arundo donax yasin

8/12/2019 Arundo Donax Yasin

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M Y D R I A S IS I N M I C Ee : C O M P O U N D 17 1 3 i i o k i ,

- 1 2 4 5 tiH O U R S

Flgl i l? 2 .Y l i c 4-halo-subhtituted coinpouiidi 9 arid 13 had t l i c 1largci-t heparation bet\\ eeii +pa-molytic id lethni(IffPCti, 9 w i i i :ilso t h c nioit potent a nd n a i tc-trtiurthcr in coinparison to :itiupinc'. Co mp o u n d 9 iva-iiot mydriatic in inice a i i t l i x t -. w l i i l c :it ropiric had :I

Gnlogogic activity of 9 w:i. \ICA i i micc. le+ thur lthat of :itropinc ('ompound 9 a- about oiic-seventh:I- potent :I- :itropine in thc teed pellet te-t. I n thvch:irco:il ineal te-t -CY> ' l'ahlc V ) , thc c>ffect- of thc trio

c quite differmt A l t r o p inc p r oduc e d :Imaximuin of :thout .SOc inhihitioil at 200 d00iiig kg , while 9 produced inhihitioil ranging f r o m -1-2to 9 4 9 at h i e s of 94-lh5 rng LgY o n Oettiiigen16 pointed o i i t t h t the para-yni-patholytic :xtioii of' :itropiiir uid it- malogs \\ :I-

poterit effect :1t do-e- of 1 mg kg o r me i l l ~

Arundo donax L. Grarninae). Phytochemical and Pharmacological Evalua Lion

k'ive iiidole-8-alk .lariiiiie liases, vii., S; -clirnethyltrypt aliiirie, 5-niet hosy-X-nie1 ylir>.pialnine, Irufoteniiie.dehvdrohufotenine, and bufotenidine, wx e isolated from the rhizomes of -1 iirtrio r1ona.c I,. This is the firstreported occurrence of hufot enidine and dehydroliufiitenine in i plant species. .i defatted ethanoliv estra rt ofthe rhizomes prodwed hyp(iten;iive and ant ispasniodic etf erts ag ai n~ t istamine-, serotonin-, and aretylcholine-induced spasms. Bufotenidine showed three main pllarmac~ologic.ala('tions, r i z . antinc,etylch(ilineeft'ect whichappears t o he more specsifir against skeletal muscle t hnn against musc*arinic it e*, h i p t amine release, and uterinestimulant. S o n ? i i f these n r t i o n s of t h i s compound had heen reported prrviouely.

. l r u t d o dor1a.r I,. Grarriznue), :L all, stout. pereiiiiialdi r i i i ) , often woody below, is -\viclely distr ibuted iiiIiiclia. -1 ltwwtion ot its rhizomes has heeii used in the.iyurvctlic. system of medicine1 as an emollient aiidcliurrtic ~ i i ds said to stimulate menstrual disrharge:i11(1 t o tliiiiiiiish thc secretioii of iiiilk.k'roni t tic. Irvives of this dirub ?\ladiiiaveitia p r e ~-orisly reported2 the isolatioii of thrce iiiclolic bases, P ~ Z . .cloiiaxariiit~,('ll3Hl8S2OL,111) 217 , gramiiie. aiicl a i l


