Exploration of Pharmacophore in Chrysosplenol C as Activator in Ventricular Myocyte Contraction

Article abstract of DOI:10.1021/acsmedchemlett.5b00043

Chrysosplenol C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) isolated from Miliusa balansae has unique structural features as a reversible inotropic agent independent of β-adrenergic signaling and with selective activation of cardiac myosin ATPase. Hence, a series of chrysosplenol analogues were synthesized and explored for identification of pharmacophore that is essential for the increasing contractility in rat ventricular myocytes. Analogue 7-chloro-2-(3-hydroxyphenyl)-3-methoxy-4H-chromen-4-one showed highly potent contractility (54.8% at 10 μM) through activating cardiac myosin ATPase (38.7% at 10 μM). Our systematic structure-activity relationship study revealed that flavonoid nucleus of chrososplenol C appears to be an essential basic skeleton and hydrophobic substituent at position 7 of chromenone such as methoxy or chloro enhances the activity. Additionally, our ATPase study suggested that these chrysosplenol analogues have selectivity toward cardiac myosin activation. Thus, the novel flavonone with 3-/7-hydrophobic substituent and 3'-hydrogen bonding donor function is a novel scaffold for discovery of a new positive inotropic agent.

Full text of DOI:10.1021/acsmedchemlett.5b00043

Letter
pubs.acs.org/acsmedchemlett
Exploration of Pharmacophore in Chrysosplenol C as Activator in
Ventricular Myocyte Contraction
Eeda Venkateswararao, Min-Jeong Son, Niti Sharma, Manoj Manickam, PullaReddy Boggu,
Young Ho Kim, Sun-Hee Woo, and Sang-Hun Jung*
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, South
Korea
S
* Supporting Information
ABSTRACT: Chrysosplenol C (4′,5,6-trihydroxy-3,3′,7-tri-
methoxyflavone) isolated from Miliusa balansae has unique
structural features as a reversible inotropic agent independent
of β-adrenergic signaling and with selective activation of
cardiac myosin ATPase. Hence, a series of chrysosplenol
analogues were synthesized and explored for identification of
pharmacophore that is essential for the increasing contractility
in rat ventricular myocytes. Analogue 7-chloro-2-(3-hydrox-
yphenyl)-3-methoxy-4H-chromen-4-one showed highly potent
contractility (54.8% at 10 μM) through activating cardiac
myosin ATPase (38.7% at 10 μM). Our systematic structure−
activity relationship study revealed that flavonoid nucleus of
chrososplenol C appears to be an essential basic skeleton and
hydrophobic substituent at position 7 of chromenone such as methoxy or chloro enhances the activity. Additionally, our ATPase
study suggested that these chrysosplenol analogues have selectivity toward cardiac myosin activation. Thus, the novel flavonone
with 3-/7-hydrophobic substituent and 3′-hydrogen bonding donor function is a novel scaffold for discovery of a new positive
inotropic agent.
KEYWORDS: Chrysosplenol C, positive inotrope, myosin activation, contractility
eart failure (HF) is an enormous health problem
regulated through the cycle of a weakly actin−myosin bound
state to a strongly actin−myosin bound state.
1
Hworldwide.
