Synthesis of a Conformationally Locked Version of Puromycin Amino Nucleoside

A conformationally locked carbocyclic version of puromycin amino nucleoside was synthesized via Mitsunobu coupling of a 3-azido-substituted carbocyclic moiety with 6-chloropurine without interference from the azido group reacting with triphenylphosphine. The requisite 3-azido-substituted carbocyclic pseudosugar was prepared by a double inversion of configuration at C3‘ (nucleoside numbering) involving a nucleophilic displacement with azide

Copper-Catalyzed C–C Cross-Couplings of Tertiary Alkyl Halides with Anilines Enabled by Cyclopropenimine-Based Ligands

Catalytic cross-couplings of tertiary alkyl electrophiles with carbon nucleophiles offer a powerful platform for constructing quaternary carbon centers, which are prevalent in bioactive molecules. However, these reactions remain underdeveloped primarily because of steric challenges that impede efficient bond formation. Herein, we describe the copper-catalyzed synthesis of such centers through the C­(sp3)–C­(sp2) bond-forming reaction between tertiary alkyl halides and arene rings of aniline derivatives, enabled by the strategic implementation of bidentate bis­(cyclo­propenimine) ligands. The copper catalyst bound by two imino-nitrogen atoms of these ligands, which have never been employed in metal catalysis previously, is highly effective in rapidly activating tertiary halides to generate alkyl radicals, allowing them to react with aryl nucleophiles under mild conditions with remarkably short reaction times (1–2 h). Various tertiary halides bearing carbonyl functional groups can be coupled with secondary or primary anilines, furnishing a range of quaternary carbon centers in good yields. Several mechanistic observations support the generation of copper­(II) species and alkyl radicals which as a result elucidate the steps in the proposed catalytic cycle

Synthesis of Conformationally Locked Versions of Puromycin Analogues

Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger−Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3′-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-l-tyrosine. North azides 2 and 3 were reported earlier. The 3′-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time

Synthesis, Anti-HIV Activity, and Molecular Mechanism of Drug Resistance of l-2‘,3‘-Didehydro-2‘,3‘-dideoxy-2‘-fluoro-4‘-thionucleosides

β-l-2‘,3‘-Didehydro-2‘,3‘-dideoxy-2‘-fluoro-4‘-thionucleosides (β-l-2‘-F-4‘-S-d4Ns) have been synthesized and evaluated against HIV-1 in primary human lymphocytes. The key intermediate 8, which was prepared from 2,3-O-isopropylidene-l-glyceraldehyde 1 in 13 steps, was condensed with various pyrimidine and purine bases followed by elimination and deprotection to give the target compounds, β-l-2‘-F-4‘-S-d4Ns (17−20 and 27−30). The antiviral activity of the newly synthesized compounds was evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which the cytosine 17, 5-fluorocytosine 18, and adenine 27 derivatives showed potent anti-HIV activities (EC50 = 0.12, 0.15, and 1.74 μM, respectively) without significant cytotoxicity up to 100 μM in human PBM, CEM, and Vero cells. The cytosine derivative 17 (β-l-2‘-F-4‘-S-d4C), however, showed cross-resistance to a 3TC-resistant variant (HIV-1M184V). Molecular modeling studies suggest that the pattern of antiviral activity, similar to that of β-l-2‘-F-d4N, stemmed from their conformational and structural similarities. The isosteric substitution of sulfur for 4‘-oxygen was well tolerated in the catalytic site of HIV-1 reverse transcriptase in the wild-type virus. However, the steric hindrance between the sugar moiety of the unnatural l-nucleoside and the side chains of Val184 of M184V RT in 3TC-resistant mutant HIV strains destabilizes the RT-nucleoside triphosphate complex, which causes the cross-resistance to 3TC (M184V mutant)

Stereoselective Synthesis and Antiviral Activity of d-2‘,3‘-Didehydro-2‘,3‘-dideoxy-2‘-fluoro-4‘-thionucleosides

