Synthesis of 9-ethyl[1,2,5]selenadiazolo[3,4-h ]quinolones by the application of modified Gould-Jacobs reaction to N -ethyl-2,1,3-benzoselenadiazol-4-amine

An effective method for the synthesis of N -ethyl-2,1,3-benzoselenadiazolamines 11 and 15 has been described. Modified Gould-Jacobs reaction of N -ethylbenzoselenadiazol-4-amine 15 provided 9-ethylselenadiazoloquinolone derivatives 2 and 19 in high yields. Acid-promoted ring closure of enamine 18c unexpectedly afforded completely deacetylated product – 9-ethylselenadiazoloquinolone 2 . Identical product was obtained by ethylation of selenadiazolo[3,4-h ]quinolone 1 and by basic hydrolysis of ethyl ester 19b followed by thermal decarboxylation of the corresponding acid 20 .


Introduction
Antibacterial agents containing 4-oxoquinoline (4-quinolone) moiety found a broad application in the treatment of bacterial infections in human and veterinary medicine. 1,2In the last decades, anti-cancer activity of 4-quinolone derivatives has been revealed and studied intensively. 3,4The presence of selenium in the structure of 4-quinolones can bring new characteristics, and plenty of selenaheterocyclic compounds showed positive biological impact. 5,60][11][12] Antimicrobial activity of 7-substituted selenadiazolo [3,4-h]quinolones was demonstrated on Gram positive and Gram negative bacteria, yeasts and filamentous fungi. 7Ultraviolet A photoexcitation of all selenadiazoloquinolones in DMSO or acetonitrile led to the formation of paramagnetic intermediates coupled with activation of molecular oxygen generating the superoxide anion or singlet oxygen.The ability to form paramagnetic species was evidenced by means of EPR spectroscopy employing the spin-trapping technique or oxidation of sterically hindered amines.The cytotoxic/photocytotoxic effect on murine and human cancer cell lines was demonstrated on 7-acetyl-6H,9H- [1,2,5]selenadiazolo [3,4-h]quinolin-6-one. 9Upon anodic oxidation of selenadiazoloquinolones in alkaline solutions, the selenadiazole ring was replaced with paramagnetic ortho semiquinone radical anions which was confirmed by EPR spectroscopy and quantum chemical calculations. 10ased on our previous results obtained for 7-and 8-substituted selenadiazoloquinolones, we decided to synthesize their analogues ethylated at nitrogen atom of the pyridone ring to improve their biological/photobiological and physicochemical properties.Prepared ethylselenadiazoloquinolones showed different redox behaviour which was studied in detail by in situ EPR/UV-vis cyclovoltammetric experiments. 12Recently, the cytotoxic/photocytotoxic effects of ethylselenadiazoloquinolones on cancer human (HeLa) and murine (L1210) and noncancer (NIH-3T3) cell lines were investigated. 13In this paper, the synthesis of ethylselenadiazoloquinolones by ethylation of selenadiazoloquinolones and modified Gould-Jacobs reaction applied to N-ethylbenzoselenadiazolamines is discussed in detail.

Results and Discussion
Usually, 4-oxoquinolone derivatives are N-ethylated with an excess of iodoethane in DMF at room or elevated temperature in the presence of K2CO3 or NaH as a base. 14Rarely, diethylsulfate, 15 triethylphosphate 16 and ethyl tosylate 17 are employed as the ethylating agents.Hence, the most straightforward route towards 6-and 9-ethylselenadiazoloquinolones seemed to be the ethylation of selenadiazoloquinolones prepared in our previous papers. 7,8However, the ethylation of selenadiazoloquinolone 1, as a representative compound, with the excess of EtI (3-4 eq.) in DMF at 50 °C in the presence of K2CO3 led to a mixture of polyethylated products in a very low yield.When NaH was used as the base instead of K2CO3 and the amount of EtI was reduced to 1.5 equivalents, the mixture of N-and O-ethylated products 2 and 3 was formed in the 3:2 ratio (Scheme 1).

Scheme 1
Analogously, treatment of selenadiazoloquinolone 4 with EtI in the presence of K2CO3 afforded a mixture of N-and O-ethylated products 5 and 6 in almost the same ratio as in the previous case.Despite the fact that formation of selanadiazoloquinoline 6 was not observed when NaH was applied as the base, desired 6-ethylselenadiazoloquinolone 5 was isolated only in 26% yield (Scheme 2).

