1963 irving johnson vinca alkaloids (paper)

The Vinca Alkaloids : A New Class of Oncolytic Agents IRVING S. JOHNSON, JAMES G. ARMSTRONG, MARVIN GORMAN, AND J. PAUL BURNETT, JR. (Lilly Laboratoriesfor Research and the Lilly Laboratotiesfor Clinical Research, Indianapolis, Indiana) SUMMARY A phytochemical investigation of the plant Vincaro@eaLinn. has demonstrated that a number of alkaloidal substances can be obtained with antitumon activity. Over 30 alkaloids have been obtained, of which four—vinbiastine, vinleunosine, vincnistine, and vinrosidine—are known definitely to be active. Chemically these compounds are closely related to one another and to two monomeric alkaloids, vindoline and catharan thine. The structure of these latter two compounds has been determined, and partial structures for the biologically active alkaloids have been proposed. They represent a new class of large complex dimenic alkaloids containing both indole and dihydnoindole moieties. Experimentally, a strain-specific, transplantable, acute, lymphocytic leukemia (P-1534) carried in DBA/@mice served as a bioassay for obtaining these compounds and for predicting their clinical activity. Vinblastine, vincnistine, and vinrosidine are capable of prolonging and/on “curing―mice of the P-1534 leukemia even when therapy is delayed until a near-terminal state of generalized disease. Resistance to an additional challenge of leukemic cells has been observed in these “cured―animals. Parenteral ad ministration of vincnistine has been demonstrated to “cure―mice given intracranial implants of the P-1534. The experimental tumor spectrum and toxicological studies are presented and discussed. Biochemical studies performed to date do not reveal any effect on cellular nespira tion, glycolysis, protein or nucleic acid synthesis. The mechanisms of action of these compounds, which may differ within the group as well as from those of other known agents, remain to be determined. Only two of these compounds, vinbiastine and vincnistine, have received extensive clinical evaluation. In spite of their close similarity, chemically, a somewhat different group of human neoplasms responds to these compounds, and there has been a singular lack of cross-resistance between these two drugs and any other oncolytic drug now in wide use. Vinblastine has proved effective in chonioepithelioma, Hodgkin's disease, and other lymphomas, and a number of beneficial results have been obtained in car cinoma of the breast and bronchus. In addition, there have been smaller numbers of a variety of other neoplasias reported as responding to this compound. Vincnistine has been striking in its ability to induce complete hematological remission of the acute leukemias of childhood, both lymphocytic and myelogenous in type. Re sponses have also been reported in a number of other malignancies. The problems and obstacles encountered in obtaining a full realization of the clinical efficacy of these new types of oncolytic compounds are discussed in addition to areas of clinical application other than those previously reported. The plant Vinca rosea Linn. (periwinkle) of the the natural state pink and white color varieties are family Apocynaceae is an ever-blooming pubescent found, and hybrids such as blush pink with red herb on sub-shrub which is widely cultivated as an eye, crimson, and white with red eye are commen ornamental in gardens throughout the world. In cially available. In a recent review (77) of current 1390 JOHNSON et al.—Vinca Alkaloids . 