Pseudogymnoascus is known as a psychrophilic pathogenic fungus which is ubiquitously distributed in Antarctica. While the studies of its secondary metabolites are infrequent. Systematic research of the metabolites of the Antarctic fungus Pseudogymnoascus sp. HSX2#-11 led to the isolation of one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester (1), together with one pyrimidine, thymine (2), and eight diketopiperazines, cyclo- (3), cyclo-(dehydroAla-l-Ile) (4), cyclo-(dehydroAla-l-Leu) (5), cyclo-(dehydroAla-l-Phe) (6), cyclo- (7), cyclo-(l-Leu-l-Phe) (8), cyclo-(l-Trp-l-Ile) (9) and cyclo-(l-Trp-l-Phe) (10). The structures of these compounds were established by extensive spectroscopic investigation, as well as by detailed comparison with literature data. This is the first report to discover pyridine, pyrimidine and diketopiperazines from the genus of Pseudogymnoascus.The species  Peganum harmala exhibited strong antiviral activity against herpes simplex virus-2 + peak at m/z 246.0742 , indicating six degrees of unsaturation  spectra , δC 52.0; δH 3.98 , δC 53.1), two methylenes, , δC 34.0; δH 3.03 , δC 30.1), three aromatic methines, , δC 127.4; δH 8.00 , δC 125.4; δH 8.66 , δC 149.6), two aromatic quaternary carbon signals , and two carbonyls  (11H13O4N indicated a pyridine substructure in 1. The splitting effects of 1H-NMR were undesirable in CDCl3 of the aromatic methines of 1. So the 1H-NMR spectrum was measured again in DMSO-d6. The coupling constants of 5.0 Hz (H-5/H-6), 1.7 Hz (H-3/H-5) and 0.8 Hz (H-3/H-6) revealed the ortho-position of H-5/H-6, meta-position of H-3/H-5 and para-position of H-3/H-6, respectively  cross peak of H-9/H-10, and key heteronuclear multiple-bond correlation (HMBC) correlations of H-9/C-3, H-9/C-5, H-10/C-4, H-10/C-11 and H-12/C-11 elucidated the methyl propionate substituent located at C-4 (1 was identified as 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester.4-(2-Methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester (turation . The 1H- spectra  exhibiteC 172.4) . The fivectively . The 1H-d at C-4 . The metd at C-4 . Thus coCyclo-(dehydroAla-l-Ile) (4) was obtained as a white powder. The HRESIMS spectrum of 4 led to the molecular formula of C9H14O2N2 , indicating 4 degrees of unsaturation , δC 11.7; δH 0.88 , δC 14.8), two methylenes, , 1.41–1.33, , δC 24.1; δH 4.76, , 5.17, , δC 98.8), two methines, , δC 40.7; δH 3.91 , δC 59.6), one aromatic quaternary carbon signals (δC 134.7), and two carbonyls  , δH 10.53, ) combined with two carbonyls indicated the diketopiperazine structure of 4. The 1H-1H COSY correlations of H-5/H-7, H-7/H-10, H-7/H-8 and H-8/H-9 revealed the substitute group of 1-methyl-propyl. And the group was located at C-5 determined by the 1H-1H COSY cross peak of H-5 and N-4, and the HMBC correlation from H-10 to C-5. The dehydro-methyl substitute was located at C-2 by the HMBC signal from H-11 to C-2. Thus the planar structure of 4 was confirmed as cyclo-(dehydroAla-Ile) which was first isolated from marine bacteria Claviceps purpurea in 1994, while its detailed NMR data were not reported + .4-(2-Methoxycarbonylethyl)-pyridine-2-carboxylic acid methyl ester (Cyclo-(dehydroAla-l-Ile) (4): white powder; + .Escherichia coli, Staphylococcus aureus, P. aeruginosa and Bacillus subtilis, and nine marine fouling bacteria, P. fulva, Aeromonas hydrophila, A. salmonicida, Vibrio anguillarum, V. harveyi, Photobacterium halotolerans, P. angustum, Enterobacter cloacae and E. hormaechei, were used, and cipofloxacin was used as a positive control.The antibacterial activities were evaluated by the conventional broth dilution assay . Four paThe cytotoxicities against human breast cancer , colorectal cancer , lung carcinoma , pancreatic carcinoma  and hepatoma  cell lines were evaluated using the sulphorodamine B (SRB) method . Adriamy1), together with one pyrimidine, thymine (2), and eight diketopiperazines 3–10, were isolated from the Antarctic fungus Pseudogymnoascus sp. HSX2#-11. All the isolated compounds showed no antibacterial or cytotoxic activities. More bioactivity evaluating models should be needed to find the effects of these secondary metabolites. This is the first time to find pyridine, pyrimidine and diketopiperazines from the genus of Pseudogymnoascus. Our chemical investigation of the Antarctic fungus Pseudogymnoascus sp. HSX2#-11 enriches the chemical diversity of this fungal species.In summary, one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester (


N-benzyl-C-(diethoxyphosphoryl)nitrone to cis-1,4-dihydroxybut-2-ene and dimethyl maleate, respectively. O,O-Diethyl 3-carbamoyl-4-hydroxy(5-oxopyrrolidin-2-yl)phosphonate was obtained from O,O-diethyl 2-benzyl-4,5-dimethoxycarbonyl(isoxazolidin-3-yl)phosphonate by hydrogenation and subsequent treatment with ammonia, whereas transformation of O,O-diethyl 2-benzyl-4,5-dihydroxymethyl(isoxazolidin-3-yl)phosphonate into O,O-diethyl 3-aminomethyl-4-hydroxy(pyrrolidin-2-yl)phosphonate was accomplished by mesylation followed by hydrogenolysis to undergo intramolecular cyclization and the introduction of amino group via ammonolysis. Stereochemistry of the isoxazolidine cycloadducts, as well as the final functionalized (pyrrolidin-2-yl)- and (5-oxopyrrolidin-2-yl)phosphonates were established based on conformational analyses using vicinal H–H, H–P, and C–P couplings and supported by the observed diagnostic NOESY correlation signals.Short and efficient syntheses of functionalized (pyrrolidin-2-yl)phosphonate and (5-oxopyrrolidin-2-yl)phosphonate have been developed. The synthetic strategy involved the diastereospecific 1,3-dipolar cycloaddition of  Colorless oil. IR : νmax= 3312, 2980, 2933, 1743, 1703, 1519, 1279, 1254, 1165, 1028. 1H NMR : δ = 5.40–5.00 , 4.80–4.72 , 4.25–4.15 , 4.10–4.00 , 3.48–3.40 , 3.28–3.20 , 2.85–2.70 , 1.50 3C), 1.48 3C), 1.46 3C), 1.35 . 13C NMR : δ = 165.12 (C=O), 153.75 (C=O), 152.98 (C=O), 82.86 (C(CH3)3), 80.78 (C(CH3)3), 79.37 (C(CH3)3), 76.01  and 75.13 , 62.83 , 62.53 , 55.76  and 54.75 , 50.47  and 50.11 , 47.00  and 45.45 , 42.46  and 41.93 , 28.37, 28.26, 27.71, 16.48 , 16.43 . 31P NMR : δ = 24.06. Analysis Calculated for C24H45N2O10P: C, 52.16; H, 8.21; N, 5.07. Found: C, 52.32; H, 8.14; N, 5.02.N-benzyl-C-(diethoxyphosphoryl)nitrone 22 with dimethyl maleate and cis-1,4-dihydroxybut-2-ene, as well as maleic anhydride proceeded diastereospecifically to give cycloadducts 20, 21 and 23, respectively. Isoxazolidine 20 was smoothly hydrogenated to substituted (5-oxopyrrolidin-2-yl)phosphonate 18 and subsequently transformed into derivative 24 by exchanging of COOMe at C3 into amido function. For transformation of isoxazolidine 21 into functionalized derivative of (pyrrolidin-2-yl)phosphonate 28, reaction sequence consisted of a standard mesylation of both hydroxy groups, a hydrogenolytic cleavage of the N–O bond, removal of benzyl group followed by spontaneous formation of the pyrrolidine ring by intramolecular SN2 reaction and finally exchanging the other mesyloxy group to amino function. Since 3-methoxycarbonyl-(5-oxopyrrolidin-2-yl)phosphonate 18 and 3-mesyloxymethyl(pyrrolidin-2-yl)phosphonate 27 contain reactive groups, i.e., COOMe at C3 in 18 and MsO at C3 in 27, studies on their further functionalization based on their reactions with other nucleophiles are underway in our laboratory. The presented methodology could also be adopted for the synthesis of other stereoisomeric isoxazolidines via application of respective trans-alkenes in 1,3-dipolar cycloaddition to C-phosphorylated nitron 22. Moreover, the syntheses elaborated herein pave the way for new enantiomerically pure functionalized phosphonate analogues of prolines, substituted glutamic acid by application of the N-chiral C-phosphorylated nitrones.The 1,3-dipolar cycloadditions of


