JPS5937077B2 - 6-deoxyneomycin, its production method, and antibacterial agents containing it as an active ingredient - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel aminoglycoside aminocyclitol derivatives, specifically 6-1, which have pharmacological activity.

] It is a mixture of epimers represented by The present invention also includes pharmaceutically acceptable acid addition salts of the above compounds.
The compounds of the invention of formula (1) exist in the form of a mixture of epimers.

The compound of the present invention has a chemical structure similar to neomycin.

For this reason, the compound of formula (1) is hereinafter referred to as 6-deoxyneomycin. 6-deoxyneomycin is a mixture of the following two types of epimers.

6-Deoxyneomycin B...When R represents a hydrogen atom and R2 represents CH2NH2.

6-Deoxyneomycin C...R1 relates to deoxyneomycin, its production method, and an antibacterial agent containing it as an active ingredient.

The novel aminoglycoside aminocyclitol derivative according to the present invention has the formula [wherein R1 and R2 are different and represent a hydrogen atom or CH2NH2,
ureus), Sarcina lutea (Sarcinal
utea), Klebsiella edwardsii (Kleb
siellaadwardsii), Shigella sonnei (ShigellasOnnei). Salmonella teifimurium
It has beneficial antibiotic effects that may be useful in the treatment of diseases caused by the proliferation of P. um).

Growth of pathogenic bacteria in a host can be inhibited by administering at least one compound of formula (1) of the present invention or a pharmacologically acceptable acid addition salt thereof. Over the years, certain strains of bacteria have developed resistance to most commercially available antibiotics, reducing the therapeutic value of these antibiotics by more than half, so that some strains of bacteria are now unable to tolerate conventional antibiotics. He has developed a tolerance to all substances. Therefore, the creation of new antibiotics that can destroy bacteria that have become resistant to currently used antibiotics is extremely necessary and has attracted widespread interest.

The above-mentioned inactivation of antibiotics is due to enzymes destroying or denaturing certain parts of the antibiotic molecule that are susceptible to attack.

Aminoglycoside aminocyclitol antibiotics, which are currently used in large quantities, are particularly susceptible to inactivation by resistant bacteria.

The above-mentioned resistance of certain bacteria that are normally sensitive to these aminoglycoside aminocyclitol antibiotics is
Due to phosphorolytic, acylating, or adenylating enzymes that attack the free hydroxyl and amino groups of antibiotics (Ann.Rev.BiOchem.242247
, l973) 0 When the hydroxyl group is removed, the antibiotic is less likely to be inactivated at that site.

Therefore, it is preferable to create antibiotics with fewer unnecessary hydroxyl and amino groups. US Patent No. 366
According to No. 9838, mutants of microorganisms known to produce antibiotics containing aminocyclitol subunits, such as neomycin, paromomycin, etc., can be generated.

This mutant strain is incapable of biosynthesizing aminocyclitol subunits due to a defect in the aminocyclitol biosynthetic pathway and therefore does not produce antibiotics. However, these aminocyclitol negative (AminO
cyclitOl-Negative) mutant bacteria are grown by adding appropriate aminocyclitol molecules to a normal medium (Nutri-Negative).
(entmedium), it has the ability to utilize the molecule and thus produce an antibiotic. or,
It is known that this method can also be used using pseudodisaccharides in place of the aminocyclitol subunit. However, from the above disclosure it cannot be predicted whether diaminocyclitol or pseudodisaccharide will be incorporated into antibiotics by the above biochemical conversion methods.

In fact, it has been extensively documented that this approach to producing new antibiotics is not effective. Therefore, the above method is not widely applicable for producing new semi-synthetic or fully synthetic antibiotics. This is illustrated, for example, in The Journal of Antibiotics.
JournaIO of AntibiOtics) VOl.
XXVI,. /F6lO, pages 551-561 (197
This is clear from 3). That is, this document describes a test to determine whether 29 different aminocyclitols were incorporated into antibiotics by the method of the above-mentioned US patent. Among the 29 aminocyclitols tested above, only streptamine and 2-epistreptamine were found in Streptomysis fradiae (
StreptOmycesfradiae), Streptomysis rimosus forma Palomomycinus (
StreptOmycesrimOsusfOrmap
arOmOmycinus) and Streptomysis kanamyceticus (StreptOmyceskanam)
yceticus') 0) 2-deoxystreptamine mononegative (D-) mutant bacteria.

