Sulfamide antifolates inhibiting thymidylate synthase: synthesis,enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.     

Stereoelectronic Properties of N-acetyl-α,β-dehydroamino acid N′-methylamides

Summary α,β-Dehydroamino acids are useful peptide modifiers. However, their stereoelectronic properties still remain insufficiently recognized. Based on FTIR experiments in the range ofv _s(N-H), AI, AII andv _s(C^α=C^β) and ab initio calculations with B3LYP/6–31G*, we studied the solution conformational preferences and the amide electron density perturbation of Ac-ΔXaa-NHMe, where ΔXaa=ΔAla, (E)-ΔAbu, (Z)-ΔAbu, (Z)-ΔLeu, (Z)-ΔPhe and ΔVal. Each of these dehydroamides adopts a C₅ structure, which in Ac-ΔAla-NHMe is fully extended and accompanied by the strong C₅ hydrogen bond. Interaction with bond C^α=C^β lessens the amidic resonance within the flanking amide groups. TheN-terminal C=O bond is noticeably shorter, both amide bonds are longer than the corresponding bonds in the saturated entities and the N-terminal amide system is distorted. Ac-ΔAla-NHMe constitutes an exception. ItsC-terminal amide bond is shorter than the standard one and both amide systems are ideally planar. Ac-(E)-ΔAbu-NHMe shares stereoelectronic features with both Ac-ΔAla-NHMe and (Z)-dehydroamides.     

Conformational investigation of alpha,beta-dehydropeptides. X. Molecular and crystal structure of Ac-DeltaAla-NMe2 compared with those of Ac-L-Ala-NMe2, Ac-DL-Ala-NMe2 and other dimethylamides.

A series of three homologous dimethyldiamides Ac-DeltaAla-NMe2, Ac-L-Ala-NMe2 and Ac-DL-Ala-NMe2 has been synthesized and the structures of these amides determined from single-crystal X-ray diffraction data. To learn more about the conformational preferences of compounds studied, the fully relaxed (phi-psi) conformational energy maps in vacuo (AM1) of Ac-DeltaAla-NMe2 and Ac-L-Ala-NMe2 were obtained, and the calculated minima reoptimized with the DFT/B3LYP/6-31G** method. The crystal-state results have been compared with the literature data. Ac-DeltaAla-NMe2 and other alpha,beta-dehydroamino acid dimethyldiamides, Ac-DeltaXaa-NMe2 adopt the conservative conformation of the torsion angles phi, psi = approximately -45 degrees, approximately 130 degrees, which are located in the high-energy region (region H) of Ramachandran diagram. Ac-L-Ala-NMe2 and Ac-DL-Ala-NMe2, as well as other saturated amino acid dimethylamides Ac-L/DL-Xaa-NMe2, present common peptide structures, and no conformational preferences are observed. Molecular packing of the amides analysed reveals two general hydrogen-bonded motifs. Dehydro and DL-species are paired into centrosymmetric dimers, and L-compounds form catemers. However, Ac-DeltaAla-NMe2 and Ac-DL-Ala-NMe2 constitute exceptions: their molecules also link into catemers.     

Acetylation of methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate

Acetylation with acetic anhydride of methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate, one of the hetareneamino acids, was studied using HPLC, H NMR, FTIR and GC-MS. The compound has a significantly decreased susceptibility to acetylation compared to 5-amino-1H-[1,2,4]triazole itself. Two isomeric diacetylated products were found.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

Conformational investigation of alpha, beta-dehydropeptides. Part III. Molecular and crystal structure of acetyl-L-prolyl-alpha, beta-dehydrovaline methylamide.

The crystal structure of Ac-Pro-delta Val-NHCH3 was examined to determine the influence of the alpha,beta-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4 degrees in needle-shaped form in orthorhombic space group P2(1)2(1)2(1) with a = 11.384(2) A, b = 13.277(2) A, c = 9.942(1) A, V = 1502.7(4) A3, Z = 4, Dm = 1.17 g.cm-3 and Dc = 1.18 g.cm-3. The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a beta-bend between the type I and the type III conformation with phi 1 = -68.3(4) degrees, psi 1 = -20.1(4) degrees, phi 2 = -73.5(4) degrees and psi 2 = -14.1(4) degrees. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i + 3)th residue stabilizes the beta-bend. An additional intermolecular N...O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single alpha,beta-dehydroamino acid residue in the (i + 2) position and forming a beta-bend reveal a type II conformation.     

Increased LH and FSH release from the anterior pituitary of ovariectomized rat, in vivo, by copper-, nickel-, and zinc-LHRH complexes.

The effect of Cu2+, Ni2+, Zn2+ and their complexes with LHRH on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) was estimated in in vivo experiments with the use of the method proposed by Ramirez and McCann. Ovariectomized, estradiol, and progesterone pretreated rats were injected intravenously either with LHRH alone, a metal ion alone, a mixture of metal and hormone, or a metal-LHRH complex. A metal alone or a mixture of it with LHRH did not affect gonadotropin release at all or no more than LHRH alone. However, the complex of Cu2+ with LHRH brought about a high release of LH and even higher release of FSH. This indicates that copper complex is more effective than metal-free LHRH. The nickel complex showed a similar although lesser effect. The zinc complex had similar potency to free LHRH though higher FSH-releasing ability was noticed. We conclude that copper-, nickel-, and zinc-LHRH complexes were more potent than the peptide hormone itself and promoted the FSH release in the ovariectomized, estradiol, and progesterone pretreated rats.     

Metal complexes of luteinizing hormone-releasing hormone (LHRH). potentiometric and spectroscopic studies

The results are reported of a potentiometric and spectroscopic study of the H+, Cu2+, and Ni2+ complexes of luteinizing hormone-releasing hormone (LHRH, HL) at 25 degrees C and an ionic strength 0.10 mol dm-3 (KNO3), since there is much evidence that the in vivo release of LHRH is influenced by the concentration of copper ions. With Cu2+ the hormone has been shown to behave similarly to the thyrotropin releasing factor, forming a very stable [CuH-1L] complex involving coordination of three nitrogen donors: the Nim atom of the imidazole side chain and the two amido-N atoms of the pyroglutamylhistidyl unit. With Ni2+, coordination proceeds differently to give four nitrogen coordination.     

Synthesis, crystal structure and molecular conformation of the tBuCO-D,L-Ala-delta Z-Phe-NHiPr alpha,beta-unsaturated dipeptide

The crystal structure of the tBuCO-D,L-Ala-delta Z-Phe-NHiPr dipeptide has been solved by X-ray diffraction. The peptide crystallizes in monoclinic space group P2(1)/c with a = 13.445 (3) A, b = 35.088 (4) A, c = 14.755 (3) A, beta = 116.73 (1) degree, Z = 12 and dc = 1.151 g.cm-3. The three independent molecules per asymmetric unit accommodate a beta II-folded conformation, but only one of them contains the typical i + 3----i interaction characterizing a beta-turn. In the other two molecules, the N...O distance exceeds 3.2 A, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the beta II-turn conformation is largely predominant.