Design, synthesis, and biological evaluation of novel pyrrolidinone small-molecule Formyl peptide receptor 2 agonists

Article abstract of DOI:10.1016/j.ejmech.2021.113805

A series of Formyl peptide receptor 2 small molecule agonists with a pyrrolidinone scaffold, derived from a combination of pharmacophore modelling and docking studies, were designed and synthesized. The GLASS (GPCR-Ligand Association) database was screened using a pharmacophore model. The most promising novel ligand structures were chosen and then tested in cellular assays (calcium mobilization and β-arrestin assays). Amongst the selected ligands, two pyrrolidinone compounds (7 and 8) turned out to be the most active. Moreover compound 7 was able to reduce the number of adherent neutrophils in a human neutrophil static adhesion assay which indicates its anti-inflammatory and proresolving properties. Further exploration and optimization of new ligands showed that heterocyclic rings, e.g. pyrazole directly connected to the pyrrolidinone scaffold, provide good stability and a boost in the agonistic activity. The compounds of most interest (7 and 30) were tested in an ERK phosphorylation assay, demonstrating selectivity towards FPR2 over FPR1. Compound 7 was examined in an in vivo mouse pharmacokinetic study. Compound 7 may be a valuable in vivo tool and help improve understanding of the role of the FPR2 receptor in the resolution of inflammation process.

Full text of DOI:10.1016/j.ejmech.2021.113805

European Journal of Medicinal Chemistry 226 (2021) 113805
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, and biological evaluation of novel pyrrolidinone
small-molecule Formyl peptide receptor 2 agonists
, b,
Monika Maciuszek a *, Almudena Ortega-Gomez c, Sanne L. Maas c, Jose Garrido-Mesa b,
Bartolo Ferraro c, Mauro Perretti b, Andy Merritt a, Gerry A.F. Nicolaes f,
, d,
Oliver Soehnlein c e, Timothy M. Chapman a
a LifeArc, Accelerator Building, Open Innovation Campus, Stevenage, UK
b The William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London, London, UK
c Institute for Cardiovascular Prevention (IPEK), LMU Munich Hospital, Munich, Germany
d Department of Physiology and Pharmacology (FyFa), Karolinska Institute, Stockholm, Sweden
e Institute for Experimental Pathology (ExPat), Centre for Molecular Biology of Inflammation, University of Münster, Münster, Germany
f CARIM e School for Cardiovascular Sciences Department of Biochemistry, Maastricht University, Maastricht, Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Formyl peptide receptor 2 small molecule agonists with a pyrrolidinone scaffold, derived from
a combination of pharmacophore modelling and docking studies, were designed and synthesized. The
GLASS (GPCR-Ligand Association) database was screened using a pharmacophore model. The most
promising novel ligand structures were chosen and then tested in cellular assays (calcium mobilization
Received 9 April 2021
Received in revised form
23 August 2021
Accepted 24 August 2021
Available online 2 September 2021
and b-arrestin assays). Amongst the selected ligands, two pyrrolidinone compounds (7 and 8) turned out
to be the most active. Moreover compound 7 was able to reduce the number of adherent neutrophils in a
human neutrophil static adhesion assay which indicates its anti-inflammatory and proresolving prop-
erties. Further exploration and optimization of new ligands showed that heterocyclic rings, e.g. pyrazole
directly connected to the pyrrolidinone scaffold, provide good stability and a boost in the agonistic ac-
tivity. The compounds of most interest (7 and 30) were tested in an ERK phosphorylation assay,
demonstrating selectivity towards FPR2 over FPR1. Compound 7 was examined in an in vivo mouse
pharmacokinetic study. Compound 7 may be a valuable in vivo tool and help improve understanding of
the role of the FPR2 receptor in the resolution of inflammation process.
Keywords:
FPR2
Pharmacophore
Agonists
Neutrophils
Resolution of inflammation
© 2021 Published by Elsevier Masson SAS.
1. Introduction
of the protein family of G protein-coupled receptors (GPCRs),
abundantly expressed in neutrophils [6e10], monocytes [11,12],
Chronic inflammation has a crucial role in the development of
many cardiovascular diseases such as atherosclerosis [1], myocar-
dial ischaemiaereperfusion [2e4] and thrombosis [5]. In general,
desirable physiological inflammation is a response of our immune
system to harmful stimuli and it is contained in time and space.
However prolonged and unresolved inflammatory response leads
to chronic inflammation. Resolution-based treatments can reduce
harmful effects, enhancing the self-recovery abilities of tissues and
assisting the return of homeostasis. One of the most studied pro-
resolving receptors is Formyl peptide receptor 2 (FPR2), a member
epithelia, endothelia, smooth muscle cells [1], and fibroblasts. FPR2
exhibits numerous physiological and pharmacological responses
and can be activated by ligands of diverse origins such as peptides,
lipids and small molecules [13e16]. The receptor has a dual impact
on the inflammatory response which varies depending on the na-
ture of the ligand [17]. FPR2 can be activated by agonists like
annexin A1 or LXA4 to resolve the inflammation. These types of
agonists promote apoptosis of neutrophils, macrophage-mediated
phagocytosis, and inhibition of epithelial activation as a mecha-
nism of resolving acute inflammatory responses. In contrast, the
FPR2 receptor may be activated by proinflammatory molecules
such as Serum amyloid A (SAA), LL-37 (the cleaved antimicrobial
peptide from Cathelicidin) and mitochondrial hexapeptides and
trigger the transmigration and survival of neutrophils, epithelial
* Corresponding author. LifeArc, Accelerator Building, Open Innovation Campus,
Stevenage, UK.
E-mail address: maciuszekmonika@gmail.com (M. Maciuszek).
https://doi.org/10.1016/j.ejmech.2021.113805
0223-5234/© 2021 Published by Elsevier Masson SAS.

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
activation and monocytes recruitment [18]. Since 2004 research
groups have concentrated on the proresolving properties of the
FPR2 receptor and several synthetic small molecules have been
submitted for Phase I clinical trials [19,20], but none of them have
yet moved beyond this. Nevertheless, FPR2 is one of the key targets
in the resolution of inflammation [14,21,22]. In this work, we pre-
sent a novel pyrrolidinone series, where the first stage was to
develop pharmacophore models and to investigate a homology
model obtained from GPCRdb [23,24] for molecular docking; these
were then used in the structure-based pursuit of novel agonists. To
discover novel FPR2 agonist structures we screened the GLASS
(GPCR-Ligand Association) database. Each compound was evalu-
ated for its agonistic activity to stimulate calcium mobilization and
b
-arrestin recruitment in CHOeK1 FPRL1 cells (DISCOVERx) over-
expressing hFPR2. The PathHunter™ GPCR -Arrestin assay was
applied in our studies to test the ligands’ ability to activate the
arrestin signalling cascade [25]. Ligand-induced -arrestin
b
b-
b
recruitment is involved in numerous processes such as kinase
activation, desensitization, and receptor internalization [26]. Upon
activation of the GPCR, if the receptor couples to Gaq, it produces an
increase in intracellular Ca2þ. In these studies, the increase in
cytosolic Ca2þ was measured by the FLIPR® Tetra® High-
Throughput Cellular Screening System [27]. The increase in levels
of intracellular Ca2þ is initiated by the activation of phospholipase C
enzymes and a boost in protein kinase C (PKC) production is
observed [28]. FPR2 activation is initiated by binding of the ligand
from the extracellular side and classic agonists have been perceived
as ligands with the ability to trigger numerous signalling pathways
downstream of a receptor [29]. However, depending on the nature
of the ligand, the G protein or b-arrestin signalling can be inde-
pendently activated, or the ligand can simultaneously trigger both
pathways [30,31]. Therefore, we considered it worthwhile to use
both of these assays, where we can monitor activation of two
different pathways. The knowledge gained from these two
distinctive assays can bring an insight into whether we have full or
biased agonists. Moreover, both assays are convenient for high
throughput screening, because of their robustness and reproduc-
ibility. Compounds were also tested in in vitro absorption, distri-
bution, metabolism and excretion and toxicity (ADMET) assays to
bring greater understanding to their potential for further devel-
opment. Two lead compounds with the best profiles (7 and 30)
were then tested in an ERK phosphorylation assay (FPR1 and FPR2
HEK cell lines). The activation of ERK signalling in cardiomyocytes
promotes beneficial and prosurvival effects [32e34]. Finally, com-
pound 7 was tested in an in vivo mouse pharmacokinetic study,
which demonstrated its potential for use as an in vivo tool.
Fig. 1. A) Selection of the compounds using Phase Ligand screening B) Hit compounds
and their characterization; Ks and LogD were measured at pH 7.4.
2. Results and discussion
2.1. In silico studies
In order to discover novel FPR2 agonists we decided to use the
GLASS (GPCR-Ligand Association) database [35] which is a re-
pository for experimentally validated GPCR-ligand interactions. The
workflow is summarised in Fig. 1. A: as a first stage we excluded
possible duplicates and structures which do not meet the Lipinski
rule of 5 using Pipeline Pilot. The next step was the generation of 3D
conformers of all molecules in the GLASS-db employing the Ligprep
Fig. 2. Five point pharmacophore model of FPR2 agonists. H-hydrophobic R-ring; D-
hydrogen bond donating and A - accepting sites in the receptor.
the pharmacophore model was established using the ‘multiple li-
gands’ option with the method ‘best alignment and common fea-
tures’ A shortlist of 330 compounds was selected based on the
results of pharmacophore screening. In the next stage these ligands
were docked into the FPR2 receptor homology model with the aim
of assessing their potential binding poses. Glide SP-protocol was
selected, and all ligands were treated as flexible molecules. The
docking poses were manually analysed for their interactions with
key amino-acid residues such as His-102, Arg-205, Cys-176, Phe-
257, Phe-292, Phe-163 and Tyr-175. From this, a set of 13 novel
€
tool (Schrodinger software). Then the compounds were screened
using a five-point pharmacophore model ADHHR_7 previously
described by our group [36]. The ADHHR_7 pharmacophore model
€
was developed (Fig. 2) employing the Phase tool in Schrodinger
Maestro [37]. The examples of FPR2 small molecule agonists used in
pharmacophore studies were included in the supporting informa-
tion. Because of the heterogeneity among the examined agonists,
2

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
€
ligands was selected and functionally tested. Amongst these li-
gands, two pyrrolidinone compounds were the most promising, the
Schrodinger software. The docking studies suggested that mole-
cules that resemble 7 (blue) are likely to occupy subpockets I and II,
whereas compounds containing an additional linker attached to
the pyrrolidinone core such as 16 (pink) and 15 (green) can
potentially bind into subpockets III and I with formation of
hydrogen bonds with amino-acid residues such as Cys-176 and His-
102 (Fig. 4).
compounds 7 and 8 showed nanomolar agonistic activity in
b-
arrestin (EC50 ¼ 302 nM and 510 nM respectively). However, in
calcium mobilization the 4-fluoro analogue 8 exhibited a distinct
drop-off in the potency compared 7 (Fig. 1B).
With the introduction of the linkers to the pyrrolidinone core,
we could obtain improved ligand fitting as well as enhanced three-
dimensionality in the structures. A variety of linkers was proposed
including alkyl moieties and ring features, for example: piperidine,
pyrrolidine, azetidine, and heterocyclic aromatic rings such as
pyrazoles and oxadiazoles. In Table 1, the first structural modifi-
cations of compound 7 are presented. The benzyl analogue com-
pound 9 unexpectedly showed no activity in both functional assays.
However, it did show improved solubility and good microsomal
stability across two species. The additional synthesized compounds
10e21 in Table 1 contain heterocycles such as piperidine, pyrroli-
dine, azetidine, and pyrazole, and were designed to increase the
polarity and aqueous solubility of the molecules. Compounds 15
and 16 showed well balanced profiles with respect to activity and
properties and both contain a piperidine ring as a linker group; the
piperidine ring is a frequent motif in numerous natural alkaloids
and synthetic small molecules. The introduction of the benzyl
moiety in compound 16 allowed us to maintain the basic nitrogen
of piperidine, which resulted in good kinetic solubility
2.2. Design and development of pyrrolidinone FPR2 agonists
Compound 7 was selected for further optimization based on the
outcomes of the cellular assays and in-house in vitro ADME assays.
The stability of the compound was determined in human and
mouse liver microsomes, where it was extremely stable (T
1/2
>400 min). In addition, the hit compounds did not show a cytotoxic
effect in a cell health assay (supporting information). The low
kinetic solubility and high logD value of compound 7 were two
properties to be improved, while preserving or increasing the
agonistic activity (Fig. 1 B). Furthermore, encouraging results from
human neutrophil adhesion assays were an additional factor which
fed into the selection of compound 7 for further optimization. Fig. 3
shows the results obtained from a known active FPR2 agonist and
novel pyrrolidinone compound 7. Compound A, which is one of the
Allergan small molecule agonists described in patent application
US20170320897 covering the treatment of ocular inflammation,
showed significant inhibition of neutrophil adhesion in the
range (Fig. 3). Similarly, compound 7 decreased the number of
adherent neutrophils in a concentration-dependent manner with a
significant reduction from 10 mM onwards, hence revealing prom-
mM
(Ks ¼ 152
mM). An additional eCH2 moiety between the pyrroli-
dinone core and piperidine linker (compound 15), led to a decrease
of one unit in LogD compared to 16 and a decrease in microsomal
clearance of around 2-fold in both HLM and MLM. Replacement of
the piperidine linker with pyrrolidine produced another stereo
centre in the molecule which resulted in two diastereoisomers 17
and 18, which displayed inferior microsomal stabilities relative to
ising anti-inflammatory properties [38e40].
In the next step of compound optimization, around 2000 virtual
compounds were proposed in which the phenyl ring attached to
the pyrrolidinone core was replaced, and their predicted binding to
the homology model was examined after docking using
Fig. 3. Anti-adhesive properties of FPR2 agonists A and 7 in a human neutrophil static adhesion assay. Data are depicted as mean, with 3 independent experiments and 3 technical
replicates; The differences between the control group, and treatment groups were evaluated by one-way ANOVA followed by Dunnett's multiple comparison test, and the dif-
ferences were considered significant for a P value < 0.05: *P < 0.05, **P < 0.01, ***P < 0.001; ns e not significant.
3

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
Fig. 4. Binding poses of pyrrolidinone agonists. Compound 7 (blue) occupies subpockets I and II where compounds 16 (pink), 15 (green), and (grey) occupy subpockets I and III.
15 and 16. Compounds 19 and 20, with a 3-piperidine linker, also
showed higher microsomal clearance. Compound 21 with a 3,5-
dimethyl-pyrazole linker displays acceptable stability in HLM,
although poorer stability in MLM. It exhibited full activation in both
functional assays in the nanomolar range with a promising ADME
profile, although the LogD requires improvement. The last ligand
described in Table 1 is compound 22, in this case we replaced the
benzyl ring with pyrimidine. It displayed a good activity profile in
pyrazole moiety which is linked directly to pyrrolidinone core. In
these examples, a flip into the subpocket II is observed. This sub-
pocket is built with amino acid residues for example Ser 84, Val
284, Tyr 175. The imidazole ring is not buried in the subpocket I but
occupies the subpocket II, making a p-p interaction with the phenyl
ring of Tyr-175 sidechain. Finally, the NH groups of the urea moiety
act as hydrogen bond donors and interact with hydroxy groups of
Ser-288 located in second polar cluster (Fig. 5).
both cellular assays (Calcium EC50 ¼ 0.008
m
M and
b
-arrestin
Amongst compounds 23e29, only 29 showed a hint of agonistic
activity in the calcium mobilization assay. This might be explained
by the fact that 29 is the only molecule where the phenyl ring is
attached directly to the urea group. This makes the molecule less
flexible and perhaps it is responsible for better fitting in the binding
pocket. The lack of activity could be triggered also by poor solubility
of the molecules.
Having demonstrated that an additional linker helps the com-
pound fit better into sub-pocket I of the FPR2 binding site and
improves the solubility while maintaining good agonistic potency,
our efforts moved to further optimizing the linkers. Based on the
information gained from the ADME and functional assays, molecule
21 was chosen for further modification. Four additional analogues
were synthesized and tested (Table 3). Ligands 30e33 were
designed to explore the effect of varying substitution pattern
around the pyrazole. Compounds 32e33 lack activity in the calcium
mobilization assay. The conformational twist induced by the 3,5-
dimethyl group in 21 may in itself be beneficial for potency, but it
can also aid solubility, which could be a limiting factor in 32 and 33.
The dimethyl substituted example 30 compared to 21, showed
increased microsomal stability including both species and gave a
good LogD value (2.6). The introduction of dimethylpyrazole
EC50 ¼ 0.385 M). However, the logD value increased by a half log
m
unit and the solubility was lost. The introduction of pyrimidine did
not improve the microsomal stability, which might indicate a
possible metabolic hotspot. Across these new compounds, we
observed good metabolic stability in vitro and activity in the
nanomolar range, yet the agonistic SAR is relatively flat (see
Table 2).
In the second phase of structural optimization, we focused on
the evaluation of the para-bromo-phenylureidic part of compound
7. Seven novel compounds were proposed: four replacements of
the para-bromo moiety with eCN (29), -Et (25), eOCH3 (27) or 3,5-
difluoro substituents (26), and three alternatives for the phenyl
ring: imidazole (23) and two 4-pyridine containing analogues (24
and 28). Based on docking studies, we decided to introduce two
compounds with a small modification on the core. Ligands 23 and
28 contain the extra methyl group in the pyrrolidinone core
showed plausible binding poses. The results from in silico SAR
studies were used to guide these modifications. Docking studies
suggested that in the case of compounds 29 (grey) and 24 (pink),
the phenyl ring connected directly to the pyrrolidinone core is
located in the deepest pocket I, created by hydrophobic amino acid
residues such as Phe-292, Phe-257. An aromatic ring attached to the
resulted in a 1.5-fold decrease (EC50 ¼ 0.740
recruitment but it improved the calcium mobilization
M) activity by 2-fold compared with 21. For com-
mM) in b-arrestin
urea moiety forms the aromatic
p-stacking interaction with Tyr-
175. The NH groups of the urea moiety act as hydrogen bond do-
nors and interact with Cys-176 and His-102. Moreover, in silico
studies showed a possible second binding mode represented by the
compounds 26 (orange) and 25 (blue). These ligands contain a
(EC50 ¼ 0.142
m
pound 31, the isopropyl moiety enhanced the microsomal stability
and lowered the LogD value, however 31 exhibits 3-fold decrease in
the calcium mobilization assay (EC50 ¼ 0.884
mM) compared to the
4

