Design and synthesis of novel glycyrrhetin ureas as anti-inflammatory agents for the treatment of acute kidney injury

Article abstract of DOI:10.1016/j.bioorg.2021.104755

To develop new anti-inflammatory drugs for the prevention and treatment of acute kidney injury, a series of novel glycyrrhetic ureas were designed, synthesized and evaluated for anti-inflammatory activity using RAW264.7 cells. Compounds 5r-5u (2.04, 2.50, 3.25 and 2.48 μM, respectively) with acidic or neutral amino acid showed potent anti-inflammatory activity (IC₅₀ = 2–3 μM for NO inhibition), amongst them, compound 5r also inhibited tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a dose-dependent manner. In cisplatin-induced AKI mice model, compound 5r significantly reduced the level of pro-inflammatory factors, ameliorated the pathological damage of kidney tissue, and maintained the normal metabolic capacity.

Full text of DOI:10.1016/j.bioorg.2021.104755

Bioorganic Chemistry 110 (2021) 104755
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel glycyrrhetin ureas as anti-inflammatory
agents for the treatment of acute kidney injury
,
,c,
,*
Hongbo Wanga 1, Jiawei Zuob 1, Liang Zhac, Xia Jiangc, Caixia Wua, Yong-An Yangd,
,
c
,
Wenjian Tanga *, Tianlu Shib
a Department of Pharmacy, Shandong Medical College, Linyi 276000, China
b First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China
c School of Pharmacy, Anhui Medical University, Hefei 230032, China
d Elion Nature Biological Technology Co., Ltd, Nanjing 210038, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To develop new anti-inflammatory drugs for the prevention and treatment of acute kidney injury, a series of
novel glycyrrhetic ureas were designed, synthesized and evaluated for anti-inflammatory activity using
Glycyrrhetinic acid
Urea
RAW264.7 cells. Compounds 5r-5u (2.04, 2.50, 3.25 and 2.48
μ
M, respectively) with acidic or neutral amino
Amino acid
acid showed potent anti-inflammatory activity (IC50 = 2–3
μM for NO inhibition), amongst them, compound 5r
Anti-inflammatory activity
Inflammatory factor
Acute kidney injury
also inhibited tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a dose-dependent manner. In cisplatin-
induced AKI mice model, compound 5r significantly reduced the level of pro-inflammatory factors, ameliorated
the pathological damage of kidney tissue, and maintained the normal metabolic capacity.
1. Introduction
kidney stress or injury, it protects the kidney from further damage [8].
Clinical manifestations have proved that AKI is associated with intra-
Acute kidney injury (AKI) is a global public health problem that af-
fects millions of patients worldwide. Approximately 20% of hospitalized
patients develop this type of injury and are accompanied by volume
overload, electrolyte disturbances, uremia complications, and drug
toxicity [1]. As a clinical multiple syndrome, AKI is the rapid loss of
renal function that is characterized by oliguria, nephritis, and renal vein
thrombosis due to the abrupt decline of glomerular filtration [2–4]. In
ischemia, sepsis and nephrotoxic models, the morphological and/or
functional changes are observed in vascular endothelial cells and/or in
tubular epithelium. Then, leukocytes including neutrophils, macro-
phages, natural killer cells, and lymphocytes infiltrate into the injured
kidneys. The injury induces the generation of inflammatory mediators
like cytokines and chemokines by tubular and endothelial cells which
contribute to the recruiting of leukocytes into the kidneys. Inflammation
is a reaction of the immune system, which activates many enzymes and
cellular processes to protect the body from various injuries [5]. Thus,
inflammation plays an important role in the initiation and extension
phases of AKI [6,7]. Kidney inflammation is the initial response to
renal and systemic inflammation, which is essential for eliminating
microbial pathogens and repairing tissue after diverse forms of injury
[9–11]. Continued active research in the fields of chemistry and biology
has contributed to pharmaceuticals to alleviate pain and other associ-
ated damage, which is reported in 90% of cases according to World
Health Organization (WHO) statistics [12]. To date, no effective
chemical agents have been developed to prevent or alleviate AKI in the
clinic. Therefore, it is urgent to develop novel therapies to improve renal
regeneration capacity, preserve renal function and prevent AKI.
Inflammation is the basic defense mechanism of the immune system
and bound up with a great range of diseases, which can prevent damage
caused by harmful stimuli such as pathogens and poisons. Non-steroidal
anti-inflammatory drugs (NSAIDs) are the most commonly used anti-
inflammatory agents, but gastrointestinal complications such as gastric
ulcer and duodenal ulcer cannot be avoided, hence, there is necessary to
find new drug molecules that effectively reduce inflammation and pain
relief [13,14]. Renal inflammation is not only a response to pathogens,
but also to sterile stimuli such as ischemia, hyperglycemia, and lipids
* Corresponding authors at: School of Pharmacy, Anhui Medical University, Hefei 230032, China (W. Tang); First Affiliated Hospital of USTC, Division of Life
Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China (T. Shi).
E-mail addresses: ahmupharm@126.com (W. Tang), tianlu828@163.com (T. Shi).
1
H.B. Wang and J.W. Zuo contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104755
Received 24 December 2020; Received in revised form 6 February 2021; Accepted 17 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
[15,16]. The effective treatment was achieved by overcoming the
serious AKI complications through the strategy of controlling the
development of inflammation. Perindopril protected AKI caused by
septic shock, FTY720 prevented kidney ischemic reperfusion injury by
regulating mitochondria biogenesis in dendritic cells, omeprazole
attenuated cisplatin-induced kidney injury through suppression of the
TLR4/NF-κB/NLRP3 signaling pathway [17–19]. While 7-hydroxycou-
marin protected cisplatin-induced AKI by inhibiting necroptosis and
promoting Sox9-mediated tubular epithelial cell proliferation, a rutae-
carpine derivative alleviated AKI by targeting PDE4B, which is the key
enzyme mediating inflammation in cisplatin nephropathy [20,21].
Although marked drugs and natural products would help to understand
the mechanism of AKI, it is significant for finding new scaffolds with
high efficiency and low toxicity for the treatment of AKI.
optimization of natural product toward new skeleton with target
bioactivity. Herein we proceeded with modification of the GA structure
using following strategies: (a) converting carboxyl acid group at C30
into a urea moiety in ring E of GA, namely glycyrrhetin urea (5c); (b)
amino of urea modified with alkyl, cycloalkyl or amino acid function-
ality (Fig. 1). Thus, twenty-four compounds were designed, synthesized
and evaluated for their anti-inflammatory activity in lipopolysaccharide
(LPS)-induced RAW264.7 cells. Further, potent compound 5r with
acidic amino acid were used to study the preliminary mechanism of anti-
inflammatory effect for the treatment of AKI in cisplatin-induced AKI
mice models.
2. Results and discussion
Glycyrrhetinic acid (GA), one of the main active ingredients of Lic-
orice, exhibited a variety of pharmacological activities such as anti-
inflammatory, antioxidant, antiviral and hepatoprotective activities,
etc [22–25]. Renal protective effects of GA may be relevant to its anti-
inflammatory activity, and recent studies have shown that GA im-
proves nephrotoxicity and kidney injury caused by multiple etiologies
through various pathways [26–28]. Interestingly, almost half of the
approved new molecular entities were derived from natural products,
which play a pivotal role in the search for new anti-inflammatory drugs
with improved drug properties and reduced side effects [29,30]. The
semi-synthesis and synthetic synthesis of natural compounds show a
variety of biological applications [31]. The structural modification of
biologically active natural products according to molecular character-
istics was an important way to find new lead compounds, for example,
structural changes of GA can improve its efficiency and decrease its
toxicity [32–37].
2.1. Chemistry
The preparation of the key intermediate 3 was prepared as previ-
ously reported [33] and outlined in Scheme 1. Acylation reaction of 3-
OH of glycyrrhetinic acid with acetic anhydride gave 3-acetyl-glycyrrhe-
tinic acid (2). Substitution reaction of 30-carboxylic acid of compound 2
and thionyl chloride in chloroform gave 3-acetyl glycyrrhetinic 30-acyl
chloride, and then which was converted to the acyl azide with NaN3 in
acetone and water. The key intermediate isocyanate 3 was obtained
from 3-acetyl glycyrrhetinic 30-acyl azide by Curtius rearrangement
reaction in anhydrous toluene. Glycyrrhetin urea derivatives 5 were
obtained from the nucleophilic addition reaction of intermediate 3 with
various substituted amines or amino acids.
Reagents and conditions: (i) Ac2O-C5H5N, rt, 2 h; refluxed, 2 h; (ii)
SOCl2, CHCl3, refluxed, 2 h: NaN3, acetone-H2O, 5 ◦C, 1 h; toluene,
75 ◦C, 6 h; (iii) substituted amines or amino acids, ethyl acetate and
methanol, rt, 1 h; (iv) NaOH, CH3OH, rt, 12 h.
Minor structural changes in triterpenoids can cause extensive
changes in biological activity in medicinal chemistry. The therapeutic
application of amino acids has received great attention in renal failure,
respiratory tract, cardiology, neurological disorders and congenital de-
fects [38]. Most of structural modifications of GA at 3-hydroxy (or 3-
oxo) or/and 30-carboxylic groups exhibited a better activity than GA
[32–39]. Urea scaffold (Fig. 1) was often incorporated to modulate drug
potency and selectivity and improve drug properties in drug design and
medicinal chemistry due to the capability of the urea functionality to
form multiple stable hydrogen bonds with protein and receptor targets
[40,41]. The first soluble epoxide hydrolase (sEH) inhibitor AR9281
showed anti-inflammatory activity through the urea moiety binding to
the catalytic residue of sHE, while protein–ligand complexes between
vascular endothelial growth factor receptor 2 (VEGFR2) and lenvatinib
highlighted the importance of the urea functionality involving critical
hydrogen bonding with Asp1046 and Glu885 of VEGFR2 [42,43].
