I. Studies on the Mescal Alkaloids and Related Compounds. II. Preparation of Several Phospho-Organic Compounds Citation Saunders, Paul Rome (1943) I. Studies on the Mescal Alkaloids and Related Compounds. II. Preparation of Several Phospho-Organic Compounds. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1jdk-2j68. https://resolver.caltech.edu/CaltechTHESIS:03132025-173850426 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Haagen-Smit, Arie Jan Thesis Committee: Unknown, Unknown Defense Date: 1 January 1943 Record Number: CaltechTHESIS:03132025-173850426 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:03132025-173850426 DOI: 10.7907/1jdk-2j68 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17060 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 13 Mar 2025 18:06 Last Modified: 13 Mar 2025 18:07 Thesis Files PDF - Final Version See Usage Policy. 22MB Repository Staff Only: item control page

I. STUDIES ON THE MESCAL ALIG-\.LOIDS AND RELATED COMPOUNDS II. PREPARATION OF SEVERAL PHOSPHO-ORGANIC COMPOUN DS The sis by P au l R. Saunder s In P art~a l Fu lfilment of t h e Re qu ire ments for the De gree of Doct or of Philosophy C~ lifor n i~ Institute of Technology P a s adena, Ca liforn i a 1 91'.2: 3 Ac knowle dgment The E',uth or wishes to express his gr a tituc.e to Dr. A. J. Haagen-Smit, Dr. Gordon A. Alles, and Dr. Henry Borsook, for their personal interest and encouragement of this work. I also am grateful to Dr. Alles for supplying the mesc a l buttons a nd for making available to me the equipment of his laboratory at severa l st ages of the work. I wish to especially thank Dr. Gertrude Opp enheimer for her va luable advice on microane,l ytical problems, and for her close coope r a tion throu ghout the course of the rese arch. TABLE OF CONTE NTS PART I. STUDIES ON THE MESCAL ALKALOIDS AND RELATED COMPOUN DS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . page 1 Experi men tal section . . . . . . . . . . . . . . . . . . page 20 .......... pag e 20 ( 2 ) Syn thetic work .................. page 29 ( 3 ) Action of amine oxida se . . . . . . . . . pag e 47 (1) Isol a,ti on of alka loi ds Sun1n1ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 50 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . pag e 51 PART II. PREPARATION OF SEVERAL PHOSPHO-ORGANIC COM- POUNDS Introduct ion page 54 Experimental section ................. . page 55 Summary • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 63 Refere 11ces . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 64 PART I STUDIES ON THE LIESCAL ALK..t1.LOIDS AND RELATED COMPOUNDS 1. INTRODUCTION Pellote (peyote, peyotl, mescal buttons) is the drug used by the Indians of northern Mexico and the southwestern United St a tes for t he hallucinatory effects produced by it when e a ten. The actions of the drug are probably entirely due to the alkaloids present, eleven of which have been isolated to da te. Pellote is obtained from the cactus which is now most comm onl y classified as Lopho·ohora willi am sii (Fam. Cactace a e). It has also been classified as Anhalonium Lewinii, which was for merly most generally accepted, Echinocactus williamsii, and others. The tops of the p l crnt, which is a small spineless cactus covered with wooly hairs, are cut off and e a ten either fresh or dried, and are comm only known as 11 rnesca.l buttons 11 • It is found chiefly in northern Jilexico a nd ne a r the Rio Gr a nde River in Texas, and though its use has spre ad somewhat, it is still confined to a comparatively few tribes of this general are a . The term mesca l button s hould not be confused with t he fer mented drink made in Mexico known a s mescal, which is a distilled beverage prepared from va rious species of Agave. The use of p ellote in religious ceremonies by the Indians of the Rio Grande valley has been known for many ye a rs, and an interesting a ccount of the actual ceremony is given by James Uooney (1). scription: 11 Q,uoting briefly from his de- The ceremony occupies from t welve to fourteen hours, be g innin i;,: a. t about 9 or 10 o I clock and lastin g: 2. sometimes until nearly noon the next day. The exercises open with a prayer by the leader, who then hands each man four mescals, which he takes and eats in rapid succession. In eating, the dry mescal is first chewed in the mouth, then rolled into a large pellet between the hands, and swallowed. After this first round the leader takes the rattle, while his assistant takes the drum, and together they sing the first song four times, with full voices, at the same time beating the drum and shaking the rattle with all the strength of their arms. The drum and rattle are then handed to the next couple, and so the song goes on round and round the circle - until perhaps 9 o'clock the next morning, when the instruments are passed out of the tepee, the sacred foods are eaten, and the ceremony is at an end. 11 There appear to be no undesirable after-effects, and the drug is often used • by the Indians for therapeutic purposes, as in the treatment of fevers and headaches. There are many descriptions of the effects produced by pellote in man, among which might be mentioned those of Mitchell (2), Havelock Ellis (3), Prentiss and Morgan (4), and Fernberger (5). The general picture of the effects of the crude drug is well-known, but the actions of the various alkaloids oresent have not been studied at all thoroughly, and a great deal of work remains to be done on this subject. The outstanding action of pellote and the one probably chiefly responsible for its use by the Indians is the production of color. hallucinations. The first phase of the 3. intoxication is marked by a general feeling of euphoria, and often stimulation is observed, leading to the loss of a sense of fatigue. When the second stage is reached, the in- dividual begins to have the characteristic color visions, and in some ca ses other sensory effects. Descriptions of the visions indicate a great individual variation in response to the drug, but in general they are characterized by an incess ~nt flow of hallucinations of great variety of form and color, sometimes being composed of geometric figures or familiar objects, usually in constant motion, and sometimes various grotesque figures are observed, all intensely colored. Th»e visions are most often obtained with the eyes shut or in the dark, but are sometimes seen in the light. During this period the subject is completely con- scious and aware of his surroundings. In addition, there are sometimes hallucinations of hearing and an increase in auditory acuity. The general sensibility may be affected in such a manner that the subject has the illusion of being without weight, or of the doubling of the ego. There may be kinesthetic disturbances leading to difficulty in ascertaining the location of the arms or legs. Accompanying these sensations are various physical changes, such as mydriasis, drop in pulse rate, and reddening of the face. The subjective sensations experienced are illustrated by the following quotation from Mitchell (2). After taking six buttons he "soon became aware of a violet haze, and also had the subjective sensation of an increased competence in 4. dealing with problems, which was not born out in pra.ctice 11 • On lying in a darkened room, he saw 11 delicate floating films of color •.••• zig-zag lines of very bright colors, in r apid motion ••••• I began to see for the first time definite objects associated with colors. A white spear of grey stone grew up to huge height, and became a tall Gothic tower of elaborate design ..... covered with clusters of what se emed to be huge precious stones. and orange. Th ese were gr een, purple, red, All the colors I have ever beheld are dull as coraoared with these ..... the vas t pendant masses of emeraid green, ruby red, a.nd arange began to drip a slow rain of color 11 • A series of visions followed, until the effects of the drug had. disappeared in a fe w !nore hours. In some de- scriptions it is stated tha t sensory and auditory stimuli often have a n effect on the intensity an d nature of the visions produced. Extensive studies on the 11 mescal p sychosis 11 are de- scribed by Berin ge r (6), Rouh ier (7), and Kl~ver (8). The three main constituents of the psychosis seem to be (1) sens ory abnormalities, (2 ) a cha nge in conscious a ttitude and coridition, an d (3) emotional abnormalities. Actions of mescal alkaloids on man ------ -Mescaline. The subjective sensations of mescal in- toxication in man are fairly closely duplic a ted by the action of the chief alkaloid, mescaline, at doses of about 200 mgm. or larger. Inasmuch as it is customary to take about five buttons to obtain the typical hallucinations, -- 5. and as about 20 0 mg m. of mescaline is p resent in this number of the buttons, it is probable that mescaline is respo nsible for the major portion of the acti on. Mesc a line has been studied quite intensively, particularly in re gard to t h e g eneration of v a rious p s y chotic state s. Its use in p sychi a try (Stockin g s (9)) is based on its ability to reproduce in a norma l subject phenomena closely related to those of certain p sychopathol◊gical st a tes (esp ecially sch izophrenia), and also upon the fact tha t these cha n ge s occur in a st a te of clear consciousness, enab li ng the subject to describe t h e effects to a n observer. Some of the sens a, ti ons experienced by schizop hrenics are k nown fr om thei~ dr aw in g s and t h e d escriptions g iven by the m c1,ft e r t h e administration of so d ium amy t a l, which often produces a t empor a ry lucid pe riod in a pa tient who is ordinarily uncomm unicative. Th e hallucinations resultin g from me sca line (visua l, a uditor y i and somatic) a re very si milar to t ho se described by these patients. The dissoci a tion of the p e rs on a lit y whi ch is t h e mo st c har a ct e ristic fe a ture of sc h izop h reni a is often e xp erienced by ;J ersons who have t a ken mescaline. Othe r p oints of similarity include t h e c a t a - toni a p roduced, disturbances of mood ass oci a t ed with the feelin g of unreality, difficulties in carrying out a lo g ical tr a in of t ho u ght, a n d other manifestations. Th e mecha nis m of a cti on c f mes c a line is un kn own. From t h e work of Quas tel a nd Whe a tle y (1 0 ) on brain r esp iration, it is known t ha t me sca line, as well a s a numb e r of rela ted 6. amines (t yramine , p h enethylamine, a nd others) and t yp ic a l narcotics, has the p r ope rt y of inhib itin g the oxidation of glucose, l a ct a te, and pyruv a te. Richt er (11) found tha t 58% of the mescaline fed to man c ould be r e covered in t he urine, and t ha t t he remainder was una cc oun t ed for . I t is unlikely t hat am ine oxidase is re sponsib le for any appreci ab l e destruction of me scaline, for Blaschke , Richt er, a.nd Schloss mann ( 12 ) found that it was a tt a cked only very sli gh tly in the p re se nce of this enzyme. Sl ot ta a nd Miiller (13) f ound t ha t 50% of t he mesc a line fe d to guinea p i gs and dogs c ou ld be recov ered fr om t h e urine as 3,4,5-trimethoxy- phenylacetic a cid, but none of t his compound coul d be isolated in human experiments. Mention is made in the literature of some visual disturbances with some of t he other alkaloids, a lthough their actions ha,/e not been inv e sti ga ted in t his respect at al l t horoughly. Heffter (14) rep orted the followin g effects after t aking several of t he other alkaloi ds by mouth : Anhalo!