I and II. Studies on Dichroa Febrifuga. III. A Method for Estimating Chloride. IV. Studies on Cambarus Clarkii Citation Brockman, John A., Jr. (1948) I and II. Studies on Dichroa Febrifuga. III. A Method for Estimating Chloride. IV. Studies on Cambarus Clarkii. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/k2bc-dx13. https://resolver.caltech.edu/CaltechTHESIS:05062025-212625604 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Chemistry and Animal Physiology) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Minor Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Koepfli, Joseph Blake (advisor) Lucas, Howard J. (advisor) Wiersma, Cornelius A. G (advisor) Thesis Committee: Unknown, Unknown Defense Date: 1 January 1948 Record Number: CaltechTHESIS:05062025-212625604 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:05062025-212625604 DOI: 10.7907/k2bc-dx13 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17205 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 07 May 2025 22:01 Last Modified: 07 May 2025 22:02 Thesis Files PDF - Final Version See Usage Policy. 37MB Repository Staff Only: item control page

I and II. STUDIES ON DICEROA FEBRIFUGA III. A MNrHOD FOR ESTIMATilJG CHLORIDE TV. STUDIES ON CAMBARUS CLARKII Thesis by John A. Brockman, _Jr. In Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy, California Institute of Technology, Pasadena, California. 1948 Surmna!"J Two crystalline, optically active alkaloid~,named febrifugine and isofebrifugine, have been isolated from extracts of the roots of Dichroa febrifuga. The method of isolation involves selective adsorption, and is capable of showing the presence of a fev-r milligrams of alkaloid. One of these alkaloids, febrifugine, is a powerful antimalarial, having more than 100 times the activity of quinine against bird malaria. Stu~ies on the structures of these alkaloids indicate them to be 3-substituted-4quinazolones of empirical formula C16H1903N3. A method is described for detenrining the chloride concentration in an aqueous solution by measuring the potential of a cell composed of a reference half cell, a.nd a silver, silver chloride, chloride ion half cell. The determination takes cao 2 to 3 minutes, is me.de with 10 ml. of solution, and is reproducible to ca. 4% for chloride concentratiom above 10-4 formal. Experiments on the fast closer system of Crunbarus clarkii are described, and show that magnesium ion blocks the muscle contraction without blocking the muscle action potential. Other experiments are described, indicating that the twitch height of the muscle contraction is dependent on the rate of perfusion, when the perfusing solution is allowed to flffi'r through the propodite. - i - ~cknowledgements My deepest thanks go to the many people and institutions which made possible the researches described in this thesis. The investigations of Parts I and II were carried out in pa.rt under a contract reco!lllD.ended by the Committee on Medical Research bet.~een the Office of Scientific Research a.:ild Development and the California Institute of Technology, and in pa.rt under a Research Grant from the United States Public Health Service. Most of the E,• febrif'uga root material and technical assistance was furnished by Eli Lilly and Company. : Botanically identified D. febrifuga roots and leaves were furnished by the Abbott Laboratories. The phar:ma.co- logical results reported here were obtained from Dr. K.K. Chen of Eli Lilly and Co., Dr. A.P. Richardson of E.R. Squil:band Sons, Dr. E.K. Marshall, Jr. of Johns Hopkins University School of Medicine, and Dr. L.H. Schmidt of Christ Hospital, Cincinati. My especial thanks go to Dr. J.B. Koepfli who introduced me to a very exciting branch of chemistry and who guided the largest part of the work described in this thesis. I would like to express my deep appreciation to Prof. How-a.rd J. , Lucas who has been a true friend and inspiration to me throughout my studies at the Institute. I regret that the recent war brought to a sudden close the researches we had only begun. To Prof. C.A. Wiersma of the Division of Biology I would express rrry regrets that I was not able to spend more time in his laboratories, studying a branch of science which I found most fascinating. - ii - TABLE OF CONI'ENI'S Page I. ISOLA.T ION OF POrENl' IAL ANT IMALARIA.IS :FR.OM D. FEBRIFUGA 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . • . l 2. Early D1vestigations of Ch'ang Shan and D. Febrifuga. 9 3. Isolation of Inactive Isofebrifugine and of a Crude Active Fraction ...........•....................... •~• 4. Introduction to the Chromatography of the Alkaloids of D. Febrifuga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. 6. Silicic Acid as a Chromatographic Adsorbent for the 2:71 Calcium Hydroxide, Talc, and Calcium Carbonate as Chromatographic Adsorbents ~or the Alkaloids and the Purification of Isofebrifugine ••••••••••••••••••••o• 32 7. Alumina as a Chromatographic Adsorbent for the Alkaloids and the isolation of Pure Febrifugine ••••••••••••••• 35 8. Effect of Heat on Febrifugine and some Distribution Coefficients of Febrifugine and Isofebrifugine •••••o 51 9. Fuller's Earth as an Adsorbent of the Alkaloids ••••• 55 10. Final Procedure for Isolating the Alkaloids••·•••••• 61 11. A:wlication of the Isolation Method to various Plant 12. Sum.rna.ry of the Properties of Febrifugine and Isofe brifugine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. 66 69 STUDIES ON THE STRUCTURES OF :FEBRIFUGINE AND ISOFEBRIFUGINE 1. Empirical Formula and Functional Group Tests•••••••• 72 2. Hydrolytic Degradation • • . . . . . • . . . . . . . . . . . . . . . . . . . . . . 89 3. Summary and Conclusions••••••••••••••••••••••••••••• 95 THE ESTIMATION OF CHLORIDE BY MEASURING TllE E.M.F. OF A SILVER, SILVER CHLORIDE, CHLORIDE IOH HALF CELL ••..•...• • IV. 23 Alkaloids • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tura. terials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • II. 13 104 STUDIES ON CAMBARUS CLARKII 1. Effect of Magnesium on the Heuro-muscular Junction •• 2. Effect of Perfusion on the Tost Closer Response••••• 114 119 - iii - !.!.'ABLES Page I. Effect of Different Developers on the Movement of Zones from 5- to 6-mg. Sa.mples of Crude Isofebrifugine Adsorbed from Chloroform on a Number 2, 15 cm. Column of Silicic Acid••••••••••••••••••••••••••••••••••••••••••••• 29 Effectiveness of Different Eluents for Isofebrifugine Ads orbed on Sili ci c Acid • • • • • . • . • • • • • • . . . • • . • • . . . . . . • • . . • 30 III. Summary of Some Experiments Showing the Double Zone Effect. 39 IV. Spectral Data for Fractions Obtained in Experiment 2 II~ v. VI. (Table III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ~uinine Coefficients of Febrifugine after Heating in Solution at 80° for Six Hours•••••••••••••••••••••••••••• 52 Distribution Coefficients for Isofebrifugine and Fabrifu.gine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Febrifugine and Isofebrifugine from Different Plant Material by the Fuller's Earth Method (Section 10) ••••••• 67 VIII. Results of Micro-analyses of the Alkaloids••••••••••••••• 73 rx. Molal Extinctions for Spectra Taken in Ethanol••••••••••• 81 x. Molal Extinctions for Spectra Taken in N/20 HCl •••••••••• 82 VII. PI.ATES I. II. Diphasic Action Potentials from Tust Closer Perfused with lOX ].iia.gries i \llll • •••••••••••••••••••••••••••••••••••• , •••• • o 117 Tracings of Records which Shovr the Effect of Perfusion••• 120 - i"lr - FIGURES 1. The isolation of isofebrifugine (exp. 2) •••••••••••••••••o Fage 14 2. Apparatus for the decomposition of isofebrifugine ••••••••• 16 3. The isolation of isofebrifugine and a crude active fraction (exp. 18) •• .... ••• .. . . . . . .. . . . .. . . . . . . .. . . . . . . . . . . . .. . . . .. 19 The isolation of isofebrifugine and crude active fractions, showing how the activity is spread throughout the various fractions (exp. 24) ••••••••••••••·•••••••••••••••••·•••••• 21 A typical chromatogram of the crude active fraction on recovered alumina ( exp. 70) . . . . . . . . . . . . • • . . . • . . . . . • • • . . • • • 37 , A typical chromatogram of the crude active fraction on new alumina {exp. 91) •••••••••••••••• ! ·•••••••••••••••·••••••• 44 Recrystallizations of febrifugine indicating the dimorphic nature of the compound•••••••••••~•••••••••••••••••••••••• 46 8. Chroroa:togra.m indicating the homogeneity of febrifugine..... 48 9. Adsorption isotherm for the crude alkaloids adsorbed on fuller's earth from E/20 hydrochloric acid•••••••••••••••• 57 Curves indicating the amount of fuller's earth needed to adsorb one gram of crude a l kaloids at different eff iciencies. 59 11. Absorption spectrum of febrifugine in ethanol•••••••••••••• 79 12. Absorption spectrum of febrifugine in y'20 HCl ••••••••••••• 80 4. 5. 6. 7. 10. - 1 P.ARI' I.- ISOLATION OF POTENI'IAL ANI'IMALA.RIA.LS EROM DICBROA FEBRIFUGA. Section 1. Introduction In the search for new antimalarials carried out in the United States during the recent war, interest in iftdigenous Chinese drugs was stimulated by the appearance of two papers in 1941 by S. K. Liu 112 . In these papers Liu gives considerable details regarding the isolation of a crystalline alkaloid, sinine, from Fraxinus malacophylla Hemsley, and gives pharmacological data indicating good antimalarial activity (presumably from the alkaloid) in extracts _of the plant. In the closing paragraph of each of his papers Liu mentions another anti-malarial herb also found in Yunnan and called "Chunine tree", the leaves of which contain a.n effective ingredient ne.med 11 Chunine"• He says "the fever relieving action seems stronger than (that of) the sinine leaf". Reputable workers in China 3 , Great Britain415 , e.nd the United States 6 were unable to duplicate Liu~s work with Fraxinus. No trace of alkaloids could be found, and no anti-plasmodial activity could be demonstrated. This of course threw considerable doubt on the ve r acity of 1. Liu, Shao-Kv.rang, Chang, Yao-teh, Ch' uri., Tz '-e-kwang, and Tan, Shih-chieh , Chine se Medical Jour. 59, 575-77 (June 1941). 2. Liu,Shao-Kwang, National Medical Jour. of China, 27, 327 (1941). 3. Letters from Dr. Needham to Dr. Woric"inEngland. 4. Tomkin and Work, nature., 156, 630 (1945). 5. Mukerji, Nature., 158, 170 (1946). 6. Koepfli and Mead, futerim Report to Board for the Coordination 0£ Malarie.l Studies , June 1945.-- - - any claims made by Liu. However, samples of Chanine obtained from Liu were found to possess considerable antimalarial activity. Chunine is the proprie- tary name given by him to the ground leaves of a plant identified as "from Saxifragaceae 11 • Further botanical identity is lacking although 8 some Chinese workers ( see for example Chang-Shaw Jang, ) think 1 t is the leaves of some Ch'ang Shan, probably ll• Febrifuga. The material was tested by Dr. Richardson for the Abbott Laboratories and fomlii to have in the F-1 test a .Q. of 0.1 to 0.25*. They report (see 6) obtaining a fraction from Ohunine with a g. of 5 to 10 in F-1. Koepfli and Mead 6 working independently also were able to obtain a fraction ot .Q. 4 to 8. Both groups agreed that the active fraction did not contain alkaloids. A scarcity ot material and the lack of botan- ical identity made a thorough investigation of Ohunine impossible. During the first half of 1943 Dr. Joseph Needham, on a British scientific mission in China, reported 3 that the Chinese workers were quite interested in another indigeneous drug called "Ch'ang Shan". This appeared to be a mixture of plants, including Dichroa febrifpga --- - - - - -- --- -------- -- -- - - - - - - -- ~ * ~ Q is the "quinine equivalent" which indicates the activity of the material relative to the activity of quinine. The letter and number of the test (F-1 in this ease) indicate the laboratories in which the test was carried out, and the conditions employed (species of Plasmodium, test animal, method of administ :e.rlhg ., the drug, etc). Most of the testing for the isolation work described later in this thesis was carried out under the supervision of Dr. K.K. Chen at Eli Lilly and Company. The test animal was the duck, infected with l• lophurae; and, in the main, samples were in~ected intravenously, l.• i.• S• For a discussion of the quinine aqui v·alent and the methods for testing see Wiselogle, Survey of !!u,.i malarial Drugs, 1941-1945, Edwards (1947). - 3 - and Clerodendron yunnanense (a detailed discussion will be given later) and was actually in use in the Chinese Army. Szechuan and Kweichow. It is said to be common in According to Dr. Yang Shih-Hsien, plant biochemist at the National United Southwestern University (formed from Peiping, Chinghya, and Nankai Universities), this drug contains only glucosides. Other Chinese workers, Dr. Tang Tang-Han of the Pharmacological Institute, West China Union Unive~sity, Chengtu, and Dr. Yeh Chiao of the Organic Chemistry Department, Wuhan University, Chiating, confirmed the absence of any alkaloids in Ch'ang Shan. Ch'ang Shan was relatively available, the Chinese workers he.d considerable hope for it, Chunine migh~ be the leaves of the same plant, and tests indicated Q' s of Oo05 to 0.15 for the crude ground roots. These facts made an investigation of Ch'ang Shan in this country appear to be a promising field of research. Since Ch'ang Shan is a generic name, and more than six different plants are known by this name in different parts of China, the question of botanical identity was a serious one during the early work on Ch'ang Shan. The supply of material was limited to such an extent that the isolation and especially the structure determination of the active principle might not be complete before additional plant material would be needed. With no certainty regarding the botanical identity, it might be very difficult to get additional samples of the same material. Furthermore, even if the isolation and structure determination could be completed, the value of the work as a contribution to the permanent literature would be considerably lessened if the identity of the plant was unknown. - 4 - Fortunately, by the middle of 1945 the botanical identity of Chungking Ch'ang Shan had been quite well established as Dichroa febrifuga, both by comparisons made at the Smithsonian by Dr. Walker, and by ini'erence from reports received from China. A brief summary of two communications from China, one by Pei Chien 7 and the other by Chang-Shaw Jani, is given below, to indicate the salient facts concerning Ch' e.ng Shan. 11 The antimalarial virtue of Ch'e.ng Shan was discovered about four thousand years ago by Emperor Sheng J\Tung, the King of Herbs". The root is recow.mended, but the leaves and shoots (called Shu Chi) are also effective against malaria. On account of its unpleasant smell and stronger irritant action to the stomach, Shu Chi is not so often used. Most authorities in China believe this Ch'ang Shan to be a Saxifragaceae, Dichroa febrifuga Lour. Because of the appearance of the roots it is also lmown as Huang (yellow) Ch'ang Shan and Chi Chi Kuo (chicken-bone) Ch'ang Shan. (Other Chinese names include Hengshan, Hytsao., Chi-shih-tsao, Ya-shih-tsao.) This Ch'e.ng Shan is a perennial sew~-evergreen shrub, something more than 9 feet high with many branches. The lee.ves are "simple, opposite, with or without fine hairs on the vein above and beneath the leaf, oblong-lanceolate, about 7. 8. Chien, The Chinese Anti-Tuia.la.rial Drug--Changshan, sent to Abbott Laboratories by A.G. Saunders from Sino-British Science Cooperation Office, Chungking (June 16, 1945). Jang, Chinese Medical Jour. April-June, 1944. - 5 - 8 inches long, acute on the base up to the petiole, with teeth along the margin". The plant bears a small, light blue flower and a blue, ellips- oidal berry. It grows mostly in the southern provinces of China along the Yangtze river, and is also found in India, Java, and the Phillipine Islands. It is abundant in Szechuan Province, but "it is said that those grown in Kwei chow are more precious". In addition to this yellow or chicken-bone Oh'ang Shan there are several others. Pei (white) Ch'ang Shan is identified as Mtlssaenda divaricata Hutchinson. This Ch'ang Shan and the Cinchona tree belong to , the same family, Rubiaceae, and it 1s not too sur:prizing to find anti- malarial activity reported. Hou (Japanese) Ch'ang Shan has been identified as Orixa japonica Thunberg. It belongs to the family Rutaceae, and grows in Korea, Japan, and in China south of t he Yangtze and along the sea coast. 11several Chi- nese botanists misus·ed Orixa .1aponica as the scientific name of yellow Ch' ang Shan." Haiohow (name of a city) Ch'ang Shan has been identified as Clerodendron trichotomum Thunberg . This plant belongs to the family Vergenaeeae, and is a small tree, about 30 feet high. It is very ord- inary in Kiangsu and Chekiang, but rare in the southwest provinces. There 1s a variety in Szechuan Province. Reported from Kunming in addition to yellow Ch'ang Shan. called there Shu or T'u Ch'ang Shan, are Chi Gu Ch'ang Shan, an Alstonia from the family Apooyanaoeae; and Dien (the old name for Yunnan) Oh'ang Shan, Olerodendron yunnanense from the Verbenaceae family. Still another, Tou (local} Ch'ang Shan, is reported as Hytlrangea thunbergii. - 6 The literal translation of Ch'ang Shan is "mountain herb". The above discussion indicates just how apropos this translation is. A sample of Ch'ang Shan obtained from one part of China may be very different from one obtained in another part. Furthermore, it is by no means certain that a given sample of Ch'ang Shan is botanically homogeneous. The isolation experiments described in this thesis were carried out for the most part with Ch'ang Shan obtained from Chu.ngking, and fairly well identified as R• febrifuga. It is to be emphasized, however, that the samples were not identified at the time of collection, but were i n the main bought in t he open market in Chungking fr om Chinese herbalists , either as chopped roots or as whole roots. Further- more, any chopped root material may have been treated with hot water _or steam in order to facilitate chopping. Such a treatment may very well alter the active principle present in the roots. The amount of Oh'ang Shan available in this country has been strictly limited. For the sake of a complete record it seems desira- ble to list here the material available for the work reported in this thesis: I&!.A:- five pounds of chopped roots sent to the N.R.C. by Dr. P.Z. King of the National Health Administration, Chungking. This material was labelled Ch'ang Shan,~- febrifuga. Lot l:- about 300 pounds consisting of six pooled samples which Eli Lilly and Company obtained from Chinese drug stores in various parts of this count ry and abroad~ This material was mostly in the fo1-m of chopped roots, and although bought as Ch'ang Shan, there i s no knowledge concerning the original source or its botanical iden- tity. This material is referred to as San Francisco Ch'ang Shan and had a .Q. of o.oa in I-5 test. lQl..Q.:- about 500 pounds of whole roots purchased in Chungking by an agent of Eli Lilly and Company. had .Q. 0.16 in I-5. A sample of the powdered roots Most of the work reported in this thesis was done with this material. Lot D:- approximately 100 pounds of whole root material label- led Ch'ang Shan, ll• febrifuga sent to the N.R.C. by Dr. P.Z. King of the National Health Administration of Chungking. Presumably this material was bought in the open market in Chungking. In summary then, at the beginning of 1945 indigenous Chinese herbs appeared to offer at least two fruitful avenues of research aimed at finding new antimalarials. The first was Chunine, the leaves of a plant of obscure botanical identity (possibly 12• febri£~) containing an active principle probably non-alkaloidal in nature, and being available in quantities far too small for a satisfactory investigation. The second was a Ch'ang Shan fairly well identified as the roots of 12• ..!fil2.rifµga, available in sufficient quantity for a significant amount of research to be carried out, and containing an active principle probably non-alkaloidal in nature. The work of this thesis has further indicated that Chungking Ch'ang Shan is indeed ;Q_. febrifuga, and that the active principle is in fact an alkaloid. Furthermore, both Chunine and J!