Synthesis and evaluation of panaxatriol derivatives as Na+, K+-ATPase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.07.027

Panaxatriol, a triterpene bearing a steroid-like structure similar to cardiac glycosides, was presumed to share the same bioactivity with cardiac glycosides, and may be a potential Na⁺, K⁺-ATPase inhibitor. In this paper, a series of panaxatriol derivatives were synthesized and evaluated for Na⁺, K⁺-ATPase inhibitory activities. The results of biological tests showed that more than half of the synthesized derivatives presented increased inhibitory activities compared with panaxatriol. Of these compounds, 13a with a 3, 4-seco skeleton showed the most potent inhibitory activity, which was equal to that of the standard drug digoxin. To understand the binding mode of the most active compound, molecular docking study of 13a with Na⁺, K⁺-ATPase was conducted. Therefore, 13a may serve as a new lead compound for the development of novel Na⁺, K⁺-ATPase inhibitors.

Full text of DOI:10.1016/j.bmcl.2018.07.027

Accepted Manuscript
Synthesis and evaluation of panaxtriol derivatives as Na+, K+-ATPase inhibitors
Qiong Wu, Peng Chen, Guangzhong Tu, Meng Li, Bowen Pan, Yan Guo, Jinbi
Zhai, Hongzheng Fu
PII:
S0960-894X(18)30590-0
https://doi.org/10.1016/j.bmcl.2018.07.027
BMCL 25958
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
23 March 2018
12 June 2018
16 July 2018
Please cite this article as: Wu, Q., Chen, P., Tu, G., Li, M., Pan, B., Guo, Y., Zhai, J., Fu, H., Synthesis and evaluation
of panaxtriol derivatives as Na+, K+-ATPase inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi:
https://doi.org/10.1016/j.bmcl.2018.07.027
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Synthesis and evaluation of panaxtriol derivatives as Na+, K+-ATPase
inhibitors
Qiong Wua, Peng Chena, Guangzhong Tub, Meng Lic, Bowen Pana, Yan Guod, Jinbi
Zhaid and Hongzheng Fua,*
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Science, Peking University, Beijing 100191, China
b Beijing Institute of Microchemistry, Beijing 100091, China
c University of Warwick, Coventry CV4 7AL, United Kingdom
d
College of Traditional Chinese Medicine, Shenyang Pharmaceutical University,
Shenyang 110016, China
*Corresponding author. Hongzheng Fu, E-mail: drhzfu@sina.com
Keywords: Na+, K+-ATPase, panaxtriol, seco-A ring derivatives, molecular docking.
Abstract: Panaxatriol, a triterpene bearing a steroid-like structure similar to cardiac
glycosides, was presumed to share the same bioactivity with cardiac glycosides, and
may be a potential Na+, K+-ATPase inhibitor. In this paper, a series of panaxtriol
derivatives were synthesized and evaluated for Na+, K+-ATPase inhibitory activities.
The results of biological tests showed that more than half of the synthesized derivatives
presented increased inhibitory activities compared with panaxatriol. Of these
compounds, 13a with a 3, 4-seco skeleton showed the most potent inhibitory activity,
which was equal to that of the standard drug digoxin. To understand the binding mode
of the most active compound, molecular docking study of 13a with Na+, K+-ATPase
was conducted. Therefore, 13a may serve as a new lead compound for the development
of novel Na+, K+-ATPase inhibitors.
Heart failure (HF) is a serious condition characterized by the incapability of the heart
to supply sufficient blood flow to meet the body’s needs.1 Across the globe, about 26
million adults worldwide are living with HF.2 However, survival rates remain poor and
the five-year mortality rate for HF is nearly 50%.3 Digitalis cardiac glycosides, such as
digoxin, have been used as positive inotrope for the treatment of HF for more than 200
years. Their mechanism of action is digitalis reversibly inhibits the membrane bound
alpha subunits of the Na+, K+-ATPase in cardiomyocytes.4 Cardiac glycosides’
aglycones, the steroidal structures, are considered to be responsible for their inhibitory
activities. 5 Despite cardiac glycosides are extensively used in clinical therapy, its safety

