U.S. patent application number 13/460472 was filed with the patent office on 2012-11-01 for pharmaceutical composition useful as acetylcholinesterase inhibitors.
This patent application is currently assigned to Council of Scientific and Industrial Research. Invention is credited to Katragadda Suresh Babu, Janaswamy Madhusudana Rao, Chandiswar Nath, Kondapuram Vijaya Raghvan, Bhimapaka China Raju, Hemant Kumar Singh, Pullela Venkata Srinivas, Jhillu Singh Yadav.
Application Number | 20120277297 13/460472 |
Document ID | / |
Family ID | 38179526 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120277297 |
Kind Code |
A1 |
Madhusudana Rao; Janaswamy ;
et al. |
November 1, 2012 |
Pharmaceutical Composition Useful as Acetylcholinesterase
Inhibitors
Abstract
The present invention relates to pharmaceutical composition
comprising the naturally occurring compounds selected from (.+-.)
Marrnesin, Columbianetin, Dihydroxanthyletin and substituted
coumarin derivatives of 7-allyloxy coumarin, 7-benzyloxy coumarin,
7 -methoxycoumarin, 7-acetyloxy coumarin, 4-methyl-7-hydroxy
coumarin and 4-methyl-7-acetyloxy coumarin. The said compositions
possess a high degree of acetylcholinesterase inhibitory (AChE)
property.
Inventors: |
Madhusudana Rao; Janaswamy;
(Hyderabad, IN) ; Raju; Bhimapaka China;
(Hyderabad, IN) ; Srinivas; Pullela Venkata;
(Hyderabad, IN) ; Babu; Katragadda Suresh;
(Hyderabad, IN) ; Yadav; Jhillu Singh; (Hyderabad,
IN) ; Raghvan; Kondapuram Vijaya; (Hyderabad, IN)
; Singh; Hemant Kumar; (Lucknow, IN) ; Nath;
Chandiswar; (Lucknow, IN) |
Assignee: |
Council of Scientific and
Industrial Research
New Delhi
IN
|
Family ID: |
38179526 |
Appl. No.: |
13/460472 |
Filed: |
April 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11688359 |
Mar 20, 2007 |
8188143 |
|
|
13460472 |
|
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Current U.S.
Class: |
514/455 ;
514/457; 549/282; 549/289 |
Current CPC
Class: |
A61K 31/37 20130101;
A61P 25/28 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/455 ;
514/457; 549/289; 549/282 |
International
Class: |
A61K 31/37 20060101
A61K031/37; C07D 493/04 20060101 C07D493/04; A61P 25/28 20060101
A61P025/28; C07D 311/20 20060101 C07D311/20 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 2006 |
IN |
0735/DEL/2006 |
Claims
1. A pharmaceutical composition comprising an effective amount of
compound of formula 1, analogs and pharmaceutically acceptable
salts thereof; ##STR00010## (i) wherein R1 and R2 is linked with
each other via following moiety and collectively makes fused
system, and R3 is H; ##STR00011## (ii) wherein R1 and R3 is linked
with each other via following moiety and collectively makes fused
system, and R1 is H; ##STR00012## (iii) wherein R1 and R2 is linked
with each other via following moiety and collectively makes fused
system, and R3 is H; ##STR00013## (iv) wherein the value of R, R1,
R2, and R3 is selected from the group consisting of: a. R=R2=R3=H;
R1=OH; b. R=R2=R3=H; R1=Prenyl; c. R=R2=R3=H; R1=Allyl; d.
R=R2=R3=H; R1=2,2-dimethyl alkyne; e. R=R2=R3=H; R1=2,2-dimethyl
alkene; f. R=R2=R3=H; R1=Benzyl; g. R=R2=R3=H; R1=Acetyl; h.
R=R2=R3=H; R1=Methyl; i. R=R1=R3=H; R2=Prenyl; j. R=R3=H;
R1=R2=Prenyl; k. R=R1=R2=H; R3=Prenyl; l. R=Methyl; R1=R2=R3=H; m.
