Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Article abstract of DOI:10.1021/acs.jmedchem.7b00925

Na,K-ATPase α4 is a testis-specific plasma membrane Na⁺ and K⁺ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca²⁺, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

Full text of DOI:10.1021/acs.jmedchem.7b00925

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Article
Design, Synthesis, in vitro and in vivo Evaluation
of Ouabain Analogs as Potent and Selective Na,K-
ATPase #4 Isoform Inhibitors for Male Contraception
Shameem Sultana Syeda, Gladis Sánchez, Kwon Ho Hong,
Jon E. Hawkinson, Gunda I. Georg, and Gustavo Blanco
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00925 • Publication Date (Web): 01 Jan 2018
Downloaded from http://pubs.acs.org on January 1, 2018
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Design, Synthesis, in vitro and in vivo Evaluation of Ouabain Analogs as Potent and Selective
Na,KꢀATPase α4 Isoform Inhibitors for Male Contraception
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Shameem Sultana Syeda, Gladis Sánchez, Kwon Ho Hong,
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Jon E. Hawkinson, Gunda I. Georg, * and Gustavo Blanco *
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Department of Medicinal Chemistry and Institute for Therapeutics Discovery and Development,
College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA
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Department of Molecular and Integrative Physiology, University of Kansas Medical Center,
Kansas City, KS 66160, USA
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ABSTRACT: Na,KꢀATPase α4 is a testis specific plasma membrane Na and K transporter
expressed in the sperm flagellum. Deletion of Na,KꢀATPase α4 in male mice results in
complete infertility, making it an attractive target for male contraception. Na,KꢀATPase α4 is
characterized by high affinity for the cardiac glycoside ouabain. With the goal of discovering
selective inhibitors of the Na,KꢀATPase α4 and of sperm function, ouabain derivatives were
modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analog 25, carrying a
benzyltriazole moiety at C17, is a picomolar inhibitor of Na,KꢀATPase α4, with an
outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility
2
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in vitro and in vivo, and affected sperm membrane potential, intracellular Ca , pH and
hypermotility. These results proved that the new ouabagenin triazole analog is an effective
and selective inhibitor of Na,Kꢀ ATPase α4 and sperm function.
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INTRODUCTION
Unintended pregnancies have been on the rise in the past years and their management
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represents a priority and a challenge for any public health program. Many of these pregnancies
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end in elective abortions and are frequently associated with physical and emotional
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complications and high economical costs.
It is clear that developing safe, effective and
reversible methods of contraception are needed to enhance birth control options. Currently,
several contraceptive methods are available for women, including hormonal treatment,
intrauterine devices and implants. These approaches place a disproportionate responsibility for
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ꢀ8
birth control on women and the potential risk for complications. It is clear that a more
comprehensive and sustainable family planning program requires extending contraception to
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males. However, male contraceptive methods are basically limited to the use of condoms and
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vasectomy. A safe, effective and reversible contraceptive for men is still unavailable.
An attractive approach to develop a male contraceptive is the targeting of proteins that are
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essential for sperm fertility.
The finding that some proteins are specifically expressed in
sperm provides the additional opportunity to interfere with male fertility, minimizing other toxic
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side effects.
Evidence from our laboratory has shown that Na,KꢀATPase α4 is an attractive
19, 20
target for male contraception.
Na,KꢀATPase is an active ion transport system of the cell
plasma membrane, which utilizes the energy from the hydrolysis of ATP to exchange
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intracellular Na for extracellular K . Structurally, Na,KꢀATPase is a heterodimeric molecular
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complex, constituted by α and β subunits. The α subunit, considered the catalytic subunit of the
enzyme, is a multiꢀpass transmembrane protein of 110ꢀ112 kDa, which contains the binding sites
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for ATP, Na , K , and the cardiotonic inhibitor ouabain. The β peptide is a 40ꢀ60 kDa single
membrane spanning protein, which plays an important role in the folding, stability and targeting
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of the α subunit to the plasma membrane. Several genes, encoding a family of α (α1, α2, α3,
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and α4) and β (β1, β2 and β3) peptides, have been identified in mammals.
Both α and β
subunits are expressed in different combinations, in a cell type specific and developmentally
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regulated manner. Each Na,KꢀATPase αβ pair has different functional characteristics with
respect to their affinities for ions, ATP and ligands. Na,KꢀATPase functional properties mainly
depend on the α subunit composition of the transporter, with each α isoform exhibiting distinct
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functional characteristics. The α4 isoform is the Na,KꢀATPase isoform with the most restricted
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pattern of expression, being uniquely present in male germ cells of the testis. Its expression is
upꢀregulated at postꢀmeiotic stages of spermatogenesis, becoming abundant in the sperm
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flagellum.
Activity of Na,KꢀATPase α4 is essential for maintaining sperm intracellular Na
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levels ([Na ] ), and for the control of several other vital sperm parameters, including membrane
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potential (V ), intracellular Ca ([Ca ] ), and pH. Importantly, Na,KꢀATPase α4 is crucial for
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sperm motility and sperm hyperactivation, a key event associated with sperm capacitation.
