Metal complexation by a,-mercapto- p-aryl acrylic acids

JOSEPH WAGNER,' PIERRE VITALI, JOSIANE SCHOUN, AND EUGENE GIROUX~ Centre de Recherche Merrell International, 16, rue &Ankara, 67084 Strasbourg, Cedex, France

Received June 21, 1977

JOSEPH WAGNER, PIERRE VITALI, JOSIANE SCHOUN, and EUGENE GIROUX. Can. J. Chem. 55, 4028 (1977).

Protonation constants of several a-mercapto-a-aryl acrylic acids and constants for formation of complexes between these ligands and bivalent metal cations were determined by computer- assisted analysis of pH-titration data. Ligand-metal ion mixtures in 50% (v/v) dioxane-water and in water, I = 0.1 M (NaC104) were titrated at 25'C with sodium hydroxide. Initial esti- mates of complex formation constants were refined with the SCOGS program. The ligands were shown to conlplex zinc(I1) and nickel(I1) metal ions avidly. These ligands potently inhib- ited the metal-dependent enzymes alkaline phosphatase, carboxypeptidase A, coeruloplasmin, and thermolysin. Partition of zinc(I1) between aqueous and n-butanol or n-octanol phases was enhanced in favor of the organic phase by several of these complexants.

JOSEPH WAGNER, PIERRE VITALI, JOSIANE SCHOUN et EUGENE GIROUX. Can. J. Chem. 55,4028 (1977).

Les constantes de protonation et les constantes deformation decomplexes demetaux bivalents avec une skrie d'acides a-mercapto-P-aryl acryliques ont et6 diterminees par analyse B l'ordina- teur des courbes de titration pH-metriques. Les titrations ont kt6 effectutes sur des melanges de ligand et du cation mktallique dans des solutions de 50% (v/v) dioxane-eau et dans I'eau, .I = 0.1 M (NaCIO,) B 25°C avec de la soude. Les valeurs initiales des constantes ont CtC deter- minkes ti partir des courbes de formation et ont et6 affinkes par utilisation du programme SCOGS. Les constantes obtenues montrent que les ligands prksentent une grande affinitC specialement pour le zinc(I1) et le nickel(I1). Ces ligands inhibent fortement les metalloenzymes, phosphatase alcaline, carboxypeptidase A, ceruloplasmine et thermolysine. Plusieurs de ces ligands augmentent I'extraction du zinc dans la phase organique dans des essais de partition du zinc(I1) entre I'eau et le n-butanol ou le n-octanol.

Introduction Some P-aryl derivatives of a-mercaptoacrylic

acid influence the distribution and metabolism of zinc in the rat. Following administration of a single dose of a-mercapto-P-(2-fury]) acrylic acid or a-mercapto-P-phenyl acrylic acid, blood plasma zinc concentration increased many-fold within a few hours and remained elevated for as long as 5 days (1). Evidence for formation of a ternary complex of plasma albumin, zinc, and the fury1 derivative was obtained (2). While the complexation of metal ions by many a- and P-mercapto acids and amino acids has been well studied (3), few data of a similar nature are avail- able for the a-mercapto-P-aryl acrylic acids. Foye and Lo (4) reported stability constant data for complexes of the phenyl derivative with A13 +, Cu2 +, and Fe3 + ; they also found this com- poundto have antimicrobial activity. In this report we present data characterizing complexation by several a-mercapto-P-aryl acrylic acids of

'Author to whom correspondence should be addressed. 'Present address: Merrell-National Laboratories,

Cincinnati, OH 54215, USA.

zinc and of other metal cations and data on the inhibition of four metal-dependent enzymes by these ligands and other metal-complexing agents.

Experimental Syntheses

The a-mercapto-P-aryl acrylic acids depicted in Scheme 1 were prepared by alkaline hydrolysis of the correspond- ing rhodanine derivatives obtained from the condensa- tion of the appropriate aldehyde with rhodanine, fol- lowing the procedure of Campaigne and Cline (5). Melt- ing points were determined on a Kofler hot stage ap- paratus and are uncorrected. The nmr spectra were recorded on a Varian T60 using tetramethylsilane (TMS) or 3-(trimethylsily1)-1-propane sulfonic acid as internal standards. Chemical shifts are given in ppm (6, TMS = 0) and coupling constants in Hz. The abbreviations s, d, m, and br refer to singlet, doublet, multiplet and broad- ened, iespectively. Compounds 1, 2, 5, 6, 7, and 10, al- ready described in the literature (6-9) presented analytical data in agreement with their s t r~c ture .~ Thiosalicylic acid 9 (Fluka) was recrystallized from methanol.