• febrifuga leaf material have been shown to contain this same alkaloid, so presumably Chunine is at least in part the leaves of D. febrifuga. A recent publication by Tsu9 indicates the potentialities of 9. Tsu, Cheng Fang, Jour. Tropical Medicine~ Hygiene , £,Q, 75 (1947) Chtang Shan as an antimalarial. Tsu gives clinical data on the use of water extracts of Ch'ang Shan for treatment of 587 out-patients and 81 hospitalized patBnts suffering from malaria. Of 322 out- patients with vivax malaria, 67% were cured, and of 253 with faleiparum malaria, 63% were cured. There were only six relapses out of 67 hospitalized cases of tertian vivax malaria, and 11 of 14 cases of ~aloiparum malaria were cured. Pharmacological studies on the pure, active principle are reported in Section 12. .,. 9 - Section 2 Early Investigations of Ch'ang Shan and D. Febrifuga. In 1945, since sufficient material was available, Drs. Koepfli and Mead at this institution began an investigation of Chungking Ch'ang Shan (D. febrifuga). It was my pleasure to join them in this work in November of that year. Before giving a detailed account of the work which followed, it would be desirable to examine the small amount of existing chemical literature on Dichroa febrifuga Lour. The first chemical investigations on the plant were made by Hartwich10 who in 1897 reported the isolation of e. glucoside "Dichroin". No details are available to this writer. In 1945 Tomkin and Work 4 report- ed that e.n acid extract of Ch'ang Shan (reputed to be D. febrifuga) g~ve no sign of alkaloids, but ga-ve considerable activity age.inst P. galinacemn. A few months later in 1946, Hooper 11 reported that some fifty years earlier he had examined D. febrifuga and found no alkaloids. At about the same time Jang, Fu, Wang, Huang, and Chou12 reported the isolation from Ch'ang Shan (D. febrifuga) of four substances, two neutral principles, Dichroin A (m. 228 to 2300) and Dichroin B (m. 179 to 181°), and two alkaloids, Dichroine A (m. 230° dee.) and Dichroine B (m. 237 to 238° dee.). Of the four, Dichr- oine B was the only one active against P. galinaceum in chicks. This is the extent of the literature concerning the chemical investigations of the plant, except for a brief communication by Koepfli, Mead, and 10. 11. 12. Hartwich, Neue Arzneidrog, 1897, 125 Hooper, D.~ture, !.§.?, 106 (1946). Jang, Fu, Wang, Huang, Chou, Science, 103, ____,... 59 (1946). - 10 - Brockman13 which reports in brief some of the work reported in this thesis. The work carried out by Mead prior to November 1945 and the conclusions drawn from it may be sUJm11arized as follows. 1.Tne11ewere two approaches to this problem; either attempts could be made to isolate pure compounds from the plant and test such compounds for activity, or the various crude fractions obtained in different steps of a schema.tic isolation could be tested and attempts made to isolate pure compounds from those fractions which had a high concentration of activity. The latter approach was chosen because it is the more direct, because Eli Lilly and Compe.ny kindly offered to carry out the necessary testing, ~nd because preliminary experiment had indicated that crystalline material of any sort was difficult to obtain. The bio-assays were of course time consuming and tended to delay the progress of the work. 2. The ground root material is difficult to exhaust of ac~ivity by extractions with various solvents such as methanol, ether, water, and mixtures of these. The order of 10 to 20% of the activity was extracted by repeated extractions with alcohol and water. There was the definite possibility that activity was being destroyed during these extractions. 3. Working with these alcohol and water extracts, it was po~s~ble to obtain a concentrate with a Q of 40. (The procedure for obtaining the concentrate will not be given here, since it was, with minor variations, 13. Koepfli, Mead, and Brockman, Jour. Amer. Chem.~•,~, 1837 (1947). - 11 - t 'he same as those given in Section 3.) This fraction gave a. strong test for alkaloids ,vi th Mayer's Rea.gent*. From this fraction a. crystal- line substance was obtained, melting crude at 150 to 152°. When recrys- tallized from hot acetone the melting behavior changed, the sample now partially melting at 136 to 137° and completely melting at 150 to 151°. This material was not tested as an antimalarial, because further work indicated that it was an artifact produced by heat (see 4 below). 4. Working with a. similar extra.ct it was possible to isolate a base melting at 128°, and from a crude mixture a hydrochloride which melted at 210 to 212° (dee.). The 128°-melting base was tested and found to be inactive (~ < l.2i). (This. base was later shOl/m to have some antimalarial activity; see Section 12.) Furthermore., when this base was heated for a short time above its melting point, and the mel-:J; · recrystallized, a new substance melting at ca. 150° was: obtained. During this heating a. loss of ~• 1% of the weight was observed. This new material gave a. hydrochloride which melted at ~• 212° (dee.). The 1280..ba.se formed a very hygroscopic hydrochloride which could not be characterized. The ultraviolet adsorption spectra of the two bases were essentially the same. Attempts to recrystallize the 12·a 0 -base were unsuccessful., the material was slowly soluble in hot benzene, alcohol., and acetone., but would not crystallize out on cooling. When the solvent was evaporated from such solutions, a gummy residue was obtained. This gum would go particulate when triturated with acetone * A solution of 13.55 g. of mercuric chloride and 50 g. of potassium iodide in one liter of water. - 12 - in the cold. After such treatment, the melting point behavior might change, the compound partially melting at 128° then apparently resolidifying and finally melting at 150°. 5. The general behavior of the compounds isolated and the fact that only a small fraction of the activity expected from the Q of the original material is isolated in these active concentrates indicate that the active principle is a very labile molecule, that the isolation procedure is a poor one, or both. 6. Since the 128°-base was inactive, end since the 137°-and 151°- bases are artifacts of the 128°-base and were presumed to be inactive (it was later shown that this is not th~ case) there were several possibilities regarding the active principle: the 128°-base might be an artifact of the still unisolated active baxe (the implication being that the active principle is an extremely labile substance); the active principle might not be an alkaloid at all; the active principle might be a base, but present in such small quantities that its; isolation is difficult. - 13 - Section 3 Isolation of Inactive Isofebrifugine and of a Crude Active Fraction When I began working on this investigation, Dr. Mead was leaving for Occidental College, so in order to avoid unnecessary duplication it was decided that he would work with the leaf material and I would work with the root me.terial of the plant. Dr. Mead's exper- iments had indicated that the active principle present in the roots probably has basic propertie& and probably is quite labile. It seem- ed pertinent then to duplicate, more or less, his isolation procedure, in order to become acquainted with some · of the properties of the material to be worked with in a new attempt to find the active principle. Eli Lilly and Co. was kind enough to carry out large scale extractions of the crude root material with various solvents, and after evaporating to dryness send us the residues for further work. Such dried extracts were used for the isolation indicated schematically in Fig. 1. Emulsion formation was ver-J troublesome during these operations. The butanol extractions in steps 3 and 6 were re- commended by Mead because they seemed to remove undesirable amorphous material and make the final basic fraction cleaner. The particulate base obtained in step 8 was washed with butanol and then acetone. melting behavior was as follows: Its darkened and melted at 126 to 127° but not to a clear liquid, rather to a viscous cloudy gel which seemed to clear and flow at ca. 1470. This 1270 - base was apparently the same one isolated by Mead, and found to be inactive as an antimalarial. About 168 mg. of this - 14 - Fig,ure l. The isolation of isofebrifugine (exp. 2). 1. Metb.anol--vra.ter extracts of Lot C Ch' ang Shan: and HCl to make pH 2 to 3. Filter. 2. Aqueous solution., pH 2 to 3: Extract with CHC1 3 . Solid: 3. Aqueous phase., pH 2 to 3: BuOH. Extra.ct with CHCl3 Phase: Discard. 4. Aqueous phase., pH 2 to 3: Ms.ke basic (pH~• 8) with NazC03 and extract with CHCl3. BuOH phase: Discard. 5. CHCl3 phase: Extract with 0.1 N HCl. Aqueous phase: Discard. 6. Aqueous phase., pH l: CHCl3 phase: Discard. 7. Aqueous phase, pH 1: Make basic with Na2 C◊.3 and extract with CHCl3. 8. CHCl3phase: Dry with Na2S04 and evaporate until~• 1 ml. of d~rk oil remains. Triturate with acetone to obtain particulate base, m. 127° (see text). Extract with BuOH. Shake with water Discard. BuOH phase: Discardo Aqueous phase: Discard. - 15 - crude base was obtained, and although·it was not the active principle being sought, it seemed desirable to investigate its properties to a lim1.ted extent. Some solubilities were determined roughly on 1-mg. samples. At room temperature the compound vras not very· soluble in acetone, butanol, isopropyl alcohol, ethanolp benzene, dioxane, or ligroin. At 100° it was quite soluble in benzene, dioxa.ne, and slOilfly in acetone, but it would not crystallize out on cooling and agitating. A little of the free base was dissolved in absolute alcohol with the aid of heat, and dry HCl was passed into the solution until it was saturated. No precipitate fo~med, and the addition of benzene followed by evaporation to a small volume gave a very hygroscopic jelly, which could not be crystallized. A 150-mg. sample of this 127°-base was decomposed in the apparatus diagrammed in Fig.2. The sample was heated for tvventy-five minutes a.t 135 to 140°, after the apparatus had been evacuated to 0.8 nun. pressure. The pressure increased to ca. 40 mm. during the heating, but when a cooling bath (dry ice in isopropyl ether) was placed around receiver~, the pressure decreased again to~• 2 mm., and a white crystalline solid collected in the cooled tube. room temperature. This receiver was sealed off and allowed to warm to The solid melted to a clear liquid. A freezing point determination was made, and the material was fotu1d to melt at -15 to -14°. The amount of liquid appeared to be~• 1 to 2 microliters, which correspond• ed to the loss in weight of ·bhe sample in the boiler (1.92 mg. loss., 1.2%). The boiling point of the liquid vras taken by the Emich method, and found to be 83° the first time, and 87° the second time on the same sample. The - lG- Micro stopc.ock Tygon / t u u i ng 1 ~/1 3 atc~o taper --- B Ce.pill a r y n1f!X;.017i.eter Scale: - 17 - liquid gave an iodoform test rapidly in the cold, and is believed to have been a mixture of acetone and water. The residue in the boiler was taken up in hot acetone and allowed to crystallize. The crystalline sample obtained gelled at 134° and melted and darkened at 151°. When this material was recrystallized from hot acetone it melted completely at 134 to 136°. The mother liquors from these crystallizations were taken to dryness a.nd heated again, this time for~• 30 min. at 140 to 150°. It was possible to isolate from the residue a small amount of light colored solid . melting at ~• 192°. : On evaporation, the mother liquors gave a very dark oil which could not be crystallized. The results of the isolation and heat treatment described above were, in the main, in agreement with the earlier results obtained by Dr. Mead~ First, a base melting at ~• 127° had been isolated. to be inactive as an antimalarial. This base was known It has since been isolated in a pure state and has been named 11 isofebrifugine" (m. 129 to 130° cor.). For convenience this na.me will be used in the following discussions. Second, isofebrifugine is labile to heat, being converted to other compounds (presumably inactive too) melting at~• 136, 151, and 192°. Third, no satisfactory method for recrystallization of isofebrifugine had been found. Since isofebrifugine isolated by this above procedure was not . the active principle, and since it was assumed that artifacts formed from it by heat were likewise not the desired principle, it remained to examine the mother liquors, to determine whether the activity was still there, or whether it had been destroyed. The acetone soluble - 18 - ·portion of the mother liquors was taken to dryness, and then taken up in dilute hydrochloric acid. The filtered acid solution was made basic with sodium carbonate and extracted with chloroform. The chloroform extract was dried with sodium sulfate, taken to dryness, and sent for testing. This crude material was found to have a~ of 10. The amount of :material obtained indicated that only a small fraction of the activity of the original extract had been concentrated in this fraction, but at least all of the activity had not been destroyed. Since, these mother liquors had stood around for several days before being worked up, it was easily possible that considerable decomposition had taken place if the active principle was as labile as it was believed to be. Attempts to obtain a crystalline base from this active fraction isolated from the mother liquors were unsuccessful. Amorphous precip~ itates were obtained with chloroplatinic acid, picric acid, and mercuric chloride. The material was dissolved in acetone and fractionally precip- itated with ether, but this too led to amorphous precipitates. The amount of crude active fraction available above was the order of a few hundred milligrams, and it was soon necessary to isolate another batch. The method is schematically indicated in Fig. 3. Because of the suspected labile nature of the active principle, all operations were carried out as rapidly as possible in order to keep the material in solution a minimum length of time; and where convenient (always when the solutions were basic) the operations were carried out in a cold room at ca. 4°. Again isofebrifugine, the 127°-base, was isolated along with its heat artifacts, melting approximately at 134 and 150°; and again the activity was found to remain in the mother liquors from these bases (step 9). - 19 • Figure~. The isolation of isofebrifugine and a crude active fraction (exp. 18). 1. Water extracts of Lot C Ch'ang Shan: Shake with water and HCl to make pH 2 to 3. Filter off and discard solid. Extract first with CHCl3, then with BuOH and discard extracts. Make aqueous phase basic with Na2C03 and extract with CHC1 3 • 2. H20phase, pH 8 to 9: Extract with BuOH. 6. 3. BuOH phase: Extract with 0.2 N HCl. H20 phase: 7. Discard. 4. 5. 9. CHCl3 phase: 0.2 N HCl. Extract with CHCl3 phase:i H20 phase: II/Take Discard. ' basic with Na2C03 t::=:======t and extract with CHCl3. BuOH phase: 8. CHCl3 phase: Dry H20phase: H20 phase: Make Disc~rd. with Na2S04 and Discard. basic with Na2C03 evaporate to and extract with BuOH. brown-o~a.:gge oil. When triturated with acetone H2o phase: this gave 246 mg. of base m. BuOH phase: Dry Discard. 126 to 151°, and a second with Na2S04 and crop of 80 mg. m. 1340. evaporate to dryness-(41 mg.) as 18-43. _g > 30. The mother liquors were taken to dryness, taken up in dilute HCl, made basic with Na2C03, extracted with CHCl3. This was taken to dryness, triturated with acetone to get 38 mg., m. 127°. The acetone mother liquors were taken to dryness (124 mg.), taken up in HCl (pH 3), and sent for testing. A Q of 25 to 40 was found. - 20 - -A nother very significant fact was observed during this experiment, namely, All the activity was not extracted from the basic aqueous solution by chloroform (end of step 1) for additional activity, highly concentrated (g > 30), was extracted later by butanol (steps 2 to 5~. The small a.mount of active concentrates again did not account for more than a small fraction of the original activity. Since a large portion of the above concentrates were used for the testing it was necessary to carry out another extraction. lined in Fig. 4. This is out- Fraction C was triturated with acetone and yielded~• 70% of isofebrifugine. In this experiment the total recovered activity (based on Q values) was ca. 36% (45.7/128). Since the~ of a sample may be in error by as nruch as a factor of 2, this recovery is fairly satisfactory. Although the activity was spread throughout the various frac- tions, evidently a considerable concentration had been effected in E which had a Q of 100 (one of the highest values of Q every observed for a naturally occuring substance). However, this fraction amounted to only 153 mg. of crude material. Root material was strictly limit- ed; and if any work were to be done on structure determination after the isolation was completed, it seemed undesirable to sacrifice any more root material than necessary to complete the isolation of a sample of the active principle. If even a small sample of the pure active principle could be isolated, then enough of its properties probably could be determined to work out a better scheme for its isolation. The most powerful method known for the separation of small - 21 - Figure 4. The isolation of isofebrifugine and crude active fractions, showing how the activity is spread throughout the various fractions (exp. 24). 1. Water extract of Lot.£ Ch'ang Shan: ma.lfe pH 2 to 3. Filter. Shake with water and HCl to 2. Solution, pH 2 to 3: 3. H20 phase, pH 2 to 3: Make basic with Na2co3 and extract with CHCl3. 4. H20phase; pH 8 to 9: Bu0H. j Solid: Discard. Extract with CHC1 3 Extract with 7. CHCl3 phase: Take to dryness·as A. CHCl3 phase: Extract with N HCl. 8. , CHCl3 phase: H20 phase: !Discard. Make basic with Na2co3 and extract with CHCl3. CHCl3 phase: Evaporate to dryness as c. H20 phase: Discard. - 5. Bu0H phase: N HCl. Extract with Make acid with HCl and extract with Bu0H. 9. H20 phase: 6. H20 phase: Bu0H phase: Make basic Take to with Na2C03 dryness as B. and extract with CHC13. Bu0H •phase: Take to dryness as F. H20 phase: Discard. - - Take to dryness as E. H20 phase: Discard. CHC1 3 phase: Sample Weight (g.) Q Q. x Weight. Totals Starting material 400 0.32 128 128 2 6.8 16 7.6 15.3 45.7 A B c 'E" F 3.4 1.9 0.761 o.n.s3 11.4 8 15 100 toxic ' - 22 - quantities of organic compounds with a high degree of purity is chromatography. This method appeared to have the greatest possibility of success for the a.mount of material available, and after many disappointing experiments, it finally led to the isolation of the pure active principle. ;;. 23 - Se-ction 4 Introduction to the Chromatography* of the Alkaloids of D. Febrifuga. The development of a satisfactory chromatographic method for the s19paration and isolation of the active principle required considerable time and effort. In the first place, the technique was entirely new to me, and although I was quite aware of its potentialities, I was not fully cognizant of its pitfalls and obstacles. In the second place., the very nature of the method, which requires an empirical search for a suitable adsorbent, developer, and eluent, is such that considerable time may be spent more or less fruitlessly. In the third place, this particular problem was complicated by the fact that the compounds being sought were colorless, and essentially non-fluorescent, l•~•, the fluorescence in concentrated solution was so weak that it did not show at all when the substance was spread out on a column. Moreover, no specific color tests could be found which would be suitable for locating a chromatographic zone. In the fourth place., the substances were very insoluble in ligroin and benzene, two of the most desirable solvents for chromatography. This meant that the more polar solvent., chlorofonn.., had to be used., with the possible disadvantage that com- * I would like to express here my appreciation both to Dr. W.A.Schroe.der who kindly introduced me to the techniques of chromatography and who guided my early experiments in that field, and to Prof. L. Zechmeister who was always willing to discuss the problems which arose during the course of the chromatographic investigation and who offered many timely suggestions which helped lead these experiments to a successful conclusion. -- 24 - -pounds might be too weakly adsorbed from such solutions. On the other hand., a method of analysis for the alkaloids seemed available, viz., the ultraviolet absorption spectra. Although at this time it was not certain that isofebrifugine was pure, itdi.d have a very characteristic absorption spectrum (see Part II), and furthermore, the most active crude fraction, E, Fig. 4 (Q. 100) although non-crystalline had an absorption spectrum very similar to that of isofebrifugine. Thus, unless the active principle composed only say 10% of the fraction and did not have a very strong absorption in the ultraviolet, it seeme~ reasonable to associate the characteristic spectrum of isofebrifugine with the unknown active principle. Because the methods for locating zones and analyzing eluates, were common to all the chromatographic experiments., a brief discussion of these methods will be given here to avoid unnecessary repitition later~ 1. Possible zones were located by three different methods: The columns were examined in ultraviolet light for possible fluorescence. The crude material being chromatographed fluoresced brightly under ultraviolet light (a G.E. Mazda Purple X bulb with a Corning 5840 filter was used as a light source), and some fluorescence could be seen on the columns. However, it came to light during the investigation that the active principle had practically no fluorescence. 