Discovery and Early Clinical Development of an Inhibitor of 5-Lipoxygenase Activating Protein (AZD5718) for Treatment of Coronary Artery Disease

Article abstract of DOI:10.1021/acs.jmedchem.8b02004

5-Lipoxygenase activating protein (FLAP) inhibitors attenuate 5-lipoxygenase pathway activity and reduce the production of proinflammatory and vasoactive leukotrienes. As such, they are hypothesized to have therapeutic benefit for the treatment of diseases that involve chronic inflammation including coronary artery disease. Herein, we disclose the medicinal chemistry discovery and the early clinical development of the FLAP inhibitor AZD5718 (12). Multiparameter optimization included securing adequate potency in human whole blood, navigation away from Ames mutagenic amine fragments while balancing metabolic stability and PK properties allowing for clinically relevant exposures after oral dosing. The superior safety profile of AZD5718 compared to earlier frontrunner compounds allowed us to perform a phase 1 clinical study in which AZD5718 demonstrated a dose dependent and greater than 90% suppression of leukotriene production over 24 h. Currently, AZD5718 is evaluated in a phase 2a study for treatment of coronary artery disease.

Full text of DOI:10.1021/acs.jmedchem.8b02004

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Drug Annotation
Discovery and Early Clinical Development of an Inhibitor of 5-Lipoxygenase-
Activating-Protein (AZD5718) for Treatment of Coronary Artery Disease
Daniel Pettersen, Johan Broddefalk, Hans Emtenäs, Martin A Hayes, Malin Lemurell, Marianne Swanson,
Johan Ulander, Carl Whatling, Carl Amilon, Hans Ericsson, Annika Westin Eriksson, Kenneth Granberg, Alleyn
T. Plowright, Igor Shamovsky, anita dellsén, Monika Sundqvist, Mats Någård, and Eva-Lotte Lindstedt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02004 • Publication Date (Web): 14 Mar 2019
Downloaded from http://pubs.acs.org on March 14, 2019
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Discovery and Early Clinical Development of an
Inhibitor of 5-Lipoxygenase-Activating-Protein
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(AZD5718) for Treatment of Coronary Artery
Disease
*†
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‡
║
Daniel Pettersen , Johan Broddefalk , Hans Emtenäs , Martin A. Hayes , Malin
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Lemurell , Marianne Swanson , Johan Ulander , Carl Whatling , Carl Amilon , Hans
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Ericsson , Annika Westin Eriksson , Kenneth Granberg , Alleyn T. Plowright , Igor
£
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†¥
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Shamovsky , Anita Dellsèn Monica Sundqvist , Mats Någård , Eva-Lotte Lindstedt
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‡
Cardiovascular, Renal and Metabolism IMED Biotech Unit; Pharmaceutical Sciences
║
#
IMED Biotech Unit Discovery Sciences IMED Biotech Unit; Early Clinical Development
§
£
IMED Biotech Unit; Drug Safety and Metabolism IMED Biotech Unit; Respiratory,
Inflammation and Autoimmunity IMED Biotech Unit; AstraZeneca, SE-43183
Gothenburg, Sweden.
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See Present Address.
KEYWORDS: FLAP, 5-lipoxygenase activating protein, inhibitor, 5-LO pathway,
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Leukotriene B LTB , Leukotriene E , LTE , AZD5718, inflammatory disease, coronary
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artery disease (CAD).
ABSTRACT: 5-lipoxygenase activating protein (FLAP) inhibitors attenuate 5-
lipoxygenase pathway activity and reduce the production of pro-inflammatory and
vasoactive leukotrienes. As such, they are hypothesized to have therapeutic benefit for
the treatment of diseases that involve chronic inflammation including coronary artery
disease. Herein, we disclose the medicinal chemistry discovery and the early clinical
development of the FLAP inhibitor AZD5718 (12). Multiparameter optimization included
securing adequate potency in human whole blood, navigation away from Ames mutagenic
amine fragments whilst balancing metabolic stability and PK properties allowing for
clinically relevant exposures after oral dosing. The superior safety profile of AZD5718
compared to earlier frontrunner compounds, allowed us to perform a Phase 1 clinical
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study in which AZD5718 demonstrated a dose dependent and greater than 90%
suppression of leukotriene production over 24 h. Currently, AZD5718 is evaluated in a
Phase 2a study for treatment of coronary artery disease.
