Michael J. Abrams

Effect of heat on the cytotoxicity and interaction with DNA of a series of platinum complexes

The effect of elevated temperature on the cytotoxicity and interaction with DNA of a series of platinum(II) complexes was examined. CDDP showed greater enhancement in cell killing with heat than the other platinum(II) complexes. There were approximately 2 decades enhancement in cell killing by 10 microM CDDP at 42 degrees C compared to 37 degrees C. The other potential cross-linking agents also showed increasing cytotoxicity with increasing temperature. K2PtCl4 (500 microM) killed about 15 times more cells at 43 degrees C than at 37 degrees C and KPt(NH3)Cl3 (500 microM) killed about 18 times more cells at 43 degrees C than at 37 degrees C. The cytotoxicity of the triammine and tetraammine complexes was less influenced by temperature. There was no significant difference in the cytotoxicity of [Pt(NH3)3Cl]Cl at any of the temperatures examined. The cytotoxicity of [Pt(NH3)4]Cl2 (500 microM) was increased about 7-fold at 43 degrees C compared to 37 degrees C, but the total cell killing by this complex at 43 degrees C was less than 1 log. Carboplatin (250 microM) was about 5 times more toxic at 42 degrees C and killed about 2.5 decades more cells at 43 degrees C than at 37 degrees C. Although there was little enhancement in the cytotoxicity of trans-Pt(NH3)2Cl2 at 42 degrees C compared to 37 degrees C trans-Pt(NH3)2Cl2 (500 microM) was about 7 times more cytotoxic than at 37 degrees C. The interaction of the various drug/temperature treatments with supercoiled pBR322 plasmid DNA was examined to assess the effect of heat on the reaction of these agents with DNA. At 42 degrees C, CDDP was able to gradually alter the gel electrophoretic mobility of the plasmid DNA to near that of the linear form. This change also occurred at 37 degrees C but at a much slower rate. Carboplatin effected similar changes in the superhelical pBR322 DNA, and the effect of temperature appeared to increase the rate of the reaction. Trans-Pt(NH3)2Cl2 also interacted with the supercoiled DNA, but at a slower rate than CDDP even under hyperthermic conditions. These results indicate that neutral platinum complexes capable of cross-linking DNA interact positively with temperature elevation to increase cytotoxicity, and, that of the platinum complexes that meet these criteria, the effect of hyperthermia is greatest with CDDP.

Synthesis and Reactions of a New Class of Orally Active Pt(IV) Antitumor Complexes

The advent of cisplatin was a breakthrough in the chemotherapy of certain cancers. Its success, in spite of adverse effects such as nephrotoxicity, nausea and vomiting, ototoxicity and myelosuppression, attests to its efficacy1. Still, the cost of treatment, in terms of patient quality of life, underscores the need for an efficacious drug with milder side effects. Carboplatin is an example of an agent specifically developed to reduce side effects while retaining the antitumor activity of cisplatin2. Its tremendous success, following its introduction in Europe and the US, attests to the importance of addressing patient quality of life. Although, oral chemotherapeutic agents are not presently a significant factor in cancer treatment, a properly designed agent could offer significant advantages in terms of a patients’ comfort and convenience, and anticipates the possibility of outpatient chemotherapy. At Johnson Matthey, in conjunction with the Institute of Cancer Research and Bristol-Myers Squibb, a portion of our platinum antitumor drug discovery program is devoted to the design and development of an orally active platinum antitumor drug. This paper describes the synthesis, reactions, and a few of the biological properties of a new class of antitumor agents that possess many characteristics required of an orally active antitumor agent.

Intracellular distribution of a platinum-rhodamine 123 complex in cis-platinum sensitive and resistant human squamous carcinoma cell lines

