Synthesis, modeling and biological studies on 4-2'(2,3-dihydrobenzofuranyl)coumarins

Reaction of 4-bromomethyl coumarins with schiff’s bases derived from salal and heterocyclic amines resulted in a tandem sequence leading to the formation of 3-heteroarylamino 2-4'- coumarinyl 2,3-dihydrobenzofurans. The cis / trans diastereomeric ratio was estimated by 1 H-NMR. Quantum chemical modeling studies provided a proof for very low energy difference between them and accounted for the inseparable mixture of the diasteromers. The synthesized compounds have been subjected to DNA cleavage studies.


Introduction
2][3][4][5][6] Recently 2,3-dihydrobenzofuran-5-ol has been shown to be more promising than vitamin E2 in inhibition of lipid per oxidation. 7This skeleton has been a part of number of naturally occurring oxygen heterocycles. 8In view of their secondary metabolite property and bio compatibility a number of synthetic approaches have been designed for 2,3-dihydrobenzofurans which make use of biomimetic approach, 9 reductant metals, 10 Lewis acids 11 and oxidation of phenol. 12Recently we have reported first thermal method for 2,3-dihydro-3-benzofuranols, 13 linked to a coumarin moiety under metal free conditions.During the present work aldimines derived from salal and pharmacophoric moieties like 2-amino thiazole and 2-amino pyridine have been reacted with 4-bromomethyl coumarins to obtain 3-heteroarylamino 2,3dihydrobenzofurans which have been screened for their ability to interact with DNA.

Results and Discussion
The required 4-bromomethylcoumarins were prepared using phenols and 4bromoethylacetoacetate under Pechmann cyclization conditions. 14The heteroaryl aldimines were obtained from salal and 2-amino pyridine/2-amino thiazole according to reported methods. 15The reaction of 1 and 2 in acetone at room temperature resulted in the formation of colorless crystalline solids isolated by the usual work up (Scheme 1).The absence of the methylene protons around 5.2 -5.4 δ ppm lead us to conclude that the initially formed ethers probably underwent a further intramolecular carbanion addition across the azomethine group located at close spatial proximity (ortho position) leading to the formation of 2,3-Dihydrobenzofurans (Table 1).The proposed intermediacy of ethers is supported by our earlier work. 16The reactivity of active C4-methylene group in 4-aryloxymethyl coumarins has been used to synthesize biologically active 4-2' benzofuranyl coumarins by an intramolecular aldol addition followed by dehydration [17][18][19] by our group.A plausible mechanistic pathway for the formation of coumarinyl dihydrobenzofurans 4 is indicated in (Scheme 2).The initially formed ethers 3 (which were not isolated) can easily generate a carbanion under the experimental conditions (acetone/K2CO3) which is stabilized as the enolate due to its conjugation with the lactone carbonyl.In the next step, formation of C-C bond is proposed due to the nucleophillic attack on the azomethine carbon.Due to the lack of control of stereochemistry of the carbanion, it is likely that the coumarinyl 2,3-dihydro benzofurans are obtained as mixture of cis and trans diastereomers.The 1 H NMR of all the compounds, 4 exhibited two sets of peaks for C2' and C3' protons with J values in the range of 2.4-4.3 and 7.4-8.4Hz which are characteristic of cis and trans isomers reported for 2, 3-dihydrobenzofurans. 20,21 IR spectrum of 7-methyl-4-[3-(pyridin-4-ylamino)-2,3-dihydro-benzofuran-2-yl]-chromen-2one 4b exhibited carbonyl frequency at 1712 cm -1 , NH stretching at 3432 cm -1 .Two doublets obtained for compound 4b recorded on a 400 MHz 1 H-NMR spectrometer (CDCl3) at 6.28 and 6.14 δ ppm were due to C2' H and C3' H of trans isomer with coupling constants 8.5 and 8. 4 Hz and additional two doublets obtained at 5.97, 5.86 ppm with coupling constants 4.3 and 4.4 Hz, were due to C2'H and C3'H of the cis isomer.In addition two singlets for the C6-CH3 groups at 2.38 and 2.31 ppm and two singlets for the C3-H of coumarin at 6.43 and 6.23 ppm.The aromatic region from 7.90 to 6.54 ppm integrated to a total of 24 protons thus indicating the presence of two diasteromers.The ratio estimated from the peak heights in the 1 H-NMR spectra was (1:1).The LCMS spectrum exhibited a m/z peak at 370.9 which confirmed the formation of compound.In some of the compounds the C2' and C3' protons were not distinguishable and appeared as complex multiplet.Experimental methods to separate these diastereomers by techniques like TLC, column chromatography, HPLC did not yield any fruitful results.In view of this, it was thought worthwhile to estimate the energy difference between these diastereomers and it was observed that the energy difference between cis-and trans-diastereomers is too close to each other, very less in comparison with the energy difference between the chair and boat conformations of cyclohexane (7.61 kcal/mol) and hence cis and trans diasteromers were found to be inseparable.It can be seen from (Table 2) that the coupled C2' and C3' proton chemical shifts differed by 0.04-0.18ppm with the J values varying from 2.4-8 Hz in cis and trans diasteromers.

