Euphorbia stracheyi Boiss was used for hemostasis, analgesia, and muscular regeneration in traditional Chinese medicine. To study the chemical constituents of E. stracheyi, the ethyl acetate part of the methanol extract of the whole plant was separated by silica gel, sephadex LH-20 column chromatography, and semi-preparative HPLC. The isolation led to the characterization of a new lathyrane type diterpenoid, euphostrachenol A (1), as well as eleven known compounds (2–11), including a lathyrane, three ingenane-type and two abietane-type diterpenoids, two ionones, and two flavonoids. The structures of these compounds were established using 1D- and 2D-NMR experiments, mass spectrometry, and X-ray crystallographic experiments. The MTT method was used to determine the cytotoxic activity of five cancer cell lines  on the isolated compounds. However, only compound 4 showed moderate cytotoxicity against these cell lines, with IC50 values ranging from 10.28 to 29.70 μM, while the others were inactive. Our chemical investigation also confirmed the absence of jatrophane-type diterpenoids in the species, which may be related to its special habitat. Euphorbia (Euphorbiaceae) is the third largest genus in flowering plants, second only to Astragalus (Fabaceae) and Psychotria (Rubiaceae) +, giving the molecular formula C20H30O3Na calculated as 341.2093, and six degrees of unsaturation. The IR spectrum showed absorption bands at 3434, 1682, and 1631 cm−1, which indicate the presence of hydroxyl and carbonyl functionalities. The 1H NMR spectrum of compound 1 showed two double-peaked methyl signals at δH 0.99  and 1.23 , a broad double-peaked methyl signal at δH 1.53 , two single-peaked methyl signals at δH 0.81  and 1.08 , two oxymethine protons at δH 4.40  and 4.94 , an exchangeable proton at δH 1.61 , and a tridisubstituted double bond at δH 6.05 . The 13C NMR data , and five quaternary carbons , which suggests a typical lathyrane-type diterpene with a 5/11/3-membered ring system [Compound NMR data  showed 21 was determined through careful analysis of its 2D NMR data. The HMBC spectrum of 1 showed correlations of H-1  to C-2 (δC 39.9), C-3 (δC 210.1), C-4 (δC 136.7), C-15 (δC 177.4), and C-16 (δC 16.4); and H-2 (δH 2.46) to C-1 (δC 35.5) and C-3. These data, together with the 1H-1H COSY cross-peak correlations of H2-1/H-2 and H-2/H3-16 (δH 1.23), indicated the existence of a five-membered ring A consisting of C-1, C-2, C-3, C-4, and C-15. The downfield-shifted carbon signals of the α, β-unsaturated ketone  further confirmed the presence of this five-membered ring involving C-3, C-4, and C-15. Furthermore, the 1H-1H COSY spectrum of 1 gave successive H-7 (δH 4.40)/H-8 /H-9 (δH 0.93)/H-11 (δH 0.49)/H-12 /H-13 (δH 1.98)/H3-20 (δH 0.99) correlations. The HMBC cross-peaks of H-5 (δH 6.05) to C-6 (δC 145.3), C-7 (δC 73.1), C-15, and C-17 (δC 17.8), as well as H-14 (δH 4.94) to C-15, linked the above structure fragment to C-5, C-6, and C-15 to afford ring B. The presence of the C ring was determined by correlation of two methine methyls, H3-18 (δH 1.08) and H3-19 (δH 0.81), with two methines, C-9 (δC 20.0) and C-11 (δC 22.6), and one quaternary carbon, C-10 (δC 16.2), respectively, in the HMBC spectrum. The 1H-1H COSY correlation of OH-14 (δH 1.61) with C-14 (δC 70.9) determined the location of this hydroxyl group, while another hydroxyl group was assigned at C-7 (δC 73.1) by its chemical shift of δC 73.1, albeit in the absence of resonances for the hydroxyl group.The planar structure of compound 1 was determined by the ROESY experiments, which showed the following correlation signals (H 4.94)/H-11 (δH 0.49), H-11/H-9 (δH 0.93), H-11/H3-20 (δH 0.99), H-14/H-5 (δH 6.05), H-11/H3-18 (δH 1.08), and H-9 (δH 0.93)/H3-18. These data suggested that H-14, H3-20, H-9, H-11, and H3-18 are cofacial and assigned as α-oriented and determined the β-configurations of H-13 and OH-14 simultaneously. The ROESY cross-peaks of H-13 (δH 1.98)/H-1β (δH 2.91) and H-1β/H-2 (δH 2.46) determined H-2 as a β-configuration and H3-16 as an α-configuration. However, the H-7 (δH 4.40)/H3-17 (δH 1.53) cross-peaks were insufficient to determine their conformations. Fortunately, the crystals of compound 1 were obtained, which confirmed the correctness of the relative conformation determined by the ROESY spectrum described above and assigned H-7 as a β-configuration and OH-7 as an α-configuration. Interestingly, although the planar structures of compound 1, kansuingols A and B, are quite similar, there are many differences in their relative conformations, that is, methyl-16, methyl-20, and hydroxyl-7 of compound 1 are α-oriented, while the corresponding groups of kansuingols A and B are β-oriented [1 was 0.03(9), which led to the determination of its absolute configuration, as shown in The relative conformation of compound  signals : H-14 (δ2) [3) [4) [(5) [6) [7) [9) [10) [Furthermore, based on the comparison of the NMR data with those reported in the literature, the known compounds euphopepluanones D (2) , 5-O-ben2) [3) , 3-O-ben [3) [4) , ingenol[4) [(5) , heliosc[(5) [6) , 7α-hydr [6) [7) , 5,6-epo [6) [7) , PBI 344 [7) [9) , kaempfe[9) [10) , and nar[9) [10)  were ide4 showed moderated cytotoxic activity against HL-60, A-549, SMMC-7721, MCF-7, and SW480 cell lines, with IC50 values of 10.5 ± 0.18, 21.47 ± 0.17, 18.36 ± 1.17, 18.82 ± 0.84, and 16.25 ± 0.71 μM, respectively; while DDP has IC50 values of 10.28 ± 0.21, 29.70 ± 0.72, 13.66 ± 0.76, 27.28 ± 1.23, and 24.13 ± 1.09; Taxol has IC50 values of <0.008, 0.127 ± 0.007, <0.008, <0.008, and <0.008, respectively. The other compounds were inactive at a concentration of 40 μM.The isolated compounds were screened and evaluated for their cytotoxicities against five human cancer cell lines , with DDP and Taxol as two positive controls. Only compound Euphorbia plants are known for their irritant milky latex [Euphorbia, jatrophane types are the most common [E. stracheyi have included ent-kaurane, ent-abietane, ent-atisane, ent-isopimarane, labdane, tigliane, lathyrane, and ingenane-type diterpenoids [E. stracheyi, all of which are known compounds and were discovered from other species of Euphorbia, suggesting a similar diterpenoid profile of these species. Importantly, our phytochemical investigation of E. stracheyi reported here also yielded no jatrophane-type diterpenoids, in accordance with the previous results. This type of diterpenoid is found exclusively in the Euphorbiaceae family, and it has a 5/12 bicyclic pentadecane skeleton, in which the absence of a cyclopropane ring is the most obvious structural feature that differentiates it from other macrocyclic and polycyclic diterpenoids from Euphorbia, such as tigliane, lathyrane, and ingenane types. The absence of jatrophanes in the species may be related to the special environment in which E. stracheyi lives, namely, the high-altitude region, which is often associated with high loads of UV radiation and low temperatures. Jatrophane-type diterpenoids exhibit many different activities, including antitumor, antiviral, antifungal, and anti-inflammatory effects [E. stracheyi may be influenced. Furthermore, the lack of jatrophanes implied that its biosynthetic pathway may be lost in the plant of E. stracheyi. Currently, jatrophanes were suggested to be derived from lathyrane-type diterpenoids by combined transcriptomic, genomic, and metabolomic investigation of E. peplus, a species harboring many jatrophane-type diterpenoids [ky latex  and chart common ,29,30,31rpenoids ,12,13,14rpenoids . Thus, cEuphorbia [E. stracheyi [E. stracheyi, in agreement with previous studies. Ionones are degraded from carotenoids that play an important role in helping plants adapt to high UV light and low temperatures. During photosynthesis, carotenoids are known to protect chlorophylls and bacteriochlorophylls from sensitizing deleterious photodestructive reactions [E. stracheyi in the present and previous studies strongly suggested that this type of secondary metabolite should be the result of high-altitude adaptation of the species. Further work should be undertaken to reveal whether other Euphorbia plants adapted to the harsh environments of Tibetan peaches also harbor ionones.Notably, ionones are rarely isolated from the species of uphorbia ; howevertracheyi ,12. We aeactions . The det1 shows high similarity to those of kansuingols A and B, except for a β–D-glucose group and a hydroxyl group present at C-15 and C-19 of kansuingol A, as well as a β–D-glucose group at C-19 of kansuingol B, respectively [1 were supported by X-ray diffraction analysis, those of kansuingols A and B were not. After checking the key ROESY correlations that determined the relative conformation assignment of methyl-16, methyl-20, and hydroxyl-7 in kansuingols A and B, we found that there are no ROESY correlations to support the claimed β-orientation of methyl-16 in both compounds. Therefore, the structures of kansuingols A and B remain to be determined.Notably, the planar structure of compound ectively . Notably


