CN1012172B

CN1012172B - Process for preparing pharmic component of sigma-butylmalonamide and its use for pharmacy

Info

Publication number: CN1012172B
Application number: CN 85106973
Authority: CN
Inventors: 陈延镛; 杨明河; 黄量; 刘耕陶; 乌尔里克·本茨
Original assignee: Pharmacology Institute of CAMS; Bayer AG
Current assignee: Pharmacology Institute of CAMS; Bayer AG
Priority date: 1984-08-24
Filing date: 1985-09-17
Publication date: 1991-03-27
Also published as: CN85106973A

Abstract

Pure clausenamide is obtained by that coconut Chinese wampee leaves are extracted, and then purification is carried out. The molecular formula of the clausenamide comprises: reduzate, ether, etc. can also be produced. The compound is effective on curing anoxia and amnesia.

Description

Process for preparing pharmic component of sigma-butylmalonamide and its use for pharmacy

The present invention relates to the preparation of the δ-butyrolactam (being called " Clausenamide " hereinafter) of active phenyl of a kind of novel pharmacology and benzyl replacement; comprise: this compounds separates from Rutaceae Clausena class plant; some derivative of Clausenamide and they are as the application of hypoxia protection agent and anti-forgetful dose, the invention still further relates to the pharmaceutical composition that contains Clausenamide and their preparation method.

Existing report Rutaceae Africa Calusena lansium in a certain area in Africa as herbal medicine people such as (, Planta Medica 32(1) I.Mester 81,1977).Also have the crude extract of report India Calusena lansium effect to be arranged, and isolating two coumarin derivatives clausmarins A and B have spasmolysis (people such as Dhan Prakash, Phytochem.17,1194,1978 from five leaf Calusena lansium to cardiovascular; People such as Aboo Shoeb, J.C.S.Chem.Commun.281,1978).From the Radix clausenae lansii of different genera, stem etc., about 50 kinds of components have been isolated.The great majority of these components are tonka bean camphor, carbazole and terpenic derivative; Up to the present only there are two straight-chain carboxylic acid's acid amides to be present in (people such as S.R.Johns, Aust.J.Chem.20,2795,1967 in the leaf of clausena plant according to reports; People such as Dhan Prakash, Indian J.Chem.Sect.B19B(12), 1975).

The Clausenamide compound that contains a kind of δ of having-butyrolactam structure in the existing known bulky look Leaf of Chinese Wampee, it contains a phenyl and a benzyl substituent, has the Clausenamide compound (compound (0)) that also contains a kind of structurally very close two ring butyrolactam structures in (" Clausenamide " and compound (9)) bulky look Leaf of Chinese Wampee with two kinds of steric isomers.Find that also Clausenamide and derivative thereof have multiple valuable pharmacological character.Chemical derivatization and spectroscopic data have confirmed that these compounds have following structure.

(X-ray crystal diffraction has confirmed this stereochemical structure)

The present invention proposes the separation method of Clausenamide compound with following formula I,

" Clausenamide "

This method comprises the following steps:

A) soak into the leaf of bulky look Calusena lansium with boiling water.

B) spissated extracting solution is mixed with sorbent material (as: silica gel, aluminum oxide, sand, diatomite, Mierocrystalline cellulose or multi-polyamide).

C) use organic solvent,, be preferably chloroform, extract above-mentioned sorbent material as chloroform, ethyl acetate, ether, methylene dichloride and vinylchlorid.

D) concentrate machine eluate is arranged.

E) use refrigerative C ₁-C ₆-alcohol or C ₂-C ₆-ketone (for example methyl alcohol) washing concentrating thing.

The crude product that obtains with above-mentioned separation method is preferably in recrystallization in the alcohol (for example methyl alcohol or ethanol).

The invention still further relates to and contain with the formula I compound as the pharmaceutical composition and the medicament of effective constituent and prepare these method for compositions.

The compound that the invention allows for formula I is used for the treatment of anoxia and amnesia.

In animal experiment, the compound of formula I has big cerebral anoxia of significant protection and anti-forgetful effect, and this effect obviously is better than 2-Pyrrolidone ethanamide (Piracetam).2-Pyrrolidone ethanamide (Piracetam) is structurally the most approaching at the treatment of brain and the allied compound in the nootrpics field.

2-Pyrrolidone ethanamide (Piracetam)

Even be applied to animal with high dosage, their behavior aspect does not show any material alterations yet.This hypoxia protection effect obviously is not (therefore the latter causes the demand of reduction to oxygen) that causes owing to a kind of non-specific sedative effect.We find: the acute toxicity of formula I compound is very low.

