H-chromenes 4a–o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a–h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a–o and 6a–h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a–o and 6a–h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4 H-chromenes have attracted attention because of containing a pharmacophore group that is found in many molecules with pharmacological effects, including anticancer + calcd for C17H11N2O3F: 310.0754; found: 310.0754 , 2 , 3 , and piperidine  were mixed in ethanol, delivering 0.433 g (61%) of 4c as a beige solid; mp 202–204 °C , 2 , 3 , and piperidine  were combined in ethanol, obtaining 0.572 g (71%) of 4d as a pale orange solid; mp 220–223 °C. FT-IR (CH2Cl2) vmax 3450, 3338, 3222, 2193, 1671 cm−1. 1H NMR  δ 4.68 , 5.96 , 6.21 , 6.49 , 6.58 , 7.21 , 7.50 . 13C NMR  δ 41.7 (C-4), 58.3 (C-2), 98.5 (C-5), 102.8 (C-13), 108.2 (C-8), 115.8 (C-5a), 120.0 (CN), 121.1 (C-9), 130.5 (C-10), 132.5 (C-11), 144.0 (C-8a), 145.6 (C-7), 146.2 (C-12), 148.3 (C-6), 161.0 (C-3). HRMS (EI): m/z [M]+ calcd for C17H11N2O3Br: 369.9953; found: 369.9968.Following the general method, 1e , 2 , 3 , and piperidine  were mixed in ethanol, giving 0.47 g (68%) of 4e as a beige solid; mp 250–252 °C. FT-IR (CH2Cl2) vmax 3422, 3297, 3178, 3178, 2155, 1655 cm−1. 1H NMR  δ 4.81 , 5.97 , 6.24 , 6.51 , 6.60 , 7.46 , 7.74 . 13C NMR  δ 42.3 (C-4), 58.0 (C-2), 98.7 (C-5), 102.9 (C-13), 108.1 (C-8), 111.6 (C-9), 115.1 (C-5a), 119.2 (Ar-CN), 119.8 (CN), 129.5 (C-10), 133.5 (C-13), 144.2 (C-8a), 145.8 (C-7), 148.5 (C-6), 152.0 (C-12), 161.3 (C-3). HRMS (EI): m/z [M]+ calcd for C18H11N3O3: 317.0800; found: 317.0801.According to the general method, 1f , 2 , 3 , and piperidine  were combined in ethanol, generating 0.47 g (64%) of 4f as a pale orange solid; mp 258–260 °C  δ 41.2 (C-4), 59.260.5 (C-2), 98.3 (C-5), 102.0 (C-13), 104.8 (C-8), 113.2 (C-5a), 119.3 (CN), 124.3 (C-11), 128.8 (C-10), 143.2 (C-8a), 145.2 (C-6), 147.3 (C-12), 147.9 (C-7), 151.7 (C-9), 159.5 (C-3).Following the general method, 1g  2 , 3 , and piperidine  were mixed in ethanol, producing 0.56 g (84%) of 4g as a cream-colored solid; mp 208–210 °C , 2 , 3 , and piperidine  were combined in ethanol, yielding 0.336 g (48%) of 4h as a pale orange solid; mp 190–192 °C , 2 , 3 , and piperidine  were mixed in ethanol, leading to 0.35 g (55%) of 4i as a yellow solid; mp 225–227 °C. FT-IR (CH2Cl2) vmax 3309, 3081, 2159, 1660, 1597 cm−1. 1H NMR  δ 4.71 , 5.98 , 6.27 , 6.54 , 6.61 , 7.22–7.24 , 8.50–8.53 . 13C NMR  δ 41.7 (C-4), 57.5 (C-2), 98.7 (C-5), 102.8 (C-12), 108.0 (C-8), 114.9 (C-5a), 119.8 (CN), 123.4 (C-10), 144.3 (C-8a), 145.7 (C-7), 148.5 (C-6), 151.1 (C-11), 154.8 (C-9), 161.5 (C-3). HRMS (EI): m/z [M]+ calcd for C16H11N3O3: 293.0800; found: 293.0805.According to the general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.32 g (57%) of 4j as a pale-yellow solid; mp 194–196 °C. FT-IR (CH2Cl2) vmax 3452, 3330, 3213, 2903, 2192, 1660 cm–1. NMR 1H  δ 4.69 , 6.00 , 6.60 , 6.69 , 6.90–7.20 , 7.33–7.46 . NMR 13C  δ 40.8 (C-4), 55.6 (C-2), 98.3 (C-5), 102.2 (C-15), 107.7 (C-8), 114.1 , 114.4 , 115.3 (C-5a), 120.9 (CN), 123.8 (C-14), 131.2 , 143.2 (C-8a), 144.6 (C-7), 147.4 (C-6), 149.3 , 161.0 (C-3), 162.7 . HRMS (EI): m/z calcd for C17H11N2O3F [M]+ 310.0754 found; 310.0756.According to general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.4 g (70%) of 4k as a pale-yellow solid; mp 232–234 °C. FT-IR (CH2Cl2) vmax 3441, 3342, 2905, 2228, 2188, 1656 cm–1. NMR 1H  δ 4.77 , 6.00 , 6.58 , 6.70 , 7.00 , 7.50–7.60 , 7.68 , 7.69–7.75 . NMR 13C  δ 40.6 (C-4), 55.2 (C-2), 98.3 (C-5), 102.2 (C-15), 107.7 (C-8), 112.0 (C-11), 114.7 (C-5a), 119.2 (CN’), 120.7 (CN), 130.6 (C-13), 131.3 (C-10), 131.3 (C-12), 132.8 (C-14), 143.2 (C-8a), 144.7 (C-7), 147.5 (C-6), 147.8 (C-9), 161.0 (C-3). HRMS (EI): m/z calcd for C18H11N3O3 [M]+ 317.0800 found; 317.0801.According to general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.47 g (84%) of 4l as a beige solid; mp 198–200 °C. FT-IR (CH2Cl2) vmax 3459, 3328, 3197, 2887, 2197, 1663 cm–1. NMR 1H  δ 4.91 , 5.98 , 6.48 , 6.69 , 6.91 , 7.13–7.24 , 7.26–7.35 . NMR 13C  δ 35.6 (C-4), 54.6 (C-2), 98.2 (C-5), 102.2 (C-15), 107.4 (C-8), 114.5 (C-5a), 116.2 , 120.8 (CN), 125.2 (C-13), 129.5 , 130.2 (C-12), 132.7 , 143.5 (C-8a), 144.6 (C-7), 147.4 (C-6), 160.4 , 161.2 (C-3). HRMS (EI): m/z calcd for C17H11N2O3F [M]+ 310.0754 found; 310.0753.According to general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.3 g (51%) of 4m as a pale yellow solid; mp 218–220 °C. FT-IR (CH2Cl2) vmax 3442, 3327, 3201, 2899, 2204, 1664 cm–1. NMR 1H  δ 5.14 , 5.98 , 6.39 , 6.69 , 6.92 , 7.19–7.23 , 7.25–7.35 , 7.42–7.46 . NMR 13C  δ 38.5 (C-4), 54.8 (C-2), 98.2 (C-5), 102.2 (C-15), 107.0 (C-8), 114.5 (C-5a), 120.6 (CN), 128.3 (C-14), 129.2 (C-12), 130.3 (C-11), 131.2 (C-13), 132.2 (C-10), 142.8 (C-9), 143.4 (C-8a), 144.6 (C-7), 147.5 (C-6) 161.0 (C-3). HRMS (EI): m/z calcd for C17H11N2O3Cl [M]+ 326.0458 found; 326.0460.According to the general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.46 g (68%) of 4n as a dark yellow solid; mp 223–225 °C. FT-IR (CH2Cl2) vmax 3442, 3327, 3201, 2899, 2204, 1664 cm–1. FT-IR (CH2Cl2) vmax 3444, 3326, 3199, 2204, 1664 cm–1. NMR 1H  δ 5.16 , 5.98 , 6.37 , 6.70 , 6.93 , 7.16–7.25 , 7.33–7.40 , 7.58–7.64 . NMR 13C  δ 40.6 (C-4), 55.1 (C-2), 98.3 (C-5), 102.2 (C-15), 106.8 (C-8), 114.6 (C-5a), 120.5 (CN), 122.6 (C-10), 129.0 (C-12), 129.5 (C-13), 131.4 (C-14), 133.4 (C-11), 143.3 (C-7), 144.6 , 147.5 (C-6), 160.9 (C-3). HRMS (EI): m/z calcd for C17H11N2O3Br [M]+ 369.9953 found; 369.9954.According to the general method, 1a , 2 , 3  and piperidine  were mixed in ethanol, leading to 0.43 g (70%) of 4n as a pale yellow solid; mp 208–210 °C. FT-IR (CH2Cl2) vmax 3442, 3350, 2185, 1656 cm–1. NMR 1H  δ 4.67 , 5.98 , 6.58 , 6.69 , 6.96 , 7.15–7.20 , 7.24 , 7.27–7.32 , 7.33–7.40 . NMR 13C  δ 40.7 (C-4), 55.6 (C-2), 98.3 (C-5), 102.2 (C-15), 107.7 (C-8), 115.2 (C-5a), 120.8 (CN), 126.5 (C-14), 127.3 (C-12), 127.5 (C-13), 131.1 (C-11), 133.7 (C-9), 143.2 (C-8a), 144.6 (C-7), 147.4 (C-6), 148.9 (C-10), 161.0 (C-3). HRMS (EI): m/z calcd for C17H11N3O5 [M]+ 337.0699 found; 337.0694.According to the general method, 1a , 2 , 5 , and piperidine  were combined in ethanol, resulting in 0.23 g (57%) of 6a as a beige solid; mp 176–178 °C : m/z [M]+ calcd for C18H16N2O3: 308.1161; found: 308.1159.Following the general method, °C , 2 , 5 , and piperidine  were mixed in ethanol, providing 0.192 g (45%) of 6b as a beige solid; mp 188–190 °C. FT-IR (CH2Cl2): vmax 3475, 3329, 2962, 2196, 1658, 1582, 1505, 1399, 1223, 1204, 1147, 1105, 1049, 822, 798 cm−1. 1H NMR  δ 3.67 , 3.78 , 4.62 , 6.15 , 6.24 , 6.30 , 6.98–7.02 , 7.22–7.17 . 13C NMR  δ 36.3 (C-4), 55.1 (7-OCH3), 55.4 (5-OCH3), 59.6 (C-3), 93.1 (C-8), 95.1 (C-6), 105.1 (C-4a), 114.8, 114.9 , 119.5 (CN), 129.1, 129.2 , 142.2 , 150.4 (C-8a), 157.7 (C-5), 160.3 (C-2), 160.7 (C-7), 162.3 . HRMS (EI): m/z [M]+ calcd for C18H15N2O3F: 326.1067; found: 326.1070.According to the general method, 1c , 2 , 5 , and piperidine  were combined in ethanol, furnishing 0.191 g (43%) of 6c as a white solid; mp 193–195 °C (Lit. + calcd for C18H15N2O3Cl: 342.0771; found: 342.0768.Following the general method, °C , 2 , 5 , and piperidine  were mixed in ethanol, affording 0.238 g (47%) of 6d as a cream-colored solid; mp 192–194 °C. FT-IR (KBr): vmax 3472, 3328, 3207, 2196, 1660, 1587, 1400, 1205, 1147, 1101, 1058, 825, 806, 790 cm−1. 1H NMR  δ 3.67 , 3.78 , 4.60 , 6.17 , 6.24 , 6.30 , 7.12 , 7.43 . 13C NMR  δ 37.2 (C-4), 55.8 (7-OCH3), 56.1 (5-OCH3), 59.9 (C-3), 93.8 (C-8), 95.8 (C-6), 105.35 (C-4a), 120.1 (CN), 120.4 (C-12), 130.2 (C-10), 132.0 (C-11), 146.2 (C-9), 151.2 (C-8a), 158.4 (C-5), 161.0 (C-2), 161.5 (C-7). HRMS (EI): m/z [M]+ calcd for C18H15N2O3Br: 386.0266; found: 386.0262.According to the general method, 1e , 2 , 5 , and piperidine  were combined in ethanol, delivering 0.2 g (46%) of 6e as a white solid; mp 243–245 °C. FT-IR (KBr): vmax 3400, 3331, 3217, 2232, 2190, 1662, 1633, 1585, 1500, 1405, 1204, 1149, 1103, 1055, 820, 795 cm−1. 1H NMR  δ 3.67 , 3.79 , 4.71 , 6.20 , 6.26 , 6.32 , 7.36 , 7.67 . 13C NMR  δ 37.9 (C-4), 55.9 (7-OCH3), 56.2 (5-OCH3), 59.3 (C-3), 94.0 (C-8), 95.9 (C-6), 104.7 (C-4a), 111.0 (C-12), 119.3 (Ar-CN), 119.9 (CN), 129.2 (C-10), 133.0 (C-11), 151.2 (C-8a), 152.2 (C-9), 158.5 (C-5), 161.1 (C-2), 161.7 (C-7). HRMS (EI): m/z [M]+ calcd for C19H15N3O3: 333.1113; found: 333.1108.Following the general method, 1f , 2 , 5 , and piperidine  were mixed in ethanol, obtaining 0.207 g (45%) of 6f as a cream-colored solid; mp 230–232 °C : m/z [M]+ calcd for C18H15N3O5: 353.1012; found: 353.1014.According to the general method, °C , 2 , 5 , and piperidine  were combined in ethanol, giving 0.184 g (44%) of 6g as a pale-yellow solid; mp 172–174 °C : m/z [M]+ calcd for C19H18N2O3: 322.1318; found: 322.1318.Following the general method,  0.184 g % of 6g a1h , 2 , 5 , and piperidine  were mixed in ethanol, generating 0.132 g (30%) of 6h as a cream-colored solid; mp 186–188 °C : m/z [M]+ calcd for C19H18N2O4: 338.1267; found: 338.1268.According to the general method, °C  ). Therefore, it considers the affinity and size of the ligand molecule, but not the size and topological properties of the molecular target [4a, 4i and 4j exhibited the highest ligand efficiency value in the series (LE = 0.407 and 0.512), which was above that of 7 , evaluates the effectiveness of a drug in its interaction with the active site of a therapeutic target. It is obtained by dividing the free binding energy of each molecule by the number of heavy non-hydrogen atoms in the structure  [The ligand-lipophilicity efficiency (LLE) is also a relevant parameter for designing new drugs. It correlates the in vitro potency of a drug with the partition coefficient (log P) or the distribution coefficient (log D). A molecule with an LLE value of 0  has the same affinity for a therapeutic target as does and 6a–h . These c enzyme) . H-chromenes can be considered as drug candidates [Ligand efficiency-dependent lipophilicity (LELP) is a global descriptor involving lipophilicity, molecular size and potency. The optimal range of LELP values is −10 to 10 and corresponds to a range of Log P values of −3 to 3. The values of the test compounds are within the optimal range. Taking all the parameters evaluated into account, the two series of 2-amino-3-cyano-4ndidates .H-chromenes in the two series, 4a–o and 6a–h, comply with Lipinski’s rule of five (RO5) by meeting the following requirements: (a) molecular weight <500 g/mol; (b) <5 hydrogen bond donors ; (c) <10 hydrogen bond acceptors; and (d) Log P < 4.15 [The analysis of the data  demonstrP < 4.15 . Hence, P < 4.15 . H-chromenes based on WLog P in the range of −0.4 < WLog P < 5.6 and PSA < 131. The steric descriptors of the test compounds, like those of topotecan (7), are within the desirable values: (a) 120 < molecular weight < 480, (b) 40 < molar refractivity < 130, and (c) the number of rings <7 [H-chromenes 4a–o and 6a–h should have good absorption and an appropriate steric volume. Because of complying with the acceptable range of values of key parameters established for screening compounds in the drug development process, 4a–o and 6a–h can be considered drug candidates.The drug-like properties were also determined ,75, predrings <7 ,75. Acco7, thus allowing for oral administration. Most should be able to cross the blood–brain barrier and reach targets in the central nervous system. However, this is not the case for the compounds with a greater number of polar functional groups, such as 4e, 4k and 6e (R = CN), 4f, 4o and 6f (R = NO2), and 4h, 6h (R = OMe), due to their strong inductive effect.Regarding the pharmacokinetic properties , all the4e, 4k and 6e) and R = NO2  gave more negative values, while those with a substituent R = Cl  and R = Me (4g and 6g) had less negative values and therefore a lower permeability [4a–o and 6a–h to