N-tert-butyl substituted 2-aminothiazol-4(5H)-one (pseudothiohydantoin) derivatives, differing in the substituents at C-5 of the thiazole ring. The inhibitory activity and selectivity of the obtained derivatives in relation to two isoforms of 11β-HSD were evaluated. The highest inhibitory activity for 11β-HSD1 showed compound 3h, containing the cyclohexane substituent at the 5-position of the thiazole ring in the spiro system (82.5% at a conc. 10 µM). On the other hand, the derivative 3f with the phenyl substituent at C-5 showed the highest inhibition of 11β-HSD2 (53.57% at a conc. of 10 µM). A low selectivity in the inhibition of 11β-HSD2 was observed but, unlike 18β-glycyrrhetinic acid, these compounds were found to inhibit the activity of 11β-HSD2 to a greater extent than 11β-HSD1, which makes them attractive for further research on their anti-cancer activity. Tumors are currently more and more common all over the world; hence, attempts are being made to explain the biochemical processes underlying their development. The search for new therapeutic pathways, with particular emphasis on enzymatic activity and its modulation regulating the level of glucocorticosteroids, may contribute to the development and implementation of new therapeutic options in the treatment process. Our research focuses on understanding the role of 11β-HSD1 and 11β-HSD2 as factors involved in the differentiation and proliferation of neoplastic cells. In this work, we obtained the 9 novel  Cancer is an important public health threat, becoming an increasingly widespread disease worldwide. Despite years of research, the biochemical processes underlying the phenomenon of neoplasms are still not fully understood . ParticuType 1 11β-hydroxysteroid dehydrogenase (11β-HSD1) and type 2 (11β-HSD2) play an important role in the local inactivation of glucocorticosteroids. In addition, they catalyze the interconversion of active glucorticoids to their inactive counterparts, such as the conversion of cortisol to cortisone by 11β-HSD2 and a reverse reaction catalyzed by 11β-HSD1 leading to the formation of active cortisol .The immune system plays an extremely important role during the disease process, including the formation of cancer cells, for example, macrophages, show the promoting effects on tumors through many different mechanisms, including promotion of tumor cell invasion and metastasis formation, stimulation of angiogenesis, and immunosuppressive effects . The stuGlucocorticoids are known for their antiproliferative function. Hence, the regulation of their secretion is crucial for the maintenance of normal cellular processes within the human body. An imbalance in their secretion may promote the formation of cancer cells or tumors and their spread in the process of metastasis . In viewA known substance that inhibits the activity of 11β-HSD2 is glycyrrhetinic acid . However, it is a non-selective inhibitor as it also significantly inhibits the enzyme isoform 1 activity ,16. An eH)-one derivatives have been shown to be selective inhibitors of 11β-HSD1. During these studies, we became interested in those compounds that did not inhibit the activity of isoform 1, but were selective 11β-HSD2 inhibitors  . Rf : 0.33.2-(tert-Butylamino)-5-phenylthiazol-4(5H)-one (3f)–Yield: 79%. M.p. 222–227 °C. 1H-NMR : 11.90 , 7.41–7.44 , 7.35-7.37 , 5.58 , 1.60 3). 13C-NMR : 171.16 (C-4), 169.30 (C-2), 130.95 , 129.71 , 129.32 , 128.34 , 58.01 (C(CH3)3), 53.63 (C-5), 27.91 (C(CH3)3). HR-MS m/z 249.1071 [M+ + 1] . Rf : 0.45.2-(tert-butylamino)thiazol-4(5H)-one (3g)–Yield: 78%. M.p. 206–207 °C. 1H-NMR : 7.56A , 7.45C , 7.23A , 7.20C , 6.10A , 5.50C , 5.47A , 5.16C , 1.59A 3), 1.52C 3). 13C-NMR : 187.72 (C-4), 177.28 (C-2), 134.91A , 132.42A , 131.62C , 130.59C , 129.86A , 129.64C , 123.86C , 121.89A , 58.31A (C(CH3)3), 57.44C (C(CH3)3), 56.42C (C-5), 53.80A (C-5), 28.41C (C(CH3)3), 28.10A (C(CH3)3). HR-MS m/z 327.0174 [M+ + 1] . Rf : 0.47.5-(4-Bromophenyl)-2-(N-(tert-butyl)thiourea (1) and 0.011 mol of the corresponding 2-bromoester (2b–2e) were added. The mixture was heated for 10-18 h (TLC control-AcOEt:hexane 1:1). The solvent was evaporated on a rotary evaporator and the residue was dissolved in 20 mL of water and neutralized with 2M HCl to pH = 7–8. The crude product was extracted with chloroform. The organic layer was dried with MgSO4, filtered and after solvent evaporated crystallized from diethyl ether  . Rf : 0.40.2-(tert-Butylamino)-5-propylthiazol-4(5H)-one (3c)–Yield: 37%. M.p. 131–132 °C. 1H-NMR : 5.66 , 4.17 , 2.15–2.19 , 1.75-1.79 , 1.48 3), 1.39–1.47 , 0.95 . 13C-NMR : 190.61 (C-4), 177.84 (C-2), 56.40 (C(CH3)3), 55.97 (C-5), 35.19 (C5-CH2CH2CH3), 28.43 (C(CH3)3), 21.16 (C5-CH2CH2CH3), 13.19 (C5-CH2CH2CH3). HR-MS m/z 215.1219 [M+ + 1] . Rf : 0.48.2-(tert-Butylamino)-5-(propan-2-yl)thiazol-4(5H)-one (3d)–Yield: 27%. M.p. 175–177 °C. 1H-NMR : 5.83 , 4.25 , 2.55–2.60 , 1.47 3), 1.03 , 0.99 . 13C-NMR : 190.28 (C-4), 178.29 (C-2), 63.65 (C(CH3)3), 55.79 (C-5), 30.30 (C5-CH), 28.35 (C(CH3)3), 22.09 (CH(CH3A), 15.74 (CH(CH3B). HR-MS m/z 215.1222 [M+ + 1] . Rf : 0.46. 2-(tert-Butylamino)-5,5-dimethylthiazol-4(5H)-one (3e)–Yield: 40%. M.p. 212–217 °C. 1H-NMR : 5.61 , 1.64 2), 1.49 3). 13C-NMR : 193.38 (C-4), 176.12 (C-2), 60.65 (C(CH3)3), 55.79 (C-5), 28.46 (C(CH3)3), 27.59 2). HR-MS m/z 201.1064 [M+ + 1] . Rf : 0.44.2-thiourea (1) and 0.01 mol of the 2-bromoester (2h-2i) were dissolved in 3.5 mL of ethyl alcohol. It was heated to reflux for 7-14 days (TLC control-AcOEt:hexane 1:1). After the solvent was evaporated, the residue was dissolved in 10 mL of water. The product was extracted with chloroform. The organic layer was dried with anhydrous MgSO4. After filtered off and evaporated of the solvent the reaction mixture was purified by column chromatography [2 mL of tography .tert-Butylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one (3h)–Yield: 12%. M.p. 218–220 °C. 1H-NMR : 5.57 , 2.08–2.12 , 1.90–1.93 , 1.82–1.87 , 1.72–1.74 , 1.49 3), 1.27–1.40 . 13C-NMR : 192.91 (C-4), 177.13 (C-2), 70.21 (C-5), 55.77 (C(CH3)3, 36.34 , 28.84 (C(CH3)3), 25.07 , 24.47 . HR-MS m/z 241.1376 [M+ + 1] . Rf : 0.5.2-(tert-Butylamino)-5-thia-7-azaspiro[3.4]oct-6-en-8-one (3i)–Yield: 16%. M.p. 211–213 °C. 1H-NMR : 5.78 , 2.78–2.86 , 2.46–2.50 , 2.26–2.30 , 1.98–2.04 , 1.47 3). 13C-NMR : 193.46 (C-8), 176.50 (C-6), 62.01 (C-4), 56.17 (C(CH3)3), 34.28 , 28.87 (C(CH3)3), 16.93 . HR-MS m/z 213.1062 [M+ + 1] . Rf : 0.5.6-, 10 μL of microsomes (1.13 μg/mL 11β-HSD1) in PBS , 60 μL of phosphate buffer (pH 7.4) and 10 µL of inhibitor solution  was placed in the well. The resulting solution was incubated for 150 min at 37 °C. The addition of 10 µL of a 100 µM solution of 18β-glycyrrhetinic acid in PBS stopped the reaction [β-HSD2 in a total volume of 100 μL. Then, 20 μL of substrate mixture cortisol/NAD+ , 10 μL of microsomes (0.127 μg/mL 11β-HSD2) solution in PBS , 60 μL of phosphate buffer (pH 7.4) and 10 μL of inhibitor solution  were mixed in the well. The resulting solutions were incubated for 150 min at 37 °C. The addition of 10 μL solution containing 100 μM carbenoxolone in PBS stopped the reaction [Transformation of cortisol to cortisone was performed on 96-well microtiter plates using human kidney microsomes as a source of 1150 values were obtained by performing similar assay as described in ICN-tert-butyl substituted 2-aminothiazol-4(5H)-one (pseudothiohydantoin) derivatives, differing in the substituents at C-5 of the thiazole ring. The inhibitory activity and selectivity of the obtained derivatives in relation to two isoforms of 11β-hydroxysteroid dehydrogenase (type 1 and type 2) were evaluated. The 3h derivative, containing the cyclohexane substituent at the 5-position of the thiazole ring in the spiro system which inhibits the enzyme isoform 1 activity by 82.5% at a concentration of 10 µM, has the highest inhibitory activity in relation to 11β-HSD1. The derivative with the phenyl substituent at C-5 showed the highest inhibition of 11β-HSD2 (53.57% at a concentration of 10 µM). A low selectivity in the inhibition of 11β-HSD2 was observed, but unlike 18β-glycyrrhetinic acid these compounds inhibit the activity of 11β-HSD2 to a greater extent than that of 11β-HSD1.In conclusion, we obtained the nine novel The obtained results may constitute an innovative approach in cancer therapy, aimed especially at the action of 11βHSD1 and 11βHSD2. It may have a significant impact on the correct response and action of glucocorticoids, which in turn may be of key importance in inhibiting the proliferation of neoplastic cells.


