Among recent advances in the identification of anti-inflammation agents, anti-cytokines (like Interleukin-1), related to p38 MAPK families play an important role; Here in we designed new effective and low toxic anti-cytokine agents based on 1-hydroxy-2,4,5-triaryl imidazole derivatives. The reaction of oximoinoketone intermediate with ten different aromatic aldehyde and ammonium acetate in refluxing acetic acid condition give imidazole derived product, the IL-1β inhibitory assay were performed on Human PBMCs  using an enzyme-linked immunosorbent assay (ELISA) kit and then in computational part the binding mode of the best compound was accomplished by docking in Crystal structure of p38 MAP kinase (PDB ID: 1A9U) compared with SB202190 as standard drug. All compounds were synthesized and evaluated in biological assay showing the inhibitory activity from 28% to 82% compared to SB202190 and binding mode analysis revealed that the hydrogen-bond interactions with residues  were key point in inhibitor binding. Compound  P38MAPK consists of four different isoforms α, β, γ, and δ. p38α is believed to be the predominant isoform involved in the inflammatory response , 2. The 50 . The imi50 , 15.5 to enhance the accessibility and orient to the sub pocket of the active site. In addition, the role of incorporation of different aldehydes (aryl group (Ar)) at the 2-position of the imidazole were employed to investigate the importance of flexibility -5-phenylimidazole substructure with p38 depicted an H-bond between the nitrogen of pyridinyl and the amide NH of Met109 in the adenine binding pocket of ATP. This H bond is necessary for inhibition and also reported that is an important factor contributing to P450 inhibition -18. Unfoxibility .ChemistryReagents1HNMR spectra were measured on Brucker DPX-250 MHZ in δ scale. The spectra were obtained with solutions of CDCl3 or DMSO-d6 with TMS as internal standard, and the values of the chemical shifts (δ) were given in ppm, coupling constants (J) given in Hz. The mass spectra were run on Hewlett Packard (HP) 6890. All commercially available reagents and solvents were used without further purification from commercial sources. Reactions were routinely monitored by thin-layer chromatography (TLC) in silica gel . IR spectra were obtained on a FT-IR Perkin-Elmer Precise system spectrophotometer (potassium bromide disks), General chemistry procedures for 1-(4-fluorophenyl)-2-(hydroxyimino)-2-(quinolone-4-yl) ethanone (4)1)  and ethyl 4- fluorobenzoate  stir at 0 °C in dry THF. In this condition 100 mL of sodium bis (tri methylsiyl) amide (NaHMDS) 2 M were added drop wise in 20 min and continued stirring about 2 h, after that reaction stirred with saturated NaNO2 in room temperature for 24 h. The mixture was washed with 30 mL water. The organic layer was dried over anhydrous MgSO4 .The solvent was then removed and the yellow pale residue was the desired compound. The A mixture of 4- methylquinoline (lepidine) (1H NMR (CDCl3): δ ppm 13.05 , 9.0092 , 8.19 – 8.16 , 8.12 , 7.82–7.79 , 7.64–7.59 , 7.52 , 7.45–7.41 ; MS: m/z (%) 294.1 (M+41), 154.1 (9.1), 123.1 (100), 95.1 (38.18), 75.1 (11), 140.1 (1.81).Yield: 73%. Mp: 214-216 °C. General Chemistry procedures for 5-(4-fluorophenyl)-2-(aryl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5a-j)4OH and the precipitate was filtered off, washed with water and was purified by column chromatography on silica diluted with EtOAc/petroleum ether (70:30) then recrystallized from suitable solvent to give the desirable pure products 5a-j. The data of the pure products has been shown in The appropriate aldehyde (1 mmol) were added to 4  and ammonium acetate (3.14 g) refluxed in 20 mL glacial acetic acid for 72 h, after cooling dish in ice water, neutralized with saturated NH5-(4-fluorophenyl)-2-(4-methoxyphenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5a)-1 3419.25 (OH), 2926.31 (C-H aromatic), 1609.60 (C=N imidazole), 1500.05 (C=C aromatic), 1HNMR (CDCl3): δ ppm 11.80 , 8.78-8.77 , 8.12-8.10 , 7.94-7.92 , 7.68-7.65 , 7.42-7.39 , 7.32-7.31 , 7.14-7.11 , 6.96-6.95 , 6.88-6.86 , 3.86 , MS: m/z (%) 411.4 (M+ 26.31), 395.4 (100), 380.3 (13.04), 350.3 (8.7), 261.1 (27.17), 154.1 (7.6), 135.1 (26.1), 119.1 (14.13).The titled compound was synthesized by the described procedure from 4-methoxy benzaldehyde  as colorless yellow solid. Yield: 64%. Mp: 240-243 °C. IR (KBr): νcm2-(4-chlorophenyl)-5-(4-fluorophenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5b)5b was prepared in similar described manner using 4- chlorobenzaldehyde , Yield: 71%. Mp: 221-223 °C. 1H NMR: δ ppm 11.78 , 8.38 , 8.06 , 7.80 , 7.55 , 7.34 , 7.27 , 7.14-7.04 , 6.72 .The orange solid product 2-(3-fluorophenyl)-5-(4-fluorophenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5c)5c was prepared by the described procedure using 124 mg (1 mmol) of 3-fluorobenzaldehyde as pale yellow solid, Yield: 58%. Mp: 238-240 °C. IR(KBr): νcm-1 3420.28 (OH), 2476.17 (C-H aromatic), 1612.35 (C=N imidazole), 1587.33 (C=N imidazole), 1517.97 (C=C aromatic), 1H NMR(DMSO): δ ppm 12.01 , 9.00 , 8.15 , 8.03 , 7.94 , 7.81 , 7.68 , 7.53 , 7.36 , 7.31 , 7.04 , MS: m/z (%) 399.3 (M+ 86.66), 382.3 (100), 261.1 (55.55), 234.2 (12.62), 107.1 (38.33).Compound 2--5-(4-fluorophenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5d)-1 3394.15 (OH), 2926.70 (C-H aromatic), 1680.84 (C=N imidazole), 1509.66 (C=C aromatic), 1H NMR (CDCl3): δppm 12.01 , 8.50 , 8.04- 8.02 , 7.84-7.73 , 7.67-7.62 , 7.35 , 7.19-7.10 , 6.91-6.68 , 3.99 ; MS: m/z (%) 441.4 (M+ 30), 425.4 (100), 408.3 (13.33), 261.1 (17.77), 165.2 (17.77).Compound 5d was obtained according to the described general procedure using 3,4 dimethoxybenzaldehyde  as orange oily, Yield: 49%. IR(KBr): νcm5-(4-fluorophenyl)-2-phenyl-4-(quinoline-4-yl)-1H imidazole-1-ol (5e)-1 3063.56 (OH), 2568.39 (C-H aromatic), 1701.77 (C=N quinoline), 1586.68 (C=N imidazole), 1497.79 (C=C aromatic), 1HNMR (CDCl3): δ ppm 12.00 , 8.31-8.29 , 8.11-8.09 , 7.90-7.84 , 7.78-7.75 , 7.48-7.43 , 7.39-7.34 , 7.22-7.19 , 6.92-5.87 , 6.66-6.61 ; MS: m/z (%) 381.4 ([M+] 86.66), 364.3 (100), 261.1 (46.66), 234.2 (11), 140.2 (11), 107.1 (12.22), 89.2 (16.60).The titled compound 5e was synthesized according to the general procedure using benzaldehyde  as pale yellow solid, Yield: 73%. Mp: 212-214 °C. IR(KBr): νcm5-(4-fluorophenyl)-4-(quinoline-4-yl)-2-(thiophen-2-yl)-1H imidazole-1-ol (5f)1H NMR(CDCl3): δ ppm 11.83 , 8.98 , 8.38-8.34 , 8.08-8.02 , 7.80 , 7.54-7.48 , 7.43 , 7.25-7.17 , 7.08 , 7.01-6.94 , 6.61-6.68 .The titled compound 5f was synthesized according to the general procedure using thiophen-2-carbaldehyde  as brown oily, Yield: 48%. 5-(4-fluorophenyl)-2-(4-hydroxy-3-methoxyphenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5g)1H NMR (CDCl3): δ ppm 11.69 , 9.22 , 8.58 , 7.99-8.03 , 7.81 , 7.54-7.51 , 7.46-7.41 , 7.29 , 7.19-7.15 , 6.84 , 6.77 , 3.94 .The yellow solid product 5g was prepared from 4-hydroxy-3-methoxybenzaldehyde  according to the general procedure, Yield: 61%. Mp: 198-200 °C. 2-(4-bromophenyl)-5-(4-fluorophenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5h)-1 3062.99 (OH), 2563.14 (C-H aromatic), 1587.84 (C=N imidazole), 1511.49 (C=C aromatic), 1H NMR (CDCl3): δ ppm 11.98 , 8.20 , 8.06-8.04 , 7.75 , 7.53-7.51 , 7.44-7.43 , 7.427-7.40 , 7.30-7.28 , 7.22-7.21 , 6.83 , 6.63 .The titled compound 5h was synthesized according to the general procedure from 4-bromobenzaldehyde  as orange solid, Yield: 65%. Mp: 208-210 °C. IR(KBr): νcm2--5-(4-flourophenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5i)1H NMR (CDCl3): δ ppm 11.98 , 8.95 , 8.22-8.19 , 8.04-7.98 , 7.74-7.68 , 7.49-7.44 , 7.37 , 7.18-7.14 , 6.95-6.93 , 6.88-6.86 .The titled compound 5i was synthesized according to the general procedure using 2,4-dichlorobenzaldehyde  as orange solid, Yield: 56%. Mp: 211-213 °C. 5-(4-fluorophenyl)-2-(2-hydroxy-3-methoxyphenyl)-4-(quinoline-4-yl)-1H imidazole-1-ol (5j)1H NMR (CDCl3): δ ppm 12.00 , 8.84 , 8.514-8.509 , 8.11-8.14 , 7.98-8.05 , 7.87-7.94 , 7.66-7.73 , 7.57-7.55 , 7.33-7.39 , 7.17-7.23 , 6-79-6.89 , 3.79.Compound 5j was obtained according to the described