Selected 1,3-Benzodioxine-Containing Chalcones as Multipotent Oxidase and Acetylcholinesterase Inhibitors

Chalcones are considered effective templates for the development of monoamine oxidase (MAO) and cholinesterase (ChE) inhibitors. The present work describes the syntheses of selected 1,3-benzodioxine-containing chalcones ( CD3, CD8 and CD10 ), and their inhibitory activities against MAO-A, MAO-B, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Compound CD8 most potently inhibited MAO-B with an IC₅₀ value of 0.026 μM, followed by CD10 and CD3 (1.54 and 1.68 μM, respectively). CD8 potently and non-selectively inhibited MAO-A (IC₅₀ value of 0.023 μM). On the other hand, CD10 and CD8 inhibited AChE with IC₅₀ values of 5.40 and 9.57 μM, respectively. Kinetics and reversibility experiments showed that all synthesized molecules were competitive and reversible inhibitors, and the K_i values of CD8 for MAO-A and MAO-B were 0.018 and 0.0019 μM, respectively. By in vitro and in silico analyses, all compounds were found to have high passive human gastrointestinal absorptions, blood-brain barrier permeabilities, and non-toxicities. Molecular docking simulations revealed that docking affinity of each compound for MAO-B was higher than that for MAO-A. The results indicate that CD8 is a potent non-selective MAO inhibitor, and CD10 is an effective selective MAO-B inhibitor, and both possess AChE inhibitory activity. Therefore, we suggest that CD8 and CD10 be considered potential dual-targeting inhibitors of MAO and AChE for the treatment of various neurodegenerative disorders.     

Exploration of a Library of 3,4‐(Methylenedioxy)aniline‐Derived Semicarbazones as Dual Inhibitors of Monoamine Oxidase and Acetylcholinesterase: Design,Synthesis, and Evaluation

A library of 3,4‐(methylenedioxy)aniline‐derived semicarbazones was designed, synthesized, and evaluated as monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitors for the treatment of neurodegenerative diseases. Most of the new compounds selectively inhibited MAO‐B and AChE, with IC₅₀ values in the micro‐ or nanomolar ranges. Compound 16 , 1‐(2,6‐dichlorobenzylidene)‐4‐(benzo[1,3]dioxol‐5‐yl)semicarbazide presented a balanced multifunctional profile of MAO‐A (IC₅₀=4.52±0.032 μm ), MAO‐B (IC₅₀=0.059±0.002 μm ), and AChE (IC₅₀=0.0087±0.0002 μm ) inhibition without neurotoxicity. Kinetic studies revealed that compound 16 exhibits competitive and reversible inhibition against MAO‐A and MAO‐B, and mixed‐type inhibition against AChE. Molecular docking studies further revealed insight into the possible interactions within the enzyme–inhibitor complexes. The most active compounds were found to interact with the enzymes through hydrogen bonding and hydrophobic interactions. Additionally, in silico molecular properties and ADME properties of the synthesized compounds were calculated to explore their drug‐like characteristics.     

Homarine Alkyl Ester Derivatives as Promising Acetylcholinesterase Inhibitors

Reversible acetylcholinesterase (AChE) inhibitors are key therapeutic tools to modulate the cholinergic connectivity compromised in several degenerative pathologies. In this work, four alkyl esters of homarine were synthesized and screened by using Electrophorus electricus AChE and rat brain AChE-rich fraction. Results showed that all homarine alkyl esters are able to inhibit AChE by a competitive inhibition mode. The effectiveness of AChE inhibition increases with the alkyl side chain length of the homarine esters, being HO−C₁₆ (IC₅₀=7.57±3.32 μM and K_i=18.96±2.28 μM) the most potent inhibitor. The fluorescence quenching studies confirmed that HO−C₁₆ is the compound with higher selectivity and affinity for the tryptophan residues in the catalytic active site of AChE. Preliminary cell viability studies showed that homarine esters display no toxicity for human neuronal SH-SY5Y cells. Thus, the long-chain homarine esters emerge as new anti-cholinesterase agents, with potential to be considered for therapeutic applications development.     

