Novel unsaturated glycyrrhetic acids derivatives: Design, synthesis and anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2017.08.002

To develop novel anti-inflammatory agents, a series of unsaturated glycyrrhetic acids were designed, synthesized and evaluated for anti-inflammatory activity using RAW264.7 cells. The structure-activity relationship (SAR) of NO inhibitory activity was analyzed. α,β-Unsaturated glycyrrhetic acids showed better activity, among them, compounds 6k and 6l with piperazine unit exhibited the most potent nitric oxide (NO) and interleukin-6 (IL-6) inhibitory activity (IC₅₀ = 13.3 and 15.5 μM respectively). Furthermore, compound 6k could also significantly suppress LPS-induced iNOS and COX-2 expression and IL-6 production through MAPKs and NF-kB signaling pathway.

Full text of DOI:10.1016/j.ejmech.2017.08.002

Accepted Manuscript
Novel unsaturated glycyrrhetic acids derivatives: Design, synthesis and anti-
inflammatory activity
Bo Li, Shi Cai, Yong-An Yang, Shi-Chao Chen, Rui Chen, Jing-Bo Shi, Xin-Hua Liu,
Wen-Jian Tang
PII:
S0223-5234(17)30605-0
10.1016/j.ejmech.2017.08.002
EJMECH 9646
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2017
Revised Date: 31 July 2017
Accepted Date: 1 August 2017
Please cite this article as: B. Li, S. Cai, Y.-A. Yang, S.-C. Chen, R. Chen, J.-B. Shi, X.-H. Liu, W.-J.
Tang, Novel unsaturated glycyrrhetic acids derivatives: Design, synthesis and anti-inflammatory activity,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.002.
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Graphical abstract
Compound 6k was much more potent than Glycyrrhetic acid for the
anti-inflammatory activity. It could also significantly suppress LPS-induced iNOS and
COX-2 expression and IL-6 production through MAPKs and NF-kB signaling
pathway.

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Novel unsaturated glycyrrhetic acids DERIVATIVES:
design, synthesis and anti-inflammatory activity
Bo Lia,1, Shi Caia,1, Yong-An Yangb, Shi-Chao Chena, Rui Chena, Jing-Bo Shia, Xin-Hua Liua,*,
Wen-Jian Tanga,*
a School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui
Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China.
b Elion Nature Biological Technology Co., Ltd, Nanjing 210038, China.
Abstract: To develop novel anti-inflammatory agents, a series of unsaturated
glycyrrhetic acids were designed, synthesized and evaluated for anti-inflammatory
activity using RAW264.7 cells. The structure-activity relationship (SAR) of NO
inhibitory activity was analyzed. α,β-Unsaturated glycyrrhetic acids showed better
activity, among them, compounds 6k and 6l with piperazine unit exhibited the most
potent nitric oxide (NO) and interleukin-6 (IL-6) inhibitory activity (IC50 = 13.3 and
15.5 µM respectively). Furthermore, compound 6k could also significantly suppress
LPS-induced iNOS and COX-2 expression and IL-6 production through MAPKs and
NF-kB signaling pathway.
Keywords: glycyrrhetic acid; anti-inflammatory activity; structure-activity
relationship; unsaturated carbonyl
_________________
*Corresponding author.
Tel. & fax: +86 551 65161115. E-mail: xhliuhx@163.com; ahmupharm@126.com.
1 B. Li and S. Cai contributed equally to this work.

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1. Introduction
Inflammation, the fundamental defense mechanism of the immune system, can
protect against injuries caused by harmful stimuli, such as pathogens and poisons.
However, the inflammatory process itself may cause tissue damage and lead to
numerous diseases and even cancer [1,2]. Nonsteroidal anti-inflammatory drugs
(NSAIDs) are therapeutically important in the treatment of pain and inflammation, but
the long-term therapy with NSAIDs was soon associated with high incidences of
adverse events in the gastrointestinal tract [3,4]. Therefore, the search for novel
anti-inflammatory agents, possessing improved pharmaceutical profiles and reduced
adverse effects, is still urgently needed. Due to benign therapeutic potency and fewer
adverse effects, natural products played a significant part in medicine producing many
of the early new molecular entities (NMEs) and nearly half of all approved NMEs
[5,6].
Licorice, the roots and rhizomes of Glycyrrhiza species, has widely used as a
traditional Chinese medicine due to its antibacterial, anti-viral, anti-inflammatory, et
al [7–10]. 18β-glycyrrhetic acid (18β-GA, glycyrrhetin) is the main active component
with diverse pharmacological activities, including anti-inflammatory, antioxidant,
antibacterial, anti-viral and hepatoprotective activities [11–15]. Its anti-inflammatory
effect is particularly well-known and has been applied to clinical therapy, because its
structure is similar to that of cortisone, a steroid hormone that exerts a strong
anti-inflammatory effect [11]. 18β-GA exhibited anti-inflammatory effects by
inhibiting the production of inflammatory cytokines and reducing the expression of
pro-inflammatory genes [11,16], for example, inhibiting macrophage inflammatory
protein (MIP)-1α in a mouse model of acute Propionibacterium acnes-induced
inflammatory liver injury [17], inserting the immunomodulatory effects by enhancing
DC maturation and modulating Th1/Th2 response in Th1 responses [18]. These
results suggested that 18β-GA could be used as a potent anti-inflammatory agent for
the treatment and prevention of inflammation-related diseases.
Structural modification of bioactive natural products according to molecular
characteristics was an important approach in search for new lead compounds [19–21].
The fact that minor structural changes in triterpenoids can cause extensive changes in
biological activity has long intrigued medicinal chemists. The most actively explored

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‘simple’ derivatives of GA, obtained by modification of 3-hydroxy (or 3-oxo) with
aminoacids or/and 30-carboxylic groups by esterification, exhibited a higher activity
than GA [22–27]. Natural products containing an α,β-unsaturated carbonyl moieties
exhibit diverse pharmacological activities, and the most important and widely studied
natural compounds include curcumin, chalcone, zerumbone, and so on [28−30]. It was
found that the introduction of a 1-en-3-one functionality in several synthetic
triterpenoids greatly improved their anti-inflammatory activities [26,27,31,32]. The
immunosuppressive effects of the α,β-unsaturated carbonyl moiety on the cells play
key role in inflammation, which were partially mediated by their effects on the
immune cells specifically on inhibition of pro-inflammatory cytokines expression
[30].
Based on the above observations, we proceeded with modification of the GA
structure using following strategies: (a) building up an α,β-unsaturated carboxylic acid
moiety at C-30 in ring E of GA, namely glycyrrhetenoic acid (5); (b) converting C-32
carboxyl group to an ester or amide functionality (Fig. 1). Thus, nineteen compounds
were synthesized and evaluated for their anti-inflammatory activity with
lipopolysaccharide (LPS)-induced RAW264.7 cells through inhibiting NO production.
Further, the most potent compounds 6k and 6l with piperazine moiety were used
study the preliminary mechanism of anti-inflammatory action.
(Fig. 1)
2. Results and discussion
2.1. Chemistry
The introduction of an α,β-unsaturated carbonyl in natural products could improve
their pharmacological activities [30]. The preparation of the key intermediate 5
containing an α,β-unsaturated carbonyl unit was outlined in Scheme 1. Compound 2
was prepared by treating the acetate of glycyrrhetic acid with ethyl chloroformate and
subsequently reducing it with NaBH4 as previously described [33]. Compound 2 was
oxidized with pyridinium chlorochromate (PCC) in CH2Cl2 to obtain aldehyde 3.
Z-α,β-unsaturated carbonyl compound 5 was obtained from Wittig-Horner reaction of
aldehyde 3 and next hydrolysis reaction.

