Metal‐based antibacterial and antifungal amino acid derived Schiff bases: their synthesis,characterization and in vitro biological activity

A new series of antibacterial and antifungal amino acid derived Schiff bases and their cobalt(II), copper(II), nickel(II) and zinc(II) metal complexes have been synthesized and characterized by their elemental analyses, molar conductances, magnetic moments, IR and electronic spectral measurements. The spectral data indicated the Schiff base ligands ( L ₁– L ₅) derived by condensation of salicylaldehyde with glycine, alanine, phenylalanine, methionine and cysteine, to act as tridentate towards divalent metal ions (cobalt, copper, nickel and zinc) via the azomethine‐N, deprotonated carboxyl group of the respective amino acid and deprotonated oxygen atom of salicylaldehyde by a stoichiometric reaction of M: L (1:2) to form complexes of the type K₂[M( L )₂] [where M = Co(II), Cu(II), Ni(II) and Zn(II)]. The magnetic moments and electronic spectral data suggested that all complexes have an octahedral geometry. Elemental analyses and NMR spectral data of the ligands and their Zn (II) complexes agree with their proposed structures. The synthesized ligands, along with their metal complexes, were screened for their in‐vitro antibacterial activity against four Gram‐negative (Escherichia coli, Shigella flexeneri, Pseudomonas aeruginosa and Salmonella typhi) and two Gram ‐ positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains and for in‐vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. The results of these studies show the metal complexes to be more antibacterial/antifungal against one or more species as compared with the uncomplexed Schiff base ligands. The brine shrimp bioassay was also carried out to study their in‐vitro cytotoxic properties. Only three compounds ( 2, 11 and 17 ) displayed potent cytotoxic activity as LD₅₀ = 8.196 × 10^?4, 7.315 × 10^?4 and 5.599 × 10^?4 M /ml respectively, against Artemia salina. Copyright © 2007 John Wiley & Sons, Ltd.     

Metal‐based isatin‐bearing sulfonamides: their synthesis,characterization and biological properties

A new series of antibacterial and antifungal isatin bearing sulfonamides and their cobalt (II), copper (II), nickel (II) and zinc (II) metal complexes have been synthesized, characterized and screened for their in vitro antibacterial activity against Bacillus cereus, Corynebacterium diphtheriae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigilla dysentriae and Staphylococcus aureus and for in vitro antifungal activity against Trichophyton schoenleinii, Candid glabrata, Pseudallescheria boydii, Candida albicans, Aspergillus niger, Microsporum canis and Trichophyton mentagrophytes. The results of these studies revealed that all compounds showed moderate to significant antibacterial activity. The brine shrimp bioassay was also carried out to study their in vitro cytotoxic properties. Only three compounds, 2, 11 and 22 displayed potent cytotoxic activity as LD₅₀ = 1.56 × 10^?7, 1.59 × 10^?7 and 1.67 × 10^?7 M /ml respectively, against Artemia salina. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis,characterization and biological properties of sulfonamide‐derived compounds and their transition metal complexes

Sulfonamide‐derived compounds and their first row d‐transition metal chelates [cobalt(II), copper(II), nickel(II) and zinc(II)] have been synthesized and characterized. The nature of bonding and structure of all the synthesized compounds have been proposed from magnetic susceptibility and conductivity measurements, IR, ¹H and ¹³C NMR, electron spectra, mass spectrometry and CHN analysis data. The structure of ligand, 4‐{[(E)‐(5‐chloro‐2‐hydroxyphenyl) methylidene] amino}‐N‐(4,6‐dimethyl pyrimidin‐2‐yl) benzene sulfonamide has also been determined by X‐ray diffraction method. An octahedral geometry has been suggested for all the complexes. The ligands and metal complexes have been screened for their in vitro antibacterial, antifungal and cytotoxic activity. The results of these studies revealed that all compounds showed moderate to significant antibacterial activity against one or more bacterial strains and good antifungal activity against various fungal strains. Copyright © 2009 John Wiley & Sons, Ltd.     

