Synthesis of Novel 1 , 3-Diacetoxy-Acridones as Cytotoxic Agents and their DNA-Binding Studies

A series of novel substituted acridones (1–15) have been synthesized. Their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7) and human promyelocytic leukemia (HL-60) cell lines has been investigated. The compounds 11, 12, 14 and 15 showed moderate activity against MCF-7 cell lines with IC50 value < 5.83 μM. The compounds 8, 10–12, and 15 showed moderate activity against HL-60 cell lines with IC50 value < 1.75 μM. The DNAbinding properties of the compounds were evaluated based on their affinity or intercalation with CT-DNA measured with absorption titration. The compound 12 bearing planar diacetoxy tricyclic ring linked with butyl piperidine side chain showed highest binding affinity with binding constant (Ki) 10.38 ×10 ×M. The examination of the relationship between lipophilicity and cytotoxic properties of acridones showed a poor correlation.


Introduction
Cytotoxic drugs remain the mainstay of cancer chemotherapy and are being administered with novel ways of therapy such as inhibitors of signals.It is therefore important to discover novel cytotoxic agents with spectra of activity and toxicity that differ from those current agents [1].Acronycine also known as acronine is an acridone alkaloid which was first isolated in 1948 from the bark of the Australian tree Acronychia baurri [2].In subsequent investigation it was found that acronycine possess anticancer activity against a wide spectrum of experimental neoplasms in laboratory animals [3].Glyfoline, another natural acridone alkaloid isolated from Glycosmis citrifolia, was found to be active molecule (IC 50 =2.2µM) for inhibition of human leukemic HL-60 cells [4,5].
A series of thioacridone derivatives was synthesized by Dheyongera et al.The binding constant of these with DNA was measured to determine their degree of intercalation with DNA [6].
Triazoloacridone (C 1305 and C 1533 ) (Fig. 1) binds to DNA and induces very specific and unusual structural changes in DNA, which plays an important role in the cytotoxic activity of this unique compound [7].
The imidazo acridones are a new class of antitumor agents.The most promising imidazo acridinone derivative C 1311 (Fig. 1) is currently under phase II clinical trials for colon and breast cancers.C 1311 damages cellular DNA in various ways.The drug binds to DNA noncovalently (by intercalation) and covalently, following oxidative metabolic activation [8].
The design of these compounds was based on structure activity relationship studies of the chemotherapeutic agent mitoxantrone.The diamino alkyl group in the side chain of mitoxantrone had previously been found to be prerequisite for biological activity of the drug.The attachment of diaminoalkyl group to known DNA-intercalating acridinone moieties resulted in the C 13xx imidazo acridinone series of the compounds which showed good activity in vitro and in vivo in various tumor model systems [9].In the view of above literature, we have designed 1,3-diacetoxyacridone ring nucleus substituted at N 10 -position with propyl and butyl side chains followed by tertiary amino groups for better cytotoxicity and DNA-binding properties (1-15, Tab. 1).Their cytotoxicity Sci Pharm.2009; 77; 19-32.
has been tested against MCF-7 and HL-60 cell lines and tried to correlate the lipophilicity, DNA-binding properties with cytotoxicity.Usually N-alkylation with alkyl halides is difficult due to the weak basic nature of nitrogen of the acridone nucleus.However, it can be achieved in the presence of a strong base like sodium amide or sodium hydroxide under anhydrous conditions.The reaction of parent acridone with chlorobromoalkanes in the presence of sodium amide in anhydrous condition gave respective N 10 -(chloroalkyl)acridones.Besides requiring drastic experimental conditions, the N 10 -alkylation using sodium amide resulted in a very low yield.

Chemistry
To overcome this drawback, N 10 -alkylation was carried out in the presence of phase transfer catalyst (PTC), which is easier to work with and gives better yield than the previously described methods.
Stirring of the compound 1 at room temperature with alkylating agent 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two phase system consisting of an organic solvent and aqueous potassium hydroxide solution in the presence of tetra butyl ammonium bromide (PTC) leads to the formation of respective compounds 2 and 9 in good yield.Here, catalyst (PTC) transports the OH -ion from the aqueous phase to organic phase where actual reaction takes place.The ion formed may be regarded as phenolate stabilized anion, which subsequently undergoes alkylation to form the aromatized system.
All the products were separated and purified by column chromatography and recrystallization method and dried under high vacuum for more than 12 h.The purified compounds were characterized by 1 H-NMR, 13 C-NMR, Mass spectral methods and elemental analysis.The assignment of protons is fully supported by the integration curves and all the derivatives showed the characteristic chemical shifts for the acridone nucleus.The assignment of the 13 C-resonance of acridone derivatives is in close agreement with an analogous compound N 10 -substituted acridone.

