Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase

The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure–activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.


Introduction
Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of fat in liver independent of alcohol consumption [1,2]. NAFLD is the most common chronic liver disease in the Western world, and is associated with the development of cardiovascular diseases and type 2 diabetes [3]. The overall global prevalence of NAFLD is approximately 25%, and it is estimated to grow in coming years [3,4]. However, no therapy is currently available to treat this disease; the only available options have been weight loss (e.g., calorie restriction, exercise) and supplementation of vitamins, e.g., vitamin E [5].
Recently, several groups identified liver pyruvate kinase (PKL) as a possible target to halt the progression of NAFLD [2,6,7]. Pyruvate kinase is responsible for the final step in glycolysis converting phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to pyruvate (PYR) and adenosine triphosphate (ATP). Additionally, pyruvate kinase is present in four different isoforms, PKM1, PKM2, PKR, and PKL, each with multiple designations over time [8]. PKR is predominantly expressed in red blood cells, PKM1 is present in the skeletal muscle, brain, and heart, and PKM2 is expressed in proliferating cells, embryonic tissues, and tumors [9,10]. PKL is expressed mainly in the liver but also in pancreatic β-cells, the small intestine, and the renal proximal tubule [11]. Hence, development of PKL-specific inhibitors may be extremely beneficial for treating NAFLD.
Our group recently started an extensive program aimed at identifying new possible compounds to inhibit PKL [12][13][14]. These efforts resulted in the identification of two

Deconstruction of Urolithin C
Previously, we identified compound 1 as a non-competitive inhibitor with a sub-micromolar activity for PKL (Table 1) but different selectivity for the PKR, PKM1, and PKM2 isoforms [14]. Considering that our previous effort to co-crystallize compound 1 with the enzyme failed, we decided to pursue a different approach. In particular, a deconstruction strategy to identify the key features for the activity of 1 was performed. The hydroxyl group and the catechol were replaced one at a time by a hydrogen atom. The lactone moiety was removed to give a biaryl compound. Compound 1 was synthesized as previously described [14], while 2 (urolithin A) was commercially available. The other compounds were synthesized as described below. Briefly, compound 6 was obtained by a Hurtley coupling from 3 and 4 and subsequent deprotection with BBr3 (Scheme 1) [15,16].

Deconstruction of Urolithin C
Previously, we identified compound 1 as a non-competitive inhibitor with a submicromolar activity for PKL (Table 1) but different selectivity for the PKR, PKM1, and PKM2 isoforms [14]. Considering that our previous effort to co-crystallize compound 1 with the enzyme failed, we decided to pursue a different approach. In particular, a deconstruction strategy to identify the key features for the activity of 1 was performed. The hydroxyl group and the catechol were replaced one at a time by a hydrogen atom. The lactone moiety was removed to give a biaryl compound. Compound 1 was synthesized as previously described [14], while 2 (urolithin A) was commercially available. The other compounds were synthesized as described below. Briefly, compound 6 was obtained by a Hurtley coupling from 3 and 4 and subsequent deprotection with BBr 3 (Scheme 1) [15,16]. The effects of derivatives on PKL activity were determined by an in vitro biochemical assay using recombinant PKL and PKR enzymes. The results are reported in Table 1. A significant decrease in the ability to inhibit PKL was observed upon the removal of the hydroxyl groups and the lactone moiety from 1, indicating that all these features are essential for the activity. Interestingly, 2 and 17 showed weak PKL activation activity.

SAR Strategy
Based on the deconstruction of 1, all the chemical features explored were necessary for the inhibitory activity vs. PKL. For these reasons, we explored and synthesized three series of urolithin C derivatives by modifying: (a) the phenol, (b) the catechol, and (c) the lactone ( Figure 2). This strategy allowed us to study the impact of every single structural change on inhibition.

Replacement of the Phenol Group
Several substituents with differing electronic and steric properties were explored to replace the OH at position 3. A total of 19 analogues were obtained and tested for their inhibitory activity vs. PKL and PKR. The nitrogen analogues 26, 28, 33-36 were synthesized as described in Scheme 4. Compound 9 was coupled under Pd catalysis with 18 or 19. The corresponding biaryl derivatives were deprotected under basic hydrolysis and subjected to K2S2O8 or N-iodosuccinimide-mediated oxidative lactonization [17,18]. Compound 24 was then deprotected to obtain 26. In a different process, 25 was reduced to result in the intermediate 27, which was deprotected to afford the target compound 28. The aniline 27 was further acylated with various reagents and deprotected with BBr3 to give 33-36.

