The association constants determined in aqueous solution  point to a stronger association with PPi, ADP, and ATP as compared with the anions containing a single phosphate unit. The [CuL4]2+ complex shows important selectivity for pyrophosphate (PPi) over ADP and ATP.We report a series of Cu(II) and Zn(II) complexes with different ligands containing a dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function. These complexes were designed for the recognition of phosphorylated anions through coordination to the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes were isolated and several adducts with pyrophosphate were characterized using X-ray diffraction measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with  Phosphorylated metabolites are involved in biologically relevant processes , such as2+ or Zn2+ complexes, with acyclic  and Zn(II) ions with nucleoside 5′-polyphosphates  were studied in detail by Sigel et al. in the early 90’s. ,25,26,27ivatives ,40,41,42ivatives ) or macrivatives ,45,46;  coordination of the anion to the metal ions, and (ii) hydrogen-bonding interactions involving the coordinated anion and the NH groups of the urea moiety as hydrogen-bond donors [2]2+ complex binds sulphate through such cooperative binding [2]2+ complex and related derivatives could be useful for the recognition of phosphorylated anions. Herein, we report a series of ligands containing a bis(pyridin-2-ylmethyl)amine unit for coordination to Cu2+ and Zn2+, and a urea moiety that is expected to reinforce anion binding through hydrogen-bonding interactions. The flexibility of the receptor was modulated by including a benzyl (1L and 2L) or ethyl (3L and 4L) spacer connecting the urea and bis(pyridin-2-ylmethyl)amine units. When considering the ability of phenylboronic acids to bind cis-diol groups (ClO4)2 that was prepared in situ for 1H-NMR measurements). In the case of 4L, the [CuL4(SO4)] complex was also prepared by a similar procedure in methanol. The complexes were isolated in 82–97% yields, with the exception of those derived from 3L, which were obtained in lower yields (49% and 59% for [ZnL3](ClO4)2 and [CuL3](ClO4)2, respectively).The reaction of 1 equivalent of ligand  and ν(O-B-O) modes are also observed when the boronic moiety is present . In all cases, the bands corresponding to the ν(Ph/Py) mode are present, and the coordination of the metal ions induces significant shifts with respect to the corresponding free ligands . It is noticeable that the bands corresponding to the νas(Cl-O) (stretching) and δas(O-Cl-O) (bending) modes of the perchlorate groups appear without splitting for all complexes at ca. 1100 and 625 cm−1, respectively, as befit uncoordinated anions [4(SO4)], the bands corresponding to the νa(S-O) and νa(O-S-O) modes are splitted, as expected for a coordinated sulphate anion with low symmetry. This is in agreement with UV-vis diffuse reflectance spectroscopy, which provided different positions for the d-d absorption band observed for [CuL4(SO4)] and [CuL4](ClO4)2 .As expected, the IR spectra (KBr discs) feature bands due to ν(N-H) and ν(C=O) modes associated to the presence of the urea group in d anions ,55. In t−3 M methanol solutions at 25 °C. The molar conductivity of the perchlorate salts (154.9–171.3 cm2 Ω−1 mol−1) are in the range reported for 2:1 electrolytes [4(SO4)], the low solubility of this charge-neutral complex prevented us from determining its molar conductivity value, confirming that the sulphate anion is directly bonded to the Cu2+ ion.Conductivity measurements were carried out in 10trolytes , indicat4L were investigated by using X-ray diffraction measurements. 2+ or Cu2+) and the anion (sulphate or pyrophosphate). The Cu2+ ions in [CuL4(SO4)] and [CuL4(H2PPi)] complexes present octahedral coordination with a strong Jahn-Teller distortion [2+ complexes of L2 [4(SO4)] is defined by the donor atoms of the dipicolylamine unit  and an oxygen atom of the anion [O(4)] (mean deviation from planarity: 0.04 Å), with the metal ion being placed 0.01 Å below this plane in [CuL4(SO4)] and 0.12 Å above it in [CuL4(H2PPi)]. The apical positions in [CuL4(SO4)] are occupied by one of the nitrogen atoms of the urea subunit [N(3)] and an oxygen atom of a sulphate anion of an adjacent complex molecule [O(5b)] (2–η1:η1-sulphate anions bridging two neighboring complex molecules leads to the formation of a coordination polymer (4(H2PPi)], the apical positions are delineated by one of the nitrogen atoms of the urea subunit [N(3)] and an oxygen atom of the H2PPi2− anion [O(8)], which coordinates in a κ2-H2PPi2− mode forming a six-membered chelate ring.The solid state structures of different complexes of stortion . The liges of L2 . The equ [O(5b)] . Thus, t polymer . In [CuLcis angles of the coordination polyhedron (81.0°–98.8° in [CuL4(SO4)] and 82.4°–98.4° in [CuL4(H2PPi)]) are relatively close to the one expected for a regular octahedral coordination (90°), whereas the corresponding trans angles (162.7°–178.0° for [CuL4(SO4)] and 164.4–173.8 for [CuL4(H2PPi)]) deviate up to 18° from the ideal one (180°) (The e (180°) .4(SO4)] interacts with the metal ion via a coordinate bond and with the urea moiety through a moderate hydrogen-bonding interaction, the latter involving one of the NH groups of the urea unit as hydrogen-bond donor and an oxygen atom of the anion as hydrogen-bond acceptor . The value of the S(1)-O(6)-H(2N) angle (116.8°) is close to the ideal one (122° ± 10°) [The sulphate anion in [CuL° ± 10°) . The int4}2(μ-PPi)] and [{CuL4}2(μ-PPi)] (2κ4-PPi4− ligand forming two six-membered chelate rings [2+ complexes derived from di(2-picolyl)amine [4}2(μ-PPi)] adopts an ′open wing butterfly′ conformation , N(5), and N(6) for Cu(1) and N(10), N(11) and N(12) for Cu(2)] and an oxygen atom of pyrophosphate [O(4) or O(9)] [mean deviation from planarity: 0.03 Å for Cu(1) and 0.05 Å for Cu(2)], while the apical position is occupied by another oxygen atom of the anion [O(8) or O(5)]. The metal ion is placed 0.29 Å [Cu(1)] or 0.28 Å [Cu(2)] above the basal plane. The cis angles of this plane (82.8°–95.7°) and those involving the oxygen atom occupying the apical position, the metal centre and the donor atoms of the equatorial plane (93.3°–102.5°), as well as the trans angles (157.4°–166.8°), are relatively close to the ideal ones . The index of trigonality τ, which amounts to 0.12 for Cu(1) and 0.16 for Cu(2)  [We have also obtained single crystals suitable for X-ray diffraction measurements of [{ZnL(μ-PPi)] , in whicte rings ,60. Thisyl)amine ,62. The ormation , which rronment)  is in ag4L in [{ZnL4}2(μ-PPi)] adopts a ′bent propeller′ conformation and wraps around the metal ions providing trigonal bipyramidal coordination environments. The equatorial plane of the bipyramid is defined by the nitrogen atoms of the pyridine units [N(5) and N(6) for Zn(1) and N(11) and N(12) for Zn(2)] and an oxygen atom of the pyrophosphate anion [O(8) for Zn(1) and O(5) for Zn(2)]. The apical positions are occupied by the amine nitrogen atom and one of the oxygen atoms of the anion [N(4) and O(4) for Zn(1) and N(10) and O(9) for Zn(2)]. The Zn2+ ion is placed 0.24 Å [Zn(1)] or 0.25 Å [Zn(2)] above the equatorial plane. The angles involving the donor atoms of this plane (113.9°–124.7°), as well as the trans angle (170.4°–170.6°) are close to the ones expected for a regular trigonal bipyramid . The index of trigonality τ amounts to 0.76 for both metals  and [{CuL4}2(μ-PPi)] show a cooperative binding in which the anion interacts simultaneously with the metal ion via coordinate bonds, and with the urea moiety through Y-shape hydrogen-bonding interactions. The N-H(N)-O angles and distances  angles (109°–122°) are close to the ideal value for a tetrahedral anion (122° ± 12°) 2+ and [ZnL2]2+ complexes hydrolyse in aqueous solution to form the [ZnL6]2+ complex (2(μ-SO4)]2 reported recently also points in this direction [2](ClO4)2, new resonances appear in the 1H-NMR spectrum from 7.35 to 6.60 ppm, none of which correspond to the complex or the free ligand. The presence of a signal at 6.94 ppm due to 3-nitroaniline confirmed the hydrolysis of the complex. The relative intensity of the signals attributable to the [ZnL2]2+ species and 3-nitroaniline indicate that 6% of the complex has hydrolysed after four hours at room temperature, while after 48, 96, and 168 h this percentage increases to 28%, 43%, and 49%, respectively. A similar situation was observed for [ZnL1]2+, which hydrolysed to a slightly lower extent after 4 h (~5%). 1H DOSY experiments performed for [ZnL2]2+ 168 h after dissolution of the complex allowed discriminating the three species that are present in solution on the basis of their different diffusion coefficients (d5 as a reference (D = 6.81 × 10−10 m2·s−1) 2+, [ZnL6]2+, and 3-nitroaniline, respectively.The  signals . This su complex , 2-aminoirection . In the ficients . Taking  m2·s−1) , these s2]2+ was monitored in aqueous solution by following the absorption band of 3-nitroaniline  over time and employing the method of initial rates (kobs) and half-life times (t1/2) were determined by fitting the data obtained at 390 nm using two temperatures  and two pH values (5.5 and 7.0). In line with previous studies 2+ complex experiences significant hydrolysis in aqueous media at room temperature. Analogous studies performed for the [CuL4]2+ complex provided rate constants of 3.12 ± 0.01 × 10−9·s−1 and 1.265 ± 0.002 × 10−7·s−1 at 25 and 50 °C, respectively, which correspond to half-life times of ~89 (25 °C) and ~2 (50 °C) months. Thus, the replacement of the 1-(3-nitrophenyl)-3-phenylurea unit of L2 by a 1-ethyl-3-(3-nitrophenyl)urea group increases the stability toward hydrolysis of the corresponding Cu2+ complex by 3–4 orders of magnitude.The hydrolysis of the urea unit in 2+, [ZnL2]2+, and [CuL2]2+ are related to the coordination of a nitrogen atom of the urea unit to the metal ion.Most of the studies regarding the decomposition of ureas focused on its hydrolysis to ammonium and carbamate ions ,67 . Thef urease . Thus, w3](ClO4)2, [CuL4](ClO4)2, and [CuL5](ClO4)2 was investigated by using spectrophotometric titrations in aqueous solutions . The absorption spectra of the complexes show a d-d absorption band with a maximum at 615 ([CuL3]2+), 654 ([CuL4]2+), and 643 nm ([CuL5]2+), typical of dxz, dyz → d2−y2x transitions in a tetragonal ligand field  shown in The binding of phosphorylated anions phosphate, AMP, CMP, UMP, PPi, ADP, and ATP to [CuLnd field . This isficients . This ingen atom . This