in vivo SAR study of glabridin and its derivatives identifies biologically relevant components and the lead compound HSG4112
We first attempted to overcome the problem of chemical instability of glabridin, whose structure is shown in Figure 1A . Its low stability can be attributed to the pyranobenzene structure in ring A, which is labile in acidic condition or light, and the resorcinol structure shown in ring B, which is labile in basic condition. For allin vivo tests in this paper, we used male C57BL/6J mice fed with HFD for 10 weeks (hereinafter HFD-mice) before administration and continually during administration.
To overcome low stability in acidic condition, we modified the pyranobenzene structure by hydrogenating the double bond between the carbon atoms at 3” and 4” in ring B to create 3”,4”-dihydro-glabridin, using previously reported protocol’s hydrogenation reaction (Jirawattanapong, Saifah and Patarapanich, 2009). We found that 3”,4”-dihydro-glabridin induced greater body weight reduction in HFD-mice than glabridin (Figure 1B) . This shows that the double bond of pyranobenzene group is not required for the pharmacological action of glabridin. Given this improvement in chemical stability and efficacy, we performed hydrogenation for all following synthetic derivatives.
Next, in order to further increase stability, we modified the resorcinol structure while retaining the -oxy backbone of glabridin. We etherified C-2’, C’4, or both carbons by attaching methoxy groups, using the typical methylation (MeI, K2CO3 in acetone) process and column separation of the resulting mixture; this was to test whether the hydroxy groups are necessary for the efficacy, and if so, to determine which of them is critical and which of them can be modified to increase stability. We found that C-2’ hydroxy to methoxy modification (R1) induced lower weight loss than glabridin (Figure 1C) ; this demonstrates that C-2’ hydroxy group in ring A is a critical pharmacophore. Surprisingly, while glabridin led to 13.2% weight loss after 5-week administration, C-4’ hydroxy to methoxy modification (R2) led to 25.5% weight loss, approximately two-fold greater in percentage. We report for the first time that C-4’ methoxy attachment remarkably improves weight-reducing action of glabridin.
Given this much higher beneficial effect of hydroxy-to-methoxy modification at C-4’, we tested whether further chain elongation of C-4’ alkoxy group would have differential effect. We found that ethoxy group outweighed methoxy group and all other tested alkoxy groups in weight-reducing efficacy (Figure 1D) . This compound with optimized chain length was termed (R )-HSG4112 (Hydrogenated Synthetic Glabridin) by the authors. Further lengthening of the alkoxy substituents had retained but reduced weight-reducing effect. While the biological significance of C-4’ alkoxy group length remains unknown, we concluded that (R )-HSG4112 presents the most optimal chain length and efficacy.
Lastly, since chiral synthesis of glabridin-derivatives presents a serious challenge in methodology and productivity (Simmler, Pauli and Chen, 2013), we synthesized and tested racemic HSG4112’s effect on body weight. Glabridin in nature exists in (R ) form, and the effect of (S )-glabridin and its derivatives was unknown and predictably null; for most cases of small molecular drugs, only one enantiomer is pharmacologically active while the other enantiomer is either inactive or toxic (Hutt and Tan, 1996; Kasprzyk-Hordern, 2010). Surprisingly, we found that at equivalent dose level, (S )-isomer surpassed both (R )-isomer and racemic HSG4112 in body weight reduction effect (Figure 1E) . This presents a remarkable discovery of a glabridin-derivative with both active enantiomers and with more potent enantiomer being unnatural, synthetic (S ) form. Given the similar pharmacology of both enantiomers and the inefficient and elaborate protocol needed for chiral HSG4112, racemic HSG4112 – simply termed HSG4112 – was chosen as our lead compound.
In order to confirm increased chemical stability of HSG4112 relative to glabridin, both compounds were placed in both acidic and basic conditions (Figure 1F, G) . HSG4112 proved to be dramatically more stable, especially in basic condition where glabridin reached almost complete degradation at 72hr timepoint while HSG4112 remained completely unbroken. Expectedly, improvement on pyranobenzene and resorcinol part significantly increased the stability.