36-derivatives of rifamycins and their use as antimicrobial agents

Rifamycin antibiotic derivatives of formulae (I) and (Ia) bearing at the position 36 a substituent selected from (C.sub.1 -C.sub.8)alkyl, halo, hydroxy, (C.sub.1 -C.sub.4)acyloxy, (C.sub.1-C.sub.4)alkoxy, (C.sub.1 -C.sub.4)alkylthio, (C.sub.1 -C.sub.4)alkylamino, di(C.sub.1-C.sub.4)alkylamino and substituted 4-oxo-3-pyridinyl carbonyloxy of formula (1) obtained by reacting rifamycin with suitably substituted malonic acid. The compounds of the invention are antimicrobial agents mainly active against gram positive bacteria and fastidious gram negative bacteria showing the considerable antimicrobial activity against the rifamypicin resistant microbial strains.

This invention relates to novel rifamycin antibiotic derivatives of general 
formula I: 
##STR2## 
and to the oxidated derivatives thereof of formula Ia: 
##STR3## 
wherein: R represents, halo, hydroxy, thio, (C.sub.1 -C.sub.4)alkoxy, 
(C.sub.1 -C.sub.4)alkylthio, (C.sub.1 -C.sub.4)acyloxy, (C.sub.1 
-C.sub.4)alkylamino, di(C.sub.1 -C.sub.4)alkylamino or a group of formula: 
##STR4## 
wherein: R.sup.3 represents (C.sub.1 -C.sub.4)alkyl or (C.sub.3 
-C.sub.6)cycloalkyl; 
R.sup.4 represents a group of formula 
##STR5## 
wherein: R.sup.6 and R.sup.7 independently represent hydrogen or (C.sub.1 
-C.sub.4)alkyl or 
R.sup.6 and R.sup.7 together with the adjacent nitrogen atom form a five or 
six membered heterocyclic ring, optionally containing one further 
heteroatom selected from oxygen, nitrogen and sulfur, wherein one of the 
carbon or nitrogen atoms of the ring is optionally substituted by a 
(C.sub.1 -C.sub.4)alkyl moiety; 
R.sup.5 is hydrogen or halo; 
or R.sup.4 together with R.sup.5 form a bifunctional alkylenic chain, 
optionally containing 1 or 2 nitrogen atoms, of the following formula: 
##STR6## 
wherein: R.sup.8 represents hydrogen or halogen; 
R.sup.9 represents (C.sub.1 -C.sub.4)alkyl, or a six membered heterocycle 
ring containing one or two nitrogen atoms, wherein the carbon and nitrogen 
atoms of the ring are optionally substituted with one or two (C.sub.1 
-C.sub.4)alkyl moieties; 
R.sup.1 is hydroxy in formula I or oxygen formula Ia; 
R.sup.2 represents hydrogen, a five or six membered heterocyclic ring 
containing one or two heteroatoms selected from oxygen, nitrogen and 
sulfur, wherein one of the carbon or nitrogen atoms of the ring is 
optionally substituted by a (C.sub.1 -C.sub.4)alkyl moiety, or a group of 
formula 
--CH.dbd.N--R.sup.10 
wherein R.sup.10 represents a six membered heterocycle ring containing one 
or two nitrogen atoms, wherein one of the carbon or nitrogen atoms of the 
ring is optionally substituted by (C.sub.1 -C.sub.4)-alkyl or (C.sub.5 
-C.sub.6)cycloalkyl; 
or R.sup.1 and R.sup.2 taken together form a group of formula 
.dbd.N--(CHR.sup.11)--X--, --NH--(CHR.sup.11)--X--, or 
--N.dbd.(CR.sup.11)--X--, 
wherein: 
X represents a sulfur atom or a --NH--group and 
R.sup.11 represents hydrogen, (C.sub.1 -C.sub.4)alkyl, (C.sub.1 
-C.sub.4)alkylamino or di(C.sub.1 -C.sub.4)alkylamino; 
and the pharmaceutically acceptale base addition salts thereof. 
in-the present description, the terms used above in defining the meanings 
of the substituents R.sup.1 to R.sup.14 are intended to have the meanings 
commonly assigned to them in the art. Accordingly: 
(C.sub.1 -C.sub.4)alkyl represent a linear or branched hydrocarbon moiety 
containing carbon atoms respectively, such as: 
--CH.sub.3, 
--CH.sub.2 --CH.sub.3, 
--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--CH--(CH.sub.3).sub.2, 
--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--CH(CH.sub.3)--CH.sub.2 --CH.sub.3, 
--C(CH.sub.3).sub.2 --CH.sub.2 --CH.sub.3, 
--CH.sub.2 --CH(CH.sub.3)--.sub.3, 
--C--(CH.sub.3).sub.3, 
halo represents fluoro, chloro, bromo or ioda; 
(C.sub.1 -C.sub.4) alkoxy represents a linear or branched ether moiety 
containing 1, 2, 3 or 4 carbon, such as: 
--O--CH.sub.3, 
--O--CH.sub.2 --CH.sub.3, 
--O--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--O--CH--(CH.sub.3).sub.2, 
--O--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--O--CH(CH.sub.3)--CH.sub.2 --CH.sub.3, 
--O--CH.sub.2 --CH(CH.sub.3)--.sub.3, 
--O--C--(CH.sub.3).sub.3, 
(C.sub.1 -C.sub.4)acyloxy represents a carboxylic moiety containing 1 to 4 
carbon atoms, such as: 
--O--CO--H 
--O--CO--CH .sub.3, 
--O--CO--CH .sub.2 --CH.sub.3, 
--O--CO--CH .sub.2 --CH.sub.2 --CH.sub.3, 
--O--CO--CH ---(CH.sub.3).sub.2 ; 
(C.sub.1 -C.sub.4)alkylthio represents a linear or branched thioether 
moiety with 1 to 4 carbon atoms, such as: 
--S--CH.sub.3, 
--S--CH.sub.2 --CH.sub.3, 
--S--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--S--CH--(CH.sub.3).sub.2, 
--S--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--S--CH(CH.sub.3)--CH.sub.2 --CH.sub.3, 
--S--CH.sub.2 --CH(CH.sub.3)--.sub.3, 
--S--C--(CH.sub.3).sub.3, 
(C.sub.1 -C.sub.4)alkylamino represents an amino moiety substituted with a 
linear or branched alkyl containing 1 to 4 carbon atoms, such as: 
--NH--CH.sub.3, 
--NH--CH.sub.2 --CH.sub.3, 
--NH--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--NH--CH--(CH.sub.3).sub.2, 
--NH--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--NH--CH(CH.sub.3)--CH.sub.2 --CH.sub.3, 
--NH--CH.sub.2 --CH(CH.sub.3)--.sub.3, 
--NH--C--(CH.sub.3).sub.3. 
(C.sub.1 -C.sub.4) dialkylamino represents an amino moiety substituted with 
two linear or branched alkyl moieties containing 1 to 4 carbon atoms such 
as: 
--N--(CH.sub.3).sub.2, 
--N(CH.sub.3)--CH.sub.2 --CH.sub.3, 
--N(CH.sub.2 --CH.sub.3).sub.2, 
--N(CH.sub.3)--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--N(CH.sub.2 --CH.sub.3)--CH.sub.2 --CH.sub.2 --CH.sub.3, 
--N(CH.sub.2 --CH.sub.2 --CH.sub.3).sub.2, 
--N(CH.sub.3)--CH--(CH.sub.3).sub.2, 
--N(CH.sub.2 --CH.sub.3)--CH--(CH.sub.3).sub.2, 
--N(CH.sub.3)--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--N(CH.sub.2 --CH.sub.3)--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3, 
--N(CH.sub.2 --CH.sub.2 --CH.sub.3)--CH.sub.2 --CH.sub.2 --CH.sub.2 
--CH.sub.3, 
--N(CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.3).sub.2, 
--N(CH.sub.2 --CH.sub.2 --CH.sub.2 --.sub.3)--CH--(CH.sub.3).sub.2. 
(C.sub.3 -C.sub.6)cycloalkyl represent a cyclic hydrocarbon moiety 
containing from .sub.3 to 6 carbon atoms such as: 
cyclopropyl, 
cyclobutyl, 
cyclopentyl, 
cyclohexyl. 
A five or six membered heterocyclic ring containing one or two heteroatoms 
selected from oxygen, nitrogen and sulfur (according to the definition of 
substituents R.sup.6 and R.sup.7 or R.sup.9) is an heterocycle ring such 
as: 
##STR7## 
wherein W represents hydrogen or the various possible substituents of the 
5 or 6 membered heterocycle ring, according to the definitions set out 
above; 
A bifunctional alkylenic chain, optionally containing 1 or 2 nitrogen 
atoms, according to the meaninings of R.sup.2 and R.sup.3 taken together, 
is a group which forms, with the two adjacent carbon atoms, a six membered 
aromatic heterocyclic ring, such as: 
##STR8## 
wherein the substituents R.sup.8 and R.sup.9 has the same meanings as in 
formula I; 
A group of formula .dbd.N--(CHR.sup.11)--X--, --NH--(CHR.sup.11)--X--or 
--N.dbd.(CR.sup.11)--X--, is a group which forms, with the adjacent carbon 
atoms in position 3 and 4,an heterocycle ring such as; 
##STR9## 
wherein R.sup.11 is as above defined; obviously, the double bond between 
the nitrogen atom and the carbon atom in position 4 is only possible when 
the rifamycin is in the oxidated form. 
Di(C.sub.1 -C.sub.4)alkylamino represents an amino moiety substituted with 
two linear or branched alkyl groups containing 1, 2, 3 or 4 carbon atoms 
such as: --N--(CH.sub.3).sub.2, --N(CH.sub.3)(CH.sub.2 --CH.sub.3), 
--N(CH.sub.2 --CH.sub.3 ).sub.2, --N(CH.sub.3)(CH.sub.2 --CH.sub.2 
--CH.sub.3), --N(CH.sub.2 --CH.sub.3)(CH.sub.2 --CH.sub.2 --CH.sub.3), 
--N(CH.sub.2 --CH.sub.2 --CH.sub.3).sub.2, 
--N(CH.sub.3)CH--(CH.sub.3).sub.2 !, --N(CH.sub.2 
--CH.sub.3)CH--(CH.sub.3).sub.2 !, --N(CH.sub.3 )(CH.sub.2 --CH.sub.2 
--CH.sub.2 --CH.sub.3 ), --N( CH.sub.2 --CH.sub.3 )(CH.sub.2 --CH.sub.2 
--CH.sub.2 --CH.sub.3), --N( CH.sub.2 --CH.sub.2 --CH.sub.3 )(CH.sub.2 
--CH.sub.2 --CH.sub.2 --CH.sub.3), --N( CH.sub.2 --CH.sub.2 --CH.sub.2 
--CH.sub.3).sub.2, --N(CH.sub.2 --CH.sub.2 --CH.sub.2 
--CH.sub.3)CH--(CH.sub.3).sub.2 !. 
Pharmaceutically acceptable base addition salts of the compounds of formula 
I are the rifamycin salts formed with alkali metal, earth-alkali metal, 
(C.sub.1 -C.sub.4)alkylamines, (C.sub.1 -C.sub.4)alkanolamines or basic 
aminoacids. 
As known in the art, the hydroxy groups linked in positions 1 and 4 on the 
naphtalenic ring of the compound of formula I, may be both in the reduced 
form (in such case R.sup.1 is hydroxy) or oxidated form (R.sup.1 is oxo in 
this case). 
These compounds are derivatives of rifamycin SV and of rifamycin S, 
respectively. The conversion from one form of rifamycin to the other, and 
viceversa, is easily carried out by means of oxidating or reducing 
reactions well known in the art; for instance the oxidation reaction may 
be carried out with manganese dioxide or potassium hexacyanoferrate(III) 
in chloroform, while the reducing reaction with ascorbic acid or sodium 
ascorbate in an hydroalcoholic solution. 
The hydroxy moiety in position 1 is also in the oxidated form when the 
substituent R.sup.1 together with R.sup.2 (in the compound of formula I) 
form a group of formula .dbd.N--(CHR.sup.11)--X--, wherein X and R.sup.11 
are as above defined; in this case the formation of the oxo moiety in 1 is 
a consequence of the double bonding between the nitrogen atom and the 
carbon atom in position 4. 
Therefore, when particular substituents are not present, which would 
prevent the conversion from the reduced form into the oxidated one, or 
viceversa, in the continuing of this specification the derivatives of 
rifamycin S are to be considered as being covertable in the SV form and 
viceversa. 
In the following specification, the term "rifamycins" is intended to 
comprise within its meanings all the suitable rifamycin and rifamycin-like 
compounds known in the art, such as rifamycin S. SV, P, the 3- and/or 
4-derivatives thereof, and the pharmaceutically acceptable salts thereof. 
The rifamycin antibiotic compounds are well known in the art, as being 
widely used for long time in the treatment of infections caused by 
Mycobacteria and Gram positive microorganisms and prophylaxis for certain 
Gram negative infections. The best known member of this antibiotic family 
is Rifampicin, which is one of the antibiotics of choice in the treatment 
of tuberculosis, whose main causative agent is Mycobacterium tuberculosis. 
