Synthesis, Characterization, and Antitumor Activity of New Chloroethylamine Platinum Complexes Abdul R. Khokhar, Quanyun Xu, Robert A. Newman, Yuichiro Kido, and Zahid H. Siddik Department of Medical Oncology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas

ABSTRACT A series of cis-bis-(2-chloroethylamine)platinum(II) and platinum(IV) complexes were synthesized and characterized by elemental analysis, IR, and ‘H and 195PtNMR spectroscopic techniques. Complexes were tested in vitro against murine L1210 leukemia and human ovarian A2780 cell lines and in vivo against the L1210 leukemia model. Some of these complexes showed excellent antitumor activity in both systems. However, all were inactive against cisplatin-resistant A2780/CP cells.

ABBREVIATIONS Ox, oxalic acid; Mal, malonic acid; CBDCA, 1,l-cyclobutanedicarboxylic acid; HPLC, highperformance liquid chromatography; IR, infrared; NMR, nuclear magnetic resonance; DMF, N,N-dimethylformamide; IC s,,, concentration of drug required to inhibit cell growth by 50%; CP, cisplatin; tetraplatin, tetrachloro( trunsdl-1,2-diaminocyclohexane) platinum(IV).

INTRODUCTION The discovery by Rosenberg et al. [l] that some platinum complexes, particularly cis-diarnminedichloroplatinum(I1) (cisplatin), have antitumor activities has led to the commercialization of cisplatin in the treatment of advanced metastatic testicular and ovarian cancers. Like other antitumor agents, cisplatin has side effects; foremost among these are nephrotoxicity, nausea and vomiting, mylosuppression, and ototoxicity. All toxicities appear to be dose related, among which nephrotoxicity and

Address reprint requests and correspondence to: Abdul R. Khokhar, Department of Medical Oncology, Box 52, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, US. Journal of Inorganic Biochemistry, 0 1992 Elsevier Science Publishing

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A. R. Khokhar et al,

ototoxicity appear to be cumulative [2-51. However, development of resistance to cisplatin appears to be a more serious impediment at the clinical level [6]. For more than two decades, attempts have been made to enhance the antitumor activity of cisplatin and widen the spectrum of drug-responsive tumors while lowering its toxicity [7-91. These efforts have been aimed at: i) modifying the drug itself by varying the attached amine ligands and leaving groups, ii) altering treatment methods by administering large volumes of fluids, and iii) combination with other drugs or radiotherapy. With regard to modification of the drug, the literature documenting substitution of both the labile ligands or the stable (nonleaving) amine ligands in the cisplatin molecule is extensive ]lC- 131. Analysis of such literature indicates that labile ligands generally modulate toxicity, while nonlabilc amine ligands modulate both spectrum of activity and toxicity. Chemically, the leaving ligands are displaced by aquation reaction to convert the nonlabile platinum complex into an alkylatmg species [ 141. We hypothesized that by incorporating a potentialiy alkylating function in the nonleaving amine part of the molecule, the mechanism of interaction of the drug molecule with the target DNA may be different than to that with cisplatin, and this would extend activity of the molecule to drug-resistant ceils. In this report, we have selected chloro-substituted alkylamine groups as the stable ligand to provide alkylating functions similar to those present in classical alkylating agents, such as with different leaving liganda and platinum melphalan [ 1.51. Such compounds, valence state to provide a limited structure-activity study? were synthesized, characterized, and evaluated for antitumor activity in both sensitive and cisplatin-resistant tumor models. This study extends our previous preliminary report on the activity of cis-bis(2-chloroethylamine)dichloroplatinum(II) against the highly- sensitive ADJ :‘PCh plasma cell tumor [ 16I

EXPERIMENTAL Chemical 2-Chloroethylamine monohydrochloride, from Aldrich Chemical Co. (Milwaukee, Matthey (Seabrook, NH). All chemicals used as received. The dicarboxylic acids treatment with sodium hydroxide. Physical

CBDCA, Mal, and Ox were purchased WI). K,PtCl, was obtained from Johnson obtained from commercial suppliers were were converted to their disodiurn salts by

measurements HPLC was performed on a Waters 600 System using a model 48 1 detector set at 2 10 mn. The mobile phase (consisting of 91% aqueous solution of 0.01 M ammonium phosphate, 8.5% MeOH, and 0.5% 2-propanol) eluted the Hypersil ODS Cl8 column at a flow rate of 1 ml/mm. Elemental analyses were performed by Robertson Laboratories (Madison, NJ). IR spectra were recorded as KBr pellets using a Beckman 250MX spectrophotometer. I H and lg5Pt NMR spectra were measured on an IBM NR 2OOJAF NMR spectrometer. A 5 mm NMR tube containing a II),0 solution of Na,PtCl, was inserted into the 10 mm sample tube during acquisition of the 195Pt NMR spectra. The D,O solution was used as a deuterium lock. and the Na,PtCl, served as reference i- 1621.0 ppm).