}ine (100 mgm . dose) - slight na rcosis. It is reported tha t lassitude is expe rienced, toe-ether with a sens a ti on of g liding . Loohe>ph orine (20 mgm . dose ) - he ada che , reddening of the f a ce. Anhalonidine (100- 250 mgm. dose) - sli gh t nar cosis. Pellotine (80- 240 mgm aver- . dose) - na rcosis, fatigue, si on to mental an d phys ic a l l ab or . Pell otine has been studie d more t h orough ly than a ny of the ot her a l ka loids with t h e 7. exception of mescaline, particularly by Robles and Robleda (15). They reported that it caus es men t a l confusion, visual hallucinations without the production of color, auditory and kinesthetic hallucinations, and disturbances of concepts of space and time. In doses of 15-30 mgm. it was formerly used clinic a lly as a narcotic (Hutchings (16)). There are no data a vailab le on the actions of t he other known a l ka loids of mes ca l on man. Pharma cologic a l a ctions of me sc a l alkaloids The pharmacological effects of the various alkaloids on animals have been studied very little, with the exce p tim of mescaline and pellotine, and even in t he case of the la~ ter compounds it is not p os s ible at the present time to draw many conclusions from the data which have been obt a in~. Mescaline. Grace (17) made a careful study on sever~ animals of the pharmacological actions of mescaline, and some of his findings are reported below: (1) Letha lity. The letha l dose in fro gs by injection into the dorsal lymph sac is 750 mgm. pe r kilogram body wei ght, and in mice by intrap eritone a l injection is 500-600 mgm. per kilogram. The symptoms observed in fro gs were pro- gressive motor par a lysis, arrest of resp iration, and abolition of reflexes. Death was due to respiratory failure, and the main action seemed to be depre ssion of the central nervous system. These results agree with those of Heffter on fro gs , but a re opposed to those of Dixon (18), who st ate s that mescaline pos sesses i strychnine-like convulsant action. 8. (2) Action on voluntary muscle. Mescaline at a con- centration of 1:500 paralyzed the isolated fro g gastrocnemius to electrical sti mul a tion. (3) Circulation and resp ira tion. (a) Ten mgm. per kilogram of mescaline given intrav~nously to a ca t a nesthetized with ether-chloroform resulted in a blood pressure fall from 110 to 50 mm. Hg, which was aocomp anied by slowing of the resp iration. The pulse r a te was unchan ged, although the strength of the beat was lessened. This a ction on the blood pressure and t he respir a tion appe ars to be of a central rather than a peripher a l nature, for if the vagi are sectioned, a sli ght rise in blood pressure is observed, and the resp ira tion is unchange d. (b) The action of mescaline on t he decerebrate cat is similar to the action on an anesthetized animal. (c) Ten mgm. per kilogram of mescaline given intravenously to a dec ap itate cat c a used a rise in blood pressure of 60 mm. Hg. This pressor action is not affected when the gang lia are paralyzed by nicotine. (4) Action on the isola ted h~art. In low concen- tration (1:50,000), mescaline caused a slight decrease in the amplitude of the beat, but no change in the rate. In high concentration ( 1: l00Q), there was a p ronounced decrease in the amplitude, accompanied by a decrease in rate, and the heart stopped in diastole. This action seems to be directly on the cardiac muscle, for a tropine was without effect. The action on t he pe rfused ra.bbit heart was of the s ame nature 9. at similar concentra tions. (5) Action on j_nvoluntary muscle. (a) The is ol ated int es tine of the rabbi~ and the cat were not a ffected by concentra tions from 1:100 ,000 to 1:2500 . However, the intestine of the intact animal was strongly stimulated by the intra venous injection of 11 mg~ oer kilogram, and the contraction was no lon~e r obtained 0 ~ after an injection of a trop ine. (b) The isolated uterus was not affe cted, while the uterus in situ was strongly stimulated. Raymond-Hamet (19) studied the a ctions of mescaline on do gs under chloralose anesthesia. Artifici a l resp irati on was given to these animals, a nd both vag i were cut. He found that v1i t h doses of 2-8 mgm. per kilogram a sli gh t fall or rise in blood pres sure occurred, but that with doses of 20 mgm. per kilogram a fall was always observed, a ccompanied by slowing of t he heart . Th is a ction is opposed to the ob- s erva tions of Gr a ce, which were obtained with different animals a nd anes thetics. In addition, Raymon d-Hamet showed t ha t small doses (2-4 mgm . per kilogram) inhib ited the intestine in situ, and t ha t l arge r doses first stimulated, and t hen depressed the tonus, si u ilar l y to nicotine. However, me sc a line still pro du ce d t his effect after a large dose of s parteine, which abolishes the nicotine ganglionic stimulation, and therefore Raymond-Ha met conclude d tha t mes ca line Ects directly on t he r.1uscle. Hou ev e r, Gra ce f ound t ha t mesca line di d not produce sti mulation of t he int es ti ne 10. after the in,jection of a tropine, so it appears t ha t · some other mechanism must be involved. The f a ll in b lood pres- sure, · slowing of t h e heart, and stimulation of the intestine indicate tha t mescaline does not have appreciable sympathomimetic a ctivity. Clerc, Paris, a nd J a not (20) worked with dogs anesthetized with chlora losane, and also observed a blood pressure f a ll, which wa s less pronounced a ft e r the injection of atrop ine or section of the vagi. They n oted a n increase in both the r a te a nd a mplitude of resp ira tory move ments, which i s contradictory to the re s ults obta ined by Grace on cats under ch loroform-ether a nesthesia. It is difficult at present to suggest a ny mecha nism that will s a tisf a ctorily expla in t l'ie va rious a cti ons of mesca line observed by t hese a ut hors, a lthough t h e centra l nervous s ys te m depression is probably rela ted to t he inhibition of brain resp ira tion f ound by Quastel a nd Whe a tle y (10). Wit h re gard to t h e un i qu e a ction of mesc a line in 9roducing color hallucina tions, it s eem s likely t hat t h e mo st fruitful me t ho d of app roach to the proble m would be through objective obse r va tions on man, rather t han atte mp tin g to study the proble m by the an a l y sis of sub jective sensations or by animal experimenta tion. Pellotine. Heffter (14) observed that in the frog pellotine first produced na rcosis a nd diminished reflex activity. In about one h our the reflex activity incre a sed, a nd convulsive breathing an d tet a nus occurred, which were 11. sometimes still present twelve hours later. The toxicity for a number of animals was determined by Robles a nd Robleda (15), and the lethal dose in mgm. of pellotine hydrochloride per kilogram of body wei ght was f ound to be as follows: fro g (500), r a t (500), guinea pig (300 ), cat (150), and pigeon (100). The p igeon, which is very sensitive to the action of p ellotine, showed incoordination, loss of equilibrium, rap id breathing, mydriasis, and finally convulsions preceding death. Clerc, Janot, and Pa ris (21) found that in the rabbit anesthetized with chloral, a dose of 5 mgm. per kilogram of pellotine hyd~ochloride lowered the blood pressure by as much as 80 mm . Hg , a nd at the s ame time slowed the he a rt, and tha t this a ction wa s abolished by sections of the vagi. They also noted a hyp ergl yce mic a ction in rabbits, with a rise in blood sugar of as much as 50%. Other alkaloids. Very little work has been done on t he phar macology of the other alkaloids of mesc a l buttons. Heffter (14) performed some preli mimar y expe riments, as did Dixon (18 ), but the latt e r's work is of doubtful value, bec a use severa l of the comp ounds used by h i m were obviously imp ure. Mogilewa ( 22 ) investigated t he actions on the p er- fused intact fro g heart. (1) Anhalonine: Heffter showed that a dose of 10 mgm. of anhalonine hydrochloride in t he fro g c a used central paralysis, which disapp eare d c1-f ter ·about one hour, when a condition of incre ased reflex a ctivity began to app e ar. 12. This latter effect often persisted for 24 hours, and sometimes for several days. (2) Anhalonidine. The perfused frog heart is slowed. The actions of anhalonidine seem to be similar to those of anhalonine, and in addition it is reported to cause paralysis of motor nerve endings. ( 3) Lopho-o horine. This alkaloid does not show the paralyzing action of anhalonine and anhalonidine, but causes an immediate increase in reflex excitability in frogs in doses of 0.25-1.0 mgm., which persists for many hours. The frog heart is also slowed by lophophorine. (4) Anhalamine. Anhalamine produces in the frog a preliminary narcosis followed by increased reflex excitability, similar to that observed with several of the other alkaloids. No mention is made in the literature of pharmacological work done on any of the other alkaloids of mescal buttons, which have been isolated more recently. 13. Chemistry of mescal a lkaloids At the present time eleven alkaloids have been isolated from mescal buttons, and the structures of all of them are known (see table on page 15). Th is table indicates the close structural relationships amongst these compounds, and it is possible tha t some of the mi ssing me mbers of these series may later be found to occur in small amounts in the buttons. The theory (Robinson (23), Hahn and Rumpf (24), and Sch6pf and Bayer le (25)) that isoquinolines a re formed in p lants through ring-closure of the corresponding phenethylamines would suggest the prese nce, in small amounts at le as t, of 3-methoxy-4,5-methylenedioxyphenethylamine and 3,4-dimethoxy-5-hydroxyphenethylamine. The references to the isola tion of the alkaloids a re as follows, in the order of their discovery, from 1888 to 1939: anhalonine (Lewin (26)), pellotine (Heffter (27)), mescaline, anhalonidine, and lophophorine (Heffter (28)), anhalamine (Kauder (29)), anhalinine (Spat h and Becke (30)), anhalidine (Sp&th an d Bec ke (31)), N-methylmescaline (Spith and Bruck (32)), N- a cetylmescaline (Spath and Bruck (33)), and 0-methyl-d-anha lonidine (Sp~th and Bruc k (34)). An- halonine, lophophorine, and 0-methyl-d-anha lonidine occur in the optically active forms, while pellotine and anhalonidine, which also possess an asymmetric carbon atom, are isolated in the racemic form. The amounts of the a.lkaloids found in the dried buttons vary widely, according to the different investigators. 