Lipophilicity
The compounds lipophilicity was determined using the software ALOGPS and SPARC v4.2 .
The efficiency of a cytotoxic drug will depend in part on its ability to accumulate in cells.any of this substituted derivatives, yet these were not very effective at increasing cytotoxic activity.In contrast, compound 13 with log 10 P (3.88) and logD (4.01) values did not show maximum activity.However, compound 12 with log 10 P (4.06) and logD (4.43) showed maximum activity.Therefore, the degree of lipophilicity of each drug would seem to be important, but it is not the sole determinant for cytotoxicity of acridone derivatives.

Tab. 1.
Lipophilicity values of different N-substituted Acridone Derivatives: The cytotoxicity of fifteen compounds was examined on MCF-7 and HL-60 cell lines by Trypan blue exclusion method with several concentrations of acridones.The IC 50 values of N 10 -chloropropyl substituted and chlorobutyl substituted 1,3-diacetoxy acridone derivatives against MCF-7 and HL-60 cells revealed that cytotoxic activity relatively increased as the chain length increased from three to four suggesting that hydrophobicity plays an important role in biological activity.The presence of acetoxy groups and increase of distance between the ring nucleus and amino group, increased the cytotoxic activity of these compounds.It is clear from the data, the comparison of the cytotoxicity against MCF-7 cell lines (Tab.2) of the diacetoxy butyl derivatives has shown that the cell killing potency follows the order, 12 > 14 > 11 > 15 > 10 > 13 > 9 and diacetoxy propyl derivatives 7 > 4 > 5 > 8 > 6 > 3 > 2. The cytotoxicity against HL-60 cell lines (Tab.2) of the diacetoxy butyl derivatives has shown that the cell killing potency follows the order, 15 > 11 > 12 > 10 > 14 > 13 > 9 and diacetoxy propyl derivatives 8 > 7 > 4 > 5 > 6 > 3 >2.However, comparison of IC 50 values with in the series revealed that the diacetoxy butyl derivatives have higher potency than diacetoxy propyl derivatives.Among this series, the compound 12 showed moderate cytotoxic activity against MCF-7 cell line with IC 50 value 3.15 μM and compound 15 against HL-60 with IC 50 value 0.45 μM.
Therefore, it can be concluded that the structural features required with in the series to cause a maximum cytotoxic activity in MCF-7 and HL-60 cell lines, include hydrophobic acridone ring with electron withdrawing diacetoxy groups and alkyl side chain preferably four methylene units with substitution positively charged tertiary amino group preferably piperidino and diethyl amino groups.

DNA-binding properties
The DNA-binding properties of the compounds 3-5, 7, 8, 10-12, 14 and 15 (1, 2, 6, 9 and 13 not analyzed due to poor solubility) were studied by monitoring the changes in the UV-Visible absorption spectra of the acridone derivatives up on addition of CT-DNA [10].In the range from 260 to 275 all the acridone derivatives exhibited strong absorption peaks with maxima near 266-268nm.Progressive addition of DNA led to strong hypochromism in the absorption intensities in all the compounds studied.The percentage hypochromism were found to be 50.0,58.6, 57.2, 62.6, 61.8, 54.1, 57.8, 53.5, 60.4 and 43.1.The Fig. 2 shows the representative absorption spectrum of the compound 11 (15 μM) in the presence of increasing concentration of CT-DNA (0-100 μM).The Fig. 3 shows Half-reciprocal plot for binding of 11 with CT DNA.The compound exhibited the similar absorption spectra pertaining to the chromophore but with the hypochromicity and isobastic point depending on the alkyl amino side chains.These results were consistant with the previous reports on the absorption titration of acridine derivatives and the hypochromicity of acridine in the presence of DNA is believed to be a result of their intercalation with the DNA [11].
The selection of ionic strength (150mM NaCl) in the absorption titration experiment was mainly based on the avoidance of DNA deposition in all drug solution (15μM).The Tab. 3 summarizes the DNA-binding constants and related properties of acridones after intercalation with CT-DNA.The relative binding affinities as indicated by the binding constant K i were in the order of 12 > 14 > 11 > 4 > 10 > 5 > 7 > 15 > 3 > 8.Among the derivatives those with strong DNA-binding affinities 11, 12 and 14 exhibited hypochromicity, isobastic points.However, a highest binding affinity and cytotoxic activity were observed for compound 12 bearing planar tricyclic ring with electron withdrawing diacetoxy groups, linked with butyl piperidine side chain.

