Synthesis and Antimicrobial Activity of the Hybrid Molecules between Sulfonamides and Active Antimicrobial Pleuromutilin Derivative

A series of novel hybrid molecules between sulfonamides and active antimicrobial 14‐o‐(3‐carboxy‐phenylsulfide)‐mutilin were synthesized, and their in vitro antibacterial activities were evaluated by the broth microdilution. Results indicated that these compounds displayed potent antimicrobial activities in vitro against various drug‐susceptible and drug‐resistant Gram‐positive bacteria such as Staphylococci and streptococci, including methicillin‐resistant Staphylococcus aureus, and mycoplasma. In particular, sulfapyridine analog ( 6c ) exhibited more potent inhibitory activity against Gram‐positive bacteria and mycoplasma, including Staphylococcus aureus (MIC = 0.016–0.063 μg/mL), methicillin‐resistant Staphylococcus aureus (MIC = 0.016 μg/mL), Streptococcus pneumoniae (MIC = 0.032–0.063 μg/mL), Mycoplasma gallisepticum (MIC = 0.004 μg/mL), with respect to other synthesized compounds and reference drugs sulfonamide (MIC = 8–128 μg/mL) and valnemulin (MIC = 0.004–0.5 μg/mL). Furthermore, comparison between MIC values of pleuromutilin‐sulfonamide hybrids 6a–f with pleuromutilin parent compound 3 revealed that these modifications at 14 position side chain of the pleuromutilin with benzene sulfonamide could greatly improve the antibacterial activity especially against Gram‐positives.     

Synthesis and Antimicrobial Evaluation of 6‐Alkylamino‐N‐phenylpyrazine‐2‐carboxamides

This work presents synthesis and antimicrobial evaluation of nineteen 6‐alkylamino‐N–phenylpyrazine‐2‐carboxamides. Antimycobacterial activity was determined against Mycobacterium tuberculosis H37Rv, M. kansasii and two strains of M. avium. Generally, the antimycobacterial activity increased with prolongation of simple alkyl chain and culminated in compounds with heptylamino substitution ( 3e , 4e ) with MIC = 5–10 μm against M. tuberculosis H37Rv. On the contrary, derivatives with modified alkyl chain (containing e.g. terminal methoxy or hydroxy group) as well as phenylalkylamino derivatives were mainly inactive. The most active compounds (with hexyl to octylamino substitution) were evaluated for their in vitro activity against drug‐resistant strains of M. tuberculosis and possessed activity comparable to that of the reference drug isoniazid. None of the tested compounds were active against M. avium. Some derivatives exhibited activity against Gram‐positive bacteria including methicillin‐resistant Staphylococcus aureus (best MIC = 7.8 μm ), while Gram‐negative strains as well as tested fungal strains were completely unsusceptible. Active compounds were tested for in vitro toxicity on various cell lines and in most cases were non‐toxic up to 100 μm .     

Metronidazole containing pyrazole derivatives potently inhibit tyrosyl‐tRNA synthetase: design,synthesis, and biological evaluation

As an important enzyme in bacterial protein biosynthesis, tyrosyl‐tRNA synthetase (TyrRS) has been an absorbing therapeutic target for exploring novel antibacterial agents. A series of metronidazole‐based antibacterial agents has been synthesized and identified as TyrRS inhibitors with low cytotoxicity and significant antibacterial activity, especially against Gram‐negative organisms. Of the compounds obtained, 4f is the most potent agent which inhibited the growth of Pseudomonas aeruginosa ATCC 13525 (MIC = 0.98 μg/mL) and exhibited TryRS inhibitory activity (IC₅₀ = 0.92 μm ). Docking simulation was performed to further understand its potency. Membrane‐mediated apoptosis in P. aeruginosa was verified by flow cytometry.     

