Acylated Flavonol Glycosides and Terpenoids from the Leaves of Alibertia sessilis

In the course of our con tin u ing search for new ac tive antifungal com pounds from Alibertia (Rubiaceae) found in “Cerrado” re gion of S. Paulo, we stud ied Alibertia sessilis (Vell.) K. Schum. col lected in Itirapina, in the state of São Paulo, Brazil. Pre vi ous in ves ti ga tions of the leaves of A. macrophylla re sulted in the iso la tion of fungitoxic non-glycosidic iridoids 1β and 1α-hydroxydihydrocornin aglycones, and the caffeic acid es ters: 2-phenylethyl caffeoate and 2-methyl-4-hydroxybutyl caffeoate. A. edulis, an other spe cies col lected in the same re gion, was also in ves ti gated, and no ac tive com pound was de tected. Rel a tively few chem i cal stud ies of the Alibertia ge nus have been re ported, de spite ev i dence of its rich terpenoidic con sti tu tion. In the pres ent study, from the leaves of A. sessilis we ob tained large amounts of the triterpenes oleanolic, ursolic, and epi-betulinic acid, and small amounts of the iridoids gardenoside, deacetylasperuloside, and 10-dehydrogardenoside, to gether with the fungitoxic αand β-gardiol. Be sides iridoids and triterpenes, the flavonols quercetin-3-Orutinoside, quercetin-3-O-β-D-(2”-O-trans-p-coumaroyl) -rutinoside (1) and kaempferol-3-Oβ-D-(2”-O-transp-coumaroyl)-rutinoside (2) were iso lated. These last two glycosides are be ing re ported for the first time from a nat u ral source.


In tro duc tion
In the course of our con tin u ing search for new ac tive antifungal com pounds from Alibertia (Rubiaceae) found in "Cerrado" re gion of S. Paulo, we stud ied Alibertia sessilis (Vell.)K. Schum.col lected in Itirapina, in the state of São Paulo, Brazil.Pre vi ous in ves ti ga tions of the leaves of A. macrophylla re sulted in the iso la tion of fungitoxic non-glycosidic iridoids 1 β and 1 α-hydroxydihydrocornin aglycones, and the caffeic acid es ters: 2-phenylethyl caffeoate and 2-methyl-4-hydroxybutyl caffeoate 1 .A. edulis , an other spe cies col lected in the same re gion, was also in ves ti gated, and no ac tive com pound was de tected 2 .Rel a tively few chemi cal stud ies of the Alibertia ge nus have been re ported, de spite ev i dence of its rich terpenoidic con sti tu tion 3 .

Re sults and Dis cus sion
The hexanic ex tract of the leaves of Alibertia sessilis was sub mit ted to a chro mato graphic pro cess to af forded sitosterol and α-tocopherolquinone, iden ti fied by com par i son with authen tic sam ples.Af ter methylation, the CH 2Cl 2 ex tract of the leaves gave ursolic, oleanolic, and epi -betulinic acid methyl es ters 2,4 .Af ter chro mato graphic sep a ra tions, the butanolic sol u ble part of the hydroalcoholic ex tract af forded a mix ture of the iso meric iridoids α and β gardiol 5 , in ad di tion to gardenoside and deacetylasperulosidic acid methyl es ter 6,7 .These com pounds were iden ti fied by com par ing the spec troscopic data with the lit er a ture [5][6][7] .
Gel per me ation chro ma tog ra phy of the EtOAc sol u ble part of the hydroalcoholic ex tract gave quercetin-3-O-rutinoside 8 (rutin) and a mix ture of the new acylflavonol glycosides 1 and 2. These were pu ri fied by sub se quent pre para tive TLC.The 1 H-NMR of 1 and 2 showed sig nals that sug gested the struc ture of quercetin and kaempferol, re spec tively, for the aglycones of the compounds.In both spec tra sig nals cor re spond ing to the trans p-coumaroyl group and a rhamnose sugar por tion were also found (Ta ble 1).The anomeric pro ton of the rhamnose ap -

