Field of invention
The present invention relates to an improved process for the manufacture of Rifabutin.
Background of the invention
Rifabutin (I) (CAS No. 72559-06-9) is chemically known as 1',4-didehydro-1-deoxy-1,4-
dihydro-5'-(2-methylpropyl)-1-oxo rifamycin XIV.

Rifabutin is a potent antimycobacterial agent and is useful in treatment of tuberculosis. It is
available commercially as a pharmaceutical composition under the brand name
MYCOBUTIN®, marketed by Pharmacia and Upjohn, and is first described in Italian patent
application 1056272, GB1542063, and US4219478.
US 4219478 discloses preparation of Rifabutin/compounds like Rifabutin via condensation
of 4-imino-3-amino rifamycin-S with ketones (including substituted piperidones) in
presence of ammonium acetate and Zinc. US'478 further discloses in the examples use of
polar solvents such as tetrahydrofuran, dioxane for carrying out this condensation reaction.
However, there are no methods described for synthesis of 3-amino-rifamycin-S and 4-
imino-3-amino-rifamycin-S.
Similarly, Canadian Patent 1089453 discloses synthesis of Rifabutin via condensation of 3-
amino-4-imino-rifamycin-S with 1-isobutyl-4-piperidone in presence of zinc and ammonium
acetate in tetrahydrofuran as a solvent the product was isolated by extraction in ether and
further in dilute acetic acid, however, the patent do not disclose the process for preparation
of the starting materials 3-amino-rifamycin-S and 4-imino-3-amino-rifamycin-S.
Among the derivatives of Rifamycin S, DE 1,670,377 and Helvetica Chimica Acta, 56,
2368(1973) disclose the 3-amino derivatives of Rifamycin-S, compounds having higher
antibacterial property, and obtained by direct amination. However, yields obtained in the
methods described in DE 1,670,377 and Helvetica Chimica Acta, 56, 2368(1973) are not
only low but also variable.
US4007169 describes preparation of 3-amino-rifamycin-S from rifamycin-S by reaction
with sodium azide in polar solvents such as formamide, at temperature of about 30 - 35°C.
However, such reactions as described in the patent and in scheme-!, beside the desired
product always give two side products 3-azido-rifamycin-S (e) and rifamycin-SV (d) as
shown in scheme-I. This has been discussed in-depth in US4007169.

Although, yields are superior to that of direct amination described in DE 1,670,377 and
Helvetica Chimica Acta, 56, 2368(1973) but the process as such is not viable because of
both technical and economical uneasiness.
Further, the present inventors have observed that the process described in the US patent
4007169 suffers from disadvantage of variable rate of reaction as would be evident for the
time required completion of reaction ranges from seven to thirteen hours.
Moreover, the process generates almost equal amount of undesired side product mainly
rifamycin-SV as would be evident from table 1.
Table 1: reaction monitoring of the process described in the '169 patent.
Hence, it is obvious from the prior art that there is a need for developing an improved
process commercially viable for manufacture of 3-amino-rifamycin-S, which in turn
derivatized to potent antibacterial/anitmycobacterial rifabutin and compounds having
similar structure.
Object of the invention
Thus, one object of the present invention is to provide a process for the synthesis of 3-
amino-rifamycin-S which is not only cost effective but also uses easily available chemicals,
which in turn gives superior material balance and quality of the product.
A further object of the present invention is to provide an improved commercially viable
process for the synthesis of rifabutin.
Summary of the invention
The invention provides a process for manufacture of rifabutin comprising steps of:
(i) Conversion of rifamycin-S to 3-amino-rifamycin-S in presence of sodium
azide in an organic solvent such as formamide or methyl formamide at a
temperature of about 33-35°C, wherein the byproduct rifamycin-SV was
simultaneously converted to rifamycin-S by adding suitable oxidizing agent
such as ammonium persulfate and further reacted with sodium azide to
form 3-amino-rifamycin-S in situ; followed by isolation of the product using
acetic acid and 2-methoxy ethanol.
