in solution. Anti-diarrhea activity was evaluated in mice by the intravenous route for anti-DL-5-hydroxytryptophan (5-HTP) induced diarrhea activity. Analgesic activity was assayed by the method of Nilsen in mice using the intravenous route, and by a modified tail flick test in rats and the acetic acid writhing test in mice following subcutaneous administration. Within the constraints of the assays the two analogs are approximately equipotent. Both are less active than [D-Ala², MePhe⁴, Met(0)ol]-enkephalinol (XII). Earlier receptor binding studies of compound XI indicated enhanced affinity for the μ receptor and little for the δ receptor. By comparison this may also be the case for compound VI.     

Synthesis and receptor binding analysis of dermorphin hepta-, hexa- and pentapeptide analogues

Sixteen dermorphin analogues were synthesized and characterized for μ- and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or the C-terminal residue; deletions of Gly⁴ or Pro⁶-Ser⁷; inclusion of Z or an acetyl group on the β-amino group of Lys⁷; and the presence of either a C-terminal amide or free acid group. Two peptides, [Lys7-OH]- and [Lys⁷-NH₂]dermorphin, had μ-affinities (K_iμ= 0.15–0.13 nm ) and μ-selectivities (K_iδ/K_iμ= 1158–1482) higher than dermorphin (K_iμ= 0.28 nm ; K_iδ/K_iμ= 295) and best fitted a one-site binding model similar to dermorphin. Significantly better (P <0.0001) fits to a two-site binding model vs. a one-site model were observed with four dermorphin analogues: [Lys(Z)⁷-OH]heptapeptide, [des-Gly⁴(Tyr⁴,Pro⁵,Asn⁶-OH)]hexapeptide and two pentapeptides, [Tyr⁵-NH₂] and [Trp⁴,Asn⁵-OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain μ-receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of μ-receptors; however, only detailed analyses of the data derived from the non-linear regression fits for one- or two-components gave evidence for the possible existence of two separate [³H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two-site model (P <0.05), but only four seemed to have two distinct Kⁱ, values (P <0.0001).     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

Synthesis and biological activity of dynorphin-(1 - 13) and analogs substituted in positions 8 and 10

Dynorphin-(1–13) (Dyn-(1–13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [³H]-ethylketocyclazocine (EKC, k ligand), [³H]-[D-Ala², MePhe⁴-Gly-ol⁵]-enkephalin (DAGO, μ ligand) and [³H]-[D-Ser², Thr⁶]-Leu-enkephalin (DSLET, δ ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the k receptor ([³H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1–13)). On the other hand, the ability of [Ala⁸]-Dyn-(1–13) to displace the binding of [³H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the μ receptor was not greatly affected ([³H]-DAGO displacement; Ki of 0.44nM as compared with 0.50nM for Dyn-(1–13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the k site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro¹⁰]-Dyn-(1–13) was equipotent to [Ala⁸]-Dyn-(1–13) in the GPI but its affinity for the μ binding site was decreased by a factor of 2.7 as compared with Dyn-(1–13). The affinity of [D-Pro¹⁰]-Dyn-(1–13) for the other binding sites (k and δ) was not greatly affected. Replacement of positions 8 or 10 by Trp or D-Trp decreased the activity on the GPI by more than 50% as well as the affinity for most receptor types. These data indicate that the selectivity of Dyn(1–13) for the k opioid receptor can be increased either by increasing its affinity for the K binding site ([Ala⁸]-Dyn-(1–13)) or lowering its potency for other binding sites, [Ala⁸]-Dyn-(1–13) being less potent on δ sites and [D-Pro¹⁰]-Dyn-(1–13) less potent on μ sites.     

Synthesis,biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp⁸-Lys¹²)-[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, i.e. cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp⁸ to Lys¹² proceeded rapidly (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.     