R=R1=Methyl; R2=R3=H; n. R=R1=Acetyl; R2=R3=H; o. R=Methyl;
R1=R3=H; R2=OH; p. R=Methyl; R1=Benzyl, R2=Benzyloxy, R3=H; q.
R=Methyl; R1=Methyl, R2=Methyloxy, R3=H; and r. R=Methyl;
R1=Acetyl, R2=Acetyloxy, R3=H, optionally along with the
pharmaceutically acceptable carrier, or diluents, wherein the
effective dose of composition is ranging between 50 to 100 mg/kg
body weight.
2. A composition as claimed in claim 1, wherein the compound of
general formula I further comprising: ##STR00014##
3. A composition as claimed in claim 1, wherein the compound of
general formula 1 further comprising: ##STR00015##
4. A composition as claimed in claim 1, wherein the compound of
general formula 1 further comprising: ##STR00016##
5. A composition as claimed in claim 1, wherein the compound of
general formula 1 further comprising: ##STR00017##
6. A composition as claimed in claim 1, wherein the compound of
general formula 1 further comprising: ##STR00018## wherein the
value of R, R1, R2, and R3 is selected from the group consisting
of: a. R=R2=R3=H; R1=OH; b. R=R2=R3=H; R1=Prenyl; c. R=R2=R3=H;
R1=Allyl; d. R=R2=R3=H; R1=2,2-dimethyl alkyne; e. R=R2=R3=H;
R1=2,2-dimethyl alkene; f. R=R2=R3=H; R1=Benzyl; g. R=R2=R3=H;
R1=Acetyl; h. R=R2=R3=H; R1=Methyl; i. R=R1=R3=H; R2=Prenyl; j.
R=R3=H; R1=R2=Prenyl; k. R=R1=R2=H; R3=Prenyl; l. R=Methyl;
R1=R2=R3=H; m. R=R1=Methyl; R2=R3=H; n. R=R1=Acetyl; R2=R3=H; o.
R=Methyl; R1=R3=H; R2=OH; p. R=Methyl; R1=Benzyl, R2=Benzyloxy,
R3=H; q. R=Methyl; R1=Methyl, R2=Methyloxy, R3=H; and r. R=Methyl;
R1=Acetyl, R2=Acetyloxy, R3=H.
7. A composition as claimed in claim 1, wherein the compound used
for the preparation of composition is selected from the group
consisting of (.+-.) Marmesin, Columbianetin, dihydroxanthyletin,
7-methoxy coumarin, 7-acetyloxy coumarin, 4-methyl-7-hydroxy
coumarin, 7-Allyloxycoumarin, and 7-Benzyloxycoumarin.
8. A composition as claimed in claim 1, wherein the compounds used
for the preparation of composition may be from natural source or
synthesized.
9. A composition as claimed in claim 1, wherein the composition
exhibit more percent memory retention than standard drug Donepezil,
in scopolamine induced memory deficit mice.
10. A composition as claimed in claim 1, wherein the composition
inhibits cholinesterase up to 49%.
11. A composition as claimed in claim 1, wherein the composition is
administered by oral route using a carrier selected from gum acacia
and Methyl cellulose.
12.-15. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a pharmaceutical
composition useful as acetylcholinesterase inhibitor (AChE). The
present invention particularly relates to the use of natural
compounds selected from (.+-.) Marmesin, Columbianetin,
Dihydroxanthyletin and substituted coumarin derivatives for the
preparation of composition useful as acetylcholinesterase
inhibitors.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease (AD) (J. Med. Chem. 46, 2279, 2003) is a
chronic neuro degenerative disorder, which finds severe behavioral
abnormalities and loss of cognitive ability. Alzheimer's disease is
associated with cerebral cholinergic hypo function and
characterized by plaques of the amyloid .beta.-peptide,
neurofibrillary tangles and degeneration or atrophy of the basal
forebrain cholinergic neurons. The loss of forebrain cholinergic
cells results reduction in acetylcholine, which plays an important
role in the cognitive impairment associated with Alzheimer's
disease. One of the most promising approaches for the treatment of
Alzheimer's disease is to increase in the levels of acetyl choline
by inhibition of acetycholinesterase.
