Method of production of essentially pure melatonin and the method of solubilizing melatonin in water

Abstract

A method of synthesizing an indole derivative of the tryptamine type particularly melatonine, melatonin comprising the steps of 1) reacting potassium phthalimide and 1,3-di-bromopropane to obtain 3-bromopropylphthalimide; 2) reacting 3-bromopropylphthalimide with sodium acetoacetic ester in ethanol to obtain ethyl-2-acetyl-5-phthalimidopentanoate; 3) reacting the product from step 2) with diazo-p-anisidine to obtain 2-carboxyethyl-3-(2-phthalimidoethyl)-5-methoxy-indole; 4) reacting the 2-carboxyethyl-3-(2-phthalimidoethyl)-5-methoxy-indole with 2N/NaOH and then 20% H₂ SO₄ to obtain impure 5-methoxytriptamine, which is purified by means of hexamethyldisilazane. The mono and disubstituted derivatives are obtained and the monosubstituted derivative is hydrolyzed with aqueous methanol and then recrystallized from ethanol. The N-acetyl derivative is prepared by reaction with acetic anhydride. Melatonine Melatonin of high purity is obtained for prophylaxy and also against AIDS (Acquired Immuno Deficiency Syndrome).

Description

BACKGROUND OF THE INVENTION

The present invention relates to a total synthesis method for preparing an indole structure derivative product class, of the tryptamine type, in particular of crystalized crystallized twice from ethanol, so as to remove the small amount of formed diphthalimidopropane.

There are thus obtained 48.2 g of a white crystaline crystalline solid, melting point 72°C, with a yield of 72%.

Purity is controlled for TLC on silica gel, using as eluent benzene-acetone (45:5), freshly prepared, Rf of about 0.95 (diphthalimidopropane having a lower Rf).

By analogous, reactions, in which K-phthalimide is added once, there is obtained a product which contains greater amounts of diphthalimidopropane, thereby it is necessary to purify by distillation (e.g. 150° C./0.25 mm) by using a Vigreux device without cooling, since the distillate tends toward solidification. The yield is substantially equal to the above disclosed yield.

Preparing Preparation of ethyl-2-acetyl-5-phthalimido-pentanoate ##STR2##

In a three-neck flask having a capacity of 500 ml, provided with CaCl₂ cooling there are dissolved 4.60 g (0.2 g/A) Na in 100 ml anhydrous ethanol. To the solution, at room temperature, there are added 27.32 (0.21 moles) of acetacetic esther acetoacetic ester and then, after ten minutes, 40 g of 3-bromopropylphthalimide and, after one hour, further 12.5 g (in total 52.5 g corresponding to 0.196 moles), by holding the reflux processing and continuing for further three hours.

At the end of this period, sodium bromide is filtered, the solution is neutralized by 2N HCl and ethanol is evaporated under reduced pressure. The residue is recovered by ether, washed by H₂ O×2, dried on anhydrous Na₂ SO₄ and the solvent is evaporated, thereby providing a light yellow oil which is crystalized crystallized by dissolving it in a minimum ethanol amount by adding a small amount of ether and upon ageing aging for a night.

There are thus obtained 45 g (yield 72%) of a white crystalline solid, with m.p. 60°C Upon recrystalisation recrystallization there is obtained a m.p. of 63°C Product also crystalizes crystallizes from benzene-petroleum ether.

TLC on silica gel, benzene-acetone (45:5), Rf about 0.70.

Purification of 4-anisidine

A sample of 4-anisidine, of a very dark colour, is dissolved in an excess of 2N HCl and the solution is repeatedly extracted by chloroform as far as the colour is no longer extracted.

