Reversibility of the covalent reaction of CC-1065 and analogues with DNA.

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.

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

Adozelesin (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.

Molecular basis for sequence-specific DNA alkylation by CC-1065.

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoelectronic factors influencing the biological activity and DNA interaction of synthetic antitumor agents modeled on CC-1065.

The synthesis, physicochemical properties, and biological activities of a series of novel spiro cyclopropyl compounds, modeled on the potent antitumor antibiotic CC-1065 (1), are described. Many of these synthetic analogues are significantly more effective than 1 against murine tumors. In particular, compound 27 exhibits high activity and potency. Structure-activity analysis supports a molecular mechanism for biological action involving hydrophobic interaction of the drug with DNA and acid-catalyzed alkylation of DNA.

Structure and activity relationship of several novel CC-1065 analogs.

CC-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-P388 leukemia activity. At the optimal dosage, U-68,415 produced 4 out of 6 long-term (greater than 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 greater than U-66,694 greater than U-68,819 greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of organic matrix in calcium oxalate renal calculi.

The quantity of protein and carbohydrate comprising the matrix of calcium oxalate monohydrate (COM) renal stones was found to decrease with distance from the surface of the stone. The average organic concentration of stones 3 to 30 mm in diameter ranged from 5.7% at the surface to 2.7% at the core. This concentration gradient suggests matrix involvement in a "growth front" on stone surfaces with migration of organic material from the "older" interior. The matrix distribution was not readily correlated with density variations or with the presence of hydroxyapatite or calcium oxalate dihydrate. Surface matrix concentrations were greater than amounts predicted by physical adsorption. Electron microscopy confirmed the presence of the organic-rich surface layer and also suggested that increase in stone size occurs predominantly by crystal growth with microcrystal aggregates as growth centers.