Rifamycin SV corresponds to the compound of formula I wherein the 
substituent R at position 36 is replaced by a hydrogen atom, R.sup.2 is 
hydrogen and R.sup.1 is hydroxy; rifamycin S is the oxidated form of 
rifamycin SV, as stated above; rifamycin P is the 
4-desoxy-thiazolo-5,4-c! rifamycin S; rifampicin is the 
3-{(4-methyl-1-piperazinyl)imino!-methyl} rifamycin SV. 
The production of rifamycin SV may be obtained by fermenting variant 
cultures of the strain ATCC 13685 Nocardia mediterranei (previously named 
Streptomyces mediterranei, now renamed as Amycolatopsis mediterranei); for 
instance from Nocardia mediterranei ATCC 21271, as described in "The 
Journal of Antibiotics vol. 22, 12, 637, (1969)". 
U.S. Pat. No. 3,884,763 discloses a process for preparing rifamycin SV or 
rifamycin S by aerobically fermenting an aqueous nutrient medium 
containing a strain of Micromonospora chalcea ATCC 21994. 
Rifamycin S and SV may also be obtained by chemical modification of the 
rifamycin B as described in U.S. Pat. No. 3,301,753. Rifamycin B was first 
obtained as a component of a rifamycin complex by fermenting the 
Streptomyces mediterranei strain ATCC 13685 as described in U.S. Pat. No. 
3,150,046; rifamycin B may also be obtained as a single component by 
adding sodium ethyl barbiturate to the culture medium of Streptomyces 
mediterranei strain ATCC 13685, as described in U.S. Pat. No. 2,988,490 or 
by fermenting a variant culture of the strain ATCC 13685, i.e. the strain 
ATCC 21796, as described in U.S. Pat. No. 3,871,965. 
Rifamycin P may be obtained either by fermentation of Streptomyces 
mediterranei ATCC 31064, ATCC 31065, ATCC 31066, as disclosed in U.S. Pat. 
No. 4,263,404, or by chemical modification of rifamycin S, as described in 
U.S. Pat. No. 4,144,234. 
U.S. Pat. No. 4,880,789 discloses the preparation of the 
2'-N,N-dialkylamino derivatives of rifamycin P, by treating rifamycin P 
with dialkylamine in ethyl acetate; such derivatives may also be obtained 
by treating the 3-bromorifamycin S with N,N-dialkylthiourea, as described 
by "Cavalleri B. et al., J. of Med. Chem., 1990, 33, 1470-1476". 
Rifampicin, which is a rifamycin SV bearing a 
(4-methyl-1-piperazinyl)-imino!methyl group in position 3, may be 
obtained by reacting rifamycin SV with N-methylene-t-butylamine (obtained 
by reacting formaldehyde with t-butylamine) in the presence of manganese 
dioxide and then with 1-amino-4-methyl piperazine, as described in U.S. 
Pat. No. 3,542,762. 
Pharmaceutically acceptable salts of rifamycins are also well known in the 
art, and are easily obtainable by contacting the unsalified rifamycin 
derivative with the desired base. 
For instance, U.S. Pat. No. 3,301,753 discloses the alkali and earth-alkali 
metal salts of rifamycin SV; U.S. Pat. No. 4,312,866 discloses the 
preparation of rifamycin SV salts with basic aminoacids, such as arginine, 
lysine and histidine. 
The reaction of 25-O-Deacetyl-3-morpholino rifamycin S-21,23-acetonide with 
malonic acid was described by W. Wehrli et al. (Journal of Antibiotics, 
Tokyo, 1987; 40, 1733). The obtained 25-O-Deacetyl-25-O-malonic 
acid-3-morpholino rifamycin S-21,23-acetonide was further reacted with 
hydroxybenzotriazole in THF and then with 
3-hydroxymethyl-1-methylpiperidine in dicyclohexilcarbodiimmide, thus 
obtaining the 36-(1-methyl-3-piperidinyl)methoxy!carbonyl!-3-morpholino 
rifamycin S. 
25-O-deacetyl-25-O-propionyl and 25-O-deacetyl-25-O-pyvaloyl derivatives of 
rifamycin are described by Kump W. et al., Helv. Chem. Acta, 1973, 56, 
2323. 
It is known in the art that prolongated and extensive use of an antibiotic 
substance in the treatment of infections has the general drawback of the 
development of mutant strains of the pathogenic microorganism, which 
strains may show resistance against the specific antibiotic. It would 
therefore be desirable to produce new antibiotic substances, which are 
active both against the main causative agent, as well as against the 
mutant strains thereof which have developed resistance against the 
original antibiotic. 
With the present invention, new antibiotic derivatives of rifamycin are 
provided which are mainly active against gram positive bacteria and gram 
positive as well as gram negative anaerobes, and show anti-microbial 
activity against rifamycin resistant strains. 
Preferred compounds of formula I are those compounds wherein: 
R is, halo, hydroxy, (C.sub.1 -C.sub.4)acyloxy, (C.sub.1 -C.sub.4)alkoxy, 
(C.sub.1 -C.sub.4)alkylthio, di(C.sub.1 -C.sub.4)alkylamino or a group of 
formula: 
##STR10## 
wherein: R.sup.3 represents (C.sub.1 -C.sub.4)alkyl or (C.sub.3 
-C.sub.6)cycloalkyl; 
R.sup.4 represents a group of formula 
##STR11## 
wherein R.sup.12 represents hydrogen or (C.sub.1 -C.sub.4)alkyl; R.sup.5 
is hydrogen or halo; 
or R.sup.4 together with R.sup.5 form a bifunctional alkylenic chain, 
optionally containing 1 or 2 nitrogen atoms, of the following formula: 
##STR12## 
wherein R.sup.9 represents (C.sub.1 -C.sub.4)alkyl, a group of formula, 
##STR13## 
wherein R.sup.13 is hydrogen or (C.sub.1 -C.sub.4)alkyl, or a group of 
formula 
##STR14## 
wherein R.sup.14 and R.sup.15 independently represent hydrogen or (C.sub.1 
-C.sub.4)alkyl; 
R.sup.1 is hydroxy in the reduced form or oxygen in the oxydated form; 
R.sup.2 represents hydrogen, a six membered heterocyclic ring containing 
one or two heteroatoms selected from oxygen, nitrogen and sulfur, wherein 
one of the carbon or nitrogen atoms of the ring is optionally substituted 
by a (C.sub.1 -C.sub.4)alkyl moiety, or a group of formula: 
##STR15## 
wherein R.sup.16 represents (C.sub.1 -C.sub.4)alkyl or (C.sub.5 
-C.sub.6)cycloalkyl; 
or R.sup.1 and R.sup.2 taken together form a group of formula 
--N.dbd.CR.sup.11 --S--wherein R.sup.11 represents hydrogen, (C.sub.1 
-C.sub.4)alkyl or di(C.sub.1 -C.sub.4)alkylamino. 
Among the above compounds, further preferred compounds are those of formula 
I wherein: 
R is propyl, butyl, octyl, fluoro, bromo, chloro, iodo, hydroxy, formyl, 
acetyl, thiomethyl diethylamino or a group of formula: 
##STR16## 
wherein: R.sup.3 is ethyl or cyclopropyl, R.sup.4 is 
4-methyl-1-piperazinyl and R.sup.5 is hydrogen; 
or R.sup.4 together with R.sup.5 form a bifunctional alkylenic chain, 
optionally containing 1 or 2 nitrogen atoms, of formula: 
##STR17## 
R.sup.1 is hydroxy in the reduced form or oxygen in the oxydated form; 
R.sup.2 is hydrogen, 4-morpholinyl, 
{(4-methyl-1-piperazinyl)imino!methyl} or 
{(4-cyclopentyl-1-piperazinyl)imino!methyl}; 
or R.sup.1 and R.sup.2 together form a group of formula 
##STR18## 
Particularly preferred compounds are those compounds of formula I wherein: 
R is bromo, chloro, iodo, hydroxy or a group of formula: 
##STR19## 
wherein: R.sup.3 is ethyl, R.sup.4 is 4-methyl-1-piperazinyl and R.sup.5 
is hydrogen; 
or R.sup.4 together with R.sup.5 form a bifunctional alkylenic chain of 
formula: 
##STR20## 
R.sup.1 is hydroxy in the reduced form or oxygen in the oxydated form and 
R.sup.2 is hydrogen, 4-morpholinyl or 
{(4-methyl-1-piperazinyl)-imino!methyl}. 
Representative examples of the compounds of the invention are: 
36-bromorifamycin S 
36-fluororifamycin S 
36-chlororifamycin S 
36-iodorifamycin S 
36-methylthiorifamycin S 
36-ethylthiorifamycin S 
36-hydroxyrifamycin S 
36-methoxyrifamycin S 
36-ethoxyrifamycin S 
36-formylrifamycin S 
36-acetylrifamycin S 
36-diethylaminorifamycin S 
3,36-dibromorifamycin S 
3,36-dichlororifamycin S 
36-bromo-3-cyanorifamycin S 
36-butyl-3-bromorifamycin S 
36-hydroxy-3-cyanorifamycin S 
36-methylthio-3-cyanorifamycin S 
36-chloro-3-methylrifamycin S 
36-bromo-3-ethylrifamycin S 
36-acetyl-3-ethoxyrifamycin S 
36-chloro-3-butoxyrifamycin S 
36-bromo-3-methylthiorifamycin S 
36-methylthio-3-ethoxycarbonylrifamycin S 
36-butyl-3-(dimethylamino)rifamycin S 
36-chloro-3-(ethylpropylamino)rifamycin S 
36-bromo-3-(diethylamino)rifamycin S 
36-butyl-3-ethylthiorifamycin S 
36-chloro-3-cyanorifamycin S 
36-methylthio-3-(dimethylaminomethylene)rifamycin S 
36-bromo-3-(ethylmethylaminomethylene)rifamycin S 
36-bromo-3-(4-morpholinyl)rifamycin S 
36-bromo-3-(4-(2-ethyl)-morpholinyl)rifamycin S 
36-chloro-3-(4-(2-ethyl)-morpholinyl)rifamycin S 
36-bromo-3-(1-piperidyl)rifamycin S 
36-bromo-3-(3-(1-methyl)piperidyl)rifamycin S 
36-iodo-3-(1-(3-methyl)-piperidyl)rifamycin S 
36-chloro-3-(1-(3-methyl)-piperidyl)rifamycin S 
36-acetyl-3-(1-piperazinyl)rifamycin S 
36-bromo-3-(1-(3-methyl)piperazinyl)rifamycin S 
36-hydroxy-3-(1-(3-methyl)piperazinyl)rifamycin S 
36-fluoro-3-(4-morpholinyl)rifamycin S 
36-chloro-3(4-morpholinyl)rifamycin S 
36-methylthio-3-(4-morpholinyl)rifamycin S 
36-hydroxy-3-(4-morpholinyl)rifamycin S 
36-formyl-3-(4-morpholinyl)rifamycin S 
36-acetyl-3-(4-morpholinyl)rifamycin S 
36-diethylamino-3-(4-morpholinyl)rifamycin S 
36-bromo-3-(4-thiomorpholinyl)rifamycin S 
36-iodo-3-(4-thiomorpholinyl)rifamycin S 
36-bromo-3-(4-(3-ethyl)thiomorpholinyl)rifamycin S 
36-fluoro-3-(3-thiomorpholinyl)rifamycin S 
36-chloro-3-(2-thiomorpholinyl)rifamycin S 
36-methylthio-3-(4-thiomorpholinyl)rifamycin S 
36-formyl-3-(4-thiomorpholinyl)rifamycin S 
36-bromorifamycin P 
36-fluororifamycin P 
36-chlororifamycin P 
36-iodorifamycin P 
36-hydroxyrifamycin P 
36-methylthiorifamycin P 
36-formylrifamycin P 
36-acetylrifamycin P 
36-diethylaminorifamycin P 
36-bromo-2'-(methyl)rifamycin P 
36-fluoro-2'-(diethylamino)rifamycin P 
36-chloro-2'-(diethylamino)rifamycin P 
36-formyl-2'-(diethylamino)rifamycin P 
36-bromo-2'-(diethylamino)rifamycin P 
36-hydroxy-2'-ethylrifamycin P 
36-chloro-2'-(ethylamino)rifamycin P 
36-bromo-2'-butylrifamycin P 
36-acetyl-2'-(butylamino)rifamycin P 
36-bromo-2'-(ethylmethylamino)rifamycin P 
36-chloro-2'-ethylrifamycin P 
36-(1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy! rifamycin S 
36-{1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl)carbonyloxy} 
rifamycin S 
36-{8-ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy} rifamycin S 
36-{1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo- 
3-quinolinyl!carbonyloxy} rifamycin S 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy} rifamycin S 
36-(1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy!-3-(4-morpholinyl) rifamycin S 
36-{1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl)carbonyloxy}- 
3-(4-morpholinyl) rifamycin S 
36-{8-ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy}-3-(4-morpholinyl) rifamycin S 
36-{1-cyclopropyl-6-fluoro-1,4 
-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo-3-quinolinyl!carbonyloxy}-3-(4 
-morpholinyl) rifamycin S 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy}-3-(4-morpholinyl) rifamycin S 
36-(1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy!-3{(4-methyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl) 
carbonyloxy}-3-{(4-methyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{8-ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy}-3-{(4-methyl-1-piperazinyl)imino!methyl} rifamycin 
SV 
36-{1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo- 
3-quinolinyl!carbonyloxy}-3{(4-methyl-1-piperazinyl)imino!methyl} 
rifamycin SV 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy}-3{(4-methyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-(1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy!-3{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin 
SV 
36-{1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl) 
carbonyloxy}-3-{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{8-ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy}-3{(4-cyclopentyl-1-piperazinyl)imino!methyl} 
rifamycin SV 
36-{1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo- 
3-quinolinyl!carbonyloxyl}-3{(4-cyclopentyl-1-piperazinyl)imino!methyl} 
rifamycin SV 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy}-3{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{1-ethyl-1,4-dihydro-6-(dimethylamino)-4-oxo-3-pyridinyl!carbonyloxy}-3 
{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{1-ethyl-1,4-dihydro-6-(dimethylamino)-5-fluoro-4-oxo-3-pyridinyl!carbo 
nyloxy}-3-{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin SV 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-5-fluoro-4-oxo-3-pyridi 
nyl!carbonyloxy}-3-{(4-cyclopentyl-1-piperazinyl)imino!methyl} rifamycin 
SV 
36-(1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy!-2'-(diethylamino) rifamycin P 
36-{1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl)carbonyloxy}- 
2'-(diethylamino) rifamycin P 
36-{8-ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy}-2'-(diethylamino) rifamycin P 
36-{1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo- 
3-quinolinyl!carbonyloxy}-2'-(diethylamino) rifamycin P 
36-{1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy}-2'-(diethylamino) rifamycin P. 
