Synthesis, metabolism and in vitro cytotoxicity studies on novel lavendamycin antitumor agents
Graphical abstract
Introduction
Lavendamycin (1), a naturally occurring 7-aminoquinoline-5,8-dione antitumor antibiotic, was first isolated from the fermentation broth of Streptomyces lavendulae by Balitz et al.1 and its structure was determined by Doyle et al.2 The latter study determined that lavendamycin is a pentacyclic structure with two moieties including quinoline-5,8-dione and indolopyridine (β-carboline) (Chart).2 Lavendamycin is similar to another potent antibiotic antitumor agent streptonigrin (2).1, 2, 3, 4, 5 The clinical use of both of these antibiotics has been precluded because of their toxicity toward human cells.2, 6, 7, 8 Initial structure–activity relationship (SAR) studies have demonstrated that the essential moiety for the cytotoxic activity of lavendamycin and streptonigrin is the 7-aminoquinoline-5,8-dione moiety.7
As it was thought that some analogues of the naturally occurring antibiotic 1 might show activity toward human tumors, efficient methods were needed to prepare these derivatives. There were two previous reports on the synthesis of lavendamycin methyl ester (3) (above and Table 1) by Kende9 and Boger10 but both were impractical, since they produced compound 3 in an overall yield of less than 2% and in 9- and 20-steps, respectively. Our group was able to develop two synthetic methods11, 12 that produced 3 in an overall yield of about 40% in only 5-steps. These efficient methods, particularly those reported in 1996,12 made it possible to prepare a large number of substituted lavendamycins needed for various in vitro and in vivo screening tests.
We previously reported that a significant number of these lavendamycin analogues have potent biological activity including anti-HIV reverse transcriptase13 and antitumor activity.14, 15, 16 More importantly, several screening tests on a number of lavendamycin analogues have shown that a significant number of these compounds possess low animal toxicity.13, 17 The NCI in vivo studies have shown that the maximum tolerated dose of three derivatives 3, 4 and 5 (Table 1) in mice is 400 mg/kg which is 31 and 1000 times higher than that for lavendamycin and streptonigrin, respectively.13, 17 The lavendamycin analogue 6 reduced tumor volume (up to 80%) in mice bearing tumor xenografts at a daily dose of 300 mg/kg for 10 days without exhibiting drug-related weight loss or lethality.17 A recent study also demonstrated that the normal rat kidney epithelial cell line (NRK-52E) exhibited much less sensitivity to several lavendamycin analogues compared to the tumor cells with the same origin.17 When lavendamycin analogues 7 and 6 at a daily dose of 300 mg/kg and 8 at a daily dose of 100 mg/kg were administered to nude mice for eight and seven days, respectively, no drug-related deaths or toxicity were observed.17 Analogues 8, 7 and 6 (Table 1) inhibited tumor growth in nude mice by 69%, 88% and 78%, respectively, when administered at 100, 150 and 300 mg/kg for 7, 8 and 8 days, respectively.17
NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) is a two-electron reductase, characterized by its capacity for utilizing either NADH or NADPH as reducing cofactors and by its inhibition by dicoumarol.18 It is primarily located in the cytosol (>80%), but a recent study has reported a substantial pool of NQO1 in the nuclei of cancer cells.19 NQO1 is generally categorized as a detoxification enzyme, and it can protect the cell from a broad range of chemically reactive metabolites including electrophiles and reactive oxygen species.20 NQO1 can also function as an activating enzyme, specifically for the reductive activation of antitumor quinones and other bioreductive anticancer agents.21
NQO1 can be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons.21 Induction by procarcinogens and the capacity of NQO1 for detoxifying reactive metabolites suggest that changes in expression of NQO1 may occur during carcinogenesis.22 Marked elevations in NQO1 activity and mRNA content have been documented in both preneoplastic tissues and established tumors. Tumors or cancer cell lines where increased NQO1 expression has been observed include those from the lung, liver, colon, and breast.23 In addition, advanced, metastatic tumors appear to express higher levels of NQO1 than non-metastatic tumors.24
Correlations between NQO1 activity in cancer cells and cytotoxicity of antitumor quinones to those cells have been reported.25, 26, 27 In our previous studies with several series of indole- and quinolinequinone antitumor agents, including analogues of lavendamycin, those compounds that were the best substrates for NQO1 were also found to be selectively toxic to cell lines with high levels of NQO1 compared to cell lines that were deficient in NQO1.15, 16, 28, 29, 30 In this report, we describe the synthesis, metabolism by recombinant human NQO1 and anticancer activity of novel analogues of lavendamycin with functional group substitutions on both the quinoline-5,8-dione and indolopyridine moieties.
Section snippets
Synthetic chemistry
Table 4 presents the structures of a total of 25 lavendamycins that were the subject of our biological studies in this report. As shown in Scheme 1, Pictet–Spengler (P–S) condensation of the quinolinedione aldehydes (Table 2) with tryptophans (Table 3) yielded the target lavendamycins 40–58 listed in Table 4. The amino lavendamycins 59–64 were prepared respectively by the acid hydrolysis of the corresponding 7-acylamino lavendamycins 40–45.
High resolution mass spectroscopy of 67 (Scheme 2) did
Conclusions
Twenty-five novel lavendamycin analogues were synthesized, characterized and evaluated as NQO1-directed antitumor agents. In line with our earlier computational and structure–activity studies,15, 16 analogues with small substituents at the R1 position and groups capable of hydrogen bonding/van der Waals interactions at the R3 position generally made the best substrates for NQO1. Consequently, many of these compounds were also selectively toxic to the cancer cells with elevated NQO1 activity.
General methods
For general methods, see Ref. 16. In nearly all of the experimental work-ups, solvents were evaporated under reduced pressure and heat using a rotaevaporator. In each experiment, the progress of the reaction was monitored by TLC and the reaction stopped when TLC showed its completion. In some instances, no NMR signals were observed for active H’s, especially when DMSO-d6 was the solvent of use.
7-N-Formamido-2-formylquinoline-5,8-dione (20)
In a 25 mL two-necked round-bottomed flask, equipped with a stirring bar, a condenser and an argon
Acknowledgments
The financial support by the National Institutes of Health is greatly appreciated (NIH Grants: R15CA74245, M.B.; R15CA78232 and P20RR017670, H.D.B.). We also thank professor David Williams and the staff at the mass spectroscopy laboratory at Indiana University for their help in obtaining mass spectral data.
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