CN1706853A - Akaloid reaction process and the application of reaction product - Google Patents

Abstract

The present invention relates to the technological process of adding one kind of phosphorus derivative into some organic solution with alkaloid for reaction, and the organic solution ,which contains reaction product of the alkaloid and un-reacted phosphorus derivative residue, is then washed with water. The obtained reaction product is suitable for being used as medicine.

Description

The effectiveness of alkaloid reaction process and reaction product

Invention field

The present invention is about a kind of novel reaction technology between alkaloid and the phosphorus derivant and the effectiveness situation of the product that generates.

Invention and prior art background

Nicotine because of its outside the organism internal memory in biological activity by known to the people.For example, people know that for a long time Chelidonium majus L (" pilewort ") juice is a kind of good verrucosis medicine.Chelidonium majus L. comprises more than 30 kind of alkaloid, and wherein a kind of Stylophorine that is has accounted for 80% of whole alkaloid component greatly.

If add a kind of cell growth or carcinogenic phosphorus derivant restrained on alkaloid, then institute's synthetic product is compared the toxicity reduction with start material matter, but can obtain to resist the cytotoxic activity of some cancerous cell line.

By some Chelidonium majus L. alkaloid and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide is reacted in organic solvent, DE 2 028 330 and US 3 865 830 have disclosed the preparation of thio-phosphamide-isoquinoline 99.9 adducts.

By reacting with the phosphorus compound of system cancer (salt by converting it into them and provide with water-soluble form), AT 354 644 and AT 377 988 have described alkaloidal phosphorus derivant preparation technology.This openly shortcoming of technology is that reaction product does not change into water-soluble salt fully, and most of reaction product is still water insoluble.

US 5 981 512 has disclosed the effectiveness of material when the treatment radiation injury that is drawn in AT 377 988 and AT 354 644.

The compound of describing in the above-mentioned patent has different supression cells and system cancer activity.Alkaloid mixture in alkaloid mixture, the especially total alkaloids of Chelidonium majus L. has been proved to be and has had unique pharmacological agent prospect, and verified its has pharmacological activity aspect cancer therapy in several researchs.Research has been found that said preparation has very important clinical effect.According to vegetable chemistry and plant treatment theory, suppose that the alkaloid that is not only single composition but all reacts has pharmacological activity.

The reactant that reaction will not react after finishing is removed from synthetic mixture.Because three (1-acridyl) phosphuret-(t)ed hydrogen sulfide can be dissolved in organic solvent, such as benzene again or chloroform, and according to the prior art that unreacted three (1-acridyl) phosphuret-(t)ed hydrogen sulfide is removed from synthetic mixture, we advise finishing the removal process with above-mentioned two kinds of solvent wash reaction product.

Figure of description

Fig. 1 is the HPLC collection of illustrative plates that all biological alkali composition of Chalidonium majus L. root place acquisition constitutes.

The preparations by HPLC finger printing of Fig. 2 for making according to example 1.

Fig. 3 is some phosphorus derivant that can be used as reactant.

Invention is introduced

Present invention is intended to provide a kind of novel process for the preparation of alkaloid and phosphorus derivant reaction product, and this technology makes alkaloid be converted into a kind of water-soluble substances.Especially therefore, except that selected alkaloid, the total nicotine that comes from ChelidoniummajusL. also can be converted, and obtains a kind of hypotoxicity and the wide as far as possible preparation of action spectrum scope.

This target is finished in 1 description as requested.This invents more specifically information and exploitation is set forth in the child requirement.

The technology of this invention comprises reacts a kind of alkaloid or multiple alkaloid mixture in certain organic solvent with a kind of phosphorus derivant, wash reaction product then with water.This kind phosphorus derivant preferably comprises at least one acridyl.Reaction product through washing then can be converted into a kind of water-soluble form.Especially when phosphorus derivant was toxic substance, it was preferably water miscible.

Water-washing step can fully be simplified preparation process, because this need not the explosion hazard of ether is taked complicated safe precaution measure.Therefore, this process can be amplified to technical scale without a doubt.And the composition of reaction product of decision pharmacological action is also because of washing process changes, and under the effect that liquid liquid distributes, water soluble component is dissolved from reaction product and removes.Surprisingly, have been found that now water also has catalytic effect in addition concerning reaction product, thereby influence the structure and the composition of synthetic product, and, then can cause 10-15 original reaction product amount doubly can convert water-soluble form to if wash with organic solvent.

