NO151088B - ANALOGY PROCEDURE FOR THE PREPARATION OF PHARMACOLOGICALLY EFFECTIVE SILYL-GLUCOSAMINE DERIVATIVES - Google Patents

Description

The invention relates to an analogue method for the production of pharmacologically active silyl-glucose derivatives of formula I

wherein R 1 , R 4 and R 8 are tri-lower alkylsilyl, primarily

trimethylsilyl, R2 means lower alkyl or phenyl, R3 means hydrogen or alkyl with 1-3 carbon atoms, R7, Rg and R13 independently mean hydrogen or lower alkyl, RtQ i means lower alkyl, R1Q means unsubstituted or carbamoylmethyl substituted carbamoyl and R12 means trilower alkylsilyloxycarbonyl or carboxy.

Alkyl is straight or branched as desired

position bonded alkyl.

Those in connection with the present description

and claims with "lower" designated residues and compounds preferably contain up to and including 7, primarily up to and including 4 carbon atoms.

In the above as in the following, can

the general terms have the following meaning:

Lower alkyl is e.g. n-propyl, n-butyl, iso-butyl, sec.-butyl, or tert.-butyl, further n-pentyl, n-hexyl, isohexyl or n-heptyl and primarily methyl or ethyl.

The new compounds produced according to the invention can be in the form of mixtures or isomers or of pure isomers.

The compounds of formula I have valuable pharmacological properties, in particular a pronounced immunopotentiating effect. This can be demonstrated with the help of the following mentioned experimental device: 1. Potentiation of the cellular immunity in vivo: Increase of late-type hypersensitivity to ovalbumin in guinea pigs.

Pirbright guinea pigs are immunized on day 0 with

10 mg Ovalbumin in complete Freudian adjuvant by injecting each time 0.1 ml of an antigen-adjuvant mixture into the two hind paws. 4 weeks, the skin reaction is triggered by intracutaneous injection

tion of 100 ug Ovalbumin in 0.1 ml of buffered physiological salt solution and is quantified due to 24 hours afterwards using the Erythem surface and the increase in skin thickness calculated reaction volume. The antigen-specific increase in reaction volume observed after 24 hours (late-type reaction) is used as a measure of cell-mediated immunity. Ovalbumin is too weak an immunogen for alone or in a water-oil emulsion incomplete Freud's adjuvant (10 parts Ovalbumin solution in 0.9% NaCl mixed with 8.5 parts "BayolF" and 1.5 parts "Arlacel A") to induce a sen-type reaction , but for effective immunization must be applied in complete adjuvant with added mycobacteria (5 mg killed and lyophilized M butyricum per 10 ml "Bayol F"/"Arlacel A").

For the detection of the immunopotentiating effect of the test substances, these can reach the site of the mycobacteria in doses of 10 to 100

yg is added to the antigen-oil mixture.

The glucosamine peptides of formula I are capable of

to mimic the effect of the mycobacteria in the mentioned experimental device and to surpass it quantitatively.

Thus it is shown that the compound of the mentioned type is able to considerably increase cellular immunity, namely both in mixture with the antigen itself (adjuvant effect in a narrower sense) or also by temporal and local administration separate from the antigen injection (systemic immunopotentiation).

2. Potentiation of humoral immunity in vivo: Increase in antibody production against bovine serum albumin. n (BSA)

in mice.

NMRI mice are immunized by intraperitoneal (i.p.) injection of 10 µg of precipitate-free BSA on day 0. 9, 15 and 29 days later, serum samples are taken and examined for their content of anti-BSA antibodies with a passive Haemagglutination technique. In the dose used, soluble BSA is subimmunogenic for the recipient animal, i.e. it fails to trigger or only triggers a very small production of antibodies. Additional treatment of the mouse with certain immunopotentiating substances before or after the administration of the antibody leads to an increase in the antibody titer in the serum. The effect of the treatment is expressed by the obtained score value, i.e. by the sum of the Log2 titer difference on the three bleeding days.

The compounds of formula I are capable, upon intraperitoneal or subcutaneous (s.c.) application of 100-300 mg/kg/animal on five consecutive days (day 0 to 4) after immunization with BSA, to markedly increase antibody production against BSA.

