Tricyclic 1 , 2 , 4-triazine bearing heterosystem : directed synthesis of new bioactive compounds

A new se ries of tricyclic heteroaromatic com pounds was pre pared by cyclization of N2-sub sti tuted-6-bromo-3-oxo(amino)-1,2,4-tri azin-5(4Í)-ones with 2-aminobenzothiol. The struc ture of bioactive tricyclic glycosides, ob tained ear lier by the sim pli fied silylic method, was con firmed as well as ex pe di ence and ad e quacy of this method for di rected glycosylation of triazine bear ing tricyclic bases.

In tro duc tion.The pres ent work is a con tin u a tion of our study on syn the sis and bi o log i cal prop er ties of a num ber of de riv a tives of tricyclic heteroaromatic system, which con tains bioactive 1,2,4-triazine, to cre ate com pounds with use ful prop er ties as well as to es tablish the in flu enc ing of struc tural al ter ation on their bi olog i cal ac tiv ity.
The com pounds from the 1st se ries (tricyclic heterobases and their glycosidic de riv a tives) have shown an ti vi ral ac tiv ity, in par tic u lar, to wards the herpes sim plex vi rus type 2 (HSV-2) and Ep stein-Barr virus [1,2].The glycosides have been syn the sized by the di rect ribosylation of tricyclic heterobases us ing the silylic method [1].
The aim of this work is to ex tend the range of condensed triazine-con tain ing heterosystems us ing another syn thetic ap proach, where triazine frag ment is rep re sented by N2-alkyl-, N2-tetrahydrofuranyl-and N2-ribofuranosyl-de riv a tives of 6-bromo-3-oxo(amino)-1,2,4-tri azin-5-one, as a re sult en sur ing pro duc tion of com pounds with the be forehand pre as signed structures.
Chem i cal syn the sis.The de riv a tives of con densed triazine (Scheme, com pounds 3a-e, ta ble 1) have been syn the sized by the method, de scribed in the pre vi ous work [1].
The syn the sis of N2-b-D-ribofuranoside of condensed triazine (3f', 3g').To a sus pen sion of 1.0 mmol N2-(2',3',5'-tri-0-acetyl)-ribofuranoside of cor respond ing 6-bromo-triazine (1f, 1g) in 5 ml of eth anol/dimethylformamide mix ture were added 0.12 ml (1.5 mmol) of pyridine and 0.15 ml (1.5 mmol) of 2-aminobenzothiol.The re ac tion mix ture was heated sev eral hours at 100 0 Ñ, mon i tor ing pro cess by TLC.Af ter a stan dard treat ment the re ac tion mix ture prod uct (oily) was pu ri fied on the sil ica gel chro mato graphic col umn.The obtained glycoside tri ace tate was crys tallized from eth a nol.Deacylation of glycoside de riv atives was car ried out for 20 hours with aque ous-al coholic solution of ammonia under room temperature.
Physicochemical char ac ter is tics of the glycosides pro duced are rep re sented in the ta bles 2 and 3.