Pharmaceutical composition of the present invention can be made: ointment, colloid, paste, emulsion, sprays (comprising aerosol), lotion, emulsion, solution and emulsion active ingredient, syrup, granule or pulvis in water or non-diluent water.

Said composition preferably make the sterile isotonic aqueous solution or make contain independent or with tablet, capsule, pill and the suppository of the compound of the present invention of mixing diluents.

Can be applied to the thinner that being suitable in the pharmaceutical composition (for example granulate) should form tablet, drageeing, capsule and pill comprises:

(a) filler, for example, starch, sugar, silicic acid;

(b) tackiness agent, for example, derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone;

(c) wetting Agent for Printing Inks, for example, glycerine;

(d) disintegrating agent, for example, agar, lime carbonate and sodium bicarbonate;

(e) absorption enhancer, for example, quarternary ammonium salt compound;

(f) tensio-active agent, for example, hexadecanol;

(g) absorption carrier, for example, kaolin and wilkinite;

(h) lubricant, talcum powder for example, calcium stearate and Magnesium Stearate and solid-state polyethylene glycol.

Tablet, dragee, capsule and the pill made by medicinal compositions of the present invention can have common dressing, wrap and protection matrix, and they can contain opalizer.These medicaments can be made its active ingredient only or more fortunately the privileged site of enteron aisle discharge, and can continue for some time.Dressing, wrap and protection matrix can prepare with polymeric material and paraffin.

The component of medicine also can be made the form of micro-capsule with one or more above-mentioned thinners.

The production of aforementioned pharmaceutical compositions and medicament can be undertaken by any method known to the people on technology.For example, the composition (for example, granulous) that activated component and mixing diluents are formed a kind of pharmacology (for example: tablet) is made medicament with this composition again.

Pharmaceutical composition of the present invention preferably contains 0.1% to 99.5% effective constituent of total composition weight, and the best is 0.5% to 95%.

The optimal dose that medicament of the present invention is used for the treatment of is 0.001 milligram to 0.2 milligram activeconstituents every day.

With example an explanation is done in this invention below.

Example 1

The separation of Clausenamide

At first 80 kilograms of bulky look Leaf of Chinese Wampee of exsiccant SKeels (Lour) and water boil were boiled 4 hours with 800 premium on currency for three times at every turn.This extracting solution concentrated obtains 18 kilograms syrupy shape crude product, 1 kilogram of this syrupy shape crude product liquid is mixed with the silica gel of its double weight, 70-80 ℃ of drying also with 4.5 liters of chloroform extraction.Extracting solution taken out and adds fresh chloroform in per 6 hours.Repeat this process 4 times.These chloroform extracted solutions are merged and concentrate obtain a brown syrup, wash this brown syrupy shape liquid, in methyl alcohol, make 3.33 gram yellow powder recrystallizations of acquisition like this, obtain the white, needle-shaped crystals of Clausenamide with cold methanol.

Clausenamide: white, needle-shaped crystals, m.p.239-40 ℃

Output: 1.72 grams (0.17% raw sugar soup compound) α] ²¹ _D0.00(0.53 in methyl alcohol), measure (M:297,1364) through ultimate analysis and high resolution mass spectrum; Molecular formula is: C ₁₈H ₁₉NO ₃Dissolve in the hot methanol, DMSO and DMF only are slightly soluble in general organic solvent, trichloromethane for example, and ethylene dichloride, ether, ethyl acetate, etc.

IR γ ^KBr _MaxCm ^-1: 3400,3310(OH), the 1680(acid amides), 1600,1580,1490,1450,740, the mono-substituted phenyl ring of 690().

UVλ ^MeOH _maxnm（1gε）：257（2.70）。

High resolving power MS m/z:297.1364(M ⁺, C ₁₈H ₁₉NO ₃),

298.1448（C ₁₈H ₂₀NO ₃），191.0946（C ₁₁H ₁₃NO ₂），190.0881（C ₁₁H ₁₂NO ₂），174.0912（C ₁₁H ₁₂NO），162.0924（C ₁₀H ₁₂NO），144.0815（C ₁₀H ₁₀N），134.0687（C ₉H ₁₀O），133.0646（C ₉H ₉O）。

Ultimate analysis records: C=72.39, H=6.39, N=4.51

Table 1, ¹H-NMR; The ownership of chemical shift and Clausenamide

Ppm hydrogen

3.05（S;3H） N-CH ₃

3.50（dd，J＝10.8;1H） C ₄-H

3.90（d，J＝10;1H） C ₃-H

4.30（dd，J＝8，2;1H） C ₅-H

4.65（d，J＝2;1H） C ₇-H

4.70-5.40（m;2H） OH

6.50-6.70(m; 2H) fragrant H

6.90-7.30(m; 8H) fragrant H

¹³The C-NMR data provide in the following Table 2, and the X-ray diffraction data have confirmed the chemical structure of Clausenamide equally.