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May 1969 INDOLEASES noM Arundo dm x L. 48 1and its Nb-oxide from the leaves of Alundo donux. I naddition, paper and thin layer chromatography revealedthe presence of several quaternary indolic bases. Thepresent paper deals with the chemistry and pharma-cology of the indole-3-alkylamines in general and bufo-tenidine in particular, occurring in the Indian reed.The utilization of the pH-gradient separation andcolumn chromatography on partially deactivatedBrockmann alumina have resulted in the isolation ofthree previously k n ~ w n ~ . ~econdary and tertiary indole-3-alkylamines, viz., N,N-dimethyltryptamine, 5-me-thoxy-N-methyltryptamine, nd bufotenine, and twoquaternary indolic bases, viz. bufotenidine and dehydro-bufotenine, from the alcoholic extract of the rhizomesof this plaut. In addition, the total alkaloidal extractrevealed, on paper and thin layer chromatograms, thepresence of a number of 5-hydroxyindole bases (colorreaction with cr-nitroso-0-naphthol reagent5 positive)of low Rr values. Gam ine, the major alkaloid of theleave^,"^ was uot found in the rhizomes. This is thefirst reported occurrence of bufotenidine and dehydro-bufotenine h i a plant species. The only other naturalsource knowu for these compounds so far was the toadpoison."JAs the rhizomes coiitained very little fat the alkaloidswere extracted directly with alcohol without priordefatting with petroleum ether. This simple hut impor-tant modification in the usual extractiou procedure keptthe formation of a melanin-like polymer to a minimum.The dark brown polymer showed Dragendorff-positiveand Ehrlich-negative color reactions. The N analysisof this compound was low, indicating thereby a veryhigh state of oxidation of the polymer. Autoxidation,a serious problem during isolatiou and preservatiou oft,he chloroform-soluble bases also led to similar productswhich were finally converted to a black melanin.Pharmacology.-The observations made by us duriiigthe pharmacological testing that certain fractions pre-pared from the rhizomes of A . d o n m producrd hypo-tensive and antispasmodic effects against histamine-,serotonin-, and acetylcholine-induced spasms led us tosubmit the defatted ethanolic extract of the plant to amore thorough pharmacological screeniug program.During these investigations, the extract was found toexhibit a strong curare-like action on frog rectus ahdom-inis muscle. It was sooil determined that these bio-logical activities resided iii the alkaloidal entities coii-tained in the extract.Detailed pharmacological studies have, however,been made with hufotenidine only as its yield wasmaximum and as very few studies seem to have beenmade8 with this alkaloid. Routine pharmacologicalmethods have beeii employed to study its actioii on thecardiovascular system, CNS, smooth and skeletalmuscles, and urinary and fecal output.The drug was found to have characteristic actions onthe dog's blood pressure, skeletal muscle, and smoothmuscles. I t showed no significant effect on barbi tu ra teinduced hypnosis or as an analgetic (tested by rat tailhot, wire method) or as an anticonvulsant (against

4) 8. G h a d idH. hl ukl i r r je r , Clem. I n d . 1,ondsn) 181UJ (1964): 7 $:l15) S. Udenfrimd. H. W e i ~ h : t c L . i ic l C. P. Climh. J . R id . Clism.. 215.1965,: J .Org .Chcm. . 81, 2284 (I l66).

337 19551.6)H. ensen and K . K . Chen, {bid. . 116, 87 (1936).(7) H. Wielnnd and T. Widand, Ann., 528. 234 (1937).8) V Erapnmer. Nolure. 170. 281 (1952).

maximal electroshock and pentylenetetrasole-inducedconvulsions). It showed some cardiotonic activity onthe frog's perfused heart bu t this was not considered ofsignificant importance as it failed to show a similareffect on the hypodynamic heart (produced by perfusingthe heart with Ringer's solution containing 25y Ca*+).Effect on Blood Pressure.-Intravenous administretion of the drug (0.2-0.5 mg/kg) produced varyingdegrees of fall in blood pressure. The fall was sharpand the recovery was slow and gradual. The durationof hypotension after the first dose varied from 0.5 to1.5 hr. Repeated injections of the same dose of thedrug showed tachyphylaxis (Figure 1) . Further, the

I'iguw 2 . 1Xw1 I l ~ ~ ~ f , ~ ~ ~ , ~ ~ i ~ i i ~ ~ ~ ~ng kgi 11t I j ~ : i i i i i l i : ~ ~ l i i t : ~ l -arirst I i r i s r d dog I h i d prwsuw :after ~ ~ r i o r,~l,,,i,,ist~:,li,,,, d w odoses (0.2mg/kg) o r rCIulmmriwii)c. . \ I t h e w h i t ? liiies the drumW:LS s l ~ p p c do r :10 mi .

