Synthesis of bis[benzyl‐N′‐hydrazinecarbodithioato‐κ2 N′,S]nickel(II) complex as a novel lead molecule for cancer treatment

A novel bis[benzyl‐N′‐hydrazinecarbodithioato‐κ² N′,S]nickel(II) complex was synthesized and characterized by means of various physical, chemical, and spectroscopic techniques. The X‐ray single crystal diffraction analysis indicated two independent close comparable bis‐chelated square planar complexes of trans‐configuration, where S‐benzyl dithiocarbazate (SBDTC) ligand is coordinated via N,S‐donor set. The complex is able to inhibit Ehrlich ascites carcinoma (EAC) cell proliferation by 51.81% and 75.75%, with 0.3 and 50 mg kg^?1 (dose adjusted) dose, respectively, administered intraperitoneally for five successive days in mice model. Apoptotic cell morphological changes were examined using optical and fluorescence microscopy techniques. Expression pattern of apoptosis regulatory genes in EAC cells treated with the synthesized nickel(II) complex for five consecutive days showed an increased expression of P⁵³, Bax, Cas‐8, Cas‐9, Cas‐3, Cyt‐c, and TNF‐α proapoptotic genes and decreased expression of antiapoptotic Bcl‐2 gene. The Ni(II) complex and Bleomycin (standard drug) were used in molecular docking coupled with molecular dynamics simulation studies with the aim to support the experimental results and to investigate the apoptotic effect towards the targeting apoptotic genes. Both experimental and computational studies reveal that the nickel(II) complex inhibits EAC cells growth successfully, suggesting a potential compound for cancer treatment.     

Synthesis,characterization and biological evaluation of a cobalt(II) complex with 5‐chloro‐8‐hydroxyquinoline as anticancer agent

A new cobalt(II) complex ( 1 ) of 5‐chloro‐8‐hydroxyquinoline was prepared and structurally characterized using infrared spectroscopy, electrospray ionization mass spectrometry, elemental analysis, single‐crystal X‐ray diffraction as well as powder X‐ray diffraction. Its biological activities including DNA binding and anticancer activity were investigated. The DNA binding study of complex 1 suggested that it interacted with calf thymus DNA mainly via an intercalative binding mode. The in vitro anticancer activity of complex 1 was screened against a series of tumor cell lines as well as the normal liver cell line HL‐7702 using MTT assay. complex 1 showed much higher cytotoxicity than corresponding metal salt and ligand towards the five tested tumor cell lines, in which T‐24 was the most sensitive tumor cell line towards 1, with IC₅₀ value of 7.04 ± 0.06 μM. complex 1 was found to greatly induce cell cycle arrest in T‐24 cells at S phase, and consequently to induce cell apoptosis in a dose‐dependent mode suggested by cell apoptosis analysis via Hoechst 33258 and acridine orange/ethidium bromide staining assays. The cell apoptosis mechanism of 1 was studied targeting the mitochondrion‐mediated pathway, since the apoptotic mechanism in the T‐24 cells treated by 1 was investigated by reactive oxygen species (ROS) detection, intracellular calcium concentration measurement and caspase‐9/3 activity assay. The results suggested that the cell apoptosis induced by 1 was closely related to the loss of mitochondrial membrane potential, ROS production and enhancement of intracellular [Ca²⁺], which would trigger the caspase‐9/3 activation via a mitochondrial dysfunction pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

DNA interaction,cytotoxicity and molecular structure of cobalt complex of 4‐amino‐N‐(6‐chloropyridazin‐3‐yl)benzene sulfonamide in the presence of secondary ligand pyridine

