Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1 S,2 S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma

Article abstract of DOI:10.1021/acs.jmedchem.9b00555

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC₅₀ = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC_50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC_50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

Full text of DOI:10.1021/acs.jmedchem.9b00555

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Article
Discovery of the Oral Leukotriene-C4 Synthetase Inhibitor (1S,2S)-2-
(
{5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-
methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic
acid (AZD9898) as a New Treatment for Asthma
Magnus Munck af Rosenschöld, Petra Johannesson, Antonios Nikitidis, Christian Tyrchan, Hui-Fang
Chang, Robert Rönn, Dave Chapman, Victoria Ullah, Grigorios Nikitidis, Pernilla Glader, Helena Käck,
Britta K. Bonn, Fredrik Wågberg, Eva Björkstrand, Ulf Andersson, Linda Swedin, Mattias Rohman,
Theresa Andreasson, Eva Lamm Bergström, Fanyi Jiang, Xiaohong Zhou, Anders Lundqvist,
Anna Malmberg, Margareta E Ek, Euan Gordon, Anna Pettersen, Lena Ripa, and Andrew Mark Davis
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00555 • Publication Date (Web): 15 Aug 2019
Downloaded from pubs.acs.org on August 15, 2019
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of their duties.

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Gordon, Euan; AstraZeneca Pharmaceuticals, Inovative Medicines, DECS
Pettersen, Anna; AstraZeneca FoU Goteborg,
Ripa, Lena; AstraZeneca R&D Mölndal, Respiratory and Inflammation
Innovative Medicines
Davis, Andrew; AstraZeneca R&D Mölndal, Respiratory and Inflammation
Innovative Medicines
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Discovery of the Oral Leukotriene-C4 Synthetase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-
difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-
yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) as a New Treatment for Asthma.
0
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a
a
a
a
Magnus Munck af Rosenschöld , Petra Johannesson , Antonios Nikitidis , Christian Tyrchan ,
a
f
a
a
b
Hui-Fang Chang , Robert Rönn , Dave Chapman , Victoria Ullah , Grigorios Nikitidis , Pernilla
a
c
a
c
f
d
Glader , Helena Käck , Britta Bonn , Fredrik Wågberg , Eva Björkstrand , Ulf Andersson , Linda
a
c
a
a
a
Swedin , Mattias Rohman , Theresa Andreasson , Eva Lamm Bergström , Fanyi Jiang , Xiao-
a
a
a
c
c
Hong Zhou , Anders J. Lundqvist , Anna Malmberg , Margareta Ek , Euan Gordon , Anna
e
*a
*a
Pettersen , Lena Ripa , Andrew M. Davis
a
Early Respiratory, Inflammation & Autoimmunity, AstraZeneca Biopharmaceuticals SE-43183,
Mölndal, Sweden.
bEarly Chemical Development, Pharmaceutical Sciences, AstraZeneca Biopharmaceuticals, SE-
4
3183, Mölndal, Sweden.
cDiscovery Sciences, AstraZeneca Biopharmaceuticals, SE-43183, Mölndal, Sweden.
Sweden.
dClinical Pharmacology and Safety Sciences, AstraZeneca Biopharmaceuticals, SE-43183,
Mölndal, Sweden.
eEarly Product Development, Pharmaceutical Sciences, AstraZeneca Biopharmaceuticals SE-
4
3150, Mölndal, Sweden.
fOrexo AB, Virdings allé 32A, 754 50 Uppsala, Sweden
*Corresponding authors
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andy.davis@astrazeneca.com
lena.e.ripa@astrazeneca.com
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Abstract
While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to
0% of asthmatics remain uncontrolled. Many studies show the cysteine leukotriene cascade
5
remains highly activated in some asthmatics, even those on high dose inhaled or oral
corticosteroids. Hence inhibition of LTC4S enzyme could provide a new and differentiated core
treatment for patients with a highly activated cysteine leukotriene cascade. Starting from a
screening hit 3, a program to discover oral inhibitors of LTC4S led to AZD9898 (36), a
picomolar LTC4S inhibitor (IC = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5),
5
0
which displays nanomolar potency in cells (PBMC IC50,free = 6.2 nM) and good in vivo
pharmacodynamics in a calcium ionophore stimulated rat model after oral dosing (in vivo IC50,free
=
34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo
toxicology findings of an early lead compound 7, with a human dose predicted to 30 mg q.d.
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Introduction
While inhaled corticosteroids are the mainstay controller treatment for asthma, many patients
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,2
remain uncontrolled even on escalation to high dose inhaled corticosteroids . Opportunities exist
for new therapies which could be targeted towards these patients, and fill a much-needed
therapeutic niche between inhaled steroids and progression to oral steroids or parenteral antibody
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therapies . Many studies demonstrate that in patients on high dose inhaled or even oral steroids,
4
,5
the arachidonic acid cascade remains highly activated , and new treatments targeting key points
on the arachidonic acid cascade could provide benefit to asthmatics with a highly activated
arachidonic acid cascade, despite current medication.
The arachidonic acid cascade forms a plethora of proinflammatory and pro-resolution lipid
metabolites including prostaglandins, thromboxanes and leukotrienes involved in inflammation,
6
bronchoconstriction and pain . In particular, the cysteinyl leukotrienes are strongly associated
7
with asthma , as demonstrated by the clinical efficacy of cysteinyl leukotriene modifier drugs
8
,9
10
such as the cysteinyl leukotriene receptor 1 antagonists, montelukast 1, zafirlukast and
1
1
12
pranlukast , and the 5-lipoxygenase inhibitor zileuton 2. While these leukotriene modulators
have achieved clinical utility, they have not succeeded in becoming central to the treatment
13
pathway in the global asthma treatment guidelines but remain an option. With such a complex
cascade, it remains an open question whether the optimum point to intervene has been found with
the current drugs.
