[CONTRIBU~ON FROM
TEIIDDIUPARTMENT OF BIOCHEMIBTRY, COLLEQEOF AQRICULTUBE, UNIVERBITY O F WIBCONSIN 1.
THE PREPARATION O F 2-(ALDO-POLYHYDROXYALKYL)BENZIMIDAZOLES' STANFORD MOORE'
AND
KARL PAUL LINK
Rscsivsd July 19, 1 0 4
Griess and Harrow (1)in 1887 studied the condensation of carbohydrates with o-phenylenediamine and were able to isolate three types of reactionproducts. When an aqueous solution of o-phenylenediamine and two equivalents of aldose was evaporated to dryness, simultaneous oxidations and condensations occurred with the formation of a Schiffbase (I),oxidation on carbon two to give a quinoxalink (11),and oxidation on carbon one to produce a 2-(aldo-polyhydroxyalkyl)benzimidazole (111).*
- 0 0 N
N
II
I
CH CH HCOH HCOH HOCH HOCH HCOH HCOH HCOH HCOH HCOH HCOH H H I
N N
N NH
II II
\/
HC-C
HOCH HCOH HCOH HCOH H I1
C HCOH HOCH HCOH HCOH HCOH
H I11
The preparation of compounds of the quinoxaline type from osones is a general reaction, and the structure of the hydroxyquinoxalines from osonic acids has recently been studied by Ohle (3, 4). But the derivatives 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Based on a thesis submitted by Stanford Moore to the Graduate Faculty of the University of Wisconsin in partial fulfillment of the requirements for the degree of Doctor of Philosophy, June 1938. S.M. is indebted to the Wisconsin Alumni Research Foundation for a fellowship in 1935-36 and to the Graduate Research Fund for assistantships for 1936-39. We also wish to thank Dr. R. J. Dimler for the valuable assistance that he has given to this study. * The imidazole structure was assigned by Hinsberg and Funcke (2) and confirmed by Ohle (3) in 1934. In the same year Kuhn and Bar ( 5 ) prepared d-galacto-benzimidazole to test its reaction to sunlight irradiation as in the lumichrome and lumiflavin cleavages. Karrer and coworkers (6) encountered I-arabo-benzimidazole as a side product in the synthesis of flavins.
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STANFORD MOORE AND KARL PAUL LINK
with the bengimidazole heterocyclic nucleus have received less attention and methods for their preparation in satisfactory yields have been lacking. The benzimidazole derivatives show chemical and physical properties which are superior from a characterization standpoint to those of the hydrazine derivatives of aldo-monosaccharides. The objective in the following investigation has been the development of a satisfactory method of preparation for aldo-benzimidazoles in order to utilize the favorable properties inherent in this type of carbohydrate derivative. An extension of the present study has been its application in a procedure for the identification of a series of naturally occurring aldoses (7). The direct oxidative condensation of aldehyde and o-phenylenediamine, as used in the experiments of Griess and Harrow, gives very low benzimidazole yields. A more promising method of preparation, and the one which has proved satisfactory, would appear to be offered by the preliminary oxidation of aldose to aldonic acid and formation of the benzimidazole by the carboxylic acid-0-phenylenediamine condensation (7, 8, 9). The first and earlier method, however, is not to be discarded in favor of the second path without consideration, because of the advantage it possesses of general applicability to disaccharides as well as to monosaccharides. Therefore the possibility of increasing the yields in this direct aldose condensation has been investigated. In general, with simple aldehydes (acetaldehyde or benzaldehyde), in which there is only one path for oxidation, the condensation with o-phenylenediamine gives fair yields of benzimidazoles (Landenburg-Hinsberg synthesis). The yields in this reaction were increased by Weidenhagen (10) in 1936 by introducing cupric acetate into the solution to serve as an immediately available oxidizing agent.