Ar ti cle
Present address: Departamento de Química, UFMa.peared as a sin glet ( δ 4.36) in ac cor dance with the α con figu ra tion.An other sig nal, which could be as signed to an anomeric pro ton of an other sugar ap peared at δ ~ 5.5 (d, J = 8 Hz) in both spec tra.
The 13 C-NMR spec tra (Ta ble 2) con firmed the structure of quercetin and kaempferol aglycones to 1 and 2, respec tively.The pres ence of rhamnose and coumaroyl moi eties was also con firmed.The lo cal iza tion of the car bohy drates in the flavonols at C-3 was de duced by the chem ical shifts of C-3, C-2, and C-4, the first be ing shielded and the lat ter two deshielded in re la tion to the free aglycones 8 .The rhamnose sig nals are un changed, so the acyl group should be lo cated on the other sugar moi ety.The methylenic car bon of that sugar is deshielded by 6 ppm when com pared with that of glu cose.A COSY 1 H -1 H spectrum of 1 showed cor re la tions be tween the anomeric pro ton sig nal at δ 5.56 and a sig nal at δ 4.87 (t, J = 8.4 Hz).These val ues sug gested an acylation of the hydroxyl at C-2 and a diaxial hy dro gen cou pling be tween H-2/H-1 and H-2/H-3.These data are in agree ment with an acylated C-2 glu cose.
Com par a tive anal y ses of the 13 C-NMR car bo hy drate data of 1 and 2 with those of the glu cose in rutin 8,9,10 re vealed that the C-2 of the glu cose in 1 and 2 was deshielded by +2 ppm, and that C-1 and C-3 are shielded by -2 ppm.These ob ser va tions are in agree ment with the lo ca tion of the coumaroyl group at C-2 of the glu cose moi ety.Thus, the struc tures of the two new acylated flavonol glycoside iso lated from Alibertia sessilis were es tablished as quercetin-3-O-β-D-(2"-O-trans -p-coumaroyl) -rutinoside ( 1) and kaempherol-3-O-β-D-(2"-O-trans -pcoumaroyl)-rutinoside ( 2).The oc cur rence of iridoids and triterpenes in Alibertia is in agree ment with chemosystematic cor re la tions and botan i cal po si tion ing of this ge nus in Gardenieae3 .How ever, the iso la tion of this type of flavonol is un usual if we consider the chem i cal com po si tion of other Alibertia al ready de scribed in the lit er a ture.

Ex per i men tal
Gen eral ex per i men tal pro ce dures NMR spec tra were mea sured at 200 MHz for 1 H, with TMS as the in ter nal stan dard and 50 MHz for 13 C, us ing the sol vent sig nal as ref er ence.

Plant ma te rial
Alibertia sessilis (Vell.)K. Shum.(Rubiaceae) was collected in the Itirapina Bo tanic Re serve, in the Cerrado re gion of São Paulo, Brazil.A voucher spec i men is de pos ited at the Bo tan i cal In sti tute of São Paulo (num ber S.P. 110683).

Ex trac tion and iso la tion of the con stit u ents
Dried and pow dered leaves (515 g) of A. Sessilis were suc ces sively ex tracted with hex ane, meth y lene chlo ride and eth a nol/wa ter (6:4).The crude hex ane ex tract (10.4 g) was frac tion ated on sil ica gel col umn chro ma tog ra phy.Fur ther pu ri fi ca tion of the frac tions by pre para tive TLC, eluted with C 6 H 6 :EtOAc (9:1) yielded sitosterol (86 mg), α-tocopherolquinone (29 mg), and fatty ma te rial.
The CHCl 3 in sol u ble frac tion (1.2 g) of the crude dichloromethanic ex tract (9.5 g) was meth yl ated with CH 2 N 2 .Pre para tive TLC eluted with CHCl 3 :MeOH (97:3) of this mate rial gave three frac tions: the first was a mix ture of ursolic acid and oleanolic acid methyl es ters (300 mg), the sec ond was the methyl es ter of ursolic acid (800 mg), and the third was the methyl es ter of epi -betulinic acid (30 mg).
The crude eth a nol/wa ter (6:4) ex tract (48 g) was par titioned into ethyl ac e tate and then into n-butanol.The sol u ble part of n-Butanol (2.5 g) was sub mit ted to col umn chro ma togra phy on sil ica gel, and eluted with CHCl 3 with in creas ing amounts of MeOH.Af ter anal y sis by TLC some frac tions were com bined.Fur ther pre para tive TLC eluted with CHCl 3 :MeOH (85:15) of the less po lar frac tions gave a mixture (5 mg) of the iso meric iridoids -gardiol and β-gardiol.An other frac tion of that col umn was sub mit ted to reversed-phase HPLC [col umn C-8 (25 x 0.4 cm) and elu tion with a H 2O -MeOH gra di ent, at a flow rate of 8 mL/min, and de tec tor UV (240 nm)], af ford ing the iridoids gardenoside (17 mg) and deacetylasperulosidic acid methyl es ter (4 mg).
The sol u ble part of ethyl ac e tate (200 mg) was pre cip itated with CHCl 3. The pre cip i tate was dis solved in MeOH and sub mit ted to col umn chro ma tog ra phy on Sephadex LH-20, and eluted with MeOH.Af ter TLC, some frac tions were com bined and one of them was iden ti fied as quercetin-3-O-rutinoside (50 mg).An other frac tion was sub mit ted to pre para tive TLC, and eluted with CHCl

Ac knowl edge ments
The au thors are grate ful to CNPq for fi nan cial sup port and schalarships.

R
1a ble 1.1H-NMR spec tral data for com pounds 1 and 2 in DMSO-d 6*.Ta ble 2.13C-NMR spec tral data for com pounds 1 and 2 in DMSO-d 6.