1391 phytochemical research on this plant, Svoboda has pointed out that the probable correct botanical name for this plant is Catharanthus roseus G. Don, but, owing to the frequent and prevalent use of Vinca ro8ea, the latter name will be used synony mously. This plant has enjoyed a popular reputation in indigenous medicine in various parts of the world. Peckholt (63) in 1910 described the use in Brazil of an infusion of the leaves to control hemorrhage and scurvy, as a mouthwash for toothaches, and for the healing and cleaning of chronic wounds. In the British West Indies it has been used to treat diabetic ulcer (86) and in the Philippines has been reported as being an effective oral hypoglycemic agent (fl). The plant is also used in South Africa as a hypoglycemic, and, in fact, a preparation un der the name of “Covinca―is marketed for this purpose. In England the plant has been sold for diabetic treatment under the name “Vin-q-lin.― The folklore reputation which this plant enjoyed as an oral hypoglycemic agent independently stim ulated its phytochemical investigation in two dif ferent laboratories, unknown to each other. One of these groups included Noble, Beer, and Cutts, then at the Collip Laboratories, University of Western Ontario, and the other a group in the Lilly Re search Laboratories including Svoboda, Johnson, Neuss, and Gonman. Although neither group could substantiate the hypoglycemic activity, the Ca nadian group observed a peripheral granulocyto penia and bone marrow depression in rats asso ciated with certain fractions (15, 18). Continued investigation led to their preparation of vincaleu koblastine (VLB) sulfate, an alkoloid capable of producing severe leukopenia in rats (17, 60, 61). During this period the Lilly group had demon strated that certain alkaloidal fractions gave a no producible prolongation oflife of DBA/@ mice given implants of the acute lymphocytic neoplasm, the P-1534 leukemia (41). The detection of activity against the P-1534 leu kemia was considered particularly significant, ow ing to the fact that this tumor system has detected other clinically useful antitumor agents in our lab oratory (40) and had been sensitive enough to study structure-activity relationships of active compounds which correlated with the clinical ac tivity (@3,46, 53). An intensive phytochemical in vestigation resulted in the obtaining by Svoboda (7@) of leunosine, a new dimeric alkaloid closely related chemically to VLB, as well as VLB sulfate. The effectiveness of both of these alkaloids against the P-1534 leukemia was first demonstrated in the Lilly Laboratories. Early in this investigation it became apparent that “indefinite―survival of animals implanted with the P-1534 leukemia was being obtained by tneatment with crude fractions of the plant which were chemically free of both leurosine and VLB (38). Further investigation of those fractions ne sulted in Svoboda's obtaining still two other new active alkaloids, leurocnistine and leurosidine (74). The A.M.A. Council on Drugs has approved yin blastine' (VLB), vinleurosine (VLR), vincnistine2 (VCR), and vinnosidine(VRD) as generic names for these alkaloids; and they will be referred to by these names in the balance of this report. The purpose of this report is to summarize our current knowledge of this new class of antineoplas tic agents, including the chemistry, pharmacology, possible mechanisms of action, and experimental and clinical activity. ISOLATION AND CHEMISTRY Following the initial observation that extracts of Vinca rosea Linn. produced prolongation of life in mice with P-1534 leukemia, a detailed fractiona tion of the plant was undertaken. It was shown (41) that the activity was found entirely in the alkaloidal constituents of this material, and that the alkaloids of the leaves were far more active than those contained in either the stems or the roots (73). The leaf material was therefore used for the preparation of the compounds described hero in, and a procedure of differential extractions was developed (78) which separated the alkaloids into several groups, as shown in Chart 1, according to their varying basicities This procedure, which in volves the conversion of the bases to their tartrates followed