Weidenhagen, however, reported no experiments on the preparation of aldo-benzimidazoles. Under the cupric acetate conditions, which give a sixty per cent yield of 2-phenylbenzimidazole from benzaldehyde, we have been able to obtain only a trace of the desired heterocyclic derivative from glucose. This same procedure, when applied to galactose, gave a forty per cent yield of galacto-benzimidazole. This fair result with galactose, which is known to be less sensitive to keto-oxidation than the stereoisomeric structure of glucose, suggested that minor changes in Weidenhagen’s experimental conditions might also bring the glucose reaction into a satisfactory range. Gradual addition
2- (ALDO-POLYHYDR0XYALKYL)BENZIMIDAZOLES
639
of the oxidizing agent, decrease in the temperature of the reaction, and increase in the acidity of the reaction-mixture were tried. The yield of gluco-benzimidazole was increased to twenty-five per cent by carrying out the reaction in dilute acetic acid for twelve hours a t 50'. But this yield is still low and side reactions are prominent. Therefore, the direct oxidative condensation must be considered impractical for aldo-monosaccharides, at least for the present, in view of the seventy to eighty per cent yields which are obtained by the following procedure involving the aldonic acid condensation. In the aldonic acid reaction, as an application of the condensation of a carboxylic acid with o-phenylenediamine, the oxidative step in the preparation can be made essentially free from side reactions. The several available methods for the oxidation of aldoses to aldonic acids may be used (7).
HO\
/H
do
"."O
A\OH HN HEZi-l H OH H HCOH HOCH 0 HOCH HOCH HCOH ! oxid. HCOH HCOH HC------! HCOH ( - 2 ~ 0 ~ ) ) HCOH HCOH HCOH HCOH H H H Glucose Gluconic acid Gluco-benzimidazole +
The general procedures for the condensation of carboxylic acids with o-phenylenediamine cannot be satisfactorily transferred to the carbohydrate group without modification. With aliphatic and aromatic acids the procedure most frequently used for the o-phenylenediamine condensation has been the heating of a mixture of the acid with the base at looo, 150', or 180'. More recently the refluxing of an excess of the carboxylic acid with o-phenylenediamine in 4 N hydrochloric acid has been used by Phillips (11) in the aliphatic series. It is first to be noted that with the stereoisomeric hexonic or pentonic acids, the fusion method, in the absence of acid catalysts, fails to work in certain cases. We have found that xylonic acid, for example, cannot be converted to its corresponding benzimidazole by this method. Haskins and Hudson (9), in the preparation of a series of aldo-benzimidazole bases for use in the resolution of racemic acids, have encountered similar difficulty in the condensation of idonic and D-manno-D-galaheptonic acids. The addition of hydrochloric acid to the reaction-mixtures gave them thirty and fifty-eight per cent yields of benzimidazoles from the two aldonic acids. Another factor which arises in the carbohydrate series, and is not present
640
S T A N m R D MOORE AND KARL PAUL L3NX
in the fusion method on optically inactive aliphatic acids, is the consideration of racemization or epimerization. It is well known that when carbohydrate acids are heated to 100' (or above) with organic bases, epimerization may take place a t an appreciable rate. In the o-phenylenediamine condensation (in the absence of hydrochloric acid) the amount of epimerization appears to be negligible a t 100". However, if an attempt is made to induce condensation of unreactive lactones by increasing the temperature to 135-150', which is the range in which the majority of aliphatic acids react most satisfactorily, we have found that