and Rhodibinamide, the Rhodium Analogues of Vitamin B12 and Cobinamide*

Abstract Monocyano-α-(5,6-dimethylbenzimidazolyl)rhodibamide, dicyano-α-(5,6-dimethylbenzimidazolyl)rhodibamide, and dicyanorhodibinamide have been prepared by insertion of rhodium into the metal-free analogue of vitamin B12 using rhodium carbonyl chloride. The monocyano form of the rhodium analogue of vitamin B12 was also obtained by treatment of the corresponding dicyano form with silver nitrate. The new compounds are characterized by their spectral and electrophoretic properties and their biological activity. While dicyano-α-(5,6-dimethylbenzimidazolyl)rhodibamide is biologically inactive, the corresponding monocyano form is active as antimetabolite to vitamin B12 in suppressing the growth of Lactobacillus leichmanii (ATCC 7830).

The monocyano form of the rhodium analogue of vitamin Blz was also obtained by treatment of the corresponding dicyano form with silver nitrate. The new compounds are characterized by their spectral and electrophoretic properties and their biological activity. While dicyano-ar-(5,6-dimethylbenzimidazolyl)rhodibamide is biologically inactive, the corresponding monocyano form is active as antimetabolite to vitamin Blz in suppressing the growth of Lactobacillus leichmanii (ATCC 7830).
The only metals so far incorporated into naturally occurring descobaltocorrinoids are cobalt (3-5), copper (5,6), and zinc (5,6). Attempts to insert other metals have been without success, although the insertion of metals into a synthetic corrin has not presented any major difficulty (7). Thus Eschenmoser has reported the successful incorporation of cobalt, nickel, palladium, rhodium, zinc, and lithium into the metal-free 1,2,2,7,7,12,12heptamethyl-15.cyan-corrin hybrobromide (7). Of principal interest in the natural descobaltocorrinoid series is the insertion of a transition metal, which would allow the synthesis of the corresponding cobamide coenzyme analogue.
Such an analogue may be an interesting inhibitor of vitamin Bls coenzyme. We wish to report the preparation of cr-(5,6dimethylbenzimidazolyl)rhodibamide and rhodibinamide, the rhodium analogues of vitamin Brz and cobinamide.
The metal-free analogue of vitamin Brz was prepared from Chromatium (ATCC 17899), which was grown as described  previously (6). The method first used to isolate and purify this compound involved repeated extraction with phenol, ion exchange treatment, and paper electrophoresis at pH 2.5 (6).
Presently the aqueous extract of Chromatium is passed through a column of XAD-21 and the retained descobaltocorrinoids are eluted separately with aqueous tert-butyl alcohol (8), as outlined in Fig. 1. The descobaltocobalamin thus obtained still contains small amounts of phenylhydrogenobamide.
The compound is therefore retained on a small bed of CM-cellulose in the hydrogen form, eluted with 0.5 N acetic acid, and recycled through a small column of XAD-2.
Rhodium Co&no&-The rhodium analogues of vitamin Bn and cobinamide were prepared by insertion of rhodium into the metal-free analogue of vitamin Brz using rhodium carbonyl chloride [Rh(CO)L$.
The use of this reagent has been indicated for the insertion of rhodium into a synthetic corrin (7) and for the preparation of rhodium porphyrins (9). A solution of ar-(5,6-dimethylbenzimidazolyl)hydrogenobamide (30 mg) in ethanol-glacial acetic acid (3 : 1, v/v) (30 ml) was allowed to react with rhodium carbonyl chloride (150 mg) for 24 hours at room temperature.
The reaction mixture was diluted with 150 ml of water and the pH was adjusted to 9.5 using solid KCN. Ethanol was distilled off under reduced pressure and the remaining solution was kept at room temperature for 10 hours. From the reaction product thus obtained rhodium corrinoids were isolated as outlined in Fig. 2. After the product was desalted by adsorption and elution using a column (2.2 x 6 cm) of XAD-2 (50 to 100 pm), basic corrinoids, mainly yellow descobaltocorrinoids, were retained on a column (2.2 x '5 cm) of CM-cellulose in the hydrogen form. The aqueous pass-through contains a mixture of rhodium-containing corrinoids which was separated into a neutral and an acid fraction by treatment with DEAEcellulose in the acetate form. Both fractions were chromatographed on columns (1.3 x 5 cm) of XAD-2 (30 to 50 pm) using aqueous tert-butyl alcohol as eluant.
After elution of small amounts of rhodium-containing byproducts with 6 volume 7. were crystallized by addition of 10 volumes of acetone to a concentrated aqueous solution.
The former forms thin orange-red and the latter deep red needles. The total yield of rhodium-containing corrinoids is about 42%. The relative yields are as follows: cyanorhodibalamin (970), dicyanorhodibinamide (20 %), and dicyanorhobidalamin (71%). The structural relation among the three compounds was established by the following reactions: cerous hydroxide hydrolysis of dicyanorhodibalamin according to the method of Friedrich and Bernhauer (10) yields equimolar amounts of dicyanorhodibinamide and a-ribazole.
The latter was identified by ultraviolet spectroscopy, paper chromatography, and paper electrophoresis. The dicyanorhodibinamide obtained is identical with the neutral 10% zone (Fig. 2).