Additional information on the role of Na,KꢀATPase α4 in male fertility was obtained through
experiments in genetically modified mice, in which this particular ion transporter was deleted.
The Na,KꢀATPase α4 knockout mice are overall phenotypically normal, and their testes that are
indistinguishable in size and morphology from those of wild type mice. Also, male mice lacking
Na,KꢀATPase α4, are able to produce normal sperm numbers. However, the male mice are
completely infertile due to defects in sperm morphology, motility and hyperactivation. In
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contrast, female mice from the Na,KꢀATPase α4 null colony are fertile.
This shows that,
while α4 is not needed for sperm production, it is an absolute requirement for male fertility. In
addition, this provides strong evidence for the suitability of Na,KꢀATPase α4 as a
pharmacological target for the control of male fertility.
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From a biochemical standpoint, Na,KꢀATPase α4 has functional characteristics that are highly
unique and different from those of the other Na,KꢀATPase isoforms. Compared to Na,KꢀATPase
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α1, α2 and α3 isoforms, α4 has a relatively higher apparent affinity for Na , a lower apparent
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affinity for K and an intermediate affinity for ATP. In addition, Na,KꢀATPase α4 is less
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sensitive to voltage.
The Na,KꢀATPase is a receptor for cardenolides and cardiac glycosides. The cardenolides are a
family of compounds that consist of a steroidal nucleus or aglycone and a fiveꢀmembered
unsaturated lactone ring attached at C17 of the steroid backbone. Cardenolides that contain a
specific sugar moiety attached at C3 of the aglycone are known as cardiac glycosides. Ouabain is
a cardiac glycoside that can be isolated from Strophanthus gratus and Acokanthera schimperi. It
is used extensively in biomedical research and has been used for the therapy of heart attacks and
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for the treatment of patients with left ventricular insufficiency. Ouabain is an endogenous
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steroidal hormone of mammals that is synthesized in the adrenal glands and the hypothalamus.
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An intriguing characteristic of the Na,KꢀATPase α4 isoform is its high affinity for several
cardiac glycosides, including ouabain (Figure 1).
According to our results, ouabain has an IC value in the low nanomolar range for Na,Kꢀ
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ATPase α4, and it is 10,000ꢀfold selective for Na,KꢀATPase α4 over the ubiquitously expressed
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Na,KꢀATPase α1 isoform, which is the only other Na,KꢀATPase present in sperm. These
values correspond to the Na,KꢀATPase of rat, in which the α1 isoform also has low affinity for
ouabain. In humans, the ouabain affinity for Na,KꢀATPase α4 is also high and similar to that of
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rat, but because the human Na,KꢀATPase α1 has a higher sensitivity to ouabain than in rat, the
difference in ouabain affinity between human Na,KꢀATPase α1 and α4 isoforms is narrower.
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Despite this, human Na,KꢀATPase α4 is ~100ꢀfold more sensitive to ouabain than α1. This
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distinct sensitivity for ouabain has been used as a tool to selectively inhibit Na,KꢀATPase α4 and
explore its physiological relevance, independently from that of α1 and other Na,KꢀATPase
isoforms. Thus, blocking Na,KꢀATPase α4 with relatively low concentrations of ouabain
provided the first evidence as to the role of this isoform in sperm motility. The preferential
inhibition of Na,KꢀATPase α4 with ouabain impairs sperm total motility and multiple parameters
of sperm movement, including progressive motility, straight line, curvilinear and average path
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velocities, lateral head displacement, beat cross frequency, and linearity, both in rat and human
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sperm.
The use of higher ouabain concentrations that also inhibited Na,KꢀATPase α1 did not
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cause additional reduction in sperm motility.
These results showed the specific role that
Na,KꢀATPase α4 has in sustaining multiple aspects of sperm flagellar movement. Additional
evidence for the effect of ouabain on male fertility comes from a clinical study suggesting a
possible correlation between endogenous ouabain levels and reduced fertility in humans. Patients
with high endogenous ouabain levels in seminal plasma (26.52 ± 1.82 ꢁg/L) displayed severe
asthenozoospermia compared to normal fertile subjects, who have lower levels of ouabain in
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semen (19.31 ± 1.45 ꢁg/L).
Taken together, these results suggest that ouabain is an attractive chemical scaffold to develop
compounds which can specifically target Na,KꢀATPase α4. Since ouabain itself exerts toxic
effects in the heart, new compounds with greater isoform specificity toward α4 are needed for
the development of safe male contraceptives. Here, we describe a new ouabagenin triazole
analog, which is an effective and selective inhibitor of Na,Kꢀ ATPase α4 and sperm function.