epimerization may occur. From the fusion of xylonic acid and o-phenylenediamine at 150°, in the absence of mineral acids, we have isolated the epimeric lyxo derivative. Addition of acid catalysts prevents this change. In any condensation method to be applied to the carbohydrate series, the question of epimerization must be under complete control. Therefore, from the standpoints both of facility of reaction and elimination of epimerization, the use of acid catalysts in a general procedure for the aldonic acid condensation is to be preferred. In use of the acid catalyzed condensation, it has been found that the procedure of refluxing the reactants with 4 N hydrochloric acid gives less satisfactory yields with carbohydrate acids than with the fatty acid series. For example, the yield from galactonic acid was 24%. If the reactionmixture was allowed to concentrate to a syrup during the boiling, the yield of the same aldo-benzimidazole was 70%. The use of phosphoric acid (in addition to hydrochloric) has been found to provide a reaction medium which gives more uniformly complete condensation with most aldonic acids. Concentration of the solution of aldonic acid and a slight excessof o-phenylenediamine to a syrup at 135' in the presence of hydrochloric and phosphoric acids gives SO-SO% yields of aldo-benzimidazoles from arabonic, galactonic, gluconic, lyxonic, mannonic, and rhamnonic acids. Xylonic acid, however, is an exception to the generality of this 135' procedure. The difference in ease of benzimidazole formation shown by stereoisomeric acids is remarkably great for this aldonic acid and its epimer, lyxonic. Under the above conditions no xylo-benzimidazole is formed, but one equivalent of o-phenylenediamine has reacted with xylonic acid and is non-extractable from the reaction-mixture with ether after neutralization with ammonia. Isolation and analysis of the intermediate proves the preliminary reaction to involve condensation with the elimination of one molecule of water. Removal of the second molecule of water to give the heterocyclic benzimidazole structure can be accomplished in this case at a higher temperature. An acid reaction medium at 180" gives xylobenzimidazole in good yields. The catalyst which has proved most satisfactory for the 180' condensation is a mixture of zinc chloride and
641
2-(ALDO-POLYHYDR0XYALKYL)BENZIMIDAZOLES
hydrochloric acid. However, the phosphoric and hydrochloric acid catalyst can be used. Together with these differences in the reactivity of aldonic acids with o-phenylenediamine, differences are observed in the stability of the benzimidazoles which result from the reaction. Xylo-benzimidazole forms at 180" and is relatively stable at that temperature in the presence of hydrochloric acid and zinc chloride. Gluco-benzimidazole, on the other hand, which forms at 100-135' undergoes appreciable decomposition at 180". In general, the six aldo-benzimidazoles which can be prepared at the lower temperature are relatively stable up to about 165' in the two hour condensation period. An indication of the stability of the gluco derivative can be observed are satisfactorily near in Table I. From 100' to 165" the values forDI.[ to the figure of +9.4' which would be obtained if benzimidazole formation were quantitative. However, when a sample of gluco-benzimidazole is heated for two hours with hydrochloric acid and zinc chloride at 180" the calculated rotation rises to +43.4'. TABLE I TBMPERATURE-ACID STABILITY OF d-GLUCO-BENZIMIDAZOLE [OID
CATALTST
&Gluco-benzimidazole.............. d-Glucono-lactone and o-phenylenediamine. ......................... 4i
I1
Il
dl
il
ld
d-Glueo-benzimidaiole . . . . . . . . . . . . . .
TWP!BATUBE
C
cmu-
LATnD *OM IIOTATION OB BEACTIORMXTWlIB.
