New antitumoral cyclopeptides

Summary Chlamydocin is a powerfulin vitro antitumoral agent, quickly inactivatedin vivo. A series of cyclic tetrapeptides related to chlamydocin or HC toxin and bearing a bioactive alkylating group on an-amino-lysyl function have been examined for their antitumoral activity on L1210 and P388 murine leukemia cell lines. One analog was found to be potent at inhibiting L1210 cell proliferation and had a higher therapeutic index than the reference compound bis--chloroethylnitrosourea on thein vivo P388-induced leukemia model.     

Rational design, synthesis, and in vivo evaluation of the antileukemic activity of six new alkylating steroidal esters

The synthesis and the in vivo evaluation against leukemias P388 and L1210 of six new alkylating steroidal esters are described. The esteric derivatives incorporating the 17β-acetamido-B-lactamic steroidal skeleton exhibited increased antileukemic activity and lower toxicity, compared to the 17β-acetamido-7-keto analogs. Among the 17β-acetamido-B-lactamic steroidal esters, the most potent compound afforded four out of six cures in leukemia P388 and was measured to be almost non-toxic, producing significant low levels of toxicity.     

In vivo growth kinetics of P388 and L1210 leukemias

The P388 lymphocytic leukemia and the L1210 lymphoid leukemia are used as test systems for putative cytotoxic drugs. These leukemias are also used to investigate the perturbation of cell cycle progression of various chemical compounds in more detail. There is little information on the normal growth kinetics in vivo of these leukemias. In the present report we therefore present the results from growth kinetic studies of P388 and L1210 leukemic cells growing in ascites form in mice. We used 3H-TdR autoradiography, DNA flow cytometry and the stathmokinetic method. During exponential growth both leukemias showed a growth fraction of unity. Whereas no significant cell loss was observed during the early growth phase of P388 cells, cell loss was indicated by a discrepancy between potential and actual doubling times during exponential growth of L1210 cells. During the phase of growth retardation, the proportion of G1 and G2 cells increased at the expence of a reduced S phase fraction in the P388 leukemia, whereas only small changes in cell cycle distributions were seen with time after inoculation of L1210 cells. An increasing discrepancy in the reduction of the S phase fraction and the 3H-TdRLI was seen in the P388 cells with time after inoculation. Thus, a majority of P388 cells with S phase DNA content were unlabelled during the late phase of growth restriction, indicating resting cells in S phase. A good correlation was found between the 3H-TdR LI and S phase fraction throughout the life history of L1210 cells, revealing considerable differences in in vivo growth kinetics between the two leukemias. Such differences should be considered when evaluating test results.     

Shikimic acid complexes of platinum. Preparation, reactivity, and antitumor activity of (R,R-1,2-diaminocyclohexane) bis(shikimato) platinum(II). Evidence for a novel rearrangement involving platinum-carbon bond formation

The complex (R,R-1,2-diaminocyclohexane)bis(shikimato)platinum(II) (shikimato = the anion of 3R,4S,5R-trihydroxy-1-cyclohexene-1-carboxylic acid), I, has been synthesized and purified by high performance liquid chromatography (HPLC). The complex is only moderately stable in aqueous solution. Its major hydrolysis product, also purified by HPLC, is proposed to be a unique complex type in which a single shikimate group is coordinated through both the carboxylate oxygen and the C(2) vinylic carbon of the shikimate moiety [Pt(R,R-dach)(O,C-shikimato)], II. In vitro, complex I is active against L1210 leukemia and against an L1210 cell line with acquired resistance to cisplatin. In vivo, the complex is active against L1210, P388, and B16 melanoma; this activity is highly schedule-dependent. Complex II is also active against L1210 leukemia.     

Factors responsible for immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The serum of mice hyperimmune to L1210 leukemia was cytotoxic to L1210 cells and, to a much lesser extent, to P388 cells in the presence of complement. However, it did not suppress in vitro growth of L1210 cells, nor did it endow a recipient mouse with immunity to inoculated L1210 cells. This indicates that the serum did not play a significant role, if any, in immune protection of hyperimmune mice.Spleen and peritoneal exudate cells of hyperimmune mice suppressed the in vitro growth of L1210 but not of P388 cells. This is consistent with the fact that hyperimmune mice did not survive the inoculation of P388 cells. The immunocytes failed to suppress the in vitro growth of L1210 cells when preincubated with anti-Thy-1.2 antisera and complement. This, together with the finding that cell populations not adherent to a plastic dish suppressed in vitro growth of L1210 cells, indicates that T cells of immune spleen and peritoneal exudate cell populations were the effectors that suppressed in vitro growth of L1210 cells. Hyperimmune mice lost their immune protection in vivo following the administration of anti-thymocyte antisera, but not with carrageenan or silica, which resulted in the lethal growth of the inoculated L1210 cells. This indicates that T cells were in vivo effectors in immune protection.Hyperimmune spleen T cells endowed a recipient with immunity to L1210 leukemia when transferred in vivo. This confirmed the above results and suggests the applicability of immune cells in an adoptive immunotherapy approach.     

