BENZOCOUMARIN AMPK ACTIVATOR COMPOUNDS, COMPOSITIONS, METHODS AND USES THEREOF

Abstract

The present invention relates to a compound having general formula (I) for use in the activation of AMPK. A composition comprising said compound for use in the activation of AMPK is also provided.

Claims

1. A method for the activation of AMPK compound having the general formula I, ##STR00023## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH.sub.3; CH.sub.2OH; CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3; a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof, for use in the activation of AMPK.

2. A method according to claim 1 wherein said compound is a compound of Formula II ##STR00024## wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl; R6, and R7 are each independently selected from the group consisting of H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl, and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof.

3. A method according to claim 1 wherein said compound is a compound of Formula III ##STR00025## wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl; R6, and R7 are each independently selected from the group consisting of H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl, and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof.

4. A method according to claim 1 wherein said compound is a compound of Formula IV ##STR00026## wherein R1, R2, and R3 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl; R4, and R5 are each independently selected from the group consisting of H, OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl, and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof.

5. A method according to claim 1 wherein said compound is a compound of Formula V ##STR00027## wherein R1, R2, R3, and R4 are each independently selected from the group consisting of OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl; R5 and R6 are each independently selected from the group consisting of H; OH; OCH.sub.3; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a halogen; a primary, secondary, or tertiary alcohol; a ketone; an aldehyde; a carboxylic acid; an ester; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; substituted and/or branched C1 to C20 alkyl; substituted and/or branched, C2 to C20 alkenyl; substituted and/or branched, C4 to C20 polyalkenyl; substituted and/or branched C2 to C20 alkynyl, or substituted and/or branched C4 to C20 polyalkynyl, and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof.

6. A method according to claim 1 wherein said compound is compound 1: ##STR00028## which is 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one.

7. A method according to claim 1 to treat or prevent a condition, disorder, or disease related to cardiometabolic health, obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and/or cancer in a subject.

8. A method according to claim 7, wherein the subject is a human.

9. A for use method according to claim 1, wherein the activation of AMPK is through a direct activation mechanism.

10. A method according to claim 1, wherein the activation of AMPK is in muscle, liver and/or kidney tissues.

11. A method according to claim 1, wherein the activation of AMPK is AMPK which comprises an α2 subunit, a β1 subunit, and a γ1 subunit.

12. A method according to claim 1, wherein the compound is obtained from a plant or plant extract.

13-14. (canceled)

15. A method according to claim 1, wherein the composition is a food, beverage, or dietary supplement.

16. A method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

17. A method according to claim 1, wherein the compound of general formula I is ##STR00029## which is 3,10-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,10-Dihydroxy-8-methoxy; 3,10-Dihydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one.

18. A pharmaceutical composition comprising a therapeutically effective amount of the compound of general formula I, ##STR00030## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH.sub.3; CH.sub.2OH; CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3; a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof, for use in the activation of AMPK, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient, and a pharmaceutically acceptable carrier, for use in the activation of AMPK.

19. (canceled)

20. A method of treatment or prevention of a condition, disorder, or disease related to cardiometabolic health, obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and/or cancer comprising administration to a subject in need of same a composition having the general formula I, ##STR00031## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; CH.sub.3; CH.sub.2OH; CHO; COOH; OH; OCH.sub.3; CO—(CH.sub.2).sub.2—CH.sub.3; O—CO—CH.sub.3; a halogen; O-glycoside; C-glycoside; acylated O-glycoside; acylated C-glycoside; sulfated O-glycoside; sulfated C-glycoside; a sulfate; and the OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge; and/or a derivative or analogue thereof, for use in the activation of AMPK.

Description

BRIEF DESCRIPTION OF FIGURES

[0599] FIG. 1—Compound 1 increases the phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC), in U-2 OS Flp-In T-REx mammalian cells.

[0600] FIG. 2—Compound 1 activates AMPK in cells expressing β1 WT, but does not activate AMPK in cells stably expressing an ADaM-binding site AMPK mutant (β1 S108A).

[0601] FIG. 3—Compound 1 activates AMPK in cells stably expressing the β1 isoform, but does not activate AMPK in cells stably expressing the 32 isoform.

