Commercial-Scale Synthesis of Protected 2′-Deoxycytidine and Cytidine Nucleosides

Abstract

Transformation of 2′-deoxyuridine and uridine analogs to protected 2′-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude that the triazolation method is better and suitable for commercial scale--up.     

Specificity of the enzymes of Salmonella O-antigen biosynthesis. 6. Synthesis of sugar nucleotides with glycosyl phosphate activation. Analogs of guanosine diphosphate mannose modified at position 6 ofthe purine ring

Interaction of alpha-D-mannopyranosyl phosphate with diphenyl phosphochloridate gave the trisubstituted pyrophosphate which was converted through the reaction with nucleoside 5'-phosphates into nucleoside 5'-(alpha-D-mannopyranosyl)pyrophosphates. The method was used for preparation of guanosine diphosphate mannose analogs derived from adenine, purine, 2-aminopurine, 2-amino-6-methoxypurine, 2-amino-6-chloropurine, and 2-amino-6-mercaptopurine. These analogs are necessary for study on substrate specificity of mannosyltransferases of Salmonella O-specific polysaccharides biosynthesis.     

Superactive analogs of the atrial natriuretic peptide (ANP)

Three analogs of the atrial natriuretic peptide ANP(105-126), lacking the N-terminal exocyclic peptide segment and containing 2-mercaptoacetic acid, 3-mercaptopropionic acid or 4-mercaptobutyric acid in place of the cysteine residue in position 105 of the peptide sequence, were synthesized by the solid-phase method. The resulting des-amino analogs showed 2 to 4 times higher diuretic/natriuretic activity than the most active natural ANP and displayed a potent hypotensive effect as well. All three analogs were relatively less potent in various in vitro bioassays and in a binding assay, indicating that their high activities in vivo may be due to resistance to enzymatic degradation and to reduced non-specific tissue adsorption. These compounds not only will serve as useful pharmacologic tools but also represent prototypes for the development of further reduced-size ANP analogs.     

Vitamin D: structure-function analyses and the design of analogs.

There is continuing and emerging new interest in the development of vitamin D analogs resulting from the recognition that analogs of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] may be therapeutically useful. Side chain analogs of this steroid hormone are of particular interest because a family of lead structures have recently emerged for possible use in the treatment of certain types of cancers and skin diseases. Because of the chaotic array of side chain structures which exhibit useful therapeutic indices for these purposes, a more systematic approach towards developing intelligible structure-function information needs development. Accordingly, a method has been devised to analyze analogs as to their side chain topology based on identifying specific occupancy volumes through conformational analysis. Dot maps have been constructed as an indication of the volume in space which the side chain of 1 alpha,25-(OH)2-D3 or analogs is permitted to occupy. Volume exclusion analyses based on comparison of structural and biological data for 1 alpha,25-(OH)2-D3 and analogs are anticipated to lead to a more cogent model for drug design. A cautionary note on the limitations of this approach is discussed.     

Alkyl lysophosphatidic acid and fluoromethylene phosphonate analogs as metabolically-stabilized agonists for LPA receptors

We describe an efficient method for the synthesis of alkyl lysophosphatidic acid (LPA) analogs as well as alkyl LPA mono- and difluoromethylene phosphonate analogs. Each alkyl LPA analog was evaluated for subtype-specific LPA receptor agonist activity using a cell migration assay for LPA(1) activation in cancer cells and an intracellular calcium mobilization assay for LPA(2) and LPA(3) activation. Alkyl LPAs induced pronounced cell migration activity with equivalent or higher potency than sn-1-oleoyl LPA, while the alkyl LPA fluoromethylene phosphonates proved to be less potent agonists in this assay. However, each alkyl LPA analog activated Ca(2+) release by activation of LPA(2) and LPA(3) receptors. Interestingly, the absolute configuration of the sn-2 hydroxyl group of the alkyl LPA analogs was not recognized by any of the three LPA receptors. The use of alkyl LPA analogs further expands the scope of structure-activity studies, which will better define LPA-LPA receptor interactions.     

Solid-phase synthesis of backbone-modified DNA analogs by the boranophosphotriester method using new protecting groups for nucleobases

Backbone-modified DNA analogs were synthesized in good yields by the boranophosphotriester method on a solid support. The oligodeoxyribonucleoside boranophosphates, protected with 2-(azidomethyl)benzoyl groups for nucleobases, were converted into DNA and its backbone-modified analogs via the corresponding H-phosphonate intermediates. A new protecting group for the O6 position of 2'-deoxyguanosine, 4-azidobenzyl (ABn) group, was also developed. The ABn group can be quickly removed by treatment with MePPh2 and H2O in the presence of 2-mercaptoethanol.     

A solid-phase approach to novel purine and nucleoside analogs

This paper describes a method for the preparation of purine analogs using the solid-phase approach. Nucleoside bases were constructed on Merrifield resin by sequential displacement of purine dichloride with amines, and after detachment, the purine analogs were condensed with d,l-ribofuranoside compounds by the Vorbrüggen method. Thereof, l-ribofuranoside was prepared from l-arabinose via the selective oxidation-reduction procedure of the 2-OH group. Some compounds exhibited moderate activity against HIV-1 in PBM cells.     

