Synthesis of 6-(2-thienyl)purine nucleoside derivatives toward the expansion of the genetic code.

Unnatural bases specifically pairing with pyridin-2-one, 2-amino-6-(2-thienyl) purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace 2-amino-6-(N,N-dimethylamino)purine. It was expected that these novel purine analogues, as compared with 2-amino-6-(N,N-dimethylamino)purine, might reduce the interference in the stacking interactions with the neighboring bases in a duplex and improve the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite these unnatural bases. The syntheses of these nucleoside derivatives and the DNA fragments were examined.     

Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

The unnatural base, 2-amino-6-(2-thienyl)purine (designated as s), instead of 2-amino-6-(N,N-dimethylamino)purine (designated as x), was designed in order to improve the specificity and efficiency of the base pairing with pyridin-2-one (designated as y). DNA fragments containing s were chemically synthesized, and the thermal stability and the enzymatic reactions involving the s-y pairing were examined. Thermal denaturation experiments showed that the DNA duplex (12-mer) containing the s-y pair was more stable than that containing the x-y pair. The incorporation of dyTP was also more advantageous to the s-y pairing than the x-y pairing in single-nucleotide insertion experiments using the Klenow fragment of Escherichia coli DNA polymerase I.     

Chemical synthesis of novel base pairs and their enzymatic incorporation into DNA.

A novel base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and the counter part, pyridin-2-one (denoted y) were designed. The bulky 6-dimethylamino group of x is expected to eliminate base pairing with all natural bases. The phosphoramidite of x for DNA templates and the 2'-deoxyribonucleoside triphosphate of y (dyTP) for a substrate were synthesized, and the selectivity of the enzymatic incorporation of dyTP opposite x in the templates was examined. dyTP was preferentially incorporated opposite x than canonical dNTPs by Klenow fragment of Escherichia coli DNA polymerase I. While dyTP was also incorporated opposite A and G, the misincorporation was suppressed in the presence of dTTP and dCTP, respectively.     

Cytostatic evaluations of nucleoside analogs related to unnatural base pairs for a genetic expansion system

The introduction of an unnatural base pair into DNA enables the expansion of genetic information. To apply unnatural base pairs to in vivo systems, we evaluated the cytostatic toxicity of several nucleoside analogs by an MTT assay. Several nucleoside analogs based on two types of unnatural base pairs were tested. One is a hydrogen-bonded base pair between 2-amino-6-(2-thienyl)purine (s) and pyridin-2-one (y), and the other is a hydrophobic base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa). Among the nucleoside analogs, the ribonucleoside of 6-(2-thienyl)purine possessed the highest cytostatic activity against CCRF-CEM and especially HT-1080, as well as the normal fibroblast cell line, WI-38. The other analogs, including its 2'-deoxy, 2-amino, and 1-deazapurine nucleoside derivatives, were less active against CCRF-CEM and HT-1080, and the toxicity of these nucleosides toward WI-38 was low. The nucleosides of y and Pa were inactive against CCRF-CEM, HT-1080, and WI-38. In addition, no cytostatic synergism was observed with the combination of the pairing nucleosides of s and y or Ds and Pa.     

Efficient cross-linking to cytidine by functional nucleobases capable of in situ activation.

We have previously demonstrated that the ODNs with 2-amino-6-(2-phenylsulfoxyethyl)purine nucleoside derivative were capable of efficient interstrand cross-linking with cytidine selectively. In this new strategy, less reactive precursor was auto-activated within a duplex to generate 2-amino-6-vinylpurine derivative. However, it turned out that 2-amino-6-(2-phenylsulfinyl)-ethylpurine nucleoside was not applicable as the precursor for the synthesis of DNA oligomers with G-rich sequences. In this report, 2-amino-6-(2-methylsulfinylethyl)purine nucleoside has been proven to be more suitable as a precursor for DNA synthesis. In addition, the ODNs incorporating either 2-amino-6-(2-phenylsulfoxy ethyl)purine or 2-amino-6-vinylpurine showed high reactivity toward the cytidine at the target site but quite less reactivity was observed for it at non-target site, demonstrating high site-selectivity.     

