Synthesis of luteinizing hormone-releasing hormone containing tritium-labelled pyroglutamic acid

A method of preparing luteinizing hormone-releasing hormone (LH-RH) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, by a combination of solid-phase and classical reactions was employed to conveniently synthesize a tritium-labelled hormone by incorporation of 4-[³H]-pyroglutamic acid into position I of the peptide chain. The tritiated LH-RH possessed a specific radioactivity of 18.3 Ci/mmole and a maximal biological potency.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

The luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, has no direct effect on spermatogenesis in the rat

Treatment of intact rats with luteinizing hormone-releasing hormone (LHRH) agonists has been shown to produce atrophy of a variable number of testicular seminiferous tubules. These findings raised the question of a possible direct versus indirect action of LHRH agonists on spermatogenesis. To answer this question, we treated hypophysectomized rats with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, dihydrotestosterone (DHT), or a combination of these two compounds for a period of 1 mo. Treatment of hypophysectomized animals with the LHRH agonist alone had no significant effect on the atrophy of seminiferous tubules found after hypophysectomy. DHT, however, maintained spermatogenesis at 80% of the level seen in intact animals. When DHT and the LHRH agonist were administered in combination, the stimulatory effects of DHT were observed with no significant interference caused by the LHRH agonist. This study shows that an LHRH agonist has no direct effect on the morphology of the seminiferous tubules in the absence of the pituitary gland and strongly suggests that the atrophy observed in the testis after LHRH agonist treatment in intact animals is mediated by the LHRH agonist-induced changes in luteinizing hormone secretion and/or direct action of the peptide on Leydig cells.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). I. Synthesis

Poly(L -histidyl-L -alanyl-α-L -glutamic acid) has been prepared in order to test the acid–base catalytic ability of a carboxyl-imidazole hydrogen-bonded system. Two different blocked histidyl-alanyl-glutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a t-butyloxycarbonyl group. The γ-carboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the N-hydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the t-butyloxycarbonyl and the benzyl groups. The water-soluble unblocked polymers obtained were fractionated on Sephadex G-50 or Bio-Gel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.     

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 of a mucin-type O-glycosylated amino acid, beta-Gal-(1----3)-[alpha-Neu5Ac-(2----6)]-alpha-GalNAc-(1----3)- Ser

Total synthesis of O-beta-D-galactopyranosyl-(1----3)-O-[(5-acetamido-3,5-dideoxy- D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2----6)]-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1----3 )-L- serine was achieved by use of the key glycosyl donor O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O- [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha-D- galactopyranosyl trichloroacetimidate and the key glycosyl acceptor N-(benzyloxycarbonyl)-L- serine benzyl ester in a regiocontrolled way.     

Analogues of luteinizing hormone-releasing hormone (LH-RH) containing dehydroalanine in 6th position

(Phe5, delta Ala6)-LH-RH and des-Gly10(Phe5, delta Ala6)-LH-RH ethylamide, two analogues of luteinizing hormone-releasing hormone (LH-RH), have been synthesised using fragment condensation approach in solution phase with minimum protection of the side chains. The presence of dehydroalanine in peptide fragments was confirmed by 1H n.m.r. and chemical analysis. Both the analogues were found to be inactive in comparison to LH-RH, indicating that alpha,beta-dehydrogenation of alanine in 6th position is not tolerated and suggesting that flexibility at this position may be crucial for the retention of biological activity.     

Viscosity and potentiometric measurements of poly(L-histidyl-L-alanyl-α-L-glutamic acid) and poly(L-lysyl-L-alanyl-α-L-glutamic acid)

Poly(His-Ala-Glu) and poly(Lys-Ala-Glu) were examined by viscosity and potentiometric titration. These measurements were interpreted in terms of the hydrodynamic size of the above sequential polypeptides. Effects of polymer, size and concentration, and solution-salt concentration were demonstrated. Although the sequential polypeptides generally behave like polyampholytes, they do demonstrate some differences. These differences my be attributed to the ability of ionized side chains three residues apart to repel themselves, in the order His < Glu < Lys.     

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.     

Effect of luteinizing hormone releasing hormone (LHRH) and [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide on alpha-subunit and LH beta messenger ribonucleic acid levels in rat anterior pituitary cells in culture

The effect of incubation with LHRH and its agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide has been measured on the concentrations of mRNAs for the common alpha-subunit of glycoprotein hormones and beta-LH in rat anterior pituitary cells in primary culture. After incubation, total RNA was analyzed by Northern blot or dot blot hybridization with alpha- and LH beta 32P-labeled cRNA probes and mRNA levels were quantified by autoradiography. Short-term treatment (4-6 h) of pituitary cells with 100 nM LHRH led to a marked stimulation of LH release but no effect was observed on alpha-subunit or LH beta mRNA levels. Longer (24-72 h) incubation periods with LHRH led to complete desensitization of the LH response to the neurohormone and induced 2- to 3-fold increases in alpha-mRNA cell content while LH beta mRNA levels remained unchanged. Maximal induction of alpha mRNA accumulation was observed with an LHRH concentration as low as 0.1 nM. Incubation with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide for 24-72 h also increased alpha mRNA but did not modify LH-beta mRNA levels. It is concluded that long-term exposure of anterior pituitary cells to LHRH or to an LHRH agonist positively regulates alpha-subunit gene expression in the absence of change in LH beta mRNA levels. This observation can provide an explanation for the high plasma levels of free alpha-subunits found in patients treated chronically with LHRH agonists.     