Wash with water reaction product is transferred in the attitude, this can help then to convert other products to water-soluble form greatly.This method both had been applicable to the alkaloid of Chelidoniummajus L., also was applicable to the alkaloid that other sources obtain.This technology can be used at present, such as, can adopt for the reaction between alkaloid and the system cancer phosphorus compound, as AT 377 988 require 1 mention, the phosphorus derivant that shows in Fig. 3, especially those have the phosphorus derivant of acridyl, are specially adapted to adopt this kind technology.

The organic solvent that is suitable for refers to any solvable reagent that can be used for the alkaloid reaction.Such as, these alkaloids can be dissolved in methylene dichloride or the chloroform.

Alkaloidal being reflected under the comparatively high temps reacted, best results when solvent reaches boiling point.

After the washing, reaction product is converted to water miscible.This can carry out according to the technology of describing among AT 377 988 and the AT 354 644, by changing into water miscible salt, especially change into hydrochloride, for example, feed HCl gas or add HCl solution in the organic solvent of the reaction product of washing, then hydrochloride precipitates.The water-soluble salt of reaction product is adapted at using in the injection liquid.

The present invention embodies, and reaction needed three (1-acridyl) phosphuret-(t)ed hydrogen sulfide (CAS numbers 52-24-4) just can be finished, and the conduct of this material is exactly known tespamin.It also has more title, comprises ledertepa, Oncothiotepa, TESPA, tespamine, the sulphur phosphamide, sulfo--TEPA, sulfo-triethylene phosphoramide (TEPA), tifosyl, three acridyl phosphuret-(t)ed hydrogen sulfide, N, N ', N "-three-1,2-ethanediylphosphorothioine triamide; N, N ', N ' '-three-1,2-ethanediylthiophosphoramide, tri-(ethyleneimino) thio-phosphoramide; N, N ', N ' '-triethylenethiophosphoramide, triethylenethiophosphorotriamide, m-triethylenethiophosphoramide, m-three (acridine-1-yl) phosphuret-(t)ed hydrogen sulfide, triethylenethiophosphoramide, tris (1-aziridinyl) phosphinesulphor, tris (ethylene imino) thiophosphate, TSPA and WR 45312.

The present invention further embodies, alkaloid extraction thing, any alkaloid of selecting from Chelidonium majus L., in organic solvent, can react with three (1-acridyl) phosphuret-(t)ed hydrogen sulfide, the reaction product that produces, as a kind of mixture, water washs once at least.Because three (acridine-1-yl) phosphuret-(t)ed hydrogen sulfide is removed from organic phase.。Preferably washing comprises the organic solution several of reaction product, and each water carries out saturated.Be more preferably, washing constantly repeats up to highly toxic three (acridine-1-yl) phosphuret-(t)ed hydrogen sulfide is removed from reaction product fully.

In addition, poisonous alkaloids may cause medical side effect, even can produce the liver sclerosis, therefore also it should be removed or reduce their concentration from the aqueous phase of synthetic mixture.By the method for Ames test, the reaction product that check prepares according to invention can not have mutagenesis.

Reaction product is a kind of alkaloidal complex mixture, with high-molecular weight three (1-acridyl) phosphuret-(t)ed hydrogen sulfide and alkaloidal reactant are arranged, and high-molecular weight three (1-acridyl) phosphuret-(t)ed hydrogen sulfide degraded product.After this synthesis technique was finished, change had taken place in alkaloidal solubility.The alkaloidal concentration of tertiary amine will rise, and quaternary amine alkaloid concentration then can descend.Contain the pilewort alkaloid of about 60-70-% in the reaction product, and contain three (1-acridyl) phosphuret-(t)ed hydrogen sulfide of about 30-40%.

Tertiary amine pilewort alkaloid is a main component.Such as, in synthetic mixture, comprise following tertiary amine alkaloid: Stylophorine, Protopine, stylopin, Yin not product, α-homochelidonine, chelamidine, chelamine, L-Tocosamine, Chelidimerine, dihydrosanguinarine, Oxysanguinarine, Oxychelidonine and methoxychelidonine.

