Synthesis and evaluation of novel cyclopentane urea FPR2 agonists and their potential application in the treatment of cardiovascular inflammation

Article abstract of DOI:10.1016/j.ejmech.2021.113194

The discovery of natural specialized pro-resolving mediators and their corresponding receptors, such as formyl peptide receptor 2 (FPR2), indicated that resolution of inflammation (RoI) is an active process which could be harnessed for innovative approaches to tame pathologies with underlying chronic inflammation. In this work, homology modelling, molecular docking and pharmacophore studies were deployed to assist the rationalization of the structure-activity relationships of known FPR2 agonists. The developed pharmacophore hypothesis was then used in parallel with the homology model for the design of novel ligand structures and in virtual screening. In the first round of optimization compound 8, with a cyclopentane core, was chosen as the most promising agonist (β-arrestin recruitment EC₅₀ = 20 nM and calcium mobilization EC₅₀ = 740 nM). In a human neutrophil static adhesion assay, compound 8 decreased the number of adherent neutrophils in a concentration dependent manner. Further investigation led to the more rigid cycloleucines (compound 22 and 24) with improved ADME profiles and maintaining FPR2 activity. Overall, we identified novel cyclopentane urea FPR2 agonists with promising ADMET profiles and the ability to suppress the inflammatory process by inhibiting the neutrophil adhesion cascade, which indicates their anti-inflammatory and pro-resolving properties.

Full text of DOI:10.1016/j.ejmech.2021.113194

European Journal of Medicinal Chemistry 214 (2021) 113194
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and evaluation of novel cyclopentane urea FPR2 agonists
and their potential application in the treatment of cardiovascular
inflammation
, b,
Monika Maciuszek a *, Almudena Ortega-Gomez c, Sanne L. Maas c, Mauro Perretti b,
, d,
Andy Merritt a, 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 German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance (MHA), Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of natural specialized pro-resolving mediators and their corresponding receptors, such as
formyl peptide receptor 2 (FPR2), indicated that resolution of inflammation (RoI) is an active process
which could be harnessed for innovative approaches to tame pathologies with underlying chronic
inflammation. In this work, homology modelling, molecular docking and pharmacophore studies were
deployed to assist the rationalization of the structure-activity relationships of known FPR2 agonists. The
developed pharmacophore hypothesis was then used in parallel with the homology model for the design
of novel ligand structures and in virtual screening. In the first round of optimization compound 8, with a
Received 17 November 2020
Received in revised form
8 January 2021
Accepted 10 January 2021
Available online 16 January 2021
Keywords:
FPR2
cyclopentane core, was chosen as the most promising agonist (
b
-arrestin recruitment EC50 ¼ 20 nM and
calcium mobilization EC50 ¼ 740 nM). In a human neutrophil static adhesion assay, compound 8
decreased the number of adherent neutrophils in a concentration dependent manner. Further investi-
gation led to the more rigid cycloleucines (compound 22 and 24) with improved ADME profiles and
maintaining FPR2 activity. Overall, we identified novel cyclopentane urea FPR2 agonists with promising
ADMET profiles and the ability to suppress the inflammatory process by inhibiting the neutrophil
adhesion cascade, which indicates their anti-inflammatory and pro-resolving properties.
© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Homology modelling
Agonists
Neutrophils
Resolution of inflammation
1. Introduction
neutrophils [19e23] and monocytes [24,25] but it can also be
located on the surface of various types of cells for example endo-
Currently there is increased interest in the exploitation of the
processes that characterise the resolution phase of inflammation
[1e4]. It is a novel approach, hence developing modulators as an
effector of resolution has the potential to treat not only the car-
diovascular inflammatory pathologies [5e7], but also for example
bowel disease [8], chronic obstructive pulmonary disease (COPD)
[9e11], rheumatoid arthritis [12,13], Alzheimer’s Disease [14],
neuroinflammation [15e17] and allergies [18]. FPR2 is a target of
interest for pro-resolving treatment of chronic inflammation. FPR2
is a G-protein coupled receptor which is highly expressed in
thelia, epithelia and smooth muscle cells. FPR2 can be activated by
agonists of diverse origins such as lipid (LXA4 [23,25e28], Resolvin
D1 [29e31]), proteins (Annexin A1 [AnxA1] [11,24]), synthetic
peptides (WKYMV [32,33]) and small molecule ligands (Compound
43 [7,34]). Depending on the nature of the agonist, FPR2 can have a
dual effect on the inflammatory response [2]. As an example,
activation of FPR2 by serum amyloid A (SAA) triggers pro-
inflammatory responses [35,36]. On the other hand, activation of
FPR2 by pro-resolving mediators such as lipoxin A4 (LXA4), resolvin
D1 or AnxA1 enables the cascade of anti-inflammatory and pro-
resolving responses e.g. inhibition of neutrophil trafficking and
transmigration and promotion of macrophage-mediated phagocy-
tosis [1,4,7,37]. Current efforts are focused on untangling the
complexity of this receptor. In 2012 Perretti et al. created FPR1/
* 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.2021.113194
0223-5234/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
FPR2 chimera proteins to determine the regions which are impor-
tant for FPR2 receptor activation using AnxA1, SAA and Compound
43 [38]. The extracellular loop I, transmembrane region III and
intracellular loop II were identified as the domains taking part in
the binding of small molecule agonist Compound 43. From muta-
genesis studies and later docking studies, four amino acid residues
(Arg-26, Asp-106, Arg-205 and Asp-281) were suggested as
important for the binding of peptide agonists [39,40]. In 2020 the
first crystal structure of human FPR2 in complex with WKYMVm
was published [41]. These discoveries increased our knowledge
regarding ligand recognition and possible binding modes of the
formyl peptide receptor ligands. In the last 10 years research groups
have focused on pro-resolving properties of the FPR2 receptor and
several synthetic small molecules have been submitted for Phase I
clinical trials [42,43], but none of them has yet become the first-in-
class pro-resolving drug. Nevertheless, each synthesized small
molecule agonist has made its contribution to the progress in
pharmacology and molecular biology of FPR2. In this work, we
demonstrate that homology modelling, followed by a combination
of ligand and structure-based design employing bioisosteric
replacement and synthesis, supported with ADME profiling is a
rational option for the development of novel agonists. Using this
approach various N-urea-substituted non-natural amino acids
moieties were explored. All synthesized compounds were assessed
aromatic stacking was observed with at least one of the amino
residues: Phe-257, Phe-292, Phe-163, Tyr-175 and His-102. This
concept corresponds partially with binding modes suggested by
research groups from Montana University and Warsaw University
[35,39,61]. In all six families, the hydrophobic pocket I is always
occupied by a phenyl ring or in the case of compound 5 by the
bridged spiro[2.4]heptane core and they make
p-p stacking and
hydrophobic interactions. The urea or amide groups in compounds
1e5 occupy polar clusters in the active site and form hydrogen
bonding interactions with the backbone of amino acid residues
such as Cys-179, Ser-288 or His-102.
Development of a pharmacophore hypothesis was performed
€
using the Phase tool in Schrodinger Maestro. The five-point phar-
macophore hypothesis has been chosen among 32 hypotheses with
PhaseHypoScore equals 1.17 and ROC value 0.9. This pharmaco-
phore model emphasized a substituted ring (Ring R9) at the para
position, preferably with a lipophilic and electronegative moiety,
for instance halogens (Hydrophobic H6). The results from the
pharmacophore studies established the importance of the NH
moiety (Donor D4) and the oxygen atom from the carbonyl group
(Acceptor A3). The hydrophobic feature (H8) covers alkyl chains
and the three-dimensional cores of the bridged spiro[2.4]heptane
derivatives and the Kyorin compounds. A pharmacophore model
derived from the binding modes of the FPR2 agonists in the ho-
mology model studies is shown in Fig. 3.
for their agonistic activity to induce calcium mobilization and b-
arrestin recruitment in CHOeK1 FPRL1 cells (DISCOVERx) over-
expressing hFPR2. Moreover, all synthesized compounds were
assessed in in vitro absorption, distribution, metabolism and
excretion and toxicity (ADMET) assays such as kinetic solubility and
microsomal stability to bring better insight to their potential for
further development.
2.2. Design and development of novel agonists
Following the development of both the homology and phar-
macophore models, they were then deployed in the search for
novel ligands. As the starting point we proposed compound 7 with
an
L-leucine motif (R1) which is known from the N-urea-
2. Results and discussion
substituted amino acids [48e51], the largest family of FPR2 agonists
developed by Allergan. From the in silico studies performed (section
2.1. In silico studies
2.1) we observed that the L-leucine alkyl chain is located in the
cavity formed with Met-85, Ser-84, Leu-81 and stabilizes the ligand
in the pocket. The presence of an aromatic ring in the R2 position is
common for FPR2 agonists (Fig. 1: 1 and 4). We decided to use a 3-
pyridyl group (R2) as the starting point in order to add a weakly
basic centre. Analysis of the binding modes showed that the 3-
pyridyl ring was oriented towards the solvent-exposed space in
subpocket 2, suggesting there may be scope to further vary this
group to tune the physicochemical properties. A series of urea
compounds was designed and synthesized via introducing an alkyl
In these studies, a ‘ready-to-use’ homology model was adopted:
an agonist (BU72) bound
m-opioid based structure (5C1M) [44]
from GPCRdb [45], allowing us to develop a hypothesis for the
binding sites of the ligands. Sitemap was used to predict possible
druggable regions in the receptor with the volume 641.5 Å3. In
order to predict probable binding modes, compounds with known
agonistic properties were selected from literature based on their
activity and the diversity of the chemical structures. For this pur-
pose, six key families were chosen to explore possible binding
poses: pyrazolone urea [46,47], N-urea-substituted amino acids
[48e51], oxazoles [52e54], N-urea-substituted furans [55], bridged
spiro[2.4] heptanes [56e58] and pyridazinones [59,60]. All litera-
ture EC50 values presented are from the calcium mobilization as-
says. In the figure below representative compounds with their
chemical structures used in modelling the active site of FPR2 are
presented (Fig. 1).
or cyclic moiety as replacements for L-leucine. For this purpose,
commercially available unnatural amino acids were chosen. We
decided to maintain two key structural features: the urea moiety
and the para-bromo substituted phenyl ring, as both play an
important role in the ligand binding according to the in silico
studies (Fig. 4).
When a ligand activates the receptor, it might trigger the G-
protein and/or
b-arrestin signalling cascade. Accordingly, in this
The final receptor grid generation was performed using the re-
sults from SiteMap using the Glide SP-protocol. To facilitate the
examination of the receptor grid, test virtual screening of actives
and decoys was performed. The decoys were generated for known
active agonists using the DUDE database. The enrichment factor
was calculated, and the value of receiver operating characteristic
(ROC) was 0.70 with all active molecules placed in the first 35% of
screened compounds. These results gave us confidence that we can
distinguish actives from inactive compounds. This model high-
lighted the concept of the binding pocket made up with three hy-
drophobic subpockets with two polar clusters. FPR2 agonists do not
need to occupy all three proposed subpockets (Fig. 2). Analysis of
the binding modes showed that for each molecule an important
study two functional assays were adopted:
b
-arrestin recruitment
and calcium mobilization. In the calcium mobilization assay we
measure the release of calcium from the endoplasmic reticulum
driven by activation of the G-protein. Using both assays we were
able to determine if the developed ligands are balanced agonists, G-
protein biased agonists or
b-arrestin biased agonists. The results
from the -arrestin and calcium mobilization assays revealed the
b
importance of a bulky substituent at R1 in order to retain the ac-
tivity. With R1 as hydrogen (6), this compound is completely
inactive in the
EC50 value is 0.73
b
-arrestin assay, and in calcium mobilization the
M, however the efficacy (Emax) is below 50%. Our
m
starting molecule, compound 7, only partially activated the recep-
tor in both cellular assays, though it has good metabolic stability
2