Sulfonamide‐derived compounds and their transition metal complexes: synthesis,biological evaluation and X‐ray structure of 4‐bromo‐2‐[(E)‐{4‐[(3,4‐dimethylisoxazol‐5 yl)sulfamoyl]phenyl} iminiomethyl] phenolate

Sulfonamide‐derived new ligands, 4‐({[(E)‐(5‐bromo‐2‐hydroxyphenyl)methylidene]‐amino}methyl)benzenesulfonamide and 4‐bromo‐2‐((E)‐{4‐[(3,4‐dimethylisoxazol‐5‐yl)sulfamoyl]phenyl}iminiomethyl)phenolate and their transition metal [cobalt(II), copper(II), nickel(II) and zinc(II)] complexes were synthesized and characterized. The nature of bonding and structure of all the synthesized compounds were deduced from physical (magnetic susceptibility and conductivity measurements), spectral (IR, ¹H and ¹³C NMR, electronic, mass spectrometry) and analytical (CHN analysis) data. The structure of the ligand, 4‐bromo‐2‐((E)‐{4‐[(3,4‐dimethylisoxazol‐5‐yl)sulfamoyl]phenyl} iminiomethyl)phenolate was also determined by X‐ray diffraction method. An octahedral geometry was suggested for all the complexes. In order to evaluate the biological activity of the ligands and the effect of metals, the ligands and their metal complexes were screened for in vitro antibacterial, antifungal and cytotoxic activity. The results of these studies revealed that all compounds showed moderate to significant antibacterial activity against one or more bacterial strains and good antifungal activity against various fungal strains. Copyright © 2011 John Wiley & Sons, Ltd.     

New anti‐bacterial polychelates: synthesis,characterization, and anti‐bacterial activities of thiosemicarbazide–formaldehyde resin and its polymer–metal complexes

New polymeric ligand (resin) was prepared by the condensation of thiosemicarbazides with formaldehyde in the presence of acidic medium. Thisemicarbazide–formaldehyde polymer–metal complexes were prepared with Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) in 1:2 metal:ligand molar ratio. The polymeric ligand and its polymer–metal complexes were characterized by elemental analysis, thermogravimetric analysis (TGA), FTIR, ¹³C NMR and ¹H NMR. The geometry of central metal ions was conformed by electronic (UV–vis) and EPR spectra. The antibacterial activities of all the synthesized polymers were investigated against Bacillus subtilis and Staphylococcus aureus (Gram‐positive) and Escherichia coli and Salmonella typhi (Gram‐negative). These compounds showed excellent activities against these bacteria using the shaking flask method. Copyright © 2008 John Wiley & Sons, Ltd.     

Microwave‐Assisted Synthesis and Spectral Characterization of Biological Activity Heterocyclic Thiosemicarbazone and Its Organometallic Derivatives

The thiosemicarbazone coordinates to metal through the thionic sulfur and the azomethine nitrogen. The thiosemicarbazone was obtained by the thiosemicarbazide with 3‐acetyl‐2,5‐dimethylfuran by microwave irradiation. Metal complexes were prepared by the reaction of thiosemicarbazone with metal salts. IR, ¹H, ¹³C NMR, FAB‐mass spectroscopic methods, and elemental analysis elucidated the structure of these compounds. The antibacterial activity of these compounds were first tested in vitro by the disk diffusion assay against two Gram‐positive and two Gram‐negative bacteria, and then minimum inhibitory concentration was determined, chloramphenicol as reference drug. The results showed that copper ( II ) metal complex of the thiosemicarbazone ( 1.1 ) is a better inhibitor as compared with chloramphenicol.     

