Synthesis and investigation of a new cyclo (Nalpha-dipicolinoyl) pentapeptide of a breast and CNS cytotoxic activity and an ionophoric specificity.

A new acylated cyclopentapeptide namely, Cyclo-( N(alpha)-dipicolinoyl)- bis-[ L-Leu-DL-Nval]- L-Lys OMe (5) was suggested and synthesized. The structural conception of 5 was rationalized by analogy to the structural features of some known cyclodepsipeptides exemplified by the antibiotic and DNA intercalator actinomycin D (NSC: 3053), the ionophore and anti-HIV enniatin B (NSC: 692895) and the ionophore and antibiotic valinomycin (NSC: 630175). The cyclopeptide 5 was chemically synthesized, starting from its linear tetrapeptide ester precursor 2 by coupling L-lysine methyl ester to the prepared tetrapeptide acid 3 or hydrazide 4 via the mixed anhydride or azide method, respectively. A cytotoxic activity (cell killing) in both breast (NCF7) and CNS (SF-268) cell lines NCI, USA) was realized for 5, while less active cytotoxic profile was determined for 2. Moreover, we have recently reported general ionophoric and sensor characteristics particularly, for Pb (II) ions for both 5 and 2. Correlation between the cytotoxic activity and the ionophoric potency is a matter of future investigations.

Optimized conventional synthesis of "RGD" and "RGDS" peptides and their sarcosine mimics as integrin GP IIb/IIIa antagonists.

Synthetic arginyl-glycyl- alpha-aspartyl "RGD" and arginyl-glycyl- alpha-aspartyl-serinyl "RGDS" peptide sequences, which are originally located in matrix proteins, are confirmed to be as versatile integrin GP IIb/IIIa antagonists. Since integrins, as cell surface glycoprotein receptors are implicated in several physiological mechanisms, these peptides are recently specially considered in the design of new therapeutics. Replacing glycine by sarcosine, as its more lipophilic isomer, in RGD peptides seemed, accordingly, interesting in revealing some structural/biological activity relationships. To render "RGD" peptides more conveniently available, an ameliorated quasi-gram yield conventional synthesis in solution of the parent "RGD" and "RGDS" [8, Scheme 1A & 15, Scheme 1B] and their sarcosine analogues, [8', Scheme 1A & 15', Scheme 1B] respectively, is herein described.A compilation of the mild hydrogenolysis removable Z and NO(2) groups and/or the acidiolytic removable Boc group were manipulated for the amino temporary protecting steps. Both the DCCI/HOBt and MA methodologies served well as peptide coupling methods.

Synthesis of a new antischistosomally active and toxicologically tolerant C-12 monothione surrogate of the universal antihelmintic praziquantel.

A new C-12 monothione mimic (III) of the universal antihelmintic Praziquantel (I) namely, 2-cyclohexylthiocarbonyl( 1,2.3,6,7,11b)-hexahydro-4H-pyrazino[2-1a] isoquinoline-4-one was chemically synthesized and structurally elucidated (Elemental analysis. El-Mass, 13C-NMR and IR spectroscopy). Antischistosomal potency in the order of -76% comparable to that for our newly reported C-12 and C-4 dithion mimic II (-70%) and Praziquantel. Praziquantel (100%, mice infected with S. mansoni cercariae), was realized. Toxicological evaluation (mice liver and kidney functions) and biochemical parameters (cholesterol, triglycerides, albumin, total serum proteins and amino acid profile of liver protein homogenate) were also assayed. Comparable to the parent drug, general insignificant toxicological diferences could be attributed for III. Interestingly, III exhibited intermediate biological figures between I and II. An order of IIIII>I, for the other tested biochemical parameters was observed. A consideration of obtained results could indicate that, structurally, an intact glycine amide segment of the pyrazine moiety, as it is the case in both I and III, and not in II (glycine thioamide) seemed now more crucial for exhibiting an optimum antihelmintic potency as well as a more tolerant toxicity characteristics. Additionally, the obtained comparable amino acid profile of mice liver protein homogenate after the treatment by III, could suggest similar biochemical, lethal mechanistic and metabolic routes for II, III and I. The new lipophilic candidatee III seems to merit more profound chemical, biological, and pharmaceutical investigations.

Synthesis and study of the antischistosomal potency and induced biological parameters of a new 2-palmitoyl analogue of the universal antihelminthic praziquantel.

2-Palmitoyl[1,2,3,6,7,11b]hexahydro-4H-pyrazino[2-la]isoquinoline-4-one [III] a highly lipophilic analogue of the universal antihelminthic PRAZIQUANTEL [I] was rationally multi-stepwise synthesized and antischistosomally and biochemically screened. The 2-palmitoyl conjugation was hypothesized to be an antischistosomal adjuvant (Tween 40 mimicry), to the reported crucial pyrazino-isoquinoline moiety. On a constant weight doses bases of I and III (500 mg/kg mouse body weight), the activity of III was found to be approximately 70% of I (mice infected with S. mansoni cercariae) and with satisfactory toxicological and biochemical profile (mice liver and kidney functions). Equivalent molar weight assay (M 1:1.4 for I and III, respectively), which could further plead in favour of the potency of III, was not yet tested. The analogue III, which favourable incorporates the human metabolically and physiologically compatible, palmitic acid segment, seems to be an antischistosomally promising candidate for more integrated studies.

Synthesis and anti-phlogistic potency of some new non-proteinogenic amino acid conjugates of "Diclofenac".

