We designed, synthesized and tested novel 2,6-disubstituted-anthraquinones able to bind dynamic secondary structures of nucleic acids, such as TAR RNA and its reverse transcript cTAR, leading to inhibition of the chaperone activities of the nucleocapsid NCp7, a highly conserved viral protein implied in crucial steps of HIV-1 replication
2,6-Dipeptidyl-anthraquinones are a promising class of nucleic acid-binding compounds that act as NC inhibitors in vitro. We designed, synthesized, and tested new series of 2,6-disubstituted-anthraquinones, which are able to bind viral nucleic acid substrates of NC. We demonstrate here that these novel derivatives interact preferentially with noncanonical structures of TAR and cTAR, stabilize their dynamics, and interfere with NC chaperone activity.
Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues.
Bis-3-chloropiperidines are a new class of DNA-active compounds capable of alkylating nucleobases and inducing strand cleavage. In this study, we investigated the reactivity of these mustard-based agents with both single- and double-stranded DNA constructs. Polyacrylamide gel electrophoresis (PAGE) and electrospray ionization mass spectrometry (ESI-MS) were used to obtain valuable insight into their mechanism at the molecular level and to investigate their time- and concentration-dependent activity. The results revealed the preferential formation of mono- and bifunctional adducts at nucleophilic guanine sites. In a stepwise fashion, alkylation was followed by depurination and subsequent strand scission at the ensuing apurinic site. We demonstrated that the covalent modifications introduced by this new class of compounds can inhibit the activity of essential DNA-processing proteins, such as topoisomerase IIα, thereby suggesting that bis-3-chloropiperidines may have excellent anticancer potential.
The
pressing demand for sustainable antitumor drugs prompted us
to investigate 3-chloropiperidines as potential mustard-based anticancer
agents. In this study, an explorative set of variously decorated monofunctional
3-chloropiperidines (M-CePs) was efficiently synthesized through a
fast and affordable route providing high yields of pure racemates
and enantiomers. Consistently with their reactivity, M-CePs were demonstrated
to alkylate DNA
in vitro
. On a panel of carcinoma
cell lines, M-CePs exhibited low nanomolar cytotoxicity indexes, which
showed their remarkable activity against pancreatic cancer cells and
in all cases performed strikingly better than the chlorambucil control.
Very interestingly, stereochemistry modulated the activity of M-CePs
in unexpected ways, pointing to additional molecular mechanisms of
action beyond the direct damage of genomic DNA. This encouraging combination
of efficacy and sustainability suggests they are valid candidates
for anticancer agent development.
In this study, we describe the synthesis and biological evaluation of a set of bis‐3‐chloropiperidines (B−CePs) containing rigid aromatic linker structures. A modification of the synthetic strategy also enabled the synthesis of a pilot tris‐3‐chloropiperidine (Tri‐CeP) bearing three reactive meta‐chloropiperidine moieties on the aromatic scaffold. A structure–reactivity relationship analysis of B−CePs suggests that the arrangement of the reactive units affects the DNA alkylating activity, while also revealing correlations between the electron density of the aromatic system and the reactivity with biologically relevant nucleophiles, both on isolated DNA and in cancer cells. Interestingly, all aromatic 3‐chloropiperidines exhibited a marked cytotoxicity and tropism for 2D and 3D cultures of pancreatic cancer cells. Therefore, the new aromatic 3‐chloropiperidines appear to be promising contenders for further development of mustard‐based anticancer agents aimed at pancreatic cancers.
The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.
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