C. albicans is the most frequently occurring fungal pathogen, and is becoming an increasing public health problem, especially in the context of increased microbial resistance. This opportunistic pathogen is characterized by a versatility explained mainly by its ability to form complex biofilm structures that lead to enhanced virulence and antibiotic resistance. In this context, a review of the known C. albicans biofilm formation inhibitors were performed and a new N-(oxazolylmethyl)-thiazolidinedione scaffold was constructed. 16 new compounds were synthesized and characterized in order to confirm their proposed structures. A general antimicrobial screening against Gram-positive and Gram-negative bacteria, as well as fungi, was performed and revealed that the compounds do not have direct antimicrobial activity. The anti-biofilm activity evaluation confirmed the compounds act as selective inhibitors of C. albicans biofilm formation. In an effort to substantiate this biologic profile, we used in silico investigations which suggest that the compounds could act by binding, and thus obstructing the functions of, the C. albicans Als surface proteins, especially Als1, Als3, Als5 and Als6. Considering the well documented role of Als1 and Als3 in biofilm formation, our new class of compounds that target these proteins could represent a new approach in C. albicans infection prevention and management. Candida spp. are normally commensals found in the gastrointestinal tract, genitourinary tract or oropharyngeal tract of healthy people, but can become opportunistic pathogens that cause superficial infections , deep-seated infections or systemic infections. Candidiasis diagnosis have increased recently due to disproportionate use of broad spectrum antibiotics, use of immunosuppressive drugs, malnutrition, aging population and the amplified use of medical devices [ devices ,2.C. albicans is the most prevalent and problematic of all Candida species, as it is responsible for 50% of the cases of candidiasis and is the fourth most common cause of nosocomial infections in the USA [ the USA .C. albicans bloodstream infections [The pathogenic potential of this microbial strain is explained by its ability to adapt to various habitats and to form surface-attached microbial communities (biofilms) . Biofilmfections . Also, bfections ,11.C. albicans and bacteria, can lead to enhanced virulence, increased biofilm formation, increased pathogenicity and thus more severe infections, increased antimicrobial resistance and even increased mortality. Most frequently, dual-species biofilm formed between C. albicans and Streptococcus mutans or Streptococcus gordonii have been isolated from denture stomatitis, peritonitis, periodontitis and dental caries, while C. albicans and S. aureus dual-biofilms are associated with vaginal, oral or blood stream infections, as well as medical-devices related biofilms . C. albicans can also form a dual-biofilm with P. aeruginosa  or E. faecalis, C. difficile  [In the human body, biofilm is rarely the product of a single microbial species, instead polymicrobial biofilms are frequently present. This microbial synergy, between ections) ,12,13,14C. albicans biofilm is a complex structure that incorporates round yeast cells (blastospores), pseudohyphal cells  and hyphal cells , both of which are interspersed with a polymeric extracellular matrix (ECM), which covers and protects the cells [he cells . Biofilmhe cells ,13. The he cells ,16. Thishe cells . The finhe cells ,13,15.C. albicans biofilm formation are members of the agglutinin-like sequence proteins family (Als) [S. gordonii) and thus is key for the formation of co-infections and polymicrobial biofilms [C. albicans virulence by acting as an invasin at the level of epithelial cells  or the endothelial cells lining the vasculature (key for deep tissues infections) [C. albicans metabolic flexibility as it serves as a receptor for ferritin and thus mediates iron acquisition from the host [The key molecules in ly (Als) ,15,17. Tly (Als) ,19. Althly (Als) ,20,21,22ly (Als) ,15,20,23biofilms ,13,20. Aections) ,20,24,25C. albicans infections prevalence, as well as the increase in antifungal drug resistance, anti-biofilm therapeutic strategies have become sorely needed [Because of the increase in the y needed ,26.Candida biofilm identified a series of natural compounds that could interfere with various stages of the process including: caffeic acid derivatives [Daemonorops draco [Penicillium sp. isolate [The search for efficient inhibitors of ivatives , usnic aivatives , variousivatives , plant eivatives ,31, probivatives , 5-hydroivatives , magnoloivatives ,35, dracps draco , shearin isolate , and oth isolate . HoweverIn the series of small molecules with anti-biofilm activity we can include: Alizarin and chrysazin , miltefoCandida biofilm inhibitors, our research efforts focused on obtaining a new scaffold that encompasses various structural moieties contained in different active molecules, as shown in Based on the structure of known Candida biofilm formation with no effects against microbial cell viability or other microbial biofilm formation. In order to propose a possible mechanism for this biological activity, we also conducted a series of in silico determinations that suggest that our compounds act as Als inhibitors. Subsequently, we evaluated the general antimicrobial potential, as well as the general anti-biofilm activity. Our compounds proved to be selectively active against 6a–d, 7a–d, 8a–d, 9a–d) have been synthesized by the N-alkylation of various previously reported intermediates (5a–d) [2a–d).A total of 16 new compounds (s (5a–d) ,49 with 2a–d) were obtained using the cyclisation of an amide with a α-haloketone to an oxazole, as shown in The 4-(chloromethyl)-2-phenyloxazoles  and the endocyclic νC=N bond, with a medium-strong signal between 1586 and 1593 cm−1. The aliphatic νC-Cl bond gives a strong signal between 690 and 702 cm−1. Signals that are specific to the intermediate compound 2d are due to the nitro moiety that gave two characteristic signals caused by the asymmetric, respectively symmetric, stretching of the νN=O bond at 1522 and 1327 cm−1.The MS spectra of intermediates 2a–d were used in alkylation reactions with previously described [3a–d), as can be observed in O-alkylation  or N-alh-CH2-Cl ). Our spThe MS spectra of all final compounds confirmed the presence of the molecular ions.−1, that has led us to confirm that a N-alkylation took place, and not an O-alkylation. The presence of the oxazole ring was confirmed by a sharp signal between 3106–3166 cm−1 (νC5-H) and a strong signal between 1515–1521 cm−1 (νC=N). The thiazolidinedione was characterized by 2 νC=O, in two groups, shown as strong signals at 1755–1717 cm−1 and 1664–1693 cm−1. Specific signals were also identified corresponding to the NO2 contain compounds  and the ether vanillin derivatives .By analyzing the IR spectra, we were able to identify a phenolic νO-H stretching, as a broad band between 3369–3523 cmN-alkylation is also supported by 1H-NMR data. As such, we can observe the lack of a broad signal from a very deshielded proton (N-H from thiazolidinedione) at >12 ppm, and the presence of a broad signal corresponding to the OH proton between 9.90–10.67 ppm. 13C-NMR is also consistent with the proposed structures, as it shows 2 groups of C=O signals between 167.11–167.91 ppm and 165.87–165.06 ppm (corresponding to the thiazolidinedione), 3 carbon atoms from oxazole , and a -CH2- bridge between 37.72–37.53 ppm.The Antimicrobial activity was evaluated using a series of Gram-positive strains, Gram-negative strains and a fungal strain. This determination aimed at establishing the biological profile of the newly synthesized compounds, namely their antimicrobial potential. Results of the initial in vitro qualitative screening are shown in C. albicans by compounds 4a, 4c, 6a, 7a that had an inhibition of growth zone diameter of 16 mm. However, this action is mediocre compared with the fluconazole standard (24 mm).The best antimicrobial activity was obtained against 7c and 7d, against the E. coli strain. A mediocre effect was observed also against E. faecalis, while most compounds had a negligible activity against S. aureus.Considering antibacterial activity, the compounds seem to be more active against Gram-negative strains compared with Gram-positive strains. The most significantly active compounds were The quantitative assay was performed in order to more precisely evaluate the direct antimicrobial effects, as initial screenings determined a potentially moderate antimicrobial effect. The results from these investigations, shown in C. albicans biofilm, the new agents could be used without risk of causing imbalances of the commensal flora.Being deprived of direct antimicrobial effects, these compounds do not create an increase in bacterial resistance by increasing the selection pressure via bacteriostatic or bactericidal effects. This could be viewed as a positive feature, if these compounds possess anti-biofilm effects, as initially assumed when designing the scaffold. Also, by specifically targeting The crystal violet staining method provides a total quantification of biofilm biomass, as it includes various cells and extracellular matrix .Anti-biofilm activity can be independent of the direct antimicrobial activity; as a consequence, new compounds must be evaluated in parallel for both properties.C. albicans biofilm formation. Out of a total of 16 compounds, 14 of them were active against biofilm formation at minimal biofilm eradication concentrations (MBEC), smaller than the standard used (berberine). The most active compound was 5d, which still maintained anti-biofilm effects even at concentrations as low as 0.038 mg/mL. 10 of the compounds were active at concentrations (0.078 mg/mL) four times smaller than the standard.Our anti-biofilm screening results, presented in 6b, 6c and 7c) seem to be moderately active against E. faecalis biofilm formation with MBEC values equal to that of the standard. Activity against the biofilm of Gram-negative strains appears to be manifested only at high concentrations, or absent. Concerning Gram-positive strains, some