Vijay Satam

Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)•pyrrole and Hx•imidazole pairings

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.

Development of a Scalable Process for the Synthesis of DNDI-VL-2098: A Potential Preclinical Drug Candidate for the Treatment of Visceral Leishmaniasis

A process suitable for kilogram-scale synthesis of (2R)-2-methyl-6-nitro-2-{[4-(trifluoromethoxy)phenoxy]methyl}-2,3-dihydroimidazo[2,1-b][1,3]oxazole (DNDI-VL-2098, 2), a preclinical drug candidate for the treatment of visceral leishmaniasis, is described. The four-step synthesis of the target compound involves the Sharpless asymmetric epoxidation of 2-methyl-2-propen-1-ol, 8. Identification of a suitable synthetic route using retrosynthetic analysis and development of a scalable process to access several kilograms of 2 are illustrated. The process was simplified by employing in situ synthesis of some intermediates, reducing safety hazards, and eliminating the need for column chromatography. The improved reactions were carried out on the kilogram scale to produce 2 in good yield, high optical purity, and high quality.

Orthogonally Positioned Diamino Pyrrole- and Imidazole-Containing Polyamides: Synthesis of 1-(3-Substituted-propyl)-4-nitropyrrole-2-carboxylic Acid and 1-(3-Chloropropyl)-4-nitroimidazole-2-carboxylic Acid

Abstract Pyrrole- and imidazole-containing polyamides can be tailored to recognize the DNA 6–8 base pair sequence. We found that adding a second amino group via the N1-position of pyrrole or imidazole in polyamides could enhance their DNA binding affinity and water solubility while retaining sequence specificity. Synthesis of the key 1-substituted-4-nitropyrrole (and imidazole)-2-carboxylic acid building blocks are described. [Supplementary materials are available for this article. Go to the publishers online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.] GRAPHICAL ABSTRACT

Conformational modulation of DNA by polyamide binding: structural effects of f-Im-Py-Im based derivatives on 5′-ACGCGT-3′

The DNA sequence 5′‐ACGCGT‐3′ is in the core site of the Mlu 1 cell‐cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido‐imidazole‐pyrrole‐imidazole‐amine+ (f‐Im‐Py‐Im‐Am+) can target the minor groove of 5′‐ACGCGT‐3′ as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f‐Im‐Py‐Im‐Am+ derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis–ligation ladders assay was used to evaluate the conformational effects of f‐Im‐Py‐Im‐Am+ and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide–DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B‐form like overall structure. After binding with f‐Im‐Py‐Im‐Am+ derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed. Copyright

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′.

Design, Synthesis, and Cytotoxicity of Novel 3‐Arylidenones Derived from Alicyclic Ketones

Forty‐four novel chalcone‐inspired analogs having a 3‐aryl‐2‐propenoyl moiety derived from alicyclic ketones were designed, synthesized, and investigated for cytotoxicity against murine B16 and L1210 cancer cell lines. The analogs belong to four structurally divergent series, three of which (series g, h, and i) contain differently substituted cyclopentanone units and the fourth (series j) contains a 3,3‐dimethyl‐4‐piperidinone moiety. Of these, the analogs in series j showed potential cytotoxic activity against murine B16 (melanoma) and L1210 (lymphoma) cells. The most active compounds 5j, 11j, 15j, and 12h produced IC50 values from 4.4 to 15 μm against both cell lines. A single‐crystal X‐ray structure analysis and molecular modeling studies confirmed that these chalcones have an E‐geometry about the alkene bond and possess a slightly ‘twisted’ conformation similar to that of combretastatin A‐4. At a concentration of 30 μm, compounds 5j, 11j, and 15j did not cause microtubule depolymerization in cells, suggesting that they have a different mechanism of action.

5-Benzyl-idene-3-phenyl-2-phenyl-imino-1,3-thia-zolidin-4-one.