[0003] Several approaches have been developed for the treatment of
Alzheimer's disease by inhibiting the AChE. The most used AChE
inhibitors are Galanthamine, donepezil, rivastigmine, tacrine and
2H-3,4-tetrahydroquinazoline-2-one & 2H-3,4-tetrahydro
quinazoline-2,4-dione (U.S. Pat. No. 5,504,088, 1996) were also
reported.
[0004] Pyrano coumarins, furo coumarins and substituted coumarins
are natural compounds possessing biological activities like
purgative (J. Indian. Chem. Soc. Vol 66, 66, 1989), insecticidal
(Jpn. Appl. 7 973 12, 1977; Chem. Abstr Vol 91, p152771u, 1977),
antimicrobial (Chem. Abstr. Vol 56, 1835b, 1962), anti feedant (J.
Agric. Food. Chem. Vol 37, 1435, 1989), antiulcer (Fitoterapia Vol
68, 410, 1997; Chem. Abstr Vol 128, 268242j, 1998), anti cancer (J.
Nat. Prod. Vol 57, 518, 1994) and anti HIV (U.S. Pat. No.
5,637,589; Chem. Abstr. Vol 127, 104326t, 1997). Coumarin
derivatives were also exhibited monoamine oxidase (MAO-A& B)
inhibitory properties (J. Med. Chem. 43, 4747, 2000, J. Med. Chem.
44, 3195, 2001, Arkivoc, 272, 2004).
[0005] The present invention relates to the compounds of natural
sources (.+-.) Marmesin, Columbianetin (Phytochemistry, Vol 39(6),
1347, 1995) Dihydroxanthyletin (Phytochemistry, Vol 34(3), 819,
1993), coumarin derivatives of 7-allyloxy coumarin, 7-benzyloxy
coumarin, 7-methoxy coumarin, 7-acetyloxy coumarin,
4-methyl-7-hydroxy coumarin and 4-methyl-7-acetyloxy coumarin are
potent highly selective towards the AChE in vitro and in vivo.
These compounds are highly effective for the treatment and
prevention of AChE.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1a: Effect of AP 20am Samples (100 mg/Kg, Po.times.3
Days) on Scopolamine induced Deficit in Passive Avoidance Test in
Mice (p**<0.001, *<0.01 Significant difference from 1st
Trial, p value determined by student's t Paired test).
[0007] FIG. 1b: Effect of Ap 20am14.15 & 16. Samples (50 mg/Kg,
po.times.3 Days) on Scopolamine induced Deficit in Passive
Avoidance Test in Mice (p**<0.001, *<0.01 Significant
difference from 1st Trial, p value determined by student's t Paired
test).
OBJECTIVE OF THE INVENTION
[0008] The main objective of the present invention is to provide a
pharmaceutical composition useful as acetylcholinesterase
inhibitor.
[0009] Another object of the present invention is to provide the
composition wherein the composition exhibit more percent memory
retention than standard drug Donepezil, in scopolamine induced
memory deficit mice.
[0010] One of the objectives of the present invention is to use the
naturally occurring compounds for the preparation of (.+-.)
Marmesin and Columbianetin.
[0011] It is another objective of the present invention is to
provide composition using dihydroxanthyletin, useful as AChE
inhibitor.
[0012] It is further objective of the present invention is to
provide composition using compounds 7-allyloxy coumarin,
7-benzyloxy coumarin and 4-methyl-7-acetyloxy coumarins, useful as
AChE inhibitor.
[0013] It is yet further objective of the present invention is to
provide composition using compounds selected from 7-methoxy
coumarin, 7-acetyloxy coumarin and 4-methyl-7-hydroxy coumarins,
useful as AChE inhibitor.