Sulfonamide Inhibitors of β-Catenin Signaling as Anticancer Agents with Different Output on c-MYC

The Wnt/β-catenin pathway is often found deregulated in cancer. The aberrant accumulation of β-catenin in the cell nucleus results in the development of various malignancies. Specific drugs against this signaling pathway for clinical treatments have not been approved yet. Herein we report inhibitors of β-catenin signaling of potential therapeutic value as anticancer agents. Ethyl 4-((4-(trifluoromethyl)phenyl)sulfonamido)benzoate (compound 14 ) inhibits the effect on Wnt reporter with an IC₅₀ value of 7.0 μM, significantly reduces c-MYC levels, inhibits HCT116 colon cancer cell growth (IC₅₀ 20.2 μM), does not violate Lipinski and Veber rules, and shows predicted Caco-2 and MDCK cell permeability P_app>500 nm s⁻¹. Compound 14 seems to have potential for the development of new anticancer therapies.     

Engineered s-Triazine-Based Dendrimer-Honokiol Conjugates as Targeted MMP-2/9 Inhibitors for Halting Hepatocellular Carcinoma

Despite the advances in developing MMP-2/9 inhibitors, off-target side effects and pharmacokinetics problems remain major challenges hindering their clinical success in cancer therapy. However, recent targeting strategies have clearly revitalized MMP research. Herein, we introduce new s-triazine-based dendrimers endowed with intrinsic MMP-2/9 inhibitory potential and tetherable to hepatocellular carcinoma-specific targeting ligands and anticancer agents via biodegradable linkages for targeted therapy. The designed dendrimeric platform was built with potential zinc-binding branching linkers (hydrazides) and termini (carboxylic acids and hydrazides) to confer potency against MMP-2/9. Preliminary cytotoxicity screening and MMP-2/9 inhibition assay of the free dendrimers revealed promising potency (MMP-9; IC₅₀=0.35–0.57 μM, MMP-2; IC₅₀=0.39–0.77 μM) within their safe doses (EC₁₀₀=94.15–42.75 μM). The hydrazide dendrimer was comparable to NNGH and superior to the carboxylic acid analogue. MTT assay showed that the free dendrimers were superior to the reference anticancer agent honokiol. Their anticancer potency was enhanced by HK conjugation, targeting ligands installation and PEGylation as exemplified by the hydrazide dendrimer conjugate (TPG₃−NH₂)-SuHK-FA-SuPEG (Huh-7; IC₅₀=5.54 μM, HepG-2; IC₅₀=10.07 μM) being 4 folds more active than HK, followed by the carboxylic acid conjugate (TPG₃−OH)-HK-LA-PEG (Huh-7; IC₅₀=14.97, HepG-2; IC₅₀=21.29 μM). This was consistent with apoptosis studies.     

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC₅₀=0.78±0.01 μM), HT29 (IC₅₀=0.92±0.15 μM) and K562 (IC₅₀=47.25±1.24 μM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G₁/G₀ phase and decreased cell population in G₂/M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg⁻¹ dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.     

Allelochemical Activities of Pyrrolizidine Alkaloids: Interactions with Neuroreceptors and Acetylcholine Related Enzymes

Thirteen pyrrolizidine alkaloids (PAs) 3′-acetylheliosupine, echihumiline, echihumiline N-oxide, echimidine, heliosupine, heliosupine N-oxide, heliotrine, monocrotaline, pycnanthine, retronecine, riddeline, senecionine, and seneciphylline) were analyzed for their interactions with acetylcholine-related enzymes, such as acetylcholine esterase (AChE), butyrylcholinesterase (BChE), choline acetyl transferase (ChAT), and neuroreceptors, such as α₁- and α₂-adrenergic, nicotinergic (nACh), muscarinergic (mACh) and serotonin₂ (5-HT₂) receptors. Whereas most PAs did not affect the enzymes, they show significant binding activities to mACh and 5-HT₂ receptors: Twelve PAs exhibited a 50% inhibition of the specific binding of the radioligand [³H]quinuclidinyl benzilate (QNB) at the mAChR, i.e., IC₅₀ values were between 8.7 μM and 512.5 μM, and 10 PAs exerted a 50% inhibition of the specific binding of the radioligand [³H]ketanserine at the 5-HT₂R with IC₅₀ values between 23.2 μM and 608.6 μM. The most active compound was 3′-acetylheliosupine, which was able to bind to all of the studied receptors with IC₅₀ values in the range between 2.9 μM and 159.7 μM. The data imply that free PAs and PA N-oxides can affect several molecular targets: Besides long-term toxicity through DNA alkylation (by PA metabolites generated in the liver), liver and pneumotoxicity, neuroreceptors (among other molecular targets) may be modulated. The interference of PAs with neuronal signal transduction could mediate adverse physiological responses in herbivores and could thus contribute to chemical defense in plants and animals against herbivores and predators.     