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(Scheme 1)
According to recent review [34], elongation of the GA carbohydrate chains or
introduction of amino acids or heterocyclic fragments has previously been shown to
significantly affect the anti-inflammatory activities. In this regard, we chose to
explore whether chemical modifications of the C-30 carboxylic acid affect the
anti-inflammatory activity. Consequently, we synthesized a series of glycyrrhetenoic
acid derivatives 6a−6q and 7a−7b containing an alkyl, aromatic ring or heterocycle
residue with amide bond or ester bond (Scheme 2 and 3).
(Scheme 2)
(Scheme 3)
2.2. Crystal structure analysis
The structure of compound 6b was determined by X-ray crystallography. As
shown in Fig. 2, the absolute configuration of compound 6b was determined using
CuKα radiation (λ = 1.54184 Å), such as C3(S), C5(R), C8(R), C9(R), C10(S),
C17(R), C18(R) and C20(S) (Scheme 1). In compound 6b, the cis D/E
stereochemistry was consistent with that of natural 18β-GA. Crystallographic data
(excluding structure factors) for the structures have been deposited in the Cambridge
Crystallographic Data Center as supplementary publication No. CCDC 1418308.
(Fig. 2)
2.3. Cytotoxicity of glycyrrhetin derivatives
To investigate whether the inflammatory inhibitory activities of glycyrrhetin
derivatives were related to cell viability, their cytotoxicity were evaluated by MTT
assay in RAW264.7 cells. As shown in Table 1, compounds GA, 5, 6a-p, 7a-b and
LPS at the test concentrations (40 µM or 20 µM for low solubility) had no obviously
cytotoxicity in RAW264.7 cells, and the relative cell viabilities of the treated cells
were all more than 90%. These results indicated that the non-toxic concentrations

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were further used in subsequent experiment processes.
(Table 1)
2.4. Inhibition of NO production in (LPS)-stimulated RAW264.7 cells
NO is a significant pro-inflammatory mediator. Excessive production of NO was
found to be associated with the pathogenesis of inflammation diseases. It is generally
accepted that NO inhibitors may offer potential opportunity to identify new
therapeutic method for the inflammatory diseases [35,36]. To evaluate the
anti-inflammatory effect of glycyrrhetin derivatives, Griess reagent was used to detect
the level of lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells. As
shown in Fig. 3, LPS treatment caused significant increase of NO release compared to
blank, after glycyrrhetin derivatives treatment, the increase of LPS-induced NO
release could be alleviated in RAW264.7 cells. Among them, compounds 5, 6a-6e, 6k,
6l and 6n significantly alleviated the increase of LPS-induced NO release. As
expected [30], glycyrrhetenoic acid (compound 5) with α,β-unsaturated carbonyl
moiety exhibited better NO inhibitory activity than glycyrrhetic acid. The order of NO
inhibitory activity of 32-substituted glycyrrhetenoic acid 5 was: (i) heterocycle amide
(6k, 6l, 6n except for 6m) > linear alkyl amide (6a-6e; 6d, 6e at 20µM) > branched
alkyl amide (6f; 6g at 20µM); (ii) amide > ester (6i > 7b); (iii) no significant NO
inhibitory activity for compounds with aromatic ring (6i, 6j, 6o, 7b). Overall,
glycyrrhetin derivatives displayed preferable anti-inflammatory activity, particularly,
compounds 6k and 6l with piperazine moiety exhibited the most potent inhibitory
activity and their NO inhibition rate exceeded 70% at the concentration of 40 µM.
These results could be very useful for our future SAR study.
(Fig. 3)
2.5. Compound 6k and 6l inhibited IL-6 and TNF-α production in RAW264.7 cells
LPS-induced activated macrophages produce a variety of inflammatory cytokines,
including tumor necrosis factor (TNF-α), interleukins (ILs), and inflammatory
mediators nitric oxide (NO), prostaglandins (PGs), which have a host defensive effect
during inflammatory situations and also maintain normal cellular conditions [37]. To

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further evaluate the effects of compounds 6k and 6l on LPS-induced IL-6 and TNF-α
production, RAW264.7 cells were cultured with LPS (1 µg/mL) in the presence of
compounds 6k and 6l for 24 h, and the levels of IL-6 and TNF-α in the supernatant
were determined by ELISA. As shown in Fig. 4, the LPS stimulation significantly
elevated the productions of TNF-α and IL-6 compared to the normally basal level of
the cytokines in macrophages. Treatment with compounds 6k and 6l inhibited the
LPS-induced production of IL-6 and TNF-α in a concentration-dependent manner
(Figs. 4A and 4B). After compounds 6k and 6l treatment, the augmented levels of
IL-6 by LPS were more markedly decreased than that of TNF-α. Data analysis
showed that IC50 values of inhibitory effect of compounds 6k and 6l on IL-6 were
13.3 µM and 15.5 µM respectively. These results showed that compounds 6k and 6l
certainly attenuate an excessive immune reaction in RAW 264.7 macrophages by
blocking the increase of pro-inflammatory cytokines stimulated by LPS. Thus,
compound 6k was selected to further explore the mechanisms of the
anti-inflammatory effect.
(Fig. 4)
2.6. Compound 6k inhibited LPS-induced inflammatory mediators in RAW264.7 cells
The pro-inflammatory mediators (NO, PGE2) play important roles in
inflammation-related diseases, which are also importantly related to the modulation of
iNOS and COX-2 expression [38]. To evaluate the impact of compound 6k treatment
on protein expression in LPS-induced RAW 264.7 cells, COX-2 and nitric oxide
synthase (iNOS) protein levels were analyzed after exposure to LPS for 24 h in
presence or absence of compound 6k (10, 20, or 40 µM) by Western blot. Bay
11-7082 (20 µM) was used as a positive control. The results showed that LPS (1
µg/mL) stimulation could markedly increase COX-2 and iNOS protein expression.
Pretreatment with compound 6k significantly inhibited LPS-induced COX-2 and
iNOS production by 55.6% and 76.9% at 40 µM respectively (Fig. 5). Also,
compound 6k inhibited COX-2 and iNOS protein expression in LPS-induced cells in
a dose-dependent manner (Fig. 5). These preliminary results demonstrated that
compound 6k may participate in signaling pathways activated by LPS in
macrophages.

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(Fig. 5)
2.7. Compound 6k inhibits LPS-induced NF-κB activation in RAW264.7 cells
Stimulation with LPS leads to the activation of NF-κB, which is a well-known
transcription factor that positively regulates inflammatory genes including iNOS and
COX-2 in many human diseases [39]. NF-κB activation is controlled by
phosphorylation and degradation of IκB-α, a cognate regulatory subunit of NF-κB
[40]. Therefore, western blotting and Immunofluorescence were used to examine the
effects of compound 6k on LPS-induced transcriptional activity of NF-κB in
LPS-stimulated RAW 264.7 cells. As shown in Fig. 6A, LPS significantly increased
the levels of phosphorylated NF-κB p65, IκBα and compound 6k treatment displayed
a concentration-dependent inhibition of LPS-induced NF-κB transcriptional activity
after 30 min of LPS exposure. Furthermore, the nuclear translocation of NF-κB was
examined in LPS-stimulated RAW264.7 cells. Immunofluorescence analysis showed
that LPS stimulation increased nuclear translocation of the p65 subunit, and
compound 6k clearly inhibited NF-κB p65 nuclear translocation from the cytosol to
the nucleus (Fig. 6B). Taken together, we suggest that compound 6k could regulate
the expression of pro-inflammatory proteins through inhibition of NF-κB signaling
pathways.
(Fig. 6)
2.8. Compound 6k suppresses LPS-induced MAPK activation in RAW264.7 cells
The mitogen-activated protein kinases (MAPKs) are a family of signal transduction
proteins that include extracellular signal-regulated kinase (ERK), c-Jun N-terminal
kinases (JNK), and the p38 isoform (p38), which are classical pathways that modulate
immune-mediated inflammatory responses and cellular activities involved in cell
survival and apoptosis through the corresponding signaling cascades [41‒43].
Activated MAPKs modify the phosphorylation on the threonine/tyrosine motif,
accelerating iNOS, COX-2, and pro-inflammatory cytokine expression in activated
macrophages [42,44]. To determine the effects of compound 6k on the LPS-induced
phosphorylation of MAPKs, the expression of ERK, JNK, and p38 was examined. As

