NO144632B - ANALOGUE PROCEDURE FOR THE PREPARATION OF NEW THERAPEUTICALLY ACTIVE DIANHYDROHEXITID ACYL DERIVATIVES - Google Patents

Description

The present invention relates to an analogue method for the preparation of new therapeutically active compounds with the formula -

IN

wherein Q represents an acetyl, chloroacetyl, p-carbomethoxy-propionyl, 3-phenyl-propionyl or γ-phenyl-butyryl group.

The bianhydro-sugar alcohol derivatives of the diepoxide type, such as L-1,2-3,4-diepoxy-butane, 1,2-5,6-dianhydro-D-mannitol and 1,2-5,6-dianhydro-dulcitol are known agents with anticarcino-

gen action (Jarman, M. Ross. W.X.j.: The formation of epox-

ides from substituted hexitols. Carbohydrate Research 9,139.

1969, Jarman, M. Ross, W.CJ.: Formation of 1,2-5,6-dianhydro-D-mannitol from 1,6-dibromo- and 1,6-dimethanesulphonoxy-1,6-dideoxy-D- mannitol. Dhem.Ind. 42, 1789. 1967, Elson, A.L. ,

Jarman, M. Ross, W.C.J.: Toxicity, haematological effects and antitumour activity of epoxides derived from disubstituted hexitols. Europ.J.Cancer 4, 617. 1968.)

Among these compounds, 1,2-5,6-dianhydro-

dulcitol the most favorable pharmacological properties.

(Nemeth, L., Institåris, L., Somfai, S., Gal, F., Pålyi, I.,

Sugér, J., Csuka, 0., Szentirmay, Z. , Kellner, B.:. Pharmaco-

logy and antitumoral effect of dulcitol diepoxide. (1,2-5,6-dianhydro-galactitol, NSC-132313). Cancer Chemotherapy Rep.

1.56, 593. 1972, Institåris, L., Nemet, L., Somfai, S., Gål,

F., Hercsel, I., Zaka, S., Kellner, B.: Investigation into

the Correlation of Cytostatic Activity with the in vitro Di-

epoxide Formation on.some. Terminally Substituted Hexitols.

Neiplasma 17, 1, 15-24. 1970).

The intramolecular reactions between the free hydroxyl groups

and the epoxy rings cause, however, in buffered aqueous solution as well

in the absence of biological media transformations of the compounds.

These transformations give unfavorable or toxic metabolites with regard to the cytostatic effect.

To avoid the aforementioned consequences, corresponding protective groups are taken

for help that is broken down under biological conditions, whereby the diepoxy compounds arise as active metabolites. Dec-

suten, the protecting groups of the hydroxyl group modify the solubility character of the extremely water-insoluble hexitol diepoxide with a view to the cytostatic effect mostly favorably.

The use of such compounds also proves advantageous

based on other pharmacological considerations. The previous tumor experiments indicate this: which were carried out using individual members of this compound series. Results show e.g. the significant lowering of the toxicity and the effect changes that are exerted on the tumor spectrum (difference

equal tumor types).

Investigation of the toxicological and anticar. cinoqene the action of 1, 2- 5„ 6- dianhvdro- 3, 4- diacyl- dulcitol

The anti-tumor effect of the produced diepoxy-diacyl-dulcitol derivatives was investigated on transplanted tumors of animal origin. Each of the prepared compounds inhibited the growth of the transplanted tumors in a dose-dependent manner.

The toxicological and anticarcinogenic effect of 4,6-diepoxy-diacyl-dulcitol was compared to the obtained effect of 1,2-5,6-dianhydro-dulcitol (starting dulcitol) in different test systems.

Designation of the compounds

DAD = 1,2-5, 6-dianhydrodulcitol

Diac-DAD = 1,2-5,6-dianhydro-3,4-di-(acetyl)-dulcitol DS - DAD = 1,2-5,6-dianhydro-3,4-di-(p-carbomethoxy- propionyl)-dulcitol

FB - DAD = 1,2-5,6-dianhydro-3,4-di(jf-phenyl-butyryl)-dulcitol FP - DAD = 1,2-5,6-dianhydro-3,4-di-( (3-phenyl-propionyl)-dulcitol

CA-DAD = 1,2-5,6-dianhydro-3,4-di-(chloroacetyl)-dulcitol

Acute toxicity

The acute toxicity was determined on normal Swiss mice. Mortality was determined after single dosing after 21 days.

The toxicity decreases in parallel with the increase in molecular weight. Mortality is achieved both after intraperitoneal and after oral poisoning on the 5th-9th. day. This also shows that all agents (active substances) are also well absorbed in the gastrointestinal tract.

