DNA Groove Binding Research Articles

DNA groove-binding and acid-base properties of a Ru(II) complex containing anthryl moieties

DNA groove binders have been poorly studied as compared to the intercalators. A novel Ru(II) complex of [Ru(aeip)2(Haip)](PF6)2 {Haip = 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline and aeip = 2-(anthracen-9-yl)-1-ethyl-imidazo[4,5-f][1, 10]phenanthroline} is synthesized and characterized by elemental analysis, 1H NMR spectroscopy and mass spectrometry. The complex is evidenced to be a calf-thymus DNA groove binder with a large intrinsic binding constant of 106 M−1 order of magnitude as supported by UV–visible absorption spectral titrations, salt effects, DNA competitive binding with ethidium bromide, DNA melting experiment, DNA viscosity measurements and density functional theory calculations. The acid-base properties of the complex studied by UV–Vis spectrophotometric titrations are reported as well.

Structural and Biological Investigations for a Series of N-5 Substituted Pyrrolo[3,2-d]pyrimidines as Potential Anti-Cancer Therapeutics.

Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds for the development of antiproliferative agents. Much of the focus has been on modifications to the pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast, this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or RNA alkylators. In addition, N5 substitution reduced the EC50 against CCRF-CEM leukemia cells by up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.

Spectroscopic and In Silico DNA Binding Studies on the Interaction of Some New N-Substituted Rhodanines with Calf-thymus DNA: In Vitro Anticancer Activities.

In this era of science, cancer is a black dot on the face of humankind. Consequently, the search of promising anticancer agents continues. Here we designed and synthesized new N-substituted rhodanines (RD1-7), evaluated their multispectroscopic interaction with calf thymus DNA, in silico and anticancer studies against MDA-MB-231cancer cell line. By MTT assay rhodanine RD1 was found to be the most potent with IC50 value of 72.61 μM. In addition, DNA binding studies (UV-vis and fluorescence) revealed strong binding affinity of RD1-7 with DNA (Kb in the range of 1.5-7.4 × 105 M-1). Moreover, molecular docking study, experimental DNA binding and anticancer studies are all well agreed to each other. It was observed that H-bonding and hydrophobic attractions were responsible for stability of DNAcompound adducts. Besides, the reported rhodanines (RD1-7) were found as minor groove binders of DNA. Concisely, RD1-7 indicated promising pharmacological properties and hence, shows auspicious future for the development of novel anticancer agents. The reported rhodanines showed excellent anticancer properties. Therefore, the described rhodanines may be used as potential anticancer agents in the future.

Synthesis, characterization and DNA binding studies of a new ibuprofen–platinum(II) complex

The study is focused on the synthesis of a novel complex of ibuprofen and Platinum(II). The formation of the product was characterized through analytical tools including Fourier-transform infrared spectroscopy, proton nuclear magnetic resonance, ultraviolet–visible spectroscopy, mass spectrometry as well as density functional theory. Using the continuous variation method, the stoichiometry of Pt(HIb)2(Cl)2 binding on DNA (ct-DNA) determines as a single class of binding. Based on the results of Stern–Volmer analysis on the fluorescence quenching data, the quenching mechanism was determined to be static in nature. The studies indicated that the complex could bind DNA molecules via groove binding for four major reasons. Initially, the complex–DNA binding constant determined based on spectrophotometric data were found to be comparable to those of groove-binding drugs. In addition, the competitive fluorimetric based on the applications of Hoechst 33258 proved the ability of Pt(HIb)2(Cl)2 to displace with Hoechst in its DNA-bounded form, reflecting the competition between Pt(HIb)2(Cl)2 and Hoechst for groove binding. Further, no considerable changes were observed in the intensity of the methylene blue (MB)-DNA system after adding the Pt(HIb)2(Cl)2 complex, reflecting the stability of MB molecules in the DNA helix and a non-intercalative bonds of Pt(HIb)2(Cl)2 interaction on DNA. Finally, minor changes in the viscosity of DNA in the presence of Pt(HIb)2(Cl)2, indicated that changes in the length of DNA in the presence of the complex are negligible, supporting the assumption of DNA groove-binding. Also induced CD spectral changes and docking simulations were in favor of the groove mechanism for the Pt(HIb)2(Cl)2–DNA binding.Communicated by Ramaswamy H. Sarma

