Determination of d-Cycloserine Impurities in Pharmaceutical Dosage Forms: Comparison of the International Pharmacopoeia HPLC-UV Method and the DOSY NMR Method.

d-cycloserine is a broad-spectrum antibiotic that is currently being used as a secondary choice in the treatment of tuberculosis. In recent years, it has become more popular, due to its effect on the nervous system. In this current study, we provide evidence that The International Pharmacopoeia HPLC-UV method for d-cycloserine impurity profiling is not repeatable due to the variable response of cycloserine dimer, one of d-cycloserine impurities. Therefore, we introduced the DOSY (diffusion ordered spectroscopy) NMR (nuclear magnetic resonance) technique to determine the levels of d-cycloserine impurities in pharmaceutical dosage forms. The DOSY NMR technique allowed separation of d-cycloserine, its degradation products, and key process impurities in concentrations below pharmacopoeial specification limits. The proposed DOSY NMR method allowed accurate identification and quantification of the cycloserine dimer, which was not possible through the use of the pharmacopoeial HPLC method. The current method has the potential for practical use in analytical laboratories of the pharmaceutical industry.

Probing the Effects of Pyrimidine Functional Group Switches on Acyclic Fleximer Analogues for Antiviral Activity.

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as "fleximers" have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

Small molecule purine and pseudopurine derivatives: synthesis, cytostatic evaluations and investigation of growth inhibitory effect in non-small cell lung cancer A549.

Novel halogenated purines and pseudopurines with diverse aryl-substituted 1,2,3-triazoles were prepared. While p-(trifluoromethyl)-substituted 1,2,3-triazole in N-9 alkylated purine and 3-deazapurine was critical for strong albeit unselective activity on pancreatic adenocarcinoma cells CFPAC-1,1-(p-fluorophenyl)-1,2,3-triazole derivative of 7-deazapurine showed selective cytostatic effect on metastatic colon cancer cells SW620. Importantly, 1-(p-chlorophenyl)-1,2,3-triazole-tagged benzimidazole displayed the most pronounced and highly selective inhibitory effect in nM range on non-small cell lung cancer A549. This compound revealed to target molecular processes at the extracellular side and inside the plasma membrane regulated by GPLD1 and growth factor receptors PDGFR and IGF-1R leading to the inhibition of cell proliferation and induction of apoptosis mediated by p38 MAP kinase and NF-κB, respectively. Further optimisation of this compound as to reduce its toxicity in normal cells may lead to the development of novel agent effective against lung cancer.

1H NMR metabolomics of microbial metabolites in the four MW agricultural biogas plant reactors: A case study of inhibition mirroring the acute rumen acidosis symptoms.

In this study, nuclear magnetic resonance (1H NMR) spectroscopic profiling was used to provide a more comprehensive view of microbial metabolites associated with poor reactor performance in a full-scale 4 MW mesophilic agricultural biogas plant under fully operational and also under inhibited conditions. Multivariate analyses were used to assess the significance of differences between reactors whereas artificial neural networks (ANN) were used to identify the key metabolites responsible for inhibition and their network of interaction. Based on the results of nm-MDS ordination the subsamples of each reactor were similar, but not identical, despite homogenization of the full-scale reactors before sampling. Hence, a certain extent of variability due to the size of the system under analysis was transferred into metabolome analysis. Multivariate analysis showed that fully active reactors were clustered separately from those containing inhibited reactor metabolites and were significantly different. Furthermore, the three distinct inhibited states were significantly different from each other. The inhibited metabolomes were enriched in acetate, caprylate, trimethylamine, thymine, pyruvate, alanine, xanthine and succinate. The differences in the metabolic fingerprint between inactive and fully active reactors observed in this study resembled closely the metabolites differentiating the (sub) acute rumen acidosis inflicted and healthy rumen metabolomes, creating thus favorable conditions for the growth and activity of pathogenic bacteria. The consistency of our data with those reported before for rumen ecosystems shows that 1H NMR based metabolomics is a reliable approach for the evaluation of metabolic events at full-scale biogas reactors.

A Kinetic Approach in the Evaluation of Radical-Scavenging Efficiency of Sinapic Acid and Its Derivatives.

