A variety of 2-bromo-4-chlorophenyl-2-bromobutanoate derivatives (5a–f) were synthesized with moderate to good yields via a Pd-catalyzed Suzuki cross-coupling reaction. To find out the reactivity and electronic properties of the compounds, Frontier molecular orbital analysis, non-linear optical properties, and molecular electrostatic potential studies were performed.In the present study, 2-bromo-4-chlorophenyl-2-bromobutanoate ( Carboxylic esters are an important class of organic compounds, having various biological activities such as antifungal , antioxiFor the synthesis of biphenyl compounds, the Suzuki–Miyaura reaction is one of the best methods. In organic synthesis, regioselective functionalization plays a progressively important role . In such3) was synthesized by the reaction of 2-bromo-4-chlorophenol (1) with 2-bromobutryl bromide (2) in the presence of pyridine and acetonitrile, and good yield of 2-bromo-4-chlorophenyl-2-bromobutyrate (3) was observed  with different phenyl boronic acids was accomplished, which ultimately led to the formation of the corresponding 2-bromo-4-chlorophenyl-2-bromobutyrates (5a–f) in moderate to good yields (64–81%) (3)4, the other bromo group present at the alpha position could not be replaced. The chloro group at the phenyl ring was not replaced due to the C-Cl bond strength that obstructs the reactivity of aryl chlorides, thus, they are resistant to the oxidative addition to Pd(0) because activation of aryl chlorides is much more difficult than that of aryl bromides. The oxidative addition step is a selectivity-determining step in Suzuki cross-coupling, and it is broadly controlled by the bond dissociation energies of the carbon–halogen bond that predictably differs as a function of the aryl halides: Ar-I > Ar-Br > Ar-Cl > Ar-F -2-yl 2-bromobutanoate (5f) has the highest N value (5.19 eV), which shows the strong nucleophilic nature of this compound.Nucleophilicity is an important parameter for understanding the reactivity of compounds. All the compounds had nucleophilicity values in the range of 4.27 eV to 5.19 eV. The compound Molecular electrostatic potential (MEP) is used to evaluate and predict the reactive sites of a molecule . MEP is −2 to 4.75 × 10−2 was observed for compound 5d. In contrast, the lowest electronic density distribution of −5.22 × 10−2 to 5.22 × 10−2 was observed for compound 5e.o). In organic molecules, the movement of electrons from donor to acceptor group is responsible for the enhancement of βo, which ultimately results in a strong NLO response.For the last decade, scientific society has been pursuing the pathways to synthesized non-linear optical (NLO) materials with large second-order nonlinear optical properties. Non-linear optical materials have various applications in laser devices, optical communication, optical data storage, optical limiting, optical computing, and medical imaging ,29. The 5a–f), the para chloro-phenyl ring (p-CPR) acts as electron-withdrawing group and influences different substitutions on the NLO response were studied at the ortho position of phenyl ring. The ester group is not involved due to the resonance between the two oxygen atoms and the electrons are unable to delocalize inside the phenyl ring. When the electron donating groups are attached to the p-CPR, the electron push full mechanism is the operative one and a large βo value is obtained. However, if the electron-withdrawing groups are attached to the p-CPR, then the electron push full mechanism is not operating and the small βo value is obtained in that case. The compound 5c has a strong electron donating group (MeS-) at the para position of the phenyl ring and we noticed a large βo value (1373.76 au) for it according to the push full mechanism. The lower βo value (218.51 au) was observed for compound 5f where an electron-withdrawing chloro group (Cl−) is present on the para position of the phenyl ring. On both sides of the phenyl rings, similar chloro-groups are attached which make these rings strongly electron-withdrawing, and the net effect is zero. The moderate electron donating methoxy group at the para position of the phenyl ring (in 5e) had a reasonable NLO response compared to compound 5b where the acetate group has an internal resonance, which has a negative impact on the NLO response. The βo values of 5e and 5b compounds were 954.47 au and 284.34 au, respectively. Compounds 5a and 5d had almost comparable βo values (in the range of 510 au −550 au). The reason may be that the presence of halogen groups on the meta and para positions of the ring nullified the effects of each other and the phenyl ring acted as moderately electron-donating. Based on these results, we suggest that compound 5c shows a better NLO response and can be used as an active NLO material in the future.In the synthesized compounds (5a–f) was also supported by the polarizability values  whereas 5f had a low αo value (235 au). The rest of the compounds had moderate values of αo in the range of 236 au to 252 au.The hyperpolarizability of these compounds  was calculated by using degenerated four waves mixing gamma values (γDFWM (ω) =  as proposed by Tarazker et al. + 353.8909; found 353.8945.3PO4 (2 equiv.) and aryl boronic acids and 2 mL distilled water were added and the reflux reaction continued under an inert argon atmosphere for 18–25 h at 80–90 °C. During the reaction, the estimation of the reaction was determined by a thin layer chromatography technique using TLC cards. When the reaction was completed, the reaction mixture was filtered using Whatman filter paper and the solvent was evaporated by a rotary evaporator. The separation and purification of the impure products were carried out using the column chromatography technique. 1H-NMR was used to find out the structure of the newly synthesized compounds -2-yl 2-bromobutanoate  8.04 , 8.00 , 7.55 , 7.41 , 7.38 , 7.35 , 4.2 , 2.11–2.17 , 1.11 . 13C-NMR  δ 10.2 (CH3), 25.4 (CH2), 42.7 (CH), 117.6 (CH), 122.4 (C), 125.6 (CH), 130.5 (CH), 131.6 (CH), 132.5 (CH), 133.2 (CH), 132.9 (C), 133.4 (C), 133.9 (C), 148.1 (C), 162.3 (C), 170.1 (C). HR-ESI-MS: m/z Calcd for C16H12BrCl2FO2, [M]+ 406.0722; found 406.0742.5b),3′-Acetoxy-5-chloro--2-yl 2-bromobutanoate  δ 8.00 , 7.50 , 7.46 , 7.42–7.34 , 7.35 , 7.28 , 4.20 , 2.35 , 2.10–2.15 , 1.10 . 13C-NMR  δ 10.1 (CH3), 19.4 (CH3), 25.6 (CH2), 45.4 (CH), 120.4 (CH), 121.1 (CH), 122.3 (CH), 128.7 (CH), 129.1 (CH), 131.2 (C), 132.7 (CH), 134.8 (CH), 136.3 (C), 137.5 (C), 145.1 (C), 148.1 (C), 170.1 (C), 171.2 (C). HR-ESI-MS: m/z Calcd for C18H16BrClO4, [M]+ 411.6808; found 411.6892.5c),5-Chloro-4′-(methylthio)--2-yl 2-bromobutanoate  δ 8.00 , 7.65 , 7.45 , 7.35 , 7.25 , 4.19 , 2.53 , 2.05–2.11 , 1.10 . 13C-NMR  δ 10.1 (CH3), 19.4 (CH3), 25.6 (CH2), 45.4 (CH), 121.1 (CH), 124.3 (CH), 125.4 (2CH), 128.1 (C), 130.2 (CH), 131.1 (C), 132.7 (C), 134.8 (CH), 135.4 (CH), 136.1 (C), 146.1 (C), 170.1 (C). HR-ESI-MS: m/z Calcd for C17H16BrClO2S, [M]+ 399.7145; found 399.7119.3′,4′,5-Trichloro--2-yl 2-bromobutanoate (5d), white crystals, m.p: 276–277 °C. 1H-NMR  δ 7.96 , 7.83 , 7.51–7.43 , 7.40 , 7.33 , 4.20 , 2.15–2.20 , 1.10 .13C-NMR  δ 10.1 (CH3), 25.6 (CH2), 45.4 (CH), 121.1(CH), 126.5 (CH), 128.1 (CH), 130.2 (CH), 131.2 (CH), 132.7 (C), 135.8 (CH), 136.2 (C), 137.2 (C), 138.5 (C), 141.5 (C), 148.1 (C), 170.1 (C). HR-ESI-MS: m/z Calcd for C16H12BrCl3O2, [M]+ 422.5233; found 422.5289.5-Chloro-4′-methoxy--2-yl 2-bromobutanoate (5e), light brown crystal, m.p: 237–238 °C. 1H-NMR  δ 7.98 , 7.65  7.49–7.35 , 7.10 , 4.22 , 3.93 , 2.08–2.15 , 1.10 . 13C-NMR  δ 10.1 (CH3), 25.6 (CH2), 45.4 (CH), 60.2 (CH3), 116.1 (CH), 116.5 (CH), 124.5 (CH), 125.3 (C), 128.4 (CH), 130.3 (CH), 131.4 (C), 132.2 (CH), 13.3 (CH), 132.7 (C), 135.8 (C), 151.2 (C), 170.1 (C). HR-ESI-MS: m/z Calcd for C17H16BrClO3, [M]+ 383.4515; found 383.4565.3′,5-Dichloro--2-yl 2-bromobutanoate (5f), white crystal, m.p: 232–233 °C. 1H-NMR  δ 8.01 , 7.52 , 7.55 , 7.42–7.41 , 7.34 , 4.22 , 2.08–2.15 , 1.10 . 13C-NMR  δ 10.1 (CH3), 25.6 (CH2), 45.4 (CH), 121.1 (2CH), 122.4 (CH), 121.9 (CH), 125.2 (CH), 128.7 (2CH), 130.2 (C), 132.7 (C), 133.2 (C), 134.7 (C), 151.3 (C), 171.2 (C). HR-ESI-MS: m/z Calcd for C16H13BrCl2O2, [M]+ 388.0861; found 388.0839.5a–f) were optimized with the hybrid functional (B3LYP) of the density functional theory (DFT). B3LYP is a widely used functional for the optimization of organic molecules because the minimization of errors in the geometrical parameters is lower for B3LYP compared to that of other density functionals. The split valence basis set developed by Pople’6-311G  was implemented for molecular orbital descriptions. For further confirmation of these minimum energy structures obtained from optimization  were synthesized and frontier molecular orbitals (FMOs) and molecular electrostatic potential (MEP) analysis were done. FMOs analysis revealed that among all the derivatives, 5c is most reactive, having a HOMO–LUMI band gap of 4.27 eV, whereas the HOMO–LUMO band-gap for 5f was 5.19 eV, and was the most stable. The chemical hardness of 5f displayed the highest value (2.60 eV), whereas 5c has the lowest value and was chemically soft and reactive. Among all the newly synthesized compounds, 5d displayed the highest chemical potential value. The molecular electrostatic potential (MEP) investigation gave us the idea of electrophilicity and nucleophilicity of the synthesized compounds, and it was envisaged that depression of electron density is highly dependent on the substituent present on an aromatic ring. The first hyperpolarizability values showed that all synthesized derivatives has a potential non-linear optics (NLO) response, but 5c has a significant NLO potential due to its large βo (1373.76 au) value.In conclusion, a series of 2-bromo-4-chlorophenyl-2-bromobutyrate derivatives (


