Study of the synthesis of novel trisubstituted acridines

An efficient methodology has been developed for the synthesis of 3,6-diamino-9[(phenylalkyl)amino]acridines by heating 2-propanol solutions of 3,6-di(butanoylamino)-9[(phenylalkyl)amino]acridines in the presence of NaOH. 3,6-Diamino-9[(phenylalkyl)amino]acridines were used in the synthesis of final products 3,6-bis{3-[2(dimethylamino)ethyl]ureido}-9-[(phenylalkyl)amino]acridines. Herein we rationalize the formation of 3,6,9-triaminoacridine and propose the reaction mechanism for the observed transformation. Keyword: Acridine, acridone, thiourea, urea


Introduction
Acridine derivatives have been extensively studied for their chemotherapeutic properties due to their ability to intercalate into DNA and disrupt unwanted cellular processes. 1,20][11] Beyond these classical applications as drugs and as ligands, acridine derivatives have also been used as fluorescent probes and chemosensors. 12aking this into consideration, structural modifications of acridine scaffold became widely studied by organic chemists.An extensive work in this field led to new synthetic inventions enabling the preparation of acridine skeleton with a broad structural diversity.Here, we would like to present results related to an attempt to prepare a new series of trisubstituted acridines based on BRACO-19 framework as G-quadruplex targeting compounds (Figure 1).Structural similarities between BRACO-19 framework and our new structures are based on the 3,6,9-derivatisation of acridine scaffold.Whereas positions 3,6-are modified by aminoaliphatic groups, the position 9-is substituted by phenylalkylamino moiety to provide better flexibility.

Results and Discussion
Preparation of acridone A synthetic pathway described herein starts by the preparation of 3,6-diaminoacridone 4 and provides access to the trisubstituted acridine framework.Preparation of 3,6-diaminoacridone 4 was done in accordance to previously described procedure based on diphenylmethane (1) (Scheme 1). 13The overall synthetic protocol was adjusted to simplify each reaction step and to enhance the overall yield.The initial step, nitration of diphenylmethane (1), proceeded in a yield similar to published results.The subsequent oxidation of 2 was modified due to poor solubility of reactant in acetic acid as stated in the original report.The amount of acetic acid had to be increased at least three times.Subsequent cyclization of compound 3 was improved substantially.Shortly, the amount of hydrochloric acid and ethanol was reduced, anhydrous stannous chloride was replaced by the cheaper dihydrate, and the reaction mixture was continuously refluxed for 18 hours.With these modifications, previously reported yield increased from 60% to 80%.Modified synthetic methodology provided 20% increase of the overall yield.
A key intermediate 6 was synthesized by the reaction of POCl 3 with precursor 5 which was in turn obtained by the reaction of acridone 4 with butyric anhydride at elevated temperature.Installation of the butanoyl protective group was performed with the aim to improve the solubility of the intermediate 6. Resulting derivative 6 was then purified by crystallization from ethyl acetate.The synthesis of trisubstituted compounds 7a-c involved treatment of 6 with phenylalkyl amines (Scheme 2).Next, there was intended to remove the protecting groups from derivates 7a-c with the intention to obtain amines.There was considered the use of acidic conditions to obtain the free amine 8a.Interestingly, this reaction afforded 3,6,9-triaminoacridine Scheme 1. Synthesis of 3,6-diamino-9(10H)acridone (4). ii. iii.

Basic hydrolysis
Overall, this approach proved to be unsuccessful to produce the desired product 8a despite extensive experimentation.To circumvent this issue, alkaline conditions was used instead, a mixture of aliphatic alcohols with sodium hydroxide.The superior level of conversion was obtained in the case of 2-propanol (Scheme 4, Table 1, entry 5), the use of other alcohols (Table 1) resulted only to partial formation of derivate 8a.The worst result was achieved when a mixture of 2-propanol/water (50% each) was used as a solvent leading to a complex mixture containing the substrate 7a along with the desired free amine 8a and acridones 4 and 5 (Scheme 4).To understand this reaction better, 9-(benzylamino)acridine (10) was used as the model compound in order to prevent any possible side reactions.
The derivative 10 was subjected to a mixture of ethanol/NaOH or 2-propanol/NaOH under the same reaction conditions.In the case of ethanol the reaction produced the acridone 10a predominately (Scheme 5).On the other hand, the use of 2-propanol resulted in minimal conversion to acridone 10a (Scheme 5).Observed outcome of the reaction with model substrate might be attributed to different kinetics of the reaction with more sterically hindered 2propanolate versus less crowded ethanolate.This observation might also provide rational for the reaction shown in the Scheme 4. In a mixture of 2-propanol/water, bulky 2-propanolate is formed that attacks predominately 3,6-positions of the acridine ring while avoiding the position 9, whereas rather small hydroxyl ion reacts with no chemoselectivity.