general procedure using 2-hydroxy-3-methoxybenzaldehyde  as orange solid, Yield: 52%. Mp: 235-237 °C. Determination of IL-1β inhibitoryReagents and methodRPMI 1640 media, FBS , trypsin, penicillin, and streptomycin were purchased from Biosera (Austria). SB202190 and Ficoll-hypaque were purchased from Sigma (USA). ELISA kit was from Enzo® Life Science International Inc.2. Following incubation, the culture medium was harvested (100 µL) and stored at -70 °C. IL-1b concentration in the medium, quantified using an enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions (assay designs). The absorbance in each well was measured with a micro plate reader at 450 nm and corrected at 570 nm. The concentrations of cytokines released were obtained and the percentage of inhibition of cytokines production was calculated as compared to the control conditions and were displayed in Inhibition of IL-1β production in a cell-based assay: Human peripheral blood mononuclear cells (PBMC) in RPMI 1640 medium containing 2.5 mM EDTA, and Ficoll-Hypaque (sigma) were overlaid with the mixture and centrifuged at 1500 rpm for 30 min, after collecting the buffy coat and cell washing, the cells was plated in 96-well plates at a concentration of 1 × 106 cells/mL in assay medium  and incubated with test compounds for 30 min and then stimulated by the addition of LPS (1 µg/mL) for a total of 6 h at 37 °C, 5% COMolecular docking+ force field. Molecular docking 4 studies of the compounds 5g and SB202190 were performed using PYRX . Crystal structure of MAP kinase was downloaded from the Protein Data Bank (PDB ID: 1A9U resolution: 2.50Å). Water molecules and cognate ligand were removed from the receptor. The active sites were defined based on the center (box: x: 2.25 y: 15.49 z: 28.18). Docking computations were performed using the Lamarkian genetic algorithm, the docking procedure was conducted according to previous work and the lowest free binding energy with the most favorable docked poses was selected for analyzing enzyme-inhibitor interactions. The chemical structures of the desired ligands were created using HyperChem software version 8.0.10 and energetically minimized using MMChemistry2 aqua solution, oximinoketone (4) formed, while the second coupled were cyclization reaction of 4 with different aromatic aldehyde and ammonium acetate in refluxing acetic acid to give imidazole derived product 5a-j.; The spectral data of synthesized compounds were in agreement with the assigned structure  (1) to a carbonyl core (2) by sodium bis (tri methylsiyl) amide (NaHMDS) gives compound (3) and then in presence of NaNOtructure , 25. Biologypara-hydroxy-meta- methoxyphenyl pendant demonstrated the most inhibition percentage of cytokines production equal to 82.17% at concentrations of 10 µM. Furthermore the presence of different aryl substituted with electron-donating or electron-withdrawing group on the phenyl ring may result in improved cytokines production inhibitory activity.All Target compounds were evaluated for their ability to inhibit IL-1β release in lipopolysaccharide (LPS) stimulated in PBMC , LPS is major constituent of the outer membrane of gram-negative bacteria that induces a systemic inflammation response, and it is a well-studied immunostimulator, particularly, the expression of proinflammatory cytokines such as IL-1β representative results with their percentage for inhibition of IL-1β existing in para- methoxyphenyl counterpart (81.61% inhibition) and 5b para- chlorophenyl derivative (78.64% inhibition) also exhibited significant activity at the concentrations of 10 µM. The obtained results, revealed that the unsubstituted phenyl derivative 5e and thiophene 5f counterparts showed lower activity (32.2 and 28.3% inhibition) in comparison with the substituted moiety on phenyl ring.In this regard, compounds such as 5a bearing Molecular docking studyTo rationalize our design and our biological activity assay, docking study was carried out for the most bioactive compounds 5g and SB202190 to elucidate the molecular reasons behind the observed inhibition profile, including which amino acids in the p38α MAP kinase active site would be involved in the anticipated hydrogen bonding interactions. According to the results obtained with the rigid docking approach, SB202190  has polaIn-silico metabolism predictionin-silico prediction of CYP450 metabolism are set in Nowadays, numerous approaches have been developed to estimate drug-likeness of bioactive compounds based on topological descriptors of molecular structure or other properties such as molecular weight, water solubility, and cLogP. In this study, the open-source program admetSAR was used to evaluate chemical metabolism properties of the most potent compounds to compare them with SB202190. The admet SAR provides a database including FDA approved and experimental investigated drugs, pesticides, environmental agents, industrial chemicals that can predict absorption, distribution, metabolism, excretion and toxicity profiles , here inHere, we have generated a novel molecular framework, 1-hydroxy-2,4,5-triaryl imidazole derivatives by utilizing the chemical lead based approach and concept of pharmacophore of 4-(pyridin-4-yl)-5-phenylimidazole derivatives. Compounds based on this framework have found to be as anti-cytokine and p38α MAP kinase inhibitory action with a convenient approach. The results indicated that the potent analog of this scaffold, 5g bearing para-hydroxy-meta- methoxyphenyl pendant with superior inhibitory action among all compounds could serve as a suitable candidate for further modification as a lead anti cytokine structure.


N-heterocyclic carbene (NHC) ligands that display biological activity is an active research field in organometallic chemistry. One of the possible approaches consists of the use of NHC ligands functionalized with a carbohydrate moiety. Two novel Au(I)–Au(I) dinuclear complexes were synthesized; they present a neutral structure with one bridging diNHC ligand, having one or both heterocyclic rings decorated with a carbohydrate functionality. With the symmetric diNHC ligand, the dicationic dinuclear complex bearing two bridging diNHC ligands was also synthesized. The study was completed by analyzing the antiproliferative properties of these complexes, which were compared to the activity displayed by similar mononuclear Au(I) complexes and by the analogous bimetallic Au(I)–Au(I) complex not functionalized with carbohydrates.The design of novel metal complexes with  N-heterocyclic carbene ligands (NHC) . Al. AlL2∙2H1∙2HPF6L and 2∙2HPF6L were characterized by 1H- and 13C{1H}-NMR spectroscopy, as well as electrospray ionization mass spectrometry (ESI-MS), and both salts appear to be spectroscopically pure. In the case of compound 1∙2HPF6L, the acidic protons of the imidazole C2-H hydrogens give two signals in the 1H-NMR spectrum at 8.72 and 8.73 ppm, indicating the lack of symmetry of the system. Conversely, the 1H-NMR spectrum of symmetric 2∙2HPF6L presents only one peak at 8.82 ppm. Anomerization processes were reported in the literature during the quaternization of the imidazole ring or during the synthesis of carbene complexes with carbohydrate functionalized NHC ; furthermore, it was reported by Nolan and co-workers that the anion which usually coordinates to gold(I) in the complexes is that of the starting azolium salt [2Br2L] complexes  center trans to a bromide ligand [3JHH coupling constant of the anomeric proton in the glucopyranose ring [2BrL]+  and the [Au2Br2LK]+ cations .Compounds terature  in whichium salt . The neuomplexes  were thee ligand ,38. In bose ring . From th2L2]2+ having two ligands bridging the two gold centers. Considering the asymmetric nature of the proligand 1∙2HPF6L, at least two configurational isomers can be isolated: one with the carbohydrate-imidazoles facing each other and one with each carbohydrate-NHC facing a methylimidazole-2-ylidene. Furthermore, we recently reported that, in similar [Au2L2]2+ complexes with heteroditopic ligands, the number of possible products is also increased by the different conformations of the propylene