A Rationally Designed Genotoxin that Selectively Destroys Estrogen Receptor-Positive Breast Cancer Cells

Abstract

The persistence of genetic damage produced by alkylating agents 1 as well as the antagonism of essential biochemical processes such as transcription can have lethal consequences for malignant cells. 2 Both mechanisms have been identified in studies to uncover the reasons for the efficacy of cisplatin in the treatment of several cancers. 2a, 3 We describe a synthetic strategy to create bifunctional molecules that produce DNA adducts capable of binding the estrogen receptor (ER), which is aberrantly expressed in many breast cancer cells. 4 It is speculated that DNA adducts that form complexes with the ER will be poorly repaired in these cells because they are camouflaged from detection by DNA repair enzymes. Consequently, the DNA lesions persist. Furthermore, the DNA adducts would be expected to act as "molecular decoys" capable of displacing the ER from its natural targets and antagonizing its role in malignant growth. In healthy cells, where the abundance of the ER is minimal, no such ER-DNA adduct complexes will be present, and the cell should survive. 5 In this report we describe the design and synthesis of compound 1, a bifunctional agent that can form covalent DNA adducts capable of binding the ER with high affinity and specificity. We show that 1 has selective toxicity toward ER+ breast cancer cells compared to ER-cells in vitro.Compound 1 consists of a bis-chloroethyl aniline mustard as the DNA alkylating unit tethered to estradiol, the natural ligand for the ER. The site of substitution of estradiol in 1 was based on reports that relatively large alkyl groups can be attached at the 7α position with retention of high affinity for the ER 6 . The synthetic strategy for 1 is shown in Scheme 1. Compound 7, a key compound in the synthesis, was prepared by a modification of a published strategy. 7 Briefly, 3 was functionalized with a 6-carbon chain at the 7-position in α-stereochemistry to provide the alkenyl steroid 4. Efficient reduction of the 6-oxo group in 4 was achieved with Et 3 SiH/BF 3 .Et 2 O; however, this treatment also caused the loss of 3,17-tetrahydropyranoxy (THP) groups producing diol 5. The 3,17-OHs of 5 were reprotected with THP groups to afford 6, followed by oxidation of the alkene at the terminus of the linker to provide alcohol 7. Steroid alcohol 7 was converted to bromide 8, which was subsequently allowed to react with a protected ethanolamine to give 9. Compound 9 was desilylated with tetrabutylammonium fluoride (TBAF) and converted to a carbonate Next, the ability of 1 to modify DNA covalently was investigated. Plasmid DNA was incubated with 100 μM [ 14 C]-1 10 at 37 °C for up to 6 h. After unbound 1 was removed by phenol-CHCl 3 extraction and ethanol precipitation, the radioactivity associated with DNA was measured. The amount of radioactivity bound to DNA increased at a constant rate over the 6-h period indicating the formation of covalently bound 1 (see Supporting Information). Based on previous studies on DNA alkylation by nitrogen mustards, 11 it is likely that covalent adducts of...

Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents

“…6 Compounds 13 and 15 containing amino groups showed toxicity similar to that of l . Both of these compounds also showed greater toxicity toward the ER-positive MCF-7 cells than towards the ER-negative MDA-MB231 cells.…”

“…6 The syntheses utilized 3,17β-bis(2-tetrahydropyranyloxy)-7α-(6-hydroxyhexan-1-yl)-estra-1,3,5(1O)triene 2 as the starting compound (see box in Scheme 1); its preparation has also been described. 6 Construction of linkers proceeded by linear additions to 2 with final addition of the N,Nbis(2-chloroethyl)aniline moiety. Compound 5 was prepared by conversion alcohol 2 to the bromide, which was allowed to react with a protected ethanolamine providing 3 as described in reference 10.…”

“…Compound 9 was prepared by conversion of 2 to the p-nitrophenyl carbonate 8, which was then allowed to react with (N,N-bis-2-chloroethylaminophenyl)-propylamine. 6 Removal ofTHP groups under acidic conditions produced 9. Compound 10, the starting material for compounds 12-15, was prepared by hydrazinolysis of the phthalimide of 2 that was formed under Mitsunobu conditions.…”

“…A similar result has been repmied for a conjugate of chlorambucil with the polyamine spetmidine. 12 Using an electrophoretic gel mobility shift assay 6 , we observed that covalent DNA adducts of 1, 5, 7, 13 and 15 form complexes with the portion of the ER containing the ligand binding domain (ER-LBD) (Table 1, column 4). Under conditions that allowed complex formation, addition of the ER to the modified DNAs resulted in the appearance of a slowly migrating band by electrophoresis that was eliminated by addition of excess competitor, estradiol (data not shown).…”

“…A compound with greater affinity for the ER was obtained by replacing the 2-PI group with that of 17β-estradiol c01mected via the 7α position. 6 Both the 2PI-and estradiol-based compounds showed increased toxicity toward breast cancer cells that express the ER. Evidence was presented that the differential toxicity of the DNA damaging agents was not due to an antihonnonal mechanism.…”