GC-Targeted C8-Linked Pyrrolobenzodiazepine–Biaryl Conjugates with Femtomolar in Vitro Cytotoxicity and in Vivo Antitumor Activity in Mouse Models

Abstract

DNA binding 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) building blocks have been developed that span two DNA base pairs with a strong preference for GC-rich DNA. They have been conjugated to a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) molecule to produce C8-linked PBD-MPB hybrids that can stabilize GC-rich DNA by up to 13-fold compared to AT-rich DNA. Some have subpicomolar IC50 values in human tumor cell lines and in primary chronic lymphocytic leukemia cells, while being up to 6 orders less cytotoxic in the non-tumor cell line WI38, suggesting that key DNA sequences may be relevant targets in these ultrasensitive cancer cell lines. One conjugate, 7h (KMR-28-39), which has femtomolar activity in the breast cancer cell line MDA-MB-231, has significant dose-dependent antitumor activity in MDA-MB-231 (breast) and MIA PaCa-2 (pancreatic) human tumor xenograft mouse models with insignificant toxicity at therapeutic doses. Preliminary studies suggest that 7h may sterically inhibit interaction of the transcription factor NF-κB with its cognate DNA binding sequence.

2-(4-(2-Chloroacetamido)-1-methyl-1H-pyrrole-2-carboxamido)ethyl Acetate

“…We have synthesized the amide 3 starting from 2-trichloroacetyl-1-methyl-4-nitropyrrole (1) [12,13] according to Scheme 1, in two steps; the first was performed by acyl nucleophilic substitution of the OPEN ACCESS trichloromethyl leaving group by ethanolamine to afford amide 2 and the second by protecting the hydroxyl group with an acetyl group to furnish ester 3 [14].…”

“…After completion of the reaction (by TLC) the reaction mixture was concentrated and then the residue was dissolved in degassed CH 2 Cl 2 containing Et 3 N. Finally, the reaction mixture was concentrated and the title compound 5 was then purified by silica gel chromatography. The required compound 5 obtained in 70% overall yield from 3 was characterized by 1 H-NMR, 13 C-NMR and mass spectral data.…”

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G–A cross-linking PBD–duocarmycin dimers

“…Based on the results of a thermallyinduced cleavage assay, they reported that 3 forms cross-links at the 5'-AATTTTCC(G)-3' sequence. During the past six years we have developed molecular dynamics simulation methodologies [11,13,48] that allow us to accurately predict and interpret how both mono-alkylating and DNA cross-linking agents of this type interact in the minor groove of DNA. As the hybrid dimer 3 reported by Lee [47] had a significantly lower DNA cross-linking potency and in vitro cytotoxicity compared to the Hurley (1) and Denny (2) dimers, we decided to use our simulation methodologies to compare the interaction of the three molecules at their proposed DNA recognition sites.…”

“…Through the years, many novel cross-linking agents have been designed, synthesized and evaluated [4] , [5] in an attempt to (a) improve on cross-linking efficiency [6], (b) modify the sequence-selectivity of cross-linking [7], (c) change the distribution of inter-versus intrastrand cross-links [8][9][10], and/or (d) target different base pair combinations [11][12][13]. For example, the naturally occurring pyrrolobenzodiazepine (PBD) molecules, derived from Streptomyces species [14][15][16][17][18], are characterized by an N10-C11 imine group that enables formation of a covalent bond to the C2-amino group of a guanine base [19], and these have been successfully incorporated into PBD dimer structures such as DSB-120 [20] and SJG-136 [21] (Figure 1A) that can form sequence-selective G-G cross-links in the DNA minor groove.…”

Intramolecular Azide to Alkene Cycloadditions for the Construction of Pyrrolobenzodiazepines and Azetidino-Benzodiazepines

“…Synthetic hybrids [5][6][7][8] and dimers [5,[9][10][11] have also shown significant biological activity and members of the dimer class have entered Phase II clinical development as antitumour compounds. PBDs with additional fused rings such as the circumdatin (4) [12], bretazenil (5) [13] and the 1,2,3-triazolo-fused system 6 [14] are attractive as potential antitumour compounds, neurological agents, and protease inhibitors, respectively.…”