Effects of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, cellular uptake, and gene expression.

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.

In Vitro and In Vivo Activity of Peptidomimetic Compounds That Target the Periodontal Pathogen Porphyromonas gingivalis.

The interaction of the periodontal pathogen Porphyromonas gingivalis with oral streptococci is important for initial colonization of the oral cavity by P. gingivalis and is mediated by a discrete motif of the streptococcal antigen I/II protein. A synthetic peptide encompassing this motif functions as a potent inhibitor of P. gingivalis adherence, but the use of peptides as topically applied therapeutic agents in the oral cavity has limitations arising from the relatively high cost of peptide synthesis and their susceptibility to degradation by proteases expressed by oral organisms. In this study, we demonstrate the in vitro and in vivo activity of five small-molecule mimetic compounds of the streptococcal peptide. Using a three-species biofilm model, all five compounds were shown to effectively inhibit the incorporation of P. gingivalis into in vitro biofilms and exhibited 50% inhibitory concentrations (IC50s) of 10 to 20 µM. Four of the five compounds also significantly reduced maxillary alveolar bone resorption induced by P. gingivalis infection in a mouse model of periodontitis. All of the compounds were nontoxic toward a human telomerase immortalized gingival keratinocyte cell line. Three compounds exhibited slight toxicity against the murine macrophage J774A.1 cell line at the highest concentration tested. Compound PCP-III-201 was nontoxic to both cell lines and the most potent inhibitor of P. gingivalis virulence and thus may represent a novel potential therapeutic agent that targets P. gingivalis by preventing its colonization of the oral cavity.

DNA-Binding Properties of New Fluorescent AzaHx Amides: Methoxypyridylazabenzimidazolepyrroleimidazole/pyrrole.

DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.

Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA.

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis.

Alternate pathway for the click reaction of 2-(2-azidophenyl)-4,5-diaryloxazoles.

The CuSO4/ascorbate-mediated 'click' reaction of 2-(2-azidophenyl)-4,5-diaryloxazoles and arylacetylenes proceeded through an alternate pathway whereby reduction of the azide predominated over formation of the 1,2,3-triazole-forming cycloaddition. The unimolecular product, 2-(2-aminophenyl)-4,5-diphenyloxazole, was isolated which appears to be a formal reduction of the arylazide to the corresponding arylamine. A series of oxazoles which possessed various substituents (F, Cl, Br, OCH3) on the 4,5-diaryl rings and having the 2-azido group on the 2-oxazolylphenyl position were submitted to the same 'click' conditions and gave the corresponding arylamine products (73-99%). The reaction appears to be specific toward the ortho-azido substitution of the polycyclic system, as the corresponding azidomethyl-substituted phenyl oxazoles do not give the 'reduction' products but gave the expected click products with the acetylenic co-reactants.

The intramolecular click reaction using 'carbocontiguous' precursors.

The synthesis and utilization of all carbon-chain 'carbocontiguous' azidoalkynyl precursors for an intramolecular click reaction is described. The substrates contain both azidoalkyl and ethynylmethyl groups which are conjoined by a 2-(phenylsulfonylmethyl)-4,5-diphenyloxazole lynchpin and are suitably disposed for ring closure. On promotion by copper salts, a number of cyclic click products having the 1,4-disubstituted endo-fused triazole component and the 4,5-diphenyloxazole component are obtained. In one case, removal of the phenylsulfonylmethyl group from the substrate prior to cyclization gave the 1,5-disubstituted exo-fused triazole. The utilization of CuSO4/sodium ascorbate system appears to be the optimal conditions for closure/cyclization and afforded the cyclized products in yields of 84-95%.

m-Chloroperbenzoic acid-oxchromium (VI)-mediated cleavage of 2,4,5-trisubstituted oxazoles.

An array of 2-substituted-4,5-diphenyloxazoles were found to be cleaved to triacylamines and diacylamines (imides) using a reagent system composed of 3-chloroperbenzoic acid (MCPBA) and 2,2'-bipyridinium chlorochromate (BPCC). The 2-alkyl-4,5-diphenyloxazoles give imides (38-60%) as the predominant cleavage product while the 2-aryl-4,5-diphenyloxazoles give triacylamines (44-71%). Two mechanisms involving intermediates such as cyclic endoperoxides or oxachromacycles were proposed. An application of the oxidative cleavage to the multi-step synthesis of (±)-phoracantholide I seco acid is detailed.

