Lead chelates of meso- and racemic-dimercaptosuccinic acid

Mario Rivera, H. Vasken Aposhiaa, and Quintus Fernando Demrtment MR, QF. Demrtment of C?zemistry.- WA. Biology, Wniversity of Arizona, Fi.ucon, Arizona

of Molecular

and cehdar

Lead chelates of racer&- and meso-dimercaptosuccinic acid (DMSA) were synthesized and isolated from aqueous solutions and characterized by potcntiometric measurements and infrared spectroscopy Two types of lead chelates of sacernic DMSA were isolated: one in which racemic-DMSA is coordinated to Pb+2 via one oxygen and one sulfur atom ‘and the oEher in which the Pb2+ is coordinated via two sulfur atoms. The latter form of the chelate is converted into the former upon dissolution in dimethylsulfoxide. Only one type of Pb2+ &elate of the meso form of the ligand was form&. In this case, meso-DMSA is coordinated to Pbf2via one oxygen and one sulfur atom. Meso- and racemic-DMSA have very different solubilities in aqueous solutions. Meso-DMSA is slightly soluble in water, whereas racer&-DMSA is very soluble in water even in the presence of strong acids. The solubilities of the chelates were found to be pX dependent. V&en the unc~ordiuz&d sulfbydryl and carboxylic acid groups dissociate, the cheiates dissolve Ad remain in aqueous solution. The infiared spectra of meso- and racemic-DMSA show distinct features that can be used to detect the presence of either diasteroisomer.

INTRODUC’HON Meso-dimercaptosuccinic

acid (meso-DMSA)

has been officially

classified

as an or-

phan drug by the U.S. Food and Drug Administration [I] and is expected to become the drug of choice for the treatment of lead intoxication. It has been extensively studied in the People’s Republic of China, the Soviet Union, and more recently in the United States [2]. The racemic form of DMSA, however, has been studied to a lesser extent [3-81. The differences in the physical properties ol’ meso- and racemic-DMSA have been well documented. The meso form melts at 210-211°C, whereas the racemate melts at 124-125°C and rearranges to the meso form when heated above its melting point [8]. Although meso-DlVISA is only slightly soluble in water, its disodium salt is

Address reprint requests to: Quintus Fernando, Departmentof Chemistry, University of Arizona, ‘Ikcson, XZ 85721.

284

M. Rivera et al. very soluble. On the other hand, racemic DMSA is very soluble in water and remains in sulution even in the presence of strong acids. The formation of metal complexes by racemic- and meso-DMSA has been studied by a conventional potentiometric method [S]. The most stable complexes were formed with Znf2, Cd+2, and Hg +2; less stable ‘compiexes were formed with CO+~ and Ni+2, and the least stable with Mn $-2. No important differences between the metal complexes of racemic- and meso-DMSA were found, ‘and complexes of either diasteroisomer were reported to have the same stability [S]. It is surprising that lead detoxification by racemic-DlvlSA and the properties of the Pb2+ racer&-DMSA chelates have not been reported to date. We .will present evidence that shows that meso-DMSA interacts with Pb+2 to form one type of chelate, while racemic-DMSA forms two types of lead chelates. Meso-DMSA folrms a lead chelate in which the ligand is coordinated to the metal via one oxygen and one sulfur atom. Chelates with two different structures are formed when racemic DlVlSA and lead interact. Gne is similar to the meso-DMSA-Pb2+ complex in which the ligand is coordinated to the metal via one oxygen and one sulfur atom. The other has the ligand coordinated to the metal via two sulfur atoms. The latter chelate of Pb2+ with racer&-DMSA is converted to the former upon dissolution in dimethyl sulfoxide (DMSO) and precipitation with acetcne or chloroform. In all three lead chelates, the uncoordinated sulfhydryl and carboxyl groups play an important role in maintaining the lead chelates in solution at physiological pII.

