THE SYNTHESIS, ANTI-INFLAMMATORY, ANALGESIC AND ANTIMICROBIAL ACTIVITIES OF…

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22.08.2018

THE SYNTHESIS, ANTI-INFLAMMATORY, ANALGESIC AND ANTIMICROBIAL ACTIVITIES OF ETHYL 2-AMINO-4-ALKYL-4,6-DIHYDROPYRANO[3,2-c][2,1]BENZOTHIAZIN-3-CARBOXYLATE 5,5-DIOXIDES AND TRIETHYLAMMONIUM 3-[(4-HYDROXY- 1-ETHYL-2,2-DIOXIDO-1H-2,1-BENZOTHIAZIN-3-YL)ALKYL]- 1-ETHYL-1H-2,1-BENZOTHIAZIN-5-OLAT 2,2-DIOXIDES

A.Lega, N.I.Filimonova, I.A.Zupanets, S.K.Shebeko, V.P.Chernykh, L.A.Shemchuk

The search for new groups of anti-inflammatory and analgesic drugs is a topical issue of the current medici-nal chemistry. It is caused by numerous diseases that are accompanied by pain and inflammation, as well as by imperfection of the existing drugs aimed to provide treatment of these pathological conditions. Derivatives of 1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide are promising chemicals to search and develop drugs with the pharmacological properties required. This heterocyclic system is structurally close to 2H-1,2-benzothiazin-4-one 1,1-dioxide, which is the core of the famous non-steroidal anti-inflammatory drugs related to the “oxicam” group. Moreover, derivatives of 1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide are also considered to be promising structures for searching effective antimicrobial substances among them. The present article is devoted to the synthesis of new derivatives of 1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide, namely ethyl 2-amino-4-alkyl-4,6-dihydropyrano[3,2-c][2,1]benzothiazin-3-carboxylate 5,5-dioxides and triethylammonium 3-[(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)alkyl]-1-ethyl-1H-2,1-benzothiazin-5-olat 2,2-dioxides. Condensed 2-amino- 4-alkyl-4H-pyran-3-carboxylates were synthesized via the three-component one-pot interaction of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide with ethyl cyanoacetate and aliphatic aldehydes. The abovementioned tri-ethylammonium salts were obtained by the two component interaction of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide with aliphatic aldehydes in the presence of the equimolar amount of triethylamine. The study of the anti-inflammatory and analgesic activity has demonstrated high prospects of new effective drugs when searching among two classes of the compounds synthesized. The screening of the antimicrobial activity has shown that the compounds synthesized are the most active against the fungal strain of C. albicans.

The inflammatory process is a natural host-defen-sive process in the innate immunity response, and it is usually associated with pain as a secondary process resulting from release of pain mediators [1]. These disease states involve a series of events that can be elicited by numerous stimuli such as infectious agents, ischemia, antigen-antibody interaction and a thermal or physical injury.

The most common drugs currently used for the treatment of pain and inflammatory conditions are non-steroidal anti-inflammatory drugs (NSAIDs). Over the past years a lot of NSAIDs have been prepared and marketed through design and development of new drug substances. These drugs play the immense role in management of various inflammatory conditions such as rheumatism, arthritis and others associated with pain. Currently, more than 30 million people world-wide take NSAIDs every day, and 40% of these pa-tients are aged over 60 years; about 20% of inpatients receive NSAIDs [2, 3]. The high prevalence of NSAIDs is caused by the unique combination of analgesic and anti-inflammatory activities. However, these drugs are known to provoke numerous adverse effects, among them gastrointestinal irritation is the most widespread. In particular, 30-40% of patients taking NSAIDs note the presence of dyspeptic disorders, 10-20% of pa-tients have erosions and ulcers of the stomach and duodenum, and 2-5% of them have gastrointestinal bleeding and perforation [4, 5]. In this regard, inten-sive studies concerning design and development of new highly efficient and safe NSAIDs continue worldwide.

Derivatives of 1H-2,1-benzothiazin-4(3H)-one 2,2- dioxide are very promising for searching new substances to treat inflammatory disorders and states ac-companied with pain.

This heterocyclic system is struc-turally close to 2H-1,2-benzothiazin-4-one 1,1-dioxide, which is the core of famous NSAIDs related to the “oxicam” group. N-R-1H-2,1-benzothiazin-3-carbox-amides have been shown to possess a high level of the analgesic activity [6]. Moreover, 1H-2,1-benzo-thiazin-4(3H)-one 2,2-dioxides are a prospective core structure for creating new antimicrobial drugs as pre-viously reported [7, 8]. Therefore, the present article is devoted to the synthesis of new derivatives of 1H- 2,1-benzothiazin-4(3H)-one 2,2-dioxide and evalua-tion of their anti-inflammatory, analgesic activities and antimicrobial properties.

