Synthesis, Characterization, and Antitumor Activity of Hodouracil Complexes Udai P. Singh, Badri N. Singh, Animesh IL Ghose, Rakesh K. Singh, and Ajit Sodhi UPS, BNS, AKG. Department of Chemistry, and-MU, AS. Department Faculty of Science, Banaras Hindu University, Varanasi, India
of Zoology,
ABSTRACT Complexes of 5-iodouracil (5IU) with Mn(II), Co@), Cu(II), Zn(II), and Cd@) ions have been prepared, characterized, and subjected to a screening system for evaluation of antitumor activity against Sarcoma-180 (S-180) and L 929 tumor cells. The complexes were characterized by their elemental analysis, infrared spectra, electronic spectra, magnetic measurements, and powder x-ray diffraction. The antitumor activity results indicate that some complexes have good antitumor activity both in vivo and in vitro against S-180 and L 929 tumor cells.
INTRODUCTION 5Iodouracil (5IU) has lethal and mutagenic effects on bacteriophage T4 [ 11. Apparently the lethal effect of 5IU is a consequence of its incorporation into DNA. It has a lethal effect on the adult Drosophila melanogaster also, by inhibiting egg-laying [2]. Very few studies on 5IU complexes with transition metal ions have been reported in the literature. The stability constant of 51U with Cu(II), Hg(II), and Be(I1) have been studied by several workers [3, 41. Villa et al. reported its complex with Cu(II) ion [5]. Such scanty information regarding its complexation has encouraged us to undertake a systematic study of its complexes with various transition metal ions and to test the antitumor activity of synthesized compounds. In this paper we describe the synthesis, characterization, and antitumor activity of 51U complexes with Mn(II), Co(II), Cu(II), Zn(II), and Cd(I1) ions. EXPERIMENTAL All the chemicals used were of reagent grade. Metal nitrates were from E. Merck and 5-iodouracil was from Aldrich. 3H-thymidine was from Bhabha Atomic Research Center, Bombay.
Address reprint requests and correspondence to: Dr. Udai P. Singh, Department of Science, Banaras Hindu University, Varanasi-22100.5, India.
of Chemistry,
Faculty
Journal of Inorganic Biochemistry, 44, 211-282 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
271 0162-0134/91/$3.50
278
U. P. Singh et al.
Synthetic
Procedures I m mol of the hydrated metal nitrates were dissolved in a mixture of 15 ml triethyl orthoformate plus 35 ml ethanol and refluxed for 8 hr. Then, I m mol 5-iodouracil was added and the resultant mixture was refluxed for 30-40 days (depending on the metal ions), after which the volume of the reaction mixture was reduced to about one-third of its original volume by heating. The new complexes were separated by filtration, washed several times with ethanol and ether.. then finall>- with ether. and dried at 50 to 60°C. The metal ions were determined by volumetry after dissolving the complexes in dilute nitric acid [6]. The analyses of carbon, hydrogen, and nitrogen were carried out with a Perkin-Elmer model 240C elemental analyzer. Infrared spectra were 783 recorded in nujol mull between 4000 and 200 cm ! with ;I Perkin-Elmer spectrophotometer. Solid-state UV/VIS spectra were obtained with a C.iV VIS- 16XA Shimadzu spectrophotometer. The room temperature (299.5 E;) magnetic susceptibiiity measurements were performed using a Faraday magnetic susceptibility balance. X-ray powder data were obtained on a Philips PW 17 i0 diffractometer using Cu-K ,j radiation.
