DE1768656C3 - - Google Patents

Description

The invention relates to the neutral ethylenediamine-dj- or d-2-oxobornane sulfonate (10) and a process for its preparation that thereby is characterized in that one d, l- or d-2-oxobornanesulfonic {10) acid in a manner known per se in aqueous or methyl alcoholic solution Room temperature affect ethylenediamine EQt.

The ethylenediamine can be used as a solution in methyl alcohol or water. This solution is slowly added to the 2-oxobornanesulfonic (10) acid, which is also dissolved in methyl alcohol or water.

The new compounds are effective as antivirals and are excellent for combating of diseases of the nasopharynx caused by viruses, e.g. B. as an aerosol. You are, as the test report below shows, compared to adamantamine hydrochloride, which is known as an influenza drug think.

Structural formula:

example 1

Neutral ethylenediamine-dJ ^ -oxobornanesulfonate- {10)

169 g (2,82MoI) ethylenediamine are dissolved in 1,2] methyl alcohol, the resulting solution is cooled aul 18 to 20 ⁰ C and under stirring with a solution containing 1.3 kg (5 moles) of d, l-2- Oxobornane sulfonic acid (10) dissolved in 1.2 1 methyl alcohol, added.

The whole is refluxed for 30 minutes and then to room temperature cooled down.

After the precipitate has been separated off, the compound thus obtained is recrystallized from methyl alcohol; 1.65 kg of neutral ethylenediamine-2-oxo-bomansulfonat- (10), melting point: 302 bis, are obtained 303 ° C with decomposition.

Molecular formula: C ₂₂ H ₄₀ O ₈ S ₂ N ₂ .

CH

H ₂ N-CH ₂ -NH ₂

Molecular weight: 524.7.

Composition:

d, l-2-oxobornane sulfonic acid- (10) = 85.55%,
Ethylene diamine = 11.45%.

Properties: White, microcrystalline powder that is soluble in water, fairly soluble in ethanol and methanol soluble and insoluble in acetone, chloroform and ether.

Example 2

Neutral
Ethylenedidmin-d-2-oxobornane sulfonate (10)

If you proceed as in Example 1, but d-2-Oxobornanesulfonsäure- (lO) used, one obtains the neutral Äthylend.'amin-d-2-oxo-bornanfulfonat- (10), which melts at about 284 ° C with decomposition.

Test report

Comparison of the effectiveness of the neutral ethylenediamine according to the invention - d, l - 2 - oxo - bomansulfonat- (10) (AMS 3) with regard to its pharmacological activity with the well-known AdamantaminhyHrochlorid (Symmetrel ·) (see Reallexikon der Medizin, 1st vol., 1966, A 152, left column, line 7 of below).

I. Study of activity

to a deoxyribonucleic acid virus

(Bacteriophage strain Twort)

Under the conditions described in Annales Pharmaceutiques Francaises 1967, 25, pp. 783 to 796 has been the role that AMS 3 plays in the development of Staphylococcus aureus in the presence or absence of his phages, examined with the help of the self-writing biophotometer.

60

1 763656

1.Neutral ethylenediamine-2-oxo-bomansulfonat- (10) (AMS 3)

In FIG. 1 the values are recorded as an example, those at concentrations of 10, 30, 40 and 80 mg / ml, in the experiment on the culture and in the Attempt with normal lysis were obtained.

In table 1 the results are compiled, those obtained with the weakest doses, namely 0-7.5-10-20-30-40 mg / ml. For this purpose, the percentage of light that the kilvettes received at the moment of bacteriophage lysis is given is let through. This ad is important and deserves to be recorded because this measure, as Zekam has shown for the same number of germs, on the number of those present Phage depends: the larger this number, the earlier the lysis occurs and the larger it is percentage of light transmission; the lower the number of phages, the lower the light permeability, and the percentage of light transmitted approaches zero. The percentage of permeability thus makes it possible to track the amount of phages present at the moment of lysis.