Decreased systolic function is a central
factor in the pathogenesis of heart failure.2−4 Systolic
dysfunction is better characterized as a decrease in cardiac
contractility that can be measured by a reduction in the left
ventricular ejection fraction (LVEF).5−9 The contractility
function of the cardiac sarcomere mainly depends on the six
thin filaments of actin arranged hexagonally around a central
thick filament of myosin.10 Actin, which acts as a binding site
for myosin heads, is surrounded by a coil of tropomyosin which
prevents binding with myosin heads. The three subunits of
troponin (troponin I, troponin C, and troponin T)11 and
tropomyosin complex regulates actin−myosin binding inter-
action.12 At the initiation of contraction, intracellular calcium
increases. Binding of calcium on the troponin C weakens the
troponin I−actin interaction and causes tropomyosin to move
to expose the hydrophobic residues on the actin filament. The
myosin heads are then able to bind to actin, initiating
contraction. The myosin heads latch on to the actin and then
rotate at the neck, resulting in fiber shortening. The myosin
sliding movement is fueled by chemical energy released from
adenosine triphosphate (ATP) during the chemical degradation
of ATP to adenosine diphosphate and inorganic phosphate
(ADP-Pi) and subsequent release of Pi by myosin ATPase. The
power-stroke, velocity, and duration of filament shortening are
Inotropic agents increase myocardial contractility through
different pathways that in most cases lead to a final increment
of intracellular cyclic adenylate monophosphate (cAMP) levels,
which in turn induces calcium release from the sarcoplasmic
reticulum, hence enhancing the contractile force generation by
the contractile apparatus.13
There are three classes of inotropic agents that are in use at
present: β-agonists, phosphodiesterase inhibitors, and calcium
sensitizers. Classical inotropes, such as β-agonists and
phosphodiesterase inhibitors, acutely improve the hemody-
namic and clinical status of acute decompensated heart failure
patients, but frequently promote and accelerate some
pathophysiologic mechanisms causing further myocardial injury
and leading to increased short- and long-term mortality.
However, calcium-sensitizing inotropic agents such as levosi-
mendan14 have been developed to increase the calcium
sensitivity of myofibrils without increasing calcium transients
for treating systolic heart failure. Unfortunately, in the later
stages, it was found that levosimendan also has significant
Received: January 28, 2015
Accepted: May 20, 2015
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
phosphodiesterase inhibition activity.15 This mixed mechanism
may be the reason for levosimendan failure in improvement on
mortality. A recent breakthrough came from identifying
omecamtiv mecarbil (Figure 1)16,17 as a selective cardiac
Miliusa balansae and Pterocaulon sphacelatum, which induced
positive inotropic effect in rat ventricular myocytes by
reversibly increasing ventricular cell shortening with an EC50
value of about 45
7.8 μM independent of β-adrenergic
signaling20 and showed the activation (28.1% at 10 μM) of
cardiac myosin ATPase as presented in Table 1. Thus, the
positive inotropic effect of Chrysosplenol C was considered to
be originated from the activation of cardiac myosin ATPase.
Chrysosplenol C possesses flavonoid scaffold, which is the
unique structural feature of an inotropic agent. Therefore, to
explore the pharmacophore responsible for its contractile
activity, we systematically studied the structure−activity
relationship of chrysosplenol C using cell contractility test of
myocytes. Later, the activation of the cardiac myosin ATPase of