by extraction into organic solvents, was first used by Svoboda (71) to prepare alkaloids from another plant of the same family, ALitonia con stricta F. Muell. It was found that the antitu mor activity was primarily located in fraction A (Chart 1). Fraction A was partially purified by chromatog raphy on alumina, deactivated by treatment with 10 per cent acetic acid. This yielded, in addition to a number of inactive alkaloids (the results of purification of fraction A are shown in Chart @), two pure compounds possessing antitumor activity (7@), vinleurosine and VLB (isolated as sulfate). When the noncnystalline residues obtained from this chromatography were tested for antitumor ac tivity, certain of the post-VLB fractions appeared to be more active than either vinleurosine or VLB (74). These materials were subjected to a gradient pH technic involving partition of the mixtures between benzene and buffers from pH @.8to 7.5 1 Velban® (Vinblastine, Lilly). 2 OncovinTM (Vincristine, Lilly). “AlkalineBenzeneExtract 1) 2% Tartaric Acid 2) EtCI2 GroundPlant I SkellyB Extract 1) HCI(2N) 2) NH4OH.CHCI3 Drug 1) NH3 2) Benzene 1) NH4OH Alkaline EtOH Extract Phenolic Alkaloids (C,D) Benzene Benz-CHCI,(1:1) CHCI, I I@ 1 I@ @.9.4.4pH4.@.6.4 [CATHARICINEJ IVINDOLININE(.2HCl)@ISOLEUROSINBenzBenz(deactivated)FVIROSINE11CHCI,residues I CATHARANTHINE LEUROSINE Moth Llq. Al,03 LOCHNERIDINE Combined 1 IA.JMALICINE@j VLB(-H,S04) chromatography@ chromatography I Ai,O, . CHCI3 Benz CHCI3 CHCI3 I I ________________________________________ Benz.CHCI, CHCI3 OSINEJ (1:3)@ Gradient @ GradIent@ pH ____________I2.1.3.4 Benz@CHCI3 IVINDOUDINEI (1:3) ICAROSID1N.!JICombined I1:3ResIdues1 ‘Gradient IpH(4.4.5.4) Ichromatog. [VINCARODINEI CHCI3. CH3OH (99:1) , [NE0LEuR0sIDINE] CHART 2.—Isolation of alkaloids from fraction A Phase 1) NaOH (pH 11) 2) EtCI, 2) chromatography Gradient pHofmoth. Iiq.(3.4) [itEUROCRI@@j DetailedDrug 1) 2% Tartarlc Acid 2) Benzene CHART 1.—Extraction scheme for Vinca rosea L. leaves [Frac@on@ Ai,03 Chromatography @ii@iy-SolubleI @lkalolds(E)] .L@ I I Benz-CHCI3Benz@CHCl,CHCI, (3:1) (1:1) I I LAjmaucinel[LOIHNERI1@iE]___________ r—1-PERIVINEI @ Moth.Liq. Benzene I (3:1) IS1TSIRIKINE( Y2H2S04)@ Catharanthinetlne@1@ndolinej H,S04 NameFormulaAjmalicine Tetrahydroalstonine Serpentine Lochnerine Akuammine* Reserpine*C,1H,4N@O@ C,1H34N,O, C21H,2N20, C20H24N,02 C,@N@.N@O4 C@H@N2O, JOHNSON et al.—Vinca Alkaloids 1393 in 0.5 pH unit increments. The resulting materials were then crystallized as the bases or sulfate salts as shown in Chart @.By this procedure two addi tional biologically active alkaloids, vincnistine and vinrosidine, were obtained pure (74) . These com pounds differed in their activity from the previous two in that they routinely gave survivors (38), rather than just limited prolongation of life in P-1534 leukemia. The remaining fractions (Chart 1) were purified as described above for fraction A, and the results obtained are outlined in Chart 3 (75, 76). In addition to the alkaloids listed in Chant 3 from fraction E, Moza and Trojanek (54) At the time of our entry into this area a number of well-known alkaloids had been reported from this plant (78) ; these are listed in Table 1. They are all found in other genera and species of the family Apocynaceae, and their structures are well documented. No direct chemical relationship could be seen between the alkaloids in Table 1 and the four active alkaloids. The remaining alkaloids which are listed in Table@ with their tentative empincal formulae, melting points, rotations, and pK'@ values were all of unknown structure but were, by and large, indole and dihydroindole in nature (77). A comparison of physical properties [FractionB1I Al,O, Chromatography Benz@CHCl3 (3:1) (Ajmalicine] I Fraction Al,03 Chromatography Benz.CHCI3 (3:1) IVindolineI ITETRAHYDROALSTONINEI Benzene [FractionB IA120,Chromatography CHART 3.