RESULTS
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First, we tested cardiac glycoside containing carbohydrate moieties of different lengths,
including, cymarin and digoxin and the cardenolide strophanthidin (Figure 1). We examined
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their capacity to inhibit the enzymatic function of Na,KꢀATPase α4 by measuring the Na , K
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and Mg dependent hydrolysis of ATP that is sensitive to ouabain. As the source of the enzyme,
we prepared recombinant rat Na,KꢀATPase α4 by expression in Sfꢀ9 insect cells using
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baculoviruses as described previously. This expression system has been extensively used in the
past to study Na,KꢀATPases. It provides the advantage that Sfꢀ9 insect cells have no endogenous
Na,KꢀATPase, therefore, activity of the expressed foreign protein can be studied in an
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environment free of any contaminating Na,KꢀATPase. We infected Sfꢀ9 cells with
baculoviruses that direct the expression of the Na,KꢀATPase α4 and β1 subunits. Whole lysates
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from the cells were used and Na,KꢀATPase activity under saturating amounts of Na , K and
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ATP was measured as previously described. The results showed that strophanthidin (without a
sugar moiety attached at 3ꢀOH), cymarin (one sugar moiety attached at 3ꢀOH) and digoxin (three
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sugar rings attached to 3ꢀOH) inhibited Na,KꢀATPase α4 with similar IC values (10 –10 M)
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compared to ouabain. This indicated that the sugar moiety in cardiac glycoside is a structural
feature that does not significantly influence binding of the compounds to the Na,KꢀATPase α4
isoform.
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Figure 1. A) Structures of ouabain, strophanthidin, cymarin, and digoxin and their IC values
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for Na,KꢀATPase isoform inhibition. IC values for ouabain are taken from reference 35. B)
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Dose response curves for the inhibition of Na,KꢀATPase α4 activity by strophanthidin, cymarin
and digoxin. Values are the mean of two experiments performed in quadruplicate.
Based on these results and the wellꢀknown fact that the C17 substituent is an important
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moiety for binding to Na,KꢀATPase, which forms a hydrogen bond with the receptor, we
designed and synthesized new ouabain analogs in which the aglycone (C3) and the lactone ring
(C17) domains were modified. We investigated the 1,2,3ꢀtriazole moiety as a replacement for the
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lactone moiety. This replacement seemed promising as triazoles can function as hydrogen bond
47
acceptors, and in addition can engage in dipole and π−π interactions. We prepared triazole
analogs in which the steroidal moiety was directly connected to a triazole or linked via a
methylene or a hydroxymethylene spacer (Figure 2). Our efforts to modify the C17 domain also
included the replacement of the 5ꢀmembered α,βꢀunsaturated butyrolactone ring of ouabain with
hydroxymethyl, oxime, nitrile and acid groups. These analogs could be easily prepared from
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intermediates generated during the synthesis of the triazole analogs. As we show in the present
work, the synthetic ouabain derivatives that we have generated inhibit Na,KꢀATPase α4 and
affect sperm motility, both in vitro and in vivo after oral administration to rats.
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Figure 2. Design of ouabain analogs.
The syntheses of the ouabain analogs are shown in Schemes 1ꢀ5. As depicted in Scheme
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1
, commercially available ouabain was converted into the corresponding diacetonide 1,
followed by protection of the hydroxyl groups of 1 with methoxymethyl chloride and
diisopropylethylamine (MOMꢀCl/DIPEA) to yield intermediate 2 in 68%. Ozonolysis of the
lactone olefin in compound 2 followed by hydrolysis of the resulting ester provided an unstable
hydroxymethyl ketone. The crude hydroxymethyl ketone was subsequently reduced with NaBH4
to form a diastereomeric mixture of diols 3 in 42% yield over three steps, which were
subsequently subjected to oxidative cleavage with NaIO to furnish aldehyde 4. Treatment of
4
aldehyde 4 with ethynylmagnesium bromide generated alkynols 5 as an inseparable
diastereomeric mixture (3:1 ratio). Next, the alkynes 5 were subjected to 1,3ꢀcycloaddition with
the respective azides (benzyl and 4ꢀflurobenzyl azides) using click reaction conditions to form
triazoles 6 and 7. Exposure of 6 and 7 to 4N HCl in MeOH resulted in the removal of the
acetonide and MOM groups to yield triazoles 8 and 9 in 42% and 40% yield respectively.
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5
5
5
5
5
5
6
Scheme 1. Synthesis of C17 hydroxymethylene triazole analogs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (a) acetone, conc. HCl, rt, 84%; (b) MOMꢀCl, DIPEA, CH Cl , rt,
2
2
68%; (c) (i) O , –78 °C then Zn/AcOH, CH Cl (ii) KHCO , MeOH, rt (iii) NaBH , MeOH, 42%
3
2
2
3
4
(
for 3 steps); (d) NaIO , THFꢀH O (8:2), rt, 63%; (e) ethynylmagnesium bromide, THF, –78 °C,
4 2
7
4%; (f) benzyl azide or 4ꢀfluorobenzyl azide, Cu SO ⋅5H O (20 mol%), sodium ascorbate (40
2
4
2
mol%), DMF:H O, 6 40%, 7 64%; (g) 4N HCl in MeOH, rt, 8 42%, 9 40%.
2
The C17 hydroxymethyl analog 10 and the nitrile analogue 11 were prepared from aldehyde 4
by reduction of the aldehyde and nitrile formation respectively as shown in Scheme 2. Attempts
for the global deprotection (acetonide and MOMꢀethers) of these compounds were unsuccessful.