+9.4
HCl-H s P 0 4 HCI-HJ'Or HCl-H ,PO, HC1-ZnClr HCl-ZnCll
lo0 135 165 165 180
+9.8
+10.0 +10.2 +10.0 +43.4
* The specific rotations of the reaction-mixtures (decolorized and made tovolume) have been calculated in terms of the benzimidazole equivalents of the weighed samples of glucono-lactone or benzimidazole used as starting material. The general chemical properties of this class of derivatives can be briefly summarized. The 2-(sldo-polyhydroxyalkyl)benzimidazoles obtained from aldoses or aldonic acids are amphoteric compounds. As bases (tertiary amines) they dissolve in aqueous acids but may be precipitated by the addition of a stronger base (ammonium hydroxide). Crystalline salts of strong acids (e.g., benzimidaaolium chloride) may be readily prepared. The hydrogen on the secondary nitrogen is weakly acidic and aldobenzimidazoles dissolve in an excess of a strong base (sodium hydroxide)
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STANFORD MOORE AND KARL PAUL LINK
but not in ammonium hydroxide. They may be precipitated from solution in sodium hydroxide by the addition of carbon dioxide. Ammoniacal silver, zinc, and copper solutions cause the formation of insoluble complex salts. In the absence of excess ammonium hydroxide the precipitation of aldo-benzimidazoles as copper salts is quantitative as shown by an [a],, of 0.00' on the filtrate. The secondary nitrogen can be alkylated. Glucobenzimidazole reacts with benzyl bromide in aqueous alcohol to give 1 - benzyl - 2 - (d - gluco - pentahydroxyamy1)benzimidazole. The physical properties of individual derivatives (m.p., [aID,m.p. of picrate and hydrochloride) are tabulated by Moore and Link (7) and by Haskins and Hudson (9) and are therefore not included here. EXPERIMENTAL
Cupric acetate procedure on glucose. The conditions for the direct oxidative condensation of glucose with o-phenylenediamine which have given the best yields in our experiments are as follows. One gram of o-phenylenediamine, 4 g. of Cu(0Ac)t. H20, and 3 cc. of glacial acetic acid were dissolved in 80 cc. of water in a 100 cc. centrifuge tube. To the solution 2 g. of glucose was added and the stoppered tube was placed in a 53" oven for 14 hours. A small amount of grey precipitate waa formed, along with a thin red film on the wall of the tube. Concentrated ammonium hydroxide was added dropwise to the tube from a dropping-funnel until addition failed to cause further precipitation. Adjustment to complete precipitation was made by testing the supernatant liquid with a drop of ammonia or with dilute acetic acid if an excess of ammonia had been added. The voluminous precipitate of copper hydroxide and copper salt of the benzimidazole was finally centrifuged, the supernatant solution decanted, and the precipitate washed twice with 40 cc. of water by suspending and centrifuging. Twentyfive cubic centimeters of water and 2 cc. of glacial acetic acid were added and the suspension was decomposed with hydrogen sulfide. After addition of carbon and removal of copper sulfide and hydrogen sulfide, the filtrate was reheated on the steam-bath and made alkaline with ammonia. From the concentrated filtrate d-gluco-benzimidazole crystallized to give a yield of 0.6 g. (a%), m.p. 215". Similarly, galactose gave 40-50% yield of d-galacto-benzimidazole (m.p. 245"). Aldonic acid condensation. Representative is the preparation from gluconic acid (or lactone). To 1.6 g. (9 mM) of gluconolactone (y or 6) were added 1.1 g. (10 mM) of o-phenylenediamine, 4 cc. of water, 1cc. of ethanol, 0.9 cc. of conc'd hydrochloric acid, and 0.9 cc. of syrupy phosphoric acid. The mixture (with boiling-chip) was warmed in a test tube until in solution, and heated for two hours on an oil-bath kept a t 135' f5". During the condensation, water boils off to leave a thick syrup, which was dissolved while still warm in about 10 cc. of water, and filtered with carbon through asbestos. The filtrate (about 30 cc.) was made basic with ammonia. The yield of gluco-benzimidazole (washed with water, acetone, ether) was 1.9 g. (80%). Calcium, barium, or potassium salts of gluconic, arabonic, galactonic, lyxonic, mannonic, and rhamnonia acids serve equally well as starting products (7). Reaction of xylonic acid. Under the above conditions a t 135' xylonic acid gave no benzimidazole. All products of the reaction remained water-soluble after addition of ammonia. Extraction of unreacted o-phenylenediamine with ether gave a recovery of less than 0.1 g. The addition of ammoniacal copper solution (7), how-