Synthesis, characterization, DNA-binding and photocleavage of complexes [Ru(phen)2(6-OH-dppz)]2+ and [Ru(phen)2(6-NO2-dppz)]2+

Two new complexes, ([Ru(phen)(2)(6-OH-dppz)](2+)) (1) and ([Ru(phen)(2)(6-NO(2)-dppz)](2+)) (2) (phen=1,10-phenanthroline; 6-OH-dppz=6-hydroxyl-dipyrido[3,2-a:2',3'-c]phenazine; 6-NO(2)-dppz=6-nitro-dipyrido[3,2-a:2',3'-c]phenazine), have been synthesized and characterized by elemental analysis, ES-MS (electrospray mass spectra), (1)H NMR, UV-Vis (UV-visible) and CV (cyclic voltammetry). The DNA-binding behaviors of both complexes have been studied by spectroscopic methods and viscosity measurements. The results indicate that the two complexes all bind to calf thymus DNA (CT-DNA) in an intercalative mode, and the DNA-binding affinity of complex 2 is greater than that of complex 1. In addition, complex 1 can promote photocleavage of pBR322 DNA upon irradiation, whereas complex 2 can promote cleavage of pBR322 DNA both upon irradiation and in the dark, with more efficient cleavage occurring upon irradiation. Theoretical studies for these complexes have been also carried out with the density functional theory (DFT) method. The difference in the DNA-binding behaviors of the two complexes can be reasonably explained by the DFT calculations.     

Radioimmunoassay of nafarelin ([ 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin [( 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately 50%. The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, 125I-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 ng/ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coefficient of 0.997. At 0.050 ng/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [3H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLC-radiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.     

Synthesis of [26-2H(3)]brassinosteroids

A number of [26-2H(3)]brassinosteroids were prepared for biochemical studies. The parent, nondeuterated compounds were considered to be biosynthetic intermediates in brassinosteroid biosynthesis. Claisen rearrangement was used to construct the steroidal side chain. Deuterium was introduced by reducing the corresponding intermediates with lithium aluminium deuteride.     

Modulation by sex steroids and [D-Trp6, Des-Gly-NH2(10)]luteinizing hormone (LH)-releasing hormone ethylamide of alpha-subunit and LH beta messenger ribonucleic acid levels in the rat anterior pituitary gland

LHRH and sex steroids play a major and direct regulatory role in the secretion of LH by the anterior pituitary gland. The aim of the present study was to investigate the interactions between sex steroids, more especially the potentiating effect of progesterone (P) in the presence or absence of a low dose of 17 beta-estradiol (E2) and/or dihydrotestosterone (D) on mRNA levels encoding the alpha- and beta-subunits of LH in both female and male rats. We also studied the effect of 2-week treatment with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide on the same parameters. After treatment with the LHRH agonist (5 micrograms daily), the accumulation of mRNA encoding the alpha-subunit was stimulated by approximately 3-fold while the LH beta mRNA concentration remained unchanged. Ovariectomy performed 14 days earlier, increased pituitary alpha and LH beta mRNA levels by 3.7- and 8.8-fold, respectively, while orchiectomy performed 14 days earlier increased alpha and LH beta mRNA levels by 6- and 6.5-fold, respectively. The present data demonstrate that although P alone exerts no effect on alpha and LH beta mRNA levels in castrated animals, treatment with P markedly potentiates the inhibitory effect of E2 on both mRNA levels in female as well as male rats. In addition, P potentiates the inhibitory effect of D on LH beta mRNA levels in castrated female rats. Furthermore, the present study illustrates the importance of the cumulative inhibitory effects of relatively low doses of E2 and D on mRNAs encoding both LH subunits. Moreover, the present observation of a differential modulation of alpha-subunit and LH beta mRNA levels after chronic treatment with an LHRH agonist offers an explanation for the high plasma levels of free alpha-subunit found in patients treated with LHRH agonists.     

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.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

p-Trifluoroacetamidophenyl O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-beta-D-mannopyranoside

p-Nitrophenyl 2-O-benzyl-4,5-O-cyclohexylidene-beta-D-mannopyranoside (4) was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide. The resulting, protected disaccharide was converted into p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-4-O-benzoyl-2-O- benzyl-beta-D-mannopyranoside (8), which was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide to give p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-O -[2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1----6)]-4-O-benzoyl-2-O -benzyl-beta-D-mannopyranoside (9) in 75% yield. Conversion of the p-nitrophenyl group followed by deprotection then yielded the title compound, whose structure was confirmed by 1H- and 13C-n.m.r. spectroscopy.     

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.     

Benzimidazoles as non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 3: Discovery of 1-(1H-benzimidazol-5-yl)-3-tert-butylurea derivatives

1-(1H-Benzimidazol-5-yl)-3-tert-butylurea derivatives have been identified as a novel class of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Herein, we disclose the synthesis and structure-activity relationships (SAR) of this class resulting in the identification of compound 12c, with dual functional activity on human and rat receptors (rat LHRH: IC50=120 nM; human LHRH: IC50=18 nM). These SAR studies suggest that 1-(1H-benzimidazol-5-yl)-3-tert-butylurea is a new pharmacophore for small molecule LHRH antagonists.