Gianfranco Rapi

Structure of the Adduct of Alantolactone with (Z)-L-Cys-Ala-OMe; 1H and 13C Assignment of the Alantolactone Moiety by NMR at 14 T

Analysis of the 1H–1H NOE effects observed for S‐(11,13‐dihydroalantolacton‐13‐yl)‐L‐cysteinyl‐L‐alanine methyl ester in (CD3)2SO at 14 T showed that the 7‐ and 11‐protons are in cis positions. The distances between 7‐, 8‐ and 11‐protons of the adduct were derived by a semi‐quantitative approach based on analysis of NOESY cross peaks and a computed molecular model. The simulated 1H spectrum of the alantolactone moiety is in excellent agreement with the experimental data. The complete assignments of the 13C signals of alantolactone, its peptide adduct and four dipeptide precursors are based on one‐ and two‐dimensional 1H–13C NMR techniques.

GLYCOSYL DERIVATIVES OF NK2 TACHYKININ RECEPTOR ANTAGONISTS

The influence of glycosylation on the structure-activity relationship of the tachykinin antagonist MEN 10376 and of its minimal active fragment MEN 10414 was investigated. The antagonist activity was preserved only when β-d-glucosylated Tyr, Ser and Asn were added to the N-terminal position of MEN 10376, while the modification of the side chain of the Tyr2 induced a dramatic decrease in affinity.

Synthesis of N-acetyl derivatives of 17β-(2-amino-oxazol-4-yl)-steroids and revised structure of N-acetylated 4- or 4,5-disubstituted 2-amino-oxazoles

Chemical and spectroscopic data clearly demonstrate that all the title compounds are acetylated only at the exocyclic nitrogen.

Synthesis of 17β-(2-amino-oxazol-4-yl)-steroids

The reaction of cyanamide with the 20-oxo-21-hydroxy-side-chain of corticosteroids in methanolic aqueous ammonia yields the title compounds.

Suppression of the skin response to alantolactone in alantolactone-sensitized guinea pigs treated with N-acetyl-l-cysteinyl-l-alanine methyl ester

Abstract Induction of a state of tolerance (hyposensitization) in alantolactone-sensitized guinea pigs was attempted by subcutaneous injections of S -(dihydroalantolacton-10-yl)- l -cysteinyl- l -alanine methyl ester 7 , N -acetyl- l -cysteinyl- l -alanine methyl ester 11 or N -acetyl- l -cysteine. Compound 7 was prepared by addition of N -benzyloxycarbonyl- l -cysteinyl- l -alanine methyl ester 5 to alantolactone, followed by the removal of the Z-group. Dipeptide 11 was obtained from N -acetyl- S -ethylcarbamoyl- l -cysteinyl- l -alanine methyl ester. Treatment of dipeptide 5 with HBr-AcOH mixture afforded mainly the S -acetyl derivative 10 , from which dipeptide 11 was also obtained. Biological assays showed that the alantolactone-adduct 7 or N -acetyl- l -cysteine did not significantly modify the positive skin response to alantolactone in alantolactone-sensitive guinea pigs. In marked contrast, dipeptide 11 significantly decreased the skin reaction to alantolactone tested at either 0.25 or 0.08 μg. Control animals did not show skin responses to alantolactone neither after treatment with dipeptides 7,11 or N -acetyl- l -cysteine. The data suggest that dipeptide 11 is an efficient and non-toxic tolerogen in the case of guinea pigs sensitized to alantolactone.

Reaction of some antiinflammatory 17β-(2-aminooxazol-4-YL) steroids with hydrogen peroxide. Synthesis of steroid-17- spiro-5'-oxazolidine-2',4'-diones

The heterocyclic moiety of 17 beta-(2-aminooxazol-4-yl) steroids is sensitive to the oxidizing action of hydrogen peroxide and yields products mainly from the opening of the amino-oxazole ring. Unlike simple 2-aminooxazoles, it does not rearrange to 2-imidazolone and the expected steroidal hydroperoxyimidazolidinones were not detected. Among the substances we isolated, N-(aminocarbonyl)-17 alpha-hydroxy-17-carboxamides (2a) and (3a) undergo spontaneous cyclization, in the reaction conditions, giving steroid-17-spirooxazolidinediones (2d) and (3d). Spirane (2d) was synthesized in high yields from (2a) in strongly alkaline medium.

Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74-85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitisin vivo strongly increased the T cell proliferative responsein vitro. We extended this study to the human immunodominant epitope hMBP(83-99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83-99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited ‘superagonist’ activity.

Amino- and dienamino-derivatives formed from adrenocortical steroids and heterocyclic bases

Pyrrolidine, morpholine, and piperidine react directly with the 20-oxo-17α, 21-dihydroxy-side-chain of corticosteroids to give 21-amino-derivatives. l.r. and n.m.r. data indicate the presence of an internal hydrogen bond between the 17α-hydroxy-group and the C-21 nitrogen atom. The reaction at the 4-en-3-one system leads, as expected, to 3-amino-3,5-dienes. Reactivity at C-3 follows the order pyrrolidine > morpholine > piperidine and can be explained on the basis of resonance theory and steric effects. It is possible to prepare either 21-amino-or 3,21-diamino-derivatives. An s-trans-structure is postulated for the 3,5-diene system; a cross-conjugated system is excluded on the basis of n.m.r. data. Corticosteroids without a 17α-hydroxy-group react at C-21 only, under more drastic conditions. A possible explanation of the function of the 17-hydroxy-group is advanced.

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

The adamantane moiety was introduced in the tachykinin NK2 receptor-selective agonist [β-Ala8]-NKA(4–10) (H-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2, MEN 10210) and in different positions of the NK2 receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) in order to investigate how this substitution affects their biological activity at tachykinin NK1, NK2 and NK3 receptors. 1-Adamantaneacetic acid (1-Ada-CH2COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH2CO-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH2CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH2CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH2CO)-NH2]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate β-COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK1 and NK2 receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK3 receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK1, NK2 and NK3 receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK2 receptors of the rabbit isolated pulmonay artery.

X-ray structure analysis of 3,11,17-trioxoandrostane-5α-carbonitrile and of 17α-ethoxycarbonyloxy-3-oxoandrost-4-ene-17-carbonitrile

The crystal and molecular structures of the title compounds were determined by x-ray diffractometric analysis. Torsion angles and puckering parameters are reported for both compounds. In 1 the 5 alpha-cyano group influences the A-ring conformation. The carbonate ester 3 crystallizes in the monoclinic P2(1) space group with two molecules (I and II) in the asymmetric unit. The D-ring conformation is to some extent different between I and II.