Biological Activities of Polymers - American Chemical Society

18

Downloaded by CORNELL UNIV on October 3, 2016 | http://pubs.acs.org Publication Date: May 12, 1982 | doi: 10.1021/bk-1982-0186.ch018

Controlled Release of Anticancer Agents That Are Complexes of cis-Diamminedichloroplatinum(II) and α-Hydroxyquinones SEYMOUR YOLLES, ROSETTE M. ROAT, MARIO F. SARTORI, and CATHARINE L . WASHBURNE University of Delaware, Department of Chemistry, Newark, D E 19711

Complexes between the square planar anticancer agent cis-diamminedichloroplatinum (II), ( c i s - P t a C l ) , and α-hydroxyquinones are reported. A complex between c i s - P t a C l and the anthracycline antibiotic, doxoru bicin, contains five cis-Pta Cl units per doxorubi cin molecule. Ratios of cis-Pta Cl to α-hydroxy quinones range from 3/1 for cis-Pta Cl /juglone com plexes to 6/1 for cis-Pta Cl /quinizarin complexes. Ultraviolet and infrared data are reported for the complexes as well as thin layer chromatography, con ductivity, and molecular weight results. The re sults indicate chelation of platinum to oxygen atoms of the α-hydroxyquinones with concomitant stacking of c i s - P t a C l units. The reaction leading to dimer ization has the potential for further chain exten sion although no such compounds have as yet been isolated. In vivo experiments with c i s - P t a C l / doxorubicin and with cis-Pta Cl /quinizarin show the efficacy of these complexes as a new time-release system for delivering anticancer agents. 2

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Several papers (1, 2^ _3) from this laboratory have dealt with the development of a system for delivering anticancer agents at a constant rate over a prolonged period, perhaps as long as several months. This system consists of incorporating the drug i n a poly meric matrix, shaping the composite into convenient form (such as beads or powder) and implanting the composite into the body tissue of animals. The drugs diffuse continuously from the interior of the polymer to the outer surface, where they are mechanically swept away by body f l u i d . The mechanism of migration of the drug i s that of diffusion and the thermodynamic driving force i s the concentration gradient (3).

0097-6156/82/0186-0233$5.00/0 © 1982 American Chemical Society Carraher and Gebelein; Biological Activities of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


234

BIOLOGICAL ACTIVITIES OF POLYMERS

Previously reported (2) i n v i t r o and i n vivo experiments with composites of poly(lactic acid) (PLA) with cyclophosphamide, with cis-diamminedichloroplatinum (II) (cis-Pta2Cl2) (4), and with cis-Pta2Cl2-doxorubicin (DOXO) (5) have demonstrated the feasib i l i t y of this system, especially their drug release a b i l i t y and the reduced toxicity of the platinum compound. During the preparation of the composite containing PLA, c i s Pta2Cl2 and DOXO, a distinctive color change to intense purple was observed (2). In view of the enhanced anticancer activity shown by this composite i n animals over those containing the separate drugs, i t was decided to isolate and characterize the purple material, believed to be a chemical complex of the p l a t i num with DOXO and, i n addition, to investigate the efficacy of this complex i n i n vivo experiments. To elucidate the binding site, several a-hydroxyquinone analogues of DOXO were complexed with cis-Pta2Cl2This paper reports the syntheses and characterization of complexes of cis-Pta Cl2 with 5-hydroxy-l,4-naphthoquinone (HNQ), 5,8-dihydroxy-l,4-naphthoquinone (DHNQ), 1,4-dihydroxy-9,10anthraquinone (DHAQ) and with DOXO. In addition i t reports preliminary results of i n vivo experiments performed with complexes cis-Pta Cl2/DOXO and cis-Pta Cl /quinizarin. Several metal complexes (Be, Co, Cu, Fe, Mg, Ni) with DOXO (6, 1) and with hydroxy-naphthoquinones and -anthraquinones (8, 10) have been recently reported. To our knowledge no study of platinum complexes with a-hydroxyquinones has been published to date, with the exception of an unsuccessful attempt to prepare a Pt/5,8-dihydroxy-l,4-naphthoquinone (8) . 2

