DNA Sequence-Specific Ligands: XII. Synthesis and Cytological Studies of Dimeric Hoechst 33258 Molecules

Abstract

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.

Structure–Activity Studies with Bis-Amidines That Potentiate Gram-Positive Specific Antibiotics against Gram-Negative Pathogens

“…The synthetic route used to access bis-amidines 21–24 is shown in Scheme 2 and was based largely on the published preparation of these and similar compounds previously evaluated as anti-parasitic agents. 22 , 34 − 39 The meta-oriented linker in compound 22 most closely mimics the 5-carbon spacer found in pentamidine, while analogues 21 and 23 differ slightly due to the ortho- and para-orientations of the benzene core. In the case of compound 24 , a 2,7-disubstituted naphthalene motif was envisioned to mimic the 7-carbon spacer found in heptamidine ( 10 ).…”

“…As shown in Scheme 1 A, the dibenzonitrile intermediates were prepared from the commercially available α,ω-dibromo-alkanes via a Williamson ether synthesis according to literature protocols. 22 Crystallization from ethanol resulted in the pure intermediates 4–8 in good to excellent yields. The transformation of the nitrile groups into the corresponding amidine is classically performed via the Pinner reaction followed by treatment with ammonia.…”

“…As shown in Scheme A, the dibenzonitrile intermediates were prepared from the commercially available α,ω-dibromo-alkanes via a Williamson ether synthesis according to literature protocols . Crystallization from ethanol resulted in the pure intermediates 4–8 in good to excellent yields.…”

“… 32 , 69 Compounds 1 , 2 , 4–6 , 8–11 , 15 , 18 , 19 , 21–23 , 1b , 21b–23b , 39 , and 40 were synthesized using optimized protocols as described below and gave NMR spectra and mass spectra consistent for the same compounds previously described in the literature. 22 , 29 , 32 , 34 , 38 , 39 , 70 − 72 Purity of the final compounds 1–3 , 9–12 , 15 , 16 , 21–24 , 1b , 21b–23b , 1c , 21c–23c , 37 , 38 , 43 , and 44 was confirmed to be ≥95% by analytical RP-HPLC using a Shimadzu Prominence-i LC-2030 system with a Dr. Maisch ReproSil Gold 120 C18 column (4.6 × 250 mm, 5 μm) at 30 °C and equipped with a UV detector monitoring at 214 nm. The following solvent system, at a flow rate of 1 mL/min, was used: solvent A, 0.1% trifluoroacetic acid (TFA) in water/acetonitrile, 95/5 and solvent B, 0.1% TFA in water/acetonitrile, 5/95.…”

“…These conditions were based on literature protocols. 22 4-cyanophenol (0.29 g, 2.4 mmol, 2.4 equiv) was suspended in dry DMF (3 mL) under an argon atmosphere. The suspension was cooled to 0 °C using an ice bath and NaH (96 mg, 60% dispersion in mineral oil, 2.4 equiv) was slowly added.…”