is still a big problem due to the life-threatening cardiac arrhythmias toxicity and the
narrow therapeutic index. 6
Panax ginsing has been widely used in Chinese medicine for the promotion of
physical strength and resistance to diseases for thousands of years. 7 In addition, ginseng
has been used for the treatment of HF, 8 and generally has a good safety profile and low
adverse effects. 9, 10 Ginsenosides are the main components of ginseng, 11 and have been
reported to inhibit Na+, K+-ATPase. 12 However, ginsenosides were poorly absorbed in
the gastrointestinal tract, 13 and were tend to be metabolized to their final aglycones by
intestinal bacterial deglycosylation after oral administration. 14 The main aglycones of
ginsenosides, such as protopanaxatriol (PPT) and protopanaxadiol, are dammarane-
type tetracyclic triterpenes.
20(R)-panaxatriol (PT) is a pseudo-aglycone of PPT-type ginsenosides, and can be
obtained by conversion of PPT during the acid hydrolysis. 15 PT was similar structurally
16, 17
to uzarigenin, which was a potent NKA inhibitor and 5αH-cardiotonic steroid
.
Thus, we presumed that PT may share the same inhibitory activity towards Na+, K+-
ATPase. To our best knowledge, the inhibitory activities of PT and its derivatives on
Na+, K+-ATPase have not been reported yet. With the aim to search for novel Na+, K+-
ATPase inhibitors with low toxicity and high affinity from natural resources, a series
of PT derivatives were synthesized for Na+, K+-ATPase inhibitory activities evaluation.
Figure 1. Structures and stereo 3D stick model of the superposition of uzarigenin (red) and PT
(blue).
Panaxtriol was prepared by acid hydrolysis of total ginsenosides derived from the
stem and leaf of panax ginseng. Whereas PT has three hydroxyl groups at C-3, C-6 and
C-12, oxidation and etherification reactions were introduced to evaluate the role of
hydroxyl groups as showed in Scheme 1. PT 1 was oxidized by equivalent Dess-Martin
periodinane to produce 6-keto-PT 2 and 3-keto-PT 3, which were further etherified by
different haloalkanes to give the ethers 4a-4d and 5a-5d, respectively. Besides,
treatment of 2 with ethoxycarbonyl isothiocyanate resulted in 4e, which was followed
by a ring closure reaction to afford 4f. 18 The 3-keto-PT 3 was converted to 3, 12-keto-
PT 6 in three steps (see supporting information). Oxidized compounds 7 and 8 were
accomplished by reacting 1 with Jones reagent and pyridinium chlorochromate,
respectively. To explore the role of A-ring, lactones 9, 10 and 11 were synthesized by
Baeyer–Villiger reaction of 6, 7 and 8 using 3-chloroperoxybenzoic acid, respectively.

In addition, lactam 12 was synthesized from 8 in two steps by first treating with
hydroxylammonium chloride and subsequent with SOCl2 through Beckmann
rearrangement reaction.
Scheme 1. Reagents and conditions: (a) Dess-Martin Periodinane, DCM, rt, 3 h; (b) (i) NaH, THF,
rt, 0.5 h; (ii) RBr, 70℃, 12 h; (c) (i) Ac2O, pyridine, reflux, 12 h; (ii) Jones reagent, 3 h; (iii) KOH,
MeOH/H2O, 1 h; (d) Jones reagent, rt, 12 h; (e) PCC, rt, 6 h; (f) m-CPBA, Li2CO3, DCM, 12 h; (g)
(i) NH2OH·HCl, NaHCO3, reflux, 8 h; (ii) SOCl2, DCM, 0℃, 1 h; (h) ethoxycarbonyl isothiocyanate,
CHCl3, 60℃, 13 h; (i) NH2OH·HCl, LiOH, EtOH, rt, 5 h.
Considering that a seco-A ring structure may provide an altered pattern of
functionalities and new scaffold with conformational flexibility, 19 a group of A-ring
opening analogs were synthesized as presented in Scheme 2. Hydrolysis of lactone 11
with p-toluenesulfonic acid in MeOH gave 3, 4-seco methyl ester 13a and 13b, while
treatment of 11 with KOH in MeOH/H2O generated 14, which was further esterified to
give ester 15. 14 was supposed to be a carboxylic acid similar to 13a, however it lost
C-4, C-28 and C-29, which was abnormal. The fact is that a retro aldol reaction took
place during the hydrolysis with the presence of KOH (mechanism was showed in
Figure 2). Indole has been known to be a very important segment in biologically active
molecules, thus was incorporated at A-ring of 8 to afford 16a-16c by Fischer indole
synthesis using different phenyldrazines. The structures of all synthesized compounds
were elucidated by 1H NMR, 13C NMR and MS (see supporting information).