14. Kauder (29) reported: mescaline (0.gfa), anhalonine (0. 25% ), lophoph orine (0. 25% ), p ellotine (0.2%), anhalonidine (0.2~; ), and anhalamine (small amount). When Spa th first worked on t he isolation of the a.lkaloids (35), he obtained: total non- phe nolic bases (0. 21~), includin12: me scaline (0.1~,~ ), lophophorine (0.00155~ ), and anhalinine (0.0075%); total phenolic bases (0.4%), includin g anha l am ine (0.1%), an d anhalidine (O.OOl~b ). The yields of the ot he r alkalo ids are not given. In his later work (3 2 ) h e obtained what are des cribed as much fresher buttons, and was ab le to increase the yields t en ti mes over those of the older ma teri a l. The only fig- ures he gives are for mesc a line (1. 25% ), a nha lonine (0.35'%,), and N-methylmescaline (0.025% ). The yi elds obtained in the present work. from mes cal buttons which were several years old were : mesca,line (0. 85% ), anhalonine (0.12%), loph- ophorine (0.07%), pellotine (0.15%), an d anhalonidine (0.02% ). Rouhier (7) also st a tes tha t the proportion and amounts of the a l ka loids v: 1-ry b oth seasonally a,nd environmentally, and th is is of c ourse par tiall y resp ons ible for the gre a t vari at ion in t he phy siolog ica l responses to t he crude drug . Anha line, which was isol a ted from the closely related c a ctus Anhalonium fissur a tum by Heffter (27), has not been found in me scal buttons. It wa s shown by Sps,th (47) to be identical with horden ine, isolated from barley malt ge rms, and p ossessing the formula 4-hydroxyphenethyldimethylamine. The present wor k was undertaken with the view to - - ..., -· .M'ETHiJXY-MET//YL E /VE - 0/MET/.IOXY.:/-/YO,~OXV TR/MET/-IOXY 0/0XY-COA-JPOUNas COMPOl.h'Vi:JS ....- '""" COA4'POUN£JS C:'//2 c100~ 'f/lz c40 ~ c~o N~ me5cavhe 'r~ L/1 /t;O(YC/lz y C,½0 ~ I N'/IC~ C,¼O /[/,mc!'/hy./ hfesca/;ne ~o(r c.1/z. \ "c,½ Ci, C/60 ~ I ¢'0 /V#C-c~ ~o #ace6,/ ,mescabne 11 ckoo:c r.. 3 ~ "c~ . C,¼ 0 "'-- c~o (J(I'~/{ C/efO ~ CJII //0 c~o-'½. _/V#' ~z a a /4"7 /a /7l / n e a/'lha/n-2✓ne J-c~ CdO((h'L 'J -!If c:40 ">.. \ HO c hz an.lt"'.a /;dne c~o~c1~ fXOo:~//, I C"/io '.3 c."' ~ c~o Jv; 0 ~ I , an/2a lo ,udu-z e I ,..N'-C~ "f~ o ~ c~o « '.3 fizc . . 0 i C(# /-anha/o,,;-vne c/1,o~c,½, ~ _).1// /lo C. '3 'C-./ne,1(>/.d anAa/._,,,_,~-: dt,--z e c40 ~ I ,Al'# C# ';¼ C. '3 t½C-O ,~'# C/i{O(Kc~,½ C/60 Ch' ,~ //0 C '3 /-/90/2C7?/2or/ne ~-I i c~ cl½: '----cw;.z:. ,,k-c~ . C[H c,¾ /'e //c /✓.ri e j 16. preparing a number of the mesc a l a lkaloids a nd rela ted substances in order to be able to make a study of the physiological properties of the compounds. The comp lete series of the tri @ethoxy-substituted compounds was p repared, including N-dimethylmescaline. Homomyristicylamine (3-methoxy-4,5- methylenedioxyphenethylamine) has been synthesized by a new method, a nd mesc a line, a nhalonine, lophop horine, p ellotine, and anhalonidine have been isolated from the mesc a l buttons in sufficient qua ntities for p reliminary study. The action of a mine oxidase 1 on a number of these compounds was investi ga ted. N-dimethylmesc a line has not been found in Lophop hora . - Ludue ~a (36) states that the cactus a lkaloid trichocereine from Trichocereus terscheki has been identified as N-dimethylmescaline, but no chemic a l da ta on this substance could be found. Gug genheim (37) refers to a publication by Reti (38) in which it is stated that Trichocereus terscheki contains on a dry weight basis 0.05% mescaline and 0.2% trichocereine. Unfortunately this paper could not be obtained at the present time. This compound h a s been synthesized in the present work, and the two N-methyl-tetrahydroisoquinolines of the 6,7,8-tri methoxy series have been prepared. Homomyristicylamine has been prepared by Salway (39), Decker and Becker (40), S p~th and Gangl (41), Sp~th and Kesztler (42), and Hahn and Schales (43). Their methods are re p orted to g ive moderate yields, but were not considered l This work was made possible through the kindness of Dr. G.A. Alles. 17. suitable for the convenient p re paration of lar g e quantities of the compound, and therefore the homoamine synthesis of Julian and Stur g is (44 ), which was known to g ive g ood results when verat raldehyde or p i p eronal were used as intermediates, was app lied using 3-methoxy-4,5-methylenedioxybenzaldehyde. Th is synthesis p roceded by the followin g reactions: <ll,;ocl-1,-CN CH2.~0 The nitrile p re pared in this manner was reduced a t high hydrogen p ressure, in the p resence of met hano lic ammon ia in order to d ecre a se formation of seconda ry amine. Sp ath and Gangl (41), and Sp at h a nd Kesztler (42 ) have reported the synthesis of dl-anhalonine by ring closure of N-acetylhomomyristicylamine with P 2 o 5 , followed by reduction of the 3,4-dihydroisoquinoline. They obtained a dihydroisoquinoline which was apparently entirely the desired 6-methoxy-7,8-methylenedioxy compo und. However, in this v ork a mixture of this substance a nd t h e isomeric 6, 7- methylenedioxy-8- me thoxy comp ound was obt a ine d , which co ul d not be separa ted, s o the hydrogenation was conducted 18. on the mixture. By fractional crystallization of the hydro- chloride of the reaction product it was possible to obtain pure dl-anhalonine. The p reparation of 3,4-dimethoxy-5-hydroxyphenethylamine requires 3,4-dimethoxy-5-hydroxybenzaldehyde as an intermediate, and an attempt was made to develop a method of preparing this substance in the large quantities necessary for subsequent syntheses. Spath and Rader (45) attempt- ed its preparation by the Sn-HCl reduction of 5-nitroveratraldehyde, and subsequent diazotization of the amino aldehyde and decomposition of the diazonium salt to the ohenolic comp ound. However, the only substance which they could isolate from the reaction mixture was 3-methoxy-4, 5-dihydroxybenzaldehyde. They succeeded in preparing the desired compound by the rather laborious method of separating the various products resulting from the methylation of gallie acid, and in this way accumulated a sufficient quantity of 3,4-dimethoxy-5-hydroxybenzoic acid to p repare the corresponding aldehyde through the Rosenmund reduction of the acid chloride. The only other preparation of the aldehyde is that given by Llauthner (46), who used a similar method~ It was thought that the compound mi ght be prepared in large amounts conveniently by the following series of reactions, where the aldehyde group is bloc.ked during the hydrogenation by for ming the dimethylacetal: 19. The reactions ap parently proceeded satisfactorily up to the decomposition of the diazonium salt formed in the presence of either HCl or H2 so 4 , but none of the desired compound could be isolated from the reaction mixture after the decomposition of the diazonium salt. However, when HCl was used in the diazotisation and Cuso 4 solution in the decomposition, 3,4-dimethoxy-5-chloro-benzaldehyde could be isolated in good yield. 21. solution with 50% aqueous KOH to separa te the non-phenolic bases from the phenolic bases. When this was tried it was found to be impossible to isolate appreciable quantities of the phenolic compounds. Titra tion experiments were carried out to investi ga te this point, and it was found tha t about 50% of the ba sic material was removed fro m the ether by one shaking with 50% KOH solution. When the KOH fraction was neutralized with HCl, followed by the addition of 2% K2C0 3 , and extracted with ether, only about 10% of this phenolic fraction could be recovered. In a preliminary experiment using 25% KOH solution, 85% of t he phenolic fraction could be recovered. When this method was applied to the large batch, only 50% of the phenolic bases could be recovered by ether extraction, and extraction with several other solvents did not increase the yield of alkaloids obtained from this fraction. 0.34 equivalents of base were present in the ether (Fraction A) a fter shaking with 25% KOH solution, representing 52% of the original basic fraction. 0.14 equivalents of base could be recovered fro m the 25% KOH solution by ether extraction after neutralizing with HCl and making a l .k aline with ~co 3 . 'rhis represents 45% of the ba sic ma terial which had been removed by the alkali tre a tment, a nd it was worked up separ EJ_tely as vJill be described l a ter (Fra ction B). It was though t p ossib le tha t t he KOH solution had resulted in the re a ction of the phenolic compounds with some other substance presen t in the extr a ct, giving a 22. product which was insolub le in ether. Therefore, the resi- due from t he eth er ext ra ction of the 25% KOH s olution was neutralized a nd mad e 2N with HCl, and refluxed 4 hours. ~co 3 was t hen added until the solution ws,s allrn,line, and another ether extra ction was carried out. In t h is way 0.046 equivalents of base were obtained (Fraction C), which re presents 16% of the tot a l p henolic base fraction. Isol a tion of non-phenolic bases from fraction A The ether solution of fraction A was concentrated to abo ut 1 liter and the basic material was extracted by repeated shakings with 2% acetic 2cid. The acetic acid solu- tion was made a lkaline with K2 co 3 and extracted with ether. 0.29 equivalents of base were recovered, representin g 85% of the original non-phenolic fraction. Isolation of mescaline sulfate. The ether was removed at 20 mm. Hg, and an oily brown residue was obtained, which was dissolved in 250 cc. of 95% EtOH, and a 10% solution of H2 S04 in EtOH was added to a sli ght acidity to litmus. A heavy precipitate was obtained, which was dried over CaCl2, and weighed 70 gm. It was recrystallized from 220 cc. of boiling water, the crystals filtered at 5° C., and washed with 95% EtOH. The mother liquor from the recrystallization and the wash solution were added to the original. The crys- tals wei ghed 41.0 gm., and possessed m.p. 186-9° C. (All melting po ints reported were taken on a Fisher hot-stage apparatus, an d are corrected.) 6°0. for mescaline sulfate. Spath (47) gives m.p. 183- The picrate was prepared from 23. equivalent amounts of mescaline sulfate and picric acid in water, and melted at 217-8°0. Spath gives m.p. 216-8°0. The hydrochloride was prepared from an aqueous solution of the sulfate by decomposition with excess NaOH, followed by benzene extraction. The benzene was removed at 20 mm. Hg, the base neutralized with methanolic HCl, and precipitated with absolute ether. 