CUGBMF180003. Their chemical structures were determined using spectroscopic data, including HRESIMS and 1D and 2D NMR techniques. Furthermore, the stereogenic carbons in 5 and 14 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. The carbon skeleton of 12–14 was identified as the first example of isocoumarin coupled with phenylethylamine derivatives. All of these compounds were examined for antimicrobial activities against Candida albicans and Staphylococcus aureus. Both 1 and 6 showed antibacterial activity against S. aureus with MIC values of 12.5 μg/mL.Nine new secondary metabolites, including six isocoumarin analogues, 7-hydroxyoospolactone ( Paraphoma fungi are commonly isolated from soil samples or plants and recognized as soil-borne pathogens , two methyl singlets at δH 2.24  and 2.06 , and two phenolic hydroxyl groups at δH 9.69  and 11.12 . The 13C and HSQC spectra of 1 (δC 166.2 (C-1), three oxygenated sp2 carbons at δC 146.7 (C-3), 143.8 (C-7), and 148.6 (C-8), two nonprotonated sp2 carbons at δC 129.6 (C-4a) and 105.9 (C-8a), and two protonated sp2 carbons at δC 113.3 (C-5) and 124.2 (C-6). Detailed analysis of the 2D NMR data , accounting for seven degrees of unsaturation  in 2. The HMBC spectrum , accounting for seven degrees of unsaturation . Detailed analysis of 13C and 2D NMR data , accounting for eight degrees of unsaturation , 7.70 , and 7.06 , two hydroxymethyl protons at δH 4.82 , and two oxymethylene protons at δH 5.09 , as well as one methyl singlet at δH 2.12 . The 13C and HSQC spectra of 4 (δC 57.1 (C-9) and 60.1 (C-10), as well as two signals for acetyl groups at δC 171.8 (C-11) and 21.0 (C-12). The presence of the hydroxymethyl group on C-3 was confirmed by the HMBC correlations , accounting for seven degrees of unsaturation , 7.66 , and 7.02 , one olefinic proton signal at 7.37 , one methyl doublet at δH 1.60 , and one oxygenated sp3 methine at δH 5.06 . The 13C and HSQC spectra (δC 166.4 (C-1), four protonated sp2 at δC 141.2 (C-3), 113.4 (C-5), 137.4 (C-6), 116.0 (C-7), four nonprotonated sp2 carbon signals at δC 122.1 (C-4), 135.7 (C-4a), 162.4 , and 106.8 (C-8a), as well as one signal for oxygenated methine sp3 at δC 65.0 (C-9) and one methyl carbon at δC 23.3 (C-10). Detailed analysis of the 2D NMR data , accounting for nine degrees of unsaturation , 7.69 , and 7.04 , two methylene sp3 protons at δH 3.06  and 2.76 , and one oxygenated methine sp3 proton at δH 5.83 . The 13C NMR and HSQC spectra (δC 196.4 (C-11), one carboxyl at δC 163.2 (C-1), three protonated sp2 at δC 118.9 (C-5), 137.5 (C-6), 119.0 (C-7), three nonprotonated sp2 at δC 127.3 (C-4), 133.9 (C-4a), and 107.8 (C-8a), two oxygenated sp2 at δC 151.1 (C-3) and 163.2 (C-8), and one methylene sp3 at δC 39.9 (C-10) and one oxymethine sp3 at δC 75.3 (C-9). Detailed analysis of the 2D NMR data of 6  and did not display ECD absorptions; therefore, 6 was elucidated as a racemic mixture. Therefore, the structure of 6 was defined as shown in Compound turation . The 1H ata of 6  displaye spectra  revealedata of 6  revealed12 was isolated as a light yellow powder. The molecular formula of 12 was determined to be C19H15NO4 based on the HRESIMS spectrum , accounting for thirteen degrees of unsaturation , δC 45.6 (C-9) and one conjugated amide carbonyl at δC 160.9 (C-10). Detailed analysis of 2D NMR data (19H15NO4) of 12 and downfield shift of C-9 (δC 45.6), combined with the HMBC correlations from H-9 to C-3, C-4, C-10, and C-1′, and from H-1′ to C-9 and C-10 confirmed the connection of C-1′, C-9, and C-10 to N, forming a cyclopentenamide ring. Therefore, the structure of 12 was elucidated as shown in Compound turation . The 1H ta of 12  displayeNMR data  confirme13 was isolated as a light yellow powder. The molecular formula of 13 was determined to be C19H15NO5 based on the HRESIMS spectrum , accounting for thirteen degrees of unsaturation  and molecular formula. Thus, the structure of 13 was elucidated as shown in Compound turation . The 1H ra of 13  resembleNMR data  revealed14 was isolated as a light yellow powder. The molecular formula of 14 was determined to be C21H17NO6 based on the HRESIMS spectrum , accounting for fourteen degrees of unsaturation , δH 3.76 , and δC 52.9 (C-2″). This moiety was confirmed by HMBC correlations from H-1′, H-2′, and H-2″ to C-1″. The absolute configuration of C-1′ was also determined by comparison of experimental and calculated ECD spectra  + .7-Hydroxyoospolactone (2): Light yellow powder; 1H and 13C NMR data, m/z 221.0811 [M + H]+ .7-Methoxyoospolactone (3): Light yellow powder; 1H and 13C NMR data, m/z 241.0705 [M + H]+ .7-Methoxy-9-hydroxyoospolactone (4): Light yellow powder; 1H and 13C NMR data, m/z 265.0708 [M + H]+ .10-Acetoxy-9-hydroxyoospolactone (5): Light yellow powder; c 0.20, MeOH); 1H and 13C NMR data, m/z 207.0648 [M + H]+ .6-Dehydroxysescandelin (6): Light yellow powder; c 0.11, MeOH); 1H and 13C NMR data, m/z 233.0443 [M + H]+ .Parapholactone (12): Light yellow powder; 1H and 13C NMR data, m/z 322.1069 [M + H]+ .Paraphamide A (13): Light yellow powder; 1H and 13C NMR data, m/z 338.1022 [M + H]+ .Paraphamide B (14): Light yellow powder; c 0.06, MeOH); 1H and 13C NMR data, m/z 380.1130 [M + H]+ .Paraphamide C  [C. albicans ATCC 10231 was inoculated on potato dextrose agar plate and cultured for 24 hours at 35 °C. Five colonies of about 1 mm in diameter were picked and suspended in 5 mL of physiological saline. The suspension was then adjusted to approximately 106 CFU/mL with RPMI 1640. For the antibacterial assay, S. aureus ATCC 25923 was inoculated on a Mueller–Hinton broth agar plate and cultured for 24 hours at 37 °C. Five colonies of about 1 mm in diameter were then picked and suspended in 5 mL of physiological saline. The suspension was then adjusted to approximately 106 CFU/mL with Mueller–Hinton broth and 2 μL of 2-fold serial dilution of each compound (in DMSO) was added to each row in the 96-well microplate, which contained 78 μL of microbe suspension in each well. Amphotericin B and vancomycin were used as positive controls for fungi and bacteria, respectively; DMSO was used as negative control. The 96-well plates were incubated at 35 °C aerobically for 24 hours. The MIC was defined as the minimum concentration of the compound that prevented visible growth of the microbes.Compounds 7 (CLSI) . Briefly1), 7-methoxyoospolactone (2), 7-methoxy-9-hydroxyoospolactone (3), 10-acetoxy-9-hydroxyoospolactone (4), 6-dehydroxysescandelin (5), and parapholactone (6), three compounds with a rare skeleton of isocoumarin coupled with phenylethylamine, namely paraphamide A (12), paraphamide B (13), and paraphamide C (14), together with five known compounds, oospolactone (7), 8-O-methoxyoospolactone (8), 10-hydroxyoospolactone (9), 9,10-dihydroxyoospolactone (10), and oospoglycol (11), were isolated from the marine-derived fungus Paraphoma sp. CUGBMF180003. The carbon skeleton of 12–14 was identified as the first example of a coupled structure of isocoumarin and phenylethylamine. Natural isocoumarin analogues display a variety of bioactivities, including inhibitory activity against α-glucosidase [1 and 6 showed inhibitory activity against S. aureus with MIC values of 12.5 μg/mL, but were devoid of growth-inhibitory activity against C. albicans activity at a concentration of 200 μg/mL.In summary, nine new secondary metabolites, including six oospolactone analogues, 7-hydroxyoospolactone (cosidase , cytotoxcosidase ,18, anticosidase  and anticosidase ,22,23,24cosidase . The new


