DE1142170B - Process for the preparation of a new compound of phenoxymethylpenicillin - Google Patents

Description

GERMAN

PATENT OFFICE

S67727IVd / 12p

REGISTRATION DATE: MARCH 24, 1960

NOTICE DEK REGISTRATION AND ISSUE OF THE EDITORIAL:

JANUARY 10, 1963

The invention relates to a process for the production of a new chemical compound of phenoxymethylpenicillin with remarkable antibiotic effects.

The compound obtainable according to the invention is the phenoxymethyl penicillinate of the tetracycline substituted on the carboxamide nitrogen atom or in the 9 or 7 position by a 4 '- (ß - hydroxyethyl) - diethylenediaminomethyl - radical, for which an acceptable formula can be given as follows:

et

HC

O C ^ ^ CfT
CH

CH ₃ CH ₃

NH-CO —CH ₂ -O method of manufacture

a new connection

of phenoxymethylpenicillin

Applicant:

Societe d'ßtudes,

de Recherches et d'Applications

et Medicales, E.R.A., S.M.E., Paris

Representative: Dipl.-Chem. Dr. I. Maas, patent attorney, Munich 23, Ungererstr. 25th

Claimed Priority: Great Britain March 31, 1959 (No. 10 887)

Boris Gradnik and Andrea Pedrazzoli,

Milan (Italy) have been named as inventors

H ₃ C

CONH-CH ₂

OH O OH O

"N"

HOH ₂ C-CH ₂

It should be noted that it has not been possible to prove this overall formula with certainty, in particular what the substitution site on the tetracycline ring system is concerned, which is why the above formula with a bond of the substituent for illustrative purposes only to the carboxamide nitrogen atom.

The main physical, chemical and biological properties of the new compound are given below.

This new compound has characteristic properties insofar as it is on the one hand in water is considerably soluble and, on the other hand, practically none

Shows sensitivity to penicillinase. This ensures the effectiveness of the penicillin part of this compound, which belongs to both the tetracycline series and the penicillin series, against staphylococci, which were previously considered to be resistant to the known biosynthetic penicillins, in particular to Staphylococcus aureus, the producer of penicillinase.
It is known that the tetracyclines and the penicillins each have their own particular advantages and disadvantages, the latter considerably limiting their use. It is known that tetracycline and most of its derivatives are practically insoluble in water.

4 »On the other hand, the well-known penicillins are in certain cases Penicillinase-producing staphylococci have no effect.

It has already been thought of linking tetracycline with certain penicillins, but that is only compound of this kind, the phenoxymethylpenicillinate of tetracycline, that has a certain activity to certain penicillin-resistant staphylococci, also practically insoluble in Water, making it difficult to use medicinally.

The product obtainable according to the invention is free from both of these disadvantages and provides the first connection

209 757/159

dung, which is at the same time water-soluble and therefore therapeutically usable with advantage and in the presence active by penicillinase, d. H. Penicillinase is active even against such strains of staphylococci produce.

The process for the preparation of the new compound of phenoxymethylpenicillin consists in either a) equimolar amounts of the reaction products of N- (β-hydroxyethyl) -diethylenediamine. The solution is filtered through a Buchner funnel and the filtrate is then subjected to lyophilization at -40 ⁰ C. The lyophilized product obtained in this way shows the same properties as the products obtained according to the preceding examples.

In all three cases, the end product is in the form of a whitish-yellow, odorless, slightly bitter one Powder that is highly soluble in water (> 1.5 g / ccm), soluble in methanol and ethanol and insoluble in ether

g g

F. = 143 to 155 ° C (decomposition, uncorrected). [«] ² _D ° = -50.5 ± 2 ° (in 2% solution in methanol).

and formaldehyde is soluble with tetracycline and phenoxymethyl- io. The pH of a 2% aqueous solution

penicillin or b) equimolar amounts of after the solution is 5.6.

Mannich reaction obtained isolated N- or 9- or 7- [4'-QS-HydroxyäÄyl) -diäthylenendiaminomethyl] tetracycline with phenoxymethylpenicillin in a common solvent to react and isolate the reaction product. The reaction proceeds preferably under heat at a temperature in the range of 4O ⁰ C.

The procedure used to isolate the product depends on the nature of the product used Solvent. When water is used as the common solvent, the reaction product becomes isolated by lyophilization while using an organic solvent such as methanol or isopropanol, the product is obtained by precipitation with ether and filtration.

