Synthesis and cytotoxic evaluation of benzoxazole/benzothiazole-2-imino-coumarin hybrids and their coumarin analogues as potential anticancer agents.

Two series of 2-imino-coumarin based hybrids: 3-(benzoxazol-2-yl)-2H-chromen-2-imines 3-9 (series A-I) and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 10-16 (series A-II), as well as their coumarin analogues: 3-(benzoxazol-2-yl)-2H-chromen-2-ones 17-21 (series B-I) and 3-(benzothiazol-2-yl)-2H-chromen-2-ones 22-28 (series B-II) were prepared as potential antitumor agents. The in vitro cytotoxic potency of the synthesized compounds was evaluated against five human cancer cell lines: DAN-G, A-427, LCLC-103H, RT-4 and SISO, and relationships between structure and anticancer activity are discussed. Among the compounds tested, 3-(benzo[d] oxazol-2-yl)-N,N-diethyl-2-imino-2H-chromen-7-amine (6, series A-I) and 3-(benzo[d]thiazol-2-yl)-6-fluoro-2H-chromen-2-one (26, series B-II) exhibited the most potent cytotoxic activity with IC50 values ranging from <0.01 µM to 1.1 µM. In particular, compound 6 demonstrated remarkable cytotoxicity against the A-427 ovarian cancer, the lung cancer LCLC-103H, urinary bladder cancer RT-4 and cervical cancer SISO cell lines with IC50 <0.01-0.30µM, inducing apoptosis in two representative cell lines.

Studies of the inhibitory activities of tiopronin and mercaptosuccinic acid on glutathione peroxidase and their cytotoxic and antioxidant properties.

Glutathione peroxidase (GPx), an important antioxidative enzmye, can be inhibited by various thiols, including of tiopronin and mercaptosuccinic acid (MSA). Recently, there has been discussion regarding the combination of tiopronin in anticancer therapy to overcome acquired resistance to anticancer drugs. However, thiols are also known to act as antioxidants, which can be contraindicated in cancer chemotherapy. This article focuses on the inhibitory effects of tiopronin and MSA on bovine and human glutathione peroxidase activities, and their effects on the redox status of cancer cells. IC50 values for the inhibition for the bovine erythrocyte enzyme were 356 and 24.7 µM for tiopronin and MSA, respectively, with the corresponding Ki values of 343 µM and 14.6 µM, respectively at pH 7.4 and 25 °C. MSA inhibited human GPx activity in human cancer cell lysates at its IC50 while tiopronin did not. Both compounds were cytotoxic to human cancer cell lines GUMBUS and HL-60, with IC50 values between 42.7 and 149.4 µM. Neither had an effect on cell cycle. Only MSA induced apoptosis in HL-60 cells but not in GUMBUS cells, while tiopronin resulted in no apoptosis in either cell line. Combination studies of the MSA with hydrogen peroxide in living cells enhanced the production of reactive oxygen species in GUMBUS cells while tiopronin acted as antioxidant in HL-60 cells. MSA and tiopronin antagonized the cytotoxic effect of cisplatin, doxorubicin and methotrexate in combination studies. Our findings indicate that the antioxidant properties of both thiols prevail over their GPx inhibitory activity in human cancer cell lines.

Cytotoxic activities of hydroxyethyl piperazine-based σ receptor ligands on cancer cells alone and in combination with melphalan, PB28 and haloperidol.

σ Receptor ligands are attracting interest as possible anti-cancer agents because of their ability to induce cell death by different mechanisms. In this study we investigated the cytotoxic effects of 12 recently developed σ-receptor ligands in a panel of eight different human tumor cell lines by either the crystal violet or MTT assays. The results show that σ ligands have broad cytotoxic activity on a number of human cancer cell lines with IC50 values in the low µM range. In addition, apoptosis was observed by the annexin-V/PI double staining method when RPMI 8226 human multiple myeloma cells were treated with a representative σ ligand, (R)-2b. Combination of (R)-2b with melphalan led to a higher apoptotic rate than with the drug alone. Likewise, combined treatment of (R)-2b with the known high affinity σ2-agonist PB28 showed an additive effect on the induction of apoptosis in the RPMI 8226 line. In contrast, combinations of (R)-2b with the known σ1-antagonist haloperidol lead to a significant reduction in the cytotoxic activity of (R)-2b. These results support the idea that (R)-2b acts as a σ-agonist to cause the death of RPMI 8226 cells.

