Patent Application
20170283366
TECHNICAL FIELD
This invention relates to benzoquinone derivatives and to methods of making the benzoquinone derivatives. The invention also relates to the use of the benzoquinone derivatives in the treatment of cancer and to pharmaceutical compositions containing the benzoquinone derivatives.
Quinones refer to the dihydro aromatic compounds whose basic sub-unit is 1,4-benzoquinone. They are naturally found in flowering plants, bacteria, fungi, arthropods and Lichens. Quinones are antioxidant reducing agents that share in electron-transfer reactions in respiratory and photosynthesis processes. Because of their natural bright colors and biological activities, several quinone compounds have been used in traditional medicine. Since the discovery of vitamin K in 1929, many quinones have been extracted from plants or fungi, Some extracted quinones showed antibiotic, antitumor, antimalarial, anti-inflammatory, and anticoagulant properties. Nowadays, natural and synthetic quinones comprise the second largest class of anti-tumor agents currently in use. Examples include Doxorubicin, Mitoxantrone and Mitomycin C. Several mechanisms have been proposed for the anticancer effects of Quinones that include apoptosis, abrogation of cell cycles, activation of caspases, stimulating the production of reactive oxygen species (ROS), inhibition of topoisomerases I and II, activation of intracellular secondary messengers and production of free radicals to attack DNA. The anticancer activity of quinoids has been attributed to their ability to undergo a biochemical reduction by one or two electrons that are catalyzed by flavoenzymes in the organism using NADPH as an electron donor producing semiquinone radical intermediates and subsequent reactions with oxygen. These forms are believed to be responsible for most of the quinoids anticancer activity. Quinones with amino-substituted side chains have shown antitumor activity. More particularly, WO2011/126544 discloses analogues of thymoquinone for the treatment of pancreatic cancer.
Another mechanism for cancer and antitumor treatment, which is a different mechanism from that described herein above, relies on the effect which a compound may have on DNA. Prior to explaining the effect and the mechanism of the treatment, a few basic features of DNA and of chromosomes, are first described herein below.
The end regions or so-called “end caps” of chromosomes have a region of repetitive nucleotide sequences called telomeres. These telomeres protect the end of chromosomes from deterioration or from fusion with neighbouring chromosomes. More specifically, telomeres consist of so-called “tandem repeats” comprising a sequence of two or more DNA base pairs that are repeated in such a way that the repeats lie adjacent to each other on the chromosome. These tandem repeats are enriched in guanine bases and fold up under physiological conditions to form so-called “four-stranded G-quadruplex structures”. Furthermore, telomeres capping the ends of human chromosomes and promoter regions of some oncogenes (such as c-myc, k-ras and c-kit) are enriched in guanine bases. Telomeres fold up forming G-quadruplex DNA structures which inhibit telomerase enzyme which is over-expressed in cancer cells. Telomerase enzyme re-elongates telomeres causing cancer cells to continue to divide indefinitely. Formation of intramolecular G-quadruplex in the promoter region of some oncogenes seems to play an important role in regulating transcription of the corresponding gene.
It has also been hypothesized that formation of G-quadruplex DNA structures in the promoter region of some oncogenes plays an important role in regulating the transcription of the corresponding gene. Consequently, small molecules that selectively bind and stabilize G-quadruplex DNA have potential for the development of new selective and efficient anticancer therapeutic agents. Molecules which stack on the face or interact with grooves and loops of quadruplex were reported as good G-quadruplex's stabilizers.
As mentioned above, DNA sequences enriched in guanine can form high order structure called G-quadruplex DNA. This G-quadruplex DNA consists of four strands connected by eight Hoogsteen hydrogen bonds. G-quadruplex DNA formation has been reported in human telomere (chromosome's ends) and in promoter regions of some oncogenes such as c-myc, k-ras and c-kit. In normal cells the telomeres are shortened after each cell division until the apoptosis of the cell occurs at short telomere length. Telomerase enzyme re-elongates the telomeres in stem and cancer cells. Furthermore, Telomerase has been found active in almost all cancer cells and contributes to the indefinite division of tumor cells. Formation of telomere quadruplex structures has shown to inhibit telomerase enzyme in breast, prostate, lung, liver, pancreatic and colon cancers. Therefore, it was assumed to control cell transcriptions and expression.
Several compounds have been reported as G-quadruplex's stabilizers, telomerase's inhibitors and/or oncogene regulators. These included some porphyrines, porphyrazines, phtalocyanines, cyclopyrroles, telomestatin, and porphyrin TMPyP4 derivatives. 5,10,15,20-tetra [4-hydroxyl-3-9-trimethyl-ammonium) methyl-phenyl] showed higher selectivity due to the presence of four positively charged groups and four hydroxyl groups that can interact with the G-quadruplex loops and grooves. Acridines, acridones, perylene, corolone, anthraquinones and quinolone derivatives were reported to strongly interact with the G-tetrads of quadruplex DNA though π-π stacking and to duplex DNA through intercalation between duplex base pairs or grooves of DNA. The length between the aromatic central core and the positively charged nitrogen atom of the lateral side chain as well as the basicity of the system were assumed responsible for giving rise to better quadruplex stabilization by perylene.
A need exists for an antitumor agent which effectively targets telomeric G-quadruplex DNA and which stabilize G-quadruplex DNA. A need further exists for compounds having molecules with high preferential affinity to stabilize G-quadruplex structures which can inhibit/stop cancer proliferations and provide potential for developing new selective genetic anticancer therapeutic agents. Furthermore a need exists for an antitumor agent which shows a better selectivity towards G-quadruplex DNA over ct-DNA (tumor DNA circulating freely in the blood of a cancer patient). Furthermore a need exists for an antitumor agent which has a high antitumor effect and/or efficacy. Also, a need exists for an antitumor agent having improved biological potency, efficacy and selectivity. A need also exists for a synthetic benzoquinone analogue which is cost effective to synthesise, non-toxic and effective in treatment of cancer.
The present invention relates to compounds of formula (I):
and to pharmaceutically acceptable salts or solvates thereof wherein:
one of X and Y is hydrogen and the other one of X and Y is selected from the group consisting of flouroaryl amines, biphenyl amines, amino-pyrrolidines and methoxyphenyl amines. More particularly, said other one of X and Y is selected from the group consisting of 3-Trifluoro-methylaniline; 3,4,5-trifluoroaniline; 4-methoxylaniline; 4-fluoroaniline; 3,3′-Dimethyl-1,1′-Biphenyl-4,4′-diamine; 2-(pyrrolidin-l-yl)ethyl)amine; 4-trifluoromethyl-benzylamine ; 4-fluorobenzyl-amine ; 3,4-dimethoxybenzylamine and 3,5-ditrifluoromethyl-benzylamine.