Similarly, diaminocyclitol described in French Patent No. 2203808 is also used in the same S
．． Frasiae and S. When a mutant strain of Limosusforma paromomycinus was used, it was not incorporated into antibiotics.

Biochemistry 01
13, Ya 25, pp. 5073-5078 (1974), Neamine, ParOmamine
) or the pseudodisaccharade known as 6-canosaminide-2-deoxystreptamine. Fraziae, S. Limosus forma palomomycinus and S. Attempts to uptake using Kanamytheticus were unsuccessful.

Similarly, TheJOwnalOfAntibiOti
cstsu01. XXI,. 7i68, pp. 573-579 (1975), pseudodisaccharides, Gentamines, are produced by Micromonospora inyoensis (MicrOmOnOspOr
When the D1 mutant strain of OinyOensis was used, it was not incorporated into the antibiotic.

Also, according to the above method, S. Frasiae and S. rimosas
When the D1 mutant of P. paromomycinus was used, no antibiotic could be produced from 5-0-βD-ribopyranosyl-2,4-dideoxystreptamine. According to the prior art, already known antibiotics such as streptomycin, blueensomycin
It has been found that diaminocyclitol or pseudodisaccharide present in hygromycin, etc., is not incorporated into antibiotics when the above-mentioned mutants are used.

For example, the above-mentioned The Journal of Antibiotics VOI. According to XXVI, Seat 10, BIuensamine, Streptidine, Blueensidine, HyOsamine, Actin.
amine) et al. Fraziae, S. Limosus forma palomomycinus and S. When D1 mutant bacteria of each of Kanamyceticus are used, they are not incorporated into antibiotics. Similarly, neamine and paromamine, which are found in neomycin and paromomycin, are also found in S. D of Frasiae
When a single mutant bacterium is used, it is not incorporated into antibiotics (see "Biochemistry 1" above).

Furthermore, the diaminocyclitol or pseudodisaccharide that is incorporated into antibiotics when using the mutant bacteria according to the method of the above-mentioned US patent is different from that when using other mutant bacteria.
It has been found that it is not always incorporated into antibiotics. For example, the above-mentioned The Journal of Antibiotics Vol. According to XXVI, Fu 10, S.
The D- mutant of S. limosusforma paromomycinus does not produce antibiotics in the presence of 2-epistreptamine; D1 mutant of S. frasiae and S. frasiae. Kanamystheticus D1 mutant incorporates this subunit into antibiotics. Moreover, the above S. The D1 mutant of S. kanamyceticus does not produce antibiotics in the presence of streptamine, but the S. D1 mutant of S. frasiae and S. frasiae. The D1 mutant of Limosus forma paromomycinus incorporates the above diaminocyclitol into antibiotics.

The Journal of Antibiotics Vol.
XXVI, Seat 12, pp. 784-785 (1973), S. Ribosidifucus (S.r.
When using the D1 mutant of ibOsidificus,
Neamine is ribostamycin (RibOstamycin)
n), but for example S. When mutants of Frasiae are used, neamine is not incorporated into antibiotics.

Finally, according to the prior art, it is not possible in any case to predict the structure of the antibiotic that will be produced when diaminocyclitol or pseudodisaccharide is used with variants.

That is, the above-mentioned The Journal of Antibiotics Vol. According to XXVI, /F6l2, S. When the D-mutant strain of S. kanamyceticus is used in the presence of 1-N-methyl-deoxystreptamine or myoinosa-1,3-diamine, the product obtained is not the expected product but other compounds. It was hot. Similarly, Neamin and M. The D1 mutant of M. inyOensis gives SisOmicin, which also gives SisOmicin in the presence of paromamine, a different pseudodisaccharide (The Journal of Antibiotics). Biotics 01.XXVIII, Taki 8). Also, S. Contrary to expectations, the D1 mutant of Ribosidifucus does not give a compound similar to neomycin in the presence of neamine, but gives only ribostamycin (The Journal of Antibiotics Vol. XXVI, rot 12). It is clear that no accurate predictions can be made regarding the incorporation of specific diaminocyclitols or pseudodisaccharides into antibiotics using variants according to the method of the above-mentioned US patent.