—Isolation of alkaloids from fractions A1, B1, B (A + B), E and F have recently reported two additional compounds tentatively named vindolidine3 and locherinine. The biological comparison of the four active alkaloids from this plant (38) indicated consider able differences in the nature and scope of their activities, and it was felt that an understanding of the chemistry of this apparently new class of on colytic agents was important to the eventual un derstanding of their mode of action as well as to enable one to study chemical modifications of these compounds which might show enhanced activities. 3 Since the name vindolidine had previously been given to another alkaloid (Table 2) Prof. Trojanek has suggested the alternate name vindorosine for his compound. TABLE 1 ALKALOIDS FROM Vinca roses LINN. a Akuammine (vincamajoridine) and reser pine have not been encountered in our investiga tion, whereas the presence of the other four has been confirmed. IFractionE IAl,03 IChromatography F I Benzene Benz-CHCI, I (3:1) I LOCHNERICINEI [Tetrahydroalstonine@ [VlndOJifld IFractionF] IA120, @J@hromatography CHC1, ISERPENTINE( HJ1O,I Benz@CHCl3 (2:1) I-CATHARINE IVINDOLICINE NameFormulaM.P., °C.*(a)@ CHCIspK'@ in DMFEtOH U.v.)@max.,m@Vinleurosine Vinblastine Virosine Perivine Catharanthine Lochnericine Vindolinine-2HC1 Vindoline Isoleurosine Lochnenidine Sitsirikine' 1/2 H@SO4 Vincamicine Catharine Vindolicine Vinrosidine VincristineC4H@N4O,t C4H@N4O, C@H,.N@O4f CHH@N,O$ C@H24N,O, C,iHUN@O@ C@H@N@O,•2HCl C,@HI,N,O C4@HoN4O,t C,OHUN,OS@ @ 1/2 H@SO4 (Dimeric) C@.H@N4O,.CU,OH (C@H32N2O.2) (Dimeric) C@.H@N4O@0202—205 (decompn. 211—216(decompn. 258—264(decompn. 180—181 126—128 190—193(decompn.) 210-212 (decompn.) 154—155 202—206(decompn. 211—214(decompn. 289-241 (decompn. 224—228(decompn. 271—275(decompn. 248-251 (melts, re cryst.) 265—267(decompn. 208—211(decompn. 218—220(decompn.+ 72 + 42@ —160 .5 —121.4 + 29 .8 —482 — 9 (H,O) —18 + 61 .2 —607.5 + 23 (Base) +418 —54 .2 —48 .4 + 55.8 + 17 .0 (EtCh)5 .5, 7 .5 (H20) S .4, 7 .4 (H,O) 5 .85 (66%) 7.5(66%) 6 .8 (66%) 4 .2 (66%) 3 .8, 7 . 1 (66%) 5 .5 (66%) 4 .8, 7.3 (66%) 5 .5 (66%) 7 .6 (66%) 4 .80, 5 .85 (66%) 5 .34 (66%) 5 .4 (66%) 5 .0, 8 .8 (33%) 5 .0, 7 .4 (38%)214, 259 214, 259 226, 270 226,314 226, 284, 292 226, 297, 827 245, 300 212, 250, 304 214,261,287 230, 298, 328 224,282,288 214, 264, 815, 341 222, 265, 292 212,257,308 214, 265 220, 255,296Carosidine Carosine Pleurosine Neoleurosidine Vincarodine Catharicine Vindolidine Neoleurocristine Vindolidi@ie#(Dimeric) C@H@N4O@0f C4H,sN4Oiot C@4@H@N4Oi@t @ C441L,N4O@ot C@H,,N4O,ot C4,H.@N4O@ot C4,H,N4O@,t C,,H,0N@O5263-278, 288(de compn.) 214-218 191—194 (decompn. 219—225(decompn. 258—256(decompn. 281—284(decompn.) 244—250(decompn.) 188—196(decompn.) 167— 89 .8 + 6 .0 + 61 .0 + 41 .6 —197.4 + 34 .8 —113.2 —57 .87 —814 .4, 5 .5 (88%) 4 .4, 5 .55 (33%) 5 .1 (88%) 5 .8 (66%) 5 .8, 6 .3 (88%) 5 .3 (83%) 4 .68 (38%)212,254,308 255, 294 267,308 214, 268 280, 272, 298 214, 268, 293, 315 261, 311 220, 257, 298 250,302Lochneridinine#C@H,sN@O4163-169424247, 326 Cancer Research Vol. @3,September 19631394 such as titration, infrared and ultraviolet spectra, and nuclear magnetic resonance spectra of the four active alkaloids indicated first of all that they are closely related chemically, and secondly that they are nonsymmetrical “dimeric―alkaloids (30). Thus, for vinleurosine and VLB titration showed the presence of two basic nitnogens, pK'a 5.5 and 7.5, one of which would quatennize when treated with methyl iodide or similar reagents. Analyses of Comparison of these spectra with those of sev eral other Vinca rosea alkaloids, notably catharan thine and vindoline (31) (Table @),clearly mdi cated a close interrelationship between these com pounds. When the infrared spectrum of a solution containing equimolar quantities