The immunostimulatory effect of said compound is, in contrast to other bacterial immunoleptics (e.g. LPS from E. coli) antigen-dependent: injection of the new compounds has only in BSA-immunized, however, not in non-immunized mice due to an increase of the anti-BSA titer. Remarkable is the so-called the filing of the aforementioned compounds as effective as in op. application, i.e. the observed immunopotentiating effect is systemic and does not depend on the stimulus having to be administered via the same route as the antigen, i.e. among the antigen as is the case with classic adjuvants.

In the experiments mentioned, it is shown that the compounds of the above type specifically manage to increase the humoral immunity,

that it improves the immunological irritation response, and that its immunopotentiating effect is due to a systemic activation of the immune system. 3. Potentiation of humoral immunity in vitro: T-cell-substituting effect by antibody response of mouse spleen cells • against sheep erythrocytes (SE).

In many cases, thymus-derived lymphocytes (T cells) are required for the induction of an antibody response. These cells cooperate in the processes of antibody-forming lymphocytes (B cells) and help them on stimulation to react with so-called T-dependent antigens such as proliferation, differentiation and anti-substance syntheses. The spleen cell suspension of congenital antimystic nu/nu mice contains no functional T cells and is capable of e.g. in vitro unlike SE not to form any anti-SE antibodies. The compound of formula I is surprisingly capable of functionally replacing T cells in such cultures and enabling an anti-substance response against SE. Addition of these substances to nu/nu spleen cell cultures in the presence of SE leads within 4 days to a considerable increase in the number of antibody-forming cells. The findings show that the aforementioned compounds are able to increase humoral antibody formation in vitro and are able to compensate for a defect in the T-cell system. 4. Selective mitogenicity for B cells: Proliferation-promoting effect in B-lymphocyte cultures

Suspensions of highly enriched B-lymphocytes (lymph node cells congenital athymische nu/nu mice), as well as pure immature and mature T-lymphocytes (Thymus cells or cortisone-resistant,

i.e. 48 hours after a cortisone injection, persistent thymus cells of Balb/c mice) are incubated in the presence of the test substances during three days. The incorporation of H 3-thymidine into the lymphocytes during the last 18 hours of the culture period applies as a measure of the proliferation activity.

The compounds of formula I are mitogenic for B-lymphocytes (ie for the course of the antibody-forming cells), but not for T-lymphocytes.

They are thus able to energize the proliferation of lymphocytes that participate in the humoral immune response.

5. Tolerability

Although the compounds of the mentioned type show their potentiating effect on guinea pigs, for example, already after a single dose of 0.05 mg/kg s.c., on mice after application of 5 times 10 mg/kg s.c, is also observed with application of 5 times 300 mg/kg i.p. no toxic effect on mice.

The aforementioned substances therefore have an excellent therapeutic range.

The compounds of formula I have the ability, on the one hand, when mixed with another antigen, to increase its immunogenicity,

on the other hand, by systemic application to increase the immunological reactivity of the treated organism. Thereby, the mentioned substances are able to promote both the cellular and the human

moral immunity and to activate the lymphocytes responsible for antibody formation.

The new compounds can thus be used as adjuvants in a mixture with grafting substances to improve the grafting result and to improve the infection protection against bacterial, viral or parasitic agents mediated by humoral antibodies and/or cellular immunity.

Finally, the compounds mentioned are suitable in admixture with various antigens as adjuvants in the experimental and industrial production of antisers for

therapy and diagnostics and in the induction of immunologically activated lymphocyte populations for the cell transfer procedure.

Moreover, the new compounds can also be used without simultaneous antigen delivery to promote immune reactions that are already underdeveloped in humans and animals. The compounds are therefore particularly suitable for stimulating the body's natural defences, e.g. in chronic and acute infections or in selective (antigen-specific) immunological defects, as well as in congenital but also acquired general (i.e. not antigen-specific) immunological defect conditions, which appear during age, during major primary diseases and above everything

after therapy with ionizing rays or with immunosuppressive hormones. The aforementioned substances can thus preferably also be administered in combination with anti-infective antibiotics, chemotherapeutics or other healing methods to counteract immunological damage. Finally, the mentioned substances are also suitable for general prophylaxis of infectious diseases in humans and animals.

Particularly valuable are compounds of the above-mentioned formula I.