Acidic hydrolysis
To find an explanation for the formation of derivative 9 under acidic conditions (Scheme 3), there was made an attempt to elucidate the reaction mechanism.There is outlined two possible reaction pathways which might explain the loss of the benzyl group (Scheme 6.).The first one suggests the formation of benzyl alcohol (12) as the consequence of nuclephilic displacement by water molecule (tautomeric form B, Scheme 6).The second proposal is based on the formation of benzaldehyde (13), the expected product of the hydrolysis of Schiff base-like tautomer C (Scheme 6).To distinguish between these two possibilities, there was made an attempt to isolate the products of acidic hydrolysis of derivative 10.There were used 50 % aqueous H 2 SO 4 , 50% aqueous H 3 PO 4 and 35% aqueous HCl.Substrate 10 was heated for 3 hours at 80 °C, followed by cooling of the mixture, and extraction by diethylether.The residues obtained upon removal of diethylether were analyzed by NMR.Benzyl alcohol (12) was found to be the product of the reaction in all cases as confirmed by NMR (Scheme 6).Benzoic acid (14) was found as the minor product in the case of sulfuric acid likely formed as a product of the oxidation of benzaldehyde (13), that might be produced by hydrolysis of tautomer C (Scheme 6.).ii.
The rational for the loss of the benzyl group is outlined in Scheme 7.Under acidic conditions, nucleophilic displacement (water as a nucleophile) takes place with 9-aminoacridine (11) as the leaving group.Protonation makes of 9-aminoacridine (11) better leaving group, and the reaction likely occures by the mixed or monomolecular type of mechanism.It is important to stress that above discussed reaction mechanism is drawn with the delocalisation of a partial charge on the phenyl ring, generated during nuclephilic attack on methylene carbon (Scheme 7).This assumption is supported by the fact that derivatives 7b and 7c possessing one or two carbon atoms longer side chain do not form triaminoacridine 9 under acidic conditions (Scheme 8).

Mechanistic studies
Although there was not detected the presence of derivative 9 as one of products of the reaction described in the Scheme 4, this alternative reaction pathway, nucleophilic displacement of acridine framework by a hydroxyl/2-propanolate ion, could not be excluded completely.Computational chemistry calculation was performed to understand the reaction in more details.There was intended to study the bimolecular nucleophilic displacement on the methylene carbon of the model compound 9-(benzylamino)acridine (10) as the alternative reaction mechanism relevant under acidic conditions (Figure 2, Table 2).The attention was focused on the competition between 2-propanolate and hydroxyl anion, reactive species present in the reaction mixture.From the energy point of view it could be concluded that bulkiness of iso-propyl group has a significant influence.From kinetic as well thermodynamic point of view, it becomes evident that the reaction with 2-propanolate is disfavored, and should the bimolecular nucleophilic substitution take place, hydroxyl ion will be preferred as a nucleophile.
When sodium hydroxide was replaced by ammonium hydroxide, the product of the reaction was derivative 15 (Table 1) likely arising from intermolecular amine exchange via hemiaminal intermediate (Scheme 9).

