NO174808B - Process for the preparation of therapeutically active methylenedioxyphenanthrene derivatives - Google Patents

Description

The present invention relates to a method for the preparation of compounds of formula (I)

wherein means hydrogen, hydroxy, methoxy or ethoxy, and their pharmaceutically acceptable salts.

As immunity stimulants with prophylaxis therapy

of infectious diseases are the aristolochic acids of formula (II)

known, which has a phagocytosis-increasing and antiviral effect, and can increase healing results in general infections, and also

can be successfully used for the treatment of tumors and tumors.

During pharmacological investigations in higher dose ranges, however, a cellular transformation of the forage in rats was determined a short time ago, so that one must be reluctant to give these in and of themselves very valuable active substances for the treatment.

Surprisingly, however, it was now found that the compound of the general formula (I) in which the Rx is as indicated above, even in very high doses (compared to the usual doses of 1-10 ug/kg in human therapy) to rats (10 mg /kg) gave neither macroscopic nor histological signs of a squamous hyperplasia. No mutagenic effects were determined either.

They are very suitable for prophylaxis therapy of infectious diseases, as they exhibit a phagocytosis-increasing and antiviral effect.

Of these compounds, 1-carboxy-3,4 methylenedioxy-8-methoxy-phenanthrene is certainly known from J. Nat. Products 46 (1983) 507 fr., where it is stated that it has an abortifacient effect in doses of 90 mg/kg (in rabbits), and in 60 mg/kg (in mice) has an implantation-inhibiting effect. An immunity-stimulating effect of this was therefore not to be expected.

The compounds with the general formula (I) are prepared according to the invention by reacting 6-bromopiperonyl bromide with the formula

with triphenylphosphine in an anhydrous solvent to a phosphonium salt of formula reacts the latter compound with benzaldehyde, o-methoxybenzaldehyde or o-ethoxybenzaldehyde in the presence of a strong base to a stilbene of formula wherein R^ is H, OCH3 or OC2H5, transfer the above-mentioned stilbene bromide by UV irradiation into the phenanthrene derivative of formula and convert this compound either with a metal cyanide, especially copper cyanide, in a polar aprotic solvent, especially in dimethylformamide, into the corresponding nitrile, and transfer this by hydrolysis in an acid of formula (I), or transfer the above-mentioned compound with n-butyllithium and carbon dioxide in an acid of formula (I), and, if desired, convert a compound of formula (I) in which R-1 is a methoxy group, by ether cleavage into a compound in which R- ^ means hydroxyl, or denitrates a nitro compound of formula:

wherein R;1 is H, OCH3 or OC2H5 with a polysulfide to one

compound of formula (I), and, if R-^ is OCH3, if desired converts the OCH3 group by ether cleavage into an OH group.

In the preparation of the phosphonium salt, an anhydrous solvent is used, e.g. benzene or an alkylbenzene, preferably toluene or xylene.

In the condensation of the phosphonium salt with the aromatic aldehyde, a strong base is used, e.g. an alkali metal alcoholate, preferably lithium methylate. The styloid thus obtained is 85% in Z-shape and 15% in E-shape. The two isomers can be separated by treatment with diethyl ether. The trans compound passes into the ether, while the desired cis compound remains undissolved.

With the UV irradiation of the stilbene bromide, one works e.g. a solvent mixture of absolute THF and absolute cyclohexane in mixing ratio e.g. 1:12 during the addition of iodine and supply of nitrogen. For the denitration, ammonium polysulphide is preferably used as polysulphide in weakly alkaline aqueous solution.

The 8-methoxy or 8-ethoxy-phenanthrene compound

(R2 and R3 together form an aromatic C-C bond; Rx means methoxy or ethoxy) can be transferred by ether cleavage in the corresponding 8-hydroxy compounds, e.g. by hydrolysis with pyridine hydrochloride.

The products produced serve to increase resistance to infection. Thus, they cause a significant activation of leukocyte phagocytosis. The products produced are therefore applicable for the treatment of general infections, e.g. for mycobacteria or pneumococci, and for the treatment of local infections. The prepared agents are also applicable when there is a depression of phagocytosis, e.g. after the use of corticosteroids or cytostatics. When using the prepared agents, the depression of phagocytosis can be normalized again.