SUMMARY OF THE INVENTION
[0014] Accordingly the present invention provides a pharmaceutical
composition comprising an effective amount of compound of formula
1, analogs and pharmaceutically acceptable salts thereof;
##STR00001## [0015] (i) wherein R1 and R2 is linked with each other
via the following moiety and collectively makes fused system, and
R3 is H;
[0015] ##STR00002## [0016] (ii) wherein R1 and R3 is linked with
each other via following moiety and collectively makes fused
system, and R1 is H;
[0016] ##STR00003## [0017] (iii) wherein R1 and R2 is linked with
each other via following moiety and collectively makes fused
system, and R3 is H;
[0017] ##STR00004## [0018] (vi) wherein the value of R, R1, R2, and
R3 is selected from the group consisting of
[0019] 1. R=R.sup.2=R.sup.3=H; R.sup.1=OH.
[0020] 2. R=R.sup.2=R.sup.3=H; R.sup.1=Prenyl
[0021] 3. R=R.sup.2=R.sup.3=H; R.sup.1=Allyl
[0022] 4. R=R.sup.2=R.sup.3=H; R.sup.1=2,2-dimethyl alkyne
[0023] 5. R=R.sup.2=R.sup.3=H; R.sup.1=2,2-dimethyl alkene
[0024] 6. R=R.sup.2=R.sup.3=H; R.sup.1=Benzyl
[0025] 7. R=R.sup.2=R.sup.3=H; R.sup.1=Acetyl
[0026] 8. R=R.sup.2=R.sup.3=H; R.sup.1=Methyl
[0027] 9. R=R.sup.1=R.sup.3=H; R.sup.2=Prenyl
[0028] 10. R=R.sup.3=H; R.sup.1=R.sup.2=Prenyl
[0029] 11. R=R.sup.1=R.sup.2=H; R.sup.3=Prenyl
[0030] 12. R=Methyl; R.sup.1=R.sup.2=R.sup.3=H
[0031] 13. R=R.sup.1=Methyl; R.sup.2=R.sup.3=H
[0032] 14. R=R.sup.1=Acetyl; R.sup.2=R.sup.3=H
[0033] 15. R=Methyl; R.sup.1=R.sup.3=H; R.sup.2=OH
[0034] 16. R=Methyl; R.sup.1=Benzyl, R.sup.2=Benzyloxy,
R.sup.3=H
[0035] 17. R=Methyl; R.sup.1=Methyl, R.sup.2=Methyloxy,
R.sup.3=H
[0036] 18. R=Methyl; R.sup.1=Acetyl, R.sup.2=Acetyloxy,
R.sup.3=H
[0037] optionally along with the pharmaceutically acceptable
carrier, or diluent, wherein the effective dose of composition is
ranging between 50 to 100 mg/kg body weight.
[0038] In an embodiment of the present invention, wherein the
compound of general formula 1 further comprising:
##STR00005##
[0039] In an embodiment of the invention, wherein the compound of
general formula 1 further comprising:
##STR00006##
[0040] In another embodiment of the invention, wherein the compound
of general formula 1 further comprising:
##STR00007##
[0041] In yet another embodiment of the invention, wherein the
compound of general formula 1 further comprising:
##STR00008##
[0042] In a further embodiment of the invention, wherein the
compound of general formula 1 further comprising:
##STR00009##
Wherein
[0043] R=R.sup.2=R.sup.3=H; R.sup.1=OH.