cross membranes was evaluated in relation to two types of transporters: the P450 cytochrome enzyme superfamily (CYP) and the ATP binding transporters (P-gp substrate). Almost all the compounds in the 4a–o series are substrates of CYP transporters, allowing them to be metabolized and excreted by these transporters . Nevertheless, their excretion can be favored because they have affinity for the other four CYP transporters . For its part, topotecan (7) is a substrate of the ATP binding transporters (P-gp substrate), leading to its elimination through the membrane.Log Kp  is a pareability . Additiosporters . Similar4a–o and 6a–h  score. The overlap of the CYP51 models with the S. cerevisiae template demonstrated an identity of over 60%  [Fifteen models of the CYP51 enzyme of each over 60% . CYP51CGDB 4WMZ) . The graDB 4WMZ) .H-chromenes, 4a–o and 6a–h, at the active site of the CYP51 of Candida spp. Some of these compounds with high binding energy values and the reference drug 8 are illustrated in  (The orientation in relation to each of the CYP51 enzymes herein tested was determined for the 2-amino-3-cyano-4zole (8) . Many ofH-chromenes than the reference drug 8. Among all the test compounds, the best one  exhibits the best binding energy with the CYP51 of three Candida species . In the series 6a–h, a similar result is found for 6d (with R = Br on the phenyl ring). Accordingly, the substituents R = Me and R = Br could be involved in the inhibitory effect on the yeasts (vide infra). The highest binding energy shown by most of the 2-amino-3-cyano-4H-chromenes 4a–o  and 6a–h  was in relation to the CYP51 enzyme of C. kefyr.In the series H-chromenes (4a–o and 6a–h) and fluconazole (8) with the CYP51 enzymes of Candida spp., the amino acid residues involved, the binding mode, and the protein–ligand interactions are shown , and they have influence in the interaction of the CYP51 of C. albicans with other azole derivatives [Candida spp., their mutation is known to cause yeast resistance mechanisms to the inhibitory effects of azoles [Most ligand–protein interactions include Tyr and Thr, hydrophilic amino acid residues that form carbon–hydrogen and conventional hydrogen bonds. Tyr118 and Tyr132 are observed in three of the ivatives . As addif azoles ,85.C. albicans [Three amino acids with basic side chains, His, Arg and Lys, also greatly influence the mechanism of action of CYP51. The most frequent positions for such interactions are 310, 312, 372, 462, 463 and 468 for His, 379 and 381 for Arg, and 143 for Lys. In this research, as well as in previous studies, we corroborated that the following amino acids: Tyr118, Phe145, Ile231, Met306, His310, Thr311, Ser378, Val509 and Val511 are involved in the active site of CYP51 of albicans ,83,86.H-chromenes 4a–o and 6a–h could possibly be administered to target the CYP51 enzymes of many Candida species, given that they bind to the active site. Hence, further research is warranted on their antifungal activity.According to the present findings, the 2-amino-3-cyano-44a–o and 6a–h with the active site of the topoisomerase I enzyme. Some key amino acids of the active site of the enzyme that are known to interact with 7 (the most common topoisomerase I inhibitor) also interact with 4a–o and 6a–h. Among such residues are Arg364, Arg488, Lys532, Asp533, Ile535, His632 and Thr718. Additionally, both series of 2-amino-3-cyano-4H-chromenes interact with certain DNA fragments, including TGP11, DG12 and DT10 [4a–o . Indeed, the binding energy was higher for 4c, 4d and 4g than 7 . Like 7, these three compounds interact with most of amino acid side chain of the active site of topoisomerase I.Compounds 4g and 7, there was a similar π–alkyl hydrophobic coupling with the TGP11 nucleotide of the complex. With a binding energy of −9.27 kcal/mol, 4b displayed most of the same interactions as 4c, 4d and 4g. Although the binding energy was lower  for 4a, this compound also exhibited similarities with the binding interactions of 7, such as hydrophilic interactions with the TGP11 DNA fragment and π–anion electrostatic interactions with the residue Asp533.For 4a–c, 4g, as well as of the compounds 4j–o, where we can observe a greater affinity shown by the S series  for the active site of the enzyme topoisomerase I with respect to the R series   . MoreoveH-chromenes 6a–h, the best binding energies were found for 6a, 6c and 6d  with the DNA fragment DG12. Overall, the 4H-chromene derivatives and 7 act in a very similar manner on the active site of topoisomerase I, revealing that they may all have the same mechanism of action. The interactions of the 2-amino-3-cyano-4H-chromenes 4e–f, 4h–i and 6a–h are illustrated in The structures of e enzyme . For 4b,7 and 4a–o at the active site of topoisomerase I , PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia), HCT-15 , MCF-7 (human mammary adenocarcinoma), and SKLU-1 (human lung adenocarcinoma). However, only four compounds  showed an inhibition percentage greater than 50% in the SK-LU-1 and PC-3 cell lines. More detailed studies on the action of these compounds are shown in In order to evaluate the cytotoxic activity of compounds, we carried out the determination at 25 μM for H-chromenes, 4a–o and 6a–h, were synthesized through an efficient and environmentally friendly methodology. This is the first report, to our knowledge, of 4d, e, i–o, as well as 6b, d and e. Regarding the evaluation of the physicochemical, pharmacokinetic, toxicological and drug-like properties of the test compounds, the values of key parameters fall within the acceptable range established for screening compounds in the drug development process. Moreover, the in silico data revealed an ample margin of safety between the effective dose of the series of compounds 4 and 6 and any possible toxicity.Two series of 2-amino-3-cyano-44a–o, 6a–h, and fluconazole (8) (the reference drug) interact with the active site of the CYP51 enzymes of C. albicans, C. dubliniensis, C. glabrata, C. kefyr, C. krusei and C. parapsilosis. CYP51 is vital for the production of ergosterol in the yeast cell membrane. The test compounds appear to have the same mechanism of action as the reference drug, since they interact with the same amino acids in the active site of the CYP51 enzyme. Better in silico binding energy was found for 4a–o and 6a–h than 8 on this enzyme, and the in vitro MIC50 of the test compounds was less than or equal to that of 8 .The molecular docking studies provided evidence that 4a–o and 6a–h interact with the same amino acids of the active site of the enzyme as one of the reference drugs 7. Compounds 4c, 4d and 4g showed better binding energy than 7. The experimental assays revealed that 4a–o were more cytotoxic than 6a–h. Furthermore, 4a and 4b were more active than topotecan (7) and cisplatin (9). This is the first report, to our knowledge, of molecular docking studies on 2-amino-3-cyano-4H-chromenes 4a–o and 6a–h with the active site of CYP51 of Candida spp., and topoisomerase I. Due to the encouraging results, in which some chromene compounds displayed better binding energy, inhibition and cytotoxicity than the reference compounds, the two series of derivatives are good candidates for future research on the design and development of new 2-amino-3-cyano-4H-chromenes, with dual activity against cancer and fungal infections.According to the results of molecular docking on topoisomerase I (involved in the growth of cancer cells),


S)- and (R)-PF74 and further enrichment of the samples (≥98%) using chiral HPLC resolution. The biological activities of each enantiomer were then evaluated, which determined (S)-PF74 (IC50 1.5 µM) to be significantly more active than (R)-PF74 (IC50 19 µM). Computational docking studies were then conducted to rationalise this large discrepancy in activity, which indicated different binding conformations for each enantiomer. The binding energy of the conformation adopted by the more active (S)-PF74  was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer  in agreement with experimental observations.PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess) of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples of this compound. Herein is described the synthesis of enantiomerically enriched ( Human Immunodeficiency Virus (HIV) is a pathogenic retrovirus that infects cells of the human immune system and, if left untreated, can result in Acquired Immunodeficiency Syndrome (AIDS). An estimated 38 million people are infected worldwide and, since the epidemic began in the early 1980s, approximately 33 million people have died from HIV-related deaths . CombinaThe HIV-1 capsid protein (CA) multimerizes to form a fullerene-like shell that encapsidates the viral RNA genome and is essential for both early and late stages of the viral replication cycle . Small mPF74 is a non-natural derivative of phenylalanine, a sub-structure that has been further optimised in lenacapavir. However, crucial information regarding the stereogenic centre present in PF74 is scarce in published syntheses and studies on the biological activities of this compound, and is not readily available from commercial suppliers of PF74 ,12,13,14The scope of this project was to study stereochemical integrity in the synthesis of PF74, and evaluate the biological activity of the two enantiomers in both cell assay and through computational docking.S)-, (R)- and racemic PF74 using enantiomerically enriched starting materials, which allowed us to assess any epimerisation and to compare our synthetic material with commercially available PF74. A PyBop-mediated amide coupling between 2-methylindole-3-acetic acid (1) and either (S)-, (R)- or racemic phenylalanine methyl ester hydrochloride  was utilised to generate the amide containing methyl ester 3  with N-methylaniline generated PF74 .Our improved synthesis of PF74 was conducted based on a previously reported literature synthesis, with some optimisation . Due to  ester 3 . Yields 4 + m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651.HRMS (ESI) exact mass calculated for Cd-phenylalaninateMethyl (2-(2-Methyl-1H-indol-3-yl)acetyl)- (3b)3b was obtained as a viscous oil. Following the general procedure outlined above, 719 mg (78%) of compound 1H NMR  δ 7.92 , 7.42 , 7.31 , 7.18 , 7.13 , 7.09 , 6.09 , 6.70 , 6.00 , 4.90–4.76 , 3.64 , 3.64 , 3.03–2.85 , 2.28 . 13C NMR  δ 171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C), 133.4 (Ar-C), 129.1 (2 × Ar-CH), 128.5 (2 × Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.6 (Ar-C), 52.8 (-CH), 52.3 (-OCH3), 37.7 (-CH2), 32.1 (-CH2), 11.6 (-CH3). 21H22N2O3Na [M + Na]+ m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651.HRMS (ESI) exact mass calculated for Cdl-phenylalaninateMethyl (2-(2-Methyl-1H-indol-3-yl)acetyl)- (3c)3c was obtained.Following the general procedure described above, 925 mg (quantitative) of 1H NMR  δ 7.89 , 7.42 , 7.31 , 7.18 , 7.15–7.11 , 7.11–7.05 , 7.00 , 6.70 , 5.99 , 4.88–4.80 , 3.64 , 3.64 , 3.00–2.86 , 2.28 . 13C NMR  δ 171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C), 133.4 (Ar-C), 129.2 (2 × Ar-CH), 128.5 (2 × Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.7 (Ar-C), 52.8 (-CH), 52.3 (-OCH3), 37.7 (-CH2), 32.2 (-CH2), 11.6 (-CH3). 21H22N2O3Na [M + Na]+ m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651. Melting point 126–130 °C.HRMS (ESI) exact mass calculated for C3 (1 equiv) in tetrahydrofuran (0.23 M) and water (0.34 M) was added a solution of sodium hydroxide (1.5 equiv) in methanol (1 M NaOH) and the resulting solution was stirred vigorously at room temperature for 18 h. The reaction mixture was diluted with water and the crude biphasic mixture was washed with dichloromethane to remove organic impurities. The aqueous phase was then acidified with 2 M aqueous hydrochloric acid forming a thick white precipitate. The aqueous phase was extracted with dichloromethane and the combined organic phases were dried with magnesium sulfate, filtered, and concentrated in vacuo. The compound was used without further purification.To a stirred solution of d-phenylalanine(2-(2-Methyl-1H-indol-3-yl)acetyl)- (4a)4a was obtained. Following the general procedure described above, 989 mg (97%) of 1H NMR  δ 8.14 , 7.34 , 7.26 , 7.16 , 7.09 , 7.09 , 6.99 , 6.74 , 6.15 , 4.76 , 3.61 , 3.00 , 2.89 , 2.13 . 