The first step of our investigations was to synthe-size 1H-2,1-benzothiazine 2,2-dioxides condensed with 4-alkyl substituted ethyl 2-amino-4H-pyran-3-carboxy-late core via the three-component interaction of 1- ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide (1) with ethyl cyanoacetate (2) and aliphatic aldehydes 3 (Scheme 1). In our previous works [9, 10] we sho-wed that application of malononitrile in the three-component interaction with 1-ethyl-1H-2,1-benzo-thiazin-4(3H)-one 2,2-dioxide and aldehydes unam-biguously led to 2-amino-3-cyano-4H-pyrans conden-sed with 1-ethyl-1H-2,1-benzothiazine 2,2-dioxide core, while utilization of ethyl cyanoacetate in this reaction was accompanied with side processes and lower yields of target products, namely fused ethyl 2-amino-4H-pyran-3-carboxylates.

The results of the three-component interaction of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide (1) with ethyl cyanoacetate (2) and aliphatic aldehydes 3a-i are presented in Scheme 1.

As one can see, we managed to obtain the target 2-amino-4H-pyrans 4 only in the cases of aldehydes 3c-g. In the cases of acetaldehyde 3b and glutaric aldehyde 3i we were unable to identify isolated products. The desired 2- amino-4H-pyranes 4c-g were formed at the room tem-perature in the presence of catalytic amounts of tri-ethylamine, but apparently in low to moderate yields. We failed in our attempts to increase the yields of the products 4 despite the application of different reac-tion conditions.

When formaldehyde 3a was introduced in the three- component interaction with 1 and 2 under conditions shown in Scheme 1, the isolated product was bis(1- ethyl-1H-2,2-dioxido-2,1-benzothiazin-4(3H)-on-3-yl) methane 5. It is interesting that product 5 was ob-tained as a dicarbonyl form though one could expect to isolate it as a triethylammonium salt since the equi-molar amount of triethylamine was applied in the reaction, and it was in our results previously reported [10]. If the interaction of 3a with 1 and 2 was car-ried out at 35-40°C, the admixture of triethylammo-nium salt 7a was observed in the isolated product (not shown in Scheme 1).

The structure of bis-product 5 was proven by 1H NMR and 13C NMR-spectroscopic data (Fig. 1).

As one can see, protons of the CH2 group bridging appear in the 1H NMR-spectrum as a set of signals in the spectral range of 2.77-3.10 ppm (Fig. 1a), and it can be explained by the existence of compound 5as two diastereomers (Fig. 2). This also explains the appearance of the signals in the 13C NMR-spectrum (Fig. 1b) having highly similar chemical shifts, which relate to the same carbons of two diastereomeric forms.

Based on the 1H NMR-spectroscopic data it has been also found that compound 5 exists exclusively in the diketoform in solutions of compounds unable to form hydrogen bonds (such as chloroform, see Fig. 1a), while such solvent as dimethylsulfoxide (DMSO) entails formation of the equilibrium between diketo-form 5 and keto-enol form 6 (Scheme 2). The molar ratio of these forms according to the 1H NMR (DMSO-d6) spectrum is about 1:0.55.

According to our research plan, we also aimed to obtain triethylammonium salts of 3-[(4-hydroxy-1- ethyl-2,2-dioxido-1H-2,1-benzothiazin-3-yl)alkyl]- 1-ethyl-1H-2,1-benzothiazin-5-olat 2,2-dioxides. These salts are new derivatives of 1H-2,1-benzothiazine-4 (3H)-one 2,2-dioxide and, so, they are of interest for evaluation of their biological activity in order to de-velop new effective anti-inflammatory, analgesic and antimicrobial substances.

In our previous works [9, 10], similar triethylam-monium salts were synthesized in high yields via the interaction of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide with (het)arylcarbaldehydes (the molar ratio – 2:1) in the alcoholic solution in the presence of the equimolar amount of triethylamine. Such con-ditions were applied for the interaction of 1-ethyl- 1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide 1 with aliphatic aldehydes 3. Thus, when formaldehyde 3a was introduced into the reaction, the mixture of tri-ethylammonium salt 7a and bis-product 5 in the ap-proximate molar ratio of 1:0.2 was isolated (Scheme 3). To avoid formation of undesirable products 5 the ten-fold excess of triethylamine was used; the reaction was carried out under reflux for 7 h. But the mixture of 7a and 5 was isolated in this case too. This result serves as evidence that a formaldehyde derived pro- duct is not inclined to easy enolization and thereby to formation of enol salts.

