Direct AMPK activator compounds

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. Said compounds are 9,10 dihydrophenanthrenes, in particular selected from the group consisting of: (i) Lusianthridin i.e. 7-Methoxy-9,10-dihydrophenanthrene-2,5-diol, (ii) 7-Methoxy-9,10-dihydrophenanthrene-2,3,5-triol, (iii) 9,10-dihydrophenanthrene-2,5-diol, (iv) 9,10-Dihydrophenanthrene-2,4,7-triol (v) 9,10-Dihydro-7-methoxy-3,5-phenanthrenediol, and the activation of AMPK improves the condition, disorder, or disease related to cardiometabolic health, obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and cancer.

Claims

1. A method for activation of AMPK to treat, and/or reduce a severity of non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, the method comprising administering to the subject a compound having the general formula I, ##STR00016## wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; OH; OMe; 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; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20 alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.20 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.20 polyalkynyl, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

2. The method according to claim 1, wherein R1, R2, R3, R6, R7, and R8 are each independently selected from the group consisting of H; OH; OMe; 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; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20 alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.20 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.20 polyalkynyl; R4 and R5 are each independently H; OH; 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; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.20 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.20 polyalkynyl, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

3. The method according to claim 1, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20 alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.20 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.2 polyalkynyl, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

4. The method according to claim 1, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H; Me; OH; OMe; OCH.sub.2CH═CH.sub.2; O-glycoside; a sulfate; a halogen; CHO; CH.sub.2OH; COOH, CONH.sub.2, COCH.sub.3; CH═CH.sub.2; CH.sub.2—CH═C(CH.sub.3).sub.2; CH(CH.sub.3).sub.2; CH═CH—CHO; CH(CH.sub.3)—OH; CH(CH.sub.3)—OMe; CH(CH.sub.3)—OC.sub.2H.sub.5; CH(CH.sub.3)—O—CH.sub.2—CH═C(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3).sub.2; 4-hydroxybenzyl; 4-hydroxy-3-methoxybenzyl; 4-hydroxybenzoyl; 4-hydroxybenzoyl glycoside, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

5. The method having the general formula I according to claim wherein R1, R2, R3, R6, R7, and R8 are each independently selected from the group consisting of H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20 alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.2 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.20 polyalkynyl; R4 and R5 are each independently H; OH; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; an optionally substituted and/or optionally branched C.sub.1 to C.sub.20 alkyl; an optionally substituted and/or optionally branched, C.sub.2 to C.sub.20 alkenyl; an optionally substituted and/or optionally branched, C.sub.4 to C.sub.20 polyalkenyl; an optionally substituted and/or optionally branched C.sub.2 to C.sub.20 alkynyl, or an optionally substituted and/or optionally branched C.sub.4 to C.sub.20 polyalkynyl, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

6. The method according to claim 1, wherein R1, R2, R3, R6, R7, and R8 are each independently selected from the group consisting of H; Me; OH; OMe; OCH.sub.2CH═CH.sub.2; O-glycoside; a sulfate; a halogen; CHO; CH.sub.2OH; COOH, CONH.sub.2, COCH.sub.3; CH═CH.sub.2; CH.sub.2—CH═C(CH.sub.3).sub.2; CH(CH.sub.3).sub.2; CH═CH—CHO; CH(CH.sub.3)—OH; CH(CH.sub.3)—OMe; CH(CH.sub.3)—OC.sub.2H.sub.5; CH(CH.sub.3)—O—CH.sub.2—CH═C(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3).sub.2; 4-hydroxybenzyl; 4-hydroxy-3-methoxybenzyl; 4-hydroxybenzoyl; 4-(addedhydroxybenzoyl glycoside; R4 and R5 are each independently H; Me; OH; OCH.sub.2CH═CH.sub.2; O-glycoside; a sulfate; a halogen; CHO; CH.sub.2OH; COOH, CONH.sub.2, COCH.sub.3; CH═CH.sub.2; CH.sub.2—CH═C(CH.sub.3).sub.2; CH(CH.sub.3).sub.2; CH═CH—CHO; CH(CH.sub.3)—OH; CH(CH.sub.3)—OMe; CH(CH.sub.3)—OC.sub.2H.sub.5; CH(CH.sub.3)—O—CH.sub.2—CH═C(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3).sub.2; 4-hydroxybenzyl; 4-hydroxy-3-methoxybenzyl; 4-hydroxybenzoyl; 4-hydroxybenzoyl glycoside, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

7. The method according to claim 1, wherein R1, R2, R3, R6, R7, and R8 are each independently selected from the group consisting of H; Me; OH; OMe; OCH.sub.2CH═CH.sub.2; O-glycoside; a sulfate; Br; CHO; CH.sub.2OH; COOH, CONH.sub.2, COCH.sub.3; CH═CH.sub.2; CH.sub.2—CH═C(CH.sub.3).sub.2; CH(CH.sub.3).sub.2; CH═CH—CHO; CH(CH.sub.3)—OH; CH(CH.sub.3)—OCH.sub.3; CH(CH.sub.3)—OC.sub.2H.sub.5; CH(CH.sub.3)—O—CH.sub.2—CH═C(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3).sub.2; 4-hydroxybenzyl; 4-hydroxy-3-methoxybenzyl; 4-hydroxybenzoyl; 4-hydroxybenzoyl glycoside; R4 and R5 are each independently H; Me; OH; OCH.sub.2CH═CH.sub.2; O-glycoside; a sulfate; Br; CHO; CH.sub.2OH; COOH, CONH.sub.2, COCH.sub.3; CH═CH.sub.2; CH.sub.2—CH═C(CH.sub.3).sub.2; CH(CH.sub.3).sub.2; CH═CH—CHO; CH(CH.sub.3)—OH; CH(CH.sub.3)—OCH.sub.3; CH(CH.sub.3)—OC.sub.2H.sub.5; CH(CH.sub.3)—O—CH.sub.2—CH═C(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3)—(CH.sub.2).sub.3—CH(CH.sub.3).sub.2; 4-hydroxybenzyl; 4-hydroxy-3-methoxybenzyl; 4-hydroxybenzoyl; 4-hydroxybenzoyl glycoside, and/or optionally a OCH.sub.3 group can cyclize with a neighboring OH group to form a methylene dioxy bridge.