Therefore, the urea moiety could be introduced into the structure of GA,
which may improve potency and drug-like properties of GA.
2.2. Cytotoxicity evaluation of glycyrrhetin ureas
The effects of glycyrrhetin ureas at the same concentration (20 µM)
on RAW264.7 cells were examined by MTT assay. At this concentration,
the effects of glycyrrhetin ureas on cell viability was not obvious, indi-
cating that the toxicity of synthesized compounds was low (Table 1). It
can be seen from the table that the cell viability percentage of tested
compounds is above 80%. Thus, all compounds at this concentration are
suitable for subsequent biological evaluation.
2.3. Suppression of NO production in LPS-stimulated RAW264.7 cells
Nitric oxide (NO) plays an important role in the immune system and
is involved in the pathogenesis and control of infectious diseases, tu-
mors, chronic degenerative diseases and autoimmune processes [44]. To
evaluate the anti-inflammatory effect of glycyrrhetin ureas, Griess re-
agent was used to detect the level of the LPS-induced NO release in the
Fragment splicing is
a primary strategy in the design and
Fig. 1. The general design strategy in this study.
2

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
Scheme 1. Synthesis of glycyrrhetin urea derivatives 4 and 5.
with acidic or neutral amino acids showed significant NO inhibitory
activity (IC50 = 14.85, 2.04, 2.50, 3.25 and 2.48 µM, respectively), while
compound 5v with basic amino acids had weak NO inhibitory activity
(inhibition rate = 2.6% at 20 µM); (iv) compounds 5j and 5 k with 3- and
4-pyridylmethyl at the R2 position displayed potent anti-inflammatory
activity (IC50 = 14.47 and 3.17 µM, respectively). Compound 5r was
the most potent for further study of the anti-inflammatory mechanism.
Table 1
Effect of glycyrrhetin ureas on the viability of RAW264.7 cells.
Compound
Cell viability (%) a
Compound
Cell viability (%) a
control
GA
4a
100
5j
96.8 ± 14.1
87.4 ± 4.6
90.7 ± 2.5
89.3 ± 3.6
102.8 ± 17.5
85.6 ± 4.2
82.7 ± 9.4
92.8 ± 5.2
81.7 ± 8.6
95.3 ± 5,5
85.8 ± 14.0
87.0 ± 8.5
91.3 ± 2.1
85.6 ± 4.0
93.4 ± 11.4
86.1 ± 8.0
87.3 ± 1.1
83.7 ± 13.1
101.5 ± 9.0
90.3 ± 10.3
97.4 ± 7.4
103.7 ± 2.3
109.6 ± 13.7
97.3 ± 11.2
93.5 ± 3.1
5k
5l
5a
5m
5n
5o
5p
5q
5r
5b
5c
2.4. Compound 5r inhibited TNF-α and IL-6 production
4d
4e
LPS-activated macrophages produce a variety of inflammatory cy-
5e
tokines, including tumor necrosis factor (TNF- ) and interleukins (ILs),
α
5f
5 s
5 t
5u
5v
5 g
5 h
5i
which have a host defense effect under inflammatory conditions and
maintain normal cell conditions [45]. The special plasticity of macro-
phages determines that it may be related to renal fibrosis and renal
tissue repair, so controlling their balance is crucial. To further evaluate
the effects of compound 5r on the pro-inflammatory factors induced by
LPS, in the presence of compound 5r, RAW264.7 cells were incubated
a
The results were showed as means ± SD of at least three independent
experiments.
with LPS (0.5 µg/mL) for 24 h, and the TNF-α and IL-6 levels in the
RAW264.7 cells. LPS treatment caused significant increase of NO release
compared to blank, after pre-treatment with glycyrrhetin ureas at the
supernatant were determined by ELISA. As shown in Fig. 2, compared
with the basal level of cytokines in macrophages in the control group,
concentration of 20 μM, the production of LPS-induced NO could be
LPS stimulation significantly increased the levels of TNF-α and IL-6.
reduced in the supernatant. As shown in Table 2, most of glycyrrhetin
ureas exhibited benign anti-inflammatory activity, amongst them,
compounds 5i, 5 k, 5 l, 5r, 5 s, 5 t and 5u significantly alleviated the
increase of LPS-induced NO release (IC50 = 3.05, 3.17, 3.02, 2.04, 2.50,
After treatment with compound 5r, the production of LPS-induced
TNF-
α and IL-6 was inhibited in a concentration-dependent manner
(Fig. 2). These results indicated that compound 5r reduced the excessive
immune response in RAW 264.7 cells by blocking the increase of pro-
inflammatory cytokines stimulated by LPS. Excessive pro-
3.25 and 2.48
μM, respectively). The structure–activity relationship
(SAR) of NO inhibitory activity of glycyrrhetin urea was followed as
bellow: (i) the NO inhibitory activity decreased when the 3-OH was
acetylated (5a > 4a; 5e > 4e); (ii) for the R2 position of urea unit,
smaller substituent showed better NO inhibitory activity, for example,
when the R2 substituent was the cyclamine, the activity of compound 5i
with the five-member ring is better than those of compounds 5 g, 5 h and
5f with the six-member ring (IC50 values, µM: 5i (cyclopentamine), 3.05
> 5 g (cyclohexane), 16.27; 5 h (morpholine), 17.84; 5f (piperazine-
NMe), 18.24); when the R2 substituent was the aliphatic amine, the
activity of compounds with methyl was more potent (IC50 values, µM: 5 l
(-NHMe), 3.02 > 5p (-NMe2), 9.15 ≫ 5 m (-NHEt), 1.3%; 5n (-NHPr-i),
13.9%; 5o (-NHBu-n), 19.0%); (iii) compounds 5a, 5r, 5 s, 5 t and 5u
inflammatory factors such as TNF-
α and IL-6 led to severe systemic
diseases and high mortality [46], while compound 5r showed fine
inhibitory activity.
2.5. In vivo activity evaluation of AKI mice
2.5.1. Assessment of the Kidney/Body mass index and renal function
All mice were weighed before anesthesia, and after euthanasia, the
kidneys were surgically removed and weighed. The kidney/body mass
index is calculated as follows: kidney weight/body weight × 100% [47].
Compared with the blank control group, cisplatin caused kidney tissue
3

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
injury and the kidney/body mass index increased significantly. How-
ever, compound 5r obviously reduced the index value (Fig. 3).
Table 2
The inhibitory effects of glycyrrhetic ureas on NO production in LPS-stimulated
RAW 264.7 cells.
Clinical data have shown that about one-third of patients develop
AKI after cisplatin treatment, accompanied by decreased glomerular
filtration rate, increased blood urea nitrogen (BUN) and serum creati-
nine (Scr), and electrolyte imbalance [48,49]. Urea and creatinine are
common indicators of renal function tests, reflecting the degree of
impaired glomerular filtration function and basal metabolism. As diag-
nostic criteria for nephrotoxicity, urea and creatinine can reflect the late
manifestations of the disease, but early intervention is essential for
effective treatment of AKI [50]. Serum markers of renal function were
determined with BUN and Scr assay kits, according to the manufac-
turer’s instructions. Cisplatin-treated group significantly increased Scr
and BUN levels compared with normal group. Renoprotective effect of
compound 5r (30 mg/kg) was further confirmed by detecting Scr and
BUN which indicated renal function (Fig. 4C and 4D).
Compound
R1
R2
IC50
(μ
M) a,b or
Inhibition rate % a,b,c
GA
4a
5a
5b
5c
4d
4e
5e
5f
12.5 ± 2.8%
18.7 ± 3.1%
14.85 ± 1.92
33.5 ± 2.1%
na d
–
–
–
–
–
–
–
CH3CO
NHCH2COOH
NHCH2COOH
NHCH2COOCH3
NH2
2.5.2. Pro-inflammatory mediators’ levels in serum
H
The release of TNF-α induced other inflammatory cytokines and led
H
to an inflammatory response [51,52]. These inflammatory mediators
further caused damage to renal tubular cells and renal tissues, so, the
activity of compound 5r to suppress inflammatory levels in cisplatin-
induced mice was evaluated. In model group, there was marked in-
H
CH3CO
CH3CO
H
NHOH
12.03 ± 2.70
na
NHCH2CH2OH
NHCH2CH2OH
34.8 ± 3.5%
18.24 ± 1.75
H
crease of TNF-α and IL-6 after administration of cisplatin (20 mg/kg),
compared with normal group (Fig. 4A and 4B). On the other hand,
pretreatment and treatment with compound 5r (30 mg/kg) antagonized
5g
5h
H
H
16.27 ± 3.94
17.84 ± 2.83
cisplatin-induced elevation of TNF-α and IL-6 compared with cisplatin-
treated group (Fig. 4A and 4B). Compound 5r reduced proin-
flammatory response during cisplatin nephrotoxicity and the ensuing
acute kidney injury and renal failure, through decreasing the levels of
5i
5j
H
H
3.05 ± 0.66
inflammatory mediators TNF-α and IL-6.
14.47 ± 3.84
2.5.3. Histopathological Analysis.
Tubular injury is an important feature of AKI, which may eventually
lead to tubulointerstitial fibrosis [53,54]. We assessed the treatment
effect of compound 5r in cisplatin nephropathy. Kidney tissue was fixed
in 10% formalin and embedded in paraffin. Cut sections with a thickness
5k
H
3.17 ± 1.12
of 5 μm and stain with H&E and PAS, and then take a photo with an
–
–
–
–
–
–
5l
H
H
H
H
H
H
H
NHCH3
3.02 ± 0.92
1.3 ± 0.7%
13.9 ± 2.6%
19.0 ± 3.1%
9.15 ± 1.80
13.10 ± 1.69
2.04 ± 1.47
optical microscope. On examination of kidney section, cisplatin caused
necrosis of renal tubular epithelial cells and degenerative changed in the
cortical and corticomedullary portions. As shown in Fig. 5, in the blank
control group, the photograph clearly showed the normal shape of the
glomerulus and proximal tubules. In cisplatin-induced AKI mice, renal
tissues were displayed severe pathological damage, leading to tubular
cell damage, inflammatory cell infiltration, and tubular dilatation.