The following examples illustrate the invention.

_f .. _{ru M n Q}

Calculated

C 57.67%, H 6.02%, N 8.96%, S 3.42%;

^{ge un 6} ^ ₁

C 56.41 J ₀ , H 6.11 / ₀ , N 6.11 / ", S 3.36 / ₀ .

Calculated

found

Components

Tetracycline derivative (D

62.6%
61.7%

Penicillin derivative

αϊ)

37.4 »/ ο 37.2%

bond and the pH of their solution allow their administration on intramuscular and even intravenous route.

The phenoxymethylpenicillinate obtained by the process according to the invention of the tetracycline substituted on the carboxamide nitrogen atom or in the 9- or 7-position by a 4 - (β - hydroxyethyl) - diethylenediaminomethyl radical has exceptional

^Bei P ^{iel l}

6.63 g of N - ((5-hydroxyethyl) diethylenediamine and 3 °
3.8 g of 38% formalin are dissolved in 200 ecm Me- The proportion (I) is determined on the basis of iodo

ethanol heated to 60 to 70 ° C. After a 1-hour metric route, the determination of the portion (II) is added to the solution thus obtained by spectrophotometry by determining E 22.2 g of anhydrous tetracycline base and 17.5 g of water at 356 ΐημ for a solution with a content of free Phenoxymethylpenicillin approximately at the same time as 35 10 γ per ecm in n / 10-HCl.

and heats the reaction mixture to 90 minutes. The extraordinary solubility of the new

an oil bath at 4O ⁰ C. After about 30 minutes a complete solution occurs.

The reaction mixture is then poured with stirring
in 600 ecm of ethanol. The precipitated product turns 40
filtered, washed twice with ether and in vacuo
Dried for 5 hours at 40 ° C. 40 g are obtained in this way
of the phenoxymethylpenicillinate of 4 '- ( ₍ 8-hydroxyethyl) -diethylenediaminomethyltetracycline, what a
Yield of about 85 to 90% corresponds. The antibiotic properties that differ from those of this product are given below to the antibiotics of which it is composed. clearly distinguish.

Example 2 - ° * ^ε strongest activity is on penicillin-resistant

Staphylococci exercised. Despite the proven

58.6 g of the resistance of these strains to phenoxy from N- (jS-hydroxyethyl) -diethylenediamine, formaldehyde-methylpenicillin, according to the Mannich reaction, are dissolved over that of the 7 - [A '- (ß - hydroxyethyl) - diethylenediamine] - carboxyamide nitrogen atom or in 9- or 7-Steltetracyclins and 35 g of phenoxymethylpenicillin under ment by a 4- (jS-hydroxyethyl) -diethylendiami stirring in 300 ecm of methanol, filtered through a 55 nomethyl radical substituted tetracycline [in the Büchner funnel, the filtrate is mixed briefly with 4 '- (3-hydroxyethyl) diethylenediamino stirring with 990 ecm of anhydrous ether, filtered lblih

again under pressure and washes the filter cake twice with 50 ecm anhydrous ether each time. After drying the product in vacuo, 84 g of a yellowish-white powder are obtained, which is also those given below
having shafts.

physicochemical intrinsic

Example 3

The same amounts of the same reactants as described in Example 2 are dissolved in 500 ecm of distilled water at 5 ° C.

methyltetracycline] and of course is also above that of all known tetracycline derivatives.

The new compound is bactericidal, while the 4 ' - (ß -hydroxyethyl) - diethylenediaminomethyltetracyeline only has a bacteriostatic effect. The effect of the new compound on bacteria that are not resistant to penicillin is also considerably greater than that of the basic part alone.

Determined on groups of white mice infected with penicillin-resistant staphylococci Mortality is in those groups with the

Phenoxymethylpenicillinate of 4 '- (/ 3-hydroxyethyl) -diethylenediaminomethyltetracycline were treated, considerably less than in those groups treated with 4 '- (/? - hydroxyethyl) - diethylenediaminomethyltetracycline had been treated.