Quaternary amine alkaloid (such as berberine) distributes it is fully removed from synthetic mixture by adopting water to carry out liquid liquid.Under the reaction conditions of regulation, berberine can not form the mixture that contains three (1-acridyl) phosphuret-(t)ed hydrogen sulfide.

The reaction product that this method obtains and wash the reaction product that obtains with ether and compare has wider medical functions scope.It destroys cancer cells-compare with the most cells inhibitor-can not attack normal cell by apoptosis.This makes that adopting said preparation to treat has good tolerance to diseases, and the prevention use is had logical suitable property.Easy to operate and when taking therapeutic dose, do not have a reflection of big pair.The interaction of these materials in synthetic mixture makes and produced medical effect.

The reaction product of Chelidonium majus L. total alkaloids has shown biologic activity aspect metabolic regulating, and can be fit to the prevention and the treatment of metabolic trouble, these diseases comprise osteoporosis, rheumatism, allergy, virus infection, epilepsy, multiple sclerosis, scar, dermatoma and wound after surgery.

Can use the extract of the dry root of Chelidonium majus L. to be used as the synthetic initial reactant.These roots contain more alkaloid than leaf and stem.

Conventional medicine vehicle, especially liquor (for example injection liquid or immersion liquid), ointment, compress or sling are applicable to the medicine that comprises according to the reaction product of the present invention's preparation.

These following examples are for example understood process of the present invention:

Example 1

A) alkaloidal extraction:

A. 25 gram alkaloid salt mixtures are placed water, pour in the separating funnel then.After adding 100 milliliters of methylene dichloride, shake separating funnel.Organic phase just can be separated, and then it is filled in the vial.

B. add 1N sodium hydroxide (pH8-9) to aqueous phase, muddy until occurring.After adding 100 milliliters of methylene dichloride, shake mixture.Organic phase just can be separated, and its methylene dichloride with step a is mixed mutually.Repeat this process, for example repeat 3 times.Filter and the mixing organic phase.

C. by adding sodium hydroxide the pH value of water is transferred to 10.After adding methylene dichloride, shake mixture.Organic phase just can be separated, and mixes with its filtration and with other organic phases.Utilize sodium hydroxide that the pH value of water is transferred to 13 now, utilize methylene dichloride fully to extract then.

D. the organic phase with these merging is evaporated to a kind of oily brown material of generation.

B) and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide react:

The alkaloid resistates is dissolved in methylene dichloride, add three (1-acridyl) phosphuret-(t)ed hydrogen sulfide then.80 ℃ temperature this mixture is carried out refluxing in 2 hours.After being cooled to room temperature, clarify this reaction mixture.Filter then, in separating funnel, utilize 250 milliliters water washing to leach thing number time afterwards, for example 3 times or multipass more.

C) and HCl reaction

To transfer in the glass beaker through the solution of washing, feeding HCl gas constantly stirs and makes and reaches capacity, and so just will form the hydrochloride mixture precipitation.Sedimentary product leached and with diethyl ether wash, then dry.Be dissolved in the water then.

In mouse body, for the reaction product that example 1 makes, the LD that is measured to ₅₀Value is 485mg/kg.The machine of testing in mouse and rat shows, the hormone level that the product that makes according to invention has been regulated thymus gland, and in the animal body of those thymectomies, induce to have synthesized and have the material that similar thymosin alpha 1 active is arranged.This effect is based on dosage and obtains.Said preparation has increased peripheral blood T-lymphocytic quantity 50% (4.04 ± 0.43 * 10 nearly ⁹Before/l handles, 6.24 ± 0.73 * 10 ⁹/ l handles the back), regulate the natural killer cell activity (198.20 ± 17.69%, control group is 71.50 ± 9.10%) that penetrates antigenic humoral immunization reflection and spleen cell, and improved leukocytic Interferon, rabbit releasability in the animal experiment.Zooperal result is through clinical observation and definite.Therefore, also observing index of immunity on one's body at cancer patient is improved.

Use for prevention and immunology, the usage quantity of the preparation in the example 1 is 5 milligrams/70 kg body weight.For cancer therapy, dosage is 5 milligrams/20 kg body weight.