13C NMR  δ = 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C), 133.8 (Ar-C), 129.2 (2 × Ar-CH), 128.6 (2 × Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8 (Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH2), 31.8 (-CH2), 11.4 (-CH3). 20H20N2O3Na [M + Na]+ m/z 359.1366, found m/z 359.1355. IR (thin film) 2916, 1724, 1618.HRMS (ESI) exact mass calculated for C(2-(2-Methyl-1H-indol-3-yl)acetyl)-l-phenylalanine (4b)4b was obtained as a solid. Following the general procedure outlined above, 358 mg (74%) of 1H NMR  δ 7.89 , 7.36–7.29 , 7.18 , 7.13 , 7.11 , 7.07–7.00 , 6.75 , 5.99 , 4.69 , 3.64 , 3.04 , 2.94 , 2.20 . 13C NMR  δ = 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C), 133.8 (Ar-C), 129.2 (2 × Ar-CH), 128.6 (2 × Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8 (Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH2), 31.8 (-CH2), 11.4 (-CH3). 20H20N2O3Na [M + Na]+ m/z 359.1366, found m/z 359.1355. IR (thin film) 2916, 1724, 1618. Melting point 140–143 °C.HRMS (ESI) exact mass calculated for Cd-phenylalanine(2-(2-Methyl-1H-indol-3-yl)acetyl)- (4c)4c was obtained.Following the general procedure outlined above, 491 mg (quantitative) of 4a.Spectroscopic data matched that of acid 4  in N,N-dimethylformamide (0.3 mL) was added N-methylaniline , HATU  and N,N-diisopropylethylamine  and the resulting solution was heated to 65 °C and stirred for 48 h. To the reaction mixture was added dichloromethane and half saturated aqueous sodium hydrogen carbonate solution and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 5% aqueous lithium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude mixture was purified by column chromatography on silica gel using an eluent of 1–5% ethanol in dichloromethane.PF74 was prepared according to a modified literature procedure . To a stNatural (S)-PF74 (5a)5a was obtained.Following the general procedure described above, 63 mg (49%) of 1H NMR  δ 8.26 , 7.39–7.31 , 7.26 , 7.18–7.04 , 6.97–6.89 , 6.71–6.65 , 6.22 , 4.81 , 3.59 , 3.58 , 3.17 , 2.74 , 2.54 , 2.26 . 13C NMR  δ 171.4 (-CONR2), 170.9 (-CONH), 142.6 (Ar-C), 136.2 (Ar-C), 135.5 (2 × Ar-C), 129.9 (2 × Ar-CH), 129.3 (2 × Ar-CH), 128.4 , 128.2 (Ar-CH), 127.5 (2 × Ar-CH), 126.8 (Ar-CH), 121.6 (Ar-CH), 119.9 (Ar-CH), 117.9 (Ar-CH), 110.6 (Ar-CH), 104.6 (Ar-C), 51.2 (-CH), 38.9 (-CH2), 37.8 (-NCH3), 32.3 (-COCH2), 11.7 (-CH3). 27H27N3O2Na [M + Na]+ m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 mL min−1, λ 208 nm) tRnatural = 32.511 min (54.8%), tRunnatural = 40.831 min (45.2%). + m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 mLmin−1, λ 208 nm) tRnatural = 32.550 min (35.0%), tRunnatural = 40.678 min (65.0%). + m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 ML min−1, λ 208 nm) tRnatural = 32.227 min (50.4%), tRunnatural = 40.120 min (49.6%). [αc = 1.00, CHCl3).HPLC  and were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with 10% FCS  and 10 μg/mL gentamicin. The VSV-G pseudotyped single-cycle HIV-1 reporter lentiviral vector CSGW is self-inactivating, and encodes GFP expressed from an internal spleen focus-forming virus (SFFV) long terminal repeat (LTR) , packageCells were seeded in 96-well plates  immediately prior to infection or treatment. Briefly, suspension HEK293T cells were infected with a titrated challenge of serially diluted CSGW-containing supernatant. At 48 h post infection, cells were trypsinized and incubated at 37 °C to enable a single cell suspension and fixed . The fixed cells were incubated to allow complete virus inactivation prior to analysis. Quantification of GFP positive cells (representative of GFP-reporter CSGW infection) was carried out using flow cytometry with 10,000 events acquired per well (Guava EasyCyte cytometer). From the titration performed with CSGW, the concentration of virus-containing supernatant required to produce 15–20% infection of cells was calculated. This dilution was then used in in fixed CSGW dose assays.For fixed compound dose assays, a CSGW titration as described above was performed with one exception. The final addition of 50 µL fresh media described above was substituted with the addition of 50 µL of a 4% DMSO solution of compound  in media. The plates were then incubated for 48 h before being fixed  and analysed by flow cytometry.m (×4 dilution series). In addition to the compound, each well contained 100 µL of a suspension of HEK293T cells in fresh media  and 50 µL of diluted CSGW  in fresh media with a final concentration of 1% DMSO per well. The plates were then incubated for 48 hours before being fixed  and analysed by flow cytometry.For compound dose response assays, each compound was titrated across a 96-well plate to give a 50 µL solution with a maximum final concentration of 100 µM to a minimum final concentration of 95 pAll structure preparation and docking was performed using the Schrödinger suite (build 2019-1) The enantiomers of PF74 were drawn with ChemDraw and converted to a 3D structure and energy minimized using the OPLS3e forcefield in LigPrep . The hexameric quaternary structure of capsid PDB: 2XDE  was geneS)-, (R)- and racemic PF74 were based on a previously published procedure [S)- and (R)-PF74. Evaluation of the biological activities was conducted in HEK293T cells to assess the ability of each enantiomer to inhibit the infectivity of the HIV viral vector CSGW-VSV-G. In broad agreement with recently published work [S)-PF74 to be significantly more active (IC50 = 1.5 µM) than (R)-PF74 (IC50 = 19 µM). In an effort to rationalise this observation, docking studies were conducted which showed the two enantiomers adopted different conformations in the PF74 binding site. The more active (S)-PF74 had additional favourable interactions compared to (R)-PF74, which was reflected in the calculated binding energies of each conformation, the more active (S)-PF74  being significantly lower in energy than (R)-PF74 .To summarise, our improved syntheses of (rocedure . Chiral hed work , this te


Structural analysis of the top scoring hits and a docking study were used to select the best derivatives for the synthesis. Chemical synthesis and structural elucidation of compounds 16a–h were discussed. The antiproliferative activity of 16a–h was evaluated against three cancer  and one normal MRC5 (IC50 = 1.27–24.06 μM) cell lines using the MTT assay. The results revealed the highest antiproliferative activity against MCF7 cells for 16g (IC50 = 0.01 μM) with an exceptionally high selectivity index of (SI = 578). Cell cycle analysis of MCF7 cells treated with compound 16g revealed a cell cycle arrest at the G2/M phase. In addition, compound 16g induced a dose-dependent increase in apoptotic events in MCF7 cells compared to the control. In silico target prediction of compound 16g showed six potential targets that could mediate these activities. Molecular docking analysis of compound 16g revealed high binding affinities toward COX-2, MAP P38α, EGFR, and CDK2. The results of the MD simulation revealed low RMSD values and high negative binding free energies for the two complexes formed between compound 16g with EGFR, and CDK2, while COX-2 was in the third order. These results highlighted a great potentiality for 16g to inhibit both CDK2 and EGFR. Taken together, the results mentioned above highlighted compound 16g as a potential anticancer agent.In the current study, a simple in silico approach using free software was used with the experimental studies to optimize the antiproliferative activity and predict the potential mechanism of action of pyrrolizine-based Schiff bases. A compound library of 288 Schiff bases was designed based on compound  Cancer is still one of the leading causes of death in the world . Among f1–5 (1–5 have similar pharmacophoric features which include three aromatic rings and hydrophobic atom/group attached to the five-membered ring. The aryl rings in these compounds are either unsubstituted or substituted at the para-position with hydrophobic groups (CH3/Br) or hydrogen bond-forming groups (SO2CH3/SO2NH2) which occupy the side pocket in COX-2.Before thirty years, the antiproliferative effect of nonsteroidal anti-inflammatory drugs (NSAIDs) was discovered . Since t1–5 , attract2 was extensively studied for its antiproliferative, either alone or in combination with anticancer drugs pyridine nucleus of vemurafenib, Contrary to the docking results for EGFR and CDK2, the results of the docking study of compound b: 4RZV)  revealed16g was able to form one conventional hydrogen bond with the key amino acid Cys532 like the pyridine nitrogen in vemurafenib. However, 16g displayed only two conventional hydrogens with BRAF compared to six hydrogen bonds for vemurafenib, In addition, the nitrogen atom of the cyano group in compound 16g  compared to –12.06 kcal/mol for the co-crystallized ligand, 8ST. The best fit conformation of compound 16g superposed only partially with the chlorophenyl-carboxamide moiety of 8ST, 16g also showed a fewer number of hydrogen bonds with VEGFR1 than 8ST, 16g into BRAF.The docking study into VEGFR1 (pdb: 3HNG) also revealed a lower binding affinity for compound 16g toward four  of the six targets compared to their co-crystallized ligands. However, additional in silico studies are needed to support these results.In conclusion, the results of the docking study revealed higher binding affinities for compound 16g in complex with each of the six potential targets as generated from the docking study. Based on the calculated RMSD values for the enzyme Cα atoms as well as compound 16g heavy atoms, the two complexes of CDK2 and EGFR bound to 16g were predicted to be the most stable as depicted from Molecular dynamic simulations (MDS) have proven to have a high value in many computational studies especially in the accurate determination of the binding affinity and the stability of the ligand-protein complexes. Accordingly, six molecular dynamic experiments were conducted for the synthesized compound 16g reached 2.6, 2.7, 3 and 3.2 Å for COX-2, MAP p38α, BRAF and VEGFR1, respectively. Furthermore, the stability of the hydrogen bond interactions between the lead compound 16g, and the six potential targets were monitored through the entire MDS. Generally, a hydrogen bond is considered stable and valid when the distance between the hydrogen bond donor and acceptor is kept less than 3.5 Å. This criterion was maintained only in all the formed hydrogen bonds of 16g-CDK2 and 16g-EGFR complexes. Some of the hydrogen bonds in the other four complexes were significantly unstable 16g to inhibit both CDK2 and EGFR.The maximum RMSD values of the two complexes mentioned only reached 1.5 and 1.7 Å for CDK2 and EGFR, respectively. In contrast, the maximum RMSD values of the other four targets in complex with 16g to each of the potential targets were calculated using the MM-PBSA approach. In this approach, the calculations are based on all trajectories extracted from the MDS, in contrast to the docking score that is based on a single conformation. Accordingly, this technique is considered as a more reliable indicator compared to the energy score obtained from the docking studies. As part of these calculations, the free energy of each component  was calculated by summing its free energy of solvation and molecular mechanics’ potential energy in vacuum. The free energy of solvation includes both the polar solvation energy and nonpolar solvation energy . Finally, the free energy of binding was calculated by subtracting the energy of the receptor and ligand from the energy of the complex. The calculated types of energies and binding free