183-4°0. The white hydrochloride melted at Hahn and Wassmuth (48) give m.p. 182°0. for mes- caline hydrochloride. It was recrystallized from absolute ether•-IdeOH ( 2: 1), and dried at room temperR.ture over P20 5 . C H Calculated for C1iH18 0 3 NCl 53.33 7.32 Found 53.48 7.35 Isolation of anhalonine. Anhalonine was isolated from the filtrate from the mescaline sulfate precipitation in the fallowing manner: the alcorwl was removed in vacuo, 2% K2 co 3 added, and the solution extracted with ether. 0.063 equivalents of base were obtained. -- --- The ether was r~ moved in vacuo, and an oil weighing 15 gm. was obtained. - - After the addition of 50 cc. of 2N HCl, a crystalline precipitate wa s i mmediately formed, which was filtered at 5°C., washed with 5% HCl and dried. Five and one-half grams of t h is crude s a lt was obtained, and it was purified by liber~ing the free base, extracting with ether, and distilling. The base distilled at 144-6°0./0.07 m~. Hg, and the distillate crystallized at once, with m.p. 84-5.5°0. Kesztler (42) give m.p. 84-5.5°0. Sp~th and The quaternary iodide 24 . wa s pre par ed. a ccordi ng t o Spat h a nd Ga ngl (41), recrystallized from water containing a little Na I and then fro m MeOH. It melted a t 222-4°0., then solidified, and remelted a t 23841°0., as described by Splt h an d Gangl. Anha lonine hydro- chloride wa,s prepared from the free b as e in absolute ether b y fla ssing in a nhydrous HOl gas . It melted a t 256-9°0. (d.), and by recryst a lliza tion from MeOH needles were obte.ined, which were dried at roo m te mp erature over P 2 o5 . C H Ca lcula ted f or 012H16 o3 N01 55.92 6.26 Found 55.87 6.37 Optical rotation of a nha lonine: c = 2 . 6 (MeOH, 1 dm. tube) _yJ•c.. [oe_, = 100(-1.68) = -64.6 0 ~ 2.6 Spath a,nd Kesztler (42 ): c = 2.62 (MeOH, ldm. tube). [o<:JU't = - I, f: IO S> Isol a tion of loohoohorine hydroch lori de. The fil- tra te fr om the a nhaloni ne hydrochloride p recipit a tion was made a,l kal ine wi t h K2C0 3 and extracted with ether (0.013 e quiv a lents of base). Th e ether was removed in va cuo, a nd the residue taken up dilute HOl. This solution was de- c oloriz ed with 100 mgm . of Llerc k an i ma l cha rcoa l an d concentr ated in a de sicc a tor, until white cry s t a ls formed, wh ic h were filtered a nd washed with 95% EtOH. Th e fil- tr a te was concentra ted furt her to a syrup , wh ich was dis- 25. solved in hot 95% Et0H, and on cooling this solution 1.4 gm. of white crystals .. (needles) were dep osited. These two crys- tal fractions were combined, and wei ghed 3.2 gm. The pie- rate was p repared from equivalent amounts of the hydrochloride a nd p icric acid in hot absolute Et0H, and possessed m.p. 161-2.5°0. Spath and Kesztler (42) give 162-3°0. as the melting p oint of lophophorine picra te. The hydrochlor- ide was decomp osed with Na0H solution, the base extracted with ether and distilled after the removal of the ether in · vacuo. A colorless oil was obtained at 120-5°0./0.05 mm. Hg. The quaternary iodide was prepared in the s a me manner as anha lonine methiodide. After recrystallization from Lle0H, it melted p artially a t 225°0., and completely at 23840°0. Spith and Gangl (41) give p artial melting at 223°0., and comp lete melting at 239-41°0. The hydrochloride was prepared from the base with methanolic H0l, and precipitated with ether. It was re- crystallized from methyl ethyl ketone for analysis, a nd dried a t roo m temperature over P 2 o5 . Calculated for c13H190 3 NC1 Found C H Methoxyl 57.45 6.68 11.42 57.96 6.67 11.11 Optical rotation of lophoohorine hydrochloride: c = 4.2 (water, 1 dm. tube) Lo<J= 100 (-0.62) = -14.s 0 ~oC. " 4.2 26. Splth and Kesztler (42 ): c = 4.425 (wa ter, 1 dm. tube) No further cryst a lline ma t e ri a l could be obtained fro m the non- phenolic base fr a ction. Is ola tion of pe llotine hydrochloride from fracti on B. After re moval of the et h er from fraction B, a bl a ck oily residue wa.s obtained. Excess aqueous HCl was then ad- ded, an d t h e solution was k e p t a t o0 c. for 3 days. A s ma ll amount of non-basic material 9 recipit a ted a nd was filtered off. Th e f il trc1, te we.s made 0, l ka line with ¾C0 3 , a resinous precipitate filtered, and the filtr a te extr a cted wit h ether (0.046 e q uivalents b a se). The ether wa s re moved in vacuo, t h e residue dissolved in 150 cc. of dilute HCl, an d after decolorization with Me rc k a ni ma l charcoal the solution was evapor a t ed t o dryness in a desiccator ov e r KOH. Th e residue was tre c1,ted with absolute EtOH, a,nd a white insoluble residue wa,s obtained 1Hh ich weighed 7.3 gm, and wa s shown to be pellotine hydrochloride. The picrate was pre pared from equivalent amounts of the hydrochloride and picric a cid in water. rated, which partially cryst a llized a t o0 c. An oil se paAfter recrys- t a lliz a tion of this ma t er i a l from wa ter, it o ossessed m.p. 1 67- 8 °0. Su& t h and Becke ( 3 5) g ive t h e melting point of p ellotine n icra te as 165-7°0. Th e free base wa s p re par e d by extraction with ether of a n aqueous solution of the hydrochlori d e which was made 27. al kaline with NH 4 0H, and after removal of the ether the base was distilled. The fraction boiling at 135-40°0./ 0.06 mm. Hg wa s collected, and this fraction crystallized i mmedi a tely a nd showed m.p. 111-2°0. Sp&th a nd Becke (49 ) give t he melting po int of p ellotine as 110-2°0. The methi odide was prepared with methyl iodide in MeOH, and the re a ction mixture a llowed to sta nd at r o om te mp era ture for t wo da ys. The lvleOH and methyl iodide were re moved in vacuo and the residue, after recrystallization fro m MeOH, p ossessed a melting p oint of 197-98°0. The mel~ ing po int given by Ka uder (29) for p e l lotine methiodide is 198°0. Pellotine hydrochl oride was prepared from the base diss olved in ether b y pass i ng HCl ga s thr ough t he solution, After filtr a ti on, it was recr y st a llized from MeOH-ether (1:3) f or a nalysis, and dried a t room temp era ture over Found C H 57.01 7.36 57.17 No further cryst a lli ne subst an ces could be isolated fro m t h is fraction. Isolation of Anhalonidine fr om fr a ction C The ether wa s re moved fr om fr a ction C (0.046 equivalents) in va cuo, a nd t he bla ck oily residue wa s dissolved in 50 cc. of ho t 2N HCl. On cooling , 8 .7 gm . of non-cr ys- . t a lline ma, teri a l separ a ted, wh ich was diss olved in 25 cc. 28. of 0.5N HCl. This solution was decolorized with Me rck ani~ mal charcoal, concentrated, and cooled. In this way 3.7 gm. of i m~u re hydrochloride was obtained, whic h was dissolved in water an d the base extra cted with ether after making the solution alkaline with K2C03, Th e ether was removed in vacuo, and the residue subJected to slow sub limation a t a ba th te mperature of 120-60°C./0.05 mm. Hg . A fraction was obt a ined melting a t 145-55°0., which was extracted t wice with boiling absolute ether, and the residue, which weighed 220 mgm. then melted a t 15 9-61°0. According to Sp~ th and Becke (35), anhal- onidine melts a t 157- 6 1°0. The picrate wa s p re pared from equivalent amounts of t h e hydrochloride an d p icric a cid in water, a nd a fter recryst a llization from water p ossessed m.~. 200-2°0. Sp lth (50) gives m.p. 201-8°0. Anhalonidine hydrochloride was 9 repared from the base with methanolic HCl, p reci p itated with ether, and recryst a llized for a n a lysis from MeOH-ether (1:2). C H Ca.lculated for C1 2 H190 3 NC1 55.49 6.98 Found 55.69 7.14 Only a small amount of b a sic material was present in the filtrate from the anhalonidine hydrochloride precipitation, and no other p ure subst an ces could be isolated from t h is fr a ction. 29. II - SYNTHETIC WORK - N-methylmesc a line (Sp~th and Bruck (32)) To 4.6 gm. of freshly distilled mesc a line was ad ded 2. 22 gm . of b enzaldehyde, and the solution warmed on the water bath for a few minutes. Th e water formed was rem oved at 50°0. in vacuo, 3. 2 gm . of methyl iodide added, and the solution a ll owe d to st and for 19 h ours a t room tempera ture and then he a ted a t 60-70°0. f or 9 hours. Th e viscous g l a ss y p r oduct was dissolved in 40 cc. of MeOH, 5 cc. of wa ter a dded, and t h e solution r e fluxed for 15 minutes. Fifty cubic centi meters of water was t h en a dd ed, t h e MeOH a nd benzaldeh yde were boiled of f, t h e soluti on made a cid with acetic acid a nd extracted with ether. Th e acid s oluti on wa s t h en mad e a l ka line with NaOH s olution, extra cted with ether, a nd t h e p roducted distilled af ter re mov a l of t h e ether. N- me thylc esc a li n e distilled a t 130-40°0./1.0 mm . Hg , and t h e colorles s oil obtained wei gh e d 2.9 gm . Th e hydroc h l ori d e was 9 rep a red in abso lute ether with HOl ga s, a nd me lted a t 187- 8° 0. After recryst a lliza tion fr om a cetone-MeOH (9:1), it melted a t 201-201.5°0. Sp~ t h and Bruc k (32 ) g ive m.p. 201- 2 °0. Th e pi cra te wa s p re pare d from t h e hydroc h loride in wa ter, a nd p ossessed m.p. 174-6°0. N-di methyl~esc~line An unsuccessful a tte mp t was mad e to p repare the compo und fro m N-methylmescaline, usin g a n e q uiv a lent amount of 30. methyl i odide, but the qu~ ternary s a lt was formed chiefly, le aving c onsiderab le unre a cted N- me t hy lmesc a li n e. Th e f ol- l owing method of nre~ a r a ti on was found to g ive satisfactory results: 3. 0 gm . of N-methylmescaline was dissolved in 5 cc. of ab solute Et OH, and l. 1 cc. of 4(Y;; for ma l d ehyde and 40 mgm. of Adams c a t a l ys t (Pt02 ·H20 ) were a d ded. Th is mixture was reduced with hydro ge n in a P aar bomb e, t room te mp er a ture ard 42 pound s p ressure, f or 2 hours. Th e c a t a l y st was filtered off, t b e MeOH re moved in vacuo, and water ao.ded. Th e solu- tion wa s maae alka line and extrac ted with ether, a nd distille d a fter re mova l of t he ether. One a nd one-ha lf g r am s of N-6i ~ ethy l mes c a l in e was ob t a ined a t 1 28-31 8 0./ 0 . 6 mm . Hg~ Th e hyd roc h l oride was ~re par ed with HCl gas in ether, recr ys t a l l ize d from a cet on e-MeOH ( 9 :1), an d had m.p. 2 08-9°0. A mixed melti ng p oint d etermin a ti on with 2-methyl-6,7,8- tri ~ ethoxy-1,2,3,4-tetrahydroisoquinoline hydroc h loride, which melts a t 2 16- 8 °0., gave a melting do int of 175- 85°0. A sample was dried at room temperature over P 2 0 5 for analysis. C Calcula ted for C:13 H22 o3 NC1 56.60 Found 56.71 H Th e p icrate wa s pr e pared fro m t h e base in 95% EtOH by addin g an e qu iv a lent amount of p icric a cid in water, and a~ ter recrystallization fro m 95% EtOH, it melted at 171-2°0. It was dried at so 0 c. over P 2 0 5 for 2 4 hours. C H Calculate d for C19 H24 o10N 4 4 8 .7 2 5.16 Fo und 48 .77 5.13 31. 