Synthesis and biological activities of analogs of luteinizing hormone-releasing hormone (LH-RH) substituted in position 1 or 2

Syntheses are described of [Pro¹]-LH-RH, [Orotic acid¹]-LH-RH, [Glu¹]-LH-RH, [Ser²]-LH-RH, [Leu²]-LH-RH, [Gln²]-LH-RH and [Phe²]-LH-RH. The LH-releasing hormone (LH-RH) activity of each of these peptides was compared with that of natural LH-RH $\text{in vivo}$. [Glu¹]-LH-RH and [Phe²]-LH-RH had significant LH-RH activity, while all the other analogs possessed extremely low activities. These findings are briefly discussed in the light of the structure-activity relationship for LH-RH.     

Synthesis and biological activity of position 1 analogs of LH-RH.

(Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.     

Structure-activity relationships of luliberin substituted at position 8.

Substitution of arginine at position 8 of luliberin by the basic amino acids homoarginine, lysine and diaminobutyric acid resulted in analogues in which the luteinizing hormone-releasing activity is markedly reduced, whereas the cross reactivity with specific antibodies to luliberin is preserved. Fluorimetric titrations of these analogues, carried out as with luliberin, revealed pK values of 6.00 +/- 0.05 and of 9.75 +/- 0.15 for His 2 and Try 5 respectively which are essentially the same as in luliberin. However, the rate of collisions between the side chains of His 2 and Trp 3 in these analogues was found to decrease by 36-39%. Substitution at position 8 with the non-basic amino acid omega-nitro arginine yielded an analogue possessing a very low hormonal activity as well as poor recognition of antibodies specific to luliberin. The fluorescence properties of this peptide are markedly different from those of luliberin and its three basic analogues. These results indicate that the functional integrity of the active unit His 2 . . . Tyr 5 . . . Arg 8 in luliberin depends both on size and basicity of the amino acid side chain at position 8.     

Syntheses and biological activities of sandostatin analogs containing stereochemical changes in positions 6 or 8

In a continuation of our research efforts on the design and synthesis of novel peptidomimetic structures, we have synthesized a series of sandostatin amide analogs in which stereoisomers of threonine and beta-hydroxyvaline(beta-Hyv) are employed. The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) explore the effects on biological activity of stereochemical modifications and beta-methylation at positions 6 or 8. By these modifications, we examine the role of the two residues in binding to somatostatin receptors. We describe the synthesis and biological activity of these analogs. In combination with the results of the conformational analysis, this study provides new insights into the structural requirements for the binding affinity of somatostatin amide analogs to somatostatin receptors [Mattern et al., Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8].     

Syntheses and biological activities of atrial natriuretic polypeptide analogs

Recently several peptides with natriuretic and diuretic potencies were isolated from human and rat atrial extract, and the precursors of the peptides were sequenced. Of the peptides, -human and rat atrial natriuretic polypeptides (-hANP, -rANP), consisting of 28 amino acids, are thought to be essential to the potency and to play an important role in the blood pressure regulation system. The amino acid sequence of -hANP is different from that of -rANP only at the position 12 (isoleucine in -rANP). In the present study, we synthesized ANPs and their analogs using a new deprotection procedure based on the concept of push-pull mechanism. Using the synthetic ANP analog, we also developed a radioimmunoassay for -ANP and examined the structure-activity relationship. Synthetic -hANP caused potent, rapid, and short-acting increases in Na⁺ and Cl^– excretion, and also an increase in urine flow and K⁺ excretion of lesser magnitude, when injected into rat. Also, we synthesized a cyclic part of -hANP, -ANP(7–23)-NH₂. Since this peptide had a little diuretic and natriuretic potency, we attempted to synthesize a chemically stable -hANP analog. We considered that the disulfide bond would be equivalent to propylene with regard to interatomic distance and employed 8-aminocaprylic acid instead of cystine. This cyclic peptide, named cyclonatrin-54, had a somewhat higher potency than -hANP(7–23)-NH₂ for diuresis and natriuresis, as expected. Furthermore, using a synthetic intermediate of cyclonatrin-54, we prepared a linear ANP analog, -hANP(8–22), Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly. This linear 15-amino acid peptide had a dose-dependent natriuretic and diuretic activity, but no hypotensive effect. It was surprising that a linear peptide exhibited a potent natriuretic activity. For the first time, a linear peptide has been prepared that has substantial natriuretic and diuretic potency. We synthesized some analogs of this 15-amino acid peptide and investigated the structure-activity relationship.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Syntheses and biological evaluation of analogs of luteinizing hormone-releasing hormone (LH-RH) modified in position 2, 3, 4 or 5