3These data have been deposited in the Depository of Unpublished Data. Copies may be obtained,at a nominal charge, from the Depository of Unpublished Data, CISTI, National Research Council of Canada, Ottawa, Canada KIA 0S2.

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WAGNER ET AL. 4029

R was recovered by filtration. The material was washed by f stirring with 50 ml of water under nitrogen at room tem- R-CHO + CHI-C R-CH=C-C

/ \ - perature, acidified with concentrated hydrochloric acid

S\C/NH ' \ and filtered to yield 0.8 g (60%) of slightly brown crystals, S\C /NH mp 158°C; nmr (acetone-d6): 7.1 (m, 2H, ring protons),

1 1 1 1 7.6 (s, lH, exocyclic -CH=). Anal. calcd. for S S C8H,F303S: C 40.34, H 2.1 1, S 13.46; found: C 40.26,

H 2.11, S 13.60.

5- (5-Sodiumsulfbnate-2-fury!methylene) Rhodanine 5-Sodiumsulfonate-2-furanaldehyde was converted as

above to the rhodanine derivative. Recrystallization in /SH water gave yellow crystals (73% yield), mp 280°C (dec.); R-CH=C \COOH nmr (DMSO-d6): 6.62 (d, J = 3, 1H, ring proton), 7.03

(d, J = 3, lH, ring proton), 7.4 (s, lH, exocyclic-CH=).

1 R = 0- 6 R = Anal. calcd. for C8H4N05S3Na: C 30.67, H 1.29, N 4.47; found: C 30.63, H 1.32, N 4.51.

4 R = Na03S a 9 msH

5 R = COOH

,COOH

I COOH

5-(5-TriJluoromethyl-2-furylmethylene) Rhodanine 5-Trifluoromethyl-2-furfural(3.4g, 2.1 mmol) (lo), rho-

danine (2.92 g, 2.2 mmol), and dry sodium acetate (5.16 g) in 35 ml of glacial acetic acid were stirred together and heated to reflux. After 10 min, a precipitate occurred and heating was continued for 2 h. The reaction mixture was cooled, 30 ml of water was added, and the precipitate was filtered, washed with water, and dried. Chromatog- raphy over silica gel (eluent CH2C12), followed by recrys- tallization in dichloromethane-pentane yielded 3.7 g (63%) of yellow crystals, mp 174°C; nmr (CDCI,): 6.80 (m, 2H, ring protons), 7.38 (s, lH, exocyclic -CH=). Anal. calcd. for C,H4F3N02S2: C 38.71, H 1.44, N 5.01, S 22.96; found: C 38.70, H 1.56, N 5.10, S 22.91.

a-Mercapto-[3-(5-triJ(uorometlzy1-2-furyl) Acrylic Acid (3)

5-(5-Trifluoromethyl-2-furylmethylene) rhodanine (1.58 g, 5.66 mmol), 25 ml of 1 N sodium hydroxide solution, and 25 ml of water were stirred under nitrogen at room temperature for 10 h. After cooling and acidification at p H 1.5 with concentrated hydrochloric acid, a precipitate

a-Mercapto-0-(5-sodiumsulfonate-2-fury) Acrylic Acid (4)

This compound was obtained from the corresponding rhodanine derivative (89% yield); mp >220"C (dec.); nmr (D20): 6.88 (s, 2H, ring protons), 7.45 (s, lH , exocyclic -CH=). Anal. calcd. for C7H506S2Na: C 30.88, H 1.85; found: C 30.69, H 1.81.

