Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: From hit to clinic

Article abstract of DOI:10.1021/jm5012885

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

Full text of DOI:10.1021/jm5012885

Article
pubs.acs.org/jmc
Discovery and Optimization of an Azetidine Chemical Series As a
Free Fatty Acid Receptor 2 (FFA2) Antagonist: From Hit to Clinic
Mathieu Pizzonero,† Sonia Dupont,† Marielle Babel,† Stephane Beaumont,† Natacha Bienvenu,†
́
Roland Blanque,† Laetitia Cherel,† Thierry Christophe,‡ Benedetta Crescenzi,† Elsa De Lemos,†
́
̈
Philippe Delerive,† Pierre Deprez,† Steve De Vos,‡ Fatoumata Djata,† Stephen Fletcher,†
Sabrina Kopiejewski,† Christelle L’Ebraly,† Jean-Michel Lefrancois,† Stephanie Lavazais,†
̧
́
Murielle Manioc,† Luc Nelles,‡ Line Oste,‡ Denis Polancec,§ Vanessa Quenehen,† Florilene Soulas,†
́
́
̀
Nicolas Triballeau,† Ellen M. van der Aar,‡ Nick Vandeghinste,‡ Emanuelle Wakselman,† Reginald Brys,‡
and Laurent Saniere*,†
†Galapagos, 102 Avenue Gaston Roussel, 93230 Romainville, France
‡Galapagos, Generaal De Wittelaan L11A3, 2800 Mechelen, Belgium
§Fidelta, Prilaz Baruna Filipovica 29, 10000 Zagreb, Croatia
S
* Supporting Information
ABSTRACT: FFA2, also called GPR43, is a G-protein
coupled receptor for short chain fatty acids which is involved
in the mediation of inflammatory responses. A class of
azetidines was developed as potent FFA2 antagonists.
Multiparametric optimization of early hits with moderate
potency and suboptimal ADME properties led to the
identification of several compounds with nanomolar potency
on the receptor combined with excellent pharmacokinetic
(PK) parameters. The most advanced compound, 4-[[(R)-1-
(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able
to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based
pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data
supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.
It was demonstrated that neutrophilic granulocytes isolated
from FFA2-deficient mice lost the ability to migrate in response
to SCFAs, indicating that FFA2 has a major role in neutrophilic
granulocyte migration.6 As neutrophil migration into the gut
mucosal tissue is considered a driver of tissue damage in IBD
and hence neutrophilic markers (myeloperoxidase (MPO),
calprotectin) are also used as markers for disease progression
in IBD, reducing neutrophil migration by FFA2 inhibition
could be considered as a therapeutic option in IBD. Reports on
the outcome of models for IBD in GPR43-deficient mice,
however, describe conflicting results, ranging from reduced
disease severity, associated with reduced polymorphonuclear
leukocytes (PMN) migration in the inflamed gut of mice
subjected to the chronic dextran sodium sulfate (DSS)-induced
colitis model, to exacerbated or unresolving inflammation in
the DSS and trinitrobenzoic sulfonic acid (TNBS)-induced
model for IBD.6,7 These different outcomes can be explained
by different parameters (genetic background of the mice used,
differences in model setup, food).8 The potential of FFA2
INTRODUCTION
■
Lipids play an important role in many biological processes and are
crucial in the pathogenesis of numerous common diseases.
Especially, fatty acids (FAs), which are derived from triglycerides
and phospholipids, are essential sources of energy in complex
organisms and animals, yielding large quantities of ATP when
metabolized. FAs are also key signals for metabolic regulation,
acting through enzymatic and transcriptional networks to modulate
gene expression, growth, and survival pathways, as well as inflam-
matory and metabolic responses. There is now a substantial body of
evidence that disturbances in FAs are strongly involved in several
chronic pathologies including metabolic syndrome, inflammatory
diseases, and some cancers.1 Fatty acid chains differ by length, often
categorized as short to very long. Short-chain fatty acids (SCFA,
carbon length C2−C6: acetate, propionate, and butyrate) are
byproducts of anaerobic bacterial fermentation of fibers in the gut.2
It was discovered that SCFAs bind to FFA2 receptor, also known
as GPR43.3 FFA2 is highly expressed in immune cells (e.g.,
neutrophils, peripheral blood mononuclear cells (PBMC), purified
monocytes, eosinophils, and B lymphocytes)3,4 and also in other
tissues that may play a role in the propagation of the inflammatory
response (e.g., intestinal cells or endothelial cells).5
Received: August 26, 2014
© XXXX American Chemical Society
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Figure 1. IC50 (CaFlux assay) of three representative compounds from HTS (1, 2, 3) and the N-methylated analogue 4.
a
Scheme 1. Synthesis of the Common Azetidine Core
a
Reagents and conditions: (a) K2CO3, MeCN, reflux, 4 h, 97%; (b) chloroacetaldehyde, MgSO4, sodium triacetoxyborohydride, AcOH, DCM, 0 °C,
1 h, 85%; (c) KHMDS −78°C/−70°C, THF, 1 h, 79%; (d) H2, Boc2O, Pd/C 10%, EtOH, 20 °C, 18 h, 79%.
inhibition for the treatment of IBD, is currently explored in
pharmacological testing.
methyl-azetidine-2-carboxylic acid ethyl ester 10. Benzyl group
was removed by hydrogenolysis and directly replaced by a Boc-
protecting group to give 5.
Considering the key function of FFA2 in inflammatory
processes, blocking the downstream signaling of this receptor by
a small molecule could be useful in the treatment of inflammatory
conditions, infectious diseases, autoimmune diseases, and impair-
ments of immune cell functions.9 At the moment we started our
investigations, only FFA2 agonists were reported in the
literature.10 Since that time, several agonist compound series
have been published as well as one series of FFA2 antagonists.11,12
The strategy developed to discover a new FFA2 antagonist was
based on a high throughput screening of a diverse compound
collection. Compounds were evaluated in a calcium flux assay
performed in antagonist mode, measuring the change, induced
by sodium acetate (FFA2 agonist), in free intracellular calcium
concentration in a HEK293 cell line stably overexpressing
the human FFA2 receptor. This calcium flux assay was successful
in identifying different compounds bearing an azetidine scaffold,
and displaying moderate potencies in the micromolar range
(compounds 1, 2 and 3, Figure 1). Interestingly, it was observed
that simple methylation of the secondary carboxamide
remarkably increased potency (3 vs 2). The same modification
was also considered for compound 1. Compound 4 was
synthesized for that purpose and evaluated, leading to a first
breakthrough with a potency of 274 nM. With a submicromolar
starting point, the azetidine scaffold was therefore considered
as a tractable series for SAR exploration, enabling possible access
to three substitutions of the core to increase the chemical space.
Boc-protected central core 5 was a pivotal intermediate in our
different synthetic strategies that allowed us to explore indif-
ferently R1, R2, and R3.13
Different substitutions at R1 were introduced by amide
coupling of the corresponding carboxylic acids and the HCl salt
of azetidine 11 obtained by the removal of the Boc-protecting
group under acidic conditions. R2 and R3 functionalities were
installed by amide coupling of secondary amines having cor-
responding moieties or a direct precursor already in place with
carboxylic acid 11 obtained by saponification of azetidine ethyl
ester 5 (Scheme 2).
With two orthogonal protecting groups installed on the central
core, the order of the two amide couplings provided a flexible
access to a wide range of compounds for a broad range of SAR
modulations. When necessary, final modifications on the R3 side
chains were performed to set the appropriate functionality.
IDENTIFICATION OF LEAD COMPOUNDS
■
Early in the discovery process, the hits were tested against mouse
and rat FFA2 receptors. Unexpectedly, consistently across our
series, the compounds were found inactive on the rodent
orthologues in different cellular assays. A recent publication
reports differences in potencies for SCFA between species
orthologues, pointing to an “ionic lock” between extracellular
loop 2 and the ligand binding pocket regulating constitutive
activity.14 This could rationalize the absence of activity of our
series on rodent FFA2 receptors. As a consequence of the lack of
orthology, the efficacy of the FFA2 inhibitors generated could
not be assessed in rodent disease models. The drug discovery
program was run by in vitro/ex vivo optimization of the
compound potencies, in addition to in vivo pharmacokinetics
parameters with the purpose of achieving a very long predicted
target coverage in human. To this end, during the lead
optimization process, the in vitro liver microsomal stability was
first evaluated in rat and further checked in human microsomes.
CHEMISTRY
■
Different synthetic approaches were developed to facilitate the
SAR exploration of the three main structural subunits.
Synthesis of the Boc-protected central core 5 was performed in
four steps following a straightforward sequence depicted in
Scheme 1. Benzylamine 6 was condensed with 2-bromo-propionic
acid ethyl ester 7 to yield secondary amine 8. Reductive amination
with chloroacetaldehyde gave tertiary amine 9, which was then
cyclized under basic condition to give the racemic 1-benzyl-2-
B
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a
Scheme 2. General Synthetic Access to Azetidine FFA2 Antagonists
a
Reagents and conditions: (a) NaOH, MeOH; (b) EDC/HOBt, chlorobenzylamine; (c) HCl, dioxane; (d) R1CO2H, EDC/HOBt, NEt3; (e)
(COCl)2, NEt3, DCM, DMF.
Table 1. Optimization of R3
compd
R1
R3
IC50 (nM) RLM (% remaining) compd
R1
R3
IC50 (nM) RLM (% remaining)
b
b
2
R1a
R1b
R1a
R1b
R1a
R1b
R1a
R1a
R1a
R1b
R1b
R1a
R1a
H
10000
1498
1434
274
0
11
0
24
25
26
27
28
29
30
31
32
33
34
35
36
R1a
R1a
R1a
R1a
R1a
R1a
R1b
R1a
R1a
R1a
R1b
R1b
R1a
CH2CONHMe
442
1584
158
510
4673
388
76
5
0
b
b
b
b
b
b
a
1
H
CH2CONMe2
3
Me
Me
Et
(CH2)3CONH2
22
4
0
(CH2)2CONH2
a
a
a
a
15
16
17
18
19
20
21
22
23
473
7
CH2CO(morpholine)
(CH2)3CO(morpholine)
(CH2)3CO(morpholine)
(CH2)2NHCOMe
(CH2)2NHSO2Me
(CH2)3SO2NH2
38
31
2
(CH2)2OH
3350
10000
10000
1208
247
1
(CH2)2NH2
(CH2)2NHCONHMe
(CH2)2OMe
CH2CN
a
0
337
429
245
76
12
b
a
b
b
a
a
a
a
2
0
0
0
11
0
(CH2)3CN
143
(CH2)3SO2NH2
(CH2)3CO2Me
CH2CONH2
361
(CH2)3SO2NHMe
(CH2)3SO2NHMe
44
0
123
263
16
a
b
Rat liver microsomal (RLM) stability % remaining after 30 min. Rat liver microsomal (RLM) stability % remaining after 60 min.