Preparation of trisubstituted acridines
Having resolved above mentioned problems, free amines 8a-c were utilized for the preparation of isothiocyanates 16a-c which might serve as key synthons in the synthesis of novel 3,6,9trisubstituted acridines.8][19] Unfortunatelly, the preparation of dithiocarbamates by the reaction of amines 8a-c with carbon disulfide was not successful, probably due to low nucleophilicity of amines 8a-c.Therefore the thiophosgene-mediated formation of isothiocyanates had to be used (Scheme 10). ii. iii.
i. CSCl An attempt to use two-phase system (dichloromethane/water), the standard routine in the synthesis of isothiocyanates, proved unsuccessful owing to low solubility of amines 8a-c.Luckily enough there was found, that amines 8a-c are soluble in mixtures of acetone or acetonitrile with water, while they are not in above mentioned individual solvents.This finding prompted us to find a suitable combination of reactants and solvents to obtain homogenous reaction mixture.The best choice turned out to be the combination of acetone/water in a ratio 7:1 as the solvent for amines 8a-c.The solution of amine was added dropwise to thiophosgenecontaining solution of sodium bicarbonate in acetone/water (1:3).Obtained isothiocyanates 16ac were not purified (expected low stability).Crude isothiocyanates 16a-c were reacted with N,Ndimethylethylenamine in methanol at 0 °C.There should be pointed out that attempts to prepare thioureas 17a-c in commonly used organic solvents led to a mixture of unidentified products.As the reaction progressed, the resulting mixture became homogenous and the formation of corresponding thioureas 17a-c was observed by TLC analysis.Thioureas 17a-c were purified by gradient chromatography and then used in the reaction with mesityl nitriloxide providing ureas 18a-c.

Conclusions
We have developed a synthetic methodology for the preparation of 3,6-urea substituted 9-[(phenylalkyl)amino]acridines as a prospective G-quadruplex stabilizing agents.We rationalized the unexpected results observed for some reactions described within the body of this paper and we proposed reasonable reaction mechanisms.We found reasonable reaction conditions for the transformation of poorly soluble amines 8a-c with low nucleophilicity into corresponding isothiocyanates 16a-c, key intermediates toward the desired products.

Experimental Section
General.All chemicals and reagents were reagent grade and were used without further purification. 1H (400 MHz, 600 MHz) and 13 C (100 MHz, 150 MHz) NMR spectra were measured on a Varian Mercury Plus or a Varian VNMRS NMR spectrometers at room temperature in CD 3 OD or DMSO-d 6 using TMS as an internal standard (0 ppm for both nuclei).Melting points were determined with a Koffler hot-stage apparatus and are uncorrected.Elemental analyses were performed on a Perkin-Elmer analyzer CHN 2400.Reactions were monitored by thin-layer chromatography (TLC) using Silufol plates with detection at 254 nm.Preparative column chromatography was conducted using Kiesegel Merck 60 column, type 9385 (grain size 250 nm).9-Benzylaminoacridine (10) and 9(10H)acridone (10a) were synthetized according to the published procedure, and all physical-chemical characteristic were in accordance to previously published results. 20,219-Aminoacridine (11) was commercially available.

2,2´,4,4´-Tetranitrodibenzophenone (3).
CrO 3 (17 g, 0.17 mol) was slowly added (over 10 min period) to a refluxing solution of 2 (10 g, 28.7 mmol) in 98% acetic acid (50 mL), and the mixture was refluxed for 3 hr.Once cooled to room temperature, the resultant mixture was poured into water (200 mL), the precipitated product was filtered off with suction, and was consecutively washed with water (10 mL), ethanol (10 mL) and diethyleter (10 mL) affording 9.8 g of product 3 as a pale yellow solid.Yield 94%.mp 234-236 o C. 1 4).SnCl 2 .2H 2 O (94 g, 0.417 mol) was slowly added to a refluxing solution of 3 (10 g, 27.6 mmol) in 35% hydrochloric acid (150 mL), followed by the addition of ethanol (25 mL).The mixture was heated for 18 hr at 90 °C.On cooling to room temperature, the precipitate was filtered off with suction, and was allowed to dry under air over night.Resulting solid was disolved in hot 0.1 M hydrochloric acid (50 mL), and the solution was alkalized with 80% NaOH.On cooling, the precipitated product was filtered off with suction to give 5 g of the product 4 as a light red solid.Yield 80%, mp >300 o C. 1

3,6-Di(butanoylamino)-9(10H)acridone (5).
Diaminoacridone 4 (10 g, 44.4 mmol) was added to 70 mL of butyric anhydride (0.428 mol).The reaction mixture was heated at 100 °C while being stirred vigorously for 1.5 hr.Once the resulting mixture had cooled to room temperature, 30 mL of acetone was added.The mixture was poured into 200 mL of brine with an additive of 15 mL of saponate.A concentrated solution of sodium hydroxide was added to the mixture in 25 mL portions over 15 min, until an alkaline pH had been achieved.The precipitated product was filtered off with suction.Then, product was crystallized from ethanol.Light brown solid, yield 49%, 8 g, mp > 300 °C. 1