Design and synthesis of 4′‐((5‐benzylidene‐2,4‐dioxothiazolidin‐3‐yl)methyl)biphenyl‐2‐carbonitrile analogs as bacterial peptide deformylase inhibitors

Herein, we report the synthesis and screening of 4′‐((5‐benzylidene‐2,4‐dioxothiazolidin‐3‐yl)methyl)biphenyl‐2‐carbonitrile analogs 11(a–j) as bacterial peptide deformylase (PDF) enzyme inhibitors. The compounds 11b (IC₅₀ value = 139.28 μm ), 11g (IC₅₀ value = 136.18 μm ), and 11h (IC₅₀ value = 131.65 μm ) had shown good PDF inhibition activity. The compounds 11b (MIC range = 103.36–167.26 μg/mL), 11g (MIC range = 93.75–145.67 μg/mL), and 11h (MIC range = 63.61–126.63 μg/mL) had also shown potent antibacterial activity when compared with standard ampicillin (MIC range = 100.00–250.00 μg/mL). Thus, the active derivatives were not only PDF inhibitors but also efficient antibacterial agents. To gain more insight on the binding mode of the compounds with PDF enzyme, the synthesized compounds 11(a–j) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. The results suggest that this class of compounds has potential for development and use in future as antibacterial drugs.     

Design,synthesis, and in vitro antimicrobial activity of hydrazide–hydrazones of 2‐substituted acetic acid

In this study, 30 hydrazide–hydrazones of phenylacetic ( 3 – 10 ) and hydroxyacetic acid ( 11 – 32 ) were synthesized by the condensation reaction of appropriate 2‐substituted acetic acid hydrazide with different aromatic aldehydes. The obtained compounds were characterized by spectral data and evaluated in vitro for their potential antimicrobial activities against a panel of reference strains of micro‐organisms, including Gram‐positive bacteria, Gram‐negative bacteria, and fungi belonging to the Candida spp. The results from our antimicrobial assays indicated that among synthesized compounds 3 – 32 , especially compounds 6 , 14, and 26 showed high bactericidal activity (MIC = 0.488–7.81 μg/ml) against reference Gram‐positive bacteria, and in some cases, their activity was even better than that of commonly used antibiotics, such as cefuroxime or ampicillin.     

Discovery of the Highly Potent Fluoroquinolone‐Based Benzothiazolyl‐4‐thiazolidinone Hybrids as Antibacterials

A new series of fluoroquinolone‐based benzothiazolyl‐4‐thiazolidinone hybrids has been yielded via sulfated tungstate‐promoted highly accelerated N‐formylation at a piperazine residue of ciprofloxacin and norfloxacin entities. The formylated fluoroquinolone moieties were then coupled with substituted 2‐aminobenzothiazoles, which were generated from their respective para‐substituted amines to form corresponding Schiff base intermediates. The Schiff bases were then treated with thioglycolic acid to equip a new class of 4‐thiazolidinones to be analyzed for their antibacterial effects against two Gram‐positive (Staphylococcus aureus and Bacillus subtilis) and two Gram‐negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and were found highly potent with lowest Minimum inhibitory concentrations (MIC), 1–2 μg/mL, that is, more potent than control drugs ciprofloxacin (3.12–6.25 μg/mL). Initial outcomes provided for these novel molecular systems will aid researchers to design and develop new antibacterial drugs. The structural assignments of the new products were done on the basis of FT‐IR, ¹H NMR and ¹³C NMR spectroscopy, and elemental analysis.     

A Facile One‐Pot Synthesis of 2‐Arylamino‐5‐Aryloxylalkyl‐1,3,4‐Oxadiazoles and Their Urease Inhibition Studies

A one‐pot method for the synthesis of structural type urease inhibitors, 2‐amino‐1,3,4‐oxadiazoles, was developed. The structures of the compounds were established using spectroanalytical techniques and unambiguously confirmed by single‐crystal X‐ray analysis of compound 3o . The synthesized compounds were tested against jack beans urease, and most of the compounds ( 3c , 3g , 3j , 3k , 3n , 3r – 3v ) were found more active than the standard. The most potent compound ( 3u ) had an IC₅₀ value of 6.03 ± 0.02 μm as compared to the IC₅₀ value of the standard (thiourea; 22.0 ± 1.2 μm ). The prominent urease inhibition activity of these compounds may serve as an important finding in the development of less toxic and more potent antiulcer drugs. The compounds were also investigated against four bacterial strains, and some of the compounds ( 3g and 3r ) were found more potent than the standard drug (ciprofloxacin) against all the tested strains. The MIC value for compound 3g was 0.156 μmol/mL against the tested bacterial strains.     