As a first step in our hit series optimization, a wide range of side
chains in the R3 position was explored (Table 1). On the basis of
HTS results, dimethylphenylacetamido and benzothiophene
3-acetamido substitutions at R1 were chosen. Short alkyl chains
substitutions in R3 seemed to improve significantly potency
(compound 15). Adding polar groups like ethylhydroxy (16),
ethylamino (17), or urea (18) led to a drop in potency, whereas
the ethylmethoxy derivative was moderately active (19).
Introduction of a nitrile side chain resulted in a potency
improvement comparable to simple methylation (20 and 21). A
large variety of esters, amides, and sulfonamides was explored, all
showing moderate to high potency (IC50 below 100 nM for
C
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Table 2. Introduction of a Carboxylic Acid Side Chain at R3
c
compd
R1
R3
IC50 (nM)
RLM (% remaining)
AUC (0−24 h) (ng·h/mL)
b
37
38
39
40
R1a
R1b
R1b
R1b
CH2COOH
CH2COOH
(CH2)2COOH
278
36
100
1160
362
b
56
a
317
42
100
a
(CH2)3COOH
68
542
a
b
c
Rat microsomal stability % remaining after 30 min. Rat microsomal stability % remaining after 60 min. Oral plasma exposure, 5 mg/kg, Sprague−
Dawley rat.
Scheme 3. Synthesis of Both Enantiomers of 37 and Determination of Their Absolute Configuration
several compounds like 30, 34). The optimal length of this spacer
emerging from this SAR exploration was one or three carbon
atoms, depending on the functionality and also on R1. Overall,
compounds showed a low stability when incubated (30 or
60 min) in rat liver microsomes. Carboxamides and sulfonamides
were also found generally unstable with few noticeable
exceptions (28, 29).
acceptable for 37, 38 and 40, demonstrating that an increase in
microsomal stability translated well to improved in vivo oral
exposure.
Among the initial compounds evaluated, compounds 38 and
40 displayed attractive profiles, both in terms of potency and oral
exposure. In addition, no undesired inhibition of CYP450
isoforms or of the hERG channel was reported.
To tackle this issue, introduction of carboxylic acid side chains
was considered. The carboxylic acid moiety is known to be
poorly accommodated by the CYP450 active site and therefore
often leads to compounds with improved metabolic stability.15 In
addition, as a short-chain fatty acid receptor is targeted, an
additional interaction could be obtained by introduction of a
carboxylic acid functionality on the molecule.
Our hypothesis was validated by a series of compounds
depicted in Table 2. Good potencies as well as good microsomal
stabilities (more than 50% remaining after 30 or 60 min
incubation) were obtained (37, 38, 39, and 40). This key finding
turned out to be a general feature throughout the series for
compounds bearing a carboxylic acid moiety. Three compounds
were evaluated in pharmacokinetic studies (PO administration in
Sprague−Dawley rats at 5 mg/kg). Oral plasma exposure was
Data generated with this subset of compounds were strong
enough to further explore the series by keeping the carboxylic
acid as a key residue in the R3 position.
For synthetic ease, all the SAR studies previously described
were performed on racemic mixtures. To assess the influence of
the absolute configuration of the quaternary center on the
activity of our FFA2 antagonists, we prepared and evaluated
potency of both enantiomers of compound 37.
For that purpose, separation of racemic intermediate 11 was
performed by preparative chiral chromatography, thus affording
11a and 11b in their enantiopure forms (Scheme 3); synthesis of
both enantiomers of 37 was then carried on following the
previously described pathway. They were both evaluated in the
CaFlux assay. R enantiomer 37a was found active (214 nM),
whereas S enantiomer 37b was found totally inactive.
D
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Table 3. Optimization of R1
Table 4. Optimization of R1
a
b
Rat liver microsomal (RLM) stability % remaining after 30 min. Rat
c
liver microsomal (RLM) stability % remaining after 60 min. Plasma
exposure in rat after oral administration at 5 mg/kg.
methyl group for exploration of the SAR. Replacing R1 by benzyl
residues or biphenyl moieties did not lead to any affinity
improvement (compounds 43, 48). Heterocyclic replacements
of R1 were investigated, substituted heterocycles like thiophene
49 were moderately potent, whereas 5−6 fused heterocycles
50 and 51 were inactive. An interesting improvement in potency
could be achieved with benzothiophene analogue 56. The
position of the heteroatom as well as substitutions on the
benzothiophene moiety proved crucial for potency and could
lead to reduction in activity as demonstrated by compounds 54,
55, and 57. Adding a carbon between the amide linkage and the
benzothiophene (compound 59) was detrimental to activity.
Because these SAR studies allowed us to identify some
benzothiophene patterns as key R1 subunits to improve potency,
we decided to further explore fused heterocycles bearing an acid
moiety on the R3 side chain. Results are depicted in Table 4. As
previously observed, this substitution had a positive impact on
potency and microsomal stability (compounds 37, 60).
Interestingly, benzofuran derivatives (compounds 61, 62) were
found to be active, whereas other 5−6 fused heterocycles like
benzimidazole 64 and oxindole 63 were much less active.
Different potent and stable compounds bearing this key
carboxylic side chain were evaluated in pharmacokinetic studies
Absolute configuration of active enantiomer was determined
by converting its precursor 11b in methyl ester 41 whose [α]D
and absolute configuration are described in the literature.16
Absolute configuration was finally unambiguously confirmed
by an X-ray structure of a single crystal of compound 42 derived
from compound 11b (see Supporting Information)
LEAD OPTIMIZATION
■
Considering the profile of lead compounds 38 and 40, we
focused our efforts on both potency and oral exposure
improvements. Lead optimization was directed by modifications
of the two other subunits R1 and R2.
A wide variety of changes to the R1 part of the molecule was
explored, and a selection of the most informative is included in
Table 3. For synthetic reasons, we decided first to set R3 as a
E
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Table 5. Optimization of R1 and R2
(oral administration in Sprague−Dawley rats at 5 mg/kg). Good
profiles were generally observed, and interestingly the lon-
gest acid side chain in the R3 position (3 carbons vs 1 carbon)
seemed to have a positive impact on oral exposure (40 vs 38
and 61 vs 62).
However, the differences in both activity and oral exposure
found for the different potent moieties identified at R1 ((3,5-
dimethyl-phenyl)-acetamide, benzothiophene-3-carboxamide,
benzothiophene-3-acetamide, and benzofuran-3-acetamide)
were not significant enough to restrict our lead optimization
strategy to one of these patterns. Therefore, for the optimization
of the R2 subunit, we decided to keep these four interesting
groups in R1 in combination with the 3C-long acid side chain in
R3 identified from the previous SAR.
of potency (compounds 65−67). Compared to 60, extending the
linker (68) reduced significantly activity whereas removing the
benzylic CH2 (69) slightly improved potency. However, this was
not observed for 3-Cl phenyls (70 being 10-times less potent
than 72). Different substitutions patterns on the benzylamine
and introduction of heterocycles were also evaluated. We ob-
served that indole substitution led to a potent analogue (73), as
well as benzofuran and indazole moieties (74, 75). Neither
benzothiazole (76) nor 4-trifluoromethylbenzyl derivative (71)
showed any increase on potency.
The most striking aspect of this study was the unexpected
results observed for the 3-chloro benzyl substitution at R2
(compound 72). It appeared to be, by far, the most potent
pattern when combined to benzothiophene-3-acetamide, where-
as it proved to be detrimental when combined to other R1
substitutions. However, other combinations of the three other
R1 groups with the best R2 moiety identified confirmed that
6-indole, indazole, and benzofuran could replace 4-chlorobenzyl
R2 moiety and retain potency.
A large exploration of the SAR in R2 was undertaken and
summarized in Table 5.
First, several R2 substitutions were explored having R1 set as
benzothiophene-3-carboxamide (first column). Replacement of
the 4-Cl benzylamine moiety by alkyl groups led to complete loss
F
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a
A selection of the best molecules was evaluated in PK in
Sprague−Dawley rats at 5 mg/kg. Oral plasma exposure of these
compounds can be found in Table 5. Heterocyclic substitutions
at R2 had a detrimental impact on oral exposure as demonstrated
with compounds 91 and 92, whereas the position of the chlorine
atom had no impact on PK parameters (72 vs 60).
It appears that both in terms of oral exposure and potency, a
single compound clearly emerged from this table. Compound 72
was the most potent compound and one of the best orally
exposed in rat.
The corresponding R-enantiomer of 72 was prepared.
Enantiopure compound 99 was synthesized following the usual
route starting from chiral Boc-protected azetidine carboxylic acid
11b. As expected, an improvement in potency for 99 was
observed, with an IC50 of 9 nM. A thorough investigation of the
selectivity profile of 99 was performed. The close receptor
homologue FFA3 (also called GPR41) is not inhibited by 99 up
to 30 μM. Less than 50% inhibition at 10 μM was observed
against all the 55 receptors, ion channels, and transporters from
the Cerep ExpresSProfile. A full evaluation of its ADMET
parameters was performed (Figure 2). Solubility, microsomal
Table 6. Plasma PK Parameter of 99 in Sprague−Dawley Rat
IV
PO
PO
rat PK parameters
C0 (ng/mL)
(1 mg/kg)
(5 mg/kg)
(30 mg/kg)
968
1.7
2.1
3.2
half-life (h)
1.7
2.8
clearance (L/h/kg)
V
ss (L/kg)
F (%)
47
1213
705
0.4
40
AUC (0−24 h) (ng·h/mL)
517
6170
2230
0.25
C
max (ng/mL)
Tmax (h)
a
PEG400/MC 0.5% (10/90; v/v) for oral route (PO), PEG200/NaCl
0.9% (60/40; v/v) for intravenous route (IV).
recruitment during intestinal inflammation. It has been shown
that sodium acetate and sodium propionate, two natural ligands
of FFA2, act as chemoattractants and induce neutrophil
migration.6 Consequently, several FFA2 antagonists were
evaluated for their ability to inhibit human neutrophil migration
induced by acetate (Table 7). Most of the compounds displayed
Table 7. Potency of Representative Compounds for Inhibition
of Acetate-Induced Human Neutrophil Migration
compd
61
60
73
99
75
38
40
IC50 (nM)
561
75
58
27
24
23
5
a good ability to block neutrophil migration in this phenotypic
assay, thus demonstrating their potential toward inhibition of
inflammatory processes.
Moreover the specificity of this mechanism has been assessed
by determining the impact of FFA2 antagonists on both fMLP-
and IL8-mediated chemotaxis. None of the compounds inhibited
the neutrophil migration, induced by these two chemotactic
agents (data not shown).
A general trend throughout our chemical series was to exhibit a
high protein binding, typically higher than 95% for most of our
compounds bearing a carboxylic acid side chain at R3. As
mentioned earlier, the lack of an animal model to assess efficacy
did drive the decision to aim for a very long target coverage in
human. To figure out whether plasma protein binding might
limit the efficacy at the target level in vivo, the human neutrophil
migration assay previously set up was performed in plasma
instead of chemotaxis buffer (compound preincubation in whole
blood and then incubation with neutrophils in 90% plasma).