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Fig. 1. Selected examples of the existing FPR2 small molecule agonists used in in silico studies
All literature EC50 values presented are from calcium mobilization assays.
Fig. 3. The five-point pharmacophore hypothesis.
concentrations. Various non-natural amino acid cores are well
tolerated in the microsomal stability assays, showing good stability
in human liver microsomes (HLM). Introduction of a larger sub-
stituent than cyclopentane 8, for example tetrahydropyran 9 or
methylcyclohexyl 10 led to reduced efficacy or a decrease in agonist
potency in calcium mobilization, respectively. Moreover, the
structural modifications in compound 10 had a negative effect on
metabolic stability and kinetic solubility. Overall, the cyclopentane
8, methylcyclopropyl 11 and cyclopropyl 12 have promising in vitro
ADME profiles which can serve as a good basis for further optimi-
zation (Table 1).
Fig. 2. Model of the compounds (Allergan urea and pyrazolone) docked to FPR2
and proposed three hydrophobic subpockets model with two polar clusters.
Compound 8 was chosen for further structural modification due
to its balance of potency, efficacy, and properties. Furthermore,
positive results from human neutrophil adhesion assays demon-
strated the anti-inflammatory properties of compound 8, so this
was an additional factor which fed into the selection [data are
presented in section 2.4]. Attention turned to replacement of the
aromatic amine with more hydrophilic moieties (Table 2). The
azetidine 14 and methyl-piperidine 15 showed markedly better
kinetic solubility and LogD values, though simultaneously we
and reasonable kinetic solubility. In the calcium mobilization assay
ligands 8 and 10e12 exhibited full agonistic efficacy, showing that
these cyclic alkyl substituents have a positive effect, and this sup-
ports our finding from docking studies. Among the newly devel-
oped ligands, compound
8 exhibited the best overall FPR2
activation profile across both functional assays at sub-micromolar
3

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Fig. 4. Starting point for the design and development of novel FPR2 agonists
Table 1
ADME properties and EC50 values for urea derivatives.
ID
R1
-H
Kinetic Solubility [
mM]
LogDa
Microsomal stability
Ca2þ EC50
[m
M]f (Emax/%g)
b-arrestin EC50 [m
M]f (Emax/%g)
t1/2 [min]b
CLint
[
m
L/min/
c
mg]
HLMd
MLMe
HLMd
MLMe
6
7
27
34
3.6
4.2
112
95
22
65
14
16
71
24
0.73 (49)
0.03 (38)
NA
4.26 (82)
(S)
8
9
17
32
>4
83
33
90
19
8
47
17
0.02 (137)
0.072 (77)
0.74 (98)
4.7
188
4.17 (93)
2.15 (77)
10
0
>4
61
11
25
142
1.3 (150)
11
12
11
41
>4
>4
91
80
44
41
17
19
35
38
0.068 (90)
1.35
0.097 (100)
7.8 (93)
NA - very low response (Emax <30% of max response) or weak activation EC50 > 10
m
M.
a
LogD - distribution coefficient.
t1/2 e half-life [min].
Clint - intrinsic clearance [mL/min/mg].
HLM - human liver microsomes.
MLM - mouse liver microsomes.
EC50 - half maximal effective concentration [
Emax efficacy- [%]
b
c
d
e
f
m
M].
g
observed a drop-off in potency. Additionally, the 3-pyridine (8) was
exchanged for cyclic ethers: oxetane (16), tetrahydropyran (17) and
2-methyltetrahydrofuran (18). The cyclic ether analogues displayed
good microsomal stability, except 18 which is extremely unstable.
The cyclic ether analogues 16e18 exhibit sub-micromolar activity
in the calcium mobilization assay, however only 18 had the ability
4

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Table 2
Summary showing ADME properties and EC50 values of selected compounds from cyclopentyl-acetamide series.
b
ID
R2
Ks [
m
M]
LogDa
T1/2 [min]
Clintc
(
m
L/min/
Ca2þ EC50
[m
M]f (Emax/%)g
b
-arrestin recruitment EC50
0.74 (98)
[m
M]f (Emax/%)g
mg)
HLMd
MLMe
33
HLMd
19
MLMe
47
8
17
47
>4
83
0.02 (100)
0.8 (120)
13
4
184
92
8
17
6.33 (100)
14
15
178
200
1.6
2.5
121
110
65
13
14
24
3
3.85
0.50 (97)
NA
477
3.67 (130)
16
17
26
1
4.3
>400
>400
<3
<3
0.62 (95)
0.6 (132)
NA
NA
>4
207
104
7
14
18
22
4.7
13
25
121
62
0.54 (96)
0.94 (101)
19
20
0
0
>4
>4
72
55
83
12
22
28
19
0.48 (100)
1.24 (111)
1.9 (86)
126
7.45 (43)
21
59
>4
16
11
99
133
0.07 (68)
1.41 (84)
NA - very low response (Emax <30% of max response) or weak activation EC50 > 10
m
M.
a
LogD - distribution coefficient.
b
t
1/2 e half-life [min].
c
d
e
f
Clint - intrinsic clearance [
m
L/min/mg].
HLM - human liver microsomes.
MLM - mouse liver microsomes.
EC50 - half maximal effective concentration [
Emax efficacy- [%].
m
M].
g
5

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Table 3
Summary showing ADME properties and EC50 values of selected compounds from cycloleucine series.
g
g
ID
R2
Ks [
m
M]
LogDa
4.1
T1/2 [min]b
HLMd
Clint
HLM
(
m
L/min/mg)c
Ca2þ EC50
[
m
M]f (Emax
)
b
-arrestin recruitment EC50
1.3 (99)
[m )
M]f (Emax
HLM
108
MLMe
79
HLMd
14
MLMe
22
103
108
14
19
0.250 (110)
23
24
214
225
3.7
3.1
110
156
110
156
104
227
14
10
14
10
15
7
0.366 (90)
0.089 (86)
7.27 (92)
1.85 (89)
25
26
223
226
1.1
1.2
520
157
520
157
419
194
3
3
4
0.272 (88)
1.6 (95)
NA
NA
10
10
7.9
27
33
4.2
130
130
90
12
12
17
1.1 (96)
4.15 (51)
28
29
43
31
4.3
3.9
8
8
9
186
16
186
16
180
42
6.07 (49)
0.60 (68)
NA
100
100
36
9.9 (60)
30
31
30
0
3.7
4.4
86
52
86
52
39
32
18
30
18
30
39
47
1.75 (79)
0.11 (60)
9.8 (60)
2.58 (71)
NA - very low response (Emax <30% of max response) or weak activation EC50 > 10
m
M.
a
LogD - distribution coefficient.
b
t
1/2 e half-life [min].
c
d
e
f
Clint - intrinsic clearance [
m
L/min/mg].
HLM - human liver microsomes.
MLM - mouse liver microsomes.
EC50 - half maximal effective concentration [
Emax efficacy- [%].
m
M].
g
to activate FPR2 through the
b
-arrestin pathway (EC50 ¼ 0.95
m
M).
SAR, it was decided to synthesise three additional analogues of 8
with the pyridine replaced by alternative heterocycles. This
included pyrazole variants (19e20) and pyridazine (21), which
were expected to represent a good compromise for the overall
The distinct difference in metabolic stability with 18 suggests that
either a CH2 in the tetrahydrofuran ring or the eCH2 linker can act
as a metabolic soft-spot in this example. Based on the observed
6