2. The columns were streaked with alkaline permanganate; 0.5% Na0H and 0.1% IOm04 were the concentrations chosen after a series of different concentrations were tried. Isofebrifugine rapidly gives a green color with this strength of the reagent. The columns were post- washed with ligroin or chloroform to remove from the column the developer - 25 - which usually contained enou gh alcohol or acetone to interfere with t he streak. This streak was not particularly good, but no other color test could be found. The test was frequently indistinct, and when small a.mounts were being looked for, they often did not show up at all by streak. 3. The columns were arbitrarily sectioned and eluted into sep- arate flasks. The eluates were evaporated to dryness at room temper- ature (to avoid possible decomposition from heat), and the residues were taken up in ethanol, !",ethanol, or y20 HCl and examined spectro- photometrically, in a Beckman, Model 1 DU, Quartz Spectrophotometer. This method was always used as a check on the first two methods, as a means of quantitative analysis, and as a means of partial identification. In genoral, the entire spectrum of a fraction was taken to detect any . changes which mi·g ht take place on chromatographing. In the main, no changes were observed, and in later experiments only a few points of the spectrum were taken near the important maxima and minima, so that the ratios of t he extinctions at t hese various extremes could be compared. If these ratios were not si gn ificantly different from those of the spectrum of isofebrifugine, it was assumed that the spectrum of the fraction bein g tested we.s also not significantly different from that of isofebri fu gine. pared, i.~., Where it seemed advisable, blank colunms were pre- du plicate colurrms with the omission of t he sample to be· chromato graphed, and corrections for the spectra. obta ined were applied. Knowing the 1% extinction of the starting material at a given wavelength, and assumin g Beer's law to hold, it was possible to determine t he r ec overy of material in a rou ghly quantitative way from the extinctions -- 26 - and volumes of the various eluates. Using these methods, more than 100 columns were prepared and exa.rnined before the crystalline active principle was finally isolated. A detailed account of these experiments seems unwarranted here, and only a brief resume of the work will be given to indicate the nature of the experiments and the results. Several adsorbents were tried, and they will be considered separately in the following sections. A careful characterization of isofebrifugine seemed desirable since it was one of the substances lmown to contaminate the active principle, and since it was probably structurally related to the active principle and any facts concerning it ntlght help later in the structure determination. Also, since there was available more isofebrifugine than crude active fraction, preliminary experiments were usually carried out with isofebrifugine. The aim of the follovdng chromatographic experiments was first to find the conditions under which one or more well defined zones could be developed. Then, if any such zone could be eluted, evaporated, tak- en up again in chloroform, and rechromatographed under the same conditions to give a similar zone, it would be assumed that a pure compound had been isola.ted. - 27 - Section 5 Silicic Acid as a Chromatographic Adsorbent for the Alkaloids The adsorbent used in these experiments was Merck Rea.gent Silicic Acid mixed with one-half part of Celite. Nmnber 2 columns, 15 cm. long were used, and were prewashed with one of two standard prewashes to give a reproducible adsorbent. The strong prevrash consisted of 10 ml. of an- hydrous ethyl ether, 30 ml. of 1:1 acetone-ether mixture, 25 ml. of ether, 30 to 60 ml. of ligroin, and 2 ml. of chloroform, reproducible, strong adsorbent. This prewash gives~ The weak prewash consisted of 30 ml. of ether, 30 ml. of absolute ethanol, 60 ml. of ligroin, end 2 ml. of chloroform. This prawash gives a much weaker adsorbent than the strong pre- wash. This method for obtaining a standard adsorbent has been thoroughly tested14 , and was recommended to me by Dr. W.A. Schroeder. Approximately 5-mg. samples were introduced to the column from 5 ml. of chloroform. The sample was washed on with 2 ml, of chloroform, and this was followed by the desired developer. After the desired amount of developer had been dravm through the column, it was post\vashed with 60 ml, of ligroin (60-70). The results obtained using silicic acid as an adsorbent were in , general no:t encouraging. The :main difficulty was simply that this ad- sorbent was too strong, even when prewashed to be weak. 14. A large percent- (a) Le Rosen, A.L., Jour. Amer. Chem. Soc., [l, 1683 (1945) and (b) Schroeder, W.A., "Annals of the New York Academy of Sciences", (in press). (c) Trueblood, K.N., Thesis , Calif. Inst. of Tech. fill, 87 (1947). - 28 - age of the material was adsorbed at the very top of the colUlll!l, and was very difficult to move. Solvents which are normally strong eluents were found to be weak developers. Some typical results indicating the types of developers and eluents tried are given in Table I and Table II. None of the solvents tried as developers showed any great promise. Even pure acetone moved the top zone onlyver--J slowly. The material which appeared in the zone between 50 and 70 mm. on the strong adsorbent with 3:1 chlorofonn-ligroin developer fluoresced blue under ultraviolet light, had a slight, non-characteristic adsorption in the ultraviolet, and a.mounted to less than 5% of the isofebrifugine used as starting material. These chromatogrruns indicated, then, that the isofebrifugine isolated by the methods of Section 3 was not pure, but contained at least traces of another compound. Two eluents, a mixture of -ethanol, chloroform, and concentrated hydrochloric acid, and a mixture of diethyla.mine and benzene, appeared to have some promise, but both had drawbacks. The mixture of ethanol, chloroform, and concentrated hydrochloric acid was found to elute as high as 60% (see TableII) of the material. Evaporation of the resulting so- lutions, which were apparently colorless, gave colored residues, which were of course hydrochlorides if the material was indeed an alkaloidal base. Before this material could be rechromatographed it would be ne - cessary to convert it back to the free base. of this step were unknown. The quantitative aspects Furthermore, the a.ppearance of color in the residue seemed to be an artifact and not something eluted from the column; therefore, it seemed wise to look for a simpler method which had less chance of altering the active principle. - 2.9 - TABIB I Effect of Different Developers on the Movement of Zones from 5- to 6-mg. Samples of Crude Isofebrifugine Adsorbed from Chloroform on a Number 2, 15 cm. Column of Silicic Acid. Prevmsh Developer Vol. (ml.) strong 3:1 CHCl 3-ligroin 30 0-2 25-40 strong 3:1 CHC1 3-1igroin 50 0-2 50-70 strong 3:1 CHC1 3-ligroin 50 0-2 50-70* wee.k 3:1 CHCl 3-ligroin 30 0-5 weak 3:1 CHCl3-ligroin 50 0-4 weak 1:4 EtOAc-benzene 100 0-4 weak acetone 45 0-10 weak acetone 250 10-15 Zone positions (mm. from top) * This 50-70 zone was rechromatographed under duplicate conditions, and was found to appear in the same place on the column. ' - 30 - -TABLE II Effectiveness of Different Eluents for Isofebrifugine Adsorbed on Silicic Acid Prewash strong weak Sample mg. Eluted mg. percent 1:9 EtOH-EtzO 5.7 o.o 0 1:4 EtOH-CHCl3 5.7 0.5 9 EtOAc 5.2 0.5 10 1:4:0.007 EtOH-CHC1 3-12N HCl 4.8 2.0 42 1:4:0.015 EtOH-CHC1 3-12N HCl 5.0 3.0 60 1:4 EtOH-EtzO 5.0 0.5 10 acetone 6ol 1.9 31 acetone (let stand 1 hr.) 5.8 1.1 19 1:1 Et2NH-CHCl3 5.0 3.3 66 1:1 EtzNH-benzene 5.9 4.1 70 Eluent - · 31 - The mixture of diethyla.mine and benzene eluted 70% of the material, but again evidence of artifact formation was found. A colored substance was formed in sufficient quantity to cbscure the spectrum of moderate quantities of the original material. This artifact was found to arise from the inter- action of the diethylamine and the benzene. Diethylamine, carefully fraction- ated through a 60 cm. column of glass rings, left no visible residue on evaporation., e.nd alcohol used to rinse the flask gave a very slight ab.sorption in the ultraviolet. Benzene similarly fractionated likewise gave no res- idue on evaporation, and alcohol used to rinse the flask gave very slight : absorption. When a mixture of these two was evaporated, however, a brown oily residue was left in the flask, and this material in alcohol had a very considerable ausorption both in the ultraviolet and in the visible. The use of this eluent, then, would contarninate any product isolated, and render uncertain the results of any spectrophotometric analysis. Most of the experiments discussed above were done with inactive, non-recrystallized isofebrifugine. A trial with a very active crude frac- tion indicated that similar results would be forthcoming. The scarcity of material made further experimentation with silicic acid seem undesirable, at least until other adsorbents had been given a trial. - 32 - • Section 6 Calcium Hydroxide, Talc, and Calcium Carbonate as Chromatographic Adsorbents for the Alkaloids and the Purification of Isofebrifugine The adsorbents used for these experiments were Shell calcium hydroxide, talc from an unirlentified source, gfild Merck Heavy Powder Calcium Carbonate, each mixed wi)th one-half part of Celi te. A brief examination of these adsorbents indicated that the compounds being worked with are weakly adsorbed from chloroform. Calcium hydroxide and talc were not investigated beyond a preliminary trial. Calcium carbonate was investigated briefly, and a few useful facts were indicate.m. The ma~erial is quantitatively eluted from calcium carbonate by 1:1 ethanol-chloroform mixture. Several diffuse fluorescent zones were found to be poorly developed by chloroform. These zones on rechromatographing did not appear in corresponding positions, but in the main were found to spread throughout the column. Some fluorescent mate- rial was strongly adsorbed and stayed at the top of the colUlll!l. when developed with chloroform; some fluorescent material was not adsorbed and ran through into the filtrate when sufficient chloroform had been added to wet the entire column. It was also found that isofebrifugine was rapidly washed into the filtrate by chloroform. A 100-mg. sample of impure isofebrifugine was dissolved in chloroform, and this solution passed through a number 2 column of calcium carbonate and washed through with an additional volume of chloroform. The colu.lTlll retained 3 mg. of 'fluorescent material. The filtrate was ... 33 - Fecrystallized by dissolving the material in chloroform, adding 1½ volumes of ethanol, and evaporating the solution slowly with nitrogen until the volume was reduced to about one-quarter of the original. This solution on standing in the ice box gave colorless crystals, which after two recrystallizations amounted to 40 mg. and melted sharply to a clear, colorless liquid at 128.7 to 12·9.5o (car.). This was: the first time a satisfactory method for recrystallizing iso~ebrifugine had been found. The base was inadvert- ently lost, but was believed to be pure isofebrifugine. later (see Section 7) substantimted this belief. Evidence obtained A chloroform solution of this compound had practically no fluorescence under the ultraviolet light. Certain anomalous results were obtained when the crude active fraction was chromatographed on calcium carbonate, and developed with chloroform. Three fractions with the absorption spectrum of isofebrifugine were apparent: one adsorbed at the top, one adsorbed about half way down the column, and one appearing in the filtrate (presuma.b°Iy isofebrifugine). When either the top zone or the middle zone was rechromatogra.phed under similar conditions, the same three fractions were observed, with a large percentage of the material appearing in the top and middle zones on the column. Such an effect might be observed on an overloaded column, but subsequent work with alumina. (see Section 7) indica.ted that this; was very likely a "double zone" effect. (For a discussion of the double zone effect on silicic acid see reference 14 b.) About 10 mg. of crude active fraction was dissolved in chloroform and passed through a number 2, 15 cm. column of calcium carbonate and the column developed with 60 ml. of chloroform. The eluate from the column was found to contain ca. 5 mg. of material having a. _9. of 100, and the - 34 - filtrate, .Q!.• 5 mg. of material with a ,9. of 20. Evidently the active principle was adsorbed preferentially. Although in these preliminary trials ealeium carbonate showed some promise as an adsorbent, it had its drawbacks, and it was considered desirable to try some preliminary experiments with alumina before making a thorough investigation of calcium carbonate. As indicated in the next section, alumina turned out to be a very fine adsorbent for the separation desired; and as a result, calcium carbonate was not further investigated. - 35 - Section 7 Alumina as a Chromatographic Adsorbent for the Alkaloids and the Isolation of Pure Febrifugine The discussion of this adsorbent will be somewhat more detailed than those of the other adsorbents, because alumina was found to be by far the best adsorbent for the problem at hand. The adsorbent used was ALORCO, Grade F, minus 80 mesh alumina, reground to pass 200 mesh, and mixed with one-half part of Celite. At the beginning o~ this in- vestigation there was no alumina in stock, and a very limited quantity of used adsorbent was kindly made available to me by Dr. Zechmeister's group. It was necessary to clean up and activate this mmterial before use. This was carried out by a procedure recommended by Dr. John Sease. The material was washed thoroughly with methanol, partially dried, placed in a porcelain dish, and set afire. After the methanol was burned out, th~ dish was heated with a Meeker burner until the pov1der no longer bubbled, then for an additional 30 to 60 minutes. The body of the povrder was at ca. 300 to 350°, and the bottom of the dish was heated to a dullred color. This ignited alumina was then partially deactivated by adding to it the desired amount of water and shaking it for several hours in a sealed bottle on a mechanical sh~er. Some preliminary experiments indicated that the best adsorbent was one containing 4 g. of water per 100 g. of alumina. The alkaloids were reasonably strongly adsorbed by this adsorbent :from chloroform, and could be more than 70 to 75% recovered by elution with 1:1 methanol- - 36 • c~oroforrn mixture. Most of the colored material in the crude fractions of alkaloid was adsorbed at the very top of the column and could easily be removed by shaving off the top 1 to 2 mm. of the column. The general method of the investigation of alumina was very nearly the same as that described for silicic acid. Somewhat smaller samples (2 to 3 mg.) were used on number~ columns to avoid any possibility of overloading the column. Examination under the ultraviolet was of little aid, since the fluorescence either staye~ at thewry top or moved through the column at the solvent rate into the filtrate. The alkaline perman- ganate streak, although not particularly distinct or sensitive, was the best method available for locating zones on the column. Any zones in- dicated were always eluted, and the eluates, were examined spectrophotometrically. Spectra were taken in methanol so that the samples could be easily recovered by evaporation, and could be rechromatographed if desired. After some experimentation using different developers, appropriate conditions were found which separated the crude active fraction into three main divisions: a zone at the top of the column, a zone about half way down the column, and the filtrate. Each of these three divisions had an absorption spectrum very similar to that of isofebrifugine (see Part II). The conditions and results for a typical chromatogram are outlined in Fig. 5. The three divisions which resulted from chromatographing the crude material looked most encouraging, but as these fractions were . rechromatographed a most distressing phenomenon was observed. A chromato- gram of zone A (Figo 5) under the same conditions as the original chromatogram gave the original three divisions,~. top zone, a middle zone, and a· small amount of material in the filtrate. Likewise, a chromatogram of - 37 - -Figure 5. A typical chromatogram of the crude active fraction on recovered alumina (exp. 70). Column: Prewash: 30 ml. CHCl3. Sample: 3.0 mg. crude active fraction in 2 ml. CHCl3. Development: Wash on with 10 ml. CHC1 3 , dev. with 90 ml. 2% methanol in CHC1 3 • Postli,rash: 30 ml. CHC1 3 . Results: Permanganate streak indicated zones shown by shading; eluates were analyzed spectrophotometrically. mg. Zone(mm.) A(0-25) B C(60-95) D o.o F 0.36 Eluates from sections A, £, and F were chrome.togre.phed a.gain under identical conditions and gave the following results. A ?? C 0.29 o.o . (0-25) o.o ?? F .o o.o 0.22 o.o ( 60-90) o.o o.o 0.09 0.19 o.o - 0.36 - 38 - -C, the original middle zone , gave rise to divisions correspondi ng to the original three. A chromatogram of F, the material in the filtrate , showed that this material again appeared in the filtrate. Table III gives the results, of a few experiments which sh~red quite clearly this "double zone" effect. It will be noticed that the total of the material in the two zones and the filtrate does: not equal the tdal sample started with. factors: This is accounted for by f'our principal 1) some colored material is strongly adsorbed at the very top of the column, and this is cut off and discarded; 2) after the column is streaked with alkaline permanganate , the streaked portion of the column is cut out and rejected, the amount discarded may amount to 5 to 10% of the col'Ulllll; 3) the alkaloids may not be quantitatively eluted from this adsorbent; 4) no attempt was· made to keep the i n- accuracies of the analytical method below ca. 5%. In experiment 2 (Table III) after the material in the two zones and in the filtrate was determined gravimetrically it was investigated spectrophotometrically. Af'ter corrections for a blank column were applied to the results, it was found that the spectra were indeed so similar (see Tabla IV) that any differences could be accounted for by experimental difficulties. The maxima and minima for each of the three fractions oc- curred at the same wavelengths , and even the 1% extinctions were very nearly the same , particularly for T and B. The ratio of the 1% extinc- tion of a maximum to that of another maximum or minimum is a quantity independent of concentration, and hence void of errors in weighing. Such ratios are giV'8n in Table IV, and they show how nearly the same the spectra of these thr ee fractions are. - 39 - TABLE III Summary of Some Experiments Showing the Double Zone Effect Sample Top zone (T) Bottom zone (B) Filtrate (F) Ratio T/B Crude: 3.0 mg. 0.75 1.21 o.36 0.62 2. 10.2 mg. 2.0* 3.8* 1.2* 0.53 3.1 mg. 0.52 (1.4a) 0.49 0.37 1. 3. Rechromatographed: 4. T from 1 0.29 0.22 0.09 1.31 5. B from 1 0.20 0.57 0.19 0.35 6. T from 2 0.56 0.79 0.20 0.71 7. B from 2 0.52 2.2 0.23 0.24 a. Ba from 3 0.21 1.23 0.01 0.17 * Opantities marked by an asterisk:r (*) were determined gravimetrically~ all others were determined spectrophotometrically. a) During elution and evaporation this fraction was kept cooled to o0 in a.n ice bath. -- 40 - TABLE IV .Spectral Data for Fractions Obtained in Experiment 2. {Tabla III) Extremes 1% Extinction mµ :type T B F 225 maximum 740 765 800 250 minimum 131 12:3 147 266 maximum 2-11 213 232 302 maximum 104 103 119 Ratios of 1% Extinctions 225/266 3.50 3.45 266/250 1.61 1. 