Similar potencies were obtained in both assays, in buffer or in
plasma (compound 99 IC50 being 27 and 43 nM, respectively).
To support clinical development, a biomarker was developed
enabling monitoring of target engagement in healthy volunteers
and patients in future studies. Efforts were focused on CD11b
activation-specific epitope (AE), considered as a hallmark of
neutrophil activation during the migration process. Indeed,
CD11b forms together with CD18 the membrane activated
complex-1 (Mac-1), a cell surface receptor found on neutrophils.
Through interaction with ICAM-1, MAC-1 mediates firm
adhesion and arrest of neutrophils on endothelium. This is an
essential step in neutrophil extravasation, leading to neutrophil
migration into tissues at foci of inflammation and infection. For
interaction with ICAM-1, a conformational change of CD11b
(CD11b activation-specific epitope [AE]) is required. Different
inflammatory stimuli up-regulate CD11b[AE] expression, which
is considered a more relevant marker of neutrophil activation in
blood than the total C11Db level.18−20
Figure 2. ADME parameters of 99.
stability, and plasma protein binding were all in the acceptable
range. No inhibition of CYP450 isoforms was observed, and no
time-dependent inhibition of CYP3A4 was noted. Compound 99
did not activate pregnane X receptor (PXR), suggesting a low
induction potential for CYP3A4.17 Compound 99 was found
negative in the AMES-II test and exhibited no inhibition of the
hERG channel.
Moreover, 99 showed excellent pharmacokinetic properties
in rat with a bioavailability of 47% and a linear increase of the
plasma exposure after oral dosing at 5 and 30 mg/kg (Table 6).
The extended half-life observed following the increase of oral
dose was consistent with the project objective to obtain long
target coverage in human. Moreover, this assumption was
further strengthened by allometric human prediction using
additional pharmacokinetics studies in nonrodent species (data
not shown).
NEUTROPHIL BIOLOGY
■
The potential of our FFA2 antagonists for inhibiting inflam-
matory processes has been further assessed. FFA2 was described
as a potential essential player in the process of neutrophil
G
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compounds, compound was injected to the well and the Ca2+-flux was
measured simultaneously to the injection of compound.
Hence, the capacity of acetate to increase CD11b[AE] expression
on neutrophils in human whole blood by flow cytometry was
identified as a potential readout. Our FFA2 antagonists dose-
dependently inhibited the acetate-induced conformational active
CD11b[AE] expression on neutrophils, indicating that this event is
driven by FFA2 (Figure 3). This observation represents the basis
Neutrophil Migration Assay. In the neutrophil migration assay,
neutrophils, isolated from buffy coats from human volunteers, were
treated with a compound for 30 min in chemotaxis buffer (RPMI 1640
medium, supplemented with 10 mM HEPES). Subsequently, the
neutrophils were transferred to the upper wells of a HTS transwell
96 permeable support system (with 5.0 μm pore size polycarbonate
membrane (Corning)), of which the lower wells were filled with
chemotaxis buffer containing compound and sodium acetate solution.
After 1 h of incubation, migration of the neutrophils toward the sodium
acetate was quantified by measuring the ATP-content of the lower wells
using the ATPlite luminescence ATP detection assay system
(PerkinElmer, cat. no. 436110). The detected luminescent signal was
linearly related to the number of migrated cells.
Whole Blood CD11b[AE] Assay. Human blood was collected by
venipuncture from human volunteers, who gave informed consent, in
acid-citrate-dextrose (ACD) vacutubes, mixed well, and processed as
soon as possible, the latest within 90 min after blood collection. Blood
was diluted 1:1 in RPMI1640. Diluted blood was treated simultaneously
with a compound in DMSO solution or with DMSO (max 0.3% of
DMSO) and with 20 μg/mL cytochalasin B, 2 ng/mL TNFalpha at
37 °C for 15 min without shaking. Then GPR43 agonist (sodium
acetate) was added to blood. Blood was gently mixed and incubated at
37 °C for 30 min without shaking. Neutrophil surface expression of
CD45, CD66b, and CD11b[AE] markers was determined by flow
cytometry. Unspecific binding of antibodies was blocked by addition of
2 μg/sample of normal mouse IgG (Invitrogen) and incubation for
10 min at +4 °C in the dark. Then 2 μL of CD45 V450 antibody (BD
Biosciences), 2.5 μL of CD11b APC Mouse Anti-Human CD11b[AE]
1 antibody (eBiosciences), and 10 μL of CD66b FITC antibody (BD
Biosciences) were added to blood at the same time and samples
incubated for 30 min at +4 °C in the dark. Red blood cells were lysed and
samples fixed by addition of RBC lysing Buffer (1× solution from BD
FACS Lysing Solution 10×, BD Pharmigen) and incubated for 10 min at
room temperature in the dark. The cells were centrifuged at 550g for
10 min at +4 °C and washed once with PBS. The cells were resuspended
in cold 300 μL of wash/staining buffer (PBS + 2% FBS), transferred to
polystyrene 5 mL tube, and placed on ice protected from light until
analyzed with FACScan/CytekDev XDP8 and FlowJo Collector’s,
FlowJo V7.6 software. Neutrophils were gated and selected according to
morphological characteristics using FSC and SSC parameters and
immunophenotype as CD45high CD66bhigh CD11b[AE]+ cells. Cellular
expression of CD11b[AE] was measured by determination of mean
fluorescence intensity units (MFI).
Aqueous Solubility. A serial dilution of test compound was
prepared in DMSO and further diluted in 0.1 M phosphate buffer,
pH 7.4, or 0.1 M citrate buffer pH 3.0 to obtain final concentrations of
299, 149, 74.6, 37.3, and 18.8 μM, with all solutions having a total
percentage of DMSO of 3% (v/v). The assay plates were incubated for
1 h at 37 °C while shaking at 230 rpm. The plates were scanned with a
white light microscope, yielding individual pictures (50×) per
concentration. The precipitate was analyzed and converted into a
number by a custom-developed software tool. The lowest concentration
at which the compound appears completely dissolved is reported.
Thermodynamic Solubility. Two individual solutions of about
2 mg/mL test compound were prepared in a 0.1 M phosphate buffer pH
7.4 or a 0.1 M citrate buffer pH 3.0 at room temperature in glass vials.
The samples were stirred for 24 h at room temperature. The vials were
centrifuged shortly, and the supernatant was filtered. Per sample, two
dilutions (factor 10 and 100) were made in DMSO and analyzed on
LC/MS-MS (API2000 from Applied Biosystems). A serial dilution of
the compound was made in DMSO and diluted in 50−50 water/
methanol to obtain final concentrations of 400, 200, 100, 50, 10, 2, 1, and
0.4 ng/mL with a total percentage of DMSO of 1.25% (v/v). These
dilutions were also analyzed on LC/MS-MS. The peak areas of the serial
dilutions were plotted in a graph, and a linear or polynomial of the
second-order equation was used to calculate the unknown concen-
trations of the test compound.
Figure 3. Dose-dependent inhibition of acetate-induced CD11b[AE]
expression by compound 99, measured by flow cytometry in human
whole blood obtained from 16 different donors.
for the first biomarker assay described to date for FFA2.21 In this
assay, compound 99 is a potent inhibitor of the FFA2-induced
CD11b[AE] expression (IC50 = 483 nM 72, n = 16).
As such, compound 99 showed high potency in all in vitro
assays, even in whole blood context. On the basis of its overall
profile, this molecule 99, 4-[[(R)-1-(benzo[b]thiophene-3-
carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-
amino]-butyric acid, was selected as preclinical candidate
(GLPG0974), predicted to be able to block the acetate-induced
neutrophil migration process in human for a long period of time.
Additional pharmacokinetics and safety studies were performed
after candidate selection to allow progression of 99 to clinical
phases, becoming the first FFA2 antagonist reported to reach
the clinic. From a drug design point of view, the course of the
project illustrated once again that the combination of SAR
improvements, although often not additive, could lead to
unpredicted potent compounds. At the late stage of a lead
optimization program, a systematic combination approach must
not be neglected.
EXPERIMENTAL SECTION
■
Calcium Mobilization Assay. The following assay can be used for
determination of GPR43 activation. The assay measures intracellular
calcium release induced by activation of GPR43. On day 1, GPR43
stably expressing cells (Multispan) were seeded at 6000 cells/25 μL
medium (DMEM supplemented with 10% FBS, 10 μg/mL puromycin,
and 1% Pen/Strep) in poly-D-lysine coated 384-well plates and
incubated overnight at 37 °C 10% CO2. On day 2, 25 μL of a
fluorescent Ca2+ indicator (Calcium 4 assay; Molecular Devices)
supplemented with 2.5 mM probenecid was added on top of the cells
and incubated for 2h at 37 °C. Subsequently, compounds at increasing
concentration (10 μL/well) were added to the plate and incubated for
15 min at 37 °C. Finally the cells were triggered with sodium acetate at
EC80 concentration (concentration which gives 80% of the activity of the
GPR43) and the Ca2+-flux was measured simultaneously to the agonist
injection on a fluorescence plate reader (FlexStation3), measuring the
antagonist activity of the compounds. To measure agonist activity of the
H
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Microsomal Stability. A 10 mM stock solution of test compound in
DMSO was diluted to 6 μM in a 105 mM phosphate buffer pH 7.4 and
prewarmed at 37 °C. This dilution was added to microsomal/
cofactormix in the same proportion (50 μL of compound diluted in
buffer and 50 μL of the mix of microsome and cofactor) at 37 °C under
shaking at 300 rpm. Final reaction conditions were: 3 μM of compound
(n = 2), 0.5 mg/mL microsomes, 0.4 U/mL GDPDH, 3.3 mM MgCl2,
3.3 mM glucose-6-phosphate, and 1.3 mM NADP+, incubation volume
100 μL, with all solutions having a total percentage of DMSO of 0.2%
(v/v). After 0 and 30 min of incubation, the reaction was stopped with
MeOH (1:1). The samples were mixed, centrifuged, and filtered and the
supernatant analyzed by LC-MS/MS (API2000 from Applied
Biosystems). The instrument responses (i.e., peak areas) were
referenced to the zero time-point samples (considered as 100%) in
order to determine the percentage of compound remaining.
Hepatocyte Stability. Test compound (1 μM initial concentration,
n = 2) was incubated in Williams’ Medium E, containing 4 mM
L-glutamine and 2 mM magnesium sulfate, with pooled cryopreserved
hepatocytes (Celsis International) in suspension at cell density of 0.5
million viable cells/mL. The incubations were performed at 37 °C in a
shaking water bath, with samples taken from the incubation at 0, 10, 20,
45, and 90 min, and reactions terminated by addition of an equal amount
of acetonitrile containing carbamazepine as analytical internal standard.