The antibacterial spectra given below show that the minimum inhibitory Dose (M.I.D.) has an extremely low value. To assess this dose must be considered that the compound obtainable according to the invention has a penicillin component and a tetracycline component contains and that therefore in the comparison tests in a predetermined amount of the product only half of the amount of the corresponding proportion is included, as in those used for comparison Single components. When evaluating the results, it must also be taken into account that the toxicity of the new compound is due solely to its tetracycline portion, d. i.e., the toxicity of the product obtainable according to the invention is approximately half as great as the toxicity of an equally great one Amount by weight of tetracycline.

In the table below, the minimum inhibitory doses in micrograms per cubic centimeter (y / ccm) are given, the product obtainable according to the invention with PHT is soluble made tetracycline part of this product is designated with T and the phenoxymethylpenicillin part with pmp.

Antibacterial spectrum in vitro

Bacterial strains

Staph. p. aureus penicillin-resistant, lOOI.E./ccm ..
Staph. p. aureus penicillin-resistant, 2001. E./ccm ..
ίο staph. penicillin resistant,
3 IU / cc, tetracycline resistant 8 y / cc

MID in y / ccm
Phenoxymethylpenicillin

0.66
0.80

62
124

tribe

Staphylococcus aureus Ox ...

Sarcina lutea

Streptococcus viridans

Streptococcus Hem. A.

Diplococcus pneum

Nesseria gonorrhea

B. Subtilis

E. Coli O

E. Coli 12

E. CoIiN '.

Staphylococcus aureus 209PFDA

Staphylococcus aureus penicillin resistant, 62 y / cc

Staphylococcus aureus, penicilin resistant, 60 y / cc

Staphylococcus aureus Silver 2, penicillin resistant, 6 y / ccm ....

B. Subtilis, penicillin resistant,
100 y / cc

MI.D in y / cc T I pmp I PHT

0.66 0.02

0.40

0.40

0.40

0.45

0.30

0.65

0.4

0.2

0.2

0.35 0.48 0.16 0.24

0.04 0.05 0.02 0.03 0.01 0.08

0.03 62 60

6 100

0.03 0.06 0.08 0.03 0.05 0.01 0.13 1.00 0.38 0.25 0.05

0.25 0.48 0.16 0.24

In vitro activity of PHT versus various

Strains by the method of continuous dilution

in a liquid environment

The bacteria used were isolated from sick people or from the collection of strains in the laboratory taken. The results were noted 18 hours after inoculation.

Results of the bacteriological measurements in the Warburg apparatus

Experiments in the Warburg apparatus showed the curves of the oxygen consumption of the bacteria that based on the breathability of the bacteria, and at the same time determines the reproduction of the bacteria. Phenoxymthylpenicillin, 4 '- (β-hydroxyethyl) - diethylenediaminomethyltetracycline were used for comparison and a blind test in which no antibiotic was used. The obtainable according to the invention The product shows a completely different and incomparably stronger effect. This effect is in the 1 and 2 shown graphically.

Fig. 1 shows the change in the oxygen consumption of the bacteria, shown as a logarithm the amount of oxygen consumed by the bacteria (in cubic millimeters) versus time (in Hours);

Figure 2 depicts the growth of Staphylococcus aureus expressed in terms of the logarithm of Number of living microorganisms as a function of time (in hours).

In the two families of curves shown in FIGS. 1 and 2, these are in the following four cases observed results are designated with 1, 2, 3 or 4: Curve 1:

Without antibiotic (blind sample),
Curve 2:

Use of phenoxymethylpenicillin at a concentration of 0.23 micrograms per cubic centimeter.

Curve 3:

Use of 4 '- (/? - hydroxyethyl) -diethylenediaminoethyltetracycline at a concentration of 0.4 micrograms per cubic centimeter.

Curve 4:

Application of the process product in a concentration of 0.63 micrograms per cubic centimeter (corresponding to 0.23 + 0.4 y / ccm of the two above notes).

From Fig. 1, the improved results can be seen with the inventive Product can be achieved in the inhibition of the respiration of Staphylococcus aureus.

In the same way, the growth of Staphylococcus aureus by the obtainable according to the invention Product impaired in a noticeable way:

While curves 1 and 2 show an increase in the number of microorganisms and curve 3 only a constant number, d. H. show only one bacteriostasis, curve 4 shows an increasing one Reducing this number; d. H. a bactericidal effect. The compound obtainable according to the invention

therefore acts, as already stated above, not only bacteriostatic, but also bactericidal, which is im With regard to the properties of its individual components is extremely surprising in itself.