Replacement of the Phenol Group
Several substituents with differing electronic and steric properties were explored to replace the OH at position 3. A total of 19 analogues were obtained and tested for their inhibitory activity vs. PKL and PKR. The nitrogen analogues 26, 28, 33-36 were synthesized as described in Scheme 4. Compound 9 was coupled under Pd catalysis with 18 or 19. The corresponding biaryl derivatives were deprotected under basic hydrolysis and subjected to K 2 S 2 O 8 or N-iodosuccinimide-mediated oxidative lactonization [17,18]. Compound 24 was then deprotected to obtain 26. In a different process, 25 was reduced to result in the intermediate 27, which was deprotected to afford the target compound 28. The aniline 27 was further acylated with various reagents and deprotected with BBr 3 to give 33-36.
Derivatives 61-66 were similarly synthesized, as reported in Scheme 5. Bromide 9 was coupled with the selected boronic acid, deprotected, and cyclized to give the corresponding lactones 55-60. Deprotection of the methoxy groups with BB 3 afforded the desired target molecules 61-66.
Compounds 81-83 were obtained with minor modifications of the previous synthetic strategies, as shown in Scheme 6. Aldehyde 67 was brominated and reacted under Suzuki coupling conditions to give 69-71. These intermediates were oxidized and cyclized using AgNO 3 /K 2 S 2 O 8 or Pd-catalyzed C-H activation [19]. Finally, deprotection yielded the urolithin derivatives 81-83.
The methoxy derivative 90 and the acetyl ester 94 required a different approach as shown in Scheme 7. Compound 8 was deprotected and alkylated with benzyl bromide to give 85. The latter was coupled with 86, deprotected, and cyclized to give 89. Finally, removal of the benzyl groups with H 2 /Pd afforded 90. The acetyl ester 94 was instead obtained by hydrolysis of 85 in basic media followed by Hurtley coupling, esterification, and finally, H 2 /Pd deprotection.
The methoxy derivative 90 and the acetyl ester 94 required a different approach as shown in Scheme 7. Compound 8 was deprotected and alkylated with benzyl bromide to give 85. The latter was coupled with 86, deprotected, and cyclized to give 89. Finally, removal of the benzyl groups with H2/Pd afforded 90. The acetyl ester 94 was instead obtained by hydrolysis of 85 in basic media followed by Hurtley coupling, esterification, and finally, H2/Pd deprotection.
The final two urolithin derivatives, 98 and 103, bearing an additional ring were synthesized as shown in Scheme 8. Compound 98 was obtained by esterification of 91 and Heck intramolecular cyclization coupling followed by deprotection. Compound 103 Pharmaceuticals 2023, 16, 668 7 of 50 instead was obtained again from 9 and the corresponding boronic acid with a four-analogue synthetic pathway similar to that shown above.  The effects of derivatives modified at the phenolic group are reported in Table 2. Most of the modifications and substitutions resulted in a marked loss of activity vs. PKL. Some compounds (e.g., 28) showed a weak potentiation of PKL activity as observed for the deconstructed analogues of 1. The IC 50 values of 81 and 82 were similar to the IC 50 of urolithin C, indicating that a carboxylic acid and an amide are tolerated.