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Introduction
The 5-lipoxygenase (5-LO) pathway is responsible for the production of leukotrienes,
lipid mediators derived from arachidonic acid that have inflammatory and vasoactive
actions and that are involved in the innate immune response.1, 2 The pathway is primarily
expressed by a subset of white blood cells including neutrophils, eosinophils,
monocyte/macrophages, and mast cells. Biological actions of leukotrienes include
stimulation of leukocyte chemotaxis, neutrophil activation, promotion of vasopermeability
and vasoconstriction.
The formation of leukotrienes involves oxidation of membrane-released arachidonic
acid by 5-LO to the unstable intermediate Leukotriene A (LTA ) that can subsequently
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be converted via Leukotriene A Hydrolase to Leukotriene B (LTB ) or via Leukotriene
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C Synthase to leukotriene C (LTC ) (Figure 1). LTC may subsequently be converted to
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leukotriene D (LTD ) and the end-metabolite leukotriene E (LTE ) by the action of
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different peptidases.
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FLAP (5-lipoxygenase activating protein) is critical for the production of leukotrienes by
intact cells. Under resting conditions 5-LO is located in the cytoplasm but following an
inflammatory stimulus and release of intracellular calcium it can translocate to the nuclear
membrane where it interacts with FLAP. FLAP facilitates the transfer of arachidonic acid
released from membrane phospholipids to the active site of 5-LO to initiate the production
of leukotrienes.
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Figure 1. The 5-lipoxygenase pathway and the biosynthesis of leukotrienes. FLAP is
located in the nuclear membrane and mediates transfer of the arachidonic acid substrate
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AA) to the active site of 5-LO. cPLA , cytosolic phospholipase A2.
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The 5-LO pathway has been a target for drug development for a number of diseases in
which chronic inflammation is involved in the pathophysiology. This includes respiratory
diseases such as asthma and chronic obstructive pulmonary disease (COPD), but also
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cardiovascular diseases such as atherosclerotic coronary artery disease (CAD). Of
particular interest, treatment with the 5-LO inhibitor VIA-2291 for 6 months resulted in an
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improvement in cardiovascular parameters in CAD patients that had experienced an
acute coronary syndrome (ACS) event around 1 month prior to treatment start.4, 5 This
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included evidence of reduced coronary lesion formation and improvement in left
ventricular ejection fraction (LVEF). On the other hand, in a separate study run as part of
the same Ph2 program, VIA-2291 did not reduce arterial inflammation in an index vessel
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measured indirectly by FDG-PET. Based on this limited clinical experience, more studies
are required to fully address whether 5-LO pathway inhibition can have a therapeutic
benefit in CAD patients.
Inhibition of leukotriene production can be achieved by targeting either 5-LO or FLAP
to block the generation of the leukotriene precursor LTA4.7, 8 FLAP may be a particularly
attractive drug target since FLAP inhibitors block activity of the 5-LO pathway at the initial
step of leukotriene production. Moreover, it has been suggested that FLAP inhibition
might deliver a more robust and sustained suppression of 5-LO pathway activity in vivo
than can be achieved by 5-LO inhibition itself.9
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FLAP inhibitors were first studied in human clinical trials in the 1990s. Following the
initial clinical studies, a number of more recent compounds have entered clinical trials or
have been proposed as clinical candidates.10-13 However, no FLAP inhibitor has
progressed beyond Phase 2 clinical trials. To date, most of the FLAP inhibitors that have
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entered into clinical development, including AM803 , have originated from the chemical
series exemplified by MK-59114, 15 (Figure 2). Development of compounds based on this
compound series were stopped due to safety concerns, low efficacy or sub-optimal ADME
properties.10 AstraZeneca’s earlier explorations of FLAP inhibitors led to the clinical
candidate AZD6642 that due to cardiovascular safety concerns, vide infra, did not
progress to clinical evaluation.16
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S
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Cl
MK-591
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AM803
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Figure 2. FLAP inhibitors
We recently published details of a novel chemical series of FLAP inhibitors, with two
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representative compounds (1 and 2) depicted in Figure 2. Compound 1 was shown to
be a potent FLAP inhibitor with potency and pharmacokinetic (PK) properties suitable for
further preclinical testing. As such, 1 was subjected to 14-day rat and dog toxicity studies
as well as a dog cardiovascular telemetry study. To our disappointment, administration of
compound 1 increased heart rate, blood pressure and cardiac contractility in dogs and
provoked coronary arteritis with a 20-fold estimated margin between NOAEL (No
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Observed Adverse Effect Level) and predicted plasma exposure in humans at an
efficacious dose (vide infra). An association between arteritis and heart rate could be
observed on a group level but the increases in heart rate on an individual level were small.