Cumulative properties (toxic cumulation)

The cumulative residual was determined according to Druckrey's method.

t

Effect on transplant tumors

a) Ehrlich Ascites Carcinoma

The tumor with 5 million tumor cells was injected i.p. in a mouse.

The treatment was started 24 hours after the tumor injection and repeated daily over 6 and 8 days respectively.

b) Walker 256 Carcinosarcoma Intramuscularis

After 24 hours from the injection of 20 million tumor cells,

it underwent a one-time treatment. For the determination of the therapeutic indices, the smallest dose was taken which, on the 10th day, resulted in a judgment of tumor inhibition exceeding 90%.

All investigated means are very effective. The therapeutic index for the new agents is more favorable than that for the unsubstituted DAD, the limiting dose for the compounds DAD, Diac-DAD and CA-DAD is 8-10 mM, for DS-, FB- and FP-DAD it is twice that.

The significant reduction in toxicity brought about by the diacyl diepoxides is accompanied by only a moderate reduction in the anticarcinogenic effect.

c) Effect on Harding-Passey s. c. Melanoma

The slow-growing, less susceptible tumor was known

induced through s.c. transplantation of a piece of tumor tissue. Treatment was started 24 hours after transplantation.

The measurements took place on the 21st day of the experiment.

The effect of the proposed compounds on these slow-growing, less sensitive tumors to anticarcinogenic agents significantly exceeds the effect of the epoxides that serve as controls.

d) Effect on Guérin Carcinoma

The effect of Diac-DAD is dose dependent. The effect of

the other remedies at the same dose are likewise beneficial.

In summary, it can be stated that the toxicity of the examined diepoxy-diacyl-dulcitol compounds in comparison with the original epoxide has decreased. The anticarcinogenic effect of the agent could be demonstrated on several transplantable tumors (Ehrlich ascites, Walker Carcinosarcoma, Harding-Passey Melanoma, Guérin Carcinoma), in some cases these even have a better tumor inhibition than the original diepoxide

(Harding-Passey Melanoma).

The calculated therapeutic index for Walker Carcinosarcoma is better with all new agents than that for DAD. The agents are well absorbed in the gastrointestinal tract.

With regard to the order of introduction of the acyloxy group and the epoxidation, two principal paths are possible: a) Subsequent construction of the epoxidation from 3,4-diacyl derivatives

b) Acylation of 1,2-5,6-dianhydro-hexitol.

For the production of the new compounds, route b) was chosen.

because oxirane ring formation after the acylation is associated with difficulties. The ring closure takes place in an alkaline medium, in which the substituents can be split off.

1,2-5,6-Dianhydrohexitol Compound of formula I

The present invention is thus characterized by reacting the corresponding 1,2-5,6-dianhydrohexitol with an acylating agent which indicates the Q groups defined at the outset.

In more detail, the new compounds are prepared as follows: 1 mol of 1,2-5,6-dianhydrohexitol is dissolved in an anhydrous, organic solvent, advantageously in benzene or in a benzene homolog at a volume/weight ratio of 40. Then one adds the solution 2.0-3.01 mol of an organic acid-binding base or an inorganic acid-binding agent. In some cases, the acid-binding base (e.g. triethylamine or pyridine) can be added in large excess. Thereby, this surplus serves as a solvent. To the solution containing the acid-binding agent, 2 mol of acylating agent are added in the form of a diluted, approx. 0.5 m solution, such that the reaction temperature in any case remains below the boiling point of the reaction mixture. An addition temperature of 20-50°C is appropriate. After the end of the addition, the reaction mixture is stirred for 10-180 min. at this temperature. In many cases, it is not necessary to stir 10-24 hours afterwards at room temperature.

The preparation of the reaction mixture can take place in two different ways, depending on whether an acid halide, acid anhydride or ester is used as the acylating agent.

a) During the acylation with acid halides, the precipitated salts are filtered after the reaction or the afterstirring from the reaction mixture and washed with an anhydrous organic solvent, advantageously with benzene. The pure solution is evaporated at a temperature of 50°C to dryness, the residue is mixed with methyl alcohol or other solvents (preferably with ether, ethyl acetate or other mixtures, etc.), and then cooled. After crystallization, the product obtained is filtered off. Additional crystalline product can be recovered from the mother liquor by concentration or precipitation with a solvent

(II generation). The product accounts for close to 10-20% of the originating

same product (I generation).

The obtained products, both I and II generation, are optionally purified by recrystallization, possibly several times of recrystallization (mainly in II generation).

b) During the acylation with acid anhydrides or esters, the reaction mixture is evaporated to half after the end of the reaction

of its original volume and the evaporated reaction mixture is poured with stirring into 2-3 times the volume of ice water. The aqueous phase is separated from the organic phase. If the aqueous phase has an acidic pH, the organic phase is washed neutrally

preferably by shaking with a 5-10% sodium bicarbonate solution. After the separation that follows the final washing, the organic phase is evaporated under reduced pressure and the residue is further worked up according to the regulations in point a). (Mixing with solvent, crystallization and possible recrystallizations).