The acid solution is boiled by with decolorizing charcoal and hot filtered. The strongly cooled filtrate is processed by concentrated NaOH and extracted by chloroform. The chloroform solution is dried on anhydrous Na₂ SO₄ and evaporated under reduced pressure. The residue is crystalized crystallized from benzene thereby providing a white lamellae product with a melting point of 57°C

Preparing Preparation of 2-carboxyethyl-5-(2-phthalimidoethyl)-5-methoxy-indole ##STR3##

24.64 g (0.2 moles) p-anisidine in 80 ml ethanol 120 ml water and 80 ml (0.96 moles) 37% HCl are diazotized at 0°-5°C by 14.5 g (0.21 moles) NaNO₂ in 40 ml water; at the end the reaction is continued for other 30 minutes at the same temperature.

The thus obtained diazonium salt solution is added to a solution (stirred and held at 0°C) of 63.46 g (0.2 moles) of ethyl-2-acetyl-5-phthalimidopentanoate and of 130.64 g (0.96 moles) of sodium acetate trihydrate in 700 ml ethanol. The reaction is continued for 1 hour (the end pH must be included in the 5-6 range); then the solution is brought to room temperature under stirring for other three hours.

At the end of this period, the mixture is diluted with 2 l water and extracted by CH₂ Cl₂ three times; the organic phase, after washing by water and drying on anhydrous Na₂ SO₄, is evaporated, thereby providing 89.2 g of a dark red oil which is dissolved in a minimum amount of ethanol and introduced into a 3-neck 1 liter capacity flask, provided with stirrer, cooler and loading funnel. By stirring and heating there are added in 20 minutes 480 ml of a 10% solution of gaseous HCl in ethanol, by refluxing for 2 hours.

At the end of this period, the mixture is cooled down (for a night in a refrigerator or for 3 hours in an ice bath) and filtered by fully washing with methanol, water and methanol again. The dry solid material has a weight of 57.3 g (yield 73%), with a m.p. of 234°-7°C

By recrystalisation recrystallization from glacial acetic acid there are obtained 54.9 g (yield 70%) with m.p. 239°-40°C

TLC on silica gel, concentrated benzene-methanol-ammonia (50:10:1), Rf about 0.80.

Preparing Preparation of 5-methoxytryptamine ##STR4##

Into a 3-neck 3 liter flask, provided with stirrer, cooler and loading funnel, there are introduced 58.86 g (0.15 moles) of 2-carboxyethyl-3-2-phthalimidoethyl-5-methoxy-indole and 187.5 ml (15 g; 0.375 moles) of 2N NaOH and the mixture is refluxed at 135°C for 2.5 hours, thereby providing a complete solution.

By holding stirring and temperature, there are added, in 30 minutes, 750 ml of H₂ SO₄ (at 20%) (v/v), by further reflux processing for 4 hours.

At the end, the solution is cooled (for a night in a refrigerator or for 3 hours in an ice bath), by removing by filtration the precipitated phtalic phthalic acid. The solution is made alkaline by cooling with 30% NaOH and extracted by CH₂ Cl₂ ×3; the collected extracted materials are washed with water, dried on anhydrous Na₂ SO₄ and evaporated, thereby providing 20.25 g (yield 71%) of crude 5-methoxytryptamine.

TLC on silica gel, sat. CHCl₃, NH₄ OH-methanol (50:2), Rf about 0.65.

Purifying Purification of 5-methoxytryptamine ##STR5##

To purify 5-methoxytriptamine, 19 g (0.1 moles) of 5-methoxytriptamine (in a raw condition) and 76 ml (58.86-0.36 moles) of hexamethyldisilazane are refluxed for a night in a flask with sodium hydroxide protected cooling.

The solution is firstly distilled under normal pressure for recovering excess HMDS (43.6 g; 0.27 moles; m.p. 124°-5°C) and then under a reduced pressure, thereby providing a mixture of biderivative (20.26 g; m.p 135°-40°C at 0.1 Torr) and monoderivative material (5.25 g; m.p. 165°C at 0.1 Torr).