prior treatment of thc animal with oiie or two doses ofd-tubocurarine (0.2-0.4 mg/kg). The reverse phenom-enon was also observed, i .e., prior administration of thealkaloid reduced or abolished the hypotensive responseof &tubocurarine. Interestingly, after tachyphylaxisthe hypotensive response could be produced if thealkaloid was administered after slow histamine perfu-sion 5 pg/ml'min for 30 miu) which itself did notproduce any appreciablc hypotciisivc response (Figure3). Ttiesc observat,ioiis suggest tha t the mode ofhypoteiisivc action and tachyphylaxis are due to histamine release from some stores which could be replenishedhy the slow histamine perfusion t,o be agaiu released bya histamine releaser.Effect on Skeletal Muscle.-The alkaloid was foundt u block spccifically the acetylcholine-induced (5 pgiml)


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482 GHOSAL,DUTTA,ANYAL,N D BHAWACHAHYA Vol. 12Dog's Tracheal Chain.-The alkaloid (up to 80pg/ml) had no effect of its own nor did it modify thcAcCh-induced (1 pgiml) spasm.

Experimental SectionYThe general procedure for the separation and Identiticalion ofthe alkaloids involved column chromatography ov0r Broekmann

spasm in isolated frog rectus abdominis muscle. It didnot alter the KC1-induced spasm. This blocking effectwas compared against d-tubocurarine, and ED,, werecalculated by drawing regression curves. The ED,,values of the alkaloid and d-tubocurarine were deter-mined as 1.4 and 1.05 pg/ml, respectively.I n wivo, when administered to albino mice and ratsin doses of 10 mg/kg ip and PO respectively, the alkaloidcaused the death of all five mice and ataxia in all tenrats. The death was due to respiratory failure, evidentby deep cyanosis and hecausc the heart was still beatingwhen respiration had ceased. There was no death orataxia when the alkaloid was administered to mice indoses of 2 mg/kg ip. The alkaloid produced head drop

followed by death in rabbits in a 5-6-mg/kg iv doseadministered against 0.3-0.5 mg/kg iv of d-tubocurarine.The EDaocould not be determined due to paucity ofmaterial. These studies suggest that the alkaloid is apotent neuromuscular blocking agent. Though its EDaoin vitro was not significantly lower than th at of d-tubo-curarine, the latter was found to be at least eight totwelve times more potent in vivo experiments. Furtherstudy is needed to resolve this discrepancy in theresults of in vivo and in vitro experiments.Effect on Smooth Muscles.-The isolated loop ofguinea pig and dog intestine in situ, the isolated uterusof albino rats and guinea pigs, and the dog's trachealchain were employed to study the effectof the alkaloidon smooth muscles.Intestine.-In situ experiments with the alkaloidproduced spasm and repeated doses resulted in tachy-phylaxis. This was thought to he due to histamincrelease. In an isolated loop of intestine the alkaloidrelaxed the muscle in concentrations of 1-10 pg/ml. Inhigher concentrations, above 20 pg/ml, the alkaloidantagonized the spasmodic effects of histamine, AcCh,and RaCL The spasmolytic action, therefore, isnonspecific in character .Uterus.-The drug produced its own spasm, moreconsistently, in isolated guinea pig utcrus than inisolated albino rat uterus. This spasm was only seenup to a concentration of 20 and 10 pglml in guinea pigand albino ra t uterus, respectively. There was notachyphylaxis. In higher concentrations the drugblocked AcCh-induced spasm. I t is possible th at thebasis of use of the plant extract (rhizomes) in indigenoussystems of medicine' "as a stimulant of menstrual

discharge" was due to the stimulant action of thealkaloid.