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
Table 1
ADME properties and EC50 values for pyrrolidinone derivatives.
ID
7
R
KS [
m
M]
LogD
Microsomal stability
[min]
Ca2þ EC50
[m
M] (Emax
)
b arrestin EC50 [mM] (Emax)
T
CLint (
mg)
mL/min/
½
HLM
MLM
HLM
2
MLM
15
>4
>400
>400
2
0.43 (73)
0.30
8
0
>4
4.4
4.6
>4
0.8
0.3
>400
168
250
19
33
127
31.3
19
3
9
46
12
49
80
11
<2
1.97
0.51
N.A.
9
75
61
43
N.A.
10
11
12
13
6
0.73 (42)
0.40 (86)
0.27 (84)
0.23 (68)
6.3 (93)
80
4
2.16 (92)
3.29 (102)
1.03 (100)
227
234
381
e
140
e
<2
14
15
222
192
>4
113
227
112
121
14
7
14
12
0.41 (67)
0.25 (99)
2.35 (103)
2.25 (93)
2.5
16
152
3.5
112
70
13
21
0.35 (76)
2.87 (96)
17
18
19
210
207
71
3.3
3.0
>4
78
56
35
35
36
19
20
28
44
44
43
80
2.7 (128)
0.37 (84)
0.13 (71)
1.37 (100)
0.70 (99)
1.38 (89)
diastereomer I
diastereomer II
diastereomer I
diastereomer II
20
54
4.1
71
30
22
50
0.06 (73)
0.55 (86)
21
22
57
0
4.6
3.9
189
57
38
44
8
40
35
0.28 (150)
0.008
0.40 (95)
27
0.385 (96)
N.A. - very low response (Emax <20% of max response) or weak activation EC50 > 10 mM; Ks and LogD were measured at pH 7.4.
5

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
Table 2
The replacement of phenylureidic group in pyrrolidinone series.
Compound
LogD
Microsomal stability
T1/2[min]
Ca2þ EC50
[
mM]
b arrestin EC50 [mM]
Clint (mL/min/mg)
HLM
MLM
HLM
MLM
1.0
e
e
<2
<2
N.A.
N.A.
23
24
2.0
2.1
410
584
3.8
2.6
<2
N.A.
N.A.
N.A.
N.A.
<2
25
not tested
not tested
not tested
N.A.
N.A.
26
27
2.8
329
505
357
151
4.7
3.0
4.3
N.A.
N.A.
N.A.
N.A.
1.99
10.2
28
2.7
85
172
17.94
8.9
15 (123)
N.A.
29
N.A. - very low response (Emax <30% of max response) or weak activation EC50 > 10
m
M; Ks and LogD were measured at pH 7.4.
lead compound 21; this can be explained by the lack of a methyl
2.4. The ERK phosphorylation assay
group and its insolubility (see Table 4).
The two lead compounds (7 and 30) were tested in an ERK
Phosphorylation Assay, using W peptide as a positive control. To
this end, we used HEK cells stably transfected with human FPR1 or
FPR2 [41], on the notion that activation of ERK phosphorylation is a
tissue-protective proresolving signalling, as demonstrated for pro-
survival effects in cardiomyocytes [32-34], [42]. At this stage we
wanted to investigate the selective agonistic activity of the pyrro-
lidinone series and elucidate whether the proresolving strategy will
contribute to regulate human inflammatory diseases and their
implications. When tested in a concentration-response manner,
2.3. Chemistry
The synthesis of the pyrrolidine series was started with N-
acylation of the desired amine with 34 resulting in formation of N-
substituted 2,4-dibromo-butanamide intermediate. The crude
product was used without any further purification in the cycliza-
tion (35e50). Total yield after two steps was in the range 30e60%.
The introduction of the amine group is performed by stirring the
substrate in a mixture of acetonitrile: aqueous ammonia 1:1 at
40 ꢀC overnight to form products 51e65 in good yield. The urea
derivatives were obtained by reacting intermediates with 4-
bromophenyl isocyanate at ambient temperature overnight
(9e11, 14, 19e22, 30e33, 66e67). Removal of the BOC protecting
group was performed with 4 M HCl in dioxane at room temperature
to obtain the products as free bases in quantitative yield using SPE
cartridges (12e13, 68e69). N-alkylation, employing classic condi-
tions with Hünig's base and benzyl bromide, yielded the final
compounds 15e18 (Scheme 1).
ranging from 1 nM to 30 mM, both FPR agonists did not show any
significant effects in hFPR1-HEK cells, demonstrating their lack of
functional responses at FPR1. In contrast a positive stimulation of
ERK phosphorylation was observed in FPR2 HEK cells, with calcu-
lated EC50 of 7.36 and 4.88 mM, for compounds 7 and 30 respec-
tively. The normal mock-transfected HEK cell line was used as a
control in order to examine any possible off-target effects of the
compounds. No off-target effect was observed (supporting
information).
6

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
Fig. 5. Proposed binding modes for non-para-bromo-phenylureidic pyrrolidinone analogues: 29 (grey), 24 (pink), 26 (orange) and 25 (blue).
Table 3
The additional analogues of pyrrolidinone series.
ID
R
Ks [m
M]
LogD
Microsomal stability
T1/2 [min]
Ca2þ EC50
[m
M] (Emax
)
b-arrestin EC50 [mM] (Emax)
Clint (mL/min/
mg)
HLM
183
MLM
172
HLM
8
MLM
30
31
9
2.6
3.7
9
0.142
0.884
0.74 (98)
1
3
322
658
159.3
526
10
NA
32
33
4.3
2
N.A.
N.A.
3.76 (103)
0
>4
209
107
7
14
N.A.
N.A. - very low response (Emax <30% of max response) or weak activation EC50 > 10 mM, Ks and LogD were measured at pH 7.4.
2.5. PK studies
administration at 10 mg/kg. Following a single IP dose, compound 7
was rapidly absorbed (Tmax ¼ 0.25 h) and showed a good Cmax and
half-life (9.42 h) along with high exposure (Table 5).
Compound 7 was chosen for pharmacokinetic studies which
were performed in male C57BL6 mice with intraperitoneal
7

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
Table 4
ERK phosphorylation assay.
heterocyclic rings such as pyrazole directly attached to the pyrro-
lidinone core gave rise to good stability and increase of potency
(30). Finally, two lead compounds 7 and 30 were tested in an ERK
Phosphorylation Assay which demonstrated their lack of functional
responses at FPR1, and selectivity towards FPR2. Moreover com-
pound 7 showed a promising pharmacokinetic profile during
intraperitoneal administration at 10 mg/kg in male C57BL6 mice.
For future work, we would like to perform chiral separation of the
lead compounds and test the two enantiomers in the cellular as-
says, to reveal the preference in the chirality of the molecules in
terms of receptor activation and their ADME profiles.
Compound
FPR1 HEK
FPR2 HEK
a
a
Emax
EC50
(
m
M)
Emax
EC50 (mM)
7
30
15
N.A.
100
1.74
N.A.
0.28
69
90
100
7.36
4.88
0.008
W peptide
a
Emax is described as percentage of Emax obtained from W peptide (3
not active/very weak.
mM), N.A. -
3. Conclusions
4. Materials and methods
4.1. In silico studies
In summary, our goal was to identify, develop and synthesize
novel FPR2 small molecule agonists, using a ‘ready-to-use’ ho-
mology model, molecular docking, and pharmacophore modelling
studies. Over 210,000 ligands were screened from the GLASS
database using our pharmacophore model. Thirteen selected li-
gands were tested in the functional assays to examine their
agonistic activity. The initial hit compounds 7 and 8 with a pyrro-
lidinone core were identified. Compound 7 displayed nanomolar
4.1.1. Homology model
An agonist bound
m-opioid based receptor structure was ob-
tained from GPCRDB [http://gpcrdb.org/protein/fpr2_human/] Full
protein sequence alignment can be found in the supporting infor-
mation [23,24].
activity
EC50 ¼ 430 nM); its 4-fluoro analogue 8 showed only a minor drop-
off in the
-arrestin recruitment assay (EC50 ¼ 510 nM), but a sig-
(b
-arrestin EC50
¼
302 nM, Calcium mobilization
4.1.2. Docking studies
Protein-Ligand docking was performed using the Glide SP-
protocol. All ligands were treated as flexible molecules. Binding
b
nificant decrease in potency in calcium mobilization. Moreover,
promising results from human neutrophil adhesion assays
demonstrated the anti-inflammatory properties of compound 7.
Further optimization was then carried out, the first round of which
involved the introduction of a range of linkers. This showed that
increased polarity and increased sp [3]-character was tolerated and
resulted in compounds with lower log D and enhanced solubility,
such as the piperidines 15 and 16. Also notably the dimethylpyr-
azole compound 21 displayed a promising ADME profile and
maintained activity in the nanomolar range. The compounds
exhibited no cytotoxic effects in a cell health assay. Further opti-
mization focused on the evaluation of the para-bromo-phenyl-
ureidic part of compound 7, but changes in this region were not
well tolerated. The final round of investigation confirmed that
€
site was identified using by Sitemap (Schrodinger Release 2018e2:
€
Schrodinger, LLC, New York, 2018.) Ligands were prepared and
visualized using Maestro 11.5. To generate decoys for active ago-
nists DUDE database (http://dude.docking.org/) was used. The
collection of compounds was implemented in Pipeline Pilot to
eliminate possible duplicates and compounds which do not meet
the Lipinski rule of 5 (Pipeline Pilot BIOVIA, Dassault Systemes, San
Diego, 2018.)
ꢀ
4.1.3. Pharmacophore studies
Pharmacophore hypothesis was obtained from multiple active
€
€
ligands using Phase (Schrodinger Release 2018e2: Schrodinger,
LLC, New York, 2018).
Scheme 1. Synthetic pathway for pyrrolidinone series.
Conditions: A) 1) 2 eq. TEA, 1 eq. amine, Et2O or THF, 0 ꢀC to RT, overnight; 2) 1.5 eq. sodium hydride (60 mass%) in mineral oil, THF, 0 ꢀC to RT, 2e6 h; B) 35% aq. Ammonia/
acetonitrile 1/1, 40 ꢀC, overnight; C) 1.1 equiv. isocyanate, DCM/DMF 9/1, RT, overnight D) 4 M HCl in dioxane, DCM, 0 ꢀC to RT; 2e4 h; E) 2.5 eq. DIPEA, 1.1eq. benzyl bromide, DMF,
RT, overnight.
Table 5
In vivo mouse PK parameters for compound 7 with intraperitoneal administration.
Dose [mg/kg]
10
Cmax [ng/mL]
3608
Tmax [h]
0.25
T1/2 [h]
9.42
AUC0-last [ng$h/mL]
AUC0-inf [ng$h/mL]
AUCExtra [%]
18.17
MRT0-last [h]
9.20
28615
34968
8

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
4.2. Chemistry
obtain desired product. Yield: 200 mg, 75%; Appearance: yellow oil;
LCMS: 1.78 min, m/z 254.0, 256.1 ((M þ H)þ 1:1, 79/81Br, 1H NMR
All reagents were purchased from Sigma-Aldrich, Enamine,
Fluorochem and other major suppliers and used without further
purification. Solvents were purchased from Fisher Scientific (HPLC
grade) or Sigma-Aldrich (anhydrous). Flash column chromatog-
raphy was carried out with Biotage KP-Sil Snap cartridges using a
Biotage Isolera-One and HPLC grade solvents. Reverse phase col-
umn chromatography was carried out on a Biotage Isolera with
prepacked columns of C18 silica and HPLC grade solvents and
buffers (Buffer A: 0.1% Ammonium Hydroxide in water; Buffer B:
0.1% Trifluoroacetic acid in water). Purification of compounds by
preparative HPLC was carried out on an Agilent Reverse Phase Mass
Directed Prep HPLC system by a member of the purification team.
LC-MS was carried out on an Agilent 6120 quadrupole LC-MS with
(500 MHz, CDCl3) d ppm 7.38e7.26 (m, 4 H), 7.25e7.23 (m, 1 H),
4.57e4.52 (m, 1 H), 4.47 (dd, J ¼ 7.45, 2.29 Hz, 1 H), 4.46e4.41 (m,
1 H), 3.46e3.38 (m, 1 H), 3.20 (ddd, J ¼ 9.88, 7.88, 2.29 Hz, 1 H),
2.60e2.51 (m, 1 H), 2.29 (ddt, J ¼ 14.54, 6.66, 2.58, 2.58 Hz, 1 H); 13
C
NMR (126 MHz, CDCl3)
d ppm 170.7, 135.6, 128.8, 128.1, 127.9, 47.1,
44.5, 44.2, 30.1; Data corresponded to that reported in the literature
[43].
4.2.1.2. tert-Butyl 4-(3-bromo-2-oxo-pyrrolidin-1-yl)piperidine-1-
carboxylate (36). Prepared according to general procedure A with
tert-butyl 4-aminopiperidine-1-carboxylate (2.5 mmol) and 2,4-
dibromobutanoyl chloride (1.2 equiv., 3 mmol); Crude product
was dry loaded on silica and purified by flash column chromatog-
raphy (50 g silica gel, PE/EA 1/1 1, gradient) to obtain desired
product. Yield: 600 mg, 70%; Appearance: yellow oil; LCMS:
1.86 min, m/z 247.1, 249.1 ((M þ H)þ 1:1, 79/81Br), Purity>95%,
an Xbridge C18 column (3.5
mm particle and 4.6 ꢁ 30 mm dimen-
sion) and a diode array UV detector. Four methods were used
depending on the nature of the compound: Method A: flow rate:
3 mL/min; Run time: 3.2 min: Solvent A: 0.1% Trifluoro Acetic acid
in water, Solvent B: Acetonitrile; Gradient - 10e100%B; Gradient
time: 2.35 min; Method B: flow rate: 3 mL/min; Run time: 3.2 min:
Solvent A: 0.1% Ammonium Hydroxide in water, Solvent B: Aceto-
nitrile; Gradient - 10e100%B; Gradient time: 2.35min; Method C:
flow rate: 3 mL/min; Run time: 3.2 min: Solvent A: 0.1% Trifluoro
Acetic acid in water, Solvent B: Methanol; Gradient - 10e100%B;
Gradient time: 2.35min; Method D: flow rate: 3 mL/min; Run time:
3.2 min: Solvent A: 0.1% Ammonium Hydroxide in water, Solvent B:
Methanol; Gradient - 10e100%B; Gradient time: 2.35min. HRMS
were recorded on a Thermo-Fisher Q-Exactive operating at 70,000
Resolution. NMR solvents were obtained from Cambridge Isotope
Labs. All 1H NMR (500 MHz) and 13C NMR (126 MHz) spectra were
recorded on a JEOL ECA-500 spectrometer at 21 ꢀC. Chemical shifts
Method D; 1H NMR (500 MHz, CDCl3)
d
ppm 4.43 (dd, J ¼ 7.16,
2.58 Hz, 1 H), 4.22 (br. s, 2 H), 4.15e4.07 (m, 1 H), 3.47 (ddd, J ¼ 9.74,
8.02, 6.87 Hz, 1 H), 3.31 (ddd, J ¼ 9.88, 7.88, 2.29 Hz, 1 H), 2.79 (br. s,
2 H), 2.60e2.49 (m, 1 H), 2.33 (ddt, J ¼ 14.54, 6.52, 2.36, 2.36 Hz,
1 H), 1.79e1.71 (m, 1 H), 1.71e1.63 (m, 1 H), 1.62e1.50 (m, 2 H), 1.46
(s, 9 H); 13C NMR (126 MHz, CDCl3)
44.8, 43.0, 41.2, 30.5, 29.3, 28.7, 28.5.
d ppm 170.5, 154.7, 79.9, 49.9,
4.2.1.3. tert-Butyl 4-[(3-bromo-2-oxo-pyrrolidin-1-yl)methyl]piperi-
dine-1-carboxylate (37). Prepared according to general procedure A
with
tert-butyl
4-(aminomethyl)piperidine-1-carboxylate
(2.332 mmol) and 2,4-dibromobutanoyl chloride (1.2 equiv.,
2.8 mmol). Crude product was dry loaded on silica and purified by
flash column chromatography (50 g silica gel, PE/EA 1/9, gradient)
to obtain desired product. Yield: 440 mg, 40%; Appearance: yellow
oil; LCMS: 1.92 min, m/z 361.1, 363.1 ((M þ H)þ 1:1, 79/81Br), method
(d) are in ppm and are relative to residual undeuterated NMR sol-
vent. Spectra are referenced to a singlet at 7.27 ppm (CDCl3), the
centreline of quintet at 2.50 ppm ((CD3)2SO), quintet at 3.31 ppm
(CD3OD) for 1H NMR and to the centreline of a triplet at 77.23 ppm
(CDCl3), septet at 39.7 ppm ((CD3)2SO), septet at 49.15 ppm
(CD3OD) for 13C NMR. All 13C NMR spectra were proton decoupled.
C; 1H NMR (500 MHz, CDCl3)
d
ppm 4.42 (dd, J ¼ 6.87, 2.29 Hz, 1 H),
4.19e4.02 (m, 2 H), 3.66e3.59 (m, 1 H), 3.44e3.24 (m, 2 H),
3.22e3.00 (m, 1 H), 2.69 (br. s., 2 H), 2.64e2.55 (m, 1 H), 2.34 (ddt,
1 H), 1.85 (ttd, 1 H), 1.71e1.52 (m, 2 H), 1.45 (s, 9 H), 1.24e1.14 (m,
2 H); 13C NMR (126 MHz, CDCl3)
d ppm 171.1, 154.7, 79.4, 48.8, 46.2,
4.2.1. General procedure A for amide formation from acyl chloride
and cyclization of pyrrolidinone
44.3, 43.4, 34.7, 30.5, 29.7, 29.5, 28.4.
To a solution of amine (1 equiv.) in anhydrous diethyl ether
(12 mL) was added, under inert atmosphere, triethylamine (2
equiv.) followed by a 2,4-dibromobutanoyl chloride (1.1 equiv.).
After 16 h at room temperature, the solution was filtered, and the
filtrate concentrated under reduced pressure. The solvent was
evaporated, and the crude product was used in the next step
without further purification. A sodium hydride (60 mass%) in
mineral oil (1.2e1.5 equiv.) was added to a stirred solution of in-
termediate (1 equiv.) in tetrahydrofuran at 0 ꢀC. The reaction
mixture was allowed to warm up to room temperature and stir for
an additional 2 h. The reaction was carefully quenched with ice and
diluted with water. The resulting mixture was extracted with
EtOAc. The combined organic layers were washed with brine and
water. The organic phase was filtered through a phase separator
and concentrated under reduced pressure. Crude product was dry
loaded on silica and purified by flash column chromatography to
obtain desired product.
4.2.1.4. 3-Bromo-1-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrrolidin-2-one
(38). Prepared according to general procedure A with 5-phenyl-
1,3,4-oxadiazol-2-amine (1.24 mmol). and 2,4-dibromobutanoyl
chloride (1.2 equiv., 1.48 mmol). Crude product was dry loaded on
silica and purified by flash column chromatography (25 g silica, PE/
EA 0/70%, gradient) to obtain desired product. Yield: 70 mg, 16%;
Appearance: yellow oil; LCMS: 1.72 min, m/z 308.0, 310.0 ((M þ H)þ
1:1, 79/81Br), Purity ¼ 80%, Method C; 1H NMR (500 MHz, CDCl3)
d
ppm 8.11e8.05 (m, 2 H), 7.57e7.47 (m, 3 H), 4.60 (dd, J ¼ 6.87,
2.86 Hz, 1 H), 4.29e4.18 (m, 2 H), 2.90e2.81 (m, 1 H), 2.60e2.53 (m,
1 H); 13C NMR (126 MHz, CDCl3)
ppm 167.8, 162.5, 156.9, 131.7,
129.0, 126.7, 123.3, 45.5, 42.2, 30.3.
d
4.2.1.5. 3-Bromo-1-(5-phenyloxazol-2-yl)pyrrolidin-2-one
(39).
Prepared according to general procedure A with 5-phenyloxazol-2-
amine (1.24 mmol). and 2,4-dibromobutanoyl chloride (1.2 equiv.,
1.48 mmol). Crude product was dry loaded on silica and purified by
flash column chromatography (25 g silica, PE/EA 0/100%) to
obtain desired product. Yield: 75 mg, 18%; Appearance: yellow oil
which solidified after few hours; LCMS: 1.89 min m/z 307.0, 309.0,
((M þ H)þ 1:1, 79/81Br), Purity ¼ 91%, Method C; 1H NMR (500 MHz,
4.2.1.1. 1-Benzyl-3-bromo-pyrrolidin-2-one (35). Prepared accord-
ing to general procedure A with phenylmethanamine (0.93 mmol).
and 2,4-dibromobutanoyl chloride (1.2 equiv., 1.12 mmol). Crude
product was dry loaded on silica and purified by flash column
chromatography (25 g silica gel, PE/EA 0/70% 15CV gradient) to
CDCl3)
d ppm 7.66e7.63 (m, 2 H), 7.44e7.39 (m, 2 H), 7.32 (tt,
J ¼ 7.45, 1.70 Hz, 1 H), 7.25 (s, 1 H), 4.60 (dd, J ¼ 6.87, 2.29 Hz, 1 H),
9