In search for more potent, particularly less ulcerogenic gastritis that hopefully replace the universal NSAID "Diclofenac", (2-[(2,6-dichlorophenyl)amino]-phenylacetic acid, C.A.S. 15307-86-5), twelve new non-proteinogenic amino acid conjugates of the drug, namely that of sarcosine, beta-alanine, D-leucine and D-phenylalanine, were synthesized and biologically screened for their anti-inflammatory, analgesic and ulcerogenic activity in rats. "Diclofenac" amino acid esters (IIa-d), were synthesized via the corresponding HOSu or HOBt active esters. Alkaline hydrolysis (NaOH) followed by acidification (KHSO4) or thioamide formation (Lawsson's Reagent, C.A.S. 19172-47-5), afforded the corresponding free acids IIIa-d or the thioamides IVa-d respectively. Interestingly, in contrary to the parent "Diclofenac", the synthesized candidates (except IIId), were entirely nonulcerogenic in rats. Further, they considerably retained a generalized anti-phlogistic activity. The major "Diclofenac" irritating gastric side effect was thus eliminated. Particularly, the sarcosine conjugate IIa and its thiomimic IVa exhibit promising therapeutic perspectives.

Synthesis and antischistosomal activity of a new 2-nicotinoyl dipeptide analogue of praziquantel.

As an approach to a structure-antischistosomal activity-relationship with possible pharmacological potentiation of the anthelmintic drug praziquantel, a new dipeptide analogue, namely, N alpha-nicotinoyl-L-aspartyl-beta-(1,2,3,6,7,11b-hexahydro-4H-pyrazino[2- 1a]isoquinoline-4-one)-L-phenylalanine methyl ester, was synthesized and antischistosomally investigated in mice infected with S. mansoni cercariae. Parallely, its simple 2-nicotinoyl analogue was synthesized and tested. Both compounds were less, but still, interestingly active (approximately 63 and approximately 66%, respectively) compared to praziquantel (approximately 90%).

Synthesis of 3,5-disubstituted pyridines as antimicrobial agents.

3,5-Diacetylpyridine (1) reacted with hydroxylamine hydrochloride, thiourea or phenylhydrazine affording the corresponding carbaldoximo- (2) aminothiazolyl-(3) and phenylhydrazono- (4) derivatives, respectively. Cyclization of 4 by the action of PPA or thionyl chloride afforded the corresponding indolyl- (5) and thiadiazolyl-(6) derivatives. Condensation of 1 with aldehydes yielded bis-(beta-acryloyl) derivatives 7, which on further treatment with malononitrile or ethyl cyanoacetate afforded cyanopyridines of the types 8 and 9. The latter was successfully utilized in the synthesis of thieno[2,3-b]pyridines 13. Some of the obtained compounds showed remarkable antimicrobial activity comparable to oxytetracycline.

Synthesis of inhibitors of the meso-diaminopimelate-adding enzyme from Escherichia coli.

In order to obtain inhibitors of the meso-diaminopimelate-adding enzyme, which participates in the biosynthesis of bacterial peptidoglycan, several N alpha-propionyl-dipeptides of the general formula Pr-L-Ala-ambo-Xaa-OH were synthesized. Xaa represented methionine S,S-dioxide, methionine S-oxide, methionine sulfoximine, and 2-amino-4-phosphonobutyric acid, i.e. transition state analogs of glutamine synthetase and gamma-glutamyl-cysteine synthetase, which catalyze the same type of reaction as our target enzyme. After synthesis, the diastereoisomers were separated by preparative HPLC or t.l.c.; those containing methionine derivatives could be identified thanks to previously synthesized reference compounds. After preincubation with the meso-diaminopimelate-adding activity from Escherichia coli, the LD diastereoisomers displayed moderate inhibitory effects, whereas the LL ones were inefficient. The best inhibition was obtained with one diastereoisomer of Pr-L-Ala-zeta-2-amino-4-phosphonobutyrate, presumably the LD one. A chloromethylketone derivative Pr-L-Ala-D-Glu(CH2Cl)-OH, potential affinity labeler of the meso-diaminopimelate-adding enzyme, was also synthesized. In the assay with preincubation, this compound behaved as the best inhibitor.

Specificity of the uridine-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate synthetase from Escherichia coli.

To investigate the specificity of the uridine-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate synthetase, various compounds mimicking more or less different parts of the UDP-MurNAc-L-Ala-D-Glu substrate were prepared. Their size ranged from that of uridine or L-Ala-D-Glu to that of the whole nucleotide substrate. Chemical synthesis led to N alpha-acyl-dipeptides, in which the acyl group mimicked the MurNAc moiety, and to glycopeptides MurNAc(alpha or beta-Me)-L-Ala-D-Glu, in which the anomeric function is blocked. Partial degradation or chemical modification of the substrate UDP-MurNAc-L-Ala-D-Glu afforded: MurOHNAc-L-Ala-D-Glu, P1-MurNAc-L-Ala-D-Glu, and DDP-MurNAc-L-Ala-D-Glu (DDP = dihydrouridine-diphosphate). All these compounds were tested as substrates or (and) inhibitors of the reaction catalyzed by the A2pm-adding enzyme, which, after partial purification, was obtained in two active forms. Among the compounds tested as substrates, only DDP-MurNAc-L-Ala-D-Glu was a good one. The Km for this compound was 97 microM versus 55 microM for the natural substrate. Among the various compounds tested as inhibitors, only P1-MurNAc-L-Ala-D-Glu and MurNAc(alpha or beta-Me)-L-Ala-D-Glu had a significant inhibitory effect at 1mM. Apparently, no particular portion of the molecule is predominantly responsible for its recognition by the enzyme. In other words, multiple sites located over the whole molecule are required for a proper recognition and determine the high specificity of this activity. Therefore, to obtain efficient competitive inhibitors it is necessary to synthesize molecules very similar in size and structure to the natural substrate.