Vibrio fischeri-quorum sensing was evaluated. The compounds in the series exhibit variable side chain length and the possible presence of a diversely substituted phenyl substituent. Biological evaluation on the Vibrio fischeri quorum sensing system revealed that the ethyl substituted carbamate (1) display a weak agonistic activity whereas compounds with longer chain length or benzyl substituents display significant antagonistic activity. The most active compounds in the series were the 4-nitrobenzyl carbamate and thiocarbamate 7 and 11 which exhibited an IC50 value of about 20 µM. These activities are in the range of other reported of AHL-structurally related quorum sensing (QS) inhibitors. Docking experiments conducted on the LuxR model showed that, compared to the natural ligand OHHL, the additional heteroatom of the carbamate group induces a new hydrogen bond with Tyr70 leading to a different global hydrogen-bond network. Tyr70 is an important residue in the binding site and is strictly conserved in the LuxR family. For the 4-nitrobenzyl carbamate and thiocarbamate analogues, the docking results highlight an additional hydrogen bond between the nitro group and Lys178. For hydrazide analogues, which are deprived of any activity, docking shows that the orientation of the carbonyl group is opposite as compared with the natural ligand, leading to the absence of a H-bond between the C=O with Tyr62. This suggests that, either this later interaction, or the influence of the C=O orientation on the overall ligand conformation, are essential for the biological activity.A series of carbamate, thiocarbamate, and hydrazide analogues of acylhomoserine lactones (AHLs) were synthesized and their ability to modulate  Vibrio fischeri-quorum sensing modulation using urea-type AHLs analogues [Acylhomoserine lactones (AHLs) are molecular signals used by Gram negative bacteria to communicate. They are biosynthesized by proteins of the LuxI family and are recognized by their cognate transcriptional regulators belonging to the LuxR family. This system referred to as quorum sensing (QS) allows bacteria to coordinate their behaviors according to their environment ,2,3,4,5.nalogues , we repo4. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVL300 or a Bruker AV400 or a Bruker AV500 spectrometer , operating respectively at 300, 400, and 500 MHz for the proton (1H) NMR. Carbon (13C) NMR spectra were recorded on a Bruker AVL300 or a Bruker AV400 spectrophotometer or a Bruker AV500 spectrometer, operating, respectively, at 75, 100, and 125 MHz. Chemical shifts were reported in parts per million (ppm) in the scale relative to residual solvent signals. Multiplicities are abbreviated as follows: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; dt, doublet of triplts, m, multiplet; br, broad. Coupling constants were measured in Hertz (Hz). High-resolution mass spectra (HRMS) and low-resolution mass spectra were performed by the Centre Commun de Spectrométrie de Masse (CCSM), University of Lyon 1, Lyon, France. l- or d-homoserine lactone hydrobromide was synthesized as previously described [All commercial materials were used without further purification. Flash chromatography was carried out using Macherey-Nagel  Kieselgel 60 M silica. Analytical thin layer chromatography was realized using aluminum-backed plates coated with Macherey-Nagel Kieselgel 60 XtraSIL G/UV254. Compounds were visualized under UV light (at 254 nm) or stained using KMnOescribed .l-α-amino-γ-butyrolactone hydrobromide  in anhydrous THF (2 mL) N,N-diisopropylethylamine  at 0 °C was added, and the appropriate commercially available benzyl chloroformate or ethyl chloroformate (1.2 eq) was added dropwise. The mixture was stirred overnight while the temperature was warmed up to room temperature. The mixture was then diluted with EtOAc (10 mL) and washed with brine (10 mL). The organic phase was collected, dried over anhydrous Na2SO4, concentrated, and the residue was purified by flash chromatography to give compounds 1 and 5 as white solids.To a solution of N-(ethoxycarbonyl)-l-homoserine lactone (1). Flash chromatography of the crude product (1:1 EtOAc-pentane) afforded 1 (72%) as a white solid. IR (cm−1): 3332 (N-H), 1785 , 1679 , 1537 (-NHCO-). [αterature .N-(benzyloxycarbonyl)-l-homoserine lactone (5). Flash chromatography of the crude product (1:3 EtOAc-pentane) afforded 5 (48%) as a white solid. IR (cm−1): 3324 (N-H), 1776 , 1686 , 1539 (-NHCO-). [αterature .N-(4-nitrobenzylthiocarbonyl)-l-homoserine lactone (11). To a solution of triphosgene (1 mmol) in DCM (2 mL) 4-nitro benzylmercaptan (2.4 mmol) was added slowly which was dissolved in DCM (2 mL) at 0 °C, followed by addition of pyridine  dissolved in DCM (2 mL). After stirring for 2 h at 0 °C, the reaction was quenched by addition of water (2 mL). The reaction mixture was diluted with DCM (16 mL) and washed with ice-cold water (8 mL). The organic layer was dried over anhydrous Na2SO4, concentrated to afford the S-4-nitrobenzyl chlorothioformate  as a colorless oil, used immediately without further purification. 1H NMR  δ 8.21 , 7.51 , 4.22 . Following the general procedure for the route A, flash chromatography of the crude product (1:1 EtOAc-pentane) afforded 11 (51%) as a white solid. IR (cm−1): 3307 (N-H), 1782 , 1640 , 1513 (-NHCO-), 1491 (Ar-NO2), 1345 (Ar-NO2). [α1H NMR  δ 8.15 , 7.49 , 6.24 , 4.57 , 4.46 , 4.27 , 4.19 , 2.79 , 2.22 . 13C NMR  δ 174.8 (CO-lactone), 167.1 (SCONH), 147.1 (Ph), 145.8 (Ph), 129.7 , 123.8 , 66.0 (OCH2-lactone), 50.8 (CH-NH), 33.4 (SCH2), 30.1 (CH2-lactone). ESI-HRMS (M + Na)+: 319.0359; found: 319.0355.p-nitrophenyl chloroformate (1 eq) at 0 °C were added. The reaction mixture was allowed to warm up to room temperature and was stirred overnight. The mixture was then diluted with EtOAc (20 mL) and filtered. The solution was washed with brine (2 × 10 mL); the organic phase was collected, dried over anhydrous Na2SO4, concentrated to afford the desired carbonates. (ii) To a solution of l-α-amino-γ-butyrolactone hydrobromide (1 eq) in dry THF (2 mL), triethylamine (1 eq) and various p-nitrophenyl carbonates (1.2 eq) dissolved in THF (1 mL) were added at room temperature. The reaction mixture was stirred at 40 °C overnight. The solution was filtered and the solvent was evaporated. The residue was purified by flash chromatography to give the corresponding carbamates as white solids.Carbamates: (i) To a stirred solution of different alcohols (1.05 eq) in THF, triethylamine (2.3 eq) and p-nitrophenyl chloroformate (1.05 eq) at 0 °C were added, respectively. The reaction mixture was allowed to warm up to room temperature and stirred overnight. The mixture was diluted with DCM (20 mL) and washed with water (2 × 10 mL), the organic phase was collected, dried over anhydrous Na2SO4, concentrated, and purified by flash chromatography to get the desired thiocarbonate. (ii) To a solution of l-α-amino-γ-butyrolactone hydrobromide (1 eq) in dry THF (2 mL), triethylamine (1 eq or 1.2 eq) and p-nitrophenyl thiocarbonates (1.2 eq) dissolved in THF (1 mL) at room temperature were added. The reaction mixture was then stirred at 60 °C overnight. The reaction mixture was cooled to room temperature, filtered, and the solvent was evaporated. The residue was purified by flash chromatography to give the corresponding thiocarbamates as white solids.Thiocarbamates: (i) To a solution of different thiols (1 eq) in dry DCM, triethylamine (1.05 eq), 4-dimethylaminopyridine (DMAP) (1.05 eq), and N-(butoxycarbonyl)-l-homoserine lactone (2). From butyl alcohol the Butyl 4-nitrophenyl carbonate was obtained (48%) as a yellow liquid. 1H NMR  δ 8.18 , 7.30 , 4.21 , 1.80–1.56 , 1.44–1.26 , 0.89 . According to the general procedure B, flash chromatography of the crude product (1:2 EtOAc-pentane) afforded 2 (68%) as a white solid. IR (cm−1): 3333 (N-H), 1775 , 1688 , 1537 (-NHCO-). [α1H NMR  δ 5.39 , 4.56–4.33 , 4.24 , 4.07 , 2.73 , 2.21 , 1.59 , 1.36 , 0.91 . 13C NMR  δ 175.2 (CO-lactone), 156.5 (OCONH), 65.8 (OCH2-lactone), 65.5 (OCH2), 50.4 (CH-NH), 30.9 (CH2), 30.3 (CH2-lactone), 19.0 (CH2), 13.7 (CH3). ESI-HRMS (M + Na)+: 224.0893; found: 224.0892.d-homoserine lactoneN-(butoxycarbonyl)- (3). Compound 3 was obtained from Butyl 4-nitrophenyl carbonate and d-α-amino-γ-butyrolactone hydrobromide. Flash chromatography of the crude product (1:3 EtOAc-pentane) afforded 3 (74%) as a white solid. IR (cm−1): 3329 (N-H), 1775 , 1687 , 1536 (-NHCO-). [α1H NMR  δ 5.49 , 4.40 , 4.22 , 4.05 , 2.69 , 2.21 , 1.57 , 1.40–1.24 , 0.94–0.84 . 13C NMR  δ 175.4 (CO-lactone), 156.6 (OCONH), 65.8 (OCH2-lactone), 65.5 (OCH2), 50.4 (CH-NH), 31.0 (CH2), 30.1 (CH2-lactone), 19.1 (CH2), 13.7 (CH3). ESI-HRMS (M + Na)+: 224.0893; found: 224.0897.l-homoserine lactoneN-(hexyloxycarbonyl)- (4). From hexyl alcohol Hexyl 4-nitrophenyl carbonate was obtained (56%) as a yellow liquid. 1H NMR  δ 8.25 , 7.36 , 4.27 , 1.83–1.61 , 1.51–1.19 , 0.89 . According to the general procedure B, flash chromatography of the crude product (1:2 EtOAc-pentane) afforded 4 (63%) as a white solid. IR (cm−1): 3332 (N-H), 1775 , 1688 , 1539 (-NHCO-). [α1H NMR  δ 5.40 , 4.41 , 4.24 , 4.05 , 2.72 , 2.21 , 1.59 , 1.43–1.19 , 0.86 . 13C NMR  δ 175.2 (CO-lactone), 156.5 (OCONH), 65.8 (OCH2-lactone), 65.8 (OCH2), 50.4 (CH-NH), 31.4 (CH2), 30.3 (CH2-lactone), 28.8 (CH2), 25.5 (CH2), 22.5 (CH2), 14.0 (CH3). ESI-HRMS (M + Na)+: 252.1206; found: 252.1207.N-(4-bromobenzyloxycarbonyl)-l-homoserine lactone (6). From 4-bromobenzyl alcohol (4-Bromoophenyl)methyl 4-nitrophenyl carbonate was obtained by flash chromatography of the crude product (1:8 EtOAc-pentane) in 47% yield as a white solid. IR (cm−1): 3306 (N-H), 1776 , 1695 , 1538 (-NHCO-). [α1H NMR  δ 8.27 , 7.54 , 7.43–7.28 , 5.24 . 13C NMR  δ 155.4 (OCO), 152.4 (Ph), 145.5 (Ph), 133.2 (Ph), 132.0 , 130.3 , 125.3 , 123.3 (Ph), 121.8 , 70.1 (OCH2Ar). ESI-HRMS (M + Na)+: 373.9635; found: 373.9637. According to the general procedure B, flash chromatography of the crude product (1:3 EtOAc-pentane) afforded 6 (40%) as a white solid. 1H NMR  δ 7.48 , 7.22 , 5.39 , 5.07 , 4.42 , 4.25 , 2.88–2.66 , 2.21 . 13C NMR  δ 174.9 (CO-lactone), 156.0 (OCONH), 134.9 (Ph), 131.7 , 129.8 , 122.37 (Ph), 66.5 (OCH2Ar), 65.8 (OCH2-lactone), 50.5 (CH-NH), 30.4 (CH2-lactone). ESI-HRMS (M + Na)+: 335.9842; found: 335.9843.N-(4-nitrobenzyloxycarbonyl)-l-homoserine lactone (7). From 4-nitrobenzyl alcohol (4-Nitrophenyl)methyl 4-nitrophenyl carbonate was obtained in 78% yield as a yellow solid. 1H NMR data were consistent with the literature [7 (52%) as a white solid. IR (cm−1): 3339 (N-H), 1783 , 1692 , 1533 (-NHCO-), 1512 (Ar-NO2), 1345 (Ar-NO2). [αterature . AccordiN-(4-phenylbutoxycarbonyl)-l-homoserine lactone (8). From 4-phenyl butanol, flash chromatography of the crude product (1:8 EtOAc-pentane) afforded (4-Phenyl)butyl 4-nitrophenyl carbonate (48%) as a white solid. 1H NMR  δ 8.24 , 7.34 , 7.32–7.11 , 4.29 , 2.67 , 1.94–1.68 . 13C NMR  δ 155.6 (OCO), 152.6 (Ph), 145.4 (Ph), 141.7 (Ph), 128.5 , 128.4 , 126.0 (Ph), 125.3 , 121.9 , 69.4 (OCH2), 35.4 (CH2), 28.1 (CH2), 27.5 (CH2). ESI-HRMS (M + Na)+: 338.0999; found: 338.0999. Following general procedure B, flash chromatography of the crude product (1:2 EtOAc-pentane) afforded 8 (40%) as a white solid. IR (cm−1): 3336 (N-H), 1776 , 1687 , 1537 (-NHCO-). [α1H NMR  δ 7.33–7.23 , 7.22–7.12 , 5.25 , 4.41 , 4.25 , 4.12 , 2.77 , 2.64 , 2.20 , 1.69 . 13C NMR  δ 175.1 (CO-lactone), 156.4 (OCONH), 142.1 (Ph), 128.5 , 128.4 , 125.9 (Ph), 65.9 (OCH2-lactone), 65.6 (OCH2), 50.5 (CH-NH), 35.5 (CH2), 30.6 (CH2-lactone), 28.5 (CH2), 27.7 (CH2). ESI-HRMS (M + Na)+: 300.1206; found: 300.1210.N-(butylthiocarbonyl)-l-homoserine lactone (9). From butyl mercaptan, flash chromatography of the crude product (1:10 EtOAc-pentane) afforded Butyl 4-nitrophenyl thiocarbonate (62%) as a light yellow liquid. 1H NMR  δ 8.27 , 7.35 , 2.96 , 1.82–1.63 , 1.53–1.33 , 0.95 . 13C NMR  δ 169.77 (SCO), 155.65 (Ph), 145.33 (Ph), 125.29 , 122.06 , 31.46 (CH2), 31.20(CH2), 21.81 (CH2), 13.54 (CH3). ESI-HRMS (M + Na)+: 278.0457; found: 278.0462. Following general procedure B, flash chromatography of the crude product (1:3 EtOAc-pentane) afforded 9 (46%) as a white solid. IR (cm−1): 3315 (N-H), 1775 , 1643 , 1522 (-NHCO-). [α1H NMR  δ 6.12 , 4.56 , 4.50–4.37 , 4.27 , 2.91 , 2.80 , 2.22 , 1.61 , 1.39 , 0.91 . 13C NMR  δ 175.0 (CO-lactone), 168.9 (SCONH), 66.0 (OCH2-lactone), 50.6 (CH-NH), 32.2 (CH2), 30.4 (CH2-lactone), 29.8 (SCH2), 21.9 (CH2), 13.6 (CH3). ESI-HRMS (M + Na)+: 240.0664; found: 240.0653.N-(benzylthiocarbonyl)-l-homoserine lactone (10). From benzyl mercaptan, flash chromatography of the crude product (1:11 EtOAc-pentane) afforded Benzyl 4-nitrophenyl thiocarbonate (39%) as a white solid. 1H NMR  δ 8.27 , 7.43–7.27 , 4.20 . 13C NMR  δ 169.24 (SCO), 155.57 (Ph), 145.43 (Ph), 136.15 (Ph), 128.97 , 128.85 , 127.90 (Ph), 125.32 , 122.07 , 35.84 (SCH2Ph). ESI-HRMS (M + Na)+: 312.0301; found: 312.0302. Following general procedure B, flash chromatography of the crude product (1:3 EtOAc-pentane) afforded 10 (27%) as a white solid. IR (cm−1): 3321 (N-H), 1776 , 1661 , 1518 (-NHCO-). [α1H NMR  δ 7.35–7.26 , 7.26–7.21 , 6.12 , 4.55 , 4.44 , 4.25 , 4.17 , 2.79 , 2.21 . 