H-benzo[f]chromene-2-carbonitriles (4a–4q) were designed and synthesized via reaction of 6-methoxy-2-naphthol with a mixture of appropriate aromatic aldehydes and malononitrile under microwave conditions. The structures of the novel compounds 4b, 4c, 4f, 4g, 4i, 4l, 4m, and 4o–4q were established according to IR, 1H-NMR, 13C-NMR/13C-NMR-DEPT, and MS. The benzochromene derivative 4c with a single chlorine at the meta position of the phenyl ring and, to a lesser extent, other benzochromenes with monohalogenated phenyl ring  exhibited the highest cytotoxicity against six human cancer cell lines MDA-MB-231, A549, HeLa, MIA PaCa-2, 5,637, and Hep G2. The mechanisms of the cytotoxic activities of benzochromenes with monohalogenated phenyl ring  were further analyzed using triple-negative breast cancer cell line MDA-MB-231. Cell cycle analysis showed accumulation of the treated cells in S phase for 4a, 4d–4f, and S-G2/M phases for 4c. In vivo, 4a and 4c–4f inhibited growth, proliferation, and triggered apoptosis in preestablished breast cancer xenografts grown on the chick chorioallantoic membranes while exhibiting low systemic toxicity. Compounds 4a and 4c–4f increased levels of mitochondrial superoxide and decreased mitochondrial membrane potential resulting in initiation of apoptosis as demonstrated by caspase 3/7 activation. In addition, 4c induced general oxidative stress in cancer cells. The SAR study confirmed that halogens of moderate size at meta or para positions of the pendant phenyl ring enhance the cytotoxic activity of 3-amino-1-aryl-8-methoxy-1H-benzo[f]chromene-2-carbonitriles, and these compounds could serve as leads for the development of novel anticancer therapies.A series of aryl-substituted 3-amino-1-aryl-8-methoxy-1 Cancer is still one of the leading cause of death and among the major public health concerns . In 2020Chromene and benzochromene scaffolds are well known as important components of biologically active synthetic or natural compounds. Indeed, these compounds display a wide variety of biological activities . MoreoveH-chromene-3-carbonitrile, A] has progressed to the NCI phase I/II clinical studies for the treatment of patients with advanced solid tumors  and related compounds act as tubulin destabilizers -4d tumors . Crolibud tumors . MX-5815bilizers , and HA1poptosis  -4H-benzo[h]chromene-3-carbonitrile (LY290181) (D) inhibited the proliferation of mammalian cells at prometaphase by an apparent action on microtubules (H-benzo[h]chromene-3-carbonitrile (E) exhibited cytotoxic and proapoptotic effects on a variety of human cancer cell lines  (H-benzo[h]chromene-3-carbonitrile (G), ethyl 4-aryl-2,7-diamino-4H-benzo[h]chromene-3-carboxylate  , in addiboxylate , and theboxylate .H-benzo[f]chromene derivatives are regarded as promising lead candidates for anticancer drug development. For example, some derivatives of 3-amino-1-aryl-1H-benzo[f]chromene-2-carbonitrile including 3-amino-8-bromo-1-(4-chlorophenyl)-1H-benzo[f]chromene-2-carbonitrile (H) exhibited c-Src kinase inhibitory and proapoptotic activities (H-benzo[f]chromene-3-amines (I), some derivatives of 3,5-diamino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile including 3,5-diamino-1-(p-tolyl)-1H-benzo[f]chromene-2-carbonitrile (J), and 3-amino-1-(4-chloro/-bromophenyl)-1H-benzo[f]chromene-2-carbonitriles (K) have been reported to exhibit cytotoxic and apoptotic effects against a variety of human cancer cell lines (H-benzo[f]-chromene-2-carbonitrile (L) have been shown to induce cell cycle arrest and apoptosis in human cancer cells via dual inhibition of topoisomerases and tubulin , which exhibited the most pronounced cytotoxicity against the human cancer cell lines tested, were analyzed in vitro and in vivo.Considering low systemic toxicity and diverse biological activities of benzochromene derivatives, we have used the 11H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on BRUKER AV 500 MHz spectrometer  in DMSO-d6 as a solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts were expressed as δ (ppm). The microwave apparatus used is Milestone Sr1, Microsynth . The mass spectra were measured using Shimadzu GC/MS-QP5050A spectrometer . Elemental analysis was carried out at the Regional Centre for Mycology and Biotechnology , and the results were within ± 0.25%. Reaction courses, product mixtures, and purity of compounds were monitored by thin layer chromatography (TLC) performed on Merck precoated silica gel 60 F254 aluminum sheets.All chemicals were purchased from Sigma-Aldrich . All melting points were determined on a Stuart™ digital melting point apparatus  and are uncorrected. The optical rotation was measured using Carl Zeiss polarimeter . The IR spectra were recorded on a Jasco FT/IR 460 plus spectrophotometer using KBr discs. The A reaction mixture of 6-methoxy-2-naphthol (1) (0.01 mol), different aromatic aldehydes (2a-2q) (0.01 mol), malononitrile (3) (0.01 mol), and piperidine (0.5 ml) in ethanol (25 ml) was heated under microwave irradiation conditions at 140°C for 2 min. After completion of the reaction, the reaction mixture was cooled to room temperature, and the precipitated solid was filtered off, washed with methanol, and recrystallized from suitable solvent. The physical and spectral data of compounds 4a–4q are as follows:H-benzo[f]chromene-2-carbonitrile (4a). Compound 4a was prepared according to the literature procedure (3-Amino-1-(4-fluorophenyl)-8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4b). Colorless needles from ethanol; yield 82%; m.p. 265–266°C; IR (KBr) υ (cm−1): 3,407, 3,330, 3,210 (NH2), 2,200 (CN); 1H-NMR δ: 7.87–6.99 , 7.00 , 5.67 , 3.82 ; 13C-NMR δ: 159.96 (C-3), 156.49 (C-8), 145.64 (C-4a), 130.97 (C-6a), 129.50 (C-10a), 128.15 (C-6), 125.01 (C-10), 119.87 (C-10b), 119.35 (C-9), 117.09 (C-7), 114.92 (CN), 107.57 (C-5), 56.11 (C-2), 55.21 (CH3), 35.17 (C-1), 142.66, 132.14, 130.00, 128.58, 128.44, 124.09 (aromatic); MS m/z (%): 364 , 362  with a base peak at 251 (100); Anal. Calcd for C21H15ClN2O2 (362.81): C, 69.52; H, 4.17; N, 7.72. Found: C, 69.47; H, 4.13; N, 7.68%.3-Amino-1-(2-chlorophenyl)-8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4c). Colorless needles from ethanol; yield 86%; m.p. 215–216°C; IR (KBr) υ (cm−1): 3,468, 3,324, 3,198 (NH2), 2,197 (CN); 1H-NMR δ: 7.87–7.11 , 7.03 , 5.36 , 3.83 ; 13C-NMR δ: 159.94 (C-3), 156.52 (C-8), 148.19 (C-4a), 132.21 (C-6a), 128.48 (C-10a), 126.63 (C-6), 124.99 (C-10), 120.31 (C-10b), 119.21 (C-9), 117.11 (C-7), 115.11 (CN), 107.35 (C-5), 57.18 (C-2), 55.20 (CH3), 37.60 (C-1), 145.36, 133.18, 130.66, 126.61, 125.65 (aromatic); MS m/z (%): 364 , 362  with a base peak at 208 (100); Anal. Calcd for C21H15ClN2O2 (362.81): C, 69.52; H, 4.17; N, 7.72. Found: C, 69.57; H, 4.23; N, 7.77%.3-Amino-1-(3-chlorophenyl)-8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4d). Compound 4d was prepared according to the literature procedure (3-Amino-1-(4-chlorophenyl)-8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4e). Compound 4e was prepared according to the literature procedure (3-Amino-1-(4-bromophenyl)-8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4f). Pale yellow needles from ethanol; yield 89%; m.p. 227–228°C; IR (KBr) υ (cm−1): 3,455, 3,322, 3,190 (NH2), 2,201 (CN); 1H-NMR δ: 7.85–6.99 , 6.98 , 5.27 , 3.82 ; 13C-NMR δ: 159.80 (C-3), 156.48 (C-8), 145.31 (C-4a), 132.20 (C-6a), 128.37 (C-10a), 125.01 (C-6), 120.37 (C-10), 119.11 (C-10b), 117.08 , 115.22 (CN), 107.35 (C-5), 57.29 (C-2), 55.21 (CH3), 37.69 (C-1), 145.54, 137.43, 129.32, 92.40 (aromatic); MS m/z (%): 454 ; Anal. Calcd for C21H15IN2O2 (454.26): C, 55.52; H, 3.33; N, 6.17. Found: C, 55.65; H, 3.46; N, 6.34%.3-Amino-1-(4-iodophenyl)-8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4g). Colorless crystals from ethanol; yield 81%; m.p. 301–302°C; IR (KBr) υ (cm−1): 3,476, 3,335, 3,291 (NH2), 2,201 (CN); 1H-NMR δ: 7.83–7.05 , 7.09 , 5.67 , 3.82 ; 13C-NMR δ: 160.71 (C-3), 149.61 (C-8), 145.61 (C-4a), 131.44 (C-6a), 129.11 (C-10a), 128.39 (C-6), 123.52 (C-10), 120.18 (C-9), 120.04 (C-7), 119.92 (C-10b), 116.94 (CN), 107.57 (C-5), 55.19 (C-2), 53.58 (CH3), 28.39 (C-1), 161.40, 156.14, 129.39, 125.59, 116.64, 112.82 (aromatic); MS m/z (%): 364  with a base peak at 251 (100); Anal. Calcd for C21H14F2N2O2 (364.34): C, 69.23; H, 3.87; N, 7.69. Found: C, 69.29; H, 3.92; N, 7.73%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4h). Compound 4h was prepared according to the literature procedure -8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4i). Colorless needles from ethanol; yield 87%; m.p. 255–256°C; IR (KBr) υ (cm−1): 3,423, 3,335, 3,206 (NH2), 2,196 (CN); 1H-NMR δ: 7.88–6.96 , 7.07 , 5.74 , 3.83 ; 13C-NMR δ: 160.08 (C-3), 156.53 (C-8), 145.23 (C-4a), 131.98 (C-6a), 129.16 (C-10a), 128.79 (C-6), 124.92 (C-10), 119.77 (C-10b), 119.50 (C-9), 117.09 (C-7), 114.44 (CN), 107.65 (C-5), 