JPS5810390B2 - Rifamycins and their manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel famycins and a method for producing the same.

It is well known that rifampicin is a compound with very good antibiotic properties and is especially used as an antituberculous compound.

At present, only three methods are known for the production of rifampicin, US Pat.
It is described in the specifications of No. 42762 and No. 3963705.

According to U.S. Pat. No. 3,342,810, rifampicin is mildly oxidized to the Mannich base of rifamycin SV, and the resulting mixture is then mildly reduced to give 3-formylrifamycin SV, which is then combined with 1- It is produced by reacting with amino-4-methylpiperazine.

On the other hand, according to US Pat. No. 3,542,762, rifampicin is produced by reacting rifamycin S with formaldehyde and a primary aliphatic amine or a condensation product thereof in the presence of manganese dioxide, and then using the resulting reaction mixture. is prepared by reacting with about 2 equivalents of 1-amino-4-methylpiperazine.

Also, according to U.S. Pat. No. 3,963,705, rifampicin is produced by reacting rifamycin S with N-bis-alkoxymethyl-amine or N-bis-hydroxymethyl-amine, using known intermediates, i.e. The 1,3-oxazino(5,6-C)rifamycin contained in the known further compounds described was obtained and reacted with 1-amino-4-methylpiperazine in a clearly basic medium. Manufactured by

The process described in US Pat. No. 3,342,810 has the disadvantage of requiring four consecutive reactions.

That is, starting from the starting material rifamycin S, two intermediates have to be isolated, namely the Mannich base of rifamycin SV and the Mannich base of 3-formylrifamycin SV, which are involved in a number of reactions in industrial production. This means that a container must be used, which is associated with high production costs and low yields.

This has also been confirmed by the patentee of this U.S. patent, who in his subsequent U.S. Pat. “It is even simpler” is stated in the 53rd line of the first column.

The process of US Pat. No. 3,542,762 has the disadvantage that the reaction has to be carried out in two different stages.

That is, in the first step, rifamycin S is condensed to form a Mannich base, then oxidized to form a Schiff base, and then manganese dioxide is converted into Sawabetsu (even traces of manganese dioxide cause an explosion when they come into contact with 1-amino-4-methylpiperazine). After this, two further reactions are carried out as a second step, namely, reduction of the quinone form of Schick base, and then transimination of the hydroquinone Schiff base to obtain fanpicin. However, this method is also associated with high costs and low yields.

U.S. Pat. No. 3,342,810 and U.S. Pat. No. 3,542,762 both involve multiple oxidation reactions followed by reduction reactions with sifting of intermediates, thus requiring the use of separate reaction vessels. There is.

According to U.S. Pat. No. 3,963,705, 1,3-oxazino(5,6-C)rifamycin is formed and at the same time necessarily forms two molecules of alcohol or water which impair this reaction.

This is explained by the fact that it is preferable to use aprotic polar solvents.

In fact, when the same reaction is carried out in the presence of an aprotic polar solvent (Example 1), when n-propanol is used (
The yield was three times that of Example 15).

According to the above US patent specification, the intermediate compound 1,3-
Oxazino(5,6-C)rifamycins are isolated either in the solid state or by extraction with water-immiscible solvents and then reacted with 1-amino-4-methylpiperazine in a basic medium. This means that the entire process is in fact carried out in two separate stages.

Journal of Medical Chemistry, No. 11
Vol., p. 936 (1968), the use of basic media results in deacetylation and/or transacetylation reactions of rifampicin, rendering derivatives of rifampicin without useful antimicrobial activity. generate.

In fact, transacetylated derivatives of rifampicin have virtually no antibiotic activity in vitro, and deacetylated derivatives have antibiotic activity in vitro but are not absorbed.・See Engineering Chemotherapy, Vol. 16, p. 317 (1970)].

The use of basic media therefore results in impure rifampicin, requiring subsequent crystallization and purification, resulting in lower yields.

Moreover, apart from the above-mentioned invention, again US Patent No. 39637
As mentioned in the specification of No. 05, when the production method described in the examples was repeated, the yield was substantially lower than the reported yield, even though the same experimental conditions were used. I got it.

Thus, the present invention provides an intermediate for an industrially advantageous method for producing rifampicin in high yield and purity, and a method for producing the same.