13C NMR  δ 174.8 (CO-lactone), 168.1 (SCONH), 137.6 (Ph), 128.8 , 128.6 , 127.4 (Ph), 66.0 (OCH2-lactone), 50.7 (CH-NH), 34.3 (SCH2Ph), 30.4 (CH2-lactone). ESI-HRMS (M + Na)+: 274.0508; found: 274.0511.2SO4, concentrated, and the residue was purified by flash chromatography to give corresponding hydrazide compounds.(i) To a 25 mL flask, 4 mL of hydrazine (80% in water) and carboxylic esters (1 g) were added. The mixture was stirred at 70 °C for 45 min and then cooled to room temperature. The reaction mixture was then extracted with EtOAc (30 mL) and washed with brine (30 mL). The organic phase was dried over anhydrous Na(ii) To a solution of different hydrazides (1.0 eq) in dry DCM, DIEA (2.0 eq), α-Bromo-γ-butyrolactone (2.0 eq), and TBABr (0.3 eq) were added, respectively. The reaction mixture was stirred at 40 °C overnight. The solvent was evaporated. The residue was purified by flash chromatography to give the corresponding hydrazide AHL analogues.N’-(2-oxotetrahydrofuran-3-yl)butyrohydrazide (12). From methyl butanoic ester, Butyrohydrazide was obtained by flash chromatography of the crude product (95:5 EtOAc-MeOH) in 90% yield. NMR data were consistent with the literature [12 (82%). IR (cm−1): 3299 (N-H), 1774 , 1637 , 1538 (-NHCO-). 1H NMR  δ 7.60 , 4.40 , 4.21 , 4.12 , 3.91 , 2.48 , 2.29–2.08 , 1.68 , 0.94 . 13C NMR  δ 176.0 (CO), 173.2 (CO), 65.8 (OCH2-lactone), 57.9 (CH-NH), 36.3 (CH2CO), 27.5 (CH2-lactone), 18.9 (CH2CH3), 13.7 (CH3). ESI-HRMS (M + Na)+: 209.0897; found: 209.0900.terature . AccordiN’-(2-oxotetrahydrofuran-3-yl)hexanehydrazide (13). From methyl hexanoic ester Hexanehydrazide was obtained by flash chromatography of the crude product (95:5 EtOAc-MeOH) in 74% yield. NMR data were consistent with the literature [13 (48%). IR (cm−1): 3326 (N-H), 1762 , 1641 , 1525 (-NHCO-). 1H NMR  δ 7.56 , 4.41 , 4.22 , 3.93 , 3.84 , 2.49 , 2.33–2.09 , 1.62 , 1.41–1.18 , 0.97–0.78 . 13C NMR  δ 175.9 (CO), 173.3 (CO), 65.8 (OCH2-lactone), 57.9 (CH-NH), 34.5 (CH2CO), 31.4 (CH2CH2CH3), 27.5 (CH2-lactone), 25.1 (CH2CH2CH2CH3), 22.3 (CH2CH3), 13.9 (CH3). ESI-HRMS (M + H)+: 215.1390; found: 215.1394.terature . AccordiN’-(2-oxotetrahydrofuran-3-yl)-4-phenylbutanehydrazide (14). From ethyl 4-phenylbutanoic ester, 4-Phenylbutanehydrazide was obtained as a white solid (96%). NMR data were consistent with the literature [14 (63%). IR (cm−1): 3235 (N-H), 1769 , 1640 , 1539 (-NHCO-). 1H NMR  δ 7.57 , 7.32–7.26 , 7.22–7.12 , 4.39 , 4.20 , 3.89 , 3.78 , 2.65 , 2.47 , 2.27–2.13 , 2.06–1.88 . 13C NMR  δ 175.8 (CO), 172.8 (CO), 141.1 (Ph), 128.5 , 128.5 , 126.1 (Ph), 65.8 (OCH2-lactone), 57.9 (CH-NH), 35.1 (CH2CO), 33.7 (CH2Ph), 27.5 (CH2-lactone), 26.8 (CH2CH2Ph). ESI-HRMS (M + Na)+: 285.1210; found: 285.1203.terature . AccordiEscherichia coli strain NM522 with the sensor plasmid pSB401 was used to measure the induction of luminescence. Bacterial cultures were grown in Luria broth (50 mL) at 30 °C. After 18 h, this bacterial culture was diluted 10 times to be used in the assay using 96-well plates (100 µL per well). For agonistic activity, compounds were tested at increasing concentrations in DMSO . For antagonistic activity, compounds were tested in competition with OHHL (200 nM) at increasing concentrations in DMSO . This 200 nM concentration of OHHL is fully pertinent since concentrations as low as 10 or 50 nM are already valid protocols as reported in meaningful studies [The recombinant  studies ,28. BiolTridimensional structures of compounds were obtained from Chem3D Pro . The luxR model  complexe1) and benzyl (5) carbamate analogues . In other cases, para-nitrophenyl chloroformate was first reacted with various alcohols and thiols to produce activated para-nitrophenyl carbonates or thiocarbonates which were further involved in reaction with l-homoserine lactone or (d-homoserine lactone for compound 3) (route B) [2–4 and 6–10 were synthesized in two steps with overall yields ranging from 11% to 41%. For hydrazide analogues, the reaction of hydrazine with various esters gave the corresponding hydrazides which were used in a nucleophilic substitution reaction with α-bromo-γ-butyrolactone furnishing hydrazides AHLs analogues 12–14 in good yield over two steps.The synthesis of ethyl  . Thus, cl-homoserine lactone (OHHL) at 200 nM (antagonistic activity) [1) exhibited very weak agonistic activity by inducing at 200 µM 18% of the bioluminescence obtained with the natural ligand OHHL at a concentration of 200 nM.All compounds were evaluated for the ability to induce bioluminescence (agonistic activity) or to decrease bioluminescence in the presence of 3-oxo-hexanoyl-ctivity) . Hydrazi2, 4–7, and 9–11 were found to considerably decrease the bioluminescence  and hexyl (4) derivatives, or the benzyl and 4-bromobenzyl substituents.To determine IC of OHHL . Values 7 and 11 were identified as the most active compounds in the series with and IC50 value of 22–23 µM. However, no additive effect of the presence of the nitro group and of the sulfur atom in the thiocarbamate was observed, although LogP of the 4-nitrobenzyl thiocarbamate is higher than the 4-nitrobenzyl carbamate like for other compounds in the series. For these two compounds, the presence of the 4-nitro substituent is therefore the key point.The 4-nitrobenzyl derivatives 1 bearing a small ethyl substituent, a particularly high level of flexibility was observed, leading to a binding mode comparable with the natural ligand in terms of hydrogen bonds network, however with some differences in terms of distances. For longer chain lengths , the alkyl chain together with the supplementary oxygen atom of the carbamate function orientate the carbamoyl group towards Tyr70 leading to hydrophobic interactions. A supplementary hydrogen bond with this amino acid is also observed , and the hydrazide 12 within the binding site of the LuxR model.Vibrio fisheri system. Both agonistic and antagonistic activities were found depending on chain length and substitution. The binding mode within the LuxR binding site unveils a new hydrogen bond between the supplementary oxygen or sulfur atoms of all compounds with Tyr70, an important conserved residue in the LuxR family. The most active compounds are the p-nitro benzyl derivatives that exhibit additional interactions with Lys178. This study showed the ability of the carbamate/thiocarbamate functions to mimic the amide group of AHLs for fine-tuning QS modulation.A novel family of carbamate/thiocarbamate analogues of AHLs was studied as quorum sensing modulators on the H NMR and 13C NMR data are available in


Plasmodium falciparum resistance to widely-used antimalarials, such as chloroquine (CQ). Current antimalarial drugs are aromatic heterocyclic derivatives, most often containing a basic component with an added alkyl chain in their chemical structure. While these drugs are effective, they have many side effects. This paper presents the synthesis and preliminary physicochemical characterisation of novel bioinspired imidazolidinedione derivatives, where the imidazolidinedione core was linked via the alkylene chain and the basic piperazine component to the bicyclic system. These compounds were tested against the asexual stages of two strains of P. falciparum—the chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains. In parallel, in vitro cytotoxicity was investigated on a human keratinocyte cell line, as well as their hemolytic activity. The results demonstrated that the antiplasmodial effects were stronger against the W2 strain (IC50 between 2424.15–5648.07 ng/mL (4.98–11.95 µM)), compared to the D10 strain (6202.00–9659.70 ng/mL (12.75–19.85 µM)). These molecules were also non-hemolytic to human erythrocytes at a concentration active towards the parasite, but with low toxicity to mammalian cell line. The synthetized derivatives, possessing enhanced antimalarial activity against the CQ-resistant strain of P. falciparum, appear to be interesting antimalarial drug candidates.Malaria is an enormous threat to public health, due to the emergence of  Anopheles mosquitoes. Malarial parasites belong to the Plasmodium genus, which include different species, with two of these species posing the greatest threat, namely, Plasmodium falciparum and Plasmodium vivax. According to the World Health Organization (WHO), in 2018, P. falciparum accounted for 99.7% of the estimated malaria cases in the African Region, 50% of cases in the South-East Asia Region, 71% of cases in the Eastern Mediterranean and 65% in the Western Pacific . In . In P. fP. berghei  . Sys. SysP. blciparum  7]. Apl. AplP. bng/mL 1.6 μM [9]. 1.69 μM) ,11.The hydantoin  core is an important pharmacophore that exhibits many biological properties. Diverse hydantoin derivatives are well-known as anticonvulsive , antiarrhythmic (azimilide), antimicrobial agents (nitrofurantoin), skeletal muscle relaxants (dantrolene), and nonsteroidal antiandrogen (nilutamide) drugs. Moreover, hydantoin derivatives  have been further explored with the view of their potential medicinal and pharmaceutical properties . HydantoZ-axinohydantoin and debromo-Z-axinohydantoin from the Sponge Stylotella aurantium, which are potent inhibitors of protein kinase C imidazole was substituted at the piperazine moiety Mendoza et al. imidazole, 1--2′,3′-dihydrospiro-2,5-dione (1), -1′,3′-dihydrospiro-2,5-dione (2), and their detailed analytical data were described previously -2,5-dione (3)(fR = 0.76 (S1); HPLC: Rt = 1.613; ESI–S [M + H]+ calcd for 365.08, found: 365.12, 367.12; 1H NMR  δ ppm 1.43–1.56  1.70  1.79–2.03  2.21–2.38  2.87  3.40  3.57  5.90  7.04  7.12–7.25 .Creamy powder. Yield: 72%; TLC: R,S)-1-(5-bromopentyl)-2′,3′-dihydrospiro-2,5-dione (4)(fR = 0.73 (S1); HPLC: Rt = 1.529; ESI-MS [M + H]+ calcd for 351.06, found: 351.16, 353.16; 1H NMR  δ ppm 1.40–1.53  1.63–1.72  1.83–1.94  2.19–2.31  2.65–2.76  2.99–3.11  3.18–3.32  3.39  3.55  5.89  7.12  7.20–7.27  7.28–7.33 .Creamy powder. Yield: 57%; TLC: The appropriate bromoalkyl derivatives of spirohydantoin (0.3 mmol), and triethylamine  were dissolved in 4 mL of acetonitrile and arylpiperazine (0.33 mmol) was added. The reaction mixture was heated to 100 °C in a microwave synthesizer . After 1 h of heating, the reaction mixture was concentrated under vacuum. The final, racemic compounds obtained were further purified by column chromatography using the following eluent systems: (S1) methylene chloride/methanol (9:0.7), and (S2) methylene chloride/methanol (9:1.2).R,S)-1-(5-(4-(1H-Benzo[d]imidazol-2-yl)piperazin-1-yl)pentyl)-3′,4′-dihydro-2′H-spiro-2,5-dione (5)(fR = 0.29 (S2); HPLC: tR = 0.948; ESI-MS [M + H]+ calcd for 487.27, found: 487.15; 1H NMR  δ ppm 1.29–1.34  1.45–1.52  1.63–1.70  1.73–1.81  1.90–1.98  2.18–2.26  2.28–2.32  2.46  2.75–2.88  3.51–3.56  6.29  6.97–7.04  7.08–7.16  7.16–7.20 . 13C NMR  δ ppm 19.13 (s) 24.53 (s) 26.07 (s) 27.98 (s) 28.86 (s) 34.28 (s) 38.60 (s) 46.55 (s) 52.34 (s) 58.29 (s) 62.78 (s) 112.50 (s.) 121.14 (s) 126.43 (s) 126.95 (s) 128.78 (s) 129.91 (s) 133.07 (s) 138.24 (s) 155.39 (s) 156.92 (s) 176.43 (s).Light-yellow solid. Yield: 71%; TLC: R,S)-1-(5-(4-(1H-Benzo[d]imidazol-2-yl)piperazin-1-yl)pentyl)-2′,3′-dihydrospiro-2,5-dione (6)(fR = 0.27 (S2); HPLC: tR = 0.928; ESI-MS [M + H]+ calcd for 473.26, found: 473.19; 1H NMR  δ ppm 1.29–1.37  1.48–1.56  1.63–1.71  2.31–2.37  2.51  3.03  3.50–3.64  5.87–5.98  7.02–7.06  7.20  7.30 . 