of catharanthine and vindoline was compared with those of vinleu rosine and VLB, it was virtually supenimposable from@ to 8@ and quite similar up to 16@ (Chant TABLE 2 NEW ALKALOIDS FROM Vinca rosea LINN. a The melting points were determined on a Kofler microstage. The ultraviolet absorption spectra were obtained with a Cary model 14 spectrophotometer. t Whilethesemolecularformulasagreewellwiththeanalyticalresultsforeachparticularalkaloid,it shouldbenotedthatthey are to be considered as proximate at this time, in light of our experience with the other dimeric alkaloids (57). @ Determined on VLB etherate. §OnthebasisofmassspectrometricevidenceobtainedbyH.BudzildewiczandI. M.Wilsonof Stanford,wenowpreferthe C,@,formulationratherthanthe C1,firstreported(54). #SeeRef.58. the free bases, sulfates, dihydrochlonides, and qua ternary salts indicated that they are C@H@_58N4O9 compounds (57). A study of the infrared spectra (Chart 4, A—D)of the bases further supported this contention (thus the intensity of the indole N-H was approximately one-half that expected for a typical indolic C21 alkaloid) and indicated that VLB contains a hydroxyl group (X CHC13 @.8, 1O.0@)that is not present in vinleunosine. Aside from this difference, the infrared spectra are very similar (Chart 4, A—D)(30). 5). It was assumed that the double molecules yin leurosine and VLB were composed of catharan thine and vindoline moieties, with minor molecular modification, bonded together in some unique manner (30) . It was therefore possible to investi gate the structures of the more plentiful smaller molecules and then relate this informatioa to the dimenic compounds. Cathananthine (31, 56) (I)@ was found to be a C21H2@N@O2compound and to form a methiodide 4 Catharanthine and derivatives are shown in Chart 8. FREQUENCY(CM@') WAVELENGTH (MICRONS) I WAVELENGTH (MICRONS) CHART 4.—Infrared spectra of biologically active Vines alkaloids A. Vinleurosine C. B. Vinblastine D. 1396 Cancer Research Vol. p23, September 1963 readily. The physical characteristics of the quater nary salt showed that the basic nitrogen (pK'a 7.0) was analogous to the one forming this salt in the dimeric compounds (77). Spectral data indicated that the alkaloid was a simple @,3-substituted pen tacyclic indole containing an isolated double bond, ester group, and closely related to the isoquinucli dine alkaloid coronanidine5 (II), C21H26O2N2(@7). The N.M.R. spectrum (Chart 6B) confirmed the above assignments and indicated that the double bond contained one vinyl proton at 5.Sö,and that a C-ethyl group characterized by a methyl triplet a 1.1öand an allylic methylene quartet at 1.9öwas present (56). These data clearly suggested structure (I) for cathananthine (Chant 8). Supporting cvi dence for the position of the double bond was ob double bond at C3-C4—viz., voacangine (III), di hydrocatharanthine (IV) (vide infra)—the Ci-C18 bond is broken to afford 3-methyl-S-ethyl pynidine (83). Mild hydrogenation of I afforded only one iso men, dihydrocatharanthine (IV). Its infrared spec trum was strikingly similar to that of coronar idine. The differences in these compounds could be explained by examining Dreiding models which showed that the hydrogens could only have en tered the molecule from the side nearest to Nb to give the axial ethyl group in dihydnocatharanthine. The ethyl group in the Iboga alkaloids has been shown to be equatorial (@).7 The reduction of catharanthine and dihydro catharanthine with LiA1H4 afforded corresponding FREQUENCY (CM1) CHART 5.—Infrared spectral comparison of vinleurosine and an equimolar solution of catharanthine and vindoline z 0 WAVELENGTH (MICRONS) alcohols. The formation of the tetrahydro-1,3- oxazine derivative (70) from cathananthinol gave additional evidence for the position of canbometh oxyl at C18. The Iboga alkaloids possessing a carbomethoxy function at C13are known to decarboxylate smooth 7 The following evidence can also be correlated with this assignment: a) The pK'a values of the axial series are higher than those in the equatorial series; dihydrocatharanthine pK'@ 6.4, coronaridine pK'. 