8. The method according to claim 1, wherein said compound is selected from the group consisting of: (i) Lusianthridin known as 7-Methoxy-9,10-dihydrophenanthrene-2,5-diol; (ii) 7-Methoxy-9,10-dihydrophenanthrene-2,3,5-triol; (iii) 2,5-Phenanthrenediol, 9,10-dihydro, 9,10-Dihydrophenanthrene-2,5-diol; (iv) 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro; and (v) Cannithrene 1, Cannabidihydrophenanthrene, 9,10-Dihydro-7-methoxy-3,5-phenanthrenediol, 3,5-Phenanthrenediol, 9,10-dihydro-7-methoxy, 7-Methoxy-9,10-dihydrophenanthrene-3,5-diol.

9. The method according to claim 1, wherein the subject is a human or a companion animal.

10. The method according to claim 9, wherein the subject is a human.

11. The method according to claim 9, wherein the activation of the AMPK further improves a condition, disorder, or disease selected from the group consisting of cardiometabolic health, obesity, type 2 diabetes, cardiovascular disease, and cancer.

12. The method according to claim 1, wherein the activation of the AMPK is a direct activation mechanism.

13. The method according to claim 1, wherein the activation of the AMPK occurs in muscle and/or liver tissues.

14. The method according to claim 1, wherein the AMPK comprises an α2 subunit, a β1 subunit, and a γ1 subunit.

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

16. A method according to claim 1, wherein the compound of formula I is administered as a medicament.

17. The method according to claim 1, wherein the compound is administered in a composition selected from the group consisting of a food, a beverage, and a dietary supplement.

18. A method according to claim 16, wherein the compound of general formula I is selected from the group consisting of: (i) Lusianthridin known as 7-Methoxy-9, 10-dihydrophenanthrene-2,5-diol; (ii) 7-Methoxy-9,10-dihydrophenanthrene-2,3,5-triol; (iii) 2,5-Phenanthrenediol, 9,10-dihydro, 9,10-Dihydrophenanthrene-2,5-diol; (iv) 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro; (v) Cannithrene 1, Cannabidihydrophenanthrene, 9,10-Dihydro-7-methoxy-3,5-phenanthrenediol, 3,5-Phenanthrenediol, 9,10-dihydro-7-methoxy, 7-Methoxy-9,10-dihydrophenanthrene-3,5-diol.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. AMP and Lusianthridin activation of bacterially-expressed AMPKα2β1γ1 (211) or α2β2γ1 (221) complexes. Lusianthridin (7-Methoxy-9,10-dihydrophenanthrene-2,5-diol, CAS number 87530-30-1) does not activate β2-containing complexes. It has been previously reported that activators at the Allosteric Drug and Metabolic (ADaM) binding site in AMPK, have weaker activation of β2-containing AMPK complexes. In contrast, regulators at the AMP-binding site are not influenced by the β-iso form composition of AMPK.

(2) FIG. 2. AMP and Lusianthridin activation of bacterially-expressed AMPKα1β1γ1 (111) or α2β1γ1 (211) complexes. Lusianthridin activates α1- and α2-containing complexes.

(3) FIG. 3. AMP and Lusianthridin activation of bacterially-expressed AMPKα2β1γ1 (211) or α2β1γ1γ1R298G (211 R298G) mutant complexes. Lusianthridin still activates AMPK complexes harbouring a mutation in the γ1 AMP binding pocket. In contrast, AMP regulation of this mutant is impaired. This suggests that Lusianthridin does not activate AMPK through the nucleotide-binding sites in the AMPKγ subunit.

(4) FIG. 4. AMP and Lusianthridin activation of bacterially-expressed AMPKα2β1γ1 (211) or α2β1γ1 complexes with the carbohydrate-binding module (CBM) deleted from the complex (ΔCBM) (211 ΔCBM). Lusianthridin does not activate the α2β1γ1 ΔCBM mutant. The CBM forms an integral part of the ADaM binding site and mediates binding of activators at this site. These data suggest that Lusianthridin activates AMPK at this site.

(5) FIG. 5. AMP and Lusianthridin activation of bacterially-expressed AMPKα2β1γ1 (211) or α2β1γ1 complexes harbouring the β1 S108A (211 S108A) mutation. Lusianthridin does not activate AMPK complexes with the S108A mutation. Previously, it has been shown that the S108A mutation interferes with regulation of AMPK at the ADaM binding site. Importantly, the phosphorylated S108 residue forms important interactions between the CBM of the β1 subunit and the kinase domain of the α subunit.

(6) FIG. 6. Lusianthridin increases the phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC), in U2OS Flp-In T-REx mammalian cells.

(7) (a) HTRF assay

(8) (b) Western blot analysis

(9) FIG. 7. Lusianthridin increases the phosphorylation of the AMPK substrate, ACC, in mouse primary hepatocytes.

(10) FIG. 8. Lusianthridin displays a dose-dependent inhibition of lipogenesis in primary hepatocytes.