However, after treatment with compound 5r, the nephrotoxicity caused
by cisplatin in C57BL/6 mice was significantly alleviated.
5m
5n
5o
5p
5q
5r
NHCH2CH3
NHCH(CH3)2
NH(CH2)3CH3
N(CH3)2
NH(CH2)2N(CH3)2
5s
5t
H
H
2.50 ± 1.15
3.25 ± 0.52
3. Conclusions
Cisplatin treatment increases expression of inflammatory cytokines
and chemokines in the kidney, and the inflammatory response acceler-
ates the development of renal tissue damage and renal failure. To dis-
covery better anti-inflammatory agents, a series of glycyrrhetic ureas 4a,
4d, 4e and 5a–5v were designed and synthesized. Most of compounds
had anti-inflammatory activity, amongst them, compounds 5i, 5 k, 5 l,
5r, 5 s, 5 t and 5u exhibited significant anti-inflammatory activity (IC50
5u
5v
H
H
2.48 ± 1.33
2.6 ± 1.4%
= 3.05, 3.17, 3.02, 2.04, 2.50, 3.25 and 2.48 μM, respectively). SAR
a
analysis showed: (i) when the 3-OH was acetylated, the NO inhibition
decreased; (ii) for whether the cyclamine or the aliphatic amine at the
R2 position, smaller substituent showed better NO inhibition; (iii) the
acidic or neutral amino acid at the R2 position significantly increased the
NO inhibition, so did the pyridylmethyl. Compound 5r had the most
potent anti-inflammatory activity and selected to further study the ac-
tion mechanism. Compound 5r also reduced the level of proin-
The results were showed as means ± SD of at least three independent
experiments.
b
c
Griess reagent was detected NO production.
The percentages of NO inhibition were measured at the concentration of 20
μ
M.
d
na = no activity at the concentration of 20 μM.
flammatory factors (TNF-α and IL-6) in a dose-dependent manner. In
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H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
Fig. 2. Compound 5r inhibited the cytokine production of RAW264.7 cells stimulated by LPS. RAW264.7 cells were pretreated with compound 5r , at concentrations
of 20, 10, 5
μ
M for 1 h, then incubated with LPS (0.5
μ
g/mL) for 24 h. The levels of TNF-
α
and IL-6 in the culture medium were measured by ELISA kits. ***p <
0.001vs control group, ##p < 0.01, #p < 0.05 vs model group.
4.2. The synthesis of glycyrrhetic isocyanate 3
This compound was prepared as previously reported [33]: A solution
of glycyrrhetic acid (2.5 g, 5.3 mmol) in pyridine (10 mL) and 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 2. (white power, 2.3 g, 85%)
Thionyl chloride (0.58 mL, 8.0 mmol) was added to a solution of 2
(1.0 g, 2.0 mmol) in CHCl3 (60 mL), and the mixture was heated to reflux
for 2 h. The reaction solution was evaporated in vacuum, and the residue
triturated using diethyl ether. The precipitate was filtered and dried in
vacuo to yield acyl chloride of 3-acetyl glycyrrhetinic acid, which was
dissolved in acetone (200 mL).
Sodium azide (0.39 g, 6.0 mmol) in water (50 mL) was dropwise
added to the solution of acyl chloride of 3-acetyl glycyrrhetinic acid at
0–5 ◦C over 30 min. Then the reaction mixture was stirred at 0–5 ◦C for
1 h and twice extracted with methylbenzene. The combined organic
phase was washed three times with water, then dried over anhydrous
Na2SO4 and filtered. The filtrates were heated to 75 ◦C and stirred for 6 h
at the same temperature. The reaction solution was evaporated in vac-
uum, and the residue was recrystallized from EtOAc to obtain isocyanate
3. White powder, 67%; 13C NMR (100 MHz, CDCl3) δ 200.0 (C-11),
171.2 (CH3CO), 168.4 (C-13), 128.8 (C-12), 122.3 (NCO), 80.8 (C-3),
61.9 (C-9), 58.8 (C-20), 55.2 (C-5), 47.4 (C-18), 45.6 (C-19), 44.7 (C-
14), 43.4 (C-8), 39.0 (C-4), 38.2 (C-1), 37.2 (C-10), 36.4 (C-16), 35.0 (C-
17), 32.9 (C-7), 32.1 (C-22), 32.0 (C-29), 28.4 (C-23), 28.2 (C-28), 26.5
(C-21), 26.4 (C-2), 23.7 (C-15), 23.5 (C-27), 21.5 (CH3CO), 18.9 (C-25),
17.6 (C-6), 16.9 (C-24), 16.6 (C-26).
Fig. 3. The effect of compound 5r on kidney/body mass index (%). The mice
and kidney tissues of each group were weighed and recorded.
cisplatin-induced AKI mice, compound 5r not only lowered the levels of
serum pro-inflammatory factors, but also improved the renal function
markers including serum creatinine and urea nitrogen. Pathologically,
compound 5r reduced inflammatory cell infiltration, relieved renal
tubular cell damage and tubular dilatation.
4. Experimental section
4.1. Chemistry
4.3. General procedures for converting the glycyrrhetic isocyanate to urea
All reagents were purchased from commercial sources and were used
without further purification. Melting points (uncorrected) were deter-
mined on a XT4MP apparatus (Taike Corp., Beijing, China). 1H NMR and
13C NMR spectra were recorded on Bruker AV-500 or AV-400 MHz in-
struments in DMSO‑d6 or CDCl3. Chemical shifts are reported in parts
per million (δ) downfield from the signal of tetramethylsilane (TMS) as
internal standards. Coupling constants are reported in Hz. The multi-
plicity 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 chroma-
tography (CC and TLC, resp.) were performed on silica gel (200–300
mesh) and silica gel GF254 (Qingdao Marine Chemical Factory)
respectively.
for compounds 4 and 5.
Method A: To the solution of the isocyanate 3 (1.0 mmol) in EtOAc
(60 mL), the substituted amine (1.2 mmol) was added and the reaction
mixture was stirred at room temperature. After 2 h, water (50 mL) was
added to the reaction mixture and the organic phase was separated. The
aqueous layer was twice extracted with EtOAc (2 × 30 mL). The com-
bined organic phase was washed twice with water (2 × 30 mL), dried
over anhydrous Na2SO4, filtered and concentrated under the reduced
pressure. The residue 4 was added methanol (30 mL) and NaOH (20
mmol), and the reaction mixture was stirred at room temperature for 12
h. Water (50 mL) was added and the pH was adjusted to 3 using diluted
HCl. The reaction mixture was twice extracted with EtOAc (2 × 60 mL).
The combined organic phase was washed twice with water (2 × 30 mL),
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H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
Fig. 4. The effect of compound 5r on cisplatin-induced AKI mice. After pre-treatment with compound 5r (30 mg/kg), the mice were injected intraperitoneally with
cisplatin (20 mg/kg) and sacrificed 72 h later. Detection of kidney function markers (BUN and Scr) and pro-inflammatory factors (TNF-α and IL-6) levels in serum.
***p < 0.001 compared with control group; ###p < 0.001 compared with model group.
Fig. 5. Effect of compound 5r on cisplatin-induced AKI in C57BL/6 mice. Kidney tissue was stained by H&E and PAS for histological examination.
dried over anhydrous Na2SO4, filtered and concentrated under the
reduced pressure. The residue was purified by flash chromatography
(SiO2, MeOH-CH2Cl2, gradient elution) to give the urea product 5.
Method B: The solution of the substituted amine hydrochloride or
amino acid (1.2 mmol) in water (30 mL) and methanol (30 mL) was
alkalified with diluted NaOH to pH 11–12. The solution of the isocya-
nate 3 (1.0 mmol) in EtOAc (60 mL) was added to the reaction mixture
and stirred for 2 h at room temperature. The reaction mixture was
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H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
acidified with diluted HCl to pH 3 and twice extracted with EtOAc (60
mL). The combined organic layer was washed twice with water (2 × 30
mL), dried over anhydrous Na2SO4, filtered and concentrated under the
reduced pressure. The residue 4 was added methanol (30 mL) and NaOH
(20 mmol), and the reaction mixture was stirred at room temperature for
12 h. Water (50 mL) was added and the pH was adjusted to 3 using
diluted HCl. The reaction mixture was twice extracted with EtOAc (2 ×
60 mL). The combined organic phase was washed twice with water (2 ×
30 mL), dried over anhydrous Na2SO4, filtered and concentrated under
the reduced pressure. The residue was purified by flash chromatography
(SiO2, MeOH-CH2Cl2, gradient elution) to give the urea product 5.
557.4400; found: 557.4404.
