Synthesis and Evaluation of the Plant Growth Regulatory Activity of 8-oxabicyclo [ 3 . 2 . 1 ] oct-6-en-3-one Derivatives

A síntese de vários análogos do 8-oxabiciclo[3.2.1]oct-6-en-3-ona é relatada. O efeito desses compostos e do ácido 4-oxoexanóico sobre a germinação e crescimento radicular do Sor ghum bicolor foi avaliado. Na concentração de 100 ppm os compostos 3-(metoxicarbonilmetil)-8-oxabiciclo[5.3.0]dec4-eno-2,9-diona (13) e ácido 4-oxoexanóico (17) apresentaram efeito estimulador do crescimento radicular de 33-35% e a 1000 ppm um efeito inibitório foi observado em ambos os casos (29% ( 13 ) e 80,2% ( 17). Todos os outros compostos inibiram o crescimento radicular a 100 e 1000 ppm. Nenhum efeito significativo foi observado sobre a taxa de germinação.


In tro duc tion
A num ber of sesquiterpene lac tones af fect plant growth, al though the na ture and ex tent of the ef fects produced de pend on a num ber of fac tors, in clud ing the lactone tested, its con cen tra tion, and the spe cies on which it acts 1 .Some sesquiterpene lac tones have been re ported to be respon si ble for the allelopathic prop er ties of cer tain plants by af fect ing the ger mi na tion and growth of other spe cies 2 .The po ten tial allelopathic ac tiv ity of sev eral nat u ral and synthetic sesquiterpene lac tones has been in ves ti gated and the pres ence of an α-meth y lene-β-butyrolactone has been shown be im por tant for the bi o log i cal ac tiv ity 3 .The presence of other re ac tive cen tres such as α,β-un sat u rated ketone, chloro hydrins, epoxide, hemiacetal, and also the mol e cules spa tial ar range ment is normaly im por tant for the bi o log i cal ac tiv ity pre sented by those lac tones [4][5][6] .
As part of our re search on the syn the sis of new compounds with her bi cidal and/or plant growth reg u la tory activ ity, de rived from the eas ily avail able 8-oxabicyclo [3.2.1]oct-6-en-3-ones 1 7 , we de vised a plan that would allow the prep a ra tion of sev eral lac tones 3-6 for bi o log i cal eval u a tion 23 (Scheme 1).

Ex per i men tal
Syn the sis IR spec tra were re corded on a Perkin-Elmer 881 dou ble beam grat ing spectrophotometer.NMR spec tra were recorded on a Perkin-Elmer R34 (220 MHz) in stru ment, a Bruker WH 400 spec trom e ter (400 MHz) or on a Varian T-60 (60 MHz) in stru ment, us ing tetramethylsilane as inter nal stan dard.Mass spec tra were ob tained on a VG ZAB-E high res o lu tion mass spec trom e ter.Flash chro matog ra phy was per formed us ing Crosfield Sorbsil C60 (40-60 µm).Sol vents were pu ri fied ac cord ing to Perrin and Armarego 26 , and pe tro leum re fers to the frac tion with b.p. 40-60 °C, ether re fers to di ethyl ether.

Bioassays
The bioassays were car ried out ac cord ing to the method of Einhelling et al. 23 with seeds of Sor ghum bicolor.Dichloro methane so lu tions of com pounds 7, 13, 17, 20a, 22 and 23 were pre pared at con cen tra tions of 100 and 1000 ppm.
As says were con ducted in a 100 x 15 mm glass Petri dishes lined with 1 sheet of Whatman No. 1 fil ter pa per and sealed with parafilm.To each dish was added 2 mL of each so lu tion and the sol vent was evap o rated be fore ad di tion of 2 mL of wa ter fol lowed by 20 seeds of Sor ghum bicolor.As says were car ried out at 25 °C un der ar ti fi cial flu o res cent light (8 x 40W) in a in cu ba tor dur ing three days, af ter which ger mi na tion was scored and the rad i cle lenth was measured.Seeds were con sid ered to be ger mi nated if a rad i cle protuded at least 1 mm.A controling ex per i ment was carried out un der the same con di tions de scribed, us ing only wa ter.Each bioassay was rep li cated 5 times in a com plete ran dom ized de sign.

