Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators

Abstract

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90-to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxiaselective generation of potent DNA minor groove alkylating agents.

Preparation and Antitumour Properties of the Enantiomers of a Hypoxia‐Selective Nitro Analogue of the Duocarmycins

“…For related nitroCBIs, we have demonstrated that the aminoCBI is a diffusible metabolite that could be responsible for a bystander effect. [6] The current study provides further evidence for aminoCBI diffusing from the cells in which it is generated, but also supports a mechanism (oxygen-insensitive reduction) for direct action against well-oxygenated tumour cells. Whatever the basis for the single agent activity, the pronounced antitumour activity in combination with radiation at nontoxic doses makes 1, and particularly the enantiomerically pure (+)-S-1, an attractive drug development candidate.…”

“…[5] We have shown that simple aminoCBIs (analogues of 3 lacking the sulfonamide substituent) act like the natural products in that they alkylate exclusively at the N3 of adenine in the minor groove of DNA in a highly sequence-specific manner. [6,7] The lesions are poorly recognised by repair enzymes and the monoadducts are very cytotoxic. [6,8,9] NitroCBIs such as 2 are, in contrast, considerably less toxic, presumably because they are unable to form a spirocyclic intermediate analogous to 4 and are thus unable to alkylate DNA.…”

“…[6,7] The lesions are poorly recognised by repair enzymes and the monoadducts are very cytotoxic. [6,8,9] NitroCBIs such as 2 are, in contrast, considerably less toxic, presumably because they are unable to form a spirocyclic intermediate analogous to 4 and are thus unable to alkylate DNA. [10] These observations provided the basis for exploring nitroCBIs as hypoxia-selective prodrugs, but in order to produce in vivo active compounds a number of structural Figure 1.…”

Pseudomonas aeruginosa NfsB and nitro-CBI-DEI – a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy

“…It has been shown that the Escherichia coli nitroreductase NfsB (NfsB_Ec) can reduce nitro-CBIs in an oxygen-independent fashion, generating highly cytotoxic metabolites that alkylate the N3 of adenine in the minor groove of DNA [12]. However, it was inferred that the lead nitro-CBI prodrug in that study, nitro-CBI-5-[(dimethylamino)ethoxy]indole (nitro-CBI-DEI; Figure 1A, structure inset) is a poor substrate for NfsB_Ec relative to CB1954 or the DNBMs [12]. In this work we sought to identify more active nitroreductases for metabolism of nitro-CBI-DEI, reasoning that superior enzymes will be required to extend the therapeutic index of nitro-CBI prodrugs in GDEPT.…”

Preparation and Properties of Clickable Amino Analogues of the Duocarmycins: Factors That Affect the Efficiency of Their Fluorescent Labelling of DNA

“…These include very similar DNA sequence selectivity, [4] toxicity (for the racemic form and individual enantiomers), [3] and exclusive DNA alkylation at N3 of adenine. [5] The last was demonstrated by reaction of 3 (in which R is the trimethoxyindole side chain common to the duocarmycins) with calf thymus DNA (ctDNA) and subsequent thermal depurination, leading to the isolation, in practically quantitative yield, of adenine adduct 4.…”