aluminn and paper and t,hin layer chromatography of the indi-vidual components in the presence uf marker compounds; deter-minat,ion of uv ahsorption spec tra; and preparation of picrate,HCI, HI, and Me1 salts where possible. Descending paperchromatography wm used on Whatman KO. 1 paper using~ - B u O A ~ ~ L - B U O H - A ~ O H - H ~ ~85:15:40:22). In the tlc, silicagel G was used a8 the adsorbant and MeOH as the developer.The indolir hasea showed pale yellow to yellow-green fluorescenceunder uv light on paper and did not respond to the Jepson andSteven's testlo for Kb-unsubstituted tryptamines indicatingthereby t,hat the basic N in these compounds ie substituted.Isolation of the Alkaloids.---Dried and milled rhizomes (700 g)were extracted EtOH j, in a percolator, at room temperature,for 4 weeks. The EIOH extract was concentrated under reducedpressure to give a brown viscous consistency (112 g) which waspoured into 2 b AcOH (100 ml j with stirring, and the mixture wmkept overnight at ordinary temperature. Suspended impuritieswere filtered off and the filtrate wm shaken with CHCla (three50-ml port,ions) which removed the CHClr-soluble a ce ta ta(10.3 g, fraction A). The pH of the aqueous solution wasbrought t,o 9 with KHa and the liberated bases were extracted(CHCI,, threo 50-ml portions). The polymeric material remainedinsoluble in CHCL and was filtered, washed (H,O), and dried(4.1 g , mp 80 and 235-240"; Amsx 300-305 mp (sh); Xmax (Nujol)2.95 p (KH); Rr ( t l c ) 0.0; N, 4.82 and 5.1'?,.The CHCla solution containing the strong bases was washed(HeO) and dried, and the solvent was removed under reducedpressure when a brawn basic gum (3.7 g, fraction B) was obtained.The aqueous alkaline mother liquor was treated with a saturatedaqueous solution of ammonium reineckate and the light pinkreineekate complex was kept far subsequent regeneration ofwater-soluble st,roug bases (fraction Cj.Fraction A. CHCL-Soluble Acetates:-The residue from thisfraction was taken in PhH and the PhH solution was ektractedwith 0.1 ill aqueous citric acid (100 ml). The aqueous acidiclayer was basified (NH.), the liberated bases were extracted(CHCI,), and the solvent was removed from the organic layerwhen a thick brown oil (0.04 g) was obtained.N,H-Dimethyltryptamine.-The identity of the above hase,Ri 0.74, was established by cochromatography with authe-,ticN,N-dimethyltryptamine [Ri .74; uv, Am= 222 mp (log 4.481,277 (3.77j, and 288 (3.75)] and from the formation of picratefrom EtOH; mp 168" undepressed on admixture with theauthentic N,N-dimethyltryptamine picrate, mp 168 '.CHCL-Soluble Strong Hases.--The total basicgum (3.7 g) was dissolved in CHClr (10ml and chromatographedon partially deactivated Broekmann alumina (35 X 4 em) .Elution was carried out with 100-ml portions of petroleum ether,(tlp 40-60"), petroleum ether-PhH (90:10, 8020 50:50) , PhH,PhH-CHCB 95:5, 90:10, 8020 50:5I 25:75j, CHClrMeOH(90:1, 8 8 9 , 95:5j, and MeOH.5-Methoxy-N-methyltryptamine.-The CHClaPhH (7525)eluates were combined and concentrated under reduced pressure.The residue (0,016 g), Rr 0.5X, was cochromatographed withauthentic 5-methoxy-K-methyll~y~t~mine,i 0.58, which showeda ringle Spot at the expected R r uv, Xmax 2'4 mp (log e 4.53)and 288 (3.82).The base formed a ed picrate from MeOH; mp 220",mmp 222"with authentic S.methory-S-methyltrptamine picrate, remain-

Fraction B.

ing undepressed.Bufotenine.-The CHClsMeOH (99:l) eluates were combinedand the solvent was removed under reduced pressure when abrown amorphous mas8 (0.18 g) was obtained, Rr 0.27; co-chromatography with suthentic bufotenine showed t 027; uv,Am- 224 mp (log c 4.46), 278 (3.Y3), 204 (3.90),and 305 (3.52).

