Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reissue Patent
2001-03-06
2004-03-09
Huang, Evelyn Mei (Department: 1625)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C546S093000, C546S097000
Reissue Patent
active
RE038460
ABSTRACT:
This application is the national phase of PCT/CN95/00100, filed Dec. 26, 1995, published as WO96/20176 on Jul. 4, 1996.
The present invention relates to semi-synthesis of natural product, and particularly to alkaloid and analogues thereof.
In the past ten years a lot of researches have been made in foreign countries about application of choline esterase inhibitor to enhance the function of the intracerebral cholinergic system for the treatment of presenile dementia. Although delightful results of research have been obtained, there still exist some defects; at the time of producing the treatment effect, there occurs a relatively serious toxic side reaction; and the duration of the effect is relatively short.
In recent years China has isolated from a Chinese herb Lycopodium serratum Thunb., a new alkaloid huperzine A (5R, 9R, 11E)-5-amino-11-ethylidene-5,6,9,10-tetrahydro-7-methyl-5,9-methylene cycloocta [b] pyridyl-2(1H)-one having the formula (I)
Upon a pharmacological study it is proved that it exhibits highly effective reversible anticholinesterase activity, and has selective inhibitory effect on intracerebral acetylcholinesterase (U.S. Pat. No. 5,177,082). In foreign countries modification of the structure of huperzine A has been carried out and analogues of huperzine A have been synthesized in hopes of discovering therefrom compounds having anticholinesterase activity [J. Org. Chem. 56, 1991(4636-4645)] and finding a method of synthesizing huperzine A [U.S. Pat. No. 4,929,731]. However, neither a good method nor an analogue having a better effect has been found so far.
The present invention has made use of the excellent resources of Chinese herbs in China to design a semi-synthesis starting from huperzine A, in the hope of finding among a great variety of huperzine A derivatives compounds having better treatment effect and lower toxicity than the existing huperzine A.
The present invention is carried out through the following steps:
1. Using alcohols such as ethanol as extraction solvent, from the plant Lycopodium serratum Thunb., the residue obtained is concentrated, and then treated with an inorganic acid (such as hydrochloric acid). The aqueous layer is neutralized with alkali (such as ammonia water, NaOH), after which an organic solvent (such as chloroform) is used to extract the total alkaloid. After the treatment, separation by layer chromatography is carried out to produce the compound of the formula (I).
2. After condensation is carried out between the compound of the formula (I), and the corresponding substituted aldehyde or the corresponding substituted acyl chloride or acid anhydride in anhydrous solvent, a compound of the formula (II) is obtained.
When Y is —C═O or —R″, Y is ═CH, R is C
1
-C
5
lower alkyl;
n=0,1, X is hydrogen, C
1
-C
5
lower alkyl, C
1
-C
5
lower alkyloxy, nitro, halogen, carboxy, alkyloxycarbonyl, hydroxymethyl, hydroxy, amino substituted by bis-C
1
-C
5
lower alkyl; —(CH
2
)
m
COOZ group, m=0-5, Z is hydrogen or C
1
-C
5
lower alkyl; —CH═CH—G group, G is phenyl, furanyl, carboxy, alkyloxycarbonyl; dihydro or tetrahydropyridyl substituted by C
1
-C
5
lower alkyl at the nitrogen atom;
R′ is hydrogen, C
1
-C
5
lower alkyl, pyridoyl, benzoyl substituted by C
1
-C
5
lower alkyloxy;
R″ is hydrogen and C
1
-C
5
lower alkyl.
When Y is C═O, R is C
1
-C
5
lower alkyl,
n=0,1, X is hydrogen, C
1
-C
5
lower alkyloxy, carboxy, alkyloxycarbonyl, pyridyl, dihydro or tetrahydropyridyl substituted by C
1
-C
5
lower alkyl at the nitrogen atom; —(CH
2
)
m
COOZ group, m=0-5, Z is hydrogen or C
1
-C
5
lower alkyl; —CH═CH—G group, G is phenyl, furanyl, carboxy, alkyloxycarbonyl;
R′ is hydrogen, C
1
-C
5
lower alkyl, pyridoyl, benzoyl substituted by C
1
-C
5
lower alkyloxy;
R″ is hydrogen and C
1
-
5
lower alkyl.
When R″, Y are ═CH, R is
n=0, X is hydrogen, C
1
-C
5
lower alkyl, C
1
-C
5
lower alkyloxy, nitro, halogen, hydroxy, hydroxymethyl, amino substituted by bis-C
1
-C
5
lower alkyl; —CH═CH—G group, G is phenyl, furanyl;
R′ is hydrogen and C
1
-C
5
lower alkyl.
Huperzine A derivatives:
No.
