Discovery of TD-0212, an Orally Active Dual Pharmacology AT1 Antagonist and Neprilysin Inhibitor (ARNI)

Article abstract of DOI:10.1021/acsmedchemlett.8b00462

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy relative to ACE inhibitors but is associated with a higher risk of life-threatening angioedema due to bradykinin elevations. We hypothesized that dual AT₁ (angiotensin II type 1 receptor) blockade and NEP inhibition with a single molecule would produce similar antihypertensive efficacy to omapatrilat without the risk of angioedema since ACE (the rate limiting enzyme in bradykinin metabolism) would remain uninhibited. Merging the structures of losartan (an AT₁ antagonist) and thiorphan (a NEP inhibitor) led to the discovery of a novel series of orally active, dual AT₁ antagonist/NEP inhibitors (ARNIs) exemplified by compound 35 (TD-0212). In models of renin-dependent and -independent hypertension, 35 produced blood pressure reductions similar to omapatrilat and combinations of AT₁ receptor antagonists and NEP inhibitors. Upper airway angioedema risk was assessed in a rat tracheal plasma extravasation (TPE) model. Unlike omapatrilat, 35 did not increase TPE at antihypertensive doses. Compound 35 therefore provides the enhanced activity of dual AT₁/NEP inhibition with a potentially lower risk of angioedema relative to dual ACE/NEP inhibition.

Full text of DOI:10.1021/acsmedchemlett.8b00462

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of TD-0212, an Orally Active Dual Pharmacology AT1
Antagonist and Neprilysin Inhibitor (ARNI)
R. Murray McKinnell,* Paul Fatheree, Seok-Ki Choi, Roland Gendron, Keith Jendza,
Brooke Olson Blair, Joe Budman, Craig M. Hill, Laxminarayan G. Hegde, Cecile Yu, Donavon McConn,
Sharath S. Hegde, Daniel G. Marquess, and Uwe Klein
Theravance Biopharma US Inc., 901 Gateway Boulevard, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat
produces superior antihypertensive efficacy relative to ACE inhibitors but is associated with a higher risk of life-threatening
angioedema due to bradykinin elevations. We hypothesized that dual AT1 (angiotensin II type 1 receptor) blockade and NEP
inhibition with a single molecule would produce similar antihypertensive efficacy to omapatrilat without the risk of angioedema
since ACE (the rate limiting enzyme in bradykinin metabolism) would remain uninhibited. Merging the structures of losartan
(an AT1 antagonist) and thiorphan (a NEP inhibitor) led to the discovery of a novel series of orally active, dual AT1 antagonist/
NEP inhibitors (ARNIs) exemplified by compound 35 (TD-0212). In models of renin-dependent and -independent
hypertension, 35 produced blood pressure reductions similar to omapatrilat and combinations of AT1 receptor antagonists and
NEP inhibitors. Upper airway angioedema risk was assessed in a rat tracheal plasma extravasation (TPE) model. Unlike
omapatrilat, 35 did not increase TPE at antihypertensive doses. Compound 35 therefore provides the enhanced activity of dual
AT1/NEP inhibition with a potentially lower risk of angioedema relative to dual ACE/NEP inhibition.
KEYWORDS: Angiotensin, AT1 antagonist, neprilysin inhibitor, dual pharmacology, heterodimer
ngiotensin II is a potent vasoconstrictive peptide in the
Arenin angiotensin system (RAS) that activates the G-
protein coupled AT1 receptor.1 Other primary actions of
angiotensin II include stimulation of aldosterone secretion by
the kidneys, renal reabsorption of sodium,2 and cardiac
stimulation.3 An AT1 receptor antagonist (ARB) blocks the
vasoconstrictor and aldosterone-secreting effects of angiotensin
II by selectively blocking binding of angiotensin II to the AT1
receptor.4 Numerous ARBs (Figure 1) have been described in
the literature, and several have been approved by the FDA for
Figure 1. Selected FDA-approved ARBs.