In a preferred embodiment, the compounds of formula (I) are selected from:
The compounds of formula (I) are synthetic analogues of benzoquinone.
Suitable pharmaceutically acceptable salts may include salts of acidic or basic groups present in compounds of formula (I). The compounds of formula (I) that are basic in nature are capable of forming a variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of formula (I) are those that form non-toxic acid addition salts. Suitable pharmaceutically acceptable salts may include acetate, benzensulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edentate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride edentate, edisylate, estolate, esylate, ethylsuccinate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylat, nitrate, oleate, oxalate, pamoate, palmitate, pantothenate, phosphate, diphosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate and valerate.
The compounds of formula (I) may be synthesised by a number of synthetic routes. In one method of making the compounds of formula (I), the compounds of formula (I) can be synthesised as shown in Scheme (I).
Scheme (I)
Compounds of formula (I) may be synthesised in a one pot reaction by reacting benzoquinone in a suitable solvent with a compound of formula (II) dissolved in the solvent, wherein the compound of formula (II) is R-NH2, wherein R is selected from the group consisting of flouroaryl amines, biphenyl amines, amino-pyrrolidines or methoxyphenyl amines. More particularly, R is selected from the group consisting of F-Z-NH2; CF3-Z-NH2, (Me)2-Z-NH2, MeO-Z-NH2 or (MeO)2-biphenyl-NH2, where Z is selected from the group consisting of phenyl, benzyl, biphenyl, ethylpyrrolidine and biphenyl amine.
More specifically, the compound of formula (II) may be selected from the group consisting of 3-(trifluoromethyl)aniline; 3,4,5-trifluoroaniline; ρ-anisidine (4-methoxyaniline); 4-fluoroanaline; o-tolidine (4-(4-Amino-3-methylphenyl)-2-methylaniline); 1-(2-aminoethyl)pyrrolidine; 4-trifluoromethylbenzyl amine; 4-fluorobenzyl amine; 3,4-dimethoxybenzylamine veratrylamine and 3,5-bis-(trifluoromethyl)benzylamine.
The suitable solvent may be methanol.
The reaction may be achieved by introducing the compound of formula (II) in a drop by drop manner.
The reaction may occur in the presence of air at room temperature. The solvent may be concentrated and purified after the reaction. The resultant products may be crystallised after the reaction.
The compounds:
Preferably the compound 2,5-bis-(4-methoxylanilino)-1,4-benzoquinone (BQ3) is used for the treatment of pancreatic cancer, lung cancer, or breast cancer.
Preferably the compound 2,5-bis-((3,3′-Dimethyl-1,1′-Biphenyl-4-amine)-4′-amino)-1,4-benzoquinone (BQ6) is used for the treatment of pancreatic cancer, lung cancer, or breast cancer.
Preferably the compound 2,5-bis-(4-trifluoromethylbenzylamino)-1,4-benzoquinone (BQ10) is used for the treatment of pancreatic cancer, lung cancer, or breast cancer.
Preferably the compound 2,5-bis-(4-fluorobenzylamino)-1,4-benzoquinone (BQ11) is used for the treatment of pancreatic cancer, lung cancer, or breast cancer.
The compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, have been found to bind G-quadruplex DNA, thereby to stabilise G-quadruplex DNA. Stabilisation of the G-quadruplex DNA can inhibit telomerase enzyme. Inhibition of telomerase enzyme may be beneficial in the treatment of cancer. The compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, have been found to inhibit telomerase enzyme.
The invention also provides a method of treating cancer in a mammal, particularly a human, comprising administering to the mammal an amount of a compound of formula (I), as defined above, or pharmaceutically acceptable salts or solvates thereof. The mammal may be in need of cancer treatment. The method may be, more particularly, for the treatment of pancreatic cancer, lung cancer, or breast cancer. The compound may be administered in a therapeutically effective amount. The method may further include administering the compound of formula (I), or the pharmaceutically acceptable salts or solvates thereof, in combination with at least one suitable anti-tumor or neoplastic agent for the treatment of cancer, in particular for the treatment of pancreatic, lung, or breast cancer.
More particularly, the compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, may be beneficial in the treatment of cancer due to the ability of the compounds of formula (I) to stabilize G-quadruplex DNA. The method may be, more particularly, for use in the treatment of pancreatic cancer, lung cancer, or breast cancer. As such, the compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof may be used in a method of stabilizing G-quadruplex DNA in a mammal, preferably in a human. The mammal or human may be in need of cancer treatment. The method may include stabilizing the G-quadruplex DNA by binding the compounds of formula (I), or the pharmaceutically acceptable salts or solvates thereof, to the G-quadruplex DNA. More specifically, the method may include stabilizing the G-quadruplex DNA by binding the compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, to the G-quadruplex DNA.
In vitro, an affinity of the compounds of formula (I) for binding to G-quadruplex DNA are greater than an affinity of the compounds of formula (I) for binding to ct-DNA. The compounds of formula (I) may be beneficial in the treatment of cancer due to the higher affinity of the compounds of formula (I) for binding G-quadruplex DNA, when compared to the affinity of the compounds of formula (I) for binding ct-DNA.
The invention further relates to a method of stabilizing G-quadruplex DNA that may be formed in a telomere region of a mammalian chromosome, more particularly in a telomere region of a human chromosome, the method comprising binding the compound of formula (I), or a pharmaceutically acceptable salts or solvates thereof, to the G-quadruplex DNA. The mammalian chromosome or the human chromosome may be from a mammal, or human, respectively, in need of cancer treatment. The mammal or human may be in need of cancer treatment for one of pancreatic cancer, lung cancer, or breast cancer. The method may, more particularly, comprise binding a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salts or solvates thereof, to the G-quadruplex DNA.