4-Acetamidophenol Binding Mechanism with DNA by UV-Vis and FTIR Techniques Based on Binding Energy, LUMO and HOMO Orbitals and Geometry of Molecule

Abstract Present study was conducted to appraise the interaction mechanism of 4-acetamidophenol (4-AP) with DNA based on UV-Vis and FTIR techniques based on binding energy, isolated atomic energy, LUMO and HOMO orbitals gap and geometry of molecule. Analysis revealed the groove binding and intercalation mode of interaction between 4-AP and DNA since hyperchromic and bathochromic shifts were observed in response of interaction of DNA. The planar part of interacting molecule intercalated with DNA and non-planar part of 4-acetamidophenol bounded with DNA (groove binding). The constants for binding between 4-AP and DNA were calculated and 20.12 × 103 mol−1 dm3 binding constant was recorded at pH 4.7, whereas this value was 5.32 × 103 mol−1 dm3 for the pH 7.4. The binding constant value for interaction of 4-AP with DNA revealed the possibility of oral administration of 4-AP. The 4-AP binding with DNA is spontaneous process, which was confirmed from negative value of free energy at room temperature. FTIR study revealed that C–H and C=C (aromatic) functional groups were involved in binding at pH 4.7 and C=O (amide) was involved in groove binding, whereas C–H (aromatic) was responsible for intercalation at pH 7.4 and C–H (alkaline) and C=O (amide) were responsible for groove binding at pH 4.7.

A PROMISING DNA GROOVE BINDER AND PHOTOCLEAVER BASED ON A DINUCLEAR RUTHENIUM(II) COMPLEX

A dinuclear ruthenium complex [Ru2(bpy)4(bip-phenol)](ClO4)4 {bpy = 2,2′-bipyridine, bip-phenol = 2,4-bis(1 H -imidazo[4,5- f ][1,10]phenanthrolin-2-yl) phenol} has been synthesized and characterized. The calf thymus (ct) DNA binding properties of the complex are investigated by means of DNA viscosity and optical spectroscopic techniques of UV-visible absorption and emission spectral titrations, steady-state emission quenching with ferrocyanide, ethidium bromide competitive binding, DNA thermal denaturation and reverse salt effect, together with molecular simulation technology. The results suggest that the complex is a promising DNA groove binder with a large DNA binding constant on 106 M−1 order of magnitude. The fluorescence of the complex manifests by 6.3-fold upon binding saturately to DNA. The complex is also demonstrated to be an efficient photocleaver of pBR 322 DNA.

Design, synthesis, and DNA interaction studies of furo-imidazo[3.3.3]propellane derivatives: Potential anticancer agents