13C NMR  δ ppm 24.46 (s) 25.85 (s) 27.90 (s) 30.15 (s) 36.82 (s) 38.39 (s) 45.90 (s) 52.31 (s) 58.38 (s) 71.23 (s) 112.29 (s) 120.76 (s) 122.54 (s) 125.43 (s) 127.29 (s) 129.52 (s) 140.07 (s) 144.17 (s) 155.81 (s) 157.26 (s) 176.33 (s).Light-yellow solid. Yield: 65%; TLC: R,S)-1-piperazin-1-yl)pentyl)-3′,4′-dihydro-2′H-spiro-2,5-dione (7)(fR = 0.24 (S1); HPLC: tR = 1.002; ESI-MS [M + H]+ calcd for 487.29, found: 487.29; 1H NMR  δ ppm 1.23–1.40  1.57  1.69  1.76–1.83  1.93–2.00  2.22–2.29  2.35–2.40  2.60  2.83–2.93  3.03–3.09  3.19  3.56  5.71  7.03  7.10–7.13  7.14–7.18  7.20–7.24 . 13C NMR  δ ppm 19.17 (s) 24.72 (s) 26.12 (s) 28.08 (s) 28.89 (s) 34.31 (s) 37.03 (s) 38.67 (s) 51.18 (s) 52.93 (s) 58.38 (s) 62.72  66.98 (s) 124.47  126.54 (s) 127.00 (s) 128.78 (s) 129.89 (s) 133.11 (s) 138.22 (s) 141.49 (s) 156.86 (s) 176.26 (s).Light-yellow solid. Yield: 61%; TLC: R,S)-1-piperazin-1-yl)pentyl)-2′,3′-dihydrospiro-2,5-dione (8)(fR = 0.24 (S1); HPLC: Rt = 0.937; ESI-MS [M + H]+ calcd for 473.26, found: 473.19; 1H NMR  δ ppm 1.32  1.52  1.66  2.19–2.26  2.30–2.35  2.36–2.77  2.87  2.98–3.08  3.12–3.19  3.24  3.52  5.93  7.08–7.13  7.13–7.18  7.20–7.24  7.28–7.32 . 13C NMR  δ ppm 24.55 (s) 25.49 (s) 27.97 (s) 36.82 (s) 38.61 (s) 44.44 (s) 50.42 (s) 52.47 (s) 58.07 (s) 66.72 (s) 68.43 (s) 124.49 (s) 124.73 (s) 126.00–127.91 (m) 127.15–128.67 (m) 139.00 (s) 141.14 (s) 156.39 (s) 176.01 (s).Light-yellow solid. Yield: 59%; TLC: Ka) and the decimal logarithm distribution coefficient (log D) were calculated using the MarvinSketch software .The negative logarithm of the acid dissociation constant . Drugs were placed in 96-well flat-bottomed microplates starting from the concentration of 20 µg/mL and 8 serial two-fold dilutions were made. Asynchronous cultures with parasitaemia of 1–1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 h at 37 °C. Parasite growth was determined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of the method of Makler in control and drug-treated cultures imidazole or 1-piperazine, whose analytical data were in line with previously described procedures imidazole-2-yl-piperazine moieties were necessary for the most promising antiplasmodial activities.The chloroquine sensitive (D10) and chloroquine resistant (W2) strains of  as well . AnotherP. falciparum strains have been described almost everywhere.One of the greatest public health problems in the control of malaria is the the spread of resistance to antimalarial drugs used on a large scale . Therefo50 values on the human cells and that on the P. falciparum strains, was calculated and reported in 5), in relation to the CQ-resistant strain. Again, (5) had the highest selectivity index which, together with its low toxicity, makes this agent the most promising compound amongst the studied series.As the studied agents have shown encouraging antiplasmodial activities, we further assessed their cytotoxic effects using the MTT assay. Additionally, for each compound, the selectivity index (SI), namely the ratio between the IC5–8) for 30 min, at a concentration of 20 µg/mL. It was shown that compound (5) caused minimal hemolysis, on the level of 5.6%, at a concentration corresponding to 3.2-fold (D10) and 8.2-fold (W2) of the IC50 of the respective Plasmodium strains. The other derivatives (6–8) had a hemolysis level of less than 2.7% at the same concentration. In summary, we can conclude that compounds (5–8) are not toxic to human erythrocytes at a concentration at which they effectively inhibit the growth of P. falciparum. This is a very valuable observation, especially considering that some artemisinin derivatives can cause hemolysis [The synthesized compounds were subsequently evaluated for their effects on human erythrocytes. In order to estimate this, fresh erythrocytes were treated with compounds (emolysis . Moreove5) turned out to be the best hit compound in the series, due to the highest activity against P. falciparum strains, especially the CQ-resistant strain, and low toxicity to mammalian cells. Due to its low solubility in water, further work will be carried out to develop an appropriate formulation that will improve its bioactivity and bioavailability.In this work, we synthesized a series of bioinspired hydantoin derivatives and further studied their antimalarial activities, as well as their cytotoxicity and hemolytic effects. Compound (


Dactylospongia elegans, yielded five new γ-oxygenated butenolide sesterterpene derivatives, dactylospenes A–E (1–5), as well as two known biosynthetically related compounds, luffariellolide (6) and furospinosulin B (7). The structures of these compounds were elucidated on the basis of their spectroscopic data, experimental and calculated electronic circular dichroism (ECD) analysis, as well as comparison of the NMR data with those of known analogs. These metabolites are the first γ-oxygenated butenolide sesterterpenes to be reported from this genus. These compounds were evaluated in antimicrobial, anti-inflammatory, and cytotoxic assays. Only compounds 1, 3, and 6 exhibited moderate cytotoxicity against DU145, SW1990, Huh7, and PANC-1 cancer cell lines with IC50 values in the range of 2.11–13.35 μM. Furthermore, compound 2, without cytotoxicity, exhibited significant inhibitory effects (inhibitory rate 77.5%) on nitric oxide production induced by lipopolysaccharide at 10 μM.Chemical investigation on a marine sponge,  Dactylospongia +, requiring seven degrees of unsaturation. The IR spectrum showed absorption bands for hydroxy (3342 cm−1) and ester carbonyl (1760 cm−1) groups. Comparison of the 1H and 13C NMR data of 1 -5-hydroxy-4-methylfuran-2(5H)-one (4S-8)  . Consequ2) was also purified as a light red oil and exhibited a pseudomolecular ion [M + NH4]+ peak in the positive HRESIMS at m/z 418.3308, consistent with the molecular formula C26H40O3, which was supported by the 1H and 13C NMR data. The molecular formula indicated seven degrees of unsaturation. The IR spectrum of 2 showed strong absorption at 3465 and 1766 cm−1, assignable to hydroxy and ester carbonyl functionalities, respectively. The 13C NMR and DEPT spectra , three sp2 quaternary carbons , three sp2 methine carbons , one sp3 oxymethine (δC 103.9), one methoxy (δC 56.1), two sp3 quaternary carbons (δC 42.0 and 33.2), two methine carbons (δC 43.9 and 41.8), eight methylene carbons , and five methyl carbons , which accounted for four degrees of unsaturation. The remaining three degrees of unsaturation were caused by the presence of three rings in the molecule.Dactylospene B , C-5−C-6−C-7−C-8−C-18 (b), C-11−C-12 (c), and C-20−C-21−C-22 (d), which were accomplished with the assistance of the HMBC experiment . Moreover, HMBC correlations from H2-3 in fragment a, and H-5 and H2-6 in fragment b to the same carbon C-4, as well as HMBC correlations from H3-17 to C-3, C-4, and C-5, allowed the linkage of fragments a and b via C-4 and the assignment of the methyl group CH3-17 at C-4. The dimethylhomoallyl group was attached to C-4 based on the HMBC correlations from H2-20 to C-3, C-4, and C-5. Further HMBC correlations from H3-18 to C-9, and from H3-19 to C-8, C-9, and C-10, revealed the connectivity of C-8 and C-10 through C-9 and placed the methyl group H3-19 at C-9 as well. Another two groups of HMBC cross-peaks from H-1 to C-5 and C-9, from H2-2, H-5 and H-6b to C-10, supported the linkage of C-1 and C-5 via C-10. Moreover, a suite of resonances at δC 170.3 (C-15), 168.6 (C-13), 116.9 (CH-14), 103.9 (CH-16), and 56.1 (16-OCH3), could be assigned to an α,β-unsaturated-γ-methoxy-γ-lactone moiety, which was further supported by HMBC correlations from H-16 to C-13, C-14, and C-15, from H-14 to C-13, C-15, and C-16, and from 16-OCH3 to C-16. This moiety was further linked to C-9 through fragment C-11–C-13, based on the HMBC correlations from H-14 to C-12 and from H-11 to C-9, C-10, and C-13. Thus, the planar structure of 2 was determined as depicted.Interpretation of the 2D NMR data, including COSY, HMQC, and HMBC spectra, led to the construction of the planar structure of periment . HMBC co2. The NOESY cross-peaks of H-5/H-20a and H-5/H3-18 indicated the cofacial orientation of these protons and methyl group, whereas the NOESY correlations of H-8/H3-19 indicated that these protons were oriented in the other direction. Therefore, we established the relative stereochemistry of 2 as 4R*,5S*,8R*,9R*.A NOESY experiment was also performed to determine the relative configuration of 3) was also deduced as C26H40O3 by HRESIMS. Analysis of the 1D and 2D NMR data  of 2 and 3 indicated that these two compounds are a pair of diastereomers.The molecular formula for dactylospene C  was isolated as a yellow oil and assigned the molecular formula of C27H44O4, based on HRESIMS data for the [M + NH4]+ ion at m/z 450.3581, which is consistent with six degrees of unsaturation. The 1H and 13C NMR signal patterns , three sp3 methylenes , one methoxy (δC 49.1), and one sp3 oxygenated quaternary carbon (δC 74.6) were deduced. The attachment at C-4 was a 2-methoxy-2-methylpentane unit, instead of the dimethylhomoallyl moiety in 2 and 3, supported by the COSY correlations of H-20a/H-21a and H-21b/H2-22, in addition to the HMBC correlations from H-20a to C-3, from H3-24 and H3-25 to C-22 and C-23, and from 23-OCH3 to C-23. Moreover, NOESY correlations of H-5/H-20a, H-5/H3-18, and H-8/H3-19 revealed the relative configurations of 4, which were the same as compounds 2 and 3. Finally, the similarity of their CD spectra between 2 and 4 suggested that compound 4 shared the same absolute configurations as those of 2 (Dactylospene D (patterns  suggesteose of 2 .5) was also obtained as a light yellow oil. The molecular formula of C27H44O4 was deduced from its HRESIMS data (m/z 450.3578 [M + NH4]+). Compound 5 showed nearly the same chemical shifts as those of compound 4. Correlations from the 2D NMR spectra confirmed the same planar structure between 5 and 4 and the same configuration of epi-halimane core in 5 and 4. A comparison with CD spectra and specific rotation values obtained for 5 and 3 unambiguously assigned absolute configuration as 4R,5S,8R,9R,16R.Dactylospene E  and furospinosulin B (7), were also isolated as metabolites of D. elegans. These compounds were identified by comparing their spectral data with the spectroscopic data reported in the corresponding literature + .Dactylospene A (2): light red oil; c 0.20, MeOH); UV (MeOH) (log ε) λmax 201 (4.47), 258 (4.02); IR (KBr) νmax 3465,2959, 2925, 2858, 1795, 1766, 1652, 1454, 1375, 1309, 1120, 959, 897, 860 cm−1; CD (MeCN) (∆ε) 202 (+4.1), 216 (+8.9), 254 (−3.8); 1H and 13C NMR data, see m/z 418.3308 [M + NH4]+ .Dactylospene B (3): light red oil; ε) λmax 213 ; IR (KBr) νmax 3466, 2959, 2926, 2858, 1795, 1765, 1651, 1454, 1376, 1310, 1203, 1119, 955, 897, 863, 804, 736, 647 cm−1; CD (MeCN) (∆ε) 201 (+31.6), 221 (+4.2), 245 (+20.1); 1H and 13C NMR data, see m/z 418.3314 [M + NH4]+ .Dactylospene C (4): light yellow oil; c 0.12, MeOH); UV (MeOH) (log ε) λmax 198 (4.36); IR (KBr) νmax 3360, 2960, 2924, 2853, 1795, 1766, 1738, 1462, 1374, 1261, 1093, 1021, 800, 700 cm–1; CD (MeCN) (∆ε) 206 (+2.5), 217 (+5.9), 251 (−3.9); 1H and 13C NMR data, see m/z 450.3581 [M + NH4]+ .Dactylospene E (5): light yellow oil; c 0.10, MeOH); UV (MeOH) (log ε) λmax 206 (4.58); IR (KBr) νmax 2926, 2860, 1795, 1767, 1650, 1460, 1373, 1309, 1260, 1202, 1118, 955, 896, 861, 804, 735 cm−1; CD (MeCN) (∆ε) 200 (+43.1), 223 (+0.3), 246 (+11.2); 1H