Furthermore, the compounds of formula (I), as defined above, or the pharmaceutically acceptable salts or solvates thereof, may be beneficial in the treatment of cancer due to the ability of the compounds of formula (I) to inhibit telomerase enzyme. As such, the compounds of formula (I), as defined above, or the pharmaceutically acceptable salts or solvates thereof, may be used in a method of inhibiting telomerase enzyme in a mammal, preferably a human. More particularly, the invention relates to a method of inhibit telomerase enzyme in a mammalian cell, particularly a human cell, comprising administering to the cell an amount of a compound of formula (I), as defined above, or the pharmaceutically acceptable salts or solvates thereof. The mammalian cell may be in need of cancer treatment. The method may be, more particularly, for the treatment of pancreatic cancer, lung cancer, or breast cancer. The compound may be administered in a therapeutically effective amount.
The method may comprise binding the compound of formula (I), or the pharmaceutically acceptable salts or solvates thereof, to G-quadruplex DNA that may be formed in the mammalian cell or in the human cell.
The invention relates also to the use of the compound of Formula (I), as defined above, or the pharmaceutically acceptable salts or solvates thereof, in the manufacture of a pharmaceutical composition for treating cancer. More particularly, the pharmaceutical composition may be for the treatment of pancreatic cancer, lung cancer, or breast cancer.
The invention further relates to the compounds of formula (I), as defined above, or the pharmaceutically acceptable salts or solvates thereof, in combination with at least one suitable anti-tumor or neoplastic agent for the treatment of cancer, in particular for the treatment of pancreatic, lung, or breast cancer.
The invention also relates to a pharmaceutical composition comprising a compound of formula (I) as described above, or the pharmaceutically acceptable salts or solvates thereof and a pharmaceutically acceptable diluents or carrier. The pharmaceutical composition may comprise an additional therapeutic agent. The pharmaceutical composition may be for treating a mammal, preferably a human, in need of cancer treatment. The pharmaceutical composition may be used in the treatment of pancreatic cancer, lung cancer, or breast cancer.
The term “treatment” is intended to include curing, reversing, alleviating, palliative and prophylactic treatment of the condition.
A “therapeutically effective amount” of a compound is an amount of the compound, which when administered to a subject, is sufficient to confer the intended therapeutic effect. A therapeutically effective amount can be given in one or more administrations.
Common cancers would include bladder, breast, colon, rectal, endometrial, kidney (renal cell), leukaemia, lung, melanoma, non-Hodgkin lymphoma, pancreatic, prostate, brain, skin, liver and thyroid cancers.
Patients suffering from cancer are commonly co-administered additional therapeutic agents, in particular suitable antineoplastic or anti-tumor agents. Suitable co-administrants would include:
1. Alkylating antineoplastic agents: such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide.
2. Plant alkaloids and terpenoids. These include:
Other therapeutic agents are commonly administered to patients to deal with the side effects of chemotherapy. Such agents might include anti-emetics for nausea, or agents to treat anaemia and fatigue. Other such medicaments are well known to physicians and those skilled in cancer therapy.
Such agents may be administered sequentially, simultaneously or concomitantly.
Suitable composition forms include forms suitable for oral administration such as tablets, capsule, pills, powders, sustained release formulations, solutions, and suspension, for parental injection such as sterile saline solutions, suspensions or emulsion; for topical administration such as ointments or creams; or rectal administration such as suppositories.
Exemplary parenteral administration forms include suspensions or solutions in sterile aqueous solutions, for example aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. Compositions may also include additional ingredients such as flavouring, binders, and excipients. Tablets may include: disintegrates such as starch, alginic acid and complex silicates; binding agents such as sucrose, gelatine and acacia, and lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc.
Solid compositions may also include soft and hard gelatin capsules. Preferred materials include lactose, milk sugars and high molecular weight polyethylene glycols.
Aqueous suspensions or elixirs may include sweetening or flavouring agents, colours and dyes, emulsifying agents, suspending agents as wells as diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof.
Pharmaceutical forms suitable for the delivery of the compounds of the present invention and methods of preparing the various pharmaceutical compositions will be readily apparent to those skilled in the art. Such compositions and methods for their preparations may be found, for example in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Described hereinbelow are the materials, reagents and apparatus, used:
in the synthesis of various benzoquinone derivatives (“BQ1-14”, “BQ”, “BQ derivatives” or “BQ's”), in accordance with the invention;
to examine the benzoquinone derivatives synthesized, in accordance with the invention, and the effect of the benzoquinone derivatives on cancer cell;
to investigate interactions between the BQ derivatives and telomeric G-quadruplex DNA and selectivity of the BQ derivatives towards G-quadruplex DNA over duplex DNA; and
to study binding affinity, binding stoichiometry and invitro anticancer effects of the BQ derivatives.
All chemicals were purchased from Sigma-Aldrich, Germany (Taufkirchen, Munchen). The following chemicals were of the highest purity grade and used without further purification; 1,4-Benzoquinone, 3-(trifluoromethyl)aniline, 3,4,5-trifluoroaniline, p-anisidine (4-methoxyaniline), 4-fluoroanaline, aniline, o-tolidine (4-(4-Amino-3-methylphenyl)-2-methylaniline), 4-(2-aminoethyl)morpholine, cyclopentylamine, 1-(2-aminoethyl)pyrrolidine, 4-trifluoromethylbenzyl amine, 4-fluorobenzyl amine, 3,4-dimethoxybenzylamine, Veratrylamine, benzylamine, 3,5-bis-(trifluoromethyl)benzylamine, methanol and Calf thymus DNA. Human telomeric DNA was purchased from Alpha DNA (Canada).
Human cancer cell lines were purchased from Hyclone Laboratories, Utah, USA. Pancreatic cancer cells (L3.6pl and MiaPaCa-2) and breast cancer cells (MCF-7) were maintained in DMEM while lung cancer cells (H1299) and prostate cancer cells (C42B) were maintained in RPMI 1640 media. All media were supplemented with antibiotics (penicillin 50 U/ml; streptomycin 50 μg/m1) and 10% fetal bovine serum (FBS, Biowest, Nouaille, France).
Thin-layer chromatography (TLC) was performed on glass-silica gel plates (Silica gel, 60 F254, Fluka) and ethyl acetate-hexane (1:1) as mobile phase. Spots were visualized under UV lamp. Column chromatography was performed on Kieselgel-S (Silica gel-S, 0.063-0.1mm). A Gallen kamp melting point apparatus was used for recording the melting points of synthesized compounds and an Euro Vector EA-3000 CHNS analyzer was used for their elemental analyses.
