Synthesis and biological evaluation of Ginsenoside Compound K analogues as a novel class of anti-asthmatic agents

Article abstract of DOI:10.1016/j.bmcl.2018.11.016

Ginsenoside Compound K (CK) showed potent activity against IgE for the treatment of asthma. A series of CK analogues were then synthesized by straightforward procedures. The in vivo anti-IgE activity evaluations using the OVA-induced asthmatic mouse model revealed preliminary SARs of the CK analogues, which showed that the sugar type, modifications on A-ring and the C20 side chain of CK all affected much on the activities. Primary SARs optimization led to the discovery of compounds T1, T2, T3, T8 and T12, which displayed superior or comparable anti-asthmatic effects (IgE value = 1237.11 ± 106.28, 975.82 ± 160.32, 1136.96 ± 121.85, 1191.08 ± 107.59 and 1258.27 ± 148.70 ng/mL, respectively) in comparison with CK (1501.85 ± 184.66 ng/mL). These potent compounds could serve as leads for further development.

Full text of DOI:10.1016/j.bmcl.2018.11.016

Accepted Manuscript
Synthesis and Biological Evaluation of Ginsenoside Compound K Analogues
as a Novel Class of Anti-asthmatic Agents
Sumei Ren, Ruiqi Liu, Yujie Wang, Ning Ding, Yingxia Li
PII:
S0960-894X(18)30881-3
https://doi.org/10.1016/j.bmcl.2018.11.016
BMCL 26127
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
15 September 2018
6 November 2018
8 November 2018
Please cite this article as: Ren, S., Liu, R., Wang, Y., Ding, N., Li, Y., Synthesis and Biological Evaluation of
Ginsenoside Compound K Analogues as a Novel Class of Anti-asthmatic Agents, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Biological Evaluation of Ginsenoside Compound
K Analogues as a Novel Class of Anti-asthmatic Agents
*
*
Sumei Ren, Ruiqi Liu, Yujie Wang, Ning Ding , Yingxia Li
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
ABSTRACT
Ginsenoside Compound K (CK) showed potent activity against IgE for the treatment
of asthma. A series of CK analogues were then synthesized by straightforward proce-
dures. The in vivo anti-IgE activity evaluations using the OVA-induced asthmatic
mouse model revealed preliminary SARs of the CK analogues, which showed that the
sugar type, modifications on A-ring and the C20 side chain of CK all affected much
on the activities. Primary SARs optimization led to the discovery of compounds T1,
T2, T3, T8 and T12, which displayed superior or comparable anti-asthmatic effects
(
1
IgE value = 1237.11±106.28, 975.82±160.32, 1136.96±121.85, 1191.08±107.59 and
258.27±148.70 ng/mL, respectively) in comparison with CK (1501.85±184.66
ng/mL). These potent compounds could serve as leads for further development.
Keywords: Ginsenoside Compound K, asthma, glycosylation, saponin, IgE
Asthma is a common, chronic inflammatory disease, characterized by lung in-
1
flammation, airway hyperresponsiveness (AHR) and mucus hypersecretion. It is con-
sidered to be an important public health issue which affects more than 300 million
2
people with a strong personal, social and economic impact. However, there is no cure
3
developed for asthma so far, which invariably relapse on ceasing the treatment. The
goal of asthma treatment is to achieve good control of symptoms, reduce exacerba-
tions and improve quality of life. Now the Inhaled corticosteroids is still the
4
pharmacotherapeutic cornerstone, while systemic exposure to corticosteroids suffers
from some adverse effects, including diabetes, hypertension, myopathies, and osteo-
5
porosis. Therefore, it is necessary to find new drugs for the treatment of asthma. Al-
lergic inflammation in asthma is associated with T-helper (Th2) cells and their cyto-
kines (IL-4, -5, and -13). Both IL-4 and IL-13 induce the secretion of IgE by B cells,
which mediates the type 1 hypersensitivity reactions and the orchestration of local
6
chronic allergic inflammation. 80% of childhood asthma and more than half of adult
7
asthma were potentially mediated by IgE. And anti-IgE omalizumab has been suc-
8
cessfully used for more than 10 years in the treatment of asthma. These facts validate
IgE as an ideal target in the treatment of asthma.
*Corresponding author: For Y. L. email: liyx417@fudan.edu.cn; For N. D. email: dingning@fudan.edu.cn
1

