Toni Brown

SYNTHESIS AND CYTOTOXIC PROPERTIES OF NITRO- AND AMINOCHALCONES

Twenty-two nitro- and aminochalcones were synthesized and characterized. Their cytotoxic properties were determined by a 3-day continuous exposure MTT assay with murine melanoma B16 cells. The aminochalcones were generally more cytotoxic than their nitro precursors. Aminochalcone 18, which has an IC50 value of 0.24 μM, was the most potent compound. The results demonstrated that the number and position of methoxy groups in ring A enhanced cytotoxicity of the chalcones. The location of the amino group on ring B also affected cytotoxicity.

1,2,3,4-Tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4

Eleven 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4 (CA-4) were synthesized and their cytotoxicity against the growth of two murine cancer cell lines (B16 melanoma and L1210 leukemia) in culture was determined using an MTT assay. Two 2-thioxopyrimidine analogs 8f and 9a exhibited significant activity (IC50<1 microM for L1210 and <10 microM for B16 cells). Exposure of A-10 cells to 8f and 9a produced a significant reduction in cellular microtubules in interphase cells, with an EC50 value of 4.4 and 2.9 microM, respectively, for microtubule loss. Molecular modeling studies using MacSpartan indicated that the two active 2-thioxopyrimidine analogs preferably adopt a twisted conformation, similar to CA-4, affirming that conformation and structure are connected to activity.

Modifying the N-terminus of polyamides: PyImPyIm has improved sequence specificity over f-ImPyIm.

Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm (1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5microM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm (4) produced a footprint of 0.5microM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm (4) was interesting as the DeltaT(m) for this compound was only 4 degrees C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (DeltaC(p)) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (-146calmol(-1)K(-1)). SPR results provided confirmation of the sequence specificity of PyImPyIm (4), with a K(eq) value determined to be 7.1x10(6) M(-1) for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm (4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm (4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm (1) is significantly improved.

Syntheses and Cytotoxic Properties of the Curcumin Analogs 2,6‐Bis(benzylidene)‐4‐phenylcyclohexanones

Fifteen curcumin analogs were synthesized and tested for in‐vitro cytotoxicity towards B16 and L1210 murine cancer cell lines using an MTT assay. Significant activity was discovered for two analogs: 8 (B16 IC50 = 1.6 μM; L1210 IC50 = 0.35 μM) and 9 (B16 IC50 = 0.51 μM; L1210 IC50 = 1.2 μM). Several other analogs exhibited notable cytotoxicity. The data from quantitative structure‐activity relationships suggest that large electron‐withdrawing substituents placed in the meta‐position of the arylidene aryl rings enhance potencies. Compounds 8 and 9 were found using a cell‐based assay to have virtually no effects on microtubules at concentrations up to 40 μM. These results suggest that tubulin inhibition is not the principal mechanism by which the curcumin analogs act.

Solution Phase Synthesis of Imidazole- and Pyrrole-containing Hairpin Polyamides

Hairpin imidazole (I)and pyrrole (P)-containing polyamide analogs of distamycin that bind at specific sequences in the minor groove of DNA are potentially useful gene control agents. There is a pressing need for efficient syntheses of such polyamides. The syntheses of four specific hexaheterocyclic hairpin polyamides (PIPγ -PPP 1, PIPγ -PII 2, PIPγ -III 3, and PIPγ -IPI 4, γ represents 4-aminobutyrate) using a solution phase approach are reported.

2-Aminopurine/cytosine base pair containing oligonucleotides: fluorescence spectroscopy studies on DNA-polyamide binding.

Studies on the binding of a triamide f-IPI (1) to its cognate sequence labeled with a 2-aminopurine (2AP or G( *)) group are described. ITC studies showed that f-IPI (1) bound to the cognate site (ACG( *)CGT) with only 3.5-fold lower affinity than binding to the unlabeled DNA (ACGCGT) (K(eq)=2 x 10(7) and 7 x 10(7)M(-1), respectively). Titration of f-IPI (1) to both sequences gave strong induced bands at 330 nm via circular dichroism studies. The compound also gave comparable DeltaT(m) values of 5.0 and 7.8 degrees C, respectively. These techniques also proved that the sequence selectivity of f-IPI (1) was uncompromised, as only limited binding to the non-cognate sequence ACCG( *)GT was observed. Fluorescence studies demonstrated a 2:1 ligand:DNA binding motif as anticipated, and indicated that the limit of detection for this technique was 20muM DNA concentration. The results demonstrate that 2-aminopurine is a sufficient substitute for guanine in a G.C base pair useful in DNA binding studies.

