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BACKGROUND: In the light of the recent findings concerning the role of apoptosis and of tumor cell enzymes in cancer chemotherapy, the interest in pyrimidine derivatives has greatly increased. Thio- and hydrazine- pyrimidines were synthesized as potential antimetabolites inhibiting the biosynthesis of nucleic acids. Some of them demonstrated biological activity, including antibacterial and antitumor action. The aim of this study was to analyze the cytotoxic activity and the ability of some derivatives to induce apoptosis in murine leukemia cells. METHODS: Exponentially growing cells were incubated with compounds and after 24, 48, and 72 hours were stained with trypan blue and counted hemocytometrically. For detection of the cell fraction undergoing apoptosis, a morphological analysis was made using fluorescent dye propidium iodide. RESULTS: Eight thio- and hydrazine- pyrimidine derivatives were investigated. 2-Thiouracil and 6- hydrazinouracil did not influence the cell growth. 2,4-dithiouracil, 2-thio-4-hydrazinouracil, 2-hydrazinouracil, and 2-thio-5-fluorouracil decreased cell proliferation, but even at the highest studied concentration (1000 µM) had no cytostatic action. Only high concentrations of 2,4-dihydrazinouracil and 2- chloro-4-hydrazinouracil showed a strong cytotoxic activity. The treatment with 2,4-dihydrazinouracil as well as with 5-fluorouracil caused the appearance of apoptotic cells with typical fragmented condensed nuclei, ghosts and apoptotic bodies. In contrast, dead cells treated with 2-chloro-4-hydrazinouracil did not show apoptotic morphology. CONCLUSION: Among studied eight thio- and hydrazine- pyrimidine derivatives only 2,4-dihydrazinouracil demonstrated strong apoptogenic activity. Its active concentrations were about 100 times higher than apoptogenic concentrations of 5-fluorouracil which points to different mechanisms of cytotoxic action.

Novel 5-ring heterocyclic building blocks are synthesized. These can be incorporated into analogs of peptide antibiotics such as microcin B17, which is a potent DNA-gyrase inhibitor that exhibits eight thiazole and oxazole moieties. In particular, the syntheses of imidazole and bisoxazole amino acids as novel peptidomimetics are reported, this includes a new procedure for the oxidative conversion of the intermediates oxazoline, imidazoline as well as oxazole-oxazoline into the corresponding heteroaromatic compounds. A mixture of 1,8-diazabicyclo-[5.4.0.]-undec-7-ene carbon tetrachloride/acetonitrile and pyridine proved to be a very effective and mild agent.

New 3'-, 5'-, 5-bromo-2'-deoxyuridine (3a-g) and 3'-, 5'-thymidine (4a-i) analogues with amino acid and peptide residues were synthesized and evaluated for antiviral activity. The influence of long peptide chains, essential amino acids and the effect of this structural modification on the antiviral activity has been also reported. Three 5-bromo-2'-deoxyuridine derivatives containing glycyl-, glycyl-glycyl- and glycyl-glycyl-glycyl- residues (3a, 3b, 3c) showed a strong activity against the herpes virus PsRV and a moderate one vs. HSV-1. The corresponding thymidine analogues were considerably less effective, and only compounds 4d and 4h showed a borderline effect against PsRV.