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Fig. 5. Optimization of cyclopentane urea FPR2 agonists.
molecular properties. The pyrazole derivatives 19 and 20 were less
active than compound 8, however both ligands exhibit low
micromolar potency in the calcium mobilization assay (Ca2þ
In summary, as the starting point we proposed compound 7
with an L-leucine core, which is known from N-urea-substituted
amino acids [48e51]. Investigation of alternative R1 substituents
based on compound 7 resulted in the enhancement of the FPR2
agonistic activity. Compound 8 was chosen for further structural
modification due to its balance of potency, efficacy, and properties.
In the next step of optimization, we introduced a geminally
substituted cyclopentane ring (22). Finally, re-optimization of the
R2 position with this core in place led to the identification of the
dimethylpyrazole-substituted example 24 (Fig. 5). For the leading
compounds, in order to determine whether any cytotoxic effects
are observed, a cell health assay was performed using HepG2 cells,
a human hepatocyte carcinoma line. None of the compounds
appeared to show any signs of toxicity at any of the parameters
EC50 ¼ 0.481 and 1.24
mM respectively). The dimethylpyrazole
analogue 19 showed moderate microsomal stability, however both
19 and 20 suffered from poor solubility. The pyridazine 21 was the
most active amongst these three modifications, nevertheless it
experiences a low metabolic stability. Compound 21 undergoes a
rapid in vitro intrinsic clearance in comparison to pyrimidine
compound 13 (HLM/MLM Clint 8/13
m
L/min/mg). The additional
M and 21 Ks ¼ 59 M)
M),
nitrogen atom in the ring (13 Ks ¼ 47
m
m
m
improves the kinetic solubility compared to 8 (Ks ¼ 17
nevertheless it has a negative impact on activity. (Table 2). In
summary, the aromatic ring at the R2 position appears to be
beneficial but it is not absolutely crucial to maintaining agonistic
activity.
tested up to 30 mM (supplementary data).
On the basis of the results described above, the next step was
further improvement of the scaffold. It was decided to introduce a
geminally substituted cyclopentane ring to the alpha-C atom of the
amino acid. Gratifyingly, this cycloleucine series showed signifi-
cantly improved solubility and the potency was maintained in the
nanomolar range in calcium mobilization (Table 3, 22e23). The SAR
studies were then focused on the replacement of pyridine ring. The
dimethylpyrazole 24 has a low clearance amongst both species
with LogD slightly above 3, increased kinetic solubility and
improved activity in the calcium mobilization assay. Another
compound with an encouraging profile is the tetrazole 25: the
acidic proton of the tetrazole results in low log D, high metabolic
stability and high solubility; however, compared to 24, it is 4-fold
2.3. Chemistry
Pathway 1 was used when starting with the alpha amino acid
ester and commercially available 4-bromophenyl isocyanate 33 to
obtain the urea derivatives 34e40 with good yield. Cleavage of the
ester group with formic acid or 2M aqueous solution of sodium
hydroxide at room temperature generated an acid 41e47 in quan-
titative yield. Final amide coupling was performed in the presence
of triethylamine and T3P as a coupling agent 7e10; 13, 15e31 and
48. When the alpha amino acid ester is not available pathway 2 was
used, the first step was amide coupling with the BOC-protected
amino acid 49e50 in the presence of triethylamine and T3P as a
coupling agent. Then deprotection of BOC protected amine was
performed using 4M HCl in dioxane in quantitative yield. Finally,
the urea derivative 11e12 was obtained in the coupling with 4-
bromophenyl isocyanate (Scheme 1).
weaker in calcium mobilization (272 nM) and inactive in the b-
arrestin assay. The weaker performance of the 1-isopropyl-1H-
pyrazole 27 in the functional assays could be justified by steric
clashes compared to 24 and 25. Compound 28, containing the 2-
pyridine, showed weaker potency and approximately a 10-fold
shorter half-life than 22, highlighting the 3-pyridine’s superior
stability. The 1-methylpiperidine derivative 26 was synthesized to
study the impact of non-aromatic rings on the agonistic activity.
The outcome correlates with the results for the cyclopentyl-
acetamide series, where substitution of the aromatic ring for an
alicyclic group caused a drop-off in activity. Introduction of a
methyl group adjacent to the pyridine nitrogen (29) reduced sol-
ubility and stability in MLM, but still sustained activity in the
nanomolar range. By contrast a methyl group at the pyridine 3-
position (30) led to a significant drop-off in activity in the cal-
cium mobilization assay. Replacing this methyl with a chlorine (31)
led to improved activity in the calcium mobilization assay
(EC50 ¼ 107 nM) although it suffers from poor solubility (Table 3).
Although compounds 29e31 display promising stability in HLM,
their stability is significantly lower in MLM.
2.4. Determination of the anti-inflammatory properties of the FPR2
agonists
To assess their possible therapeutic activity for the treatment of
cardiovascular inflammatory diseases, a human neutrophil static
adhesion assay was performed on the leading compounds.
Fig. 6 shows the results obtained from profiling two known
active FPR2 agonists. They were used as a benchmark for the novel
agonists. Compound 2, which is one of the Allergan small molecule
agonists described in patent application US20170320897 covering
treatment of ocular inflammation, showed good inhibition of
neutrophil adhesion. The second compound is Compound 43 (32),
developed by Amgen, which also shows anti-inflammatory prop-
erties in a dose-dependent manner (Fig. 6).
The functional assays described earlier had shown that in the
non-natural amino acid series, the presence of a bulky substituent
7

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Scheme 1. Two synthetic pathways used for non-natural amino acid series. Conditions: Pathway 1: (a) 1 eq. amino acid., 1.5 eq. TEA, DCM, RT, 4 h; (b) formic acid, RT, 4 h; (c) 1.5 eq
T3P, 1.1 eq. amine, ethyl acetate, RT, overnight; * in addition compound 48 was deprotected to form compound 14 using 4M HCl in dioxane, DCM, RT 4 h; Pathway 2: (d) 1.5 eq T3P,
1.1 eq. amine, ethyl acetate, RT, overnight; (e) 4M HCl in dioxane, DCM, 0 ꢁC to RT, 2e4 h or overnight; (f) 1.2 eq. 4-bromophenyl isocyanate., 1.5 eq. TEA, DCM, RT, 4 h.
in the position R1 is crucial to maintaining the activity. Leading
examples were tested in primary cell assays and in Fig. 6 three
selected compounds from this series are presented. The parent L-
(24) and tetrazoles (25) showed the most promising ADME and
activity profiles in the cycloleucine series. The dimethylpyrazole-
substituted ligand 24 showed reduced lipophilicity (LogD ¼ 3.1),
leucine compound 7 showed around a 50% decrease relative to the
control and this value is perpetual across entire concentration
range; this could be triggered by desensitization of the receptor.
Compound 11, with the cyclopropylmethyl core, showed minimal
reduction of adherent neutrophils. The cyclopentane analogue 8
decreased the number of adherent neutrophils in a concentration
dependent manner (Fig. 6) and showed enhanced anti-
inflammatory properties compared to compound 7.
high solubility (Ks ¼ 225
(HLM/MLM Clint 10/7
mM) and low in vitro intrinsic clearance
mL/min/mg). Leading exemplars were tested
in a static adhesion assay, and compound 8 in particular demon-
strated the ability to reduce the neutrophil adhesion to proteins
such as P-selectins and ICAM. The binding of anti-inflammatory
agonists might prevent binding of the other pro-inflammatory li-
gands like Serum Amyloid A (SAA), or control release of pro-
inflammatory cytokines. Future work will involve testing the later
molecules in the static adhesion assays, especially compounds 22
and 24. Finally, compound 8 also showed a promising PK profile
and the small molecule FPR2 agonists described here might be
valuable tools for the further study of the molecular mechanisms
involved in atherosclerosis or ischemia reperfusion injury.
2.5. PK studies
Compound 8 was chosen for pharmacokinetic studies which
were performed in male C57BL6 mice with intraperitoneal
administration at 10 mg/kg (Table 4). Following a single dose, 8 was
rapidly absorbed (Tmax ¼ 0.5 h), showed a high Cmax, along with a
good half-life (2.08 h) and high exposure (area under the
4. Materials and experimental methods
concentration-time
curve
from
0
to
infinity
(AUC0
4.1. In silico studies
einf) ¼ 68961 ng h/mL).
Homology model: An agonist bound m-opioid based structure
receptor was acquired from GPCRDB [http://gpcrdb.org/protein/
fpr2_human/].
3. Conclusions
Docking Studies: Protein-Ligand docking was performed using
the Glide SP-protocol with an outer box 20x20 ꢀ 20 Å. All ligands
were treated as flexible molecules. Binding site was identified using
In these studies, novel urea-based FPR2 agonists were designed
and a structure-activity relationship study was conducted, based on
insights provided from in silico studies including homology
modelling, docking and pharmacophore studies. Based on the re-
sults from the in silico studies, the introduction of different moieties
in the R1 position, for example alkyl, cycloalkyl and heterocyclic
substituents, offered the chance to improve the fit into the binding
pocket and gain activity. Compound 8 was chosen for further
structural modification due to its balance of potency, efficacy, and
properties. Further investigation led to the more rigid cycloleucine
(compound 22). The cycloleucine series resulted in an improve-
ment in solubility and the activity was sustained in the nanomolar
or submicromolar range. Leading examples showed good in vitro
ADMET profiles and no cytotoxicity in HepG2 cells, a human he-
patocyte carcinoma cell line. Through building the structure-
activity relationships (SAR), we discovered the benefit of an aro-
matic ring at the R2 position such as 3-pyridine, pyrimidine or
substituted pyrazoles. The five-membered rings such as pyrazoles
€
€
by Sitemap (Schrodinger Release 2018-2: Schrodinger, LLC, New
York, 2018.) Ligands were prepared and visualized using Maestro
11.5. To generate decoys for active agonists DUDE database (http://
dude.docking.org/) was used.
Pharmacophore Studies: Pharmacophore hypothesis was ob-
tained from multiple active ligands using Phase with the ‘multiple
ligands’ option and the method ‘best alignment and common fea-
€
€
tures’. (Schrodinger Release 2018-2: Schrodinger, LLC, New York,
2018).
4.2. Chemistry
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
8

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Fig. 6. Anti-adhesive properties of FPR2 agonists 2 (Allergan) and 32 (compound 43) and non-natural amino acids series
All data were shown as means SEM., 3e4 independent experiments with 3 technical replicates. The difference between control group, and treatment groups was evaluated via
one-way Anova followed by Dunnett’s multiple comparison test, and the difference was considered significant for a P value < 0.05: *P < 0.05, **P < 0.01, ***P < 0.001; ns e not
significant.
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
9