Utilization of other aliphatic aldehydes 3b-i al-lowed to obtain the desired triethylammonium salts 7b-i in good yields. Reactions in all cases were con-ducted in refluxing alcohol (ethanol or 2-propanol) in the presence of the equimolar quantity of triethyl-amine. The yields of compounds 7b-i synthesized are presented in Scheme 3.

The next stage of our investigations was evalu-ation of the anti-inflammatory (AIA) and analgesic (AA) activity. Compounds 4g and 7g as representa-tives of each group of the derivatives synthesized were used for this purpose. The anti-inflammatory and anal-gesic properties of 4g and 7g were studied in albino adult rats weighing 200-240 g in full compliance with the Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the pro-tection of animals used for scientific purposes [11] and with the Ukrainian Law No. 3447-IV “On protec-tion of animals from severe treatment” [12].

AIA was studied on the model of carrageenan-in-duced paw edema [13, 14], and AA was determined on the model of local inflammatory hyperalgesia [14, 15]. Pathology in both cases was reproduced by the intra-plantar injection of 0.1 mL of 1% solution of λ-car-rageenan (“Fluka”, Switzerland) into the right hind limb of the rats [14, 15]. Piroxicam (Sopharma, Bul-garia) was used as a reference drug because of its

2H-1,2-benzothiazin-4-one 1,1-dioxide core being struc-turally similar to the compounds tested. The substances under research and the reference drug were intro-duced orally one hour before the carrageenan injec-tion in the form of fine aqueous suspensions stabi-lized with Tween-80 (0.5 mL/100 g). The screening dose of Piroxicam was 2 mg/kg, the compounds stu- died were introduced in doses that were equimolar to Piroxicam. The control group received the equivalent amount of Tween-80 water solution. Seven experi-mental animals were involved in each experimental group to obtain statistically reliable results.

The initial and final paw volumes were measured by the water displacement method using a plethys-mometer, the final value of the paw volume was ob-tained in three hours after the phlogogen agent in-jection. The anti-inflammatory activity (%) was cal-culated as the percentage of edema inhibition in the animals treated with the substances studied and Pi- roxicam compared to the control rats.

The analgesic effect (in %) was assessed by the change of the pain threshold (PT) checked on the in-flamed paw in rats receiving 4g, 5g and Piroxicam in three hours after administration of the test subs- tances compared to the vehicle-treated animals.

The comparative analysis of the experimental data is given in Tab. 1. The results were calculated using standard math procedures and presented in the form of arithmetic mean±standard error of the mean. The results of biological tests were also processed by the method of variation statistics using Student’s t-criterion.

Carrageenan-induced pathology is commonly used as an experimental model in animals for acute inflam-mation and indicated the anti-exudative activity of the compounds synthesized, as well as their ability to inhibit the action of pain mediators. This is con-nected with biphasic evolution of the disease process in this case [16, 17]. The initial phase of the carra-geenan model is mainly mediated by histamine, sero-tonin and the increased synthesis of prostaglandins in the damaged tissue. The next phase is supported by prostaglandin release and mediated by bradykinin, leukotrienes, polymorphonuclear cells and prostaglan-dins produced by tissue macrophages.

leukotrienes, polymorphonuclear cells and prostaglan-dins produced by tissue macrophages. All of the me-diators released are the main inflammation factors and act at their receptors to increase permeability of small blood vessels and thereby to support the pro-gress of exudation. The chemosensitivity of nocicep-tors allows the released mediators to act on these neurons and causes the appearance of hyperalgesia. These features allow to determine the inhibition ef-fect of the test compounds at the second (exudative) stage of inflammation, as well as to identify the pe-ripheral component affecting the nociceptive system in the mechanism of the analgesic effect of the sub-stances studied.

During pharmacological studies of anti-inflam-matory and analgesic agents (especially during the screening phase), a substantial level of the pharma-cological activity must be not less than 20% [7]. In this regard, the results of AIA of the compounds as-sessed are promising (Tab. 1). Compound 4g admi- nistered in the dose of 2.4 mg/kg moderately decre- ased development of edema (29.6±6.3%) in 3 h after the phlogogen injection, whereas triethylammonium salt 7g was slightly more active and in the equimolar dose of 3.7 mg/kg decreased development of edema at the level of 35.9±4.9%.