Colletotrichum fragariae and the invasive red imported fire ant, Solenopsis invicta. C. fragariae is known to cause anthracnose disease in fruits and vegetables, while S. invicta is known for its aggressive behavior and painful stings and for being the cause of significant damage to crops, as well as harm to humans and animals. Many plants have been studied for potential activity against C. fragariae and S. invicta. Among the studied plants, Houttuynia cordata Thunb has been shown to contain 2-undecanone, which h is known for its antifungal activity against Colletotrichum gloesporioides. Based on the mean amount of sand removed, 2-undecanone showed significant repellency at 62.5 µg/g, similar to DEET , against S. invicta. The 2-Undecanone with an LC50 of 44.59 µg/g showed toxicity against S. invicta workers. However, neither H. cordata extract nor 2-undecanone had shown activity against C. fragariae despite their known activity against C. gloesporioides, which in turn motivates us in repositioning 2-undecanone as a selected candidate for a Claisen–Schmidt condensation that enables access to several analogs (2a–f). Among the prepared analogs, (E)-1-(3-methylbenzo[b]thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f) showed promising activity against C. fragariae, revealing a distinctive structural activity relationship (SAR). The generated analogs revealed a clear regioselectivity pattern through forming the C=C alkene bond at position C-1. These data open the window for further lead optimization and product development in the context of managing C. fragariae and S. invicta.The U.S. Department of Agriculture (USDA) has established research programs to fight the phytopathogen  Colletotrichum fragariae, which is known to cause anthracnose disease in crucial crops such as strawberries.The U.S. Department of Agriculture (USDA) is actively engaged in ongoing research programs to control the spread of the aggressive red imported fire ant (RIFA) and to find alternative treatments for the fungal phytopathogen, Solenopsis invicta Buren; RIFA) (Hymenoptera: Formicidae), native to South America, are serious exotic pests of quarantine significance thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f), demonstrated faint clear zones on the TLC plate, which in turn highlighted the importance of such reactions in the development of fungicidal products. Moreover, compounds 2a–b showed a distinctive SAR, where the introduction of a methyl group at C-3 of the benzothiophene ring (2b) is a promoter for increasing activity against C. fragariae in comparison to the unsubstituted derivative (2a).Insecticidal, repellent, and behavioral effects of 2-undecanone have been well established ,30. Whani et al.  reported Schmidt . The rea Schmidt . An examn-3-one) , where dn-3-one) . Using o1H and 13C-NMR spectra were recorded in DMSO-d6 and CDCl3 on a Bruker 400 and 500 MHz spectrometer operating at 400, 500 MHz for 1H and 100, 150 MHz for 13C NMR. Chemical shift (δ) values are presented in ppm and in reference to the residual solvent signals of DMSO-d6 and CDCl3 at δH/δC 2.50/39.5 and 7.25/70.2, respectively. The coupling constant (J) is reported in Hz.The 2a–2f) was conducted using an Agilent 1100 HPLC system, RP-C18 column  with column oven temperature set at 25 °C, and a gradient system of eluent water (A) and acetonitrile (B). The gradient condition was as follows: 0–2 min (5% B), 2–5 min (5% B → 50% B), 5–10 min (50% B → 100% B), and 10–15 min (100% B). The flow rate of the solvent was 1.0 mL/min, and the injection volume was 25 μL. All analyses were carried out at a wavelength of 254 nm with a run time of 15 min. HPLC-grade acetonitrile and water solvents were used. Acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent.The LC analysis of the generated analogs (2a–2f) was carried out using an Agilent 1100 HPLC system, RP-C18 column  with column oven temperature set at 25 °C and a gradient system of eluent water (A) and acetonitrile (B). The gradient condition was as follows: 0–2 min (5% B), 2–5 min (5% B → 50% B), 5–10 min (50% B → 100% B), and 10–15 min (100% B). The flow rate of the solvent was 3.0 mL/min, and the injection volume was 25 μL. All analyses were carried out at a wavelength of 254 nm with a run time of 15 min. HPLC-grade acetonitrile and water solvents were used. Acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent.Preparative HPLC purification of the generated analogs  on precoated silica gel G2a–2f. Benzothiophene-2-carboxaldehyde, benzothiophene-3-carboxaldehyde, and indole-5-carboxaldehyde were purchased from TCI AMERICA  with purity ≥98%. 3-Methylbenzothiophene-2-carboxaldehyde and 5-bromothiophene-2-carboxaldehyde were purchased from Alfa Aesar  with purity ≥98% and ≥98%, respectively. 1-methylindole-3-carboxaldehyde was purchased from ACROS ORGANICS  with purity ≥97%.The 2-Undecanone (1.0 mmol), , was add2a. Yield of 35 mg (72.0%); 1H-NMR : δ = 0.90 , 1.32 , 1.70 , 2.66 , 6.62 , 7.39 , 7.52 , and 7.79 ; 13C-NMR : δ = 14.13 (C-12), 22.69 (C-11), 24.33 (C-5), 29.30 (C-6), 29.35 (C-7 and C-9), 29.48 (C-8), 31.90 (C-10), 41.35 (C-4), 122.49 (C-3′), 124.41 (C-4′), 124.91 (C-7′), 126.33 (C-5′), 127.14 (C-6′), 129.30 (C-1), 135.18 (C-2), 139.66 (C-3a’), 139.99 (C-7a’), 140.19 (C-2′), and 199.98 (C=O); HRMS m/z calcd. for C20H27OS [M + H]+ 315.17826, found 315.17826;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered, and dried to afford a yellow solid of 5.17826; .2b. Yield of 33 mg (70.0%); 1H-NMR : δ = 0.91 , 1.32 , 1.71 , 2.55 , 2.66 , 6.62 , 7.41 , 7.73 , 7.79 , and 7.96 ; 13C-NMR : δ = 12.22 (C-8′), 14.13 (C-12), 22.69 (C-11), 24.40 (C-5), 29.31 (C-8), 29.36 (C-6), 29.48 (C-7 and C-9), 31.90 (C-10), 41.89 (C-4), 122.46 (C-4′), 124.55 (C-7′), 126.18 (C-5′), 126.64 (C-2), 133.11 (C-1), 134.13 (C-3′), 137.17 (C-6′), 139.17 (C-7a’), 140.68 (C-3a’), and 200.03 (C=O); HRMS m/z calcd. for C21H29OS [M + H]+ 329.19391, found 329.19391;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered off, and dried to afford a yellow solid of 9.19391; .2c. Yield of 36 mg (75.0%); 1H-NMR : δ = 0.90 , 1.29 , 1.73 , 2.71 , 6.62 , 7.47 , 7.82 , 7.88 , 7.92 , and 8.05 ; 13C-NMR : δ = 14.12 (C-12), 22.69 (C-11), 24.47 (C-5), 29.31 (C-6), 29.39 (C-8), 29.49 (C-7 and C-9), 31.90 (C-10), 41.30 (C-4), 122.07 (C-2′), 123.09 (C-7′), 124.98 (C-4′), 125.12 (C-5′), 126.61 (C-6′), 128.18 (C-2), 131.66 (C-3′), 133.83 (C-7a’), 137.28 (C-1), 140.59 (C-3a’), and 200.61 (C=O); HRMS m/z calcd. for C20H27OS [M + H]+ 315.17826, found 315.17826;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered off, and dried to afford a yellow solid of 5.17826; .2d. Yield of 34 mg (67.0%); 1H-NMR : δ = 0.90 , 1.29 , 1.72 , 2.69 , 6.61 , 6.77 , 7.27 , 7.45 , 7.74 , and 7.86 ; 13C-NMR : δ = 14.13 (C-12), 22.69 (C-11), 23.90 (C-5), 24.72 (C-8), 29.45 (C-6), 29.50 (C-7 and C-9), 31.90 (C-10), 40.89 (C-4), 103.51 (C-3′), 111.67 (C-7′), 121.75 (C-4′), 122.77 (C-6′), 123.77 (C-2′), 125.38 (C-2), 126.64 (C-3a’), 128.26 (C-7a’), 137.16 (C-5′), 144.47 (C-1), and 201.13 (C=O); HRMS m/z calcd. for C20H28NO [M + H]+ 298.21709, found 298.21709;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered off, and dried to afford a yellow solid of 8.21709; .2e. Yield of 35 mg (71.0%); 1H-NMR : δ = 0.91 , 1.33 , 1.73 , 2.66 , 3.84 , 6.80 , 7.34 , 7.40 , 7.82 , and 7.96 ; 13C-NMR : δ = 14.12 (C-12), 22.70 (C-11), 24.96 (C-5), 29.33 (C-8), 29.52 (C-6), 25.54 (C-7 and C-9), 31.91 (C-10), 33.25 (C-8′), 40.99 (C-4), 110.04 (C-7′), 112.26 (C-3′), 120.65 (C-4′), 121.36 (C-5′), 121.66 (C-6′), 123.08 (C-3a’), 133.69 (C-2), 135.97 (C-7a’), 138.19 (C-1), and 200.99 (C=O); HRMS m/z calcd. for C21H30NO [M + H]+ 312.23274, found 312.23274;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered off, and dried to afford a yellow solid of 2.23274; .2f. Yield of 35 mg (78.0%); 1H-NMR : δ =0.89 , 1.29 , 1.66 , 2.59 , 6.44 , 7.03 , and 7.54 ; 13C-NMR : δ = 14.12 (C-12), 22.65 (C-11), 24.33 (C-5), 29.29 (C-8), 29.31 (C-6), 29.46 (C-7 and C-9), 31.88 (C-10), 41.36 (C-4), 116.08 (C-5′), 125.09 (C-4′), 131.20 (C-1), 131.76 (C-2), 133.75 (C-3′), 141.57 (C-2′), and 199.76 (C=O); HRMS m/z calcd. For C16H24BrOS [M + H]+ 343.07312, found 343.07312;  was used, and the reaction mixture was stirred by refluxing overnight for 15 h. Neutralization was performed using HCl, filtered off, and dried to afford a yellow solid of 3.07312; .RIFA used in these bioassays were collected from Washington County, MS 38748 (33°09′31.2″ N 90°54′56.4″ W). A plastic tray was used to contain the colony, and the tray walls were coated with Insect-a-Slip  to prevent the fire ants from crawling out. A 25% solution of water and sugar was provided to the ant colonies, and crickets were added weekly as food sources. A water-filled test tube plugged with cotton was added to serve as a moisture source. The laboratory conditions were set at 27 ± 2 °C and 60 ± 10% RH with a photoperiod regimen of 12:12 h (L:D). Ants were maintained under this setting for one month before starting the bioassays. The venom alkaloid and hydrocarbon indices of the workers were used for the identification of the imported fire ant species ,38.N,N-diethyl-meta-toluamide) were tested. DEET was tested as a positive control for comparison whereas ethanol was included as a negative control. Serial doses were started from a dose of 125 µg/g until the failure of the treatment. The stock solution and concentrations were prepared in ethanol. Overall, three sets of experimental replicates were run on 3 different days. Sand removal data were analyzed using Analysis of Variance (ANOVA) and means were separated using Ryan–Einot–Gabriel–Welsch multiple range test , , .50 values were calculated using probit analysis (SAS 2012), , .C. fragariae. First, the inoculum of C. fragariae was prepared by harvesting fresh conidia from a 7–10-day old fungal culture grown on ½ strength potato dextrose agar (PDA). The conidia were collected by flooding the plate with 10 mL of sterile water and scrapping it with a sterile L-shaped spatula. The resulting spore suspension was filtered through a sterile double Mira cloth  to remove mycelia and agar pieces, and the spores were counted using a Countess 3 cell counter. The spore suspension was centrifuged at 1968 RCF for 10 min, and the supernatant was discarded. The spore concentration was then adjusted to 3 × 105 spores mL−1 by adding the required volume of PDB-TLC media  to obtain the inoculum for the bioautography assay.The direct bioautography assay was conducted to assess the antifungal activity of 2-undecanone and its six analogs against the phytopathogen 2a–f) was spotted twice onto a silica gel plate . The inoculum of C. fragariae was then uniformly sprayed onto the silica gel plate using a hand sprayer, ensuring that the spores were evenly distributed across the plate. The TLC plate was placed in a moisture chamber box with 99.9% humidity and incubated at approximately 26 °C for 4 days, which provides optimal conditions for the growth of C. fragariae. After incubation, the antifungal activity of the compounds was evaluated by the presence of clear zones on the TLC plate, which indicates the inhibition of fungal growth. Captan  and fludioxonil , which are technical-grade fungicides, were used as positive controls at a concentration of 2 mg/mL in ethanol, with 1 µL of each control applied on the plate to compare the antifungal activity of the test compounds.Subsequently, 10 µL of each compound . Among these analogs, (E)-1-(3-methylbenzo[b]thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f) showed promising activity against C. fragariae, revealing an interesting SAR and clear regioselectivity pattern through the formation of the C=C alkene bond at position C-1. Alongside that, 2-Undecanone significantly suppressed the digging activity at 62.5 µg/g, similar to DEET, against red imported fire ants, and it showed toxicity with an LC50 of 44.59 µg/g against fire ant workers. These data suggested that 2-undecanone may play a useful role in the integrated management of C. fragariae and S. invicta.


