d4TMP delivery from 7-substituted cycloSal-d4TMPs

Benzyl-substituted cycloSal-d4T monophosphates were prepared and evaluated for their ability to release d4TMP selectively. In contrast to previously reported derivatives, two of the new compounds release d4TMP as the sole product while two others gave the expected benzyl phosphate diesters. However, these diesters were surprisingly stable against degradation to release d4TMP.     

Study of Different Substituted Cyclic and Acyclic Benzylpronucleotides of d4T Relative to Their Hydrolytic Stability and Antiviral Activity

Abstract

CycloSal-d4TMP and two different bis(benzyl) phosphate triesters of the antivirally active nucleoside analog d4T were studied with regard to their chemical hydrolysis behavior at pH 7.3, in CEM/0 cell extracts, and their anti-HIV activity. In contrast to triesters 2–4, bis-(o-AB)-d4TMP 1 was found to be chemically exquisitely stable. All compounds led to the formation of d4TMP in cell extracts and all triesters achieved the TK-bypass.     

Cyclo-saligenyl-2′,3′-dideoxy-2′,3′-didehydroythymidinemonophosphate (cycloSal-d4TMP) — A New Pro-Nucleotide Approach

Abstract

The synthesis of cycloSal-d4TMP 3a-g as new pro-nucleotide approach for d4TMP 2 is described. Phosphotriesters 3 release the d4TMP 2 selectively by a controlled, chemically induced tandem reaction. CycloSal-phosphotriesters 3 exhibited high biological activity against HIV-1/HIV-2 in CEM cells which was completely retained in CEM TK^? cells.     

A New Cyclic Phosphoramidate D4T Prodrug Approach cycloAmb-D4T-Phosphoramidates

Abstract

A new potential phosphoramidate prodrug approach for d4T 1 is described. In hydrolyses studies the cycloAmb-d4T-phosphoramidates 2 and 3 proved to deliver d4TMP following a tandem reaction.     

Effects of cycloSal-D4TMP Derivatives in H9 Cells with Induced AZT Resistance Phenotype

Abstract

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9^rAZT²⁵⁰). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9^rAZT²⁵⁰ cells, which exert decreased thymidine kinase (TK) gene expression.     

BENZYL-FUNCTIONALIZED cycloSal-d4T MONOPHOSPHATES

Synthetic routes to benzyl-functionalized cycloSal-d4T monophosphates (7CH₂X-cycloSal-d4TMP) have been developed. Their hydrolytic behavior in basic aqueous solution (pH = 7.3) was studied and their hydrolysis half-lives were determined. It turned out that two different degradation pathways are leading to different products: beside the formation of the expected d4TMP and a styrene type derivative, a phenyl-d4T-phosphodiester was obtained as well. The product distribution was specified.     

New Lipophilic Derivatives of AZT and d4T 5′-Phosphonates

Abstract

5′-Aminocarbonylphosphonyl and aminocarbonylmethylphosphonyl diesters of AZT and d4T were synthesized as potential anti-HIV agents.     

SYNTHESIS AND ANTI-HIV ACTIVITY OF [D4U]-[TROVIRDINE ANALOGUE] AND [D4T]-[TROVIRDINE ANALOGUE] HETERODIMERS AS INHIBITORS OF HIV-1 REVERSE TRANSCRIPTASE

ABSTRACT

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a–e) and (N-3)d4T-Trovirdine conjugates (10a–f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.     

Synthesis,and in vitro Enzymatic and Antiviral Evaluation of d4T Polyphosphate Derivatives as Chain Terminators

A series of d4T di‐ or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV‐1 RT, and also tested for their in vitro anti‐HIV activity. The steady‐state kinetic study of compounds 1 – 4 in an enzymatic incorporation assay by HIV‐1 RT follows Michaelis? Menten profile. In addition, compounds 2 – 4 are able to inhibit HIV‐1 replication to the same extent as d4T and d4TMP in MT‐4 cells, as well as in CEM/0 cells and CEM/TK^? cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.     