6.1 (38% DMF). b) The rate of methiodide formation has been found to be a sensitive indicator of the configuration of the C-ethyl group, the rate being much faster in the axial series (private commu nication, Prof. M. Shamma, Penn. State Univ., State College, Pennsylvania). c) The Re values of these compounds vary when they are run by thin-layer chromatography on silica gel plates. (RT of dihydrocatharanthine 0.2, coronaridine 0.8, solvent ethyl ace tate-chloroform, 1: 1.) This difference may be ascribed to the acidity of the silica. tamed recently from decoupling experiments per formed on the vinyl proton of the alkaloid.6 Saturation of this proton led to changes in the shape of the C-@proton at 2.6@6,and, in addition, small decoupling effects were seen on the protons at 4.18@(C-5 proton) and 1.96 (methylene of ethyl group). As expected, dehydrogenation of catharan thine with Pd on carbon yielded 3-ethyl pyridine by the cleavage of the allylic C1-C2 bond. In iso quinuclidine Iboga alkaloids which do not have a 6 The correlation of isoquinucidine alkaloids (with no aro matic enethexyl groups) is readily found by the observance of a triplet centered at 6.8@ in the infrared spectrum. N. Neuss, “LillyCollection of Physical Data on Indole and Dihydroin dole Alkaloids,― Eli Lilly and Company, Indianapolis, Indiana (1961). 6 The decoupling experiments were carried out by Mr. Paul Landis, Eli Lilly and Company, on a proton-proton decoupler patterned after that described by Mr. L. F. Johnson, Varian Associates, Palo Alto, California (private communication). JOHNSON et al.—Vinca Alkaloids 1397 ly on saponification (@9), followed by mild acid treatment, or alternately after prolonged refiuxing of the ester with hydrazine (65) in ethanol. Anal ogously, dihydrocatharanthine afforded the con responding descarbomethoxy base epi-ibogamine (V), differing from the known ibogamine (VI) only in the orientation of the C-4 ethyl group as pre viously mentioned. The final confirmation of the nature of the ring system was given by the isola tion of 4-ethyl-@,6-dimethyl-11-H-indolo (@, 3-C) quinoline from Se-dehydrogenation of epi-iboga mine. The identical compound has been obtained B @@@ @u ‘ ‘ ‘ I I 400 300 260 100 0 5@O C.Ps. CHART 6.—N.M.R. spectra in CDC13 (60 mc.) A, vindoline; B, catharanthine 1398 Cancer Research Vol. @3,September 1963 earlier by the same treatment of ibogamine (57). Catharanthine (I) must therefore be &-dehydno coronanidine. The nature of the second half of the dimeric compounds could best be understood by a study of the dihydroindole alkaloid vindoline (VII) (@6, 31),a C@H@N2O6substance.8Its basicnitrogen did not form a methiodide in analogy to the nitrogen with PK'a 5.4 in the dimenic alkaloids. The nature of the six oxygens was determined by the forma tion of suitable derivatives (31). Thus, brief treat ment of vindoline with acid afforded desacetylvin doline, C2@H30N2O5,corresponding to the loss of an a Chart 9 shows vindoline and related compounds. acetyl group. This was further corroborated in the infrared spectrum by the appearance of a hydnoxyl band (@.7@i)and corresponding reduction of inten sity of the canbonyl band. The presence of the methyl ester was shown by lithium aluminum hydride reduction of the base with simultaneous removal of the acetyl to yield vindolinol, C@H30N2O4. The infrared spectrum of vindoline, in which a broad band at 3.5—4.0@twas conspicuous, showed that the fifth oxygen was a hydrogen-bonded hydroxyl. Formation of the di acetate C27H3@N2O7(acetic anhydnide in pynidine) was accompanied by the disappearance of this broad band in the infrared spectrum. From the 4. VL.B CHART 7.