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
4.23e4.16 (m,1 H), 4.16e4.10 (m,1 H), 2.80 (dtd, J ¼ 14.61, 8.16, 8.16,
J ¼ 14.32, 2.29 Hz, 1 H), 1.93e1.80 (m, 1 H), 1.77e1.69 (m, 1 H),
1.68e1.49 (m, 2 H), 1.45e1.42 (m, 9 H); 13C NMR (126 MHz, CDCl3)
6.87 Hz, 1 H), 2.54e2.47 (m, 1 H); 13C NMR (126 MHz, CDCl3)
d ppm
167.4, 152.0, 148.4, 128.6, 128.1, 127.1, 123.6, 120.9, 45.2, 42.9, 29.8.
d ppm 170.6, 154.5, 80.0, 48.9, 48.7, 44.4, 44.4, 42.1, 30.5, 28.3, 27.9,
23.9.
4.2.1.6. 3-Bromo-1-(3,5-dimethyl-1-phenyl-pyrazol-4-yl)pyrrolidin-
2-one (40). Prepared according to general procedure A with 5-
phenyloxazol-2-amine (1.24 mmol). and 2,4-dibromobutanoyl
chloride (1.2 equiv., 1.48 mmol). Crude product was dry loaded on
silica and purified by flash column chromatography (silica gel, PE/
EA 0/100% 20CV) to obtain desired product. Yield: 200 mg, 50%;
Appearance: brown oil which solidified after few hours LCMS:
1.75 min m/z 334.1, 336.0 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 95%,
4.2.1.10. tert-Butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)pyrrolidine-1-
carboxylate (44). Prepared according to general procedure A with
tert-butyl 3-aminopyrrolidine-1-carboxylate (1.6 mmol) and 2,4-
dibromobutanoyl chloride (1.2 equiv., 1.92 mmol). Crude product
was dry loaded on silica and purified by flash column chromatog-
raphy (silica gel, PE/EA 0 / 100%, gradient) to obtain desired
product (first fraction). Yield: 166 mg, 66%; Appearance: yellow oil;
Method D; 1H NMR (500 MHz, CDCl3)
d ppm 7.49e7.41 (m, 4 H),
LCMS: 1.76 min m/z 332.1, 334.1 ((M
þ
H)þ 1:1, 79/81Br),
Purity ¼ 80e85%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm 4.70
7.41e7.35 (m, 1 H), 4.56 (dd, J ¼ 6.87, 1.72 Hz, 1 H), 3.89 (ddd,
J ¼ 10.17, 8.74, 6.30 Hz, 1 H), 3.66e3.61 (m, 1 H), 2.82e2.75 (m, 1 H),
2.47 (ddt, J ¼ 14.39, 6.23, 1.72, 1.72 Hz, 1 H), 2.25 (s, 3 H), 2.23 (s,
(td, J ¼ 6.87, 13.75 Hz, 1 H), 4.41 (dd, J ¼ 2.29, 6.87 Hz, 1 H),
4.03e4.03 (m, 1H), 3.63e3.43 (m, 4H), 3.36 (ddd, J ¼ 2.00, 7.73,
9.74 Hz, 2 H), 2.56 (dd, J ¼ 7.16, 14.61 Hz, 1 H), 2.34 (ddd, J ¼ 2.86,
5.73, 11.46 Hz, 1 H), 2.22e2.07 (m, 1 H), 2.05e1.94 (m, 1 H), 1.47 (s,
3 H); 13C NMR (126 MHz, CDCl3)
d ppm 171.0, 145.3, 139.6, 136.0,
129.2, 127.8, 124.8, 117.5, 48.6, 44.2, 31.0, 11.7, 10.8.
9 H); 13C NMR (126 MHz, CDCl3)
d ppm 170.9, 154.4, 79.9, 51.6, 50.7,
4.2.1.7. tert-Butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)azetidine-1-
carboxylate (41). Prepared according to general procedure A with
tert-butyl 3-aminoazetidine-1-carboxylate (1.74 mmol) and 2,4-
dibromobutanoyl chloride (1.1 equiv., 1.9 mmol). Crude product
was dry loaded on silica and purified by flash column chromatog-
raphy (silica gel, PE/EA 0/80%, gradient) to obtain desired product.
Yield: 368 mg, 60%; Appearance: yellow oil which solidified after
few hours; LCMS: 1.73 min m/z 319.1, 321.1 ((M þ H)þ 1:1, 79/81Br),
48.3, 47.8, 44.4, 41.9, 30.5, 28.6, 28.1.
4.2.1.11. tert-Butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)pyrrolidine-1-
carboxylate (45). Prepared according to general procedure A with
tert-butyl 3-aminopyrrolidine-1-carboxylate (1.6 mmol) and 2,4-
dibromobutanoyl chloride (1.2 equiv., 1.92 mmol). Crude product
was dry loaded on silica and purified by flash column chromatog-
raphy (silica gel, PE/EA 0 / 100%, 15CV gradient) to obtain desired
product (second fraction). Yield: 193 mg, 66%; Appearance: yellow
oil; LCMS: 1.792 min m/z 332.1, 334.1 ((M þ H)þ 1:1, 79/81Br),
Purity ¼ 95%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm 4.94 (tt,
J ¼ 8.09, 5.37 Hz, 1 H), 4.43 (dd, J ¼ 7.16, 2.58 Hz, 1 H), 4.19 (td,
J ¼ 8.88, 4.01 Hz, 2 H), 4.01 (dd, J ¼ 9.45, 5.44 Hz, 1 H), 3.95 (dd,
J ¼ 9.74, 5.16 Hz, 1 H), 3.71 (ddd, J ¼ 9.74, 7.73, 6.59 Hz, 1 H), 3.58
(ddd, J ¼ 10.02, 7.73, 2.86 Hz, 1 H) 2.68e2.59 (m, 1 H), 2.40 (ddt,
J ¼ 14.32, 6.59, 2.72, 2.72 Hz, 1 H), 1.45 (s, 9 H); 13C NMR (126 MHz,
Purity ¼ 80e85%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm
4.76e4.66 (m, 1 H), 4.42 (dd, J ¼ 2.29, 6.87 Hz, 1 H), 3.65e3.44 (m,
4 H), 3.43e3.32 (m, 2 H), 2.58 (qt, J ¼ 7.35, 14.46 Hz, 1 H), 2.39e2.31
(m, 1 H), 2.19e2.10 (m, 1 H), 2.04e1.93 (m, 1 H), 1.47 (s, 9 H); 13C
CDCl3)
28.4.
d ppm 171.03, 156.1, 80.3, 46.2, 43.9, 41.8, 41.3, 35.2, 30.4,
NMR (126 MHz, CDCl3)
30.5, 28.6, 28.4.
d ppm 171.0,154.4, 79.9, 51.6, 47.6, 44.2, 41.9,
4.2.1.8. tert-Butyl
(3S)-3-(3-bromo-2-oxo-pyrrolidin-1-yl)piperi-
4.2.1.12. 3-Bromo-1-(1,3-dimethylpyrazol-4-yl)pyrrolidin-2-one
(46). Prepared according to general procedure with 1,3-
dine-1-carboxylate (42). Prepared according to general procedure A
with tert-butyl (3S)-3-aminopiperidine-1-carboxylate (1.99 mmol)
and 2,4-dibromobutanoyl chloride (1.2 equiv., 2.4 mmol). Crude
product was dry loaded on silica and purified by flash column
chromatography (silica gel, PE/EA 0 / 100%, gradient) to obtain
desired product (first fraction). Yield: 300 mg, 80%; Appearance:
yellow oil; LCMS: 1.87 min m/z 347.1, 349.1 ((M þ H)þ 1:1, 79/81Br),
A
dimethylpyrazol-4-amine (1.8 mmol) and 2,4-dibromobutanoyl
chloride (1.2 equiv., 2.16 mmol). Crude product was dry loaded on
silica and purified by flash column chromatography (silica gel, PE/
EA 0 / 100%, and then EA/MeOH 9/1) to obtain desired product
Yield: 230 mg, 60%; Appearance: yellow oil; LCMS: 0.93 min, m/z
258.1, 260.1 ((M þ H)þ 1:1, 79/81Br), Purity>85%, Method D; 1H NMR
Purity ¼ 90%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm 4.41 (dd,
(500 MHz, CDCl3)
d
ppm 7.38 (s,1 H), 4.52 (dd, J ¼ 6.87, 2.29 Hz,1 H),
J ¼ 6.87, 2.29 Hz, 1 H), 3.95 (br. s., 3 H), 3.58e3.46 (m, 1 H),
3.43e3.30 (m, 1 H), 2.87 (d, J ¼ 10.88 Hz, 1 H), 2.71 (t, J ¼ 11.74 Hz,
1 H), 2.60e2.50 (m, 1 H), 2.38e2.26 (m, 1 H), 1.94e1.74 (m, 1 H), 1.74
(d, J ¼ 10.31 Hz, 1 H), 1.68e1.52 (m, 2 H), 1.45 (d, J ¼ 3.44 Hz, 9 H);
3.87 (ddd, J ¼ 10.02, 8.31, 6.30 Hz, 1 H), 3.77 (s, 3 H), 3.64 (ddd,
J ¼ 10.17, 7.88, 2.00 Hz, 1 H), 2.81e2.70 (m, 1 H), 2.45e2.38 (m, 1 H),
2.21 (s, 3 H); 13C NMR (126 MHz, CDCl3)
117.9, 49.0, 44.1, 36.8, 30.6, 9.8.
d ppm 169.9, 134.1, 133.4,
13C NMR (126 MHz, CDCl3)
d ppm 170.6, 154.58, 80.0, 48.9, 48.7,
44.5, 44.4, 42.2, 30.6, 28.3, 27.9, 23.9.
4.2.1.13. 3-Bromo-1-(1-isopropylpyrazol-4-yl)pyrrolidin-2-one (47).
Prepared according to general procedure with 1-
4.2.1.9. tert-Butyl
(3S)-3-(3-bromo-2-oxo-pyrrolidin-1-yl)piperi-
A
dine-1-carboxylate (43). Prepared according to general procedure A
with tert-butyl (3S)-3-aminopiperidine-1-carboxylate (1.99 mmol)
and 2,4-dibromobutanoyl chloride (1.2 equiv., 2.4 mmol). Crude
product was dry loaded on silica and purified by flash column
chromatography (silica gel, PE/EA 0 / 100%, gradient) to obtain
desired product (second fraction). Yield: 250 mg, 80%; Appearance:
yellow oil; LCMS: 1.75 min m/z 347.1, 349.1 ((M þ H)þ 1:1, 79/81Br),
isopropylpyrazol-4-amine (2.0 mmol) and 2,4-dibromobutanoyl
chloride (1.2 equiv., 2.4 mmol). Crude product was dry loaded on
silica and purified by flash column chromatography (silica gel, PE/
EA 0 / 100%, and then EA/MeOH 9/1) to obtain desired product.
Yield: 384 mg, 67%; Appearance: yellow oil; LCMS: 1.44 min, m/z
272.1, 274.1 ((M þ H)þ 1:1, 79/81Br), Purity>90%, Method D; 1H NMR
(500 MHz, DMSO‑d6)
d ppm 8.07 (s, 1 H), 7.68 (s, 1 H), 4.83 (dd,
Purity ¼ 90%, Method D; 1HNMR (500 MHz, CDCl3)
d
ppm 4.40 (dd,
J ¼ 7.16, 3.15 Hz, 1 H), 4.49 (quin, J ¼ 6.59 Hz, 1 H), 3.70e3.80 (m,
2 H), 2.79 (dq, J ¼ 14.89, 7.45 Hz,1 H), 2.35 (ddt, J ¼ 14.46, 6.44, 3.29,
3.29 Hz, 1 H), 1.39 (d, J ¼ 6.87 Hz, 6 H); 13C NMR (126 MHz,
J ¼ 7.16, 2.00 Hz, 1 H), 4.07 (m, 3 H), 3.85e3.34 (dtd, J ¼ 9.17, 5.73,
5.73, 2.86 Hz,1 H), 3.57e3.45 (m,1 H), 2.97e2.79 (m, 1 H), 2.69 (br t,
J ¼ 11.74 Hz, 1 H), 2.54 (dq, J ¼ 14.75, 7.30 Hz, 1 H), 2.30 (dq,
DMSO‑d6) d ppm 167.3, 128.5,122.1,117.8, 53.3, 45.7, 43.5, 29.8, 22.6.
10