(ii) Conversion of 3-amino-rifamycin-S to 4-imino-3-amino-rifamycin-S by
treatment of 3-amino-rifamycin-S with ammonia gas in an organic solvent
such as tetrahydrofuran; isolation of the product in 2-methoxy ethanol,
followed by optional purification using methanol,
(iii) Condensation of 4-imino-3-amino-rifamycin-S with isobutyl-4-piperidone in
presence of ammonium acetate and zinc in an organic solvent such as
tetrahydrofuran; followed by isolation of the rifabutin by extraction in ether
and then in dilute acetic acid.
(iv) Optional purification of rifabutin obtained in step (iii) using acetone and
water, wherein the product was isolated at pH of about 7.3 to 7.5.
Other aspect of the present invention is to provide a process for manufacture of 3-amino-
rifamycin-S comprising steps of:
(a) Treatment of rifamycin-S with about 4 mole-equivalents of sodium azide in
formamide at 33-35°C.
(b) Stirring the mixture at 33-35°C till concentration of rifamycin-S in the mixture
goes below 3%.
(c) Addition of about 0.5 Mole equivalent of ammonium persulfate to the mixture
and stirring at 33-35°C.
(d) Extraction of the product in chloroform, followed by isolation using acetic acid
and 2-methoxy ethanol.
Detailed description of the invention:
US4007169 describes reaction of rifamycin-S with sodium azide in formamide/methyl
formamide, leading to formation of three products viz. 3-amino-rifamycin, 3-azido-
rifamycin-S and rifamycin-SV (hydroquinone form of rifamycin-S). As rifamycin-SV is a
major side product it is prudent to convert it back to rifamycin-S by oxidation so that it can
be re-used. US4353826 describes oxidation of rifamycin-SV and its derivatives to
corresponding rifamycin-S derivatives using oxidizing agents such as ammonium
persulfate, potassium ferricyanide, hydrogen peroxide or air. The '169 patent also
describes isolation and recyclability of rifamycin-SV formed during the reaction by
oxidation with nitrous acid, however, the recycled rifamycin-S would again yield the stated
three products. Though this process would be suitable for continuous production of 3-
amino-rifamycin-S, would not be suitable for batch processing or where the production is
limited to few batches. The process is bound to keep lot of rifamycin-SV back log waiting
till next production batch, thereby increasing burden on temporary storage zone of the
manufacturing plant.
It would have been a better commercial process if there would have been absolutely no
formation of the side product, or the side product formed would have been in situ
converted to the product. However, considering the conversions taken place in the process
(scheme-1), it is very difficult to get a product without formation of side product.
The present inventors have found that in the manufacturing process of getting 3-amino-
rifamycin-S from rifamysin-S, after the complete consumption of the starting material i.e.
rifamycin-S in the reaction with sodium azide, when the reaction mass was treated with
ammonium persulfate in situ without isolation of formed reaction products and unreacted
sodium azide, gives desired product viz. 3-amino-rifamycin-S without any formation of side
product thereby giving the product with high yield and quality.
3-Amino-rifamycin-S thus obtained gives excellent quality rifabutine when it is
subsequently transformed to rifabutin.
The process of the present invention for the manufacture of Rifabutin is as presented in
Scheme 2, and comprises following chemistry.
Rifamycin S was treated with 4 mole-equivalents of sodium azide in Formamide at 33-
35°C, the mixture was stirred for about six hours at 33-35°C. 0.5 Mole equivalent of
ammonium persulfate were added to the reaction mixture and it was stirred at 33-35°C.
The product 3-amino-rifamycin-S, was isolated by extraction in chloroform, and further the
product was crystallized from acetic acid and 2-methoxy ethanol.
Ammonia gas was passed through chilled 8-12°C solution of 3-amino rifamycin S in THF
for 22 to 24 hours. The product was isolated by distillation of the solvent followed by
crystallization from 2-methoxy ethanol.
4-imino-3-amino-rifamycin S was condensed with isobutyl-4-piperidone in presence of
ammonium acetate and zinc in THF as solvent. The product was isolated by extraction in
ether and further in dilute acetic acid.
