Discovery of the fibrinolysis inhibitor AZD6564, acting via interference of a protein-protein interaction

Article abstract of DOI:10.1021/ml400526d

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC₅₀ of 0.44 μM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Full text of DOI:10.1021/ml400526d

Letter
pubs.acs.org/acsmedchemlett
Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via
Interference of a Protein−Protein Interaction
Leifeng Cheng,†,⊥ Daniel Pettersen,*,† Bengt Ohlsson,† Peter Schell,†,# Michael Karle,†
Emma Evertsson,†,○ Sara Pahlen,† Maria Jonforsen,† Alleyn T. Plowright,† Jonas Bostrom,† Tomas Fex,†
́
̈
Anders Thelin,‡ Constanze Hilgendorf,§ Yafeng Xue,∥ Goran Wahlund,‡ Walter Lindberg,§,∇
̈
Lars-Olof Larsson,§ and David Gustafsson‡
‡
§
∥
†Medicinal Chemistry, CVMD iMED, Bioscience, CVMD iMED, DMPK, CVMD iMED, and Discovery Sciences, AstraZeneca
Molndal, SE-43183 Molndal, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: A class of novel oral fibrinolysis inhibitors has
been discovered, which are lysine mimetics containing an
isoxazolone as a carboxylic acid isostere. As evidenced by X-ray
crystallography the inhibitors bind to the lysine binding site in
plasmin thus preventing plasmin from binding to fibrin, hence
blocking the protein−protein interaction. Optimization of the
series, focusing on potency in human buffer and plasma clotlysis
assays, permeability, and GABAa selectivity, led to the discovery
of AZD6564 (19) displaying an in vitro human plasma clot lysis
IC50 of 0.44 μM, no detectable activity against GABAa, and with
DMPK properties leading to a predicted dose of 340 mg twice a
day oral dosing in humans.
KEYWORDS: Fibrinolysis, plasminogen, inhibitor, protein−protein interaction, isoxazolone, tranexamic acid
he physiological role of plasma coagulation is to prevent
Texcessive blood loss from injured vessels, and the role of
fibrinolysis is to dissolve fibrin clots after wound repair.
Fibrinolysis is a cascade reaction with zymogene-to-enzyme
conversion, feedback loops, and inhibitors.1 The key protein in
fibrinolysis is plasminogen, which is transformed into plasmin
by the plasminogen activators (tPA and uPA). The localized
activation of plasminogen and action of plasmin on the fibrin
surface is facilitated by binding of C-terminal lysine residues on
fibrin to lysine binding sites (LBS) in the kringle domains in
plasminogen or plasmin, respectively.2 Inhibition of fibrinolysis
through blockade of LBS on plasminogen is an effective and
safe treatment of bleeding conditions.3 Two LBS inhibitors that
are used clinically, ε amino caproic acid (EACA) and
tranexamic acid (TXA), elicit their effects by preventing the
protein−protein interaction between plasminogen and fibrin
(Figure 1).4
The main use of EACA and TXA are in the treatment of
heavy menstrual bleeding, although reduction of blood loss
during surgery and trauma are other application areas.4 In the
recent CRASH-2 trial, it was demonstrated that TXA can be
administered safely to a wide spectrum of patients with
traumatic bleeding.6 EACA and TXA have also been shown to
have an effect as add-on treatment of mild hemophilia and in
von Willebrands disease.7
Figure 1. ε-Amino caproic acid (EACA), tranexamic acid (TXA), and
4-PIOL.5
attempts to reduce the dose by novel LBS inhibitors8 or
prodrugs of TXA,9 no other LBS inhibitors have reached the
market. Oral dosing of TXA is associated with dose-dependent
gastrointestinal (GI) side effects, including nausea, vomiting,
diarrhea, and cramping. This has limited the use and efficacious
dosing in menorrhagia. There are also indications that
treatment of menorrhagia in von Willebrands disease requires
higher doses, and as a consequence, the dose-dependent side
effects may become efficacy limiting.10 In an attempt to prolong
the exposure of TXA, it has been formulated as a modified
release tablet, Lysteda (1.3 g three times daily), and this has
recently received approval by the FDA.11 Interestingly, GI side
Received: December 20, 2013
Accepted: February 18, 2014
Published: February 18, 2014
TXA is more potent than EACA, but typical TXA efficacious
doses in humans are 1−1.5 g given 3−4 times per day. Despite
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
effects in the treatment groups were not more frequent than in
the placebo control group, indicating that the local amount of
TXA released in the GI lumen may be causative for the GI side
effects.12
Finally, there are rare reports of seizures during treatment
with TXA, probably caused by a weak GABAa antagonistic
activity.12−14 It can be speculated that GI side effects associated
with high dose TXA is also GABAa mediated. Thus, a novel
oral fibrinolysis inhibitor, with the same mechanism of action as
TXA but with more convenient dosing and higher selectivity
over GABAa is hypothesized to be advantageous.