N-acylation/aza Diels–Alder cycloaddition/decarboxylation/dehydration) was performed to synthesize a series of pyrrolopyridin-5-ones in 20% to 92% overall yields using ytterbium triflate as a catalyst, toluene as a solvent, and microwaves as a heat source. The synthesized molecules were evaluated in vitro against breast cancer cell lines MDA-MB-231 and MCF-7, finding that compound 1f, at a concentration of 6.25 μM, exhibited a potential cytotoxic effect. Then, to understand the interactions between synthesized compounds and the main proteins related to the cancer cell lines, docking studies were performed on the serine/threonine kinase 1 (AKT1) and Orexetine type 2 receptor (Ox2R), finding moderate to strong binding energies, which matched accurately with the in vitro results. Additionally, molecular dynamics were performed between proteins related to the studied cell lines and the three best ligands.An Ugi–Zhu three-component reaction (UZ-3CR) coupled in a one-pot manner to a cascade process ( Breast cancer is the most common invasive neoplasm and the leading cause of death in women worldwide. According to the World Health Organization, 2.5 million new cases and 685,000 deaths were reported worldwide in 2020 . Furtherb]pyridin-5-ones 1a–1k, molecular docking assays were performed using 20 protein targets related to breast cancer: Serine/Threonine-protein kinase AKT (AKT1), Troponin cardiac muscle (cTn), Cytochrome P450 2C9 (CYP2C9), Cytochrome P450 3A4 (CYP3A4), MAP kinase p38 alpha (p38α), Cathepsin S (CTSS), Cyclin-dependent kinase 2/cyclin A (Cdk2/CA), Insulin-degrading enzyme (IDE), Cyclin-dependent kinase 2/cyclin E (Cdk2/CE), Cyclin-dependent kinase 4 (CDK4), Mitogen-activated protein kinase 8 (MAPK8), Cathepsin L (CTSL), Serotonin 6 receptor (5-HT6), Dual specificity protein phosphatase 3 (DUSP3), GTPase Kras (KRAS), MAP kinase-activated protein kinase 3 (MAPK3), Metabotropic glutamate receptor 2 (mGluR2), Ghrelin receptor (ghrelinR), Histamine H3 receptor (H3), and Orexin-2 receptor (Ox2R). The best hits were confirmed using molecular dynamics (MD). Based on available information, there are no prior investigations into the potential of this compound series to target AKT1 pyridin-5-ones [Multicomponent reactions are privileged synthetic tools due to the high atom economy observed when assembling polyheterocyclic cores in a one-pot manner . Within n-5-ones .b]pyridin-5-ones 1a–1k, a one-pot process was performed by coupling an UZ-3CR to a cascade sequence: aza Diels–Alder/N-acylation/aromatization (decarboxylation/dehydration). In some of our previous reports, it was found that toluene and ytterbium triflate were the best solvent and catalyst, respectively, to perform this process pyridin-5-one could interact with a significant number of proteins implicated in cancer-related disorders. In this context, it is plausible that compounds 1a–1k may exhibit inhibitory properties akin to those observed in antineoplastic drugs. Therefore, these compounds may be identified as potential candidates for utilization in breast cancer therapy.A thorough target analysis demonstrates that pyrrolopyridin-5-one nucleus. The consistency of this interaction is observed across all inhibitors, with the sole difference being the way in which residue PHE2717.34 is accommodated on the pyrrolopyridin-5-one nucleus. This adjustment is contingent upon the orientation of the inhibitors, as illustrated in 6.58, it mostly engages in hydrogen bonding and π–cation interactions, either with the pyrrolopyridin-5-one core of 1f or the amino pyridine substituent of 1h. In the present context, the observed variability in energy contributions pertaining to ligand 1h may provide insights into the factors underlying the more favorable interaction energy between inhibitor 1h and Ox2R, as opposed to AKT1.The energy increase experienced by each residue is primarily determined by the specific contact exerted by each ligand . Regardi7.31 in the activation mechanisms of Ox2R, as well as its apparent inhibition by ligands 1f, 1h, and 1k through favorable interactions, it can plausibly be suggested that these ligands have the potential to interfere with the interactions between transmembrane helices 5 (TM5) and 6 (TM6). The potential intermembrane contact described may give rise to an inhibitor outcome due to the interconnected inward motion of TM5 and TM6 helices, which is also associated with the activation of Ox2R pyridin-5-ones 1g–1k.General procedure (GP): The corresponding aldehydes H-pyrrolopyridin-5-one (1g)2-benzyl-3-(diethylamino)-7-(pyridin-3-yl)-6-(pyridin-3-ylmethyl)-6,7-dihydro-5N,N-diethyl-2-isocyano-3-phenylpropanamide (289.0 mg), and maleic anhydride (143.0 mg) were reacted together in toluene (1.0 mL) to afford 1g  as a yellow oil; Rf = ; FT-IR (ATR) νmax/cm−1 1693 (C = O); 1H NMR : δ 8.65 , 8.56 , 8.51 , 8.47 , 7.92 , 7.62–7.59 , 7.41–7.37 , 7.33–7.30 , 7.29–7.26 , 7.19–7.13 , 5.38 , 5.31 , 4.32 , 4.17 , 4.00 , 3.03 , 0.97  ppm; 13C NMR : δ 167.4, 164.1, 158.7, 150.2, 149.6 (2), 149.3, 146.9, 139.2, 136.1, 135.3, 132.2, 131.2, 128.9 (2), 127.9 (2), 125.9, 125.7, 123.8, 123.7, 122.8, 62.3, 47.6 (2), 41.7, 39.8, 12.0 (2) ppm; HRMS (ESI+): m/z calcd for C29H29N5O [M + H]+ 464.2450, found 464.2445.According to the GP, 3-Pyridinecarboxaldehyde (100.0 μL), 3-Picolylamine (107.0 μL), ytterbium(III) triflate (19.0 mg), H-pyrrolopyridin-5-one (1h)2-benzyl-3-(piperidin-1-yl)-6-(pyridin-3-ylmethyl)-7-(pyridin-4-yl)-6,7-dihydro-51h  as a yellow oil; Rf = ; FT-IR (ATR) νmax/cm−1 1694 (C = O); 1H NMR : δ 8.62–8.60 , 8.53 , 8.42–8.40 , 7.84 , 7.56–7.53 , 7.23 , 7.17–7.12 , 7.10–7.08 , 5.40 , 5.18 , 4.23 , 4.12 , 3.91 , 2.82–2.78 , 1.75–1.69 , 1.62–1.57  ppm; 13C NMR : δ 167.6, 162.7, 157.9, 150.5 (2), 149.8, 149.6, 149.4, 144.6, 139.1, 136.2, 132.1, 128.8 (2), 128.1 (2), 126.1, 123.8, 123.3, 122.9, 122.6 (2), 63.3, 54.2 (2), 41.8, 39.7, 26.3 (2), 23.9 ppm; HRMS (ESI+): m/z calcd for C30H30N5O [M + H]+ 476.2437, found 476.2445.According to the GP, 4-Pyridinecarboxaldehyde (100.0 μL), 3-Picolylamine (106.0 μL), ytterbium(III) triflate (19.0 mg), 2-isocyano-3-phenyl-1-(piperidin-1-yl)propan-1-one (300.0 mg), and maleic anhydride (141.0 mg) were reacted together in toluene (1.0 mL) to afford H-pyrrolopyridin-5-one (1i)2-benzyl-3-(piperidin-1-yl)-7-(pyridin-2-yl)-6-(pyridin-3-ylmethyl)-6,7-dihydro-51i  as a yellow oil; Rf = ; FT-IR (ATR) νmax/cm−1 1691 (C = O); 1H NMR : δ 8.58 , 8.46 , 8.41–8.39 , 7.87 , 7.61–7.57 , 7.24–7.19 , 7.17–7.06 , 7.05–7.01 , 5.46 , 5.20 , 4.22 , 4.20–4.16 , 2.81–2.75 , 1.74–1.65 , 1.61–1.53  ppm; 13C NMR : δ 167.5, 162.2, 158.3, 155.4, 149.9, 149.8, 149.6, 148.9, 139.4, 136.9, 136.3, 132.7, 128.8 (2), 128.0 (2), 125.9, 123.8, 123.5, 123.4, 123.4, 123.0, 66.2, 54.2 (2), 42.1, 39.8, 26.3 (2), 23.9 ppm; HRMS (ESI+): m/z calcd for C30H30N5O [M + H]+ 476.2442, found 476.2445.According to the GP, 2-Pyridinecarboxaldehyde (100 μL), 3-Picolylamine (108 μL), ytterbium(III) triflate (19 mg), 2-isocyano-3-phenyl-1-(piperidin-1-yl)propan-1-one (306 mg), and maleic anhydride (144 mg) were reacted together in toluene (1.0 mL) to afford H-pyrrolopyridin-5-one (1j)2-benzyl-3-(diethylamino)-7-(pyridin-2-yl)-6-(pyridin-3-ylmethyl)-6,7-dihydro-5N,N-diethyl-2-isocyano-3-phenylpropanamide (291 mg), and maleic anhydride (144 mg) were reacted together in toluene (1.0 mL) to afford 1j  as a yellow oil; Rf = ; FT-IR (ATR) νmax/cm−1 1692 (C = O); 1H NMR : δ 8.60 , 8.47 , 8.41 , 7.88 , 7.61 , 7.23 , 7.19 , 7.14–7.11 , 7.03 , 5.47 , 5.22 , 4.25 , 4.23–4.17 , 2.97 , 0.92  ppm; 13C NMR : δ 167.5, 163.6, 158.4, 155.3, 149.9, 149.8, 148.9, 146.7, 139.4, 136.8, 136.3, 132.6, 129.5, 128.9 (2), 127.8 (2), 125.8, 123.5, 123.4, 122.9, 66.2, 47.7 (2), 42.1, 39.8, 12.0 (2) ppm; HRMS (ESI+): m/z calcd for C29H30N5O [M + H]+ 464.2445, found 464.2545.According to the GP, 2-Pyridinecarboxaldehyde (100 μL), 3-Picolylamine (108 μL), ytterbium(III) triflate (19 mg), H-pyrrolopyridin-5-one (1k)2-benzyl-7-(pyridin-2-yl)-6-(pyridin-3-ylmethyl)-3-(pyrrolidin-1-yl)-6,7-dihydro-51k  as a yellow oil; Rf = ; FT-IR (ATR) νmax/cm−1 1693 (C = O); 1H NMR : δ 8.57 , 8.45 , 8.41 , 7.67 , 7.58 , 7.23–7.09 , 7.05–7.02 , 7.01 , 5.44 , 5.19 , 4.24 , 4.18 , 3.20–3.09 , 1.92–1.82  ppm; 13C NMR : δ 167.7, 156.4, 155.6, 155.1, 149.7, 149.6, 148.8, 146.1, 139.4, 136.7, 136.1, 132.6, 128.3 (2), 127.9 (2), 125.7, 123.5, 123.3, 123.1, 122.8, 118.5, 66.0, 51.8 (2), 42.0, 41.5, 25.0 (2) ppm; HRMS (ESI+): m/z calcd for C29H28N5O [M + H]+ 462.2298, found 462.2288According to the GP, 2-Pyridinecarboxaldehyde (100 μL), 3-Picolylamine (108 μL), ytterbium(III) triflate (19 mg), 2-isocyano-3-phenyl-1-(pyrrolidin-1-yl)propan-1-one (288 mg), and maleic anhydride (144 mg) were reacted together in toluene (1.0 mL) to afford ® (5240-062). MTT (M2128) was purchased from Sigma-Aldrich .Culture Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Sigma-Aldrich , antibiotics/antimycotics GIBCO2 at 37 °C. The MCF-7 and MDA-MB-231 breast cancer cells were serum-deprived for 24 h before treatment.The MCF-7 and MDA-MB-231 breast cancer cells lines were obtained from ATCC , and were cultured in DMEM/F12 medium  with 5% fetal bovine serum (FBS) and 1% antibiotic . Cells were maintained in a humidified atmosphere with 5% CO4 cells were seeded and grown overnight in a 96-well plate. Treatments with the synthesized compounds were performed by employing different concentrations ranging from 6.25 to 200 mM. After 24 h and 48 h of treatment, 0.5 mg/mL MTT was added to each well and incubated at 37 °C for 4 h, protected from light. Formazan crystals were dissolved in DMSO, and absorbances were measured at 570 nm in a MultiskanTM GO microplate spectrophotometer . The percentage of cell viability was calculated as the ratio of optical densities of the control and experimental groups.Viability was measured by MTT  assay. A quantity of 2 × 10p < 0.05 was considered significant.The results are presented as the mean ± SD. Data were analyzed statistically by one-way ANOVA, and comparisons were performed by Dunnett’s multiple comparison test. A statistical probability of The assessment of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMETox) characteristics was carried out. The assessment of ADMETox characteristics was conducted using the SwissADME (Ver. 2017)  and ProT1a–1k was conducted via SwissTargetPrediction (Ver. 2019) . Th. Th68]. Autodock Vina version 1.2.5  was usedTo improve the accuracy of the docking analysis, the ligands were subjected to a redocking procedure and subsequently classified using a clustering, with a cutoff of 0.5 Å. The ligands selected for subsequent MD analyses were those with binding scores that were one standard deviation below the mean and belonged to the most densely populated clusters .50 values were chosen for subsequent MD simulations using the GROMACS program [https://server.poissonboltzmann.org/) (accessed on 14 August 2023). The construction of input files was carried out with the Input Generator CHARMM-GUI [The molecular complexes that displayed the most favorable energy scores and exhibited the lowest IC program . The pro program  availablARMM-GUI . The CHAARMM-GUI  was usedARMM-GUI  was used1), while the other nine systems included the Orexin-2 receptor (Ox2R). The systems underwent a sequential process, which included an initial minimization phase consisting of 10,000 iterations using the steepest descent method. This was followed by two subsequent phases of MD simulations, with the first phase lasting 10 ns and conducted in the NVT ensemble, and the second phase lasting 20 ns and conducted in the NPT ensemble. These MD simulations were performed to achieve system equilibration. Following that, MD simulations lasting 100 ns were conducted. In the case of AKT1 systems, a timestep of 4 femtoseconds was used together with Hydrogen-Mass-Repartitioning (HMR) [2R, a timestep of 2 femtoseconds was utilized. The use of periodic boundary conditions (PBC) and Particle-Mesh-Ewald (PME) was employed for the treatment of electrostatic interactions. Inside the NPT ensemble, the temperature coupling was performed using the velocity rescale method, and the pressure coupling was performed using the Parrinello–Rahman method. The system was maintained at a temperature of 310.15 K and a pressure of 1 bar.The orthorhombic cell, with a thickness of 15 Å, was used to enclose each system. TIP3P water molecules were employed to surround the cell and, to neutralize the system’s charge, 0.15 M NaCl was introduced. A total of eighteen systems were constructed, with nine of them using AKT kinases (AKTng (HMR) . Convers2R, a membrane protein, using the parameters established by Marrink et al. (2014) [The simulation of Ox. (2014)  and Im e. (2014) , specifi. (2014)  inside t1f, 1h, and 1k) and two target proteins (AKT1 and Ox2R), yielding a total of six systems, each with three replicas. The total time of the overall simulation effort was 1.8 µs. The trajectory analysis was conducted using the MDAnalysis software (Ver. 2.6.1) [The MD simulations were carried out in triplicate, with each run being independent. The simulations encompassed three distinct ligands (. 2.6.1) , and the. 2.6.1) .The molecular mechanics generalized Born surface area (MM/GBSA) approximation was used to obtain the interaction energy ,89,90. TΔG binding) was determined according to the equation:The binding free energy  [Since entropy was not calculated during the experiment, the reported binding free energies only show the enthalpic part of the binding free energy . To examry 2023) .The investigation of molecules potentially possessing pharmacological properties that may effectively engage multiple protein receptors offers a promising strategy for addressing sophisticated medical problems, such as breast cancer. The pharmaceutical compounds synthesized for the present study exhibited interesting properties and numerous benefits, including the abilities to enhance therapeutic efficacy through synergistic interactions with various targets, enhance safety profiles, and decrease required dosages. Then, computer simulations were performed to enhance the progress of multi-target molecules for the treatment of cancer and accelerate the identification and progression of innovative therapeutic treatments.1f showed the greatest potential as a viable and promising candidate for cancer treatment in triple-negative breast cancer, exhibiting the lowest IC50 at 14.8 mM and morphological changes, indicative of decreased cell viability in the triple-negative breast cancer cell line MDA-MB-231. Nevertheless, there is a dearth of comprehension around the precise pathways through which 1f exhibits its antineoplastic properties, underscoring the imperative for additional investigation in the realm of cancer treatment.Interestingly, of all compounds evaluated here, compound


