Esters and amides of 3-R-2,8-dioxo-7,8-dihydro-2 H -pyrrolo[1,2- a ][1,2,4] triazino[2,3- c ]quinazolin-5 a (6 H )-carboxylic(-propanoic) acids: synthesis and biological activity

bonded to or moieties a of the “pharmacophore” frag- ment in most NSAID molecules. the carboxyl group may cause the appearance of toxic effects and is characterized by unsatisfactory pharmacokinetic properties. The structural modification of the carboxyl group, including its bioisosteric replacement, is among the most widely used approaches in medicinal chemistry to improve pharmacodynamic, pharmacokinetic and technological characteristics. Aim. To develop the synthetic procedures for functional derivatives of 3-R-2,8-dioxo-7,8-dihydro-2 Н -pyrrolo[1,2- а ]- [1,2,4]triazino[2,3- с ]quinazoline-5 а (6 Н )-carboxylic(-propanoic) acids, study the effect of the carboxyl group chemical modification on the LOX-inhibiting and antiradical activity as a possible mechanism of the pharmaco - logical activity. Results and discussion. The synthesis of esters of 3-(2,8-dioxo-3-R 1 -7,8-dihydro-2 H -pyrrolo[1,2- a ][1,2,4]- triazino[2,3- c ]quinazolin-5 a (6 H )-yl)carboxylic(propanoic) acids was conducted by esterification of the corresponding acids and tandem heterocyclization of 2-(6-R 1 -2,5-dihydro-5-оxo-1,2,4-triazino-3-yl)anilines with diethyl 4-oxo - heptanedioate. The synthesis of amides was conducted by aminolysis of N- acylimidazolides generated in situ . The of approach for the synthesis of the target esters via condensation of 2-(6-R 1 -2,5-dihydro-5-oxo-1,2,4-triazino-3-yl)anilines with diethyl 4-oxoheptanedioate has been proposed. It has been found that the highest radical scavenging and LOX-inhibiting activities are characteristic for hetarylpropa noic acids that contain electron withdrawing substituents in position 3, as well as fluorine atoms in positions 11 and 12. The chemical modification of the carboxylic group in most cases results in a decrease or the loss of the activity.

Recently, the pyrrolo[1,2-а][1,2,4]triazino[2,3-с]quinazoline heterocyclic system was described as a promising "scaffold" for construction of novel bioactive compounds [8]. Additionally, the concept of the purposeful structural optimization of the abovementioned heterocyclic system previously developed [9] allowed us to synthesize the novel 3- quinazolin-5a(6H)-carboxylic(-propanoic) acids with the promising anti-inflammatory activity. However, one of the possible approaches for the chemical modification of the abovementioned acids, namely functionalization of carboxylic group, has not been described.
Hence, the present study aimed at the development of the synthetic procedures towards functional derivatives of 3- [2,3-с]quinazoline-5а(6Н)carboxylic(-propanoic) acids, as well as the study of the effect of the carboxyl group chemical modification on the LOX-inhibiting and antiradical activity as a possible mechanism of the pharmacological activity.