55.59 (C-2), 55.21 (CH3), 36.22 (C-1), 145.63, 132.16, 129.01, 128.93, 128.64, 123.95 (aromatic); MS m/z (%): 400 , 398 , 396  with a base peak at 110 (100); Anal. Calcd for C21H14Cl2N2O2 (397.25): C, 63.49; H, 3.55; N, 7.05. Found: C, 63.54; H, 3.60; N, 7.10%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4j). Compound 4j was prepared according to the literature procedure -8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4k). Compound 4k was prepared according to the literature procedure -8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4l). Colorless crystals from ethanol; yield 85%; m.p. 314–315°C; IR (KBr) υ (cm−1): 3,466, 3,318, 3,198 (NH2), 2,193 (CN); 1H-NMR δ: 7.84–7.09 , 7.05 , 6.09 , 3.82 ; 13C-NMR δ: 160.41 (C-3), 156.22 (C-8), 146.39 (C-4a), 134.33 (C-6a), 132.01 (C-10a), 128.86 (C-6), 125.22 (C-10), 123.83 (C-10b), 119.56 (C-9), 119.18 (C-7), 116.78 (CN), 107.69 (C-5), 55.18 (C-2), 52.59 (CH3), 35.19 (C-1), 137.45, 135.04, 130.96, 129.57 (aromatic); MS m/z (%): 400 , 398 , 396  with a base peak at 251 (100); Anal. Calcd for C21H14Cl2N2O2 (397.25): C, 63.49; H, 3.55; N, 7.05. Found: C, 63.46; H, 3.52; N, 7.02%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4m). Colorless crystals from ethanol/benzene; yield 85%; m.p. 240–241°C; IR (KBr) υ (cm−1): 3,412, 3,328, 3,200 (NH2), 2,198 (CN); 1H-NMR δ: 7.88–6.96 , 7.08 , 5.76 , 3.84 ; 13C-NMR δ: 160.08 (C-3), 156.53 (C-8), 145.63 (C-4a), 129.01 (C-6a), 128.79 (C-10a), 128.64 (C-6), 123.95 (C-10), 119.77 (C-10b), 119.50 (C-9), 117.09 (C-5), 114.44 (CN), 107.65 (C-7), 55.59 (C-2), 55.21 (CH3), 36.23 (C-1), 145.23, 132.16, 131.98, 129.15, 128.93, 128.79, 124.92 (aromatic); MS m/z (%): 400 , 398 , 396  with a base peak at 208 (100); Anal. Calcd for C21H14Cl2N2O2 (397.25): C, 63.49; H, 3.55; N, 7.05. Found: C, 63.46; H, 3.52; N, 7.02%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4n). Compound 4n was prepared according to the literature procedure -8-methoxy-1rocedure .H-benzo[f]chromene-2-carbonitrile (4o). Colorless crystals from ethanol; yield 84%; m.p. 239–240°C; IR (KBr) υ (cm−1): 3,424, 3,381, 3,322 (NH2), 2,192 (CN); 1H-NMR δ: 7.77–6.55 , 6.94 , 5.31 , 3.80 , 3.78 , 3.71 ; 13C-NMR δ: 159.98 (C-3), 156.79 (C-8), 145.65 (C-4a), 131.83 (C-10a), 128.25 (C-6), 125.74 (C-6a), 125.03 (C-10), 121.54 (C-10b), 119.39 (C-7), 117.59 (C-9), 116.88 (CN), 107.62 (C-5), 60.91 (CH3), 60.54 (C-2), 57.53 (C-2), 55.98 (CH3), 55.41 (CH3), 33.01 (C-1), 152.86, 140.92, 136.11, 112.64, 104.62 (aromatic); MS m/z (%): 418  with a base peak at 387 (100); Anal. Calcd for C24H22N2O5 (418.44): C, 68.89; H, 5.30; N, 6.69. Found: C, 68.81; H, 5.29; N, 6.61%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4p). Colorless crystals from ethanol; yield 84%; m.p. 259–260°C; IR (KBr) υ (cm−1): 3,469, 3,350, 3,225 (NH2), 2,197 (CN); 1H-NMR δ: 7.78–6.60 , 6.92 , 5.35 , 3.81 , 3.79 , 3.72 , 3.66 ; 13C-NMR δ: 160.60 (C-3), 156.80 (C-8), 145.70 (C-4a), 131.85 (C-10a), 128.23 (C-6), 125.75 (C-6a), 125.00 (C-10), 121.53 (C-10b), 119.46 (C-7), 117.55 (C-9), 116.86 (CN), 107.60 (C-5), 61.75 (CH3), 60.62 (CH3), 57.62 (C-2), 56.05 (CH3), 55.57 (CH3), 33.04 (C-1), 152.49, 150.24, 141.71, 132.45, 123.80, 108.77 (aromatic); 13C-NMR-DEPT spectrum at 135°: CH, CH3 [positive (up)] and CH2 [negative (down)], revealed the following signals at δ: 128.23 (C-6 ↑), 125.00 (C-10 ↑), 123.80 (aromatic ↑), 119.46 (C-7 ↑), 117.55 (C-9 ↑), 108.77 (aromatic ↑), 107.60 (C-5 ↑), 61.75 (CH3 ↑), 60.62 (CH3 ↑), 56.05 (CH3 ↑), 55.57 (CH3 ↑), 33.04 (C-1 ↑); MS m/z (%): 418  with a base peak at 40 (100); Anal. Calcd for C24H22N2O5 (418.44): C, 68.89; H, 5.30; N, 6.69. Found: C, 68.95; H, 5.37; N, 6.76%.3-Amino-1--8-methoxy-1H-benzo[f]chromene-2-carbonitrile (4q). Colorless crystals from ethanol; yield 84%; m.p. 259–260°C; IR (KBr) υ (cm−1): 3,441, 3,333, 3,210 (NH2), 2,183 (CN); MS m/z (%): 436 , 434 , 432 , 430 ; Anal. Calcd for C21H13Cl3N2O2 (431.70): C, 58.43; H, 3.04; N, 6.49. Found: C, 58.49; H, 3.10; N, 6.54%.3-Amino-1--8-methoxy-1All cell lines were purchased from the American Type Culture Collection , except for the human urinary bladder carcinoma cell line 5,637, which was obtained from the German Collection of Microorganisms and Cell Cultures  and cultured as recommended. Cells were used in all experiments without synchronization.® 200 PRO microplate reader   and GR50 , alternative small-molecule drug-response metrics  measures cellular metabolic activity. Cells were treated with various concentrations of the respective compounds for 24 h and analyzed as recommended by manufacturer using a TECAN Infinitezerland) . Etoposi metrics .Following a 24-h treatment of MDA-MB-231 cells with either the most active benzochromene derivatives , etoposide, or camptothecin , all at 10 μM, cells were permeabilized, stained with propidium iodide, and analyzed by using flow cytometry and FACSVerse cytometer  . Further2O2 (50 µM) was used as positive control  in serum-free medium for 20 min at 37°C, excited by the 488-nm laser wavelength, and analyzed at 527 and 590 nm by using flow cytometry . Cells t control .2DCFDA) (2DCFDA (10 μM) in HBSS for 20 min at 37°C. The stained cells were analyzed by using flow cytometry. Cells treated with H2O2 (50 μM) were used as positive control.Cellular production of reactive oxygen species (ROS) was determined by the cell-permeable fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) , which u® 3/7 activity assay , which measures caspase 3/7 activity in cell lysates using a luminogenic caspase 3/7 substrate DEVD  in medium/Matrigel  8 days after fertilization. The next day, the xenografts were topically treated daily for three consecutive days with 20 μl of benzochromenes 4a, 4c–4f, or etoposide (each at a concentration of 10 µM). Tumor volume was calculated with the formula: V = (L × W × W)/2, where V is the tumor volume, W is the tumor width, and L is the tumor length. On day 12 after fertilization, the xenografts were excised, fixed, and paraffin embedded for histological analysis. Serial sections (5 µm) were deparaffinized before staining with either hematoxylin and eosin (HE) or a specific antibody against the human nuclear proliferation antigen Ki-67 . DNA strand breaks in apoptotic cells were visualized by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method using a TUNEL assay kit (Roche Diagnostics) . The secpost-hoc test for multigroup comparisons by using SigmaPlot 14.0 .All values are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using the Newman–Keuls H-benzo[f]chromene-2-carbonitriles (4a–4q), which was prepared as depicted in H-benzo[f]chromene-2-carbonitrile (4a–4q) was obtained with high yield by condensation of 6-methoxy-2-naphthol (1) with appropriate aromatic aldehydes (2a–2q) and malononitrile (3) in ethanolic piperidine solution under microwave irradiation at 140 °C for 2 min. The optimum condition was determined to be 400 W and 2 min after yield analysis at different irradiation power  and time . All the reactions were monitored using TLC technique. It is also important to note that the 1 position of compounds 4a–4q is a chiral center and, as the observed optical rotation of compounds 4a–4q demonstrated, they are optically inactive, which indicates that they are present in the form of racemic (±) mixtures , and number of hydrogen bond donors ≤5 . CompounH-benzo[f]chromene-2-carbonitriles derivatives (4a–4q) differ in the substitution pattern on the phenyl ring at the 1-position of 1H-benzo[f]chromene moiety. The phenyl ring could be monohalogenated substituted (4a–4f), dihalogenated substituted (4h–4l), or trisubstituted (4n–4q) with either electron-withdrawing groups like halogens and/or electron-donating groups, like methoxy groups. The antiproliferative and cytotoxic effects of the synthesized benzochromene compounds were further analyzed using cell lines originating from common advanced human cancers. The tested cancer cell lines were the triple-negative breast cancer (TNBC) MDA-MB-231, the non-small cell lung cancer (NSCLC) A549, the cervical cancer HeLa, the pancreatic adenocarcinoma MIA PaCa-2, the urinary bladder carcinoma 5,637, and the hepatocellular carcinoma Hep G2. The clinical anticancer drug etoposide was used as a positive control. 