The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are formed from arachidonic acid in several
steps, Figure 1. The enzymes 5-lipoxygenase (5-LO) and 5-lipoxygenase activating protein
(FLAP) catalyze the formation of leukotriene A4 (LTA4) from arachidonic acid, which is
converted into LTC4 through the conjugation with glutathione by the enzyme LTC4S 14, 15, 16, 17.
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LTC4 is exported from cells and is rapidly metabolized into LTD4 by a gamma-glutamyl-
1
8
transpeptidase and LTD4 into LTE4 by dipeptidases . Instead of conjugation to glutathione, the
reactive epoxide of LTA4 can also be opened by the enzyme LTA4H to generate LTB4.
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Cysteinyl leukotrienes LTC4 and LTD4 bind with high affinity to specific receptors (cysteinyl
leukotriene receptor 2 [CYSLTR2] and cysteinyl leukotriene receptor 1 [CYSLTR1]
respectively) on various target cells (leukocytes, macrophages, airway and vascular smooth
muscle, endothelium, brain, adrenal gland, cardiac Purkinje cells), and elicit their well-
recognized actions of anaphylaxis, smooth-muscle constriction and vascular edema by increasing
post-capillary venule endothelial cell permeability and inflammation. While much focus has been
given to selective inhibition of CYSLTR1 by drugs such as montelukast 1, dual inhibition of
CYSLTR1 and CYSLTR2, blocking the effects of LTC4 and LTD4, has been investigated
19
clinically by the dual CysLTR1/2 antagonist gemilukast (Ono-6950). So far in clinical studies,
2
0
dual inhibition has not shown increased efficacy over selective inhibition of CYSLTR1 . While
2
1
LTE4 can elicit bronchoconstriction through CYSLTR1 , evidence indicates that LTE4 can also
2
2
act independently of CYSLTR1 and CYSLTR2 . Although its receptor has yet to be identified,
2
3
24
25
LTE4 has been directly implicated in driving eosinophilia , bronchial hyperreactivity , edema
2
6
and mucus production in asthmatics. It has been suggested that the LTE4 may act through the
G-protein coupled receptor 99 (GPR99) 27,28. Cysteinyl leukotriene antagonists such as
montelukast 1 are thought to primarily block the effects of the cysteinyl leukotrienes acting
through CYSLTR1, due to their high affinity for CYSLTR1 and selectivity over CYSLTR2. It is
unknown if they can block the effects of LTE4 mediated through its own receptor.
Arachidonic acid can also be metabolized by 15-lipoxygenase-1, present at high levels in airway
epithelial cells, eosinophils, alveolar macrophages dendritic cells and reticulocytes, to 14,15
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leukotriene A4 (EXA4) . LTC4S can conjugate glutathione to this 14,15-epoxide of arachidonic
2
9
acid to generate eoxin C4 (EXC4), the 14,15-regioisomer of LTC4 . EXC4 is similarly exported
from cells and can further be metabolized to eoxin D4 (EXD4) and eoxin E4 (EXE4). It is
3
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emerging that EXC4/D4/E4, have their own proinflammatory roles , and evidence is increasing
that eoxins are raised in subpopulations of asthma including aspirin intolerant asthma 31 and
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childhood asthma .
EXA4, from oxidation of arachidonic acid by 15-lipoxygenase can also be further oxidized by 5-
lipoxygenase to generate lipoxin A4 (LXA-4), one of the leukotrienes most strongly linked to
33
resolution of inflammation . LXA4 is recognized as a pro-resolving mediator through its actions
in inhibition of leukocyte recruitment and activation including neutrophils and eosinophils34 , 35,36
and in restoration of injured airway epithelium by blocking the release of pro-inflammatory
37
38
cytokines . Lipoxin A4 has been shown to be depressed in severe asthma .
Zileuton as a 5-lipoxygenase inhibitor can inhibit the synthesis of the cysteinyl leukotrienes, and
therefore block their pharmacology irrespective of which receptor they act through. Even though
at clinically approved doses zileuton 2 only partially inhibits the synthesis of the cysteinyl
3
9 40
leukotrienes , some clinical evidence suggests that zileuton 2 does indeed have superior
efficacy to montelukast 1 in acute asthma41, 42 . However, zileuton’s 2 dosing regimen43, and
4
4
requirements for liver transaminase monitoring have limited its clinical use . More effective
inhibition of the synthesis of the cysteinyl leukotrienes has been achieved with improved 5-LO
45
inhibitors and FLAP inhibitors which have entered clinical development. Surprisingly, in
4
6
clinical trials, improved inhibition of FLAP/5-LO has not led to increased efficacy . FLAP/5-LO
inhibitors also block the production of LTB4. While LTB4 acts as a neutrophil chemoattractant,
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the role of the neutrophil in asthma remains unclear. Neutrophils may drive pathology in certain
4
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asthma subtypes , but the lack of clinical efficacy observed with a number of neutrophil targeted
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drugs including LTA4H inhibitors and CXCR2 inhibitors may question the neutrophils
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pathogenic role in asthma. Neutrophils also have a protective role against bacterial colonization
5
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in the asthmatic lung . Hence completely inhibiting LTB4 formation may be undesirable.
Furthermore, FLAP/5-LO inhibitors also inhibit the production of pro-resolution lipids such as
lipoxin A4 (LXA4).
The 5-lipoxygenase inhibitor zileuton has also been shown not to inhibit the production of EXA4,
51
so 5-lipoxygenase inhibition will not block the pathways to the eoxins . Hence more complete
inhibition of FLAP/5-LO, which may change the balance of inhibition of downstream pro
inflammatory and pro-resolution pathways, may be undesirable.