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Experimental Section General Information. A l l reactants were dried at room temperature under vacuum i n an Abderhalden apparatus to constant weight. A l l experiments were carried out i n a dry box; continuously under nitrogen. Elemental analyses were performed by Microanalysis, Wilmington, DE, a l l compounds prepared yielded satisfactory analyses (see Table I ) . The UV spectra were obtained on samples dissolved i n DMF with a NSI Hitachi computerized #100-80 Ultraviolet Spectrophotometer. The IR spectra were recorded on a Perkin Elmer 180 Recording IR Spectrophotometer i n Csl pellets (except for DOXO and DOXO complex where KBr discs were used). Conductivity measurements were taken on a Beckman Model RC16B2 conductivity bridge using a Fisher conduct i v i t y c e l l ( c e l l constant - 0.1799, determined on standard KC1 solutions). *H NMR spectra were obtained i n dyDMF at 90MHz on a Bruker HFX-10 spectrometer. Thin layer chromatographs on s i l i c a gel 60 supports from 0.1 to 1.0 mM DMF solutions of starting materials and products were developed i n 9/1 acetone/HCl, and visualized with 2% SnCl i n 1M HC1 spray. 2

Carraher and Gebelein; Biological Activities of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

YOLLES ET AL.

18.

Controlled

Release of Anticancer

235

Agents


Experiments In Vivo. The standard assay of the Ascites Sarcoma i n the ICR mice was used (2). There were six mice i n each cage, with two cages for the negative controls, two cages for the positive controls and one cage for each dose level of drug tested. Tumor c e l l injections were performed on day 0 and drug injection on day 1, both given intraperitoneally. Suspensions i n carboxymethyl cellulose (CMC-7LF, Hercules, Inc., Wilmington, DE) of complexes, as l i s t e d i n Table IV, with saline as a carrier were used. Complex I: cis-Pta2Cl2/5-hydroxy-l,4-napthoquinone. In a typical preparation, cis-Pta2Cl2 (u.MJ g, l.bb mmol; was' dissolved in 30 ml N,N-dimethylformamide (DMF) and added to HNQ (0.14 g, 0.80 mmol) dissolved i n 10 ml DMF. The reactants were combined i n a 120 ml brown glass j a r which contained 83.5 gms of 5 mm glass beads. The j a r was sealed under N2, then placed on a b a l l m i l l (1750 rpm) to rotate for 120 hrs. The b a l l milled suspension (brown-purple i n color) was centrifuged to remove a small amount of solids. The main product was precipitated by addition of 80 ml methylene chloride (MeCl2) to yield a brown-purple solid collected on a frit-glass f i l t e r . Crude product was recrystallized from 10-15 ml DMF with precipitation by excess MeCl2- Yield: 0.59 gm (90%) of brown-purple product. Table I Elemental Analyses

Complex I cis-Pta Cl /HNQ 10 22Cl4.5N O Pt3 2

c

2

2.27 2.69

15.63 15.71

8.24 8.61

57.37 55.51

a b

9.22 8.68

2.16 2.40

14.97 14.75

8.60 7.51

59.94 58.68

a b

8.38 8.68

1.90 2.48

21.20 20.10

6.98 6.52

58.33 56.76

a b

16.56 15.86

3.32 3.21

14.39 14.15

7.85 7.52

49.50 47.64

a b

2

H

cl

N

Pt

10 30 5.5 8°4 4

Complex III cis-Pta2Cl2/DHAQ H

C1

N

Pt

14 36 12 10°4 6

Complex IV cis-Pta Cl /DOXO 2

C

11.77 11.68

3

Complex II cis-Pta Cl2/DHNQ

C

Pt

N

H

6

c

H

CI

C

H

cl

2

N

Pt

27 56 8 ll°ll 5

a b

Calculated Found

Complex IT: cis-Pta2Cl2/5,8-dihydroxy-l,4-naphthoquinone. Following the procedure described above, 5,8-dihydroxy-l,4naphthoquinone (0.039 g, 0.20 mmol) and cis-Pta2Cl2 (0.373 g, 1.24 mmol) were condensed to give 0.40 g (yield 97%) of compound II as navy blue product.