Antimicrobial and cytotoxic abietane diterpenoids from the roots of Meriandera benghalensis (Roxb.) Benth.

Four abietane diterpenoids were isolated from the methanolic extract of the roots of Meriandera benghalensis and tested for their biological activity. Cryptotanshinone (2) and 17-hydroxycryptotanshinone (4) are known metabolites however the occurrence of tanshinone IIA (1) and przewaquinone A (3) from Meriandera benghalensis is reported for the first time. The four diterpenoids were identified by MS and one- and two dimensional NMR experiments. The isolated compounds were tested for their in vitro antiproliferative activity against three human cancer cell lines (a lung cancer (A-427), a urinary bladder cancer (5637) and a breast cancer (MCF-7) cell line) and for their antibacterial effect against three Gram-positive bacterial strains. All four abietanes showed potent cytotoxic effect against all cancer cell lines (IC50 between 1 and 8 microM) as well as antibacterial effect against the bacteria tested (MIC values between 33 and 70 microM).

Synthesis and cytotoxic activity of 5,6-heteroaromatically annulated pyridine-2,4-diamines.

A series of 5,6-heteroaromatically annulated pyridine-2,4-diamines have been synthesized and their in vitro cytotoxic activities evaluated against six human cancer cell lines. Benzo[g] annulated pyrido[2,3-b]indolediamines 7a-b and 8 showed relatively high cytotoxic activity as well as most of the diamines with pyrrolo[2,3-b]pyridine 17, thieno[2,3-b]pyridine and furo[2,3-b]pyridine 26-28, 1,8-naphthyridine 32 and 34 and benzo[h]quinoline 37 skeletons. Surprisingly, pyrido[2,3-b]indolediamines 13 and 14 without benzo[g] annulation were inactive. None of the new compounds were as potent as ellipticine, the reference compound.

Evaluation of the in vitro anticancer, antimicrobial and antioxidant activities of some Yemeni plants used in folk medicine.

The present research study deals with the evaluation of sixty four methanolic and aqueous extracts of thirty Yemeni plants used in traditional medicine for their in vitro antiproliferative activity against three human cancer cell lines in a microtiter plate assay based on cellular staining with crystal violet, for their antimicrobial activity against antibiotic susceptible three Gram-positive, three Gram-negative bacterial and one fungal stains and three multiresistant Staphylococcus strains by the agar diffusion method and the determination of MIC against three Gram-positive bacteria with the broth micro-dilution assay, as well as for their antioxidant activity using the DPPH radical scavenging method. Furthermore the chemical composition of the methanolic extracts was determined by using chromatographic methods. As a result of this work, 12 Yemeni herbs namely Centaurothamus maximus, Costus arabicus, Cupressus sempervirens, Dichrocephala integrifolia, Euphorbia schimperi, Gomphocarpus fruticosus, Kanahia laniflora, Meriandera benghalensis, Pulicaria inuloides, Solanum glabratum, Tarconanthus camphoratus and Vernonia leopoldii demonstrated a noteworthy growth inhibitory effect against all cancer cell lines with IC50 values <50 microg/ml. Pronounced antimicrobial activity was observed only against Gram-positive bacteria among them multiresistant bacteria with inhibition zones >15 mm and MIC values <500 microg/ml, by 9 plants especially Centaurothamus maximus, Cupressus sempervirens, Enicostemma verticillare, Meriandera benghalensis, Nepeta deflersiana, Pulicaria inuloides, Tarconanthus camphoratus, Teucrium yemense and Vernonia leopoldii. Moreover, the methanolic extracts of Cupressus sempervirens, Meriandera benghalensis, Pulicaria inuloides and Rhus retinorrhaea showed a remarkable radical scavenging effect at low concentrations.