Novel Glycoconjugate of 8‐Fluoro Norfloxacin Derivatives as Gentamicin‐resistant Staphylococcus aureus Inhibitors: Synthesis and Molecular Modelling Studies

Antibiotic resistance has been the subject of interest in clinical practice due to high prevalence of antibiotic‐resistant pathogenic organisms. In view of the prevalence of lesser resistance in antibiotics belonging to aminoglycoside class of compounds viz. Food and Drug Administration‐approved gentamicin for the treatment of Staphylococcus infections, which also has instances of resistance in the clinical isolates of Staphylococcus aureus, a series of novel glycoconjugates of 8‐fluoro norfloxacin analogues with high regio‐selectivity by employing copper (I)‐catalyzed 1, 3‐dipolar cycloaddition of 1‐O‐propargyl monosaccharides has been synthesized and evaluated for the antibacterial activity against gentamicin resistance Staphylococcus aureus. Among these compounds, the compound 10g showed better antibacterial activity (MIC = 3.12  μ g/ml) than gentamicin (Escherichia coli (12.5  μ g/ml), Staphylococcus aureus (6.25  μ g/ml) and Klebsiella pneumonia (6.25  μ g/ml), including gentamicin resistant (>50  μ g/ml) strain in vitro). The docking studies suggest DNA gyrase of Staphylococcus aureus as a probable target for the antibacterial action of compound 10g .     

Synthesis and In Vitro Antibacterial Activity of 7‐(3‐Alkoxyimino‐4‐methyl‐4‐methylaminopiperidin‐1‐yl)‐fluoroquinolone Derivatives

A series of novel 7‐(3‐alkoxyimino‐4‐methyl‐4‐methylaminopiperidin‐1‐yl)fluoroquinolone derivatives were designed, synthesized, and characterized by ¹H‐NMR, MS, and HRMS. These fluoroquinolones were evaluated for their in‐vitro antibacterial activity against representative Gram‐positive and Gram‐negative strains. Generally, all of the target compounds have considerable antibacterial activity against the tested forty strains, and exhibit exceptional potency in inhibiting the growth of methicillin‐sensitive Staphylococcus aureus (MSSA) and methicillin‐resistant S. aureus (MRSA) ATCC33591 (MICs: 0.06 to 2 μg/mL). In particular, compounds 14 , 19 , 28 , and 29 are fourfold more potent than ciprofloxacin against MSSA 08‐49. Compounds 23 , 26 , and 27 are twofold more potent than ciprofloxacin against MRSA ATCC33591 and MSSA ATCC29213. In addition, compound 14 exhibits excellent activity (MIC: 0.06 μg/mL) against Acinetobactes calcoaceticus, which is two‐ to 16‐fold more potent than the reference drugs gemifloxacin, levofloxacin, and ciprofloxacin.     

Synthesis,Antibacterial Activities,and 3D‐QSAR of Sulfone Derivatives Containing 1, 3, 4‐Oxadiazole Moiety