Furthermore, it is to highlight compounds of the above-mentioned formula I, in which R^, and Rg means trilower alkylsilyl, primarily trimethylsilyl, means lower alkyl or phenyl,

R^ means hydrogen or methyl, <R>^, R^ and R^ mean hydrogen,

Rg means lower alkyl, R-^q means carbamoyl and R^ means

carboxy or trimethylsilyloxycarbonyl, .as well as the new i

the examples discussed compounds.

The compounds of formula I are obtained when, in a manner known per se, a compound of formula

in which the substituents have the above-mentioned meaning, are reacted with a silylating agent which introduces a trilower alkylsilyl residue and the formed silyl esters of formula I are optionally transferred with water to silyl ethers of formula I. As silylating agents, special mention should be made of: trilower alkyl silyl halides, especially -chlorides or -bromides, hexalaverealkyldisilazane, trilaverealkylsilylamine, trilaverealkylsilyldiethylamine, trilaverealkylsilylacetamide, bis-(trilaverealkylsilyl)acetamide or trilaverealkylsilylsulfamide, especially the corresponding trimethylsilyl compounds.

The reaction is preferably carried out in a solvent which does not contain reactive hydroxy or amino groups such as dimethylformamide, dimethylsulfoxide or acetonitrile, dioxane, tetrahydrofuran, dimethoxyethane or chloroform. One can also work in the presence of an anhydrous base such as triethylamine, piperidine, pyridine or imidazole. If a free carboxyl group is present in the starting materials, this can also be silylated under the reaction conditions. The silyl esters formed are, however, very easily split, e.g. on contact with water.

The above-mentioned method is carried out according to methods known per se in the absence or preferably in the presence of diluents or solvents, if necessary under cooling or heating under elevated pressure and/or in an inert gas such as nitrogen atmosphere.

The starting materials used and their preparation

is known.

The compounds of formula I can be used in the form of pharmaceutical preparations, which contain compounds of formula I. As such, these pharmaceutical preparations concern enteral, especially parenteral administration to warm-blooded animals, and which contain the pharmacologically active substance alone or together with a pharmaceutical applicable carrier material. Dosage-

one of the active substances depends on warm blood type. the age and the individual condition, as well as the method of application.

The new pharmaceutical preparations contain from approx.

10% to approx. 95%, preferably from approx. 20% to approx. 90% of the active substance. Pharmaceutical preparations according to the invention can be present, e.g. in dosage unit forms, such as suppositories or ampoules.

The pharmaceutical preparations are prepared in a manner known per se, e.g. by conventional mixing, granulating, coating, dissolving or lyophilizing methods. Preferably, the new compounds are dissolved in lecithins, even

tuled together with cholesterol, in a halogenated hydrocarbon, such as chloroform or methylene chloride, it then evaporates and

dividing the resulting mixture into an aqueous buffer solution, e.g. phosphate-buffered common salt of pH 7.7, e.g. by means of sound influence.

It can also be processed together with a suitable carrier for solid forms of administration. Suitable carriers are especially fillers, such as sugar, e.g. lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate, further binders, such as starch pastes using e.g. corn, wheat, rice or potato starch, gelatins, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/

or polyvinylpyrrolidone, and/or if desired, explosives,

such as the above-mentioned starches, further carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate, auxiliaries are primarily flow regulating and lubricating agents, e.g. silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Dragon cores are equipped with suitable, possibly gastric juice-resistant covers, using, among other things, conc. sugar solutions which optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions in suitable organic solvents or solvent mixtures or, for the production of gastric juice-resistant coatings, solutions of suitable cellulose preparations, such as acetyl cellulose phthalate or hydroxypropyl methylcellulose phthalate . Dyes or pigments can be added to the tablets or dragon-overcoats, e.g. for the identification or characterization of different active drug doses.

The invention shall be explained in more detail by means of

some examples.

Example 1

Dissolve 0.3 g of 2-benzamido-2-deoxy-3-O-^/~L-1-(D-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl-7-carbamoyl-methyl^-D-glucose in 3 ml dimethylformamide and mix with 0.4

ml of bis-trimethylsilylacetamide. After 5 hours at 35°C, it is evaporated in a vacuum to syrup, from which you can separate by dissolving abs. ether or abs. acetic ester formed acetamide. After renewed evaporation, a colorless syrup with [_ = + 15° is obtained

(c = 0.8 dioxane) of 2-benzmido-2-deoxy-4-O-£/~L-1-(D-1-carbamoyl-3-trimethylsilyl-carboxypropyl)-carbamoyl-ethyl-7-carbamoyl-methylj -1, 4,6-O-tris-trimethylsilyl-α,B-D-glucose.