58 266/302 2.04 1.95 - 41 - A possible explanation for this double zone effect is the f ollowing. ,! and Ji represent a compound which exists in solution in two readily interconvertible forms, for example, lactam-lactim, keto-enol, or cis-trans, and furthermore these forms can be separated on the alumina column. When either form is eluted, however, the solution quickly becomes . a mixture of the two forms so that a rechromatogram gives two zones. The material appearing in the filtrate, 1, might represent a decomposition product or' more probably a more stable isomer of the compound represented by l, and .Ji• If this theory is correct, it is possible that a change in temperature would change the ratio of 1 to Ji in solution. An attempt was made to carry out cbromatograms in a cold room kept at Q!.• 4°, but it was found that under the conditions indicated in Fig. 5 no zone appeared on the column and most of the material appeared in the filtrate. In experiments 3 and 8, Table III, the eluate from the original chromatogram was kept cold during the evaporat ion to note the effect on the ratio of 1 to B, in t he rechromatogram. The ratio was quite small, but the significance of this is not too clear considering the wide variation observed in the experiments carried out at room temperature. The room temperature experiments were carried out for the mos t part i n August, in a laboratory on the west end of the building, so that the changes in temperature throughout the day were often as large as 7°. No record ,of this factor was kept, so that no correlation can be attempted. Since so little was known of the nature of the mixture being chromatographed, it was considered unwise to spend more time investigating such factors extensively. It did seem pertinent to prepare one - 42 - large chromatogram and attempt to isolate crystalline material from these three fractions if possible. Such an experiment was carried out with 78 mg. of cru.de starting material. This gave a column on which permanganate indicated a single zone corresponding to B (Table III). Elution of the column and evaporation gave 11 mg. of a dirty oily residue corresponding to L. and 40 mg. of slightly colored solid corresponding to]., and a spectrwn of the filtrate indicated 15 mg. of material with the characteristic spectrum of isofebrif'ugine. From the 40 mg. of colored solid it was possible to obtain 20 ~• of colorless needles by taking the material up in chloroform, adding benzene and evaporating (at room temperature with nitrogen) to about three-fourths of the original volume and letting the solution stand in the ice box over night. This material sintered at .QA• 120° and finally melted at .QI!• 145°. Since there was -,no guarantee that this material was the active principle, a sample was sent to Eli Lilly and Co. for a duck test. The results were most gratifying, 9. 100 to 150. It was now certain that the active principle is an alkaloid, and the name "febrifugine" was given to the substance. Although the melting behavior indicated that the material might not be pure, a definite step forward had been made. As pointed out earlier, a very limited amount of alumina was available for these experiments, and any large scale operations would be made very tediuus by the necessity of recovering the adsorbent after each chromatogram. Fortunately, at about this time, a back or- der of alumina arrived and solved this problem of supply. It was nec- essary, of course, to test the new adsorbent to ascertain if its properties were the same as those of the recovered adsorbent which had been :. 43 - used up to this time. It was very soon discovered that the new adsorbent was not identical with the old recovered adsorbent. Figure 6 shows in outline a twical chromatogram using the new alumina as an adsorbent. The streak gave no indication whatever that the double zone effect was being ob- served. On the contrary, a single zone was evident, and remained fairly constant on rechromatographing. Several experiments to change the activ- ity of the new alumina were tried involving various heat treatments and water deactivations similar to the recovery process tor the old alumina, but none of them gave an adsorbent which gave the double zone effect described above for the old recovered alumina. A 100-:mg. sample of crude material was chromatographed on the new alumina and yielded 43 mg. of needle-like crystals (from chloroform and benzene as described above) softening and sintering at M• 120, and finally melting at 135°. It was noticed in this experiment that con- siderable oil was eluted from the eolunm. (probably machine oil from the mill used to grind the alumina). It was thought that this might be re- sponsible in part for the poor melting range. With the material avail- abie at this time several distribution coefficients and sol ubilities ~ were determined, in order to work out a methgd for freeing the active principle of extraneous material from the column, and also in order to get a more satisfactory method for the isolation of the crude material from the plant. (Section 8) . The details of these determinations will be given later Suffice it to say here, that these determinations led to a better isolation method which will be described below. - 44 - Fi~e 6. A typical chromatogram of the crude active fraction on new alumina (exp. 91). Column: #2, 15 cm., new Al2o3-Celite, 2:1. Prewash: 10 ml. CHCl3. Sample: 4.1 mg. crude active fraction in 2 ml. CHC1 3 • Development: Wash on with 5 ml. CHC1 3 , dev. with 60 ml. 2% MeOH in CRC1 3 • Postwash: 30 ml. CHC1 3 • Results: Permanganate streak indicated single zone (shaded). Zone(mm.) mg. A 0.52 B(50-90) 2.03 C 0.19 D 0.43 Eluate B was rechromatographed under duplicate conditions to give the following results. 0.08 (40-75) 1.40 0.12 - - 45 - The problem of the poor melting behavior of febrifugine was also cleared up by several recrystallization experiments. line of these recrystallizations is given in Fig. 7. An out- All crystal- lizations were carried out at room temperature and below. The gen- eral procedure was to dissolve the material in some solvent at room temperature and evaporate with frequent stirring until the volume was considerably reduced and crystals had begun to form. Then the mixture was placed in an ice box for a few hours to allow the material to crystallize. When desired, a seeorul solvent in which the material was not very soluble was added before the material was placed in the ice box. The crystals were collected by centrifugation, washed with the same solvent (sometimes finally with ether}, and dried at reduced pressure and room temperature. The melting points were not changed when the samples were dried in an Abderhalden at~• eo 0 , lmm., over P205. It can be seen from Fig. 7 that the material crystallizes from ethanol, acetone-chloroform mixture, and ethyl acetate in a form which melts quite sharply at .rua• 137°. ll.• 152°. From chloroform, the material melts at The evidence from these experiments seems to indicate that the compound is dimorphic. It was later demonstrated by analysis that the compound was not solvated. Furthermore, material melting sharply at .rua• 138 0 was obtained from chloroform when the solution was seeded with the 138° material obtained from ethanol. It has also since been found that when the calculated amount of sodium hydroxide is added to a saturated solution of febrifugine dihydrochloride the free vase crystallizes out in the form melting at 152° unless the solution is seeded - 46 - Figure 7. Recrystallizations of febritugine indicating the dimorphic nature of the compound. {All melting points are uncorrected.) 65 mg. crystalline febrifugine: Stir w1 th CHC1 3 • Soln: Evap. to small vol. Add benzene. Solid: Dissolve in CHCl3• Evap. to small vol. Add Et 2o. Hairlike needles: 30 mg. sinter 120, m. 149-52. Stir with acetone. Hairlike needles: 10 mg. sinter 120 m. 152-5. Dissolve in CHC1 3 . Evap. iuntil crystals form. Solid: Soln.: Evap. to Solid: 5 mg. $olJn.: Evap. to dryness. soft 145, dryness. Dissolve m. 137-8 !Dissolve: in EtO.Ac. Evap. m. 151-3 in CHOl3• Evap. to Dissolve in ltmtil crystals form. small vol. Add OHCl3. Evap. Et20• to small vol. Crystals: m. 134-36. Dissolve Add Et20• in CHCl3. Evap. until crystals Orys tals: 20 mg. form. Sinter 120, m. Crystals: 132-45. Dissolve sinter 120, in const. boiling m. 145-7. Crystals: soft 144, m. 148-lSO. CHOl3-aeetone ( 66:34). Evap. to small vol. Crystals: 9 mg. m. 136-7. Mo. liq.: Evap. to small vol. Dissolve in OHCl3. Evap. until Crystals : 5 mg. m. 135-6. crys tale form. Crystals: m. 150-52. Combine all crystalline fractions: Dissolve in EtOH. Evap. to dryness. Dissolve in EtOH. Evap. to small vol. Add Et20• Crystals: m. 137-8. • 47 - freely with the lower melting modification. Both forms give the same dihydro compound in the same yield when reduced in ethanol in the presence of platinum. Both forms give the same zone when chromatographed under the same conditions. This additional evidence confirms the con- clusion that febrifugine is dimorphic. The recrystallizations outlined in Fig. 7 also give some evidence that the material being worked with is homogeneous. The first step was to stir the material with chloroform, and then separate the saturated solution from the solid. The solution gave the same crystalline ma- terial that the solid gave on recrystallization. This is reasonable evidence that the starting material is a single substance. As confirmatory evidence for the purity of the material the chromatogram outlined in Fig. 8 was prepared. It can be seen from the figure that a single zone wasindicated by alkaline permanganate, and that this zone was cut into three parts and each worked up separately to yield the same product. This would seem to indicate quite con- clusively that the material isolated from the column is indeed a single, pure compound. The following method for isolating material from the column was ' used because it freed the material from machine oil which was present in the eluates. mixture. The elution was carried out with 1:1 chloroform-methanol The eluates were evaporated to dryness at room temperature and at reduced pressure. and The residues were taken up in 10 ml. of !/20 RCl, the aqueous solution washed once with 10 ml. of ligroin and once with 10 ml. of chloroform. The ligroin and chloroform contained the - 48 - Figure 8. Chromatogram indicating the homogeneity of tebrifugine. Column: #6, 24 cm., new 2:1 Al203-Celite. Prewash: 100 ml. CHCl3• Sample: 167 mg. crune active traction in 40 ml. CHC1 3 . Development: Wash on with 75 ml. CHC1 3 , dev. with 1000 ml. 2% MeOH in CHC1 3 • Postwash: 500 ml. CHC1 3 • Streak: 0.1% KMn04 - 1% NaOH. Elution: 250 ml. of 1:1 Me0H-CHC1 3 per section. Results: (m. P• 's uncorrected.} A 9.0 mg. crystals m. 136.3-7.2° B 24.0 mg. crystals m. J.36.9-7.9° C 11.1 mg. crystals m. 136.5-7.5° Crystals from A, ~, e.nd C were combined and recr-y-sta.llized from ethanol to give 38.9 mg. of crystals m. 139 to 140° (cor.). - 49 - oil and were discarded. The acid solution was next made basic with sodium carbonate and extracted three times with 5-ml. portions of butanol. The butanol phases were combined and taken to dryness at reduced pressure at room temperature. The crystalline residues were next dissolved in a small amount of absolute ethanol. of insoluble sodium carbonate was left. A. small amount The alcohol was evaporated to a small volume with frequent stirring, and allowed to stand in the ice box for several hours. The crystalline solid was then collected by centrif'Ugation and washed with ethanol, ethanol-ether, and ether. At the time this method for recrystallization was used it was known that the activity of febrifugine was decreased significantly by heat (see Section 8). Material at this time was very scarce, and it did not seem wise to take the chance of destroying material by looking for a more suitable method for crystallization. It has since been found that small quantities of the alkaloid may be crystallized satisfactorily from hot ethanol provided the operations are carried out with reasonable dispatch. Analytical samples have been prepared both ways (analytical results are given in Part II), but the recrystallization from hot ethanol is superior because it facilitates the removal of ash from the sample. Under the conditions of the chromatogram indicated in Fig. 8, isofebrifugine appears in the filtrate. Pure isofebrifugine can be ob- tained from this filtrate in the following way. ed with dilute hydrochloric acid. The aqueous phase is made basic with sodium carbonate and extracted with chlorotorm. with sodium sulfate The filtrate is extract- The chloroform is dried and evaporated to dryness at room temperature. The - 50 - residue can be recrystallized by dissolving it in chloroform, adding two volumes of ethanol, evaporating at room temperature with frequent stirring until the volume is reduced to QA• one-fourth, and letting the mixture stand in the ice box to crystallize. Isofebrifugine can also be crystallized from methanol by slow evaporation at room temperature. A 120-mg. sample of isofebrifugine (m.* 126.5 to 127.5°) was dissolved in methanol. The solution was evap- orated with nitrogen, with frequent stirring until crystals formed. After cooling in the ice box for several hours, a first crop was obtained, amounti:ng to 33 mg. melti:ng at 127.0 to 12e.0°. The mother liquors were further evaporated, and a second crop was obtained, amount to 31 mg., melting at 127.0 to 12a.0°. These two crops were combined and crystal- lized from chloroform-ethanol mixture as described above. 57 mg., melting at 127.2 to 12e.2°. The yield was This material was agiin crystallized from chloroform-ethanol mixture and yielded 43 mg. of material melting at 127.1 to 12a.1°. rial is pure. This behavior seems sufficient evidence tbat the mate- (.Analytical results are given in Part II.) It has recently been found that isofebrif'ugine can be satisfactorily crystallized from hot methanol if the operations are carried out rapidly. * Melting points given in this paragraph are uncorrected. - 51 - Seetion 8 Effect ot Heat on Febr1fug1ne and some Distribution Coefficients of Febritugine and Isofebrifugine As soon as .QA• 20 mg. of febrifugine was available in crystalline form, several experiments were carried out to determine enough of its properties to aid in devising a better isolation scheme. From re- crystallization experiments it was lmown that febrifugine is quite insoluble in ligroin, benzene, and ether. It is somewhat soluble in water, ethanol, ethyl acetate, acetone, methanol, and chloroform. It 1s very mt1ch more soluble in a mixtUl'e of chloroform and methanol than it is in either solvent alone, and likewise much more soluble in a mixture of water and ethanol than in either alone. Up to the time that crystalline febritugine was first isolated, extrac·.t i\ons of the root material had been made with hot solvents, ~• ~•• alcohol an4 water; therefo re it seemed desirable to investigate the effect ot heat on febrifugine in solution. Two milligram samples were sealed in glass tubes with the desi red solvents and heated in a bath of boiling ethanol for six hours. The tubes were openen , evaporated to dryness with nitrogen at 40 to 50°, and the residues taken up in "flj50 HOl. There waa no change in the adsorption spectrum in any of the trials. The samples were sent to Eli Lilly and Co. who tested the antimalarial activity in ducks. The results are shown m Table v. It is quite apparent from the results, that at ao 0 the activity is markedly decreased in six hours. This fact showed in a very clear manner that a different method was needed for the isolation, :. 52 - TABLE V Quinine Coefficients of Febrifugine after Heating in Solution at eo 0 for Six Hours Solvent Abs. ethanol 25 Abs. ethanol 1 Min NH3 25 6 H HCl Control (no heating) 189 and led to the preliminary experiments with fuller's earth (see Section 9) which ultimately developed into a very satisfactory isolation procedure. The other information which seemed desirable in working out an • • isolation procedure was the distribution coefficients of the alkaloids between various solvents and water. This information could be obtained with very little expenditure of material, for small quantities of the alkaloi!s could be determined by means of the spectrophotometer. The procedure for determining the distribution coefficients was the following. A stock solution ot; the alkaloid was made up in chloroform at a concentration of .QA• 0.2 mg. per ml. Aliquots of this solution con~.~ taining £a.• l mg. were pipetted into mixing cylinders. If it was desired to carry out the experiment using some solvent other than chloroform, the chloroform was evaporated and 10 ml. of the desired solvent added. wise, the volume of chloroform was brought up to 10 ml. Other- Ten milliliters of the desired aqueous phase was next added, and the mixture was shaken on on a mechanical shaker for 30 to 60 min. The phases were then separated • 53 - and analyzed spectrophotometrically. The extinctions (at the 266-m)-C -maximmn) of the aqueous phases were taken in acid solution. The organic phases were analyzed differently depending on their constitution. Chloroform solutions were evaporated to dr~ess, an4 the residues taken up in methanol before the extinctions were measured. Butanol solutions were diluted with methanol before the extinctions were taken, and the 1% extinction was assumed to be the same in this solvent as it is in methanol. Since the amount of starting material was known the analyses could be checked by comparing the total alkaloid found with the amount started with. The agreement was usually quite good, and was always within .ru!.• 10%. Suitable blanks were prepared, of course, and a correction applied to the extinction where necessary. The results of these determinations are given in Table VI. It can be seen there that febrifugine is best extracted from aqueous basic solution by butanol. In practice it has been found convenient to use 1:4 butanol-chloroform because less inorganic matter contaminates the product, although the distribution is not quite so favorable. For the results for aqueous solutions at different pH's an4 chloroform, it is possible to calculate a value for PKi3 tor ~ebrifugine, assuming that at pH's higher than 8.5 the compound exists as the tree base, and that the salt form is quantitatively in the aqueous phase at lower pH's. The value obtained for the data at pH 5.87 is <6.9, and at pH 7.13 is 6.25. Dr. Mead had previously carried out a potentiometric titration of isofebritugine, and found two basic groups, pK 6.3 and 12. .;. 54 - .TABLE VI Distribution Coefficients tor Isotebritugine and Febrifugine Coefficients, (c 1/c 2 ) Solvents 2 l Febritugine Isotebrifugine '.i/20 HCl CHC1 3 50 68 'flj20 HCl BuOH 4 4 ".i/20 HCl 1 :4 BuOH-CHC1 3 47 'lI/20 HCl 2:1 BuOH-ligroin 30 CHC1 3 J/20 NaOH 10 CHC1 3 Na 2co3 , pH 9.8 10 BuOH JY20 NaOH 6 1:19 BuOH-CHC13 !/20 Na 2co 3 2.4 1:9 BuOH-CHOl3 !/20 Na 2co 3 5.1 1:4 BuOH-CHCl3 H/20 Na2003 ll BuOH Na2C03, pH 10.l 31 EtOAc Na2C0 3 , pH 9.47 1.2 pH 5.87 * CHC1 3 28 pH 7.13 * 7 . :1, pH 8.51 ** 1.6 pH 10.l ** 1.3 pH 11.4 *** l.6 pH 11.5 *** l.5 • Citrate buffer. ** Sodium carbonate-bicarbonate buffer. *** Sodium hydroxide. ;.. 55 - Section 9 Fuller's Earth as an Adsorbent of the •lkaloids The eX1periments described in the previous section on the effect of heat on febr1fug1ne made clear the need for a new isolation method which does not require heat. ~he obvious choice of solvent to extract basic compounds from plant material is dilute mineral acid, provided decompos1 tion is not promoted. lation method because: is an alkaloid; and It had not been used in the early iso- ' (l} it was not known that the active principle (2} fairly large volumes of solvent are employed in the original extraction of the plant material, and some sort ot concentration is necessary. Evaporation, the usual method of con- centration, leads to the formation of a strongly acidic solution which may destroy the substance being isclated, or if the acid is neutralized, large quantities of inorganic salt mast be contended with. There is again, however, a method of obvious choice for concentration a small amount of material dissolved in a large amount of solvent, namely, adsorption. The choice of adsorbent is perhaps not quite so obvious, although it has been lrnown for some time that charcoal, Lloyd's Reagent,, and clays strongly adsorb many alkaloids from aqueous solution15 • A few preliminary experiments with fuller's earth indicated that febrifugine is strongly adsorbed from acid solution azw can be removed by. shaking with aqueous sodium carbonate and butanol. 