Example 2:HPLC finger printing

This mensuration is by ion pair reverse phase gradient chromatography and use a DAD detector to finish in 285 nanometers measurement absorbancy.Simultaneously, color atlas is drawn with reference to alkaloid by using.In addition, carry out a kind of HPLC-MSD and analyze, which peak value can draw by this analysis does not have outside these alkaloid scopes.Can obtain the HPLC collection of illustrative plates of Fig. 1 and 2 based on following experimental data:

Chromatographic parameter:

Post: LiChrospher 60 RP Select B, 5 μ m, 125 * 24mm ID

Elutriant: A) 200ml (acetonitrile)+800ml (water)+1.5g (hexane sulfonic acid)+0.3ml (85% phosphoric acid)

B) 900ml (acetonitrile)+100ml (water)+1.5g (hexane sulfonic acid)+0.3ml (85% phosphoric acid)

Gradient: 5 minutes isocratic elution 100%A;

24 minutes to 40%B

1 minute to 100%B

5 minutes 100%B;

5 minutes 100%A balances

Survey: UV lamp 285 nanometers

Wash-out flow rate: 1ml/min stopped after 35 minutes

Sampling volume: 10 μ l

Specimen preparation:

Extraction (Fig. 1) before the reaction: 25 milligrams of alkaloids are dissolved in 40 ml methanol by ultrasonic wave,, filter by membrane filter then near 50 milliliters.

Reaction product (Fig. 2): reaction product is converted into hydrochloride, is dissolved in the water and regulates pH between 2.5 and 6.5 with the concentration of 1mg/ml.

Following example has shown that the different application of this compound (is called Ukrain below ^), it is made by the program of describing in the example 1.

Example 3:I and II type osteoporosis:

With Ukrain ^Be used in 30 patients that suffer from I and II type osteoporosis, dosage is 5 milligrams, and concentration is that 1mg/ml carries out intravenous injection, carries out once weekly in ten weeks for all patients.Haematol and biochemical parameter and the prolactin antagonist concentration of the progress of treatment by measuring serum FSH, LH is monitored.Measure calcium and phosphorus in serum and the urine.Through after the trimestral treatment, all found the body movement of the improvement and the increase of clinical manifestation on one's body all patients.Do not observe the side effect reflection of processing.

Example 4: skeleton development is unusual:

It is unusual to make a definite diagnosis I type skeleton development on one's body an aeg of 14 years old.X-ray figure transmission sheet shows osteoporosis diffusion, blue look sclera, milky white tooth (development abnormalities of teeth), taper thoracic cavity, scoliosis and muscle hyperpiesia.Patient's dosage of 5 milligrams weekly carries out Ukrain ^Intravenous injection.Treatment is carried out three courses of treatment, three months at interval per course of treatment.Treatment has obtained significant improvement aspect muscle function; Sclera and tooth also begin to bleach again.The degree that scoliotic situation has obtained remarkable improvement and treatment physical exercise also will significantly increase.Patient's health begins to coordinate again.X-ray figure transmission sheet shows that osseous tissue is recovered; In the transmission sheet, no longer find any osteoporosis symptom that detects.

Example 5: spondylarthritis:

71 years old an aeg complaint is at her back lower region, L ₁-L ₃The zone suffers from serious pain, and it can not be moved.Patient's dosage of 5 milligrams weekly carries out Ukrain ^Intravenous injection, and at surface use compress 4 time-of-weeks.Patient's the state of an illness improves.Also can re-move.

Example 6: ulceration skin injury:

7 years old spadger ulceration skin injury after its shoulder blade left field has an operation.For adopting cosmetic surgery, this scar is too big.Patient adopts contains Ukrain ^Compress carry out surface treatment 12 time-of-weeks.The scar state of an illness has obtained very big improvement.Ulcer disappears and does not have other inflammation to occur.

Example 7: asthma is irritated

Diagnose a kind of serious allergy on one's body an Aeg of 12 years old, and with cough and asthma.By using 5 milligrams of Ukrain every day ^Continue to treat in 3 weeks.His state of an illness be significantly improved and nowadays patient be considered to not need to treat.

Example 8: milk allergy

An aeg of 12 years old is suffering serious milk allergy, and this is irritated for to contain the allergy that causes in the milk foods, such as chocolate.Patient carried out vein Ukrain in per two days ^Injection continues two time-of-weeks.Nowadays patient has been considered to not need to treat.