Organometallic based biologically active compounds: synthesis of mono‐ and di‐ethanolamine derived ferrocenes with antibacterial,antifungal and cytotoxic properties

Condensation reactions of 1,1′‐diacetylferrocene with ethanolamine were studied. The obtained compounds were further investigated for their ligation and biological properties with Co(II), Cu(II), Ni(II) and Zn(II) metal ions. The synthesized compounds were characterized by their physical, spectral and analytical properties and screened for their antibacterial properties against pathogenic bacterial strains, e.g. Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and Salmonella typhi and for antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata using the agar‐well diffusion method. All the compounds have shown good antibacterial and antifungal activity, which increased on coordination with the metal ions, thus introducing a potential class of organometallic‐based antibacterial and antifungal agents. Brine shrimp bioassay was also carried out for in vitro cytotoxic properties against Artemia salina. Copyright © 2007 John Wiley & Sons, Ltd.     

Organometallic compounds with biologically active molecules: in vitro antibacterial and antifungal activity of some 1,1′‐(dicarbohydrazono) ferrocenes and their cobalt(II), copper(II), nickel(II) and zinc(II) complexes

Some 1,1′‐(dicarbohydrazono) ferrocenes have been prepared by condensing 1,1′‐diacetylferrocene with either 2‐furoic hydrazide, 2‐thiophenecarboxylic hydrazide or 2‐salicylic hydrazide. All the ligands synthesized were characterized by IR and NMR spectroscopy and elemental analysis data (carbon, hydrogen, nitrogen) and then were used as ligands to react with cobalt(II), copper(II), nickel(II) and zinc(II) metals as chlorides to afford metal complexes having the general formula M(L)Cl₂. IR and electronic spectral data, magnetic moment and elemental analyses were used in the structural investigation of the metal complexes synthesized. The ligands synthesized and their metal(II) complexes have been screened for their in vitro antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, Bacillus cereus, Corynebacterium diphtheriae, Staphylococcus aureus and Streptococcus pyogenes bacterial strains and for in vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. The results of these studies show the metal complexes to be more antibacterial and antifungal than the uncomplexed ligands. However, the potency of all the ligands synthesized and their metal complexes was lower than that of the standard drugs. Copyright © 2005 John Wiley & Sons, Ltd.     

Dicoumarol complexes of Cu(II), Fe(II) and Fe(III): preparation,characterization, in‐vitro antibacterial and DNA binding activity

3‐3′‐Benzylidenebis[4‐hydroxycoumarin] or 4‐nitro,3‐3′‐benzylidenebis[4‐hydroxycoumarin] or 4‐methoxy,3‐3′‐benzylidenebis[4‐hydroxycoumarin] and their complexes with Cu(II), Fe(II) and Fe(III) were synthesized and characterized using ¹H‐NMR, ¹³C‐NMR, IR spectra, electronic spectra, magnetic measurements and elemental analyses. The ligands, metal salts, complexes, control and standard drug were tested for their in‐vitro antibacterial activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi, and Serratia marcescens. The metal complexes exhibit good activity against bacterial strains compared with parental compounds and moderate compared with the standard drug (ciprofloxacin). In‐vitro DNA‐binding activity was carried out using agarose gel electrophoresis. The synthesized compounds show effective DNA‐binding activity. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and Antimicrobial Activity of Some New N‐substituted‐5‐arylidene‐thiazolidine‐2,4‐diones

A total of 17 new N‐substituted derivatives ( 2b , 2c , 2d , 2e , 2f , 2g , 2h , 2i , 2j , 2k and 3b , 3c , 3d , 3e , 3f , 3g , 3h ) of 5‐((2‐phenylthiazol‐4‐yl)methylene) thiazolidine‐2,4‐dione ( 2a ) and 5‐(2,6‐dichloro‐ benzylidene)thiazolidine‐2,4‐dione ( 3a ) were synthesized. The structural elucidation of the newly synthesized compounds was based on elemental analysis and spectroscopic data (MS, ¹H NMR, ¹³C NMR), and their antimicrobial activities were assessed in vitro against several strains of Gram‐positive and Gram‐negative bacteria and one fungal strain (Candida albicans) as growth inhibition diameter. Some of them showed modest to good antibacterial activity against Gram‐negative Escherichia coli and Salmonella typhimurium and Gram‐positive Staphylococcus aureus, Bacillus cereus, and Enterococcus fecalis bacterial strains, whereas almost all the compounds were inactive against Listeria monocytogenes. All of the synthesized compounds showed moderate to very good activity against C. albicans.     