Reactivity of coumarinyl dihydrobenzofurans (4a-4p)
Coumarinyl dihydrobenzofurans 4 when refluxed in acetone K2CO3 quickly underwent βelimination leading to the formation of 4-2'-benzofuranyl coumarins 22 5.The identity of the product 5 (Scheme 3) was confirmed by an independent synthesis using 4-bromomethyl coumarins 1 and salal.The m.p, IR, 1 H NMR was identical with the sample obtained in the earlier route.Similarly, compounds 4i-4p also yielded identical compound 5 under refluxing conditions in acetone and anhydrous potassium carbonate.
It can be seen that all the compounds exhibited the expulsion of Het-NH2 fragment in their GCMS spectra leading to the formation of benzofuranyl coumarins.Scheme 3. Formation of 4-2'-benzofuranyl coumarins 5 from diasteromeric dihydrobenzofuranyl coumarins 4.

Quantum-chemical modeling studies
Quantum-chemical modeling studies were performed by robust DFT method utilizing B3LYP hybrid exchange-correlation functional, consisting of non-local hybrid exchange part as defined by Becke's three parameters equation 23 and the nonlocal Lee-Yang-Parr correlation functional. 24plit-valence Gaussian basis sets 6-311G (d) and 6-311+G (d, p) were used.Geometry optimization of the studied molecules was performed without applying any restrictions on the molecular symmetry.All calculations were done with the GAUSSIAN 03 program. 25o ensure good quality of the quantum-chemical modeling, we used DFT method at the B3LYP/6-311G-(d) level of theory for energy minimization.Energies for several isomers were calculated and are presented hereunder (Figures 1-8).Rows differ by conformation of coumarin moiety (as it is seen from the table orientation of H3' towards furan oxygen is more preferable than of H5' both for cis-and trans isomers).Energies are given (in kcal/mole) relative to the most stable isomer in the series.
Minimum energy conformations in (Figure 1) and (Figure 3) for the cis diasteromers indicate the dihedral angle to be very close to 0 o .Whereas (Figure .2) and (Figure 4) for the trans diasteromers show the anti periplanar relation between H2 and H3 indicating the dihedral angle to be around 180 o .Energy difference between the most stable cis-and trans-isomers is small -0.42 kcal/mole only.For thiazole derivative picture is the same, but energy difference between cis-and transisomers is slightly higher (+0.88 kcal/mole).The conformation of coumarin moiety was chosen the same as in the most stable isomers of pyridine derivative.Isomers in rows differ in orientation of thiazole ring.The energies of the most stable structures were refined employing 6-311+G (d, p).For energy difference between I-cis and I-trans isomers in this basis set the value obtained is 0.25 kcal/mole and it is even lower than calculated with 6-311G (d) basis set.For the thiazole derivative with the extended basis set 6-311+G (d, p) extended with polarization p-functions on hydrogen atoms and diffuse functions.For energy difference between cis and trans isomers in this basis set the value obtained is 0.25 kcal/mole and it is even lower than calculated with 6-311G (d) basis set.For the thiazole derivative with the extended basis set 6-311+G (d, p) the relative stability is also lower and is equal to 0.56 kcal/mole.Lower energy is for trans-isomer.