5- (1,2,3,4-Tetrahydro-6-methyl-2,4-dioxo-5- pyrimidylmethylene) Rhodanine

This compound was obtained from 5-carbaldehyde-6- methyluracil (11) (83% yield); orange crystals, mp > 260°C; nmr (DMSO-d6): 2.33 (s, 3H, CH,), 7.21 (s, IH, exocyclic -CH=), 11.46 (s, lH, NH), 12.33 (b s, 1H, NH). Anal. calcd. for C9H7N303S2: C 40.13, H 2.62, N 15.60; found: C 40.20, H 2.74, N 15.64.

a-Mercapto-[3 (1,2,3,4-tetrahydro-6-methyl-2,4-dioxo- 5-pyrimidyl) Acrylic Acid ( 8 )

This compound was obtained from the preceding com- pound (68% yield); yellow crystals, mp 240°C (dec.); nmr (DMSO-d6): 2 (s, 3H, CH,), 7.2 (s, 1 H, -CH=), 11.1 (b s, 3H, NH, COOH). Anul. calcd. for C8H8N2O4S: C42.09, H 3.54,N 12.27; found: C41.99,H 3.72,N 12.31.

Potentiometric Titrations Water with greater than 10 MIL cm-' resistivity was

prepared using a Millipore 'Milli-Q' system. Dioxane (Baker AR) was refluxed 48 h over sodium and distilled through a Vigreux column. Solutions (0.1 IM) of sodium perchlorate, zinc(I1) nitrate, nickel(I1) chloride (all from E. Merck) or of the perchlorates of cobalt(II), iron(II), manganese(II), nickel(II), or zinc(I1) (all from Pierce Chemical) in water or in 50% (v/v) dioxai~e-water were slightly acidified with perchloric acid (pH = 3) to sup- press hydrolysis; concentrations were determined by Schwarzenbach's procedure (12). Sodium hydroxide solu- tions were standardized by titration with hydrochloric acid (Titrisol, E. Merck). Solutions (0.1 M ) of ligands in 50% (v/v) dioxane-water were prepared by dissolving the ligands in 5 ml of dioxane (degassed by bubbling of nitrogen) and adjusting to 10 ml with degassed water. One half equivalent of HCIOl was added in preparation of solutions of ligands 1, 2, 5, and 9. Ligand solutions were stored under nitrogen at 5°C. Fresh stock was pre- pared each day, as some ligands in solution, especially 6 and 7, oxidized to disulfide (5-10%) after 6-10 h.

Mixtures (20 ml) of ligand and metal ion were titrated

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4030 CAN. 5. CHEM. VOL. 55. 1977

with NaOH (0.3 M in water, or in 50% (v/v) dioxane- water). At each of two ligand concentrations (2 and 4 mM), metal ion concentrations were employed to yield ligand-metal stoichiometries of 8 : 1 ,4 : 1, and 2 : 1. Ligand solutions also were titrated in the absence of metal ion. All titrations were carried out in a water-jacketed vessel thermostated at 25 0.l0C. The titration cell was tightly covered and nitrogen saturated with solvent was bubbled through the solution.

A Mettler semi-automatic titration system consisting of modules DK 10, 12, 13, and 15 connected to a DV 11 burette and equipped with a Metrohm glass electrode EA 109 and an E 430 saturated calomel electrode was used. The electrodes and p H meter were calibrated to read p H with four aqueous buffer solutions (pH = 4.01 to 10.01). For measurements in 50% aqueous dioxane, the glass electrode was conditioned for several davs in

tained. Experimental and calculated formation curves were compared on a Hewlett-Packard 9820 desk calcu- lator fitted with a HP 9862 plotter.

Ligands and Zinc Partition Experitnents Two millilitres of a solution of zinc sulfate (33.5 pM),

prepared in 0.05 M 3-(N-morpholino)propanesulfonic acid, 0.15 M NaCI, p H 6.0, was shaken with complexant and n-octanol (8 ml) or n-butanol (2.5 ml). The com- plexant (4 mol/mol ZnS04) was freshly dissolved either in the organic solvent or in the zinc sulfate solution. After 5 min of contact the phases were separated by centrifuga- tion. Zinc concentration in the aqueous phase was deter- mined by atomic absorption spectroscopy. Zinc standards were prepared by dilution of Titrisol standard (E. Merck), using aqueous solvent contacted with organic solvent. Each partition mixture was analyzed in triplicate.

the mixed solvent (1 3). The sleeve-type calomel electrode was refilled with saturated KC1 after five to six titrations. Inhibition ofEnzymes by Complexants

The pH values were measured to +0.01 unit. E. Coli alkaline phosphatase (E.C.3.1.3.1., Sigma Chemical) was assayed s~ectro~hotometrical~y (18).