A large number of natural products containing the propellane scaffold have been reported to exhibit cytotoxicity against several cancers; however, their mechanism of action is still unknown. Anticancer drugs targeting DNA are mainly composed of small planar molecule/s that can interact with the DNA helix, causing DNA malfunction and cell death. The aim of this study was to design and synthesize propellane derivatives that can act as DNA intercalators and/or groove binders. The unique structure of the propellane derivatives and their ability to display planar ligands with numerous possible geometries, renders them potential starting points to design new drugs targeting DNA in cancer cells. New substituted furo-imidazo[3.3.3]propellanes were synthesized via the reaction of substituted alkenylidene-hydrazinecarbothioamides with 2-(1,3-dioxo-2,3-dihydro-1H-2-ylidene)propanedinitrile in tetrahydrofuran at room temperature. The structures of the products were confirmed by a combination of elemental analysis, NMR, ESI-MS, IR and single crystal X-ray analysis. Interestingly, 5c, 5d and 5f showed an ability to interact with Calf Thymus DNA (CT-DNA). Their DNA-binding mode was investigated using a combination of absorption spectroscopy, DNA melting, viscosity, CD spectroscopy measurements, as well as competitive binding studies with several dyes. Their cytotoxicity was evaluated against the NCI-60 panel of cancer cell lines. 5c, 5d and 5f exhibited similar anti-proliferative activity against the A549 non-small cell lung cancer (NSCLC) cell line. Further mechanistic studies revealed their ability to induce DNA damage in the A549 cell line, as well as apoptosis, evidenced by elevated Annexin V expression, enhanced caspase 3/7 activation and PARP cleavage. In this study, we present the potential for designing novel propellanes to provoke cytotoxic activity, likely through DNA binding-induced DNA damage and apoptosis.

Synthesis and Molecular Docking Studies of Novel 5-Arylideneaminouracils

A series of novel 5-arylideneaminouracils is synthesized from 5-aminouracil and substituted salicylaldehydes, and characterized by spectroscopic methods. DNA interactions of model molecules are explored using molecular docking simulations that reveal that these types of molecules can act as DNA groove binders.

Elucidating DNA binding of dithienylethenes from molecular dynamics and dichroism spectra.

DNA binding modes of the stereoisomeric rotamers of two dithenylethene derivatives (DTE1 and DTE2) representing candidate molecular photoswitches of great promise for photopharmacology and nanotechnology have been identified and characterized in terms of their binding energies and electronic circular dichroism (CD) responses. In the open form, two binding modes are identified namely minor-groove binding of the lowest-energy conformer with an anti-parallel arrangement of methyl groups and major-groove double-intercalation of the P-enantiomers of an intermediate-state rotamer. Only the latter binding mode is found to be enantiomerically selective and expected to have an overall negative linear dichroism (LD) as observed in the experiment for DTE1 (Angew. Chem., Int. Ed., 2013, 52, 4393). In the closed form, the most favorable binding mode is found to be minor groove binding. Also this binding mode is found to be enantiomerically selective and for DTE1, it is the M-enantiomer that binds the strongest, showing a positive theoretical signature CD band in the long wavelength region with origin in pyridinium ligands. The theoretical CD spectrum is found to be in good agreement with the experimental one, which provides an indirect evidence for a correct identification of the binding mode in the closed form.

Cyclic Voltammetric DNA Binding Investigations on Some Anticancer Potential Metal Complexes: a Review.

Cancer is developed by rapid, uncontrolled, and abnormal cell proliferation and one of the leading causes of deaths worldwide in human beings. For the remedial measures of preventing different types of cancers, one of the research domains that have gained substantial importance in medical science is the development of new metallo-drugs and their investigations as potential anticancer drug agents by using various analytical techniques. Since metal-based complexes show weak absorption bands, electrochemical methods are considered more feasible and preferable over spectroscopic methods for easy characterization. Due to closer resemblance of electrochemical and biological processes, cyclic voltammetry among different electrochemical methods is considered the most versatile for the study of in-vitro metal-based drug-DNA interactions in terms of changes in the redox activities. Current potential data of a metal complex leads to determine binding kinetics in terms of binding constant and binding site size that involve determining the binding mode of drug with DNA, i.e., electrostatic interactions, intercalation, or minor-major groove binding. Binding parameters and modes of interactions, further, help to develop the mechanism of action of drug with the DNA. In this review, we emphasize on cyclic voltammetric DNA binding studies on some metal complexes that have been carried out in the last three decades for the investigation of their anticancer potentials.

