NMR studies of chromomycin A3 interaction with DNA

The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.     

2-(3'-Hydroxypropylidene)-1alpha-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3'-hydroxypropylidene) analogs of 1alpha,25-dihydroxy-19-norvitamin D(3) showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1alpha-hydroxy-19-norvitamin D analogs characterized by the presence of a 3'-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8beta-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig-Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7-11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1alpha,25-(OH)(2)D(3) (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

ent-Pimarane Diterpenes from the Aerial Parts of Siegesbeckia pubescens

Five new ent-pimarane diterpenes ( 1 – 5 ) and five known analogs ( 6 – 10 ) were isolated from the aerial parts of Siegesbeckia pubescens. Their structures, including absolute configurations, were determined by comprehensive spectroscopic methods especially 1D and 2D NMR and quantum chemical electronic circular dichroism calculations. All the isolated compounds were evaluated for their cytotoxicity against human BT549, A549 and H157 cancer cell lines. Among them, compounds 1 and 2 showed mild cytotoxicity against lung cancer cell lines H157 with IC₅₀ values of 16.35±2.59 and 18.86±4.83 μM, respectively.     

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing ¹H-¹⁵N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting ¹⁵N 1D and 2D ¹H-¹⁵N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built ¹H-¹⁵N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.     

Synthesis and antiviral activity of some 2-substituted 3-formyl- and 3-cyano-5,6-dichloroindole nucleosides

A series of dichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The isopropylidene-protected analogs of the previously reported 3-formyl-2,5,6-trichloro-1-(beta-Dribofuranosyl)indole (FTCRI) and 3-cyano-2,5, 6-trichloro-1-(beta-D-ribofuranosyl)indole (CTCRI) were modified by nucleophilic displacement of the 2-chloro substituent using secondary amines. Deprotection of the intermediates provided 2-substituted analogs of FTCRI and CTCRI in good yield. There was a significant difference in reactivity between the isopropylidene-protected and the fully deprotected FTCRI and CTCRI with respect to nucleophilic displacement of the 2-chloro substituent using dialkylamines. This difference in reactivity was not observed with monoalkylamines or with alkoxides, and the corresponding 2-alkylamino- and 2-methoxy substituted analogs were synthesized from FITCRI and CTCRI directly. None of the synthesized analogs demonstrated potent antiviral activity without some corresponding cytotoxicity.     

Design, synthesis, and biological evaluation of achiral analogs of duocarmycin SA

The design, synthesis, as well as biochemical and biological evaluation of two novel achiral analogs of duocarmycin SA (DUMSA), 1 and 2, are described. Like CC-1065 and adozelesin, compounds 1 and 2 covalently reacted with adenine-N3 in AT-rich sequences and led to the formation of DNA strand breaks upon heating. The cytotoxicity of compounds 1 and 2 against human cancer cells (K562, LS174T) was determined using a MTT assay giving IC(50) values in the low nanomolar. Further cytotoxicity screening of compound 2 conducted by the NCI against a panel of 60 different human cancer cell lines indicated that it was particularly active against several solid tumor cells lines derived from the lung, colon, CNS, skin, and breast.     

Heterochromatin composition and nucleolus organizer activity in four canid species

Sequential staining with a counterstain-contrasted fluorescent banding technique (chromomycin A3-distamycin A-DAPI) revealed the occurrence of distamycin A-4,6-diamidino-2-phenylindole (DA-DAPI) staining heterochromatin in the centromeric regions of chromosomes 33, 36, 37, and 38 in the wolf (Canis lupus pallipes) and of chromosomes 13, 16, and 23 in the blue fox (Alopex lagopus). The red fox (Vulpes vulpes) lacked such regions. Staining with DAPI--actinomycin D produced a QFH-type banding pattern with clearcut differences in the staining behaviour of DA-DAPI positive regions between these three canid species. Staining with the fluorochrome D 287/170 did not preferentially highlight any of the DA-DAPI positive regions in any of them. Counterstain-enhanced chromomycin A3 R-banding and studies of nucleolus organizer region location and activity confirmed a close relationship between the karyotype of the wolf and the domestic dog. Few heterochromatic marker bands were encountered in these two species, but heterochromatin polymorphism was evident in the blue fox.     