Replacement of the Catechol Moiety
To further improve our understanding of the SAR of urolithin C as a PKL inhibitor, we then investigated the modifications of the catechol moiety. A total of 14 compounds were designed and synthesized to explore this part of the molecule. The dimethoxy analogue 118 and the two monomethoxy positional isomers 119 and 120 were obtained from the corresponding 2-bromobenzoic acid and resorcinol through an Hurtley coupling (Scheme 9). The 2-bromobenzoic acids employed, 113 and 115, were synthesized from the corresponding aldehyde by oxidation and deprotection. Differently, the 9-and 8-amino analogues (124 and 126) were obtained by Pd-mediated reduction and BBr 3 deprotection of the key nitro intermediates 121 and 122. The latter were synthesized again through Hurtley coupling from the respective acids. Compounds 127 and 128 were then isolated by simply acetylation/deprotection from 124 and 126.
The last four analogues with modifications of the catechol moiety were synthesized as reported in Scheme 11. Methylenedioxy derivative 144 was obtained by alkylation of 84 with diiodomethane, followed by hydrolysis and Hurtley coupling. In a different process, compounds 150, 153, and 154 were synthesized from the key intermediate 136 by converting it into its 2-hydroxybenzimidazole, benzimidazole, and benzotriazole analogues (145-147). Analogue 145 was hydrolyzed to its corresponding acid and reacted with resorcinol to produce 150. In another synthesis, 147 was further protected to give 148. Both 148 and 146 were transformed under Suzuki coupling conditions and finally deprotected and cyclized to yield 153 and 154.
The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC 50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC 50 of 14 µM. Again, some small modifications (e.g., OH vs. NH 2 ) resulted in the compounds being weak activators. afford the desired biaryl compound 132. Hydrolysis of the methyl ester followed by cyclization and reduction/deprotection yielded 135. Its acetylated derivative 140 and the 2methyl-1H-imidazole analogue 141 were synthesized similarly. Reduction of methyl 2bromo-4,5-dinitrobenzoate (110) followed by acetylation and Suzuki coupling with the selected boronic acid gave 139. One-step deprotection/cyclization yielded the desired compound 140. Condensation of the latter in HCl afforded 141. Scheme  The last four analogues with modifications of the catechol moiety were synthesized as reported in Scheme 11. Methylenedioxy derivative 144 was obtained by alkylation of 84 with diiodomethane, followed by hydrolysis and Hurtley coupling. In a different process, compounds 150, 153, and 154 were synthesized from the key intermediate 136 by converting it into its 2-hydroxybenzimidazole, benzimidazole, and benzotriazole analogues (145-147). Analogue 145 was hydrolyzed to its corresponding acid and reacted with resorcinol to produce 150. In another synthesis, 147 was further protected to give 148. Both 148 and 146 were transformed under Suzuki coupling conditions and finally deprotected and cyclized to yield 153 and 154.  The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC50 of 14 µM. Again, some small modifications (e.g., OH vs. NH2) resulted in the compounds being weak activators. The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC50 of 14 µM. Again, some small modifications (e.g., OH vs. NH2) resulted in the compounds being weak activators. The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC50 of 14 µM. Again, some small modifications (e.g., OH vs. NH2) resulted in the compounds being weak activators. The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC50 of 14 µM. Again, some small modifications (e.g., OH vs. NH2) resulted in the compounds being weak activators.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid 8 into the corresponding amide. The latter was coupled with 4-methoxyboronic acid, then 164 was obtained via copper-catalyzed cyclization [20]. This was either directly deprotected or methylated and deprotected to give the two target compounds.