Synthesis of steroidal imidazolidinthiones as potential apoptotic agents: Investigation by theoretical and experimental studies

New steroidal imidazolidinthione derivatives (4–6) were synthesized from steroidal thiosemicarbazones and dichloroethane. The synthesized compounds were characterized using spectral data analysis. Theoretical DFT involving B3LYP/6-31G∗∗ level of theory was employed to gain insights into the molecular structure of the target compounds. MEPS and FMO analysis were carried out. HOMO-LUMO energy gap was determined which helped to evaluate various global descriptors like hardness, chemical potential, electronegativity, nucleophilicity and electrophilicity index, etc. The calculated properties established that the synthesized products are more or less similar in their reactivity behaviour. To explore their biological potential, interaction studies of compounds (4–6) with DNA were carried out using various biophysical techniques. The compounds bind DNA preferentially through electrostatic and hydrophobic interactions with Kb of 3.21 × 103 M−1, 2.79 × 103 M−1 and 2.26 × 103 M−1, respectively indicating the higher binding affinity of compound 4 towards DNA. Gel electrophoresis of compound 4 demonstrated strong interaction during the concentration dependent cleavage activity with pBR322 DNA. It was observed that these steroidal imidazolidinthiones are minor groove binders of DNA which was validated using molecular docking studies. An in vitro cytotoxicity screening using MTT assay revealed that the compounds (4–6) exhibit potential toxicity against different human cancer cells. Highest antiproliferative effect was observed on HeLa cells by compound 4. The results suggested that compounds 4–6 cause apoptotic cell death by cleaving apoptotic protein caspase-3 and suppress anti-apoptotic protein Bcl-2 in HeLa cancer cells.

Capecitabine as a minor groove binder of DNA: molecular docking, molecular dynamics, and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 104 M−1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.

Understanding of the interactions of ctDNA with an antioxidant flavone analog: Exploring the utility of the small molecule as fluorescent probe for biomacromolecule

Spectroscopic, viscometric and molecular docking studies were done to ascertain the occurrence and nature of binding interactions between 4′-N,N-dimethylamino-3-hydroxyflavone (DMAHF), a potent antioxidant flavone analog having intrinsic fluorescence activity and calf thymus deoxyribonucleic acid (ctDNA) in Tris-HCl buffer (pH 7.4) medium. Results indicate that DMAHF adopted a completely non-planar structure due to its hydrogen bonding interactions with B-form of ctDNA at its minor groove. DNA-groove binding activity of DMAHF was also confirmed by performing its competitive binding studies with EtBr, a perfect intercalator. The structure of DMAHF showed strong influence on its mode of binding as evident from its single crystal XRD and DFT based optimized geometry. Moreover, the structural changes of DMAHF on binding with ctDNA greatly influenced its ESIPT based dual emission fluorescence behavior. This opens up its utility as fluorescence probe of biomacromolecules, like DNA.

The effect of dimerization on the interaction of ibuprofen drug with calf thymus DNA: Molecularmodeling and spectroscopic investigation

ABSTRACTThe interaction between the dimer structure of ibuprofen drug (D-IB) and calf thymus DNA under simulative physiological conditions was investigated with the use of Hoechst 33258 and methylene blue dye as spectral probes by the methods of UV-visible absorption, fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling study.Using the Job's plot, a single class of binding sites for theD-IB on DNA was put in evidence. The Stern–Volmer analysis of fluorescence quenching data shows the presence of both the static and dynamic quenching mechanisms. The binding constants, Kb were calculated at different temperatures, and the thermodynamic parameters ∆G∘, ∆H∘ and ∆S∘ were given. The experimental results showed that D-IB molecules could bind with DNA via groove binding mode as evidenced by: I. DNA binding constant from spectrophotometric studies of the interaction of D-IB with DNA is comparable to groove binding drugs. II. Competitive fluorimetric studies with Hoechst 33258 have shown that D-IB exhibits the ability of this complex to displace with DNA-bounded Hoechst, indicating that it binds to DNA in strong competition with Hoechst for the groove binding. III. There is no significantly change in the absorption of the MB-DNA system upon adding the D-IB, indicates that MB molecules are not released from the DNA helix after addition of the D-IB and are indicative of a non-intercalative mode of binding. IV. Small changes in DNA viscosity in the presence of D-IB, indicating weak link to DNA, which is consistent with DNA groove binding. As well as, induced CD spectral changes, and the docking results revealed that groove mechanism is followed by D-IB to bind with DNA.