Stereochemistry of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates

Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ₃₁P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arising from R _P and S _P diastereomers were reversed.     

蝇蛹俑小蜂对寄主及果实的趋性反应

【背景】蝇蛹俑小蜂是实蝇类害虫蛹期重要的寄生蜂之一。有关植物果实和寄主挥发物在蝇蛹俑小蜂寻找寄主过程中的作用尚不明确。【方法】采用"Y"型嗅觉仪测试了寄主和果实(健康果实和虫害诱导果实)对蝇蛹俑小蜂的引诱作用。【结果】虫伤1 d的番石榴和杨桃果实对蝇蛹佣小蜂雌蜂具有显著的引诱作用;寄主蛹——3日龄橘小实蝇蛹和4日龄瓜实蝇蛹对蝇蛹俑小蜂雌蜂也具有显著的引诱作用。蝇蛹俑小蜂雌蜂对雄蜂有显著的吸引作用。橘小实蝇性诱剂甲基丁香酚对蝇蛹俑小蜂的搜索行为则无显著影响。【结论与意义】寄主蛹和幼虫危害1 d的果实能释放对蝇蛹俑小蜂有引诱作用的物质,这可为利用蝇蛹俑小蜂防治实蝇类害虫提供基础信息。     

Shewanella putrefaciens CN32对粘土附着态铵氮释放的影响

【目的】研究产胞外分泌物微生物Shewanella putrefaciens CN32对土壤中常见粘土矿物附着态NH_4~+的释放效果及影响机制。【方法】以吸附NH_4~+的蒙脱石、蛭石、伊蒙混层矿物和黑云母为对象,通过监测S. putrefaciens CN32作用下不同粘土释放的NH_4~+含量及过程,以及监测微生物量及释放的胞外聚合物(Extracellular Polymeric Substances,EPS)的含量变化,研究S. putrefaciens CN32作用下不同粘土矿物类型附着态NH_4~+释放的差异性。【结果】粘土矿物附着态NH_4~+含量从高到低依次为蒙脱石蛭石伊蒙混层矿物黑云母(黑云母NH_4~+吸附量极低,会在非生物作用下几乎完全释放),CN32作用下粘土附着态NH_4~+相对释放量依次为蒙脱石伊蒙混层矿物蛭石;然而,尽管CN32有效促进了粘土附着态NH_4~+释放,但释放的NH_4~+并未在溶液中大量累积,而是多被微生物同化吸收转化为生物有机氮(EPS为主)并吸附在粘土表面,且粘土对EPS的吸附能力表现为蒙脱石伊蒙混层矿物蛭石黑云母;由于粘土吸附NH_4~+及EPS都与矿物中的羟基(结构水或层间水)关系密切,推测EPS对矿物羟基的竞争吸附可能是CN32促进NH_4~+释放的重要原因之一。【结论】以上结果表明,产EPS微生物S. putrefaciens CN32能够促进各类粘土矿物的附着态NH_4~+释放,但释放的NH_4~+可以通过微生物作用转化为有机氮,从而在减少NH_4~+流失的同时增加土壤氮肥的生物可利用性,因此微生物在降低土壤氮肥流失、转化土壤氮肥污染过程中可能起到了重要作用,也揭示了深入系统地分析不同类型土壤(粘土类型不同)中粘土附着态NH_4~+在不同功能微生物作用下的迁移转化过程,是精准评估土壤氮肥施用效率及流失风险的前提之一。     

5-Diacetoxymethyl-cycloSal-d4TMP—A prototype of enzymatically activated cycloSal-pronucleotides

A new class of “lock-in”-modified cycloSal-pronucleotides has been synthesized. On the example of 5-diacetoxymethyl-cycloSal-d4T-monophosphate (5-di-AM-cycloSal-d4TMP), the concept of enzymatically activated cycloSal-pronucleotides is elucidated. Synthesis, hydrolysis studies, and antiviral activities against HIV are presented.     