energy values for the six complexes are summarized in 16g with the two targets. On the other hand, 16g achieved average binding free energy of −360, −354, −337, and −328 KJ/mol with COX-2, p38α, BRAF, and VEGFR-1. We believe that the results from the MD simulations validated our design and supported our hypothesis for CDK2 and EGFR as potential targets for 16g.The binding free energy that resulted from the binding of http://www.swissadme.ch) [10, and 16a–h, http://molsoft.com/mprop).To calculate physicochemical properties related to drug-likeness, several web-based tools are available for free. Of these tools, SwissADME (adme.ch)  was used16a–h was in the range of 386.42–427.50 daltons, which fall within the limits of Lipinski’s rule. They also showed comparable or slightly higher molecular volumes compared to compound 10. In addition, the number of the hydrogen bond acceptors (HA)/donors (DH) were also within the limits of Lipinski’s rule. Accordingly, no violations from Lipinski’s rule was observed for any of the new compounds 16a–h.The molecular weight of all the new compounds 10 (84.79%). The new compounds also exhibited drug-likeness score in the range of 0.25–0.95 compared to 0.80 for compound 10. Among the new compounds, compound 16g exhibited the highest DLS.All of the new compounds exhibited similar bioavailability scores with 81.60–84.79% oral absorption compared to compound http://biotransformer.ca) [16g. The test compound was submitted to the server. Thereafter, phase I metabolic transformation was selected. The output results in the form of metabolic pathways were obtained describing the expected metabolites and the transforming phase I enzymes which could perform this action in humans. The results were collected in one figure, In the current study, Biotransformer (rmer.ca)  was used16g revealed five potential metabolic pathways and eight expected metabolites. The metabolic pathways included epoxidation of the aromatic rings, aromatic hydroxylation, O-demethylation of the methoxy group, N-hydroxylation of the carboxamide nitrogen, and oxidation of one of the secondary carbons of the pyrrolidine ring, The results of the metabolic study of compounds 16g did not show any results. This was due to the absence of the polar functional groups (OH and COOH), which can undergo conjugation metabolism. However, most of the expected phase I metabolites of compound 16g . Absorption bands in the infrared (IR) spectra were recorded on a RUKER TENSOR 37 FTIR spectrophotometer. Molecular ions and mass spectra (MS) of compounds 16a–h were analyzed using the Shimadzu Qp-2010 Plus mass spectrometer (EI ionization mode). The elemental analyses  of compounds 16a–h were measured in a Microanalytical Center, Cairo University. 1H-NMR, 13C-NMR, and DEPT C135 spectra were recorded using BRUKER AVANCE III at 500 MHz, 125 and 125 MHz, respectively.Chemical reagents and solvents were purchased from Sigma Aldrich . Melting points (uncorrected) of compounds 12, 14a,b, and 15a,b was achieved following the previous reports [Preparation of the starting compounds  reports ,33,34.1H-NMR, 13C-NMR, and DEPT C135 spectra of compounds 16a–h are provided in Copies of the spectra data including IR, mass, The readers are advised to consider that the two phenyl rings are assigned as phenyl ring A and B .15a,b according to the reported procedures [15a,b (3.4 mmol), the appropriate aldehyde (4.4 mmol), and glacial acetic acid (0.5 mL) in absolute ethanol (30 mL) was refluxed for 4–6 h. The reaction mixture was then concentrated and set aside to cool. The solid product obtained was recrystallized from acetone-chloroform.The new compounds were prepared from compound ocedures . A mixtu15a  was refluxed with 4-(dimethylamino)benzaldehyde  according to the general procedure A to afford compound 16a as a yellow solid product, m.p. 226-8 °C, yield 58%. IRʋmax/cm−1 3218 (NH), 3076, 3002, (aromatic C-H), 2958, 2904, 2830 , 2209 (CN), 1656 (C=O), 1583, 1539, 1509 . 1H-NMR : δ 2.55 , 3.04 , 3.13 2), 3.83 , 4.52 , 6.79 , 6.92 , 7.62 , 7.82 , 8.98 , 10.82 . 13C-NMR : δ 24.59 (CH2-2), 25.43 (CH2-2), 40.27 (N(CH3)2), 49.98 (CH2-3), 55.53 (OCH3), 111.85 (phenyl B CH-3 + CH-5), 114.22 (phenyl A CH-3 + CH-5), 116.71 (C-7), 116.76 (CN), 121.25 (phenyl A CH-2 + CH-6), 122.06 (phenyl B CH-2 + CH-6), 123.15 (C-6), 130.90 (phenyl A C-1), 131.87 (phenyl B C-1), 147.61 (C-5), 153.17 (C-7a), 155.94 (phenyl B C-4), 158.75 (phenyl A C-4), 159.59 (N=CH), 163.43 (CO). DEPT C135 : δ 24.59 (CH2-2), 25.43 (CH2-2), 40.28 (N(CH3)2), 49.98 (CH2-3), 55.54 (OCH3), 111.86 (phenyl B CH-3 + CH-5), 114.22 (phenyl A CH-3 + CH-5), 121.25 (phenyl A CH-2 + CH-6), 122.06 (phenyl B CH-2 + CH-6), 159.59 (N=CH). MS (EI): m/z (%) 427 , 411 , 402 (33), 312 , 287 (88), 261 (81), 256 (85), 227 (38), 218 (42), 191 (67), 147 (33), 72 (57), 52 (100). Anal. Calcd. for C25H25N5O2 (427.50): C, 70.24; H, 5.89; N, 16.38. Found: C, 69.85; H, 5.62; N, 16.84.Compound 15b  was refluxed with 4-(dimethylamino)benzaldehyde  according to the general procedure A to afford compound 16b as a yellow solid product, m.p. 244-6 °C, yield 54%. IRʋmax/cm−1 3236 (NH), 3061 (aromatic C-H), 2997, 2907, 2822 , 2213 (CN), 1660 (C=O), 1611, 1580, 1529 . 1H-NMR : δ 2.57 , 3.05 , 3.17 2), 4.49 , 6.86 , 7.05 , 7.72 , 7.95 , 8.77 , 11.00 . 13C-NMR : δ 24.72 (CH2-2), 25.48 (CH2-1), 40.67 (N(CH3)2), 49.98 (CH2-3), 111.19 (phenyl B CH-3 + CH5), 112.56 (phenyl B CH-2 + CH-6), 115.57 , 115.92 (C-7), 116.54 (CN), 117.21 (C-6), 121.58 , 130.46 (phenyl B C-1), 132.34 (C-5), 134.59 , 147.62 (C-7a), 152.24 (phenyl B C-4), 158.43 (N=CH), 159.11 , 160.48 (CO). DEPT C135 : δ 24.72 (CH2-2), 25.48 (CH2-1), 40.67 (N(CH3)2), 49.98 (CH2-3), 111.19 (phenyl B CH-3 + CH5), 112.61 (phenyl B CH-2 + CH6), 115.57 , 121.58 , 158.43 (N=CH). MS (EI): m/z (%) 415 , 412 , 395 (37), 384 (100), 346 (34), 301 (48), 295 (86), 253 (49), 223 (37), 199 (49), 173 (62), 144 (68), 105 (79). Anal. Calcd. for C24H22FN5O (415.46): C, 69.38; H, 5.34; N, 16.86. Found: C, 68.91; H, 5.77; N, 17.14.Compound 15a  was refluxed with 4-methylbenzaldehyde  according to the general procedure A to afford compound 16c as a yellow solid product, m.p. 231-3°C, yield 61%. IRʋmax/cm−1 3230 (NH), 3061, 3000 (aromatic C-H), 2948, 2830 , 2209 (CN), 1663 (C=O), 1600, 1549, 1509 . 1H-NMR : δ 2.47 , 2.54 , 3.01 , 3.83 , 4.51 , 6.91 , 7.34 , 7.58 , 7.80 , 9.11 , 10.60 . 13C-NMR : δ 21.78 (CH3), 24.50 (CH2-2), 25.41 (CH2-1), 50.08 (CH2-3), 55.53 (OCH3), 114.25 (phenyl A CH-3 + CH-5), 116.40 (C-7), 117.75 (CN), 121.08 (phenyl A CH-2 + CH-6), 128.75 (phenyl B CH-2 + CH-6), 129.73 (C-6), 129.90 (phenyl B CH-3 + CH-5), 131.61 (phenyl A C-1), 132.79 (phenyl B C-1), 139.10 (C-5), 143.24 (C-7a), 148.04 (phenyl B C-4), 156.07 (phenyl A C-4), 158.32 (CO), 159.49 (N=CH). DEPT C135 : δ 21.78 (CH3), 24.50 (CH2-2), 25.41 (CH2-1), 50.08 (CH2-3), 55.53 (OCH3), 114.25 (phenyl A CH-3 + CH-5), 121.08 (phenyl A CH-2 + CH-6), 128.75 (phenyl B CH-2 + CH-6), 129.90 (phenyl B CH-3 + CH-5), 159.49 (N=CH). MS (EI): m/z (%) 399 , 393 , 382 , 377 (56), 364 (31), 336 (37), 318 (89), 287 (100), 275 (34), 240 (13), 212 (26), 198 (31), 171 (23), 154 (22), 92 (24). Anal. Calcd. for C24H22N4O2 (398.46): C, 72.34; H, 5.57; N, 14.06. Found: C, 72.81; H, 5.69; N, 13.84.Compound 15b  was refluxed with 4-methylbenzaldehyde  according to the general procedure A to afford compound 16d as a yellow solid product, m.p. 241-3 °C, yield 59%. IRʋmax/cm−1 3235 (NH), 3066 (aromatic C-H), 2955, 2924, 2858 , 2214 (CN), 1664 (C=O), 1601 1551, 1506 , 1208 (C-F). 1H-NMR : δ 2.47 , 2.56 , 3.02 , 4.51 , 7.05 , 7.34 , 7.62 , 7.80 , 9.11 , 10.71 . 13C-NMR : δ 21.79 (CH3), 24.53 (CH2-2), 25.40 (CH2-1), 50.11 (CH2-3), 115.71 , 116.25 (C-7), 117.46 (CN), 121.09 , 128.75 (phenyl B CH-2+ CH-6), 129.73 (C-6), 129.95 (phenyl B CH-3 + CH-5), 132.69 (phenyl B C-1), 134.46 , 139.37 (C-5), 143.45 (C-7a), 148.23 (phenyl B C-4), 158.10, 158.46 (CO), 159.07 , 159.77 (N=CH). DEPT C135 : δ 21.79 (CH3), 24.53 (CH2-2), 25.40 (CH2-1), 50.11 (CH2-3), 115.71 , 121.09 , 128.76 (phenyl B CH-2+ CH-6), 129.95 (phenyl B CH-3 + CH-5), 159.77 (N=CH). MS (EI): m/z (%) 386 , 383 , 370 , 356 , 341 (65), 326 (22), 308 (33), 285 (29), 247 (26), 216 (17), 193 (100), 165 (61), 133 (44), 110 (58). Anal. Calcd. for C23H19FN4O (386.42): C, 71.49; H, 4.96; N, 14.50. Found: C, 71.62; H, 4.69; N, 14.43.Compound 15a  was refluxed with 4-flourobenzaldehyde  according to the general procedure A to afford compound 16e as a yellow solid product, m.p. 239-41 °C, yield 67%. IRʋmax/cm−1 3233 (NH), 3070, 3007 (aromatic C-H), 2955, 2890 , 2210 (CN), 1662 (C=O), 1600, 1584, 1509 , 1229 (C-F). 1H-NMR : δ 2.57 , 3.05 , 3.84 , 4.54 , 6.93 , 7.23 , 7.56 , 7.93 , 9.14 , 10.46 . 13C-NMR : δ 24.54 (CH2-2), 25.45 (CH2-1), 50.15 (CH2-3), 55.54 (OCH3), 114.33 (phenyl A CH-3 + CH-5), 114.80 (C-7), 116.50 , 117.97 (CN), 121.19 (phenyl A CH-2 + CH-6), 129.59 (C-6), 130.73 , 131.39 (phenyl C-1), 131.80 , 132.23 (C-5), 138.80 (C-7a), 148.10 (phenyl A C-4), 156.22 (CO), 158.16 (N=CH), 165.27 . DEPT C135 : δ 24.54 (CH2-2), 25.45 (CH2-1), 50.15 (CH2-3), 55.54 (OCH3), 114.34 (phenyl A CH-3 + CH-5), 116.51 , 121.19 (phenyl A CH-2 + CH-6), 130.73 , 158.16 (N=CH). MS (EI): m/z (%) 403 , 402 , 401 , 386 , 375 , 356 (68), 331 (23), 315 (64), 252 (58), 226 (17), 178 (23), 165 (23), 137 (49), 99 (24). Anal. Calcd. for C23H19FN4O2 (402.42): C, 68.65; H, 4.76; N, 13.92. Found: C, 68.31; H, 4.32; N, 13.84.Compound 15b  was refluxed with 4-flourobenzaldehyde  according to the general procedure A to afford compound 16f as a yellow solid product, m.p. 253-5°C, yield 66%. IRʋmax/cm−1 3222 (NH), 3072, 3003 (aromatic C-H), 2966, 2905 , 2212 (CN), 1664 (C=O), 1600, 1585, 1553, 1507 , 1230 (C-F). 1H-NMR : δ 2.59 , 3.08 , 4.55 , 7.07 , 7.25 , 7.61 , 7.95 , 9.16 , 10.57 . 13C-NMR : δ 24.60 (CH2-2), 25.46 (CH2-3), 50.18 (CH2-3), 115.82 , 116.15 (C-7), 116.31 (CN), 116.60 , 117.69 (C-6), 121.27 , 130.50  C-1), 130.83 , 131.65 (C-5), 132.27 , 134.23 (C-7a), 158.39 (CO), 158.63 (N=CH), 159.21  C-4), 163.42  C-4). DEPT C135 : δ 24.60 (CH2-2), 25.46 (CH2-3), 50.18 (CH2-3), 115.82 , 116.60 , 121.27 , 130.83 , 158.63 (N=CH). MS (EI): m/z (%) 391 , 388 , 352 (12), 341 (100), 328 (15), 300 (34), 287 (24), 257 (35), 208 (26), 192 (32), 164 (48), 149 (38), 110 (20), 83 (27). Anal. Calcd. for C22H16F2N4O (390.39): C, 67.69; H, 4.13; N, 14.35. Found: C, 68.16; H, 3.67; N, 14.76.Compound 15a  was refluxed with 4-chlorobenzaldehyde  according to the general procedure A to afford compound 16g as a yellow solid product, m.p. 241-3 °C, yield 72%. IRʋmax/cm−1 3281, 3238, 3133 (NH), 3057, 3000 (aromatic C-H), 2970, 2907, 2830 , 2209 (CN), 1663 (C=O), 1592, 1550, 1509 , 825 (C-Cl). 1H-NMR : δ 2.56 , 3.02 , 3.84 , 4.52 , 6.92 , 7.51 , 7.55 , 7.83 , 9.11 , 10.42 . 13C-NMR : δ 24.50 (CH2-2), 25.41 (CH2-1), 50.17 (CH2-3), 55.53 (OCH3), 114.33 (phenyl A CH-3 + CH-5), 116.30 (C-7), 118.17 (CN), 121.07 (phenyl A CH-2 + CH-6), 129.51 (phenyl B CH-3 + CH-5), 129.69 (phenyl B CH-2 + CH-6), 130.93 (C-6), 131.37 (phenyl A C-1), 133.88 (C-5), 138.47 (phenyl B C-1), 138.48 (C-7a), 148.26 (phenyl B C-4), 156.20 (phenyl A C-4), 157.89 (N=CH), 158.10 (CO). DEPT135 : δ (ppm): 24.49 (CH2-2), 25.41 (CH2-1), 50.17 (CH2-3), 55.53 (OCH3), 114.33 (phenyl A CH-3 + CH-5), 121.06 (phenyl A CH-2 + CH-6), 129.51 (phenyl B CH-3 + CH-5), 129.69 (phenyl B CH-2 + CH-6), 157.89 (N=CH). MS (EI): m/z (%) 418 , 417 , 413 , 412 , 368 (88), 354 (100), 328 (49), 317 (26), 292 (19), 278 (13), 229 (11), 179 (10), 165 (11), 113 (10), 97 (9). Anal. Calcd. for C23H19ClN4O2 (418.88): C, 65.95; H, 4.57; N, 13.38. Found: C, 66.34; H, 4.93; N, 13.24.Compound 15b  was refluxed with 4-chlorobenzaldehyde  according to the general procedure A to afford compound 16h as a yellow solid product, m.p. 262-4 °C, yield 63%. IRʋmax/cm−1 3274 (NH), 3073, 3005 (aromatic C-H), 2955 , 2209 (CN), 1669 (C=O), 1612, 1554, 1504 , 1205 (C-F), 835 (C-Cl). 1H-NMR : δ 2.56 , 3.06 , 4.54 , 7.09 , 7.53 , 7.60 , 7.85 , 9.15 , 10.54 . 