E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain‐like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule‐associated serine proteases. Its exopeptidase activity is structurally explained by the so‐called exclusion domain, which blocks the active‐site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)‐trans‐epoxysuccinyl‐L ‐leucylamido‐3‐methylbutane (E‐64c) (k₂/K_i=140±5 M ^?1 s^?1) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′–S2′ area with its leucine‐isoamylamide moiety. Furthermore, structure–activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n‐butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56 000±1700 M ^?1 s^?1).     

Polysubstituted Pyrimidines as Potent Inhibitors of Prostaglandin E2 Production: Increasing Aqueous Solubility

Water solubility is one of the key features of potential therapeutic agents. In order to enhance the low water solubility of the parent 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine, a potent inhibitor of prostaglandin E₂ (PGE₂) production, we synthesized and evaluated a new series of derivatives in which the butyl group at the C5 position of the pyrimidine ring was replaced with a less lipophilic substituent, preferably with a hydrophilic aliphatic moiety. Except for the 5-cyanopyrimidine derivative, all target compounds exhibited increased (2.7–87-fold) water solubility relative to the parent compound. Although nontoxic in mouse peritoneal cells, the prepared compounds were either equipotent or weaker inhibitors of PGE₂ production than the parent compound. The most promising compound from the series was found to be the 5-(2,5,8,11-tetraoxadodecyl)pyrimidine derivative (with three polyethylene glycol units at the C5 position), which exhibited 32-fold higher water solubility and only slightly weaker inhibitory activity (22 % of remaining PGE₂ production) compared with the parent compound (15 % of remaining PGE₂ production).     

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies

We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2′ ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC₅₀ value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme K_i but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIV_DRV^R_P20 and HIV_DRV^R_P30 viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.     

Adenine Modification at C7 as a Viable Strategy to Potentiate the Antimalarial Activity of Quinolones

Although many quinolones have shown promise as potent antimalarials, their clinical development has been slow due to poor performance in vivo. Insights into structural modifications that can improve their therapeutic potential will be very valuable in this vibrant area of research. Our studies involving a library of quinolones which vary in substitution pattern at N1, C3, C6 and C7 positions have shown that the presence of adenine moiety at C7 can bring a noticeable improvement in activity compared to other heterocyclic groups at this location. The most potent compound emerged from this study showed IC₅₀ values of 0.38 μM and 0.75 μM against chloroquine-sensitive and -resistant (W2) strains, respectively. Docking analysis in the Q_o site of cytochrome bc1 complex revealed the contribution of a key H-bonding interaction from the adenine unit in target binding. This corroborates with compound-induced loss of mitochondrial functions. These findings not only open avenues for further exploration of antimalarial potential of adenine-modified quinolones, but also suggests broader opportunities during lead-optimization against other antimalarial targets.     

Bisamidate Prodrugs of 2‐Substituted 9‐[2‐(Phosphonomethoxy)ethyl]adenine (PMEA,adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

Novel small‐molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2‐substituted derivatives of 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L ‐phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2‐fluoro PMEA derivative (IC₅₀=0.145 μM ). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.     

Unusual Lipid A from a Cold‐Adapted Bacterium: Detailed Structural Characterization

Colwellia psychrerythraea 34H is a Gram‐negative cold‐adapted microorganism that adopts many strategies to cope with the limitations associated with the low temperatures of its habitat. In this study, we report the complete characterization of the lipid A moiety from the lipopolysaccharide of Colwellia. Lipid A and its partially deacylated derivative were completely characterized by high‐resolution mass spectrometry, NMR spectroscopy, and chemical analysis. An unusual structure with a 3‐hydroxy unsaturated tetradecenoic acid as a component of the primary acylation pattern was identified. In addition, the presence of a partially acylated phosphoglycerol moiety on the secondary acylation site at the 3‐position of the reducing 2‐amino‐2‐deoxyglucopyranose unit caused tremendous natural heterogeneity in the structure of lipid A. Biological‐activity assays indicated that C. psychrerythraea 34H lipid A did not show an agonistic or antagonistic effect upon testing in human macrophages.     