N ‐Formamido‐Containing Mono‐ and Diheterocyclic Pyrrole‐and Imidazole‐2‐carboxylic Acids as Building Blocks for Polyamide Synthesis

Abstract Four N‐formamido‐containing mono‐and diheterocyclic pyrrole‐ and imidazole‐2‐containing acids 1–4 were synthesized as intermediates for the preparation of polyamide molecules. The N‐formamido‐moiety forces the compounds to bind strongly as a stacked dimer, and in a staggered fashion, at specific sequences in the minor‐groove of DNA. The acid moiety at the C‐terminus of compounds enables these molecules to be coupled to amine‐containing intermediates to form the amide linkages of the target polyamide. This convergent approach increases the synthetic diversity in polyamide chemistry by enabling one acid to be used with a variety of different C‐terminus‐functionalized intermediates.

DNA Recognition: Design, Synthesis and Biophysical Characteristics of Pyrrole(H) Based Polyamides

N-Methyl imidazole (Im) and N-methyl pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific DNA sequences in the minor groove of DNA and control gene expression. Polyamides are being investigated as potential medicinal agents for treating diseases including cancer. The naturally occurring polyamide distamycin binds as a dimer in the minor groove of DNA and recognizes sequences rich in A/T and T/A base pairs indiscriminately. Synthetic analogs of distamycin that incorporate N-methylimidazole into the heterocyclic core have been shown to bind to G/C rich sequences with a high degree of specificity. The purpose of this study is to investigate the behavior of polyamides containing the 2,5-linked N-methylpyrrole-2-carboxamide or pyrrole(H) [Py(H)] moiety upon binding to DNA. The synthesis and biophysical characteristics of two polyamides PyPyPyPy(H) 2 and ImPyPyPy(H) 3 designed to test the binding preference of a Py/Pyrrole(H) pairing [Py/Py(H)] and a [Im/Py(H)] is described. Studies utilizing circular dichroism, thermal denaturation (ΔT(M)), biosensor-surface plasmon resonance and DNase I footprinting show that an [Im/Py(H), 3] pairing prefers a G/C or C/G pairing whilst a [Py/Py(H), 2] pairing tolerates A/T or T/A base pairs and avoids a G/C base pair.

Abstract 3521: Pharmacological modulation of Topoisomerase IIα expression and confluence-induced resistance to etoposide by a novel fluorescent HxIP polyamide

Pyrrole (P)-Imidazole (I) containing polyamides are small synthetic molecules which can target predetermined DNA sequences with high affinity and modulate gene expression by interfering with the binding of transcription factors to DNA. As a model system we have previously used the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα promoter and shown that targeted polyamides inhibit the binding of the transcription factor NF-Y and induce expression of topo IIα in confluent cancer cells. Critical to this approach is the need for low molecular weight polyamides that readily enter in the nucleus. In this study, the fluorophore p-anisylbenzimidazolecarboxamido (Hx) moiety was rationally designed to mimic the recognition of A/T base pairs by two consecutive pyrrole units. The hybrid polyamide HxIP is the first example of a small fluorescent molecule that fluoresces significantly more intensely upon binding to its target sequence, 5′-TACGAT-3′ of the 5′-flank of ICB2. Sequence specificity was confirmed by DNAse I footprinting, with HxIP binding with increased affinity compared to the corresponding triamides/tetraamides (f-PIP/PPIP). Thermal denaturation, circular dichroism, and Surface Plasmon Resonance studies confirmed the sequence specificity of HxIP. A dose-dependent inhibition of protein binding to ICB2 by HxIP with complete abolition at concentrations >3 μM was seen in an Electrophoretic Mobility Shift Assay (EMSA). A supershift assay confirmed NF-Y binding to the target sequence. HxIP was also able to displace bound protein factors from DNA at the same concentrations. The incorporation of Hx in the hybrid molecule provides an intrinsic probe to directly monitor cellular uptake and migration into the nucleus. Confocal microscopy in both fixed and live NIH3T3 and A549 cells showed that HxIP is readily taken up by cells and quickly localises in the cell nucleus within 5 minutes. Exposure of confluent cells to 20-40 μM HxIP resulted in time-dependent upregulation of topo IIα expression, reaching levels comparable to those of proliferating cells within 24h as shown by immunoblotting analysis. Confluence-associated repression of topo IIα expression contributes to cellular resistance to agents such as etoposide. De-repression of topo IIα by pre-incubation with HxIP was shown to resensitise NIH3T3 cells to the cytotoxic effect of etoposide (MTT assay) and enhance its DNA damaging effects by increasing levels of etoposide-induced DNA strand breaks as assessed by the single cell gel electrophoresis (comet) assay and the phosphorylation of H2AX. Subcellular localisation of HxIP and potential synergies with etoposide treatment is currently being investigated in vivo against human tumour xenografts. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3521.