Platinum Complexes of p- and m-Aminobenzamidine . Synthesis, Characterization, and Cytotoxic Activity C. Navarro-Ranninger, P . Amo Ochoa, J . M . Perez, J . H . Rodriguez, J. R . Masaguer, and C . Alonso CNR, PAO, JHR, JRM . Departamento de Quimica, Universidad Autonoma de Madrid, Madrid, Spain .-JMP, CA . Centro de Biologia Molecular, Universidad Autonoma de Madrid, Madrid, Spain
ABSTRACT Four platinum(II) aminobenzamidine complexes have been prepared and characterized by IR and 'H and 13 C NMR spectroscopy, and tested for their ability to interact with the nicked and closed circular forms of the pUC8 plasmid DNA . The results show that the complexes of formula [Pt(LH)2C1 2 ]2X have a cis- geometry with an amino-Pt bonding, where L is either p- or m-aminobenzamidine and where 2X is 2C1 - or PtCl 42- . It was observed that these complexes significantly alter the electrophoretic mobility of nicked and closed circular forms of DNA and that the alteration in electrophoretic mobility due to Pt(II)-p-aminobenzamidine binding is higher than that due to Pt(II)-m-aminobenzamidine . No difference in mobility was observed whether the DNA interacted with complexes having as counteranion Cl - or PtCl 42- . The synthesized compounds were, in addition, assayed for antitumor activity in vitro against colon (CX-1), lung (LX-1), and mammary (MX-1) human tumor cells . The results show that these complexes inhibited the multiplication of the tumor cells and that they show higher specificity for lung cells .
INTRODUCTION The widespread success of cis-Pt(NH 3 )2 C1 2 , cis-DDP, in clinical treatment of testicular and ovarian cancers has stimulated research in the area of metal-bases anticancer drugs [1, 2, 31 and spurred the search for compounds with improved therapeutic properties . One class of active platinum complexes that has emerged
Address reprint request to : Dr. C . Navarro-Ranninger, Departamento de QuImica (C-VIII), Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spain and Dr . Carlos Alonso, Centro de Biologia Molecular, Facultad de Ciencias C-X, Universidad Autonoma de Madrid, 28049 Madrid, Spain . Journal of Inorganic Biochemistry, 48, 163-171 (1992) © 1992 Elsevier Science Publishing Co ., Inc., 655 Avenue of the Americas, NY, NY 10010
163 0162-0134/92/$5 .00
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from these efforts are platinum complexes with ligands that contain amidine groups [4, 5, 6] . Since it is believed that the antitumor mode of action of the cis-Pt(II) complexes involves the formation of platinum-DNA adducts which introduce profound changes in the structure of the double helix [7, 8] and it is known, also, that the nature of the amino group in complexes of cis-PtA,Cl, has a primary influence on the antitumor activity of the products, we have synthesized Pt(II) complexes having as ligands p- and m-aminobenzamidine residues . The rationale of this approach is also based in the fact that the p- and m-aminobenzamidine compounds have important biological activities [9, 10] such as being inhibitors of proteolitic enzymes, have the ability to stimulate cell growth, enhance cellular migration, and modify cellular and extracellular protein components that could be responsible for some of the phenotypic features of malignant cells . In the present paper we show that in the cis-Pt(II) compounds we have synthesized, the amidino group of each one of the ligands remains protonated while the amino group is coordinated to the Pt(II) atom forming a square-planar complex . We observed, moreover, that the complexes induce important conformational changes in the OC (open circular) and CCC (covalently closed circular) forms of the pUC8 plasmid DNA . The DNA conformational changes due to the interaction of the DNA with Pt(II) :p-aminobenzamidine complexes is more profound than those due to the interaction with complexes of Pt(II) :m-aminobenzamidine. We observed, on the other hand, that whether the counteranion in every complex was Cl - or PtC1 42- they had no influence in the capacity of each compound for DNA interaction . Analysis in vitro of the cytotoxic activity of these compounds against different kinds of carcinoma cells indicate that the cis-Pt(II) :p- and m-aminobenzamidine complexes inhibit tumoral cell growth and that they have similar cytotoxic activity .
EXPERIMENTAL Materials p-Aminobenzamidine and m-aminobenzamidine dihydrochloride compounds were purchased from Aldrich Chemical Co . The K 2 PtC1 4 was from Johnson Mathey Co . The ligands and salt were used as received from the suppliers . Analytical Methods IR spectra were recorded on a Nicolet model 5DX in KBr pellets and Nujol in 4000-200 cm -' . ' H and "C NMR spectra were recorded on a Bruker WP-200-SY spectrometer in DMSO-d 6 solutions at 200 .1 and 50.3 MHz, respectively . The residual undeuterated DMSO signal was used as reference for chemical shifts (ppm) for proton (2 .49 ppm) and carbon (39 .5 ppm) spectra . The elemental analyses (C, H, and N) of the complexes was performed on a Perkin-Elmer analyzer, model 2400 CNH . Analysis of Drug-DNA Interactions PUC8 plasmid DNA was isolated from the JM83 strain of E. Coli according to the modified alkaline lysis method of Birboin et al . [11].