Figure 1. The structures of compound K and 20S-protopanaxadiol
The current anti-IgE drugs are all biological agents, while there is no small mol-
ecule one reported. Fortunately, we recently discovered that a saponin, Ginsenoside
Compound K (CK) (Figure 1), could significantly reduce IgE level in the serum and
decrease the airway resistance on OVA (ovalbumin)-induced mouse model with asth-
ma. CK, one of the active metabolites of Panaxnotoginseng saponins, has been re-
9
, 10
ported to exhibite potent anti-inflammatory effect.
In 2008, Yang et al reported that
CK was a novel agonist of glucocorticoid receptor, which could compete with the
synthetic glucocorticoid dexamethasone for binding to glucocorticoid receptor and
activated glucocorticoid responsive element-containing reporter plasmids in a
11
dose-dependent manner. In addition, Ling et al reported that elevating glucocorti-
coid receptor level and promoting glucocorticoid receptor translocation by increasing
1
2
the glucocorticoid effect may be the common mechanism of ginsenosides effects.
The potent anti-asthmatic mechanism of CK and its analogous may be related to glu-
cocorticoid receptor. These findings encouraged us to make a systematic structure and
activity relationship (SAR) studies on the potential effects of the CK analogues as an-
ti-asthmatic agents. Herein we described our efforts on this project.
A series of CK analogues that all with a sugar head were designed as shown in
Figure 2. To show if the sugar type plays a role on the activity, T1-T5 that bearing
different sugar rings were prepared. In addition, it's also easy to modify on the 3-OH,
1
2-OH and C20 side chain of CK, resulting designed compounds T6-T14. We believe
that the syntheses and evaluations of these CK analogues would give us a preliminary
but useful body of knowledge to tell if these kinds of compounds are worthy of fur-
ther studies.
2

Figure 2. Designed CK analogues for biological evaluations.
Scheme 1. Synthesis of aglycones 1-6. Reagents and conditions: (a) Ac O, DMAP, pyridine; (b)
2
3

NaOCH , CH OH-CH Cl ; (c) PDC, Ac O, CH Cl , 49% for three steps; (d) NaOCH ,
3
3
2
2
2
2
2
3
CH OH-CH Cl , 50 C, 96%; (e) CH I, NaH, DMF, 78%; (f) 4-Toluene sulfochloride, CH Cl ,
3
2
2
3
2
2
pyridine, 80 C, 88%; (g) LiBr, Li CO , DMF, 153 °C, 84%; (h) PDC, Ac O, CH Cl , 61%; (i)
2
3
2
2
2
PivCl, Et N, CH Cl ; (j) PDC, Ac O, CH Cl ; (k) NaOCH , CH OH-CH Cl , 50 C, 40% for three
3
2
2
2
2
2
3
3
2
2
steps; (l) O-Methylhydroxylamine hydrochloride or O-Allylhydroxylamine hydrochloride, pyri-
dine, 80 C; (m) PDC, Ac O, CH Cl , 46% for two steps for 5, 65% for two steps for 6.
2
2
2
Our synthetic work commenced from (20S)-protopanaxadiol (PPD) which is
much cheaper than CK and is more flexible to be modified. Scheme 1 described the
syntheses of several aglycons that modified on the 3-OH of A-ring (compounds 1-6).
The common feature of compounds 1-6 is that they all have the 12-keto group, which
is the key for the successful 20-OH glycosylation in the later stage. First, PPD was
transformed into compound 1 by blocking the 3-OH with an acetyl group followed by
13
oxidation of the 12-OH. Replacing of the 3-O-acetyl group by methyl afforded
compound 2. Compound 7 was selectively tosylated on 3-OH, which was then elimi-
nated to provide compound 8. On the other hand, blocking of the 12-OH of PPD first
with pivaloyl (Piv) followed by oxidation afforded compound 9. Removal of the Piv
group gave 10. The 3-keto group of 10 further reacted with O-methylhydroxylamine
1
4
or O-allylhydroxylamine to provid oxime derivative 5 or 6, respectively.
Scheme 2. Synthesis of compounds T1, T6, T13 and T14. Reagents and conditions: (a) TMSOTf,
CH Cl , -40 °C; (b) NaOCH , CH OH-CH Cl , rt, 81% for two steps; (c) NaOCH ,
2
2
3
3
2
2
3
CH OH-CH Cl , 50 C, 75% for two steps; (d) Pd-C, H , CH OH, 61%; (e) NBS, H O, THF; (f)
3
2
2
2
3
2
4