The platinum(II) tetrachlorodianion and two molecules of rhodamine-123 associate to form a neutral tight ion pair. To examine the intracellular fate of this ionic complex, the levels of uptake after a 1-hr exposure to a 100 microM concentration of each component of the complex, the complex itself and cis-diamminedichloroplatinum(II) (CDDP) were measured in SCC-25 cells. The uptake of Pt(Rh-123)2 was measured by two independent methods: fluorescence and 195mPt gamma-counting. There was excellent agreement between these two methods as to the amount of Pt(Rh-123)2 which was taken up by the cells, indicating that the Pt(Rh-123)2 is probably entering cell intact. Association with Rh-123 increased the amount of platinum which entered the cells by about 70-fold compared to CDDP and increased by about 700-fold the amount of platinum which entered the cells compared to K2PtCl4. The subcellular distributions of Pt(Rh-123)2, Rh-123, CDDP and K2PtCl4 were also examined. When measured by fluorescence or 195mPt gamma-counting, 40-54% of the Pt(Rh-123)2 was in the nuclei of the SCC-25 or SCC-25/CP cells and 27-35% was in the cytosol of the cells. There was excellent agreement between the findings of fluorescence and 195mPt gamma-counting regarding the amount of Pt(Rh-123)2 in each of the subcellular fractions immediately after incubation with the drug and over the time course of observation after drug removal, indicating that the Pt(Rh-123)2 is probably remaining largely intact intracellularly. On a per mg protein basis, there was about a 55-fold greater amount of platinum in the nuclei of the SCC-25 cells exposed to Pt(Rh-123)2 compared to cells exposed to CDDP. In the SCC-25/CP cells, there was about 258-fold greater platinum in the nuclei of cells exposed to Pt(Rh-123)2 than those exposed to CDDP because CDDP was taken up to a much lesser extent by the SCC-25/CP cells. Association of Rh-123 with potassium tetrachlorodianion forms a tight ion pair, which enters cells in relatively high amounts and is selectively concentrated in the nuclei of the cells.

Investigations of technetium-organohydrazine coordination chemistry. The crystal and molecular structures of [TcCl2(C8H5N4)(PPh3)2]·0.75C7H8 and [TcNCl2(PPh3)2]·0.25CH2Cl2

Abstract Reaction of [TcOCl4]− with hydralazine·HCl in methanol at room temperature yields the Tc(V)-hydrazido(3−) complex [Tc(C8H5N4)Cl2(PPh3)2]·0.75C8H7 (1). In contrast, the reaction in refluxing methylene chloride produces the Tc(V)-nitrido complex [TcNCl2(PPh3)2]·0.25CH2Cl2 (2). Crystal data. 1: triclinic P 1 , a=11.592(3), b=12.105(3), c=17.884(4) A, α= 107.76(2)°, β=99.03(2)°, γ=106.11(2)°, V=2232.7(9) A3, Z=2, Dcalc=1.40 g/m3; stucture solution and refinement based on 2623 reflections (F⩾6σ(Fo); Mo Kα, λ=0.71073 A) converged at R=0.058. 2: monoclinic C2/c, a=24.081(9), b=9.539(4), c=15.650(6) A, β=116.18(1)° V=3226.8(11) A3, Z=4, Dcalc=1.37 g/cm3; 3118 reflections; R=0.052.

Potentiation of radiation-induced cell kill by synthetic metalloporphyrins☆

The effects of the combination of several meso-substituted, water soluble metalloporphyrins with ionizing radiation on hypoxic and oxic monolayers of Chinese hamster fibroblast (V79N) cells were studied. The metalloporphyrins tested included a series of cationic metalloporphyrins complexed with Co(III), Zn(II), Fe(III), Cu(II), Pd(II) or Mn(III) and a series of anionic porphyrins chelated with Co(III), Fe(III), Cu(II), Rh(III), Mn(III) or Sn(IV). Both cationic and anionic free porphyrins were also tested. Cationic ligands were tetrakis(4N-methylpyridyl)porphine [TMPyP], tetrakis(4N-trimethylamino phenyl)porphine [TMAP], tetrakis(4N-butylpyridyl)porphine [TBPyP] and tetrakis(3N-methylpyridyl)porphine [3TMPyP]. Anionic ligands tested were tetrakis(4-sulfonato phenyl)porphine [TPPS], tetrakis(biphenyl)porphine sulfonate [TBPS] and tetrakis(4-carboxyphenyl)porphine [TCPP]. SER calculated from survival curves and SFR from one radiation dose were used to assess the relative effectiveness of this class as non-cytotoxic hypoxic and oxic cell-kill potentiators. Comparisons were made at 100 microM, which was essentially non-toxic (greater than 70% survival) for all porphyrins tested except for Co[TMPyP] (approximately 50% survival after 1 hour at 37 degrees C under oxic conditions). The greatest effects on radiation-induced cell kill were achieved with Co[TPPS] and Co[TMPyP] with SER values of 2.3 and 2.4 respectively. Porphyrin analogs with no coordinated metal were found to be less active than the same compound with metal. The overall charge on the molecule did not systematically relate to the biological activity of the compounds tested.

