Chem. Res. Toxicol. 1991,4,692-698
692
Structure-Activity Relationships for in Vivo Cadmium Mobilization by Dithiocarbamates Derived from Lactose and Maitotriose Mark M. Jones,* Pramod K. Singh, and Shirley G. Jones Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235
Glen R. Gale, Loretta M. Atkins, and Alayne B. Smith Veterans Affairs Medical Center and Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29403 Received July 17,1991 The relationships between chemical structure and the relative ability to mobilize cadmium in vivo from its aged renal and hepatic deposits have been examined in a series of newly synthesized dithiocarbamates derived from lactose and maltotriose. The results suggest that, in the selection of hydrophobic groups to counter the hydrophilicity contributed by the disaccharides, aromatic groups provide compounds which have a superior efficacy t o compounds containing aliphatic groups. The compounds derived from trisaccharides are much less effective than those derived from disaccharides, suggesting that there is a practical size limit to the hydrophilic groups which can be used in the structures of such compounds. With both di- and trisaccharides, aliphatic derivatives with straight chains containing more than eight carbon atoms tend to be less effective than the ones with seven or fewer carbon atoms in the alkyl chain. The three compounds prepared from lactose which contain a benzyl or a methyl-substituted benzyl group are the most effective compounds reported to date for the reduction of whole-body cadmium levels.
I ntroductlon After parenteral administration, cadmium which remains in the body becomes rapidly and progressively more difficult to remove with the passage of time (1-4).This has been shown to be due to the movement of cadmium from extracellular to intracellular sites (5).One result of this is that typical chelating agents such as EDTA and its derivatives are quite ineffective as antagonists if administration is delayed by more than an hour. In cases of human intoxication, there is presently no clinically available treatment which can be used to effectively reduce such deposits (6). The ability of dithiocarbamates to act as antagonists for acute cadmium intoxication as well as to mobilize cadmium from its aged intracellular deposits in the kidney and liver of animals has been reported in some detail (3,7-9). The use of structural modifications which introduce hydrophilic groups into the dithiocarbamate has previously been shown to prevent the concurrent redistribution of mobilized cadmium to the brain (10,ll).These and related studies have also allowed the development of a systematic approach to the synthesis of more effective agents for the removal of cadmium from its aged renal and hepatic deposits in rodent models (12, 13). During the last five years the development of compounds of successively greater effectiveness has been pursued quite successfully (11,13,14)and the major factors governing this development have been outlined. The action of various dithiocarbamates in removing such cadmium deposits in rodent models has been demonstrated clearly for a number of analogues which contain a group derived from *Address correspondence to this author at Box 1583, Station B, Vanderbilt University, Nashville, TN 37235. 0893-228~ f 91f 2704-0692$02.50f 0
a sugar such as glucose (9,10,12,14) or lactose (11,13). The present study was undertaken to examine a number of topics related to these earlier observations: (a) Can derivatives of the sugars of even higher molecular weight such as maltotriose be prepared which are effective for this purpose; (b) are the alkyl derivatives of dithiocarbamates derived from lactose as effective as the aralkyl derivatives previously prepared; and (c) what are the factors governing the overall efficacy of dithiocarbamates as agents for the mobilization of intracellular cadmium?
Experlmental Section Maltotriose (approximately 95% purity) and a-lactose were purchased from Sigma Chemical Co., St. Louis, MO. All the primary amines, 2a-m (Figure l),were obtained from Aldrich Chemical Co., Milwaukee, WI. The solvents were predegassed with N2or Ar. Melting points were determined on a ThomasHoover stirred-liquid apparatus. 'H NMR spectra were recorded on an IBM 30-MHz FT NMR spectrometer in D20 using 3(trimethylsily1)-1-propanesulfonicacid sodium salt (DSS) as an internal reference. The chemical shifta have been reported in ppm (a). Elemental analyses were performed on a Carlo Erba Strumentazione Model 1106 elemental analyzer. Sodium N-benzyl4-0-(~-~galactopyranosyl)-~glucamine-N-car~ithi~te (BLDTC) which served as a standard drug was prepared according to a published procedure (11). Preparation of N-Alkylmaltotriosamines (3a-m). The synthesis of the secondary amines was primarily based on the preparation of N-aralkyl- and alkyllactamines (II,13). In a typical reaction (Figure 1) 5.00 g of maltotriose (1) (9.91 mmol) was mechanically stirred with 1molar excess of alkylamine 2a-m (19.82 mmol) and 1 mL of H20 under N2on a steam bath for 20-25 min to give a transparent yellowish oil. The oil was then dissolved in methanol (250 mL) and hydrogenated using either Pt02(ca. 0.20 g) a t 60 psi and a t 22 "C for 3-4 days or Raney Ni (ca. 10.0 g wet weight) at 900 psi and at 40 "C for 7-10 days. The com0 1991 American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 693
Dithiocarbamates in Calcium Mobilization MdDW
+
HZN-K
Om!j-y-! a 2.-m
1
?-':A OH
HO H
OH
1
i. Steam brth N2 ii. Crulyrie hydrogmation