Y
R
R′
R″
No. 1
C═O
HOOC—CH
2
CH
2
—
H
H
No. 2
C═O
C
6
H
5
CH
2
—
H
H
No. 3
C═O
CH
3
CH
3
═
No. 4
C═O
H
H
═
No. 5
C═O
H
═
═
No. 6
C═O
H
H
═
No. 7
C═O
4-CH
3
OC
6
H
4
—
H
H
No. 8
C—O
2,3,4-(CH
3
O)
3
C
6
H
2
—
H
H
No. 9
C═O
2,3,4-(CH
3
O)
3
C
6
H
2
—
H
—
No. 10
C═O
(CH
3
)
2
CH—
H
H
No. 11
C═O
C
6
H
5
—
H
H
No. 12
C═O
2-py
H
H
No. 13
C═O
3-py
3-py-CO—
H
No. 14
C═O
4-py
H
H
No. 15
C═O
2-HOOC—C
6
H
4
—
H
H
No. 16
C═O
trans-HOOCCH
2
CH═CH—
H
H
No. 17
CH
H
—
No. 18
CH
H
—
No. 19
CH
H
—
No. 20
CH
H
—
No. 21
CH
H
—
No. 22
CH
H
—
No. 23
CH
H
—
No. 24
CH
H
—
No. 25
CH
H
—
No. 26
CH
H
—
No. 27
CH
H
—
No. 28
CH
H
—
No. 29
CH
H
—
No. 30
CH
H
—
No. 31
CH
H
—
No. 32
CH
H
—
No. 33
CH
H
—
No. 34
CH
H
—
No. 35
CH
H
—
No. 36
CH
H
—
No. 37
CH
H
—
No. 38
CH
H
—
Pharmacological effect of huperzine A derivatives:
In the present invention a calorimetric method reported by Ellman was used to determine the effect of inhibition of the pharmaceutical on enzyme activity. The total volume of the solution of the enzyme activity reaction was 4 ml, which contained 0.3 mmol/L of acetylcholin iodide (acetylcholinesterase substrate), or 0.4 mmol/L of butyrylcholin iodide (butyrylcholinesterase substrate), 1 ml of the buffer solution of 25 mmol/L of phosphate of pH 7.4, and water to make up to 4 ml (including the amount of the enzyme solution and the testing reagent added afterwards). After the solution was kept at 37° C. for 5 minutes, 0.1-0.2 ml of enzyme solution (red cell membrane or serum of rat) was added, or 0.1-0.3 ml of the testing reagent was added at the same time. The solution was kept at the same temperature for another 8 minutes. Then 1 ml of 3% of sodium lauryl sulphate was added to terminate the reaction. Finally 1 ml of 0.2% 5,5-dithio-2,2′-nitro-benzoic acid solution was added for developing. “721” spectrophotometer was used to determine the density of light at 440 nm. A solution without addition of the pharmaceutical to inhibit the enzyme activity was used as a control (100%). A diagram was plotted with the percentage of the remaining enzyme activity against gram molecule concentration of the pharmaceutical, so as to obtain the dose of the pharmaceutical at 50% of inhibition of the enzyme activity, i.e., IC
50
. The results of the test showed that all the derivatives exhibited inhibition effect on acetylcholinesterase; derivatives No. 1, No. 17, No. 18 and No. 19 exhibited remarkable inhibition effect on acetylcholinesterase; their effect was slightly weaker than that of huperzine A, but apparently stronger than that of physostigmine and galanthamine. They exhibited weaker inhibition effect on butyrylcholinesterase (pseudo enzyme) than huperzine A. Derivatives No. 1 and No. 17 exhibited greater selective inhibition effect on acetylcholinesterase than huperzine A (see Tables 1, 2 and 6). A study on kinetics of enzyme indicated that the combination of derivatives No. 17, No. 18 and No. 19 respectively with acetylcholinesterase was reversible.
The two memory model test, mouse passive escape operation (Platform jumping method) and 8-arm maze spatial distinction operation of rat, indicated that both derivatives No. 18 and No. 19 exhibited very strong effect, similar to that of the compound of formula (I), on improving memory (see Tables 3 and 4).
The critical toxicity test on mice indicated that LD50 of derivatives No. 18 and No. 19 were smaller than, only ⅓ of, that of the compound of formula (I) (see Table 5).
TABLE 1
In-vitro anticholinesterase effect of huperzine
A derivatives (determined by colorimetric method)
concentration for inhibition
of 50% of enzyme activity
(IC
50
&mgr;M)
acetylcholin-
butyrylcholin-
esterase
esterase
IC
50
ratio
Tang Xi-can
Zhu Dayuan
Huang Evelyn Mei
Shanghai Institute of Materia Medica Chinese Academy of Sciences
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