A series of sulfone derivatives containing 1, 3, 4‐oxadiazole moiety were prepared and evaluated for their antibacterial activities by the turbidimeter test. Most compounds inhibited growth of Ralstonia solanacearum (R. solanacearum) from tomato and tobacco bacterial wilt with high potency, among which compounds 5a and 5b exhibited the most potent inhibition against R. solanacearum from tomato and tobacco bacterial wilts with EC₅₀ values of 19.77 and 8.29 μg/mL, respectively. Our results also demonstrated that 5a, 5b , and a number of other compounds were more potent than commercial bactericides Kocide 3000 and Thiodiazole Copper, which inhibited R. solanacearum from tomato bacterial wilt with EC₅₀ values of 93.59 and 99.80 μg/mL and tobacco bacterial wilt with EC₅₀ values of 45.91 and 216.70 μg/mL, respectively. The structure–activity relationship (SAR) of compounds was studied using three‐dimensional quantitative structure–activity relationship (3D‐QSAR) models created by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) based on compound bioactivities against tomato and tobacco bacterial wilts. The 3D‐QSAR models effectively predicted the correlation between inhibitory activity and steric–electrostatic properties of compounds.     

Synthesis and anti‐Candida activity of novel benzothiepino[3,2‐c]pyridine derivatives

A novel series of thiepine derivatives were synthesized and evaluated as potential antimicrobials. All the synthesized compounds were evaluated for their antimicrobial activities in vitro against the fungi Candida albicans (ATCC 10231), C. parapsilosis (clinical isolate), Gram‐negative bacterium Pseudomonas aeruginosa (ATCC 44752), and Gram‐positive bacterium Staphylococcus aureus (ATCC 25923). Synthesized compounds showed higher antifungal activity than antibacterial activity, indicating that they could be used as selective antimicrobials. Selected thiepines efficiently inhibited Candida hyphae formation, a trait necessary for their pathogenicity. Thiepine 8‐phenyl[1]benzothiepino[3,2‐c]pyridine ( 16 ) efficiently killed Candida albicans at 15.6 μg/mL and showed no embryotoxicity at 75 μg/mL. Derivative 8‐[4‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)phenyl][1]benzothiepino[3,2‐c]pyridine ( 23 ) caused significant hemolysis and in vitro DNA interaction. The position of the phenyl ring was essential for the antifungal activity, while the electronic effects of the substituents did not significantly influence activity. Results obtained from in vivo embryotoxicity on zebrafish (Danio rerio) encourage further structure optimizations.     

Design,synthesis, and antimicrobial activities of novel functional peptides against Gram‐positive and Gram‐negative bacteria

The extensive use of antibiotics in medicine results in the multidrug resistance of bacteria, making the development of new antimicrobial agents an urgent need. Antimicrobial peptides (AMPs) are considered as a new class of antibiotic with characteristics including an ability to kill target cells rapidly and a broad spectrum of activity. We have developed a potent antimicrobial peptide MAP‐0403 (MIC = 5 μM), but it exhibited a high hemolytic side‐effect (70.7%). To reduce its hemolytic effect and increase antimicrobial activity, three peptides derivatives of MAP‐0403 (J‐1, J‐2, and J‐3) were designed, synthesized by solid phase synthesis, purified by RP‐HPLC, and characterized by MALDI‐TOF MS. Structure–activity relationships of these peptides were studied by using circular dichroism and antimicrobial assays. The percentage of helical structure in J‐1, J‐2, and J‐3 was lower than that of MAP‐0403. The antimicrobial activity of J‐1 was the same as that of MAP‐0403 (MIC = 5 μM), J‐2 exhibited the highest activity (MIC = 2.5 μM), while J‐3 showed decreased activity (MIC = 10 μM). Compared to MAP‐0403, J‐2 showed significantly reduced hemolytic effect (3.4%), while J‐1 and J‐3 showed slightly decreased hemolytic effect (46.2%, 55.6%, respectively). Peptide J‐2 was discovered as a novel and potent antimicrobial agents.     