Some complexes of cobalt(III) and iron(III) are radiosensitizers of hypoxic EMT6 cells

The radiosensitizing potential in hypoxic EMT6 cells of several complexes of Co(III) and Fe(III) has been examined. The cytotoxicity of each of the agents toward oxygenated and hypoxic EMT6 cells was tested over the concentration range of 1 to 500 micron for 1-h drug exposure. There was no statistically significant difference between the cytotoxicity of these complexes toward oxygenated and hypoxic cells. Based on these findings, 100 micron was selected as the drug concentration for the initial assessment of radiosensitizing potential. The radiation survival of EMT6 cells in the presence of 100 microM drug for a series of Co(III) complexes in which the number of nitro ligands was varied showed that the hexanitro and the triamine-trinitro complexes are very effective radiosensitizers. The trans-tetrammine dinitro complex was a more effective radiosensitizer than the corresponding cis-dinitro complex. The diethylenetriamine and 1,10-phenanthroline complexes were very effective radiosensitizers, producing dose-modifying factors of 2.4. The trans-tetrammine dichloro complex was moderately effective, giving a dose-modifying factor of 1.9. On the other hand, the hexammine and triammine tricyano complexes and the trans-dinitro complex with negatively charged acetylacetonate ligands were ineffective as radiosensitizers in this system. Finally, three complexes with cyclopentadienyl ligands were examined. The ferricenium salt itself was a moderately effective radiosensitizer, giving a dose-modifying factor of 2.0. However, both the dimethylferricenium salt and the analogous cobalt complex were ineffective. The FSaIIC fibrosarcoma was used to study radiosensitizing potential in vivo. The trans-tetramminedinitro complex was administered at doses of 100, 200, or 300 mg/kg as a single ip injection 1 h prior to irradiation or as three daily ip injections. There was increasing dose modification with increasing drug dosage. With a fractionated radiation protocol in which five daily fractions of 2, 3, or 4 Gy were administered to the tumor-bearing limb with ip drug injections of 100 or 200 mg/kg given 1 h prior to irradiation, a dose-modifying effect of 1.6 was observed with 5 X 200 mg/kg of the drug.

Synthesis and characterization of oxorhenium(V)-catecholate complexes. Crystal and molecular structures of (CH3)4N[ReO (O2C6H4)2] and (CH3)4N[ReO(PPh3) (O2C6H4)2]·CH3OH

Abstract The reactions of ReOCl 3 (PPh 3 ) 2 with catechol and substituted catechols in methanol in the presence of triethylamine and tetramethylammonium chloride under N 2 yield the green complexes [(CH 3 ) 4 N] + [ReO(cat) 2 (PPh 3 )] − (cat=O 2 C 6 H 4 ( 2 ), 4CH 3 O 2 C 6 H 3 ( 3 ), O 2 C 6 Cl 4 ( 4 )). The reactions of 2 and 3 with pyridine in methylene chloride yield [(CH 3 ) 4 N] + [ReO(cat) 2 (py)] − ( 5 ) and [(CH 3 ) 4 N] + [ReO(4Mecat) 2 (py)] − ( 6 ). Variable temperature 1 H and 31 P NMR spectroscopy studies on these complexes indicate that the ancillary ligands (PPh 3 : 2 , 3 ; py: 5 , 6 ) undergo a dissociation-association process in solution along with concomitant cis-trans isomerization of the catecholate ligands. The reaction of [(CH 3 CH 2 ) 4 N] + [ReO 2 (cat) 2 ] − ( 8 ) with triphenylphosphine in refluxing ethanol yields the reduced rhenium(V) square pyramidal complex [(CH 3 CH 2 ) 4 N] + [ReO(cat) 2 ] − ( 7 ) as an air and moisture-sensitive tan solid where triphenylphosphine acts as a reducing agent. These complexes were characterized by elemental analysis, variable temperature 1 H and 31 P NMR spectroscopy and IR and UV-Vis spectroscopies. Complexes 2 and 7 were also characterized by X-ray crystallography. Crystal data: C 20 H 28 NO 5 Re ( 2 ): tetragonal space group I 4/ m , a =19.890(3), c =10.438(2) A, V =4129(2) A 3 , Z =8, D calc =1.766 g cm −3 ; structure solution and refinement based on 976 reflections (Mo Kα, λ=0.71073 A) converged at R =0.043. C 36 H 43 NO 7 PPe ( 7 ): monoclinic space group C 2/ c , a =25.485(5) A, b =9.127(2), c =30.590(6) A, β=101.84(3)°, V =6964(3) A 3 , Z =8, D calc =1.563 g cm −3 ; 3298 reflections, R =0.047.