isn]2+: Anion, n = 3, 4 or 5). The high resolution mass spectra recorded by electrospray ionization (ESI+) for solutions of [CuL4]2+ in the presence of one equivalent of PPi, ADP, or ATP show peaks due to the 2:1 and 1:1 entities . The definition of pA is similar to that of pM often used to assess the relative stabilities of metal complexes in aqueous media  and [{CuL4}2(μ-PPi)] ] . It is a process .31P NMR spectra recorded in the presence of phosphate shows two very broad signals, which evidences the presence of different species in solution involved in dynamic processes. In the case of AMP, the 31P NMR spectra show at least three signals for both [ZnL3]2+ and [ZnL4]2+ complexes, none of which coincides with that of free AMP, again suggesting the presence of several species in solution.The 4(H2PPi)] and [{ZnL4}2(μ-PPi)] complexes correspond to those observed in solid state, the geometries of both monomeric and dimeric species were modeled by means of DFT calculations (TPSSh/SVP) in dmso solution ] species is in agreement with the one previously described in the solid state, where identical conformations of the ligands backbone (‘bent propeller’), coordination polyhedron around the Zn(II) metal ion , and intramolecular hydrogen-bonding interactions are present. In the case of the [ZnL4(H2PPi)] species, the obtained geometry differs considerably from that observed for the [CuL4(H2PPi)] complex in the solid state. The coordination polyhedron around the Zn2+ metal ion can be now best described as a square pyramid, with the ligand in an ‘open wing butterfly‘ conformation. Despite this, it is noticeable to remark that in both geometries the pyrophosphate anion is coordinated to the Zn(II) metal ions forming six-membered chelate rings in κ2-H2PPi2− or μ2κ4-PPi4− modes for [ZnL4(μ-PPi)] and [{ZnL4}2(μ-PPi)], respectively. Hydrogen bond interactions between the oxoanion and the urea moieties are observed in both cases.To check whether the structures in solution of [ZnLsolution . In bothVP level . The min4}2(μ-PPi)] dimeric complex, and with a doubly protonated form for the [ZnL4(H2PPi)] monomeric complex. The good agreement between experimental and calculated data suggests that both complexes adopt the calculated minimum energy geometries in solution.The comparison of the calculated chemical shifts with the experimental values recorded in the presence of 0.5 or 1 equivalents of pyrophosphate anion are in agreement with those calculated for a fully deprotonated form in the case of the (ClO4)2 or [CuL5](ClO4)2 complexes in H2O at pH 7.0 upon addition of a 0.1 M solution of the corresponding sodium salt containing a 10−3 M concentration of the corresponding complex at pH 7.0. For the [CuL4](ClO4)2 complex, the concentrations of the titration solutions and the solutions of the sodium salts were 5 × 10−3 M and 0.5 M, respectively. The pH was buffered with MOPS in all cases. Binding constants were obtained by using a simultaneous fit of the UV/vis absorption spectral changes at 7–12 selected wavelengths in the range 500–1200 nm. A minimum of 26 absorbance data points at each of these wavelengths was used, and all spectrophotometric titration curves were fitted with the HYPERQUAD 2008 (HypSpec) program +, 164.1 (47%). Elem. Anal. Calcd for C8H10BNO2: C, 59.0; H, 6.2; N, 8.6%. Found: C, 58.6; H, 6.3; N, 8.5%. IR: 3444, 3361, 3215 ν(N-H), 2981, 2910 ν(C-H), 1625, 1579, 1484, 1443 ν(C=C), 1338 ν(C-BO2), 1210 ν(O-B-O) cm−1.2-(3-Tolyl)-1,3,2-dioxaborolane (4). This compound was prepared by adapting the experimental procedure found in the literature +, 163.1 (38%). Elem. Anal. Calcd for C9H11BO2: C, 66.7; H, 6.8%. Found: C, 66.6; H, 6.8%. IR: 3023, 2974, 2908 ν(C-H), 1607, 1583, 1481 ν(C=C), 1335 ν(C-BO2), 1206 ν(O-B-O) cm−1.terature . A solut1-phenyl)-3-(2-(chloromethyl)phenyl)urea (5). A solution of compound 3  and 1-(chloromethyl)-2-isocyanatobenzene  in diethyl ether (40 mL) was stirred at room temperature for 16 h. The precipitate formed was isolated by filtration and washed with diethyl ether (3 × 5 mL) to give 2.594 g of the desired compound (91%) as a white solid. δH : 9.38 (s), 9.20 (s), 8.25 (s), 8.21 (s), 7.88 (m), 7.69–7.64 (m), 7.61 (m), 7.43 (m), 7.34–7.31 (m), 7.27–7.19 (m), 7.06 (m), 4.85 (s), 4.35 (s), 4.32 (s), 3.39 (s). δC : 152.7, 139.4, 137.5, 130.6, 129.2, 128.5, 127.9, 127.6, 124.0, 123.1, 122.4, 121.1, 65.9, 65.7, 62.8, 43.5. MS-ESI+, m/z (%BPI): [5 + H]+, 331.1 (7%); [5 − PG + H]+, 305.1 (23%); [5 − Cl]+, 295.1 (9%); [5 − PG − Cl]+, 269.1 (85%). Elem. Anal. Calcd for C16H16BClN2O3: C, 58.1; H, 4.9; N, 8.5%. Found: C, 58.4; H, 4.9; N, 8.3%. IR: 3285 ν(N-H), 2979, 2912 ν(C-H), 1637 ν(C=O), 1606, 1586, 1489, 1477 ν(C=C), 1560 δ(N-H), 1335 ν(C-BO2), 1214 ν(O-B-O), 672 ν(C-Cl) cm−1.1-phenyl)-3-(2-chloroethyl)urea (7). A solution of compound 3  and 1-chloro-2-isocyanatoethane  in diethyl ether (40 mL) was stirred at room temperature for 16 h. The precipitate formed was isolated by filtration and washed with diethyl ether (3 × 5 mL) to give 2.052 g of the desired compound (89%) as a white solid. δH : 8.70 , 7.76 , 7.55−7.52 , 7.27–7.25 , 6.39 , 4.31 , 3.66 , 3.42 . δC : 155.1, 155.0, 139.9, 139.4, 128.4, 127.7, 127.4, 127.2, 123.8, 123.7, 120.9, 119.8, 65.6, 62.8, 44.5, 44.4, 41.2. MS-ESI+, m/z (%BPI): [7 + H]+, 269.1 (48%); [7 − PG + H]+, 243.1 (100%). Elem. Anal. Calcd for C11H14BClN2O3: C, 49.2; H, 5.3; N, 10.4%. Found: C, 49.3; H, 5.2; N, 10.4%. IR: 3329 ν(N-H), 2978, 2914 ν(C-H), 1629 ν(C=O), 1605, 1486, 1476 ν(C=C), 1564 δ(N-H), 1333 ν(C-BO2), 1214 ν(O-B-O), 614 ν(C-Cl) cm-1.2-(3-(Bromomethyl)phenyl)-1,3,2-dioxaborolane (9). This compound was prepared by adapting the experimental procedure found in the literature +, 161.1 (100%). Elem. Anal. Calcd for C9H10BBrO2: C, 44.9; H, 4.2%. Found: C, 45.0; H, 4.3%. IR: 2976, 2908 ν(C-H), 1605, 1583 ν(C=C), 1338 ν(C-BO2), 1216 ν(O-B-O), 602 ν(C-Br) cm−1.terature . A mixtutert-Butyl-(2-bromoethyl)carbamate (12). This compound was prepared according to the literature procedure +, 246.0 (1%); [12 − Boc + H]+, 124.0 (68%). Elem. Anal. Calcd for C7H14BrNO2: C, 37.5; H, 6.3; N, 6.3%. Found: C, 37.1; H, 5.7; N, 6.8%. IR: 3336 ν(N-H), 2977, 2932 ν(C-H), 1689 ν(C=O), 1507 ν(N-H), 1162 νa(N-C) cm−1.rocedure . A suspetert-Butyl-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate (13). A solution of compound 12 , bis(pyridin-2-ylmethyl)amine , N,N-diisopropylethylamine  and a catalytic amount of KI in acetonitrile (100 mL) was heated to reflux with stirring for three days. The resulting solution was filtered while hot and the filtrate concentrated to dryness. The residue was extracted with chloroform (5 × 50 mL) and water (25 mL), the organic layers combined and dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using a 48 g-neutral alumina column and CH2Cl2 as eluent to obtain pure product 13 as an orange oil which was dried under vacuum for several hours . δH : 8.51 , 7.60 , 7.38 , 7.12 , 5.80 , 3.83 , 3.20 , 2.67 , 1.41 . δC : 159.3, 156.3, 149.2, 136.6, 123.2, 122.2, 78.8, 60.3, 53.7, 38.6, 28.6. MS-ESI+, m/z (%BPI): [13 + Na]+, 365.2 (100%); [13 + H]+, 343.2 (19%). Elem. Anal. Calcd for C19H26N4O2: C, 66.6; H, 7.7; N, 16.4%. Found: C, 66.3; H, 7.7; N: 15.6%. IR: 3315 ν(N-H), 3051–2821 ν(C-H), 1699 ν(C=O), 1590, 1569 ν(Py), 1520 ν(N-H), 1167 νa(N-C), 757 ν(C-H) cm−1.N′,N′-Bis(pyridin-2-ylmethyl)ethane-1,2-diamine (14). A solution of compound 13  and trifluoroacetic acid  in chloroform (10 mL) was stirred at room temperature for 16 h. The solvent was evaporated under reduced pressure and the residue was dissolved in chloroform (30 mL). The acid excess was neutralized with an aqueous solution of NaOH (1 M) and the organic layer washed with distilled water (2 × 15 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent removed by rotary evaporation to give the pure product 14 as an orange oil that was dried under vacuum for several hours . δH : 8.50 , 7.62 , 7.45 , 7.12 , 3.82 , 2.77 , 2.65 , 2.06 . δC : 159.7, 149.2, 136.5, 123.1, 122.1, 60.8, 57.4, 39.7. MS-ESI+, m/z (%BPI): [14 + H]+, 243.2 (100%). Elem. Anal. Calcd for C14H18N4: C, 69.4; H, 7.5; N, 23.1%. Found: C, 69.1; H, 6.9; N, 22.8%. IR: 3360 ν(N-H), 3054–2823 ν(C-H), 1589, 1568, 1473, 1432 ν(Py), 756 ν(C-H) cm−1.(3-(3-(2-((Bis(pyridin-2-ylmethyl)amino)methyl)phenyl)ureido)phenyl)boronic acid (1L). A solution of compound 5 , bis(pyridin-2-ylmethyl)amine , N,N-diisopropylethylamine  and a catalytic amount of KI in acetonitrile (50 mL) was heated to reflux with stirring for three days. The resulting solution was filtered while hot and the filtrate concentrated to dryness. The residue was extracted with chloroform (5 × 50 mL) and water (25 mL), the organic extracts combined and dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using a 48 g-neutral alumina column and CHCl3/MeOH as eluent  to obtain pure product 1L as a pale brown solid . δH : 10.30 , 8.96 , 8.50 , 8.03 , 8.01 , 7.77 , 7.76–7.73 , 7.71 , 7.46 , 7.40 , 7.30 , 7.27–7.20 , 6.93 , 3.80 , 3.72 . : 157.7 C5, 152.6 C15, 149.0 C1, 139.4 C13, 139.2 C17, 136.9 C3, 129.9 C9, 127.9 C19, 127.9 C18, 127.8 C11, 125.6 C8, 124.2 C23, 123.6 C4, 122.5 C2, 121.5 C10, 120.2 C20, 119.4 C12, 58.5 C6, 56.4 C7. MS-ESI+, m/z (%BPI): [1L + H]+, 468.2 (100%). Elem. Anal. Calcd for C26H26BN5O3: C, 64.3; H, 5.8; N, 14.4%. Found: C, 64.5; H, 5.1; N: 14.6%. IR: 3290 ν(N-H/O-H), 3050–2830 ν(C-H), 1695 ν(C=O), 1593, 1571, 1477 ν(Ph/Py), 1538 ν(N-H), 1207 ν(O-B-O), 749 ν(C-H) cm−1.(3-(3-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)ureido)phenyl)boronic acid (3L). A solution of compound 7 , bis(pyridin-2-ylmethyl)amine  and N,N-diisopropylethylamine  in acetonitrile (30 mL) was heated to reflux with stirring for four days. The resulting solution was filtered while hot and the filtrate concentrated to dryness. The residue was extracted with chloroform (5 × 20 mL) and water (10 mL). During this process a brown solid insoluble in both phases was formed. This solid was filtered off, the organic extracts were combined, dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using an 8 g-neutral alumina column and CH2Cl2/MeOH as eluent  to obtain pure product 3L as a pale yellow solid . δH : 8.55 , 8.48 , 7.96 , 7.72 , 7.60–7.56 , 7.33 , 7.23 , 7.18 , 6.15 , 3.81 , 3.25 , 2.59 . : 159.0 C5, 155.3 C10, 148.8 C1, 139.7 C12, 136.5 C3, 134.7 C16, 127.7 C14, 127.0 C13, 123.8 C15, 122.8 C4, 122.2 C2, 119.8 C18, 59.6 C6, 53.8 C7, 37.0 C8. MS-ESI+, m/z (%BPI): [3L + H]+, 406.2 (100%). Elem. Anal. Calcd for C21H24BN5O3: C, 62.2; H, 6.0; N, 17.3%. Found: C, 61.9; H, 6.1; N: 17.7%. IR: 3303 ν(N-H/O-H), 3054–2849 ν(C-H), 1659 ν(C=O), 1593, 1477 ν(Ph/Py), 1551 ν(N-H), 1338 ν(C-BO2), 757 ν(C-H) cm−1.1-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)-3-(4-nitrophenyl)urea (4L). A solution of compound 14  and 1-isocyanato-4-nitrobenzene  in chloroform (40 mL) was stirred at room temperature for two hours. The solvent was evaporated under reduced pressure and the residue was purified on a CombiFlash RF-200, using a 12 g-silica gel column and CHCl3/MeOH as eluent  to give 4L as a yellow oil that became a solid after remaining under vacuum for several days . δH : 8.95 , 8.46 , 8.00 , 7.56–7.51 , 7.26 , 7.09 , 6.99 , 3.73 , 3.26 , 2.61 . : 158.1 C5, 155.3 C10, 149.1 C1, 146.7 C12, 141.3 C15, 136.9 C3, 125.1 C14, 123.9 C4, 122.5 C2, 117.2 C13, 60.0 C6, 53.0 C7, 37.3 C8. MS-ESI+, m/z (%BPI): [4L + H]+, 407.2 (100%). Elem. Anal. Calcd for C21H22N6O3: C, 62.1; H, 5.5; N, 20.7. Found: C, 62.4; H, 5.3; N, 20.0%. IR: 3317 ν(N-H), 3078–2804 ν(C-H), 1643 ν(C=O), 1614, 1591, 1475, 1433 ν(Ph/Py), 1553 νa(NO2), 1328 νs(NO2), 749 ν(C-H) cm−1.