In addition, an antiviral effect was found in the manufactured agents.

The compound produced according to the invention was examined, among other things, with regard to their macrophage-activating effect. The stimulation of monocytes and macrophages in the abdominal cavity of mice was used as a parameter.

For the production of an increased chemotactic immigration of monocytes into the abdominal cavity and the differentiation of monocytes into macrophages with an increased ability for phagocytosis, an intraperitoneal injection of the test substance is appropriate.

The stimulation is a process that requires a certain amount of time. The testing of the phagocytosis activity was therefore carried out as a normal pharmacological model after a three-day pre-treatment of the mice with the compounds produced according to the invention. In some experimental groups, the intraperitoneal cell population was taken immediately after substance release to show that the observed stimulation only appeared after a certain incubation time.

Substances and materials

The substances to be tested were dissolved at 0.5 mg/ml in water and diluted as necessary.

May Griinwald solution

modified (Merck, Art. 1424)

Sheep blood

No. 5, 30.08.84, MPI Freiburg

Antiserum

Anti-sheep erythrocyte serum from rabbits No. 308,

IKA 468/6-11 days; 18.07.78, MPI Freiburg.

Laboratory animals

The investigation was carried out with 18-week-old hybrid female mice (Balb/c x C57B16). The animals were acclimated to laboratory conditions for 5 days before the beginning of the experiments. They received pelleted standard feed for rats and mice (Altromin no. 1324) and tap water ad libitum. They were tested with a thioglycolate standard for macrophage stimulability and showed good positive reactions.

Dosage and application

The substances were applied in aqueous solution (0.5 mg/ml) in the following doses:

The dilution of the mother solution for the application of the different doses was at: always below 0.4 ml.

The production of macrophage cell suspension and phagocvtosis Experimental principles ipp

After application of immunologically activating substances, the macrophages in the experimental animals are stimulated, and monocytes are produced for differentiation in the macrophage. The undifferentiated, stimulated macrophages are also able, after isolation, to phagocytose with antigenic structures opsonized with corresponding antibodies, in this case sheep erythrocytes in vitro. The phagocytosed erythrocytes can be seen microscopically.

The execution

After corresponding pretreatment, the mice are killed by neck-vertebral fracture. The peritoneum is exposed and 4 ml of DMEM (5% fetal calf serum, 0.2% heparin) is applied intraperitoneally. After approx. 30 second massage, 3 ml of cell suspension is taken out with a syringe and placed in a polypropylene tube in a cold bath (0°C). From each experimental group, one mouse was selected to measure the cell concentration in the exudate; a concentration around 4 x 10<5> cells/ml was approximately set.

Isolation of adherent macrophages:

Macrophages and monocytes are selected from the isolated cell population by incubating it on coverslips; macrophages and monocytes adhere to it, but other cells do not. In tissue culture dishes with 24 cups (polystyrene, Fabrikk Costar) are placed in round coverslips and overlaid with 0.25 ml DMEM (with 5% fetal calf serum). To redistribute the cells, the dishes are placed in a shaker, and each insert is pipetted while shaking (5 min., approx. 200 rpm) 0.25 ml of cell suspension. The tissue culture dish is then incubated for 1 hour at 37°C and 8% C02 in an incubator. After incubation, the non-adherent cells are aspirated and then washed twice with 0.25 DMEM (plus 5% fetal calf serum).

Opsonization of sheep erythrocytes:

1 ml sheep blood is washed twice with 4 ml PBS/BSA (centrifugation for 10 min. at approx. 500xg). The washed blood is diluted 1:50 with PBS/BSA. The antiserum against sheep erythrocytes is diluted with PBS/BSA 1:200. Diluted sheep blood and diluted antiserum are combined 1:1 and incubated for 1.5 hours under gentle shaking (approx. 80 rpm).

Phagocytosis:

Pipette 0.5 ml of opsonized erythrocyte suspension into each cup of the tissue culture dishes with adherent macrophages on the coverslips. The dishes are incubated for 20 min. at 37°C and 8% C02. The excess erythrocytes are then suctioned off, and the coverslips are washed twice with DMEM (0.5% FCS).

