ACETYLMIGRATIOX IN RIFAMPICIN
September 196s
937
1
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Figure 1.-Smr
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combined extraction, crystallization, and column chromatography. One of the products (Rf 0.23) was identified as I1 by comparison with an authentic samle.^ The other two products, I11 ( R f 0.35) and IT'
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spectrum of 25-desacetyl-2l-acetylrifampicin (111).
0 I,Ri=COCH,; RL=Rj=H 11, R, =R1= R,= H 111, R,= COCH,; R, = R2= H IV, R2= COCH,; R, = R, = H
( R f 0.621, showed most physical properties identical with those of I, cy., color, water solubility, ionization behavior, electronic spectrum, and polarographic oxidation wave. Elemental analyses (C, H, N) gave for both the same C.13Hj8K4012formula, like I. Yo useful information could be derived from the ir spectra which, however, showed some differences from that of I. Nmr spectra (CDCl, solution) of I11 and IT' (Figures 1 and 2 ) revealed the presence of all the proton signals previously reported for rifampicin3 with changes in cheniical shifts for some of them.
The chemical behavior of I11 and I V was therefore investigated by subjecting them to a mild acidic and basic treatment under controlled conditions. (a) After heating separately I11 and IT.' at 90" for 2 hr in a buffered aqueous solution (pH 6.0), two distinct products were found which on nmr analysis were found to be 3-formyl derivatives, different from the hydrolysis product of I, i e . , 3-formylrifamycin ST',4 thus indicating the persistence of the structural differences after the removal of the basic side chain at C-3. (b) When heating pH 8.5 buffered solutions of I11 and IT' for a few hours at 90-95", a mixture of I, 111, and IV along with small amounts of I1 was detected by tlc in both cases. Examination of the reaction solutions at various time intervals indicated that when I11 was the starting substance, the transformation into IT' was initially detected, whereas I was formed subsequently. Starting from IV, the formation of I and I11 was practically simultaneous. Since, starting from I, with the same treatment, an early formation of IT' followed by I11 was observed, the equilibria I IT' and IT' I11 can be considered as effective. (c) Stronger basic conditions, e . y . , treatment in a water-ethanol (1: 1)-1 N S a O H solution, induced a rapid transformation of both I11 and IV into desacetylrifampicin (11),formerly obtained from rifampicin7 (I). The chemical data referred to above are thus consistent with a reversible isomerism of 111, IT', and rifampicin (I). Since the physicochemical data do not provide evidence for differences in the chromophoric moiety of 111, IT-, and I, the only possible interpretation is the migration of the acetyl group during the mild alkaline treatment. It is noteworthy that the
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Figure 2.--?;mr spectrum of 2.j-desacetyl-%:~-acet~lrifanipic~i11 ( I \ .1. o t i l ~ .fra/rs acetylation
reported for aii inactive series of rifamyciiis iq described to occur under acidic conditions.8 T o ascertain whether the position of the acetyl group in I11 and IY rvab at the 21- arid 23-OH1respectively, or vice ce/'sa, iinir qpiri decouplirig experiments were performed. baied on the fact that the hydrogen at the carh i atom bearing the acetoxv group is at lower field than the hJ drogeii at the carbon atom bearing the h?,drox) 1.' In 1"igures 1 and 2 the iimr spectra of I11 and IT' are bhown together with the principal spin decouplings, which allowed us to assign the reported structures. The signals of the C-26, C-24, arid C-22 protons of 111 are almost at the same field, as shown by the excitation of the C-24 proton which causes, besides the complete decoupling of 33-CHd, also the partial decoupling of 34- arid 32-CH3 (lcigure l ) . For this reason it partial decouplirig of the signal of the C-21 proton i y observed by exciting the C-24 proton. The excitation of thc C-20 proton confirms the above attributions arid permit5 the location of the acetoxy group a t C-'21. Concerning IT'% the assignment of the signal o f the proton at C-24 arid its excitation permits the location of the acetoxF group at C-23 (Figure 2). The 2 1 ) v z t i o antibacterial teht. carried out by Dr. Istem of the hacterial c(,]l 111
Experimental Section Thiii layer c.hrciniatography (tlr j was perforn~ed(111 cilic:i gel I1F (E. AIewk, A . ( ;. 1)armstadt ) plates, using CI1Cl8-,\leOT1 ( 9 : I ) as solvent: the riiiining distance W R S 10 cni. lfelriiig points (open capillai method) are iiiicwrected. Column chrw rrieti oiit on silica gel 0.01-0.2 nim (E. matographies were lIerck, A,(;. I)arin-;tadt) prewitshed with CHC1,. Avidic. pk',, valrie. were deterinined spec.1rophotonietrically i n aqiieorii scilulion. Basic: pk', values ( p K ~ ( . were s ) obtained by potentiometric. titration i n lICS-H& (4:I ) > o l i i t i c i i i , Xmr apectra were recwrded oii it \'aria11 11.4-100 iiiAti.riment i l l CI)Cla solution (Tl18 as internal reference), by cwiirtesy of l h . A. Segre of the Politecnicci. AIilaii. Isolation of 111 and IV.- T o n tvarm (60-TO") ~ o l ~ t i oofi i 30 g of I in 3.0 1. of Sijrenson phosphate buffer, pH 8.2, :in excess (30 g ) of I-amino-l-mrthylpiperazine \vas added in ordw to prevent the hydrolysis t o the aldehyde and the oxidation 1)). atmospheric oy.gen to I quinone. After adjusting the pH to the appropriate valiir 18.2) with 2 3 SaOH, t h r solution W I S
September 1968
SAXGIVAMITCIN
stirred on a steam bath for 5 hr. Tlc analysis of the solution showed the presence of four major orange-yellow spots: Rf 0.23 (11), Rf 0.3.3 (111), Rr 0.52 (I), and Rf 0.62 (I\.) in the approximate ratio 2: 1 : 3.5: l.:, besides other colored by-products a t Rf