Even though the mechanisms behind the cardiovascular effects for AZD6642 and 1 may
be different, both compounds induced heart rate increases in dogs. Despite detailed
investigations, the mechanism behind the increases in heart rates is still unknown and no
in vitro signal could be identified. To our knowledge, previous FLAP compounds have not
been reported to have general cardiovascular safety concerns and even though
compound 1 displayed sub-optimal cardiovascular safety in vivo, its cardiovascular safety
profile was better than AZD6642. This suggested that the observed safety concerns were
not due to inhibition of FLAP per se. We therefore decided to continue lead optimization
with the aim of identifying a compound with improved potency and metabolic stability and,
as a result, a lower predicted human dose and increased safety margin. Moreover, the
desired candidate drug target profile demanded a compound without any significant
effects on functional cardiovascular parameters, with a focus on heart rate, and a 100-
fold margin to the predicted free maximum concentration, Cmax, in human. To confirm lack
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of treatment related histopathology findings, such as coronary arteritis, selected
compound was subjected to a thorough in vivo safety profiling in repeated dose toxicology
studies.
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The structure-activity relationship (SAR) previously generated for this chemical series
provided insights and learnings that were beneficial for further optimization.17 For
example, metabolism of the cyclohexyl core via cytochrome P450 (CYP450) metabolism
proved problematic and this could be mitigated by reducing lipophilicity (log D < 3.0). The
presence of the ketone functionality initially caused some concern due to its potential
electrophilic and reactive nature. However, no or low levels of adducts using nucleophiles
such as cyanide, glutathione or methoxylamine, with and without pre-incubation in human
microsomes, were observed for this chemical series indicating no increased risk for
reactive metabolite formation. In addition, the early SAR data generated indicated that
the amino-pyrazole fragment heavily impacted the potency, lipophilicity and metabolic
stability of the compounds and it was therefore decided to focus our optimization efforts
on that specific moiety. As there were no high-resolution crystal structures available for
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the FLAP protein, the optimization work at this stage was driven mainly by structure-
activity and structure-property relationships and ligand-based strategies.14, 18 As part of
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our screening and profiling cascade we utilized a competition binding assay using H-MK-
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91 as the tracer followed by a human whole blood (hWB) assay to study the inhibition
of the 5-LO pathway by measuring the levels of LTB after calcium ionophore stimulation.
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To enable a comparison of the compounds intrinsic activity in the cellular system the
potency was corrected for the protein binding to establish the free potency. In the human
whole blood assay, we and others noticed a time course dependency, especially for
poorly permeable compounds, and we therefore measured LTB inhibition after a 4 h
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compound incubation.11, 17 Besides cellular activity, metabolic stability in human
hepatocytes and permeability in Caco-2 cells were key for compound optimization clearly
motivating a multiparameter design approach to identify a clinical candidate. To select
compounds to progress into further studies, an early dose to man prediction (eD2M) was
used as a ranking tool. We aimed to inhibit the FLAP 5-LO pathway activity at 80% over
a 24 h period and 3xIC50 in the hWB assay was used as the minimum efficacious
concentration (C ) over 24 h. Predicted human clearance (CLhum, pred) was scaled from
ss
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intrinsic clearance data (Cl ) generated in human hepatocytes using well established
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methods (see Supporting Information for details). Bioavailability (F) was estimated
considering hepatic first pass effects only, since absorption was predicted to be complete
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based on the high Caco-2 apparent permeability (P ) values. Scaling factors, constants
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and equations for eD2M prediction are described in the Supporting Information.