Platinum coordination complexes which circumvent cisplatin resistance

In the search for a platinum complex capable of oral administration, the poor bioavailability of established drugs has been circumvented by the discovery of a novel class of platinum (IV) ammine/amine dicarboxylate dichlorides. These compounds, when administered orally to mice carrying the ADJ/PC6 plasmacytoma, exhibit antitumor selectivities far superior to those of cisplatin or carboplatin (given intraperitoneally). Oral activity comparable to that of intraperitoneal cisplatin and carboplatin has also been demonstrated in a panel of human ovarian tumor xenografts. Platinum (IV) ammine/amine dicarboxylates retain cytotoxicity in cultures of L1210/cisplatin and L1210/tetraplatin acquired resistant cells. This property does not translate into a cisplatin-resistant variant of the ADJ/PC6 tumor, in the example of JM221. This result reflects experience with tetraplatin, a drug currently in phase I study, which is comparably ineffective against an ADJ/PC6/cisplatin variant. It is a moot point whether either L1210 or ADJ/PC6/cisplatin-resistant variants are clinically predictive screening models, since this issue must be determined ultimately by clinical study. We have attempted to resolve this dichotomy through the establishment of human ovarian carcinoma lines, both in vitro and in vivo, where there is evidence that response to platinum coordination complexes in the several models reflects that of the donor patients tumor to platinum-based clinical therapy. The data herein for platinum (IV) ammine/amine dicarboxylates in these models gives encouragement to the notion that these novel compounds may be of value as oral therapeutic agents, whilst also providing an important lead to the discovery and development of a new generation of platinum drugs possessing broad clinical utility.

High oxidation state rhenium complexes with the hydridotris(1-pyrazolyl)borato-oxorhenium(V) core. Crystal and molecular structures of [HB(pz)3ReO(HNNC8H5N2)] and [HB(pz)3ReO(2-SC5H3N)]

Abstract The reactions of [HB(pz)3ReOCl2] (1) with chelating hydrazines were studied. In contrast to the common substitution pattern which occurs through a condensation type reaction to displace the oxo group in forming the metal-hydrazido functionality, 1 reacts with hydrazinophthalazine (hydralazine) to give [HB(pz)3ReO(HNNC8H5N2)] (2). Similarly, reaction with bishydrazinophthalazine produces the binuclear species [{HB(pz)3ReO}2{(HNN)2C8H4N2}] (3). The relative ease of substitution of the chloride groups in 1 is evident in the reactions of 1 with thiolate donors to yield complexes of the type [HB(pz)3ReO(2-pyridinthiol)2] (4). Crystal data: 2 · CH2Cl2, monoclinic P2 1 /c, a = 12.820(3), b = 11.616(2), c = 15.626(3) A , β = 104.93(3)°, V = 2248(1) A 3 , Z = 4, D calc = 1.171 g cm −3 ; structure solution and refinement based on 2316 reflections converged at R = 0.0395. 4 · 1.5CH2Cl2, triclinic P 1 , a = 10.181(2), b = 14.159(3), c = 9.622(2) A , α = 95.51(3), β = 93.25(3), γ = 88.18(3)°, V = 1377.9(7) A 3 , Z = 2, D calc = 1.718 g cm −3 ; R = 0.0351 for 3291 reflections.

THE SYNTHESIS AND IN VITRO PHOTODYNAMIC ACTIVITY OF A SERIES OF NOVEL RUTHENIUM(II)-2,3-NAPHTHALOCYANINES

Abstract A simple synthetic route to a ruthenium(II)-2,3-naphthalocyanine-bis-benzonitrile complex is presented. A series of ligands can be substituted in the axial position of this complex, which confers water solubility on the ruthenium(II)-2,3-naphthalocyanines (Ru(II) -2,3-Ncs). The ability of these complexes to function as potential sensitizers for photodynamic therapy was investigated. The sensitizers presented in this paper have Q-band absorption maxima in the 710–760 nm range, with extinction coefficients around 1 × 10 5 M −1 cm −1 . The activity of these complexes was tested in vitro against the HeLa cell line and was found to be in the micromolar range. The photobleaching of the complexes was also studied.