Full length plasminogen (Glu-plasminogen) may exist in a
compact closed conformation and as an open extended
confirmation.15 There are five kringle domains in plasmin-
(ogen), and each has an LBS. The amino acid sequence identity
between the kringle domains are around 70%. The X-ray crystal
structure of the full length plasminogen (pdb code: 4A5T)
revealed that only kringle-1 (K1) is exposed on the surface of
the closed complex, as such available for ligand binding. The
respective LBS from K2, K4, and K5 are engaged in
interdomain interactions contributing to the formation of the
closed conformation, whereas the LBS on K3, being less
exposed, does not engage any lysine from the neighboring
domain. However, in the N-terminal truncated Lys-plasmi-
nogen, having an open conformation, all LBS should be
available for binding. Full length Glu-plasminogen with the
closed conformation has one strong affinity binding site for
TXA (Kd = 1.1 μM, K1) and several low affinity binding sites
(Kd = 750 μM), while elongated Lys-plasminogen has two high
affinity binding sites (Kd = 2.2 μM, K1, and Kd = 36 μM,
probably K4).16,17 TXA binds to plasmin(ogen) noncovalently
by a charge interaction (pdb code: 1CEB). The carboxylic acid
of TXA interacts with two arginines, ARG34 and ARG70, and
the basic piperidine binds to two aspartate residues, ASP54 and
ASP56, in the small and shallow pocket.18 Thus, a novel ligand
would require both positive and negative charges, i.e., to be a
zwitterion, to effectively bind to K1. Although other kringles
may be important in the binding and activation of plasminogen
and subsequent fibrinolysis, this report focuses on the K1
ligand−protein interactions.
Figure 2. Overlay of TXA (light green) and 4-PIOL (dark green) in
the binding site of plasmin(ogen).19,21 The position of 4-PIOL is
generated by docking into the binding pocket using Glide from
Schrodinger.
̈
Table 1. Initial SAR Development around 4-PIOL
a
clotlysis IC50 (μM)
buffer/plasma
GABAa IC50
Caco-2 Papp A to B
b
compd
(μM)
(10−6 cm s−1
)
EACA
105/40
12/3.1
2.8/0.8
100/100
31/40
TXA
1600
35
4-PIOL
<0.01
1.0
1
2
3
4
0.4
5.7/2.0
146/−
6.3/2.1
12.2/−
51
0.4
0.6
0.5
c
In the search for new chemical leads as plasmin(ogen)
inhibitors, we previously reported that 4-PIOL (Figure 1) was
identified via a small-scale virtual screening campaign using an
electrostatic similarity approach using TXA as a seed molecule
(Figure 2).19 4-PIOL was also found to bind to the shallow but
very polar pocket of the K1 domain of plasmin. This interaction
occurs in a similar way to TXA, with ionic interactions to the
basic nitrogens in the two aspartates (ASP54 and ASP56) and
acidic interactions to the two arginine residues (ARG34 and
ARG70).
5
>2000
6
267
a
b
Values are means of three experiments. Top concentration tested
c
was 2000 μM. Compound 5 is the racemic cis-isomer. − = not
measured.