(11) FIG. 9. Lusianthridin increases glucose uptake into differentiated C2C12 cells.

(12) FIG. 10. Compound 2, Compound 3, Compound 4 and Compound 5 activation of bacterially-expressed AMPKα2β1γ1 complexes.

(13) Compound 2, Compound 3, Compound 4 and Compound 5 activate AMPKα2β1γ1 complexes.

(14) Compound 2 is known as 7-Methoxy-9,10-dihydrophenanthrene-2,3,5-triol.

(15) Compound 3 is known as 2,5-Phenanthrenediol, 9,10-dihydro-, 9,10-Dihydrophenanthrene-2,5-diol, with a CAS number 1071055-42-9.

(16) Compound 4 is known as 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro, with a CAS number 70205-52-6.

(17) Compound 5 is known as Cannithrene 1, Cannabidihydrophenanthrene, 9,10-Dihydro-7-methoxy-3,5-phenanthrenediol, 3,5-Phenanthrenediol, 9,10-dihydro-7-methoxy, 7-Methoxy-9,10-dihydrophenanthrene-3,5-diol, with a CAS number 71135-80-3.

(18) FIG. 11. Compound 4 activation of various bacterially-expressed AMPK complexes and mutants.

(19) 211 represents AMPK α2β1γ1; 221 represents AMPK α2β2γ1; 111 represents AMPK α1β1γ1;

(20) 211 S108A represents the AMPK α2β1γ1 S108A mutant;

(21) 211 ΔCBM represents the AMPK α2β1γ1 ΔCBM mutant

(22) Compound 4 is known as 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro, with a CAS number 70205-52-6.

(23) Compound 4 activates α1- and α2-containing complexes but does not activate β2-containing complexes, the α2β1γ1 ΔCBM mutant and AMPK complexes with the S108A mutation. Taken together, this suggests that Compound 4 activates AMPK through binding to the ADaM pocket of AMPK.

(24) FIG. 12. Compound 2, Compound 3, Compound 4 and Compound 5 increase the phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC), in U2OS Flp-In T-REx mammalian cells.

(25) Compound 2 is known as 7-Methoxy-9,10-dihydrophenanthrene-2,3,5-triol.

(26) Compound 3 is known as 2,5-Phenanthrenediol, 9,10-dihydro-, 9,10-Dihydrophenanthrene-2,5-diol, with a CAS number 1071055-42-9.

(27) Compound 4 is known as 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro, with a CAS number 70205-52-6.

(28) Compound 5 is known as Cannithrene 1, Cannabidihydrophenanthrene, 9,10-Dihydro-7-methoxy-3,5-phenanthrenediol, 3,5-Phenanthrenediol, 9,10-dihydro-7-methoxy, 7-Methoxy-9,10-dihydrophenanthrene-3,5-diol, with a CAS number 71135-80-3.

(29) FIG. 13. Compound 4 displays a dose-dependent inhibition of lipogenesis in primary hepatocytes.

(30) Compound 4 is known as 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro, with a CAS number 70205-52-6.

(31) FIG. 14. Compound 4 increases glucose uptake into differentiated C2C12 cells.

(32) Compound 4 is known as 9,10-Dihydrophenanthrene-2,4,7-triol, 9,10-Dihydro-2,4,7-phenanthrenetriol, 2,4,7-Trihydroxy-9,10-dihydrophenanthrene, 2,4,7-Phenanthrenetriol, 9,10-dihydro, with a CAS number 70205-52-6.

(33) FIG. 15. Lusianthridin does not increase glucose uptake in C2C12 lacking the AMPKα1/α2 subunits.

(34) FIG. 16. Insulin can increase glucose uptake in C2C12 lacking the AMPKα1/α2 subunits.

(35) FIG. 17. Wortmanin does not affect Lusianthridin-stimulated glucose uptake in C2C12.

(36) FIG. 18. Lusianthridin did not display a cytotoxic effect on mouse primary hepatocytes.

(37) Mouse primary hepatocytes were treated with the indicated concentrations of Lusianthridin, Oligomycin and FCCP for 1 h at 37 C. Cell viability was determined by using an MTT assay as per the manufacturer's protocol. The results are displayed as the percentage of viable cells after treatment with these compounds.

(38) FIG. 19. Lusianthridin does not activate AMPK in cells stably expressing an ADaM-binding site AMPK mutant (β1 S108A) as assessed by the pACC HTRF assay.

(39) The β1 WT and β1 S108A mutant were stably expressed in AMPKβ1β2 double knockout cells and treated with varying concentrations of Lusianthridin for 30 mins at 37 C. Lusianthridin did not increase pACC in cells expressing the β1 S108A mutant. In contrast, Lusianthridin was capable of activating the β1 WT-expressing cells.

(40) FIG. 20. Lusianthridin does not activate AMPK in cells stably expressing an ADaM-binding site AMPK mutant (β1 S108A) as assessed by western blot analysis.

(41) In contrast to β1 WT-expressing cells, Lusianthridin did not increase phosphorylation of ACC in cells expressing the β1 S108A mutant-expressing cells. Taken together, this demonstrates that the ability of Lusianthridin to activate AMPK in cells is through its ability to bind to the ADaM site and not the nucleotide-binding site of AMPK.

EXAMPLES

Example 1

(42) Chemical Synthesis of Lusianthridin by Wittig Reaction Between (3-(benzyloxy)-5-methoxybenzyl)triphenylphosphonium bromide and 5-(benzyloxy)-2-iodobenzaldehyde

(43) Part 1: Synthesis of (3-(benzyloxy)-5-methoxybenzyl)triphenylphosphonium bromide.