A kinetic approach was used to determine the radical scavenging activities of sinapic acid and its derivatives: sinapine, 4-vinylsyringol, syringic acid, syringaldehyde, and ethyl, propyl and butyl sinapate. The responses were expressed as rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) scavenging (RS), superoxide radical (O2˙-) scavenging (RFF), and ß-carotene bleaching in the emulsion system (RB). For RS and RB, the esters of sinapic acid showed the highest responses while, for RFF, this was seen for syringic acid. The effectiveness of the selected compounds for scavenging these free radicals was also determined at a fixed endpoint. The early response parameters were demonstrated to be good discriminators in assessing differences for antioxidants with comparable fixed endpoint activity. The primary feature that ranks the kinetic data and the endpoint determinations is interpreted in terms of the mechanisms of the reactions involved in each of the assays conducted.

Synergistic complex from plants Solanaceae exhibits cytotoxicity for the human hepatocellular carcinoma cell line HepG2.

BACKGROUND: It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells. METHODS: Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells. RESULTS: Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01-1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se. CONCLUSION: In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.

Conformational NMR Study of Bistriazolyl Anion Receptors.

Conformational features of pyridine- and pyrimidine-based bistriazolyl anion receptors dissolved in acetonitrile-d3 were assessed by multidimensional, heteronuclear NMR spectroscopy. NOESY correlation signals suggested preorganization of both host molecules in solution in the absence of anions. In addition, only a single set of signals was observed in the 1H NMR spectra, which suggested a symmetrical conformation of anion receptors or their conformational exchange that is fast on the NMR time-scale. Furthermore, the predominant conformations of the pyridine- and pyrimidine-based anion receptors are preserved upon addition of chloride, bromide, and acetate anions. Chemical shift changes observed upon addition of anions showed that the NH (thio)urea and triazole protons are involved in anion-receptor interactions through hydrogen bonding.

UV-visible and (1)H-(15)N NMR spectroscopic studies of colorimetric thiosemicarbazide anion sensors.

Four model thiosemicarbazide anion chemosensors containing three N-H bonds, substituted with phenyl and/or 4-nitrophenyl units, were synthesised and studied for their anion binding abilities with hydroxide, fluoride, acetate, dihydrogen phosphate and chloride. The anion binding properties were studied in DMSO and 9 : 1 DMSO-H2O by UV-visible absorption and (1)H/(13)C/(15)N NMR spectroscopic techniques and corroborated with DFT studies. Significant changes were observed in the UV-visible absorption spectra with all anions, except for chloride, accompanied by dramatic colour changes visible to the naked eye. These changes were determined to be due to the deprotonation of the central N-H proton and not due to hydrogen bonding based on (1)H/(15)N NMR titration studies with acetate in DMSO-d6-0.5% water. Direct evidence for deprotonation was confirmed by the disappearance of the central thiourea proton and the formation of acetic acid. DFT and charge distribution calculations suggest that for all four compounds the central N-H proton is the most acidic. Hence, the anion chemosensors operate by a deprotonation mechanism of the central N-H proton rather than by hydrogen bonding as is often reported.

Hyaluronic acid conjugates of glycine peptides and L-tryptophan.

This work reports about the conjugation of glycine C-terminal ethyl and methyl ester peptides and L-tryptophan methyl ester with sodium hyaluronate in aqueous solutions using the peptide coupling agent DMTMM (or short DMT, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride). Detailed infrared (IR) absorbance and 1H and 13C (2D) NMR studies (heteronuclear multi-bond correlation spectroscopy, HMBC) confirmed covalent and regioselective amide bonds with the D-glucuronate, but also proves the presence of DMT traces in all conjugates. The ethyl ester`s methyl protons on the peptides` C-terminal could be used to quantify the degree of substitution of the peptide on the hyaluronate scaffold by NMR. The ester group also proved stable during conjugation and work-up, and could in some cases be selectively cleaved in water whilst leaving the amide bond intact as shown by potentiometric charge titration, NMR and IR. The conjugates did not influence the capability of human umbilical vein endothelial cells (HUVECs) to reduce MTS (5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) to a formazan dye, which points towards a low cytotoxicity for the obtained products. The conjugation method and products could be tested for tissue engineering gels or drug delivery purposes with alternative, biologically active peptides.