Antitumor
Activity
Evaluation
C,H/He mice of either sex. 8- 10 weeks old (average weight, 20-22 gm) were used in this study. Sarcoma-180 (ascites) is being maintained in our laboratory by serial transplantation in C,H/He mice by intraperitoneal (ip) injection of :! x 10" ascites tumor cells at weekly intervals. Six mice were used per test compound. The compound suspensions were fresh11 prepared in 0.89% NaCl and were injected only once ip at the doses of 12.5 and 25.0 mg/kg body weight 48 hr after tumor transplantation. The same volume of sterile saline (without any test compound) was injected in control mice. Therapeutic effectiveness of the various compounds against tumor bearing mice was assessed from their T./C percentage which was calculated as follows Mean life span of treated mica” T/C s = -.-____--_--.____... X 100 Mean life span of untreated mice (* excluding tumor free survivors). A T/C value of 115 indicates significant activity, whereas a value of > 125 indicates that the complex is worthy of testing in other tumor Fystems 17. 8). Effect of Compounds
on DNA Replication
in vitro
The tumor cell (S-180/1_. 929) suspension was prepared in complete medium (RPM1 medium supplemented with antibiotics, penicillin, streptomycin. and 10% heat inactivated fetal calf serumj at a concentration of 10h cellQm1. as described earlier [9]. To determine the effect of various compounds, 2 x 10’ cells; well were added in a 96 well plate (NUNC. Denmark) in duplicate, the test compounds were added at different doses (5, 10, and 20 pg/ml) and incubated for 23 hr at 37°C in ;I Co, incubator. In control sets, cells were incubated with medium alone (without any compound). After 24 hr of incubation, the tumor cells were washed three times with balanced salt solution and the cell pellets were resuspended in 0.2 ml complete medium containing 1 pCi/ml ‘H-thymidine and pulse-labeled for 1 hr. The cells
5IU AND ANTITUMOR
ACTIVITY
279
were then washed three times with balanced salt solution. The cells were lysed in 0.5% SDS and the lysate was counted for radioactivity in a LKB P-liquid scintillation counter. The percentage inhibition of 3-thymidine incorporation in DNA was calculated as follows CPM in treated tumor cells % Inhibition
RESULTS
= 1-
CPM in untreated
tumor cells
x loo.
AND DISCUSSION
The new metal complexes involve 1:2 metal-to-ligand ratio. These compounds contain two molecules of coordinated water per metal ions. All the complexes are colored except Zinc(I1) and Cadmium(I1) (Table 1). These complexes have melting points in the 230-270°C range. Infrared
Spectra Some important IR frequencies of 5IU [5] and its complexes are given in Table 2. There are several absorption bands in the region of 3000-3500 cm-r. The vO-H(,,,,, bands at 3250-3500 cm-’ suggest the coordination of water [lo]. The presence of VM-O@quo)bands in the lower region of the infrared spectra and absence of HOH bending bands due to lattice water in the 1630- 1600 cm- ’ region confirm binding of water [ 111. The vNH band of free 51U (3 145 cm- ’ ) is also seen to exhibit shifting in all these complexes. Regarding the NH deformation modes, the 6N,-H band of 51U at 1430 cm-’ does not exhibit a substantial shift in any case whereas the 6N,-H band (1512 cm- ‘) exhibits significant shifts toward lower wave numbers upon formation of the new complexes. These results indicate that coordination occurs at the N, nitrogen of 5IU. The vC, = 0 band remains unchanged in intensity and is only slightly shifted in position whereas the vC, = 0 band shows sizeable shifts toward lower frequency, indicating the participation of the C, = 0 group in coordination. The relative insensitivity of the vC, = 0, 6N,-H, vC-I bands to metal complex formation demonstrates that none of these potential ligand sites (O,,, N(3), I) are coordinated. In the lower frequency IR region, bands attributable to M-O stretching modes were assigned on the basis of previous studies [12, 131 and favor a coordination number of six for the metal ions in these complexes. The tentative
TABLE 1. Analytical Data and Physical Properties
of the Complexes
Amount Found (Calc.) (%) Complexes Mn(SIU),.2H,O Co(5IU),.2H,O
M
C
H
N
(“C)
Light brown Violet
(9.68) 9.62 (10.31) 10.34 (11.03) 11.05 (11.32) 11.35 (18.00) 18.04
(16.93) 16.90 (16.81) 16.86 (16.68) 16.69 (16.62) 16.66 (15.37) 15.33
(1.76) 1.71 (1.75) 1.73 (1.73) 1.75 (1.75) 1.76
(9.87) 9.83 (9.80) 9.75 (9.73) 9.78 (9.69) 9.65 (8.96) 8.99
231
Zn(SIU),.2H,O
Bluish green White
Cd(5IU),.2H,O
White
Cu(XU),.2H,O
Melting Point
Color
(1.60) 1.64
263 257 255 252
280
CT.P. Singh et al.
