Synthesis of Lipid Derivatives of Pyrrole Polyamide and Their Biological Activity

Novel fatty acyl and phospholipid derivatives of pyrrole polyamide were synthesized. Their cytotoxicity against a cancer cell line of MT-4 cells and those infected by human immunodeficiency virus (HIV) was examined. Although no anti-HIV activity was found, their cytotoxicitty against the cancer cells was significantly enhanced by introducing a lipophilic group into the pyrrole polyamide.     

Synthesis of pyrrole-imidazole polyamide oligomers based on a copper-catalyzed cross-coupling strategy

Pyrrole-imidazole (Py-Im) polyamides are useful tools for chemical biology and medicinal chemistry studies due to their unique binding properties to the minor groove of DNA. We developed a novel method of synthesizing Py-Im polyamide oligomers based on a Cu-catalyzed cross-coupling strategy. All four patterns of dimer fragments could be synthesized using a Cu-catalyzed Ullmann-type cross-coupling with easily prepared monomer units. Moreover, we demonstrated that pyrrole dimer, trimer, and tetramer building blocks for Py-Im polyamide synthesis were accessible by combining site selective iodination of the pyrrole/pyrrole coupling adduct.     

Modulation of the EMT/MET process by pyrrole–imidazole polyamide targeting human transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial–mesenchymal transition (EMT). Mesenchymal–epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole–imidazole (PI) polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

Synthesis and evaluation of oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids as novel gene expression control compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T(m) values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.     

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.     

Synthesis and Evaluation of Oligonucleotide-Conjugated Pyrrole Polyamide-2′-Deoxyguanosine Hybrids as Novel Gene Expression Control Compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2′-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T_m values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.     

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.     

Alkylation of template strand of coding region causes effective gene silencing

We recently developed a new type of pyrrole (Py)–imidazole (Im) polyamide–tetrahydrocyclopropabenzindolone (CBI) conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing activities of polyamides A, B and C, which selectively alkylate specific sequences in the promoter region, non-coding strand and coding strand, respectively, of the green fluorescent protein (GFP) gene. GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells were treated with 100 nM of the polyamides for 24 h. Fluorescence microscopy indicated that a significant reduction of GFP fluorescence was only observed in the cells that were treated with polyamide C. In clear contrast, polyamides A and B did not show such activity. Moreover, real-time PCR demonstrated selective reduction of the expression of GFP mRNA following treatment with polyamide C. These results suggest that alkylating Py–Im polyamides that target the coding strand represent a novel approach for sequence-specific gene silencing.     

Synthesis and DNA binding properties of 1-(3-aminopropyl)-imidazole-containing triamide f-Im(∗)PyIm: A novel diamino polyamide designed to target 5'-ACGCGT-3'

A novel diamino/dicationic polyamide f-Im(?)PyIm (5) that contains an orthogonally positioned aminopropyl chain on an imidazole (Im(?)) moiety was designed to target 5'-ACGCGT-3'. The DNA binding properties of the diamino polyamide 5, determined by CD, ΔT(M), DNase I footprinting, SPR, and ITC studies, were compared with those of its monoamino/monocationic counterpart f-ImPyIm (1) and its diamino/dicationic isomer f-ImPy(?)Im (2), which has the aminopropyl group attached to the central pyrrole unit (Py(?)). The results gave evidence for the minor groove binding and selectivity of polyamide 5 for the cognate sequence 5'-ACGCGT-3', and with strong affinity (K(eq)=2.3×10(7)M(-1)). However, the binding affinities varied according to the order: f-ImPy(?)Im (2)>f-ImPyIm (1)?f-Im(?)PyIm (5) confirming that the second amino group can improve affinity, but its position within the polyamide can affect affinity.     