With the contribution of all three cysteinyl leukotrienes to the pathology of asthma, the equivocal
role of LTB4, the importance of LXA4 to resolution of inflammation, and the emerging role of
the proinflammatory eoxins, we proposed that inhibiting the enzyme LTC4S may be a
differentiated point to block the leukotriene cascade (Figure 1), as it would block the synthesis of
LTC4, LTD4, LTE4 and EXC4, EXD4 and EXE4 , while preserving LTB4 and pro-resolution
LXA4.
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Figure 1. The differentiation in the arachidonic acid cascade of an LTC4S inhibitor (marked with
a red ×) compared to a cysteinyl leukotriene receptor 1 antagonist, montelukast 1 and a 5-
lipoxygenase inhibitor zileuton 2.
Therefore, we embarked on a campaign to discover potent LTC4S inhibitors which could test the
clinical hypothesis that inhibiting LTC4S is a preferred point of intervention in the leukotriene
cascade.
Results and Discussion
Di-benzoic acid 3 (Figure 2) was identified as an IC = 1.9 µM hit in a focused screen of 400
5
0
commercially available compounds in the LTC4S enzyme assay. However, compound 3 suffered
from poor physicochemical properties and did not inhibit cysteinyl leukotriene production in the
zymosan stimulated peripheral blood mononuclear cell (PBMC) assay. Rational design focused
on increasing potency and drug like properties of 3, resulted in a mono-benzoic acid series
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exemplified by compound 4 . Compound 4 was potent in the LTC4S enzyme assay (IC = 35
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0
nM). By adding a methoxy substituent to the core pyridyl ring 5 and extending the alkyl chain on
the amine to a methyl cyclopropyl, the large 2-methoxy benzamide side chain could be removed
keeping potency in the LTC4S enzyme assay (IC = 27.4 nM), improving physiochemical
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properties, and achieving activity in the PBMC cell assay (IC = 46.7 nM).
5
0
O
O
O
O
O
O
O
HO
HN
OH
NH
HO
HN
HO
N
O
N
N
N
O
O
O
3
4
5
Cl
Cl
Figure 2. Original hit di-benzoic acid 3 and mono-benzoic compound 4, and lead compound 5.
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Table 1. Initial SAR exploration
O
O
O
O
N
O
N
HO
R
N
N
N
Ar
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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
5-10
11
Cl
huHepd
Clint
LTC4Sa,b
IC50 (nM) IC50 (nM)
HuPBMCc
Compound
R
Ar
LogD
(l/min/
6
1
0 cells)
5
6
7
8
9
3-benzoic acid
4-chlorophenyl
27.4
24.3
1.41
4.94
158
46.7
58.0
9.41
27.7
1.7
1.3
2.4
1.6
0.65
2.8
2.9
28
2-cyclopropanecarboxylic acid 4-chlorophenyl
1.8
5.0
4.3
3.0
6.8
15
2
-(1S,2S)-
4-chloronaphthyl
cyclopropanecarboxylic acid
2-(1S,2S)-
cyclopropanecarboxylic acid
2-(1S,2S)-
3,4-
dichlorophenyl
phenyl
1640
cyclopropanecarboxylic acid
NDe
1
1
0
1
4-butanoic acid
4-chloronaphthyl
109
NDe
2-cyclopropanecarboxylic acid 4-chloronaphthyl
15.5
aInhibitory effect of compounds on LTC4 methyl ester production in presence of glutathione.
bValues are mean of at least two experiments with two replicates each, unless otherwise noted.
c
9
5% confidence interval of IC values was +/-1.7 fold Inhibitory effect of compounds on LTC4
5
0
d
production in peripheral blood mononuclear cells (PBMCs) activated with zymosan. Metabolic
stability of compound in human hepatocytes measured as disappearance of compound over time
e
in presence of human hepatocytes in suspension. ND - not determined.
Having established cell potency, the focus was to further improve properties for oral
administration. Exchanging the benzoic acid to a cyclopropane carboxylic acid 6 further lowered
lipophilicity to 1.3 while retaining potency. The low lipophilicity of 6 suggested that a more
lipophilic substituent on nitrogen would be tolerated and 4-chloronaphthyl was introduced to give
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two separated enantiomers. The S,S enantiomer 7 was the most active enantiomer with further
improved potency both in the enzyme assay IC = 1.41 nM and in the PBMC assay IC = 9.41
5
0
50
nM. Introducing more lipophilicity by adding an extra chlorine atom to the phenyl ring 8 also
increased potency but not to the same extent as the naphthyl. Unsubstituted phenyl 9 led to a
large drop in potency. Opening the cyclopropyl to the 5-oxopentanoic acid 10 gave a 78-fold
drop in potency compared to 7.
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
To support the design towards improved metabolic stability, metabolite identification studies in
human hepatocytes were performed on selected compounds. This revealed that reduction of the
ketone was a key site of metabolism. The pyrimidine analog 11 with the methoxy group shifted
one position away from the keto group showed increased keto reduction and human hepatocyte
6
Clint (15 µl/min/10 cells) compared to 7 having the methoxy in ortho position (Clint 5.0
6
µl/min/10 cells). The cyclopropyl and the ortho-methoxy on the flanking pyridyl group thus
appeared to protect the keto function from reduction, minimizing the overall hepatocyte Clint.
The high potency of compound 7 together with its reasonable stability in human hepatocytes, low
percentage of keto reduction, excellent solubility (576 µM) and permeability (Caco-2 apical-basal
6
permeability at pH6.5 105×10 cm/s) prompted us to profile it further. In vivo rat
pharmacokinetics (Table 4) for compound 7 showed moderate clearance (12 ml/min/kg) and a
5
3
high volume of distribution of 2.8 L/kg, which is unusual for an acid , yielding an effective half-
life of 2.7 hours. The rat oral bioavailability was 46%. The crossover of potency from human to
rat was excellent and 7 showed a good pharmacokinetic-pharmacodynamic (PKPD) profile in a
calcium ionophore challenge rat model (Figure 3). Furthermore, compound 7 had no significant
cytochrome P450 inhibition (>20 µM for CYP1A2, CYP2C9, CYP2C19, CYP3A4 and
CYP2D6), no hERG inhibition and was negative in the 5 strain Ames genotoxicity assays54.