The intermediate is novel and, in addition to its usefulness as an intermediate, has antimicrobial activity itself.

The object compound according to the invention is prepared with 3-tertiary aminoalkyl-1,3-oxazino(5,6-C)rifamycin as follows.

That is, rifamycin S and 1,3,5-trisubstituted hexahydro-1,3,5-triazine are mixed in an aprotic polar solvent, optionally in the presence of formaldehyde,
preferably in the presence of some acid without changing the pH of the medium,
It is obtained by controlling the reaction temperature and reaction time.

The 1,3,5-tri-substituted hexahydro-1,3,5 used
As the 5-triazine, 1,3,5-tri(2-morpholinoethyl) or 1,3,5-tri(1-ethyl-3-piperidyl)hexahydro-1,3,5-triazine is preferable.

According to this new method, rifamycin S and 1.3.
5-Tri-tertiary aminoalkyl hexahythrot 3,5
3-tertiary aminoalkyl 3-oxazino(
5.6-C) Rifamycins are produced.

These products may differ from those listed in US Pat. No. 3,963,705.

Note that the number of water molecules produced in this reaction is only half that of the method described in US Pat. No. 3,963,705.

However, when rifamycin S is reacted with an excess of 1,3,5-tritertiary aminoalkylhexahythroto 3,5-triazine without formaldehyde, the reaction is similar if appropriate reaction conditions are adopted. proceed to form the corresponding 3-tertiary aminoalkyl root 3-oxazino (5,6
-C) produces rifamycin, but in this case no water is produced.

This indicates that 1 of N-bis-hydroxymethyl-amine
This is surprising considering that it was disclosed in US Pat. No. 3,963,705 that one molecule of famycin S reacts and as a result two molecules of water are produced.

Also l,3,5-trialkyl-substituted hexahydro-1,
3,5-triazine (condensation product of an equivalent amount of formaldehyde and a primary alkylamine) and famycin S0
Unlike the prior patent, in which a Mannich base is necessarily formed upon reaction with an active hydrogen atom, such as the hydrogen atom in the 3-position, the new method instead forms a 3-tertiary aminoalkyl-1,3- It is surprising that oxazino(5,6-C)rifamycins are formed.

Tertiary aminoalkyl groups are selected in particular for their basicity and structure to give the best results, and are generally tertiary amines having an alkyl group with an open ring structure, preferably a cyclic structure, and having up to 3 carbon atoms. Representative groups are 2-morpholinoethyl group and 1-ethyl-3-piperidyl group.

In this reaction, the 1,3-oxazino ring is formed using formaldehyde, which may be paraformaldehyde or gaseous monomeric formaldehyde.

Surprisingly, when special hexahydrotriazines such as rifamycin S are reacted under special experimental conditions, the addition of formalin is completely unnecessary, regardless of the amount of N. We have discovered that it is possible.

This is because when azines are present in a moderate excess, they easily liberate formaldehyde and exhibit a similar effect.

Generally speaking, p of the 1,3,5-trialkyl-substituted hexahydro-1,3,5-triazine ζ reaction medium
It can react as described above regardless of the H value, but on the other hand, 1.3
- 5-triaminoalkyl compounds can react in this way only in somewhat acidic media.

The pH of the medium also plays an important role more generally.

Thus, the addition of a medium strength acid such as acetic acid or oxalic acid not only increases the reaction rate (because it suppresses the formation of certain by-products, especially rifamycin SV).

Otherwise, the famycin S will be found unchanged when the synthesis is finished, possibly compromising the purity and/or yield of the final product and requiring additional recrystallization. It will stop happening.

The above discovery also constitutes an important feature of the present invention, which requires the addition of oxalic acid when producing 3-aminoalkyl-substituted l,3-oxazino(5,6-C)rifamycins. is particularly important for obtaining good results.

In this reaction, the choice of solvent is very important.

The solvent used is preferably an aprotic polar solvent, such as dimethylformamide, dimethylacetamide or dimethylsulfoxide.

These solvents are inert towards the reaction components and
It has a very high solvent power, which is necessary to carry out the reaction at very high concentrations and can increase the reaction rate.

Furthermore, these solvents can selectively guide the ζ reaction in the desired direction, but on the other hand, when non-polar, almost non-polar, or protic solvents are used, many undesirable by-products are generated. There is.