l!MATEalLL%ILS AND

METHODS

Materials Acetylenedicarboxylic acid, thiolacetic acid and dimethylsulfoxide were obtained from Aldrich Chemical Co. Inc., Milwaukee, WI; lead nitrate was purchased from Mallinckrodt, Inc., Paris, KY. Meso-dimercaptosuccinic acid was a gift from Johnson & Johnson Baby Products Co., Skills, NJ. Synthesis of Racemk-DMSA Racemic-DMSA was synthesized from acetylenedicarboxylic acid and thiolacetic acid, according to the procedure described by Gerecke et. al. [8]. The purity of the product was confirmed by its melting point, 123.5-124.5°C (literature, 124-125OC), potentiometric titration, infrared, and NMR spectroscopy. ’

Synthesis

ol Lead Complexes

of Meso-

and Racemic-DMSA

To prevent oxidation of the mercapto groups to disulfides, the syntheses were carried out in a Schlenk line under nitrogen. All solvents were freed from oxygen by freezethaw cycles. All solvents were transferred from one flask to another with the aid of a caxmulum to minimize contact with oxygen in the air. MESO-DMSA-Pb

COMPLEXES

a suspension of 10 mm01 meso-DMSA in 25 mL of water, 2(4 mm01 of Na0l-I (0.25 M) was added. To the resultant solution of the disodium salt of meso-DMSA, 10 mm01 lead nitrate in 20 rnL of water was added. A yellow precipitate formed almost immediately. The compound was filtered under xdtiogan, w&i& -with S.5

To

Pb CHELATES

OF DMSA DIASTEREOISOMERS

285

M percbloric, acid, then with methanol and dried in vacua. The dried compound is stable in air. It was suspended in dimethylsulfoxide (DMSO) in order to dissolve any unreacted meso-DMSA, ffitered, washed with 0.5 M perchloric acid, then with methanol, and the last traces of solvent were removed in vacua. The lead content was determined as PbO after calcination of +&echelate, Pb(C&+0&).2H20. It was found to be 49.06%; the calculated value is 48.95%. Racemic

DMSA

Lead Complexes

(A) The synthesis was carried out in a manner similar to that described for the mesoDMSA-Pb complex, except that ‘the product was not suspended in DMSO since unreacted racemic DMSA is soluble in water. The product was dried in vacua. The lead content in the chelate, Pb(C&IIO&), was determined to be 53.8%; the calculated value is 53.5%. (B) The yellow compound obtained from (A) was stirred in DMSO for 2 hr under nitrogen. The yellow compound partially dissolved to give a yellow solution. Addition of acetone or chloroform to the yellow filtrate resulted in the formation of a yellow precipitate. The precipitate was washed With methanol and dried in vacua. The lead content in the chelate Pb(C4&0&) was determined to be 53.7%; the calculated value is 53.5%. (C) Raccmic DMSA (5 mmol) was dissolved in 20 mL 0.5 M perchloric acid. Lead nitrate (5 mmol) dissolved in 5 mL 0.5 M perchloric acid was added rapidly. A yellow precipitate was formed imme$ately, filtered, washed with methanol, and dried in vacua. The dry compound is stable in air. The lead content was found to be 53.0%; the calculated value is 53.5%.

Potentiometry Poteutiometric measurements of hydrogen ion concentration scribed previously [9]. Infrared

were performed as de-

Spectra All compounds were suspended were obtained

a Perkin

KBr and PIE 983

to f&m

pellet. Infkared

NMR Spectra

The proton NMR spectrum of racer& spectrometer

DMSA was obtained with a &ulcer WM-250

operating in the l?T mode.

REZWLTS

Infrared Spectroscopy:

Meso- versus Racetic-DIMSA

The infrared spectra of meso- and racemic-DMSA (‘Fig. la, b) are clearly different. The O-H stretching frequency in meso-DMSA is centered at 2900 cm-l (Fig. la) and is much broader than the corresponding stretching frequency in the racemate, which is centered ‘at 3200 cm- 1 (Pig. lb). Another region in the two spectra that shows differences between the meso and racer& forms of DMSA is that corresponding to the S-H stretching frequency. In the meso isomer, two sharp peaks at 2561 and 2535 cm-r are present. The spectrum of the racemate shows a single less intense and broader p& at 2556 cm-l.