15. , Weiser, Colloid. Chemistry. P• 72, New York: In Wiley (1939}. - 56 - order to have some basis for estimating the amount of adsorbent needed for a given amount of .alkaloid, an approximate adsorption isotherm for the adsorption of a mixture of the crude alkaloids on fuller's earth was determined. So that the acidity could be controlled, acid washed fuller's earth was prepared by treating Mefford coso Fuller's Earth with hydrochloric acid until the water remained acidic to Oongo Red Paper. The solid was then filtered off, washed With distilled water, and dried at 100°. The loss in weight during this treatment was .Q.!• 36.5%. Samples of this adsorbent were weighed into conical flasks, a stock solution of the alkaloids in 'II}20 HCl was pipetted into each, and after 30 to 60 min. of intermittent shaking the solutions were filtered through a pad ot Oelite, and the extinctions of the solutions at the 266-mfl maximmn were taken with a Beekman spectrophotometer. Suitable blanks were prepared, and the extinctions corrected for adsorption caused by material from the adsorbent. A preliminary eXJ)eriment in which similar samples were treated in the same way except for the time of shaking indicated that the adsorption is complete in less than 10 min. From the extinctions it was possible to calculate the amount of alkaloid still in the solution, and by difference the amount adsorbed on the fuller's earth. The results were calculated in terms of the weight of the original fuller's earth before the acid wash and are shown graphically in Fig. 9. The valuesof concentration in the :region Oto 5 p. g. per ml. are not very accurate because the extinctions at these concentrations are small, and the blanks were relatively large, so that the corrected extinctions might be in considerable error. For the purpose at hand, - Fit,1.tre g . 5 '/ - Ads orption is otherm for t he or;.;.de slkr.loid.s a.dso:ct od on .f\ ;_ ller ' s earth from _!tl,;o h:rd.rochl oric: a.cic:.• Adsorpti on L • G·• m·~1m- (x m) 20 0 4 60 Co!!cen-t ration, ~ c •/ 1. ( r;) r, (b) n GoV c/(x/r:,) 1.0 0 n V 40 20 C 60 8 - 58 - nowever, these inaccuracies are not serious. ~he plot of c/(x/m} versus c, given in Fig. 9 b, indicates that the adsorption isotherm is probably of the t~e to which the Langnmir equation, c/(x/m} = 1/ab + c/b, is applicable. This shows the adsorbent to be saturated with respect to the alkaloids at .ru!• 43 ~• per gram, so that the least amount of tuller•s earth which could adsorb one gram of alkaloids is .Q.!• 23 g. In Fig~ 10 a more useful plot of the curve in Fig. 9 is given in terms of the amount of adsorbent needed to adsorb f~om a solution of given , concentration one gram of alkaloid with a loss (material remaining in solution} of 1, 5, or 10%. It can be seen from this graph that at initial concentrations greater than ~- 100 µ g./ml. the adsorption 1s quite efficient. If 9o% adsorption is considered sufficient, the initial conc~n- tration may be as low as 10 µ g./ml. and still not require undue amounts of adsorbent. In order to make use of the graph in Fig. 10, it is necessary to know the concentration of alkaloid in the solution being work with. A convenient and satisfactory analysis can be made with Dragendorff's * Reagent. In 0.1 J! HCl, 3 drops of Dragendorff's Reagent adde4 to l ml. of solution will give an immediate cloudiness with 12 µ. g. of alkaloid, a barely perceptible clowiiness in 1/2 to l min. with 6 µ. g., and a clear solution with 3 µ, g. ( If allowed to stand for several minutes the solution may become cloudy.) An approximate value of the conoentration of alkaloid in a solution can be obtained by serially diluting a sample * This reagent consists essentially of an acidic solution of potassitm1 and bismuth iodides. See The Merck Index, P• 696, 5th ed. (1940 ). ?i ,:~ure 10 . c,_,.rres :b,di ce.t1rqi the e.L.(Yunt of fuller's c1a.l·th needed to v.c.sor ·, c-ne ran of c:::-u.d e alkaloid,,; at different effj_ciencies. 400 1% loss 300 5% loss I 10% loss I Gl"&.I: s of F\1ller' s '3::trth to adsor \., 1 · • Al ke.loid fr~m !!/20 lICl I I I I I I 200 .I I I \I I I \ lCO \ 1 \ \ '',, ... --- --- --::--------------=--=----,f-- -- o + - - - - - , - - - - -.......- - - - - - - - -4u0 ----0 1 80 200 3 0 Ini tla l concor:trct:i.on ( µ g ./rJ.. ) ~ 60 - with e.1 E HCl until no test is obtained in 1/2 to 1 min., assuming the concentration to be 5 ~g./ml~, and maltiplying by the dilution faetor. The plant extract sometimes gives a pronounced deepening in color with Dragendorff's Reagent, but this is easily distinguished from the cloudiness, which is the alkaloid te~t. The test 1s not critically dependent on the acid concentration, but the sensitivity decreases with increasing acidity. The solution mast be acidic to prevent the precipitation of basic salts of bismth. From the data accumulated at this point it was possible to devise an isolation procedure. following section. The procedure is given in some detail in the It was found to be quite satisfactory, anil is the method used to isolate the material being used for the structure determination of the alkaloids (discussed in Part II). -: 61 - Section 10 Final Procedure for Isolating the Alkaloids The crude alkaloids are obtained by extracting the ground plant material with dilute hydrochloric acid, adsorbing the alkaloids on fuller's earth, removing them from the fuller's earth with butanol un4er alkaline conditions, concentrating them by distribution between immiscible solvents, and finally evaporating to dryness. Febrifugine and isofebrifugine are separated by means of the dihydrochlorides or by chromatography. Extraction .Q!.ll!! Roots.- The roots are ground to 8 to 10 mesh and packed in a percolator. They are next soaked in 0.1 ,n HCl for 48 hours or longer, the HCl is drained off, and fresh acid is added to the roots. This process is repeated until the roots are essentially exhausted of alkaloid, as indicated by the Dragendorff test (Section 9). Extraction of the root material should be complete before the concentration of alkaloid in the final solution is less that .QA• 75 to 100 µ.g ./ml. The combined extracts are next filtered through a pad of Celite to clarify the solution. The concentration (and total amount) of alkaloid is determined by serial dilution and Drageruiorff's Reagent as described in Section 9. From this data and the graph given in Fig. 10, the amount of fuller's earth necessary for 99% adsorption is found, and this amount of adsorbent plus a 5o% excess (to allow for errors in the determinations involved} is added to the clear acid extract. ' Sufficient 12 _H HCl i s added to make the mixture strongly acidic to Congo Red. The equivalent weight ~ 62 - of fuller's earth (reacting as a base) was found to be fil!• 100 to 150, so the amount of acid needed could be calculated roughly. The mixture is stirred for S8"f'eral hours, and the pH is tested oocasiona:tcywith Congo Red Paper, additional HOl being added if necessary. Although it is known that the adsorption takes place rapidly, the mixture is stirred for several hours because it is desirable both to insure good contact between the relatively small amount of adsorbent and large amount of liquid, and to dissolve out the acid-soluble compounds in the fuller's earth. At the beginning of the last hour of stirring, enough Celite to equal '. one-third the weight of fuller ts earth is added. When the stirring is complete, the mixture is filtered through a pad of Celite weighing about one-sixth the weight of fuller's earth. The filtrate is tested with Dragendorff's Reagent, and if found to contain very little alkaloid is discarded. Elution~ Butanol.- The filter cake is now mixed with enough water to make a thin paste. It was found that too much water leads to undesirable fuller's earth-butanol emulsions later, and it was felt that too little water might make the extraction unduly slow because of poor contact between solid and butanol. An amount of water approximately equal in weight to the fuller's earth and Celite has been found to be satisfactory. water is added. Next, 6 g. of sodium carbonate per 100 ml. of This should make the mixture decidedly basic. In the event that the pH is less than 8.5, more sodium carbonate is added. To the resulting basic paste is added an amount of butanol equal to three ttmes the amount of water added. This mixture is vigorously stirred or - 63 - shaken for about two hours. On standing•:• , if the proper proportions of solid, water, and butanol were attained, the mixture separates into a top butanol phase and a bottom solid-aqueous phase. The butanol is siphoned off, and more (one-third the original amount) is added for a second extraction. It the proper proportions of solid, water, and butanol were not attained, a solid-butanol emulsion may form the bottom layer. It is then necessary to filter the m1xttll"e and separate the water arui butanol pbases of the filtrate. Water-butanol emulsions may be encountered, but they are easily broken by centrifugation. The aqueous phase is returned to the solid, and additional butanol is added for a second extraction. A total of two or three extractions with butanol elutes most of the alkaloids from the fuller's earth. The solid can then be dis- carded, and the combined butanol extracts treated as indicated below. Concentration~ Distribution Between Immiscible Solvents.To the combined butanol extracts are added 1/2 its volume of ligroin (60-70) and 1/2% of its volume of 6 ,N HCl. That is, to one liter of butanol extract · is added 500 ml. of ligroin and 5 ml. of RCl. After shaking, the mixture is allowed to stand until the phases can be separated. The butanol-ligroin phase is extracted three more times with 0.11i HCl, using portions equal to 8% of the original butanol volume. The combined acid extracts, which contain the alkaloids, are made basic with sodium carbonate and extracted three times with 1:4 bu.tanol-cbloroform mixture, using portions equal to one-third the volume of the aqueous phase. The extraction into acid and back into butanol-chloroform - 64 - mixture from basic solution is repeated. The butanol-chloroform solution 1s then dried with sodium sulfate, and evaporated almost to dryness at room temperature. A light colored solid is left as a residue, and it is mixed with a little ether and filtered off. gives a second crop. Evaporation of the filtrate This combined material is the cruile mixture of alka- loids from the plant. Separation~ Febrifugine ~ Isofebrifugine.- To separate isofebrifugine and febritugine from this mixture, use is made of the fact that the dihydrochloride of febri:f'ugine is easily crystallizable from 90 to 95% ethanol, whereas the corresponding salt of isofebrifugine is very hygroscopic, and is soluble in this solvent. The crude alkaloids are taken up in hot ethanol and 10% excess of 12 N HCl is added to convert the material to dihydrochlorides. The mix- ture is seeded with febrifugine dihydrochloride and allowed to crystallize over night in the ice box. second crop ot crystals. The mother liquors are concentrated for a The febrifugine dihydrochloride which is obtain- ed from this step is recrystallized by dissolving in a minimwn of hot 1:1 water-ethanol mixture, and adding sufficient absolute alchhol to make the final concentration of alcohol 90%. The mother liquors from the original preparation of febritugine dihydrochloride are next taken almost to dryness at room temperature, the residue is taken up in a small amount of water, sufficient sodium carbonate is added to make the solution basic, and it is extracted with chloroform. The chloroform is evaporated at room temperature until the mixture becomes semisolid. and ether. The solid is then filtered off and washed with ethanol This solid recrystallized quickly from hot methanol gives pure isofebrifugine. - 65 - The combined mother liquors from the above several crystallizations are best worked up for additional material by ohromatographing them from chloroform on alumina as described earlier in Section 7. To obtain the free base from febrifugine dihydrochloride the material is dissolved in a minimam amount of water, and the calculated amount of 2.5 .I NaOM is added in the cold. The tree base slowly crystallizes out on standing a few hours in the ice box. ethanol. It may be recrystallized from hot .; 66 - Section 11 Application of the Isolation Method to Various Plant Materials Essentially the method outlined in the previous section was applied to several batches of root material and to one small sample of Ohunine (see Section 1). VII. The results obtained are given in Table The following significant results have been found: 1. The yield of alkaloids is much higher by this fuller's earth method than it was by the original, hot alcohol or hot water extraction method. 2. (Compare 3 with 5, Table VII.) A sample of botanically identified Dichroa febri:tuga has been shovm to contain the same alkaloids that Ch'ang Shan (Chungking) contains. (See 2, Table VII.) This is convincing evidence that the botanical identity of the Ch'ang Shan being worked with is in fact~febrifuga. 3. A sample of Ohunine {Section 1) has been found to contain tebrifugine. The fact that 25 g. of plant material is all that was necessary to demonstrate conclusively the presence of febrifugine indicates how powerful the isolation method is. The fact that Ohanine is admittedly leaf material from Saxifragaoea combined with the faot that it contains febrifugine seems quite conclusively to indicate that Chunine is at least in part the leaves of~- febrifuga. Using this same isolation method, Dr. Mead has found that the leaves of botanically identified J2.• febrifuga also contain febrifugine. 4~ The alkaloidal content of the root material varies between 0.05 and Gel%• - 67 - -TABLE VII Febrifugine and Isofebrifugine from Different Plant Material by the Fuller's Earth Method {Section 10) Alkaloids 8 Material Run wt. Description kg. crude g. febrifugine g. isofebrifugine g. 1. 2.9 Chungking Ch' ang Shan, Lot~• 2.54 1.27 0.11 2. 2.9 Dichroa Febrituga, root material from Indiab. 1.50 o.95 0.03 3~ 23 Oh'ang Shan, LOt C (Lilly P-44899) 17.6 4.9 d (6.9) 4.6 d (5.4) 4. 0.025 Chunine 0.017 o.oos 5~ 23 Ch! ang Shan, Lot .Q. 4 o.54 o.75 a. As the free base. b. :Material botanically identified at the time of collection, and kindly furnished by the Abbott Laboratories. c. The first steps of this large scale isolation were carried out at Eli Lilly and Co. d. Recrystallized. e. This material was worked iP by the method of Section 3. The original extraction was maae with hot alcohol at Eli Lilly and Co. The quality of the crude base is much poorer than that of the crude base from the fuller's earth method. The figure in parentheses is the yield once crystallized. ~ 5. 68 - Runs 1 ana 2 (Table VII) indicate that febrifugine makes up by far the largest part of the alkaloids present in the plant. Runs 3 and 5, in which more heat was used, indicate the alkaloids to be about half' febrifugine and half' isof'ebrifugine. Since it is known that the•e two alkaloids are interconvertible (see Part II) when heated in solution, it would appear quite possible that the plant actually does not contain isofebrifugine, and that this alkaloid is formed as an artifact during the isolation. 6. ln addition to febri:f.'ugine and isofebrifugine, the fraction , of crude allurloids contains small amounts of other basic substances, some dark oils awl some high melting solids (m. P• 's .Q.!• 200 and > 240). It is notknown whether these are artifacts or if they actually represent bases present in the original plant material. terise them has been made. No extensive attempt to charac- ·- 69 - Section 12 Summary of the Properties of Febrifugine and Isofebrifugine Febrifugine crystallizes from ethanol in colorless needles, m. 139 to 140° (cor.). A second form can be obtained from chloroform or 0 water, m. 152 to 154 (.g_ (cor.) . . The specific rotation , [~ 25' 0 • o.s, chloroform) and t 2a0 {c o.s, ethanol). This base is approximately 1 to 3% soluble at room temperature in water, ethanol, acetone, and chloroform; very soluble in methanol- chloroform mixtures and in water-ethanol .mixtures; insoluble in ether, benzene, and petroleum ether. The base is very active as an antimalarial. Dr. Richardson reports a .2., of 100 in test G-5 (,!:. lophurae in ducks) both by intravenous injection and orally. Dr. Coatney reports a .2., of 64 in test A-1 (1:• galinaceum in chicks) both intravenously and orally. The base is very toxic. Dr. Schmidt reports an acute oral toxicity for mice as 2.0 mg. of febrifugine per kg. body weight. Single oral doses of 2 to 8 mg. per kg. kill mice within 24 to 72 hours. It is notable that even at such large doses as 1600 mg. per kg. death rarely occurs before 12 hours. The only noteworthy finding in autopsies of mice at any doses is the presence of erosion in the pyloric region of the stomach. A monkey given o.75 mg. per kg. daily lost weight to a marked extent, and was definitely 111, although the symptoms were not well-defined except for the loss in appetite. Isofebrifugine crystallizes from methanol in colorless ohanky - 70 - prisms, m. 129 to 130° (eor.}, 3 [a:]~ + 131° (.Q. 0.35, ehloro:form). At room temperature this base is approximately o.5% soluble in water, 3 to 4% in methanol, 7% in chloroform, very soluble in methanolchloroform mixtures, and insoluble in ether, acetone, benzene, and petroleum ether. Thi s base has given different results as an antimalarial in different laboratories. Dr. Marshall found it to be inactive at the maximum tolerated dose in test D-l (,1:. lophurae in ducks) when given intravenously two times a day. The Q is reported then as less than 1, toxic. Dr. Chen reports a .Q. of 1 in the I-2 test (~. lophurae in ducks). when the drug is given intravenously, !•i•_g,• Dr. Richardson reports~ 1 in test G-5 (1:• lophurae in ducks} intravenously, !•i•.£• - 71 - PART II. STUDIES OH THE STRUCTURES OF :IBBRIFUGINE AND ISOFEBRIFUGTiiJE The structures of febrifugine and isofebrifugine are unknmm. Studies beyond those given in this thesis are being continued, and it is hoped they will lead to a thorough elucidation of the structures. The work is being done in co-operation with Dr. Mead at Occidental College, and to avoid unnecessary duplication the studies have been divided so that Dr. Mead is studying oxidative degradation, and I am studying the functional groups and hydrolytic degradation. It is to be emphasized that any conclusions indicated in this part of this thesis are tentative. Any interpretation of the facts · offered at this time may require revision later, because of newly discovered information. Because of this state of flux, the experimental facts Wlill be presented in the first tvro sections with little or no interpretation. • In the last section a sunnnary of the facts and a possible interpretation will be given. Early in the structure determination Dr. Mead found that both alkaloids when oxidized with basic permanganate at room temperature give good yields of 4-quinazolone (I). I ~ 72 - ~action 1 Empirical Forrnula and Functional Group Tests In this section are given the results of many micro-analyses and many micro-tests,_!