—N.M.R. spectrum vinbiastine in CDC1, (60 mc.) I R :COOCH3 II R1 : COOCH3; R2: C2H5; R3 H;R4 : H; Coronaridine ill R1 : COOCH3; R2 : C2 H5; R3 H; R4 : OCH3; Voacangine IV R1@ COOCH3; R2: H; R3@ C2H5; R4@ H; Dihydro cat haranthine V R1:H;R2:H;R3:C2H5; R4 H; Epi-ibogamine VI R1 : H; R2 : C2H5; R3 : H; R4 H; lbogamine CHART 8.—Structures of catharanthine and related compounds JOHNSON et al.—Vinca Alkaloids 1399 study of the ultraviolet and infrared spectra, the sixth oxygen was shown to be present as an ano matic methoxyl on a dihydroindole chromophore. The confirmation of the assignment of the oxygen functional groups was obtained from the N.M.R. spectrum (Chart 6A). Vindoline consumed 1 mole of hydrogen at at inosphenic pressure, showing it to be a pentacyclic compound and afforded dihydrovindoline (VIII) which could be converted to an amorphous hy groscopic hydrochloride (Chart 9). Pyrolysis of this salt at 195°—@00°C. in vacuum gave a distil late from which a C21H2@N@O2compound (IX) was obtained in a 15 per cent over-all yield by direct crystallization from hexane. The methoxydihydno (8).Thecleavagecanberationalizedasoccurring in the manner shown in Chant 10.@ The position of the carbonyl in the ketone (IX) was assigned on the basis of the presence of the peak at m/e @98(M-4@ is equivalent with loss of ketene in IX or ethylene in XI) which suggested that this group involved either C-3 or C-4. Position 4 was selected for two reasons; the typical ABX pattern at low field (3—3.58)in the N.M.R. spec trum which was consistent with structure VIII in dicated three protons between the canbonyl group and nitrogen; and equilibration with CH3OD/ methoxide resulted in the introduction of only two deuterium atoms per molecule (M = 34@); where as if the carbonyl was at C-3 there would be three I DELTA VALUES 2.65 3.4 5.u 2.1—3.0 6.91 5.23 6.30 5.43 3.75@ __ 900 3.80 2.68 VII CH3O R p x R:CH3C@- XI R=CH3 H CH)O@@@J1@H CH3 XII VIII H COOCH3 XIV CH3d''211@ CH3 XIII CHART 9.—Structures of vindoline and related compounds indole portion of this compound was the same as in vindoline, and the second oxygen was found to be present as a ketonic canbonyl (X@fi'c',5.85j@)(@8). Vindoline (VII) was found to possess the same ring system as the alkaloid aspidospermine (X) (7), obtained from plants of the Aspidosperma genus which is in the same family as Vinca. This observation was made by comparing the mass spectral fragmentation patterns of the pyrolysis ketone and dihydnovindoline with that of the aspi dospermine (X) derivative desacetyl-N-methyl as pidospermine (XI). In each compound the identical intense peaks were found at rn/c 1@4,174, 188, and @98(@8). A study by Biemann and co-workers has shown that these four peaks are uniquely formed by cleavage of this type of a pentacyclic system B @N@YD CH3Q IC CH3 Ix XI CLEAVAGE AT A,C rn/c •174 B.C rn/c =188 C.D rn/c =298 (HEAVY LINES) A,D rn/c :124 CHART 10.—Mass spectral fragmentation of the vindoline pyrolysis ketone (IX, Y =C= 0) and desacetyl-Nmethyl aspidospermine (Y [...]... were 3 CLASSIFICATION OF THE ALKALOIDS FROM Vines Vol @3, eptember 1963 S rosea LINN @1Band Leurosine-like Alkaloids: 1 Vincaleukoblastine (Vinblastine) 2 Leurosine (Vinleurosine) 3 Leurocristine (Vincristine) 4 Leurosidine (Vinrosidine) 5 Isoleurosine 6 TABLE 7 Carosine 8 9 Catharicine 10 Neoleurocristine 11 Neoleurosidine Monomeric VBL IN P.1534 LEUKEMIA ActivityAlkyl Alkaloids: 2 Virosine 8 Catharanthine... chromatography of the reac tion mixture an indole compound (vide infra) , fol lowed by vindoline derivatives (Chart 11) Vin JOHNSON et al.