Byron H. Long

Discovery of antitumor indolocarbazoles: rebeccamycin, NSC 655649, and fluoroindolocarbazoles.

A fermentation directed product search for potential anticancer drugs conducted by Bristol-Myers in the 1970s and early 1980s resulted in the identification of a novel indolocarbazole (IC) rebeccamycin (RBM) as a potential drug development candidate. Subsequently, an analog program designed to impart distal site in vivo antitumor activity resulted in the discovery of diethylaminoethyl analog of RBM (DEAE-RBM), which is presently undergoing clinical evaluation as NSC 655649 and BMY-27557. Strong DNA intercalation is the primary mechanism of action of DEAE-RBM resulting in the potent catalytic inhibition of both topoisomerases I and II. Precursor feeding fermentation experiments with fluorine-substituted tryptophans yielded novel fluoroindolocarbazoles (FICs). These FICs were identified as targeting topoisomerase (topo) I in a mechanism-based screen and their action on topo I was confirmed by production of topo I-mediated single-strand breaks in DNA at sites essentially identical to those induced by camptothecin. Topo I dependent cytotoxicity was demonstrated for specific FICs using a P388 and camptothecin-resistant P388/CPT45 pair of cell lines, the latter expresses little or no functional topo I. For example, topo I selectivity was greatest with 3,9-difluoro substituted FIC and was least significant and least cytotoxic with 4,8-difluoro substituted FIC. The review focuses on the discovery of the rebeccamycin class of compounds and their structure-activity relationships leading to the development of the clinical candidate BMY-27557 (NSC 655649), as well as the lead identification of the fluoroindolocarbazole class of compounds.

Diastereoselective addition of Grignard reagents to azetidine-2,3-dione: Synthesis of novel Taxol® analogues

Abstract Synthesis and cytotoxicity properties of novel C-2′ analogues of paclitaxel are described. The analogues were synthesized using Holtons β-lactam approach to append the side chain on baccatin III. The key intermediate to the synthesis of novel analogues was prepared employing an unprecedented stereocontrolled addition of Grignard reagent to a chiral azetidine-2,3-dione.

Inhibitors of topoisomerase II: Structure-activity relationships and mechanism of action of podophyllin congeners

The specific inhibition of eukaryote DNA topoisomerase II by the anti-cancer drugs VP16, VM26, and 21 other congeners of podophyllotoxin has been extensively studied in this laboratory through the use of alkaline elution and other techniques. A structure-activity relationship has been established for cytotoxicity, single and double strand DNA breakage, and inhibition of the DNA strand passing activity of topoisomerase II. Furthermore, topoisomerase inhibition was measured in four naturally sensitive and resistant human lung carcinoma cells by quantifying the amount of single and double strand DNA breakage produced by VP16 and VM26 in cells and isolated nuclei. A direct correlation between double but not single strand DNA breaks and cytotoxicity was observed for the analogs in A549 human lung adenocarcinoma cells. In fact, some analogs were capable of producing substantial single strand DNA breakage without producing cytotoxicity. A similar correspondence was observed between double strand DNA breaks and cytotoxicity produced by VP16 and VM26 in the naturally sensitive and resistant cell lines. Evidence is also presented suggesting that the association of the drug with enzyme-DNA intermediate complex and the formation of the enzyme-DNA complex alone both reflected equilibrium governed conditions that were readily reversible. These studies support a model based on the proposal that the actual cytotoxic events are genetic alterations caused by possible heterologous subunit exchanges occurring between adjacent enzyme molecules, which result from the stabilization of the intermediate complex, rather than the actual loss of topoisomerase II activity caused by the inhibition. The resistance of normal cells and cells with acquired resistance to the possible clastogenic effects of topoisomerase inhibition may be, in part, related to the low topoisomerase II levels found in such cells. Topoisomerase II may also play a role in gene amplification and tumor cell heterogeneity by serving as a vehicle through which genetic recombination events may occur.

Non-camptothecin topoisomerase I active compounds as potential anticancer agents

The success of two camptothecin analogues against human cancers has prompted pharmaceutical companies and academic laboratories to not only initiate camptothecin analogue programs of their own, but also to either screen for or to rationally design novel topoisomerase (topo) I active agents. The primary focus of this review is on those agents that most closely mimic the actions of camptothecin, the prototype topo I acting agent. Indolocarbazoles are the most exploited and advanced chemotype, being led by NB-506 and J-107088, both of which have entered Phase I/II clinical evaluations by Merck/Banyu. Kyowa Hakko Kogyo, while expanding the K252a/staurosporine series, identified topo I active agents but continued to the clinic with the protein kinase inhibitor UCN-01. Approximately 100 non-proprietary indolocarbazoles have been described in the literature by a consortium of academic scientists working in conjunction with scientists at Aventis and Novartis. Their intent appears to be more academic than commercial and this association has now ended. An academic group, through an agreement with Avax Technologies is commercially exploring benzimidazoles, terbenzimidazoles, coralyne and protoberberines. Indenoisoquinoline NSC 314622 and anthracenyl-amino acid conjugate NU/ICRF 505 are interesting and novel structures that have not yet progressed to the clinic. The naphthacenedione family includes saintopin, which was discovered by Kyowa Hakko Kogyo. This company appears to be focusing on UCE6. Other agents include nogalamycin, dexniguldipine HCl (B859-35), ecteinascidin 743 (Et 743) and phthalascidin (Pt 650). 2280-DTI and 2890-DTI may be true catalytic inhibitors of topo I. Only NB-506, J-107088, dexniguldipine and Et 743 have undergone or are presently undergoing Phase I/II clinical evaluation with little information coming forth, except for Et 743, which is yielding responses against sarcomas.