Replacement of the Lactone Moiety
Finally, we explored the impact of the lactone moiety on PKL inhibitions. We initially designed and prepared six analogues by switching the lactone position (161) or converting it to its corresponding lactam or methyl lactam (165, 167). Alternatively, we removed the moiety altogether but kept the planarity (178) and H-bond acceptor properties (179, 180), and increased the bulk (183). The lactone and lactam derivatives were obtained as shown in Scheme 12. Compound 161 was obtained by esterification of the corresponding acid 155 and subsequent Suzuki coupling. Hydrolysis followed by cyclization and deprotection afforded the desired compound. Analogues 165 and 167 were synthesized converting acid Pharmaceuticals 2023, 16,  The synthesized compounds were then assessed using the in vitro biochemical assay (Table 3). Very low or no inhibition of enzyme activity was observed for most molecules with few exceptions. The two monomethoxy derivatives 119 and 120 showed almost full inhibition at 10 µM but with a dramatic reduction in their IC50 (1.9 µM and 1.5 µM, respectively). Even compound 141 retained some weak activity with an IC50 of 14 µM. Again, some small modifications (e.g., OH vs. NH2) resulted in the compounds being weak activators. The synthesis of phenanthrene 178 and phenanthridines 179 and 180 is reported in Scheme 13. The corresponding benzaldehyde 68 was obtained as reported above while 169 was isolated after bromination of 3-methoxybenzaldheyde. These compounds were reacted with the selected boronic acids to produce 170 and 171. The first compound was converted to 172 by a Wittig reaction and subjected to dehydrative cycloaromatization [21]. Deprotection with BBr 3 gave the desired compound 178. The analogues 179 and 180 were instead obtained converting the aldehyde to the phenanthridine core through ironcatalyzed intramolecular N-arylation [22]. Finally, deprotection with HBr in acetic acid gave 179 and 180.
Finally, compound 183 was obtained from 1 by protecting the hydroxyl groups, reacting with CH 3 MgBr and final deprotection with H 2 /Pd (Scheme 14). The compounds were then evaluated using an in vitro biochemical assay ( The synthesis of phenanthrene 178 and phenanthridines 179 and 180 is reported in Scheme 13. The corresponding benzaldehyde 68 was obtained as reported above while 169 was isolated after bromination of 3-methoxybenzaldheyde. These compounds were reacted with the selected boronic acids to produce 170 and 171. The first compound was converted to 172 by a Wittig reaction and subjected to dehydrative cycloaromatization [21]. Deprotection with BBr3 gave the desired compound 178. The analogues 179 and 180 were instead obtained converting the aldehyde to the phenanthridine core through ironcatalyzed intramolecular N-arylation [22]. Finally, deprotection with HBr in acetic acid gave 179 and 180. Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)  Finally, compound 183 was obtained from 1 by protecting the hydroxyl groups, reacting with CH3MgBr and final deprotection with H2/Pd (Scheme 14). The compounds were then evaluated using an in vitro biochemical assay (Table 4).  Finally, compound 183 was obtained from 1 by protecting the hydroxyl groups, reacting with CH3MgBr and final deprotection with H2/Pd (Scheme 14). The compounds were then evaluated using an in vitro biochemical assay (Table 4).  The benzophenone compounds were easily isolated in two steps by Friedel-Crafts acylation of 1,2-dimethoxybenzene with the corresponding acid and subsequent deprotection.
Interestingly, the two fluorenone analogues of 1, compounds 192 and 194, retained most of the activity of their parent molecule (Table 5). In particular, the position of the phenolic group plays a relevant role since the 6-OH derivative (194) is slightly more active than 1 and twice as active as 192. Adding an additional -OH (196) resulted in an 8-10-fold drop in the IC 50 value. Alkylation of the catechol moiety resulted in completely inactive compounds (197,198). Similarly, removing the carbonyl group as in 202 resulted in a drop in activity. Finally, all the benzophenone analogues 209-211 showed no significant inhibitory effect. Interestingly, switching the position of the lactone (161) resulted in a tremendous loss of activity. Similarly, the lactam derivatives (165, 167) showed at least a 10-fold drop in IC50 vs. PKL. Removing the moiety but keeping the planarity and/or an H-bond acceptor group (178-180) afforded compounds with a very weak inhibitory activity. Surprisingly, the introduction of a geminal-dimethyl group instead of the carbonyl gave a quite potent activator (EC50 = 3 µM).