9)All meltmg points were taken in open espillar~ nd are unoorreoted.Uv spectra we_ recorded in B Carl Zeiss Universal swetrophotometer, \'SUI.mmg aoeotral EtOH. Mioroanalyaes were performed by Dr. A . Bernhardt,M a x Plank h a t i t o t e , Mulheim (Ruhrl. Germany.

10) J. B.Jepson and B. I tevens. Nature, 172. 172 (1953).


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May 1969 BENZYLPENICILLENICCID N D THIOLS 483The base af forded a yellow picrate from E tOH; mp 178", mmp177-178' with the authentic bufotenine dipicrate, remainingundepressed. The Xb-oxide was crystallized from ?\IezCO-EtOHas rods, mp 217" (lit." mp 217").Rufotenidine.--AIeOH washings of the alumina column onevaporation left a brown basic gum ( 2 .3 g), R f 0.18 (as the majorarea of intensity wi th both Dragendorff and Ehrlich reagen ts)plus three other spots, Rr 0.0, 0.08, and 0.22. The major compo-nent was separated by preparative tlc over silica gel G usingNeOH as the developer; Rr 0.16; uv, Xmax 218-220 and 284-288mr; red picrate from aqueous Et OH; mp 198-200", mmp 198-

200" (lit.i mp 198")with authentic bufotenidine picrate, remain-ing undepressed.A portion of t he basic gum was directly converted to the picrate.The picrate was crystallized from aqueous EtOH as red needles,mp 198". A n a l . Calcd for C13H18SZ0.C~H3S30,: 15.67.Found: K 5.82.The base hydriodide crystallized from EtOH as light grayneedles, mp 209-210 , mmp 209-210 with bufotenine methiodide,remaining undepressed. A n a l . Calcd for CI~HI~SLOHI: S,b.09. Found: S , 7.93.Fraction C. Water-Soluble Bases.-The pink reineckate com-11) ill . Fish, N. AI. Johnson, and E. C. Horning, J . Am. Chem. Soc., 78,36iO 1956).

plex, mp 168-170" dec, &-as dissolved in A1e2CO and passedthrough a column of De-acidite FF (pH 8).l2 The E tOH eluateson evaporation yielded a crystalline alkaloid (0.44 g), Rr 0.06(plus two other minor components, Rr 0.16 and 0.31).Dehydrobuf0tenine.-The crystalline compound, obtainedabove, had a double melting point, 202 and 217", R t 0.06 (paper)and 0.04 (tlc) . The compound was identified as dehydrobufo-tenine,6s7 mp 198 and 218', on the basis of i ts physical andchemical properties and those of its sa lts; uv, Xmax 218-220 mp(log c 4.55) and 285 (3.98) ;violet color with a-nitroso-@-naphtholreagent;5 and negative Jepson and Stevens testlo for Sb-unsub-stituted tryptamines. A n a l . Calcd for CHHIJIO .HzO: C,65.45; H, 7.27; S 2.72. Found: C, 64.98; H, 6.99; K, 2.81.The HCl salt crystallized from EtOH as needles, mp 237-238 O(lit.; mp 237-238'), and the H I salt crystallized from aqueousEtOH as light gray rods, mp 243-245" (lit.: mp 243-245').The picrate crystallized from aqueous EtOH as yellow needles,mp 182-184" (lit.; mp 183-184'). d n a l . Calcd for C12H14SgO.C6H3K307: N , 16.27. Found: S 5.98.

Acknowledgment.-The authors are grateful to theC.S.I.R., New Delhi, for awarding a Junior ResearchFellowship to one of them (S. K . D.).12) P. K. Banerjee and S Ghosal, Aust . J.C h em . , 28, 265 1969).