Syntheses by the conventional methods as well as the chemical, physical and biological properties are described of the following analogs of the LH-releasing hormone (LH-RH): [Leu³]-LH-RH, [Phe³]-LH-RH, [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH, Des-His²-[Phe⁵]-LH-RH, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH. $\text{In vivo}$ assays showed that [Leu³]-LH-RH did not release LH in doses as high as 5 – 25 μg, having less than 0.0008% of LH-RH activity, while [Phe³]-LH-RH had 0.43% of the LH-RH activity of natural LH-RH. The LH-RH activities of [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH and Des-His²-[Phe⁵]-LH-RH were extremely low. On the other hand, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH had significant LH-RH activity. The structure-activity relationship of LH-RH is discussed on the basis of these findings.     

Syntheses and biological activities of deoxy-d-allose fatty acid ester analogs

We describe the syntheses of three different deoxy-D-allose analogs [2-deoxy-D-allose (2-DOAll), 1,2-dideoxy-D-allose (1,2-DOAll), and 1,2-didehydro-1,2-dideoxy-D-allose (1,2-DHAll)] and their fatty acid esters via regioselective lipase-catalyzed transesterification. Among them, 2-DOAll and its decanoate (2-DOAll-C10) showed higher inhibitory activity on plant growth, which is similar to D-allose (All) and its decanoate (All-C10). Bioassay results of deoxy-All-C10 on four plant species suggest that the hydroxy group at the C-1 position might be important showing growth inhibitory activity. In addition, co-addition of gibberellin (GA₃) with 1,2-DHAll-C10 and 2-DOAll-C10 recovered plant growth, suggesting that they might mainly inhibit biosynthesis of gibberellin.     

Syntheses and biological activities of daunorubicin analogs with uncommon sugars

To study the effects of the sugar structure on the activity of anthracycline against cancer cells, six daunorubicin analogs containing different uncommon sugars were synthesized. Their cytotoxicities were tested against colon cancer cells by MTS assay. The results showed that the aglycon without sugar moiety has 70-100-fold lower activity against cancer cells than daunorubicin derivatives with various uncommon sugars. It suggests that the sugar structure in daunorubicin plays a critical role in determining its anticancer activity. In the compounds with various sugars, the 4'-OH of the sugar is an important determinant for their activity, while the axial-3'-substituent in the sugar interferes with the binding of daunorubicins to DNA. Therefore, 2,6-dideoxy sugars are a better choice for generating biologically active daunorubicin analogs than 6-deoxysugars, 2,3,6-trideoxysugars, or 2,3,4,6-tetradeoxysugars.     

Vasopressin antagonist analogs modified at position 7

Vasopressin antagonist analogs having alanine or glycine at position 7 were essentially equipotent with analogs with proline, N-methylalanine or sarcosine at position 7. This demonstrates that the conformational constraint imposed by an N-alkyl residue at position 7 is not necessary for binding of antagonist to the receptor, the exact opposite of what is seen in agonists. This suggests that antagonists bind to the receptor in a manner which is very different from that of agonists.     

Activities of monomeric insulin analogs at position A8 are uncorrelated with their thermodynamic stabilities

Previous studies have demonstrated that the potency and thermodynamic stability of human insulin are enhanced in concert by substitution of Thr(A8) by arginine or histidine. These surface substitutions stabilize the N-terminal alpha-helix of the A chain, a key element of hormone-receptor recognition. Does enhanced stability necessarily imply enhanced activity? Here, we test by structure-based mutagenesis the relationship between the stability and activity of the hormone. To circumvent confounding effects of insulin self-association, A chain analogs were combined with a variant B chain (Asp(B10), Lys(B28), and Pro(B29) (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and (1)H NMR spectra of the DKP analogs are essentially identical to those of DKP-insulin, indicating a correspondence of structures. Receptor binding affinities were determined by competitive displacement of (125)I-insulin from human placental membranes. Thermodynamic stabilities were measured by CD titration; unfolding was monitored as a function of guanidine concentration. In this broader collection of analogs receptor binding affinities are uncorrelated with stability. We suggest that receptor binding affinities of A8 analogs reflect local features of the hormone-receptor interface rather than the stability of the free hormone or the intrinsic C-capping propensity of the A8 side chain.     