A chemoselective approach to functionalize the C-10 position of 10-deacetylbaccatin III. Synthesis and biological properties of novel C-10 Taxol® analogues

Abstract A chemoselective approach to functionalize the C-10 position of 10-deacetyl baccatin III, a key intermediate for the semi-synthesis of paclitaxel, is described. The chemistry provides an easy access to a variety of C-10 hydroxyl derivatives, such as, ethers, esters, carbonates, carbamates, and sulfonates under mild conditions. The C-10 modified baccatin derivatives were further employed in the synthesis of novel biologically active Taxol® analogues.

Synthesis and antitumor evaluation of 2′-oxycarbonylpaclitaxels (paclitaxel-2′-carbonates)

Abstract A number of 2′-oxycarbonylpaclitaxels (paclitaxel-2′-carbonates) 3 have been prepared and evaluated for their cytotoxicity and in vivo antitumor activity. Most of these paclitaxel-2′-carbonates were found to exhibit in vivo antitumor activity in the i.p. M109 murine tumor model system, comparable to that of the parent drug.

NOVEL C-4 PACLITAXEL (TAXOL) ANALOGS : POTENT ANTITUMOR AGENTS

Abstract A large number of C-4 paclitaxel analogs have been prepared in the course of our systematic C-4 modification. These include C-4 esters, carbonates, carbamates as well as a C-4 deacetyl derivative. All of these analogs were evaluated in a tubulin polymerization assay as well as in a cytotoxicity assay against a human colon cancer cell line. The potent analogs emerging from these in vitro assays were further evaluated in vivo. With the exception of paclitaxel side chain bearing C-4 carbamates and C-4 aromatic esters, most of the C-4 aliphatic esters and carbonates were found to possess comparable or superior activity to paclitaxel in vitro. Several C-4 aliphatic esters and carbonates also exhibited in vivo activities against i.p. implanted murine M-109 lung carcinoma.

Taxol® structure-activity relationships: synthesis and biological evaluation of taxol analogs modified at C-7

Abstract A series of taxol derivatives, modified at C-7, is described. This includes sulfonate, silylether, ester, carbonate, carbamate, fluoro, dehydro and deoxy derivatives. Biological evaluation shows that these modifications do not usually significantly compromise activity. However, none of the C-7 analogs prepared thus far have been shown to be better than taxol in both in vitro and in vivo assays.

Cellular uptake profile of paclitaxel using liquid chromatography tandem mass spectrometry.

A new method for studying cellular uptake has been developed. This method is based on selected reaction monitoring liquid chromatography tandem mass spectrometry analysis of preparations from cell culture. The limit of detection for paclitaxel was approximately 0.1 microM intracellular concentration. This method has been utilized to study the uptake of paclitaxel and an analog (BMS-190616) in normal and multidrug resistant (MDR) cell lines. Paclitaxel and the analog, that had been noted to overcome MDR in animal models, were incubated with normal cells (HCT116) and MDR cells (HCT116(VM)46) at therapeutic concentrations. Intracellular drug concentrations were assayed at intervals from 0 to 1.0 h. Results show that paclitaxel accumulates to a level 12 times greater and BMS-190616 to a level 5 times greater in the normal cells as compared to MDR cells suggesting that paclitaxel is more sensitive to MDR than the analog. Furthermore, the steady state level of BMS-190616 was 4 fold greater than paclitaxel in the MDR cell line suggesting that at least part of this compounds increased therapeutic effect can be attributed to processes of uptake and efflux at the cellular level. These data show that the method is rapid, sensitive and presents a unique advantage over traditional radioisotopic methods in that it can readily be employed on a range of analogs without any additional synthetic effort.

Paclitaxel analogs modified in ring C: Synthesis and biological evaluation

Abstract Lead tetracetate oxidation of 6α-hydroxy-7-epi-paclitaxel leads to C-nor-paclitaxel and C-seco-paclitaxel derivatives. Tetrapropylammonium perruthenate (TPAP) oxidation of a 6α-hydroxy-7-epi-paclitaxel derivative leads to a 6-formyl-C-nor-paclitaxel derivative. Reaction of a 6α-O-trifluoromethanesulfonyl-7-epi-paclitaxel derivative with DMAP yields a 20-O-acetyl-4-deacetyl-5,6-dehydro-6-formyl-C-nor-paclitaxel derivative. C-nor-paclitaxel analogs are less active than paclitaxel.