ANTITUMOR

PLATINUM(II) AND (IV) COMPLEXES

213

Animals Male BDFl mice were purchased (Wilmington, MA).

from Charles River Breeding

Laboratories,

Inc.

Cell lines Cisplatin-sensitive murine leukemia L1210/0 cells were obtained from Dr. Alan Eastman [ 171. The cisplatin-sensitive human ovarian A2780 and cisplatin-resistant A278O/CP cell lines [18] were obtained from Drs. Hamilton and Ozoles. Synthesis

of Platinum

Complexes

A series of cis-bis-(2-cbloroetbylamine)platinum(II) and (IV) complexes has been prepared. Synthetic routes used to obtain the desired product are shown in Scheme 1.

Preparation

of cis-(CICHJH, NH,), PtZ2 (1). cis-(ClCH,CH,NH,),P$ was prepared according to a previously reported method [19]. K,PtCl, (4.1511 g, 10 mmol) was converted into K,PtI, and then mixed with a freshly prepared solution of ClCH,CH,NH, [C1CH,CH,NH2 * HCl (2.3198 g, 20 mmol) was dissolved in 15 ml water and 20 ml of 1 N NaOH was added to it; the resulting solution ended up alkaline]. Immediately, a yellow precipitate formed. The solid was collected by filtration, dissolved in DMF (20 ml), and then filtered. Water (400 ml) was added to the filtrate. A yellow precipitate was obtained. This was filtered, washed with water, and dried in a vacuum desiccator. The complex was purified from DMF/water (yield 80%). Preparation of cis-(CICH,CH, NH,), PtCI, (21, cis-(ClCH,CH, NH,), Pt(Ox) (3), cis-(ClCH,CH, NH,), Pt(Ma1) (4), and cis-(ClCH,CH, NH,), Pt(CBDCA) (5). Silver nitrate solution (2.2014 g, 12.9 mmol) was added to an aqueous suspension of cis-(ClCH,CH,NH,),PtI, (1) (4.0197 g, 6.6 mmol). The reaction mixture was stirred overnight in the dark at room temperature. The mixture was filtered through Celite; this gave a colorless solution of cis[(C1CH2CH,NHz),Pt(H,0),1(N0,)2, to which a solution of disodium oxalate (0.8682 g, 6.5 mmol) was added. Gradually, a precipitate formed. After stirring at room temperature overnight, the volume of solution was reduced to 50 ml on a rotary evaporator at 40°C. The off-white solid was filtered, washed with water, and dried in vacua to give cis-(ClCH,CH ,NH,), Pt(Ox) (3). The complex was recrystallized from water (yield 50%). Complexes 4 and 5 were prepared in a similar manner using disodium salts of the corresponding acids, while an aqueous solution of hydrochloric acid was used in the preparation of cis-(ClCH,CH,NH,),Ptr& (2).

Preparation of cis-(ClCH,CH, NH,), Pt(OH),CI, (6), cis-(CICHJH, NH,), Pt(OH),(Ox) (7), cis-(ClCH,CH, NH,), Pt(OH),(Mal) (8), and cis(ClCH,CH, NH,), Pt(OH),(CBDCA). H,O (9). Platinum complexes 6-9 were prepared by oxidizing their corresponding platinum (II) complexes with hydrogen peroxide in water. In a typical procedure, cis-(ClCH,CH,NH,),Pt(Ox) (3) (1.8712 g, 4.2 mmol) was suspended in 100 ml of water. To the suspension, an aqueous hydrogen peroxide solution (14 ml, 30% w/w) was added. The reaction mixture was stirred at 60°C for 3 hr and a yellowish solution was obtained. The solution was filtered and evaporated to dryness under reduced pressure at 40°C and cis(C1CH,CH,NH,),Pt(OH)2(0x) (7) was obtained as a yellow solid. The complex was purified from acetone/ether (yield 93%).

216 A. R. Khokhar

et al.

TABLE 1. Elemental Analysis of Cis-Bis(2-Chloroethyhunine)Platinum(IK) and Platinum(IV) Complexes Compound No.

%C

1 2 3 4 5 6 7 a 9 10 11 12 13

R.lX II.41 16.3’3 18.47 23.81 10.56 14.91 16.58 2i.68 11.26 1.5.73 19.98 9.66

Calc.

