Medicinal Chemistry - Volume 1, Issue 5, 2005

Genetically engineered herpes simplex virus ICP34.5 null mutants replicate only in dividing cells and have shown potential for the treatment of malignant disease, including glioma. Phase I trials have demonstrated the safety of these viruses in various clinical settings but it is envisaged that for full efficacy they will be used in combination with other therapeutic modalities. To enhance virus-induced tumour cytotoxicity, we have engineered an ICP34.5 null mutant (HSV1716) of HSV1 which expresses the noradrenaline transporter gene (NAT). This virus is designated HSV1716/NAT. We have shown previously that introduction of the NAT gene into a range of tumour cells, via plasmid-mediated transfection, conferred the capacity for active uptake of the radiopharmaceutical [131I]MIBG and resulted in dosedependent toxicity. In this study, combination therapy utilising HSV1716/NAT and [131I]MIBG was assessed in vitro by the MTT assay. We demonstrate that the NAT gene, introduced by HSV1716/NAT into cultured glioma cells, was expressed 1 h after viral infection, enabling active uptake of [131I]MIBG. The combination of viral oncolysis and induced radiopharmaceutical uptake resulted in significantly enhanced cytotoxicity compared to either agent alone and the response was dose- and time-dependent. These studies show that the combination of oncolytic HSV therapy with targeted radiotherapy has the potential for effective tumour cell kill and warrants further investigation as a treatment for malignant glioma.

Overexpression of membrane bound, ATP-dependent transport proteins is one of the predominant mechanisms leading to multiple drug resistance in tumor therapy as well as in the treatment of bacterial and fungal infections. In tumor therapy, P-glycoprotein (P-gp, ABCB1) is responsible for transport of a wide variety of natural product toxins out of tumor cells leading to decreased accumulation of cytotoxic drugs within the cells. Inhibition of P-gp thus gives rise to a resensitization of multidrug resistant tumor cells and represents a versatile approach for modulation of multidrug resistance. Within this paper, a set of propafenone-type inhibitors of P-gp were analyzed using both interaction field based methods such as CoMFA and CoMSIA and Hologram QSAR. With both methods, highly predictive models with q2-values > 0.65 were obtained. Models using logP as additional descriptor generally yielded higher predictive power. On basis of unfavorable steric and favorable electrostatic and hydrophobic interaction fields, these models were able to explain all outlayers identified in previous Hansch-analyses. For HQSAR analysis, models with q2-values up to 0.72 were obtained. Positive influences were found for electron donating groups on the aromatic systems. Highly negative influences were found for diphenylalkylamine substituents, which is a further hint for steric hindrance. The models with highest predictive power were used for screening of a small virtual library. Synthesis and pharmacological testing of a sub set of this library showed that the external predictivity of the HQSAR models generally is lower than the internal one.

The syntheses of (R)- and (S)-norcarnitine ethyl esters are described starting with an optimized, chiral chemical reduction of ethyl 4-chloroacetoacetate followed by azide substitution, reduction, and dimethylation. The reaction of (R)- and (S)-norcarnitine ethyl esters with 1-bromoheptadecan-2-one gives (+)- and (-)-6-[(methoxycarbonyl)methyl]-2- pentadecyl-4,4-dimethylmorpholinium bromide, respectively, which hydrolyzes to (+)- and (-)-6-(carboxylatomethyl)-2- pentadecyl-4,4-dimethylmorpholinium (hemipalmitoylcarnitinium, (+)- and (-)-HPC), respectively, upon treatment with a hydroxide resin. (+)- and (-)-HPC are reversible active-site directed inhibitors of hepatic mitochondrial CPTs. Both stereoisomers inhibit CPT I and CPT II in control and streptozotocin diabetic rat to the same extent (Imax = 100%). Using intact mitochondria (CPT I), I50 values for (-)-HPC and (+)-HPC were 15.5 μM and 47.5 μM, respectively. The I50 values for CPT II were 6.7 μM and 38.5 μM for (-)-HPC and (+)-HPC, respectively. The mode of inhibition was uncompetitive for CPT I with respect to acyl-CoA. The apparent Ki for (-)-HPC is about 5 μM. These data suggest that (-)-HPC may be useful for further evaluation as an antidiabetic agent.

Free energy perturbation studies have been performed on Glucoamylase II (471) from Aspergillus awamori var. X100 complexed with three different inhibitors: (+)lentiginosine, (+)(1S,2S,7R,8aS) 1,2,7-trihydroxyindolizidine, (+)(1S,2S,7S,8aS) 1,2,7-trihydroxyindolizidine and the inactive compound (+)(1S,7R,8aS)-1,7-dihydroxyindolizidine. Molecular dynamic simulations were carried out using a recently developed procedure for fast Free Energy Perturbation calculations. In this procedure only a sphere of 1.8 nm around the central atom of the inhibitor is considered in the calculations. Crystallographic restraints are applied over this reduced system using a generated electron density map. The obtained values for the free energy differences agree with experimental data showing the importance of fast calculations in drug design even when the crystallographic structure of the complex is not available. As the method uses only the crystallographic structure of the receptor, it is possible to test the possible efficiency of even still not synthesised ligands, making the pre-selection of compounds much easy and faster.