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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
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 3 Effect of 7 on inhibition of CysLT production in the rat calcium ionophore challenge
model. Compound 7 was administered orally two hours prior to a calcium ionophore challenge
with A23187 via terminal bronchoalveolar lavage (BAL), and plasma concentration of 7 was
determined at time of PD (CysLT in BAL fluid) measurement. Data from two consecutive
experiments (1-90 mg/kg in study 1, triangles; 0.1-90 mg/kg in study 2, circles) were pooled and
fitted to a sigmoid Emax model, yielding an EC of 1.21 µM (CI 95% 0.89-1.5).
5
0
To further evaluate 7 the compound was progressed to a rat 7-day oral toxicology study at 30
mg/kg and 300 mg/kg (6 animals per group including controls 3 males + 3 females) and was well
tolerated at both doses. A complete histochemical assessment was undertaken and unilateral
sperm granulomas in the epididymal sperm duct were found in the high dose male group of rats.
These findings could potentially be attributed to potent inhibition of organic anion transporting
polypeptide 1B1 (OATP1B1) (0.6 µM) and/or other ATP-binding cassette (ABC) transporters, as
5
5
these have been shown to protect sperm cells from xenobiotics. According at the exposures
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achieved in the safety study, approaching 140 µM (~1 µM free) at Cmax, OATP1B1 would have
been inhibited.
In a secondary pharmacology assessment in a panel of >150 in vitro radio ligand binding and
enzyme assays covering a diverse set of enzymes, receptors, ion channels, and transporters it was
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
-
56
found that 7 overall had a clean profile but was a potent GABA gated Cl channel (rat cerebral
cortex) binder with IC 0.074 µM. GABA agonists are anxiolytic, anticonvulsant,
5
0
antihyperalgesic, can cause sedation, would impair motor function, cause muscle relaxation,
ataxia, dizziness, depression, anterograde amnesia and antagonists could be excitatory, pro-
convulsant and lead to seizures57.
Due to the moderate metabolic stability, the transporter interactions, the GABA binding and the
testicular toxicity findings in rat, compound 7 was not progressed further. A further optimisation
campaign was launched targeting high PBMC potency and low hepatocyte turnover together with
a low logD and high ligand lipophilic efficiency to reduce a general logD driven toxicology risk,
including the interactions with transporter proteins and GABA binding.
A screening cascade to mitigate the deficiencies of compound 7 was established, and assays
conducted in parallel defined as “waves”. Wave 1 contained the critical assays guiding compound
design including, LTC4S enzyme and PBMC cell potencies, physicochemical properties,
metabolic stability, as well as GABA binding. For compounds progressing to wave 2,
permeability and rat pharmacokinetics were included to explore in vivo pharmacokinetic
properties and in vitro to in vivo scaling of clearance. Finally, compounds were assessed for dog
pharmacokinetics as a second pharmacokinetic species to provide additional confidence in
predicting human pharmacokinetics. They were also assessed for efficacy in the rat calcium
ionophore challenge PKPD model, in which the degree of inhibition of cysteinyl leukotriene
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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
production measured in rat bronchoalveolar lavage after calcium ionophore challenge was
measured after oral administration of the LTC4S inhibitor. For shortlisting of potential clinical
candidates, an integrated in vitro approach to risk assess the potential for liver toxicity and
5
8
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
idiosyncratic drug reactions in humans was undertaken . This consisted of assays addressing 1)
covalent binding of parent compound or metabolites to human hepatocytes, 2) toxicity to
transformed human liver epithelial 2 (THLE) cells with and without metabolic activation, 3)
mitochondrial toxicity in HepG2 cells, and 4) inhibition potential against human bile salt export
pump (BSEP) and multidrug resistance associated protein 2 (Mrp2). Additional safety liabilities
were assessed through Ames assays, and pharmaceutical developability was assessed through
crystallinity and melting point.
An apo structure of human LTC4S (2IIU), and a structure in complex with glutathione (2UUH)
59
exist in the literature . To support structural based design, we determined the structure of
6
0
compound 12 (LTC4S IC = 2.4 nM) bound to LTC4S (Figure 4). The crystal structure
5
0
revealed the binding mode of 12 in the substrate pocket of LTC4S and suggested that the activity
resides in the S,S enantiomer (Supporting Figure S3). However, the data cannot unambiguously
define the stereochemistry of the bound compound. The acidic side chain points towards the
glutathione binding site and forms hydrogen bonds to the side chain of Arg104 and the phenol
oxygen on Tyr93. Furthermore, the side chain of Arg90 was arranged to coordinate the keto-
group and one of the pyrimidine nitrogens (Figure 4A). Tyr59 π-stacked with the pyrimidine ring
(Figure 4B) The methoxy group and the naphthalene ring reside in enclosed cavities while the N-
cyclopropyl methyl side chain faces the entrance of the binding pocket (Figure 4A). Compound
S,S-12 takes advantage of the polar groups available to it while matching the shape of the binding
pocket well. The structure also suggested that the naphthyl pocket would allow for a small
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substituent of the size of fluorine, chlorine or methyl in the 2-position and the N-cyclopropyl
methyl was expected to tolerate modifications as it pointed out of the binding pocket.
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
Figure 4. X-ray crystal structure of 1S,2S-12 complexed with human LTC4S (pdb 6R7D). (A) The
acid forms hydrogen bonds to the side chain of Arg104 and the phenol oxygen on Tyr93. The side
chain of Arg90 is arranged to coordinate the keto-group and one of the pyrimidine nitrogens. N-
cyclopropyl methyl side chain faces the entrance of the binding pocket (B) Tyr59 π-stacked with
the pyrimidine ring.
