308-1
G. EMERSON, W . G. BRINK,F. Itr.HOLLY,F. KONIUSZY, D. HEYLAND K . FOLKERSVol. 72
[CONTRIBUTION FROM
Vitamin Bl,.
THE
VIII.
MERCKINSTITUTE FOR THERAPEUTIC RESEARCH AXD MERCK& CO., INC.]
THE
RESEARCH LABORATORIES OF
Vitamin B ,,-Like Activity of 5,6-Dimethylbenzimidazole and Tests on Related Compounds
BY GLADYS EMERSON, NORMAN G. BRINK,FREDERICK W. HOLLY,FRANK KONIUSZY, DOROTHEA HEYL AND KARLFOLKERS The degradation of vitamin B I ~by acid hy- zimidazoles and diamines might lead to the syndrolysis to give a new basic compound which was theses of vitamin BIZantagonists which are desired identified by its reactions and by synthesis as for various biological problems. A summary of 3,6-dimethylbenzimidazole (I) has been de- these combined chemical and biological results is scribed. l It was concluded that 5,6-dimethyl- as follows. benzimidazole is a component of vitamin Blz The data in Table I show that 5,6-dimethyland is terminal and linked through nitrogen as benzimidazole and 1,2-diamino-4,5-dimethylbenin the provisional formula (11). Further deg- zene have vitamin BIZ-like activity when fed to rats radation of 5,6-dimethylbenzimidazolewith ben- on the above described diet.a Daily quantities zoyl chloride and alkali gave 1,2-dibenzamido- of 2-5 mg. of 5,6-dimethylbenzimidazole and 4,5-dimethylbenzene (111). Thus, 1,2-diamino- 2-3 mg. of 1,2-diamino-4,5-dimethylbenzene were 4,Ei-dimethylbenzene (IV) may be considered a comparable to 0.125-0.250 pg. of vitamin BIZ. Although these milligram-amounts of 5,6-dimethylbenzimidazole and 1,2 - diamino - 4,5 - dimethylbenzene are approximately 10,000-20,000 times greater than the microgram quantities of vitamin BIZ necessary to produce comparable growth responses in the test animals, they show I1
TABLE I VITAMINB I z - L 1 ACTIVITY ~ DATA Wt.
I11
IV
degradation product of 5,6-dimethylbenzimidazole or vitamin BIZ,and it may be contemplated as a “precursor” of vitamin Biz. I n another laboratory,2 spectroscopic evidence and paper chromatographic data indicated 5,6dimethylbenzimidazole as an acid degradation product of vitamin Biz. In addition to its high therapeutic activity for pernicious anemia and other anemias, vitamin B12 .has shown APF (“animal protein factor”) activitya when fed a t a level of 0.063 pg./day to rats on a diet devoid of animal protein and containing 0.25% of thyroid powder. It is considered important t o determine whether parts of the large vitamin Biz molecule, which may be readily synthesizable, show useful biological activity. Therefore, 5,6-dimethylbenzimidazole and 1,2-diamino-4,5-dimethylbenzene, as such degradation products, have been tested for APF activity or vitamin BIZ-likeactivity. Since this test gave a positive and interesting result, several alkyl-substituted benzimidazoles were prepared and tested to gain knowledge of the specificity of the vitamin BIZ-like activity of 5,6-dimethylbenzimidazole and its related diamine. It was considered possible that an investigation of ben(1) Brink a n d Folkera, Tms JOURNAL, 71, 2961 (1949). (2) Holliday a n d Pctror, J . Pharm. ard Pharmacol., 1,734 (1940). (8) Emcrwn, Pw. SOC.E#). B W . Idbd,, TS, 80P (1049),
Substance
(Negative controls) Vitamin BIZ Vitamin Blz Vitamin B12
5,6-Dimethylbenzimidazole 5,6-Dirnethylbenzimidazole 1,2-Diamin0-4,5-dimethyl-
f%%:,’ ,,.. 0.0625pg. 0.125 pg. 0.250fig. 2.0 mg. 5.0 mg.
benzene 2.0 mg. 1,2-Diamino-4,5-dimethyl3.0 mg. benzene
increment No. of g.-15 rats days
57 50 20 10 10 10
28 54 64 72 48 84
9
60
9
78
that these compounds compare on a weight-basis with certain vitamins such as vitamin E, pantothenic acid and choline in their respective tests. The vitamin requirements of the rat per 100 g. of ration are as follows for these substances: vitamin E, 3 mg.; pantothenic acid, 1 mg.; choline, 100 For better comparison per 100 g. of ration, the level of 5,6-dimethylbenzimidazolemay be estimated as about 12-30 mg. and that of 1,2diamin0-4~5-dirnethylbenzene as 12-18 mg. The extremely potent biological activity of vitamin BIZ tends to over-shadow the potency of these degradation products a t their respective levels. Benzimidazole (V), four mono-methylbenzimidazoles, and two other dimethylbenzimidazoles were assayed for vitamin Bln-like activity in rats (4) “Vitamins and Hormonea,” Vel, VII, Academic Prem, Inca,
New York, N. Y., 1040, D. 197.