The title compound, C22H16N2OS, is a chalcone analog with a thiazolidinone core that was synthesized as a potential cytotoxic and anticancer agent. The structure is commensurately modulated by unit-cell doubling along the direction of the a axis of the cell. The two crystallographically independent molecules are differerentiated by the dihedral angle between the mean planes of the benzylidene phenyl group against the thiazolidin-4-one moiety, which is 5.01 (7)° in one molecule, and 17.41 (6)° in the other. The two molecules are otherwise close to being indistinguishable and are related by crystallographic pseudo-translation. The two molecules are not planar but are slightly bent with the benzylidene and phenylimino substituents being bent upwards with respect to the center planes of the two molecules. The degree of bending of the two halves of the thiazolidin-4-one moieties (defined as the planes that intersect at the S atom) are 11.08 (7) and 15.88 (7)°. Packing of the molecules is facilitated by C—H⋯π interactions and slipped π–π stacking between one of the phenyl rings and a neighboring ethylene π system [distance between the centroid of the ethylene group and the closest phenyl C atom = 3.267 (2) Å, Cg(phenyl)⋯Cg(ethylene) = 3.926 Å].

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.

Abstract 3755: Genome-wide transcriptome analysis reveals the distinct gene expression profiles and specific biological effects of fluorescent polyamides, HxIP and azaHxPI

DNA-binding polyamides target predetermined sequences to inhibit specific transcription factor-DNA interactions and modulate gene expression. Inherently non-genotoxic, these non-covalent binding small molecules exert their biological activity without inflicting DNA damage and provide the basis for potentially less toxic DNA-targeting anticancer therapeutics. However, the genome-wide specificity of polyamide binding remains an outstanding issue in delivering the desired biological response without inducing widespread off-target effects. In this study, we used RNA-seq transcriptome analysis to assess the global effects of fluorescent polyamides HxIP and azaHxPI, having previously shown that they bind with high affinity and selectivity to their respective target DNA sequences 5’-WWCGWW-3’ and 5’-WCGCGW-3’ (W = A/T)1,2. Changes to the MDA-MB-231 breast adenocarcinoma transcriptome were measured following 24 h treatment with 2 and 5 μM HxIP or azaHxPI. At 5 μM, HxIP affected 349 genes by two-fold (p Following a 96 h treatment HxIP had no effect on cell growth (GI50 > 100 μM), whereas azaHxPI causes growth inhibition (GI50 = 20.3 μM) and induces apoptosis. Immunoblotting analysis of γ;H2AX showed no evidence of DNA damage, further corroborated by the absence of chk2 phosphorylation. In addition, no ATR-chk1 activation was detected suggesting that azaHxPI does not induce replication stress, despite inhibiting DNA synthesis as shown using the BrdU incorporation assay. Levels of phospho-AKT (Ser472) decreased in response to polyamide treatment, whilst p21 is significantly upregulated and both may be implicated in the mechanism of azaHxPI induced-apoptosis. Taken together these results emphasise the potential application of polyamides as apoptosis-inducing DNA-targeting agents benefiting from enhanced sequence selectivity which in turn confers specific biological activity. References: 1. Kiakos. et al Chem. Biol. (2015) 22(7):862-75. 2. Satam. et al Bioorg. Med. Chem. Lett. (2015) 25(17):3681-5. Citation Format: Luke Pett, Vijay Satam, Pravin Patil, Moses Lee, John A. Hartley, Konstantinos Kiakos. Genome-wide transcriptome analysis reveals the distinct gene expression profiles and specific biological effects of fluorescent polyamides, HxIP and azaHxPI. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3755.

Synthesis of 2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-methylpropanoic Acid Ethyl Ester

The title compound was synthesized by N-alkylation of 4-(4-chlorophenyl)piperazine with ethyl 2-bromo-2-methylpropanoate and its IR, 1H NMR, 13C NMR and Mass spectroscopic data are reported.