It is not possible to predict whether the method will be useful or what the exact structure of the antibiotic to be produced will be. Therefore, the Journal of Organic Chemistry (J.Org.Chem.) VO
l. 4O, Taki 4, pp. 456-461 (1975) states that 2,4-dideoxystreptamine can be incorporated into neomycins, paromomycins and ribostamycin by bioconversion methods. However, it is considered to be extremely vague and unclear, and lacks a valid and reliable basis. 1. However, according to the present invention, quite unexpectedly, S. It has been found that 2,4-dideoxystreptamine can be incorporated into a new antibiotic by the D1 mutant of S. frasiae. This discovery was made by S.
This is quite surprising considering that Kanamyceticus D-mutant does not produce antibiotics in the presence of 2,4-dideoxystreptamine. The results of the tests performed on 2,4-dideoxystreptamine all indicate that the present invention is truly unexpected in view of the current state of the art.

Two compounds of the present invention include soluble carbohydrates, assimilable nitrogen sources and essential inorganic salts, and a) 1D-(1,3:
5/2)-1,5-diamino-2,3-cyclohexanediol, i.e. 2,4-dideoxystreptamine or an acid addition salt thereof, e.g. dihydrochloride, in a suitable medium containing
It can be produced by culturing a mutant strain of Streptomyces that is negative for deoxystreptamine.

In the production method of the present invention, the antibiotic is obtained in the form of a free base, whether the diaminocyclitol of general formula () is in the form of a free base or a salt. If it is desired to use the resulting antibiotic in the form of a salt, the antibiotic may be reacted with a suitable organic or inorganic acid, such as sulfuric acid, to form its pharmacologically acceptable acid addition salt. The microorganism used in the present invention is D-streft and is disclosed in US Pat. No. 3,669,838.

D-Streptomysis Fradiae ATCC2l4O
The 6-deoxyneomycin of the present invention can be produced by using 1. According to known methods, the microorganisms have a suitable pH value, such as glucose, casein hydrolyzate,
(NH4)2HP04, MgS04・7H20, FeS
It is cultured in a normal medium containing 04.7H20, cus04.5H20, cac03, etc. This culture is then prepared containing a diaminocyclitol of formula () in the form of the free base or its acid addition salt and, for example, in soybean porridge (SOyabea
nmeal), yeast extract, NaCbcacO3, glucose, etc., and added to a separate growth medium with an appropriate pH value. The culture is shaken, well aerated, and cultured at 28-30° C. for at least 5 days in order to produce the desired 6-deoxyneomycin in maximum yield. 6-
Deoxyneomycin B and 6-deoxyneomycin C are obtained from 6-deoxyneomycin and separated by known methods such as paper talomatography.

Diaminocyclitol of formula () is a known compound,
It is disclosed in British Patent No. 1445675.

The process of this British patent describes the preparation of 1D-(1,3,5/2)-
1,5-Diazido-2,3-cyclohexane diol is hydrogenated in the presence of a catalyst such as Raney nickel. As mentioned above, 6-deoxyneomycin of the present invention has a useful antibiotic effect.

The antibacterial effects against various test bacteria are as follows.

a) Antibacterial effect of 6-deoxyneomycin To test the antibacterial effect, add a weighed amount of the compound to be tested to sterile water,
This was done by adding this to ordinary agar.

Agar plates prepared with increasing concentrations of the compound to be tested are then placed in Petri dishes, grown in advance in a suitable medium, and mixed with a 1:1 mixture of growth medium and glycerol.
Several different bacteria stored at -20 C were mechanically added to the mixture using a multi-inoculator.

The Petri dishes were incubated at 37° C. for 14 hours and then observed for growth. The concentration of antibiotic that immediately inhibits bacterial growth is recorded and is referred to as the "minimum inhibitory concentration," or M. I. C. shall be.

The results obtained in this test are shown in Table 1 in comparison with neomycin sulfate.

The numbers in the table correspond to the amount of free base. According to these results, even if the hydroxyl group at the 6-position of the aminocyclitol moiety of neomycin is removed (corresponding to 6-deoxyneomycin), the antibacterial activity of neomycin does not substantially change as a whole.