Samples were centrifuged and the supernatant fractions analyzed by
LC-MS/MS (Waters TQD instruments). The instrument responses
(i.e., peak heights) were referenced to the zero time-point samples
(considered as 100%) in order to determine the percentage of
compound remaining. Ln plots of the % remaining for each compound
were used to determine the half-life for the hepatocyte incubations.
Plasma Protein Binding (Equilibrium Dialysis). A 5 μM solution
of test compound was made in freshly thawed human, rat, mouse, or dog
plasma plasma (BioReclamation INC) with a total percentage of DMSO
of 0.5% (v/v). A Pierce Red Device plate with inserts (ThermoScien-
tific) was filled with 450 μL of PBS (buffer chamber) and 300 μL of the
spiked plasma (plasma chamber) and incubated at 37 °C while shaking
at 100 rpm. After 4 h incubation, 120 μL from each chamber was
transferred into 480 μL of methanol. The samples were mixed and
immediately centrifuged for 30 min at 1400 rcf at 4 °C. The supernatant
was filtered and analyzed by LC-MS/MS (API2000 from Applied
Biosystems). Addition of test compound peak area in the buffer chamber
and the plasma chamber were considered to be 100% compound.
The percentage bound to plasma is derived from these results and is
reported as percentage bound to plasma.
cannulated rats or at the retro-orbital sinus with lithium heparin as
anticoagulant at 4−7 time points in the following range: 0.05−8 h
(intravenous route) and 0.25−6 or 24 h (oral route). Whole blood
samples were centrifuged at 5000 rpm for 10 min, and the resulting
plasma samples were stored at −20 °C pending analysis. Quantification
of compound levels in plasma: An API4000 mass spectrometer
(ABSciex) operating in positive TurboIonSpray mode was used for
the detection of the test compound in plasma, and Analyst 1.4.2 software
(ABSciex) was used for samples injection, peaks integration, and plasma
level quantification. Pharmacokinetic parameters were calculated using
Winnonlin (Pharsight, US, version 5.2.1).
Chemistry. All reagents were of commercial grade and were used as
received without further purification unless otherwise stated.
Commercially available anhydrous solvents were used for reactions
conducted under inert atmosphere. Reagent grade solvents were used in
all other cases unless otherwise specified. Column chromatography was
performed on silica standard (35−70 μm). Thin layer chromatography
was carried out using precoated silica gel 60 F-254 plates (thickness
0.25 mm). 1H NMR spectra were recorded on a Bruker Advance
400 NMR spectrometer (400 MHz). Chemical shifts (δ) for 1H NMR
spectra are reported in parts per million (ppm) relative to
tetramethylsilane (δ 0.00) or the appropriate residual solvent peak as
internal reference. Multiplicities are given as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad (br). 13C NMR
spectrum of 99 was recorded on a Varian Unity Inova 400 NMR
spectrometer at 80 °C. Electrospray MS spectra were obtained on a
Waters platform LC/MS spectrometer. Unless otherwise specified the
purity of all intermediates and final compounds was determined to be
>95% by analytic LCMS: columns used, Waters Acquity UPLC BEH
C18 1.7 μm, 2.1 mm ID × 50 mm L or Waters Acquity UPLC BEH C18
1.7 μm, 2.1 mm ID × 30 mm L. All the methods are using MeCN/H2O
gradients. MeCN and H2O contain either 0.1% formic acid or NH3 (10
mM). Preparative LCMS: column used, Waters XBridge Prep C18 5 μm
ODB 30 mm ID × 100 mm L. All the methods are using MeOH/H2O
gradients. MeOH and H2O contain either 0.1% formic acid or 0.1%
siethylamine. CHN analysis was determined using the PerkinElmer
2400 CHNS/O series II elemental analyzer, which uses a combustion
method in a pure oxygen environment to convert the accurately weighed
sample into the simple gases CO2, H2O, and N2. After reduction through
pure copper, the resulting CHN gases are then controlled to exact
conditions of pressure, temperature, and volume whereupon the system
uses a steady state wavefront approach to separate the controlled gases.
This approach involves separating a continuous homogenized mixture
of gases through a chromatographic column. The gases eluting off the
column are measured as a function of their thermal conductivity.
Microwave heating was performed with a Biotage Initiator.
hERG Patch Clamp. Whole-cell patch-clamp recordings were
performed on HEK293 cells stably expressing hERG channels. Cells
were cultured on 12 mm round coverslips (German glass, Bellco)
anchored in the recording chamber using two platinum rods
(Goodfellow). All compound concentrations were made in DMSO
prior to being added to extracellular buffer with all solutions having a
total percentage of DMSO of 1% (v/v). The external bathing solution
contained: 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 5 mM glucose,
10 mM HEPES, pH 7.4. The internal patch pipet solution contains:
100 mM potassium gluconate, 20 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
5 mM Na2ATP, 2 mM glutathione, 11 mM EGTA, 10 mM HEPES, pH
7.2. Compound was perfused using an Biologic MEV-9/EVH-9 rapid
perfusion system. hERG currents were evoked using an EPC10 amplifier
controlled by Pulse v8.77 software (HEKA) with an activating pulse to
+40 mV for 1000 ms followed by a tail current pulse to −50 mV for
2000 ms, holding potential was −80 mV. Pulses were applied every 15 s,
and all experiments were performed at room temperature.
2-Methyl-azetidine-1,2-dicarboxylic Acid 1-tert-Butyl Ester 2-
Ethyl Ester (5). Step 1: 2-Benzylamino-propionic Acid Ethyl Ester (8).
To a solution of 2-bromo-propionic acid ethyl ester 7 (17.9 mL, 1 equiv)
in MeCN (250 mL) was added benzylamine 6 (13.5 mL, 0.9 equiv) and
potassium carbonate (28.6 g, 1.5 equiv). The reaction was refluxed for
4 h then cooled to 20 °C and diluted with EtOAc. The crude was filtered,
and the filtrate was concentrated under reduced pressure then purified
by chromatography on silica gel (elution with heptane/EtOAc: 70/30)
to afford 2-benzylamino-propionic acid ethyl ester 8 (24.7 g, 97% yield).
1H NMR δ (ppm) (CDCl3): 7.37−7.29 (4H, m), 7.28−7.22 (1H, m),
4.20 (2H, q), 3.82 (1H, d), 3.69 (1H, d), 3.38 (1H, q), 1.33 (3H, d), 1.29
(3H, t). MW (calcd): 207.2. MW (obsd): 208.1 (M + 1).
Step 2: 2-[Benzyl-(2-chloro-ethyl)-amino]-propionic Acid Ethyl
Ester (9). To a solution of chloroacetaldehyde (56 mL, 45% w/w in
water, 3 equiv) in DCM (75 mL) under nitrogen was added MgSO4
(55 g, 4 equiv). The mixture was stirred for 15 min at 20 °C under
nitrogen. The solid was filtered, washed with dry DCM, and the resulting
filtrate (solution I) rapidly used in the following reaction. To a solution
of 8 (27.7 g, 1 equiv) in dry DCM (114 mL) was added MgSO4 (10.8 g,
0.75 equiv). The reaction was cooled to 0 °C then the above solution of
chloroacetaldehyde in dry DCM (solution I) and acetic acid (1 equiv)
was added. Sodium triacetoxyborohydride (38 g, 1.5 equiv) was added
portionwise. The reaction was stirred for 1 h at 0 °C. The crude was
carefully quenched with a saturated aqueous solution of NaHCO3. Then
Single Dose Pharmacokinetic Study in Rats. All animal
protocols were reviewed and approved by Galapagos Animal Care and
Use Committee registered at “Comite
́ ́
National de Reflexion Ethique
sur l’Experimentation Animale” (France). Compounds were formulated
́
in PEG200/NaCl 0.9% (60/40; v/v) for the intravenous route and
in PEG400/0.5% methylcellulose (10/90 v/v) for the oral route.
Test compounds were orally dosed as a single esophageal gavage at
5−10 mg/kg and intravenously dosed as a bolus via the caudal vein at
1 mg/kg to male Sprague−Dawley rats. Each group consisted of three
rats. Blood samples were collected either via the jugular vein using
I
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aqueous NaOH (2N) was added. The aqueous layer was extracted with
DCM. The combined organic layers were washed with an aqueous
saturated solution of NaHCO3, then dried over MgSO4 and
concentrated under reduced pressure. The crude was purified by
chromatography on silica gel (elution with heptane/EtOAc 98/2 to
95/5) to afford 2-[benzyl-(2-chloro-ethyl)-amino]-propionic acid ethyl
(2H, m), 4.77−4.44 (2H, m), 3.94−3.74 (2H, m), 2.94 (3H, s), 2.55−
2.39 (1H, m), 2.31−2.18 (1H, m), 1.82 (3H, s), 1.48 (9H, s).
Steps 3 and 4: Synthesis of 3.
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (4-Chloro-benzyl)-methyl-amide (3). Step 3: BOC removal.
To a solution of 12 (283 mg, 1 equiv) in dioxane under argon was added
a solution of HCl (4N in dioxane, 10 equiv). The reaction was stirred for
16 h at 20 °C, then concentrated under reduced pressure. The crude was
triturated with a mixture of DCM/iPr2O, and the resulting solid was
filtered and dried to afford the corresponding free amine as a hydro-
chloride salt (230 mg, quantitative yield). 1H NMR δ (ppm) (MeOD-
d4): 7.46−7.40 (2H, m), 7.38−7.32 (2H, m), 4.78−4.58 (2H, m), 4.21−
4.07 (1H, m), 3.87−3.77 (1H, m), 3.11−2.98 (1H, m), 2.93 (3H, s),
2.76−2.65 (1H, m), 1.97 (3H, s).
1
ester 9 (27.4 g, 85% yield). H NMR δ (ppm) (CDCl3): 7.40−7.29
(4H, m), 7.29−7.22 (1H, m), 4.24−4.14 (2H, m), 3.90 (1H, d), 3.77
(1H, d), 3.50 (1H, q), 3.43−3.36 (2H, m), 3.13−2.94 (2H, m), 1.34
(3H, d), 1.31 (3H, t). MW (calcd): 269.8. MW (obsd): 270.1 (M +
1, 35Cl).
Step 3: 1-Benzyl-2-methyl-azetidine-2-carboxylic Acid Ethyl Ester
(10). A solution of 9 (27.4 g, 1 equiv) in dry THF (253 mL) was cooled
to −78 °C under nitrogen. KHMDS (170.4 g, 15% w/w in toluene,
1.25 equiv) was slowly added so that temperature was kept below
−65 °C. The reaction was stirred for 1 h at −70 °C, then acetic acid
(1.75 mL, 0.3 equiv) was added. The reaction was warmed to 20 °C and
stirred for 10 min, then quenched with a saturated aqueous solution of
NaHCO3 and partially concentrated under reduced pressure. The crude
was extracted twice with DCM. The organic layers were combined, dried
over MgSO4, filtered, and concentrated under reduced pressure. The
crude was purified by chromatography on silica gel (elution with
heptane/EtOAc 100/0 then 95/5) to afford 1-benzyl-2-methyl-
azetidine-2-carboxylic acid ethyl ester 10 (18.75 g, 79% yield).
