Synthesis of 1,4-benzothiazinones from acylpyruvic acids or furan-2,3-diones and o-aminothiophenol

Two synthetic approaches to enaminones fused to 1,4-benzothiazin-2-one moiety, which can be interesting in studies on biological activity, chemosensors, and fluorescence, were developed via the reaction of furan-2,3-diones or acylpyruvic acids in the presence of carbodiimides with o-aminothiophenols. The target enaminones were formed together with pharmaceutically interesting 2-hydroxy-2H-1,4-benzothiazin-3(4H)-ones. A selective synthetic approach to 2-hydroxy-2H-1,4-benzothiazin-3(4H)-ones was developed via the solvent-switchable reaction of furan-2,3-diones with o-aminothiophenol. Preliminary biological assays (antimicrobial, acute toxicity) of the new compounds were carried out.


Enaminones fused to the 1,4-benzothiazin-2-one moiety (BTAs) 3a-n. General procedure.
To a cooled (to 0-5 °C) stirring suspension of acylpyruvic acid 2 (5 mmol) and DCC (5 mmol) in acetonitrile (10 mL), oaminothiophenol 1 (5.1 mmol) was added. The mixture was allowed to reach ambient temperature (25 °C) overnight. Then the formed yellow precipitate was filtered off and washed with toluene (50-100 mL; it is necessary to remove precipitated orange crystals of the target compound). The yellow precipitate was disposed of. The collected mother liquor was evaporated to dryness under vacuum. The solid residue was stirred with ethanol (10 mL) for 1 h (it is necessary to remove dicylohexylurea). Then, the orange precipitate was filtered off and recrystallized from toluene (5-10 mL) to give the target BTA 3.
This procedure can be modified to save acetonitrile. The modified variant affords slightly lower yields of BTAs 3. Modified procedure: To a cooled to 0-5 °C stirring suspension of acylpyruvic acid 2 (5 mmol) and o-aminothiophenol 1 (5.1 mmol) in toluene (10 mL), DCC (5 mmol) was added. The mixture was allowed to reach ambient temperature (25 °C) overnight. Then the formed yellow precipitate was filtered off and washed with toluene (50-100 mL; it is necessary to remove precipitated orange crystals of the target compound). The yellow precipitate was disposed of. The collected mother liquor was evaporated to dryness under vacuum. The solid residue was stirred with ethanol (10 mL) for 1 h (it is necessary to remove dicylohexylurea). Then, the orange precipitate was filtered off and recrystallized from toluene (5-10 mL) to give the target BTA 3.
It should be mentioned that compounds 4a-n are thermally stable. They did not eliminate water even when they were heated up to 110 °С (boiling toluene).

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The growth inhibition of C. albicans was determined measuring absorbance at 530 nm (OD530), while the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD600-570), after the addition of resazurin (0.001% final concentration) and incubation at 35 °C for additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. (Table 1) Percentage growth inhibition of an individual sample is calculated based on negative controls (media only) and positive controls (bacterial/fungal media without inhibitors). Please note negative inhibition values indicate that the growth rate (or OD600) is higher compared to the negative control (Bacteria/fungi only, set to 0% inhibition). The growth rates for all bacteria and fungi has a variation of -/+ 10%, which is within the reported normal distribution of bacterial/fungal growth. Any significant variation (or outliers/hits) is identified by the modified Z-Score, and actives are selected by a combination of inhibition value and Z-Score.

Z-Score
Z-Score analysis is done to investigate outliers or hits among the samples. The Z-Score is calculated based on the sample population using a modified Z-Score method which accounts for possible skewed sample population. The modified method uses median and MAD (median average derviation) instead of average and sd, and a scaling factor

Quality Control
All screening is performed as two replica (n = 2), with both replicas on different assay plates, but from single plating and performed in a single screening experiment (microbial incubation). Each individual value is reported in the table (see ..1 and ..2). In addition, two values are used as quality controls for individual plates: Z'-Factor [ 1 -(3 * (sd(NegCtrl)+sd(PosCtrl))/(average(PosCtrl)-average(NegCtrl))) ] and standard antibiotic controls at different concentrations (>MIC and <MIC). The plate passes the quality control if Z'-Factor >0.4 and Standards are active and inactive at highest and lowest concentrations, respectively. Data not supplied.

Selection of Actives
A -[Active] Samples with inhibition values equal to or above 80% and abs(Z-Score) above |2.5| for either replicate (n=2 on different plates) were classed as active. P -[Partial Active] compounds with inhibition values between 50.9% -79.9% or abs(Z-Sore) below |2.5|. I -Inactive compounds with inhibition values below 50% and/or abs(Z-Sore) below |2.5|.