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
4.2.1.14. 3-Bromo-1-(1-phenylpyrazol-4-yl)pyrrolidin-2-one
(48).
4.2.2.2. tert-Butyl 4-(3-amino-2-oxo-pyrrolidin-1-yl)piperidine-1-
carboxylate (52). Product was synthesized according to general
procedure B with tert-butyl 4-(3-bromo-2-oxo-pyrrolidin-1-yl)
piperidine-1-carboxylate (36) (0.79 mmol). Yield: 440 mg, 72%;
Appearance: yellow oil; LCMS: 1.57 min m/z 284.0, (M þ H)þ Pu-
rity>90%, weak UV active, Method D; 1H NMR (500 MHz, CDCl3)
Prepared according to general procedure A with 1-phenylpyrazol-
4-amine (0.95 mmol) and 2,4-dibromobutanoyl chloride (1.2 equiv.,
1.13 mmol). Crude product was dry loaded on silica and purified by
flash column chromatography (silica gel, PE/EA 0 / 100%, and then
EA/MeOH 9/1) to obtain desired product. Yield: 177 mg, 66%;
Appearance: white solid; LCMS: 1.84 min, m/z 306.1, 308.1
((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz,
d
ppm 4.21 (br. s., 2 H), 4.14e4.05 (m, 1 H), 3.54 (dd, J ¼ 9.74,
8.59 Hz, 1 H), 3.30 (td, J ¼ 9.17, 1.72 Hz, 1 H), 3.20 (td, J ¼ 9.45,
6.87 Hz, 1 H), 2.79 (br. s., 2 H), 2.44 (dddd, J ¼ 12.67, 8.23, 6.59,
1.72 Hz, 1 H), 1.80e1.73 (m, 2 H), 1.73e1.68 (m, 1 H), 1.68e1.53 (m,
DMSO‑d6)
d
ppm 8.67 (s, 1 H), 8.08 (s, 1 H), 7.81 (d, J ¼ 7.45 Hz, 2 H),
7.47 (t, J ¼ 8.02 Hz, 2 H), 7.29 (t, J ¼ 7.34 Hz, 1 H), 4.86 (dd, J ¼ 7.45,
3.44 Hz, 1 H), 3.87e3.76 (m, 2 H), 2.82 (dq, J ¼ 14.46, 7.40 Hz, 1 H),
4 H), 1.46 (s, 9 H); 13C NMR (126 MHz, CDCl3)
79.7, 53.4, 49.3, 39.6, 29.1, 28.4.
d ppm 175.3, 154.6,
2.41e2.34 (m, 1 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 167.9,
139.5, 131.9, 129.6, 126.4, 125.3, 118.2, 117.2, 45.9, 43.7, 29.9.
4.2.2.3. tert-Butyl 4-[(3-amino-2-oxo-pyrrolidin-1-yl)methyl]piperi-
dine-1-carboxylate (53). Product was synthesized according to
general procedure B with tert-butyl 4-[(3-bromo-2-oxo-pyrrolidin-
1-yl)methyl]piperidine-1-carboxylate (37). Yield: 128 mg, 70%;
Appearance: yellow oil; LCMS: 1.67 min m/z 298.2, (M þ H)þ Pu-
rity>90%, weak UV active, Method D; 1H NMR (500 MHz, CDCl3)
4.2.1.15. 3-Bromo-1-[1-(4-pyridyl)pyrazol-4-yl]pyrrolidin-2-one
(49). Prepared according to general procedure A with 1-(4-pyridyl)
pyrazol-4-amine (0.65 mmol) and 2,4-dibromobutanoyl chloride
(1.2 equiv., 0.8 mmol). Crude product was dry loaded on silica and
purified by flash column chromatography (silica gel, PE/EA
0 / 100%, and then EA/MeOH 9/1) to obtain desired product Yield:
60 mg, 31%; Appearance: white solid; LCMS: 1.56 min, m/z 307.0,
309.1 ((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
d
ppm 4.20e4.05 (m, 2 H), 3.58 (dd, J ¼ 8.59, 9.74 Hz, 1 H),
3.35e3.29 (m, 2 H), 3.17 (br. s., 2 H), 2.73e2.63 (m, 2 H), 2.49e2.41
(m, 1 H), 2.27e2.07 (m, 2 H), 1.85e1.73 (m, 2 H), 1.64e1.54 (m, 2 H),
1.45 (s, 9 H), 1.21e1.12 (m, 2 H); 13C NMR (126 MHz, CDCl3)
d ppm
(500 MHz, DMSO‑d6)
d
ppm 8.86 (s, 1 H), 8.61 (dd, J ¼ 6.30, 1.72 Hz,
175.9, 154.7, 79.4, 52.9, 48.7, 44.9, 42.9, 34.6, 29.7, 28.8, 28.4.
2 H), 8.21 (s, 1 H), 7.85 (dd, J ¼ 6.30, 1.72 Hz, 2 H), 4.87 (dd, J ¼ 7.45,
3.44 Hz, 1H), 3.88e3.77 (m, 2 H), 2.88e2.78 (m, 1 H), 2.42e2.34 (m,
1 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 168.3, 149.4, 134.7, 126.0,
4.2.2.4. tert-Butyl 3-(3-amino-2-oxo-pyrrolidin-1-yl)pyrrolidine-1-
carboxylate (54). Product was synthesized according to general
procedure B with tert-butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)
pyrrolidine-1-carboxylate (44) (0.45 mmol); Yield: 86 mg, 71%;
Appearance: light orange oil; LCMS: 1.26 min, m/z 170.2 (M þ H)þ,
mass without BOC group Purity>90%, weak UV active, Method D;
117.6, 112.4, 45.8, 45.6, 29.8.
4.2.1.16. 3-Bromo-1-(1-pyrimidin-2-yl-4-piperidyl)pyrrolidin-2-one
(50). Prepared according to general procedure A with 1-pyrimidin-
2-ylpiperidin-4-amine; dihydrochloride (0.88 mmol) and 2,4-
dibromobutanoyl chloride (1.2 equiv., 1.05 mmol). Crude product
was dry loaded on silica and purified by flash column chromatog-
raphy (silica gel, PE/EA 0 / 100%, and then EA/MeOH 9/1) to obtain
desired product Yield: 80 mg, 44%; Appearance: white solid; LCMS:
1.60 min, m/z 325.1, 327.1 ((M þ H)þ 1:1, 79/81Br), Purity>85%,
1H NMR (500 MHz, CDCl3)
d ppm 4.75e4.64 (m, 1 H), 3.66e3.42 (m,
3 H), 3.41e3.18 (m, 4 H), 2.51e2.35 (m, 3 H), 2.08 (d, J ¼ 5.73 Hz,
1 H), 2.03e1.89 (m, 1 H), 1.87e1.70 (m, 1 H), 1.45 (s, 9 H); 13C NMR
(126 MHz, CDCl3)
29.0, 28.4, 28.0.
d ppm 175.6,154.3, 79.7, 53.4, 53.1, 50.9, 47.7, 44.7,
Method D; 1H NMR (500 MHz, DMSO‑d6)
d
ppm 8.35 (d, J ¼ 4.58 Hz,
4.2.2.5. tert-Butyl 3-(3-amino-2-oxo-pyrrolidin-1-yl)pyrrolidine-1-
carboxylate (55). Product was synthesized according to general
procedure B with tert-butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)
pyrrolidine-1-carboxylate (45) (0.54 mmol); Yield: 100 mg, 70%;
Appearance: orange oil; LCMS: 1.47 min, m/z 170.2 (M þ H)þ, mass
without BOC group Purity>90%, weak UV active, Method D; 1H
2 H), 6.60 (t, J ¼ 5.16 Hz, 1 H), 4.81e4.74 (m, 2 H), 4.66 (dd, J ¼ 10.31,
3.44 Hz, 1 H), 4.03 (tt, J ¼ 11.74, 4.30 Hz, 1 H), 3.33e3.29 (m, 2 H),
2.95e2.87 (m, 2 H), 2.58e2.57 (m, 1 H), 2.19e2.12 (m, 1 H),
1.67e1.52 (m, 4 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 169.4,
161.0, 158.0, 109.9, 49.8, 46.8, 42.4, 40.9, 29.9, 27.9.
NMR (500 MHz, CDCl3)
d 4.76e4.65 (m, 1 H), 3.63e3.44 (m, 3 H),
4.2.2. General procedure B
3.42e3.18 (m, 4 H), 2.52e2.41 (m, 1 H), 2.16e2.04 (m, 1 H),
2.01e1.91 (m, 1 H), 1.88 (br. s, 2 H), 1.79e1.66 (m, 1 H), 1.46 (s, 9 H);
3-bromopyrrolidin-2-one derivative (1 eq.) was dissolved in
acetonitrile (4 mL). Under nitrogen atmosphere ammonia (32 mass
%) in water (2 mL) was added to the reaction mixture. The RM was
stirred overnight at 40 ꢀC. LCMS showed only partially formed
product. Acetonitrile was evaporated and remaining aq. phase was
extracted with DCM. Organic phase was filtered through a phase
separator and concentrated under reduced pressure to obtain
desired 3-aminopyrrolidin-2-one derivative. The crude product
was used in the next step without any purification step.
13C NMR (126 MHz, CDCl3)
47.1, 44.2, 28.8, 28.4, 28.0.
d ppm 175.9, 154.3, 79.7, 53.4, 53.1, 50.3,
4.2.2.6. tert-Butyl
(3S)-3-(3-amino-2-oxo-pyrrolidin-1-yl)piperi-
dine-1-carboxylate (56). Product was synthesized according to
general procedure B with tert-butyl (3S)-3-(3-bromo-2-oxo-pyr-
rolidin-1-yl)piperidine-1-carboxylate (42) (0.75 mmol). Yield:
200 mg, 94%; Appearance: orange oil; LCMS: 1.60 min, m/z 184.2
(M þ H)þ mass without BOC group, Purity>80%, weak UV active,
4.2.2.1. 3-Amino-1-benzyl-pyrrolidin-2-one (51). Product was syn-
thesized according to general procedure B with 1-benzyl-3-bromo-
pyrrolidin-2-one (35) (0.4 mmol). Crude product was used in the
next step without any purification step. Yield: 39 mg, 60%;
Appearance: yellow oil; LCMS: 1.25 min m/z 191.1, (M þ H)þ Pu-
Method D; 1H NMR (500 MHz, CDCl3)
d ppm 4.05e3.88 (br. m, 2 H),
3.43e3.25 (br. m, 2 H), 3.16 (br. s, 2 H), 2.88e2.76 (m, 1 H), 2.66 (br.
s,1 H), 2.52e2.42 (m, 1 H),1.84 (br. s, 2 H),1.72 (dd, J ¼ 6.30, 2.86 Hz,
1 H), 1.64e1.51 (m, 2 H), 1.44 (s, 9 H).
rity>80%, Method D; 1H NMR (500 MHz, CDCl3)
d
ppm 7.35e7.26
4.2.2.7. tert-Butyl
(3S)-3-(3-amino-2-oxo-pyrrolidin-1-yl)piperi-
(m, 3 H), 7.24e7.20 (m, 2 H), 4.45 (d, J ¼ 1.72 Hz, 2 H), 3.56 (dd,
J ¼ 9.74, 8.59 Hz, 1 H), 3.21e3.12 (m, 2 H), 2.43e2.35 (m, 1 H),
1.77e1.64 (m, 3 H); Compound has been reported in the literature
[44].
dine-1-carboxylate (57). Product was synthesized according to
general procedure B with tert-butyl (3S)-3-(3-bromo-2-oxo-pyr-
rolidin-1-yl)piperidine-1-carboxylate (43) (0.6 mmol). Yield:
107 mg, 70%; Appearance: orange oil; LCMS: 1.63 min, m/z 184.2
11

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
(M þ H)þ, mass without BOC group, Purity>80%, weak UV active,
4.2.2.13. 3-Amino-1-(1-phenylpyrazol-4-yl)pyrrolidin-2-one
(63).
Method D; 1H NMR (500 MHz, CDCl3)
d
ppm 4.11e3.75 (m, 3 H),
Product was synthesized according to general procedure B with 3-
bromo-1-(1-phenylpyrazol-4-yl)pyrrolidin-2-one (48) (0.2 mmol).
Yield: 50 mg, 95%; Appearance: orange oil; LCMS: 1.46 min, m/z
243.2 (M þ H)þ Purity>95%, Method D; 1H NMR (500 MHz,
3.58e3.46 (m, 1 H), 3.42e3.32 (m, 1 H), 3.31e3. 18 (m, 1 H),
2.86e2.77 (m, 1 H), 2.67 (br. s, 1 H), 2.47e2.38 (m, 1 H), 1.93 (br. s,
2 H), 1.86e1.78 (m, 1 H), 1.77e1.66 (m, 2 H), 1.65e1.50 (m, 2 H), 1.43
(s, 9 H).
DMSO‑d6)
d ppm 8.61 (s, 1 H), 8.09 (s, 1 H), 7.86e7.80 (m, 2 H),
7.54e7.47 (m, 2 H), 7.34e7.27 (m, 1 H), 3.70 (td, J ¼ 9.45, 2.29 Hz,
1 H), 3.65e3.53 (m, 1 H), 2.45e2.38 (m, 1 H), 1.81 (dq, J ¼ 12.32,
4.2.2.8. tert-Butyl 3-(3-amino-2-oxo-pyrrolidin-1-yl)azetidine-1-
carboxylate (58). Product was synthesized according to general
procedure B with tert-butyl 3-(3-bromo-2-oxo-pyrrolidin-1-yl)
azetidine-1-carboxylate (41) (1.1 mmol). Yield: 220 mg, 79%;
Appearance: yellow oil; LCMS: 1.37 min, m/z 156.2 (M þ H)þ mass
without BOC group, Purity>90%, weak UV active, Method D; 1H
9.26 Hz, 1 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 174.8, 140.1,
132.4, 130.2, 126.8, 126.0, 118.5, 116.9, 53.4, 44.7, 29.0.
4.2.2.14. 3-Amino-1-[1-(4-pyridyl)pyrazol-4-yl]pyrrolidin-2-one
(64). Product was synthesized according to general procedure B
with 3-bromo-1-[1-(4-pyridyl)pyrazol-4-yl]pyrrolidin-2-one (49)
(0.16 mmol). Yield: 50 mg, 95%; Appearance: orange oil; LCMS:
1.46 min, m/z 243.2 (M þ H)þ Purity>95%, Method D; 1H NMR
NMR (500 MHz, CDCl3)
d ppm 4.99e4.91 (m, 1 H), 4.19e4.09 (m,
2 H), 3.97 (dd, J ¼ 6.87, 5.73 Hz, 2 H), 3.56 (dd, J ¼ 9.74, 8.02 Hz, 2 H),
3.42 (td, J ¼ 9.74, 6.87 Hz, 1 H), 2.51 (dddd, J ¼ 12.67, 8.09, 6.73,
1.72 Hz, 1 H), 1.84 (br. s, 2 H), 1.81e1.72 (m, 1 H), 1.44 (s, 9 H); 13C
(500 MHz, DMSO‑d6) d ppm 8.90 (s,1 H), 8.67e8.63 (m, 2 H), 8.28 (s,
1 H), 7.94e7.84 (m, 2 H), 4.25 (t, J ¼ 9.45 Hz,1 H), 3.91e3.82 (m,1 H),
NMR (126 MHz, CDCl3)
39.7, 28.6, 28.2.
d ppm 176.0, 156.0, 80.0, 53.2, 52.6, 41.1,
3.81e3.72 (m,1 H), 2.64e2.55 (m, 1 H), 2.21e2.08 (m, 1 H); 13C NMR
(126 MHz, DMSO‑d6)
d ppm 167.8, 151.3, 145.2, 133.8, 125.3, 117.3,
111.9, 50.2, 44.8, 24.0.
4.2.2.9. 3-Amino-1-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrrolidin-2-one
(59). Product was synthesized according to general procedure B
with 3-bromo-1-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrrolidin-2-one
(38). Yield: 12 mg, 20%; Appearance: white solid; LCMS:
1.336 min, m/z 245 (M þ H)þ Purity>80%, Method D; 1H NMR
4.2.2.15. 3-Amino-1-(1-pyrimidin-2-yl-4-piperidyl)pyrrolidin-2-one
(65). Product was synthesized according to general procedure B
with 3-bromo-1-(1-pyrimidin-2-yl-4-piperidyl)pyrrolidin-2-one
(50) (0.2 mmol). Yield: 60 mg, 90% Appearance: off-white solid;
LCMS: 1.18 min, m/z 262.2 (M þ H)þ Purity>90%, Method D; 1H
(500 MHz, CD3OD)
d ppm 7.91e7.85 (m, 2 H), 7.58e7.49 (m, 3 H),
4.69 (d, J ¼ 4.58 Hz, 1 H), 3.58e3.50 (m, 1 H), 3.42e3.34 (m, 1 H),
NMR (500 MHz, DMSO‑d6)
d
ppm 8.31 (d, J ¼ 4.58 Hz, 2 H), 6.56 (t,
2.25e2.19 (m, 1 H), 2.10e2.01 (m, 1 H).
J ¼ 4.58 Hz, 1 H), 4.77e4.70 (m, 2 H), 4.03e3.93 (m, 1 H), 3.27 (dd,
J ¼ 9.74, 8.02 Hz, 1 H), 3.18 (td, J ¼ 9.17, 2.29 Hz, 1 H), 3.05 (td,
J ¼ 9.45, 6.87 Hz, 1 H), 2.92e2.81 (m, 2 H), 2.16 (dddd, J ¼ 12.17, 8.16,
6.59, 1.72 Hz, 1 H), 1.79 (br s, 1 H), 1.61e1.43 (m, 4 H); 13C NMR
(126 MHz, DMSO‑d6)
42.5, 42.5, 28.6, 28.5.
4.2.2.10. 3-Amino-1-(3,5-dimethyl-1-phenyl-pyrazol-4-yl)pyrroli-
din-2-one (60). Product was synthesized according to general
procedure B with 3-bromo-1-(3,5-dimethyl-1-phenyl-pyrazol-4-
yl)pyrrolidin-2-one (40) (0.39 mmol). Yield: 40 mg, 38%; Appear-
ance: yellowish oil; LCMS: 1.39 min m/z 271.2, (M þ H)þ Pu-
d ppm 175.0, 161.0, 158.0, 1099, 53.0, 49.2,
rity>90%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm 7.50e7.41
4.2.3. General procedure C
To a solution of amine (1 equiv.) in dichloromethane at room
temperature was added the appropriate isocyanate (1.1 equiv.) and
triethylamine (1.5 equiv.). The resulting mixture was stirred at
room temperature for 3 h. RM was concentrated and purified by
either flash column chromatography or by preparative HPLC
(m, 4 H), 7.40e7.34 (m, 1 H), 3.74 (dd, J ¼ 10.31, 8.02 Hz, 1 H), 3.65
(td, J ¼ 9.74, 6.87 Hz, 1 H), 3.56 (td, J ¼ 9.45, 1.72 Hz, 1 H), 2.63e2.55
(m, 1 H), 2.23e2.22 (s, 3 H), 2.21e2.19 (s, 3 H), 1.97 (dq, J ¼ 12.60,
9.36 Hz, 1 H), 1.82e1.65 (m, 2 H); 13C NMR (126 MHz, CDCl3)
d ppm
176.0, 145.0, 139.5, 135.5, 129.1, 127.6, 124.6, 118.3, 52.8, 47.1, 29.6,
11.7, 10.9.
(19 ꢁ 100mm (5 m) C-18 Waters Xbridge, MeOH pH ¼ 10) to yield
m
final urea product as a white solid.
4.2.2.11. 3-Amino-1-(1,3-dimethylpyrazol-4-yl)pyrrolidin-2-one (61).
Product was synthesized according to general procedure B with 3-
4.2.3.1. 1-(1-Benzyl-2-oxo-pyrrolidin-3-yl)-3-(4-bromophenyl)urea
(9). Prepared according to modified procedure C with intermediate
3-amino-1-benzyl-pyrrolidin-2-one (51) (0.18 mmol) and 4-
bromophenyl isocyanate (1.1 equiv., 0.20 mmol). Crude product
was purified by reverse phase column chromatography (C18,
acetonitrile/water pH ¼ 10). Yield: 42 mg, 62%; Appearance: white
solid; LCMS: 2.06 min m/z 388.1, 390.1 ((M þ H)þ 1:1, 79/81Br),
bromo-1-(1,3-dimethylpyrazol-4-yl)pyrrolidin-2-one
(36),
(0.73 mmol). Yield: 150 mg, 84%; Appearance: yellowish oil; LCMS:
0.29 min m/z 195.2, (M þ H)þ Purity>90%, Method C; 1H NMR
(500 MHz, DMSO‑d6)
d 7.34e7.32 (m, 1H), 3.71e3.68 (s, 3H),
3.57e3.51 (m, 1H), 3.48e3.46 (m, 1H), 3.46e3.43 (m, 1H), 2.31
(dddd, J ¼ 2.00, 6.59, 8.31, 12.32 Hz, 1H), 2.11 (s, 3H), 1.82e1.69 (m,
Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.81 (s, 1 H),
1H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 175.2, 133.2, 133.0, 119.2,
7.42e7.33 (m, 6 H), 7.31e7.21 (m, 4 H), 6.60 (d, J ¼ 6.87 Hz, 1 H),
4.47e4.36 (m, 2 H), 4.36e4.27 (m, 1 H), 3.26e3.14 (m, 2 H),
2.41e2.33 (m, 1 H), 1.88e1.78 (m, 1 H); 13C NMR (126 MHz,
52.4, 46.9, 36.5, 28.9, 9.5; The NMR analysis showed rotamers.
4.2.2.12. 3-Amino-1-(1-isopropylpyrazol-4-yl)pyrrolidin-2-one (62).
DMSO‑d6) d ppm 172.3, 154.8, 139.7, 136.6, 131.4, 128.6, 127.6, 119.6,
Product was synthesized according to general procedure B with 3-
119.5, 112.5, 51.1, 46.1, 43.0, 26.9; HRMS: Exact Mass: 387.0582;
bromo-1-(1-isopropylpyrazol-4-yl)pyrrolidin-2-one
(47)
(M þ H)þ: 388.0662; Observed mass: 388.0668; 1.45 ppm.
(0.57 mmol). Yield: 125 mg, 94%; Appearance: off-white solid;
LCMS: 0.98 min, m/z 209.2 (M þ H)þ Purity>90%, Method D; 1H
4.2.3.2. tert-Butyl 4-[3-[(4-bromophenyl) carbamoylamino] -2-oxo-
pyrrolidin-1-yl] piperidine-1-carboxylate (10). Prepared according
to modified procedure C with intermediate tert-butyl 4-(3-amino-
2-oxo-pyrrolidin-1-yl)piperidine-1-carboxylate (52) (0.25 mmol)
and 4-bromophenyl isocyanate (1.1 equiv., 0.27 mmol); Crude
compound was dry loaded on silica and purified by flash column
NMR (500 MHz, DMSO‑d6)
d ppm 7.97 (s, 1 H), 7.61 (s, 1 H), 4.45
(quin, J ¼ 6.73 Hz, 1 H), 3.65e3.57 (m, 2 H), 3.56e3.48 (m, 1 H),
2.41e2.33 (m, 1 H), 1.85e1.75 (m, 1 H), 1.35 (d, J ¼ 6.30 Hz, 6 H); 13
C
NMR (126 MHz, DMSO‑d6)
d ppm 171.8, 128.3, 122.5, 117.2, 53.2,
52.1, 44.2, 27.3, 22.6.
12