Novel cobalt complex of 4‐amino‐N‐(6‐chloropyridazin‐3‐yl)benzene sulfonamide (sulfachloropyridazine) has been synthesized and characterized by elemental analysis, FT‐IR spectroscopy and magnetic susceptibility (VSM). Cobalt complex of Sulfachloropyridazine (Co‐SCP) crystallized in monoclinic space group P2₁/n with Z = 4. The structure is solved by direct method and refined to R = 0.099 for 4720 reflections with I ?4σ(I). The results of FT‐IR spectra suggest the binding of cobalt atom to the sulfonamide ligand which is in agreement with the crystal structure determination. In crystal structure, molecule is linked via, C‐H … π, C‐Cl … π and π … π intermolecular interactions. The computational studies like the optimization energy and root means square deviation compare with single crystal structure, frontier molecular orbital (Homo‐Lumo energy) and binding energy of the Co‐SCP has been carried out using DFT/B3LYP level of theory in gaseous phase. Hirshfeld surfaces and the 2D‐fingerprint analysis are performed to study the nature of interactions and their measurable contributions towards crystal packing. The interaction of the complex with DNA is investigated using viscosity measurement and absorption titration studies. The result shows the complex bind to DNA with intercalative mode with high DNA‐binding constant (K_b). Also, in vivo and in vitro cytotoxic studies are performed using S. pombe cells and brine shrimp lethality bioassay. DNA‐cleavage study shows better cleaving ability of the complex.     

Design and Synthesis of α,β‐Epoxyketones as New Anticancer Agents

As epoxy functional group has high anticancer activity, α,β‐epoxyketones were designed and synthesized as new anticancer agents, and their structures were confirmed by UV, ¹H NMR, IR, MS technigeces and elemental analysis. Their in vitro anticancer activities were evaluated by MTT method and the results showed that the compound 4c exhibited good activity with IC₅₀ of 17.8, 22.0 and 24.1 µg/mL against A‐549, Hela and HepG2 cells, respectively. The dose of LD50 of the mice by intragastric administration was 1864.4 mg/kg. Therefore, the α,β‐epoxyketones could potentially provide as new anticancer agents.     

Palladium Catalyzed Tricyclohexylphosphine Ligand Associated Synthesis of N‐(2‐(pyridine‐4‐yl)‐1H‐pyrrolo[3,2‐c]‐pyridin‐6‐yl‐(substituted)‐sulfonamide Derivatives as Antiproliferative Agents

A series of novel N‐(2‐(pyridine‐4‐yl)‐1H‐pyrrolo[3,2‐c]‐pyridin‐6‐yl‐(substituted)‐sulfonamide derivatives were synthesized from 2‐bromo‐6‐nitro‐1H‐pyrrolo[3,2‐c]pyridine through a series of reactions including Suzuki reaction, reduction, protection, and sulfonamide coupling. All the synthesized compounds were screened for anticancer activity against MCF‐7, HeLa, A‐549, and Du‐145 cancer cell lines by the MTT assay. The preliminary bioassay suggests that most of the compounds show antiproliferation with different degrees. Doxorubicin was used as a positive control. Among the synthesized compounds, 8d and 8h were most active compared with the standard in cell line data. The synthesized compounds 8d and 8h show IC₅₀ values in the range of 1.88–5.16 μM for all the cell lines. Compounds 8d and 8h were further studied for a panel of eight human kinase at 10 μM concentrations and the result shows 64% to 70% inhibitions for both Aurora‐A and Aurora‐B kinase.     

Separation of α‐ from β‐arylalanines by nickel nitrilotriacetate chromatography

A method is described to separate α‐ from β‐arylalanines by ligand exchange chromatography on a nickel nitrilotriacetate agarose column with UV monitoring of the effluent. Separate mixtures containing an α‐ and β‐arylalanine pair (1 mg of each) were individually loaded onto the nickel resin pre‐equilibrated with the mobile phase at room temperature, and the amino acids were eluted from the column with a gradient from pH 12.0–8.0. The β‐arylalanines eluted first, followed by the α‐isomers. The four α/β‐amino acid pairs tested were well separated with baseline resolution. An aliquot of each fraction was chemically treated to derivatize the amino acids to their N‐acyl methyl ester analogs, and their identities were confirmed by GC/MS analysis. The sample recovery was quantitative (>98%), and the column matrix was very resilient, as demonstrated by consistent separation of the solutes after ～100 preparative cycles.     