of the compounds  tend to have a lesser impact on binding than those from the benzylidene moiety. The position of the OH group on the benzylidene seems to have the highest influence on binding affinity: Optimal affinity is achieved by inserting the OH in the 3rd or 4th position, while the insertion of an extra methoxy group is unfavorable.When interpreting these results, we considered that a higher standard deviation for the binding affinity shows that the variation of substituent induces bigger differences in binding mode, while a reduced standard deviation translates by a decreased influence of the substituent on the binding affinity. As such, we can observe that the substituents on the 2-phenyloxazole residue , as well as multiple adhesins  and other factors. It is important to note that these molecules perform complementary functions and work together for biofilm formation [ormation ,15,21. A4b and 5d, which have good inhibition potential against 6 of the 9 Als targets, and especially against Als3, that is believed to be the most important Als protein for biofilm development and a key element of overall Candida albicans virulence.As such, the most interesting compounds from our series seem to be 5d, against Als1 and Als3 is shown in The docking pose of one of the most promising new compounds, The binding pocket of Als1 is a rather flattened surface, shaped as 2 adjacent Y. In other words, each of its’ distal and proximal extremities present 2 sub-pockets formed by protruding moieties that subdivide the central binding site.5d. The gateway to the large distal sub-pocket is an Arg168 residue that interacts with the π electrons from the phenol aromatic ring, via the guanidine moiety.The distal extremity is characterized by a protruding moiety made up by the vicinal joint of Tyr243 and Ala186. As a result, 2 distal sub-pockets are formed: A small pocket with very limited use (non-druggable) and a larger, predominantly lipophilic one, which accommodates the phenol ring of compound 5d is situated in the central part of the binding pocket, away from residues capable of interaction, with the notable exception of a hydrogen bond formed between the Tyr243 phenol OH group and a C=O group from the thiazolidinedione.The thiazolidinedione ring of compound The proximal extremity of the Als1 binding site also presents a protruding moiety formed by the spatial proximity of the C-terminal end (Gly314-Tyr315) and a loop (Ala35-Ala36-Asn37). The 2 resulting proximal sub-pockets are mostly lipophilic, but each has at least one hydrophilic residue: The small sub-pocket has Tyr35, while the more voluminous sub-pocket has Asn37.5d acts primarily as a spacer, with the two rings being co-planar, and forming a large entity that cannot fit in the small proximal sub-pocket. However, this makes it capable of favoring the spatial orientation of the nitro group in the vicinity of the peptide bond Ala36-Asn37, found in the larger proximal sub-pocket. Also, the methylene moiety from 5d acts as a hinge and helps to direct the nitro-phenyl residue towards the more voluminous sub-pocket. As a result, polar interactions take place between the NO from the nitro group and the peptide bond Ala36-Asn37.The oxazolyl-phenyl structure from In the case of Als3, the predicted binding site is located in the core of the protein. It is shaped like a channel running across the protein. Access to this area is controlled by 2 entities: A beta-strand formed by Leu293-Arg294-Trp295-Thr296 and then by Gly297-Phe298-Arg299, that border and provide lining to the pocket and an omega loop Val268-Asn269-Ser270 that partially obstruct the binding site entrance. 5d is conveniently oriented towards Ser170, with whom it forms a hydrogen bond, but also near Tyr271, thus allowing for supplementary interaction. The thiazolidine ring of 5d sits in a polar region and interacts via a hydrogen bond formed between a C=O group and the Arg171 residue. The p-NO2-phenyl-oxazole form 5d lingers towards the exit of the binding pocket, and because of the widening of the binding site, the nitro group does not manage to form reliable interactions.The OH phenol group from compound A phylogenetic analysis of Als proteins reveals that these surface proteins are related, which was to be expected, as they are part of the same superfamily . HoweverThe degree of similarity was assessed by calculating a similarity matrix, presented in To better understand the degree of similarity, a phylogenetic tree was generated using phylogeny.fr , and is Both the similarity matrix and the phylogenetic tree indicate that, despite the fact that studied ALS sequences have a common ancestry, ALS7 has a different evolutionary path to the other ALS proteins. Also, Als6 seem to have evolved separately, whereas Als9 and Als1-5 share a common evolutionary ancestor. As in the case of the similarity matrix, the phylogenetic tree underlines the close connections between Als1, Als3 and Als5 which is also supported by the known biologic roles of these proteins .N-(oxazolylmethyl)-thiazolidinediones .A comparative analysis of the active sites’ binding pockets from different Als, shown in All chemicals were of analytical reagent grade purity, and have been purchased from Merck  or Sigma-Aldrich .The uncorrected melting points were obtained by the open glass capillary method, using a MPM-H1 melting point apparatus .MS spectra were obtained by using an Agilent 1100 series, in positive ionization with an Agilent Ion Trap SL mass spectrometer (70 eV) instrument . IR spectra were recorded after compression of the samples in KBr pellets, under vacuum, using a FT/IR 6100 spectrometer . The device was controlled using the computer interface software Spectra Manager. Assignment of IR signals was made using Know It All 7.8 by Bio-Rad Laboratories .1H-NMR and 13C-NMR were recorded on an Avance NMR spectrometer  using DMSO-d6 as solvent. Chemical shift values are reported in δ units, relative to TMS as internal standard. All spectral data were in accordance with the proposed chemical structures.The Elemental analysis was performed by Vario El CHNS analyzer . The results obtained for all synthesized compounds were in agreement with the calculated values.2a–d). 10 mmol of benzamides 1a–d and 10 mmol 1,3-dichloroacetone were mixed well in a round bottom flask with 5 mL of propylene glycol and 0.5 mL of dimethylsulfoxide. Reactions were performed in an open vessel, under condenser, with vigorous magnetic stirring. Between the reaction flask and the condenser, a valve was designed from a small inner diameter conical glass adaptor, equipped with a small glass ball that moved vertically freely within, in order to reduce the volatilization of dichloroacetone. The mixture was refluxed for one hour. Upon completion of the reaction, the mixture was cooled to room temperature, 5 mL of methanol was added and the mixture was stirred well. Further, water was added carefully dropwise, in order to obtain a precipitate. The resulted solid was filtered under vacuum. The impure 4-(chloromethyl)-2-aryloxazoles 2a–d were recrystallized twice from methanol-water and activated a charcoal mixture to give the appropriate pure 2a–d intermediate products.General Procedure for the synthesis of the 4-(chloromethyl)-2-aryloxazoles (4-(Chloromethyl)-2-phenyloxazole (2a): white solid; mp = 55 °C (lit. 55.5–56 °C [max cm−1: 3091 (C5-H oxazole), 1593 (C=N), 702 (C-Cl); MS: m/z = 194.1 and 196.3 (M + 1 and M + 3 due to 35Cl and 37Cl); 1H NMR  δ: 8.23 , 7.94 , 7.51–7.49 , 4.31 ; 13C NMR  δ: 161.27 (oxazole C2), 138.02 (oxazole C4), 136.58 (oxazole C5), 130.74, 129.55, 127.01, 126.39 (4 aromatic carbons), 41.13 (-CH2-)..5–56 °C ); yield 4-(Chloromethyl)-2-(p-tolyl)oxazole (2b): white solid; mp = 94–95 °C (lit. 94–95 °C [max cm−1: 3128 (C5-H oxazole), 1587 (C=N), 693 (C-Cl); MS: m/z = 208.1 and 210.3 (M + 1 and M + 3 due to 35Cl and 37Cl); 1H NMR  δ: 8.29 , 7.71 , 7.29 , 4.38 , 2.59 ; 13C NMR  δ: 160.59 (oxazole C2), 138.21 (oxazole C4), 135.73 (oxazole C5), 140.98, 130.65, 122.73, 120.84 (4 aromatic carbons), 42.01 (-CH2-).94–95 °C ); yield 4-(Chloromethyl)-2-(4-chlorophenyl)oxazole (2c): white solid; mp = 97 °C (lit. 97–98 °C [max cm−1: 3128 (C5-H oxazole), 1586 (C=N), 690 (C-Cl); MS: m/z = 228, 230, 232 (M + 1 and M + 3 due to 35Cl and 37Cl); 1H NMR  δ: 8.24 , 7.92 , 7.49 , 4.39 ; 13C NMR  δ: 160.44 (oxazole C2), 138.20 (oxazole C4), 135.91 (oxazole C5), 136.89, 129.49, 128.31, 124.42 (4 aromatic carbons), 41.63 (-CH2-).97–98 °C ); yield 4-(Chloromethyl)-2-(4-nitrophenyl)oxazole (2d): brown-yellow solid; mp = 138–139 °C (lit. 140 °C [max cm−1: 3148 (C5-H oxazole), 1587 (C=N), 1522, 1337 (N=O nitro), 702 (C-Cl); MS: m/z = 239.1 and 239.4 (M + 1 and M + 3 due to 35Cl and 37Cl); 1H NMR  δ: 8.23 , 8.12 , 8.05 , 4.32 ; 13C NMR  δ: 160.29 (oxazole C2), 138.27 (oxazole C4), 135.99 (oxazole C5), 145.91, 128.52, 126.58, 124.22 (4 aromatic carbons), 41.55 (-CH2-).. 140 °C ); yield 3a–d (Z isomers) was based on a Knoevenagel condensation in alkaline medium, provided by the anhydrous sodium acetate. The condensation between the corresponding phenolic aldehydes and thiazolidine-2,4-dione was made under microwave irradiation in acetic acid. The synthetic protocol and the characterization of the intermediate compounds 3a–d were previously reported [General Procedure for the Synthesis of the Intermediate Compounds reported ,49.4a–d, 5a–d, 6a–d and 7a–d). 5-(hydroxybenzylidene)-thiazolidine-2,4-diones intermediates (3a–d) were selectively alkylated on the nitrogen atom from the thiazolidine-2,4-dione ring, in alkaline medium using a modified protocol [3a–d and 1 mmol of intermediate compound 2a–d, dimethylformamide (DMF) was added dropwise until their dissolution, in order to obtain the highest possible concentration of the intermediate compounds, to ensure a high reaction rate. After that, 2 mmol of anhydrous potassium carbonate and 1 mmol of anhydrous potassium iodide were added to this solution. The mixture was stirred overnight at room temperature. Upon completion of the reaction, the mixture was poured over ice cold saturated brine. A 10% sulfuric acid solution was added dropwise until complete precipitation of the product had occurred. The resulted solid was filtered under vacuum and dried. The remaining residue was crystallized twice from acetone, giving the pure final compounds 4a–d, 5a–d, 6a–d and 7a–d.General Procedure for the Synthesis of the Final Compounds (protocol . Over 1.