Renate Grünert

Chiral platinum(II) metallointercalators with potent in vitro cytotoxic activity

Four platinum(II) metallointercalating complexes of 1,10‐phenanthroline (phen) with the chiral ancillary ligands trans‐R,R‐ and trans‐S,S‐1,2‐diaminocyclohexane (R,R‐ and S,S‐dach, respectively), and N,N′‐dimethyl‐R,R‐ and N,N′‐dimethyl‐S,S‐1,2‐diaminocyclohexane (Me2‐R,R‐dach and Me2‐S,S‐dach, respectively) have been synthesised and characterised. The crystal structure of [Pt(Me2‐S,S‐dach)(phen)](ClO4)2⋅1.5 H2O (C20H26Cl2N4O9.5Pt) has been determined; orthorhombic, space group P212121(No. 19), a=23.194(8), b=25.131(9), c=8.522(3) Å. In vitro cytotoxic assays (IC50) in the human bladder cancer cell line 5637 and in the murine leukemia L1210 cell line revealed that [Pt(S,S‐dach)(phen)](ClO4)2 (0.091 and 0.13 μM, respectively) and [Pt(R,R‐dach)(phen)](ClO4)2 (0.54 and 1.50 μM, respectively) were more cytotoxic than cisplatin (0.31 and 0.50 μM, respectively) and considerably more cytotoxic than their methylated counterparts, [Pt(Me2‐R,R‐dach)(phen)](ClO4)2 and [Pt(Me2‐S,S‐dach)(phen)](ClO4)2 (both>23 μM). Chiral discrimination for [Pt(S,S‐dach)(phen)](ClO4)2 over its R,R‐enantiomer was observed in all 13 cancer cell lines investigated. Moreover, [Pt(S,S‐dach)(phen)](ClO4)2 was more active than cisplatin in all cell lines tested and shows only partial cross‐resistance to cisplatin in two cisplatin resistant cell lines.

Trans,trans,trans-[PtIV(N3)2(OH)2(py)(NH3)]: A Light-Activated Antitumor Platinum Complex That Kills Human Cancer Cells by an Apoptosis-Independent Mechanism

Photoactivatable PtIV diazido complexes have unusual photobiologic properties. We show here that trans,trans,trans-[PtIV(N3)2(OH)2(py)(NH3)] complex 3 is a potent photoactivated cytotoxin toward human cancer cells in culture, with an average IC50 value in 13 cell lines of 55 ± 28 μmol/L after 30 minutes (0.12 mW/cm2) photoactivation with UVA, although visible light was also effective. Photoactivated complex 3 was noncross-resistant to cisplatin in 3 of 4 resistant cell lines. Cell swelling but very little blebbing was seen for HL60 cells treated with irradiated complex 3. Unlike cisplatin and etoposide, both of which cause apoptosis in HL60 cells, no apoptosis was observed for UVA-activated complex 3 by the Annexin V/propidium iodide flow cytotometry assay. Changes in the levels of the autophagic proteins LC3B-II and p62 in HL60 cells treated with UVA-activated complex 3 indicate autophagy is active during cell death. In a clonogenic assay with the SISO human cervix cancer cell line, 3 inhibited colony formation when activated by UVA irradiation. Antitumor activity of complex 3 in mice bearing xenografted OE19 esophageal carcinoma tumors was photoaugmented by visible light. Insights into the novel reaction pathways of complex 3 have been obtained from 14N{1H} nuclear magnetic resonance studies, which show that photoactivation pathways can involve release of free azide in buffered solution. Density functional theory (DFT) and time-dependent DFT calculations revealed the dissociative character of singlet and triplet excited states of complex 3, which gives rise to reactive, possibly cytotoxic azidyl radicals. Mol Cancer Ther; 11(9); 1894–904. ©2012 AACR.