Tab. 3.
Binding constant (K i ) and photometric properties of acridones in contact with CT-DNA:

Conclusion
The new diacetoxyacridone derivatives derived from acridone with tertiary amines group at the terminal end of the alkyl side chains had strong inhibiting activity against MCF-7 and HL-60 cell lines, which may be associated with their DNA-binding capacity.In particular, the effect is more pronounced when acridones have propyl and butyl side chain.
Comparison of the derivatives for their ability to bind with DNA revealed that they largely follow the order N 10 -butyl side chain > N 10 -propyl side chain.The substitution of hydrogens by OCOCH 3 increased the ability to bind DNA.Careful examination of the results obtained, revealed that the diacetoxy butyl derivatives have higher activity than diacetoxy propyl derivatives.With respect to these observations, we concluded that this series could be developed as a promising cytotoxicity as DNA-Intercalators.

Experimental
Reactions were monitored by TLC.Column Chromatography utilized silica gel Merck Grade 60 (230-400 mesh, 60 Å).Melting points were recorded on a Tempirol hot-stage with microscope and are uncorrected.Elemental analysis was performed and found values are ±0.4% of theoretical values unless otherwise noted. 1 H-NMR, 13 C-NMR spectra were recorded in CDCl 3 , and DMSO-d 6 solution in a 5-mm tube on a Bruker DRX 400 Fourier transform spectrometer with tetramethylsilane as internal standard.Chemical shifts are expressed as δ (ppm) values.Mass spectra were recorded on Thermo Finnigan trace DSQ GC-Mass Spectrometer.DNA-binding studies of synthesized compounds were performed by Nano Drop ND-1000 UV Spectrophotometer.

General method for the synthesis of 10-(chlorobromoalkyl)-1,3-diacetoxy acridones (2) and (9)
One gram (0.0044 mol) of 1,3-diacetoxyacridone (1) was dissolved in 25 mL tetrahydrofuran and then 20 mL (0.05 mol) of potassium hydroxide and 0.5 g (0.015 mol) of tetrabutylammonium bromide was added to it.The reaction mixture was stirred at room temperature for 30 min and added Chlorobromoalkanes (0.015 mol) slowly into the reaction mixture and stirred for 24 h at room temperature.Tetrahydrofuran was evaporated and the aqueous layer was extracted with chloroform.The chloroform layer was washed with water and organic layer dried over anhydrous sodium sulfate and rotavaporated.The crude product was purified by column chromatography by using the solvent system chloroform/methanol (9:1) to give a green solid of (2) and (9).

General procedure for the synthesis of 10-(N-substituted alkyl)-1,3-diacetoxy acridones (3-8 and 10-15)
10-(Chloroalkyl)-1,3-diacetoxyacridone (0.0044 mol) was dissolved in 30 ml of anhydrous acetonitrile and 1.68 g potassium iodide and 3.3 g of potassium carbonate were added and refluxed for 30 min.Then added (0.0044 mol) different secondary amines into it slowly and refluxed for 15 h until a substantial amount of the product was formed as evidenced by TLC.The contents were cooled, diluted with water and extracted with chloroform.The chloroform layer was washed with water thrice, dried over anhydrous sodium sulfate and evaporated to give an oily product.The semi solid residue was purified by column chromatography using the solvent system chloroform/methanol (9:1) to give a light yellow product of 10-(3′-[N-substituted]alkyl)-1,3-diacetoxyacridone.An acetone solution of the free base was treated with ethereal hydrochloride to give the hydrochloride salt, which was dried over high vacuum to get pure solid.

Cytotoxicity assay against MCF-7 and HL-60 cell lines
The trypan blue dye exclusion test was used to determine drug-mediated cytotoxicity as described previously [13].Briefly, 1 x 10 4 target tumor cells resuspended in 1 ml.Two ml of cell suspension were distributed into each well of a 6-well plate, and medium at the desired concentration was added into each well.Each plate was incubated for 48 h at 37°C and 5% CO 2 atmosphere.Following the incubations, 100 µl of the trypan was added Fig. 1.