CYCLOALKANECARBALDEHYDES IN SYNTHESIS OF NOVEL 1,2-BENZOXATHIIN- 4(3H)-ON 2,2-DIOXIDE DERIVATIVES AND STUDY OF THE ANTIMICROBIAL ACTIVI- TY OF SYNTHESIZED COMPOUNDS

Мета: Метою даної роботи було дослідження взаємодії циклоалканкарбальдегідів, 1,2-бензоксатіїн4(3Н)-он 2,2-діоксиду та метиленактивних нітрилів та вивчення антимікробних властивостей одержаних сполук. Методи: Як вихідні речовини були використані циклоалканкарбальдегіди, 1,2-бензоксатіїн-4(3Н)-он 2,2діоксид та метиленактивні нітрили. В ході дослідження застосовувались методи органічного синтезу. Структуру синтезованих сполук було підтверджено елементним аналізом та Н ЯМР-спектроскопією. Антимікробну активність вимірювали методом дифузії в агар. Результати: Нові 2-аміно-4Н-пірани синтезували шляхом трикомпонентної взаємодії циклоалканкарбальдегідів, 1,2-бензоксатіїн-4(3Н)-он 2,2-діоксиду та малонодинітрилу. Заміна останнього на етилціаноацетат у випадку циклогексанкарбальдегіду призвела до виділення відповідної триетиламонієвої солі. Виходячи з цього результату та з огляду на його новизну було одержано ряд амонієвих солей циклогексанкарбальдегіду з іншими вторинними та третинними амінами. Синтезовані сполуки виявили більш високу антимікробну активність, ніж препарати порівняння щодо грампозитивних штамів. Висновки: Дані дослідження показали перспективний шлях розширення існуючої різноманітності 2аміно-4Н-піранів з використанням в їх синтезі такого енолнуклеофілу та карбонільних сполук як 1,2бензоксатіїн-4(3Н)-он 2,2-діоксид та циклоалканкарбальдегіди відповідно. Виявлена антимікробна активність отриманих сполук проти грампозитивних мікроорганізмів дає можливість для подальших досліджень щодо створення антибіотиків вузького спектру дії Ключові слова: 1,2-бензоксатіїн-4(3Н)-он 2,2-діоксид, 2-аміно-4Н-піран,багатокомпонентні реакції, циклоалканкарбальдегіди, амонієві солі, антимікробна активність


Introduction
Nowadays the variety of organic compounds is absolutely tremendous. In 2015 the world's largest database of unique chemical substances CAS registered the 100 millionth chemical substance [1], and thousands of new molecules are still generated by scientists all over the world each year. Among this number the compounds with prospective biological properties are of the greatest interest, because they usually give rise to the development of novel medicines. 2-Amino-4H-pyrans, that were for the first time synthesized nearly 60 years ago, can be considered as such group, because of the known antimicrobial [2] and antitumor [3] activities for this class of compounds on the one hand and convenient chemical pathways to their synthesis on the other hand. According to this, the extension of the condensed 2-amino-4Hpyranseries is justified, especially by means of utilization of novel unexplored molecules in order to investigate the influence of this one on the biological activity. In this regard 1,2-benzoxathiin-3(4H)-on 2,2-dioxide is one of insufficiently studied compounds, that may be used for construction of novel condensed 2-amino-4H-pyrans as prospective antimicrobial agents.

Formulation of the problem in a general way, the relevance of the theme and its connection with important scientific and practical issues
Multicomponent reactions are an effective and convenient tool in organic synthesis, because they provide rapid way to the creation of large novel series of compounds through applying of wide range of initial compounds. The most common and attractive route towards 2amino-4H-pyrans E represents the three-component reaction of active methylene nitriles A, carbonyl compounds B and enolnucleophiles C (Fig. 1) and includes the hetero-Thorpe-Ziegler cyclization of adducts D [4]. According to the recent literature data such interaction proceeds perfectly as multicomponent reaction, when adducts D are generated in situ from the mixture of starting compounds. Formation of D and heterocyclization processes occurs sequentially and is considered as Knoevenagel-Michaelhetero-Thorpe-Ziegler domino type reaction. 5 Among carbonyl compounds cycloalkanecarbaldehydes were not previously widely used in such interactions type. Utilization of latter along with 1,2benzoxathiin-3(4H)-on 2,2-dioxide as enolnucleophile gives the possibility to obtain novel condensed 2-amino-4H-pyrans, the evaluation of the antimicrobial activity of which are of the greatest interest due to the current necessity of the search of new core-structures with such properties.