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101–126) and of the analog [3-Mpr¹⁰⁵]ANF(105–126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr¹⁰⁵]ANF(105–126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the α-carbon in position 105. The analog [3-Mpr¹⁰⁵, Nva¹⁰⁹]ANF(105–126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105–126) analogs may have a somewhat longer half-life in vivo or, alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.     

Synthesis and properties of equine β-melanotropin analogs with substitution in residue position 1

Five analogs of equine β-melanotropin have been synthesized by the solid phase method. The NH₂-terminal aspartic acid was substituted with amino acids (Gly, Trp, Ile, Lys and Nα-acetyl-Asp) differing widely in physicochemical properties. On the basis of their lipolytic potencies it was concluded that this position plays a negligible role in this activity.     

Potent agonists of growth hormone-releasing hormone

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr¹, or N-MeTyr¹. Some peptides contained one or more additional l - or d -amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH₂, [Dat′,Ala¹⁵]GH-RH(1-28)Agm (MZ-3-191) and [d -Ala²,Ala¹⁵]GH-RH(l-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met²⁷. Their Nle²⁷ analogs, [Dat^I,Ala¹⁵,Nle²⁷]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [^D-Ala²,Ala¹⁵,Nle²⁸]GH-RH(l-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met²⁷ by Nle²⁷ enhanced the GH-releasing activity of the analog when the molecule contained Dat¹-Ala² residues at the N-terminus, but peptides containing Tyr¹-D-Ala² in addition to Nle²⁷ showed decreased potencies. Replacement of Ser²⁸ with Asp in multi-substituted analogs of GH-RH(l-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser²⁸-containing MZ-2-51, and [Dat¹,Ala¹⁵,d -Lys²¹,Nle²⁷]GH-RH(l-28)Agm, its Asp²⁸ homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively. In vivo after the iv injection, the analogs [Dat¹,Ala¹⁵,Nle²⁷,Asp²⁸]GH-RH(l-28)Agm (MZ-3-149), [Dat¹, Ala¹⁵]GH-RH(l-28)Agm, (MZ-3-191) and [d -Ala²,Ala^,5]GH-RH(l-28)Agm (MZ-3-201) showed a potency equivalent to 7.6, 4.9 and 3.3 times that of GH-RH(1-29)NH₂, respectively, at 5 min and 20.3, 4.3 and 1.7 times higher, respectively, at 15 min. After sc administration, analogs MZ-3-149, MZ-3-191, and MZ-3-201 were shown to be 63.7, 55.2 and 56.8 times more potent than the parent hormone at 15 min and 57.6, 60.6, and 42.6 times more active, respectively, at 30 min. In addition, MZ-3-149 had prolonged GH-releasing activity as compared to the standard, and proved to be more potent than MZ-2-51, the most active member of our previous series (8, 9). Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Synthesis and biological investigations of new tuftsin analogs with elongated peptide chain

In this paper the synthesis of the following elongated tuftsin analogs: Thr-Lys-Pro-Lys-Thr-Lys-Pro-Lys (I), Thr-Lys-Pro-Lys-Thr-Lys-Pro-Arg (II) and Ala-Lys-Thr-Lys-Pro-Arg-Glu-Gln (III) by the classical method is described. The compound II markedly inhibited the growth of murine sarcoma viruses (MSV).     

Synthesis and biological activities of ψ(CH2NH) pseudopeptide analogues of the C-terminal hexapeptide of neurotensin

Each peptide CO-NH function in the biologically important C-terminal 8-13 sequence of neurotensin was replaced by the reduced CH₂-NH isostere using the rapid in situ solid phase procedure developed by Sasaki & Coy. In general this modification resulted in a drop in receptor affinity except for the [Argψ(CH₂NH)Arg]-NT_8–13 analogue (PIC₅₀ 9.23 vs. NT_8–13 PIC₅₀ 8.03). This analogue also showed enhanced enzymatic stability, but acted as a full agonist as shown by the observation of relaxations of guinea-pig colon ascendens.