Results and Discussion
When comparing compound 1 and the primary amide 2 we realized that the lipophilicity
was alike for the two compounds despite 2 having a more polar substituent on the amino-
pyrazole with compound 2 containing a primary amide and compound 1 containing a
methyl substituent (Table 1). We hypothesized that the lipophilicity could be reduced,
leading to higher metabolic stability, by moving the N-methyl substituent in the amino-
pyrazole ring closer to the primary amide substituent forcing the primary amide slightly
out of plane due to steric effects resulting in a more exposed primary amide. We were
therefore pleased to see that compound 3 had a reduced lipophilicity value, as measured
by log D (2.6 instead of 3.1) whilst maintaining a similar potency to 2 in the competition
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Page 13 of 72
Journal of Medicinal Chemistry
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9
binding assay (IC value = 35 nM). The increased polarity also translated to improved
50
solubility and a higher metabolic stability in human and rat hepatocytes, with the
permeability in the Caco-2 assay remaining at an acceptable level (Table 1) to reach a
reasonable fraction absorbed in rat and dog PK experiments (data not shown). The
overall improved properties for 3 resulted in the eD2M prediction to be considerably lower
compared to previous compounds 1 and 2. Elongating the N-methyl substituent on the
amino-pyrazole ring to an ethyl group resulted in the equipotent derivative 4 suggesting
that expansion in this direction was tolerated. However, the switch to an ethyl substituent
in 4 caused an increase in log D relative to 3 and a dramatic increase in the eD2M
prediction due to the reduced metabolic stability but retained potency. Instead we
introduced a polar group such as an elongated primary amide to give compound 5, which
although active was significantly less active than 3. We reasoned that the primary amide
functionality was worth further explorations and as we had observed that modification of
the angle between the amino-pyrazole ring and the primary amide substituent could have
a large impact on both potency and lipophilicity (cf. compound 2 vs. 3) we explored if the
torsion angle between the amino-pyrazole ring in relation to the cyclohexyl carboxamide
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
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Journal of Medicinal Chemistry
Page 14 of 72
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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
bond could have a similar effect. As a result, a methyl group was introduced into the 3-
position of the amino-pyrazole of 3 to give compound 6 which proved to have a very
promising profile. The potency of 6 in the competition binding assay was slightly reduced
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
(IC50 = 101 nM) but the free potency observed in the human whole blood assay was
significantly improved (IC = 8.0 nM). The metabolic stability was also improved further
50
compared to 3, which resulted in a reduced eD2M prediction. We also prepared the
primary amide 7 having no N-substitution on the amino-pyrazole ring, but this compound
exhibited reduced metabolic stability in human and rat hepatocytes, despite its reduced
lipophilicity compared to 3 and 6.
Metabolic activation of primary aromatic and heteroaromatic amines, such as N-
hydroxylation by P450 enzymes, primarily CYP1A2, and further formation of
bioconjugates by Phase II enzymes, may lead to mutagenic and carcinogenic activity.20,
21
Since hydrolysis of the cyclohexyl carboxamide motif would result in the release of
primary amino-pyrazoles we were keen to understand their potential for mutagenic
activity. Mutagenicity of the primary amino-pyrazoles was evaluated in an Ames bacterial
mutagenicity assay in strains TA98 and TA100, with and without metabolic activation
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Page 15 of 72
Journal of Medicinal Chemistry
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9
2
2
using rat S9 fraction (Table 1). As part of our project strategy we decided to not progress
any FLAP inhibitors having an Ames positive masked amino-pyrazole moiety. To our
disappointment we realized that several of the amino-pyrazoles were Ames mutagenic,
thus having an increased risk for carcinogenic activity. For example, the corresponding
amino-pyrazoles in 3 and 6 where both Ames mutagenic. There are reports suggesting
that genotoxicity of aromatic amines is influenced by their geometric fit to the active site
of CYP1A2 and stability of the anionic intermediates23, 24 as well as by the chemical
reactivity of hydroxylamines and their bioconjugates for DNA basepairs under slightly
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
25
acidic conditions. Therefore, modulation of the electronic character and steric effects of
the amino-pyrazoles could be one way to modulate genotoxicity. However, due to little
genotoxicity data being available for amino-pyrazoles used in the chemical series we
could not verify this hypothesis. We were instead pleased to see some of the amino-
pyrazoles tested to be negative in the Ames test indicating that Ames negative aromatic
amines could be identified within the scope of this chemical series and we adopted the
Ames test of the amino-pyrazoles as part of our screening strategy going forward.