the treatment of conditions such as hypertension and heart
failure.5
is a zinc metalloprotease enzyme,6 inhibition of which
Natriuretic peptides [atrial natriuretic peptide (ANP), brain-
potentiates the activity of the cardiac natriuretic peptide system
derived natriuretic peptide (BNP), and C-type natriuretic
peptide (CNP)] effect peripheral vasodilation, increased renal
sodium excretion, reduction of RAS activity, and promotion of
antihypertrophic and antifibrotic effects, which are mediated
through activation of guanylate cyclase signaling pathways. NEP
Received: October 4, 2018
Accepted: December 3, 2018
Published: December 3, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00462
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Table 1. Exploration of S1′ and Biphenyl Substitution
Figure 2. Selected NEP and ACE/NEP inhibitors.
a
Scheme 1. Synthesis of Losartan−Thiorphan Heterodimer 1
a
Reagents and conditions: (a) EDC, HOBt, DIPEA, DMF, rt; (b)
NaOH, MeOH, rt.
(CNPS) by inhibiting breakdown of the natriuretic peptides,
mainly ANP.7,8 To date, only one NEP inhibitor (sacubitril,
Figure 2) has been approved by the FDA for clinical use (in a
fixed-dose combination with the ARB valsartan).
Since the RAS and the CNPS are interconnected and both are
critical in cardiovascular homeostasis, simultaneous inhibition of
both systems has the potential to provide superior reductions in
blood pressure, increased responder rates, improved cardiac
performance, reduction of target-organ damage, and improve-
ments in long-term outcomes in hypertensives and chronic heart
failure (CHF) patients.
Dual inhibition of the RAS and CNPS with a single molecule
has been attempted by different companies in a number of R&D
programs. Omapatrilat is an agent that inhibits both ACE (RAS)
and NEP (CNPS). Clinical hypertension trials established its
efficacy in patients with mild to moderate hypertension, and the
25,000 patient OCTAVE study demonstrated broadly superior
antihypertensive efficacy of omapatrilat relative to the ACE
inhibitor enalapril.9 Heart failure trials showed comparable
efficacy between omapatrilat and lisinopril (another ACE
inhibitor) in improving exercise tolerance in patients with
NYHA class II/III heart failure, with a strong suggestion that
omapatrilat improved clinical outcomes in heart failure to a
greater extent than lisinopril (IMPRESS trial).10 However, the
3-fold higher incidence of angioedema observed for omapatrilat
relative to lisinopril and the incidence of life threatening
angioedema in the OCTAVE study led to an unfavorable risk−
benefit profile and the program was discontinued in 2002. While
ACE is the predominant rate limiting enzyme in the metabolism
of bradykinin, NEP, APP, DPP-IV, and other zinc metal-
lopeptidases also play a role in bradykinin breakdown. The
prevailing hypothesis is that dual inhibition of ACE and NEP by
omapatrilat results in significantly greater elevation of
bradykinin levels relative to ACE inhibition alone, which in a
subset of patients can lead to excessive vascular leakage, fluid
extravasation, and angioedema.11
B
DOI: 10.1021/acsmedchemlett.8b00462
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Table 2. Exploration of Imidazole Headgroup and Biphenyl Substitution
AT1
pKi
NEP
pIC50
% inhibition of ang-II pressor response (3 urinary cGMP fold increase over vehicle
compound
R3
R4
R5
R6
n
mg/kg, iv)
(3 mg/kg, iv)
losartan
omapatrilat
7
8.3
<5
<5
9.7
7.2
9
67
5.3
2.2
Cl
Cl
Me
Me
Et
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
PrO
PrO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
0
7.8
7.2
7.3
6.7
8.3
7.4
8.2
7.5
8.1
7.5
8.6
7.7
8.9
8.2
9.1
8.9
8.9
8.3
8.3
11
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
7.1
9.2
6.9
8.5
6.9
9.0
6.9
7.7
7.3
9
68
18
55
5
2.6
3.2
1.8
4
Et
Cyclopropyl
Cyclopropyl
i-Bu
i-Bu
Et
Et
Et
Et
Et
Et
Et
Et
Et
32
65
23
67
57
85
76
1.9
1.9
3.8
2.9
3.6
1.8
3.5
7.5
9
7.8
9.2
7.1
8.5
7.8
F
H
H
F
F
F
57
3.3
Our expectation was that inhibition of the RAS with an AT1
antagonist/NEP inhibitor (ARNI) rather than an ACE/NEP
inhibitor would result in an efficacy profile similar to that of
omapatrilat without the risk for bradykinin elevation since ACE
would remain uninhibited. A similar strategy led to the approval
in 2015 of Entresto (LCZ696, a fixed dose 1:1 coformulation of
the ARB valsartan with the NEP inhibitor prodrug sacubitril) for
the treatment of CHF.12 An alternative to coformulation is to
design a molecule with potent activity at two different targets.13
Potential benefits of this approach include matched pharmaco-
dynamic (PD) effects on the targets of interest, thereby
maximizing the potential synergy between the two mechanisms,
and reduced potential for drug−drug interactions compared to
drug cocktails. We therefore set out to design an ARNI by
chemically merging the structure of an ARB with that of a NEP
inhibitor.