Conformational analysis of geometric isomers of pitavastatin together with their lactonized analogues.

Super-statins are synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate-limiting enzyme responsible for the biosynthesis of cholesterol. All of the super-statins with a C=C double bond spacer between the heterocyclic and the dihydroxycarboxylic moiety that are currently on the market exist as E-isomers. To extend the understanding of conformational and thermodynamic preferences of Z-isomeric super-statin analogues, this study focused on analyzing pitavastatin and its lactonized derivatives via NMR spectroscopy and ab initio calculations. Z-isomeric pitavastatin analogues exist in solution as a pair of interconverting rotamers, where the Gibbs free energies between the major and minor rotamers are within 0.12 and 0.25 kcal mol-1 and the rotational energy barriers are between 15.0 and 15.9 kcal mol-1. The analysis of long-range coupling constants and ab initio calculations revealed that rotation across the C5'-C7 single bond is essential for generating a pair of atropisomers. The overall comparison of the results between Z-isomeric pitavastatin and rosuvastatin analogues demonstrated that the former are to some extent more flexible to attain numerous conformations. Demonstrating how structural differences between super-statin analogues induce distinctive conformational preferences provides important insight into the super-statins' conformational variability and may well improve future drug design.

The novel [4,5-e][1,3]diazepine-4,8-dione and acyclic carbamoyl imino-ureido derivatives of imidazole: synthesis, anti-viral and anti-tumor Activity evaluations.

In the present paper, we report on the synthesis, and in vitro antiviral and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4,8-dione (compounds 9-11) and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13) derivatives. These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM) with no concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 µM) with no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.

Cytotoxicity and Antibacterial Efficacy of Betaine- and Choline-Substituted Polymers.

Cationic charge has been widely used to increase polymer adsorption and flocculation of dispersions or to provide antimicrobial activity. In this work, cationization of hydroxyethyl cellulose (HEC) and polyvinyl alcohol (PVA) was achieved by covalently coupling betaine hydrochloride and choline chloride to the polymer backbones through carbonyl diimidazole (CDI) activation. Two approaches for activation were investigated. CDI in excess was used to activate the polymers' hydroxyls followed by carbonate formation with choline chloride, or CDI was used to activate betaine hydrochloride, followed by ester formation with the polymers' hydroxyls. The first approach led to a more significant cross-linking of PVA, but not of HEC, and the second approach successfully formed ester bonds. Cationic, nitrogen-bearing materials with varying degrees of substitution were obtained in moderate to high yields. These materials were analyzed by Fourier transform infrared spectroscopy, nuclear magnetic resonance, polyelectrolyte titration, and kaolin flocculation. Their dose-dependent effect on the growth of Staphylococcus aureus and Pseudomonas aeruginosa, and L929 mouse fibroblasts, was investigated. Significant differences were found between the choline- and betaine-containing polymers, and especially, the choline carbonate esters of HEC strongly inhibited the growth of S. aureus in vitro but were also cytotoxic to fibroblasts. Fibroblast cytotoxicity was also observed for betaine esters of PVA but not for those of HEC. The materials could potentially be used as antimicrobial agents for instance by coating surfaces, but more investigations into the interaction between cells and polysaccharides are necessary to clarify why and how bacterial and human cells are inhibited or killed by these derivatives, especially those containing choline.

Efficiency of Neat and Quaternized-Cellulose Nanofibril Fillers in Chitosan Membranes for Direct Ethanol Fuel Cells.