4.3.4. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)urea (5c)
Mehtod A; white powder, 80%; m.p. 228–230 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 5.60 (s, 1H, CONH), 5.46 (s, 1H, 12-H), 5.21 (s, 2H, NH2),
4.30 (d, J = 5.2 Hz, 1H, OH), 3.01 (m, 1H, 3-H), 2.58 (m, 1H, 9-H), 2.30
(s, 1H, 18-H), 2.10 (m, 2H), 1.87–1.36 (m, 14H), 1.32 (s, 3H, 29-H), 1.19
(s, 3H, 27-H), 1.14 (m, 2H), 1.04, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.94
(m, 2H), 0.90 (s, 3H, 25-H), 0.80 (s, 3H, 28-H), 0.69 (s, 1H), 0.68 (s, 3H,
26-H); 13C NMR (126 MHz, DMSO‑d6) δ 199.0 (C-11), 170.0 (C-13),
158.1 (C-30), 127.1 (C-12), 76.5 (C-3), 61.1 (C-9), 54.1 (C-20), 51.1 (C-
5), 46.0 (C-18), 44.8 (C-19), 42.9 (C-14), 41.9 (C-8), 38.8 (C-4), 38.5 (C-
1), 36.6 (C-10), 35.4 (C-16), 32.1 (C-17), 31.5 (C-7), 31.4 (C-22), 28.3
(C-29), 28.3 (C-23), 28.1 (C-28), 26.9 (C-21), 25.8 (C-15), 22.9 (C-2),
18.4 (C-27), 17.1 (C-25), 17.0 (C-6), 16.2 (C-26), 16.0 (C-24). TOF-
HRMS: m/z [M + H]+ calcd for C30H49N2O3: 485.4148; found:
485.4149.
4.3.1. 1-((3β,20β)-3-acetoxyl-11-oxo-olean-12-en-20-yl)-3-(acetic acid-
2-yl)urea (4a)
Method B; white powder, 72%; m.p. 213–215 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.90 (t, J = 5.7 Hz, 1H, CH2NH), 5.85 (s, 1H, NH), 5.50 (s,
1H, 12-H), 4.42 (dd, J = 11.8, 4.5 Hz, 1H, 3-H), 3.71 (dd, J = 18.0, 4.9
Hz, 1H, NCH2-Ha), 3.63 (dd, J = 18.0, 4.5 Hz, 1H, NCH2-Hb), 2.63 (m,
1H, 9-H), 2.38 (s, 1H, 18-H), 2.20–2.08 (m, 2H), 2.00 (s, 3H, CH3CO),
1.88–1.35 (m, 12H), 1.34 (s, 3H, 29-H), 1.20 (s, 3H, 27-H), 1.13 (m, 3H),
1.06, 1.04 (2 × s, 2 × 3H, 23-H, 24-H), 0.90 (m, 2H), 0.82 (s, 6H, 25-H,
28-H), 0.79 (s, 3H, 26-H); 13C NMR (100 MHz, DMSO‑d6) δ 199.1 (C-11),
172.7 (COOH), 170.3 (C-13), 170.2 (CH3CO), 157.2 (C-30), 127.1 (C-
12), 79.7 (C-3), 60.8 (C-9), 53.7 (C-20), 51.4 (C-5), 45.9 (C-18), 44.9 (C-
19), 43.0 (NCH2), 42.1 (C-14), 41.4 (C-8), 37.9 (C-4), 37.6 (C-1), 36.5
(C-10), 35.3 (C-16), 31.9 (C-17), 31.5 (C-7), 31.3 (C-22), 28.4 (C-29),
28.3 (C-23), 27.7 (C-28), 26.1 (C-21), 25.8 (C-2), 23.2 (C-15), 23.0 (C-
27), 21.0 (CH3CO), 18.4 (C-25), 17.0 (C-6), 16.6 (C-24), 16.2 (C-26).
TOF-HRMS: m/z [M + H]+ calcd for C34H53N2O6: 585.4341; found:
585.4340.
4.3.5. 1-((3β,20β)-3-acetoxyl-11-oxo-olean-12-en-20-yl)-3-hydroxylurea
(4d)
Method B; white powder, 67%; m.p. 231–233 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 8.63 (s, 1H, NHOH), 8.17 (s, 1H, NHOH), 5.82 (s, 1H,
CONH), 5.47 (s, 1H, 12-H), 4.30 (dd, J = 11.8, 4.5 Hz, 1H, 3-H), 2.63 (m,
1H, 9-H), 2.39 (s, 1H, 18-H), 2.05 (m, 3H), 2.00 (s, 3H, COCH3),
1.92–1.14 (m, 10H), 1.35 (s, 3H, 29-H), 1.23 (m, 5H, overlap 27-H), 1.14
(m, 2H), 1.06, 1.04 (2 × s, 2 × 3H, 23-H, 24-H), 0.92 (m, 2H), 0.82 (s,
6H, 25-H, 28-H), 0.80 (s, 3H, 26-H). TOF-HRMS: m/z [M + H]+ calcd for
C32H51N2O5: 543.4326; found: 543.4324.
4.3.6. 1-((3β,20β)-3-acetoxyl-11-oxo-olean-12-en-20-yl)-3-(2-
hydroxyethyl)urea (4e)
4.3.2. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(acetic acid-
2-yl)urea (5a)
Method A; white powder, 77%; m.p. 224–227 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.76 (t, J = 5.6 Hz, 1H, NHCH2), 5.65 (s, 1H, 12-H), 5.48 (s,
1H, CONH), 4.70 (t, J = 5.0 Hz, 1H, 3-H), 4.42 (dd, J = 11.8, 4.5 Hz, 1H,
OH), 3.37 (m, 2H, CH2OH), 3.02 (m, 2H, NHCH2), 2.63 (m, 1H, 9-H),
2.39 (s, 1H, 18-H), 2.10 (m, 1H), 2.00 (s, 3H, OCH3), 1.90 (d, J =
13.2 Hz, 1H), 1.76–1.36 (m, 10H), 1.34 (s, 3H, 29-H), 1.20 (s, 3H, 27-H),
1.06, 1.04 (2 × s, 2 × 3H, 23-H, 24-H), 0.90 (m, 2H), 0.82 (s, 6H, 25-H,
28-H), 0.79 (s, 3H, 26-H). TOF-HRMS: m/z [M + H]+ calcd for
Method B; white powder, 65%; m.p. 221–223 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.91 (t, J = 5.6 Hz, 1H, NHCH2), 5.85 (s, 1H, NH), 5.49 (s,
1H, 12-H), 4.33 (brs, 1H, OH), 3.70 (dd, J = 17.9, 4.8 Hz, 1H, NCH2-Ha),
3.62 (dd, J = 18.0, 4.5 Hz, 1H, NCH2-Hb), 3.01 (dd, J = 17.2, 4.4 Hz, 1H,
3-H), 2.57 (m, 1H, 9-H), 2.30 (s, 1H, 18-H), 2.19–1.33 (m, 14H), 1.32 (s,
3H, 29-H), 1.20 (s, 3H, 27-H), 1.14 (m, 2H), 1.03, 1.02 (2 × s, 2 × 3H,
23-H, 24-H), 0.93 (m, 2H), 0.90 (s, 3H, 25-H), 0.79 (s, 3H, 28-H), 0.69 (,
1H), 0.68 (s, 3H, 26-H); 13C NMR (100 MHz, DMSO‑d6) δ 199.2 (C-11),
172.7 (COOH), 170.1 (C-13), 157.2 (C-30), 127.2 (C-12), 77.6 (C-3),
61.2 (C-9), 54.1 (C-20), 51.4 (C-5), 45.9 (C-18), 44.9 (C-19), 43.0
(NCH2), 42.1 (C-14), 41.4 (C-8), 38.8 (C-4), 38.6 (C-1), 36.7 (C-10), 35.4
(C-16), 32.1 (C-17), 31.5 (C-7), 31.3 (C-22), 28.4 (C-29), 28.3 (C-23),
28.2 (C-28), 27.0 (C-21), 26.1 (C-15), 25.6 (C-2), 23.0 (C-27), 18.4 (C-
25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-24). TOF-HRMS: m/z [M + H]+
calcd for C32H51N2O5: 543.4228; found: 543.4230.
C34H55N2O5: 571.4322; found: 571.4318.
4.3.7. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(2-
hydroxyethyl)urea (5e)
Method A; white powder, 66%; m.p. 229–230 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.76 (t, J = 5.5 Hz, 1H, CH2NH), 5.65 (s, 1H, 12-H), 5.47 (s,
1H, NH), 4.70 (t, J = 5.0 Hz, 1H,CH2OH), 4.32 (d, J = 5.1 Hz, 1H, OH),
3.37 (m, 2H, CH2OH), 3.01 (m, 3H, 3-H), 2.57 (d, J = 13.3 Hz, 1H, 9-H),
2.30 (s, 1H, 18-H), 2.15 (m, 2H, NHCH2), 1.89 (d, J = 12.7 Hz, 3H),
1.79–1.35 (m, 11H), 1.31 (d, J = 13.8 Hz, 3H, 29-H), 1.20 (s, 3H), 1.15
(m, 2H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.94 (s, 2H), 0.90 (s, 3H,
25-H), 0.78 (s, 3H, 28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR
(101 MHz, DMSO) δ 199.1 (C-11), 170.1 (C-13), 157.5 (C-30), 127.1 (C-
12), 76.6 (C-3), 61.1 (C-9), 60.9 (CH2OH), 54.1 (C-20), 51.2 (C-5), 45.9
(C-18), 44.9 (C-19), 42.9 (C-14), 42.4 (C-8), 41.7 (C-4), 38.8 (C-1), 38.5
(C-10), 36.7 (C-16), 35.4 (C-17), 32.1 (NCH2), 31.5 (C-7), 31.2 (C-22),
28.4 (C-29), 28.3 (C-23), 28.1 (C-28), 27.0 (C-21), 26.0 (C-15), 25.8 (C-
2), 23.0 (C-27), 18.36 (C-25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-24). TOF-
HRMS: m/z [M + H]+ calcd for C32H53N2O4: 529.4312; found:
529.4315.