Study of the anticancer potential of Yemeni plants used in folk medicine.

The present work evaluated the anticancer activity of methanol extracts from 24 plants used in Yemeni traditional medicine. To evaluate the in vitro cytotoxic potency of the investigated extracts, an established microtiter plate assay based on cellular staining with crystal violet was used with 5 human cancer cell lines: two lung cancer (A-427 and LCLC-103H), two urinary bladder carcinoma (5637 and RT-112) and one breast cancer (MCF-7) line. The methanolic extracts of Dendrosicyos socotrana, Withanina aduensis, Withania riebeckii, Dracena cinnabari and Buxus hildebrandtii exhibited the highest toxicity on all tumor cell lines with IC50 values ranging between 0.29 and 5.54 microg/ml. The extracts of Jatropha unicostata and Punica protopunica showed a moderate potency on the most tumor cell lines.

New synthetic route to [bis-1,2-(aminomethyl)benzene]dichloroplatinum(II) complexes, screening for cytotoxic activity in cisplatin-sensitive and resistant human cancer cell lines, and reaction with glutathione.

A new synthetic route to [bis-1,2-(aminomethyl)benzene]dichloroplatinum(II) complexes is described. o-Xylene and the 4-methoxy substituted derivative were used as starting points for the synthesis: benzylic bromination with N-bromosuccinamide/benzoylperoxide followed by the substitution of the benzyl bromides for azide and finally a catalytic hydrogenation with Pd/C of the diazides gave the desired diamines ligands. An attempt to synthesize the 4,6-dimethoxy derivative was unsuccessful due to the bromination of the aromatic ring. The diamines were complexed with K2PtCl4 to give the target Pt(II) complexes: [1,2-bis(aminomethyl)benzene]dichloroplatinum(II) (4a) and [1,2-bis(aminomethyl)-4-methoxy-benzene]dichloroplatinum(II) (4b). Screening for cytotoxic activity was done in comparison to cisplatin in a panel of eight human cancer cell lines; in all cases, the 4-methoxy derivative 4b was less active than the unsubstituted analog, 4a. In four cell lines 4a was as potent as cisplatin, while in the other four lines cisplatin was considerably more potent then 4a. The 5637 bladder cancer cell line was made 4-5 fold resistant to either cisplatin or [d,l-trans-1,2-diaminocyclohexane]dichloroplatinum(II); 4a showed some cross resistance (2-3 fold) to both resistant cell lines. The reactivity of 4a towards substitutions with glutathione (GSH), a biological thiol involved in intrinsic and acquired resistance to Pt-complexes, was measured by a RP-HPLC method. It was found that the second-order rate constant for the reaction of 4a with GSH was similar to that that reported for CDDP, indicating that reactivity towards GSH does not explain the different levels of cross resistance.

Effect of thiols exported by cancer cells on the stability and growth-inhibitory activity of Pt(IV) complexes.

PURPOSE: The role of thiols in the reduction of Pt(IV) antitumor agents to Pt(II) is well recognized and it is widely thought that this reaction is required for activity. The sources of extracellular thiols in cell culture have been less studied. The purpose of the present work was to determine whether the stability of Pt(IV) complexes in culture medium can be affected by thiols that are released by cancer cells. METHODS: A two-column HPLC assay with UV/visible detection was used to determine the stability of two Pt(IV) complexes in culture medium with and without cells. The kinetics of the thiol release from a human ovarian cancer cell line SK-OV-3 and a human glioblastoma cell line U-87 MG were determined by a modification of the Ellman's method. RESULTS: The stability of a Pt(IV) complex with equatorial iodo ligands, trans, cis-[Pt(en)(OAc)(2)I(2)], was dramatically lower in culture medium in the presence of cells than in fresh culture medium, whereas the half-life of the dichloro analog, trans,cis-[Pt(en)(OAc)(2)Cl(2)], was somewhat increased. Although both complexes showed similar in vitro cell-growth-inhibitory activity, trans, cis-[Pt(en)(OAc)(2)Cl(2)] required a longer incubation time than the iodo analog to reach its maximal effect. The thiol content of the culture medium in the presence of cells was measured after 2 days: the concentrations from cultures of U-87 MG and SK-OV-3 cells were 3. 6 +/- 0.1 microM and 9.3 +/- 0.1 microM respectively, compared to 0. 07 +/- 0.04 microM in fresh medium. During the rapid growth phase, the extracellular thiol content reached a maximum of 20.0 +/- 0.5 microM and 47.8 +/- 0.2 microM for U-87 MG and SK-OV-3 cells respectively. CONCLUSIONS: These findings show that the culture medium conditioned by cancer cells can influence the stabilities of certain Pt(IV) complexes in cytotoxicity studies.

