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

Abstract

Racemic 2-{[1-(chloromethyl)-5-nitro-3-{5-[2-(dimethylamino)ethoxy]indol-2-carbonyl}-1,2-dihydro-3H-benzo[e]indol-7-yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia-selective prodrug active against radiation-resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X-ray crystallography. The intermediate was used to prepare the prodrug's enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia-selective cytotoxins, but the "natural" S enantiomer is at least 20-fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia-selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa-tumour bearing animals, the (-)-R enantiomer of the prodrug was inactive, while the (+)-S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)-S-prodrug and radiation eliminated detectable colony-forming cells in four out of five treated tumour-bearing animals.

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy

“…For example, nitroCBIs 12 – 14 and 16 – 19 were prepared by 1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide hydrochloride (EDCI)-mediated coupling of the alkylating subunit 87 to the appropriate side chain acid under acidic conditions (in the presence of toluenesulfonic acid). This approach is generally high-yielding, with the only difficulty being the tendency of the product to eliminate HCl, giving an exomethylene byproduct hard to separate from the desired material, particularly if basic conditions are used to remove the excess acid side chain (see Supporting Information). A slight modification of this method employs a TBDMS protecting group on the alkylating subunit, previously reported in the synthesis of the enantiomers of 11 to give more soluble and more easily purified intermediates .…”

“…This approach is generally high-yielding, with the only difficulty being the tendency of the product to eliminate HCl, giving an exomethylene byproduct hard to separate from the desired material, particularly if basic conditions are used to remove the excess acid side chain (see Supporting Information). A slight modification of this method employs a TBDMS protecting group on the alkylating subunit, previously reported in the synthesis of the enantiomers of 11 to give more soluble and more easily purified intermediates . For the current application, TBDMS-protected sulfonamide and carboxamide subunits 89 and 92 were prepared as shown and used to make 6 nitroCBI analogues (four sulfonamide and two carboxamide examples), with a subsequent HCl-mediated deprotection of the TBDMS group.…”

“…The particular example 9 is highly active against hypoxic cells in human tumor xenografts at well-tolerated doses in mice and provides a substantial increase in tumor growth delay when combined with γ irradiation. Although 9 is racemic and the enantiomers differ at least 20-fold in potency, there is no enantioselectivity in the hypoxia-selective activation step in vitro; both enantiomers show clean metabolism from nitro to amino in the presence of hypoxic tumor cells and selectively kill cells under hypoxic conditions . Hypoxia-selective metabolism of 9 was subsequently demonstrated in a panel of 23 human tumor cell lines in a study which identified three flavoreductases capable of performing the one-electron reductive activation step …”

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

“…[9] Hypoxic and oxic conditions refer to the following: hypoxic (95 % N 2 , 5% CO 2 ); oxic (95 % air, 5 % CO 2 ), except for Figure 8 and Figure S13 (95 % O 2 , 5% CO 2 ).…”

“…[6a,b] The particular example 5, when administered as a water-soluble phosphate pre-prodrug, was found to selectively eliminate radiation-resistant cells in human tumour xenografts in mice. [8] Substantial cell killing was observed at nontoxic doses (in particular for the "natural" S enantiomer), [9] whereas the corresponding aminoCBI 6 was tolerated only at much lower doses and, using systemic administration, was completely inactive against hypoxic tumour cells. Notably, in combination with fractionated radiation, 5 (as the phosphate pre-prodrug) significantly prolonged the life of tumour-bearing mice relative to radiation treatment alone.…”