(Z)-5-(4-Hydroxybenzylidene)-3-((2-phenyloxazol-4-yl)methyl)thiazolidine-2,4-dione (4a): C20H14N2O4S, calcd: C 63.48%, H 3.73%, N 7.40%, S 8.47%, found: C 63.45%, H 3.74%, N 7.44%, S 8.46%; yellow solid; mp = 223–224 °C; yield = 57%; FT IR (KBr) νmax cm−1: 3410 (O-H), 3145 (C5-H oxazole), 1755 (C=O), 1675 (C=O), 1590 (C=N); MS: m/z = 379.3 (M + 1); 1H NMR  δ: 10.43 , 8.25 , 7.92 , 7.88 , 7.58 , 7.52–7.50 , 6.93 , 4.82 ; 13C NMR  δ: 167.19 (C=O), 165.11 (C=O), 161.29 (oxazole C2), 159.38 (ArC-OH), 138.01 (oxazole C4), 136.55 (oxazole C5), 134.57 (-CH=), 133.29, 130.99, 129.63, 127.05, 126.41, 125.61, 115.82 (7 aromatic carbons), 116.22 (TZD C5=), 37.61 (-CH2-).(Z)-5-(4-Hydroxybenzylidene)-3-((2-(p-tolyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (4b): C21H16N2O4S, calcd: C 64.27%, H 4.11%, N 7.14%, S 8.17%, found: C 64.21%, H 4.01%, N 7.09%, S 8.05%; yellow solid; mp = 229–230 °C; yield = 53%; FT IR (KBr) νmax cm−1: 3403 (O-H), 3140 (C5-H oxazole), 1737 (C=O), 1675 (C=O), 1593 (C=N); MS: m/z = 393.1 (M + 1); 1H NMR  δ: 10.28 , 8.28 , 7.94 , 7.75 , 7.51 , 7.28 , 6.96 , 4.80 , 2.61 ; 13C NMR  δ: 167.24 (C=O), 165.29 (C=O), 160.26 (oxazole C2), 160.23 (ArC-OH), 138.16 (oxazole C4), 135.90 (oxazole C5), 134.41 (-CH=), 140.11, 133.47, 130.84, 126.54, 125.69, 122.80, 116.81 (7 aromatic carbons), 116.32 (TZD C5=), 37.55 (-CH2-), 23.48 (-CH3).(Z)-3-((2-(4-Chlorophenyl)oxazol-4-yl)methyl)-5-(4-hydroxybenzylidene)thiazolidine-2,4-dione (4c): C20H13ClN2O4S, calcd: C 58.18%, H 3.17%, N 6.79%, S 7.77%, found: C 58.21%, H 3.19%, N 6.82%, S 7.80%; yellow solid; mp = 236 °C; yield = 62%; FT IR (KBr) νmax cm−1: 3410 (O-H), 3150 (C5-H oxazole), 1739 (C=O), 1685 (C=O), 1595 (C=N); MS: m/z = 413.2 and 415.5 ; 1H NMR  δ: 10.40 , 8.25 , 7.94 , 7.89 , 7.58 , 7.50 , 6.94 , 4.82 ; 13C NMR  δ: 167.58 (C=O), 165.81 (C=O), 160.86 (oxazole C2), 160.37 (ArC-OH), 138.28 (oxazole C4), 135.93 (oxazole C5), 134.35 (-CH=), 136.82, 133.15, 129.79, 128.23, 125.86, 124.20, 116.94 (7 aromatic carbons), 116.82 (TZD C5=), 37.53 (-CH2-).(Z)-5-(4-hydroxybenzylidene)-3-((2-(4-nitrophenyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (4d): C20H13N3O6S, calcd: C 56.73%, H 3.09%, N 9.92%, S 7.57%, found: C 56.70%, H 3.07%, N 9.91%, S 7.55%; yellow solid; mp = 257 °C; yield = 64%; FT IR (KBr) νmax cm−1: 3504 (O-H), 3166 (C5-H oxazole), 1717 (C=O), 1664 (C=O), 1600 (C=N), 1521, 1336 (N=O); MS: m/z = 424.5 (M + 1); 1H NMR  δ: 10.47 , 8.25 , 8.17 , 8.09 , 7.88 , 7.59 , 6.93 , 4.83 ; 13C NMR  δ: 167.39 (C=O), 165.18 (C=O), 160.22 (oxazole C2), 160.55 (ArC-OH), 138.54 (oxazole C4), 135.99 (oxazole C5), 134.26 (-CH=), 145.10, 133.54, 128.35, 126.72, 125.73, 124.16, 117.09 (7 aromatic carbons), 116.35 (TZD C5=), 37.33 (-CH2-).(Z)-5-(3-Hydroxybenzylidene)-3-((2-phenyloxazol-4-yl)methyl)thiazolidine-2,4-dione (5a): C20H14N2O4S, calcd: C 63.48%, H 3.73%, N 7.40%, S 8.47%, found: C 63.46%, H 3.74%, N 7.43%, S 8.45%; white solid; mp = 217 °C; yield = 74%; FT IR (KBr) νmax cm−1: 3460 (O-H), 3143 (C5-H oxazole), 1734 (C=O), 1670 (C=O), 1599 (C=N); MS: m/z = 379.4 (M + 1); 1H NMR  δ: 9.90 , 8.23 , 7.89 , 7.96 , 7.54–7.51 , 7.34 , 7.09 , 7.03 , 6.90 , 4.83 ; 13C NMR  δ: 167.47 (C=O), 165.65 (C=O), 161.30 (oxazole C2), 158.44 (ArC-OH), 138.03 (oxazole C4), 136.52 (oxazole C5), 134.04 (-CH=), 134.55, 131.26, 130.94, 129.65, 127.04, 126.45, 121.90, 121.38, 118.54 (9 aromatic carbons), 116.52 (TZD C5=), 37.72 (-CH2-).(Z)-5-(3-Hydroxybenzylidene)-3-((2-(p-tolyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (5b): C21H16N2O4S, calcd: C 64.27%, H 4.11%, N 7.14%, S 8.17%, found: C 64.31%, H 4.08%, N 7.19%, S 8.21%; white solid; mp = 229 °C; yield = 70%; FT IR (KBr) νmax cm−1: 3432 (O-H), 3142 (C5-H oxazole), 1735 (C=O), 1680 (C=O), 1592 (C=N); MS: m/z = 393.2 (M + 1); 1H NMR  δ: 10.01 , 8.21 , 7.89 , 7.77 , 7.35 , 7.10 , 7.04 , 6.93 , 4.84 , 2.58 ; 13C NMR  δ: 167.24 (C=O), 165.32 (C=O), 160.38 (oxazole C2), 158.31 (ArC-OH), 138.21 (oxazole C4), 135.87 (oxazole C5), 133.85 (-CH=), 141.15, 134.26, 131.25, 130.70, 126.99, 122.79, 122.03, 121.30, 118.45 (9 aromatic carbons), 116.52 (TZD C5=), 37.58 (-CH2-), 23.47 (-CH3).(Z)-3-((2-(4-Chlorophenyl)oxazol-4-yl)methyl)-5-(3-hydroxybenzylidene)thiazolidine-2,4-dione (5c): C20H13ClN2O4S, calcd: C 58.18%, H 3.17%, N 6.79%, S 7.77%, found: C 58.25%, H 3.29%, N 6.70%, S 7.76%; white solid; mp = 227 °C; yield = 73%; FT IR (KBr) νmax cm−1: 3407 (O-H), 3159 (C5-H oxazole), 1736 (C=O), 1684 (C=O), 1592 (C=N); MS: m/z = 413.4 and 415.5 ; 1H NMR  δ: 9.93 , 8.23 , 7.93 , 7.87 , 7.51 , 7.37 , 7.09 , 7.02 , 6.92 , 4.81 ; 13C NMR  δ: 167.51 (C=O), 165.26 (C=O), 160.31 (oxazole C2), 158.21 (ArC-OH), 138.20 (oxazole C4), 135.99 (oxazole C5), 133.91 (-CH=), 137.21, 134.87, 131.17, 129.69, 128.35, 124.61, 121.85, 121.18, 118.37 (9 aromatic carbons), 116.56 (TZD C5=), 37.68 (-CH2-).(Z)-5-(3-Hydroxybenzylidene)-3-((2-(4-nitrophenyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (5d): C20H13N3O6S, calcd: C 56.73%, H 3.09%, N 9.92%, S 7.57%, found: C 56.73%, H 3.08%, N 9.91%, S 7.58%; yellow solid; mp = 255 °C; yield = 72%; FT IR (KBr) νmax cm−1: 3369 (O-H), 3164 (C5-H oxazole), 1731 (C=O), 1669 (C=O), 1593 (C=N), 1515, 1342 (N=O); MS: m/z = 424.3 (M + 1); 1H NMR  δ: 9.95 , 8.24 , 8.15 , 8.09 , 7.88 , 7.38 , 7.09 , 7.02 , 6.91 , 4.83 ; 13C NMR  δ: 167.29 (C=O), 165.24 (C=O), 160.29 (oxazole C2), 158.56 (ArC-OH), 138.29 (oxazole C4), 136.09 (oxazole C5), 134.26 (-CH=), 146.01, 134.51, 131.06, 128.61, 126.65, 124.29, 122.01, 121.35, 118.61 (9 aromatic carbons), 116.33 (TZD C5=), 37.60 (-CH2-).(Z)-5-(2-Hydroxybenzylidene)-3-((2-phenyloxazol-4-yl)methyl)thiazolidine-2,4-dione (6a): C20H14N2O4S, calcd: C 63.48%, H 3.73%, N 7.40%, S 8.47%, found: C 63.46%, H 3.75%, N 7.42%, S 8.44%; yellow solid; mp = 217 °C; yield = 40%; FT IR (KBr) νmax cm−1: 3409 (O-H), 3152 (C5-H oxazole), 1731 (C=O), 1668 (C=O), 1596 (C=N); MS: m/z = 379.1 (M + 1); 1H NMR  δ: 10.59 , 8.24 , 8.12 , 7.94 , 7.54–51 , 7.38–7.34 , 6.97–6.96 , 4.83 ; 13C NMR  δ: 167.72 (C=O), 165.87 (C=O), 161.28 (oxazole C2), 157.85 (ArC-OH), 138.00 (oxazole C4), 136.60 (oxazole C5), 133.09 (-CH=), 131.25, 129.64, 129.16, 129.11, 126.45, 127.05, 120.31, 120.07, 120.05 (9 aromatic carbons), 116.71 (TZD C5=), 37.62 (-CH2-).(Z)-5-(2-Hydroxybenzylidene)-3-((2-(p-tolyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (6b): C21H16N2O4S, calcd: C 64.27%, H 4.11%, N 7.14%, S 8.17%, found: C 64.38%, H 4.18%, N 7.15%, S 8.28%; yellow solid; mp = 205 °C; yield = 38%; FT IR (KBr) νmax cm−1: 3432 (O-H), 3151 (C5-H oxazole), 1730 (C=O), 1686 (C=O), 1590 (C=N); MS: m/z = 393.2 (M + 1); 1H NMR  δ: 10.28 , 8.30 , 8.05 , 7.68 , 7.31–7.35 , 6.96 , 4.79 , 2.56 ; 13C NMR  δ: 167.11 (C=O), 165.06 (C=O), 160.17 (oxazole C2), 157.32 (ArC-OH), 138.19 (oxazole C4), 135.89 (oxazole C5), 132.84 (-CH=), 141.29, 130.71, 129.05, 128.99, 126.01, 122.68, 120.87, 120.64, 120.36 (9 aromatic carbons), 116.93 (TZD C5=), 37.61 (-CH2-), 23.52 (-CH3).(Z)-3-((2-(4-Chlorophenyl)oxazol-4-yl)methyl)-5-(2-hydroxybenzylidene)thiazolidine-2,4-dione (6c): C20H13ClN2O4S, calcd: C 58.18%, H 3.17%, N 6.79%, S 7.77%, found: C 58.17%, H 3.16%, N 6.76%, S 7.74%; yellow solid; mp = 257 °C; yield = 41%; FT IR (KBr) νmax cm−1: 3413 (O-H), 3157 (C5-H oxazole), 1731 (C=O), 1668 (C=O), 1595 (C=N); MS: m/z = 413.4 and 415.2 ; 1H NMR  δ: 10.61 , 8.25 , 8.18 , 7.93 , 7.50 , 7.37–7.35 , 6.97–6.99 , 4.83 ; 13C NMR  δ: 167.41 (C=O), 165.87 (C=O), 160.54 (oxazole C2), 158.09 (ArC-OH), 138.27 (oxazole C4), 135.81 (oxazole C5), 132.95 (-CH=), 137.09, 129.71, 129.44, 129.14, 128.37, 124.15, 120.99, 120.90, 120.23 (9 aromatic carbons), 116.77 (TZD C5=), 37.72 (-CH2-).(Z)-5-(2-Hydroxybenzylidene)-3-((2-(4-nitrophenyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (6d): C20H13N3O6S, calcd: C 56.73%, H 3.09%, N 9.92%, S 7.57%, found: C 56.76%, H 3.11%, N 9.88%, S 7.60%; yellow solid; mp = 255–256 °C; yield = 40%; FT IR (KBr) νmax cm−1: 3503 (O-H), 3165 (C5-H oxazole), 1718 (C=O), 1665 (C=O), 1600 (C=N), 1520, 1336 (N=O); MS: m/z = 424.6 (M + 1); 1H NMR  δ: 10.67 , 8.25 , 8.17 , 8.10 , 8.06 , 7.37–7.35 , 6.98 , 4.81 ; 13C NMR  δ: 167.81 (C=O), 165.31 (C=O), 160.01 (oxazole C2), 157.03 (ArC-OH), 138.35 (oxazole C4), 135.98 (oxazole C5), 132.25 (-CH=), 145.84, 129.13, 128.67, 128.57, 126.66, 124.15, 121.44, 121.09, 120.11 (9 aromatic carbons), 117.03 (TZD C5=), 37.54 (-CH2-).(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-3-((2-phenyloxazol-4-yl)methyl)thiazolidine-2,4-dione (7a): C21H16N2O5S, calcd: C 61.76%, H 3.95%, N 6.86%, S 7.85%, found: C 61.76%, H 3.92%, N 6.87%, S 7.88%; yellow solid; mp = 190 °C; yield = 63%; FT IR (KBr) νmax cm−1: 3523 (O-H), 3140 (C5-H oxazole), 1733 (C=O), 1684 (C=O), 1594 (C=N), 1284 (C-O-C); MS: m/z = 409.3 (M + 1); 1H NMR  δ: 10.26 , 8.24 , 7.93 , 7.85 , 7.65 , 7.60 , 7.54–7.52 , 6.92 , 4.82 , 3.73 ; 13C NMR  δ: 167.59 (C=O), 165.13 (C=O), 161.30 (oxazole C2), 156.09 (ArC-OCH3), 155.27 (ArC-OH), 137.98 (oxazole C4), 136.61 (oxazole C5), 133.17 (-CH=), 133.01, 131.09, 130.08, 129.61, 128.21, 127.03, 126.43, 125.72 (8 aromatic carbons), 116.36 (TZD C5=), 55.89 (-CH3), 37.55 (-CH2-).(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-3-((2-(p-tolyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (7b): C22H18N2O5S, calcd: C 62.55%, H 4.29%, N 6.63%, S 7.59%, found: C 62.60%, H 4.35%, N 7.58%, S 8.51%; yellow solid; mp = 205 °C; yield = 57%; FT IR (KBr) νmax cm−1: 3412 (O-H), 3132 (C5-H oxazole), 1742 (C=O), 1693 (C=O), 1590 (C=N), 1266 (C-O-C); MS: m/z = 423.2 (M + 1); 1H NMR  δ: 10.32 , 8.26 , 7.89 , 7.69 , 7.61–7.63 , 7.27 , 6.94 , 4.77 , 3.78 , 2.64 ; 13C NMR  δ: 167.34 (C=O), 165.16 (C=O), 160.81 (oxazole C2), 155.91 (ArC-OCH3), 155.62 (ArC-OH), 138.28 (oxazole C4), 135.79 (oxazole C5), 133.67 (-CH=), 141.80, 134.29, 130.71, 129.21, 128.26, 126.81, 125.89, 124.19 (8 aromatic carbons), 116.19 (TZD C5=), 55.34 (-CH3), 37.61 (-CH2-), 23.51 (-CH3).