13C-NMR : δ 24.57 (CH2-2), 25.45 (CH2-1), 50.20 (CH2-3), 115.84 , 116.15 (C-7), 117.88 (CN), 121.20 , 129.49 (C-6), 129.59 (phenyl B CH-3 + CH-5), 129.72 (phenyl B CH-2 + CH-6), 130.94 (C-5), 133.83 (C-7a), 143.22 , 138.86 (phenyl B C-1), 148.42 (phenyl B C-4), 158.32 (CO), 158.33 (N=CH), 159.20 . DEPT C135 : δ 24.57 (CH2-2), 25.45 (CH2-1), 50.20 (CH2-3), 115.84 , 121.20 , 129.60 (phenyl B CH-3 + CH-5), 129.72 (phenyl B CH-2 + CH-6), 158.33 (N=CH). MS (EI): m/z (%) 406 , 403 , 387 (14), 365 (16), 341 (100), 317 (24), 273 (68), 260 (36), 202 (28), 136 (35), 121 (52), 95 (44). Anal. Calcd. for C22H16ClFN4O (406.84): C, 64.95; H, 3.96; N, 13.77. Found: C, 64.79; H, 4.28; N, 13.92.Compound In the current study, the three cancer cell lines  used were obtained from the ATCC. The cell lines were cultured following our previous report . On the 3/well) separately. The cells were treated by tested compounds 16a–h in final concentrations of 0, 0.1, 1, 10, 25, and 50 μM and incubated at 37 °C for 72 h. The MTT was added and the antiproliferative activities  of the new compounds were calculated.The antiproliferative activity was measured using the MTT assay according to the previous report . Briefly16g on the cell cycle distribution of MCF7 was performed following the previous report [16g. Next, the cells were washed with PBS x1 and trypsinized. The collected cells were spinned and fixed with 70% ethanol. Ribonuclease A was added (15 min) after suspending the cells in cold PBS x1. Following the addition of propidium iodide, analysis of the ice-cold cells was done by flow cytometry .The effect of compound s report . Briefly16g at 0.00, 0.01, 0.05 and 0.10 μM final concentrations for 72 h. Briefly, the superanent of the cells was collected in ice-cold tubes, trypsinized and incubated (37 °C). The procedures were completed following the previous report [MCF7 cells were treated by compound ncubated  °C. The http://www.swisstargetprediction.ch) [16a–h. The test compounds were submitted one by one to the server. After running the prediction process, the results were obtained in the form of a pie chart for each compound including the major classes of the potential targets ranging with their percent. In addition, a detailed report for the first 100 entry of these targets was also obtained.The SwissTargetPrediction (tion.ch)  was used16g into COX2 (pdb: 3LN1) [The molecular docking study of compound b: 3LN1) , MAP p38b: 3LN1) , EGFR (pb: 3LN1) , CDK2 (pb: 3LN1) , BRAF (pb: 3LN1) , and VEGb: 3LN1) . Preparab: 3LN1) , grid, ab: 3LN1) ,50 was db: 3LN1)  was used16g bound to a different target . The Automated Topology Builder (ATB) and Repository version 3 [In this section, GROMACS 5.1 software was used to conduct all the molecular dynamics simulations . Six MDSersion 3  were impersion 3 . Solvatiersion 3 . After tersion 3 . The sixersion 3 ,56. The MM-PBSA which applies the following equation:Binding free energy calculations were performed using the (Complex) is the total free energy of the ligand–protein complex. G(Receptor) and G(Ligand) are total free energies of the isolated protein and ligand in solvent, respectively. The total free energy was calculated for all MD trajectories from its molecular mechanics potential energy plus the energy of solvation using the g_mmpbsa package implemented in GROMACS software [Gsoftware . Individ16a–h were calculated by the SwissADME webserver (http://www.swissadme.ch/) [http://molsoft.com/mprop/) was used in the calculation of drug-likeness scores (DLSs) of the final compounds.The physicochemical properties of compounds dme.ch/) . Each cohttp://biotransformer.ca) [16g. The test compound was submitted in the server. Thereafter, the task of metabolic transformation was selected (phase I/II), and the number of reaction steps was set to one. The output results in the form of metabolic pathways/expected metabolites were obtained.Biotransformer (rmer.ca)  was used10. A pharmacophore of the compound library search was performed. Structural analysis of the top-scoring hits was conducted, and a preliminary docking study into COX-2 was performed to select the promising hits for the synthesis. The chemical synthesis and structural elucidation of the new compounds 16a–h were discussed. The MTT assay was used to evaluate the antiproliferative activity of compounds 16a–h against MCF-7, A2780, and HT29 cancer lines (IC50 = 0.01–40.50 μM). Amongst the new compounds, compound 16g exhibited the highest antiproliferative activity against MCF7 cells (IC50 = 0.01 μM). To assess the toxicity and selectivity of the new compounds, their growth inhibitory activity was evaluated against normal MRC5 cells (IC50 = 1.27–24.06 μM). Compound 16c showed the highest selectivity index against MCF7 and A2780 cells, while 16g was the most selective for the HT29 cell line. To investigate the potential mechanism of action of the new compounds, 16g, the most active in the MTT assay was evaluated for its effects on the cell cycle distribution of MCF7 cells. The results revealed cell cycle arrest at the G2/M phase. Compound 16g also induced a dose-dependent increase in the apoptotic events in MCF7 cells compared to the control (7.9–17.8%). SwissTargetPrediction was used to predict the potential molecular targets which could mediate the anticancer potential of 16g. The results revealed six potential targets including COX-2, MAP P38α, EGFR, CDK2, BRAF, and VEGFR1. A comparative molecular docking study of compound 16g was performed into the six targets, where the results revealed high binding affinities for 16g toward four of these targets . The molecular dynamic simulation also revealed favored stability and binding energy for 16g in complex with CDK2 and EGFR, while COX-2 was in the third order. These findings suggested that compound 16g could serve as a potential anticancer agent.In the current study, an in silico approach based on free software was used to optimize the antiproliferative activity and investigate the potential mechanism of action of a series of pyrrolizine-based Schiff bases. A compound library of 288 Schiff base derivatives was designed based on compound


The latter, containing a sterically hindered cyclic N-hydroxy group, upon oxidation with lead dioxide in acetone, virtually quantitatively form stable nitroxyl bicyclic radicals of a new class, which are derivatives of both 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPON) and 3-imidazolines.In recent decades, bicyclic nitroxyl radicals have caught chemists’ attention as selective catalysts for the oxidation of alcohols and amines and as additives and mediators in directed C-H oxidative transformations. In this regard, the design and development of synthetic approaches to new functional bicyclic nitroxides is a relevant and important issue. It has been reported that imidazo[1,2- Among various types of organic radicals, nitroxyl radicals (NRs) are the most stable, most robust, and best-studied class of paramagnetic species. In addition to numerous publications on their synthesis and properties ,6,7,8,9,N-centered radicals during electrosynthesis. For example, TEMPO-mediated electrolysis of anilides or ketoximes generates amidyl or iminoxy radicals that trigger subsequent cyclization via an interaction of the forming radical with an unsaturated part of the molecule thereby leading to the closure of a pyrroline-type structure or cyclic nitrone -6-octene 8-oxyl, combining 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPON) and 3-imidazoline NR moieties  as the main product and 3-acetyl-1-hydroxypyrrole 4a–d as a minor product. We found that this alkali-catalyzed reaction for compounds 2a,c,d under much milder conditions  yields a completely different result: bicycle 5 becomes the main product. Its structure was proved on the basis of spectral and analytical data. For instance, IR spectra of compounds 5a,c,d (in KBr) contain intense bands at 1714–1716 and 1629–1639 cm−1, corresponding to stretching vibrations of isolated C=O and C=N bonds, as well as a band at 3600 cm−1 (in CHCl3), corresponding to the presence of an OH group. Compounds 5a,c,d do not have absorption maxima in the UV spectrum above 220 nm, indicating the absence of a nitrone group or a conjugated system of unsaturated moieties in the molecule. Analysis of their 1H and 13C NMR spectra points to the existence of bicycle 5 in the form of a mixture of three isomers, one of which is cyclic hemiacetal 5C and the other two are the respective hydroxyamino derivatives in which the N-OH bond is oriented either toward the keto group (compound 5O-cis) or away from it (compound 5O-trans) , corresponding to protons of the C-OH group of the hemiacetal and N-OH protons for isomers 5O-cis and 5O-trans. The ratio of these species is shifted toward the prevailing, hypothetically, 5O-trans conformation (47–65%), while the proportion of 5C is 22–44%, and the minor component is always 5O-cis (1–13%). This structural assignment is confirmed by the 13C NMR spectra of bicycles 5a,c,d, in which carbonyl C atoms for 5O-cis and 5O-trans isomers are clearly distinguished at ~206 and 207 ppm, whereas the unique quaternary carbon atom of 5C, linked to two oxygen atoms, appears at 114 ppm  . Accordi 114 ppm .5, encumbered with a sterically hindered N-OH group, can be oxidized by lead dioxide to form a stable NR  as the only reaction product. Acetone is a solvent of choice for the preparation of BNRs; in this case, the yields of compounds 6a,c,d could reach 80–87% , 207 (6c), or 221 (6d), M + 1 ions are present, and the most intense peak belongs to the M − 30 ion corresponding to a loss of the N-O group  constant aN of ~1.855 mT, for one of which , a fine structure is observed due to the presence of a hyperfine interaction with hydrogen atoms in the CH2− and CH3− groups (The EPR spectra of BNRs − groups .D), in which the hydrogen atoms located on the α- and α′-carbon atoms of the carbonyl group  interact with an unpaired electron, with the aH constant of 0.25 mT (2H) or 0.12 mT (2H) depending on whether they are in the axial or equatorial position of the six-membered ring . Al. Al7, whion data , later, trone 10 . They al8–2.0066 . Neverth2. The mechanism consists of the transformation of 2 into an intermediate  or to the product of thermodynamic control (4-pyridone-2-ketoxime 3 in the latter case). Indeed, in the control experiment, when we introduced a crystallized sample of bicycle 5d into the reaction, with short heating (60–65 °C) of this aqueous solution in 0.25N NaOH, 5d virtually quantitatively turned into a single product, pyridone 3d, which was isolated and characterized by NMR spectroscopy.According to our observations and similar data in the literature, now we can propose a mechanism of base-catalyzed recyclization of imidazoisoxazoles 6a,c,d using a Hewlett-Packard HP 8453 spectrophotometer. 