The syn the sis of N2-(2',3',5'-tri-0-acetyl-b-D-ribofuranosyl)-3-amino-6-bromo-1,2,4-triazine-5(4H)-one (1g).To the sus pension of of 3-amino-6-bromotriazine-5(2H)-one (1b) (900 mg, 2 mmol) and tetraacetylribose ( 700 mg, 2.2 mmol) in 15 ml of ab so lute acetonitrile were added 0.4 ml (3.2 mmol) of trimethylchlorosilane, 0.34 ml (1.6 mmol) of hexamethylsilazane and 0.3 ml (3.2 mmol) of tin chlo ride.The re ac tion mix ture was stirred in ar gon at mo sphere un der the room tem per a ture for 4 hours.De po si tion was re moved, fil trate was va por ized down to oily res i due.The lat ter was solved in 70 ml of chlo roform, flushed and af ter chlo ro form re moval ap plied onto the sil ica-gel col umn for chro mato graphic pu ri fica tion.An a lyt i cally pure acylated N2-glycoside was ob tained af ter reprecipitation from ethylacetate (the char ac ter is tics are rep re sented in ta ble 2.) Re sults and dis cus sion.The con densed heterocyclic sys tems, based par tic u larly on py rimidines, imidazoles, tri azines, take a sig nif i cant place in stud ies, ded i cated to the de vel op ment of chem i cal meth ods for pro duc tion of bioactive polycyclic heterosystems, re lated to the nat u ral an ti bi ot ics and alka loids, which have antitumor and an ti vi ral ac tiv ity [8][9][10] and are also im por tant for the con struc tion of diag nos tic flu o res cence probes [11][12][13][14].Our pre vi ous work dem on strated that the most ef fec tive pro ce dure for form ing a lin ear triazine-con tain ing sys tem appeared to be the annelation of 1,2,4-triazine de riv a tives by 2-aminobenzothiol.Heteroaromatic bases, obtained by this method, were used for the synthesis of their nucleoside analogs.
The com pounds struc ture was de ter mined by physi cal and chem i cal ap proaches.However, sometimes the an a lyt i cal char ac ter is tics are not enough to in ter -pret un am big u ously the struc tural mod i fi ca tions of heterosystem , e.g. in the case of its glycosylation.
There fore, this work in ad di tion to ob tain ing novel com pounds with sub stitu ents in var i ous po si tions of triazine cy cle was aimed to con firm the ear lier produced tricyclic glycosides struc tures us ing proper azanucleosides (1f, g) with fixed po si tion of sugar moiety upon syn the sis.
The choice of com pounds 1a, 1d -f and 3-amino-re placed triazine (1b, c, g) is de ter mined by the pres ence of two vic i nal electrophylic cen ters (carbon at oms in po si tions 5  triazine heterocycle.These at oms are con sis tently connected with ox y gen and halogen atoms. The pro cess of con densed triazine for ma tion (as well as py rim i dine) starts from sub sti tu tion of bro mine atom for aryl frag ment.The fur ther intramolecular cyclization of arylation prod uct [6-phenylthio-triazin-3,5(2H,4H)-dione] leads to the ex pected lin ear sys tem formation [1,8].
Con sid er ing a more pro nounced ar o matic char ac ter of triazine bases in com par i son with py rim i dine bases [15], we pre dicted that the syn the sis of con densed system to in volve N2-sub sti tuted de riv a tives would not re quire an ad di tional ac ti va tion of re ac tive cen ters in a triazine ring and pro ceed with a sat is fac tory yield.This as sump tion ap peared true only par tially.
The heterosystem for ma tion us ing azanucleosides (1f, 1g) oc curred with some com pli ca tion be cause of strict re quire ments for re ac tion, con trary annelation of non-glycosylated tri azines (1a -e), with for ma tion of side products.
The pu ri fi ca tion of acylated glycosides and furanyl de riv a tive of con densed triazine was car ried out by the col umn chro ma tog ra phy on the sil ica gel with fur ther crys tal li za tion.The yield made up 45-50%.The compounds 3f, 3g having fixed po si tion of sugar moi ety were pro duced in this way.The NMR spec tra, cor re lat -ing with cur rent pro ton sig nals in the spec tra of ini tial acylglycosides 1f, 1g and tricyclic aglycones 1a, 1b (table 1, 2), indicate this definitely.
The most typ i cal are the sig nals of car bo hy drate frag ment (the lo ca tion of anomeric pro ton and pro ton sin glet of acetyl groups with to tal in ten sity 9H) and signals of phenyl nu cleus in re gion 7.0 -6.8 ppm for both com pounds.The 3f-com pound is dis tin guished by the sig nal of cy clic ni tro gen pro ton at triazine ring in region 11.57ppm whereas 3g-glycoside is char ac ter is tic by the pro ton sig nal of exoaminogroup to be located at 7.23 ppm.