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Table 4
intermediate 33 (3 mmol) and methyl 2-amino-2-cyclopentyl-ace-
tate HCL (1 equiv., 3 mmol); Yield: 640 mg, 70%; Appearance: white
solid; LCMS: 1.77 min, m/z 356.0, 358.0 ((MþH)þ 1:1, 79/81Br), Method
In vivo PK parameters of compound 8 with intraperitoneal administration.
Dosea Cmaxb Tmaxc T1/2
AUC0-last
68961
AUC0-inf
68978
AUCExtra
0.02
MRT0-last
2.41
c
d
d
e
c
B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.70 (s, 1 H), 7.47e7.29 (m,
10 31905 0.5 2.08
4 H), 6.62 (d, J ¼ 8.02 Hz, 1 H), 4.13 (t, J ¼ 7.73 Hz, 1 H), 3.64 (s, 3 H),
a
b
c
Dose is expressed in mg/kg.
Cmax is expressed in ng/mL.
Tmax, T1/2 and MRT0-last are expressed in hours [h].
AUC0ꢂlast and AUC0ꢂinf are expressed in ng$h/mL.
AUC0ꢂlast is expressed in %.
2.21e2.10 (m, 1 H), 1.69e1.46 (m, 6 H), 1.36e1.23 (m, 2 H); 13C NMR
(126 MHz, DMSO‑d6)
d ppm 173.18 (1 C, C]O), 154.75 (1 C, C]O),
d
e
139.47 (1 C, ArC), 131.52 (2 C, ArCH), 119.54 (1 C, ArCH), 112.69 (1 C,
ArC), 55.60 (1 C, CH), 51.82 (1 C, CH3), 41.59 (1 C, CH), 28.63 (1 C, CH2),
28.22 (1 C, CH2), 24.89 (1 C, CH2), 24.67 (1 C, CH2).
Rac-methyl
2-[(4-bromophenyl)carbamoylamino]-2-
Directed Prep HPLC system by a member of the purification team.
LC/MS purity analysis was performed on an Agilent 6120 quadru-
cyclohexyl-acetate (36) Prepared according to procedure A with
intermediate 33 (3 mmol) and methyl 2-amino-2-cyclohexyl-ace-
tate (1.1 equiv., 3.3 mmol). Crude product was dry loaded on silica
and purified by flash column chromatography (silica gel, PE/EA 10–
>60%, gradient). The beige solid was washed with diethyl ether
(15 mL), filtered and dried under high vacuum. Yield: 682 mg, 70%;
Appearance: white solid; LCMS: 1.87 min, m/z 369.1, 371.1 ((MþH)þ
pole LC-MS with an Xbridge C18 column (3.5
mm particle and
4.6 ꢀ 30 mm dimension) 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: Acetonitrile; Gradient - 10e100%B; Gradient time:
2.35min; Method C: flow rate: 3 mL/min; Run time: 3.2 min: Sol-
vent 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: 00.1% 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
1:1, 79/81Br), Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.67 (s,
1 H), 7.40e7.28 (m, 4 H), 6.57 (d, J ¼ 8.59 Hz, 1 H), 4.12 (dd, J ¼ 8.59,
5.73 Hz, 1 H), 3.62 (s, 3 H), 1.73e1.63 (m, 3 H), 1.58e1.52 (m, 2 H),
1.25e0.96 (m, 6 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 172.67
(1 C, C]O), 154.68 (1 C, C]O), 139.49 (1 C, ArC), 131.48 (2 C, ArCH),
119.44 (2 C, ArCH), 112.60 (1 C, ArC), 57.10 (1 C, NCH), 51.79 (1 C,
CH3), 29.16 (1 C, CH), 28.57 (1 C, CH2), 27.95 (1 C, CH2), 25.64 (1 C,
CH2), 25.49 (2 C, CH2).
Rac-methyl
2-[(4-bromophenyl)carbamoylamino]-3-
cyclohexyl-propanoate (37) Prepared according to procedure A
with intermediate 33 (3 mmol) and methyl 2-amino-3-cyclohexyl-
propanoate HCl (1 equiv. 3.3 mmol). Crude product was dry loaded
on silica and purified by flash column chromatography (silica gel,
PE/EA 10–>60%, gradient). The beige solid was washed with diethyl
ether (15 mL), filtered, and dried under high vacuum to afford
desired product as a white solid. Yield: 410 mg, 40%; Appearance:
white solid; LCMS: 1.97 min, m/z 383.2, 385.1 ((MþH)þ 1:1, 79/81Br),
spectrometer at 21 ꢁC. Chemical shifts (
d) are in ppm and are
relative to residual undeuterated NMR solvent. Spectra are refer-
enced 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. Structural assignments for
13CNMR were denoted as follows: eOCH3 ¼ C atom of methoxy
group, C]O ¼ C atom of carbonyl, ArCH ¼ aryl carbon bearing a
hydrogen atom, ArC ¼ aryl carbon, Cgem ¼ the alpha-C atom of the
amino acid attached directly to cyclopentane, CH ¼ alkenyl carbon,
CH2 ¼ methylene carbon, CH3 ¼ methyl carbon, COOH ¼ carbon
atom of carboxylic acid, C(CH3)3 ¼ carbon atom of tertbutyl group,
C(CH3)3 ¼ tertiary carbon atom of tertbutyl group.
Purity 90%, Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.68 (s,
1 H), 7.39e7.30 (m, 4 H), 6.57 (d, J ¼ 8.02 Hz, 1 H), 4.22 (td, J ¼ 8.31,
5.73 Hz, 1 H), 3.61 (s, 3 H), 1.70 (br. d, J ¼ 12.60 Hz, 1 H), 1.66e1.54
(m, 5 H), 1.50 (ddd, J ¼ 8.74, 5.58, 3.44 Hz, 2 H), 1.22e1.03 (m, 4 H),
0.96e0.79 (m, 2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 173.86
(1 C, C]O), 154.66 (1 C, C]O), 139.50 (1 C, ArC), 131.43 (2 C, ArCH),
119.57 (2 C, ArCH), 112.63 (1 C, ArC), 51.88 (1 C, CH3), 50.14 (1 C, CH),
33.53 (1 C, CH2), 33.07 (1 C, CH2), 31.70 (1 C, CH), 25.96 (1 C, CH2),
25.76 (1 C, CH2), 25.57 (1 C, CH2).
General procedure A for urea formation: Prepared according
to the literature procedure [49,50,62]. To a solution of amino acid
(1.1 equiv.) in dichloromethane at room temperature was added the
appropriate isocyanate (1 equiv.) and triethylamine (1.5 equiv.). The
resulting mixture was stirred at room temperature for 3 h. RM was
concentrated and purified by flash column chromatography using
as an eluent PE/EtOAc to obtain the desired product as white solid.
Tert-butyl 2-[(4-bromophenyl)carbamoylamino]acetate (34)
Prepared according to procedure A with intermediate 33 (1 equiv.,
3 mmol) and glycine tert-butyl ester hydrochloride (1.1 equiv,
3.3 mmol). Crude product purified by flash column chromatog-
raphy (silica gel) using PE/EtOAc 1/1. Yield: 460 mg, 90%; Appear-
Rac-methyl
2-[(4-bromophenyl)carbamoylamino]-2-
tetrahydropyran-4-yl-acetate (38) Prepared according to proced-
ure A with intermediate 33 (3 mmol) and methyl 2-amino-2-
tetrahydropyran-4-yl-acetate 56 (3 mmol). Crude product was
purified via FCC (PE/EA 0–>70%) then additionally washed with
diethyl ether to remove impurities from aliphatic region. Yield:
510 mg, 70%; Appearance: white solid; LCMS: 1.93 min, m/z 371.1,
373.0 ((MþH)þ 1:1, 79/81Br), Method B; 1H NMR (500 MHz, CHLO-
ROFORM-d)
d ppm 7.43e7.37 (m, 2 H) 7.25e7.19 (m, 2 H) 6.92 (s,
1 H) 5.61 (d, J ¼ 8.59 Hz, 1 H) 4.57 (dd, J ¼ 8.59, 5.16 Hz, 1 H) 3.78 (s,
3 H) 4.04e3.95 (m, 2 H) 3.44e3.32 (m, 2 H) 2.13e2.03 (m, 1 H)
1.61e1.42 (m, 4 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 172.29
79/
ance: white solid; LCMS: 1.61 min, m/z 330, 332 ((MþH)þ 1:1,
(1 C, C]O), 154.68 (1 C, C]O), 139.42 (1 C, ArC), 131.48 (2 C, ArCH),
119.52 (2 C, ArCH), 112.70 (1 C, ArC), 66.62 (2 C, CH2), 56.57 (1 C,
CH3), 51.91 (1 C, CH), 37.22 (1 C, CH), 29.03 (1 C, CH2), 27.99 (1 C,
CH2).
81Br), Purity >95%, Method B 1H NMR (500 MHz, CHLOROFORM-d)
d
ppm 7.37e7.32 (m, 2 H), 7.13e7.21 (m, 2 H), 5.66 (t, J ¼ 5.16 Hz,
1 H), 3.96 (d, J ¼ 5.16 Hz, 2 H), 1.47 (s, 9 H); . 13C NMR (126 MHz,
DMSO‑d6) d ppm 169.91 (1 C, C]O), 155.03 (1 C, C]O), 139.74 (1 C,
Tert-Butyl
(2S)-2-[(4-bromophenyl)carbamoylamino]-4-
ArC), 131.42 (2 C, ArCH), 119.63 (2 C, ArCH), 112.55 (1 C, ArC), 80.57
(1 C, C(CH3)3), 41.96 (1 C, CH2), 27.77 (3 C, CH3).
methyl-pentanoate (39) Prepared according to procedure A with
33 (2.7 mmol) and H-Leu-OtBu HCl (1.1 equiv., 3 mmol); Crude
product was dry loaded onto silica and purified by flash column
chromatography (EA/PE 1:1 gradient). Yield: 700 mg, 70%;
Rac-methyl
2-[(4-bromophenyl)carbamoylamino]-2-
cyclopentyl-acetate (35) Prepared according to procedure A with
10

