Synthesis and using of 10-hydroxy-3,3,6,6-tetramethyl-9-(4-hydroxy-3-methoxyphenyl)-1,2,3,4,5,6,7,8,9,10- decahydroacridin-1,8 - dion as acid base titration indicator

: The aim of this work is the synthesis of new 10-hydroxydecahydroacridine-1,8-dione derivative, determination of the structure and to study the possibility of using this compound as an indicator of acid-base titration. Environmentally friendly synthesis of 10-hydroxy-3,3,6,6-tetramethyl-9-(4-hydroxy-3-methoxyphe-nyl)-1,2,3,4,5,6,7,8,9,10-decahydroacridin-1,8-dion has been developed by one pot interaction of dimedone, hydroxylamine and 4-hydroxy-3-methoxybenzoic aldehyde in water-alcohol or water solution using citric acid or sodium dodecyl sulfate as catalysts, respectively. Purification of the synthesized compound was carried out by crystallization from ethanol. The obtained compound was characterised by 1 H NMR , 13 C NMR and UV-Vis spectroscopies. This substance in water-alcohol solution shows intense violet light absorption. Addition alkali induces red shift of absorption maximum to the blue region. UV irradiation of solution of this substance in alcohol induces two-band fluorescence in the visible region. One band disappears upon addition of a base in solution. The structure of the obtained compound was confirmed by high resolution mass-spectrometry analysis. In the mass-spectrum of 10-hydroxy-3,3,6,6-tetramethyl-9-(4-hydroxy-3-methoxyphe-nyl)-1,2,3,4,5,6,7,8,9,10-decahydroacridin-1,8-dion observed [M+1] + ion peak. The base peak corresponds to tricyclic fragment due to the elimination aromatic cycle from molecular ion. This substance is colorless in acidic and neutral and pink in base solutions. The acid dissociation constant of this compound in a water-alcohol solution was determined by the UV-Vis spectroscopic technique. It was shown that the obtained compound can be used as an indicator for the titration of strong acids and bases.

This substance is colorless in acidic and neutral and pink in base solutions. That is why it can be suitable acid-base titration indicator. In water-alcohol solution hydroxydecahydroacridinedione 1 shows intense absorption with band maximum at 401.5 nm. Addition alkali induces red shift of absorption maximum to 502.5 nm. It is obvious that observed changes in the electronic absorption spectrum result from formation of anion (II) in alkaline solution (Fig. 2). Both absorption bands are wide and correspond to intramolecular charge transfer between donor and acceptor groups in the molecule. The presence of a negative charge in the donor part of the molecule (NO -) increases the energy of the highest occupied molecular orbital (HOMO), which is responsible for red shift of charge-transfer band. Electron acceptors are two carbonyl groups.
By measuring the change in the optical density of absorption, proportional to the concentration of the painted form (II) depending on the pH-value of water or water-alcohol solutions of several hydroxydecahydroacridinediones (I), we calculated the values of acid dissociation constant (К Ind ) of this compound, which allows you to set the color changing pH range of the indicator. It is determined by the value of the constants: ΔрН Ind = рК Ind ±1.  are molar concentrations of proton, acid and its conjugate base (salt), respectively. To use this formula, it is necessary to determine the concentrations of all three components. Proton concentration was determined with a pH meter, a concentration of the base form -using spectrophotometric measurements. The mathematical expression for calculating the constants obtained as follows.
Denote the total concentration of both forms of C 0 . The concentration of the main (colored) form denoted C, then the concentration of the acid form is C 0 -C expression for the dissociation constant is: Bouguer -Lambert -Beer law [20] relates the molar concentration of the substance with the optical density of the maximum absorption of the solute (form): where D max.the optical density of the absorption maximum; C Mits molar concentration; lcell width, cm; εmolar extinction. Using the expression of the optical density concentration, denoting C 0 · l · ε = D 0 in the equation for the equilibrium constants have: If during the measurement the solution volume and the temperature does not change, then a number of successive measurements of pH values and corresponding densities longwave absorption maximum core mold (II) left side of the equation remains constant. Then, for two consecutive measurements (1 and 2) can be written: In the resulting expression is absent D 0 , which means there is no need to prepare a certain concentration of solution of the substance (C 0 ) and using the resulting expression eliminates the need to weigh the samples and measuring the volume of the solution, and thus eliminates the associated measurement errors. Obviously, the accuracy of determining the dissociation constant depends on the range of measurement. At high pH, the concentration of the acid form is insignificant and low-basic. In the first case, the denominator in the expression for the dissociation constant tends to zero in the second the numerator. Comparable amounts of both forms are solutions in which the pH is close to pKdiss. (at pH = pK concentrations of both forms of the same, that is, C = 0,5C 0 ).