In the first attempt to reduce lipophilicity, a set of analogues to 7 were prepared with a 4-
fluoronaphthyl (Table 2) instead of the 4-chloronaphthyl and with variable alkyl side chains
(cyclopropylmethyl 13, 2-methylpropyl 14, propyl 15 and ethyl 16). This revealed that the human
hepatocyte intrinsic clearance was lowered by changing the N-alkyl side chain from 2-
6
6
methylpropyl as in 14 (56.4 µl/min/10 cells) to a n-propyl 15 (7.57 µl/min/10 cells) and to ethyl
6
1
6 (3.8 µl/min/10 cells) following the reduction in lipophilicity. Acceptable cellular PBMC
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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
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5
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5
5
6
potency (17 nM) was achieved also with compound 16 with the lowest logD in the comparison.
Reducing logD also had a clear effect on the GABA binding potency, that was reduced from IC50
= 0.64 µM for compound 13 to IC = 2.9 µM for compound 16. In a reactive metabolite study of
5
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
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compound 13 with added gluthathione (GSH) to trap formed electophilic species, GSH adducts
were found. However, in this series of compounds no GSH adducts could be identified for
compounds 14-16.
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Table 2. Optimization of N-alkyl and N-aryl groups. Reducing GABA binding and formation of
glutathione adducts.
O
O
O
O
O
O
O
O
O
HO
HO
HO
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
N
R
N
R
N
N
F
N
N
Ar
13-16
17-24
25-28
F
F
F
f
huHep Clint
LTC4Sa,b
IC50 (nM)
HuPBMCc
IC50 (nM)
GSH
adducts
GABA
(µM)
6
Compound
R or Ar
LogD
(l/min/10
d
e
cells)
13
14
cyclopropylmethyl
2-methylpropyl
propyl
0.758
0.381
0.626
1.05
6.42
4.74
31.8
8.65
7.90
13.0
11.5
13.5
2.08
3.71
5.26
1.97
26.5
2.26
7.09
16.8
52.4
20.6
NDg
55.5
58.4
113
1.9
2.2
1.8
1.4
1.4
1.6
0.0
0.9
1.2
0.7
1.1
1.9
1.2
1.1
1.2
1.1
Yes
No
0.64
NDg
3.5
5.4
56
7.6
1
1
1
1
1
2
2
2
2
2
5
6
7
8
9
0
1
2
3
4
No
ethyl
No
2.9
3.8
cyclopropylmethyl
2-methylpropyl
No
11
2.3
NDg
NDg
No
NDg
>100
54
2.9
2-hydroxy-2-methylpropyl
2-fluoro-2-methylpropyl
propyl
NDg
<1.5
7.2
NDg
NDg
Yes
No
19
ethyl
20
3.1
NDg
NDg
2,2,2-trifluoroethyl
4-fluorobenzyl
156
NDg
157
4.2
<1.1
1.3
2
-fluoro-5-
25
26
27
14.0
34.3
42.3
27.6
No
8.2
NDg
3.0
(
trifluoromethyl)phenyl
5-chloro-2-fluorophenyl
3-chloro-4-fluorophenyl
No
NDg
3.1
2
8
5-chloro-2,4-difluorophenyl
No
5.4
<1.5
aInhibitory effect of compounds on LTC4 methyl ester production in presence of glutathione.
bValues as mean of at least two experiments with two replicates each unless otherwise noted.
c
95% confidence interval of IC values was +/-1.7-fold. Inhibitory effect of compounds on LTC4
5
0
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1
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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
5
6
d
production in peripheral blood mononuclear cells (PBMCs) activated with zymosan. Compounds
were incubated in human liver microsomes in the presence glutathione and NADPH at pH 7.4
e
-
56
f
and 37°C. Binding to GABA gated Cl channel (rat cerebral cortex) Metabolic stability of
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
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9
0
1
2
3
4
5
6
7
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0
compound in human hepatocytes measured as disappearance of compound over time in presence
g
of human hepatocytes in suspension. ND - not determined
Next, to further reduce the lipophilicity we replaced the 4-fluoronapthyl with 3-
(
trifluoromethyl)phenyl. On that scaffold the N-alkyl group was optimized with the primary aim
to afford low human hepatocyte Clint. The N-cyclopropyl methyl derivative 17 with logD of
7
.4
1
1
.4 was synthesized as a reference for the N-alkyl optimization and displayed a GABA binding of
1µM and no GSH adducts. It displayed a LTC4S potency of IC = 6.42 nM and a lower human
5
0
6
6
hepatocyte Clint (2.3 µl/min/10 cells) as compared to the matched pair 13 (5.4 µl/min/10 cells).
Further, compared to 7, compound 17 afforded only a 5-fold reduced LTC4S potency
accompanied with a 10-fold decrease of lipophilicity. Changing to N-2-methylpropyl (18) gave a
compound with similar LTC4S potency (IC = 4.74 compared to 6.42 nM) but with two-fold
50
increased PBMC potency (IC = 20.6 compared to 52.4 nM).
5
0
Similar to the structure activity relationship for the 4-fluoronapthyls 13-16, decreasing the size of
the R-group from 2-methylpropyl to propyl and ethyl lead to a 5-fold decrease of the PBMC
potency. The 3-(trifluoromethyl)phenylamine 18 bearing a N-2-methylpropyl displayed a reduced
human hepatocyte Clint compared to the matched pair fluoro naphthyl 14, 2.9 and 56 µl/min/106
cells respectively.