Thunbergia erecta L. contains cytotoxic and liver-protective compounds. Thunbergia erecta L. leaves were macerated in 70% aqueous ethanol, then fractionated with ethyl acetate (9.3 g) and butanol (12.7 g), and attenuated Den-induced liver cancer in a Wistar rat experimental model. Ethyl acetate and butanol fractions were chromatographed using column chromatography and solid-phase extraction (SPE); Vicenin-II (1), kaempferol (2), biochanin A, sissotrin 7-O-β-glucopyranoside (3), gentianose (4), acacetin 7-O-β-glucopyranoside (5), apigenin 7-O-β-glucopyranoside (6), and rosmarinic acid (7) were extracted, and their structures were determined using NMR spectroscopy and ESI-mass spectrometry. Sixty rats were divided into six groups (ten each): control group, Den group, doxorubicin/Den-treated group, butanol fraction/Den-treated group, and isolated acacetin 7-O-β-glucopyranoside/Den-treated group. The liver enzymes and proinflammatory biomarkers were used to estimate the liver function. In addition, liver tissues were collected for analysis of oxidative stress markers, gene expression, and histopathology. There is a significant increase in the levels of liver enzymes, AFP, and TNF-ἁ. This was conveyed by a significant increase of IL-1 and caspase-3, elevation of MDA and reduction of GSH, and suppression of Bcl2 and elevation of Bax expression. All parameters in butanol, ethyl acetate fractions, and isolated acacetin 7-O-β-glucopyranoside (major constituents) of T. erecta L. were significantly improved to values close to those of the control group.The online version contains supplementary material available at 10.1007/s12010-022-04292-x. Thunbergia erecta L. is vigorous, a woody shrub which belongs to Acanthaceae family. This shrub has small, ovate leaves with entire margins borne opposite on thin, brown stems − at m/z 593.271 and 201.78, 125.0014 Da. 1H NMR ; δPPM 13.6 , 8.01 , 6.91 , 6.56, 5.46 , 5.11 , 3.85–3.16 remaining of two sugar protons. 13CNMR  &DEPT.Q 100 MHz); δPPM 182.7 (C-4), 164.4 (C-2), 159.0 (C-7) 161.6 (C-4`), 159.1 (C-5), 155.6 (C-9), 129.4 (C-2`/6`), 122.0 (C-1`), 116.1 (C-3`/5`), 107.9 (C-6), 105.6 (C-8), 102.9 (C-3), 82.2, 81.2 , 78.2 , 73.8, 72.2 , 71.4, 71.0 , 69.5  and 61.7, and 60.3 .Yellowish white amorphous powder (10 mg). Chromatographic properties dark purple fluorescence spot with Rf values; 0.80 and 0.04 in solvent systems BAW (4:1:5 v:v:v) & (15% aqueous acetic acid). 1H NMR ; δPPM 8.03 , 6.92 , 6.44 , 6.19 .yellow amorphous powder chromatographic properties dull yellow spots changed to bright yellow with ammonia vapors, green color with FeCl3 and negative Molisch’s test. 3 and weak unclear change with ammonia vapors. 1HNMR  δPPM 8.52 , 7.97 , 7.09 , 6.66 , 5.95 ,4.91 , 3.85 . Negative ESI/MS: m/z 445.19 [M-H]−, 281 [M-glucosyl-2H]−.Yellowish white amorphous powder and show purple fluorescence (UV light) and light green color with FeCl2SO4) spray reagent, gave reddish violet spot-on TLC with diphenylamine positive ESI-MS/MS: m/z 525.47 [M + 4H + NH4]+, 365.10, 237.05, and 147.23. 1HNMR ; δ PPM fructose moiety 3.6 , 4.19 , 4.17 , 3.69 (H5), 3.62  for β-glucose moiety 4.31 , 3.25 , 2.96 , 3.70 , 3.72 . For α-glucose moiety 4.89 , 3.15–3.17 , 3.25 , 2.96 , 3.70 , 3.72 . 13CNMR spectral data : δPPM for fructose 60.0 (C1), 104.3 (C2), 81.4 (C3), 75.9 (C4), 81.8 (C5), 61.0 (C6), for β-glucose 97.1 (C1`), 72.5 (C2`), 75.4 (C3`), 71.0 (C4`), 75.1 (C5`), 60.6 (C6`) for α-glucose 92.5 (C1``), 71.7 (C2``), 73.7 (C3``), 70.2 (C4``), 74.9(C5``), 68.5 (C6``).Colorless crystalline powder, chromatographic properties, no fluorescence (UV light), give positive Molisch’s test and on TLC gave sharing spot after spraying with 10%  & ethyl acetate: methylene chloride:methanol:water (80:20:18:2 v:v:v:v) respectively. Dull yellow florescence (UV light/ammonia vapor), give green color with FeCl3 spray reagent and gives positive Molisch’s test. 1H NMR ; δPPM 12.93 , 8.07 , 67.13 , 6.97, 6.87 6.46 , 5.08 , 3.87 , 3.74–3.17  remaining of sugar protons. 13CNMR  & DEPT-Q 100 MHz); δ PPM 182.1 , 163.8 , 163 , 162.5 ,  161.1, 157 , 128.5 , 122.7 (C-1′ Q), 114.68 , 105.4 (C-10 Q), 103.8 , 99.8 , 99.5 , 94.9 , 77.1 , 76.4 , 73.1 , 69.5 , 60.5(C6′′ CH2), and 55.6 (OCH3). Figure Off-white amorphous powder (0.5 g). Chromatographic properties dark purple fluorescence spot with ms BAW 4::5 v:v:v p ≤ 0.05) was observed in the Den group when compared to control group. In contrast, Dox / Den-treated animal caused decrease in their levels when compared to Den-treated rat. Both fractions of T. erecta L. supplementation resulted in a moderate reduction in the activity of ALT, AST, and ALP, while ethyl acetate fraction showed a significant reduction in ALT as compared to the Den-treated group. Acacetin glycoside was able to restore liver enzyme activity as shown in Table Significant increase in liver enzymes   level was significantly increased in Den-treated group when compared to the normal group Table . SignifiT. erecta L. Ethyl acetate fraction was more effective in decreasing the expression of Bax gene than other treatments. And, the most effective fraction was acacetin 7-O-β-glucopyranoside in increasing the expression of Bcl2 gene  but significant decrease in Bcl2 (anti-apoptotic gene) gene when compared to the control group, while there were significant differences between all treated groups, but the treatment with Histopathological examination of liver sections from different groups showed the highest score of liver fibrosis in the control-positive group with portal tract deposition of collagen fibers group 2) ..2B2 spinH 7.09 (~ ∆ + 0.2 ppm) was indicative for 4′-O-methoxy, which was assigned as singlet at 3.85. The isoflavone was also supported by the characteristic location of H-2 resonance at about δH 8.52 ppm. As well as the downfield shift of H-8 proton (≈ ∆ + 0.25 ppm) together with the presence of a β-anomeric proton signal at 4.91 (H1′′), were two evidence of the attachment of β-glucopyranoside moiety on OH-7 as O-glycoside. Negative ESI/MS showed a molecular ion peak at m/z 445.19 [M-H]− corresponding to mol. weight 446 assigned for molecular formula C22H22O10, together with other fragment ion at 281[M-hexose-2H]− assigned for a glycone + characteristic for C12H22O11 which representative for sucrose moiety, confirmed by 1HNMR spectrum, α-G1 at 4.89 , in addition to characteristic chemical shift of F-H-3′, F-H-4′ resonances at δH 4.19  key proton signals for sucrose, in addition to finger print region for sucrose in 13CNMR at δC 92.50,104.34, 60.01, and 61.07assigned for C1-α-G1, C2-F, C1-F, and C-6-F respectively [from chromatographic properties expected to be sugar with no florescence under UV light then gave black spot-on TLC after spraying with , A2B2-spin coupling system protons at δ 8.07 and 7.13  assigned for (H-2′/H-6′) and (H-3′/H-5′) of B-ring respectively, two meta doublet protons at δH 6.87 and 6.46 with J value 2.55 Hz assigned for H-8, H-6 respectively on A-ring, together with an olefinic protons at δH 6.97 singlet as signal for H-3 on a flavone C-ring in addition, signal at δH 5.08  assigned for β glucopyranosyl anomeric proton (H-1′′). Finally presence of s at δH 3.87 characteristic for the presence of OCH3. And from 13C NMR, the spectrum showed 13 carbon resonance agreement with apigenin aglycone; the most characteristic signals were 182.1, 163.8, 163.0, 162.5, 161.1, 157.0, 122.7, and 105.4 assigned for quaternary C-4, C-2, C-7, C-4′, C-5, C-9, C-1′, and C-10 respectively and olefinic carbon resonance at δC 128.5, 114.6, and 103.8 assigned for C-2′/6′, 3′/5′, and C-3 respectively to confirm the aglycone as apigenin and the presence of carbon resonance at δC 55.6 characteristic for OCH3 at C4′-methyl ether apigenin which is known as acacetin and the value of anomeric carbon at δC 99.8 and the carbon resonance of remaining sugar agreement with β-d-glucopyranoside and sugar part attached to C-7 confirmed by the downfield shift of H-8 and H-6 approximately (≈ ∆ + 0.2–0.4 ppm) and by the value of C-7 in 13C NMR spectrum, and the data was agreed with data published by [O-β-d-glucopyranoside which known as acacetin 7-O-β-d-glucopyranoside  [. (2021) .T. erecta L. ethyl acetate, butanol fractions, and the major isolated metabolite; acacetin 7-O-glucopyranoside against diethylnitrosamine induced liver toxicity. The polyphenolic compounds and flavonoids are identified in T. erecta L. and the presence of these biomolecules in erecta leaves makes them a potential bioantioxidant source. A chronic hepatocellular injury is the main cause of liver fibrosis, a serious condition with considerable morbidity and mortality. Patient mortality is increasing with liver fibrosis because it manifests a variety of pathological conditions, sometimes leading to liver cancer [T. erecta L. bioactive constituents initiate cell proliferation and survival possible by overexpressing Bcl2—this is attributed to phenolic acids and flavonoids, which have anti-apoptotic properties as has been described previously [O-glucopyranoside is the most active ingredient isolated from this plant and it provides powerful healing properties. Our current study showed that Den treatment resulted in reduced GSH levels and higher MDA levels in liver tissue of rats diethylnitrosamine causes fibrotic lesions to form in the liver during oxidative damage [T. erecta L. arises from its antioxidants content as quercetin-3-O-xyloside [O-glucoside [O-glucoside). As a result of this inhibition, hepatic fibrosis and hepatocellular toxicity associated with Den may have been reduced. In conjunction with these findings, recent studies have shown that herbal bioactive molecules can be effective in preventing liver fibrosis [O-glucoside markedly alleviated and achieved marked improvements in reversing this pathological injury the Den induced in the rats’ livers ( reduction in fibrosis scoring). Furthermore, chromatin fragmentation was much less or absent in the Den-treated DoX. EE, EB, and acacetin 7-O-glucoside rats. This histopathological analysis is in accordance with the results of the biochemical diagnostic indicators. This could be due to the observed ability of extracts to recover endogenous antioxidant mechanisms, resulting in the free radicals being scavenged to allow hepatocyte regeneration. This antioxidant effect breaks the vicious cycle of continuous inflammation fibrosis resulting from the continuous activation of KCs, causing a release of pro-inflammatory factors and aiding fibrosis.The study aims to evaluate the antioxidant, anti-inflammatory, anti-apoptotic, and antifibrogenic effect of r cancer . Hepatocr cancer . Furtherr cancer  and in tr cancer . In thisr cancer . Reactivr cancer . Hepatocr cancer . Accordir cancer . Caspaser cancer . The prer cancer ; as a rer cancer , 25. . Ieviously . Due to eviously . Acacetie damage . The antxyloside , kaempfexyloside , and acalucoside  which prlucoside . A markefibrosis , 54. . HO-β-glucopyranoside isolated from T. erectaL. leaf extract, ethyl acetate, and butanol fractions have beneficially hepatoprotective and antifibrotic impact against oxidative injury induced by diethylnitrosamine. T erecta L. has a defensive outcome on hepatic fibrosis may be due to its free radical scavenging, antioxidant, and anti-inflammatory effects that are attributed to polyphenoliccontents, especially acacetin glucoside. These outcomes may be valuable in developingnovel hepatic fibrosis prevention approaches. The present study revealed that acacetin 7-Supplementary file1 (DOCX 723 KB)Below is the link to the electronic supplementary material.