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Scheme 2. Synthesis of C17 hydroxymethyl and nitrile analogs
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (a) NaBH , MeOH, 78%; (b) (i) NH OH⋅HCl, NaOAc (ii) CDI,
4
2
CH Cl , 70% (for 2 steps).
2
2
Scheme 3 describes the synthesis of ouabain analogs with modifications at the C3 and C17
positions. The synthesis began by simultaneously removing the sugar and introducing the
4
9
acetonide protecting group with HCl in acetone to generate ouabagenin monoacetonide 12 in
5% yield. Intermediate 12 was transformed to aldehyde 16 in an analogous manner to that
5
described in Scheme 1 (conversion of 2 to 4). The three hydroxyl groups of intermediate 12 were
protected as MOM ethers to obtain 13 in 75% yield. Ozonolysis of 13 followed by hydrolysis of
the resulting ester provided the unstable hydroxymethyl ketone 14 in 63% yield over two steps.
Reduction of 14 followed by oxidative cleavage of the resulting diols 15 with NaIO provided
4
aldehyde 16 in 62% yield. Reduction of aldehyde 16 with NaBH in MeOH furnished alcohol 17
4
in 72% yield, which was converted to the corresponding tosylate in 73% yield. Nucleophilic
displacement of the tosylate group with NaN in DMF provided azide 18 in 70% yield. Click
3
reaction between azide 18 and 4ꢀmethoxyphenyl acetylene provided the corresponding triazole
derivative, which was deprotected with 4N HCl in MeOH to yield the target compound 19 in
5
1% yield.
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of C3 and C17 modified triazolylmethyl analogs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (a) acetone, conc. HCl, rt, 55%; (b) MOMꢀCl, DIPEA, rt, 75%; (c) (i)
O , –78 °C then Zn/AcOH, CH Cl (ii) KHCO , MeOH, rt, 63% (for 2 steps); (d) NaBH ,
3
2
2
3
4
MeOH, 76%; (e) NaIO , THFꢀH O (8:2), rt, 62%; (f) NaBH , MeOH, 72%; (g) TsCl, pyridine,
4
2
4
73%; (h) NaN , DMSO, 60 °C, 70%; (i) 4ꢀmethoxyphenyl acetylene, Cu SO ⋅5H O (20 mol%),
3
2
4
2
sodium ascorbate (40 mol%), DMF:H O, 68%; (j) 4N HCl in MeOH, rt, 51%.
2
The synthesis of ouabain analogs, in which the triazole moiety is directly attached to C17 was
accomplished as shown in Scheme 4. Aldehyde 16 upon reaction with the BestmannꢀOhira
reagent 20 (dimethyl (1ꢀdiazoꢀ2ꢀoxopropyl)phosphonate) furnished alkyne 21 in 71% yield. The
triazole ring was installed by click chemistry between alkyne 21 and benzyl azide, 4ꢀ
1
chlorobenzyl azide, and 4ꢀfluorobenzyl azide in 58%, 56% and 66% yield respectively. H NMR
analysis revealed the formation of triazole diastereomers, indicating that partial epimerization
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occurred at the C17 position during the introduction of the alkyne. The major diastereomers of
5
0
triazoles 22 and 23 could be separated by multiple flash column chromatography.
Diastereomerically pure intermediates 22 and 23 and the diastereomeric mixture 24 were
deprotected with 4N HCl to provide target compounds 25, 26 as single isomers and 27 as a
diastereomeric mixture.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Synthesis of C17 triazole analogs
Reagents and conditions: (a) K CO , MeOH, 71%; (b) benzyl azide, (22, 58%), 4ꢀchlorobenzyl
2
3
azide, (23, 56%), or 4ꢀfluorobenzyl azide (24, 66%), Cu SO ⋅5H O (20 mol%), sodium ascorbate
2
4
2
(
40 mol%), DMF:H O; (h) 4N HCl in MeOH, rt, 25, 47%, 26, 51%, 27, 53%.
2
Scheme 5 shows the synthesis of additional ouabain analogs that were readily accessible from
aldehyde 16. Aldehyde 16 was converted to oxime 28 in 81% yield by reaction with
hydroxylamine and then dehydrated to furnish nitrile 29 in 78% yield. Reaction of aldehyde 16
with TMSCF provided trifluoroethanol analog 30. The propynyl analog 31 was obtained by
3
reaction of aldehyde 16 with ethynylmagnesium bromide in 79% yield. Oxidation of
hydroxyethanone intermediate 14 provided the C17 acid analog 32 in 52% yield.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Synthesis of C17 substituted analogs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (a) NH OH⋅HCl, NaOAc, EtOH, rt, 81%; (b) CDI, CH Cl rt, 78%;
2
2
2
(
c) TMSCF , TBAF, THF, rt, 46%; (d) ethynylmagnesium bromide, THF, –78 °C, 79%; (e)
3
NaIO , H OꢀAcOH (2:1), EtOH, rt, 52%.
