Evaluation of Lactam Protection for Synthesis of 2′-O-Alkylated Uridines

Two different classes of protection for the uridine lactam function have been evaluated. These are benzoyl protections and different acetal functions. In particular the triisopropylsiloxymethyl protection is a most promising lactam protecting group for use in synthesis of 2′-O-alkyl-uridines.     

One-Step Protection of the Guanine Moiety in 2′-Deoxyguanosine and Guanosine

Abstract

2′-Deoxyguanosine reacts with 4-nitrophenylsulphonylethene to give a protected nucleoside derivative. Deprotection can be achieved by treatment with concentrated aqueous ammonia. The applicability of the protective group is shown by the synthesis of dT₄G.     

7-Deaza-2′-deoxyinosine: A Stable Nucleoside with the Ambiguous Base Pairing Properties of 2′-Deoxyinosine

Abstract

The base pairing ambiguity of 7-deaza-2′-deoxyinosine (c⁷I_d, 2) was studied and was found to be the same as that of 2′-deoxyinosine. The duplex stability decreases in the order [d(c⁷I-C) > d(c⁷I-A) > d(c⁷I-T) > d(c⁷I-G)]. Modified nucleosides were used to probe the various base pair motifs which were the same for dl and c⁷I_d. The 7-deazapurine nucleoside (2) is extremely stable against acid or base. As oligonucleotides can be prepared using phosphoramidite chemistry and DNA is accessible by enzymic polymerisation of the triphosphate of 2, the latter can be used as an universal nucleoside for the sequencing of DNA by chemical degradation and is otherwise a facile substitute of 2′-deoxyinosine when stability in acidic or alkaline solution is required.     

Synthesis of 2′-Deoxynucleoside 5′-Methylenebis-(phosphonate)s Using 2-(4-Nitrophenyl)ethyl Methylenebis(phosphonate) as the Phosphonylating Agent.

Abstract

2-(4-Nitrophenylethyl) methylenebis(phosphonate) (1) has been prepared by reaction of 2-(4-nitrophenyl)ethyl alcohol with methylenebis(phosphonyl) tetrachloride. Compound 1 was treated with diisopropylcarbodiimide (DIC) to give bicyclic intermediate 2, which in reaction with suitably protected 2′-deoxynucleosides 3 gave P¹,P²-disubstituted methylenebis(phosphonate)s 4. Removal of the nitrophenylethyl group by β-elimination with DBU afforded the corresponding 2′-deoxynucleoside 5′-methylenebis(phosphonate) analogues 5.

     

Synthesis of RNA Fragments Using the H-Phosphonate Method and 2′-(2′-Chlorobenzoyl) Protection

Abstract

The performance of 2′-(2-chlorobenzoyl) protected ribonucleoside H-phosphonates in the synthesis of oligoribonucleotides has been studied.     

A Convergent Regiospecific Synthesis of the Lariat-Trinucleotides and A2′p 5′G and A2′p 5′G 3′p 5′C from A O4-2-Nitrophenyl) -Uridine Building Block

Abstract

Total synthesis of title compounds 1_ and 2_ from a common intermediate 7 is reported using the phosphotriester-phosphiteamidite approach. Appropriate NMR evidence has been presented in support of the regiospecific synthesis of target molecules in addition to enzymatic analysis. Present work clearly shows that the NMR evidence is mandatory to establish the isomeric purity of branched RNA molecules; enzymatic or/and electrophoretic analysis alone as tools for confirmation of branched RNA structures can be misleading.     

A One-Pot Preparation of 1-Benzyl-2′-deoxyinosine from ionized 2′-Deoxyinosine

Abstract

A simple and one step synthetic method for the formation of 1-benzyl-2′-deoxyinosine was developed by direct benzylation of ionized 2′-deoxyinosine. Treatment of 2′-deoxyinosine, in the presence of NaOH, with benzyl bromide in 2, 2, 2–trifluoroethanol (TFE) or N, N–dimethylaetamide (DMA) gave 1-benzyl-2′-deoxyinosine in 35% and 80% yields, respectively.     