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
Appearance: white solid; LCMS: 2.00 min, m/z 329.9, 331.9
((MþH)þ 1:1, 79/81Br, without tert-butyl group); Purity>95%,
Appearance: white solid; LCMS: 1.16 min, m/z 369.1, 371.1 ((MþH)þ
1:1, 79/81Br), Purity>95%, Method B; 1H NMR (500 MHz, DMSO‑d6)
Method D; 1H NMR (500 MHz, CHLOROFORM-d)
d
ppm 7.33e7.28
d
ppm 8.93 (s, 1 H), 7.34 (m, 4 H), 6.57 (br d, J ¼ 8.02 Hz, 1 H), 4.11
(m, 2 H), 7.24e7.20 (m, 2 H), 7.11 (s, 1 H), 5.86 (d, J ¼ 8.02 Hz, 1 H),
4.43 (ddd, J ¼ 9.74, 8.02, 5.16 Hz, 1 H), 1.81e1.72 (m, 1 H), 1.67e1.59
(m, 1 H), 1.52 (s, 9 H), 1.50e1.43 (m, 1 H), 0.97 (dd, J ¼ 6.59, 4.87 Hz,
(td, J ¼ 8.59, 5.16 Hz, 1 H), 1.73 (br d, J ¼ 12.03 Hz, 1 H), 1.61 (m, 3 H),
1.59e1.41 (m, 3 H), 1.34 (br d, J ¼ 2.86 Hz, 1 H), 1.20e1.04 (m, 3 H),
0.96e0.77 (m, 2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 174.96
6 H); 13C NMR (126 MHz, DMSO‑d6)
d
ppm 172.53 (1C, C]O),154.62
(1 C, C]O), 154.72 (1 C, C]O), 139.79 (1 C, ArC), 131.41 (2 C, ArCH),
119.41 (2 C, ArCH), 112.34 (1 C, ArC), 50.35 (1 C, CH), 39.51 (1 C,
CHCH2), 33.63 (1 C, CH2), 33.19 (1 C, CH2), 31.91 (1 C, CH), 26.02 (1 C,
CH2), 25.80 (1 C, CH2), 25.65 (1 C, CH2); once carbon (CHCH2)
hidden under DMSO peak.
(1 C, C]O), 139.57 (1C, ArC.), 131.44 (2C, ArCH), 119.54 (2C, ArCH),
112.57 (ArCBr), 80.61 (1C, C(CH3)3), 51.39 (CH), 40.85 (1C, CH2),
27.65 (3C, C(CH3)3), 24.41 (1C, CH), 22.69 (1C, CH3), 21.67 (1C, CH3);
Data corresponded to that reported in the literature [49].
General ester hydrolysis procedure B1:
A solution of ester in formic acid was stirred at room tempera-
ture for 3 h. The resulting mixture was quenched with water then
extracted with ethyl acetate. Crude product was purified by flash
column chromatography (PE/EtOAc) to obtain desired product as
white solid.
2-[(4-bromophenyl)carbamoylamino]-2-tetrahydropyran-4-
yl-acetic acid (45) Prepared according to procedure B2 with in-
termediate
methyl
2-[(4-bromophenyl)carbamoylamino]-2-
tetrahydropyran-4-yl-acetate (38). The crude product was used
for next reaction without further purification. Yield: 286 mg, 90%;
Appearance: white solid; LCMS: 1.36 min, m/z 357.1, 359.1 ((MþH)þ
1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
2-[(4-bromophenyl)carbamoylamino]acetic acid (41) Pre-
pared according to procedure B1 with intermediate tert-butyl 2-
[(4-bromophenyl) carbamoylamino]acetate (34). Crude product
was dry loaded onto silica and purified by flash column chroma-
tography (silica gel, PE/EtOAc 6/4). Yield: 460 mg, 80%; Appearance:
white solid; LCMS: 1.09 min, m/z 273.1, 275.1 ((MþH)þ 1:1, 79/81Br),
d
ppm 12.83 (br. s, 1 H), 7.40e7.34 (m, 4 H), 6.62 (d, J ¼ 8.59 Hz, 1 H),
4.13 (dd, J ¼ 8.59, 5.16 Hz, 1 H), 3.89e3.83 (m, 2 H), 3.27 (qd,
J ¼ 11.74, 2.00 Hz, 2 H), 2.01e1.93 (m, 1 H), 1.51e1.29 (m, 4 H); 13
C
NMR (126 MHz, DMSO‑d6) d ppm 173.19 (1 C, C]O),154.80 (1 C, C]
O), 139.65 (1 C, ArC), 131.70 (2 C, ArCH), 119.45 (2 C, ArCH), 112.48
(1 C, ArC), 66.78 (2 C, CH2), 56.40 (1 C, CH), 37.30 (1 C, CH), 29.21
(1 C, CH2), 27.83 (1 C, CH2).
Purity >95%, Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 12.56
(br. s, 1 H), 8.90 (s, 1 H), 7.39e7.27 (m, 4 H), 6.37 (t, J ¼ 5.73 Hz, 1 H),
3.75 (d, J ¼ 5.73 Hz, 2 H); 13C NMR (126 MHz, DMSO‑d6)
d
ppm
(2S)-2-[(4-bromophenyl)carbamoylamino]-4-methyl-penta-
noic acid (46) Prepared according to procedure B1 with interme-
diate tert-butyl (2S)-2-[(4-bromophenyl)carbamoylamino]-4-
methyl-pentanoate (39) (2 mmol). Crude product was purified by
flash column chromatography (silica gel, PE/EtOAc 2/8, gradient).
Yield: 440 mg, 83% Appearance: white solid; LCMS: 0.90 min, m/z
329.0, 331.0 ((MþH)þ 1:1, 79/81Br), Purity>95%, Method B; 1H NMR
172.13 (1 C, C]O), 155.01 (1 C, C]O), 139.78 (1 C, ArC), 131.43 (2 C,
ArCH), 119.57 (2 C, ArCH), 112.51 (1 C, ArC), 41.33 (1 C, CH2).
General ester hydrolysis procedure B2: Methyl 2-[(4-
bromophenyl)carbamoylamino]-2-tetrahydropyran-4-yl-acetate
(1 eq.) was suspended in the mixture methanol (3 mL)/water (2 mL)
and 2M solution of sodium hydroxide (1 mL) was added. RM was
stirred for 2 h at room temperature. Methanol was evaporated, the
remaining solution was acidified with 1M HCl (pH ¼ 2e3). The
precipitation was filtered washed with water and dried to give final
product as a white powder.
(500 MHz, DMSO‑d6)
d ppm 12.66 (s, 1 H), 8.71 (s, 1 H), 7.43e7.32
(m, 4 H), 6.45 (d, J ¼ 8.59 Hz, 1 H), 4.17 (td, J ¼ 8.59, 5.73 Hz, 1 H),
1.67 (dquin, J ¼ 13.75, 6.73 Hz, 1 H), 1.57e1.46 (m, 2 H), 0.90 (dd,
J ¼ 10.31, 6.30 Hz, 6 H); Data corresponded to that reported in the
literature [49].
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-acetic
acid (42) Prepared according to procedure B2 with intermediate
Methyl 2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-acetate
(35). Yield: 479mg, 79%; Appearance: white solid;LCMS: 1.55 min, m/
z 341.1, 343.1 ((MþH)þ 1:1, 79/81Br), Purity ¼ 90%, Method D; 1H NMR
1-[(4-bromophenyl)carbamoylamino]cyclo-
pentanecarboxylic acid (47) 1-bromo-4-isocyanato-benzene
(0.76 mmol) was dissolved in dichloromethane (10 mL) at room
temperature and methyl 1-aminocyclopentanecarboxylate HCl
(0.76 mmol, 1.0 equiv.) was added in one portion. Then triethyl-
amine (1.15 mmol,1.5 equiv.) was added to the solution and RM was
stirred at room temperature overnight. The RM was diluted with
DCM and washed twice with water. Organic layer was passed
through the phase separator and concentrated under reduced
pressure. The intermediate 40 was used directly in the next step. A
2-(4-bromoanilino)-3-oxa-1-azaspiro[4.4]non-1-en-4-one
(0.42 mmol) was suspended in the mixture methanol (2 mL)/water
(3 mL) and 2M solution of sodium hydroxide (0.847 mmol,
0.424 mL) was added. RM was stirred for 4 h at room temperature.
Methanol was evaporated, the remaining solution was acidified
with 1M HCl (pH ¼ 2e3). The precipitation was filtered washed
with water and dried to give final product as a white powder. Yield:
119 mg, 85%; LCMS:1.35 min, m/z 327.1, 329.1 ((MþH)þ 1:1, 79/81Br),
(500 MHz, DMSO‑d6)
d ppm 9.03 (s,1 H), 7.37 (m, 4 H), 6.65 (d,
J ¼ 8.02 Hz, 1 H), 4.06 (dd, J ¼ 8.02, 6.30 Hz, 1 H), 2.26e2.16 (m, 1 H),
1.68e1.27 (m, 8 H); .13C NMR (126 MHz, DMSO‑d6)
d
ppm 174.40 (1 C,
C]O), 154.98 (1 C, C]O), 140.15 (1 C, ArC), 131.30 (2 C, ArCH), 119.34
(2 C, ArCH),112.02 (1 C, ArC), 55.74 (1 C, CH), 42.20 (1 C, CH), 28.76 (s,
1 C, CH2), 27.72 (1 C, CH2), 24.80 (2 C, CH2).
2-[(4-bromophenyl)carbamoylamino]-2-cyclohexyl-acetic
acid (43) Prepared according to procedure B2 with intermediate
methyl 2-[(4-bromophenyl)carbamoylamino]-2-cyclohexyl-ace-
tate (36). The crude product was not purified further and was taken
for next step. Yield: 480 mg, 97%; Appearance: white solid; LCMS:
1.04 min, m/z 355, 357 ((MþH)þ 1:1, 79/81Br), Purity>90%, Method
B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 12.69 (br. s, 1 H), 8.72 (s,
1 H), 7.40e7.30 (m, 4 H), 6.43 (d, J ¼ 8.59 Hz, 1 H), 4.07 (dd, J ¼ 8.59,
4.58 Hz, 1 H), 1.75e1.48 (m, 6 H), 1.27e1.13 (m, 2 H), 1.13e0.96 (m,
Purity> 95%, Method C; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.56
3 H); 13C NMR (126 MHz, DMSO‑d6)
d
ppm 173.60 (1 C, C]O),
(s, 1 H), 7.39e7.31 (m, 4 H), 6.59 (s, 1 H), 2.11e2.03 (m, 2 H),
154.76 (1 C, C]O), 139.65 (1 C, ArC), 131.55 (2 C, ArCH), 119.42 (2 C,
ArCH), 112.46 (1 C, ArC), 56.91 (1 C, NCH), 29.31 (1 C, CH), 27.67 (2 C,
CH2), 25.64 (3 C, CH2).
2-[(4-bromophenyl)carbamoylamino]-3-cyclohexyl-prop-
anoic acid (44) Prepared according to procedure B2 with inter-
1.89e1.82 (m, 2 H), 1.70e1.66 (m, 4 H); 13C NMR (126 MHz,
DMSO‑d6) d ppm 176.52 (1 C, C]O), 155.05 (1 C, C]O), 140.26 (1 C,
ArC.), 131.88 (2 C, ArCH), 119.87 (2 C, ArCH), 112.82 (1 C, ArC.), 65.30
(1 C, Cgem.), 37.35 (2 C, CH2), 24.61 (2 C, CH2).
General procedure C for amide coupling using T3P: SM was
dissolved in ethyl acetate, then amine (1 equiv), TEA, (1.5 equiv.),
and T3P (1.5 equiv.) were added. Reaction mixture was stirred
overnight at room temperature, then EtOAc was added and washed
mediate
methyl
2-[(4-bromophenyl)carbamoylamino]-3-
cyclohexyl-propanoate (37). The crude product was not purified
further and was taken for next step. Yield: 200 mg, 60%;
11