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expected (Fig. 7), levels of phosphorylation of p38, JNK, and ERK were increased
after LPS-stimulated for 30 min. Compound 6k suppressed LPS-induced
phosphorylation of ERK in 40 µM, but had little effect on phosphorylation of JNK or
p38 in RAW264.7 cells. These results suggested that the anti-inflammatory activity of
compound 6k might be associated with its negative effects on ERK activation.
(Fig. 7)
3. Conclusion
In conclusion, based on the structure characteristics, a series of glycyrrhetic acid
derivatives as anti-inflammatory agents were designed and synthesized. In vitro
bioassays showed that title compounds could be alleviated the increase of
LPS-induced NO release in RAW264.7 cells. The order of NO inhibitory activity was:
(i) compound 5 with α,β-unsaturated carbonyl moiety > glycyrrhetic acid; (ii)
heterocycle amide (6k, 6l, 6n) > linear alkyl amide (6a-6c) > branched alkyl amide
(6f); (iii) amide > ester (6i > 7b). Compounds 6k and 6l with piperazine moiety
exhibited the most potent nitric oxide (NO) and interleukin-6 (IL-6) inhibitory activity
(IC50 = 13.3 µM and 15.5 µM for IC50 values on IL-6 inhibition respectively).
Western blotting and immunofluorescence showed that compounds 6k reduced
decreased the expression of iNOS, COX-2, and MAPKs, as well as activation of
NF-κB in LPS-stimulated RAW264.7 cells. Taken together, compounds 6k exerted its
anti-inflammatory activity through inhibition of NO generation as a result of
inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. These results
could be very useful for the SAR study in the future.
4. Experimental section
4.1. Chemistry
All reagents were purchased from commercial sources and were used without further
purification. Melting points (uncorrected) were determined on a XT4MP apparatus
(Taike Corp., Beijing, China). 1H NMR and 13C NMR spectra were recorded on Bruker
AV-400 or AV-300 MHz instruments in CDCl3. Chemical shifts are reported in parts
per million (δ) downfield from the signal of tetramethylsilane (TMS) as internal

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standards. Coupling constants are reported in Hz. The multiplicity is defined by s
(singlet), d (doublet), t (triplet), or m (multiplet). High resolution mass spectra (HRMS)
were obtained on an Agilent 1260-6221 TOF mass spectrometry. Column and
thin-layer chromatography (CC and TLC, resp.) were performed on silica gel (200-300
mesh) and silica gel GF254 (Qingdao Marine Chemical Factory) respectively.
4.2. The synthesis of title compound glycyrrhetenoic acid 5
4.2.1. (3β,20β)-3-Acetoxyl-11-oxo-olean-12-en-29-dehyde (3)
This compound was prepared as previously reported [33]: A solution of
glycyrrhetic acid (2.5 g, 5.3 mmol) in pyridine (10 mL) THF (20 mL) was added
acetic anhydride (1.0 mL, 11 mmol) over 30 min, and the mixture was stirred and
heated to reflux for 2 h. The reaction solution was evaporated in vacuum, and the
residue was recrystallized from ethanol to obtain glycyrrhetic acid acetate (1). Ethyl
chloroformate (2 mL, 21 mmol) was dropped to a solution of 1 (2.26 g, 4.4 mmol) and
Et3N (2 mL) in THF (10 mL) over 30 min at −5 °C, and the mixture was stirred for 30
min at the same temperature. The precipitate was filtered and washed with THF, and
the combined filtrates were added over 30 min to a solution of NaBH4 (1.0 g, 25
mmol) in H2O (5 mL) in ice bath. Then the reaction mixture was stirred at room
temperature for 4 h and acidified with diluted HCl to pH 5−6, the reaction mixture
was twice extracted with EtOAc. The organic phase was washed twice with water,
then dried over anhydrous Na2SO4, filtered, and concentrated under the reduced
pressure. The residue was recrystallized from ethanol to obtain compound 2 (white
crystal powder, 1.46 g, yield 55% for two-step reaction, no purification for the next
reaction).
A solution of 2 (1.45 g, 2.9 mmol) in CH2Cl2 (30 mL) was added pyridinium
chlorochromate (PCC, 1.50 g, 7.0 mmol) in an ice bath for 1 h, and the mixture was
stirred for 2 h at room temperature. The precipitate was filtered over a celite pad and
washed with EtOAc, and the combined filtrate was washed twice with water, then
dried over anhydrous Na2SO4, filtered, and concentrated under the reduced pressure.
The residue was purified by flash column chromatography (petroleum/EtOAc = 1/1,
v/v) to obtain aldehyde 3 as white powder (0.52 g, 47%), which was used directly for
1
the next step reaction [45]. M.p. 235‒238 °C; H-NMR (300 MHz, CDCl3), δ (ppm):
9.42 (s, 1H, O=CH), 5.67 (s, 1H, H-12), 4.52 (dd, 1H, J = 11.3, 5.2 Hz, H-3), 2.80 (m,

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1H, H-18), 2.36 (s, 1H, H-9), 2.05 (s, 3H, OAc), 1.39 (s, 3H), 1.16 (s, 3H), 1.13 (s,
3H), 0.98 (s, 3H), 0.88 (s, 2×3H), 0.82 (s, 3H).
4.2.2. Ethyl (3β,20β)-3-acetoxyl-11-oxo-olean-12,30(31)-dien-32-oate (4)
This compound was obtained from Wittig-Horner reaction of compound 3 [46].
Triethyl phosphonoacetate (1.40 g, 6.0 mmol) was dropwise added to a stirred
mixture of 80% NaH (250 mg, 6.0 mmol) in dry THF (100 mL) over 30 min in
nitrogen at 0–5°C. After addition of triethyl phosphonoacetate is completed, the
mixture is stirred for 1 h at 0–5°C. To the reaction mixture was dropwise added a
solution of compound 3 (2.50 g, 5.0 mmol) in THF (30 mL) over 30 min in an ice
bath, the mixture was then stirred at room temperature for 2 h. The precipitate was
filtered and washed with EtOAc, and the combined filtrate was washed twice with
water, then dried over anhydrous Na2SO4, filtered, and concentrated under the
reduced pressure. The residue was recrystallized from ethanol to obtain compound 4
1
as white crystal powder (1.83 g, 64%), m.p. 254‒257 °C; H-NMR (600 MHz,
CDCl3), δ (ppm): 6.89 (d, 1H, J = 16.2 Hz, H-30), 5.79 (d, 1H, J = 16.2 Hz, H-31),
5.63 (s, 1H, H-12), 4.52 (m, 1H, 3-H), 4.21 (q, 1H, J = 7.1 Hz), 2.80 (m, 1H, H-18),
2.36 (s, 1H, H-9), 2.05 (s, 3H, Ac), 1.37 (s, 3H), 1.31 (q, 1H, J = 7.1 Hz), 1.16 (s, 3H),
1.12 (s, 3H), 1.02 (s, 3H), 0.88 (s, 6H), 0.82 (s, 3H); 13C-NMR (125 MHz, CDCl3), δ
(ppm): 200.2 (C-11), 171.2 (Ac-C=O), 169.7 (C-13), 167.1 (C=O), 155.7 (C-30),
128.6 (C-12), 120.4 (C-31), 81.8 (C-3), 62.0 (C-9), 60.6 (OCH2), 55.3 (C-9), 47.6
(C-5), 45.7 (C-18), 43.5, 43.0, 39.0, 38.3, 38.0, 37.2, 37.0, 33.4, 32.9, 32.5, 31.1, 28.7,
28.3, 26.65, 26.60, 23.8, 23.7, 21.5, 18.9, 17.6, 16.9, 16.6, 14.5 (CH2CH3).
TOF-HRMS: m/z [M + Na]+ calcd forC36H54NaO5: 589.3863; found: 589.3862.
4.2.3. (3β,20β)-3-Hydroxyl-11-oxo-olean-12,30(31)-dien-32-oic acid (5)
A NaOH solution (50 mL, 5%) was added to a solution of 4 (1.80 g, 3.2 mmol) in
EtOH/CHCl3 mixed solvent (5/1, 60 mL) at room temperature, and the mixture was
stirred for 2 h. The reaction mixture was acidified with diluted HCl to pH 4−5,
extracted twice with CHCl3. The organic phase was washed with water and brine
solution, then dried over anhydrous Na2SO4, filtered, and concentrated under the
reduced pressure. Compound 5 was obtained as white powder (1.44 g, 91%), m.p.
1
259‒262°C; H-NMR (300 MHz, CDCl3), δ (ppm): 7.00 (d, 1H, J = 16.2 Hz, H-30),
5.82 (d, 1H, J = 16.2 Hz, H-31), 5.66 (s, 1H, H-12), 3.73 (q, 2H, J = 7.0 Hz), 3.24 (m,