(3-((Bis(pyridin-2-ylmethyl)amino)methyl)phenyl)boronic acid (5L). A solution of compound 9 , bis(pyridin-2-ylmethyl)amine , N,N-diisopropylethylamine  and a catalytic amount of KI in acetonitrile (50 mL) was heated to reflux with stirring for 16 h. The resulting solution was filtered while hot and the filtrate concentrated to dryness. The residue was extracted with chloroform (5 × 25 mL) and water (15 mL), the organic layers combined and dried over anhidrous sodium sulfate, and the solvent evaporated under reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using a 48 g-neutral alumina column and CH2Cl2/MeOH as eluent  to obtain pure product 5L as a pale yellow solid . δH : 8.48 , 8.04 , 7.80 , 7.77 , 7.67 , 7.59 , 7.47 , 7.31 , 7.24 , 3.70 , 3.62 . : 159.2 C5, 148.8 C1, 137.5 C8, 136.6 C3, 134.6 C14, 134.1 C12, 132.9 C11, 130.4 C9, 127.4 C10, 122.5 C4, 122.2 C2, 59.1 C6, 57.9 C7. MS-ESI+, m/z (%BPI): [5L + Na]+, 356.2 (67%); [5L + H]+, 334.2 (100%). Elem. Anal. Calcd for C19H20BN3O2: C, 68.5; H, 6.1; N, 12.6%. Found: C, 68.2; H, 6.0; N: 11.0%. IR: 3049–2850 ν(C-H), 1593, 1571, 1475 ν(Ph/Py), 1362 ν(C-BO2), 758 ν(C-H) cm−1.1](ClO4)2 (M = Cu or Zn).General procedure for the preparation of (ClO4)2·H2O[CuL ([CuL1](ClO4)2). Dark green solid. Yield: 0.077 g, 96%. MS-ESI+, m/z (%BPI): [CuL1(ClO4)]+, 629.1 (3%); [Cu(L1-H)]+, 529.1 (41%). Elem. Anal. Calcd for C26H26BCl2CuN5O11·H2O: C, 41.8; H, 3.8; N, 9.4%. Found: C, 42.1; H, 3.8; N, 9.1%. ΛM : 154.9 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3358 ν(N-H/O-H), 1651 ν(C=O), 1614 ν(Ph/Py), 1549 ν(N-H), 1345 ν(C-BO2), 1053 νa(Cl-O), 619 νa(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy: 633 nm.1](ClO4)2[ZnL ([ZnL1](ClO4)2). Light brown solid. Yield: 0.073 g, 97%. MS-ESI+, m/z (%BPI): [ZnL1(ClO4)]+, 630.1 (4%); [Zn(L1-H)]+, 530.1 (100%); [ZnL1]2+, 265.6 (71%). Elem. Anal. Calcd for C26H26BCl2N5O11Zn: C, 42.7; H, 3.6; N, 9.6%. Found: C, 42.6; H, 3.7; N: 9.3%. ΛM : 164.0 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3354 ν(N-H/O-H), 1612 ν(Ph/Py), 1559 ν(N-H), 1343 ν(C-BO2), 1055 νa(Cl-O), 620 νa(O-Cl-O) cm−1. δH : 8.60 , 8.42 , 8.15 , 8.01 (s), 7.97 (m), 7.60–7.55 , 7.49–7.35 , 7.22 , 4.22 , 3.82 , 3.67 . δC : 154.4, 153.6, 147.6, 140.5, 138.6, 138.5, 134.8, 133.3, 129.4, 128.3, 127.9, 127.8, 127.5, 125.7, 124.9, 124.7, 124.4, 120.4, 55.4, 51.7.3](ClO4)2 (M = Cu or Zn).General procedure for the preparation of (ClO4)2·2H2O[CuL ([CuL3](ClO4)2). Dark green solid. Yield: 0.052 g, 59%. MS-ESI+, m/z (%BPI): [CuL3(ClO4)]+, 567.1 (16%); [Cu(L3-H)]+, 467.1 (8%); [CuL3]2+, 234.1 (100%). Elem. Anal. Calcd for C21H24BCl2CuN5O11·2H2O: C, 35.8; H, 4.0; N, 10.0%. Found: C, 35.7; H, 3.6; N, 10.1%. ΛM : 170.2 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3364 ν(N-H/O-H), 1613 ν(Ph/Py), 1556 ν(N-H), 1341 ν(C-BO2), 1052 νa(Cl-O), 619 νa(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy: 585 nm.3](ClO4)2·H2O[ZnL ([ZnL3](ClO4)2). Light brown solid. Yield: 0.041 g, 49%. MS-ESI+, m/z (%BPI): [Zn(L3-H)(OMe)]+, 482.1 (2%); [ZnL3(OMe)]2+, 241.6 (100%). Elem. Anal. Calcd for C21H24BCl2N5O11Zn·H2O: C, 36.7; H, 3.8; N, 10.2%. Found: C, 36.5; H, 4.0; N: 9.8%. ΛM : 167.2 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3363 ν(N-H/O-H), 1612 ν(Ph/Py), 1565 ν(N-H), 1341 ν(C-BO2), 1054 νa(Cl-O), 619 νa(O-Cl-O) cm−1. δH : 8.60 (m), 8.52 (s), 8.08 (m), 7.95 (s), 7.62–7.49 , 7.37 , 7.20 , 6.24 , 4.37 , 4.05 , 3.60 (m), 2.83 (m). δC : 155.7, 154.6, 147.6, 140.6, 139.2, 134.8, 127.8, 127.5, 124.7, 124.5, 124.3, 120.3, 57.2, 54.9, 34.7.4](ClO4)2 (M = Cu or Zn) and [CuL4(SO4)].General procedure for the preparation of (ClO4)2·2H2O[CuL ([CuL4](ClO4)2). Light blue solid. Yield: 0.075 g, 86%. MS-ESI+, m/z (%BPI): [CuL4(ClO4)]+, 568.0 (83%); [Cu(L4-H)]+, 468.1 (22%); [CuL4]2+, 234.6 (100%). Elem. Anal. Calcd for C21H22Cl2CuN6O11·2H2O: C, 35.8; H, 3.7; N, 11.9%. Found: C, 35.9; H, 3.4; N, 11.5%. ΛM : 144.5 cm2·Ω−1·mol−1. IR: 3584, 3410, 3342 ν(N-H), 3089–2780 ν(C-H), 1697 ν(C=O), 1613, 1597 ν(Ph/Py), 1550 ν(N-H), 1504 νa(NO2), 1334 νs(NO2), 1093, 1061 νa(Cl-O), 620 νa(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy: 633 nm.4](SO4)·1.5H2O[CuL ([CuL4(SO4)]). Light blue solid. Yield: 0.047 g, 64%. MS-ESI+, m/z (%BPI): [Cu(L4+H)(SO4)]+, 566.1 (20%); [Cu(L4-H)]+, 468.1 (3%); [CuL4]2+, 234.6 (52%). Elem. Anal. Calcd for C21H22CuN6O7S·1.5H2O: C, 42.5; H, 4.3; N, 14.2%. Found: C, 42.7; H, 3.9; N, 13.9%. ΛM : the low solubility of this complex in methanol has prevented us from determining the conductivity value for this compound. IR: 3254 ν(N-H), 3117–2943 ν(C-H), 1692 ν(C=O), 1612, 1595 ν(Ph/Py), 1562 ν(N-H), 1502 νa(NO2), 1324 νs(NO2), 1106, 1049, 1029 νa(S-O), 600, 584 νa(O-S-O) cm−1. UV/vis diffuse reflectance spectroscopy: 706 nm. Slow evaporation of a solution of the complex in a dimethylformamide/water mixture provided dark blue single crystals suitable for X-ray diffraction analysis.4](ClO4)2·CHCl3[ZnL ([ZnL4](ClO4)2). Light yellow solid. Yield 0.092 g, 95%. MS-ESI+, m/z (%BPI): [ZnL4(ClO4)]+, 569.1 (74%); [Zn(L4-H)]+, 469.1 (100%); [ZnL4]2+, 235.1 (83%). Elem. Anal. Calcd for C21H22Cl2N6O11Zn·CHCl3: C, 33.4; H, 2.9; N, 10.6%. Found: C, 33.8; H, 3.1; N: 10.7%. ΛM : 161.9 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3372 ν(N-H), 1647 ν(C=O), 1614 ν(Ph/Py), 1556 ν(N-H), 1506 νa(NO2), 1329 νs(NO2), 1094, 1051 νa(Cl-O), 619 νa(O-Cl-O) cm−1. δH : 9.39 , 8.61 , 8.16–8.06 , 7.63–7.57 , 6.52 , 4.38 , 4.06 , 3.45 (m), 2.82 . δC : 154.6, 154.5, 147.5, 146.9, 140.6, 125.1, 124.7, 124.4, 117.0, 56.8, 54.0, 34.5.5](ClO4)2.Procedure for the preparation of (ClO4)2·H2O[CuL ([CuL5](ClO4)2). Dark blue solid. Yield: 0.075 g, 82%. MS-ESI+, m/z (%BPI): [CuL5(ClO4)]+, 495.1 (70%); [Cu(L5-H)]+, 395.1 (100%). Elem. Anal. Calcd for C19H20BCl2CuN3O10·H2O: C, 37.2; H, 3.6; N, 6.9%. Found: C, 37.4; H, 3.8; N, 7.1%. ΛM : 171.3 cm2·Ω−1·mol−1 (2:1 electrolyte). IR: 3441 ν(O-H), 1613, 1576, 1487 ν(Ph/Py), 1346 ν(C-BO2), 1048 νa(Cl-O), 619 νa(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy: 603 nm.4(SO4)] were obtained by evaporation of a solution of the isolated complex, as described above. Crystals of [CuL4(H2PPi)] were grown by slow evaporation of an acetonitrile–methanol (1:1) mixture containing stoichiometric amounts of [CuL4](ClO4)2 and sodium pyrophosphate. Finally, [{CuL4}2(μ-PPi)] and [{ZnL4}2(μ-PPi)] were crystallized by slow evaporation of solutions of [CuL4](ClO4)2 (acetonitrile-methanol mixture) or [ZnL4](ClO4)2 (deuterium oxide) in the presence of 0.5 equiv. of sodium pyrophosphate.Crystals of [CuL4}2(µ-PPi)], [{CuL4}2(µ-PPi)] and [CuL4(H2PPi)]) or BRUKER-NONIUS X8 APEX CCD ([CuL4(SO4)]) diffractometers. The data of [{CuL4}2(µ-PPi)], [CuL4(H2PPi)]) and [CuL4(SO4)] were corrected for Lorentz and polarization effects and for absorption by semiempirical methods based on symmetry-equivalent reflections [4}2(µ-PPi)] was a twin, so that the correction for absorption was made with TWINABS [® computer [4}2(µ-PPi)] or Patterson methods with DIRDIF2008 [4}2(µ-PPi)], [CuL4(H2PPi)] and [CuL4(SO4)]). All of the structures were refined by full-matrix least-squares on F2 (SHELXL-2017) [4(H2PPi)] and six molecules per unit cell in crystals of [{ZnL4}2(µ-PPi)]) were located in a difference electron-density map and the H⋯H and O-H distances restrained.Three-dimensional X-ray data were collected on Bruker X8 APEXII CCD  . Hydroge4}2(µ-PPi)], the data were cleaned up with SQUEEZE  and [{ZnL4}2(μ-PPi)] complexes were carried out in dmso solution employing DFT within the hybrid meta-GGA approximation with the TPSSh exchange-correlation functional [4(H2PPi)] and [{ZnL4}2(μ-PPi)] by replacing the Cu(II) metal ion in the first one. For geometry optimization purposes we used the standard Ahlrichs’ valence double-ξ basis set including polarization functions (SVP) 2+ and [CuL5]2+ complexes show important selectivities towards PPi, ADP, and ATP over anions containing a single phosphate group. The results indicate that the cooperative binding is established for the complexes derived from L4 during the anion recognition and that it is necessary to consider the N-type coordination mode of the urea moiety to prevent the receptor hydrolysis when designing urea based receptors with different delocalized withdrawing groups in their backbones.We have presented a series of Cu