Syn the sis
The oxabicyclic ketone 7, has al ready been trans formed into a num ber of nat u ral prod ucts and their an a logues 8,9 , and we thought to ex plore fur ther its chem is try by us ing it as start ing ma te rial for the syn the sis of sesquiterpene lactone 4 (R 1 = H or CH 3 ), ac cord ing to the strat egy shown on Scheme 1.
The ketone 7 was pre pared on a large scale us ing Sato and Noyori's meth od ol ogy 10 .Cat a lytic hy dro ge na tion of 7 us ing 10% Pd-C af forded the oxabicyclic ketone 8 in almost quan ti ta tive yield.The enol silyl ether 9 was pre pared in about 95% yield us ing trimethylsilylchloride (TMSCl) in the pres ence 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU).The al most quan ti ta tive con ver sion of the par ent ketone 8 into the re quired enol ether 9 was con firmed by the vir tual dis ap pear ance of the car bonyl stretch ing at around 1715 cm -1 (in the in fra red spec trum) with con com i tant appear ance of a very strong band at ~1640 cm -1 cor re sponding to the enol trimethylsilyl group.
The enol ether 9 was treated with methyl lith ium and the enolate formed was trapped with methyl bromoacetate (Scheme 2).It was ob served that when the enolate was gener ated at -78 °C/1.5 h, the re ac tion was in com plete re sulting in 29% re cov ery of the start ing enol ether.Raising the tem per a ture to -40 °C re sulted in com plete trans for ma tion of 9 into the cor re spond ing enolate.In gen eral, typ i cal over all yields for this alkylation was 30-45% for the monoalkylated com pound 10a, 15-20% for the cor respond ing dialkylated methyl es ter 10b and 20-30% re covery of the start ing ketone 8.It was found that sep a ra tion of the monoalkylated 10a from the dialkylated methyl es ter 10b was ex tremely dif fi cult, and 10b was not ob tained in a pure form.Some re ac tions were car ried out with 10a con-Scheme 2.
Al though a high de gree of exo-stereoselectivity for this alkylation has been claimed 11 , the com plex ity of the sig nals for H-1 and H-5 be tween δ 4.50 and δ 4.70 as so ci ated with two sig nals for methoxy group at δ 3.72 and δ 3.80 (ra tio 5:1) showed that a con sid er able amount (~20%) of the endo-alkylated prod uct was formed (the sam ple ana lysed was not con tam i nated with 10b as judged by the mass spectrum).
It was en vis aged that the trans for ma tion of 10a into lactone 12 could be achieved via an in ter me di ate like 11, formed by the cleav age of the ether bridge (Scheme 3).
An at tempt to pro duce lactone 12 was made by treat ing com pound 10a with trimethylsilyltrifluoromethanesulfonate (TMSOTf) and triethylamine (TEA) 20 , for two hours at room tem per a ture.In this case all the start ing ma te rial was con sumed and a very com plex mix ture was formed.How ever when a mixture of 10a+10b was treated with TMSOTf/TEA, the only prod uct iso lated was the lactone 13 (Scheme 4).
The struc ture of the lactone 13 was de duced by spec troscopic means.In the high res o lu tion mass spec trum, there was a peak at m/z 238.0827 cor re spond ing to the pro posed for mula C 12 H 14 O 5 .The in fra red spec trum showed a very strong ab sorp tion at 1781 cm -1 due to the γ-butyrolactone, and an other band at 1717 cm -1 for the ketone su per im posed with the es ter group.Spe cial fea tures in the 13 C-NMR spectrum are the ab sorp tions at δ 172, 175 (lactone and es ter), and δ 195 (ketone).Sig nals cor re spond ing to three CH2, one CH3, and five CH were ob served.The 220 MHz 1 H-NMR spec trum showed a sin glet at δ 3.72 for the methoxy group, and a multiplet at δ 5.80-5.90for alkene pro tons (Fig. 1).
The for ma tion of lactone 13 prob a bly in volves the inter me di ate 14 18 , and it shows the fea si bil ity of our ini tial syn thetic pro posal (Scheme 1).
The for ma tion of a com plex mix ture of prod ucts from this re ac tion is prob a bly due to the fact that keto es ter 10a was a mix ture of α and β-alkyl iso mers and also be cause the cleav age of the ether bridge was not regioselective.A fur ther in ves ti ga tion on the prep a ra tion of the 10b and its re ac tion un der the con di tions de scribed should be car ried out, since one can en vis age the trans for ma tion of lactone 13 into a pseudoguaianolide skel e ton 4.
Due to the prob lems with the stereoselective monoalkylation of 8 and pu ri fi ca tion of 10a, an al ter na tive route lead ing to lac tones 4 and 6 was in ves ti gated (Schemes 5 and 6).
Succinic an hy dride was con verted into the keto acid 17 in 69% yield 21 .Af ter methylation with CH3OH/H2SO4, the es ter 18 formed was brominated with Br 2 /HBr to af ford the re quired dibromoketone 19 in 42% yield.
The cycloaddition be tween the dibromoketoester 19 and furan was car ried out in the pres ence of Cu/NaI.The re-Scheme 3.
In or der to ac com plish the strat egy pre sented on route 1, com pound 20b was re quired, and since this was the mi nor iso mer formed, we used the ma jor iso mer 20a to fol low the syn the sis ac cord ing to route 2 (Scheme 1).
The bicyclic ketone 20a was treated with NaBH 4 /MeOH and the in ter me di ate formed by the re duction of the keto group re acted in a intramolecular fash ion with the carbomethoxy group re sult ing in the for ma tion of lactone 22 in 59% yield (Scheme 6).
The hy dro ge na tion of the oxabicyclo 20a fol lowed by sim i lar treat ment with NaBH 4 /MeOH led to the iso la tion of the lactone 23 in 50% yield.
Work is know in prog ress to trans form com pounds 22 and 23 in to more com plex and functionalized lac tones.