linkers between the carbene units  = 1.2:1 and PhICl2:[Au] = 1.5:1)  complex was immediately formed, and no further evolution of the product was detected. The reactivity of the gold(I) complex = 1.5:1) . Many po= 1.5:1)  46]. Th. Th3 in 1H-NMR spectra which present only one set of peaks relative to the sugars and imidazole rings. This suggests that the two metal centers are equivalent and, therefore, present the same oxidation state. The 13C-NMR spectra show more definite proof that the oxidation state of the gold centers changed from gold(I) to gold(III); the carbene carbons present, in fact, a peak at 145.6 ppm for complex 4 and 154.6 ppm for 5. These values of chemical shifts are 20–30 ppm lower than the value (δ 183.2 ppm) found for complex 3. This is usually explained taking into consideration the more pronounced Lewis acidic behavior of gold(III), causing an extended delocalization of the π electron density of the imidazole C=C double bond toward the carbene carbon [13C-NMR carbene chemical shifts in 4 and 5 is ca. 10 ppm and is due to the different nature of the two halide ligands [2X4L2]2+ ion, at m/z 1287.0697 for 4 and 1103.1974 for 5, both for monoisotopic peaks.In both cases, the symmetry of the complex is maintained, as shown by the e carbon ,48. The  ligands . The def1–3 and 6 was tested on different eukaryotic cell lines. Complex 6  complexes omplex 6 , bearing4 and 5, as it is well known that similar dicationic gold(III) complexes easily undergo reduction to the corresponding gold(I) species in a physiological environment  imidazole-2-ylidene) ,,a +, 983 [H2L2PF6]+, 1479 [(H2L1)2(PF6)3]+. Elemental analysis C24H34N4O9P2F12. Calculated: C, 35.48%; H, 4.22%; N, 6.90%. Found: C, 34.96%; H, 3.73%; N 5.57%. Compound a  was dissolved in 50 mL of acetonitrile at 0 °C. A solution of 1,3-propylenebistriflate  in 7 mL of acetonitrile was prepared and added dropwise, over one hour, to the solution containing a. The resulting solution was warmed to room temperature and left stirring for 16 h. The solvent was removed and the obtained solid was dissolved in 5 mL of distilled water. A saturated aqueous NH4PF6 solution (5 mL) was added, and the final solution was left stirring for an hour until the formation of a white solid precipitate was observed. The solid was filtered, washed with distilled water, and then dried under reduced pressure (yield 48%). 1H-NMR  δ 8.82 , 7.67 , 7.50 , 5.78 , 5.52 , 5.38–5.25 , 4.28–4.22 , 4.22–4.17 , 2.47–2.36 , 2.05 , 2.05 , 2.01 . 13C{1H}-NMR  δ 171.2 (CO), 170.6 (CO), 170.5 (CO), 136.5 (NCHN-Im), 124.2 (Im), 121.8 (Im), 85.4 (1-Glu), 75.7 (3-Glu), 72.2 (Glu), 71.9 (Glu), 68.0 (Glu), 62.1 (Glu), 47.6 (NCH2), 30.4 (CH2), 20.7 (CH3-Ac), 20.7 (CH3-Ac), 20.5 (CH3-Ac). ESI-MS : m/z 983 [H2L2PF6]+, 507 [H2L2-Glu]+, 419 [H2L2]2+. Elemental analysis C37H50N4O18P2F12. Calculated: C, 39.37%; H, 4.46%; N, 4.96%. Found: C, 39.07%; H, 4.24%; N, 4.36%.Compound 2) (0.172 mmol), K2CO3 (1.89 mmol), LiBr (0.86 mmol), and 40 mL of acetonitrile were added to a round-bottom flask. The mixture was heated to 60 °C and left stirring for 18 h, then filtered on Celite to remove excess salts. The solvent was removed under reduced pressure. The solid was finally recrystallized with chloroform/n-hexane (for 1) or acetonitrile/diethyl ether (for 2), obtaining a white solid product which was filtered and dried under vacuum. The proper bis(imidazolium) salt (0.086 mmol), AuCl δ 7.43 , 7.29 , 7.20 , 7.17 , 6.12 , 5.52 , 5.33–5.19 , 4.27–4.17 , 4.17–4.02 , 3.85 , 2.50–2.35 , 2.06 , 2.04 , 1.98 , 1.93 . 13C{1H}-NMR  δ 175.07 (NCN-MeIm), 172.91 (NCN-GluIm), 171.27 (CO), 170.66 (CO), 170.58 (CO), 169.99 (CO), 123.93 (MeIm), 122.30 (GluIm), 120.73 (MeIm), 119.62 (GluIm), 86.64 (1-Glu), 75.13 (3-Glu), 72.64 (Glu), 72.24 (Glu), 68.51 (Glu), 62.34 (Glu), 48.31 (NCH2), 47.50 (NCH2), 38.75 (CH3-MeIm), 30.96 (CH2), 21.08 (CH3-Ac), 20.95 (CH3-Ac), 20.82 (CH3-Ac), 20.75 (CH3-Ac). ESI-MS : m/z 993 [Au2L1Br]+, 1113 [Au2L1Br2K]+. 2. White solid, yield 89%. 1H-NMR  δ 7.45 , 7.30 , 6.14 , 5.54 , 5.26 , 4.34–4.18 , 4.18–3.98 , 2.49–2.35 , 2.06 , 2.04 , 1.98 , 1.92 . 13C{1H}-NMR  δ 174.64 (NCN-Im), 171.24 (CO), 170.65 (CO), 170.59 (CO), 170.01 (CO), 122.34 (Im), 119.67 (Im), 86.76 (1-Glu), 75.20 (Glu), 72.65 (Glu), 72.33 (Glu), 68.55 (Glu), 62.36 (6-Glu), 48.22 (NCH2), 21.05 (CH3-Ac), 20.97 (CH3-Ac), 20.85 (CH3-Ac), 20.78 (CH3-Ac). ESI-MS : m/z 1311 [Au2L2Br]+, 1429 [Au2L2Br2K]+. 2∙2HPF6L , AuCl(SMe2) , K2CO3 , and 40 mL of acetonitrile were added to a round-bottom flask; the mixture was heated to 60 °C and left stirring for 18 h, then filtered on Celite to remove excess salts. The solvent was removed at reduced pressure. The residue was recrystallized with acetonitrile/diethyl ether, obtaining a white solid which was filtered and dried under vacuum (yield 69%). 1H-NMR  δ 7.58 , 7.42 , 6.08 , 5.63 , 5.35 , 4.47–4.31 , 4.31–4.13 , 2.54–2.38 , 2.05 , 2.01 , 2.01 , 1.92 . 13C{1H}-NMR  δ 183.17 (Im), 171.16 (CO), 170.73 (CO), 170.55 (CO), 170.09 (CO), 126.84 (Im), 123.80 (Im), 87.14 (1-Glu), 75.51 (Glu), 72.70 (Glu), 72.58 (Glu), 68.29 (Glu), 62.26 (Glu), 50.11 (NCH2), 32.53 (CH2), 21.04 (CH3-Ac), 20.83 (CH3-Ac), 20.80 (CH3-Ac). HRMS : m/z 991.2506 [Au2L22(-2CH2CO)]2+ , 1012.2554 [Au2L22(-CH2CO)]2+ , 1033.2594 [Au2L22]2+ .The salt 3 was added to an NMR tube and dissolved in CD3CN; the oxidant (I2 or PhICl2) was then added in a [I2]/[Au] = 1.2 and [Cl2]/[Au] = 1.5 ratio. NMR spectra were recorded before the addition of the oxidant, immediately after, and three hours and 24 hours later to monitor the reaction. In the case of the [Au2L22Cl4](PF6)2 complex, the product was isolated as a white solid by removal of the deuterated solvent and by treatment of the residue with diethyl ether to remove the coproduct of the oxidant (PhI). These reactivity tests were run on NMR scale and, for this reason, the characterization of the complexes involved only NMR and MS analysis.Complex 4. 1H-NMR  δ 7.77 , 7.62 , 5.90 , 5.66 , 5.40 , 5.24 , 4.28–4.00 , 2.66–2.43 , 1.96 , 1.94 , 1.89 . 13C{1H}-NMR  δ 171.06 (CO), 171.01 (CO), 170.66 (CO), 170.64 (CO), 145.63 (NCN-Im), 126.58 (Im), 123.81 (Im), 85.81 (1-Glu), 75.96 (Glu), 72.69 (Glu), 72.29 (Glu), 68.11 (Glu), 62.43 (Glu), 50.02 (NCH2), 30.45 (CH2), 22.13 (CH3-Ac), 20.96 (CH3-Ac), 20.80 (CH3-Ac), 20.75 (CH3-Ac). HRMS : m/z 1160.1664 [Au2L22I2]2+ , 1287.0697 [Au2L22I4]2+ , 1266.0660 [Au2L22I4(-CH2CO)]2+ .5. 1H-NMR  δ 7.84 , 7.68 , 6.39 , 5.80 , 5.45–5.19 , 4.61–4.42 , 4.42–4.27 , 4.27–4.05 , 2.71–2.48 , 2.05 , 2.01 , 1.93 , 1.90 . 13C{1H}-NMR  δ 171.37 (CO), 171.01 (CO), 170.66 (CO), 170.63 (CO), 154.57 (NCN-Im), 126.14 (Im), 122.99 (Im), 85.68 (1-Glu), 76.16 (Glu), 73.56 (Glu), 72.41 (Glu), 68.10 (Glu), 62.40 (6-Glu), 49.46 (NCH2), 31.56 (CH2), 21.50 (CH3-Ac), 20.80 (CH3-Ac), 20.76 (CH3-Ac). HRMS : m/z 1033.2615 [Au2L22]2+ , 1103.1970 [Au2L22Cl4]2+ .l-glutamine and antibiotics. Cells were grown in a 5% CO2 humidified atmosphere at 37 °C and seeded in 96-well plates at a density of 2 × 103 cells per well. Cells were incubated with increasing concentrations of each compound (from 10 to 200 µg·mL−1). After 4 h of incubation, cell viability was measured using the MTT -2,5-diphenyltetrazolium bromide) assay, which measures mitochondrial functionality. Briefly, the MTT reagent was dissolved in DMEM in the absence of phenol red (Sigma-Aldrich) and added to the cells . Following 4 h of incubation at 37 °C, the culture medium was removed and the resulting formazan salts were dissolved by adding isopropanol containing 0.01 mol·L−1 HCl (100 μL per well). Absorbance values were determined at 570 nm using an automatic plate reader . To assess the cytotoxicity of the compounds, both immortalized and tumorigenic cells were chosen. Immortalized human keratinocytes , immortalized murine fibroblasts , human epidermoid carcinoma cells , and BALB/c-3T3 transformed with simian virus 40 (SV40)  were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum , 2 mM 2Br2(diNHC) and, with the symmetric ligand, also the dicationic complex [Au2(diNHC)2](PF6)2 were synthesized. The antiproliferative properties of these complexes were investigated. Results suggest that the complexes appear rather inert and the introduction of a carbohydrate moiety does not significantly improve their performance. The investigation of the coordinating properties of the new ligands described in this work will be extended to other metal centers in the future.In this work, we reported two novel diNHC precursors with one or both the heterocyclic rings functionalized with a carbohydrate moiety. The corresponding neutral complexes of the type Au