†Vinca Alkaloids 1401 blastine (XV),9 vinleurosine, and vinnosidine af forded desacetylvindoline, thus proving the iden tity of the dihydroindole portion of these alkaloids The corresponding fraction from the cleavage of vincristine (XVI) yielded des-N(a@-methyldesace tylvindoline,... iodide45 128N-Propyl iodide33Ally! iodide71Bencyl Alkaloids: Vindolicine Vindolidine Vincamicine Vincarodine Carosidine 44 bromide Carbethoxymethyl bromide @9-Hydroxyethyl bromideInactive 45VLB:Methyl Inactive iodide Ethyl iodide26 25Propy! iodide attention was turned toward the effect of struc tuna! changes on biological activity One can con sider the four active alkaloids as minor modifica tions of one another,... only the method 1403 Alkaloids EXPERIMENTAL of at BIOLOGICAL PROPERTIES tachment This is most probably photochemical since, as mentioned above, the major portion of the dimenic alkaloids are found in the leaves of the plant The first step can be visualized as an oxida tion of vindoline to a free radical by a peroxidase The most striking experimental biological effect of the four Vinca alkaloids under discussion... of solid tumors of > 100% prolongation of leukemias; N.D = not done 0 0 0 0 JOHNSON as maintained response in our laboratories to vinleurosine and 196@2,but the response et al.†Vinca has changed vinblastine since to vincristine in greater therapeutic effect in the combination then apy Combinations of the various active Vinca alkaloids themselves gave no evidence of any greater activity in this tumor... the alkaloids of Vinca rosea comprise a occasional elevation of serum transaminase new class of antitumon agents, it was of consider Autopsy of these monkeys revealed no charac able interest to us to study the effect of these com tenistic gross findings Histological studies showed pounds on various biochemical reaction sequences reduced celluanity of myeloid and lymphoid tissue JOHNSON et al.†Vinca. .. cannot be equated with in vivo activi ty That is, the injected alkaloid may be modified in some way by the host to provide an “active component.― CLINICAL STUDIES WITH THE VINCA ALKALOIDS This clinical discussion of the Vinca alkaloids is confined to vinblastine sulfate and vincnistine sul fate, which are the only members of this family of drugs which at this time have undergone extensive clinical... Leurocristine, A New Alkaloid from Vinca rosea Linn Proc Am Assoc Cancer lIes., 3:301, 1962 4 ARMSTRONG, G.; Dvxn, R W.; Foirra, P J.; and G@s.m J MER, J E Hodgkin's Disease, Carcinoma of the Breast, and Other Tumors ‘Treated with VinblaátineSulfate Cancer Chemother Rep., 18:49—71, 1962 JOHNSON et al.†Vinca 5 BARTLETT,M F.; DIcxEz@, F.; and TAYLOR, W I Alka D :l4@5 Alkaloids Quelques aspects de... NEUSS, N.; and SvOBODA, G H Ymca Alka bids IV Structural Features of Leurosine and Vincaleuko blastine J Am Chem Soc., 81:4745-46, 1959 31 GORMAN,M ; NEUSS, N ; SVOBODA,G H ; BARNES, A J.; and CONE, N J Note on Alkaloids of Vinca rosea L II Catharanthine, Lochnericine, Vindoliine and Vindoline J.Pharm 48:256—57, Sci., 1959 J Vincaleukoblastine ClinicalTrialwith OralPrepa III ration Cancer Chem Rep., 14:... BOND, W H ; YARDLEY, J M.; and CORPENING, S Vincaleukoblastine A W IV Summary of Two and One-Half Years' Experience in the Use of Vinblastine Cancer Chem Rep., 16:401—6, 962 1 38 JOHNSON, I S ; Sv0B0DA, G H ; and WRIGHT, H F Ex perimental Basis for Clinical Evaluation of Two New Al kaloids from Vinca rosea Linn Proc Am Assoc Cancer lIes., 3:381, 1962 39 JOHNSON, I S ; Vz@'nxs, J ; MArr@is, B ; and . well documented. No direct chemical relationship could be seen between the alkaloids in Table 1 and the four active alkaloids. The remaining alkaloids which are listed in Table@ with their tentative empincal. concerning the chemical nature of the dimenic alkaloids our TABLE 3 CLASSIFICATION OF THE ALKALOIDS FROM Vines rosea LINN. @1Band Leurosine-like Alkaloids: 1. Vincaleukoblastine (Vinblastine) 2 to two monomeric alkaloids, vindoline and catharan thine. The structure of these latter two compounds has been determined, and partial structures for the biologically active alkaloids have been