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
with water. An organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. Crude product was purified
by reverse phase flash column chromatography.
d
ppm 10.51 (s, 1 H), 8.87 (s, 1 H), 8.77 (d, J ¼ 3.44 Hz, 1 H), 8.28 (dd,
J ¼ 4.58, 1.72 Hz, 1 H), 8.10e8.02 (m, 1 H), 7.41e7.34 (m, 5 H), 6.69
(br d, J ¼ 8.59 Hz,1 H), 4.35 (dd, J ¼ 8.59, 6.87 Hz,1 H), 3.91e3.82 (m,
2 H), 3.31e3.22 (m, 2 H), 1.98e1.88 (m, 1 H), 1.57 (br d, J ¼ 11.46 Hz,
1 H), 1.48e1.40 (m, 2 H), 1.30 (qd, J ¼ 12.51, 4.30 Hz, 1 H); 13C NMR
2-[(4-bromophenyl)carbamoylamino]-N-(3-pyridyl)acet-
amide (6) Prepared according to procedure C with intermediate 2-
[(4-bromophenyl) carbamoyl amino]acetic acid (41) (1 equiv.,
0.15 mmol) and 3-aminopyridine (1.1 equiv., 0.17 mmol). Yield:
40 mg, 80%; Appearance: white solid; LCMS:1.21 min, m/z 349.2,
351.1 ((MþH)þ 1:1, 79/81Br), Purity> 95%, Method B; 1H NMR
(126 MHz, DMSO‑d6)
d ppm 170.77 (1 C, C]O), 154.76 (1 C, C]O),
144.26 (1 C, ArCH), 140.89 (1 C, ArCH), 139.60 (1 C. ArC), 135.30 (1 C,
ArC), 131.43 (2 C, ArCH), 126.35 (1 C, ArCH), 123.86 (1 C, ArCH),
119.41 (2 C, ArCH),112.54 (1 C, ArC), 66.59 (2 C, CH2), 57.48 (1 C, CH),
38.14 (1 C, CH), 29.21 (1 C, CH2), 28.30 (1 C, CH2); HRMS: Exact
Mass: 432.0797; (MþH)þ: 433.0877; Observed Mass: 433.0879;
0.46 ppm.
(500 MHz, DMSO‑d6)
d ppm 10.28 (br. s., 1 H), 9.02 (s, 1 H), 8.74 (d,
J ¼ 2.86 Hz, 1 H), 8.26 (dd, J ¼ 4.87, 1.43 Hz, 1 H), 8.03 (dq, J ¼ 8.16,
1.48 Hz, 1 H), 7.41e7.38 (m, 4 H), 7.38e7.34 (m, 1 H), 6.52 (t,
J ¼ 5.44 Hz, 1 H), 3.96 (d, J ¼ 5.73 Hz, 2 H); 13C NMR (126 MHz,
2-[(4-bromophenyl)carbamoylamino]-3-cyclohexyl-N-(3-
pyridyl)propenamide (10) Prepared according to procedure C with
intermediate 2-[(4-bromophenyl)carbamoylamino]-3-cyclohexyl-
propanoic acid (44) (0.087 mmol) and 3-aminopyridine (1.1 equiv.,
0.1 mmol). Crude product was purified by reverse phase column
chromatography (C18 12 g, Water pH-10/MeOH 90/10–>100) to
give final product. Yield: 8 mg, 21%; Appearance: white solid;
LCMS: 2.31min, m/z 445.2, 457.2 ((MþH)þ 1:1, 79/81Br), Purity>95%,
DMSO‑d6) d ppm 169.13 (1 C, C]O), 155.09 (1 C, C]O), 144.26 (1 C,
ArCH), 140.77 (1 C, ArCH), 139.77 (1 C, ArC), 135.54 (1 C, ArC), 131.42
(2 C, ArCH), 126.06 (1 C, ArCH), 123.68 (1 C, ArCH), 119.50 (2 C,
ArCH), 112.49 (1 C, ArC), 43.20 (1 C, CH2); HRMS: Exact Mass:
348.0222; (MþH)þ: 349.0300; Mass Observed: 349.0292;
2.33 ppm.
(2S)-2-[(4-bromophenyl)carbamoylamino]-4-methyl-N-(3-
pyridyl)pentanamide (7) Prepared according to procedure C with
intermediate tert-butyl (2S)-2-[(4-bromophenyl)carbamoyla-
mino]-4-methyl-pentanoic acid (46) (0.14 mmol) and 3-
aminopyridine (1.3 equiv., 0.18 mmol). Yield: 34 mg, 61%; Appear-
ance: white solid; LCMS: 1.54 min, m/z 405.2, 407.1 ((MþH)þ 1:1, 79/
Method D; 1H NMR (500 MHz, CHLOROFORM-d)
d ppm 9.91 (br. s,
1 H), 8.72 (d, J ¼ 2.29 Hz, 1 H), 8.31 (dd, J ¼ 4.58, 1.15 Hz, 1 H),
7.88e7.82 (m, 1 H), 7.80 (br s, 1 H), 7.19 (d, J ¼ 9.17 Hz, 2 H), 7.11 (dd,
J ¼ 8.31, 4.87 Hz, 1 H), 6.94 (d, J ¼ 9.17 Hz, 2 H), 6.77 (br d,
J ¼ 7.45 Hz, 1 H), 4.84e4.74 (m, 1 H), 1.81e1.51 (m, 8 H), 1.43 (br s,
1 H), 1.21e0.84 (m, 4 H); HRMS: Exact Mass: 444.1161; (MþH)þ:
445.1239; Observed Mass: 445.1234; 1.15 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-pyr-
imidin-5-yl-acetamide (13) Prepared according to procedure C
with
81Br), Purity>95%, method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm
10.38 (s, 1 H), 8.77e8.69 (m, 2 H), 8.24 (dd, J ¼ 4.87, 1.43 Hz, 1 H),
8.02 (dq, J ¼ 8.16, 1.48 Hz, 1 H), 7.40e7.27 (m, 5 H), 6.52 (d,
J ¼ 8.02 Hz, 1 H), 4.49e4.33 (m, 1 H), 1.73e1.59 (m, 1 H), 1.56e1.44
(m, 2 H), 0.91 (d, J ¼ 6.30 Hz, 6 H); 13C NMR (126 MHz, DMSO‑d6)
intermediate
2-[(4-bromophenyl)carbamoylamino]-2-
d
ppm 172.46 (1 C, C]O), 154.64 (1 C, C]O), 144.41 (1 C, ArCH),
cyclopentyl-acetic acid (42) (0.15 mmol) and pyrimidin-5-amine
(1.2 equiv., 0.18 mmol). The crude product was purified by flash
column chromatography (silica gel, PE/EA 10–>100%, then EA/
MeOH 2% gradient). Yield: 29 mg, 65%; Appearance: white solid;
LCMS: 2.15 min, m/z 418.1, 420.1 ((MþH)þ 1:1, 79/81Br), Purity>95%,
140.94 (1 C, ArCH), 139.60 (1 C ArC), 135.56 (1 C, ArC), 131.47 (2 C,
ArCH), 126.30 (1 C, ArCH), 123.69 (1 C, ArCH), 119.48 (2 C, ArCH),
112.55 (1 C, ArC), 52.09 (1 C, CH), 41.76 (1 C, CH2), 24.43 (1 C, CH),
23.05 (1 C, CH3), 21.82 (1 C, CH3); HRMS: Exact Mass: 404.0848;
(MþH)þ: 405.0926; Observed Mass: 405.0921; 1.27 ppm.
Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 10.66 (s, 1 H), 9.02
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-(3-
pyridyl)acetamide (8) Prepared according to procedure C with
intermediate 2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-
acetic acid (42) (0.11 mmol) and 3-aminopyridine (1.1 equiv.,
0.122 mmol). Crude product was purified by reverse phase column
chromatography (C18 12 g, Water pH-10/MeOH 90/10 / 100) to
give final product. Yield: 22 mg, 47%; Appearance: white solid;
LCMS: 1.57 min m/z 417.2, 419.1 ((MþH)þ 1:1, 79/81Br), Purity>95%
(s, 2 H), 8.90 (s, 1 H), 8.79 (s, 1 H), 7.40e7.34 (m, 4H), 6.64 (d,
J ¼ 8.59 Hz, 1 H), 4.33 (t, J ¼ 8.02 Hz, 1 H), 2.21 (sxt, J ¼ 8.60 Hz, 1 H),
1.75e1.57 (m, 4 H), 1.57e1.47 (m, 2 H), 1.46e1.28 (m, 2 H); 13C NMR
(126 MHz, DMSO‑d6)
d ppm 172.22 (1 C, C]O), 154.76 (1 C, C]O),
153.28 (1 C, ArCH), 147.21 (2 C, ArCH), 139.54 (1 C, ArC), 134.17 (1 C,
ArC), 131.61 (2 C, ArCH), 119.47 (2 C, ArCH) 112.59 (1 C, ArC.), 56.57
(1 C, CH), 42.48 (1 C, CH), 28.69 (1 C, CH2), 28.19 (1 C, CH2), 24.65
(2 C, CH2); HRMS: Exact mass: 417.0800; (MþH)þ: 418.0880;
Observed mass: 418.0877; 0.81 ppm.
method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 10.44 (s, 1 H), 8.80
(s, 1 H), 8.77 (d, J ¼ 1.72 Hz, 1 H), 8.27 (dd, J ¼ 4.58, 1.15 Hz, 1 H),
8.07e8.03 (m,1 H), 7.41e7.31 (m, 5 H), 6.61 (d, J ¼ 8.59 Hz,1 H), 4.33
(t, J ¼ 8.02 Hz, 1 H), 2.20 (sxt, J ¼ 7.90 Hz, 1 H), 1.72e1.55 (m, 4 H),
1.54e1.45 (m, 2 H), 1.43e1.31 (m, 2 H); 13C NMR (126 MHz,
Tert-butyl
3-[[2-[(4-bromophenyl)carbamoylamino]-2-
cyclopentyl-acetyl]amino] azetidine-1-carboxylate (48) was
prepared according to procedure C with intermediate 2-[(4-
bromophenyl)carbamoylamino]-2-cyclopentyl-acetic acid (42)
(0.11 mmol) and tert-butyl 3-aminoazetidine-1-carboxylate (1.1
equiv., 0.122 mmol). Crude product was purified by reverse phase
column chromatography (C18 12 g, Water pH-10/MeOH 90/
10 / 100) to give final product. Yield: 27 mg, 49%; Appearance:
white solid; LCMS: 1.76 min, m/z 495.1, 497.1 ((MþH)þ 1:1, 79/81Br),
DMSO‑d6) d ppm 171.80 (1 C, C]O), 154.76 (1 C, C]O), 144.39 (1 C,
ArCH), 140.92 (1 C, ArCH), 139.62 (1 C, ArC), 135.50 (1 C, ArC), 131.53
(2 C, ArCH), 126.24 (1 C, ArCH), 123.77 (1 C, ArCH), 119.46 (2 C,
ArCH), 112.53 (1 C, ArC), 56.57 (1C, CH), 42.73 (1 C, CH), 28.65 (1 C,
CH2), 28.22 (1 C, CH2), 24.85 (1 C, CH2), 24.67 (1 C, CH2); HRMS:
Exact Mass: 416.0848; (MþH)þ: 417.0926; Observed Mass:
417.0922; 0.99 ppm.
2-[(4-bromophenyl)carbamoylamino]-N-(3-pyridyl)-2-
tetrahydropyran-4-yl-acetamide (9) Prepared according to pro-
cedure C with intermediate 2-[(4-bromophenyl)carbamoylamino]-
2-tetrahydropyran-4-yl-acetic acid (43) (0.14 mmol) and 3-
aminopyridine (1.1 equiv., 0.16 mmol). Crude product was puri-
fied by reverse phase column chromatography (C18 12 g, Water pH-
10/MeOH 90/10–>100) to give final product. Yield: 25 mg, 41%;
Appearance: white solid; LCMS: 1.99 min, m/z 433.1, 435.1 ((MþH)þ
1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
Purity>90%, Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.74 (s,
1 H), 8.73 (s, 1 H), 7.40e7.27 (m, 4 H), 6.41 (d, J ¼ 8.59 Hz, 1 H),
4.41e4.33 (m, 1 H), 4.09e3.95 (m, 2 H), 3.72e3.57 (m, 2 H),
2.12e1.99 (m, 1 H), 1.63e1.41 (m, 6 H), 1.35 (s, 9 H), 1.32e1.23 (m,
2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 172.40 (s, 1 C, C]O),
156.00 (1 C, C]O), 155.13 (s, 1 C, C]O), 140.25 (1 C, ArC.), 132.01
(2 C, ArCH), 119.91 (2 C, ArCH), 112.91 (1 C, ArC.), 79.22 (1 C,
C(CH3)3), 56.21 (1 C, CH), 43.36 (1 C, CH), 39.13 (2 C, CH2), 29.17 (1 C,
CH), 28.59 (3 C, C(CH3)3), 28.55 (2 C, CH2), 25.26 (2 C, CH2).
General procedure D for BOC deprotection: Starting material
was dissolved in dichloromethane and hydrochloric acid (4 mol/l)
12