Protonation and Formation Constants using the'kit supplied by ~ o e h r i n ~ e r - ~ a n n h e i m . ~nz;r& Formation constants P p q , correspond to the general was incubated with inhibitor (freshly dissolved in di-

eauation methylsulfoxide or in buffer) for 15 min at 30°C in v H

'Practical' protonation constants of the ligands are

Initial estimates for plol and plO2 were obtained from titration curves and formation curves (6, = f(pH)) and were refined using the computer program SCOGS (14) on a UNIVAC 11 10 computer. For the calculation of H + and OH- concentrations from H+ activities as measured by the glass electrode, a correction factor of -0.06 + 0.02 was determined by titrating at a constant ionic strength of 0.1 at 25°C a 0.01 M solution of HC1 in 50% (v/v) dioxane-water with a solution of NaOH in 50% (vlv) dioxane-water. The pK, was calculated to be 15.38 + 0.02, in agreement with previous results (15, 16). For n1easuren1ents in water, the mean activity coefficient f * was taken as 0.79 and the pK, as 13.996 (17) at 25°C and I = 0.1 M (NaC104). Approximate values of forma- tion constants pilo and pZl0 were obtained at R = f(pL), given by [4] and [5]. CM and CL are total ligand and

metal concentrations, respectively, [EXacid] is the excess of perchloric acid added in preparation of stock solutions and Chase is the concentration of added base. Constants were refined over the entire range of experimental data by the use of the SCOGS program. If by adding other species, no further reduction in the standard deviation of titre could be achieved, the last set of values was re-

10.5 buffer before initiation of the enzymatic reaction-by addition of substrate. In all assays dimethylsulfoxide was present at a final concentration of 5% (v/v). Bovine car- boxypeptidase A (E.C.3.4.12.2, Sigma Chemical) was assayed by a pH-stat technique (19). Enzyme was incu- bated with inhibitor (freshly dissolved in polyethylene glycol 300 or in buffer) for 10 min at 30°C at pH7.5 before initiation of the enzymatic reaction by addition of sub- strate, hippuryl-D,L-P-phenyllactic acid (Fox Chemical). The final concentration of polyethylene glycol 300 was 5% (v/v). The pH of the reaction mixture was maintained at 7.5 with a Mettler titration system. Human coerulo- plasmin (E.C.1.16.3.1., Sigma Chemical) was assayed spectrophotometrically using o-dianisidine (Sigma Chem- ical) as substrate (20). The rate of reaction was deter- mined after preincubation of enzyme with inhibitor (freshly dissolved in ethanol; final ethanol concentra- tion 2.5% (v/v)) for 15 min at 30°C in p H 5.0 buffer. Assay mixtures contained 0.1 mM disodium EDTA (21) to suppress enhancement of activity due to traces of iron. The possibility of decolorization of the assay mixture by inhibitor was checked by the method of Curzon and Cumings (22). Thermolysin (E.C.3.4.24.4., Sigma Chem- ical) was assayed fluorimetrically using enzyme freed of low molecular weight contaminants by gel chromatog- raphy on sephadex G-25 (Pharmacia - ~ k e chemicals). Enzyme solution was stored at -25°C and thawed prior to assay. An aliquot was diluted to 1.8 ml with a buffer of 10 mM 5,5-diethylbarbituric acid, 90 mM NaCI, p H 7.5. (23). To the enzyme solution was added inhibitor freshly dissolved in buffer or in ethanol (a total of 0.1 ml ethanol in all assay tubes). The mixture was heated for 10 min in a water bath at 40°C. The enzymatic reaction was initiated by addition of 0.1 ml of a 20 m M solution of carbobenzoxyglycyl-L-leucineamide (Protein Research Foundation, Osaka, Japan) in ethanol. The reaction mix- ture was maintained at 40°C and six aliquots of 0.2 ml were removed at 5 min intervals. Hydrolysis of the pep- tide bond was measured in these samples by the fluo- rescamine technique (24).

In all inhibition studies the effect of four or more con-

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WAGNI <R ET AL. 403 1

centrations of a given inhibitor was assessed. Remaining enzyme activity was plotted as a function of inhibitor concentration. From a smooth curve drawn through the data, the concentration of inhibitor necessary to reduce activity to 50% of the control value was obtained by interpolation.