As 2‘,3‘-didehydro-2‘,3‘-dideoxy-2‘-fluoronucleosides have exhibited interesting antiviral effects against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4‘-thionucleosides in which 4‘-oxygen is replaced by a sulfur atom. To study structure−activity relationships, various pyrimidine and purine nucleosides were synthesized from the key intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylselenyl-4-thio-β-d-ribofuranoside 8, which was prepared from the 2,3-O-isopropylidene-d-glyceraldehyde 1 in 13 steps. The antiviral activity of the synthesized compounds were evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which cytidine 17, 5-fluorocytidine 18, adenosine 24, and 2-fluoroadenosine 32 showed moderate to potent anti-HIV activities (EC50 1.3, 11.6, 8.1, and 1.2 μM, respectively). It is noteworthy that 2-fluoroadenosine analogue 32 showed antiviral potency as well as high cytotoxicity (IC50 1.5, 1.1, and 7.6 μM for PBM, CEM, and Vero, respectively) whereas no other compound showed cytotoxicity up to 100 μM. The cytidine 17 and 5-fluorocytidine 18 analogues showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1M184V), whereas the corresponding d-2‘-Fd4 nucleosides showed limited cross-resistance. Molecular modeling studies demonstrated that the larger van der Waals radius as well as the close proximity to Met184 of the 4‘-sulfur atom of d-2‘-F-4‘-Sd4C (17) may be the reasons for the decreased antiviral potency of synthesized 4‘-thio nucleosides against the lamivudine-resistant variants (HIV-1M184V)

Understanding How the Herpes Thymidine Kinase Orchestrates Optimal Sugar and Nucleobase Conformations To Accommodate Its Substrate at the Active Site: A Chemical Approach

The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme but also on the ability of the compound(s) to interact effectively with cellular kinases and with the target viral or cellular DNA polymerases. Herein, we describe the synthesis and study of two nucleoside analogues built on a conformationally locked bicyclo[3.1.0]hexane template designed to investigate the conformational preferences of HSV-tk for the 2‘-deoxyribose ring. Intimately associated with the conformation of the 2‘-deoxyribose ring is the value of the C−N torsion angle χ, which positions the nucleobase into two different domains (syn or anti). The often-conflicting sugar and nucleobase conformational parameters were studied using North and South methanocarbadeoxythymidine analogues (6 and 7), which forced HSV-tk to make a clear choice in the conformation of the substrate. The results provide new insights into the mechanism of action of this enzyme, which cannot be gleaned from a static X-ray crystal structure

Understanding How the Herpes Thymidine Kinase Orchestrates Optimal Sugar and Nucleobase Conformations To Accommodate Its Substrate at the Active Site: A Chemical Approach

The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme but also on the ability of the compound(s) to interact effectively with cellular kinases and with the target viral or cellular DNA polymerases. Herein, we describe the synthesis and study of two nucleoside analogues built on a conformationally locked bicyclo[3.1.0]hexane template designed to investigate the conformational preferences of HSV-tk for the 2‘-deoxyribose ring. Intimately associated with the conformation of the 2‘-deoxyribose ring is the value of the C−N torsion angle χ, which positions the nucleobase into two different domains (syn or anti). The often-conflicting sugar and nucleobase conformational parameters were studied using North and South methanocarbadeoxythymidine analogues (6 and 7), which forced HSV-tk to make a clear choice in the conformation of the substrate. The results provide new insights into the mechanism of action of this enzyme, which cannot be gleaned from a static X-ray crystal structure

Machine Learning-Based Approach to Developing Potent EGFR Inhibitors for Breast CancerDesign, Synthesis, and In Vitro Evaluation