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Hydroxylation 19 and fluorination 20 of the 2-methylpropyl led to decreased LTC4S potency
(
IC = 32 and 8.65 nM respectively) and the human hepatocyte Clint was reduced to below the
50
6
detection limit (<1.5 µl/min/10 cells) for compound 20. Exchanging N-ethyl (22) to trifluoro
ethyl (23) had no effect on the LTC4S binding (13.0 versus 11.5 nM), and increasing the size of
the R-group to N-4-fluoro benzyl (24) did not contribute to an improvement in potency (IC =
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
5
0
1
3.5 nM).
In parallel to exploring the best option for the N-alkyl group, the phenyl substitution pattern was
explored (Table 2). Modelling suggested that the 6-position of the phenyl ring may tolerate a
small substituent such as a fluorine. Compound 25 displayed an improved potency in both the
LTC4S binding assay and in the PBMC assay together with a reduced logD compared to the des-
fluoro matched pair 17. By changing the 3-trifluoromethyl substituent in 25 to a 3-chloro
substituent 26 the low logD was retained together with a slight decrease in potency. Moving the
fluorine substituent to the 4-position 27 resulted in a further decrease in potency and an increase
of the logD. However, combining the 4- and 6-fluoro substituent as in 28 led to an increase in
LTC4S binding and PBMC potency IC = 1.97 and IC = 28 nM respectively, together with
5
0
50
6
reduction of human hepatocyte Clint <1.5 µl/min/10 cells similar to 25.
In conclusion N-2-methylpropyl 18 and N-2-fluoro-2-methylpropyl 20 were identified as the
preferred N-alkyl substituents in combination with the 3-trifluoromethyl phenyl due to the
favourable PBMC potency and human hepatocyte Clint profiles. Of significance is the low logD
(0.9) for 20 where the fluorine-carbon bond dipole reduces the lipophilicity approximately 7-fold
compared to N-2-methylpropyl, leaving flexibility for further optimisation. The 5-chloro-2,4-
difluorophenyl and the 2-fluoro-5-(trifluoromethyl)phenyl represented the two most optimal
substituted phenyls identified.
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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
The preferred N-aryl and N-alkyl groups identified above (Table 2) were combined to give
pyridines 29-32 (Table 3). The N-2-methylpropyls 29 and 30 were more potent in the PBMC than
the N-2-fluoro-2-methylpropyls 31 and 32, IC = 6.8 and IC = 8.5 nM compared to IC = 31
5
0
50
50
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
and IC = 16 nM. However, both had higher logD and displayed a higher human hepatocyte
5
0
6
6
Clint (3.9 and 4.9 µL/min/10 cells) that the matched pairs (<1.0 and <1.7 µL/min/10 cells).
From the X-ray crystal structure of 12 complexed with human LTC4S (Figure 4B) a π-stacking
interaction between the central pyridyl with Tyr59 was identified. We hypothesized that the
affinity to the central core could be improved by the introduction of alternative electron poor
aryls. Quantum Mechanical (QM) calculations on a representative system (Figure 5) were
performed with a number of selected core replacements. Relative energies are given between the
reference core ring from 12 (C-Ref), whereas higher energies suggest a more favorable
interaction. The high relative solvation energy consistent with the relative gas phase energy
suggested that the pyridine core ring C-E could be replaced by pyrazine C-F, to improve affinity
to LTC4S.
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1
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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
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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
Figure 5. (A) Reduced system for QM calculations with the core ring (magenta) of ligand 12
(blue) with selected amino acids (grey) based on the X-ray of 12. Atoms fixed during the
geometry optimization are colored green. (B) Relative energies are given between the reference
core ring from 12 (C Ref) and the modelled variants. (C) Core ring models used in the QM
calculations.
Combining the pyrazine core C-F with the same preferred N-aryls and N-alkyls as above gave
compounds 33-36. As predicted the pyrazine core displayed a 2-fold increase in the LTC4S
binding and PBMC potency compared to the pyridine matched pairs. Again, the N-2-fluoro-2-
6
methylpropyls 35 and 36 display low human hepatocyte Clint, 1.5 and <1.2 µL/min/10 cells,
respectively. Due to the lower in human hepatocyte Clint of 36 relative to 35, 36 was chosen for
progression.
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1
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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
Table 3. Combining optimal phenyl and N-alkyl groups with either pyridyl or pyrazine central
aryl.
O
O
O
O
O
O
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
HO
HO
N
N
N
N
N
X
X
Ar
Ar
29-32
33-36
huHep
LTC4Sa,b
IC50 (nM)
HuPBMCc
IC50 (nM)
d
6
Compound
X
Ar
LogD
Clint (l/min/10
cells)
2
-fluoro-5-
trifluoromethyl)phenyl
-chloro-2,4-
difluorophenyl
-fluoro-5-
2
3
3
3
3
3
3
3
9
0
1
2
3
4
5
6
H
H
F
0.66
0.70
0.63
0.60
0.36
0.28
0.25
0.28
6.8
8.5
31
1.4
1.3
0.8
0.8
2.0
1.8
1.2
1.1
3.9
4.9
(
5
2
<1.0
<1.7
2.9
(trifluoromethyl)phenyl
5-chloro-2,4-
difluorophenyl
2-fluoro-5-
(trifluoromethyl)phenyl
5-chloro-2,4-
F
16
H
H
F
3.5
4.1
7.8
10
3.0
difluorophenyl
2
-fluoro-5-
trifluoromethyl)phenyl
-chloro-2,4-
difluorophenyl
1.5
(
5
F
<1.2
aInhibitory effect of compounds on LTC4 methyl ester production in presence of glutathione.
bValues as mean of at least two experiments with two replicates each unless otherwise noted.
c
9
5% confidence interval of IC values was +/-1.7-fold Inhibitory effect of compounds on LTC4
5
0
d
production in peripheral blood mononuclear cells (PBMCs) activated with zymosan. Metabolic
stability of compound in human hepatocytes measured as disappearance of compound over time
in presence of human hepatocytes in suspension.
