Jan., 1951
APO- A N D ISOAPO-p-ERYTHROIDINE
crystalline residue. The diamine was dissolved in 120 ml. of 4 N hydrochloric acid containing 4 ml. of 90% formic acid. The solution was refluxed for 40 minutes, cooled, and neutralized to pH 8 with 28% aqueous ammonia. The crystalline product was collected on a filter and washed with water. Recrystallization of the product from methanolwater (Dare" was used) gave 5.5 g. of l-(l'-D-sorbitYl)5,6-dic~lorobenzimidazole, m.p. 198-200 with a transition A sample recrystallized three times from $ethanol a t 147 melted a t 205-207" after a transition below 200 . After two additional crystallizations from acetic acid-water the melting point was 206-207 O after a transition that began a t 145". The 1-(1'-glycityl) -5,6-dichlorobenzimidazolesand 1-
.
[CONTRIBUTION FROM
THE
333
(1 I-D-ribityl) -5,6-dimethylbenzimidazole are reported in Table I.
Summary 1-(l'-~-Ribityl)-5,6-dimethylbenzimidazole, 1(1'-~-sorbityl)-j,6-dichlorobenzimidazole,I-(1/-L)arabityl) -5,6-dichlorobenzimidazoleand 1-(l'-D-xy 1ity1)-~,6-dichlorobenzimidazo1e have been synthesized and have been found to be ineffective in producing regression of established lymphosarcoma implants in mice. RAHWAY, NEWJERSEY
RECEIVED JUNE29, 1950
RESEARCH LABORATORIES O F MERCK& CO.,INC.]
Erythrina Alkaloids. XX.
Apo- and Isoapo-p-erythroidine
BY FRANK KONIUSZY AND KARL FOLKERS Considerations of structure I for @-erythroidine and structure I1 for desmethoxy-P-erythroidine have been described.l @-Erythroidine was converted into desrnethoxy-p-erythroidineby reaction with 30y0 sulfuric acid at 100'. The degradation product was isolated as a perchlorate. The reactions of @-erythroidineunder other conditions of acid hydrolysis were also examined, and the results of these concomitant studies are summarized herein.
OI
\&It 0
I
l
methoxy-P-erythroidine. Structure I11 may be envisaged for apo-@-erythroidineto account for its properties, and because the weak nitrogen-carbon bond in the
II
0 I1
The reaction of &erythroidine with concentrated hydrobromic acid a t looo, followed by fractional crystallization of the resulting free-base products, gave a crystalline compound, m. p. 144') which has the composition C16H16N02. This product is isomeric with desmethoxy-@-erythroidine and was designated apo-0-erythroidine. It reacts with ferric chloride in aqueous solution to give an intensely purple-colored product ; consequently, apo-@-erythroidine was used as the basis for the development of a colorimetric determination2 of @-erythroidine. The positive color reaction of apo-@-erythroidine with ferric chloride and the corresponding negative reaction with desmethoxy-&erythroidine can be interpreted on the basis of a structural difference in the nitrogen-containing ring of apo-@-erythroidine. Although the hydrogenation of desmethoxy@-erythroidine gave a hexahydro- derivative, the hydrogenation of apo-&erythroidine has been found to give an octahydro- derivative. Octahydro-apo-@-erythroidine is a crystalline compound, m. p. 135-13601 which was also characterized as a crystalline hydrochloride. Thus, there is an additional double bond in apo-b-erythroidine, which differentiates it from the isomeric des(1) Koniuszy and Folkers, THISJOURNAL, 73, 6579 (1950). (2) Dietz and Folkers, J . Am. Pharm. Assoc., 35, 48( 1948).
I
I
1
of structure
I could be expected to be cleaved under appropriate conditions with either acid or alkali. Structure IV may be considered for the octahydro-apo-perythroidine.