O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (1), (6Z)-3-hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β-d-glucopyranoside (2), (6E)-3-hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β-d-gluco-pyranoside (3), and 7-[(β-d-apiofuranosyl-(1→6)-β-d-glucopyranosyl)oxymethy]-8,8-dimethyl-4,8-dihydrobenzothiazine-3,5-dione (4), were isolated from the fruits of Xanthium sibiricum Patr together with three known compounds, xanthiside (5), adenosine (6), and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one (7). The structures of the new compounds were determined on the basis of detailed spectroscopic analyses.Four new glycosides, namely 3β-norpinan-2-one 3- Xanthium sibiricum Patr. (Compositae), hereafter defined as “Fructus Xanthii”, known as Cang er zi in Traditional Chinese Medicine, are used for treating nasal sinusitis, numbness of limbs, arthritis, ulcer, pruritus, cancer, and herpes -8,8-dimethyl-4,8-dihydrobenzothiazine-3,5-dione (4), as well as three known compounds: xanthiside (5) , two methylene , two oxygenated methylenes , one methyl [C-9 (δ 21.9)] moieties were observed, together with a quaternary carbon signal at δ 76.7 (C-3). In the HMBC spectrum, the key correlations were observed from H3-9 to C-7 and C-8; H2-10 to C-2, C-3, and C-4; and H-1' to C-8. Therefore, the glycosyl group is located at C-8. A diagnostic cross peak was observed between the methyl hydrogen (H3-9) and the olefinic hydrogen (H-6) in the NOESY spectrum -3-hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β-d-glucopyranoside.Compound etection . The 1H-spectrum . Thus, t3 was also obtained as a yellow amorphous powder. The positive-ion HRESIMS of 3 exhibits a quasi-molecular ion peak at m/z 371.1685 [M+Na]+. The molecular formula of 3 (C16H28O8) was determined from the quasi-molecular ion peak and HRESIMS measurements. Acid hydrolysis of 3 liberated d-glucose, which was identified by HPLC with optical rotation detection , [δ 5.88 ], , , [δ 5.48 ], , , [δ 3.41 ] and a β-d-glucopyranosyl moiety [δ 4.23 ]. All 1H-NMR and 13C-NMR data were similar to those for 2. However, an important difference observed for this part was the chemical shift value of the vinyl methyl carbon, δ 14.1 (C-9). Analysis of the 13C-NMR spectra revealed signal values similar to those reported for creoside II -8,8-dimethyl-4,8-dihydrobenzothiazine-3,5-dione.Compound d-sugars . The glyguration ,26. Ther1H-NMR and 100 MHz for 13C-NMR), respectively. Chemical shifts are given as δ values, with reference to that of tetramethylsilane (TMS), which was used as an internal standard. Coupling constants are given in Hz. Preparative HPLC  was performed on a Hypersil-ODS II column . Semipreparative HPLC  was performed using a SunFireTM-C18 column . Silica gel  and ODS-A  were employed for column chromatography (CC).IR spectra were recorded on an IR-47 spectrometer. An optical-rotation detector  was used. The optical rotation was recorded on a Perkin-Elmer 241 polarimeter. The melting points (uncorrected) were measured on a Kofler micromelting point apparatus. The HRESIMS analyses were conducted on an IonSpec Ultima 7.0T FTICR. The UV and NMR spectra were recorded on a Shimadzu UV-1601 and Bruker DPX 400  was deposited at the herbarium of Heilongjiang University of Chinese Medicine.Fructus Xanthii (2.7 kg) was partitioned between EtOAc and H2O and between n-BuOH and H2O. The n-BuOH extract (59.4 g) was subjected to silica gel chromatography using CH2Cl2–MeOH mixtures  to afford fractions (Fr.) A–D. Fr.A was stored and Fr.B (12.1 g) was repeatedly subjected to CC on an ODS column using MeOH–H2O solution [1:7 (3 L)–5:6 (4 L) v/v] as the mobile phase to afford compounds 1 (9 mg) and 6 (24 mg). Fr.C (20.3 g) was subjected to silica gel chromatography using CH2Cl2–MeOH mixtures  to afford subfractions C1 (8.2 g) and C2 (9.7 g). Fr.C2 was repeatedly subjected to separation using Sephadex LH-20 and CH2Cl2–MeOH solution [1:1 (0.7 L) v/v] to yield Fr.C2-1 (4.6 g) and Fr.C2-2 (3.7 g). Fr.C2-2 was subjected to isocratic elution on an ODS column using MeOH–H2O solution [2:1 (0.5 L) v/v] to afford Fr.C2-2-1 (1.0 g). Fr.C2-2-1 was finally purified by preparative HPLC using MeOH–H2O solution  to afford compounds 2 (3.2 mg) and 3 (4.1 mg). Fr.D (10.7 g) was loaded on a Sephadex LH-20 column and eluted with CH2Cl2–MeOH solution [1:1 (0.8 L)] to yield five fractions, D1–D5. D2 (300 mg) was purified by semipreparative HPLC (isocratic elution) using MeOH–H2O solution  to afford compounds 4 (10.0 mg), 7 (19.0 mg), and 5 (57.1 mg).A 70% aqueous EtOH extract of d-apiofuranosyl-(1→3β-Norpinan-2-one 3-O-β-d-glucopyranoside6)-β- (1): white amorphous powder  −10.2  UV λmax (MeOH) nm (log ε): 201(3.18) IR (KBr): 3560, 3495, 2980, 1713, 1075, 1042 cm−11H-NMR: 13C-NMR: m/z [M+Na]+ Calcd for C20H33O11Na 471.1842, Found: m/z 471.1847.d-glucopyranoside(6Z)-3-Hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β- (2): yellow amorphous powder  −6.7  UV λmax (MeOH) nm (log ε): 202(2.10) IR (KBr): 3378, 2980, 2830, 1050, 917; cm−11H-NMR: 13C-NMR: m/z [M+Na]+ Calcd for C16H28O8Na 371.1682, Found: m/z 371.1680.d-glucopyranoside(6E)-3-Hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β- (3): yellow amorphous powder  −8.0  UV λmax (MeOH) nm (log ε): 201(2.10) IR (KBr): 3380, 2979, 2833, 1056, 919; cm−11H-NMR: 13C-NMR: m/z [M+Na]+ Calcd for C16H28O8Na 371.1682, Found: m/z 371.1685.d-Apiofuranosyl-(1→7-[(β-d-glucopyranosyl)oxymethy]-8,8-dimethyl-4,8-dihydrobenzothiazine6)-β--3,5-dione (4): colorless needle crystals mp 190–192 °C  −10.7  UV λmax (MeOH) nm (log ε): 201(2.18), 246(2.87) IR (KBr): 3462, 1690; 1658, 1215, 1177, 929 cm−11H-NMR: 13C-NMR: m/z [M+H]+ Calcd for C22H32NO12S 534.1645, Found: m/z 534.1650.1 (3.0 mg) was dissolved in 2 M HCl (2.0 mL) and then heated at 80 °C in a water bath for 2 h. The reaction mixture was diluted with H2O (20 mL) and then extracted with n-BuOH (20 mL). The aqueous layer was neutralized with Amberlite MB-3 , and then dried under reduced pressure to afford the monosaccharide fraction. The monosaccharide fraction was extracted with MeOH, and the MeOH extract was analyzed using HPLC under the following conditions: HPLC column, Kaseisorb LC NH2-60-5 ; detection, optical rotation ; mobile phase, CH3CN–H2O ; flow rate, 1.0 mL/min. Identification of d-glucose and d-apiose from 1 was carried out by comparing their retention times and optical rotations with those of authentic samples. The values of tR were 11.5 min  and 5.8 min . Through the same method, the monosaccharides were identified as d-glucose for 2 and 3, and d-glucose and d-apiose for 4.Compound Fructus Xanthii, four new glycosides, namely 3β-norpinan-2-one 3-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (1), (6Z)-3-hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β-d-glucopyranoside (2), (6E)-3-hydroxymethyl-7-methylocta-1,6-dien-3-ol 8-O-β-d-glucopyranoside (3), and 7-[(β-d-apiofuranosyl-(1→6)-β-d-glucopyranosyl)oxymethy]-8,8-dimethyl-4,8-dihydrobenzothiazine-3,5-dione (4) were isolated. Their structures were established on the basis of spectroscopic evidence. This discovery represents a further addition to the number and diversity of glycosidic compounds.As a part of our chemical investigation on


Their structures and stereochemistries were elucidated by spectroscopic methods, including 1D- and 2D-NMR and HR-ESI-MS techniques.Two new dibenzocyclooctadiene lignans, neglschisandrins C-D ( Schisandra neglecta were reported +). The UV spectrum, with absorption maxima at 217, 252 and 280 nm, along with the 1H-NMR and 13C-NMR data mentioned below indicated that compound 1 was a dibenzocyclooctadiene lignan cycloocten-1-yl ester.The circular dichroism (CD) spectrum showed a negative guration . The NOEene ring . The ste2), obtained as a colorless powder, had the molecular formula C30H34O7 according to HR-ESI-MS [m/z 529.2180 ([M+Na]+)]. The UV absorptions  and NMR spectra  were assigned to 7-Me and 8-Me, respectively. This suggested that there was no substitution at C-7 and C-8, and these two methyl groups were in cis-orientation cycloocten-1- yl ester.The circular dichroism (CD) spectrum showed a negative ss peaks  for H-4 -OMe see . From th254 . Column chromatography (CC): Silica gel . Prep HPLC: Amersham UV-900, with RP-C18 column (250×10 mm). UV : Hitachi U-3010 spectrophotometer. CD Spectra [λ in nm (∆ε in mdeg)]: Jasco-810 spectropolarimeter. Optical rotation (ORD): JASCO P-1020 spectropolarimeter. IR Spectra : Avatar 360-ESP spectrophotometer (Thermo Nicolet). 1H- (400 MHz) and 13C-NMR (100 MHz) spectra in CDCl3 soln.; δ in ppm rel. to Me4Si, J in Hz): Bruker DRX400 Spectrometer. ESI-MS (m/z): Bio TOF Q spectrometer; HR-ESI-MS (m/z): Bruker Dalonics-BioToF Q spectrometer.TLC: Silica-gel plates GFSchisandra neglecta were collected in Lin-zhi County, Xi-zang Autonomous Region, People’s Republic of China, in September of 2004, and identified by Associate Professor Hong-ping Deng of the School of Life Sciences, SouthWest University. A voucher specimen (MC-LZ-040901) is deposited in the Herbarium of Medicinal Plant, School of Life Sciences, SouthWest University, Chongqing, People’s Republic of China.Stems of Schisandra neglecta (5 kg) were ground and extracted exhaustively with 95% ethanol at room temperature. The EtOH extract was evaporated in vacuo to yield a semisolid (430 g), which was suspended in H2O (1 L) and extracted with Et2O (5×1L). This ether solution was concentrated to yield 112 g of residue, which was subjected to CC . Fraction 4 (eluted with PE/acetone 9:1) was subjected to repeated CC (eluted with PE/EtOAc 15:1) and prep. RP-HPLC (MeOH/H2O 70:30) to yield compound 2 (2 mg). Fraction 5 (eluted with PE/acetone 8:2) was subjected to repeated CC (eluted with PE/CHCl3 1:1) and prep. RP-HPLC (MeOH/H2O 80:20) to give compound 1 (29 mg).Air-dried stems of -3-phenylacrylic acid-7-hydroxy-2,3,10,11,12-pentamethoxy-6,7-dimethyl-5,6,7,8-tetra-hydrodibenzocycloocten-1-yl ester . Colorless powder; UV: 217 (4.44), 252 (4.05), 280 (3.23); CD : nm (Δε) 249 (+39.76), 234 (+55.71), 215 (-76.79); =+72.1°; IR: 3415, 1726, 1636, 1594, 1494;  1H-NMR and 13C-NMR: see m/z: 571.1 ([M+Na]+); HR-ESI-MS: found 571.2290 .-benzoic acid-2,3,10,11,12-pentamethoxy-6,7-methyl-5,6,7,8-tetrahydrodibenzo-cycloocten-1-yl ester . Colorless powder; UV: 226 (4.42), 250 (4.12), 279 (3.71); CD : nm (Δε) 247 (+312.52), 233 (+389.88), 211 (-278.28); =+79.6°; IR: 1738, 1597, 1493, 707; 1H-NMR and 13C-NMR: see m/z: 529.5 ([M+Na]+); HR-ESI-MS: found 529.2180 .


D. divaricata collected off the coast of Yantai (P.R. China) was dried, powdered, and extracted with the mixture of CHCl3 and MeOH . By a combination of silica gel and Sephadex LH-20 column chromatography and preparative TLC, a new cadinane sesquiterpene 1,4-epoxymuurolan-5β-ol (1) was isolated from this species. Its structure was established by detailed MS and NMR spectroscopic analysis, as well as comparison with literature data.A sample of the marine brown alga  Dictyopteris are prolific sources of sesquiterpenes, and cadinane is a main carbon skeleton type +. The molecular formula was determined as C15H26O2 on the basis of HRESIMS , suggesting three degrees of unsaturation. The 1H-NMR spectrum of 1   to give ten fractions, I-X. Fraction VII, eluted with PE/EtOAc 2:1, was further purified by Sephadex LH-20 (CHCl3/CH3OH) and RP-18 (CH3OH/H2O 3:1) column chromatography and preparative TLC (PE/EtOAc 3:1) to afford 1,4-epoxymuurolan-5β-ol  as a colorless oil; [α]25D –29.2° ; IR (KBr) cm-1: 3,452, 2,962, 2,954, 2,870, 1,458, 1,377, 1,065; 1H-NMR and 13C-NMR: see m/z: 261 [M+Na]+; HRESIMS m/z: 261.1829 [M+Na]+, calcd. for C15H26O2Na, 261.1830. Dried and powdered alga Brine shrimp assays were performed as previously described .