Results Protonation Constants

Computer-refined values of protonation con- stants of the thiol and carboxyl groups of the ligands are presented in Table 1. In the series of cl-mercapto-P-aryl acrylic acids the nature of the aryl substituent did not influence strongly the ionization of the carboxyl group, but ionization of the thiol group was influenced by the electron donation ability (25) of the aryl group (compare 1,2, and 5). For the furan derivatives, ionization of the carboxyl and thiol groups varied in the same order as the inductive effect of the sub- stituent (26) in position 5 of the furan ring (com- pare 2, 3, and 4). The pKa values found for ligands 1 and 9 are in agreement with those of other workers (4, 27, 28) if we take into account that our results are 'practical' pKa values.

Metal Complex Formation Constants Computer-refined values of formation con-

stants of metal-ligand complexes are presented in Table 2. An emphasis was placed upon the study of zinc complexes because of the effect these ligands have upon zinc metabolism. In Fig. 1 are illustrated titration curves of two concen-

TABLE 1. Protonation constants ((3,,,) of ligands at 25°C and I = 0.1 M (NaC104) in 50% (v/v) aqueous dioxane

Ligand ~ O I ( 3 1 0 ~ nb mc

afl,,, refers to the general complex L,M,H,, where L = ligand, M = metal, and H = proton; o is the standard deviation.

bNumber of experimental observations. =Number of titration curves analyzed. *pKa1 = 10.22, pKa2 = 4.72 in 50% ethanol-water at 25'C (4). .Values determined in aqueous medium. JpKa1 = 4.93, pKa2 = 9.96 in 50% (v/v) dioxane-water at 25"C,

I = 0.1 M (NaC104) (27). pK,, = 6.50, pK,, = 11.95 i n 75% ((rv) dioxane-water at 30°C, I = 0.02 (KCI) (sto~ch~ometr~c stab~llty constants) (28).

trations of a-mercapto-P-fury1 acrylic acid 2 in the absence and in the presence of zinc(I1) at ligand-metal ratios of 8 : 1, 4 : 1, and 2 : 1. Similar curves were prepared for the other ligands. The formation curves (Fi versus log [L2-1) corre- sponding to the titration curves of Fig. 1 for CL = 4 m M were all superimposable. This indi- cated that for the zinc(I1)-ligand system only the mononuclear species ZnL and ZnLz2- were present. Approximate values of the stability constants log PI,, (11.1) and log Pzl0 (20.5) ob- tained at ii = 112 and Fi = 312 were refined with the SCOGS program over the entire p H range (2.8-9.7) to an estimated standard deviation in titre of 4.1 x (6 curves and 161 points). The value found for the estimated standard devi- ation of titre in calculation of the protonation constants was 3.8 x lop3. Qualitatively similar conclusions were reached for all ligands listed in Table 2: only mononuclear zinc(I1) complexes appeared to be formed.

Only mononuclear complexes appeared to form between nickel(I1) and the ligands. Titra- tion curves obtained with a-mercapto-P-thienyl acrylic acid 5 and several bivalent metal ions at a ligand-metal ratio of 2 : 1 are illustrated in Fig. 2. In titrations with iron(I1) and manganese(II), hydrolysis occurred at high p H values, especially at lower ligand-metal ratios. Attempts to titrate ligand-copper(I1) mixtures failed; dark-colored solutions occurred upon adding copper(I1) salt to carefully degassed ligand solutions, indicating, presumably, oxidation-reduction involving the thiol group (29). Cadmium(I1) and lead(I1) precipitated with ligands 1 to 8 in the mixed solvent when the p H was raised to 3-5. No for- mation constants were obtained. The values ob- tained for formation of calcium(I1)-ligand 5 complexes indicated, as expected, that affinity of these ligands for alkaline earth cations was relatively low.

Using data in Tables 1 and 2, species distribu- tions in metal-ligand systems were calculated as a function of pH. In Fig. 3 are illustrated con- centrations across the p H range of zinc ion, ligand 2 species, and zinc-ligand complexes in 50% aqueous dioxane at two ligand-metal ratios. In Fig. 4 are illustrated species distribu- tions of the zinc ion - ligand 4 system in two media, an aqueous and a mixed solvent. Under any of these conditions essentially all of the metal is complexed by these ligands at neutral pH.