To lyse non-phagocytosed erythrocytes, these are treated by passing 1 ml of NH 2 Cl-tris-buf f er (an action time of 3.75 min. was measured to be suitable). After aspirating the buffer, the slides were washed twice with PBS/BSA.

Faaocvtose production (staining techniques)

After phagocytosis, the macrophages are stained according to Pappenheim according to the combined May-Griinwald-Giemsa method. Staining is performed in the dishes of the tissue culture dishes.

- 5 min. May-Grunwald, concentration suction

- 3 min. H20 double distilled, with H3P0A minimum

85% adjusted to pH 7.0; extraction

- 9 min. Giemsa solution 1:40 diluted and filtered before use; extraction - post-rinse with H20 dest. from spray bottle.

The cell nuclei are red violet after staining, and the cytoplasm is light blue. The phagocytosed erythrocytes are pale pink to look at. After air-drying, the microscope preparations are glued with "Eukitt" onto slides

(3 preparations/mouse).

Microscopic assessment

Of the three microscope preparations from one mouse, two were taken out. A Zeiss microscope with 1000 times magnification and oil immersion was used. The third preparation was used when the result from the two preparations was not clear. Per preparation, 300 cells were counted and, depending on the phagocytosis activity, divided into classes with increasing numbers of erythrocytes/macrophages.

+ For the calculation, it was assumed that those > 11 erythrocytes/MPH had the distribution in class 12 a center of gravity at 12 erythrocytes/macrophage. The macrophage from class 12 appeared relatively rarely.

Data processing and graphic representations

The counting of microscopic preparations was carried out on a Wang LVP 2200 counting system with a special feeding program that allowed the counting classes to be taken separately. The mean value of individual data was calculated with the program "MA 1", and the graphical representations with the program "NPL".

The stimulation effect was shown for each test substance and dose in two ways: 1. Distribution of the macrophages on the classes (erythrocytes/macrophage) in % in relation to 300 cells as 100%. This also includes the number of macrophages in the graphical representation that had not phagocytised any erythrocytes. 2. Distribution of phagocytosed erythrocytes (number of NPH in a class x number of erythrocytes/MPH) on the classes in % in relation to the total number of phagocytosed erythrocytes as 100%. Here, the number of macrophages that did not have is not taken into account

phagocytosed some erythrocytes.

Parameters

As a parameter for macrophage stimulation, the rise in phagocytosis activity expressed as the number of phagocytosed erythrocytes per macrophage (= class). A stimulation manifests itself in a reduction of macrophages that had phagocytosed no or few erythrocytes, and in an increase of macrophages with more erythrocytes.

Result

MPH stimulation with l-carboxy-3,4-methylenedioxy-8-methoxyphenanthrene (i.p.):

Fig. 1: 3 x 20 µg/kg i.p.

MPH stimulation after 3 days (x ± SEM)

a) Distribution of 300 cells in classes 1 to 12 in %

b) Distribution of phagocytosed erythrocytes in classes 1 to 12 in %.

12

Figure 2: 3 x 500/4^x)/kg i.p.

MPH stimulation after 3 days (x + SEM)

a) Distribution of 300 cells in classes 1 to 12 in %

b) distribution of phagocytosed erythrocytes in classes 1 to 12 1%

Table: 3 x 500 /*g/kg i.p.

MPH stimulation after 3 days

Figure 3: 3x5 mg/kg i.p.

MPH stimulation after 3 days (x + SEM)

a) Distribution of/ 300 cells on clusters 1 to 12 in %

b) Distribution of phagocytosed erythrocytes among the classes

1 to 12 in .%

Table: 3x5 mg/kg i.p.

MPH stimulation after 3 days

Dose dependence of st imulerinctsef fect

The control groups according to i.p. respectively p.o. administration of physical NaCl solution shows that the i.p. application itself already causes a weak macrophage activation. A large proportion of macrophages have phagocytosed 2 erythrocytes.

An increase in the macrophages by more than 2 erythrocytes must therefore be perceived as a substance effect. The percentage of the erythrocytes from class 4 (macrophages with 3 erythrocytes and more) was therefore set in relation to the dose.