(Z)-3-((2-(4-Chlorophenyl)oxazol-4-yl)methyl)-5-(4-hydroxy-3-methoxybenzylidene)thiazolidine-2,4-dione (7c): C21H15ClN2O5S, calcd: C 59.95%, H 3.41%, N 6.33%, S 7.24%, found: C 59.95%, H 3.43%, N 6.35%, S 7.20%; orange-yellow solid; mp = 247 °C; yield = 63%; FT IR (KBr) νmax cm−1: 3421 (O-H), 3149 (C5-H oxazole), 1727 (C=O), 1670 (C=O), 1589 (C=N), 1239 (C-O-C); MS: m/z = 443.2 and 445.6 ; 1H NMR  δ: 10.20 , 8.24 , 7.94 , 7.89 , 7.64–7.61 , 7.50 , 6.93 , 4.81 , 3.72 ; 13C NMR  δ: 167.91 (C=O), 165.83 (C=O), 160.61 (oxazole C2), 156.01 (ArC-OCH3), 155.19 (ArC-OH), 138.31 (oxazole C4), 135.99 (oxazole C5), 132.88 (-CH=), 137.18, 132.80, 129.45, 129.16, 128.44, 128.19, 125.60, 124.53 (8 aromatic carbons), 116.91 (TZD C5=), 55.60 (-CH3), 37.59 (-CH2-).(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-3-((2-(4-nitrophenyl)oxazol-4-yl)methyl)thiazolidine-2,4-dione (7d): C21H15N3O7S, calcd: C 55.63%, H 3.33%, N 9.27%, S 7.07%, found: C 55.59%, H 3.32%, N 9.28%, S 7.08%; yellow solid; mp = 233–234 °C; yield = 75%; FT IR (KBr) νmax cm−1: 3398 (O-H), 3106 (C5-H oxazole), 1739 (C=O), 1669 (C=O), 1588 (C=N), 1512, 1341 (N=O), 1271 (C-O-C); MS: m/z = 454.5 (M + 1); 1H NMR  δ: 10.35 , 8.23 , 8.14 , 8.07 , 7.88 , 7.62 , 7.57 , 6.95 , 4.81 , 3.70 ; 13C NMR  δ: 167.41 (C=O), 165.09 (C=O), 160.54 (oxazole C2), 155.74 (ArC-OCH3), 155.38 (ArC-OH), 138.37 (oxazole C4), 136.07 (oxazole C5), 133.80 (-CH=), 145.77, 135.13, 129.17, 128.49, 128.38, 126.39, 125.31, 124.99 (8 aromatic carbons), 116.52 (TZD C5=), 55.12 (-CH3), 37.66 (-CH2-).4a–d, 5a–d, 6a–d and 7a–d was assessed by using 3 distinct approaches. The antimicrobial potential was determined for all compounds via an initial in vitro qualitative screening study, followed by an in vitro quantitative assay. Furthermore, we investigated the anti-biofilm, and thus antipathogenic potential of the new compounds.The biologic activity of the final compounds Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212), 2 Gram-negative strains  and 1 fungal strain . These strains were reference strains and their identity was confirmed using the VITEK 1 automatic system. Anti-biofilm tests were performed for all compounds investigated, regardless of their activity as direct antimicrobial agents, as it was this paper’s aim to obtain and to prove that the new molecules selectively inhibit Candida biofilm formation and do not affect other microbial biofilm, nor do they have direct antimicrobial action.Our aim was to investigate the specificity of the compounds in their predicted biologic activity as anti-Candida biofilm agents. In order to prove the lack of direct antibacterial or antifungal effect, we selected an array of 2 Gram-positive strains . Culture positive controls and blank DMSO dilution were used. The plates were incubated for 24 h at 37 °C for bacterial strains and 48 h at 28 °C for the fungal strain. The minimal inhibitory concentration (MIC) values were determined as the lowest concentration of the investigated compound that inhibited the growth of the microbial cultures, compared to the positive control, as established by a decreased value of absorbance at 600 nm  [The quantitative assay was performed using 96-wells plates containing liquid Mueller-Hinton medium seeded with 20 μL microbial inoculum. The stock solutions of the tested compounds were prepared at concentrations of 5 mg/mL in DMSO. They were applied as two-fold serial dilutions ranging from 2500 µg to 2 µg mLGermany) ,63,64.The microtiter plate method, previously reported ,65, was C. albicans, in order to understand the differences between compounds in terms of interaction with the microorganism’s adhesion proteins. Our in silico study focused on finding differences of interactions between our compounds and the target proteins from the point of view of the various substitutions and isomerism in our molecules and from the point of view of the differences between the macromolecular targets.The tested compounds were docked in the binding site of the most important adhesion proteins of For Als3 and Als9, 3D structures obtained by X-ray diffraction were deposited in Protein Data Bank (PDB), as presented in 4a–d, 5a–d, 6a–d and 7a–d) and the macromolecular targets were prepared as reported [The files of ligands -thiazolidindione. A series of 16 new compounds bearing this moiety were synthesized and their structures were confirmed using physicochemical parameters and spectral data.In an effort to obtain new agents that target A general antimicrobial activity screening was performed using both qualitative and quantitative methods against Gram-positive and Gram-negative bacteria, as well as fungi. Results showed that the compounds do not possess significant direct antimicrobial activity and thus are not estimated to determine selection pressure or to affect non-pathogenic commensal flora.C. albicans biofilm formation. In order to provide a possible mechanism of action, we performed a docking study that proved these compounds have a very good binding potential against most of the Als surface proteins of C. albicans. All compounds seem to be able to bind to Als1, Als5 and Als6, while some are also capable of good interactions with Als3. Considering the well documented role of Als1, Als3 and Als5 as adhesins and key agents in biofilm formation, we postulate that these compounds selectively inhibit C. albicans biofilm formation most likely by interfering with the Als proteins.The biologic anti-biofilm evaluation demonstrated that, as hypothesized when constructing this scaffold, the compounds are very active selectively against


The synthesis is based on domino ring-closure followed by microwave-induced retro Diels-Alder (RDA) protocols, where the chirality of the desired products is transferred from norbornene derivatives. The stereochemistry of the synthesized compounds was proven by X-ray crystallography. The absolute configuration of the product is determined by the configuration of the starting amino hydroxamic acid.From 2-aminonorbornene hydroxamic acids, a simple and efficient method for the preparation of pyrrolo[1,2- The first hydroxamic acid  was disAccording to the literature data, aromatic hydroxamic acids are useful compounds ,21,22,23a]quinazolines and pyrimidoisoindoles quinazolinones and pyrrolopyrimidines starting from hydroxamic acids. Pyrroloquinazolines and pyrrolopyrimidines are important heterocyclic ring systems that occur as a core structure in a variety of naturally occurring alkaloids and synthetic compounds. Pyrroloquinazolines are tricyclic compounds with great potential and combine the quinazoline substructure, a privileged structure in medicinal chemistry, with a pyrrole [a]quinazolines in the near future, mainly for their potential applications in medicinal chemistry pyrimidines starting from 2-aminonorbornene hydroxamic acids pyrimidine derivatives.Our present aim was (i) to examine the domino ring-closure reaction of diendo- and diexo-2-aminonorbornene hydroxamic acids (±)-1 and (±)-2 were prepared from the appropriate ester bases with aqueous hydroxylamine solution according to an earlier procedure pyrimidine 5, the retro Diels-Alder reaction of the methanopyrroloquinazoline derivatives was examined in the microwave reactor under varied conditions. The best results for the cycloreversion were achieved in 1,2-dichlorobenzene (DCB) at 250 °C (20 min).In order to produce racemic pyrrolopyrimidine (+)-5 and (–)-5 -5 .8. In this case, however, the ring-closure reaction with levulinic acid resulted in only a small quantity of the minor diastereomers (±)-6b and (±)-7b, which could not be isolated. The stereochemistry of (±)-6a was confirmed by X-ray diffraction analysis. The relative configuration of the CH3 group at the C-3a asymmetric centre and the bridgehead hydrogens of C-5a and C-9a have the same steric orientation in (±)-6a (We attempted to use the same methodology to synthesize racemic n (±)-6a .8 and (–)-8. Namely, we carried out the cycloreversions of the major methanopyrroloquinazoline derivatives (+)-6a, (–)-6a, (+)-7a, and (–)-7a and isolated the pyrrolopyrimidines (+)-8 and (–)-8 in 55–57% yields -elds see .7a and (–)-7a was determined by chemical correlation: the absolute configurations of the starting 2-aminonorbornene hydroxamic acids, and the relative configuration of (±)-6a from the X-ray diffraction analysis was known. When (+)-7a was heated under the RDA reaction conditions, (–)-8 was gained. In addition, the RDA product of (+)-6a also afforded (–)-8. In contrast, when (–)-7a and (–)-6a were treated under the RDA reaction conditions, (+)-8 was formed. The NMR and HPLC data (retention times: (–)-8: 41.91 min, opposite enantiomer (+)-8: 34.19 min) and comparison of the optical rotations revealed that the final compound was identical to that of (R)-1-methoxy-8a-methyl-1,7,8,8a-tetrahydropyrrolopyrimidine-2,6-dione (–)-8. Since this asymmetric centre was not affected during the RDA reaction, these results allow for the assumption that the absolute configuration of position 3a of (+)-7a is R, and that of (–)-7a is S.The absolute configuration of the newly built asymmetric centre of (+)-1H-NMR spectra were recorded at 400.13 MHz or 600.20 MHz and the 13C-NMR spectra were recorded at 100.62 MHz or 150.92 MHz in CDCl3 at ambient temperature, with a Bruker AM 400 or Bruker AV 600 spectrometer . Chemical shifts are given in δ (ppm) relative to TMS as the internal standard. Microwave-promoted reactions were performed in sealed reaction vials (10 mL) in a microwave  cavity . Optical rotations were measured with a Perkin-Elmer 341 polarimeter . Mass spectra were recorded with a Micromass Q-TOF Premier mass spectrometer . Melting points were measured with a Hinotek-X4 micro melting point apparatus  and are uncorrected. Racemic 2-aminonorbornene hydroxamic acids (±)-1 and (±)-2 and enantiomeric 2-aminonorbornene hydroxamic acids (+)-1, (–)-1, (+)-2, and (–)-2 were prepared by a literature method :Methyl -4-methoxy-1,5-dioxo-1,2,3,3a,4,5,5a,6,9a-decahydro-6,9-methanopyrroloquinazoline-3a-carboxylate  m/z = 307.1294, measured: m/z = 307.1288.3b)]:Methyl -4-methoxy-1,5-dioxo-1,2,3,3a,4,5a,6,9a-decahydro-6,9-methanopyrroloquinazoline-3a-carboxylate  m/z = 307.1294, found m/z = 307.1288.4a)]:Methyl -4-methoxy-1,5-dioxo-1,2,3,3a,4,5a,6,9a-decahydro-6,9-methanopyrroloquinazoline-3a-carboxylate  