Pd-C, H , DIEA, EtOAc; (g) NaOCH , CH OH-CH Cl , 50 C, 37% for four steps.
2
3
3
2
2
With the six aglycones in hand, the final products were synthesized according
to the procedures as depicted in Schemes 2-3. The preparations of target compounds
T1, T6, T13 and T14 were shown in Scheme 2. Glycosylation of 1 with
2
,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate 11 catalyzed by
1
5
TMSOTf led to 12 in a good yield. Treated 12 with sodium methoxide in MeOH at
room temperature only afforded compound T6 with 3-O-acetyl intact, while pro-
longed reaction time at 50 C further cleaved the 3-O-acetyl group, resulting T1. Hy-
drogenation of the double bond of T1 gave T13. Treatment of 12 with NBS in wa-
ter/THF mixture, debromination with Pd-C/H , and removal of all acetyl groups pro-
2
vided T14.
Scheme 3. Synthesis of compounds T2-T12. Reagents and conditions: (a) TMSOTf, CH Cl ,
2
2
-
40 °C-0 °C; (b) NaOCH , CH OH-CH Cl , 50 °C, 70% for two steps for T2, 66% for two steps
3 3 2 2
for T3, 63% for two steps for T4, 67% for two steps for T5; 52% for two steps for T7, 80% for
two steps for T8, 39% for two steps for T9, 59% for two steps for T11; (c) Pd(OAc) , PPh ,
2
3
EtOH-H O, 40% for three steps; (d) Hydroxylamine hydrochloride, pyridine, 80 °C, 70%.
2
5

As shown in scheme 3, the syntheses of T2-T11 are straightforward. In detail,
aglycone 1 was glycosylated by 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl
trichloroacetimidate 13, or the corresponding mannosyl, xylosyl and rhamnosyl type
1
6, 17
donor (14-16), providing T2, or T3-T5, respectively.
trichloroacetimidate 11 was employed as the glycosyl donor to react with aglycone
-6, the expected glycosides were all furnished in good yields. Removal the acyl pro-
On the other hand, when
2
tecting groups with sodium methoxide and allyl group with Pd(OAc) (if needed) gave
2
the designed CK analogues T7-T11. Subsequent condensation of T9 with hydroxyla-
mine hydrochloride in pyridine provided T12.
4
000
*
*
3
3
500
000
*
*
2
2
500
000
*
1
500
000
*
*
*
*
*
*
**
**
*
*
1
5
00
0
*
*
Figure 3. In vivo efficacies of all compounds on IgE level in plasma. CK and its analogues were
orally administered daily for 7 consecutive days, Dex was orally given as a positive control. IgE
level was measured 24 hours after the final OVA challenge. Results are expressed as the mean ±
SEM (n = 5/group). SEM, standard error of the mean. *p<0.05, ** p<0.01 compared to OVA
group.
The synthesized CK analogues were evaluated for their in vivo efficacies in mice
1
8, 19
that had been sensitized to an allergen (OVA).
Dexamethasone acetate (Dex) was
selected as the positive control. The results are summarized in Figure 3 (See details
in ESI). It is worth to mention that compound T1 with 12-carbonyl group exhibited
enhanced potency of reducing IgE level in comparison with CK. Comparison of the
activities of T1-T5 bearing different sugar types revealed that the sugar moiety do
play an important role. The galactopyranosyl and mannopyranosyl containing com-
pounds T2 and T3 exhibited better activities than the glucopyranoside T1. While the
rhamonopyranosy and xylopyranose containing compounds T4 and T5 showed al-
most no anti-IgE effect. In addition, modifications on A-ring also led to different ac-
tivities. 3-O-acetyl and 3-O-methoxyl compounds T6 and T7 showed obviously in-
creased effect on the IgE, while 3-keto (T9) and 3-oxime (T10 and T11) analogues
almost lost the activity. The 3 (T8) and dioxime (T12) compounds played a similar
activity to CK. Furthermore, the importance of the double bond on the C20 side chain
of CK was clearly revealed. Compounds T13 and T14 both displayed very weak an-
ti-IgE activity.
6