The epidermal growth factor receptor (EGFR) is vital for regulating cellular functions, including cell division, migration, survival, apoptosis, angiogenesis, and cancer. EGFR overexpression is an ideal target for anticancer drug development as it is absent from normal tissues, marking it as tumor-specific. Unfortunately, the development of medication resistance limits the therapeutic efficacy of the currently approved EGFR inhibitors, indicating the need for further development. Herein, a machine learning-based application that predicts the bioactivity of novel EGFR inhibitors is presented. Clustering of the EGFR small-molecule inhibitor (∼9000 compounds) library showed that N-substituted quinazolin-4-amine-based compounds made up the largest cluster of EGFR inhibitors (∼2500 compounds). Taking advantage of this finding, rational drug design was used to design a novel series of 4-anilinoquinazoline-based EGFR inhibitors, which were first tested by the developed artificial intelligence application, and only the compounds which were predicted to be active were then chosen to be synthesized. This led to the synthesis of 18 novel compounds, which were subsequently evaluated for cytotoxicity and EGFR inhibitory activity. Among the tested compounds, compound 9 demonstrated the most potent antiproliferative activity, with 2.50 and 1.96 μM activity over MCF-7 and MDA-MB-231 cancer cell lines, respectively. Moreover, compound 9 displayed an EGFR inhibitory activity of 2.53 nM and promising apoptotic results, marking it a potential candidate for breast cancer therapy

Selective binding of <i>trans</i>- and <i>cis</i>-hinokiresinols on CB1R and CB2Rs.

<p>(A) Competitive binding experiments against [³H]CP55,940 (³H-CP) were performed using either membrane fractions from mouse brain (mCB1R) or CHO cells stably expressing human CB2R (hCB2R) in the presence of <i>trans</i>- or <i>cis</i>-hinokiresinols (<i>trans</i>- or <i>cis</i>-HR). Data are presented as mean ± SEM. N = 6. (B) Calculated EC₅₀ and IC₅₀ values of antagonistic and agonistic activities from CB1R reporter gene assay and forskolin-stimulated cAMP accumulation assay in CHO-hCB2R cells. Hu-210 was used as a positive control for agonistic activity.</p

Non-Selective Cannabinoid Receptor Antagonists, Hinokiresinols Reduce Infiltration of Microglia/Macrophages into Ischemic Brain Lesions in Rat via Modulating 2-Arachidonolyglycerol-Induced Migration and Mitochondrial Activity

<div><p>Growing evidence suggests that therapeutic strategies to modulate the post-ischemic inflammatory responses are promising approaches to improve stroke outcome. Although the endocannabinoid system has been emerged as an endogenous therapeutic target to regulate inflammation after stroke insult, the downstream mechanisms and their potentials for therapeutic intervention remain controversial. Here we identified <i>trans</i>- and <i>cis</i>-hinokiresinols as novel non-selective antagonists for two G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 and type 2. The Electric Cell-substrate Impedance Sensing and Boyden chamber migration assays using primary microglial cultures revealed that both hinokiresinols significantly inhibited an endocannabinoid, 2-arachidonoylglycerol-induced migration. Hinokiresinols modulated 2-arachidonoylglycerol-induced mitochondrial bioenergetics in microglia as evidenced by inhibition of ATP turnover and reduction in respiratory capacity, thereby resulting in impaired migration activity. In rats subjected to transient middle cerebral artery occlusion (1.5-h) followed by 24-h reperfusion, post-ischemic treatment with hinokiresinols (2 and 7-h after the onset of ischemia, 10 mg/kg) significantly reduced cerebral infarct and infiltration of ED1-positive microglial/macrophage cells into cerebral ischemic lesions <i>in vivo</i>. Co-administration of exogenous 2-AG (1 mg/kg, i.v., single dose at 2 h after starting MCAO) abolished the protective effect of <i>trans</i>-hinokiresionol. These results suggest that hinokiresinols may serve as stroke treatment by targeting the endocannabinoid system. Alteration of mitochondrial bioenergetics and consequent inhibition of inflammatory cells migration may be a novel mechanism underlying anti-ischemic effects conferred by cannabinoid receptor antagonists.</p></div