4.3.3. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(methyl
acetate-2-yl)urea (5b)
Method B; white powder, 51%; m.p. 226–228 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 6.00 (t, J = 6.1 Hz, 1H, CH2NH), 5.87 (s, 1H, NH), 5.49 (s,
1H, 12-H), 4.32 (d, J = 5.2 Hz, 1H, OH), 3.78 (dd, J = 17.8, 6.2 Hz, 1H,
NCH2-Ha), 3.71 (dd, J = 17.8, 5.9 Hz, 1H, NCH2-Hb), 3.62 (s, 3H,
OCH3), 3.01 (m, 2H, 3-H), 2.58 (m, 1H, 9-H), 2.30 (s, 1H, 18-H), 2.13
(m, 2H), 1.84–1.34 (m, 12H), 1.32 (s, 3H, 29-H), 1.19 (s, 3H, 27-H), 1.14
(m, 2H), 1.04, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.95 (m, 2H), 0.90 (s,
3H, 25-H), 0.79 (s, 3H, 28-H), 0.68 (s, 3H, 26-H), 0.68 (m, 1H); 13C NMR
(100 MHz, DMSO‑d6) δ 199.2 (C-11), 171.9 (COO), 170.1 (C-13), 157.2
(C-30), 127.2 (C-12), 76.6 (C-3), 61.1 (C-9), 54.1 (C-20), 51.5 (C-5),
51.4 (OCH3), 45.9 (C-18), 44.9 (C-19), 42.9 (NCH2), 41.8 (C-14), 41.2
(C-8), 38.8 (C-4), 38.5 (C-1), 36.7 (C-10), 35.3 (C-16), 32.1 (C-17), 31.5
(C-7), 31.4 (C-22), 28.4 (C-29), 28.3 (C-23), 28.2 (C-28), 27.0 (C-21),
26.1 (C-15), 25.8 (C-2), 23.0 (C-27), 18.4 (C-25), 17.2 (C-6), 16.2 (C-
26), 16.0 (C-24). TOF-HRMS: m/z [M + H]+ calcd for for C33H53N2O5:
4.3.8. N-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)piperazidine-1-
carboxamide (5f)
Mehtod A; white powder, 72%; m.p. 169–171 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.64 (s, 1H, CONH), 5.60 (s, 1H, 12-H), 4.32 (d, J = 5.1 Hz,
1H, OH), 3.27 (t, J = 5.0 Hz, 4H, piperazine), 3.01 (m, 2H, 3-H), 2.58
(m, 1H, 9-H), 2.30 (s, 1H, 18-H), 2.23 (t, J = 5.0 Hz, 4H, piperazine),
7

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
2.15 (s, 3H, NCH3), 2.12–1.90 (m, 15H), 1.87–1.22 (m, 12H, overlap
1.31, s, 3H, 29-H), 1.20 (s, 3H, 27-H), 1.17–1.06 (m, 3H), 1.03, 1.02 (2
× s, 2 × 3H, 23-H, 24-H), 0.95 (m, 2H), 0.90 (s, 3H, 25-H), 0.78 (s, 3H,
28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR (101 MHz, DMSO‑d6) δ
199.2 (C-11), 169.9 (C-13), 157.0 (C-30), 127.4 (C-12), 76.6 (C-3), 61.1
(C-9), 54.7 (2C, piperazine), 54.1 (C-20), 52.1 (C-5), 46.0 (C-18), 45.9
(2C, piperazine), 44.8 (C-19), 44.1 (NCH3), 43.0 (C-14), 41.7 (C-8), 38.8
(C-4), 38.6 (C-1), 36.7 (C-10), 35.7 (C-16), 32.1 (C-17), 31.4 (C-7), 30.8
(C-22), 28.6 (C-29), 28.3 (C-23), 28.1 (C-28), 26.9 (C-21), 26.1 (C-15),
26.0 (C-2), 22.9 (C-27), 18.3 (C-25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-
24). TOF-HRMS: m/z [M + H]+ calcd for C35H58N3O3: 568.4432; found:
568.4428.
4.3.12. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(3-
pyridylmethyl)urea (5j)
Method A; white powder, 70%; m.p. 227–229 ◦C; 1HNMR (400 MHz,
DMSO‑d6) δ 8.45 (dd, J = 2.3, 0.9 Hz, 1H), 8.43 (dd, J = 4.8, 1.7 Hz, 1H),
7.64 (m, 1H), 7.34 (m, 1H), 6.23 (t, J = 6.1 Hz, 1H, NHCH2), 5.69 (s, 1H,
NH), 5.42 (s, 1H, 12-H), 4.36 (d, J = 5.1 Hz, 1H, OH), 4.20 (m, 2H,
CH2NH), 3.00 (m, 2H, 3-H), 2.56 (m, 1H, 9-H), 2.30 (s, 1H, 18-H), 2.08
(m, 2H), 1.86–1.35 (m, 12H), 1.31 (s, 3H, 29-H), 1.22 (s, 3H, 27-H), 1.14
(m, 2H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (s, 2H), 0.89 (s, 3H,
25-H), 0.78 (s, 3H, 28-H), 0.69 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR
(101 MHz, DMSO‑d6) δ 199.3 (C-11), 170.1 (C-13), 157.4 (C-30), 148.5
(pyridine), 147.9 (pyridine), 136.7 (pyridine), 134.8 (pyridine), 127.2
(C-12), 123.5 (pyridine), 76.7 (C-3), 61.2 (C-9), 54.2 (C-20), 51.6 (C-5),
46.2 (C-18), 45.0 (C-19), 43.1 (NCH2), 41.9 (C-14), 40.4 (C-8), 40.1 (C-
4), 38.9 (C-1), 38.6 (C-10), 36.7 (C-16), 35.5 (C-17), 32.2 (C-7), 31.6 (C-
22), 28.5 (C-29), 28.4 (C-23), 28.2 (C-28), 27.0 (C-21), 26.1 (C-15), 25.9
(C-2), 23.0 (C-27), 18.5 (C-25), 17.3 (C-6), 16.3 (C-26), 16.1 (C-24).
TOF-HRMS: m/z [M + H]+ calcd for C36H54N3O3: 576.4540; found:
576.4538.
4.3.9. N-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)piperidine-1-
carboxamide (5 g)
Method A; white powder, 78%; m.p. 157–159 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.57 (s, 1H, CONH), 5.56 (s, 1H, 12-H), 4.37 (brs, 1H, OH),
3.24 (m, 4H, piperidine), 3.01 (m, 1H, 3-H), 2.58 (m, 1H, 9-H), 2.31 (s,
1H, 18-H), 2.10 (m, 4H), 1.76–1.34 (m, 16H), 1.32 (s, 3H, 29-H), 1.19 (s,
3H, 27-H), 1.15 (m, 2H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.94 (s,
2H), 0.90 (s, 3H, 25-H), 0.78 (s, 3H, 28-H), 0.70 (m, 1H), 0.68 (s, 3H, 26-
H); 13C NMR (101 MHz, DMSO‑d6) δ 199.4 (C-11), 170.2 (C-13), 157.1
(C-30), 127.4 (C-12), 76.7 (C-3), 61.2 (C-9), 54.2 (C-20), 52.1 (C-5),
46.3 (2C, piperidine), 45.3 (C-18), 44.9 (C-19), 43.1 (C-14), 41.6 (C-8),
38.9 (C-4), 38.7 (C-1), 36.8 (C-10), 35.9 (C-16), 32.3 (C-17), 31.5 (C-7),
31.2 (C-22), 28.7 (C-29), 28.5 (C-23), 28.2 (C-28), 27.0 (C-21), 26.2 (C-
15), 26.1 (C-2), 25.6 (2C, piperidine), 24.4 (piperidine), 23.0 (C-27),
18.4 (C-25), 17.3 (C-6), 16.3 (C-26), 16.1 (C-24). TOF-HRMS: m/z [M +
H]+ calcd for C35H57N2O3: 553.4849; found: 553.4848.
4.3.13. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(4-
pyridylmethyl)urea (5 k)
Method A; white powder, 71%; m.p. 228–230 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 8.48 (m, 1H), 7.23 (d, J = 5.8 Hz, 2H), 6.28 (t, J = 6.1 Hz,
1H, NHCH2), 5.76 (s, 1H, 12-H), 5.45 (s, 1H, CONH), 4.32 (d, J = 5.1 Hz,
1H, OH), 4.20 (m, 1H), 3.00 (m, 1H, 3-H), 2.58 (m, 1H, 9-H), 2.31 (s, 1H,
18-H), 2.11 (m, 2H), 1.88–1.35 (m, 18H), 1.32 (s, 3H, 27-H), 1.23 (s,
3H), 1.15 (m, 3H), 1.04, 1.03 (2 × s, 2 × 3H, 23-H, 24-H), 0.92 (m, 2H),
0.90 (s, 3H, 25-H), 0.80 (s, 3H, 28-H), 0.70 (m, 1H), 0.68 (s, 3H, 26-H);
13C NMR (101 MHz, DMSO) δ 199.1 (C-11), 170.0 (C-13), 157.3 (C-30),
150.5 (pyridine), 149.4 (2C, pyridine), 127.1 (C-12), 121.8 (2C, pyri-
dine), 76.6 (C-3), 61.1 (C-9), 54.1 (C-20), 51.5 (C-5), 46.2 (C-18), 44.9
(C-19), 43.0 (NCH2), 41.7 (C-14), 41.6 (C-8), 38.8 (C-4), 38.5 (C-1), 36.6
(C-10), 35.4 (C-16), 32.1 (C-17), 31.6 (C-27), 31.5 (C-22), 28.4 (C-29),
28.3 (C-23), 28.2 (C-28), 27.0 (C-21), 26.1 (C-15), 25.8 (C-2), 22.9 (C-
27), 18.4 (C-25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-24). TOF-HRMS: m/z
[M + H]+ calcd for C36H54N3O3: 576.4606; found: 576.4608.