Entry
Interestingly, switching the position of the lactone (161) resulted in a tremendous loss of activity. Similarly, the lactam derivatives (165, 167) showed at least a 10-fold drop in IC50 vs. PKL. Removing the moiety but keeping the planarity and/or an H-bond acceptor group (178-180) afforded compounds with a very weak inhibitory activity. Surprisingly, the introduction of a geminal-dimethyl group instead of the carbonyl gave a quite potent activator (EC50 = 3 µM). Interestingly, switching the position of the lactone (161) resulted in a tremendous loss of activity. Similarly, the lactam derivatives (165, 167) showed at least a 10-fold drop in IC50 vs. PKL. Removing the moiety but keeping the planarity and/or an H-bond acceptor group (178-180) afforded compounds with a very weak inhibitory activity. Surprisingly, the introduction of a geminal-dimethyl group instead of the carbonyl gave a quite potent activator (EC50 = 3 µM). Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)202). Moreover, further simplification of the fluorenone resulted in few benzophenone analogues (209-211). The synthesis of all these analogues is shown in Scheme 15; Scheme 16. The corresponding methyl ester was reacted with the selected boronic acids to afford the key intermediates 186-190. These intermediates were converted into the corresponding fluorenones by intramolecular Friedel-Crafts acylation catalyzed by triflic acid. Deprotection of the methoxy groups gave 192, 194, and 196, while 197 and 198 did not require any additional steps. Compound 202 was instead obtained from 192 by benzyl protection, reduction, and alkylation of the resulting alcohol. Finally, deprotection/reduction by H2/Pd gave the desired fluorene compound. Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)202). Moreover, further simplification of the fluorenone resulted in few benzophenone analogues (209-211). The synthesis of all these analogues is shown in Scheme 15; Scheme 16. The corresponding methyl ester was reacted with the selected boronic acids to afford the key intermediates 186-190. These intermediates were converted into the corresponding fluorenones by intramolecular Friedel-Crafts acylation catalyzed by triflic acid. Deprotection of the methoxy groups gave 192, 194, and 196, while 197 and 198 did not require any additional steps. Compound 202 was instead obtained from 192 by benzyl protection, reduction, and alkylation of the resulting alcohol. Finally, deprotection/reduction by H2/Pd gave the desired fluorene compound. Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)  Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)202). Moreover, further simplification of the fluorenone resulted in few benzophenone analogues (209-211). The synthesis of all these analogues is shown in Scheme 15; Scheme 16. The corresponding methyl ester was reacted with the selected boronic acids to afford the key intermediates 186-190. These intermediates were converted into the corresponding fluorenones by intramolecular Friedel-Crafts acylation catalyzed by triflic acid. Deprotection of the methoxy groups gave 192, 194, and 196, while 197 and 198 did not require any additional steps. Compound 202 was instead obtained from 192 by benzyl protection, reduction, and alkylation of the resulting alcohol. Finally, deprotection/reduction by H2/Pd gave the desired fluorene compound. 16 N.D. 13 N.D.

180
Pharmaceuticals 2023, 16 Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)  Subsequently, we decided to explore a molecular simplification of urolithin C to produce a fluorenone nucleus. A few analogues were synthesized with a different substitution pattern (192,194,(196)(197)(198)202). Moreover, further simplification of the fluorenone resulted in few benzophenone analogues (209-211). The synthesis of all these analogues is shown in Scheme 15; Scheme 16. The corresponding methyl ester was reacted with the selected boronic acids to afford the key intermediates 186-190. These intermediates were converted into the corresponding fluorenones by intramolecular Friedel-Crafts acylation catalyzed by triflic acid. Deprotection of the methoxy groups gave 192, 194, and 196, while 197 and 198 did not require any additional steps. Compound 202 was instead obtained from 192 by benzyl protection, reduction, and alkylation of the resulting alcohol. Finally, deprotection/reduction by H2/Pd gave the desired fluorene compound.
The compound behaves as an activator. a The value refers to an EC 50. in activity. Finally, all the benzophenone analogues 209-211 showed no significant inhibitory effect.
* The compound behaves as an activator.

SAR Summary
Three major SAR series were explored by modifying the phenol, catechol, and lactone moiety (Figure 3). At the phenolic position, a carboxylic acid or an amide seems to be tolerated while any other substitution led to a loss of activity.
* The compound behaves as an activator. The benzophenone compounds were easily isolated in two steps by Friedel-Crafts acylation of 1,2-dimethoxybenzene with the corresponding acid and subsequent deprotection.  (Table 5). In particular, the position of the phenolic group plays a relevant role since the 6-OH derivative (194) is slightly more active than 1 and twice as active as 192. Adding an additional -OH (196) resulted in an 8-10-fold drop in the IC50 value. Alkylation of the catechol moiety resulted in completely inactive compounds (197,198 The benzophenone compounds were easily isolated in two steps by Friedel-Crafts acylation of 1,2-dimethoxybenzene with the corresponding acid and subsequent deprotection.  (Table 5). In particular, the position of the phenolic group plays a relevant role since the 6-OH derivative (194) is slightly more active than 1 and twice as active as 192. Adding an additional -OH (196) resulted in an 8-10-fold drop in the IC50 value. Alkylation of the catechol moiety resulted in completely inactive compounds (197,198). Similarly, removing the carbonyl group as in 202 resulted in a drop

SAR Summary
Three major SAR series were explored by modifying the phenol, catechol, and lactone moiety (Figure 3). At the phenolic position, a carboxylic acid or an amide seems to be tolerated while any other substitution led to a loss of activity.
* The compound behaves as an activator.