Treatment of dicyanorhodibalamin with silver nitrate gives in almost quantitative yield the monocyano form of rhodibalamin. An aqueous solution of the acid 10% zone was allowed to react with silver nitrate for 10 hours at room temperature. The identity of the formed product with the neutral 8% zone (Fig.   2) was established, after it was desalted by XAD-2 treatment and passed through a small bed of DEAE-cellulose in the acetate form (see Scheme 1).
All three compounds were found to contain 1 mole of rhodium, as determined by atomic absorption.
Based on the similarity of their absorption spectra (Figs. 4 and 5) with those of the corresponding Co"' analogues the Rhr" oxidation state is assigned to the rhodium in these complexes.
The presence of the cyano ligands is indicated by the infrared spectra (Fig. 3) of cyanorhodibalamin, dicyanorhodibalamin, dicyanorhodibinamide, and aquocyanorhodibinamide, which show absorption maxima at 2137 cm-l, 2119 cm-l, 2119 cm-l, and 2133 cm-l, respectively. The differences in the stretching frequences of the cyano ligand can be attributed to the different axial ligand in the second axial position.
The same effect has been observed in the corresponding cobalt corrinoid series (11). The similarity of the spectrum of cyanorhodibalamin with that of cyanocobalamin further indicates an identical structure of the peripheral corrin moiety of both compounds.
The assigned structures are further confirmed by the electrophoretic behavior and the absorption spectra of these compounds.
Monocyanorhodibalamin and dicyanorhodibinamide are neutral in 0.1 N KCN, at pH 7 and 2.7. Dicyanorhodibalamin is negatively charged in 0.1 N KCN and at pH 7, and it is neutral at pH 2.7. The charge properties are explained by the formulae in Scheme 2. The neutral behaviour of dicyanorhodibinamide at pH 2.7 indicates that the second cyan0 group is not exchanged as in the corresponding dicyanocobinamide. (2.54 X lo+ M) in water at pH 2.5 and 6.5 (-*)* 345 run). The spectra of dicyanorhodibalamin and dicyanorhodibinamide are identical in the region from 300 to 600 nm; the only difference is the appearance of a narrow band at 289 mu (pH 7 and 11) or 285 nm (pH 1) which can be attributed to the 5,6-dimethylbenzimidazole moiety. This difference is not as obvious because the spectrum of dicyanorhodibinamide contains a band at 291 nm (pH 1,7, and 11). In the curve of monocyanorhodibalamin the benzimidazole band is replaced by a shoulder at 286 nm (pH 1, 7, and 11) which is similar to the spectrum of the monocyano form of vitamin B12, where the absence of this band is explained by a coordinate linkage between N-3 of 5,6dimethylbenzimidazole and cobalt (12). That the marked decrease in the resolution of the corresponding maximum in the spectrum of the rhodium analogue may be interpreted as a similar coordination is indicated by a hypsochromic shift of the main peaks in the visible and ultraviolet regions as compared with the corresponding bands in the spectrum of dicyanorhodibalamin at pH 1, 7, and 11 (528 nm --f 514 nm, 497 nm + 486 nm, and 350 nm -+ 345 nm).
Compared with the spectrum of dicyanocobalamin ( Fig. 4) that of the corresponding rhodium analogue shows a general hypsochromic shift of the (Y-, fl-, and y-bands. Similar observations have been made in the coordination chemistry of metalloporphyrins where in the series of Co"', Rh"', and IrlI1 the band shift to shorter wave length increases in the order Ir > Rh > Co (9).
A striking property of the dicyanorhodium corrinoids is their conversion into yellow products at a pH below 2.5. If dicyanorhodibinamide is kept in 0.01 N HCl for 15 hours at room temperature a product is obtained with properties expected for cyanoaquorhodibinamide.
The compound is neutral at pH 11 and positively charged at pH 6.5 and 2.5. The absorption spectrum (Fig. 5) shows a hypsochromic shift of the a-, /3-, and y-bands as compared with the spectrum of dicyanorhodibinamide (528 nm -+ 499 nm, 497 nm + 481 nm, and 350 nm + 340 nm). At pH 11 these bands shift to longer wave length (499 nm --t 510 nm, 481 nm -+ 492 nm, and 340 nm -+ 343 nm), which can be explained by the loss of 1 proton of the aquo ligand. This interpretation is confirmed by the neutral charge of this compound at pH 11. Addition of KCN reconverts this product into dicyanorhodibinamide.
The biological activity of mono-and dicyanorhodibalamin was tested with Lactobacilhs Zeichmanii (ATCC 7830) according to the United States Pharmacopeia method (13). In the absence of vitamin BIZ neither compound showed signifiant growth-promoting activity.
The activity was less than 0.005% that of cyanocobalamin.
The highest level tested was 20 pmoles of rhodium corrinoid per ml of medium.
Further tests were carried out to determine whether these compounds exhibited any anti-vitamin Biz activity. The effect was determined on a culture of Lactobadus leichmanii (ATCC 7830) supplemented with cyanocobalamin and grown by the standard method (13). The vitamin Bi2 concentration was 0.1 pmole per ml of test medium.
While dicyanorhodibalamin showed no anti-vitamin effect, the corresponding monocyano form was active as antimetabolite of vitamin Blz with a 50% inhibition index of 65: 1. The 50% inhibition index is defined as the ratio of inhibitor to cyanocobalamin which reduces the growth response to 50% of that obtained with the vitamin alone.