As starting materials, 1,2-5,6-dianhydro-D-mannitol is used (melting point: 64-66°C, [oc]^ = +40° in water, c = 1.25) or 1,2-5,6-dianhydro-dulcitol (melting point: 98.5°C , [oc]D = +2°

in water, c = 1.25). The preparation of these compounds is described in the literature. (Jarman, M. Ross, W.C.J.: The formation of epoxides from substituted hexitols. Carbohydrate Research 9, 139.1969, jarman, M. Ross, W.C.J.: Formation of 1,2-5,6-dianhydro-D-mannitol from 1,6 -dibromo- and 1,6-dime-thanesulphonoxy-1,6-dideoxy-D-mannitol. Chem. Ind. 4_2, 1789. 1967).

As a medium or solvent, the anhydrous organic solvents are used which are not in any chemical interaction with the reaction components, i.e. do not react with them. Suitable solvents are the homologues of aliphatic, cycloalkyl and aromatic hydrocarbons or halogenated hydrocarbons. As a medium, even the organic acid-binding bases (e.g. triethylamine, pyridine, etc.) are suitable, when they are used in a corresponding excess. For washing the filtered salt, the amount of anhydrous solvent is suitably taken which corresponds to the original amount of solvent used to avoid the formation of a solvent mixture. Such solvent mixtures make it difficult to work up the reaction mixture.

Acid halides, acid anhydrides, mixed acid anhydrides or esters, preferably compounds with the general formula R-CO-X, are mentioned as acylating agents, where R means an optionally substituted alkyl-, aryl-, aralkyl-, cycloalkyl-, alkylidene-cycloalkyl-, heterocyclic or alkylidene heterocyclic group; X a halogen atom or an OR' group - in which R' stands for the alkyl, aryl, acyl or aralkyl group.

Tertiary amines, trialkyl amines, their homologues, further alkali carbonates can be used as acid binding agents. The use of triethylamine, which dissolves 1,2-5,6-dianhydrohexitides well and from which the formed salts could be easily isolated, proved advantageous.

Reaction conditions: The acylation generally takes place at a temperature of 20-50°C. An exception is the acid halides, which in the oc position are equipped with large space-filling groups. When using them at 50°C, a reaction time of 1-3 hours is necessary to carry out the reaction with good yield. The maximum temperature can reach the boiling point of the solvent used, or the reaction mixture. The temperature is normally regulated by the rate of addition

of the acylating agent. An external cooling is hardly necessary.

It is only used when the addition of the acylating agent takes place

too fast in the beginning.

It is particularly important that the starting compounds, solvents, acid-binding agents and the reagents (acylating agent) are completely anhydrous. This condition must be checked before use and, if necessary, the compounds must be subjected to further water removal and cleaning.

The pharmacological application of the anticarcinogenic new acylated dianhydro-hex±ol compounds can take place in different ways: Per os: the pure active substance itself or the pure active substance with the usual excipients for tableting (starch, lactose, talc) mixed in the form of tablets.

Intravenous: in aqueous solution or in solution in a physiologically inert organic solvent (various glycols, etc.)

Intramuscular: in the form of a solution as indicated above or

in the form of suspension.

Intraperitoneal or intracavitalis: also in the form of solutions or suspensions as above.

Locally: (in loco): in the skin or at the site of the operation, the active substance is used by itself or mixed with common antibacterial and wound agents (sulphonamides, corticoids, vitamins, etc.).

Active substance concentration in the various preparations:

The production of the anticarcinogenic compounds according to the invention is illustrated through the following examples.

Example 1

Preparation of 1, 2- 5, 6- dianhydro- 3, 4- diacetyl- dulcitol

1.46 g (10 minol) of dianhydrodulcitol and 60 ml of abs. benzene and stir for 10 min. at room temperature.

Add 2.8 ml (20 mmol) of abs. triethylamine (or 2.45 gd.v.s. 20 m ol anhydrous potassium carbonate) and 2.45 g i.e. 1.4 7 ml (20 nmol) freshly distilled acetyl bromide dissolved in 5 ml abs. benzene, stir for 20 min. at room temperature, then the precipitated triethylamine hydrobromide is suctioned off on a glass filter G3 and washed thoroughly with abs. benzene. The combined filtrates are evaporated in vacuo, the remaining syrup is added to 15 ml abs. ether and 5 ml abs. methyl alcohol and cooled, whereby a crystalline precipitation immediately occurs. The mixture is left overnight in a refrigerator, the precipitated crystals are suctioned off and then recrystallized from methyl alcohol. White needle-like crystals are obtained. Yield: 1.65 g ( 71 ,!/ <>) . Melting point: 91°C$