[0044] R=R.sup.2=R.sup.3=H; R.sup.1=Prenyl
[0045] R=R.sup.2=R.sup.3=H; R.sup.1=Allyl
[0046] R=R.sup.2=R.sup.3=H; R.sup.1=2,2-dimethyl alkyne
[0047] R=R.sup.2=R.sup.3=H; R.sup.1=2,2-dimethyl alkene
[0048] R=R.sup.2=R.sup.3=H; R.sup.1=Benzyl
[0049] R=R.sup.2=R.sup.3=H; R.sup.1=Acetyl
[0050] R=R.sup.2=R.sup.3=H; R.sup.1=Methyl
[0051] R=R.sup.1=R.sup.3=H; R.sup.2=Prenyl
[0052] R=R.sup.3=H; R.sup.1=R.sup.2=Prenyl
[0053] R=R.sup.1=R.sup.2=H; R.sup.3=Prenyl
[0054] R=Methyl; R.sup.1=R.sup.2=R.sup.3=H
[0055] R=R.sup.1=Methyl; R.sup.2=R.sup.3=H
[0056] R=R.sup.1=Acetyl; R.sup.2=R.sup.3=H
[0057] R=Methyl; R.sup.1=R.sup.3=H; R.sup.2=OH
[0058] R=Methyl; R.sup.1=Benzyl, R.sup.2=Benzyloxy, R.sup.3=H
[0059] R=Methyl; R.sup.1=Methyl, R.sup.2=Methyloxy, R.sup.3=H
[0060] R=Methyl; R.sup.1=Acetyl, R.sup.2=Acetyloxy, R.sup.3=H
[0061] In yet another embodiment of the invention, wherein the
compound used for the preparation of composition is selected from
the group consisting of (.+-.) Marmesin, Columbianetin,
dihydroxanthyletin, 7-methoxy coumarin, 7-acetyloxy coumarin,
4-methyl-7-hydroxy coumarin, 7-Allyloxycoumarin, and
7-Benzyloxycoumarin.
[0062] In an embodiment of the invention, wherein the compounds
used for the preparation of composition may be from natural source
or synthesized.
[0063] In still another embodiment of the invention wherein the
composition is useful as acetyl cholinesterase inhibitor.
[0064] In a further embodiment of the invention wherein the
composition inhibits cholinesterase up to 49%
[0065] In still another embodiment of the invention wherein the
composition is administered by oral route.
[0066] Accordingly the present invention provides a method of
treating Alzheimer's disease and related chronic neuron
degenerative disorders comprising administering the composition in
a subject need thereof wherein the composition comprising an
effective amount of compound of formula 1, analogs and
pharmaceutically acceptable salts thereof optionally along with the
pharmaceutically acceptable carrier, or diluents.
[0067] In an embodiment of the invention, wherein the composition
is administered by oral route.
[0068] In another embodiment of the invention, wherein the
composition exhibit more percent memory retention than standard
drug Donepezil, in scopolamine induced memory deficit mice.
[0069] In a still another embodiment of the invention, wherein the
composition comprising the compound selected from group consisting
of (.+-.) Marmesin, Columbianetin, Dihydroxanthyletin,
7-Hydroxy-6-prenylcoumarin, 7-Methoxycoumarin, 7-Acetyloxycoumarin,
4-Methyl-7-hydroxycoumarin, 4-Methyl-7-methoxycoumarin,
4-Methyl-7-acetyloxy coumarin, 4-Methyl-7,8-dihydroxycoumarin,
4-Methyl-7,8-dibenzyloxycoumarin, 4-Methyl-7,8-dimethoxycoumarin,
and 4-Methyl-7,8-diacetyloxycoumarin inhibiting the AChE in
vitro.
[0070] In an embodiment of the invention, wherein the composition
comprising the compound selected from the group consisting of
7-allyloxy coumarin, 7-benzyloxy coumarin, 7-Methoxycoumarin,
7-Acetyloxycoumarin, 4-Methyl-7-hydroxycoumarin, and
4-methyl-7-acetyloxy coumarins, is inhibiting acetylcholinesterase
in vivo
DETAILED DESCRIPTION OF THE INVENTION
[0071] The invention is further described in the following examples
that are given by the way of illustration and therefore should not
be construed to limit the present invention in any manner.
Example-1
Preparation of (.+-.) Marmesin, Columbianetin, Dihydroxanthyletin,
Seselin and Coumarin Derivatives
[0072] The synthetic (.+-.) Marmesin and columbianetin were
prepared starting from 7-Hydroxy coumarin (Umbelliferone).
