Synthesis of Certain Acyclic Nucleoside Analogs of 1,2,4-Triazolo[3,4-f][1,2,4]triazine and Pyrimido[5,4-d]pyrimidine

Abstract

Synthesis of 2-penten-1-yl (8a) and ganciclovir analog (8b) of 1,2,4-triazolo[3,4-f][1,2,4]triazine was accomplished by the ring annulation of the corresponding hydrazides (6a and 6b), which in turn was obtained by the dehydrative coupling of 4 with 5a or 5b. Base catalysed ring expansion of N9-alkylpurine-6-carbonitriles (10a 10c 10e) provided the acyclic analogs of 4-aminopyrimido-[5,4-d]pyrimidines (13a 13d 13e). Debenzylation of 13e afforded the ganciclovir analog (13f) of 4-amino-8-(β-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine. However, compound 10b did not undergo the expected rearrangement but resulted in the formation of the methyl formimidate derivative (12).     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat.

1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.     

Synthesis of the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine dimer

We report the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 16 synthesised through a new and efficient route, thus establishing that C2-C3-endo unsaturation enhances both cytotoxicity and DNA-binding affinity in A-Ring-linked PBD dimers but to a lesser extent than C2/C2'-exo-unsaturation. This new route has allowed the preparation of multi-gram quantities of the related clinical candidate 1 and should lead to more structurally diverse PBD dimer analogues.     

Acyclic Nucleosides. Synthesis and Antiherpetic Activity of 9-[[[2-Hydroxy-1-(Hydroxymethyl)Ethyl]Thio]Methyl]Guanine and 1-[[[2-Hydroxy-1-(Hydroxymethyl)Ethyl]Thio]Methyl]Cytosine

Abstract

The synthesis and antiherpetic activity of 9-[[[2-hydroxy-1-(hydroxymethyl)ethyl]thio]methy1]guanine (4) and 1-[[[2-hydroxy-1-(hydroxymethyl)ethyl]thio]methy]cytosine (6), the side-chain thio analogues of ganciclovir (3) and BW A1117U (5), are described. The sidechain synthon 1,3-bis(benzyloxy)-2-[(chloromethyl)thio]propane (11) was prepared in four steps from 1,3-bis(benzyloxy)-2-propanol (7). Alkylation of 2-amino-6-chloro-9H-purine with 11 provided the intermediate 9-substituted-2-amino-6-chloropurine 12, which was conveniently converted to 4 in two steps. Reaction of a fivefold excess of cytosine with 11 provided the desired 1-isomer 14, which was debenzylated to give 6. In contrast with ganciclovir (3) and BW A1117U (5), neither 4 nor 6 had significant in vitro activity against human cytomegalovirus.     

Synthesis of imidazo[4,5-e][1,4]diazepine and 3-substituted 3-deazapurine nucleosides from 1-substituted inosines.

New synthetic methods of imidazo[4,5-e][1,4]diazepine nucleosides 8-11 and 3-substituted 3-deazainosines 14 from inosine have been reported. Treatment of N1-substituted inosines 1-3 with aqueous NaOH gave 5-amino-4-(N-substituted carbamoyl)imidazole ribosides 5-7, followed by appropriate manipulations to afford ring-expanded guanosine, inosine, and xanthosine analogues. Additionally, the 5-amino group of 4-N-allylcarbamoyl derivative 12 was converted into corresponding 5-iodo nucleoside 13. We found that 13 was cyclized to form 3-deaza-3-methylinosine (14) in the presence of Pd catalysts.     

Synthesis and Biological Evaluation of Some Acyclic 4,6-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidine Nucleosides

Abstract

The chemical synthesis and biological evaluation of some acyclic α-[6-(1′-carbamoylalkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylamide nucleosides are described.     