Control
OVA
1
00
OVA+Dex 0.6 mg/Kg
OVA+CK 20 mg/Kg
OVA+T1 20 mg/Kg
OVA+T2 20 mg/Kg
OVA+T3 20mg/Kg
OVA+T8 20mg/Kg
OVA+T12 20 mg/Kg
80
8
6
4
*
*
** **
*
*
**
*
*
**
*
***
**
**
**
**
**
2
0
*
*
*
*
**
6.25
12.5
25
50
100
Methacholine (mg/mL)
Figure 4. In vivo efficacies of some representative compounds on AHR to methacholine. Results
are expressed as the mean ± SEM (n = 5/group). SEM, standard error of the mean. *p<0.05, **
p<0.01 compared to OVA group.
Airway hyperresponsiveness (AHR) is referred as one of the major hallmark
features of asthma. Therefore, decreasing airway resistance is one of the goals to treat
2
0
asthma. Methacholine was used to induce AHR. The bronchomotor response to
methacholine leads to contraction of distal airways and focal airway closure in select
lung territories. As shown in Figure 4 (See details in ESI), the OVA group developed
significantly greater airway resistance than the control group at methacholine doses of
2
5, 50 and 100 mg/mL. Based on the above ant-IgE evaluation, compounds T1, T2,
T3, T8, T12 as well as CK were selected to conduct the AHR experiment. To our de-
light, all of these compounds (20 mg/Kg) remarkably decreased airway resistance in
mice as efficacious as 0.6 mg/Kg of DEX.
In summary, CK was first discovered to show potent activity against IgE for the
treatment of asthma. A series of CK analogues were synthesized by straightforward
procedures. The synthesized compounds were evaluated in vivo on the anti-IgE activi-
ties using OVA-induced asthmatic mouse model and the in vivo airway
hyperresponsiveness experiment. We found that compounds T1, T2, T3, T8 and T12
led to a significant decrease in IgE and airway resistance. Mode of action and further
optimization of this novel class of anti-asthmatic agents are currently under investiga-
tion.
Acknowledgements
We would like to thank Prof. Quanhai Liu (Shanghai Institute of Pharmaceutical In-
dustry) for providing the PPD, and Dr. Shiqiang Yan for helpful discussion. We grate-
fully acknowledge the financial support from the National Major Scientific and
Technological Special Project of China for “Significant New Drugs Development”
(
(
2018ZX09711002-006-008) and National Natural Science Foundation of China
81773576).
Supplementary data
7

Supplementary data is available on the publishers’ web site along with the published
article.
References:
1
. Bao W, Zhang Y, Zhang M, Bao A, Fei X, Zhang X, Zhou X. Effects of ozone
repeated short exposures on the airway/lung inflammation, airway
hyperresponsiveness and mucus production in a mouse model of ovalbumin-induced
asthma. Biomed Pharmacother. 2018;101:293.
2
. Dominguez-Ortega J, Phillips-Angles E, Barranco P, Quirce S. Cost-effectiveness
of asthma therapy: a comprehensive review. J Asthma. 2015;52:529.
. Zhu W, Liu X, Wang Y, Tong Y, Hu Y. Discovery of a novel series of
3
α-terpineol derivatives as promising anti-asthmatic agents: Their design, synthesis,
and biological evaluation. Eur J Med Chem. 2018;143:419.
4
. Ghidini E, Marchini G, Capelli A. M, Carnini C, Cenacchi V, Fioni A,
Facchinetti F, Rancati F. Novel Pyrrolidine Derivatives of Budesonide as Long Act-
ing Inhaled Corticosteroids for the Treatment of Pulmonary Inflammatory Diseases. J
Med Chem. 2018;61:4757.
5
. Ernst P, Suissa S. Systemic effects of inhaled corticosteroids. Curr Opin Pulm
Med. 2012;18:85.
. Matucci A, Vultaggio A, Maggi E, Kasujee I. Is IgE or eosinophils the key player
in allergic asthma pathogenesis? Are we asking the right question? Resp Res.
018;19:113.
. Johansson SGO, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey RF,
6
2
7
Motala C, Ortega Martell JA, Platts-Mills TAE, Ring J. Revised nomenclature for al-
lergy for global use: Report of the Nomenclature Review Committee of the World
Allergy Organization, October 2003. J Allergy Clin Immun. 2004;113:832.
8
. Richards ML, Lio SC, Sinha A, Tieu KK, Sircar JC. Novel 2-(Substituted phe-
nyl)benzimidazole Derivatives with Potent Activity against IgE, Cytokines, and CD23
for the Treatment of Allergy and Asthma. J Med Chem. 2004;47:6451.
9
. Chen J, Wang Q, Wu H, Liu K, Wu Y, Chang Y, Wei W. The ginsenoside me-
tabolite compound K exerts its anti-inflammatory activity by downregulating memory
8