The similar results were obtained for the study of AA. Both of the compounds studied showed the high level of AA (Tab. 1). While 4H-pyran annulated derivative 4g showed AA of 41.4±4.8%, triethylam-monium salt 7g (48.5±6.2%) was almost as good as the reference drug Piroxicam (54.2±5.3%).

The results obtained allow considering 4H-pyran annulated derivatives 4 and triethylammonium salts 7 as the basis for design of highly effective substances which will be useful for treatment of various diseases accompanied by inflammation and pain such as in-flammatory arthropathies, injuries, etc.

The antimicrobial activity of 2-amino-4H-pyranes 4 and triethylammonium enolates 7 in vitro was stu- died according to the requirements of the State Phar-macopoeia of Ukraine (1 ed.) by the double serial di-lution method in the liquid growth medium. The com-pounds synthesized were tested against Pharmaco-poeial Gram positive (S. aureus – АТСС 6538, B. sub-tilis – АТСС 6633) and Gram negative (E. coli – АТСС 8739, P. aeruginosa – АТСС 9027) strains of bacteria, as well as against the fungal strain of C. albicans (АТСС 10231) [18, 19]. The solutions of the compounds studied with the concentrations of 500, 250, 125, 62.5, 31.25, 15.62 μg/mL were prepared using dimethylsulfoxide (DMSO) as a solvent and the broth as a growth medium. Since DMSO exhibits a moderate antimicrobial activity [20], it is used as a reference drug. Inocula of the bac-terial and fungal cultures were prepared according to the optical turbidity standard of 0.5 ME from a daily agar culture. The microbial load was 150×106 microbes per mL. The test-tubes containing bacterial cultures were kept in the thermostat at 37°C for 24 h, and test- tubes containing C. albicans culture were kept in the thermostat at 25°C for 48 h and observed for the pre-sence of turbidity. The lowest concentration when no growth of microorganisms was observed was taken as the minimum inhibitory concentration (MIC) value. The MIC values detected for the solutions studied are presented in Tab. 2.

From the activity report it can be noticed that most of the test compounds did not reveal any activity or displayed a slight antimicrobial activity against the bacterial strains. Furthermore, it is interesting that DMSO solutions of the derivatives studied showed the higher MIC values against bacterial strains compared to the reference DMSO.

At the same time, all of the compounds studied exhibited a moderate or high antifungal activity against C. albicans. Among 2-amino-4H-pyrans 4 the most ac-tive were isobutiric and isovaleric aldehyde derived products 4f,g; in addition, compound 4g was also mo-derately active against P. aeruginosa strain. Isobutiric and isovaleric aldehyde derived products 7f,g and pro-pionaldehyde derived compound 7c displayed the lo-west values of MIC among triethylammonium salts 7. Thus, 4H-pyran derivatives 4 and triethylammonium salts 7 containing the branched alkyl chain were the most active compounds studied. These results are of interest for discovering a new class of compounds to treat fungal related diseases.


Chemical Part

The starting aldehydes and ethyl cyanoacetate were obtained from commercial sources and used without further purification. The starting 1-ethyl-1H-2,1-ben-zothiazin-4(3H)-one 2,2-dioxide was obtained accor-ding to the procedure previously described [21]. The new compounds are characterized by the data of mel-ting points (obtained on a Gallenkamp melting point apparatus, Model MFB-595 in open capillary tubes), 1H and 13C NMR-spectroscopic data (recorded on a Varian WXR-400 spectrometer in DMSO-d6 or CDCl3using TMS as an internal standard, chemical shifts in parts per million) and elemental analysis (carried out using a Carlo Erba CHNS-O EA 1108 analyzer).

The general procedure for the synthesis of ethyl 2-amino-6-ethyl-4,6-dihydropyrano[3,2-c][2,1]benzothiazin-3-carboxylate 5,5-dioxides (4c-g).To the solution of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide 1 (0.225 g, 0.001 Mol), ethyl cyanoacetate 2 (0.112 g, 0.001 Mol) and appropriate aldehyde 3c-g (0.001 Mol) in ethanol (5-10 mL) add the catalytic amount of triethylamine. Allow the mixture to stand at the room temperature. Filter the resulting precipi-tates of 4c-g, wash with cold ethanol and dry on the air.