GABAa selectivity and increased permeability. These parame-
ters became the important key factors in our optimization
program described below.
4-PIOL is a very interesting lead compound as it displays
approximately 4-fold improvement of potency in both human
buffer and plasma clot lysis assays compared with TXA (Table
1). However, 4-PIOL has also been reported to be a GABAa
partial agonist.20 In an in-house GABAa binding assay,
measuring displacement of radiolabeled muscimol from rat
brain vesicles, the IC50 was 35 μM. This can be compared to
the IC50 of TXA in the same assay of 1600 μM. Although 4-
PIOL serves as a promising lead from a potency perspective, it
has poor permeability properties as measured in a Caco-2 cell
permeability assay (Papp A to B < 0.01 × 10−6 cm s−1) and
development of a new oral pharmaceutical compound will
require not only optimization of potency but also increased
First, a methyl scanning strategy was performed to evaluate
structure and activity/property relationships (Table 1, synthetic
details for compounds 1−6 can be found in the Supporting
Information).
N-Substitution of 4-PIOL either at the piperidine (1) or
isoxazolone (2) resulted in a drastic decrease in potency
suggesting a more optimal geometry and zwitterionic character
for 4-PIOL as compared to 1 and 2. Further substitution on the
isoxazolone ring, as in 3, was tolerated from a potency
perspective, but no improvement on the GABAa selectivity
could be seen. Methyl substitution in the 4-position of the
piperidine ring (4) decreased potency. However, methyl
substitution in the 2-position of the piperidine ring as in 5
539
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ACS Medicinal Chemistry Letters
Letter
gave a potency comparable with 4-PIOL, and most importantly,
compound 5 dramatically improved the selectivity over GABAa
with no detectable activity at a concentration of 2000 μM,
hence revealing the 2-position as a site for further exploration.
Finally, the regioisomeric isoxazolone 6 also lost potency
compared with 4-PIOL.
Table 2. Stereoisomeric Evaluation of 2,4-Disubstituted
Piperidines 14−17
Encouraged by the improved GABAa selectivity and
equivalent potency of 5 as compared with 4-PIOL, further
substituents in the 2-position of the piperidine ring were
investigated. In Scheme 1, the synthetic route to the benzyl
substituted compounds 14−17 is depicted.
a
clotlysis IC50, (μM)
GABAa IC50 Caco-2 Papp A to B
b
compd
buffer/plasma
(μM)
(10−6 cm s−1
)
14 (2S,4R)
15 (2S,4S)
16 (2R,4R)
17 (2R,4S)
150/−
2.9/0.58
1350
4.1
1.2
1.0
1.9
>2000
>2000
a
Scheme 1. General Synthesis for 14−17
169/−
0.95/0.28
>2000
a
b
Values are means of three experiments. Top concentration tested
was 2000 μM.
a
Reagents and conditions: (a) (i) BnMgCl, THF, (ii) methyl
chloroformate, (iii) HCl; (b) Pd/C, EtOAc; (c) TOSMIC, KtBuO;
(d) conc. HCl; (e) (i) CDI, THF, (ii) MgCl2, potassium ethyl-
malonate, (iii) diastereoisomer separation; (f) (i) NaOH, MeOH,
H2O, −20 °C, (ii) NH2OH, −20 °C, (iii) 6 N HCl, 80 °C; (g) (i)
chiral HPLC, (ii) 33% HBr in HOAc.