Novel Acyclic Analogues of Purine Nucleosides: 2,3-Dihydroxy-1-Methoxypropyl and 3-Hydroxy-1-Methoxypropyl Derivatives

Abstract

Novel purine nucleoside analogues in which the N-9 ribosyl moiety is replaced by a 2,3-dihydroxy-1-methoxypropyl or 3-hydroxy-1-methoxypropyl substituent and their N-7 substituted isomers have been synthesized and tested for antiviral activity.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Design,synthesis and biological evaluation of 2H-benzo[b][1,4] oxazine derivatives as hypoxia targeted compounds for cancer therapeutics

A small library of 2H-benzo[b][1,4] oxazine derivative was synthesized and their biological activity was tested on HepG2 cells under normoxic and hypoxic conditions. From preliminary screening, we found compound 10 and 11 specifically inhibit hypoxic cancer cell growth IC₅₀ 87 ± 1.8 μM and IC₅₀ 10 ± 3.7 μM while sparing ‘normoxic’ cells IC₅₀ >600 M and >1 mM (not applicable), respectively. We tested the effect of 10 on MTT, clonogenic and hypoxia induced genes. The MTT correlates with clonogenic assays and most importantly compound 10 down regulates hypoxia induces genes (HIF-1α, P21 and VEGF) appropriately. We are in the process to explore the molecular mechanism of action of oxazine derivative compounds on hypoxia tumor cells.     

Synthesis and antitubercular evaluation of amidoalkyl dibenzofuranols and 1H-benzo[2,3]benzofuro[4,5-e][1,3]oxazin-3(2H)-ones

A new class of amidoalkyl dibenzofuranols and 1H-benzo[2,3]benzofuro[4,5-e][1,3]oxazin-3(2H)-ones was synthesized in very good yields through polyphosphoric acid supported on silica (PPA-SiO₂) catalyzed one-pot three component condensation of 2-dibenzofuranol; aromatic aldehydes and acetamide or benzamide or urea under solvent free conditions. At 125 °C the reaction led to the formation of amidoalkyl dibenzofuranols 5a-k where as at 160 °C cyclization take place to give oxazin-3(2H)-one analogues 6a-e. Screening all the 16 compounds for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (MTB) resulted 1-((4-chlorophenyl)(2-hydroxydibenzo[b,d]furanyl)methyl)urea 5h; 1-((4-bromophenyl)(2-hydroxydibenzo[b,d]furanyl)methyl)urea 5i; 1-phenyl-1H-benzo[2,3]benzo furo[4,5-e][1,3]oxazin-3(2H)-one 6a (MIC 3.13 μg/mL) and 1-(4-chlorophenyl)-1H-benzo[2,3]benzofuro[4,5-e][1,3]oxazin-3(2H)-one 6b; 1-(4-bromophenyl)-1H-benzo[2,3]benzofuro [4,5-e][1,3]oxazin-3(2H)-one 6c (MIC 1.56 μg/mL) as most active antitubercular agents.     

Synthesis,Antiproliferative, and Antiviral Evaluation of Certain Acyclic 6-Substituted Pyrrolo[2,3-D]-pyrimidine Nucleoside Analogs Related to Sangivamycin and Toyocamycin

Abstract

A number of 6-substituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine and 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidine derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their biological activity. Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole (2) with triethylorthoformate, followed by alkylation via the sodium salt method with either 2-(acetoxyethoxy)methyl bromide or (1,3-diacetoxy-2-propoxy)methyl bromide, furnished the corresponding N-substituted pyrroles 3a and 3b. These compounds were then smoothly converted to the requisite deprotected 4-amino-6-bromopyrrolo[2,3-d]-pyrimidine-5-carbonitriles 5a and 5b (toyocamycin analogs) by methanolic ammonia. The 6-amino-derivatives were obtained by a displacement of the bromo group with liquid ammonia. Conventional functional group transformations involving the 5-cyano group furnished the 5-carboxamide (sangivamycin) and 5-thioamide analogs. Compounds substituted at the 7-position with a ribosyl moiety were active against human cytomegalovirus (HCMV) at micromolar concentrations, but the apparent activity was not selective. The 7-ribosyl compounds also had no activity against human immunodeficiency virus (HIV), though they were all cytotoxic. The new compounds were also evaluated against HCMV, herpes simplex virus type I (HSV-1), HIV, and also for their ability to inhibit the growth of L1210 murine leukemic cells in vitro. None of these compounds with (2-hydroxyethoxy)methyl substituents or 7-(1,3-dihydroxy-2-propoxy)methyl substituent at N-7 showed significant cytotoxicity toward L1210, or toward uninfected human foreskin fibroblasts (HFF cells), and KB cells. Nor were they cytotoxic in human lines CEM or MT2. Only compound 4a was found to be active against HCMV, having an IC₅₀ of 32 μM.     