Ethyl 2-amino-4,6-diethyl-4,6-dihydropyrano [3,2-c][2,1]benzothiazin-3-carboxylate 5,5-dioxi-de (4c). Yield – 0.11 g (29%), colourless prisms. M. p. – 163-165°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.67 (t, J = 7.34 Hz, 3H, CHCH2CH3), 1.12-1.24 (m, 6H, NCH2CH3, OCH2CH3), 1.58-1.73 (m, 2H, CHCH2CH3), 3.90 (t, J = 3.67 Hz, 1H, CHCH2CH3), 3.97-4.19 (m, 4H, NCH2CH3, OC H2CH3), 7.33 (t, J = 7.52 Hz, 1H, Ar-H), 7.51-7.56 (m, 1H, Ar-H), 7.58-7.64 (m, 1H, Ar-H), 7.71 (s, 2H, NH2), 7.92 (d, J = 8.07 Hz, 1H, Ar-H). Anal. Calcd for C18H22N2O5S: C, 57.13; H, 5.86; N, 7.40; S, 8.47. Found: C, 57.25; H, 5.71; N, 7.54; S, 8.23.

Ethyl 2-amino-4-propyl-6-ethyl-4,6-dihydropy-rano[3,2-c][2,1]benzothiazin-3-carboxylate 5,5- dioxide (4d). Yield – 0.17 g (43%), colourless prisms. M. p. – 180-182°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.77 (t, J = 7.17 Hz, 3H, CHCH2CH2CH3), 1.03-1.27 (m, 8H, NCH2CH3, OCH2CH3, CHCH2CH2CH3), 1.50-1.69 (m, 2H, CHCH2CH2CH3), 3.88 (t, J = 3.73 Hz, 1H, CHCH2 CH2CH3), 3.96-4.20 (m, 4H, NCH2CH3, OCH2CH3), 7.32 (t, J = 7.48 Hz, 1H, Ar-H), 7.49-7.56 (m, 1H, Ar-H), 7.57-7.64 (m, 1H, Ar-H), 7.69 (s, 2H, NH2), 7.92 (d, J = 7.63 Hz, 1H, Ar-H). Anal. Calcd for C19H24N2O5S: C, 58.15; H, 6.16; N, 7.14; S, 8.17. Found: C, 58.30; H, 6.03; N, 7.08; S, 8.41.

Ethyl 2-amino-4-butyl-6-ethyl-4,6-dihydropy-rano[3,2-c][2,1]benzothiazin-3-carboxylate 5,5- dioxide (4e). Yield – 0.12 g (30%), colourless prisms. M. p. – 131-133°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.69-0.81 (m, 3H, CHCH2CH2CH2CH3), 0.98-1.32 (m, 10H, NCH2CH3, OCH2CH3, CHCH2CH2CH2CH3), 1.53-1.73 (m, 2H, CHCH2CH2CH2CH3), 3.86-3.93 (m, 1H, CHCH2 CH2CH2CH3), 3.97-4.22 (m, 4H, NCH2CH3, OCH2CH3), 7.33 (t, J = 7.14 Hz, 1H, Ar-H), 7.50-7.57 (m, 1H, Ar-H), 7.58-7.65 (m, 1H, Ar-H), 7.69 (s, 2H, NH2), 7.93 (d, J = 7.68 Hz, 1H, Ar-H). Anal. Calcd for C20H26N2O5S: C, 59.09; H, 6.45; N, 6.89; S, 7.89. Found: C, 59.23; H, 6.70; N, 7.07; S, 7.52.

Ethyl 2-amino-4-(propan-2-yl)-6-ethyl-4,6-di-hydropyrano[3,2-c][2,1]benzothiazin-3-carboxy-late 5,5-dioxide (4f ). Yield – 0.19 g (48%), colour-less prisms. M. p. – 160-162°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.70 (d, J = 6.60 Hz, 3H, CHCH (CH3)2), 0.79 (d, J = 6.97 Hz, 3H, CHCH(CH3)2), 1.11-1.24 (m, 6H, NCH2CH3, OCH2CH3), 1.88-2.02 (m, 1H, CHCH (CH3)2), 3.84 (d, J = 2.93 Hz, 1H, CHCH(CH3)2), 3.96-4.19 (m, 4H, NCH2CH3, OCH2CH3), 7.33 (t, J = 7.52 Hz, 1H, Ar-H ), 7.50-7.56 (m, 1H, Ar-H), 7.57-7.64 (m, 1H, Ar-H), 7.70 (s, 2H, NH2), 7.95 (d, J = 7.70 Hz, 1H, Ar-H). Anal. Calcd for C19H24N2O5S: C, 58.15; H, 6.16; N, 7.14; S, 8.17. Found: C, 58.34; H, 6.21; N, 7.27; S, 8.30.