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1H, H-3), 2.79 (m, 1H, H-18), 2.36 (s, 1H, H-9), 2.34 (s, 1H, H-9), 1.38 (s, 3H), 1.13
13
(s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H); C-NMR
(75 MHz, CDCl3), δ (ppm): 200.3 (C-11), 170.4 (C-13), 169.5 (C-32), 157.9 (C-30),
128.3 (C-12), 119.4 (C-31), 78.8 (C-3), 61.8 (C-9), 58.4 (C-22), 54.9 (C-5), 47.4
(C-18), 45.4, 43.3, 42.8, 39.1 (2C), 38.0, 37.1, 36.7, 32.7, 32.2, 30.7, 28.4, 28.0, 26.4,
26.3, 23.5, 18.6, 18.4, 17.4, 16.3, 15.5. TOF-HRMS: m/z [M + H]+ calcd for C32H49O4:
497.3625; found: 497.3628.
4.3. General procedures for converting the carboxylic acid to an amide for
compounds 6a‒6p.
To a solution of glycyrrhetenoic acid 5 (0.50 mmol, 250 mg), EDC·HCl (230 mg,
1.2 mmol), HOBt (165 mg, 1.2 mmol) and corresponding amine (1.0 mmol, 2 equiv.)
in CH2Cl2 (20 mL), was added NEt3 (2.0 mmol, 0.3 mL). The reaction mixture was
stirred overnight at room temperature. The reaction mixture was added to ice-water
(50 mL) and the two layers of the reaction mixture were subsequently separated. The
organic layer was washed twice with water and then dried over anhydrous Na2SO4.
The solvent was evaporated to yield a residue, which was crystallized from ethanol to
obtain the amides 6a‒6p.
4.3.1. N-Methyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6a).
White powder, yield, 67%; m.p. 269‒271°C; 1H-NMR (300 MHz, CDCl3), δ (ppm):
6.78 (d, 1H, J = 15.8 Hz, H-30), 5.68 (d, 1H, J = 15.8 Hz, H-31), 5.55 (s, 1H, H-12),
3.23 (m, 1H, H-3), 2.90 (d, 3H, J = 4.9 Hz, NCH3), 2.79 (m, 1H, H-18), 2.34 (s, 1H,
H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H),
0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.2 (C-11), 169.6 (C-13), 166.5
(C-32), 150.9 (C-30), 128.2 (C-12), 121.9 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5),
47.2 (C-18), 45.4 (C-8), 43.3, 42.6, 39.1 (2C), 37.4, 37.1, 36.8, 35.8, 33.6, 32.7, 31.2,
28.4, 28.1, 27.3, 26.44, 26.36, 23.5, 18.6, 17.4, 16.4, 16.3, 15.5. TOF-HRMS: m/z [M
+ H]+ calcd for C33H52NO3: 510.3942; found: 510.3940.

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4.3.2. N,N-Dimethyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6b).
1
White powder, yield, 70%; m.p. 265‒268°C; H-NMR (300 MHz, CDCl3), δ (ppm):
6.78 (d, 1H, J = 15.8 Hz, H-30), 5.68 (d, 1H, J = 15.8 Hz, H-31), 5.55 (s, 1H, H-12),
3.23 (m, 1H, H-3), 2.90 (s, 2×3H, NCH3), 2.79 (m, 1H, H-18), 2.34 (s, 1H, H-9), 1.38
(s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H);
13C-NMR (75 MHz, CDCl3), δ(ppm): 200.2 (C-11), 169.6 (C-13), 166.5 (C-32), 150.9
(C-30), 128.2 (C-12), 121.9 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 47.4 (C-18),
45.4 (C-8), 43.3, 42.8, 39.1 (2C), 37.7, 37.5, 37.1, 36.8, 33.7, 32.8, 32.3, 31.2, 28.5,
28.1, 27.3, 26.44, 26.39, 23.1, 18.7, 17.4, 16.4, 16.3, 15.5. TOF-HRMS: m/z [M + H]+
calcd for C34H54NO3: 524.4098; found: 524.4096.
4.3.3. N-Ethyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6c).
1
White powder, yield, 60%; m.p. 265‒269°C; H-NMR (300 MHz, CDCl3), δ (ppm):
6.92 (d, 1H, J = 15.6 Hz, H-30), 6.07 (d, 1H, J = 15.6 Hz, H-31), 5.55 (s, 1H, H-12),
4.62 (m, 2H), 3.14 (m, 2H, CH2CH3), 2.77 (m, 1H, H-18), 2.34 (s, 1H, H-9), 1.38 (s,
3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H);
13C-NMR (75 MHz, CDCl3), δ (ppm): 200.3 (C-11), 170.0 (C-13), 165.7 (C-32),
150.9 (C-30), 128.2 (C-12), 122.1 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 53.4
(CH2CH3), 47.2 (C-18), 45.4 (C-8), 43.3, 42.5, 39.1 (2C), 37.4, 37.1, 36.8, 34.5, 33.7,
32.7, 31.2, 28.5, 28.1, 27.3, 26.43, 26.36, 23.5, 18.6, 17.4, 16.4, 15.6, 14.8 (CH2CH3).
TOF-HRMS: m/z [M + H]+ calcd for C34H53NaNO3: 546.3918; found: 546.3916.
4.3.4.
N-(2-Hydroxyl)ethyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6d). White powder,
yield, 50%; m.p. 278‒281°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.82 (d, 1H, J =
15.7 Hz, H-30), 5.77 (d, 1H, J = 15.5 Hz, H-31), 5.59 (s, 1H, H-12), 3.78 (m, 2H,
CH2O), 3.52 (m, 2H, NCH2), 3.23 (m, 1H, H-3), 2.79 (m, 1H, H-18), 2.34 (s, 1H,
H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H),