TABLE 2. Some Characteristic Band Assignments
IR Bands for S-Iodouracil 5IU
VO-H 3 145w 1710s 1657s 1512m 1430m ._
vN-H YC, = 0 vC, = 0, PC = C in phase 6N,-H 6N,-H “M-%,uo, uM-N
_.
vM-0
and its Complexes
Mn(I1)
Co(II)
Cu(II)
Zn(I1)
Cd@)
3450W
3120~ 169Om 165% 149sm 1430s 375s 263m
347ow 3120~ 1685m 1652s 1498m 143Om 41sm ?Shm
346Ow 312OW 1690s 165?\ 1482m 143Om 375m ‘6.5\,
346Ow 313ovI 1693s 16.53s 1490m 143ow 3.5sm 272m
243m
240m
3420~ 3 12ow 1687s 1650s 1480s 14305 46Om 29Om 280~ 23%
228m
242m
PM-N band assignments [lo, 12) also support the coordination number suggested by the location of vM-0 bands. On the basis of the above infrared spectral studies, it may be suggested that the ligand 51U behaves as bidentate, binding through its C z = 0 and N ,-H groups.
Electronic
Spectra
and Magnetic
Properties
The magnetic moments of the Mn(I1) and Cu(I1) complexes, 5.90 B.M. and 2.20 B.M., show the presence of five and one unpaired electrons. respectively. Their electronic spectra show bands at 300 nm, 399 nm, and 537 nm for Mn(II)-SIU and at 630 nm for Cu(II)-SIU suggest octahedral geometry for Mn(I1) complex [14] and distorted octahedral geometry for Cu(I1) compound [ 151. The magnetic moment value of the Co(I1) complex is 5.14 B.M. and its electronic spectrum exhibits a d-d transition band at 520 nm and 820 nm and the band due to ‘LT!,(F) -+J’Tzg is absent. It favors octahedral structure for Co(II)-SIU [ 151. X-Ray
Diffraction
studies
X-Ray diffraction data (not shown) of the complexes were indexed according to the method of, Ito [16]. The ifdexing pattern yields the lat$ce constan? a = 14.08 A, b, = 3.78 A, and c = 3.54 A for I$n(II)-SIU; aO= 14.06 A. b = 7.$6 A. and c = 4.68 A for CoQI)-SIU; a ~~13.79 A. b = 60.83 A, and c =L-4.64 A f$r Cu(II)-5II-J; a = 14.08 A, b = 4.66 A, and c = 4.58 A for Zn(II)-5IU: a -= 14.15 A, h = 6.03 A. and c = 4.69 i for Cd(II)-SIU indicating orthorhombic symmetry for all these complexes. On the basis of analytical data, magnetic data and other spectral studies, the following structure may be proposed for the complexes:
M = MnlII),
Antitumor
CdII),
Cu(II),
Zn(II),
CdlII)
Properties The ligand 51U has significant antitumor activity against Sarcoma-180 tumor with T/C value 487.5 at a dose of 25.0 mg/kg body weight. Among the synthesized
51U AND ANTITUMOR
ACTIVITY
281
TABLE 3. Screening Data for Antitumor Activity Against Sarcoma- 180 in vivo Dosage ip Injection Compound
Mean Life Span of Nonsurvivors T/C=
mglkg Body Weight’
No. of Mice Surviving > 6 Monthsb
T/C %
5IU
12.5 25.0
718 3918
-
87.5 487.5
Mn(SIU),.2H,O
12.5 25.0 12.5 25.0 12.5 25.0 12.5 25.0 12.5 25.0
1218 10/8 1418 58/8 8/8 918 918 10/8 8/8 718
3(50) -
150.0 125.0 175.0 725.0 100.0 112.5 112.5 125.0 100.0 87.5
Co(SIU),.2H,O Cu(SIU),.2H,O Zn(SIU), .2H s0 Cd(5IU),.ZH,O
In each set of experiments six mice were used; T/C = 6/6, T = tumored, C = control. “, In calculating the average survival time, mice surviving > 6 months were not included. Number in parenthesis indicates percentage of mice surviving six months or more. ’ A single injection of the reported dose was given.