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The ligands were dissolved in water whereas the rest of the compounds were dissolved in dimethyl sulfoxide (DMSO) . DNA aliquots (100 µm/ml) were incubated with the drugs in a buffer solution containing 50 mM NaCl, lOmM TrisHCl, pH 7 .4, and EDTA 0 .1 mM, at different input Pt/nucleotido molar ratios (ri). Incubations . were carried out in the dark at 37°C for 24 hr . 2Oµ1 aliquots of drug:DNA complexes containing 1µg of DNA were subjected to 1 .5% agarose gel electrophoresis for 16 hr at 1 V/cm in 40 mM Tris acetate buffer (pH 8) containing EDTA 2 mM . The gels were stained with ethidium bromide and photographed with an MP-4 Polaroid camera on 665 Polaroid film using an orange filter. Cytotoxicity Tests Colon (CX-1), lung (LX-1), and mammary (MX-1) human cancer cell lines were incubated with various concentrations of the p- and m-aminobenzamidine Pt(II) complexes as indicated in the results section to determine the ID 50 for each drug . All experiments were performed in triplicate . Control cells were incubated in medium without the drugs . Drug activity was determinated by a crystal violet colorimetric assay as described by Flick and Gifford [12]. After incubation with the drugs the proteins from the surviving adherent cells were stained with dye . The test cells were plated in the wells of 96-well flat bottom microtiter plates at a density of 2-3 10 3 cells/well and incubated for two days under standard culture conditions (37°C, 5% CO 2 ) in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% nonessential amino acids . When the cells had adhered to the wells and ressumed exponential growth they were incubated with the drugs . After an incubation period of 72 hr the culture medium was removed by flicking and 50 µl of a crystal violet staining solution were added to each well . After a staining period of 20 min the staining solution was removed and the plates were washed vigorously with water until all unbound dye was removed . The insoluble dye crystals were dissolved by the addition to each well of 100 µl of 50% ethanol and 0 .1% acetic acid . The absorbance at 540 nm was determinated in an Elisa microtiter plate reader (Titertec Multiscan, Plow Lab, Meckenhe) . The ID 50 for each drug was calculated from the dose-response curves . SYNTHESIS OF THE COMPLEXES A. Platinum p-Aminobenzamidine Complexes [Pt(p-ambH) 2 C1 2 ]PtC1 4 (1) . Equivalent amounts (0 .5 mmols) of K 2 PtC1 4 and p-aminobenzamidine .2HC1 were stirred in aqueous solution at room termperature for 6 hr . Then the insoluble yellow product was filtered off and washed with HC1 (1 :1), H 2 O, methanol and ether, and dried in vacuo . Yield was 87% . (Found: C, 19 .01 ; H, 2.36 ; N, 9 .22 . Calc . : C, 19 .21 ; H, 2 .30 ; N, 9.59 .) [Pt(p-ambH)2 C1 2 ]Cl 2 (2). To a solution of 0.5 mmols of p-aminobenzamidine.2HC1 in 3 ml of water two equivalents of NaHCO 3 were added . After stirring for 2 hr, 0 .5 mmols of K 2 PtC1 4 dissolved in 2 ml of water were added to the solution. Six hr later the brown product obtained was filtered off and washed with H 2O, methanol and ether, and dried in vacuo . Yield was 65% . (Found: C, 27 .47; H, 3 .33 ; N, 13.58 . Calc. : C, 27 .60; H, 3 .31 ; N, 13 .79 .)
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B . Platinum m-Aminobenzamidine Complexes [Pt(m-ambH) 2 CI 2 ]PtCI 4 (3). Equivalent amounts (0.5 mmols) of K 2 PtCl 4 and m-aminobenzamidine .2HCI were stirred in aqueous solution at room temperature for 6 hr . The insoluble yellow product obtained was filtered off and washed with HCI (1 :1), H 2 O, methanol and ether, and dried in vacuo. Yield was 78% . (Found : C, 19 .11 ; H, 2 .24; N, 9.53 . Calc . : C, 19 .21 ; H, 2 .30; N, 9.59 .) [Pt(m-ambH) 2 C1 2 ]C1 2 (4). To a solution of 0 .5 mmols of m-aminobenzamidine .2HCI in 1 ml of water, two equivalents of NaHCO 3 were added . After stirring for 3 hr, 0.5 mmols of K 2 PtCl 4 dissolved in 2 ml of water were added to the solution . After stirring for 3 hr the pink product was filtered off and washed with water, methanol and ether, and dried in vacuo . Yield was 70% . (Found : C, 27.78, H, 3 .02; N, 13 .60. Calc . : C, 27 .60; H, 3 .31 ; N, 13 .79 .) RESULTS AND DISCUSSION The reaction of K 2 PtC1 4 with the p- and m-aminobenzamidine dihydrocloride compounds in aqueous solution and at different pH resulted in the isolation in high yield of four complexes with formula (Pt(LH),C1 2 )2X, where L is the ligand and 2X is 20 - (in complexes 2 and 4) and PtCl 42- (in complexes 1 and 3) . IR and NMR spectroscopy was used to determine the structure of the synthesized compounds since the p- and m-aminobenzamidine ligands present two functional amidine and amino groups with two possibilities for metal binding, either through the amidino or the amino group, and because it is known that when the metal bonding occurs through the amidine group monodentate, bidentate and binding modes of bonding are possible [13, 14] . IR Spectra In order to interpret the IR data the spectra were divided in two groups . The wave numbers of the absorption bands in the far-IR below 600 cm --t characterize the ligand metal bonding . The wave numbers from 4000 to 600 cm - ' belong to the absorption of the ligands (Table 1) . The IR spectra of compounds 1, 2, 3, and 4 indicate that the bonding of the pand m-aminobenzidine ligands to the Pt(II) atom does not take place through the amidine group since there is no reduction in intensity of bands either in the region between 1520 and 1390 cm -', corresponding to amidine II, or in the region between 1400 and 1240 cm ' corresponding to amidine III . Baker et al . [14] have shown that when the amidine group binds to metal there is considerable reduction in the number of absorption bands, compared with the parent amidine, and also a reduction in intensity of many other absorptions . In our
TABLE 1 . Stretching Frequencies of the Complexes in the Range 600-200 cm -1 Complexes [Pt(p-ambH) 2 C1 2 ][PtCl 4 ] ( 1) [Pt(p-ambH)2CI 2 ]Cl2 (2) [Pt(m-ambH) 2 C1 2 ][PtCl 4 ] (3) [Pt(m-ambH) 2C1 2 ]C1 2 (4) * w = weak, sh = shoulder, s = strong .