Example 9: to cat allergy

An aeg of 9 years old suffers from cat symptom hypersensitive, and syndrome is serious asthma.Patient is with 5 milligrams of Ukrain ^Treat twice weekly.Her state of an illness obtains taking a turn for the better, and nowadays patient has been considered to not need to treat.

Example 10: zoster

An old aeg of 64 years old is diagnosed as suffers from the zoster infection and recurrence.Along the Th that has ulcer ₃-Th ₄Typical blister appears in a breach on the zone.Patient is with 5 milligrams of Ukrain ^Carry out intravenous injection and continue 8 time-of-weeks twice weekly.Do not observe recurrence in 4 years.

11: the third livers of example

Elderly patients of 42 years old suffer from chronic hepatitis C, positive HCV-RNA, 1b type HCV, and are often tired, follow pain and physical efficiency poor.He is with 5 milligrams of Ukrain ^Carry out intravenous injection and continue 8 time-of-weeks twice weekly.The result of treatment is, patient's pain relief, and the improvement of overall physical appearance, physical efficiency obtains increasing.Nowadays patient HCV-RNA presents feminine gender.

Example 12: allergy

Patient A.N., 12 years old, serious allergic symptom is arranged, and with cough, and have asthma.He is oral Ukrain ^, 5 milligrams of every days.His patient's condition has obtained noticeable improvement after 4 week, and does not have those syndromes to take place.

Example 13: operation back injured (having the skin lesion of ulcer)

Patient S.D., 7 years old, because operation has a great skin lesion in its shoulder zone, left side, to such an extent as to can not adopt cosmetic surgery.Patient carries out local Ukrain ^-The compress surface treatment continues 3 months.The skin lesion area dwindles.Do not have ulcer, do not have inflammation.

Example 14: osteoporosis

Patient H.J., 68 years old, walking was had any problem, and is tired easily.After carrying out clinical fully and laboratory study, be diagnosed as I type osteoporosis.Use Ukrain ^Treated for ten weeks, 5 milligrams of intravenous injections weekly, patient is responsible for and feels that improvement is arranged, and the result by relevant skeleton density, calcium and hormone level confirms to have taken place major improvement.

Example 15: zoster

Patient K.R., 37 years old, in the time of 7 years old, once suffered from varicella, typical pain skin eruption-zoster has appearred at the rib position now.With 5 milligrams of Ukrain ^Intravenous injection treatment, in 4 weeks biweekly.Not recurrence in 6 years.

Example 16: hepatitis B

Patient G.H., 48 years old, weakness appears, and tired and vigor reduces.Be diagnosed as through clinical fully and laboratory: chronic viral hepatitis B, positive HB _s-Ag, the blood enzyme level of increase.In 3 months, carry out 5 milligrams of Ukrain once in a week ^After the intravenous injection treatment, subjective sensation obtains very big improvement and can enhance vigour.Lab investigation show that viral load increases love greatly and enzyme level is tending towards normalizing.

Example 17: rheumatosis, osteoarthritis

Patient I.N., 71 years old, in the L1-L3 zone serious back pain is arranged, it is restricted to take action.Be diagnosed as through clinical fully and laboratory: osteoarthritis (joint disease).Treatment: Ukrain ^, 5 milligrams of intravenous injections biweekly continued for 6 weeks.Clearly reduced pain, and patient's moving range increases.

Example 18: epileptic seizures

Patient D.R. 46 years old, has complicated focal epilepsy and the trend of deterioration is arranged.The side effect that the Phenytoin Sodium of high dosage can cause adenopathy to take place.10mg Ukrain biweekly in 6 weeks ^Intravenous injection, symptom have obtained very big alleviating, and it is also fewer and feweri and not obvious to show effect.Also can use town's epilepsy agent medicine of more low dosages.

Example 19: multiple sclerosis

Patient M.G., 36 years old, it was unaccountable weak to feel that shank has, then can be dizzy.Outbreak becomes shorter and shorter at interval.After checking, be diagnosed as multiple sclerosis comprehensively.Adopted a physiatrics, but failed.Through 5mgUkrain ^Intravenous injection biweekly continues 4 months treatment, notices that outbreak increases at interval, and patient feels that the strong disease of shank does not have dizzy phenomenon.