236


Complex III: cis-Pta2Cl2/l,4-dihydroxy-9,10-anthraquinone. Following the procedure described above, l,4-dihydroxy-9,10anthraquinone (0.062 g, 0.26 mmol) was reacted with cis-Pta Cl2 (0.50 g, 1.66 mmol) to give 0.29 g (yield 52%) III as purple product.


2

Doxorubicin Base from Doxorubicin Hydrochloride. Doxorubicin hydrochloride was converted to the free base by bubbling gaseous ammonia through a capillary pipet into a stirred suspension of doxorubicin hydrochloride (0.050 g, 0.08 mmol) i n 15 ml of an 8:1 mixture of methylene chloride and methanol at 0°C. The bubbling of ammonia and the s t i r r i n g were continued u n t i l the mixture was red-orange. After the ammonium chloride was removed by centrifugation, excess solvent and ammonia were removed on a Rotovap. DMF was added to give a solution of 0.047 g (0.086 mmole) of doxorubicin base i n 100 ml of solvent. Complex IV: cis-Pta2Cl2/Doxorubicin. Following the procedure described above tor complex I, cis-Pta2Cl2 (0.104 g, 0.34 mmol) was condensed with doxorubicin (0.047 g, 0.086 mmol) to give 0.103 g (68.7% yield) of compound IV as purple product. Results and Discussion Complexes of cis-Pta2Cl2 with HNQ, DHNQ, DHAQ and DOXO have been obtained. A l l these complexes are similar i n physical form: fine powder (solids) of intense purple to blue color; poorly soluble i n common organic solvents, giving purple or blue solutions; none sublime on heating; a l l decompose before melting. U.V. and I.R. spectra of the parent ligands and platinum complexes are reported i n Tables II and III. a-Hydroxyquinones exhibit a so-called chelated H absorbance in the UV, caused by H bonding of the a-hydroxyl H to the keto oxygen of the quinone (11). In our complexes with platinum, this absorbance disappears, with concurrent appearance of a new absorbance at longer wavelengths characteristic of the a-hydroxyquinone dianion (11). Retention of the 302 nm band supports the presence of c i s Pta2Cl2 units i n the structures of these complexes. It i s assumed that these units are stacked i n a way similar to that of the Magnus green salt (12). Hydrolysis of a cis-Pta2Cl2/DHAQ complex in dilute HC1, followed by thin layer chromotography has i d e n t i fied 75% of the Pt containing fraction as pure cis-Pta2Cl2, the other 25% being the fractions which have lost chloride ligand. Pure quinizarin was also identified as a component of the hydrolyzate by thin layer chromotography. The IR spectra of the quinone complexes show a shift of the v(C=0) band stretching frequency to lower wavenumbers i n comparison 1


18.

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with the corresponding parent hydroxyquinones. This shift i n d i cates coordination or chelation of Pt(II) to oxygen of the hydroxyquinone as shown by Pierpont (10) for the copper (II) complexes of 1,5-dihydroxy-l,4-naphthoquinone. Table II Ultraviolet Spectra of Hydroxyquinone Ligands and Their Corresponding Pt(II) Complexes


Compounds

Absorption (log e) nm

HNQ HNQ Complex DHNQ DHNQ Complex DHAQ DHAQ Complex DOXO DOXO Complex

425(3.6), 260(4.2) 485, 380,260(4.2) 515(4.2), 482(4.2), 270(4.4) 620, 570,302, 271(4.4) 478(3.8), 278(4.0) 580, 540,302, 278 530(2.7), 495(2.9), 475(2.9), 265(3.0) 592(2.7), 535(2.8), 506(2.8), 265(3.3)

•Absorption maxima cis-Pta Cl2 370,302 nm. 2

1

Table III IR Absorption (cm" ) of hydroxyquinone ligands and their Pt(II) complexes