Based on the hypothetical biogenesis of the 5-oxo-5H-benzo[7]annulene skeleton of the natural product purpurogallin  of KDM4A in enzymatic assays and in cells by increasing levels of its specific targets, H3K9me3/2 and H3K36me3. However, these two compounds/derivatives did not induce cell death. We then synthesized a further set of analogs of these two compounds with greater structural diversification. The most potent of these analogs, 9bf, displayed the highest KDM4A inhibitory enzymatic activity in vitro (IC50 of 10.1 and 24.37 μM) in colon cancer cells, and the strongest antitumor action in several solid and hematological human cancer cell lines with no toxic effect in normal cells. Our findings suggest that further development of this compound and its derivatives may lead to the identification of new therapeutic antitumor agents acting through inhibition of KDM4A.Aberrant epigenetic modifications are involved in cancer development. Jumonji C domain-containing histone lysine demethylases (KDMs) are found mainly up-regulated in breast, prostate, and colon cancer. Currently, growing interest is focusing on the identification and development of new inhibitors able to block the activity of KDMs and thus reduce tumor progression. KDM4A is known to play a role in several cellular physiological processes, and was recently found overexpressed in a number of pathological states, including cancer. In this work, starting from the structure of purpurogallin Salfeld,  the pyro Epigenetic modifications, in other words changes occurring in the phenotype without altering the genotype, are known to be responsible for a myriad of biological processes that can result in the development of fatal diseases such as cancer  enzymes, a class of ~60 proteins in humans , which targets the substrate binding site  was discovered after docking 600,000 fragments into KDM4A, and linking other fragments to the initial 5-aminosalicylate hit   was shown to inhibit KDM6 subfamily member JMJD3 and to induce the cell cycle arrest in the S-phase on A375 melanoma cells , an antagonist of transcription factor 4 (TCF4)/β-catenin signaling, has been recently shown to inhibit KDM4A  was shown to potently inhibit KDM4A in vitro, and some of its derivatives with improved cell permeability were shown to significantly upregulate histone trimethylation and act as potent cancer cell growth inhibitors. In addition, deferasirox (6) potently inhibited human 2OG-dependent hipoxia inducible factor prolyl hydroxylase activity  was reported to inhibit KDM4 with high efficacy in breast and colon cancer PDX models , the first natural histone lysine demethylase inhibitor  selective KDM inhibitors identified to date .254  and visualized by UV irradiation (254 nm) or by staining with an acid solution of phosphomolybdic acid and ethanol. Flash column chromatography was carried out in a Combiflash system using Merck Kieselgel 60 (230–400 mesh) . Infrared (IR) spectra were obtained on a JASCO FTIR 4200 spectrophotometer  from either NaCl window or a diamond ATR probe. Melting points were determined on a Stuart SMP10 apparatus . High Resolution Mass Spectrometry  was measured with an Apex III FT ICR mass spectrometer . 1H- Nuclear Magnetic Resonance (NMR) spectra were recorded in CDCl3, acetone-d6, and DMSO-d6 at room temperature with a Bruker AMX-400 spectrometer  operating at 400.16 MHz with residual protic solvent as the internal reference ; chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) are given in Hertz (Hz). The proton spectra are reported as follows: δ . 13C-NMR spectra were recorded in CDCl3, acetone-d6, and DMSO-d6 at room temperature with the same spectrometer operating at 101.62 MHz with the central peak of CDCl3 (δ = 77.00 ppm), acetone-d6 (δ = 29.84 ppm), and DMSO-d6 (δ = 39.50 ppm) as the internal reference. DEPT-135 pulse sequences were used to aid in the assignment of signals in the 13C-NMR spectra.Analytical thin-layer chromatography was performed on aluminum plates with Merck Kieselgel 60F11b  and pyrogallol 10a  were dissolved in a mixture of acetone and freshly prepared pH 5 phosphate-citrate buffer , which contained 0.1 mg of horseradish peroxidase. Freshly prepared 3% H2O2 (10 mL) was added over 40 min by mechanical syringe and the mixture was stirred at room temperature overnight. The resulting precipitate was filtered off, washed with water (3x), and dried under reduced pressure. The obtained solid was purified by column chromatography to yield 14 mg (7%) of 3,4,6-trihydroxy-5H-benzo[7]annulen-5-one 9ab and 35 mg (14%) of purpurogallin 9aa.Sublimed catechol H-benzo[7]annulen-5-one . Orange solid. 1H-NMR  δ 7.35 , 7.07 , 6.90 , 6.74  ppm. 13C-NMR  δ 182.24 (s), 154.70 (s), 152.31 (s), 151.60 (s), 134.73 (s), 134.33 (d), 133.01 (s), 123.58 (d), 116.52 (d), 114.85 (s), 110.27 (d) ppm. ATR-FTIR: ν 3,600–3,400 (br), 3,400–3,200 (br), 2,973 (m), 1,585 (s), 1,419 (s), 1,376 (s), 1,330 (m), 1,196 (s), 1,046 (s) cm−1. HRMS: (ESI+) Calcd. for C11H9O5 ([M+H]+), 221.0445; found, 221.0443.2,3,4,6-tetrahydroxy-5H-benzo[7]annulen-5-one (9ab) Orange solid. 1H-NMR  δ 8.80 , 8.48 , 7.59 , 7.55–7.47 , 7.31 , 6.89  ppm. 13C-NMR  δ 185.04 (s), 155.66 (s), 151.11 (s), 147.11 (s), 136.84 (d), 133.26 (s), 126.31 (d), 124.00 (d), 122.78 (d), 120.94 (s), 119.04 (d) ppm. ATR-FTIR: ν 3,600–3,400 (br), 3,400–3,200 (br), 2,918 (m), 1,584 (s), 1,414 (s), 1,327 (s), 1,236 (m), 1,159 (s), 1,093 (s) cm−1. HRMS: (ESI+) Calcd. for C11H9O4 ([M+H]+), 205.0495; found, 205.0504.3,4,6-trihydroxy-511c , pyrogallol 10a , 3% H2O2 (21.3 mL), and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  afforded 44 mg (6%) of an orange solid identified as 1-bromo-3,4,6-trihydroxy-5H-benzo[7]annulen-5-one (9ac). 1H-NMR  δ 9.00 , 8.07 , 7.92 , 7.34 , 7.05  ppm. 13C-NMR  δ 185.21 (s), 156.40 (s), 152.09 (s), 147.24 (s), 133.78 (d), 129.80 (s), 127.48 (d), 125.63 (d), 121.80 (s), 118.88 (d), 116.79 (s) ppm. ATR-FTIR: ν 3,600–3,400 (br), 3,400–3,200 (br), 1,585 (s), 1,480 (m), 1,419 (s), 1,376 (s), 1,196 (s) cm−1. HRMS: (ESI+) Calcd. for 4 ([M+H]+), 282.9601; found, 282.9608.Following the general procedure previously described for the formation of benzotropolones, the reaction of 4-bromocatechol 11b , gallic acid 10b , 3% H2O2 (21.3 mL), and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  afforded 300 mg (46%) of an orange solid identified as 3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylic acid (9bb). 1H-NMR  δ 13.55 , 10.38 , 9.69 , 8.33 , 7.66 , 7.64 , 7.49  ppm. 13C-NMR  δ 184.96 (s), 167.64 (s), 153.13 (s), 151.24 (s), 148.64 (s), 138.80 (d), 128.74 (s), 128.25 (d), 123.44 (s), 122.00 (d), 120.36 (s), 115.96 (d) ppm. ATR-FTIR: ν 3,600–3,400 (br), 3,400–3,200 (br), 2,973 (m), 1,585 (s), 1,480 (m), 1,419 (s), 1,197 (s) cm−1. HRMS: (ESI+) Calcd. for C12H9O6 ([M+H]+), 249.0394; found, 249.0388.Following the general procedure previously described for the formation of benzotropolones, the reaction of catechol 11c , gallic acid 10b , 3% H2O2 (21.3 mL), and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  afforded 200 mg (23%) of an orange solid identified as 1-bromo-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylic acid (9bc). 