286 Ad. River-aet al.

3200

b 4000

F’IGUIUI I.

1129-

. 3000

I&ared

. 2000

I

1600

I

1200

800

cm”

spectrum of (a) neso-DMSA

and (b) racemic-BMSA.

The carbonyl-stretching fiecpuency in meso-DMSA occurs ,as a single band at 1693 cm-* (Fig. la), whereas the same type of vibrational mode occurs as a double peak at 1729 and 1682 cm-’ in. the spectrum of racemic DMSA (Fig. lb). This type of behavior of *he main carbonyl abso-rptisn band seems to be chqracteristic of the s&re&Oir;eg$c fQm* -* ~--~s a& ‘ r* G;L derived Fgorn the same dicarboxylic acid with different substituents [lo]. The double ‘peaks are characteristic of the racemic fo&rms of CX:, p‘UlsUu3uLULGu _.?_.....,*!r_.._.. siicciiiic. a&%, C.k iiXs0 fGrziX, CY however, show a sing+ band [lo].

Pb CHELATES

I

I

4000

3000

I

.

2000

1600 cm

FIGURE 2, !!kcxxe

OF D&ISA DIASTERE0PSOMERS

.

I

1200

800

287

.

400

-1

Infrared spectrum of the meso-DMSA-Pb chelate.

the infrzred

spectra

of &UIb

diasteroisomers

are

quitedi*YererLt,

tile presence

of either diasteroisomer can be verified by infrared spectroscopy. Meso-DMSA-Pb

Chelate

In the infrared spectrum of the lead chelate of meso-DMSA (Fig. 2), the band at 1695 cm-’ corresponds to the carbonyl-stretching frequency of a protonated carboxylic acid group. In addition to this band, the antisymmetric coordinated carboxylate stretch is located at 1540 cm-l and the symmetric coordinated carboxylate stretch at 1366 cm-*. It is also possible to observe the two sharp peaks at 2562 and 2537 cm-’ that correspond to the S-H frequencies in meso-DMS.4. The information obtained from the infrared spectrum leads to the conclusion that one of the two carboxylic groups is not coordinated to Pb*+ . The presence of the S-H frequencies in the spectrum indicates that one sulfhydryl group is free; hence, the second sulfur atom must be coordinated to lead. Therefore, meso-QMSA coordinates lead via one sulfur and one oxygen atom, Racemic DMSA-Pb

Chelates

When the racemic DMSA-Pb &elate was prepared using the same procedure that was, used for the preparation oft tie *meso-DMSA-Pb complex, Procedure A (see Methods), a yellow precipitate was obtained. The lead content of this precipitate corresponds to that expected for the molecular formula of racemic DMSA-Pb. The infrared spectrum of the compound (Fig. 3a) shows a band at 1695 cm-l that corresponds to the protonated carbonyl-stretching frequency. In addition to this band, the antisymmetric carboxylate stretch occurs at 1540 cm-l, and the symmetric carboxylate stretch is observed at 1372 cm- I. In contrast to the infrared spectrum of the meso-DMSA

288

M. Riveia ‘et al.

0

4000

.

3000

.

I

2000

1000

.

1280

I

000

em-’

FIGURE 3. Infrared spectrum of the Pb+2 chelate of racemic-DMSA Procedure A and (b) synthesized by Procedure B.

(a) synthesized

by

complex (Fig. 2), the protonated carbonyl-stretching frequency at 1695 cm-’ is substantially more intense than the antisymmetric, carboxylate stretching at 1549 cm-*. This indicates that a mixture of two types of metal chelates is obtained if Procedure A is employed in the synthesis. To corroborate this hypothesis, Procedure B was devised (see Methods). The infrared spectrum of thz product obtained with Procedure B
Pb CHELATES

QF DMSA DIASTEBEOISOMERS

259

t 1606 -UUIu..a....T”

, 4000

3000

.