Infrared and NMR spectra were performed using a Thermo Nicolet model 470 FT-IR and a Varian 400 MHz FT-NMR spectrometers, respectively. IR spectra were recorded in KBr solid pellets while NMR spectra were recorded in DMSO-d6 and CDCl3 solutions with tetramethylsilane (TMS) as an internal reference. Mass spectra were performed using Finnigan-Trace GC 2000 (Thermo Quest, USA) equipped with a split-splitless injector, AS-3000 autosampler (Thermoelectron Corporation, USA) and a quadruple mass spectrometer (Trace-MS Finnigan) detector with mass range of 1 to 1050 a.m.u. for detecting derivatized phenoxy herbicides.
Buffer solution (Tris-KCl buffer)
A 0.01 M Tris-KCl buffer solution-pH 7.4, was prepared by dissolving 10.00 mM of tris-hydroxymethylaminomethane hydrochloride (1.576 g), 1.00 mM Na2EDTA (0.3722 g) and 100.00 mM KCl (7.455 g) into 1.0 L of deionized water. The pH was adjusted using glass electrode. A 1.00 mL of Tween-80 was added to the solution and shacked well.
Stock solutions (2×10−3 M) of benzoquinone derivatives were prepared in DMSO. Solutions having lower concentrations were prepared by appropriate dilution into DMSO.
Calf Thymus DNA (ct-DNA)
Calf thymus ds-DNA (1000 μg/ml; 8×10−8 M) was prepared by dissolving 10.0 mg of DNA into 10.0 ml Tris-KCl buffer, pH 7.4, without sonication or stirring. To prevent shearing of the large genomic DNA, the solution was gently inverted overnight at 4.0° C. to completely solubilize the DNA. Solutions of DNA are stable for several months at 4.0° C. in Tris-KCl buffer pH 7-8.
Purchased synthetic nucleic acids primers with human telomere sequence; 5′-AGGGTTAGGGTTAGGGTTAGGG-3′, its fluorescein labeled 5′ primer Fl-5′-AGGGTTAGGGTTAGGGTTAGGG-3′ or its complementary strand 3′-TCCCAAT-CCCAATC-CCAATCCC-5′ were reconstituted by centrifugation for 10 min at 7000 rpm to collect DNA in the bottom of the vials. Tris-KCl buffer (2.00 ml) were added and left 2 min for rehydration then vortexed for 30 s. Reconstituted primers were kept overnight at 4.0° C. Stability of reconstituted primers is more than 6 months.
Telomeric G-quadruplex DNA was prepared by heating gently 2.0 ml of stock single stranded 5′-AGGGTTAGGGTTAGGGTTAGGG-3′ DNA up to 95.0° C. Resultant solution was incubated at 95.0° C. for 10 min. The solution was left to cool gently down to room temperature, then kept in fridge at 4.0° C. overnight before use. A 10−5 M fluorescein labeled G-quadruplex DNA was prepared similarly using 5′-Flu-telomeric DNA.
A 1×10−4 M telomeric ds-DNA was prepared by mixing equimolar amounts of 5′-AGGGTTAGGGTTAGGGTTAGGG-3′ (268.80 μL of 7.44×10−4 M) with its complementary strand 3′-TCCCAATCCCAATCCCAATCCC-5′ (738.00 μL of 2.71×10−4 M). The solution was made up to 2000.0 μl using KCl-Tris-Cl buffer pH 7.4, vortexed for 15 s and incubated at 95.0° C. for 10.0 min then left to cool to room temperature. Resultant hybridized ds-DNA was kept in refrigerator at 4.0° C. till use.
To determine the concentrations of prepared DNA stock solutions, a 10.0 μl DNA solution was diluted using Tris-KCl-buffer solution, pH 7.4, to 1.0 ml. Resultant solution was vortexed for 15 s, followed by measuring absorbance at 260 and 280 nm. Concentration in μg/ml was calculated using the following equation:
C(μg/ml)=A260×weight per OD×dilution factor
where OD is the optical density at 260 nm. The ratio A260/A280 was used to estimate the purity of each oligonucleotide. Ratios ≧1.8 were considered enough to indicate high purity for synthetic and calf thymus DNAs.
Stability and aggregation of BQs were studied by following changes in absorbance of 10−5 M solution of each BQ in 5.0% DMSO Tris-KCl buffer, pH 7.4 and 0.1% Tween-80 over 48 hrs.
Interactions of BQs with G-quadruplex
Interactions of the BQs with G-quadruplex DNA were studied using UV-Vis, fluorescence, fluorescence quenching and circular dichroism spectroscopies as well as melting temperature. Binding parameters such as binding constant, stoichiometry, selectivity towards G-quadruplex over duplex DNA were evaluated.
Successive amounts of G-quadruplex DNA (AGGG(TTAGGG)3 , 1.44×10−4 M) were added to 1.00 ml of each BQ (5×10−6 M) in Tris-KCl buffer, pH 7.4. Solution was shacked well after each addition, incubated for 3 min at room temperature and its absorbance was scanned in the range 200-600 nm. Titration was stopped when no change in absorbance was observed. The experiment was reversed by adding successive amounts of the each BQ (1×10−3 M) to 1.0 ml of 4×10−6 M G-quadruplex DNA.
Binding affinity of BQs towards human telomeric G-quadruplex DNA was further confirmed using fluorescence quenching assay. Successive amounts of each BQ (1×10−3 M) were added to 3.00 ml of Fl-labeled G-quadruplex (Fl-5′-AGGGTTAGGGTTAGGGTTAGGG-3′) (1×10−7 M) in Tris-KCl buffer, pH 7.4. After each addition, the solution was stirred for 20 s, incubated for 3 min and scanned for fluorescence using λmax=518 nm as excitation wavelength.
Additional evidences on interactions of the BQs with telomeric G-quadruplex DNA were obtained using circular dichroism. Successive amounts of each BQ (1×10−3 M) were added to 1.0 ml telomeric G-quadruplex DNA (4×10−6 M) in Tris-KCl buffer, pH 7.4. After each addition, solution was shacked, incubated for 3 min at room temperature and scanned in the range 200-400 nm using scan speed of 50.0 nm/min and band width of 1.0 nm. Averages of at least 3 accumulation scans were considered.
