Aloe vera (L.) Burm.f. and Aloe plicatilis (L.) Miller and includes their 5-LOX/COX-1 inhibiting potency. After initial Sephadex-LH20 gel filtration and combined silica gel 60- and RP18-CC, three dihydroisocoumarins (nonaketides), four 5-methyl-8-C-glucosylchromones (heptaketides) from A. vera, and two hexaketide-naphthalenes from A. plicatilis have been isolated by means of HSCCC. The structures of all polyketides were elucidated by ESI-MS and 2D 1H/13C-NMR  techniques. The analytical/preparative separation of 3R-feralolide, 3′-O-β-d-glucopyranosyl- and the new 6-O-β-d-glucopyranosyl-3R-feralolide into their respective positional isomers are described here for the first time, including the assignment of the 3R-configuration in all feralolides by comparative CD spectroscopy. The chromones 7-O-methyl-aloesin and 7-O-methyl-aloeresin A were isolated for the first time from A. vera, together with the previously described aloesin  and aloeresin D. Furthermore, the new 5,6,7,8-tetrahydro-1-O-β-d-glucopyranosyl- 3,6R-dihydroxy-8R-methylnaphtalene was isolated from A. plicatilis, together with the known plicataloside. Subsequently, biological-pharmacological screening was performed to identify Aloe polyketides with anti-inflammatory potential in vitro. In addition to the above constituents, the anthranoids (octaketides) aloe emodin, aloin, 6′-(E)-p-coumaroyl-aloin A and B, and 6′-(E)-p-coumaroyl-7-hydroxy-8-O-methyl-aloin A and B were tested. In the COX-1 examination, only feralolide (10 µM) inhibited the formation of MDA by 24%, whereas the other polyketides did not display any inhibition at all. In the 5-LOX-test, all aloin-type anthranoids (10 µM) inhibited the formation of LTB4 by about 25–41%. Aloesin also displayed 10% inhibition at 10 µM in this in vitro setup, while the other chromones and naphthalenes did not display any activity. The present study, therefore, demonstrates the importance of low molecular phenolic polyketides for the known overall anti-inflammatory activity of Aloe vera preparations.The present study aims at the isolation and identification of diverse phenolic polyketides from  Aloe are characterized by various polyketide constituents, such as naphthalenes (hexaketides), chromones (heptaketides), anthrones/anthraquinones (octaketides), and nonaketides such as feralolide +, 367 [M + Na]+, 381 [M + K]+.O-β-d-glucopyranosyl-3R-feralolide; (3R-(2′-acetyl-5′-hydroxyphenyl-3′-O-β-d- glucopyranosyl)methyl-3,4-dihydro-8-hydroxy-2(1H)-benzopyran-1-on) (2):3′-1H-NMR : 10.93, 9.96 ; 6.53 ; 6.41 ; 6.18 (s(br), 1H, H-5); 6.12 (s(br), 1H, H-7); 4.86 ; 4.55 ; 3.69, 3.49 ; 3.50 ; 3.23 ; 3.21 ; 3.19 ; 2.92, 2.83 ; 2.81, 2.80 ; 2.49 . 13C-NMR : 204.34 (C-7′); 168.60 (C-1); 163.03 (C-6); 162.56 (C-8), 158.90 (C-5′); 157.17 (C-3′); 141.75 (C-8a); 137.06 (C-1′); 120.99 (C-2′); 109.60 (C-6′); 107.21 (C-5); 99.44 (C-7); 101.75 (C-4′); 101.11 (C-1″); 99.33 (C-5a); 79.66 (C-3); 76.66 (C-3″); 75.85 (C-2″); 72.75 (C-5″); 69.33 (C-4″); 60.37 (C-6″); 37.23 (C-9); 31.91 (C-8′); 31.85 (C-4). ESI-MS: 529 [M + Na]+, 345 [M-Glucose]+.O-β-d-glucopyranosyl-3R-feralolide; ((3R)-3,4-dihydro-8-hydroxy-3-methyl-1H-[2]-benzopyran-1-on) (3):6-1H-NMR : 11.04 ; 10.02, 9.80 ; 6.52 ; 6.47 ; 6.30 ; 6.23 ; 5.33 (s(br), 1H, OH-2″); 5.10 (s(br), 1H, OH-3″); 5.04 (s(br), 1H, OH-4″); 5.00 ; 4.72 ; 4.56 (s(br), 1H, OH-6″); 3.67, 3.46 ; 3.39 ; 3.22 ; 3.27 ; 3.16 ; 2.92, 2.90 ; 2.97, 2.89 ; 2.49 . 13C-NMR : 203.37 (C-7′); 168.80 (C-1); 163.23 (C-6); 162.89 (C-8); 159.39 (C-5″); 157.78 (C-3″); 141.69 (C-4a); 137.00 (C-1″); 120.70 (C-2″); 109.79 (C-6″); 107.17 (C-5); 102.40 (C-8a); 101.81 (C-7); 101.37 (C-4″); 99.52 (C-1′); 79.67 (C-3); 76.39 (C-3′); 77.03 (C-5′); 73.02 (C-2′); 69.49 (C-4′); 60.53 (C-6″); 37.65 (C-9); 32.44 (C-8′); 31.91 (C-4). ESI-MS: 529 [M + Na]+, 345 [M-Glucose]+ (see se]+ see .1) from A. ferox +O-methyl-aloeresin A; (2-acetonyl-8-C-β-d-(2-O-(E)-p-coumaroyl)glucopyranosyl- 7-methoxy-5-methylchromone) (6):7-1H-NMR : 9.90 (s(br), 1H, OH-7″); 7.44 ; 7.28 ; 6.85 ; 6.74 ; 6.19 ; 6.05 ; 5.46 ; 5.23 (s(br), 1H, OH-3′); 5.17 (s(br), 1H, OH-4′); 4.94 ; 4.42 (s(br), 1H, OH-6′); 3.84 ; 3.80 (s(br), 2H, H-9); 3.74, 3.41 ; 3.51 ; 3.25 ; 3.34 ; 2.76 ; 2.26 . 13C-NMR : 204.63 (C-10); 182.06 (C-4); 168.06 (C-1″); 162.93 (C-2); 162.08 (C-7); 161.25 (C-7″); 159.59 (C-8a); 145.25 (C-3″); 144.67 (C-5); 130.81 (C-5″); 127.00 (C-4″); 117.11 (C-4a); 116.66 (C-6″); 115.11 (C-2″); 113.76 (C-3); 112.22 (C-6); 111.97 (C-8); 82.80 (C-5′); 77.81 (C-3′); 73.89 (C-2′); 72.53 (C-4′); 72.14 (C-1′); 63.33 (C-6′); 57.12 (O-CH3); 49.10 (C-9); 29.89 (C-11); 23.67 (CH3). ESI-MS: 555 [M + H]+C-glucosyl compounds of the aloin-type included in this biological screening have been preliminarily published +, 379 [M + Na]+, 395 [M + K]+.4 production. Among the tested chromones, only aloesin (4) displayed any inhibitory activity that was also limited to the LOX system. All tested naphthalene derivatives were inactive in both applied test systems. Among the tested substances, only feralolide (1) (at a concentration of 10 µM) achieved a 24% reduction of MDA production in the COX-1 test. All other tested substances were inactive in this test system. Feralolide did not, however, show any effect on the production of LTB4. Even if the glycosylated feralolide derivatives were inactive in the applied test system, they may very well be active in the human body, as the function of glycosides as inactive prodrugs that are transformed into active components by the human metabolism after ingestion is a well-documented fact in pharmacokinetics [10) have already been identified as lipoxygenase inhibitors [4) itself [In the applied 5-LOX screening system  the aloikinetics . The anthibitors . In part) itself , in cont) itself ,20,21,32Hydrangea macrophylla Seringe var. thunbergii Makino) [O-glucoside, (−)-hydrangenol-4′-O-glucoside, thunberginol C, thunberginol D, thunberginol E, and thunberginol G—have been described to exert substantial anti-allergic and antihistaminic effects [R-phyllodulcin, thunberginol C, thunberginol D, and thunberginol G, have been found to display differentiation-inducing activities against leukemic cells [R,4R)-(−)-6-methoxy-1-oxo-3-pentyl-3,4-dihydro-1H-isochromen-4-yl-acetate, isolated from the Brazilian medicinal plant Xyris pterygoblephara Steud., which showed strong aromatase inhibitory activity in an in vitro breast cancer model [R,4R)-(−)-6- methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one against dermato- parasitic fungi [Haloxylon scoparium Pomel has also been reported [Most of these plant polyketides have been isolated, together with related isocoumarins from the Japanese medicinal and recreational tea plant, amacha, meaning “sweet tea” in Japanese ( Makino) ,34, the  Makino) . In acco effects ,37. Furtic cells , hintinger model . In the ic fungi . Most rereported .Aloe species. The presented proof of its anti-inflammatory activity is of high relevance in the context of the typical application of A. vera in wound healing and a variety of inflammatory diseases, all of which have been validated by successful clinical studies [Hydrangea macrophylla Seringe var. thunbergii Makino) [Xyris pterygoblephara Steud. [Aloe spp. dihydroisocoumarins were isolated from the laxative aloe resin drug in the present research project, there is no reason to assume that the presence of these compounds in the living plants should be limited to the resin-storing vessels as described above. They may therefore very well be of high interest in the context of the numerous dermatological applications of the Aloe spp. gel drugs. Furthermore, anticancer activities reported from Brazilian traditional medicine for dihydroisocoumarins in the literature [Aloe spp., as the use of whole leaf macerates of Aloe arborescens Mill. as an anticancer drug is one of its most famous applications in this ethnopharmacological tradition [The documented reduction of MDA production in the COX-1 test by feralolide constitutes the first attribution of biological activity to a dihydroisocoumarin of an  studies ,43,44,45 studies . Feralol Makino) ,37 and ta Steud. , both ofterature ,39 mightradition . HoweverAloe barbadensis (syn. A. vera) drug (Ph.Eur.) (Lot No. Tot.J/W27.02.84/038) was obtained from Müggenburg Pflanzliche Rohstoffe . For the analysis, 100 g of the powdered drug were extracted with 2 l of a mixture of EtOAc/H2O  under constant shaking for 17 h. After filtration through a frit for removing insoluble components, the organic and aqueous phases were separated. Afterward, the organic phase was extracted again, first with 100 mL of water, then with 100 mL of a saturated aqueous NaCl solution, and then once again with 100 mL of water. Finally, the organic phase was dried over Na2SO4 and subsequently evaporated to dryness under reduced pressure, resulting in 22.8 g of dry extract. A part of the organic fraction (20 g) was adsorbed to 80 g of silica gel (40–63 µm) and subjected to normal phase silica gel CC ) to give nine fractions (Fr.A1 (2.2 g), Fr.A2 (3.5 g), Fr.A3 (4.6 g), Fr.A4 (7.2 g), Fr.A5 (0.8 g), Fr.A6 (0.7 g), Fr.A7 (0.4 g), Fr.A8 (0.3 g), Fr.A9 (0.1 g)). The entire 2.2 g of Fr.A1 were subjected to HSCCC   7:13:8; 1 mL/min) to give three fractions (Fr.B1 (1.22 g), Fr.B2 (0.75 g), Fr.B3 (0.58 g)). After this, 255 mg of Fr.B2 were subsequently adsorbed to 0.4 g of silica gel (40–63 µm) and subjected to normal phase silica gel CC ), eluting feralolide (1) (114 mg) as a pure compound. Fr.A5 and Fr.A6 were pooled, adsorbed to 5 g of silica gel (63–200 µm) and subjected to normal phase silica gel CC ) to give seven fractions (Fr.C1 (95 mg), Fr.C2 (180 mg), Fr.C3 (80 mg), Fr.C4 (210 mg), Fr.C5 (120 mg), Fr.C6 (350 mg), Fr.C7 (450 mg)). Fr.A5 and Fr.A6 were pooled and the entire 800 mg subjected to HSCCC  7:13:8; 2 mL/min) to give three fractions (Fr.D1 (235 mg), Fr.D2 (90 mg), Fr.D3 (198 mg)). In a final step, fraction Fr.D2 was purified by gradient semipreparative HPLC  ; H2O–MeOH (6:4 → 4:6) 30 min), 6-O-β-d-glucopyranosyl-feralolide (3) (36 mg) and 3′-O-β-d-glucopyranosyl-feralolide (2) (13 mg) as pure compounds, at Rt = 15 min and Rt = 19 min, respectively.254 TCL plates . Depending on the chromatographic properties of the extract fractions encountered during the isolation procedure, either  or  was used as mobile phase A and B, respectively. Detection took place under UV light (quenching at 254 nm and fluorescence at 366 nm) and in the daylight after derivatization with either 5% methanolic KOH (after incubation at 105 °C for 10 min) or with a 0.5% aqueous solution of fast blue B zinc salt , depending on the properties of the individual extract. When using mobile phase A, 1, 2, and 3 displayed Rf values of 0.96, 0.66, and 0.63, while the corresponding values for mobile phase B were 0.69, 0.32, and 0.33, respectively.All extract fractions in the above-described isolation protocol were controlled for the presence of desirable substances using silica gel 60 F1H: 300.075 MHz, 13C: 75.462 MHz), a Bruker  Advance DRX-400 Spectrometer , and a Bruker DRX-600 Spectrometer  were used. An internal reference relative to the respective solvent system was used as a standard. The structures of all three isolated compounds were additionally identified via Nuclear Overhauser Effect (NOE) experiments. All mass spectra were recorded using a Sciex 365 API No. 99 MS-MS instrument from Perkin-Elmer  in electrospray ionization mode. CD spectra were recorded on a Jasco715-spectrometer .As far as the applied NMR instruments are concerned, a Varian  Gemini 300 Spectrometer . The remaining plant material is homogenized for 1 h with 250 mL water and 250 mL methanol. After filtration, the resulting extract is evaporated to dryness in a vacuum . Then, 4.2 g of fraction A were suspended in 50 mL methanol. After removing the insoluble parts, the liquid