Ethyl 2-amino-4-(2-methylpropyl)-6-ethyl-4,6- dihydropyrano[3,2-c][2,1]benzothiazin-3-carboxy-late 5,5-dioxide (4g). Yield – 0.20 g (49%), colour-less prisms. M. p. – 155-157°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.78 (d, J = 6.24 Hz, 3H, CHCH2 CH(CH3)2), 0.85 (d, J = 6.24 Hz, 3H, CHCH2CH(CH3)2), 1.13-1.24 (m, 6H, NCH2CH3, OCH2CH3), 1.32-1.41 (m, 1H, CHCH2CH(CH3)2), 1.46-1.63 (m, 2H, CHCH2CH(CH3)2), 3.84 (dd, J = 6.42, 4.22 Hz, 1H, CHCH2CH(CH3)2), 3.97-4.23 (m, 4H, NCH2CH3, OCH2CH3), 7.32 (t, J = 7.52 Hz, 1H, Ar-H ), 7.50-7.54 (m, 1H, Ar-H), 7.57-7.63 (m, 1H, Ar-H), 7.71 (s, 2H, NH2), 7.95 (d, J = 7.70 Hz, 1H, Ar-H). Anal. Calcd for C20H26N2O5S: C, 59.09; H, 6.45; N, 6.89; S, 7.89. Found: C, 59.01; H, 6.32; N, 7.13; S, 7.72.

The synthesis of bis(1-ethyl-2,2-dioxido-1H- 2,1-benzothiazin-4(3H)-on-3-yl)methane (5). To the solution of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2-dioxide 1 (0.225 g, 0.001 Mol) in 2-propanol (10 mL) add 40% water solution of formaldehyde 3a (0.075 g, 0.001 Mol), ethyl cyanoacetate (0.112 g, 0.001 Mol) and triethylamine (0.101 g, 0.001 Mol). Allow the so-lution to stand at the room temperature for 48 h. After that evaporate the solvent in vacuum, dissolve the re-sidue in methanol, and cool the solution to 5°C. Fil-ter the precipitate formed, wash with cold methanol and dry on the air to obtain a pure product. Yield – 0.09 g (39%), a white powder. M. p. – 147-149°C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.43 (t, J = 6.79 Hz, 6H, 2×NCH2CH3), 2.77-3.10 (m, 2H, CH2 bridged), 4.01-4.17 (m, 4H, 2×NCH2CH3), 4.81 (t, J = 6.79 Hz, 2H, 2×SO2CHCO), 7.13-7.23 (m, 4H, Ar-H), 7.64 (t, J = 7.70 Hz, 2H, Ar-H), 8.11 (t, J = 6.97 Hz, 2H, Ar-H). 13C NMR (400 MHz, CDCl3): δ (ppm) 11.93 (2×NCH2CH3), 17.26 (CH2 brid-ged), 39.29 (NCH2CH3), 39.25 (NCH2CH3), 64.74 (SO2 CHCO), 64.87 (SO2CHCO), 115.13, 115.22, 120.36, 120.41, 121.21, 121.27, 127.35, 127.39, 133.79, 140.10, 140.12, 183.61 (SO2CHCO), 183.70 (SO2CHCO) Anal. Calcd for C21H22N2O6S2: C, 54.53; H, 4.79; N, 6.06; S, 13.86. Found: C, 54.42; H, 4.85; N, 5.91; S, 13.58.