Relationships between reduction properties and cancer cell growth inhibitory activities of cis-dichloro- and cis-diiodo-Pt(IV)-ethylenediamines.

The chemical reactivities and cancer cell growth inhibitory activities of a new series of cis-diiodo-Pt(IV)-ethylenediamines were compared and contrasted with their cis-dichloro-Pt(IV)-counterparts, cis-Diiodo-Pt(IV)-ethylenediamines bearing various axial ligands (i.e., OH, OAc, OCOCF3, OSO2CH3) were prepared by oxidizing [PtI2(en)] with 30% H2O2 to yield trans,cis-[PtOH2I2(en)], which was then reacted with either Ac2O, (CF3CO)2O, or (SO2CH3)2O in CH2Cl2. The cis-diiodo-Pt(IV) complexes were readily reduced by biological thiols such as L-cysteine, glutathione (GSH), and bovine serum albumin (BSA) at pH 6.9 and 37 degrees C; the kinetics of reduction were second-order with respect to thiol concentration. In contrast, the cis-dichloro analogues were stable in the presence of GSH. The reduction potentials estimated by means of cyclovoltammetry for the Pt(IV) complexes are useful for obtaining a ranking order of reactivity towards biological thiols; however, the reduction potentials alone cannot be used to predict whether a Pt(IV) complex will be reduced by GSH at biologically relevant concentrations. GSH greatly facilitated the platination of calf thymus DNA by the diiodo-Pt(IV) complexes, which was > 90% complete after 24 h at 37 degrees C when the ratio of GSH to Pt(IV) was 2:1. DNA-platination by trans,cis-[Pt(OH)2I2(en)] and trans,cis-[Pt(OAc)2I2(en)] were dependent on the presence of GSH while trans,cis-[Pt(OSO2CH3)2I2(en)] showed 23% DNA platination after 24 h in the absence of GSH. In contrast, the dichloro analogues trans,cis-[Pt(OH)2Cl2(en)] and trans,cis-[Pt(OAc)2Cl2(en)] failed to react with DNA in the presence of either low (0.015 mM) to high (3.0 mM) concentrations of GSH. Cell culture experiments with four human cancer cell lines showed that the maximal growth inhibitory activity of the cis-diiodo-Pt(IV)-ethylenediamines was reached within a 24 h exposure to platinum complex, while the dichloro-Pt(IV) analogues required a much longer drug-exposure time (i.e., 96 h) to reach maximal activity.

Unusual reactivity of cisplatin analogs that bear o-phenylenediamine ligands: insights for the design of more effective cytotoxic agents.