,/Pt-N
VPt-Cl
572wsh, 554w 582w, 549w 557w, 524w 576w
334s, 323sh, 328s 326s, 323sh 338s, 326sh, 322s 326sh, 322s
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studies the spectra of the p- and m-aminobenzidine compounds are very closely similar to the IR spectra of all the Pt(II) complexes (1, 2, 3, and 4) . The IR spectra of all the aminobenzamidine complexes show, moreover, the existence of a strong vibrational band at 1600 cm -1 , assigned to das(NCN) which has been designated as amidine I [15, 16] . This fact suggests that the Pt(II) atom does not coordinate through the amidino group of the aminobenzamidines. The presence of two Pt-Cl stretching vibrations between 330-315 cm - ' and two Pt-N stretching vibrations at 580-500 cm - ' (A 1 and B 1 in C 2V symmetry) confirms the cis-geometry of the compounds since they indicate the presence of an aminoPt(II) bonding . On the other hand, complexes 1 and 3 have another additional 2band at 338-334 cm -1 which may correspond to the absorption of the PtC1 4 ions (Eu under D4h symmetry) [17]. 13 C NMR Spectroscopy Data In order to confirm the structure given by IR an analysis of complexes 1, 2, 3, and 4 was carried out using 1 H and 13 C bidimensional NMR . The 1 H NMR chemical shift (S ppm) data for ligands and complexes are summarized in Tables 2 and 3 . By comparison with the observed signals of the protonated ligands, the two broad singlets detected in the complexes between 9 .15-8.40 ppm should be attributed to the protonated amidine residues [18, 19] (see Tables 2 and 3) . The chemical shifts of the signals corresponding to the aromatic protons in the complexes can be explained by inductive effects . By comparison .with the free ligands, the upfield shifts of amino protons detected in complexes 2, 3, and 4 may result from the shielding of the these protons by the positively charged Pt(II) atom . The upfield shifts confirm the existence of an NH 2 -* Pt coordination bond. Although the ' H NMR spectrum of complex 1 is similar to that of the parent ligand, the 13 C NMR data indicates the existence of coordination through the amino group of the p- or m-aminobenzamidines . Tables 4 and 5 show the 13 C NMR chemical shifts of complexes 1, 2, 3, and 4 . It may be observed that the formation of the complexes produces produces an
TABLE 2. 1 H NMR, Compounds 1 and 2
1
Amidino Protons Complexes p-amb.2HC1 [Pt(p-ambH) 2C1 2 ][PtCl 4 ](1) [Pt(p-ambH) 2C1 2 ]C1 2 (2) bs = broad singlet . Solvent : DMSO d 6 and 0 in ppm .
Aromatic Protons AA'BB' System
Amino Protons
H1
Hl'
H2
H3
H4
9.10bs 9.25bs 8.80bs
8 .85bs 8.85bs 8.40bs
7.70 7.70 7.60
6.85 7.35 6.65
7.25bs 7.20bs 6.25bs
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TABLE 3 . ' H NMR, Compounds 3 and 4
Aromatic Protons
Amidino Protons Complexes m-amb .2HCl [Pt(m-ambH) 2 Cl,][PtCl 4 ] (3) [Pt(m-ambH),Cl 2 ]C1 2 (4)
Amino Protons
HI
Hl'
H2
H4
H5
H6
H3
9 .64bs 9 .15bs 9 .15bs
9 .34bs 8 .83bs 8 .85bs
7.72m 6.86m 6.85m
7.64m 7.22m 7.22m
7.64m 6.86m 6 .85m
7 .72m 6.86m 6.85m
7 .28bs 5.60bs 5.60bs
bs = broad singlet ; m = multiplet . Solvent: DMSO d,, and d in ppm .
important downfield shift at the C4 carbon atom of complexes 3 and 4 and at the C5 carbon atom of complexes 1 and 2 which are bound to the amino group . Upfield shifts at the C3, C5, and C7 carbon atoms, for complexes 3 and 4, and downfield shifts at C2 and C4, for complexes 1 and 2, were detected as a consequence of their position with respect to the amino group which we think are involved in the coordination . The signal assigned to Cl is also indicative that the amidine group is not bound to the Pt(II) atom because it appears in the same range as the free ligand (166-164 ppm in both cases) [13, 14] . Thus, the IR and NMR data all together indicate that complexes 1, 2, 3, and 4 bind to the platinum atom through the amino group . Drug: DNA Interaction Since it has been described that the DNA damage caused by different platinum compounds not only alters DNA functions but that this alteration may determine their specificity as anticancer drugs, we have investigated the biochemical
TABLE 4 . 13 C NMR, Compounds I and 2
NH 2
3
2HCI
4
Assignment (ppm)
p-amb .2HCl
[Pt(p-ambH) 2 C1 2 ][PtCl 4 1(1)
[Pt(p-ambH) 2 C1 2 ]C1 2 (2)
Cl C2 C3 C4 C5
165 .0 121 .6 130.0 120.2 144.0
165 .0 114 .5 130.0 112.8 154.2
164 .5 113 .0 130.0 111 .0 154.5
Solvent: DMSO d 6 .
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TABLE 5. 13 C NMR, Compounds 3 and 4
Assignment (ppm)
m-amb.2HC1
[Pt(m-ambH) 2 C1 2 ][PtCl 4 1(3)
[Pt(m-ambH) 2 C1 2 ]C1 2 (4)
166.0 129.6 122.1 134.4 127.8 130.6 126.8
166 .2 129.5 115 .1 148 .8 112.2 129 .1 118 .9
166.7 129.0 114.9 149.0 112.3 129.0 118.8
C1 C2 C3 C4 C5 C6 C7
Solvent : DMSO d 6 .