Solution of rifabutin crude in acetone was added to water, pH of the mixture was adjusted
to 7.3 to 7.5, further, and the solid product was filtered to obtain pure rifabutin.
In an endeavor to know this process in-depth the process was monitored by HPLC.
Complete consumption of the starting material takes around six hours at this point the
chromatograph of the reaction mixture shows presence of product i.e. 3-amino-rifamycin-S
and a by-product i.e. rifamycin-SV as the major components. After addition of ammonium
persulfate to reaction mixture at this stage the by-product gets oxidized to rifamycin-S,
which further reacts with excess of sodium azide to form the product i.e. 3-amino-
rifamycin-S, hence, to facilitate conversion of this additional rifamycin-S to the product
excess of sodium azide needs to be added during the reaction. Table 2 shows HPLC
analysis results before and after addition of ammonium persulfate for three batches during
the reaction monitoring.

It is evident from the reaction monitoring data provided in table 2 that all the by-product
fonned gets converted to the product after addition of ammonium persulfate. Table 3
summarizes the quality and yield in the batches with and without addition of ammonium
persulfate.

These modifications effectively lead to the improvement in overall yield of rifabutine, the
summary of comparison is provided in table 4.

Merits of the present invention are:
1) Improvement in yield and quality of 3-amino-rifamycin-S.
2) Reduction in number of batches due to total consumption of starting material and
by-product.
3) Clean reaction and no need to recover the by-product, hence ease in operability
and reduction in batch time-cycle.
4) Increase in overall output of rifabutin.
NMR spectra were obtained at 200 and 400 MHz Bruker instruments, with CDCI3 as
solvent; chemical shifts (6) are given in ppm relative to tetramethylsilane (d = 0 ppm). IR
spectra were recorded by mixing a compound homogeneously with anhydrous potassium
bromide and compressing a mixture into a thin pellet to mount on sample cell of Perkin
Elmer Spectrum (Model: Spectrum 100) and absorption bands are given in cm-1.
The following examples illustrate the practice of the invention without being limiting in any
way.
Example 1: Preparation of 3-amino rifamycin S.
Dry Rifamycin-S (100 g) was added to clean and dry round bottom flask containing 300 ml
of formamide at 25-30 °C. The mixture was stirred and 37.5 g of sodium azide was added
to it under stirring in lots maintaining the temperature about 25 to 30oC. The reaction
mixture was then heated to 30-35°C and stirred for 6 hrs maintaining the same
temperature. Ammonium persulfate (6 g) was added to the reaction mass in one lot. For
further five hours 2 g of ammonium persulfate was added for each hour while continuing
stirring at 33 - 35°C. Completion of reaction was monitored on HPLC till concentration of
rifamycin-S was below 3% and rifamycin-SV (side product) below 1%. The reaction mass
was then cooled to 25-30 °C and 700 ml of chloroform and 1500 ml of water was added to
it. The reaction mixture was then stirred for 30 minutes at 25 - 30°C, and then allowed to
settle for 60 minutes. Aqueous layer was re-extracted with 300 ml chloroform. Chloroform
layers were combined together and washed with twice with 500 ml 10 % w/v sodium
chloride solution at 25-30 °C. The chloroform layer so obtained was concentrated under
vacuum at 35 to 40°C till about 100 to 150 ml remained in the flask. Acetic acid (40 ml)
was added to the concentrate mass at 35-40 °C and it was subjected for distillation under
vacuum at 40-45 °C to remove traces of Chloroform. 2-Methoxy ethanol (100 ml) was
charged to the reaction mass at 40-45 °C. The mixture was then slowly stirred for 30
minutes at 40-45 °C to allow the product to get crystallized. The reaction mass was then
gradually cooled to 0-2 °C and stirred maintaining the temperature to about 0-2 °C for 1 to
2hrs. The solid obtained was filtered at 0-2 °C and washed iniatially with 10 ml chilled 2-
methoxy ethanol followed by 100 ml of diisopropyl ether two times at 20 to 22°C. The
product was then dried under vacuum at temperature of 55 - 60°C till moisture content is
below 2%. Yield; 55 g.