Condensation of 7-hydroxy coumarin with 2-chloro-2-methyl but-3-yne
in the presence of base gave propargylether. The obtained ether on
catalytic hydrogenation afforded alkene. The alkene on claisen
rearrangement gave two isomeric products. Oxidation of
7-Hydroxy-6-Prenyl coumarin with m-chloroperbenzoic acid in ethyl
acetate solvent gave (.+-.) Marmesin. Oxidation of
7-Hydroxy-8-Prenyl coumarin with m-chloroperbenzoic acid in ethyl
acetate solvent gave columbianetin. 7-Hydroxy-6-Prenyl coumarin on
oxidation with m-chloroperbenzoic acid in chloroform solvent gave
dihydroxanthyletin. (Tetrahedron, 27, 1247, 1971, Tetrahedron, 27,
4901, 1971).
[0073] The synthetic seselin was prepared starting from 7-Hydroxy
coumarin (Umbelliferone). Condensation of 7-hydroxy coumarin with
2-chloro-2-methyl but-3-yne in the presence of base gave
propargylether. The propargylether on heating at 200.degree. C.
with N,N-dimethyl aniline gave seselin. (Tetrahedron, 27, 1247,
1971, Tetrahedron, 27, 4901, 1971).
Example-2
Experimental Procedures for In vitro and In vivo evaluation of
(.+-.) Marmesin, Columbianetin, Dihydroxanthyletin, Seselin and
Coumarin Derivatives
Acetylcholinesterase Inhibiting Potency:
[0074] Single trial passive avoidance is widely used as
experimental test to assess learning memory functions in rodents.
Scopolamine induced impairment in passive avoidance (in vivo) and
inhibition of acetylcholinesterase (in vitro) in rodents are
commonly employed and screening test to predict potential of an
acetylcholinesterase inhibitor as cognitive enhancer
(anti-dementic) drug.
Passive Avoidance Test (In Vivo):
[0075] The study was conducted in adult Swiss male mice of 3-4
months (wt. 20-25 g) were kept in standard housing condition with
12 h light and dark cycle. The food and water were available ad
libitum.
[0076] The mice were subjected to single trial passive avoidance
test as described by Brioni.
[0077] The passive avoidance test was studied by a computerized
shuttle box (Columbus Instruments, Ohio, USA) provided with a
software program PACS 30. The shuttle box is comprised of two
compartments. An automated door was used to isolate the
compartments. After an exploration period of 30 s for
acclimatization the animal was subjected to a trial of 270 seconds.
Each mouse was placed in the bright compartment and on transfer
into the dark compartment it was given an electric shock (0.5 mA
for 5 s) through floor grid. The transfer of mice from the bright
to dark compartment was recorded as transfer latency time (TLT) in
seconds. TLT was recorded in control and treated groups (1.sup.st
Trial, acquisition) and then after 24 h (2.sup.nd Trial,
retention). An increase in the TLT on 2.sup.nd Trial (retention) as
compared to 1.sup.st Trial (acquisition) was taken as the criterion
for successful learning and memory (cognitive activity).
Scopolamine Induced Deficit (Dementia)
[0078] Scopolamine, a muscarinic antagonist known to produce
impairment in cognitive functions (dementia) in human as well as in
experimental animals, was used to produce deficit (no significant
increase on 2.sup.nd trial) in passive avoidance learning.
Scopolamine was administered 5 min prior to 1.sup.st trial.
Reversal of scopolamine induced deficit i.e. significant increase
in 2.sup.nd trial by test substance indicates potential
anti-dementia activity. Scopolamine was administered 5 min prior to
1.sup.st trial.
Drug Administration
[0079] The test substance Columbianetin was administered orally in
dose of 100 mm/kg (1% aqueous suspension in gum acacia) for 3 days.
The treated animals were subjected to 1.sup.st trial after 60 min
of 3.sup.rd dose of test substance. Scopolamine was administered 5
min prior to 1.sup.st trial in test group.