1H NMR δ (ppm) (CDCl3): 7.34−7.19 (5H, m), 4.24−4.16 (2H, m),
3.79 (1H, d), 3.58 (1H, d), 3.30−3.22 (1H, m), 3.15−3.05 (1H, m),
2.63−2.54 (1H, m), 1.97−1.88 (1H, m), 1.49 (3H, s), 1.29 (3H, t).
MW (calcd): 233.3. MW (obsd): 234.1 (M + 1).
Step 4: 2-Methyl-azetidine-1,2-dicarboxylic Acid 1-tert-butyl
Ester 2-Ethyl Ester (5). To a solution of 10 (18,75 g, 1 equiv) in
EtOH (1,500 mL) was added Boc2O (75g, 1.2 equiv) and Pd/C (7.3 g,
0.05 equiv). The flask was evacuated and backfilled with argon, then the
reaction was evacuated and backfilled with H2 and stirred for 15 h at
20 °C under atmospheric pressure. The crude was filtered through a pad
of Celite and washed with EtOH and DCM. The filtrate was
concentrated under reduced pressure to afford 2-methyl-azetidine-1,2-
dicarboxylic acid 1-tert-butyl ester 2-ethyl ester 5 (57 g, 79% yield). 1H
NMR δ (ppm) (CDCl3): 4.12−3.92 (2H, m), 3.86−3.71 (1H, m),
3.66−3.54 (1H, m), 2.17−2.00 (1H, m), 1.99−1.83 (1H, m), 1.33 (3H,
s), 1.21 (9H, s), 1.11 (3H, t). MW (calcd): 243.3. MW (obsd): 266+
(M + Na+).
Step 4: Amide coupling. To a solution of (3,5-dimethyl-phenyl)-acetic
acid (65 mg, 1.5 equiv) in DCM (1 mL) and THF (1 mL) was added
HOBt (35 mg, 1.1 equiv) and EDC·HCl (77 mg, 1.5 equiv). Then the
above prepared azetidine intermediate as a hydrochloride salt (75 mg,
1 equiv) and TEA (70 μL, 3 equiv) were added. The reaction was stirred
at 20 °C for 15 h. The crude was concentrated under reduced pressure
and partitioned between a saturated aqueous solution of NaHCO3 and
EtOAc. The aqueous layer was extracted three times with EtOAc. The
organic layers were combined, washed with water then with brine, dried
over MgSO4, filtered, and concentrated under reduced pressure to afford
1-[2-(3,5-dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carboxylic
1
acid (4-chloro-benzyl)-methyl-amide 3 (77 mg, 74% yield). H NMR
δ (ppm) (MeOD-d4): 7.49−7.25 (4H, m), 7.02−6.89 (3H, m), 4.75−
4.49 (2H, m), 4.22−4.05 (1H, m), 3.95−3.81 (1H, m), 3.51−3.35 (2H,
m), 3.00−2.88 (3H, m), 2.70−2.36 (2H, m), 2.35−2.30 (6H, m), 2.00−
1.87 (3H, m). MW (calcd): 398.2. MW (obsd): 399.4 (M + 1).
General Procedure A2 for the Synthesis of Compounds 45, 46, and
47. Step 1: Following general procedure A1 and using (4-iodo-phenyl)-
acetic acid or (3-iodo-phenyl)-acetic acid in step 4 gave the cor-
responding iodo intermediate.
Step 2: Suzuki coupling. To a solution of aryl boronic acid
(1.5 equiv) in dioxane/H2O (9/1) were added Na2CO3 (4 equiv) and
the corresponding iodo intermediate (1 equiv). The flask was
evacuated and backfilled with argon. Then Pd(PPh3)4 was added
and the reaction was heated at 90 °C until completion. The crude was
partitioned between water and EtOAc. The organic layer was washed
with water and brine, dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude was purified by chromatography on
silica gel (elution with heptane/EtOAc: 100/0 to 20/80) to afford the
corresponding carboxamide.
First Route: Introduction of R2 First. General Procedure A1 for
Synthesis of Compounds 3, 4, 12, 43, 44, 48, 49, 51, 52, 53, 54, 55,
56, 57, 58, and 59. Steps 1 and 2: Synthesis of 12.
1-(2-Biphenyl-4-yl-acetyl)-2-methyl-azetidine-2-carboxylic Acid
(4-Chloro-benzyl)-methyl-amide (45). Following general procedure
A2 and using (4-iodo-phenyl)-acetic acid (30 mg, 1 equiv) in step 1
and phenyl boronic acid (11 mg, 1.5 equiv) in step 2 gave 1-(2-biphenyl-
4-yl-acetyl)-2-methyl-azetidine-2-carboxylic acid (4-chloro-benzyl)-
methyl-amide 45 (12 mg, 35% yield over two steps). 1H NMR
δ (ppm) (MeOD-d4): 7.88−7.68 (4H, m), 7.68−7.36 (9H, m), 4.89−
4.61 (2H, m), 4.42−4.19 (1H, m), 4.13−3.63 (3H, m), 3.06 (3H, d),
2.87−2.49 (2H, m), 2.08 (3H, d). MW (calcd): 446.2. MW (obsd):
447.4 (M + 1).
General Procedure A3 for the Synthesis of Compounds 74, 76, 85,
87, 93, and 97. Step 1: Following general procedure A1 and using
the corresponding secondary amine intermediate in step 2 and the cor-
responding R1 carboxylic acid in step 4 gave the corresponding ester
intermediate.
Step 2: Saponification. To a solution the above prepared ester
(1 equiv) in EtOH was added an aqueous solution of 2N NaOH
(2 equiv). The reaction was stirred at 20 °C for 15 h. The solvent was
removed under reduced pressure, and the crude was partitioned
between water and EtOAc. The organic layer was discarded, and the
aqueous layer was acidified by addition of a solution of citric acid 10% in
water until pH = 3 and thoroughly extracted by EtOAc. The organic
layers were combined, dried over MgSO4, filtered, and concentrated
under reduced pressure to afford the corresponding carboxylic acid.
4-{[1-(Benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-car-
bonyl]-benzofuran-6-ylmethylamino}-butyric Acid (74). Following
2-[(4-Chloro-benzyl)-methyl-carbamoyl]-2-methyl-azetidine-1-
carboxylic Acid tert-Butyl Ester (12). Step 1: To a solution of 5 (63 g,
1 equiv) in EtOH was added an aqueous solution of NaOH 2N (260 mL,
2 equiv). The reaction was stirred at 20 °C for 15 h. The solvent was
removed under reduced pressure, and the crude was partitioned
between water and EtOAc. The organic layer was discarded, and the
aqueous layer was acidified by addition of a solution of citric acid 10% in
water until pH = 3 and thoroughly extracted three times with EtOAc.
The organic layers were combined, dried over MgSO4, filtered, and
concentrated under reduced pressure to afford 2-methyl-azetidine-1,2-
1
dicarboxylic acid 1-tert-butyl ester 11 (50 g, 89% yield). H NMR
δ (ppm) (CDCl3): 3.93−3.81 (1H, m), 3.81−3.68 (1H, m), 2.85−2.71
(1H, m), 2.12−2.00 (1H, m), 1.72 (3H, s), 1.48 (9H, s).
Step 2: Amide coupling. To a solution 11 (250 mg, 1 equiv) in DCM
(2 mL) and THF (2 mL) was added HOBt (157 mg, 1 equiv) and EDC·
HCl (334 mg, 1.5 equiv), then (4-chloro-benzyl)-methyl-amine
(270 mg, 1 equiv) and TEA (660 μL, 4 equiv) were added. The
reaction was stirred at 20 °C for 15 h. The crude was concentrated under
reduced pressure and partitioned between a saturated aqueous solution
of NaHCO3 and EtOAc. The aqueous layer was extracted three times
with EtOAc. The organic layers were combined, washed with water
then with brine, dried over MgSO4, filtered, and concentrated under
reduced pressure to afford 2-[(4-chloro-benzyl)-methyl-carbamoyl]-2-
methyl-azetidine-1-carboxylic acid tert-butyl ester 12 (283 mg, 70%
1
yield). H NMR δ (ppm) (CDCl3): 7.39−7.31 (2H, m), 7.26−7.15
J
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Journal of Medicinal Chemistry
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general procedure A3 and using 4-[(benzofuran-6-ylmethyl)-amino]-
butyric acid ethyl ester and benzo[b]thiophene-3-carboxylic acid in step
1 gave the corresponding carboxylic acid 4-{[1-(benzo[b]thiophene-3-
carbonyl)-2-methyl-azetidine-2-carbonyl]-benzofuran-6-ylmethylami-
pressure. The crude was purified by chromatography on silica gel to
afford the desired compound.
1-(2-Benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-2-carbox-
ylic Acid 4-Chloro-benzylamide (1). Following general procedure B1
and using intermediate 13c (100 mg) and 4-chloro-benzylamine
(63 μL) gave 1-(2-benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-
2-carboxylic acid 4-chloro-benzylamide 1 (64 mg, 45% yield). 1H NMR
δ ppm (CDCl3): 8.74 (1H, br s), 7.91 (1H, d), 7.74 (1H, d), 7.45−7.35
(2H, m), 7.27−7.21 (3H, m), 7.15 (2H, d), 4.39 (2H, ddd), 4.10−3.94
(2H, m), 3.71 (2H, s), 2.96−2.86 (1H, m), 2.15−2.06 (1H, m), 1.81
(3H, s). MW (calcd): 412.1. MW (obsd): 413.3 (M + 1).
General Procedure B2 and Synthesis of Compound 32. To a
solution of 1-chloro-N,N-2-trimethylpropenylamine (2 equiv) in DCM
under nitrogen was added the corresponding carboxylic acid (1 equiv).
The solution was stirred at 20 °C for 1 h, then added to a solution of
appropriate amine (1.2 equiv) in DCM at 0 °C. TEA (2 equiv) was then
added, and the mixture was stirred at 0 °C for 3 h. The crude was diluted
with DCM, washed twice with a saturated aqueous solution of NaHCO3,
twice with aqueous HCl (0.5N), twice with water, dried over MgSO4,
filtered, concentrated under reduced pressure, and purified by
chromatography on silica gel to afford the corresponding carboxamide.
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (4-Chloro-benzyl)-(2-methanesulfonylamino-ethyl)-amide
(32). Following general procedure B2 and using intermediate 13a
(120 mg) and N-[2-(4-chloro-benzylamino)-ethyl]-methanesulfona-
mide gave (130 mg) 32 (80 mg, 35% yield). 1H NMR δ ppm (CDCl3):
7.45−7.31 (2H, m), 7.27−7.11 (1H, m), 7.10−6.85 (4H, m), 5.25−4.70
(1H, m), 4.64−4.21 (2H, m), 4.16−3.63 (3H, m), 3.50−3.21 (4H, m),
3.03−2.89 (4H, m), 2.77−2.49 (1H, m), 2.33 (6H, s), 1.86 (3H, s). MW
(calcd): 505.2. MW (obsd): 506.1 (M + 1).