The preferred pharmaceutically acceptable salts of the compounds of formula 
I are the rifamycin salts formed with alkali metal or basic aminoacids; 
most preferred are the salts with sodium, arginine, lysine or histidine. 
A-further object of the present invention is to provide a process for 
preparing the compounds of general formula I. With said process it is also 
possible to prepare the rifamycin derivatives of formula I wherein R is a 
(C.sub.1 -C.sub.8)alkyl group. Thus, only when referring to the 
preparation method, the meaning of R will encompass also this further 
group. 
All the suitable rifamycins known in the art may in general be used as 
starting materials for producing the compounds of the present invention. 
Further chemical modifications of the above rifamycins, which allow to 
obtain the suitable starting material for producing the compounds of the 
invention, are known in the art, or can easily be carried out by the 
skilled man, according to the common general knowledge. 
For instance, U.S. Pat. No.4,086,225 discloses the preparation of 
4-desoxy-imidazolo4,5-c! rifamycin derivatives from Rifamycin S; U.S. 
Pat. No.4,880,789 describes the preparation of 2'-dialkylamino derivatives 
of Rifamycin P. 
Alternatively, 36-substituted rifamycin S or SV may be obtained according 
to the present process while the desired substituents are introduced in 
the other positions of the molecule only afterwards. This procedure is 
preferably followed when said further substituents would prevent the 
normal course of reaction or could itself undergo unwanted chemical 
modifications during the preparation of the 36-derivatives according to 
the process of the present invention. 
For instance the presence of the group --CH.dbd.N--R.sup.10, wherein 
R.sup.10 is as defined in formula I, in position 3 would prevent the 
protection of positrons 21 and 23 of the rifamycin molecule (i.e. the 
formation of the cyclic-21,23-(1-methylethylidene acetal)) which 
protection is necessary for obtaining the starting material of the present 
process. Therefore, after the desired 36-derivative of rifamycin SV has 
been obtained as described hereinafter, it will be further reacted, 
for instance with N-methylene-t-butylamine in the presence of t-butylamine 
and manganese dioxide and then with a compound of formula NH.sub.2 
-R.sup.10, in order to insert at the 3- position the desired substituent 
of formula --CH.dbd.N--R.sup.10. 
According to the common general knowledge, the skilled man will decide 
whether introduce such further substituents before or after the process of 
the present invention, depending on the specific characteristics of the 
desired substituent. 
The compounds of formula I may be obtained by reacting the corresponding 
25-O-deacetylated rifamycin with a suitable malonic acid derivative in the 
presence of a condensing agent. 
The deacetylation in position 25 of rifamycin, is easily achieved according 
to the common hydrolysis techniques known in the art; for instance, U.S. 
Pat. No.4,188,321, discloses the preparation of 25-O-Deacetylrifamycin S, 
or derivatives thereof, by reacting rifamycin S, or derivatives thereof, 
with sodium hydroxide or sodium bicarbonate, respectively. 
For the process of the invention, the above rifamycin starting materials 
may not be reacted as such, but has to be protected on the positions 21 
and 23, before reacting it. Such protection is performed according to the 
methods known in the art for protecting geminal hydroxy groups and results 
in a ciclyzation of the two oxygen atoms of the hydroxy functions in 
position 21 and 23. As a general procedure, it is preferred to perform the 
deacetylation in position 25 after the protection reaction. 
For instance, 25-O-deacetylrifamycin S cyclic-21,23-(1-methylethylidene 
acetal)--wherein R.sup.1 is oxo and R.sup.2 is hydrogen--may be prepared 
by reacting rifamycin S with acetone and anhydrous cupric sulfate or with 
2,2-dimethoxypropane and sulfuric acid, and then hydrolyzing with NaOH, 
according to "W. Kump and H. Birchel, Helv.Chim. Acta, 1973, 56, 2323"; 
the preparation of 25-O-deacetyl-3-(4-morpholinyl)rifamycin S 
cyclic-21,23-(1-methyl-ethylidene acetal)--wherein R.sup.1 is oxo and 
R.sup.2 is morpholinyl--by reacting 3-(4-morpholinyl)rifamycin S with 
2,2-dimethoxypropane in anhydrous acetone and then hydrolyzing with NaOH, 
is described in "W. Wehrli et al., Journal of antibiotics, 1987, 40, 
1733". 
The protected rifamycin P may be prepared by reacting the 
25-O-Deacetylrifamycin S cyclic-21,23-(1-methylethylidene acetal) with 
N-bromosuccinimide and 1,1-diethylthiourea in dimethylformamide, thus 
obtaining the corresponding 25-O-Deacetylrifamycin P 
cyclic-21,23-(1-methyl-ethylidene acetal). 
The process of the present invention therefore comprises: 
a) reacting a compound of general formula II 
##STR21## 
wherein R.sup.1 and R.sup.2 have the same meanings as in formula I, with 
the proviso that R.sup.2 is not a group of formula: 
--CH.dbd.N--R.sup.10, 
with a malonic acid derivative of formula III: 
##STR22## 
wherein R is as above defined, in the presence of a condensing agent; b) 
removing the protecting group in positions 21 and 23 by means of an acidic 
cleavage of the acetonidic moiety; 
c) contacting the deprotected compound with a cuprous salt or oxide or a 
mixture thereof in the presence of an inert organic solvent. 
According to the first step of the above process one of the two malonic 
carboxylic moieties reacts with the hydroxy moiety in position 25 of the 
compound of formula II, thus forming an ester moiety in such position. The 
second carbon atom of this moiety is conventionally numbered as C.sup.36. 
This reaction is generally conducted in the presence of an inert organic 
solvent which do not unfavorably interfere with the reaction course and is 
capable of at least partially solubilizing the antibiotic material. 
Said inert organic solvents are those commonly used in the art and comprise 
alkylamides, alkylnitriles, saturated linear or cyclic ethers, glycol 
ethers, phosphoramides, sulfoxides, chlorinated solvents or mixtures 
thereof. 
Preferred inert organic solvents are: dimethylformamide, acetonitrile, 
dimethoxyethane, tetrahydrofuran (THF), hexamethyl-phosphoramide, 
dimethylsulfoxide, chloroform and dichloroethane or mixtures thereof; most 
preferred is tetrahydrofuran. 
The condensing agent may be any substance commonly used in the art for the 
esterification reactions selected from carboxydiimides, 
dialkylaminopyridines, carbonylimidazoles, triphenilphosphine in carbon 
tetrachloride, substituted dithiocarbonates and diphenylphosphorilazides. 
Examples of said condensating agents are: 4-dimethylaminopyridine, 
N,N'-carboyl-bis imidazole, 
S,S'-bis-1-(phenyl-1H-tetrazol-5-yl)-dithiocarbonate and 
1,3-Dicyclohexylcarbodiimmide (DCC). 
Preferred condensing agents are carboxydiimmides derivatives, 
1,3-Dicyclohexylcarbodiimmide being the most preferred one. 
The above reaction is preferably conducted at temperatures from about 
0.degree. C. to 35.degree. C., while the reaction time may vary from 1 to 
2 hours. More preferably, the mixture is reacted at 0.degree. C. for about 
15 minutes and then for about one hour at room temperature. 
The removal of the acetonidic moiety bridging positions 21 and 23 is 
performed under mild acidic conditions in the presence of an inert organic 
solvent, as above defined. The acids commonly used for the cleavage of 
such moiety can be here conveniently utilized; these acids have to be in 
diluted form, to avoid the demolition of the substrate. Suitable acids are 
mineral acids (e.g. hydrochloric acid, sulfuric acid) or organic sulfonic 
acids (e.g. p-toluensulfonic acid), the preferred one being sulfuric acid. 
The reaction is conducted between 30.degree. C. and 50.degree. C., while 
the reaction time may vary from 14 to 18 hours. It is generally preferred 
to carry it out at a temperature of about 40.degree. C. for about 16 
hours. 
Step c of the present process permits the removal of the free carboxy 
moiety linked to the C.sup.36,for obtaining the corresponding compound of 
general formula 1. 
For the decarboxylation reaction any salt or oxide or a mixture thereof 
containing the cuprous ion can be employed. Examples of these compounds 
are: Cu.sub.2 O, Cu.sub.2 S, CuCl, CuBr and Cu.sub.2 SO.sub.4, most 
preferred being cuprous oxide. 
Although all the cuprous compounds work well as decarboxylating agents, the 
use of CuCl or CuBr should be avoided when the malonic acid substrate is 
substituted with a bromine or chlorine atom respectively, because of a 
possible ion exchange side reaction. Of course, this problem arises only 
if the product has to be obtained in a pure form, while if a mixture of 
halogenated derivatives is desired, the use of CuCl or CuBr in this 
specific case is also possible. 
The inert organic solvent to be used should be capable of at least 
partially solubilizing the antibiotic material and should not unfavorably 
interfere with the reaction course. The skilled man would be able to 
choose the most appropriate solvent among those commonly used in the art. 
Suitable solvents are those previously listed and, among those, the 
preferred solvent for this reaction is acetonitrile. 
The reaction temperature is from 50.degree. C. to 75.degree. C., preferably 
from 60.degree. C. to 70.degree. C. 
The reaction time may vary depending on the nature of substituent on the 
malonic moiety. For the most reactive compounds it is about one hour, 
while for the less reactive ones it raises up to 20 hours. 
Although with the above described process it is possible to directly obtain 
the new rifamycin derivatives of the invention, some of these compounds 
are preferably obtained according to an alternative procedure, which 
comprises contacting a 36-halo rifamycin derivative obtained according to 
the above process with an appropriate reactant, in order to insert the 
desired substituent R in the position 36. 
With this alternative procedure it is therefore not necessary to prepare 
the specific substituted malonic acid derivative of formula III according 
to each meaning of R, but starting from halo-malonic acid most of the 
compounds of the invention are easily obtainable. 
This procedure is particularly suitable when the desired substituent R in 
the position 36 of rifamycin is hydroxy, iodo, (C.sub.1 -C.sub.4)acyloxy, 
(C.sub.1 -C.sub.4)alkylamino, di(C.sub.1 -C.sub.4)alkylamino or a group of 
formula: 
##STR23## 
Such further reactions for preparing these new rifamycin derivatives are 
all subsequent to the previous steps a to c described above and may be 
either alternative or subsequent one to each other; these further steps 
are illustrated hereinafter for each kind of substituent. 
When R is iodo, the 36-chloro or 36-bromo rifamycin derivative obtained 
according to steps a to c of the above process, is contacted with an 
acetonic solution of an alkali metal iodide. 
The halogen ion exchange reaction for obtaining the 36-iodo rifamycin 
derivatives is easily accomplished by contacting the corresponding 36-halo 
rifamycin derivative with a metal alkali iodide, preferably sodium iodide, 
in the presence of acetone, as commonly known in the art. 
Such reaction is generally conducted at room temperature for about 3 to 5 
hours. 
When R is (C.sub.1 -C.sub.4)acyloxy, the 36-chloro, -bromo or -iodo 
rifamycin derivative obtained as above described, is contacted with a 
(C.sub.1 -C.sub.4)acylate salt, in the presence of an inert organic 
solvent. 
The (C.sub.1 -C.sub.4)acylate salt is preferably an alkali metal, most 
preferred is potassium, or a silver salt, while the inert organic solvent 
is selected among those listed above, preferably being anhydrous 
dimethylformamide. 
The reaction is generally conducted at room temperature for about 16 to 26 
hours. 
When R is (C.sub.1 -C.sub.4)alkylamino or (C.sub.1 -C.sub.4)dialkylamino 
the 36-chloro, -bromo or -iodo rifamycin derivative obtained as above 
described, is contacted with the correspondent alkyl or dialkylamine in 
the presence of an inert organic solvent. 
The inert organic solvent is preferably tetrahydrofuran and the reaction is 
generally conducted at room temperature for about 2 to 6 hours. 
When R is hydroxy, the 36-formyloxy rifamycin derivative obtained as above 
described, is hydrolized underbasic conditions. 
The hydrolisis is conducted in an hydroalcoholic solution under mild basic 
conditions, at room temperature, for about 10 to 16 hours. Preferably, the 
reaction is performed with potassium bicarbonate in a water/methanol 
mixture (rate from 1.div.2 to 1.div.3 v/v). 