Gel electrophoresis technique is based on the migration of DNA under the influence of electric potential.The photograph (Figures 9 and 10) show the molecular weight difference compared to control and is the differentiating criterion for the DNA cleaving ability of the tested compound with E. coli, control experiments using DNA alone does not indicate any significant cleavage of DNA even after long exposure time.After marker M and control C, the first six lanes correspond to 4-[3-(pyridin-4-ylamino)-2, 3-dihydro-benzofuran-2-yl]-chromen-2-ones 4a-4f, (Figure 9) and in this series the gel used for the analysis of DNA treated with 4a-4f samples show streak indicating only a partial cleavage.But an extract band of high molecular weight DNA was found in all these samples treated DNA.This is because of strong binding of the compounds to DNA by which its molecular weight was found to be increased.In all samples two bands were seen, compounds corresponding to the band and free DNA which is concentration dependent.The DNA bound with compound formed a high molecular weight band and unbound DNA migrated as in control.In 4f, only high molecular weight band was observed possibly because the concentration of compound used was enough to bind with the DNA used for treatment.Like 4f, in 4c treated DNA lane also, the low molecular weight band was missing, but high molecular weight band was could not be clearly seen because of the fluorescence of compound left in wells under UV.
After marker M and control C, the first six lanes in Figure 10 correspond to 4-[3-(Thiazol-2ylamino)-2, 3-dihydro-benzofuran-2-yl]-chromen-2-ones 4i-4n, and in this series the similar behavior of samples was noted as compared with 4a-4f.In conclusion it can be seen that all the synthesized compounds which were screened against E. coli for DNA studies show, that they have the ability to partially cleave the DNA and as well strongly bind with DNA.In conclusion all compounds have better DNA binding property than DNA cleavage.

Conclusions
A series of 4-[3-(pyridin/thiazol-4-ylamino)-2,3-dihydro-benzofuran-2-yl]-chromen-2-ones 4a-4p were synthesized via intermediates 3 and were well characterized and evaluated for DNA cleavage studies by agarose gel electrophoresis method against E. Coli.The results show that compounds exhibit both DNA cleavage and binding activity against E. Coli, in specific DNA binding was prominently observed with all the compounds.There ability to exist in the conformers makes them good candidate for interactive studies with DNA.

Experimental Section
General.The melting points were determined by open capillaries and are uncorrected.All the commercial samples were purified before use.TLC analyses were performed on commercial Kieselgel 60 F254 silica gel plates.IR Spectra were recorded on a Bruker EQUINOX 55 FTIR.NMR spectra were obtained on Bruker spectrometer using CDCl3 as solvent, with proton resonances at 300 and 400 MHz, respectively.Mass spectral data (LCMS) were recorded on Agilent Single Quartz mass spectrometer.The elemental analysis was carried out using Heraus CHN rapid analyzer.Quantum-chemical modeling studies were performed by robust DFT method utilizing B3LYP hybrid exchange-correlation functional, consisting of non-local hybrid exchange part as defined by Becke's three parameters equation [23] and the nonlocal Lee-Yang-Parr correlation functional.Split-valence Gaussian basis sets 6-311G (d) and 6-311+G (d, p) were used.Geometry optimization of the studied molecules was performed without applying any restrictions on the molecular symmetry.All calculations were done with the GAUSSIAN'03 program.