General Procedure B3 for the Synthesis of Compounds 33, 34, 35,
and 36. 1-(2-Benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-2-
carboxylic Acid (4-Chloro-benzyl)-(3-sulfamoyl-propyl)-amide (34).
Step 1: Following general procedure B2 using intermediate 13c (50 mg)
with 3-(4-chloro-benzylamino)-propane-1-sulfonic acid 2,4-dimethoxy-
benzylamide (70 mg) gave the corresponding sulfonamide intermediate
(60 mg, 52% yield).
Step 2: Sulfonamide cleavage. The above prepared protected
sulfonamide (52 mg) was dissolved in TFA (10 mL) and stirred at
20 °C for 15 h. The crude was concentrated under reduced pressure
then partitioned between water and EtOAc. The organic layer was
washed twice with water, then dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude was purified by
chromatography on silica gel to give 1-(2-benzo[b]thiophen-3-yl-
acetyl)-2-methyl-azetidine-2-carboxylic acid (4-chloro-benzyl)-(3-sulfa-
moyl-propyl)-amide 34 (16 mg, 39% yield). 1H NMR δ (ppm)
(CDCl3): 7.90−7.71 (2H, m), 7.46−7.26 (5H, m), 7.19−6.89 (2H, m),
5.20−4.88 (2H, m), 4.70−4.34 (1H, m), 4.08−3.85 (2H, m), 3.80−3.44
(3H, m), 3.29−2.88 (3H, m), 2.59−2.37 (1H, m), 2.36−2.13 (1H, m),
2.09−1.98 (1H, m), 1.86 (3H, br s). MW (calcd): 533.1. MW (obsd):
534.4 (M + 1).
1
no}-butyric acid 74 (36 mg, 35% yield over four steps). H NMR
δ (ppm) (MeOD-d4): 8.33−8.04 (1H, m), 8.02−7.90 (1H, m), 7.81
(1H, d), 7.72−7.62 (1H, m), 7.61−7.40 (4H, m), 7.35−7.14 (1H, m),
6.89 (1H, m), 5.12−4.69 (2H, m), 4.39−3.95 (2H, m), 3.82−3.18 (2H,
m), 2.85−2.46 (2H, m), 2.45−2.28 (2H, m), 2.23−1.86 (5H, m). MW
(calcd): 490.2. MW (obsd): 491.4 (M + 1).
Second Route: Introduction of R1 First. Synthesis of 13a, 13b,
13c, and 13d. Step 1: BOC removal. To a solution of intermediate 7
(1 equiv) in dioxane under argon was added a solution of HCl (4N in
dioxane,10 equiv). The reaction was stirred for 16 h at 20 °C, then
concentrated under reduced pressure. The crude was triturated with a
mixture of DCM/iPr2O, and the resulting solid was filtered and dried to
afford 2-methyl-azetidine-2-carboxylic acid ethyl ester hydrochloride.
1H NMR δ (ppm) (CDCl3): 9.48 (1H, br s), 4.35 (2H, q),
4.16−4.00 (2H, m), 2.82−2.70 (1H, m), 2.60−2.45 (1H, m), 1.95
(3H, s), 1.36 (3H, t).
Step 2: Amide coupling. To a solution of the corresponding carboxylic
acid (1 equiv) in either DCM or THF was added HOBt (1.1 equiv),
EDC·HCl (1.5 equiv). Then the above prepared azetidine intermediate
as a hydrochloride salt (1 equiv) and TEA (4 equiv) were added. The
reaction was stirred at 20 °C for 15 h. The crude was concentrated under
reduced pressure and partitioned between a saturated aqueous solution
of NaHCO3 and EtOAc. The aqueous layer was extracted three times
with EtOAc. The organic layers were combined, washed with water then
with brine, dried over MgSO4, filtered, and concentrated under reduced
pressure to afford the corresponding carboxamide.
Step 3: Saponification. To a solution of the corresponding above
prepared carboxamide (1 equiv) in EtOH or MeOH was added an
aqueous solution of 2N NaOH (2 equiv). The reaction was stirred at
20 °C for 15 h. The solvent was removed under reduced pressure, and
the crude was partitioned between water and EtOAc. The organic layer
was discarded, and the aqueous layer was neutralized by addition of
aqueous HCl (2N) and thoroughly extracted with EtOAc. The organic
layers were combined, dried over MgSO4, filtered, and concentrated
under reduced pressure to afford the corresponding carboxylic acid
intermediates 13a, 13b, 13c, and 13d.
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
1
ylic Acid (13a). H NMR δ (ppm) (MeOD-d4): 6.98−6.90 (3H, m),
4.26−4.12 (2H, m), 3.47 (2H, bs), 2.53−2.43 (1H, m), 2.34 (6H, bs),
2.29−2.18 (1H, m), 1.73 (3H, s). MW (calcd): 261.3. MW (obsd):
262.0 (M + 1).
1-(2-Benzofuran-3-yl-acetyl)-2-methyl-azetidine-2-carboxylic
Acid (13b). 1H NMR δ (ppm) (DMSO): 7.86 (1H, s), 7.65−7.52 (2H,
m), 7.34−7.19 (2H, m), 4.24−4.13 (2H, m), 3.49 (2H, dd), 2.39−2.26
(1H, m), 2.21−2.07 (1H, m), 1.56 (3H, s). MW (calcd): 273.3. MW
(obsd): 274.1 (M + 1).
1-(2-Benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-2-carbox-
ylic Acid (13c). 1H NMR δ (ppm) (CDCl3): 87.92 (1H, d), 7.78 (1H,
d), 7.50−7.40 (2H, m), 7.35 (1H, s), 4.05−3.93 (2H, m), 3.79 (2H, s),
2.96−2.76 (1H, m), 2.19−2.08 (1H, m), 1.80 (3H, s). MW (calcd):
289.3. MW (obsd): 290.0 (M + 1).
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (3-Carbamoyl-propyl)-(4-chloro-benzyl)-amide (26). Step 1:
Following general procedure B1 using intermediate 13a (300 mg) and
4-(4-chloro-benzylamino)-butyric acid ethyl ester (353 mg) in the last
step gave the corresponding ester intermediate.
1-(2-Benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-car-
boxylic Acid (13d). 1H NMR δ (ppm) (CDCl3): δ 8.41 (1H, d), 7.92
(1H, d), 7.87 (1H, s), 7.57−7.44 (2H, m), 4.49−4.39 (1H, m), 4.31−
4.21 (1H, m), 3.10−3.00 (1H, m), 2.36−2.25 (1H, m), 2.01 (3H, s).
General Procedure B1 for the Synthesis of Compounds 1, 2, 15, 16,
19, 20, 21, 22, 23, 27, and 50. To a solution of the relevant carboxylic
acid (13a, 13b, 13c, or 13d) (1 equiv) in DCM under nitrogen was
added DMF (0.01 equiv) then oxalyl chloride (2 equiv). The solution
was stirred at 20 °C for 30 min, then cooled to 0 °C. A solution of the
corresponding amine (1.5 equiv) and either TEA or DIPEA (2 to
7 equiv) in DCM was then added to the previous mixture. The solution
was stirred at 0 °C for 1 h then at 20 °C until completion (1 h). The
reaction was quenched with a saturated aqueous solution of NaHCO3
and extracted three times with DCM. The organic layers were
combined, dried over MgSO4, filtered, and concentrated under reduced
Step 2: Amide synthesis. To a solution of the above prepared ester
intermediate in MeOH (2 mL) was added an aqueous solution of NH3
(20% in water). The reaction was stirred at 60 °C for 15 h then cooled to
20 °C. The solvents were evaporated under reduced pressure, and the
crude was purified by preparative LCMS to afford 1-[2-(3,5-dimethyl-
phenyl)-acetyl]-2-methyl-azetidine-2-carboxylic acid (3-carbamoyl-
propyl)-(4-chloro-benzyl)-amide 26 (100 mg, 18% yield over two
1
steps). H NMR δ (ppm) (CDCl3): 7.47−7.27 (2H, m), 7.24−6.98
(2H, m), 6.97−6.56 (3H, m), 5.61−5.20 (1H, m), 4.97−4.52 (1H, m),
4.49−4.06 (1H, m), 4.04−3.52 (3H, m), 3.43−2.97 (2H, m), 2.62−2.43
(1H, m), 2.40−2.10 (7H, m), 2.09−1.73 (5H, m), 1.72−1.51 (2H, m).
MW (calcd): 469.2. MW (obsd): 470.1 (M + 1).
General Procedure B4 for the Synthesis of Compounds 37, 38, 39,
40, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 81, 83, 88,
89, 90, 91, 95, 96, and 98. Step 1: Following general procedure B1 or
K
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B2 and using the corresponding acids 13a−13d and secondary amines
gave the corresponding ester intermediate.
δ (ppm) (CDCl3): 8.67−8.58 (1H, m), 7.31−7.27 (1H, m), 7.23−7.16
(2H, m), 6.91 (1H, s), 6.83 (2H, s), 4.48 (2H, dd), 4.06−3.87 (2H, m),
3.37 (2H, s), 3.23−3.06 (4H, m), 2.88 (3H, s), 2.85−2.74 (1H, m), 2.29
(6H, s), 2.10−1.99 (1H, m), 1.70 (3H, s). MW (calcd): 485.0. MW
(obsd): 486.2 (M + 1).
General Procedure B6 for the Synthesis of Compounds 29 and 30.
Step 1: Following general procedure B4 and using the corresponding
acid 13a−13d and secondary amine gave the corresponding acid
intermediate.
Step 2: Amide formation. To a solution of the above prepared
carboxylic acid (1 equiv) in either DCM or THF was added HOBt
(1.1 equiv) and EDC·HCl (1.5 equiv). Then the corresponding amine
(1 equiv) and TEA (4 equiv) were added. The reaction was stirred at
20 °C for 15 h. The crude was concentrated under reduced pressure and
partitioned between a saturated aqueous solution of NaHCO3 and
EtOAc. The aqueous layer was extracted three times with EtOAc. The
organic layers were combined, washed with water then with brine, dried
over MgSO4, filtered, and concentrated under reduced pressure to afford
the corresponding carboxamide.
Step 2: Saponification. To a solution of the above prepared ester
(1 equiv) in EtOH was added an aqueous solution of 2N NaOH
(2 equiv). The reaction was stirred at 20 °C for 15h. The solvent was
removed under reduced pressure and the crude was partitioned between
water and EtOAc. The organic layer was discarded, and the aqueous
layer was acidified by addition of a solution of citric acid 10% in water
until pH = 3 and thoroughly extracted by EtOAc. The organic layers
were combined, dried over MgSO4, filtered, and concentrated under
reduced pressure to afford the corresponding carboxylic acid.