and 13C NMR data, see m/z 450.3595 [M + NH4]+ .Dactylospene D  using Merck Molecular Force Field (MMFF) applying a 21 kJ/mol energy window. Subsequently, the conformers with a Boltzmann population of over 1% were re-optimized at the B3LYP/6-31G(d) level with Gaussian 09 by employing the polarizable continuum model (PCM) in MeCN, which generated two conformers for model 8 and four conformers for model 9. The theoretical calculations of ECD for simplified models 8 and 9 were calculated at the CAM-B3LYP/TZVP (PCM/MeCN) level. The ECD spectra were generated by the program SpecDis 1.6 applying a Gaussian band shape with the width of 0.35 eV, from dipole-length rotational strengths [Conformational searches for simplified models trengths .1–7 against two strains of hospital-acquired, methicillin-resistant Staphylococcus aureus (MRSA H0556 and MRSAH0117) were evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines [90 2 μg/mL), while methicillin was used as the negative control (MIC90 128 μg/mL). The cytotoxic activity of compounds 1–7 against DU145, SW1990, Huh7, and PANC-1 cell lines was performed by the Cell Counting Kit-8 (CCK-8) assay, as described before [1–5 was measured using the Griess reagent following the reported method [1–5 were evaluated by the CCK-8 assay, as above.The antimicrobial activities of compounds idelines ,17, and d before . Each caD. elegans, led to the isolation and structure elucidation of a series of γ-hydroxybutenolide sesterterpene derivatives, dactylospenes A–E (1–5), together with two known biosynthetically related compounds 6–7. From a biosynthetic perspective, compounds 2–5 may be generated from the possible precursor 1 by cyclization and methoxy-substitution reactions. These compounds were evaluated in antibacterial and cytotoxic activities. Only compounds 1, 4, and 6 exhibited moderate cytotoxicity against DU145, SW1990, Huh7, and PANC-1 cancer cell lines with IC50 values ranging from 2.11 to 13.35 μm. Compound 2 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide, with an inhibitory rate of 77.5%. Interestingly, the anti-inflammatory activity of compound 2 was not achieved through cytotoxic activity, indicating that compound 2 deserves further study for its therapeutic potential to develop new anti-inflammatory drugs.Investigation on the secondary metabolites from the marine sponge,


E)-3-bromobenzylidene)piperidin-4-one 3 displayed dual p300/EZH2 inhibition joined to cancer-selective cell death in a panel of tumor cells and in in vivo xenograft models, we prepared a series of bis((E)-2-bromobenzylidene) cyclic compounds 4a–n to test in biochemical  and cellular  assays. The majority of 4a–n exhibited potent dual p300 and CARM1 inhibition, sometimes reaching the submicromolar level, and induction of apoptosis mainly in the tested leukemia cell lines. The most effective compounds in both enzyme and cellular assays carried a 4-piperidone moiety and a methyl (4d), benzyl (4e), or acyl (4k–m) substituent at N1 position. Elongation of the benzyl portion to 2-phenylethyl (4f) and 3-phenylpropyl (4g) decreased the potency of compounds at both the enzymatic and cellular levels, but the activity was promptly restored by introduction of a ketone group into the phenylalkyl substituent (4h–j). Western blot analyses performed in NB4 and MCF-7 cells on selected compounds confirmed their inhibition of p300 and CARM1 through decrease of the levels of acetyl-H3 and acetyl-H4, marks for p300 inhibition, and of H3R17me2, mark for CARM1 inhibition.Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us as epigenetic multiple ligands, but different substitutions at the two wings provided analogues with selective inhibition. Since the 1-benzyl-3,5-bis(( Polypharmacology is a new emerging approach for chemotherapy, particularly useful when the single-targeted therapy tailored for a specific disease remains without or loses its effect . This caTranscriptional dysregulations and epigenetic dysfunctions are common and recurring themes in cancer. Alterations in histone acetylation and/or methylation or in DNA methylation modify the transcription regulation leading to aberrant events characterized by either gene silencing or activation ,6,7,8,9.1 , U937 AML, MCF-7 breast cancer and SH-SY5Y neuroblastoma cells, to determine their effects in cell cycle phases and apoptosis (sub-G1 peaks) induction. Western blot analyses of the levels of acetyl-H3, acetyl-H4, acetyl-H3K9/14, and acetyl-α-tubulin, marks of p300 activity in cells, as well as of those of H3K27me3 (trimethylated histone 3 lysine 27) and H3R17me2 (dimethylated histone 3 arginine 17), histone marks of cellular activity of EZH2, and CARM1, respectively, have been performed for selected derivatives in NB4 and MCF-7 cells to correlate the observed biological effects with the modulation of the cited epigenetic targets.The considerable anticancer activity observed with E)-2-bromobenzylidene)cyclohexan-1-one 4a  in dry dichloromethane (DCM) (4j) for 2 h at room temperature  in dry DCM for 1 h at room temperature  and anacardic acid (for PCAF) were added as reference compounds. To determine the activity against SIRT1 and -2, 4a–n were tested starting from 200 μM solutions using a modified Fluor de Lys fluorescence-based assay with amino acids 379–382 (Arg-His-Lys-Lys(Ac)) of human p53 conjugated with aminomethylcoumarin as substrate and NAD+ as co-substrate. The SIRT1 inhibitor selisistat was used as internal reference compound. Furthermore, 4a–n were tested against a human five-membered Polycomb repressive complex 2 (PRC2) containing EZH2, embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), RbAp48, and adipocyte enhancer-binding protein (AEBP2), to assess their capability to inhibit the catalytic unit EZH2. According to a 2-fold serial dilution pattern, 4a–n were tested in a 10-dose IC50 evaluation in duplicate starting from 400 μM as initial concentration. The core histone and 3H-SAM were used as substrate and co-substrate, respectively, whereas S-adenosyl-l-homocysteine (SAH) was used as reference compound. Finally, 4a–n were screened against CARM1 in agreement to the experimental protocol already reported above for p300 and PCAF, but using histone H3 and 3H-SAM as substrate and co-substrate, respectively, and SAH as the internal reference. The newly synthesized compounds 4a–n demonstrated to consistently inhibit the HAT p300, with some compounds displaying a submicromolar activity. Differently, 4a–n were completely ineffective against PCAF as well as SIRT1/2  significantly decreased the acetyltransferase inhibition, that was promptly restored when a carbonyl function was added to the N1-alkylphenyl chain . The replacement of the alkylphenyl group at the N1 4-piperidone position with an acyl moiety, such as in 4k–n, gave a gain of potency against p300 in particular with 4l and 4m .When tested toward histone (de)acetylation enzymes, the majority of  SIRT1/2 . In part4a–n generally displayed low, if any, inhibition  and partially restored by the introduction of the ketone function in the alkylaryl chain.Towards PRC2/EZH2, hibition . Only N150 values at low micromolar/submicromolar levels (4d and 4e), while 2-phenylethyl (4f) and 3-phenylpropyl (4g) substitution were less effective. Single-digit micromolar IC50 values were shown by compounds bearing a carbonyl group (ketone or amide function) in the N1-side chain, while 4l and 4m were the most potent .Against CARM1, the N1-substituted 4-piperidone compounds displayed the highest activity, reaching in some cases ICr levels . The ins4a–n were tested at 5 μM for 30 h in NB4 PML and U937 AML cells among hematological cancers, and in MCF-7 breast cancer and SH-SY5Y neuroblastoma cells among solid cancers, to determine their effects in cell cycle and apoptosis induction. In NB4 cells, the majority of compounds  gave a block in G2/M phase, while in U937 cells only 4f, 4l, and 4m displayed the same effect, and 4g and 4n arrested the cell cycle at the G1/S phase  or in the S phase   C. SH-SY5ectively D.4l and 4m displayed the highest induction  (4l) and 3-phenylpropionyl (4m) moiety at the N1 4-piperidone position to elicit this cellular effect. It is noteworthy that 4l and 4m are also the most potent among the described dual p300/CARM1 inhibitors, reaching IC50 values at submicromolar level in both the enzymatic assays  A, demonsc assays . In the over 30% B, the otG1 peaks C, and inn of 17% D.4l and 4m, were tested in mesenchymal progenitor MePR2B cells, an immortalized non-cancer cell system +, Found: 524.02. Calculated for M.W. 523.27, %: C 59.68; H 4.05; N 2.68; Br 30.54. Found, %: C 59.44; H 3.96; N 2.85; Br 30.68.3,5-Bis((4-one 4c  (0.46 mmE)-2-bromobenzylidene)-1-phenethylpiperidin-4-one (4f). M.p.: 105–107 °C (cyclohexane). Yield: 65%. 1H-NMR (CDCl3) δ ppm: 7.98 , 7.69 , 7.36 , 7.28–7.27 , 7.25–7.20 , 7.16 , 7.09 , 3.76 , 2.75–2.73 , 2.70–2.65 . 13C-NMR (CDCl3) δ ppm: 182.6, 146.4 (2C), 140.0 (2C), 136.9, 135.2 (2C), 133.4 (2C), 129.9 (2C), 129.3 (2C), 128.9 (2C), 128.5 (2C), 127.0 (2C), 126.0, 125.1 (2C), 60.2, 52.4 (2C), 32.8. MS (ESI-MS): Calculated: 538.02 for C27H24Br2NO [M + H]+, Found: 538.03. Calculated for M.W. 537.30, %: C 60.36; H 4.31; N 2.61; Br 29.74. Found, %: C 60.59; H 4.39; N 2.42; Br 29.51.3,5-Bis((E)-2-bromobenzylidene)-1-(3-phenylpropyl)piperidin-4-one (4g). M.p.: 93–95 °C (cyclohexane). Yield: 67%. 1H-NMR (CDCl3) δ ppm: 7.97 , 7.68 , 7.36 , 7.29–7.26 , 7.25–7.20 , 7.16 , 7.06 , 3.68 , 2.56 , 2.49 , 1.65 . 13C-NMR (CDCl3) δ ppm: 182.5, 146.7 (2C), 140.3 (2C), 137.4, 135.1 (2C), 133.9 (2C), 129.5 (2C), 129.4 (2C), 128.9 (2C), 128.6 (2C), 127.7 (2C), 126.4, 125.0 (2C), 58.2, 53.6 (2C), 32.0, 29.8. MS (ESI-MS): Calculated: 552.04 for C28H26Br2NO [M + H]+, Found: 552.07. Calculated for M.W. 551.32, %: C 61.00; H 4.57; N 2.54; Br 28.99. Found, %: C 60.85; H 4.49; N 2.62; Br 29.11.3,5-Bis((E)-2-bromobenzylidene)-1-(2-oxo-2-phenylethyl)piperidin-4-one (4h). M.p.: 109–111 °C (cyclohexane). Yield: 78%. 1H-NMR (CDCl3) δ ppm: 7.99 , 7.88 , 7.65 , 7.57 , 7.44 , 7.34 , 7.24–7.21 , 4.01 , 3.98 . 13C-NMR (DMSO-d6) δ ppm: 197.3, 186.6, 136.0 (2C), 135.2 (2C), 134.7, 134.7 (2C), 133.8, 133.4 (2C), 131.4 (2C), 131.3 (2C), 129.1 (2C), 128.3 (2C), 128.3 (2C), 125.0 (2C), 62.2, 53.9 (2C). MS (ESI-MS): Calculated: 551.99 for C27H21Br2NO2 [M + H]+, Found: 552.00. Calculated for M.W. 551.28, %: C 58.83; H 3.84; N 2.54; Br 28.99. Found, %: C 58.99; H 3.91; N 2.32; Br 28.79.3,5-Bis((E)-2-bromobenzylidene)-1-(3-oxo-3-phenylpropyl)piperidin-4-one (4i). M.p.: 95–97 °C (cyclohexane). Yield: 69%. 7.89 , 7.75 , 7.56–7.51 , 7.44 , 7.32–7.31 , 7.29 , 3.88 , 3.13 , 2.60 . 13C-NMR (CDCl3) δ ppm: 196.4, 182.1, 144.8 (2C), 140.5 (2C), 137.2, 135.5 (2C), 134.0, 132.3 (2C), 130.5 (2C), 129.3 (2C), 128.5 (2C), 127.3 (2C), 127.0 (2C), 125.0 (2C), 59.3, 52.4 (2C), 40.7. MS (ESI-MS): Calculated: 566.01 for C28H23Br2NO2 [M + H]+, Found: 566.02. Calculated for M.W. 565.31, %: C 59.49; H 4.10; N 2.48; Br 28.27. Found, %: C 59.27; H 3.96; N 2.61; Br 28.45.3,5-Bis((E)-2-bromobenzylidene)piperidin-4-one (4c) +, Found: 580.01. Calculated for M.W. 579.33, %: C 60.12; H 4.35; N 2.42; Br 27.58. Found, %: C 60.32; H 4.45; N 2.29; Br 27.34.To a stirring solution of the 3,5-bis((one (4c)   in anhydrous dichloromethane (5 mL). The resulting mixture is then allowed to stir at room temperature. After 1 h the reaction was quenched with water (50 mL) and extracted with dichloromethane (3 × 30 mL). The collected organic layers were washed with 2N HCl (3 × 30 mL) and then with a saturated solution of NaCl (30 mL). The organic phase was dried with anhydrous Na2SO4, filtered and evaporated under vacuum to afford a crude that was then purified on silica gel (AcOEt/n-hexane 1:3) to afford the desired product . M.p.: 98–100 °C (cyclohexane). 