Table 2: ¹³C-NMR(is at CDCl ₃In); The chemical shift and the ownership of " Clausenamide ", compound (O) and compound (9)

Carbon " Clausenamide " is (9) (O)

（ppm）（ppm）（ppm）

2 173.6 172.2 172.7

3 68.9 80.3 69.3

4 49.9 50.7 46.7

5 65.3 70.1 68.4

6 30.4 27.3 28.2

7 71.9 82.5 77.3

1′ 136.0 133.3 140.5

2′

1″ 140.5 139.1 141.8

126.3 125.4 125.9 of fragrance

Carbon 128.9 128.5 128.5

Example 2

Compound of the present invention is to the influence of liver function

Adopting body weight in whole experiment is the male Kunming mouse subspecies of 18-22 gram.Compound to be tried is suspended in 5% tween 80, uses the oral gavage administration then, give mouse with control group with identical method the carrier of 5% tween 80 solution.In experiment in vitro, compound dissolution, is directly added in the culturing mixt in the dinethylformamide then at N.

The parameter that liver poison (hepatotoxicity) is adopted comprises: serum transaminase (SGPT), the pathological examination of liver triglyceride level and liver slice.Liver injury is mainly kept the score by inflammation and downright bad degree, and is divided into 0 to 4 grade.

A) effect of protection liver

The test mouse is divided into three groups, feeds control group with carrier.Be spaced apart 8 hours for respectively other two groups take medicine twice (250 milligrams/kilogram) with each compound at every turn.Fed for the second time behind the evolution compound 24 hours, and be dissolved in 0.1% tetracol phenixin in the vegetables oil by 10 milliliters of/kilogram subcutaneous injections, fasting 16 hours, broken end kills then.Measure SGPT and liver fat thing.Be used as liver slice for pathological observation with one.

As shown in the following Table 3, " Clausenamide " and compound (O) both has reduced the level of the SGPT of carbon tetrachloride poisoning mouse significantly.

B) Clausenamide is to the toxic effect of protection tetracol phenixin, thioacetamide and Paracetamol.

The toxicologic experimental arrangement of anti-tetracol phenixin liver is identical with said process.The dosage of the active compound that is adopted is 125 milligrams/kilogram and 250 milligrams/kilogram.The data of listing in table 4 show, have reduced the rising of the SGPT that causes owing to tetracol phenixin significantly with the Clausenamide of 125 milligrams/kilogram and 250 milligrams/kilogram dosage.With the mouse of 250 milligrams/kilogram compounds for treating, the seriousness of the damage of its liver such as hepatitis and hepatic necrosis is lower than the contrast mouse, and liver fat does not reduce.

In another experiment, at first every other day to three 10 milligrams of/kilogram 0.15% tetracol phenixin in vegetables oil of mouse injection.After for the first time injecting tetrachloroization, treat mouse every day since second day to the 5th day with Clausenamide (250 milligrams/kilogram).Carried out the mensuration of SGPT and the pathological examination of liver slice at the 7th day that tests.Gained is the result show: this compound has the effect that SGPT is reduced, but constantly influences liver injury (seeing Table 5).

In thioacetamide liver toxicological experiment, according to the programmed treatment mouse of testing for the first time, but use is thioacetamide (50 milligrams/kilogram) rather than tetracol phenixin.We find that Clausenamide has obviously reduced SGPT level (table 6).

Resist-the toxicologic experiment of Paracetamol liver with following method, gave and same dosage for twice Clausenamide of mouse (250 milligrams/kilogram) in subsequent second day in first day.After the last administration 6 hours, give mouse subcutaneous injection Paracetamol by 150 milligrams of/kilogram dosage.Behind the injection Paracetamol 20 hours, to measure SGPT and also detect hepatic tissue, Clausenamide has obviously reduced the level of SGPT and has alleviated liver injury (seeing Table 7).

C) to the influence of the serum and the liver transaminase (GPT) of normal mice.

The Clausenamide of feeding carrier or 250 milligrams/kilogram for respectively two groups of mouse, once a day, continuous seven days.Twenty four hours behind the last medicine feed is measured serum and liver GPT.As shown in table 8, the SGPT of a little higher than contrast of the SGPT level of the mouse for the treatment of with Clausenamide mouse, but difference is not obvious.To liver GPT, also obtained similar results.