Determination of extracellular and intracellular enrichments of [1-C]-α-ketoisovalerate using enzymatically converted [1-C]-valine standardization curves and gas chromatography–mass spectrometry

We report a validated method for the determination of extra- and intracellular [1-¹³C]-α-ketoisovalerate ([1-¹³C]-KIV) enrichments by gas chromatography–mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-¹³C]-valine enriched standards of known composition. Slopes of [1-¹³C]-valine standardization curves (mean±SD: 0.99±0.02, n=5) and [1-¹³C]-KIV standardization curves (mean±SD: 0.98±0.01, n=7) were not significantly different. The method was applied for the determination of [1-¹³C]-KIV enrichments in plasma and tissues during [1-¹³C]-valine infusion in a piglet. [1-¹³C]-KIV enrichment could be determined±0.1 MPE (C.V. 1%), and extracellular [1-¹³C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-¹³C]-KIV enrichment.     

Synthesis and biological evaluation of an N10-Psec substituted pyrrolo[2,1-c][1,4]benzodiazepine prodrug

The first example of an N10-protected (e.g., Psec, 15) pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogue that retains significant cytotoxicity in a number of tumour cell lines is reported. This prototype could lead to a new generation of clinically useful N10-protected PBD prodrugs.     

Synthesis and characterization of 3-ketohexadecanoic acid-1-14-C, DL-3-hydroxyhexadecanoic acid-1-14-C, and trans-2-hexadecenoic acid-1-14-C

The chemical synthesis and characterization of three intermediates in the Beta oxidation of palmitic acid-1-(14)C by rat liver mitochondria, namely, 3-ketohexadecanoic acid-1-(14)C, DL-3-hydroxyhexadecanoic acid-1-(14)C, and trans-2-hexadecenoic acid-1-(14)C, are described.     

Synthesis of novel C2 and C2-C8 linked pyrrolo[2,1-c][1,4]benzodiazepine-naphthalimide hybrids as DNA-binding agents

Synthesis of C2 and C2-C8 linked pyrrolobenzodiazepine-naphthalimide hybrids have been prepared that exhibit significant DNA-binding affinity and cytotoxicity.     

Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as anti-cancer agents

A new series of novel Podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones possessing structural elements of 4-aza-2,3-didehydropodophyllotoxins with central diazepine ring was designed and synthesized as anti-cancer agents. In initial assessment, the cytotoxic activity of the synthesized compounds was evaluated against three cancer cell lines including MCF-7, PC3 and B16-F10 employing the MTT assay. Some of compounds (12h, 13a, 13c and 14b) showed significant cytotoxic activity. So, we investigated the cytotoxicity of compounds 12h, 13a, 13c and 14b, along with podophyllotoxin as the reference drug in different cancer cell lines including A549, A2780, DU145, HeLa, and normal Huvec cell line. Among these four compounds, 13c showed promising antiproliferative activity against all cancer cells stronger than the other compounds and comparable to reference drug podophyllotoxin in some cancer cells. All these four compounds did not show significant cytotoxicity on normal Huvec cell line. The flow cytometry analysis of the MCF-7, PC3 and A2780 human cancer cell lines treated with 13c showed that 13c, induced apoptosis in the MCF-7, PC3 and A2780 human cancer cell lines, which is in good agreement to its cytotoxic activity as well. Compound 13c did not show significant influence on tubulin assembly and exert its cytotoxic effects via induction of apoptosis and has potent and selective cytotoxic effects in cancer cells.