It is noteworthy that the antibacterial activity of 6-deoxyneomycin against E. coli W3llO is superior to that of neomycin. Antibacterial activity of 6-deoxyneomycin B and 6-deoxyneomycin C D. S. The antibacterial activity of 6-deoxyneomycin B and 6-deoxyneomycin C was tested using T agar in the same manner as above.

M. neomycin and 6-deoxyneomycin. I. C. are shown in Table 2. The numbers in the table correspond to the amount of free base. These results show that 6-deoxyneomycin B is somewhat inferior to neomycin B, but 6-deoxyneomycin C is superior to neomycin C. When the compounds of the present invention are used therapeutically, they are usually administered in the form of antimicrobial agents, which are pharmaceutical or veterinary compositions formed into dosage units suitable for the intended mode of administration.

The composition contains at least one compound of the present invention as an active ingredient together with a pharmacological carrier or excipient. For oral administration, the composition is in the form of, for example, coated or uncoated tablets, hard or soft gelatin capsules, suspensions, syrups and the like. Alternatively, suppositories for rectal or vaginal administration;
Solutions or suspensions may be used for parenteral administration, and creams or ointments for topical administration. The method for producing the compound of the present invention will be described in detail with reference to Examples below.

Example 1 Preparation of 6-deoxyneomycin and its sulfate a) 6-deoxyneomycin in free base form in a flask;
D-Streptomysis Fradiae ATCC2l4
Ol is cultured for 48 hours in the following medium adjusted to pH 7.2 to 7.3 in advance.

The cultures are shaken and kept at 28°C for the indicated periods.

A 10% inoculum of the culture was then pre-adjusted to pH 7.2.
Add to the following growth medium adjusted to ~7.3. The culture is grown at 28.degree. C. to 30.degree. C. in earthen Erlenmeyer flasks on a rotary shaker with sufficient aeration.

Production of 6-deoxyneomycin begins after 2 days and reaches maximum production after 5 days.

Control cultures without 2,4-dideoxystreptamiJ. tuna salt did not produce any 6-deoxyneomycin.

The culture was then centrifuged, and the soil clear liquid was mixed with a moderately porous, weakly acidic carboxyl (polymethacryno) type cation exchange resin (AInberlit).
e) Pass through a column containing IRC-50 (trade name) in the form of ammonium. This operation is repeated twice to extract 6-deoxyneomycin and unchanged 2,4-dideoxystreptami Z dihydrochloride. Wash the column with water,
6-deoxyneomycin is eluted with 2N aqueous ammonium hydroxide. The eluate is concentrated on a rotary evaporator under vacuum and applied to a Sephadex G-10 column. Elution with 0.1M ammonium hydroxide solution yields 6-deoxyneomycin as the first fraction and 2,4-dideoxystreptamine dihydrochloride as the second fraction.

By this method, 83 units (1 unit = 1 μg/
An amount of 6-deoxyneomycin is obtained in the form of the free base with an activity of ml neomycin). This is about 2.5% of the 2,4-dideoxystreptamiJ tuna_salt used at the beginning.
This corresponds to 2% incorporated into the 6-deoxyneomycin obtained as described above. This was the maximum activity that could be obtained, and further culturing did not increase 6-deoxyneomycin. Another method is to use crude 6
- deoxyneomycin in a 4:1 solution of methanol/ammonium hydroxide by Whatman (What)
Man) Purify by paper chromatography on 7f63MM paper soil.

Spots of 6-deoxyneomycin are developed using an aqueous solution of 0.25% sodium hypochlorite, absolute ethanol, 1% soluble starch, and 1% potassium iodide, with air drying between each run.

As a result, the Rf of 6-deoxyneomycin was 0.30. For comparison, R of neomycin, 2-deoxystreptamine and 2,4-dideoxystreptamine
The values of f in the same solvent system are as follows.

The results of elemental analysis of the free base of 6-deoxyneomycin obtained above are shown.

b) 6-deoxyneomycin sulfate 6-deoxyneomycin obtained as above is reacted with an equal amount of dilute sulfuric acid to form its sulfate.

[α]5Mc)6- of 6-deoxyneomycin sulfate
Structure of deoxyneomycin The structure of the antibiotic of formula (1) obtained as above in the free base form is as follows:
The determination was made by comparing it with neomycin, an antibiotic obtained by culturing 2-deoxystreptamine in an appropriate medium.