The addition of benzyl magnesiumchloride to 4-methox-
ypyridine 7 via the in situ formed 1-acyl pyridinium salt gave,
after acidic workup, the 2-substituted dihydropyridone 8 in a
method analogous to the one pioneered by Comins et al.22
Hydrogenation over Pd/C gave the corresponding piperidone
9 that could be converted to the nitrile 10 by treatment with
TOSMIC.23 Hydrolyis of the nitrile in the presence of the
methylcarbamate could be achieved by conc. HCl to yield the
carboxylic acid 11. The 3-isoxazolone moiety could be prepared
in analogy to the method reported by Krogsgaard-Larsen et
al.24 via formation of the β-keto-ester 12, and subsequent
treatment of 12 with NaOH and then cyclization at −20 °C
using hydroxylamine for the formation of 13. The cis- and
trans-diastereomers were conveniently separated by chroma-
tography on the stage of the β-keto-esters 12, whereas
separation of the enantiomers by chiral HPLC was best
undertaken with intermediates 13 before final removal of the
methyl carbamate with HBr in acetic acid to give the benzyl
substituted isoxazolone derivatives 14−17.
Figure 3. X-ray crystal structure of 17 (blue) binding to plasminogen
(pdb code: 4CIK). The structure was overlaid with the kringle 1/TXA
complex (pdb code: 1CEB). The position of TXA (gray) is shown for
clarity.
The structure shows that 17 binds in the same binding
pocket as TXA, interacting with the same key amino acids. The
isoxazolone carbonyl overlays well with the carboxylic acid
moiety of TXA and is interacting with Arg34, Tyr63, and
Arg70. The piperidine nitrogen of 17 takes a similar position as
the amino group of TXA (about 1 Å apart between the
respective nitrogen atoms), both are within H-bond distance to
Asp54 (2.8 Å) and Asp56 (2.7 Å). Evidently, the benzyl
substituent does not make any specific interactions with the K1
kringle, which is in line with the observed small difference in
potency between the two stereoisomeric compounds 15 and
17. Nevertheless, as indicated by the lower GABAa affinity for
2-substituted 4-PIOL analogues as 14−17, it suggests that the
benzyl groups populate an unfavorable region in the GABAa
binding site. Similar substitution effects on the GABAa affinity
has been observed for a related structural series indicating the
generality of this finding.25
To understand the stereoisomeric significance of substitution
in the 2-position of the piperidine ring, the four stereoisomers
of the benzyl substituted piperidines (14−17) were studied in
more detail (Table 2). It appears that an (S)-configuration at
the 4-position is critical for potency as highlighted with 15 and
17, while the 2-position is more open for both configurations
with 17 (2R,4S), having a cis-conformation, being somewhat
more potent than the corresponding trans-analogue 15.
Introduction of a larger group such as benzyl into the 2-
position resulted in the desired selectivity over GABAa, which
is in line with what was seen for methyl-substituted 5, but also
gave an increased permeability as shown for 14−17.
In order to assess the scope of the 2-substituted 4-PIOL
derivatives, we further evaluated a range of analogues focusing
on the 2R,4S piperidine configuration (Table 3, synthetic
details for compounds 18−25 can be found in the Supporting
Information). Introduction of larger aliphatic groups in the 2-
A crystal structure was obtained for 17 binding to K1 of
plasminogen (pdb code: 4CIK; Figure 3).
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ACS Medicinal Chemistry Letters
Letter
permeability increases with reducing zwitterionic character.
This effect has also been showed for other molecular series.27
For more details on the relationship between pKa and
permeability for several compounds in this article, please see
the Supporting Information.
Table 3. Optimization of the Substituent in the 2-Position of
the Piperidine
Compounds in this series, including 17, 19, and 22, showed
very high metabolic stability in human hepatocytes (<1 μL/
min/106 cells) and liver microsomes (<5 μL/min/mg), as well
as displaying no notable inhibition of CYP450s and no
inhibition of hERG. Biotransformation experiments in human
hepatocytes supported the observed low metabolism. The only
observed metabolism of these compounds, if any could be seen,
was trace amounts of glucuronidation on the isoxazolone ring.
Compounds 17, 19, and 22 were selected for further
exploration in vivo based on the balanced potency, perme-
ability, and GABAa selectivity properties. The pharmacokinetic
profiles of 17, 19, and 22 were studied in rat and dog after i.v.
and oral dosing (Table 4).