Dancing of the Second Aromatic Residue around the 6,8-Diazabicyclo[3.2.2]nonane Framework: Influence on σ Receptor Affinity and Cytotoxicity

A series of 6,8-diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, the affinity toward sigma(1) and sigma(2) receptors was investigated, and the growth inhibition of six human tumor cell lines was determined. The enantiopure bicyclic ketones 5a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane-2,7,9-trione) and 5b ((+)-(1S,5S)-6-allyl-8-(2,4-dimethoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane-2,7,9-trione) as well as their enantiomers ent-5a and ent-5b served as chiral building blocks, which were derived from (S)- and (R)-glutamate, respectively. Structure-affinity relationships revealed that 11a (K(i) = 154 nM), ent-11a (K(i) = 91 nM), and ent-17a (K(i) = 104 nM) are the most potent sigma(1) ligands. High sigma(2) affinity was achieved with 17b (K(i) = 159 nM) and 8b (K(i) = 400 nM). The bicyclic sigma ligands showed a selective growth inhibition of the small cell lung cancer cell line A-427 with the benzyl ethers 11 and the benzylidene derivatives 17 being the most potent compounds. 11a has a cytotoxic potency (IC(50) = 0.92 muM), which exceeds the activity of cisplatin and interacts considerably with both sigma(1) and sigma(2) receptors.

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.

Nicotinamide adenine dinucleotide phosphate-regenerating system coupled to a glutathione-reductase microtiter method for determination of total glutathione concentrations in adherent growing cancer cell lines

Improvements in the traditional glutathione (GSH)-reductase recycling method for determining total glutathione levels in adherent growing cells have been achieved by eliminating the direct use of expensive nicotinamide adenine dinucleotide phosphate (NADPH) and normalizing the levels of GSH to moles/liter instead of the more usual but more error-prone method of normalizing with cellular protein. A glucose-6-phosphate-dehydrogenase auxiliary reaction has been added to the microtiter-adapted enzyme method of Tietze; thus NADP(+) and glucose-6-phosphate replace NADPH in the method. This modification lowers the possibility for substrate inhibition of the reductase by high levels of NADPH during the initial phase of the reaction while at the same time reducing the assay costs by 75-85%. To calculate the cellular concentration of GSH, the number of cells used for the GSH determination, estimated by counting cell nuclei of benzalkonium chloride-lysed cells with a Coulter Counter Z2, and the average cell volume, also determined with the Coulter Counter, are multiplied to give the total sample volume. The quotient of the amount of GSH found in the cells and the total sample volume yields the GSH concentration in moles/liter. The assay has been validated with respect to precision (+/-2.6%), relative accuracy (-4.2 %), linearity (r(2)=0.98), linear range (0.5-10 microM), and limit of detection (80 pmol). Recovery was cell line dependent and ranged between 70 and 103% in the six cell lines. As an application of this method, the GSH concentrations in six human cancer cell lines were determined, without and with a 24-h preincubation with 200 microM D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis. As expected, BSO lowered GSH levels on the average 85%.

Effects of light-activated diazido-PtIV complexes on cancer cells in vitro

Various PtIV diazides have been investigated over the years as light-activatable prodrugs that interfere with cell proliferation, accumulate in cancer cells and cause cell death. The potencies of the complexes vary depending on the substituted amines (pyridine=piperidine>ammine) as well as the coordination geometry (trans diazide>cis). Light-activated PtIV diazides tend to be less specific than cisplatin at inhibiting cancer cell growth, but cells resistant to cisplatin show little cross-resistance to PtIV diazides. Platinum is accumulated in the cancer cells to a similar level as cisplatin, but only when activated by light, indicating that reactive Pt species form photolytically. Studies show that Pt also becomes attached to cellular DNA upon the light activation of various PtIV diazides. Structures of some of the photolysis products were elucidated by LC–MS/MS; monoaqua- and diaqua-PtII complexes form that are reactive towards biomolecules such as calf thymus DNA. Platination of calf thymus DNA can be blocked by the addition of nucleophiles such as glutathione and chloride, further evidence that aqua-PtII species form upon irradiation. Evidence is presented that reactive oxygen species may be generated in the first hours following photoactivation. Cell death does not take the usual apoptotic pathways seen with cisplatin, but appears to involve autophagy. Thus, photoactivated diazido-PtIV complexes represent an interesting class of potential anti-cancer agents that can be selectively activated by light and kill cells by a mechanism different to the anti-cancer drug cisplatin.

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 (1), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24, with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: Ki=24 nM. Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ1 and/or σ2 affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ2: Ki=83 nM; δ: Ki=75 nM). In [35S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25. The enantiomeric 4‐benzylidene derivatives 15 and ent‐15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.