Example 20: scar

Patient I.N. stands after the abdominal operation, in abdomen area a big scar is arranged.Through 5mg Ukrain ^Intravenous injection biweekly continues the treatment and the local compress that uses in 4 weeks, then notices to have certain cosmetic result.

Example 21: influenza

Patient B.N. 24 years old, suffers from influenza infection every year, sometimes with pneumonia.Ukrain ^Use as a kind of preventive preparation: twice intravenous injection of all 5mg, total dose reaches 50mg in the outbreak period.Nowadays patient did not suffer from this disease in 4 years.

Example 22: type-II diabetes

Patient B.L., 62 years old, suffer from type-II diabetes (non insulin dependent diabetes), with glipizide (to the sulfourea antidiabetic medicine) every day 10 milligrams of dosage treat.Although patient shown hypertension go on a diet but still lay particular stress on, tired, tired and weak fast.Use 5mg Ukrain ^Intravenous injection biweekly continues 2 months, and overall physical state improves, and vigor increases, loses weight.Patient reduces the use glipizide gradually, and patient feels that health is comfortable.

Example 23: recover

Patient O.L. 74 years old, suffers from left articular fracture.After the main joint transplantation operation through lasting a few hours, patient is arranged on one's sick bed.Use 5mg Ukrain ^Intravenous injection biweekly continued for 6 weeks, and overall physical appearance obviously improves, and decubation shortens greatly.

Example 24: to chocolate irritated

Patient V.P., 8 years old, to chocolate irritated.5mg Ukrain ^Oral 3 times one weeks continue the treatment of 4 weeks, do not have more allergic symptom outbreak.

Example 25: recover

Patient J.R., 78 years old, because gallbladdergallstonecholetithiasis and cholecystitis and undergo surgery (cholecystectomy).After the operation, oral Ukrain ^, 3 lasting 2 weeks of one week of 5mg.Notice recovery quick and without a doubt, do not have any complication.Patient can leave hospital sooner than expeced time.

It is to have shown slight invalid one in some cases for those compounds of describing at AT 377 that all application in routine 3-35 have all obtained one of success.

Claims (14)

1. the technology of an alkaloid reaction comprises the following steps:

A) react in the organic solvent environment of comparatively high temps (especially under the boiling temperature at solvent) by at least a alkaloid and a kind of phosphorus derivant of containing an acridyl at least and prepare a kind of reaction product, and

B) reaction product that obtains in a) of washing step;

Water comes the washing reaction product in this process.

2. according to right 1, this phosphorus derivant is three (1-acridyl) phosphuret-(t)ed hydrogen sulfide (CAS numbers 52-24-4).

3. according to right 1, choose in the alkaloid of alkaloid from be included in Chelidonium majus L..

4. according to right 1, wherein a kind of alkaloid mixture reacts.

5. according to right 4, wherein a kind of all alkaloid mixtures of Chelidonium majus L. react.

6. according to right 1, wherein solvent is a methylene dichloride.

7. according to right 1, wherein reaction product is converted into a kind of salt, behind the process washing step, and especially a kind of water miscible salt.

8. according to right 7, salt is muriate.

9. according to right 1, reaction product is made by this technology.

10. according to right 9, a kind of mixture of reaction product comprises the degradation production of reaction product and three (1-acridyl) the phosphuret-(t)ed hydrogen sulfide of alkaloid and alkaloid and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide.

11. according to right 10, mixture contains a kind of alkaloid of selecting from following each material at least: Stylophorine, Protopine, stylopin, Yin not product, α-homochelidonine, chelamidine, chelamine, L-Tocosamine, Chelidimerine, dihydrosanguinarine, Oxysanguinarine, Oxychelidonine and methoxychelidonine.

12. according to right 1, the reaction product that this technology obtains is as drug use.

13. according to right 12, this pharmaceutical preparation is as the prevention or the treatment of following disease or organ disorder: cancer, Immunological diseases and metabolic trouble.

14. according to right 13, that applicable disease comprises is injured after allergy, osteoporosis, dermatoma, virus infection, rheumatism, scar, the operation, epilepsy and multiple sclerosis.

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