Scheme 2. Reagents and conditions: (j) p-toluenesulfonic acid, MeOH, rt, 2 h; (k) KOH,
MeOH/H2O, rt, 1 h; (l) DCM/MeOH, H2SO4, 3 h; (m) phenylhydrazine derivatives, AcOH, reflux,
3 h.
Figure 2. Mechanism for the retro aldol reaction of 11 to 14.
All the synthetic derivatives were evaluated in the enzyme inhibition assay against
Na+, K+-ATPase by using a known colorimetric method. 20, 21 Digoxin was chosen as
reference compound because it is the most common drug for the treatment of HF. As
can be seen in Table 1, more than half of tested PT derivatives showed increased
inhibitory activities. Oxidation of hydroxyl groups of 1 on C-3 or C-6 exhibited no
enhancement of inhibitory activity, while oxidation of hydroxyl group on C-12 slightly
increase the inhibitory activity (6 vs 3). As to ethers 4a-4d and 5a-5d, only the mono-
benzyl ether derivatives, such as 4c, 4d and 5d, significantly improved the inhibition.

These results revealed that ethyl ether and allyl ether were not preferable substitutions.
Among them, the most potent compound (4d) was about 3.6-fold more potent than 1.
Compared with 4d, 4c with a trifluoromethoxy group substituted on its C-4 position of
the benzene ring showed a decrease of the potency. In addition, 4f containing an
oxadiazolone group showed a slight improvement of activity (4f vs 2). Besides, the
benzyl ether group at C-12 in 5c decreased the inhibitory on Na+, K+-ATPase compared
with 5d, suggesting that benzyl ether group at C-12 is not beneficial to the affinity to
certain enzyme site.
Table 1
Inhibitory activities of PT derivatives against Na+, K+- ATPase
Compound
R1
H, β OH
R2
R3
R4
R5
R6
X
IC50 (μM)
1.09±0.11
1.18±0.11
1.14±0.12
1.14±0.12
1.05±0.11
0.45±0.03
0.30±0.03
1
2
H, α OH
H, β OH
H, β OH
H, β OH
H, β OH
H, β OH
H, β OH
H, β OH
H, β OH
O
3
O
H, α OH
4a
4b
4c
4d
H, β OC2H5
H, β OCH2CH=CH2
H, β O4(CF3O)Bn
H, β OBn
O
O
O
O
4e
O
H, β OH
1.38±0.14
H, β
H, β
4f
O
H, β OH
0.96±0.09
5a
5b
5c
5d
6
O
O
O
O
O
O
O
H, α OCH2CH=CH2 H, β OCH2CH=CH2
0.75±0.08
0.91±0.09
0.87±0.08
0.33±0.03
0.70±0.06
0.76±0.08
0.81±0.07
1.23±0.13
1.13±0.11
1.18±0.11
H, α OCH2CH=CH2
H, β OH
H, β OBn
H, β OH
O
H, α OBn
H, α OBn
H, α OH
7
O
O
8
O
H, β OH
O
9
H, α OH
O
O
O
10
11
12
O
O
O
O
H, β OH
H, β OH
NH 0.41±0.03