II

1600

2000

I

1200

800

cm-

FIGURE 4.

Infrared spectrum of the Pb +2 chelate of racemic-DMSA

synthesized by Proce-

dure C.

the antisymmetric carboxylate-stretching frequency at 1540 cm-!. The intensity ratio of these two bands is very similar to that obtained for the meso-DMSA chelate. In addition, a low-intensity broad S-II stretch is observed. at 2555 cm-‘. This supports the hypothesis that the product obtained by Procedure A is a mixture consisting of two types of chelates. Furthermore, Procedure B gives only one type of chelate in which one sulfur and one oxygen act as donor atoms. The evidence presented above suggested that under the appropriate conditions, the isomer in which two sulfur atoms of racemic DMSA are coordinated to lead could be obtained in a pure form, if the dissociation of the carboxylic acid groups was prevented. Procedure C (see Methods) was employed to synthesize this isomer. The infrared spectrum of the product (Fig. 4) shows a band at 1696 cm-’ that corresponds to the presence of a protonated carboxylic acid group. Bands corresponding to the antisymmetric and symmetric carboxylate-stretching frequencies are absent. This indicates that under the .conditions described in Procedure C, the chelate In which racemic DMSA uses the two sulfur atoms as donors can be obtained free of the isomer in which’racemic DkISA uses one oxygen and one sulfur as the donorsPotentiometry:

Meso-DMSA-Pb

When equimolar amounts of Pb+2 and the disodium salt of meso-DMSA (Compound 1, Scheme A) are mixed in an aqueous solution, the pH decreased from 7.2 to 2.4 with the formation of a precipitate of Compound 2 in Scheme A (Fig. 5, Curve I). When 1 mol NaOH is added in small increments to a suspension of 1 mol of the mesoD&ISA-Pb complex (Compound 2), the pII of ‘ithesolution, which is in equilibrium with the solid, increases gradually from pH 2.4 to 6.2 (Fig. 5, Curve 1). In this

290

M. Ri’vem et al.

_w-

+

Pb’+

Ifi

_)

2

1

1

OH’

H

S’ 0+ OH-

0

*‘Pb’ 4

SCHEME

A

region, one carboxylic acid group is being neutralized to form Compound 3. Upon addition of a second mole of NaOH, the pH of the solution increases from 6.2 to 10.9; in this pH range, a sulfbydryl group is being neutralized, with the formation of Compound 4. This is in good agreement with the evidence obtained from infrared spectroscopy, that meso-DMSA is coordinated to Pb+2 via one sulfur and one oxygen. lacemic-DMSA-Pb The behavior of the racemic-DMSA-Pb che!zre, in which the ligand is coordinated to Pb+Z via two sulfur atoms, upon titration with base is shown in Figure 5, Curve 2, and Scheme B. When 1 mol of NaOH is added in small increments to a suspension of 1 mol of the racemic-DMSA-Pb chelate 6, the pH of the solution, which is in equilibrium with the solid, increases gradually from 2.3 to 4.3 (Fig. 5, Curve 2).

Pb”

OH

HO

__) HCIO, 0.5 WI

5

1

OH'

-+om

Howe_

*OH-

‘Pb’

‘Pb’ 7

8

SCHEME

B

Pb CHELATES

OF DMSA D;1#STEREQHSOMi§

291

&jIB=* 8

8.0

6.0

5.0 4.0 3.0

9

W

2.8

1.Q mmol

id+/

mmol

D#LSA

FIGURE 5. Potentiometrictitration of: Curve 1: 1 mm01 of meso-DMSA+mmol Ph(NO&. Curve 2: 1 mm01 of the racer&-DMSA-Pb chdate synthesized by Procedure C. Curve 3: 1. mm01 racemic-DMSA disodium salt + mm01 Pb(N03)2.