Design,synthesis and biological evaluation of novel quinoline‐based carboxylic hydrazides as anti‐tubercular agents

In this study, seventeen novel quinoline‐based carboxylic hydrazides were designed as potential anti‐tubercular agents using molecular hybridization approach and evaluated in‐silico for drug‐likeness behavior. The compounds were synthesized, purified, and characterized using spectral techniques (like FTIR, ¹H NMR, and Mass). The in‐vitro anti‐tubercular activity (against Mycobacterium tuberculosisH37Ra) and cytotoxicity against human lung fibroblast cells were studied. Among the tested hydrazides, four compounds ( 6h , 6j , 6l, and 6m ) exhibited significant anti‐tubercular activity with MIC values below 20 μg/mL. The two most potent compounds of the series, 6j and 6m exhibited MIC values 7.70 and 7.13 μg/mL, respectively, against M. tuberculosis with selectivity index >26. Structure–activity relationship studies were performed for the tested compounds in order to explore the effect of substitution pattern on the anti‐tubercular activity of the synthesized compounds.     

Synthesis,biological evaluation,and molecular docking studies of novel 3‐aryl‐5‐(alkyl‐thio)‐1H‐1,2,4‐triazoles derivatives targeting Mycobacterium tuberculosis

A small library of new 3‐aryl‐5‐(alkyl‐thio)‐1H‐1,2,4‐triazoles was synthesized and screened for the antimycobacterial potency against Mycobacterium tuberculosis H₃₇Ra strain and Mycobacterium bovis BCG both in active and dormant stage. Among the synthesized library, 25 compounds exhibited promising anti‐TB activity in the range of IC₅₀0.03–5.88 μg/ml for dormant stage and 20 compounds in the range of 0.03–6.96 μg/ml for active stage. Their lower toxicity (>100 μg/ml) and higher selectivity (SI = >10) against all cancer cell lines screened make them interesting compounds with potential antimycobacterial effects. Furthermore, to rationalize the observed biological activity data and to establish a structural basis for inhibition of M. tuberculosis, the molecular docking study was carried out against a potential target MTB CYP121 which revealed a significant correlation between the binding score and biological activity for these compounds. Cytotoxicity and in vivo pharmacokinetic studies suggested that 1,2,4‐triazole analogues have an acceptable safety index, in vivo stability and bio‐availability.     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Novel 3‐Substituted Ocotillol‐Type Triterpenoid Derivatives as Antibacterial Candidates

Plant‐derived triterpenoid saponins are involved in the plant defense system by targeting bacterial membranes. A series of ocotillol‐type triterpenoid derivatives were synthesized starting from PPD, one of the main components of Panax ginseng and their antibacterial activity against several representative bacteria were evaluated. Compounds 5 and 11 exhibited excellent antibacterial activity with MIC values of 1 μg/mL against Staphylococcus aureus and 8 μg/mL and 4 μg/mL against Bacillus subtilis, respectively. Furthermore, when compounds 5 and 11 were combined with two commercial antibiotics kanamycin and chloramphenicol, they showed strong synergistic activity at sub‐MIC levels against S. aureus USA300 and B. subtilis 168. Moreover, chloramphenicol turned from a bacteriostatic to a bactericidal agent when combined with compound 11 against B. subtilis 168.     

Synthesis and biological evaluation of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides as dual inhibitors of α‐glucosidase and protein tyrosine phosphatase 1B

α‐Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the dephosphorylation of the insulin receptor and regulation of insulin sensitivity. Therefore, dual antagonists by targeting both α‐glucosidase and PTP1B may be potential candidates for type 2 diabetes therapy. In this work, three series of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides were synthesized and assayed for their α‐glucosidase and PTP1B inhibitory activities, respectively. Compound 3l , exhibiting the most effective α‐glucosidase inhibitory activity (IC₅₀ = 10.96 μm ( 3l ), IC₅₀ = 51.32 μm (Acarbose), IC₅₀ = 18.22 μm (Ursolic acid)) and potent PTP1B inhibitory activity (IC₅₀ = 13.46 μm ( 3l ), IC₅₀ = 14.50 μm (Ursolic acid)), was identified as a novel dual inhibitor of α‐glucosidase and PTP1B. Furthermore, 3l is a highly selective PTP1B inhibitor because no inhibition was showed by 3l at 100 μm against PTP‐MEG2, TCPTP, SHP2, or SHP1. Subsequent kinetic analysis revealed 3l inhibited α‐glucosidase in a reversible and mixed manner. Molecular docking study indicated that hydrogen bonds, van der Waals, charge interactions and Pi‐cation interactions all contributed to affinity between 3l and α‐glucosidase/PTP1B.     