Chemoenzymatic synthesis of HIV-1 gp41 glycopeptides: effects of glycosylation on the anti-HIV activity and alpha-helix bundle-forming ability of peptide C34

C34 is a 34-mer peptide derived from the C-terminal ectodomain of HIV-1 envelope glycoprotein, gp41. The C34 region in native gp41 carries a conserved N-glycan at Asn637 and the sequence is directly involved in the virus-host membrane fusion, an essential step for HIV-1 infection. This paper describes the synthesis of glycoforms of C34 which carry a monosaccharide, a disaccharide, and a native oligosaccharide moiety. The synthesis of the glycopeptide which carries a native high-mannose type N-glycan was achieved by a chemoenzymatic approach by using an endoglycosidase-catalyzed oligosaccharide transfer as the key step. The effects of glycosylation on the inhibitory activity and the helix-bundle forming ability of C34 were investigated. It was found that glycosylation moderately decreases the anti-HIV activity of C34 and, in comparison with C34, glyco-C34 forms less compact six-helix bundles with the corresponding N-terminal peptide, N36. This study suggests that conserved glycosylation modulates the anti-HIV activity and conformations of the gp41 C-peptide, C34.     

Modulation on C‐ and N‐Terminal Moieties of a Series of Potent and Selective Linear Tachykinin NK2 Receptor Antagonists

Herein we describe the synthesis of a series of new potent tachykinin NK₂ receptor antagonists by the modulation of the C‐ and N‐terminal moieties of ibodutant (MEN 15596, 1 ). The N‐terminal benzo[b]thiophene ring was replaced by different substituted naphthalenes and benzofurans, while further modifications were evaluated at the C‐terminal tetrahydropyran moiety. Most compounds demonstrated a high affinity for the human NK₂ receptor and high in vitro antagonist potency, indicating that a wide range of substituents at both termini can be incorporated in the molecule without detrimental effects on the interactions with the NK₂ receptor. Selected compounds were tested in vivo confirming their activity as NK₂ antagonists. In particular, after both iv and id administration to guinea pig, compound 61 b was able to antagonize NK₂‐induced colonic contractions with a potency and duration‐of‐action fully comparable to the reference compound 1 (MEN 15596, ibodutant).     

Synthesis and anti-HIV activity of aryl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydrazones as potent non-nucleoside reverse transcriptase inhibitors

A series of novel diarylpyrimidines (DAPYs) with a ketone hydrazone substituent on the methylene linker between the pyrimidine nucleus and the aryl moiety at the C‐4 position were synthesized, and their antiviral activity against human immunodeficiency virus (HIV)‐1 in MT‐4 cells was evaluated. Most compounds of this class exhibited excellent activity against wild‐type HIV‐1, with EC₅₀ values in the range of 1.7–13.2 nM . Of these compounds, 2‐bromophenyl‐2‐[(4‐cyanophenyl)amino]‐4‐pyrimidinone hydrazone ( 9 k ) displayed the most potent anti‐HIV‐1 activity (EC₅₀=1.7±0.6 nM ), with excellent selectivity for infected over uninfected cells (SI=5762). In addition, the 4‐methyl phenyl analogue 9 d (EC₅₀=2.4±0.2 nM , SI=18461) showed broad spectrum HIV inhibitory activity, with EC₅₀ values of 2.4±0.2 nM against wild‐type HIV‐1, 5.3±0.4 μM against HIV‐1 double‐mutated strain RES056 (K103N+Y181C), and 5.5 μM against HIV‐2 ROD strain. Furthermore, structure–activity relationship (SAR) data and molecular modeling results for these compounds are also discussed.     

Design,Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2′ ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2′ ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.     

Stepwise Post-glycosylation Modification of Sugar Moieties in Kanamycin Biosynthesis

Kanamycin A is the major 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6-amino-6-deoxy-d -glucose (6ADG) at O-4 and 3-amino-3-deoxy-d -glucose at O-6. Because the 6ADG moiety is derived from d -glucosamine (GlcN), deamination at C-2 and introduction of C-6-NH₂ are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C-6-NH₂, although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3′′-deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C-2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C-6-NH₂ is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo-trisaccharide formation, followed by the introduction of C-6-NH₂ by KanQ/KanB and deamination at C-2 by KanJ.     

Synthesis and Pharmacological Evaluation of Fluorinated Quinoxaline-Based κ-Opioid Receptor (KOR) Agonists Designed for PET Studies

