Süreyya Ölgen

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17α-hydroxylase-17.20-lyase (CYP17)-Part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.

The Role of Fluorine Substitution in Biphenyl Methylene Imidazole-Type CYP17 Inhibitors for the Treatment of Prostate Carcinoma

It has been established that the growth of most prostate carcinomas depends on androgen stimulation. The inhibition of cytochrome P450‐17 (CYP17) to block androgen biosynthesis is therefore regarded as a promising approach to therapy. Based on our previously identified lead compound Ref 1, a series of fluorine‐substituted biphenyl methylene imidazoles were designed, synthesized, and evaluated as CYP17 inhibitors to elucidate the influence of fluorine on in vitro and in vivo activity. It was found that meta‐fluoro substitution at the C ring improved activity, whereas ortho substitution decreased potency. Docking studies performed with our human CYP17 homology model suggest the presence of multipolar interactions between fluorine and Arg109, Lys231, His235, and Glu305. As expected, introduction of fluorine also prolonged the half‐life in plasma. The SARs obtained confirm the reliability of the protein model; compound 9 (IC50=131 nM) was identified as a strong CYP17 inhibitor, showing potent activity in rat, high bioavailability, and a long plasma half‐life: 12.8 h.

Site-specific modification of Shigella flexneri virF mRNA by tRNA-guanine transglycosylase in vitro

Shigella flexneri is an enteropathogen responsible for severe dysentery in humans. VirF is a key transcriptional regulator that activates the expression of the downstream virulence factors required for cellular invasion and cell-to-cell spread of this pathogen. There are several environmental factors that induce the translation of VirF including temperature, pH, osmolarity and post-transcriptional RNA modification. Durand and colleagues (vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase of Escherichia coli K-12. J. Bacteriol., 176, 4627–4634) have demonstrated a correlation between VirF and tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of the hypermodified base queuine for the guanine in the wobble position of certain tRNAs. They characterized tgt- mutant S. flexneri strains in which the translation of VirF is markedly reduced and the bacteria are unable to invade host cells. Although the function of TGT is to modify tRNA, we report that the virF mRNA is recognized by the Escherichia coli TGT (99% identity to the S. flexneri TGT) in vitro. Further, we show that this recognition results in the site-specific modification of a single base in the virF mRNA. In the context of previous reports that small molecule binding motifs (‘riboswitches’) in mRNAs modulate mRNA conformation and translation, our observations suggest that TGT may modulate the translation of VirF by base modification of the VirF encoding mRNA.

Synthesis and receptor docking studies of N-substituted indole-2-carboxylic acid esters as a search for COX-2 selective enzyme inhibitors

A series of N-substituted indole-2-carboxylic acid esters have been prepared by replacing the benzoyl group of indomethacin with a benzyl and a phenyl group. The carbocyclic acid side chain was extended via creating an ester structure by using several dialkylaminoalkyl groups. The receptor docking studies were performed to investigate the docking mode of each compound by using DOCK 4.0. All the compounds were shown to be docked at the site where intact flurbiprofen was embedded for COX-1 and s-58 (1-phenylsulphonamide-3-trifluoromethyl-5-para-bromophenylpyrazole) for COX-2. It was predicted that N-phenyl-indole-2-carboxylic acid piperazine ester 22 can be a fairly strong COX-2 selective compound which was compared to the others. Other predicted COX-2 selective compounds included are N--H indole-2-carboxylic acid diethyl 30 and piperazine 34 esters. In view of these findings, compounds 22, 30 and 34 were chosen for the in vitro biological assays.

Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors

Hyaluronic acid (HA) is the major biopolymer of the extracellular matrix and contributes significantly to cell proliferation and migration. Human hyaluronidase hPH-20 has been identified as a tumor marker for breast and laryngeal cancer. A hPH-20-autotransporter fusion protein for cell surface display was transformed into Escherichia coli BL21 (DE3) and hPH-20 was displayed on the surface of E. coli. Enzymatic activity, however, was not detectable due to competitive inhibition by lipopolysaccharide (LPS). Finally, expression in E. coli F470, a strain missing the O-polysaccharide of LPS, yielded cells with sufficient hyaluronidase activity. 6-Palmitoyl-l-ascorbic acid (Vcpal) and two indole-carboxamides, N-(4-fluorobenzyl)-1-benzyl-1H-indole-2-carboxamide (1) and N-(4-chlorobenzyl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide (2), were tested on inhibition of hPH-20. Vcpal with a concentration of 5 μM inhibited hPH-20 to 93% at pH 7, compounds 1 and 2 showed 61% and 21% inhibition at a concentration of 50 μM. At the same inhibitor concentrations the most frequently used bovine testes hyaluronidase (BTH) was inhibited by Vcpal to a similar extent (95%), whereas compound 1 (80%) and compound 2 (66%) showed much differing inhibition. Thus it can be assumed that BTH is not applicable as an alternative to human PH-20. These results indicate that Autodisplay enables the expression of human target enzymes normally forming inclusion bodies in E. coli and accelerates inhibitor testing as shown by the example of human hyaluronidase PH-20.