Melting temperature curves for telomere G-quadruplex, ct-DNA and their BQs' adducts were constructed using CD spectral measurements. A 1.0 ml telomeric G-quadruplex (3.93×10−6 M) or ct-DNA (100.00 ppm) in Tris-KCl-buffer, pH 7.4 was heated in the range 25-95° C. applying 2.0-5.0° C. increments and using 5 min incubation time intervals. The CD spectra in the range 200-400 nm were recorded at each temperature using the scan parameters described in previous section.
BQs complexes with G-quadruplex or ct-DNA were prepared by mixing equimolar amounts of G-quadruplex (1.44×10−4 M) with BQs (1×10−4 M) or ct-DNA (1000 ppm) with BQs (1×10−4 M) solution in 1.0 ml KCl-buffer pH 7.4. Solutions were incubated for 30 min before scan.
Collected CD spectra were smoothed and baseline corrected against blank solution. Intensities of CD peaks for G-quadruplex, ct-DNA and their BQs' complexes at 293 and 282 nm were recorded. Plots of CD intensities versus temperature were constructed.
Selectivity of BQs towards G-quadruplex over duplex DNAs was investigated fluorometrically using duplex telomere DNA. A 3.0 ml solution that is 1×10−7 M in 5′-Fl-G-quadruplex and 1×10−7 M in BQs was mixed with 10.00, 50.00 or 100.00 folds of telomere dsDNA in Tris-KCl-buffer pH 7.4. Solutions were vortexed for 10 s, incubated for 30 min at room temperature and scanned for their fluorescence spectra in the range 500-600 nm using excitation λmax of 494 nm.
The molar ratio method based on measuring UV-Vis absorption was used for determining stoichiometry of G-quadruplex DNA interactions with BQs. A 1.00 ml BQs (5×10−6 M) was titrated with telomere G-quadruplex (1.44×10−4 M) in Tris-KCl buffer, pH 7.4. The solution was shacked well after each addition, incubated for 3.0 min at room temperature and scanned in the range 200-600 nm. A plot of absorbance versus molar ratio [BQs]/[DNA] was constructed.
Binding affinity of the BQs towards telomere G-quadruplex DNA were estimated using Scatchard model based on the above UV-Vis absorption titration. Scatchard plot was
constructed as versus r according to the Scatchard equation;
Wherein r is the number of moles of the BQ bound to one mole of telomere G-quadruplex DNA (Cb/[G-quadruplex DNA], Cf is the free BQ's concentration, n is number of equivalent binding sites per G-quadruplex molecule and K is the binding constant. The free and bound concentrations of BQ (Cf and Cb) are calculated using Cb=Ctotal−Cf, where Ctotalis the concentration of BQ at zero addition of G-quadruplex and Cf is calculated using Cf=Ctotal(1 −α). The fraction of BQ bound to G-quadruplex (α) is calculated using
where Af, A and Ab are the absorbance at zero addition, after each addition and at saturation, respectively.
Linear plots of versus r give slope and intercept equals to K and n, respectively. For nonlinear plots, the modified Scatchard equation was used. Plots of r against Cf
were subjected to nonlinear fitting to get the values of K and n.
BQs were tested for their antiprolifrative activity in vitro using MTT assay on human pancreatic cancer cells (L3.6pl and MiaPaCa-2), human lung cancer cells (II1299), human breast cancer cells (MCF-7) and human prostate cancer cells (C42B).
The cell viability test was conducted by seeding pancreatic (L3.6pl and MiaPaCa-2), lung (H1299), breast (MCF-7) and prostate (C42B) cancer cells into 96-well culture plates at a density of 3×103 cells per well. Cells were then exposed to increasing doses (0-25 μM) of BQs for 72 hrs and MTT assay was performed using thymoquinone as a positive control in all experiments. The results were plotted as means ±SD of at least three separate experiments using six determinations per experiment. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells whose viability is assumed to be 100%. IC50s of the BQs were estimated from the linear plots of concentrations versus cell viabilities.
The synthesis of various benzoquinone derivatives (BQ1-14), in accordance with the invention; is described herein below by way of example.
Benzoquinone analogs were synthesized according to the following general procedure. Into a two necks flask (100 mL), 5.0 mmol benzoquinone (541 mg) were dissolved in 50.0 ml methanol (95%). Resultant solution was stirred and air bubbled followed by adding the corresponding amine solution (15.0 mmol) in methanol (5 ml) drop by drop. Stirring and air bubbling of the reaction mixture at room temperature continued overnight during which the reaction's progress was monitored by TLC. Formation of a precipitate was an indication for the reaction progress. When the reaction is completed, the solution was filtered off, and washed by methanol. The product obtained was recrystallized from methanol.
More particularly, fourteen benzoquinone analogs BQ(1-14) were synthesized by coupling benzoquinone with selected aromatic and alicyclic amines in one pot reaction under air bubbling at room temperature (Scheme 1 and
Reaction's progress was monitored by TLC. The weak solubility of products in methanol protected them from further rearrangement. Structures of resulted compounds were confirmed using IR, 1H-NMR, 13C-NMR, MS and elemental analyses.
Scheme 1 (Repeated in
An example is the reaction between 3,3′-dimethyl-(1,1′-biphenyl)-4,4′-diamine and benzoquinone resulted in symmetrically di-substituted benzoquinone (BQ6). Referring also to
The gCOSY, gHSQC, and gHMBC 2D-NMR spectra gave additional conformation on BQ6 structure. The gCOSY spectra of BQ6 in
Short-rang HSQC of BQ6 collected in DMSO-d6 is given in
Long-range 1H-13C gHMBC showed a strong correlation between BQ protons resonate at δ=5.11 ppm and the C=O and C-2 resonated at δ=182.14 ppm (2%) at δ=152.22 ppm (3%), respectively (
Interaction of obtained BQs with G-quadruplex DNA AGGG(TTAGGG)3 as examined using UV-Vis, fluorescence, NMR and CD spectroscopy, will be explained in more detail hereinbelow. Binding parameters and melting temperatures will also be investigated hereinbelow. Effects of the synthesized BQ derivatives on human pancreatic cancer cells (L3.6pl and MiaPaCa-2), human lung cancer cells (H1299), human breast cancer cells (MCF-7) and human prostate cancer cells (C42B) cancer cells were investigated in vitro using MTT assay, as will be explained in more detail hereinbelow.
Chemical analysis data for all synthesized compounds are described below.