4.3.10. N-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)morpholine-4-
carboxamide (5 h)
Method A; white powder, 80%; m.p. 226–228 ◦C; 1HNMR (400 MHz,
DMSO‑d6) δ 5.69 (s, 1H, CONH), 5.59 (s, 1H,12-H), 4.36 (brs ,1H, OH),
3.55–3.52 (m, 4H, morpholine), 3.22 (dd, J = 5.8, 3.9 Hz, 4H, mor-
pholine), 3.01 (m, 1H, 3-H), 2.58 (m, 1H, 9-H), 2.30 (s, 1H,18-H), 2.07
(m, 2H), 1.81–1.34 (m, 12H), 1.31 (s, 3H, 29-H), 1.20 (s, 3H, 29-H), 1.14
(m, 2H), 1.02, 1.01 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (m, 2H), 0.89 (s,
3H, 25-H), 0.77 (s, 3H, 28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR
(101 MHz, DMSO‑d6) δ 199.4 (C-11), 170.1 (C-13), 157.4 (C-30), 127.5
(C-12), 76.7 (C-3), 66.2 (2C, morpholine), 61.2 (C-9), 54.2 (C-20), 52.2
(C-5), 46.1 (C-18), 44.9 (C-19), 44.8 (2C, morpholine), 43.1 (C-14), 41.8
(C-8), 38.9 (C-4), 38.7 (C-1), 36.8 (C-10), 35.8 (C-16), 32.2 (C-17), 31.5
(C-7), 30.8 (C-22), 28.7 (C-29), 28.3 (C-23), 28.2 (C-28), 27.0 (C-21),
26.1 (C-15), 26.1 (C-2), 23.0 (C-27), 18.4 (C-25), 17.3 (C-6), 16.3 (C-
26), 16.1 (C-24). TOF-HRMS: m/z [M + H]+ calcd for C34H55N2O4:
555.4156; found: 555.4154.
4.3.14. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-methylurea
(5 l)
Method B; white powder, 75%; m.p. 187–189 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 6.25 (t, J = 4.0 Hz, 1H, CH3NH), 5.94 (s, 1H, NH), 5.59 (s,
1H, 12-H), 5.82 (brs, 1H, OH), 3.50 (dd, J = 11.3, 4.0 Hz, 1H, 3-H), 3.18
(m, 1H, 9-H), 2.88 (d, J = 4.6 Hz, 3H, NCH3), 2.47 (s, 1H, 18-H), 2.35
(m, 2H), 2.12–1.62 (m, 8H), 1.60 (s, 3H, 29-H), 1.47 (m, 2H), 1.33 (s,
3H, 27-H), 1.23 (m, 3H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 1.06 (m,
2H), 1.11 (s, 3H, 25-H), 1.09 (s, 3H, 28-H), 0.88 (m, 2H), 0.83 (s, 3H, 26-
H); 13C NMR (100 MHz, DMSO‑d6) δ 200.0 (C-11), 170.0 (C-13), 159.8
(C-30), 128.8 (C-12), 78.3 (C-3), 62.6 (C-9), 55.7 (C-20), 52.9 (C-5),
47.5 (C-18), 46.0 (C-19), 44.0 (C-14), 43.6 (C-10), 40.2 (C-8, C-4), 38.1
(C-1), 36.7 (C-16), 33.4 (C-17), 32.7 (C-7), 32.5 (NCH3), 28.6 (C-22),
29.6 (C-29), 29.2 (C-23), 29.1 (C-28), 27.3 (C-21), 27.1 (C-15), 27.0 (C-
2), 23.8 (C-27), 19.3 (C-25), 18.4 (C-6), 17.3 (C-26), 17.1 (C-24). TOF-
HRMS: m/z [M + H]+ calcd for C31H51N2O3: 449.3894; found:
449.3899.
4.3.11. N-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)pyrrolidine-1-
carboxamide (5i)
Method A; white powder, 77%; m.p. 220–222 ◦C; 1HNMR (400 MHz,
DMSO‑d6) δ 5.57 (s, 1H, CONH), 4.95 (s, 1H, 12-H), 4.36 (d, J = 5.2 Hz,
1H, OH), 3.20 (m, 4H, pyrrolidine), 3.01 (m, 1H, 3-H), 2.58 (m, 1H, 9-
H), 2.30 (s, 1H, 18-H), 2.07 (m, 4H, pyrrolidine), 1.76 (m, 5H),
1.63–1.35 (m, 12H), 1.31 (s, 3H, 29-H) 1.20 (s, 3H, 27-H), 1.15 (m, 2H),
1.02,1.01 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (m, 2H), 0.89 (s, 3H, 25-H),
0.78 (s, 3H, 28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR (101 MHz,
DMSO‑d6) δ 199.4 (C-11), 170.1 (C-13), 157.4 (C-30), 127.5 (C-12), 76.7
(C-3), 61.2 (C-9), 54.2 (C-20), 52.2 (C-5) 46.1 (C-18), 44.9 (C-19), 44.8
(2C, pyrrolidine), 43.1 (C-14), 41.8 (C-8), 38.9 (C-4), 38.7 (C-1), 36.8
(C-10), 35.8 (C-16), 32.2 (C-17), 31.5 (C-7, C-22), 30.8 (C-29), 28.7 (C-
23), 28.3 (C-28), 28.2 (C-21), 27.0 (C-15), 26.1 (C-2), 26.0 (2C, pyrro-
lidine), 23.0 (C-27), 18.4 (C-25), 17.3 (C-6), 16.3 (C-26), 16.1 (C-24).
4.3.15. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-ethylurea
(5 m)
Mehtod B; white powder, 81%; m.p. 220–223 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.60 (t, J = 5.5 Hz, 1H, NHCH2), 5.45 (s, 2H, CONH, 12-H),
4.37 (d, J = 5.2 Hz, 1H, OH), 2.98 (m, 4H, 3-H), 2.56 (m, 1H, 9-H), 2.30
(s, 1H, 18-H), 2.08 (m, 2H), 1.92–1.33 (m, 11H), 1.31 (s, 3H, 29-H) 1.19
(s, 5H), 1.03, 1.01 (2 × s, 2 × 3H, 23-H, 24-H), 0.97 (t, J = 7.2 Hz, 3H),
0.93 (s, 1H), 0.89 (s, 3H, 25-H), 0.78 (s, 3H, 28-H), 0.69 (s, 1H), 0.68 (s,
3H, 26-H); 13C NMR (101 MHz, DMSO‑d6) δ 199.3 (C-11), 170.3 (C-13),
157.5 (C-30), 127.2 (C-12), 76.7 (C-3), 61.2 (C-9), 54.2 (C-20), 51.4 (C-
TOF-HRMS: m/z [M
+
H]+ calcd for C34H55N2O3: 539.4415;
found:539.4413.
8

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
5), 46.1 (C-18), 45.0 (C-19), 43.1 (C-14), 42.3 (C-8), 38.9 (C-4), 38.6 (C-
1), 36.7 (C-10), 35.5 (C-16), 33.9 (C-17), 32.2 (C-7), 31.6 (C-22), 31.5
(NCH2), 28.6 (C-29), 28.4 (C-23), 28.2 (C-28), 27.0 (C-21), 26.1 (C-15),
25.9 (C-2), 23.1 (C-27), 18.5 (C-25), 17.3 (C-6), 16.3 (C-26), 16.1 (C-
24), 15.8 (CH2CH3). TOF-HRMS: m/z [M + H]+ calcd for C32H53N2O3:
513.4051; found: 513.4048.
(101 MHz, DMSO‑d6) δ 199.3 (C-11), 170.3 (C-13), 157.5 (C-30), 127.2
(C-12), 76.7 (C-3), 61.2 (C-9), 59.1 (NCH2), 54.2 (C-20), 51.4 (C-5), 46.0
(C-18), 45.2 (N(CH3)2), 45.0 (C-19), 43.0 (C-14), 42.4 (C-8), 38.9 (C-4),
38.6 (C-1), 37.0 (C-10), 36.7 (C-16), 35.5 (C-17), 32.2 (C-7), 31.5 (C-
22), 31.3 (NHCH2), 28.6 (C-29), 28.4 (C-23), 28.2 (C-28), 27.0 (C-21),
26.1 (C-15), 25.9 (C-2), 23.1 (C-27), 18.5 (C-25), 17.3 (C-6), 16.3 (C-
26), 16.1 (C-24). TOF-HRMS: m/z [M + H]+ calcd for C34H58N3O3:
556.4473; found: 556.4478.
4.3.16. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-
isopropylurea (5n)
Mehtod B; white powder, 80%; m.p. 224–226 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.50 (d, J = 7.8 Hz, 1H, CHNH), 5.44 (s, 1H, 12-H), 5.38 (s,
1H, NH), 4.32 (d, J = 5.2 Hz, 1H, OH), 3.60 (m, 1H), 3.0(m, 1H, 3-H)
2.57 (m, 1H, 9-H), 2.30 (s, 1H, 18-H), 2.08 (m, 2H), 1.84–1.34 (m,
12H), 1.31 (s, 3H, 29-H), 1.20 (s, 3H), 1.15 (m, 3H), 1.07–1.00 (m, 12H,
23-H, 24-H, NH(CH2)3), 0.93 (m, 2H), 0.90 (s, 3H, 25-H), 0.79 (s, 3H,
28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR (101 MHz, DMSO) δ
199.1 (C-11), 170.1 (C-13), 156.8 (C-30), 127.1 (C-12), 76.6 (C-3), 61.1
(C-9), 54.1 (C-20), 51.2 (C-5), 46.1 (C-18), 44.9 (C-19), 43.0 (C-14),
42.1 (C-8), 40.6 (NHCH), 38.8 (C-4), 38.5 (C-1), 36.7 (C-10), 35.4 (C-
16), 32.1 (C-17), 31.5 (C-7, C-22), 28.5 (C-29), 28.3 (C-23), 28.1 (C-28),
27.0 (C-21), 26.0 (C-15), 25.8 (C-2), 23.3 (CHCH3), 23.2 (CHCH3), 23.0
(C-27), 18.4 (C-25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-24). TOF-HRMS: m/
z [M + H]+ calcd for C33H55N2O3: 527.4640; found: 527.4639.