The Reaction of Benzylpenicillenic Acid with Thiol-Containing Compounds.The Formation of a Possible Penicillin Antigenic Determinant1

EUGENE WAGNER,WILLIAMW . DAVIS, ND MARVINGORMANYhe Lilly Research Laboratories, Eli Lilly and Company, Indianapolas, Indiana

Received October 4, 1968I t has been shown tha t the penicilloyl determinant in penicillin allergy can be formed by the reaction of the

penicillin degradation product, benzylpenicillenic acid, with free amino groups in proteins. This study was con-ducted to determine whether benzylpenicillenic acid could react similarly with another functional group inproteins, the SH group. The reactivity of benzylpenicillenic acid a t pH 7.5 was measured spectrophotometricallyand was found to be ten times greater with compounds having free sulfhydryl groups than those having freeamino groups. Experiments using compounds with missing or blocked SH groups indicated the sulfhydryl groupwas the reactive species and suggested the possible formation of a product different from those reported previ-ously. TICof the products of the reaction of benzylpenicillenic acid and ethanethiol at pH 7.5 disclosed thepresence of four components. Characterization of these components by nmr and mass spectrometry revealed thatthey are stereoisomers of the thiol ester of benzylpenicilloic acid, a-ethylthiobenzylpenicilloate. These resultsindicate th at benzylpenicillenic acid can react with free SH groups of pro tein to form thioesters in much the sameway it reacts with free amino groups to form amides.

In allergenic responses to simple compounds it isknow n that irreversible binding to protein is necessaryfor the simple chemical compound to sensitize andelicit an allergic reaction. Benzylpenicillenic acid,which forms spontaneously from benzylpenicillin,28 canreact irreversibly with proteins2band is believed t o playa role in penicillin allergy. 3 , 4

It has been demonstrated that penicilloyl compoundsmay be formed by reaction of either penicillenic acid516or by direct interaction of p e n i ~ i l h - ~ith free aminogroups of protein. The penicilloyl group is believed to

(1) Presented in part at the 155th National Meeting of the BmericanChemical Society, Sen Francisco, Calif., 1968.

2) (a) F. H. Carpenter, R. A . Turner, and 1 . du Vigneaud,J. B i d . Chem. ,176, 893 1948) ; (b) I. M. Klotz, J. M. Urquhart. and UT. .Weber, Arch .Biochem. , 26, 420 1950).(3) B. B. Levine , J . Ezp . Med . , 112, 1131 1 960).4) A. L. De Weck and H. N. Eisen. ibid., 112, 1227 1 960).5 ) B. B. Levine and Z. Ovary, ibid., 114, 875 1961).(6) 4 . L. De Weck In tern . Arch . A l l erg y A p p l . Zmmuno l . , 21. 20 1962).

(7) C. W. a r k e r and J. A . Thiel, J . Lab . Clin. Med . , 62, 482 1963).8) F. R. Batchelor.J.bl . Dewdney, and D. Gazzard, Nature . 206,362 1965).9) C . H. Schneider and A . L. De Weck, ibid. 208, 57 1965).

be the major antigenic determinant in penicillinallergy. o, l1The present work is a study of the reactivity of benzyl-penicillenic acid with another functional group found inproteins, the sulfhydryl group. Cysteine, N-acetyl-cysteine, serine, penicillamine, homocysteine, methio-nine, glutathione, e-aminocaproic acid, 2-mercapto-ethanol, and ethanethiol serve as model compounds forfree amino groups and free as well as blocked SH groupsof proteins. The reactivity of penicillin and penicillenicacid with these model compounds is compared, theimplication being that penicillenic acid reacts with freeSH groups of proteins to form thioesters in much thesame way it reacts with free amino groups to formamides. The possibility that the product of thisreaction, a penicilloyl thioester, may be a determinantof penicillin allergy is discussed.

(10) B. B. Levine. Federation Proc., 24, 45 1965).(11) A. L. De Weck and G. Blum, Intern. Arch . . l l / e r g y d p p l . Zmmunol..27, 221 1963).

arundo donax yasin

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