•~•, spot tests, hydrolytic tests on 1-mg. samples, and classification tests done on 1-mg. samples. places spot tests will be mentioned briefly. In various The detailed procedures which were followed in carrying out these spot tests are given in Feigl, Spot Tests, 3rd. Ed., New York: Elsevier (1946). The micro-analyses were carried out by Wm. Saschek, G. Oppenheimer, and G.A. Swinehardt. The micro-analysis of febrifugine and isofebrifugine proved to be somewhat difficult in that carbon and hydrogen values fluctuated considerably. of drying. This difficulty was traced at least in part to the method Samples dried to constant weight at room temperature over P205 at reduced pressure gave good analytical results, but samples dried at 78° were more likely to give variable results, and a sample dried at 110° was found to lose weight continually, and a sublimate could be seen collecting on surrounding vessels. This sublimate had been observed earlier when a sample of isofebrifugine was heated at ca. for some time in vacuo in a sublimation apparatus. in ca. 135 to 140° The sublimate formed 24% yield (based on weight of original material), melted at ca. 212°, and is nmv believed to have been 4-quinazolone. Some typical analyses for the alkaloids are given in Table VIII. The results for both free bases and for febrifugine dihydrochloride, coupled with the fact tha~ a potentiometric titration of isofebrifugine - 73 - TABLE VIII Results of Micro-analyses of the Alkaloids Compound prying temp. C H N febrifugine 80° 80° 80° 110° 110° 25° 25° 25° 25° 63.51* 63.15* 63.3 6.07* 5.97* 5.6 13.94* 13.76* 14.0 13.86* 13.82* 63.30* 63.47* 64.2 64.14 6.22* 6.34* 6.6 6.34 a a b a a a a b b 14.04 14.07 14.2 a a b a i sofebrifugine febrifugine dihydrochloride 80° 80° 25° 25° 25° 63.20 63.3 63.50 5.82 6.4 5.96 51.17 5.63 51.39 51.26 5.85 5.82 64.24 63.78 63.33 5.72 6.36 6.98 5.14 250 C16H1703N3 C16H1903N3 C16Hz103N3 C16H19.03N3Cl2 C16H2;r03N3Cl2 Cl6H2303N3Cl2 51.63 51. :3"4 51.ll Cl 10087 10.97 11.30 11.15 11.16 11.16 18.40d 18.62d 18. 57 8 18.648 18.91d 1s.9od Anal. ·Lab. a a a a a a 14.02 13.95 13.85 11 . 29 11 . 2:s 19.05 18.95 11.16 18 . 85 a. Wm. Saschek, College of Physici ans and Surgeons, Columbia University. b. G. Oppenheimer and G.A. Swinehardt, California Institute of Technology. c. Pregl dry combustion. d. Ionic. e. Carius. * Values marked with an asterisk were corrected for ash. - 74 - had indicated an equivalent weight of 2!:_• 300, leave little doubt that the correct empirical formula for both febrifugine and isofebrifugine is C16H1903N3. The possibility that the number of hydrogen atoms is incorrect by 2 is not ruled out, but analyses of several derivatives made subsequently are consistent with the choice of 19. The Hertzig-Meyer N-methyl determination indicated that no methoxy groups are present, but gave values for N-methyl of 0.75% for febrifugine and 5.3% for isofebrifugine, determined gravimetrically. value for one N-n~thyl group is 5.0%. The theoretical The value for isofebrifugine was redetermined by the volumetric procedure, and this time was found to give The determination has since been carried out on the dihydro-alkaloids 1.1;1o. with negative results. It seems quite definite that there is no _!-methyl group, as such, present in either febrifugine or isofebrifugine, but 'perhaps some other grouping which on heating with hydriodic acid gives some methyl iodide. For example, the grouping =C-0-CH2-N = has been postulated to account for the formation of methyl iodide in methyl-imino determinations of vomicine16 , and varying amounts of methyl iodide have been observed in N-methyl determinations of 4-quinazolone-3-acetic acid17 • Micro-catalytic hydrogenations using platinum oxide (Adam's catalyst) and hydrogen at one atmosphere and room temperature showed that fabrifugine takes up one mole of hydrogen in ethanol or glacial acetic acid; isofebrifugine takes up one mole of hydrogen in ethanol, 16. Henry, Plant Alkaloids, P• 539, 3rd. Ed., Philadelphia: Blakiston. 17. SpHth and Nikawitz, ~ • , QI, 45 (1934). - 75 - and 2 moles (or more) in glacial acetic acid; 4-quinazolone takes up no hydrogen (in 6 hrs.) in ethanol, and ~-~mole in glacial acetic acid. Preparation of crystalline dihydro-compoundsin ethanol has been successfully carried out on a larger scale, but no crystalline product could be obtained from isofebrifugine reduced in glacial acetic acid (2 moles of hydrogen). Yields of the crystalline dihydro-compounds are low (~• 70% for febrifugine and less than 50% for isofebrifugine), and it seems indicated that the reduction leads to the formation of more than one compound in each case. Both febrifugine and isofebrifugine fo:mn crystalline dibenzene.;. sulfonyl derivatives when treated with benzenesulfonyl chloride in pyridine. These derivatives are non-basic (insoluble in 0.1, 1.0, and 6.0 N HCl). The acetyl and benzoyl derivatives are oils. As yet no pure, crystalline derivatives of the dihydro-compounds have been prepared. Attempts with benzenesulfonyl chloride and tosyl chloride gave compounds (presumably lmv melting) which could not be satisfactorily crystallized. A derivative with 3, 5-dinitrobenzoyl chloride has so far not been obtained pure. The Zerwitinoff active hydrogen micro-determination has not given satisfactory results. Tobrifugine and isofebrifugine fol7Ill crystalline compounds when treated with hydrochloric acid and sodium nitrite in aqueous solution. The compounds are difficult to purify and sufficient quan- ti t,es for analysis have not been prepared. The compounds formed give a Libermann test for the nitroso group, and are presumed to be N-nitroso derivatives of a secondary aliphatic amine. The derivative - 76 - from febrifugine melts at 161 to 169° with the slow evolution of a gas; the derivative from isofebrifugine melts at 178.5 to 180° with the slow evolution of a gas. The alkaloids do not react at room temperature in alcoholic solution with carbon disulfide to form dithiocarbamates (spot test for primary and secondary aliphatic amines). A solution of sodium nitro- prusside and acetaldehyde gives no color with febrifugine when sodium carbonate is added, but isofebrifugine under these conditions gives a weak purple color (spot test for secondary a.mine). With a saturated solution of chloranil in dioxane, isofebrifugine gives a violet color in ca. 15 sec. and febrifugine a violet color in ca. 60 sec. (most amines give red, blue, or green colorations). Febrifugine forms a crystalline oxime and seJD.icarbazone, but , isofebrifugine and dihydrofebrifugine under the same conditions do not. A spot test involving the formation of a bisulfite addition compound is negative for febrifugine and isofebrifugine• No color is formed with febrifugine and isofebrifugine in a saturated solution of o-dianisidine in glacial acetic acid (negative color test for aldehydes). Both alka- loids reduce Tollen's Reagent in l to 5 min., but the dihydro-compoun.ds do not, even on standing 3 hours. The solubility of febrifugine and isofebrifu.gine in water and in 0.1 N NaOH were determined by stirring a few mg. of each alkaloid with a faw drops of solvent for 5 min., centrifuging, drawing out a sample in a weighed capillary, weighing the sample of solution, and determining the amount of alkaloid spectrophotometrically. I The values obtained were - 77 - the following: febrifugine 21 mg./ml. of water, and 21 mg./ml. of 0.1 N NaOH; isofebrifugine 5.2 mg./ml. of water, and 13 mg./ml. of O.l N NaOH. 4-Quinazolone's unsubstituted in the three position are in general quite soluble in dilute sodium hydroxide whereas these alkaloids are not. When a few milligrams of febrifugine or isofebrifugine is heated with strong sodium hydroxide solution (30%) a blob of resin appears very quickly, and there is given off a basic gas which was shown to consist of ammonia and some strong smelling secondary amine. On heating in more dilute (2.5 N) sodium hydroxide, a dark color forms but no resin separates. The resulting hydrolyzates were shown to contain formic acid (spot test using chromatropic acid) and a diazotizable aromatic amine (diazotization was carried out in HCl solution with sodium nitrite., and the presence of a diazonium salt was demonstrated by adding the solution to a basic solution of (3 -naphthol whereupon a bright orange color developed). amine has since been shown to be anthranilic acid. This When dihydrofebrifugine is treated with strong base no resin forms and no basic gas is liberated. The hydrolyzate gives the tests for anthranilic acid and formic acid. When the alkaloids are heated in concentrated hydrochloric acid at 100° for 30 min. no a.nthra.nilic acid can be demonstrated. 4-~inazolone's in general are stable toward acid hydrolysis. 4-~uinazolone and 2-substituted-4-quinazolone's are also quite stable toward basic hydrolysis. However, 4-quinazolone's substituted in the 3-position are evidently more easily hydrolyzed. A 1-mg. sample of 3-allyl- ,4 -quinazolone heated with 10 µ1. of 2.5 N NaOH in a bath of boiling water gave a strong test for anthranilic acid after heating 5 min., - 78 - even though most of the sample did not dissolve in the aqueous base. A 1-mg. sample of 2-methyl-4-quinazolone under the same conditions is completely soluble, and heated for 30 min. gave a very weak test for anthra.nilic acid. A sample of 4-quinazolone under these conditions ga.ve no test for anthranilic acid after heating for 30 min. The absorption spectra of febrifugine in ethanol and in E/20 HCl are given in Fig. 11 and in Fig. 12, resp. The spectra of iso- febrifugine, dihydrofebrifugine, dihydroisofebrifugina, 2-methyl-4quinazolone, and 3-allyl-4~quinazolone are remarkably similar to that ✓ l of febrifugine. Separate curves for all these compounds are not given because the shape and general characteristics are the same, however, in Tables IX and X the molar extinctions at the wavelengths of principal maxima and minima are gi van. It can be seen in these tables that th;e· 3-subst:ituted-4-quinazolone has maxima and minima. within 1 m~ of those of the alkaloids. The 2-substituted-4-quinazolone has maxima and minima shifted in general a f mv mp, toward the shorter wavelengths, compared with the alkaloids. The molecular extinctions of all these compounds are remarkably constant, varying generally less than 10% from the average at any given extreme. These spectra were taken at concentrations of ca. 10-4 to 10-5 .~ molal which gave extinctions between~• 0.3 and 0.9 as read on the Beclonan spectrophotometer. To get values for the 225-mµ. maxima it· was necessary to use more dilute solutions than the ones used for the other extremes. There has been some evidence that solutions of these compounds do not obey Beer's Law. However, this evidence has not been convincing, and for extinctions in the range 0.3 to 0.9 the variance - 79 - GOO Absorptior. specb :•um of f e '.~·; r ifti i~.ir,e i :s etl:.ar~ol. 700 • 500 4,CO 300 200 220 24.-0 2 60 2SO 300 320 lllf-L - 00 - 900 Fi CJ.l'e 12. A°: ;sorpticn speotrum of fe~Tifu "-;ii~e in. E/20 HCl. '100 soo 500 4-00 300 200 100 0 220 240 260 280 300 320 mp, 26.l 24.6 26.3 26.l 24.2 225 225 226 226 225 225 febrifugine i sofe brifugine dihydro-fe b rifugine dihydroisofebrifugine 3-allyl-4-quinazolone 2-methyl-4quinazolone 26.2 Em l'fJa.ximum mµ Compound 247 249 249 249 249 250 mµ 4.84 3.78 4.02 4.58 4.09 4.21 Em mininn.un 263-4 267 267 267 26606 266 mµ 7.60 7.44 7. 91 8033 7.10 7 .34 ·Em rnaximllln (molal extinction x 10- 3 ) 7.41 - - 3.65 303-4 4.06 301-2 3.61 302 3.91 302. 7.75 3.46 303 6.62_ 3.49 Em 302: mµ maximum 6.68 E·m 275,.6 6.88 275 275 275 275 mµ maximum Molal Extinctions for Spectra Taken in Ethanol TABLE IX 315 313 314 314 314 314 mp, 3.30 2.94 2.92 3.12 2.77 3.04 Em maximum co I-' .. 82 - TABLE X 1blal Extinctions for Spectra Taken in M/20 HCl (molal extinction x 10-3 ) Compound minimum I!linimum maximum maximum Em Ein mµ maximum Em mµ. Em m IL Bin mµ 214 18.9 227 27.4 257 4.94 266 6.04 274 6.Z6 215 16.9 229 24.8 257 4o69 266 5.78 274 6.08 f ebrifugine 216 18.3 229 232- 25. 6 257 4.60 274 6.48 dihydroisofebrifugine 217 160 7 229 232 24.7 256 4.64 275 6.63 232- 28.4 256 4.47 270 5.45 mµ. febrifugine isofebrifugine dihydro- 2-methyl-4.. quina,z olone ... 83 - has not been greater than a ffffl percent. It is believed that the values of molar extinctions reported in Tables IX and X are not in error by more than 5:2:_• 5% from the combined inaccuracies of weighing, dilution, instrumental difficulties, and any possible deviation from Beer's Law. Isofebrifugine and febrifugine are interconvertible. If iso- febrifugine is refluxed in ethanol for about 2 hours, a yield of ca. 50% of febrifugine can be isolated by chromatography or through the dihydrochloride. Samples of isofebrifugine heated neat above the melt- ing point for several minutes appear to give higher yields of febrifugine dihydrochloride (~. 80%), A sample of febrifugine was refluxed for 3 hours in chloroform and the solution then chromatographed. of isofebrifugine was isolated. A 43% yield A solution of febrifugine in chloroform containing a small amount of iodine was illuminated with an arc lamp for 1 hour, and then chromatographed. Small quantities of isofebrifugine were found(~. 2 or 3%). Dr. Mead has recently found that in basic solution the alkaloids take up four oxidizing equivalents of periodate, and a crystalline product can be isolated in good yield. formula C11H1202N2, This material appears to have the empirical Dihydrofebrifugine does not take up any periodate under these conditions. This oxidation product has some remarkable properties. Its ab- sorption spectrum in dilute hydrochloric acid is very nearly the same as that of the original alkaloids. In ethanol or in alkaline aqueous solution the absorption spectra are quite different, having a strong absorption with a maximum between ca. 280 mµ. and 315 mtJ, depending on the pH. If - 84 - this colorless oxidation product is dissolved in a small amount of dilute hydrochloric the solution soon turns a bright yellow, and when sodium bicarbonate is added a yellow amorphous precipitate is obtained. If the solution is made more strongly basic (sodium carbon- ate or sodium hydroxide) a very strong a.mine smell is noticed. If a very dilute solution of the compound in water is made acidic with hydrochloric acid no yellow color develops. This oxidation product takes up hydrogen in ethanol in the presence of platinum (Adam's catalyst). The rate of hydrogen uptake isir- regular and slow 1 but the amount of hydrogen taken up becomes constant after about six hours 1 and corresponds to an uptake of one mole for a compound with a molecular weight of ca. 200. Evaporation of the alcohol leaves a colorless oil which no longer gives a bright yellow solution in acid 1 and which has an absorption spectrum similar to that of a 4... quinazolone with regard to the positions of the extrames 1 but differing considerably in relative extinctions. Since the spectrum vras taken on a sample of unpurified material a useful interpretation can not be given. It does seem certain that the absorption spectrum of the reduced compound is different from that of the oxidation product. Alkaline hydrolysis of this oxidation product on a micro-scale closely parallels the hydrolysis of febrifugine; anthranilic acid 1 forrnic acid, and a.mmonia are observed 1 and the solution darkens considerably. Oxidation of the compound with alkaline permanganate gives 4- quinazolone. The preparations and analyses of the derivatives discussed above ":" 85 - El.re given below. Preparation of Dihydrofebrifugine.- 409 mg. (1.36 millimoles) of febrifugine was dissolved in 50 ml. of absolute ethanol, 50 mg. of platinum oxide (Adam's catalyst) was a.dded, and the solution was shaken in an atmosphere of hydrogen (at one atm. pressure) until no more hydrogen was taken up. About 86% of the theoretical amount of hydrogen (1.36 millimoles) was taken up• A sample of maleic acid hydrogenated under the same conditions took up 87% of the theoretical amount of hydrogen, so it is believed that actually the theoretical runount is taken up, but the method for measuring the uptake is inexact. hour. The reduction took ca. one The catalyst wa.s filtered off, and the solution evaporated to dry- ness at reduced pressure. The crystalline residue was recrystallized from hot ethanol. Yield was 263 mg. (64%) of colorless crystals melting at 187 to 192°. A second crop of 34 mg. was obtained, melting at 163 to 184°. The mother liquors taken to dryness a.mounted to 107 mg. of oil. The dihydrofebrifugine was recrystallized two times from hot ethanol to constant melting point of 192 to 193°. C, 63.34; H, 6.99; N, 14.07. A second reduction of a larger se..mple gave similar results. of febrifugine in 75 ml. absolute ethanol in the presence of 102 mg. catalyst gave a yield of 565 mg. (55%) first crop melting at 191.5 to 192.5°. A second crop amounted to 161 mg. melting at 175 to 182_0 • Preparation 2.£. Dihydroisofebrifugine.- The same method was used here as that given under the preparation of dihydrofebrifugine. 400 mg. .- 86 - •· (1.33 millimoles) of isofebrifugine in 50 ml. of absolute ethanol in the presenc~ of 50 mg. of Adam's catalyst took up essentially the theoretical a.mount of hydrogen (1.33 mol.) in~• three hours. The catalyst was filtered off, and the solution taken to dryness at reduced pressure. The residue was an oil which could be crystallized from acetone or acetone-benzene mixture. The first crop from acetone amounted to 182 mg. (45%) melting at 151 to 154°. A second crop from acetone-benzene amounted to 97 mg. melting at 86 to 124°. The mother liquors evaporated to dryness amounted to 127 mg. of oil. The dihydroisofebrifugine was recrystallized from acetone to constant melting point. Three recrystallizations were required, the pure compound melting at 156.5 to 157.5°. Anal. Cale. for c16H2 1 o3N3 : C, 63.41; H, 6.93; C, 63.33; H, 6.98; N, 13.85. Found: N, 13.77. Preparation of the Benzenesulfonyl Derivatives ~ Febrifugine and Isofebrifugine.- 52 mg. (0.17 millimola) of isofebrifugine, 104 mg. (0.59 millimole) of benzenesulfonyl chloride, and 114 mg. (1.9 millimoles) of pyridine were mixed in a centrifuge tube and allowed to .stand overnight. The mixture was now stirred with 0.25 ml. 6 N HCl. separated and was washed with water. alcohol. A thick oil The oil was crystallized from aqueous Samples recrystallized from chloroform-ethanol mixture and from ethanol-water mixture had identical sharp melting points of 182.5 to 183.5° (dee.). The compound is not very soluble in aqueous alcohol even when hot. The yield was 48 mg. of colorless crystals. ~•Cale.for c28 H27 o7s2N3 : C, 57.91; H, 4o64; N, 7.29. C, 57.85; H, 4.68; N, 7.23. Found: - 87 - The same method was applied to 49 mg. (0.16 millimole) febrifugine 101 mg. (0.57 millimole) benzenesulfonyl chloride, and 115 mg. (1.9 millimoles) pyridine. The resulting compound was recrystallized three times from aqueous alcohol to constant melting point. The yield was 41 mg. of colorless crystals, melting at 148.0 to 148.5°. This compound was much more soluble in hot aqueous a.lcohol than the isofebrifugine derivative. Anal. Found: Cale. for C2aH2 7o7s2N3 : C, 57.80; H, 4.98; C, 57.85; H, 4.68; N, 7.23. N, 7.31. Preparation of Febrifugine Semicarbazone.- 98 mg. (0.26 millimo~) of febrifugine dihydrochloride, 100 mg. (0.90 millimole) semi-carbazide hydrochloride, and 255 mg. (3.80 millimoles) sodium acetate• (anhydrous) were weighed into a small centrifuge tube. About 1 ml. of water was added, and the mixture stirred until a clear solution resulted. This was allowed to stand over night at room temperature. The solution was now ma.de just basic with sodium hydroxide solutiono A colorless compound soon began to crystallize. After several hours in the ice box the solid was collected, washed with water, and recrystallized from aqueous alcohol. The compound partially melted at 110-1150 and finally at ca. 185 to 190°. When recrystallized from ethyl acetate or absolute alcohol, the compound melted sharply at 187 to 188° with the evolution of a gas. The yield was 26 mg. Anal. C, 57.07; Cale. for C17H2303Ne: H, 6.48; N, 23.14. C, 56.90; H, 6.43; N., 23.40. Found: .. 88 - . Preparation 2!_ Febrifugine Oxime.