6,7,8-tri met hoxy -1, 2 ,3,4-tetrahydroisoauinoline (Anhalinine) (Spath (51)) To 13.5 gm . of freshly distilled mescaline was added 15 cc. of water and 7.0 cc. of 40fo for ma ldehyde with shaking. The sol u tion was heated for 1 hour on the water bath, and then t h e separated aq ueous l ay er was re moved, and t h e residue washed with 20 cc. of water. Th e white solid p roduct was dissolved in 95 cc. of hot 2N HCl, and allowed to cryst a llize. After recrystallization fro m 6N HCl, 7.0 gm. of the hydrochloride was obta ined, which melt ed in an evacuated tube at 247- 9 °0. A s ampl e was dr ied at so 0 c. over P 2 o5 for 24 hours for analysis. C H Calcula ted for C12 H18 o3 NC1 55.49 6.98 Found 55.65 6.81 Th e p icra te was p re p ared a ccording to Sp ith, and me lted a t 1 83-4°0. 2 - me thyl-6,7,8-tri methoxy-1,2,3,4-tetrahydroisoquinoline To 3.6 gm . of fres hly distilled 6 , 7 , 8-trimeth oxy-l,2, 3,4-tetrahydroisoquinoline in a tube were add ed 2 .2 cc. of 40o/~ for maldehyde and 1. 2 4 cc. o f 9 E3)i for mic acid. Th ere wa s an i mm ediate evolu tion of ga s, and t h e s olution was he a t ed on t h e wa ter b a th for one -half hour , until ga s evoluti on h a. d ceased. The tube was then se a led a.nd h e a ted at 100°0. for 1 2 houi" s, a fter wh ich t h e re a ction p roduct was made a. l ka line with NaOH soluti on , and extra.cted with ether. Th e residue af ter the remov a l 123-6 ° 0. / 0. 06 mm. Hg . of t h e et h er distilled a t Th e hydroch loride was p repared in ether with HCl ga s, and recr y st a llized from a solution of 32. ethyl acetate-abs olute EtOH (1:1). This matericJ,l, which melted at 216-8°0., was dried at room temp erature over P 2 05. C H Calculated for C13 H2 oo 3 NC1 57.02 7.36 lt,ound 57.18 7.57 The p icrate wa s 9 re9 ared from the hydrochloride and an equivalent amount of p icric acid in EtOH-water (1:1), and recrystallized from MeOH-water (1:1). It possessed a meltin g point of 136-7.5°0., and was dried for analysis at so 0 c. over P205. C H Calculated for C19H 22 010N4 48.93 4.76 Found 49.66 4.72 l-methyl-6,7,8-trimethoxy-l,2,3,4-tetrahydroisoquinoline (Sp~th (51), and Sp ~th and Bruck (34)). To 17.6 gm. of mesc a line was added 10.0 .cc. of freshly distilled acetic an hydride, and this solution heated at 15060°0. for 1.5 hours. The v ol a tile material was removed at 150°0. at 20 mm. Hg , and a portion of the residue distilled at 185-90°0./0.05 mm. Hg. The distilla te crystallized im- medi a tely, and a fter recr y st a lliza tion from ether containing 10}~ ivi eOH the p roduct melted at 90-1°0. Th e re ma inder of the N-acetylmescalin~ (18.0 gm.) was dissolve d in 160 cc. 6 f a nhydr o us toluene, a nd 70 g m. of P 2 o5 ad ded in sma ll portions t o t h e g entl y refluxin g solution. Th e r eaction mixt u re w&s cooled, and a fter the a d- dition of wa ter wa s he a ted on t h e wa t er ba t h for 10 ~inutes, 33. and then a llowed to st a nd overnight. The toluene layer was washed twice with 50 cc. of 3N HOl, and the washings added to the aqueous solution. The latter was made alkaline with KOH solution, extra cted with ether, the ether removed in y~Q, a nd the residue distilled. The fraction boiling a t 135-40°0./0.02 mm. Hg was collected, and a light yellow oil was obtained, ~hich weighed 14.0 gm. The p icrate was p re- pared in benzene solution, and melted at 181-2°0. Spith (51) also gives 181-2°0. Reduction of the 3,4-dihydro-isoquinoline with granul a ted zinc in HOl solution containing a little CuS0 4 (Spath and Bruck (34)) gave a low yield of t he corresponding tetr~ hydroi soquinoline, and the ref ore a catalytic 1ae thod was adcp ted, which gave a quantita tive yield. F ourteen grams of the dihydroisoquinoline was dissolved in 75 cc. of MeOH, 42 mgm. of Adam s c a tal yst added, and the hydrogenation conducted at room te mpe r a ture an d 1 a tmosphere pressure. The t heor et- ic a l ar:i ount of hydrogen wa s taken up in 1 hour and 20 minutes. The cat a lyst was filt ered off, the LleOH removed a t 20 mm. Hg , a nd the hydrochloride prepared fro m the residue with metho.nolic HOl and p recipit a ted with absolute ether. After recrystallization fro m methyl ethyl ketone, it melted at 153-8°0., and wa s dried for analysis at room tempe rature ov er P 2 05. 0 H Calculated for 013H200 3N01 57.02 7.36 Found 56.93 7.14 34. The picrate was prepared from the free base in 95% EtOH, and precipita ted through the addition of an equal volume of water. After recrystallization fro m EtOH-water (1:1), the melting po int wa s 172-4°0. Sp£th (51) gives the melting point as 172-3°0. l,2-di methyl-6,7,8-trimethoxy-1,2,3,4-tetrahydroisoquinoline Four a nd one- ha lf gr am s of freshly distilled l-methyl6,7,8-trimeth oxy-l,2,3,4-tetrahydroisoquinoline was mixed with 1.45 cc. of 981~ formic acid a nd 2.6 cc. of 40% for ma l dehyd e in a tube. The solution was heated on the water ba th f or 1 h our until gas _evolution had ceased, a nd t h e tube was then se a led a nd he a ted for 13 hours a t 100°c. The re a.ction p roduct was made a lka li ne with NaOH solution, extracted with ether, and a fter remov a l of the ether 1E vacuo, the product distilled. Four and one-half grams of a color- le~s oil was obtained a t 108-10°C./0.12 mm. Hg. In order to re move a ny unie a cted secondary am ine, the base was dissolved in 5 cc. of absolute ether a nd 10 cc. of freshly distilled a cetic anhydride was added. After standing for one hour, the solution was tre a ted with NaOH solution and extracted with ether. After re mova l of the ether, the hydro- chlori de was prepared fro m the residue with methanolic HCl and ;)·recip i t e. ted with 4 volumes of a b solute ether at o0 c., while scra tching with a gl a ss rod. In this way the hydro- chloride could be p repared in white crystalline form. It is quite hy groscop ic, absorbing 1 mol of water from the atmosphere in about 10 minutes. The salt was recrystallized 35. by dissolving in the minimum amount of hot MeOH and adding absolute ether until a turbidity was produced. The crystals obtained on co oling were exp osed to the a tmos phere until a consta nt weight was obtained, a nd the comp ound then p ossessed m.p. 125-8 °0. The analysis wa s run on t he n ono- h ydrate, dried in air. C H Calculat e d for c14 H2203NCl•H20 54.__98 7.91 Found 55.22 7.88 The p icra te was 9re pared from e quiv a lent amounts of t he b as e a nd p icric acid in EtOH, a nd precipitated with an equal volume of water. An oil sep ar a. ted, which crystallized. o0 c. This material was filtered, and af- in a few hours a t ter recr yst a llization fro m EtOH-water (1:1), with scra tching to avoid oil form a tion, it melted a t 122-3.5°0, and was dried ·at 80°0. over P 2 o5 for analysis. C H Ca lcul a ted for C20H24010N 4 50.00 5.04 Found 50.57 5.27 Pre-oarati on of homomyris ticylamine ( 3-me thoxy-4, 5-me thylen~ dioxyphenethylamine) Myristicin Liyristicin was obtained by t he fr a ctiona tion (8 inch column pac ked with helices) of 3.15 K;;i;m . of oil nut meg (he a.v y ) obta ined from t he Dod~e a nd Olcott Co. 1.6 Kgm. 36. of myristicin wa s collected at 1 54-7°0./lBmm. Hg . Isomyristicin A solution of 540 gm. of myristicin in 1400 cc. of 95% EtOH c ont a inin g 2 50 gm . of KOH wa s refluxed for 24 hours, t hen neutraliz e d wit h HCl, a nd t h e EtOH distilled off a t 2 0 mr:1 . H,2; . Th e re s idue wa s po ure d into 1 liter of water, t h e se par u ted oil wa she d t Kice with 500 cc. of wa t e r, and fraction a ted. Iso myristicin was collected a t 170-3°0./ 15 mm . Hg , a nd a ft e r sta n d in g a f ew hours cr y sta llized in needles wit h m. p . 41°0. Powe r and Salway (5 2 ) g ive m.p. Myristici nal d e h yde (S a lwa y (53)) Thirty gr am s of isomyristicin wa e stirre d vi g orously wit h 150 cc. of wat er a t 60 ° 0., an d a solution of 60 gm. of Kh1n0 4 in 1. 5 liters of wa ter was then a dded dro_p wise over a period of 1. 5 hours, while maint a ining t h e temp erature at 0 60~4 O. Th e p recip ita ted Mn02 was filtered off a ft e r co o l- ing, wa shed with 500 cc. of ice-water, and dried at 70°0. Several such b a tc h es were run, and the combined Mn02 was then extracted for 12 hours with chloroform in a Soxlet extractor. The chloroform was evap orated off on the water bath, a nd the solid r es idue wa shed with cold ether. Th e yield was usua lly about 45~0 of the theoretical, and the a la.e 'ny de . r."r elted. c9.t l '-'<: 2 °0. Sp ath a nd Gan g l (41) g ive m.p. 37. Prep a ration of r hodanine (Julian and Stur g is ( 44 )) Anhydrous NH 3 wa s passed into of cs 2 , 200 cc. a solution of 250 gm. of abso lute EtOH, a n d 200 cc. of a bsolute et her for 3.5 hours, at 0-5°0. Th e ammo nium dithiocar- barnate was filt e red off, a nd washed with c old EtOH and et her . A solution of sodium c h loroa cet a te was ~oreoared fro m 84 gm . of NaOH in 1 40 cc. of wa ter and 205 gm . of . c h loroacetic acid in 175 cc. of water. Napo 3 was added until t h e soluti on wa s neutral to lit mus, an d then the a mm oniumdith i oc arbarnat e was added to t his solution with goo d cooling and stirring. Th e res u ltin g s o lution was a d- ded to 275 c c . of concent ra te d HCl a t 85°0., an d t h e p r e cip it a ted mate ri a l recrystallized fr om 200 cc. of g l a ci a l a cetic a cid. It wei gh ed 1 75 gm ., and melted at 1 63- 6° 0. Myristicinalrhodanine Fifty gr am s of myristicin alde hyde and 37 gm. of r h od a nine we re dissolved in 95 cc. of g l a ci a l a cetic a cid, and 69 gm . of anhydrous sodi um a cet a te dissolved in 95 cc. of g laci a l ace tic a cid added . An or ange p reci p it a te for med at once, and t he mixture wa s refluxed for 15 minutes. It wa s t h en poured into 1.5 liters of wa ter, filtered, an d washed with water, EtOH, an d fin a lly ether. 75 gm . of ma terial wit h m.p. 252-5°0. Th e yield wa s It was recryst a llized for a nalysis from a cetone-pyridine (3:1), and dried a t 110°0. over · P 2 o 5 . 38. C H Calculated for C1 2 H90 4 S2 N 48.80 3.07 Found 49.00 3.14 o:.