Under the conditions mentioned above, at a temperature of 20 to 100 °C, preferably 40 °C
When reacted at ~80°C, the first step of the reaction generally consists of 3
Complete in 0 minutes to 4 hours.

The reaction mixture is then treated with water at moderately acidic pH, followed by direct filtration or extraction with a suitable solvent to isolate the 3-tertiary alkylamino 3oxazino (5,6-c ) Rifamycins are obtained.

3-Aminoalkyl-1.3 of the novel compound of this invention
-Oxazino(5,6-C) rifamycins have high antibiotic activity against many bacterial strains and have similar
- more potent than alkyl-substituted derivatives.

On the other hand, if rifampicin is the desired product, the reaction proceeds to the second step and the 3-alkyl or 3-aminoalkyl-1,3-oxazino(5,6-C)rifamycin is added to the 1-amino-4- Reaction with methylpiperazine yields rifampicin.

. One of the features of this new manufacturing method is U.S. Patent No. 3963
705, or by isolating the intermediate oxazino derivative, or by replacing the aprotic polar solvent used in the first step with an inert, water-immiscible organic solvent in the second step. It means there is no need.

Rather, ■-amino-4-methyl-piperazine can be added directly to the crude intermediate in the production medium without the need to replace the solvent.

In this regard, the use of the same aprotic polar solvent already used in the first stage of the production not only improves the process industrially and economically, but also increases the reaction rate. It becomes more layered.

If the pH value of the medium is suitably acidic, the reaction will proceed more favorably as far as purity is concerned, although this is not related to the reaction rate (the formation of deacetylated and/or transacetylated products of rifampicin can be avoided).

) was found to be quite surprising.

Therefore, if the first stage of the reaction is carried out in an acid solution (which is the preferred condition), the reaction with 1-amino-4-methylpiperazine does not require additional acid to reach a pH of 5-7. The reaction must continue as is or the required amount of acid must be added at that point.

In either case, it is possible to avoid adding a basic substance to create alkaline reaction conditions.

As mentioned above, a number of organic acids can be used, particularly acetic acid.

In special cases, the best results are obtained with oxalic acid, but in any case the best results are obtained at pH values of 5 to 7 O.

Under these experimental conditions, 20°C to 80°C, preferably 40°C to
At a temperature of 50 °C, the reaction to give rifampicin is 20
Completes easily and quickly in ~60 minutes.

The final product is then isolated in the usual manner, for example by treating the reaction mixture with water under mildly acidic conditions and then extracting with a suitable water-immiscible organic solvent and washing the organic extract thoroughly. There is.

It was then dried and evaporated to dryness to obtain the desired product in high purity or almost quantitative yield.

Some of the following examples, such as Example 4, use rifamycin S as a starting material to obtain sufficiently pure fanpicin in almost quantitative yields, and optionally It is described that a yield of 90% or more of the theoretical value was obtained by recrystallization.

This yield is the actual yield obtained by repeating the known method.
This is much higher than the rate.

The following examples illustrate the invention but do not limit it.

Example 1 0,72 y-oxalic acid, 0.489 paraformaldehyde and 2.28 f of 1,3,5-tri(2-morpholinoethyl)-hexahydro-1,3,5-triazine, 5.62 2'O containing glue famycin S
ml of dimethylformamide solution.

This mixture was heated to 75°C until the reaction was completed, i.e., the rifamycin S disappeared, and the mixture was heated to 75°C.
, 3-(2-morpholinoethyl)-1,3-oxazino(5,6-C) rifamycin (Merck silica gel 60F2., thin layer chromatography, Rf value 0.
．． The temperature was maintained at 75° C. for about 1 hour until 37 blue spots, 1 methanol (9:IV/V') were formed.

The reaction mixture was cooled to 50 DEG C. and a solution containing 3.42 of 1-amino-4-methylpiperazine in 5 rul of dimethylformamide, pH-valued with acetic acid, was added directly.

It was then heated with stirring for an additional hour, until the reaction was complete and the blue spot on the thin layer chromatograph disappeared.

After diluting with a 2% aqueous acetic acid solution and extracting the resulting suspension with chloroform, the organic solution was thoroughly washed with water and evaporated to dryness.

The residue (5,59) was then crystallized from acetone-ethyl acetate to yield 4 g of pure fanpicin.