Calophyllum genus has led us toa detailed study on the chemistry of the stem bark of Calophyllum soulattri. This gave one new pyranoxanthone, soulattrin (1), together with three other xanthones, caloxanthone B (2), caloxanthone C (3), macluraxanthone (4), the triterpene friedelin (5) and the steroid stigmasterol (6). The identities of these compounds were established using analyses of 1D and 2D NMR data.Our interest on xanthones from the  Calophyllum is widely distributed in Asia, Australia, Africa and Polynesia. Some species of Calophyllum have been used in folk medicine. Previous studies indicated the existence of a variety of secondary metabolites such as xanthones xanthone and named soulattrin  as internal standard. Ultraviolet spectra were recorded in EtOH on a Shimadzu UV-160A UV-Visible Recording Spectrophotometer. Infrared spectra were measured using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 Series FT-IR spectrometer. Melting points were measured using Leica Galen III microscope, equipped with Testo 720 temperature recorder.Calophyllum soulattri was ground into fine powder and extracted successively in a Soxhlet apparatus with n-hexane, dichloromethane, ethyl acetate and methanol for 72 hours. The extracts were evaporated to dryness under vacuum to give 101.2 g of hexane extract and 15.3 g of dichloromethane extract. Part of each extract was subjected to column chromatography over silica gel and eluted with a stepwise gradient system using n-hexane, dichloromethane, ethyl acetate and methanol. Further purifications of the hexane extract afforded friedelin . Meanwhile, purification of the dichloromethane extract afforded the new xanthone, soulattrin , caloxanthone B , caloxanthone C , macluraxanthone  and stigmasterol .Approximately 1 kg of air-dried stem bark of Soulattrin (1) Yellow crystals; m.p. 180–181 °C; UV (EtOH) λmax nm (log ε): 336 (5.42), 291 (5.36), 281 (5.35), 242 (5.28); IR νmax cm−1: 3300, 2969, 1640, 1583; EIMS m/z (rel. int.): 394 [M+] (33), 379 (100), 365 (6), 339 (6), 321 (6), 182 (11), 162 (18); HRESIMS: 394.1550 ; 1H-NMR (CDCl3): δ 13.86 , 6.95 , 7.54 , 6.66 , 5.64 , 6.46 , 4.99 , 4.84 , 1.70 , 1.45 ; 13C-NMR (CDCl3): δ 181.1 (C-9), 158.8 (C-3), 156.5 (C-1), 155.3 (C-4a), 151.8 (C-2’), 151.2 (C-6), 146.0 (C-5a), 133.0 (C-4), 127.5 (C-11), 116.2 (C-8), 115.6 (C-10), 113.5 (C-5), 112.9 (C-7), 107.0 (C-3’), 104.9 (C-2 & C-9a), 102.8 (C-8a), 78.2 (C-12), 41.0 (C-1’), 151.8 (C-2’), 106.7 (C-3’), 29.3 (C-4’ & C-5’), 27.2 (C-13 & C-14).Caloxanthone B (2) Yellow needles; m.p. 157–158 °C (Lit. 160.5 °C); spectral data are consistent with literature [15].Calophyllum soulattri furnished one new prenylated pyranoxanthone, soulattrin (1), together with three other xanthones, caloxanthone B (2), caloxanthone C (3) and macluraxanthone (4) the common triterpene friedelin (5) and the steroid stigmasterol (6).The stem bark of


Compound 3c, 6-(4-methoxyphenyl) benzo[d]thiazole-2-amine, exhibited the highest nitric oxide percentage scavenging at 100 µg/mL.In general, benzothiazole derivatives have attracted great interest due to their pharmaceutical and biological importance. New 2-amino-6-arylbenzothiazoles were synthesized in moderate to excellent yields via Suzuki cross coupling reactions using various aryl boronic acids and aryl boronic acid pinacol esters and the antiurease and nitric oxide (NO) scavenging activity of the products were also examined. The most active compound concerning urease enzyme inhibition was 6 Results of the Suzuki cross coupling reactions under optimized condition are summarized in Firstly, we studied the optimization of the reaction solvent. Thus, the effect of solvents on the coupling reactions between aryl boronic acids/esters and 2-amino-6-bromobenzothiazole (2) (2.183 mmol) was coupled with tolyl boronic acids (2.401 mmol) using a 4:1 toluene/water ratio in the presence of 5 mol% Pd(0) catalyst using K3PO4 (4.366 mmol) as base, product 3a was obtained with moderate yield. However, when 1,4 dioxane was used in the the reaction solvent, 6-tolybenzo-[d]thiazole-2-amine (3a) was obtained after 31 h heating (Table the Suzuki cross coupling of 2-amino-6-bromobenzothiazole (2) with 4-methoxyphenyl boronic acid (4.366 mmol) in refluxing with DMF using 5 mol% of Pd(0) catalyst this resulted in the conversion into 6-(4methoxyphenyl)benzo[d]thiazol-2-amine (3c) in 64% isolated yield) (When 2-amino-6-bromobenzothiazole (d yield) , Table 12) was smoothly coupled with 1,3-trifluorobenzene boronic ester in 1,4-dioxane under heating conditions with conversion into the corresponding 6-phenyl)benzo[d]thiazol-2-amine (3d) in high yield  acetamide (4) in excellent 85% isolated yield. The reaction was carried out by the acylation of 2-amino-6-bromobenzothiazole (2) using a previously reported method thiazol-2-amine (3a) proved to be the most potent urease inhibitors, with activities of 95 ± 0.005 and 90.49 ± 0.002 at 50 μg/mL with IC50 values of 28.4 and 27.27, respectively.All synthesized compounds showed very good to excellent urease inhibition activity. The compounds 2-amino-6-bromobenzothiazole (N-(6-bromobenzo[d]thiazol-2-yl)acetamide (4) also showed comparatively good activity, with a percentage inhibition of 39 ± 0.001 at 25 μg/mL and 89 ± 0.009 at 50 μg/mL with an IC50 value of 30.5. Munakata et al. showed that carbon chain and branch chain structure of the aryl residues apparently have no effect on activity. The additional fall in inhibitory potency may be attributed to less interaction of the surrounding residues of urease active site with the non- aromatic cyclohexane functional group thiazol-2-amine (3e). The IC50 values were 28.57 and 26.35, respectively. Contribution of various functional groups present on 2-amino-6-bromobenzothiazole (2) in urease inhibition was also investigated. In general, compounds containing electron-donating functional groups exhibited high anti-urease activity. 2-Amino-6-bromobenzothiazole (2) and compound 3a containing an electron-donating methyl functional group exhibited high urease activity, whereas compound 3c containing methoxy functional group also showed good uease anti-urease activity. These observations are in agreement with the expected results. Since urease needs Ni ions as a cofactor, compounds containing electron donating groups would be expected, though neighbouring group participation to facilitate substituent amino or imino groups to chelate these ions thereby inactivating the urease enzyme.Urease inhibitions of 86.24 ± 0.001 and 85 ± 0.002 percent were seen at 50 μg/mL for 6-(4-methoxyphenyl)benzo[d]thiazol-2-amine (Nitric acid (NO) is a free radical causing cancer  and infl50 values are shown in The newly synthesized compounds showed very good to excellent ability to scavenge NO radical ,36. Asco3c) and 6-p-tolylbenzo[d]thiazol-2-amine (3a) were found to be the most potent NO scavengers with percentage activities of 67 ± 0.002 and 67 ± 0.009 at 50 μg/mL with IC50 values of 68.3 and 38.19 μg/mL each, respectively. N-(6-bromobenzo[d]thiazol-2-yl) acetamide (4) also exhibited comparatively good activity showing a percentage NO scavenging of 54 ± 0.002 at 50 μg/mL and 92 ± 0.002 at 100 μg/mL, with an IC50 of 46.5. Compounds 3a, 3c, 4 presented significant percentages of NO scavenging  and IC50 values were 68.3, 38.9 and 46.5, respectively. 2-Amino-6-bromobenzothiazole (2) also exhibited good NO scavenging activity, 48 ± 0.0007 at 50 μg/mL and 90 ± 0.007 at 100 μg/mL with an IC50 of 57.8. Moderate NO scavenging percentages of 34 ± 0.005, 23 ± 0.009 and 22 ± 0.001 at 50 μg/mL were observed for 6-(4-chlorophenyl)benzo[d]thiazol-2-amine (3b), 6-phenyl)benzo[d]thiazol-2-amine (3d) and 6-phenylbenzo[d]thiazol-2-amine (3e) with IC50 values of 75, 83.75 and 96.66, respectively.The compounds 6-(4-methoxyphenyl)benzo[d]thiazol-2-amine  and silica gel (230–400 mesh) were used. The reaction were monitored on TLC, using Merk Silica gel 60PF254 cards. The compound were visualized by UV lamp . Melting points were determined using a Buchi Melting Point B-540 apparatus. All reagents were purchased from Alfa Aesar and Sigma Aldrich company. 3a–e, to 2-amino-6-bromobenzothiazole , under a nitrogen atmosphere Pd(PPh3)4 (5 mol%) was added and the resulting mixture stirred for 30 min with the addition of the appropriate solvent (3 mL). After 30 min the aryl boronic acid (2.401 mmol), and K3PO4 (4.366 mmol) were added, following the addition of water (1.5 mL). The solution was stirred for 31 h at 95 °C, removed from heating and cooles to room temperature. After work up with ethyl acetate, the organic layer was separated, dried with magnesium sulfate and the solvent was removed under vacuum. The crude solid residue obtained was purified by column chromatography using a 50% ratio of ethyl-acetate and n-hexane to give the desired products, which were further analyzed by using different spectroscopic techniques thiazole-2-amine (2). M.p. 215–217 °C; 1H-NMR : δ = 7.80 , 7.67–7.42 , 7.30 . 13C-NMR : δ = 119.86, 120.35, 123.58, 124.45, 129.78, 154.0, 167.0; EIMS (m/z -ion mode): 228.92 [M−H]; [M-bromine and 2H fragment]− = 147.00. Anal. Calcd for C7H5 N2S: C, 36.70; H, 2.20; N, 12.24. Found: C, 36.63; H, 2.15; N, 12.20.N-(6-Bromobenzo[d]thiazol-2-yl)acetamide (4). M.p. 193–195 °C; 1H-NMR : δ = 7.94 , 7.61–7.53 , 12.20 , 2.31 . 13C-NMR : δ = 23.50, 117.12, 121.62, 124.17, 129.95, 146.22, 148.71, 159.20, 168.37; EIMS (m/z -ion mode): 271.08 [M−H−]; [M−Acetyl fragment]− = 229.00. Anal. Calcd for C9H7Br N2OS: C, 39.88; H, 2.59; N, 10.34. Found: C, 39.56; H, 2.43; N, 10.29.6-p-Tolylbenzo[d]thiazole-2-amine (3a). M.p. 184–186 °C; 1H-NMR : δ = 8.11 , 7.7–7.30 , 7.25 . 13C-NMR : δ = 21.46, 116.01, 119.20, 128.57 (2C), 128.69, 132.09, 132.81 (2C), 134.0, 135.72, 140.81, 145.96, 167.85; EIMS (m/z -ion mode): 239.08 [M−H−]; [M-2H fragment]− = 237.08; [M-benzene-CH3 fragment]− = 149.17; [M−CH3 fragment and 2-H]− = 221.25. Anal. Calcd for C14H12 N2S: C, 69.97; H, 5.03; N, 11.66. Found: C, 69.75; H, 4.95; N, 11.46.6-(4-Chlorophenyl)benzo[d]thiazole-2-amine (3b). M.P, 194–196 °C; 1H-NMR : δ = 7.59 , 7.43–7.26 . 13C-NMR : δ = 114.65, 119.27, 122.96, 123.42 (2C), 128.51, 128.91, 129.32 (2C), 132.03, 134.96, 150.02, 167.48; EIMS (m/z -ion mode): 259.08 [M-H−]; [M-NH2 fragment]− = 243.00; [M-benzene and Cl-fragment]− = 149.08 Anal. Calcd for C13H9Cl N2S: C, 59.88; H, 3.48; N, 10.74. Found: C, 59.78; H, 3.33; N, 10.69.6-(4-Methoxyphenyl)benzo[d]thiazole-2-amine (3c). M.p. 165–167 °C; 1H-NMR : δ = 7.56–7.40 , 7.38 , 7.25 (2H-NH2), 3.48 . 13C-NMR : δ = 49.57, 114.51, 118.13, 122.96, 123.37 (2C), 129.24 (2C), 131.88, 132.28, 149.38, 167.73; EIMS (m/z -ion mode): 255.08 [M−H−]; [M−OCH3 fragment]− = 237.17; [M-CH3 and N fragment]− = 227.08; [M-NH fragment]− =242.0. Anal. Calcd for C14H12N2OS: C, 65.60; H, 4.72; N, 10.93. Found: C, 65.54; H, 4.57; N, 10.79. 6-phenyl)benzo[d]thiazole-2-amine (3d). M.p. 180–182 °C; 1H-NMR : δ = 8.22 , 8.08 , 7.86–7.55 , 7.26 . 13C-NMR : δ = 116.6, 117.5, 122.6, 123.5, 124.72 (2C), 128.5, 129.73, 132.03 (2c), 133.5, 134.50, 152.02, 168.50; EIMS (m/z -ion mode): 361.08 [M−H−]; [M-NH2 fragment]− = 346.2; [M-2-aminobenzothiazole fragment]− = 214.11. Anal. Calcd for C15H8 F6N2S: C, 49.73; H, 2.23; N, 7.73. Found: C, 99.35; H, 2.19; N, 7.69.6-Phenylbenzo[d]thiazole-2-amine (3e). M.p. 175–177 °C; 1H-NMR : δ = 7.59 , 7.57–7.32 , 7.27 . 13C-NMR : δ = 115.01, 118.70, 123.57, 128.56, 128.68 (2C), 129.57 (2C), 131.91, 132.24, 132.01, 147.61, 167.85; EIMS (m/z -ion mode): 225.08 [M−H−]; [M-benzene and 2 H fragment]− = 147.08; [M-benzene and NH2 fragment]− = 135.17. Anal. Calcd for C13H10N2S: C, 69.43; H, 4.45; N, 12.38. Found: C, 69.39; H, 4.43; N, 12.36.N-1-naphthylethylenediamine dihydrochloride under acidic conditions to detect NO2− generated at the expense of NO by the antioxidant system. Briefly, a known quantity of the test compounds 2, 3a–e, 4 in solution form was mixed with 20 mM sodium nitroprusside solution (100 µL). Total volume was made up to 1,000 µL with 200 mM Phosphate buffer, pH 7.4. The contents were mixed well and incubated at 37 °C for 2 h followed by addition of Griess reagent (100 µL). The mixture was kept at room temperature for 20 min. Optical density (OD) of the colored solution formed was measured at 528 nm. Ascorbic acid was used as the positive control while the negative control was used to develop the standard curve thiazol-2-yl)acetamide starting from 2-amino-6-bromobenzothiazole. A palladium catalyst was used in Suzuki coupling reactions in the presence of different boronic acids/esters. The results of this study reveal that 2-aminobenzothiazole derivatives can be used explored as anti-urease and antioxidant molecules.In summary we have reported an efficient synthesis of 2-amino-6-arylbenzothiazoles and