- 96 mg. (0.26 millimole) of febrifugine dihydrochloride, 104 mg. (1.51 millimoles) of hydroxylrunine hydrochloride, end 299 mg. (4.45 millimoles) of sodium acetate (anhydrous) were weighed into a small centrifuge tube. About 1 mlo of water was added, e.nd the mixture stirred until a clear solution resulted. This was allowed to stand over night at room temperature. The solution was now made just basic with sodium hydroxide solution. On cooling and scratching, a white crystalline compound separated. This was collected, washed l'li th water, and recrystallized twice from aqueous, alcohol. The yield was 45 mg. of colorless crystals, darkening slightly above 210° and melting with decomposition at 224 to 225°. Anal. C, 60.85; Cale. for C16H2003N4: H, 6.22; N, 17.45. C, 60.80; H, 6.31; N, 17.69. Found: Section 2 Hydrolytic Degradation The preliminary micro-tests indicated that the alkaloids are cleaved by aqueous base to anthranilic acid and ammonia. Larger exper- iments were set up using~• 100-mg. samples of the alkaloids, 2.0 ml. of 2.5 N NaOH, and allowing the mixtures to stand one to two days at room temperature. In two experiments nitrogen was passed through the mixture and the effluent gas was passed through a known a.mount of standard hydrochloric acid. Ba.ck titration of this acid indicated that from isofeb- rifugine a relatively small runount of ammonia is formed,~•, 0.044 milli-equivalent of base from 0.30 millimole of alkaloid. From feb- rifugine 0.015 milli-equivalent of base was obtained from 0.27 millimole of alkaloid. When the reaction mixture of febrifugine was heated at 100° for two hours, an additional 0.031 milli-equiva.lent of base was found, and the hydrolyze.ta darkened consider~bly. After the back titration to determine the amount of volatile base collected, the titration solutions were evaporated to dryness on a steam bath. A sample of the residue l was placed in the depression of a micro-culture slide, a drop of strong sodium hydroxide solution added, and a hanging drop of platinum chloride solution was placed above the solution. In a few moments crystals formed in the hanging drop, and microscopic examination showed them to be the characteristic octahedra of the ammonium salt. That the volatile base .; 90 - was not entirely ammonia was evident from a strong eharacteristio odor above the basic solution, suggesting a combination of piperidine and impure acetamide (mousy}. The alkaline hydrolyzates were next brought to pH 8 to 9 by adding hydrochloric acid, and the mixtures extracted with 1:4 butanol-chloroform. These extracts on drying and evaporating to dryness yielded about 20% of the original weight of alkaloid as a dark brown oil, soluble in dilute hydrochloric acid, and precipitated from the acid solution by base. Attempts to prepare the acetyl derivative, the picrate, and the chloroplatinate led , to amorphous, colored precipitates. When this material was heated with 20% aqueous sodium hydroxide, a globe of resin collected rapidly. When this resin was heated i n a dry test tube, the vapors above it gave a pine splinter test for pyrrole. The characteristic amine smell was present duri~ this treatment. The hydrolyzate was next acidified with hydrochloric acid until the pH was between 3 and 4, and the mixture was extracted with chloroform. This chloroform extract when taken to dryness gave a light colored crystalline solid amounting to~• 40% of the original weight of alkaloid. This material was sublimed, and then recrystallized from aqueous alcohol, and it proved to be anthranilic aci d {m. 145 to 146°, no depression in mixed melting point with an authentic sample}. On the basis of one mole of anthranilic aoid per mole of alkaloid, the yield is.£.!.• 88% of theoretical. The aqueous phase from these extractions was next taken to dryness or to a small volume in a vacuum desiccator over sodium hydroxide. This residue if warmed with 20% aqueous sodium hydroxide gave very rapidly a blob of resin which gave a pine splinter test when heated with zinc dust. The material given off during this heating with zinc could be collected in dilute HCl, and the resulting solution gave a positive test with Ehrlich's Reagent (test for pyrroles and indoles) and a positive test for secondary amine with sodium nitro-prusside and acetaldehyde (Simon test). dn other experiments attempts were made to prepare an acetyl der- ivative, a picrate, a chloroplatinate, an oxime, and a phenacyl ester from this residue, but in every case the only isolable material was a dark oil or resin. Briefly then, alkaline hydrolysis of febrifugine and isofebrifugipe gives a good yield (88%) of anthranilic acid, a poor yield (5 to 10%) of ammonia (plus some other volatile base), an undetermined amount of formic acid, and unidentified resins and oils. Since the supply of alkaloids is - strictly limited, ' it seemed unwise to expend more material on such hydro~ lyses, at least until more information is available on the structure of the "other half". Preliminary experiments had indicated that alkaline hydrolysis of the dihydro-alkaloids was not accompanied to any great extent by the resin fo~tion or the liberation of a volatile base. Because of this ~ apparent increase in stability of the dihydro-comp¢,unds, hydrolytic experiments were started with dihydrofebrifugine. These experiments have , not been completed, but encouraging results have been obtained. The hydrolysis of dihydrofebrifugine was carried out in a manner very similar to that described above for febrifu gine. The compound was mixed with 2.5 !!_ Na.OH (100 mg. of dihydrofebrifugine to 2 ml. Na.OH) and allowed to stand at room temperature. The dihydrofebrifugine slowly -- 92 - dissolved, taking 12 to 16 hours for complete solution. After compl ete solution had taken place the mixture was allowed to stand for an additional 12 to 16 hours. At no time during the hydrolysis would a piece of moist neutral litnrus turn blue when held in the space above the mixture, and no color developed ' in the alkaline solution. After the hydrolysis, the solution was diluted and extracted with chloroform. residue. Evaporation of the chloroform gave essentially no The solution was then acidified to pH 3 to 4, and again extract- ed with chloroform. Evaporation of the chloroform gave a good yield of , anthranilic acid. The a ~ueous phase was treated in two different ways in an attempt to isolate a derivative of the hydrolyzed fragment. An attempt to pre- pare an acetyl derivative led to the isolation of a basic, hygroscopic glass, while an attempt to preps.re a benzenesulfonyl derivative led to the isolation of a crystalline compound. The methods employed are indi- cated briefly below. 1. The aqueous phase was made basic with NaOH, and shaken in the cold with a large excess of acetic anhydride. No crystalline derivative separated on cooling a..-r1.d scratching so the mixture was made acidic with hydrochloric acid and taken to dryness in a vacuum desiccator over NaOH. The solid (mostly sodium chloride) was extracted with absolute ethanol. The ethanol on evaporating left a very hygroscopic oil, which ~asted very acidic on moist pH paper. This was stirred with freshly prepared silver oxide in water, and the water removed in a vacuum desiccator over Na.OH. The residue was an oil, very soluble in water, ethanol, and methanol, quite .. 93 - insoluble in diethyl ether and benzene, and soiuble in hot acetone and ethyl acetate, separating as an oil on cooling. No derivative could be obtained with ethanolic picric acid, aqueous chloro-platinic acid, Reinecke salt in dilute hydrochloric acid, or on heating with methyl iodide. 'When the material(~• 30 mg.) was distilled in a micro-sublimation apparatus, it darkened on heating but about one-third of the material distilled at ca. 150° and 0.6 mm. The distillate was a hygro- scopic glass, which gave a pH of 8 to 9 on moist p..'9: pe.per. It gave no spot tests for primary and secondary amine, gave a positive test for ester (or amide) by forming a hydroxrunic acid with hydroxylamine in basic solution, gave a positive test for nitrogen by sodium fusion, end gave no characteristic absorption spectrum in ethanol in the region _ 220 to 300 mµ. and essentially no absorption at all in the region 240 to 300 m~ at a concentration of ca. 0.25 mg. per ml. 2. A crystalline derivative was obtained from the alkaline hydro- lyzate in the following manner. concentration was ca. To the solution was added NaOH until the 15%, then excess benzenesulfonyl chloride was added and the mixture shaken vigorously. Soon a pasty solid separated. This solid was insoluble in water, and in dilute hydrochloric acid, and could be .; recrys·l;e.llized from acetonitrile to yield fine crystals, melting and decomposing at 211 to 213° (cor.). C, 54.29; H, 5.09; S, 16.73; H, ~.89; S, 16.7; N, 4.86. The analyses for this compound were N, 5.16 and 5,06. These analyses a.re in ~ 94 - Vfhen this compound was heated for one-half hour with alcoholic KOH, a new compound was obtained and could be crystallized from ethanol. It melted at 220 to 222° (cor.) and contained 6.63% N. rial was not available for other analyses. Sufficient mate- The difference in nitrogen content corresponds to the loss of one benzenesulfonyl group on hydrolysis. - 95 - Section 3 Summary and Conclusions It is to be emphasized again that any conclusions drawn from the experimental work of the prev-lous sections are tentative and ve~y likely to cha.~ge as addit:ioml.information is obtained. However, a sum- mary of the pertinent facts and a possible interpretation seems in order here. 1. The empirical formula.- Both febrifugine and isofebrifugine have the srune empirical formula, C16H1903N3. Further.more, the alkaloids are easily interconvertible on warming in solution, a property believed to be unique among the lmovm. alkaloids . . 2. Presence of~ Secondary Aliphatic Amine.- Evidence that both alkaloids contain a secondary aliphatic amine is quite strong. a basic group of pKB 6.3. Both have Both form dibenzenesulfonyl derivatives which are insoluble in dilute hydrochloric acid and dilute sodium hydroxide. Both give nitroso compounds with sodium nitrite and hydrochloric acid. The Simon color test for secondary amines is positive but weak with isofebrifugine and negative with febrifugine. Both alkaloids give a color with chloranil in dioxane, but febrifugine responds more simrly. This might mean that the febrifugine is being converted to isofebrifugine under the conditions of the test. On the other hand, it might mean that febrifugine (and perhaps isofebrifugine) is actually a tertiary amine which ha~ a labile carbon-nitrogen bond which is broken under the conditions of • 96 - these various . .tests. 3. The aromatic Portion ,2! the !(olecule 1s A A-substituted-!- guinazolone.- There is excellent evidence that the alkaloids are 3-substituted-4-quinazolone •s. a) Both are oxidized by aqueous alkaline permanganate at room temperature to 4-quinazolone. When the alkaloids are heat- ed above the melting point 1n vacuo for sometime, 4-quinazolone sublimes out. The alkaloid, vasicine {II), has been oxidized with permanganate to both 4-qu1nazolone18 and to 4-quinazolone- II 3-acetic acidl7. b) The ultraviolet absorption spectra of the alkaloids and of 4-quinazolone 's are very similar. The positions of maxima and minima agree within a few m~ , and the molal extino ti ons are very nearly the same. The absorption spectrum of a 3-substituted-4- quinazolone gives the best agreement, the position of the extrem,s agreeing within l mµ, • c) Alkaline hydrolysis of either alkaloid gives a good yield of anthranilic acid, a low yield of amnonia, an unknown yield of formic acid, and oils and resins 18. Ghose, Krishna, Narang, and Ray, Jour. Chem • .§,Q,Q_., 2740 (1932). of unknown constitution. Since at least one of the alkaloids, febrifugine, has been shown to contain a ket o group, and since hydrolysis of a 3-substituted-4-quinazolone would give a primary amine, it is reasonable to interprete these oils and resins as condensation products of the primary hydrolysis product.from the alkaloids. The fact that ~apors from these resins (when heat- ed) give a positive pine splinter test for pyrrole may indicate that an a: -aminoketone is formed during the original hydrolysis and, among other condensation products, a small amount of some pyrrole derivative is formed. The appearance of small amounts of ammonia is also probably the result of some side reaction. The presence of formic acid can be demonstrated in the alkaline hydrolyzates of not only febrifugine and isofebrifugine, but also each of the dihydro-alkaloids and the c11Hi 20;!2 oxidation product from periodate oxidation. Although formic acid might conceivably come from some other portion of the molecule, a very likely source is the 2-position of the 4-quinazolone. This im- plies of course tbatthe 2-position is unsubstituted. d) The alkaloids and the dihydro-alkaloids are not readily soluble in dilute sodium hydroxide. 4-Quinazolone's unsubstituted in the 3-position are commonly soluble in dilute base. e) The alkaloids and the dihydro-alkaloids are quite easily hydrolyzed in 2.5 Ji NaOH. Hydrolysis of 4-quinazolone's unsub- stituted in the 3-position is normally much slower. f) An alkaloid with 14% nitrogen arui 16% oxygen might be expected to melt much higher than febrifugine or isofebrifugine. The melting points of 3-substituted-4-qulnazolone's are consistently mt1ch lower than those of the 2-substituted isomers. For example, 3-phenyl-4-quinazolone melts at 139° while the 2-phenyl compound melts at 234°; the 3-propyl compound melts at 83° while the 2-propyl compound melts at 205°. The low melting points of the alkaloids would appear to indicate a 3-substituent on the quinazolone ring. 4. Ox;rgen Functional Groups.- The presence of a secondary amine and of a qulnazolone ring system shows the function of all three nitrogens and one oxygen in the alkaloids. This leaves unexplained the function of two oxygen atoms. Neither alkaloid gives a positive spot test for aldehyde or methyl ketone. Febrifugine shows a reactive carbonyl group by the formation of an oxime and a semicarbazone; isofebrifugine does not. Both compounds reduce Tollen's Reagent. Both compounds give only dibenzenesulfonyl derivatives. Presum- ably one benzenesulfonyl group is attached to the secondary amine nitrogen, and the other to one of two atoms, either an oxygen which was originally a hydroxyl group or the 1-nitrogen of the quinazolone ring. This possibility that the 1- nitrogen is involved is indicated by the completely non-basic behavior of the dibenzenesulfonyl derivatives. either dilute or strong hydrochloric acid. They are insoluble in Quinazolone's are not strong bases {probable pKB's .ru!• 11 to 13) but in general they are soluble in dilute acid. Aoetyl and benzoyl derivatives involving the 1-nitrogen of a 4-quinazolone are known in the case of the alkaloid, rutacarpine (III), which is known to form a monoacetyl {and benzoyl) derivative {IV). The - 99 - postulated mechanism is 19 tbat the acid anhydride {or acid chloride and water} adds to the 1,2 double bond, and then a molecule of acid is split out with the indole hydrogen at the same time a hydrogen shifts to the 2-position, thus ► III IV If such a reaction is taking place between febrifugine or isofebrifugine and benzenesulfonyl chloride the most likely source for hydrogen would be a tertiary alcohol. A primary or secondary alcohol would probably just form the benzene-sulfonate. In summary then, in addition to the quinazolone oxygen there is in febrifugine a keto-group and a hydroxyl-group. The oxygen functional groups in isofebrifugih~ are less clearly indicated, but a hydroxyl-group seems fairly certain. The keto-group present in feb rifug ine has not been demonstrated in isofebritugine. ' This would seem to link the keto-group with the reversible isomerization of the two alkaloids. 5. Catalytic (.f1) Hytlrogenation.- Both alkaloids in ethanol take up one mole of hydrogen, but crystalline dihydro-products are iso- 19. Asahina, ~ - Zentr., "-"' 94 (III), 248 (1923). l~ted in only 70% yield for febrifugine and 45% yield for isofebrifugine. Other reducti on products appear to be formed. In glacial acetic acid febrifugine takes up one mole of hydrogen but isofebrifugine takes up 2 or more. No crystalline products have been isolated from this mixture. The crystalline dihydro-compounds have absorption spectra very similar to the original alkaloids, so presumably the reduction has not taken place in the quinazolone portion of the molecule. Neither dihydro- compound reduces Tollents Reagent, and dihydrofebrifugine does not give an oxime, a semicarbazone, nor reduce periodate as does febrifugine. Although normally under these reducing conditions only ethylenio double bonds are reduced, it would appear that in the case of febrifugine a keto-group is beillg reduced. In the molecules of febrifugine and iso- febrifugine there would seem to be a very labile center which is the point of alteration in the isomerization (interconversion), the oxidation by periodate or Tollen•s Reagent, the reduction with hydrogen and platinum, and in the formation of resins on alkaline hydrolysis. Since these proP- erties disappear on reduction (the possible interconversion of dihydrofeb- rifugine and dihydroisofebrif'tlgine has not been studied} at the same time the keto-group can no longer be demonstrated, it seems logical to associate this labile center and consequently the point of reduction with the ketogroup. 6. Hydrolysis~ Dihydrofebrifugine.- Alkaline hydrolysis of dihydrofebrifugine gives anthranilic acid, formic acid, no ammonia or other volatile base, and a very water soluble fragment which gives a crystalline benzenesulfonyl derivative of the empirical formula c26H28o7s3N2 ltentative}. The identity of this compound is not lmown, - 101 - but it would appear to be a definite lead to the structure of the "other half". 7. Periodate Oxidation.- Oxidation of febrifugine or iso- febrifugine with periodate in basic solution uses four equivalents of oxidizing agent. and yields a crystalline product which is tentatively assigned the formula C11H1202N2• It appears to be a 3-substituted""4- quinazolone. 8. Best Formulae.- The best partial formulae which ca.n be written for febrifugine and isofebrifugine from the data given above are the following: Febrifugine Isofebrifugine -:- 102 - In conclusion, febrifugine and isofebrifugine occupy a singular position among the known alkaloids. They ar e uni que in being readily inter convertible, optically active alkaloids. They are further distin- guished, if the partial for!lllll.ae given above are correct, by the fact that the known ring. system is linked to the other portion of the molecule through a single bond to a nitrogen atom, and not by a typical fused ring system. Leuceno1 20 (V) is the only other plant constituent known to the 0 Cr V N CH2CH1ili2C001I writer which has this structural feature. There are of course _N-methyl groups in several alkaloids, an ]I-ethyl group in ac onitine, and a simple amide in piperi ne. In addition to these interesting chemical features, febrifugine shows remarkable anti- plasmodial ac tivity. Although numerous claims have been made for antimalarial activity of many alkaloidal compounds, no activity has been demonstrated for any naturally occuring alkaloids except those from Cinchona and the closely related Remijia. Very recently a slight activity has been claimed for alstonine in bird malaria. F~brifugine, on the other hand, is extremely active against bird' malaria, having a potency of at least 100 times that of quini ne ; and it is known that the crude drug, containing febrifugine, is active in hwnan malaria. 2·0 . Although febrifugi ne is very toxic, and may not make a Adams , J ones, and Johnson.~• Amer. Chem • .§.Q_Q,., ~• 1810 (1947). ~ 103 - good therapeutic agent for malaria, still the structure determination may furnish a model which will lead to the syntheses of new and useful agents. PART Illa THE ESTIMATION OF CHLORIDE BY MEASURING TEE E.M.F. OF A SILVER, SILVER CHLORIDE, CHLORIDE ION HALF CELL* John A. Brockman, Jr. and Thomas s. Lee** This work was done under Contract OEMsr 325 between the Office of Scientific Research and Development and the California Institute of Technology. ** Present address: University of Minnesota. A rapid procedure is described for the routine determination of small quantities of chloride. The measurements are made with 10 ml. of solution and are reproducible to .Ql!.• 4% for chloride concentrations above 10-4 formal and to .ru!• 4-8% for concentrations between 5 x 10-5 and 10-4 formal. This method for determining chloride was developed t o fill the need for a rapid, routine procedure for detel'lllining small quantities of bis(2-chloroethyl)sulfide and tris(2-ohloroethyl)amine. Since the method is applicable to other compounds which readily give chloride ion in aqueous solutions, the results are reported here, although time was not available for a comprehensive study. In principle , the method is similar to a method used by Furman and Low (1) who employed a concentration cell to determine a. This part of the thesis is a copy of a manuscript which will be sub·m1tted to Analytical Chemistry. The work was done in cooperation with Thomas s. Lee. ~ 105 - small concentrations of chloride in the p~esence of other salts. However. for one half of the conventional concentration cell we have substituted a reference half cell consisting of a Beclanan oalomel electrode. The com- plete cell can be represented as follows: The silver. silver chloride. chloride ion half cell did not contain the conventional silver. silver chloride electrode, but instead a sflver electrode and a suspension of finely divided silver chloride were used. This modification appears to have been first used in measurements made in an undergraduate physical chemistry laboratory course given under the supervison of Prof. R. M. Badger and Dr. P.A. Shaffer. Jr. at this institution. The two half cells were connected by a salt bridge of saturated ammonium nitrate solution. The potential of the cell was measured with a Beckman pH meter, and the concentration of chloride was found by comparing the potential obtained with a calibration curve. Reagents C.P. nitric acid, 10 formal and 2.5 formal. A solution 0.01 formal in silver chloride an:! 