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
chromatography (silica gel, PE/EtOAc 0e100%). Yield: 65 mg, 49%;
Appearance: white solid; LCMS: 2.15 min m/z 481.1, 483.1
((M þ H)þ 1:1, 79/81Br), Purity ¼ 90%, Method D; 1H NMR (500 MHz,
154.8, 153.9, 139.7, 131.4, 119.6, 112.5, 78.9, 54.9, 51.3, 48.6, 48.2,
28.1, 27.4, 27.3, 24.0; HRMS: Exact Mass: 480.1372; (M þ H)þ
481.1452; Observed mass: 481.1443; 1.87 ppm.
CDCl3)
d
ppm 7.96 (br. s,1 H), 7.28e7.26 (m, 2 H), 7.14 (d, J ¼ 8.59 Hz,
2 H), 6.33 (br. s, 1 H), 4.39e4.27 (m, 1 H), 4.27e4.13 (m, 2 H),
4.12e4.04 (m, 1 H), 3.46e3.38 (m, 1 H), 3.34e3.27 (m, 1 H), 2.74 (br.
s, 2 H), 2.68e2.60 (m, 1 H), 2.07e1.96 (m, 1 H), 1.69e1.56 (m, 4 H),
4.2.3.6. tert-Butyl (3S)-3-[3-[(4-bromophenyl)carbamoylamino]-2-
oxo-pyrrolidin-1-yl] piperidine-1-carboxylate (20). Prepared ac-
cording to modified procedure C with intermediate tert-butyl (3S)-
3-(3-amino-2-oxo-pyrrolidin-1-yl)piperidine-1-carboxylate (57)
(0.3 mmol) and 4-bromophenyl isocyanate (1.1 equiv., 0.33 mmol);
Crude compound was dry loaded on silica and purified by flash
column chromatography (silica gel, DCM/MeOH 97/3). Yield:
125 mg, 54%; Appearance: beige solid; LCMS: 2.13 min m/z 481.1,
483.1 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 85%, Method D; 1H NMR
1.47 (s, 9 H); 13C NMR (126 MHz, CDCl3)
d ppm 173.6, 155.6, 154.5,
138.2, 131.5, 120.6, 114.8, 80.0, 53.07, 50.1, 42.9, 40.5, 29.1, 28.7, 28.4;
HRMS: Exact mass: 480.1372; (M þ H)þ:481.1452; Observed mass:
481.1446; 1.29 ppm.
4.2.3.3. tert-Butyl 4-[[3-[(4-bromophenyl)carbamoylamino]-2-oxo-
pyrrolidin-1-yl]methyl] piperidine-1-carboxylate (11). Prepared ac-
cording to modified procedure C with intermediate tert-butyl 4-[(3-
amino-2-oxo-pyrrolidin-1-yl)methyl]piperidine-1-carboxylate
(53) (0.37 mmol) and 4-bromophenyl isocyanate (1.1 equiv.,
0.41 mmol); Crude compound was dry loaded on silica and purified
by flash column chromatography (silica gel, DCM/MeOH 97/3).
Yield: 160 mg, 83%; Appearance: white solid; LCMS: 2.20 min m/z
495.1, 497.1 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 95%, Method D; 1H
(500 MHz, DMSO‑d6)
d ppm 8.78 (s, 1 H), 7.41e7.35 (m, 4 H),
6.55e6.49 (m, 1 H), 4.32e4.22 (m, 1 H), 3.83 (br.d, 2 H) 3.74e3.65
(m, 1 H) 3.39e3.34 (m, 1 H) 3.30e3.19 (m, 1 H) 2.96e2.59 (m, 2 H)
2.41e2.32 (m, 1 H) 1.81e1.64 (m, 4 H) 1.64e1.51 (m, 1 H) 1.40 (d,
J ¼ 2.29 Hz, 9 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 172.0, 154.8,
153.8, 139.7, 131.4, 119.6, 112.5, 78.9, 51.4, 51.3, 48.2, 28.1, 27.4, 27.3,
27.0; 1 carbon hidden under DMSO peak; HRMS: Exact Mass:
480.1372; (M þ H)þ 481.1452; Observed mass:481.145; 0.42 ppm.
NMR (500 MHz, CDCl3)
d
ppm 7.93 (br. s, 1 H), 7.23 (d, J ¼ 8.59 Hz,
2 H), 7.10 (d, J ¼ 8.59 Hz, 2 H), 6.50 (br. s., 1 H), 4.39e4.23 (m, 1 H),
4.13 (d, J ¼ 6.87 Hz, 2 H), 3.51e3.39 (m, 2 H), 3.32e3.13 (m, 2 H),
2.69 (br. s., 2 H), 2.61 (br. s, 1 H), 2.22e2.05 (m, 1 H), 1.81 (dtt,
J ¼ 11.17, 7.45, 7.45, 3.87, 3.87 Hz, 1 H), 1.68e1.56 (m, 2 H), 1.45 (s,
4.2.3.7. tert-Butyl 3-[3-[(4-bromophenyl)carbamoylamino]-2-oxo-
pyrrolidin-1-yl] pyrrolidine-1-carboxylate (66). Prepared according
to modified procedure C with intermediate tert-butyl 3-(3-amino-
2-oxo-pyrrolidin-1-yl)pyrrolidine-1-carboxylate (54) (0.3 mmol)
and 4-bromophenyl isocyanate (1.1 equiv., 0.33 mmol); Crude
compound was dry loaded on silica and purified by flash column
chromatography (silica gel, DCM/MeOH 97/3). Yield: 90 mg, 60%;
Appearance: beige solid; LCMS: 2.12 min m/z 467.1, 469.1 ((M þ H)þ
1:1, 79/81Br), Purity ¼ 85%, Method D; 1H NMR (500 MHz, CDCl3)
9 H), 1.20e1.08 (m, 2 H); 13C NMR (126 MHz, CDCl3)
d ppm 174.6,
155.7, 154.8, 138.3, 131.6, 120.8, 114.9, 79.7, 52.7, 49.2, 45.6, 43.2,
34.5, 29.8, 28.6, 28.5; HRMS: Exact Mass: 494.1529; (M þ H)þ:
495.1609; Observed mass: 495.1599; 2.02 ppm.
4.2.3.4. tert-Butyl 3-[3-[(4-bromophenyl)carbamoylamino]-2-oxo-
pyrrolidin-1-yl]azetidine-1-carboxylate (14). Prepared according to
modified procedure A with intermediate tert-butyl 3-(3-amino-2-
oxo-pyrrolidin-1-yl)azetidine-1-carboxylate (58) (0.29 mmol) and
4-bromophenyl isocyanate (1.1 equiv., 0.32 mmol); Crude com-
pound was dry loaded on silica and purified by flash column
chromatography (silica gel, DCM/MeOH 97/3). Yield: 62 mg, 44%;
Appearance: beige solid; LCMS: 2.090 min m/z 453, 455 ((M þ H)þ
1:1, 79/81Br), Purity ¼ 95%, Method D; 1H NMR (500 MHz, CDCl3)
d ppm 7.98 (br. s., 1 H), 7.25 (br. s, 2 H), 7.13 (br. s., 2 H), 6.51e6.32
(m, 1 H), 4.72e4.63 (m, 1 H), 4.32e4.21 (m, 1 H), 3.61e3.51 (m, 2 H),
3.50e3.43 (m, 2 H), 3.43e3.28 (m, 3 H), 2.68e2.58 (m, 1 H), 2.11 (br.
s, 1 H), 2.08e1.93 (m, 2 H), 1.47 (s, 9 H), broad peaks, possible
rotamers; 13C NMR (126 MHz, CDCl3)
d ppm 174.2, 155.6, 154.3,
138.1, 131.6, 120.6, 114.9, 80.0, 52.9, 51.6, 51.1, 47.7, 44.6, 29.1, 28.4,
27.9.
4.2.3.8. tert-Butyl 3-[3-[(4-bromophenyl)carbamoylamino]-2-
d
ppm 7.79 (br. s., 1 H), 7.28e7.27 (m, 1 H), 7.27e7.25 (m, 1 H),
oxo-pyrrolidin-1-yl]
Prepared according to modified procedure C with intermediate
tert-butyl 3-(3-amino-2-oxo-pyrrolidin-1-yl)pyrrolidine-1-
pyrrolidine-1-carboxylate
(67).
7.13e7.09 (m, 2 H), 6.27 (d, J ¼ 5.73 Hz, 1 H), 4.89 (tt, J ¼ 8.16,
5.59 Hz, 1 H), 4.31 (dd, J ¼ 7.45, 5.16 Hz, 1 H), 4.17 (td, J ¼ 8.88,
5.16 Hz, 2 H), 4.03 (dt, J ¼ 9.88, 5.08 Hz, 2 H), 3.67 (td, J ¼ 8.02,
2.86 Hz, 1 H), 3.51 (td, J ¼ 9.59, 7.16 Hz, 1 H), 2.72e2.63 (m, 1 H),
carboxylate (55) (0.24 mmol) and 4-bromophenyl isocyanate (1.1
equiv., 0.27 mmol); Crude compound was dry loaded on silica and
purified by flash column chromatography (silica gel, DCM/MeOH
97/3). Yield: 50 mg, 44%; Appearance: beige solid; LCMS:
2.13 min m/z 467.1, 469.1 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 85%,
2.16e2.05 (m, 1 H), 1.45 (s, 9 H); 13C NMR (126 MHz, CDCl3)
d ppm
174.3, 156.0, 155.5, 137.9, 131.6, 120.8, 115.1, 80.3, 52.9, 52.4, 41.8,
40.8, 28.3, 27.2; HRMS: Exact mass: 452.1059; 453.1139; (M þ H)þ
Observed mass: 453.1127; 2.65 ppm.
Method D; 1H NMR (500 MHz, CD3OD)
d ppm 7.37e7.27 (m, 4 H),
4.65e4.55 (m, 1 H), 4.43e4.30 (m, 1 H), 3.60e3.46 (m, 2 H),
3.45e3.40 (m, 1 H), 3.40e3.31 (m, 3 H), 2.54e2.44 (m, 1 H),
2.16e1.85 (m, 3 H), 1.45 (s, 9 H), broad peaks, possible rotamer; 13C
4.2.3.5. tert-Butyl (3S)-3-[3-[(4-bromophenyl)carbamoylamino]-2-
oxo-pyrrolidin-1-yl] piperidine-1-carboxylate (19). Prepared ac-
cording to modified procedure C with intermediate tert-butyl (3S)-
3-(3-amino-2-oxo-pyrrolidin-1-yl)piperidine-1-carboxylate (56)
(0.6 mmol) and 4-bromophenyl isocyanate (1.1 equiv., 0.66 mmol);
Crude compound was dry loaded on silica and purified by flash
column chromatography (silica gel, DCM/MeOH 97/3). Yield:
150 mg, 49% Appearance: beige solid; LCMS: 2.129 min m/z 481,
483 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 85%, Method D; 1H NMR
NMR (126 MHz, CD3OD)
d ppm 173.9, 156.1, 154.9, 138.8, 131.4,
120.4, 114.3, 79.9, 52.1, 51. 8, 44.5, 44.0, 40.5, 28.3, 27.4, 26.7.
4.2.3.9. 1-(4-Bromophenyl)-3-[1-(3,5-dimethyl-1-phenyl-pyrazol-4-
yl)-2-oxo-pyrrolidin-3-yl]urea (21). Prepared according to modified
procedure
C
with intermediate 3-amino-1-(3,5-dimethyl-1-
phenyl-pyrazol-4-yl)pyrrolidin-2-one (60) (0.11 mmol) and 4-
bromophenyl isocyanate (1.1 equiv., 0.12 mmol); Crude com-
pound was purified by reverse phase column chromatography
(MeOH/water pH ¼ 10). Yield: 47 mg, 90%; Appearance: white
solid; LCMS: 2.094 min m/z 468, 470 ((M þ H)þ 1:1, 79/81Br),
(500 MHz, DMSO‑d6) d ppm 8.83e8.72 (m, 1 H), 7.41e7.34 (m, 4 H),
6.56e6.48 (m, 1 H), 4.30e4.22 (m, 1 H), 3.83 (d, J ¼ 9.74 Hz, 2 H),
3.76e3.65 (m, 1 H), 3.39e3.34 (m, 1 H), 3.33e3.30 (m, 1 H),
3.30e3.19 (m, 1 H), 2.95e2.74 (m, 1 H), 2.41e2.32 (m, 1 H),
1.80e1.65 (m, 3 H), 1.59 (qd, J ¼ 12.03, 3.44 Hz, 1 H), 1.41e1.38 (m,
Purity ¼ 95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.81
9 H), 1.37 (br. s, 1 H); 13C NMR (126 MHz, DMSO‑d6)
d
ppm 172.0,
(s, 1 H), 7.48 (d, J ¼ 4.58 Hz, 4 H), 7.42e7.34 (m, 5 H), 6.63 (d,
13