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PL^Pro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PL^Pro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PL^Pro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PL^Pro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC₅₀ value below 40 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC₅₀ value below 10 μM. Among four inhibitors with an IC₅₀ value below 10 μM, SJB2-043 is the most unique in that it does not fully inhibit PL^Pro but has a noteworthy IC₅₀ value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PL^Pro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PL^Pro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.     

2-Hydroxy-4-benzyloxylimine Resveratrol Derivatives as Potential Multifunctional Agents for the Treatment of Parkinson's Disease

A series of 2-hydroxy-4-benzyloxylimine resveratrol derivatives was designed, synthesized and evaluated as multifunctional agents for the treatment of Parkinson's disease. The results revealed that most derivatives possessed good multifunctional activities. Among them, representative compound (E)-5-[(4-fluorobenzyl)oxy]-2-{[(4-hydroxyphenyl)imino]methyl}phenol ( 7 h ) exhibited excellent MAO-B inhibition (IC₅₀=8.43×10⁻³ μM) and high antioxidant activity (ORAC=3.45 Trolox equivalent). Additionally, 7 h displayed good metal chelating ability, appropriate blood–brain barrier (BBB) permeability, significant neuroprotective effect, and great anti-neuroinflammatory activity. Furthermore, 7 h can also ameliorate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease symptoms in mice. Therefore, compound 7 h was found to be a promising candidate for further development against PD.     

2-amino-3,4,5-trimethoxybenzophenones as potent tubulin polymerization inhibitors

A series of novel 2‐amino‐3,4,5‐trimethoxybenzophenone analogues exhibited excellent activity as tubulin polymerization inhibitors by targeting the colchicine binding site of microtubules. The lead compound 17 exhibited an IC₅₀ value of 1.6 μM , similar to that of combretastatin A‐4 (IC₅₀=1.9 μM ). It also displayed remarkable anti‐proliferative activity, with IC₅₀ values ranging from 7–16 nM against a variety of human cancer cell lines and one MDR(+) cancer cell line. SAR information indicated that the introduction of an amino group at the C2 position of benzophenone ring A and the C3’ position of benzophenone ring B play important roles in maximizing activity.     

Experimental and Computational Evaluation of Piperonylic Acid Derived Hydrazones Bearing Isatin Moieties as Dual Inhibitors of Cholinesterases and Monoamine Oxidases

A set of piperonylic acid derived hydrazones with variable isatin moieties was synthesized and evaluated for their inhibitory activity against the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A/B). The results of in vitro studies revealed IC₅₀ values in the micromolar range, with the majority of the compounds showing selectivity for the MAO-B isoform. N-[2-Oxo-1-(prop-2-ynyl)indolin-3-ylidene]benzo[d][1,3]dioxole-5-carbohydrazide ( 3 ) was identified as a lead AChE inhibitor with IC₅₀=0.052±0.006 μm . N-[(3E)-5-chloro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-2H-1,3-benzodioxole-5-carbohydrazide ( 2 ) was the lead MAO-B inhibitor with IC₅₀=0.034±0.007 μm , and showed 50 times greater selectivity for MAO-B over MAO-A. The kinetic studies revealed that compounds 2 and 3 displayed competitive and reversible inhibition of AChE and MAO-B, respectively. The molecular docking studies revealed the significance of hydrophobic interactions in the active site pocket of the enzymes under investigation. Further optimization studies might lead to the development of potential neurotherapeutic agents.     