ACCEPTED MANUSCRIPT
0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.7 (C-11), 170.6 (C-13), 166.6
(C-32), 151.4 (C-30), 128.0 (C-12), 122.0 (C-31), 78.7 (C-3), 66.9 (CH2O), 61.8 (C-9),
54.9 (C-5), 47.9 (NCH2), 47.1 (C-18), 45.5 (C-8), 43.3, 42.2, 39.1 (2C), 37.5, 37.1,
37.0, 33.7, 32.7, 32.3, 31.2, 28.5, 28.1, 27.2, 26.40, 26.34, 23.5, 18.7, 17.4, 16.4, 15.6.
TOF-HRMS: m/z [M + H]+ calcd for C34H54NO4 : 540.4047; found: 540.4045.
4.3.5.
N-(2-Hydroxyl)propyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6e). White powder,
yield, 60%; m.p. 280‒284°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.78 (d, 1H, J =
15.8 Hz, H-30), 6.57 (d, 1H, NH), 5.80 (d, 1H, J = 15.8 Hz, H-31), 5.62 (s, 1H, H-17),
1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H);
13C-NMR (75 MHz, CDCl3), δ (ppm): 200.7 (C-11), 170.6 (C-13), 166.6 (C-32),
151.4 (C-30), 128.0 (C-12), 122.0 (C-31), 78.7 (C-3), 66.9 (OCH2), 61.8 (C-9), 54.9
(C-5), 47.9 (NCH2), 47.1 (C-18), 45.5 (C-8), 43.3 (C-14), 42.2, 39.1 (2C), 37.5, 37.1,
36.8, 33.7, 32.7, 32.2, 31.2, 28.5, 28.1, 27.2, 26.4, 26.3, 23.5, 23.1, 18.6, 17.0, 16.3,
15.6. TOF-HRMS: m/z [M + H]+ calcd for C35H56NO4: 554.4204; found: 554.4202.
4.3.6. N-Isopropyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6f).
1
White powder, yield, 86%; m.p. 254‒257°C; H-NMR (300 MHz, CDCl3), δ (ppm):
6.78 (d, 1H, J = 15.8 Hz, H-30), 5.68 (d, 1H, J = 15.8 Hz, H-31), 5.61 (s, 1H, H-12),
5.58 (d, 1H, NH), 3.23 (m, 1H, H-3), 2.90 (m, 1H, i-Pr-CH), 2.79 (m, 1H, H-18), 2.34
(s, 1H, H-9), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s,
3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.4 (C-11), 170.2 (C-13), 165.0 (C-32),
150.9 (C-30), 128.2 (C-12), 122.3 (C-31), 78.7 (C-3), 61.9 (C-9), 55.0 (C-5), 47.2
(C-18), 45.5 (C-8), 43.3, 42.8, 42.2, 39.2 (2C), 37.4, 37.1, 36.8, 33.7, 32.7, 32.3, 31.2,
28.5, 28.1, 27.3, 26.42, 26.36, 23.5, 22.8 (2C), 18.7, 17.4, 16.3, 15.5. TOF-HRMS:
m/z [M + H]+ calcd for C35H56NO3: 538.4255; found: 538.4254.
4.3.7. N,N-Diisopropyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide

ACCEPTED MANUSCRIPT
1
(6g). White powder, 76%; m.p. 264‒268°C; H-NMR (300 MHz, CDCl3), δ (ppm):
6.78 (d, 1H, J = 15.8 Hz, H-30), 5.68 (d, 1H, J =15.8 Hz, H-31), 5.55 (s, 1H, H-12),
3.94 (s, 1H, NH), 3.23 (m, 1H, H-3), 2.90 (m, 2H, H-33), 2.79 (m, 1H, H-18), 2.34 (s,
1H, H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s,
13
3H), 0.80 (s, 3H); C-NMR (75 MHz, CDCl3), δ (ppm): 200.2 (C-11), 169.6 (C-13),
166.5 (C-32), 150.9 (C-30), 128.2 (C-12), 121.9 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9
(C-5), 47.4 (C-18), 45.4 (C-8), 43.3, 43.0 (2C), 42.7, 39.1 (2C), 37.6, 37.4, 37.1, 36.7,
33.6, 32.2, 31.2, 28.4, 28.1, 27.3, 26.4, 26.4, 23.5, 23.1 (4C), 18.7, 17.5, 16.3, 15.5.
TOF-HRMS: m/z [M + H]+ calcd for C38H62NO3: 580.4724; found: 580.4726.
4.3.8.
N-(3-Chloro)propyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6h). White powder,
yield, 67%; m.p. 243‒246°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.78 (d, 1H, J =
15.8 Hz, H-30), 5.68 (d, 1H, J =15.8 Hz, H-31), 5.55 (s, 1H, H-12), 3.62 (t, 2H, J =
6.8 Hz, ClCH2), 3.50 (m, 2H), 3.23 (m, 1H, H-3), 2.79 (m, 1H, H-18), 2.34 (s, 1H,
H-9), 2.07 (m, 4H), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H),
0.81 (s, 3H), 0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.4 (C-11), 170.1
(C-13), 166.2 (C-32), 151.5 (C-30), 128.2 (C-12), 121.9 (C-31), 78.7 (C-3), 61.9
(C-9), 54.9 (C-5), 47.2 (C-18), 45.4 (C-8), 43.3, 42.6, 39.1 (2C), 37.5, 37.1 (2C), 36.8,
33.6, 32.7, 32.3, 32.2, 31.2, 29.7, 28.5, 28.1, 27.3, 26.42, 26.38, 23.5, 18.7, 17.4, 16.3,
15.5. TOF-HRMS: m/z [M + H]+ calcd for C35H55ClNO3: 572.3865; found: 572.3863.
4.3.9. N-(2-Phenyl)ethyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide
1
(6i). White powder, yield, 50%; m.p. 287‒291°C; H-NMR (300 MHz, CDCl3), δ
(ppm): 7.33 (m, 2H, H-Ar), 7.23 (m, 3H, H-Ar), 6.80 (d, 1H, J = 15.8 Hz, H-30), 5.65
(d, 1H, J = 15.8 Hz, H-31), 5.57 (s, 1H, H-12), 3.61 (m, 2H, NCH2), 3.23 (m, 1H,
H-3), 2.88 (t, 2H, J = 7.1 Hz, PhCH2), 2.80 (m, 1H, H-18), 2.34 (s, 1H, H-9), 1.38 (s,
3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H);
13C-NMR (75 MHz, CDCl3), δ (ppm): 200.2 (C-11), 169.8 (C-13), 165.8 (C-32),