Cell viability studies for benzotriazin-7-ones and 1,2,4-benzotriazinyl (Blatter-type) radical precursors are described with comparisons made with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). All of the stable free radicals were several orders of magnitude less cytotoxic than the benzotriazin-7-ones. The synthesis and evaluation of two new pyrid-2-yl benzotriazin-7-ones are described, where altering the 1,3-substitution from phenyl to pyrid-2-yl increased cytotoxicity against most cancer cell lines, as indicated using National Cancer Institute (NCI) one-dose testing. COMPARE analysis of five-dose testing data from the NCI showed very strong correlations to the naturally occurring anti-cancer compound pleurotin. COMPARE is program, which analyzes similarities in cytotoxicity data of compounds, and enables quantitative expression as Pearson correlation coefficients. Compounds were also evaluated using the independent MTT assay, which was compared with SRB assay data generated at the NCI. Incorporating pyridine is reported to significantly alter cytotoxicity against certain cancer cell lines, hence we made the choice of replacing phenyl in the parent structure 1 with the pyrid-2-yl substituent in 4a and 4b triazin-7-ones 1 and 2  showed v,4triazin disease , in thisa and 4b ,7,8.3a and 3b has previously been reported triazin-4-yl (3a) and 1-phenyl-3-(pyrid-2-yl)-1,4-dihydrobenzo[e]triazin-4-yl (3b) were prepared according to literature procedures [Reactions were protected from atmospheric moisture using CaClGermany) . Meltingocedures .3-Phenyl-1-(pyrid-2-yl)benzo[e]triazin-7(1H)-one (4a). To a stirred solution of 3-phenyl-1-(pyrid-2-yl)-1,4-dihydro-1,2,4-benzotriazin-4-yl (3a)  in dichloromethane (10 mL), MnO2  was added and the reaction mixture was stirred at ca. 20 °C for two days. The reaction mixture was filtered through Celite® , rinsed with additional dichloromethane (50 mL), and volatiles were removed in vacuo. The residue was chromatographed on a short pad of silica with tert-butyl methyl ether (TBME) to give the title compound 4a  as purple needles, m.p. (hot-stage): 210.1–213.8 °C (from PhH); m.p. (DSC) onset: 209.9 °C, peak max: 210.5 °C, decomp. onset: 225.8 °C, decomp. peak max: 227.7 °C (from PhH); Rf 0.58 ; found: C, 71.87; H, 4.12; N, 18.70. C18H12N4O requires C, 71.99; H, 4.03; N, 18.66%; λmax(DCM)/nm 295 (logε 4.58), 310 inf (4.46), 339 inf (3.91), 356 inf (3.91), 544 (3.75), 582 (3.70), 635 inf (3.33); νmax/cm−1 3073w (aryl C-H), 1624s, 1599s, 1587s, 1570m, 1547s, 1526m, 1497w, 1468m, 1437s, 1398w, 1385w, 1333w, 1310w, 1281w, 1238m, 1192m, 1150w, 1115w, 1103w, 1094w, 1072w, 1028w, 995w, 974w, 905w, 856s, 787s,781m, 760s, 737s; δH  8.63 , 8.26–8.25 , 8.02 , 7.80 , 7.66 , 7.48–7.46 , 7.25 , 6.78 ; δC  183.2 , 156.5 (s), 154.6 (s), 150.0 (s), 148.5 (d), 141.5 (d), 139.5 (d), 134.3 (s), 133.7 (s), 132.7 (d), 130.6 (d), 128.8 (d), 126.7 (d), 124.3 (d), 120.0 (d), 99.9 (d); m/z (MALDI-TOF) 302 , 301 , 300 , 299 , 272 (16).1-Phenyl-3-(pyrid-2-yl)benzo[e]triazin-7(1H)-one (4b). To a stirred solution of 1-phenyl-3-(pyrid-2-yl)-1,4-dihydrobenzo[e]triazin-4-yl (3b)  in dichloromethane (10 mL), MnO2  was added and the reaction mixture was stirred at ca. 20 °C for two days. Upon completion, the reaction mixture was filtered through a short pad of Celite®, rinsed with additional dichloromethane (50 mL), and volatiles were removed in vacuo. The residue was chromatographed on a short pad of silica (acetone) to give the title compound 4b  as purple needles; m.p. (hot-stage): decomp: 199.8–204.0 °C (from PhH); m.p. (DSC) decomp. onset: 205.8 °C, peak max: 210.8 °C (from PhH); Rf 0.69 ; found: C, 71.85; H, 4.13; N, 18.48. C18H12N4O requires C, 71.99; H, 4.03; N, 18.66%; λmax(DCM)/nm 240 (logε 4.15), 300 (4.46), 310 inf (4.41), 338 inf (4.02), 351 inf (4.05), 374 inf (3.75), 534 (3.64), 572 (3.60), 628 inf (3.24); νmax/cm−1 3040w (aryl C-H), 1624m, 1611m, 1589m, 1584m, 1541s, 1537s, 1522m, 1493m, 1477m, 1456m, 1435m, 1395m, 1331m, 1304m, 1233m, 1200m, 1150w, 1117m, 1096w, 1072w, 1045w, 1026w, 995w, 978w, 928w, 907w, 856s, 822m, 814m, 800m, 779s, 764m, 745m; δH 8.84 , 8.31 , 7.88–7.83 , 7.65–7.55 , 7.41 , 7.32 , 6.09 ; δC 182.2 (s), 155.3 (s), 151.6 (s), 150.2 (d), 149.8 (s), 142.5 (d), 140.7 (s), 137.0 (d), 136.6 (s), 132.4 (d), 130.3 (d), 130.1 (d), 125.7 (d), 124.7 (d), 122.3 (d), 98.4 (d); m/z (MALDI-TOF) 302 , 301 , 300 , 285 (19), 273 (28), 242 (35).1 was previously reported triazin-7-one DU-145 was grown in RPMI-1640 medium and supplemented with 1% 2 Mm L-glutamine, 1% penicillin-streptomycin, and 10% non-heat inactivated fetal bovine serum (FBS). MCF-7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose (4.5 g/mL) and supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (FBS). All cells grew as adherent cultures. Cell culture reagents were obtained from Sigma-Aldrich. Disposable sterile plastic ware was obtained from Sarstedt . v/v final concentration in well). The control cells were exposed to the same concentration of the vehicle control alone (DMSO). All cells were incubated at 37 °C and 5% CO2 (humidified atmosphere) for 72 h. MTT  was added and the cells were incubated for a further 3 h. The supernatant was then removed using a multi-transfer pipette and DMSO (100 µL) added to dissolve the MTT formazan crystals. The absorbance was determined using a plate reader at 550 nm with a reference at 690 nm. Cell viability is expressed as a percentage of the vehicle-only treated control (DMSO). Dose-response curves were analyzed by non-linear regression analysis and IC50 values were determined using GraphPad Prism software, v 8.0 . The in vitro activity of the drugs towards all cell lines is expressed as IC50 .The MTT colorimetric assay was used to determine cell viability . Cells w3a and 3b were significantly less cytotoxic than their oxidation products, benzotriazin-7-ones 4a and 4b, towards the cancer cell lines evaluated. Pyridyl-substituted benzotriazin-7-ones exhibited submicromolar cytotoxicity using the MTT assay on par with 1,3-bisphenylbenzotriazin-7-one 1. The variable DTP-NCI one-dose testing cytotoxicity profiles for 4a and 4b led to their selection for five-dose testing. COMPARE analysis demonstrated very strong correlations to pleurotin, despite the overall greater cytotoxicity of the pyrid-2-yl-substituted compounds compared to 1 after one-dose testing.All three stable free radicals evaluated were significantly more cytotoxic towards DU-145 than the MCF-7 cell line. Benzotriazin-4-yl radicals


Phospholipids with NSAIDs at sn-1 and palmitic acid at sn-2 (9 and 10) were also less toxic against Caco-2 and normal cells (IPEC-J2).In this study, novel phosphatidylcholines containing ibuprofen or naproxen moieties were synthesized in good yields and high purities. Under the given synthesis conditions, the attached drug moieties racemized, which resulted in the formation of phospholipid diastereomers. The comperative studies of the cytotoxicity of ibuprofen, naproxen and their phosphatidylcholine derivatives against human promyelocytic leukemia HL-60, human colon carcinoma Caco-2, and porcine epithelial intestinal IPEC-J2 cells were carried out. The results of these studies indicated that phospholipids with NSAIDs at both  NSAIDs are a large heterogeneous group of compounds and their classification is based mainly on their chemical structure. The most well-known group of NSAIDs are derivatives of 2-arylpropionic acid (profens) such as ibuprofen (1) and naproxen (2)  exhibit anti-inflammatory, analgesic, and antipyretic effectsbuprofen  and naprS isomers of profens consistently bind to proteins and inhibit prostaglandin synthesis4. In-vivo studies have shown that in some animal organisms, including humans, the inactive R enantiomer can be converted to a pharmacologically active S isomer by 2-arylpropionyl-CoA epimerase5. Since the synthesis of optically active drugs is costly, the production and sale of racemic mixtures has been approved. However, preparations containing drugs as (S)-isomers are also available, e.g., dexibuprofen and dexketoprofen7.Only 1) and naproxen (2), show lack of selectivity and inhibit the cyclooxygenase-1 (COX-1) enzyme, which is involved in maintaining the normal homeostasis of the body8. Therefore, long-term use of ibuprofen (1) or naproxen (2) has negative effects on the gastrointestinal tract (GI), leading to damage of the mucous membrane and consequent bleeding and perforation9.The mechanism of action of all NSAIDs is closely related to the inhibition of the enzyme cyclooxygenase-2 (COX-2). Unfortunately, most drugs, including ibuprofen (11 and the derivatives of NSAIDs with phosphate moieties (phospho-NSAIDs)12. Moreover, it has been reported that phospho-NSAIDs are safer than normal drugs because of their reduced gastrointestinal toxicity13. In addition to their anti-inflammatory properties, it has been found that phospho-NSAIDs exhibit anticancer properties against colon and breast cancer16.Because of this GI toxicity, there is a clear need to create new generations of NSAIDs. In recent years, many NSAID derivatives have been found to exhibit similar activities but with less side effects than standard drugs. From these, noteworthy are the nitric oxide-releasing prodrugs of NSAIDs (NO-NSAIDs)19. One reason for this is that they are non-toxic and biocompatible. Studies that were carried out on animal and human cells indicated that the mixture of NSAIDs and phosphatidylcholine exhibits stronger activity at identical concentrations and lower GI toxicity than NSAIDs alone22. Moreover, phospholipids with linked drugs mediate the transport of compounds and increase their bioavailability23. Our studies showed that compounds with anticancer activity linked covalently with phosphatidylcholine in the place of natural fatty acids lose their toxicity toward normal cells25.Among different approaches to improving drug quality, there has been a huge potential for drugs combined with phospholipids1) or naproxen (2) at sn-1 and/or sn-2 positions. Phosphatidylcholines with ibuprofen have previously been reported26 but herein we applied a different, shorter synthetic pathway. Additionally, the cytotoxicity of the newly synthesized phospholipid derivatives against human promyelocytic leukemia HL-60 cells, human colon carcinoma Caco-2 cells, and porcine epithelial intestinal IPEC-J2 cells was studied.In this paper, we report the synthesis of phosphatidylcholines containing ibuprofen (1) or naproxen (2) moieties at the sn-1 position (7–10) were synthesized according to Fig. 3 and 4, and stannylene acetal (6) were obtained. Acyl chlorides resulted from the reaction of acids 1 and 2 with oxalyl chloride in the presence of catalytic amounts of anhydrous DMF. Then, according to known procedures28, the reaction of stannylene acetal (6) and acyl chloride (3 or 4) gave the corresponding 2-lysophosphatidylcholine (7 or 8) in good yields . In the next step, lysophosphatidylcholines 7 and 8 were esterified with palmitic acid using the Steglich conditions to give phosphatidylcholines 9 and 10, respectively.Phosphatidylcholines containing ibuprofen  for the methyl groups and at 2.41 ppm  for the methylene group. Moreover, the isobutyl methine proton was observed as a multiplet at δ = 1.81, CH3-3′ was detected as a doublet (J = 7.2 Hz) at 1.46 ppm, and the H-2′ methine proton was identified as a quartet (J = 7.2 Hz) at 3.72 ppm. Additionally, in the 1H NMR spectrum of lysophosphatidylcholine 8 a characteristic singlet of the methoxy group at δ = 3.88 was visible, and the H-2′ and H-2 methine protons overlapped and gave multiplets in the range of 3.89–3.93 ppm (Supplementary Information).The structures of lysophosphatidylcholines sn-2 position was confirmed by the appearance of signals from fatty acid residue in the 1H NMR spectra of phosphatidylcholines 9 and 10. Triplets from terminal methyl groups at δ = 0.84 (J = 7.1 Hz) for 9 and at δ = 0.85 (J = 7.0 Hz) for 10 were observed. Additionally, the structures of 9 and 10 were confirmed by the shift in the methine proton H-2 signals from δ = 3.92 (7) to δ = 5.19 (9) and from δ = 3.91 (8) to δ = 5.19 (10), respectively. Moreover, phosphatidylcholines 9 and 10 were obtained as mixtures of diastereomers, which was a result of the enolization of the carboester group with contribution of H-2′ from the ibuprofen or naproxen moiety under mildly alkaline conditions29. However, in acidic solutions, NSAIDs are reportedly highly stable and do not undergo racemization30.The introduction of palmitic acid into the 9 was confirmed by the two quartets (J = 7.2 Hz) of H-2′ observed at 3.66 and 3.69 ppm and the two doublets (J = 7.2 Hz) of CH3-3′ at 1.43 and 1.44 ppm in the 1H NMR spectrum. Double signals were also observed for CH2-1 and CH2-3, and in the 13C NMR spectrum: C-1, C-1′, C-2′, C-3′, C-1″′, C-2″′, and C-3″′. The composition of the phosphatidylcholine isomers mixture (9) was determined by integrating one of the CH2-1 protons. Assuming that there was a