SAR Summary
Three major SAR series were explored by modifying the phenol, catechol, and lactone moiety (Figure 3). At the phenolic position, a carboxylic acid or an amide seems to be tolerated while any other substitution led to a loss of activity. Modifications of the catechol group with standard and non-standard bioisosteres and other chemical modifications resulted in a loss of activity, suggesting that this group is needed for the activity. The most promising group for further substitution seems to be the lactone. Molecular simplification to obtain a carbonyl resulted in 194, which was slightly more active than the parent compound. In general, we discovered that these molecules need fine-tuning, and even slight modifications might lead to inactive compounds ormore surprisingly-to an activation of the enzyme. Modifications of the catechol group with standard and non-standard bioisosteres and other chemical modifications resulted in a loss of activity, suggesting that this group is needed for the activity. The most promising group for further substitution seems to be the lactone. Molecular simplification to obtain a carbonyl resulted in 194, which was slightly more active than the parent compound. In general, we discovered that these molecules need fine-tuning, and even slight modifications might lead to inactive compounds or-more surprisingly-to an activation of the enzyme.

General Information
All reagents were purchased from Sigma-Aldrich and Fluorochem and were used without further purification unless noted otherwise. Solvents were dried on a solvent purification system (PS-MD-5/7 Inert technology). Microwave reactions were performed in capped vials using a Biotage Initiator Sixty instrument with fixed hold time. The reactions were monitored by LC-MS (Perkin Elmer Series 200; Waters Symmetry C8 column 3.5 µm, 4.6 × 50 mm; water:CH 3 CN (0.1% formic acid)) or by thin-layer chromatography (TLC) on silica-plated aluminum sheets (Silica gel 60 F254, E. Merck, (Rahway, NJ, USA)) and detecting spots by UV light (λ = 254 nm). Flash column chromatography was performed on silica gel 60 (0.040−0.063 mm), manually or using a Biotage SP4 Flash instrument, using silica gel SNAP KP-Sil FSK0-1107 cartridges. NMR spectra were recorded on a Varian NMR 400, Brucker 700 or Oxford 800 (Brucker, (Billerica, MA, USA)) spectrometer at 25 • C unless noted otherwise, using CDCl 3 , CD 3 OD, or DMSO-d 6 as solvent as indicated. Chemical shifts are reported in ppm with the solvent residual peak as internal standard: 1 H-residual CHCl 3 (δ H 7.26), CD 3 OD (δ H 3.31) or DMSO-d 6 (δ H 2.50) and 13 C-CDCl 3 (δ C 77.16), CD 3 OD (δ C 49.80) or DMSO-d 6 (δ C 39.52). NMR data are reported as follows: chemical shift, number of protons/carbons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplot; br, broadened), coupling constants (Hz). Melting points were recorded on a Büchi melting point apparatus B-545 or a Mettler FP82 hot stage equipped with a FP80 temperature controller and are uncorrected. High resolution mass spectra (HRMS) were recorded on an Agilent 6520 quadrupole time of flight instrument coupled to an Agilent 1290 infinity ultra-performance liquid chromatography instrument (Santa Clara, CA, USA). Samples were dissolved in acetonitrile and eluted using isocratic elution (100% acetonitrile) with a flow rate of 0.4 mL/min. A mass spectrometer was operated in positive electrospray ionization scanning mode between 50 and 1200 m/z. Ion source parameters were as follows: drying gas flow 10 L/min and temperature 325 • C and nebulizer pressure 35 psig. The mass spectrometer was calibrated before analyses.