The stabilities of dichloro(o-phenylenediamine)platinum(II) (1) and several 4,5-disubstituted analogs [i.e., with: Cl (2), Br (3), Me (4), or MeO (5)] were investigated under various aqueous conditions. The Pt complexes 1-5- decomposed by reactions which were independent of the amount of chloride in the medium. The poor aqueous stabilities of 1-5 were attributed to two factors: 1) The compounds underwent facile oxidation reactions in aqueous solution at pH 7.4 and 37 degrees C, resulting in the formation of intensely colored Pt-species as well as H2O2. Compounds 2 and 3 oxidized considerably faster than 1, 4, and 5. Based on the redox behavior and UV-Vis spectra of the decomposition products, it is proposed that they are o-benzoquinonediimine Pt complexes. 2) Compound 4 underwent an unusually rapid substitution reaction with L-methionine, a component of the culture medium, whereby both of the chloro ligands of platinum were replaced by an N,S-chelated methionine. At an L-methionine concentration of 0.5 mM, the reaction ran to completion within 1 min. Thus, the weak growth inhibitory activities of 1-5 on human cancer cells in vitro was likely a result of their poor chemical stability in the culture medium. Based on a knowledge of the decomposition pathways, analogs were designed to be resistant to these types of reactions. Dichloro(o-aminomethylaniline)platinum(II) (6) and [bis-1,2(aminomethyl)benzene]-di-chloroplatinum(II) (7) were synthesized and their aqueous stabilities investigated. Both 6 and 7 were considerably more stable than 1-5 under aqueous conditions, as well as being more effective in inhibiting the growth of human cancer cells in vitro.

Synthesis and X-ray crystal structure of trans,cis-[Pt(OAc)2I2(en)]: a novel type of cisplatin analog that can be photolyzed by visible light to DNA-binding and cytotoxic species in vitro.

An original approach intended to facilitate the intratumoral activation of Pt(IV) diamines by illumination with visible light to form photolysis products that irreversibly bind to DNA and are cytotoxic to human cancer cells is reported. The novel Pt(IV) complex trans,cis-[Pt(OAc)2I2-(en)] was prepared by the acetylation of trans,cis-[Pt(OH)2I2(en)] with acetic anhydride in CH2-Cl2; trans,cis-[Pt(OH)2I2(en)] was synthesized by oxidation of [PtI2(en)] with 30% aqueous H2O2. trans,cis-[Pt(OAc)2I2(en)] crystallized from methanol as deep-red needles with a = 9.029(4) A, b = 11.443(2) A, c = 12.822(2) A, beta = 95.48(3) degrees, monoclinic space group Cc, and Z = 4. The conformation of the acetato groups around the O-Pt-O axis deviated significantly from the conformation of the acetato groups in the X-ray crystal structure reported for the cis-dichloro analog, which may explain the very different aqueous solubilities of the two compounds. trans,-cis-[Pt(OAc)2I2(en)] and trans,cis-[Pt(OH)2I2(en)] displayed broad ligand-to-metal charge-transfer bands centered at lambda = 389 and 384 nm, respectively (epsilon = 1372 and 1425 M-1 cm-1, respectively), with tailing out to ca. 550 nm. When trans,cis-[Pt(OAc)2I2(en)] was incubated with calf thymus DNA in the absence of light, no covalent binding of Pt to DNA was measurable after 6 h; however, irradiation with light of wavelengths > 375 nm resulted in 63 +/- 13% of the platinum being covalently bound to DNA after 6 h, suggesting that a photoreduction to Pt(II) species took place. Although trans,cis-[Pt(OH)2I2(en)] was also labile to visible light, only 10 +/- 2% DNA platination was observed after 6 h of illumination; however, covalent binding of Pt to DNA took place quantitatively when a reducing agent such as glutathione was added to the photolyzed incubations. These results provide evidence that the photolysis of the trans-dihydroxo analog resulted predominately in the substitution of the iodide ligands for water rather than a reduction of Pt(IV) to Pt(II). When protected from light, trans,cis-[Pt(OAc)2I2-(en)] and trans,cis-[Pt(OH)2I2(en)], both at a concentration of 10 microM, had half-lives of 6.6 +/- 0.5 and 46.8 +/- 8.8 h, respectively, at 37 degrees C in Eagle's minimum essential medium (EMEM) containing 5% fetal calf serum. When irradiated with light lambda(irr) > 375 nm, the half-lives were decreased by 24- and 53-fold for the diacetato- and dihydroxoplatinum(IV) complexes, respectively. Compared to the "dark" control, the in vitro treatment of TCCSUP human bladder cancer cells with trans,cis-[Pt(OAc)2I2(en)] resulted in 35% greater growth inhibitory activity when during the first 1.5 h of drug exposure the cells were irradiated with light lambda irr > 375 nm. The photolysis of trans,cis-[Pt(OH)2I2(en)] with visible light resulted in a 22% enhancement of antiproliferative activity.