activity of the synthesized cis-Pt(II) :p- and m-aminobenzamidine complexes with respect to DNA interaction . In this study we have compared the electrophoretic mobility of the circular and supercoiled forms of native plasmid DNA with the electrophoretic mobility of these forms of the DNA after interaction with complexes 1, 2, 3, and 4 and with cis-DDP because alteration in electrophoretic mobility of plasmid DNA is indicative of perturbation in DNA conformation . Figure 1 shows that indeed incubation of pUC8 DNA with compounds 1 and 2 [A] and 3, 4 [B] at increasing Pt/nucleotide ratios leads to the induction of drastic changes in the mobility of the CCC and OC forms . We observed that when assayed at the same Pt/nucleotide ratio, complexes 1 and 2, having as ligand p-aminobenzamidine, induce stronger alterations in DNA mobility of both OC and CCC forms than the m-aminobenzamidine complexes 3 and 4 . The electrophoretic mobility of the OC forms increases while the mobility of the CCC forms decreases . Relative to the alteration in DNA mobility due to cis-DDP binding, complexes 1 and 2 modify DNA at a significant lower r ; (0.05 for complexes 1 and 2 against 0 .25 for cis-DDP) . This result was unexpected since the p- and m-aminobenzamidine ligands did not induce any alteration in DNA mobility of the OC and CCC forms of pUC8 DNA. At the highest molar ratio (r i = 0.25) the alteration in DNA mobility due to binding of complexes 1-4 was very drastic . As has been described for the interaction of DNA with Pt(II) compounds [20, 21, 22] the alteration in mobility due to binding of complexes 1 and 4 is probably due to the formation of DNA microloops . Thus, we think that the alteration in conformation due to the binding of complexes 1, 2, 3, and 4 with the DNA is not exclusively directed by Pt(II) :DNA adducts but that a new type of adducts are formed, influenced by the covalent binding of the ligands to Pt(II) atoms. Cytotoxicity Tests In order to determine whether or not the modification of the DNA conformation observed in in vitro experiments has any influence in their biological
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UC
CCC
Pt /nucleotide
0C
CCC 0
.25 0 .25
01
Pt/nucleotide
0 15
0 .25 005 0-05 0 25 FIGURE 1 . Changes in the electrophoretic mobility of the CCC and OC forms of pUC8 plasmid DNA after incubation with compounds 1, 2, 3, 4 and controls . (A) Control (lane 1), compounds : 1 (lanes 2, 3, 4), 2 (lanes 8, 9, 10), p-amb .2HCI (lane 5), and cis-DDP (lane 6) . (B) Control (lane 1), compounds : 3 (lanes 2, 3, 4), 4 (lanes 8, 9, 10) m-amb .2HCI (lane 5), and cis-DDP (lane 6) .
activity, complexes 1, 2, 3, and 4 were assayed against colon, lung, and mammary human tumor cells to calculate the ID 50 (Table 6) . The results show that all the cis-Pt(II):p- and m-aminobenzamidine complexes inhibit the growth of these cells within the same range of concentration and that they have similar cytotoxic activity. However, the inhibitory effect against lung cells (4 .7-8 .7 x 10-6 M) is
TABLE 6. ID S0 , Values Obtained for Pt(II)-Aminobenzamidine Complexes Against the Tumor Colon (CX-1), Lung (LX-1), and Mammary (MX-1) Human Cells In Vitro, Respectively
Complexes
Colon ID50 (M)
[Pt(p-ambH) 2 C1 2 1[PtC1 4 1(1) [Pt(p-ambH) 2 C1 2 1 Cl 2 (2) [Pt(m-ambH)2 C1 2 l[PtC1 4 ](3) [Pt(m-ambH)2C1 2l C1 2 (4)
2.5 2 .5 2.6 2.6
10 -5 10 -5 10 -5 10 -5
Lung ID50 (M) 4 .710 -6 -6 8.0 10 5.3 10 -6 8.7 10 -6
Mammary ID50 (M) 3 .2 4.2 2.1 3.4
10 -5 10 -5 10 -5 10 -5
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-5 about 10-fold higher than against the other two types of cells (2 .1-4.2 10 M) . It seems, however, that the cytotoxicity against lung cells of the complexes carrying PtC1 4 2- as counteranion is higher than that of the complexes carrying Cl - . In fact, the ID50 against lung cells of the p- and m-aminobenzamidine complexes having as counteranion PtC1 4 2- is of similar order of magnitude than that of cis-DDP [23] . Since complexes 1, 2, 3, and 4 alter the conformation of DNA more than cis-DDP it is likely that the low cytotoxicity shown by these complexes, relative to the cytotoxicity of cis-DDP [23], may be explained by differential transport mechanisms of the molecules inside the cells . We thank Johnson Matthey Chem, Ltd . for their generous loan of K 2 PtC14 and Dr. M. Fernandez Brana (Knoll-Made Laboratories, Madrid, Spain) for his help in obtaining the cytotoxicity data . We also acknowledge the technical assistance of M . A. Fuertes. The work was supported by CICYT grants No . FAR 516 / 90 and PB88-0011 and Fundacion Ramon Areces.