Novel coumarin rhenium(I) and -(V) complexes: Formation, DFT and DNA binding studies

Herein, we report the formation and characterisation of novel rhenium(I) and -(V) compounds with coumarin bidentate chelates: trans-[ReOBr2(PPh3)(hbc)] (1) (Hhbc = 7-(2-hydroxybenzylideneamino)-4-(trifluoromethyl)-2H-chromen-2-one), fac-[Re(CO)3Cl(aomc)] (2) (aomc = 7-(((2-amino-4-oxo-4H-chromen-3-yl)methylene)amino)-4-(trifluoromethyl)-2H-chromen-2-one) and fac-[Re(CO)3Cl(moac)] (3) (moac = 7-(((2-methoxy-4-oxochroman-3-yl)methylene)amino)-4-(trifluoromethyl)-2H-chromen-2-one). The coumarin free-ligands and the metal complexes 1–3 were characterized by NMR, UV–Vis and FTIR spectroscopy, melting point and molar conductivity measurements as well as time-of-flight mass spectrometry. Their structural elucidations were supported by the respective solid state structures of 1, Hhbc and moac. DNA binding studies conducted using the facial tricarbonylrhenium(I) complexes 2 and 3, revealed that they are DNA groove binders with intrinsic binding constants in the order of 105 and 104 M bp, respectively. This study was also complemented using density functional theory (DFT) and time-dependent DFT methods to attain a deeper understanding into the structural parameters, infrared and electronic spectra of these coumarin free-ligands and their metal complexes 1–3.

Turning intercalators into groove binders: synthesis, photophysics and DNA binding properties of tetracationic mononuclear ruthenium(ii)-based chromophore-quencher complexes.

The synthesis of two new tetracationic mononuclear RuII complexes containing the tetrapyridyl [3,2-a:2',3'-c:3'',2''-h:2''',3'''-j] phenazine ligand in which the uncoordinated site has been converted into a dicationic ethylene-bipyridyldiylium unit is reported. The structure of the complexes is fully assigned through detailed NMR studies and, in one case, through an X-ray crystallography study. Voltammetry, optical spectroscopy and computational studies confirm that the bipyridyldiylium moiety has a low-lying reduction that quenches the 3MLCT-based emission usually observed in such systems. The new complexes interact with DNA in a quite different manner to their dicationic analogues: they both bind to duplex DNA with micromolar affinity through groove binding. These observations are rationalized through a consideration of their structural and electronic properties.

Synthesis, antitumor activity evaluation, and DNA-binding study of coumarin-based agents.

A novel series of coumarin-thiadiazole heterocycle derivatives was synthesized by the nucleophilic substitution reaction. The synthesized compounds were structurally verified by IR, 1 H NMR, 13 C NMR, mass spectra, and elemental analyses. The antitumor activity of the synthesized compounds was evaluated through DNA binding assays and the 60-cell line panel according to the US NCI-DTP protocol or a selection of human tumor cell lines: breast cancer (MCF-7), liver cancer (HepG-2), and colorectal cancer (HCT-116). Most of the compounds had better DNA/ethidium bromide fluorescence quenching rather than methyl green displacement, suggesting superior DNA intercalation over DNA groove binding. Compounds 8 and 14b showed the best quenching effect with KSV = 4.27 × 105 M-1 . Moreover, the results for compounds 8, 4c, and 4e revealed a possible dual DNA binding mode with the intercalation to be superior, with KSV 4.27 × 105 , 3.96 × 105 , and 3.51 × 105 M-1 , respectively, compared to 42%, 45%, and 43% methyl green displacement, respectively. Out of the 60-cell line panel, the leukemia HL-60 cell line was the most susceptible to growth inhibition when treated with 14a, resulting in 61% growth, followed by the lung carcinoma cell line NCI-H522 showing 67% growth when treated with 9. Moreover, compound 10c had an IC50 value of 24.9 μg/mL against the HepG-2 cell line.