Synthesis and characterization of transition metal complexes of hydrochloride salt of 3‐chlorobenzaldehyde hydralazine hydrazone: a new class of possible anti‐cariogenic agents

A novel hydrazone, formed by the condensation of the hydrochloride salt of hydralazine with 3‐chlorobenzaldehyde, and its Co(II), Ni(II), Cu(II) and Zn(II) complexes have been synthesized. Their structures have been elucidated on the basis of elemental analyses, conductance measurements, magnetic moments, and spectral (infrared, ¹H NMR, UV–visible, electrospray ionization (ESI) mass) and thermal studies. The bidentate behaviour of the ligand is proposed on the basis of spectral studies. Interestingly, all four complexes exhibit different geometry around the metal centre. The conductance data of the complexes suggest them to be non‐electrolytes. The ESI mass spectra of the complexes support their monomeric nature. The compounds were tested against two Gram‐positive and three Gram‐negative bacterial strains and three fungal strains. Excellent inhibitory activity is observed against the Gram‐positive bacteria Streptococcus mutans and Enterococcus faecalis which play major roles in tooth decay. Among the fungal strains used, Candida albicans is inhibited predominantly. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and Antimicrobial Evaluation of Some New 4,5′‐Bisthiazoles

Thiazole and bisthiazole derivatives represent a prevalent scaffold in the antimicrobial drug discovery. Therefore, we have decided to synthesize some new series of 4,5′‐bisthiazoles. A total of 17 compounds were synthesized, their structural elucidation being based on elemental analysis (C,H,N,S) and spectroscopic data (MS and ¹H NMR). Their in vitro antimicrobial activities were assessed against several Gram‐positive and Gram‐negative bacteria strains and also against one fungal strain (Candida albicans) using the difusimetric method. Some of the compounds showed modest to good antibacterial activity against Gram‐negative Escherichia coli and Salmonella typhimurium and Gram‐positive Staphylococcus aureus and Bacillus cereus bacterial strains. All of the synthesized compounds showed moderate to very good antifungal activity against C. albicans.     

Synthesis,characterization and catalytic,cytotoxic and antimicrobial activities of two novel cyclotriphosphazene‐based multisite ligands and their Ru(II) complexes

Two novel cyclotriphosphazene ligands ( 2 and 3 ) bearing 3‐oxypyridine groups and their corresponding Ru(II) complexes ( 4 and 5 ) were synthesized and their structures were characterized using Fourier transform infrared, ¹H NMR and ³¹P NMR spectroscopic data and elemental analysis. The Ru(II) complexes were used as catalysts for catalytic transfer hydrogenation of p‐substituted acetophenone derivatives in the presence of KOH. Additionally, the cytotoxic activities of compounds 2 , 3 , 4 , 5 were evaluated against PC3 (human prostate cancer), DLD‐1 (human colorectal cancer), HeLa (human cervical cancer) and PNT1A (normal human prostate) cell lines. Finally the antimicrobial activities of compounds 2 , 3 , 4 , 5 were evaluated against a panel of Gram‐positive and Gram‐negative bacteria and yeast cultures. The complexes showed efficient catalytic activity towards transfer hydrogenation of acetophenone derivatives, especially those bearing electron‐withdrawing substituents on the para‐position of the aryl ring. The compounds were found to have moderate to high cytotoxic and antimicrobial activities, and Ru(II) complexation enhanced both cytotoxic and antimicrobial activities in comparison with the parent compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis,spectral, DFT calculations and antibacterial studies of Fe(III) complexes of new fluorescent Schiff bases derived from imidazo[4',5':3,4]benzo[1,2‐c]isoxazole