κ-Opioid receptors (KORs) play a predominant role in pain alleviation, itching skin diseases, depression and neurodegenerative disorders such as multiple sclerosis. Therefore, imaging of KOR by a fluorinated PET tracer was envisaged. Two strategies were followed to introduce a F atom into the very potent class of cis,trans-configured perhydroquinoxalines. Whereas the synthesis of fluoroethyltriazole 2 has already been reported, fluoropyrrolidines 14 (1-[2-(3,4-dichlorophenyl)acetyl]-8-[(R)-3-fluoropyrrolidin-1-yl]-perhydroquinoxalines) were prepared by S_N2 substitution of a cyclic sulfuric acid derivative with hydroxypyrrolidine and subsequent transformation of the OH moiety into a F substituent. Fluoropyrrolidines 14 showed similar low-nanomolar KOR affinity and selectivity to the corresponding pyrrolidines, but the corresponding alcohols were slightly less active. In the cAMP and β-arrestin assay, 14b (proton at the 4-position) exhibited similar KOR agonistic activity as U-50,488. The fluoro derivatives 14b and 14c (CO₂CH₃ at the 4-position) revealed KOR-mediated anti-inflammatory activity as CD11c and the IFN-γ production were reduced significantly in mouse and human dendritic cells. Compounds 14b and 14-c also displayed anti-inflammatory and immunomodulatory activity in mouse and human T cells. The PET tracer [¹⁸F]- 2 was prepared by 1,3-dipolar cycloaddition. In vivo, [¹⁸F]- 2 did not label KOR due to very fast elimination kinetics. Nucleophilic substitution of a mesylate precursor provided [¹⁸F]- 14c . Unfortunately, defluorination of [¹⁸F]- 14c occurred in vivo, which was analyzed in detail by in vitro studies.     

The Pharmacological Profile of Second Generation Pyrovalerone Cathinones and Related Cathinone Derivative

Pyrovalerone cathinones are potent psychoactive substances that possess a pyrrolidine moiety. Pyrovalerone-type novel psychoactive substances (NPS) are continuously detected but their pharmacology and toxicology are largely unknown. We assessed several pyrovalerone and related cathinone derivatives at the human norepinephrine (NET), dopamine (DAT), and serotonin (SERT) uptake transporters using HEK293 cells overexpressing each respective transporter. We examined the transporter-mediated monoamine efflux in preloaded cells. The receptor binding and activation potency was also assessed at the 5-HT_1A, 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors. All pyrovalerone cathinones were potent DAT (IC₅₀ = 0.02–8.7 μM) and NET inhibitors (IC₅₀ = 0.03–4.6 μM), and exhibited no SERT activity at concentrations < 10 μM. None of the compounds induced monoamine efflux. NEH was a potent DAT/NET inhibitor (IC₅₀ = 0.17–0.18 μM). 4F-PBP and NEH exhibited a high selectivity for the DAT (DAT/SERT ratio = 264–356). Extension of the alkyl chain enhanced NET and DAT inhibition potency, while presence of a 3,4-methylenedioxy moiety increased SERT inhibition potency. Most compounds did not exhibit any relevant activity at other monoamine receptors. In conclusion, 4F-PBP and NEH were selective DAT/NET inhibitors indicating that these substances likely produce strong psychostimulant effects and have a high abuse liability.     

Silsesquioxane chemistry, 6 : The first beryllium silsesquioxane: synthesis and structure of [Cy7Si7O12BeLi]2·2THF

The compound [Cy₇Si₇O₁₂BeLi]₂·2THF (3) was prepared in high yield by reacting in situ prepared Cy₇Si₇O₉(OLi)₃ (2) with anhydrous BeCl₂ in THF at 20°C. The molecular structure of 3 has been determined by X-ray diffraction and is the first structurally characterized silsesquioxane derivative of beryllium. In the solid state, self-assembly leads to the formation of a dimeric molecule.     

1,2,4-Oxadiazole Topsentin Analogs with Antiproliferative Activity against Pancreatic Cancer Cells,Targeting GSK3β Kinase

A new series of topsentin analogs, in which the central imidazole ring of the natural lead was replaced by a 1,2,4-oxadiazole moiety, was efficiently synthesized. All derivatives were pre-screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. The five most potent compounds were further investigated in various pancreatic ductal adenocarcinoma (PDAC) cell lines, including SUIT-2, Capan-1, and Panc-1 cells, eliciting EC₅₀ values in the micromolar and sub-micromolar range, associated with significant reduction of cell migration. These remarkable results might be explained by the effects of these new topsentin analogues on epithelial-to-mesenchymal transition markers, including SNAIL-1/2 and metalloproteinase-9. Moreover, flow cytometric analysis after Annexin V-FITC and propidium iodide staining demonstrated that these derivatives enhanced apoptosis of PDAC cells. Keeping with these data, the PathScan intracellular signaling and ELISA array revealed cleavage of caspase-3 and PARP and a significant inhibition of GSK3β phosphorylation, suggesting this kinase as a potential downstream target of our novel compounds. This was further supported by a specific assay for the evaluation of GSK3β activity, showing IC₅₀ values for the most active compounds against this enzyme in the micromolar range.     