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Journal of Medicinal Chemistry
Page 16 of 72
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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
To build our understanding further we investigated if other polar groups could be used
to replace the primary amide on the amino-pyrazole ring. The carboxylic acid 8 and the
hydroxamic acid 9 both displayed good FLAP binding activity but the potency in the
human whole blood assay was reduced, even if protein binding was compensated for. As
a consequence of their high polarity the acids did not display sufficient Caco-2
permeability, but the data suggested that various polar motifs were tolerated in this area
of the molecule from a potency perspective. To modulate the permeability of 9 we
evaluated less polar and alkylated hydroxamic acids such as 10 which displayed high
potency in both the competition binding assay and in the human whole blood assay in
combination with good Caco-2 permeability, even at the expense of a somewhat reduced
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
ligand lipophilicity efficiency (pIC50 hWBfree - LogD ). The chemical stability for the
7.4
hydroxamic ether 10, containing an O-tBu residue, was surprisingly high and no chemical
decomposition was observed at pH 1, 7 or 10. In addition, 10 was found to be
metabolically stable and no cleavage of the hydroxamic ether could be identified in rat or
human hepatocytes. The sulfonamide 11 also displayed high binding affinity (IC = 34
50
nM) which translated into good inhibition of LTB production in the human whole blood
4
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Page 17 of 72
Journal of Medicinal Chemistry
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9
assay. Following the promising profiles of 10 and 11, both compounds displayed low
eD2M predictions and gratifyingly the amino-pyrazoles in the hydroxamic ester 10 and
sulfonamide 11 were found to be Ames negative.
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
Overall, the SAR data suggested that the use of polar moieties in the 2-position of the
amino-pyrazole such as primary amides, carboxylic acids, sulfonamides and other polar
groups (not listed here) were well tolerated from a potency perspective. However,
analyzing the overall profile and the eD2M predictions, the primary amides 3 and 6 had
preferred profiles but failed the screening criteria as they contained Ames mutagenic
amino-pyrazoles. We expected the amino-pyrazoles in 3 and 6 to have some flexibility
and rotational character around the primary amide bond leading to some degree of
conformational freedom. To reduce the conformational degrees of freedom, the more
conformationally restricted bicyclic amino-pyrazole 12 (AZD5718) was made, which
demonstrated good FLAP binding affinity displaying an IC value of 6.0 nM. Compound
50
12 also showed excellent potency in inhibiting LTB production in the human whole blood
4
assay (IC50free value = 2.0 nM). Overall, compound 12 displayed a promising profile from
a metabolic stability and Caco-2 permeability perspective which was reflected in the
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Journal of Medicinal Chemistry
Page 18 of 72
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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
reduced eD2M prediction and we were pleased to see the corresponding amino-pyrazole
contained in compound 12 to be Ames negative.
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
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Page 19 of 72
Journal of Medicinal Chemistry
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9
Table 1. Profiling of compounds 1-12.
O
R
N
H
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
O
N
N
H
Amino-
LogDa Solub
FLAP
LLEd
Hep.Clinte
h/r
(μL/min/ 10-
Pappf
eD2M Amesh
Bindb/
g
pyrazole
Aq.