4.3.20. (S)-1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-
(propanoic acid-2-yl)urea (5r)
Method B; white powder, 59%; m.p. 226–229 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.97 (d, J = 7.8 Hz, 1H, NHCH), 5.75 (s, 1H, NH), 5.46 (s,
1H, 12-H), 4.32 (brs, 1H, OH), 4.10 (dd, J = 7.2 Hz, 1H, NHCH), 3.00
(m, 1H, 3-H), 2.58 (m, 1H, 9-H), 2.31 (s, 1H, 18-H), 2.08 (m, 2H),
1.81–1.34 (m, 11H), 13.1 (s, 3H, 29-H), 1.17 (s, 3H, 27-H), 1.26–1.11
(m, 7H), 1.04, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (m, 2H), 0.90 (s,
3H, 25-H), 0.79 (s, 3H, 28-H), 0.70 (m, 1H), 0.68 (s, 3H, 26-H); 13C NMR
(101 MHz, DMSO‑d6) δ 199.1 (C-11), 175.4 (COOH), 170.0 (C-13),
156.7 (C-30), 127.1 (C-12), 76.6 (C-3), 61.1 (C-9), 54.1 (C-20), 51.3 (C-
5), 47.9 (C-18), 45.9 (C-19), 44.0 (NHCH), 42.9 (C-14), 42.2 (C-8), 38.8
(C-4), 38.5 (C-1), 36.6 (C-10), 35.3 (C-16), 32.1 (C-17), 31.2 (C-7), 31.1
(C-22), 28.4 (C-29), 28.3 (CHCH3), 28.1 (C-28), 26.9 (C-21), 26.0 (C-
15), 25.8 (C-2), 22.9 (C-27), 18.6 (C-32), 18.3 (C-25), 17.1 (C-6), 16.2
(C-26), 16.0 (C-24). TOF-HRMS: m/z [M + H]+ calcd for C33H53N2O5:
557.3949; found: 557.3953.
4.3.17. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-n-butylurea
(5o)
Mehtod A; white powder, 76%; m.p. 227–230 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.63 (t, J = 5.5 Hz, 1H, NHCH2), 5.44 (s, 2H, CONH, 12-H),
4.37 (d, J = 5.2 Hz, 1H, OH), 3.00 (m, 3H, 3-H), 2.56 (m, 1H, 9-H), 2.30
(s, 1H, 18-H), 2.08 (m, 6.5 Hz, 2H), 1.92–1.34 (m, 11H), 1.31 (s, 3H, 27-
H) 1.19 (s, 5H), 1.02, 1.01 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (m, 2H),
0.89 (s, 3H, 25-H), 0.86 (t, J = 7.2 Hz, 3H, CH2CH3), 0.78 (s, 3H, 28-H),
0.69 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR (101 MHz, DMSO‑d6) δ 199.3
(C-11), 170.3 (C-13), 157.6 (C-30), 127.2 (C-12), 76.7 (C-3), 61.2 (C-9),
54.2 (C-20), 51.3 (C-5), 46.2 (C-18), 45.0 (C-19), 43.1 (C-14), 42.1 (C-
8), 38.9 (C-4), 38.6 (C-1), 38.6 (NCH2), 36.7 (C-10), 35.5 (C-16), 32.3
(C-17), 32.2 (C-7), 31.6 (C-22), 31.6 (NCH2CH2), 28.6 (C-29), 28.4 (C-
23), 28.2 (C-28), 27.0 (C-21), 26.1 (C-15), 25.9 (C-2), 23.0 (C-27), 19.6
(CH2CH3), 18.5 (C-25), 17.3 (C-6), 16.3 (C-26), 16.1 (C-24), 13.8
(CH2CH3). TOF-HRMS: m/z [M + H]+ calcd for C34H57N2O3: 541.4364;
found: 541.4359.
4.3.21. (S)-1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(4-
methylpentanoic acid-2-yl)urea (5 s)
Method B; white powder, 55%; m.p. 231–233 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.93 (d, J = 8.7 Hz, 1H, NHCH), 5.70 (s, 1H, NH), 5.44 (s,
1H, 12-H), 4.31 (d, J = 4.9 Hz, 1H,OH), 4.11 (m, 1H, NHCH), 3.02 (m,
1H, 3-H), 2.58 (m, 1H, 9-H), 2.32 (s, 1H, 18-H), 2.08 (m, 2H), 1.82–1.35
(m, 14H), 1.32 (s, 3H, 29-H), 1.19 (s, 3H, 27-H),1.15 (m, 2H), 1.04, 1.02
(2 × s, 2 × 3H, 23-H, 24-H), 0.89 (m, 6H), 0.78 (s, 3H, 28-H), 0.70 (m,
1H), 0.68 (s, 3H, 26-H); 13C NMR (101 MHz, DMSO‑d6) δ 199.1 (C-11),
175.4 (COOH), 170.0 (C-13), 156.8 (C-30), 127.1 (C-12), 76.6 (C-3),
61.1 (C-9), 54.1 (C-20), 51.3 (C-5), 50.4 (C-18), 46.2 (C-19), 44.8
(NCH), 43.0 (C-14), 41.6 (C-8), 38.8 (C-4), 38.5 (C-1), 36.6 (C-10), 35.2
(C-16), 32.1 (C-17), 31.6 (C-7), 31.5 (C-22), 28.4 (C-29), 28.3 (CHCH2),
28.1 (C-28), 26.9 (C-21), 26.0 (C-15), 25.8 (C-2), 24.4 (CHCH3), 23.0
(CHCH3), 22.9 (C-27), 21.8 (CHCH3), 18.4 (C-32, C-25), 17.2 (C-6), 16.2
(C-26), 16.0 (C-24). TOF-HRMS: m/z [M + H]+ calcd for C36H59N2O5:
599.4400; found: 599.4403.
4.3.18. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-
dimethylurea (5p)
Mehtod B; white powder, 65%; m.p. 151–153 ◦C; 1H NMR (400 MHz,
CDCl3) δ 5.56 (s, 1H, CONH), 4.03 (s, 1H,12-H), 3.18 (m, 1H, 3-H), 2.86
(s, 6H, NH(CH3)2), 2.72 (m, 1H, 9-H), 2.30 (m, 1H, 18-H), 2.04 (m, 3H),
1.84–1.54 (m, 10H), 1.42–1.27 (m, 12H) 1.16 (m, 1H), 1.10, 1.09 (2 × s,
2 × 3H, 23-H, 24-H), 1.00 (m, 2H), 0.97 (s, 3H, 25-H), 0.83 (s, 3H, 28-
H), 0.77 (s, 3H, 26-H), 0.66 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 200.3
(C-11), 169.1 (C-13), 157.5 (C-30), 128.6 (C-12), 78.9 (C-3), 62.0 (C-9),
55.1 (C-20), 52.9 (C-5), 47.4 (C-18), 45.6 (C-19), 43.5 (C-14), 43.0 (C-
8), 39.3 (C-4), 39.3 (C-1), 37.2 (C-10), 36.6 (C-16), 36.1 (N(CH3)2), 33.0
(C-17), 32.2 (C-7), 32.0 (C-22), 28.9 (C-29), 28.8 (C-23), 28.3 (C-28),
27.4 (C-21), 26.6 (C-15), 26.5 (C-2), 23.5 (C-27), 18.8 (C-25), 17.7 (C-
6), 16.6 (C-26), 15.8 (C-24). C32H53N2O3: 513.4240; found: 513.4235.
4.3.22. (S)-1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-
(succinic acid-2-yl)urea (5 t)
Mehtod B; white powder, 59%; m.p. 217–220 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 6.09 (d, J = 6.0 Hz, 1H, NHCH), 6.02 (s, 1H, CONH), 5.50 (s,
1H, 12-H), 4.16 (s, 2H), 3.00 (m, 2H, 3-H), 2.54 (m, 1H, 9-H), 2.29 (s,
1H, 18-H), 2.18 (m, 1H), 2.05 (m, 2H), 1.90 (d, J = 18.3 Hz, 1H),
1.73–1.31 (m, 15H), 1.30 (s, 3H, 29-H), 1.18 (m, 3H), 1.14 (m, 3H),
1.02, 1.01 (2 × s, 2 × 3H, 23-H, 24-H), 0.91 (s, 2H), 0.89 (s, 3H, 25-H),
0.77 (s, 3H, 28-H), 0.68 (s, 1H), 0.67 (s, 3H, 26-H); 13C NMR (126 MHz,
DMSO‑d6) δ 199.4 (C-11), 174.6 (COOH), 173.4 (COOH), 170.3 (C-13),
156.9 (C-30), 127.3 (C-12), 76.8 (C-3), 61.3 (C-9), 54.2 (C-20), 51.5 (C-
5), 49.5 (NCH), 45.9 (C-18), 45.0 (C-19), 43.1 (C-14), 42.5 (C-8), 38.9
(CH2CO), 38.7 (C-4), 36.7 (C-1), 35.5 (C-10), 32.3 (C-16), 31.6 (C-17),
31.1 (C-7), 28.5 (C-22), 28.4 (C-29), 28.3 (C-23), 27.1 (C-28), 26.2 (C-
21), 25.9 (C-15), 23.1 (C-2), 21.3 (C-27), 18.5 (C-25), 17.3 (C-6), 16.3
(C-26), 16.1 (C-24). TOF-HRMS: m/z [M + H]+ calcd for C34H53N2O7:
601.3847; found: 601.3845.