This table shows the constant change in the observed phenomena as a function of those used Dose. This always proves to be quite weak bacteriostatic, while compared to the Phages, the phenomenon becomes more pronounced: while namely at a concentration of 7.5 mg / ml practically no effect occurs, there is no primary lysis and also no alternating lysis at 40 mg / ml Lysing more. Any manifestation of any phage present has disappeared. To hold fast is already that from 20 mg / ml on the secondary cultivation is not followed by a phenomenon of alternating lyses.

In order to study the mechanism of action of the product more closely, the experiments were continued that the applied doses increased from 50 over 60 to 80 mg / ml and the properties that are affected by the Bacteria and the phage obtained at the end of the experiment, d. H. 24 hours after the start of breeding, were acquired.

a) To check whether the obstruction of the lysis is caused by the presence of the product becomes and disappears by simply diluting the medium, this 24-hour culture became in one fresh nutrient medium diluted to 1/100 °. This diluted approach was repeated with the self-recording Instrument measured.

b) To examine the presence of active phages in the cuvette at the end of the experiment, the contents of the cuvette were centrifuged. 1 ml of the liquid floating on top was used for inoculation of 9 ml of fresh medium containing the usual concentration of staphylococci. the Examination of the recording curve obtained allows the presence or absence at least of a phage strain to control.

c) The bacterial strain obtained was examined for its lysogenic properties. The phenomenon the alternating lysis normally leads to staphylococci at the end of the experiment which are resistant to Twort phage by lysogenization and are strongly lysogenized. The residue obtained from centrifuging the contents of the cuvette is mixed with 1 ml of antiphage serum, which is diluted in 10 ml nutrient medium, mixed and continued for 1 hour at + 37 ° C emotional. The contact achieved in this way enables all external phages to be eliminated. 0.5 ml of this Bacterial suspension in a dilution of 1/100 ° with 0.5 ml of a suspension of fresh Staphylococci mixture !, and the whole thing is placed on the surface of a nutrient medium in a Petri dish spread out. After 24 hours of incubation, the presence of lysis zones indicates the presence of lysogenic bacteria in the investigated residue that have released their phages.

The results obtained are shown in Table 2.

This table 2 shows the regular increase in the activity of the AMS 3 as a function of the dose. This activity has its effects on the phages, the number of which is constantly decreasing and from 80 mg / ml it is zero will. At the smaller doses, the bacteria show a pronounced tendency to lysogenization, without suffering a lysis. Since AMS 3 reduces the virulence of the phages, it can therefore be in the prophage state can be added.

The AMS 3 according to the invention thus shows opposite the Twort phage strain a remarkable one even in the presence of its sensitive bacterial strain virulicidal effect.

Incidentally, this effect can be found again. if you put the phages (100 / ml) and the product in Brings together doses of 60 or 80 mg / ml directly inside the culture medium; after a period of contact for 24 hours, the added staphylococcus experiences no lysis, and the phages have consequently been inactivated.

The AMS 3 thus presents itself as a viruloid that destroys a deoxyribonucleic acid virus and is able to reduce its virulence in lower doses.

2. Adamantamine hydrochloride (Symmetrel ®)

The product tested was a white, water-soluble powder. The investigations were under carried out under the conditions described above.

In FIG. 2 the values are recorded that were obtained at concentrations of 0.5, 1 and 2 mg / m) with or were obtained without phage.

Table 3 summarizes the results obtained with the various tested doses the following were obtained: 0-0.5-1-2-3-5 mg / ml. In addition the percentage of light that was passed through the cuvette at the moment of bacteriophage lysis is shown passes through.

A clear antibacterial effect can be seen from 3 mg / ml. As has been verified, it is a simple bacteriostatic effect that disappears when you wash the Symmetrel ® of the bacteria that are in contact with it in the culture medium, eliminated.

In the presence of the phages, the percentage that failed at the moment of the first lysis is found Amount of light compared to the test tube, in which there is no product, increased. This Measure reflects a decrease in the bacterial concentration at the moment of this lysis, i.e. i.e. that the product promotes virus maturation.

This observation indicates that the Symmetrel® has no reproductive antiviral activity of bacteriophages, while AMS 3 is clearly virulicid under the same conditions works.

II. Study of the activity against a ribonucleic acid virus

The concentration gradient method was used (cf. Annales Pharmaceutiques Francaises 1969, 27, pp. 705 to 710).