1H NMR and 13C NMR spectra were recorded on Bruker AV-300, AV-400, DRX-500, and AV-600 spectrometers at 300/400/500/600 and 75/100/125/150 MHz, respectively, for 3–10% solutions of compounds 2, 3, and 5 in DMSO-d6; the positions of signals were determined relative to a residual proton signal [DMSO-d6 (2.50 ppm) for 1H spectra] or a carbon signal [DMSO-d6 (39.4 ppm) for 13C spectra] of the deuterated solvent. The analyses were performed on a Thermo Scientific DFS (Double Focusing System) high-resolution mass spectrometer ; the recording mode was electron ionization with an ionizing electron energy of 70 eV, and exact mass was measured relative to the lines of the standard perfluorokerosene.Fourier transform infrared spectra (FT-IR) were recorded in KBr pellets on a Bruker Vector-22. UV-Vis spectra were obtained for EtOH solutions of bicyclic nitroxides 3-MeOH.Elemental analyses were carried out on a Euro EA 3000 automatic CHNS analyzer. Melting points were determined by means of an FP 81 HT instrument . Column chromatography and thin-layer chromatography (TLC) were conducted using Acros silica gel 60A (0.035–0.070 mm) and Sorbfil PTLC-AF-UV 254 , respectively, with eluents EtOAc and CHCl6a,c,d were acquired by means of a Bruker Elexsys E540 X-band continuous-wave EPR spectrometer at 295 K for diluted (10−4 M) and oxygen-free toluene solutions. Experimental settings for 6c were as follows: microwave power, 0.63 mW; modulation frequency, 100 kHz; modulation amplitude, 0.002 mT; the number of points, 2048; sweep magnetic field, 6 mT; the number of scans, 160; and the time constant, 20 ms. Experimental settings for 6a,d were as follows: microwave power, 2.0 mW; modulation frequency, 100 kHz; modulation amplitude, 0.05 mT; the number of points, 1024; sweep magnetic field, 10 mT; the number of scans, 8; and the time constant, 20 ms. The following experimental settings for the center line of 6c were applied: microwave power, 0.63 mW; modulation frequency, 100 kHz; modulation amplitude, 0.002 mT; the number of points, 1048; sweep magnetic field, 1.5 mT; the number of scans, 196; and the time constant, 20 ms. To determine giso values, X-band continuous-wave EPR spectra of a mixture of the analyzed radical  with TEMPO were recorded. Then, the known giso of TEMPO was used for a spectrum simulation, and the target giso value was excluded. Simulations of solution EPR lines were carried out in the Easy Spin software, which is available at www.easyspin.org (accessed on 18 May 2021).EPR spectra of nitroxides 2a,c,d were synthesized via a reaction of 2-hydroxyamino oximes 1a,c,d isoxazole 1-oxide (2a) (a mixture of three diastereomers). 13C NMR , δ, ppm: 9.8, 10.17*, 10.2 (C(2)-CH3); 12.2, 16.8*, 18.5 (C(3)-CH3); 19.2, 23.7*, 24.7, 24.8, 25.3*, 25.9 (C(7a)-CH3 and C(6)-CH3); 48.9, 49.6, 50.6* (C-7); 62.2, 66.3*, 70.5 (C-3); 98.3, 98.9*, 99.2 (C-7a); 100.4, 101.3*, 104.9 (C-6); 137.1, 138.3*, 139.3 (C-2). (The asterisk denotes a minor isomer).2-Hydroxy-2,3a-dimethyl-2,3,3a,5,6,7,8,8a-octahydrobenzo[d]isoxazoloimidazole 4-oxide (2c) (a mixture of two diastereomers). 13C NMR , δ, ppm: 23.6, 23.7*, 23.8*, 24.7, 25.2* ; 24.7*, 24.8, 25.1*, 25.6 (C(3a)-CH3 and C(2)-CH3); 32.5*, 34.6 (C-5); 48.8, 50.4* (C-3); 69.0*, 72.7 (C-8a); 99.6, 100.0* (C-3a); 102.2*, 106.0 (C-2); 140.9*, 141.7 (C-4a); (The asterisk denotes a minor isomer).2-Hydroxy-2,3a-dimethyl-3,3a,5,6,7,8,9,9a-octahydro-2H-cyclohepta[d]isoxazoloimidazole 4-oxide (2d) (a mixture of six diastereomers). 13C NMR , δ, ppm: 12.8, 13.5, 18.6*, 18.7, 19.9, 23.6, 24.7, 24.9, 25.3, 25.4, 25.96, 26.03* (C(3a)-CH3 and C(2)-CH3); 24.0, 24.2, 24.3, 24.5*, 24.7, 25.5, 26.6*, 26.8, 26.9, 27.0, 27.7, 27.8, 27.9, 27.94, 28.7, 28.8, 28.9*, 29.2, 30.5*, 30.53, 30.58, 33.9, 34.1, 34.8, 36.4* ; 48.7, 49.1, 49.8*, 50.6, 56.3 (C-3); 66.7, 68.1*, 69.6, 71.2, 75.4, 77.8 (C-9a); 98.3, 98.7, 99.0, 99.6*, 100.7*, 101.7, 102.8, 105.4, 105.9, 106.8 (C-3a and C-2); 141.2, 141.6, 142.8*, 143.7, 144.2, 145.3 (C-4a); (The asterisk denotes the main isomer).2 (10 mmol) in 10–15 mL of water, a solution of 10 mmol of NaOH in 14 mL of water was added with stirring for 20 min and was kept on ice for 3 h. The reaction mixture was neutralized with 10% hydrochloric acid in an ice-water bath, and NaCl was added until saturation at room temperature. The precipitate of bicycle 5 was filtered off, washed with a small amount of ice-cold water, and dried until constant weight.To a cold solution (0–5 °C) of imidazoisoxazole 8-Hydroxy-1,5,7-trimethyl-6,8-diazabicyclo[3.2.1]oct-6-en-3-one (5a), colorless crystals, isolated yield 0.72 g (40%), m.p. 175–177 °C (i-PrOH). Elemental analysis: found: C, 59.42; H, 7.90; N, 15.45; calcd. for C9H14N2O2: C, 59.32; H, 7.74; N, 15.37%. IR spectrum, ν, cm−1, (KBr): 3124, 2978, 2847, 1714 (C=O), 1637 (C=N), 1456, 1375, 1315, 1242. 1H NMR , δ, ppm : A: Main isomer (keto form): 1.16 ; 1.29 ; 1.87 ; 1.95 ; 1.99 ; 2.47 ; 2.57 ; 8.33 . B: Cyclic hemiacetal: 1.21 ; 1.34 ; 1.90 ; 1.49 ; 1.57 ; 1.67 ; 1.80 ; 7.45 . C: Minor isomer (keto form): 1.29 ; 1.41 ; 1.91 ; 2.29 ; 2.43 ; 7.88 . 13C NMR , δ, ppm: A: Main isomer (keto form): 16.4 (CH3); 18.8 (CH3); 23.1 (5-CH3); 40.7 (CH2); 42.0 (CH2); 69.8 (C-1); 85.8 (C-5); 177.8 (C=N); 206.9 (C=O). B: Cyclic hemiacetal: 16.7 (CH3); 21.5 (CH3); 25.8 (5-CH3); 44.5 (CH2); 45.9 (CH2); 76.1 (C-1); 93.6 (C-5); 114.0 (NOCO); 177.3 (C=N). C: Minor isomer (keto form): 16.4 (CH3); 17.1 (CH3); 21.3 (CH3); 49.0 (CH2); 50.3 (CH2); 75.1 (C-1); 90.6 (C-5); 175.1 (C=N); 205.8 (C=O).8-Hydroxy-5-methyl-1,7-tetramethylene-6,8-diazabicyclo[3.2.1]oct-6-en-3-one (5c), colorless rhombic crystals, isolated yield 1.04 g (50%), m.p. 188–190 °C (abs EtOH). Elemental analysis: found: C, 63.50; H, 7.90; N, 13.33; calcd. for C11H16N2O2: C, 63.44; H, 7.74; N, 13.45%. IR spectrum, ν, cm−1, (KBr): 3402, 3099, 2941, 2862, 1715 (C=O), 1639 (C=N), 1452, 1313, 1238, 1009, 955, 812. 1H NMR , δ, ppm : A: Main isomer (keto form): 1.25–1.87 3); 1.30 ; 1.91 ; 1.97 ; 2.14–2.70 ; 2.49 ; 2.56 ; 8.29 . B: Cyclic hemiacetal: 1.25–1.87 3 and CH2COCH2); 1.37 ; 2.12–2.70 ; 7.54 . C: Minor isomer (keto form): 1.25–1.87 3); 1.45 ; 2.14–2.32 ; 2.43 ; 2.57 ; 2.60–2.70 ; 7.89 . D: tetrahydropyridone 3c (as an impurity): 1.25–2.25 4); 1.88 ; 2.16 ; 2.49 ; 4.57 ; 7.27 ; 10.75 . 13C NMR , δ, ppm: A: Main isomer (keto form): 23.0 (Me); 21.0, 24.3; 29.2, 34.5 ((CH2)4); 42.5, 46.2 (CH2-C=O); 68.9 (N-C-C=N); 85.9 (N-C-N); 178.7 (C=N); 207.0 (C=O). B: Cyclic hemiacetal: 25.7 (CH3); 21.6, 24.2, 29.7, 36.9 ((CH2)4); 44.0, 44.8 (CH2-C=O); 76.2 (N-C-C=N); 94.2 (N-C-N); 114.1 (NOCO); 178.2 (C=N). C: Minor isomer (keto form): 21.5 (Me); 21.2, 24.9, 28.2, 29.0 ((CH2)4); 48.2, 50.3 (CH2-C=O); 74.9 (N-C-C=N); 91.0 (N-C-N); 175.7 (C=N); 205.8 (C=O). D: tetrahydropyridone 3c (as an impurity): 20.6 (CH3); 20.1, 20.4, 25.4, 38.2 ((CH2)4); 44.8 (C-5); 58.6 (C-6); 96.9 (C-3); 156.9, 160.4 (C-2 and C-7); 190.0 (C-4).8-Hydroxy-5-methyl-1,7-pentamethylene-6,8-diazabicyclo[3.2.1]oct-6-en-3-one (5d), colorless rhombic crystals, isolated yield 1.11 g (50%), m.p. 203–204 °C (abs EtOH). Elemental analysis: found: C, 65.10; H, 8.31; N, 12.62; calcd. for C12H18N2O2: C, 64.84; H, 8.16; N, 12.60%. IR spectrum, ν, cm−1, (KBr): 3400, 3093, 2926, 2852, 1716 (C=O), 1630 (C=N), 1448, 1313, 1182, 991, 825. 1H NMR , δ, ppm : A: Main isomer (keto form): 1.06–1.90 4); 1.30 ; 1.94 ; 2.00 ; 2.17–2.42 ; 2.46 ; 2.61 ; 8.28 . B: Cyclic hemiacetal: 1.06–1.90 4 and CH2COCH2); 1.34 ; 2.17–2.42 ; 7.51 . 13C NMR , δ, ppm: A: Main isomer (keto form): 23.4 (CH3); 23.4, 28.4, 30.7, 32.0, 32.1 ((CH2)5); 41.2, 42.8 (CH2-C=O); 73.5 (N-C-C=N); 85.9 (N-C-N); 182.2 (C=N); 207.3 (C=O). B: Cyclic hemiacetal: 26.0 (CH3); 24.3, 27.8, 32.1, 32.5, 35.4 ((CH2)5); 44.5, 46.2 (CH2-C=O); 80.1 (N-C-C=N); 93.8 (N-C-N); 113.9 (NOCO); 181.9 (C=N).5 (0.55 mmol) was stirred in 10 mL of acetone for 5 min; after that, 2.0 mmol of PbO2 was added, and the suspension was stirred for 3 h at room temperature and passed through a fine pore glass filter, and the precipitate was washed thoroughly with Me2CO (3 × 5 mL). Combined filtrates were evaporated, and the solid residue was diluted with 2 mL of EtOAc and flash chromatographed with an ethyl acetate on a column with silica to obtain a colored fraction containing radical 6. The solvent was evaporated, the residue was treated with cold hexane, and the precipitate of NR 6 was filtered off.An amount of 8-hydroxydiazabicyclooctene 1,5,7-Trimethyl-6,8-diazabicyclo[3.2.1]oct-6-en-3-one 8-oxyl (6a). Light purple fine crystals, isolated yield 81 mg (81%), m.p. 66–68 °C (hexane), fR 0.45 (EtOAc). Elemental analysis: found: C, 59.42; H, 7.60; N, 15.41; calcd. for C9H13N2O2: C, 59.65; H, 7.23; N, 15.46%. IR spectrum, ν, cm−1, (KBr): 2991, 2937, 2893, 1718 (C=O), 1626 (C=N), 1450, 1371, 1313, 1236. UV (EtOH), λmax nm, (lg ε): 298 (2.65), 312 (2.65). High-resolution mass spectrometry (HRMS), electrospray ionization (EI): Observed 181.0970; C9H13N2O2 ([M]+); calculated 181.0972. ESR (PhMe): br. triplet; aN1 = 1.852 mT; giso = 2.0054.5-Methyl-1,7-tetramethylene-6,8-diazabicyclo[3.2.1]oct-6-en-3-one 8-oxyl (6c). Purple needle crystals, isolated yield 92 mg (81%), m.p. 102–104 °C (hexane), fR 0.45 (EtOAc). Elemental analysis: found: C, 63.55; H, 7.31; N, 13.30; calcd. for C11H15N2O2: C, 63.75; H, 7.30; N, 13.52%. IR spectrum, ν, cm−1, (KBr): 2951, 2862, 1716 (C=O), 1628 (C=N), 1448, 1392, 1373, 1317, 1238, 1167. UV (EtOH), λmax nm, (lg ε): 235 (3.30). HRMS (EI): Observed 207.1126; C11H15N2O2 ([M]+); calculated 207.1126. ESR (PhMe): aN1 = 1.855 mT; aH1 = 0.217 mT; aH2 = 0.1798 mT; aH3 = 0.119 mT; aH4 = 0.095 mT; aH5,H6,H7 = 0.041 mT; aH8 = 0.054 mT; aH9 = 0.025 mT; aH10 = 0.022 mT; aH11 = 0.019 mT; aH12 = 0.015 mT; aN2 = 0.009 mT; aH13 = 0.004 mT; aH14 = 0.002 mT; aH15 = 0.001 mT; giso = 2.00664.5-Methyl-1,7-pentamethylene-6,8-diazabicyclo[3.2.1]oct-6-en-3-one-8-oxyl (6d). Dark purple needles, isolated yield 106 mg (87%), m.p. 98–100 °C (hexane), fR 0.45 (EtOAc). Elemental analysis: found: C, 65.22; H, 8.01; N, 12.57; calcd. for C12H17N2O2: C, 65.14; H, 7.74; N, 12.66%. IR spectrum, ν, cm−1, (KBr): 2941, 2929, 2856, 1722 and 1711 (C=O), 1616 (C=N), 1446, 1383, 1313, 1317, 1227, 1182. UV (EtOH), λmax nm, (lg ε): 238 (3.30). HRMS (EI): Observed 221.1286; C12H17N2O2 ([M]+); calculated 221.1285. ESR (PhMe): br. triplet; aN1 = 1.841 mT; giso = 2.00667.6 (1.89 mmol) in 15 mL of acetone, zinc powder  was added, followed by dropwise addition of a solution of NH4Cl  in 1 mL of water during 15 min. The mixture was incubated for 25 min, the precipitate was filtered off, and the filtrate was evaporated. The residue was treated with cold water, and the precipitate was filtered off, washed with a minimal amount of water and diethyl ether, and dried until constant weight. The yield of bicyclic hydroxylamine 5 depends on its solubility in water and varies from 48% (for compound 5a) up to 79% (for 5d). IR spectra of the obtained compounds are consistent with the IR spectra of those synthesized through the recyclization of 2.To a stirred solution of nitroxide 5d in 1 mL of water, and the resulting solution was stirred at 65 °C for 150 min. The reaction mixture was cooled to ambient temperature, neutralized with 10% aq HCl and kept at 3 °C for 15 h. The precipitate of 3d was filtered off, washed with 1 mL of ice water, and dried to constant weight. The aqueous filtrate was saturated with NaCl and kept at room temperature for 2 d; the precipitate was filtered off and washed with a minimal volume of ice water to isolate an additional amount of 3d.A solution of 21 mg (0.525 mmol) of NaOH in 1 mL of water was added to a suspension of 111 mg (0.5 mmol) of bicycle 7-(Hydroxyimino)-2-methyl-1-azaspiro[5.6]dodec-2-en-4-one (3d). Pale fine crystal powder, total isolated yield 84 mg (76%). Its IR and 1H NMR spectra are consistent with those described in the literature isoxazoles formed by the reaction of acetylacetone with 2-hydroxyaminooximes having a secondary hydroxyamino group are key compounds for the synthesis of sterically hindered hydroxylamines, precursors of the corresponding BNRs containing two functional groups in a paramagnetic molecule. It should be pointed out that these nitroxides simultaneously contain TEMPON and 3-imidazoline frames in the molecular core; this dualism may open up new possibilities for the synthesis of functional DNP agents, radical polymerization mediators, catalysts of one-electron oxidation, spin-labeled chelating ligands, and other constructs. Being aza-analogs of NRs of the nortropinone series, radicals 6 can become convenient starting compounds for the synthesis of polyfunctional hydrophilic nitroxides of the 3-imidazoline series .Considering the increased interest of researchers in BNRs as promising agents for the oxidation of various alcohols, amines, and related compounds , further work is being planned regarding the synthesis, reactivity evaluation, transformation of the keto function, and determination of electrochemical characteristics of BNRs of the 6,8-diazabicyclo [3.2.1]-6-octene 8-oxyls series.