[0080] The control group received 1 ml/kg of vehicle (1% aqueous
suspension in gum acacia) orally for 3 days. Among the control mice
half (n=5) received scopolamine 5 min prior to 1.sup.st trial
(Control--dementia) and the remaining half were subjected to
passive avoidance test (Control--trained).
[0081] The control group showed significant increase in TLT on
2.sup.nd trial as compared to 1.sup.st trial indicating the
successful learning and memory function. Mice treated with
scopolamine did not show significant increase in TLT on 2.sup.nd
trial as compared to 1.sup.st trial indicating the deficit in
learning and memory function. In the animals pretreated with
7-Allyloxycoumarin (AP20am12), 7-Benzyloxycoumarin (AP20am13),
7-Methoxycoumarin (AP20am14), 7-Acetyloxycoumarin (AP20am15),
4-Methyl-7-hydroxycoumarin (AP20am16) and
4-Methyl-7-acetyloxycoumarin (AP20am19) treatment with scopolamine
failed to cause memory deficit as indicated by significant increase
in TLT on 2.sup.nd trial as compared to 1.sup.st trial. See FIGS.
1a and 1b.
[0082] Table 1 shows the comparison between of AP20am samples 14,
15 and 16 (50 mg/kg, po) and Standard Drug Donepezil (10 mg/kg, po)
on scopolamine induced memory deficit in passive avoidance test on
basis of memory retention i.e. % increase in TLT of 2.sup.nd trial
from TLT of 1.sup.st trial. The passive avoidance test is for
testing learning and memory functions. Scopolamine is used to
produce memory deficit in this test. Inhibition of scopolamine
induced memory deficit by a test substance indicate potential of
that test sample as memory enhancer. Among these compounds
7-Methoxycoumarin, 7-Acetyloxycoumarin, and
4-Methyl-7-hydroxycoumarin were most effective. The prevention of
scopolamine induced memory deficit suggests that 7-Methoxycoumarin,
7-Acetyloxycoumarin, and 4-Methyl-7-hydroxycoumarin have potential
for anti-dementia activity.
[0083] Level of p value<0.05 indicates statistically significant
difference. Lowering in the p value means more significant
difference suggesting very significant increase in from TLT from
1.sup.st trial.
TABLE-US-00001 TABLE 1 comparison data of coumarin compounds with
Standard Drug Donepezil Groups % Memory retention Control trained
(No scopolamine) 483 Scopolamine (Memory deficit) 33 Donepezil +
Scopolamine 288 AP20am 14 + Scopolamine 295 AP20am 15 + Scopolamine
321 AP20am 16 + Scopolamine 376
Acetylcholinesterase (AChE) Assay in Brain (In Vitro)
[0084] The study was conducted in adult SD male rats (200-250 g).
The rats were perfused under mild ether anesthesia through heart
with ice cooled normal saline (0.9% NaCl) to reduce blood-borne
cholinesterase from the brain. After perfusion the whole brain was
taken out. A 10% (w/v) homogenate of brain was prepared first by
homogenizing in an Ultra-Turrax T25 homogenizer at a speed of 9500
rpm thrice giving intervals for few seconds in between the runs,
with sodium phosphate buffer (30 mmol/lit, pH 7.0). Sodium
phosphate buffer was taken in a volume half to the final volume
required for 10% homogenate.
[0085] 1% Triton X-100 (1% w/v in 30 mmol/lit. sodium phosphate
buffer, pH 7.0) is then added in a volume to make the final volume
for 10% homogenate, slowly while stirring the homogenate on ice.
The homogenate was centrifuged at 100,000.times.g at 4.degree. C.
in a Beckman Ultracentrifuge (LE 80) using a fixed angle rotor (80
Ti) for 60 min. Supernatant was collected and stored at 4.degree.
C. Aliquots of this supernatant was diluted in the ratio of 1:10
and used as a source of enzyme for the assay.