The silyl derivative is hydrolized hydrolyzed by aqueous methanol, thereby providing 15.36 (0.08 moles) with a yield of 80%. The mixture is crystalized crystallized from ethanol, so as to provide a white product having a m.p of 120°-1°C

Preparing Preparation of N-acetyl-5-methoxytryptamine

PAC Method A ##STR6##

To a suspension, cooled in ice, of 20 g (0.105 moles) of pure 5-methoxytryptamine in 400 ml methylene chloride there is slowly added, under stirring, a cold solution of 20 ml (0.21 moles) of acetic anhydride in 200 ml methylene chloride. Stirring and cooling are continued for 1 hour (the reaction progression can be controlled by TCL) so as to obtain a full complete solution; then the solution is washed with Na₂ CO₃ ×2, under long stirring, and then with water. The organic phase, dried on Na₂ SO₄ and evaporated, provides 24 g (yield 98%) of lightly colored melatonine melatonin.

In order to obtain a white product it is sufficient to process, if necessary, by charcoal in acetone and then crystalize crystallize from acetone-water. There are obtained 20 g (yield 83%) with m.p 116°-7°C (Tottoli).

TLC on silica gel, chloroform-ethanol (9.1). Rf of about 0.60.

Yield 46.5%, calculated on 2-carboxyethyl-3-(2-phthalimidoethyl)-5-methoxy-indole.

Method B

To a suspension, cooled in ice, of 5 g (26.3 moles) of 5-methoxytryptamine (in a raw condition) in 100 ml methylene chloride there is slowly added, under stirring, a cold solution of 5 ml (52.6 moles) of acetic anhydride in 50 ml of methylene chloride. Stirring and cooling are continued for 1 hour (the reaction progression can be controlled by TLC), so as to obtain a full complete solution; then the solution is washed by Na₂ CO₃ 2N×2, under strong stirring, and then by water. The organic phase, dried on anhydrous Na₂ SO₄ and evaporated, provides 6 g of raw melatonine, crude melatonin which is purified by chromatography on column (4 cm; diameter/length ratio 1:5) of Merck silica gel (70-230 mesh) (120 g); the solution is eluted by methylene chloride in order to remove the scarcely polar products thereby providing pure melatonine melatonin by eluting with methylene chloride-acetone (8.2). There are obtained 4 g of product (purifying yield 65%) which are crystalized crystallized from acetone-water. Melting point 116°-7°C

TLC on silica gel, chloroform-ethanol (9:1), Rf of about 0.60.

Yield of 46% calculated on 2-carboxyethyl-3-(2-phtalimidoethyl)-5-methoxy-indole.

In order to better disclose the total synthesis synthetic method according to the present invention, reference is now made to the accompanying drawing, in which there is shown the diagram of the several steps of this method.

The thus obtained melatonine melatonin has such a purity that it can be used, in suitable packages, both in the tumoural prophylaxis and in the tumoural therapy, as well as against AIDS.

In fact it has been found that this product, administered in suitable doses and with suitable procedures, provides, in addition to the above mentioned effects, also specific effects, such as:

a calming and slightly hypnotic action (which is useful for improving the antipain effect) and an antispasm effect (which is indispensable in the therapy of primitive tumours and brain metastasis tumours);

a mielotropic action, thereby it is possible to use comparatively high doses of radiation and chemical therapeutical substances;

an antimitotic action, perhaps of the same type of those found on the microtubule arrangement and eyelash regeneration;

a modulating action on the NK cell activity.

In this connection it should be pointed out that, in order to obtain the above mentioned effects, there are sufficient very small doses by os or by i.m injection or endovenous injection: from 2 mg/day to 20 mg/day; higher doses should be avoided in order to prevent the antiaggregating action of melatonine melatonin on the circulating platelets.

In addition there has been recently found a possible relationship between opium peptides and the action mode of melatonine melatonin.

This fact is very important, since antagonists are able of slowing neoplastic growth, whereas opium agonists seem to have an antimithotic action, both in vivo and in vitro.