This study aims at exploring the structure–activity relationship (SAR) and improving the CXCR2 antagonistic potency of this pyridopyrimidine via systematic structural modifications of the substitution pattern. Almost all new analogues completely lacked the CXCR2 antagonism, the exception being a 6-furanyl-pyridopyrimidine analogue (compound 17b) that is endowed with similar antagonistic potency as the original hit.Upregulated CXCR2 signalling is found in numerous inflammatory, autoimmune and neurodegenerative diseases, as well as in cancer. Consequently, CXCR2 antagonism is a promising therapeutic strategy for treatment of these disorders. We previously identified, via scaffold hopping, a pyrido[3,4- Upon detection of a pathogen or in response to trauma an acute inflammatory response is mounted by tissue-resident immune cells, such as macrophages, dendritic cells and mast cells. It involves the release of a number of pro-inflammatory chemokines such as CXCL1, CXCL2 and CXCL8, resulting in the chemo-attraction of circulating immune cells . NeutropCXCR2 upregulation has been linked to a myriad of inflammatory disorders , autoimmune diseases , and even neurodegenerative diseases (such as multiple sclerosis and Alzheimer’s disease) . Hence, Furthermore, elevated expression and signaling of CXCR2 is correlated with aggressive cancer phenotypes and a poor prognosis in various cancers, such as lung cancer, colorectal cancer, pancreatic cancer, prostate cancer, breast cancer, gastric cancer and squamous cell carcinoma . In addiCXCR2 recruits myeloid derived suppressor cells (MDSCs) to the tumor microenvironment . High MDBecause of the crucial role of CXCR2 in inflammation and oncology, various small molecule CXCR2 antagonists have been developed . SeveralBesides the urea and urea isosteres class, several derivatives with an aromatic ring core have been explored, such as SX-682 , AZD-830d]pyrimidine core of the known CXCR2 antagonist AZD-8309 [d]pyrimidine analogue 2 emerged as a promising hit, displaying an IC50 value of 0.11 µM as CXCR2 antagonist, as measured in a kinetic fluorescence-based calcium mobilization assay [d]pyrimidines with CXCR2 antagonistic properties are not known, this scaffold has been associated with various other bioactivities. Pyridopyrimidin-4-ones were shown to be potent and selective matrix metalloproteinase-13 inhibitors [d]pyrimidin-4(3H)-ones were potent inhibitors of histone lysine demethylase [d]pyrimidines displayed potent inhibition of monopolar spindle kinase 1 pyrimidine scaffold is pursued in an effort to explore the structure–activity relationship (SAR) of pyridopyrimidine 2 and to improve its CXCR2 antagonistic potency.In this study, a systematic exploration of the substitution pattern of the pyridopyrimidine was synthesized starting from commercially available 3,5-dichloropyridine-4-carbonitrile 3 -alaninol gave compound 9 in low yield, because of its low solubility resulting in product losses during purification.The 5-chloropyridopyrimidine 9 with phenylboronic acid gave no conversion when standard reaction conditions were applied, i.e., tetrakis(triphenylphosphine)palladium(0) as catalyst and using potassium carbonate as a base in a mixture of water and dioxane. When transitioning to a more catalytically active system pyrimidine derivative 8. The poor outcome of these Suzuki reactions can be ascribed to different factors. Lewis-basic heterocycles or compounds having coordinating groups are known to behave poorly in palladium-catalyzed cross-coupling reactions pyrimidine scaffold prevents an efficient Suzuki reaction.The Suzuki-coupling of 5-chloropyridopyrimidine analogue was prepared from commercially available 5-bromo-4-methyl-3-nitropyridine 10  pyrimidine-4-one 14a pyrimidine scaffold via palladium-catalyzed cross-coupling reactions, the 6-bromo analogue is preferred. Therefore, several attempts to prevent this transhalogenation were made. Performing the Béchamp reduction of the nitro group of compound 11 prior to oxalyl chlorination should greatly reduce the chance of transhalogenation, since the aminopyridine is far less electrophilic than the corresponding nitropyridine pyrimidin-4-one 14b was followed by benzylation yielding compound 15b. Next, the one-pot benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)-mediated amination with (R)-alaninol gave the 4-amino-6-imidazolyl-pyridopyrimidine 16b in good yield pyrimidine analogue were unsuccessful, the derivatization of the 6-chloropyridopyrimidine 16a via palladium-catalyzed cross-coupling was considered -alaninol group towards 15a. For the Suzuki coupling with 2-thienylboronic acid, the more active catalyst system, Pd2dba3/Xphos [d]pyrimidine 17d in fair yield.Since various attempts to synthesize the 6-bromopyridopyrimidine scaffold was performed using benzophenone imine as ammonia surrogate (15a was protected with p-methoxybenzyl chloride which proceeded quantitatively to compound 18.pyrimidine-4-one 20 which was isolated by simple filtration. Finally, compound 21 was obtained via a BOP-mediated amination with (R)-alaninol.Finally, the formal introduction of ammonia at position 6 of the pyrido. Th. Thd]pyrpound 18. Buchwaldd]pyrimidine scaffold, 2,4-dichloropyridopyrimidine 22, prepared according to a literature procedure ethanol 23e was only moderate. Finally, introduction of the 2,3-difluorobenzylthio moiety on 2-chloropyridopyrimidines 23a–e proceeded in high yields affording target compounds 24a–e.For the introduction of structural modifications at positions 2 and 4 of the pyridopyrimidine 26a in good yield. Introduction of 2,3-difluorobenzylalcohol required using the alcohol nucleophile as cosolvent and a long reaction time. Even then, the reaction did not proceed to full conversion, resulting in tedious purification and a low yield of compound 26b. For the thiol nucleophiles (thiophenol (compound 26c), phenylethanethiol (compound 26d), and hexanethiol (compound 26e)), the nucleophilic aromatic substitution proceeded smoothly and the target compounds 26c–e were isolated in high yields.In another series, the 2-position substituent was varied while retaining the  when compared to the original hit 2. Synthesis problems (vide supra) prevented us from further elaboration of this particular position. The 6-chloro analogue 16a was completely devoid of CXCR2 antagonism. Further elaboration at the 6-position by the synthesis of a 6-N-imidazolyl, 6-phenyl- and 6-thienyl-pyridopyrimidine analogue  did not have any beneficial effect on CXCR2 antagonism. Remarkably, the 6-furanyl congener 17b (IC50 = 0.54 µM) showed comparable activity to its unsubstituted congener 2 (IC50 = 0.11 µM). However, introduction of a methyl group on the furanyl group (compound 17c) led again to complete loss of activity. It is well known that an amino group on the thiazolo moiety of thiazolopyrimidine yields potent CXCR2 antagonists.[d]pyrimidine series, the presence of an amino group at position 6 (compound 21) led to a complete loss of CXCR2 antagonistic activity. This suggests that the structure–activity relationship (SAR) in the thiazolopyrimidines and pyridopyrimidines is different and does not run in parallel. Although compounds 2 and 17b are the most potent CXCR2 antagonists within this series, they are still clearly much less active than navarixin and the reference thiazolopyrimidine analogue.In a first round of SAR, the substitution pattern of the pyridine moiety of the pyridopyrimidine 2, structural modifications of the substituents at positions 2 and 4 were explored. Subtle structural variations of the (R)-alaninol group of hit compound 2 (24a), insertion of an additional methyl group yielding the gem-dimethyl analogue 24b, introducing a hydroxyl functionality (compound 24c) or removal of the methyl group (compound 24d), afforded pyridopyrimidines that were completely devoid of CXCR2 antagonism, as evidenced by IC50 values exceeding 30 µM. Similarly, an ethyleneglycol linker at position 4 gave compound 24e also lacking CXCR2 antagonism. This situation is different from the thiazolopyrimidine series, since for this latter scaffold, CXCR2 antagonism tolerates structural variation at this position pyrimidine scaffold. Shortening (compound 26c) or elongation (compound 26d) of the linker between the sulfur and the phenyl moiety furnished analogues that were inactive as CXCR2 antagonists. Since it has been shown before that in the thiazolopyrimidine series, an aliphatic side chain is tolerated for CXCR2 antagonism, an n-hexyl linker was installed (compound 26e), which was inactive as CXCR2 antagonist, with an IC50 value exceeding 30 µM , iodine coated silica or KMnO4-staining. Column chromatography was performed via standard column chromatography or using an MPLC apparatus. For column chromatography, 70–230 mesh silica 60  was used as the stationary phase. MPLC was performed using a CombiFlash EZ prep apparatus with BGB Scorpius Silica 60Å Irregular—50 µm cartridges. 1H NMR spectra were recorded on a Bruker Avance 300 (300 MHz working frequency) or Bruker Avance 400 (400 MHz working frequency). Proton-decoupled 13C NMR spectra were recorded on a Bruker Avance 400 (101 MHz working frequency). 