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4032 CAN. J. CHEM. VOL. 5 5 , 1977

TABLE 2. Formation constants (P,,,) of metal(I1) cation-ligand complexes at 2S°C and I = 0.1 M (NaCI04) in 50% (v/v) aqueous dioxane

Ligand Metal - P I I O P210 nb me

ap,,, refers to the general complex L,M,H,, where L = ligand, M = metal, and H = proton; a is the standard deviation.

bNumber of experimental observations. <Number of titiation curves analyzed. Values determined in aqueous medium. .Log Dl = 9.95; log a 2 = 18.52 in 757' (v/v) dioxane-water at 30DC, I = 0.02 M (KCI) (28). ,Log KI = 8.1; log K2 = 5.25 in 50% :v/v) dioxane-water at 2S0C, I = 0.1 M (NaCIO,) (27).

Partition Studies of Zinc (11) -Ligand Complexes Data on extraction of zinc(I1) from an aqueous

phase into immiscible alcohols are presented in Table 3. In the absence of complexant, little zinc (added as zinc sulfate) partitioned into the organic phase. Some water-soluble complexants such as EDTA did not induce transfer of zinc into either alcohol. Other complexants did solubilize zinc(I1) in the alcohols. Alcohol- soluble species were presumably those in which complexation was involved, although com- plexant concentration was not determined in any phase. Ligands 1, 2 and 5 appeared to extract zinc ion well, relative to most of the complexants tested.

Inhibition of Metalloenzymes Also presented in Table 3 are data on inhi-

bition by complexants of enzymatic activity of the zinc-dependent enzymes, alkaline phosphatase and carboxypeptidase A, and the copper-depen- dent enzyme, coeruloplasmin. The tabled values were obtained from plots of inhibition vs. con- centration curves, but the precision with which such concentrations were determined was not evaluated by statistical techniques, since in sev-

eral cases the curves appeared to indicate com- plex inhibition kinetics. However, the mercapto acrylic acids were judged to be potent inhibitors. Results of the study of inhibition of thermolysin, a zinc-dependent enzyme, are plotted in Fig. 5, in which the concentration of complexant neces- sary to inhibit half of the total enzymatic activity appears related to the value of the formation constant of the 1: 1 complex of zinc ion and inhibitor.

Discussion Compared to many a- and P-mercapto acids

and amino acids described in the literature (3), a-mercapto-P-aryl acrylic acids complex bivalent metal cations with relatively high affinity. Re- sults obtained for P2 ,, complexes of a-mercapto- P-thienyl acrylic acid 5 with several metals showed that the affinity of complexation fol- lowed the Irving-Williams order, Mn2+ < Fez+ < Co2+ < Ni2+ > Zn2+. With a given metal ion, but different a-mercapto-P-aryl acrylic acids, values of the complex formation constants varied with values of the protonation constants, as expected. Protonation constants of ligand 4 in aqueous solvent and in 50% (v/v) aqueous

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b a s e O M )

FIG. 1. Titration curves for a-mercapto-0-fury1 acrylic acid - Zn(N03), in 0.1 M NaC104 in 50% (v/v) dioxane water. Curve I, CL = 2.0 mM, CM = 0.0 M ; 2, CL = 2.0 mM, CM = 0.25 mM; 3, CL = 2.0 mM, CM = 0.5 mM; 4, CL = 2.0 mM, CM = 1.0 mM; 5, CL = 4.0 mM, Chl = 0.0 M ; 6, CL = 4.0 mM, CM = 0.5 mM; 7, CL = 4.0 mM, CM = 1.0 mM; 8, CL = 4.0 mM, CM = 2.0 mM.

1 1 1 I I 1 I 1 1 I

0 20 40 60 80 100 120 140 160 180 200

b a s e (4M)

FIG. 2. Titration curves for a-mercapto-0-thienyl acrylic acid alone (L) and in the presence of cations in 50% (v/v) dioxane-water. CL = 4.0 mM; Ch, = 2.0 mM.