Synthesis, X-ray crystal structural study, antiviral and cytostatic evaluations of the novel unsaturated acyclic and epoxide nucleoside analogues

A series of the novel purine and pyrimidine nucleoside analogues were synthesised in which the sugar moiety was replaced by the 4-amino-2-butenyl (2-6 and 10-18) and oxiranyl (8 and 20) spacer. The Z- (2-6) and E-isomers (10-18) of unsaturated acyclic nucleoside analogues were synthesized by condensation of 2- and 6-substituted purine and 5-substituted uracil bases with Z- (1) or E-phthalimide (9) precursors. The oxiranyl nucleoside analogues (8 and 20) were obtained by epoxidation of 1 and 9 with m-chloroperoxybenzoic acid and subsequent coupling with adenine. The new compounds were evaluated for their antiviral and antitumor cell activities. Among the olefinic nucleoside analogues, Z-isomer of adenine containing 4-amino-2-butenyl side chain (6) exhibited the best cytostatic activities, particularly against colon carcinoma (SW 620, IC50 = 26 microM). Its E-isomer 15 did not show any antiproliferative activity against malignant tumor cell lines, except for a slight inhibition of colon carcinoma (SW 620, IC50 = 56.5 microM) cells. In general, Z-isomers showed better cytostatic activities than the corresponding E-isomers. (Z)-4-Amino-2-butenyl-adenine nucleoside analogue 6 showed albeit modest but selective activity against HIV-1 (EC50 = 4.83 microg mL(-1)).     

2'-Deoxy-3,7-dideazaguanosine and related compounds. Synthesis of 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl) and 1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]pyridin-4(5H)-one via direct glycosylation of a pyrrole precursor.

The synthesis of two new analogs of 2'-deoxyguanosine, 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H-pyrrolo[3,2-c] pyridin-4(5H)-one (8) and 6-amino-1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]-pyridin-4(5H)-one (13) has been accomplished by glycosylation of the sodium salt of ethyl 2-cyanomethyl-1H-pyrrole-3-carboxylate (4c) using 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose( 5) and 1-chloro-2,3,5-tri-O-benzyl-alpha-D-arabinofuranose (9), respectively. The resulting blocked nucleosides, ethyl 2-cyanomethyl-1-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro- pentofuranosyl)-1H-pyrrole-3-carboxylate (6) and ethyl 2-cyanomethyl-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)- 1H-pyrrole-3-carboxylate, were ring closed with hydrazine to form 5-amino-6-hydrazino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H- pyrrolo[3,2-c]-pyridin-4(5H)-one (7) and 5,6-diamino-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)-1H- pyrrolo[3,2-c]pyridin-4(5H)-one (11), respectively. Treatment of 7 with Raney nickel provided the 2'-deoxyguanosine analog 8 while reaction of 11 with Raney nickel followed by palladium hydroxide/cyclohexene treatment gave the 2'-deoxyguanosine analog 13. The anomeric configuration of 8 was assigned as beta by proton NMR, while that of 13 was confirmed as beta by single-crystal X-ray analysis of the deblocked precursor ethyl 2-cyanomethyl-1-beta-D-arabinofuranosyl-1H-pyrrole-3-carboxylate (10a).     

New pregnane-type steroidal alkaloids from Sarcocca saligna and their cholinesterase inhibitory activity

Five new steroidal alkaloids, 5,14-dehydro-N(a)-demethylsaracodine [3beta-N(a)-methyl-20S-N(b)-acetyl-N(b)-methylamino-pregn-5,14-diene] (1), 14-dehydro-N(a)-demethylsaracodine [3beta-N(a)-methyl-20S-N(b)-acetyl-N(b)-methylamino-5alpha-pregn-14-ene] (2), 16-dehydrosarcorine [(20S)-20-(N,N-dimethylamino)-3beta-(N(a)-acetylamido)-5alpha-pregn-16-ene] (3), 2,3-dehydrosarsalignone [(20S)-20-(N,N-dimethylamino)-3beta-(tigloylamino)-pregn-2,5-diene-4-one] (4), and 14,15-dehydrosarcovagine-D [(20S)-20-(N,N-dimethylamino)-3beta-(tigloylamino)-5alpha-pregn-2,14-diene-4-one] (5), were isolated from the ethanolic extract of Sarcococca saligna, along with two known bases, sarcovagenine-C (6) and salignarine-C (7). Their structures were elucidated on the basis of spectroscopic methods. All seven compounds were found to possess cholinesterase inhibitory potential in a concentration-dependent manner with the IC50 values ranging from 12.5 to 200 microM against acetylcholinesterase and from 1.25 to 32.2 microM against butyrylcholinesterase.