The analogs of oxytocin and D-homoarginine vasopressin with bulky substituted phenylalanine in position 2

Solid phase technique on p-methylbenzhydrylamine resin wasused for the synthesis of eight analogs of oxytocin and 8-D-homoarginine vasopressin with the non-coded amino acids L- or D-2,3,4,5,6-pentamethylphenylalanine and L- or D-4-phenylphenylalanine in position 2. The preparation of theabove mentioned non-coded amino acids is described as well.All eight analogs were found to be potent inhibitors ofoxytocin activity in the uterotonic in vitro test in theabsence of Mg²⁺ ions. In the uterotonic test invitro in the presence of Mg²⁺ and in the test invivo, their potency is strongly decreased or completelyabolished. The substances are also weak pressor inhibitors.The L or D configuration does not seem to influence theactivity significantly.     

New analogs of acyclovir substituted at the side chain

A series of novel analogs of acyclovir, substituted with an alkyl (methyl, ethyl, n-butyl) or phenyl group at the positions 1', 4', and/or 5', has been obtained in a direct one-pot coupling reaction of guanosine and the respective 1,3-dioxolanes. The new acyclonucleosides were essentially inactive in antiviral (HSV, VV, VSV, HBV) evaluation in vitro.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

The analogs of oxytocin and D-homoarginine vasopressin with bulky substituted phenylalanine in position 2

Summary Solid phase technique on p-methylbenzhydrylamine resin was used for the synthesis of eight analogs of oxytocin and 8-D-homoarginine vasopressin with the non-coded amino acids L- or D-2,3,4,5,6-pentamethylphenylalanine and L- or D-4-phenylphenylalanine in position 2. The preparation of the above mentioned non-coded amino acids is described as well. All eight analogs were found to be potent inhibitors of oxytocin activity in the uterotonicin vitro test in the absence of Mg²⁺ ions. In the uterotonic testin vitro in the presence of Mg²⁺ and in the testin vivo, their potency is strongly decreased or completely abolished. The substances are also weak pressor inhibitors. The L or D configuration does not seem to influence the activity significantly. Abbreviations: All the chiral amino acids unless otherwise stated are of the L-series. Phe(4-Ph) denotes the 4-phenylphenylalanine, Phe(pentaMe) the 2,3,4,5,6-pentamethylphenylalanine, Har the homoarginine, DMF dimethylformamide, OHBT 1-hydroxybenzotriazole and DCC dicylohexyl-carbodiimitz. The Nomenclature and symbols of the amino acids and peptides obey the published recommendations: IUPAC-IUB Joint Commission on Biochemical Nomenclature: Eur. J. Biochem., 138 (1984) 9.     

LH- and FSH response to long-term application of LH-RH analogue in normal males.

The effect of luteinizing hormone-releasing hormone (LHRH) analogue after subcutaneous application over a 12-day period was evaluated in 15 normal males to determine whether gonadotropin secretion is influenced by longterm application. After 1.25 mcg/day, LH increases were measurable 3 hours after daily receipt of the analogue. After 2.5 mcg/day, the release of LH secretion was even greater, although this stimulation was markedly reduced in the 2nd week. After 5 mcg in 2-day intervals, a clearly induced LH increase was observed on the day of administration and was maintained for 10 hours. Similar results were noted for follicle-stimulating hormone (FSH). There was a response of FSH after 1.25 mcg and 2.5 mcg, but this response decreased in the 2nd week. After application of 5 mcg every other day, there was a consistent FSH increase that subsided on the treatment-free day and continued into the 2nd week of treatment. These findings suggest that LHRH analogues should be administered in low doses subcutaneously or intranasally at daily intervals to enable a longterm effect on gonadotropin release.     

Pharmacological profile of 8-amino octanoic acid substituted atrial natriuretic factor analogs

A series of six analogs of rat atrial natriuretic factor have been prepared by the solid-phase method. The modified analogs contain 8-amino octanoic acid (a simple alkyl spacer) in place of selected tripeptides. Binding affinity to cultured aortic smooth muscle cell membranes suggests that the sequence Arg11-Gly16 is important for binding. Vasorelaxant activity on serotonin contracted rabbit aortic rings indicates that the Phe8-Gly16 sequence must be present for vasorelaxation. In anesthetized rats, the natriuretic and diuretic effects of an IV bolus dose correlate with in vitro vasodilatory activity. The alkyl spacer approach provides a facile method to quickly determine key regions of a large peptide involved in molecular recognition.