This product was dissolved in phosphate buffer (pH 7,38) for 23 hours.
It exhibited maximum absorption of ultraviolet light with a wavelength of 7.255.344°475 nm.

In addition, the above 3-(2-morpholinoethyl)-1.3-
The physical properties of oxazino(5.6-C)rifamycin were as follows.

Solubility: Insoluble in water; soluble in chloroform, methylene chloride, methanol, ethanol, dimethyl sulfoxide, formamide and pyridine.

Ultraviolet absorption λmax (ethanol solution): 274°36
0 and 605 nm Melting point: 177-180°C (decomposition) Elemental analysis: Example 2 2.8 g Norifamycin S was dissolved in 10 ml dimethylformamide, then 0.249 of paraformaldehyde and 1.42 of 1.3. 5-tri(2-morpholinoethyl)-hexahydro-1,3,5-triazine oxalate was added.

The reaction mixture was kept at 75°C until the reaction was complete (approximately 4 hours).
1-amino-4-methylpiperazine, heated to 50° C. and then cooled to 50° C. and suitably acidified with acetic acid, was added directly according to the method described in Example 1 to yield the desired rifampicin as described above. , this was determined by microbiological quantification (micr).
The result of the obiological assay was 99.7%.

Example 3 14 g of rifamycin S was dissolved in 50 ml of dimethylformamide at 20°C.

Next, 1.8 g of oxalic acid and 5.5 g of 1.3.5-(2-
Morpholinoethyl)-hexahydro-1,3,5-triazine was added to the solution and a gas stream of pure formaldehyde and dry nitrogen was bubbled for 20 minutes.

The reaction mixture was then heated to 70°C to complete the reaction.

10 ml of a dimethylformamide solution containing 8.5 g of 1-amino-4-methylpiperazine and brought to a pH value of 5 with acetic acid were added to the resulting solution, then until the blue spot in the thin layer chromatogram disappeared. The mixture was stirred at 40°C.

To the resulting solution, 150 rnl of dichloromethane were added and the mixture was washed several times with water and dried over anhydrous sodium sulfate.

After filtering, evaporate to dryness, and crystallize the residue from acetone.
10.7 g of chromatographically pure fanpicin was obtained.

When this product was subjected to thin layer chromatography under normal experimental conditions, a reddish-orange spot was observed at a position with an Rf value of about 0.7.

Example 4 0.72S' oxalic acid and 3.42 1,3,5-tri(
2-morpholinoethyl)-hexahydro-1,3,5-
Triazine was added to 20 ml of dimethylformamide solution containing 5.6 g of rifamycin S.

The reaction mixture was then heated at 75° C. for about 3 hours and 1-amino-4-methylpiperazine in acidic medium was added in a manner similar to that described in Example 1.

The resulting chloroform solution is distilled to yield the desired rifampicin (5,8P), which can be easily crystallized from acetone.

The product was freely soluble in chloroform, soluble in methanol and ethyl acetate, slightly soluble in water and acetone, and its physicochemical properties were consistent with the reference sample of fanpicin.

Example 5 Rifamycin S is reacted with ■.3.5-tri(2-morpholinoethyl)-hexahydro-1.3.5-triazine and paraformaldehyde in the presence of oxalic acid, especially dimethyl as a solvent. The procedure was as described in Example 1, except that acetamide was used.

1-Amino-4-methylpiperazine was added and treated as described above to isolate substantially pure fanpicin in similar yields.

This product melted at 183-188°C (decomposition) even in a mixture with a reference sample of fanpicin.

Example 6 A solution of 10 ml of dimethylformamide containing 2.8 g of rifamycin S was mixed with 1.0.36 g of oxalic acid, 0.36 g of oxalic acid, and 0.36 g of oxalic acid.
24g rose formaldehyde and 1.125g 1
- Treated with 3,5-tri(1-ethyl-3-piperidyl)-hexahydro-1,3,5-triazine.

The reaction was then completed and 3-(1-ethyl-3-piperidyl)-1,3-oxazino(5,6-C)rifamycin [thin layer chromatography using Merck silica gel 60F254, Rf value 0 The reaction mixture was heated at 75° C. for about 1 hour until a blue spot of .40, developer chloroform:methanol 9:1 v/v] was formed.