its potent analogues was demonstrated.
Figure 1. Known myosin activators.
myosin adenosine triphosphatase (ATPase) activator, which is
currently completing late phase II trials on the verge of phase
III trials for treatment of systolic heart failure, demonstrating
the enormous potential of myosin-targeting drugs.
Thus, on the basis of ATPase activation concept, we
screened18 compounds from the natural sources. Our efforts
brought to light chrysosplenol C19 (4′,5,6-trihydroxy-3,3′,7-
trimethoxyflavone, Figure 1), a known flavonoid isolated from
Designed chrysosplenol C analogues 5 and 6 (Table 1) were
synthesized as outlined in Scheme 1. The key intermediate
chalcones 321 were prepared in excellent yields by aldol
condensation of substituted hydroxyacetophenones 1 with
corresponding substituted benzaldehydes 2 in the presence of
KOH in aqueous ethanol under reflux conditions. Chalcone
Table 1. Summary of the Changes in Ventricular Cell Contractility and Cardiac Myosin ATPase Activity by Chrysosplenol C
a
Analogues
ventricular cell contractility (% of
change)
cardiac myosin ATPase % activation
compd
R1
R2
R3
R4
10 μM
50 μM
at 10 μM
5a
5-OH
6-OH
7-OMe
7-Cl
OMe
OMe
OMe
OMe
OMe
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Cl
4.20 2.10 (3)
3.90 2.30 (3)
12.3 4.02 (3)
9.30 1.89 (3)
0
5b
5c23
5d
5e
c
c
60.7 3.10 (3)
45.3 2.09 (3)
33.7 2.64
36.4 1.32
c
b
43.9 6.83 (3)
41.8 5.25 (3)
44.9 4.27 (3)
0 (1)
c
c
H
30.5 5.10 (3)
5f
5-OH
6-OH
7-OMe
7-Cl
0.00 0.00 (3)
0.00 0.00 (3)
8.17 3.17 (3)
5g
OH
0 (1)
5h23
5i
OH
3.98 2.28 (3)
57.3 9.65 (3)
10.3 2.08 (3)
0 (1)
0
c
b
OH
23.5 2.40 (3)
5j
H
OH
10.5 3.10 (3)
0.00 0.00 (3)
5k
5l24
7-Cl
H
c
c
7-OMe
7-Cl
OMe
OMe
OMe
OMe
OMe
OH
42.1 2.54 (3)
38.6 5.49 (3)
62.1 4.75 (3)
0 (1)
30.9 1.01
28.9 2.29
5m24
5n
H
49.6 4.67 (3)
c
b
7-OMe
7-Cl
H
0.00 0.00 (3)
0 (1)
5o24
5p
H
Cl
0 (1)
c
c
7-Cl
OH
H
H
54.8 2.76 (3)
53.9 2.79 (3)
38.7 1.09
5q25
5r26
5s
7-OMe
7-Cl
OH
OH
H
8.87 3.87 (3)
0.00 0.00 (3)
23.10 3.17 (3)
11.0 2.51 (3)
b
OH
H
b
b
7-Cl
OMe
OH
OMe
OMe
16.6 4.30 (3)
0.00 0.00(3)
0 (1)
23.6 3.29 (3)
5t
7-Cl
H
0.00 0.00(3)
0.00 0.00 (3)
6
727
chalcone
methylated chrysosplenol C
c
8.70 3.00 (3)
16.6 3.05 (3)
21.6 3.90 (3)
b
c
chrysosplenol C 5-OH,6-OH,7-
OMe
OMe
OMe
OH
53.0 4.07 (3)
28.1 1.20
80.4 2.89
omecamtiv mecarbil
400 nM
1 μM
d
b
59.3 2.60 (6)
33.8 4.10 (5)
a
b
c
d
Values represent mean SEM. *P < 0.05. **P < 0.01. ***P < 0.001 vs control (one sample t test). The number in the parentheses indicates
number of cells tested. ATPase activation measurements were done twice.
B
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Letter
a
2.13, ClogP = 2.782), 6-hydroxy (5b, % change in contractility
3.91 2.27, ClogP = 1.882), and 7-methoxy (5c, % change in
Scheme 1. Synthesis of Chrysosplenol C Analogues 5
contractility 60.7
3.10, ClogP = 2.298) analogues were
prepared and studied for their rat ventricular cell contractility
activity (assay briefed in the Supporting Information). Among
these derivatives, 7-methoxy analogue 5c (solubilty in DMSO/
tyrode solution, 17.06 μg/mL) showed the strong contractility
activity and improved aqueous solubility profile compared with
chrysosplenol C (solubilty in DMSO/tyrode solution, 14.08
μg/mL, and unlike 5c chrysosplenol C was partially
precipitated on long-standing in in vitro DMSO/tyrode stock
solution), while 5a and 5b remarkably diminished the activity.
The above results implicated that hydroxy functional groups on
positions 5 and 6 of chromenone ring of chrysosplenol C are
not essential.
a
Reagents and conditions: (a) KOH,90% EtOH, reflux; (b) H2O2,10%
NaOH, dioxane/ethanol (1:1) 5 °C−rt; (c) CH3I, NaH, DMF; (d)
TFA/thioanisole at rt; (e) I2, DMSO, 1 h/100 °C.