HOCH 2-
OH
0
H
-CH
OH I
2 -N H
-R
OH
H
OH
j&&csz
H W H 2 - i < - k - ~ - c H 2 -OH N-R H
OH
OH H
HO
OH
OH
! I
AH
L1 s @N#
0
H
d
H
OH 40-m
4j (HHxMDTC);j, R = (CH2)60H 4a (n-CsMDTC);a, R = (CH2)4CH3 4k (CMMDTC); k, R = cyclohexylmethyl 4 b ( n - C W T C ) ;b, R = (cH2)sCH3 4 1 (MEtMDTC); I, R = 2-morpholinoethyl 4C (~I-C~MDTC); C, R = (CH2)6CH3 4m (MRMDTC);m,R = 3-morpholinopropyl 4d (n-CgmTC);d, R = (CH2)7"j 4e (n-CgMDTC); e, R = (CH2)8CH3 4f (br-CsMDTC);f, R = CH(CHzCH3)z 4g (br-C6mTC);g, R = CH(CH~)CHZCH(CH~)~ 4 h (br-C7MDTC); h, R = CH(CH3)(CH2)4CH3 4i (br-CgMDTC); i, R = CH2CH(CH2CH3) (CH2)3CH3 Figure 1. Scheme used for the preparation of maltotriose-based dithiocarbamates 4a-m. (s, br, 10 H), 0.85 (t, J = 13.1 Hz,3 H). Anal. Calcd for pletion of reduction was verified by 'H N M R . After workup and C26H51N015~2H20 C, 47.77;H,8.48;N,2.14. Found: C, 47.51; purification, which involved a t least two charcoal treatments of H,8.18;N,1.88. the methanolic solution of the crude product followed by pre(E) N - ( n-Nonyl)-4-O-(a-D-glucopyranosyl)-4-O-(a-Dcipitation with Et,O and finally freeze-drying (II), the product glucopyranosy1)-D-glucamine (38): yield 77.1% (white (3a-m)was obtained as a fairly nonhygroscopic white solid in amorphous solid); 'H NMR (DzO/DSS)6 5.39 (d, J = 3.5 Hz,1 71-82% yield. None of the amines (3a-m)could be crystallized, and they all melted with foaming around 100-120 "C. H),5.10 (d, J = 3.5 Hz,1 H),4.02-3.60 (m, 17 H),3.41 (t, 1 H), 2.77 (d, J = 6.2 Hz,2 H),2.67-2.64 (m,2 H), 1.50(m, 2 H),1.28 (A) N-( II -Pentyl)-40-(a-~-glucopyranosyl)-40-(U-D(br, s, 12 H), 0.87 (t, J = 11.5 Hz,3 H). Anal. Calcd for glucopyranosy1)-D-glucamine (3a): yield 71.0% (white C2,H63N015~2Hz0 C, 48.57;H,8.60;N,2.10. Found: C, 48.38; amorphous solid); 'H NMR (D,O/DSS)6 5.39 (d, J = 3.9 Hz,1 H),5.09 (d, J = 3.8 Hz,1 H),4.02-3.52 (m, 17 H),3.40 (t, 1 H), H,8.21;N,1.90. (F)N-(l-Ethylpropyl)-40-(a-~glucopyranosyl)-40 a( 2.76 (d, J = 6.3 Hz,2 H),2.66-2.59 (m, 2 H),1.52-1.47 (m, 2 H), D-glucopyranosy1)-D-glucamine(3f): yield 71.0% (white 1.31 (br. m, 4 HI.0.87 (t, J = 13.5 Hz.3 H). Anal. Calcd for C23HuN01i.1H20:C, 46.54;H,7.63;N, 2.36. Found: C, 46.45; amorphous solid); 'H NMR (D,O/DSS)6 5.40(d, J = 3.9 Hz,1 H,7.88;N,1.93. H),5.10(d, J = 3.8 Hz,1 H),4.00-3.58 (m, 17 H),3.40 (t, 1 H), (B) N-(a-Hexyl)-4-O-(u-D-glucopyranosyl)-4-0-(a-D- 2.86-2.70 (m, 2 H),2.53 (t, 1 H), 1.54-1.42 (m, 2 H),0.87 (t,J = 14.8 Hz,6 H). Anal. Calcd for C23HuN015*2H20: C, 45.17;H, glucopyranosy1)-D-glucamine (3b): yield 72.3% (white amorphous solid); 'H NMR (D,O/DSS)6 5.39 (d, J = 3.8 Hz,1 8.05;N,2.29. Found: C, 45.57;H,7.77;N,1.80. H),5.09 (d, J = 3.8 Hz,1 H), 4.02-3.54 (m, 17 H),3.40 (t, 1H), (G)N-( 1,3-Dimethylbutyl)-40-(u-~-glucopyranosyl)-42.75 (d, J = 6.3 Hz,2 H),2.65-2.59(m, 2 H),1.55-1.46 (m, 2 H), O-(u-Dglucopyranoeyl)-Dglucamine (38): yield 72.0% (white 1.27 (s, br, 6 H), 0.85 (t, J = 13.2 Hz,3 H). Anal. Calcd for amorphous solid); 'H NMR (D,O/DSS)6 5.39 (d,J = 3.9 Hz,1 CUH4,N015a1.5H20: C, 46.74;H,8.17;N,2.27. Found C, 46.58; H),5.9 (d, J = 4.0 Hz,1 H),4.02-3.54 (m, 17 H),3.40 (t, 1 H), H,7.99;N,1.95. 2.88-2.80(m,2 H),2.74-2.67 (m, 1 H),1.65-1.59 (m,1 H),1.36-1.20 (C)N-(~-Heptyl)-4-O-(u-~-glucopyranosyl)-4-O-(u-D- (m, 2 H), 1.07-1.40 (t, J = 13.8 Hz,6 H). Anal. Calcd for glucopyranosy1)-D-glucamine (3c): yield 82.0% (white C24H47N01b-2Hz0: C, 45.81;H,8.23;N,2.23. Found: C, 45.70; amorphous solid); 'H NMR (D,O/DSS)6 5.40 (d, J = 3.5 Hz,1 H,7.77;N,1.80. H),5.10 (d, J = 3.5 Hz,1 H),4.02-3.55 (m, 17 H),3.40 (t, 1 H), (H)N -( 1-Methylhexy1)-4-0 (u-~-glucopyranosyl)-40 2.76 (d, J = 6.1 Hz,2 H),2.66-2.62 (m, 2 H),1.50 (m, 2 H), 1.28 (a-D-glucopyranosy1)-D-glucamine (3h): yield 76.0% (white (br, s, 8 H), 0.85 (t, J = 12.7 Hz,3 H). Anal. Calcd for amorphous solid); 'H NMR (D20/DSS)6 5.40 (d, J = 3.8 Hz, 1 Cz,H,9N0,5.2H,0: C, 46.94;H,8.35;N,2.19. Found: C, 46.49; H),5.10 (d, J = 3.8 Hz,1 H),4.03-3.53 (m, 17 H),3.41 (t, 1 H), H,7.99;N,1.92. 2.88-2.68(m, 3 H), 1.53 (m, 7 H), 1.07-1.04 (m, 2 H),0.84 (t, J = 13.5 Hz,3 H). Anal. Calcd for CzaH49N015.2Hz0C, 46.94; (D) N -(L) -0ctyl)-40-(a-~-glucopyranosyl)-40- (a-Dglucopyranosy1)-D-glucamine (3d): yield 74.2% (white H,8.35;N,2.19. Found: C, 46.80;H,8.00,N,1.71. amorphous solid); 'H NMR (D20/DSS)6 5.40(d, J = 3.8 Hz,1 (I) N -(2-Ethylhexy1)-4-0 (a-~-glucopyranosyl)-40 -(a+ H),5.10 (d, J = 3.7 Hz,1 H),4.03-3.55 (m, 17 H),3.41 (t, 1 H), glucopyranosy1)-D-glucamine (3i): yield 78.7% (white 2.77 (d, J = 6.4Hz,2 H),2.66-2.63 (m, 2 H),1.50 (m, 2 H), 1.29 amorphous solid); 'H NMR (D,O/DSS)6 5.40 (d, J = 3.8Hz,1
Jones et al.
694 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
R5 a-e a, R = cyclohexylmethyl; CMLDTC
b, R = 2-morpholinoethyl; MEtLDTC
c, R = 3-morpholinopropyl; MPrLDTC d,R=3,4-MezC+jH3CH;!; DMeBLDTC e, R = 4-MeCa4CHz; MeBLDTC (standard) Figure 2. Structures of lactose-based dithiocarbamates 5a-e. H), 5.10 (d, J = 3.8 Hz, 1 H), 4.03-3.55 (m, 17 H), 3.41 (t, 1 H), 2.77 (d, J = 6.9 Hz, 2 H), 2.60-2.53 (m, 2 H), 1.50 (br, m, 1 H), 1.37-1.27 (m, 8 H), 0.86-0.82 (m, 6 H). Anal. Calcd for C28HllN015-2H20:C, 47.77; H, 8.48; N, 2.14. Found: C, 46.55; H, 8.48; N, 1.74. (J)N-(6-Hydroxyhexyl)-4-O -(a-~-glucopyranosy1)-40(a-D-glucopyran0syl)-D-glucamine (3j): yield 74.4% (white solid); 'H NMR (D,O/DSS) 6 5.39 (d, J = 3.8 Hz, 1 H), 5.19 (d, J = 3.9 Hz, 1H), 4.45-4.20 (m, 2 H), 4.03-3.38 (m, 23 H), 1.76-1.54 (m, 4 H), 1.39-1.35 (br, m, 4 H). Anal. Calcd for C25H48N0160.5Hz0: C, 42.13; H, 6.65; N, 1.97. Found: C, 41.99; H, 6.94; N, 2.20.