The general procedure for the synthesis of tri-ethylammonium 3-[(4-hydroxy-1-ethyl-2,2-dioxido-1H-2,1-benzothiazin-3-yl)alkyl]-1-ethyl-1H-2,1-benzothiazin-4-olat 2,2-dioxides (7b-g). To the so-lution of 1-ethyl-1H-2,1-benzothiazin-4(3H)-one 2,2- dioxide 1 (0.450 g, 0.002 Mol) and aldehyde 3b-g(0.001 Mol) in 2-propanol (10 mL) add triethylamine (0.14 mL, 0.001 Mol). Stir the solution and reflux for 2 h, and cool the mixture to the room temperature. Filter the precipitates formed, wash with 2-propanol and dry on the air. Recrystallize the crude products from 2-propanol to obtain pure products 7b-g.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)ethyl]-1- ethyl-1H-2,1-benzothiazin-4-olate 2,2-dioxide (7b). Yield – 0.42 g (73%), a white crystalline powder. M. p. – 110-112°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.06-1.16 (m, 15H, 2×NCH2CH3, HN+(CH2CH3)3), 1.57 (d, J = 7.32 Hz, 3H, CH3CH), 3.00 (q, J = 7.32 Hz, 6H, HN+(CH2CH3)3), 3.86 (q, J = 6.92 Hz, 4H, 2×NCH2CH3), 7.12 (t, J = 7.48 Hz, 2H, Ar-H), 7.22 (d, J = 7.93 Hz, 2H, Ar-H ), 7.35-7.41 (m, 2H, Ar), 7.92 (d, J = 7.63 Hz, 2H, Ar-H ), 17.58 (br. s, 1H, OH). Anal. Calcd for C28H39 N3O6S2: C, 58.21; H, 6.80; N, 7.27; S, 11.10. Found: C, 58.34; H, 6.93; N, 7.38; S, 11.32.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)propyl]-1- ethyl-1H-2,1-benzothiazin-4-olate 2,2-dioxide (7c). Yield – 0.34 g (57%), a white crystalline powder. M. p. – 121-123°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.73 (t, J = 7.32 Hz, 3H, CH3CH2CH), 1.05-1.15 (m, 15H, 2×NCH2CH3, HN+(CH2CH3)3), 2.09 (quin, J = 7.48 Hz, 2H, CH3CH2CH), 2.98 (q, J = 7.12 Hz, 6H, HN+(CH2CH3)3), 3.85 (q, J = 7.02 Hz, 4H, 2×NCH2CH3), 4.20 (t, J = 8.09 Hz, 1H, CH3CH2CH), 7.09 (t, J = 7.48 Hz, 2H, Ar-H), 7.20 (d, J = 8.24 Hz, 2H, Ar-H), 7.33-7.39 (m, 2H, Ar), 7.89 (d, J = 7.93 Hz, 2H, Ar-H), 17.53 (br. s, 1H, OH). Anal. Calcd for C29H41N3O6S2: C, 58.86; H, 6.98; N, 7.10; S, 11.84. Found: C, 58.70; H, 7.11; N, 7.21; S, 11.93.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)butyl]-1- ethyl-1H-2,1-benzothiazin-4-olate 2,2-dioxide (7d). Yield – 0.33 g (55%), a white crystalline powder. M. p. – 118-120°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.79 (t, J = 7.34 Hz, 3H, CH3CH2CH2CH), 1.06-1.18 (m, 17H, 2×NCH2CH3, HN+(CH2CH3)3, CH3CH2CH2CH), 2.06 (q, J = 7.99 Hz, 2H, CH3CH2CH2CH), 3.01 (q, J = 7.34 Hz, 6H, HN+(CH2CH3)3), 3.85 (q, J = 6.85 Hz, 4H, 2×NCH2CH3), 4.34 (t, J = 8.19 Hz, 1H, CH3CH2CH2CH), 7.09 (t, J = 7.46 Hz, 2H, Ar-H), 7.20 (d, J = 8.07 Hz, 2H, Ar-H ), 7.34-7.40 (m, 2H, Ar-H), 7.87-7.93 (m, 2H, Ar-H), 17.47 (br. s, 1H, OH). Anal. Calcd for C30H43N3O6S2: C, 59.48; H, 7.15; N, 6.94; S, 10.59. Found: C, 59.39; H, 7.27; N, 7.03; S, 10.75.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)pentyl]-1- ethyl-1H-2,1-benzothiazin-4-olate 2,2-dioxide (7e). Yield – 0.25 g (40%), a white powder. M. p. – 170-172°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.75 (t, J = 7.15 Hz, 3H, CH3CH2CH2CH2CH), 1.07-1.26 (m, 19H, 2×NCH2CH3, HN+(CH2CH3)3, CH3CH2CH2CH2CH), 2.08 (q, J = 7.70 Hz, 2H, CH3CH2CH2CH2CH), 3.29-3.36 (m, 6H, HN+(CH2CH3)3), 3.85 (q, J = 6.97 Hz, 4H, 2×NCH2CH3), 4.31 (t, J = 8.07 Hz, 1H, CH3CH2CH2CH2CH), 7.09 (t, J=7.34 Hz, 2H, Ar-H), 7.20 (d, J = 8.07 Hz, 2H, Ar-H), 7.33-7.40 (m, 2H, Ar-H), 7.89 (d, J = 7.70 Hz, 2H, Ar-H), 17.54 (br. s, 1H, OH). Anal. Calcd for C31H45N3O6S2: C, 60.07; H, 7.32; N, 6.78; S, 10.35. Found: C, 59.93; H, 7.48; N, 6.59; S, 10.53.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)-2-methyl-propyl]-1-ethyl-1H-2,1-benzothiazin-4-olate 2,2- dioxide (7f ). Yield – 0.45 g (74%), white prisms. M. p. – 157-159°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.81 (d, J = 6.50 Hz, 6H, (CH3)2CHCH), 1.10-1.19 (m, 16H, 2×NCH2CH3, HN+(CH2CH3)3, (CH3)2CHCH), 3.02-3.10 (m, 6H, HN+(CH2CH3)3), 3.82-3.90 (m, 5H, 2×NCH2CH3, (CH3)2CHC H), 7.08 (t, J = 7.34 Hz, 2H, Ar-H), 7.19 (d, J = 8.07 Hz, 2H, Ar-H), 7.33-7.39 (m, 2H, Ar-H), 7.86-7.90 (m, 2H, Ar-H), 17.65 (br. s, 1H, OH). Anal. Calcd for C30H43N3O6S2: C, 59.48; H, 7.15; N, 6.94; S, 10.59. Found: C, 59.58; H, 7.14; N, 7.10; S, 10.41.