Bactericidal activity by BACTEC
Thw bactericidal activity of chemical compounds against Mtb (H37Rv) was studied by a standard ВАСТЕС MGIT 960 radiometric growth system (Becton Dickinson).
Initial stock solutions of test compounds (10 mg/mL) were prepared in DMSO. Then they were diluted with Middlebrook 7H9 sterile nutrient broth (9 mL) to result in the concentration of test compounds of 1000 μg/mL. The aliquots of obtained solutions were treated by two-fold serial dilutions and added to MGIT tubes in quantities that provide the final concentrations (µg/mL): 20.0, 10.0, 5.0, 2.5, 1.25, 0.6, 0.31. Each MGIT tube contained 7 mL of Middlebrook 7H9 sterile nutrient broth. Then 0.8 mL of ВАСТЕС MGIT OADC (oleic acid, albumin, dextrose, and catalase) growth supplement was added to each tube. In addition to the liquid medium, the tubes contained an oxygen-free fluorochrome, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium chloride pentahydrate [Ru(dpp)], placed on the bottom of the tube and coated with silicone. A 1.0 McFarland suspension of Mtb (5 × 10 8 microbial cells per 1 mL) was prepared using a densitometer. Next, a solution was prepared by diluting the initial suspension 10 times with sterile physiological saline solution, thus resulting in 5 × 10 7 microbial cells per 1 mL. Then a suspension of Mtb (0.5 mL) was added to each of the above prepared MGIT tubes. In parallel, the inoculum of Mtb was loaded into control MGIT tubes with Middlebrook 7H9 broth (0.5 mL) each containing no test compounds. For control, similar experiments were carried out with isoniazid (isonicotinic acid hydrazide, 99%, Sigma-Aldrich).
All tubes were incubated at 37 °C and analyzed by ВАСТЕС MGIT 960. If the compound is active against Mtb, it inhibits its growth and suppresses fluorescence of Ru(dpp), while in the control tube growth is not inhibited and, accordingly, the level of fluorescence in this tube is pronounced. During bacterial growth, free oxygen is consumed inside the tubes and replaced with CO2. As free oxygen is consumed, inhibition of the fluorochrome, Ru(dpp), is stopped. Fluorescence becomes detectable when the test tube is irradiated with UV light and is automatically registered by photosensors of ВАСТЕС MGIT 960. The minimum dilution of the examined compound, in which the growth was not registered by ВАСТЕС, was taken as MBC. All assays were carried out in duplicate.
The results are given in Table 3. The plates were incubated at 37 °C for 7 days. Then 50 μL of a resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) solution (with the addition of Tween 80) was added to the wells and the incubation was continued at 37 °C. The results were monitored after 24, 48 and 72 h. The minimum dilution of the examined compound, in which there was no change in the blue-violet color of resazurin in all replicates, was taken as MIC. All assays were carried out in triplicate.

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The results are given in Table 4.

Acute toxicity in mice
Adult Balb/C mice (22-28 g) were used for acute toxicity studies (male). Test animals were kept under vivarium conditions (with natural lighting at 22-24°C and relative humidity 40-50%) on a standard diet (GOST R 50258-92). Experiments were conducted according to good laboratory practice (GLP) rules for preclinical studies in the RF (GOST 3 51000.3-96 and 1000.4-96) and rules of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986). Animals were quarantined for 10-14 d before the experiments.
The test compound 3a was suspended in a 1% starch solution and administered to the animals orally (per os) at doses of 100, 500, 1000 mg/kg. As an equistress effect, the animals in the control group were administered with 1% starch solution. The animals had unlimited access to water and food.
The results are given in Table 5.

Crystal structure determination
The unit cell parameters and the X-ray diffraction intensities were measured on a Xcalibur Ruby diffractometer. The empirical absorption correction was introduced by multi-scan method using SCALE3 ABSPACK algorithm [7]. Using the Olex2 [8], the structures were solved with the SHELXS [9] or SUPERFLIP [10] programs and refined by the full-matrix least-squares method in the anisotropic approximation for all non-hydrogen atoms with the SHELXL program [11]. Hydrogen atoms bound to carbon were located from the Fourier synthesis of the electron density and refined using a riding model. The hydrogen atoms of NH and OH groups were refined independently with isotropic displacement parameters.

Partial atomic charges comparison in intermediate XI.
Partial atomic charges of carbonyl carbon atoms in intermediate XI (Table 11) were calculated semi-empirically by PM7 method [12] in MOPAC2016 program [13].