complexes only Mn(II)-5IU and Co(R)-5IU complexes display significant antitumor activity at the reported doses as shown by their T/C values (Table 3). Zn(LI)-5IU was also found to be therapeutically effective when administered at a dose of 25.0 mg/kg body weight. With Co(R)-5IU complex treatment, 50% of the mice survived beyond six months at a dose of 25.0 mg/kg body weight. Further, it was found that none of the test compounds except Cd(I1) showed toxicity up to a dose of 50.0 mg/kg body weight. The Cd(E) complex was found to
TABLE 4. Percentage Inhibition of 3H-Thymidine Tumor Cells in vitro
Incorporation
in L 929/Sarcoma-180
Dose Compounds
SIU Mn(SIU),.2H,O Co(SIU),.2H,O Cu(SIU),.2H,O Zn(51U),.2H20 Cd(SIU),.2H,O
5IU Mn(SIU),.2H,O Co(SIU),.2H,O Cu(SIU),.2H,O Zn(SIU),.2H,O Cd(SIU),.2H,O
Gg/ml
10 pg/ml
20pgw
19.04 14.99 55.43 -
L 929 28.20 28.03 50.73 -
38.65 6.07 43.11 -
43.44 -
48.46 -
26.58 -
7.54 46.90 50.08 -
Sarcoma- 180 18.35 37.54 40.20 46.90 -
25.80 -
282
U. P. Singh et al.
be toxic even at a dose of 2.5 mg/kg body weight. The details of the toxic effect of this compound remain under investigation. Ligand 51U and its transition metal complexes were also tested for their inhibitory effect on 3H-thymidine incorporation in S-180 and L 929 tumor cells in vitro. Most of the compounds that caused inhibition of “H-thymidine incorporation in S-180 and L 929 tumor cells also showed antitumor activity in vivo (Table 4). The maximum inhibition was produced by Co(II)-SIU and Zn(II)-SIIJ in both S-l 80 and I_. 929 tumor cells; the percentage of inhibition decreases as the dose increases. The mechanism of antitumor action of these compounds is not known. it is probable from the results of this study that these agents may be effective antitumor compounds due to their inhibitory action on the replication of DNA in tumor. c&is. The authors are thankful to C. S. I. R. and li. G. C., New Delhi ,for ,finuncial assistance.
REFERENCES 1. D. M. Byrd and W. H. Pusoff‘, Antimicroh.
Agents Chemother.
8. 5.58 (197%: 11.
312 (1977). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
M. M. Clynes and E. J. Duke, J. Insect. Physiol. 22, 1709 (19761. L. Kaminski and H. Altmann, Experientia 23, 599 (1967’~ B. S. Sekhon, J. Indian Chem. Sot. 64, 308 (1987).