Antifungal,anticancer, and docking studies of colchiceine complexes with monovalent metal cation salts

Complexes of colchiceine with monovalent cation perchlorates and iodides have been obtained and characterized by spectroscopic methods. DFT and spectroscopic studies reveal that the dihedral angle ω_{1‐1a‐12‐12a}, crucial for colchicine biological mechanism of action, that is, binding to tubulins depends on the diameter of the complexed metal cation. Biological tests indicated no antifungal properties of colchicine (it was active only toward A.pullulans), in contrast to its derivative—(colchiceine). Complexation of colchiceine with metal cations improved significantly the antifungal potency, even below MIC <1 μg/ml. The colchiceine complexes were more potent than colchiceine, and some of them were even more potent than the fungicidal standard IPBC. The highest potency of colchiceine complexes was noted against A. pullulans (MIC = 0.5 μg/ml). In contrast to the findings concerning antifungal potency, the anticancer studies showed complexes of colchicine more active (~IC₅₀ = 2 nM) than those of colchiceine (~IC₅₀ = 6 μM). MDA‐MB‐231 breast cancer cell lines and human lung fibroblasts CCD39Lu were also tested.     

Synthesis and Anticholinergic Activity of 4‐hydroxycoumarin Derivatives Containing Substituted Benzyl‐1,2,3‐triazole Moiety

A series of 4‐hydroxycoumarin‐derived compounds 8a‐p containing N‐benzyl‐1,2,3‐triazole motif were designed as AChE inhibitors. The title compounds were obtained conveniently using multicomponent click reaction. The in vitro anticholinesterase evaluation of synthesized compounds against AChE and BuChE showed that some of them are potent and selective inhibitors of AChE. Among them, 2‐chlorobenzyl derivative 8k showed the most potent activity against AChE (IC₅₀ = 0.18 μm ). Its activity was also superior to that of standard drug tacrine. The kinetic study and molecular docking simulation of the most potent compound 8k were also described.     

Budesonide use and misuse in sports: Elimination profiles of budesonide and metabolites after intranasal,high‐dose inhaled and oral administrations

Budesonide (BUD) is a glucocorticoid (GC) widely used in therapeutics. In sports, the World Anti‐doping Agency (WADA) controls the use of GCs, and WADA‐accredited laboratories use a reporting level of 30 ng/mL for 6β‐hydroxy‐budesonide (6βOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6βOHBUD, BUD, and 16α‐hydroxy‐prednisolone (16αOHPRED) after intranasal (INT), inhaled (INH) (at high doses) and oral administrations in male and female volunteers. BUD was administered to healthy volunteers using INT route (256 μg/day for three days, n = 4 males and 4 females), INH route (800 μg/day for three days, n = 4 males and 4 females, and 1600 μg/day for three days, n = 4 males) or oral route (3 mg, n = 8 females). Urine samples were collected before and after administration at different time periods, and were analyzed by liquid chromatography–tandem mass spectrometry. 6βOHBUD and BUD concentrations were very low after INT treatment (0.0–7.1 and 0.0–8.1 ng/mL, respectively), and higher after INH treatments (0.0–35.4 and 0.0–48.3 ng/mL, respectively). For 16αOHPRED, elevated concentrations were detected after INT and INH treatments (2.6–66.4 and 3.4–426.5 ng/mL, respectively). Concentrations obtained following oral administration were higher than after therapeutic administrations (2.8–80.6, 1.5–36.1, and 10.4–532.2 ng/mL for 6βOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6βOHBUD is the best discriminatory marker and a reporting level of 40 ng/mL was found to be the best criterion to distinguish allowed from forbidden administrations of BUD.