New fluorescent heterocyclic ligands were synthesized by the reaction of 8‐(4‐chlorophenyl)‐3‐alkyl‐3H‐imidazo[4',5':3,4]benzo [1,2‐c]isoxazol‐5‐amine with p‐hydroxybenzaldehyde and p‐chlorobenzaldehyde in good yields. The coordination ability of the ligands with Fe³⁺ ion was examined in an aqueous metanolic solution. Schiff base ligands and their metal complexes were characterized by elemental analyses, IR, UV–vis, mass, and NMR spectra. The optical properties of the compounds were investigated and the results showed that the fluorescence of all compounds is intense and their obtained emission quantum yields are around 0.15 – 0.53. Optimized geometries and assignment of the IR bands and NMR chemical shifts of the new complexes were also computed by using density functional theory (DFT) methods. The DFT‐calculated vibrational wavenumbers and NMR chemical shifts are in good agreement with the experimental values, confirming suitability of the optimized geometries for Fe(III) complexes. Also, the 3D‐distribution map for HOMO and LUMO of the compounds were obtained. The new compounds showed potent antibacterial activity and their antibacterial activity (MIC) against Gram‐positive and Gram‐negative bacterial species were also determined. Results of antibacterial test revealed that coordination of ligands to Fe(III) leads to improvement in the antibacterial activity.     

Design,synthesis, characterization and antibacterial properties of copper(II) complexes with chromone‐derived compounds

A new series of six chromone‐derived compounds and their Cu(II) complexes was synthesized and characterized by their physical, spectral and analytical data The elemental analysis data of the complexes agree well with the proposed composition of the compounds, which were found to be dimeric in nature with two hydrazine molecules bridging the two copper atoms through coordination. The ligands and their Cu(II) complexes were screened for their in‐vitro antibacterial activity against four Gram‐negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri) and two Gram‐positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains by the agar‐well diffusion method. The ligands were found to exhibit either no or low to moderate activities against one or more of the bacterial species, whereas all the metal complexes exhibited moderate to high activities against different bacterial species. The ligands which were inactive before complexation turned active and less active ones became more active upon coordination with copper ions. Overall, the complexes 7–12 showed comparatively much higher activities than the ligands. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,spectral characterization and biological studies of transition metal(II) complexes with triazole Schiff bases

New metal based triazoles (1–12) have been synthesized by the interaction of novel Schiff base ligands (L¹–L³) with the Co(II), Ni(II), Cu(II) and Zn(II) metal ions. The Schiff base ligands and their all metal(II) complexes have been thoroughly characterized using various physical, analytical and spectroscopic techniques. In vitro bacterial and fungal inhibition studies were carried out to examine the antibacterial and antifungal profile of the Schiff bases in comparison to their metal(II) complexes against two Gram‐positive, four Gram‐negative and six fungal strains. The bioactivity data showed the metal(II) complexes to have more potent antibacterial and antifungal activity than their uncomplexed parent Schiff bases against one or more bacterial and fungal species. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and In Vitro Antibacterial Activity of Novel Steroidal (6R)‐Spiro‐1,3,4‐thiadiazoline Derivatives

Novel steroidal (6R)‐spiro‐1,3,4‐thiadiazoline derivatives were synthesized by the cyclization of steroidal thiosemicarbazones with acetic anhydride, screened in vitro against antibacterial activity using disc‐diffusion method and the minimum inhibitory concentration. The results showed that steroidal thiadiazoline derivatives exhibited better antibacterial activity than the steroidal thiosemicarbazone derivatives. Chloro and acetoxy substituents on the 3β‐position of the steroidal thiadiazoline ring increased the antibacterial activity. Among all the compounds, compound 7 and 8 were found better inhibitors of both types of bacteria (Gram‐positive and Gram‐negative) as compared to the respective drug amoxicillin. All the synthesized compounds were well characterized by spectroscopic methods such as IR, ¹H‐NMR, ¹³C‐NMR mass, and elemental analysis and their stereochemistry was also discussed.     