The 5-position of the imidazole ring in the ARB losartan has
been shown to accommodate a wide range of substituents,
including some that provide a second pharmacological
activity.14−18 Using a similar strategy, we linked a losartan
analog (Intermediate A) to the terminal carboxylic acid of
thiorphan, a thiol-based inhibitor of NEP. While thiol-based
drugs are associated with complex PK/PD relationships and
development risks,19 thiorphan was preferred over other NEP
inhibitors due to its low molecular weight and high potency
(Scheme 1). Heterodimer 1 maintained potent binding affinity
at the AT1 receptor relative to losartan (Table 1) but was
significantly less potent than thiorphan in the NEP inhibition
assay.
This data suggested that the AT1 active site was more tolerant
than that of NEP with respect to addition of a second
pharmacophore. Removing the glycine spacer (compound 2)
led to a minor loss of potency at both targets. However, the
lower molecular weight and reduced conformational flexibility
of 2 was considered more conducive to achieving oral activity,
and so 2 became the focal point of our early optimization efforts.
Assuming the thiorphan fragment of 2 binds to NEP in a
similar fashion to thiorphan itself, the benzyl substituent (R2) of
2 would occupy the S1′ subsite of the enzyme. Replacement of
the benzyl group with another S1′ substituent commonly utilized
in NEP inhibitors (rac-isobutyl, 3) had little impact on potency
at either target (Table 1). Surprisingly, the first significant
improvement in NEP potency was achieved by modification of
the ARB region of the molecule. Replacing the tetrazole moiety
(R1) on the biphenyl fragment with a carboxylic acid (5 vs 3)
afforded a 1.7 log unit gain in NEP pIC50 when the S1′ group was
rac-isobutyl. Synthesis of the individual enantiomers of 5
revealed that the AT1 receptor had a clear preference for S-
enantiomer 7. An extensive survey of other alkyl and aromatic
groups in the S1′ pocket (compounds 8−19) demonstrated that
the S-isobutyl group maintained the best balance of potency at
the two targets. Replacing the β-thiol zinc-chelator (7) with an
C
DOI: 10.1021/acsmedchemlett.8b00462
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a
Scheme 2. Synthesis of 35 (TD-0212)
Figure 3. Comparison of 24 h average MAP reductions following oral
administration of 35, omapatrilat, valsartan, or a valsartan/candoxatril
combination in conscious SHR.
a
Reagents and conditions: (a) SEMCl, DIPEA, DCM, rt; (b) KOt-
Bu, EtOH, reflux; (c) DMF, n-BuLi, THF, −78 °C; (d) TFA, 0 °C;
(e) K2CO3, DMF; (f) Pd(PPh3)4, (C2H3)Sn(n-Bu)3, DMF, 90 °C;
(g) NH2OH.HCl, pyridine, H2O; (h) H2, Pd(OH)2, EtOH; (i)
NaBH3CN, TiCl3, NH4OAc, MeOH, 0 °C; (j) (S)-2-(acetylthio)-4-
methylpentanoic acid, HATU, DIPEA, DMF, rt; (k) TFA, DCM, rt;
(l) NaOH, MeOH, H2O, rt.