Photolysis of an iodoplatinum(IV) diamine complex to cytotoxic species by visible light.

The feasibility of photolyzing the Pt(IV) complex trans,cis-[PtCl2I2(en)] to cytotoxic species by visible light was evaluated. The synthesis of trans, cis-[PtCl2I2(en)] was achieved by the oxidation of [PtI2(en)] with PCl5 in tetrahydrofuran at room temperature for 30 min in the dark. The UV-Vis spectrum of trans, cis-[PtCl2I2(en)] in water showed a broad ligand-to-metal charge-transfer (LMCT) band with lambda(max) = 396 nm (epsilon = 1191/M/cm). Although trans,cis-[PtCl2I2(en)] was relatively stable in water in the dark, irradiation at lambda(irr) = 410 nm brought about its rapid decomposition. A detailed analysis of the photodecomposition products was not carried out, but two lines of evidence suggest that I2 and [PtCl2(en)], a known antitumor agent, may be formed as a result of a reductive-elimination type reaction: (i) irradiation of trans, cis-[PtCl2I2(en)] in water at lambda(irr) = 410 nm led to the same spectral changes as when [PtCl2(en)] and I2 together were irradiated at the same wavelength; (ii) the photoinduced loss of trans,cis-[PtCl2I2(en)] was accompanied by the covalent binding of Pt to DNA at a rate comparable to that of [PtCl2(en)] at 37 degrees C, and the presence of 100 mM chloride suppressed this DNA platination. On the other hand, the combined photolysis products, formed when trans,cis-[PtCl2I2(en)] was irradiated in culture medium at lambda(irr) > 375 nm for 60 min, were less potent than [PtCl2(en)] at inhibiting the growth of two human cancer cell lines. Two limitations make the use of trans,cis-[PtCl2I2(en)] in the therapy of cancer impractical: (i) trans,cis- [PtCl2I2(en)] was relatively unstable in the presence of serum: however, [PtI2(en)] did not appear to be a product of the reaction; (ii) the LMCT band extends only weakly into the region of the electromagnetic spectrum (i.e. lambda > 600 nm) where maximal tissue penetration would be expected. In conclusion, these investigations demonstrate that iodo-Pt(IV) diamines can be photolyzed to cytotoxic species by visible light, but the aforementioned limitations must be overcome before this new class of Pt(IV) complexes can be used as antitumor agents.

A novel approach to preparing water soluble prodrug forms of cisplatin analogues bearing chelating diamines.

A novel method for creating water soluble prodrugs of cisplatin analogues bearing chelating diamines is introduced. When 2-(amino-methyl)aniline is reacted with K2PtCl4 between a pH of 6 and 7, the neutral chelated complex [2-(aminomethyl)aniline)dichloroplatinum(II) (1) is isolated. On the other hand, when the complexation occurs under acidic conditions (i.e. pH 3), the zwitterionic, "open-ring" form [2-(ammonio-methyl)aniline-N1]trichloroplatinate(II) (2) is obtained, whereby only the aniline nitrogen is coordinated to platinum. Compound 2 has a solubility of 10 mM in acidic aqueous medium; that is ca. 20 times greater than that of 1. However, 2 rapidly converts to compound 1 at physiologic pH; thus 2 functions as a water soluble prodrug of 1. Both 1 and 2 are equally effective at halting the growth of three different human cancer cell lines in vitro, indicating that the prodrug is quantitatively converted to the parent drug in a complex, biologically relevant medium. In animal experiments, the prodrug form, when given at a dose of 25 mumol/kg three times a week for 6 weeks, significantly inhibits the growth of the MXT (M3.2) mammary tumor in BDF mice while the same dose of the parent drug has no antitumor activity.