In this work, fully polysaccharide based membranes were presented as self-standing, solid polyelectrolytes for application in anion exchange membrane fuel cells (AEMFCs). For this purpose, cellulose nanofibrils (CNFs) were modified successfully with an organosilane reagent, resulting in quaternized CNFs (CNF (D)), as shown by Fourier Transform Infrared Spectroscopy (FTIR), Carbon-13 (C13) nuclear magnetic resonance (13C NMR), Thermogravimetric Analysis (TGA)/Differential Scanning Calorimetry (DSC), and ζ-potential measurements. Both the neat (CNF) and CNF(D) particles were incorporated in situ into the chitosan (CS) membrane during the solvent casting process, resulting in composite membranes that were studied extensively for morphology, potassium hydroxide (KOH) uptake and swelling ratio, ethanol (EtOH) permeability, mechanical properties, ionic conductivity, and cell performance. The results showed higher Young's modulus (119%), tensile strength (91%), ion exchange capacity (177%), and ionic conductivity (33%) of the CS-based membranes compared to the commercial Fumatech membrane. The addition of CNF filler improved the thermal stability of the CS membranes and reduced the overall mass loss. The CNF (D) filler provided the lowest (4.23 × 10-5 cm2 s-1) EtOH permeability of the respective membrane, which is in the same range as that of the commercial membrane (3.47 × 10-5 cm2s-1). The most significant improvement (~78%) in power density at 80 °C was observed for the CS membrane with neat CNF compared to the commercial Fumatech membrane (62.4 mW cm-2 vs. 35.1 mW cm-2). Fuel cell tests showed that all CS-based anion exchange membranes (AEMs) exhibited higher maximum power densities than the commercial AEMs at 25 °C and 60 °C with humidified or non-humidified oxygen, demonstrating their potential for low-temperature direct ethanol fuel cell (DEFC) applications.

Nitroreductase-sensitive fluorescent covalent organic framework for tumor hypoxia imaging in cells.

Covalent organic frameworks (COFs) have been used in cell imaging, but very rarely for imaging specific cell conditions. Herein, a ß-ketoenamine-based fluorescent COF was post-synthetically modified to incorporate a hypoxia-targeting molecule. Fluorescence microscopy imaging shows that the material discriminates between HeLa cells grown under hypoxia and those cultured under normoxia.

Novel 1,2,4-triazole and imidazole derivatives of L-ascorbic and imino-ascorbic acid: synthesis, anti-HCV and antitumor activity evaluations.

Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC(50)s of 10 ± 4 and 7.3 ± 0.1 µM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead.

Glasdegib Dimaleate: Synthesis, Characterization and Comparison of Its Properties with Monomaleate Analogue.

Glasdegib is a recently approved drug for the treatment of acute myeloid leukemia. It is formulated and marketed in monomaleate salt form. In our investigation, we were able to prepare a glasdegib dimaleate form, which could, in theory, exist in double-salt form or as a mixture of salt and co-crystal species. Therefore, the obtained crystals of glasdegib dimaleate were characterized via 15N ssNMR and single-crystal X-ray diffraction, which revealed that the obtained glasdegib dimaleate exists in double-salt form. This is a surprising finding based on the pKa values for glasdegib and maleic acid. Furthermore, we fully characterized the new dimaleate form using thermal analyses (DSC and TGA) and spectroscopy (IR and Raman). Finally, the physicochemical properties, such as solubility and chemical stability, of both forms were determined and compared.

Children gut microbiota exhibits a different composition and metabolic profile after in vitro exposure to Clostridioides difficile and increases its sporulation.

Clostridioides difficile (Clostridium difficile) infection (CDI) is one of the main public health concerns in adults, while children under 2 years of age are often colonized asymptomatically. In both adults and children, CDI is strongly associated with disturbances in gut microbiota. In this study, an in-vitro model of children gut microbiota was challenged with vegetative cells or a conditioned media of six different toxigenic C. difficile strains belonging to the ribotypes 027, 078, and 176. In the presence of C. difficile or conditioned medium the children gut microbiota diversity decreased and all main phyla (Bacteroidetes, Firmicutes, and Proteobacteria) were affected. The NMR metabolic spectra divided C. difficile exposed children gut microbiota into three clusters. The grouping correlated with nine metabolites (short chain fatty acids, ethanol, phenolic acids and tyramine). All strains were able to grow in the presence of children gut microbiota and showed a high sporulation rate of up to 57%. This high sporulation rate in combination with high asymptomatic carriage in children could contribute to the understanding of the reported role of children in C. difficile transmissions.

Organic acid cross-linked 3D printed cellulose nanocomposite bioscaffolds with controlled porosity, mechanical strength, and biocompatibility.