(K)N-(Cyclohexylmethyl)-4-O-(a-~-glucopyranosyl)-40-(a-D-glucopyranosyl)-D-glucamine (3k): yield 81% (white solid); 'H NMR (D,O/DSS) 6 5.40 (d, J = 3.8 Hz, 1 H, 5.10 (d, J = 4.0 Hz, 1 H), 4.03-3.55 (m, 17 H), 3.41 (t, 1 H),2.73 (d, J = 6.7 Hz, 2 H), 2.53-2.40 (m, 2 H), 1.71-0.85 (m, 11H). Anal. Calcd for C2SH,7N015-2H20C, 47.09; H, 8.06;N, 2.20. Found C, 46.32; H, 7.59; N, 1.84.
(L)N-(2-Morpholinaethyl)-4-O-(a-~-glucopyranosyl)-4O-(a-D-glucopyranosyl)-D-glucamine (31): yield 67% (white solid); 'H NMR (D,O/DSS) 6 5.39 (d, J = 3.8 Hz, 1 H), 5.10 (d, J = 3.8 Hz, 1H), 4.02-3.37 (m, 22 H), 2.79-2.44 (m, 10 H). Anal. Calcd for CuHleNzOls.0.6H20: C, 45.80; H, 7.56; N, 4.45. Found: C, 45.82; H, 8.05; N, 5.40. (M) N-(2-Morpholinopropyl)-40 -(a-D-g~ucopyranosy~)4-O-(a-~glucopyrano~yl)-~glucamine (3m): yield 66% (white solid); 'H NMR (D,O/DSS) 6 5.39 (d, J = 3.3 Hz, 1 H), 5.09 (d, J = 3.2 Hz, 1 H), 4.02-3.54 (m, 21 H), 2.71-2.38 (m, 10 H), 1.71-1.61 (m, 2 H). Anal. Calcd for C25HaN2016-0.25H20:C, 47.13; H, 7.67; N, 4.40. Found C, 47.16; H, 8.05; N, 5.65. P r e p a r a t i o n of N - A l k y l m a l t o t r i o s a m i n e Dithiocarbamates (4a-m). The dithiocarbamates were synthesized by treating the stirred methanolic solution of the parent amine (3a-m) with an equimolar quantity of aqueous NaOH followed by addition of excess carbon disulfide at 0-5 OC (Figure 1). The reaction mixture was stirred overnight at 22 "C, the solvent was removed under reduced pressure leaving ca. 5 mL, and the dithiocarbamate was precipitated with acetone. The crude material was further purified as described earlier for substituted lactamine dithiocarbamates (11, f3). The dithiocarbamates (4a-i) were obtained in good yields (79-92%) as fluffy white solids and melted with foaming (at ca. 100-110 "C) followed by decomposition (at h a . 150 "C). (A) Sodium N-(n-pentyl)-4-O-(a-~-glucopyranosyl)-4O-(a-D-glucopyranosyl)-D-glucamine-N-carbithioate (4a, n-CSMDTC): yield 86.2% (fluffy white solid); 'H NMR (D,O/DSS) 6 5.40 (d, J = 3.8 Hz, 1H), 5.19 (d, J = 3.8 Hz, 1 H), 4.45-4.20 (m, 3 H), 4.03-3.55 (m, 18 H), 3.38 (t, 1H), 1.73 (quintet, 2 H),1.37-1.25 (m,4 H),0.88 (t, J = 13.8 Hz, 3 H). Anal. Calcd for CuH,,NNaO15Sz-1.5Hz0: C, 41.14; H, 6.76; N, 2.00. Found: C, 40.74; H, 6.44; N, 1.88. (€3) Sodium N-(n -hexyl)-4-O-(a-D-glucopyranosyl)-4(4b, O-(a-D-glucopyranosyl)-Dglucamine-N-carbithioate
n-C6MDTC): yield 79.5% (fluffy white solid); 'H NMR (D20/DSS) 6 5.40 (d, J = 3.8 Hz, 1H), 5.19 (d, J = 3.8 Hz,1 H), 4.42-4.21 (m, 3 H), 4.02-3.55 (m,18 H), 3.41 (t, 1 H), 1.80-1.66 (br, m, 2 H), 1.17 (br, s, 6 H), 0.86 (t, J = 13.2 Hz, 3 H). Anal. Calcd for CaHaNNaO15S2.3H,0: C, 40.47; H, 7.07; N, 1.89. Found: C, 40.04; H, 6.67; N, 1.42. (C) Sodium N - ( n-heptyl)-4-O-(a-~-glucopyranosyl)-4O-(a-D-glucopyranosyl)-D-glucamine-N-carbithioate (4c, n-C,MDTC): yield 86.9% (white fluffy solid); 'H NMR (DzO/DSS) 6 5.40 (d, J = 3.9 Hz, 1H), 5.19 (d, J = 3.8 Hz, 1 H), 4.41-4.20 (m, 3 H), 4.03-3.55 (m, 18 H), 3.41 (t, 1 H), 1.80-1.65 (quintet, 2 H), 1.30 (8, br, 8 H), 0.86 (t,J = 12.9 Hz, 3 H). Anal. Calcd for CzsH48NNa01sS2.1.5Hz0:C, 42.33; H, 7.10; N, 1.90. Found: C, 42.41; H, 6.99; N, 1.54. (D) Sodium N -(m-octyl)-4-0-(a-~-glucopyranosyl)-40(a-D-glucopyranosyl)-D-glucamine-N-carbodithioate (4d, n-C8MDTC): yield 92.2% (white fluffy solid); 'H NMR (D,O/DSS) 6 5.40 (d, J 3.8 Hz, 1H), 5.18 (d, J = 3.8 Hz, 1H), 4.43-4.19 (m, 3 H), 4.03-3.54 (m,18 H), 3.41 (t, 1 H), 1.79-1.67 (m, br, 2 H), 1.30 (8, br, 10 H), 0.85 (t, br, J = 12.2 Hz,3 H).Anal. C, 42.14; H, 7.33; N, 1.82. Calcd for C2,HSONNaOlSS2~3H20: Found: C, 41.92; H, 6.97; N, 1.25. (E)Sodium N-( n -nonyl)-4-0-(a-~-glucopyranosyl)-4O-(a-D-glucopyranosyl)-D-glucamine-N-carbodithioat (4e, n-C9MDTC): yield 90.7% (white fluffy solid); *H NMR (D,O/DSS) 6 5.40 (d, J = 3.6 Hz, 1 H), 5.19 (d, J = 3.7 Hz,1 H), 4.46-4.20 (m, 3 H), 4.03-3.55 (m, 18 H), 3.41 (t, 1 H), 1.80-1.65 (m, 2 H), 1.28 (8, br, 12 H), 0.88 (t, br, J = 13.7 Hz, 3 H). Anal. Calcd for C28Hs2NNa015S2~3H10:C, 42.90; H, 7.46; N, 1.79. Found: C, 42.90; H, 6.99; N, 1.20. (F) S