Finally, when R is a group of formula 
##STR24## 
the 36-chloro, -bromo or -iodo rifamycin derivative obtained as above 
described is contacted with a salt of a 4-oxo-3-pyridinyl carboxylic acid 
derivative of general formula IV 
##STR25## 
wherein R.sup.3,R.sup.4 and R.sup.5 are as defined in formula I, in the 
presence of an inert organic solvent. 
Preferably a litium, sodium, potassium or silver salt of the 
4-oxo-3-pyridinyl carboxylic acid derivative is employed. 
The 4-oxo-3-pyridinyl carboxylic acid derivatives, may be commercially 
available compounds such as Pefloxacin.RTM., Nalidixic acid or 
N-methyl-pipemidic acid, known compounds such as those described in U.S. 
Pat. No.5,075,319, or new compounds derived from the compounds of general 
formula Va or Vb: 
##STR26## 
wherein Hal represents chloro or bromo and Y is hydrogen or lower alkyl. 
The compounds of formula IVa and IVb may be prepared by saponification of 
the corresponding 4,6-di-halo-nicotinic acid or lower alkyl 
4,6-di-halo-nicotinate, as disclosed in Israeli Patent 44327/2. 
The compounds of formula Va are preferably reacted first with an alkylating 
agent, so to obtain the corresponding N-(C.sub.1 -C.sub.4)alkyl or 
N-(C.sub.3 -C.sub.6)cycloalkyl derivatives; afterwards this N-alkylated 
compound is reacted with a suitable amine in order to substitute the 
halogen with the desired moiety of formula --NR.sup.6 R.sup.7, wherein 
R.sup.6 and R.sup.7 are as defined in formula I. 
For instance, suitable starting materials for the process of the present 
invention may be prepared by reacting a N-alkyl derivative of a compound 
of general formula IVa with N-methylpiperazine. 
The novel compounds of general formula VI 
##STR27## 
wherein Y is hydrogen or (C.sub.1 -C.sub.4)alkyl and R.sup.3, R.sup.6 and 
R.sup.7 have the same meanings as in formula I, also fall within the scope 
of the present invention. 
The inert organic solvent that may be used in the present process should be 
capable of at least partially solubilizing the antibiotic material and 
should not unfavourably interfere with the reaction course. The skilled 
man would be able to choose the most appropriate solvent among those 
commonly used in the art, partcularly in view of the teachings of the 
present disclosure. Suitable solvents are those listed before when dealing 
with reaction of the malonic acid derivative with rifamycin. Particularly 
preferred is dimethylformamide. 
The reaction is conducted generally at a temperature between 15.degree. C. 
and 40.degree. C. 
The reaction time may vary from 10 to 24 hours. 
Preferably, the salt of the compond of formula IV is first added to the 
inert organic solvent and stirred at room temperature for about 30 
minutes, optionally in the presence of an activated molecular sieve, such 
as Union Carbide type 4 .ANG.(FLUKA); afterwards, the suitable 36-chloro, 
-bromo or -iodo rifamycin derivative is added to the solution and the 
mixture is stirred for from 14 to 20 hours at room temperature. 
Preferred 36-halo rifamycin derivatives which may be employed for the 
present process are the 36-iodo derivatives, whilst preferred carboxylic 
acid salt is potassium salt. 
Separation and purification of the reaction products obtained according to 
the various steps of the present process is made according to the known 
per se techniques. 
The separation of the reaction products is preferably accomplished by means 
of extraction with water-immiscible organic solvents or by adding 
non-solvents. 
The term "water-immiscible solvent" as used in this application, is 
intended to have the meaning currently given in the art to this term and 
refers to solvents that at the conditions of use are slightly miscible or 
practically immiscible with water in a reasonably wide concentration 
range, suitable for the intended use. 
Examples of water-immiscible organic solvents that can be used in the 
extraction of the antibiotic substances of the invention from an aqueous 
phase are: the usual hydrocarbon solvents which may be linear, branched or 
cyclic such as hexane or cyclohexane; halogenated hydrocarbons such as 
chloroform, carbon tetrachloride, dichloromethane, dichloroethane, 
fluorobromoethane, dibromoethane, trichloropropane, chlorotrifluorooctane 
and the like; aromatic hydrocarbons such as benzene, toluene, xylene and 
the like; esters of at least four carbon atoms, such as ethyl acetate, 
propyl acetate, ethyl butyrate, and the like; alkanols of at least four 
carbon atoms which may be linear, branched or cyclic such as butanol, 
1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 
3,3-dimethyl-1-butanol, 4-methyl-1-pentanol; 3-methyl-1-pentanol, 
2,2-dimethyl-3-pentanol, 2,4-dimethyl-3-pentanol, 4,4-dimethyl-2-pentanol, 
5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5-methyl-1-hexanol, 
2-ethyl-1-hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, 
cyclopentanol, 2-cyclopentylethanol, 3-cyclopentyl-1-propanol, 
cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 
4-ethylcyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 
1-nonanol, 2-nonanol, 1-decanol, 2-decanol and 3-decanol; straight or 
branched alkyl ethers and mixture thereof such as petroleum ether, ethyl 
ether, propyl ether, butyl ether, etc; and mixtures or functional 
derivatives thereof; the preferred one being ethyl acetate. 
Examples of precipitating agents are petroleum ether and lower alkyl 
ethers, such as ethyl ether, propyl ether and butyl ether; the preferred 
one being petroleum ether. 
Purification of the reaction products may be obtained by precipitation with 
non-solvents or by chromatographic techniques. 
Precipitating agents suitable for purification are those listed above. 
The chromatographic techniques suitable for purifying the reaction products 
of the present process are those commonly known in the art and comprise 
partition chromatography, reverse-phase partition chromatography, 
ion-exchange chromatography, flash chromatography, affinity 
chromatography, HPLC techniques and the like. the preferred one being 
flash chromatography. Preferably the purification of the residue is 
accomplished by means of flash-chromatography; particulary preferred as 
stationary phase is silica gel, while preferred eluent is methanol in 
dichloroethane. 
The antimicrobial activity of the compounds of the present invention was 
demonstrated by a series of standard in vitro tests. 
The minimal inhibitory concentration (MIC) for the microorganisms was 
determined by broth microdilution methodology. Inocula were approximately 
10.sup.4 colony-forming units per ml (CFU/ml), except for Bacteroides 
fragilis, Clostridium perfringens and Propionibacterium acnes (10.sup.5 
CFU/ml). 
Incubation was at 37.degree. C. Neisseria gonorrhoeae and Haemophilus 
influenzae were incubated in 5% carbon dioxide in air; C. perfringens, P. 
acnes and B. fragilis in nitrogen-carbon dioxide-hydrogen (80:10:10); 
other organisms in air. Incubation times were 48 hours for N. gonorrhoeae, 
H. influenzae, P. acnes and B. fragilis; 20-24 hours for other organisms. 
The growth media were: Iso-Sensitest broth (Oxoid) for staphylococci, 
Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Klebsiella 
pneumoniae, and Pseudomonas aeruginosa; Todd Hewitt broth (Difco) for 
streptococci; GC Base broth (Difco)+1% (v/v) IsoVitaleX (BBL) for N. 
gonorrhoeae; Brain Heart Infusion broth (Difco)+1% (v/v) Supplement C 
(Difco) for H. influenzae; Wilkins-Chalgren broth (Difco) for C. 
perfringens, P. acnes and B. fragilis. 
The following table I reports the antimicrobial activity of representative 
compounds of the invention. 
TABLE I 
__________________________________________________________________________ 
Internal 
MIC of the compounds (.mu.g/ml) 
STRAIN code 
C1 C2 C3 C4 C8 C9 C10 C12 C13 C15 C22 
__________________________________________________________________________ 
Staphylococcus aureus 
L165 
0.004 
0.015 
0.004 
0.008 
0.002 
0.06 
0.06 
0.03 
0.002 
0.002 
0.06 
Staph. aureus rifa-r.sup.(1) 
L721 
0.25 
n.t. 
1 n.t. 
0.5 8 n.t. 
n.t. 
0.25 
2 0.13 
Staph aureus rifa-r.sup.(2) 
L1282 
2 64 16 32 0.5 &gt;128 
32 32 4 4 1 
Staph aureus Smith 
L819 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
0.06 
Staph epidermidis ATCC 12228 
L147 
0.004 
0.015 
0.008 
0.001 
0.001 
0.06 
0.06 
0.008 
0.001 
0.001 
0.06 
Staph haemolyticus rifa-r.sup.(2) 
L602 
32 .gtoreq.128 
64 128 16 .gtoreq.128 
64 64 32 4 0.5 
Streptococcus pyogenes 
L49 0.03 
0.008 
0.016 
0.008 
0.06 
0.13 
0.015 
0.06 
0.008 
0.03 
0.06 
Strept. pneumoniae 
L44 0.03 
0.015 
0.03 
0.002 
0.03 
0.13 
0.03 
0.03 
0.008 
0.016 
0.06 
Enterococc. faecalis ATCC 7080 
L149 
1 1 0.25 
0.5 0.5 2 2 0.25 
0.13 
0.5 2 
Propionibact. acnes ATCC 6919 
L1014 
0.016 
0.13 
0.03 
0.06 
0.004 
0.06 
0.13 
0.5 0.03 
0.016 
n.t. 
Clostridium perfringens 
L290 
0.0003 
n.t. 
0.0003 
n.t. 
0.001 
n.t. 
n.t. 
n.t. 
0.005 
0.001 
n.t. 
Bacteroides fragilis ATCC 23745 
L1010 
n.t. 
2 n.t. 
0.5 n.t. 
0.13 
0.5 2 n.t. 
n.t. 
0.13 
Bacteroides fragilis ATCC 25285 
L1011 
1 n.t. 
0.13 
n.t. 
0.03 
n.t. 
n.t. 
n.t. 
0.5 0.03 
n.t. 
Neisseria gonorrheae 
L997 
1 2 0.13 
0.5 0.03 
0.25 
16 1 0.25 
0.03 
0.25 
Moraxella catarr. ATCC 8178 
L76 0.008 
n.t. 
0.008 
n.t. 
0.002 
n.t. 
n.t. 
n.t. 
0.004 
0.002 
n.t. 
Haemoph. influenzae ATCC 19418 
L970 
1 2 1 1 0.13 
0.25 
4 1 0.5 0.06 
0.5 
Escherichia coli 
L47 32 128 32 64 8 16 64 32 16 8 .gtoreq.128 
Esch. coli K12 hyperpermeable 
G1640 
0.25 
1 0.25 
0.51 
.ltoreq.0.13 
2 1 .ltoreq.0.13 
.ltoreq.0.13 
.ltoreq.0.13 
n.t. 
Esch. coli K12 rifa-r.sup.(1) 
G1064 
&gt;128 
&gt;128 
&gt;128 
&gt;128 
&gt;128 
&gt;128 
.gtoreq.128 
&gt;128 
&gt;128 
&gt;128 
n.t. 
Esch. coli K12 wild type 
G210 
n.t. 
.gtoreq.128 
n.t. 
64 n.t. 
n.t. 
128 64 n.t. 
n.t. 
n.t. 
Proteus vulgaris ATCC 881 
L79 16 32 8 8 8 32 32 8 8 8 64 
Klebsiella pneumoniae 
L142 
128 n.t. 
64 n.t. 
16 n.t. 
n.t. 
n.t. 
64 8 n.t. 
Pseudomonas aerug. ATCC 10145 
L4 32 64 32 32 16 16 32 32 32 16 32 
__________________________________________________________________________ 
Internal 
MIC of the compounds (.mu.g/ml) 
STRAIN code 
C23 
C24 
C25 
C26 
C27 
C28 
C29 
C30 
__________________________________________________________________________ 
Staphylococcus aureus 
L165 
0.06 
0.06 
0.06 
0.06 
0.03 
0.06 
0.03 
0.016 
Staph. aureus rifa-r.sup.(1) 
L721 
0.06 
0.06 
0.13 
0.13 
0.25 
0.13 
0.5 
0.5 
Staph. aureus rifa-r.sup.(2) 
L1282 
1 0.13 
1 2 16 2 2 1 
Staph. aureus Smith 
L819 
n.t. 
0.06 
0.06 
0.13 
0.016 
0.06 
0.03 
0.016 
Staph. epidermidis ATCC 12228 
L147 
0.06 
0.008 
0.13 
0.06 
0.016 
0.06 
0.03 
0.016 
Staph. haemolyticus rifa-r.sup.(2) 
L602 
4 0.13 
1 0.5 
16 4 8 4 
Streptococcus pyogenes 
L49 0.004 
0.06 
0.03 
0.03 
0.008 
0.016 
0.016 
0.016 
Strept. pneumoniae 
L44 0.004 
0.008 
0.016 
0.03 
0.008 
0.008 
0.016 
0.008 
Enterococc. faecalis ATCC 7080 
L149 
0.13 
0.13 
2 4 2 8 2 0.25 
Propionibact. acnes ATCC 6919 
L1014 
2 0.06 
n.t. 
0.13 
0.03 
0.06 
0.06 
0.03 
Clostridium perfringens 
L290 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Bacteroides fragilis ATCC 23745 
L1010 
8 0.13 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Bacteroides fragilis ATCC 25285 
L1011 
n.t. 
n.t. 