DNA/BSA‐binding property and in vitro anticancer activity of a new dicopper(II) complex with N‐(2‐hydroxy‐5‐nitrophenyl)‐N′‐[3‐(diethylamino)propyl]oxamide as bridging ligand: Synthesis and crystal structure

A new oxamido‐bridged dicopper(II) complex formulated as [Cu₂(ndpox)(bpy)(CH₃OH)₂]‐ (ClO₄), where H₃ndpox is N‐(2‐hydroxy‐5‐nitrophenyl)‐N′‐[3‐(diethylamino)propyl]oxamide; and bpy represents 2,2′‐bipyridine, was synthesized and structurally characterized using X‐ray single‐crystal diffraction and other methods. In the molecule, the endo‐ and the exo‐copper(II) ions bridged by the cis ‐ndpox³⁻ ligand are in {N₃O₂} and {N₂O₃} square‐ pyramidal environments, respectively. There is a three‐dimensional hydrogen bonding network dominated by O‐H···O and C‐H···O interactions in the crystal. The reactivity toward DNA/protein bovine serum albumin (BSA) revealed that the complex could interact with herring sperm DNA (HS‐DNA) through the intercalation mode, and effectively quench the intrinsic fluorescence of BSA via a static process. Cytotoxicity studies suggest that the complex displays selective cancer cell antiproliferative activity. The present investigation confirmed that the combined effects of both electron‐withdrawing and hydrophobic groups on the bridging ligand in the dicopper(II) complex systems can increase DNA/BSA‐binding ability and in vitro anticancer activity.     

Antibacterial,antifungal and in vivo anticancer activities of chitin-binding lectins from Tomato (Solanum lycopersicum) fruits

A mixture of chitin-binding lectins from Tomato (Solanum lycopersicum) fruits, designated as Tomato chitin-binding lectins (TCLs), was isolated through affinity chromatography using an acetylated chitin column. Molecular weights of TCLs were determined to be 30 to 115 KDa which possessed mild toxicity with an LC₅₀ value of 521 µg/ml examined by the brine shrimp nauplii toxicity assay. Strong antibacterial activity of TCLs was found against Escherichia coli, Staphylococcus aureus and Shigella boydii at a concentration of 500 µg/ml by using disc diffusion method. Minimum inhibitory concentrations (MIC) of TCLs against Staphylococcus aureus and Escherichia coli were found to be 200 µg/ml and 140 µg/ml, respectively whereas minimum bactericidal concentrations (MBC) against the same bacterial species were 840 and 600 µg/ml, respectively. TCLs also exerted antibiofilm activity (53.32% at 250 μg/ml) against Escherichia coli. Strong antifungal activity of TCLs against Aspergillus niger was found at 600 µg/ml whereas the lectin mixture agglutinated A. niger spores at 200 µg/ml. TCLs exhibited 19.63% and 59.91% anti-proliferative activity against Ehrlich ascites carcinoma (EAC) cells in vivo in Swiss albino mice when intraperitonealy injected at doses 1.0 mg/kg/day and 2.0 mg/kg/day, respectively for five consecutive days. Morphological changes of apoptosis in EAC cells under fluorescence microscope and alteration of the expression of apoptosis-related genes (Fas, Caspase 8 and Caspase 3) had also been observed. MTT assay showed 27.61%, 38.74% and 49.23% of in vitro anticancer activity of the tomato lectins at concentrations of 37.5, 75 and 150 µg/ml, respectively.     

Iron(III) diacetylmonoxime‐2‐hydrazinopyridine complex: A new prospective antitumor drug

Because of the side effects and drug resistance of cisplatin, a basic clinically approved chemotherapeutic drug, a new attempt is reported to develop a novel antitumor drug based on complexation of iron metal ion with organic moiety that may be effective and safer. A newly synthesized iron(III) diacetylmonoxime‐2‐hydrazinopyridine complex was tested firstly for its cytotoxicity and superoxide dismutase (SOD)‐mimic activity in vitro then for its antitumor activity against Ehrlich ascites carcinoma (EAC) and the related biochemical alterations in vivo in comparison with cisplatin. The complex showed 80.88% SOD‐mimic activity and IC₅₀ of 2.6 μg ml⁻¹. In EAC‐bearing mice, in a dose‐dependent manner, Fe(III) complex treatment exhibited significant hematological profile improvements, tumor volume, viable cell count and hepatic lipid peroxidation level decreases, life span extension, hepatic glutathione and total antioxidant capacity levels enhancements, hepatic SOD and catalase activities augmentations, liver function tests alterations attenuations, and hepatocyte nucleic acids content normalization. Thus, the Fe(III) diacetylmonoxime‐2‐hydrazinopyridine complex is a novel, promising, less toxic antitumor agent. Its killing of tumor cells may be via a reactive oxygen species scavenging mechanism.     