(44) After suitable protection, 3,5-dihydroxybenzoic acid methyl ester was reduced to a primary alcohol, and converted to its corresponding alkyl halide before reaction with triphenylphosphine to give the desired triphenylphosphonium ylide reagent (Scheme 1).

(45) ##STR00013##

(46) Step a. To a solution of methyl 3,5-dihydroxybenzoate 1 (300 g, 1784.12 mmol) in acetone (7200 mL) was added potassium carbonate (271.22 g, 1962.53 mmol). The suspension was stirred at room temperature for 10 min. Benzyl bromide (222.50 mL, 1873.32 mmol) was added, and the resultant suspension was heated at 60° C. for 12 h. After cooling to room temperature, the suspension was filtered, the filter cake washed with acetone, and the filtrate was concentrated to a residue. The residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give methyl 3-(benzyloxy)-5-hydroxybenzoate 2 as an off-white solid. (144 g, 31% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 9.89 (s, 1H), 7.33-7.46 (m, 5H), 7.01 (dd, J=6.30, 0.90 Hz, 2H), 6.67 (t, J=2.40 Hz, 1H), 5.11 (s, 2H), 3.82 (s, 3H); MS (ES+) m/z 257.1 [M−H]+; HPLC-UV analysis: retention time=13.35 min; detection: 190-400 nm: peak area, 99.81%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(47) Step b. To a solution of methyl 3-(benzyloxy)-5-hydroxybenzoate 2 (140 g, 542.06 mmol) in acetone (7000 mL) was added potassium carbonate (224.74 g, 1626.20 mmol). The suspension was stirred at room temperature for 10 min. Iodomethane (168.73 mL, 2710.34 mmol) was added, and the resultant suspension was stirred at room temperature for 16 h. The suspension was filtered, the filter cake washed with acetone, and the filtrate was concentrated to a residue. The residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give methyl 3-(benzyloxy)-5-methoxybenzoate 3 as liquid. (125 g, 94% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 7.33-7.48 (m, 6H), 7.16 (t, J=2.10 Hz, 1H), 7.08 (d, J=1.20 Hz, 1H), 6.87 (t, J=2.40 Hz, 1H), 5.15 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H); MS (ES+) m/z 273.1 [M+H]+; HPLC-UV analysis: retention time=15.31 min; detection: 190-400 nm: peak area, 99.78%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(48) Step c. Lithium aluminium hydride (16.86 g, 444.36 mmol) in THF (605 mL) was added to methyl 3-(benzyloxy)-5-methoxybenzoate 3 (121 g, 444.36 mmol) in THF (1600 mL) at 0° C. The suspension was stirred at 0° C. for 20 min, at room temperature for 1 h. The reaction mixture was diluted with THF and quenched by addition of water. The resultant mixture was filtered through a pad of celite, and washed with ethyl acetate. The filtrate was concentrated in vacuo to give (3-(benzyloxy)-5-methoxyphenyl)methanol 4 as liquid. (100 g, 92% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 7.30-7.46 (m, 5H), 6.59 (d, J=0.60 Hz, 1H), 6.51 (s, 1H), 6.45 (d, J=2.40 Hz, 1H), 5.19 (t, J=5.70 Hz, 1H), 5.07 (s, 2H), 4.44 (d, J=5.70 Hz, 2H), 3.72 (s, 3H); MS (ES+) m/z 245.1 [M+H]+; HPLC-UV analysis: retention time=12.86 min; detection: 190-400 nm: peak area, 99.64%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(49) Step d. To a solution of (3-(benzyloxy)-5-methoxyphenyl)methanol 4 (100 g, 409.34 mmol) in 1,4-dioxane (1000 mL) was added phosphorous tribromide (50.54 mL, 532.15 mmol). The reaction mixture was stirred at 40° C. for 1 h and quenched by addition of water. The aqueous phase was extracted with ethyl acetate, and the combined organic extracts were washed with water, brine and concentrated to give 1-(benzyloxy)-3-(bromomethyl)-5-methoxybenzene 5 as pale yellow solid. (100 g, 80% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 7.33-7.46 (m, 5H), 6.72 (s, 1H), 6.63 (s, 1H), 6.54 (d, J=1.80 Hz, 1H), 5.09 (d, J=5.40 Hz, 2H), 4.62 (d, J=5.70 Hz, 2H), 3.74 (s, 3H); MS (ES+) m/z 309 [M+2H]+; HPLC-UV analysis: retention time=15.77 min; detection: 190-400 nm: peak area, 99.71%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(50) Step e. To a solution of 1-(benzyloxy)-3-(bromomethyl)-5-methoxybenzene 5 (100 g, 325.53 mmol) in toluene (2488 mL) was added triphenylphosphine (85.38 g, 325.53 mmol). The reaction mixture was stirred at 100° C. for 6 h, then allowed to cool to room temperature. The solid was collected by filtration, washed with ether, and dried under vacuum to give (3-(benzyloxy)-5-methoxybenzyl)triphenylphosphonium bromide 6 as an off-white solid. (150 g, 82% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.89-7.91 (m, 3H), 7.65-7.75 (m, 12H), 7.28-7.37 (m, 5H), 6.51 (s, 1H), 6.23 (s, 1H), 6.12 (s, 1H) 5.07 (d, J=15.60 Hz, 2H), 4.82 (s, 2H), 3.48 (s, 3H); MS (ES+) m/z 489.2 [M−HBr]+; HPLC-UV analysis: retention time=14.19 min; detection: 190-400 nm: peak area, 95.51%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(51) Part 2: Synthesis of 5-(benzyloxy)-2-iodobenzaldehyde

(52) 3-Hydroxybenzaldehyde was protected before ortho iodination, as displayed in Scheme 2.

