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.     

Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet

We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure–activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.     

Synthesis, biological activity, and SAR of antimycobacterial 2- and 8-substituted 6-(2-furyl)-9-(p-methoxybenzyl)purines

A number of 6-(2-furyl)-9-(p-methoxybenzyl)purines carrying a variety of substituents in the 2- or 8-position have been synthesized and their ability to inhibit growth of Mycobacterium tuberculosis in vitro has been determined. It is demonstrated that sterical hindrance in the purine 8-position reduces activity and that C-8 should be unsubstituted. In the purine 2-position small, hydrophobic substituents are beneficial. The electronic properties of the 2-substituents appear to have only a minor influence on bioactivity. The compounds studied exhibit low toxicity toward mammalian cells (VERO cells) and are essentially inactive toward Staphylococcus aureus and Escherichia coli. The most active and selective antimycobacterial in the series detected to date is the novel 2-methyl-6-furyl-9-(p-methoxybenzyl)purine with MIC=0.20 microg/mL against M. tuberculosis and IC(50) against VERO cells >62.5 microg/mL. Also the novel 2-fluoro analog and the previously known 2-chloro compound, both with MIC=0.39 microg/mL, are highly interesting drug candidates.     

Formation of 3-(2'-deoxyribofuranosyl) and 9-(2'-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.     

Synthesis of (Z)-(1-Fluoro-2-hydroxymethyl-cyclopropylmethyl)purines

Abstract

(Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines were designed, synthesized and evaluated their antiviral activity against poliovirus, HSV, and HIV.     

Synthesis of (Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines

(Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines were designed, synthesized and evaluated their antiviral activity against poliovirus, HSV, and HIV.     

Synthetic approaches to 6-substituted 9-(3-azido-3,4-dideoxy-β-d,l-erythro-pentopyranosyl)purines

Methyl 3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranoside (6) was synthesized through two routes in five steps from methyl 2,3-anhydro-4-deoxy-β-dl-erythro-pentopyranoside (1). The first route proceeded via selective azide displacement of the 3-tosyloxy group of methyl 4-deoxy-2,3-di-O-tosyl-α-dl-threo-pentopyranoside, followed by detosylation and benzoylation. The second route consisted, with a better overall yield, in the azide displacement of the mesyloxy group of methyl O-benzoyl-4-deoxy-3-O-methylsulfonyl-α-dl-threo-pentopyranoside (10), obtained by benzylate opening of 1, followed by benzoylation, debenzylation, and mesylation. Compound 6 was transformed into its glycosyl chloride, further treated by 6-chloropurine to give the nucleoside 9-(3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranosyl)-6-chloropurine (13). When treated with propanolic ammonia, 13 yielded 9-(3-azido-3,4-dideoxy-β-dl-erythro-pentopyranosyl)adenine.     

Cytokinins: Synthesis of 2-, 8-, and 2,8-substituted 6-(3-methyl-2-butenylamino)purines and their relative activities in promoting cell growth

We have synthesized 2- and 8-monosubstituted and 2,8-disubstituted derivatives of the cytokinin 6-(3-methyl-2-butenylamino)purine (N⁶-isopentenyladenine) and have shown the dependence of growth-promoting activity in the tobacco bioassay upon the position, number, and type of substituent. The representative substituent groups were MeS, Me, MeSO₂, C₆H₅CH₂S, HS and Cl. The 8-methyl derivative was exceptional in being more active than the unsubstituted parent compound. In general, substitution in the 8-position decreases activity less than substitution in the 2-position, with the exception of the electron-attracting methylsulfonyl. Substitution in both the 2- and 8-positions lowers the activity more than substitution at either single position on the adenine nucleus, with the exception of the 2,8-dimethyl derivative. The chloro and methylthio derivatives show activity in the same range as the methyl derivatives, and the mercapto compounds, which exist mainly as CS tautomers, show somewhat less activity than the corresponding methylthio compounds. Bulky (C₆H₅CH₂S and MeSO₂) and strongly electron-attracting (MeSO₂) substituents cause relatively great reduction in cytokinin activity.     

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.     

Mechanisms for the solvolytic decompositions of nucleoside analogues. X. Acidic hydrolysis of 6-substituted 9-(beta-D-ribofuranosyl)purines.