Studies on Reactions of Nucleoside H-Phosphonates with Bifunctional Reagents. Part VI. Reaction with Diols

Abstract

Reactions of nucleoside H-phosphonates with various diols using different types of condensing agents have been studied. Depending on the coupling procedure and the length of a polymethylene chain of the diol, acyclic H-phosphonate diesters or cyclic phosphite triesters were formed. The course of oxidation with iodine to produce cyclic nucleoside alkyl phosphotriesters or hydroxyalkyl nucleoside phosphodiesters can be controlled by the amount of water present in the reaction medium.     

Effect of H4R antagonist N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamides and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on histamine and 4-methylhistamine-induced mast cell response

Context: The histamine plays a decisive role in acute and chronic inflammatory responses and is regulated through its four types of distinct receptors designated from H1 to H4. Recently histamine 4 receptor (H4R) antagonists have been reported to possess various pharmacological effects against various allergic diseases.

Objective: To investigate the inhibitory effect of N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamide (Compound A) and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole (Compound L) on H4R-mediated calcium mobilization, cytokine IL-13 production, ERK1/2, Akt and NF-κB activation in human mastocytoma cells-1 (HMC-1).

Materials and methods: Compounds A and L were synthesized chemically and their inhibitory effect on intracellular calcium release was analyzed by Fluo-4 calcium assay, cytokine measurement through ELISA and activation of signaling molecules by western blot.

Results: Pre-treatment with compounds A and L significantly reduced the H4R-mediated intracellular calcium release. Histamine and 4-methylhistamine (4-MH) induced Th2 cytokine IL-13 production in HMC-1 cells, was inhibited by compound A (77.61%, 74.25% at 1?μM concentration) and compound L (79.63%, 81.70% at 1?μM concentration). Furthermore, histamine induced the phosphorylation of ERK1/2, Akt and NF-κB was suppressed by compounds A and L at varying levels, ERK1/2 (88%, 86%), Akt (88%, 89%) and NF-κB (89%, 87%) in HMC-1 cells.

Discussion and conclusions: Taken together these data demonstrate that compound A and compound L may block H4R-mediated downstream signaling events.     

Conditional control of chimeric antigen receptor T-cell activity through a destabilizing domain switch and its chemical ligand

Background aimsDespite the impressive efficacy of chimeric antigen receptor (CAR) T-cell therapy, adverse effects, including cytokine release syndrome and neurotoxicity, impede its therapeutic application, thus making the modulation of CAR T-cell activity a priority. The destabilizing domain mutated from Escherichia coli dihydrofolate reductase (DHFR) is inherently unstable and degraded by proteasomes unless it is stabilized by its chemical ligand trimethoprim (TMP), a Food and Drug Administration-approved drug. Here the authors reveal a strategy to modulate CAR T-cell activity at the protein level by employing DHFR and TMP as a chemical switch system.MethodsFirst, the system was demonstrated to work in human primary T cells. To introduce the system to CAR T cells, DHFR was genetically fused to the carboxyl terminal of a third-generation CAR molecule targeting CD19 (CD19-CAR), constructing the CD19-CAR-DHFR fusion.ResultsThe CD19-CAR-DHFR molecule level was shown to be modulated by TMP. Importantly, the incorporation of DHFR had no impact on the recognition specificity and normal function of the CAR molecule. Little adverse effect on cell proliferation and apoptosis was detected. It was proved that TMP could regulate cytokine secretion and the in vitro cytotoxicity of CD19-CAR-DHFR T cells. Furthermore, the in vivo anti-tumor efficacy was demonstrated to be controllable through the manipulation of TMP administration. The approach to control CD19-CAR also succeeded in 19-BBZ(71), another CD19-targeting CAR with a different structure.ConclusionsThe proposed approach based on DHFR and TMP provides a facile strategy to bring CAR T-cell therapy under conditional user control, and the strategy may have the potential to be transplantable.     