m/z = 307.1294, found m/z = 307.1288.4b]:Methyl -4-methoxy-1,5-dioxo-1,2,3,3a,4,5a,6,9a-decahydro-6,9-methanopyrroloquinazoline-3a-carboxylate  m/z = 307.1294, found m/z = 307.1288.3a, (–)-4a, or (–)-3b and (+)-4b  was dissolved in 2 mL DCB in a 10 mL sealed reaction vial. The solution was stirred at 250 °C for 20 min at max 300 W microwave irradiation. Then the solvent was evaporated, the residue was dissolved in 2 mL EtOAc/MeOH = 9:1, and was purified by column chromatography on silica gel eluted by EtOAc/MeOH = 9:1. The product was crystallized from iPr2O to produce white crystals.Tetracyclic compound (–)-5]:Methyl (R)-1-methoxy-2,6-dioxo-1,2,6,7,8,8a-hexahydropyrrolopyrimidine-8a-carboxylate  m/z = 241.0825, found m/z = 241.0819.5]:Methyl (S)-1-methoxy-2,6-dioxo-1,2,6,7,8,8a-hexahydropyrrolopyrimidine-8a-carboxylate :-4-methoxy-3a-methyl-2,3,3a,4,5a,6,9a-octahydro-6,9-methanopyrroloquinazoline-1,5-dione  m/z = 263.1396, found m/z = 263.1390.6a]:-4-methoxy-3a-methyl-2,3,3a,4,5a,6,9a-octahydro-6,9-methanopyrroloquinazoline-1,5-dione :-4-methoxy-3a-methyl-2,3,3a,4,5a,6,9a-octahydro-6,9-methanopyrroloquinazoline-1,5-dione  m/z = 263.1396, found m/z = 263.1390.7a]:-4-methoxy-3a-methyl-2,3,3a,4,5a,6,9a-octahydro-6,9-methanopyrroloquinazoline-1,5-dione :(S)-1-methoxy-8a-methyl-1,7,8,8a-tetrahydropyrrolopyrimidine-2,6-dione  m/z = 197.0926, measured: m/z = 197.0916.8(R)-1-methoxy-8a-methyl-1,7,8,8a-tetrahydropyrrolopyrimidine-2,6-dione[(–)-]: White crystals, (57% yield), m.p. 139–142 °C; c = 0.23, EtOH), ee 99%, 1H- and 13C-NMR is similar to (+)-8.a]pyrimidines were synthesized starting from diendo- and diexo-2-aminonorbornene hydroxamic acids. Their domino ring closure reactions with α-ketoglutaric acid and levulinic acid, and the formation of two diastereomers were observed in each case. After separation by column chromatography, single diastereomers were subjected to a microwave-mediated RDA reaction and gained bicyclic pyrrolopyrimidines through the loss of cyclopentadiene. When enantiomeric diexo- or diendo-3-amino-N-hydroxybicyclo[2.2.1]hept-5-ene-2-carboxamides were used, the products were enantiomeric heterocycles with ee = 95–99%, demonstrating that the starting compounds are excellent chiral sources, and the stereochemical information can be effectively transferable to the newly formed stereogenic centre.In conclusion, new racemic and enantiomeric pyrrolo[1,2-


These meso-trifluoromethyl derivatives of β-octaalkylporphyrins underwent smooth metalation, similar to other common porphyrins, however, the corresponding zinc complexes underwent a type of solvolysis, whereby the trifluoromethyl groups were converted into methoxycarbonyl groups by the methanol used as solvent. UV-visible absorption spectra and X-ray crystal structure analyses revealed that the presence of a methoxycarbonyl substituent did not influence the deformation of the molecular framework and its absorption properties; this is because the methoxycarbonyl has a planar and perpendicular geometry, as opposed to the relatively bulky trifluoromethyl substituent.5,10-Bistrifluoromethyl substituted β-octamethylporphyrins were synthesized via a scrambling side reaction of a dipyrromethane precursor in the presence of a large excess of trifluoroacetic acid. Compared with the  A solution of 7 and large excess of zinc acetate in CHCl3 and MeOH (3:1 v/v) zinc acetated was stirred at room temperature until 7 was completely consumed. The resulting solution was washed with water twice and dried over anhydrous sodium sulfate. After removal of the solvent, 7Zn was obtained almost quantitatively. Recrystalization from CHCl3–MeOH gave an analytical sample of 7Zn as purple solids 1H-NMR (CDCl3): δ = 1.70 , 1.80 , 1.82–1.87 , 3.89–3.96 , 4.00 , 4.05–4.10 , 9.83  and 9.92  ppm; 19F-NMR (CDCl3): δ T = −31.77 (CF3) ppm; UV-vis (CH2Cl2) : λmax (ε [M−1cm−1]): 416 , 550 , 567 , and 611  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C37H43F2N4Zn ([M − F]+), calcd: 645.2742, found: 645.2733 (100%); Elemental analysis calcd for C37H43F3N4Zn: C 66.70, H 6.77, N 8.69; found: C 66.71, H 6.51, N 8.41.5,10-Bis(trifluoromethyl)-2,3,7,8,12,13,17,18-octamethylporphyrin zinc complex (9Zn). A solution of 9 and large excess of zinc acetate in CHCl3 and MeOH (3:1 v/v) zinc acetated was stirred at room temperature until 9 was completely consumed. The resulting solution was washed with water twice and dried over anhydrous sodium sulfate. After removal of the solvent, 9Zn was obtained almost quantitatively. Recrystalization from CHCl3-MeOH gave an analytical sample of 9Zn as purple solids 1H-NMR (CDCl3): δ = 3.31 , 3.33 , 3.40 , 3.46 , and 9.67  ppm; 19F-NMR (CDCl3): δ T = −35.12 (CF3) ppm; UV-vis (CH2Cl2): λmax (ε [M−1cm−1]): 360 , 429 , 578 (9300), and 623  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C30H26F6N4Zn ([M]+), calcd: 620.1348, found: 620.1350 (100%); Elemental analysis calcd for C30H26F6N4Zn: C 57.94, H 4.21, N 9.01; found: C 57.73, H 4.32, N 8.89.5-Methoxycarbonyl-2,3,7,8,12,13,17,18-octaethylporphyrin zinc complex (11Zn). A solution of 7Zn (10.0 mg) in CHCl3 (5 mL) and MeOH (5 mL) was refulxed for 1 day. After evaporated to dryness, recrystalization from CHCl3/MeOH afforded 11Zn as purple solids . 1H-NMR (CDCl3): δ = 1.71 , 1.91–1.96 , 3.80–3.84 , 4.08–4.15 , 4.53  10.13 , and 10.21  ppm; UV-vis (CH2Cl2): λmax (ε [M−1cm−1]): 338 , 406 , 537 , and 574  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C38H46N4O4Zn ([M]+), calcd: 654.2907, found: 654.2911 (100%); Elemental analysis calcd for C38H46N4O2Zn: C 69.56, H 7.07, N 8.54; found: C 69.72, H 6.73, N 8.60.5,10-Bis(methoxycarbonyl)-2,3,7,8,12,13,17,18-octamethylporphyrin zinc complex (12Zn). A solution of 9Zn (10.9 mg) in CHCl3 (2 mL), MeOH (10 mL), and H2O (0.5 mL) was refulxed for 1 day. After evaporated to dryness, recrystalization from CHCl3/MeOH afforded 12Zn as purple solids . 1H-NMR (CDCl3): δ = 3.33 , 3.35 , 3.41 , 3.54 , 4.51  and 9.74 ppm; UV-vis (CH2Cl2): λmax (ε [M−1cm−1]): 345 , 409 , 541 , and 577  nm. Elemental analysis calcd for C32H32N4O4Zn: C 63.84, H 5.36, N 9.31; found: C 63.21, H 5.17, N 9.14.3 substituted β-octamethylporphyrins in a cis-arrangement and the metal-dependent solvolysis of meso-CF3 functionalities. The synthesis of such a cis-configured porphyrin is usually challenging because more complicated oligo-pyrrolic precursors containing CF3 substituent may be required. Compared with its trans-analogue 5, the cis-configured 9 showed more red-shifted absorption bands, suggesting that a cis-arrangement worked more effectively than a trans-arrangement for extension of the absorption range meso-positions, and even fewer examples with β-octaalkylporphyrins [3 into CO2Me including the role of the metal is still unclear (We have demonstrated the formation of bis-CFrphyrins ,20,21. A unclear , our syn


Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I–IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a–e). All newly prepared compounds have been screened for selected human breast cancer  and ovarian cancer  cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.The vacuolar (H The V-ATPase proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V1 and V0. The domain V1 is responsible for ATP hydrolysis and V0 provides the transmembrane proton channel imidazol]-2′-yl)phenol (compound 1) with appropriate substituted alkyl halides in the presence of cesium carbonate in dimethyl formamide (DMF) at reflux temperature (2a–e) using methanol and chloroform (3:7). We quickly developed a fast and efficient synthetic procedure to prepare novel bisbenzimidazole analogs using the one pot procedure. A general synthesis of bisbenzimidazole derivatives (perature . The cru+)-ATPase in breast as well as ovarian cancer cell lines -6-(1-methyl-4-piperazyl)benzimidazole trihydrochloride (compound 6-(4-Methylpiperazin-1-yl)-2'-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H,3'H-2,5'-bibenzo[d]imidazole (2a) Yield: 12%, brown solid powder. m.p.: 165–166 °C; 1H-NMR  δ 2.06–2.18 , 2.55 , 2.70 , 2.89-3.00 , 3.19–3.28 , 4.74 , 7.06-7.09 , 7.18 , 7.53 , 7.72 , 7.97 , 8.09 , 8.29 ; MS (ESI): m/z 522.6 [M + H]+; HRMS (ESI): m/z calcd for C31H35N7O [M + H]+ 522.2981; obsd 522.2971.N,N-Dimethyl-3-(4-(6-(4-methylpiperazin-1-yl)-1H,3'H-imidazol]-2'-yl)phenoxy)propan-1-amine (2b). Yield: 15%, brown solid powder. m.p.: 171–172 °C; 1H-NMR  δ 1.90 , 2.20 2), 2.24 , 2.44 , 3.08–3.16 , 3.36–4.02 , 4.09 , 6.91–6.95 , 7.12 , 7.36–7.49 , 7.58–7.72 , 7.95–8.03 , 8.14 , 8.20–8.33 , 12.60 , 12.96 ; MS (ESI): m/z 510.6 [M + H]+; HRMS (ESI): m/z calcd for C30H35N7O [M + H]+ 510.2984; obsd 510.2983.N,N-Dimethyl-2-(4-(6-(4-methylpiperazin-1-yl)-1H,3'H-imidazol]-2'-yl)phenoxy)ethanamine (2c). Yield: 13%, yellow solid powder. m.p.: 197–198 °C; 1H-NMR  δ 2.44 3), 2.65 , 3.10–3.13 , 3.34–3.39 , 4.13 , 6.91–6.93 , 7.12 , 7.38–7.46 , 7.65 , 7.98 , 8.15 , 8.27 , 12.60 , 12.96 ; MS (ESI): m/z 496.4 [M + H]+; HRMS (ESI): m/z calcd for C29H33N7O [M + H]+ 496.2825; obsd 496.2820.2'-(4-(2-Methoxyethoxy)phenyl)-6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzo[d]imidazole (2d). Yield: 22%; yellow solid powder; m.p.: 208–209 °C; 1H-NMR  δ 2.35 , 2.62–2.65 , 3.16–3.18 , 3.39 , 3.68–3.70 , 4.05–4.08 , 6.98–7.00 , 7.07 , 7.46 , 7.52 , 7.57–7.58 , 7.95 , 8.16 ; 13C-NMR  δ 44.59, 50.25, 54.68, 57.85, 67.05, 70.63, 100.75, 114.57, 114.90, 120.98, 121.65, 124.17, 128.10, 134.5, 138.0, 148.06, 152.24, 153.70, 160.79, 171.56; MS (ESI): m/z 483.5 [M + H]+; HRMS (ESI): m/z calcd for C28H30N6O2 [M + H]+ 483.2508; obsd 483.2498.2'-phenyl)-6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzo[d]imidazole (2e). Yield: 16%; pale yellow solid powder; m.p.: 247–248 °C; 1H-NMR  δ 0.98 3), 1.70 , 2.24 , 3.12–3.30 , 4.11 , 6.91-6.96 , 7.13 , 7.58-7.72 , 7.28–7.49 , 7.94–8.02 , 8.13 , 8.20–8.35 , 12.58 , 12.94 ; 13C-NMR  δ 28.74, 29.23, 44.55, 46.94, 50.28, 54.68, 65.24, 100.10, 114.59, 114.97, 121.02, 121.32, 124.21, 128.14, 148.08, 152.32, 153.92, 161.14; MS (ESI): m/z 509.7 [M + H]+; HRMS (ESI): m/z calcd for C31H36N6O [M + H]+ 509.3029; obsd 509.3028.V), and bisbenzimidazole analogs 2a–e in the selected human breast cancer cell lines MDA-MB-231 , MDA-MB-468 , MCF-7  and the normal breast epithelial cell line, MCF10A, and the ovarian cancer cell lines, A2780, Cis-A2780, and PA-1 . Human breast cancer cell lines and the human ovarian cancer cell line (PA-1) were purchased from ATCC . The cisplatin-sensitive human ovarian carcinoma cell line, A2780, was obtained from Sigma-Aldrich. The cisplatin-resistant human ovarian cell line, Cis-A2780, was produced in our laboratory, as described in the literature [v/v) heat-inactivated fetal bovine serum , 100 U/mL penicillin G and 100 U/mL streptomycin . Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and were passaged by trypsinization after reaching 80–90% confluence.We studied the antiproliferative activity of an initial hit (compound terature . MDA-MB-3) were seeded into each well of 96-well plates and incubated overnight. Dimethyl Sulfoxide (DMSO) stock solutions of the compounds  were diluted in corresponding media and exposed to different concentrations for 48 h. All three independent experiments were performed in quadruplicates. Untreated cells were used as a negative control and data were presented as the mean ± standard deviation (S.D.). The absorbances at 492 nm were measured using a Microplate Reader . The compound concentration that inhibited cell growth by 50% of the untreated control (IC50) was calculated from the dose response curves constructed by plotting percentages of cell viability versus drug concentrations using nonlinear regression analysis by GraphPad Prism Software 7 .CellTiter 96 AQueous One Solution cell proliferation assay (MTS) ,30 was u6 cells per mL in the same buffer and placed on ice. H+ uptake was initiated by the addition of MDA-MB-231 cells  to pre-warmed HBSS (2.4 mL) containing AO  and placed in a cuvette. AO fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 540 nm using the LS-50B Luminescence Spectrometer . Nigericin  was added to collapse the pH gradient and unquench the AO fluorescence (See 1a and 1b). MDA-MB-231 cells  were treated with the standard V-ATPase inhibitor Baf A1 (1 μM), along with compounds V, 2d, and 2e (10 μM), for 20 min followed by addition of AO  for the proton pump activity . A decrease in the fluorescence intensities per min of Baf A1 and the potent compounds  was calculated and is expressed as a percentage of the control (mean ± SD of two independent experiments) (The proton pump activity was assessed by using the AO fluorescence quenching method . Bafilomriments) . V) as an initial hit compound. Synthesis and anticancer screening of a focused set of novel bisbenzimidazole derivatives indicated that they were potent against selected breast and ovarian cancer cell lines in vitro. The selected bisbenzimidazole analogs (2d and 2e) have demonstrated selective potency towards the TNBC cell line, MDA-MB-468. The compound 2e potently inhibited the proton pump activity, which is largely responsible for the acidification, and it showed selective anticancer activity. To the best of our knowledge, the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. Together, our findings demonstrated that the bisbenzimidazole analog (2e) could be further developed as an anticancer vacuolar (H+)-ATPase inhibitor for the treatment of breast cancer.In conclusion, starting from the virtual screening practice using known small molecule V-ATPase inhibitors, we identified a bisbenzimidazole analog (


Their structures were unambiguously determined on the basis of spectroscopic analyses using one-dimensional (1D) and two-dimensional (2D) NMR techniques and High resolution Fast Atom Bombardment Mass Spectrometry (HRFABMS). Compounds 1–3 were evaluated for their effects on the production of nitric oxide (NO) in Lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that compounds 2 and 3 have high potency as inhibitors of macrophage activation.A novel  Extracts from its flowers, bark, and seeds have been used for centuries by the Amazonian people and exhibit analgesic ; three acetyls , a propanoyl , a methyl ester , an 1,8,9-orthoacetyl group  , six sp3 quaternary carbons including two oxycarbons , a furan , and a lactone [δC 174.2 (s)]. Analysis of the 1H-1H COSY spectrum of 1 revealed the partial structure shown in bold face in 3-18 [δH 1.23 (s)]/C-12, C-13, C-14 and C-17 [δC 69.8 (d)]; between H-3 [δH 5.26 (s)]/C-1 [δC 84.6 (s)], C-2 [δC 86.1 (s)], C-4, C-5, C-28, C-29, and C-30 [δC 68.7 (d)]; between H2-6 [δH 2.52 (d)], 2.68 (dd)]/C-4, C-5, C-7 [δC 174.2 (s)], and C-10 [δC 86.4 (s)]; between H-14 [δH 2.36 (dd)]/C-8 [ δC 85.3 (s)], C-9 [δC 84.0 (s)], C-12, C-13, C-15, C-16 [δC 173.9 (s)], and C-30; between H-17 [δH 5.68 (s) ] /C-12, C-13, C-14, C-20 [δC 122.5 (s)], C-21 [δC 142.0 (d)], and C-22 [δC 109.2 (d)]; between H2-29 [δH 1.7 8 and 1.91 (each d)]/C-1, C-2, C-3, C-4, C-5, and C-10; and between H-30 [δH 5.94 (s)]/C-2, C-3, C-8, C-9, C-1 4, and C-1′′′′′ [δC 172.0 (s)] were obtained (Carapanosin D ( spectra  exhibiteobtained .1 were similar with those of andirolide O , respectively. Therefore 1 would be a 19-nor limonoid, and the E ring has a γ-lactone. On the other hand, C-16–C-17 was opened and attached to methylester and acetate, respectively. Thus, the framework of 1 could be a C-19-nor, C-16,17-seco-phragmalin-1,8,9-orthoacetate. The relative configuration of 1 was determined by the NOESY spectrum, in which significant nuclear Overhauser effect (NOE) were observed between H-3 and H2-29; between H-5 and H-12β, H-30, and CH3-28; between H-14 and H-11β; between H-17 and H-12β, H-30; between H-30 and H-5, H-12β, H-15β, and H-17; and between CH3-18 and H-11, H-12α, and H-22. Therefore, the relative structure of 1 was confirmed as shown in Nor-phragmalin was first isolated from Chukrasia tabularis by Yin, J-L., et al., who described Tabulvelutin A as a unique 7,10-γ-lactone , 2-methylpropanoyl , 2-methylbutanoyl , a methylester , an αβ-unsaturated δ-lactone , a six-membered ring ketone [δC 204.1 (s)], two tertiary hydroxyls that disappear by heavy water processing , and a β-substituted furan ring , therefore, 2 could be suggested as a mexicanolide-type limonoid. The HMBC connectivities between H3-18 [δH 1.28 (s)]/C-12, C-13, C-14 [δC 165.8 (s)], and C-17 [δC 78.9 (d)]; H3-19 [δH 1.09 (s)]/C-1 [δC 204.1 (s)], C-5, C-9, and C-10; H-3 [δH 5.15 (s)]/C-1, C-2 [δC 86.3 (s)], C-4, C-5, C-28, C-29, and C-30 [δC 73.9 (d)]; H-15 [δH 6.34 (s)]/C-8 [δC 80.6 (s)], C-13, C-14, and C-16 [δC 164.9 (s)]; H-17 [δH 5.44 (s)]/C-12, C-13, C-16, C-20 [δC 120.3 (s)], C-21 [δC 141.6 (d)], and C-22 [δC 110.5 (d)]; H-30 [δH 6.51 (s)]/C-1, C-2, C-3 [δC 79.7 (s)], C-8, C-9, and C-14; 2-OH (δH 4.08 (s)/C-1, C-2, and C-30; 8-OH (δH 2.84 (s)/C-8, C-9, C-14, and C-30 were observed. In the 1H-1H COSY spectrum, five distinct spin sets of H-5–H-6; H-9–H2-11–H2-12; H-22–H-23; H3-3′–H-2′–H3-4′; and H3′′′′-5–H-2′′′′–H2-3′′′′–H3-4′′′′ were observed ; δC 16.7 (q)] were in accordance with those of carapanolide F  . NMR data were very similar to those of 2 except for a tigroyl group  at C-3. NOESY spectrum revealed the relative stereochemistry of 3 to have the same conformation as 2.Carapanosin F (G-monomethyl-L-arginine acetate (l-NMMA), which is an inducible nitric oxide synthase (iNOS) inhibitor, were assayed for their inhibitory effects on NO production in LPS stimulated RAW 264.7 cells. Cytotoxicities of limonoids tested were evaluated by the  (MTT) assay for determination of safe concentrations. Mexicanolide-type limonoids 2 and 3 exhibited stronger inhibitory activities  than the positive control, l-NMMA (IC50 of NO produced 47.6 μM) without cytotoxicities , 6α-acetoxygedunin (IC50 7.9 μM), 7-deacetoxy-7-hydroxygedunin (IC50 8.7 μM), and 6α-acetoxy-7α-deacetoxy-7α-hydroxygedunin (IC50 9.4 μM) from the flower oil of C. guianensis +: 745.2693; found 745.2707; IR (KBr) νmax cm−1; 2975, 1747(O–C=O), 1633; for 1H and 13C-NMR spectroscopic data, see m/z (rel. int.): 745 (100), [M + H]+, 685 [M + H − HOAc]+ (72), 449 (33).Carapanosin D (2): Colorless amorphous solid; m.p. 96–98 °C; c 0.1, CHCl3); HRFABMS: m/z calcd for C36H49O12 [M + H]+: 673.3224; found 673.3224; UV λmax (CH3CN) nm (log ε): 219 (3.76); IR (KBr) νmax cm−1: 3489 (OH), 2974, 1727 (O–C=O), 1710 (six membered ring ketone), 1670 : 673 (27) [M + H]+, 57 (100). Carapanosin E (3): Colorless amorphous solid; m.p. 83–85 °C; c 0.1, CHCl3); HRFABMS m/z calcd. for C37H49O12 [M + H]+: 685.3224; found 685.3224; UV λmax (CH3CN) nm (log ε): 232 (3.82), IR (KBr) νmax cm−1: 3462 (OH), 2970, 1727 (O–C=O), 1707 (six membered ring ketone), 1670 (αβ-unsaturated δ-lactone), 1549, and 1461; for 1H and 13C-NMR spectroscopic data, see m/z (rel. int.): 685 (11) [M + H]+, 83 (100). Carapanosin F (2 humidified incubator at 37 °C. RAW264.7 cells (mouse macrophages) purchased from DS Pharma Biomedical Co., Ltd.  were incubated in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) in a 5% CORAW264.7 cell proliferation was examined as described previously .An inhibitory assay of nitric oxide production was performed as describe previously .nor-phragmalin-type limonoid, named carapanosin D (1), and two new mexicanolide-type limonoids, named carapanosins E and F  were isolated from the seeds of Carapa guianensis (andiroba). Their structures were elucidated by extensive spectroscopic techniques. Carapanosin D (1) is the second example of 19-nor-phragmalin. Compounds 1–3 showed non-toxicities at 0–30 μM. Of these, compounds 2 and 3 showed superior inhibitory activities  compared to the positive control, l-NMMA (IC50 of NO produced 47.6 μM). These results suggest that compounds 2 and 3 have high potency as inhibitors of macrophage activation.A novel


Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one  were obtained as the tautomeric mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis. In addition, the absolute configurations of these three pairs of new enantiomers were determined by quantum chemical ECD calculations.Three pairs of new isopentenyl dibenzo[ Up to now, about nine isopentenyl dibenzooxepinones had been reported from fungi in Nature, including arugosins A–D from Aspergillus rugulosus oxepin-11(6H)-one  from Penicilium sp. oxepinone enantiomers ((+)-(5S)-arugosin K (1a), (−)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (−)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M (3a), (−)-(5R)-arugosin M (3b)), a new isopentenyl dibenzooxepinone, arugosin N (4), and two known compounds 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) and chrysophanol (6) (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) were a pair of tautomers, and they were isolated as mixtures. Detail of the isolation and structural elucidations of compounds 1–6 are presented herein.ompounds ,9. In ouompounds ,13,14,15ompounds ,13,14,15anol (6) . The abs1 was obtained as a yellow solid. The quasi-molecular ion at m/z 355.1550 [M + H]+ obtained by HRESIMS indicated the molecular formula of 1 was C21H22O5 with 11 degrees of unsaturation. The 13C-NMR spectrum combined with the DEPT-135 spectrum showed 21 signals (2 quaternary carbons + by HRESIMS indicated the molecular formula of 2 was C22H24O5 with 11 degrees of unsaturation. Except for the loss of an oxygenated methyl at δC 56.9 (5-OCH3) and the appearance of an additional oxygenated methylene at δC 65.3 (C-1′′) and methyl at δC 14.8 (C-2′′), the NMR data of 2   , the plaablished .1 and 2 coexist in the same strain, 2 was also a racemic mixture, and it displayed two peaks in a Phenomenex Lux Cellulose-2 chiral column (2a (tR = 22.6 min) and (−)-2b (tR = 26.6 min) were isolated from 2 by a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as eluent at a flow rate of 0.7 mL/min. The ECD spectra and 2a ) and 2b ) showed their enantiomeric relationship. Since the ECD data of 2a were similar to that of 1a  as shown in 1H–1H COSY experiment and molecular formula, the HMBC correlations  and (−)-3b (tR = 22.1 min) were isolated from 3 by a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as eluent at a flow rate of 0.7 mL/min. The ECD spectra and 3a ) and 3b ) showed their enantiomeric relationship. The absolute configurations of 3a and 3b were determined by quantum chemical ECD calculations at the APFD/6-311++g level. The predicted ECD curves of (5S)-3 and (5R)-3 were in good accordance with the experimental ECD for 3a and 3b, respectively , the 1H NMR and 13C-NMR data of 4   , the pla5 was fourteen atomic mass units less than 1. Except for the loss of an oxygenated methyl at δC 56.9 (5-OCH3), the 1H-NMR and 13C-NMR data of 5  -dibenzoxepin-11(6H)-one +; ESI-MS (negative): m/z 253 [M − H]− indicated the molecular weight was 254. It was identified as chrysophanol by comparing its data  with literature values oxepin-11(6H)-one (5). Then, arugosin K (1) and arugosin L (2) may originate from 5 by methylation and ethylation, respectively. Arugosin M (3) may originate from 4 by methylation.Based on the structural features of compounds proposed . Chrysopdization ,18, ringdization ,18, redudization ,18, and dization , which mMethanol (MeOH) was purchased from Yuwang Industrial Co. Ltd. . Cyclohexane, ethyl acetate (EtOAc), and petroleum ether (PE) were purchased from Tianjin Fuyu Fine Chemical Co. Ltd. .3: δH 7.26/δC 77.0) as internal standard. The analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector (DAD), an Ultimate 3000 Column Compartment, an Ultimate 3000 autosampler  using a Gemini C18 column  . The semi-preparative HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Gemini C18 column (Phenomenex Inc.) . The chiral HPLC was performed on a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Lux Cellulose-2 chiral column .Optical rotations were measured on a P1020 digital polarimeter . UV data were recorded using a Jasco V-550 UV/Vis spectrometer. IR data were recorded on a Jasco FT/IR-480 plus spectrometer. ECD spectrum was recorded on a Jasco J-810 spectrophotometer using MeOH as the solvent. The ESI-MS spectra were recorded on a Finnigan LCQ Advantage MAX mass spectrometer . The HR-ESI-MS spectra were obtained on a Micromass Q-TOF mass spectrometer . The NMR spectra were measured with Bruker AV-400 and Bruker AV-600 spectrometers  using the solvent signals  and the 5.8S rRNA gene sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as KX011167. The fungus was cultured on slants of potato dextrose agar at 25 °C for 5 days. Agar plugs were used to inoculate four Erlenmeyer flasks (250 mL), each containing 100 mL of potato dextrose broth. Four flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 200 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 20 Erlenmeyer flasks (500 mL), each containing 70 g of rice. Distilled H2O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving at 120 °C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the seed culture containing mycelia and incubated at room temperature for 50 days.The strain AHK07-3 was isolated from the soil, collected at the wetland of Ahongkou, Sinkiang Province, China. The strain was identified as v/v aqueous MeOH (400 mL). The methanolic solution was further extracted with cyclohexane (400 mL) to afford a cyclohexane fraction C (4.6 g). Fraction C was subjected to silica gel (200–300 mesh) column chromatography initially eluting with cyclohexane (500 mL) and then with MeOH (500 mL) to afford subfractions CC and CM. Subfraction CC (3.3 g) was further separated by open silica gel column chromatography eluting with cyclohexane–EtOAc (100:0 (150 mL), 99:1 (150 mL), 98:2 (150 mL), 97:3 (150 mL), 96:4 (150 mL), 95:5 (150 mL), 94:6 (150 mL), 93:7 (150 mL), 92:8 (150 mL), 91:9 (150 mL), 90:10 (150 mL), 50:50 (150 mL), and 0:100 (150 mL), v/v) to yield four sub-subfractions (CC1 to CC4). Sub-subfraction CC2 (2.8 g) was subjected to open silica gel column chromatography eluting with PE (petroleum ether)–EtOAc (100:0 (100 mL), 99:1 (100 mL), 98:2 (100 mL), 97:3 (100 mL), 96:4 (100 mL), 95:5 (100 mL), 94:6 (100 mL), 93:7 (100 mL), 92:8 (100 mL), 91:9 (100 mL), 90:10 (100 mL), 50:50 (100 mL) and 0:100 (100 mL), v/v) to yield four sub-subfractions (CC2a to CC2d). Sub-subfraction CC2b (1.1 g) was isolated by semi-preparative HPLC purification using MeOH–H2O  at a flow rate of 3 mL/min to yield 1 (617.4 mg), 2 (2.6 mg), and 3 (24.7 mg). Sub-subfraction CC2c (0.6 g) was purified by semi-preparative HPLC purification using MeOH–H2O  at a flow rate of 3 mL/min to yield 6 (1.1 mg). Sub-subfraction CC4 (220.1 mg) was isolated by semi-preparative HPLC purification using MeOH–H2O  at a flow rate of 3 mL/min to yield the mixtures of 4 and 5 (12.5 mg). Enantioseparation of 1–3 was carried out on a Phenomenex Lux Cellulose-2 chiral column using CH3OH/H2O  as mobile phase.The culture was extracted with EtOAc (3 × 5000 mL). Removal of EtOAc under reduced pressure yielded a crude extract (19.7 g) that was dissolved in 90% (±)-Arugosin K (1): yellow solid; UV (MeOH) λmax (log ε) 205 (3.75), 221 (3.63), 304 (3.35), 359 (3.29) nm; IR (KBr) νmax 3438, 2920, 1708, 1619, 1590, 1421, 1206, 1052 cm−1; ESI-MS (positive): m/z 355 [M + H]+, ESI-MS (negative): m/z 353 [M − H]−; HRESIMS (positive) m/z 355.1550 [M + H]+ ; 1H- and 13C-NMR data see (+)-S-Arugosin K (1a): c 0.5, CHCl3); ECD  λmax (Δε) 208 (+12.50), 294 (−5.71), 334 (+3.76), 382 (−1.77) nm.(−)-R-Arugosin K (1b): c 0.5, CHCl3); ECD  λmax (Δε) 208 (−11.38), 295 (+6.96), 334 (−3.88), 382 (+2.82) nm.(±)-Arugosin L (2): yellow solid; UV (MeOH) λmax (log ε) 206 (3.70), 222 (3.57), 304 (3.30), 359 (3.23) nm; IR (KBr) νmax 3440, 2916, 1624, 1590, 1422, 1206, 1057 cm−1; ESI-MS (positive): m/z 369 [M + H]+; ESI-MS (negative): m/z 367 [M − H]−; HRESIMS (positive) m/z 369.1695 [M + H] + ; 1H- and 13C-NMR data see (+)-S-Arugosin L (2a): c 0.5, CHCl3); ECD  λmax (Δε) 208 (+13.90), 295 (−6.07), 335 (+5.07), 382 (−1.91) nm.(−)-R-Arugosin L (2b): c 0.5, CHCl3); ECD  λmax (Δε) 208 (−13.79), 295 (+7.98), 335 (−3.44), 382 (+3.40) nm.(±)-Arugosin M (3): yellow solid; UV (MeOH) λmax (log ε) 206 (3.85), 221 (3.73), 299 (3.42), 357 (3.36) nm; IR (KBr) νmax 3441, 2913, 1620, 1464, 1202, 1050 cm−1; ESI-MS (positive): m/z 355 [M + H]+, ESI-MS (negative): m/z 353 [M − H]−; HRESIMS (positive) m/z 355.1541 [M + H]+ ; 1H- and 13C-NMR data see (+)-S-Arugosin M (3a): c 0.5, CHCl3); ECD  λmax (Δε) 209 (+14.24), 290 (−6.29), 334 (+6.67), 381 (−3.30) nm.(−)-R-Arugosin M (3b): c 0.5, CHCl3); ECD  λmax (Δε) 209 (−12.84), 292 (+7.26), 334 (−5.93), 381 (+4.27) nm.Arugosin N (4): 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5): yellow solid; UV (MeOH) λmax (log ε) 206 (3.75), 222 (3.65), 286 (3.29), 356 (3.16) nm; IR (KBr) νmax 3443, 2917, 1628, 1589, 1420, 1353, 1173, 1052 cm−1; ESI-MS (positive): m/z 341 [M + H]+, m/z 363 [M + Na]+; ESI-MS (negative): m/z 339 [M − H]−; HRESIMS (positive) m/z 363.1213 [M + Na]+ ; 1H- and 13C-NMR data see S)-1, (5R)-1, (5S)-3, and (5R)-3 were converted into SMILES codes, respectively. Conformer databases were generated in CONFLEX version 7.0  using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal·mol−1 above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5 Å. Then each conformer of the acceptable conformers was optimized with HF/6-31G(d) method in Gaussian09 oxepinones with intact 6-7-6 tricyclic systems have been reported from the genera of Aspergillus, Emericella, Pestalotiopsis, Graphiopsis, and Penicilium. Except the absolute configuration of cephalanone D that had been verified by X-ray crystallography oxepinones established by ECD experiments. Furthermore, 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenzoxepin-11(6H)-one (5) is a known compound, but no NMR data was previously reported. The assignments of NMR data of 5 are thus provided for the first time.Up to now, about nine isopentenyl dibenzo[