In particular, melatonine melatonin has been found to be of essential importance in the following cases:

neuroblastoms, glioblastoms and astrocytoms;

leio and rabdomions;

condro-osteo-mixo-liposarcomas;

melanomas;

tumours of the respiratory paths and lungs:

tumours of the digestive apparatus;

tumours of the man and woman genital apparatus, loins, and prostate;

spino and basocellular epitheliomas:

malignant lymphomas and, with a less efficacy, in the Hodgkin lymphoma;

plasmocytoms;

thyroid tumours;

mamma mammary tumours;

linphoblastic leukaemia and cronic limphoides;

mieloblastic leukaemia and cronic mieloid.

Thus, we can reasonably think that the efficacy of melatonine melatonin in the above very different nature tumours is such as to advise its use it because of its general indirect and not specific action, which, on the other hand, is very essential.

In this connection it should be moreover pointed out that in the last ten years, experimental reports have stressed the fact that the neuroendocrin neuroendocrinologic system and immunity seem to be mutually related and that some diseases, characterized by immunitary disorders, may be due to alterations of this interrelationship.

Among the several modulating neuroendocrin neuroendocrinologic factors affecting the immunitary system pineal secretions and endogen opium peptides seem to have a very important function.

In fact there has been demonstrated that both pineal gland and opium system are involved in the control of cellular growth and tumoural growth.

At the immunitary system level, the endogen opium peptides seem to provide a stimulating-action; in particular endorphine may, under given conditions, stimulate the NK activity and the interleukine production.

On the other hand, basic data seem to suggest that melatonine melatonin, i.e. N-acetyl-5-methoxytryptamine has a very important function in maintaining an efficient immunologic immunological response in rats, under induced immunitary experimental stimulation.

The effect provided by melatonine melatonin, under the disclosed experimental conditions, is hindered by the simultaneous administration of naltrexone; this suggests that the immunomodulating action of melatonine melatonin can be controlled by opium mechanisms.

Under basal condition and in the absence of the immunity activity, in rats, the melatonine melatonin administration has no efficacy.

There has been moreover demonstrated that repeated administrations of pineal extracts induce lymphocytopoiesis and timic hyperplasia, whereas pinealhectomy causes timic atrophy.

In this connection it should moreover be stressed that pineal endocrin endocrinologic function itself seems to be modulated by opioid tone and that, vice versa, some typical actions of opium substances, such as analgesic action, are controlled by the activity of pineal gland and follow a circadic rythm circadian rhythm.

Thus, one may reasonably think that the pineal gland, through its main melatonine melatonin hormone, as a structure involved into the modulation of the neuroendocrin neuroendocrine activities, is able of controlling the effects exerted by phychoemotional effects on the immunitary system.

In fact, documented circadic circadian variations of the NK activity could be related to the circadic rythm circadian rhythm of melatonine melatonin, as demonstrated by some recent results.

From a lot of experimental tests, it has been found that surprising results has been obtained in the treatment of patients affected by AIDS.

These patients have been treated by melatonine with melatonin with doses of 20 mg per day and, after a long therapy, it has been demonstrated that they had a less amount of infections, with a significative increase of the "null cells", as determined by an examination of peripheral blood.

Melatonine Melatonin, or N-acetyl-5-methoxytryptamine, which has a formulation which constitutes the subject matter of the Italian Patent Application No. 23,323 A/79 in the name of the same applicant applicants, and which his is herein included by reference, has been found to provide significative improvements in the treatment of patients affected by AIDS.

The effect of melatonine melatonin is further increased as melatonine melatonin is used together with azidotimidine.

In particular, patients affected by AIDS, who were treated by azidotimidine with a dose of 3 mg/kg each four hours, and who required weekly blood transfusions because of the alteration of the coagulation processes, and subjected to a simultaneous treatment by melatonine melatonin, with a dose of 20 mg per day, provided greatly improved collateral effects affording the possibility of performing blood transfusions at 8 week intervals.