Cytometric study of the effects of destruxin E on leukemic cells in mice

The activity of destruxin E, a cryptogamic toxin isolated from the hyphomycete Metarhizium anisopliae, was studied on mouse leukemia cells (L 1210 and P 388) in culture. Besides a cytotoxic effect, a cytostatic effect (increase of diplo?d cells proportion) was observed with all doses for P 388 (from 10 micrograms/ml to 0.001 microgram/ml) and to a concentration of 0.1 microgram/ml for L 1210. At the lower doses, the effect on cellular DNA content appears distinctly.     

Some pharmacological activities of novel adenine-related compounds isolated from a marine sponge Agelas mauritiana

Agelasimine A and agelasimine B, two novel compounds related to adenine, have been isolated from the orange sponge, Agelas mauritiana, and have been tested for a variety of biological activities. Both compounds inhibited proliferation of cultured L1210 leukemia cells at nanomolar concentrations with accumulation in the G1 stage of the cell cycle. However, no prolongation of life was observed in mice bearing P388 leukemia treated with these compounds. In the rat isolated aorta, micromolar concentrations of agelasimines were very effective in inhibiting contractions elicited by potassium chloride but had little or no effect on responses for prostaglandin F2 alpha and had modest effects on the responses to noradrenaline and significant effects on 5-hydroxytryptamine. Agelsamines A and B appeared to be equipotent in causing relaxation in rabbit jejunum and bovine coronary artery, and they also inhibited nucleoside transport into rabbit erythrocytes in micromolar concentrations.     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Synthesis, structures and antitumor activity of the first crown ester-linked bipyridyl platinum complexes.

Three new crown ester-linked bipyridine homologs with three, four or five ethylene glycol units, which are bulky and soluble in both hydrophilic and lipophilic media, were synthesized. The reaction of the appropriate macrocycles with K2PtCl4 in water gave yellow cisplatin analogs in good yield. These complexes were converted to carboplatin analogs by exchange of the leaving group. All the compounds were characterized by elemental analysis and various spectroscopic methods. Carboplatin analogs showed good solubility in both hydrophilic and lipophilic media. The crystal structure of 2c, the carboplatin analog with macrocycles containing five ethylene glycol units, was determined by X-ray diffraction: space group P1, a = 9.798(1), b = 12.580(3), c = 13.945(2) A, alpha = 108.61(2), beta = 94.59(1), gamma = 97.42(2) degrees, Z = 2, R = 0.0618. Some of platinum complexes showed a moderate cytotoxic effect on both murine leukemia L1210 and P388 even though they do not have any NH proton.     

Effect of schisanhenol on the antitumor activity of adriamycin

Schisanhenol (Sal) did not diminish the antitumor activity of adriamycin in mice bearing P388 ascites tumor. Sal did not antagonize the suppressive effect of adriamycin on DNA synthesis and cell proliferation in an L1210 ascitic tumor cell culture. Furthermore, Sal at the concentration of 0.1, 0.25, or 1 mM accelerated adriamycin-dependent DNA damage in the presence of Fe3+ in vitro. It appears that Sal was able to protect against adriamycin induced heart mitochondrial toxicity, while it did not antagonize the antitumor activity of adriamycin.     

Synthesis and inhibition of cancer cell proliferation of (1,3')-bis-tetrahydroisoquinolines and piperazine systems

Some (1,3')-bis-tetrahydroisoquinolines were reported as scaffold intermediates for the synthesis of pentacyclic piperazine core alkaloids and their cytotoxicity against cancerous cell lines was evaluated. The NMR and X-ray structural assignments revealed an anti C3-C11 backbone stereochemistry of piperazine structures. Inhibition of cancer cell proliferation of (1,3')-bis-tetrahydroisoquinoline scaffolds and pentacyclic piperazine systems was assessed against three human cancer cell lines (K562 myelogenous leukemia, A549 lung carcinoma, MCF-7 breast adenocarcinoma) and both mouse tumor cell lines of blood (P388) and lymphocytic (L1210) leukemia with considerable activity against the latter. The cell cycle analysis was also studied by flow cytometry measurement on K562 cell line.     