higher content of phosphatidylcholine with S-ibuprofen, which was the starting enantiomer in the synthesis, the quantitative composition was defined as 77% 2 R,2′S isomer and 23% 2 R,2′R isomer.The presence of two diastereomers of phosphocholine 10) with a naproxen moiety at the sn-1 position and palmitoyl residue at the sn-2 position was also obtained as a mixture of diastereomers. However, a pronounced advantage of the 2 R,2′S isomer was observed in this case. The intensities of the 1H NMR signals from the 2 R,2′R isomer was so low (below 5%) that the percentage composition of the mixture could not be accurately determined. The smaller degree of racemization of the S-naproxen can be explained by the electron-donating effect of the methoxy group on the enol formation process.Phosphatidylcholine (sn-1 position and ibuprofen (12) or naproxen (13) at the sn-2 position were synthesized as depicted in Fig. 11 was prepared according to a known procedure by reacting stannylene acetal (6) and palmitoyl chloride31. Then, 2–lysophosphatidylcholine 11 was esterified with ibuprofen (1) or naproxen (2) using the Steglich conditions.Phosphatidylcholines containing palmitoyl residue at the aproxen 1 at the s12 and 13 were confirmed by NMR spectroscopy. In the 1H NMR spectrum of 12 all signals of the palmitoyl residue were visible. Signals from the isobutyl group were observed at 2.41 ppm  for the methylene protons, at 1.81 ppm (multiplet) for the methine proton, and at 0.86 ppm  for the methyl groups. The presence of a multiplet at 5.20 ppm for H-2 indicated the attachment of the ibuprofen molecule onto the sn-2 position. Also, in the range of 7.03–7.17 ppm multiplets of the ibuprofen aromatic protons were visible. In addition, the structure of phosphatidylcholine 13 was confirmed by the presence of the following signals: 1.53 ppm  from CH3-3′, 3.88 ppm (singlet) from the methoxy group, 5.21 ppm (multiplet) from H-2 and multiplets in the range of 7.08–7.70 ppm from the aromatic protons (Supplementary Information).The structures of phosphatidylcholines 9, phosphatidylcholine 12, was obtained as a mixture of diastereomers with similar quantitative composition: 73% of the 2 R,2′S isomer and 27% of the 2 R,2′R isomer. The presence of two diastereomers of 12 was indicated by the doubled signals for C3′ (17.57 and 17.63 ppm), C-2″′ (33.25 and 33.52 ppm), and C-3″′ (24.14 and 24.77 ppm) in the 13C NMR spectrum. Interestingly, the rest of the carbon atoms from phosphatidylcholine 12 were present in the 13C NMR spectrum as single signals. In contrast to compound 12, phosphatidylcholine 13 containing naproxen at the sn-2 position was detected as one isomer .Similar to compound sn-1 and sn-2 positions (15 and 16) were synthesized in a one-step synthesis from the highly pure sn-glycero-3-phosphocholine (GPC) and ibuprofen (1) or naproxen (2), respectively  was prepared according to a previously published procedure24. In the synthesis of 15 and 16, the Steglich esterification of two free hydroxyl groups in the presence of a fourfold molar excess of ibuprofen (1) or naproxen (2) was applied.Phosphatidylcholines containing ibuprofen or naproxen moieties at both ely Fig. . In orde15 and 16, since it detected intensive signals with m/z 634.3510 for 15 and m/z 682.2760 for 16. Additionally, the structures of these compounds were confirmed by 1H, 13C, and 31P NMR spectroscopy. In the 1H NMR spectra of 15 and 16, multiplied signals of acid moieties, glycerol, and choline were observed, which suggested the presence of four diastereomers in the product mixtures. Due to the substitution of identical molecules at the sn-1 and sn-2 positions and the racemization of acids bonded to GPC, the interpretation of the spectral data was complicated and very difficult. The racemization caused a formation of four diastereomers for both 15 and 16, respectively  of CH3-3′a-b could clearly be observed. Their differentiation was caused by the direct attachment of the methyl group to the chiral C-2′a-b carbon atom of the ibuprofen moiety. Moreover, the methine protons of H-2′a-b were detected as multiplets in the range of 3.43–3.69 ppm. The separation of signals was imperceptible due to the large distance from the ibuprofen isobutyl moiety to the stereogenic center. In addition, in the range of 0.84–0.88 ppm doublets (J = 6.6 Hz) from the isobutyl methyl groups were detected and at 3.11, 3.12, 3.15, and 3.16 ppm, four singlets from the choline methyl groups were detected. This indicated the formation of four diastereomers of compound 15. Two-dimensional HSQC spectroscopy was instrumental in elucidating the structure of 15. The measurements detected signals of the CH2-α (δ = 4.11 and 4.19) and CH2-β (δ = 3.49 and 3.55) protons, multiplets of the CH2-1 protons in the range of 3.98–4.42 ppm, and multiplets of the CH2-3 protons in the range of 3.84–3.96 ppm. Multiplets of the methine proton H-2 were detected at δ = 5.15 and 5.20 ppm. In the 13C NMR spectrum of phosphatidylcholine 15, signals of C-3′a-b and C-2′a-b in the ranges of 17.44–17.87 and 44.24–44.58 ppm, respectively, were observed. Multiplied signals of the glycerophosphocholine carbon atoms were detected at 58.54 and 58.60 ppm , as well as in the ranges of 62.29–63.11 ppm (C-1 and C-3) and 65.75–65.90 (C-β). Interestingly, a single doublet (JC-P = 8.0 Hz) from the chiral C-2 carbon atom appeared at 70.21 ppm. The carbon atoms from the aromatic ring did not show any differentiation with exception of C-1″a-b, which was closest to the chiral C-2′a-b carbon atom and gave 8 signals in the range of 136.78–136.97 ppm. The signal of the ester carbon atom C-1′a-b was also multiplied eight times and appeared in the range of 173.86–174.36 ppm. In the 31P NMR spectrum, only one signal from the phosphorus atom of 15 was visible at δ = –1.02.When looking at the 16, a racemization of naproxen was also expected. The 1H NMR spectrum clearly indicated the formation of diastereomers and the multiplication of almost all signals. Signals of CH3-3′a-b appeared as eight doublets (J = 7.2 Hz) in the range of 1.23–1.49 ppm, while H-2′a-b were visible as overlapping quartets (J = 7.2 Hz) at 3.39–3.81 ppm. The signals of the methoxy group were observed at 3.86 and 3.87 ppm as two singlets. Hydrogen atoms from the glycerophosphocholine fragment were also distinctly differentiated. The CH2-1 groups of each diastereomer gave rise to two doublet of doublets  with signals at 3.98, 4.11, 4.15, and 4.16 ppm from one of the protons, and a doublet of doublets  with signals at 4.23, 4.33, 4.41, and 4.44 ppm from the second. The CH2-3 groups appeared as two multiplets in the range of 3.80–3.95 ppm. Moreover, at 2.90, 2.91, 3.01, and 3.03 ppm four singlets that were assigned to the choline methyl groups were detected. Particularly interesting was the chemical shift of the first two singlets, because these proton signals have never been observed at such a high field for phosphatidylcholines. The CH2-β methylene groups appeared on the spectrum as two multiplets at 3.08 and 3.36 ppm, and the CH2-α protons appeared as three multiplets in the range of 3.72–3.84 ppm and at 4.05 ppm. The shifts of the N(CH3)3 and CH2-β signals toward the higher field, as well as the differentiation of the CH2-α protons, may indicate the influence of the naproxen moiety from the sn-2 position on the hydrophilic choline part of the molecule. Modeling of the phosphatidylcholine 16 structure showed that this may be the result of the rotation around the C2-C3 bond and the existence of two distinctive rotamers: one with the phosphocholine moiety in syn conformation to the naproxen at the sn-2 position and the second one in the anti. In the first conformation, which was stabilized by the nucleophilic interaction of the –N+(CH3)3 group and naphthalene electrons, the choline moiety was located in the shielding cone of the magnetic field induced by the electrons of the naphthalene ring. In the second conformation, the electrons of the aromatic ring did not affect the chemical shifts of the hydrogen atoms of the choline moiety.In the synthesis of phosphatidylcholine 13C NMR spectrum of phosphatidylcholine 16, a differentiation of the signals of C-3′a-b (17.22–17.66 ppm) and C-2′a-b (44.42–44.83 ppm) was observed. A doubling of the signals from the carbon atoms was observed: C-α at 58.35  and at 58.59 ppm , C-β in the range of 65.41–65.77 ppm (two m), and C-3 at 62.96 and 63.03 ppm  and at 63.13 and 63.18 ppm . In the range of 62.13–62.42 ppm, differentiated signals of the C-1 carbon atom were detected. Signals of the C-1′a-b carbonyl atoms were visible at: 173.73, 173.82, 173.92, 174.09, 174.11, 174.21, and 174.25 ppm. The phosphorus atom was also differentiated, and the signals were registered at −1.01 and −1.19 ppm.In the 32. Therefore, HL-60 cells, gastrointestinal origin - human colon cancer Caco-2 cells and normal porcine epithelial intestinal IPEC-J2 cells were used in these studies. Phosphatidylcholine derivatives were dissolved in dimethyl sulfoxide (DMSO) prior to examination. Unfortunately, due to the poor solubility of compound 13 in DMSO, its cytotoxicity was not tested. The solvent did not show cytotoxicity against analyzed cell lines.Taking into consideration the idea of the future application of novel phosphatidylcholines containing ibuprofen or naproxen moieties as anti-inflammatory drugs with reduced gastrointestinal toxicity, it is necessary to analyze the cytotoxicity of these derivatives against the sensitive and normal cells. Then, the cytotoxicity of ibuprofen, naproxen, and their phospholipid derivatives was analyzed by a WST-1 cell proliferation assay. Our earlier studies indicated that among different cell lines, human promyelocytic leukemia HL-60 cells are the most sensitive to modified phosphatidylcholines1) was non-toxic up to concentration of 100 µM. In contrast to ibuprofen (1), 2-lysophosphatidylcholine 7 was non-toxic even at concentration 200 µM  showed higher toxicity in comparison to 1. Analysis of results of cytotoxic activity against normal porcine IPEC-J2 cells showed that compounds 7 and 9 were less toxic than ibuprofen (1)  and with two residues of ibuprofen (15) were more cytotoxic.In the case of human leukemic HL-60 cells, the experiments revealed that ibuprofen  Fig. , Table 12) was non-toxic toward these cells up to concentration 100 µM. Among phosphatidylcholines containing naproxen, only compound 8 was significantly less cytotoxic (up to concentration of 200 µM) in comparison to free naproxen  was less toxic in comparison to leukemic HL-60 cells. Interestingly, also in contrast to leukemic cells, compound 10 was non-toxic up to concentration of 200 µM against the colon carcinoma cells. These cells were only sensitive to compound 16  towards these cell lines at concentration 200 µM.Incubation of HL-60 cells revealed that naproxen  were more toxic than ibuprofen or naproxen themselves. 2-Lysophosphatidylcholines (7 and 8) were less toxic against all tested cell lines. Phospholipids with NSAIDs in sn-1 and palmitic acid in sn-2 (9 and 10) were also less toxic against Caco-2 and normal cells (IPEC-J2).The results of these studies showed that phosphatidylcholines containing NSAIDs at both sn-1, sn-2 or sn-1 and sn-2 positions, and the cytotoxicity of the resulting compounds towards two cancer cells (HL-60 and Caco-2) and normal IPEC-J2 cells was evaluated. The results from the biological studies indicated that 2-lysophosphatidylcholines with NSAIDs at sn-1 position (7 and 8) and phosphatidylcholines with NSAIDs at sn-1 and palmitic acid residue at sn-2 (9 and 10) were less toxic against Caco-2 and normal cells (IPEC-J2). Compounds 7 and 8 were also less toxic than ibuprofen or naproxen against HL-60 cells. These results confirmed our earlier observations that phospholipid derivatives of active compounds are less cytotoxic than the compounds themselves. These data indicate also that phospholipids could be used as carriers of NSAIDs. However, further biological studies are necessary in order to confirm this proposal.In this study, we synthesized eight phosphatidylcholines with ibuprofen or naproxen in their sn-Glycero-3-phosphocholine (GPC) was purchased from Bachem . S-Ibuprofen (1), S-naproxen (2), N,N′-dicyclohexylcarbodiimide (DCC), 4-pyridine (DMAP), dibutyltin(IV) oxide (DBTO), triethylamine (TEA), palmitoyl chloride, oxalyl chloride, Dowex® 50WX8 H+ form (an ion-exchange resin), hexane, methanol, 2-propanol for liquid chromatography, and chloroform (CHROMASOLV®) were purchased from Sigma-Aldrich . Chloroform and methanol for column and thin-layer chromatography (TLC) were purchased from Stanlab . TLC was performed on Merck Kieselgel 60 F254 plates (0.2 mm silica gel with fluorescent indicator UV254). As a visualization reagent, primuline spray  was used. Visualization was determined using UV light (λ = 365 nm). Column