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.