To further understand the role of electronic effect of
substituent on A ring, methoxy group of 5c was replaced with
mild electron withdrawing chloro group as shown in 5d (%
change in contractility 43.9 6.83, ClogP = 3.043), and the
activity was retained. However, omission of all the substituents
on A ring as shown in 5e decreased (% change in contractility
30.5 5.10, ClogP = 2.294) the activity. These results indicate
that lipophilic substituents on position 7 of chromenone ring
enhanced the activity.
intermediates 3 were cyclized using Algar−Flynn−Oyamada
reaction22 in the presence of hydrogen peroxide in aq. NaOH
in a mixture of ethanol and dioxane solution at 5−10 °C, which
afforded chromenones 4. Then, methylation of chromenones 4
using methyl iodide and NaH in DMF followed by
deprotection of benzyl group using thioanisole as a catalyst in
trifluoroacetic acid furnished desired compounds 5a−e, 5l−p,
and 5s. Analogues 5f−j, 5q, 5r, and 5t were prepared by
deprotection of benzyl group of the corresponding 4 as shown
in Scheme 2. Compound 5k was obtained by the treatment of
chalcone 3 with iodine in DMSO at 100 °C. Chalcone 6 was
obtained by debenzylation of the corresponding 3.
Next our attention was turned to explore the role of methoxy
group on B ring. This prompted the synthesis and evaluation of
analogues 5f−j. Compounds 5f, 5g, 5h (% change in
contractility 8.2
3.2), and 5j (% change in contractility
a
10.5 3.1) with 3-hydroxy group on the B ring did not show
any activity or showed negligible activity. However, 3-hydroxy
analogue 5i with 7-chloro group exhibited moderate potency
Scheme 2. Synthesis of Compounds 5f−j; 5q,r,t
(% change in contractility 23.5
2.40; ClogP = 3.033).
Elimination of 3-methoxy group as shown in the compound 5k
on the B ring did not show any inotropic activity. These results
indicate that 3-methoxy group is preferred.
Compounds 5c and 5d were considered as lead compounds
for further optimization on the C ring of chrysosplenol C.
Removal of the methoxy group on the C ring of lead
compounds 5c and 5d provided compounds 5l (% change in
contractility 42.1 2.54, ClogP = 2.455) and 5m (% change in
a
Reagents and conditions: (a) TFA/thioanisole at rt.
Compound 7 was prepared as shown in Scheme 3 by
methylating chrysosplenol C using methyl iodide in the
contractility 49.6
4.67, ClogP = 3.216), respectively. Both
a
Scheme 3. Synthesis of Compound 7
compounds 5l and 5m showed nearly 3-fold greater potency
(Table 1) than chrysosplenol C and comparable activity to 5c
and 5d. These results implied that the methoxy substituent is
not necessary for the activity.
Replacement of the hydroxy group on the C ring of 5c and
5d with chloro gave inactive compounds 5n and 5o,
respectively. These results implicate that the hydrogen bonding
donor at C ring should be very important for the activity.
Encouraged by these findings, we pursued further to find the
effect of the substituents on ring C. Hence we prepared
compound 5p by changing the position of the hydroxy of C
ring in 5m from position 4′ to 3′. This compound showed
a
Reagents and conditions: (a) CH3I, NaH, DMF at 5−10 °C.
presence of NaH in DMF solvent. The structures of the
compounds were confirmed by physical and spectral analysis
(see synthesis and characterization of compounds in the
Supporting Information). All the synthesized compounds were
measured for cell shortening activity,18 and the results are listed
in Table 1. The time courses of induced positive inotropic
effect and the effect of the kinetics of contraction and relaxation
for potent compounds 5c, 5l, 5m, and 5p are illustrated in
Figure 2.
In order to identify a potential pharmacophore of
chrysosplenol C, our attempt was begun by investigating the
effect of substituents on A ring (Figure 1) of chrysosplenol C.