After cooling to 50°C, 1,72 1-amino-4-methylpiperazine adjusted to pH 6 with acetic acid was added and the reaction was continued according to Example 1 to obtain 3.1 g of crude rifampicin, which was A high yield of pure rifampicin (2.5 g) was obtained by crystallization from a suitable solvent.

The obtained rifampicin was dissolved in phosphate buffer (pH.

7.38) in 237.255.334 and 475
It showed maximum absorption of ultraviolet light with a wavelength of nm.

In addition, the above 3-(1-ethyl-3-piperidyl)-1
- The physical properties of 3-oxazino (5, 6-C) rifamycin were as follows.

Solubility: Insoluble in water; soluble in methanol, ethanol, methylene chloride, chloroform, pyridine, formamide and dimethyl sulfoxide.

Ultraviolet absorption λmax (ethanol solution): 276°3
62 and 604 nm Example 7 0.9j? 1,3,5-tri(2-morpholinoethyl)hexahydro-1,3,5-triazine,
72 of rifamycin S was added to a solution of 25 ml of dimethylformamide.

The reaction mixture was then heated at 75° C. for 1 h until the rifamycin S was completely reacted ((thin layer chromatography)).

Ten times the volume of 2% aqueous acetic acid was added, the resulting suspension was extracted with chloroform, and the extract was washed repeatedly with water.

After drying with anhydrous sodium sulfate and distilling off the solvent, 7.
Obtaining a residue consisting of 52 3-(2-morpholinoethyl)-1,3-oxazino(5,6-C)rifamycins,
This was then purified with a suitable solvent mixture (a small amount of rifamycin SV was formed during the preparation).

). Alternatively, after diluting the reaction mixture with acidic water, the resulting suspension is directly filtered and thoroughly washed.
The collected material was then vacuum dried to isolate the product.

Purification can also be carried out using standard methods such as those described below.

Namely, it was subjected to column chromatography using silica gel (O,OS~0.2 mm, Merck & Co.), first eluting with chloroform alone to remove impurities, and then containing up to 5%
The pure product was recovered after elution with a suitable solvent mixture containing up to v/v methanol and evaporation of the solvent.

The obtained 3-(2-morpholinoethyl)-1,3-oxazino(5,6-C) rifamycin was prepared using Merck's silica gel 60F254 and chloroform:methanol (9:1 v/v) as a developing agent. It is a blue crystal with an Rf value of 0.37 in the thin layer chromatogram, and its characteristic absorption peak in the infrared absorption spectrum (nujol mul) is 3440.1720~1708.1650°1606.1550.1520.1258° 1230.
1160.1118.1063.970 and 900c
It was m-1.

This substance has high antibiotic activity against some bacterial strains and is more potent than the similar 3-alkyl-substituted 1,3-oxazino(5,6-C) rifamycins.

For example, the following minimum inhibitory concentration values are shown.

Claims (1)

[Claims] 13-tertiary aminoalkyl-1,3-oxazino (5
・6-C) Rifamycin (However, a tertiary aminoalkyl group means a cyclic alkyl group with up to 3 carbon atoms)
. 23-(2-morpholinoethyl)-1,3-oxazino(5,6-C)rifamycin or 3-(1-ethyl-3-piperidyl)-i,3-oxazino(5,6-
C) A compound according to claim 1 which is a rifamycin. 3 Rifamycin S and 1,3,5-tritertiary aminoalkyl hexahythroto 3,5-triazine (however,
A tertiary aminoalkyl group is a cyclic alkyl group having up to 3 carbon atoms) in an aprotic polar solvent at a temperature of about 20 to 100°C to form 3-tertiary aminoalkyl-1. - A method for producing rifamycins, which comprises obtaining 3-oxazino(5.6-C)rifamycins (tertiary aminoalkyl is the same as defined above). 4 Claims in which the reaction between rifamycin S and 1,3,5-tri-tertiary aminoalkylhexahythroto-3,5-triazine (tertiary aminoalkyl is the same as defined above) is carried out in the presence of formaldehyde. The method described in Section 3. 5. A patent claim in which the reaction between rifamycin S and 1,3,5-tri-tertiary aminoalkylhexahythroto-3,5-triazine (tertiary aminoalkyl is the same as defined above) is carried out in the presence of an organic acid. The method according to scope 3 or 4.