Triethylammonium 3-[1-(4-hydroxy-1-ethyl- 2,2-dioxido-1H-2,1-benzothiazin-3-yl)-3-methyl-butyl]-1-ethyl-1H-2,1-benzothiazin-4-olate 2,2-di-oxide (7g).Yield – 0.48 g (78%), white prisms. M. p. – 120-122°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 0.80 (d, J = 6.71 Hz, 6H, (CH3)2CHCH2CH), 1.07-1.16(m, 15H, 2×NCH2CH3, HN+(CH2CH3)3), 1.28-1.39 (m, 1H, (CH3)2CHCH2CH), 1.97 (t, J = 7.32 Hz, 2H, (CH3)2CHC H2CH), 3.02 (q, J = 7.32 Hz, 6H, HN+(CH2CH3)3), 3.85 (q, J = 6.92 Hz, 4H, 2×NCH2CH3), 4.43 (t, J = 8.09 Hz, 1H, (CH3)2CHCH2CH), 7.09 (t, J = 7.48 Hz, 2H, Ar-H), 7.20 (d, J = 8.24 Hz, 2H, Ar-H), 7.33-7.39 (m, 2H, Ar-H), 7.89 (d, J = 7.93 Hz, 2H, Ar-H), 17.49 (br. s, 1H, OH). Anal. Calcd for C31H45N3O6S2: C, 60.07; H, 7.32; N, 6.78; S, 10.35. Found: C, 59.91; H, 7.48; N, 6.54; S, 10.50.

The procedure for the synthesis of di(triethyl-ammonium) 3,3′-[1,5-bis(4-hydroxy-1-ethyl-2,2- dioxido-1H-2,1-benzothiazin-3-yl)pentane-1,5-diyl]bis(1-ethyl-1H-2,1-benzothiazin-4-olat 2,2- dioxide) (7i). To the solution of 1-ethyl-1H-2,1-ben-zothiazin-4(3H)-one 2,2-dioxide 1 (0.450 g, 0.002 Mol) and 50% water solution of glutaric aldehyde 3i (0.105 g, 0.0005 Mol) in ethanol (10 mL) add triethylamine (0.101 g, 0.001 Mol). Stir the solution at 50°C for 30 min, cool to the room temperature and allow it to stand overnight. Treat the oily precipitate formed with wa-ter until it becomes solid. Filter it, wash with water and dry on the air to obtain a pure product 5i. Yield – 0.42 g (73%), a pink powder. M. p. – 125-127°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.05-1.17 (m, 32H, 4×NCH2CH3, 2×HN+(CH2CH3)3, CHCH2CH2CH2CH), 2.02 (q, J = 7.32 Hz, 4H, CHCH2CH2CH2CH), 3.00 (q, J = 7.21 Hz, 12H, 2×HN+(CH2CH3)3,), 3.63-3.73 (m, 4H, 2×NCH2CH3), 3.76-3.86 (m, 4H, 2×NCH2CH3), 4.19 (t, J = 7.89 Hz, 2H, C HCH2CH2CH2CH), 7.08 (t, J = 7.34 Hz, 4H, Ar-H), 7.16 (d, J = 8.07 Hz, 4H, Ar-H), 7.34 (t, J = 7.52 Hz, 4H, Ar-H), 7.85 (d, J = 7.70 Hz, 4H, Ar-H), 17.48 (br. s, 2H, OH ). Anal. Calcd for C57H78N6O12S4: C, 58.64; H, 6.73; N, 7.20; S, 10.99. Found: C, 58.52; H, 6.81; N, 7.03; S, 11.12.

Resources:

Journal of Organic and Pharmaceutical Chemistry. – 2016. – Vol. 14, Iss. 4 (56)

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