α-thiol (21) increased NEP potency to a level similar to that of
thiorphan, but simultaneously reduced AT1 potency. Since the
α-thiols (low AT1, high NEP activity) provided a distinct profile
to the β-thiols (high AT1, low NEP activity), both series were
progressed to SAR studies focused on the imidazole headgroup
substituents.
Replacement of the imidazole 4-Cl substituent [R3, (Table
2)] with a methyl group (23, 24) reduced the AT1 activity in
both the α- and β-thiol series. The bulky i-Bu (28) group was not
tolerated by NEP in the α-thiol series. Intermediate-size alkyls
such as ethyl (24) and cyclopropyl (26) were relatively well-
tolerated by both active sites. At the 2-position of the imidazole
(R4), insertion of an oxygen atom into the alkyl chain (30 vs 24)
was slightly beneficial in terms of both AT1 and NEP potency.
An additional gain in AT1 potency was realized by shortening the
alkoxy chain from four atoms to three atoms (32 vs 30). The
AT1 potency of this ARNI series was now greater than that of the
parent ARB, losartan. Using compounds 32 and 33 as starting
points, a limited substitution scan on the biphenyl fragment was
performed. Adding a fluorine atom ortho- to the headgroup
maintained NEP activity and significantly boosted AT1 potency
in the α-thiol series (35 vs 33). Alternative fluorine substitution
Figure 4. Comparison of antihypertensive efficacy and duration
following oral administration of 35 or omapatrilat in conscious
DOCA-salt hypertensive rats.
patterns were neutral at AT1 but deleterious to NEP relative to
the parent molecule. Biphenyl substitutions did not have a
significant effect in the β-thiol analogs.
To determine the in vivo activity of these novel dual
pharmacology molecules, a rat pharmacodynamic (PD) assay
was developed to simultaneously measure AT1 antagonism via
inhibition of the angiotensin-II-evoked pressor response and
D
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35 (ED10 = 13 mg/kg) was comparable to that of omapatrilat,
Table 3. Selectivity Profile of Compound 35, Valsartan, and
Omapatrilat against Related Targets
valsartan, and the valsartan/candoxatril combination (ED10
15, 17, and 15 mg/kg respectively).
=
potency
In vivo NEP inhibition after oral dosing was determined using
the deoxycorticosterone acetate (DOCA) salt rat model of
hypertension. The DOCA model is insensitive to ARBs and
therefore considered a model for low-renin hypertension.
Compound 35 produced a dose-dependent reduction of blood
pressure in the DOCA model (Figure 4). The potency and
duration of effect of 35 (ED10 = 44 mg/kg) were comparable to
omapatrilat (ED10 = 66 mg/kg).
target
35
valsartan
omapatrilat
a
AT2
5.6
<5.0
ND
ND
ND
ND
10
6.7
ND
b
ACE
APP
<5.0
<5.0
7.1
b
b
ECE-1
a
b
pKi. pIC50.
Additional in vitro studies confirmed that 35 is a potent,
competitive antagonist of the AT1 receptor, with a selectivity for
AT1 over AT2 of approximately 2000-fold (Table 3). In contrast
to losartan, which is classified as a surmountable AT1
antagonist,22 35 was found to be partially insurmountable in
an inositol phosphate accumulation assay (<5% vs 87%
accumulation, respectively). Compound 35 is also a potent,
selective, and competitive inhibitor of NEP, exhibiting 120-fold
selectivity for inhibition of human recombinant NEP over
human recombinant ECE-1, and no measurable activity at
human ACE or APP at a concentration of 10 μM.
In order to assess the risk of angioedema with 35 relative to
that with omapatrilat, a rat tracheal plasma extravasation (TPE)
model was developed based on a method described by Sulpizio
et al.23 In this model, TPE was used as a surrogate to assess the
propensity of compounds to promote upper airway angioedema
by measuring the extent of Evans Blue dye leakage into
peritracheal tissue at different antihypertensive doses. Omapa-
trilat produced a robust increase in TPE at low oral subactive
doses of 0.3 and 3 mg/kg. In contrast, 35 had no effect at doses
of up to 100 mg/kg (Figure 5).