2 1 0.13 
1 0.13 
0.06 
Neisseria gonorrheae 
L997 
8 0.06 
0.5 
0.13 
0.13 
0.25 
0.25 
0.13 
Moraxella catharralis ATCC 8178 
L997 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Haemoph. influenzae ATCC 19418 
L970 
2 0.13 
1 1 0.25 
2 0.25 
1 
Escherichia coli 
L47 128 
16 64 2 32 64 16 16 
Esch. coli K12 hyperpermeable 
G1640 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Esch. coli K12 rifa-r.sup.(1) 
G1064 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Esch. coli K12 wild type 
G210 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
n.t. 
Proteus vulgaris ATCC 881 
L79 128 
16 64 2 16 64 128 
16 
Klebsiella pneumoniae 
L142 
n.t. 
n.t. 
n.t. 
n.t. 
n.t 
n.t. 
n.t. 
n.t. 
Pseudomonas aerug. ATCC 10145 
L4 &gt;128 
16 128 
32 32 128 
&gt;128 
32 
__________________________________________________________________________ 
rifa-r = rifampicin resistant strain; .sup.(1) moderately resistant 
strain, .sup.(2) highly resistant strain; n.t. = not tested 
Compounds C22, C24, C25 and C30 have also been tested on various clinical 
isolates of rifampicin resistant strains of S. aureus, showing in the most 
of cases a MIC of from 0.125 to 2 .mu.g/ml. 
Compound C30 has further been tested against Mycobacterium tuberculosis and 
Mycobacterium avium, according to the following methodology: 
M. tuberculosis was grown for 2-3 weeks on Lowenstein-Jensen medium 
(Sclavo) and M. avium on 7H10 agar (Difco) for 2 weeks. The cultures were 
suspended in 7H9 broth (Difco), diluted in Becton Dickinson diluting 
fluid, and inoculated into Bactec 12B vials (Becton Dickinson). 
The inocula for control vials (without the compound to be tested) were 
10.sup.2 -10.sup.3 cfu/ml; vials with compound C30 were inoculated with 
100 times this number of cells. The vials were incubated at 37.degree. C. 
and read daily in a Bactec 460 machine. 
M. tuberculosis was considered sensitive to a given concentration of 
antibiotic if the difference between growth indexes on successive days in 
the vial was less than that of the control. M. avium was considered 
sensitive to a given antibiotic concentration if the growth index was less 
than that of the control. 
The inhibitory concentration of compound C30 was found to be less than 0.2 
against both the strains. 
Some compounds of the invention were also tested in experimental septicemia 
in mice infected with Staphylococcus aureus Smith (Int. code L 819). 
For this purpose, groups of five mice were infected intraperitoneally with 
about 1.times.10.sup.6 CFU of S. aureus Smith suspended in 0.5 ml of Difco 
bacteriological mucin. 
Untreated animals died within 48 hours. 
The other animals were treated once immediately after infection. On the 7th 
day the value of ED.sub.50 (expressed in mg/kg) was calculated by the 
method of Spearman-Karber (Finney, D. J., Statistical method in biological 
assay, page 254, C. Griffin Ltd., London, 1952), on the basis of the 
percentage of animals surviving at each dose. 
The results are reported in the following table II: 
TABLE II 
______________________________________ 
Experimental septicemia in mice infected with S. aureus Smith (L 819) 
ED.sub.50 (mg/kg) 
Compound SC route IV route 
______________________________________ 
C22 5.3 0.7 
C23 32.9 n.t. 
C24 &gt;50 3.8 
C25 4.7 2.9 
C26 1.6 0.95 
C30 1.6 0.72 
______________________________________ 
SC = Subcutaneous; IV = Intravenous; n.t. = not tested 
As shown in table II, when the compounds of the invention are administered 
by subcutaneous or intravenous routes, a good activity is observed in 
general. The activities of the above compounds were however negligible 
when administered orally. 
In view of their properties, the compounds of the invention can be used as 
active ingredients in the preparation of medicaments for human or animal 
treatment. 
In particular, the antibiotic compounds of general formula I are 
antimicrobial agents mainly active against gram positive bacteria, and 
fastidious gram negative bacteria. 
Furthermore, the compounds of the invention show a considerable 
antimicrobial activity against those strain which have developed 
resistance to rifampicin. 
The main therapeutic indication of the antibiotic substances of the 
invention is thus in the treatment of infections related to the presence 
of a microorganism susceptible to it. 
The term "treatment" is intended to encompass also prophylaxis, therapy and 
cure. 
The patient receiving this treatment is any animal in need, including 
primates, in particular humans, and other mammals such as equines, cattle, 
swine and sheep; and poultry and pets in general. 
The compounds of the invention can be administered as such or in admixture 
with pharmaceutically acceptable carriers and can also be administered in 
conjunction with other antimicrobial agents generally employed in the art 
along with rifamycin antibiotics. Conjunctive therapy, thus includes 
sequential, simultaneous or separate administration of the active compound 
in a way that the therapeutical effects of the first administered one has 
not entirely disappeared when the subsequent one is administered. 
The amount of active substance in the finished dosage form is related to a 
certain extent to the minimal inhibitory concentration of active substance 
against the infection causative agents and its particular type of 
formulation. 
The dosage may obviously be adjusted according to the severity of the 
infection, the type, age and conditions of the patient, the formulation 
selected for the administration, the administration schedule, etc. 
Experimental tests for determining the sensitivity of the microorganisms 
isolated from the patient may also offer useful indication to select the 
appropriate dosage. 
In general, effective antimicrobial dosages are employed per single unit 
dosage form. 
Repeated administrations, e.g. from 2 to 6 times a day, are in general 
preferred. An effective dosage may be in general in the range 0.5-100 
mg/kg body weight/day, preferably 5-50 mg/kg body weight/day. 
Anyway, the prescribing physician will be able to determine the optimal 
dosage for a given patient in a given situation. 
The antibiotic compounds of the invention may be administered by parenteral 
(intramuscular, intravenous, subcutaneous, etc.) or oral route; the nature 
of the compound will determine the specific route of administration that 
may be employed. When the compound is not inactivated and is absorbed from 
the gastro-intestinal tract, the oral route is generally preferred; 
otherwise, and also in the case of the unconscious or uncooperative state 
of the patient, parenteral administration of the active substance may 
conveniently be employed. 
For oral administration the compounds of the invention can be formulated 
into solid or liquid preparations such as capsules, pills, tablets, 
troches, powder, solutions, suspensions or emulsions. 
For instance, the solid unit dosage form can be a capsule of the ordinary 
gelatin type containing lubricants and inert filler, such as lactose, 
sucrose and cornstarch. In another embodiment, the compounds of general 
formula I can be tableted with conventional tablet bases such as lactose, 
sucrose and cornstarch in combination with binders, such as acacia, 
cornstarch or gelatin, disintegrating agents such as potato starch or 
alginic acid and a lubricant such as stearic acid or magnesium stearate. 
A unit dosage for oral administration may contain, for instance, from 50 to 
700 mg of the active ingredient, preferably about 150 to 500 mg of the 
active ingredient. 
For parenteral administration the compounds of the invention may be 
formulated into suitable injectable preparations containing a liquid 
vehicle. Such vehicle normally has no therapeutic effect and should not be 
toxic. Examples of suitable vehicles for preparing injectable dosage forms 
of the compounds of the invention are water, aqueous vehicles (e.g. Sodium 
chloride injections, Dextrose injections, etc.), water miscible solvents 
(e.g. ethyl alcohol, polyethylene glycol, propylene glycol, etc.) and 
non-aqueous vehicles (e.g. "fixed oils" such as corn oil, cottonseed oil, 
peanut oil and sesame oil). Optionally, the injectable preparation may 
further contain buffers for stabilizing the solution (e.g. citrates, 
acetates and phosphates) and/or antioxidants (e.g. ascorbic acid or sodium 
bisulfite). 
Also in the case of parenteral administration of the compounds of the 
invention, the nature of the product will determine the specific route of 
administration (e.g. intramuscular, intravenous or subcutaneous) that may 
be employed. Conversely, the desired route of administration will place 
requirements on the formulation. For example, suspensions would not be 
administered directly in the blood stream because of the danger of 
insoluble particles blocking capillaries, whilst solutions to be 
administered subcutaneously would require strict attention to tonicity 
adjustment, otherwise irritation of the nerve endings in the anatomical 
area would give rise to pronounced pain. 
Useful indications for the preparations of suitable parenteral and oral 
dosage forms can be found in: Remington's Pharmaceutical Sciences, 17th 
Edition, 1985 (Mack Publishing Company, Easton, Pa.). 
Besides their use as medicaments in human and veterinary therapy, the 
compounds of the invention may also be used as animal growth promoters. 
For this purpose, a compound of the invention is administered orally in a 
suitable feed. The exact concentration employed will reflect the amount of 
active agent needed for a growth promotant effect and the amount of feed 
normally consumed. 
The addition of the active compound of the invention to animal feed is 
preferably accomplished by preparing an appropriate feed premix containing 
the active compound in an efficacious amount and incorporating the premix 
into the complete ration. Alternatively, an intermediate concentrate or 
feed supplement containing the active ingredient can be blended into the 
feed. 
The way in which such feed premixes and complete rations can be prepared 
and administered, are described in reference books (such as "Applied 
Animal Nutrition", W. H. Freedman and CO., S. Francisco, U.S.A, 1969 or 
"Livestock Feeds and Feeding", O and B books, Corvallis, Oreg., USA, 
1977).

For better illustrate the present invention, examples of synthesis of 
compounds of general formula I are given hereinafter. 
For the sake of clarity, the preparation of the starting material, of the 
intermediates and of the compounds of formula I has been so divided: 
Preparations A to G refer to the preparation of the starting materials (the 
compounds obtained are marked with letter P, i.e. P1 to P7); 
Examples A to I refer to the preparation of intermediate compounds (the 
compounds obtained are marked with letter E, i.e. E1 to E9); 
Examples 1 to 30 refer to the preparation of the compounds of formula I, 
and also of those compounds wherein R is (C.sub.1 -C.sub.8)alkyl (the 
compounds obtained are marked with letter C, i.e. C1 to C30). 
In the following examples, the reaction is followed by means of TLC plates 
(silica-gel 60 F.sub.254 pre-coated, 5.times.10 cm, thickness 0.25,Merck); 
the purification of the products obtained by flash-chromatography is 
performed on silica gel (32-63, 60 .ANG.; 
ICN Biomedicals GmbH), with methanol in dichloromethane as the eluent. 
Preparation A: 25-O-Desacetyl-3-(4-morpholinyl)rifamycin S 
cyclic-21,23-(1-methylethylidene acetal) (Compound P1) 
a) A mixture of rifamycin S (15 g), morpholine (40 ml) and dioxane (40 ml) 
is stirred 2 hours at room temperature. The mixture is cooled in icy water 
and 5N hydrochloric acid (45 ml) is added. Extraction with ethyl acetate 
(2.times.100 ml) and evaporation of the solvent give an oily residue which 
is dissolved in chloroform (100 ml) and stirred 2 hours with a 25% 
solution of potassium hexacyanoferrate (III) in water (100 ml). The 
organic phase is separated, washed with water, dried and evaporated to 
dryness. The residue is purified by flash-chromatography; elution with 2% 
methanol in dichloromethane gives pure 3-(4-morpholinyl) rifamycin S, of 
which, after crystallization from ethyl ether/ petroleum ether, 13.8 g are 
obtained; m.p. 180.degree.-185.degree. C. (dec). TLC 
(methanol:dichloromethane, 5:95): black spot, Rf 0.39. 
b) Two drops of concentrated sulfuric acid are added to a mixture of 
3-(4-morpholinyl) rifamycin S (11 g), 2,2-dimethoxypropane (11 ml) and dry 
acetone (120 ml). The reaction mixture is stirred 45 minutes at room 
temperature. Anhydrous sodium carbonate (1 g) is added and stirring 
continued for 5 min. The solution is filtered and evaporated to dryness. 
The residue is purified by flash-chromatography; elution with 1% methanol 
in dichloromethane affords 7.3 g of pure 3-(4-morpholinyl)rifamycin S 
cyclic-21,23-(1-methylethylidene acetal) which is dissolved in a cold 
solution of 5% NaOH in methanol (100 ml). The resulting mixture is stirred 
18 hours at room temperature then diluted with icy water (100 ml), 
acidified (about pH 4) with citric acid and extracted with dichloromethane 
(3.times.100 ml). The combined extracts are dried and evaporated to 
dryness. The residue, by crystallization from ethyl ether/petroleum ether, 
gives the pure title compound (6.4 g) m.p. 171.degree.-174.degree. C. 
(dec). TLC (methanol:dichloromethane, 5:95): black spot, Rf 0.33. Analysis 
for C.sub.42 H.sub.54 N.sub.2 O.sub.12, MW=778.904; Calculated C 64.76 H 
6.99 N 3.59; Found C 64.57 H 7.01 N 3.39 
Preparation B: 25-O-Desacetylrifamycin S cyclic-21,23-(1-methylethylidene 
acetal) (Compound P2) 
The title compound is prepared as described by W. Kump and H. Birchel; 
Helv. Chim. Acta, 1973, 56, 2323. 
