NO120416B - - Google Patents

Description

Process for preparing a new antibiotic agent from spiramycins.

In patent no. 89771, the preparation is described

of an antibiotic agent given the name

spiramycin. It is obtained by cultivating the strepto-mycete strain S-3486 (NRRL No. 2420). As

indicated in the said patent, this product consists of

three components with properties that are close

up to each other. For the sake of convenience, these components are called spiramycins I, II and III

and has distinguishing characteristics as indicated in

the following.

Spiramycin I is a basic substance that is soluble in chlorinated solvents, alcohols,

hexane, aromatic hydrocarbons, ketones,

ethyl acetate and amyl acetate. It is able to

form salts with acids. Spiramycin I contains

the elements carbon, hydrogen, oxygen and

nitrogen essentially in the following weight ratio:

In ethanol solution, spiramycin I has a maximum absorption at 232 m/ in. It has a molecular weight, determined by an ebullioscopic method, of about 800, a neutral equivalent of

463, a dissociation constant pKb of 7.7 and et

melting point determined on Maquenne block at,

134—137° C. Its optical rotatability

~T20

a D in methanol (c—1%) = -57° C. In the solid state, the substance shows characteristic absorption i

the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3470,

2970, 2940, 1735, 1455, 1378, 1317, 1275, 1237,

1160, 1112, 1090, 1052, 1015, 993, 905, 865, 840,

810, 782.

Spiramycin II is a basic substance which is soluble in chlorinated solvents, alcohols, hexane, aromatic hydrocarbons, ketones, ethyl acetate and amyl acetate. It is capable of forming salts with acids. Spiramycin II contains the elements carbon, hydrogen, oxygen and nitrogen in essentially the following weight ratio.

In ethanol solution, the substance has a maximum absorption at 232 m/f. Its molecular weight determined by the ebullioscopic method is about 800, its neutralization equivalent is 464, its dissociation constant pKb is 7.6. Spiramycin II's melting point determined on the Maquenne block is 130—133° C, its optical rotation is « -pi methanol (C — 1%) = -86°, V ~T20 in ethanol (c — 1%) = -80° in chloroform ( c—1%) = -55°. in the solid state, the substance shows characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3460, 2970, 2940, 1740, 1457, 1372, 1300, 1275, 1232, 1160, 1122, 1085, 1052, 1015, 993, 940, 905, 860, 840, 810, 782, 685.

Spiramycin III is a basic substance which is soluble in chlorinated solvents, alcohols, hexane, aromatic hydrocarbons, ketones, ethyl acetate and amyl acetate. It is capable of forming salts with acids. Spiramycin III contains the elements carbon, hydrogen, oxygen and nitrogen in essentially the following weight ratio:

In ethanol solution, the substance has a maximum absorption at 232 m/f. Its molecular weight determined by the ebullioscopic method is about 900, its neutralization equivalent is 473, its dissociation constant pKb is 7.6 and its melting point determined on the Maguenne block is 128—131° C. The substance's optical rotation r ~f2o a j-j is methanol (c—1 %) = -83°, in ethanol (c—1%) = -70° and in chloroform (c—1%) = -50°. In the solid state, spiramycin III shows characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3470, 2970, 2940, 1740, 1460, 1380, 1370, 1300, 1280, 1240, 1185, 1162, 1122, 1085, 1052, 1015, 995, 906, 865, 842, 810, 782, 695, 685.

In the present specification and claims, "spiramycin" denotes the mixture of spiramycins I, II and III obtained by culturing the streptomyces strain S-3486 (NRRL No. 2420).

It has now been found that the compounds obtained by controlled hydration of any of the aforementioned products have antibacterial properties that are stronger than those the product had before hydration. The characteristic main feature of the method according to the present invention is that spiramycin or one of its constituents is hydrogenated as described above, so that no more than four hydrogen atoms are introduced into the spiramycin molecule (with a molecular weight of 800-900).

It is an important feature of the present invention that the hydrogenation must be carried out under conditions which allow a maximum of four hydrogen atoms to be introduced into the spiramycin molecule. Products that are more hydrated turn out to be of little or no value as antibiotics.

According to the invention, the spiramycin complex or any mixture of spiramycin's components in the form of the base or a salt thereof is preferably subjected to catalyzed hydrogenation. The usual catalysts for such hydrogenation rings can be used, e.g. Raney nickel, but it is preferable to use a noble metal, e.g. palladium or platinum as a catalyst. When you use a palladium catalyst (on a carrier consisting of e.g. aluminum oxide, coal or barium sulphates) you find that the absorption of hydrogen stops automatically after four hydrogen atoms have been absorbed per molecule spiramycin or spirarnycin component. When treating the component spiramycin I, there is a very sharp reduction in the absorption rate for hydrogen when two atoms of hydrogen are absorbed per molecule spiramycin I. This greatly facilitates the preparation of dihydrospiramycin I as the reaction can conveniently be stopped at this stage.