1H-NMR (CDCl3) δ ppm: 7.95 , 7.71 , 7.54 , 7.44–7.40 , 7.39–7.31 , 7.20 , 6.38 , 4.80–4.78 . 13C-NMR (CDCl3) δ ppm: 185.6, 165.3, 145.5 (2C), 143.5, 141.9 (2C), 137.0, 135.1 (2C), 133.6 (2C), 129.2 (2C), 128.7 (2C), 128.3, 127.9 (2C), 127.6 (2C), 126.0 (2C), 125.6 (2C), 117.1, 48.6 (2C). MS (ESI-MS): Calculated: 563.99 for C28H21Br2NO2 [M + H]+, Found: 564.01. Calculated for M.W. 563.29; %: C 59.70; H 3.76; N 2.49; Br 28.37. Found, %: C 59.49; H 3.65; N 2.70; Br 28.54.Cinnamoyl chloride  was slowly added at 0 °C to a stirring solution of 4c  (0.45 mmE)-2-bromobenzylidene)piperidin-4-one (4k). M.p.: 124–126 °C (cyclohexane/toluene). Yield: 76%. 1H-NMR (CDCl3) δ ppm: 8.01 , 7.69 , 7.23–7.21 , 7.14–7.11 , 4.75–4.45 . 13C-NMR (CDCl3) δ ppm: 182.5, 169.1, 144.8 (2C), 139.8 (2C), 136.1, 133.8 (2C), 132.1 (2C), 128.9, 128.5 (2C), 128.3 (2C), 127.8 (2C), 126.1 (2C), 125.8 (2C), 124.7 (2C), 50.1 (2C). MS (ESI-MS): Calculated: 537.98 for C26H19Br2NO2 [M + H]+, Found: 538.00. Calculated for M.W. 537.25, %: C 58.13; H 3.56; N 2.61; Br 29.75. Found, %: C 58.32; H 3.68; N 2.49; Br 29.67.1-Benzoyl-3,5-bis((E)-2-bromobenzylidene)-1-(2-phenylacetyl)piperidin-4-one (4l). M.p.: 126–128 °C (cyclohexane/benzene). Yield: 67%. 1H-NMR (CDCl3) δ ppm: 7.95 , 7.76 , 7.68 , 7.45–7.36 , 7.26–7.24 , 7.20–7.18 , 7.08 , 6.84–6.82 , 4.77 , 4.45 , 3.50 . 13C-NMR (DMSO-d6) δ ppm: 186.1, 169.8, 135.2 (2C), 134.4, 134.0, 133.6 (4C), 131.8 (2C), 131.4 (2C), 128.8 (5C), 128.4 (2C), 127.0 (2C), 125.5 (1C), 47.8, 44.5, 42.8. MS (ESI-MS): Calculated: 551.99 for C27H21Br2NO2 [M + H]+, Found: 552.00. Calculated for M.W. 551.28, %: C 58.83; H 3.84; N 2.54; Br 28.99. Found, %: C 59.04; H 3.97; N 2.35; Br 28.75.3,5-Bis((E)-2-bromobenzylidene)-1-(3-phenylpropanoyl)piperidin-4-one (4m). M.p.: 95–97 °C (cyclohexane). Yield: 69%. 1H-NMR (CDCl3) δ ppm: 7.94 , 7.68 , 7.45–7.39 , 7.25–7.21 , 7.19–7.14 , 6.98 , 4.76 , 4.41 , 2.83 , 2.39 . 13C-NMR (CDCl3) δ ppm: 182.3, 168.4, 144.3 (2C), 142.1, 140.1 (2C), 134.1 (2C), 133.0 (2C), 128.6 (2C), 128.3 (2C), 127.3, 128.0 (2C), 126.6 (2C), 125.0 (2C), 124.7 (2C), 48.5 (2C), 35.7, 32.9. MS (ESI-MS): Calculated: 566.01 for C28H23Br2NO2 [M + H]+, Found: 566.01. Calculated for M.W. 565.31, %: C 59.49; H 4.10; N 2.48; Br 28.27. Found, %: C 59.29; H 3.97; N 2.72; Br 28.45.3,5-Bis((3H]-Acetyl Coenzyme A (3 µM) as an acetyl donor in reaction buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO) for 1 h at 30 °C in the presence or absence of a dose titration of the compounds. The reaction mixture was delivered to filter-paper for detection. IC50 values were analyzed using Excel and GraphPad Prism 6.0 software . The experiments for p300 were performed in triplicate.The effect of tested derivatives on the catalytic activity of p300 and PCAF was determined in a HotSpot HAT activity assay by Reaction Biology Corporation  according to the company’s standard operating procedure. In brief, the recombinant catalytic domains of PCAF  or p300  were incubated with histone H3 as substrate (5 µM) and [Acetyl-E. coli. Compounds were tested using a modified Fluor de Lys fluorescence-based assay kit based on the method described in the BioMol product sheet . The assay procedure has two steps: in the first part the SIRT1/2 substrate, an acetylated Lys side chain comprising amino acids 379-382 (Arg-His-Lys-Lys(Ac))  of human p53 conjugated with aminomethylcoumarin is deacetylated during incubation at 37 °C for 1 h by SIRT1 or SIRT2 in the presence of NAD+ and the tested compounds. The second stage is initiated by the addition of the Developer II (KI176), including nicotinamide (NAM), nicotinamide that stops the SIRT1/2 activity, and the fluorescent signal is produced. The fluorescence was measured on a fluorometric reader  with excitation set at 360 nm and emission detection set at 460 nm.The SIRT inhibition assay was performed using human recombinant SIRT1 and SIRT2 produced in 3H-SAM was added into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h at 30 °C and then it was delivered to filter-paper for detection. The data were analyzed using Excel and GraphPad Prism software for IC50 curve fits. The experiments were performed in duplicate.The EZH2 substrate (0.05 mg/mL core histone) was added in the freshly prepared reaction buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO). The PRC2 complex  was delivered into the substrate solution and the mixture was mixed gently. Afterwards, the tested compounds dissolved in DMSO were delivered into the enzyme/substrate reaction mixture by using Acoustic Technology  in nanoliter range, and 2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT, 1% DMSO). CARM1 was delivered into the substrate solution and the mixture was mixed gently. Afterwards, the tested compounds dissolved in DMSO were delivered into the enzyme/substrate reaction mixture by using Acoustic Technology  in nanoliter range, and 1 µM 3H-SAM was also added into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h at 30 °C and then it was delivered to filter-paper for detection. The data were analyzed using Excel and GraphPad Prism software for IC50 curve fits. The experiments were performed in triplicate.Histone H3 (5 μM) was added in freshly prepared reaction buffer (50 mM Tris-HCl (pH 8.5), 5 mM MgCl2. The human leukemia cells were grown in RPMI-1640  while human breast cancer and neuroblastoma cells in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich) culture media, 1% l-glutamine , 10% heat-inactivated Fetal Bovine Serum (FBS)  and antibiotics. MePR2B cells were grown as previously reported [NB4 tumor cell line was purchased by DSMZ, Braunschweig Germany, and U937, NB4, MCF7, and SH-SY5Y tumor cell lines by American Type Culture Collection . Cell lines have been tested and authenticated following manufacturer’s instruction. All cell lines were maintained in an incubator at 37 °C and 5% COreported .5 cells/mL) were plated in multiwells in triplicate. After stimulations at different times and concentrations, cells were diluted in the ratio 1:1 in Trypan Blue (Sigma) and counted with an optical microscope. For cell cycle analyses, cells were plated (2 × 105 cells/mL) and were harvested, centrifuged at 1200 rpm for 5 min and resuspended in 500 μL of a hypotonic solution containing 1× PBS, Sodium Citrate 0.1%, 0.1% NP-40, RNAase A and 50 mg/mL Propidium Iodide (PI). After 30 min at room temperature (RT) in the dark, samples were acquired by FACS-Calibur  using CellQuest software (BD Biosciences). The percentage in different phases of the cell cycle was determined by ModFit LT V3 software (Verity). All experiments were performed in triplicate. Cell death was measured as percentage of cells in sub-G1 phase as in [For colorimetric exclusion, the cells -3-bromobenzylidene)piperidin-4-one 3 behaved as dual p300/EZH2 inhibitor and displayed cancer-selective growth arrest and cell death in hematological malignancies as well as solid tumors, in in vitro, ex vivo, and in vivo models [Curcuminoids are a wide family of variegated compounds bearing a plethora of therapeutic activities, including anti-inflammatory, antidiabetic, neuroprotective, and anticancer activities ,36,37,38o models ,22.H-pyran-4-one or piperidin-4-one moiety (compounds 4a–n). Such compounds were evaluated in p300, PCAF, SIRT1/2, EZH2 and CARM1 enzyme assays, and in NB4 APL, U937 AML, MCF-7 breast cancer and SH-SY5Y neuroblastoma cells, to determine their effects in cell cycle phases and apoptosis induction when used at 5 μM for 30 h. Generally, in biochemical assays 4a–n exhibited high inhibitory activities against p300 and CARM1, weak activity (if any) against PRC2/EZH2, whereas were totally inactive against PCAF and SIRT1/2. In particular, compounds carrying the cyclohexanone (4a), tetrahydro-4H-pyran-4-one (4b), and piperidin-4-one substituted at N1 with a methyl, benzyl, oxophenylalkyl and acyl moieties (4d–n) displayed the highest inhibition, with 4l and 4m reaching submicromolar IC50 values for both p300 and CARM1 enzymes. In cellular assays, the majority of 4a–n gave an alteration of the cell cycle with pro-apoptotic effect in the tested cell lines, with the leukemia cells being generally more sensitive than the solid cancer cells. In NB4 cells, 4l and 4m induced the strongest apoptosis (near 70%). In U937 cells, in addition to 4l and 4m also the N1 benzyl (4e) and benzoyl (4k) analogues exhibited near or over 40% apoptosis induction. In MCF-7 and in SH-SY5Y cells the effect is lower: the N1 2-oxo-2-phenylethyl 4h and the N1 acyl derivatives 4k–m were the most potent but the induction of apoptosis was around 25% (MCF-7) or less than 20% (SH-SY5Y) at the tested conditions. Despite some apparent discrepancies, due to the differences between the in vitro enzymatic assays and the more complex cellular system, Western blot analysis of selected derivatives in NB4 and MCF-7 cells, performed to detect the levels of acetyl-H3/acetyl-H4/acetyl-α-tubulin, H3K27me3 and H3R17me2 as histone marks for cellular activity of p300, EZH2, and CARM1, respectively, supports the role of the recruitment and inhibition of these epigenetic targets, mainly p300 and CARM1, in the mechanism of anticancer activities of the studied compounds.Starting from these data, we decided to explore the effect of the shift of the bromine substitution at the two phenyl ring from the 3 to the 2 position, using as connection unit cyclohexanone, tetrahydro-4


Matrix metalloproteinases (MMPs) are a family of enzymes involved at different stages of cancer progression and metastasis. We previously identified a novel class of bisphosphonic inhibitors, selective for MMPs crucial for bone remodeling, such as MMP-2. Due to the increasing relevance of specific MMPs at various stages of tumor malignancy, we focused on improving potency towards certain isoforms. Here, we tackled MMP-9 because of its confirmed role in tumor invasion, metastasis, angiogenesis, and immuno-response, making it an ideal target for cancer therapy. Using a computational analysis, we designed and characterized potent MMP-2/MMP-9 inhibitors. This is a promising approach to develop and clinically translate inhibitors that could be used in combination with standard care therapy for the treatment of skeletal malignancies. Matrix metalloproteinases (MMPs) are a family of 23 enzymes that control extracellular matrix (ECM) remodeling and act as key regulators of cancer-bone interactions in skeletal malignancy . NumerouAn imbalance of the MMP enzymatic activity/endogenous inhibitor ratio leads to irregular bone remodeling and tissue destruction. These processes can occur under inflammatory conditions (periodontitis and rheumatoid arthritis) , in metaTumor–bone interaction has been classically described as a “vicious cycle” in which cancer cells promote the expression of potent osteoclastogenic factors such as RANK ligand by bone-lining osteoblasts ,8. This Another factor that negatively influenced the development of MMPIs is the presence of the hydroxamic function in the clinically studied compounds. This functional group, in fact, is a good zinc binding group (ZBG) but possesses a detrimental effect on selectivity and