One carboxylic acid group is being neutralized in this region, with the formation of Compound 7. Addition of a second mole of NaOH increases the pH fram 4.3 to 10.8. A second carboxylic acid group is being neutralized in this region; Compound 8 is formed in this region. This is strong evidence that racemic DMSA is coordinated to Pb+2 via two sulfur atoms. ‘When an equimolar amount of PbWk2and. the disodium salt of racemic-DMSA are added to water, the pH decreases from 6.7 to 2.2 (Fig. 5, Curw: 3), with the formation of a mixture of the two lead chelates of racemic-DMSA. The titration of this mixture (Fig. 5, Curve 3) shows an identical behavior to Ckves 1 and 2 durin$gthe addition of the first equivalent of base. The addition of a second equivalent of base produces a curve whose pH is in-between that of Curve 1 and Curve 2. This indicates that the precipitate that is in equilibrium with the solution is a mixture of a chelate in which racemic-DMSA is coordinated to lead via two sulfur atoms (Compound 6, Scheme B) and another chelate in which racemic-DMSA is coordinated to lead via one oxygen and one sulfur atom (Compound 2, Scheme A).

292

M. Rivera et d.

DISCUSSION The Three Pb Chelates

of DMSA

When meso-DMSA forms a chelate with Pb+2 , it is coordinated to lead via one oxygen and one sulfur atom. It appears that only this Pb2+ chelate of meso-DMSA is formed. Racemic-DMSA, however, appears to form two different chelates with Pb+2. In one of them, racer&-DMSA, is coordinated to lead via one oxygen and one sulfur atom. In the other chelate, racemic-DMSA is coordinated to lead via two sulfur atoms. A mixture of the last two chelates is obtained when Pb+* interacts witt the disodium salt of racer&-DMSA. When the mixture is treated with dimethylsulfoxide, the chelate, in which the two sulfur atoms are coordinated, dissolves. The addition of acetone causes the precipitation of the chelate in which one oxygen atom and one sulfur atom are coordinated. The other chelate, in which two sulfur atoms are co&dinzted (Compound 6)) is obtained in plure fo_rm as a precipitate after the addition of a solution of lead nitrate in 0.5 M perchloric acid to a stoichiometric amount of racemic-DMSA, dissolved in 0.5 M perchloric acid. The Pb Chelates

of Meso-

and. Racemic-DMSA

Have Different

Solnbilities

Important qualitative information can be obtained from potentiometric titrations of insoluble chelates that are in equilibrium with aqueous solutions [9]. The potentiometric titrations of the chelates of meso- or racemic-DMSA indicate that the lead chelates of both ligands have different solubiiities that depend on the pH of the solution. In the case of meso-DMSA, it is necessary to neutralize one carboxylic acid group and one sulfhydryl group in crder to dissolve the Pb2.+ chelate (Fig. 5, Curve I), On the other hand, it is necessary to neutralize two carboxylic acid groups to dissolve the lead chelate of racemic-DMSA (Fig. 5, Curve 2). This deprotonation of two functional groups b&low pH 7.0 imparts to the lead chelates of DMSA their solubility at physiological pH and, hence, their usefulness in solubilizing and excreting lead in the urine. This property appears to be of importance in the case of meso-DMSA-Pb chelate. Upon chelation, the uncoordinated sulfhydryl group becomes more acidic and dissociates in the neighborhood of pH 7.0 (Fig. 5, Curve 1). The reported p& values for the f&c ligand, meso-DMSA, are 2.71, 3.48, 8.89 and 10.79 [ll]. In the case of the racemic-DMSA-Pb chelate, the dissociation of the second carboxylic acid group occurs in the neighborhood of pH 5.5. This solubilizes its Pb2+ chelate at a pH significantly lower than the physiological pH and closer to the pH that is found in the kidney. The Potency