0.2 formal in ammonium hydroxide, prepared by dissolving 0.07 g. of silver chloride in 1.7 ml. of 6 formal ammonium hydroxide an:! diluting with water to 50 ml. - 106 - -Apparatus The apparatus is sho'W?l in Figure 1. The sample solution was contained in a 20 ml. glass vessel with a convenient drain and plug, l., at the bottom. The solution was stirred by a motor driven glass propeller, .Q.• The reference half cell,!, was a No. 270 Beckman calomel electrode, and was connected to the lower jack of a Beckman Model G pH Meter. The ammonium nitrate salt bridge, B, was used to prevent chloride from the calomel electrode from leaking into the main cell. The silver electrode,!., was a 5~7 cm. length of 12 gauge silver wire. It was held at the upper end by an alligator clip which was con- nected by a shielded cable to the top jack of the pH meter. The lower end of the wire dipped into the solution to a depth of M• 2 om. Procedure The silver wire is cleaned by dipping it into 10 formal nitric acid for 15 to 30 seconds and thoroughly rinsing it with distilled water. It was found that the potential of the cell is affected by failure to clean the silver electrode. Other methods, such as heating the electrode to glowing in a gas flame, washing with dilute nitric acid, or washing with strong ammonium hydroxide did not give satisfactory results. The recommended treatment with nitric acid was found to give reproducible cell potentials. - l01Z - C---- -A D -B E -- CM Figure 1. App.sratu s . Calo:me1 ref erence h ,:i l f c ell. " · sa tur ~i ted e m..'T!o nium nl tr ~te srilt brl d_:;,: e with 31nt,sred f'l<is s pl up . C. N'o tor f riven s tir rer. D. Ther~ometer. F. Silver wirP. F . Gl9ss d r ,:iin pluf he l a in p l ~c e by rubber tubln r . 1 \ . - 108 - To the solution to be analyzed 1.0 ml. of 2.5 formal nitric acid is added, the solution is diluted to 10 ml. and poured into the cell, the stirrer is started, two drops of the silver chloride-ammonium hydroxide reagent are added to produce a finely divided suspension of silver chloride, and after 1 to 3 minutes the potential of the cell is measured with the pH meter. The amount of chloride is determined by reading from a calibration curve the value corresponding to the potential of the cell. Calibration From potentials determined for samples of known chloride concentrations the calibration curve is constructed by plotting the potential against the logarithm of the chloride concentration of the sample. Examples are shown in Figure 2. The calibration curve is a straight line for chlor- ide concentrations above 10-4 formal. Below 10-4 the curve is not linear, and below 5 x 10-5 the potential cha?Jges very slowly with the concentration of chloride, because the amount of chloride from the dissolved silver chloride is comparable to the amount from the sample. The potential observed when a certain sample of chloride was analyzed varied from day to day by as much as 3 millivolts even though the temperature at which the measurements were made was the same. Per- , haps this was due to slow changes in the liquid junction potential of the ammonium nitrate bridge. The effect of this variation was nullified by constructing a new calibration cu~e each day. The ~aw curve was made merely by measuring the potential of a known concentration of chloride, plotting the corresponding point on semilog coordinate paper, and draw, - 109 -2 ,o-5 25°C-·· rl gj ·- rl 0 e Q) 'Cl ..-t ~ 0 rl .c 0 c... 0 s:: 0 ..-t '-' gj ~ -'-' s::Q) (.) s:: 0 0 ,o.. Figure 2. 8AlibrAtion curves qt di fferent tem p eratures. Jci 160 180 200 220 Poten ti2l (millivo lts) 240 260 280 ·- 110 - il:18 a curve parallel to a calibration ourve already established. That is, the slope of the curve was found to be independent of day to day changes, and the interoept was determined by analyzing a chloride solution of known ooneentration. One standardization for each day was sufficient. Factors Affecting the Determination The sensitivity of the Beclonan pH meter when used oarefully is .QA• 1 millivolt. method. This is the limiting factor in the accuraoy of the In the linear portion of the calibration curve it corresponds to a relative error of 4% in the determination of ohloride ooncentration. Between 10-4 and 5 x 10-5 formal it corresponds to an error of from 4 to 8%. The ionic strength and the hydrogen ion concentration of the solution being analyzed should be the same as those of the solutions used for preparing the calibration curve. However, it was found that the potential of the cell is not critically dependent on the ionic strength in the range 0.25 to 1.00 molal, although there is a gradual shift of a few millivolts. Likewise, the hydrogen ion concentration is not critioal between 0.05 and 0.25 molal. Certain ions or molecules when present in , the solution interfere with the determination. For example, traces of iron salts or permanganate were found to affect the potential markedly. The determination could not be carried out in solutions of acetic acid at concentrations greater than .QA• 3.5 formal because of anomalous effects. Amounts of thiodiglycol equivalent to the amount of chloride present, moderate amounts of manganous ion, and small amounts of oxalic acid did ._ 111 - - not interfere. Temperature affects the potential of the cell. (See Figure 2.} Both the slope and intercept of the calibrati on curve are changed. For a few degrees chaDge in temperature the change in slope is not appreciable (less than 1 mv. per 10 fold change in chloride}, but the intercept changes .QA• l rove per degree. If calibration curves are constructed for five degree intervals, interpolation gives sufficient accuracy for intermediate temperatures. If the temperature differences are small the method for constructing a daily standardization curve discussed under "calibration" may be used to constro.ct a curve for the new temperature. This method was applied to the analysis of solutions of bis(2ohloroethyl }sulfide (I } and tris{ 2-chloroethyl )ami ne {II} in diethyl phthalate,. To obtain an aqueous solution of chloride from I, the diethyl phthalate solution was stirred with an excess of aqueous acidic permanganate, made basic with sodium hydroxide, heated 15 minutes in a bath of boi.ling water, made acidic with nitric acid, and the excess permangana.t e and manganese di oxi de reduced with oxalic acid. To obtain an aqueous solution of chloride from I I , the diethyl phthalate solution was extracted with nitric acid, the resulting aqueous solution made basic with sodium hydroxide and heated on a bath of boiling water for 5 minutes. The cooled soluti on was acidified with nitric acid. The chloride i on in the resulting solutions was then determined - 112 - ,in the manner described. Blanks were prepared by each method, and potentials corresponding to 20 micrograms of I and II ware found. This correction has been applied to the results shown in Table I. Aclmowledgement The OSRD project under which this work was carried out was uruier the joint supervision of Prof's. Carl Niemann and E.H. SWift. Especial thanks goes to Prof. SWift for his timely suggestions during the course of the work and for his aid in preparing this manuscript. References Cited l) N.H. Furman and G.W. Low, Jr., J. Am. Chem. Soc. fil, 1585 (1935). ;;. 113 - TABLE I Substance Amount present l'I!icrograms -b1s(2-ohloroethyl }sulfide tris( 2-chloroethyl }amine A)Dount found micrograms 156 391 1172 1953 50 83 158 358 1157 2055 27 32 49 65 69 118 167 39 78 54 99 157 391 782 1173 3910 380 770 1130 4140 % 128 106 101 92 99 105 137 133 128 119 106 97 99 96 106 -- 114 - PART IV. STUDIES ON C.AMBARUS CLARKII Section I Effect of Magnesium on the Neuro-Muscular Junction Introduction.- The narcotic action of magnesium on Orustaoea is we-l:•l known. point. The site of depression is, however, still a controversial There is general agreement that the site of action is not the nerve l4,6}. Katz l4) claims a "curare effect" at the neuro-mu.scular junction, but he gives little evidence to support this claim. Further- more, his preparations are to be oritisized since he stimu.lated the whole nerve o:f' crab walking legs w1 th pulsating cur1·ents, a procedure which mu.st at times lead to spurious results because o:f' inhibition, and perhaps even because o:f' heterofacilitation effects. The present work was urulertaken to reinvestigate this problem. Time was net available for a thorough study, so experiments were confined to the fast closer system of the crayfish, Cambarus clarkii. In this pre- paration it is not necessary to isolate the single nerve fibre for stimulation, since single shockS given to the whole nerve activate only the fast closer system. Methods.- Non-regenerated claws were used. To study the effect of magnesium on the nerve, the method of Ellis, Thienes, and Wiersma ll) was employed. The whole nerve, isolated from the meropodite, was led through a small celluloid chamber and sealed in with vaseline. Various solutions were then introduced into the chamber, and the effect on the contracti on of the muscle was noted by recording the movement of the dactylopodi te, auxotonically on a smoked drum. - 115 - To study the effect of magnesium on the muscle and the neuroma.seular junction, the method of Waterman (6} was used. Here the whole nerve was isolated in the meropodite according to the technique of van Harreveld and Wiersma (3}. After the tip was cut off, the propodite was joined by a rubber tube to a funnel. Perfusion solutions were run in from Mariotta's bottles, allowed to flow through the claw, and finally to overflow from the meropodite. Perfusion rates depended on the size of the claw, and varied from 1 to 10 ml.. per min. Movement of the daotylopodite was recorded auxotonically on a smoked drum. The basic perfusion solution was that of van Harreveld (2}. The magnesium concentration, normally 0.25 g. per liter, was varied in simple multiples and is recorded as 2X, 5X, etc. Thus, 0.50 g. per liter is recorded as 2x. Stimlation was supplied at regular intervals in the form of single, break, induction shocks, at near-threshold strength. For a few experiments direct current stimulation was obtained with a Lucas pendulum, at roughly chronaxie values. Platinum stimulating electrodes were used throughout the work. In recording muscle action potentials it was desirable to record isometrically at times, and perfusion was carried out by periodically injecting the desired solutipns into the tip of the propodite with a syringe. The potentials were led off from the mu.sole by drilling small holes in the chitin of the propodite and inserting cotton moistened with van Harreveld's solution. These potentials were amplified and recorded photographically with a Mathews' oscillograph. - 116 - Results .Q! Bathing ~ Nerve 1n Magnesium Rich Solutions.specific effect of magnesium on the nerve was observed. No Even when the nerve was bathed in a solution containing 40X magnesium, there was no significant change in the muscle response. As woul d be expected, iso- tonic magnesium chloride (0.21 ,M) rapidly stopped the response. However, van Harreveld 's solution to which was added enough magnesium chloride to maka the concentration 0.21 M stopped the action only after about ten minute& 7 even though this solution is strongly hypertonic. Clearl y then the effect of isotonic magnesium chloride was not a specific effect of . magnesium, but rather the effect of the lack of other ions such as potassium, calcium, and sodium. Results .Q! Muscle Perfusi on.- I ncreasing the magnesium concentration caused a reversible decrease in the ampli tude of the muscle response. In many cases 5X magnesium would stop the muscle response, and lOX always rapi dly stopped the mechanical action when recording was auxotonic. These results confirm the observations of Waterman (6). Several attempts were made to record muscle ~ction potentials, but the results ware not altogether sa tisfactory. In those cases where mono- phasic action potentials were obtained it was not always possible to distinguish with certainty between the shock artifact and the action potential. On the other hand, when diphasic action potentials were obtained the interpretation is complicated by the fact that a relatively slight movement of the lead-off electrodes may change the shape of the potentials markedly (7). However, the results seem to indicate quite clearly that the muscle action potential is not completely blocked by oonventrations of magnesium w~ch definitely block the muscle contraction. Plate I shows the results -11'1- norm.t1 I I • a:1 9 13 r I JO X 3 2. r ! JII-• / N#Villt!ll4i rtC6{; !) 6 7 10 /J l'f_J &Uh A 4 £lP! 8 n~rma/ yest PLATE I Diphasic Action Potentials from Fast Closer Perfused with lOX Magnesium ;,. 119 - Section 2 Effect of Perfusion on the Fas t Closer Response Introduction.- In the course of the investigation discussed in the previous section, a very interesting observati on was made regarding the change in mechanical response when the rate of perfusion was changed. This effect was investigated briefly; and, although no definite conclusions can be drawn, there was a very clear indication that perfusion was washing away a "facilitating substance". There was also an effect which might be interpreted as the washing away of a "fa tigue substanc ett but as indicated below, it is b:elieved that this effect is real ly the result of multiple discharges~ Method.- The same perfusion techni que as described in Section l was used. The perfusion solution was that of van Rarreveld. was by induction shock or with a Lucas pendulum. Stimulation Platinum electrodes were used throughout. Results. - In many preparations the mechanical response was much greater when the perfusion was on, than it was when the perfusion was off. In other preparations just the opposite effect was observed. It was not possible to predict which effect would be observed, and indeed one pre- , paration showed both effects at different times. Plate II shows tracings made from two records which illustrate the effects descrt~ed above. The record of A is typical of the change which appears to be the washing out of a fatigue substance. The magnitude of the change in response on changing the perfusion from on to off is so g~eat and the rate of change is so rapid that an explanation based on A I 1 .1 I I I I .' I I I / ,· / I j I l, l, • // : .' _: / I •I II ; I I I I ; i / . ,· .: / j I / / I iii,/ I! /,I I I 'i I • ,' ; : I '1 'IJr+-+ , 11 , , 1 I ' • I Ttttn I i 1. • j I I !I I I I ' I on I off I j I ' Ii I i : j i I I I I I I I I I I I O!"l 1- ---- 1 ♦ on off 1 5 s s c. :\.nt e ri/Tl.l St i rr:Hl t•.t:lon by indt:. ction shoo..'.c B I I I I ! I ' I ! I I ! i I : I I! ! ' on off i iI' on ' I I I ! ! 'I :!' of f I ' I of f 1 5 sa c. ir,tenm.l Dire ct ourrer;:t stim:...lation f or O. 966'" on off 30 sec . b.terval ( koas penduh@) P-.LATE II Tracings o f reco rds which sho~·i the effect o f porf'usian r -121 - multiple discharges seems most tenable. It is very unlikely that a chemical substance could be washed away so quickly and completely. In,i (Plate II), however, di rect current stimulation was applied for 0.96 <r with a Lucas pendulum. This is a condition under which multiple discharge is very unlikely. It is also clear from the record that the change in twitch height is gradual and not sudden. In this case, then~ there appears to be a real effect of perfusion. Conclusions.- Marmont and Wiersma (5} have suggested that step 2 (see previous section) of the neuro-museular transmission process might be under the control of two chemical mediators, one to account for facilitation and the other to account for inhibition. The present experiments indicate the presence of at least one such substance, a faeilitati11g substance, which can be washed away by perfusion. The pos- sibility that a fatigue or inhibiting substance is also present can not be substantiated by these experiments, It is believed that those records which could be explained as the washing out of a fatigue substance are better explained by multiple discharges which arise because of s ome increased excitability caused by the perfusion, perhaps because of mechan= ical irritation. These experiments further indicate that this preparation, in which ! the perfusion solution is flowed through the propodi te, is a rather poor one because of unpredictable responses to changes in the perfusion rate. Perfusion experiments for the mu.soles of the propodite would probably be more satisfactory if carried out by injecting the desired soluti ons. ,. 122 - References ' 1. •Ellis., Thienes., and Wiersma., 1942., Biol. Bull. 83., 334-352. --- ~ 2. van Harreveld., 1936., Proc. Soco Exp. Biol. Med,., 34., 428-432. 3. van Harreveld, and Wiersma, 1936, J. Physiol • ., ~, 78-99. 4. Katz, 1936, J. Physiol., 87, 199-221. -------- - -"'"--- -- - ~ - 5o :Ma.rmont and Wiersma., 1938, Jo Physiol • ., 93., 173-193. 6. Waterman., 1941., i.• Cell. and Comp. Physiol • ., ;)&, 109-126. 7. Wiersma and Wri ght, 1947, ;!,· Exp. Biol • ., ~. 205-212. -"'"--- .,. I - PROPOSITIONS 1. The Structures of 4-quinazolones are analogous to those of the GC - and ! -hydroxypyridinesa, being resonance-hybrids of structures of the following types: o- c~1-"IR+ cx and I ✓,:: CR Rand R l = Hor alkyl. l N ::;,- a. 2. Taylor and Baker, PP• 530 to 533, Sidgwick's Organic Chemistry of Nitrogen, Oxford, 1942. The stability of 4-quinazolones to acid hydrolysis, and the stability of 3-tuisubsti tuted-4-quinazolones to alkaline hydrolysis compared with the 3-substituted compoundsb can be accounted for by the increased resonance possible in the ions formed in acid or alkaline solution. b. This thesis, PP• 77 and 78. 3. Febrifugine and isofebrifugine are 3-substituted-4-quinazolonesc. c. 4. This thesis, PP• 96 to 98. The theoretically interesting compound, bromochloroiodo- methane, might be prepared in the optically active state by the follm;ring steps: (1) successive iodination, bromination, and chlorination of 3-acetyl-2,4,6-tribromobenzoic acid; resulting from (l); and (3) basic solution. (2) resolution of the ena.ntiomorphs hydrolytic cleavage of the triha.loketone in ":" II - 5. Alkyl chlorosulfinates, formed from thionyl chloride and prime.ry or secondary alcohols, could be reacted with hydroxy acids to give sulfites containing a carboxyl group. Such derivatives might be useful for the resolution of optically active alcohols. 6. The synthesis of alkyl thiolsulfinates from thionyl chloride d proposed by Small, et. al. , was found to be unsuccessful. Better results would have been obtained using thionyl chloride in the following manner, sirnils.r to the known synthesis for the oxygen analoge: S0Clz + RSH ---- RSS0Cl + HCl RSS0Cl + R' MgX - d. e. 7o RSS0R' + MgClX Small, Baile;/, and Cavallito, Jour. Amer. Che2::_. Soc • ., 1710 (1947). Carre and Libermru.m, Compto rend • . , ~ , 2086 (1935). ~~ The theory for olfaction proposed by Dysonf is inadequate, and overlooks some significant physiological factors. f. 8. Dyson, Chem.~ Ind., !f!, 647-51 (1938). Magnesium ion blocks the chain of events between motor impulse and muscle contraction in the fast closer of the crayfish at the saine step that simple inhibitiong does. g. 9. Marmont and Wiersma, J. Physiol., ~, 173-93 (1938)0 Wiersma and van Harreveldh propose a cha.in of events connecting the nerve impulse with the muscle :response in the slow and fast contractions in Crustacea. An additional common step between the muscle action potentials (of both the fa.st and slow systems) and the muscle contraction would help - III - explain some of the phenomena observed in: inhibitiong and heterofacilitation. h. 10. - Wiersma and van Harreveld., Fhysiol. Zool.., 12., 43, (1939). ,_ Candidates for the degree of Master of Science are required to complete a course in the humanities. Candidates for the Degree of Doctor of Philosophy should not be irrunu..-rie to the humanistic. A few well chosen lecture courses requiring at most a few hours per week might form a much needed cultural supplement to a candidate's scientific training.