D) to hepatomicrosome cytochrome p-450 inducing action

In the detoxification processes of (Xenobitics) of heteroplasia, the hepatomicrosome cytochrome p-450 plays a key effect.Give and test the Clausenamide that mouse feeds 250 milligrams/kilogram, once a day, for three days on end.The contrast mouse is then accepted carrier.Overnight fasting kills the test mouse.The preparation hepatomicrosome.Measure microsomal monoxygenase then.

Data are shown in table 9.Liver cell pigment p-450, cytochrome b ₅, NADPH-cytochrome C-reductase, pyramidon piptonychia enzyme and benzo hydroxylase activity all obviously improve.

In other experiment, give the Clausenamide of 250 milligrams/kilogram of test mouse.After obeying this compound, carried out subcutaneous injection vetanarcol (50 milligrams/kilogram) in back 1 hour and 24 hours taking medicine.The length of one's sleep is estimated in disappearance and recovery by the record regular reflection at interval.Data are listed in table 10.Advance Clausenamide at preceding 24 hours clothes of injection Sodital, obviously shorten the length of one's sleep of mouse, otherwise took this compound in last hour in the Sodital injection, and obviously prolong rather than shorten the length of one's sleep of mouse.Yet obeying this compound did not in advance influence by veronal inductive mouse length of one's sleep.Because veronal is not by liver metabolism.The prolongation of the Sodital length of one's sleep of this explanation due to Clausenamide is by having suppressed hepatic drug metabolizing enzyme.So this compound has the two-phase effect to the hepatomicrosome cytochrome p-450, promptly; Earlier suppress afterwards to induce.

E) acute poisoning experiment

10 mouse are pressed the single oral dose administration of 3 gram/kilogram Clausenamides, do not have in 7 days to cause death.

Table 3: the influence of carbon tetrachloride poisoning mouse on the SGPT level

(every group of 9 mouse)

The % P of SGPT unit

X±SE

Control group 1678 ± 261＜0.01

Compound (O) 391 ± 94

" Clausenamide " 617 ± 323＜0.01

The protective effect of the test mouse liver poisoning due to the table 4 pair tetracol phenixin

The liver injury of the % of group SGPT unit liver lipoid

The downright bad rank of X ± SE mg/g liver inflammation rank

X±SE

Contrast 3016 ± 23 21.0 ± 4.2 1.70 2.33

Clausenamide

125 milligrams/kilogram

×2 2365±245＊ 21.6±4.4 - -

250 milligrams/kilogram

×2 1900±257＊＊ 13.4±3.0 0.38 1.33

Every group of 9 mouse

＊p＜0.05;＊＊p＜0.01

The therapeutic action of the test mouse liver poisoning due to the table 5 pair tetracol phenixin

The % P of number of elements SGPT unit of group mouse

X±SE

Contrast 8 2695 ± 110

Clausenamide

(250 milligrams/

Kg/day * 4) 8 1718 ± 224＜0.01

The provide protection of the test mouse liver poisoning due to the table 6 pair thioacetamide

The % of group SGPT unit liver lipoid (milligram/gram)

X±SE X×SE

Contrast 1696 ± 231 61 ± 11.7

Clausenamide 718 ± 229* 39 ± 3.9

(250 milligrams/kilogram

×2）

Every group of 9 mouse

＊P＜0.01

Test due to table 7 acetylaminobenzene (acetaminophen)

The provide protection of mouse liver poisoning

Parameter contrast Clausenamide

250 milligrams/kilogram * 3

The % of SGPT unit 2778 ± 270 697 ± 163**

Liver lipoid 79 82 ± 17

Milligram/kilogram

Liver inflammation (rank) 0.5 0.1

Hepatic necrosis (rank) 1.4 0

Every group of 9 mouse

＊＊P＜0.01

The serum of table 8 pair normal mice and the influence of liver transaminase

% LGPT unit of group SGPT unit/100 milligrams

X×SE X×SE

Contrast 217 ± 17.5 260 ± 11.6

Clausenamide 252 ± 22.1 292 ± 8.0*

(250 milligrams/kg/day * 7)

Every group of 8 mouse

＊P＞0.05

The inducing action of table 9 pair test mouse hepatomicrosome cytochrome P-450

The yellow acid amides of contrast

250 milligrams/kilogram

/ day * 3

Liver weight in grams % 4.0 ± 0.2 5.4 ± 0.2**

Microsomal protein matter 6.3 ± 0.3 9.1 ± 0.6**

(milligram/gram liver)