Synthesis,characterization and cytotoxicity of new 2‐isonicotinoyl‐N‐phenylhydrazine‐1‐carbothioamide and its metal complexes

The reaction of the newly synthesized ligand, 2‐isonicotinoyl‐N‐phenylhydrazine‐1‐carbothioamide (H₃L), with acetate salt of Co (II), Cu (II),Ni (II) and Zn (II) led to isolation of four solid complexes. The ligand and complexes structure elucidation were based on elemental analyses, spectral analyses (IR, UV–Visible, ¹H and¹³C‐NMR, MS and ESR), TGA, molar conductivity and magnetic moments measurements. The results indicated that the ligand exists in the thioketo form, while on coordination to the metal ions; it behaves as mono‐negative bidentate chelate and exists in enol form. The optical band gap measurements of the ligand and its metal complexes are in the range 3.83–4.48 eV indicating their semi‐conducting character. The cytotoxicity examination of H₃L and its Zn (II) complex showed that the ligand have very strong cytotoxicity against both HCT‐116 and HEPG‐2 cell lines while, Zn (II) complex has moderate activity.     

Divalent manganese,cobalt, copper and cadmium complexes of (Z)‐N‐benzoyl‐N′‐(1H‐1,2,4‐triazol‐3‐yl)carbamimidothioic acid: Preparation,characterization, computational and biological studies

In this work, (Z)‐N‐benzoyl‐N′‐(1H‐1,2,4‐triazol‐3‐yl)carbamimidothioic acid and its Mn(II), Co(II), Cu(II) and Cd(II) complexes were introduced for the first time. This carbonyl thiourea ligand was prepared by the reaction of 1H‐1,2,4‐triazol‐3‐amine with benzoyl isothiocyanate. The structural elucidation of these compounds was performed using elemental analysis and spectral and magnetic measurements. Octahedral structures of all complexes, except Cd(II) complex with a tetrahedral geometry, were confirmed by applying DFT structural optimization. The thermal decomposition behaviour of metal complexes of carbonyl thiourea ligand is discussed. The calculation of kinetic parameters for prepared complexes (E_a, A, ΔH*, ΔS* and ΔG*) of all thermal degradation stages has been evaluated using two comparable approaches. Antimicrobial and ABTS‐antioxidant studies indicated potent activity of Cd(II) complex compared with the other investigated compounds. The cytotoxic activity of the prepared compounds was investigated in vitro. The results indicated potent activity of Mn(II) complex against both HePG2 (liver carcinoma) and MCF‐7 (breast carcinoma) cancer cells.     

立方烷型2-羟甲基-N-邻羟基苯亚甲基苯胺四核镍配合物的合成与晶体结构

The tetranuclear cubane-like complex, [NiL（EtOH）]4·0.5EtOH （1） with tridentate Schiff base ligand （H2L= 2-Hydroxymethyl-N-salicylideneaniline） has been synthesized and its crystal structure and spectroscopic properties have been studied. The complex consists of a tetranuclear （NiO）4 cubane core, of which four nickel（Ⅱ） ions are bridged by μ3-alkoxide group and each nickel（Ⅱ） ion is coordinated to three μ3-alkoxide oxygen atoms, one imino nitrogen atom and one phenoxide oxygen atom from Schiff base ligand, and further ligated by one EtOH molecule, completing a distorted octahedral geometry.     

Design,Synthesis and Antifungal Evaluation of N‐Substituted‐1‐(3‐chloropyridin‐2‐yl)‐N‐(pyridin‐4‐yl)‐5‐(trifluoromethyl)‐1H‐pyrazole‐4‐carboxamide Derivatives

A series of 1‐(3‐chloropyridin‐2‐yl)‐5‐(trifluoromethyl)‐1H‐pyrazole‐4‐carboxamide derivatives which have di‐substituents on nitrogen were designed and synthesized. Bioassay results showed that all the synthetic compounds exhibited lower antifungal activities against Gibberella zeae, Cytospora mandshurica, and Fusarium oxysporum than T ₃ (14.7, 21.1, and 32.7 μg/mL), but some of them exhibited better activities against Botrytis cinerea, Phytophthora infestans, and Sclerotinia sclerotiorum than T ₃ (>200, >200, and >200 μg/mL); the EC₅₀ values of 7d and 7c against B. cinerea were 94.9 and 56.2 μg/mL, respectively. The EC₅₀ values of 7a , 7d , and 7c against S. sclerotiorum were 73.5, 78.7, and 68.5 μg/mL, respectively.     