The combined results from in vivo pharmacology studies
indicate that 35 is as effective as omapatrilat in terms of
antihypertensive activity. Unlike omapatrilat, 35 does not
increase tracheal plasma extravasation in rats, which is indicative
of a low risk for causing angioedema. Taken together, 35 can be
described as an ARB-equivalent of omapatrilat with a lower risk
of angioedema. Additional studies with 35 will be reported in
future publications.
Figure 5. Comparison of tracheal plasma extravasation following
administration of vehicle, omapatrilat, or 35 in normotensive rats.
NEP inhibition via potentiation of ANP-induced elevation of
urinary cyclic guanosine monophosphate (cGMP) output
relative to vehicle.20 Compounds were dosed IV in the PD
assay to determine the intrinsic activity of the parent molecule
without oral absorption playing a role. In general, in vivo activity
correlated with in vitro activity (Table 2). Compounds with pKi
values >8.0 at AT1 inhibited the angiotensin-II blood pressure
response to a similar extent as losartan. With respect to NEP
inhibition, pIC50 values of >8.0 generally produced more than 3-
fold increases in urinary cGMP relative to vehicle. None of the
compounds were as active as omapatrilat in this assay, in keeping
with their reduced in vitro potency relative to omapatrilat.
On the basis of its activity in the PD assay and rat
pharmacokinetics (see Supporting Information), compound
35 (Scheme 2) was advanced to a dose−response study in the
spontaneously hypertensive rat (SHR) model, which is known
to be sensitive to ARBs.21 In this model, telemetry is used to
monitor blood pressure for a period of 24 h after oral dosing of
the compound. Efficacy was determined by the peak percent fall
in mean arterial pressure (MAP) from baseline, while duration
of action was reflected by the vehicle-adjusted % change in AUC
over 24 h. Administration of sequential escalating doses of 3, 10,
30, and 100 mg/kg of 35 at 24 h intervals between the successive
doses produced dose-dependent reductions in MAP (Figure 3).
At doses of 10 mg/kg and above, the duration of effect was
sustained for 24 h. In comparison to vehicle treatment the effect
was statistically significant at 10 mg/kg and higher doses (p <
0.05, two way ANOVA with Bonferroni post hoc analysis).
Omapatrilat and valsartan produced a dose-dependent
reduction in MAP similar in magnitude to 35. The addition of
the NEP inhibitor prodrug candoxatril (at a dose of 100 mg/kg)
to valsartan did not result in further blood pressure lowering in
the SHR model relative to valsartan alone, which is consistent
with its non-RAS action. The estimated ED10 (dose required to
produce an average 10% reduction in the 24 h average MAP) for
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00462.
In vitro and in vivo assay procedures; representative
synthetic procedures and compound characterization
data (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mmckinnell@theravance.com.
ORCID
R. Murray McKinnell: 0000-0003-2828-1046
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
E
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ACKNOWLEDGMENTS
The authors would like to thank David Beattie, Venkat Thalladi,
and Zhengtian Gu for assistance preparing this manuscript.
■
ABBREVIATIONS
■
AT1, angiotensin II type 1 receptor; ACE, angiotensin-
converting enzyme; NEP, neprilysin; TPE, tracheal plasma
extravasation; RAS, renin angiotensin-system; ARB, AT1
receptor antagonist; ANP, atrial natriuretic peptide; BNP,
brain-derived natriuretic peptide; CNP, C-type natriuretic
peptide; CNPS, cardiac natriuretic peptide system; APP,
aminopeptidase P; DPP-IV, Dipeptidyl Peptidase IV; CHF,
chronic heart failure; SHR, spontaneously hypertensive rat;
MAP, mean arterial pressure; DOCA, deoxycorticosterone
acetate; cGMP, guanosine monophosphate; ED10, dose required
to produce an average 10% reduction in the 24h average MAP;
ECE, endothelin converting enzyme 1
́
Sanchez-Alonso, P.; Griera, M.; Vaquero, J. J.; Rodriguez-Puyol, D.;
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DOI: 10.1021/acsmedchemlett.8b00462
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