Analysis of recent studies and publications in which a solution of the problem is foundand which draws on the author
The structural diversity of 2-amino-4H-pyrans depends on the initial compounds that are used in their synthesis: active methylene nitriles, carbonyl compounds and enolnucleophiles.
The most well-known active methylene nitriles applied in the interaction described above are malononitrile A1 and esters of cyanoacetic acid A2. Cyanacetamide A3 and thiocyanacetamide A4 are not so widely used. As for carbonyl compounds they are represented with different types of aldehydes, the most common of which are aromatic B1 and hetarenecarbaldehydes B2. In the case of ketones (isatins B5 and ningidrin B6) the products of the interaction are spirocondensed compounds. Enolnucleophiles is the widest group in the 2-amino-4H-pyrans synthesis and is represented with diketones C1, ketoesters C2-C4, cyanoketones C5-C6, nitroketones C7and many other related compounds that are shown on Fig. 2. Corresponding multicomponent interactions generally proceed under reflux in polar solvents medium in the presence of basic catalysts, such as triethylamine, morpholine, piperidine [4].
Current investigations in this synthetic area are mainly focused on the selection of new reaction conditions and catalysts, and to search of some novel suitable components, that could replace well-known ones in order to expand the existing 2-amino-4H-pyrans diversity.

Allocation of unsolved parts of the general problem, which is dedicated to the article
As it was mentioned above, the huge variety of enolnucleophiles have been already used in multicomponent 2-amino-4H-pyrans synthesis. That is why searching of some new compounds of this group is challenging and interesting at the same time.
Previously we reported the utilization of 1H-2,1benzothiazin-4-on 2,2-dioxide in 2-amino-4H-pyrans synthesis [5,6]. This "newcomer" turned out to be the prospective core structure for such interactions due to the presence of SO 2 CH 2 CO moiety. Moreover the series of obtained compounds were screened for antibacterial, antifungal, analgesic and anti-inflammatory activities [7], and among them some promising substances with moderate to high levels of them were found.
As the next consequential step of the research we drew our attention to the other almost unknown enolnucleophile -1,2-benzoxathiin-4(3H)-on 2,2-dioxide, that is an isostere of 1H-2,1-benzothiazin-4-on 2,2dioxide.This approach gave us the opportunity not only to compare the chemical properties of such close related compounds, but also to investigate the structure/bioactivity relationships for the obtained substances.
We also decided to use cycloalkanecarbaldehydes as a carbonyl component for the 2-amino-4H-pyrans synthesis, because this group was not broadly employed in such interactions before.

Formulation of goals (tasks) of the article
According to the information given above we aimed to synthesize 2-amino-4H-pyrans with the use of cycloalkanecarbaldehydes, 1,2-benzoxathiin-4(3H)-on 2,2-dioxide and active methylene nitriles threecomponent interaction, to determine the most suitable reaction conditions and to evaluate the antimicrobial properties of the obtained compounds.

