Chem. Res. Toxicol. 1990, 3, 248-253
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Optimization of Chelating Agent Structure for the Mobilization of Aged Renal and Hepatic Cadmium Deposits: Sodium N-Benzyl-4- 0 -(,t?-~-galactopyranosyl)-~-glucamineN-ca rbodit hioat e Mark M. Jones,* Pramod K. Singh, and Shirley G. Jones Department of Chemistry and Center in Molecular Toxicology, Vanderbilt Uniuersity, Nashuille, Tennessee 37235 Received December 7, 1989 An examination of the structure-activity relationships for the mobilization of intracellular cadmium by previously synthesized dithiocarbamates indicates that superior compounds can be prepared by increasing the molecular weight by using bulky polar but nonionic substituents. This suggested the synthesis and characterization of sodium N-benzyl-4-O-(@-~-galactopyranosy1)-D-glucamine-N-carbodithioate.T h e preparation of this compound and its characterization as an agent for the mobilization of aged renal and hepatic cadmium deposits in mice show it to be superior to the compounds reported earlier which have been used for this purpose. Five ip injections a t 0.40 mmol/kg reduced hepatic cadmium levels to 35% of their initial level and renal cadmium levels to 37%. The nature of the structural factors responsible for the efficacy of such agents is outlined as well as the relationship of these to the toxicity of the compounds and their cadmium complexes. These factors indicate clearly the differences in the molecular requirements for the mobilization of extracellular and intracellular toxic metal ions. T h e requirement that intracellular toxic metals be mobilized places several constraints on the structures of potentially efficacious compounds in addition to the thermodynamic and kinetic restrictions imposed by the requirements for extracellular efficacy.
Introduction Cadmium intoxication in humans may arise from environmental or industrial exposures and results in damaging effects on the kidneys, lungs, bone, and cardiovascular system, among others ( I ) . Chronic exposure frequently results in a nephropathy (2,3),and for this reason, the cadmium level of the kidney is considered to be a critical index of exposure. It is generally considered that there is no clinically acceptable method for the treatment of cadmium intoxication (3),though animal studies have shown that 2,3-dimercaptopropan-l-o1(4-6) and dithiocarbamates (7-10) can mobilize cadmium from intracellular deposits in mammals. A key problem in the formulation of a potentially useful clinical procedure for the treatment of chronic cadmium intoxication is the preparation of a compound that can effect a substantial reduction in renal cadmium levels without causing untoward damage as this reduction occurs (9, IO). The problem of the design of new molecular structures for toxic metal antagonists, which are more efficacious in vivo than those currently known, has been examined previously by several investigators. The m o r e purely chemical viewpoint has been presented by Catsch and Harmuth-Hoene ( I I ) , Crumbliss et al. ( I 2 ) ,Martell (131, and Raymond (14), who have emphasized the steps necessary to prepare chelating agents with larger effective stability constants for the toxic metal ion, i.e., an approach emphasizing the in vitro thermodynamic behavior of the chelating agent. Martell (13)has also discussed the importance of the kinetic aspects of the interaction of the toxic metal ion and the chelating agent in vitro. Such treatments ignore the problems that arise when a toxic metal is stored primarily in intracellular sites, as is the case with cadmium ( 1 5 ) . An alternative pharmacological viewpoint has been succinctly put by Danielli (16), who
emphasized the need for controlling the toxicity of the chelating agent and stressed the importance of designing an antagonist capable of functioning effectively in vivo in the mobilization of intracellularly deposited toxic metals. A major difference between the chemical and the pharmacological approaches is the emphasis which the latter places on structural features that affect the biological properties greatly but have little or no influence on the stability constants of any metal complexes that are formed. Since Danielli’s work, the description of these other factors has been put on a much more systematic basis, primarily as a result of the work of Hansch and his collaborators (17-19). More detailed considerations of the processes involved in the chelate decorporation of toxic metals (20) allow at least a partial joining of the two approaches. The purpose of the present study was to examine the use of an approach based primarily on the ideas of Danielli and Hansch to simplify the search for a more effective agent for the mobilization of aged renal and hepatic deposits of cadmium.