I
-id-
I
> N --C-C-CO-grouping
I1
0
I11
YJ---CHI It
0 IV
If apo-/3-erythroidine has structure 111, it would be expected that by suitable oxidation conditions, formic acid would be formed. The oxidation of apo-@-erythroidine with potassium permanganate in sulfuric acid solution did result in the production of formic acid. The formic acid was removed from the oxidation solution by distillation and was isolated as a 9-bromophenacyl ester. This specimen of the derivative was identical with an authentic one. It is evident from structure I11 that apo-perythroidine might also be prepared directly from desmethoxy-@-erythroidine, and that the latter compound may be an intermediate in the degradation of @-erythroidine to apo-$erythroidine. From the mother liquors of the crystallization of apo-@-erythroidine,there was obtained another crystalline compound, m. p. 154-155', which also has the composition C15Hd?Os; this compound was designated isoapo-@-erythroidine. The hydrogenation of isoapo-@-erythroidinealso resulted in the characterization of an octahydro- derivative, m. p. 134-136') which is identical with octahydroapo-@-erythroidine. If apo-@-erythroidinehas an exocyclic bond as shown in structure I11 to account for the additional hydrogen absorption and degradation to formic acid, it is understandable that the exocyclic bond might shift, as in structure V,
Vol. 7s
FRANKKONIUSZY AND KARLPOLKERS
334
so that it is conjugated with both the carbonyl and 138 mg., m.p. 140-141 '. After three recrystallizations from methanol-ethyl ether, the isoapo-B-erythroidine melted conbenzenoid groups. Accordingly, isoapo-@-erythroi- stantly at 154-155"; [(Y]=D f7.1 (c, 1.4, CH30H). dine appears to have structure V (or possibly VI). Anal. Calcd. for C I ~ H L ~ N O c,~74.65; : H,6.26; S ,
5.80. Found: C, 74.49; H , 6.09; N, 5.72; C-CHa, 1.65. Octahydro-opo-p-erythroidine.-A 200-mg. sample of apo-@-erythroidine was dissolved in 15 ml. of water and the solution was acidified with 0.5 ml. of concd. hydrochloric acid. A suspension of 25 mg. of Adams platinum catalyst in 45 ml. of water was added, and hydrogenation a t atmospheric pressure resulted in the absorption of four moles of hydrogen. The solution was filtered, made alkaline with I/ C, sodium bicarbonate, and extracted with chloroform. The crystalline residue, which was left after solvent removal, I' VI weighed 118 mg. and was recrystallized twice from ethanol; Oxidation of isoapo-@-erythroidine, under the m.p. 134-136". The octahydro-apo-,9-erythroidine did not same conditions applied to apo-@-erythroidine,did give a color reaction with ferric chloride or Ehrlich reagent. Anal. Calcd. for C,sHpaNO2: C, 72.26; H , 9.29; Iv, not yield formic acid; this result is consistent with structure V. However, a Kuhn-Roth determina- 5.61; C-CHa, 6.01. Found: C, 72.28; H , 9.34; h', 5.21; tion on isoapo-&erythroidine did yield acetic acid C-CHI, 2.75. Octahydro-apo- &erythroidine Hydrochloride .-The octashowing the presence of a carbon-methyl group in hydro-apo-8-erythroidine was dissolved in ethanol and a this compound. A Kuhn-Roth determination on slight excess of hydrogen chloride was added. The hydroapo-@-erythroidine also yielded acetic acid, but chloride crystallized afteor the solution was refrigerated overthis result is quite probably due to the rearrange- night; m.p. 250-252.5 Anal. Calcd. for C I ~ H ~ ~ K O ~ .C, H C63.03; ~: H , 8.46. ment of apo-p-erythroidine to isoapo-@-erythroidine under the conditions of acid and temperature Found: C, 63.03, 63.27; H, 8.23, 8.52. Octahydro-apo-@-erythroidine from Hydrogenation of used in this determination. A Kuhn-Roth deter- Isoapo-8-erythroidine.-Twenty ml. of water and 5 ml. of mination on octahydro-apo-@-erythroidine also concd. hydrochloric acid weore mixed and 1 g. of isoapo-pyielded acetic acid, a result which is consistent with erythroidine, m.p. 154-155 , was added. Hydrogenation was carried out with 100 mg. of Adams platinum catalyst structure IV. a t 40 lb. pressure. The filtrate from the catalyst was made Sauvage, Berger and Boekelheide3 have also alkaline with sodium bicarbonate and extracted with ten prepared apo-@-erythroidineand isoapo-@-erythroi- 25-ml. portions of chloroform, Evaporation of the chlorodine, and have converted desmethoxy-@-erythroi- form yielded 949 mg. of a colorless gum which solidified. dine into apo-/3-erythroidine. The slight differ- This residue was crystallized twice from methanol-petroleu? m.p. 134-136 . to give octahydro-apo-8-erythroidine, ences of melting points for the apo- and isoapo- ether A sample of this hydrogenation product was mixed with a compounds are possibly due to the ease of re- sample of octahydro-apo-8-erythroidinewhich melted a t arrangement of the apo- to the isoapo-compound 134-136 and was obtained from the hydrogenation of and to the difficulty of separating them. We have apo-&erythroidine. There was no depression of the meltfound that apo-@-erythroidine tends to isomerize ing point of the mixture. Anal. Calcd. for C16H23N02: C, 72.26; H, 9.29; S, in hot methanol. 