50) calculated using the above data are shown in H-benzo[f]chromene scaffold  of the benzo[f]chromene scaffold revealed the highest in vitro cytotoxicity with IC50 range from 3.0 to 6.8 μM toward all the investigated cancer cell lines. Concerning the cytotoxic activity of the synthesized benzochromene derivatives with dihalogenated phenyl ring, compounds 4i–4k exhibited higher cytotoxic activity than compounds 4h and 4l. Finally, we demonstrated that among the benzochromene derivatives with trisubstituted phenyl ring, compounds 4o and 4p showed weaker cytotoxic activity than compounds 4n and 4q. Thus, compounds 4b, 4h, 4o, and, except for one cancer cell line, 4p exhibited rather low toxicity against cancer cells lines with IC50 >30 μM.The structures of the synthesized 3-amino-1-aryl-8-methoxy-1scaffold , most of50. Different from IC50 values, GR50 is the cell division rate and, hence, assay duration independent (max) indicates the maximal drug response on growth rate, and it scores between −1 and 1. A value of 0 corresponds to a fully cytostatic response, whereas positive and negative values indicate partial inhibition or partial cytotoxic response, respectively. The normalized growth rate inhibition metrics, GR50 and GRmax values of the synthesized 1H-benzo[f]chromene derivatives (4a–4q), are presented in 50 range of 1.0–8.1 μM and GRmax range of −0.76–−1, and compound 4c showed the greatest in vitro antiproliferative/cytotoxic activities against the six different types of cancer cell lines with a GR50 range of 1.0–4.9 μM and GRmax range of −0.9–−1. These data obtained from the normalized growth rate inhibition metrics were in accordance with those obtained from IC50 metrics, indicating the accuracy of both parameters in the determination of drug potency.The cytotoxic effects of benzochromene derivatives on cancer cell lines were further normalized to respective cell line doubling time and are presented as GRependent . EfficacThe difference in cytotoxic activity of the synthesized benzochromene derivatives with different substitution patterns at the phenyl ring prompted us to further study the mechanisms that impede proliferation and induce cell death.50 values of tested compounds. The same concentration was used for all compounds to enable their comparison. Compound 4c with the highest cytotoxic activity was the only compound that induced, in addition, a strong increase in cell number in the G2/M phase. The positive controls, etoposide, and camptothecin, at this time point mostly increased the number of cells in the S phase of the cell cycle.Cell cycle analyses  revealedThe mitochondria is a critical regulator of many cellular processes such as cell death, cell signaling circuitry, and energetic homeostasis. The mitochondria clearly have essential roles in the apoptotic cell death by integrating and propagating cell death signals originating from both the extrinsic and intrinsic apoptotic signaling pathways . To deliThe mitochondrial membrane potential is controlled by electron flux through the respiratory chain and proton circuit across the mitochondrial inner membrane. Electrons leaked in the mitochondria are a major source for superoxide anions, which might be converted to other types of reactive oxygen radicals (ROS) leading to the initiation of cellular oxidative damage and cancer cell death . Indeed,The predominant form of programmed cell death is apoptosis, a process that requires mostly the activation of the caspase proteases . Caspase50 values, suitable for comparison between different compounds, compound 4c showed the highest cytotoxic activity against all cancer cell lines tested compared with other benzochromene derivatives, but this compound had less pronounced effects on mitochondrial membrane integrity or on caspase 3/7 activation in cancer cells. Intriguingly, among the active benzochromene derivatives with a monohalogenated phenyl ring , only compound 4c induced a pronounced increase in cytosolic ROS level . All compounds tested  induced reduction of tumors at least by half , which wβ-enaminonitrile linked to 8-methoxy-1H-benzo[f]chromenes. The variations in the substitution pattern, including the type of substituents  and their position on the phenyl ring at the 1-position of 1H-benzo[f]chromene moiety, affected the cytotoxic activity of the synthesized benzochromene derivatives toward the tested cancer cell lines. Interestingly, we observed that the grafting of one lipophilic electron-withdrawing group, such as a halogen at either the meta or the para position of the phenyl ring at the 1-position of 1H-benzo[f]chromene moiety, led to a pronounced cytotoxic activity toward all tested cancer cell lines. However, the presence of the lipophilic electron-withdrawing group at the ortho position did not result in any significant cytotoxic activity at the tested concentration range. More interestingly, we noticed a difference in cell death mechanisms when the grafting of the halogen atom of moderate size occurs at either the meta or the para position of the phenyl ring. Among the benzochromene compounds with monohalogenated phenyl ring, only compound 4c has a chlorine atom at the meta position of the phenyl ring, and its mechanism of action in inhibition of the proliferation and induction of death seems to be different from that of compounds 4d–4f, which show halogen atoms of moderate size, like chlorine, bromine, and iodine at the para position of the phenyl ring. Only compound 4c accumulated the treated MDA-MB-231 cells in the S and G2/M phases of the cell cycle, induced ROS, and increased cell granularity/internal complexity. In addition, its effect on mitochondrial depolarization and caspase 3/7 activation was less pronounced than that of compounds 4d–4f, which induced accumulation of cancer cells only in the S phase of the cell cycle. Among benzochromene compounds with a para-monohalogenated phenyl ring, compound 4f with para-monoiodinated phenyl ring had the strongest effect on mitochondrial integrity and membrane potential, as well as on mitochondrial superoxide anions, indicating its mitochondrial-targeting ability, while compounds 4c and 4d with a para-monochlorinated and a para-monobrominated phenyl ring, respectively, had the strongest effect on the caspase 3/7 activity, and their effect on mitochondrial integrity and membrane potential was lower than that of compound 4f indicating that a death receptor signaling pathway might be involved.The structure–activity relationship (SAR) study revealed several crucial structural requirements, which enhanced the potency of the In the benzochromene compounds with dihalogenated phenyl ring, the presence of two electron-withdrawing groups, with one of them at the ortho position, decreased their cytotoxic activity in comparison with the benzochromene compounds with monohalogenated phenyl ring with an electron-withdrawing group at either the meta or the para position. In addition, the presence of lipophilic electron-donating substituents like methoxy groups in compounds with trisubstituted phenyl ring decreased pronouncedly the cytotoxic activity of benzochromene compounds toward all the tested cell lines. This decrease in the activity occurred independently of the position of substituents on the benzene ring. We could hypothesize that the presence of either lipophilic electron-donating substituents at any position of the phenyl ring or electron withdrawing group at the ortho position in combination with two electron withdrawing groups at either the meta or the para position would decrease the cytotoxic activity of the benzochromene compounds, in comparison with the benzochromene compounds with dihalogenated or trisubstituted phenyl ring with electron-withdrawing groups at either the meta or the para position.H-benzo[f]chromene-2-carbonitriles (4a–4q) were synthesized and evaluated for their cytotoxic and apoptosis-inducing activity. Benzochromene compounds with a monohalogenated phenyl ring  exhibited the most pronounced cytotoxicity, with 4c displaying the highest cytotoxic activity in vitro and in vivo. Compound 4c with one chlorine atom at the meta position of the phenyl ring induced accumulation of breast cancer cells in the S and G2/M phases of the cell cycle, induced ROS, and increased cell granularity/internal complexity. Differently, compounds 4d–4f with halogen atoms at the para position of the phenyl ring were stronger inducers of mitochondrial membrane depolarization and caspase 3/7 activation compared with 4c. Compounds 4d–4f induced accumulation of breast cancer cells only in the S phase of the cell cycle, and compound 4f had the strongest effect on mitochondrial integrity and membrane potential dissipation, as well as on the induction of mitochondrial superoxide anions, indicating its mitochondrial-targeting ability. The structure–activity relationship (SAR) study of the synthesized benzochromene compounds confirmed that the presence of lipophilic electron-withdrawing group, like halogens at meta or para positions of the phenyl ring at the 1 position of 1H-benzo[f]chromene nucleus, is responsible for the enhancement of cytotoxic activity against different cancer cell lines and define the molecular mechanism involved in cell death.In summary, 17 aryl-substituted 3-amino-1-aryl-8-methoxy-1