Accordingly, 5-hydroxy (5a, % change in contractility 4.23
more potent activity (% change in contractility 54.8
2.75,
ClogP = 3.216) than 5m. This result indicates that hydrogen
bonding donor at position 3′ of C ring may be ideal for the
activity. Even though compounds have a hydroxy group at
position 4′ of the C ring, the existence of hydroxyl group on B
ring resulted in very weak activity as shown in compounds 5q
and 5r. Once again, these confirm that methoxy group on B
ring is very crucial. Replacement of hydroxyl function on
position 3′ of C ring in 5p with methoxy as shown in 5s (%
change in contractility 16.60
4.27) markedly reduced the
C
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Figure 2. Time course of chrysosplenol C analogue-induced positive inotropic effect and the effect of kinetics of contraction and relaxation. (A)
Time course of change in the peak values of cell shortenings in the presence of corresponding compounds. (B) Traces recorded at the time periods
indicated by (1), (2), and (3) in panel A.
activity. This result indicates that the hydrogen bonding
donating property of the hydroxy group on C ring is important.
Replacement of the methoxy group of 5s with hydroxy on
position 3 of chromenone ring as shown in 5t completely
demolished the activity. This again proves that 3-methoxy
function of chromenone ring of these analogues is important
for the activity.
To find the mechanism of action of cardiac contractility of
chrysosplenol C and its analogues, active compounds 5c, 5d, 5l,
5m, and 5p were assessed by sarcomere ATPase assay,28 and
the results are listed in Table 1. All of these analogues showed
significant myosin activation activity compared to chrysosplenol
C. The compound 5c exhibiting the best contractile activity
showed moderate ATPase activation activity and better than
chrysosplenol C. The chloro substituted compound 5d
exhibited most potent activity in ATPase activation as well as
highly potent contractility. Even though 5l and 5m with only 4-
hydroxy substituent on C ring showed the highest level of
cardiac contractility activity, their ATPase activities were
moderate. Interestingly, the compound 5p showed the best
performance in contractility activity as well as ATPase
activation. Although the ATPase activation of chrysosplenol
C analogues are not entirely parallel to their activity in cell
contractilities, cardiac myosin ATPase activation of these
Methylation of all hydroxy groups in chrysosplenol C gave
compound 7 (% change in contractility 8.73
3.00), which
showed less activity. This might be due to masking of hydrogen
bonding donor capability of hydroxyl group at C ring of
chrysosplenol C.
The next set of experiments studied the importance of
chromenone backbone by mimicking it into chalcone 6, which
resulted in complete loss of activity. This might indicate that
planar chromenone with α,β-unsaturated ketone combination is
very important for the activity.
D
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Letter
analogues might be the potential mode of action of their
positive inotropic effect. Thus, chrysosplenol C analogues are
quite unique scaffolds for finding a novel inotropic agent.
The compounds that showed good cell contractility and
myosin activity were analyzed for selectivity in myosins at 10
μM concentration18 (Results are presented in the Supporting
Information). The experiment was done in duplicate and
repeated twice, none of the tested compounds showed
significant activity for myosin ATPase using skeletal and
smooth myosin S1. Thus, from these results it can be assumed
that these compounds are selective for Cardiac myosin S1.
However, more detailed in vivo studies are required for the
same.
(NRF) funded by the Ministry of Education, Science and
Technology (2009-0093815).
Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and spectroscopic data for the compounds
described in this Letter. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acsmedchemlett.5b00043.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jungshh@cnu.ac.kr. Tel: +82 42 821 5939. Fax: +82
42 823 6566.
■
(16) Morgan, B. P.; Muci, A.; Lu, P.-P.; Qian, X.; Tochimoto, T.;
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Tomasi, A.; Valdez, S. C.; Wang, W.; Jiang, H.; Hartman, J.; Rodriguez,
H. M.; Kawas, R.; Sylvester, S.; Elias, K. A.; Godinez, G.; Lee, K.;
Anderson, R.; Sueoka, S.; Xu, D.; Wang, Z.; Djordjevic, N.; Malik, F. I.;
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Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
This work was supported by Priority Research Centers
Program through the National Research Foundation of Korea
(17) Malik, F. I.; Hartman, J. J.; Elias, K. A.; Morgan, B. P.;
Rodriguez, H.; Brejc, K.; Anderson, R. L.; Sueoka, S. H.; Lee, K. H.;
E
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(18) Please see the Supporting Information for the contraction assay
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DOI: 10.1021/acsmedchemlett.5b00043
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