-thioketo-p- ( 3-methoxy-4, 5-methylenedioxyphenyl )-prop ionic acid Seventy-six grams of myristicinalrhodanine and 350 cc. of 15% NaOH were heated on the water bath for one hour with stirring, until com J lete solution occur::'ed, and then the contents of the flask were heated to 100°0. for 10 minutes. After cooling, the product was J recipitated by the rapid addition of 350 cc. of 10% HCl. The yellow product was washed with water and after drying in air weighed 58 gm. This thioketo acid could not be crystallized, and the next step was carried out without further purification of this intermedia.te. ~-oximino-~-(3-methoxy-4,5-methylenedioxyphenyl)-pyruvic acid A solution of hydroxylamine was prepared by adding a solution of sodium ethylate prepared from 16.5 gm. of sodium and 475 cc. of absolute EtOH to a warm solution of 49.5 gm. of hydroxylamine hydrochloride in 44 cc. of water, followed by filtration of th,e precipitated NaCl. To this solution was added 58 gm. of the above thioketo acid, and the solution was heated on the water bath for 40 minutes, and the alcohol then removed in vacuo. The oily residue was dissolved in 120 cc. of 5f~ NaOH, the solution fl 1 tered, 39. and then precipitatea. slowly with 120 cc. of lo% HOl, scratching as the precipitation proceeded to avoid oil formation. In this way 48.2 gm. of a yellow-brown solid was obtained, which was dried for 48 hours over KOH and Oa012The compound could be obtained in crystalline form by dissolving in toluene at 85-90°0. and allowing to cool slowly. Microscopic needles which were washed with petroleum ether .were obtained, with m.p. 145-6°0. The compound was dried at room temperature over P20 5 and paraffin. Calculated for c11H11 o6N Found C H 52.17 4.38 52.57 4.85 3-methoxy-4,5-methylenedioxyphenyl-acetonitrile Forty-eight grams of the crude oximino acid was dropped into 150 cc. of acetic anhydride at a temperature of 60-80°0., at a rate which go.ve moderate foaming. At the end of the reaction the acetic anhydride w2-.s removed in vacuo, the residue taken up in ether and water, and the ether solution washed three times with one-half volume of lOo/o Na 2 Co 3 solution. The ether was removed 1:_Q vacuo, and the residue distilled. The nitrile was collected at 170- s°C./l.5 mm. Hg, and weighed 22 gm. It solidified quickly, and after recrystallization from MeOH melted at 88-9°0. Hahn and Scha les (43) give m.p. 90°0. for this compound, prepared by another met h od. 40. Homomyristicylamine Twent y-three g r am s (0.12 mol) of the above nitrile ,;,-Jas reduced in an Americ a n Instrument Co. high pressure hydro g enation bomb. Two cubic centimeters of Raney Ni c a t a lyst was used, a t 600 pounds p ressure a nd 12 5°C., in the p r e sence of 72 cc. of methanolic NH 3 (9-llN). The h y dro g ena tion wa s run for 4 h ours, and a ft e r cooling, the catalyst we s filtered off, and the Me OH a nd wc1.ter were removed in vacuo. The residue was distilled, and 16.0 gm. of homomyristic y lamine wa s collected at 120-4°0./0.2 mm. Hg. Th is colorless oil wa s dissolved in 30 cc. of 10% HCl, and evapora ted to dryness in a desiccator over KOH. After re- cryst a llization from 95% EtOH-ethyl acetate (1:1), the hydrochloride was dried at 56°C. over P 2 o5 , and melted at 164-5°0. _ Salway (39) g ives the melting point of homomyristic y lamine hydrochloride as 165°C. Ca lculated for c10 H14o3 NC1 Found C H 51.84 6.09 51.93 6.32 Th e o icrate wa s p re pared from the hy d r o chloride in wa ter, and melted a t 1 93-4°C. dl- a nha lonine (Spltth a nd Ga n g l (41), and Sp ath a nd Keszt- ler ( 4 2 )) To a solution of 6 .7 gm. of homomyristicylamine in 1 4 cc. of anhy dr ous b enze n e wa s added drop wise 5.5 cc. of fres h l y d istilled a cetic a nh ydride. Re a ction occurred im- medi a tel y , an d t h e vol a tile ma terial wa s t h en r e moved in 41. _ a desiccator con taini ng H2 so 4 , KOH, a nd paraffin . The im- pure N-acetylhomomyristicylamine me lted a t 12 2-3°0., an d after recrystallization from li groin (S0-100°0.) melted at 133-4°0. Sp ath and Gang l give m.p. 130 ° 0. Ca lcula ted for C1 2 H15 o4 N 60.75 6.37 Found 61.23 6.27 Ei gh t gr am s of t he a cetyl compound was dissolved in 110 cc. of anhydrous toluene, and 32 gm. P 2 o5 was added in about 2 gm . portions to the refluxing solution, over a period of 30 minutes. After al l t he P 2 o5 was added, the re- action mixture was cooled, water added, and the solution extracted with ether. After making the solution alkaline with NaOH the base was extracted with ether. The ether was removed 1!'! vacuo, t he residue fractionally di-stilled, and . the product boiling at 146-9°0./0.075 mm. Hg was collected. The distillate solidified i mmedi a tely, 67°0. ~ftd melted at 55- Sp ath and Ga ngl (41) give m.p. 60-2°0. A solution of 5.2 gm . of the above 3,4-dihydroiso- quinoline in 50 cc. of IvleOH was hydrogenated at room te112Perature an d 1 atmosphere pressure, in the presence of 23 mgm. of Adams c a talyst. The theoretica l amount of hydrogen was taken up in 45 minutes. The catalyst was removed by filtration, t he MeOH rem oved in ~£:d2, e.nd the material distilling- at 144-6°0./0.08 mm . Hg- collected. The quaterna ry methiodide prepared a ccordin g to Sp~ t h and Gang l melted at 205-225°0. The hydrochloride 42. of the base was prepared with methanolio HCl, and p recipitated with ether. This salt, which weighed 4.1 gm., wa s recrystallized from 1N HCl, and the first crystal fraction then recryst a llized from MeOH, g iving a product which melted at 241-5°C. The qua ternary iodide of this comp ound melted at 245-8°C. Sp&th a nd Ga ng l g ive m.p. 242 -~ 0 c. 1-Anhalo- nine methiodide p repared from 1- a nhalonine isolated from t~ mescal buttons i,181 ted s. t 222-4 °c., then recrystallized aind remelted a t 238-41°0. Spith a nd Gang l showed that racemi- zation occurred a t a temp er&ture of a bout 2 30 o C., and that the higher temperature represents t h e meltin g point of dlanhalonine methiodide. A sample of 1-anhalonine methiodide was heated at 235°0. for three minutes, and the product recryst a llized from MeOH-water (1:1). It then melted at 240- 5°0., and a mixture of this materi a l and the methiodide p repared from the synthetic product melted at 242-6°0. i ?nrneric cp1 ;::i The ternRJ'.l me thi odide of 1, 2-dime thyl-6, 7-methylene- dioxy-8-me t h oxy-l, 2, 3, 4-tetrahydroi soquinoline (prepared by t h e action of s ethy l magnesium iodide on cot a rnine iodide) melts at 228-9°0. (Freund (54)). ~ h e dl-anhalonine hydro- chloride was recrystallized fro m MeOH for a n a lysis, and dried a t roo m t emn era ture over P 2 o5 . Calculated for Found c12 H16 o3 NC1 C H 55.92 6.26 56.37 6.30 43. Atte mp ted ureparation of 3,4-dimethoxy-5-hydroxy-benzaldehy;de • 5-nitro-va nillin (Bentle y (55 )) To a solution of 455 gm . of •vanillin (Mo nsanto U.S.P.) in 1800 cc. of g l a ci a l a c e tic DCid at 10° c. was added dropwi se a solution of 135 cc. of fumi ng HllJ 0 3 ( sp . gr. 1. 5 ) in 500 cc. of gl a ci a l a cetic a c id . The te m9 era ture was al l owed to rise s l owl y to 30°0. a t the end of the reaction, and after cooling- to 15°0. the yellow-green product was filt ered, a nd was hed free of a cetic acid with hl eOH. The dr ied product we i ghed 414 gm ., and melted a t 174-5° 0. Pota ssium salt of 5-nitro-vanillin One hundred and fort y gr am s of 5-nitro-vanillin was susp e nded in 3.5 lit ers of wa ter, and 49 gm . of KOH in 150 cc. of w1:\ ter was added. A clear, dark red s olution was ob- t a i ned , t o which was adde d 50;0 KOH s olution, until no further p reci p it a tion of reddish-orange crys t a ls was obtained. On ac i dify ing t he filtrate with HCl, a s mall am ount of 5nitro-va nillin remaining in solution was p recipitated.· The po t a s s ium s a lt, af ter washing with 95;S EtOH and dr ying a t 130°0. for 4 hotirs, we i ghed 140 gm . 5-nitro-v er a tr a l dehyde (Sp& t h a nd Roder ( 45)) Spith a nd Roder st a te tha t the po t a ssium s a lt of 5nitro-v a nillin s hould be he a ted with di methyl sulf a te a t 170- 5°0. Only a very low yield wa s ob t aine d under t hese conditi ons, so a l ower t emper a ture was used in this preparat i on . Two hundr ed grams of t he p ot a ssium salt was 44. suspended in 625 cc. of freshly distilled dimethyl sulfate, and heated with stirring. At ap p roximately 145°0. the color chang ed abruptly to yellow, and the reaction mixture was cooled i mmediately to room temperature. The dimethyl sul- fate was distilled off in vacuo, the residue dissolved in ether, and the ether solution washed with 5% NaOH to remove unreacted 5~ nitro-vaqillin. The ether was removed by dis- tillation, the residue recrystallized from EtOH-water (2:1), and dried over OaOl2- The yield of 5-nitro-veratraldehyde was 144 gm., m.p. 88-90°0. The oxime was prepared and re- crystallized from EtOH, and melted at 150-1°0. Brady and Manjunath (56) give rn.p. 151°0. for this derivative. 5-nitro-veratraldehyde-dimethylacetal To a solution of 118 gm. of 5-nitro-veratraldehyde in 250 cc. of MeOH was added concentrated HOl to give a concentration of 0.5%. This solution was allowed to stand at roofil temperature for 1 week, and the HCl was then neutralized with a solution of sodium methylate in MeOH, and the MeOH was distilled off l!:! va~- On fractional distilla- tion, 113 gm. of the dimethylacetal was collected at 13844°0./0.2 mm. Hg. Crystals formed on standing, with m.p. 31-6°0., which after recrystallization from petroleum ether (60-70°0.) melted at 34-6°0. A sample was dried over P 2 o5 and paraffin for analysis. C H Calculated for C11H 15 o6 N 51.36 5.88 Found 51.93 6.27 45. Reduction of 5-nitro-veratraldehyde-dimethylacetal Fifty g rams of 5-nitro-veratraldehyde-dimethylacetal was dissolved in 100 cc. of MeOH, and 10 cc. of Raney Ni c a talyst in EtOH added. The hydrogenation was conducted in the hi g h p ressure hydrogenation bomb a t an initial hydrogen pressure of 1000 pounds, at 23°C. The reduction was stopped when t h e theoretical amount of hydro g en ha d been a bsorbed, a nd the c a t a l y st wa s filtered off. No at- tempt was made to isolate t h e a mino-aldehyde. Diazotization of a mino- a l dehyde a nd d ecomp osition of diazonium salt The MeOH was distilled off in vacuo from a sample (one-tenth of the total) of the above solution, and t h e resi d ue dissolved in 200 cc. of ice-water containing 5 cc. of concentra ted H2 S0 4 • Th e calculated amount of NaN02 dis- solved in a little water was added slowly, until the diazotization was comp leted, as indicated by the starch-iodide re a ction. Th e excess nitrous acid wa s d ecomp osed with urea, and t h e soluti0n heat e d o n t h e water t at h in a stre a m of CO 2 for 1 hour. A d a r k s olution resulted, and a fter cool- ing , was extracted with ether, and the ether removed in v a cuo. No crystalline compounds could b e isolated from the residue, a n d a ll furt h er atte mpts to p rep a re 3,4-dimethoxy5-hydro ~ybenz a l d e hyd e b y diazotiza tion with other acids and decomp osition under mild er c onditi ons were unsuccessfuL However, a nother substance could be prepared in reasonable yield by decomp osition of t h e di a zonium compoun d 46. formed in the presence of HCl, by means of Cuso 4 solution. A quantity of the amino-aldehyde solution corresponding to 5.0 gm. of 5-nitro-veratraldehyde-dimethylacetal before reduction, was evaporated in vacuo. The resid.ue was taken up in 50 cc. of 4N HCl, and diazotized with the calculated 0 amount of NaN0 2 at 0-5 C. This solution was then added dropwise to a solution of 75 gm. of Cuso 4 ·5H 2 0 in 100 cc. of water, at a temper a ture of 85-90°C. A smooth evolution of nitro g en occurred, and at the end of the reaction the solution was extracted with benzene. The benzene was dis- tilled off, and the residue distilled in v a cuo. A color- less oil wei gh ing 2.3 gm . was obtained at 110-2°0./0.75 mm. Hg, 2nd this product immed iately crystallized. After re- crystalliza tion from li g roin, it ~ elted at 57°0. It was dried at room temperature over P20 5 and paraffin for the 1:,.n&lys-is, which showed the compound to be 3,4-dimethoxy5-chlorobenzaldehyde. C H Cl Calculated for C9 H9 0 Cl 3 53.88 4.52 17.68 Found 53.94 4.66 18.27 This substance has been prepared by another niethod by Hann (57), and its melting point is given as 57°0. 