Light brown crystals, yield 88% (1.876 g), mp 280-282.5° C.; IR (KBr, ν cm−1): 3257 (N−H), 1641 (C=0); 1H-NMR (400 MHz, DMSO-d6) δ ppm 5.86 (2 H, s, ethylene-H-3,6) 7.54-7.72 (8 H, m, (Ar-H)8) 9.51 (2 H, s, (NH)2); 13C NMR (400 MHz, DMSO-d6) δ ppm 96.90, 120.69, 122.15, 127.59, 130.98, 139.30, 147.01, 180.60. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C20H12F6N2O2:: C, 56.35%; H, 2.84%; N, 6.57%. Found: C, 55.61%; H, 2.51%; N, 6.31%.
Brown crystals, yield 92% (1.832 g), mp>350° C.; IR (KBr, ν cm−1): 3227 (N−H), 1642 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 5.31 (2 H, s, ethylene-H-3,6) 7.52-7.64 (4 H, m, (Ar-H)2) 8.93 (2 H, s, (NH)2); 13C NMR (400 MHz, DMSO-d6) δ ppm 96.90, 120.69, 122.14, 125.63, 127.60, 130.97, 139.23, 146.96, 180.60. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C18H8F6N2O2: C, 54.28%; H, 2.02%; N, 7.03%. Found: C, 55.64%; H, 2.16%; N, 4.25%.
Dark brown crystals, yield 86% (1.507 g), mp 323-325° C.; IR (KBr, ν cm−1): 3457, 3062, 1664 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 3.76 (6 H, s, 20CH3) 5.59 (2 H, s, ethylene-H-3,6) 6.97 (4 H, d, 4Ar-H, J=7.2) 7.26 (4 H, d, 4Ar-H, J=7.2) 9.02 (2 H, s, NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 55.87, 94.94, 115.07, 125.79, 130.95, 148.68, 157,69, 179.68. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C20H18N2O4: C, 68.56%; H, 5.18%; N, 8.00%. Found: C, 69.68%; H, 4.57%; N, 6.92%.
Dark brown crystals, yield 83% (1.354 g), mp>350° C.; IR (KBr, νcm−1): 3467, 3228 (N−H), 1640 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 5.66 (2 H, s, ethylene-H-3,6) 7.22-7.27 (4 H, 4Ar-H, m) 7.36-7.40 (4 H, 4Ar-H, m) 9.34 (2 H, s, 2NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 95.51, 105.00, 116.38, 116.60, 126.43, 126.51, 134.47, 148.13, 180.09. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C18H12F2N2O2: C, 66.26%; H, 3.71%; N, 8.59%. Found: C, 67.45%; H, 3.46%; N, 8.54%.
Brown crystals, yield 93% (1.350 g), mp 342-345° C.; IR (KBr, ν cm−1): 3466, 3233 (N−H), 1639 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 5.76 (2 H, s, ethylene-H-3,6) 7.32-7.44 (10 H, 10Ar-H, m) 9.33 (2 H, s, 2NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 99.96, 123.71, 129.69, 133.61, 139.27, 144.80, 186.35. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C18H14N2O2: C, 74.47%; H, 4.86%, N, 9.65%. Found: C, 75.05%; H, 4.54%; N, 10.05%.
Dark brown crystals, yield 78% (2.063 g), mp>350° C.; IR (KBr, νcm−1): 3436, 3225 (N−H), 1633 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 2.10 (6 H, s, 2CH3) 2.19 (6 H, s, 2CH3) 5.02 (4 H, s, 2NH2) 5.09 (2 H, s, ethylene-H-3,6) 6.65 (2 H, d, 2Ar-H, J=8) 7.17 (2 H, d, 2Ar-H, J=7.6) 7.24 (2 H, d, 2Ar-H, J=8) 7.28 (2 H, s, 2Ar-H) 7.42 (2 H, d, 2Ar-H, J=7.6) 7.50 (2 H, s, 2Ar-H) 9.11 (2 H, s, 2NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 18.05, 94.76, 114.70, 121.78, 124.07, 125.20, 126.83, 127.29, 127.33, 128.23, 128.63, 134.05, 134.63, 140.10, 146.99, 150.10, 179.18. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C34H32N4O2: C, 77.25%; H, 6.10%; N, 10.60%. Found: C, 78.49%; H, 5.95%; N, 11.21%.
Brown crystals, yield 55% (1.002 g), mp 184-188° C.; IR (KBr, ν cm−1): 3354, 2980, 2957, 2922, 2869, 2813, 1644 and 1613 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 2.46 (8 H, s, 4CH2) 2.64 (4 H, t, 2CH2, J=6) 3.20 (4 H, m, 2CH2) 3.72 (8 H, t, 4CH2, J=4.4) 5.28 (2 H, s, ethylene-H-3,6) 7.00 (2 H, s, 2NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 38.55, 53.20, 55.56, 66.85, 93.15, 151.05, 178.31. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C18H28N4O4: C, 59.32%; H, 7.74%; N, 15.37%. Found: C, 58.52%; H, 7.64%; N, 13.69%.
Brick red crystals, yield 73% (1.001 g), mp 289-291° C.; IR (KBr, ν cm−1): 3467, 3252, 2956, 2867, 1634 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 1.48-1.64 (12 H, m, 6CH2) 1.88 (4 H, m, 2CH2) 3.72 (2 H, m, 2CH) 5.24 (2 H, s, ethylene-H-3,6) 7.33 (2 H, d, 2NH, J=7.2); 13C NMR (400 MHz, DMSO-d6) δ ppm 24.28, 32.01, 53.76, 93.28, 151.09, 177.77. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C16H22N2O2: C, 70.04%; H, 8.08%; N, 10.21%. Found: C, 72.00%; H, 8.27%; N, 10.16%.
Dark brown crystals, yield 53% (0.881 g), mp 159-162° C.; IR (KBr, ν cm−1): 3465, 1643 and 1621 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 1.65 (8 H, s, 4CH2) 2.43 (8 H, s, 4CH2) 2.59 (4 H, t, 2CH2, J=6.4) 3.20 (4 H, m, 2CH2) 5.23 (2 H, s, ethylene-H-3,6) 7.47 (2 H, s, 2NH); 13C NMR (400 MHz, DMSO-d6) δ ppm 23.57, 41.18, 53.33, 53.85, 92.57, 151.53, 177.64. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C18H28N4O2: C, 65.03%; H, 8.49%; N, 16.85%. Found: C, 65.00%; H, 8.25%; N, 16.72%.