ACCEPTED MANUSCRIPT
151.2 (C-30), 138.0 (C-Ar), 128.7 (2C-Ar), 128.6 (2C-Ar), 128.2 (C-12), 126.5
(C-Ar), 122.0 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 47.1 (C-18), 45.4 (C-8), 43.3,
40.8, 39.1 (2C), 37.4, 37.1, 36.8, 35.7, 33.6, 32.7, 32.3 (2C), 31.1, 28.5, 28.1, 27.3,
26.44, 26.38, 23.5, 18.7, 17.4, 16.3, 15.5. TOF-HRMS: m/z [M + H]+ calcd for
C40H58NO3: 600.4411; found: 600.4414.
4.3.10. N-Phenyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-acylhydrazine
1
(6j). White powder, yield, 70%; m.p. 281‒285°C; H-NMR (300 MHz, CDCl3), δ
(ppm): 8.26 (s, 1H, NH), 7.27‒7.19 (m, 2H, H-30, NH), 6.85 (m, 5H, Ar-H), 5.80 (d,
1H, J = 15.8 Hz, H-31), 5.67 (s, 1H, H-12), 3.23 (m, 1H, H-3), 2.77 (m, 1H, H-18),
2.34 (s, 1H, H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H),
0.81 (s, 3H), 0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.6 (C-11), 170.3
(C-13), 166.1 (C-32), 153.0 (C-30), 148.1 (C-Ar), 129.1 (2C-Ar), 128.2 (C-12), 121.1
(C-31), 119.3 (C-31), 113.8 (2C-Ar), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 47.1 (C-18),
45.4 (C-8), 43.3, 42.3, 39.1 (2C), 37.7, 37.1, 36.8, 33.6, 32.7, 32.2, 31.0, 28.5, 28.1,
27.2, 26.39, 26.35, 23.5, 18.6, 17.4, 16.3, 15.6. TOF-HRMS: m/z [M + H]+ calcd for
C38H55N2O3: 587.4207; found: 587.4205.
4.3.11.
N-(4-Methyl)piperazinyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6k). White powder,
yield, 78%; m.p. 254‒258°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.84 (d, 1H, J =
15.7 Hz, H-30), 6.19 (d, 1H, J = 15.7 Hz, H-31), 5.55 (s, 1H, H-12), 3.64 (m, 4H,
NCH2), 3.23 (m, 1H, H-3), 2.77 (m, 1H, H-18), 2.43 (m, 4H, NCH2), 2.33 (s, 1H,
H-9), 2.33 (s, 3H, NCH3), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s,
13
3H), 0.81 (s, 3H), 0.80 (s, 3H); C-NMR (75 MHz, CDCl3), δ (ppm): 200.2 (C-11),
169.8 (C-13), 165.6 (C-32), 152.9 (C-30), 128.3 (C-12), 118.3 (C-31), 78.7 (C-3),
61.9 (C-9), 54.9 (2C), 54.8 (C-5), 47.4 (C-18), 46.0 (2C), 45.4 (C-8), 43.4, 43.0, 39.1
(2C), 37.7, 37.1, 36.8, 33.6, 32.7, 32.3 (2×NCH2), 31.4, 28.5, 28.1, 27.3, 26.4 (2C),
23.5, 18.7, 17.5, 16.4, 15.6 TOF-HRMS: m/z [M + H]+ calcd for C37H59N2O3:

ACCEPTED MANUSCRIPT
579.4520; found:579.4523.
4.3.12.
N-(4-Ethyl)piperazinyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6l). White powder, yield,
1
74%; m.p. 257‒262°C; H-NMR(300 MHz, CDCl3), δ (ppm): 6.84 (d, 1H, J = 15.7
Hz, H-30), 6.19 (d, 1H, J = 15.7 Hz, H-31), 5.55 (s, 1H, H-12), 3.64 (m, 4H, NCH2),
3.23 (m, 1H, H-3), 2.77 (m, 1H, H-18), 2.45 (m, 6H, CH2CH3, NCH2), 2.34 (s, 1H,
H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H),
0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3), δ (ppm): 200.2 (C-11), 169.8 (C-13), 165.5
(C-32), 152.8 (C-30), 128.3 (C-12), 118.3 (C-31), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5),
52.2 (2C), 47.5 (C-18), 45.9 (2C), 45.4 (C-8), 43.3, 43.0, 39.1 (2C), 37.7, 37.1, 36.8,
33.6, 32.7, 32.3 (2C), 31.1, 28.5, 28.1, 27.3, 26.44, 26.40, 23.5, 18.7, 17.5, 16.4, 15.6,
11.8 (CH2CH3). TOF-HRMS: m/z [M + H]+ calcd for C38H61N2O3: 593.4677; found:
593.4674.
4.3.13. N,N-Morpholinyl (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide
(6m). White powder, yield, 76%; m.p. 265‒268°C;1H-NMR (300 MHz, CDCl3), δ
(ppm): 6.78 (d, 1H, J = 15.8 Hz, H-30), 6.57 (d, 1H, J = 7.4 Hz), 5.80 (d, 1H, J =
15.8 Hz, H-31), 5.62 (s, 1H, H-12), 4.13‒3.57 (m, 4H, 2×CH2O), 3.23 (m, 1H, H-3),
2.77 (m, 1H, H-18), 2.34 (m, 3H, H-9, NCH2), 2.21 (m, 2H, NCH2), 1.38 (s, 3H), 1.13
13
(s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H); C-NMR
(75 MHz, CDCl3), δ (ppm): 200.2 (C-11), 169.7 (C-13), 165.8 (C-32), 153.6 (C-30),
128.3 (C-12), 117.8 (C-31), 78.7 (C-3), 66.7 (2CH2O), 61.9 (C-9), 54.9 (C-5), 47.4
(C-18), 45.4 (C-8), 43.4, 43.0, 42.4, 39.1 (2C), 37.8, 37.1, 36.8, 33.5 (2C), 32.3 (2C),
31.1, 28.5, 28.1, 27.3, 26.41, 26.38, 23.5, 18.7, 17.5, 16.4, 15.6. TOF-HRMS: m/z [M
+ H]+ calcd for C36H56NO4: 566.4204; found: 566.4206.
4.3.14.
N-(3-Morpholinyl)propyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6n). White powder,

ACCEPTED MANUSCRIPT
yield, 56%; m.p. 272‒276°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.89 (d, 1H, J =
4.9 Hz, NH), 6.78 (d, 1H, J = 15.8 Hz, H-30), 5.68 (d, 1H, J = 15.8 Hz, H-31), 5.55
(s, 1H, H-12), 3.73 (m, 4H, OCH2), 3.43 (m, 4H, NCH2), 3.23 (m, 1H, H-3), 2.78 (m,
1H, H-18), 2.49 (m, 6H, NCH2), 2.34 (s, 1H, H-9), 1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s,
3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H); 13C-NMR (75 MHz, CDCl3),
δ (ppm): 200.1 (C-11), 169.8 (C-13), 165.8 (C-32), 150.6 (C-30), 128.2 (C-12), 122.3
(C-31), 78.7 (C-3), 67.1 (2×OCH2), 61.8 (C-9), 57.5 (2×NCH2), 54.9 (C-5), 53.6 (2C),
47.3 (C-18), 45.4 (C-8), 43.3, 42.9, 39.1 (2C), 37.3, 37.0, 36.7, 33.5, 32.7, 32.2, 31.2,
29.7, 28.0, 27.2, 26.4, 26.3, 26.3, 25.0, 23.5, 18.6, 17.4, 16.3, 15.6. TOF-HRMS: m/z
[M + H]+ calcd for C39H62NaN2O4: 645.4602; found:645.4603.
4.3.15.
N-(3-Methyl)phenyl
(3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6o). White powder,
yield, 56%; m.p. 260‒265°C; 1H-NMR (300 MHz, CDCl3), δ (ppm): 7.87 (s, 1H, NH),
7.50 (s, 1H, H-Ar), 7.44 (m, 1H, H-Ar), 7.21 (m, 1H, H-Ar), 6.95 (s, 1H, H-Ar), 6.78
(d, 1H, J = 15.8 Hz, H-30), 5.92 (d, 1H, J = 15.7 Hz, H-31) , 5.60 (s, 1H, H-12), 3.23
(m, 1H, H-3), 2.80 (m, 1H, H-18), 2.34 (s, 1H, H-9), 2.34 (s, 3H, Ar-CH3), 1.38 (s,
3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s, 3H);
13C-NMR (75 MHz, CDCl3), δ (ppm): 200.4 (C-11), 170.1 (C-13), 164.0 (C-32),
152.7 (C-30), 138.8 (Ar-C), 138.1 (Ar-C), 128.8 (Ar-C), 128.1 (C-12), 125.0 (Ar-C),
122.6 (Ar-C), 122.0 (C-31), 116.9 (Ar-C), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 47.1
(C-18), 45.4 (C-8), 43.3, 42.4, 39.2, 39.1, 37.6, 37.1, 36.8, 33.8, 32.2 (2C), 31.1, 28.5,
28.1, 27.3, 26.3 (2C), 23.5, 21.5 (Ar-CH3), 18.6, 17.4, 16.4, 15.5. TOF-HRMS: m/z
[M + H]+ calcd for C39H56NO3: 586.4255; found:586.4253.
4.3.16. N-Thiourea (3β,20β)-3-hydroxyl-11-oxo-olean-12,30(31)-dien-32-amide (6p).
1
White powder, yield, 58%; m.p. 243‒247°C; H-NMR (300 MHz, DMSO-d6), δ
(ppm): 9.7 (s, 1H, NH), 6.57 (d, 1H, J = 16.0 Hz, H-30), 5.96 (s, 1H), 5.94 (d, 1H, J
= 15.8 Hz, H-31), 5.68 (s, 1H, H-12), 4.31 (m, 1H), 3.02 (m, 1H, H-3), 2.59 (m, 1H,