On contact with water, the trimethylsilyl ester group is quickly saponified, giving 2-benzamido-2-deoxy-3-0-L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl/-carbamoyl-methylJ-1, 4,6-tris-O-trimethylsilyl-α,B-D-glucose.

Analogously, syrupy 2-acetamido-2-deoxy-3-0-^~L-1-(D-1-carbamoyl-3-trimethylsilyl-carboxy-propyl)-carbamoyl-ethyl7-carbamoylmethyl 1,4,6-tris-O -trimethylsilyl-α,3-D-glucose with </~a7p°= -10°C (c = 0.91, dioxane) .

Example 2

Dissolve 3 g of 2-benzamido-2-desoxy-3-0-£/~L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl-7-carbamoyl-methyl^-D-glucose in 20 ml of dimethylformamide and mix with 4 ml of bis-trimethylsilylacetamide. After 5 hours at 35°C, it is evaporated in a vacuum to syrup. Take up in chloroform, shake out briefly with ice water and dry the organic phase. After renewed evaporation, a mixture of the corresponding 1,4,6-tris-trimethylsilyl ether trimethylsilyl ester and the associated tris-trimethylsilyl ether is obtained as free carboxylic acid.

Example 3

Analogous to the above-mentioned example, the following trimethylsilyl ether esters are obtained, from which trimethylsilyl ethers are formed on contact with water: 2-acetamido-2-desoxv-3-0 carbamoylethylJ -1,4,6-tris-trimethylsilyl-a,3-D-glucose, / S/q0 = + 1°° (c = 1 dioxane) Decomposition range: 99-106°C, = 0.52 (chloroformrmethanol = 7:3

in mixture with 1.5% triethylamine.

2-acetamido-2-deoxy-3-O-[D-1-(L-1-/~D-1-carbamoyl-3-trimethyl-silyl-carboxy-propyl-7-carbamoyl-propyl)-carbamoylethyl)J-1,4 , 6-O-tris -trimethylsilyl-α,3-D-glucose, decomposition point 102°C,

t ^/q 0 = + 54°C (c = 0.91 pyridine), Rf = 0.27 (chloroform:ethanol = 1:1).

2-acetamino-2-desoxy-3-0-^D-1-(L-1-(D-1-carbamoyl-3-trimethyl-silyl-carboxypropyl/-carbamoyl-2-methyl-propyl)-carbamoylethyl) - 1,4,6,0 -tris-trimethylsilyl-α,3-D-glucose, syrup 2-propionamino-2-deoxy-3-0- [<_> £L-l-(D-l-carbamoyl-3-trimethyl-silyl-carboxy-propyl)- carbamoyl-ethylJ -carbamoyl-methyl7-1,4,6-O-tris-trimethylsilyl-α,3-D-glucose, decomposition 91-96°C £~ u7p° = Rf = 0.25 (chloroform:ethanol= 1 :1).

2-benzoylamino-2-desoxy-3-O-/ £ L-l-(D-l-carbamoyl-3-trimethyl-silyl-carboxypropyl)-carbamoyl-propyl^j -carbamoyl-methyl7 -1,4,6-O-tris- trimethylsilyl-α,B-D-glucose, decomposition range 93-98°C «7^° = +17° Rf = °'33 (chloroform:ethanolrl:l).

2-acetamido-2-deoxy-3-0-/| L-1-(D1-carbamoyl-3-trimethylsilyl-carboxy-propyl)-carbamoyl-propylj -carbamoylmethyl/-1,4,6-0-tris-trimethylsilyl-α,3-D-glucose, syrup.

2-acetamino-2-desoxy-3-0-/ ^L-1-(D-1-carbamoyl-3-trimethylsilyl)-carboxypropyl)-carbamoyl-2-methylpropyl3-carbamoyl-methyl7-1,4,6-0-tris-trimethylsilyl -α,3-D-glucose, syrup.