4
2
Activity of ouabain analogs as inhibitors of Na,KꢀATPase α4
4
3
The activity of the synthetic ouabain analogs was examined as reported previously, using
recombinant Na,KꢀATPase α4 and β1 subunits expressed in insect cells. Ouabain analogs
ꢀ
5
ꢀ12
inhibited the activity of Na,KꢀATPase α4 with a broad range of potencies, from 10 to 10
M
(Table 1).
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Table 1. IC values for the inhibition of Na,KꢀATPase α4 activity by ouabain analogs
50
a
compound IC (M)
5
0
ꢀ9
ꢀ9
ꢀ6
ꢀ9
ꢀ8
ꢀ8
ꢀ6
ꢀ9
ꢀ8
ꢀ11
ꢀ5
ꢀ9
ꢀ12
Ouabain
4.3, 8.5 x 10
4.0, 4.3 x 10
2.1, 3.7 x 10
4.9, 7.1 x 10
1.2, 7.0 x 10
1.3, 4.4 x 10
1.2, 1.5 x 10
0.6, 1.6 x 10
3.3, 6.2 x 10
1.1, 1.8 x 10
1.5, 3.9 x 10
5.0, 6.9 x 10
1.7, 3.2 x 10
3.2, 4.3 x 10
3.2, 4.9 x 10
1.4, 2.9 x 10
1.6, 3.7 x 10
5.6, 5.9 x 10
1.0, 6.0 x 10
3.9, 7.7 x 10
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
4
6
8
9
1
1
1
1
2
2
2
2
2
2
3
3
3
0
1
7
9
2
5
6
7
8
9
0
1
2
ꢀ8
ꢀ8
ꢀ9
ꢀ5
ꢀ6
ꢀ8
ꢀ8
a
IC values were calculated from dose response curves for inhibition of Na,KꢀATPase α4β1
5
0
expressed in Sf9 insect cells. The values shown are the results of the best fit of the data obtained
from two independent experiments.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
Of the 19 compounds tested, most retained significant inhibitory activity. The most potent
inhibitors were analogs 25, 17 and 10. These three compounds are differently substituted at C3.
Compound 10 carries a protected carbohydrate, compound 17 a methoxymethyl protecting
group, and analog 25 a hydroxyl group, supporting the finding from testing the cardenolides
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 1) that the C3 carbohydrate group is not required for activity and furthermore indicating
that this site can tolerate a variety of groups without reduction in potency. Compound 25 is a
picomolar inhibitor, carrying a C17 Nꢀbenzyltriazole moiety that we introduced as a replacement
of the cardenolide C17 lactone. The introduction of this group enhanced potency significantly,
and as will be shown below, selectivity was enhanced substantially as well (Table 2). The 4ꢀ
chloroꢀ and 4ꢀfluoroꢀbenzyl analogs 26 and 27 were significantly less potent than 25, indicating
that substitution is unfavorable at that position, but they were still 30 nM inhibitors. Compounds
1
0 and 17 that carry a C17ꢀhydroxymethyl group are nanomolar and subnanomolar inhibitors,
respectively, demonstrating that this group effectively replaced the lactone and furthermore
conferred excellent selectivity for the inhibition of the α4ꢀisoform (Table 2). The outstanding
inhibitory properties of compounds 10 and 17 was surprising because they both carry acetonide
and methoxymethyl protecting groups, indicating that modifications at the C1, C19, C11, and
C14 hydroxyl groups unexpectedly did not negatively influence the inhibitory effectiveness of
these compounds. Other potent analogs are the C17 aldehyde 4 and the C17 oxime 28, which
have potency similar to ouabain. Although a range of C17 modifications could be made at C17
without loss of inhibitory activity, other C17 modifications did lead to reduced activity. The C17
modified hydroxymethyl spacer analogs with and without C3 modifications (3, 9, and 30)
showed a significant decrease in activity, except compounds 6, 8 and 31, which displayed double
digit nanomolar activities. The C17 modified nitrile 11 and the C17 carboxylic acid analog 32
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showed about 20ꢀfold reduced activity compared to ouabain. The comparison between C17
carboxylic acid analog 32 and the corresponding nitrile analog 29 demonstrated that the
introduction of the nitrile moiety reduced activity by three orders of magnitude. Similarly, a
comparison between the picomolar inhibitor 25 and the corresponding methylene bridged
triazole analog 19 led to a six orders of magnitude loss of activity. Based on these results we
selected compounds 10, 17 and 25 for further study, because those exhibited high inhibitory
activity for Na,KꢀATPase α4.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Due to their high inhibitory activity, we tested whether compounds 10, 17 and 25 had
preferential effect on the Na,KꢀATPase α4 isoform by measuring their inhibitory activity against
the other Na,KꢀATPase isoforms (α1, α2 and α3), also obtained after expression in Sfꢀ9 cells. As
shown in Figure 3A, 3B and 3C, the ouabain analogs exhibited much weaker inhibitory activity
against the Na,KꢀATPase α1, α2 and α3 isoforms than against Na,KꢀATPase α4.