J. F. Villa and H. C. Nelson, J. Indian Chem. Sot. 55, 63 I (1978). H. A. Flaschka, EDTA Titration, Pergamon, New York, 1964. S. E. Livingstone, Proceeding of the 20th Corzference on Coordination Chemistr.y, Calcutta, India, 1980, p. 14 1. S. Agrawal, N. K. Singh. R. C. .4ggarwal, A. Sodhi.. and P Tandon, J. Med. Chem. 29, 199 (1986). A. Sodhi and S. M. Singh, [nt. 1. Immunopharmacol. 8, 701 (1986). A. N. Specs, C. M. Mikulski, F. J. Iaconianni, L. L. Pyttewski, and N. M. Karayannis. Inorg. Chim. Acfa 37, L55 1 (1979) and references therein. K. Nakamoto, Infrared Spectra qf Inorganic and Coordination Compounds, 2nd Ed..
Wiley Interscience, New York, 1970. 12. U. P. Singh, R. Ghose, and A. K. Ghose,
13. 14. 15. 16.
Inorg. Chim. Acta 136, 21 (1987); Trans. Met. Chem. 13, 50 (1988); J. Inorg. Biochem. 36. 325 (1989) R. Ghose, Inorg. Chim. Acta 156, 303 (1989). B. N. Figgis, Introduction to Ligand Field, Wiley Eastern. Banglore, 1976. A. B. P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, 1968. L. V. AzarofT, Elements qf X-Ra.y CrystaIlography. New York. 1968.
Received March 25, 1.991: accepted May 30, 1991
of Zoology,
ABSTRACT Complexes of 5-iodouracil (5IU) with Mn(II), Co@), Cu(II), Zn(II), and Cd@) ions have been prepared, characterized, and subjected to a screening system for evaluation of antitumor activity against Sarcoma-180 (S-180) and L 929 tumor cells. The complexes were characterized by their elemental analysis, infrared spectra, electronic spectra, magnetic measurements, and powder x-ray diffraction. The antitumor activity results indicate that some complexes have good antitumor activity both in vivo and in vitro against S-180 and L 929 tumor cells.
INTRODUCTION 5Iodouracil (5IU) has lethal and mutagenic effects on bacteriophage T4 [ 11. Apparently the lethal effect of 5IU is a consequence of its incorporation into DNA. It has a lethal effect on the adult Drosophila melanogaster also, by inhibiting egg-laying [2]. Very few studies on 5IU complexes with transition metal ions have been reported in the literature. The stability constant of 51U with Cu(II), Hg(II), and Be(I1) have been studied by several workers [3, 41. Villa et al. reported its complex with Cu(II) ion [5]. Such scanty information regarding its complexation has encouraged us to undertake a systematic study of its complexes with various transition metal ions and to test the antitumor activity of synthesized compounds. In this paper we describe the synthesis, characterization, and antitumor activity of 51U complexes with Mn(II), Co(II), Cu(II), Zn(II), and Cd(I1) ions. EXPERIMENTAL All the chemicals used were of reagent grade. Metal nitrates were from E. Merck and 5-iodouracil was from Aldrich. 3H-thymidine was from Bhabha Atomic Research Center, Bombay.
Address reprint requests and correspondence to: Dr. Udai P. Singh, Department of Science, Banaras Hindu University, Varanasi-22100.5, India.
of Chemistry,
Faculty
Journal of Inorganic Biochemistry, 44, 211-282 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
271 0162-0134/91/$3.50
278
U. P. Singh et al.
Synthetic
Procedures I m mol of the hydrated metal nitrates were dissolved in a mixture of 15 ml triethyl orthoformate plus 35 ml ethanol and refluxed for 8 hr. Then, I m mol 5-iodouracil was added and the resultant mixture was refluxed for 30-40 days (depending on the metal ions), after which the volume of the reaction mixture was reduced to about one-third of its original volume by heating. The new complexes were separated by filtration, washed several times with ethanol and ether.. then finall>- with ether. and dried at 50 to 60°C. The metal ions were determined by volumetry after dissolving the complexes in dilute nitric acid [6]. The analyses of carbon, hydrogen, and nitrogen were carried out with a Perkin-Elmer model 240C elemental analyzer. Infrared spectra were 783 recorded in nujol mull between 4000 and 200 cm ! with ;I Perkin-Elmer spectrophotometer. Solid-state UV/VIS spectra were obtained with a C.iV VIS- 16XA Shimadzu spectrophotometer. The room temperature (299.5 E;) magnetic susceptibiiity measurements were performed using a Faraday magnetic susceptibility balance. X-ray powder data were obtained on a Philips PW 17 i0 diffractometer using Cu-K ,j radiation.