Chemistry. Compound 36 and analogues described above were derived from three main building
blocks by the general methods depicted in Scheme 1. First the carboxylic chain was introduced
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on the corresponding iodo bromo substituted heterocycle through a Grignard reaction on either
the Weinreb amide (method A) or the aldehyde followed by oxidation to the ketone (method B).
The formed heterocyclic bromide was further coupled to secondary amines (method C) or
primary amines followed by alkylation (method D) under Buchwald conditions. Alternatively, in
the case of 3-methoxypyrazines the secondary amine was deprotonated with a base and added
through nucleophilic aromatic substitution to the electron poor 3-methoxypyrazine (method E).
Ester hydrolysis and chiral separation of enantiomer mixtures then gave the final products. Non-
commercial secondary amines used in method C and E were synthesized from commercial
primary amines by acylation with acyl halides followed by reduction with borane.
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
Scheme 1. General method for preparation of compounds 5-36a
O
I
N
O
H
O
O
O
N
O
O
Br
Ar
R2
ester hydrolysis
chiral separation
R1
O
R1
5 - 36
or
R1
N
N
N
R1
H
C, D or E
A or B
R2
Br
N
Ar
aFor R1, R2 and Ar see Table 1-3 ans experimental. Method A: Weinreb amide,
(CH ) CHMgCl∙LiCl; Method B: aldehyde, (CH ) CHMgCl∙LiCl followed by MnO Method C:
3
2
3 2
;
2
secondary amine, Pd-catalyst, ligand Cs CO Method D: primary amine, Pd-catalyst, ligand,
2
3;
Cs CO followed by NaH and alkyl bromide; Method E: secondary amine, ((CH ) Si) NK.
3
3
2
2
3
The detailed synthesis of compound 16 and 36 is outlined below and described in the
experimental part. Compound 16 (Scheme 2) was prepared according to a combination of route B
and D. Trans-ethyl 2-formylcyclopropanecarboxylate was reacted with 5-bromo-2-iodo-3-
methoxypyridine in the presence of iso-propyl magnesium chloride. The formed alcohol rac-37
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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
was oxidized with manganese dioxide to form the ketone rac-38 that following a palladium
catalysed coupling with 4-fluoronaphthalen-1-amine gave rac-39. Alkylation with ethyl bromide
followed by saponification with sodium hydroxide and chiral separation afforded compound 16.
For some chiral compounds a standard procedure was developed where intermediate rac-38 was
submitted to chiral purification followed by mixing the enantiomers as a 90/10 mixture of
active/inactive component. This mixture was used for synthesis of test compounds where the
contamination of up to 10% inactive enantiomer in the final compound was utilized to secure
chiral separation of target compound enantiomers and determination of enantiomer excess (% ee).
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
a
Scheme 2 . Synthesis of 16.
O
OH
O
O
O
O
O
O
O
I
O
O
a
b
+
O
N
N
Br
Br
N
Br
rac
rac-37
rac-38
O
O
O
O
O
O
c
d,e and f
O
HO
NH2
N
N
NH
F
N
rac-39
S,S-16
F
F
a(a) (CH ) CHMgCl∙LiCl, tetrahydrofuran; (b) MnO , tetrahydrofuran; (c) Pd(OAc) , racemic
3 2
2
2
BINAP, Cs CO , toluene; (d) NaH, ethyl bromide, N,N-dimethylformamide; (e) NaOH; (f) chiral
2
3
chromatography
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In the case of 36 (Scheme 3), the secondary amine 41 was prepared from the 5-chloro-2,4-
difluoro-aniline and 2-fluoro-2-methyl-propanoic acid with the help of isobutyl chloroformate
and 4-methylmorpholine followed by reduction of the amide 40 with borane tetrahydrofuran
6
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
complex. The coupling partner rac-43 was prepared through a Weinreb−Nahm ketone synthesis
reacting 5-bromo-2-iodo-3-methoxypyrazine with the Weinreb amide rac-42 obtained from
trans-2-methoxycarbonylcyclopropanecarboxylic acid under standard conditions. Compound 41
was deprotonated with potassium bis(trimethylsilyl)amide at -20ºC and reacted with compound
rac-43 to yield rac-44 that after saponification with sodium hydroxide and separation of the
enantiomers gave compound 36.
Scheme 3. Synthesis of 36.a
O
F
F
NH2
HN
F
HN
a
b
F
F
F
OH
F
Cl
O
Cl
Cl
O
F
F
4
0
f
41
O
O
O
O
O
O
c and d
e
O
O
OH
O
N
O
N
N
Br
rac
rac-42
rac-43
O
O
O
F
O
O
O
O
N
N
HO
N
g and h
N
F
N
F
N
F
rac-44
S,S-36
Cl
Cl
F
F
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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
a(a) isobutyl chloroformate, 4-methylmorpholine; (b) borane tetrahydrofuran complex,
tetrahydrofuran, 60ºC; (c) Oxalyl chloride; (d) N,O-dimethyl hydroxylamine hydrochloride; (e)
(CH ) CHMgCl∙LiCl, 5-bromo-2-iodo-3-methoxypyrazine, tetrahydrofuran; (f) ((CH ) Si) NK, -
3
2
3 3
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
2
0ºC; (g) NaOH; (h) chiral chromatography
The protein crystal structure of 12 did not conclusively define the absolute stereochemistry of the
potent enantiomer of the compound. Therefore, the structure and absolute stereochemistry of 36,
were generated by single crystal X-ray diffraction and confirmed the S,S configuration of the
active enantiomer (Figure 6 and supplementary pages for detail).
Figure 6. Single crystal X-ray diffraction structure of 36. Thermal ellipsoids drawn at the 50%
probability level. Hydrogen atoms and methanol moiety are omitted for clarity.
Preclinical pharmacokinetic profile, in vitro pharmacology and off-target safety. Pyrazine
3
6 was selected for progression into preclinical development. Its profile is shown in Table 4.