Study on Reactivity and Protection of the α-Hydroxyphosphonate Moiety in 5′-Nucleotide Analogues: Formation of the 3′-O-P-C(OH)-C4′ Internucleotide Linkage

Abstract

The recently described epimeric nucleosidyl-5′-C-phosphonates (α-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2′-deoxythymidine and 2′-deoxythymidinyl-5′-C-phosphonate, both as model compounds, in dependence on the nature of the 5′-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analogues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.     

The Protection of The 2′-Hydroxyl Function in Oligoribonucleotide Synthesis

Abstract

A new, easily accessible and achiral 2′-ketal protective group has been designed for the use in the chemical synthesis of oligoribonucleotides; the proposed 2′-ketal group⁽¹⁾ has the additional advantage that it could be easily functionalized to the diamide (6) with aq. ammonia at the penultimate step of deblocking of oligoribonucleotides which makes it more acid-labile than the parent 2′-ketal group during the final acid-promoted deprotection step.     

A Simple Synthesis of N4 -(6-Aminohexyl)-2′-Deoxy-5′-O-(4,4′-Dimethoxytrityl)Cytidine

Abstract

The title compound was synthesized by a transamination reaction between N⁴ -benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)cytidine and hexane-1,6-diamine in the presence of 1,5,7-triazabicyclo(4.4.0)dec-5-ene (TBD).     

Simultaneous Protection of 3′- and 5′-Hydroxyls of Ribonucleosides with Di-t-Butoxydichlorosilane

Abstract

Di-t-butoxydichlorosilane was found to protect simultaneously 3′- and 5′-hydroxyls of uridine. The preliminary results on the dialkoxysilanediyl group introduction, properties and applications are presented.     

Efficient Syntheses of (E)-5-(2-Bromovinyl)-2′-deoxy-4-thiouridine; A Nucleoside Analogue with Potent Biological Activity

Abstract

(E)-5-(2-Bromovinyl)-2′-deoxy-4′-thiouridine (S-BVDU) is a potent antiherpesvirus agent and its use in gene therapy as an anticancer agent has recently been described. We here outline 2 efficient methods for the synthesis of S-BVDU. The decision as to which method is to be used depends upon the starting materials available but starting from BVU, an overall yield of β-nucleoside of 35% can be expected. From 5-ethyl-2′deoxy-4-thiouridine, radical bromination using bromine will give a quantitative conversion to S-BVDU if unreacted starting material is recycled (50%) or using N-bromosuccinimide, a one step yield in excess of 80% can be obtained.     

Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation

A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4′-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential antitumor prodrug. Firstly, according to the structure–activity relationship, drug combination principle and bioisosterism, the –COO– and the –NH– bond substituents at the 4 position of cycloparaffin would be a great modification direction to improve antitumor activity of 4′-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view, the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a series of heterocyclic with pharmacological activity were chosen as module for improving antitumor activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell line HeLa (i.e., the IC₅₀ value of 0.60 ± 0.20 μM), A549 (i.e., the IC₅₀ value of 3.83 ± 0.08 μM), HepG2 (i.e., the IC₅₀ value of 1.21 ± 0.05 μM), and BGC-823 (i.e., the IC₅₀ value of 4.15 ± 1.13 μM) was significantly improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer drug etoposide (i.e., the IC₅₀ values of 15.32 ± 0.10, 59.38 ± 0.77, 67.25 ± 7.05, and 30.74 ± 5.13 μM), respectively. Compound 1 could arrest HeLa cell cycle G₂/M and induce apoptosis by strongly diminishing the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly demonstrated by the bioactivity test.     

A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor,NR4A2 (Nurr1)

Background

The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.

Methods

In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.

Results

The IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.

Conclusion

Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.