1H NMR: d 8.43 (s), 5 7.28 (s), 5 6.06 - 6.45 (m), 5 4.83 - 5.11 (q), 5 3.02 - 3.76 (m), 5
1.62 - 2.37 (m), 5 1.05 - 1.14 (q) and <1.0 (s).
IR (cm-1): 3412, 3333, 2973, 2940, 2882, 2827, 2361, 2350, 1731, 1643, 1613, 1566,
1512, 1462, 1420, 1380, 1355, 1328, 1291, 1259, 1174, 1156, 1070, 1016, 973, 945, 917,
887, 836, 759, 719, 600, 535, 478, 460, 446, 434, 419 and 408.
HPLC Parameters: Column - C-8 (250 X 4.6, 5 µ), Mobile phase (pH 7.2) - Buffer (0.025
molar phosphate):acetonitrile: 10:8.1 (v/v). Flow rate 1.5 mL/minute (isocratic), detection
at 235 nm (UV), injection volume: 20 µL, Column temperature ~ 25 ° C and sample
temperature: <10 °C with the run time of 50 minutes. The retention time of 3-amino-
rifamycin-S is about 16.5 minutes and an average purity is (on HPLC) = 99 %.
Example 2: Preparation of 4-imino-3-amino rifamycin-S.
3-Amino rifamycin S (100 gm) was added to a clean and dry round bottom flask equipped
with gas purging assembly containing 500 ml of tetrahydrofuran at 25 - 30°C under
stirring. The mass was further stirred for 5 - 10 minutes maintaining the temperature at
about 25 - 30°C. The reaction mass was cooled to about 8-12°C. The reaction mixture
was then purged with ammonia gas continuously for 18 to 30 hours maintaining the
temperature at 10 - 15°C. Completion of reaction was monitored on HPLC till
concentration of 3-amino rifamycin S was below 2%. After completion of the reaction
excess of ammonia gas in the reaction mixture was degassed with nitrogen for 30 to 60
minutes at a temperature of about 8 - 30°C. The reaction mass was then concentrated
under vacuum at 30 - 35°C. Traces of tetrahydrofuran were removed by adding 150 ml of
2-methoxy ethanol to the reaction mass and subjecting the mass to distillation under
vacuum at a temperature of about 35 - 40°C. The reaction mass was then stirred at 35 -
40°C for about 30 minutes. The reaction mass was then cooled gradually to 0 - 5°C and
stirred at the same temperature for 2 - 4 hours to obtain solid product. The product was
filtered at 0 - 5°C, washed with 50 ml of chilled 2-methoxy ethanol. Then slurred and
filtered twice using 150 ml hot (45 - 50°C) water each time and finally spay washed with
50 ml of hot (45 - 50°C) water. The product was then dried under vacuum at temperature
of about 70 - 75°C till water content was below 0.5%. Yield: 90 g.
Example 3: Purification of 4-innino-3-amino rifamycin-S
4-imino-3-amino rifamycin-S (100 gm) was added to a clean and dry round bottom flask
containing 400 mi of methanol at 25 - 30°C under stirring. The reaction mass was then
heated to 55-60°C & maintaining the temperature for 15-30 minutes. The reaction mass
was then cooled to about 25-30°C & filtered pure 4-imino-3-amino rifamycin-S which was
then washed with 50 ml methanol. The product was then dried under vacuum at
temperature of about 70 - 75°C till Loss on drying was below 2.0% & water content was
below 2.0%. Yield: 95 g.
1H NMR: d 14.65 (s), d 14.22 (s), d 8.27 (s), d 7.26 (s), d 6.06 - 6.43 (m), d 4.81 - 5.14 (q),
d 2.88 - 3.85 (m), d 1.59 - 2.36 (m), d 0.64 - 1.02 (t) and d 0.04 - 0.05 (d).