These patients have been treated by melatonine with melatonin, with the mentioned doses of 20 mg per day, and after a long therapy it has been demonstrated that they had a less amount of infections, with a significative increase of the null cells, as determined by an examination of peripheral blood.

Thus it has been found that melatonine melatonin can efficiently treat patients affected by AIDS, mainly in combination with other known treatment methods.

In this connection, it should be apparent that all of the administering details and the used doses can be suitably changed depending on each patient.

In particular, for a better use of melatonine, melatonin the present invention suggests to solubilize it with in water in order to facilitate the therapeutical applications, by using a particular method.

In fact, as is known melatonine melatonin is a substance scarcely soluble in water, and satisfactorily soluble at 40°-45°C

After long experimentation applicant has found that adenosine is adapted to easily dissolve melatonine melatonin in water.

In particular an optimal ratio has been found i.e.:

for a mole of melatonine melatonin (252.27 g) must be used four moles of adenosine (267.26 g).

From the above disclosure it should be apparent that the invention fully achieves the intended objects.

While the invention has been disclosed and illustrated with reference to some embodiments thereof, it should be apparent that the disclosed embodiments are susceptible to several modifications and variations all of which will come within the spirit and scope of the invention, as defined in the accompanying claims.

Claims

1. The method of solubilizing melatonine melatonin in water which consists of mixing melatonine melatonin with adenosine in a ratio of one mole of melatonine melatonin to four moles of adenosine whereby a water soluble product is obtained. 2. A method of preparation of melatonin having a high degree of purity which consists of the steps of:

a) reacting potassium phthalimide with dibromopropane, whereby 3-bromopropylphthalimide is obtained;

b) reacting 3-bromopropylphthalimide from step a) with acetoacetic ester in the presence of sodium ethoxide whereby ethyl 2-acetyl phthalimido-pentanoate is obtained;

c) reacting said ethyl 2-acetyl phthalimido pentanoate from step b) with the diazonium salt of p-anisidine whereby 2-carboxyethyl 3-(2-phthalimidoethyl) 5-methoxy indole is obtained;

d) reacting said 2-carboxyethyl 3-(2-phthalimidoethyl) 5-methoxy-indole from step c) first with sodium hydroxide and then with sulfuric acid whereby crude 5-methoxy-tryptamine is obtained;

e) reacting said crude 5-methoxy-tryptamine from step d) with hexamethyl disilazane to obtain a mixture of mono- and disubstitution products and hydrolyzing said mixture with aqueous methanol to obtain essentially pure 5-methoxy-tryptamine;

f) reacting said essentially pure 5-methoxy-tryptamine from step c) with acetic anhydride to obtain crude melatonin and purifying said crude melatonin by chromatography on silica gel and first eluting with methylene chloride followed by eluting with methylene chloride and acetone to obtain a solution, concentrating said methylene chloride and acetone solution to obtain a solid and recrystallizing said solid whereby purified melatonin

is obtained. 3. The method according to claim 2 wherein said step d) is carried out by refluxing at 135°C for 21/2 hours until complete solution is obtained, then adding a 20% (v/v) H₂ SO₄ solution and further refluxing for four hours. 4. The method according to claim 3 wherein after refluxing with said 20% sulfuric acid, the solution is cooled to let phthalic acid precipitate and filtering off said phthalic acid. 5. The method according to claim 4 wherein after said phthalic acid is filtered off, sodium hydroxide is added and crude 5-methoxytryptamine is extracted with

methylene dichloride. 6. The method according to claim 2 wherein said step f) is carried out by refluxing for 12-14 hours said crude 5-methoxytryptamine with hexamethyl-disilazane, to obtain the mono and di-silyl substitution products, then distilling the solution under normal pressure so as to recover excess hexamethyl disilazane and hydrolyzing the silyl substitution products with aqueous methanol whereby essentially pure 5-methoxytryptamine is provided.