(53) ##STR00014##

(54) Step a. To a solution of 3-hydroxybenzaldehyde 7 (25 g, 204.85 mmol) in acetone (250 mL) was added potassium carbonate (42.46 g, 307.27 mmol). The suspension was stirred at room temperature for 10 min. Benzyl bromide (31.38 mL, 264.25 mmol) was added, and the resultant suspension was heated at 60° C. for 12 h. After cooling to room temperature, the suspension was filtered, the filter cake washed with acetone, and filtrate concentrated to a residue. The residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give 3-(benzyloxy)benzaldehyde 8 as an off-white solid. (42 g, 96% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.98 (s, 1H), 7.27-7.50 (m, 5H), 7.25-7.26 (m, 2H), 5.14 (s, 2H); GCMS: m/z 212.1: (GCMS condition: column: HP-5 (30 m×320 μm×0.25 μm); gradient:120° C.-300° C., 40° C. min−1; HPLC-UV analysis: retention time=14.37 min; detection: 190-400 nm: peak area, 99.58%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(55) Step b. To a solution of 3-(benzyloxy)benzaldehyde 8 (42 g, 197.87 mmol) in chloroform (1050 mL) was added Silver trifluoroacetate (65.56 g, 296.81 mmol). The suspension was stirred at 0° C. for 10 min. Iodine (32.43 g, 126.90 mmol) was added at 0° C. and the resultant suspension was stirred at room temperature for 12 h and quenched by addition of water. The resultant mixture was filtered through a pad of celite, washed with dichloromethane. The aqueous phase was extracted dichloromethane, and the combined organic extracts were washed with water, brine and concentrated to a residue. The residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give 5-(benzyloxy)-2-iodobenzaldehyde 9 as an off-white solid. (40 g, 59% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 10.04 (s, 1H), 7.83 (d, J=8.40 Hz, 1H), 7.54 (s, 1H), 7.37-7.53 (m, 5H), 7.01 (dd, J=8.80, 3.20 Hz, 1H), 5.12 (s, 2H); GCMS m/z 338: (GCMS condition: column: ZB1MS (10 m×100 μm×0.1 μm); gradient:120° C.-300° C., 40° C. min−1.; HPLC-UV analysis: retention time=16.04 min; detection: 190-400 nm: peak area, 99.84%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(56) Part 3: Synthesis of Lusianthridin.

(57) Lusianthridin was prepared through a Wittig reaction between (3-(benzyloxy)-5-methoxybenzyl)triphenylphosphonium bromide and 5-(benzyloxy)-2-iodobenzaldehyde, followed by cyclization, deprotection, and reduction, as shown in Scheme 3.

(58) ##STR00015##

(59) Step a. To a solution of 5-(benzyloxy)-2-iodobenzaldehyde 9 (36 g, 106.46 mmol) in THF (3600 mL) was added (3-(benzyloxy)-5-methoxybenzyl)triphenylphosphonium bromide 6 (127.32 g, 223.57 mmol). The suspension was stirred at 0° C. Potassium tert-butoxide (26.28 g, 234.08 mmol) was added at 0° C. and the resultant suspension was stirred at room temperature for 12 h. The reaction mixture was concentrated to a residue and the residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give (Z)-4-(benzyloxy)-2-(3-(benzyloxy)-5-methoxystyryl)-1-iodobenzene 10 as an off-white solid. (50 g, 86% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.74 (d, J=8.80 Hz, 1H), 7.26-7.42 (m, 8H), 6.89 (d, J=3.20 Hz, 1H), 6.49-6.65 (m, 4H), 6.40 (s, 3H), 6.33 (t, J=1.60 Hz, 1H), 4.85 (s, 4H), 3.62 (s, 3H); MS (ES+) m/z 549.1 [M+H]+; HPLC-UV analysis: retention time=18.74 min; detection: 190-400 nm: peak area, 89.98%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

(60) Step b. To a solution of (Z)-4-(benzyloxy)-2-(3-(benzyloxy)-5-methoxystyryl)-1-iodobenzene 10 (50 g, 91.17 mmol) in toluene (1250 mL) was added tributyltin hydride (49.14 mL, 182.34 mmol) and azobisisobutyronitrile (7.48 g, 45.58 mmol). The reaction mixture was sparged with nitrogen for 5 min and heated at 100° C. for 16 h. The reaction mixture was concentrated to a residue and the residue was purified by automated normal-phase chromatography and eluted with ethyl acetate/hexanes to give 1:1 mixture of 4,7-bis(benzyloxy)-2-methoxyphenanthrene 11a and 2,7-bis(benzyloxy)-4-methoxyphenanthrene 11b as an off-white solid. (25 g, 65% yield). MS (ES+) m/z 421.3 [M+H]+; HPLC-UV analysis: retention time=(6.59 & 6.70) min; detection: 190-400 nm: peak area, 99.28%; eluent A, 0.1% TFA in water; eluent B, 0.1% TFA in Acetonitrile; isocratic/gradient over 10 min with a flow rate of 2.0 mL min−1.