Scheme 2
Because of the poor regioselectivity as well as relatively low yields of target N-ethylated derivatives 2 and 5, the ethylation of selenadiazoloquinolones did not seem to be the perspective method for the preparation of ethylselenadiazoloquinolones.
Another approach to ethylselenadiazoloquinolones could represent ethylation of precursors of selenadiazoloquinolones under the same reaction conditions which were applied to selenadiazoloquinolones 1 and 4. The products obtained after ethylation might be cyclized to target compounds in various acidic media.However, selected precursors did not undergo ethylation with an excess of EtI in the presence of K2CO3 even at higher temperature (70 °C) and only starting materials were recovered.On the other hand, using NaH as the base led to complex reaction mixtures of practically inseparable products.
In modified Gould-Jacobs reaction, 14 N-substituted (hetero)aromatic amines play the role of the starting materials.In such a case the enamines formed in nucleophilic vinylic substitution, no longer bear a hydrogen at the amino nitrogen, thus making thermal cyclization impossible.][20] Although the preparation of N-substituted (hetero)aromatic amines is more laborious, the regioselectivity of the alkylation is no longer a problem.Thus we focused our attention on the synthesis of N-ethylbenzoselenadiazolamines 11 and 15.First, we excluded classical ethylation of benzoselenadiazolamines 7 with EtI because that would probably give a mixture of polyethylated products.Since benzoselenadiazole ring is very sensitive to reductive conditions, a reductive amination of benzoselenadiazolamines 7 was not considered as a suitable method for preparation of ethylamines 11 and 15.Treatment of benzoselenadiazolamines 7 with concentrated H2SO4 in triethyl orthoformate did not provide the appropriate N-ethylformamides.
The latter ones could yield N-ethylbenzoselenadiazolamines 11 and 15 after acid hydrolysis. 21onoethylation of amines 7 according to Katritzky's protocol [22][23][24] failed already at the preparation of benzotriazole ethylating agent.Another route for preparation of ethylamines 11 and 15 could include ethylation of acetamides 8 followed by hydrolysis of the resulting Nethylacetamides 9 (Scheme 3).
Acetamides 8 were obtained by a simple acetylation of amines 7 in acetic anhydride according to procedures described in the literature. 25Before ethylation of acetamides 8, we tried to take advantage of their direct reduction into ethylamines 11 and 15.The reduction of acetamides 8 with LiAlH4 in refluxing THF resulted in a mixture of unidentified products of decomposition.On the other hand, their treatment with BH3•SMe2 in refluxing THF caused besides reduction of acetamide moiety even reductive deselenation of benzoselanadiazole ring to form corresponding benzenetriamines.These were extremely unstable and sensitive to air oxidation.Despite their instability we were able to isolate triamine 10 as a pale brown semisolid.Due to its instability it was characterized only by 1 H NMR spectroscopy (Scheme 3).To our knowledge, reductive deselenation of benzoselenadiazole ring with BH3•SMe2 was not described in the literature up to date.Immediate dissolution of triamine 10 in EtOH and treatment with stoichiometric amount of SeO2 dissolved in water resulted in a black mixture of reaction products in which desired ethylamine 15 was observed only in traces along with decomposition and oxidation products.In our next strategy, the acetamides 8 underwent deprotonation with NaH in DMF followed by treatment with EtI to access N-ethylacetamides 9 in high yields.
Alcoholysis of N-ethylacetamide 9b with one equivalent of MeONa in refluxing MeOH proceeded smoothly affording N-ethylbenzoselenadiazol-5-amine 11 in a good yield (Scheme 3).On the contrary, basic hydrolysis of N-ethylacetamide 9a with a large excess of MeONa in MeOH proceeded very slowly and starting material was still identified (TLC) in the reaction mixture even after 7 days of reflux.Moreover, hydrolysis of N-ethylacetamide 9a under basic (NaOH/H2O/EtOH/reflux, NaOH/EtOH/reflux), acidic (20% HCl/reflux, 20% HCl/dioxane/reflux) or neutral (N2H4•H2O/EtOH/reflux) conditions failed or led to complex reaction mixtures.Since hydrolysis of acetamide group was not successful, it was replaced by trifluoroacetyl group which is more susceptible to basic hydrolysis.Nevertheless, trifluoroacetamide 12 obtained by trifluoroacetylation of amine 7a did not undergo ethylation under the same reaction conditions described for acetamide 8a (Scheme 4).

Scheme 4
Finally, the ethoxycarbonyl group was successfully employed as a protecting group in the synthesis of ethylamine 15.Ethylation of carbamate 13 smoothly yielded ethylcarbamate 14 which was subjected to basic hydrolysis to provide N-ethylbenzoselenadiazol-4-amine 15.Even under very hard reaction conditions for the removal of ethoxycarbonyl group, (reflux of ethylcarbamate 14 with 10 equivalents of NaOH in EtOH for 60 h), ethylamine 15 was isolated in 90% yield over three steps (Scheme 4).
Next, ethylamines 11 and 15 entered into the nucleophilic vinylic substitution 26 with the appropriate alkoxymethylidene derivatives 17 (activated enol ethers).Generally, this substitution proceeds smoothly with primary amines in refluxing alcohol (MeOH, EtOH) with a small excess of the activated enol ether.In our case, nucleophilic vinylic substitution of diethyl 2-(ethoxymethylidene)propanedioate 17b (EMME) with ethylamines 11 and 15 in refluxing EtOH did not proceed at all, even if EMME was used in an excess.Therefore the substitution was performed under solvent free conditions by a simple heating of ethylamines 11 and 15 in an excess of EMME at 160 °C.In case of ethylamine 11, the reaction was complete within 5 hours and desired enamine 16 was isolated in 85% yield (Scheme 5).