%(ALDO-POLYHYDR0XYALKYL)BENZIMIDAZOLES
643
ever, gave a heavy precipitate of a copper salt which was centrifuged and decomposed with hydrogen sulfide t o give an aqueous solution of the reaction-product free from inorganic salts. After concentration of the solution to a thin syrup, the addition of 25 cc. of acetone caused the formation of an insoluble oil from which the supernatant solution was decanted and the oil heated with a second portion (15 cc.) of acetone t o remove water further. The oil was dissolved in a few cc. of hot butanol, acetone added until a permanent cloudiness resulted, and the solution filtered through carbon. After the acetone was removed by evaporation, the butanol solution was slowly concentrated in a vacuum desiccator, and allowed to crystallize for 48 hours. The crystals (needles) were triturated with cold butanol, filtered, and washed with butanol, acetone, and ether; m.p. 140-141". Recrystallized from butanol (m.p. 140-141", picrate, m.p. 187-189') CllH1sN206,calc'd N,10.94;found N,10.9. A sample of these crystals heated with hydrochloric acid-zinc chloride for one hour at 180" gave xylo-benzimidazole, m.p. 224". Xylo-benzimidazole is also obtained by direct condensation from xylonic acid a t 180" (7). N-Benzyl-d-gluco-benzimidazole. One gram of d-gluco-benzimidazole was dissolved in 25 cc. of hot water, and 1.8 cc. of benzyl bromide was added together with sufficient ethanol to keep the alkyl halide in solution. To the warm solution on a steam-bath, 8 cc. of 10% sodium carbonate was added dropwise over a period of about 15 minutes. The alcohol was allowed to boil off during the next hour and the benzyl alcohol which separated as an oil was shaken out with ether. The benzyl derivative crystallized during the ether extraction or upon evaporation of the aqueous solution; yield 0.4g. (30% of theory, plates, m.p. 188", [a]: f37.0"). C1PHZZN20S calc'd N, 7.82; found N, 7.74. The benzyl derivative is non-acidic (insoluble in sodium hydroxide). The second crop of crystals from the aqueous solution was completely soluble in sodium hydroxide, m.p. 215' (unreacted gluco-benzimidazole), SUMMARY
1. Study has been made of the preparation of 2-(aldo-polyhydroxyalkyl)benzimidazoles by two methods. The earlier yields in the direct oxidative condensation of aldo-monosaccharides with o-phenylenediamine have been raised to twenty-five per cent and forty per cent for the gluco and galacto derivatives. For the preparation from the aldonic acid, instead of the aldose, conditions have been developed which give seventy to eighty per cent benzimidazole yields with a series of carbohydrate acids. 2. Observation has been made of the variation in the condensation of stereoisomeric aldonic acids with o-phenylenediamine and of the differences in stability of the resulting benzimidazoles. 3. The methods of preparation and the chemical properties of the benzimidazole nucleus have been studied in reference to the use of these derivatives in carbohydrate characterization. MADISON,WIS.
REFERENCES (1) GRIESSAND HARROW, Ber., a0, 281, 2205, 3111 (1887). (2) HINSBERQ AND FUNCKE, Ber., 26, 3092 (1893).
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STANFORD MOORE AND KARL PAUL LINK
(3) OHLE,Ber., 67, 155 (1934). (4) ERLBACH AND OHLE,Ber., 67, 555 (1934). (5) KWN AND BAR,Be?., 67, 898 (1934). (6) KARBER,BECKER, BENZ,FREI,SALOMON, AND SCHBPP, Helv. Chim. Acta, 18, 1435 (1935). (7) MOOREAND LINK,J . B i d . Chem., 159, 293 (1940). (8) Summaries of Doctoral Dissertations, Univ. of Wis. Press, 3, 191 (1938). J . Am. Chem. SOC.,61, 1266 (1939). (9) HASKINSAND HUDSON, (10) WEIDENHAGEN,Ber., 69, 2263 (1936). (11) PEILLIPS,J . C h m . SOC.,1928, 2393.