(61) Step c. To a solution of 4,7-bis(benzyloxy)-2-methoxyphenanthrene 11a and 2,7-bis(benzyloxy)-4-methoxyphenanthrene 11b (25 g, 59.45 mmol) in acetic acid (2000 mL) and THF (250 mL) was added 10% Pd/C (25 g). The reaction mixture was stirred at room temperature 2 days under hydrogen balloon. The resultant mixture was filtered through a pad of Celite, washed with ethyl acetate and the filtrate was concentrated to give a 1:1 mixture of regioisomers. (11.50 g, 79% yield). This crude product (11.50 g, 1:1 mixture) was purified by SFC (SFC condition: column: YMC Amylose-C; detection: 210 nm: co-solvent: 0.5% isopropyl amine in methanol; flow rate of 4.0 mL min−1) to give 7-methoxy-9,10-dihydrophenanthrene-2,5-diol Lusianthridin as an off white solid (1.1 g). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.60 (s, 1H), 9.18 (s, 1H), 8.08 (d, J=8.80 Hz, 1H), 6.60 (s, 1H), 6.59 (d, J=7.20 Hz, 1H), 6.37 (s, 1H), 6.32 (s, 1H), 3.70 (s, 3H), 2.60 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 158.13, 155.52, 155.45, 140.32, 138.86, 129.05, 124.67, 114.86, 114.62, 113.09, 105.15, 100.97, 55.25, 30.70, 29.95; MS (ES+) m/z 243.1 [M+H]+; Elemental analysis calculated (%) for C15H14O3+0.2CH3OH: C73.41, H 6.00. Found: C73.43, H 5.84; HPLC-UV analysis: retention time=11.84 min; detection: 190-400 nm: peak area, 98.62%; eluent A, 0.1% TFA in water; eluent B, Acetonitrile; isocratic/gradient over 30 min with a flow rate of 1.0 mL min−1.

Example 2

(62) Lusianthridin Activates Bacterially-Expressed AMPKα2β1γ1 and α1β1γ1 Complexes in a Dose-Dependent Manner.

(63) Lusianthridin (CAS 87530-30-1) was isolated from Thunia alba fruit. It was sourced from Analyticon (NP-012362). The plant was purchased through Analyticon's biomaterial supplier in 2003. The country of origin is Nepal. The AMPK heterotrimers were expressed in bacteria and purified through the His-α subunit by nickel purification. AMPK complexes were purified through gel filtration and phosphorylated by incubation with CaMKKβ, and further purified with a final gel filtration purification step. Phosphorylated purified AMPK was incubated with varying concentrations of ligand for 30 mins using substrate and reagents from the HTRF-KinEASE Cisbio assay kit (STK S1 Kit). Phosphorylation of the substrate was measured by incubating with donor and acceptor antibodies for 2 h at room temperature as per the manufacturer's protocol (and Coulerie et al., (2016) PMID: 27792327) and phosphorylated peptide detected by performing HTRF. The 665 nm/620 nm ratio was determined and the results are plotted as fold activation compared to the respective AMPK complex without any compound.

(64) FIGS. 1 to 5 show that Lusianthridin (7-Methoxy-9,10-dihydrophenanthrene-2,5-diol, CAS number 87530-30-1) does not activate AMPK complexes containing the β2-subunit.

(65) Furthermore, it does not activate AMPK complexes with a mutation in the β1 subunit (S108A) or a deletion of the carbohydrate-binding module (ΔCBM). This activation profile is characteristic of activators mediating their effects through the ADaM (allosteric drug and metabolic) site. The AMPK γ1 R298G mutant which shows impaired AMP regulation has no effect on activation by Lusianthridin. These data are consistent with Lusianthridin activation at the ADaM binding pocket of AMPK formed between the kinase domain of the a subunit and the CBM of the β subunit.

Example 3

(66) Lusianthridin Increases the Phosphorylation of the AMPK Substrate, acetyl-CoA carboxylase (ACC), in U2OS Flp-In T-REx Mammalian Cells.

(67) U2OS Flp-In T-REx cells were seeded at 100 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 1 h with varying concentrations of Lusianthridin 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). For western blot analysis, SDS-sample buffer was added to the remainder of the lysate, denatured at 95 C and then subjected to western blot analysis with phospho-specific and total ACC antibodies (Cell Signalling). Bands were visualized by incubating with Li-Cor secondary antibody and scanned on a Li-Cor Odyssey machine.

(68) FIG. 6(a) shows that using a pACC HTRF assay kit (Cisbio), Lusianthridin increases the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in U2OS Flp-In T-REx mammalian cells. Phosphorylation of ACC is widely used as a cellular indicator of AMPK activity.

(69) FIG. 6(b) shows a Western blot analysis of U2OS Flp-In T-REx lysates that were treated with varying concentrations of Lusianthridin. Lysates were probed using phosphos-specific ACC and total ACC antibodies (Cell signalling). These data indicate that Lusianthridin activates AMPK in cells and this leads to phosphorylation of one its downstream substrates.

Example 4

(70) Lusianthridin Increases the Phosphorylation of the AMPK Substrate, ACC, in Mouse Primary Hepatocytes.

(71) Hepatocyte isolation: The liver was first perfused with 50 ml perfusion buffer (Krebs-Hepes buffer with 0.5 μM EDTA), followed with 50 ml collagenase A buffer (Krebs-Hepes buffer with 5 mM CaCl.sub.2 and 0.5 mg/ml collagenase). After passage through a 100 μm mesh, the cell solution was washed several times with cold media and finally the cell culture pellet was resuspended in culture medium (medium 199 (M199)+GlutaMAX, 100 U/ml penicillin G, and 100 μg/ml streptomycin, 0.1% (wt/vol) BSA, 10% FCS, 10 nM insulin, 200 nM triiodothyronine and 500 nM dexamethasone). Hepatocytes were left to attach (3-4 h) and cultured overnight in M199 supplemented with antibiotics and 100 nM dexamethasone. Cells were used for experiments the following morning.