hWBfree
Caco2
1E-
2-strain
hWBc
(amino-
mg/da
(-NR)
(µM)
free
6
pyrazole
)
cells)
6.cm/s
y OD
1900
Cmpd
(IC50 nM)
N
N
3
.1
225
60
59/7.4
5.1
4.6
4.0/12.8
20.5
22.0
NEG
NEG
N
H
1
N
N
>1800
0
N
H
3.1
36/22
12.4/14.0
NH2
O
2
N
N
N
H
2
.6
.9
475
329
35/13
29/16
5.3
4.9
3.0/6.5
4.9/8.6
2.6
9.2
170
POS
POS
NH2
O
3
N
N
N
H
2
7800
NH2
O
4
H N
2
N
O
N
2.1
496
241/487
4.2
nd/nd
nd
3500
nd
N
H
5
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Journal of Medicinal Chemistry
Page 20 of 72
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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
N
N
N
H
2.6
399
402
101/8.0
24/96
5.5
4.7
<1.0/5.7
6.7/14.4
1.4
34
POS
nd
NH2
O
6
N
N
>1800
0
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
N
H
2
.3
.2
0.15
NH2
O
7
N
N
>
1800
0
N
H
0
599
224
208
17/570
47/nd
6.0
-
3.2/nd
nd/nd
0.21
nd
nd
nd
O H
O
8
N
N
N
H
O H
2.1
3.3
-
N
H
O
9
N
N
N
H
O
5.8/1.6
5.5
6.0/19.5
14.7
55
NEG
N
H
O
10
N
N
N
H
3.4
79.7
104
34/6.5
6.3/2.0
4.8
4.9/9.7
9.0
3.7
80
44
NEG
NEG
O
S
NH2
1
1
O
N
N
N
H
2.9
5.8
4.2/13.8
N
H
O
1
2
a
See Supporting Information for full details. Octanol/water partition coefficient at pH 7.4,
bIC50 in the FLAP competition binding assay, Calculated as hWB IC50free (IC50 in the
c
human whole blood assay measuring the inhibition of the downstream LTB production
4
after 4 h incubation of the compound) × (compound’s unbound fraction in human plasma
d
(Fu) /100). Fu = 100-human protein binding(%). LLE calculated from pIC hWBfree
5
0
e
minus LogD7.4. Intrinsic clearance of compounds after incubation in human or rat
f
hepatocytes. Passive permeability measured in Caco-2 cells in the A to B direction,
g
h
pH=6.5 Early dose to man prediction based on 3xIC hWB coverage for 24 h. 2 strain
50
Ames test with and without metabolic activation using rat S9. nd = not determined.
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Page 21 of 72
Journal of Medicinal Chemistry
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8
9
Based on the overall properties and the eD2M predictions, compounds 10, 11 and 12
were selected for further profiling. The eD2M prediction, albeit preliminary, indicated a
significant lowering of the human dose which potentially could lead to higher safety
margin for compounds 10, 11 and 12 relative to our previous frontrunner 1.
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
Generally, in this chemical series no issues were observed with inhibition of CYP450
enzymes and compounds 10 to 12 did not inhibit any of the isoforms 1A2, 2C8, 2C9,
2C19, 2D6 or 3A4 (IC50 values > 20 M). No adducts could be seen for 10-12 using
glutathione, cyanide or methoxylamine as trapping agents after compound incubation in
human liver microsomes confirming the inert nature of their respective ketone moiety.
Major metabolic elimination pathways in this series were CYP450 mediated hydroxylation
in the cyclohexyl ring, ketone reduction, N-demethylation in the amino-pyrazole ring and,
less frequently, hydrolysis of the cyclohexyl carboxamide.17 The major metabolic
pathways and contribution of CYP3A4 metabolism for compounds 10-12 are listed in
Table 2. Compounds 10 and 11 were moderately dependent on CYP3A4 metabolism
(66% and 68%, respectively) as seen by the relative contribution of CYP3A4 considering
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Journal of Medicinal Chemistry
Page 22 of 72
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2
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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
a panel of six CYP isoforms 1A2, 2C8, 2C9, 2C19, 2D6 or 3A4. Compound 12 displayed
a higher degree of CYP3A4 dependence (94%) indicating a potential risk for drug-drug
interaction if co-medicated with a CYP3A4 inhibitor. The contribution of CYP3A4
mediated oxidation related to total metabolism was further investigated in hepatocytes as
fraction metabolized by CYP3A4 using ketoconazole as the CYP3A4 inhibitor. The CLint
was reduced by 54% in incubations with ketoconazole in human hepatocytes, suggesting
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
12 to have a low risk as a victim due to drug-drug interactions. This was rationalized due
to the pronounced alternative Phase 2 elimination pathways such as N-glucuronidation
of the parent molecule 12.