o d i u m N-(l-ethy1propyl)-4-~-(u-D-glucopyranosyl)-4- 0 -( a-D-glUCOpyranOSy~)-D-glUCamine-Ncarbodithioate (4f, br-CsMDTC): yield 79.8% (white fluffy solid); 'H NMR (DzO/DSS) 6 5.77-5.65 (m, 1 H), 5.40 (d, J = 3.8 Hz, 1 H), 5.15 (d, J = 3.8 Hz, 1 H), 4.29-4.22 (m, 1 H), 4.03-3.56 (m, 18 H), 3.41 (t, 1 H), 1.70-1.40 (m, 4 H), 0.86 (t, J = 7.1 Hz, 6 H). Anal. Calcd for CuH~NNaOlsS2~1.4HzO C, 41.24; H, 6.75; N, 2.00. Found: C, 41.60; H, 6.74; N, 1.52. (G) Sodium N-( 1,3-dimethylbutyl)-4-O-(a-D-glucopyranosy1)-4- 0-(U-D-glUCOpyranOSyl)-D-glUCamine-Ncarbodithioate (4g, br-C6MDTC): yield 85.2% (white fluffy solid); 'H NMR (D,O/DSS) 6 5.45-5.31 (m, 1 H), 5.40 (d, J = 3.8 Hz, 1 H), 5.16 (d, J = 3.8 Hz, 1H), 4.30-4.10 (m, 1 H), 4.03-3.38 (m, 18 H), 2.90 (t, 1 H), 1.60-1.30 (m, 1 H), 1.20-1.15 (m, 2 H), 0.94 and 0.89 (2 d, 9 H). Anal. Calcd for CzaH,,JV"N01&2.1Hz0 C, 42.55; H, 6.85; N, 1.98. Found: C, 42.81; H, 6.84; N, 1.45. ( H ) S o d i u m N-(l - m e E h y l h e x y l ) - 4 - 0 -(a-D-glucopyranosy1)-4- 0-(a-D-glucopyranosy1)-D-glucamine-Ncarbodithioate (Oh, br-C7MDTC): yield 79.0% (white fluffy solid); 'H NMR (D20/DSS) 6 5.81 (m, br, 1H), 5.41 (d, J = 3.7 Hz, 1 H), 5.16 (d, J = 4.0 Hz,1 H), 4.30-4.13 (m, 2 H), 4.03-3.56 (m, 18 H), 3.41 (t, 1H), 1.57-1.14 (m, 11H), 0.86 (s, br, 3 H). Anal. Calcd for C26Ha~NaO15S2.1H,0: C, 43.39; H, 7.00; N, 1.94. Found: C, 43.53; H, 7.00; N, 1.45.
Dithiocarbamates in Calcium Mobilization Table I. Effect of Cc and C6Dithiocarbamates Derived from Maltotriose on Cadmium Levels in Micea** whole body, liver, pg of % of kidneys, t g of Cd/g of adminis- Cd/g of tissue tissue wet group tered Cd wet wt wt 83.24 f 8.06 3.96 f 0.34 6.48 f 0.49 control 31.30 f 3.46 1.67 f 0.10 0.82 f 0.22b BLDTC 49.61 f 5.17 2.24 f 0.24 2.74 f 0.46 n-C6MDTC (4a) br-C6MDTC ( 4 0 46.43 f 6.39 2.39 f 0.15 2.69 f 0.63 36.54 f 5.13 2.37 f 0.21 1.45 f 0.52 n-CBMDTC (ab) br-CBMDTC (4g) 73.05 f 4.39 3.33 f 0.35 6.04 f 0.70 a BDFl male mice were each given 1.0 mL of 0.9% NaCl (saline) ip containing 0.03 mg of CdC12.2.5H,0 and 1.0 pCi of 'OBCdC12. Starting 14 days later, each animal was given 0.40 mmol/kg of the indicated compound ip each day for 5 days. Three days after the last injection whole-body cadmium levels were measured, animals were Sacrificed, and organs were removed for measurement of radioactivity. There were 8 animals in each group. The brain cadmium levels of these groups varied from 0.025 i 0.004 to 0.031 f 0.003 pg of Cd/g, and there were no significant differences in brain Cd levels among any of the groups. *All intercompound differences for the whole body or a given organ are significant at the p I 0.05 level except for those between 4a and 4f, the whole-body difference between BLDTC and 4b, and the difference in liver values for the control and 4g.
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 695 Table 11. Effect of C 7 C , Dithiocarbamates Derived from Maltotriose on Cadmium Levels in MiceaSb whole body, liver, pg of % of kidneys, pg of Cd/g of adminis- Cd/g of tissue tissue wet erouD tered Cd wet w t wt control 84.29 f 4.09 3.85 f 0.31 5.74 f 0.23 26.69 f 1.65 1.42 f 0.10 0.52 f 0.07 BLDTC n-C7MDTC ( 4 ~ ) 32.98 f 2.10 2.84 f 0.21 0.75 f 0.10 br-C,MDTC (4h) 64.46 f 5.05 2.94 f 0.15 3.97 f 0.31 n-CBMDTC (4d) 35.24 f 3.54 3.38 f 0.33 1.04 f 0.23 br-C,MDTC (4i) 40.45 f 6.10 2.55 f 0.35 1.50 f 0.32 n-CgMDTC (4e) 36.28 f 3.36 3.61 f 0.14 1.36 f 0.06 a BDF, male mice were each given 1.0 mL of saline ip containing 0.03 mg of CdC12-2.5H20and 1.0 pCi of 'OBCdC12.Starting 13 days later, each animal was given 0.40 mmol/kg of the indicated compound ip each day for 5 days. Three days after the last injection, whole-body Cd burdens were measured, animals were sacrificed, and organs were removed for measurement of radioactivity. There were 8 animals in each group. Brain cadmium levels of the animals in these groups ranged from 0.023 0.006 to 0.029 f 0.003 pg of Cd/g of tissue wet weight, and there were no significant differences among any of the groups. bSee Table I11 for results of statistical analyses.