The biological importance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin

To systematically evaluate the contribution of each amino acid residue of the troponin I (TnI) inhibitory region (104-115), 14 synthetic analogs were synthesized by the solid-phase method. The analogs consisted of either single glycine or multiglycine replacements. The importance of the substituted amino acid(s) was determined from the extent of inhibition of the acto-S1 ATPase activity and the strength of binding to a troponin C (TnC) high pressure liquid chromatography affinity column of each synthetic analog. Every residue of the TnI sequence (104-115) is necessary to achieve maximum inhibition of the ATPase activity. However, the analogs quantitatively differed in the amount of inhibition induced. The TnI analogs bound less tightly to the TnC affinity column than the native synthetic peptide indicating that all residues in the TnI sequence contribute to the binding of TnC in the presence of Mg2+ or Ca2+. In the presence of Ca2+, there is a definite increase in the strength of the interaction between most analogs and TnC. This is accompanied with a shift toward a more specific interaction with the C terminus of the TnI inhibitory sequence.     

Studies of peptide antibiotics. XLIV. Syntheses and biological activities of gramicidin S analogs containing delta-hydroxy-L-norvaline or glycine

The antibiotic gramicidin S (GS) has the structure of cyclo (-L-Val1-L-Orn2-L-Leu3-D-Phe4-L-Pro5-L-Val1'-L-Orn2'-L-Leu3'-D-Phe4'-L-Pro5'-) and is basic in character. Five GS analogs including [Gly1,1']-GS and the neutral [L-Hnv2,2']-GS (Hnv represents delta-hydroxynorvaline) were synthesized by the solid-phase method to evaluate the role of L-Val1,1' and L-Orn2,2' residues in GS. The hybrid analogs [( Gly1]-GS and [L-Hnv2]-GS) and [D-Tyr4,4']-GS showed high antibacterial activities, whereas [Gly1,1']-GS and [L-Hnv2,2']-GS possessed no activity. Inhibitory effects by these analogs for the adsorption of 14C-labeled GS on cells of bacteria sensitive to GS were determined. The structure-activity relationship of GS is discussed on the basis of the results on these GS analogs.     

Synthesis and Evaluation of 2′-Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs

The preparation of 2′-deoxy-2′-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2′-position. Then, a series of novel 2′-deoxy-2′-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.     

Facile synthesis of new 4-aza-podophyllotoxin analogs via microwave-assisted multi-component reactions and evaluation of their cytotoxic activity

A series of new 4-aza-podophyllotoxin analogs containing thiazole unit were synthesized via multi-component reactions of aldehydes, tetronic acid and 2-methylbenzo[d]thiazol-5-amine under microwave irradiation. The method not only provides a valuable tool in design and synthesis of new 4-aza-podophyllotoxin analogs but also has the advantages of atom-economy, environmental-friendliness, good yields and operational simplicity. More importantly, the preliminary evaluation on the cytotoxic activity of this type of new 4-aza-podophyllotoxin analogs has resulted in the finding of several compounds with potent and efficacious cytotoxicity to three carcinoma cell lines M14, MCF7 and SW1116.     

Determination of binding parameters of cyclic AMP and its analogs to cyclic AMP-dependent protein kinase by the fluorescent probe method.

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of nonfluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180,000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions.     

Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH(2) or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH(2), respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure. Dmt(1)-substituted analogs displayed high affinity at the μ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting μ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration. Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood-brain barrier permeability.     

Affinity of 5-thio-L-fucose-containing Lewis X (LeX) trisaccharide analogs to anti-LeX monoclonal antibody

5-Thiofucose-containing LeX trisaccharide analogs Gal beta(1,4)[5SFuc alpha(1,3)]GlcNAc-OMe (2) and Gal beta(1,4)[5SFuc beta(1,3)]GlcNAc-OMe (4) were synthesized via 5-thiofucosylation of methyl 2-azido-lactoside derivative 6 by the trichloroacetimidate method. Inhibitory activity of these analogs for the binding of LeX to anti-Lex antibody was evaluated by enzyme immunoassay, indicating that anti-LeX strictly recognizes alpha-configuration of the fucose moiety and its binding pocket includes no advantageous region, such as hydrophobic area, for recognizing the ring sulfur atom of 5-thiofucosyl LeX analog 2.     

A sensitive cell-based method to screen for selective inhibitors of SMS1 or SMS2 using HPLC and a fluorescent substrate

Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH₂ column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.     

Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.     

Directed synthesis of 2',5'-oligoadenylates with increased stability towards phosphodiesterases

Phosphodiesterase stability of synthetic analogs of 2',5'-oligoadenylates, the mediators of antiviral and antiproliferative action of interferons was analysed. The analogs with a 3'-terminal acyclic nucleoside residue were prepared. These analogs were treated with NIH3T3 cell lysate, mice liver homogenate and snake venom phosphodiesterase. All analogs have demonstrated a high stability as compared with the natural 2',5'-oligoadenylate and its 3'-deoxyderivative. The possible biological activity of these stable analogs of 2',5'-oligoadenylates is discussed.     

Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure–activity relationships compared to 2-methoxyestradiol

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.     

ATP analogs substituted on the 2-position as substrates for dynein ATPase activity

In contrast to previously studied ATP analogs, the two-substituted ATP analogs, 2-N3 ATP and 2-Cl ATP were good substrates for dynein ATPase. The Vmax for hydrolysis of both analogs was significantly higher than for ATP and the Km for both analogs was comparable to ATP. The higher hydrolytic rate for the analogs might be explained by a faster dissociation rate of the diphosphate product. This interpretation is supported by measurements of the dissociation rate of the inhibitor, vanadate. The estimate dissociation rate of vanadate with the analogs as substrate is approx. 2-fold higher than with ATP as substrate. These data together with previous studies on a variety of ATP analogs suggest that the 6-amino group on adenine is important for recognition by dynein and that the anti-conformation of the adenine, favored by 2-substituents, is the favored conformation of the nucleotide.