[[1-(2-Benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-2-car-
bonyl]-(4-chloro-benzyl)-amino]-acetic Acid (38). Following general
procedure B4 and using intermediate 13c (100 mg) and (4-chloro-
benzylamino)-acetic acid ethyl ester (115 mg) (B1-step1) gave [[1-(2-
benzo[b]thiophen-3-yl-acetyl)-2-methyl-azetidine-2-carbonyl]-(4-
1
chloro-benzyl)-amino]-acetic acid 38 (20% yield over two steps). H
NMR δ (ppm) (CDCl3): 7.91−7.69 (2H, m), 7.48−7.29 (5H, m), 7.17
(2H, d), 4.71−4.33 (3H, m), 4.22−3.92 (3H, m), 3.86−3.61 (2H, m),
2.79−2.17 (2H, m), 2.01−1.69 (3H, m). MW (calcd): 470.1. MW
(obsd): 471.3 (M + 1).
General Procedure B5 for the Synthesis of Compounds 77, 78, 79,
and 80. Step 1: Following general procedure B2 and using intermediate
13c and the corresponding secondary amine gave the corresponding
ester intermediate.
Step 2: Saponification. To a solution of the above prepared ester in
THF/water (1/1) LiOH monohydrate (1 equiv) was added. The
mixture was stirred at 20 °C for 16 h. The pH was adjusted to pH = 7−8
by addition of aqueous HCl 0.1N, and the mixture was concentrated.
The crude was purified by preparative LCMS to the give the
corresponding carboxylic acid.
4-(1-(Benzo[b]thiophene-3-carbonyl)-N-(6-chloropyridin-3-yl)-2-
methylazetidine-2-carboxamido)butanoic Acid (80). Following
general procedure B5 and using 4-(6-chloro-pyridin-3-ylamino)-butyric
acid ethyl ester in step 1 gave 80. 1H NMR δ (ppm) (CDCl3): 8.46 (1H,
s), 8.29 (1H, d), 7.84 (1H, d), 7.67 (1H, dd), 7.45−7.38 (2H, m), 7.12−
7.03 (HH, m), 4.03−3.92 (1H, m), 3.90−3.76 (2H, m), 3.67−3.58 (1H,
m), 2.72−2.65 (1H, m), 2.48 (2H, t), 2.45−2.18 (1H, m), 2.01−1.75
(5H, m). MW (calcd): 471.1. MW (obsd): 472.0 (M + 1).
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (4-Chloro-benzyl)-(4-morpholin-4-yl-4-oxo-butyl)-amide
(29). Following general procedure B6 and using intermediate 13a, 4-
(4-chloro-benzylamino)-butyric acid methyl ester in step 1, and
morpholine in step 2 gave 1-[2-(3,5-dimethyl-phenyl)-acetyl]-2-
methyl-azetidine-2-carboxylic acid (4-chloro-benzyl)-(4-morpholin-4-
1
yl-4-oxo-butyl)-amide 29. H NMR δ (ppm) (MeOD-d4): 7.52−7.20
(4H, m), 7.02−6.83 (3H), 4.86−4.49 (2H, m), 4.22−3.73 (2H, m),
3.73−3.43 (10H, m), 3.35−3.12 (2H, m), 2.69−2.27 (10H, m), 2.06−
1.81 (5H, m). MW (calcd): 539.3. MW (obsd): 540.4 (M + 1).
General Procedure B7 for the Synthesis of Compounds 24, 25, and
28. 1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-car-
boxylic Acid (4-Chloro-benzyl)-methylcarbamoylmethyl-amide
(24). To a solution of 37 (46 mg, 1 equiv) in DMF (1 mL) were
added a solution of methylamine in ethanol (33% w/w, 10 μL, 1 equiv),
TEA (14 μL, 1 equiv), and TBTU (33 mg, 1 equiv). The reaction was
stirred at 20 °C for 15 h. The reaction was carried on with addition of
methylamine (1 equiv), TEA (1 equiv), and TBTU (1 equiv), then
stirring at 20 °C for 15 h. The crude was concentrated under reduced
pressure then partitioned between water and EtOAc. The organic layer
was washed twice with water, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude was purified by
preparative LCMS to afford 1-[2-(3,5-dimethyl-phenyl)-acetyl]-2-
methyl-azetidine-2-carboxylic acid (4-chloro-benzyl)-methylcarbamoyl-
methyl-amide 24. 1H NMR δ (ppm) (MeOD-d4): 7.50−7.21 (4H, m),
7.01−6.85 (3H, m), 4.84−4.74 (1H, m), 4.68−4.44 (1H, m), 4.24−4.00
(2H, m), 3.95−3.71 (2H, m), 3.65−3.50 (1H, m), 3.42−3.34 (3H, m),
2.85−2.74 (3H, m), 2.32 (6H, s), 2.01−1.86 (3H, m). MW (calcd):
455.2. MW (obsd): 456.4 (M + 1).
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (2-Amino-ethyl)-(4-chloro-benzyl)-amide (17). Step 1:
Following general procedure B1 intermediate 13a (100 mg, 1 equiv)
and [2-(4-chloro-benzylamino)-ethyl]-carbamic acid tert-butyl ester
(163 mg, 1.5 equiv) gave the corresponding BOC protected amino
intermediate (139 mg, 69% yield).
Step 2: NBOC deprotection. The above prepared protected amino
compound was dissolved in DCM and TFA (30%) and stirred at 20 °C
for 15 h. The crude was then partitioned between a saturated NaHCO3
solution and DCM. The organic layer was washed with water, then dried
over MgSO4, filtered, and concentrated under reduced pressure. The
crude was purified by chromatography on silica gel to afford 1-[2-
(3,5-dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carboxylic acid
General Procedure B8 for the Synthesis of Compounds 84 and 86.
Step 1: Following general procedure B1 or B2 and using corresponding
carboxylic acids 13a−d and appropriate secondary amines gave the
related N-protected heterocycles.
1
(2-amino-ethyl)-(4-chloro-benzyl)-amide 17 (72 mg, 68% yield). H
Step 2: BOC removal. Following general procedure A1 (step 1) gave
NMR δ (ppm) (CDCl3): 8.53−8.41 (1H, m), 7.28−7.23 (3H, m), 6.89
(1H, bs), 6.85 (1H, bs), 4.06−3.87 (2H, m), 3.75 (2H, s), 3.46−3.27
(3H, m), 2.88−2.69 (3H, m), 2.28 (6H, s), 2.09−1.97 (1H, m), 1.92−1.78
(1H, m), 1.72 (3H, s). MW (calcd): 480.2. MW (obsd): 481.1 (M + 1).
1-[2-(3,5-Dimethyl-phenyl)-acetyl]-2-methyl-azetidine-2-carbox-
ylic Acid (4-Chloro-benzyl)-[2-(3-methyl-ureido)-ethyl]-amide (18).
To a solution of 17 (10 mg, 1 equiv) in MeCN (1 mL) was added N-
succinimidyl-N-methylcarbamate (4 mg, 1.1 equiv). The reaction was
stirred overnight at 20 °C before adding 2 additional equiv of
N-succinimidyl-N-methylcarbamate. Stirring was continued for 24 h
until completion. The reaction mixture was then concentrated under
reduced pressure and partitioned between water and EtOAc. The
organic layer was washed twice with water, then dried over MgSO4,
filtered, and concentrated under reduced pressure. The crude was
purified by chromatography on silica gel to afford 1-[2-(3,5-dimethyl-
phenyl)-acetyl]-2-methyl-azetidine-2-carboxylic acid (4-chloro-benzyl)-
the corresponding esters.
Step 3: Saponification. Following general procedure B4 (step 2) gave
the corresponding carboxylic acids.
4-[[1-(2-Benzofuran-3-yl-acetyl)-2-methyl-azetidine-2-carbonyl]-
(1H-indol-6-ylmethyl)-amino]-butyric Acid (84). Following general
procedure B8 and using intermediate 13c and 6-[(3-ethoxycarbonyl-
propylamino)-methyl]-indole-1-carboxylic acid tert-butyl ester in step
1 gave 82. 1H NMR δ (ppm) (MeOD-d4): 7.64−7.55 (1H, m), 7.40−
7.28 (2H, m), 7.03−6.87 (4H, m), 6.48 (1H, bs), 4.81−4.58 (2H, m),
4.12 (1H, bs), 3.91−3.85 (1H, m), 3.81−3.77 (1H, m), 3.64−3.41 (1H,
m), 3.34−3.15 (2H, m), 2.78−2.50 (1H, m), 2.43−2.23 (8H, m), 2.01−
1.92 (5H, m), 1.35 (1H, s). MW (calcd): 476. MW (obsd): 477 (M + 1).
4-[[(R)-1-(Benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-
carbonyl]-(3-chloro-benzyl)-amino]-butyric Acid (99). Step 1: Chiral
separation of 11 was performed by preparative chiral chromatography
using the following conditions: column, Chiralpak AD-H, (20 mm ×
250 mm), 5 μm; mobile phase, hexane/ethanol/formic acid (95:5:0.05);
1
[2-(3-methyl-ureido)-ethyl]-amide 18 (4 mg, 35% yield). H NMR
L
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flow rate of 9.5 mL/min, at 20 °C, thus affording 11a (S isomer, rt:
6.42 mn) and 11b (R isomer, rt: 9.00 mn) in their enantiopure forms
(ee = 99%).
3.44−3.34 (1H, m), 3.26−3.15 (1H, m), 2.58−2.51 (1H, m), 2.41−2.32
(1H, m), 2.18 (2H, t), 1.85−1.76 (5H, m). MW (calcd): 485.0. MW
(obsd): 485.4 (M + 1). 13C NMR δ (ppm) (DMSO-d6) at 80 °C: 173.1
(C), 171.4 (C), 164.2 (C), 140,0 (C), 138.7 (C), 137.1 (C), 132.9 (C),
130.0 (C), 129.7 (CH), 128.4 (CH), 126.5 (CH), 126.5 (CH), 125.1
(CH), 124.1 (CH), 123.6 (CH), 121.9 (CH), 70.6 (C), 48.4 (CH2),
46.5 (CH2), 45.9 (CH2), 30.6 (CH2), 28.9 (CH2), 23.3 (CH3), 22.4
(CH2). Anal. Calcd for C25H25ClN2O4S: C, 61.91; H, 5.20; N, 5.78.
Found: C, 61.82; H, 5.03; N, 5.72.
(R)-1-Benzyl-2-methyl-azetidine-2-carboxylic Acid Methyl Ester
(41). Step 1: To a solution of 11b (125 mg, 0.58 mmol) in MeOH
(5 mL) at 0 °C under nitrogen was added dropwise thionyl chloride
(100 μL, 2.5 equiv), and the reaction was stirred at 20 °C for 15 h. The
solvents were concentrated under reduced pressure to afford
quantitatively (R)-2-methyl-azetidine-2-carboxylic acid methyl ester
hydrochloride that was used as such without further purification.