extract was evaporated to dryness in a vacuum, yielding 2.44 g of dry extract. This extract was divided into two equal portions and separated using two identical CC setups  200 mL, EtOAc/MeOH (10:2) 200 mL, EtOAc/MeOH (10:3) 200 mL, EtOAc/MeOH (10:4) 200 mL). During this CC, two fractions could be identified via TLC detection, namely, fraction 1 (0.13 g/Rf (SiO2): 0.47 (EtOAc/MeOH/H2O 100:17:13)) and fraction 2 (0.25 g/Rf (SiO2): 0.11 (EtOAc/MeOH/H2O 100:17:13)). Subsequently, plicataloside (8) (0.12 g/Rf (SiO2): 0.09 (EtOAc/MeOH/H2O 100:20:13)/0.15 (CH3Cl3/MeOH/H2O/HCOOH 7:13:8:0.1)) was isolated via CC of 0.24 g of fraction 2 with Sephadex LH 20 and methanol as a mobile phase, as a pure, solid yellow substance. Likewise, 5,6,7,8-tetrahydro-1-O-β-d-glucopyranosyl-3,6-dihydroxy-8- methylnaphthalene (9) (58 mg/Rf (SiO2): 0.59 (EtOAc/EtOH/H2O 100:20:13)/0.19 (CH3Cl3/MeOH/H2O/HCOOH 7:13:8:0.1)) was isolated via CC of 0.11 g of fraction 1 on Sephadex LH 20 and methanol as a mobile phase, and also as a pure, solid yellow substance.Fresh leaves of 2 (prostaglandin H2) into TXA2 (thromboxane A2) by the thromboxane synthase system, malondialdehyde (MDA) is produced at a ratio of 1:1 with 12-HHT (12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid). MDA can be detected photometrically after aldol condensation with thiobutyric acid [g. The supernatant, which is rich in thrombocytes, was carefully collected using a syringe and centrifuged once again for 20 min at 200× g to remove all remaining erythrocytes. In the subsequent centrifugation step at 1000× g for 15 min, the thrombocytes sediment and can finally be re-suspended in isotonic PBS at a defined cell density of 109 cells/mL. All isolation steps can be performed in plastic vessels at room temperature. For the test, 700 µL of this thrombocyte suspension was placed in 1.5 mL Eppendorf vessels , together with 10 µL of the respective inhibitor solution, and incubated for 10 min at 37 °C. The enzyme reaction was started by adding 50 µL of calcium ionophore buffer  and terminated after 10 min at 37 °C by adding 400 µL of trichloroacetic acid. All samples were centrifuged at 4000× g for 15 min, after which the supernatant was used for determining the MDA concentration. A negative control was performed in four repetitions, in which only 10 µL of DMSO solvent were added to the reaction mixture instead of the respective isolated compounds. For the positive control (n = 2), 700 µL of thrombocyte suspension was mixed with 10 µL of solvent and incubated at 37 °C. After 10 min, 400 µL of trichloroacetic acid and subsequently 50 µL of calcium ionophore buffer were added to the mixture. The subsequent steps were identical to those described for the extracted Aloe constituents, as described above. For determining the MDA concentration, 0.5 mL of the abovementioned supernatant was mixed with 0.5 mL of thiobarbituric acid solution. This mixture was incubated in a water bath for 30 min at 70 °C. After another 30 min of incubation at room temperature, the amount of MDA was measured in a spectral fluorometer .During the catalysis of PGHric acid . For the4 (Leukotriene B4) test, the products of the arachidonic acid production can be directly measured via HPLC after the enzyme reaction in the thrombocytes and the subsequent activation reaction have taken place. The produced amounts of LTB4 and 5-HETE (5-Hydroxyeicosatetraenoic acid) were directly detected at 270 nm, revealing the inhibitory potential of the tested substance (inhibition by zileuton as positive control). For the screening, 1.0 L of cow blood was collected in a plastic bucket that already contained 100 mL of sodium-EDTA solution (0.077 M) as an anticoagulant, immediately after sacrificing the animal. The blood was centrifuged at 200× g for 20 min, after which the thrombocytes containing the supernatant were separated. In order to facilitate the lyses of the erythrocytes, the remaining pellet was re-suspended in 800 mL of water. After 30 s of incubation, 400 mL of hypertonic PBS solution was added, followed by another centrifugation step at 485× g for 10 min. The resulting cell pellet was re-suspended in 50 mL of isotonic PBS solution and transferred to 20 mL of Histo-Paque . After yet another centrifugation for 45 min at 675 g, lymphocytes and macrophages that did not sediment in this setup were removed, while the granulocyte pellet was re-suspended in 10 mL of isotonic PBS solution. After an additional washing step in isotonic PBS solution, the granulocytes were re-suspended in O2-saturated, isotonic PBS solution, which had been continuously stirred on a magnetic stirrer at maximum speed for 5 min, at an exact cell density of 6 × 107 cells per ml. Although this granulocyte isolation protocol was performed at room temperature, the light exposure of the cells had to be limited as much as possible. For the enzyme reaction, a glass centrifuge tube was filled with 2.5 µL of the Aloe constituent test solution, or 2.5 µL of DMSO for the kinetic and control measurements. Subsequently, the tubes were filled with 0.8 mL of the above-described granulocyte suspension and incubated for 5 min at 37 °C in the water bath under constant shaking. Thereafter, 0.2 mL of calcium chloride was added, followed by an additional 5 min of incubation at 37 °C. In the next step, leukotriene production was induced by the addition of 2.5 µL of calcium ionophore buffer to the reaction mixture. After an additional 5 min  of incubation at 37 °C, the enzyme reaction was terminated by adding 1 mL of a solution of acetonitrile/methanol (1:1) that contains NDGA (nordihydroguaiaretic acid) as an antioxidant and PGB2 (prostaglandin B2) as an internal standard. All glass centrifuge tubes were put into an ice-water bath immediately after termination of the reaction, sealed, and incubated in the ice-water bath for 20 min. Thereafter, centrifugation was performed for 15 min at 4000 × g under constant cooling at 4 °C. The supernatants were filled into vials, which were flanged and stored at −20 °C for further preparation. Each incubation setup contained 4 control and 8 sample measurements. Each defrosted sample was diluted with 10 mL of water and applied to an RP octadecyl extraction column , which had previously been washed successively with 10 mL MeOH, 5 mL water, and 5 mL of a 0.1% EDTA solution. After washing the column two times with 5 mL of water, the adsorbed substances were eluted with 3 mL of MeOH. This eluate was diluted with 3 mL of water. Subsequently, the concentration of LTB4 was directly measured by HPLC using a Nucleosil 7 µm C18 column (250 mm × 4.6 mm)  with tetrahydrofuran/MeOH/aqueous EDTH solution (0.1%)/acetic acid , pH 5.5 (conc. NH4 aq) as a mobile phase, at a flow rate of 0.9 mL/min. UV detection took place at 270 nm. For measuring the 5-HETE concentration, only the mobile phase was changed to MeOH/H2O/acetic acid , pH 5.5 (conc. NH4 aq) with a flow rate of 1.0 mL/min. UV detection took place at 232 nm. The inhibition of the 5-lipoxygenase systems was then calculated from the measured amounts of LTB4 and 5-HETE. The relative concentration of LTB4/5-HETE in each sample equals the ratio of the peak areas of LTB4/5-HETE, relative to the internal standard PGB2. The inhibition of the 5-lipoxygenase system, therefore, equals the ratio between the LTB4/5-HETE value in the presence and in the absence of the respective Aloe constituent.For the LTB


Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzofuran-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 μM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 μM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.Five new compounds, eupatodibenzofuran A ( Eupatorium fortunei Turcz. (Asteraceae) is a perennial herb widely distributed in China. The aerial part of E. fortunei (Chinese name: Pei-Lan) has been used as a traditional medicine for the treatment of various diseases such as flu, poor appetite, constipation, nausea, and siriasis furan derivatives, eupatodibenzofuran A (1) and eupatodibenzofuran B (2), a new 4-chromanone, 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), a new acetophenone derivative, eupatofortunone (4), a new dithiecine derivative, eupatodithiecine (5), and eleven known compounds (6–16) have been isolated and confirmed from the aerial part of E. fortunei. This report describes the structural elucidation of five new compounds 1–5 and the inhibitory activities of 1 against non-small-cell lung cancer and breast cancer cells.siriasis . Diversesiriasis ,3,4,5,6,siriasis ,7, sesqusiriasis , benzofusiriasis , and theE. fortunei by silica gel chromatography and preparative thin-layer chromatography (TLC) afforded five new (1–5) and eleven known compounds (6–16)  .1 was obtained as colorless needles. Its molecular formula C22H18O4 was deduced from a sodium adduct ion at m/z 369.11041 [M + Na]+  in the HRESIMS spectrum. The presence of hydroxyl (3439 cm−1) and conjugated carbonyl (1645 cm−1) groups were revealed from the IR spectrum. Analysis of the 1H and 13C NMR data of 1 showed the signals for an acetyl group , a chelated hydroxyl group [δH 12.65 ], a methyl group , a 2-hydroxy-4-methylphenyl group , two ortho-coupled aromatic protons , and two singlet aromatic protons  (H 7.65)/Ac-2 (δH 2.41), Me-6 (δH 2.62)/H-7 (δH 7.33), OH-2′ (δH 4.97)/H-3′ (δH 6.96), H-3′ (δH 6.96)/Me-4′ (δH 2.44), and Me-4′ (δH 2.44)/H-5′ (δH 6.93) and by HMBC correlation between ‘Ac-2 (δH 2.41)/C-2 (δC 116.6)’, ‘OH-3 (δH 12.65)/C-2 (δC 116.6), C-4 (δC 99.8)’, ‘Me-6 (δH 2.62)/C-5a (δC 155.9), C-7 (δC 128.6)’, ‘OH-2′ (δH 4.97)/C-3′ (δC 116.1)’, and ‘Me-4′ (δH 2.44)/C-4′ (δC 140.4), C-5′ (δC 121.7)’ -6-methyldibenzofuran-2-yl)ethenone, named eupatodibenzofuran A.Compound 4 (C-1)] . The pos 121.7)’ . The fulC, ROESY A, and HMC, ROESY B spectra2 was isolated as amorphous powder with molecular formula C23H20O4, as determined by positive-ion HRESIMS, showing an [M + Na]+ ion at m/z 383.12612 . The presence of a conjugated carbonyl group was revealed by a band at 1645 cm−1 in the IR spectrum, and was confirmed by the resonance at δC 203.7 in the 13C NMR spectrum. The 1H NMR data of 2 were similar to eupatodibenzofuran A (1), except that the 2′-methoxy group  of 2 replaced the 2′-hydroxy group [δH 4.97 ] of 1. This was supported by the ROESY correlations between OMe-2′ (δH 3.70) and H-3′ (δH 6.91) and by the HMBC correlations between OMe-2′ (δH 3.70) and C-2′ (δC 156.7) (H 7.59)/Ac-2 (δH 2.40), Me-6 (δH 2.60)/H-7 (δH 7.27), H-7 (δH 7.27)/H-8 (δH 7.17), OMe-2′ (δH 3.70)/H-3′ (δH 6.91), H-3′ (δH 6.91)/Me-4′ (δH 2.49), and Me-4′ (δH 2.49)/H-5′ (δH 6.95) and by HMBC correlation between ‘H-1 (δH 7.59)/MeCO-2 (δC 203.7), C-3 (δC 162.9), C-4a (δC 161.3), C-9a (δC 121.7)’, ‘Ac-2 (δH 2.40)/C-2 (δC 116.0)’, ‘OH-3 (δH 12.62)/C-2 (δC 116.0)’, ‘H-4 (δH 7.05)/C-2 (δC 116.0), C-9b (δC 117.8)’, ‘Me-6 (δH 2.60)/C-5a (δC 155.4), C-7 (δC 127.9)’, ‘H-7 (δH 7.27)/C-5a (δC 155.4)’, ‘H-8 (δH 7.17)/C-6 (δC 120.5), C-9a (δC 121.7), C-1′ (δC 125.4)’, ‘H-6′ (δH 7.28)/C-9 (δC 130.6), C-2′ (δC 156.7), C-4′ (δC 139.8)’, ‘OMe-2′ (δH 3.70)/C-2′ (δC 156.7)’, ‘Me-4′ (δH 2.49)/C-4′ (δC 139.8), C-5′ (δC 121.4)’ -6-methyldibenzofuran-2-yl)ethan-1-one, named eupatodibenzofuran B.Compound C 156.7) . The pos 121.4)’ . The fulY, ROESY A, HSQC, Y, ROESY B techniq3 was isolated as light brown amorphous powder. Its molecular formula, C14H16O4, was determined on the basis of the positive HRESIMS at m/z 249.1123 [M + H]+  and was supported by the 1H and 13C NMR data. The IR absorption bands of 3 revealed the presence of carbonyl (1686 cm−1) function. Analyses of the 1H and 13C NMR data of 3 showed the signals for two methyl groups , an acetyl group , a methoxy group , two meta-coupled aromatic protons , and two methylene protons  (H 1.55)/H-3 (δH 2.79), H-5 (δH 8.08)/Ac-6 (δH 2.60), Ac-6 (δH 2.60)/H-7 (δH 7.70), and H-7 (δH 7.70)/OMe-8 (δH 3.95) and HMBC correlations between ‘Me-2 (δH 1.55)/C-2 (δC 81.0), C-3 (δC 48.5)’, ‘H-3 (δH 2.79)/C-2 (δC 81.0), C-4 (δC 191.7), Me-2 (δC 26.5)’, ‘H-5 (δH 8.08)/C-4 (δC 191.7), MeCO-6 (δC 196.6), C-7 (δC 114.3), C-8a (δC 154.0)’, ‘H-7 (δH 7.70)/C-5 (δC 120.0), MeCO-6 (δC 196.6), C-8a (δC 154.0)’, and ‘OMe-8 (δH 3.95)/C-8 (δC 149.8)’ ] . The pos 149.8)’ . The fulve NOESY A, and HMve NOESY B data. C4 was obtained as colorless oil and the molecular formula was determined to be C14H16O3 by HRESIMS [m/z 255.10030 [M + Na]+ ]. The IR spectrum showed the presence of ester and conjugated carbonyl groups at 1736 and 1684 cm−1, respectively. Analysis of the 1H and 13C NMR data of 4 revealed the signals for an acetyl group , a methyl group , a (Z)-(2-methylbut-2-enoyl)oxy group , and three mutually coupled aromatic protons  (H 2.52)/H-3 (δH 7.74), H-3 (δH 7.74)/H-4 (δH 7.12), H-4 (δH 7.12)/Me-5 (δH 2.40), Me-5 (δH 2.40)/H-6 (δH 6.94), H-3′ (δH 6.30)/H-4′ (δH 2.08), and H-3′ (δH 6.30)/H-5′ (δH 2.08) and by HMBC correlations between ‘COMe-2 (δH 2.52)/C-2 (δC 128.3), COMe-2 (δC 197.1)’, ‘H-3 (δH 7.74)/C-1 (δC 149.3), COMe-2 (δC 197.1), C-5 (δC 144.7)’, ‘H-4 (δC 7.12)/C-2 (δC 128.3), C-6 (δC 124.4), Me-5 (δC 21.4)’, ‘Me-5 (δH 2.40)/C-4 (δC 126.7), C-5 (δC 144.7), C-6 (δC 124.4)’, ‘H-6 (δH 6.94)/C-1 (δC 149.3), C-2 (δC 128.3), C-4 (δC 126.7), Me-5 (δC 21.4)’, ‘H-4′ (δH 2.08)/C-2′ (δC 127.0)’, and ‘H-5′ (δH 2.08)/C-1′ (δC 166.1), C-2′ (δC 127.0), C-3′ (δC 141.4)’ -2-acetyl-5-methylphenyl 2-methylbut-2-enoate, named eupatofortunone.Compound 4 (C-3)] . The pos 141.4)’ . The fulC, ROESY A, and HMC, ROESY B spectra5 was isolated as light brown amorphous powder. Its molecular formula, C20H20O4S2, was determined on the basis of the positive HRESIMS at m/z 411.07026 [M + Na]+  and was supported by the 1H and 13C NMR data. The IR absorption bands of 5 revealed the presence of alkynyl (2230 cm−1) and conjugated carbonyl (1639 cm−1) functions. Analysis of the 1H and 13C NMR data of 5 revealed the signals for an acetyl group , a prop-1-yn-1-yl group , a methoxy group , and a singlet aromatic proton  (H 2.50)/C-2 (δC 122.6), COMe-2 (δC 190.2)’, ‘C≡CMe (δH 2.09)/C≡CMe (δC 73.2), C≡CMe (δC 94.8)’, ‘H-4 (δH 6.84)/C-2 (δC 122.6), C-3 (δC 130.6), C≡CMe (δC 73.2)’, ‘OMe-5 (δH 3.94)/C-5 (δC 159.3)’ and by the 1D selective NOESY correlation between H-4 (δH 6.84) and OMe-5 (δH 3.94) -5,7-dimethoxy-3,9-di(prop-1-yn-1-yl)-1,6-dithiecine-2,10-diyl)diethanone, named eupatodithiecine.Compound 1 (C-4)] . The posδH 3.94) . Accordive NOESY A, HSQC, ve NOESY B data. C1H NMR, and MS) with those of authentic samples or literature values. They include four thymol derivatives, thymyl angelate (6)  was used for column chromatography (CC). Silica gel 60 F-254  was used for thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC).Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), rotating frame nuclear Overhauser effect spectroscopy (ROESY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple-bond correlation (HMBC), and heteronuclear single-quantum coherence (HSQC) experiments, were acquired using a BRUKER AVIII-500 spectrometer , operating at 500 MHz  was pulverized and extracted three times with MeOH (30 L each) for 3 days. The MeOH extract was concentrated under reduced pressure at 35 °C, and the residue (123.7 g) was partitioned between EtOAc and H2O (1:1) to provide the EtOAc-soluble fraction . Fraction A (25.6 g) was chromatographed on silica gel , eluting with n-hexane, gradually increasing the polarity with EtOAc to give ten fractions: A1 , A2 , A3 , A4 , A5 , A6 , A7 , A8 , A9 , and A10 . Fraction A2 (1.8 g) was subjected to column chromatography (CC)  to give 11 subfractions: A2-1–A2-11. Part (125 mg) of fraction A2-2 was further purified by preparative TLC  to yield thymyl angelate (6) (5.2 mg) and 8,9-dehydrothymol 3-O-tiglate (7) (4.1 mg). Part (95 mg) of fraction A2-5 was further purified by preparative TLC  to obtain 2-hydroxy-4-methylacetophenone (10) (3.2 mg). Part (76 mg) of fraction A2-8 was further purified by preparative TLC  to afford eupatofortunone (4) (4.6 mg). Fraction A3 (1.6 g) was subjected to CC  to give ten subfractions: A3-1–A3-10. Part (135 mg) of fraction A3-9 was further purified by preparative TLC  to obtain coumarin (15) (7.8 mg). Fraction A4 (2.3 g) was purified by medium pressure liquid chromatography (MPLC)  to give eight subfractions: A4-1–A4-8. Fraction A4-5 (265 mg) was purified by MPLC  to afford four subfractions . Fraction A4-5-3 (48 mg) was further purified by preparative TLC  to obtain trans-O-coumaric acid (11) (8.9 mg). Fraction A4-7 (53 mg) was further purified by preparative TLC  to obtain 2,4-di-tert-butylphenol (13) (3.2 mg). Faction A6 (2.2 g) was subjected to MPLC  to give 13 subfractions: A6-1–A6-13. Part (92 mg) of fraction A6-6 was further purified by preparative TLC  to afford eupatodibenzofuran A (1) (4.3 mg) and eupatodibenzofuran B (2) (3.2 mg). Fraction A6-7 (226 mg) was purified by MPLC  to afford 5 subfractions . Fraction A6-7-2 (49 mg) was further purified by preparative TLC  to obtain taraxasterol (16) (4.7 mg). Part (92 mg) of fraction A6-10 was further purified by preparative TLC  to yield 9-angeloyloxythymol (8) (5.1 mg) and 9-O-angeloyl-8,10-dehydrothymol (9) (4.3 mg). Part (111 mg) of fraction A6-11 was purified by preparative TLC  to obtain 6-hydroxy-7-methoxy-2-isopropenyl-5-acetylcumaran (12) (5.3 mg). Part (75 mg) of fraction A6-13 was further purified by semipreparative normal-phase high-performance liquid chromatography (HPLC)  to afford 1-(2-hydroxy-5-methoxy-4-methylphenyl)ethanone (14) (4.2 mg). Faction A8 (1.6 g) was subjected to CC  to give ten subfractions: A8-1–A8-10. Fraction A8-4 (88 mg) was further purified by semipreparative normal-phase HPLC  to afford eupatodithiecine (5) (3.7 mg). Part (93 mg) of fraction A8-7 was further purified by preparative TLC  to yield 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3) (4.4 mg).The aerial part of 1): Colorless needles; mp 153–155 °C (CH2Cl2); UV (MeOH) λmax (log ε) 238 (4.09), 272 (3.97), 352 (1.43) nm; IR (KBr) υmax 3439 (OH), 1645 (C=O) cm−1; 1H and 13C NMR data, see m/z 369.11041 [M + Na]+ ; HRESIMS, 1D-, and 2D-NMR spectra, see Eupatodibenzofuran A (2): Amorphous powder; UV (MeOH) λmax (log ε) 238 (4.29), 271 (4.12), 348 (3.24) nm; IR (neat) υmax 3429 (OH), 1645 (C=O) cm−1; 1H and 13C NMR data, see m/z 383.12612 [M + Na]+ ; HRESIMS, 1D-, and 2D-NMR spectra, see Eupatodibenzofuran B (3): Light brown amorphous powder; UV (MeOH) λmax (log ε) 247 (4.08), 280 , 329 (3.24) nm; IR (neat) υmax 1686 (C=O) cm−1; 1H and 13C NMR data, see m/z 249.1123 [M + H]+ ; HRESIMS, 1D-, and 2D-NMR spectra, see 6-Acetyl-8-methoxy-2,2-dimethylchroman-4-one (4): Colorless oil; UV (MeOH) λmax (log ε) 211 (4.18), 246 , 284  nm; IR (neat) υmax 1736 (C=O), 1684 (C=O) cm−1; 1H and 13C NMR data, see m/z 255.10030 [M + Na]+ ; HRESIMS, 1D-, and 2D-NMR spectra, see Eupatofortunone (5): Light brown amorphous powder; UV (MeOH) λmax (log ε) 227 (4.03), 323 (4.26) nm; IR (neat) υmax 2230 (C≡C), 1639 (C=O) cm−1; 1H and 13C NMR data, see m/z 411.07026 [M + Na]+ ; HRESIMS, 1D-, and 2D-NMR spectra, see Eupatodithiecine  and human breast cancer cell (MCF-7) were obtained from American Type Culture Collection  and cultivated in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/mL penicillin and 100 µg/mL streptomycin . Cells were washed by warm phosphate buffered saline (PBS) every day and changed medium every 2–3 days [All cell lines were cultured at 37 °C under a humidified atmosphere with 5% CO2–3 days ,22.3 cells in 200 μL per well were plated in 96-well culture plates and cultured in complete medium overnight. After 24 h, cells were treated with different concentrations  of compounds 1–16. Fluorouracil (5-FU)  was used as a positive control against A549 and MCF-7 cells with IC50 values of 10.57 ± 1.89 and 8.59 ± 1.03 μM, respectively. The optical density at 570 nm was measured by ELISA plate reader (μ Quant) and the IC50 value was calculated. The optical density of formazan formed in control (untreated) cells was taken as 100% viability.The cell viability was conducted by the MTT assay as previously described method . Briefly3 cells per well and incubated for 12 h. The cells were then treated with the indicated concentrations of compound 1, and cultured for 10 days. The cells were washed three times using PBS and fixed using 95% methanol for 30 min. After washing three times with distilled water, the cells were stained using 0.2% crystal violet dye for 20 min and rinsed with distilled water to wash away the excess dye. The visible colonies were compared with the control samples and photographed using a standard camera under natural light.The colony-formation assay was determined by the reference method with a slight modification . In this6 cells/mL respectively. After 24 h incubation, the cells were treated with following concentrations of 0, 5, and 10 μM for compound 1. After 24 h, they were washed and re-suspended in PBS solution (500 μL). Then, Annexin V-FITC (5 μL) and PI staining solution (5 μL) were introduced to the mixture, and the incubation process was followed under the dark condition for 5 min at 25 °C. Finally, flow cytometer analysis  was performed using an AnnexinV-FITC Apoptosis Detection Kit  and Flowjo version 7.6.1. Software.Annexin V/PI assay was used to determine the apoptotic and necrotic cells. The A549 and MCF-7 cells were seeded on 6-well microplates at a density of 105 cells) were seeded into 6-wells plate and grown until 85–90% confluent. Then different concentrations  of compound 1 were added. Cells were collected and lysed by radioimmunoprecipitation assay (RIPA) buffer. Lysates of total protein were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated with anti-Bax, anti-Bcl-2 , anti-caspase-3, and anti-β-actin  primary antibodies at 4 °C overnight. Then each membrane was washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h while shaking. Finally, each membrane was developed using enhanced chemiluminescence (ECL) detection kit, and the images were visualized by ImageQuant LAS 4000 Mini biomolecular imager . Band densities were quantified using ImageJ software .Western blot analysis was performed according to the method previously reported ,26. Briet-test. A probability of 0.05 or less was deemed significant. The software Microsoft Excel 2016 was used for the statistical analysis.Results were expressed as mean ± SEM, and comparisons were made applying Student’s E. fortunei. Among these isolated compounds, compound 1 markedly induces apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways. The compound 1 belongs to hydrophobic molecule, so it can cross the cell membrane by passive diffusion, a nonselective process. During passive diffusion, compound 1 simply dissolves in the phospholipid bilayer, diffuses across it, and then dissolves in the aqueous solution at the other side of the membrane. During this process, no membrane proteins are involved and the direction of transport is determined simply by the relative concentrations of the molecule inside and outside of the cell. This suggests that eupatodibenzofuran A (1) is worth further investigation and may be expectantly developed as a candidate for the treatment or prevention of non-small-cell lung cancer and breast cancer.Five new (including two with new carbon skeleton) and eleven known compounds have been isolated and identified from the aerial part of


Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 μM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 μM (1a) and 23.1 μM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 μM, with no toxicity for NCTC mammalian cells (CC50 > 200 μM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, ( Leishmania sp. Different forms of this disease can be found, and the visceral is responsible for the high lethality of this disease + . S,3R,4R)-3-Hydroxy-4-methyl-2-(n-eicos-11′-ynil)butanolide (2). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 4.43 , 4.24 , 2.55 , 2.11 , 1.78 , 1.63 , 1.41 , 1.26 . 13C NMR , δ 178.0 (C-1), 139.2 (C-19′), 114.2 (C-20′), 80.3 (C-12′), 80.2 (C-11′), 79.1 (C-4), 71.3 (C-3), 47.7 (C-2), 33.8 (C-18′), 28.7–29.6 (C-3′ to C-9′ and C-14′ to C-17′), 27.7 (C-2′), 23.4 (C-1′), 18.8 (C-10′ and C-13′), 13.8 (CH3-4). ESI-HRMS (positive mode) m/z 393.3382 [M + H]+ . (21 + 2 (100 mg) dissolved in n-hexane (5 mL) and Ni-Raney catalyst (10 mg). After adding H2 (18 atm), the mixture was stirred for 3 h at 100 °C. Then, the catalyst was removed by filtration over a bed of celite. Purification of the product by silica gel chromatography (n-hexane/EtOAc 9:1) afforded compound 3 .In a high-pressure reactor (stainless steel) was added a mixture of compounds S,3R,4R)-3-Hydroxy-4-methyl-2-(n-eicosanil)butanolide (3). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 4.44 , 4.31 , 2.57 , 1.82 , 1.64 , 1.43 , 1.25 , 0.87 . 13C NMR  δ 177.6 (C-1), 79.9 (C-4), 71.4 (C-3), 47.7 (C-2), 32.0 (C-18′), 29.5–29.8 (C-3′ to C-17′), 27.7 (C-2′), 23.4 (C-1′), 22.8 (C-19′), 14.2 (C-20′), 13.8 (CH3-4). ESI-HRMS (positive mode) m/z 419.3480 [M + Na]+ .(21 (15 mg), 2 (12 mg), or 3 (58 mg) were individually dissolved in pyridine (5 mL) and cooled to 0 °C. Acetic anhydride (5 mL) was added to each solution and was stirred for 15 h at room temperature. After adding cold H2O (20 mL), the residue was extracted with CHCl3 (3 × 15 mL). Organic layers were dried over Na2SO4, filtered, and concentrated. This procedure afforded 1a , 2a , and 3a .Solutions of compounds S,3R,4R)-3-Acetoxy-4-methyl-2-(n-eicos-11′-yn-19′-enil)butanolide (1a). Amorphous white solid, 99% purity by HPLC. 1H NMR  δ 5.80 , 5.59 , 4.96 , 4.55 , 2.69 , 2.13 , 2.13 , 2.04 , 1.81 , 1.46 , 1.31 , 1.25 . 13C NMR : δ 176.4 (C-1), 170.0 (CH3COO), 139.1 (C-19′), 114.2 (C-20′), 80.2 (C-12′), 80.1 (C-11′), 77.3 (C-4), 72.4 (C-3), 45.6 (C-2), 33.7 (C-18′), 28.6–29.7 (C-3′ to C-9′ and C-14′ to C-17′), 27.3 (C-2′), 23.7 (C-1′), 20.4 (CH3COO), 18.7 (C-10′ and C-13′), 14.0 (CH3-4). ESI-HRMS (positive mode) m/z 455.3210 [M + Na]+ .-3-Acetoxy-4-methyl-2-(n-eicos-11′-ynil)butanolide (2a). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 5.59 , 4.56 , 2.69 , 2.13 , 2.13 , 1.46 , 1.32 , 1.26 , 0.87 . 13C NMR  δ 176.4 (C-1), 170.0 (CH3COO), 80.2 (C-12′), 80.1 (C-11′), 77.3 (C-4), 72.4 (C-3), 45.6 (C-2), 31.8 (C-18′), 28.6–29.7 (C-3′ to C-9′ and C-14′ to C-17′), 27.3 (C-2′), 23.7 (C-1′), 22.7 (C-19′), 20.4 (CH3COO), 18.7 (C-10′ and C-13′), 14.1 (C-20′), 14.0 (CH3-4). ESI-HRMS (positive mode) m/z 457.3006 [M + Na]+ .-3-Acetoxy-4-methyl-2-(n-eicosanil)butanolide (3a). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 5.59 , 4.55 , 2.69 , 2.12 , 1.82 , 1.31 , 1.25 , 0.87 . 13C NMR  δ 176.5 (C-1), 170.1 (CH3COO), 77.5 (C-4), 72.4 (C-3), 45.8 (C-2), 32.0 (C-18′), 29.8–29.4 (C-3′ to C-17′), 27.4 (C-2′), 23.8 (C-1′), 22.8 (C-19′), 20.5 (CH3COO), 14.2 (C-20′), 14.1 (CH3-4). ESI-HRMS (positive mode) m/z 461.3602 [M + Na]+ .(23a (30 mg) was placed on the top of an Al2O3 90  column. Elution with pentane afforded 3b . This same procedure was performed using a mixture of compounds 1a + 2a (20 mg), and obtained products were purified by HPLC  to afford pure compounds 1b (11 mg) and 2b (4 mg). Compound R-Methyl-2-(n-eicos-11′-yn-19′-enil)but-2-enolide (1b). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 6.97 , 5.80 , 5.00 , 4.94 , 2.26 , 2.13 , 2.04 , 1.40 , 1.27 . 13C NMR  δ 174.0 (C-1), 148.9 (C-3), 139.2 (C-19′),134.4 (C-2), 114.3 (C-20′), 80.4 (C-12′), 80.3 (C-11′), 77.5 (C-4), 33.8 (C-18′), 27.5 (C-2′), 25.3–29.6 (C-3′ to C-9′ and C-14′ to C-17′), 25.2 (C-1′), 18.8 (C-10′ and C-13′), 19.2 (CH3-4). ESI-HRMS (positive mode) m/z 395.2958 [M + Na]+ .4R-Methyl-2-(n-eicos-11′-ynil)but-2-enolide (2b). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 6.97 , 4.99 , 2.26 , 2.13 , 2.11 , 1.27 , 0.87 . 13C NMR  δ 174.0 (C-1), 148.9 (C-3), 134.4 (C-2), 80.4 (C-12′), 80.3 (C-11′), 77.5 (C-4), 27.5 (C-2′), 25.3–29.6 (C-3′ to C-9′ and C-14′ to C-18′), 25.2 (C-1′), 22.7 (C-19′), 18.8 (C-10′ and C-13′), 19.2 (CH3-4), 18.8 (C-20). ESI-HRMS (positive mode) m/z 397.3118 [M + Na]+ .4R-Methyl-2-(n-eicosanil)but-2-enolide (3b). Amorphous white solid, 100% purity by HPLC. 1H NMR  δ 6.97 , 4.98 , 2.25 , 1.56 , 1.40 , 1.25 , 0.87 . 13C NMR  δ 173.9 (C-1), 148.8 (C-3), 134.3 (C-2), 77.4 (C-4), 31.9 (C-18′), 29.7–29.2 (C-3′ to C-17′), 27.4 (C-2′), 25.1 (C-1′), 22.7 (C-19′), 19.2 (C-20′), 14.1 (CH3-4). ESI-HRMS (positive mode) m/z 401.3387 [M + Na]+ .41, 1a, and 1b were conducted using the online platform SwissADME (available online: http://www.swissadme.ch/ (14 January 2022)). Using this approach, different aspects associated with pharmacokinetics, drug-likeness, and medicinal chemistry parameters were determined, including: ADME , physicochemical properties , lipophilicity , pharmacokinetics , drug-likeness, especially Lipinski rule, and alert for PAINS (pan-assay interference compounds) [In silico studies for bioactive compounds mpounds) .Mesocricetus auratus). The animals were maintained in sterilized cages under a controlled environment, receiving water and food ad libitum. Animal procedures were performed with the approval of the Research Ethics Commission, which is in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences.The animal breeding facility at the Instituto Adolfo Lutz, São Paulo, Brazil supplied the BALB/c mice and Golden hamsters (L. (L.) infantum (MHOM/BR/1972/LD) was maintained through successive passages in golden hamsters up to 60–70 days post-infection. Amastigotes were harvested from spleens of infected hamsters by differential centrifugation [fugation .2. NCTC (clone 929) cells were maintained in RPMI-1640 (without phenol red and supplemented with 10% FBS) at 37 °C in the same conditions of the peritoneal macrophages [Macrophages were collected from the peritoneal cavity of female BALB/c mice by washing with RPMI-1640 without phenol red, supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% COrophages .50) against intracellular amastigotes for tested compounds were determined in infected macrophages. Peritoneal macrophages were obtained as described in the L. (L.) infantum amastigotes were obtained from spleens of infected hamsters by differential centrifugation. Peritoneal macrophages were seeded at 1 × 105 cells per well in Nunc™ 16-well slide chambers  for 24 h at 37 °C in a 5% CO2-humidified incubator. Next, amastigotes were isolated from a spleen of an infected hamster as described [2-humidified incubator, using miltefosine as a standard drug. At the end of the assay, the cells were fixed in methanol, stained with Giemsa, and observed under a digital light microscope  to determine the number of infected macrophages out of 400 cells [The 50% inhibitory concentrations  was determined in NCTC clone 929 cells. NCTC cells were seeded at 6 × 104 cells/well in 96-well microplates and incubated with serial dilutions of the tested compounds or standard drug (miltefosine) to the highest concentration of 200 μM for 48 h at 37 °C in a 5% CO2-humidified incubator. An MTT assay determined the viability of the cells at 570 nm [50 NCTC cells/IC50 parasites.The 50% cytotoxic concentration .The data obtained represent the mean and standard deviation of duplicate samples from at least three independent assays. ICP. macrocarpa (1 and 2) and seven chemically related derivatives . Among these compounds, 1 and 1b exhibited more substantial potential, with similar IC50 values of positive control (miltefosine) and reduced mammalian cytotoxicity. However, chemically related compounds 2, 2a, 2b, 3, 3a, and 3b showed no activity, indicating that a double bond at C-19 and a triple bond at C-11 are crucial to the antileishmanial effect. However, the effect observed to the mixture of natural acetogenins 1 and 2 at 2:1 proportion showed superior selectivity to those determined to the isolated compounds. Therefore, our data corroborate the promising activity of acetylene derivatives from P. macrocarpa as antiprotozoal agents, especially for the mixture of natural acetogenins 1 and 2.This work reports the isolation and antileishmanial activity of two natural acetogenins isolated from seeds of


Our work highlights the importanceof finding productive ways to perturb existing water networks whengrowing into solvent-filled protein pockets.We describe the optimizationof modestly active starting pointsto potent inhibitors of BCL6 by growing into a subpocket, which wasoccupied by a network of five stably bound water molecules. Identifyingpotent inhibitors required not only forming new interactions in thesubpocket but also perturbing the water network in a productive, potency-increasingfashion while controlling the physicochemical properties. We achievedthis goal in a sequential manner by systematically probing the pocketand the water network, ultimately achieving a 100-fold improvementof activity. The most potent compounds displaced three of the fiveinitial water molecules and formed hydrogen bonds with the remainingtwo. Compound  Germinal center B-cells are dependent on the expressionof BCL6 (B-cell lymphoma 6 protein), which is a transcriptional factorthat binds to corepressor proteins NCOR, SMRT, or BCOR via a peptide-bindingsurface on its BTB domain dimer.3 Through this interaction,BCL6 represses genes involved in cell cycle control, cell death, anddifferentiation.4 Most B-cell lymphomasarise from germinal center B-cells.7 Once oncogenic, the tumorrelies on the sustained expression of BCL6.8 In the case of DLBCL, blocking the interaction between BCL6 andits corepressors enables the re-expression of repressed genes, whichleads to the continuation of B-cell differentiation and cell death.9 This highlights potential therapeutic efficacyfor inhibitors of this BCL6 protein–protein interaction.Diffuse large B-celllymphomas (DLBCLs) are the most common subtypeof non-Hodgkin lymphoma.10 macrocycles,11 as well as small molecule inhibitors.17 Molecules, which induce degradation of BCL6, have also been reported,including monovalent degraders and a PROTAC.18Inhibitors of the BCL6-corepressor protein–protein interactionbinding to the BCL6 BTB domain have been described. These includehigh-affinity peptides,CCT365386 (1) yielding potent BCL6 degraders,including cell-active CCT369260.17 However, in this benzimidazolone series, we were only able to obtaininhibitors of modest potency. We wished to explore alternative corescaffolds with alternative vectors, which would allow us to improvepotency by growing more efficiently into subpockets on the protein.We hypothesized that replacing the benzimidazolone core with a quinolinonecould maintain the important interactions for potent binding: a hydrogen-bonddonation from the central nitrogen to Met51, with the acceptance ofa hydrogen bond from the main chain N–H of Glu115 onto thequinolinone carbonyl, matching the hydrogen-bond pattern seen withthe benzimidazolones. Additionally, a quinolinone scaffold would supplytwo new positions for vector exploration (C(3) and C(4)) that weredistinct from the benzimidazolone.We havepreviously reported the optimization of hit compound 2 as a proof-of-concept compoundand found that it inhibited BCL6 with an IC50 of 2.7 μMin our biochemical time-resolved fluorescence energy transfer (TR-FRET)assay (1.We prepared quinolinone T)assay 1, repres25, a BCL6 inhibitor witha submicromolar cellular activity.We report below the optimizationof the quinolinone chemical series,which resulted in compound 1 was prepared by a single-stepnucleophilic aromatic substitution reaction