Rf = 0.63 (benzene : methyl alcohol = 90:15) -

Example 2

Preparation of 1,2-5,6-dianhydro-3,4-diacetyl-dulcitol with acetic acid

1.46 g (10 mmol) of dianhydrodulcitol and 60 ml of abs. benzene (or anhydrous toluene) and stirred 10

my. at room temperature. Add 2.8 ml (20 mmol) of abs. triethylamine, 2.04 g i.e. 2.0 ml (20 mmol) of freshly distilled acetic anhydride dissolved in 5 ml of benzene over one hour at room temperature, then stirred for a further two hours at 50°C. The solution is evaporated to dryness, then taken up with 20 ml of water, shaken with 30 ml of benzene, the benzene is washed with a 10% sodium hydrogen carbonate solution, dried over magnesium sulphate, filtered and evaporated. Add 15 ml abs to the remaining syrup. ether

and 5 ml abs. methanol, is cooled, whereby a momentary crystalline phase occurs. The mixture is left overnight in a refrigerator, then the precipitated crystals are filtered off and recrystallized from methyl alcohol. White needle-like crystals are obtained. Yield: 1.65 g (71.7%); Melting point: 91°C-, Rf = 0.63 (benzene : methyl alcohol = 90:15).

Example 3

Preparation of 1. 2- 5, 6- dianhydro- 3, 4- diacetyl- dulcitol with p- nitrophenyl acetate

1.46 g (10 mmol) of dianhydrodulcitol and 60 ml of abs. benzene (or anhydrous dichloroethane) and stirred for 10 min. at room temperature. The solution is added to 2.8 ml (20 mmol) abs. triethylamine and 3.62 g (20 mmol) p-nitro-phenylacetate, is stirred for 6 hours at 50°C and is left to stand overnight at room temperature. The solution containing the organic solvent is shaken with 20 ml of water and dried over magnesium sulfate.

The remaining syrup is added to 15 ml abs. ether and cooled. The mixture is allowed to stand overnight in a refrigerator, then the precipitated crystals are sucked off on a glass filter G2. Yield: 2.5 g. Melting point: 72-78°c. The yellow crystalline product thus obtained is purified as follows: The crystals are dissolved in 20 ml of benzene and treated with diethylamino-propylamine. After one hour, 10 ml of water is added and the whole is filled into a rinsing funnel, the aqueous phase is separated and the operation is repeated with 10 ml of water. The benzene solution is dried over magnesium sulphate and evaporated. The remaining syrup is added to 15 ml abs. ether and 5 ml abs. methanol and is left overnight in a refrigerator, then the crystals are sucked off.

Yield: 1.58 g (69%); Melting point: 90°C.

Rf = 0.63 (benzene : methyl alcohol = 90:18).

Analysis

When 1,2-5,6-dianhydro-D-mannitol is taken as the starting material

obtained by the above method 1.65 g (70%) 1,2-5,6-dianhydro-3,4-diacetyl-D-mannitol.

Example 4

Preparation of 1, 2- 5, 6- dianhdro- 3. 4- di- chloroacetyl- dulcitol

5 g (34 mmol) of dianhydrodulcitol and 150 ml of abs. benzene. After stirring for four hours, 10 ml (70 mmol) of abs are added to the mixture. triethylamine (or 5.7 ml abs. pyridine)

and then within approx. 5 min. at room temperature, 8.94 g are added dropwise

(6 mL; 68 mmol) freshly distilled chloroacetyl chloride solution

in 5 ml abs. benzene. The mixture is stirred for 30 min. at room temperature, then the triethylamine hydrochloride is suctioned off on a glass filter G3 and the filter cake is thoroughly washed with benzene. The combined filtrates are evaporated in vacuo. The remaining syrup is added to 15 ml abs. ether and 10 ml abs. methyl alcohol, is cooled, whereby a momentary crystal precipitation occurs. The mixture is left overnight in a refrigerator, then the precipitated crystals are suctioned off and recrystallized from methyl alcohol. White needle-like crystals are obtained.

Yield: 4.4 g (43%); Melting point: 102°C.

Analysis:

The further examples 5-8 are summarized in tabular form to provide a better overview. The end products in the following examples were prepared as in the manner described in the preceding examples.

Claims (1)

Analogy method in the production of new therapies tically active compounds of formula I in which Q means an acetyl, chloroacetyl, 6-carbomethoxy-propionyl, 3-phenyl-propionyl or y-phenyl-butyryl group, characterized by reacting the corresponding 1,2-5,6-dianhydrohexitol with a acylating agent which is capable of emitting a Q group as indicated above.