Synthesis of thieno[2,3-b ]benzo[1,8]naphthyridine-2-carboxylic acids under microwave irradiation and interaction with DNA studies

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Introduction
Naphthyridine derivatives continued to be of great interest due to a wide spectrum of their biological activity.Antibiotics of this group are being widely used for the diagnostics and aromatic ring with a halomethyleniminium salt to yield the corresponding imnium species, which facilitates its inclusion into a large number of novel heterocyclic systems.
Thus, the Vilsmeier-Haack reagent was used to cyclise N-(4-methylquinolin-2-yl) acetamide 2a-d carried out by adding POCl 3 to the substrate in DMF.The reaction mixture was irradiated in microwave oven resulting in 2-chloro-3-formylbenzo [1.8]naphthyridines 3a-d with good yield of 90-95% in a short reaction time (Scheme 1).The structures of the new compounds were confirmed from the spectroscopic data and elemental analysis.
The starting compounds 2-chloro-3-formylbenzo [1.8]naphthyridines, were cyclised with thioglycolicacid/esters under microwave irradiation in one pot to furnish the title compounds (4a-g) in good yields (Scheme 1).The structures of the compounds were confirmed on the basis of elemental analysis and spectral data (Experimental section).As an example the IR (KBr) spectra of 4b exhibit an absorption band at 1678 cm −1 due to ester carbonyls and the 1 H NMR (DMSO-d 6 ) spectrum of the compound 4b displayed a triplet at δ 1.37 ppm (3H) and a quatret at δ 4.37 ppm (CH 2 ) characteristic of an ethyl group.Its mass spectrum showed the molecular ion peak at 324 (M .+2).
The reaction was proceeding through the formation of intermediate by the substitution of chlorine at C-2 of (3a-d) by the alkyl thioglycolates.In these intermediate the nucleophile generated by K 2 CO 3 base knocks on the aldehyde carbon with simultaneous elimination of water offerded (4a-g) with good yields.We synthesized nine more title compounds, which exhibited similar spectral characteristics.The IR spectra of all the compounds exhibit C=O stretching frequency in the region 1678-1700 cm −1 .In the 1 H NMR thiophene C3-H proton showed a signal at δ 8.20-8.35ppm, may be due to hydrogen bonding and anisotropy effect of the adjacent carbonyl groups and other aromatic protons resonated at δ 6.86-8.04ppm (m, Ar-H).

DNA binding studies
We studied the DNA binding properties of thieno [2,3-b]benzo [1,8]naphthyridine-2-carboxylic acids (TBNC) (4a) which features planar fused tetracyclic heterocyclic rings.The planar hydrophobic moiety of TBNC is expected to facilitate into the relatively nonpolar interior of the DNA helix.The strong absorption, viscosity and thermal denaturation characteristics of TBNC drug provide a sensitive spectroscopic handle to study its interaction with DNA.The change in the intensities of these spectra can be used to decipher the nature and strength of the stacking interaction between the chromophore and the DNA base pairs.
The UV-Vis absorption spectrum of TBNC was characterized by two maxima at 460 and 520 nm.The addition of increasingly higher concentrations of DNA led to bathochromic and hypochromic changes, as seen in Figure 1, i.e. the interaction of TBNC with DNA resulted in a strong decrease of the absorption intensity, accompanied by a shift towards higher wavelengths.A 21.8 % reduction in absorption was observed at the 520 nm peak maximum in the presence of an excess of DNA at a molar ratio of DNA nucleotide: TBNC (P/D) equal to 15. Hypochromism was suggested to be due to strong interactions between the electronic states of the intercalating chromophore and that of the DNA base pairs. 19The spectral changes that we observed (hypochromicity and red shift) were consistent with the intercalation of the chromophore into the stack DNA base pairs [27][28][29][30][31] .
The change in the obsorbance values with increasing amount of CT-DNA were used to evaluate intrinsic binding constant K b, from the compounds the values of K b are 1.8 x 10 6 M -1 .

Viscosity measurements
3][34][35] We have measured the viscosity changes in short, rod like DNA fragments.The relative length increase (η/η 0 ) of the complex formed between TBNC and DNA is shown in Figure 2. It is evident that binding of TBNC increased the viscosity of DNA, corresponding to an increase in the contour length of the DNA fragments.