Statement of the basic material of the study (methods and objects) with the justification of the results
Considering the previous research of the interaction of 1H-2,1-benzothiazin-4-on 2,2-dioxide with active methylene nitriles and cycloalkanecarbaldehydes [6] we began our investigation from the studying of the reaction of 1,2-benzoxathiin-4(3H)-one 2,2-dioxide 1 with malononitrile 2 and cyclopropanecarbaldehyde 3a.It was discovered, that as in the previous case, the reaction proceeded under room temperature in ethanol with the presence of catalytic amount of triethylamine and resulted into the formation of 2-amino-4-cyclopropyl-4,6dihydropyrano[3,2-c][2,1]benzoxathiin-3-carbonitrile 5,5-dioxide 4a in 55 % yield (Scheme 2). The same reaction conditions were successfully applied for cy-clopentane-and cyclohexanecarbaldehydes 3b and 3c respectively.
Inspired by such encouraging results we continued the research and replaced malononitrile with ethyl cyanoacetate, which is also often used in such interactions type. The reaction was carried out with equimolar quantities of all reagents in the same conditions as previous one, but unfortunately gave no rise to the desired products. We did not succeed in isolation of any product at all in this case. Simultaneously the performance of the reaction at 60-70 °C in the case of aldehyde 3c allowed us to isolate a crystalline powder that appeared to be the triethylammonium This unexpected result can be explained by two ways (Fig. 3). The first one comprises the formation of Michael adduct F which looses the molecule of ethyl cyanoacetate and is converted into enone G, that gives triethylammonium salt according to what was reported previously [6]. The second possible way involves the formation of enone G by direct interaction of 1,2benzoxathiin-4(3H)-on 2,2-dioxide 1 with aldehyde 3c, that next reacts with the second molecule of 1 forming symmetrical bis-derivative isolated as triethylammonium salt 5c. On the current stage of the research these two ways can be considered as equiprobable and further investigations are needed to disclose finally the mechanism of salts 5 formation via applying of multicomponent format of the reaction. Since no traces of triethylammonium salts were found for three-component reaction of 1,2-benzoxathiin-4(3H)-on 2,2-dioxide with malononitrile and cycloalkanecarbaldehydes, we can assume that 2-amino-4H-pyran-3-carbonitriles 4 are more stable in the applied conditions than the corresponding salts 5. Considering the possibility of the enones G to be formed into the reaction mixture we tried to convert triethylammonium salt 5c into 2amino-3-carbonitrile-4H-pyran 4c by treating the former with malononitrile. In spite of this the attempt appeared to be unsuccessful and only initial material was recovered after the reaction. In this way, we can suppose that such triethylammonium salts 5 are rather stable compounds and the retro-Michael cleavage reaction with formation of the enones G is not typical for them.
In regard to expand the range of such ammonium salts we also examined the possibility of secondary amines utilization in this reaction. The desired products 6c, 7c, 8c, 9c were isolated for aldehyde 3c in the case of piperidine, morpholine, 1,2,3,4-tetrahydroisoquinoline and dimethylamine (Fig. 5).
The reaction proceeded under gently heating and mixing for 1 h in i-PrOH with equimolar quantity of the corresponding amine.  The antimicrobial properties of the obtained compounds was studied according to the international standards [8,9] by the agar "well" diffusion method against the standard test-strains of gram-positive and gramnegative bacteria and fungi. The results revealed higher antimicrobial activity than for the reference drugs against S. aureus and E.coli. The compound 4c appeared to be the most active among all tested samples. The diameters of its growth inhibition zones were 21 mm for S. aureus, 19 mm for E. coli, 17 mm for C. albicans and 16 mm for B. subtilis, P. aeruginosa and P. vulgaris. The results for the reference drugs Synthomycine and Metronidazole did not exceed 17 mm.
There was no significant distinction of antimicrobial properties of triethylammonium salts corresponding to 2-amino-4H-pyrans. The average diameter of growth inhibition zones was 17 mm against all tested strains.
Most of the similar derivatives of 1H-2,1benzothiazin-4(3H)-on 2,2-dioxide did not possess antibacterial activity against gram-positive and gramnegative microorganisms and revealed moderate antifungal activity [6]. Thereby the isosteric replacement of 1-N-R-group to O-atom appeared to be the effective tool allowing to find more active antimicrobial agents.

Experimental chemical part
Initial aldehydes and active methylene nitriles were obtained from commercial sources and used without further purification. Melting points were determined on a Gallenkamp melting point apparatus, Model MFB-595 in open capillary tubes. 1 H NMR spectra were recorded on Varian WXR-400 spectrometer using DMSO-d 6 as solvent and TMS as an internal standard. Elemental analyses were carried out using Carlo Erba CHNS-O EA 1108 analyzer.