ACCEPTED MANUSCRIPT
H-18), 2.35 (s, 1H, H-9), 1.33 (s, 3H), 1.04 (s, 6H), 0.97 (s, 3H), 0.91 (s, 3H), 0.77 (s,
3H), 0.69 (s, 3H); 13C-NMR (75 MHz, DMSO-d6), δ (ppm): 200.4 (C-11), 174.8
(C=O), 169.8 (C-13), 163.9 (C-32), 155.2 (C-30), 132.7 (C-12), 126.0 (C-31), 81.8
(C-3), 66.4 (C-9), 59.3, 54.9, 51.7, 50.1, 48.2, 45.6 (2C, overlap), 42.4, 41.9 (2C),
38.0, 37.3, 37.1 (2C), 35.9, 33.6, 33.3, 32.2, 31.2 (2C), 28.5, 23.6, 21.4, 21.2.
TOF-HRMS: m/z [M + H]+ calcd for C33H52N3O4: 554.3952; found: 554.3955.
4.4. General procedures for converting the carboxylic acid to an ester for compounds
7a and 7b.
A solution of glycyrrhetenoic acid 5 (0.5 g, 1.0 mmol) in pyridine (2 mL) was
added acetic anhydride (0.2 mL, 2.2 mmol) over 30 min, and the mixture was stirred
and heated to reflux for 30 min. The reaction solution was evaporated in vacuum, and
the residue was recrystallized from ethanol to obtain glycyrrhetenoic acid acetate (448
mg, 82%). To a solution of glycyrrhetenoic acid acetate (160 mg, 0.3 mmol,) and
alcohol (10 equiv.) in CH2Cl2 (10 mL), was added boron trifluoride dimethyl etherate
(0.7 mmol, 0.1 mL). The reaction mixture was stirred at room temperature for 24 h.
The reaction mixture was added to ice-water (50 mL) and the two layers of the
reaction mixture were subsequently separated. The organic layer was washed twice
with water and then dried over anhydrous Na2SO4. The solvent was evaporated to
yield a residue, which was crystallized from ethanol to obtain the esters 7a and 7b.
4.4.1. Ethyl (3β,20β)-3-acetoxyl-11-oxo-olean-12,30(31)-dien-32-oate (7a). White
powder, yield, 77%; 1H-NMR (300 MHz, CDCl3), δ (ppm): 6.89 (d, 1H, J = 16.2 Hz,
H-30), 5.79 (d, 1H, J = 16.2 Hz, H-31), 5.63 (s, 1H, H-12), 4.52 (m, 1H, H-3), 4.20 (q,
2H, J = 7.1 Hz, OCH2CH3), 2.80 (m, 1H, H-18), 2.36 (s, 1H, H-9), 2.05 (s, 3H, OAc),
1.38 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.80 (s,
3H). That is compound 4.
4.4.2. Benzyl (3β,20β)-3-Acetoxyl-11-oxo-olean-12,30(31)-dien-32-oate (7b). White
1
powder, yield, 73%; m.p. 243‒247°C; H-NMR (600 MHz, CDCl3), δ (ppm):
7.41-7.32 (m, 5H, Ar-H), 6.95 (d, 1H, J = 16.3 Hz, H-30), 5.84 (d, 1H, J = 16.3 Hz,
H-31), 5.61 (s, 1H, H-12), 5.19 (dd, 2H, J = 15.4, 12.4 Hz, OCH2Ph), 4.52 (m, 1H,

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3-H), 2.80 (m, 1H, H-18), 2.36 (s, 1H, H-9), 2.05 (s, 3H, Ac), 1.37 (s, 3H, H-27), 1.16
(s, 3H, H-29), 1.12 (s, 3H, H-25), 1.01 (s, 3H, H-26), 0.88 (s, 6H, H-23, H-24), 0.82 (s,
3H, H-28). TOF-HRMS: m/z [M + H]+ calcd for C41H57O5: 629.4201; found:
629.4198.
4.5. Crystal structure determination for compound 6b
X-ray single-crystal diffraction data for compound 6b was collected on a Bruker
SMART APEX CCD diffractometer at 300.79(10) K using CuK\α radiation (
λ =
1.54184 Å) by the ω scan mode. The program SAINT was used for integration of the
diffraction profiles. The structure was solved by direct methods using the SHELXS
program of the SHELXTL package and refined by full-matrix least-squares methods
with SHELXL [47]. The corrections for LP factors were applied. Multi-scan
symmetry-related measurement was used as experimental absorption correction type.
The non-hydrogen atoms of compound 6b were refined with anisotropic thermal
parameters whereas all hydrogen atoms were generated theoretically onto the parent
atoms and refined isotropically with fixed thermal factors.
The crystal data of compound 6b was as follows: Colorless block crystals, yield,
o
76%; mp 266-268 C; C34H53NO3, Mr = 523.77, orthorhombic, space group P21 21 21
;
a = 10.0515(8), b = 15.1327(13), c = 19.9334(15) (Å);
α = 90, β = 90, γ = 90, V =
3032.0(4) Å3, T = 300.79(10), Z = 4, Dx = 1.147 g/cm3, F(000) = 1152, Reflections
collected / Independent reflections = 11269 / 4495, Data / restraints / parameters =
5452 / 0 / 353, Goodness of fit on F2 =1.127, Final R indexes [all data], R1 = 0.0646,
wR(F2) = 0.1389.
4.6. Cell Culture
RAW264.7 murine macrophages were cultured in DMEM containing 10% fetal
bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 ºC in a 5%
CO2 humidified atmosphere.

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4.7. Assay for NO Production
RAW264.7 cells were inoculated at 1×105 cells per well in 24-well plate and
cultured for 20 h. The cells were then pre-treated with different concentrations
compounds which were prepared in serum-free medium for 2 h before stimulation
with LPS (1 µg/mL). After LPS-stimulated for 24 h, the NO production was
determined by detecting the nitrite level using Griess reagent (Beyotime, China)
according to the manufacturer’s instructions, then measured absorbance of the
samples at 540 nm (OD540) in a microplate reader (MQX200, Bio-Tek, USA). NO
inhibition rate = [control (OD540) - compound (OD540)] / [control (OD540) - blank
(OD540)] × 100%.
Control: treated with LPS only.
Compound: treated with LPS and compounds.
Blank: cultured with fresh medium only.
4.8. Cell Cytotoxicity
Cell cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assay.
RAW264.7 cells were inoculated at 6 × 103 cells per well in 96-well plate. After
cultured for 24 h, the cells were treated with different compounds which were diluted
in DMEM for 24 h. Then 20 µL of 0.5 mg/mL MTT reagent was added into the cells
and incubated for 4 h. After 4 h, cell culture was removed and then 150 µL DMSO
was added to dissolve the formazan. The optical density was measured at 570 nm
(OD570). Cell viability was calculated from three independent experiments. The
density of formazan formed in blank group was set as 100% of viability. Cell viability
(%) = compound (OD570 / blank (OD570) × 100%
Blank: cultured with fresh medium only.
Compound: treated with compounds or LPS.
4.9. Measurement of IL-6 and TNF-α
RAW264.7 cells (7 × 104 cells/well) were cultured in 24-well plate. After cultured
for 24 h, and pretreated with 10µM-40 µM of compounds for 2 h, and then LPS was
added. The production of IL-6 and TNF-α was stimulated by the addition of 1 µg/mL