Preparation C: 25-O-Deacetyl-2'-(diethylamino)rifamycin P 
cyclic-21,23-(1-methylethylidene acetal) (Compound P3) 
N-Bromosuccinimide (2.2 g) in dimethylformamide (5 ml) is slowly dropped 
into a cold (5.degree. C.) solution of Compound P2 (7.7 g) and 
triethylamine (1.25 g) in dimethylformamide (25 ml). The reaction mixture 
is stirred for 3 hours at room temperature, then 1,1-diethylthiourea (1.8 
g) in dimethylformamide (4 ml) is added. Stirring is continued for 1.5 h, 
then ascorbic acid (2.6 g) in water (5 ml) is added. The mixture is 
allowed to rest overnight and then is poured into water (300 ml) and 
extracted with ethyl acetate (3.times.100 ml). The organic extracts are 
combined, washed with brine (200 ml), dried and evaporated to dryness. The 
residue is purified by flash-chromatography; elution with 1.8% methanol in 
dichloromethane gives the pure title compound (6.5 g).TLC 
(methanol:dichloromethane, 1:9): orange spot, Rf 0.54. Analysis for 
C.sub.43 H.sub.57 N.sub.3 O.sub.10 S, MW=808.031; Calculated C 63.92 H 
7.11 N 5.20; Found C 63.59 H 7.26 N 5.06 
Preparation D: 
1-Ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinecarboxylic 
acid dihydrochloride (Compound P4) 
a) ethyl 4,6-dichloronicotinate (17 g) is boiled for 4 hours with 24% 
sulfuric acid (400 ml). The white crystals that separate are filtered from 
the boiling solution and 4-chloro-6-hydroxynicotinic acid (6 g, m.p. 
299.degree.-300.degree. C.) is obtained; the acidic solution is left 
overnight in the refrigerator. The crystallized solid is collected by 
filtration and dried under vacuum, thus obtaining 
6-chloro-4-hydroxynicotinic acid (4.6 g) m.p. 231.degree.-233.degree. C. 
b) A mixture of 6-chloro-4-hydroxynicotinic acid (4 g), absolute ethanol 
(120 ml), toluene (60 ml) and sulfuric acid (13 ml) is gently refluxed for 
8 hours. After cooling the solvent is distilled off and the residue 
treated with icy water (100 ml). The solution is neutralized with a 
concentrated solution of Na.sub.2 CO.sub.3 and then extracted with 
dichloromethane (3.times.100 ml). The combined extracts are dried over 
sodium sulfate and evaporated to dryness. The residue is dissolved in 
boiling ethyl ether (10 ml) then petroleum ether is added (10 ml); on 
cooling, ethyl 6-chloro-4-hydroxy-nicotinate (3.8 g) mp 
59.degree.-61.degree. C. 
c) A mixture of ethyl 6-chloro-4-hydroxynicotinate (3.7 g), powdered 
potassium carbonate (3.7 g), iodoethane (5 ml) and dimethylformamide (50 
ml) is well stirred for 7 hours at 90.degree. C. After cooling, the 
reaction mixture is filtered and the solvent evaporated off. The residue 
is treated with icy water (30 ml) and extracted with dichloromethane 
(2.times.30 ml). The combined extract are dried and evaporated to dryness. 
The residue is boiled 90 minutes with 1N sodium hydroxide (18 ml). After 
cooling the solution is extracted with diethyl ether (30 ml), that is 
discharged. The alkaline solution is concentrated to small volume, cooled 
and acidified to pH 4 with 1N hydrochloric acid. The white material that 
precipitates is collected and crystallized from ethanol to give 
6-chloro-1,4-dihydro-1-ethyl-4-oxo-3-pyridine-carboxylic acid (3 g) m.p. 
155.degree.-57.degree. C. 
d) 6-chloro-1,4-dihydro-1-ethyl-4-oxo-3-pyridine-carboxylic acid (1.8 g) 
and N-methylpiperazine (5 ml) are stirred 5 hours at 130.degree. C. The 
excess of amine is distilled off and the residue dissolved in 1M 
hydrochloric acid (10 ml). The acidic solution is evaporated to dryness 
and the residue crystallized from ethanol (4 ml). The alcoholic solution 
is kept overnight in the refrigerator, thus obtaining the title compound 
(1.1 g), m.p. 224.degree.-226.degree. C. Analysis for C.sub.13 H.sub.19 
N.sub.3 O.sub.3.2 HCl MW=338.236; Calculated N 12.42 Cl 20.96; Found N 
11.97 Cl 20.80 
Preparation E: 
1-Cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3-qu 
inoline carboxylic acid (Compound P5) 
a) Acetyl chloride (17 ml) is slowly dropped into a stirred mixture of 
1-bromo-2,5-difluorobenzene (30 g) and aluminium trichloride (53 g) heated 
at 60.degree. C. and kept under argon. The reaction mixture is then 
stirred 90 minutes at 95.degree. C. The mixture is cooled at 40.degree. C. 
and carefully poured into crushed ice (400 g) and concentrated 
hydrochloric acid (35 ml). The resulting mixture is stirred a few minutes 
and then extracted with ethyl ether (2.times.250 ml). The ethereal 
solution is washed to neutral with brine, dried over sodium sulfate and 
the solvent is evaporated. The distillation of the oily residue gives 
4'-bromo-2 ',5'-difluoroacetophenone (21.2 g) b.p. (0.05 mm)=60.degree. C. 
b) Sodium hydride (7.1 g, 55% in mineral oil) is added in small portions to 
a well stirred solution of 4'-bromo-2',5'-difluoroacetophenone (20.8 g) in 
diethylcarbonate cooled at 5.degree. C. The resulting mixture is stirred 
10 minutes at 5.degree. C. and 90 minutes at 80.degree. C. After cooling 
the reaction mixture is poured into crushed ice (800 g) and acetic acid 
(35 ml), then extracted with ethyl ether (3.times.200 ml). The combined 
extracts are washed to neutral with brine dried over sodium sulfate and 
evaporated to dryness. The residue is purified by column chromatography 
containing 450 g of silica-gel (silica gel 60, particle size 0.063-0.200 
mm Merck). Elution with 18% ethyl acetate in petroleum ether allows the 
separation of the less polar compound that, after crystallization from 
n-hexane gives ethyl 4-bromo-2,6-difluorobenzoylacetate (5.6 g) m.p. 
49.degree.-52.degree. C. 
c) Dimethylformamide dimethylacetal (2.4 ml) is slowly dropped into a 
stirred solution of ethyl 4-bromo-2,6-difluorobenzoylacetate (5.4 g) in 
dry tetrahydrofuran (15 ml). Stirring is continued overnight at room 
temperature, then the solvent is evaporated. The reddish oily residue is 
dissolved in dry tetrahydrofuran (22 ml) and cooled to 0.degree. C.; 
cyclopropylamine (1.3 ml) is added and the resulting solution stirred 1 
hour at 0.degree. C. The solvent is evaporated under vacuum at room 
temperature. The residue, dissolved in dimethylformamide (25 ml), is 
stirred 1 hour at 100.degree. C. with anhydrous potassium carbonate (4.5 
g). After cooling the solution is filtered and evaporated to dryness. The 
residue is triturated with water, collected and recrystallized from 
ethanol to give ethyl 
7-bromo-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylate 
(3.2 g) 251.degree.-253.degree. C. 
d) A mixture of 2,6-lutidine-N-oxide hydrochloride (48 g) and phosphorus 
oxychloride (110 ml) is heated 6.5 hours at reflux. After cooling the 
reaction mixture is carefully poured into crushed ice (1 kg). While the 
temperature is maintained below 15.degree. C., concentrated ammonium 
hydroxide is added up to reach pH 8. The product that separates is 
extracted with ethyl ether (2.times.500 ml) which is dried and distilled 
off. The residue is dissolved in ethanol (400 ml) and boiled 3 hours with 
triethylamine (20 ml). After cooling, the solution is evaporated to 
dryness, treated with water (200 ml) and extracted with ethyl ether 
(4.times.150 ml). The combined extracts are dried and the solvent 
evaporated. The residue after distillation gives 
4-chloro-2,6-dimethylpyridine (25 g), b.p. (15 mm)=67.degree.-69.degree. 
C. To a mixture of sodium (10 g, 30% dispersion in toluene) in anhydrous 
dimethyoxyethane (40 ml) cooled in an ice-bath and kept under argon, 
trimethyltin chloride (12.1 g) in dimethoxymethane (6 ml) is added over a 
one hour period while keeping the temperature at 0.degree. C. The mixture 
is stirred 2.5 hours at 0.degree. C.; then 4-chloro-2,6-dimethylpyridine 
(7 g) in dimethoxyethane is slowly dropped into. Stirring is continued for 
an additional hour then the reaction mixture is allowed to stand overnight 
at room temperature. The reaction mixture is diluted with ethyl ether (100 
ml) and filtered. The solvent is evaporated, the residue dissolved in 
ethyl ether (100 ml) and filtered again. After evaporation of the solvent 
the residue is distilled giving 2,6-dimethyl-4-(trimethyl-stannyl)pyridine 
(8.8 g), b.p. (15 m)=130.degree.-140.degree. C. 
e) 2,6-dimethyl-4-(trimethylstannyl)pyridine (2.6 g) in dioxane (6 ml) is 
slowly dropped into a stirred mixture of ethyl 
7-bromo-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylate 
(3.1 g), dioxane (55 ml) and hexamethylenphosphoramide (2.6 ml) kept under 
argon. To the stirred mixture, dichlorobis (triphenylphosphine) palladium 
(0.4 g) is added. The reaction mixture is heated under reflux for 24 
hours. After cooling it is poured in water (200 ml) and repeatedly 
extracted with dichloromethane. The organic extracts are dried and 
evaporated to dryness. The residue, after trituration with ethyl ether, 
gives ethyl 
1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3-qu 
inolinecarboxylate (2.4 g) m.p. 196.degree.-199.degree. C. 
f) A suspension of ethyl 
1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3-qu 
inolinecarboxylate (2.3 g) in 1.5% NaOH (50 ml) is heated 2.5 h at reflux. 
The solution is decolorized with charcoal (1 g) filtered, cooled in an 
ice-bath and acidified with acetic acid. The separated solid is collected 
by filtration and crystallized from ethanol/chloroform to give the title 
compound (1.66 g) m.p. 300.degree.-303.degree. C. 
Preparation F: Potassium salt of 
1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinecarboxylic 
acid (Compound P6) 
The potassium salt of compound P4 is prepared by adding 1N potassium 
hydroxide (6 ml) to the free acid dihydrochloride (676 mg-2 mmol) 
dissolved in water (5 ml). The aqueous solution is evaporated to dryness 
and the residue treated with boiling ethanol (20 ml). The insoluble 
material (KCl) is filtered and the alcoholic solution re-evaporated to 
dryness. The solid residue is triturated with diethyl ether, collected by 
filtration and dried over phosphorous pentoxide under vacuum giving the 
desired potassium salt (575 mg). 
Preparation C: Potassium salt of 
1-Cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3-qu 
inoline carboxylic acid (Compound P7) 
The potassium salt of compound P5 is prepared by adding 1N potassium 
hydroxide (2 ml) to the free acid (705 mg-2mmol) dissolved in ethanol (10 
ml). The resulting solution is evaporated to dryness and the solid residue 
is triturated with a 1:1 mixture of diethyl ether/ethanol, collected by 
filtration and dried over phosphorous pentoxide under vacuum giving the 
desired potassium salt (672 mg). 
EXAMPLE A 
36-Bromo-36-carboxyrifamycin S (Compound E1) 
a) 1,3-Dicyclohexylcarbodiimide (950 mg, 4.6 mmol) dissolved in dry 
tetrahydrofuran (2 ml) is slowly dropped into a stirred mixture of 
compound P2 (940 mg, 1.4 mmol), bromomalonic acid (840 mg, 4.6 mmol) and 
dry tetrahydrofuran (15 ml) cooled at 0.degree. C. The reaction mixture is 
stirred 15 minutes at 0.degree. C., then 1 hour at room temperature. The 
dicyclohexylurea that forms is filtered off and the solution evaporated to 
dryness. The oily residue is dissolved in ethyl acetate (20 ml) and washed 
with water (2.times.20 ml). After drying over sodium sulfate, the solvent 
is evaporated and the residue purified by flash-chromatography. Elution 
with 4% methanol in dichloromethane gives pure 
(36-Bromo-36-carboxy)rifamycin S cyclic-21,23-(1-methylethylidene acetal) 
(680 mg). TLC (methanol:dichloromethane, 15:85): Rf 0.34 
b) A mixture of the above obtained compound (680 mg), tetrahydrofuran (8 
ml) and 3% sulfuric acid (3 ml) is stirred at 40.degree. C. for 16 hours. 
After cooling, the reaction mixture is poured into water (20 ml) and 
extracted with ethyl acetate (2.times.20 ml). The organic extracts are 
washed with brine to neutral, then dried and concentrated to a small 
volume. Dilution with petroleum ether gives a brownish precipitate which 
is collected and dried under vacuum yielding the title compound (490 mg). 