Antitumor
Activity
Evaluation
C,H/He mice of either sex. 8- 10 weeks old (average weight, 20-22 gm) were used in this study. Sarcoma-180 (ascites) is being maintained in our laboratory by serial transplantation in C,H/He mice by intraperitoneal (ip) injection of :! x 10" ascites tumor cells at weekly intervals. Six mice were used per test compound. The compound suspensions were fresh11 prepared in 0.89% NaCl and were injected only once ip at the doses of 12.5 and 25.0 mg/kg body weight 48 hr after tumor transplantation. The same volume of sterile saline (without any test compound) was injected in control mice. Therapeutic effectiveness of the various compounds against tumor bearing mice was assessed from their T./C percentage which was calculated as follows Mean life span of treated mica” T/C s = -.-____--_--.____... X 100 Mean life span of untreated mice (* excluding tumor free survivors). A T/C value of 115 indicates significant activity, whereas a value of > 125 indicates that the complex is worthy of testing in other tumor Fystems 17. 8). Effect of Compounds
on DNA Replication
in vitro
The tumor cell (S-180/1_. 929) suspension was prepared in complete medium (RPM1 medium supplemented with antibiotics, penicillin, streptomycin. and 10% heat inactivated fetal calf serumj at a concentration of 10h cellQm1. as described earlier [9]. To determine the effect of various compounds, 2 x 10’ cells; well were added in a 96 well plate (NUNC. Denmark) in duplicate, the test compounds were added at different doses (5, 10, and 20 pg/ml) and incubated for 23 hr at 37°C in ;I Co, incubator. In control sets, cells were incubated with medium alone (without any compound). After 24 hr of incubation, the tumor cells were washed three times with balanced salt solution and the cell pellets were resuspended in 0.2 ml complete medium containing 1 pCi/ml ‘H-thymidine and pulse-labeled for 1 hr. The cells
5IU AND ANTITUMOR
ACTIVITY
279
were then washed three times with balanced salt solution. The cells were lysed in 0.5% SDS and the lysate was counted for radioactivity in a LKB P-liquid scintillation counter. The percentage inhibition of 3-thymidine incorporation in DNA was calculated as follows CPM in treated tumor cells % Inhibition
RESULTS
= 1-
CPM in untreated
tumor cells
x loo.
AND DISCUSSION
The new metal complexes involve 1:2 metal-to-ligand ratio. These compounds contain two molecules of coordinated water per metal ions. All the complexes are colored except Zinc(I1) and Cadmium(I1) (Table 1). These complexes have melting points in the 230-270°C range. Infrared
Spectra Some important IR frequencies of 5IU [5] and its complexes are given in Table 2. There are several absorption bands in the region of 3000-3500 cm-r. The vO-H(,,,,, bands at 3250-3500 cm-’ suggest the coordination of water [lo]. The presence of VM-O@quo)bands in the lower region of the infrared spectra and absence of HOH bending bands due to lattice water in the 1630- 1600 cm- ’ region confirm binding of water [ 111. The vNH band of free 51U (3 145 cm- ’ ) is also seen to exhibit shifting in all these complexes. Regarding the NH deformation modes, the 6N,-H band of 51U at 1430 cm-’ does not exhibit a substantial shift in any case whereas the 6N,-H band (1512 cm- ‘) exhibits significant shifts toward lower wave numbers upon formation of the new complexes. These results indicate that coordination occurs at the N, nitrogen of 5IU. The vC, = 0 band remains unchanged in intensity and is only slightly shifted in position whereas the vC, = 0 band shows sizeable shifts toward lower frequency, indicating the participation of the C, = 0 group in coordination. The relative insensitivity of the vC, = 0, 6N,-H, vC-I bands to metal complex formation demonstrates that none of these potential ligand sites (O,,, N(3), I) are coordinated. In the lower frequency IR region, bands attributable to M-O stretching modes were assigned on the basis of previous studies [12, 131 and favor a coordination number of six for the metal ions in these complexes. The tentative
TABLE 1. Analytical Data and Physical Properties
of the Complexes
Amount Found (Calc.) (%) Complexes Mn(SIU),.2H,O Co(5IU),.2H,O
M
C
H
N
(“C)
Light brown Violet
(9.68) 9.62 (10.31) 10.34 (11.03) 11.05 (11.32) 11.35 (18.00) 18.04
(16.93) 16.90 (16.81) 16.86 (16.68) 16.69 (16.62) 16.66 (15.37) 15.33
(1.76) 1.71 (1.75) 1.73 (1.73) 1.75 (1.75) 1.76
(9.87) 9.83 (9.80) 9.75 (9.73) 9.78 (9.69) 9.65 (8.96) 8.99
231
Zn(SIU),.2H,O
Bluish green White
Cd(5IU),.2H,O
White
Cu(XU),.2H,O
Melting Point
Color
(1.60) 1.64
263 257 255 252
280
CT.P. Singh et al.
TABLE 2. Some Characteristic Band Assignments
IR Bands for S-Iodouracil 5IU
VO-H 3 145w 1710s 1657s 1512m 1430m ._
vN-H YC, = 0 vC, = 0, PC = C in phase 6N,-H 6N,-H “M-%,uo, uM-N
_.
vM-0
and its Complexes
Mn(I1)
Co(II)
Cu(II)
Zn(I1)
Cd@)
3450W
3120~ 169Om 165% 149sm 1430s 375s 263m
347ow 3120~ 1685m 1652s 1498m 143Om 41sm ?Shm
346Ow 312OW 1690s 165?\ 1482m 143Om 375m ‘6.5\,
346Ow 313ovI 1693s 16.53s 1490m 143ow 3.5sm 272m
243m
240m
3420~ 3 12ow 1687s 1650s 1480s 14305 46Om 29Om 280~ 23%
228m
242m
PM-N band assignments [lo, 12) also support the coordination number suggested by the location of vM-0 bands. On the basis of the above infrared spectral studies, it may be suggested that the ligand 51U behaves as bidentate, binding through its C z = 0 and N ,-H groups.
Electronic
Spectra
and Magnetic
Properties
The magnetic moments of the Mn(I1) and Cu(I1) complexes, 5.90 B.M. and 2.20 B.M., show the presence of five and one unpaired electrons. respectively. Their electronic spectra show bands at 300 nm, 399 nm, and 537 nm for Mn(II)-SIU and at 630 nm for Cu(II)-SIU suggest octahedral geometry for Mn(I1) complex [14] and distorted octahedral geometry for Cu(I1) compound [ 151. The magnetic moment value of the Co(I1) complex is 5.14 B.M. and its electronic spectrum exhibits a d-d transition band at 520 nm and 820 nm and the band due to ‘LT!,(F) -+J’Tzg is absent. It favors octahedral structure for Co(II)-SIU [ 151. X-Ray
Diffraction
studies
X-Ray diffraction data (not shown) of the complexes were indexed according to the method of, Ito [16]. The ifdexing pattern yields the lat$ce constan? a = 14.08 A, b, = 3.78 A, and c = 3.54 A for I$n(II)-SIU; aO= 14.06 A. b = 7.$6 A. and c = 4.68 A for CoQI)-SIU; a ~~13.79 A. b = 60.83 A, and c =L-4.64 A f$r Cu(II)-5II-J; a = 14.08 A, b = 4.66 A, and c = 4.58 A for Zn(II)-5IU: a -= 14.15 A, h = 6.03 A. and c = 4.69 i for Cd(II)-SIU indicating orthorhombic symmetry for all these complexes. On the basis of analytical data, magnetic data and other spectral studies, the following structure may be proposed for the complexes:
M = MnlII),
Antitumor
CdII),
Cu(II),
Zn(II),
CdlII)
Properties The ligand 51U has significant antitumor activity against Sarcoma-180 tumor with T/C value 487.5 at a dose of 25.0 mg/kg body weight. Among the synthesized
51U AND ANTITUMOR
ACTIVITY
281
TABLE 3. Screening Data for Antitumor Activity Against Sarcoma- 180 in vivo Dosage ip Injection Compound
Mean Life Span of Nonsurvivors T/C=
mglkg Body Weight’
No. of Mice Surviving > 6 Monthsb
T/C %
5IU
12.5 25.0
718 3918
-
87.5 487.5
Mn(SIU),.2H,O
12.5 25.0 12.5 25.0 12.5 25.0 12.5 25.0 12.5 25.0
1218 10/8 1418 58/8 8/8 918 918 10/8 8/8 718
3(50) -
150.0 125.0 175.0 725.0 100.0 112.5 112.5 125.0 100.0 87.5
Co(SIU),.2H,O Cu(SIU),.2H,O Zn(SIU), .2H s0 Cd(5IU),.ZH,O
In each set of experiments six mice were used; T/C = 6/6, T = tumored, C = control. “, In calculating the average survival time, mice surviving > 6 months were not included. Number in parenthesis indicates percentage of mice surviving six months or more. ’ A single injection of the reported dose was given.
complexes only Mn(II)-5IU and Co(R)-5IU complexes display significant antitumor activity at the reported doses as shown by their T/C values (Table 3). Zn(LI)-5IU was also found to be therapeutically effective when administered at a dose of 25.0 mg/kg body weight. With Co(R)-5IU complex treatment, 50% of the mice survived beyond six months at a dose of 25.0 mg/kg body weight. Further, it was found that none of the test compounds except Cd(I1) showed toxicity up to a dose of 50.0 mg/kg body weight. The Cd(E) complex was found to
TABLE 4. Percentage Inhibition of 3H-Thymidine Tumor Cells in vitro
Incorporation
in L 929/Sarcoma-180
Dose Compounds
SIU Mn(SIU),.2H,O Co(SIU),.2H,O Cu(SIU),.2H,O Zn(51U),.2H20 Cd(SIU),.2H,O
5IU Mn(SIU),.2H,O Co(SIU),.2H,O Cu(SIU),.2H,O Zn(SIU),.2H,O Cd(SIU),.2H,O
Gg/ml
10 pg/ml
20pgw
19.04 14.99 55.43 -
L 929 28.20 28.03 50.73 -
38.65 6.07 43.11 -
43.44 -
48.46 -
26.58 -
7.54 46.90 50.08 -
Sarcoma- 180 18.35 37.54 40.20 46.90 -
25.80 -
282
U. P. Singh et al.
be toxic even at a dose of 2.5 mg/kg body weight. The details of the toxic effect of this compound remain under investigation. Ligand 51U and its transition metal complexes were also tested for their inhibitory effect on 3H-thymidine incorporation in S-180 and L 929 tumor cells in vitro. Most of the compounds that caused inhibition of “H-thymidine incorporation in S-180 and L 929 tumor cells also showed antitumor activity in vivo (Table 4). The maximum inhibition was produced by Co(II)-SIU and Zn(II)-SIIJ in both S-l 80 and I_. 929 tumor cells; the percentage of inhibition decreases as the dose increases. The mechanism of antitumor action of these compounds is not known. it is probable from the results of this study that these agents may be effective antitumor compounds due to their inhibitory action on the replication of DNA in tumor. c&is. The authors are thankful to C. S. I. R. and li. G. C., New Delhi ,for ,finuncial assistance.
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Received March 25, 1.991: accepted May 30, 1991