The inhibitory effect of a series of 5'-O-amino acid and oligopeptide derivatives of uridine on rat liver UDP-glucuronosyltransferase (UGT) activities was investigated using two assay systems. A quantitative structure-activity relationship (QSAR) study was performed. The compounds include a lipophilic residue linked to the nucleoside by a variable spacer. Moreover, half of the derivatives have two spacers linked to the uridine moiety. Compound 1, a serine derivative of isopropylideneuridine, was found to be the most potent inhibitor of both 4-nitrophenol (4-NP) and phenolphthalein (PPh) glucuronidation, with an I50 of 0.45 mM and 0.22 mM, respectively. Kinetic studies with this substance revealed a mixed type of inhibition towards 4-NP and UDP-glucuronic acid, with apparent Ki values of 150 microM and 120 microM, respectively. The dipeptide derivatives 11-14 exhibited a low activity against 4-NP conjugation. However, a marked suppression of PPh glucuronidation was found with compounds 11 and 13. Generally, compounds with two spacers are more inhibitory against the UGT activities studied. The QSAR analysis outlined the significance of the spacers with a minimum length of 5 atoms and lipophilic residues linked to them for the inhibitory effect of the compounds. The most significant contribution to this effect is given by the six-atom spacer for both 4-NP and PPh substrates. 4-NP converting UGT isoforms seem to respond more specifically to the inhibitors: a five-atom for the first and six-atom for the second spacer enhance binding to both 4-NP and PPh conjugating isoenzymes, while a long second spacer contributes to inhibitor binding to UGT isoforms only converting PPH.

A study of quantitative structure-activity relationships (QSAR) in a series of carboxylic acid bis(2-chloroethyl)aminoethyl esters with potential antitumor activity was carried out. The statistical-heuristic technique was applied to estimate the contributions of the structural features of the compounds to the probability of their activity in vivo against lymphoid leukemia L1210. The results obtained were compared with those of the pharmacological screening of the esters. Some assumptions are made concerning the difference in antileukemic activity of compounds based on the computed weights of their structural features.

The in vitro inhibition of purine biosynthesis de novo by a series of cyanopyrazoles was studied. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. GAR synthesis was inhibited at a lower degree at the same range of concentrations. The compounds demonstrated a similar pattern of inhibition of the last steps, e.g. AICAR formylation and cyclization as found on the whole pathway.

The interaction of three spin-labelled compounds, derivatives of bis-(2-chloroethyl)-hydrazine (HMSL), N-methyl,N-chloroethyl-hydrazine (MCEHSL) and bis-(2-bromoethyl)-hydrazine (BEHSL) with DNA was studied by the method of electron paramagnetic resonance (EPR). It was found that HMSL (containing two chloroethyl groups) and MCEHSL (containing one chloroethyl group) gave spin-labelled dsDNA with identical strongly immobilized EPR spectra. The conclusion was drawn that only one of the alkylating groups of HMSL reacted with DNA. In contrast, the EPR spectrum of DNA spin-labelled with BEHSL was non-immobilized due to the strong destabilizing effect of this compound on the double helix. The extent of alkylation of DNA with the three hydrazine mustard derivatives was one and the same. It was found, however, that chloroethyl-containing derivatives (HMSL and MCEHSL) had an expressed base specificity and alkylated preferably the guanilic residues, and their bromo-analogue (BEHSL) did not show any base specificity and alkylated the bases of DNA at random.

The antitumor effect of some N alpha-benzyloxycarbonyl-N,N-bis-(2-halogenethyl)hydrazide derivatives of lysine, glycine, cystine, phenylalanine and p-chlorophenylalanine, was studied. Six of eight newly synthesized compounds show considerable antitumor effect on solid Walker carcinosarcoma 256 (about 95% tumor growth inhibition). Three of these compounds under study increased the lifespan of mice with leukemia L1210. The investigation of the effect of N alpha-benzyloxycarbonyl,D,L-phenylalanine-N,N-bis(2-chloroethyl)hydrazine on various mouse tumors showed remarkable growth inhibition of the Ehrlich ascites carcinoma, sarcoma 37, colon adenocarcinoma akatol and lesser antitumor effect also on solid adenocarcinoma 755, Lewis lung carcinoma and melanoma B16. All investigated compounds exhibited depression of leukocyte count--their toxicity being, however, lower than that of sarcolysine in parallel experiments.

The chemical synthesis of certain mono- and bis-dialkyltriazenopyrazoles is described. In antitumor studies it was found that none of the compounds produced increase in life span (ILS) of L 1210 bearing mice or inhibition of adenocarcinoma 755 growth above the criteria established. The introduction of a second triazenogroup increases the toxicity of the compounds tested.