Selective alkylation/oxidation of N-substituted isoindolinone derivatives: synthesis of N-phthaloylated natural and unnatural α-amino acid analogues.

The interchangeability of the isoindolinone group as a nitrogen protecting group for amino acid intermediates is demonstrated by the preparation of several natural and unnatural α-amino acid derivatives using a two-carbon N-isoindolinone (phthalimidine) scaffold. Using a selective benzylic oxidation, the N-isoindolinone group is then converted to the N-phthaloyl group for convenient removal (65-98%). For preparation of the isoindolinone products which were to be the substrates for benzylic oxidation, a range of side chains were installed on the isoindolinone-protected glycine equivalent on deprotonation to demonstrate the utility of the N-protected isoindolinone synthon (51-93%). While the ensuing benzylic oxidation is employed successfully for converting the N-isoindolinone group to the N-phthaloyl group in simple substrates, substrates bearing unsaturated or electron-rich side chains respond poorly to the oxidation.

Synthesis of Extended Oxazoles III: Reactions of 2-(Phenylsulfonyl)methyl-4,5-diaryloxazoles.

2-((Phenylsulfonyl)methyl)-4,5-diphenyloxazole is a useful scaffold for synthetic elaboration at the 2-methylene position thereby affording extended oxazoles. The corresponding α-sulfonyl anion reacts smoothly with diverse alkyl halides giving monoalkylated (47-90%), dialkylated (50-97%), and cyclic (59-93%) products. The reductive desulfonylation of the monoalkylated and selected dialkylated products was optimized with a magnesium/mercuric chloride reagent system and afforded desulfonylated products in the range of 66-97%. The anti-inflammatory Oxaprozin was prepared using the α-sulfonyl carbanion strategy along with optimized desulfonylation.

1,2,3-Triazole-based inhibitors of Porphyromonas gingivalis adherence to oral streptococci and biofilm formation.

The development and use of small-molecule inhibitors of the adherence of Porphyromonas gingivalis to oral streptococci represents a potential therapy for the treatment of periodontal disease as these organisms work in tandem to colonize the oral cavity. Earlier work from these laboratories demonstrated that a small synthetic peptide was an effective inhibitor of the interaction between P. gingivalis and Streptococcus gordonii and that a small-molecule peptidomimetic would provide a more stable, less expensive and more effective inhibitor. An array of 2-(azidomethyl)- and 2-(azidophenyl)-4,5-diaryloxazoles having a full range of hydrophobic groups were prepared and reacted with substituted arylacetylenes to afford the corresponding 'click' products. The title compounds were evaluated for their ability to inhibit P. gingivalis' adherence to oral streptococci and several were found to be inhibitory in the range of (IC50) 5.3-67µM.

Synthesis of extended oxazoles II: Reaction manifold of 2-(halomethyl)-4,5-diaryloxazoles.

2-(Halomethyl)-4,5-diphenyloxazoles are effective, reactive scaffolds which can be utilized for synthetic elaboration at the 2-position. Through substitution reactions, the chloromethyl analogue is used to prepare a number of 2-alkylamino-, 2-alkylthio- and 2-alkoxy-(methyl) oxazoles. The 2-bromomethyl analogue offers a more reactive alternative to the chloromethyl compounds and is useful in the C-alkylation of a stabilized (malonate) carbanion as exemplified by a concise synthesis of Oxaprozin.

Oxazoles for click chemistry II: synthesis of extended heterocyclic scaffolds.

New routes to 2, 4, 5-trisubstituted oxazoles were established whereby the substitution pattern was established by the structure of the starting nonsymmetrical acyloins. 2-Chloromethyl-4, 5-disubstituted oxazoles were prepared by refinements of an earlier described process whereby chloroacetyl esters of symmetrical and non-symmetrical acyloins were cyclized using an ammonium acetate/acetic acid protocol. After substitution is effected, the azide moiety is then installed by substitution under mild conditions. While dibrominated and iodinated phenyloxazoles are required for further synthetic elaboration, the cyclization reaction was found to be very sensitive to the relative positions of the halogens in the starting materials.

'Second-generation' 1,2,3-triazole-based inhibitors of Porphyromonas gingivalis adherence to oral streptococci and biofilm formation.