Microwave‐assisted Synthesis,Characterization, and Density Functional Theory Study of Biologically Active Ferrocenyl Bis‐pyrazoline and Bis‐pyrimidine as Organometallic Macromolecules

Organometallic macromolecules such as ferrocenyl bis‐pyrazoline ( 2 , 3 ) and bis‐pyrimidine ( 4 , 5 ) derivatives were synthesized by reacting ferrocenyl bis‐chalcone 1 with thiosemicarbazide/phenylhydrazine/guanidine hydrochloride/thiourea, respectively, under microwave irradiation. Ferrocenyl bis‐chalcone 1 was synthesized by reacting acetyl ferrocene with terephthalaldehyde. Synthesized compounds were characterized by using IR, ¹H NMR, ¹³C NMR, EI‐MS, and elemental analysis. In vitro antibacterial activity against two Gram‐negative and two Gram‐positive bacteria was determined by the disc diffusion assay. Moreover, minimum inhibition concentrations were also measured with reference to chloramphenicol. Thioamide functionally containing ferrocenyl bis‐pyrazoline derivative 2 shows the best antibacterial activity on Escherichia coli and Salmonella typhimurium, among all tested compounds including the reference drug chloramphenicol. The structure–activity relationship is also developed by using computational calculations with density functional theory (DFT)/B3LYP method.     

Synthesis, Characterization and Antimicrobial Activity of Potassium Hydro(benzoyl)(phthalyl)borate and Its Cobalt(II), Nickel(II), and Copper(II) Complexes

A new organoborate ligand, hydro(benzoyl)(phthalyl)borate has been synthesized as its potassium salt (KL) and treatment of KL with one equivalent of MCl2•6H2O gave complexes ML(H2O)x•Cl [x＝2, M＝Co(II), Ni(II); x＝1, M＝Cu(II)]. All compounds were characterized by elemental analysis, FTIR, 1H NMR, ESI MS, UV-Vis techniques, conductivity and magnetic data measurements. Spectroscopic results suggest a square planar geometry in the Cu(II) complex, while the Co(II) and Ni(II) complexes possess an octahedral geometry. Antibacterial activities (in vitro) of the ligand and its metal complexes were studied against two Gram positive (B. subtillis and B. magterium) and two Gram negative bacteria (E. Coli and S. boydi) at a single concentration (75 μg/mL) by using the Disc diffusion method. Antifungal activities (in vitro) were also checked for the compounds by using the same method against Candida albicans 10261, Penicillium sp. and Asperjillius niger., at a single concentration (50 μg/mL). The results showed that all the metal complexes, specially the nickel(II) complex, have higher antibacterial and antifungal activities than the corresponding potassium salt.     

DNA interaction,antimicrobial and molecular docking studies of biologically interesting Schiff base complexes incorporating 4‐formyl‐N,N‐dimethylaniline and propylenediamine

Four new transition metal complexes incorporating a Schiff base ligand derived from propylenediamine and 4‐formyl‐N ,N ‐dimethylaniline have been synthesized using transition metal salts. The characterization of the newly formed complexes was done from physicochemical parameters and using various techniques like ¹H NMR, ¹³C NMR, IR, UV, electron paramagnetic resonance and mass spectroscopies, powder X‐ray diffraction and magnetic susceptibility. All the complexes were found to be monomeric in nature with square planar geometry. X‐ray powder diffraction illustrates that the complexes have a crystalline nature. The interaction of metal complexes with calf thymus DNA was investigated using UV–visible absorption, viscosity measurements, cyclic voltammetry, emission spectroscopy and docking analysis. The results indicate that the Cu(II), Co(II), Ni(II) and Zn(II) complexes interact with DNA by intercalative binding mode with optimum intrinsic binding constants of 4.3 × 10⁴, 3.9 × 10⁴, 4.7 × 10⁴ and 3.7 × 10⁴ M⁻¹, respectively. These DNA binding results were rationalized using molecular docking in which the docked structures indicate that the metal complexes fit well into the A‐T rich region of target DNA through intercalation. The metal complexes exhibit an effective cleavage with pUC19 DNA by an oxidative cleavage mechanism. The synthesized ligand and the complexes were tested for their in vitro antimicrobial activity. The complexes show enhanced antifungal and antibacterial activities compared to the free ligand.