Experimental Section Melting points were determined by using a Thomas-Hoover stirred liquid apparatus and are uncorrected. Whatman silica gel 60A K6F plates with fluorescent indicator were used for thin-layer chromatography. ‘H NMR spectra were recorded on an IBM NR/300 FT NMR (300-MHz) spectrometer in D20 using sodium 3-(trimethylsilyl)-l-propanesulfonate(DSS) as an internal standard; the chemical shifts are reported in parts per million (6). IR spectra were recorded on a Perkin-Elmer Model 727 spectrometer; stronger bands are denoted as “s”; others were medium or weak. Elemental analyses were performed by Galbraith Laboratories, Knoxville, TN. a-Lactose monohydrate (1) was from Sigma Chemical Co., St. Louis, MO, and it contained 3% of the p-anomer. Benzylamine (2) was from Aldrich Chemical Co. Water and methanol were predegassed with NZ.
0893-228x/90/ 2703-0248~02.50/0 0 1990 American Chemical Society
Chelating Agent for Cadmium Deposits
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 249
3-day interval, 32 of the mice were divided into two groups of 16 N-Benzyl-4-O-(~-~-galactopyranosy1)-D-glucamine (4). The preparation was achieved by using the general procedures each, and each mouse was given a daily ip injection of 0.40 mmol/kg of either BLDTC or 4-MeOBGDTC in 0.5 mL of 0.9% described earlier (21, 24-26) with some specific variations as saline for five consecutive days. The 18 remaining mice were given follows (see Figure 1). ip injections of 0.5 mL of 0.9% saline each day. After a 2-day (a) Using PtO, Catalyst. a-Lactose monohydrate (1; 10.00 interval, the mice were sacrificed by cervical dislocation and g, 27.76 mmol) and benzylamine (2; 8.92 g, 83.28 mmol) were dissected. Weighed amounts of the liver, kidney, and brain stirred mechanically in the presence of water (5 mL) at 80-85 "C samples were digested in concentrated nitric acid (G. F. Smith under Nzover a period of 30-35 min until the formation of a Chemical Co., redistilled with low heavy metal content), taken transparent yellow viscous oil. The oil was hydrogenated in to dryness on a heating block, and redissolved in redistilled 1% methanol (200 mL) in the presence of PtOz catalyst (0.70 g) a t nitric acid prepared with pyrogen-free deionized water. The 45 "C and a t 55-60 psi for 72 h. After removal of the catalyst, samples were analyzed for cadmium by using a Perkin-Elmer 403 the filtrate was concentrated under reduced pressure and the atomic absorption spectrometer in the flame mode for samples resulting colorless oil was repeatedly stirred with ethyl ether (4 in the ppm range and in the flameless mode for samples in the X 150 mL) under Nz to wash off excess benzylamine, leaving the crude amine 4 as a white amorphous hygroscopic solid. The crude ppb range. Cadmium analyses on other experimental groups were also obtained in the same manner. product was dried in vacuo, redissolved in methanol, and precipitated by adding it to ethyl ether. One additional precipitation (b) Dose-Response Data and Cadmium Levels in Other Organs. Dose-response data were obtained on male ICR mice afforded a fairly nonhygroscopic material which was dissolved (Harlan Industries, Indianapolis, IN) weighing 31 f 2 g adminin water, filtered through a cellulose filter aid to remove any insoluble impurities, and then freeze-dried t o give 4.2H20 as a istered a total of 20 mg CdC1,.2.5H20/kg. The administration schedule involved ip administration of 1mg/kg on day 1,2 mg/kg white solid: yield 10.1 g (77.5%); mp 75-80 "C dec; 'H NMR on days 2 and 3, and 3 mg/kg on days 4 , 8 , 9 , 1 0 , and 11. Three (D,O/DSS) 6 7.44-7.32 (m, 5 H), 4.43 (d, J = 7.6 Hz, 1 H), 4.04-3.49 (m, 14 H), 2.83-2.80 (dd, J = 12.4 Hz, 3.0 Hz, 1 H), days after the last cadmium chloride injection, the chelating agents 2.64-2.60 (dd, J = 12.4 Hz, 9.00 Hz, 1 H); IR (Nujol) 3350 (br, were given daily, a t the indicated dosages, for a total of 5 days. All injections were given in 0.5 mL of 0.9% saline. Three days s), 1650,1510,1365,1345,1305,1220,1145,1120,1065-1040 (br, after the last injection of the chelating agent, the animals were s), 890,790,720,700,610cm-'. Anal. Calcd for C,J-131N0,0.2Hz0 sacrificed and dissected. The organs were analyzed as described C, 48.61; H, 7.52; N, 2.98. Found: C, 48.66, H, 8.13; N, 2.98. above. There were five animals in each of the ten experimental (b) Using Raney Nickel Catalyst. The oil obtained from groups. the Schiff base reaction starting from a-lactose (20.00 g, 55.51 (c) Whole-Body Cadmium Levels. The cadmium loading mmol) and benzylamine (17.85 g, 166.52 mmol) in the presence of these male ICR mice was at 10 mg of CdC1,.2.5H2O and was of water (10 mL) was dissolved in 600 mL of methanol and hycarried out as described for the first experimental group, as was drogenated a t 45 "C and a t 700 psi for 170 h in the presence of the sequence of chelate treatments. There were eight animals Raney nickel catalyst (ca. 6 g wet weight). Usual workup as in each of two groups, one of which received 0.400 mmol of described in (a) (above) afforded 19.55 g (75.2% yield) of 4.2H20 as a white nonhygroscopic amorphous solid. NMR and IR were BLDTC/kg on each of five successive days. Two days later the animals were sacrificed by cervical dislocation and analyzed for similar to those of 4 obtained in (a). Sodium N-Benzyl-4-O-(~-D-galactopyranosyl)-D-gluc-cadmium as described above. The same procedure was used to determine the whole body cadmium levels in animals treated with amine-N-carbodithioate (BLDTC) (5). The carbodithioate 4-MeOBGDTC. 5 was prepared from the corresponding amine 4 as reported earlier All animals were kept in an AAALAC-approved animal care (21, 24, 25). facility during the course of the experiments and were provided An aqueous solution (5 mL) of NaOH (0.43 g, 10.75 mmol) was with free access to food and water. added dropwise to the stirred solution of N-benzyl-4-O-(P-~The statistical analysis of the data was carried out by using galaCtOpyranOSyl)-D-glUCaminedihydrate (4; 5.00 g, 10.65 mmol) the paired, two-tailed Student's t test. in a 1:l methanol-water mixture (40 mL) at 0-5 "C under N,. After 30 min a mixture of carbon disulfide (5 mL) and dioxane (10 mL) was added to it slowly at 0 OC, followed by overnight Results stirring at room temperature. Solvents were then removed, leaving T h e reaction s c h e m e used is given in Figure 1. The some (4-5 mL). Acetone (60 mL) was added and stirred, efforts m a d e toward the synthesis of the amine 4 revealed whereupon the product separated out as a somewhat sticky that a change from a reducing monosaccharide, e.g., glucose yellowish white solid. The supernatant colored layer was dis(211,t o a reducing disaccharide (e.g., lactose in t h e present carded, and the solid was again stirred with acetone. Three or case) d i d n o t cause serious problems, although somewhat four treatments were usually necessary to discharge the yellow greater difficulties were encountered during the Schiff base color from the product. After drying, the product was redissolved formation a n d i t s s u b s e q u e n t hydrogenation. Of several in methanol (30 mL), treated with charcoal, passed through cellulose filter aid, and precipitated by adding it to the stirred variations a t t e m p t e d , the m o s t suitable was the use of a acetone. The solid thus obtained was finally dissolved in water 3-fold molar excess of benzylamine (2) i n t h e presence of and freeze-dried to give the carbodithioate 5-0.8H20as a non5 mL of w a t e r / l O g of lactose ( I ) a n d a relatively higher hygroscopic white amorphous solid: yield 4.60 g (79.0%); mp temperature (80-85 OC) for t h e reaction in which the Schiff 140-145 "C dec; 'H NMR (D,O/DSS) 6 7.45-7.27 (m, 5 H), 5.69 base is formed. The excess benzylamine is n o t removed (d, J = 15.8 Hz, 1 H), 5.31 (d, J = 15.8 Hz, 1 H), 4.55 (d, J = 7.8 until after t h e hydrogenation s t e p t o prevent the reversal Hz, 1 H), 4.45-4.35 (m, 2 H), 4.01-3.51 (m, 12 H); IR (Nujol) 3250 of t h e Schiff base formation reaction. Use of