3,6-Di(butanoylamino)-9-chloroacridine (6).
Finely powdered acridone 5 (5 g, 13.7 mmol) was slowly added to 20 mL of POCl 3 (0.214 mol) over 15 min.The mixture was heated at 70 °C while being stirred vigorously.After 90 min, the resulting mixture was cooled to room temperature and slowly poured onto 100 g of ice in 100 mL of distilled water.A concentrated solution of sodium hydroxide was added to the resultant mixture in 25 mL portions in 15 min intervals until an alkaline pH had been achieved.The precipitated product was filtered off with suction, was washed with distilled water and was dried overnight.The resultant crude product 6 was crystallized from ethyl acetate, this operation yielded 2.5 g as a light yellow solid.Yield 47%, mp 153-155 °C. 1 -c).Chloroacridine 6 (1.0 g, 2.6 mmol) was dissolved in hot acetonitrile (60 mL), followed by the addition of corresponding amine (8.0 mmol).The mixture was refluxed for 1 hr.and the precipitated product was filtered off, was washed with acetonitrile.To remove the rest of the amine, the product was suspended in acetone, the mixture was vigorously stirred under reflux for 2 hr.On cooling, the product was filtered off, and was crystallized from ethanol to afford yellow crystals.

3,6-Di(butanoylamino)-9-aminoacridine (15).
Derivative 7a (0.20 g, 0.4 mmol) was dissolved in methanol (3 mL), followed by the addition of 1 mL of concentrated amonnium hydroxide solution.The reaction mixture was heated at 50 °C for 5 hr.The resulting mixture was then poured into 30 mL of brine, and precipitated product was filtered off.Product 10 was crystallized from MeOH and dried in vacuo over P 2 O 5 affording yellow crystals.Yellow crystalline solid, yield 83%, 0.12 g, mp > 300 °C. 1  General procedure for the preparation of 3,6-bis(isothiocyanato)-9-[(phenylalkyl) amino]acridine (16a-c).Diaminoacridines 8a-c (1.6 mmol) were dissolved in a mixture acetone/water (10 mL; 7:1, v:v).Resulting solution of amines were added dropwise to the vigorously stirred suspension of thiophosgene (0.368 mL, 4.8 mmol) in a mixture of water/acetone (10 mL; 3:1; v:v) containing sodium bicarbonate (1.34 g, 16 mmol, ) at 0 °C, leading to the formation of isothiocyanates 16a-c as a yellow paste.After the addition was completed, the mixture was stirred for additional 30 min.The crude product was filtered off with suction, was washed with acetone, and was dried in vacuo over P 2 O 5 for 1 hr.Due to low stability, isothiocyanates 16a-c were used immediately for the preparation of thioureas 17a-c.

General procedure for the preparation of 3,6-bis{3-[2-(dimethylamino)ethyl]thioureido}-9-[(phenylalkyl)amino]acridine (17a-c).
Previously prepared isothiocyanates 16a-c were suspended in methanol, followed by the addition of dimethylethylenediamine (0.435 mL, 4 mmol).The reaction mixtures were stirred at 0 °C for 1 hr, clear solutions were obtained at the end of the reaction.Subsequently, the solvent was evaporated under reduce pressure providing light brown oily residue.The crude product was purified on a silica gel column by gradient elution using a mixture acetone and 12% aqueous solution of diethyl amine (from 15:1 to 5:1, v/v).Evaporation of the solvent under reduced pressure provided products 17a-c as a dark yellow semi-solid residues.

Figure 2 .
Figure 2. Plausible transition states (TS) for the theoretical reaction of derivative 10 with Onucleophiles, hydroxyl (left) or 2-propanolate (right) ion.One molecule of the solvent was added to imitate the solvent effect.Hydrogens outside of the center of nucleophilic attack are omitted for clarity.Geometries of TS were obtained on semi-empirical level of theory using PM3 (MOPAC2012 16 ) (C -light blue, N -blue, O -red, H -white).

Table 2 .
Energetic terms of the reaction of derivative 10 with O-nucleophiles a Heat of formation (Kcal.mol -1 ) obtained by PM3 calculation.b Derivative 10 and hydroxyl ion.c Derivative 10 and 2-propanolate ion.d Energy gab between transition state (TS) and reactants (RCT) or transition state (TS) and products (PDT).e Energy gab between products (PDT) and reactants (RCT).