Brick red crystals, yield 61% (1.386 g), mp 271-274° C.; IR (KBr, ν cm−1 ): 3274, 1643 and 1621 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 4.45 (4 H, d, 2benzyl-CH2, J=6.8) 5.16 (2 H, s, ethylene-H-3,6) 7.48 (4 H, d, 4Ar-H, J=8) 7.67 (4 H, d, 4Ar-H, J=8) 8.34 (2 H, t, 2NH, J−6); 13C NMR (400 MHz, DMSO-d6) δ ppm 45.01, 93.96, 125.81, 125.84, 128.31, 142.79, 151.24, 178.37. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C22H16F6N2O2: C, 58.15%; H, 3.55%; N, 6.17%. Found: C, 57.10%; H, 3.71%; N, 6.16%.
Brick red crystals, yield 86% (1.524 g), mp 248-250° C.; IR (KBr, ν cm−1): 3281, 1643 and 1606 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 4.32 (4 H, d, 2benzyl-CH2, J=6.8) 5.18 (2 H, s, ethylene-H-3,6) 7.12 (4 H, 4Ar-H, m) 7.31 (4 H, 4Ar-H, m) 8.25 (2 H, t, 2NH, J=6.8); 13C NMR (400 MHz, DMSO-d6) δ ppm 44.78, 93.72, 115.56, 115.78, 129.68, 129.77, 133.99, 134.02, 151.22, 160.54, 162.96, 178.28. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C20H16F2N2O2: C, 67.79%; H, 4.55%; N, 7.91%. Found: C, 70.28%; H, 4.78%; N, 6.46%.
Brick red crystals, yield 83% (1.820 g), mp 256.5-258.5° C.; IR (KBr, ν cm−1): 3299 (N−H), 3006, 2934, 2838, 1641 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 3.69 (12 H, s, 40CH3) 4.25 (4 H, d, 2benzyl-CH2, J=6.4) 5.19 (2 H, s, ethylene-H-3,6) 6.76-6.93 (6 H, 6Ar-H, m) 8.15 (2 H, t, 2NH, J=6.4); 13C NMR (400 MHz, DMSO-d6) δ ppm 45.41, 55.90, 55.93, 93.63, 111.82, 112.17, 119.91, 130.07, 148.43, 149.20, 151.32, 178.16. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C24H26N2O6: C, 65.74%; H, 5.98%; N, 6.39%. Found: C, 64.85%; H, 5.77%; N, 6.37%.
Brown crystals, yield 77% (1.226 g), mp 254.5-256° C.; IR (KBr, ν cm−1): 3467, 3277 (N−H), 1643 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 4.34 (4 H, d, 2 benzyl-CH2, J=6.8) 5.16 (2 H, s, ethylene-H-3,6) 7.20-7.33 (10 H, 10Ar-H, m) 8.25 (2 H, t, 2NH, J=6.8); 13C NMR (400 MHz, DMSO-d6) δ ppm 45.56, 93.70, 127.59, 127.63, 128.93, 137.82, 151.38, 178.21. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated elemental analysis for C20H18N2O2: C, 75.45; H, 5.70; N, 8.80. Found: C, 77.37; H, 5.70; N, 9.61.
Brick red crystals, yield 66% (1.948 g), mp 237-239° C.; IR (KBr, ν cm−1): 3337(N−H), 3062, 2932, 1642 (C=0); 1H NMR (400 MHz, DMSO-d6) δ ppm 4.52 (4 H, d, 2benzyl-CH2, J=6.4) 5.34 (2 H, s, ethylene-H-3,6) 7.99 (2 H, s, 2Ar-H) 8.03 (4 H, s, 4Ar-H) 8.40 (2 H, t, 2NH, J=6.4 Hz); 13C NMR (400 MHz, DMSO-d6) δ ppm 44.43, 94.05, 121.54, 122.41, 125.12, 128.78, 130.49, 130.82, 141.61, 151.00, 178.68. EI-MS: m/z 292.2, 0.9% (M+), 232.1, 23.1%, 206.1, 61.1%, 149.0, 67.6%, 100, 100%. Calculated CHN analysis for C24H14F12N2O2: C, 48.83%; H, 2.39%; N, 4.75%. Found: C, 50.11%; H, 2.77%; N, 4.71%.
Circular dichroism is a well-established method for studying changes in DNA conformation. G-quadruplex DNA showed a hybrid (parallel-antiparallel) structure characterized by a negative band at 235 nm and two positive bands at 253 nm and 293 nm. Additions of up to 5% DMSO and 0.1% Tween-80 did not change the hybrid G-quadruplex conformation indicating that they can be safely used for studying DNA interactions with the BQ compounds.
In the followings, Interactions of the BQs with DNA were studied using UV-Vis absorption, fluorescence, fluorescence quenching, circular dichroism and NMR spectroscopic techniques. The results obtained were used to evaluate binding affinities and stabilization effects towards G-quadruplex DNA.
Four absorption bands were identified in UV-Vis spectra of p-benzoquinones. A strong band at 250 nm (ε=20,000) and a weak band at 300 nm (ε=320) ascribed to π-π* transitions. The other two are assigned to the n-n* singlet-singlet and singlet-triplet transitions and shown as very weak at 400-500 (ε=20-30) and 540 nm (ε=0.2), respectively.
The BQs give absorption bands around 280, 330 and/or460 nm. To avoid interference with DNA absorption bands at 260 and 280 nm, titration of the BQs with DNA was followed at 330 or 460 nm (see
CD titration confirmed the Interactions of BQs with telomere G-quadruplex DNA. In
Additional confirmations for the interactions between the BQs with G-quaduplex were obtained from fluorescence quenching titrations.
Binding Stoichiometry and Binding affinity
Molar ratio method using UV-Vis titration was used to estimate the binding stoichiometry of the BQs to G-quadruplex DNA. The number of bound BQ molecules per G-quadruplex molecule ranged between 1 and 4 (See Table 2 of
Scatchard plots based on absorbance measurements gave estimates of the binding constants of BQs towards G-quadruplex DNA. (K). Linear plots indicate one type of equivalent or independent binding sites whereas nonlinear plots indicate more than one type of dependent binding sites that cause neighbor exclusion effect (in which binding on one site may encourage or suppress binding on the another site). Values of binding constants and number of binding sites are given in Table 2 (see
Melting temperature gives indications for the stability of DNA-ligand complex and the binding affinity of the ligand towards DNA
Melting temperatures for G-quadruplex-BQs complexes were estimated using CD spectroscopy (
Comparison of melting temperatures of G-quadruplex and duplex DNAs complexes can also give an indication on their selectivity towards both DNAs. Higher ΔTm change gives higher selectivity.