B cell in adjuvant-induced arthritis. Pharm Biol. 2016;54:1280.
0. Joh E, Lee I, Jung I, Kim D. Ginsenoside Rb1 and its metabolite compound K
inhibit IRAK-1 activation-The key step of inflammation. Biochem Pharmacol.
011;82:278.
1. Yang C, Ko S, Cho B, Shin D, Yuk J, Li S, Kim J, Evans R, Jung J, Song D, Jo E,
1
2
1
The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor,
induces tolerance to endotoxin-induced lethal shock. J Cell Mol Med. 2008;12:1739.
1
2. Cheng B, Feng Y, Du J, Ling C. Upregulation Effect of Ginsenosides on Gluco-
corticoid Receptor in Rat Liver Horm Metab Res. 2009;41:531.
3. Liao J, Sun J, Niu Y, Yu B. Synthesis of ginsenoside Rh2 and
1
chikusetsusaponin-LT8 via gold(I)-catalyzed glycosylation with a glycosyl or-
tho-alkynylbenzoate as donor. Tetrahedron Lett. 2011;52:3075.
1
4. Huong DTT, Thuy TTT, Hien TT, Tra NT,Tien NQ, Smirnova IE, Kazakova OB,
Minnibaeva E M, Tolstikov GA. Synthesis and Cytotoxicity of Derivatives of
Dipterocarpol, a Metabolite of Dipterocarpus alatus. Chem Nat Compd. 2013;49:58.
1
5. Yan S, Ren S, Ding N, Li Y. Concise total synthesis of acylated phenolic glyco-
sides vitexnegheteroin A and ovatoside D. Carbohydr Res. 2018;460:41.
6. Liu X, Ren S, Gao Q, Hu C, Li Y, Ding N. Glycosylation of a Ketone with an O
Glycosyl Trichloroacetimidate Provides an Enol Glycoside. Org Lett. 2018;20:5186.
7. Ding N, Chen Q, Zhang W, Ren S, Guo Y, Li Y. Structure-activity relationships
1
-
1
of saponin derivatives: A series of entry inhibitors for highly pathogenic H5N1 influ-
enza virus. Eur J Med Chem. 2012;53:316.
1
8. Meng X, Sun X, Zhang Y, Shi H, Deng W, Liu Y, Wang G, Fang P, Yang S.
PPARγ Agonist PGZ Attenuates OVA-Induced Airway Inflammation and Airway
Remodeling via RGS4 Signaling in Mouse Model. Inflammation. 2018.
DOI:10.1007/s10753-018-0851-2
1
9. Shin N, Park S, Ko J, Cho Y, Lee I, Kim J, Shin I, Kim J. Lobeglitazone Attenu-
ates Airway Inflammation and Mucus Hypersecretion in a Murine Model of Ovalbu-
min-Induced Asthma. Front Pharmacol. 2018. DOI:10.3389/fphar.2018.00906.
9

2
0. Choi JY, Lee HY, Hur J, Kim K H, Kang J Y, Rhee CK, Lee SY. TRPV1 Block-
ing Alleviates Airway Inflammation and Remodeling in a Chronic Asthma Murine
Model. Allergy Asthma Immunol Res. 2018;10:216.
1
0

Synthesis and Biological Evaluation of Ginsenoside Compound
K Analogues as a Novel Class of Anti-asthmatic Agents
*
*
Sumei Ren, Ruiqi Liu, Yujie Wang, Ning Ding , Yingxia Li
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
Highlights
1
2
. CK showed potent activity against IgE for the treatment of asthma.
. Fourteen CK analogues were synthesized and evaluated for their in vivo ant
i-asthmatic activity.
3
4
. Their structure-activity relationships were discussed.
. Five compounds exhibited excellent anti-asthmatic effects.
*Corresponding author: For Y. L. email: liyx417@fudan.edu.cn; For N. D. email: dingning@fudan.edu.cn
1
1

Synthesis and Biological Evaluation of Ginsenoside Compound
K Analogues as a Novel Class of Anti-asthmatic Agents
*
*
Sumei Ren, Ruiqi Liu, Yujie Wang, Ning Ding , Yingxia Li
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
Ginsenoside Compound K (CK) showed potent activity against IgE for the treatment
of asthma. A series of CK analogues were then synthesized by straightforward proce-
dures. The in vivo anti-IgE activity evaluations using the OVA-induced asthmatic
mouse model revealed preliminary SARs of the CK analogues, which showed that the
sugar type, modifications on A-ring and the C20 side chain of CK all affected much
on the activities. Primary SARs optimization led to the discovery of compounds T1,
T2, T3, T8 and T12, which displayed superior or comparable anti-asthmatic effects
(
1
IgE value = 1237.11±106.28, 975.82±160.32, 1136.96±121.85, 1191.08±107.59 and
258.27±148.70 ng/mL, respectively) in comparison with CK (1501.85±184.66
ng/mL).
*Corresponding author: For Y. L. email: liyx417@fudan.edu.cn; For N. D. email: dingning@fudan.edu.cn
1
2

1
3