Her bi cidal Ac tiv ity
The dis cov ery of new her bi cides usu ally in volves the fol low ing ap proaches: i) the ra tio nal de sign of spe cific inhib i tors of key met a bolic pro cesses; ii) an a logue syn the sis of com pounds with known her bi cidal ac tiv ity and iii) the ran dom screen ing of new chem i cals.
Al though in this work we planed to make use of strategy ii), by de vel op ing a syn thetic route for the prep a ra tion of sev eral sesquiterpene lac tones, hav ing an α,β-un sat urated car bonyl group, we de cided to carry out a ran dom screen ing on sev eral syn thetic in ter me di ates (strat egy iii).
For this screen ing the in vivo ef fect of com pounds 7, 13, 17, 20a, 22 and 23 on the ger mi na tion and rad i cle growth of Sor ghum bicolor was eval u ated ac cord ing to the meth od ology pro posed by Enhelling et al. 23 .Two con cen tra tions (100 and 1000 ppm) of each com pound were tested, since it has al ready been shown that some com pounds ex hib ited both stimulatory and in hib i tory ef fects on seed ling growth, de pend ing on the con cen tra tion 24 .
Fig ure 2 shows the rad i cle lenth (mm) of Sor ghum af ter 3 days in cu ba tion at 25 °C and the per cent age of rad i cle growth (in hi bi tion or stim u la tion) in re la tion to the con trol is pre sented in Ta ble 1.At 100 ppm all com pounds showed a condiderable in hib i tory ef fect on the rad i cle growth, specially the ketoacid 17, that caused a 80% in hi bi tion.As com pound 17 showed a re mark ably dif fer ent ef fect on plant de vel op ment at lower and higher con cen tra tion 24 , and since it can be eas ily pre pared, it becames an in ter est ing start ing ma te rial for the prep a ra tion of other prod ucts for bi o log i cal eval u a tion.Al though the lactone 13 showed a sim i lar ef fect on rad i cle growth as 17, its prep a ra tion is more la bo ri ous and this makes fur ther bi o log i cal eval u ation less ap peal ing.Com pound 7 showed no clear ef fect at 100 ppm and 34% in hi bi tion at a 1000 ppm.
Com pound 20a (at 100 ppm) was 26 times more ac tive than its sim ple an a logue 7, and this ef fect can be at trib uted to the pres ence of the sub stitu ents at the 2 and 4 po si tions.
In view of these re sults and due to the ver sa til ity of the [3+4] cycloaddition meth od ol ogy used 25 for the prep a ration of com pounds 7 and 20a, the syn the sis of other oxabicyclic com pounds like 7 hav ing dif fer ent sub stitu ents at var i ous po si tions is now our next goal.Also an in ves ti gation of the her bi cidal se lec tiv ity of the com pounds al ready dis cussed to wards a wide range of crops and weeds is under way and will be pub lished else where.Ta ble 1. Ger mi na tion and rad i cle growth in hi bi tion of Sor ghum bicolor by sev eral syn thetic com pounds af ter 3 days, in cu ba tion at 25 °C.

Fig ure 2 .
Fig ure2.Root growth of Sor ghum bicolor af ter ex po sure to var i ous com pounds (100 ppm and 1000 ppm) and wa ter, af ter 3 days in cu bation at 25 °C.
Neg a tive val ues cor re spond to rad i cle growth in duc tion.