of Meso- and Raaremic- DMSA

as Metal Antidotes

Differ

Both forms of DMSA, when given to rats, have been reported to increase the urinary excretion of ‘%d, 65Zn, and %o 133. When compared to the results with mesoDMSA, racemic-DMSA increased the 1*%d excretion by a factor of three and 65Zn excretion by a factor of two. It has been claimed that racemic-DMSA is twice as effective as meso-DMSA in increasing the urinary excretion of 203Hg in rats [4]. The latter report, however, has been questioned [7]. Neither the racetiate nor the meso-DMSA affected the elimination of 5gFk ‘%u, or “Mn [3] . There is, howevkr, some disagreement as to whether meso-DMSA affects Cu2+ excretion. It will increase the excretion of copper in the normal rat that is not given

an additional burden of copper [12]. Graziano et al. [13, 143, in their n0w classic’ articles dealing with meso-DMSA therapy of men a.n.d children with lead burdens, were unable to detect any significant change in copper excretion. Meso-DMSA has also been found to be a favorable cupruretic agent to increase copper excretion in patients with Wilson’s Disease. The EDso values of meso- and racemic-DMSA for rats receiving an LD99 dose of sodium arsenite were not significantly different [7]. Either form of DMSA was found’ to mobilize 74As in rabbits. Although the mobilization was consistently greater when the racemic form was administered, the difference between the activity of meso- and racemic-DMSA was not statistically significant except in the case of muscle and lung. The differences in the urinary excretion of heavy metals observed after treatment with racemic DMSA as compared to meso-DMSA may be related in some degree to the different solubilities of their chelates. Whether these are the chelate structures found in vivo after the use of DMSA as a metal antidote is unknown at the present time. It is surprising that we were unable to find any report in the literature comparing dl-DMSA and msso-DMSA as antidotes or mobilizers of tissue lead. The results of our experiments suggest that such an evaluation would be of interest. Of even’greater interest, the present experiments clearly demonstrate that the lead chelate of dl-DMSA is more soluble than the lead &elate of meso-DMSA at the pH normally found in the kidney. This is an important and desirable property that a chelating agent should have when used for therapeutic purposes.

This work was supported in PMrpby NIEHS Grant ES 03356. REFERENCES 1. Federal Register 54, 7103 (1989). 2. H. V. hposhian, Annu. Rev. Pharmacoi. Toxiwi. 23, 193415 (1983). 3. I. E. Qkonishnikova, Gig. Tk. Prof. Zabol. 15, 50-52 (1971). 4. I. E. Okonishnikova, Gig. 7k. Prof. Zabof. 14, 18-22. (1971). 5. L. G. Egorova, I. E. Okonishnikova, V. L. Nirenburg, and I. Y. Postovskiy, Khim. Fakz. Zh. 5, 26-30 (1971). 6. I. E. Okonishnikova, L. G. Egorova, V. L. Nirenburg, and I. Y. Postovskiy, Khim. Farm. Zh. 4, 21-24 (1970). 7. H. V. Aposhian, C. Hsu, and T. D. Hoover, Toxicol. Appl. Pharmacol. 69, 206-213 (1983). 8. M. Gerecke, E. A. H. Fricdheim, and A. Brossi, Nelv. Chim. Acta 44, 4, 955-960

(1961). 9. M. Rivera, W. Zheng, H. V. Aposhian, and Q. Fernando, Tox. Appl. PharmacolB00, 96-106 (1989). 10. A. Rosenberg and L. Schotte, Acta Chem. &and. 8, 5, 867-869 (1954). 11. G. R. Lcnz, A. E. Martell, Inorg. Chem. 4, 378-384 (1965). 12. H. V. Aposbian, R. M. Maiorino, R. C. Dart, and D. F. Perry, CYin. Pharmawi. Ther. 45, 520-526 (1989). 13. J. H. Graziano, E. S. Siris, N. Lolalcono, S. J. Silverberg, and L. ‘Itrrgeon, Clin. Pharmawf. Ther. 37, 431-438 (1985). i4. J. H. Graziano, N. J. Lolalcono, and P. A. Meyer, J. Pediatr. 113, 751-757 (1988). Received June 8, 1989; accept& June 24, 1989