from available buildingblocks,17 while its quinolinone counterpart 2 was synthesized in a three-step sequence from commerciallyavailable 6-nitroquinolin-2(1H)-one 3 -one 3 1.H)-one compounds weremade from 4-chloro-1-methyl-6-nitroquinolin-2(1H)-oneintermediate 5, which was formed via acid-catalyzed hydrolysisand subsequent nitration of 2,4-dichloroquinoline 4.Substitution in the 4-position was achieved through nucleophilic aromaticsubstitution, palladium-catalyzed Buchwald-type, or Suzuki couplingsto give nitro-intermediates 6a–k. Reduction ofthe nitro-intermediates 6a–k was either achievedthrough reduction with tin chloride or transfer hydrogenation withPd/C and ammonium formate to give aromatic amino compounds 7a–k. The final step for this group of compounds is the nucleophilicsubstitution with 2,4-dichloropyridine-3-carbonitrile and triethylamineor diisopropylethylamine (DIPEA) as a base if needed to give compounds 8a–f and 12a–e.Substitutedquinolin-2-one intermediate 5. N-Alkylationwas achieved through reaction with sodium hydride and iodomethaneor through cesium carbonate and either (bromomethyl)cyclopropane or(bromomethyl)cyclobutane to give 9, 14a,and 14b, respectively. Substitution in the 4-positionof N1-methyl-quinolinone 9 was achieved through nucleophilic aromatic substitution or Buchwald-typecouplings with primary amines. Reduction of these 4-substituted nitro-quinolinones 10a–h was achieved through the use of tin chlorideor by transfer hydrogenation with Pd/C and ammonium formate. Substitutionwith 1-pyrimidin-2-ylethanamine in the 4-position of N1-methylcyclopropyl and N1-methylcyclobutyl quinolinones 14a,b was achieved throughBuchwald-type couplings. The resulting nitro-quinolinones 15a,b were reduced via transfer hydrogenation and coupled to 2,4-dichloropyridine-3-carbonitrilevia nucleophilic aromatic substitution to give 16a,b.The final step for all N1-substitutedcompounds was nucleophilic aromatic substitution with 4,6-dichloro-5-cyanopicolinicacid or 2,4-dichloropyridine-3-carbonitrile with DIPEA as a base ifneeded H of thequinolinone either solvent-facing (blue) or protein-facing (orange)2. For boN1-position of the quinolinone core.Having validated the quinolinone core, our next aim was to improvethe potency through growing from this relatively small molecule. Weidentified a subpocket below the quinolinone core that is part ofthe peptide-binding groove on the BCL6 BTB domain but not currentlyoccupied by our ligands. Our aim was to increase potency by growinginto this pocket. Importantly, the C(4)-position of the quinolinonein the protein-facing conformation seemed well positioned to extendinto this pocket with a variety of substituents. However, the pocketwas occupied by a network of five water molecules conserved in severalin-house ligand-bound BCL6 structures. Growing into this pocket wouldinevitably perturb this water network, and this perturbation was likelyto have an effect on the potency of new ligands. We suspected thatthe key challenge in improving potency by growing into this pocketwas to discover substituents that not only engage in interactionswith the protein but also interact with and perturb the water networkin a productive way. Furthermore, we hypothesized that we can discoversuch substituents by probing the pocket by introducing a range ofsmall substituents in the C(4)-position of the core. A representativeset of carbon- and nitrogen-linked derivatives are summarized in 8a–8f showed no increase in potency inour biochemical assay.Moreover, the structure–activity relationship (SAR) was flatwith IC50s of 1.5–2.5 μM and hence did notprovide any indications as to which substituents bind preferentiallyin the targeted pocket. Despite the flat SAR, we hypothesized thatsome of the compounds may still place the newly introduced substituentsin the pocket. Furthermore, we speculated that these represent betterstarting points since the substituent can be modified further to deriveadditional interactions and more completely fill the extended pocket.Since our initial starting point bound in two conformations and theflat SAR did not reveal the binding preference, we progressed allcompounds to crystallography. To rapidly confirm the binding modeof these compounds, we solved the BCL6 protein–ligand structuresbut initially did not model and refine the bound ligand. Instead,we just checked for the presence of ligand density and, in particular,for electron density in the extended pocket. Ligand density in thepocket is consistent with the compound bound in the pocket-facingconformation, whereas the presence of electron density for the coreof the ligand, but not in the pocket, suggested that the compoundbound in the solvent-facing conformation. This crystallographic analysisgave us an insight into the different binding modes of compounds withotherwise similar biochemical potency.Disappointingly, this set of compounds 2. Secondary amines, including dimethylamine 8a, piperidine 8b, piperazine 8c, and morpholine 8d, were all found to bind solely in the solvent-facing conformation.Because the second pocket-facing conformation was not observed, wesuggest that the substituents did not interact favorably with theextended pocket despite their similar potency consistent with theflat SAR observed. There were two compounds for which ligand densitywas observed in the extended pocket: carbon-linked pyrazole 8e and ethylamine 8f. To understand how thesecompounds bound, we modeled the compounds and fully refined theirstructures. The pyrazole compound 8e was shown to bindin both orientations with comparable occupancies, in a similar fashionto 2 . Interestingly, ethylamine-substituted quinolinone 8f bound solely orientated with the 4-substituent in the extendedpocket. A detailed analysis of the 8f-bound BCL6 structure when a water moleculeis replaced by a hydrophobic probe at the coordinates of a water moleculeseen in an X-ray structure. A positive predicted free-energy difference(ΔΔG) indicates that displacing a waterwith a hydrophobic group at this location is favored, a negative thatthis is disfavored. In the case of ethylamine compound 8f, the remaining waters are all calculated to have a negative free-energydifference , adding an ET assay 3.12a was aracemic mixture, we next synthesizedthe two enantiomers. They showed different potency, with the (R)-enantiomer 12b showing an IC50 of 0.25 μM, which translates as a ninefold increase for theintroduction of the amide group. The (S)-enantiomer 12c was significantly less potent. To rationalize this SARand derive design hypotheses, we solved the crystal structures ofboth compounds. Consistent with its lower activity, the (S)-enantiomer bound in the solvent-facing orientation with the amidesubstituent pointing toward the bulk solvent with no interaction createdbetween the quinolinone C(4)-substituent and the protein. The structureof the (R)-enantiomer proved to be more interesting.It bound in the pocket-facing orientation, and the ethylamine portionof the molecule was situated in the same position as observed with 8f, displacing water 1. Not surprisingly, introducing theamide substituent substantially perturbed the water network. It displacedwater 3, positioning the carbonyl oxygen atom in this position. Thecarbonyl function also engaged in a polar interaction with water 4,with the distance (water to carbonyl oxygen of 2.73 Å) and angles(C=O···O angle of 124.6° with the wateroxygen in the plane of the carbonyl) consistent with a strong hydrogenbond. Finally, the NH moiety of the amide acted as a donor and engagedin a hydrogen bond (amide nitrogen to water oxygen distance of 2.83Å) with water 2 (As amide  water 2 4.11d resulting in 12d but in this case observeda loss of potency (IC50 = 1.08 μM), suggesting thatthe SAR for the ethyl and the cyclopropylmethyl substituents is different.We introduced the same amide group to cyclopropyl-substituted 12a led to a 30-fold loss of activity(12e). This result highlights the critical importanceof this methyl group to anchor the substituent in the pocket, likelyby displacing water 1 and engaging in hydrophobic interaction withthe side chain of Val18. Consistent with its poor potency, the crystalstructure of 12e revealed that it bound in the solvent-facingorientation, with the C4 substituent seen in multiple conformations,highlighting its poor stabilization .Another revealing observation was that the removal of the terminalmethyl group from R)-ethyl amide 12b achievinga ninefold improvement in potency, we next profiled the compound further.Not surprisingly, given that the compound featured four hydrogen-bonddonors, we observed no measurable passive permeability in a PAMPAassay (N1-position with a methylgroup (13a). While this modification maintained potency, 13a still showed no passive permeability (PAMPA Pe <0.2× 10–6 cm s–1), likely dueto the combination of three remaining hydrogen-bond donors and relativelylow lipophilicity . 1,3,4-Oxadiazole 13c also showed comparable potencyto amide 13a. Interestingly, N-Me triazole 13d brought a fourfold improvement in potency but no measurable improvementin passive permeability, suggesting that the triazole introduced toomuch polarity. Gratifyingly, the pyrimidine analogue 13e provided a significant breakthrough. It was not only sevenfold morepotent but also had a measurable permeability of 1.4 × 10–6 cm s–1 in our PAMPA assay. Thisenabled us to progress into the cellular assay. In contrast to theTR-FRET assay, which uses the BTB domain of BCL6 and a short peptidefrom the corepressor, the NanoBRET assay uses full-length BCL6 andcorepressor. Pleasingly, compound 13e showed measurableactivity (1.21 μM), which we looked to optimize by further increasingbinding affinity using a structure-based design.Consequently, we turned to amide binding isosteres,particularlyheteroaromatic groups.pothesis4. Oxazol13e. Ashypothesized, the pyrimidine adopted the position of the amide groupin 12a, displacing water 3 while acting as a double hydrogen-bondacceptor from waters 2 and 4 with water positions consistent withinteractions with the in-plane lone pairs on the pyrimidine nitrogens(N···H–O distance 2.8 Å). The ethylamineportion of the substituent maintained its position, with the terminalmethyl group displacing water 1 and forming lipophilic interactionswith Val18 (R)-enantiomer in the structure. We thereforesynthesized the single (R)-enantiomer 13f, which indeed showed the expected minor improvement of biochemicalpotency (IC50 = 0.082 μM)  4.13f, we had improved biochemicalpotencyand achieved a measurable cellular activity. However, despite thesignificant improvement, the passive permeability was still <1× 10–6 cm s–1. To increasethe permeability of 13f, we aimed to incorporate morelipophilicity into the molecule. Inspection of the crystal structureled to different hypotheses as to where lipophilic substituents couldbe tolerated. Modification at the N1-positionwith lipophilic substituents yielded N1-methylcyclopropyl and N1-methylcyclobutylcompounds 17a and 17b. These confirmed thatincreasing the lipophilicity can indeed lead to increased permeation gain in potencycompared to the (R)-enantiomer 13f.We progressed this compound to crystallography. The X-ray structureshowed that the newly introduced methyl group indeed replaced water5 and otherwise bound in an analogous fashion to 13e,displacing waters 1 and 3. Distances from pyrimidine nitrogen to waters2 (2.83 Å) and 4 (2.88 Å) are consistent with hydrogen bonding,although the position of water 4 is no longer coplanar, potentiallyweakening this interaction –NH bond and theC(α)-pyrimidine bond . This analysis suggests that introducing the methylgroup to yield 13g preorganizes the torsion angle towarda single conformation close to the bioactive conformation. This analysisis consistent with our hypothesis