47. I I I - ACTION OF AlviINE OXIDASE ON MESCAL ALKALOIDS AND RELATED COMPOUNDS A p arti a lly p urified a mine oxi dase preparation was made a s d escribed by Alles a nd Hee g aard (58): a liver (70-90 gm.) from a freshly killed rabbit was ground with ice and water in a Waring blender, the volume made up to 500 cc. and the pH adjusted to 8,0. Fiber was screened out, the mixture passed twice through a Sharples supercentrifuge, and dialyzed in Visking tubing for 20 hours in 10 liters of water at 5°C. The pH of the dialyzed solu- tion was adjusted to 6.0 with acetic acid, and the precipitate centrifuged off. This precipitate was re-sus- pended in one liter of distilled water, the pH adjusted to 6.0, the solids centrifug ed off, and the centrifugate discarded. The solids were then re-suspended in wa,ter with the addition of concentrated buffer solution and 1:1000 phenylmercuric acetate solution, and the pH adjusted. The final solution had a volume of 250 cc. and contained O.lM sodium p hosphate buffer of p H 7.0 a.nd 1:10,000 phenylmercuric acetate. The oxidation system consisted of 2.0 cc. of purified amine . oxidase in O.l~ phosphate buffer at pH 7.0 and 0.3 cc. water, to which was added 0.2 cc. O.lM amine-hydrochloride solution. The oxygen uptake at 30°c. was measured in the usual way with the Wa rburg ap p aratus. A control was run si mu ltaneously without a mine, as well as a com- 48. parison standard containing 0.2 cc. of O.J.M phenethylamine. 'P-he rate recorded was t h e maxi mum rate, because with □ a ny substrates the r a te of oxygen uptake decreases with time, possibly due to inhibition by re a ction p roducts (Hare (59)), ~ and this r a te was expressed in t e r tns of p er cent relative to t he rate of oxida ti on of y hene t hy lamine. Th e frillowin g compoun ds were tested in this manner: Mese s.line, N-methylmesc a line, N-di methy lmes c a line, homomyristicylamine, 6,7,8- trimethoxy-1,2,3,4-tetrahydroisoqui~ oline, 2- methyl-6,7,8-tri met ho x y-l,2,3,4-tetrahydroisoquinoline, l-methy l-6,7,8-tri me t h o xy-1, 2 ,3,4-tetrahydroisoquinoline, l, 2- d i methy l-6,7, 8 -tri met h oxy-l,2,3,4-tetr ahy droisoquinoline, dl-anh&lonine, 1-a nhalonine, lophophorin e, anhalonidine, and pe llotine. Mesc a li n e had been 9 revi ousl y studied in re gard to the a ction of am ine oxida se a nd t h e findin g of Bla schko , Richter, and Schlossmann (12) a nd Pugh and Quastel (60) t ha t it is not appre ci a bly oxidized in t h e oresence of amine oxid.8.s e was confirmed in the presen t work . Only one of t h e com- pounds listed c1.bove ga.ve cm upt2.ke of oxy~_:en , nam el y homomyristic y l a mine. It wa s oxi d ize d a t a r a te corr esp onding to 72% of t h e r a te of oxida tion of ) h enet hy l am i n e. It is q uite g enera. lly true t ha t co m1)ounds not a.ttacked by amine oxi da se (e phe drine, benzedrine, mesc a line) are a ctive 1./ nen g iv en by mo ut h , v, h ile subst an ces wl1 ich D.re oxidized a re not effective when adm inistered orally (Bey e r a nd Lee (61)). It t herefo re se ems li kely t ha t homomyristic y lamine 49. would · not be expected to s how strong activit y when given or a ll y , a nd in contr as t to t h e other compounds tested tha t amine oxidase is at least partially res p onsible for its destruction in the body. 50. SUMlvIARY 1. Th e a lkal o ids of mesc a l buttons have been in- vesti gate d, and five of the known a lkaloi ds have be en isol a ted and i den tified. 2. Six c omp o unds of the 3,4,5-trimethoxyphenethyl- amin e ana. t he 6, 7, 8-trimethoxy-1, 2, 3, 4-tetrahydroj_soquinoline type s have been s y nthesized fr om mesc a line. 3. Homo myr isticylamine has been p re par ed by a new me th od , a nd used to synthesize dl-anhalonine. 4, An unsuccessful attempt wa s made to develop a met hod f or the pre par a tion of l a r g e quantities of 3,4-dimeth oxy-5-hydroxy-benz a ldehyde for use a s a n intermedi a te in t h e synthesis of 3,4-dimet h oxy-5-hydroxyphe nethylamine. 5. Th e a cti on of amin e oxidase on a number of these c ompoun ds wa s studied. 51. REFERENCES 1. Mooney, Therap. Ga. zette, 20, 7 (1896) 2. Mitchell, Brit. Med. J., 2, 1625 (1896) 3. Ellis, Lancet,!, 1540 (1897) 4. Prentiss and Morgan, Therap. Gazette, 20, 4 (1896) 5. Fernber g er, Am. J. P sychol., 36, 267 ( 1923) 6. Beringer, Der Meskalinrausch, Berlin (1927) 7. Rouhier, Le Peyotl, Paris (1927) 8. Kl~ver, Mescal, London (1928) 9. Stockings, J. Mental Sci., 86, 29 ( 1940) 10. Q,uastel and Wheatley, Biochem. J., 27, 1609 (1933) 11. Richter, Biochem. J. 32, 1763 (1938) 12. Blaschko, Ric h ter, and Schlossmann, Biochem. J., 31, 2187 (1937) 13. Slotta and l~iuller, Z. physiol. Chem., 238, 14 ( 1936) 14. Heffter, Arch exp. Path. Pharme,kol., iQ., 385 (1898) 15. Robles and Robleda, Anales inst. biol. Mex., 15 (1931) g, 16. Hutchin12:s, State Hosp. Bulletin, St. Lawrence, No. 1 ( 1897) 17. Grace, J. Pharmacol., 50, 359 (1934) 18. Dixon, J. Physiol., 25, 69 (1899) 19. Raymond-Hamet, Arch, exp. Path. Phc1.rma.kol., 169, 97 (1933) 20. Clerc, Paris, and J a not, COlilpt rend. soc. biol., ]:21, 1300 (1936) 21. Clerc, Janot, EJ.nd Paris, Corno t rend. soc. biol., 119, 8 0t,_,Qn (1935) 22. (1903) Mogilev-m, Arch. exp. Path. Pharmakol., _, 49 137 52. 23. Robinson, J. Chem. Soc., 876 (1917) 24. Hahn and Rumpf, Ber., 71, 2141 (1938) 25. Schopf a,nd Ba:yerle, Ann., 513, 190 (1934) 26. 04 401 (1888) Lewin, Arch. exp. Path. Pharmalrn 1. , ;;:_' 27. Heffter, Ber., 27, 2975 (1894) 28. Heffter, Ber., 29, 216 (1896) 29. Kauder, Arch. Pharm., 30. Spe,th and Becke, Ber., 68, 501 (1935) 31. Sp1lth and Becke, Ber., 68, 944 (1935) 32. Spa,th ano. Bruck, Ber., 70, 2446 (1937) 33. Splth a.nd Bruc.k., Ber., 71, 1275 (1938) 34. Spa th and Bruck, Ber., 72, 334 (1939) 35. Sp& th and Becke, Monatsh., _, 66 327 (1935) 36. Ludue...,:.na, Com~Jt. rend. soc. biol., 121, 368 (1936) 37. Guggenhe im, Die biogenen Amine, Basel (1840) 38. Reti, X Congr. Interna t. chim., Rome (1938) 39. Salway, J. Chem. Soc., 1208 (1910) 40. Decker and Becker, Ann., 395, 328 (1913) 41. Spa,th and Gangl, Monatsh., 44, 103 (1923) 42. Spath and Kesztler, Ber., 68, 1663 (1935) 43. Hahn and Schales, Ber., 67, 1486 (1934) 44. (1935) n :z_r7 ~-' 190 (1899) Julian and Sturgis, J. Arn. Chem. Soc., 57, 1126 Sp&th and Roder, Mo natsh., 43, 93 (1922) 46. Mau thner, Ann., 449, 102 (1926) 47. Spa,th, L10natsh. 40, 129 (1919) 48. Hahn and Wassmu th, Ber., 67, 696 (1934) 49. Spath c::.nd Becke, Ber., 67, 266 ( 1934) 53. 50. Spa,._.__h u , Monatsh., 43, 477 (1923 ) 51. 42 97 (1921) ivl ona t s_h . , _, Spa''t·n, ]' 52. Power and Salway, J. Ch em. Soc., 2054 (1907) 53. Salway, J. Chem . Soc., 1204 (190 9 ) 54. Freund, Ber. 36, 4257 (1904 ) 55. Bentley, Arn er. Chem. J., 24, 171 (1900 ) 56. Bra dy a nd Llanjunath, J. Chem . Soc., 1060 (1 924 ) 57. H2-.nn, J. Wash. Acad. Sci., 24, 124 (1934 ) 58. (1943) Alles B.nd Hee gaard , J. Biol. Chem., 147, 491 59. Har e, Biochem. J., 22, 968 (1928) 60 . Pugh and Quastel, Blochem. J., 31, 2306 (1937) 61. Beyer and Lee, J. Phar ma. col. 74, 155 (1942) FART I I PREPARATION OF SEVERAL PHOSPHO-ORGANIC COMPOUN.DS 54. INTRODUCTION At the pres ent ti me it i s reco g n i zed t ha t ph osphoor ~an ic compo und s inv olved in cellula r me t ab olism may be cla ssified roug hly into t wo types . In t h e first group c a n be p laced t hose c ompounds in wh ic h t h e pho s phate i s li nked to an a lcohol ic h ydr oxyl g r oup , a nd wh ich are hydro l y zed wit h A free ener gy cha n g e of a pproxi ma tely - 3000 c a lorie s . I n t h e se c ond group 2_re incl uded cre a tine ph osphat e, a r g inin e pho s pha te, pho s pho- enolpyruv ic a ci d , )h os pho g lycery l pho s p hate, ad e nosi ne tri- ph os phate , a nd a cet y l pho s pha te, whic h D,re hyd rol y zed with a free energy c hEt n g e of approx ima tely -10 , 000 c a l ories. (1) Acet y l p ho sphate wa s s hown by Lipmann (2) t o b e a p rod u ct of t h e ox id a t ion of pyr u vic a c id by Ba cteri um Delbruec k ii! 0 " CH.fC0-COOH + H3 P0't + (1 ~ CH3-[-0-P03 Hi +[({ + H2 0-l Th e p ro9 er ties o f c a rboxy l pl:lo s pha.te sugges t t 1-u:; t c omp ound s c ont a inin g t h is etruct ur e may be u tili zed in v a ri ous biolo g ic s.l syn t heses , a s fo r exax:1p le i n t ~1.e f or ma ti on of t he ester g rou9 of fats ~nd of t h e p e p ti d e bond . it wa s c onsidered t o b e of int e r es t pha te, I n this c onn e cti on to p r epare be n zoyl phos- t h e a nhydri d e of ben z oi c a cid and pho s phoric a ci d . A metho d wa,s develo 1) ed for t h e )repG,r a t ion of t h is sub- st a nce, a ltho u gh it h a s no t been found po ssible to obtain it pure . I n add ition, ace t y l ph o s pha te has b een prepared a c- c ording to t h e metho d of Ly nen (3 ), and p h osp h o-e nolp yruv ic a cid has been p repared and par ti a lly purified by t h e met h od of Kiesslin g ( 4 ). 