Scheme 5
On the other hand, the heating of ethylamine 15 with the excess of EMME at 150-160 °C was not effective since ethylamine 15 is somewhat volatile and condenses on the walls of the reaction vessel or condenser preventing thus reaction with EMME which is much less volatile and remains at the bottom of the reaction vessel.To avoid this obstacle, xylene became the solvent of choice.Reflux of ethylamine 15 and two equivalents of suitable activated enol ether 17 during 24-48 h was found as the optimal reaction conditions for preparation of [(2,1,3benzoselenadiazol-4-ylamino)(ethyl)amino]methylidene derivatives 18 (Scheme 6).

Scheme 6
The unwillingness of ethylamines 11 and 15 to substitute the alkoxy group of the activated enol ethers 17 can be explained by the larger steric demands.Enamines 18a and 18b bear identical substituents X and Y, whereas enamine 18c with different substituents X and Y exist as a mixture of E and Z isomers.The relative ratio of the particular geometric isomers (E:Z 10:1) was estimated from its NMR spectral data considering intensities of the signals.We assume, on the basis of steric hindrance, that E-isomer strongly prevails.
Obtained enamines 16 and 18 were subjected to acid-catalysed pyridone ring closure.Heating of enamine 16 in PPA at 120 °C did not yield angularly and/or linearly annulated ethylselenadiazoloquinolones instead ethylamine 11 was isolated.Thus a cleavage of the bond between nitrogen and methylidene carbon atom occurred before the closure to pyridone ring.Our next attempts on cyclization to pyridone ring using various acidic media such as PPE, BF3•OEt2 and POCl3 also failed.In case of enamines 18 cyclization in PPA at 120 °C smoothly afforded 9ethylselenadiazoloquinolones 2 and 19 in high yields (Scheme 7).

Scheme 7
To our surprise, enamine 18c after the treatment with PPA gave completely deacetylated product 2. 1 H and 13 C NMR spectra as well as physicochemical properties of the product of deacetylation were in accordance with those of 9-ethylselenadiazoloquinolone 2 prepared by ethylation of selenadiazoloquinolone 1 (Scheme 1).Basic hydrolysis of ethyl ester 19b readily provided acid 20 in high yield (Scheme 7).Next we decided to examine decarboxylation of the acid 20.Decarboxylation promoted by cyanide ions in hot DMSO or DMF 27 did not proceed at all.Although, decarboxylation conducted in boiling quinoline was complete in about 1 hour, the isolation of the product was troublesome since it did not precipitated from the reaction mixture.Moreover, basic quinoline caused difficulties in the separation by FLC.Finally, decarboxylation was successfully performed in boiling Ph2O (Scheme 7).In this case precipitation of the product also did not occur but non-polar Ph2O can be easily removed by FLC.In this way 9ethylselenadiazoloquinolone 2 was isolated in 75 % yield and the structure of product obtained after the ring closure of enamine 18c was confirmed.

Conclusions
The ethylation of selenadiazolodiazoloquinolones 1, 4 and 21 was found to be ineffective (low yields) due to the poor regioselectivity.The synthesis of N-ethylbenzoselanadiazolamines 11 and 15 by ethylation of benzoselenadiazolamines 7 was developed.Modified Gould-Jacobs reaction of N-ethylbenzoselanadiazol-4-amine 15 gave three 9-ethylselenadiazoloquinolone derivatives 2, 19a and 19b.Surprisingly, acid-catalysed cyclization of enamine 18c resulted in totally deacetylated product -9-ethylselenadiazoloquinolone 2. Basic hydrolysis of ethyl ester 19b followed by thermal decarboxylation of the resulting acid 20 yielded identical product as the acid-promoted ring closure of enamine 18c.

Experimental Section
General.Thin-layer chromatography (TLC) was performed on aluminium plates pre-coated with 0.2 mm silica gel (25 μm) containing fluorescent indicator 254 nm (Fluka) and stains were visualized by UV light (254 nm or 366 nm).Flash column liquid chromatography (FLC) was performed on silica gel Normasil 60 (43-60 μm).Melting points were measured on Koffler block and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on Varian Mercury 300-MHz spectrometer at 25 °C.The operation frequencies were 300 MHz for 1 H and 75.5 MHz for 13 C nuclei.Chemical shifts (δ) are reported in ppm and coupling constants (J) are given in Hz.Elemental analyses were determined using a Thermo Finnigan Flash EA 1112 instrument.The starting compounds 1, 4, 7 and 21 were prepared according to procedures described in our previous papers. 7,8The alkoxymethylidene derivatives 17a and 17b are commercially available (AlfaAesar ® , Sigma-Aldrich ® ) while derivative 17c was synthesized by condensation of ethyl 3oxobutanoate with triethyl orthoformate. 28,29