Repurposing of 8-Hydroxyquinoline-Based Butyrylcholinesterase and Cathepsin B Ligands as Potent Nonpeptidic Deoxyribonuclease I Inhibitors

A library of 31 butyrylcholinesterase (BChE) and cathepsin B (CatB) inhibitors was screened in vitro for inhibition of deoxyribonuclease I (DNase I). Compounds 22 , 8 and 7 are among the most potent synthetic non-peptide DNase I inhibitors reported to date. Three 8-hydroxyquinoline analogues inhibited both DNase I and BChE with IC₅₀ values below 35 μM and 50 nM, respectively, while two nitroxoline derivatives inhibited DNase I and Cat B endopeptidase activity with IC₅₀ values below 60 and 20 μM. Selected derivatives were screened for various co-target binding affinities at dopamine D₂ and D₃, histamine H₃ and H₄ receptors and inhibition of 5-lipoxygenase. Compound 8 bound to the H₃ receptor and is highlighted as the most promising multifunctional ligand with a favorable pharmacokinetic profile and one of the most potent non-peptide DNase I inhibitors. The present study demonstrates that 8-hydroxyquinoline is a structural fragment critical for DNase I inhibition in the presented series of compounds.     

Molecular‐Target‐Based Anticancer Photosensitizer: Synthesis and in vitro Photodynamic Activity of Erlotinib–Zinc(II) Phthalocyanine Conjugates

Targeted photodynamic therapy is a new promising therapeutic strategy to overcome growing problems in contemporary medicine, such as drug toxicity and drug resistance. A series of erlotinib–zinc(II) phthalocyanine conjugates were designed and synthesized. Compared with unsubstituted zinc(II) phthalocyanine, these conjugates can successfully target EGFR‐overexpressing cancer cells owing to the presence of the small molecular‐target‐based anticancer agent erlotinib. All conjugates were found to be essentially non‐cytotoxic in the absence of light (up to 50 μM ), but upon illumination, they show significantly high photo‐cytotoxicity toward HepG2 cells, with IC₅₀ values as low as 9.61–91.77 nM under a rather low light dose (λ=670 nm, 1.5 J cm^?2). Structure–activity relationships for these conjugates were assessed by determining their photophysical/photochemical properties, cellular uptake, and in vitro photodynamic activities. The results show that these conjugates are highly promising antitumor agents for molecular‐target‐based photodynamic therapy.     

Structure‐Based Design of New KSP‐Eg5 Inhibitors Assisted by a Targeted Multicomponent Reaction

An integrated multidisciplinary approach that combined structure‐based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi+Boc removal/cyclization reaction sequence generated low‐micromolar‐range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds— 20 (IC₅₀=1.49 μM , EC₅₀=3.63 μM ) and 22 (IC₅₀=1.37 μM , EC₅₀=6.90 μM )—were synthesized with high efficiency by taking advantage of the multicomponent reactions.     

Antagonism of the Stat3-Stat3 protein dimer with salicylic acid based small molecules

More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure–activity relationship (SAR) study based on the previously identified inhibitor S3I‐201 (IC₅₀=86 μM , K_i>300 μM ). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC₅₀ range of 18.7–51.9 μM , and disruption of Stat3–pTyr peptide interactions with K_i values in the 15.5–41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.     

Design,Synthesis, Structure-Activity Relationship and Docking Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as Potent Antiplasmodial as well as Anticancer Agents

A novel series of synthetic functionalized arylvinyl-1,2,4-trioxanes ( 8 a – p ) has been prepared and assessed for their in vitro antiplasmodial activity against the chloroquine-resistant Pf INDO strain of Plasmodium falciparum by using a SYBR green-I fluorescence assay. Compounds 8 g (IC₅₀=0.051 μM; SI=589.41) and 8 m (IC₅₀=0.059 μM; SI=55.93) showed 11-fold and >9-fold more potent antiplasmodial activity, respectively, as compared to chloroquine (IC₅₀=0.546 μM; SI=36.63). Different in silico docking studies performed on many target proteins revealed that the most active arylvinyl-1,2,4-trioxanes ( 8 g and 8 m ) showed dihydrofolate reductase (DHFR) binding affinities on a par with those of chloroquine and artesunate. The in vitro cytotoxic potentials of 8 a – p were also evaluated against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines. Following screening, five derivatives viz. 8 a , 8 h , 8 l , 8 m and 8 o (IC₅₀=1.65–31.7 μM; SI=1.08–10.96) were found to show potent cytotoxic activity against (A549) lung cancer cell lines, with selectivity superior to that of the reference compounds artemisinin (IC₅₀=100 μM), chloroquine (IC₅₀=100 μM) and artesunic acid (IC₅₀=9.85 μM; SI=0.76). In fact, the most active 4-naphthyl-substituted analogue 8 l (IC₅₀=1.65 μM; SI >10) exhibited >60 times more cytotoxicity than the standard reference, artemisinin, against A549 lung cancer cell lines. In silico docking studies of the most active anticancer compounds, 8 l and 8 m , against EGFR were found to validate the wet lab results. In summary, a new series of functionalized aryl-vinyl-1,2,4-trioxanes ( 8 a – p ) has been shown to display dual potency as promising antiplasmodial and anticancer agents.     