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
J ¼ 6.87 Hz, 1 H), 4.46 (ddd, J ¼ 10.45, 8.74, 7.16 Hz, 1 H), 3.68e3.61
(m, 1 H), 3.47 (dt, J ¼ 8.59, 1.72 Hz, 1 H), 2.17 (s, 3 H), 2.09e2.01 (m,
pH ¼ 1). the compound was dissolved in MeOH and passed through
the Isolute SPE column (500 mg) to obtain desired product as free
base.; Yield: 28 mg, 51%; Appearance: white solid; LCMS: 1.92 min,
m/z 441.0, 443.0 ((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H
2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 173.0, 155.3, 145.3, 140.2,
140.0, 136.0, 131.9, 129.8, 127.8, 124.4, 120.2, 119.5, 113.1, 51.3, 47.1,
28.1, 11.9, 11.1; HRMS: Exact Mass: 467.0957; (M þ H)þ 468.1037;
Observed mass: 468.103; 1.5 ppm.
NMR (500 MHz, DMSO‑d6)
d ppm 8.88 (s, 1 H, NH) 8.77 (s, 1 H)
8.63e8.59 (m, 2 H) 8.22 (s, 1 H) 7.86e7.81 (m, 2 H) 7.40e7.34 (m,
4 H) 6.66 (d, J ¼ 7.45 Hz, 1 H) 4.51e4.43 (m, 1 H) 3.79e3.62 (m, 2 H),
2.53e2.49 (m, 1 H) 2.13e2.02 (m, 1 H); 13C NMR (126 MHz,
4.2.3.10. 1-(4-Bromophenyl)-3-[2-oxo-1-(1-pyrimidin-2-yl-4-
piperidyl)pyrrolidin-3-yl]urea (22). Prepared according to modified
procedure C with intermediate 3-amino-1-(1-pyrimidin-2-yl-4-
piperidyl)pyrrolidin-2-one (65) (0.14 mmol) and 4-bromophenyl
isocyanate (1.1 equiv., 0.155 mmol); Crude compound was puri-
DMSO‑d6)
d ppm 171.5, 155.2, 151.7, 145.8, 140.2, 134.2, 131.9, 126.8,
120.3, 117.0, 113.1, 112.3, 51.8, 44.7, 27.0; HRMS: Exact Mass:
440.0596; (M
1.59 ppm.
þ
H)þ: 441.0676; Observed Mass: 441.0669;
fied by preparative HPLC (19 ꢁ 100mm (5
mm) C-18 Waters Xbridge,
MeOH pH ¼ 10); Yield: 38 mg, 60%; Appearance: white solid; LCMS:
4.2.3.14. 1-(4-Bromophenyl)-3-[2-oxo-1-(1-phenylpyrazol-4-yl)pyr-
rolidin-3-yl]urea (33). Prepared according to modified procedure C
with intermediate 3-amino-1-(1-phenylpyrazol-4-yl)pyrrolidin-2-
one (63) (0.16 mmol) and 4-bromophenyl isocyanate (1.1 equiv.,
0.18 mmol); Crude compound was purified by preparative HPLC
1.99 min, m/z 459.0, 461.0 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 95%,
Method D; 1H NMR (500 MHz, DMSO‑d6)
d 8.80 (s, 1H), 8.35 (d,
J ¼ 4.58 Hz), 7.35e7.41 (m, 4H), 6.60 (t, J ¼ 4.58 Hz, 1 H), 6.53 (d,
J ¼ 6.87 Hz, 1 H), 4.74e4.82 (m, 2 H), 4.25 (ddd, J ¼ 6.87, 8.59,
10.31 Hz, 1 H), 4.02e4.11 (m, 1 H), 3.26e3.32 (m, 1 H), 3.15e3.22 (m,
1 H), 2.86e2.97 (m, 2 H), 2.32e2.40 (m, 1 H), 1.69e1.77 (m, 1 H),
(19 ꢁ 100mm (5 m) C-18 Waters Xbridge, MeOH pH ¼ 10).; Yield:
m
12 mg, 16%; Appearance: white solid; LCMS: 2.09 min, m/z 440.0,
1.48e1.69 (m, 4 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 171.6,
442.0 ((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
161.0, 158.2, 154.8, 139.7, 131.4, 119.3, 112.5, 109.9, 51.6, 49.5, 42.5,
28.3, 27.3; HRMS: Exact Mass: 458.1066; (M þ H)þ:459.1146;
Observed Mass: 459.1142; 0.87 ppm.
(500 MHz, DMSO‑d6) d 8.88 (s, 1 H), 8.60 (s, 1 H), 8.08 (s, 1 H), 7.80
(d, J ¼ 8.02 Hz, 2 H), 7.47 (dd, J ¼ 7.45, 8.59 Hz, 2 H), 7.39e7.33 (m,
4 H), 7.28 (t, J ¼ 7.39 Hz, 1 H), 6.65 (d, J ¼ 7.45 Hz, 1 H), 4.46 (ddd,
J ¼ 7.45, 8.88, 10.02 Hz, 1 H), 3.77e3.70 (m, 1 H), 3.69e3.63 (m, 1 H),
2.53e2.49 (m, 1 H), 2.10e2.00 (m, 1 H); 13C NMR (126 MHz,
4.2.3.11. 1-(4-Bromophenyl)-3-[1-(1,3-dimethylpyrazol-4-yl)-2-oxo-
pyrrolidin-3-yl]urea (30). Prepared according to modified proced-
ure C with intermediate 3-amino-1-(1,3-dimethylpyrazol-4-yl)
pyrrolidin-2-one (61) (0.26 mmol) and 4-bromophenyl isocyanate
(1.1 equiv., 0.28 mmol); Crude compound was purified by reverse
phase column chromatography (MeOH/water pH ¼ 10); Yield:
35 mg, 35%; Appearance: white solid; LCMS: 1.78 min m/z 392.1,
394.1 ((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
DMSO‑d6)
d ppm 170.7, 154.7, 139.7, 139.5, 131.7, 131.4, 129.6, 126.2,
125.4, 119.7, 118.0, 116.4, 112.6, 51.2, 44.2, 26.6; HRMS: Exact Mass:
439.0644; (M
2.05 ppm.
þ
H)þ:440.0724; Observed Mass: 440.0715;
4.2.4. Compound were synthesized according to general procedure
D: BOC deprotection
(500 MHz, DMSO‑d6)
d
ppm 8.92 (s, 1 H), 7.39 (s, 4 H), 7.36 (s, 1 H),
Starting material was dissolved in dichloromethane and hy-
drochloric acid (4 mol/l) in 1,4-dioxane (5e10 equiv.) was added.
Reaction mixture was stirred at room temperature for 2.5 h. A crude
product was dissolved in methanol. Methanol solution was passed
through the Isolute NH2 SPE cartridge and filtrate was concentrated
under reduced pressure to get desired product as a free base.
6.73 (d, J ¼ 6.87 Hz,1 H), 4.41 (ddd, J ¼ 10.45, 8.74, 7.16 Hz,1 H), 3.71
(s, 3 H), 3.65 (td, J ¼ 9.59, 6.59 Hz, 1 H), 3.45e3.55 (m, 1 H),
2.41e2.48 (m, 1 H), 2.14 (s, 3 H), 2.00 (dd, J ¼ 12.60, 10.31 Hz, 1 H);
13C NMR (126 MHz, DMSO‑d6)
d ppm 171.6, 154.8,139.8,133.5, 133.1,
131.4, 119.7, 119.0, 112.5, 50.9, 46.9, 36.6, 27.3, 9.4; HRMS: Exact
Mass: 391.0644; (M þ H)þ:392.0724; Observed Mass: 392.0719;
1.28 ppm.
4.2.4.1. 1-(4-Bromophenyl)-3-[2-oxo-1-(4-piperidyl)pyrrolidin-3-yl]
urea (13). Compound was prepared according to general procedure
D with intermediate: tert-butyl 4-[3-[(4-bromophenyl) carbamoy-
lamino] -2-oxo-pyrrolidin-1-yl] piperidine-1-carboxylate (10)
(0.11 mmol). A crude product was dissolved in methanol. Methanol
solution was passed through the Isolute NH2 SPE cartridge
(500 mg) and filtrate was concentrated under reduced pressure to
give the desired product as a free base. Yield: 40 mg, 92%;
Appearance: light grey solid: LCMS: 1.72 min m/z 381.1, 383.1
((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz,
4.2.3.12. 1-(4-Bromophenyl)-3-[1-(1-isopropylpyrazol-4-yl)-2-oxo-
pyrrolidin-3-yl]urea (31). Prepared according to modified proced-
ure C with intermediate 3-amino-1-(1-isopropylpyrazol-4-yl)pyr-
rolidin-2-one (62) (0.24 mmol) and 4-bromophenyl isocyanate (1.1
equiv., 0.264 mmol); Crude compound was purified by preparative
HPLC (19 ꢁ 100mm (5 m) C-18 Waters Xbridge, MeOH pH ¼ 10);
m
Yield: 38 mg, 39%; Appearance: white solid; LCMS: 1.92 min m/z
406.1, 408.0 ((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
(500 MHz, DMSO‑d6)
d
ppm 8.87 (s, 1 H), 8.01 (s, 1 H), 7.66 (s, 1 H),
DMSO‑d6) d ppm 8.86 (s, 1 H), 7.39e7.30 (m, 4 H), 6.54 (d,
7.45e7.33 (m, 4 H), 6.63 (d, J ¼ 7.45 Hz, 1 H), 4.49 (quin, J ¼ 6.59 Hz,
1 H), 4.42 (ddd, J ¼ 10.17, 8.74, 7.45 Hz, 1 H), 3.71e3.64 (m, 1 H),
3.63e3.55 (m, 1 H), 2.49e2.46 (m, 1 H), 2.01 (dd, J ¼ 12.32, 10.02 Hz,
J ¼ 6.87 Hz, 1 H), 4.20 (ddd, J ¼ 10.31, 8.59, 6.87 Hz, 1 H), 3.84 (ddd,
J ¼ 16.04, 12.03, 4.01 Hz, 1 H), 3.29e3.23 (m, 1 H), 3.18 (td, J ¼ 9.45,
6.87 Hz, 1 H), 3.07e3.00 (m, 2 H), 2.66e2.56 (m, 2 H), 2.38e2.31 (m,
1 H), 1.39 (d, J ¼ 6.30 Hz, 6 H); 13C NMR (126 MHz, DMSO‑d6)
d
ppm
1 H), 1.75e1.67 (m, 1 H), 1.67e1.58 (m, 1 H), 1.57e1.46 (m, 3 H); 13
C
170.5, 155.3, 140.2, 131.9, 128.8, 123.2, 120.2, 117.7, 113.1, 53.7, 51.8,
44.6, 27.3, 23.2; HRMS: Exact Mass: 405.0800; (M þ H)þ: 406.088;
Observed Mass: 406.0872; 1.97 ppm.
NMR (126 MHz, CD3OD) d ppm 174.9,157.8,140.5,133.0,122.1,115.9,
54.0, 51.3, 46.3, 41.5, 30.1, 28.5; HRMS: Exact Mass: 380.0848;
(M þ H)þ: 381.0928; Observed Mass: 381.0924; 1.05 ppm.
4.2.3.13. 1-(4-Bromophenyl)-3-[2-oxo-1-[1-(4-pyridyl)pyrazol-4-yl]
pyrrolidin-3-yl]urea (32). Prepared according to modified proced-
ure A with intermediate 3-amino-1-[1-(4-pyridyl)pyrazol-4-yl]
pyrrolidin-2-one (64) (0.24 mmol) and 4-bromophenyl isocyanate
(1.1 equiv., 0.264 mmol); Crude compound was purified by pre-
4.2.4.2. 1-(4-Bromophenyl)-3-[2-oxo-1-(4-piperidylmethyl)pyrroli-
din-3-yl]urea (12). Compound was prepared according to general
procedure D with intermediate: tert-butyl 4-[[3-[(4-bromophenyl)
carbamoylamino]-2-oxo-pyrrolidin-1-yl]methyl]
piperidine-1-
carboxylate (11) (0.303 mmol). A crude product was dissolved in
parative HPLC (19 ꢁ 100mm (5
mm) C-18 Waters Xbridge, MeOH
methanol. Methanol solution was passed through the Isolute NH2
14