Found ---%H 1.94 2.86 2.81 3.05 3.5x 2.88 2.98 3.25 3.73 2.56

2.84 3.27 2.16

%N

%C

4.46 6.59 6.30 0.37 5.75 6 11 5.63 5.70 S,lS 5.34 5.03 4.82 5 39

1 I .30 16.30 18.43 24.20 10.46 1s ii 17 is 2 I.90 i4.w I5.3t; x.52 9. h’i

XH

%N

7.YO

4.61 h.W h.34 6.14

5.65 6.10

5 88 5.72 5.1 I 5.46 5.32 4.78

5.06

the degeneracy of the vd (COO) band. All the dicarboxylatoplatinum complexes display one vS (COO) band in the region 1352- 1551 CIC '. The complexes also display weak N-H stretching bands in their IR spectra. The interpretation of the proton NMR spectra for the amine ligand in platinum complexes (Table 3) is straightforward. Two sets of methylene proton of the ligand appear as two triplets in the range 2.80-4.07 ppm. For complex 4, the proton NMR peak of one of two methylcne protons in amine ligand and that of the methylene proton in malonate overlap. The CBDCA ligand in the complex displays two sets of resonances, a triplet of four methylene protons, and a quintet of two methylene protons. This is in contrast to those observed for platinum complexes with chelating amine [22]. “‘Pt NMR spectroscopic data for selected platinum (II) complexes also suggest that the complexes are square planar and have either two unidentate or one bidentate leaving group. Complexes gave singlets in the range of -- 1X56 to -- 2236 ppm, which is characteristic of platinum(I1) complexes.

TABLE 2. IR data of Cis-Bis(2-Chloroethylamine)Platinum(II) ---

and Platinum(IV) Complexes

~__..~_~~~~.~_~._._.~~~~~~~~_.~~_~~~~~~~~_~__~_~____~

Compound No. ~2 3 4 s 7 8 9 10 11 12 13 --__--_

._y (NH,)

__..

IR -_-_.___-._._.._-.-__ I’a(COO)

3199

_..

3180 3187, 3109 3186, 3109 3183 3080 3080 3060 3070 3080 3224, 3166

1688. lhS7 1619 1611 1706 1636 1624 1703 1637 L694 __

)‘\ (COO) 1372 1382 1368 1363 1364 1352 I363 1360 1551

ANTITUMOR

PLATINUM(I1)

AND (IV) COMPLEXES

217

TABLE 3. NMR Data for Cis-Bis(2-Chloroethylamine)PIatinum(II)

and Platinum(IV) Complexes Compound No. Free ligand 2 3 4 5 1

‘H, ppm ClCH -

-CH -

C(CH) -

-CH -

19’Pt, ppm

3.80(t) (5.7Hz) 3.74(t) (5.9Hz) 3.94(t) (6.3Hz) 3.72(t) (5.5 Hz) 3.71(t) (5.6 Hz) 4.07(t) (5.7Hz)

3.25(t) (5.7Hz) 3.16(t) (5.7Hz) 3.02(t) (6.lHz)

-

-

-

-

-

- 2236

-

-

- 1901 - 1853

(multiplicity) 2.80(t) (5.6 Hz) 3.10(t) (6.5Hz)

2.70(t) (8.1 Hz) -

l.‘ll(quin) (7.8Hz) -

- 1856 -

‘H NMR of complexes 3 and 7 was recorded in DMF-d, and those of other complexes were recorded in CD,OD. proton chemical shift was referenced to TMS (0.00 ppm).

Biological

Activity

Against Cisplatin-Sensitive

Tumor

The antitumor activity of the complexes was determined in vitro against the murine leukemia L1210/0 and human ovarian A2780 and A278O/CP lines (Table 4) and in vivo against L1210/0 murine leukemia (Table 5). The compound was more active in the platinum(I1) form. No definitive conclusions can be drawn, however, with regard

TABLE 4. In Vitro Antitumor Activity of Platinum@)

and Platinum(IV)

Y cis-(CICH,CH,NH,),P;;II)

cis-(C1CH,CH,NH,),~~~

Complexes

7 k

Compound No.

Y

'Y

IC,, (J&u@ X

Y

L1210

A2180

A218O/CP

2 6 13 3 7 10 4 8

OH Cl OH Cl OH

11

Cl

5 9 12 Cisplatin Tetraplatin

OH Cl -

Cl Cl Cl ox ox ox Ma1 Ma1 Ma1 CBDCA CBDCA CBDCA -

2.1 20 3.4 2.9 7.8 7.2 23 >25 > 25 z 25 >25 > 25 1.6 1.1

0.33 1.12 0.39 0.30 0.14

2.3 (7.0) 9.9 (8.8) 3.5 (8.7) 2.2 (7.3) 0.16 (1.1)

Values in parentheses indicate fold resistance of the A2780/CP cell line to the platinum complex.

218

A. R. Khokhur et al.

to the axial OH vs Cl group. With regard to the equatorial posttion. complexes bearing the Cl ligands wet-c generally most potent and effcctivc against these tumor models and had high therapeutic ratios. Complex 2 was particularly impressive and its activity in viva was greater than those of the model compounds. cisplatin, and tetraplatin. Oxalate anaiogs were moderately potent in vitro,, but in viva their activity was only evident with the platinumill) complex. Convcrhely , rnnionatc analogs appeared to lack potency when tested in vitro but demnnstratcci significant activity and acceptable therapeutic ratios in viva against !he \amo I.1 JiC) ‘0 m&l. Whether The CBDC’il. this was due to greater rate or extent of activation in v’&