*
2.15-2.00 (m, 1H), 1.71-1.64 (m, 6 H), 1.29-1.01 (m, 4 H). Anal. Calcd for CzoHSsNNaOloS2~O.3Hz0: C, 44.23; H, 6.79; N, 2.58. Found: C, 44.24; H, 6.95; N, 2.38. (I) Sodium N-( l-ethylhexyl)-4- 0-(a-D-glucopyranosy1)(B) Sodium N-(2-morpholinoethyl)-4-O-(B-~-galacto(4i, 4- O-(u-Dglucopyranosyl)-Dgluca"-carbo pyranosy1)-Dglucamine-N-carbodithioate (5b, MEtLDTC): br-CsMDTC): yield 85.2% (white fluffy solid); 'H NMR yield 83% (white amorphous solid); 'H NMR (D,O/DSS) d 4.58 (D,O/DSS) 6 5.40 (d, J = 3.7 Hz, 1H), 5.19 (d, J = 4.0 Hz, 1 H), (d, J = 7.7 Hz, 1 H), 4.46-4.40 (m, 2 H), 4.25-4.12 (m, br, 1 H), 4.41-4.27 (m, 3 H), 4.03-3.54 (m, 18 H), 3.40 (t, 1 H), 2.08 (m, 4.05-3.53 (m, 16 H), 2.92-2.71 (m, 2 H), 2.63 (t,br, 4 H). Anal. 1 H), 1.36-1.29 (m, 8 H), 0.87 (br, S, 6 H). Anal. Calcd for Calcd for CleHSSNzNaOl1Sz:C, 41.15; H, 6.36; N, 5.05. Found: CnHmNNaOl6Sz-1H20: C, 42.62; H, 7.29; N, 1.84. Found: C, C, 41.08; H, 6.53; N, 4.60. 42.87; H, 6.94; N, 1.52. (C) Sodium N-(3-morpholinopropy1)-4-O-(~-D-ga~acto(J) S o d i u m N-(6-hydroxyhexyl)-4-O-(a-~-glucopyranosy1)-Dglucamine-N-carbodithioate (5c, MPrLDTC): pyranosy1)-4- 0-(a-D-glucopyranosy1)-D-glucamine-Nyield 91% (white amorphous solid); 'H NMR (D20/DSS) 6 4.58 carbodithioak. (4j, HHxMDTC): yield 75.3% (white amorphous (d, J = 7.7 Hz, 1H), 4.50-4.15 (m, 2 H), 4.04-3.53 (m, 16 HI, 2.56 solid); 'H NMR (D,O/DSS) 6 5.34 (d, J = 3.8 Hz, 1 H), 5.19 (d, (s, br, 4 H), 2.43 (t, J = 7.8 Hz, 2 H), 2.05-1.95 (m, 2 H). Anal. J = 3.8 Hz, 1H), 4.45-4.20 (m, 2 H), 4.03-3.38 (m, 23 H), 1.761.54 Calcd for CzoH37NzNa011S2~0.5H20: C, 41.59; H, 6.63; N, 4.85. (m, 4 H ) , 1.37-1.30 (m, 4 H). Anal. Calcd for Found: C, 41.80; H, 6.77; N, 4.64. CzaH~NNa01,&.0.5Hz0: C, 42.13; H, 6.65; N, 1.97. Found: C, (D) Sodium N -(3,4-dimet hylbenzyl) -4- 0-(B-~-galacto41.99; H, 6.94; N, 2.20. t hDMeBLDTC): i~~ (K) Sodium N-(cyclohexylmethyl)-4-O-(u-D-g~uco- p y r a n o s y l ) - D g l u c a m i n ~ N - ~ ~(a, yield 90% (white solid); 'H NMR (D20/DSS) 6 7.27-7.08 (m,3 pyranosyl)-4- 0 -(u-D-glucopyranosy1)-D-glucamine-NH), 5.68 (d, J = 15.9 Hz, 1 H), 5.31 (d, J = 15.7 Hz, 1 H), 4.59 carbodithioate (4k, CMMDTC): yield 87.8% (white fluffy solid); (d, J = 7.8 Hz, 1H), 4.62-4.57 (m, 2 H), 4.05-3.57 (m, 12 H), 2.32 'H NMR (D,O/DSS) 6 5.39 (d, J = 3.8 Hz, 1H), 5.19 (d, J = 3.8 and 2.31 (2 s, 6 H). Anal. Calcd for CBH3NNaOl&: C, 47.22; Hz, 1 H), 4.42-4.39 (m, 2 H), 4.23-4.17 (m, 1 H), 4.02-3.44 (m, H, 6.12; N, 2.51. Found: C, 47.06; H, 6.46; N, 2.22. 18 H), 3.37 (t, 1 H), 2.08 (m, br, 1 H), 1.72-1.64 (m, br, 4 H), 1.25-1.03 (m, 6 H). Anal. Calcd for C2sH16NNa01sSz.1.8Hz0:C, The percentage yields and characterizations of the parent amines of the above lactamine dithiocarbamates are as follows. 42.65; H, 6.83; N, 1.91. Found: C, 42.64; H, 6.72; N, 1.65. (L) Sodium N-( 2-morpholinoet hyl)-4- 0-(u-D-gluco(A) N -(Cyclohexylmet hyl)-4-0- (B-~-galactopyranosy1)pyranosy1)-4-0 -(cr-D-glucopyranosy1)-D-glucamine-N- D-glucamine: yield 85% (white solid); 'H NMR (D,O/DSS) 6 4.48 (d, J = 7.7 Hz, 1 H), 4.06-3.50 (m, 12 H), 2.81-2.77 (dd, J carbodithioate (41, MEtMDTC): yield 67% (white solid); 'H = 12.5 Hz, 2.9 Hz, 1 H), 2.61-2.57 (dd, J = 12.3 Hz, 9.2 Hz, 1H), NMR (DzO/DSS) 6 5.40 (d, J = 3.8 Hz, 1 H), 5.19 (d, J = 3.8 Hz, 1 H), 4.50-4.30 (m, 3 H), 4.20-3.34 (m, 23 H), 2.70-2.60 (m,6 H). 1.71-1.62 (m, 6 H), 1.51-1.47 (m, 1H), 1.30-1.12 (m, 2 H), 0.964.86 (m, 2 H). Anal. Calcd for C1&97N010~0.25H20C, 51.40; H, 8.51; H, 6.39; N, Anal. Calcd for C ~ H ~ N 2 N a 0 1 6 S 2 ~ 0 . 5C, H z41.38; 0 3.86. Found: C, 41.42; H, 6.80; N, 4.55. N, 3.15. Found: C, 51.37; H, 8.51; N, 2.93. (M) Sodium N-(3-morpho~~nopropy~)-4-~-(u-D-g~uco-( B ) N-(2-Morpholinoethyl)-4-0- ( B - ~ - g a l a c t o p y r a n o s y l ) -4- 0- (a-D-glucopyranosy1)-D-glucamine-N- pyranosy1)-D-glucamine: yield 78.9% (white solid); 'H NMR carbodithioate (4m, MPrMDTC): yield 71% (white solid); 'H (D20/DSS) 6 4.48 (d, J = 7.6 Hz, 1 H), 4.03-3.63 (m, 15 H), 3.53 NMR (D,O/DSS) 6 5.40 (d, J = 3.7 Hz, 1 H), 5.19 (d, J = 3.8 Hz, (dd, J = 9.8 Hz, 7.6 Hz, 1 H), 2.85-2.74 (m, 3 H), 2.66-2.49 (m, 1 H), 4.45-4.25 (m, 3 H), 4.03-3.33 (m, 23 H), 2.562.22 (m, 6 H), 7 H). Anal. Calcd for C18H36N2011:C, 47.36; H, 7.95; N, 6.14. 1.99-1.94 (m, 2 H). Anal. Calcd for C2eH47N2Na016S2-1.3H20: Found: C, 47.47; H, 7.98; N, 6.19. ( C ) N-(2-Morpholinopropyl)-4-O-(~-~-ga1actoC, 41.41; H, 6.63; N, 3.71. Found: C, 41.43; H, 6.67; N, 3.40. P r e p a r a t i o n of N-Substituted Lactamine Dithiopyranosy1)-D-glucamine: yield 82.7% (white fluffy solid); 'H carbamates (5a-d). The dithiocarbamates (Figure 2) in the NMR (D,O/DSS) 6 4.48 (d, J = 7.7 Hz, 1H), 4.04-4.00 (m,1H), prenent study were obtained from the parent-substituted lacta3.92-3.64 (m, 14 H), 3.54 (dd, 1 H), 2.84-2.39 (m, 10 H), 1.72-1.65 mines, which were in turn prepared from a-lactose and the cor(m, 2 H). Anal. Calcd for Cl&38N2011: C, 48.50; H, 8.14; N, 5.95. responding primary amines according to reported methods for Found: C, 48.32; H, 8.25; N, 5.34. other lactamine dithiocarbamates (11, 13). ( D ) N-(3,4-Dimethylbenzy1)-4-0 -(B-~-galacto(A) Sodium N-(cyc~ohexylmethy~)-4-0-(~-~-ga~actopyranosy1)-Dglucamine: yield 71.4% (white amorphous solid); pyranosy1)-D-glutamine-N-carbodithioate (5a, CMLDTC): 'H NMR (D,O/DSS) 6 7.27-7.15 (m, 3 H), 4.48 (d, J = 7.6 Hz, yield 86% (white amorphous solid); 'H NMR (D20/DSS) 6 4.58 1 H), 4.10-3.54 (m, 14 H), 2.87-2.84 (dd, 1 H), 2.68-2.65 (dd, 1 H), 2.31 and 2.30 (2 s, 6 H). Anal. Calcd for C21H35NOlo.1H20: (d, J = 7.7 Hz, 1 H), 4.47-4.46 (m, 1 H), 4.40-4.36 (2 d, J = 5.2 C, 52.60; H, 7.78; N, 2.92. Found: C, 52.25; H, 7.55; N, 2.75. Hz, 1 H), 4.18-4.14 (2 d, J = 7.5 Hz, 1 H), 4.03-3.53 (m,13 H),
696 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
Jones et al.