(72) Primary hepatocytes were seeded at 15 K cells in a 96-well plate overnight. Cells were treated for 1 h with varying concentrations of Lusianthridin in media 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. Lysates were incubated overnight with the antibody before 665 nm/620 nm ratio was determined using a MolecularDevices i3 plate reader (with a HTRF cartridge add-on).

(73) FIG. 7 shows that using a pACC HTRF assay kit (Cisbio), Lusianthridin increases the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in primary hepatocytes (1 h incubation at 37 C).

Example 5

(74) Lusianthridin Displays a Dose-Dependent Inhibition of Lipogenesis in Primary Hepatocytes.

(75) For primary hepatocyte isolation see Example 4. For lipogenesis measurements in primary hepatocytes, cells were seeded at 600 K cells per well in a 6-well plate overnight. Media was replaced with fresh M199 media alone for 2 hours prior to incubation with varying concentrations of Lusianthridin for 1 h at 37 C, in the presence of [1-.sup.14C]-acetate. The incorporation of [.sup.14C] into fatty acids was determined in the lower organic layer after separation from the aqueous phase. The results are displayed as the disintegrations per min (DPM) per μg of protein.

(76) Lipogenesis is controlled by the AMPK substrate ACC, and phosphorylation and inhibition of ACC by AMPK, leads to a decrease in lipogenesis. Lipogenesis was measured in primary hepatocytes by determining the incorporation of .sup.14C-labelled acetate into fatty acids. Lipogenesis was monitored in the presence or absence of varying concentrations of Lusianthridin for 1 h at 37 C. The results shown in FIG. 8, displayed as the DPM per μg of total protein, that Lusianthridin is able to inhibit lipogenesis in a dose-dependent manner. This observation is consistent with its ability to activate AMPK in hepatocytes, which leads to the phosphorylation and inhibition of ACC. These data are consistent with the ability to Lusianthridin to activate AMPK in both a cell line and primary hepatocytes as previously determined by a HTRF assay and western blot analysis (see examples above).

Example 6

(77) Lusianthridin Increases Glucose Uptake into Differentiated C2C12 Cells.

(78) C2C12 cells were maintained in DMEM GlutaMAX supplemented with 10% (vol/vol) FBS and 100 U/ml penicillin G, and 100 μg/ml streptomycin. C2C12 myoblasts were differentiated into myotubes by 7 days of culture in DMEM GlutaMAX supplemented with 2% (vol/vol) horse serum and antibiotics. Cells were transferred to serum-free media for 24 hours, before equilibrating with bicarbonate-free medium prior to treatment. Cells were treated with Control (DMSO 1% final concentration) or 100 μM Lusianthridin in the presence of .sup.3H-2-deoxyglucose (in Krebs-Hepes buffer containing sodium pyruvate). Cells were lysed and the quantity of .sup.3H determined in these samples using a scintillation counter.

(79) Activation of AMPK in muscle cells has been previously shown to increase glucose uptake. Therefore, it was tested whether activation of AMPK in a differentiated muscle cell line, C2C12 cells, would lead to an increase in glucose uptake. Differentiated C2C12 cells were treated with either control or 100 μM Lusianthridin for 4 h at 37 C. Glucose uptake was determined by monitoring the uptake of .sup.3H-2-deoxyglucose into cells. The results in FIG. 9 are displayed as fold increase relative to the control wells. These results show that Lusianthridin causes around a 2-fold increase in glucose uptake into C2C12 cells. These data suggest that the ability of Lusianthridin to activate AMPK in muscle leads to phosphorylation of downstream substrate(s), culminating in the increase in glucose uptake into this muscle cell line. This is consistent with the important role of AMPK in mediating glucose uptake in muscle.

Example 7

(80) Further Dihydrophenanthrene Analogues Directly Allosterically Activate AMPK and Increase Substrate Phosphorylation in Cells.

(81) For the AMPK in vitro activity assay, see Example 2. FIG. 10 shows that Compound 2, Compound 3, Compound 4 and Compound 5 activate bacterially-expressed AMPKα2β1γ1 complexes. Similar to Lusianthridin, Compound 4 did not activate β2-containing complexes, the α2β1γ1 ΔCBM mutant and AMPK complexes with the S108A mutation (FIG. 11). Taken together, this suggests that Lusianthridin and Compound 4 share a common activation mechanism and this activity profile is characteristic of ligands that regulate AMPK by binding to the ADaM pocket.

(82) For treatment of U2OS Flp-In T-REx cells see Example 3. FIG. 12 shows that using a pACC HTRF assay kit (Cisbio), Compound 2, Compound 3, Compound 4 and Compound 5 increase the phosphorylation of the AMPK substrate, ACC, in a dose-dependent manner in U2OS Flp-In T-REx mammalian cells.

Example 8

(83) Compound 4 Activates AMPK in Mouse Primary Hepatocytes and Decreases Hepatic Lipogenesis, as Well as Increasing Glucose Uptake into C2C12 Cells.

(84) Mouse primary hepatocytes were isolated (see Example 4) and treated (see Example 5) with varying concentrations of Compound 4 for 1 h at 37 C before the rate of lipogenesis was determined (see Example 5). As shown in FIG. 13, Compound 4 caused a dose-dependent decrease in the rate of lipogenesis, comparable to the effect seen with Lusianthridin.

(85) C2C12 cells were cultured and differentiated into myotubes (see Example 6), before treatment with Compound 4. FIG. 14 shows that Compound 4 caused around a 2-fold increase in glucose uptake, comparable with the effect seen with Lusianthridin.

