DE1196648B - Process for the preparation of water-soluble derivatives of tetracyclines - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

C07c

German class: 12 ο-25

Number: 1196 648

File number: L 41290IV b / 12 ο

Filing date: February 21, 1962

Opening day: July 15, 1965

The present invention relates to the preparation of water-soluble derivatives of tetracyclines by Treatment with formaldehyde. This results in methylol compounds of the general formula

R -CONH CH ₂ OH

where R is the remainder of the tetracycline used. For example, if you get from Tetracycline runs out of compound

H ₃ C

H ₃ C CH ₃
OH N

OH

CONH-CH ₂ OH

OH O OH O

Obtained from other tetracyclines such as chlortetracycline, oxytetracycline or desmethylchlortetracycline one similar products.

The reaction is carried out in such a way that the formaldehyde is added to an aqueous suspension of the tetracycline can act. One can use a solution of formaldehyde in the cold or in the heat. You can also work with gaseous formaldehyde by slowly passing it through the suspension or with solid paraformaldehyde, which releases formaldehyde when heated.

The pH of the reaction mixture can be between 5 and 9, with an optimum reaction between 7 and 8.0. During the reaction the product becomes completely water-soluble; it can then can be isolated in amorphous or crystalline form by freeze-drying or by precipitation. This precipitation can be done either by converting the aqueous solution into an organic one Solvent or a mixture of organic solvents that is completely miscible with water pouring or by adding the organic solvent or the mixture of solvents gradually added to the aqueous solution.

As a solvent, for. B. used are aliphatic alcohols such as methyl, ethyl or propyl alcohol, ketones such as acetone, methyl ethyl ketone or methyl isobutyl ketone, substituted alcohols such as ethylene chlorohydrin, esters such as methyl acetate or ethyl acetate, and also water-immiscible solvents such as ether, z. B. Diethyl Process for Making Water Soluble
Derivatives of tetracyclines

Applicant:

Leo Industrie Chimiche Farmaceutiche S. p. A.,

Rome

Representative:

Dr.-Ing. A. van der Werth, patent attorney,

Hamburg-Harburg, Wilstorfer Str. 32

Named as inventor:

Dr. Ezio Tubaro,

Dr. Feriano Banci, Rome

Claimed priority:

Switzerland of March 27, 1961 (3654)

ether, diisopropyl ether, or aromatic hydrocarbons such as benzene, toluene or xylene, or aliphatic Hydrocarbons which are so miscible with alcohols or ketones that with water completely miscible mixtures can be obtained.

Depending on whether these conditions are varied in a suitable manner, the product can be amorphous or crystalline form can be obtained.

The resulting compounds are highly soluble in water. One can find solutions with one Get a pH of about 7, which is suitable for the manufacture of injectable preparations.

The new derivatives have remarkable pharmacological properties that make their use more advantageous than those of the previously known water-soluble tetracyclines, such as those below Comparative tests show:

Acute toxicity, chronic toxicity and the blood level in animals and humans are compared (i.e. the therapeutic efficacy) and tolerability of known and claimed derivatives of the tetracycline and then of the chlortetracycline.

509 600/431

1

Acute toxicity (LD50)

Carried out at the white mouse Swiss.

All LD50 values refer to the basic stracycline, to enable a direct comparison.

Method of administration: intravenous

Tetracycline hydrochloride

Pyrrolidinomethyltetracycün

(German Patent 1,044,806) Lysinomethyltetracycline

(Belgian patent 597 187) .. N-4'-0S-hydroxyethyl) diethylenediaminomethyltetracycline

(Belgian patent 588 705) .. N-methylol tetracycline

(present invention)

Method of administration: intraperitoneally

Tetracycline hydrochloride

Pyrrolidinomethyl tetracycline

(German Patent 1,044,806) Lysinomethyltetracycline

(Belgian patent 597 187) .. N-4 '- (j8-hydroxyethyl) diethylenediaminomethyltetracycline

(Belgian patent 588 705) .. N-methylol tetracycline

(present invention)

0.155 g / kg

0.145 g / kg 15th 0.170 g / kg 0.155 g / kg 20th 0.172 g / kg 0.338 g / kg 25th 0.325 g / kg 0.355 g / kg 30th 0.340 g / kg 0.360 g / kg