The structures of the new compounds were determined by detailed analysis of the NMR and HRESIMS spectroscopic data. The absolute configurations were established by X-ray crystallography, Marfey’s method, and the ICD method. All isolates were tested for in vitro anti-food allergic bioactivities in immunoglobulin (Ig) E-mediated rat basophilic leukemia (RBL)-2H3 cells. Compound 13 significantly decreased the degranulation release with an IC50 value of 60.3 μM, compared to that of 91.6 μM of the positive control, loratadine.Ten new ( Penicillium species, which are recognized as the richest source for the discovery of biologically important and structurally unique secondary metabolites + in its (+)−HRESIMS (High Resolution Electrospray Ionization Mass Spectroscopy) spectrum, requiring ten degrees of unsaturation. The 1H and 13C NMR spectroscopic data , 7.33 , 7.46 ; δC 127.5 , 128.3 , 129.1 , 143.2 ], one ortho-disubstituted benzene moiety , one methyl , two oxygenated methines , and two carbonyls . In the 1H–1H COSY (Correlation Spectroscopy) spectrum, correlations of H-2 (H-6)/H-3 (H-5)/H-4, H-3′/H-4′/H-5′/H-6′, and H-7 /H-8  confirmed the two benzene units and deduced another fragment of C-7/C-8. By the HMBC (Heteronuclear Multiple-bond Correlation) correlations of H-7 (δH 5.16) to C-1/C-2/C-6/C-9 and H-6′ (δH 7.60)/7′-NMe (δH 2.89) to C-7′, 1 was then assigned a phenylpropionyl moiety and a benzamide groups -N-methyl-benzamide, and named penigrisamide.Compound pic data , Table 1e groups . Howevere groups , the abs2 was afforded as a colorless oil. The molecular formula C19H21N3O4 was deduced from (+)-HRESIMS data (m/z 378.1418 for [M + Na]+), indicative of eleven degrees of unsaturation. The 1H and 13C NMR spectroscopic data (11 was replaced by the methoxy unit (δC 52.2) in 2. The assumption was confirmed by the HMBC correlation of 17-OMe (δH 3.46) to C-17 (δC 173.9). Accordingly, the structure of 2 was determined as 17-deamino-17-methoxylaurantiomide C, and named aurantiomoate C.Compound pic data , Table 23 was obtained as a colorless oil. Its molecular formula was established as C17H27N3O5 on the basis of the protonated molecule peak at m/z 376.1841 [M + Na]+ in its (+)-HRESIMS spectrum, requiring six degrees of unsaturation. Diagnostic NMR data for 3 suggested the presence of a pyroglutamylleucinmethylester (20) + in its positive HRESIMS spectrum, requiring two degrees of unsaturation. Its 1H and 13C NMR spectra were very similar to those of pyroglutamylleucinmethylester (20) +. Comparison of the 1H and 13C NMR spectra of 6 with those of verrucosidinol (25) +. Its 1H and 13C NMR spectroscopic data greatly resembled those of helvafuranone [7 was then established as 8-hydroxyhelvafuranone.Compound furanone  except f8 gave a molecular formula C10H21NO3 as deduced by the protonated molecule peak at m/z 202.1504 [M − H]− in its negative HRESIMS spectrum. The 1H NMR spectrum exhibited one methyl doublet at δH 0.96 , and two methyl singlets at δH 1.12  and δH 1.16 . The 13C and DEPT spectra revealed the presence of 10 carbons, including three methyls, two methylenes, three methines, and one oxygenated and one carbonyl non-protonated carbon. In the 1H–1H COSY spectrum, correlations were found of H-6 via H-5 to H-4/H-3 and of H-3 to H3-10/H-2. By the HMBC correlations of H2-2  to C-1/C-4/C-10, H-6  to C-4/C-7, and H3-8 /H3-9  to C-6/C-7, the planar structure of 8 was then established. To determine the absolute configuration of C-6, a dimolybdenum tetraacetate [Mo2(OAc)4]-induced circular dichroism (ICD) experiment was employed. The ICD spectrum exhibited a positive Cotton effect at 310 nm -HRESIMS data at m/z 257.1237 ([M + Na]+), indicative of one degree of unsaturation. The 1H NMR spectrum showed a methyl at δH 1.14  and a methoxyl at δH 3.65 . The 13C NMR and DEPT (Distortionless Enhancement by Polarization Transfer) data displayed 11 carbons, including one methyl, one methoxyl, five methylenes, three methines, and one carbonyl. In the 1H–1H COSY spectrum, two isolated spin systems were observed as H2-2 /H-3 (δH 3.79)/H2-4 (δH 1.22)/H2-5 (δH 1.82) and H2-6 (δH 1.21)/H-7 (δH 3.54)/H2-8 /H-9 (δH 3.94)/H3-10 (δH 1.14). These two fragments could be connected by the HMBC correlations of H2-2  and H-11(δH 3.65) to C-1 (δC 173.7). Therefore, 9 was established as methyl-3,7,9-trihydroxydecanate.Compound 10 was obtained as a colorless oil. Its molecular formula was established as C10H18O4 on the basis of the protonated molecule peak at m/z 225.1109 [M + Na]+ in its positive HRESIMS spectrum, requiring two degrees of unsaturation. The 13C NMR spectrum in association with the DEPT spectrum indicated 10 carbon signals ascribed to one methyl doublet , five 3sp methylenes , three 3sp methines , and one carbonyl . In the 1H–1H COSY spectrum, a long chain of C-2/C-3/C-4/C-5/C-6/C-7/C-8/C-9/C-10 could be deduced by correlations of H2-2 (δH 2.41)/H-3 (δH 3.76)/H2-4 /H2-5 /H2-6 /H-7 (δH 3.57)/H2-8 (δH 1.48)/H-9 (δH 3.93)/H3-10 (δH 1.13). In the HMBC spectrum, H-3 (δH 3.76) was correlated to C-7 and C-1, which constructed a hexacyclic ring via an ether bond between C-1 and C-7. Accordingly, 10 was established as 9-hydroxy-3,7-epoxydecanoic acid.Compound 11) + .Penigrisamide (Aurantiomoate C (2): Colorless oil; + .3): colorless oil; + .5-Deoxypyroglutamyl-pyroglutamylleucinmethylester (4): colorless oil; + .Methyl-2-hydroxy-3-methylbutanoyl-L-leucinate (5): Colorless oil; + .Verrucosidinol A (6): Colorless oil; + .Verrucosidinol B (7): Colorless oil; + .8-Hydroxyhelvafuranone (8): Colorless oil; − .6,7-Dihydroxy-3,7-dimethyloctanamide (9): White powder; +.Methyl-3,7,9-trihydroxydecanate (10): Colorless oil; + .9-Hydroxy-3,7-epoxydecanoic acid . Space group P212121, a = 4.7555(2) Å, b = 14.7379(7) Å, c = 22.971(1) Å, α = β = γ = 90°, V = 1609.95(12) Å3, Z = 4, Dcalcd = 1.371 mg/cm3; µ = 0.847 mm−1, F (000) = 704. The final R indicates R = 0.0484 (2682), wR2 = 0.1337 (3174). Crystallographic data of 1 have been deposited in the Cambridge Crystallographic Data Center, with deposition number 2072655. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, U.K. (fax +44(0)-1233-336033; email: deposit@ccdc.cam.ac.uk).Compound 3 and 4 (each for 1 mg) were separately dissolved in HCl (1 mL) and incubated for 24 h. The hydrolysate was dried and dissolved in acetone. Then NaHCO3 and FDAA were added to incubate for 1 h. After being cooled, the mixture was dissolved in 50% aqueous CH3CN to yield FDDA derivatives. The corresponding standard amino acids were treated with the same procedures. The FDAA derivates were analyzed by HPLC at 254 and 340 nm by comparing the retention times with those of standards.As reported , compoun8 and dimolybdenum tetracetate [Mo2(OAc)4] were resolved in dried DMSO. Their CD spectra were recorded immediately. Then the ICD spectra were measured every 3 min until they were stationary. The inherent CD data of compound 8 was subtracted to provide its induced CD spectrum as described previously [Compound eviously ,42.The in vitro anti-food allergic experiment was conducted according to the reported method . BrieflyPenicillium griseofulvum MCCC 3A00225, 10 new and 26 known compounds were obtained. The structures of the new compounds were determined by extensive analysis of their NMR and HRESIMS spectra, the absolute configurations were confirmed by different methods including the single X-ray crystallography, Marfey’s method, and ICD experiment etc. (−)-Cyclopenol (13) showed the strongest in vitro anti-food allergic activity with an IC50 value of 60.3 μM in IgE-mediated RBL-2H3 cells.From the deep sea-derived fungus