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4034 CAN. J. CHEM. VOL. 5 5 , 1977

FIG. 3. Computed species distribution as a function of pH for solutions of a-mercapto-P-fury1 acrylic acid (4.0 mM) with (a) Zn2+ (2.0 mM) and (b) Zn2+ (4.0 mM) in 50% (v/v) dioxane-water.

dioxane indicated increases in pK, of 1.4 and 1.75 units in passing to the mixed solvent. One may compare pK, values of ligand 4 in aqueous solution for dissociation of the carboxyl and thiol groups, 3.2 and 7.35, respectively, with those for a molecule such as a-mercapto acetic acid, 3.5 and 10.2, respectively (29). The more facile ionization is due in part to the double bond in the a position and the inductive effect of the arvl substituent. This characteristic underlies the potent complexation of metal ions by these compounds at physiological pH. Lipid solubility of zinc - mercapto acrylic acid complexes may be another important feature underlying their physiological effects. 1 and 2 were among the most effective ligands in enhancing the alcohol solubility of zinc, compared to many ligands previously studied in the aqueous buffer - bu- tan01 system (30).

Consistent with the generalizations drawn from the potentiometric study of complex forma-

tion, results of the study of inhibition of metal- loenzymes were compatible with avid complexa- tion of metals by mercapto acrylic acids. Alkaline phosphatase contains two zinc atoms per enzyme molecule (31) and coeruloplasmin contains at least six copper atoms (32). Carboxypeptidase contains a single zinc atom per molecule, but hydrolysis by the enzyme of the ester substrate does not follow simple Michaelis-Menten kin- etics (33). Thermolysin contains a single zinc atom (34).

Relative differences in inhibitory potency may reflect characteristics of the interaction of en- zyme with inhibitor, or they may reflect less pro- found properties such as the stability of the con~pound in the assay milieu. The four enzymes were assayed at acid p H (coeruloplasmin), at neutral p H (carboxypeptidase and thermolysin), and at alkaline p H (alkaline phosphatase). At micromolar concentrations of total zinc, ranking of the degree to which zinc would be complexed

" 2 3 4 5 6 7 8 P H

FIG. 4. Computed species distribution as a funcrion of pH for solutions of a-mercapto-P-(5-sodiumsulfonate-2- furyl) acrylic acid (4.0 mM) and Zn2+ (2.0 mM) in (a) 50% (v/v) dioxane-water and (b) aqueous solvent.

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WAGNER ET AL. 403 5

TABLE 3. Effect of complexants on partition of zinc(I1) between aqueous and organic phases and as inhib- itors of metalloenzymes

PC" Ligand concentration (mM) for 50% inhibition

Alkaline Carboxy Ligand Butanol Octanol phosphatase peptidase A Coerulo-plasmin

1 2 5 6 7 9

NazEDTA 8-Thioquinoline 8-Hydroxyquinoline 1 ,lo-Phenanthroline Mercapto acetic acid

'PC = concentration of zinc in organic phase/concentration of zinc in aqueous phase. Wore than 90% of total zinc in aqueous phase. CLess than 10Z of total zinc in aqueous phase. dDecolorisation (22) of reaction mixture.

concentration in serum 10- to 15-fold, it had little or no effect on serum copper and iron con- centration (1). Presumably the avidity of metal complex formation, the lipid solubility of the complexes, the stability of the complexant, and other features as well are involved in the effects of these compounds on trace metal metabolism.

.-. r - I , , ,

Acknowledgements - n We wish to express appreciation to Miss E. n z Friderich for her valuable assistance with the

'3 syntheses and to Dr. G'. Letertre for the prepara- J L, 4 @ tion of ligands 1 and 2. We acknowledge helpful

'-1 discussions with Prof. J. M. Lehn and Dr. N. J. 4 6 8 Prakash and we thank Prof. D. R. Williams for a

log K , , PH 15 copy of the program SCOGS.

FIG. 5. Ligand concentration calculated for 50% inhibition of activity of thermolysin versus apparent formation constant at p H 7.5 for the ML complex. For- mation constants of reference inhibitors were taken from the literature: a = 8-thioquinoline (35); b = 1,lO- phenanthroline (36); c = 8-hydroxyquinoline (37); d = mercapto acetic acid (29).

at neutral p H by a considerable excess of ligand was calculated to be 5 > 4 > 2 > 3 > 6 > 8 > 1 > 7 > 9. This order bears some relationship to that observed in the inhibition studies.

We have demonstrated that mercapto acrylic acids form complexes with a variety of metal cations. Yet in rats, these compounds affect zinc metabolism most markedly. While a-mercapto- P-fury1 acrylic acid 2, for example, raised zinc

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