that the conformation preferenceintroduced by the methyl group at least partially compensates forloss due to replacing water 5. Moreover, we observed that the torsionangle slightly differed between 13e and 13g and it is possible that the angle observed for 13g leadsto improved interaction of the pyrimidine ring and thus also contributesto off-setting the penalty caused by replacing a relatively stablewater with a lipophilic moiety.The observation that replacing water 5 in the complex of ragments 7 of bothine bond 7. These 13g proved to be an effectiveinhibitor inour cellular NanoBRET assay . However, while the lipophilicity translated intoa slight increase in cellular activity, its PAMPA permeability wasstill relatively low. To optimize passive permeability further, wecombined the lipophilic substitution of the chiral carbon (13g) and the extended pyrimidine (24b). Gratifyingly, theincreased lipophilicity of the resulting compound 25 enabledsatisfactory permeability (35 × 10–6 cm s–1) for the first time in this series while maintainingan acceptable solubility of 25 (30 μM). Biochemicaland cellular potency was maintained, and binding affinity to the BCL6BTB domain was also confirmed by SPR .Compound 25 and 4 (2.96 Å). In addition, the cyclopropyl group of 25 is shown to fold down over the edge of the pocket, providing additionalhydrophobic contacts.TheX-ray structure of 25 8 shows tSupplementary Figure S4). Although this datais encouraging, with 25 fulfilling our requirements foran early lead compound, clearly more potent inhibitors of the BCL6:corepressorprotein–protein interaction are required to enable the testingof the therapeutic hypothesis in vivo. Nevertheless, to assess whetherthis series had the potential for further optimization and identifypotential issues, we then analyzed in vivo pharmacokinetic(PK) parameters in a female Balb\c mouse study , dosing at 1 mg kg–1 i.v. (n = 3) and 5 mg kg–1 p.o. (n = 3). All experiments were carried out according to the UKCC guidelinesfor animal experimentation, and no toxicity was observed. 25 (CCT369347) demonstrated moderate clearance (CL 24 mL min–1 kg–1) with a mean oral bioavailability of 29%.Protein binding measurements using equilibrium dialysis showed thatthe compound is 1.3% free in mouse plasma.To further assess the potentialof this series, we carried outadditional profiling. We conducted 14 day viability assays in a panelof BCL6-positive lymphoma cell lines and in BCL6-low or negative cell lines  to give anindication of specificity. As expected based on the NanoBRET data,we observed GI50 value ranging from 1 to 4 μM in the BCL6-dependentcell lines and weaker activity in those cell lines with low BCL6 expression bound in a single conformation withits C(4) substituent extended into the subpocket. All other compoundsbound either in the alternative or in both binding orientations. 8f provided critical information on how the pocket can bestably occupied through forming an additional hydrogen bond with Met51,hydrophobic interactions with the side chain of Val18, and displacingone water molecule. At this stage, we used SZMAP to analyze the networkof four water molecules remaining in the subpocket. This analysispredicted that all four water molecules were stably bound, suggestingthat simply displacing them with lipophilic groups was unlikely tobe beneficial for potency. Starting from 8f, we henceintroduced polar moieties to probe the subpocket and the water network.Amide 12a provided a breakthrough and was our first submicromolarcompound. X-ray analysis showed that the newly introduced amide replacedan additional water molecule and critically formed hydrogen bondswith two additional water molecules.We solved this challengeby subjecting several derivatives to arapid crystallographic analysis. This analysis revealed that onlyone compound —for example, 10–100, 40–100,60–100, or 55–80% at a flow rate of 20 mL per minute.Microwave-assisted reactions were carried out using a Biotage Initiatormicrowave system. Final compounds were purified to ≥95% purity.Product yields are quoted as . NMR data was collected on a Bruker Avance 500 spectrometerequipped with a 5 mm BBO/QNP probe or on a Bruker Avance Neo 600 spectrometerequipped with a 5 mm TCI Cryo-Probe. NMR data is presented in theform of chemical shift δ  for major diagnostic protons, given in parts per million(ppm) relative to tetramethylsilane (TMS), referenced to the internaldeuterated solvent. Where “2 min ToF” is indicated,liquid chromatography–mass spectrometry (LC/MS) and high-resolutionmass spectrometry (HRMS) analyses were performed on an Agilent 1200series HPLC and a diode array detector coupled to a 6210 time-of-flightmass spectrometer with a dual multimode APCI/ESI source. Analyticalseparation was carried out at 40 °C on a Merck Chromolith Flashcolumn  using a flow rate of 1.5 mL/minin a 2 min gradient elution with detection at 254 nm. The mobile phasewas a mixture of methanol (solvent A) and water (solvent B), bothcontaining formic acid at 0.1%. Gradient elution was as follows: 5:95(A/B)–100:0 (A/B) over 1.25 min, 100:0 (A/B) for 0.5 min, andthen reversion back to 5:95 (A/B) over 0.05 min, and finally 5:95(A/B) for 0.2 min. Where “4 min ToF” is indicated, themethod is as previous except at 30 °C, using a flow rate of 0.75mL/min in a 4 min gradient elution as follows: 5:95 (A/B)–100:0(A/B) over 2.5 min, 100:0 (A/B) for 1 min, and then reversion backto 5:95 (A/B) over 0.1 min, and finally 5:95 (A/B) for 0.4 min. Where“4 min ESI” is indicated, LC/MS and HRMS analyses wereperformed on a Waters Acquity UPLC and a diode array detector coupledto a Waters G2 QToF mass spectrometer fitted with a multimode ESI/APCIsource. Analytical separation was carried out at 30 °C on a PhenomenexKinetex C18 column using a flow rate of 0.3 mL/min in a 4 min gradient elution with detectionat 254 nm. The mobile phase was a mixture of methanol (solvent A)and water (solvent B), both containing formic acid at 0.1%. Gradientelution was as follows: 10:90 (A/B)–90:10 (A/B) over 3 min,90:10 (A/B) for 0.5 min, and then reversion back to 10:90 (A/B) over0.3 min, and finally 10:90 (A/B) for 0.2 min.H)-one 9 (1 equiv) or 4-chloro-6-nitroquinolin-2(1H)-one 5 (1 equiv) and the requisite amine(10 equiv) in NMP (0.3 M) was heated in the microwave at 160 °Cfor up to 16 h. The reaction mixture was allowed to cool to rt, andwater was added. The resulting precipitate was filtered and washedwith water to afford the desired 4-substituted-quinolinone, whichwas used without further purification. If no precipitate formed, thereaction mixture underwent HPLC purification to afford the desiredproduct.Step 1: a suspension of 4-chloro-1-methyl-6-nitroquinolin-2(12 (4 equiv) in a 6:1 mixtureof ethanol: trifluoroethanol (0.01 M) was heated in the microwaveat 120 °C for up to 2 h. The reaction mixture was loaded ontoan SCX-2 column (2 g) and washed with methanol. The desired productwas eluted with methanolic ammonia (approximately 2 M) and concentratedunder reduced pressure to give the desired amino-quinolin-2-one, whichwas used with no further purification.Step 2: a mixture of the requisite 4-substituted-6-nitroquinolin-2-onefrom step 1 (1 equiv) and SnClStep 3: the requisite4-substituted, 6-amino-1-methyl-quinolin-2-onefrom step 2 (1 equiv), 2,4-dichloropyridine-3-carbonitrile (1.5 equiv),and triethylamine (2 equiv) in NMP (0.1 M) were heated in the MW at120–160 °C for up to 1 h. The crude reaction mixture wasdiluted with dimethyl sulfoxide (DMSO) and MeCN and purified by Biotagereverse-phase chromatography [12 g C18 eluting 10–100% MeOHin water (containing 0.1% formic acid)] or via HPLC [40–100%MeOH in water] to give the desired product.H)-one 9 (1 equiv), with requisite amine (1.5equiv), cesium carbonate (3 equiv), palladium(II)acetate (20 mol %),and rac-BINAP (20 mol %) was added toluene (2 mL) and the vial waspurged with argon for 5 min. The mixture was heated in the microwaveat 120 °C for up to 4 h and then diluted with water and extractedwith ethyl acetate. The organic layers were dried over MgSO4 and concentrated under reduced pressure. The resulting residue waspurified by either normal-phase column chromatography (0–15%methanol in dichloromethane (DCM)) or reverse-phase column chromatography(10–100% methanol in water [0.1% formic acid modifier]) togive the desired 4-substituted nitroquinolin-2-one, which was subjectedto further purification by SCX-2 if necessary.Step 1: to a mixture of 4-chloro-1-methyl-6-nitroquinolin-2 in ethanol (0.05 M) or NMP (0.05 M) were added Pd/C  and ammonium formate (10 equiv) under argon. The sealedvessel was then placed into a preheated heating block at 60 °Cand stirred for 30 min to 4 h. The reaction mixture was loaded ontoan SCX-2 column (2 g) and washed with methanol. The desired productwas eluted with methanolic ammonia (3.5 M) and concentrated underreduced pressure to give the desired amino-quinolin-2-one of sufficientpurity for use in the subsequent step.Step 3: a mixture ofthe product of step 2 (1 equiv), 2,4-dichloropyridine-3-carbonitrile(1.2 equiv) or 4,6-dichloro-5-cyano-pyridine-2-carboxylic acid (1.2equiv), and DIPEA (1.4 equiv) in NMP (0.1 M) was heated to 120–160°C for 1 h in the microwave. The crude reaction mixture was dilutedwith DMSO and MeCN and purified by reverse-phase chromatography (10–100%MeOH in water [0.1% formic acid modifier]) to give the desired product.18 (1 equiv), and DIPEA (3 equiv) in NMP (0.2 M) was stirredeither at 160 °C under microwave irradiation or at 80 °Cin a heating block. Solid LiCl (6 equiv) was added to the mixture,and the reaction was further stirred under microwave irradiation at120–160 °C for 1–6 h. The residue was taken upin water and extracted twice with EtOAc. The organic extracts werecombined, washed with water and brine, then dried over MgSO4, and concentrated under reduced pressure. The crude residue waspurified by reverse-phase chromatography  to give the desired 4-substitutednitroquinolin-2-one.Step 1: a mixture of the requisiteamine (1.5 equiv), ethyl 4-chloro-1-methyl-6-nitro-2-oxo-quinoline-3-carboxylate Step 2: to the requisite 4-substituted,6-nitro-1-methyl-quinolin-2-one from step 1 (1 equiv) in ethanol (0.05M) or NMP (0.05 M) were added Pd/C  and ammoniumformate (10 equiv). The flask was sealed and evacuated and then refilledwith argon three times. The flask was then placed into a preheatedheating block at 60 °C and stirred for 30 min to 4 h. The reactionmixture was loaded onto an SCX-2 column (2 g) and washed with methanol.The desired product was eluted with methanolic ammonia (3.5 M) andconcentrated under reduced pressure to give the desired amino-quinolin-2-one,which was used with no further purification.Step 3: a mixtureof the aniline product from step 2 (1 equiv),2,4-dichloropyridine-3-carbonitrile (1.2 equiv), and DIPEA (1.4 equiv)in NMP (0.1 M) was heated under microwave irradiation to 120–160°C for 1 h. The crude reaction mixture was diluted with DMSO(0.1 mL) and MeCN (0.1 mL) and directly loaded and purified by reverse-phasechromatography [12 g C18 column eluting 10–100% MeOH in water(containing 0.1% formic acid)] to give the desired product.H)-one 9 (1 equiv) and the requisite amine(10 equiv) in NMP (0.3 M) was heated in the microwave at 160 °Cfor up to 16 h. The reaction mixture was allowed to cool to rt, andwater was added. The resulting precipitate was filtered and washedwith water to afford the desired 4-substituted-quinolinone. If noprecipitate formed, the reaction mixture underwent direct HPLC [Agilent6120 MS-Prep LC using an ACE 5 C18-PFP 250 mm × 21.2 mm columnusing a 15 min gradient of 40–100% MeOH in water (containing0.1% formic acid)].Step 1: a mixture of 4-chloro-1-methyl-6-nitroquinolin-2 in ethanol (0.05M) or NMP (0.05 M) were added Pd/C  and ammoniumformate (10 equiv). The flask was sealed and evacuated and then refilledwith argon three times. The flask was then placed into a preheatedheating block at 60 °C and stirred for 30 min to 4 h. The reactionmixture was loaded onto an SCX-2 column (2 g) and washed with methanol.The desired product was eluted with methanolic ammonia (3.5 M) andconcentrated under reduced pressure to give the required amino-quinolin-2-one.Step 3: a mixture of the 4-substituted, 6-amino-1-methyl-quinolin-2-onefrom step 2 (1 equiv) and 4,6-dichloro-5-cyano-pyridine-2-carboxylicacid (1.2 equiv) in NMP (0.1 M) was heated in a heating block at 100°C for 2 h. The crude reaction mixture was diluted with DMSO(0.1 mL) and MeCN (0.1 mL) and directly loaded and purified by reverse-phasechromatography [12 g C18 column eluting 20–100% MeOH in water(containing 0.1% formic acid)] to give the desired product.H)-one 3 , sodium hydride, and iodomethane  in DMF (8 mL) was stirred at 0 °C for 1 h. Brine wasadded, and the resulting precipitate was collected, washed with water,and dried to give 1-methyl-6-nitroquinolin-2(1H)-one as a white solid, which was used without further purificationin the subsequent step. LCMS (2 min ToF) Rt = 1.08 min, m/z 205.1 [M + H]+.Step 1: a mixture of 6-nitroquinolin-2(1H)-one  in ethanol:trifluoroethanol  was added SnCl2  and the resultingmixture was heated in the microwave at 120 °C for 1 h. The reactionmixture was purified using an SCX-2 column and then further purifiedby flash column chromatography (5–10% methanol in DCM) to give6-amino-1-methylquinolin-2(1H)-one . 