Enzyme Assay
[0086] The assay of AChE was performed according to method
described by Ellman et al. A kinetic profile of the enzyme activity
was studied spectrophotometrically at 412 nm at an interval of 15
s. The assay for each sample was run in duplicate and each
experiment was performed thrice. The specific activity of AChE is
expressed in .mu.moles/min/mg of protein. The test substance
(dissolved in DMSO) was incubated with enzyme source in
concentration of 100 .mu.g/1 ml of reaction mixture for 30 min at
37.degree. C. prior to obtain kinetic profile of AChE activity. The
AChE inhibitory activity was calculated on the basis of % decrease
change from control values.
Protein Assay
[0087] Protein was estimated in the brain samples by modified
Lowry's method. Bovine serum albumin (BSA) was used as standard in
the concentration of 1 mg/ml. was estimated in the range of
0.01-0.1 mg/ml.
Results
[0088] The results are reported in Table 2. The compounds that
showed in vitro inhibition of AChE (25-49%) are (.+-.) Marmesin,
Dihydroxanthyletin, 7-Acetyloxycoumarin and
4-Methyl-7-methoxycoumarin. The compounds showed in vitro
inhibition of AChE inhibition (20-25%) are 7-Methoxycoumarin,
4-Methyl-7-acetyloxycoumarin, 4-Methyl-7,8-dibenzyloxycoumarin and
4-Methyl-7,8-diacetyloxycoumarin. However, in passive avoidance
test (in vivo) 7-Acetyloxycoumarin and 4-Methyl-7-methoxycoumarin
were effective. The ineffectiveness of other in vitro active
compounds in in vivo passive avoidance test may be due to
pharmacokinetics factors like absorption and metabolism.
Statistical Analysis
[0089] Mean values and standard error (S.E.) of mean were
calculated for TLT and specific activity of AChE in the different
regions of brain samples of each group. The significance of
difference between the values of AChE activity and TLT of two
groups was determined by one-way ANOVA test that followed by
Dunnett's test.
TABLE-US-00002 TABLE 2 In Vitro and In Vivo data of the coumarin
compounds Compound InVitro %) In Vivo (.+-.) Marmesin 23.91
Significant activity (P < 0.01) Columbianetin 13.93 No
significant activity Dihydroxanthyletin 37.8 No significant
activity Seselin No significant activity 7-Hydroxycoumarin No
significant activity 7-Prenyloxycoumarin No significant activity
7-Hydroxy-6-prenylcoumarin 42.2 No significant activity
7-Dimethylpropargylcoumarin No significant activity
7-Dimethylalkenylcoumarin No significant activity
7-Hydroxy-8-prenylcoumarin No significant activity
7-Allyloxycoumarin Significant activity (P < 0.05)
7-Benzyloxycoumarin Significant activity (P < 0.005)
7-Methoxycoumarin 25.2 High significant activity (P < 0.0001)
7-Acetyloxycoumarin 39.13 High significant activity (P < 0.0001)
4-Methyl-7-hydroxycoumarin 17.39 High significant activity (P <
0.0001) 4-Methyl-7-methoxycoumarin 48.91 No significant activity
4-Methyl-7-acetyloxycoumarin 23.91 Significant activity (P <
0.01) 4-Methyl-7,8-dihydroxycoumarin 14.13 No significant activity
4-Methyl-7,8-dibenzyloxycoumarin 24.1 No significant activity
4-Methyl-7,8-dimethoxycoumarin 14.2 No significant activity
4-Methyl-7,8-diacetyloxycoumarin 24.01 No significant activity
Advantages:
[0090] 1. The compounds (.+-.) Marmesin, Columbianetin,
Dihydroxanthyletin and coumarin derivatives are useful as AChE
inhibitors. [0091] 2. The percentage inhibition is high when
compared to the earlier reported compounds. [0092] 3. All the above
mentioned compounds are easily extractable from the natural source
and can be synthesized efficiently in the laboratory. [0093] 4.
Coumarin derivatives were efficiently synthesized in the
laboratory.
* * * * *