L. H. Li

Adozelesin, a selected lead among cyclopropylpyrroloindole analogs of the DNA-binding antibiotic, CC-1065

SummaryAdozelesin (U-73975) is a potent synthetic cyclopropylpyrroloindole (CPI) analog of the cytotoxic DNA-binding antibiotic, CC-1065. In contrast to the natural product, adozelesin and related CPI analogs do not cause delayed death in non-tumored mice. Adozelesin, selected from a series of analogs for its superior in vivo antitumor activity and ease of formulation, is highly active when administered i.v. against i.p.- or s.c.-implanted murine tumors, including L1210 leukemia, B16 melanoma, M5076 sarcoma, and colon 38 carcinoma, and produces long-term survivors in mice bearing i.v.-inoculated L1210 and Lewis lung carcinoma. Modest activity is shown against the highly drug-resistant pancreas 02 carcinoma. Adozelesin is also highly effective against human tumor xenografts s.c.-implanted in athymic (nude) mice, including colon CX-1 adenocarcinoma, lung LX-1 tumor, clear cell Caki-1 carcinoma, and ovarian 2780 carcinoma. Its broad spectrum of in vivo activity compares favorably with three widely used antitumor drugs, i.e. cisplatin, cyclophosphamide, and doxorubicin. Adozelesin appears to be more effective than these drugs in the treatment of very resistant tumors such as s.c.-implanted mouse B16 melanoma, pancreatic 02 carcinoma, and human colon CX-1 and human lung LX-1 tumor xenografts. Based on its high potency and high efficacy against a broad spectrum of experimental tumors, adozelesin was chosen for clinical investigation and development.

Mechanism of action of didemnin B, a depsipeptide from the sea

With the brush border membrane vesicles prepared from the rat kidney cortex, didemnin B and its parent compound, didemnin A function neither as a K+-specific ionophore nor as an ionophore for Na+ ions while other depsipeptide antibiotics such as valinomycin and gramicidin promote transmembrane movement of K+ and Na+ ions, respectively. Didemnin B inhibits protein synthesis and DNA synthesis much more than RNA synthesis and is in general more potent than didemnin A. Time course studies reveal that the action of didemnin B is rapid and cannot be reversed after 2 h in contact with the cells. The inhibition of protein synthesis is almost superimposable to that of L1210 cells growth. DNA synthesis is also markedly inhibited. These results collectively suggest that didemnin B acts differently, at least in part, from other depsipeptide antibiotics and its biological effect is primarily mediated through its inhibition of protein synthesis and to a lesser extent its inhibition of DNA synthesis.

Structure and activity relationship of several novel CC-1065 analogs

SummaryCC-1065 was found to cause delayed toxicity at therapeutic doses, therefore, a large number of analogs have since been synthesized. A series of analogs with simplified but closely related structures were chosen for this investigation because some were found to be superior to CC-1065 in the treatment of several experimental tumors. The inhibition of L1210 cell growth by U-68,415 was comparable to that by CC-1065. A similar situation was true in terms of their in vivo potency; however, U-68,415 was superior to CC-1065 in terms of anti-P 388 leukemia activity. At the optimal dosage, U-68,415 produced 4 out of 6 long-term (> 30 day) survivors; whereas CC-1065 produced a mere 62% increase of life span (ILS) and no long-term survivors. The order of antitumor potency and effectiveness of the CC-1065 analogs was U-68,415 > U-66,694 > U-68,819 > U-66,664, which was parallel to the inhibition of L1210 cell growth. CC-1065 and all the analogs tested here inhibited DNA synthesis approximately 10 times more than RNA synthesis. Protein synthesis was the least inhibited. On a molar basis, U-68,415 was about 6–9 times more inhibitory toward cellular DNA synthesis than CC-1065, yet the interaction and/or binding of CC-1065 to DNA determined by circular dichroism, DNA melting or differential cytotoxicity assay was much stronger than that of U-68,415. The order of binding of these analogs to calf thymus DNA was U-68,415 > U-66,694 > U-68,819 > U-66,664, and was parallel to that of DNA synthesis inhibition which was in turn parallel to cell growth inhibition and antitumor potential. These results collectively suggest that the cellular DNA is a major site of the action of CC-1065 analogs; however, time course studies reveal that the inhibition of cellular DNA synthesis could not wholly account for their cytotoxicity. Hence, the precise mechanism of action of these agents is not yet fully understood. U-68,415, which exhibited superior activity against a number of tumors and did not cause delayed death in mice, warrants further investigation. U-68,415 is a racemate and two chiral isomers were recently isolated. Therefore, further investigation of both U-68,415 and its chiral isomers is necessary.

Adozelesin, a potent new alkylating agent: cell-killing kinetics and cell-cycle effects

SummaryAdozelesin (U-73975) was highly cytotoxic to V79 cells in culture and was more cytotoxic than several clinically active antitumor drugs as determined in a human tumor-cloning assay. Phase-specificity studies showed that cells in the M + early G1 phase were most resistant to adozelesin and those in the late G1 + early S phase were most sensitive. Adozelesin transiently slowed cell progression through the S phase and then blocked cells in G2. Some cells escaped the G2 block and either divided or commenced a second round of DNA synthesis (without undergoing cytokinesis) to become tetraploid. Adozelesin inhibited DNA synthesis more than it did RNA or protein synthesis. However, the dose needed for inhibition of DNA synthesis was 10-fold that required for inhibition of L1210 cell growth. The observation that cell growth was inhibited at doses that did not cause significant inhibition of DNA synthesis and that cells were ultimately capable of completing two rounds of DNA synthesis in the presence of the drug suggests that adozelesin did not exert its cytotoxicity by significant inhibition of DNA synthesis. It is likely that adozelesin alkylates DNA at specific sites, which leads to transient inhibition of DNA synthesis and subsequent G2 blockade followed by a succession of events (polyploidy and unbalanced growth) that result in cell death.