4.3.19. 1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(2-
dimethylaminoethyl-1-yl)urea (5q)
Mehtod A; white powder, 60%; m.p. 228–230 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 5.70 (s, 1H, CONH), 5.65 (t, J = 5.4 Hz, 1H, NHCH2), 5.48
(s, 1H, 12-H), 4.37 (d, J = 5.2 Hz, 1H, OH), 3.02 (m, 1H ,3-H), 2.56 (m,
1H, 9-H), 2.30 (s, 1H, 18-H), 2.23 (t, J = 6.2 Hz, 2H, NHCH2CH2), 2.13
(s, 8H), 2.07–1.33 (m, 13H), 1.31 (s, 3H, 29-H), 1.18 (s, 3H, 28-H), 1.15
(m, 2H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H), 0.93 (s, 2H), 0.89 (s, 3H,
25-H), 0.78 (s, 3H, 28-H), 0.69 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR
4.3.23. (S)-1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-
(pentanedioic acid-2-yl) urea (5u)
Mehtod B; white powder, 55%; m.p. 220–222 ◦C; 1H NMR (500 MHz,
9

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
DMSO‑d6) 1H NMR (500 MHz, DMSO‑d6) δ 9.27 (s, 1H, COOH), 7.67 (s,
1H, COOH), 5.96 (s, 1H,NHCH), 5.86 (s, 1H, CONH), 5.52 (s, 1H,12-H),
4.32 (d, J = 4.8 Hz, 1H, OH), 3.75 (dd, J = 6.1 Hz, 1H, NHCH), 3.02 (m,
1H, 3-H), 2.58 (m, 1H,9-H), 2.30 (s, 1H,18-H), 2.20 (m, 2H,
CHCH2CH2), 2.10 (m, 2H, CHCH2CH2). 1.84–1.36 (m, 14H), 1.31 (s, 3H,
29-H), 1.19 (s, 2H), 1.13 (m, 3H), 1.03, 1.02 (2 × s, 2 × 3H, 23-H, 24-H),
0.91 (s, 2H), 0.89 (s, 3H, 25-H), 0.77 (s, 3H, 28-H), 0.68 (s, 3H, 26-H),
0.66 (s, 1H); 13C NMR (126 MHz, DMSO‑d6) δ 199.2 (C-11), 176.9
(COOH), 170.2 (C-13), 157.4 (COOH), 157.0 (C-30), 127.1 (C-12), 76.6
(C-3), 61.1 (C-9), 54.1 (C-20), 51.2 (C-5), 45.7 (C-18), 44.9 (C-19), 42.9
(C-14), 42.7 (NCH), 40.5 (C-8), 38.8 (C-4), 38.6 (C-1), 36.6 (C-10), 35.4
(C-16), 32.2 (C-17), 31.4 (C-7), 31.3 (CH2), 31.0 (C-22), 28.5 (C-29),
28.3 (C-23), 28.2 (C-28), 27.0 (C-21), 26.0 (C-15), 25.8 (C-2), 25.4
(CH2), 23.0 (C-27), 18.4 (C-25), 17.2 (C-6), 16.2 (C-26), 16.0 (C-24).
TOF-HRMS: m/z [M + H]+ calcd for C35H55N2O7: 615.4000; found:
615.3996.
solutions were mixed and determined at 540 nm in an automated
microplate reader.
4.4.4. Measurement of inflammatory mediators
RAW264.7 cells were cultured in 48-well plates for 24 h, after gly-
cyrrhetic ureas (5, 10 and 20 μM) were added and incubated for 1 h, then
LPS solution (0.5 μg/mL) stimulated for 24 h, and the supernatant was
extracted for detection. TNF-α and IL-6 release levels from macrophage
supernatant were determined by ELISA kits (Huamei, Wuhan) according
to manufacturers’ instruction.
4.4.5. Animal experiments
Male C57BL/6 mice (6–8 weeks old, weight 18–25 g) were supplied
by Animal Center of Anhui Medical University (Hefei, China). Before the
experiment, all animals were allowed to feed and drink freely for 7 days
at 60 ± 10% relative humidity and 23 ± 2 ◦C in a 12 h light/dark cycle.
All animal experiments were conducted in accordance with the
“Guidelines for the Care and Use of Laboratory Animals” of the National
Institutes of Health. All mice were randomly divided into 3 groups (n =
10), blank control group, model group and 5r group. Mice in 5r group
were given compound 5r (30 mg/kg) with intragastric administration
for 3 consecutive days, and the other groups were given the same
amount of normal saline. On the third day, except for the normal group,
all mice were injected intraperitoneally with cisplatin solution (20 mg/
kg) (Aladdin, China) 1 h after intragastric administration. In the normal
group, mice were injected intraperitoneally with the same dose of saline.
Under the same conditions, the mice continued to be administered
compound 5r or an equal volume of saline for 3 days, and then serum
and kidney tissue were obtained.
4.3.24. (S)-1-((3β,20β)-3-hydroxyl-11-oxo-olean-12-en-20-yl)-3-(5-
guanidinopentanoic acid-2-yl)urea (5v)
Mehtod B; white powder, 52%; m.p. 220–222 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 6.00 (d, J = 7.9 Hz, 1H, C = NH), 5.79 (s, 1H, CONH), 5.50
(s, 1H, 12-H), 4.30 (brs, 1H, OH), 4.05 (m, 1H), 3.01 (m, 1H, 3-H), 2.59
(m, 1H, 9-H), 2.31 (s, 1H, 18-H), 2.27–2.07 (m, 4H), 1.94–1.35 (m,
14H), 1.32 (s, 3H, 29-H), 1.20 (m, 3H, 27-H), 1.16 (m, 2H), 1.04, 1.03 (2
× s, 2 × 3H, 23-H, 24-H), 0.94 (m, 2H), 0.91 (s, 3H, 25-H), 0.80 (s, 3H,
28-H), 0.70 (s, 1H), 0.68 (s, 3H, 26-H); 13C NMR (126 MHz, DMSO‑d6) δ
199.1 (C-11), 174.6 (COOH), 173.9 (C = NH), 169.9 (C-13), 156.9 (C-
30), 127.2 (C-12), 76.6 (C-3), 61.1 (C-9), 54.1 (C-20), 51.7 (NCH), 51.4
(C-5), 45.9 (C-18), 44.8 (C-19), 43.0 (NCH2), 42.3 (C-8, C-14), 38.8 (C-
4), 38.5 (C-1), 36.7 (C-10), 35.3 (C-16), 32.1 (C-17), 31.4 (C-7), 31.0 (C-
22), 30.2 (CH2), 28.32 (C-29), 28.27 (C-23), 28.1 (C-28), 28.0 (CH2),
27.0 (C-21), 26.0 (C-15), 25.8 (C-2), 23.0 (C-27), 18.4 (C-25), 17.2 (C-
6), 16.2 (C-26), 16.0 (C-24). TOF-HRMS: m/z [M + H]+ calcd for
According to the manufacturer’s instructions, the serum markers of
renal function BUN and Scr were determined by the BUN and Scr assay
kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).
Similarly, enzyme-linked immunosorbent assay was used to detect
C
36H60N5O5: 642.4589; found: 642.4593.
serum levels of pro-inflammatory factors TNF-α and IL-6. The absor-
bance values under different wavelengths were detected using a
microplate reader. Samples were taken from the kidney of mice in
different groups and fixed in 10% formalin in saline for 24 h. All samples
were washed in tap water for half an hour and dehydrated with ethanol.
The obtained tissue sections were collected on slides, deparaffinized,
and stained with hematoxylin-eosin (H&E) and periodic acid-schiff
(PAS) staining for examination using a photoelectron microscope.
4.4. Biological evaluation
4.4.1. Cell culture
The murine macrophage RAW 264.7 cell line obtained from BeNa
Culture Collection Company. Cells were cultured in DMEM (Hyclone,
USA) supplemented with 10% FBS (Biological Industries, Israel), 100 U/
mL penicillin and 100 mg/mL streptomycin (Beyotime) in a humidified
incubator with 5% CO2 at 37 ◦C.
4.5. Statistical analysis
Unless otherwise specified, all data are expressed as the mean ±
standard deviation (SD) of two or three independent experiments. The
statistical significance of mean values was assessed using one-way
analysis of variance (ANOVA). The SPSS 20.0 software was used to
analysis the study results. A value of p < 0.05 indicated that the dif-
ference is statistically significant.
4.4.2. Cell viability assay
Cell viability is based on cell metabolism measured by the MTT
assay. RAW264.7 cells were seeded at a density of 1 × 104 cells/mL in
96-well plates (three replicates) and allowed to attach overnight to
become almost confluent. After adherence, the cells were treated with
the same concentration of glycyrrhetic ureas (20 μM) for 12 h. Then,
MTT solution (0.5 mg/mL) was added to each well for 4 h in media. The
Declaration of Competing Interest
culture media were discarded and replaced with DMSO (150 μL/well) to
dissolve crystals, incubated for 20 min in a constant temperature shaker,
and the absorbance was read at 492 nm with a microplate reader
(MQX200, Bio-Tek, USA).
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.4.3. Griess assay
Acknowledgments
NO production from activated RAW264.7 cells was determined by
measuring the amount of nitrite, a relatively stable oxidation product of
NO, as described previously [55]. RAW 264.7 cells were cultured in 48-
well plates at a density of 7 × 104 cells/mL overnight. After adding
Financial support was provided by Natural Science Fundation of
Anhui provincial Department of Education (KJ2019ZD21), and Linyi
Science and Technology Development Program (NO. 202020070).
different concentrations of glycyrrhetic ureas (5, 10 and 20 μM) for 1 h,
cells were stimulated with LPS (500 ng/mL) for 24 h. The determination
of the nitrite concentration is based on the Griess reaction by colorim-
etry (Griess reagent, Beyotime, China). Briefly, the Griess reagents І and
Notes
II was added to 50
μL supernatant of the cultured media, and the
The authors declare no competing financial interest.
10

H. Wang et al.
Bioorganic Chemistry 110 (2021) 104755
[28] X. Cheng, L. Qiu, F. Wang, 18
α
-Glycyrrhetinic acid (GA) ameliorates fructose-
Appendix A. Supplementary material
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