GABAa
clotlysis IC50
a
IC
Caco-2 A to B
50b
compd
R
(μM) plasma
(μM)
(10−6 cm s−1
)
17
18
19
20
21
22
23
24
25
Ph
0.37
1.02
0.44
0.77
1.06
0.39
1.26
0.36
0.45
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
1090
1.9
1.0
2.6
4.5
6.2
2.6
1.8
3.1
3.7
iPr
tBu
−CH2C(CH3)3
−C6H11−
−CH2Ph
2-F−Ph
3-F−Ph
4-F−Ph
Table 4. Pharmacokinetic Profiles of Selected Compounds
a
b
Values are means of three experiments. Top concentration tested
was 2000 μM.
CLtot/CLrenal rat
a
CLtot/CLrenal dog
b
a
b
compd
(mL/min/kg)
F% rat
(mL/min/kg)
F% dog
17
19
22
46/11
13/6
24/6
33
39
19
2.9/0.2
1.1/0.8
73
53
59
position resulted in an increase in potency compared with
methyl-substituted 5 as seen for the isobutyl 18 and neopentyl
19 derivatives. Further elongation with an additional methylene
linker (20) gave a slight decrease in potency indicating a
limitation in length and volume of the side chain. Introduction
of an aliphatic ring as in 21 reduced potency. In contrast to 20,
elongation of the benzyl substituent to a phenethyl group was
tolerated as shown for 22 displaying excellent potency as well as
GABAa selectivity and permeability. Fluorine atoms in the
phenyl ring (23−25) were also tolerated, but no significant
improvement in the desired properties was achieved.
The analogues in Table 3 all show a narrow span in potency.
Despite varying the side chain from aliphatic to aromatic,
varying chain length, and introducing fluorine atoms, the
potency varies less than 5-fold. This is in agreement with the
conclusions from the crystal structure of 4-PIOL and 17
(Figure 3) that the substituent in the 2-position does not make
any specific interactions to the protein. However, some steric
limitations could be seen, for example, 20. The small variation
noted could possibly be attributed to differences in solvation
and entropic effects.
Substitution in the 2-position of the piperidine ring led to
more permeable compounds compared to 4-PIOL (Table 3).
Introduction of the 2-substituents led to an increase in
lipophilicity, which has been shown to increase membrane
permeability, especially in the low lipophilicity range of these
derivatives.26 Systematic modification on the substitution
pattern and stereochemistry revealed how the zwitterionic
nature of the compounds contribute to the permeability profile.
This is seen by stereoisomers 14 and 15 in Table 2, where the
experimentally determined pKa difference between the basic
and acidic centers falls from 4.9 for compound 15 to 4.4 for
compound 14, while Papp increases from 1.2 to 4.1 × 10−6 cm
s−1, respectively. For compounds with similar lipophilicity, the
compound with a lower difference between the acid and base
pKa values had the higher Papp value. Although not evident with
classical molecular descriptors related to permeability such as
logD or number of H-bond donors, which are identical for the
stereoisomers, the permeability is also influenced by the pKa
difference between the acid and base within the molecule, and
1.8/0.12
a
b
Doses used: i.v. 5 μmol/kg and oral 20 μmol/kg. Doses used: i.v. 3
μmol/kg and oral 6 μmol/kg.
Compound 17 had higher clearance in rat and dog compared
with 19 and 22. Compound 19 demonstrated good
bioavailability of 39% and 53% in rat and dog, respectively,
whereas compound 22 showed 19% and 59%, respectively.
Investigation of the elimination pathways revealed that between
25% (17 and 22) and 45% (19) of the dose was eliminated
renally in the rat and that between 7% (17) and 70% (19) was
eliminated renally in the dog. To confirm reduction in bleeding
in vivo, compounds 17, 19, and 22 were tested in a rat bleeding
model (please refer to Supporting Information for details). It
could be demonstrated that inhibition of fibrinolysis measured
as the prolongation of the human plasma clot lysis time in vitro
translated to a shortening of tPA-prolonged bleeding time in
vivo (Table 5). For example, compound 19 reduced bleeding
Table 5. In Vivo Fibrinolysis Inhibition EC50 and ED50
Values in a Rat Bleeding Model
a
compd
rat in vivo EC50 (μM)
rat in vivo ED50 (μmol/kg·min)
TXA
17
19.2
0.79
1.62
1.92
15.9
0.65
0.84
1.44
19
22
a
Compounds were infused at four different doses in 6 individual
animals per dose. The ED50 and EC50 were calculated as the infused
dose and plasma concentration giving 50% reduction in bleeding time,
respectively.