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
SPE cartridge and filtrate was concentrated under reduced pressure
to give the desired product as a free base. Yield: 120 mg, 100%;
Appearance: light brown solid; LCMS: 1.75 min m/z 395.1, 397.1
((M þ H)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz,
DMSO‑d6)
d
ppm 172.2, 154.8, 139.7, 138.6, 131.4, 128.7, 128.1, 126.8,
119.6, 112.5, 62.3, 52.7, 51.1, 48.0, 43.9, 33.6, 29.5, 27.5; HRMS: Exact
mass: 484.1474; (M þ H)þ: 485.1554; Observed mass: 485.1545;
1.86 ppm.
CD3OD)
d ppm 7.38e7.35 (m, 2 H). 7.33e7.28 (m, 2 H), 4.35 (t,
J ¼ 9.74 Hz, 1 H), 3.47e3.42 (m, 2 H), 3.28e3.21 (m, 4 H), 2.81
(td,J ¼ 10.30, 2.86 Hz, 2 H), 2.52e2.44 (m, 1 H), 2.09e2.00 (m, 1 H),
2.00e1.90 (m, 1 H), 1.90e1.76 (m, 2 H), 1.38e1.27 (m, 2 H); 13C NMR
4.2.5.2. 1-[1-(1-Benzyl-4-piperidyl)-2-oxo-pyrrolidin-3-yl]-3-(4-
bromophenyl)urea (16). Compound was prepared according to
general procedure P with intermediate: 1-(4-bromophenyl)-3-[2-
oxo-1-(4-piperidyl)pyrrolidin-3-yl]urea (13) (0.066 mmol) and
benzyl bromide (1.2 equiv., 0.9 mmol). Crude product was purified
(126 MHz, CD3OD)
d ppm 175.6, 157.5, 140.3, 132.8, 122.0, 115.8,
53.6, 50.2, 46.2, 45.7, 34.2, 29.2, 27.9; HRMS: Exact Mass: 394.1004;
(M þ H)þ: 395.1084; Observed Mass: 395.1081; 0.76 ppm.
by prep. HPLC (19 ꢁ 100mm (5
mm) C-18 Waters Xbridge, MeOH
pH ¼ 10). Yield: 10 mg, 30%; Appearance: white solid; LCMS:
4.2.4.3. 1-(4-Bromophenyl)-3-(2-oxo-1-pyrrolidin-3-yl-pyrrolidin-3-
yl)urea (68). Compound was prepared according to general pro-
cedure D with intermediate tert-butyl 3-[3-[(4-bromophenyl)car-
bamoylamino]-2-oxo-pyrrolidin-1-yl] pyrrolidine-1-carboxylate
(66) (0.16 mmol); A crude product was dissolved in methanol.
Methanol solution was passed through the Isolute NH2 SPE car-
tridge (500 mg) and filtrate was concentrated under reduced
pressure to give the desired product as a free base. Yield: 59 mg,
100%; Appearance: light brown solid; LCMS: 1.69 min m/z 367.1,
369.1 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 90%, Method D; 1H NMR
2.184 min m/z 471.1, 473.1 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 95%,
Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.75 (s, 1 H),
7.36e7.20 (m, 9H), 6.48 (d, J ¼ 6.87 Hz, 1 H), 4.19 (ddd, J ¼ 5.16, 6.87,
10.31 Hz, 1 H), 3.70 (tt, J ¼ 4.08, 11.96 Hz, 1 H), 3.42 (s, 2 H), 3.29 (d,
J ¼ 9.16 Hz, 1H), 3.18 (dt, J ¼ 2.86, 6.30 Hz, 1 H), 3.14e3.13 (m, 1 H),
2.86e2.78 (m, 2 H), 2.37e2.30 (m, 1 H), 1.96 (dd, J ¼ 2.29, 11.46 Hz,
2 H), 1.74e1.60 (m, 3 H), 1.53e1.42 (m, 2 H); HRMS: Exact mass:
470.1317; (M þ H)þ: 471.1397; Observed mass: 471.1395; 0.43 ppm.
(500 MHz, CD3OD)
d
ppm 7.37e7.33 (m, 2 H), 7.31e7.26 (m, 2 H),
4.2.5.3. 1-[1-(1-Benzylpyrrolidin-3-yl)-2-oxo-pyrrolidin-3-yl]-3-(4-
bromophenyl)urea (17). Compound was prepared according to
general procedure P with intermediate 1-(4-bromophenyl)-3-(2-
oxo-1-pyrrolidin-3-yl-pyrrolidin-3-yl)urea (68) (0.08 mmol) and
benzyl bromide (1.2 equiv., 1 mmol). Crude product was purified by
4.55e4.46 (m, 1 H), 4.34 (t, J ¼ 9.45 Hz, 1 H), 3.48e3.36 (m, 2 H),
3.14e3.03 (m, 2 H), 2.98e2.89 (m, 2 H), 2.51e2.44 (m, 1 H),
2.13e2.00 (m, 1 H), 1.98e1.86 (m, 2 H); 13C NMR (126 MHz, CD3OD)
d
ppm 175.3, 157.6, 140.3, 132.8, 121.9, 115.7, 54.7, 53.7, 49.9, 47.2,
42.7, 30.3, 28.2.
prep. HPLC (19 ꢁ 100mm (5
mm) C-18 Waters Xbridge, MeOH
pH ¼ 10). Yield: 16 mg, 40%; Appearance: white solid; LCMS:
4.2.4.4. 1-(4-Bromophenyl)-3-(2-oxo-1-pyrrolidin-3-yl-pyrrolidin-3-
yl)urea (69). Compound was prepared according to general pro-
cedure D with intermediate tert-butyl 3-[3-[(4-bromophenyl)car-
bamoylamino]-2-oxo-pyrrolidin-1-yl] pyrrolidine-1-carboxylate
(67) (0.1 mmol). A crude product was dissolved in methanol.
Methanol solution was passed through the Isolute NH2 SPE car-
tridge (500 mg) and tr filtrate was concentrated under reduced
pressure to afford the desired product as a free base. Yield: 29 mg,
70%; Appearance: white solid; LCMS: 1.68 min m/z 367.0, 369.0
((M þ H)þ 1:1, 79/81Br), Purity ¼ 85%, Method D; 1H NMR (500 MHz,
2.194 min m/z 457, 459 ((M þ H)þ 1:1, 79/81Br), Purity ¼ 95%,
Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.95e8.82 (br.s,
1 H), 7.55e7.43 (m, 5 H), 7.41e7.34 (m, 5 H), 6.70e6.56 (br.s, 1 H),
4.80e4.60 (m, 1 H), 4.52e4.36 (m, 1 H), 4.36e4.16 (m, 2 H),
3.53e3.39 (m, 2 H), 3.30e3.16 (m, 2 H), 2.45e2.25 (m, 2 H),
2.22e1.95 (m, 2 H), 1.94e1.63 (m, 2 H); broad peaks due to presence
of rotamers. HRMS: Exact mass: 456.1161; (M þ H)þ: 457.1241;
Observed mass: 457.1238; 0.66 ppm.
4.2.5.4. 1-[1-(1-Benzylpyrrolidin-3-yl)-2-oxo-pyrrolidin-3-yl]-3-(4-
bromophenyl)urea (18). Compound was prepared according to
general procedure G with intermediate 1-(4-bromophenyl)-3-(2-
oxo-1-pyrrolidin-3-yl-pyrrolidin-3-yl)urea (69); Crude product
CD3OD)
d ppm 7.36e7.26 (m, 4 H), 4.56e4.46 (m, 1 H), 4.35 (dd,
J ¼ 10.31, 8.59 Hz, 1 H), 3.47e3.35 (m, 2 H), 3.11e2.97 (m, 2 H),
2.95e2.82 (m, 2 H), 2.51e2.43 (m, 1 H), 2.09e1.96 (m, 1 H),
1.95e1.83 (m, 2 H); 13C NMR (126 MHz, CD3OD)
d ppm 175.0, 157.4,
was purified by prep. HPLC (19 ꢁ 100mm (5
mm) C-18 Waters
140.2, 132.7, 121.8, 115. 6, 54.3 53.5, 49.9, 47.1, 42.1, 29.8, 28.0.
Xbridge, MeOH pH ¼ 10); Yield: 12 mg, 30%; Appearance: white
solid; LCMS: 2.198 min, m/z 457, 459 ((M þ H)þ 1:1, 79/81Br),
4.2.5. General procedure E: N-alkylation
Purity ¼ 95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm
1-(4-bromophenyl)-3-[2-oxo-1-(4-piperidylmethyl)pyrrolidin-
3-yl]urea (1 eq.) was dissolved in N,N-dimethylformamide (1 mL).
To the reaction, N,N-diisopropylethylamine (0.1 mmol, 2 eq.)
following benzyl bromide (1.1 eq.) were added via syringe. The
reaction was stirred overnight. The crude product was purified by
preparative HPLC under basic conditions to give final compound.
8.97e8.80 (br.s, 1 H), 7.40e7.29 (m, 9 H), 6.66e6.51 (br.s, 1 H),
4.65e4.40 (m, 1 H), 4.49e4.30 (m, 1 H), 4.29e4.15 (m, 2 H),
3.48e3.34 (m, 2 H), 3.27e3.12 (m, 2 H), 2.39e2.26 (m, 2 H),
2.14e1.94 (m, 2 H), 1.85e1.68 (m, 2 H); broad peaks due to presence
of rotamers; HRMS: Exact mass: 456.1161; (M þ H)þ: 457.1241;
Observed mass: 457.1238; 0.66 ppm.
4.2.5.1. 1-[1-[(1-Benzyl-4-piperidyl)methyl]-2-oxo-pyrrolidin-3-yl]-
3-(4-bromophenyl)urea (15). Compound was prepared according to
general procedure P with intermediate 1-(4-bromophenyl)-3-[2-
oxo-1-(4-piperidylmethyl)pyrrolidin-3-yl]urea (12) and benzyl
bromide (1.2 equiv., 1 mmol). Yield: 10 mg, 30%; Appearance: white
solid; LCMS: 2.18 min m/z 485.1, 487.1, ((M þ H)þ 1:1, 79/81Br),
4.3. General biological procedures
4.3.1. Culture of CHOeK1 FPRL1 cells
PathHunter® CHOeK1 FPRL1 cells (DISCOVERx) were grown in
the F-12 nutrient mixture (HAM) (21765e037, Gibco®) supple-
mented with 10% Fetal Bovine Serum (A3840401, Thermo Fisher
Purity ¼ 95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.74
(s, 1 H), 7.37e7.30 (m, 4 H), 7.30e7.22 (m, 4 H), 7.22e7.18 (m, 1 H),
6.47 (d, J ¼ 6.87 Hz,1 H), 4.22 (ddd, J ¼ 10.31, 8.59, 6.87 Hz,1 H), 3.41
(s, 2 H), 3.28e3.21 (m, 2 H), 3.05 (qd, J ¼ 13.65, 7.16 Hz, 2 H), 2.73 (d,
J ¼ 10.88 Hz, 2 H), 2.37e2.30 (m, 1 H), 1.90e1.81 (m, 2 H), 1.81e1.70
(m, 1 H), 1.59e1.46 (m, 3 H), 1.15e1.03 (m, 2 H); 13C NMR (126 MHz,
Sci), Hygromycin B 800
mg/mL (10687e010, Thermo Fisher Sci),
Geneticin 300
m
g/mL (10131e027, Thermo Fisher Sci). The cells
were incubated at 37 ꢀC and passaged every 2e3 days, based on the
doubling time of the cell line. The Accutase ® (AT104-500) was
used as the cell detachment solution.
15

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
4.3.2.
To identify novel FPR2 agonists PathHunter® CHOeK1 FPRL1
cells (DISCOVERx) expressing complementing fragments of the
b
-Arrestin path Hunter® assay detection protocol
(0.5 ꢁ 106 cells/mL) and 30
mL of the agonists (final dilution 1:5)
were added. Cells were incubated 30 min 37 ꢀC with gentle
b
-
continuous mixing. From the pre-incubation plate, 125 mL of the
galactosidase (
b
-gal) enzyme were used. One day before the assay,
reaction mixture was transferred into the pre-coated assay plate,
followed by an incubation of 20 min at 37 ꢀC. Non-adherent neu-
trophils were removed, and the fixation step was performed with
PFA 4%. Cells were stained with DAPI for 10 min and then washed
the cells were diluted to a concentration of 0.5 x 106/mL in DIS-
COVERx plating media (Assay CompleteTM Cell Plating 2 Reagent;
93e0563R2A). The cells were plated out 10 mL per well of 384 well
plate (5000 cells per well) and incubated 1 h at room temperature
then overnight at 37 ꢀC. 10 mM stock solutions of compounds were
serially diluted in assay buffer (PBS using a half log dilution protocol
on a Biomek instrument (automated liquid handler). The com-
pound addition to the assay plate was performed Biomek instru-
ment. Cells were stimulated with compounds or for high controls
with W-peptide (a control ligand sequence WKYMVm;
concentration ¼ 100 nM) for 150 min at 37 ꢀC. Detection reagent
(19 parts Assay buffer, 5 parts Emerald II and 1 part Galacton Star;
gently with 200 mL PBS. Four pictures per well were taken with a
Leica microscope (DMI8, software: LAS X. V.3.4.2.18368, autofocus,
exposure 51e57 ms, gain: 1.5, DAPI). The image files were pro-
cessed using an ImageJ macro. Raw data were entered into
GraphPad Prism 7.01 analysis software for data processing and
visualisation. Each compound was tested in technical triplicates.
4.3.6. Cell culture and ERK1/2 assay
HEK293A cells and FPR1 or FPR2 stably transfected HEK293A
cells were maintained in DMEM containing 10% FCS and 1% peni-
cillin/streptomycin and kept at 37 ꢀC with 5% CO2. Cells were
seeded in 96-wells plates at 2 ꢁ 104 cells/well and allow to grow for
48 h.
Pathhunter detection kit (93e0001) DISCOVERx) was added 12 mL
per well and incubated for 60 min in the dark at room temperature.
Receptor activation was determined by chemiluminescence using a
BMG Pherastar plate reader. Raw data were entered into GraphPad
Prism 7.01 analysis software for data processing and visualisation.
4.3.7. ERK phosphorylation assay
4.3.3. Calcium mobilization assay protocol
Cells were serum-starved for 3 h to stabilize basal levels of
signalling and reduce background levels of p-ERK. Drug solutions
were prepared in 0% FCS DMEM. Compounds were tested using
The increase in cytosolic Ca2þ was measured by the FLIPR®
Tetra® High-Throughput Cellular Screening System. In the calcium
mobilization assay FLIPR calcium 6 dye (Molecular Devices) was
used. One day before the assay, the cells were diluted to a con-
centration of 0.5 ꢁ 106/mL in F-12 nutrient mixture (HAM)
(21765e037, Gibco®) containing 10% Fetal Bovine Serum
30
mM as top concentration followed by 1/3 serial dilutions to
generate concentration response curves. Cells were stimulated for
5 min with drugs and lysed immediately with ice-cold extraction
buffer supplied in the ERK1/2 (pT202/Y204) SimpleStep ELISA Kit
(Abcam ab176640), used for p-ERK quantification according to
manufacturer instructions. Optical Density (OD) measurements
were converted to calculate relative p-ERK quantity and results
were expressed as increment over basal levels for each receptor
subtype.
(A3840401, Thermo Fisher Sci) and plated out 40 mL per well of 384
well plate (20,000 cells per well) then incubated 1 h at RT and then
overnight at 37 ꢀC. On the assay day, media was removed from the
cell plate and 20 mLs of dye per well were added. The cell plate was
incubated for 2 h at 37 ꢀC and later for 30 min at RT in the dark.
10 mM stock solutions of compounds were serially diluted in assay
buffer (HBSS 20 mM HEPES) using a half log dilution protocol on a
Biomek instrument (automated liquid handler). In both assays W-
4.3.8. Statistical data analysis
GraphPad Prism 7.01 (GraphPad Software, San Diego, CA, USA)
was used for statistical analysis of the data. For comparison of
treatment groups with the control group and P- value determina-
tion, the one-way ANOVA analysis followed by Dunnett's multiple
comparison test was used. All data were shown as means SEM.
Differences were considered statistically significant for a P < 0.05. P
value; ***P < 0.001; **P < 0.01; *P < 0.05, ns e nonsignificant.
peptide was used as a control compound (100 nM). The 5
mL
compound addition was performed by the FLIPR® Tetra® High-
Throughput Cellular Screening System. The calcium mobilization
was measured for 240s (Read Mode: Exc. Wavelength 470e495 nm,
Em. Wavelength 515e575 nm, Gain: 2000). Data are processed
using ScreenWorks 4.0.0.30. Raw data were entered into GraphPad
Prism 7.01 analysis software for data processing and visualisation.
4.3.9. PK studies
4.3.4. Isolation of human neutrophils from venous blood
The pharmacokinetic studies were performed in male C57BL6
mice with intraperitoneal administration at 10 mg/kg. In total 12
mice were used: 9 received the compound and 3 mice for blank
plasma collection for bioanalysis. Compound concentration in
plasma was measured at eight time points over 24 h. Each mouse
used in the study was bled thrice with sufficient time gap. Mice
Blood was collected from healthy donors in accordance with a
protocol approved by the Ludwig Maximilian University of Munich.
Neutrophils were isolated from the blood using Polymorphprep
(Axi-Shield) following the manufacturer's instructions. Isolated
neutrophils were washed and resuspended in HBSS buffer con-
taining 20 mM HEPES, 0.25% BSA, Ca2þ pH 7.4 (0.5 ꢁ 106 cells/mL)
and used on the day of the experiment.
(n
¼
12) were treated with either the vehicle (DMSO
(10%) þ PEG400 (35%) þ PG (25%) þ water (30%)) or a clear solution
of Compound 7 (10 mg/kg in vehicle).
4.3.5. Human neutrophil static adhesion assay
The static adhesion assay was performed with the aim of
examining the small molecule agonists and their anti-
inflammatory and proresolving properties. The day before the
4.3.10. Cell health assay
In order to measure a variety of parameters which leads to cell
viability, cell death, cytotoxicity or cell proliferation, the cell health
assay was performed. A human hepatocyte carcinoma cells (HepG2
cells EACC origin) were cultured a week before assay in Eagle's
Minimum Essential Medium (EMEM) inc. NEAA (non-essential
amino acids) with 10% Fetal Bovine Serum and 2 mM GlutaMAX.
During cell plating 1% Penicillin/Streptomyocin was added to me-
assay, 96-well flat-bottomed plates were coated with 50
adhesion molecules, both with a final concentration of 1
m
L of cell
mg/mL: P-
selectin (13025-H02H-100; SinoBiological) and ICAM- 1 (10346-
H03H-100; SinoBiological). The coated plates were kept at 40 ꢀC
overnight. The excess of solution was removed with pipette and the
unspecific binding was blocked with BSA 2% for 1 h at room tem-
dia. 2000 HepG2 cells per well were plated in 25
mL media of a 384
perature. Per well of the pre-incubation plate, 120 mL of neutrophils
well plate and incubated at 37 ꢀC overnight. The compounds were
16