Chemical stability, biological activity and cellular uptake of a cisplatin analogue having a 1,2-diarylethyleneamine ligand in cultures of human breast cancer cells.

The platinum(II) complex PtCl2(meso-6), which has the estrogenic ligand meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine (meso-6), has been reported to be an effective antitumor drug for estrogen-receptor(ER)-positive tumors in animal experiments. The goal of this study was to investigate whether the observed biological effects could be ascribed to the intact PtCl2(meso-6). Cultures of the ER-positive human breast cancer cell line MCF-7 were used as the in vitro test system. In culture medium containing 10% fetal calf serum, PtCl2(meso-6) had a half-life of about 2 h, as determined by HPLC analysis, and no PtCl2(meso-6) was detectable after 10 h. The Pt complex bound irreversibly to serum protein. After 30 min, the diamine ligand was found released, with a maximum conversion of about 35% at 24 h. At this time the culture medium still had estrogenic activity, i.e. it induced ER processing in the MCF-7 cells. This indicates that the estrogenic effect was elicited by the released diamine ligand. In contrast, the growth-inhibitory activity of the medium preincubated with PtCl2(meso-6) was lost at a rate similar to the rate of loss of PtCl2(meso-6) from the medium. This accords with the platinum complex being the main cytotoxic entity. When MCF-7 cells were incubated with PtCl2([3H]meso-6), no free Pt complex could be identified in cellular extracts, and most of the cell-associated radioactivity coeluted with meso-6 in HPLC analysis. After 12 h, only 1.4% of the total cellular platinum was bound to DNA, but no tritium label could be detected. In conclusion, diamine ligand is released from the Pt(II) complex and can account for the estrogenic effects so far ascribed to PtCl2(meso-6).

Reversible and irreversible interactions of a cisplatin analog bearing a 1,2-diphenylethylenediamine ligand with plasma and plasma proteins in vitro.

The cisplatin analog [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl) ethylenediamine]dichloroplatinum(II) [PtCl2(1)], by virtue of its estrogenic 1,2-diphenylethylenediamine ligand 1, was intended to function as a cytotoxic estrogen. This article reports on the reversible and irreversible interactions of this compound with plasma and plasma proteins in vitro. At 37 degrees C [PtCl2(1)] is > 99% reversibly bound to proteins in plasma. At 0 degree C [PtCl2(1)] reversibly binds to albumin at specific binding sites not shared by 1. By use of HPLC the in vitro half-life of total [PtCl2(1)] in plasma was found to be 35 min at 37 degrees C, which is approximately 1/3 the half-life reported for cisplatin under similar conditions. To understand this decreased stability, irreversible reactions of [PtCl2(1)] with albumin and plasma globulins were investigated. The reaction rate of [PtCl2(1)] with albumin is independent of the protein concentration and is comparable to the rate of the first Pt-Cl hydrolysis reaction. Thus, [PtCl2(1)], like cisplatin, reacts irreversibly with albumin through a solvent-assisted SN2 substitution pathway. Because the hydrolysis rate for [PtCl2(1)] is 40% slower than for cisplatin, irreversible reactions of [PtCl2(1)] with albumin cannot account for the decreased stability of the compound in plasma. alpha-Globulins undergo substitution reactions with [PtCl2(1)] by both solvent-assisted and direct SN2 pathways. The half-life of [PtCl2(1)] in the presence of alpha-globulins at concentrations normally present in plasma (6-16 g/liter) is from 41 to 22 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and evaluation of sulfur-containing glutethimide derivatives for aromatase and desmolase inhibitory activity.