Study of different substituted cyclic and acyclic benzylpronucleotides of d4T relative to their hydrolytic stability and antiviral activity

CycloSal-d4TMP and two different bis(benzyl) phosphate triesters of the antivirally active nucleoside analog d4T were studied with regard to their chemical hydrolysis behavior at pH 7.3, in CEM/0 cell extracts, and their anti-HIV activity. In contrast to triesters 2-4, bis-(o-AB)-d4TMP 1 was found to be chemically exquisitely stable. All compounds led to the formation of d4TMP in cell extracts and all triesters achieved the TK-bypass.     

Benzyl-functionalized cycloSal-d4T monophosphates

Synthetic routes to benzyl-functionalized cycloSal-d4T monophosphates (7CH2X-cycloSal-d4TMP) have been developed. Their hydrolytic behavior in basic aqueous solution (pH = 7.3) was studied and their hydrolysis half-lives were determined. It turned out that two different degradation pathways are leading to different products: beside the formation of the expected d4TMP and a styrene type derivative, a phenyl-d4T-phosphodiester was obtained as well. The product distribution was specified.     

Tetramethylpyrazine inhibits CoCl2‐induced neurotoxicity through enhancement of Nrf2/GCLc/GSH and suppression of HIF1α/NOX2/ROS pathways

Hypoxia‐mediated neurotoxicity contributes to various neurodegenerative disorders, including Alzheimer's disease and multiple sclerosis. Tetramethylpyrazine (TMP), a major bioactive component purified from Ligusticum wallichii Franchat, exhibited potent neuroprotective effect. However, the mechanism of TMP‐exerted neuroprotective effect against hypoxia was not clear. In the study, we investigated the mechanism of the neuroprotective effect of TMP against hypoxia induced by CoCl₂ in vitro and in vivo. The results showed that TMP could protect against CoCl₂‐induced neurotoxicity in PC12 cells and in rats, as evidenced by enhancement of cell viability in PC12 cells and improvement of learning and memory ability in rats treated with CoCl₂. TMP could inhibit mitochondrial dysfunction, mitochondrial apoptotic molecular events, and thus apoptosis induced by CoCl₂. TMP inhibited CoCl₂‐increased reactive oxygen species (ROS) level, which may contribute to hypoxia‐related neurotoxicity induced by CoCl₂. The antioxidant and neuroprotective activities of TMP involved two pathways: one was the enhancement of nuclear factor erythroid 2‐related factor 2 (Nrf2)/catalytic subunit of γ‐glutamylcysteine ligase‐mediated regulation of GSH and the other was the inhibition of hypoxia‐inducible factor 1 α/NADPH oxidase 2 (NOX2)‐mediated ROS generation. These two pathways contributed to improvement of oxidative stress and thus the amelioration of apoptosis under hypoxic conditions. These results have appointed a new path toward the understanding of pathogenesis and TMP‐related therapy of hypoxia‐related neurodegenerative diseases.

     

Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones induced apoptosis in RKO colon cancer cells

Novel 1,5-diphenyl-6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones were synthesized and characterized. All compounds were screened for their anti-proliferative activities in five different cancer cell lines. The results showed that compounds 7a and 7b comprising aminoguanidino or guanidino moiety at position 6 inhibited proliferation of RKO colon cancer cells with IC₅₀ of 8 and 4?μM, respectively. Compounds 7a and 7b induced apoptosis in RKO cells, which was confirmed by TUNEL and annexin V-FITC assays. Flow cytometric analysis indicated that compounds 7a and 7b arrested RKO cells in the G1 phase and the most active compound 7b increased levels of p53, p21, Bax, ERK1/2 and reduced levels of Bcl2 and Akt. Compound 7b also activates release of cytochrome c, which is consistent with activation of caspase-9. Additionally, compound 7b increased caspase-3 activity and cleaved PARP-1 in RKO cells. Collectively, these findings could establish a molecular basis for the development of new anti-cancer agents.