Several 'second-generation' click inhibitors of the multi-species biofilm propagated by the adherence of the oral pathogen Porphyromonas gingivalis to Streptococcus gordonii were synthesized and evaluated. The design of the structures was based on the results obtained with the first-generation diphenyloxazole 'click' inhibitors which bear suitable hydrophobic and polar groups within a dual scaffold molecule bearing a 1,2,3-triazole spacer. The structures of the synthetic targets reported herein now consist of a triazolyl(phenylsulfonylmethyl) and a triazolyl(phenylsulfinylmethyl) spacer which joins a 4,5-diphenyloxazole with both phenyl rings bearing lipophilic substituents. The triazolyl "linker" group is formed by a click reaction between the 4-azido(phenylsulfonyl/sulfinylmethyl) oxazoles and acetylenic components having aryl groups bearing hydrophobic substituents. The 1,3,5-trisubstituted-2,4,6-triazine scaffold of the most active click compounds were modeled after the structural motif termed the VXXLL nuclear receptor (NR) box. When substituted at the 3- and 5-positions with 2- and 4-fluorophenylamino and N,N-diethylamino units, the candidates bearing the 1,3,5-trisubstituted-2,4,6-triazine scaffold formed a substantial subset of the second-generation click candidates. Four of the click products, compounds 95, 111, 115 and 122 showed inhibition of the adherence of P. gingivalis to S. gordonii with an IC50 range of 2.3-4.3 µM and only 111 exhibited cytotoxic activity against telomerase immortalized gingival keratinocytes at 60 µM. These results suggest that compounds 95, 115, 122, and possibly 111 represent the most suitable compounds to evaluate for activity in vivo.

Unnatural Amino Acid Derivatives through Click Chemistry: Synthesis of Triazolylalanine Analogues.

A novel tert-butyl 2-(1-oxoisolndolin-2-yl)acetate derivative is selectively alkylated with propargyl bromide in the presence of lithium hexamethyldisilazide. After removal of the tert-butyl protecting group, the resulting N-isoindolinyl (ethynylalanine) derivative is reacted with a series of azides under 'click conditions'. The click reactions afford an array of N-isoindolinyl-1,2,3-triazolylalanine derivatives as the free carboxylic adds. Following esterification, the N-isoindolinone protecting group is then transformed into the more easily removable phthaloyl group by selective oxidation at the benzylic position.

A Short Review on the Synthetic Strategies of Duocarmycin Analogs that are Powerful DNA Alkylating Agents.

The duocarmycins and CC-1065 are members of a class of DNA minor groove, AT-sequence selective, and adenine-N3 alkylating agents, isolated from Streptomyces sp. that exhibit extremely potent cytotoxicity against the growth of cancer cells grown in culture. Initial synthesis and structural modification of the cyclopropa[c] pyrrolo[3,2-e]indole (CPI) DNA-alkylating motif as well as the indole non-covalent binding region in the 1980s have led to several compounds that entered clinical trials as potential anticancer drugs. However, due to significant systemic toxicity none of the analogs have passed clinical evaluation. As a result, the intensity in the design, synthesis, and development of novel analogs of the duocarmycins has continued. Accordingly, in this review, which covers a period from the 1990s through the present time, the design and synthesis of duocarmycin SA are described along with the synthesis of novel and highly cytotoxic analogs that lack the chiral center. Examples of achiral analogs of duocarmycin SA described in this review include seco-DUMSA (39 and 40), seco-amino-CBI-TMI (13, Centanamycin), and seco-hydroxy-CBI-TMI (14). In addition, another novel class of biologically active duocarmycin SA analogs that contained the seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) DNA alkylating submit was also designed and synthesized. The synthesis of seco-iso-CFI-TMI (10, Tafuramycin A) and seco-CFQ-TMI (11, Tafuramycin B) is included in this review.

Recent advancement in discovery and development of natural product combretastatin-inspired anticancer agents.

The natural stilbenoids combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are potent antitubulin agents demonstrating antimitotic activity as well as tumor vascular disruption property. Due to structural simplicity and potent cytotoxicity of CA4 and CA1, they are considered as promising leads for the development of potent anticancer agents. In fact, scientific fraternity is motivated to synthesize several derivatives of CA4 and CA1 as novel therapeutic agents. In the literature, several studies have been carried out to evaluate the medicinal chemistry, pharmacology and structure-activity relationships (SAR) of a variety of modified combretastatin derivatives. The present report aimed at comprehensively revising the recent advancements (2006-2014) in the medicinal chemistry and SAR of diversified combretastatin analogues. The published data concerning new combretastatin A-4 analogues as antimitotic anticancer agents are presented and SAR is reviewed and discussed.