Novel sulfur-containing glutethimide derivatives, substituted with either thiol or methylsulfide groups in the ortho/para positions of the aromatic ring, were synthesized and tested for both human placental aromatase and bovine adrenocortical desmolase inhibitory activities. The synthesis was achieved by the chlorosulfonation of gluthethimide, which yielded a 3:1 mixture of the para to ortho sulfonyl chlorides 2a/b. The sulfonyl chlorides of gluthethimide were reduced with Zn/H2SO4 to give the thioglutethimides 3a/b, which in turn were methylated with MeI/EtOH to give the corresponding methylsulfides 4a/b. In comparison to aminoglutethimide (AG), 3a/b and 4a/b were weak inhibitors of aromatase, with 3a/b being more potent than 4a/b. Aromatase inhibition by the thiol compound was pH-dependent; 3a/b was most potent at higher pH (7.4) than at lower (6.6). This suggested that the thiolate form of 4 coordinates with the ferric heme of aromatase. Likewise, both 3a/b were less potent at inhibiting bovine adrenal desmolase than AG. Possible reasons for the surprisingly poor aromatase inhibitor activity of these compounds are discussed.

Diamine ligand release from the cisplatin analogue [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) in cell culture medium.

The stability of the five-membered chelate ring of the cisplatin analogue [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) was investigated under typical cell culture conditions (IMEM-Richter's medium with 10% fetal calf serum, 37 degrees C). For this purpose, the platinum compound was radiolabeled with tritium in the meta position of the aromatic ring by an acid-catalyzed tritium-exchange reaction, and a reversed-phase HPLC assay with radiochemical detection was developed to monitor for the presence of the free diamine ligand in the cell culture medium. A gradual increase in radioactivity attributed to the free diamine was found in medium containing the dichloroplatinum(II) complex (ca. 25% after 24 h), indicating that the diamine ligand was being released from the metal atom. When 1 mM glutathione (GSH) was included in the incubation medium, the amount of free diamine nearly doubled after 24 h, while the amount of radioactivity attributed to serum protein-platinum adducts decreased relative to incubations without GSH. On the other hand, the omission of serum from the incubations resulted in a dramatic decrease in the amount of radioactivity eluting under the diamine peak, while the concentrations of the two methionine-Pt adducts, which formed in a 1:1 ratio, rose. Through the use of liquid secondary ion mass spectroscopy, the two methionine-Pt adducts were identified as monomethionine metabolites of the title compound, whereby the two chloride ligands have been replaced by the amino acid. These compounds are probably diastereomers since the sulfur of methionine can coordinate to platinum with equal probability either cis or trans to the R-configured benzylamine carbon. On the basis of the chemical shifts of the MeS groups in the 250-MHz 1H NMR, it is concluded that a S,N-five-membered chelate ring is present in these methionine-Pt adducts.

Relationships between the aqueous chemistry and the in vitro cytotoxic activities of mixed-amine cisplatin analogues.

The possibility that variations in the cytotoxic activities of cisplatin analogues could be a result of differences in the aqueous chemistry of the compounds was investigated. A series of structurally related mixed-amine dichloroplatinum complexes (cis-coordinated with amine and various diphenylmethylamines and 1,2-diphenylethylamines) was prepared and selected physicochemical properties of the new compounds were characterized. Cytotoxicity was determined in two human breast cancer cell lines (MDA-MB-231 and MCF-7) and one human ovarian cancer cell line (SK-OV-3) by means of a microtiter assay. There is no apparent relationship between the hydrophobicities of the compounds and their cytotoxic potencies. There is no evidence for an inverse relationship between the aqueous stability of the dichloroplatinum complexes and cytotoxic potency, as has been reported for nitrogen mustards and some nitrosoureas. The differences in cytotoxic activity cannot be explained by inter-compound variations in the area under the concentration-time curves (AUC) of the dichloroplatinum complexes in culture medium. Thus, it appears that the differences in the cytotoxic potencies of this series of cisplatin analogues are related to factors other than dissimilarities in these physiochemical properties. Nevertheless, a relationship was found between the AUC of a dichloroplatinum complex in medium and the efficacy of the compound in the MCF-7 cell line. However, the AUC-efficacy relationship does not always hold in the MDA-MB-231 and SK-OV-3 cell lines. In these cells, treatment with a "high" bolus dose of platinum complex over finite exposure times is often less cytotoxic than treatment with lower doses of the same compound but over a continuous exposure time, although the cells are subjected to the same AUC of dichloroplatinum complex.