The pH-rate profiles were determined for the acidic hydrolysis of some 6-substituted 9-(beta-D-ribofuranosyl) purines. The product analyses indicated that the reactions generally proceed with formation of purine bases as initial products. However, at low oxonium ion concentrations the hydrolysis of the unsubstituted compound yields 4-amino-5-formamidopyrimidine, instead of purine formed in highly acidic solutions. The rate constants for the spontaneous and oxonium ion catalyzed heterolysis of the protonated substrates were calculated from the acidity constants and the observed rate constants. The dependence of the partial rate constants on the polar nature of the 6-substituents are consistent with rate-limiting formation of free purine bases and glycosyl oxocarbenium ions. No anomerization of the substrates was observed during the course of the hydrolysis.     

Inhibition of activity of purine nucleoside phosphorylase with N9- and N7-(beta-D-glucofuranuronosyl)purines and 8-substituted N9-(beta-D-ribofuranosyl)purines)]

In an effort to develop more potent inhibitors of purine nucleoside phosphorylase (PNP, EC 2.4.2.1) as immunosuppressive and anticancer chemotherapeutic agents, the affinity of the electrophoretically homogeneous enzyme from rabbit kidney for sixteen N9- and N7-beta-D-glucofuranuronosides and for C8-substituted beta-D-ribofuranosyl purines was determined. In all cases N7-substituted analogues of hypoxanthine and guanine were twice more active inhibitors of PNP than N9-substituted compounds. No effective inhibitors were found among the C8-substituted analogues, apparently due to the bulky C8-groups hindering rotation around the glycosidic bond and thus preventing optimal binding with the enzyme.     

Synthesis of 6-alkyl- and arylamino-9-(tetrahydro-2-pyranyl)purines via 6-methylsulfonylpurine

A series of six potential plant hormones, 6-alky- and arylamino-9-(tetrahydro-2-pyranyl)purines were prepared in three steps in 35 to 45% overall yield from 6-methylthiopurine via 6-methylsulfonylpurine.     

Synthesis of 6-Alkyl- and Arylamino-9-(tetrahydro-2-pyranyl)purines via 6-Methylsulfonylpurine

Abstract

A series of six potential plant hormones, 6-alky- and arylamino-9-(tetrahydro-2-pyranyl)purines were prepared in three steps in 35 to 45% overall yield from 6-methylthiopurine via 6-methylsulfonylpurine.     

In Vitro antifungal activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones

The in vitro antifungal activity of several N2-phenyl-3(2H)-isothiazolones substituted at C4 of the phenyl moiety with heterocyclic nucleus or groups of different physico-chemical properties against four human pathogenic fungi was determined by broth macrodilution method; results were compared with those obtained with itraconazole and ketoconazole. These isothiazolones showed moderate to high activity against some or all tested strains and in comparison with the reference drugs, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g), 5-chloro-2-phenylisothiazol-3-one (1c), 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]-1,4-dihydrotriazol-5-one (1s) and 2-(4-nitrophenyl)isothiazol-3-one (2g) against Aspergillus niger, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g) and 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]piperazine-1-carboxamide (1q) against Trichophyton mentagrophytes had comparable activity, compounds 1g and 2g showing higher activity against Microsporum canis. Antifungal activity was favored by the presence of chlorine at C5 of the isothiazolone and/or the presence of nitro group or heterocyclic nucleus at C4 of the phenyl ring and proper hydrophilicity of the molecule.     

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.     

Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.     

Novel indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbothiohydrazides as TPO receptor agonists

Synthesis and evaluation of novel series of indoline-1- or 3,4-dihydroquinoline-1(2H)-substituted carbothiohydrazide or carbohydrazide based small molecule compounds as thrombopoietin (TPO) receptor agonists are reported. Members of these compounds have been identified as full agonists of human c-mpl in BaF3/TPOR cell line. Indoline-1-carbohydrazide 9b exhibited reasonable pharmacokinetic profile.     

pH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2'-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pK_a values: 1a = 5.30; 2a = 5.71; dA = 3.50).     

Synthesis and structure-activity relationships of soluble 8-substituted 4-(2-chlorophenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of the Wee1 and Chk1 checkpoint kinases

Pyrrolo[3,4-c]carbazoles bearing solubilising basic side chains at the 8-position retain potent Wee1 and Chk1 inhibitory properties in isolated enzyme assays, and evidence of G2/M checkpoint abrogation in several cellular assays. Co-crystal structure studies confirm that the primary binding to the Wee1 enzyme is as described previously, with the C-8 side chains residing in an area of bulk tolerance.