An increased oxidative stress may contribute to the development of diabetic nephropathy. We have recently reported that high glucose level stimulated superoxide production through protein kinase C (PKC)-dependent activation of NAD(P)H oxidase in cultured vascular cells. Here we show that 3-hydroxy-3-methylglutaryl CoA reductase inhibitor (statin) attenuates both high glucose level-induced and angiotensin II (Ang II)-induced activation of p42/44 mitogenactivated kinase (MAP kinase) in cultured human mesangial cells through inhibition of NAD(P)H oxidase activity. The intracellular oxidative stress in cultured mesangial cells was evaluated by electron spin resonance (ESR) measurement. MAP kinase activity was evaluated by western blot analysis using anti phospho-specific MAP kinase antibody and anti- ERK-1 antibody. Exposure of the cells to high glucose level (450mg/dl) for 72 hrs significantly increased MAP kinase activity as compared to normal glucose level (100mg/dl). This increase was completely blocked by the treatment of pitavastatin (5x10-7M) as well as a NAD(P)H oxidase inhibitor (diphenylene iodonium, 10-5M) in parallel with the attenuation of oxidative stress. Ang II-induced activation of MAP kinase was also completely blocked by pitavastatin as well as a diphenylene iodonium in parallel with the attenuation of oxidative stress. In conclusion, pitavastatin attenuated high glucose-induced and Ang II- induced MAP kinase activity in mesangial cells through inhibition of NAD(P)H oxidase. Thus, statins may have a potential as a therapeutic tool for early diabetic nephropathy.

Inherited and somatic mutations in the APC gene, a human tumor-suppressor, occur in a large percentage of colon cancers, leading to elevated levels of nuclear β-Catenin, and to activation of TCF/ β-Catenin-responsive genes including cyclin D1 and c-myc. To identify small molecule antagonists of this pathway, we screened transformed colorectal cells with a secondary structure-templated chemical library, in search of compounds that attenuated a TCF/ β- Catenin-responsive reporter gene. From this library we selected ICG-001 (IC50 = 3 M) as a lead compound. Design and synthesis of the chemical library and some preliminary biological evaluation is described.

The enantiomers of the potent cognition-enhancer DM232 ((1), unifiram) and of its isopropylsulfonyl analog (2), which is endowed with amnesic properties, have been synthesized using (S)- and (R)-5-(hydroxymethyl)-2- pyrrolidinone as chiral precursors. The enantiomeric excess was determined by means of capillary electrophoresis, and found higher than 99.9 %. DM232 enantiomers were tested as cognition-enhancers in the passive-avoidance and social learning tests, and their ability to induce ACh release from rat cerebral cortex was also determined; in all the performed essays, (R)-(+)-(1) displayed higher potency than its (S)-(-) enantiomer, being able to elicit comparable effects at 3-fold to 10-fold lower doses. On the contrary, (R)-(+) and (S)-(-)-(2) showed the same amnesic potency when tested in the passiveavoidance test. These findings may be useful to clarify the mechanism of action of these substances.

Analogues of talampanel (1), a highly active AMPA antagonist 2,3-benzodiazepine, were synthesized, where the characteristic amino-function was either transposed or sterically shielded. For the key intermediates (hemiketals 6a, b) a new synthetic method of different mechanism was developed. The inactivity of several new compounds indicates the significance of the 4-amino(phenyl) function in BDZs of type 1.

The solution models of [Tyr3]octreotate (DPhe1-Cys2-Tyr3-DTrp4-Lys5-Thr6-Cys7-Thr8-COOH, disulfide bridged) (I), its analogs functionalized with an open chain tetraamine chelator, N4-[Tyr3]octreotate (II), and the N4- (Asp)2-[Tyr3]octreotate (III) peptide have been determined through 2D 1H NMR spectroscopy in DMSO. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring during attachment of the tetraamine to the peptide backbone. NMR-derived geometrical constraints have been used in order to calculate high resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues, have shown that these somatostatin analoges adopt a predominant antiparallel β-sheet conformation characterized by a β-like turn spanning residues DTrp4 and Lys5 which is supported in the case of N4-(Asp)2-[Tyr3]octreotate and N4-[Tyr3]octreotate by medium range NOEs. These data indicate that the above-mentioned molecules adopt a rather constrained structure in the 4-residue loop Tyr3-Thr6. Additionally, the C-terminal of [Tyr3]octreotate, comprising Cys7 and Thr8, appears to form a turn-like structure manifested by characteristic side-chain NOEs between Lys5 and Thr8, which have not been detected for the other two compounds. These data are discussed in the light of previous structural data of Sandostatin (octreotide) and suggest that attachment of the N4-chelator and two Asp residues at the N-end of [Tyr3]octreotate impose considerable structural changes and affect the binding properties of these peptides. Indeed, the IC50 values determined during competition binding assays against the sst2 (somatostatin subtype 2 receptor) suggest that the presence of the N4 group enhances receptor affinity, while extension of peptide chain by two negatively-charged Asp residues impairs receptor affinity at approximately one order of magnitude.

Starting from our lead compound, VL-0395, an anthranilic acid based CCK1 receptor antagonist, and following the well established "step by step" lead investigation strategy, we describe the final step of the anthranilic acid N-terminal optimization. Improvements for both affinity and selectivity towards CCK1 receptors have been accomplished through introduction of the fluoro substituent at C-5 and C-7 position of the indole ring together with the appropriate configuration of the aminoacidic chiral center.

The pharmaceutical industry currently suffers unsustainably high program failure rates despite our best efforts to implement drug design methods and to develop high throughput biochemical screening technologies over the past 20 years. While much of this failure is rationalized to be due to uncontrollable late stage drug development issues and clinical events, it has become increasingly clear that the choices we make in early drug discovery are vital to the ultimate failure or success outcomes of our drug discovery programs. The judicious selection of high probability of success therapeutic modalities, the rigorous determination of leadlikeness and druglikeness, and the all-important selection of high probability of success enzyme and receptor targets are the vital drivers of failure and success in small molecule drug discovery as it is performed in the age of biochemical screening. Consideration of these guiding principles will improve our chances of success in drug discovery, and increase our ability to address unmet medical need in the future.