Step 2: To a solution of (R)-2-methyl-azetidine-2-carboxylic acid
methyl ester (50 mg, 1 equiv) in THF (2 mL) were added TEA (84 μL,
2 equiv) and benzyl bromide (36 μL, 1 equiv). The reaction was heated
at 50 °C for 15 h. The solvent was concentrated under reduced pressure,
and the crude was purified by chromatography on silica gel (heptane/
EtOAc 100/0 to 50/50) to afford (R)-1-benzyl-2-methyl-azetidine-2-
carboxylic acid methyl ester 41 (31 mg, 47% yield). 1H NMR δ (ppm)
(CDCl3): 7.42−7.21 (5H, m), 3.86−3.74 (4H, m), 3.66−3.54 (1H, m),
3.34−3.07 (2H, m), 2.68−2.55 (1H, m), 2.02−1.91 (1H, m), 1.53 (3H,
s). [α]20D= +45 (c = 1.55, CHCl3). Reported value for (R) enantiomer:
[α]20D = +36° (c = 0.5, CHCl3, 91% ee).16
Step 2: to a solution of (R)-2-methyl-azetidine-1,2-dicarboxylic acid 1-
tert-butyl ester 11b (13 g, 1 equiv) in DCM (300 mL) under nitrogen
was added dropwise a solution of 1-chloro-N,N-2-trimethylpropenyl-
amine (16 mL, 2 equiv) in DCM (50 mL). The solution was stirred at
20 °C for 30 min, then cooled to −5 °C. A solution of 4-(3-chloro-
benzylamino)-butyric acid methyl ester hydrochloride (18.5 g,
1.1 equiv) and TEA (25 mL, 3 equiv) in DCM (200 mL) was added
slowly to the previous mixture. The reaction was stirred at 20 °C for 2.5
h. The mixture was added to 500 mL of a saturated aqueous solution of
NaHCO3. The crude was extracted three times with DCM. The organic
layers were combined, washed with brine, dried over MgSO4, filtered,
concentrated under reduced pressure. The crude was purified by
chromatography on silica gel (heptane/EtOAc: 90/10 to 50/50) to
afford (R)-2-[(3-chloro-benzyl)-(3-methoxycarbonyl-propyl)-carbamo-
yl]-2-methyl-azetidine-1-carboxylic acid tert-butyl ester (27 g, quantita-
tive yield). 1H NMR δ (ppm) (CDCl3): 7.35−7.04 (4H, m), 5.13−4.29
(2H, m), 3.96−3.74 (1H, m), 3.68 (3H, s), 3.13−2.90 (4H, m), 2.65−
2.40 (1H, m), 2.39−2.15 (2H, m), 2.00−1.65 (5H, m), 1.58−1.30 (9H,
m). MW (calcd): 439.0. MW (obsd): 439.4 (M + l).
Step 3: To a solution of (R)-2-[(3-chloro-benzyl)-(3-methoxycar-
bonyl-propyl)-carbamoyl]-2-methyl-azetidine-1-carboxylic acid tert-
butyl ester, (17.6 g, 1 equiv) in dioxane (200 mL) under nitrogen was
added a solution of HCl (4N) in dioxane (50 mL, 5 equiv). The reaction
was stirred for 16 h at 20 °C, then concentrated under reduced pressure.
The crude was solubilized in DCM, poured into Et2O, and the resulting
solid was filtered, washed with Et2O and pentane, and dried to afford
4-[(3-chloro-benzyl)-((R)-2-methyl-azetidine-2-carbonyl)-amino]-butyric
acid methyl ester hydrochloride (12.5 g, 81% yield). 1H NMR
δ (ppm) (CDCl3): 8.82 (1H, br s), 7.41−7.26 (2H, m), 7.25−7.11
(2H, m), 4.83−4.53 (1H, m), 4.47−4.31 (1H, m), 4.31−3.95 (2H, m),
3.72−3.64 (3H, m), 3.51−3.26 (1H, m), 3.17−3.07 (1H, m), 3.02−2.86
(1H, m), 2.78−2.52 (1H, m), 2.40−2.30 (2H, m), 2.16−2.01 (3H, m),
1.97−1.83 (2H, m). MW (calcd): 338.8. MW (obsd): 339.3 (M + 1).
Step 4: To a suspension of benzo[b]thiophene-3-carboxylic acid (7.12 g,
1.2 equiv) in DCM (210 mL) and THF (210 mL) were added HOBt
(5.4 g, 1.2 equiv), EDC·HCl (9.6 g, 1.5 equiv), and TEA (18.5 mL,
4 equiv). The reaction was stirred at 20 °C for 15 h, then a solution of
4-[(3-chloro-benzyl)-((R)-2-methyl-azetidine-2-carbonyl)-amino]-butyric
acid methyl ester hydrochloride (12.5 g, 1 equiv) in DCM (105 mL) and
THF (105 mL) was added. The reaction was stirred at 20 °C for 4.5 h. The
crude was diluted with DCM. A saturated aqueous solution of NaHCO3
was added. The aqueous layer was extracted three times with DCM. The
organic layers were combined, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude was purified by
chromatography on silica gel (elution with heptane/EtOAc: 90/10 to
50/50) to afford 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-
azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid methyl ester
(R)-1-(4-Bromo-benzoyl)-2-methyl-azetidine-2-carboxylic Acid
(42). Step 1: To a solution of 11b (550 mg, 1 equiv) in dioxane
(30 mL) was added a solution of HCl (4N) in dioxane (5 mL), and
the reaction was stirred at 20 °C for 15 h. The solvent was removed
under reduced pressure, and the crude was crystallized in a mixture of
DCM/iPr2O/pentane to afford quantitatively (R)-2-methyl-azetidine-2-
carboxylic acid hydrochloride that was used as such without further
purification.
Step 2: To a solution of (R)-2-methyl-azetidine-2-carboxylic acid
methyl ester (1 equiv) in water were added aqueous NaOH (1M)
(7.5 mL, 3 equiv) then 4-bromo-benzoyl-chloride (550 μL, 1 equiv)
dropwise. The reaction was stirred for 4 h at 20 °C. The reaction was
neutralized with acetic acid. The solvent was partially removed under
reduced pressure, and the crude was extracted with EtOAc. The organic
layer was dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude was purified by chromatography on silica gel
(elution with DCM/MeOH/AcOEt/AcOH/iPr2O: 95/5/98/2/300).
The resulting white solid was then recrystallized in isopropyl ether to
afford (R)-1-(4-bromo-benzoyl)-2-methyl-azetidine-2-carboxylic acid
1
42 (355 mg, 48% yield). H NMR δ (ppm) (CDCl3): 7.65 (2H, d),
7.59 (2H, d), 4.48−4.39 (1H, m), 4.28−4.19 (1H, m), 3.07−2.97
(1H, m), 2.34−2.24 (1H, m), 1.95 (3H, s).
1
ASSOCIATED CONTENT
* Supporting Information
(14.53 g, 87% yield). H NMR δ(ppm) (CDCl3): 8.37−8.07 (1H, m),
■
7.92−7.77 (1H, m), 7.51−7.36 (2H, m), 7.34−6.67 (5H, m), 5.49−4.72
(1H, m), 4.57−4.33 (1H, m), 4.05−3.76 (2H, m), 3.69 (3H, s), 3.29−2.66
(2H, m), 2.50−2.19 (3H, m), 2.15−1.83 (5H, m), 1.74−1.57 (1H, m).
MW (calcd): 499.0. MW (obsd): 499.3 (M + 1).
S
Experimental details, analytical data of compounds not described
in the experimental part, detailed results of Cerep ExpresSprofile
as well as crystallographical data of 42. This material is available
free of charge via the Internet at http://pubs.acs.org.
Step 5: To a solution of 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-
2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid
methyl ester, (25 g, 1 equiv) in 750 mL of MeOH was added aqueous
NaOH (2N) (75 mL, 3 equiv). The reaction was stirred at 20 °C for
15 h. The solvent was removed under reduced pressure, and the crude
was partitioned between water and EtOAc. The organic layer was
discarded. The aqueous layer was acidified by addition of HCl (2N) until
pH = 2 and thoroughly extracted three times with EtOAc. The organic
layers were combined, dried over MgSO4, filtered, and concentrated
under reduced pressure to afford 4-[[(R)-1-(benzo[b]thiophene-3-
carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-
butyric acid (23.7 g, 97% yield); [α]20D = +176 (c = 1.01, CHCl3); ee
>99.0%. 1H NMR δ (ppm) (DMSO-d6) at 80 °C: 8.14−8.08 (1H, m),
7.99−7.93 (1H, m), 7.90 (1H, s), 7.44−7.31 (3H, m), 7.30−7.25
(2H, m), 7.22−7.16 (1H, m), 4.73−4.54 (2H, m), 4.11−3.98 (2H, m),
AUTHOR INFORMATION
Corresponding Author
*Phone: +33 1 49424664. E-mail: laurent.saniere@glpg.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Anne-Laure Boutet, Annick Hagers, and Anne-Sophie
Wesse for their respective contributions in chiral chromatog-
raphy and biological data generation.
M
dx.doi.org/10.1021/jm5012885 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(11) Ulven, T. Short-chain free fatty acid receptors FFA2/GPR43 and
FFA3/GPR41 as new potential therapeutic targets. Front. Endocrinol.
2012, 3, 1−9.
ABBREVIATIONS USED
■
GPR43, G-protein coupled receptor 43; CD11b, cluster of
differentiation molecule 11B; AE, activation-specific epitope; FA,
fatty acid; SCFA, short-chain fatty acid; PBMC, peripheral blood
mononuclear cell; DSS, dextran sodium sulfate; TNBS,
trinitrobenzoic sulfonic acid; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; HOBt, hydroxybenzo-
triazole; KHMDS, potassium bis(trimethylsilyl)amide; ICAM-
1, intercellular adhesion molecule 1; MAC-1, macrophage 1
antigen; TEA, triethylamine
(12) Brantis, C. E.; Ooms, F.; Bernard, J. Novel amino acid derivatives
and their use as GPR43 receptor modulators. PCT Int. Appl.
WO2011092284, 2011.
(13) Saniere, L. R. M.; Pizzonero, M. R.; Triballeau, N.; Vandeghinste,
N., E, R.,; De Vos, S. I. J. ; Brys, R. C. X. ; Pourbaix-L’Ebraly, C. D.
Azetidine derivatives useful for the treatement of metabolic and
inflammatory disorders. PCT Int. Appl. WO2012098033, 2012.
(14) Hudson, B. D.; Tikhonova, I. G.; Pandey, S. K.; Ulven, T.;
Milligan, G. Extracellular ionic locks determine variation in constitutive
activity and ligand potency between species orthologs of the free fatty
acid receptors FFA2 and FFA3. J. Biol. Chem. 2012, 287, 41195−41209.
(15) Srepan, A. F.; Mascitti, V.; Beaumont, K.; Kalgutkar, A. S.
Metabolism-guided drug design. Med. Chem. Commun. 2013, 4, 631−
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