pharmacokinetics. For this reason, new scaffolds with alternative ZBGs have been developed—for example, potent MMPIs were obtained using phosphonate , phosphiIn recent papers, we have shown that the use of bisphosphonic-based bone-seeking MMP inhibitors (BMMPIs) makes the specific targeting of individual MMPs in the bone microenvironment a feasible strategy ,19. The Among BMMPIs, ML 115  was the These novel bone-seeking MMPIs demonstrated that metastatic breast cancer growth in bone and cancer-inducer osteolysis in multiple myeloma can be reduced in vivo ,23. FurtAlthough the inhibition of MMP-2 is undoubtedly beneficial for the reduction of tumor burden and for the prevention and treatment of metastases , a usefuIn order to obtain detailed structural information on the binding mode of the bisphosphonate group, we tried to acquire the crystal structure of MMP-9 in a complex with the inhibitor ML 115. However, the crystallization trials did not give the expected results; therefore, we decided to solve the crystal structure of the complex between that inhibitor and MMP-8, given the well-established protocols of purification and crystallization of the catalytic domain of this particular MMP isoform ,29,30 anML 115 binds the catalytic zinc ion of MMP-8 in a monodentate fashion with one phosphonate moiety . MeanwhiThe obtained binding pose overlaps very well with a prior potent phosphonic sulfonamide with nanomolar potency towards MMPs . The com2–3) or a spacer was inserted between the two phenyl rings (compounds 4–13) by synthesizing compound 1 as an intermediate.This evidence prompted us to design a new series of BMMPIs , most of1–13 were prepared by treating the appropriate commercially available sulfonyl chloride with 30% NH4OH to obtain the corresponding sulfonamides 14–15 (23–24 (16–17 and 25–26. Reduction of the nitro group of tetraethyl (4-nitro-phenylsulfonylamino)methyl-1,1-bisphosphonate (17) and dealkylation of bisphosphonate esters 19, 25, and 26 under acidic conditions or with TMSBr afforded the desired bisphosphonic acids 1–3. Bisphosphonic acids es 14–15  and 23–25  led to a loss of potency towards all tested MMPs of approximately one order of magnitude compared to the reference compound; however, relative selectivity towards MMP-2 was kept.The presence of a 4-amino group (2 and 3) resulted in a decrease of activity towards MMP-2 and -8, as expected for the long and narrow S1’ pocket of these two isozymes, although compound 3 showed a micromolar activity towards all tested MMPs without any particular selectivity. The substitution of biphenyl moiety of ML 115 with 1- or 2-naphthyl (4) or phthalic group (5) as side chain led to compounds with improved activity towards MMP-9, while retaining a five-times higher potency towards MMP-2. Compounds 4 and 5 showed similar activity profiles towards MMP-9 and -13, while being less potent towards MMP-8.The introduction of a 3-phenylureido moiety , while eliminating any kind of selectivity. The corresponding isomer 7, however, showed a significant loss of potency, with evident selectivity towards MMP-2. Compound 6 was then explored. Different groups  were evaluated, obtaining new compounds with interesting inhibition profiles. All substitutions in 4’-position (mainly compounds 9 and 11) led to a significant increase in activity towards MMP-9. Compound 9, in particular, showed the best activity profile in terms of potency (in the nanomolar range) and selectivity towards MMP-9, while maintaining a good activity for the other isoforms. The substitution of the aromatic ring of the benzamide side chain of compound 10, bearing a bromine atom in 3’ position, showed a complete loss of activity on MMP-9 and, overall, an inferior activity profile towards all tested MMPs when compared to its isomer compound 9. Compound 12 and 13, did not translate in higher activity towards MMP-9. Indeed, these compounds showed a certain selectivity towards MMP-2, with potency in the low micromolar range.The introduction of bulkier substituents, as seen for compounds To investigate the BMMPI-binding mechanism, molecular-docking studies were performed on the MMP-2, MMP-8, MMP-9, and MMP-13 catalytic sites. 3D structures used for this study were selected through crossdocking studies, as previously reported . Water mDocking calculations were carried out using Glide , applyinNotably, docking calculations carried out on the original MMP-8:ML115 complex (4KQZ) were not successful in justifying the activity of ligands with longer aromatic moieties. Indeed, MMP-8 S1’ site is hindered by the Arg222 side chain that is involved in the conformational change of the S1’ loop, leading to the disclosure of the S1’* site in the MMP-8 . Therefo9, in particular, almost perfectly fits in the hydrophobic field calculated by SiteMap for MMP-9, with the bromine substituent interacting with the Arg249 (9) towards the target isoform MMP-9 by a more effective adaptation of the ligand to the large hydrophobic field in the enzyme S1’ site.The analysis of obtained docked poses shows that all ligands bind in the expected way, with the bisphosphonic function and the sulfonamide maintaining the crystallographic geometry of ML 115. Enzyme inhibition assays confirm the validity of the suggestion derived from the SiteMap analysis  to elonge Arg249 . These p2, 1-µM ZnCl2, 0.05% Brij-35, and 1% DMSO). Incubation of the enzyme and inhibitor solutions occurred for 15 min at room temperature; fluorogenic substrate solution -Ala-Lys-NH2, Enzo Life Sciences , 1-µM final concentration) was subsequently added. The assay was incubated for 2–4 h at 37 °C, after which, a Perkin-Elmer Victor V3 plate reader  was used to measure fluorescence  Included in the assay were control wells lacking any inhibitor. MMP activity was thus determined and expressed in relative fluorescence units (RFU). Percent inhibition was calculated from control wells. IC50 values were determined using GraphPad Prism 5.0  and are shown as mean ± SEM of at least two independent measurements in triplicate.Catalytic domains of MMP-2, -8, -9, and -13 were obtained from from Enzo Life Sciences . Ninety-six-well white microtiter NBS plates  were used to carry out the assays (in triplicate). The assay measurements were performed by preparing dilutions to six different concentrations (1 nM–100 µM) of each inhibitor in a fluorometric assay buffer  with UV light (short-wave ultraviolet 254 nm and long-wave ultraviolet 365 nm). Inert atmosphere of N2 or Ar was used to carry out all reactions that required an anhydrous environment. Column chromatography was conducted using Fluka silica gel 60 Å (63–200 µm) or silica gel Si 60 (40–63 µm) . Mass spectrometry was conducted on a HP MS 6890-5973 MDS spectrometer, electron impact 70 eV, equipped with a HP ChemStation or with an Agilent 6530 Series Accurate-Mass Quadrupole Time-of-FLIFHT (Q-TOF) LC/MS . A Bruker micro-TOF QII mass spectrometer  equipped with an electrospray ion source (ESI) was used to carry out high-resolution mass spectroscopy (HRMS) analyses. 1H NMR spectra were obtained deuterated solvents on a Varian Mercury 300  or an Agilent VNMRS500 spectrometer . Chemical shifts (δ) are expressed as parts per million (ppm) and coupling constants (J) in Hertz (Hz). Melting points were measured on a Gallenkamp electrothermal apparatus  in open capillaries and are uncorrected. Chemical reagents were commercially obtained from Merck Life Science S.r.l Milano, Italy and were utilized without purification. Monitoring of the reactions occurred via thin-layer chromatography  was stirred for 15 min at 0 °C and at room temperature for 3–24 h. Subsequently, the mixture was partitioned between EtOAc and NaHCO3, and the layers were separated. The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated in vacuo to give a crude product that was used for the next step without any purification. A suspension of the appropriate sulfonyl chloride (4.41 mmol) and NH3-Nitrobenzenesulfonamide 14: Yellow solid, 50% yield. 1H NMR : δ = 7.65 , 7.88 , 8.21–8.23 , 8.43–8.45, 8.57–8.58 . GC-MS: m/z (%): 202(97), 186(33), 156(42), 138(59), 122(55), 108(25), 92(57), 75(100).4-Nitrobenzenesulfonamide 15: Yellow solid, 50% yield; 1H NMR : δ = 8.42–8.40 , 8.07–8.05 , 7.73 . GC-MS: m/z (%): 202(100), 186(44), 156(20), 138(83), 122(60), 108(25), 92(35), 75(95).Naphthalene-1-sulfonamide 23: White solid, 93% yield. 1H NMR : δ = 7.59–7.68 , 7.63 , 8.06–8.16 , 8.59–8.61 . GC-MS: m/z (%): 207 (80), 143 (68), 127 (100), 115 (33).Naphthalene-2-sulfonamide 24: White solid, 98% yield. 1H NMR : δ = 7.49 , 7.60–7.70 , 7.85–7.87 , 7.99–8.03 , 8.09–8.14 , 8.40–8.41 . GC-MS: m/z (%): 207 (35), 143 (40), 127 (100), 115 (20), 77 (10).Triethyl orthoformate, diethyl phosphite, and the opportune sulfonamide were mixed in a 1.2:3:1 stoichiometric ratio. The reaction was heated at 160 °C until the evolution of EtOH was complete; then, EtOAc was used to dissolve the residue. Distillation of the solvent afforded a crude yellow oil, which was purified by column chromatography on silica gel (eluent EtOAc). The titled compounds were obtained as white solids in 35–50% yield.Tetraethyl ((3-nitro-phenylsulfonylamino)methyl)-1,1-bisphosphonate 16: Yellow solid, 35% yield. 1H NMR : δ = 1.26–1.31 , 4.05–4.20 , 4.26 , 5.80 , 7.72 , 8.23–8.26 , 8.43–8.431 , 8.75 . MS(ESI):m/z: 511 [M + Na]+, 489 [M + H]+; MS2 (489): m/z (%): 377 (35), 298 (64), 295 (64), 204 (26), 110 (100).Tetraethyl ((4-nitro-phenylsulfonylamino)methyl)-1,1-bisphosphonate 17: Yellow solid, 47% yield; mp: 122–125 °C. 1H NMR : δ = 1.23–1.31 , 4.11–4.21 , 4.25 , 6.08 , 8.08–8.12 , 8.32–8.35 ; MS (ESI): m/z: 511 : 373 (100).Tetraethyl ((1-naphthylsulfonylamino)methyl)-1,1-bisphosphonate 25: White solid, 35% yield. 1H NMR: δ = 1.07–1.18 , 3.81–4.00 , 4.15 , 5.58 , 7.54 , 7.58–7.63 , 7.67–7.73 , 7.92–7.96 , 8.07 , 8.25 , 8.56 . MS (ESI): m/z: 516 [M + Na]+; MS2: m/z (%): 488 (39), 378 (100). 492[M − H]−; MS2: m/z (%): 191 (100). Tetraethyl ((2-naphthylsulfonylamino)methyl)-1,1-bisphosphonate 26: White solid, 50% yield. 1H NMR: δ = 1.12–1.23 , 3.90–4.12 , 4.23 , 5.29 , 7.57–7.67 , 7.85–7.97 , 8.43–8.44 . MS (ESI): m/z: 516 [M + Na]+; MS2: m/z (%): 488 (28),378 (100). 492[M − H]−; MS2: m/z (%): 191 (100).3/MeOH 9.5:0.5 v/v), affording the desired amino derivate. To a solution of the nitro compound (0.532 mmol) in 8.5 mL EtOH, 10% Pd/C (0.20 mmol) was added. The hydrogenation of the mixture was carried out at room temperature at a pressure of 3 bar for 12–24 h. The reaction mixture was filtered through a celite pad, and the resulting solution was evaporated in vacuo, giving an oil. The crude was purified by chromatography on silica gel (eluent: CHClTetraethyl ((3-amino-phenylsulfonylamino))methyl-1,1-bisphosphonate 18: White solid, 75% yield. 1H NMR : δ = 1.11–1.43 , 3.99–4.07 , 4.11–4.19 , 5.64 , 6.85–6.87 , 7.23–7.25 . MS (ESI): m/z: 481[M + Na]+. 459[M + H]+; MS2(459): m/z (%): 183(100), 138(62).Tetraethyl ((4-amino-phenylsulfonylamino)methyl)-1,1-bisphosphonate 19: White solid, 77% yield. 