Selectivity of the BQs towards G-quadruplex DNA in the presence of telomere dsDNA was examined.
Based on the differences between ΔTm (G-quadruplex) and ΔTm (ct-DNA) given in table 2 of
Thus, the results from selectivity test using fluorescent G-quadruplex DNA and melting temperatures are consistent and indicated that the BQs have shown higher selectivity towards G-quadruplex over duplex DNA. This conclusion is also supported by the binding constants listed in table 2 of
Referring to
The BQs concentrations (1.0-25.0 μM) caused concentration- and time-dependent inhibition in cellular viability of L3.6pl, MiaPaCa-2, H1299, MCF-7 and C42B cells over 72 hours. Colon and lymphoma cancer cells were found insensitive.
The IC50 concentrations lower than 10 μM are highly desirable in drug syntheses. Referring to
The high potency of the BQ derivatives against the tested cell lines is consistent with their binding constants (7.28×105-5.6×104 M−1) and melting temperature values with G-quadruplex DNA. Thus, the anticancer activity of BQs may be attributed to their ability to inhibit telomerase enzyme through stabilizing G-quadruplex DNA. Other mechanisms may also be effective.
The synthesized benzoquinone analogues showed anticancer activity against all examined cancer cell lines except prostate cancer cells (C42B). IC50 ranged between 5.0 and 30.0 μM were obtained in almost all compounds. Compounds BQ6, BQ9, BQ10 and BQ11 have shown the highest potency with IC50 less than 10.0 μM.
Interactions of obtained benzoquinone compounds with telomeric G-quadruplex DNA sequence; AGGG(TTAGGG)3; were tested. The compounds gave affinities ranged in 5.6×104 -7.3×105 M−1 towards G-quadruplex. Selectivity test indicated high selectivity towards G-quadruplex over ct-DNA. Melting temperatures indicated that BQ5, BQ6, BQ7, BQ8, BQ10, BQ12 and BQ14 have ≧3.5 folds higher selectivity towards G-quadruplex over ct-DNA. Compounds BQ1, BQ9, BQ11 and BQ13 showed moderate selectivity (1.0-3.0 folds) while BQ4 gave the least selectivity (0.6 folds).
The BQ derivatives have shown IC50s ranged in 8.04-50.49 for MCF-7, 6.18-64.61 for MiaPaCa-2, 8.94-50.41 for L3.6pl, 7.48-66.88 for H1299 and 42.06-740.48 μM. Human prostate cancer cells (C42B) gave the highest IC50s indicating low potency for tested BQs. BQ9 gave the highest loss of cell viability (92.50%) in MCF-7, BQ6 have shown the highest loss of cell viability (88.15%) in MiaPaCa-2, BQ9 have shown the highest loss of cell viability (84.40%) in H1299, BQ6 have shown the highest loss of cell viability (80.14%) in L3.6pl and BQ5 have shown the highest loss of cell viability (27.25%) in C42B. All the BQ derivatives at 25 μM gave showed higher potency and efficacy than the parental thymoquinone compound except BQ7 and BQ8 in H1299 and BQ3 in C42B.
IC50s of 8.04 and 8.42 μM were obtained for BQ10 and BQ6 in MCF-7, of 6.18 and 7.68 μM for BQ11 and BQ3 in MiaPaCa-2, of 8.94 and 9.67 μM for BQ6 and BQ11 in L3.6pl and of 7.48 μM for BQ4 in H1299. The results also showed that H1299 cells seem to be the highest sensitive cells to the cytotoxic effects of BQs whereas C42B cells are the least sensitive cells. These results are very important since IC50s less than 10.0 μM are recommended for successful development of new drugs
Interaction of synthesized BQ derivatives with G-quadruplex DNA (AGGG(TTAGGG)3) was also investigated using UV-Vis spectrophotometry, fluorescence spectrophotometry, NMR, melting temperature and CD spectroscopy. Binding parameters include binding constant, binding mode; melting temperature; selectivity and binding stoichiometry were evaluated.
The results indicated that the BQs interact with G-quadruplex DNA. Scatchard plots revealed linear and nonlinear correlations indicating binding constants in the range 7.28×105 -5.60×104 M−1 and one or two types of binding sites. BQ1 showed the highest binding constant (7.28×105 M−1) while BQ11 showed the lowest binding constant (1.40×105 M−1).
Similar findings were obtained using melting temperature curves. The BQs have shown to stabilize G-quadruplex. BQ1 gave the highest Tm and ΔTm (75.5 and 7.50° C.) while BQ4 gave the least (68.1 and 0.1° C.). With the exception of BQ1 and BQ3, all other BQs complexes with ct-DNA gave ΔTm≦0.0 indicating that The BQs are destabilizing the duplex ct-DNA. Selectivity test indicated high selectivity towards G-quadruplex over ct-DNA. BQ5, BQ6, BQ7, BQ8, BQ10, BQ12 and BQ14 showed ≧3.5 folds higher selectivity towards G-quadruplex, over ct-DNA Compounds BQ1, BQ9, BQ11 and BQ13 showed moderate selectivity (1.0-3.0 folds) while BQ4 gave the least selectivity (0.15 folds).
As stabilizing G-quadruplex structures in human telomere is expected to inhibit telomerase enzyme found active in almost all cancer cells, the BQ compounds could be good candidates for treating cancers. In additions, they have shown very good potency against tested cancer cell lines
These results revealed a number of novel benzoquinone based compounds with high antitumor efficacy that can be further processed as anticancer agents. More specifically the Benzoquinone derivatives can be used in the manufacture of a pharmaceutical composition for treating cancer.
Most of the BQ derivatives have shown higher binding affinity than parental compound and better selectivity towards G-quadruplex over ct-DNA. The results suggest that these compounds target telomeric G-quadruplex DNA. However, stabilizing DNA may not be the only mechanism by which these compounds act on cancer cells.
While the present invention has been described with respect to specific examples, it should be appreciated that the present invention is not limited to these examples. It is to be believed that one skilled in art, using the preceding description, can utilize the present invention to its fullest extent, and many variations and modifications may present themselves to those of skill in the art without diverting from the scope of the present invention.