The in vivo PK characteristics of compounds 10, 11 and 12 were assessed in the rat
and the dog, Table 2. For compound 10, the oral bioavailability in dog was high (65%) but
lower in the rat (12%) and the half-life following iv administration was 4.7 and 7.0 h for rat
and dog respectively. Compound 11 displayed similar PK properties in the dog, with a
slightly higher volume of distribution and clearance, but a similar half-life to compound 10.
A somewhat higher clearance in the rat contributed to a shorter half-life in rat compared
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Page 23 of 72
Journal of Medicinal Chemistry
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9
to the dog than what was observed for compound 10. For compound 12, the bioavailability
was in line with 10 and 11 in the dog. However, a shorter half-life was observed following
iv administration of compound 12 to the dog (2.1 h). A high clearance contributed to an
even shorter half-life in the rat.
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
Table 2. ADME data for compound 10-12.
Cmp
d
PPBa
h/r
Major
Contr.
3A4c.
OATP-
1B1
inhibd
PK rat/dog (iv)
e
elimination
pathwayb
CL
mL/min/k
g
t1/2
(h)
Vss
F%
%free
(%)
66
(L/Kg)
IC50 µM
10
11
12
0.6/<0.0
4
Hydrox.
Red+gluc.
1.4
2.5
1.2/2.0
10/2.5
36/7.6
4.7/7.0 0.18/0. 12/6
4
5
3.1/1.4
68
94
2.9/6.8 1.3/1.3 14/6
5
5.3/2.4
Hydrox + gluc.
2.3
0.45/2. 1.1/1.1 14/6
1
5
a
See Supporting Information for full details. Plasma protein binding (human or rat).
bHydrox: CYP450 oxidation in the cyclohexyl ring. Red: Ketone reduction to
c
corresponding alcohol. Gluc: Phase II glucuronidation. Contribution of CYP3A4 to
CYP450 mediated metabolism considering a panel of six CYP isoforms (CYP1A2,
d
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). Inhibition of pivastatin uptake to
e
HEK293 cells transfected with human OATP1B1. CL, t and Vss derived from i.v. data,
1/2
F% from p.o.; Doses: rat i.v. 1 µmol/kg for 10, 12; 2 µmol/kg for 11; rat p.o. 2 µmol/kg for
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Journal of Medicinal Chemistry
Page 24 of 72
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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0, 2.8 µmol/kg for 11, 2.5 µmol/kg for 12; dog i.v. 1 µmol/kg for 10, 2 µmol/kg for 11 and
2; dog p.o. 2 µmol/kg for 10 and 12; 4.2 µmol/kg for 11.
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Preclinical safety evaluation
Cell toxicity was generally low in the series and no cell toxicity as measured in THP-1
cells, or mitochondrial impairment as measured in HepG2 cells was seen for 10 to 12
(IC values > 250 µM). Compound 11 and 12 (no data for compound 10) demonstrated
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inhibition of hBSEP and hMrp2 transport proteins with IC50 values of 74 to 340 µM,
respectively, indicating sufficient margins to predicted human Cmax. No activity was
observed in a human aryl hydrocarbon receptor assay or a phospholipidosis assay.
Compounds 10 to 12 did not demonstrate any ion channel interference (hERG, hIKs, hIto,
hNav1.5) (IC values > 33 µM). Compounds 11 and 12 were further evaluated against
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additional ion channels and demonstrated IC values > 32 µM for HCN4, hCav3.2 and
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hKv1.5 (see Supporting Information). The secondary pharmacology profiles and off-target
promiscuity were evaluated using a panel of 83 targets for 10 and 190 targets for 11, 12
(Cerep, France). The three compounds displayed low promiscuity with > 30-fold margins
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to predicted therapeutic exposures except for the adenosine transporter where compound
11 and 12 demonstrated 16- and 12-fold margin, respectively. In addition, compounds in
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this series did not show any effects on the production of other eicosanoids, generated by
the cyclooxygenases COX-1 and COX-2, and compounds 10 to 12 displayed greater than
100-fold selectivity.