TLC (methanol:dichloromethane, 15:85): Rf 0.27; Analysis for C.sub.38 
H.sub.44 BrN.sub.14, MW=818.682; Calculated C 55.75 H 5.41 N 1.85 Br 9.76; 
Found C 55.85 H 5.60 N 1.70 Br 8.98 
EXAMPLE B 
36-Carboxy-36-fluororifamycin S (Compound E2) 
By following the procedure of Example A, but starting from Compound P2 (0.7 
g) and fluoromalonic acid, the title compound is obtained (0.22 g). TLC 
(methanol:dichloromethane, 15:85): Rf 0.20; Analysis for C.sub.38 H.sub.44 
FNO.sub.14, MW=757.771; Calculated C 60.23 H 5.85 N 1.71; Found C 60.00 H 
5.94 N 1.70 
EXAMPLE C 
36-Carboxy-36-chlororifamycin S (Compound E3) 
By following the procedure of Example A, but starting from P2 (9.6 g) and 
chloromalonic acid, the title compound is obtained (9.3 g). TLC 
(methanol:dichloromethane, 2:8): Rf 0.45; Analysis for C.sub.38 H.sub.44 
ClNO.sub.14, MW=774.226; Calculated C 58.95 H 5.73 N 1.81 Cl 4.58; Found C 
58.49 H 5.61 N 1.73 Cl 4.80 
EXAMPLE D 
36-Carboxy-36-methylthiorifamycin S (Compound E4) 
By following the procedure of Example A, but starting from Compound P2 
(0.96 g) and methylthiomalonic acid, the title compound is obtained (0.47 
g). TLC (methanol:dichloromethane, 2:8): Rf 0.39; Analysis for C.sub.39 
H.sub.47 NO.sub.14 S, MW=785.873; Calculated C 59.61 H 6.03 N 1.78 S 4.08; 
Found C 59.14 H 6.10 N 1.76 S 3.70 
EXAMPLE E 
36-Carboxy-36-ethylrifamycin S (Compound E5) 
By following the procedure of Example A, but starting from Compound P2 
(0.96 g) and ethylmalonic acid, the title compound is obtained (0.40 g). 
TLC (methanol:dichloromethane, 15:85): Rf 0.34; Analysis for C.sub.40 
H.sub.49 NO.sub.14, MW=767.836; Calculated C 61.01 H 6.43 N 1.82; Found C 
60.58 H 6.50 N 1.79 
EXAMPLE F 
36-Carboxy-36-butylrifamycin S (Compound E6) 
By following the procedure of Example A, but starting from Compound P2 
(0.96 g) and butylmalonic acid, the title compound is obtained (0.4 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.51; Analysis for C.sub.42 
H.sub.53 NO.sub.14, MW=795.889 Calculated for C 63.38 H 6.71 N 1.76; Found 
C 62.96 H 6.75 N 1.70 
EXAMPLE G 
36-Carboxy-36-octylrifamycin S (Compound E7) 
By following the procedure of Example A, but starting from Compound P2 
(0.96 g) and octylmalonic acid, the title compound is obtained (0.66 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.55; Analysis for C.sub.46 
H.sub.61 NO.sub.14, MW=851.998; Calculated C 64.85 H 7.22 N 1.64; Found C 
64.47 H 7.30 N 1.62 
EXAMPLE H 
36-Bromo-36-carboxy-3-(4-morpholinyl)-rifamycin S (Compound E8). 
By following the procedure of Example A, but starting from Compound P1 (32 
g) and bromomalonic acid, the title compound is obtained (26 g). TLC 
(methanol:dichloromethane, 2:8): Rf 0.43; Analysis for C.sub.42 H.sub.51 
BrN.sub.2 O.sub.15, MW=903.787; Calculated C 55.82 H 5.69 N 3.10 Br 8.84; 
Found C 55.30 H 5.70 H 3.04 Br 8.46 
EXAMPLE I 
36-Bromo-36-carboxy-2'-(diethylamino)-rifamycin P (Compound E9) 
By following the procedure described in Example A, from Compound P3 (3.3 g) 
and bromomalonic acid (2.5 g), the title compound (1.4 g) is obtained. TLC 
(methanol:dichloromethane, 25:75): orange spot Rf 0.35; Analysis for 
C.sub.43 H.sub.54 BrN.sub.3 O.sub.13 S, MW=932.894; Calculated C 53.36 H 
5.83 N 4.50 Br 8.61; Found C 54.01 H 5.93 N 4.61 Br 8.04 
EXAMPLE 1 
36-Bromorifamycin S (Compound C1) 
Compound E1 (3.6 g) dissolved in anhydrous acetonitrile (40 ml) is very 
slowly added to a well stirred suspension of cuprous (I) oxide (Cu.sub.2 
O) (100 mg) in anhydrous acetonitrile (160 ml) heated at 60.degree. C. and 
kept under argon. The resulting mixture is stirred 1 hour at 
60.degree.-70.degree. C. After cooling, it is filtered and evaporated to 
dryness. The residue, dissolved in ethyl acetate (40 ml), is washed with 
1N hydrochloric acid (3.times.20 ml) and with brine to neutral. The 
organic phase is dried, the solvent evaporated off and the residue 
purified by flash-chromatography; elution with 1.2% methanol in 
dichloromethane allowed the isolation of the pure title compound (1.6 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.61; Analysis for C.sub.37 
H.sub.44 BrNO.sub.12, MW=774.673; Calculated C 57.36 H 5.73 N 1.80 Br 
10.31; Found C 57.60 H 6.10 H 1.76 Br 10.24. 
EXAMPLE 2 
36-Fluororifamycin S (Compound C2) 
By following the procedure of Example 1, but starting from Compound E2 
(0.14 g) and Cu.sub.2 O (0.01 g), the title compound is obtained (0.04 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.65; Analysis for C.sub.37 
H.sub.44 FNO.sub.12, MW=713.763; Calculated C 62.26 H 6.21 N 1.96; Found C 
61.60 H 6.34 N 2.04 
EXAMPLE 3 
36-Chlororifamycin S (Compound C3) 
By following the procedure of Example 1, but starting from Compound E3 
(18.6 g) and Cu.sub.2 O (0.48 g), the title compound is obtained (9.3 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.68; Analysis for C.sub.37 
H.sub.44 ClNO.sub.12, MW=730.217; Calculated C 60.86 H 6.07 N 1.92 Cl 
4.85; Found C 61.00 H 6.30 N 1.76 Cl 4.73 
EXAMPLE 4 
36-Methylthiorifamycin S (Compound C4) 
By following the procedure of Example A, but starting from Compound E4 
(0.43 g) and Cu.sub.2 O (0,03 g), the title compound is obtained (0.22 g). 
TLC (methanol:dichloromethane, 1:9): Rf 0.54; Analysis for C.sub.38 
H.sub.47 NO.sub.12 S, MW=741.863; Calculated C 61.52 H 6.38 N 1.88 S 4.32; 
Found C 61.54 H 6.75 N 1.90 S 4.30 
EXAMPLE 5 
36-Ethylrifamycin S (Compound C5). 
The procedure is essentially the same of Example 1 but the reaction mixture 
is stirred for 8 hours at the reflux. From 0.23 g of Compound E5 and 0.02 
g of Cu.sub.2 O, the pure title compound is obtained (0.038 g). TLC 
(methanol:dichloromethane, 1:9): Rf 0.52; Analysis for C.sub.39 H.sub.49 
NO.sub.12, MW=723.726; Calculated C 64.72 H 6.82 N 1.93; Found C 63.98 H 
6.90 N 2.13 
EXAMPLE 6 
36-Butylrifamycin S (Compound C6). 
The procedure is as same as in Example 1 but the reaction mixture is 
stirred for 20 hours at reflux. From Compound E6 (0.40 g) and Cu.sub.2 O 
(0.035 g), the title compound is obtained (0.030 g). TLC 
(methanol:dichloromethane, 1:9): Rf 0.7; Analysis for C.sub.4 1H.sub.53 
NO.sub.12, MW=751.879; Calculated C 65.49 H 7.10 N 1.86; Found C 65.57 H 
7.40 N 2.02 
EXAMPLE 7 
36-Octylrifamycin S (Compound C7). 
By following the procedure of example 5, but starting from Compound E7 
(0.65 g) and Cu.sub.2 O (0.050 g), the title compound is obtained (0.150 
g). TLC (methanol:dichloromethane, 1:9): Rf 0.75; Analysis for C.sub.45 
H.sub.61 NO.sub.12, MW=807.988; Calculated C 66.89 H 7.61 N 1.73; Found C 
66.56 H 7.69 N 2.01 
EXAMPLE 8 
36-Bromo-3-(4-morpholinyl)rifamycin S (Compound C8) 
By following the procedure of Example 1,but starting from Compound E8 (26 
g) and Cu.sub.2 O (0.70 g), the title compound is obtained (11.8 g). TLC 
(methanol:dichloromethane, 1:9): Rf 0.61; Analysis for C.sub.41 H.sub.51 
BrN.sub.2 O.sub.3, MW=859.777; Calculated C 57.27 H 5.99 N 3.26 Br 9.29; 
Found C 56.91 H 6.03 N 3.07 Br 8.81 
EXAMPLE 9 
36-Bromo-2'-(diethylamino)rifamycin P (Compound C9) 
By following the procedure described in Example 1, from Compound E9 (1.6 g) 
and Cu.sub.2 O (80 mg), the title compound (0.69 g) is obtained. TLC 
(methanol:dichloromethane, 1:9): orange spot Rf 0.5; Analysis for C.sub.42 
H.sub.54 BrN.sub.3 O.sub.11 S, MW=888.884; Calculated C 56.75 H 6.12 N 
4.72 Br 8.99; Found C 55.89 H 6.05 N 4.68 Br 9.30 
EXAMPLE 10 
36-Iodorifamycin S (Compound C10) 
Sodium iodide (NaI) (0.47 g) dissolved in acetone (3 ml) is added to 
Compound C1 (1.12 g) dissolved in acetone (8 ml). The resulting mixture is 
stirred at room temperature for 4 hours. The solvent is evaporated off and 
the residue purified by flash-chromatogarphy. Elution with 1.4% methanol 
in dichloromethane gives pure title compound (1.1 g). TLC 
(methanol:dichloromethane, 1:9): Rf 0.59; Analysis for C.sub.37 H.sub.44 
INO.sub.12, MW=821.668; Calculated C 54.08 H 5.40 N 1.70; Found C 54.01 H 
6.00 N 1.85 
EXAMPLE 11 
36-Iodo-3-(4-morpholinyl)rifamycin S (Compound C11) 
By following the procedure of Example 10, but starting from compound 
Compound C8 (1.25 g) and NaI (0.5 g), the title compound is obtained (1.05 
g). TLC (methanol:dichloromethane, 1:9): black spot Rf 0.6; Analysis for 
C.sub.41 H.sub.51 IN.sub.2 O.sub.13, MW=906.772; Calculated C 54.31 H 5.67 
N 3.09; Found C 53.82 H 5.75 N 2.79 
EXAMPLE 12 
36-Diethylaminorifamycin S (Compound C12) 
A mixture of Compound C11 (300 mg), diethylamine (100 mg) and 
tetrahydrofuran (5 ml) is stirred 4 hours at room temperature, then poured 
into water (25 ml) and extracted with ethyl acetate (3.times.15 ml). The 
combined extracts are dried and evaporated to dryness. The residue is 
purified by flash-chromatography; elution with 2% methanol in 
dichloromethane gives the pure title compound (135 mg). TLC 
(methanol:dichloromethane, 1:9): Rf 0.5; Analysis for C.sub.41 H.sub.54 
N.sub.2 O.sub.12, MW=766.993; Calculated C 64.20 H 7.10 N 3.65; Found C 
63.64 H 6.95 N 3.38 
EXAMPLE 13 
36-Acetyloxyrifamycin S (Compound C13) 
A mixture of Compound C10 (180 mg), silver acetate (250 mg) and anhydrous 
dimethylformamide (18 ml) is stirred 18 hours at room temperature. The 
solvent is distilled off at 40.degree. C. under vacuum. The residue is 
purified by flash-chromatography; elution with 1.2% methanol in 
dichloromethane allowed the recovery of unreacted Compound C10 (66 mg) as 
less polar component and the isolation of the pure title compound (41 mg). 
TLC (methanol:dichloromethane, 1:9): Rf 0.52; Analysis for C.sub.39 
H.sub.47 NO.sub.14, MW=753.809; Calculated C 62.14 H 6.28 N 1.86; Found C 
61.48 H 6.40 N 1.85 
EXAMPLE 14 
36-Formyloxy-3-(4-morpholinyl)rifamycin S (Compound C14) 
A mixture of potassium formate (11.5 g), dimethylformamide (800 ml) and 
activated 4 .ANG.molecular sieves (30 g) is stirred for 20 minutes. 