Chloroalkyl piperazine and nitrogen mustard porphyrins: synthesis and anticancer activity

Fifteen new chloroalkyl piperazine and nitrogen mustard porphyrins have been synthesized by the direct condensation of chloroalkyl piperazine, nitrogen mustard benzaldehyde, and pyrrole. Each porphyrin bears 1-4 chloroalkyl piperazine or nitrogen mustard moieties, which have been used as drugs. The Lindsey method was modified to synthesize chloroalkyl piperazine and nitrogen mustard porphyrins. To successfully synthesize chloroalkyl piperazine and nitrogen mustard porphyrins, catalyst acidity was proved to be the key factor, while the ratio of pyrrole to aldehyde had great influence on product yield. The synthetic chloroalkyl piperazine and nitrogen mustard porphyrins were characterized by elementary analysis, MS, (1)H NMR, IR, and UV-vis. Their anticancer activity to bel-7404 liver cancer cells was tested by the MTT assay. Most of the synthetic porphyrins had good anticancer activity toward bel-7404 liver cancer cells in the absence of light. These compounds might be potential anticancer medicines.     

Design, synthesis, and biological evaluation of 4-phenylpyrrole derivatives as novel androgen receptor antagonists

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer.     

Antimycobacterial compounds. New pyrrole derivatives of BM212

We have identified BM212 as a lead compound among a series of pyrrole derivatives with good in vitro activity against mycobacteria and candidae. First studies led us to synthesize some pyrrole compounds in which the thiomorpholine fragment was present. Some compounds revealed very active and these findings prompted us to prepare new pyrrole derivatives 2-15 in the hope of increasing the activity. The microbiological data showed interesting in vitro activity against Mycobacterium tuberculosis and atypical mycobacteria.     

Synthesis and antitumor activity of duocarmycin derivatives: modification at the C-7 position of segment-A of A-ring pyrrole compounds

A series of the C7-substituted A-ring pyrrole derivatives of duocarmycin were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. All of the C7-substituted A-ring pyrrole compounds decreased potency in vitro and in vivo. However, some showed strong antitumor activity with T/C values less than 0.3. Among them, the 7-formyl compound 5d showed remarkable potent in vivo antitumor activity and low peripheral blood toxicity, which were equal to 2c.     

Potent activity against K562 cells by polyamide–seco-CBI conjugates targeting histone H4 genes

We designed and synthesized conjugates between pyrrole–imidazole polyamides and seco-CBI that alkylate within the coding regions of the histone H4 genes. DNA alkylating activity on the histone H4 fragment and cellular effects against K562 chronic myelogenous leukemia cells were investigated. One of the conjugates, 5-CBI, showed strong DNA alkylation activity and good sequence specificity on a histone H4 gene fragment. K562 cells treated with 5-CBI down-regulated the histone H4 gene and induced apoptosis efficiently. Global gene expression data revealed that a number of histone H4 genes were down-regulated by 5-CBI treatment. These results suggest that sequence-specific DNA alkylating agents may have the potential of targeting specific genes for cancer chemotherapy.     

Synthesis of indolizidinone analogues of cytotoxic alkaloids: monocyclic precursors are also active

Readily available proline derivatives can be transformed in just two steps into analogues of cytotoxic phenanthroindolizidine alkaloids. The key step uses a sequential radical scission-oxidation-alkylation process, which yields 2-substituted pyrrolidine amides. A second process effects the cyclization to give the desired alkaloid analogues, which possess an indolizidine core. The major and minor isomers (dr 3:2 to 3:1) can be easily separated, allowing their use to study structure-activity relationships (SAR). The process is versatile and allows the introduction of aryl and heteroaryl groups (including biphenyl, halogenated phenyl, and pyrrole rings). Some of these alkaloid analogues displayed a selective cytotoxic activity against tumorogenic human neuronal and mammary cancer cells, and one derivative caused around 80% cell death in both tumor lines at micromolar doses. The cytotoxicity of some monocyclic precursors was also studied, being comparable or superior to the bicyclic derivatives.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.