1H NMR : δ = 1.26–1.31 , 4.04–4.12 , 4.13–4.20 , 5.05 , 6.65 , 7.65 . MS (ESI): m/z: 481[M + Na]+, 459[M + H]+; MS2(469): m/z (%): 156(100), 138(28), 108(50).18 and 19) (1 mmol) in anhydrous THF. Stirring of the mixture occurred at room temperature under inert atmosphere (N2 or Ar) for 3–12 h. After a given time, the mixture was dried under vacuum and dissolved in EtOAc; the resulting solution was washed with NaHCO3 and then with HCl 1N, NH4Cl ss, and brine. The organic phase was finally dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated in vacuo. The residue was purified by chromatography on silica gel (eluent: CHCl3/MeOH 98: 2 v/v or AcOEt /MeOH 9: 1 v/v) or crystallized with AcOEt to give the desired product. The opportune acyl chloride (1.1–2 mmol) and triethylamine (2 mmol) were added to the solution of amino derivate (Tetraethyl((3-benzamidophenylsulfonylamino)methyl)-1,1-bisphosphonate 6a:1H NMR : δ = 1.11–1.88 , 3.87–4.03 , 4.11 , 7.50–7.61 , 7.95–7.97 , 8.30 , 8.80 , 10.50 . MS (ESI): m/z: 561[M − H]−; MS2: m/z (%): 288(54), 260(37), 134 (100), 106 (34). 563 [M + H]+.White solid, 64% yield, (AcOEt). Tetraethyl((4-benzamidophenylsulfonylamino)methyl)-1,1-bisphosphonate 7a:3/MeOH 98: 2 v/v). 1H NMR : δ = 1.13–1.17 , 3.89–4.05 , 4.14 , 7.52–7.62 , 7.78 , 7.90–7.96 , 8.63 , 10.53 . MS (ESI): m/z: 585[M + Na]+, 563[M + H]+; MS2(563): m/z (%): 351(22), 282(10), 261(20), 260(100), 196(50), 166(63), 138(74), 110(42).White solid, 65% yield phenylsulfonylamino)methyl)-1,1-bisphosphonate 8a: 1H NMR: δ = 1.25–1.34 , 2.43 , 4.06–4.20 , 4.23 , 5.31 , 7.30 , 7.78–7.88 , 8.15 . MS (ESI): m/z: 599 [M + Na]+, 577[M + H]+; MS2(577): m/z (%): 393(18), 365(23), 291(21), 275(26), 274(100), 166(87), 138(74), 138(58), 119 (96), 110 (36). 575[M − H]−; MS2: m/z (%): 437(25), 302(16), 210(32), 134(100). White solid, 79% yield (AcOEt). Tetraethyl((4-(4-bromobenzamido)phenylsulfonylamino)methyl)-1,1-bisphosphonate 9a:1H NMR: δ = 1.14–1.18 , 3.94- 4.04 , 4.14 , 7.74 , 7.79 , 7.80–7.92 , 8.55 , 10.54 . MS (ESI): m/z: 643[M + 2 + H]+, 641[M + H]+; MS2(641): m/z (%): 458(18), 431(18), 340(59), 338(53), 276 (11), 274(15), 185(24), 183(32), 166(100), 138(82), 110(39). White solid, 55% yield (AcOEt). Tetraethyl((4-(3-bromobenzamido)phenylsulfonylamino)methyl)-1,1-bisphosphonate 10a: 1H NMR: δ = 1.13–1.96 , 3.97–4.05 , 4.14 , 7.51 , 7.80–7.82 , 7.89–7.96 , 8.14 , 8.63 , 10.62 . MS (ESI): m/z: 643[M+2+H]+, 641 [M + H]+; MS2: m/z (%): 459(12), 430(12), 340(70), 338(43), 276(20), 274(18), 185(18), 183 (42), 166(88), 138(100). White solid, 73% yield (AcOEt). Tetraethyl((4-(4-nitrobenzamido)phenylsulfonylamino)methyl)-1,1-bisphosphonate 11a:1H NMR: δ = 1.15–1.17 , 3.90–4.05 , 4.14 , 7.81 , 7.92 , 8.17–8.20 , 8.36–8.39 , 8.66 , 10.85 . MS (ESI): m/z: 630[M + Na]+, 608 [M + H]+; MS2(630): m/z (%): 492(100). 606 [M − H]−; MS2: m/z (%): 468(86), 333(42), 305(45), 240 (100), 134(61). White solid, 57% yield (AcOEt). Tetraethyl((4-(1-naphthamido)phenylsulfonylamino)methyl)-1,1-bisphosphonate 12a:1H NMR: δ = 1.16–1.43 , 4.04–4.15 , 4.23 , 5.29 , 7.52 , 7.55–7.62 , 7.74 , 7.81–7.88 , 7.88–7.97 , 7.99 , 8.10 , 8.30 . MS (ESI): m/z: 635[M + Na]+, 613 [M + H]+; MS2(613): m/z (%): 310(47), 166(55), 155(100), 138(45), 110(22). 611[M − H]−; MS2: m/z (%): 473(45), 388(67), 310(42), 246 (33), 245(39), 134 (100), 106(29). White solid, 59% yield (AcOEt). Tetraethyl((4-(2-naphthamido)phenylsulfonylamino)methyl)-1,1-bisphosphonate 13a: v/v). 1H NMR : δ = 1.04–1.29 , 3.89–4.09 , 4.16 , 7.60–7.67 , 7.82 , 7.96–8.10 , 8.60 , 8.63 , 10.72 . MS (ESI): m/z: 635[M + Na]+, 613 [M + H]+; MS2(613): m/z (%): 310(76), 166(61), 134(54), 110(18). 611[M − H]−; MS2: m/z (%): 473(43), 388(70), 310(30), 246 (25), 245(33), 134 (100), 106(29). White solid, 49% yield phenylsulfonylamino)methyl)-1,1-bisphosphonate 4a:19 (1 mmol) in anhydrous toluene (2 mL), and the mixture was heated to reflux for 2 h. The eluent was evaporated in vacuo, and the residue was purified by chromatography on silica gel (eluent AcOEt/MeOH 9: 1 v/v) to obtain the desired product. A solution of phenyl isothiocyanate (1.2 mmol) in anhydrous toluene (2 mL) was added to the suspension of 1H NMR : δ = 1.17–1.81 , 4.05–4.16 , 4.31 , 5.75 , 7.03 , 7.26–7.28 , 7.42 , 7.58 , 7.72 , 8.09 , 8.39 . MS(ESI): m/z: 600[M + Na]+, 578[M + H]+; MS2(578): m/z (%): 366(13), 293(22), 275 (100), 211(36), 166 (84), 156(24), 138 (49), 108(40).White solid, 71% yield. Tetraethylphenylsulfonylamino)methyl)-1,1-bisphosphonate 5a:19 (1 mmol) and phthalic anhydride (1.07 mmol) in 6 ml of glacial acetic acid was refluxed for 4 h. The resulting solution was dried under vacuum. EtOAc was subsequently used to dilute the residue; then, 6-M NaOH was added until pH = 6. After separation of the layers, the organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was dried in vacuo. Column chromatography on silica gel (eluent: CHCl3/MeOH 9: 1 v/v) was used to purify the residue, affording the desired product. A mixture of 1H NMR : δ = 1.15–1.17 , 3.87–4.05 , 4.16 , 7.61, 7.90–7.98 , 8.89 . MS (ESI): m/z: 611[M + Na]+; MS2: m/z (%): 473 (100), 445(11), 324(10), 270(23), 160 (12). 587[M − H]−; MS2: m/z %: 287(12), 286(60), 222 (31), 137(100), 108(30). White solid, 47% yield. Method A: A solution of the appropriate tetraethyl bisphosphonate (1 mmol) in 4-mL 2N HCl solution was refluxed for 12–24 h. The aqueous phase was removed under reduced pressure, and the crude bisphosphonic acids were then triturated with the opportune solvent and filtered to afford the final compounds as white solids in 20–96% yield.Method B: Anhydrous trimethylsilylbromide (17–32 mmol) was carefully added to a solution of the corresponding tetraethyl bisphosphonate (1 mmol) in anhydrous acetonitrile (6 mL) at 0 °C under argon, and the resulting mixture was stirred at room temperature for 24–48 h. Two milliliters of MeOH were added, and the mixture was stirred for 5 min; the solvent was distilled off, and the crude bisphosphonic acids were triturated with the opportune solvent and filtered to afford the desired compounds as white solids in a 33–99% yield. (4-aminophenylsulfonylamino)methyl-1,1-bisphosphonic acid (1). Method B: 2O). 1H NMR : δ = 3.78 , 5.38–6.42 , 6.50 , 6.81, 7.45 . 31P NMR : δ = 15.03 . MS (ESI): m/z: 345 [M − H]−; MS2: m/z (%): 280 (100). HRMS [M − H]−: calculated 344.9717; found 344.9709.White solid, mp: 243°C (dec)(Acetone/ H(2-naphthylsulfonylamino)methyl-1,1-bisphosphonic acid (2). Method B:1H NMR : δ = 3.88 , 6.2–7.00 , 7.60–7.66 , 7.90–8.07 , 8.40 . 31P NMR : δ = 14.96 . MS (ESI): m/z: 380 [M − H]; MS2: m/z (%): 362 (100), 298(19). White solid, 43% yield. (IPA) mp: 107–110 °C; (1-naphthylsulfonylamino)methyl-1,1-bisphosphonic acid (3). Method A: 2O 1:1 v/v). 1H NMR : δ = 3.71 , 6.10–6.50 and 6.99–7.38 , 7.51 , 7.55–7.65 , 7.98–8.02 , 8.08 , 8.36 , 8.64 . 31P NMR (300 MHz [D6] DMSO): δ = 14.14 . MS (ESI): m/z: 380 [M − H]−; MS2: m/z (%): 362 (100), 298 (21). White solid, 20% yield; mp: 183 °C (dec) (MeOH/Et(4-(3-phenylureido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (4). Method B: 1H NMR : δ = 3.82 , 5.65–6.80 , 6.97 , 7.27 , 7.42–7.54 , 7.72 , 8.76 , 9.01. 31P NMR : δ = 15.04 . MS (ESI): m/z: 464[M − H]−; MS2: m/z (%): 382(56), 275(27), 263(95), 106(71), 79(100). HRMS [M − H]−: calculated 464.0088; found 464.0081.White solid, 33% yield; mp: > 243 °C (dec). phenylsulfonylamino)methyl-1,1-bisphosphonic acid (5). Method B:1H NMR : δ = 3.87–3.97 , 4.25–5.40 , 7.56 , 7.89–7.93 , 7.96–8.00 . 31P NMR : δ = 14.90 . MS (ESI): m/z: 475[M − H]−; MS2: m/z (%): 393(100), 286(65), 106(100). HRMS [M − H]−: calculated 474.9772; found 474.9768.White solid, 47% yield; mp: > 250 °C (MeOH). (4-benzamidophenylsulfonylamino)methyl-1,1-bisphosphonic acid (6). Method B: 1H NMR : δ = 3.83 , 4.0–5.5 , 7.53 , 7.58–7.60 , 7.79 , 7.87 , 7.94 , 10.54 . 31P NMR : δ = 14.82 (s). MS (ESI): m/z: 449[M − H]−; MS2: m/z (%): 368(20), 367(100), 260(45), 106(76). HRMS [M − H]−: calculated 448.9979; found 448.9972.White solid, 99% yield; mp: 243–245 °C (Acetone). (3-benzamidophenylsulfonylamino)methyl-1,1-bisphosphonic acid (7). Method B:1H NMR : δ = 3.75–3.97 , 5.65–6.57 , 7.41–7.65 , 7.67–7.80 , 7.95–7.98 , 8.25 , 10.43 . 31P NMR : δ = 14.87 . MS (ESI): m/z: 449 [M − H]−; MS2: m/z (%): 367(94), 260(34), 106(100). HRMS [M − H]−: calculated 448.9979; found 448.9973.White solid, 53% yield; mp: > 250 °C (IPA). (4-(4-methylbenzamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (8). Method B: 1H NMR : δ = 2.38 , 3.84 , 5.18–6.05 , 7.34 , 7.58 , 7.78–7.87 , 10.40 . 31P NMR : δ = 14.02 . MS (ESI): m/z: 463 [M − H]−; MS2: m/z (%): 382(14), 381(62), 274(34), 106(60), 79(100). HRMS [M − H]−: calculated 463.0135; found 463.0130.White solid, 61% yield; mp: > 250 °C (MeOH). (4-(4-bromobenzamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (9) Method B: 1H NMR : δ = 3.74–3.92 , 7.46–7.92 , 7.67 , 10.56 . 31P NMR : δ =14.95 . MS(ESI): m/z: 529[M + 2 − H]−, 527[M − H]−. MS2: m/z (%): 447(100), 445(69), 340(24), 338(19). HRMS [M − H]−: calculated 526.9084; found 526.9077.White solid, 80% yield; mp: > 250 °C (MeOH). (4-(3-bromobenzamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (10) Method B: 1H NMR : δ = 3.83 , 6.20–7.07 , 7.50 , 7.63 , 7.80–7.86 , 7.94 , 8.12–8.13 , 10.58 . 31P NMR (500 MHz [D6]DMSO): δ = 14.92 . MS (ESI): m/z: 529[M + 2 − H]−, 527[M − H]−; MS2: m/z (%): 447(98), 445 (69), 340(23), 338(20), 106 (72), 79 (100). HRMS [M − H]−: calculated 526.9084; found 526.9076.White solid, 68% yield; mp: >246 °C (IPA). 4-(4-nitrobenzamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (11) Method B: : δ = 3.79–3.90 , 6.00–7.20 , 7.69 , 7.81–7.88 , 8.18 , 8.37 , 10.80 . 31P NMR (500 MHz [D6]DMSO): δ = 14.96 . MS (ESI): m/z: 494[M − H]−MS2: m/z (%): 458 (8), 412(100), 305 (56), 106 (76). HRMS [M − H]−: calculated 493.9830; found 493.9824.White solid, 71% yield; mp: > 250 °C (MeOH). (4-(1-naphthamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (12) Method B: 1H NMR : δ = 3.86 , 4.25–6.22 , 7.57–7.62 , 7.76 , 7.82 , 7.88 , 8.00–8.03 , 8.09 , 8.13–8.17 , 10.85 . 31P NMR : δ = 13.91 . MS (ESI): m/z: 499 [M − H]−; MS2: m/z (%): 417 (94), 310(39), 106(66), 79(100). HRMS [M − H]−: calculated 499.0135; found 499.0130.White solid, 44% yield; mp: 237–240 °C (IPA). (4-(2-naphthamido)phenylsulfonylamino)methyl-1,1-bisphosphonic acid (13) Method B: 1H NMR : δ = 3.85 , 5.15–5.95 , 7.53–7.58 , 7.60–7.66 , 7.83 , 7.92 , 7.99–8.09 , 8.58 , 10.67 . 31P NMR : δ = 13.97 . MS (ESI): m/z: 499 [M − H]−; MS2: m/z (%): 417 (90), 310 (37), 106 (66), 79 (100). HRMS [M − H]−: calculated 499.0135; found 499.0127.White solid, 68% yield; mp: 248–250 °C (IPA). All calculations were carried out using the Schrodinger Suite 2018-3  . Ligand In conclusion, a series of novel bisphosphonic compounds was synthesized and tested on a panel of MMP isozymes with the aim of improving the activity profile of reference compound ML 115 towards MMP-9.6.The benzensulfonylamide scaffold of ML 115 was derivatized with different substituents in order to achieve the same hydrophobic interaction shown by its diphenyl moiety. The best results were obtained when an amide spacer was introduced as a structural flexibility element, as seen with compound 9 and 11, with a bulky electron-withdrawing substituent in the 4’ position  with even lower IC50 towards MMP-9. Compound 9, in particular, proved to be the best in the series, considering its high potency towards MMP-9 while maintaining a good activity for the other isoforms. On the other hand, better selectivity towards MMP-2 was reached for compounds 4 and 5, which showed a proper activity profile, sparing MMP-8. These results show that the design of BMMPIs targeting MMP-2 and -9 is indeed possible and represents a promising avenue of research for the treatment of bone metastases, which could maximize therapeutic efficacy while limiting side effects.Further developments led to the obtainment of compounds, such as