1H-NMR  δ 13.46 , 10.78 , 9.98 , 8.87 , 7.83 , 7.61  ppm. 13C-NMR  δ 185.20 (s), 167.56 (s), 154.06 (s), 151.58 (s), 148.66 (s), 135.46 (d), 126.32 (d), 125.54 (s), 124.84 (s), 121.63 (s), 118.37 (s), 115.26 (d) ppm. FTIR (NaCl): ν 3,600–3,200 , 2,923 (s), 2,853 (s), 1,699 (s), 1,540 (m), 1,420 (m), 1,310 (m), 1,234 (s) cm−1. HRMS: (ESI+) Calcd. for 6 ([M+H]+), 326.9499; found, 326.9502.Following the general procedure previously described for the formation of benzotropolones, the reaction of 4-bromocatechol 9aa  and KOH  in distilled DMSO (2.73 mL) was added CH3I , and the mixture was stirred overnight at room temperature. After this time, the reaction was quenched by addition of water and the mixture was stirred for 20 min. After addition of EtOAc, the organic layer was washed with water (3x) and the combined aqueous layers were extracted with EtOAc (3x). The organic extracts were dried (Na2SO4) and solvent was removed under reduced pressure to provide 18 mg (96%) of an orange solid identified as 2,3,4,6-tetramethoxy-5H-benzo[7]annulen-5-one (12aa). m.p.: 154–156°C (hex/EtOAc). 1H-NMR  δ 7.07–6.93 , 6.54 , 6.25 , 3.97 , 3.95 , 3.86 , 3.78  ppm. 13C-NMR  δ 185.19 (s), 159.57 (s), 156.13 (s), 153.08 (s), 144.42 (s), 133.25 (s), 129.14 (d), 126.54 (s), 124.24 (d), 107.92 (d), 105.18 (d), 62.78 (q), 60.98 (q), 56.34 (q), 56.21 (q) ppm. FTIR (NaCl): ν 3,008 (m), 2,930 (s), 2,848 (s), 1,648 (m), 1,630 (m), 1,580 (s), 1,487 (m), 1,460 (m), 1,394 (m), 1,353 (m), 1,216 (s) cm−1. HRMS: (ESI+) Calcd. for C15H16NaO5 ([M+Na]+) 299.0890; found, 299.0895.To a cooled (0°C) suspension of purpurogallin 9ab , KOH , and CH3I  in DMSO (2.0 mL) afforded 12 mg (99%) of an orange solid identified as 3,4,6-trimethoxy-5H-benzo[7]annulen-5-one (12ab). m.p.: 150–152°C (hex/EtOAc). 1H-NMR  δ 7.38 , 6.96 , 6.43 , 6.24 , 3.95 , 3.89 , 3.75  ppm. 13C-NMR  δ 186.61 (s), 158.36 (s), 153.98 (s), 147.29 (s), 132.64 (s), 129.98 (s), 128.95 (d), 126.37 (d), 122.34 (d), 116.65 (d), 105.76 (d), 62.15 (q), 56.57 (q), 56.25 (q) ppm. FTIR (NaCl): ν 2,923 (s), 2,854 (s), 1,730 (m), 1,460 (m), 1,401 (s), 1,284 (m), 1,259 (m), 1,218 (s), 1,027 (s) cm−1. HRMS: (ESI+) Calcd. for C14H14NaO4 ([M+Na]+), 269.0784; found, 269.0789.Following the general procedure previously described for KOH-mediated methylation of phenols, the reaction of 9aa  and K2CO3  in anhydrous DMF (1.82 mL) was added CH3I , and the mixture was stirred overnight at room temperature. After this time, the reaction was quenched by addition of water and the mixture was stirred for 20 min. The aqueous phase was extracted with EtOAc (3x) and the combined organic layers were washed with water (3x). The organic extracts were dried (Na2SO4) and the solvent was removed under reduced pressure. The residue was purified by column chromatography to yield 7 mg (62%) of an orange solid identified as 4-hydroxy-2,3,6-trimethoxy-5H-benzo[7]annulen-5-one (13aa) 1H-NMR  δ 7.41 , 7.09 , 6.92 , 6.83 , 3.96 , 3.83 , 3.78  ppm. 13C-NMR  δ 185.18 (s), 158.14 (s), 157.50 (s), 156.59 (s), 136.21 (s), 135.87 (s), 133.97 (d), 124.15 (d), 117.05 (s), 113.56 (d), 106.20 (d), 59.67 (q), 56.24 (q), 56.02 (q) ppm. FTIR (NaCl): ν 3,600–3,200 (br), 3,008 (m), 2,924 (s), 2,851 (s), 1,581 (s), 1,460 (m), 1,411 (s), 1,354 (s), 1,253 (m), 1,214 (s) cm−1. HRMS: (ESI+) Calcd. for C14H15O4 ([M+H]+), 263.0914; found, 263.0910.To a cooled (0°C) suspension of purpurogallin 2CO3-mediated methylation of phenols, the reaction of 9ab , K2CO3 , and CH3I  in DMF (2.0 mL) afforded 5 mg (46%) of an orange solid identified as 4-hydroxy-3,6-dimethoxy-5H-benzo[7]annulen-5-one (13ab). 1H-NMR  δ 7.61 , 7.41 , 7.40 , 6.95 , 6.69 , 3.91 , 3.82  ppm. 13C-NMR  δ 186.93 (s), 156.37 (s), 153.05 (s), 147.78 (s), 134.80 (d), 130.92 (s), 123.84 (d), 121.55 (d), 120.57 (s), 117.86 (d), 115.02 (d), 56.27 (q), 56.04 (q) ppm. FTIR (NaCl): ν 3,600–3,200 (br), 2,925 (s), 2,850 (s), 1,648 (m), 1,630 (m), 1,584 (s), 1,433 (s), 1,210 (m), 1,190 (m), 1,043 (m) cm−1. HRMS: (ESI+) Calcd. for C13H13O4 ([M+H]+), 233.0808; found, 233.0805.Following the general procedure previously described for K2SO4), and solvent was evaporated under reduced pressure to obtain 4.3 g (89%) of a white solid identified as methyl 3,4,5-trihydroxybenzoate (10c). 1H-NMR  δ 9.15 , 6.93 , 3.74  ppm. 13C-NMR  δ 166.46 (s), 145.70 , 138.53 (s), 119.46 (s), 108.66 , 51.72 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 1,669 (s), 1,609 (s), 1,537 (m), 1,461 (m), 1,436 (m), 1,300 (s), 1,254 (s), 1,190 (m), 1,095 (w), 1,033 (s), 1,000 (s) cm−1. HRMS (ESI+): Calcd. for C8H8O5Na ([M+Na]+), 207.0261; found, 207.0264.To a solution of gallic acid 10b.  in methanol (50.0 mL) was added sulfuric acid, and the solution was stirred at 80°C for 20 h. Then, water was added and the aqueous phase was extracted with EtOAc (3x). The combined organic layers were washed with water (3x), brine (1x), and dried , methyl gallate 10c , 3% H2O2 (21.3 mL), and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  afforded 612 mg (68%) of an orange solid identified as methyl 1-bromo-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylate (9cc). 1H-NMR  δ 8.80 , 7.83 , 7.53 , 3.89  ppm. 13C-NMR  δ 184.74 (s), 166.21 (s), 153.83 (s), 151.65 (s), 148.83 (s), 135.41 (d), 126.06 (d), 124.99 (s), 123.40 (s), 121.09 (s), 118.64 (s), 114.44 (d), 53.12 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 1,719 (s), 1,669 (s), 1,607 (m), 1,434 (m), 1,327 (s), 1,257 (s), 993 (s) cm−1. HRMS (ESI+): Calcd. for 6 ([M+H]+), 340.9655; found, 340.9651.Following the general procedure previously described for the formation of benzotropolones, the reaction of 4-bromocatechol 11d , methyl gallate 10c , and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  with 3% H2O2 (2.5 mL) afforded 116 mg (72%) of an orange solid identified as methyl 1-chloro-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylate (9cd). 1H-NMR  δ 10.85 , 10.05 , 8.80 , 7.65 , 7.54 , 3.88  ppm. 13C-NMR  δ 184.54 (s), 166.12 (s), 153.61 (s), 151.30 (s), 148.87 (s), 131.73 (d), 127.24 (s), 123.68 (d), 123.16 (s), 122.52 (s), 120.67 (s), 114.49 (s), 53.07 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 1,722 (m), 1,592 (w), 1,431 (m), 1,394 (w), 1,235 (s), 995 (s) cm−1. HRMS (ESI+): Calcd. for 6 ([M+H]+), 297.0160; found, 297.0158.Following the general procedure previously described for the formation of benzotropolones, the reaction of 4-chlorocatechol 11e , methyl gallate 10c , and 0.5 mg of horseradish peroxidase in a mixture of acetone and pH 5 phosphate-citrate buffer  with 3% H2O2 (2.5 mL) afforded 97 mg (65%) of an orange solid identified as methyl 3,4,6-trihydroxy-1-methyl-5-oxo-5H-benzo[7]annul-ene-8-carboxylate (9ce). 1H-NMR  δ 10.29 , 9.76 , 8.53 , 7.58 , 7.45 , 3.88 , 2.85  ppm. 13C-NMR  δ 185.28 (s), 166.86 (s), 153.46 (s), 149.65 (s), 148.45 (s), 132.99 (d), 132.38 (s), 126.09 (s), 124.60 (s), 121.84 (s), 121.41 (s), 114.74 (d), 53.03 (q), 21.58 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 1,717 (m), 1,607 (w), 1,434 (m), 1,257 (s), 1,185 (s), 1,119 (s), 993 (s) cm−1. HRMS (ESI+): Calcd. for C14H13O6 ([M+H]+), 277.0707; found, 277.0708.Following the general procedure previously described for the formation of benzotropolones, the reaction of 4-methylcatechol 9cc  and K2CO3  in DMF (10.7 mL) was added dropwise with dimethyl sulfate , and the reaction mixture stirred at 50°C for 15 h. After this time, the reaction was quenched by addition of water and EtOAc, and the layers were separated. The organic phase was washed with water (3x), the combined aqueous layers were extracted with EtOAc (3x), and the whole organic phase was washed with brine, dried, and the solvent was evaporated under reduced pressure to afford 284 mg (94%) of an orange oil identified as methyl 1-bromo-3,4,6-trimethoxy-5-oxo-5H-benzo[7]annulene-8-carboxylate (13cc). m.p.: 173–175°C (hex/EtOAc). 1H-NMR  δ 8.41 , 7.47 , 6.68 , 3.95 , 3.92 , 3.84  ppm. 13C-NMR  δ 186.03 (s), 167.65 (s), 156.79 (s), 154.55 (s), 146.03 (s), 134.22 (s), 132.41 (d), 125.51 (s), 125.09 (s), 120.73 (s), 120.23 (d), 102.27 (d), 62.82 (q), 56.64 (q), 56.38 (q), 53.04 (q) ppm. FTIR (NaCl): ν 3,077 (w), 2,991 (w), 2,941 (m), 2,845 (w), 1,715 (s), 1,681 (s), 1,623 (m), 1,577 (s), 1,539 (s), 1,472 (s), 1,445 (s), 1,290 (s), 1,252 (s), 1,223 (s), 1,093 (m), 1,033 (m) cm−1. HRMS (ESI+): Calcd. for 6 ([M+H]+), 383.0125; found 383.0121.A solution of benzotropolone 2O (0.3 mL) was added bromide 13cc , phenylboronic acid 14f , Na2CO3 , and PdCl2(dppf) , and the mixture was stirred at 90°C for 90 min. Then, the reaction was quenched by the addition of water. The aqueous layer was extracted with EtOAc (3x). The combined organic layers were dried and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography  to afford 25 mg (81%) of a white solid identified as methyl 3,4,6-trimethoxy-5-oxo-1-phenyl-5H-benzo[7]annulene-8-carboxylate (15cf). m.p.: 154–156°C (hex/EtOAc). 