Synthese und Struktur von N,N,N‴,N‴‐Tetraisobutyl‐N′,N″‐isophthaloylbis(thioharnstoff) und Dimethanol‐bis(N,N,N‴,N‴‐tetraisobutyl‐N′,N″‐isophthaloylbis(thioureato))dicobalt(II)

Synthesis and Structure of N,N,N?,N?‐Tetraisobutyl‐N′,N″‐isophthaloylbis(thiourea) and Dimethanol‐bis(N,N,N?,N?‐tetraisobutyl‐N′,N″‐isophthaloylbis(thioureato))dicobalt(II) The synthesis and the crystal structure of the ligand N,N,N?,N?‐tetraisobutyl‐N′,N″‐isophthaloylbis(thiourea) and its Co^II‐complex are reported. The ligand co‐ordinates quadridentately forming a di‐bischelate. The donor atoms O and S are arranged in cis‐position around the central Co^II ions. In addition the co‐ordination geometry is determined by methanol molecules resulting in the co‐ordination number five. The complex crystallizes in the space group P1 (Z = 1) with two additional methanol molecules per formula unit. The free ligand crystallizes in the space group P1 (Z = 2) with one methanol molecule per formula unit. It shows the typical keto form of N‐acylthioureas with a protonated central N atom. The structures of both acylthiourea fragments come close to E,Z′‐configurations.     

Nickel‐Catalyzed Asymmetric Hydrogenation of N‐Sulfonyl Imines

An efficient nickel‐catalyzed asymmetric hydrogenation of N‐tBu ‐ sulfonyl imines was developed with excellent yields and enantioselectivities using (R,R)‐QuinoxP* as a chiral ligand. The use of a much lower catalyst loading (0.0095 mol %, S/C=10500) represents the highest catalytic activity for the Ni‐catalyzed asymmetric hydrogenations reported so far. Mechanistic studies suggest that a coordination equilibrium exists between the nickel salt and its complex, and that excess nickel salt promotes the formation of the active Ni‐complex, and therefore improved the efficiency of the hydrogenation. The catalytic cycle was also investigated by calculations to determine the origin of the enantioselectivity. An extensive network of numerous weak attractive interactions was found to exist between the catalyst and substrate in the transition state and may also contribute to the high catalytic activity.     

Ammine(2,2′‐bipyridine‐κ2N,N′)silver(I) nitrate: a dimer formed by π–π stacking and ligand‐unsupported Ag...Ag interactions

Reaction of AgNO₃ and 2,2′‐bipyridine (bipy) under ultrasonic treatment gave the title compound, [Ag(C₁₀H₈N₂)(NH₃)]NO₃. The crystal structure consists of dimers formed by two symmetry‐related Ag^I–bipy monomers connected through intra‐dimer π–π stacking and ligand‐unsupported Ag...Ag interactions. A crystallographic C2 axis passes through the mid‐point of and is perpendicular to the Ag...Agⁱ(−x + 1, y, −z + ) axis. In addition, each Ag^I cation is coordinated by one chelating bipy ligand and one ammine ligand, giving a trigonal coordination environment capped by the symmetry‐equivalent Ag atom. Molecules are assembled by Ag...Ag, π–π, hydrogen‐bond (N—H...O and C—H...O) and weak Ag...π interactions into a three‐dimensional framework. Comparing the products synthesized under different mechanical treatments, we found that reaction conditions have a significant influence on the resulting structures. The luminescence properties of the title compound are also discussed.     

Synthesis,Characterization, and DNA Binding Studies of a Nickel(II) Complex Containing the 1,3‐Bis(1‐propylbenzimidazol‐2‐yl)‐2‐oxapropane

A complex of formula [Ni(pobb)₂](pic)₂, (pobb = 1,3‐bis(1‐propylbenzimidazol‐2‐yl)‐2‐oxapropane, pic = 2,4,6‐trinitrophenol), has been synthesized and structurally characterized by physico‐chemical and spectroscopic methods. The crystals crystallize in the monoclinic system, space group C2/c, a = 25.766(11) Å, b = 14.943(7) Å, c = 19.543(14) Å, α = 90°, β = 129.722(4)°, γ = 90°, Z = 4. The coordination environment around nickel(II) atom can be described as a distorted octahedral geometry. The interactions of the ligand pobb and the nickel (II) complex with calf thymus DNA (CT‐DNA) are investigated by using electronic absorption titration, ethidium bromide‐DNA displacement experiments and viscosity measurements. The experimental evidence indicated the compounds interact with calf thymus DNA through intercalation.     