1H NMR  δ7.66 , 7.25 , 6.94 ,6.78 , 6.48 , 5.09 , 3.53 . LCMS (2 min ToF) Rt = 0.11 min, m/z 175 [M + H]+.Step 2: to a solution of 1-methyl-6-nitroquinolin-2(1H)-one , 2,4-dichloropyridine-3-carbonitrile, and triethylamine  in DMF(1.5 mL) was heated in the microwave at 160 °C for 1 h. The crudereaction mixture was purified by HPLC to give the title compound . 1H NMR  δ 8.31 , 8.14 , 7.99 ,7.91–7.83 , 7.14 ,6.87 , 3.91 ; LCMS (4 minToF) Rt = 2.48 min, m/z 311.0689 [M + H]+ expected 311.0694for C16H12ClN4O.Step 3: a vial containing 6-amino-1-methylquinolin-2 in 1,4-dioxane(126 mL) was added concentratedHCl  dropwise. The reaction mixture was refluxedfor 18 h. The mixture was cooled to room temperature, poured intoexcess ice water, and allowed to stir for 1 h. The obtained solidwas filtered and dried under vacuum to afford 4-chloroquinolin-2(1H)-one  as an off-white solid. 1H NMR  δ 12.09–11.97, 7.85 , 7.62 , 7.38 , 7.30 , 6.82 ; LCMS (2 min ToF) Rt = 1.26 min, m/z 180.0 [M + H]+.Step 1: to a stirred solutionof 2,4-dichloroquinoline H-quinolin-2-one from step 1  in sulfuricacid  cooled to 0 °C was added nitric acid dropwise. The solution was stirred at 0 °Cfor 1 h and then poured onto ice. The yellow precipitate that formedwas filtered and washed with water until the pH of the filtrate was∼7. The residue was then washed with methanol and diethyl ether.This crude residue was then dried under a high vacuum in a desiccatorfor 72 h to give the title compound  as a yellow powder. 1H NMR  δ12.57 , 8.68–8.54 , 8.47–8.38 ,7.56–7.40 , 7.09–6.95 ; LCMS (2 min ToF) Rt = 1.17 min, m/z 225.0056 [M + H]+ expected 225.0061 for C9H6ClN2O3+.Step 2: to a stirred solution of 4-chloro-1General Procedure A, steps 1–2, from ethylamine . The resulting6-amino-4-(ethylamino)quinolin-2(1H)-one (18 mg)was combined with 2,4-dichloropyridine-3-carbonitrile (15 mg) andtriethylamine (25 μL) in DMF (1.5 mL) and heated in the MW at160 °C for 1 h. The crude reaction mixture was diluted with water,and the resulting precipitate was purified by HPLC to give 2-chloro-4-((4-(ethylamino)-2-oxo-1,2-dihydroquinolin-6-yl)amino)nicotinonitrile 8f (2 mg) as a yellow solid, and further elution gave 2-chloro-4-((4-(dimethylamino)-2-oxo-1,2-dihydroquinolin-6-yl)amino)nicotinonitrile 8a  as a yellow solid, the dimethylamino groupcoming from thermal decomposition of DMF solvent. 8a:1H NMR  δ 8.00 , 7.80 , 7.58–7.37 , 6.76 , 5.97 , 3.00 ; LCMS (4 minESI) Rt = 2.32 min, m/z 340.0962 [M + H]+ expected 340.0965for C17H15ClN5O.Prepared by 8f was reprepared using NMP as solvent to avoid 8a byproduct. 6-Amino-4-(ethylamino)quinolin-2(1H)-one, 2,4-dichloropyridine-3-carbonitrile , and triethylamine  in NMP (2 mL) werestirred at 140 °C under microwave irradiation for 1 h, then purifiedby an SCX-2 column, further purified by HPLC, and desalted by an SCX-2column. The title compound  was obtained as a yellow solid. 1H NMR  δ10.82 , 9.52 , 8.12–8.06 , 7.54–7.45, 6.92 , 6.79 , 5.40 , 3.27 , 1.29 . LCMS(4 min ESI) Rt = 2.43 min, m/z 340.0966 [M + H]+ expected 340.0965for C17H15ClN5O.Compound General Procedure A from piperidine  to afford the title compoundas a cream solid . 1H NMR  δ 11.41 , 9.56 , 8.03 , 7.48 , 7.42 , 7.32 , 6.79 , 5.86 ,3.01 , 1.70 , 1.60 ; LCMS (4 min ToF) Rt = 2.86 min, m/z 380.1265 [M + H]+ expected 380.1273 for C20H19ClN5O.Prepared by General Procedure A from 1-methylpiperazine  to afford the title compoundas a yellow solid . 1H NMR  δ 8.02 , 7.71 , 7.54 , 7.47 , 6.81 , 6.15 ,3.44–3.34 , 3.24–3.11 ,2.72 ; LCMS (4 min ToF) Rt = 1.43min, m/z 395.1383 [M + H]+ expected 395.1387 for C20H20ClN6O.Prepared by General Procedure A from morpholine  to afford the title compoundas a white solid . 1H NMR  δ 11.63 , 9.79 , 8.31, 7.98 , 7.78 , 7.73, 7.13 , 6.17 , 4.04 ,3.32 ; LCMS (4 min ToF) Rt = 2.51 min, m/z 382.1055 [M + H]+ expected 382.1065 for C19H17ClN5O2.Prepared by H)-one 5 , 1-methyl-4--1H-pyrazole , tetrakis(triphenylphosphine)palladium(0), DMF (3 mL), and 2 M aqueous sodium carbonate was heated in the microwave at 140 °C for1 h. The reaction mixture was partitioned between water and EtOAc,and the organic layer was washed with brine and water, dried overMgSO4, and concentrated under reduced pressure to give14 mg of a yellow solid containing 4-(1-methyl-1H-pyrazol-4-yl)-6-nitroquinolin-2(1H)-one and residualtriphenylphosphine oxide. This material was combined with SnCl2  in a 6:1 mixture of ethanol: trifluoroethanol(0.01 M) and heated in the microwave at 120 °C for 1 h. The resultingmixture was loaded onto an SCX-2 column (2 g) and washed with methanol.The desired product was eluted with methanolic ammonia (2 M) and concentratedunder reduced pressure to give 6-amino-4-(1-methyl-1H-pyrazol-4-yl)quinolin-2(1H)-one 7e  as a yellow solid, which was used without further purificationin the subsequent step. LCMS (2 min ToF) Rt = 0.22 min, m/z 241.1108 expected241.1084 for C13H13N4O+ [M + H]+.Step 1: a suspension of 4-chloro-6-nitroquinolin-2(1H-pyrazol-4-yl)quinolin-2(1H)-one fromstep 1 , 2,4-dichloropyridine-3-carbonitrile , and triethylamine  in DMF(1.5 mL) were heated in the microwave at 160 °C for 1 h. HPLCpurification [40–100% MeOH in water (containing 0.1% formicacid)] afforded the title compound  as a light brown solid. 1H NMR  8.09, 8.02 , 7.88–7.85, 7.59 , 7.53 , 6.81 ,6.70 , 4.01 ; LCMS (4 min ToF) Rt = 2.46 min, m/z 377.0888[M + H]+ expected 377.0912 for C19H14ClN6O.Step 2: 6-amino-4-(1-methyl-1H)-one 5  in DMF (60 mL) wasadded sodium hydride , andthe reaction was stirred for 10 min before iodomethane  was added portionwise. The reaction was stirred at room temperaturefor 90 min before water (80 mL) was added cautiously. After 10 min,the resulting precipitate was filtered, washed with water (80 mL)and diethyl ether (100 mL), and dried under vacuum to give the titlecompound  as a pale yellow solid. 1H NMR  δ 8.67 , 8.51 , 7.83 , 7.19 ,3.67 ; LCMS (2 min ToF) Rt = 1.32min, m/z 239.0207 expected 239.0128for C10H8ClN2O3+ [M + H]+.To an ice-cooled suspension of 4-chloro-6-nitroquinolin-2 affording thetitle compound . 1H NMR  δ 13.68 , 9.92 ,8.06–8.02 , 7.54–7.52 , 7.18 ,6.80 , 5.45 , 3.53 , 3.20–3.14 , 1.21 ; LCMS (4 min ToF) Rt = 2.45 min, m/z 398.0992 [M + H]+ expected398.1014 for C19H17ClN5O3.Prepared by General ProcedureD from propylamine  affording thetitle compound as a pale orange solid . 1H NMR δ 9.90 ,8.06–8.05 , 7.55–7.50 , 7.19 ,6.85 , 5.45 , 3.52 , 3.11 , 1.62 , 0.93 ; LCMS (4min ESI) Rt = 2.39 min, m/z 412.1162 [M + H]+ expected 412.1176for C20H19ClN5O3.Prepared by General ProcedureB from 2-fluoroethylamine hydrochloride to afford the title compound  as a yellow solid. 1H NMR  δ 9.89, 8.06 , 7.55 ,7.17 , 7.06–7.02 , 5.57 , 4.67 , 4.58 ,3.57–3.50 , 3.48 ;LCMS (4 min ToF) Rt = 2.42 min, m/z 416.0919 [M + H]+ expected416.0920 for C19H16ClFN5O3.Prepared by General ProcedureB from cyclopropylmethanamine  to affordthe title compound  as a yellow solid. 1H NMR δ 9.93 ,8.08 , 7.59–7.48 ,7.20 , 6.95 , 5.48 , 3.53 , 3.10–2.91 , 1.13 , 0.51–0.45 , 0.27–0.21 ;LCMS (4 min ToF) Rt = 2.86 min, m/z 424.1165 [M + H]+ expected424.1171 for C21H19ClN5O3.Prepared by General Procedure A from 2-amino-N-methylpropanamide  to afford the title compound as a light brown solid. 1H NMR  δ 10.96 , 8.10 , 8.10, 8.03 , 7.38 , 7.28 , 6.77 , 6.60 , 5.17 , 3.93 , 2.61 , 1.40 ; LCMS (4 min ToF) RT = 2.28 min, m/z 397.1166 [M + H]+ expected397.1174 for C19H18ClN6O2.Preparedby General Procedure A from (R)-2-amino-N-methylpropanamide  to afford the titlecompound  as a light brown solid. 1H NMR  δ 8.08 , 8.00 ,7.49 , 7.43 , 6.72 ,5.45 , 4.09 , 2.77 , 1.56 ; LCMS (4 min ToF) Rt = 2.30 min, m/z 397.1167 [M + H]+ expected 397.1174 for C19H18ClN6O2.Preparedby General Procedure A from (S)-2-amino-N-methyl-propanamide hydrochloride  toafford the title compound  as a light brown solid. 1H NMR  δ8.08 , 7.99 , 7.49 ,7.43 , 6.72 , 5.45 , 4.09 , 2.77 , 1.56 ; LCMS(4 min ToF) Rt = 2.29 min, m/z 397.1146 [M + H]+ expected 397.1174for C19H18ClN6O2.Preparedby General Procedure D from 2-amino-2-cyclopropyl-N-methylacetamide trifluoroacetic acid salt to afford the title compoundas a white solid . 1H NMR  δ 8.14 , 8.01 , 7.50 , 7.43 , 6.73 , 5.41 ,3.31 , 2.80 , 1.40–1.32, 0.76–0.66 , 0.66–0.59 , 0.46–0.39; LCMS (4 min ESI) Rt = 2.27 min, m/z 423.1334 [M + H]+ expected423.1336 for C21H20ClN6O2.Prepared by General Procedure A from 2-amino-N-methylacetamide hydrochloride  to affordthe title compound  as a pale brown solid. 1H NMR  δ 8.30 , 8.03 , 7.89 , 7.38 , 7.29 , 6.67 , 3.75 ,2.61 ; LCMS (4 min ESI) Rt = 2.17min, m/z 383.1004 [M + H]+ expected 383.1018 for C18H16ClN6O2.Prepared by General Procedure A from 2-amino-N-methyl-propanamide  to afford the title compound as a cream solid. 1H NMR  δ 8.14 , 8.01 , 7.67 , 7.61 , 6.75 , 5.56 ,4.08 , 3.69 , 2.77 , 1.57 ; 13C NMR δ 174.5,164.1, 156.9, 153.5, 150.7, 150.4, 138.5, 131.9, 128.8, 119.8, 116.6,116.1, 113.2, 106.8, 93.6, 92.1, 52.8, 28.3, 25.0, 17.4; LCMS (4 minToF) Rt = 2.42 min, m/z 411.1329 [M + H]+ expected 411.1258for C20H20ClN6O2.Preparedby General Procedure B from 1-oxazol-2-ylethanamine  to afford the titlecompound . 1H NMR  δ 8.18 , 8.05 , 8.04, 7.55–7.47 , 7.16, 7.02 , 7.65 , 6.61 , 5.56 , 5.00–4.87 , 3.50 , 1.62 ; LCMS (4 min ESI) Rt = 2.56 min, m/z 421.1169 [M + H]+ expected 421.1174 for C21H18ClN6O2.Prepared by 18 , and N,N-diisopropylethylamine in NMP (1.5 mL) was stirred at 160 °Cunder microwave irradiation for 1 h. Solid LiCl was then added to the mixture, and the reaction was further stirredunder microwave irradiation at 160 °C for 1 h. The reaction mixturewas purified by HPLC [40–100% MeOH in water (containing 0.1%formic acid)] to give 1-methyl-4-ethyl)amino)-6-nitroquinolin-2(1H)-one 19a ; 1H NMR δ 9.18 ,8.40 , 7.80 , 7.64 ,5.75 , 5.17 , 3.56, 2.48 , 1.69 ,LCMS (2 min ToF), Rt = 1.19 min, m/z 330.1178 expected 330.1197 for C15H16N5O4+ [M +H]+.Step 1: a mixture of 1-ethanaminehydrochloride , ethyl 4-chloro-1-methyl-6-nitro-2-oxo-1,2-dihydroquinoline-3-carboxylate H)-one from step 1  and SnCl2  in a 6:1 mixture of ethanol: trifluoroethanol(0.01 M) was heated in the microwave at 120 °C for 1 h. The reactionmixture was loaded onto an SCX-2 column (2 g) and washed with methanol.The desired product was eluted with methanolic ammonia (2 M) and concentratedunder reduced pressure to give 6-amino-1-methyl-4-ethyl)amino)quinolin-2(1H)-one 20a , which was used withoutfurther purification in the subsequent step; 1H NMR  δ 7.34 , 7.28 ,7.18–7.07 , 5.70 , 5.09 , 3.59 , 2.52 , 1.79 , LCMS (2 min ToF), Rt =0.47 min, m/z 300.1443 expected300.1455 for C15H18N5O2 [M + H]+.Step 2: a mixture of 1-methyl-4-ethyl)amino)-6-nitroquinolin-2(1H)-one from step 2 , 2,4-dichloropyridine-3-carbonitrile, and DIPEA  in NMP (1.5mL) was heated in the MW at 160 °C for 1 h. Purification by HPLC[40–100% MeOH in water (containing 0.1% formic acid)] affordedthe title compound . 