1H-NMR  δ 7.97 , 7.52–7.40 , 7.37–7.32 , 7.15 , 6.69 , 4.02 , 3.96 , 3.86 , 3.75  ppm. 13C-NMR  δ 187.10 (s), 167.92 (s), 156.62 (s), 154.13 (s), 145.89 (s), 140.30 (s), 139.93 (s), 134.15 (s), 132.52 (d), 130.18 , 128.46 , 128.08 (d), 124.80 (s), 123.26 (s), 117.43 (d), 102.32 (d), 62.79 (q), 56.40 (q), 56.30 (q), 52.71 (q) ppm. FTIR (NaCl): ν 2,924 (s), 2,852 (m), 1,713 (s), 1,621 (w), 1,569 (m), 1,465 (m), 1,325 (w), 1,254 (s), 1,220 (s) cm−1. HRMS (ESI+): Calcd. for C22H21O6 ([M+H]+), 381.1333; found 381.1329.To a degassed mixture of DMF (1.3 mL) and H13cc , 4-trifluorotolueneboronic acid 14g , Na2CO3 , and PdCl2(dppf)  in DMF (1.3 mL) and H2O (0.3 mL) afforded, after purification by column chromatography , 25 mg (69%) of a white solid identified as methyl 3,4,6-trimethoxy-5-oxo-1-4'-trifluoromethyl-phenyl-5H-benzo[7]annulene-8-carboxylate (15cg). m.p.: 157–159°C (hex/EtOAc). 1H-NMR  δ 7.86 , 7.74 , 7.47 , 7.11 , 6.68 , 4.02 , 3.96 , 3.86 , 3.76  ppm. 13C-NMR  δ 186.86 (s), 167.71 (s), 156.85 (s), 154.23 (s), 146.46 (s), 143.98 (s), 138.07 (s), 134.30 (s), 131.59 (d), 130.59 , 130.52 , 124.65 (s), 124.08 , 123.93 (s), 117.29 (d), 102.14 (d), 62.83 (q), 56.47 (q), 56.35 (q), 52.84 (q) ppm. FTIR (NaCl): ν 2,925 (s), 2,852 (m), 1,716 (s), 1,619 (w), 1,572 (w), 1,492 (m), 1,461 (m), 1,435 (m), 1,326 (s), 1,256 (m), 1,221 (s) cm−1. HRMS (ESI+): Calcd. for C23H20F3O6 ([M+H]+), 449.1207; found 449.1200.Following the general procedure previously described for the Suzuki coupling, the reaction of bromide 13cc , 4-fluorophenylboronic acid 14 h , Na2CO3 , and PdCl2(dppf)  in DMF (1.3 mL) and H2O (0.3 mL) afforded, after purification by column chromatography , 19 mg (59%) of a white solid identified as methyl 3,4,6-trimethoxy-5-oxo-1-4'-fluoro-phenyl-5H-benzo[7]annulene-8-carboxylate (15ch). m.p.: 158–160°C (hex/EtOAc). 1H-NMR  δ 7.91 , 7.33–7.28 , 7.16 , 7.11 , 6.67 , 4.01 , 3.96 , 3.86 , 3.77  ppm. 13C-NMR  δ 187.01 (s), 167.84 (s), 162.68 , 156.71 (s), 154.15 (s), 146.02 (s), 138.72 (s), 136.25 , 134.20 (s), 132.13 (d), 131.82 , 124.86 (s), 123.48 (s), 117.45 (d), 115.50 , 102.20 (d), 62.80 (q), 56.43 (q), 56.32 (q), 52.79 (q) ppm. FTIR (NaCl): ν 2,925 (s), 2,853 (m), 1,714 (s), 1,572 (w), 1,489 (m), 1,463 (m), 1,434 (m), 1,254 (m), 1,221 (s) cm−1. HRMS (ESI+): Calcd. for C22H20FO6 ([M+H]+), 399.1238; found 399.1234.Following the general procedure previously described for the Suzuki coupling, the reaction of bromide 13cc  in methanol (1.3 mL) and H2O (0.4 mL) was added LiOH  and the mixture was stirred at room temperature. Then, the reaction was quenched by addition of a saturated aqueous solution of NaHCO3 and the aqueous layer was extracted with EtOAc (2x). The aqueous phase was acidified to pH 1 by addition of an aqueous solution of 10% HCl and then extracted with EtOAc (3x). The combined organic layers were washed with brine (1x), dried, and the solvent was evaporated to isolate a yellow solid that was dissolved in CH2Cl2 (0.65 mL) and HATU , DIPEA , and aniline , and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography  to afford 21 mg (36% combined yield) of a yellow solid identified as 1-bromo-3,4,6-trimethoxy-5-oxo-5H-benzo[7]annulene-8-benzamide (16dc). m.p.: 181–183°C (hex/EtOAc). 1H-NMR  δ 7.92 , 7.83 , 7.66–7.64 , 7.46 , 7.42–7.38 , 7.21–7.16 , 6.52 , 3.96 , 3.94 , 3.82  ppm. 13C-NMR  δ 186.33 (s), 167.40 (s), 157.32 (s), 154.14 (s), 145.95 (s), 138.00 (s), 133.67 (s), 131.46 (s), 129.25 , 126.68 (d), 125.41 (s), 124.92 (d), 120.44 , 120.32 (d), 119.96 (s), 102.08 (d), 62.80 (q), 56.66 (q), 56.51 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 2,929 (s), 2,852 (m), 1,653 (s), 1,577 (s), 1,537 (m), 1,468 (s), 1,442 (m), 1,292 (s), 1,217 (m), 1,034 (m) cm−1. HRMS (ESI+): Calcd. for 5 ([M+H]+), 444.0441; found 444.0436.To a solution of ester 9cc  in CH2Cl2 (1 mL) was added BBr3 , and the mixture was stirred at room temperature overnight. Then, the mixture was carefully quenched by dropwise addition of a saturated aqueous solution of NaHCO3 and the resulting biphasic mixture was stirred for 10 min at room temperature. The aqueous layer was washed with EtOAc (2x) prior to bringing to pH = 1 by addition of 10% HCl. Then, the aqueous layer was extracted with EtOAc (3x), the combined organic layers were washed with brine, dried, and the solvent was evaporated under reduced pressure to render 46 mg (99%) of an orange solid identified as 1-bromo-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylic acid (9bc).To a cooled solution (0°C) of ester 3-mediated hydrolysis of methyl esters and deprotection of ethers, the reaction of ester 9cd  and BBr3  in CH2Cl2 (0.44 mL) afforded 9 mg (76%) of an orange solid identified as 1-chloro-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylic acid (9bd). 1H-NMR  δ 13.48 , 10.80 , 9.99 , 8.90 , 7.68 , 7.62  ppm. 13C-NMR  δ 185.25 (s), 167.52 (s), 154.01 (s), 151.14 (s), 148.66 (s), 131.78 (d), 126.94 (s), 124.82 (s), 124.33 (s), 122.91 (d), 121.44 (s), 115.28 (d) ppm. ATR-FTIR: ν 3,600–3,200 (br), 2,919 (m), 2,850 (m), 1,692 (s), 1,589 (m), 1,435 (s), 1,401 (m), 1,306 (s), 1,241 (s), 1,119 (m), 1,033 (s), 879 (m) cm−1. HRMS (ESI+): Calcd. for 6 ([M+H]+), 283.0004; found, 283.0005.Following the general procedure previously described for the BBr3-mediated hydrolysis of methyl esters and deprotection of ethers, the reaction of ester 9ce  and BBr3  in DCM (0.35 mL) afforded 9 mg (99%) of an orange solid identified as 1-methyl-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylic acid (9be). 1H-NMR  δ 10.23 , 9.71 , 8.62 , 7.66 , 7.47 , 2.61  ppm. 13C-NMR  δ 185.25 (s), 167.89 (s), 153.34 (s), 149.57 (s), 148.14 (s), 132.93 (d), 132.10 (s), 126.39 (s), 124.62 (d), 122.88 (s), 121.53 (s), 115.44 (d), 21.61 (q) ppm. ATR-FTIR: ν 3,600–3,200 (br), 2,919 (m), 2,850 (m), 1,692 (s), 1,588 (w), 1,435 (s), 1,401 (m), 1,307 (s), 1,241 (s), 1,195 (s), 1,033 (m), 119 (m), 1,033 (s), 879 (m) cm−1. HRMS (ESI+): Calcd. for C13H11O6 ([M+H]+), 263.0550; found, 263.0552.Following the general procedure previously described for the BBr3-mediated hydrolysis of methyl esters and ethers, the reaction of 9cf  and BBr3  in CH2Cl2 (0.44 mL) afforded 12 mg (88%) of an orange solid identified as 3,4,6-trihydroxy-5-oxo-1-phenyl-5H-benzo[7]annulene-8-carboxylic acid (9bf). 1H-NMR  δ 9.74 , 8.37 , 7.62 , 7.56–7.42 , 7.41–7.29  ppm. 13C-NMR  δ 185.48 (s), 167.64 (s), 153.22 (s), 150.99 (s), 147.72 (s), 140.75 (s), 138.10 (s), 138.08 (s), 135.51 (d), 130.16 , 128.48 , 127.72 (d), 126.19 (s), 123.70 (d), 121.27 (s), 115.82 (d) ppm. ATR-FTIR: ν 3,600–3,200 (br), 2,919 (m), 1,692 (s), 1,588 (m), 1,435 (s), 1,307 (s), 1,241 (s), 1,195 (s), 1,119 (m), 1,033 (s), 879 (m) cm−1. HRMS (ESI+): Calcd. for C18H13O6 ([M+H]+), 325.0707; found 325.0710.Following the general procedure previously described for the BBr3-mediated hydrolysis of methyl esters and ethers, the reaction of ester 15cg  and BBr3  in CH2Cl2 (0.44 mL) afforded 11 mg (63%) of an orange solid identified as 3,4,6-trihydroxy-5-oxo-1-(4'-trifluoromethyl)-phenyl-5H-benzo[7]annulene-8-carboxylic acid (9bg). 1H-NMR  δ 10.53 , 9.82 , 8.29 , 8.05 , 7.61 , 7.50 , 7.41  ppm. 13C-NMR  δ 185.54 (s), 167.44 (s), 167.02 (s), 153.49 (s), 151.36 (s), 147.82 (s), 145.11 (s), 136.92 (s), 135.09 (d), 130.51 , 129.97 (s), 129.40 , 124.55 , 123.46 (d), 121.31 (s), 119.49 (s), 115.53 (d) ppm. ATR-FTIR: ν 2,926 (m), 2,850 (m), 1,713 (s), 1,681 (m), 1,622 (w), 1,577 (w), 1,468 (m), 1,441 (m), 1,389 (w), 1,287 (s), 1,247 (s), 1,225 (s), 1,035 (w) cm−1. HRMS (ESI+): Calcd. for C19H12F3O6 ([M+H]+), 393.0581; found 393.0580.Following the general procedure previously described for the BBr3-mediated hydrolysis of methyl esters and ethers, the reaction of ester 15ch  and BBr3  in CH2Cl2 (0.44 mL) afforded 10 mg (73%) of an orange solid identified as 3,4,6-trihydroxy-5-oxo-1-(4'-fluoro)-phenyl-5H-benzo[7]annulene-8-carboxylic acid (9bh). 