Herein, we fabricated chemically cross-linked polysaccharide-based three-dimensional (3D) porous scaffolds using an ink composed of nanofibrillated cellulose, carboxymethyl cellulose, and citric acid (CA), featuring strong shear thinning behavior and adequate printability. Scaffolds were produced by combining direct-ink-writing 3D printing, freeze-drying, and dehydrothermal heat-assisted cross-linking techniques. The last step induces a reaction of CA. Degree of cross-linking was controlled by varying the CA concentration (2.5-10.0 wt.%) to tune the structure, swelling, degradation, and surface properties (pores: 100-450 µm, porosity: 86%) of the scaffolds in the dry and hydrated states. Compressive strength, elastic modulus, and shape recovery of the cross-linked scaffolds increased significantly with increasing cross-linker concentration. Cross-linked scaffolds promoted clustered cell adhesion and showed no cytotoxic effects as determined by the viability assay and live/dead staining with human osteoblast cells. The proposed method can be extended to all polysaccharide-based materials to develop cell-friendly scaffolds suitable for tissue engineering applications.

Methoxymethyl (MOM) group nitrogen protection of pyrimidines bearing C-6 acyclic side-chains.

Novel N-methoxymethylated (MOM) pyrimidine (4-13) and pyrimidine-2,4-diones (15-17) nucleoside mimetics in which an isobutyl side-chain is attached at the C-6 position of the pyrimidine moiety were synthesized. Synthetic methods via O-persilylated or N-anionic uracil derivatives have been evaluated for the synthesis of N-1- and/or N-3-MOM pyrimidine derivatives with C-6 acyclic side-chains. A synthetic approach using an activated N-anionic pyrimidine derivative afforded the desired N,N-1,3-diMOM and N-1-MOM pyrimidines 4 and 5 in good yield. Introduction of fluorine into the side-chain was performed with DAST as the fluorinating reagent to give a N,N-1,3-diMOM pyrimidine 13 with a 1-fluoro-3-hydroxyisobutyl moiety at C-6. Conformational study of the monotritylated N-1-MOM pyrimidine 12 by the use of the NOE experiments revealed the predominant conformation of the compound to be one where the hydroxymethyl group in the C-6 side-chain is close to the N-1-MOM moiety, while the OMTr is in proximity to the CH(3)-5 group. Contrary to this no NOE enhancements between the N-1-MOM group and hydroxymethyl or fluoromethyl protons in 13 were observed, which suggested a nonrestricted rotation along the C-6 side-chain. Fluorinated N,N-1,3-diMOM pyrimidine 13 emerged as a model compound for development of tracer molecules for non-invasive imaging of gene expression using positron emission tomography (PET).

Benzylidene-bis-(4-hydroxycoumarin) and benzopyrano-coumarin derivatives: synthesis, ¹H/¹³C-NMR conformational and X-ray crystal structure studies and in vitro antiviral activity evaluations.

We report on the synthesis of 4-hydroxycoumarin dimers 1-15 bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives 16-20 and on their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities and structure of compounds 1-20 were deduced from their homo- and heteronuclear NMR measurements whereas the conformational properties of 5, 14 and 20 were assessed by the use of 1D difference NOE enhancements. Unequivocal proof of the stereostructure of compounds 7, 9, 16 and 18 was obtained by single crystal X-ray diffraction method. The X-ray crystal structure analysis revealed that two 4-hydroxycoumarin moieties in the 4-trifluoromethylphenyl- and 2-nitrophenyl derivatives (compounds 7 and 9, respectively) are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms. Consequently, the compounds 7 and 9 adopt conformations in which two 4-hydroxy-coumarin moieties are anti-disposed. Antiviral activity evaluation results indicated that the 4-bromobenzylidene derivative of bis-(4-hydroxycoumarin) (compound 3) possesses inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK⁻ KOS (ACVr) at a concentration of 9-12 µM and at a minimum cytotoxic concentration (MCC) greater than 20 µM. Compounds 4-6, 8, and 20 were active against feline herpes virus (50% effective concentration, EC50 = 5-8.1 µM), that is at a 4-7-fold lower concentration than the MCC.