648

Method of administration: oral ν,

Tetracycline hydrochloride 2.55 g / kg

Pyrrolidinomethyl tetracycline

(German, Patent 1044 806) 1.32 g / kg
Lysinomethyltefracycline

(Belgian patent specification 597 187) .. 2.80 g / kg
N-.4 '- (jf-hydroxyethyl) -diethylen- ■ diaminomethyltetracycline

(Belgian patent 588 705) .. 2.70 g / kg
N-methylol tetracycline

(present invention) 3.00 g / kg

Chronic toxicity (chronic LDso; method by Beauvillaine)

Carried out at the white mouse Swiss.
All LD50 values refer to the basic tetracycline.

Method of administration: intraperitoneally

Tetracycline hydrochloride 1.4 g

Pyrrolidinomethyl tetracycline
- (German patent specification 1 044 806) .... 1.0 g
Lysinomethyl tetracycline

(Belgian patent 597 187) .. 3.0 g

N-4 '- (0-hydroxyethyl) -diethylene-

diaminomethyltetracycline

(Belgian patent 588 705) 2.2 g

N-methylol tetracycline

(present invention) 3.7 g

Blood levels in humans

The blood levels refer to a single oral administration of antibiotic equivalent to 150 mg basic stracycline.

1 hour

3 hours

6 hours

Tetracycline hydrochloride

Pyrrolidinomethyltetracycline (German patent specification 1 044 806)

Lysinomethyltetracycline (Belgian patent 597 187)

N ^ '- i / S-Hydroxyäthy ^ -diäthylenendiaminomethyltetracycrin

(Belgian patent 588 705)

N-methylol tetracycline (present invention)

0.06
0, -
0.12

0.08
0.15

0.75
0.06
1.05

0.95
1.30

0.42
0 -
0.85

0.63
0.95

The blood levels are expressed in y / ml.

The comparison shows that only the product of the Belgian patent 597 187 with can be compared to the product obtained according to the invention.

This was to be expected, because it is already known from the literature that aminomethyl derivatives given per os are hardly active because they are not absorbed (E. N. E w a 1 d and G. S. R e d i η, Antibiotics and Chemotherapy, Vol. 10, 1960, p. 418). That means a considerable limitation in the Therapy, because the oral route, which is more common in practice, is excluded for the aminomethyl derivatives is.

The product obtained according to the invention, on the other hand, is completely absorbed and gives therapeutically best blood level with perfect tolerance, no matter how it is administered.

These differences in tolerability and therapeutic effectiveness between the after Invention and the compounds obtained by known processes are even clearer, when examining the derivatives of chlortetracycline.

Many references report the greater effectiveness of chlortetracycline among antibiotics the tetracycline group. However, the use of chlortetracycline has one severe limitation experienced because of the side effects that occur with oral administration while parenteral Administration was practically completely abandoned, on the one hand because of these side effects, on the other hand, because of the low solubility of the antibiotic, administration only by means of slower Venous infusion allowed. In whatever way the remedy is given, the consequences are Dizziness, vomiting, dermatitis, itchy papules, urticaria, glossitis, stomatitis, colitis, proctitis as well Superinfections emanating from the intestine due to antibiosis of the bacterial flora, whereby Staphylococcal toxins are put into circulation and hypovitaminosis sets in. With intravenous Gifts often arise in cases of phlebitis and thrombophlebitis. (Mark H. L e ρ ρ e r, aureomycin [chlorine

tetracyclineJ-Antibiotics Monograph No. 7, Medical Encyclopedia Inc. New York, 1956, pp. 35 to 44; Finland and coworkers, American Medical Association, Archives International of Medicine, Vol. 93, 1954, p. 23; Kunin and coworkers, Clinical Pharmacology and Therapeutics, Vol. 2, 1961, p. 51).

All these serious damages were not overcome by products of the known processes, but probably through the products obtained according to the invention.

The N-methylolchlorotetracycline produced according to the invention has an antibacterial activity in vitro that is equivalent to that of chlortetracycline, an increased solubility at the pH of the physiological fluids (about 1 g / ccm) and remains for many hours in solution without chlorotetracycline separating out. Because of this, it can be on oral, intramuscular and intravenous routes.

The subsequent rapid absorption, regardless of the way in which the remedy was given, has result in increased blood levels (and therefore considerable therapeutic efficacy) and completely eliminates the side effects.