1H-NMR  δ 10.50 , 9.77 , 8.31 , 7.62 , 7.52–7.07  ppm. 13C-NMR  δ 185.48 (s), 167.52 (s), 161.69 , 153.32 (s), 151.08 (s), 147.73 (s), 137.05 , 136.88 (s), 135.25 (d), 132.21  126.27 (s), 123.75 (d), 123.13 (s), 121.26 (s), 115.67 (d), 115.36  ppm. ATR-FTIR: ν 2,924 (s), 2,851 (m), 1,714 (s), 1,681 (m), 1,622 (w), 1,576 (s), 1,468 (s), 1,442 (s), 1,389 (w), 1,288 (w), 1,249 (s), 1,225 (s), 1,095 (w), 1,035 (m) cm−1. HRMS (ESI+): Calcd. for C18H12FO6 ([M+H]+), 343.0613 found 343.0614.Following the general procedure previously described for the BBr3-mediated demethylation, the reaction of amide 16dc  and BBr3  in CH2Cl2 (0.44 mL) afforded 8 mg (47%) of an orange solid identified as 1-bromo-3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene-8-benzamide (9dc). 1H-NMR  δ 10.66 , 10.56 , 10.11 , 8.44 , 7.87 , 7.74 , 7.47 , 7.37 , 7.12  ppm. 13C-NMR  δ 185.09 (s), 166.80 (s), 154.49 (s), 151.21 (s), 147.88 (s), 139.05 (s), 132.44 (d), 130.66 (s), 128.66 , 126.50 (d), 125.93 (s), 123.88 (d), 121.46 (s), 120.24 , 117.50 (s), 115.53 (d) ppm. ATR-FTIR: ν 3,600–3,200 (br), 2,924 (s), 2,853 (s), 1,713 (s), 1,681 (m), 1,622 (w), 1,577 (w), 1,491 (m), 1,460 (m), 1,443 (w), 1,290 (w), 1,035 (w) cm−1. HRMS (ESI+): Calcd. for 5 ([M+H]+), 401.9972; found 401.9971.Following the general procedure previously described for BBrHuman colon cancer HCT-116 and HT-29, breast cancer MCF7, lung cancer A549, and pancreatic cancer MiaPaCa cells  were propagated in Dulbecco's Modified Eagle's Medium  with 10% fetal bovine serum (FBS) (Euroclone), 2 mM L-glutamine (Euroclone), and antibiotics (100 U/mL penicillin-streptomycin) (Euroclone). Acute promyelocytic leukemia (NB4), histiocytic lymphoma (U937), hepatocellular carcinoma (Hep-G2), and mesenchymal progenitor (Mepr2B) cell lines were propagated in RPMI 1640 medium containing 4.5 g/L glucose (Euroclone) supplemented with 10% FBS (Euroclone), 100 U/mL penicillin-streptomycin (Euroclone), and 2 mM L-glutamine (Euroclone).Escherichia coli BL21 bacteria after transfection with pGEX-4T-1 plasmid. One bacteria colony was grown in LB Broth medium  supplemented with antibiotics  overnight at 37°C. The enzyme expression was induced by isopropyl-β-D-1-thiogalactopyranoside  at 200 μM for 7 h. The bacteria were centrifuged at 3,000 rpm  for 20 min and after the pellet was lysated by sonication  in lysis buffer containing PBS with 1 mM dithiothreitol  and cOmplete™ Protease Inhibitor Cocktail 1x . The sonication was performed in 10 cycles for 45 s at 14,000 MHz with intervals of 30 s between each sonication. The bacteria suspension was incubated for 15 min in ice in 0.1% of Triton-X100 (Applichem) solution and centrifuged at 13,000 rpm for 30 min and then filtered with a filter of 0.45 μm. The enzymes were purified using a GSTrap 4B columns . The columns were equilibrated with 20 mL of lysis buffer. The elution carried out with elution buffer . The purified recombinant enzymes were dialyzed in water solution , 1 mM DTT and protease inhibitors) overnight at 4°C. The day after, the samples were cryopreserved in 20% glycerol (SigmaAldrich).KDM4B/C/D-GST enzymes were purified from 9bf against KDM4B/C and D. In AlphaLisa assay KDM4A enzyme was incubated with 9bf at 10 different concentrations, and the assay was done following the protocol of the kit. The IC50 was calculated by GraphPad Prism 7.0 software . The experiments were performed in triplicate.Assay was performed as previously described using a KDM4A Inhibitor Enzymatic Assay KIT   at 50 μM.9ac and 9bc at 25 μM for 24 h, and with 10 μM 9bf for 24 h, 28 h and 24 h + 24 h (compounds were added again 24 h post 24 h stimulation). PKF118-310 (5) (1 μM) and QC6352 (7) (10 μM) were used as positive controls. Cells were then lysed in Triton Extraction Buffer (TEB) containing PBS with 0.5% Triton X-100 (v/v), 2 mM phenylmethylsulfonyl fluoride, and 0.02% (w/v) NaN3 at a cell density of 107 cells/mL for 10 min on ice and centrifuged . The supernatant was removed, and the pellet washed with half the volume of TEB and once more centrifuged. The pellet was suspended in 0.2 N HCl overnight at 4°C on a rolling table. The samples were centrifuged at 2,000 rpm for 10 min at 4°C and the supernatant was recovered. The histone protein was determined using a Bradford assay .MCF7 cells were incubated with compounds Western blot analysis was performed following the recommendations of antibody suppliers, loading 8 μg of histone extracts on 15% polyacrylamide gels. Antibodies used were H3K9me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3, and H3K27me2 (Diagenode), and histone H4 . Semi-quantitative analysis was performed using ImageJ software.5 cells/well) were plated in a six-well plate and treated with 9ac and 9bc at 25 μM for 24 h, and with 9bf at 25 μM for 12 h and 24 h. In addition, NB4 and U937 cells (2 × 105 cells/mL) were incubated with the same compound at 1, 5, and 10 μM for 12, 24, and 48 h. SAHA  and PKF118-310 (5) (1 μM) were used as controls for 24 h. After treatment, cells were collected, then centrifuged  and suspended in a solution containing 1 × PBS, 0.1% sodium citrate, 0.1% NP40, and 50 mg/mL propidium iodide. After 20 min of incubation at room temperature in the dark, cell cycle was evaluated by FACS  and analyzed with ModFit v3 software (Verity Software House). Cell death was measured as a percentage of cells in pre-G1 phase and analyzed by FACS with Cell Quest software .HCT-116 cells (1 × 104 cells/well) were plated in a 24-well plate and treated, in duplicate, with 9bf at 10 different concentrations (50–0.09 μM) for 72 h. MTT solution was added for 3 h at 0.5 mg/mL, the purple formazan crystals were dissolved in DMSO (Sigma-Aldrich), and the absorbance was read at a wavelength of 570 nm with a TECAN M-200 reader (Tecan).The viability of cells was determined using MTT  assay. Briefly, HT-29, MCF7, A549, MiaPaCa, Hep-G2, and Mepr2B cells  and Dunnett's multiple-comparison test. Differences between groups were considered to be significant at a The ligands were prepared by LigPrep, a tool of MAESTRO software, using OPLS3e as force field  was chosen from the Protein Data Bank (PDB)  and pyrogallol (10) derivatives . In addition, a carboxylic acid was incorporated on the pyrogallol skeleton (10) in order to evaluate whether this group could influence the interaction with the receptor-binding pocket by hydrogen bonding interactions , blocking KDM4A and therefore reducing synthesis of the fluorescent compound. Specifically, 9bc showed a greater enzymatic inhibition than PKF118-310 (5), which was used as positive control  may also have led to the decreased activity of these derivatives.These findings prompted us to hypothesize that the higher activity against KDM4A exerted by compounds 9ac and 9bc were tested at 25 μM for 24 h on MCF7 cells, in which high levels of KDM4 gene are expressed (7)  and lysine 36 in histone 3 (H3K36me3), considered bona fide KDM4A targets .Since KDM enzymes are known to have a similar structure, it is difficult to obtain compounds displaying greater selectivity toward one isoform. Therefore, the levels of other KDM targets such as H3K4me1/2/3 and H3K27me2 were evaluated after reatment . The com9aa, 9ac, and 9bc for 24 h at 25 μM did not show any variation in cell cycle phases compared to induction with SAHA, and PKF18-310 (5), which induced 20 and 73% of cell death, respectively  (1). For compounds 9cc, 9cd, and 16dc, the presence of a halogen, Br or Cl, was maintained. For compounds 15bf, 15bg, and 15bh, this position was variously replaced with a para-substituted phenyl group in order to evaluate whether the presence of further π-π interactions could increase the binding efficacy. Hence, methyl gallate 10c, prepared in 89% yield by Fischer esterification of commercially available gallic acid 10b, was condensed with 4-bromocatechol 11c to provide the corresponding benzotropolone 9cc in 68% yield. Under the same reaction conditions, chloro- and methyl-substituted analogs (9cd and 9ce) were prepared by condensation of methyl gallate 10c with 4-chloro- and 4-methylcatechol (11d and 11e) in 72 and 65% yield, respectively  . Furtherectively . Compoun6% yield . Lastly,9bf inhibited KDM4A by 80%, comparable to 9bc  but slightly higher than KDM4B/C (IC50 of 35.9 and 38.85 μM)  , suggest9bf blocked the KDM4A enzyme with an IC50 of 10.1 μM  from Celgene and others]  in extra precision mode (XP) with and without constraints   in cell population at pre-G1 phase. This result is comparable to the activity of SAHA, a well-known anticancer drug, which exhibited its antitumoral activity through G2/M phase accumulation after 24 h at a concentration of 5 μM, especially in hematological cancer cells.Next, we performed cell cycle analyses on HCT-116 cells in order to evaluate the ability of ll death . In thesll death  and PKF19bf in two leukemia cell lines. Acute promyelocytic leukemia NB4 and histiocytic lymphoma U937 cells were treated in three doses and at three different times in order to better assess its activity. Given the sensitivity to treatment of these cell lines compared to colon and breast cancer cells, lower doses (1–10 μM) were used. Our results show that 9bf acted in a time- and dose-dependent manner, strongly inducing cell death especially in U937 cells already at 5 μM after 24 h of treatment  for 72 h. Our data show that compound 9bf was able to induce a strong cell death in all cancer cell lines, as shown by the IC50 values  ranging from 7.04 μM to 4.04 μM. This tight range of IC50 values was in line with the RNA expression analysis of KDM4A in these cancer cells (http://ist.medisapiens.com/#ENSG00000066135).To further investigate its anticancer activity, ll lines  and its 9bf did not show any antiproliferative effects in immortalized Mepr2B cells, a normal mesenchymal cell line , a known KDM4A inhibitor. In MCF7, 9ac better than 9bc selectively increased the methylation levels of lysine 9 and 36, targets of this enzyme, and did not change the levels of all off-targets tested. However, none of the compounds induced cell death.KDM4A is known to be involved in several cellular functions, including embryonic development, cell cycle regulation, and DNA damage response (Guerra-Calderas et al., 9bf. This analog shares the common benzotropolone scaffold and incorporates an additional aromatic ring at position R1 of the catechol unit that increases lipophilicity, and a carboxylic acid at position R2 of the pyrogallol fragment. This molecule strongly inhibited KDM4A in in vitro assays with an IC50 of 24.37 and 10.1 μM, and in breast cancer cells increased the levels of H3K9me3 and H3K36me3, specific targets of KDM4A, already after 24 h of treatment at 10 μM. Given the structural homology among the enzymes belonging to the KDM4 subfamily, 9bf also inhibits KDM4B/C/D but with a greater IC50, demonstrating the selectivity of this compound toward KDM4A (These precedents led to the development of a new compound, namely rd KDM4A . It also9bf displayed high toxicity in several solid tumor cell lines, with IC50 values from 7.04 to 4.04 μM, but low activity in normal cells, potentially making it an excellent chemotherapeutic agent. Our findings suggest that further development of this compound and its derivatives may lead to the identification of new therapeutic antitumor agents that act by strongly inhibiting KDM4A.Excitingly, 9bf as a potent modulator of the epigenetic enzyme KDM4A, with promising antiproliferative effects in different cell systems. Our findings highlight the key structural role of the benzotropolone core of these molecules in inhibiting the enzyme, as well as the increased inhibitory activity exhibited by the carboxylic acid at C8 containing an aryl group at C1, which appear to induce a better binding of these benzotropolone derivatives to the ligand-binding pocket of Jumonji C domain-containing KDMs.To summarize, in this work we describe the synthesis and biological characterization of a new family of epigenetic modulators based on the structure of the natural product purpurogallin. Among the analogs synthesized, we identified compound All datasets generated for this study are included in the article/in silico studies. AN and LA contributed to the conception of the biological experiments. All authors contributed to manuscript revision, read and approved the submitted version.ÁL and RÁ planned and coordinated, and JS planned and performed the organic chemical synthesis of the compounds. FS and CP performed biological experiments, and data analysis and interpretation. UP, JL, and AP performed and contributed to the conception of binding CP was employed by the company EPI-C srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Copolymerization of the esters with vinyl benzene in solution with radical initiation (ABCN) at 70°C led to formation copolymers. The products were characterized by CHN elemental analysis, IR, 1 H- and 13 C-NMR, GPC, DSC, and TGA.Condensation of isopropyl cyanoacetate and substituted benzoic aldehydes resulted in formation of novel isopropyl esters of 2-cyano-3-phenyl-2-propenoic acid, RPhCH = C(CN)CO Pharmaceutical research is reported for a number of CPP compounds  · 100%; ICPA (mol %) = 100 – VB mol %.3 and insoluble in methanol, ethyl ether, and petroleum ether.According to elemental analysis, between 9.4 and 35.2 mol% of ICPA monomer is present in the copolymers prepared at VB/ICPA = 3 (mol), which is indicative of relatively high reactivity of the monomers towards ST. The copolymers were all soluble in ethyl acetate, THF, DMF and CHCl3.4Relative reactivities of VB and the ICPA monomers in the copolymerization can be estimated by application of the copolymerization equation for the terminal copolymerization model .(1)m1/mm1 and m2 are mole fractions of VB and ICPA monomer units in the copolymer, [M1] and [M2] are concentrations of VB and an ICPA in the monomer feed, and r1 and r2 are monomer reactivity ratios, r1 = kVB-VB/kVB-ICPA and r2 = kICPA-ICPA/kICPA-VB. In the absence of self-propagation of ICPA monomers, none of them formed homopolymers,  /[M2] ratio in all the experiments was equal to 3.0, relative reactivities (1/r1) for the ICPA monomers decrease in the following row R = 5-chloro-2,3-dimethoxy (3.55) > 6-bromo-3,4-dimethoxy (3.43) > 4-bromo-2,6-difluoro (2.99) > 5-bromo-2,3-dimethoxy (2.81) > 3-bromo-4,5-dimethoxy (2.75) > 5-bromo-2,4-dimethoxy (2.69) > 3-chloro-2,6-difluoro (2.43) > 4-chloro-2,6-difluoro (2.10) > 3-chloro-4,5-dimethoxy (1.82) > 2-bromo-3-hydroxy-4-methoxy (1.48) > 2,3,4,5,6-pentafluoro (1.39) > 2,3,6-trichloro (1.21) > 2,3,4-trifluoro (1.20) > 3,4,5-trifluoro (1.17) > 2,3,4-trimethoxy (1.11) > 2,3,5,6-tetrafluoro (1.08) > 2,4,5-trifluoro (0.99) > 2,4,6-trifluoro (0.81) > 2,4,6-trimethoxy (0.73) > 2,4,5-trimethoxy (0.52) > 2-chloro-3,4-dimethoxy (0.35).These relative reactivity values can be used to predict specific copolymer composition as function of the comonomer feed. Additional research will be needed to correlate effect of phenyl ring substitution with reactivity of ICPA monomers in radical copolymerization.3.5.Tg and absence of crystalline endotherm on repeated heating and cooling cycles  [Tg which is related to segmental mobility [Thermal transitions of the VB-ICPA copolymers were analyzed by differential scanning calorimetry (DSC). The second heating results were obtained in all cases so that the samples become more dry and without ‘thermal memory’. DSC analysis confirmed amorphous morphology of the EB-ICPA copolymers showing glass transition temperatures g cycles . A singl (104ºC) . Introdumobility , whereasco-ICPA) copolymers is lower than that of poly [Thermogravimetric analysis (TGA) provided information on thermal stability of the copolymers . Thermalne, PVB) , the onsne, PVB) . TGA sho4.1 and 13 C-NMR. The thermal gravimetric analysis indicated that the copolymers decompose in two steps, first in the 200–500°C range with a residue, which then decomposed in the 500–800ºC range.Novel isopropyl esters of ring-substituted 2-cyano-3-phenyl-2-propenoic acid were prepared and copolymerized with vinyl benzene. The compositions of novel copolymers were calculated from nitrogen analysis and the structures were analyzed by IR, H