An organocatalyzed intramolecular carbonyl-ene reaction was developed to produce carbocyclic and heterocyclic 5- and 6-membered rings from a citronellal-derived trifluoroketone and a variety of aldehydes. A phosphoramide derivative was found to promote the cyclization of the trifluoroketone, whereas a less acidic phosphoric acid proved to be a superior catalyst for the aldehyde substrates. Traditihoramide . The reahoramide . While thoramide . The majlization , while l1 , induced very slow conversion of substrate, resulting in a low yield of ene product . While citronellal is less activated than the corresponding trifluoromethyl ketone, it serves as the prototypical substrate for a Type I carbonyl-ene cyclization, as it is commercially available [3b resulted in the isolation of a complex mixture of products with only a trace yield of ene product 5a and no starting material recovery (3c) successfully promoted the reaction, albeit slowly, resulting in a low yield and a 77% recovery of starting material after 24 h  was the primary diastereomer isolated from the reaction mixture in addition to a small amount of neoisopulegol (5a′), typically in a 2:1 ratio (Entries 3–5). A similar yield of product 5a was obtained when only 0.06 equivalents of catalyst were used at a higher concentration (0.5 M) compared to 0.5 equivalents at 0.1 M, but a decreased selectivity was observed in the isolated products (compare Entries 3 and 4) [5a decreased considerably  . At an eiderably , Entry 53a, 0.1 M, 24 h, rt), the scope of the reaction was explored. The carbonyl-ene reaction proceeds with excellent yield to give 3,4-disubstituted piperidine product 5b  as well as a substantial amount (~17%) of the conjugated diene, 3-isopropenyl-1,2-dihydronaphthalene, that resulted from an elimination reaction (4d) resulted in the formation of five-membered ring 5d with great diastereoselectivity, albeit in only moderate yield (Entry 3). The reaction conditions have been modified in an attempt to increase the yield of the carbonyl-ene product; however, in each case, a complex mixture of products was isolated with no starting material recovered.Once the cyclization of the aldehyde substrate was optimized  was purchased from Aldrich. Trifluoromethyl ketone 1 was prepared from citronellic acid as previously described [3b) was prepared as previously described [3b was washed with 6 M of HCl and extracted with chloroform to ensure protonation of the catalyst, as discussed for the preparation of related N-triflylphosphoramide catalysts [N-(3-methylbut-2-enyl)-N-(3-oxopropyl)benzenesulfonamide [1H-NMR spectra were obtained by using a Jeol 400 MHz spectrometer . Chemical shifts are reported in parts per million relative to TMS. Coupling constants were reported in Hertz, and multiplicities were indicated using the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), ddd (doublet of doublets of doublets), etc.13C-NMR data was obtained using Jeol 400 MHz NMR operating at 100 MHz. All products were characterized by 1H-NMR and 13C-NMR and compared with available literature data. High-resolution mass spectra (HRMS) of 2a/a′ were obtained on a Thermo LTQ-FTMS instrument .Citronellal (93%) was obtained from Acros and was purified with normal-phase column chromatography. 2,4-dimethylheptenal (80%) was purchased from Aldrich and was purified via normal phase chromatography before use. Diphenyl phosphate , catalyst 3 , and anhydrous dichloromethane  were added to a small glass vial containing a stir bar. After stirring at room temperature for 24–48 h, the reaction was concentrated and purified by column chromatography.Aldehyde or CF2a/a′ were prepared according to the above-described general procedure by stirring CF3 ketone 1  and diphenylphosphoramide 3b  in anhydrous dichloromethane (0.5 mL) at room temperature for 24 h to provide a mixture of diastereomers as a colorless oil in 86% yield (flash-chromatography: 20% diethyl ether in petroleum ether). Retention factor of 2a/2a′ = 0.3 (5% ethyl acetate: 95% hexanes). The relative stereochemical assignments of 2a/2a′ were made on the basis of coupling constants for H2 and H6ax [2a, the coupling constants for H2 = 3.6 and 12.8 Hz indicated axial orientation, and the coupling constants for H6ax = 12.4 and 14.0 Hz indicated axial-axial splitting with H5 and germinal coupling. Therefore, H5 must be in the axial position and trans to H2. For 2a′, H2 had a coupling constant of 13.2 Hz, indicating axial orientation, and one of the coupling constants for H6ax = 4.5 Hz indicated axial-equatorial splitting with H5. Therefore, H5 must be in the equatorial position and cis to H2. 1H-NMR of 2a : δ 4.94 ; 4.83 ; 2.37 ; 2.25 ; 1.96 ; 1.84 ; 1.83 ; 1.80 ; 1.73 ; 1.55 ; 1.18 ; 0.94 ; 0.93 . 13C-NMR (100 MHz): δ 147.6 (vinylic C); 126.1 ; 112.2 (vinylic C); 74.3 ; 46.3 (C2); 39.0 (C6); 34.3 (C4); 28.5 (C3); 26.5 (C5); 24.2 (vinylic Me); 21.9 (C5-Me). HRMS, APCI: Calcd. for C11H17F3O (M+): 222.12315; found: 223.13509. 1H-NMR of 2a′ (400 MHz): δ 5.01 ; 4.90 ; 2.92 ; 2.31 ; 2.25 ; 1.89 ; 1.82 ; 1.75 ; 1.73 ; 1.66 ; 1.125 ; 1.00 ; 0.94 . 13C-NMR : δ 144.1 (vinylic C); 126.6 ; 115.7 (vinylic C); 73.0 ; 53.2 (C2); 42.2 (C6); 34.1 (C4); 28.2 (C5); 26.3 (C3); 22.4 (C5-Me); 19.7 (vinylic Me). HRMS, ESI: Calcd. for C11H18F3O+ (M + H+): 223.13043; found: 223.13041.Compounds and H6ax . For 2a,5a/a′ were prepared according to the above-described general procedure by stirring citronellal  and diphenyl phosphate (3a)  in anhydrous dichloromethane (6.6 mL) at room temperature for 22 h to provide a colorless oil in a 61% yield (products were purified by flash-chromatography with 10% diethyl ether in petroleum ether). The NMR spectra of isopulegol (5a) and neoisopulegol (5a′) matched those previously reported [1H-NMR of isopulegol -5-methyl-2-(1-methylethenyl)cyclohexanol, 5a) : δ 4.91 ; 4.86 ; 3.46 ; 2.04 ; 1.89 ; 1.71 ; 1.65 ; 1.48 ; 1.34 ; 0.96 ; 0.95 . 1H-NMR of neoisopulegol -5-methyl-2-(1-methylethenyl)cyclohexanol, 5a′) : δ 4.95 ; 4.78 ; 3.98 ; 1.98 ; 1.79 ; 1.73 ; 1.55 ; 1.45 ; 1.12 ; 0.95 ; 0.88 .Compounds reported . 1H-NMR 5b/b′ were prepared according to the above-described general procedure by using 4-methyl-N-(3-methylbu-2-enyl)-N-(3-oxopropyl)benzenesulfonamide  and diphenyl phosphate (3a)  in anhydrous dichloromethane (3.8 mL) at room temperature for 24 h to provide an oil in a 89% yield (products were purified by flash-chromatography with a stepwise gradient of 20%–30% ethyl acetate in hexanes). The NMR spectra of isolated piperidines matched those previously reported [1H-NMR of trans-3-isopropenyl-1-(toluene-4-sulfonyl)piperidin-4-ol, 5b, : δ 7.64 ; 7.32 ; 5.01 ; 4.89 ; 3.84 ; 3.76 ; 3.44 ; 2.44 ; 2.37 ; 2.26 ; 2.17 ; 2.04 ; 1.71 ; 1.64 . 1H-NMR of cis-3-isopropenyl-1-(toluene-4-sulfonyl)piperidin-4-ol, 5b′ : δ 7.66 ; 7.32 ; 4.99 ; 4.59 ; 3.97 ; 3.59 ; 2.60 ; 2.57 ; 2.42 ; 2.37 ; 1.96 ; 1.87 ; 1.77 .Compounds reported . 1H-NMR 5c/c′ were prepared according to the above-described general procedure by using 2-(4-methyl-3-pentenyl)benzaldehyde  and diphenyl phosphate (3a)  in anhydrous dichloromethane (7.3 mL) at room temperature for 24 h to provide a mixture of diastereomers in a 44% yield (products were purified by flash-chromatography with a stepwise gradient of 0%–1% diethyl ether in petroleum ether). The NMR spectra of alcohol products (5c and 5c′) [1H-NMR of trans-2-isoprenyl-1,2,3,4-tetrahydro-1-naphthalenol, 5c, : δ 7.61 ; 7.23 ; 7.19 ; 7.09 ; 4.96 ; 4.89 ; 4.70 ; 2.89 ; 2.82 ; 2.39 ; 2.07 ; 1.93 ; 1.83 ; 1.81 . 1H-NMR of cis-2-isoprenyl-1,2,3,4-tetrahydro-1-naphthalenol, 5c′ : δ 7.38 ; 7.23 ; 7.22 ; 7.16 ; 5.08 ; 4.91 ; 4.76 ; 2.95 ; 2.81 ; 2.40 ; 2.06 ; 1.91 ; 1.79 . 1H-NMR of 3-isopropenyl-1,2-dihydronaphthalene : δ 7.11 ; 6.57 ; 5.22 ; 5.04 ; 2.85 ; 2.53 ; 2.05 .Compounds and 5c′) , and theand 5c′)  matched 5d/d′ were prepared according to the above-described general procedure by using 2, 6-dimethyl-5-heptenal  and diphenyl phosphate (3a)  in anhydrous dichloromethane (20 mL) at room temperature for 19 h to provide a mixture of diastereomers in a 37% yield (products were purified by flash-chromatography with a stepwise gradient of 5–20% diethyl ether in petroleum ether). 1H-NMR of 5d (major isomer), : δ 4.81 ; 3.4 ; 2.38 ; 1.78–1.95 ; 1.74 ; 1.73 ; 1.5 ; 1.15 ; 1.08 . 13C-NMR (100 MHz): δ 146.3, 110.8, 81.8, 55.0, 41.0, 29.3, 26.0, 19.7, 18.1. The NMR spectra of 5d/d′ match those previously reported for an analogous compound [5d′ overlapped with the major isomer (5d) except for H1, which appeared at 3.93 ppm, and the β-methyl group at 1.00 ppm.Compounds 2a/a′ and 5d/d′ can be found in the NMR spectra for products trans–configured carbocyclic and heterocyclic 5- and 6-membered rings in moderate-to-good yield. In addition, these reactions are complete within 7–24 h. Further exploration of the scope of the reaction as well as screening enantioselective catalysts are ongoing in our laboratory.In summary, we have reported organocatalyzed intramolecular carbonyl-ene cyclizations of a citronellal-derived trifluoroketone and several aldehydes. The scope of this reaction is more general than previous reports and produces various


N-bromosuccinimide (NBS) with (carboethoxymethylene) triphenylphosphorane (1) in CH2Cl2 followed by the addition of an alcohol in the presence of manganese dioxide under ultrasonic irradiation constitutes a stereoselective one-pot procedure for the preparation of Z-configured α–bromo-α,β-unsaturated esters in good to excellent yield.Treatment of  This method avoids the most common problems associated with the handling of intermediate aldehydes, which are often difficult to isolate due to their volatility, toxicity, penchant to polymerize or facile hydration  (54), 175 (91), 174 (100), 102 (97).oate (3a): Yellow -Ethyl 2-bromo-5-phenylpenta-2,4-dienoate (3b)  (27), 155 (47), 129 (100), 102 (25).ate (3b) : Yellow (Z)-Ethyl 3-(furan-2-yl)-2-bromo-3-propenoate (3c)  (38), 165 (58), 137 (100), 92 (30).ate (3c) : Yellow -Ethyl 2-bromohepta-2,4-dienoate (3d): Yellow oil. 1H-NMR: δ = 1.04 ; 1.31; 2.22 ; 4.25 ; 6.32-6.44 ; 7.63 ; IR  3055, 1726, 1265, 705; MS (EI), m/z (%) = [M+ cannot be detected], 208 (41), 206 (36),106 (39), 104 (39).(2Z)-Ethyl 2-bromo-5-phenylpent-2-en-4-ynoate (3e): Yellow oil. 1H-NMR: δ = 1.34 ; 4.33 ; 7.29-7.58 ; 7.50 ; IR  2980, 1718, 1265, 738; MS (EI), m/z (%) = 280 (42), 278 [M+] (42), 199 (73), 171 (100), 106 (39), 126 (97).(Z)-Ethyl 2-bromohept-2-en-4-ynoate (3f): Yellow oil. 1H-NMR: δ = 1.21 ; 1.30 ; 2.44 , 4.27 ; IR  2998, 1718, 1265, 740; MS (EI), m/z (%) = 232 (49), 230 [M+] (51), 186 (40), 151 (96),77 (100).(Z)-Ethyl 2-bromonon-2-enoate (3g) [1H-NMR: δ = 1.24 ; 1.27-1.32 , 2.33 , 4.27 ; 7.27 ; IR  2926, 1732, 1259, 1041, 802.ate (3g) : Yellow 3. These results demonstrate the value of ultrasonic- assisted chemistry.In summary, we have developed an efficient ultrasonic-assisted one-pot general procedure for a domino primary alcohol oxidation-Wittig reaction for the highly stereoselective synthesis of Z-α-bromo-α,β-unsaturated esters