Results and discussion
Previously described [8] [1,2,4]triazino-[2,3-c]quinazolin-5a(6H)-carboxylic(propanoic) acids 2 were used as substrates for the chemical modification of the carboxyl group. It was found that abovementioned compounds under esterification conditions (Scheme, Method A) resulted in esters 3 with the yields of 33 -60 %. Tandem heterocyclization of 2-(6-R 1 -2,5-dihydro-5-оxo-1,2,4-triazino-3-yl)anilines 1 with diethyl 4-oxoheptanedioate in glacial acetic acid (Scheme, Method B) was shown to be more efficient approach to obtain compounds 3 (yields of 74 -87 %). The synthesis of amides 4 was conducted by the known method [10], namely by aminolysis of N-acylimidazolides obtained in situ. It was found that the abovementioned intermediates A were synthetically available and revealed high reactivity towards benzyl-(aryl-)amines. The conversion of acids 2 in amides 4 via the method mentioned above requires anhydrous dioxane and refluxing for 3 -4 hours (Scheme). The yields of amides 4 were in the range of 30 -73 %. The attempt of the synthesis of amide 4 via the reaction of esters 3 with amines was not successful.
Elemental analysis, 1 H, 13 C NMR and LS-MS data proved the structure and purity of the substances synthesized.  3.1 -3.6 were observed as series of multiplets overlapping on the equatorial and axial protons of the pyrrole cycle at 2.93 -2.58 ppm (H-7 eq , 7 ax , -CH 2 CH 2 COOC 2 H 5 ) and 2.47 -2.06 ppm (H-6 eq , 6 ax , -CH 2 CH 2 COOC 2 H 5 ). Such complex splitting is probably associated with the presence of an asymmetric carbon atom. 1 H NMR-spectra of compounds 4 were characterized by the signals of amide NH-protons. The abovementioned signals were registered as triplets at 8.09 -6.23 ppm (4.1 -4.5) or singlet at 9.71 -9.49 ppm (4 .6 -4.10). Additionally, the signals of the methylene group protons were observed as two-proton doublets at 4.29 -4.09 pрm. As it was expected, in 1 H NMRspectra of amides 4.1 -4.3 the signals of the pyrrole cycle protons (7 eq , 7 ax , 6 eq , 6 ax ) were observed as a wide high-field multiplet in the range of 3.24 -2.64 ppm.  [11]. Besides, in 1 H NMR-spectra of compounds 3 and 4 the signals were caused by the nature of substituents in positions 3 and 5a [11].
The 13 C NMR-spectrum of compound 3.2 additionally proved its structure. The characteristic were the signals of a carbon atom of position 5a, cyclic and exocyclic ethylene fragments. The abovementioned signals were registered at 31.8, 29.5, 27.7, 27.7 and 83.5 ppm, respectively.
The antiradical and LOX-inhibiting activities of the compounds obtained were studied as possible mechanisms of the anti-inflammatory activity [12]. It was found that compounds 2, 3 and 4 revealed the antiradical activity (ARA = 0.87 -43.6 %) in the concentration of 10 -3 M. The SAR-analysis conducted showed that introduction of electron-withdrawing substituents to position 3, as well as introduction of fluorine atoms in positions 11 and 12 increased the DPPHscavenging activity of hetarylpropanoic acids 2.1 -2.6.
All compounds exhibited lower antiradical activity in the concentration of 10 -4 М, but the "structure -antiradical activity" relationship was preserved. Hetarylcarboxylic acids 2.7 and 2.8 were characterized by a moderate antiradical activity.
The chemical modification of acids 2.1 -2.6 by the carboxylic group esterification (compounds 3.1 -3.6) resulted in decreasing of the radical scavenging (0.87 -17.11 %) and the LOX-inhibiting activity (3.32 -15.78 %). Thus, fluorine-containing compound 3.5 revealed the highest LOX-inhibiting activity among esters 3. Compounds 4.2, 4.7 and 4.8 (Table) exhibited the highest radical scavenging activity among ami- Hence, the study conducted allowed us to detect the classes of effective anti-inflammatory agents, as well as to propose the effective approaches for constructing novel anti-inflammatory agents. It should be noted that the lipophilic, but not active esters obtained can not be considered as non-promising bioactive agents due to the possibility of their biotransformation in active metabolites.