Metal-Dependent DNA Recognition and Cell Internalization of Designed, Basic Peptides.

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5′-GTCAT-3′). However, addition of Pd(en)Cl2 (en, ethylenediamine) promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide–DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report (1) a designed, short peptide that displays highly selective, major groove DNA binding, (2) a reversible, metal-dependent DNA interaction, and (3) a metal-promoted cell internalization of this basic peptide.

Synthesis of a Naturally Occurring Plant Alkaloid Berberine Analogue and Its Application in Nanomolar Selective Detection of Hydrazine in Free and DNA‐Binding Situation.

AbstractA sensitive platform for in vivo selective detection of the toxic element by a DNA stabilized biomolecule is of great importance. Particularly sensing of hydrazine in the presence of DNA is very important during cell damage and post‐mortem. In this study, a new 9‐O‐(4‐nitrobenzoyl) berberine analogue was designed and synthesized. The Calf thymus DNA (CT‐DNA) binding affinity studies revealed that the analogue was excellent DNA binding efficacy with groove binding mode (K= 4.2 × 106 M−1) compared to parent berberine (K= 2.2 ×105 M−1). Importantly, our synthesized analogue for the first time a berberine analogue that can effectively use as a selective ratiometric colorimetric probe for hydrazine detection based on ICT (intramolecular charge transfer) mechanism with a good limit of detection (20nM) which is much below the threshold limiting value (TLV) of hydrazine (10 ppb or 312 nM) as regulated by Environmental protection agency (EPA). Interestingly, we report a new avenue in sensing study in the presence of DNA i. e. our DNA groove binding analogue (at maximum saturation) also can serve as a promising turn on fluorimetric sensor for hydrazine detection with high selectivity over biologically abundant amines and metal cations with well limit of detection (LOD) value (14 nM). The sensing mechanism in the absence of DNA and in the presence of DNA was established by different spectroscopic methods. In addition, the berberine analogue could also be utilized for qualitative detection of hydrazine in the vapor phase and for the imaging of hydrazine in living Michigan Cancer Foundation‐7 (MCF‐7) cells.

Novel amidino substituted benzimidazole and benzothiazole benzo[b]thieno-2-carboxamides exert strong antiproliferative and DNA binding properties

Within this manuscript design, synthesis of novel 2-imidazolinyl substituted benzo[b]thieno-2-carboxamides bearing either benzimidazole or benzothiazole subunit and biological activity are presented and described. The antiproliferative activities were assessed in vitro on a panel of human cancer cell lines. Tested compounds showed moderate activity while cytotoxicity on normal fibroblasts was lower in comparison with 5-fluorouracile. The variations of 2-imidazolinyl substituent at heteroaromatic subunits in different positions led to different cytotoxic properties. The strongest selective activity against HeLa cells was observed for the benzothiazole derivative 4d with 2-imidazolinyl group at the benzo[b]thiophene subunit with a corresponding IC50 = 1.16 μM. Additionally, several biological experiments were performed to explain the mode of biological action. Fluorescence microscopy evidenced nuclear subcellular localization of compounds 3a, 4a and 4c. Additionally, detailed DNA binding studies confirmed a strong DNA groove binding for derivatives 4a and 4c while DNase I footprinting experiments evidenced sequence-selective binding of compound 4c in the A-T rich side. Furthermore, topoisomerase suppressive effect was for compounds 4a-4c.