REFERENCES 1 . B. F. Cain, G . J. Atwell, and R. N. Seelye, J. Med. Chem. 12,199 (1969). 2. C. F. J . Barnard, M . J. Cleare, and P . C. Hydes, Chem. Brit . 22, 1001 (1986) . 3 . C . Kopf, Chem. Rev . 87, 1137 (1987) . 4. B. F. Cain, B . C . Baguley, and W . A . Denny, J. Med. Chem . 21, 658 (1978). 5. J. M . Perez, M. C. Navarro-Ranninger, M . C. Lopez, D . Craciunescu, E . Parrondo, J. M. Requena, A. J . Ruiz, and C. Alonso, Chem . Biol. Interactions 77, 341 (1991) . 6. B. G. Cain, G . J. Atwell, and R. N . Seelye, J. Med. Chem .14, 311 (1971). 7. A. L. Pinto and S . J . Lippard, Biochim . Biophys . Acta 167, 780 (1985) . 8. A. Marcelis and J . Reedijk, J. Rev. Trav . Chem . Pay Bas ., 102, (1983). 9. R. H. Goldfarb and J. P. Quigley, Biochemistry 19, 5463 (1980). 10. H . B. David, Immunochemistry 8, 539 (1971). 11 . H. C. Birnboin and J . Doly, J. Nucl. Acids. Res. 7, 1513 (1969) . 12. H. Flick and G . E. Gifford, J. Immunol . Meth . 65, 167 (1984) . 13. J. Barker, N . Cameron, M. Kilner, M . M . Mahoud, and S . C. Wallwork, J. Chem. Soc. Dalton . Trans ., 1359 (1986) . 14. J . Barker, M . Kilner, and R . 0 . Gould, J. Chem . Soc . Dalton . Trans ., 2687 (1987). 15 . C. D. Prevorseck, J. Phys. Chem . 66, 769 (1962). 16. J . W . Lown, J . Balzarini, R . A. Newman, K . Krowicki, and E . D. Clercq, J. Med. Chem . 32, 2368 (1989) . 17. C. Engelter, A. T. Hutton, and D . A. Thornton, J. Mol. Struct. 44, 23 (1978). 18. F. Bebart, C . Perigaud, G. Gosselin, D. Mrani, B. Rayner, P. L. Ber, C. Auclair, J . Balzarini, E. D . Clercq, C. Paooletti, and J. L. Imbach, J. Med. Chem. 32, 1074 (1989) . 19. S. Mong, C . H. Huang, and S . T. Crooke, Cancer . Res. 40, 3318 (1980). 20. T. S. Hermans, B. A. Teicher, V. Chan, and L. S. Collins, Cancer Res . 48, 2335 (1988) . 21 . S. Mong, C . H. Huang, and S . T. Crooke, Cancer Res . 40, 3313 (1980) . 22. G. L. Cohen, W . R. Bauer, and S . J. Lippard, Science 203, 1014 (1979). 23. N. Farrell, J . Med. Chem. 32, 2240 (1989) . Received February 10, accepted April 8, 1992
ABSTRACT Four platinum(II) aminobenzamidine complexes have been prepared and characterized by IR and 'H and 13 C NMR spectroscopy, and tested for their ability to interact with the nicked and closed circular forms of the pUC8 plasmid DNA . The results show that the complexes of formula [Pt(LH)2C1 2 ]2X have a cis- geometry with an amino-Pt bonding, where L is either p- or m-aminobenzamidine and where 2X is 2C1 - or PtCl 42- . It was observed that these complexes significantly alter the electrophoretic mobility of nicked and closed circular forms of DNA and that the alteration in electrophoretic mobility due to Pt(II)-p-aminobenzamidine binding is higher than that due to Pt(II)-m-aminobenzamidine . No difference in mobility was observed whether the DNA interacted with complexes having as counteranion Cl - or PtCl 42- . The synthesized compounds were, in addition, assayed for antitumor activity in vitro against colon (CX-1), lung (LX-1), and mammary (MX-1) human tumor cells . The results show that these complexes inhibited the multiplication of the tumor cells and that they show higher specificity for lung cells .
INTRODUCTION The widespread success of cis-Pt(NH 3 )2 C1 2 , cis-DDP, in clinical treatment of testicular and ovarian cancers has stimulated research in the area of metal-bases anticancer drugs [1, 2, 31 and spurred the search for compounds with improved therapeutic properties . One class of active platinum complexes that has emerged
Address reprint request to : Dr. C . Navarro-Ranninger, Departamento de QuImica (C-VIII), Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spain and Dr . Carlos Alonso, Centro de Biologia Molecular, Facultad de Ciencias C-X, Universidad Autonoma de Madrid, 28049 Madrid, Spain . Journal of Inorganic Biochemistry, 48, 163-171 (1992) © 1992 Elsevier Science Publishing Co ., Inc., 655 Avenue of the Americas, NY, NY 10010
163 0162-0134/92/$5 .00
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from these efforts are platinum complexes with ligands that contain amidine groups [4, 5, 6] . Since it is believed that the antitumor mode of action of the cis-Pt(II) complexes involves the formation of platinum-DNA adducts which introduce profound changes in the structure of the double helix [7, 8] and it is known, also, that the nature of the amino group in complexes of cis-PtA,Cl, has a primary influence on the antitumor activity of the products, we have synthesized Pt(II) complexes having as ligands p- and m-aminobenzamidine residues . The rationale of this approach is also based in the fact that the p- and m-aminobenzamidine compounds have important biological activities [9, 10] such as being inhibitors of proteolitic enzymes, have the ability to stimulate cell growth, enhance cellular migration, and modify cellular and extracellular protein components that could be responsible for some of the phenotypic features of malignant cells . In the present paper we show that in the cis-Pt(II) compounds we have synthesized, the amidino group of each one of the ligands remains protonated while the amino group is coordinated to the Pt(II) atom forming a square-planar complex . We observed, moreover, that the complexes induce important conformational changes in the OC (open circular) and CCC (covalently closed circular) forms of the pUC8 plasmid DNA . The DNA conformational changes due to the interaction of the DNA with Pt(II) :p-aminobenzamidine complexes is more profound than those due to the interaction with complexes of Pt(II) :m-aminobenzamidine. We observed, on the other hand, that whether the counteranion in every complex was Cl - or PtC1 42- they had no influence in the capacity of each compound for DNA interaction . Analysis in vitro of the cytotoxic activity of these compounds against different kinds of carcinoma cells indicate that the cis-Pt(II) :p- and m-aminobenzamidine complexes inhibit tumoral cell growth and that they have similar cytotoxic activity .
EXPERIMENTAL Materials p-Aminobenzamidine and m-aminobenzamidine dihydrochloride compounds were purchased from Aldrich Chemical Co . The K 2 PtC1 4 was from Johnson Mathey Co . The ligands and salt were used as received from the suppliers . Analytical Methods IR spectra were recorded on a Nicolet model 5DX in KBr pellets and Nujol in 4000-200 cm -' . ' H and "C NMR spectra were recorded on a Bruker WP-200-SY spectrometer in DMSO-d 6 solutions at 200 .1 and 50.3 MHz, respectively . The residual undeuterated DMSO signal was used as reference for chemical shifts (ppm) for proton (2 .49 ppm) and carbon (39 .5 ppm) spectra . The elemental analyses (C, H, and N) of the complexes was performed on a Perkin-Elmer analyzer, model 2400 CNH . Analysis of Drug-DNA Interactions PUC8 plasmid DNA was isolated from the JM83 strain of E. Coli according to the modified alkaline lysis method of Birboin et al . [11].