19F NMR spectra were recorded on a Bruker Avance 400 (377 MHz working frequency). Samples were dissolved in d6-DMSO or CDCl3, and chemical shifts (δ) were reported in parts per million (ppm) referenced to tetramethylsilane (1H), or the internal (NMR) solvent signal (1H and 13C) as internal standards pyrimidine-4-one (7)2-Thioxo-5-chloropyridopyrimidine-4-one (8)2--5-chloropyridopyrimidine-4-one  and dry DMF (20 mL) were added to a flame dried, N2-flushed 100 mL round-bottom flask under N2-atmosphere. The suspension was sonicated for 15 min, after which triethylamine  was added, resulting in dissolution of the starting material. Next, a solution of 2,3-difluorobenzyl bromide  in dry DMF (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 1 h, concentrated in vacuo and 20 mL H2O was added. The formed precipitate was filtered, washed with water, MeOH and Et2O, and dried under vacuum to afford the title compound  as a beige solid. mp > 300 °C.2-Thioxo-5-chloropyrido+ calcd. for C14H8Cl1F2N3O1S1: 340.01173; found: 340.0114.R)-2-pyrimidine-4-yl)amino]propanol (9)-5-chloropyridopyrimidine-4-one  and POCl3 (10 mL) were added. After heating to 120 °C for 2 h, the mixture was cooled down to room temperature, and concentrated to dryness in vacuo. The obtained material was resuspended in ethyl acetate, poured over ice and neutralized with saturated aqueous NaHCO3. The obtained aqueous solution was extracted with ethyl acetate, and the combined organic phases were dried over MgSO4 and concentrated in vacuo. The crude intermediate was then dissolved in dry DMF (6 mL), and Et3N  and (R)-alaninol  were added dropwise while cooling in an ice bath. After stirring overnight at room temperature, the reaction mixture was concentrated in vacuo. Water (20 mL) and EtOAc (20 mL) were added. Following phase separation, the aqueous phase was extracted with EtOAc (2 × 20 mL). The combined organic phases were washed with water (2 × 20 mL) and brine (2 × 20 mL), dried over Na2SO4 and coated on Celite. Filtration over silica using 1:1 EtOAc/Et2O followed by column chromatography using 40–100% Et2O/PE afforded the title compound  as a white solid. mp 146–148 °C.In a flame dried, N1H NMR : δ (ppm) 8.83 , 8.43 , 8.04 , 7.45–7.37 , 7.34–7.23 , 7.19–7.09 , 5.15 , 4.47 , 4.37–4.24 , 3.64–3.48 , 1.21 ; 13C NMR : δ (ppm) 168.0, 156.2, 149.6 , 149.5, 148.1 , 146.3, 142.9, 127.9 , 126.2 , 124 , 124.6 , 124.5, 116.2 , 114.3, 63.0, 48.9, 27.3, 16.3; 19F NMR : δ (ppm) −139.30 , −142.74 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C17H15Cl1F2N4O1S1: 397.06958; found: 397.0697.2-Bromo-5-nitropyridine-4-carboxylic acid (11)10  was dissolved in concentrated H2SO4 (100 mL). The mixture was cooled in an ice bath, and K2Cr2O7  was added portion-wise. Following complete addition, the mixture was stirred in an ice bath for 1 h and at room temperature for 24 h. The resulting green solution was slowly poured onto 1L of ice. The obtained precipitate was filtered, washed with water until the eluent was no longer green, and dried under vacuum to afford the title compound as white solid .2-Bromo-4-methyl-5-nitropyridine 1H NMR : δ (ppm) 13.03 , 9.11 , 8.14 ; 13C NMR : δ (ppm) 163.7, 146.39, 146.31, 142.2, 138.5, 127.1;2-Chloro-5-nitropyridine-4-carboxamide (12a)11  in dry DCM (200 mL) at 0 °C and under N2-atmosphere. After stirring for 5 min, dry DMF (0.2 mL) was added. The reaction mixture was stirred at 0 °C for 1 h and at room temperature overnight. The resulting solution was concentrated in vacuo. The obtained residue was dissolved in dry DCM (50 mL) and added dropwise to a heavily stirred, ice cold 25% aqueous ammonia solution (300 mL). After stirring for 15 min, the formed precipitate was filtered, washed with saturated aqueous NaHCO3 and water, and dried under vacuum overnight to afford the title compound  as a white solid that was used without further purification.Oxalyl chloride (20 mL) was added to an ice-cold solution of 2-bromo-5-nitropyridine-4-carboxylic acid 2-Imidazolyl-5-nitropyridine-4-carboxamide (12b)2-flushed round-bottom 100 mL two-necked flask, equipped with a N2-balloon, septum and stir bar, 2-chloro-5-nitropyridine-4-carboxylic acid 11 , 1,1-carbonyldiimidazole  and dry THF (20 mL) were combined. The reaction mixture was stirred at room temperature for 30 min, then aqueous ammonia  was added in one portion. The mixture was stirred overnight at room temperature, and then was transferred to a separatory funnel along with EtOAc (50 mL) and water (20 mL). Following phase separation, the aqueous phase was extracted with EtOAc (1 × 20 mL). The combined organic phases were washed with water and brine, dried over Na2SO4 and concentrated to dryness in vacuo. The obtained solid was triturated with ethanol, filtered and washed with ice cold ethanol to afford the title compound as a white solid . mp 259–261 °C.In a flame dried N1H NMR : δ (ppm) 9.20 , 8.72 , 8.31 , 8.16 , 8.11 , 8.13–8.05 , 7.20 ; 13C NMR : δ (ppm) 164.9, 151.3, 145.9, 144.0, 140.1, 136.0, 131.1, 117.0, 111.4; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C9H7N5O3: 234.06216; found: 234.0625.2-Chloro-5-aminopyridine-4-carboxamide (13a)12a  in water (15 mL), EtOH (30 mL) and AcOH (30 mL). The resulting suspension was sonicated for 1 h and then filtered over 1:1 Celite/MgSO4, which was washed with EtOAc (500 mL). The filtrate was transferred to a separatory funnel along with water (100 mL). After phase separation, the aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic phases were washed with saturated aqueous NaHCO3 (3 × 100 mL), water (3 × 100 mL) and brine (2 × 100 mL), dried over Na2SO4 and coated on Celite. Flash chromatography using 50–100% EtOAc/PE afforded the title compound  as an off-white solid.Activated iron powder  was added to a mixture of 2-chloro-5-nitropyridine-4-carboxamide 1H NMR : δ (ppm) 8.12 , 7.93 , 7.57 , 7.54 , 6.65 ; 13C NMR : δ (ppm) 168.4, 144.5, 139.3, 135.0, 121.7, 121.5.5-Amino-2-imidazolyl-pyridine-4-carboxamide (13b)12b  and Pd/C  were combined in THF (20 mL). The reaction mixture was stirred for 24 h at room temperature and filtered over Celite. The Celite was thoroughly washed with EtOAc. The solution was coated on Celite. Flash chromatography using 0–20% MeOH/EtOAc afforded the title compound  as an off-white solid. mp 248–251 °C.In a round-bottom 100 mL two-necked flask, equipped with a hydrogen-balloon, septum and stir bar, 2-imidazolyl-5-nitropyridine-4-carboxamide 1H NMR : δ (ppm) 8.28 , 8.14 , 8.05 , 7.79 , 7.75 , 7.67 , 7.08 , 6.67 ; 13C NMR : δ (ppm) 168.9, 144.1, 138.2, 137.9, 134.3, 129.4, 120.8, 116.6, 111.1; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C9H9N5O1: 204.08798; found: 204.0871.d]pyrimidine-4-one (14a)2-Thioxo-5-chloropyridopyrimidine-4-one (14b)6-Chloro-2-thioxopyridopyrimidine-4-one (15a)2--6-chloropyridopyrimidine-4-one 14a  and dry DMF (40 mL) were added to a flame dried, N2-flushed 250 mL round-bottom flask under N2-atmosphere. The suspension was sonicated for 15 min, after which triethylamine  was added, resulting in dissolution of the starting material. Next, a solution of 2,3-difluorobenzyl bromide  in dry DMF (20 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h, concentrated in vacuo and 50 mL H2O was added. The formed precipitate was filtered, washed with water (2 × 10 mL), MeOH (10 mL) and Et2O (10 mL) and dried under vacuum to afford the title compound  as a beige solid. mp 235–237 °C2-Thioxo-6-chloropyrido+ calcd. for C14H9Cl1F2N3O1S1: 340.0117; found: 340.0119.d]pyrimidine-4-one (15b)2--6-imidazolyl-pyridopyrimidine-4-one 14b , and dry DMF (2.0 mL) and Et3N  were added. A solution of 2,3-difluorobenzyl bromide  in dry DMF (1 mL) was added dropwise. After stirring at room temperature for 1 h, the reaction mixture was concentrated to dryness in vacuo, and coated on Celite. Flash chromatography using 0–10% MeOH/EtOAc afforded the title compound .To a flame dried, N1H NMR : δ (ppm) 13.06 , 8.91 , 8.64 , 8.21 , 8.08 , 7.64–7.26 , 7.25–6.93 , 4.49 ; 19F NMR : δ (ppm) −139.17 , −142.15 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C17H11F2N5O1S1: 372.07250; found: 372.0727.R)-2-pyrimidine-4-yl)amino]propanol (16a)-6-chloropyridopyrimidine-4-one 15a  and SOCl2 (10 mL) and 1 drop of DMF were added. After heating to 80 °C for 4 h, the mixture was cooled down to room temperature, and concentrated to dryness in vacuo to afford the crude intermediate as a yellow solid. The intermediate was then dissolved in dry DMF (10 mL), and Et3N  and (R)-alaninol  were added dropwise while cooling in an ice bath. After stirring for 1 h at room temperature, the reaction mixture was concentrated to dryness in vacuo, transferred to a separatory funnel along with water (10 mL) and EtOAc (20 mL). After extracting with EtOAc (3 × 20 mL), the combined organic phases were washed with water (3 × 20 mL) and brine (2 × 20 mL), dried over Na2SO4 and coated on Celite. Flash chromatography using 50% PE/Et2O–1% MeOH/Et2O, followed by trituration with 50% pentane/Et2O afforded the title compound  as a white solid. m.p. 211–213 °C.In a flame dried, N1H NMR : δ (ppm) 8.79 , 8.39 , 8.37 , 7.46–7.37 , 7.34–7.23 , 7.17–7.07 , 4.81 , 4.47 , 4.42–4.31 , 3.57–3.41 , 1.19 ; 13C NMR : δ (ppm) 168.0, 156.9, 150.2, 149.6 , 148.1 , 143.7, 143.0, 128.1 , 126.3 , 124.5 , 119.9, 116.3, 116.2, 116.1, 63.5, 48.8, 27.3, 16.4; 19F NMR : δ (ppm) −139.30 , −142.77 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C17H15Cl1F2N4O1S1: 397.06958; found: 397.0693.R)-2-pyrimidine-4-yl)amino]propanol (16b) and BOP-reagent  in dry DMF (1 mL) under N2-atmosphere. Following complete addition, the reaction mixture was stirred for 10 min, after which (R)-alaninol  was added dropwise. The reaction mixture was stirred at room temperature overnight, and then heated to 60 °C for 2–3 h. Next, the reaction mixture was concentrated in vacuo and redissolved in 20 mL EtOAc and NH4Cl (10 mL) was added. The aqueous phase was extracted with EtOAc (2 × 20 mL). The combined organic phases were washed with saturated NH4Cl (3 × 10 mL), with water (2 × 10 mL) and brine (2 × 10 mL), and dried over Na2SO4. After coating on Celite, flash chromatography using 0–5% MeOH/DCM afforded the title compound  as a white-yellow solid. mp 245–247 °C.DBU  was added dropwise to a solution of 6-chloro-2--pyrido+ calcd. for C20H18F2N6O1S1: 429.13035; found: 429.1311.R)-2-pyrimidine-4-yl)amino]propanol (17a)-2-pyrimidine-4-yl)amino]propanol 16a  K2CO3 , 2-phenylboronic acid  and Pd(PPh3)4  were added. Following three cycles of backfilling with nitrogen, 1,4-dioxane (3.0 mL) and water (1.0 mL) were added. After heating to 120 °C for 48 h, the reaction mixture was transferred to a separatory funnel and water (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated aqueous NaHCO3, water and brine, dried over Na2SO4 and coated on Celite. Flash chromatography using 20–500% EtOAc/isohexane gave a mixture of starting material and title compound . This crude product was recrystallized by dissolving in EtOAc and layering with pentane. The obtained crystals were filtered and washed with 1:1 Et2O/pentane to afford the title compound  as an off-white/light-yellow solid. mp 202–204 °C.To a flame dried, N1H NMR : δ (ppm) 9.02 , 8.75 , 8.47 , 8.24–8.16 , 7.54 , 7.49–7.41 , 7.38–7.27 , 7.20–7.10 , 4.87 , 4.52 , 4.48–4.38 , 3.60–3.46 , 1.24 ; 13C NMR : δ (ppm) 167.2, 157.9, 149.9, 149.6 , 148.2 , 143.6, 138.2, 128.7, 128.3 , 126.3, 124.6 , 119.1, 116.1 , 111.6, 63.6, 48.7, 27.2, 16.6;19F NMR : δ (ppm) −139.35 , −142.82 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C23H20F2N4O1S1: 439.13985; found: 439.1395.R)-2-pyrimidine-4-yl)amino]propanol (17b)-2-pyrimidine-4-yl)amino]propanol 16a  K2CO3 , 2-furanyl boronic acid  and Pd(PPh3)4  were added. Following three cycles of backfilling with nitrogen, 1,4-dioxane (3.0 mL) and water (1.0 mL) were added. After heating to 120 °C for 48 h, the reaction mixture was transferred to a separatory funnel, and water (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated aqueous NaHCO3, water and brine, and dried over Na2SO4. After coating on Celite, flash chromatography using 70–100% Et2O/PE to 1–2 MeOH/Et2O afforded the title compound  as a light brown solid. mp 163–165 °C.To a flame dried, N1H NMR : δ (ppm) 8.91 , 8.54 , 8.50 , 7.86 , 7.47–7.38 , 7.34–7. 