EXPERIMENTAL
Dimedone, hydroxylamine hydrochloride, 4-hydroxy-3-methoxybenzoic aldehyde, sodium acetate and citric acid were acquired from Sigma-Aldrich and used without further purification. The UV-Vis absorption spectra were recorded on a UV-2501 PC spectrophotometer. Fluorescence spectra were measured on RF-5301 PC ("Shimadzu", Japan) spectrofluorometer. The 1 Н and 13 С NMR spectra of compound were examined on a Bruker Avance 500 spectrometer at 500 and 125 MHz, respectively; tetramethylsilane was used as internal reference. Chromatographic-mass spectrometric analysis was carried out on liquid hybrid chromatography mass spectrometer LTQ Orbitrap Discovery (Thermo Electron Corporation, USA), which includes a linear Quadruple trap LTQ XL and the orbital trap of high permission. Ionization of the sample was carried out electrospray with using the source H-ESI II Ion Max. Calibration of linear and orbital traps LTQ Orbitrap Discovery was carried out using a standard solution, containing caffeine (m/z 195), L-methionyl-arginyolphenylalanine acetate (MRFA, m/z 524) and Ultramark 1621 (micsture of fluorinated phosphazines). As an internal calibrant during the removal of mass spectra indapamide was used (m/z 66.0674).
The progress of reaction and the purity of product was monitored by TLC on Silufol UV-254 plates using EtOAc-hexane (1:1) as eluent; spots were visualized under UV radiation or by treatment with iodine vapor, followed by calcination at 250-350 ºС. The melting point was determined on a Boetius hot stage.
1. A mixture of 2.8 g (20 mmol) of 5,5-dimethylcyclohexane-1,3-dione (dimedone), 1.52 g (10 mmol) of 4-hydroxy-3-methoxybenzoic aldehyde (vanillin) and 0.2 g of citric acid was stirred for one hour at room temperature in 50 ml of ethanol. Then 0.695 g (10 mmol) of hydroxylamine hydrochloride, 0.82 g (10 mmol) sodum acetate and 50 ml water were added thereto and stirred for another two hours. It was then diluted with 50 ml of water, and left to stand for 24 h. The precipitate was filtered off, washed with water (150 ml), and dried in air. Procedure for determining the dissociation constant. The total solution volume was 100 ml, wherein 50 ml buffer and 50 ml of ethanol. Buffer was Triss -1.21 g per 100 ml. Needless active substance is 5 mg. A pH meter for measuring the pH of the solution was adjusted to 6.86 by adding hydrochloric acid. Then poured into a cuvette 2 ml of the solution and the absorption spectrum was recorded on a spectrophotometer UV-2501 PC. Then, the pH was increased with concentrated KOH to pH 7.24, 7.61, 7.97, 8.41, 8.98, 9.73, 10.34, 10.79, 11.02 respectively, and re-measured with a spectrophotometer. Registration density maximum absorption was carried out at 502.5 nm. The pH measurements were taken at 20 ºC using an HI 221 pH meter. The error of the ten definitions of the dissociation constant K for solutions of compound 1a was calculated as the root mean square error of the arithmetic mean (standard deviation) taking into account the Student coefficient of 2.26 for ten determinations for the confidence probability P = 0.95.

RESULTS AND DISCUSSION
The structure of the obtained compound is confirmed by the data of the 1 H and 13 C NMR, UV spectra, elemental and mass spectrometric analysis. The 1 H and 13 C NMR spectra correspond to structure with symmetry plane passing through the C 9 and N 10 atoms [18]. Thus, in the 1 H NMR spectrum of compound four methyl groups in positions 3, 6 and four methylene groups in 2, 4, 5, 7 positions appear as two singlets (0.91;0.99 and 2.27; 2.51 ppm respectively). The 13 C NMR spectrum exhibited 15 signals of 24 carbon atoms, because the signals of equivalent atoms coincide.
The structure of compound 1 was confirmed by high resolution mass-spectrometry data (Table 1). In the spectrum observed [M+1] + (412 m/z), ion peak. The base peak (288 m/z) corresponds to tricyclic fragment due to cleavages (C 9 -C 1 arom ) bond and the elimination aromatic cycle from molecular ion. Such type fragmentation was observed for other 9-aryldecahydroacridine-1,8-dione derivatives [21].
Results of measurements and calculations are given in the table 2. The resulting value pK diss. conversion process I ↔ II is 7.425±0.016. The value of the dissociation constant indicates the possibility of using this compound as an indicator for the titration of strong acids and bases. In this case, it is a better indicator than phenolphthalein and methyl orange, since its pK is close to the pH equivalence point (neutral medium pH = 7.0).
Irradiation of solution of this substance in alcohol (λ max. 370 nm) induces fluorescence at λ max. 468 and λ max. 680 nm. First band disappears upon addition of a base in solution. Irradiation of basic solution (λ max. 500 nm) induces fluorescence at λ max. 680 nm (Fig. 3). The presence of two bands in the fluorescence spectrum of hydroacridinedion (I) in a neutral medium can be explained by its dissociation in an excited state and its transformation into an anion (II) (see Fig. 2). The long-wavelength band at 680 nm corresponds to the emission of an excited anionic form II. Since obtained hydroacridindion shows two emission bands in the visible region of fluorescent spectrum with large Stokes shift, it is of interest as fluorescent marker for studying biological molecules and supra-molecular structures.
2. The structure of obtained compound has been confirmed by the data of the 1 H and 13 C NMR, UV spectra, elemental and mass spectrometric analysis.
3. The acid dissociation constant of the resulting compound in hydroalcoholic solution was determined by the UV-Vis spectroscopic technique. 4. It has been shown that this compound can be used as an indicator for the titration of strong acids and bases.
5. Fluorescence spectra have been studied and it has been shown that this compound is of great interest as a fluorescent marker.