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
in 1,4-dioxane (5e10 equiv.) was added. Reaction mixture was
stirred at room temperature for 2.5 h. The solvent was evaporated,
and crude product was purified by preparative HPLC (19 ꢀ 100mm
(2 C, CH2), 28.58 (2 C, CH2), 24.87 (1 C, CH2), 24.64 (1 C, CH2); HRMS:
Exact Mass: 423.1158; (MþH)þ: 424.1236; Observed Mass:
424.1231; 1.13 ppm.
(5
product.
N-(azetidin-3-yl)-2-[(4-bromophenyl)carbamoylamino]-2-
m
m) C-18 Waters Xbridge, MeOH, pH ¼ 10) to obtain final
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-
(tetrahydrofuran-2-ylmethyl)acetamide (18) Prepared according
to procedure C with intermediate 2-[(4-bromophenyl)carbamoy-
lamino]-2-cyclopentyl-acetic acid (42) (0.11 mmol) and 3-
oxetanamine (1.1 equiv., 0.122 mmol). Crude product was purified
by reverse phase column chromatography (C18 12 g, Water pH-10/
MeOH 90/10 / 100) to give final product. Yield: 31 mg, 62%;
Appearance: white solid; LCMS: 2.26 min, m/z 424.2, 426.2
((MþH)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz,
cyclopentyl-acetamide (14) Prepared according to procedure D
with intermediate tert-butyl 3-[[2-[(4-bromophenyl)carbamoyla-
mino]-2-cyclopentyl-acetyl]amino] azetidine-1-carboxylate (48).
The crude product was purified by preparative HPLC (19 ꢀ 100mm
(5
m
m) C-18 Waters Xbridge, MeOH, pH ¼ 10); Yield: 10 mg, 27%;
Appearance: white solid; LCMS: 2.05 min, m/z 395.1, 397.1 ((MþH)þ
1:1, 79/81Br), Purity>90%, Method D;1H NMR (500 MHz, DMSO‑d6)
DMSO‑d6)
d ppm 8.79 (s, 1 H), 8.22e8.14 (m, 1 H), 7.42e7.30 (m,
d
ppm 8.82 (s,1 H), 8.62 (d, J ¼ 6.87 Hz,1 H), 7.40e7.31 (m, 4 H), 6.45
4 H), 6.41 (d, J ¼ 9.17 Hz, 1 H), 4.16 (ddd, J ¼ 8.74, 7.30, 2.86 Hz, 1 H),
3.85e3.78 (m, 1 H), 3.77e3.69 (m, 1 H), 3.63e3.56 (m, 1 H),
3.25e3.14 (m, 1 H), 3.11e3.03 (m, 1 H), 2.08 (sxt, J ¼ 7.79 Hz, 1 H),
1.89e1.71 (m, 3 H), 1.62e1.40 (m, 7 H), 1.39e1.24 (m, 2 H); 13C NMR
(d, J ¼ 9.17 Hz, 1 H), 4.44 (sxt, J ¼ 7.33 Hz, 1 H), 4.10 (t, J ¼ 8.31 Hz,
1 H), 3.51 (t, J ¼ 7.45 Hz, 2 H), 3.39 (t, J ¼ 7.73 Hz, 2 H), 2.05 (qd,
J ¼ 7.80, 15.54 Hz, 1 H), 1.61e1.43 (m, 6 H), 1.37e1.24 (m, 2 H);
HRMS: Exact Mass: 394.1004; (MþH)þ: 395.1083; Observed Mass:
395.1078; 1.17 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-(1-
methyl-4-piperidyl) acetamide (15) Prepared according to pro-
cedure C with intermediate 2-[(4-bromophenyl)carbamoylamino]-
2-cyclopentyl-acetic acid (42) (0.11 mmol) and 4-amino-1-
methylpiperidine (1.1 equiv., 0.122 mmol). The crude product was
purified by preparative flash column chromatography
(126 MHz, DMSO‑d6)
d ppm 172.01 (1 C, C]O), 154.58 (1 C, C]O),
139.82 (1 C, ArC.), 131.43 (2 C, ArCH), 119.30 (2 C, ArCH), 112.30 (1 C,
ArC), 76.96 (1 C, CH), 67.19 (1 C, CH2), 55.48 (1 C, CH), 43.09 (1 C,
CH), 42.58 (1 C, CH2), 28.57 (1 C, CH2), 28.43 (1 C, CH2), 27.99 (1 C,
CH2), 25.14 (1 C, CH2), 24.86 (1 C, CH2), 24.69 (1 C, CH2); HRMS:
Exact Mass: 423.1158; (MþH)þ: 424.1236; Observed Mass:
424.1234; 0.42 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-(1,3-
dimethylpyrazol-4-yl)acetamide (19): Prepared according to
procedure C with intermediate 2-[(4-bromophenyl)carbamoyla-
mino]-2-cyclopentyl-acetic acid (42) (0.11 mmol) and 1,3-
dimethyl-1h-pyrazol-4-amine (1.1 equiv., 0.122 mmol). Crude
product was washed with diethyl ether to give products. Yield:
48 mg, 99%; Appearance: beige solid; LCMS: 2.14 min, m/z 434.1,
436.1 ((MþH)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
(19 ꢀ 100mm (5 m) C-18 Waters Xbridge, MeOH, pH ¼ 10). Yield:
m
5 mg, 10%; Appearance: white solid; LCMS: 2.17 min, m/z 437.1,
439.1 ((MþH)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
(500 MHz, DMSO‑d6)
d
ppm 8.78 (s, 1 H), 8.24 (br d, J ¼ 7.45 Hz, 1 H),
7.41e7.31 (m, 4 H), 6.44 (d, J ¼ 9.17 Hz, 1 H), 4.10 (br t, J ¼ 8.02 Hz,
1 H), 2.89 (br s, 2 H), 3.75 (br. s, 1 H), 2.12e2.01 (m, 2 H), 2.73e2.55
(m, 3 H), 1.95e1.82 (m, 2 H), 1.46 (s, 1 H), 1.64e1.43 (m, 8 H),
1.38e1.22 (m, 2H); two protons are hidden under DMSO peak;
HRMS: Exact Mass: 436.1474; (MþH)þ: 437.1552; Observed Mass:
437.1549; 0.72 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-(oxe-
tan-3-yl)acetamide (16) Prepared according to procedure C with
intermediate 2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-
acetic acid (42) (0.11 mmol) and 3-oxetanamine (1.1 equiv.,
0.122 mmol). The crude product was purified by preparative flash
(500 MHz, DMSO‑d6) d ppm 9.51 (s, 1 H), 8.81 (s, 1 H), 7.45e7.43 (m,
1 H), 7.41e7.33 (m, 4 H), 6.52e6.48 (m, 1 H), 4.35 (t, J ¼ 8.31 Hz,1 H),
3.68 (s, 3 H), 2.19e2.11 (m, 4 H), 1.65e1.53 (m, 4 H), 1.53e1.32 (m,
4 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 171.11 (1 C, C]O), 155.20
(1 C, C]O), 140.29 (1 C, ArC.), 132.89 (1 C, ArC), 132.06 (1C, ArC),
130.63 (2 C, ArCH), 119.95 (2 C, ArCH), 118.21 (1 C, ArCH), 112.90
(1 C, ArC), 56.03 (1 C, CH), 43.71 (1 C, CH), 36.92 (1 C, CH3), 29.17
(1 C, CH2), 28.70 (1 C, CH2), 25.23 (2 C, CH2), 9.32 (1 C, CH3); HRMS:
Exact Mass: 433.1113; (MþH)þ: 434.1193; Observed Mass:
434.1187; 1.39 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-(1-
isopropylpyrazol-4-yl)acetamide (20): Prepared according to
procedure C with intermediate 2-[(4-bromophenyl)carbamoyla-
mino]-2-cyclopentyl-acetic acid (42) (0.11 mmol) and 1-isopropyl-
1h-pyrazol-4-amine (1.1 equiv., 0.122 mmol). Crude product was
washed with diethyl ether to give products. Yield: 50 mg, 100%;
Appearance: white solid; LCMS: 2.23 min, m/z 448.1, 450.1 ((MþH)þ
1:1, 79/81Br), Purity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
column chromatography (19 ꢀ 100mm (5
mm) C-18 Waters
Xbridge, MeOH, pH ¼ 10); Yield: 2.6 mg, 6%; Appearance: white
solid; LCMS: 1.41 min, m/z 396.1, 398.1 ((MþH)þ 1:1, 79/81Br), Pu-
rity>95%, Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.90 (d,
J ¼ 6.30 Hz, 1 H), 8.77 (s, 1 H), 7.44e7.29 (m, 4 H), 6.43 (d,
J ¼ 8.59 Hz, 1 H), 4.82e4.73 (m, 1 H), 4.70 (td, J ¼ 6.73, 2.00 Hz, 2 H),
4.47e4.34 (m, 2 H), 4.15e4.15 (m, 1 H), 2.07 (sxt, J ¼ 7.90 Hz, 1 H),
1.64e1.42 (m, 6 H), 1.39e1.24 (m, 2 H); HRMS: Exact Mass:
395.0845; (MþH)þ: 396.0923; Observed Mass: 396.0918; 1.21 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-tet-
rahydropyran-4-yl-acetamide (17): Prepared according to pro-
cedure C with intermediate 2-[(4-bromophenyl)carbamoylamino]-
d
ppm 10.18 (s, 1 H) 8.78 (s, 1 H), 7.88 (s, 1 H), 7.42 (s, 1 H), 7.40e7.33
(m, 4 H), 6.50 (d, J ¼ 8.59 Hz, 1 H), 4.43 (dt, J ¼ 13.32, 6.80 Hz, 1 H),
4.19 (t, J ¼ 8.02 Hz, 1 H), 2.14 (sxt, J ¼ 7.90 Hz, 1 H), 1.69e1.53 (m,
4 H),1.53e1.43 (m, 2 H),1.37 (d, J ¼ 6.87 Hz, 6 H),1.35e1.29 (m, 2 H);
2-cyclopentyl-acetic
acid
(42)
(0.11
mmol)
and
4-
aminotetrahydropyran (1.1 equiv., 0.122 mmol). Crude product
was purified by reverse phase column chromatography (C18 12 g,
Water pH-10/MeOH 90/10 / 100) to give final product. Yield:
22 mg, 47%; Appearance: white solid; LCMS: 2.13 min m/z 424.2,
426.2 ((MþH)þ 1:1, 79/81Br), Purity>95% method D;1H NMR
13C NMR (126 MHz, DMSO‑d6)
d ppm 169.31 (1 C, C]O),154.69 (1 C,
C]O), 139.68 (1 C, ArC), 131.54 (2 C, ArCH), 129.36 (1 C, ArCH),
120.77 (1 C, ArC), 119.43 (2 C, ArCH), 118.12 (1 C, ArCH), 112.42 (1 C,
ArC), 56.10 (1 C, CH), 52.94 (1 C, CH), 42.73 (1 C, CH), 28.66 (1 C,
CH2), 28.30 (1 C, CH2), 24.66 (2 C, CH2), 22.63 (1 C, CH3); HRMS:
Exact Mass: 447.127; (MþH)þ:448.135; Observed Mass: 448.1345;
1.12 ppm.
(500 MHz, DMSO‑d6)
d
8.76 (s, 1 H), 8.11 (d, J ¼ 8.02 Hz, 1 H),
7.40e7.27 (m, 4H), 6.38 (d, J ¼ 9.17 Hz, 1 H), 4.13e4.05 (m, 1 H),
3.83e3.68 (m, 3 H), 2.02 (sxt, J ¼ 8.02 Hz, 1 H), 1.63 (d, J ¼ 12.60 Hz,
2 H), 1.59e1.47 (m, 4 H), 1.46e1.23 (m, 6 H); 13C NMR (126 MHz,
2-[(4-bromophenyl)carbamoylamino]-2-cyclopentyl-N-pyr-
idazin-3-yl-acetamide (21) Prepared according to procedure C
DMSO‑d6) d ppm 170.95 (1 C. C]O), 154.56 (1 C, C]O), 139.81 (1 C,
ArC), 131.49 (1 C, ArCH), 119.41 (1 C, ArCH), 112.32 (1 C, ArC), 65.86
(2 C, CH2), 55.46 (1 C, CH), 44.77 (1 C, CH), 43.41 (1 C, CH), 32.52
with
intermediate
2-[(4-bromophenyl)carbamoylamino]-2-
cyclopentyl-acetic acid (42) (0.11 mmol) and 3-aminopyridazine
13