Structure–Activity Relationships of Glucosamine‐Derived Glycerolipids: the Role of the Anomeric Linkage,the Cationic Charge and the Glycero Moiety on the Antitumor Activity

The potent antitumor activity of 1‐O‐hexadecyl‐2‐O‐methyl‐3‐O‐(2′‐amino‐2′‐deoxy‐β‐D ‐glucopyranosyl)‐sn‐glycerol ( 1 ) was previously shown to arise through an apoptosis‐independent pathway. Here, a systematic structure–activity study in which the effects of the anomeric linkage, the cationic charge and the glycero moiety on the antitumor activity is described. Eight analogues of 1 were synthesized, and their antitumor activity against breast (JIMT1 and BT549), pancreas (MiaPaCa2) and prostate (DU145, PC3) cancer was determined. 1‐O‐Hexadecyl‐2‐O‐methyl‐3‐O‐(2′‐amino‐2′‐deoxy‐α‐D ‐glucopyranosyl)‐sn‐glycerol ( 2 ) consistently displayed the most potent activity against all five cell lines with CC₅₀ values in the range of 6–10 μM . However, replacement of the O‐glycosidic linkage by a thioglycosidic linkage or replacement of the amino group by an azide or guanidino group leads to a threefold or greater decrease in potency. The glycero moiety also contributes to the overall activity of 1 and 2 but its effects are of lesser importance. Investigation into the mode of action of this class of compounds revealed that, in agreement with previous findings, the cytotoxic effects arise through induction of large acid vacuoles.     

Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome

Lead (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF-7 GI₅₀=30 nM and 400-fold selective c.f. MCF10A (normal breast tissue). Acetamide moiety modification ( 13 a - g ) to introduce additional hydrophobicity was favoured with MCF-7 breast cancer cell activity enhanced at 1.3 nM. Other analogues were potent against the HT29 colon cancer cell line at 23 nM. Textbook SAR data was observed in the MCF-7 cell line, in an MTT assay, via the ortho ( 17 a ), meta ( 17 b ) and para ( 13 f ). The amino alcohol -OH moiety was pivotal, but no stereochemical preference noted. But, these data did not fit our homology modelling expectations. Aberrant MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) screening results and metabolic interference confirmed by sulforhodamine B (SRB) screening. Interfering analogues resulted in 120 and 80-fold CYP1A1 and CYP1A2 amplification, with no upregulation of SULT1A1. This is consistent with activation of the AhR pathway. Piperidine per-deuteration reduced metabolic inactivation. 3-OH / 4-OH piperidine analogues showed differential MTT and SRB activity supporting MTT assay metabolic inactivation. Data supports piperidine 3-OH, but not the 4-OH, as a CYP substrate. This family of β-amino alcohol substituted 3,4-dichlorophenylacetonitriles show broad activity modulated via the AhR pathway. By SRB analysis the most potent analogue was 23 b , (Z)-3-(4-(3-(4-phenylpiperidin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile.     

Propellanes as Rigid Scaffolds for the Stereodefined Attachment of σ-Pharmacophoric Structural Elements to Achieve σ Affinity

Following the concept of conformationally restriction of ligands to achieve high receptor affinity, we exploited the propellane system as rigid scaffold allowing the stereodefined attachment of various substituents. Three types of ligands were designed, synthesized and pharmacologically evaluated as σ₁ receptor ligands. Propellanes with (1) a 2-methoxy-5-methylphenylcarbamate group at the “left” five-membered ring and various amino groups on the “right” side; (2) benzylamino or analogous amino moieties on the “right” side and various substituents at the left five-membered ring and (3) various urea derivatives at one five-membered ring were investigated. The benzylamino substituted carbamate syn,syn-4a showed the highest σ₁ affinity within the group of four stereoisomers emphasizing the importance of the stereochemistry. The cyclohexylmethylamine 18 without further substituents at the propellane scaffold revealed unexpectedly high σ₁ affinity (K_i = 34 nM) confirming the relevance of the bioisosteric replacement of the benzylamino moiety by the cyclohexylmethylamino moiety. Reduction of the distance between the basic amino moiety and the “left” hydrophobic region by incorporation of the amino moiety into the propellane scaffold resulted in azapropellanes with particular high σ₁ affinity. As shown for the propellanamine 18, removal of the carbamate moiety increased the σ₁ affinity of 9a (K_i = 17 nM) considerably. Replacement of the basic amino moiety by H-bond forming urea did not lead to potent σ ligands. According to molecular dynamics simulations, both azapropellanes anti-5 and 9a as well as propellane 18 adopt binding poses at the σ₁ receptor, which result in energetic values correlating well with their different σ₁ affinities. The affinity of the ligands is enthalpy driven. The additional interactions of the carbamate moiety of anti-5 with the σ₁ receptor protein cannot compensate the suboptimal orientations of the rigid propellane and its N-benzyl moiety within the σ₁ receptor-binding pocket, which explains the higher σ₁ affinity of the unsubstituted azapropellane 9a.     

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.