Synthesis and study of spin-labeled nitrosoureas

For the first time we have synthesized spin-labeled nitrosoureas and have studied their properties--reduction of the iminoxyl group by vitamin C leading to the formation of the corresponding hydroxylamine derivatives and degradation in the presence of an aminoradical, leading to biradicals. The ESR spectra of biradicals in methanol have nine hyperfine resonance lines. The spin-labeled nitrosoureas have shown a high antitumor activity against the L 1210 lymphoid leukemia and P 388 lymphocytic leukemia in BDF1 mice. A study of a broad range of transplantable tumors is in progress.     

A comparison of the Antileukaemic Effects of Recombinant Human Tumour Necrosis Factor-alpha and its Muteins on Leukaemia L1210 and Leukaemia P388 in Mice

We investigated the influence of recombinant human tumour necrosis factor alpha (TNF-alpha) and its derivatives termed muteins III, V, VI-in which the first 3 to 7 amino acids of native TNF-alpha have been replaced-on the survival time of mice inoculated with leukaemia L1210 or leukaemia P338. TNF-alpha prolonged the survival of mice with leukaemia L1210 but did not have any therapeutic activity in leukaemia P388-bearing mice. Muteins-treated mice with leukaemia P388 lived longer than animals receiving TNF-alpha, while those inoculated with leukaemia L1210 did not show any significant prolongation of life compared with the TNF-alpha treated group. The results presented in this report indicate that the antileukaemic activity of TNF-alpha is governed at least in part by the nature of the N-terminal amino acids.     

Novel Bioactivities from a Coral, Galaxea fascicularis: DNase-like Activity and Apoptotic Activity Against a Multiple-Drug-Resistant Leukemia Cell Line

From the coral Galaxea fascicularis, a crude mucus-like extract (MS) and subsequently its purified component (P6) appear to contain a DNase-like activity that indiscriminately digested λDNA, as well as naked genomic DNAs isolated from a multiple-drug-resistant murine leukemia cell line, P388/VCR, and a nontransformed liver cell line, BL8L. However, MS and P6 specifically induced in situ DNA digestion in cultured P388/VCR cells from 30 minutes onward. After 3 days of incubation with MS or P6, DNA degradation coincided with complete killing of P388/VCR. In situ fluorescent labeling of fragmented DNA revealed that P6 induced apoptosis of P388/VCR cells, occurring as early at 1.5 hours. By day 3, all the P6-treated leukemia cells were apoptotic. In contrast, P6 caused neither in situ DNA digestion, nor apoptosis in the untransformed BL8L cells. Whether the DNase-like action of P6 is independent of or responsible for triggering the intrinsic endonuclease activity in the leukemia cell, thus leading to apoptosis, remains an object for further research. Nevertheless, the specificity of the apoptotic action of P6 on P388/VCR cells indicates its potential role in the development of an anticancer agent. Received July 6, 1998; accepted December 21, 1998     

Synthesis and biological evaluation of 2,7-Dihydro-3H-dibenzo[de,h]cinnoline-3,7-dione derivatives,a novel group of anticancer agents active on a multidrug resistant cell line

A series of anthrapyridazone derivatives with one or two basic side chains at various positions in the tetracyclic chromophore have been synthesized. The key intermediates in the synthesis are 2,7-dihydro-3H-dibenzo[de,h]cinnoline-3,7-diones 1, 12 and 15 monosubstituted at position 2 (4d, 16a-e), or 6 (2a-f) or disubstituted at positions 2 and 6 (4a-c) or 2 and 8 (17a-e) with appropriate alkylaminoalkylamines. All analogues showed in vitro cytotoxic activity against murine leukemia (L1210) and human leukemia (K562) cell lines. The compounds were also active against human leukemia multidrug resistant (K562/DX) cell line with resistance index (RI) in the range 1-3 depending on the compound's structure. Two of the most active in vitro compounds 4a and 11 were tested in vivo against murine P388 leukemia and displayed antileukemic activity comparable with that of Mitoxantrone. DNA-binding assays were performed and DNA affinity data were correlated with the structures of the compounds. The cytoplasmatic membrane affinity values (log k'(IAM)) have also been determined and the correlation with the resistance indexes discussed. The anthrapyridazones constitute a novel group of antitumor compounds that can overcome multidrug resistance.     