The compound assumed to be 6-deoxyneomycin will be referred to as compound X hereinafter.

Methanolysis of both neomycin and compound X using a 10% methanol solution of hydrochloric acid at reflux for 2 hours yields two major products.

One of these, methylneobisamide, was obtained from both compounds. The other product is hereinafter referred to as compound Z. This differs between the two antibiotics. According to a chromatographic assay using a 4:1 methanol/ammonium hydroxide solution as the eluent, Compound Z obtained from neomycin migrated less than Compound Z obtained from Compound X. Hateful. The structure of each compound Z was determined by mass spectrometry of its part-trimethylsilyl derivative. Although no molecular ion was obtained, the spectrum of Compound Z obtained from neomycin and the spectrum of Compound Z obtained from Compound X are very similar. The difference is that the two peaks (m/E343 and 460) in the former spectrum appeared 88 mass units lower in the latter than the two peaks (m/E2).
55 and 372). This corresponds to the absence of 0Si(CH3)3 and substitution with hydrogen. Therefore, the structure of compound Z from neomycin is neamine, compound
Compound Z from corresponds to 6-deoxyneamine. Acetylation of both compounds Z in methanol and hydrolysis using 3N hydrochloric acid at reflux for 10 hours yields two main products.

By paper chromatography, one of these appears to be the same as the one obtained from compound Z from neomycin and the one obtained from compound Z from compound X;
It can be estimated that it is 2,6-diamino-2,6-dideoxy-D-glucose. The other two compounds were identified by chromatography as 2-deoxystreptamine from neamine and 2,4-dideoxystreptamine from deoxyneamine. From these results, it is concluded that compound X corresponds to 6-deoxyneomycin.

Reference Example 1 Separation of 6-deoxyneomycin into 6-deoxyneomycin B and 6-deoxyneomycin C 6-deoxyneomycin obtained according to the method described in Example 1 was purified by tert-butanol/butanone/6.5N 6-deoxyneomycin B and 6-deoxyneomycin C were separated by paper chromatography using a 3:16:6:1 ammonium hydroxide solution/methanol solution as a solvent.

6-deoxyneomycin B and 6-deoxyneomycin C were confirmed on paper by the same method as above.

Using a 4:1 solution of methanol/ammonium hydroxide on 3MM Watman chromatography paper, 6-
Deoxyneomycin B and C(7) Rf values were obtained in comparison to the corresponding epimers of neomycin.

The results are shown below. 6-deoxyneomycin BO
．． 29 6-deoxyneomycin 0.21 Neomycin B0.25 Neomycin C0.165

Claims (1)

[Claims] 1 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) [R_ in the formula
1 and R_2 are hydrogen atoms or CH_2NH_2
, and R indicates ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ . ] and its pharmacologically acceptable acid addition salts. 2. The acid addition salt according to claim 1, wherein the pharmacologically acceptable acid addition salt is a sulfate. 3 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) [In the formula, R_1 and R_2 are different water carbon atoms or CH_2N
Indicates H_2, and R indicates ▲There are mathematical formulas, chemical formulas, tables, etc.▼. A method for producing 6-deoxyneomycin and its pharmacologically acceptable acid addition salts, which are mixtures of epimers represented by Streptomyces
fradiae) ATCC 21401 is cultured in an appropriate medium containing soluble carbohydrates, assimilable nitrogen sources, essential inorganic salts, and 2,4-dideoxystreptamine or its acid addition salt represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼ whereby a compound of formula (I) is obtained in the form of a free base, which is optionally reacted with a suitable organic or inorganic acid to obtain a pharmacologically acceptable acid addition salt thereof. Manufacturing method. 4 D^-Streptomysis frasiae ATCC21
4. The method according to claim 3, wherein 6-deoxyneomycin is obtained in the form of a free base by culturing 401 in the presence of 2,4-dideoxystreptamine dihydrochloride group. 5. The manufacturing method according to claim 3, wherein the inorganic acid is sulfuric acid. 6 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) [R_ in the formula
1 and R_2 are hydrogen atoms or CH_2NH_2
, and R indicates ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ . An antibacterial agent for humans or animals containing as an active ingredient at least one of 6-deoxyneomycin, which is a mixture of epimers represented by the following formula, and a pharmacologically acceptable acid addition salt thereof.