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The ligands were dissolved in water whereas the rest of the compounds were dissolved in dimethyl sulfoxide (DMSO) . DNA aliquots (100 µm/ml) were incubated with the drugs in a buffer solution containing 50 mM NaCl, lOmM TrisHCl, pH 7 .4, and EDTA 0 .1 mM, at different input Pt/nucleotido molar ratios (ri). Incubations . were carried out in the dark at 37°C for 24 hr . 2Oµ1 aliquots of drug:DNA complexes containing 1µg of DNA were subjected to 1 .5% agarose gel electrophoresis for 16 hr at 1 V/cm in 40 mM Tris acetate buffer (pH 8) containing EDTA 2 mM . The gels were stained with ethidium bromide and photographed with an MP-4 Polaroid camera on 665 Polaroid film using an orange filter. Cytotoxicity Tests Colon (CX-1), lung (LX-1), and mammary (MX-1) human cancer cell lines were incubated with various concentrations of the p- and m-aminobenzamidine Pt(II) complexes as indicated in the results section to determine the ID 50 for each drug . All experiments were performed in triplicate . Control cells were incubated in medium without the drugs . Drug activity was determinated by a crystal violet colorimetric assay as described by Flick and Gifford [12]. After incubation with the drugs the proteins from the surviving adherent cells were stained with dye . The test cells were plated in the wells of 96-well flat bottom microtiter plates at a density of 2-3 10 3 cells/well and incubated for two days under standard culture conditions (37°C, 5% CO 2 ) in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% nonessential amino acids . When the cells had adhered to the wells and ressumed exponential growth they were incubated with the drugs . After an incubation period of 72 hr the culture medium was removed by flicking and 50 µl of a crystal violet staining solution were added to each well . After a staining period of 20 min the staining solution was removed and the plates were washed vigorously with water until all unbound dye was removed . The insoluble dye crystals were dissolved by the addition to each well of 100 µl of 50% ethanol and 0 .1% acetic acid . The absorbance at 540 nm was determinated in an Elisa microtiter plate reader (Titertec Multiscan, Plow Lab, Meckenhe) . The ID 50 for each drug was calculated from the dose-response curves . SYNTHESIS OF THE COMPLEXES A. Platinum p-Aminobenzamidine Complexes [Pt(p-ambH) 2 C1 2 ]PtC1 4 (1) . Equivalent amounts (0 .5 mmols) of K 2 PtC1 4 and p-aminobenzamidine .2HC1 were stirred in aqueous solution at room termperature for 6 hr . Then the insoluble yellow product was filtered off and washed with HC1 (1 :1), H 2 O, methanol and ether, and dried in vacuo . Yield was 87% . (Found: C, 19 .01 ; H, 2.36 ; N, 9 .22 . Calc . : C, 19 .21 ; H, 2 .30 ; N, 9.59 .) [Pt(p-ambH)2 C1 2 ]Cl 2 (2). To a solution of 0.5 mmols of p-aminobenzamidine.2HC1 in 3 ml of water two equivalents of NaHCO 3 were added . After stirring for 2 hr, 0 .5 mmols of K 2 PtC1 4 dissolved in 2 ml of water were added to the solution. Six hr later the brown product obtained was filtered off and washed with H 2O, methanol and ether, and dried in vacuo . Yield was 65% . (Found: C, 27 .47; H, 3 .33 ; N, 13.58 . Calc. : C, 27 .60; H, 3 .31 ; N, 13 .79 .)
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B . Platinum m-Aminobenzamidine Complexes [Pt(m-ambH) 2 CI 2 ]PtCI 4 (3). Equivalent amounts (0.5 mmols) of K 2 PtCl 4 and m-aminobenzamidine .2HCI were stirred in aqueous solution at room temperature for 6 hr . The insoluble yellow product obtained was filtered off and washed with HCI (1 :1), H 2 O, methanol and ether, and dried in vacuo. Yield was 78% . (Found : C, 19 .11 ; H, 2 .24; N, 9.53 . Calc . : C, 19 .21 ; H, 2 .30; N, 9.59 .) [Pt(m-ambH) 2 C1 2 ]C1 2 (4). To a solution of 0 .5 mmols of m-aminobenzamidine .2HCI in 1 ml of water, two equivalents of NaHCO 3 were added . After stirring for 3 hr, 0.5 mmols of K 2 PtCl 4 dissolved in 2 ml of water were added to the solution . After stirring for 3 hr the pink product was filtered off and washed with water, methanol and ether, and dried in vacuo . Yield was 70% . (Found : C, 27.78, H, 3 .02; N, 13 .60. Calc . : C, 27 .60; H, 3 .31 ; N, 13 .79 .) RESULTS AND DISCUSSION The reaction of K 2 PtC1 4 with the p- and m-aminobenzamidine dihydrocloride compounds in aqueous solution and at different pH resulted in the isolation in high yield of four complexes with formula (Pt(LH),C1 2 )2X, where L is the ligand and 2X is 20 - (in complexes 2 and 4) and PtCl 42- (in complexes 1 and 3) . IR and NMR spectroscopy was used to determine the structure of the synthesized compounds since the p- and m-aminobenzamidine ligands present two functional amidine and amino groups with two possibilities for metal binding, either through the amidino or the amino group, and because it is known that when the metal bonding occurs through the amidine group monodentate, bidentate and binding modes of bonding are possible [13, 14] . IR Spectra In order to interpret the IR data the spectra were divided in two groups . The wave numbers of the absorption bands in the far-IR below 600 cm --t characterize the ligand metal bonding . The wave numbers from 4000 to 600 cm - ' belong to the absorption of the ligands (Table 1) . The IR spectra of compounds 1, 2, 3, and 4 indicate that the bonding of the pand m-aminobenzidine ligands to the Pt(II) atom does not take place through the amidine group since there is no reduction in intensity of bands either in the region between 1520 and 1390 cm -', corresponding to amidine II, or in the region between 1400 and 1240 cm ' corresponding to amidine III . Baker et al . [14] have shown that when the amidine group binds to metal there is considerable reduction in the number of absorption bands, compared with the parent amidine, and also a reduction in intensity of many other absorptions . In our
TABLE 1 . Stretching Frequencies of the Complexes in the Range 600-200 cm -1 Complexes [Pt(p-ambH) 2 C1 2 ][PtCl 4 ] ( 1) [Pt(p-ambH)2CI 2 ]Cl2 (2) [Pt(m-ambH) 2 C1 2 ][PtCl 4 ] (3) [Pt(m-ambH) 2C1 2 ]C1 2 (4) * w = weak, sh = shoulder, s = strong .