Thermal denaturation measurements
Other strong evidence for the binding of TBNC into the double helix DNA was obtained from DNA melting studies.The intercalation of small molecules into the double helix is known to increase the DNA melting temperature (Tm), at which the double helix denatures into single stranded DNA, owing to the increased stability of the helix in the presence of an intercalator. 36he molar extinction coefficient of DNA bases at 260 nm in the double helical form is much less than the single stranded form; hence, melting of the helix leads to an increase in the absorbance at 260 nm.Thus the helix to coil transition temperature can be determined by monitoring the absorbance of DNA at 260 nm as a function of temperature.The DNA melting studies were carried out with calf thymus DNA in the absence and presence of TBNC.The T m for calf thymus DNA with no TBNC present was 60°C and with TBNC a single transition at 68°C was observed (Figure 3).The advantage of this method is that it is much easier to identify when more than one transition occurs. 37These various DNA melting experiments strongly supported the intercalation of TBNC into the double helix DNA.

Conclusions
In conclusion, the presented synthetic procedure is convenient, simple and high yielding microwave-assisted method for the synthesis of benzo [1,8]naphthyridines using inexpensive 2aminoquinoline could be the economical method and more practical alternative to the existing methodologies.The study of binding behavior of structurally correlated tetracyclic thieno [2,3b]benzo [1,8]naphthyridine-2-carboxylic acid TBNC (4a) with (ds)DNA by use of absorption, viscosity and thermal denaturation studies.The results of all these studies provide coherent evidence that binding of TBNC with DNA.The absorption spectrum studies shows that, on binding of TBNC to DNA typical bathochromic and hypochromic shifts observed with a binding constant of 1.8 X 10 -6 M -1 , commonly reported for such drugs.The viscometric study shows that binding of TBNC increased the viscosity of ct-DNA.Thermal denaturation studies strongly supported the binding of ct-DNA by increased T m for ct-DNA about 8 °C in presence of TBNC.

Experimental Section
General Procedures.Melting points were determined in an open capillary tube with a Buchi melting point apparatus and are uncorrected.Elemental analyses were carried out using Perkin-Elmer 240C CHN-analyzer.IR spectra were recorded on a FT-IR spectrophotometer. 1 H-NMR spectra was run in (DMSO-d6) solvent at 300 MHz and 75 MHz on a NMR spectrophotometers (chemical shifts in δ ppm).Mass spectra were recorded on a LC MS Mass spectrometer.TLC monitored the reactions.The absorption spectral studies were carried out by Ultraviolet-visible spectrophotometer.

General procedure for the synthesis of substituted benzo[1,8]naphthyridinones (3a-d)
To a mixture of 2-amino-4-methylquinoline (5mmol), DMF (13mmol), POCl 3 (45mmol) were added and the contents irradiated in MW at 160W for 6 minutes.The completion of reaction was checked by TLC and poured in ice-cold water, the solid seperated was filted, dried, and recrystallized from acetonitrile 3a.The physicochemical data for the synthesized compounds are as shown below.The same procedure was used for the synthesis of other compounds (3b-d).

Spectral measurements
Absorption spectroscopic measurements.Ultraviolet-visible absorption spectra were determined in a Perkin-Elmer model 554 UV-Vis recording spectrophotometer using quartz cuvettes of 10 mm light-path.The parameters of interaction between TBNC and calf thymus DNA were determined spectrophotometrically using a Beckman 25 double beam spectrophotometer.Aliquots of a concentrated DNA solution (0⋅1 mM-1⋅00 mM) were added to a cuvette filled with TBNC solution (12-25 mM) and thoroughly mixed.Extreme care was taken to ensure that optical reference solutions were prepared in an identical manner.Viscosity measurements.Viscosity measurements were made according to published procedures using a semimicro dilution capillary viscometer (Viscomatic Fica MgW) with a thermostated bath D40S at 20 °C.The flow time for water was 71⋅3 s.For the viscosity experiments samples of calf thymus DNA were sonicated to fragments having an estimated molecular weight of approximately 500,000.Thermal denaturation studies.The DNA melting studies were done by controlling the temperature of the sample cell with a Shimadzu circulating bath while monitoring the absorbance at 260 nm.The temperature of the solution was continuously monitored with a thermo-couple attached to the sample holder.

Figure 2 .Figure 3 .
Figure 2. Effect of increasing amount of the TBNC (4a) on the relative viscosities of CT-DNA at 25 0 C