The high solubility and stability in solutions at the pH value of the plasma finally allow the intravenous administration without harmful side effects.

Some comparative data on toxicity and blood levels follow.

Acute toxicity (LD50)

Carried out at the white mouse Swiss.

All LD50 values refer to the basic chlorotetracycline, to enable a direct comparison.

Method of administration: intravenous

Chlortetracycline hydrochloride 0.093 g / kg

N-methylolchlorotetracycline 0.115 g / kg

Method of administration: intraperitoneally

Chlortetracycline hydrochloride 0.214 g / kg

N-methylolchlorotetracycline 0.273 g / kg

Method of administration: oral

Chlortetracycline hydrochloride 2.15 g / kg

N-methylolchlorotetracycline 2.83 g / kg

Chronic toxicity (LD50: Beauvillaine method)

White mouse Swiss.
The LD50 values will be
expressed.

1 hour
3 hours
6 hours

Chlortetracycline hydrochloride

0.85 y / ml
1.40 y / ml
1.0 y / ml

N-methylolchlorotetracycline

1.05 y / ml 1.80 y / ml 1.40 y / ml

After intravenous administration of 0.010 g / kg (always with regard to basic chlorotetracycline) are shown the following blood levels, determined in the same way as before. ■ "

15th Chlortetracycline-.
hydrochloride N-methylol-
chlortetracycline 1 hour
3 hours
²⁰ 6 hours 4.95 y / ffil
2.50 y / ml
1.30 y / ml 5.50 y / ml
2.80 y / ml
2.10 y / ml

Human blood levels Oral administration

The average values are reported for a hundred patients who received 150 mg (expressed as Basic chlorotetracycline) chlortetracycline hydrochloride and N-methylolchlorotetracycline every 6 hours over Periods of 3 to 13 days were administered. The administration had to be carried out in the majority of the patients should be discontinued from chlortetracycline in the first few days because of severe side effects. notices occurred. Patients taking N-methylolchlorotetracycline received, could continue to be treated without disturbances having occurred.

The blood levels were determined microbiologically with Bacillus subtilis. var. mycoides as a test germ determined against a standard of chlortetracycline.

40

45 1 hour *
3 hours
6 hours

Chlortetracycline hydrochloride

1.20 y / ml
0.62 y / ml
0.21 y / ml

N-methylolchlorotetracycline

2 y / ml 1.25 y / ml 0.70 y / ml

* Calculated from the last administration.

After administration of 300 mg (expressed as basic chlorotetracycline) every 6 hours, results the following values:

in basic chlorotetracycline

Method of administration: oral

Chlortetracycline hydrochloride 5.8 g

N-methylolchlorotetracycline 24 g

Blood levels in the dog

After oral administration of 0.050 g / kg (expressed in basic chlorotetracycline) of chlortetracycline hydrochloride and N-methylolchlorotetracycline, the following blood levels are shown, determined by microbiological means with Bacillus subtilis var. Mycoides against a standard of chlortetracycline. 1 hour
3 hours
6 hours

Chlortetracycline hydrochloride

1.8 y / ml
1.2 y / ml
0.5 y / ml

N-methylolchlorotetracycline

3.8 y / ml 2 y / ml 1.65 y / ml

Intravenous administration

After administration of N-methylolchlorotetracycline, equivalent to 250 mg basic chlorotetracycline every 12 hours, dissolved in 10 ecm aqua bidistillata, over a period of 20 days at fifty

Claims (1)