control +I+/+ +I+/+ +I+/+ +/+I+ +I+/+ +I-/+ BLDTC +I+/+I+/+ +I+/+ +I+/+ +I+/+ n-C,MDTC ( 4 ~ ) +I-/+ -I+/+ +I+/+ -/+I+ br-C,MDTC (4h) +I+/+ +I+/+ +I+/+ n-CBMDTC (4d) +I+/+ -/-I+ br-CBMDTC(4i) +/+I(+) indicates a significant difference, p 5 0.05; (4 indicates that the difference is not significant. Symbols are presented in the sequence: whole body values/ kidney values/liver values. Animal Studies. Male mice of the (C57BL/6 X DBA2)F1 strain were from the NCI-Frederick Cancer Research Facility, Frederick, MD. They were housed in a facility which was fully AAALAC accredited and were provided with Wayne Rodent Blox and tap water a t all times. When they attained a mean weight of about 23 g, each mouse was given an ip injection of 0.03 mg of CdC12.2.5H20 in 1.0 mL of 0.9% NaCl solution (saline) containing 1.0 pCi (37 kBq) of 10BCdC12(Du Pont NEN Products, North Billerica, MA). In this model, cadmium retained after the first 2 days postinjection is retained for an extended period of time (7),thus allowing experiments on the mobilization of such cadmium to be started a t any time a t or beyond 72 h after the injection of the cadmium. Treatment with the dithiocarbamate analogues was begun from 5 to 14 days after cadmium administration, and the treatment schedule in each case consisted of 5 consecutive daily ip injections a t a dose of 0.40 mmol/kg body weight. Three days after the last injection, whole-body cadmium burdens were measured with a Canberra modular whole-body y-counting system, and data were e x p r d as percent of retained cadmium by reference to phantoms prepared the same day the cadmium was administered. Mice were then sacrificed, kidneys, brains, and livers were excised, and radioactivity in the organs was measured with a y scintillation well counter (Beckman Biogamma I). Organ cadmium levels were expressed as micrograms of cadmium per gram of tissue wet weight by reference to standards containing 1.0 pCi of cadmiumf0.03 mg of CdC12. 2.5H20. Statistical analysis of data was done by using Duncan's multiple-range test in a statistical software package (SAS/STAT);the level of significance was designated as 50.05.
Results The dithiocarbamates (4a-m) were obtained as white, fluffy solids from maltotriose (1) and various alkylamines (2a-m) in two steps (Figure 1). They were relatively more hygroscopic compared to the analogous N-alkyllactamine dithiocarbamates. None of the parent secondary amines (3a-m) could be crystallized; purification was achieved only by repeated precipitations from methanolic solution of the amine with ethyl ether. A notable feature observed was that the hydrogenation of the imine derived from maltotriose, which is a trisaccharide, was more difficult owing to the greater bulk of the hydrophilic end than the ones from a disaccharide, e.g., lactuse, or a monosaccharide, e.g., glucose or galactose. The substituted lactamine dithiocarbamates (5a-d) were prepared on the basis of other lactamine dithiocarbamates reported earlier and are shown in Figure 2. Here, too, the purification was done by precipitation only, since neither the parent amines nor the dithiocarbamates could be crystallized. The effects of C5 and c6 maltotriose dithiocarbamates (MDTCs) (at a dose of 0.40 mmol/kg) on cadmium levels in mice are shown in Table I. These values and those presented in later tables are given as percent of administered dose (rather than percent of control dose) because they are all standardized against a sample of the cadmium present in a suitably configured model to allow corrections for the geometry of the counting apparatus for the whole-body levels. The lactose analogue, BLDTC, was included as a positive control. None of the MDTCs was
Table IV. Comparative Effects of Dithiocarbamates Derived from Lactose and Maltotriose on Cadmium Levels in MiceaPb whole body, liver, pg of 70 of kidneys, yg of Cd/g of adminis- Cd/g of tissue tissue wet group tered Cd wet w t wt 73.81 f 2.24 3.46 f 0.28 6.33 f 0.57 contro1 26.13 f 2.08 2.05 f 0.20 0.50 f 0.05 MeBLDTC (5e) 46.50 4.28 2.46 f 0.09 2.49 f 0.04 CMMDTC (4k) 71.83 f 3.47 2.99 f 0.16 5.66 f 0.33 HHxMDTC (4j) 80.08 f 1.52 3.55 & 0.29 6.66 f 0.59 MEtMDTC (41) MPrMDTC (4m) 71.12 4.28 3.45 & 0.19 6.67 f 0.52 'BDF, male mice were each given 1.0 mL of 0.9% saline ip containing 0.03 mg of CdC12.2.5H20 and 1.0 pCi of 'OgCdC12. Starting 5 days later, each animal was given 0.40 mmol/kg of the indicated compound ip daily for 5 days. Three days after the last injection, whole-body cadmium levels were measured, animals were sacrificed, and organs were removed for measurement of radioactivity. There were 8 animals in each group. The brain cadmium levels of the animals in these groups ranged from 0.030 f 0.002 to 0.040 f 0.007 pg of Cd/g of tissue wet weight with the control level at 0.033 f 0.002 pg of Cd/g of tissue wet weight. The only significant ( p C 0.05) increase over control was found with the MPrMDTC group at 0.040 0.007 rg of Cd/g of tissue wet weight. See Table V for results of statistical analyses.