13a
13b
C(CH3)2OH Me
CCH3=CH2 Me
0.26±0.03
1.09±0.08
0.54±0.05
0.41±0.03
0.77±0.07
0.91±0.08
0.36±0.04
0.27±0.03
14
H
H
H
15
Me
16a
H
F
16b
16c
OCF3
Digoxin
When it comes to the A-ring extended compounds, lactone skeleton and lactam
skeleton showed different impacts on the inhibitory activities. While lactones (9, 10,
11) showed no improvement of inhibitory activities compared with 1, lactam (12)
presented a 2.6-fold inhibitory activity more potent than 1. In addition, the nearly no
differences of activities between 9, 10 and 11 revealed that ketone group on C-6 and C-
12 had only minimal effect on inhibition. Almost all of the seco-A ring derivatives had
exhibited more potent inhibition against Na+, K+-ATPase than that of 1 except 13b.
Among these seco-compounds, 13a is the most potent inhibitor (IC50 = 0.26 μM), about
4.2-fold more potent than 1. The inhibitory effect of 4-hydroxyl-3-methyl ester 13a was
much stronger than the 4-methylene-3-methyl ester 13b, indicating that hydroxyl at C-
4 is important for binding to the enzyme in 3, 4-seco derivatives. On the other hand, the
further cleavage of 3, 4-seco compounds led to a decrease in the inhibitory activity (14,
15 vs 13a, 13b). Besides, methyl esterification of carboxylic acid 14 to 15 at C-3
position brought slightly enhanced performance. For indole-fused derivatives, 16c
exhibited a much better inhibitory activity than 16a and 16b, indicating that
trifluoromethoxy group substituted on benzene ring is a more preferable substitution
than fluorine atom and no substitution.
The arrhythmogenic activity of the most potent compound 13a was determined in
the isolated rat hearts by using a known method. 22 Digoxin was chosen as reference
compound. The results showed that no arrhythmia occurred in all 4 isolated rat hearts
when perfused with 0.26 μmol/L 13a. However, the perfusion of 0.26 μmol/L digoxin
induced arrhythmias in 2 of 4 isolated rat hearts. All these results indicated that 13a was
much safer than digoxin (see supporting information).
To understand the binding mode of the most active compound, molecular docking
study of 13a with Na+, K+-ATPase (PDB code: 4HYT) was performed with maestro
v11.1. As showed in Figure 3, 13a has a nice fit in the binding pocket. The methyl
propionate chain stretched into a specific cavity, and the carbonyl group at C-3 of 13a
formed hydrogen bonds with THR-797 (2.5 Å) and ASP-121 (2.2 Å), respectively. In
addition, the hydroxyl group at C-12 formed a hydrogen bond with GLU-116 in a
distance of 2.2 Å.

Figure 3. Predicted binding model of 13a to Na+, K+-ATPase. Key residues are highlighted in green
and the H-bonds are represented by dashed red lines.
In summary, modification of PT by oxidation, etherification of hydroxyl groups,
cleavage of A-ring and fusion of indole on A-ring, led to a series of PT derivatives.
Almost a half of them showed increased inhibitory activities on Na+, K+-ATPase, and
the most active compound 13a exhibited an inhibitory activity equal to that of the
standard drug digoxin. Moreover, the molecular docking study illustrated its potential
binding mode. Thus, 13a containing a 3, 4-seco structure provides a basis for
developing novel Na+, K+-ATPase inhibitors.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of
China (No. 30973628) and the National Science and Technology Major Project of
China (No. SQ2018ZX090301).
References and notes
1. Boxer R, Yang SX, Hager WD. Congestive heart failure and the elderly. Conn Med.
2003;67(8): 497.
2. Bui AL, Horwich TB, Fonarow GC. Epidemiology and risk profile of heart failure.
Nat Rev Cardiol. 2011;8(1): 30-41.
3. Blass BE, Huang CT, Kawamoto RM, et al. Parallel synthesis and evaluation of N-
(1-phenylethyl)-5-phenyl-imidazole-2-amines as Na+/K+ ATPase inhibitors. Bioorg
Med Chem Lett. 2000;10(14): 1543-1545.
4. Smith TW. Digitalis. Mechanisms of action and clinical use. N Engl J Med.
1988;318(6): 358-365.
5. Schönfeld W, Menke KH, Schönfeld R, Repke KR. 5 Beta,14 beta-androstane-3
beta,14-diol binds to the digitalis receptor site on Na/K-ATPase. J Enzyme Inhib.
1987;2(1): 37-45.
6. Cerri A, Serra F, Ferrari P, Folpini E, Padoani G, Melloni P. Synthesis, Cardiotonic
Activity, and Structure−Activity Relationships of 17β-Guanylhydrazone Derivatives of
5β-Androstane-3β,14β-diol Acting on the Na+,K+-ATPase Receptor. J Med Chem.
1997;40(21): 3484-3488.