7 8 Patients found the following blood levels, given a product that was precipitated in a vacuum counted from the last administration. is dried at 30 to 40 ° C. Yield 70 to 30 minutes 14.8 y / ml ⁷⁵ ° l ° - 1 hour 9.3 y / ml B e i s ρ i e 1 5 3 hours 5 y / ml ⁵ ...,, 6 hours 3 4WmI ^5il 8 chlorotetracycline hydrochloride are added in 12 hours V // ^ V ^ '.'. '.'. '.'. '.'. 0.9 _Y lnu JOecm water, which has an excess of Contains 0 25 v / ml formaldehyde for 24 hours, the pH being below ' ^{γ is set} to 7 with gentle heating. The ab- In this case, no comparative tests could be made - a cooled solution is made into a mixture of ethyl, since alcohol and ethyl ether are given in a ratio of 2: 5 under the same conditions, intravenous administration over such a long period of time. A yellow, flaky precipitate forms . It is dried submission of chlortetracycline or washed with ether and dried in vacuo at 30 to other derivative chlorotetracycline un- 40 ⁰ C. The product is very possible in water. 15 easily soluble. With slow heating in the ge-The invention is to be explained in more detail on several execution-closed tubes, it decomposes between 145 examples. and 170 ° C. Yield 75%. Example 1 analysis for C23H25O9N2CI: 4.8 g of tetracycline hydrochloride are calculated in the cold 20 suspended in 20 ecm of water containing an excess of C 54.27%, H 4.95%, N 5.5%, Cl 6.96%; contains formaldehyde; the pH is found below gentle heating with a dilute solution C 54.80%, H 4.80%, N 5.6%, Cl 7.05%. brought to 7 by sodium hydroxide in a water bath. "· · 1 <;
The resulting solution is cooled and poured into a 25 e 1 s ρ 1 e
Mixture of ethyl alcohol, acetone and ethyl ether 4.94 g of oxytetracycline hydrochloride are poured in in a ratio of 1: 1: 5. A yellow fluffy 20 ecm of water with an excess of form-precipitate is formed, which is filtered off. The product aldehyde dissolved, the pH being adjusted to 7, washed with excess ether and gently warmed in the mixture. The abge vacuum at 30 to 40 ⁰ C dried. It melts. Cooled solution is poured into a mixture of alcohol with decomposition between 150 and 180 ^° C. Aus and ether, the product yield 80%. separates, which after filtration in vacuo at 30 to 40 ⁰ C is dried. Yield 70%.
Analysis for ΟβΗ ^ Οθ ^: Calculated ... C 58.22%, H 5.22%, N 5.90%; 35 B e 1 s ρ 1 e 1 7 found ... C 59.01%, H 5.15%, N 6.01%. 4.95 g desmethylchlorotetracycline hydrochloride are . . those with an excess of formaldehyde in Example 2 20 ecm of water while adjusting the pH to 7 4.4 g of tetracycline base are dissolved with gentle warming and gentle warming. The cooled warm and while adjusting the pH with 40 solution is in a mixture of acetone and diluted alkalis to 7 dissolved in 20 ecm of water, ether added, whereby a product separates out, which contains an excess of formaldehyde. In that after filtration in vacuo. 30 to 40 ⁰ C the cooled solution is gradually dried a mixture. Yield 75%.
of ethyl alcohol, ether and acetone in proportion 1: 1: 5 until the product is precipitated, which 45 claims:
separates out in crystalline form. After filtration, the product is treated with ether 1. Process for the preparation of water-soluble washed and dried in vacuo at 30 to 40 ° C. Derivatives of tetracyclines, thereby genetized. The product is the same as in Example 1. From indicates that one is aqueous prey 75 to 80%. 50 suspension of a tetracycline in known per se. -, ο way with formaldehyde at a pH of Examplej 5 to 9 treated and the methylol formed 4.8 g of tetracycline hydrochloride become tetracycline in the cold by freeze-drying or precipitation suspended in 20 ecm of water, which is an excess with a mixture of water-miscible contains formaldehyde. The pH will be below 55 or immiscible organic solvents slight warming through a dilute solution wins. brought to 7 by sodium hydroxide. According to the second method according to claim 1, characterized by filtration, the filtrate is lyophilized. The product indicates that the precipitation of the reac is yellow and very easily soluble in water. tion products with a mixture of alcohol, _n . . . . 60 Acetone and ether in a ratio of 1: 1: 5 through ^BeIS P ^ieI4 . 4.8 g of tetracycline hydrochloride are in 20 ecm Suspended in water. Due to the suspension, considered publications: slow flow of gaseous formaldehyde up to German Auslegeschriften No. 1 044 806, Saturation passed, the pH value adjusted to 7 65 1 063 598; and the mixture until completely dissolved Austrian patent specification No. 205 168; is slightly heated. In this solution, a Belgian patent specification No. 588 705, 597 187; Mixture of alcohol, ether and acetone up to J. Am. Chem. Soc, Vol. 81, 1959, pp. 1198 to 1201. 509 600/431 7.65 © Bundesdruckerei Berlin