,/Pt-N
VPt-Cl
572wsh, 554w 582w, 549w 557w, 524w 576w
334s, 323sh, 328s 326s, 323sh 338s, 326sh, 322s 326sh, 322s
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studies the spectra of the p- and m-aminobenzidine compounds are very closely similar to the IR spectra of all the Pt(II) complexes (1, 2, 3, and 4) . The IR spectra of all the aminobenzamidine complexes show, moreover, the existence of a strong vibrational band at 1600 cm -1 , assigned to das(NCN) which has been designated as amidine I [15, 16] . This fact suggests that the Pt(II) atom does not coordinate through the amidino group of the aminobenzamidines. The presence of two Pt-Cl stretching vibrations between 330-315 cm - ' and two Pt-N stretching vibrations at 580-500 cm - ' (A 1 and B 1 in C 2V symmetry) confirms the cis-geometry of the compounds since they indicate the presence of an aminoPt(II) bonding . On the other hand, complexes 1 and 3 have another additional 2band at 338-334 cm -1 which may correspond to the absorption of the PtC1 4 ions (Eu under D4h symmetry) [17]. 13 C NMR Spectroscopy Data In order to confirm the structure given by IR an analysis of complexes 1, 2, 3, and 4 was carried out using 1 H and 13 C bidimensional NMR . The 1 H NMR chemical shift (S ppm) data for ligands and complexes are summarized in Tables 2 and 3 . By comparison with the observed signals of the protonated ligands, the two broad singlets detected in the complexes between 9 .15-8.40 ppm should be attributed to the protonated amidine residues [18, 19] (see Tables 2 and 3) . The chemical shifts of the signals corresponding to the aromatic protons in the complexes can be explained by inductive effects . By comparison .with the free ligands, the upfield shifts of amino protons detected in complexes 2, 3, and 4 may result from the shielding of the these protons by the positively charged Pt(II) atom . The upfield shifts confirm the existence of an NH 2 -* Pt coordination bond. Although the ' H NMR spectrum of complex 1 is similar to that of the parent ligand, the 13 C NMR data indicates the existence of coordination through the amino group of the p- or m-aminobenzamidines . Tables 4 and 5 show the 13 C NMR chemical shifts of complexes 1, 2, 3, and 4 . It may be observed that the formation of the complexes produces produces an
TABLE 2. 1 H NMR, Compounds 1 and 2
1
Amidino Protons Complexes p-amb.2HC1 [Pt(p-ambH) 2C1 2 ][PtCl 4 ](1) [Pt(p-ambH) 2C1 2 ]C1 2 (2) bs = broad singlet . Solvent : DMSO d 6 and 0 in ppm .
Aromatic Protons AA'BB' System
Amino Protons
H1
Hl'
H2
H3
H4
9.10bs 9.25bs 8.80bs
8 .85bs 8.85bs 8.40bs
7.70 7.70 7.60
6.85 7.35 6.65
7.25bs 7.20bs 6.25bs
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TABLE 3 . ' H NMR, Compounds 3 and 4
Aromatic Protons
Amidino Protons Complexes m-amb .2HCl [Pt(m-ambH) 2 Cl,][PtCl 4 ] (3) [Pt(m-ambH),Cl 2 ]C1 2 (4)
Amino Protons
HI
Hl'
H2
H4
H5
H6
H3
9 .64bs 9 .15bs 9 .15bs
9 .34bs 8 .83bs 8 .85bs
7.72m 6.86m 6.85m
7.64m 7.22m 7.22m
7.64m 6.86m 6 .85m
7 .72m 6.86m 6.85m
7 .28bs 5.60bs 5.60bs
bs = broad singlet ; m = multiplet . Solvent: DMSO d,, and d in ppm .
important downfield shift at the C4 carbon atom of complexes 3 and 4 and at the C5 carbon atom of complexes 1 and 2 which are bound to the amino group . Upfield shifts at the C3, C5, and C7 carbon atoms, for complexes 3 and 4, and downfield shifts at C2 and C4, for complexes 1 and 2, were detected as a consequence of their position with respect to the amino group which we think are involved in the coordination . The signal assigned to Cl is also indicative that the amidine group is not bound to the Pt(II) atom because it appears in the same range as the free ligand (166-164 ppm in both cases) [13, 14] . Thus, the IR and NMR data all together indicate that complexes 1, 2, 3, and 4 bind to the platinum atom through the amino group . Drug: DNA Interaction Since it has been described that the DNA damage caused by different platinum compounds not only alters DNA functions but that this alteration may determine their specificity as anticancer drugs, we have investigated the biochemical
TABLE 4 . 13 C NMR, Compounds I and 2
NH 2
3
2HCI
4
Assignment (ppm)
p-amb .2HCl
[Pt(p-ambH) 2 C1 2 ][PtCl 4 1(1)
[Pt(p-ambH) 2 C1 2 ]C1 2 (2)
Cl C2 C3 C4 C5
165 .0 121 .6 130.0 120.2 144.0
165 .0 114 .5 130.0 112.8 154.2
164 .5 113 .0 130.0 111 .0 154.5
Solvent: DMSO d 6 .