chromatography was performed on silica gel  using an eluent mixture of CHCl3:CH3OH:H2O . The purity of all final phosphatidylcholines was determined by HPLC to be 96% or higher. HPLC was performed on an UltiMate 3000 apparatus  with a charged aerosol detector . Nitrogen was used as a nebulizing gas at a pressure of 35 psi and at an acquisition range of 100 pA. A BETASIL Diol column  was used. The mobile phase was set at a flow rate of 1.5 mL/min and consisted of the following solvents: (A) water, (B) hexane, and (C) 2-propanol. The mobile phase was run as %A:%B:%C (v/v/v) using the following gradient timetable: 0 min - 0:43:57, 5 min -3:40:57, 8 min - 10:40:50, 13 min - 10:40:50, 13.1 min - 0:43:57, and 22 min - 0:43:57. All NMR spectra were recorded on an Avance II 600 MHz spectrometer  working at a frequency of 600 MHz for 1H, 150 MHz for 13C, and 243 MHz for 31P. Samples of all compounds were measured in a mixture of CDCl3:CD3OD . The chemical shifts were calibrated using: the methanol proton signals (δH = 3.31) in the 1H NMR spectra and the CDCl3 carbon atoms (δc = 77.0) in the 13C NMR spectra. H3PO4 (85%) was used as an external standard for the 31P NMR chemical shifts. HRMS spectra were recorded using the ESI technique on a spectrometer . Melting points  were determined on a Boetius apparatus.sn-glycero-3-phosphocholine , and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (11) were prepared according to previously reported procedures33.The complexes of cadmium chloride and 7 and 8. S-Ibuprofen  or S-naproxen  was dissolved in anhydrous methylene chloride (5 mL), and catalytic amount (2 drops) of anhydrous DMF was added to the mixture. Excess of oxalyl chloride  was added dropwise to the stirring solution. The reaction was carried out at room temperature for 2 h and the solvent and excess of oxalyl chloride were subsequently evaporated. The resulting chloride 3 or 4 was immediately used for the synthesis of 2-lysophosphatidylcholine 7 or 8.General procedure for the preparation of 2-lysophosphatidylcholines 3 or 4 were then added to it. The mixture was stirred for 30 min. After completion of the reaction (checked by TLC) the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using chloroform:methanol:water (65:25:2 → 65:25:4) to give the corresponding 2-lysophosphatidylcholine (7 or 8).GPC  and DBTO  were suspended in 15 mL of 2-propanol and refluxed for 2 h. The mixture was cooled to room temperature and TEA  and corresponding chloride 7): Colorless, waxy product; yield 68% (0.292 g). 1H NMR  δ: 0.86 2-CH-CH2-), 1.46 , 1.81 2-CH-CH2-), 2.41 2-CH-CH2-), 3.17 3), 3.58 , 3.72 , 3.81 , 3.88 , 3.92 , 4.02 , 4.18 , 4.23 , 7.04–7.08 , 7.14–7.18 . 13C NMR  δ: 17.85 (C-3′), 21.63 ((CH3)2-CH-CH2-), 29.68 ((CH3)2-CH-CH2-), 44.46 (C-2′), 44.53 ((CH3)2-CH-CH2-), 53.48, 53.51 and 53.53 (N(CH3)3), 58.67 , 64.76 (C-1), 65.87 (C-β), 66.34 , 68.11 , 126.62 (C-3″ and C-5″), 128.82 (C-2″ and C-6″), 137.06 (C-1″), 140.15 (C-4″), 174.80 (C-1′). 31P NMR  δ: –0.34. ESI-MS: m/z calculated for C21H36NO7P [M + H]+: 446.2308. Found: 446.2319.1-[2′S-(4″-Isobutylphenyl)]propanoyl-2-hydroxy-sn-glycero-3-phosphocholine (8): Light pink powder; yield 70% (0.318 g); mp 162–164 °C. 1H NMR  δ: 1.54 , 3.08 3), 3.44 , 3.79 , 3.85 , 3.88 , 3.89–3.93 , 4.07 , 4.13 , 4.18 , 7.09–7.13 , 7.37 , 7.64 , 7.67–7.70 . 13C NMR  δ: 17.84 (C-3′), 44.85 (C-2′), 53.41, 53.44 and 53.46 (N(CH3)3), 54.68 (O-CH3), 58.55 , 64.75 (C-1), 65.80 (C-β), 66.24 , 68.06 , 105.14 (C-7″), 118.47 (C-5″), 125.41 (C-2″), 125.72 (C-10″), 126.72 and 128.77 (C-4″ and C-9″), 128.48 (C-3″), 133.30 (C-8″), 135.12 (C-1″), 157.24 (C-6″), 174.63 (C-1′). 31P NMR  δ: –0.30. ESI-MS: m/z calculated for C22H32NO8P [M + H]+: 470.1944. Found: 470.1961.1-[2′S-]propanoyl-2-hydroxy-sn-glycero-3-phosphocholine  or 8  and DMAP  were dissolved in dry methylene chloride (5 mL). The, a mixture of palmitic acid  and DCC  in the same solvent (5 mL) was added. The reaction was stirred for 72 h at room temperature. The suspension was filtered off and stirred with Dowex® resin (50WX8 H+) for 30 min. The resin was then filtered off and washed with a Folch mixture (15 mL). The solvent was evaporated and the crude product was purified by column chromatography on silica gel  to give the corresponding phosphatidylcholines 9 or 10. The yields and the physical and spectroscopic data of the products are given below.General procedure for the preparation of phosphatidylcholines 9): Colorless waxy product; yield 25% (0.034 g). 1H NMR  δ: 0.84 , 0.86 2-CH-CH2-), 1.22–1.26 , 1.43 and 1.44 , 1.47–1.55 , 1.80 2-CH-CH2-), 2.15 and 2.20 , 2.40 2-CH-CH2-), 3.16 3), 3.55 , 3.66 and 3.69 , 3.91–3.99 , 4.03 and 4.09 , 4.20 , 4.25 and 4.38 , 5.19 , 7.03–7.06 , 7.11–7.15 . 13C NMR  δ: 13.44 (C-16″′), 17.76 (C-3′), 21.73 ((CH3)2-CH-CH2-), 24.36 (C-3″′), 28.68–31.46 (C-4″′-C-15″′), 29.76 ((CH3)2-CH-CH2-), 33.63 (C-2″′), 44.57 (C-2′), 44.59 ((CH3)2-CH-CH2-), 53.58, 53.60 and 53.63 (N(CH3)3), 58.67 , 62.65 (C-1), 63.28 , 65.99 (C-β), 69.79 , 126.68 (C-3″ and C-5″), 128.88 (C-2″ and C-6″), 136.94 (C-1″), 140.24 (C-4″), 173.10 (C-1″′), 174.43 (C-1′). 31P NMR  δ: –0.76. ESI-MS: m/z calculated for C37H66NO8P [M + H]+: 684.4604. Found: 684.4619.1-[2′-(4″-Isobutylphenyl)]propanoyl-2-palmitoyl-sn-glycero-3-phosphocholine (10): Colorless waxy product; yield 57% (0.081 g). 1H NMR  δ: 0.85 , 1.18–1.27 , 1.35–1.41 , 1.53 , 1.98 , 2.07 , 3.12 3), 3.47 , 3.84 , 3.88 , 3.90–3.93 , 4.14 , 4.16 , 4.38 , 5.19 , 7.09–7.12 , 7.34 , 7.61 , 7.66–7.69 . 13C NMR  δ: 13.33 (C-16″′), 17.63 (C-3′), 24.17 (C-3″′), 22.12–31.39 (C-4″′-C-15″′), 33.43 (C-2″′), 44.83 (C-2′), 53.41, 53.43 and 53.46 (N(CH3)3), 54.61 (O-CH3), 58.54 , 62.60 (C-1), 63.09 , 65.82 (C-β), 69.64 , 105.08 (C-7″), 118.50 (C-5″), 125.40 (C-2″), 125.62 (C-10″), 126.69 and 128.72 (C-4″ and C-9″), 128.46 (C-3″), 133.30 (C-8″), 134.89 (C-1″), 157.25 (C-6″), 173.00 (C-1″′), 174.27 (C-1′). 31P NMR  δ: –0.82. ESI-MS: m/z calculated for C38H62NO9P [M + H]+: 708.4240. Found: 708.4233.1-[2′-]propanoyl-2-palmitoyl-sn-glycero-3-phosphocholine  and DMAP  in anhydrous methylene chloride (5 mL), a mixture of S-ibuprofen  or S-naproxen  and DCC  in the same solvent (3 mL) was added. The reaction was stirred for 72 h at room temperature. The resulting precipitate was filtered off and a Dowex® 50WX8 H+ form was added. After 30 min of stirring, the resin was filtered off on a Shott funnel and washed with a Folch mixture (15 mL). Products 12 and 13 were separated by column chromatography .General procedure for the preparation of phosphatidylcholines 12): Colorless waxy product; yield 41% (0.113 g). 1H NMR  δ: 0.85 , 0.86 2-CH-CH2-), 1.23–1.26 , 1.45 , 1.44 , 1.53 , 1.81 2-CH-CH2-), 2.02–2.31 , 2.41 2-CH-CH2-), 3.19 3), 3.52 and 3.58 , 3.67 and 3.71 , 3.91–3.99 , 4.05 and 4.11 , 4.22 , 4.26 and 4.39 , 5.20 , 7.03–7.08 , 7.12–7.17 . 13C NMR  δ: 13.29 (C-16″′), 17.57 (C-3″), 21.59 ((CH3)2-CH-CH2-), 24.14 (C-3″′), 28.57–31.37 (C-4″′-C-15″′), 29.68 ((CH3)2-CH-CH2-), 33.25 (C-2″′), 44.44 and 44.48 (C-2′ and (CH3)2-CH-CH2-), 53.44, 53.46 and 53.49 (N(CH3)3), 58.60 (C-α), 62.03 (C-1), 63.19 , 65.86 (C-β), 70.21 , 126.56 (C-3″ and C-5″), 128.75 (C-2″ and C-6″), 136.97 (C-1″), 140.07 (C-4″), 173.35 (C-1″′), 174.10 (C-1′). 31P NMR  δ: –0.78. ESI-MS: m/z calculated for C37H66NO8P [M + H]+: 684.4604. Found: 684.4617.1-Palmitoyl-2-[2′-[4″-isobutylphenyl)]propanoyl-sn-glycero-3-phosphocholine (13): Colorless waxy product; yield 53% (0.152 g). 1H NMR  δ: 0.85 , 0.98–1.24 , 1.25–1.27 , 1.53 , 1.79 and 1.84 , 3.13 3), 3.46 , 3.85 , 3.88 , 3.95–3.99 , 4.06 , 4.13 , 4.24 , 5.21 , 7.08–7.12 , 7.36 , 7.63 , 7.65–7.69 . 13C NMR  δ: 13.42 (C-16″′), 17.56 (C-3′), 24.04 (C-3″′), 22.16–31.48 (C-4″′-C-15″′), 33.14 (C-2″′), 44.88 (C-2′), 53.50, 53.52 and 53.55 (N(CH3)3), 54.68 (O-CH3), 58.52 , 62.00 (C-1), 63.24 , 65.87 (C-β), 70.44 , 105.09 (C-7″), 118.54 (C-5″), 125.40 (C-2″), 125.72 (C-10″), 126.68 and 128.77 (C-4″ and C-9″), 128.48 (C-3″), 133.31 (C-8″), 134.99 (C-1″), 157.29 (C-6″), 173.34 and 173.99 (C-1′ and C-1″′). 31P NMR  δ: –0.73. ESI-MS: m/z calculated for C38H62NO9P [M + H]+: 708.4240. Found: 708.4230.1-Palmitoyl-2-[2′S-]propanoyl-sn-glycero-3-phosphocholine  and DMAP  were dissolved in anhydrous methylene chloride (5 mL). To this solution, a solution of DCC  and S-ibuprofen  or S-naproxen  in anhydrous methylene chloride (5 mL) was added. The mixture was stirred for 48 h at 25 °C under nitrogen atmosphere and the progress of the reaction was monitored by TLC. The precipitated dicyclohexylurea was then filtered off and a Dowex® 50WX8 H+ form was added to the mixture. The solution was stirred for 30 min, after which the resin was filtered off on a Shott funnel. Then, the resin was washed with 15 mL of Folch mixture  and the solvent was evaporated in vacuo. The residues were purified by column chromatography using a mixture of chloroform/methanol/water (65:25:4) as eluent to give a colorless waxy product 15 or 16.General procedure for the synthesis of compounds 15): Colorless waxy product; yield 61% (0.177 g). 1H NMR  δ: 0.84–0.88 2-CH-CH2-), 1.29–1.43 , 1.76–1.85 2-CH-CH2-), 2.39–2.43 2-CH-CH2-), 3.11, 3.12, 3.15 and 3.16 3), 3.43–3.69 , 3.49 and 3.55 , 3.84–3.96 , 3.98, 4.08–4.29, 4.35 and 4.42 , 4.11 and 4.19 , 5.15 and 5.20 , 7.02–7.16 . 13C NMR  δ: 17.44, 17.51, 17.60, 17.66, 17.69, 17.78 and 17.87 (C-3′a-b), 21.58 ((CH3)2-CH-CH2-), 29.65 ((CH3)2-CH-CH2-), 44.24–44.58 (C-2′a-b and (CH3)2-CH-CH2-), 53.45 and 53.47 (N(CH3)3), 58.54 and 58.60 , 62.29–63.11 (C-1 and C-3), 65.75–65.90 (C-β), 70.21 , 126.58, 126.60, 126.62 and 126.66 (C-3″a-b and C-5″a-b), 128.74, 128.76, 128.78 and 128.80 (C-2″a-b and C-6″a-b), 136.78, 136.80, 136.84, 136.85, 136.89, 136.92, 136.95 and 136.97 (C-1″a-b), 140.06, 140.10, 140.13 and 140.15 (C-4″a-b), 173.86, 173.94, 173.97, 174.05, 174.23, 174.25, 174.33 and 174.36 (C-1′a-b). 31P NMR  δ: –1.02. ESI-MS: m/z calculated for C34H52NO8P [M + H]+: 634.3509. Found: 634.3510.1,2-Di-[2′-(4″-isobutylphenyl)]propanoyl-sn-glycero-3-phosphocholine (16): Colorless waxy product; yield 72% (0.225 g). 1H NMR  δ: 1.23, 1.29, 1.33, 1.35, 1.42, 1.44, 1.48 and 1.49 , 2.90, 2.91, 3.01 and 3.03 3), 3.08 and 3.36 , 3.39, 3.41, 3.54, 3.56 and 3.72–3.81 , 3.72–3.84 and 4.05 , 3.80–3.95 , 3.86 and 3.87 , 3.98, 4.11, 4.15 and 4.16 , 4.23, 4.33, 4.41 and 4.44 , 5.14 and 5.21 , 7.05–7.69 . 13C NMR  δ: 17.22, 17.35, 17.41, 17.46, 17.57 and 17.66 (C-3′a-b), 44.42, 44.47, 44.70, 44.79 and 44.83 (C-2′a-b), 53.30 and 53.40 (N(CH3)3), 54.55, 54.57 and 54.61 (O-CH3a-b), 58.35 , 58.59 , 62.13, 62.40 and 62.42 (C-1), 62.96 and 63.03 , 63.13 and 63.18 , 65.41–65.77 , 70.24–70.47 , 105.04, 105.07 and 105.10 (C-7″a-b), 118.34, 118.43 and 118.45 (C-5″a-b), 125.27–125.74 (C-2″a-b and C-10″a-b), 126.56–128.78 (C-4″a-b and C-9″a-b), 128.32, 128.34, 128.43 and 128.45 (C-3″a-b), 133.20, 133.28 and 133.30 (C-8″a-b), 134.72, 134.87, 134.88, 134.92, 134.95 and 135.00 (C-1″a-b), 157.15, 157.21 and 157.23 (C-6″a-b), 173.73, 173.81, 173.82, 173.92, 174.09, 174.11, 174.21 and 174.25 (C-1′a-b). 31P NMR  δ: –1.01 and –1.19. ESI-MS: m/z calculated for C36H44NO10P [M + H]+: 682.2781. Found: 682.2760.1,2-Di-[2′-]propanoyl-sn-glycero-3-phosphocholine , 2 mM glutamine and antibiotics (complete DMEM).Human promyelocytic leukemia HL-60 cells and human colon cancer cell line Caco-2 were obtained from the American Type Culture Collection . The HL-60 cells were cultured in an RPMI medium supplemented with 10% fetal bovine serum , 2 mM L-glutamine, and antibiotics (complete RPMI). The Caco-2 cells were cultured in an DMED medium supplemented with 20% fetal bovine serum , 2 mM glutamine and antibiotics (complete DMED). Intestinal epithelial IPEC-J2 cells from newborn porcine, a kind gift from Prof. P. Schierack 3/well) were grown for 24 h in 96-well plates  in appropriate complete medium at 37 °C and atmosphere containing 5% CO2. Next day, increased concentrations of analyzed compounds  dissolved in dimethyl sulfoxide  were added to cells growing in successive wells, and cell cultures were continued for 72 h. Due to the poor solubility of compound 13 in DMSO, its cytotoxicity was not tested. After indicated periods of time, WST-1 mixture was added to each well, and the cells were cultured for additional 6 h. The absorbance at 450 nm was measured on an EnSpire 2300 Multilabel Reader , and data were analyzed using the program EnSpire 9.0 . The experiments were performed in triplicates and repeated at least three times independently. Results were presented as mean IC50  ± standard deviation. IC50 values were calculated using Cheburator 0.4 software35.Cells . To statistically evaluate the significance of differences between cytotoxicity of reference drugs (ibuprofen and naproxen) and phosphatidylcholines containing ibuprofen or naproxen moieties non-parametric two-way Annova and multicomparsion Bonfferoni post-test was performed. The Supplementary Information