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2
2
2
2
2
2
3
3
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3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3. Selectivity of ouabain analogs on Na,KꢀATPase α4 over other isoforms. Dose response
curves for the inhibition of Na,KꢀATPase activity by compounds 10 (A), 17 (B) and 25 (C) were
determined on rat α1β1, α2β1 and α3β1 produced in Sfꢀ9 insect cells, and was compared to that
+
+
2+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of α4β1. Hydrolysis of ATP in the presence of saturating concentrations of Na , K and Mg
32
was measured using γ[ P]ꢀATP. The curves represent the best fit of the experimental data,
assuming a single population of binding sites. Each value is the mean ± SEM of three
independent experiments. The corresponding IC values are shown in Table 2 and exhibit a
5
0
much lower affinity, in the microꢀ and millimolar range for Na,KꢀATPases α1, α2 and α3,
compared to the nanoꢀ to picomolar range observed for Na,KꢀATPase α4.
Table 2. Structures of compounds 10, 17, and 25 and their IC values for the inhibition of
5
0
different Na,KꢀATPase isoforms
a
isoform specificity of cardenolides IC (M)
50
compound
α4
α1
α2
α3
+
ꢀ9
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
1
1
0
7
1.6 0.5 x 10
>10
>10
> 10
+
ꢀ11
ꢀ4
+
ꢀ6
1.1 0.6 x 10
> 10 6.6 2.4 x 10 > 10
+
ꢀ12
ꢀ4
+
ꢀ5
25 a
3.2 2.5 x 10
> 10 2.8 1.2 x 10 > 10
The IC values were calculated from dose response curves of inhibition of Na,KꢀATPase
5
0
α1β1, α2β1, α3β1 and α4β1 expressed in Sf9 insect cells. Values are the mean ± SEM of three
independent experiments.
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In order to obtain insight concerning the binding mode of ouabain and its analogs in the
binding pocket, we constructed homology models of the rat α1 and α4 isoforms of Na,KꢀATPase
5
1
using Prime based on the cocrystal structure of the shark derived Na,KꢀATPase with ouabain
4
5, 52
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(PDB ID: 3A3Y).
The identities between the Na,KꢀATPase of the shark renal gland (uniprot
ID: Q4H132) and the human and rat α4 isoforms (uniprot IDs: Q13733 and Q64541,
respectively) are 75% and 74%, respectively. The final homology models were generated via the
5
3
relaxation of the initial models without ouabain using MacroModel, followed by the
incorporation of ouabain and protein preparation with the protein preparation wizard in the
5
4
Schrodinger Suite. Initially, we performed molecular docking studies and compared the
docking poses of ouabain and analog 25 in the Na,KꢀATPase α1 and α4 isoforms (SI Figure 1).
The static docking models did not show preferential binding of ouabain and 25 to the α4 isoform,
but instead revealed that interaction with the α1 isoform (SI Table 1) is disfavored. We therefore
performed molecular dynamics (MD) simulations, which can offer information about relative
55
binding affinities of a ligand and enzyme isoforms. As shown in Figure 4A, Na,KꢀATPase α4
and ouabain form direct Hꢀbonds between Asn129 and 19ꢀOH and Thr804 with 14ꢀOH. The
same amino acids Asp129 and Thr804 of the rat α1 isoform form waterꢀmediated Hꢀbonds with
5
6
ouabain. Thr804 is a crucial aa residue for ouabain binding (Thr797 in the reference). Thr804
of the rat α1 interacts with ouabain with lower frequencies than those of the rat α4 isoform. This
suggested that these interactions might be important for ouabain’s selectivity for the rat α4
isoform over the rat α1 isoform. With respect to compound 25, the 1ꢀOH, 11ꢀOH, and 19ꢀOH
groups maintain direct hydrogen bonding interactions with Asn129 and His118 of the rat α4
isoform with higher frequencies compared to the corresponding aa residues Asp129 and Arg118
of the rat α1 isoform (Figures 4C and 4D). These might be the key interactions for the selectivity
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2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
4
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4
4
4
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4
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5
5
5
5
6
of 25 for the rat α4 over the rat α1 isoform. An additional stabilizing effect is the interaction of
2
5 with Arg887 of the rat α4 isoform compensating for the loss of the hydrogen bonding
interaction with Thr804. The simulation results are consistent with the results from the Na,Kꢀ
ATPase inhibition assay suggesting that the homology model could serve as a tool for the design
of additional ouabain analogs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(A)
(B)
(C)
(D)
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Figure 4. Simulation interaction diagram obtained from the MD simulations of the ligandꢀ
protein complexes (from 5ns to 18ns): (A) ouabain with the rat α4 isoform, (B) ouabain with the
rat α1 isoform, (C) compound 25 with the rat α4 isoform, and (D) compound 25 with the rat α1
isoform. The amino acid residue numbers are based on those for the rat α1 in the uniprot
database (ID: P06685). The solid magenta arrows represent hydrogen bonding interactions with
the backbone of the protein, the dashed magenta arrows hydrogen bonding interactions with the
side chains of the protein, green dotted lines hydrophobic surface, and the blue hashed lines piꢀpi
interactions.