Compound C8 (11.5 g) is then added in small portions and stirring 
continued for 24 hours at room temperature. The mixture is filtered and 
the solvent removed at 40.degree. C. under vacuum. The residue is purified 
by flash-chromatography; elution with 1.5% methanol in dichloromethane 
gives the pure title compound (8.6 g). TLC (methanol:dichloromethane, 
1:9): black spot Rf 0.42; Analysis for C.sub.42 H.sub.52 N.sub.2 O.sub.15, 
MW=824.886; Calculated C 61.15 H 6.35 N 3.40; Found C 60.41 H 6.37 N 3.30 
EXAMPLE 15 
36-Hydroxy-3-(4-morpholinyl)rifamycin S (Compound C15) 
A solution of potassium bicarbonate (18 g) in water (180 ml) is slowly 
added to Compound C14 (8.2 g) dissolved in methanol (450 ml). The 
resulting mixture is stirred overnight at room temperature, then 
evaporated to dryness at 30.degree. C. and under vacuum. Citric acid (10% 
W/V) is carefully added. The mixture is extracted with ethyl acetate 
(2.times.150 ml), which is then evaporated off. The residue is purified by 
flash-chromatography; elution with 1.8% methanol in dichloro-methane, 
after crystallization from ethyl ether, gives the pure title compound (4.3 
g). M.P. 168.degree.-171.degree. C. (dec). TLC (methanol:dichloromethane, 
5:95): black spot Rf 0.33; Analysis for C.sub.41 H.sub.52 N.sub.2 
O.sub.14, MW=796.876; Calculated C 61.79 H 6.58 N 3.51; Found C 61.30 H 
6.56 N 3.22 
EXAMPLE 16 
36-Iodo-2'-diethylaminorifamycin P (Compound C16) 
Sodium iodide (NaI) (0.2 g) dissolved in acetone (2 ml) is added to 
compound C9 (0.55 g) dissolved in acetone (4 ml). The resulting mixture is 
stirred at room temperature for 4 hours. The solvent is evaporated off and 
the residue purified by flash-chromatogarphy. Elution with 1.4% methanol 
in dichloromethane gives pure title compound (0.48 g). TLC 
(methanol:dichloromethane, 1:9): orange spot Rf 0.48; Analysis for 
C.sub.42 H.sub.54 IN.sub.3 O.sub.11 S, MW=953.879; Calculated C 53.90 H 
5.82 N 4.49 S 3.42; Found C 54.04 H 5.82 N 4.48 S 3.21 
EXAMPLE 17 
36-Bromo-3-{(4-methyl-1-piperazinyl)-imino!methyl}rifamycin SV (Compound 
C17) 
a) N-methylen-t-butylamine (1.92 g) is slowly dropped into a cooled 
solution (15.degree. C.) of Compound C1 (3.3 g) in tetrahydrofuran (25 
ml). After an addition of tert-butylamine (0.4 ml), the reaction mixture 
is stirred for 5 minutes, then manganese dioxide (1.7 g) is added and 
stirring continued overnight at 48.degree. C. After cooling, the mixture 
is filtered and dropped into a cold solution (0.degree. C.) of 16% 
sulfuric acid (15 ml), ascorbic acid (3 g) and tetrahydrofuran (5 ml). The 
reaction mixture is stirred 3 hours at 45.degree. C., then is poured in 
icy water (150 ml) and finally extracted with ethyl acetate (3.times.5 ml) 
that is dried and evaporated to dryness. The residue is purified by 
flash-chromatography; elution with 3% methanol in dichloromethane gives 
pure 36-Bromo-3-formylrifamycin SV (500 mg). TLC 
(methanol:dichloromethane, 85:15): red spot Rf 0.44; Analysis for C.sub.38 
H.sub.46 BrNO.sub.13, MW=804.699; Calculated for C 56.72 H 5.76 N 1.74 Br 
9.93; Found C 56.06 H 5.94 N 2.00 Br 10.10 
b) 1-Amino-4-methylpiperazine (50 mg) is added to the above obtained 
compound (300 mg) dissolved in tetrahydrofuran (7 ml). The solution is 
stirred 30 min at room temperature. The solvent is evaporated off and the 
residue dissolved in ethyl acetate; addition of petroleum ether leads to 
the precipitation of the pure title compound (280 mg). TLC 
(methanol:dichloromethane, 15:85): orange spot Rf 0.65; Analysis for 
C.sub.43 H.sub.57 BrN.sub.4 O.sub.12, MW=901.862; Calculated C 57.27 H 
6.37 N 6.21 Br 8.86; Found C 56.45 H 6.40 N 5.90 Br 8.34 
EXAMPLE 18 
36-Chloro-3{(4-methyl-1-piperazinyl)-imino!methyl}rifamycin SV (Compound 
C18) 
a) By following the procedure of Example 17, step a, but starting from 
Compound C3 (4.1 g), N-methylen-t-butylamine (2.6 g), tert-butylamine (0.5 
ml) and manganese dioxide (2.3 g), 36-chloro-formylrifamycin SV is 
obtained (3.9 g). TLC (methanol:dichloromethane, 15:85): red spot Rf 0.47; 
Analysis for C.sub.38 H.sub.46 ClNO.sub.13, MW=760.243; Calculated C 60.03 
H 6.10 N 1.84 Cl 4.66; Found C 59.61 H 6.22 N 1.80 Cl 4.45 
b) By following the procedure of Example 17, step b, but starting from the 
above obtained compound (3.9 g) and 1-Amino-4-methylpiperazine (0.67 g), 
the title compound (2.92 g) is obtained. TLC (methanol:dichloromethane, 
15:85): orange spot Rf 0.61; Analysis for C.sub.43 H.sub.57 ClN.sub.4 
O.sub.2, MW=857.406; Calculated C 60.23 H 6.70 N 6.53 Cl 4.13; Found C 
59.94 H 6.42 N 6.36 Cl 4.40 
EXAMPLE 19 
36-Iodo-3{(4-methyl-1-piperazinyl)-imino!methyl}rifamycin SV (Compound 
C19) 
Sodium iodide (110 mg) is added to Compound C17 (300 mg) dissolved in 
acetone (3 ml). The mixture is stirred 4 hours at room temperature and 
then evaporated to dryness. The residue is purified by 
flash-chromatography; elution with 3% methanol in dichloromethane gives 
pure title compound (160 mg). 
Alternatively, Compound C18 (2.9 g) is reacted with sodium iodide (1 g) as 
above described, thus obtaining the pure title compound (2.83 g). TLC 
(methanol:dichloromethane, 15:85): orange spot Rf 0.63; Analysis for 
C.sub.43 H.sub.57 IN.sub.4 O.sub.12, MW=948.857; Calculated C 54.43 H 6.05 
N 5.90; Found C 54.70 H 6.20 N 5.49 
EXAMPLE 20 
36-Bromo-3{(4-cyclopentyl-1-pipera-zinyl)imino!methyl}rifamycin SV 
(Compound C20) 
a) By following the procedure of Example 17, step b, but starting from 
36-Bromo-3-formylrifamycin SV (0.65 g) and 1-Amino-4-cyclopentylpiperazine 
(0.14 g), the title compound is obtained (0.64 g). TLC 
(methanol:dichloromethane, 15:85): orange spot Rf 0.64; Analysis for 
C.sub.47 H.sub.63 BrN.sub.4 O.sub.12, MW=955.955; Calculated C 59.05 H 
6.64 N 5.86 Br 8.36; Found C 58.38 H 6.39 N 5.60 Br 7.61 
EXAMPLE 21 
36-Iodo-3{(4-cyclopentyl-1-pipera-zinyl)imino!methyl}rifamycin SV 
(Compound C21) 
b) By following the procedure of Example 19, but starting from Compound C20 
(730 mg) and sodium iodide (250 mg), the title compound (432 mg) is 
obtained. 
EXAMPLE 22 
36-(1-Ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl)carbonyloxy!-2'-(diethylamino)rifamycin P (Compound C22) 
(1-Ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinolinyl 
)carboxylic acid (Pefloxacin) potassium salt (440 mg) dissolved in 
dimethylformamide (44 ml) is stirred for 30 min with activated 4 
.ANG.molecular sieves (Union Carbide type 4 .ANG., Fluka) (4.4 g). 
Compound C16 (440 mg) is added in small portions and stirring continued 
overnight at room temperature. The reaction mixture is filtered and 
evaporated to dryness at 40.degree. C. under vacuum. The residue is 
purified by flash-chromatography; elution with 8% methanol in 
dichloromethane allows the isolation of the pure title compound (380 mg). 
TLC (methanol:dichloromethane, 1: 9): orange spot Rf 0.4; Analysis for 
C.sub.59 H.sub.73 FN.sub.6 O.sub.14 S, MW=1141.313; Calculated C 62.09 H 
6.45 N 7.36 S 2.81; Found C 61.20 H 6.36 N 7.05 S 2.44 
EXAMPLE 23 
36-{2-(1-Ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-qui 
nolinyl!carbonyloxy} rifamycin S (Compound C23) 
Pefloxacin potassium salt (250 mg) is reacted with Compound C10 (250 mg) in 
dimethylformamide (25 ml) as described in Example 22. In this way the 
title compound (178 mg) is obtained. TLC (methanol:dichloromethane, 1:9): 
brown spot Rf 0.38; Analysis for C.sub.54 H.sub.63 FN.sub.4 O.sub.15, 
MW=1027.120; Calculated C 63.14 H 6.18 N 5.45; Found C 62.50 H 5.98 N 5.06 
EXAMPLE 24 
36-{1-Ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl!carbonyloxy}-3-(4-morpholinyl) rifamycin S (Compound C24) 
Compound C8 (460 mg) is reacted with Pefloxacin potassium salt (460 mg) in 
dimethylformamide (46 ml) as described in Example 22. The residue is 
purified by flash-chromatography; elution with 6% methanol in 
dichloromethane gives pure title compound (354 mg). TLC 
(methanol:dichloromethane, 1:9): black spot Rf 0.42; Analysis for C.sub.58 
H.sub.70 FN.sub.5 O.sub.16, MW=1112.225; Calculated C 62.63 H 6.34 N 6.29; 
Found C 61.72 H 6.37 N 5.95 
EXAMPLE 25 
36-{1-Ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinol 
inyl!carbonyloxy}-3-{(4-methyl-1-piperazinyl)imino!methyl}rifamycin SV 
(Compound C25) 
By reacting Pefloxacin potassium salt (650 mg) with Compound C19 (650 mg) 
in dimethylformamide (65 ml), as described in Example 22, the title 
compound (480 mg) is obtained after twice purification by 
flash-chromatography (eluition with increasing percentages--4 to 15%--of 
methanol in dichloromethane). TLC (methanol:dichloromethane, 2:8): orange 
spot Rf 0.47; Analysis for C.sub.60 H.sub.76 FN.sub.7 O.sub.15, 
MW=1154.310; Calculated C 62.43 H 6.63 N 8.49; Found C 62.06 H 6.18 N 8.17 
EXAMPLE 26 
3{(4-Cyclopentyl-1-piperazinyl)-imino!methyl}-36-{1-ethyl-6-fluoro-1,4-di 
hydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinolinyl!carbonyloxy}-rifamycin 
SV (Compound C26) 
By following the procedure described in Example 25, but starting from the 
Compound C21 (400 mg) and Pefloxacin potassium salt (400 g), the title 
compound (280 mg) is obtained. TLC (methanol:dichloromethane, 2:8): orange 
spot Rf 0.60; Analysis for C.sub.64 H.sub.82 FN.sub.7 O.sub.15, 
MW=1208.403; Calculated C 63.61 H 6.84 N 8.11; Found C 63.70 H 7.00 N 8.02 
EXAMPLE 27 
36-{1-Ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl)carbonyloxy}- 
3{(4-methyl-1-piperazinyl)imino!methyl}rifamycin SV (Compound C27) 
According to the procedure of example 25, but starting from Compound C19 
(500 mg) and 
1-Ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridin-3-yl)carboxylic acid 
(Nalidixic Acid) potassium salt (500 mg), the title compound (452 mg) is 
obtained. TLC (methanol:dichloromethane, 1:9): red-orange spot Rf 0.49; 
Analysis for C.sub.55 H.sub.68 N.sub.6 O.sub.15, MW=1053.186; Calculated C 
62.72 H 6.51 N 7.98; Found C 61.86 H 6.43 N 7.79 
EXAMPLE 28 
36-{8-Ethyl-5,8-dihydro-2-(4-methyl-1-piperazinyl)-5-oxopyrido2,3-d!pyrim 
idin-6-yl!carbonyloxy}-3-{(4-methyl-1-piperazinyl)imino!-methyl}rifamycin 
SV (Compound C28) 
According to example 25, but starting from N-methyl-pipemidic acid 
potassium salt (400 mg) and Compound C19 (400 mg), the pure title compound 
(275 mg) is obtained. TLC (methanol:dichloromethane, 15:85): red-orange 
spot Rf 0.36; Analysis for C.sub.58 H.sub.75 N.sub.9 O.sub.15, 
MW=1138.295; Calculated C 61.20 H 6.64 N 11.07; Found C 60.89 H 6.60 N 
10.71 
EXAMPLE 29 
36-{1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo- 
3-quinolinyl!-carbonyloxy}-3-{(4-methyl-1-piperazinyl)imino!-methyl}rifamy 
cin SV (Compound C29) 
According to Example 25, but starting from Compound P7 (500 mg) and 
Compound C19 (500 mg), the pure title compound (420 mg) is obtained. TLC 
(methanol:dichloromethane, 1:9): red-orange spot Rf 0.45; Analysis for 
C.sub.63 H.sub.73 FN.sub.6 O.sub.15, MW=1173.313; Calculated C 64.49 H 
6.27 N 7.16; Found C 64.47 H 6.38 N 7.10 
EXAMPLE 30 
36-{1-Ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinyl!carbo 
nyloxy}-3-{(4-methyl-1-piperazinyl)imino!methyl}rifamycin SV (Compound 
C30) 
According to example 25, but starting from Compound P6 (500 mg) and 
Compound C19 (500 mg), the pure title compound (320 mg) is obtained. TLC 
(methanol:dichloromethane, 15:85): red-orange spot Rf 0.44; Analysis for 
C.sub.56 H.sub.75 N.sub.7 O.sub.15, MW=1086.260; Calculated C 61.92 H 6.96 
N 9.02; Found C 62.00 H 6.96 N 8.91;