FTIR (cm-1): 3381, 3198, 2969, 2938, 2828, 2322, 1692, 1605, 1555, 1413, 1372, 1347,
1262, 1174, 1064, 1024, 960, 890, 835, 814, 779, 766, 747, 732 and 519.
HPLC Parameters: Column - C-8 (250 X 4.6, 5 µ), Mobile phase (pH 7.2) - Buffer (0.025
molar phosphate):acetonitrile :: 10:8.1 (v/v). Flow rate 1.5 mL/minute (isocratic), detection
at 235 nm (UV), injection volume: 20 µL, Column temperature ~ 25 ° C and sample
temperature: <10 °C with the run time of 50 minutes. The retention time of 4-imino-3-amino
rifamycin-S is about 27 minutes and an average purity is (on HPLC) ~ 97 %.
Example 4: Preparation of Rifabutin
4-imino-3-amino rifamycin-S (100 g) was added to the clean and dry round bottom flask
containing 150 ml of tetrahydrofuran under stirring at a temperature of about 25 - 30°C,
the solid stuck to the charging funnel was flushed with 20 ml of tetrahydrofuran.
Ammonium acetate (2.4 g), solution of isobutyl-4-piperidone (21.8 g) in 20 ml of
tetrahydrofuran and 2.4 g of zinc powder were added to the reaction mixture and the
particles of the reagents stuck to the charging funnel were flushed with 60 ml of
tetrahydrofuran. The reaction mixture was further heated to 30 - 35°C and the temperature
was maintained with continued stirring for further 60 to 360 minutes, during which the
reaction was monitored for completion by HPLC till the concentration of starting material
was below 0.50%. After completion of the reaction the solid was filtered at 30 - 35°C and
washed with 200 ml of tetrahydrofuran. The combined filtrate was concentrated under
vacuum till thick mass was obtained. Diisopropyl ether (3500 ml) was added to the mass
slowly and the mass was stirred for 60 minutes at a temperature of about 25 - 30°C. The
solid obtained was filtered and washed with 500 ml of diisopropyl ether. The filtrates were
combined and washed four times with 1000 ml of aqueous disodium hydrogen
orthophosphate buffer with pH in the range of 7.5 - 7.6 at a temperature of about 25 to
30°C. Further the organic layer was extracted twice with 1500 ml of dilute (6%) aqueous
acetic acid solution at 25 - 30°C. The combined aqueous acetic acid extract was then
washed with 200 ml of diisopropyl ether at 25 - 30°C. The so obtained aqueous layer was
then degassed under vacuum at a temperature of about 43 - 45°C, then the vacuum was
filled with nitrogen gas at 35 - 45°C. The mass was cooled to 25 - 30°C and 300 ml of
acetone was added to it. pH of the mixture was adjusted to about 5.2 to 5.5 with the help
of 6% aqueous ammonia solution at a temperature of 25 - 30°C and the reaction mixture
was stirred for 15 - 30 minutes maintaining the same temperature. pH of the mixture was
then adjusted to 7.3 - 7.5 using 6% aqueous ammonia solution and stirred the mass for 60
minutes at 25 - 30°C. The solid obtained was filtered and washed with 800 ml water. The
product was then dried under vacuum at about 65 - 70°C till the water content was below
2.5%. Yield: 85 g.
Example 5: Purification of Rifabutin.
Rifabutin 100 g was added to a clean and dry round bottom flask containing 500 ml of
acetone at a temperature of about 25 - 30°C under stirring. The clear solution so obtained
was passed through 0.2 micron filter. The particle free clear solution so obtained was then
added slowly to a vessel flask containing 5000 ml of water at a temperature of about 25 -
30°C. pH of the reaction mixture was then adjusted to 7.3 - 7.5 with the help of 6%
aqueous ammonia solution at 25 - 30°C. The mixture was then stirred for further 30
minutes at a temperature of about 25 - 30°C. The product was then filtered at the same
temperature, washed with 400 ml water and dried under vacuum at temperature of about
65 - 70°C till the moisture content was below 2.5%. Yield: 99 g.