General Procedure A
The compound was obtained following the literature procedure [23]. To a suspension of the corresponding 2-bromo benzoic acid (1 mmol) and resorcinol (4) (3 mmol) in water Pharmaceuticals 2023, 16, 668 18 of 50 (3.0 mL), an aqueous solution of NaOH (4 M aq, 1 mmol) was added and the mixture was stirred at room temperature until it became a clear solution. Then, Na 2 CO 3 (2.2 mmol) was added, and the mixture was stirred at 50 • C for 10 min. Subsequently, CuI (0.3 mmol) was added, and the reaction mixture was stirred at 50 • C for 24 h (until full conversion, reaction monitored by LC-MS). The resulting suspension was filtered and the solid obtained dried. Crystallization or purification by flash column chromatography afforded the desired compound. To a solution of methyl ester (1 mmol) in 1,4-dioxane (10.0 mL), LiOH (2 M aq, 2 mmol) was added. The reaction mixture was stirred at reflux for 12 h (or until full conversion, reaction monitored by TLC). Then, the reaction mixture was cooled down to room temperature and dioxane was evaporated under reduced pressure. The residue was diluted with water (20.0 mL) and acidified to pH 1-2 by addition of an aqueous solution of HCl (1 M). The aqueous solution was extracted with EtOAc (3 × 40.0 mL); the combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to afford the corresponding carboxylic acid.

General Procedure F
The compound was obtained following the literature procedure [17]. To a solution of the corresponding 2-aryl benzoic acid (1 mmol) in water:CH 3 CN (1:1, 10.0 mL), K 2 S 2 O 8 (2 mmol) and AgNO 3 (0.01 mmol) were added. The reaction mixture was stirred at 50 • C for 12 h (until full conversion, reaction monitored by LC-MS). Then, CH 3 CN was removed under reduced pressure. The remaining aqueous solution was basified to pH 9 with a saturated aqueous solution of NaHCO 3 and extracted with EtOAc (3 × 10.0 mL), and the combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. Purification by flash column chromatography afforded the desired compound.

General Procedure G
This procedure was previously reported for the synthesis of dibenzopyranones [18]. To a suspension of the selected acid (1.00 mmol) in 1,2-dichloroethane (15 mL), N-iodosuccinimide (0.9 g, 4.0 mmol) was added. The reaction mixture was stirred at 80 • C for 20 h. The reaction mixture was cooled down to room temperature, EtOAc (70 mL) was added, the suspension was washed with a saturated aqueous solution of Na 2 S 2 O 3 (3 × 30 mL) and water (2 × 30 mL), and then the organic layer was evaporated under reduced pressure. Crystallization, trituration, or purification by flash column chromatography afforded the desired compound.

General Procedure H
To a solution of the selected benzylated derivative (1 mmol) in DMF/MeOH (2:1, 10.0 mL), 10% Pd/C was added (10% w/w for each group to be reduced/deprotected) and the reaction stirred at room temperature for 24 h (until full conversion, reaction monitored by LC-MS). The catalyst was then filtered on a Celite pad, and the solvent removed under reduced pressure to afford the desired compound.

General Procedure I
To a suspension of the selected aniline (1 mmol) in CH 3 CN (15 mL), DIPEA (9 mL) and appropriate acyl chloride (4.5 mmol) were added. The reaction mixture was stirred at room temperature. After complete conversion of the starting material, the reaction was quenched with MeOH, and the solvent removed under reduced pressure. The obtained crude was suspended in CH 2 Cl 2 and filtered. The resulting solid was washed with a mixture of CH 2 Cl 2 /MeOH to yield the acylated compound.

General Procedure J
To a suspension of the selected aldehyde (0.75 mmol) in t-BuOH (12 mL) and CH 3 CN (2.5 mL), 2-methyl-2-butene (0.6 mL, 5.66 mmol), and a solution of NaClO 2 (80%, 170 mg, 1.5 mmol) and KH 2 PO 4 (204 mg, 1.5 mmol) in water (6 mL) were added dropwise (exothermic). The reaction mixture was stirred at room temperature for 24 h. A second portion of NaClO 2 (85 mg) and KH 2 PO 4 (102 mg) in water (3 mL) was added and the reaction mixture was stirred for additional 24 h. Then, an aqueous solution of HCl (1 M, 2 mL), water (30 mL), and EtOAc (40 mL) were added. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude was triturated with pentane and Et 2 O to afford the desired product.

General Procedure K
To a solution of the selected phenol (1 mmol) and K 2 CO 3 (2.5 mmol per phenolic group) in acetone (30 mL), benzyl bromide was added (2.5 mmol per phenolic group). The mixture was stirred at room temperature for 12-24 h (until full conversion, reaction monitored by TLC). The reaction was then filtered, and the solvent concentrated under reduced pressure. Purification by flash chromatography afforded the desired compound.