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To determine the effect of compounds 10, 17 and 25 more directly on sperm function, we
tested their capacity to affect rat sperm motility in vitro. Sperm were isolated from the cauda
epididymis of rats, incubated in the absence and presence of 10, 17 and 25 for 1 h and then
sperm motility was determined using computer assisted sperm analysis (CASA). Compound 10,
1
7 and 25 reduced total sperm motility (Figure 5A). In agreement with their IC50 values for
inhibition of Na,KꢀATPase α4 activity, 25 displayed the largest reduction in sperm motility,
ꢀ8
decreasing sperm motility by approximately 60% at concentrations of 10 M and higher (Figure
ꢀ8
5
A). The activity of the compounds on sperm total motility, at a single concentration of 10 M,
showed that 25 displayed a timeꢀdependent reduction in motility (Figure 5B).
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Figure 5. Effect of compounds 10, 17 and 25 on rat sperm motility. A) Dose response curve for
the effect of compounds 10, 17 and 25 on total sperm motility after 1 h incubation. Sperm were
collected from the cauda epididymis of rats and after 1 h incubation, sperm movement was
determined by CASA. Values are the mean ±SEM of three determinations. B) Time
dependence for the effect of 10, 17 and 25 on total sperm motility. Rat sperm were treated
ꢀ8
in the absence (control) or presence of 10 M of the indicated compound. Sperm total
motility was assessed as mentioned in (A). Values are the mean ±SEM of three
determinations. The data points of compound 25, after treatment for 30 min and longer were
statistically different from those of compounds 10 and 17 and the control, with P < 0.001.
In addition, the compounds inhibited other parameters of sperm motility, including progressive
motility, straight line, curvilinear, and average path velocities, linearity and beat cross frequency
(Figure. 6Aꢀ6F). These results show that 10, 17 and 25 not only reduce total sperm motility, but
they also directly block all parameters of sperm movement. As observed for total motility,
compound 25 was the most effective at decreasing all parameters of sperm motility. We also
tested the potential reversibility of effect of compounds 10, 17 and 25. For this, we treated rat
sperm in the absence and presence of each compound and then measured sperm motility, before
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Page 23 of 78
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and after washing the cells three times with medium. This procedure did not significantly affect
sperm motility in the untreated samples; however, the sperm motility reduction caused by 10, 17
and 25 did not recover after the washout, at least for a period of 2 h (Figure 7). Although these
experiments do not directly quantify the reversibility of binding of the compounds to their sperm
target, they suggest that ouabain analogs have a longꢀlived effect on sperm motility, at least for
the time points used in our study.
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Figure 6. Effect of 10, 17 and 25 on different parameters of rat sperm motility. Rat sperm was
collected from the cauda epididymis and treated in the absence or presence of the indicated
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concentrations of each compound. After 1 h incubation, different patterns of sperm movement
were determined by CASA. A) progressive motility, B) straight line velocity, C) curvilinear
velocity, D) average path velocity, E) linearity, and F) beat cross frequency. Values are the mean
0
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±
SEM of three determinations. Asterisks indicate statistically differences between compound 25
vs compounds 10 and 17, with P < 0.05.
Figure 7. Persistent reduction in sperm motility by compounds 10, 17 and 25. Rat sperm,
obtained from the cauda epididymis, was treated in the absence (black circles) and presence of
ꢀ8
10 M of each of the compounds (empty circles, compound 10; grey squares compound 17; and
black triangles, compound 25). Sperm motility was measured by CASA, before and after
washing the cells three times in Tyrode’s modified medium, at the indicated times. Values are
the mean ±SEM of three determinations. Comparison of the data points for each compound and
the untreated controls, as well as among the different compounds showed statistical differences
with P < 0.05. No statistical differences were found for the values of any of the particular
compounds before and after wash.
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+
+
We have previously shown that the Na and K ion gradients created by Na,KꢀATPase α4
activity are essential to maintain vital parameters of sperm function. Na,KꢀATPase α4 is required
2
+
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to maintain sperm plasma membrane potential (V ), intracellular calcium concentration ([Ca ] ),
m
i
32
and cell pH. Accordingly, sperm from mice in which Na,KꢀATPase α4 has been knocked out
2
+
exhibit depolarization of the plasma membrane, higher [Ca ] levels and cytoplasm
i
1
9
acidification. To test whether ouabain derivatives can alter sperm parameters specifically
dependent on Na,KꢀATPase α4, we determined the effect of our most active compound 25 in rat
2
+
ꢀ8
sperm V , pH and [Ca ] . Cells were treated in the absence and presence of 10 M 25 for 1 h
m
i
2
+
19
and sperm V , pH and [Ca ] were determined with different fluorophores as described.
m
i
Compound 25 produced sperm plasma membrane depolarization, increasing Vm by
approximately 40% (Figure 8A). Compound 25 also caused intracellular sperm acidification
2
+
(Figure 8B). Finally, 25 increased [Ca ] in sperm by approximately 40% (Figure 8C). These
i
results agree with the notion that 25 specifically targets Na,KꢀATPase α4.