Couchioplanes sp. RD010705, a strain belonging to an underexplored genus of rare actinomycetes, as a producer of new metabolites. HPLC-DAD-guided fractionation of its fermentation extracts resulted in the isolation of five new methyl-branched unsaturated fatty acids, -2,4-dimethyl-2,4-octadienoic acid (1), -2,4,7-trimethyl-2,4-octadienoic acid (2), (R)-(−)-phialomustin B (3), -7-hydroxy-2,4-dimethyl-2,4-octadienoic acid (4), -7-hydroxy-2,4,7-trimethyl-2,4-octadienoic acid (5), and one prenylated tryptophan derivative, 6--N-acetyl-ʟ-tryptophan (6). The enantiomer ratio of 4 was determined to be approximately S/R = 56:44 by a recursive application of Trost’s chiral anisotropy analysis and chiral HPLC analysis of its methyl ester. Compounds 1–5 were weakly inhibitory against Kocuria rhizophila at MIC 100 μg/mL and none were cytotoxic against P388 at the same concentration.A genome mining survey combined with metabolome analysis of publicly available strains identified  Actinomycetales, have provided many important clinical drugs + calcd for C10H16NaO2, 191.1043; found, 191.1044.-2,4,7-Trimethyl-2,4-octadienoic acid (2): yellow oil; UV (MeOH) λmax (log ε) 260 (4.04) nm; IR (ATR) νmax: 2957, 1681, 1622, 1417, 1269, 1137 cm–1; 1H and 13C NMR data, see m/z): [M + Na]+ calcd for C11H18NaO2, 205.1199; found, 205.1202.(2R)-(−)-Phialomustin B (3): yellow oil; [α]D27 −12 , [α]D22 −76 ; UV (MeOH) λmax (log ε) 256 (4.19) nm; IR (ATR) νmax: 2958, 2956, 1686, 1620, 1279, 982 cm–1; 1H and 13C NMR data, see m/z): [M + Na]+ calcd for C12H20NaO2, 219.1356; found, 219.1356.-7-Hydroxy-2,4-dimethyl-2,4-octadienoic acid (4), R/S = 44:56: yellow oil; [α]D22 −2.6 ; UV (MeOH) λmax (log ε) 259 (4.20) nm; IR (ATR) νmax: 3356, 2969, 2929, 1680, 1623, 1266, 1125 cm–1; 1H and 13C NMR data, see m/z): [M + Na]+ calcd for C10H16NaO3, 207.0992; found, 207.0992.-7-Hydroxy-2,4,7-trimethyl-2,4-octadienoic acid (5): yellow oil; UV (MeOH) λmax (log ε) 258 (3.94) nm; IR (ATR) νmax: 3365, 2970, 1681, 1623, 1266, 1130 cm–1; 1H and 13C NMR data, see m/z): [M + Na]+ calcd for C11H18NaO3, 221.1148; found, 221.1149.. The fraction 4 (5:5) was evaporated, and purified by preparative HPLC operated at the same conditions as above to give 6--N-acetyl-ʟ-tryptophan .Compound N-acetyl-ʟ-tryptophan (6): yellow oil; [α]D22 +15 ; UV (MeOH) λmax (log ε) 229 (4.59), 282 (4.05) nm; IR (ATR) νmax: 3310, 2916, 1724, 1656, 1627, 1548, 1453, 1223, 806 cm–1; 1H and 13C NMR data, see m/z): [M − H]− calcd for C18H21N2O3, 313.1558; found, 313.1556.6--4  in CHCl3/MeOH (25 μL each) was added a solution of TMS-diazomethane in n-hexane  at room temperature. After stirring for 15 min, the reaction mixture was concentrated to dryness to give methyl ester 4' (0.2 mg).To a solution of 4': 1H NMR  δ 3.76 , 7.14 , 5.66 , 2.33 , 3.92 , 1.24 , 2.01 , 1.87 .Methyl ester 4' , (R)-MPA , and N,N-dimethyl-4-aminopyridine  in dichloromethane (200 μL) were added N,N’-diisopropylcarbodiimide (3 μL). After stirring for 1 h at room temperature, ice-water was poured into the reaction mixture, which was then extracted with EtOAc. After evaporation of the solvent, the residue was purified by preparative silica gel thin-layer chromatography  developed by a mixture of CHCl3/MeOH (5:1) to give (R)-MPA esters 4'a and 4'b with a slight excess yield of the former. A recursive application of Trost’s chiral anisotropy analysis allowed to identify 4'a to be (R)-MPA ester of S- and 4'b to be that of R-enantiomers, respectively.To a solution of R)-MPA ester of S-enantiomer 4'a: 1H NMR  δ 3.77 , 7.07 , 5.49 , 2.37 , 2.45 , 5.03 , 1.14 , 1.95 , 1.81 , 3.405 , 4.72 .(R)-MPA ester of R-enantiomer 4'b: 1H NMR  δ 3.76 , 6.93 , 5.26 , 2.27 , 2.31 , 5.03 , 1.26 , 1.88 , 1.68 , 3.396 , 4.71 .-MPA esters 4'c and 4'd was prepared from 4' and (S)-MPA.In the same manner as described for the preparation of S)-MPA ester of 7S enantiomer 4'c: 1H NMR data was identical to those of 4'b.(S)-MPA ester of 7R enantiomer 4'd: 1H NMR data was identical to those of 4'a. eluted with n-hexane/iPrOH (4:1) at 0.5 mL/min. Peaks for (S)- and (R)-4' were detected at 11.3 min and 14.3 min, respectively, by monitoring the absorbance at 250 nm.A 0.2 μL-portion of 6  in dry N,N-dimethylformamide (100 μL) and N,N-diisopropylethylamine (10 μL) were added (R)-PGME , PyBOP  and HOBt  at room temperature. After stirring for 3 h, ice-water was poured into the reaction mixture, which was then extracted with EtOAc. After evaporation of the solvent, the residue was purified on a silica gel thin-layer plate  developed by a mixture of CHCl3/MeOH (5:1). Extraction of the collected silica gel powder with MeOH gave (R)-PGME amide 6a (0.5 mg).In a manner similar to a procedure from , to a soR)-PGME amide 6a: 1H NMR  δ 10.61 , 7.01 , 7.46 , 6.78 , 2.85 , 2.97 , 4.72 , 8.02 , 1.79 ; HR–ESI–TOFMS (m/z): [M + Na]+ calcd for C27H31N3NaO4, 484.2207; found, 484.2209. was prepared from 1 and (S)-PGME.In the same manner as described for S)-PGME amide 6b: 1H NMR  δ 10.64 , 7.07 , 7.53 , 6.80 , 2.88 , 3.06 , 4.68 , 8.01 , 1.73 ; HR–ESI–TOFMS (m/z): [M + Na]+ calcd for C27H31N3NaO4, 484.2207; found, 484.2207..Antimicrobial assays were carried out against five bacteria, eviously . The MICFile 1Copies of NMR spectra.


We have found that the diol derived from allylated camphorquinone undergoes iodine or bromine mediated deep-seated skeletal rearrangement to provide an interesting tricyclic ring system. The iodo group in the rearranged product provided convenient leverage for further functionalization. For example, it was converted into an azide and the azide was subjected to copper( We described an unprecedented skeletal rearrangement of camphorquinone motif to an interesting tricyclic ring system.Peripheral functional group modification provided a terpene-triazole conjugate. The extraordinarily compact structure of camphor, forced to be in an uncomfortable boat form of cyclohexane, has been known to go through extraordinarily diverse skeletal rearrangements and ring opening reactions.5 Over a century back, Lowry and Armstrong noted, “No substance known to us suffers rearrangement of its parts and undergoes a complete change of type more readily than does camphor”.6 Relief of the strain and the anti-periplanar nature of the C–C bonds in the norbornane ring system are driving forces for the rearrangement reactions, possibly via the intermediates involving non-classical carbocations. The chemistry of skeletal rearrangements of camphor has been a topic of interest throughout the history of natural product chemistry. Rearrangement reactions involving camphorquinone72  1 , a quintuinone72 , its sib2 carbon C(3) in the ring. Indeed, two carbonyl groups located on C(2) and C(3) of 2 can be differentiated readily in nucleophilic reactions. Due to CMe at C(1), adjacent C(2) carbonyl group is more sterically hindered to approach of the nucleophiles than to C(3) carbonyl.17–19 Furthermore, reactions at C(3) carbonyl group with nucleophiles exhibit high stereo-selectivity due to steric hindrance from the gem-methyl groups located on C(7). High regio-, chemo- and stereo-selectivity in the reactions with nucleophiles and potential for rearrangements make it an attractive starting compound for synthetic endeavours. Surprisingly however, it did not find many applications in organic synthesis.20 Previously, we have established a method for the synthesis of 2-methylfuran appended polycyclic aromatic hydrocarbons (PAHs) from corresponding 1,2-diketones.21 Barbier type allylation on one of the two carbonyl groups, reduction of the remaining carbonyl, iodine mediated 5-exo-dig cyclization yielded 5-methylfuran appended to PAHs. Since camphorquinone 2 has 1,2-diketone functionality, we planned to synthesize 3 -camphorquinone 2 with allyl bromide in presence of zinc metal and trimethylsilyl chloride  resulted in regio- (at C(3) out of the possible C(2) and C(3)) and stereo- (endo- out of possible exo- and endo-) selective allylated product 5 in 88% yield  carbonyl group in 5 with sodium borohydride in methanol reflux provided exo, exo-diol 6 (exo- out of possible exo- and endo-) exclusively in near quantitative yield. In the 1H NMR spectrum of the diol 6, C(2)H appeared at δ 3.2 ppm which inferred its endo orientation.23 Stereochemistry of the vicinal diol 6 was further confirmed by its facile conversion to acetonoide 7.The Barbier type reaction of 18% yield . The rea1H NMR spectrum of 4, three singlet signals at δ 1.08, 0.98 and 0.96 ppm, for methyl groups, which when compared to the corresponding signals for the diol 6  were distinctly different. The 1H decoupled 13C NMR spectrum of 4 revealed 13 signals, among which signals at 96.7, 89.0 and 81.0 ppm were of oxygen-attached CH type (from DEPT spectra). The vicinal diol 6, on the other hand, exhibited two oxygen-attached-carbon signals located at 85.3 (CH) for C(2) and 78.0 (C) for C(3). Since both 1H and 13C NMR spectra of 4 were distinctly different from what is anticipated from the camphor framework, we concluded that there has been a deep-seated rearrangement of the skeleton. The structure of 4 was further delineated from the analysis of 2D NMR  spectral data. Finally, we confirmed the assigned structure 4 by the analysis of the single crystal X-ray crystallographic data -(−)-camphorquinone 2 resulted in inseparable mixture of two isomeric tert-α-hydroxy ketones, namely the propargylated 13 and the allenylated 14 products in 6 : 4 ratios in 74% yield  and HRMS data.Next, we sought to elaborate 4 into further complex products. In this quest, we planned substitution of iodo group with azide so that 1,3-dipolar cycloaddition with terminal acetylenes are possible. The reaction of 4 with sodium azide in dimethylformamide (DMF) led to the corresponding azide 16 in 81% yield . As a re3i) mediated Huisgen 1,3-dipolar cycloaddition with an acetylene furnished 1,4-disubstituted triazole 17. We anticipate that present findings will lead to further exploration in the rearrangement reactions involving camphorquinone and attachment of such rearranged products to biomolecules.In summary, we have shown that the allylated diol 6, derived from camphorquinone 2 in chemo-, regio- and stereo-selective manner in two simple steps, undergoes a deep-seated rearrangement when treated with iodine or bromine to provide 4/8 which possess interesting and unprecedented tricyclic ring system. The reaction of 2 with propargyl bromide and indium resulted in allenylated product but it failed to undergo rearrangement reaction. The functional group transformation of the iodo group in 4 into azide 16 followed by copper spectra were recorded by using Bruker Avance 400 spectrometer with tetramethylsilane (TMS) as the internal standard and  solutions; J-values are in Hz. 1H-NMR data are reported as follows: chemical shift , coupling constant (J) and integrations). High resolution mass spectra (HRMS) were recorded on Agilent 6350 B Q-TOF mass spectrometer using electro spray ionization mode.All reactions were carried out in oven dried glass-wares. Solvents were dried according to the standard procedures given in literature. Thin layer chromatography (TLC) was used for monitoring the reactions. Silica gel G and