quite as effective as BLDTC, on a molar basis, in reducing whole-body cadmium burdens or cadmium concentrations in the kidneys or liver. The n-C5 and n-C6 isomers were quite effective and were virtually identical in their actions with no significant differences between them in regard to effects on whole-body, kidney, or liver cadmium levels. In contrast, the c6 isomers had quite disparate actions. The respective reductions by n-C6MDTC and br-C6MDTCof whole-body, kidney, and liver cadmium were 56% and 12%; 40% and 16%; and 78% and 7%, revealing a strong dependency of pharmacologic action on configuration of the alkyl group. Table I shows that the differences between these two isomers in their actions of all three parameters were significant at the 95% confidence level (4b vs 4g). In regard to the C7 isomeric pair, there was also a striking difference between the actions of n-C7MDTC(4c) and br-C7MDTC(4h) (Tables I1 and 111). As with the c6 pair, the normal isomer was almost as effective on all three parameters as BLDTC, while the branched isomer was only moderately active. The respective reductions by the n-C7 and br-C7MDTC analogues of whole-body, kidney, and liver cadmium levels were 61% and 24%; 26% and 24%; and 87% and 31%. Table I11 shows that the difference between the compounds on whole-body and liver cadmium were significant at p 5 0.05. Higher molecular weight analogues in the maltotriose series were generally less active than those shown in Tables I and 11. The cyclohexylmethyl (4k) derivative had moderate activity, while the 6-hydroxyhexyl (4j), 2morpholinoethyl (41), and 3-morpholinopropyl(4m) congeners were only marginally active or were without effect (Tables IV and V). The possibility that compound 41 had
Dithiocarbamates in Calcium Mobilization
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 697
Table V. Intergroup Statistical Analyses of Results Presented in Table IVn MeBLDTC (5e) CMMDTC (4k) HHxMDTC (4j) MEtMDTC (41)
MPrMDTC (4m)
control +I+/+ +I+/+ -/+/+ +/-I-/-/MeBLDTC (5e) +/+I+ +/+I+ +/+I+ +/+I+ CMMDTC (4k) +I+/+ +I+/+ +/+I+ HHxMDTC (4j) +I+/+ -I+/+ MEtMDTC (41) +/-I(+) indicates a significant difference, p 5 0.05; (-) indicates that the difference is not significant. Symbols are presented in the sequence: whole body values/ kidney values/ liver values.
Table VI. Comparative Effects of Dithiocarbamates Derived from Lactose on Cadmium Levels in MiceaPb whole body, liver, pg of % of kidneys, pg of Cd/g of adminisCd/g of tissue tissue wet group tered Cd wet wt wt 90.25 f 3.68 3.22 f 0.13 6.37 f 0.28 control 0.37 & 0.01 MeBLDTC (5e) 24.81 f 1.72 1.83 f 0.12 25.58 f 1.56 1.89 f 0.12 0.43 f 0.06 CMLDTC (5a) 2.71 0.17 5.37 f 0.29 MEtLDTC (5b) 73.56 4.30 6.34 f 0.52 MPrLDTC (512) 77.52 f 2.49 2.83 f 0.25
*
*
Table VII. A Comparison of the Most Effective Dithiocarbamates Derived from Lactosen,b whole body, liver, pg of % of kidneys, yg of Cd/g of adminis- Cd/g of tissue tissue wet group tered Cd wet wt wt 78.90 7.89 2.93 f 0.29 5.57 0.17 control 32.91 f 4.18 1.48 f 0.06 1.04 f 0.17 BLDTC MeBLDTC (5e) 26.45 2.12 1.84 0.11 0.43 0.06 DMeBLDTC (5d) 30.87 f 2.36 1.42 f 0.08 1.13 0.38
**
"BDF1 male mice were each given 1.0 mL of 0.9% saline ip containing 0.03 mg of CdClZ.2.5Hz0 and 1.0 WCi of lWCdC12. Starting 5 days later, each animal was given 0.40 mmol/kg of the indicated compound ip daily for 5 days. Three days after the last injection, whole-body cadmium levels were measured, animals were sacrificed, and organs were removed for measurement of radioactivity. There are 8 animals in each group. The brain cadmium levels of the animals in these groups ranged from 0.021 f 0.002 to 0.028 f 0.003 pg of Cd/g of tissue wet weight; in no case was a statistically significant change in brain cadmium found. bAll intercompound differences for the whole-body cadmium levels or those of a given organ are significant at the p I0.05 level except for those between 5a and 5e, the difference in liver levels between the control and 5c, and the kidney level difference between 5b and 5c.
'BDF1 male mice were each given 1.0 mL of 0.9% saline ip containing 0.03 mg of CdClz.2.5H20 and 1.0 pCi of 'OBCdClZ. Starting 5 days later, each animal was given 0.40 mmol/kg of the indicated compound ip daily for 5 days. Three days after the last injection, whole-body cadmium levels were measured, the animals were sacrificed, and organs were removed for measurement of radioactivity. There were 8 animals in each group. The brain cadmium levels of the animals in these groups ranged from 0.026 f 0.003 to 0.028 f 0.002 pg of Cd/g of tissue wet weight; in no case was a statistically significant change in brain cadmium found. bAll differences between control and treated groups were significant at the p 5 0.05 level. All organ cadmium differences between MeBLDTC and both BLDTC and DMeBLDTC were significant except for the whole-body difference between MeBLDTC and DMeBLDTC. None of the differences between DMeBLDTC and BLDTC were significant.