Earlier experimental work with the previous lead compound 1 indicated that dog was the
most sensitive pre-clinical species with respect to increases in heart rate. Thus,
compounds 10 to 12 were assessed in anaesthetized beagle dogs for their effects on
heart rate, arterial blood pressure, electrocardiography and contractility parameters. To
our disappointment, compound 10 was found to cause a rapid increase in heart rate and
in left ventricular DP/dtmax (index of cardiac contractility) during the second infusion
period (see Supporting Information for study details). Compounds 11 and 12 did not show
any pronounced increase in heart rate or effect on contractility parameters at the doses
tested and these compounds were therefore evaluated further in conscious dogs.
Compound 11 was found to be poorly tolerated as adverse clinical signs, but not
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cardiovascular related, were observed including repeated vomiting. Compound 12, was
well tolerated and no adverse clinical signs were observed. The effects of 12 on heart
rate, arterial blood pressure, left ventricular parameters, lead II electrocardiogram and
body temperature were assessed in conscious telemetered dogs, following single
intravenous infusions at three dose levels. Intravenous infusion was used to attain a target
plasma concentration 100-fold above predicted free Cmax in humans at the predicted
therapeutic dose. We were delighted that compound 12 did not produce any effects on
the cardiovascular parameters assessed even at the highest achieved exposure (110-
fold to predicted human free Cmax). No treatment related cardiovascular findings (function
or histopathology changes) were observed in the toxicology studies in rat and dog with
up to 28-day duration of treatment.
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Pre-clinical in vivo PK/PD relationship
With the promising safety profile and no adverse cardiovascular effects observed in
dogs for compound 12, we further investigated its ability to inhibit 5-LO pathway activity
when dosed in vivo. The compounds in this series showed no inhibition of 5-LO
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pathway activity in rodent blood but potently inhibited LTB production in dog blood and
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hence dogs were utilized for preclinical PK/PD assessment. Pharmacodynamic
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actions of 12 were initially assessed by measuring ex vivo LTB production in whole
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blood. Full data sets and individual data are provided in the Supporting Information.
Following administration of a single per oral (p.o.) dose (0.9 mg/kg, n=2, Study A), 12
suppressed LTB production by > 90% with maximum inhibition reached within 2.5 h of
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administration. In a second study (B), 12 was administered as two p.o. doses separated
by 48 h (day 1: 0.18 mg/kg, day 3: 0.09 mg/kg, n=2) and a greater than 90% reduction
in LTB production was observed after each dose, with the maximum effect occurring
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between 4 and 9 h after dosing. LTB production returned towards baseline prior to the
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second dose, and reached baseline again 48 h after this dose, indicating reversibility of
FLAP inhibition. In a third study, repeated once daily p.o. administration of 12 at 0.02,
0.09 and 0.2 mg/kg for 3 consecutive days (n=3 per dose; Study C, Figure 3) caused a
concentration dependent inhibition of LTB production throughout the dosing period that
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returned to baseline 24 h after the final dose.
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Figure 3. Median plasma concentrations of 12 and LTB response after repeated once
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daily p.o. administration of 12 at 0.02, 0.09 and 0.2 mg/kg for 3 consecutive days, n=3
per dose; green 0.02 mg/kg, blue 0.09 mg/kg, red 0.2 mg/kg; n=3 per dose. No error
margins given, see Supporting Information for full data set.
In order to also evaluate the effect of 12 on cysteinyl leukotriene production,
endogenous LTE levels in urine (uLTE ) were measured at baseline and during the
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final 24 h in Study C. uLTE levels were undetectable at the two highest doses, and
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suppressed by around 60% at the lowest dose, confirming that 12 also suppresses
cysteinyl leukotriene production (Figure 4).
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Study C, urine LTE 0-24h post last dose
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Figure 4. Inhibition of urinary LTE (uLTE ) levels (Study C) after administration with
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compound 12. One dog in the medium dose group displayed uLTE below LOQ at
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baseline and is excluded from the analysis.
To compare in vitro and ex vivo potency for 12 in dog whole blood, concentration
response curves were generated for the ex vivo assays by plotting exposure against the
baseline corrected LTB level observed (Figure 5). Surprisingly, this analysis revealed
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