The biochemical pharmacology of nogalamycin and its derivatives.

This review assimilates up-to-date information on the biochemical pharmacology of nogalamycin and selected derivatives that have shown good biological activities and/or received a relatively detailed investigation. The structure and chemical preparation of these derivatives from nogalamycin is described and the nomenclature which has been rather perplexing in the literature is clarified. The interaction of this class of compounds, particularly nogalamycin, with DNA is extensively reviewed. The biochemical mechanism of action of nogalamycin and its structurally closely-related derivatives is described. Among nogalamycin derivatives, menogaril showed distinct biochemical effects as well as superior cytotoxicity and antitumor activity and also proved to be effective against breast cancer clinically.

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA) · poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.

Evaluation of DNA binding characteristics of some CC-1065 analogs

The factors influencing the binding of CC-1065 to DNA were examined using racemic analogs with varying chain lengths. The ability of these agents to bind DNA appeared to be related to cytotoxic potency, however this did not appear to be a direct quantitative correlation. Two enantiomers of a bis-indole analog of CC-1065 were studied for DNA binding and cytotoxic activity. The agent with the same stereochemical configuration as CC-1065 was a potent cytotoxin, but its enantiomer was essentially inactive. Both enantiomers showed significant binding to DNA, but the biologically less active isomer showed less overall binding. In all cases, the agents preferred AT-rich DNA, and all bound to similar regions in DNA as evidenced by positions of drug-initiated thermal breaks in single end-labelled fragments of phi X 174RF DNA. The overall similarity in site specificity for binding of the structurally diverse agents suggests that much of the specificity observed in binding of the agent to DNA lies in the DNA itself. Thus, it may be difficult to change minor groove specificity for agents of this type simply by designing structures that can encompass guanine or cytosine residues. Other modifications, such as changing the specificity of the alkylating moiety, may be required to achieve this goal.

Drug sensitivity of ten human tumor cell lines compared to mouse leukemia (L1210) cells

L1210 leukemia cells, because of their rapid growth rate in suspension culture and high growth fraction, are ideally suited to screen in vitro for cytotoxic compounds. Although L1210 cells may mimic rapidly growing tumors, they have not been effective in selecting agents active against slow growing solid tumors. We expected that cell lines originating from human solid tumors, because of their slower growth rate and lower S phase fraction, would be more drug resistant than L1210. Therefore, we compared ten human tumor cell lines (5 melanomas, 4 colon carcinomas and 1 small cell lung carcinoma) to L1210 growth inhibition by 9 anti-tumor drugs. Not one human tumor cell line was consistently more resistant to all nine drugs than L1210 when the cells were exposed to drugs for about 2 doubling times. The drug sensitivity of 2 cell lines (L1210 and SK MEL 28) was again determined after a short term (2 hr) exposure and using growth inhibition and cell survival as end points. For both end points these two cell lines exhibited a random pattern of sensitivity to the drugs tested. Cell kill showed an order of sensitivity different than growth inhibition. The implication of these findings for drug-screening is discussed.

Pharmacological evaluation of combination therapy of P388 leukemia with cyclophosphamide and pyrimidinones

The administration of pyrimidinone prior to tumor inoculation did not increase the lost resistance to tumor invasion. In addition, the administration of pyrimidinone prior to cyclophosphamide (CY) was no better than the treatment with CY alone. The best therapeutic effect was observed when the pyrimidinone was given 1 day after CY administration. A statistically significant synergism (mostly P<0.01) with CY was obtained when the pyrimidinone was given every 4 days thereafter for 1 – 7 intraperitoneal (i.p.) injections. A significant synergistic antitumor effect was also observed when the pyrimidinone was given at 4 day, 7 day, 10 day, or 14 day intervals for a maximum of 7, 4, 3 and 2 injections, respectively. For some pyrimidinones, such as ABPP, multiple injections appeared to yield better therapeutic results. This phenomenon may be in part related to the magnitude and duration of the stimulation of natural killer (NK) cell activity after mice received a single or multiple i.p. injections of ABPP. When given i.p., the pyrimidinone level in peritoneal exudates was approximately twice that found in serum. CY showed no apparent effect on the absorption and clearance of pyrimidinone. The combination therapy was also statistically synergistic (P < 0.01) when ABPP was given orally. The significance and advantage of these findings is discussed in terms of their clinical implications.

CHEMISTRY AND BIOCHEMISTRY OF THE STREPTOVARICINS PART 12, ATROPISOMERIC STREPTOVARICINS

Beim Erhitzen von Streptovaricin Cund einiger Derivate in siedenden Losungsmitteln (Toluol, Pyridin, Benzol) bilden dieseGemische mit ihren Atropisomeren.Beim Erhitzen von Streptovaricin Cund einiger Derivate in siedenden Losungsmitteln (Toluol, Pyridin, Benzol) bilden dieseGemische mit ihren Atropisomeren.