5.61. Found: C,72.36; H,9.11; N,5.64. If structure I for @-erythroidine and the correOxidation of Apo-p-erythroidine to Formic Acid.-A sponding structures for its acid degradation products solution containing 66 mg. of apo-&erythroidine in 4 ml. of are not entirely satisfactory, they are a t least 2 LV H&Od was cooled to 0" in an ice-bath. One drop of provocative of new and obvious experiments which 2% potassium permanganate solution was added, but the red color did not disappear in five minutes. The solution would either confirm these structures or lead to was allowed to warm t o room temperature and oxidation final modifications. took place slowly. Altogether, 5 ml. of 2% potassium permanganate solution was added. A few drops of 30% hyExperimental drogen peroxide solution was added t o dispel the last of the Apo-p-erythroidine.-A 750-mg. sample of p-erythroipermanganate, and the mixture was centrifuged. The superdine hydrochloride was dissolved in 5 ml. of concd. hydro- natant was filtered, combined with the washings and distilled bromic acid, and the solution was heated for one hour on t o a residual volume of 5 ml. The concentrate was diluted the steam-bath. The solution was then diluted with 50 ml. with 10 ml. of water and distilled again t o a 5 4 . volume; of water, and extracted with six portions of chloroform. this dilution and distillation process was repeated for the The chloroform extract was washed with water and dis- third time. The distillates were combined and neutralized tilled. Th: crystalline residue weighed 300 mg. and melted to pH 6.5 by the addition of 10% sodium hydroxide solution. at 130-132 After five recrystallizations from methanolAfter adding 10 ml. of methanol, 49 mg. of p-bromophenrthyl ether, apo-&erythroidine which melted a t 144' was acyl bromide was added and the solution was refluxed for obtained; [ a I z 64-26.6 ~ (c, 0.3, CHaOH). three hours. The solution was evaporated to dryness in A n a l , Calcd. for Cl6HlbNOl: c , 74.65; H , 6.26; N , vacuo, and the residue was triturated with 2 ml. of warm 5.81; C-CHI, 6.2. Found: C, 74.42; H , 6.09; N, 5.70; methanol. The sodium bromide was collected on a filter and the filtrate was diluted with petroleum ether. After C-CHI, 1.00. Apo- and Isoapo-p-erythroidine.-Four grams of p - cooling in an ice-bath, crystal: separated which weighed 36 Recrystallization of this erythroidine hydrochloride was dissolved in 24 ml. of concd. mg. and melted a t 130-133 hydrobromic acid and the solution was heated in a sealed material from methanol g:ve the p-bromophenacyl ester of A mixture of this sample with tube a t 100-110' for three hours. After cooling, the solu- formic acid, m.p. 134-135 tion was diluted with 120 ml. of water and extracted twenty- an authentic specimen showed no depression of the melting point. five times with chloroform. The chloroform extract yielded 1.57 g. of crystalline residue, which was recrystallized from Acknowledgment.-We are indebted to Messrs. ethyl alcohol. The Srst crop of crystals weighed 914 mg. D. F. Hayman, W. Reiss and H. C. Clark for the and melted at about 126", and contained apo-&erythroidine in substantial amount. The mother liquor from the first microanalyses. summary crop was concentrated t o about one-half volume and refrigerated. The yield of crude isoapo-@-erythroidine was @-Erythroidine has been degraded to apo-P-
.
.
.
.
(3) Sauvage, Berger and Boekelheide, Scirncc, 109, G27 (1949).
erythroidine and isoapo-&erythroidine by reaction
Jan., 1951
ADDITIONALDATAON VITAMIN
with hydrobromic acid. The hydrogenation of apo- and isoapo-&erythroidine yielded the same compound, octahydro-apo-@-erythroidine. Oxidation of apo-&erythroidine gave formic acid.
[CONTRIBUTION FROM THE RESEARCH
335
Interpretation of these reactions and degradation products of @-erythroidineallows one to formulate tentative structures for these products. RAHWAY,N. J.