55. EXPERIMENTAL WORK PREPARATION OF ACETYL PHOSPHA'rE (3) Dibenzyl_.2noscJhoric acid Two hundred and twenty g rams of benzyl alcohol (Merck re a~ ent) was dissolved in 200 cc. of absolute ether, and 90 gm . of P205 was added to this solution in portions of a bout 2 gm., with moderate cooling to keep t h e temperature at 2530°0. At the end of the reaction, 100 cc. of water was ad- ded, and the solution heated on the water bath, while 200 gm. of BaC0 3 and then 1 liter of saturated Ba(OH)2 solution were a dded to bring the solution to al ka linity to phenolphthalein. The hot solution was filtered and the pre- cipitate boiled twice with 500 cc. of water, filtered, and the three filtrates combined, concentrated at 100°c. to 500 cc., cooled, and the first crystal fraction collected. Further concentration yielded more product of the same purity. The total weight of Ba-salt was 26.6 gm., with m.p. 255-61°C. (a.). Twenty-five grams of the Ba-salt was dissolved in 750 cc. of hot water, and the calculated amount of AgNO~ - ._, dissolved in a little water was added. The Ag- salt crystallized at once in needles, and after filtering and drying it wei g hed 24.6 gm., and melted at 215-16.5°C. Lynen gives m.p. 216°C. The compound was dried at room temperature over P20 5 for analysis. C H Calculated for C14H14 o4PAg 43.66 3.66 Found 44.15 3.83 56. Palladium-cha rcoal catalyst A solution of 0.3 gm . of palladium chlori de in 30 cc. of 0,lN HCl was added dropwise to a shak in g suspension of 6.0 gm. of acid-washed Norite A in 300 cc. of distilled water, wh ile hydrogen was passed t h rough in a continua us stream. The c a t a lyst was centrif w~;ed off af ter shakin g for 20 minutes, washed twice wit h ab solute EtOH, centrifuged, anc,_ dried in ~ £ over H2 So 4 . It was then dried over P20 5 a t 110°0. for 4 hours in vacuo. Th is pr eparation wa s quite a ctive, as shown by t h e rapid hydrogenation of cinn am ic acid in BO% MeOH. Ag-s a lt of acetyl phosohate The re a ction was c a rried out in a wide-mouth bottle fitted with a dropping funnel and a Pyrex filter stick, an d mo ist ure wa s c are full y excluded during t h e reaction and subsequent filtration. One an d one-half g rams of the Ag-salt of dibenzyl pho s phoric a cid and 5 cc. 6f ab solute et h er were introduced into t h e bottle, and t h en 0.7 gm. of freshly distilled acetyl chloride dissolved in 2 cc. of absolute ether was added dropwise, at room temper a ture, over a period of several minutes. The mixture was allowed t o st a nd for one hour, then 20 cc. of absolute ether was adde d, ana t h e bottle was shaken vi gorousl y . The ether' solution was then filtered, a nd the ether and excess a cetyl c h loride removed by distillation in vacuo at 30°C. Th e oily residue wa.s dis- solved in 30 cc. of ab solute e t h er, and 250 mgm. of palladium-c har co a l catalyst add ed. Th e a cet y l-dib en z yl-pbo s p hate 57. wa s then hy dro g en a ted a t 2 5°0. a nd one a t mos phe r e p ressure, wit h t h e hydrog enation fl a sk p rotecte d fro m moisture b y a U-t ube c on t a i n in g Drierite. Th e up t ake of hydr og en st opp ed a ft e r t h e t h eor e tic a l amoun t of hydro g e n ha d b e e n a b sorbed in 2 5 mi nut~s. Th e c a t a l y st wa s filt ered off a t o0 c., t h e et h er so l u ti on s hake n t wice wit h 5 cc. of ice- wa t e r, a nd t h e a q ue ou s so l u ti on qu ic k l y neutra lize d v it h s a t ur a t ed Ba (OH) 2 solution to phe no l ph t ha lein. Th e s e op er a ti ons were con- ducted in t h e col d , b eca use of t h e g re a t instabilit y of a cetyl p h os pha te in a ci d solution. Th e ins oluble barium p h os p hate wa s ce n trif uge d off, and 4 cc. of 25% AgN 0 3 s o l u ti on ad ded t o t h e s up ern a t a nt, follo we d b y a n e q u a l volume of a ci d-free EtOH. T~e Ag- s alt cry st a llized a t once in ne ed les, and wa s ce n trif u ge d , washed wit h c ol d EtOH a n d et h er in t h e c en trif u g e, a nd dri~ d . Th e wei ght wa s 488 mgm . Ac cordi ng t o Lyn e n t h e Ag -s a lt of a c e t y l ph os pha te h ydr o l y zes q u a ntit a ti ve l y a s f o llows: 3 RR1. (C~-CO)PO'f- + 5 OH--+ 2 Flg 3 PO,.. + HPO: -t-J(H~-(00- -r2H2. 0 A titr a ti on c ond uct ed a ft e r hydr ol y sis of t h e Ag-s a lt in bo ili n g wa ter fo r 10 mi nu t e s i n dic a te d a purit y of 77%. Th e s ub st a nce was furt he r purifie d a s d esc r i be d b elow. Th e Ag-s a l t wa. s stirre d up wit h sli ~::nt l y l e s s t h a n th e CEtlcul a t e d amoun t of KCl solution at o0 c. a n d pH 7.0, t h e pre- cip it a te d AgCl wa s c en t r i fu g ed of f , and t h e Ag-sa lt rep reci p i t s. t ed by t he drop wise a d d iti on of 2 5~0 AgN 03 s olution. Th e cr y st a ls were wa shed wit h EtOH a n d et he r, a n d d rie d over H2 so . 4 An a cet y l d etermina ti on a cc or d i n g to Pre g l ga v e : 58. Acetvl Calculated for C2 H3 0 5PAg 2 12.15 Found 11.90 PREPARATION OF BENLOYL PHOSPHATE When t h e Ag-salt of dibenzy l pho s phR te was re a cted with fres h l y distille d b enzo y l c h loride i n t h e s ~me man ner ac wit h ace t y l c hlor i de , t ha t is by u si ng t wo ti mes t h e c a lcula ted amount of t h e acid c hl ori d e, only two-thirds of t h e calcula ted a mount of hydrogen wa s t aken up , a nd the metf.cl S?, l ts isol a t ed subs equently were obtained i n low ~'i eld a nd 1-Jere of a .9uri ty of a_bou t ;2 5o/~ , as determined by titration of a cid formed on hydrolysis, and by benzoyl determina tion. Furt h er investigation led to the isolation of one of t h e p roducts f or med from be nzo y l chloride and Ag-dibenz y l-p h osph2.te when the reaction was conducted in t h is manner. Re mo v a l of the et h er in vacuo after the re- action gave an oil, whi ch could be cryst a llized by vigorous scratchin g at o0 c. This product was washed free of benzoyl chloride with ligre>in (85-100°0. ), and dried. Two g rams were obtained from 3.0 gm . of Ag-dibenzyl-p h ospha te, with m.p. 61-75°0. By repe a ted recrystallizations through dis- solving in absolute ether and adding four volumes of ligroin, a product of const an t melting p oint of 113-6°0. was obt2ined. Re pea ted recrystallization fro m a n hydrous iso- propy l ethe r gave a p roduct of the s ame melting pain~. Severa l fr a ctions we re dried at room temperature over P 2 o5 and paraffin for an a lysis. This intermediate should be 59. C H p c21 H19 o5P 6 5 . 96 5.01 8 .12 Fracti on of m.p. 61-75°0 . 66.64 4 , 99 8 .12 Fraction of m.p. 82- S0°C . 65 .36 4,58 Fra cti on of rn . p . 1 13-6° 0. 64.08 4.61 Ca lcula te d for 5.88 The above a nal y s es indic at e cle a rl y t ha t t h e ma tericl of const an t m.p. 113-6°0 is n o t t h e des ired intermedia te. Th e metho d fin a l ly adop te d for t h e 9re paration of ben zo y l pho s phat e is d escrib e d be l ow . T:1 ree g r B,m s of dibenz y l-p ho s pha te was tre a ted d ropwise a t 2 5°0. with 0.98 cc. ( mol/mol ) benzoyl c h loride over a period of about 5 mi nutes, in t h e o resence of 10 cc. of ab s o l u te et h er. Th e mixture waE s hake n d~rin g t h is addi ti on , t h en a llowe d to 0 st a nd a t 2 5 C. for t h ree hours , 40 cc. of D.bsolute ether added , an d t h e AgCl filtered off a s pre vi ousl y described. Th er e was no de tect ab le odor of b enzo y l c h l ori de in the filtrate. Th is ether solution was then hy dro g enated a fter the add ition of 5 00 mgm . of pa lla di um-c harc oa l c a t a l y s t . Ti e uptake of hydroge n sto)p ed a fter t h e t h eoretical amo unt had been &,bsor b ed in 2 5 min u t es . l subs e qu e n t p r oce dur e s A l.L..L were c onducted a t 0 -5 ° C., in order to reduce hydrolysis . Th e cat a l y st was f ilter ed off, and t h e ether s oluti on t h en s haken t wice with 10 cc. of water, a nd t h e aqueous solution 60. neutralized at once to phenolph t halein with 60 cc. of 0.2N No.OH. Ten cubic centi ~il eters of 1 0~~ CaC1 2 solution was added, and rosettes of crystals formed imme diately, which were centrifu g ed off, washed with acid-free EtOH and et he r in the centrifug e, and dried. In t h is way was ob- tained 1. 2 5-1.50 gm. of t h e Ca -salt of benzoyl pho s phat e of a pur it y of a~ou t 75% as determined by titr a ti on of a s amp le hydrolyzed in boilin g water for 10 minutes. A benzoyl de- termin a tion a ccordin g to Pre g l on the ma teri a l combined fro m several p repar a tions gave t h e followin g : used 5.05 cc. of 0. 0 1 26N NaOH. 6.55 cc. of 0. 0 126N Na OH. 19. 8 mgm. Ca lcula te d f or 19.8 mgm.: A purit y of 77% is indicated by t hi s determination. Th e Ag-salt is ve ry insoluble, while the Ba-salt is somewha t more soluble than the Ca -salt. Th e p reparation of the pure Ag-salt t hro ug n first precipitating t h e in or ganic phosphate with Ba(OH) 2 solution was not po ssible because of the li mite d solubility of t he Ba -salt of be nzo y l pho s pha te. Th e Ca -s a lt was used because it wa s t hough t likely t ha t it c ou l d more e a sily be sep a r a ted fro m i n or gan ic impurities. Ho1'Iever, repe c:_ t ed a tte mp t s t o ob t a.i n t h e pur e Ca -s a lt by d is so l vin g t h e i mpure ma t e ri a l in wa.t er a t o0 c. and pH 7.0 (the solubility under t hese conditions is 80 mgm. per 100 cc. of water), and fractionally p recipitating with 10'/o CaCl2 s ol u tion and EtOH, wer e unsuccessful. 61. PREPARATION OF PHOSPHO-El'J OLPYRUVIC ACID (4) A solution of 18 cc. of freshly d istilled phosphorus oxychloride in 30 cc. of redistilled quinoline was added dropwise to a solution of 30 gm. of pyruvic a cid (b.p. 745°0./22 mm. Hg ) in 30 cc. of quino+ine. There was a strong evolution of he a t, and the te mp era ture was maintained a t 0 60-70 C. by cooling. Th e reaction 1Jroduct was cooled to 5°0., and dissolved by slowly adding 25% NaOH solution, avoidin g an excess of alkali. Two more p reparations were made a s described, t h e solutions combined, a nd t h e quinoline sep ar a ted off. The p H wa s a djusted to 8 .9, two volumes of 95% EtOH add ed, and a s □ a ll am ount of precip it a te filtered. The filtr a te was p reci p itated with 200 cc. of 2 5% Ba(OAc) 2 solution, a nd the precip ita te filtered and washed with 2:1 EtOH-water. Th is p recipit a te wa s suspended in 100 cc. of water, 35 cc. of 25% H2 so 4 solution a dded, and t h e mixture was stirred for 1 hour. Then the suspensi on was tre a ted with magnesia mixture in ammoni a cal solution, and allowed to stand f or 4 hours, a ft e r wh ich ti me it wa s ce n trifug ed. To t h e sup erna t a.nt ·wa,s adde d 75 cc. of 2 51~ Ba ( OAc) 2 solution and an equa l volume of 95% EtOH at pH 7.0, a n d the precipitate wh ich formed was filter e d off a nd stirred with 20 0 cc. of wa ter to which sufficient HCl was added to g ive a pH of 4.0. After centrifuging , the pH of the superna tant was ad- justed to 7.5, and on t h e add ition of a n e q u a l volume of 95% EtOH, a p reci p itate wa s obt a ined wh ich wa s dried over 62. CaC1 2 , a nd weighed 6.5 gm . The phospho-enolpyruvic a cid was determined according to t he me t ho d of Meyerhof and Kiesslin g (5). This metho d makes use of the fact that phospho-enolpyruvi c a cid is s p lit quantitatively by I2/alkali with the libera tion of inorganic phosphate, which c a n be determined colorimetrically. The difference b et ween t he 2.l ka l:l::-hydrolyzable pt1o s.9ha te, a nd t he I 0 /alka li li berated -oho sp hat e is a ~ measure of t he phospho-enolpyruvic a cid pre sent. By t h is method t he above pro duct was s hown to c on tain 35'Jo of t h e Ba-s a lt of ph ospho-e nolpyruvic a cid. SUMMARY 1. The silver s a lt of a cetyl p hosphate has been p re- pared and obtained pure . 2. A method has been develop ed for the prepara ti on of the c alcium SD,l t of benzoyl p h osphate wit h a purity of approxi mately 75%. 3. The barium s a lt of p h osph o-en olpyruvic acid has be e n p repared a nd obt a ined wit h a p urit y of 35%. 64. REFERENCES L Lipmann, Advances in En zymo l o gy, Vol. I, 99 2. Lipmann, Enz ymolo gia, i, 65 (1937) 3. Lynen, Ber., 73, 367 (1 940 ) 4. Kiessling, Ber., 69 , 2331 (1 936 ) 5. Me yerhof and Kiessling, Bioche m. z ., ~ ' 249 (194 1) (1 935)