COLORAND ACIDITYOF HYDROXYNAPHTHOQUINONES
July, 1950
3085
zene were observed to be inactive in the Lactobacillus lactis Dorner assay for vitamin Bl2 activity. They were tested a t concentrations up to 0.5 mg./ml. by Miss Muriel C. Caswell of our Microbiology Department. These benzimidazoles were prepared by the reaction of the diamine with the appropriate acid v under comparable conditions, and these results essentially as described in the literature, and the melting points were identical with the published are summarized in Table 11. constants: benzimidazole,6 l-methylbenzimidazole,' 2-methylbenzimidazole,* 4-methylbenzimiTABLE I1 d a ~ o l e ,5-methylbenzimidazole, ~ lo 2,S-dimethylRESULTSOF TESTSFOR VITAMIN B I 2 - L 1 ~ ACTIVKKY ~ benzimidazolelI and 4,6-dimethylbenzimidazole. l2 W t . increment Amounts No. of g.-15 Substituted Summary fed daily rats days benzimidazoles 5,6-Dimethylbenzimidazole and 1,2-diamino57 28 ... Negative Controls 4,5-dimethylbenzene1 which are degradation prod64 20 0,125 pg Vitamin BIZ ucts of vitamin BIZ, have been found to show 8 37 2 . 0 mg. Benzimidazole vitamin BIZ-likegrowth activity when fed to rats 9 27 2 . 0 mg. 1-Methylmaintained on a diet devoid of animal protein 10 29 2 . 0 mg. 2-Methyland containing thyroid powder. These two 7 31 2 . 0 tng. 4-Methylproducts are active a t milligram-levels in con2 . 0 mg. 10 59 5-Methyltrast to vitamin BIZwhich is active a t the micro8 17 2 . 0 mg. 2,5-Dimethyl gram-level. 10 37 2 . 0 mg. 4,6-Dimethyl5-Methylbenzimidazole showed significantly Benzimidazole, 1-methyl-, 2-methyl-, 4-methyl- high vitamin BIZ-like activity also. Benzimidaand 4,6-dimethylbenzimidazole did not elicit zole and four monomethyl and dimethyl derivaa significantly high response. 2,5-Dimethylben- tives failed to elicit significantly high activity, zimidazole appeared to act as a growth-depressant 2,5-Dimethylbenzimidazole appeared to show and may have inhibitor activity. 5-Methylben- growth-depressant or inhibitor properties. zimidazole showed significant growth activity, a (6) Pauly a n d Gundermann, Be?., 41, 4012 (1908). fact which is interesting since 6- and 7-methyl ( 7 ) Fischer and Vriel, ibid., 38, 321 (1905). (8) Hinsbera a n d I'uncke, ibid., 27, 2189 (1894). substitutions in the %(1'-D-ribityl)-isoalloxazine (9) Gabriel and Thieme, ibid.. 62, 1081 f l 9 1 9 ) . series show low riboflavin activity.j (10) Ladenburg, ibid., 10, 1123 (1877); I'ischer. i b i d . , 22, 611 Both 5,6-dimethylbenzimidazole and the corre- (188Q). sponding diamine, 1,2-dimethyl-4,3-dianiinoben(11) Ladenixira. fbid., 8, 677 (187.5). ( 5 ) Karrer, V. Euler, Malmberg a n d Schopp, Svensk. Kern. Tids., 47, 153 (1935); Karrer and Strong, Helu. Chirn. Acta, 18, 1343 (1936).
[COSTRIBUTION F R O M THE CHEMICAL
(12) Fischer a n d Rigaud, ibid.,34, 4205 (1901).
RAHWAT, 5. J.
RECEIVED JANUARY 13 1950
LABORATORY OF IIARVARD USIVERSITY]
Hydroxynaphthoquinones. I. Color and Acidity BY MARTING. ETTLINGER' I t :s well known? that 3-substituted 2-hydroxy1,4-naphthoquinones (I) are acids comparable in strength to carboxylic acids, and that their salts are deeply colored, from yellow to violet. The influence of the substituent (R) on these two properties is the subject of this paper. &H
'
-R /I 0 I
( I ) Member of t h e Society of Fellows, Harvard University. (21 l'irser, 1,eMer. ( 1 a , ,T w r s J O I l n N A r . , 70, 0 1 5 1 ( I 9 l s , .
Absorption spectra? of both the free hydroxy quinones (I) and their anions contain several bands in the ultraviolet and visible, whereof those a t longest wave lengths are most sensitive to structural change, and therefore interesting. For example, the spectra3f4of hydrolapachol (I, R = (CH2)2CH(CK&) and a-lapachone (11) are identical below 360 mp, and hence unaltered in that region by substitution of 2-alkoxy1 for hydroxyl, but differ in the position of the band extending into the visible. In an un-ionized hydroxy quinone a more intense peak a t 330-335 mp overlaps the interesting one, but in the anion the latter band is shifted on the order of 100 m p (3) Cooke, XIacheth a n d Winzor, J . Chcm. Soc., 879 (1'339). (4) R t t l i n g e r , Paper 11. 'rjl1s JurJnN.41z, 72, 3090 ( I S S O j .