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
added from the serial dilution plates using Biomek robot (auto-
mated liquid handler). The assay plates were incubated at 37 ꢀC for
72 h. Four live staining dyes were added on Biomek robot and plates
were incubate 1 h at 37 ꢀC. Cell measurements were imaged on the
InCELL Analyzer 2000 using the following four wavelength chan-
nels for each of the four dyes: Hoechst (nuclei) - DAPI channel;
Fluo-4 AM (cellular calcium) - Fluorescein isothiocyanate (FITC)
channel; Tetramethylrhodamine, methyl ester (TMRM) (mito-
chondria) - Texas Red channel; TOTO-3 (dead nuclei) e Cy® 5
channel. Raw data were processed and visualized in Genedata
Screener analysis software. The cell measurements were normal-
ised as a percentage in contradiction of low control. Any cell
parameter that deviates from 100% baseline shows a variation in
cell phenotype and cytotoxicity after drug treatment.
MS/MS (Agilent Technologies G6410 series, triple quadrupole
with MM-ESI ion source) using optimised multiple reaction
monitoring scans and a standard column gradient on an Acquity
UPLC BEH C8 1.7 mm column (mobile phase: acetonitrile and water
with 0.05% acetic). The % turnover was obtained by calculating the
percentage difference of the peak areas, normalised to the internal
standard, at t ¼ 0 and t ¼ 30. Verapamil was used as the standard
compound.
Contributors
TC supervised the findings of this work and conceived the
studies. OS conceived and designed the neutrophil studies. AM was
involved in planning and supervised the work. MP is the Coordi-
nator of the Marie Skłodowska-Curie ITN EVOluTION and was
involved in supervision the work. MM performed in silico studies,
designed and synthesized the compounds, performed and analysed
the in vitro assays. GN supervised the in-silico studies. MM per-
formed and analysed the human neutrophil static assays and
contributed to all the stages of the experimental work. AOG and
MM designed the human neutrophil static adhesion assay. SM
aided in interpreting the results. SM and BF performed in vivo
studies. JGM performed and analysed the ERK phosphorylation
assay. MM and TC prepared the first draft of the manuscript. All
authors have given approval to the final version of the manuscript.
4.3.11. Kinetic solubility measurements
The kinetic solubility was measured by diluting a 4
DMSO stock solution of the compound into PBS buffer pH 7.4 in a
filtration plate at a target concentration of 200 M giving a final
mL of 10 mM
m
solution composition of 98:2 PBS:DMSO. For each compound,
measurements were performed in triplicates with two standard
compounds (Verapamil and Ketoconazole) on the same plate. The
filtration plate was agitated at 500 rpm for 90 min and then filtered
into V-bottom plate. The filtrate was sampled and diluted further
with a DMSO:PBS mixture in a flat bottomed UV plate resulting
with a final solution composition of PBS:DMSO 80:20. A serial
dilution for each compound was then performed in flat bottomed
Declaration of competing interest
plates in PBS:DMSO 80:20 with concentrations 200
mM, 100 mM,
50 M, 25 M, 12.5 M and 6.75 M. The UV absorbance was read
m
m
m
m
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: MP is shareholder of ResoTher Pharma which is devel-
oping FPR2 peptide-agonists for clinical application. MM, TC, OS,
AM, GN, AOG, BF, JGM and SM declare that they have no known
competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.
across 230e400 nm at 1 nm intervals using a TECAN Safire II plate
reader and a suitable UV wavelength chosen around the UV
maximum of each compound. This was used to calculate the con-
centration in the filtrate for each compound, and hence amount
remaining in solution after 90 min which is reported as the kinetic
solubility.
4.3.12. LogD measurements
Acknowledgment
LogD measurements were conducted using the shake flask
method. Compound was diluted from 10 mM DMSO stock solution
into an Eppendorf containing equal amounts of octanol and phos-
The authors would like to acknowledge Dr David Tickle, Teresa
Sementa and Sadhia Khan for performing the in vitro ADME testing.
We are grateful to Anthony Weatherhead for carrying out the HRMS
measurements, to Nathalie Bouloc for her assistance in preparative
HPLC purification of the compounds and to Michelle Newman for
performing the cell health assay. We would also like to thank Dr
Kris Birchall for his assistance with the in silico studies.
phate buffered saline (PBS) to give a final concentration of 100 mM.
The tubes were shaken for 12 h, centrifuged at 10000 rpm for
10 min and samples taken from the octanol and PBS layers. The
samples from both layers were analysed in triplicate by LC-MS/MS
(Agilent Technologies G6410 series, triple quadrupole with MM-ESI
ion source) using optimised multiple reaction monitoring (MRM)
scans and a standard column gradient on an Acquity UPLC BEH C8
Abbreviations
1.7 mm column, running acetonitrile and water with 0.05% Acetic
Acid as the mobile phase. The ratios of areas of the peaks were used
to calculate the LogD in accordance with the equation:
LogD ¼ Log10(Area-TL/Area BL).
ACN
ADME
BOC
BSA
CLint
Acetonitrile
Absorption, Distribution, Metabolism, Excretion
tert-Butyloxycarbonyl
Bovine Serum Albumin
Intrinsic Clearance
4.3.13. Microsomal stability assay
The human and mouse liver microsomes (MLM) were obtained
from BD Biosciences. The compounds were pre-incubated at 37 ꢀC
for 5 min with the microsomes and the reaction was initiated by
adding an equal volume of NADPH generating solution (BD Bio-
sciences). The final compound concentration in the incubation is
CVDs
DAPI
DCC
DCM
DCE
Cardiovascular Diseases
40,6-Diamidino-2-phenylindole
N,N0-Dicyclohexylcarbodiimide
Dichloromethane
Dichloroethane
1
m
M, and the microsomal protein concentration is 0.2 mg/mL. A
DIPEA
DMAP
DMF
DMSO
EC50
N,N-Diisopropylethylamine
4-Dimethylaminopyridine
N,NeDimethylformamide
Dimethyl Sulfoxide
Half Maximal Effective Concentration
Efficacy
sample was taken at t ¼ 0 and quenched with two times volume of
ice-cold methanol containing an internal standard reference com-
pound (Carbamazepine). The reaction was agitated at 37 ꢀC for
30 min, when a further sample was taken and quenched in an
identical fashion. The samples were centrifuged at 10000 rpm for
10 min and the supernatant taken for analysis in triplicate by LC-
Emax
FPR2
Formyl Peptide Receptor 2
17

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
H.R. Brady, S.P. Colgan, J.L. Madara, Design of lipoxin A4 stable analogs that
block transmigration and adhesion of human neutrophils, Biochemistry 34
(44) (1995) 14609e14615.
GLASS database GPCR-Ligand Association database
GPCR
HBSS
HEPES
HLM
HPLC
HRMS
ICAM
Ks
G-Protein-Coupled Receptors
Hanks' Balanced Salt Solution
4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid
Human Liver Microsomes
High Performance Liquid Chromatography
High Resolution Mass Spectrometry
Intercellular Adhesion Molecule
Kinetic Solubility
[11] M.A. Sugimoto, J.P. Vago, M.M. Teixeira, L.P. Sousa, Annexin A1 and the res-
olution of inflammation: modulation of neutrophil recruitment, apoptosis,
and clearance, J Immunol Res 2016 (2016) 8239258.
[12] J.F. Maddox, M. Hachicha, T. Takano, N.A. Petasis, V.V. Fokin, C.N. Serhan,
Lipoxin A4 stable analogs are potent mimetics that stimulate human mono-
cytes and THP-1 cells via a G-protein-linked lipoxin A4 receptor, J. Biol. Chem.
272 (11) (1997) 6972e6978.
[13] M. Perretti, C. Godson, Formyl peptide receptor type 2 agonists to kick-start
resolution pharmacology, Br. J. Pharmacol. 177 (20) (2020) 4595e4600.
[14] M. Perretti, X. Leroy, E.J. Bland, T. Montero-Melendez, Resolution pharma-
cology: opportunities for therapeutic innovation in inflammation, Trends
Pharmacol. Sci. 36 (11) (2015) 737e755.
[15] M.A. Sugimoto, L.P. Sousa, V. Pinho, M. Perretti, M.M. Teixeira, Resolution of
inflammation: what controls its onset? Front. Immunol. 7 (2016).
[16] Y. Zhuang, H. Liu, X. Edward Zhou, R. Kumar Verma, P.W. de Waal, W. Jang,
T.H. Xu, L. Wang, X. Meng, G. Zhao, Y. Kang, K. Melcher, H. Fan, N.A. Lambert,
H. Eric Xu, C. Zhang, Structure of formylpeptide receptor 2-Gi complex reveals
insights into ligand recognition and signaling, Nat. Commun. 11 (1) (2020)
885.
LC-MS
LogD
MLM
NA
Liquid Chromatography-Mass Spectroscopy
Logarithm of Distribution-Coefficient
Mouse Liver Microsomes
No Activity
NMR
ON
Nuclear Magnetic Resonance
Overnight
PE
Petroleum Ether
PFA
Paraformaldehyde
[17] H.Q. He, R.D. Ye, The formyl peptide receptors: diversity of ligands and
mechanism for recognition, Molecules 22 (3) (2017).
[18] S. Bozinovski, D. Anthony, R. Vlahos, Targeting pro-resolution pathways to
PK
Pharmacokinetics
RoI
Resolution of Inflammation
Receiver Operating Characteristic
Serum Amyloid A
Structure-Activity Relationship
Triethylamine
combat chronic inflammation in COPD, J. Thorac. Dis.
1548e1556.
6 (11) (2014)
ROC
SAA
SAR
[19] Y. Asahina, N.R. Wurtz, K. Arakawa, N. Carson, K. Fujii, K. Fukuchi, R. Garcia,
M.Y. Hsu, J. Ishiyama, B. Ito, E. Kick, J. Lupisella, S. Matsushima, K. Ohata,
J. Ostrowski, Y. Saito, K. Tsuda, F. Villarreal, H. Yamada, T. Yamaoka, R. Wexler,
D. Gordon, Y. Kohno, Discovery of BMS-986235/LAR-1219: a potent formyl
peptide receptor 2 (FPR2) selective agonist for the prevention of heart failure,
J. Med. Chem. 63 (17) (2020) 9003e9019.
[20] A.K. Stalder, D. Lott, D.S. Strasser, H.G. Cruz, A. Krause, P.M. Groenen,
J. Dingemanse, Biomarker-guided clinical development of the first-in-class
anti-inflammatory FPR2/ALX agonist ACT-389949, Br. J. Clin. Pharmacol. 83
(3) (2017) 476e486.
TEA
TM
Template Model
T3P
THF
Propylphosphonic Anhydride
Tetrahydrofuran
VCAM
Vascular Cell Adhesion Molecule
[21] O. Corminboeuf, X. Leroy, FPR2/ALXR agonists and the resolution of inflam-
mation, J. Med. Chem. 58 (2) (2015) 537e559.
Appendix A. Supplementary data
[22] M. Maciuszek, A. Cacace, E. Brennan, C. Godson, T.M. Chapman, Recent ad-
vances in the design and development of formyl peptide receptor 2 (FPR2/
ALX) agonists as pro-resolving agents with diverse therapeutic potential, Eur.
J. Med. Chem. 213 (2021) 113167.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113805.
ꢁ
[23] A.J. Kooistra, S. Mordalski, G. Pandy-Szekeres, M. Esguerra, A. Mamyrbekov,
Funding
}
C. Munk, G.M. Keseru, D.E. Gloriam, GPCRdb in 2021: integrating GPCR
sequence, structure and function, Nucleic Acids Res. 49 (D1) (2021)
D335eD343.
This work was supported by the European Union's Horizon 2020
research and innovation program under the Marie Sklodowska-
Curie grant agreement No. 675111.
ꢁ
[24] G. Pandy-Szekeres, C. Munk, T.M. Tsonkov, S. Mordalski, K. Harpsøe,
A.S. Hauser, A.J. Bojarski, D.E. Gloriam, GPCRdb, Adding GPCR structure
models and ligands, Nucleic Acids Res., 2018 46 (D1) (2018) D440eD446.
[25] T. Wang, Z. Li, M. Cvijic, C. Krause, L. Zhang, C. Sum, Measurement of
b-
Arrestin Recruitment for GPCR Targets, 2019.
References
[26] P.Y. Jean-Charles, S. Kaur, S.K. Shenoy, G, Protein-coupled receptor signaling
through -arrestin-dependent mechanisms, J. Cardiovasc. Pharmacol. 70 (3)
b
(2017) 142e158.
[1] M.H. Petri, A. Laguna-Fernandez, M. Gonzalez-Diez, G. Paulsson-Berne,
G.K. Hansson, M. Back, The role of the FPR2/ALX receptor in atherosclerosis
development and plaque stability, Cardiovasc. Res. 105 (1) (2015) 65e74.
[2] N.E. Kieran, P.P. Doran, S.B. Connolly, M.C. Greenan, D.F. Higgins, M. Leonard,
C. Godson, C.T. Taylor, A. Henger, M. Kretzler, M.J. Burne, H. Rabb, H.R. Brady,
Modification of the transcriptomic response to renal ischemia/reperfusion
injury by lipoxin analog, Kidney Int. 64 (2) (2003) 480e492.
[27] M. Qiang, Y. Lingyan, L. Hongxia, S. Ying, Z. Naiming, An overview of Ca2þ
mobilization assays in GPCR drug discovery, Expet Opin. Drug Discov. 12 (5)
(2017) 511e523.
[28] B.K. Kobilka, G, Protein coupled receptor structure and activation, Biochim.
Biophys. Acta 1768 (4) (2007) 794e807.
[29] E.J. Whalen, S. Rajagopal, R.J. Lefkowitz, Therapeutic potential of b-arrestin-
and G protein-biased agonists, Trends Mol. Med. 17 (3) (2011) 126e139.
[30] J.S. Smith, T.F. Pack, A. Inoue, C. Lee, K. Zheng, I. Choi, D.S. Eiger, A. Warman,
X. Xiong, Z. Ma, G. Viswanathan, I.M. Levitan, L.K. Rochelle, D.P. Staus,
J.C. Snyder, A.W. Kahsai, M.G. Caron, S. Rajagopal, Noncanonical scaffolding of
[3] G. Leoni, O. Soehnlein, (Re) solving repair after myocardial infarction, Front.
Pharmacol. 9 (2018), 1342-1342.
[4] R.A. García, B.R. Ito, J.A. Lupisella, N.A. Carson, M.-Y. Hsu, G. Fernando,
M. Heroux, M. Bouvier, E. Dierks, E.K. Kick, D.A. Gordon, J. Chen, G. Mintier,
M. Carrier, S. St-Onge, H. Shah, J. Towne, M.S. Bucardo, X. Ma, C.S. Ryan,
N.R. Wurtz, J. Ostrowski, F.J. Villarreal, Preservation of post-infarction cardiac
structure and function via long-term oral formyl peptide receptor agonist
treatment, J Am Coll Cardiol Basic Trans Science 349 (2019).
[5] B.E. Sansbury, M. Spite, Resolution of acute inflammation and the role of
resolvins in immunity, thrombosis, and vascular biology, Circ. Res. 119 (1)
(2016) 113e130.
G
a
i and
b-arrestin by G protein-coupled receptors, Science 371 (6534) (2021),
eaay1833.
€
[31] C.A. Raabe, J. Groper, U. Rescher, Biased perspectives on formyl peptide re-
ceptors, Biochim. Biophys. Acta Mol. Cell Res. 1866 (2) (2019) 305e316.
[32] S. Gallo, A. Vitacolonna, A. Bonzano, P. Comoglio, T. Crepaldi, ERK: a key player
in the pathophysiology of cardiac hypertrophy, Int. J. Mol. Sci. 20 (9) (2019)
2164.
[33] C.X. Qin, L.T. May, P.M. Sexton, A.J. DeBono, J.B. Baell, A. Christopoulos,
R.H. Ritchie, Correspondence: reply to ‘Compound 17b and formyl peptide
receptor biased agonism in relation to cardioprotective effects in ischaemia-
reperfusion injury’, Nat. Commun. 9 (1) (2018) 530.
[6] M. Arita, T. Ohira, Y.P. Sun, S. Elangovan, N. Chiang, C.N. Serhan, Resolvin E1
selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to
regulate inflammation, J. Immunol. 178 (6) (2007) 3912e3917.
[7] C.B. Clish, K. Gronert, C.N. Serhan, Local and systemic delivery of an aspirin-
triggered lipoxin stable analog inhibits neutrophil trafficking, Ann. N. Y.
Acad. Sci. 905 (2000) 274e278.
[8] C.B. Clish, J.A. O'Brien, K. Gronert, G.L. Stahl, N.A. Petasis, C.N. Serhan, Local and
systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil
recruitment in vivo, Proc. Natl. Acad. Sci. U. S. A. 96 (14) (1999) 8247e8252.
[9] V. Kain, J.K. Jadapalli, B. Tourki, G.V. Halade, Inhibition of FPR2 impaired leu-
kocytes recruitment and elicited non-resolving inflammation in acute heart
failure, Pharmacol. Res. 146 (2019) 104295.
[34] C.X. Qin, L.T. May, R. Li, N. Cao, S. Rosli, M. Deo, A.E. Alexander, D. Horlock,
J. Bourke, Y.H. Yang, A.G. Stewart, D.M. Kaye, X.-D. Du, P.M. Sexton,
A. Christopoulos, X.-M. Gao, R.H. Ritchie, Small-molecule-biased formyl pep-
tide receptor agonist compound 17b protects against myocardial ischaemia-
reperfusion injury in mice, Nat. Commun. 8 (2017) 14232.
€
[35] W.K.B. Chan, H. Zhang, J. Yang, J.R. Brender, J. Hur, A. Ozgür, Y. Zhang, GLASS: a
comprehensive database for experimentally validated GPCR-ligand associa-
tions, Bioinformatics 31 (18) (2015) 3035e3042.
[36] M. Maciuszek, A. Ortega-Gomez, S.L. Maas, M. Perretti, A. Merritt,
[10] C.N. Serhan, J.F. Maddox, N.A. Petasis, I. Akritopoulou-Zanze, A. Papayianni,
18

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 226 (2021) 113805
O. Soehnlein, T.M. Chapman, Synthesis and evaluation of novel cyclopentane
urea FPR2 agonists and their potential application in the treatment of car-
diovascular inflammation, Eur. J. Med. Chem. 214 (2021) 113194.
2 reduces leukocyte-endothelial interactions in a murine model of stroke,
FASEB J 29 (5) (2015) 2161e2171.
[41] R.A. Teixeira, K.K. Mimura, L.P. Araujo, K.V. Greco, S.M. Oliani, The essential
role of annexin A1 mimetic peptide in the skin allograft survival, J Tissue Eng
Regen Med 10 (2) (2016) E44eE53.
[37] S.L. Dixon, A.M. Smondyrev, E.H. Knoll, S.N. Rao, D.E. Shaw, R.A. Friesner,
PHASE:
a new engine for pharmacophore perception, 3D QSAR model
development, and 3D database screening: 1. Methodology and preliminary
results, J. Comput. Aided Mol. Des. 20 (10) (2006) 647e671.
[42] A. Jovanovic, Cardioprotective signalling: past, present and future, Eur. J.
Pharmacol. 833 (2018) 314e319.
[38] J. Ansari, E.Y. Senchenkova, S.A. Vital, Z. Al-Yafeai, G. Kaur, E.M. Sparkenbaugh,
A.W. Orr, R. Pawlinski, R.P. Hebbel, D.N. Granger, P. Kubes, F.N.E. Gavins,
Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, pro-
moting thromboinflammation resolution in sickle cell disease, Blood 137 (11)
(2021) 1538e1549.
[39] F.N. Gavins, M.J. Hickey, Annexin A1 and the regulation of innate and adaptive
immunity, Front. Immunol. 3 (2012) 354.
[40] H.K. Smith, C.D. Gil, S.M. Oliani, F.N. Gavins, Targeting formyl peptide receptor
[43] M. Ibrahim-Ouali, M.-E. Sinibaldi, Y. Troin, D. Gardette, J.-C. Gramain, Syn-
thesis of b-enaminoesters and lactams by michael addition of N-benzylaniline
to new allenic esters and lactams, Synth. Commun. 27 (11) (1997)
1827e1848.
[44] X. Lin, W. Chen, Z. Qiu, L. Guo, W. Zhu, W. Li, Z. Wang, W. Zhang, Z. Zhang,
Y. Rong, M. Zhang, L. Yu, S. Zhong, R. Zhao, X. Wu, J.C. Wong, G. Tang, Design
and synthesis of orally bioavailable aminopyrrolidinone histone deacetylase 6
inhibitors, J. Med. Chem. 58 (6) (2015) 2809e2820.
19