7. Tachikawa E, Kudo K, Hasegawa H, et al. In vitro inhibition of adrenal
catecholamine secretion by steroidal metabolites of ginseng saponins. Biochem
Pharmacol. 2003;66(11): 2213-2221.
8. Wagner H, Liu X. The International Textbook of Cardiology. New York: Pergamon
Press; 1987.
9. Lee NH, Son CG. Systematic review of randomized controlled trials evaluating the
efficacy and safety of ginseng. J Acupunct Meridian Stud. 2011;4(2): 85-97.
10. Kim YS, Woo JY, Han CK, Chang IM. Safety Analysis of Panax Ginseng in
Randomized Clinical Trials: A Systematic Review. Medicines. 2015;2(2): 106.
11. Yang WZ, Hu Y, Wu WY, Ye M, Guo DA. Saponins in the genus Panax L.
(Araliaceae): a systematic review of their chemical diversity. Phytochemistry.
2014;106(10): 7.
12. Lee SW, Lee JS, Kim YH, Jin KD. Effect of ginseng saponin on the Na+, K+-
ATPase of dog cardiac sarcolemma. Arch Pharm Res. 1986;9(1): 29-38.
13. Takino Y. Studies on the pharmacodynamics of ginsenoside-Rg1, -Rb1 and -Rb2
in rats. Yakugaku Zasshi. 1994;114(8): 550-564.
14. Nah SY, Kim DH, Rhim H. Ginsenosides: Are any of them candidates for drugs
acting on the central nervous system? CNS Drug Rev. 2007;13(4): 381–404.
15. Wei Y, Ma CM, Hattori M. Anti-HIV protease triterpenoids from the acid
hydrolysate of Panax ginseng. Phytochem Lett. 2009;2(2): 63-66.
16. Brown L, Thomas R, Watson T. Cardiac glycosides with non-rotating steroid to
sugar linkages: tools for the study of digitalis structure-activity relationships. Naunyn-
Schmiedeberg's Arch Pharmacol. 1986;332(1): 98-102.
17. Cerri A, Gobbini M. Simplified digitalis-like compounds acting on Na(+), K(+)-
ATPase. J Enzyme Inhib Med Chem. 2003;18(4): 289-295.
18. Czollner L. Synthesis of new glycyrrhetinic acid derived ring Aazepanone, 29-urea
and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2
inhibitors. Biorg Med Chem. 2011;19(6): 1866-1880.
19. Mao SW, Chen H, Yu LF, et al. Novel 3,4-seco bile acid diamides as selective
anticancer proliferation and migration agents. Eur J Med Chem. 2016;122: 574.
20. Taussky HH, Shorr E, Kurzmann G. A microcolorimetric method for the
determination of inorganic phosphorus. J Biol Chem. 1953;202: 675-685.
21. Matsukawa M, Mukai T, Akizawa T, et al. Isolation and Characterization of Novel
Endogenous Digitalis-like Factors in the Ovary of the Giant Toad, Bufo marinus. J Nat
Prod. 1998;61(12): 1476-1481.
22. Zhao TC, Zhang XL. Effect of activation of Na +/H + exchange system on ischemia
reperfusion injury. Chin Appl Physiol. 1996; 12 (3): 270-272.

Highlights
• 26 panaxtriol derivatives were synthesized and evaluated for Na+, K+-
ATPase inhibitory activities.
• Compound 13a showed most inhibitory activity equal to that of the
standard drug digoxin.
• Molecular docking predicted the potential binding mode.