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Figure 8. Effect of 25 on different biomarkers of Na,KꢀATPase α4 activity. Sperm from the
cauda epididymis was isolated in modified Tyrode’s medium and treated in the absence and
ꢀ8
presence of 10 M 25 for 1 h. Then, (A) sperm V was measured using the fluorescent marker
m
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+
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DiSC3(5), (B) sperm pH was determined with SNARFꢀ1, and C) sperm [Ca ] was assessed by
i
calcium green. Bars are the mean ±SEM of three determinations and asterisks show statistically
significant differences, with P<0.001.
5
7ꢀ60
Besides being essential determinants of sperm flagellar beat,
intracellular pH, V and
m
2+
[
Ca ] , parameters controlled by α4 are required for sperm capacitation. An event associated
I
with sperm capacitation is hyperactivation, a particular pattern of motility which allows sperm to
6
1
gain their fertilizing capacity. Compound 25 significantly reduced the hyperactivation
accompanying sperm capacitation by approximately 70% (Figure 9). These results show that
compound 25 not only reduces sperm motility in general, but also specifically interferes with the
hyperactivation that sperm acquire when capacitated.
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Figure 9. Effect of compound 25 on sperm hyperactivated motility. Sperm from the cauda
epididymis were isolated and capacitated in Tyrode’s modified medium supplemented with
ꢀ8
albumin, bicarbonate and calcium and in the absence (black bar) or presence (grey bar) of 10 M
of compound 25 for 1 h. Sperm motility was determined using CASA. Bars represent the mean
±
SEM of three experiments. Statistical significance between samples treated with or without
compound 25 are indicated with an asterisk, with P values ranging between 0.05 and 0.001.
To determine whether ouabain analogs had activity in vivo, we tested the effect of our most
potent compound by administering 25 to rats by oral gavage. Prior to in vivo testing, several
assays were performed with the compound, including metabolic stability, and toxicity
assessment. Compound 25 had high metabolic stability in liver microsomes, which indicated low
metabolic turnover for the compound (Table 3). Permeability, studied in Cacoꢀ2 cell monolayers
cultured in vitro, showed very low passage of compound 25 from the apical to the basolateral
side of the cells and vice versa. This indicated that the compound has low permeability across
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this epithelium (Table 4). The low in vitro permeability suggests that compound 25 has low oral
absorption consistent with the relatively high oral doses required for in vivo studies using this
picomolar Na,KꢀATPase inhibitor. Antiproliferation dose response assays for compounds 10, 17,
and 25 against MCFꢀ7 breast cancer cells were performed to assess the toxicity of the
compounds using ouabain as the positive control. The tested compounds did not exert any
antiproliferative activity up to 100 ꢁM (SI Figure 2). The effect of ouabain and compounds 10,
0
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7, and 25 was evaluated in a competitive displacement fluorescence polarization assay using
membranes from cells stably expressing hERG (Invitrogen, Carlsbad, CA). The hERG assay was
carried out using Eꢀ4031 and fluoxetine as positive controls. Compounds 10, 17, and 25 did not
inhibit the binding of the fluorescentlyꢀtagged hERG ligand at concentrations up to 60 ꢀM,
whereas ouabain inhibited hERG binding with an IC of 5.8 ꢀM (SI Figure 3). Based on these
5
0
studies, 25 exhibits high metabolic stability, low permeability across epithelia, and it appears not
to possess hERG liability.
Table 3. Metabolic stability of ouabain analog 25 in vitro. Compound 25 was incubated
with liver microsomes and the remaining levels of compounds were determined after the
incubation for 60 min at 37 °C
species
mouse rat
dog
monkey
human
ꢀ6
Test concentration 1x10 M
Parent remaining
76%
95%
99%
89%
107%
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Reference compound data are shown in SI Table 2.
Table 4. Permeability of ouabain analog 25 across epithelia. Cacoꢀ2 cell monolayers
cultured to confluence were used to study both apical to basal and basal to apical
movement of compound 25
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assay
permeability
flag
percent
ꢀ6
st
nd
Test concentration 1x10 M
1
2
Mean
recovery
ꢀ6
ꢀ6
ꢀ6
(
10 cm/s) (10 cm/s) (10 cm/s)
AꢀB Permeability
0.19
0.19
0.47
<0.2
0.3
BLQ 94%
(
Cacoꢀ2, pH 6.5/7.4)
BꢀA Permeability
Cacoꢀ2, pH 6.5/7.4)
0
.14
98%
(
Reference compound data are shown in SI Table 3. BLQ (Below the limit of quantitation). Test
compound was detected in the donor sample but not detected in receiver sample. The
concentration of test compound in the receiver sample was below the limit of quantitation.
We next studied the in vivo effects of 25 by administering the compound to rats by oral gavage
following two different protocols. In one, male rats were treated orally with three doses of
compound 25 (5, 10 and 20 mg/kg) daily for a total of 3 days. In the other, a daily dose of 5
mg/kg weight was given for a duration of 3, 6, 9 or 12 days. After treatment, animals were
sacrificed, sperm was collected from the cauda epididymis and sperm motility was measured by
CASA. Compound 25 inhibited sperm total (Figure 10A) and progressive motility (Figure 10B)
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