1H NMR: 5 14,74 (s), 5 9.03 (s), 5 8.27 (s), 5 7,27 (s), 5 6,04 - 6.31 (m), 6 5,09 -5.19 (q), 5
4.76 - 4.81 (d), 5 2.97 - 3.70 (m), 5 1.75 - 2.70 (m), 8 0.58 - 1.02 (m) and 6 0.62 - 0.98
(d).
IR (cm-1): 3381, 3198, 2969, 2938, 2829, 2657, 2322, 1692, 1605, 1555, 1413, 1372,
1347, 1262, 1174, 1084, 1064, 1024, 960, 890, 836, 814, 779, 766 and 507.
HPLC Parameters: Column - ODS 3V (250 X 4.6, 5 µ), Mobile phase A (pH 2.5): Buffer
(0.1 % triethylamine in water, v/v) and Acetonitrile in the ratio of 80:20 (v/v). Mobile phase
B (pH 2.5): Buffer (0.1 % triethylamine in water, v/v) and Acetonitrile in the ratio of 10:90
(v/v). Flow rate 1.0 mL/minute (Gradient), detection at 254 nm (UV), injection volume: 20
µL, Column temperature ~ 30 ° C and sample temperature: <10 °C with the run time of 80
minutes. The retention time of Rifabutin is about 29 minutes and an average purity is (on
HPLC)>99 %.
We claim:
1. A process for manufacture of rifabutin comprising steps of;
(i) conversion of rifamycin-S to 3-amino-rifamycin-S in presence of sodium
azide in an organic solvent such as formamide or methyl formamide at a
temperature of about 33-35oC, wherein the byproduct rifamycin-SV was
simultaneously converted to rifamycin-S by adding suitable oxidizing
agent such as ammonium persulfate and further reacted with sodium
azide to form 3-amino-rifamycin-S in situ, followed by isolation of the
product using acetic acid and 2-methoxy ethanol
(ii) conversion of 3-amino-rifamycin-S to 4-imino-3-amino-rifamycin-S by
treatment of 3-amino-rifamycin-S with ammonia gas in an organic
solvent such as tetrahydrofuran; Isolation of the product in 2-methoxy
ethanol; followed by optional purification using Methanol
(iii) condensation of 4-imino-3-amino-rifamycin-S with isobutyl-4-piperidone
in presence of ammonium acetate and zinc in an organic solvent such
as tetrahydrofuran; followed by isolation of the rifabutin by extraction in
ether and then in dilute acetic acid;
(iv) optional purification of rifabutin obtained in step (iii) using acetone and
water, wherein the product was isolated at pH of about 7.3 to 7.5.
2. A process for manufacture of 3-amino-rifamycin-S comprising steps of
(a) treatment of rifamycin-S with about 4 mole-equivalents of sodium
azide in Formamide at 33-35°C;
(b) stirring the mixture at 33-35°C till concentration of rifamycin-S in
the mixture goes below 3%;
(c) addition of about 0.5 Mole equivalent of ammonium persulfate to
the mixture and stirring at 33-35°C;
(d) extraction of the product in chloroform, followed by isolation using
acetic acid and 2-methoxy ethanol.

The invention provides a process for manufacture of rifabutin comprising steps of: (a)
Conversion of rifamycin-S to 3-amino-rifamycin-S in presence of sodium azide in an
organic solvent such as formamide or methyl formamide at a temperature of about 33-
35°C, wherein the byproduct rifamycin-SV was simultaneously converted to rifamycin-S by
adding suitable oxidizing agent, (b) Conversion of 3-amino-rifamycin-S to 4-imino-3-amino-
rifamycin-S by treatment of 3-amino-rifamycin-S with ammonia gas in an organic solvent
such as tetrahydrofuran. (c) Condensation of 4-imino-3-amino-rifamycin-S with isobutyl-4-
piperidone in presence of ammonium acetate and zinc in an organic solvent such as
tetrahydrofuran.