Cell‐Selective,Apoptosis‐inducing Rhodium(III) Crown Thiaether Complexes

Half‐sandwich rhodium(III) polypyridyl (pp) complexes with the metal atom capped by the facial crown thiaether 1,4,7‐trithiacyclononane [9]aneS₃ represent a promising class of apoptosis‐inducing potent cytostatic agents. The necrotic damage caused by the complexes is negligible. In vitro cytotoxicity assays with the human cancer cell lines MCF‐7 and HT‐29 and immortalized HEK‐293 cells indicate that the dicationic κ²N(imino) complexes [([9]aneS₃)RhCl(pp)]²⁺ are much more active than monocationic complexes [([9]aneS₃)RhCl₂(L)]⁺ (L=imidazole, CH₃CN). Whereas the κ²N(amino) complex [([9]aneS₃)RhCl(piperazine)]²⁺ is inactive, replacing piperazine with the structurally analogous κ²S (thiaether) ligand 1,4‐dithiane restores cytotoxicity as evidenced by IC₅₀ values in the range 8.1‐11.6 μM . Spectroscopic (CD, UV/Vis, NOESY) and viscosity measurements indicate that the active dppz complex 8 (IC₅₀ values: 4.7–8.9 μM ) exhibits strong intercalative binding towards DNA whereas the even more potent bipyrimidine complex 9 (IC₅₀ values: 0.6–1.9 μM ) causes no alteration of the duplex B conformation. Weaker intercalative binding is observed for the dpq complex 7 . A comparative annexin V–propidium iodide binding assay with lymphoma (BJAB) cells and healthy leukocytes demonstrates that the cytotoxic activity of complex 8 and particularly complex 9 is highly selective towards the malignant cells.     

Design,Synthesis, and Biological Evaluation of New Urushiol Derivatives as Potent Class I Histone Deacetylase Inhibitors

In the present study, a novel series of 11 urushiol-based hydroxamic acid histone deacetylase (HDAC) inhibitors was designed, synthesized, and biologically evaluated. Compounds 1 – 11 exhibited good to excellent inhibitory activities against HDAC1/2/3 (IC₅₀: 42.09–240.17 nM) and HDAC8 (IC₅₀: 16.11–41.15 nM) in vitro, with negligible activity against HDAC6 (>1409.59 nM). Considering HDAC8, docking experiments revealed some important features contributing to inhibitory activity. According to Western blot analysis, select compounds could notably enhance the acetylation of histone H3 and SMC3 but not-tubulin, indicating their privileged structure is appropriate for targeting class I HDACs. Furthermore, antiproliferation assays revealed that six compounds exerted greater in vitro antiproliferative activity against four human cancer cell lines (A2780, HT-29, MDA-MB-231, and HepG2, with IC₅₀ values ranging from 2.31–5.13 μM) than suberoylanilide hydroxamic acid; administration of these compounds induced marked apoptosis in MDA-MB-231 cells, with cell cycle arrest in the G2/M phase. Collectively, specific synthesized compounds could be further optimized and biologically explored as antitumor agents.     

Synthesis,Cytotoxicity, and Insight into the Mode of Action of Re(CO)3 Thymidine Complexes

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of Re^I(CO)₃ core complexes of thymidine and uridine. For the binding of the Re^I(CO)₃ core, a tridentate dipicolylamine metal chelate was introduced at positions C5′, C2′, N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5′ or N3 (IC₅₀=124–160 μM ). No toxicity was observed for complexes modified at C2′ or C5. Complex 53 , with a dodecylene spacer at C5′, exhibits remarkable toxicity and is more potent than cisplatin, with an IC₅₀ value of 6.0 μM . To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)₃]⁺¹–nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK‐1), enzyme inhibition was observed for complexes modified at either N3 or C5′, but our results suggest that the toxicity cannot be attributed solely to interaction with hTK‐1.