2- acetamido-2-deoxy-3-O- (Dl-N-carbamoylmethyl-carbaroyl-3-trimethylsilyl-carboxypropyl)-carbamoyl-ethylJ -carbamoyl-methyl/- 1,_4, 6-O-tris- trimethylsilyl-α, 3-D-glucose, decomposition 93-98°C +47° (c = 0.8 pyridine) Rf = 0.24 (chloroform:ethanol= 1:1).

Example 4

The mentioned trimethylsilyl derivatives can be used as adjuvants advantageously in the following way: 1. One part of the silyl compound is dissolved either together with 10 parts of egg lecithin or together with e.g. 8 parts egg lecithin and 2 parts cholesterol in chloroform, evaporate in a rotary evaporator to a lipid film, which is mixed with a buffer solution, e.g. phosphate-buffered, physiological saline solution pH= 7.2. During slight cooling, you treat with ultrasound at 30-50 watts for approx. 3-5 minutes, so that the internal temperature does not exceed 30°C. You thus get a suspension of lipid vesicles, so-called liposomes, with a very uniform size of approx. 1-3 u diameter. Lightly lipophilic antigens are added in aqueous solution to this suspension and absorbed onto the liposomes. However, the antigens can also be added before sound treatment. Liposome suspensions thus formed can be injected, i.p. s.c., i.d. or i.m. 2. The required amount of the silyl compound is mixed with Draceol, mixed with the aqueous, buffered solution of the antigen and arlacel and sonicated as indicated above, until a stiff water-in-oil emulsion is obtained which can be sprayed . 3. The lipophilic silyl compounds can be absorbed from aqueous suspension, also on cells such as erythrocytes or tumor cells, whereby depending on the concentration, a small amount of polyethylene glycol 400 can be helpful as a dissolution agent.

Example 5

200 mg 2-acetamido-2-deoxy-3-0-[(L-1-/fD-1-carbamoyl-3-carboxy-propyl7-carbamoyl-ethyl)-carbamoylmethyl

α,3-D-glucose is allowed to react together with 283 mg of N.O-bis-trimethylsilylcarbamate in 15 ml of dimethylformamide for 18 hours at room temperature. Evaporate to dryness in a vacuum and keep the residue for a further 0.5 hours at 50°C and 0.06 torr. Amorphous 2-acetamine-2-deoxy-3-0-£(L-l-^~D-l-carbamoyl-3-trimethylsilyl-oxycarbonylpropyl7-carbamoyl-ethyl-carbamoylmethyl}-1,4,6-tris-O-trimethyl- silyl-a, 3-D-glucose with l~ a./^ = +47° (c=1.08, pyridine) and Rf = 0.27 (chloroform:ethanol = 1:1 in mixture with 1% triethylamine) on silica gel thin-layer plates from Merck.

Claims (3)

1. Analogy method for the preparation of pharmacologically active silylglucosamine derivatives of formula I in in which Ri; R4 and Rg mean trilower alkylsilyl, primarily trimethylsilyl, R2 means lower alkyl or phenyl, R^ means hydrogen or alkyl with 1-3 carbon atoms, R?, Rg and R13 independently mean hydrogen or lower alkyl, Rg means lower alkyl, R-^ q means unsubstituted or carbamoylmethyl substituted carbamoyl, and R12 means trilower alkylsilyloxycarbonyl or carboxy, characterized in that a compound of formula II in which the substituents have the above meaning are reacted with a silylating agent which introduces the trilower alkylsilyl residue, and the formed silyl ester of formula I is optionally transferred with water to silyl ether of formula I. 2. Method according to claim 1, characterized in that the starting materials are selected so that 2-acetamino-2-deoxy-3-0- ^/_ L-1-(D-1-carbamoyl-3-trimethylsilyl-carboxy-propyl)-carbamoylethyl/- is obtained carbamoyl-methyl}-1,4,6-O-tris-trimethylsilyl-α,3-D-glucose. 3. Process according to claim 1, characterized in that the starting materials are selected so that 2-benzamide-2-deoxy-3-0-L-1-(D-1-carbamoyl-3-trimethylsilyl-carboxyl-propyl)-carbamoyl-ethyl/-carbamoyl is obtained -methylJ -1,4,6-0-tris-trimethylsilyl-cx, 3-D-glucose.