In vitro anticancer activity and evaluation of DNA duplex binding affinity of phenyl-substituted indoloquinolines

Phenyl-substituted indoloquinolines were studied for their biological activity and their DNA binding affinity. Water-soluble aminoalkyl derivatives were prepared and have shown significant in vitro anticancer activity. Unlike previous reports on the potential role of duplex DNA as target for various indoloquinoline based drugs, duplex UV melting experiments and fluorescence titrations suggest only weak and moderately strong binding of the phenyl-substituted indoloquinolines at 120 mM and 20 mM Na(+) concentrations, respectively. Binding is suggested by ethidium displacement and circular dichroism experiments to be associated with drug intercalation between base pairs.

Relationships between the structure of 6-allyl-6,8-diazabicyclo[3.2.2]nonane derivatives and their σ receptor affinity and cytotoxic activity

A series of bridged piperazine derivatives was prepared and the affinity toward sigma(1) and sigma(2) receptors by means of radioligand binding assays as well as the inhibition of the growth of six human tumor cell lines was investigated. All possible stereoisomers of the 2-hydroxy, 2-methoxy, 2,2-dimethoxy, 2-oxo, and 2-unsubstituted 6,8-diazabicyclo[3.2.2]nonanes were prepared in a chiral pool synthesis starting with (S)- and (R)-glutamate. A Dieckmann analogous cyclization was the key step in the synthesis of the bicyclic framework. The configuration in position 2 was established by a diastereoselective LiBH(4) reduction and subsequent Mitsunobu inversion. Structure-affinity relationships demonstrate that substituents in position 2 decrease sigma(1) receptor affinity which might be due to unfavorable interactions with the sigma(1) receptor protein. Without a substituent in position 2 high sigma(1) affinity was obtained (23a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane): K(i)=11 nM). Experiments with six human tumor cell lines showed a weak but selective growth inhibition of the human small cell lung cancer cell line A-427 by the methyl ethers ent-16b (IC(50)=18.9 microM), 21a (IC(50)=16.4 microM), ent-21a (IC(50)=20.4 microM), and 21b (IC(50)=27.1 microM) and the unsubstituted compounds 23a and 23b (42% inhibition at 20 microM).

Unusual reactivity of cytotoxic cis-dihydrazide Pt(II) complexes in aqueous solution

Complexes of the general structure cis-[PtX(2)(hydrazide)(2)] and cis-[PtX(2)NH(3)(hydrazide)], where X=Cl(-), Br(-) and I(-), and hydrazide=cyclohexylcarboxylic acid hydrazide (chcah), cyclopentylcarboxylic acid hydrazide (cpcah), 3-aminocyclohexanspiro-5-hydantoin (achsh) and 3-aminocyclopentanspiro-5-hydantoin (acpsh), were investigated with respect to aqueous stability, DNA platination rates and cytotoxic activity on a panel of seven human cancer cell lines as well as a cisplatin-resistant cell line. Stabilities in aqueous solution, determined by RP-HPLC and UV-Vis methods, were highly dependent on the type of halide ligand, with stability decreasing in the order I(-)>Cl(-)>Br(-). Added chloride (100 mM) only stabilized the dichloro-Pt(II) complexes containing the hydrazide as part of a hydantoin ring (i.e., achsh). Platination of calf thymus DNA determined by AAS was most rapid with dichloro-Pt(II) complexes containing achsh ligand. The mixed-amine dichloro-Pt(II) complexes with either chcah or cpcah ligands also platinated DNA >80%, but at a slower rate, while dihydrazide dichloro-Pt(II) complexes with either chcah or cpcah ligands resulted in <25% DNA platination at 24 h. cis-[PtX(2)(hydrazide)(2)], where hydrazide=chcah or cpcah, were the most potent compounds (chcah>cpcah), but activity was independent of the halide ligand (I(-)=Cl(-)=Br(-)). These complexes showed no cross-resistance with cisplatin, but they also showed little differentiation in potency over the seven cell lines. Complexes with the hydantoin ligands achsh and acpsh were inactive in all cell lines. Thus, neither stability in aqueous media nor covalent binding to DNA are correlated with biological activity, suggesting that cis-dihydrazide Pt(II) complexes act by a unique mechanism of action.