Example 9

(86) Ability of Lusianthridin to Increase Glucose Uptake in C2C12 Myotubes is Dependent on AMPK

(87) In order to test whether the ability of Lusianthridin to increase glucose uptake in C2C12 myotubes is dependent on AMPK, we generated C2C12 cells lacking both isoforms of the catalytic AMPKα subunit, AMPKα1α2−/−. C2C12 AMPK α1α2 double knockout cell lines were generated by transient transfection of wild type cells with plasmids containing Cas9 enzyme linked to green fluorescent protein expression and Cas9 guide RNA sequences targeting the murine AMPK α1 subunit (sc-430618, SantaCruz) and the murine AMPK α2 subunit (sc-430803, SantaCruz). Cells positive for green fluorescent protein expression were single cell sorted by flow cytometry into 96-well plates containing growth medium and analysed for loss of AMPK signaling following expansion of the clones.

(88) Differentiated C2C12 AMPKα1α2+/+ and AMPKα1α2−/− cells were treated with either vehicle (0.2% DMSO), 30 μM or 100 μM Lusianthridin for 1 h at 37° C. In addition, the insulin-dependent/AMPK-independent glucose uptake was also assessed by treating C2C12 cells with 300 nM Insulin for 1 h in the presence or absence of the 15 nM Wortmanin, an inhibitor of insulin-signalling. Finally, differentiated C2C12 AMPKα1α2+/+ and AMPKα1α2−/− cells were treated with either vehicle (0.2% DMSO) or 100 μM Lusianthridin in the presence or absence of 15 nM Wortmanin for 1 h at 37° C. For the Wortmanin experiments, differentiated C2C12 cells were pre-incubated with DMSO or Wortmanin for 1 h prior to treatment.

(89) Glucose uptake was determined by monitoring the uptake of 3H-2-deoxyglucose into cells. C2C12 AMPKα1α2+/+ and AMPKα1α2−/− cells were maintained in DMEM GlutaMAX supplemented with 20% (vol/vol) FBS and 100 U/ml penicillin G, and 100 μg/ml streptomycin. C2C12 myoblasts were differentiated into myotubes by 5-7 days of culture in DMEM GlutaMAX supplemented with 1% (vol/vol) horse serum and antibiotics. Cells were transferred to serum-free media for 24 hours, before equilibrating with bicarbonate-free medium prior to treatment. Cells were treated in the presence of .sup.3H-2-deoxyglucose (in Krebs-Hepes buffer containing sodium pyruvate). Cells were lysed and the quantity of .sup.3H determined in these samples using a scintillation counter. Fold change in glucose uptake was determined by dividing the counts per minute obtained for each treatment condition by the counts per minute obtained in the vehicle control condition.

(90) The results in the FIGS. 15 to 17 are displayed as fold change relative to the control wells. The results in FIGS. 15 and 16 show that treatment of C2C12 AMPKα1α2+/+ cells with Lusianthridin and insulin lead to an increase in glucose uptake. Insulin-stimulated glucose uptake was unaffected in the AMPKα1α2−/− cells, but as expected its effects are abolished by treatment with Wortmanin (FIG. 16).

(91) In contrast, Wortmanin had no effect on the ability of Lusianthridin to increase glucose uptake in AMPKα1α2+/+ cells, suggesting that this compound does not increase glucose uptake through the insulin-signalling pathway (FIG. 17). In contrast, Lusianthridin-stimulated glucose uptake was abolished in AMPKα1α2−/− cells (FIG. 15). Taken together, these data suggest that the ability of Lusianthridin to increase glucose uptake into C2C12 cells is dependent on its ability to activate AMPK.

Example 10

(92) Lusianthridin Does Not Cause Cytotoxicity in Mouse Primary Hepatocytes.

(93) For cytotoxicity experiments in primary hepatocytes, cells were seeded at 20 K cells per well in a 96-well plate overnight in M199 media. Hepatocytes were incubated with varying concentrations of Lusianthridin, Oligomycin or FCCP for 1 h at 37 C. The media was changed for media without phenol red, before performing an MTT assay as per the manufacturer's instructions (Thermo fisher, V131154). The MTT assay is a calorimetric assay that is routinely used to measure the cytotoxicity of compounds in cells. The results shown in the FIG. 18 show that at all the concentrations of Lusianthridin tested, there was no change in cell viability. In contrast, two known toxic compounds, Oligomycin and FCCP (carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone), caused a robust decrease in cell viability. Taken together, this suggests that Lusianthridin does not have a cytotoxic effect on mouse primary hepatocytes up to a final concentration of 100 μM.

Example 11

(94) Lusianthridin Does Not Activate a Complex Containing a Mutation at the Allosteric Drug and Metabolite (ADaM) Site in Cells (S108A).

(95) AMPKβ1/β2 double knockout U2-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 β1 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.

(96) Cells stably expressing β1 WT or a β1 S108A mutant were treated with varying concentrations of Lusianthridin and subjected to the pACC HTRF (Cisbio) assay or western blot analysis to determine phosphorylation of the AMPK substrate, ACC, in cells lysates. Western blots were quantified using the LI-COR odyssey system and shown as the ratio of the antibody signal from pACC divided by total ACC.

(97) As shown in FIGS. 19 and 20, Lusianthridin was able to dose-dependently increase pACC in cells stably expressing the β1 WT. In contrast, Lusianthridin was not able to increase pACC in cells expressing the β1 S108A mutant. This cellular data is consistent with our in vitro data showing that Lusianthridin cannot allosterically activate recombinant α2β1γ1 S108A complexes. Taken together, we show that in vitro and in cells, Lusianthridin activates AMPK by binding to the ADaM pocket of AMPK separate from the nucleotide-binding site in the AMPKγ subunit.