GF 254 coated glass plates were used for making TLC plates. Purification was done by using column chromatography technique by using 100–200 mesh silica from Avra Synthesis Pvt. Ltd and hexanes and ethyl acetate. Melting points were uncorrected and were recorded on VEEGO VMP-DS instrument by using open-ended capillary tubes. IR spectra were recorded as KBr pellets on a Nicolet-6700 spectrometer. NMR  was suspended in THF (8 mL). To this suspension, a solution of TMSCl (3 mol%) in xylene (1 mL) was added. Resulting mixture was heated to reflux along with vigorous stirring for 30 min. To the resulting suspension, -(−)-camphorquinone 2  in THF (5 mL) and allyl bromide  in THF (5 mL) were added drop wise simultaneously by using two syringes for a period of 30 min. After addition, the reaction mixture which was orange in color was heated at 45 °C till all the camphorquinone had been consumed and the color changed to light yellow. After completion of the reaction, ) the crude product was extracted using dichloromethane . The DCM layer was washed with water (2 × 30 mL), brine solution (2 × 30 mL) and then dried over anhydrous sodium sulfate. The concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) with increasing amounts of EtOAc (2 to 4%) in hexane to afford -(+)-3-allyl-3-hydroxy-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one 5 as a light yellow colored semi-solid . [α]22D = +95° ; IR  3479, 3077, 2959, 2874, 1747, 1639, 1494, 1455, 1394, 1293, 1220, 1105, 998, 953, 916, 861, 750; 1H NMR  δ 5.92–5.81 , 5.13–5.09 , 2.75 , 2.27 , 2.19 , 1.92 , 1.85–1.35 , 0.98 , 0.92 , 0.85 ;13C NMR  δ 220.3 (C), 132.6 (CH),119.6 (CH2), 77.8 (C), 58.5 (C), 51.8 (CH), 46.4 (C), 41.1 (CH2), 30.0 (CH2), 22.6 (CH3), 22.4 (CH2), 20.6 (CH3), 9.7 (CH3); HRMS (ESI) calcd for C13H21O2 [M + H]+ 209.1542, found 209.1524.4.3.In a single necked 100 mL round bottom flask, -(+)-3-allyl-3-hydroxy-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one 5  was dissolved in MeOH (4 mL), and sodium borohydride  was added in portions slowly at room temperature and refluxed for 12 h. After completion of the reaction ), MeOH was evaporated. Then the crude product was extracted using dichloromethane (10 mL). The DCM layer was then washed with water (2 × 10 mL), brine solution (2 × 10 mL) and then dried over anhydrous sodium sulfate. Then concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) and increasing amounts of (5 to 8%) EtOAc in hexanes led to -(−)-3-allyl-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diol 6 as white semisolid ; [α]22D = −8°; IR  3479, 3077, 2959, 2874, 1747, 1693, 1494, 1455, 1394, 1293, 1220, 1154, 1044, 998, 953, 916, 861, 750, 691, 616, 562; 1H NMR  δ 5.89 , 5.18 , 3.19 , 2.59–2.58 , 2.36 , 1.68 , 1.53 , 1.38–1.30 , 1.18 , 0.91 , 0.84 ;13C NMR  δ 134.3 (CH), 118.5 (CH2), 85.5 (CH), 79.0 (C), 54.1 (CH), 49.7 (C), 48.7 (C), 44.9 (CH2), 33.7 (CH2), 22.6 (CH3), 22.4 (CH3), 22.1 (CH2), 11.6 (CH3); HRMS (ESI) calcd for C13H23O2 [M + H]+ 211.1698, found 211.1694.4.4.In a 100 mL single necked round bottom flask, -(−)-3-allyl-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diol 6  was dissolved in distilled acetone (5 mL) and was treated with 70% aqueous perchloric acid (0.2 mL) at room temperature and stirred for 24 h. After completion of the reaction ), reaction was stopped by addition of excess solid NaHCO3 and the organic layer was collected separately. The solvent acetone was evaporated and the concentrated crude product so obtained, was purified by column chromatography using silica (100–200 mesh) and increasing amount of 5 to 10% EtOAc in hexanes afforded -(−)-3a-allyl-2,2,7,8,8-pentamethylhexahydro-4,7-methanobenzo[d]dioxole 7 as a yellow semi solid ; [α]22D = −11° ; IR  2930, 2870, 1736, 1645, 1455, 1374, 1260, 1192, 1035, 998, 909, 803, 661; 1H NMR  δ 5.98–5.88 , 5.10–5.06 , 3.54 , 2.63–2.62 , 2.46–2.40 , 2.30–2.22 , 1.91 , 1.61–1.57 , 1.55 , 1.43 , 1.30–1.26 , 1.22 , 0.98 , 0.84 ; 13C NMR  δ 134.6 (CH), 117.4 (CH2), 108.8 (C),91.9 (CH), 90.2 (C), 51.4 (CH), 48.8 (C), 47.4 (C), 42.8 (CH2), 31.9 (CH2), 27.8 (CH3), 26.6 (CH3), 24.3 (CH3), 22.6 (CH2), 20.9 (CH3), 12.1 (CH3); HRMS (ESI) calcd for C16H27O2 [M + H]+ 250.1933, found 250.1998.4.5.In a 100 mL single necked round bottom flask, -(−)3-allyl-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diol 6  was dissolved in 1,4-dioxane (2 mL) and then iodine , was added to it and stirred at room temperature for 24 h. After completion of reaction ) the solvent was evaporated and the crude product was extracted using dichloromethane (20 mL). The organic layer was washed with hypo solution (2 × 20 mL), brine solution (2 × 20 mL) and then dried over anhydrous sodium sulfate. The concentrated crude product was purified by column chromatography using silica (100–200 mesh) and 9% EtOAc/hexane as eluent to afford -(−)-2-(iodomethyl)-6,7,7-trimethylhexahydro-2H-3a,6-methanobenzofuran-8-ol 4 as a white semi solid ; [α]22D = −9° ; IR  3466, 3418, 2955, 2926, 2868, 1669, 1598, 1453, 1380, 1132, 1034, 995, 761, 701, 622; 1H NMR  δ 4.48–4.41 , 3.56 , 3.29–3.26 , 3.12 , 2.38 , 2.39–2.34 , 1.68–1.65 , 1.27–1.14 , 1.08 , 0.97 , 0.96 ; 13C NMR  δ 96.7 (CH), 89.0 (CH), 81.0 (CH), 57.4 (C), 50.8 (C), 43.4 (C), 35.2 (CH2), 29.8 (CH2), 26.8 (CH3), 23.4 (CH2), 21.4 (CH3), 13.5 (CH3), 11.4 (CH2); HRMS (ESI) calcd for C13H22IO2 [M + H]+ 337.0664, found 337.0659.4.6.In a 100 mL single necked round bottom flask, -(−)-3-allyl-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diol 6  was dissolved in distilled CHCl3 (3 mL). The solution was kept in ice-bath, so that the temperature was maintained below 0 °C. Then, bromine  in CHCl3 solution (2 mL) was added to it drop-wise, and the reaction mixture was stirred at room temperature. After completion of the reaction after 8 h ), the crude product was extracted using DCM (10 mL). The DCM layer was washed with water (2 × 10 mL), brine solution (2 × 10 mL) and then dried over anhydrous sodium sulfate. Then concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) and increasing amounts of (5 to 8%) EtOAc in hexanes furnished -(−)-2-(bromomethyl)-6,7,7-trimethylhexahydro-2H-3a,6-methanobenzofuran-8-ol 8 as yellowish liquid . [α]22D = −12° ; IR  3435, 2951, 2884, 2726, 1451, 1384, 1281, 1233, 1124, 1092, 1047, 833, 657, 493; 1H NMR  δ 3.81 , 3.58 , 2.88 , 1.75 , 1.72–1.68 , 1.64 , 1.46 , 1.25 , 1.09 , 0.96 , 0.93 , 0.80 ; 13C NMR  δ 96.66 (CH), 88.99 (CH), 81.02 (CH), 57.34 (C), 50.77 (C), 43.38 (C), 35.21 (CH2), 29.77 (CH2), 26.88 (CH2), 23.78 (CH2), 21.35 (CH3), 13.50 (CH3), 11.32 (CH2); HRMS (ESI) calcd for C13H22BrO2 [M + H]+ 289.0703, found 289.0795.4.7.-(−)-Camphorquinone 2  was dissolved in dimethyl formamide (5 mL) and then 80% propargyl bromide in toluene , indium metal , sodium iodide  were added and stirred at 70 °C for 24 h and then after completion of reaction ), 1 N HCl solution was added to the reaction mixture and the organic layer was extracted using dichloromethane (30 mL) and washed with water (2 × 30 mL), brine solution (2 × 30 mL) and dried over anhydrous sodium sulfate. The concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) and increasing amounts of (2 to 5%) EtOAc in hexanes to afford -(+)-3-hydroxy-1,7,7-trimethyl-3-bicyclo[2.2.1]heptan-2-one 14 as a light yellow semi solid ; [α]22D = +70° ; IR  3385, 2939, 2885, 1956, 1745, 1465, 1382, 1287, 1097, 1052, 849; 1H NMR  δ 5.27 , 4.85 , 2.88 , 2.01 , 1.88–1.32 , 1.04 , 0.96 , 0.88 ; 13C NMR  δ 217.7 (C), 206.9 (C), 93.4 (CH), 79.3 (C), 78.4 (CH2), 58.3 (C), 52.9 (CH), 46.5 (C), 28.7 (CH2), 23.7 (CH2), 22.4 (CH3), 20.9 (CH3), 9.7 (CH3); HRMS (ESI) calcd for C13H19O2 [M + H]+ 207.1385, found 207.1380.4.8.-(+)-3-Hydroxy-1,7,7-trimethyl-3-bicyclo[2.2.1]heptan-2-one 14  was dissolved in methanol (3 mL) and then sodium borohydride  was added in portions and refluxed for 12 h and then after completion of the reaction ) MeOH was evaporated. Then the crude product was extracted using dichloromethane (30 mL). The DCM layer was washed with water (2 × 30 mL), brine solution (2 × 30 mL) and then dried over anhydrous sodium sulfate. Then concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) and increasing amounts of (5 to 10%) EtOAc in hexanes to furnish -(+)-1,7,7-trimethyl-3-bicyclo[2.2.1]heptane-2,3-diol 15 as a white semi solid ; mp 62–64 °C; [α]22D = +66° ; IR  3380, 2939, 2885, 1956, 1465, 1382, 1287, 1097, 1052, 849; 1H NMR  δ 5.36 , 4.83 , 3.54 , 3.42 , 3.40 , 1.69 , 1.55–1.49 , 1.44–1.37 , 1.19 , 0.92 , 0.85 ; 13C NMR  δ 206.2 (C), 97.7 (CH), 82.8 (CH), 79.3 (C), 78.3 (CH2), 55.3 (CH), 49.9 (C), 49.1 (C), 33.3 (CH2), 22.9 (CH2), 22.8 (CH3), 22.3 (CH3), 11.7 (CH3); HRMS (ESI) calcd for C13H20O2H [M + H]+ 209.1542, found 209.1534.4.9.-(−)-2-(Iodomethyl)-6,7,7-trimethylhexahydro-2H-3a,6-methanobenzofuran-8-ol 4  was dissolved in dimethylformamide (5.0 mL) and then to it sodium azide  was added and stirred at 60 °C for 3 h. After the completion of reaction ), DMF was evaporated. Then the crude product was extracted using DCM (30 mL). The DCM layer was washed with water (2 × 30 mL), brine solution (2 × 30 mL) and then dried over anhydrous sodium sulfate. Then concentrated crude product was purified by column chromatography using silica gel (100–200 mesh) and increasing amount of 4 to 9% EtOAc in hexanes resulted -(+)-2-(azidomethyl)-6,7,7-trimethylhexahydro-2H-3a,6-methanobenzofuran-8-ol 15 as a colorless liquid ; [α]22D = +42° ; IR  3513, 2927, 2866, 2099, 1457, 1271, 1035, 804, 692, 493; 1H NMR  δ 4.48–4.41 , 3.56 , 3.29–3.26 , 3.14–3.12 , 2.43 , 2.36 , 1.68–1.64 , 1.22–1.13 , 1.11 , 0.99 , 0.96 ; 13C NMR  δ 96.5 (CH), 89.1 (CH), 80.4 (CH), 56.8 (C), 55.1 (C), 50.8 (C), 43.2 (CH2), 31.5 (CH2), 29.9 (CH2), 26.8 (CH3), 23.7 (CH2), 21.5 (CH3), 13.6 (CH3); HRMS (ESI) calcd for C13H22N3O2 [M + H]+ 252.1706, found 252.1707.4.10.-(+)-2-(Azidomethyl)-6,7,7-trimethylhexahydro-2H-3a,6-methanobenzofuran-8-ol 16  was dissolved in dichloromethane(DCM) (3 mL) and then 4-nitrophenylpropargyl ether , copper sulphate pentahydrate , sodium ascorbate  were added and then stirred at rt for 24 h. After the completion of reaction ) the crude product was extracted using DCM (15 mL). The DCM layer was washed with water (2 × 15 mL), brine solution (2 × 15 mL) and then dried over anhydrous sodium sulfate. Then concentrated crude product was purified by column chromatography using silica (100–200 mesh) and increasing amount of 3 to 6% EtOAc in hexanes afforded -(+)-6,7,7-trimethyl-2-((4-((4-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)hexahydro-2H-3a,6-methanobenzofuran-8-ol 17 as a semisolid ; [α]22D = +10° ; IR  3416, 2953, 2872, 1597, 1509, 1339, 1259, 1173, 1112, 1049, 997, 853, 489. 1H NMR  δ 8.18 , 7.76 , 7.08 , 5.31 , 4.64 , 4.48 , 4.33 , 3.33 , 3.21 , 2.26 , 1.68–1.61 , 1.25 , 1.18–1.13 , 1.06 , 0.99 , 0.93 , 0.91 .·13C NMR  δ 163.2 (C), 143.0 (C), 142.2 (C), 126.1 (CH), 124.1 (CH), 115.1 (CH), 96.4 (CH), 88.6 (CH), 79.7 (CH), 62.6 (CH2), 56.6 (C), 54.3 (CH2), 50.9 (C), 43.4 (C), 31.4 (CH2), 29.7 (CH2), 26.7 (CH3), 23.8 (CH2), 21.5 (CH3), 13.4 (CH3); HRMS (ESI) calcd for C22H29N4O5 [M + H]+ 429.2138, found 429.2119.There are no conflicts to declare.RA-011-D0RA09839F-s001RA-011-D0RA09839F-s002