Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands.

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ1 and σ2 receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC50 values in the low µM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl2 family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.

Sustainable synthesis and automated deposition: an accessible discovery screening library of fragment-like purines.

A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays.

Synthesis and biological evaluation of conformationally restricted σ(1) receptor ligands with 7,9-diazabicyclo[4.2.2]decane scaffold.

The key step in the synthesis of the 7,9-diazabicyclo[4.2.2]decane system was a modified Dieckmann condensation of piperazinebutyrate 11, which makes use of trapping the first cyclized intermediate with TMS-Cl. Reduction of the bicyclic ketone 14 with LiBH(4) at -90 °C provided diastereoselectively (>99 : 1) the syn-configured alcohol 15a, which was converted into the final alcohol and ethers 16a-g. The configuration at the 2-position was established by X-ray structure analysis of methyl and ethyl ethers 15b and 15c. In contrast to bicyclic systems with a three-carbon bridge, inversion of the configuration at the 2-position of the alcohol 15a failed to give the inverted alcohol 19a. However, an unselective reduction of the ketone 24 with L-Selectride led to the diastereomeric alcohols 16a and 25a in the ratio 36 : 64. LiAlH(4) reduction of the tosylate 20 and the alkene 18 yielded the diazabicyclo-decane 26 and -decene 27 without further substituents at the four-carbon bridge. The σ(1) and σ(2) receptor affinities were investigated in receptor binding studies with radioligands. All test compounds showed a lower σ(1) affinity than the corresponding bicyclic derivatives with a three-membered bridge. The reduced σ(1) receptor affinity is attributed to the larger four-membered bridge. This hypothesis is supported by the alkene 27, which represents the most potent σ(1) ligand of this series (K(i) = 7.5 nM). In the alkene 27 the size and flexibility of the bridge is considerably reduced by the double bond. The methyl ether 25b and the unsubstituted derivatives 26 and 27 revealed moderate inhibition of the growth of the human tumor cell lines A-427, 5637 and MCF-7. Again, these compounds are less potent than the analogues with a three-membered bridge. The IC(50)-value of the most potent σ(1) ligand 27 against the small cell lung cancer cell line A-427 (IC(50) = 10 µM) should be emphasized, since this cell line is particularly sensitive to homologues with a three-carbon bridge.

Rigidity versus Flexibility: Is This an Issue in σ1 Receptor Ligand Affinity and Activity?

Stereoisomeric 2,5-diazabicyclo[2.2.2]octanes 14 and 15 were prepared in a chiral-pool synthesis starting from (S)- or (R)-aspartate. The key step in the synthesis was a Dieckmann-analogous cyclization of (dioxopiperazinyl)acetates 8, which involved trapping of the intermediate hemiketal anion with Me3SiCl. The σ1 affinity was tested using membrane preparations from animal (guinea pig) and human origin. The binding of bicyclic compounds was analyzed by molecular dynamics simulations based on a 3D homology model of the σ1 receptor. The good correlation between Ki values observed in the σ1 assays and calculated free binding energy, coupled with the identification of four crucial ligand/receptor interactions, allowed the formulation of structure-affinity relationships. In an in vitro antitumor assay with seven human tumor cell lines, the bicyclic compounds inhibited selectively the growth of the cell line A427, which is due to induction of apoptosis. In this assay, the compounds behave like the known σ1 receptor antagonist haloperidol.

Synthesis, pharmacological evaluation, and σ1 receptor interaction analysis of hydroxyethyl substituted piperazines.

Starting from (S)- or (R)-aspartate, three synthetic strategies were explored to prepare hydroxyethyl substituted piperazines with different substituents at the N-atoms. σ receptor affinity was recorded using receptor material from both animal and human origin. σ1 affinities determined with guinea pig brain and human RPMI 8226 tumor cell lines differed slightly but showed the same tendency. (S)-2-[4-(Cyclohexylmethyl)-1-(naphthalene-2-ylmethyl)piperazin-2-yl]ethanol (7c) revealed the highest affinity at human σ1 receptors (Ki = 6.8 nM). The potent σ1 receptor ligand 7c was able to inhibit selectively the growth of three human tumor cell lines with IC50 values in the low micromolar range. The reduced growth of the RPMI-8226 cell line was caused by apoptosis. The interaction of 7c with the σ1 receptor was analyzed in detail using the 3D homology model of the σ1 receptor. The calculated free binding energies of all hydroxyethylpiperazines nicely correlate with their recorded affinities toward the human σ1 receptor.

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

Various Pt(IV) 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 Pt(IV) diazides tend to be less specific than cisplatin at inhibiting cancer cell growth, but cells resistant to cisplatin show little cross-resistance to Pt(IV) 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 Pt(IV) diazides. Structures of some of the photolysis products were elucidated by LC-MS/MS; monoaqua- and diaqua-Pt(II) 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-Pt(II) 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-Pt(IV) 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.

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 Pt(IV) diazido complexes have unusual photobiologic properties. We show here that trans,trans,trans-[Pt(IV)(N(3))(2)(OH)(2)(py)(NH(3))] complex 3 is a potent photoactivated cytotoxin toward human cancer cells in culture, with an average IC(50) value in 13 cell lines of 55 ± 28 µmol/L after 30 minutes (0.12 mW/cm(2)) 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 (14)N{(1)H} 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.