Alteration in p53 pathway and defect in apoptosis contribute independently to cisplatin-resistance

The accumulation of molecular genetic defects selected during the adaptation process in the development of cisplatin-resistance was studied using progressive cisplatin-resistant variants (L1210/DDP2, L1210/DDP5, L1210/DDP10) derived from a murine leukemia cell line (L1210/0). Of these cell lines, only the most resistant L1210/DDP10 was cross-resistant to etoposide and deficient in apoptosis induced by these two drugs, indicating that resistance to DNA-damaging agents correlates with a defect in apoptosis. This defect was tightly associated with the loss of a Ca2+/Mg2+-dependent nuclear endonuclease activity present in the less cisplatin-resistant cells. Evidence is presented that p53-dependent function (a) is lost not only in the apoptosis defective L1210/DDP10 cells, but also in the apoptosis susceptible L1210/DDP5 cells; (b) is unrelated to drug-induced cell cycle perturbations. These results suggest that deficiency in the p53 pathway and resistance to DNA-damaging agents due to a defect in apoptosis are independent events.     

Antitumor effect of lysine-isopeptides

Isopeptides (ε-peptides) of lysine, with a given Mw and low polydispersity (10–400 units), were synthesized to study the relationship between their chemical structure and biological effect. The designed compounds were of high purity, low polydispersity and high stereochemical purity. The effect of the compounds was tested on a human erythroleukemia cell line (K-562) and on four transplantable mouse tumors (L1210 lymphoid leukemia, P38 macrophage derived tumor, Ehrlich ascites carcinoma, Lewis lung tumor /LLT/). In case of the L1210 and P388 tumors and the Ehrlich carcinoma, survival of the animals was used as an indicator of the effect. In case of the Lewis lung tumor, the number and size of metastases in the lung and/or liver of treated and untreated mice were used as indicators. The polymers of polymerisation degree 80–120 (Mw 10.2–15.4 KD) showed the strongest antiproliferative effect both on K562 cells and the tumors growing in vivo. This effect was manifest with a significantly higher survival rate as compared to the control (L1210, P38, Ehrlich ascites), furthermore, by a decrease in the number and size of liver and lung metastases (LLT).     

Dinuclear palladium(II) complexes containing two monofunctional [Pd(en)(pyridine)Cl] units bridged by Se or S. Synthesis, characterization, cytotoxicity and kinetic studies of DNA-binding

Two novel dinuclear palladium(II) complexes, {[Pd(en)Cl]₂(bpse)}(NO₃)₂ (1) and {[Pd(en)Cl]₂ (bpsu)}(NO₃)₂ (2), (where en is ethylenediamine; bpse is bis(3-methyl-4-pyridyl) selenide; bpsu is bis(3-methyl-4-pyridyl) sulfide) have been synthesized. The complexes have been characterized by elemental analysis, IR, ¹H NMR, and ¹³C NMR. They have been assayed for antitumor activity in vitro against the mice leukemia L1210 and the human coloadenocarcinoma HCT8 cell lines. The results show that compound 1 has a lower I.D.₅₀ value against the two cancer cell lines as compared to compound 2; the compounds also shows a lower I.D.₅₀ value than cisplatin against the HCT8 cell line, but a higher I.D.₅₀ value than cisplatin against the L1210 cell line. Binding studies indicate that compound 1 possibly interacts with DNA by a nonintercalative mode. Kinetics of binding of the two compounds to DNA are firstly studied using ethidium bromide as a fluorescence probe with stopped-flow spectrophotometer under pseudo-first-order condition. The stronger binding of two steps in the process of the compounds interacting with DNA are observed, and the k_obs and E_a of binding of the two steps (where k_obs is the observed pseudo-first-order rate constant, E_a is the observed energy of activation) are obtained.     

Synthesis and Biologic Evaluation of 8-Substituted Derivatives of Nebularine (9-β-D-Ribofuranosylpurine)

Abstract

A series of 8-substituted purine ribonucleosides were prepared from 2′, 3′, 5′-tri-O-acetyl-8-bromoadenosine and evaluated for cytotoxicity and antiviral activity. Four of these nucleosides (6b-9b) were significantly toxic to both HEp-2 and L1210 cells in culture but the most cytotoxic one (9b) was inactive against the P388 leukemia in mice. None of these nucleosides showed significant antiviral activity against Herpes Simplex 1 or 2, vaccinia, or influenza A.