23 , 7.17–7.09 , 7.06 , 6.68 , 6.68 , 4.84 , 4.49 , 4.45–4.36 , 3.63–3.53 , 3.53–3.44 , 1.21 ; 13C NMR : δ (ppm) 167.1, 157.7, 153.3, 150.2, 149.6 , 148.2 , 143.8, 143.5, 142.7, 128.3 , 126.3 , 124.6 , 117.9, 116.1 , 112.5, 109.6, 108.4, 63.7, 48.7, 27.2, 16.5; 19F NMR : δ (ppm) −139.32 , −142.80 ;m/z [M+H]+ calcd. for C21H18F2N4O2S1: 429.1191; found: 429.1195.HRMS (ESI-Q-TOF): R)-2-pyrimidine-4-yl)amino]propanol (17c)-2-pyrimidine-4-yl)amino]propanol 16a  K2CO3 , 2-furanyl boronic acid  and Pd(PPh3)4  were added. Following three cycles of backfilling with nitrogen, 1,4-dioxane (3.0 mL) and water (1.0 mL) were added. After heating to 120 °C for 10 h, the reaction mixture was transferred to a separatory funnel, and water (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated aqueous NaHCO3, water and brine, and dried over Na2SO4. After coating on Celite, flash chromatography using EtOAc/DCM afforded the title compound  as an off-white solid. mp 162–164 °C.To a flame dried, N1H NMR : δ (ppm) 8.89 , 8.50 , 8.37 , 7.48–7.38 , 7.36–7.25 , 7.19–7.09 , 6.96 , 6.30 , 4.83 , 4.50 , 4.47–4.35 , 3.61–3.52 , 3.51–3.44 , 2.42 , 1.21 ; 13C NMR : δ (ppm) 166.7, 157.7, 153.0, 151.8, 150.2, 149.6 , 148.2 , 143.2, 142.9, 128.3 , 126.3 , 124.6 , 118.0, 116.1 , 109.5, 108.7 , 63.6, 48.7, 27.2, 16.5, 13.7;19F NMR : δ (ppm) −139.36 , −142.83 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C22H20F2N4O2S1: 443.13476; found: 443.1345.R)-2-pyrimidine-4-yl)amino]propanol (17d)3 , XPhos  (2R)-2-pyrimidine-4-yl)amino]propanol 16a , 2-thienyl boronic acid , and K3PO4  were added. Following three cycles of backfilling with nitrogen, degassed nBuOH (2.0 mL) was added. After heating to 120 °C for 20 h, the mixture was concentrated in vacuo, and transferred to a separatory funnel along with saturated NH4Cl (8 mL), H2O (2 mL) and EtOAc (20 mL). Following phase separation, the mixture was extracted with EtOAc (1 × 20 mL). The combined organic phases were washed with water (10 mL) and brine (10 mL), and dried over Na2SO4. After coating on Celite. Flash chromatography using 5–40% EtOAc/isohexane afforded the title compound  as an off-white solid. mp 176–178 °C.To a flame dried, N1H NMR : δ (ppm) 8.89 , 8.65 , 8.35 , 7.77 , 7.63 , 7.47–7.40 , 7.36–7.26 , 7.22 , 7.18–7.09 , 4.89 , 4.50 , 4.46–4.35 , 3.62.–3.45 , 1.24 ; 13C NMR : δ (ppm) 166.9, 157.7, 149.8, 149.6 , 148.2 , 146.0, 144.3, 143.4, 128.4, 128.2 , 127.9, 126.3 , 124.6 , 124.4, 118.1, 116.2 , 109.9, 63.6, 48.7, 27.3, 16.5; 19F NMR : δ (ppm) −139.32 , −142.80 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C21H18F2N4O1S2: 445.09627; found: 445.0955.d]pyrimidine (18)6-Chloro-2--4-(4-methoxybenzyl)-pyridopyrimidin-4-one 15a , K2CO3  and dry DMF (10 mL) were added, resulting in the formation of a clear orange solution. 4-Methoxybenzyl chloride  was added dropwise while stirring at room temperature. The mixture was stirred at room temperature for 1 h. After a few minutes, a white precipitate started to form. After stirring at 60 °C for 22 h, the mixture was cooled to room temperature; 20 mL ice water was added. The resulting precipitate was filtered, and washed with water, methanol and diethyl ether. After drying in vacuo, the obtained solid was purified via flash chromatography using 40% EtOAc/PE to afford the title compound as a white solid . mp 137–139 °C.To a flame dried N1H NMR : δ (ppm) 8.89 , 7.97 , 7.51–7.42 , 7.40–7.29 , 7.21 , 7.19–7.10 , 6.89 , 5.21 , 4.61 , 3.71 ; 13C NMR : δ (ppm) 159.2, 158.9, 158.7, 149.6, 145.2, 140.8, 128.5, 127.0, 126.7, 126.6, 126.2 , 124.7, 119.0, 116.8 , 113.9, 55.1, 48.9, 29.1; 19F NMR : δ (ppm) −139.15 , −141.77 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C22H16Cl1F2N3O2S1: 460.0693; found: 460.0690.N-diphenylmethylene)-pyridopyrimidine-6-amine (19)2--4-(4-methoxybenzyl)-2 , (±)-BINAP , 6-chloro-2--4-(4-methoxybenzyl)-pyridopyrimidine 18  and Cs2CO3  were added. Following three cycles of backfilling with N2, dry 1,4-dioxane (6.0 mL) and benzophenone imine  were added. The mixture was heated to 120 °C for 24 h, then cooled to room temperature, and transferred to a separatory funnel. Water (20 mL) was added, and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with water and brine, dried over Na2SO4 and coated on Celite. Flash chromatography using 0–40% EtOAc/isohexane afforded the title compound  as an orange solid. mp 188–191 °C.To a flame dried, N1H NMR : δ (ppm) 8.73 , 7.72 , 7.63–7.55 , 7.55–7.47 , 7.46–7.40 , 7.39–7.27 , 7.25–7.09 , 6.90–6.83 , 5.14 , 4.56 , 3.71 ; 1H NMR : δ (ppm) 8.80 , 7.85 , 7.57–7.41 , 7.39–7.17 , 7.15–6.99 , 6.89–6.81 , 5.22 , 4.56 , 3.79 ; 13C NMR : δ (ppm) 171.3, 160.9, 160.5, 159.4, 155.8, 150.6 , 149.3 , 148.6, 138.8, 138.2, 136.0, 132.5, 131.5, 130.1, 129.9, 129.6, 129.4, 129.1, 128.3, 128.2, 127.0, 126.15 , 126.05 , 124.08 , 116.8 , 114.0, 109.6, 55.3, 47.3, 29.7; 19F NMR : δ (ppm) −137.73 , −141.08 ; HRMS (ESI-Q-TOF): m/z [M+H-Benzophenone]+ calcd. for C22H16Cl1F2N3O2S1: 441.1191; found: 441.1142.d]pyrimidine-4-one (20)4-Amino-2--pyridopyrimidine-6-amine 19  and TFA (6.0 mL) were combined and heated to 75 °C for 1 h. Next, the reaction mixture was concentrated in vacuo. Ethyl acetate and saturated aqueous NaHCO3 were added; the mixture was stirred and filtered. The obtained solid was washed with water and methanol, and dried under vacuum to afford impure 6-amino-2--pyridopyrimidine-4-one , which was used without further purification. mp > 300 °C.In a screw-capped reaction tube equipped with a stir bar, 2--4-(4-methoxybenzyl)-(R)-2-pyrimidine-4-yl)amino]propanol (21), BOP  and dry DMF (2.0 mL) were combined. DBU  was added dropwise, and the reaction mixture was stirred for 10 min at room temperature. Next, (R)-alaninol  was added dropwise. The reaction mixture was stirred for 23 h at room temperature and for 2 h at 60 °C. After concentrating in vacuo, EtOAc (40 mL) and saturated NH4Cl (10 mL) were added and the mixture was transferred to a separatory funnel. After phase separation, the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated NH4Cl (3 × 10 mL), water (2 × 10 mL) and brine (2 × 10 mL), dried over Na2SO4 and coated on Celite. Flash chromatography using 0–10% MeOH/DCM afforded the title compound  as an off-white solid. mp 174–176 °C.The crude 6-amino-2--pyrido+ calcd. for C17H17F2N5O1S1: 378.11945; found: 378.1200.d]pyrimidine (22)2,4-Dichloro-pyridopyrimidine-2,4-dione  was obtained as a white solid.A mixture of 3-aminoisonicotinic acid  and urea  in a screw-capped sealed 80 mL reaction tube was heated to 210 °C for 1 h. After cooling to room temperature, a solution of NaOH  in 60 mL water was added and the mixture was heated to 100 °C for 1 h. Next, the reaction mixture was cooled down to room temperature, the solid was filtered and washed with water. The obtained crude product was suspended in AcOH (60 mL) and heated to 100 °C for 1 h. After cooling to room temperature, the reaction mixture was filtered and the obtained solid was washed with a large amount of water, and a small amount of methanol and diethyl ether. After drying in vacuo (40 °C) pyridopyrimidine-2,4-dione was first finely ground with a pestle and mortar. To a flame dried two-necked 500 mL round bottom flask, equipped with an Ar-balloon, septum and stir bar, pyridopyrimidine-2,4-dione , dry toluene (100 mL) and POCl3  were added. While cooling to 0 °C in an ice bath, DIPEA  was added dropwise. After stirring at 0 °C for 30 min, the reaction mixture was stirred at room temperature for 24 h and concentrated in vacuo. To the obtained oil, diethyl ether and ice water were added. The mixture was neutralized with saturated aqueous NaHCO3 (pH = 7) while cooling in an ice bath. Due to extensive tar formation, phase separation of the aqueous and organic layers did not occur. This was solved by pouring the mixture into a large beaker (2L) and adding a lot of paper towel to adsorb the tar. This mixture was extracted 5 times with 300 mL diethyl ether, by manually stirring the contents of the beaker with a large spatula and decanting off the organic phase. The combined organic phases were washed with water (2 × 50 mL) and brine (2 × 50 mL), decolorized with activated charcoal and dried over Na2SO4. After concentrating to dryness in vacuo, while keeping the heating bath at room temperature, and drying in vacuo (30 °C), the title compound  was obtained as a beige-brown solid which was used without further purification.Pyridopyrimidine with aminesGeneral procedure A: Nucleophilic aromatic substitution of 2,4-dichloro-pyridopyrimidine 22  and dry ACN (4.0 mL) were added. While cooling to 0 °C in an ice bath, Et3N  and an amine nucleophile  were added dropwise. After stirring for 10 min at 0 °C, the reaction mixture was stirred at room temperature for the specified time. During and after the addition of Et3N and nucleophile, generally, the formation of a yellow precipitate occurred. Next, the reaction mixture was transferred to a 100 mL round bottom flask with methanol; the entire sample was dissolved under gentle heating, and coated on Celite. Flash chromatography using 0–20% MeOH/DCM afforded the title compounds as white or beige solids.To a flame dried, Nd]pyrimidine with alcoholsGeneral procedure B: Nucleophilic aromatic substitution of 2,4-dichloro-pyridopyrimidine 22  and dry ACN (3.0 mL) were added. While cooling to 0 °C in an ice bath, the solution of alcohol sodium salt was added dropwise to the 2,4-dichloro-pyridopyrimidine solution. After stirring for 10 min at 0 °C, the reaction mixture was stirred at room temperature for the specified time. Next, the reaction mixture was transferred