RECEIVED JULY 18, 1950
LABORATORIES OF MERCK& CO.,INC.]
Vitamin B12. XIII. Additional Data on Vitamin B12a BY EDWARD A. KACZKA, ROBERT G. DENKEWALTER, ARNOLDHOLLAND AND KARLFOLKERS In a recent communication, ' crystalline vitamin B*a was described as a reaction product of vitamin BU and hydrogen over a platinum catalyst. The described physical properties of vitamin B12a showed that it could be differentiated from vitamin Bls although these two compounds are obviously very closely related in composition and properties. The results of the lirst preliminary biological tests which were reported,' were not intended as a critical and final evaluation, but to show that vitamin B'Za possesses high vitamin B12 activity. Vitamin Bun has been examined further, and samples, including the one described,' have been found to be about 98% pure by solubility analyses. Vitamin B'Za separates from aqueous acetone in needle-like or bladed crystals which belong to the orthorhombic systems. The color of the crystals is somewhat darker red than those of vitamin BIZ. On the micro-block, the crystals start to darken at about 200°, with more decomposition appearing to take place than with vitamin B12, but do not melt below 300'. The refractive indices of two samples (dried in vacuo a t room temperature) which resulted from two separate hydrogenation experiments are given in Table I. When the crystals of sample 2 were heated for two hours a t 100' in vacuo, the refractive indices were found to be: CY, 1.604 * 0.004; P, 1.640 * 0.004; y, 1.654 * 0.004. This heating of crystals of vitamin Blza does not seem to alter very much the @ and y indices, but does seem to cause a progressive increase in the CY index. The refractive indices may be used as a criterion for identity and reproducibility of vitamin BIZa.
ABSORPTION SPECTRA IN WATER OF:
260
B
a f
340
-VITAMIN VlTPMlN VITAMIN (FROM
420 460 WAVELENGTH IN Mp.
380
B,* 84po
Blz0 ~.GRISE~~)
500
580
540
slight variations in the fiH of the solutions and this influence is demonstrated in Fig. 2. During the early work, the PH variations of water solutions of samples of vitamin were not determined, It is evident that vitamin B12a and the crystals from S. griseus cannot be differentiated by absorption spectrum (Fig. 1) or refractive indices (Table I). PBSORPTION SPECTRA OF VITAMIN B,,o:
----_-
WATER PHOSPHATE MIXTURE PH 5 0 ' PHOSPHATE MIXTURE PH 88'
-IN
L Y
1.580 * 0.002 1.640 A 0.002 1.657 1.580 0.002 1.640 * 0.002 1.656
300
------
Fig. 1.
TABLE I REFRACTIVE INDICES OF VITAMIN BIZ. Sample 1 2 Vitamin Blr (from s. griseus)
I\
200-
0.002
'
260
,
0
300
E
*
340
I
"
e
I
"
380 420 460 WAVELENGTH IN M,I.
I
500
"
540
"
' 500
Fig. 2.
* 0.002
A sample (NP-92-58-4)z of vitamin B12b,* has been compared with vitamin B12a obtained from Amorphous and crystalline concentrates from culture broths of Strefitomyces griseus, which the hydrogenation reaction with the following showed vitamin BIZ activity in microbiological results: The two samples cannot be differentiated assays, yielded a red crystalline compound after by absorption spectrum as illustrated by Fig. 3. further fractional crystallization. The absorption The sample of vitamin B1za used for this comspectrum of this red crystalline product is like parison was found to be 98.5% pure. When that of vitamin BIZ., but differs from that of vita- powdered samples of vitamin B12a were dried a t min B11 as shown in Fig. l. The slight variations 100' for two hours in vacuo and then dissolved in in the absorption spectra of vitamin BIZ. and the water, the spectrum was found to be, on immediate (S) Through the cowtony of Dr. T. E. Jukes of the Lederle Laboracrystals from S. griscUs in t h e regions 265-275 Division of tho Amerimn Cyanamid Company. w, 300-320 m p and 350-358 m p are influenced by tories, (8) Piorco, Page, S t o w and Jukr, TEN Jonnwu, 71, 2959 1.581 t 0 . 0 0 2
1.640h 0 . 0 0 2 1.657 a0.002
(1) k h ,Well amd Fotkw. Tma
JOURNAL,
V l , 1614 (1949).
(1949).