Synthesis,Crystal Structure and Spectroscopic Properties of [2,13‐Bis(1‐naphthalenylmethyl)‐5,16‐dimethyl‐2,6,13,17‐tetraazatricyclo(14,4,01.18,07.12)docosane]copper(II) Diperchlorate Acetonitrile Disolvate

The N‐functionalized macrocyclic ligand 2,13‐bis(1‐naphthalenylmethyl)‐5,16‐dimethyl‐2,6,13,17‐tetraazatricyclo(14,4,0^1.18,0^7.12)docosane (L³) and its copper(II) complex were prepared. The crystal structure of [Cu(L³)](ClO₄)₂·2CH₃CN was determined by single‐crystal X‐ray diffraction at 150 K. The copper atom, which lies on an inversion centre, has a square planar arrangement and the complex adopts a stable trans‐III configuration. The longer distance [2.081(2) Å] for Cu–N(tertiary) compared to 2.030(3) Å for Cu–N(secondary) may be due to the steric effect of the attached naphthalenylmethyl group on the tertiary nitrogen atom. Two perchlorate ions are weakly attached to copper in axial sites and are further connected to the ligand of the cation through NH ··· O hydrogen bonds [N ··· O 3.098 Å]. IR and UV/Vis spectroscopic properties are also described.     

Synthesis,Characterization, and Catalytic Performance in Oxidation of Styrene and α‐Methyl Styrene of a Nickel(II) Complex Derived from a Schiff Base Ligand

A tetrahedrally‐distorted square‐planar nickel(II) complex of tetradentate Schiff base ligand derived from 2‐hydroxypropiophenone and 2,2′‐dimethylpropandiamine [bis(2‐hydroxypropiophenone)2,2′‐dimethylpropylenediimine] ( H₂L ) was prepared and used as catalyst for oxidation of styrene and α‐methyl styrene with tert‐butylhydroperoxide (TBHP). Oxidation of styrene with TBHP gave benzaldehyde and styrene oxide, but in the case of α‐methylstyrene a mixture of α‐methylstyrene oxide and acetophenone was obtained. The structure of nickel(II) complex ( NiL ) was determined by X‐ray crystallography. Crystal data for NiL at –173 °C: orthorhombic, space group P2₁2₁2₁, a = 907.7(1), b = 1289.4(1), c = 1752.4(1) pm, Z = 4, R₁ = 0.0454.     

[N,N′‐Bis­(salicyl­idene)‐2,2‐di­methyl‐1,3‐propane­diaminato]­nickel(II) and [N,N′‐bis­(salicyl­idene)‐2,2‐di­methyl‐1,3‐propane­diaminato]copper(II)

In the title compounds, {2,2′‐[2,2‐di­methyl‐1,3‐propane­diyl­bis­(nitrilo­methyl­idyne)]­diphenolato‐κ⁴N,N′,O,O′}nickel(II), [Ni(C₁₉H₂₀N₂O₂)], and {2,2′‐[2,2‐di­methyl‐1,3‐propane­diyl­bis­(nitrilo­methyl­idyne)]­diphenolato‐κ⁴N,N′,O,O′}copper(II), [Cu(C₁₉H₂₀N₂O₂)], the Ni^II and Cu^II atoms are coordinated by two iminic N and two phenolic O atoms of the N,N′‐bis­(salicyl­idene)‐2,2‐di­methyl‐1,3‐propane­diaminate (SALPD^2?, C₁₇H₁₆N₂O₂^2?) ligand. The geometry of the coordination sphere is planar in the case of the Ni^II complex and distorted towards tetrahedral for the Cu^II complex. Both complexes have a cis configuration imposed by the chelate ligand. The dihedral angles between the N/Ni/O and N/Cu/O coordination planes are 17.20 (6) and 35.13 (7)°, respectively.