1H NMR  δ 8.11 , 8.00 , 7.67 , 7.60 , 6.72 , 5.78 ,5.12 , 3.68 , 2.52 , 1.78 ; LCMS (4 min ToF) Rt = 2.48 min, m/z 436.1271 [M + H]+ expected 436.1283 for C21H19ClN7O2.Step 3: a mixture of 6-amino-1-methyl-4-ethyl)amino)quinolin-2ethan-1-aminehydrochloride  was used according to GeneralProcedure C to afford the title compound  as a creamsolid. 1H NMR  δ 8.04, 7.95 , 7.56 , 7.40 , 7.35 , 7.12 ,6.58 , 5.81 , 5.78 , 4.85 , 3.89 , 3.61 , 1.66 ; LCMS (4 min ToF) Rt =2.47 min, m/z 435.1440 [M + H]+ expected 435.1443 for C21H20ClN8O.1-to afford the title compound  as a yellow solid. 1H NMR  δ9.62 , 8.79 , 8.29 , 8.05 ,7.56–7.44 , 7.40 ,7.23 , 6.61 , 5.27 , 4.74 , 3.46 , 1.60 .LCMS (4 min ToF) Rt = 2.55 min, m/z 432.1329 [M + H]+ expected432.1334 for C22H19ClN7O.Prepared by General Procedure C from (R)-1-(pyrimidin-2-yl)ethan-1-amine  to yield the title compound . 1HNMR  δ 9.62 , 8.79 , 8.29 , 8.05 , 7.56–7.44, 7.40 , 7.23 , 6.61 ,5.27 , 4.74 , 3.46 , 1.60 ; LCMS (4 min ToF) Rt = 2.53 min, m/z 432.1309 [M + H]+ expected 432.1334 for C22H19ClN7O.Prepared by General Procedure B from 2-pyrimidin-2-ylpropan-2-amine dihydrochloride  to afford the title compound  as a pale orange solid. 1H NMR  δ9.59 , 8.82 , 8.25 , 8.05 ,7.51 , 7.46 , 7.39 ,6.85 , 6.61 , 4.69 , 3.41 , 1.76 ; 13C NMR  δ 172.5, 161.2, 157.5, 156.7, 153.1,151.4, 147.4, 138.4, 131.0, 128.6, 120.6, 119.4, 116.1, 116.0, 114.3,107.5, 94.4, 92.3, 58.8, 28.4, 27.5.; LCMS (4 min ToF) Rt = 2.64 min, m/z 446.1482[M + H]+ expected 446.1491 for C23H21ClN7O.Prepared by H)-one 5  in DMF (2 mL)were added bromomethylcyclopropane  and cesiumcarbonate . The reaction was stirred at rt for42 h. The reaction was diluted with DCM and washed with 1 M aq HCland brine. The organic layer was dried and concentrated under reducedpressure and then purified by column chromatography (12–100%EtOAc in cyclohexane) affording the title compound  asa yellow solid. 1H NMR  δ8.90 , 8.46 , 7.65 , 7.00, 4.27 , 1.28–1.17, 0.57 . LCMS (2 min ToF) Rt = 1.51 min, m/z 279.0530 expected 279.0531 for C13H12ClN2O3+ [M + H]+.To a suspension of 4-chloro-6-nitroquinolin-2(1H)-one 5  and (bromomethyl)cyclobutane using method as for 14a affordingthe title compound  as a yellow solid. 1HNMR  δ 8.93 , 8.45 ,7.49 , 7.02 , 4.43 , 2.86–2.70 , 2.11–2.01, 2.02–1.84 ; LCMS (2 min ToF) Rt = 1.56 min, m/z 293.0676expected 293.0687 for C14H14ClN2O3 [M + H]+.Prepared from 4-chloro-6-nitroquinolin-2 and1-pyrimidin-2-ylethanamine hydrochloride  were usedin an analogous fashion to General Procedure B to affordthe title compound  as a white solid. 1H NMR δ 8.80 , 8.19 ,8.03 , 7.78 , 7.61 ,7.40 , 6.79 , 5.53 , 4.93–4.89 , 4.21 , 1.71 ,1.36–1.20 , 0.52–0.43 . LCMS (4 min ESI) Rt = 2.71 min, m/z 472.1650 [M + H]+ expected 472.1653 for C25H23ClN7O.14b  and1-pyrimidin-2-ylethanamine hydrochloride  were usedin an analogous fashion to General Procedure B to affordthe title compound  as a brown solid. 1H NMR δ 8.80 , 8.17 ,8.03 , 7.63 , 7.58 ,7.40 , 6.78 , 5.53 , 4.92–4.88 , 4.36 , 2.85–2.75 , 2.06–1.81, 1.71 ; LCMS (4 min ESI) Rt = 2.89 min, m/z 486.1804 [M + H]+ expected 486.1809 for C26H25ClN7O.H-benzo[d]oxazine-2,4(1H)-dione  as anorange powder. 1H NMR  δ 8.64 ,8.61 , 7.66 , 3.54 .Step 1: to a solution of 5-nitro-isatoicanhydride  in DMF (241 mL) was added sodium hydride. The solution was allowed tostir at room temperature for 15 min. Iodomethane was added, and the mixture was stirred for 4 h at room temperature.The reaction mixture was poured onto ice and then filtered and washedwith water (5 L). The solid was collected and dried under vacuum overnightaffording 1-methyl-6-nitro-2H-benzo[d]oxazine-2,4(1H)-dione  in DMF (176 mL) was added diethylmalonate . The solution was cooled to 0 °C,and sodium hydride  was addedin 4 portions over 30 min. The solution was allowed to warm to roomtemperature and stirred for 4 h. Water was added with care to thereaction mixture followed by 10% aq. HCl until the pH of the mixturewas ∼5. The resulting precipitate was filtered and washed withwater (5 L) and dried under vacuum to give ethyl 4-hydroxy-1-methyl-6-nitro-2-oxo-1,2-dihydroquinoline-3-carboxylate as a pale yellow solid, which was used without furtherpurification in the subsequent step. LCMS (2 min ToF) Rt = 1.45 min, m/z 293.0739expected 293.0768 for C13H13N2O6+ [M + H]+.Step 2: to a solutionof 1-methyl-6-nitro-2CAUTION, apply appropriateprocedures in the disposal of POCl3. The residue was diluted with water and extracted with EtOAc.The combined organic extracts were washed with brine and then driedover MgSO4. The crude residue was purified (by dry loadingonto silica) using column chromatography  to afford the title compound  as a darkorange solid. 1H NMR  δ8.95 , 8.50 , 7.53 , 4.48, 3.78 , 1.42 ; LCMS (2 min ToF), Rt = 1.42 min, m/z 311.0427expected 311.0429 for C13H12ClN2O5 [M + H]+.Step 3: a mixtureof ethyl 4-hydroxy-1-methyl-6-nitro-2-oxo-1,2-dihydroquinoline-3-carboxylate and phosphorus oxychloride was heated to 80°C under argon with stirring for 2.5 h. The reactionwas concentrated under reduced pressure. 18 , 1-(5-bromopyrimidin-2-yl)ethanamine, and DIPEA  in NMP (9.7 mL) wasstirred at 80 °C overnight. Once cooled to rt, lithium chloride was added to the mixture, which was then heatedto 160 °C for 2 h. The residue was taken up in water and extractedtwice with EtOAc. The organic extracts were washed twice with 1 Maq. NaOH and brine and then dried over MgSO4. The residuewas purified by flash column chromatography eluting 50–100%EtOAc in cyclohexane. The title compound was isolated as a mustardsolid  in 90% purity and used without further purificationin the subsequent step. LCMS (method T2) Rt = 1.38 min, m/z 404.0291 expected404.0353 for C16H15BrN5O3 [M + H]+.A mixture of ethyl 4-chloro-1-methyl-6-nitro-2-oxo-quinoline-3-carboxylate 18 , 1-(5-bromopyrimidin-2-yl)-1-methylethylaminehydrochloride , and DIPEA  wasstirred at 120 °C for 6 h. Once cooled to rt, lithium chloride was added to the mixture and heated to 160 °Covernight. Once cooled to rt, the residue was taken up in water andextracted twice with EtOAc. The combined organic extracts were washedwith water and brine and then dried over MgSO4. The residuewas purified by flash column chromatography (Biotage KP-Sil 25 g eluting0–10% MeOH in DCM). A second purification was required, sothe residue was purified again (Biotage KP-Sil 25 g eluting 20–100%EtOAc in cyclohexane) affording the title compound  asa yellow solid. 1H NMR  δ 9.27 ,9.03 , 8.37 , 7.57, 7.54 , 4.71 ,3.46 , 1.79 . LCMS (method T2) Rt = 1.42 min, m/z 418.0502expected 418.0509 for C17H17BrN5O3 [M + H]+.A suspension of ethyl4-chloro-1-methyl-6-nitro-2-oxo-quinoline-3-carboxylate H)-one 21a , bis(triphenylphosphine)palladium(II)chloride , and methylboronic acid  were added 1,4-dioxane (0.54 mL) and 2 M aq sodium carbonate. The mixture was then heated under an argon atmospherein the microwave at 120 °C for 2 h. Once cooled to rt, the reactionmixture was filtered through a syringe filter, and the resulting filtratewas concentrated under reduced pressure. DMSO (0.5 mL) was added tothe sample, which was purified using reverse-phase C18 column elutingfrom 10 to 100% MeOH in water (containing 0.1% formic acid). 1-Methyl-4-((1-(5-methylpyrimidin-2-yl)ethyl)amino)-6-nitroquinolin-2(1H)-one 22a  was obtained as an off-whitesolid. 1H NMR δ 8.68 , 8.61 , 8.39, 7.40 , 6.53 , 5.82 , 4.87 , 3.67 , 2.35 , 1.67 . LCMS(method T2) Rt = 1.30 min, m/z 340.1386 expected 340.1404 for C17H18N5O3 [M + H]+.Step 1: to 4-((1-(5-bromopyrimidin-2-yl)ethyl)amino)-1-methyl-6-nitroquinolin-2(1H)-one from step 1  in ethanol (1 mL)were added ammonium formate  and Pd/C (10 wt %)(10 mg). The resulting mixture was stirred in a sealed vial underargon at 60 °C for 1 h. 6-Amino-1-methyl-4-((1-(5-methylpyrimidin-2-yl)ethyl)amino)quinolin-2(1H)-one 23a  was obtained as a paleorange solid following SCX purification and used without further purificationin the subsequent step. LCMS (2 min ToF) Rt = 0.87 min, m/z 310.1658 expected310.1662 for C17H20N5O [M + H]+.Step 2: to a solution of 1-methyl-4-((1-(5-methylpyrimidin-2-yl)ethyl)amino)-6-nitroquinolin-2 δ 8.61 , 8.10, 7.60 , 7.48 , 7.44, 7.00 , 6.67 , 6.41 ,5.85 , 4.88 ,3.69 , 2.36  1.67 ; 13C NMR  δ 167.1, 163.2, 157.4,156.1, 153.9, 151.7, 147.7, 139.5, 129.8, 129.1, 127.8, 118.6, 116.6,116.5, 113.9, 105.7, 94.4, 94.4, 53.3, 29.1, 20.9, 15.4. LCMS (4 minToF) Rt = 2.61 min, m/z 446.1480 [M + H]+ expected 446.1491for C23H21ClN7O.Step 3: a mixture of the product from step 2 ,2,4-dichloropyridine-3-carbonitrile , NMP (3.96mL), and triethylamine  under argon washeated in the microwave for 1 h at 160 °C. DMSO (0.5 mL) wasadded, and the sample was purified by reverse-phase chromatography)to yield the title compound  as an off-white solid. 24a startingfrom cyclopropylboronic acid  to afford the titlecompound  as an off-white solid. 1H NMR  δ 8.47 , 8.06 , 7.58 , 7.44 , 7.40 , 7.09  6.63 , 6.43, 5.80 , 4.83 , 3.65 , 1.90–1.83 ,1.62 , 1.15–1.06 ,0.83–0.74 ; 13C NMR  δ 166.8, 163.2, 156.1, 155.1, 151.7, 147.7, 139.4, 135.3,129.8, 127.8, 118.6, 116.6, 116.5, 113.9, 105.7, 94.3, 94.3, 53.3,29.1, 20.8, 10.7, 8.9, 8.9. LCMS (4 min ToF) Rt = 2.73 min, m/z 472.1633[M + H]+ expected 472.1633 for C25H23ClN7O.Method as for H)-one 21b , and sodium carbonate in DMF (2.4 mL) was stirred under microwaveirradiation at 140°C for 1 h. The solid was removed by filtration,washing with DMF. Brine was then added to the filtrate, and the precipitateformed was collected, washed with water, and dried to afford 4-((2-(5-cyclopropylpyrimidin-2-yl)propan-2-yl)amino)-1-methyl-6-nitroquinolin-2(1H)-one 22c (24 mg) as a light brown solid,which was used without further purification in the subsequent step.LCMS (2 min ESI) Rt = 1.52 min, m/z 380.1714, expected 380.1717 for C20H22N5O3 [M + H]+.Step 1: a mixture of cyclopropylboronicacid , tetrakis(triphenylphosphine)palladium(0), 4-((2-(5-bromopyrimidin-2-yl)propan-2-yl)amino)-1-methyl-6-nitroquinolin-2(1H)-one 23c  as ayellow oil. LCMS (4 min ToF) Rt 1.68 min, m/z 350.1988 expected 350.1975 for C20H24N5O [M + H]+.Step 2: to a solution of the product from step 1 (24 mg)in ethanol (1 mL) were added ammonium formate  andPd/C . The suspension was stirred undermicrowave irradiation at 60 °C for 1 h, filtered, and the filtratewas purified using an SCX-2 column to afford 6-amino-4-((2-(5-cyclopropylpyrimidin-2-yl)propan-2-yl)amino)-1-methylquinolin-2 δ 9.58 , 8.56 , 8.24, 8.05 , 7.51 , 7.45, 6.81 , 6.61 , 4.71 , 3.42 , 1.96–1.89, 1.73 , 1.06–0.98 , 0.87–0.81; 13C NMR  δ 169.6, 161.2, 156.7, 154.6, 153.1, 151.4, 147.5,138.4, 134.4, 131.0, 128.6, 120.6, 116.1, 116.0, 114.3, 107.5, 94.4,92.3, 58.5, 28.4, 27.5, 10.3, 9.2; LCMS (4 min ESI) Rt = 2.73 min, m/z 486.1804[M + H]+ expected 486.1809 for C26H25ClN7O.Step3: a mixture of 6-amino-4-[[1-(5-cyclopropylpyrimidin-2-yl)-1-methyl-ethyl]amino]-1-methyl-quinolin-2-onefrom step 2 , 2,4-dichloropyridine-3-carbonitrile, and triethylamine  inNMP (0.6 mL) under argon was heated in the microwave for 1 h at 160°C. The mixture was diluted with DMSO (0.5 mL) and purified byreverse-phase chromatography ). The isolated material was then passed througha silica plug, eluting with 5% MeOH in DCM to give the title compound as an off-white solid.