The catalyzed hydration is conveniently carried out with the spiramycin complex or the spiramycin components in solution. Various solvents can be used which do not themselves absorb hydrogen under the hydrogenation conditions used, e.g. lower alcohols such as methanol, ethanol and isopropyl alcohol, ethers such as diethyl ether and dioxane and esters such as ethyl acetate.

The catalyzed hydrogenation is preferably carried out at normal pressure and room temperature, as under these conditions it can easily be controlled. However, other temperatures and pressures can also be used, but this does not achieve any significant advantages.

In the following, some embodiments of the invention are described as examples. In the examples, the absorption of the products is indicated in the infrared range of the spectrum. This is shown graphically in the attached drawings, as the wavelengths are plotted in microns (lower scale) and in reciprocal centimeters (upper scale) along the abscissas, while percent transmission is plotted on the ordinates.

Example I.

A solution of spiramycin (5 g) in ethanol (100 cm<3>) containing in suspension a catalyst (1 g) consisting of 5% palladium precipitated on aluminum oxide is stirred in a hydrogen atmosphere. After about 30 minutes, the hydration ceases and 275 cm<3> of hydrogen has then been absorbed. The catalyst is filtered off, and the solution is evaporated in vacuo at room temperature. Tetrahydrospiramycin (4.8 g) is obtained as a white powder. This is insoluble in water, and very soluble in the solid organic solvents except petroleum ether.

The product has the empirical formula

C46-48<H>83-87°ii5-ii7N2 °S its analysis is as follows:

It melted at 120-125° C and its optical rotation was « ^ = -71.5° (C 1%, r ~Ti7 methanol). The absorption of the product in the ultraviolet region of the spectrum instead of the prominent maximum at 232 m/f characteristic of the starting material spiramycin, namely a weak maximum at 820 m/f E^^<=> 14 (ethanol). Its absorption curve in the infrared region of the spectrum is shown by fig. 1 in the attached drawing and has characteristic absorptions at the following frequencies expressed in reciprocal centimeters: 3470, 2930, 2800, 1730, 1628, 1462, 1453, 1410,

1372, 1318, 1289, 1270, 1232, 1182, 1160, 1120, 1075, 1050, 1015, 991, 971, 956, 903, 841, 808, 783.

Example II.

Spiramycin sulfate (5 g) in water (100 cm<3>) is hydrated using palladium on alumina (1 g) as catalyst. When the hydration ceases, the catalyst is filtered off and tetrahydrospiramycin sulfate is isolated by evaporating the water in a vacuum at room temperature.

The product melts at 175° C, =

-58.7° (C 1%), methanol.

Example III.

Spiramycin I (1 g) in ethanol (20 cm<3>) is hydrated in the presence of a palladium catalyst (0.2 g) and the hydration is stopped when 27 cm<3> of hydrogen has been absorbed, which coincides with a very sudden decrease in the rate of absorption. This gives dihydrospiramycin I (0.9 g).

_ ~~h 27

sm.p. 128—132° C. « D = -83° (c 1

methanol.

The compound corresponds to the empirical formula <C>46-4s<H>8i-85°i6-i7N2 °S has the following analysis:

The curve for this product's absorption in the infrared region of the spectrum is shown in fig. 2 in the attached drawing and has characteristic absorptions at the following frequencies expressed in reciprocal centimeters: 3470, 2930, 2800, 1720, 1660, 1462, 1453, 1410, 1378, 1320, 1273, 1240, 1184, 1160,1122,1075,1060 ,1050,1015, 993, 965, 903, 868, 841, 808, 783.

Example IV.

One proceeds as indicated in example III, but continues the hydrogenation until hydrogen is no longer absorbed, which takes place when 52 cm<3> has been absorbed. One obtains tetrahydrospiramy-V "T27

one I (0.8 g), with sm. p. 132-135°C « D =

-79° (c = 1%, methanol).

The compound corresponds to the empirical formula C48.48H 83.87016-17N2 and has the following analysis:

The compound's absorption curve in spectrum I

3

infrared range is shown in fig. 3 on the attached drawings, and has characteristic absorptions at the following frequencies expressed in reciprocal centimeters: 3470, 2930, 2800, 1730, 1720, 1650, 1462, 1453, 1410, 1377, 1318, 1272, 1242, 1185, 1160, 1120, 1080, 1052, 1015, 991, 903, 841, 809, 783.

Example V.

Spiramycin II (1 g) in ethanol (20 cm<3>) is hydrated in the presence of 5% palladium on aluminum oxide (0.2 g) until hydration ceases, which occurs when 52 cm<3> of hydrogen has been absorbed. This gives tetrahydrospiramycin II (0.8 g)

~ ~t27 with sm.p. 125—128° C. « D = -63° (c =

1%, methanol).