Compound 36 showed low turnover in human, monkey, dog and rat hepatocyte incubations
which was also reflected in low plasma clearance observed in vivo in the preclinical species
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(
<2% of liver blood flow). The unbound steady state volume of distribution (Vss) varied between
rat and dog, which posed a risk for the prediction of the human volume of distribution. To
increase confidence in predicting human Vss, the pharmacokinetics of 36 was explored in
cynomolgus monkey, in which the unbound Vss was found to be similar to dog. As dog and
monkey were more consistent, the human Vss estimate was based on these two species.
Compound 36 had excellent pharmaceutical properties, being a non-hygroscopic crystalline
anhydrate with a melting point of 129 ºC with no other thermal events.
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 4. Comparison of preclinical profile of compounds 7 and 36.
7
36
hPBMC IC50,free
1
.1
6.2
a
(nM)
In vivo inh of
CysLT EC50,free
7.5
2.4
34
a,b
(nM)
logD7.4
1.1
Solubility
5
76
2080
pH 7.4 (µM)
PPB (%free)
hu/rat/dog/cyno
Caco-2 Papp
0.66/0.62/0.21/NDc 3.6/2.5/5.9/3.0
105 / NDc
61 / 3.2
−
6
−1
(
10 cm s ) /
efflux ratio
Pharmacokinetic parameters in rat / dog / cynod,e
f
2.7 / 4.8 / NDc
t
1/2
g
,
effective (h)
26 / 6.1/ 12
100 / 100/ 90
F %
CL
(
V
46 / 91 / NDc
h
2 / 2.4 / NDc
1
2.3 / 1.8 / 0.6
5.3 / 1.0 / 0.6
ml/min/kg)
ss (L/kg)
i
2.8 / 1.1 / NDc
Safety & Selectivity
GABA binding
0
.074
0.6
>100
3.2
(µM)
OATP1B1(M)
CYP inhibition
>
20M
>30M
No
(5 isoforms)
GSH adducts
Yes
aThe IC50 values were converted to unbound (free) values (see experimental section under
b
c
respective assay); Inhibition of CysLT in rat calcium ionophore challenge model; ND - not
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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
d
e
determined; Han Wistar rats, male Beagle dogs, cynomologous monkeys dose in rat 0.5 mg/kg
iv; 1 mg/kg po, dose in dog 1 mg/kg iv; 2 mg/kg po; dose in cyno 1 mg/kg iv; 2 mg/kg po;
ft½,effective = ln2 × V /Cl; F oral bioavailability; CL clearance; V volume of distribution at
g
h
i
ss
ss
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
steady state.
Compound 36 showed no reactivity to glutathione and did not inhibit cytochrome P450 isoforms
up to 30 M. It displayed excellent selectivity in a panel of >200 in vitro radio ligand binding and
enzyme assays covering a diverse set of enzymes, receptors, ion channels, and transporters. The
only activity identified within 100 times of the human predicted Cmax, was an interaction with
GABA-A as a positive allosteric modulator at 24 times human predicted Cmax. A positive
allosteric modulator would enhance an endogenous response to GABA. No seizures were
observed in any of the rat toxicology studies, at up to 122 times the human predicted exposure,
and the in vitro observation was assessed as a low risk for progression. Additionally, compound
3
6 had no activity below 33 µM at a panel of cardiovascular ion channels and was negative in a
5-strain Ames genetic toxicology assessment. Taken together, these data demonstrate that
compound 36 is a highly selective LTC4S inhibitor with low risk for off-target effects.
In vivo efficacy and safety. Compound 36 was evaluated head to head with the original lead 7 in
a 7-day rat toxicology study. In the study, doses were chosen so 36 achieved matching or greater
exposures than those achieved by 7 earlier (Table 5).
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a
Table 5 . Seven day rat toxicology study and results for compounds 7 and 36.
no adverse effect
level (NOAEL)
lowest adverse
effect level
findings at
highest dose
Safety
margins
study design
e
Sperm granuloma
(moderate-severe)
Bilirubinemia
Liver hypertrophy
No testicular
Rat 3M+3F/group
C
max 47 M
Cmax 144 M
AUC 1830 M.hr
7
7-16 times
200 times
3
0 & 300 mg/kg
AUC 200 M.hr
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
Rat 6M/group
C
max 349 M
3
6
NV
observations
Liver hypertrophy
1
0 & 100 mg/kg
AUC 4500 M.hr
a
e
NV no value, Margins @NOAEL to human predicted total AUC
Compound 7 again showed evidence of sperm granuloma in the high dose group providing
margins to a no adverse effect level of 7-16 times based on the predicted human exposures at the
predicted efficacious dose. Compound 36 showed no signs of testicular toxicity, and only
adaptive changes in the liver due to cytochrome P450 induction which were not judged adverse,
providing a 200 fold margin between the no adverse effect level and the predicted human
exposure at the predicted therapeutic dose. Hence 36 was chosen as a progressable candidate.
The PKPD relationship for in vivo LTC4S inhibition by 36, was explored in a calcium ionophore
challenge rat model, measuring inhibition of CysLT production in the lung following oral
delivery of 36. A dose response experiment was first performed, where 36 was administered two
hours prior to a terminal bronchoalveolar lavage with calcium ionophore A23187, followed by a
time-course experiment, where the challenge was done 0.5, 2, 6 and 24 hours post a single dose,
and 0.5, 2 and 6 hours after four days of daily oral dosing. The relationship between 36 plasma
concentration and reduction of CysLT in BAL fluid (BALF) across both experiments was well
described by a sigmoid Emax model (Figure 7), suggesting a direct relationship between inhibition
of leukotriene production in the rat lung, and the instantaneous plasma level at the timepoint of
the PD measurement. The EC for inhibition of CysLT production in rat lung was estimated to
5
0
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