Experimental part
Melting points were determined in open capillary tubes in a Stuart SMP30 apparatus and were uncorrected. The elemental analyses (C, H, N) were performed using an ELEMENTAR vario EL cube analyzer. 1 H NMR-spectra (400 MHz) and 13 C NMR (101 MHz) were recorded using a Varian-Mercury 400 spectrometer with TMS as an internal standard in DMSO-d 6 solution. LC-MS spectra were recorded using the chromatography/mass spectrometric system consisting of an Agilent 1100 Series high-performed liquid chromatograph equipped with an Agilent LC/MSD SL diode-matrix and mass-selective detector (atmospheric pressure chemical ionization -APCI). The ionization mode was a concurrent scanning of positive and negative ions in the mass range of 80 -1000 m/z. The synthetic studies were conducted according to the general approach to the search of potential biologically active substances using reagents of companies Sigma-Aldrich (Missouri, USA) and Enamine (Kyiv, Ukraine).

-a]-[1,2,4]triazino[2,3-c]quinazolin-5a(6H)-yl)propanoates 3.1 -3.6:
Method A. To 10 mmol of the corresponding acid 2.1 -2.6 in 20 mL of ethanol add 1.29 g (11 mmol) of thionyl chloride and 1 drop of DMF. Heat the resulting mixture on a water bath for 6 h. Then cool the reaction mixture and pour into 5 mL of the saturated sodium bicarbonate solution. Filter the precipitate formed and dry. If it is necessary, the compounds obtained can be recrystallized from ethanol.

Method B.
To the suspension of 10 mmol of the corresponding anilines 1.1 -1.6 in glacial acetic acid add 2.30 g (10 mmol) of diethyl 4-oxoheptanedioate. Reflux the resulting mixture for 6 h. Evaporate the solvent under vacuum, add 15 mL of methanol to the residue formed. Filter the precipitate formed, wash by diethyl ether and dry. The compounds obtained can be purified by recrystallization from methanol.
Research methodology. Dissolve the compounds in DMSO to obtain 1 mM solution. Mix 2 mL of this solution with 2 mL of 0.1 mM DPPH methanol solution and incubate for 30 min at 25 °C. Then measure the absorbance (A d ) [17]. Simultaneously determine the absorbance of 2 mL of 0.1 mM DPPH solution in 2 mM of methanol (A DPPH ). Calculate the antiradical activity (ARA) by the following formula: ARA, % = (A DPPH -A d )×100 %/A DPPH . In the case of a negative meaning ARA in % is estimated as 0.
Weighing of reagents and the compounds synthesized were conducted on ANG200C electronic scales (Axis, Gdansk, Poland), and the absorbance was measured by a ULAB 108UV spectrophotometer (Ulab, Shanghai, China).
The in vitro study of soybean LOX inhibition. The in vitro study was evaluated as it was reported previously [18,19]. To 3.88 mL of borate buffer add 40 µL of 2·10 -5 w/v solution of LOX in buffer and 40 µL of 100 µM of the compound solution studied (or nordihydroguaiaretic acid (NDGA)). Shake the resulting mixture and incubate at ambient temperature for 5 min. After incubation add 40 µL of 0.01 M solution of sodium linolenate. The intensity of absorbance at 234 nm is recovered after 20 min of the incubation at ambient temperature. Calculate the results by the formula: LOX inhibiting activity, % = (А control -А test compound )×100 %/А control .

Conclusions
The methods for the synthesis of esters and amides of 2,8-dioxo-3-R 1 -7,8-dihydro-2H-pyrrolo[1,2-a]- [1,2,4]triazino [2,3-c]quinazolin-5a(6H)-carboxilic (propanoic) acids have been developed. The abovementioned transformations were conducted by alcoholysis of generated in situ acyl halides and aminolysis of N-acylimidazolides. The more efficient alternative approach for the synthesis of the target esters via condensation of 2-(6-R 1 -2,5-dihydro-5-oxo-1,2,4- triazino-3-yl)anilines with diethyl 4-oxoheptanedioate has been proposed. It has been found that the highest radical scavenging and LOX-inhibiting activities are characteristic for hetarylpropanoic acids that contain electron withdrawing substituents in position 3, as well as fluorine atoms in positions 11 and 12. The chemical modification of the carboxylic group in most cases results in a decrease or the loss of the activity. Conflict of interests: authors have no conflict of interests to declare.