The compound corresponds to the empirical formula C46-48H 83 - 87015 16N2 and has the following analysis:

The absorption curve of the compound in the infrared region of the spectrum is shown in fig. 4 on the attached drawing and has characteristic absorptions at the following frequencies expressed in reciprocal centimeters: 3470, 2930, 2800, 1730, 1720, 1462, 1455, 1410, 1370, 1330, 1318, 1289, 1273, 1238, 1182, 1260,11 ,1080,1065,1050, 1015, 991, 965, 956, 903, 865, 841, 808, 783.

Example. WE.

One proceeds as indicated in example IV using spiramycin III as starting material and then obtains tetrahydrospiramycin III with sm. p 135—140° C. ^ = -71° (c = 1%,

methanol).

The connection corresponds to the empirical formula

C46-48H83 -87°i5-i6N2 and has the following analysis:

The absorption curve of the compound in the infrared region of the spectrum is shown in fig. 5 on the attached drawing, and has characteristic absorptions at the following frequencies expressed in reciprocal centimeters: 3470, 2930, 2800, 1740, 1730, 1640, 1462, 1453, 1410, 1375, 1315, 1290, 1270, 1230, 1182, 1160, 1120, 1075, 1050, 1018, 993, 965, 903, 860, 841, 808, 783.

Example VII.

A mixture of spiramycin II and III (10 g)

(containing about 50% of each of the

parts), in ethanol (35 cm<3>) is hydrated in the presence of 5% palladium catalyst (2 g). The hydrogenation ceases when 540 cm<3> of hydrogen has been absorbed. The catalyst is filtered off and the alcohol is removed in vacuo. A mixture of tetrahydrospiramycins II and III is obtained, which can be recrystallized from a mixture of cyclohexane and petroleum ether and has 110-115 °C a D =

1%, methanol).

The mixed product corresponds to the empirical formula C46. 48H 83 . 87016.16N2.

Example VIII.

"Adams platinum" is prepared from the oxide (0.2 g) in alcohol (20 cm<3>). Spiramycin (1 g) is added there and the mixture is hydrated until 50 cm<3> of hydrogen has been absorbed. This corresponds to about four atoms of hydrogen per molecule spiramycin. The hydration takes about 25 minutes. This gives tetra-

hydrospiramycin (1 g) with sm. p. 120-125° C, i.e. the same product obtained according to example I.

Tetrahydrospiramycins I, II and III also show, like the tetrahydrospiramycin complex, an absorption maximum in the ultraviolet region of the spectrum at 820 m/h, E = 14 (ethanol).

As indicated above, the hydrated spiramycins produced by the method according to the invention have antibacterial properties that exceed these properties of the spiramycin from which the compounds were prepared. The antibacterial spectrum of tetraspiramycin (containing all three components) has been compared in vitro with this spectrum of the spiramycin mixture used as starting material and the results, obtained in liquid medium, are given in the following Table I:

in

The compounds have also been compared in vitro with respect to their activity against staphylococcus aureus (strain FD A 209 P).

The chosen unit of activity is the smallest amount of the purified product which, dissolved in 1 cm<3> of a suitable culture medium, prevents the development of staphyloccus aureus. The results obtained are shown in the following table:

The toxicity of the tetrahydrospiramicih mixture in comparison with that of the spiramycin mixture has been determined in mice by giving the substances subcutaneously, whereby the antibiotic agent was used in the form of the sulphate. Mortality was noted over five days for groups of 10 mice. The results are compiled in the following table III.

DL60 here denotes the calculated dose as

causing a 50% mortality rate in the mice.

The compounds are further compared for their activity against streptococcal and pneumococcal peritonitis in mice.

The products were then used in the form of sulphates and were given (to groups of 10 mice) per mouth once a day for three days, beginning immediately after the mice had been inoculated into the peritoneum with the aforementioned bacteria.

Mortality was noted within 8 days.

The results are compiled in the following table IV.

DC50 in this table denotes the calculated value for the amount of the compounds that would cure 50% of the mice.

Claims (3)

1. Method for the production of antibiotic agents, characterized in that one sub- throws spiramycin or a component of the same, here designated spiramycin I, II or III, or mixtures of these components, or salts of any of these, catalyzed hydrogenation under such conditions that no more than 4 atoms of hydrogen are introduced into the spiramycin molecule. 2. Method according to claim 1, characterized in that the hydrogenation is carried out in the presence of a platinum or palladium catalyst, preferably show palladium precipitated on a support consisting of alumina, charcoal or barium sulphate. 3. Method according to claim 1 or 2, characterized in that spiramycin II, spiramycin III, or a mixture of these is used as starting material, which starting materials are essentially free of spiramycin I.