a paradoxical effect and actually retarded the excretion of cadmium (Table IV) was not investigated. In contrast to the corresponding results in the maltotriose series, (cyclohexylmethy1)lactamine dithiocarbamate (5a) was quite effective in reducing wholebody, kidney, and liver cadmium contents, educing respective reductions of 72%, 41% ,and 93%. Its action was virtually identical to that of the methylbenzyl derivative (5e). However, the 2-morpholinoethyl(5b) and 3-morpholinopropyl (5c) analogues were substantially inactive (Table VI). Table VI1 illustrates the comparative actions of three very active dithiocarbamates, the benzy, 4-methylbenzyl (b), and 3,4-dimethylbenzyl (5d) analogues, and reveals an impressive reduction of cadmium in all of the three parameters for each of these compounds. There was no significant difference between BLDTC and DMeBLDTC on any parameter and no significant difference between MeBLDTC (5e) and DMeBLDTC (5d) on whole-body cadmium levels. While all other intergroup differences were significant at the p I0.05 level, the magnitudes of these differences were small. Both BLDTC and MeBLDTC are used as reference compounds here, with the MeBLDTC being more useful with those compounds which are more effective. The relationship between these two compounds is given in Table VII. Discussion The previously reported dithiocarbamates derived from lactose were significantly superior to the corresponding compounds derived from glucose. One of the reasons for this is probably the importance of the increased molecular weight in facilitating the passage of the lactose derivatives
to the liver and their uptake by hepatocytes (5,15). The ability of compounds of this type derived from glucose to remove cadmium from the kidneys is dependent more strongly on relatively minor structural variations which appear to be related to hydrogen-bonding interactions (14). The effect of the total charge on chelation is very important for the compounds which have been reported to date. Increases in the anionic charge lead to very large decreases in the ability of the molecule to mobilize intracellular cadmium from both the liver and the kidney (14). We selected the amines used in this study with these factors in mind. One advantage of using compounds of higher molecular weight is that there is apparently an upper limit beyond which the ability of a molecule to cross the blood-brain barrier decreases greatly. This has been estimated to occur somewhere in the region of molecular weights ranging from 400 to 500 (16). The compounds reported here which have polar groups derived from either lactose or maltotriose have molecular weights above this range and thus would not be expected to be capable of penetrating the bloodbrain barrier. The fact that brain cadmium levels found here subsequent to chelate treatment of cadmium-loaded mice were essentially those of the control cadmium exposed animals supports the conclusion that neither the compounds nor their cadmium complexes were able to penetrate this barrier. The compounds derived from maltotriose were, without exception, less potent (on a molar basis) than those analogous compounds derived from lactose in the mobilization of cadmium from either kidney or liver. From the range of compounds prepared and their behavior, this reduced effectiveness of the maltotriose derivatives can be ascribed to the less than optimal balance between the
698 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
nonpolar and polar parts of the molecule and a molecular size which is sufficiently large to hinder the passage through the cellular membrane in the absence of specific receptors and/or a carrier mechanism. A point of some interest is that the compounds in a series which are optimal for the removal of cadmium from the liver are not necessarily optimal for the removal of cadmium from the kidney. An example of this is evident in the compounds listed in Table VII, which are the most effective agents for the reduction of whole-body cadmium levels reported to date (11, 13). This raises the question as to the relative importance of the cadmium deposits in these two organs in the determination of the course and severity of chronic cadmium intoxication. Numerous studies have demonstrated that the movement of hepatic cadmium to the kidney in the form of the metallothionein complex causes a drastic deterioration of renal function (17-19) as this complex is perhaps the most nephrotoxic of the cadmium compounds. On this basis, the prevention of transport of the extensive hepatic cadmium to the kidneys must be given a high priority. Since these dithiocarbamates enhance the biliary and then, in turn, the fecal excretion of hepatic cadmium (9, 20), they may be significantly superior for in vivo usage to chelating agents which enhance primarily the renal excretion of cadmium. The dosages of compounds administered here were equimolar ones in order to clearly allow the data to be compared on a molar basis. I t would also be possible to administer a constant fraction of the LD50 of the compound. This latter method was not used because of the relatively enormous number of mice which would be required to obtain LD50 values of sufficient accuracy to distinguish between those for such similar compounds. Our preliminary unpublished studies using very small numbers of mice indicate that the LD50 values of the compounds examined here are generally some value significantly in excess of 7 mmol/kg.
Acknowledgment. We acknowledge the support of this work by the National Institute of Environmental Health Sciences via Grant ES-02638 (M.M.J.) and by the Department of Veterans Affairs (G.R.G.).
References (1)Eybl, V.,Sykora, J., and Mertl, F. (1966)Wirkung von Ca-ADTA und Ca-DTPA bei der Kadmiumvergiftung. Acta Biol. Med. Ger. 17,178-185. (2) Neimeier, B. (1967)Der Einfluss von Chelatbildnern auf Verteilung und Toxicitit von Cadmium. Int. Arch. Gewerbepathol. Gewerbehyg. 24,160-169.
Jones et al. (3) Cantilena, L. R., Jr., and Klaassen, C. D. (1982)Decreased effectiveness of chelation therapy with time after acute cadmium poisoning. Toxicol. Appl. Pharmacol. 63,173-180. (4) Planas-Bohne, F., and Lehmann, M. (1983)Influence of chelating agents on the distribution and excretion of cadmium in rata. Toxicol. Appl. Pharmacol. 67,408-416. (5) Waalkes, M. P., Watkins, J. B., and Klaassen, C. D. (1983) Minimal role of metallothionein in decreased chelator efficacy for acute cadmium. Toxicol. Appl. Pharmacol. 68,392-398. (6) Klaassen, C. D. (1990)Heavy metals and heavy metal antagonists. In The Pharmacological Basis of Therapeutics (Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P., Eds.) 8th ed., pp 1592-1614,Pergamon Press, New York. (7) Gale, G. R.,Atkins, L. M., and Walker, E. M., Jr. (1982)Effecta of diethyldithiocarbamate on organ distribution and excretion of cadmium. Ann. Clin. Lab. Sci. 12,463-470. (8) Jones, S. G., Basinger, M. A., Jones, M. M., and Gibbs, S. J. (1982)Polarity and antidotal efficacy of dithiocarbamates for acute cadmium chloride intoxication. Res. Commun. Chem. Pathol. Pharmacol. 40,155-164. (9)Kojima, S.,Kaminaka, K., Kiyozumi, M., and Honda. T. (1986) Comparative effects of three chelating agenta on distribution and excretion of cadmium in rats. Toxicol. Appl. Pharmacol. 83, 516-524. (10)Shinobu, L. A., Jones, S. G., and Jones, M. M. (1984)Sodium N-methyl-D-glucamine dithiocarbamate and cadmium intoxication. Acta Pharmacol. Toxicol. 54, 189-194. (11)Jones, M. M.,Singh, P. K., and Jones, S.G. (1990)Optimization of chelating agent structure for the mobilization of aged renal and hepatic cadmium deposits: Sodium N-benzyl-4-0-(8-D-galactopyranosy1)-D-glucamine-N-cartdithioate. Chem. Res. Toricol. 3, 248-253. (12) Singh, P.K., Jones, S. G., and Jones, M. M. (1990)Structural factors in the in vivo chelate mobilization of aged cadmium deposits. Enuiron. Health Perspect. 85,361-370. (13)Jones, M. M., Singh, P. K., Jones, S. G., and Holscher, M. A. (1990)Dithiocarbamates of improved efficacy for the mobilization of retained cadmium from renal and hepatic deposits. Phurmacol. Toxicol. 68,115-120. (14)Shimada, H., Kawago, M., Kiyozumi, M., and Kojima, S. (1990) Comparative effects of three dithiocarbamates on tissue distribution and excretion of cadmium in mice. Res. Commun. Chem. Pathol. Pharmacol. 69,49-59. (15) Tiribelli, C.,Lunazzi, G. C., and Sottocasa, G. L. (1990)Biochemical and molecular aspects of the hepatic uptake of organic anions. Biochim. Biophys. Acta 1031,261-275. (16) Levin, V. A. (1980)Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J. Med. Chem. 23,682-684. (17)Johnson, D. R., and Foulkes, E. C. (1980)On the proposed role of metallothionein in the transport of cadmium. Enuiron. Res. 21, 360-365. (18)Suzuki, C.A. M., and Cherian, M. G. (1987)Renal toxicity of cadmium-metallothionein and enzymuria in rats. J. Pharmacol. Exp. Ther. 240,314-319. (19)Shaikh, Z.A., and Smith, L. M. (1984)Biological indicators of cadmium exposure and toxicity. Experientia 40,36-43. (20)Cikrt, M., Basinger, M., Jones, S. G., and Jones, M. M. (1986) Structural effects in the dithiocarbamate enhanced biliary excretion of cadmium. J. Toxicol. Enuiron. Health 17,429-439.