GXNU-Y45, together with seven previously reported metabolites. The structures of 1–4 were elucidated based on analysis of HRESIMS and NMR spectroscopic data. The absolute configurations of the stereogenic carbons in 1–3 were established through a combination of spectroscopic data and electronic circular dichroism (ECD). Compounds 1–11 were evaluated for their anti-inflammatory activity. Compounds 1, 3, 4, and 6 showed an inhibitory activity against the production of nitric oxide (NO), with the IC50 values of 14.42 ± 0.11, 18.03 ± 0.14, 16.66 ± 0.21, and 21.05 ± 0.13 μM, respectively.Four undescribed compounds, guhypoxylonols A ( Pleosporales sp., isolated from diverse marine environments has led to the discovery of broad-spectrum cytotoxic secondary metabolites, such as dipleosporalones A and B + , indicating six degrees of unsaturation. The 1H-NMR , and 3.75 , three aromatic protons at δH 7.24 , 7.14 , and 6.83 , two multiplets at δH 1.80 , and 1.51, 2.09 , and two multiplets at δH 4.41 , and 4.35 . The 13C NMR spectrum , two methoxyls (δC 55.7 and 56.7), two methylene sp3 (δC 27.2 and 24.7), and two oxygenated methine sp3 (δC 69.8 and 67.8). The COSY spectrum . The HMBC spectrum showed correlations from the proton sinal at δH 4.41  to δC 24.7 (C-3), 118.7 (C-8), and 143.1 (C-8a), from δH 4.35  to δC 157.5 (C-5), 27.2 (C-2), and 143.1 (C-8a). The 1H and 13C NMR spectra of 2 were very similar to those of nodulisporol + , indicating five degrees of unsaturation. The 1H NMR  and 4.58 , two multiplets of methine groups at δH 3.86  and 2.63 , two methyl doublets at δH 1.18  and δH 1.19 , and two methyl singlets at δH 2.10 . The 13C NMR CH(CH3)O-. The HMBC (δC 21.0 (C-11), 134.8 (C-3a), 36.4 (C-3), and 60.8 (C-8), from H-3 to δC 134.8 (C-3a), 115.9 (C-4), 114.4 (C-7a), 18.2 (C-12), and 21.0 (C-11), suggests that C-3 is connected to C-3a. The HMBC correlations from H-9 (δH 2.10) to C-4, C-5 (δC 111.3), and C-3a, from H-10 (δH 2.10) to C-4, C-5, C-6 (δC 153.3), indicate that the two methyl groups were on C-4 and C-5, respectively. Finally, the HMBC correlations from H-8 to C-3a, C-7a, C-2 (δC 76.0), and C-7 (δC 149.6), indicated that the remaining substructure of 3 was established as shown in Compound  was obtained as a white powder and the molecular formula C25H30O9 was deduced from the HRESIMS m/z 473.1816 [M − H]− , indicating 11 degrees of unsaturation. The 1H NMR (4 displayed two methyl singlets at δH 2.10 (H-9) and 2.07 (H-10), one methoxyl singlet at δH 3.67 (-OCH3-8), and two singlets at δH 3.73 (H2-7) and 2.50 (H2-11). The 13C NMR spectrum , one ester carbonyl at δC 173.8 (C-8), one methoxy at δC 52.5 (OCH3), two methyls at δC12.1, and 9.0, and the two sp3 methylene carbons at δC 36.5 (C-7) and 32.5 (C-11). The presence of six non-protonated sp2 at δC123.1, 118.7, 155.3, 112.5, 157.5, and 130.6 is an indicative of the presence of a benzene ring. The HMBC correlations (δH 3.73 (H-7) to C-8, 123.1 (C-6), 118.7 (C-1), and from δH 3.67 to C-8, confirm that a methyl acetate is connected to C-1. HMBC correlations from δH 2.07 (H-9) to C-1, 130.6 (C-2), and 157.5 (C-3), from δH 2.07 (H-10) to δC112.5 (C-4), 155.3 (C-5), and C-3, and from H-11 to C-1 and C-12, suggested that 4 contains methyl  acetate moiety, with -CH2-C=O connected to C-6. Since the molecular formula of C25H30O9, only a ketone carbonyl (δC207.9) is present in 4. Therefore, the structure of 4 is a disubstituted acetone whose substituents are methyl acetate. Since 4 has never been reported, it was named guhypoxylonol D.Compound  [6) [7) [8) [9) [10) [11) [The previously described  [6) [7) , nodulis [7) [8) , isoscle [8) [9) , xylaren[9) [10) , scytalo10) [11) , respect1–11 were evaluated for their anti-inflammatory effects on the production of the NO in the RAW 264.7 macrophage cell line exposed to the inflammatory stimulus by lipopolysaccharide (LPS)  . Compoun1H and 13C NMR spectra are given in δ (ppm) and referenced to the solvent signal . Coupling constants (J) are reported in Hz. The mass spectrometric (HRESIMS) data were acquired using a Micro Mass Q-TOF spectrometer . ECD data was recorded using a JASCO J-715 spectropolarimeter . Semipreparative HPLC was performed on an ODS column .NMR spectra were recorded on a AVANCE-400 spectrometer . The chemical shifts of Acanthus ilicifolius, October 2019, in Beihai City, China. The fungal strain GXNU-Y45 was identified as Aspergillus sp. based on the sequence of its internal transcribed spacer region (ITS) and morphology. ITS-rDNA of GXNU-Y45 was submitted to GenBank and the accession number is MT626059.The strain GXNU-Y45 was isolated from a leaf of a mangrove tree The fungus was cultured in 60 × 1000 mL Erlenmeyer flasks each containing 50 g cooked rice and 60 mL of water  or 300 mL medium . The fungus was cultured in the medium and incubated at room temperature for 35 days.v/v) to yield six subfractions (Fr. 1–Fr. 6). Fr. 3 (3 g) was applied to ODS silica gel with gradient elution of MeOH-H2O  to afford four subfractions (Fr. 3-1–Fr. 3-4). Fr. 3-2 (650 mg) was subjected to semipreparative HPLC  to obtain 1 (15.6 mg), 2 (7.5 mg), and 3 (4.4 mg). Fr. 3-3 (345 mg) was repurified by RP-18 CC  and Sephadex LH-20  to afford 5 (10.6 mg), 9 (3.3 mg), 10 (5.2 mg), and 11 (6.7 mg). Fr. 4 (1.1 g) was separated by ODS silica gel with gradient elution of MeOH-H2O  to yield four subfractions (Fr. 4-1–Fr. 4-4). Fr.4-3 (73 mg) was purified by Sephadex LH-20 eluted with CH2Cl2/MeOH (50:50) to give 4 (6.3 mg). Fr.4-4 (84 mg) was separated by semipreparative HPLC  to give 6 (5.6 mg), 7 (8.1 mg), and 8 (5.2 mg).The fermented material was extracted three times with EtOAc to obtain 16.8 g crude extract (liquid medium) and 20.2 g (solid medium). The crude extract was subjected to a silica gel VLC column, eluting with a stepwise gradient of petroleum ether-EtOAc : was obtained as a brown oil; 1H and 13C NMR data . Guhypoxylonol A : was obtained as a colorless powder; 1H and 13C NMR data .Guhypoxylonol B : white powder; 1H and 13C NMR data .Guhypoxylonol C : white powder; 1H and 13C NMR data .Guhypoxylonol D , and seven previously reported metabolites (5–11). Based on modifications of the culture medium strategy, the fungus Aspergillus sp. GXNU-Y45 was cultured in different media to stimulate a production of its metabolites. It was found that the fungus Aspergillus sp. GXNU-Y45 produced different metabolites in two culture media. The liquid medium can stimulate the fungus to produce a series of metabolites, 1, 5, 6, 7, 8, 9, 10, and 2 (a new precursor of 1). On the contrary the solid medium yeiled 3 and 4. Different compositions of the culture media represented a powerful tool to induce new metabolites from microorganisms. Preliminarily screening of 1–11 for their ability to prevent NO production of LPS-induced RAW264.7 cells showed that 1, 3, 4, and 6 exhibited significant inhibitory effects against NO release with IC50 values of 14.42 ± 0.11, 18.03 ± 0.14, 16.66 ± 0.21, and 21.05 ± 0.13 μM, respectively. The inhibition of NO production by 1 and 6 was stronger than 5 and 7, which showed the same skeleton but differ only the presence of -OCH3 at C-3. Compounds 2 and 8–11, which are precursors of 1, 5, 6, and 7, did not exhibit inhibitory effects against NO release. Compounds 3 and 4 exhibited remarkable inhibitory effects against NO release suggesting that the fully substituted benzene ring was essential for inhibition of the production of NO release. In summary, this study revealed that 1, 3, 4, and 6 could be considered as potential metabolites for further anti-inflammatory studies. The chemical investigation of a marine-derived fungus