Cmpds

DHNQ DHNQ Cmplx DHAQ DHAQ Cmplx DOXO DOXO Cmplx

Chelate OH

3010

— —

a

3000 2910

—

Chelate OX

2910 a

b

—

b

2910

—

v(c=o)

1600 1560 1626,1590 1605,1575 1620-1635 1620-1630

C-O-Pt

Pt-0

_

„

1030-1170

445

—

— 1000-1150 —

445

—

450

1050-1150

a

Hadzi, D.; Sheppard, N., Trans. Far. S o c , 1954, 50, 911 ^reference 13 Broad, strong absorption bands at 1000-1500 cm reported (13) to be indicative of ether type linkages, are tentatively assumed to be due to C-O-Pt linkages. Absorbance at 445-450 cm" i s analogous to that reported by Nakomoto for Pt-0 bonds i n Pt(II) acetylacetonates (14). A l l complexes also exhibit IR (in cm ) absorbances reported for cis-PtaoClo by Poulet (15): V (NH3): 3280, 3200; 6 (NH.): 1620, 1290; V (Pt-Cl); 315; V (Pt-N): 240. % NMR spectra of DHAQ (6 - 12.8, chelated OH; 6 = 8.3-8.4, aromatic H; 6 - 7.5, 2,3H) and c i s - P t a C l (6 - 4.2, N-H) were straightforward, however, NMR of the complex showed retention of 1

2

2


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the N-H peak of cis-Pta2Cl2 only, plus several unidentified upf i e l d resonances. Experiments are currently underway to show that these unidentified peaks are isotropically shifted DHAQ resonanc s caused by paramagnetism i n the complex. Attempts at obtaining single crystals for X-ray structure determination have failed thus far. Low s o l u b i l i t y of the complexes i n solvents suitable for vapor pressure osmometry have made molecular weight determination d i f f i c u l t . One result for complex III, cis-Pta Cl /DHAQ, i n acetone yielded a M.W. of 2270 gm/mole, vs a calculated M.W. from elemental analyses of 2006 gm/ mole. Based on data given, a probable structure of Complex IV, c i s PtaoCWDOXO, i s shown i n Figure 1. Conductivity of the c i s - P t a CI2/DHAQ complex increased from zero, immediately after solution i n DMF, to values which varied with concentration i n the manner of a 2:1 electrolyte (as. compared to R^PtC^ by the method of Feltham and Hayter (16). Probable structure of the electrolyte differs from that i n Figure 1 i n that the two five-co-ordinate platinum centers become four-co-ordinate through loss of chloride anions, resulting i n a dipositive complex ion containing platinum co-ordinated to the hydroxyquinone portion of DOXO. Fractional chlorine content from elemental analyses calculations for HNQ and DHNQ complexes indicates that either one or two chloride ligands are lost from cis-Pta2Cl2 on complexation.


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In Vivo Experimental Results Preliminary i n vivo results (see Table IV) show the f e a s i b i l i t y of this potential time-release system for delivering anticancer agents. cis-Pta Cl9/D0X0 complex was significantly better than the positive control (7 mg cis-Pta2Cl2/kg mouse). The improvement i n l i f e span compared with the positive control i s substantial and the toxic effect of the platinum compound has certainly been decreased. These tests show that i t i s possible to use a dose of complex three times the toxic level of c i s Pta Cl2» because of slow release from cis-Pta Cl /DOXO complex. Preliminary i n vivo results (see Table Iv) show that the complex formed between cis-Pta2Cl2 and quinizarin was more e f f i c a cious than the positive control. It did not have quite the degree of increased l i f e span shown by the cis-Pta2Cl2/DOXO complex. On the other hand, as before with the DOXO complex, f a i r l y high levels of cis-platinum upwards of 20 mg/Kg mouse could be used as compared to a toxic level of 7 mg/Kg mouse for cis-Pta Cl2 alone. This paper has reported only products of the reaction which were isolated and characterized. However, the potential for further chelation and therefore doubling or trebling the molecular weight to polymer proportions exists. This suggests the possib i l i t y of a polymeric drug moiety which would be inherently slow releasing i n vivo. It i s planned to continue this line of research i n the future. 2

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NH^ Downloaded by CORNELL UNIV on October 3, 2016 | http://pubs.acs.org Publication Date: May 12, 1982 | doi: 10.1021/bk-1982-0186.ch018

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Pt

/NH

Agents

3

CH OH 2

CH 0

0

3

0 .Pt.