Some penicillinase-resistant penicillins are known from Belgian patent 569728. The two most effective of these known penicillins, phenylthiomethylpenicillin sodium and the benzylpenicillin sodium, were found to be those described below Comparative experiments as the phenoxymethylpenicillinate of 4 - ((S-hydroxyethyl) diethylenediaminomethyltetracycline obtainable according to the invention (PHT) inferior.

First was the minimum inhibitory concentration determined against the following bacterial strains:

Staphylococcus aureus, penicillinase generator, phage group III.

B. substilis 6633, ATCC.

E. coli K 64.

Proteus mirabilis.

The so-called Oxford dilution method (H. W. Florey and M. A. Jennings, Brit. J. Exp. Path., Vol. 23, 1942, pp. 120 to 123) in a slightly known manner in terms of volume applied to the sample and the production of the inoculum modified form:

In each case 0.05 ecm of the freshly prepared bacterial suspension are used to inseminate a number of test tubes, each 9 ecm one

ίο liquid culture medium (antibiotic test medium Difer) and contain regularly decreasing amounts of the antibiotic to be tested. After filling up the test tubes are kept at 38 ° C. for 18 hours at 10 ecm. Then the antibiotic concentration of the first test tube found in which no growth was observed and the liquid is clearly formed.

The results obtained in this way are listed in the following table, each in brackets

zo mern also indicated the maximum active dilution is.

Bacterial strain Minimal inhibiting concentration

Phenylthiomethylpenicillin Sodium

Benzylpenicillin Sodium

PHT

Staphylococcus aureus,
Producer of penicillinase, phage group III

B. subtilis 6633, ATOC

E. coli K 46

Proteus mirabilis

Furthermore, the antipenicillinase activity of the two known penicillins and the PHT obtainable according to the invention was determined according to the procedure described by Henry and Housewright ("J. Biol. Chem.", Vol. 167, 1947, pp. 579 to 571). In this procedure, when the bacterial strain used for testing generates penicillinase, CO _{2 is} generated in an amount proportional to the amount of penicillinase generated.

The results obtained in this test are summarized in the table below.

500y / ml
(1: 2000)

0.1 y / ml
(1: 10 M)

500 y / ml
(1: 2000)

500 y / ml
(1: 2000)

500 y / ml
(1: 2000)

0.05 y / ml
(1:20 m)

500 y / ml
(1: 2000)

500 y / ml
(1: 2000)

0.3 y / ml (1: 3.3 M)

0.03 y / ml (1:33 M)

3 y / ml (1: 330000)

20 y / ml (1: 50000)

Developed CO ₃ in millimeters PHT Minutes (Induction of penicillinase) 0 Benzylthiomethyl
penicillin Benzyl penicillin 0 10 0 0 0 20th 0 0 0 30th 5 10 0 40 28 30th 0 50 70 80 70 230 230

The comparison tests carried out result in:

1. That the minimum inhibitory concentration of the new compound is less than that of the two known reference substances and

2. that the penicillinase production of a microorganism in the presence of the invention available compound is completely suppressed, whereas the two known compounds are unable to block the formation of penicillinase of the same microorganism.

Furthermore, kidney excretion is compared to that of the constituents of the new compound markedly degraded. ("Excretion" of a substance, for example urea, = the amount of plasma in Milliliters, which contains the amount of substance in grams that is removed in 1 minute of kidney activity).

Claims (3)

PATENT CLAIMS: 1. A process for the preparation of a new compound of phenoxymethylpenicillin, characterized in that either a) equimolar amounts of the reaction product of N - (/ 3-hydroxyethyl) diethylenediamine and formaldehyde with tetracycline and phenoxymethylpenicillin or b) equimolar amounts of after the Mannich reaction obtained isolated N- or 9- or 7- [4 '- (jS-hydroxyethyl) - diethylenediaminomethyl] - tetracycline with Phenoyxmethylpenicülin in a common solvent to react and isolate the reaction product 2. The method according to claim 1, characterized in that one is used as a common solvent Water is used and the product obtained is isolated by lyophilization. 3. The method according to claim 1, characterized in that there is used as a common solvent an organic solvent such as methanol, 10 and the product is isolated by precipitation with ether and filtration. Documents considered: German Auslegeschrift No. 1 044 806; published documents of Belgian patent No. 569 728. When the registration was announced, a test report (14 pages) was displayed. 1 sheet of drawings