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LPS and incubated for 24 h. The levels of IL-6 and TNF-α in the supernatant were
determined using the mouse ELISA kit (IL-6: R&D SYSTEMS, DY406-05. TNF-α:
R&D SYSTEMS, DY410-05) which is operated according to the manufacturer’s
instructions. The results were analyzed using GraphPad Prism 5.0 software and SPSS
version 19.0 statistic software.
4.10. Western Blot analysis
RAW264.7 cells were seeded into a 6-well culture plate at a density of 2 × 106 cells
per well, and then cultured for 24 h. Then, the culture medium was replaced by fresh
medium containing 10 、 20 and 40 µM compounds, or Bay 11-7082
(MedChemExpress, HY-13453) and 1 µg/mL LPS was added. After cultured for
another 30 min or 24 h, the cells were harvested and lysed with IP buffer (Beyotime,
P0013) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF: Beyotime,
ST506) and incubated on ice for 30 min. The cell lysates were centrifuged at 14,000 ×
g for 10 min at 4 ºC to remove insoluble materials and the supernatant was collected.
Total protein concentration was determined using a BCA protein assay kit (Thermo
Scientific, 23227). Each protein sample was mixed with a quarter volume of 5X
SDS-PAGE sample loading buffer (100 mmol/L Tris-HCl pH 6.8, 4% SDS, 5%
β-mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue) and boiled for 10 min.
Equal amounts of total cellular protein were loaded per well in 12.5% precast
SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes
(Bio-Rad) for over 60 min at 300 mA. The membranes were blocked with 5% non-fat
dry milk in TBS plus 0.1% Tween 20 (TBST) for 2 h at room temperature, washed 3
times in TBST for 5 min each, incubated with the primary antibody
(anti-phosphorylation of SAPK/JNK, anti-SAPK/JNK, anti-phosphorylation of
ERK1/2, anti-ERK1/2, anti-phosphorylation of p38, anti-p38, anti-phosphorylation of
iκb, anti-iκb, antiphosphorylation of NF-kB p65, and anti-NF-κB p65) at 4 ºC
overnight (all the primary antibodies were purchased from Cell Signaling Technology
and diluted at the ratio of 1:1000),washed 3 times in TBST for 5 min each, incubated
with anti-rabbit or anti-mouse secondary antibody (1:5000 in TBST, Cell Signaling
Technology) for 90 min, washed in TBST and exposed to ECL reagents.

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4.11. Immunofluorescence assay
RAW264.7 cells (7 × 104 cells/well) were cultured in 24-well plate. After cultured
for 24 h, and pretreated with 20 and 40 µM of compound 6k for 2 h, and then treated
with LPS (1 µg/mL) for 3 h. The cells were washed twice with cold PBS, fixed with
4% formaldehyde for 15 min, and then permeabilized with 0.3% Triton X-100 in PBS
for 10 min. After that, the cells were blocked for 0.5 h with 5% BSA. Cells were later
incubated with primary antibody anti-NF-κB p65 antibody for overnight, followed by
Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody. After a wash step,
stained with DAPI for 5 min and the images were acquired.
Acknowledgments
Financial support was provided by the National Natural Science Funding of China
(21572003, 20802003), Anhui University Natural Science Research Project
(KJ2016A339), and University Project of Introduction and Cultivation of Leading
Talents (gxfxZD2016044).
Notes
The authors declare no competing financial interest.
Appendix A. Supplementary material
Crystallographic data (excluding structure factors) for the structure had been
deposited with the Cambridge Crystallographic Data Center as supplementary
publication No. CCDC 1418308 (6b). These data can be obtained free of charge via
the URL http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
1
deposit@ccdc.cam.ac.uk). Representative H and 13C NMR spectra can be found at
http://
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Figure Captions
Table 1
Effect of glycyrrhetin derivatives on the viability of RAW264.7 cellsa
Fig. 1. The general design strategy in this study.
Fig. 2. ORTEP drawing of compound 6b.
Fig. 3. The inhibitory effects of glycyrrhetin derivatives on NO production in
LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with glycyrrhetin
derivatives (A: 40µM and B: 20µM) for 2 h, and then in the presence or absence of
LPS (1 µg/mL) for 24 h. The results were showed as means ± SD (n = 3) of at least
three independent experiments.# p < 0.05, ## p < 0.01, ### p < 0.001 compared with the
blank group; * p < 0.05, ** p < 0.01, *** p < 0.001 compare with LPS-stimulated group.
Fig. 4. Compounds 6k and 6l inhibited LPS-induced IL-6 and TNF-α production in
RAW264.7 cells (A and B). RAW 264.7 cells were incubated with different
concentrations of compounds 6k and 6l for 2 h, and then in the presence or absence of
#
LPS (1 µg/mL) for 24 h. p < 0.05, ## p < 0.01, ### p < 0.001 compared with the
*
**
***
control group; p < 0.05, p < 0.01,
p < 0.001 compare with LPS-stimulated
group.
Fig. 5. Effects of compound 6k on LPS-induced iNOS and COX-2 protein expression
in RAW264.7 cells. The cells were treated with different concentrations of compound
6k, and then in the presence or absence of LPS (1 µg/mL) for 24 h. Bay 11-7082 is the
NF-κB inhibitors (20 µM). The results were showed as means ± SD (n = 3) of at least
three independent experiments. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the
*
**
***
control group; p < 0.05, p < 0.01,
p < 0.001 compare with LPS-stimulated
group.

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Fig. 6. A: Compound 6k suppressed LPS-induced activation of NF-κB signaling
pathway in RAW 264.7 cells. RAW264.7 cells were treated with compound 6k (10
µM – 40 µM) and LPS (1 µg/mL) for 30 min. The levels of NF-κB p65, IκB, and their
phosphorylated forms were analyzed using western blotting. Bay 11-7082 is the
NF-κB inhibitors (20 µM). The results were showed as means ± SD (n = 3) of at least
#
three independent experiments. p < 0.05, ### p < 0.01, ### p < 0.001 compared with
*
the control group; p < 0.05, ** p < 0.01, *** p < 0.001 compared with LPS-stimulated
group.
B: Compound 6k clearly inhibited NF-κB p65 nuclear translocation. RAW264.7 were
pretreated with compound 6k for 1 h and then stimulated with LPS (1 µg/mL) for 3 h.
Fig. 7. Compound 6k suppressed LPS-induced activation of MAPK signaling
pathway in RAW 264.7 cells. RAW264.7 cells were treated with Compound 6k (10
µM – 40 µM) and LPS (1 µg/mL) for 30 min. The levels of JNK, ERK1/2, and p38
MAPK proteins, and their phosphorylated forms were analyzed using western blotting.
The results were showed as means ± SD (n = 3) of at least three independent
experiments. # p < 0.05, ### p < 0.01, ### p < 0.001 compared with the control group; * p
< 0.05, ** p < 0.01, *** p < 0.001 compared with LPS-stimulated group.
Scheme 1. Synthesis of compound 5.
Scheme 2. Synthesis of glycyrrhetenoic acid derivatives 6a‒6p.
Scheme 3. Synthesis of glycyrrhetenoic acid derivatives 7a‒7b.