Cytochrome P-450 0.87 ± 0.07 1.14 ± 0.07**

(mmole/milligram protein)

NADPH-cytochrome C-reductase 103 ± 2.5 117 ± 2.5**

(mmole reductive pigment C/ minute

/ mg protein)

Cytochrome b ₅0.15 ± 0.01 0.19 ± 0.01

(mmole/milligram protein)

Pyramidon demethylase 81 ± 6.3 126 ± 5.2**

(mmole formaldehyde/minute protein)

AHH

Mmole/minute/milligram protein 2.6 ± 0.4 5.7 ± 0.76**

＊＊P0.01

The influence of test mouse length of one's sleep due to the table 10 pair barbiturate(s)

The barbiturate(s) group is with compound and use the length of one's sleep

Barbiturate(s) it (minute)

Between interval X ± SE

Sodital contrast 71 ± 7

50 milligrams of/kilogram Clausenamide 1h 152 ± 15＜0.01

(250 milligrams/kilogram)

Clausenamide 24h 46 ± 6＜0.01

(250 milligrams/kilogram)

Veronal contrast 197 ± 27

200 milligrams of/kilogram Clausenamide 1h 172 ± 13＞0.05

250 milligrams/kilogram

Example 3

The raising of anoxic tolerance (mouse) due to the Clausenamide

A group male mice (body weight 20 grams) is placed in the plastic box that is divided into two Room (volume of case is 15 * 28 * 40 centimetres, and being equivalent to its capacity is 1.68 liters of every bos).20 mouse of each indoor placement.Charge into the mixed gas that contains 3.5% oxygen and 96.5% nitrogen in the case.The volume that charges into is 4 liters/minute.Experimentizing oral test material and carrier preceding 30 minutes.

Charge into the anoxic gas mixture and began the back about 7 minutes, the animal hypoxia death.When three animals of chamber, the left side (control animal group) only respiratory symptom occurs, stop experiment.Open chest and calculate the number that a treated animal after treatment still lives then.

According to Fisher and Yates(1963) X test evaluation difference between the surviving animals in two groups of proposing such as (Thomann people, 1975).

Table 11

Dosage contrast Clausenamide effectiveness

(milligram/kilogram is oral) survival number/total number of animals (%)

10 9/60 19/60 19.6

30 9/60 31/60 41.1

100 9/60 40/60 58.8

P＝0.05

Table 11 shows, can significantly improve the anoxybiotic tolerance with Clausenamide, only uses oral 100 milligrams/kilogram of few material (barbiturates), and survival rate just can improve 59%.

Example 4

Clausenamide is to the influence of hypomnesis (rat) under anoxia condition

Device (39 cm long, 21 centimetres are high, 21 centimetres are wide) form by two Room.Make (29 centimetres in length) by translucent plastic and another blacking (long 10 centimetres) for one.This device has at the bottom of the metal grid mesh, and aperture plate is connected with the stimulating apparatus that 20 seconds 1.6mA electric currents can be provided.

Connected by door between two Room, door can be closed.

With male rat (body weight 100-120 gram) be placed on big between, allow rat to check two Room three minutes of this device then.

Then, rat is placed into little (blacking), close the connection door, carry out end vibration, afterwards rat is put into sealed chamber, charge into the mixed gas that contains 3.8% oxygen and 96.2% nitrogen in this sealed chamber.Animal is placed this kind anaerobic environment, up to show show be about to occur the panting of respiratory insufficiency till (the longest is 15 minutes).

After 24 hours, again rat is put back to device bright.Be 3 minutes observing time.

In three groups of 15 rats, once test:

The A group: control group, without undergoing anaerobic environment.

The B group: control group, accepting anaerobic environment after the training for the first time.

The C group: the group of taking medicine, accepting anaerobic environment after the training for the first time.

Assessment: animal enters the required time of inner room with calculating second.

Regard the time difference between two control groups as 100%(A-B=100%).

Calculate control group B and the time difference of taking medicine between the group C with percentage ratio (C-B=X%), X is that material to be tested resists the tolerance of forgeing effect.

Clausenamide X

(milligram/kilogram is oral) (%)

3 47

10 65

30 100

Claims

1, the method for the Clausenamide that the isolating construction formula is following,

This method comprises following step:

A) soak into the leaf of bulky look Calusena lansium with boiling water,

B) mix spissated extracting solution and sorbent material, preferably mix with silica gel,

C) use organic solvent, chloroform extraction sorbent material preferably,

D) concentrate machine eluate arranged,

E) with cold C ₁-C ₆Alcohol or C ₂-C ₆Ketone washing concentrating thing.