N3H CI

NH NH.

HN 3

Pt H N 3

Figure 1.

/

^NH

3

cis-Diamminedichloroplatinum(II)/DOXO

complex.


239

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Table IV


In Vivo Test Results of Complexes Dose mg Compound/ kg Mouse

Compound

Number Avg. Day of Death

% Increased Life Span

o

of Cures* (out of 6)

Negative Control*"

0

17.3

—

-

Positive control cis-Pta Cl

7

31.3

80

4

97 108 101 51

4 6 1 1

2

2

cis-Pta Cl2 + doxorubicin 2

5 10 15 20

34.2 36.0 34.8 26.2

Negative control

0

13.8

—

-

Positive control cis-Pta Cl

7

25.0

81

1

107 88 16 -80 -72

1 0 0

2

cis-Pta Cl quinizarin 2

2

2

+

"TJegative control:

8 12.5 25 50 100

28.0 26.0 16.0 2.75 3.8

Mice injected only with cancer c e l l s .

2 Cure: The National Cancer Institute uses the term "cure" for those animals which l i v e beyond 60 days.


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Aolmowledgments

We acknowledge generous g i f t s of doxorubicin (Adriamycin) by Adria Laboratories, Inc.; cis-Pta2Cl2 by Johnson Matthey, Inc. Thanks to H. Blount, E. Bancroft, G. Mclntire for electrochemical analyses, J. Wells and J. Murtha for compound syntheses and TLC. Literature Cited 1.


2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16.

Yolles, S.; Leafe, T.D.; Meyer, F.J. J . Pharm. S c i . 1975, 64, 115. Yolles, S.; Morton, J.; Rosenberg, B. Acta Pharm. Suecica 1978, 15, 382. Yolles, S.; Sartori, M.F. "Degradable Polymers for Sustained Drug Release" i n Drug Delivery Systems, R.L. Juliano, Oxford University Press, Inc., New York, 1980; p. 84. This drug has recently been approved by the FDA. An anthracycline antibiotic isolated from Streptomyces peucetius. Gosalvez, M.; Blanco, M.F.; Vivero, C.; Valles, F., Europ. J . Cancer 1978, 14, 1185. Greenaway, F.T.; Dabrowiak, J.C., ACS Inorganic Abstract #169 (Sept. 1979). Bottei, R.S.; Gerace, P.L., J. Inorg. Nucl. Chem. 1961, 23, 245. Coble, H.D.; Holtzclaw, H.F., Jr., J. Inorg. Nucl Chem. 1974, 36, 1049. Pierpont, C.G.; Francesconi, L.C.; Hendrickson, D.N., Inorg. Chem. 1978, 17, 3470. Thomson, R.H. "Naturally Occurring Quinones" (2nd Ed.) Academic Press, New York, 1971; pp. 39-92. Cotton, F.A.; Wilkinson, G. "Advanced Inorganic Chemistry" (3rd Ed.) Wiley and Sons, New York, 1972; pp. 1034, 1035, 1036. Bellamy, L.J. "Infrared Spectroscopy of Complex Molecules" (2nd ed. reprint) Wiley and Sons, New York, 1959. Nakomoto, K.; McCarthy, P.J. "Spectroscopy and Structure of Metal Chelate Compounds" Wiley and Sons, New York, 1968; pp. 251 f f . Poulet, H.; Delorme, P.; Mathieu, J.P. Spectrochim. Acta 1964, 20, 1855. Feltham, R.D.; Hayter, R.G. J . Chem. Soc., 1964, 4587.

RECEIVED October 1, 1981.


Biological Activities of Polymers - American Chemical Society

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