Synthesis and biological evaluation of N-substituted indole esters as inhibitors of cyclo-oxygenase-2 (COX-2)

A series of novel N-substituted indole carboxylic, acetic and propionic acid esters have been prepared as possible cyclo-oxygenase-2 (COX-2) enzyme inhibitors. Compounds 20, 23 were found slightly active against COX-2. The synthesis of indole carboxylic, acetic and propionic acid esters were furnished by using dicyclohexyl carbodiimide (DCC), dimethylamino pyridine (DMAP) as carboxylate activators. N-substitution of indole esters was verified with several benzyl and benzoyl group in presence of NaH in DMF, respectively.

Synthesis and antioxidant properties of novel N-substituted indole-2-carboxamide and indole-3-acetamide derivatives

A series of N‐substituted indole‐2‐carboxamide and indole‐3‐acetamide derivatives have been prepared and their in vitro effects on rat liver lipid peroxidation levels and superoxide anion formation were determined. The results clearly demonstrate that indole derivatives 4, 5, 10, 15, 17 are very efficient antioxidants compared to α‐tocopherol.

Synthesis and Activity of Novel 5‐Substituted Pyrrolo[2,3‐d]pyrimidine Analogues as pp60c‐Src Tyrosine Kinase Inhibitors

Therapy with receptor tyrosine kinase inhibitors provides an improved treatment option in a number of diseases such as cancer, myocardial infection, osteoporosis, stroke, and neurodegeneration. We have designed, synthesized, and evaluated a series of novel 2‐amino‐5‐[(benzyl)imino]methyl‐3,7‐dihydro‐4H‐pyrrolo[2,3‐d]pyrimidine‐4‐one 7a and 2‐amino‐5‐[(substituted‐benzyl)imino]methyl‐3,7‐dihydro‐4H‐pyrrolo[2,3‐d]pyrimidine‐4‐one 7b‐e derivatives as potential tyrosine kinase inhibitors. These compounds were synthesized by condensation reaction using 2‐tritylamino‐4‐oxo‐4,7‐dihydro‐3H‐pyrrolo[2,3‐d]pyrimidine‐5‐carbaldehyde 5 and appropriate benzylamines followed by detritylation. Compounds were evaluated for their inhibitory activity toward tyrosine phosphorylation for the pp60c‐Src tyrosine kinase. Compounds 7a, 7d, and 7e demonstrated potent inhibitory activities against pp60c‐Src tyrosine kinase with IC50 values of 13.9, 34.5, and 78.4 μM, respectively. Dihalogenated compounds 7d and 7e have 3 to 7‐times lower IC50 values than that of the parent compound 7a.

New potent indole derivatives as hyaluronidase inhibitors.

Because of the physiologic importance of hyaluronidases, the identification of potent and selective inhibitors of hyaluronidases has become increasingly important. A variety of assay methods have been used for such a purpose, i.e. classical turbidimetric, viscometric and colorimetric. In this study, a modified enzymatic assay has been used to obtain a microtiter plate‐based sensitive activity screening. All inhibitors were tested in a stains‐all assay at pH 7 and in a Morgan‐Elson assay at pH 3.5. Among the tested compounds, 1, 2, 3, 6, 7, 8, 16, 17 and 18 showed good inhibition of more than 50%, so the IC50 values of these derivatives were determined in the range of 25–41 μm. The IC50 value of the most active hyaluronidase inhibitor Vcpal (6‐palmitoyl‐l‐ascorbic acid) was measured as 8.36 μm. All inhibitors including Vcpal showed twofold less activity at pH 3.5 in a Morgan‐Elson assay. Examination of substituent effects on the activity showed that para‐positions of benzamide needs to be chlorinated or fluorinated to obtain good inhibitory effect. It was found that the introduction of a p‐fluoro benzyl ring in the indole nitrogen has a positive effect for the inhibitory effects of both indole‐2‐ and 3‐carboxamide derivatives.

Evaluation of Indole Esters As Inhibitors of p60c-Src Receptor Tyrosine Kinase and Investigation of the Inhibition Using Receptor Docking Studies

Several indole esters were tested as inhibitors of tyrosine kinase p60c-Src. Compound (4) was found fairly active against the enzyme with IC50=1.34 μM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60c-Src tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.