2('),3(')-didehydro-2('),3(')-dideoxynucleosides are degraded to furfuryl alcohol under acidic conditions

2('),3(')-Didehydro-2('),3(')-dideoxynucleosides are clinically relevant antiviral agents. These nucleosides could be degraded under acidic conditions. Acidic stability studies showed the D4N had the following increasing stability order: D4G相似文献   

Synthesis and biological evaluation of polyhydroxycurcuminoids

A series of curcumin analogs (1-3, 5a-5t) was synthesized through the condensation of appropriately protected hydroxybenzaldehydes with acetylacetone, followed by deprotection. The antioxidant activity of these analogs was determined by superoxide free radical nitroblue tetrazolium and DPPH free radical scavenging methods and the polyhydroxycurcuminoids (5l-5s) displayed excellent antioxidant activity. These analogs showed cytotoxicity to lymphocytes and promising tumor-reducing activity on Dalton's lymphoma ascites tumor cells.     

Synthesis and Antiviral Activity of Some 2-Substituted 3-Formyl-and 3-Cyano-5,6-Dichloroindole Nucleosides

A series of dichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The isopropylidene-protected analogs of the previously reported 3-formyl-2,5,6-trichloro-1-(β-D-ribofuranosyl)indole (FTCRI) and 3-cyano-2,5,6-trichloro-1-(β-D-ribofuranosyl)indole (CTCRI) were modified by nucleophilic displacement of the 2-chloro substituent using secondary amines. Deprotection of the intermediates provided 2-substituted analogs of FTCRI and CTCRI in good yield. There was a significant difference in reactivity between the isopropylidene-protected and the fully deprotected FTCRI and CTCRI with respect to nucleophilic displacement of the 2-chloro substituent using dialkylamines. This difference in reactivity was not observed with monoalkylamines or with alkoxides, and the corresponding 2-alkylamino- and 2-methoxy substituted analogs were synthesized from FTCRI and CTCRI directly. None of the synthesized analogs demonstrated potent antiviral activity without some corresponding cytotoxicity.     

Antitumor agents 290. Design, synthesis, and biological evaluation of new LNCaP and PC-3 cytotoxic curcumin analogs conjugated with anti-androgens

In our continuing study of curcumin analogs as potential anti-prostate cancer drug candidates, 15 new curcumin analogs were designed, synthesized and evaluated for cytotoxicity against two human prostate cancer cell lines, androgen-dependent LNCaP and androgen-independent PC-3. Twelve analogs (5-12, 15, 16, 19, and 20) are conjugates of curcumin (1) or methyl curcumin (2) with a flutamide- or bicalutamide-like moiety. Two compounds (22 and 23) are C4-mono- and difluoro-substituted analogs of dimethyl curcumin (DMC, 21). Among the newly synthesized conjugates compound 15, a conjugate of 2 with a partial bicalutamide moiety, was more potent than bicalutamide alone and essentially equipotent with 1 and 2 against both prostate tumor cell lines with IC(50) values of 41.8 μM (for LNCaP) and 39.1 μM (for PC-3). A cell morphology study revealed that the cytotoxicity of curcumin analogs or curcumin-anti-androgen conjugates detected from both prostate cancer cell lines might be due to the suppression of pseudopodia formation. A molecular intrinsic fluorescence experiment showed that 1 accumulated mainly in the nuclei, while conjugate 6 was distributed in the cytosol. At the tested conditions, anti-androgens suppressed pseudopodia formation in PC-3 cells, but not in LNCaP cells. The evidence suggests that distinguishable target proteins are involved, resulting in the different outcomes toward pseudopodia suppression.     

Synthesis and antiproliferative activity of side-chain unsaturated and homologated analogs of 1,25-dihydroxyvitamin D(2). (24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol and congeners

A series of analogs of 1,25-dihydroxyergocalciferol (1-4) was synthesized and screened for their antiproliferative activity in vitro. The structure of new analogs was designed based on biological activity of the previously obtained side-chain modified analogs of vitamin D(2) and D(3). The analogs were obtained by the Julia olefination of C(22)-vitamin D sulfone 11 with side-chain aldehyde 15. The analogs were tested for their antiproliferative activity against the cells of human breast cancer lines T47D and MCF7 as well as human and mouse leukemia lines, HL-60 and WEHI-3, respectively. Analog 2 (PRI-1907) showed the strongest antiproliferative activity out of the present series of analogs of 1,25-dihydroxyvitamin D(2) with the mono homologated and double unsaturated side chain. The activity of 2 was 3-150 times stronger, depending on the cell line, than that of 1,25-dihydroxycholecalciferol (calcitriol), used as standard.     