times from an incision in the tail by 50% at a 19- and 12-fold
lower dose and plasma concentration, respectively, as compared
to TXA. The corresponding values for 17 and 22 were 25- and
11-fold reductions in dose and 24- and 10-fold decrease in
plasma concentration, respectively.
541
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Letter
From clinical experience, it is known that a total TXA plasma
concentration of at least 30 μM is necessary to achieve the
desired clinical effect suggesting that a 95% receptor occupancy
is required.28 For compound 19, the plasma and blood clot lysis
IC95 estimates fall in the range of 1.5 and 6.6 μM. These results
are used as potency estimates in the dose prediction in man. If,
as observed in rat and dog studies, hepatic metabolism and
glomerular filtration are the routes of elimination the total
human clearance is estimated to be 17.5 L h−1, a composite of
metabolism and renal filtration. Considering the 95% coverage
of LBS and the absorption and clearance properties of 19 gives
a human predicted dose estimate of twice daily dosing of 340
mg.29
drug metabolism and pharmacokinetics; GABA, γ-aminobutyric
acid; TBSCl, tert-butyl-dimethyl silyl chloride; DMF, dimethyl
formamide; DCM, dichloromethane; THF, tetrahydrofuran;
TBAF, tert-butyl ammonium fluoride; CDI, carbonyldiimida-
zole; HPLC, high performance liquid chromatography
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In summary, we have described the discovery of a novel
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and experimental details (1−25). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(D.P.) E-mail: daniel.pettersen@astrazeneca.com. Phone: +46
31 70665663.
■
Present Addresses
⊥(L.C.) E-mail: leifengcheng88@gmail.com.
#(P.S.) GPPS (Global Product and Portfolio Strategy) RiA
(13) Koster, A.; Borgermann, J.; Zittermann, A.; Lueth, J. U.; Gillis-
̈
IMED, Astrazeneca Molndal, SE-43183 Molndal, Sweden.
̈
̈
○(E.E.) Medicinal Chemistry RIA iMED, Astrazeneca Molndal,
Januszewski, T; Schirmer, U. Moderate dosage of tranexamic acid
during cardiac surgery with cardiopulmonary bypass and convulsive
seizures: incidence and clinical outcome. Brit. J. Anaesth. 2013, 110,
34−40.
̈
SE-43183 Molndal, Sweden.
̈
∇(W.L.) DMPK RIA iMED, Astrazeneca Molndal, SE-43183
̈
Molndal, Sweden.
̈
(14) Furtmuller, R.; Schlag, M. G.; Berger, M.; Hopf, R.; Huck, S.;
̈
Author Contributions
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Dagenais, F. High-dose tranexamic acid is an independent predictor of
early seizure after cardiopulmonary bypass. Ann. Thorac. Surg. 2012,
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The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. Project leader L.C.; Lead author D.P.;
Medicinal chemists P.S., D.P., B.O., M.K., S.P., M.J., A.P., and
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
Maria Thorstensson and Ahlke Hayen are acknowledged for
early lead generation work in this project. Ola Fjellstrom is
acknowledged for interesting discussions around the article
topic.
■
̈
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ABBREVIATIONS
■
(19) Bostrom, J.; Grant, A. J.; Fjellstrom, O.; Thelin, A.; Gustafsson,
̈
̈
TXA, tranexamic acid; 4-PIOL, 5-(4-piperidyl)-3-isoxazolone;
EACA, epsilon aminocaproic acid; tPA, tissue-plasminogen
activator; PLG, plasminogen; LBS, lysine binding site; DMPK,
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Cl
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