Table 4. Dependence of the % sum of erythrocytes from 4.

class and above of the dose according to i.p. administration of 20 nq/ kq, 500 /xg/kg and 5 mg/kg. Controls are at 45% ± 6% (x ±SEM). In the figure, the area ±SÉM around the mean value is indicated by the broken line.

Judgment

The substance causes an increase in macrophage activation of 38% at 20 M9/kg and 64% at 500 µg/kg. A further increase in the dose to 5 mg/kg leads to a slight weakening of the stimulation. Macrophage activation does not proceed linearly in the selected dosage range.

The experimental group that was applied intraperitoneally once immediately before the macrophage extraction showed that the absorbed effects only appeared after an incubation period. This result suggests that the substance induced in vivo the mechanisms responsible for the increase in phagocytosis in the macrophages.

This indicates the following effects:

1. Stimulation of the membrane internalization rate

2. Increase in Fc receptor density and/or

3. increasing the mobility of the C3b receptors.

The other compounds produced according to the invention show a comparable pharmacological effect. The effect could be confirmed with other pharmacological models and clinically.

The following examples illustrate the preparation according to the invention of the compounds.

Example 2:

Preparation of 1-carboxy-3,4-methylenedioxy-8-hydroxy-phenanthrene 1.Og l-carboxy-3,4-methylenedioxy-8-methoxy-phenanthrene was melted with 2.Og pyridine hydrochloride in an oil bath at 170°C , and kept under gas blowing with nitrogen at this temperature for 1 hour. After cooling, the solidified mass was taken up in 10 1 5% HCl and extracted 3 times with each time 10 1 chloroform. The extracts were combined, washed neutrally with water and filtered over silanized filter paper (Whatman 1 PS) for drying. After evaporation of the solvent at 30°C, and rotary evaporator, the mixture was esterified to separate the starting material and the hydrolyzate, distilling with a mixture of 2.5 L of methanol and 0.1 L of concentrated H 2 SO 4 for 3 hours under reflux. This was then poured into 10 liters of water and the aqueous-methanolic phase was shaken out 3 times with 10 liters of diisopropyl ether each time. The organic solvent was extracted with 10 liters of NaOH after it had been washed several times with water, during which the methyl ester of the 8-methoxy compound remained in the organic layer and the 8-hydroxy compound, during simultaneous ester hydrolysis, was transferred to the aqueous phase. After washing out with fresh diisopropyl ether, the alkaline phase was acidified and shaken out several times with chloroform, dried over phase separation paper WHATMAN 1PS and the solvent evaporated on a rotary evaporator. 1-Carboxy-3,4-methylenedioxy-8-hydroxyphenanthrene remained as thin-layer chromatographically uniform compound in solid form. Fluorescence: (MeOH; Xmax, nm): Activation: 261,302,318,329,

358, 377

Emission: 386, 402 Yield: 0.714 g (75%)

Claims (1)

Process for the preparation of compounds of formula (I) in which R-1 means hydrogen, hydroxy, methoxy or ethoxy, and their pharmaceutically acceptable salts, characterized by reacting 6-bromopiperonyl bromide with the formula with triphenylphosphine in an anhydrous solvent to a phosphonium salt of formula reacts the latter compound with benzaldehyde,. o-methoxy-benzaldehyde or o-ethoxybenzaldehyde in the presence of a strong base to a stilbene of formula wherein R-1 is H, OCH3 or OC2H5, the above-mentioned stilbene bromide converts upon UV irradiation into the phenanthrene derivative of formula and converts this compound either with a metal cyanide, especially copper cyanide, in a polar aprotic solvent, especially in dimethylformamide, to the corresponding nitrile, and transfers this by hydrolysis in an acid of formula (I), or transfers the above-mentioned compound with n-butyllithium and carbon dioxide in an acid of formula (I), and, if desired, converts a compound of formula (I) in which R^ means a methoxy group, by ether cleavage into a compound in which R^ means hydroxyl, or denitrates a nitro compound of formula: wherein R 1 is H, OCH 3 or OC 2 H 5 with a polysulfide to a compound of formula (I), and, if R 1 is OCH 3 , optionally converting the OCH 3 group by ether cleavage into an OH group.