Raney nickel (br, s), 1640,1360, 1350,1305,1200,1160,1070-1035 (br, s), 965, catalyst, even t h o u g h i t required longer t i m e a n d a m u c h 885,715,600 cm-'. Anal. Calcd for CJ&,,NNaOloS2~0.8H20:C, higher pressure (700 psi), was preferred over the use of the 44.00; H, 5.83; N, 2.57; S, 11.74. Found C, 43.65; H, 6.47; N, 2.57; s, 12.12. PtO, because of the cleaner reaction product. The prepSodium N-(4-methoxybenzyl)-~-glucamine-N-carbodithioate aration of t h e d i t h i o c a r b a m a t e 5 f r o m the a m i n e 4 was (4-MeOBGDTC) was prepared as described previously (21). successfully achieved b y using the procedure reported Animal Studies. (a) Renal, Hepatic, and Brain Cadmium earlier (21124,251. T h e dithiocarbamate, BLDTC, is stable Levels. Of 55 male ICR mice (Harlan Industries, Indianapolis, at room t e m p e r a t u r e a n d q u i t e soluble in water. IN) weighing 30 f 2 g, five were given 6 mmol/kg of BLDTC, Preliminary a p p r o x i m a t e toxicity tests indicated that ip, in 0.5 mL of 0.9% saline. For the 10 days following injections the LD50 for this compound was in excess of 6.00 mmol/kg the mice showed no ill effects. On this basis, the LD50 can be when administered ip. estimated to be in excess of 6 mmol/kg for mice. All (50) of the The comparative ability of t h e BLDTC a n d s o d i u m remaining mice were given a series of four consecutive daily ip N - (Cmethoxybenzy1)-D-glucamine-N-carbodithioate moinjections of 1, 3, 3, and 3 mg/kg of CdC12-2.5H20in 0.5 mL of 0.9% saline for each injection for a total of 10 mg/kg. After a n o h y d r a t e (4-MeOBGDTC) (21) i n the mobilization of
250 Chem. Res. Toxicol., Vol. 3, No. 3, 1990
k
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2
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Jones et al. Table 11. Effect of Chelating Agent Dosage on Residual Hepatic Cadmium Levels for 4-MeOBGDTC a n d BLDTC" 4-MeOBGDTC, BLDTC, pg of pg of Cd/g of Cd/g of tissue dosage, mmol/kg tissue wet wt wet wt 0 90.8 f 8.8 92.4 f 10.5 0.100 87.8 f 13.4 80.7 f 6.3 0.200 83.0 f 10.3 77.8 f 5.gb 0.300 73.1 f 14.5b 68.6 f 12.5b 0.400 68.0 & 15.2b,d 39.0 f 13.9'~~ "Animals were loaded with 20 mg/kg CdC12-2.5H20.There were five animals in each group and cadmium loading to a total of 20 mg of CdC1,.2.5H20/kg was carried out as described under Experimental Section. Animals were subsequently given a daily injection of the indicated compound daily on each of five consecutive days. Values given are means f standard deviations. Significantly different from controls, p < 0.05. CSignificantly different from controls, p < 0.001. dSignificantly different, p 6 0.05. Table 111. Effect of Chelating Agent Dosage on Residual Renal Cadmium Levels for 4-MeOBGDTC a n d BLDTC" 4-MeOBGDTC, BLDTC, yg of hg of Cd/g of Cd/g of tissue dosage, mmol/kg tissue wet wt wet wt 41.7 f 8.1 0 42.4 f 3.3 0.100 42.6 f 3.8 38.9 f 5.7 27.8 f 5.2b 0.200 38.7 f 5.7 0.300 33.4 f 2.4b 28.4 f 7.0b 0.400 28.7 f 5.5b*C 18.9 f 6.7*tC "Animals were loaded with 20 mg/kg CdC1,.2.5Hz0. These data were obtained on the same animals used for Table 11; there were five animals in each group. Values given are means f standard deviations. Significantly different from controls p 5 0.05. Significantly different, p 5 0.05.
H H OHH
OH
OH
I
FH20H
k
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OH 5 (BIl)TO
Figure 1. Reaction scheme used for the preparation of BLDTC (5).
Table I. Comparative Mobilization of Intracellular Cadmium by 4-MeOBGDTC a n d BLDTC" liver, kidney, brain, group N PPm PPm PPb cadmium only 18 45.1 f 6.1 21.7 f 3.9 117 f 59 Cd + 4-MeOBGDTC 16 32.6 f 3.3b 10.9 f 2.4b 134 & 54 Cd + BLDTC 16 17.0 f 5.4b9c 8.4 f 1.6b,d 154 f 42 "CdC12-2.5H20was administered at I, 3, 3, and 3 mg/kg on Monday, Wednesday, Thursday, and Friday, to give a total of 10 mg/kg. After a 3-day interval, 0.40 mmol/kg chelating agent was given ip in 0.5 mL of saline on five consecutive days. After a 2-day interval the animals were sacrified and dissected for the organs described. bp