Design,synthesis and cytotoxicity of novel 3′-N-alkoxycarbonyl docetaxel analogs

By-product 9a exhibited potent cytotoxicity against both SK-OV-3 and A549 cell lines. The structure of 9a was characterized using 1D and 2D NMR experiments and confirmed by synthesis to afford a diastereomeric mixture (16a) that was identical to 9a, as well as a pair of diastereomers (R)-16b and (S)-16c. The preliminary SAR study demonstrated that analogs with an (R)-configuration were slightly more potent than analogs with an (S)-configuration. In addition, α,α-gem-dimethyl analogs 16g–i were the most potent analogs in this series, exhibiting similar potency to docetaxel and greater potency than Taxol against the SK-OV-3 cell line. For the A549 cell line, analogs 16g–i were more potent (>65-fold) than both docetaxel and Taxol.     

Apoptotic effect of Semecarpus anacardium nut extract on T47D breast cancer cell line

There is an increasing interest in identifying potent cancer-preventive and therapeutic agents against breast cancer. A great number of reports have in recent years dealt with anticancer characteristics of Semecarpus anacardium nut extract (SA). The majority of these studies has been targeted on the protective effect rendered to the living system rather than the preventive effect on cancer cells. SA was tested for its inhibitory effect on human breast cancer cells (T47D). Cytotoxicity analyses suggested that these cells had become apoptotic. SA was discovered to induce rapid Ca(2+) mobilization from intracellular stores of T47D cell line, and its cytotoxicity against T47D was well correlated with altered mitochondrial transmembrane potential. At the molecular level, these changes are accompanied by decrease in bcl(2) and increase in bax, cytochrome c, caspases and PARP cleavage, and ultimately by internucleosomal DNA fragmentation. Taken together, our results provide unprecedented evidence that SA triggers apoptotic signals in T47D cells.     

Lipase-catalyzed preparation of chromomycin A₃ analogues and biological evaluation for anticancer activity

Several acyl derivatives of the aureolic acid chromomycin A(3) were obtained via lipase-catalyzed acylation. Lipase B from Candida antarctica (CAL-B) was found to be the only active biocatalyst, directing the acylation regioselectively towards the terminal secondary hydroxyl group of the aglycone side chain. All new chromomycin A(3) derivatives showed antitumor activity at the micromolar or lower level concentration. Particularly, chromomycin A(3) 4'-vinyladipate showed 3-5 times higher activity against the four tumor cell lines assayed as compared to chromomycin A(3).     

Dissecting the role of disulfide bonds on the amyloid formation of insulin

Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenicity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6>INS-3>INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6-A11 caused a significant cytotoxicity increase and a higher potency to form high order toxic oligomers.     

New Rosane Diterpenoids and Their Analogs from Euphorbia ebracteolata Hayata

Phytochemical investigation of the roots of Euphorbia ebracteolata Hayata resulted in the isolation of three new rosane diterpenoids, euphebracteolatins C–E ( 1 – 3 ), along with fourteen known analogs ( 4 – 17 ). Their structures were determined on the basis of extensive spectroscopic analysis including HR-ESI-MS, 1D and 2D NMR. Euphebracteolatin C ( 1 ) contains a C-1/C-10 double bond and a keto group at C-7, and euphebracteolatins D and E ( 2 – 3 ) possess an aromatic ring-A in their skeleton. The plausible biogenetic pathways of all the isolates were also proposed. Furthermore, compounds 1 and 9 showed selective cytotoxicity against HepG2 cells with IC₅₀ values of 14.29 and 12.33 μM, respectively, and 2 – 3 displayed moderate cytotoxicity against three human cancer lines, with IC₅₀ values ranging from 23.69 to 39.25 μM.     

Synthesis, metal ion binding, and biological evaluation of new anticancer 2-(2'-hydroxyphenyl)benzoxazole analogs of UK-1

UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A simplified analog of UK-1, 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1 against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg2+ and Zn2+. A series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazole analogs of this 'minimal pharmacophore' of UK-1 were prepared. The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in methanol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg2+ and Cu2+ ions. Although none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, some analogs were identified that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs bind Cu2+ ions better than Mg2+ ions, and the nature of the 4-substituent is important for the Mg2+ ion binding ability of these 2-(2'-hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg2+ ion binding ability and cytotoxicity; however, within this series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazoles the variations in cytotoxicity do not correlate with either Mg2+ or Cu2+ ion binding ability. These results, together with recent ESI-MS studies of Cu2+-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction with Cu2+ or other transition metal ions, rather than Mg2+, and that metal ion-mediated DNA binding, rather than metal ion binding affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu2+ ions in the cytotoxic action of UK-1 is further supported by the observation that UK-1 in the presence of Cu2+ displays enhanced cytotoxicity to MCF-7 and A549 cells when compared to UK-1 alone.     

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.