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TABLE 5. 13 C NMR, Compounds 3 and 4
Assignment (ppm)
m-amb.2HC1
[Pt(m-ambH) 2 C1 2 ][PtCl 4 1(3)
[Pt(m-ambH) 2 C1 2 ]C1 2 (4)
166.0 129.6 122.1 134.4 127.8 130.6 126.8
166 .2 129.5 115 .1 148 .8 112.2 129 .1 118 .9
166.7 129.0 114.9 149.0 112.3 129.0 118.8
C1 C2 C3 C4 C5 C6 C7
Solvent : DMSO d 6 .
activity of the synthesized cis-Pt(II) :p- and m-aminobenzamidine complexes with respect to DNA interaction . In this study we have compared the electrophoretic mobility of the circular and supercoiled forms of native plasmid DNA with the electrophoretic mobility of these forms of the DNA after interaction with complexes 1, 2, 3, and 4 and with cis-DDP because alteration in electrophoretic mobility of plasmid DNA is indicative of perturbation in DNA conformation . Figure 1 shows that indeed incubation of pUC8 DNA with compounds 1 and 2 [A] and 3, 4 [B] at increasing Pt/nucleotide ratios leads to the induction of drastic changes in the mobility of the CCC and OC forms . We observed that when assayed at the same Pt/nucleotide ratio, complexes 1 and 2, having as ligand p-aminobenzamidine, induce stronger alterations in DNA mobility of both OC and CCC forms than the m-aminobenzamidine complexes 3 and 4 . The electrophoretic mobility of the OC forms increases while the mobility of the CCC forms decreases . Relative to the alteration in DNA mobility due to cis-DDP binding, complexes 1 and 2 modify DNA at a significant lower r ; (0.05 for complexes 1 and 2 against 0 .25 for cis-DDP) . This result was unexpected since the p- and m-aminobenzamidine ligands did not induce any alteration in DNA mobility of the OC and CCC forms of pUC8 DNA. At the highest molar ratio (r i = 0.25) the alteration in DNA mobility due to binding of complexes 1-4 was very drastic . As has been described for the interaction of DNA with Pt(II) compounds [20, 21, 22] the alteration in mobility due to binding of complexes 1 and 4 is probably due to the formation of DNA microloops . Thus, we think that the alteration in conformation due to the binding of complexes 1, 2, 3, and 4 with the DNA is not exclusively directed by Pt(II) :DNA adducts but that a new type of adducts are formed, influenced by the covalent binding of the ligands to Pt(II) atoms. Cytotoxicity Tests In order to determine whether or not the modification of the DNA conformation observed in in vitro experiments has any influence in their biological
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UC
CCC
Pt /nucleotide
0C
CCC 0
.25 0 .25
01
Pt/nucleotide
0 15
0 .25 005 0-05 0 25 FIGURE 1 . Changes in the electrophoretic mobility of the CCC and OC forms of pUC8 plasmid DNA after incubation with compounds 1, 2, 3, 4 and controls . (A) Control (lane 1), compounds : 1 (lanes 2, 3, 4), 2 (lanes 8, 9, 10), p-amb .2HCI (lane 5), and cis-DDP (lane 6) . (B) Control (lane 1), compounds : 3 (lanes 2, 3, 4), 4 (lanes 8, 9, 10) m-amb .2HCI (lane 5), and cis-DDP (lane 6) .
activity, complexes 1, 2, 3, and 4 were assayed against colon, lung, and mammary human tumor cells to calculate the ID 50 (Table 6) . The results show that all the cis-Pt(II):p- and m-aminobenzamidine complexes inhibit the growth of these cells within the same range of concentration and that they have similar cytotoxic activity. However, the inhibitory effect against lung cells (4 .7-8 .7 x 10-6 M) is
TABLE 6. ID S0 , Values Obtained for Pt(II)-Aminobenzamidine Complexes Against the Tumor Colon (CX-1), Lung (LX-1), and Mammary (MX-1) Human Cells In Vitro, Respectively
Complexes
Colon ID50 (M)
[Pt(p-ambH) 2 C1 2 1[PtC1 4 1(1) [Pt(p-ambH) 2 C1 2 1 Cl 2 (2) [Pt(m-ambH)2 C1 2 l[PtC1 4 ](3) [Pt(m-ambH)2C1 2l C1 2 (4)
2.5 2 .5 2.6 2.6
10 -5 10 -5 10 -5 10 -5
Lung ID50 (M) 4 .710 -6 -6 8.0 10 5.3 10 -6 8.7 10 -6
Mammary ID50 (M) 3 .2 4.2 2.1 3.4
10 -5 10 -5 10 -5 10 -5
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-5 about 10-fold higher than against the other two types of cells (2 .1-4.2 10 M) . It seems, however, that the cytotoxicity against lung cells of the complexes carrying PtC1 4 2- as counteranion is higher than that of the complexes carrying Cl - . In fact, the ID50 against lung cells of the p- and m-aminobenzamidine complexes having as counteranion PtC1 4 2- is of similar order of magnitude than that of cis-DDP [23] . Since complexes 1, 2, 3, and 4 alter the conformation of DNA more than cis-DDP it is likely that the low cytotoxicity shown by these complexes, relative to the cytotoxicity of cis-DDP [23], may be explained by differential transport mechanisms of the molecules inside the cells . We thank Johnson Matthey Chem, Ltd . for their generous loan of K 2 PtC14 and Dr. M. Fernandez Brana (Knoll-Made Laboratories, Madrid, Spain) for his help in obtaining the cytotoxicity data . We also acknowledge the technical assistance of M . A. Fuertes. The work was supported by CICYT grants No . FAR 516 / 90 and PB88-0011 and Fundacion Ramon Areces.
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