1.Neutral ethylenediamine-d, l-2-oxobornane sulfonate- (10) (AMS 3)
The following scheme was obtained with AMS 3:

1 mg / ml

OQOQQ-Q-QO

O O O

0.5 mg / ml

0.1 mg / ml

10 mg / ml

Active area
toxicity

small or large zones (plages) O ^- OO

The macroscopically visible toxicity is thus zero up to 10 mg / ml and the antiviral activity is counteracted 0.5 mg / ml. It has been observed that this is precisely the threshold of activity that is also against another Virus type, namely the measles virus, was found.

2. Adamantamine hydrochloride (Symmetrel ^e )
With Symmetrel® the following scheme was obtained:

1 mg / ml

O O

OQQO-O-O

- * 0.5 mg / ml

0.1 mg / ml

10 mg / ml

In the course of this series of experiments, a cell toxicity to chicken embryo cells from 0.5 mg / ml observed: The cells have died and no longer allow the virus to multiply, while in the case of the immediate the concentration below that, the size and number of phages are slightly reduced. the Antiviral activity is zero down to 0.1 mg / ml.

I /

The two products examined therefore have different antiviral properties. AMS 3 shows effectiveness while Symmetrel® is ineffective.

Conclusion: The two methods of investigation show that the inventive MS 3 has a clear antivirus activity that cannot be found in Symmetrel *.

table

product
mg / ml without phages
observed phenomena 0 normal breeding 7.5 normal breeding 10 normal breeding 20th slightly demanding
velvet breeding 30th
40 slightly demanding
velvet breeding
slightly demanding
velvet breeding

bacteria

with phages

percentage of permeability, just before the first lysis

60% 58% 56% 50%

35% observed phenomena

first lysis, secondary cultivations and alternate lyses

slight delay in the first lysis, secondary

Cultures and alternating lyses

slight delay in the first lysis, secondary

Breeding that is delayed but lysed

first lysis delayed, secondary breeding delayed,

no alternating lyses, but presence of

Phages at the end of the experiment

first lysis delayed, second cultivation delayed,

no alternating lyses

no first lysis, no alternating lyses, but

the studies of phages and bacteria that

are sensitive to phage, at the end of the

Attempt are positive

table

product mg / ml

60

tries bacteria

Complementary examinations

without phages

with phages

slowed down a bit
breeding

somewhat slowed breeding

slowed breeding, prolonged
Gradient

greatly slowed down
breeding

no first lysis, no alternating lyses
no first lysis, no alternating lysis

no first lysis, no alternating lyses

no first lysis, no alternating lyses

1/100 dilution

Phages and sensitive bacteria

Phages and sensitive bacteria,
fewer free
Phages than before
Phages and sensitive bacteria,
some free phage

absence of
Phages

Floating on top Material + fresh germs Phage examination

Residue

Lyso-

gene bacteria

first lysis and alternate lysis

first lysis and

absence of

alternating

Lyses

first lysis and

absence of

alternating

Lyses

absence of

Phajen

product mg / ml

0 0.5

table

bacteria

without phages observed planenomeoe

normal breeding

slowed down a bit
breeding

percentage fluidity, just before the first lysis

60% 68% with phage

observed phenomena

first lysis, secondary cultivations, alternating lysis

first lysis, secondary breeding, alternating Lyses

409633/271

Product mg / ml

2 3 5

phenomena observed in the absence of phage

somewhat slowed breeding

somewhat slowed breeding

breeding slowed down by 2 hours

no bacterial development

continuation

10

percentage of permeability, just before the first lysis

70% 80% 84%

bacteria

with phages

observed phenomena

first lysis, secondary cultivations, alternating lysis

first lysis, secondary cultivations, alternating lysis

first lysis, no secondary cultivation to prevent bacterial development no bacterial development

1 sheet of drawings

Claims (2)

Patent claims: 1. Neutral ethylenediamine-d,! - or d-2-oxobornanesulfonate- {10). 2. Method of making the connections $ according to claim 1, characterized in that dai one d, l- or d-2-oxobornanesulfonic (10} acid ii in a known manner in aqueous or methyl alcoholic solution at room temperature au Ethylenediamine can act.