An im por tant in for ma tion was also re ceived ow ing to the analysis of elec tronic ab sorp tion spectra of the syn the sized com pounds.The ab sorp tion curves of the un pro tected glycosidic de riv a tives were com pa ra ble with the same spec tra for the cor re spond ing tricyclic aglycones thus sug gest ing a weak ef fect of the glycosidic res i dues on the chromophoric system of condensed triazines.
The com par i son of ex per i men tally de ter mined physico-chem i cal and spec tral char ac ter is tics of condensed triazine deacylated glycosides (3f ', 3g' ) with those ob tained via an in de pend ent syn thetic pro ce dure (tricyclic glycosides IV and VIII [1]) is pre sented in table 3. It is worth not ing that col la tion of 1   Table 3.
Comparison of the physicochemical characteristics of the condensed 1,2,4-triazine N2-b-ribofuranosides have been synthesized by spec tra re vealed con vinc ing co in ci dence in po si tion and form for sig nals from car bo hy drate frag ment and triazine ring of aglicones to in di cate en tire struc tural identity between compound pairs .The eval u a tion of other an a lyt i cal properties of these com pounds let us to claim that all of them have the struc ture of N2-glycosidic de riv a tives.
The prop o si tion in pa per [1] con cern ing glycosilation of N5-po si tion in triazine ring was based on quan tum-chem i cal cal cu la tions for the re action-com pe tent nucleofilic cen ters in orig i nal heterosystem III and sig nif i cant dis crep ancy be tween 1 HNMR data and shift of anomeric pro ton in compounds III and IV.Ad di tion ally it should be noted that prototropic tau tom er ism for a syn the sized tri azin-contain ing com pound re mains un ex plored, but it seems nec es sary to con sider the avail abil ity of tautomers.The data of quan tum-chem i cal cal cu la tions dem on strate that ni tro gen atom charges in N2 and N5 tricyclic system po si tions dif fer from the charge at ni tro gen atom N5 in tiazine ring ex ceed ing that of at ni tro gen atom (N2) in triazine cy cle.That's why by anal ogy with glycosilation of other con densed sys tem de riv a tives (gua nine and aloxasine) it seems rea son able to sup pose that N5-glycoside may pres ent a fi nal ki netic prod uct of the re ac tion, but upon strict syn the sis con di tions involved (~100°C, ac tive cat a lyst, time length) there is pref er en tially gen er ated the en er get i cally most advantageous product of thermodynamic control with glycoside bond in N2-position.
Hence, the over all struc tural iden tity be tween tricyclic nucleoside sam ples, ob tained by two syn thetic ap proaches, pro vides ev ery rea son to be lieve that un der the con di tions of sim pli fied silyl con den sa tion technique as a ma jor regioisomer the N2-riboside may be generated.
Pi lot in ves ti ga tion into the syn the sized com pounds ac tion on HCV replicon (Ph.D. S.L.Rybalko from the L.V.Gromashevskii In sti tute of Ep i de mi ol ogy and Infec tious Dis eases of Acad emy of Med i cal Sci ences of Ukraine) brought out the ef fec tive in hi bi tion of hep a titis C vi rus by some con densed triazine de riv a tives at the level of clinic prep a ra tion, rybavirin.The ma te ri als of bi o log i cal re search will be pre sented in a sep a rate publication.

Con clu sion.
The us ing of a triazine bases with fixed substituents at N2-po si tion in clud ing the ribose res i due too for generating new bi o log i cally ac tive triazine-bear ing de riv a tives al lows to avoid the problem of structural un cer tainty in the syn the sized compounds and to provide the functionalization of the triazine frag ment in tricyclic base in the al ter na tive way.Such ap proach proved the ad e quacy of the sim plified silylic con den sa tion for the di rected glycosilation of tricyclic triazine-bear ing bases.