Using the method of twofold serial dilution of substances in liquid nutrient medium with subsequent reseeding on solid nutrient medium a comparative study has been carried out of the antibacterial action in vitro of long chain saturated (C10-C18) and unsaturated (C11-C22) carboxylic acids and their bis(2-chloroethyl)aminoethyl esters against Pseudomonas aeruginosa, Klebsiella pneumoniae tp 16 Koph, Escherichia coli ATCC 25922, Staphylococcus aureus 101+ and Proteus mirabilis 56. The compounds studied have an inhibitory effect upon the development of the microorganisms. The minimum inhibitory concentration ranges from 1.8 to 19.3 mumol/ml. There exists a linear correlation between the length of the carbon chain (the number of C atoms) of the compounds and the minimum inhibitory concentration in the following cases: esters of saturated acids (C16-C24) and unsaturated acids (C11-C22) against Pseudomonas aeruginosa; unsaturated acids (C11-C22) against Klebsiella pneumoniae tp 16 Koph; esters of unsaturated acids (C17-C28) against Staphylococcus aureus 101+. This correlation has not been found in all other compounds against Escherichia coli ATCC 25922 and Proteus mirabilis 56.

It is shown that L-cystine-bis-(N,N-beta-chloroethyl)-hydrazide-hydro-bromide possesses strong (50-100%) inhibitory effect in vivo against myeloma P-8, carcinosarcoma Walker, lymphosarcoma Pliss, sarcoma Yoshida, sarcoma Jensen and sarcoma 180 in doses 5-12 mg/kg/day. No suppression of the growth of Ehrlich ascites tumor was observed. The acute toxicity (LD50) of this substance on mice and rats is 71 mg/kg and 47 mg/kg respectively.

Using a direct protentiometric method the kinetics of the intramolecular cyclization of 12 synthesized bis(2-chlorethyl)-aminoethyl esters of carboxylic acids has been studied. To determine the rate constants of hydrolysis (in 50% water-ethanol medium) the model of successive reactions of first order A----B----C has been used. An attempt has been made to relate the values obtained for the rate constants with the chemical structures of the compounds studied. The kinetic characteristics have been compared to the data of the antileucemic activity of the compounds against tumor models in vivo.

The effect of several derivatives of ureidosuccinic acid on the growth of Escherichia coli 387, Staphylococcus aureus strain 209 and its mutant UV-2, Sarcina lutea, Candida tropicalis and Neurospora crossa 9863 as pre-screening systems for antitumor activity was studied. It was found that dihydrazide of D,L-ureidosuccinic acid (DHUA) had a marked antibacterial activity. The inhibitory effect of DHUA on N. crassa could not be removed by aspartic acid, ureidosuccinic acid, dihydroorotic acid, orotic acid, uracil or cytosine. DHUA suppressed the growth of Myeloma P-8 by 38%, that of Sarcoma 180 by 12% and that of Yoshida sarcoma by 19%. No effect was found on the growth of Lymphosarcoma Pliss.

The cytostatic activity of 2-thio-4-hydrazinouracil (THU) on some transplantable tumors has been studied. A strong effect of this compound (between 60% and 100% suppression) has been found in the case of Myeloma P-8 (MOPC-21) and Sarcoma 180 (Crocker). A less pronounced effect has been observed on Yoshida sarcoma, while the development of Jensen sarcoma is not influenced.

A series of bis-beta-chlorethyl-, dimethyl- and diethyltriazenopyrazoles (I-IV) were synthesized. By the method of IR spectroscopy the most probable transformation form of I was shown. The compounds were tested as antibacterial agents on a series of bacterial species. It was established that only one of them (VI) possessed a low inhibitory effect. All the rest inhibited strongly the growth of Staphylococcus aureaus 209 and E. coli 387.