EXAMPLES

Example 1: Compound 1 Increases the Phosphorylation of the AMPK Substrate, Acetyl-CoA Carboxylase (ACC), in U-2 OS Flp-In T-REx Mammalian Cells

[0602] U-2 OS Flp-In T-REx cells were seeded at 50 K in a 96-well plate and left overnight at 37 C in DMEM GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (vol/vol) FBS and 100 U/ml penicillin G, and 100 μg/ml streptomycin. Cells were treated for 30 mins with varying concentrations of Compound 1 in media lacking FBS and then cells were lysed in 50 μl of Cisbio lysis buffer #1 supplemented with blocking solution as per the manufacturer's protocol (Cisbio). Cells were lysed for 30 mins at room temperature before 16 μl of lysate was incubated with 4 μl of the HTRF antibodies (1:40 dilution of the acceptor and donor (p)ACC antibodies, as per the manufacturers protocol). Lysates were incubated overnight with the antibodies before 665 nm/620 nm ratio was determined using a MolecularDevices i3 plate reader (with a HTRF cartridge add-on).

[0603] FIG. 1 shows that using the pACC HTRF assay kit (Cisbio), Compound 1 increases the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in U-2 OS Flp-In T-REx mammalian cells. Phosphorylation of ACC is widely used as a cellular indicator of AMPK activity.

Example 2- Compound 1 does not Activate AMPK Complexes Containing a Mutation at the Allosteric Drug and Metabolite (ADaM) Site in Cells (S108A)

[0604] AMPKβ1/β2 double knockout U-2 OS Flp-In™ T-Rex™ cell lines were generated by Horizon Discovery (Cambridge, UK). Cells were genotyped and analysed by western blotting to confirm that there was a complete knockout of AMPKβ1/β2. We took these AMPKβ1/β2 double knockout cells, and re-introduced the expression of human β1 wild-type (WT) or a 31 Serine 108 to alanine mutation (S108A). This was achieved using the Flp-In™ system (Invitrogen) present in this cell line and stable cells expressing β1 WT or a β1 S108A mutant were generated according to the manufacturers' protocols. Re-expression of the β1 subunit was confirmed by Western blot analysis. Mutation of β1 S108A has previously been shown to interfere with regulation of AMPK by compounds binding to the allosteric drug and metabolite (ADaM) binding site formed at the interface between the β subunit carbohydrate binding module (CBM) and the α subunit kinase domain. In contrast, activators through the nucleotide binding site on the AMPK γ subunit can still regulate the β1 S108A mutant comparable to β1 WT activation.

[0605] Cells stably expressing β1 WT or a β1 S108A mutant were treated with varying concentrations of Compound 1 and subjected to the pACC HTRF (Cisbio) assay to determine the level of phosphorylation of the AMPK substrate, ACC, in cell lysates. As shown in FIG. 2, Compound 1 was able to dose-dependently increase pACC in cells stably expressing the 1 WT subunit. In contrast, Compound 1 was not able to increase pACC in cells expressing the β1 S108A mutant. This suggests that compound 1 directly activates AMPK by binding to the ADaM pocket of AMPK.

Example 3—Compound 1 does not Activate AMPK Complexes Containing the β2 Isoform Subunit

[0606] AMPKβ1/β2 double knockout cells were used and re-introduced the expression of human β1 WT or β2 WT isoforms. Re-expression of the β1 and β2 subunit was confirmed by Western blot analysis and were shown to be expressed to a similar extent. Cells stably expressing β1 WT or β2 were treated with varying concentrations of Compound 1 and subjected to the pACC HTRF (Cisbio) assay to determine the level of phosphorylation of the AMPK substrate, ACC, in cell lysates, as in Example 1. As shown in FIG. 3, Compound 1 was able to dose-dependently increase pACC in cells stably expressing β1 WT.

[0607] In contrast, Compound 1 was not able to increase pACC in cells expressing the β2 WT isoform. This activation profile is characteristic of activators that bind to the ADaM site and consistent with previous studies showing that ADaM-site activators have poorer activation of β2-containing complexes in vitro and in cells. Taken together, we show that in cells, Compound 1 activates AMPK by binding to the ADaM pocket of AMPK separate from the nucleotide-binding site in the AMPKγ subunit.