to a 100 mL round bottom flask with methanol; the entire sample was dissolved under gentle heating, and coated on Celite. Flash chromatography using 0–100% EtOAc/DCM, followed by trituration with pentane and drying under vacuum (40 °C), afforded the title compounds as white or beige solids.NaH (60% dispersion) was added to a solution of alcohol nucleophile (1.1 eq.) in dry acetonitrile (2.0 mL), in a flame dried Nd]pyrimidines with thiolsGeneral procedure C: Nucleophilic aromatic substitution of 2-chloropyridopyrimidine , thiol nucleophile  and dry 1,4-dioxane (1 mL) were combined. NaH 60% dispersion in mineral oil  was added in one portion. After stirring at room temperature overnight, methanol (1.0 mL) was added and the mixture was heated to 60 °C for 30 min. After cooling to room temperature, the reaction mixture was transferred to a 100 mL round bottom flask with methanol; the entire sample was dissolved under gentle heating, and coated on Celite. Flash chromatography using 0–20 MeOH/DCM, followed by trituration with pentane and drying under vacuum (40 °C), afforded the title compounds as white solids.To a flame dried, NThe following compounds were made according to these procedures:R)-2-[amino]butanol (23a)(2R)-2-Aminobutan-1-ol  was reacted for 3h at room temperature. Flash chromatography was performed using 0–20% MeOH/DCM to afford the title compound  as an off-white solid. mp> 300 °C.This compound was prepared following general procedure A. : δ (ppm) 8.98 , 8.72–8.55 , 8.29 , 4.82 , 4.35–4.19 , 3.65–3.50 , 1.83–1.68 , 1.67–1.51 , 0.90 ; 13C NMR : δ (ppm) 160.8, 158.3, 150.5, 145.1, 144.2, 118.0, 115.9, 62.1, 54.9, 23.2, 10.6; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C11H13Cl1N4O1: 253.0851; found: 253.0848.d]pyrimidine-4-yl)amino]-2-methyl-propan-1-ol (23b)2-+ calcd. for C11H13Cl1N4O1: 253.08505; found: 253.0854.d]pyrimidine-4-yl)amino]propan-1,2-diol (23c)3-+ calcd. for C10H11Cl1N4O2: 255.06432; found: 255.0643.d]pyrimidine-4-yl)amino]ethanol (23d)2-+ calcd. for C9H9Cl1N4O1: 225.05376; found: 225.0539.d]pyrimidine-4-yl)oxy]ethanol (23e)3-pyrimidine-4-yl)amino]ethanol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as a beige-brown solid. mp 181–183 °C.This compound was prepared following general procedure B. 3-+ calcd. for C16H14F2N4O1S1: 349.09290; found: 349.0922.R)-2-pyrimidine-4-yl)amino]butanol (24a)(2R)-2-[amino]butanol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as a white solid. mp 174–176 °C.This compound was prepared following general procedure C. : δ (ppm) 8.93 , 8.50 , 8.25 , 8.19 , 7.51–7.37 , 7.36–7.23 , 7.20–7.07 , 7.77 , 4.49 , 4.37–4.17 , 3.59–3.43 , 1.79–1.65 , 1.64–1.49 , 0.86 ; 13C NMR : δ (ppm) 167.4, 158.3, 150.1, 149.6 , 148.1 , 144.4, 142.8, 128.3 , 126.3 , 124.6 , 117.3, 116.1 , 115.8, 62.2, 54.4, 27.2, 23.3, 10.6; 19F NMR : δ (ppm) −139.39 , −142.85 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C18H18F2N4O1S1: 377.12420; found: 377.1239.d]pyrimidine-4-yl)amino]-2-methyl-propan-1-ol (24b)2-pyrimidine-4-yl)amino]-2-methyl-propan-1-ol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as a white solid. mp 166–168 °C.This compound was prepared following general procedure C. 2-+ calcd. for C18H18F2N4O1S1: 377.12420; found: 377.1238.d]pyrimidine-4-yl)amino]propan-1,2-diol (24c)3-pyrimidine-4-yl)amino]propan-1,2-diol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as an off-white solid. mp = 156–158 °C.This compound was prepared following general procedure C. 3-+ calcd. for C17H16F2N4O2S1: 379.10347; found: 379.1031.d]pyrimidine-4-yl)amino]ethanol (24d)3-pyrimidine-4-yl)amino]ethanol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as a white solid. mp 181–183 °C.This compound was prepared following general procedure C. 3-+ calcd. for C16H14F2N4O1S1: 349.09290; found: 349.0922.d]pyrimidine-4-yl)oxy]ethanol (24e)3-pyrimidine-4-yl)amino]ethanol  and 2,3-difluorobenzylthiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–10% MeOH/DCM to afford the title compound  as a beige-brown solid. mp 181–183 °C.This compound was prepared following general procedure B. 3-+ calcd. for C16H14F2N4O1S1: 349.09290; found: 349.0922.R)-2-[amino]propanol (25) and (R)-alaninol  was reacted for 3h at room temperature. Flash chromatography was performed using 0–20% MeOH/DCM, followed by trituration with pentane afforded the title compound  as an off-white solid. mp > 300 °C.This compound was prepared following general procedure A. 2,4-Dichloropyrido+ calcd. for C10H11Cl1N4O1: 261.0514 found: 261.0512.R)-2-pyrimidine-4-yl)amino]propanol (26a)(22-flushed 8 mL screw-capped reaction tube equipped with a stirring bar (2R)-2-[amino]propanol 25 , 2,3-difluorobenzylamine  and dry 1,4-dioxane (5 mL) were added. The mixture was stirred at 100 °C for 22 h. After cooling to room temperature, the reaction mixture was dissolved in methanol and coated on Celite. Flash chromatography using 0–20% MeOH/DCM, followed by trituration with pentane, afforded the title compound  as a white-yellow solid. mp 136–140 °C.In a flame dried, N1H NMR : δ (ppm) 8.59 , 8.15 , 7.93 , 7.75 , 7.39 ,7.31–7.17 , 7.16–7.08 , 4.74 , 4.62 , 4.35 , 3.60–3.39 , 1.27–1.02 ; 13C NMR : δ (ppm) 160.1, 15.9, 149.5 , 148.9, 147.7 , 146.8, 138.9, 130.3, 124.4 , 115.6, 115.4 , 63.8, 48.1, 37.6, 16.8; 19F NMR : δ (ppm) −140.15 , −145.26 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C17H17F2N5O1: 346.1474; found: 346.145.R)-2-pyrimidine-4-yl)amino]propanol (26b)(22-flushed 8 mL screw-capped reaction tube equipped with a stir bar (2R)-2-[amino]propanol 25 , 2,3-difluorobenzylalcohol , dry 1,4-dioxane (4 mL) and K2CO3  were added. The mixture was stirred at 120 °C for 48 h. After cooling to room temperature, the reaction mixture was dissolved in methanol and coated on Celite. Flash chromatography using 0–10% MeOH/DCM, followed by trituration with pentane, afforded the title compound  as a light-brown solid. mp 167–169 °C.In a flame dried, N1H NMR : δ (ppm) 8.88 , 8.43 , 8.38 , 8.15 , 7.70–7.33 , 7.32–7.12 , 5.48 , 4.82 , 4.47–4.26 , 3.62–3.50 , 3.50–3.40 , 1.21 ; 13C NMR : δ (ppm) 162.7, 160.9, 149.9, 149.6 , 148.2 , 145.6, 141.7, 126.7 , 125.9 , 124.9 , 117.3 , 116.8, 115.8, 63.7, 61.4, 48.7, 16.5; 19F NMR : δ (ppm) −139.34 , −143.8 ; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C17H16F2N4O2: 347.1314; found: 347.1319.R)-2-pyrimidine-4-yl)amino]propanol (26c)(2R)-2-[amino]propanol  and thiophenol  were used and reacted at 60 °C for 1 h 30 min. Flash chromatography was performed using 0–20% MeOH/DCM to afford the title compound  as an off-white solid. mp 193–196 °C.This compound was prepared following general procedure C. : δ (ppm) 8.79 , 8.47 , 8.28 , 8.13 , 7.70–7.60 , 7.51–7.40 , 4.73 , 4.12–3.96 , 3.45–3.37 , 1.08 ; 13C NMR : δ (ppm) 168.4, 157.8, 150.1, 144.7, 142.7, 135.0, 129.4, 128.94, 128.85, 117.3, 115.7, 63.3, 48.8, 16.3; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C16H16N4O1S1: 347.1314; found: 347.1319.R)-2-pyrimidine-4-yl)amino]propanol (26d)(2R)-2-[amino]propanol  and 2-phenylethanethiol  were used and reacted at 60 °C for 1 h 30 min. Flash chromatography was performed using 0–20% MeOH/DCM, followed by trituration with pentane, to afford the title compound  as a white solid. mp 182–184 °C.This compound was prepared following general procedure C. : δ (ppm) 8.91 , 8.48 , 8.26 , 7.44–7.26 , 7.26–7.17 , 4.83 , 4.50–4.35 , 3.62–3.44 , 3.42–3.35 , 3.01 , 0.122 ; 13C NMR : δ (ppm) 168.5, 157.6, 150.1, 144.6, 142.6, 140.5, 128.6, 128.4, 126.2, 117.1, 115.7, 63.7, 48.6, 35.6, 31.5, 16.6; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C18H20N4O1S1: 341.1431; found: 341.1427.R)-2-pyrimidine-4-yl)amino]propanol (26e)(2R)-2-[amino]propanol  and hexane thiol  were used and reacted at 60 °C for 30 min. Flash chromatography was performed using 0–20% MeOH/DCM to afford the title compound  as an off-white solid. mp 147–149 °C.This compound was prepared following general procedure C. : δ (ppm) 8.85 , 8.46 , 8.23 , 8.13 , 4.81 , 4.39 , 6.62–3.42 , 3.20–3.00 , 1.67 , 1.48–1.35 , 1.34–1.24 , 1.23–1.17 , 0.85 ; 13C NMR : δ (ppm) 168.7, 157.5, 150.0, 144.6, 142.4, 117.0, 115.7, 63.6, 48.6, 30.8, 30.0, 29.4, 28.0, 22.0, 16.5, 13.8; HRMS (ESI-Q-TOF): m/z [M+H]+ calcd. for C16H24N4O1S1: 321.1744; found: 321.1747.4 cells per well in gelatin-coated black-walled 96-well plates. This coating was done via a two hour incubation of 0.1% gelatin in Dulbecco’s phosphate-buffered saline (DPBS) on each well after which they were washed with DPBS. After cell-seeding, the cells were incubated overnight at 37 °C and 5% CO2. Then, growth medium was discarded, cells were loaded with 80 µL/well Calcium 6-dye mix  and incubated for two hours at 37 °C and 5% CO2. In the meantime, compounds were serially diluted in assay buffer , 20 mM HEPES buffer (Thermo Fischer Scientific), 0.5% FCS) in a 96-well plate. Moreover, a 96-well plate containing CXCL8 was prepared. After two hours of incubation, the cells were placed into the FLIPR Tetra system along with the compound plate and the plate containing CXCL8. Compounds were automatically added to the cell plate, followed by a ten minute incubation. Subsequently, CXCL8 was added at a final concentration of 15 nM. Changes in cytosolic calcium concentrations were measured in all 96 wells simultaneously in real time. For each compound, IC50 values for inhibition of the CXCL8-induced calcium response were determined based on negative and positive controls .Cells stably expressing CXCR2 (U87.CXCR2) were cultivated in Dulbecco’s modified eagle medium  supplemented with 10% Fetal Calf Serum  and 10 µg/mL blasticidin . Intracellular calcium fluxes induced in U87.CXCR2 cells upon human CXCL8 ligand stimulation were measured at 37 °C using the FLIPR Tetra system . Therefore, U87.CXCR2 cells were seeded at 2 × 10d]pyrimidine skeleton. Since this scaffold has not been associated with CXCR2 antagonism before, we embarked on a hit-to-lead optimization campaign in order to optimize the potency of compound 2. Using different synthetic approaches, various substituents were introduced at positions 2, 4, 5 and 6 of the pyridopyrimidine scaffold  that is endowed with a five-fold reduced potency when compared to the original hit 2. Therefore, we must conclude that pyridopyrimidine 2 is a ‘singleton’ hit compound.We recently reported the discovery of a CXCR2 antagonist, based on a pyrido[3,4-scaffold . Unfortu