Hilary Mackay

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.

Synthesis and Evaluation of an Intercalator–Polyamide Hairpin Designed to Target the Inverted CCAAT Box 2 in the Topoisomerase IIα Promoter

The synthesis and DNA‐binding properties of a novel naphthalimide–polyamide hairpin (3) designed to target the inverted CCAAT box 2 (ICB2) site on the topoisomerase IIα (topoIIα) promoter are described. The polyamide component of 3 was derived from the minor‐groove binder, 2, and tailored to bind to the 5′‐TTGGT sequence found in and flanking ICB2. The propensity of mitonafide 4 to intercalate between G–C base pairs was exploited by the incorporation of a naphthalimide moiety at the N terminus of 2. Hybrid 3 targeted 5′‐CGATTGGT and covered eight contiguous base pairs, which included the underlined ICB2 site. DNase I footprinting analysis with the topoIIα promoter sequence demonstrated that 3 bound selectively to the ICB2 and ICB3 sites. Thermal‐denaturation studies confirmed these results, and the highest degree of stabilization was found for ICB2 and ‐3 in preference to ICB1 (4.1, 4.6, and 0.6 °C, respectively). CD studies confirmed minor‐groove binding and suggested a 1:1 binding stoichiometry. Emission‐titration experiments established intercalative binding. Surface plasmon resonance results showed strong binding to ICB2 (2.5×107 M−1) with no observable binding to ICB1. Furthermore, the binding constant of 3 to ICB2 was larger than that of the parent polyamide 2. The increased binding affinity was primarily due to a reduction in the dissociation‐rate constant of the polyamide–DNA complex, which can be attributed to the N‐terminal naphthalimide moiety. In addition, the binding site of 3 was larger than that of 2, which innately improved sequence selectivity. We conclude that the polyamide–naphthalimide 3 selectively binds to the ICB2 site by simultaneous intercalation and minor‐groove binding, and warrants further investigation as a model compound for the regulation of topoIIα gene expression.

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.

Synthesis and DNA-binding Properties of 1,2,3-triazole-linked H-pin Pyrrole- and Imidazole-containing Polyamides Formed by the Huisgen Reaction

Abstract Covalently linked pyrrole (Py)- and imidazole (Im)-containing H-pin polyamides bind in the minor groove of specific DNA sequences with high affinity. The synthesis of 1,2,3-triazole-linked and heterodimeric H-pin polyamides 13a,b formed from the Huisgen reaction of an alkyne-containing f-PyPyPy (6) with an azide-containing f-ImPyIm (7) is reported. The reaction proceeded smoothly under thermal conditions to give an inseparable mixture of 1,4- and 1,5-isomers (13a and 13b, respectively) by column chromatography. When the reaction was conducted under ‘click’ or Cu(I)-catalyzed conditions or in the presence of the cognate DNA sequence, no desired product was observed. Preliminary results from DNA thermal denaturation and circular dichroism titration studies provided evidence of mixture 13a,b binding to the target DNA sequence 5′-TCTCAA-3′.

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.

Synthesis and biophysical studies of hairpin polyamides targeting the Brn-3b and GATA-3 transcriptional sites

Abstract Hairpin polyamide analogs of distamycin A (1) were designed and synthesized to evaluate their ability to bind 5′-ATAGA-3′ and 5′-AGATA-3′ sequences which are important elements for controlling the function of the Brn-3b and GATA-3 transcriptional factors, respectively. The hairpin polyamides are composed of pyrrole and imidazole units linked together via a γ-aminobutyrate (GABA) unit. Hairpins 2b (Py-Py-Im-γ-Py-Py-Py) and 2c (Im-Py-Py-γ-Py-Py-Py) were synthesized to target the respective Brn-3b and GATA-3 cognate sequences. Preliminary biophysical studies including thermal denaturation and circular dichroism were performed and the results ascertained the binding of hairpins 2a and 2b to their respective cognate DNA sequences.

Synthesis and Biophysical Testing of a Novel Pyrrole-Containing Polyamide-Benzamidine Hybrid

This communication details the rationale for the design of a novel polyamide molecule, 1, in which the standard dimethylamino C-terminus functionality has been replaced with a benzamidine moiety. The synthesis of this molecule and the subsequent DNA binding properties, determined from surface plasmon resonance and DNA melts are reported. The benzamidine moiety was shown to significantly increase the binding affinity of the polyamide to its cognate A A T T T sequence compared to the parent C-terminus compound 2.