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
(1.1 equiv., 0.122 mmol). Crude product was washed with diethyl
ether to give products. Yield: 14.3 mg, 31%; Appearance: white
solid; LCMS: 2.07 min, m/z 418.1, 420.1 ((MþH)þ 1:1, 79/81Br), Pu-
66.13 (1 C, Cgem), 36.49 (2 C, CH2), 36.37 (1 C, CH3), 23.72 (2 C, CH2),
8.82 (1 C, CH3); HRMS: Exact Mass: 419.0957; (MþH)þ: 420.1037;
Observed Mass: 420.1030; 1.67 ppm.
rity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d
ppm
1-[(4-bromophenyl)carbamoylamino]-N-(2H-tetrazol-5-
ylmethyl) cyclopentanecarboxamide (25): Prepared according to
procedure C with intermediate 1-[(4-bromophenyl)carbamoyla-
mino] cyclopentane carboxylic acid (47) (0.14 mmol) and c-(2h-
tetrazol-5-yl)-methylamine (1.1 equiv., 0.16 mmol). Crude product
was purified by reverse phase column chromatography (C18 12 g,
Water pH-10/MeOH 90/10 / 100) to give final product. Yield:
20 mg, 40%; Appearance: white solid; LCMS: 1.55 min, m/z 408.1,
410.1 ((MþH)þ 1:1, 79/81Br), Purity>90%, Method D; 1H NMR
11.31e11.20 (br.s, 1 H), 8.94 (dd, J ¼ 4.58, 1.72 Hz, 1 H), 8.79 (s, 1 H),
8.27 (dd, J ¼ 9.17, 1.72 Hz, 1 H), 7.65 (dd, J ¼ 9.17, 4.58 Hz, 1 H), 7.33
(q, J ¼ 9.17 Hz, 4 H), 6.56 (d, J ¼ 8.02 Hz, 1 H), 4.46 (t, J ¼ 7.73 Hz,
1 H), 2.19 (sxt, J ¼ 7.79 Hz, 1 H), 1.73e1.53 (m, 4 H), 1.52e1.38 (m,
3 H),1.35e1.25 (m,1 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 173.17
(1 C, C]O), 155.39 (1 C, ArC), 154.77 (1 C, C]O), 148.62 (1 C, ArCH),
139.60 (1 C, ArC), 131.50 (2 C, ArCH), 128.56 (1 C, ArCH), 119.51 (2 C,
ArCH), 118.38 (1 C, ArCH), 112.53 (1 C, ArC), 56.49 (1 C, CH), 42.33
(1 C, CH), 28.55 (1 C, CH2), 28.13 (1 C, CH2), 24.68 (2 C, CH2);; HRMS:
Exact Mass: 417.0800; (MþH)þ 418.0880; Observed Mass:
418.0876; 0.96 ppm.
(500 MHz, DMSO‑d6)
d ppm 8.78 (br s, 1 H), 8.54 (br s, 1 H),
7.49e7.28 (m, 4 H), 6.74 (br s, 1 H), 4.54 (br d, J ¼ 5.16 Hz, 2 H), 2.08
(br d, J ¼ 13.17 Hz, 2 H), 1.66 (br s, 4 H) 1.81 (br d, J ¼ 12.60 Hz, 2 H);
1-[(4-bromophenyl)carbamoylamino]-N-(3-pyridyl)cyclo-
pentanecarboxamide (22) Prepared according to procedure C with
13C NMR (126 MHz, DMSO‑d6)
d ppm 174.74 (1 C, C]O), 154.99 (1 C,
C]O), 154.84 (1 C, ArC), 139.40 (1 C, ArC), 131.46 (2 C, ArCH), 119.62
(2 C, ArCH), 112.73 (1 C, ArC), 65.86 (1 C, Cgem), 36.52 (2 C, CH2),
33.18 (1 C, CH2), 23.79 (2 C, CH2);; HRMS: Exact Mass: 407.0705;
(MþH)þ: 408.0785; Observed Mass: 408.0777; 1.97 ppm.
intermediate
1-[(4-bromophenyl)carbamoylamino]cyclo-
pentanecarboxylic acid (47) (0.21 mmol) and 3-aminopyridine (1.1
equiv., 0.23 mmol). Crude product was dry loaded on silica and
purified by flash column chromatography (silica gel, PE/EA 10–
>100%, EA/MeOH 2% gradient).; Yield: 45 mg, 50%; Appearance:
white solid; LCMS: 1.99 min, m/z 403.1, 405.1 (MþH)þ (1:1, 79/81Br),
1-[(4-bromophenyl)carbamoylamino]-N-(1-methyl-4-
piperidyl) cyclopentane carboxamide (26): Prepared according to
procedure C with intermediate 1-[(4-bromophenyl)carbamoyla-
mino] cyclopentane carboxylic acid (47) (0.12 mmol) and 4-amino-
1-methylpiperidine (1.1 equiv., 0.13 mmol). The crude product was
purified by preparative flash column chromatography
Purity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 9.75 (s,
1 H), 8.75 (d, J ¼ 2.29 Hz, 1 H), 8.74e8.71 (m, 1 H), 8.23 (dd, J ¼ 4.58,
1.72 Hz, 1 H), 8.06e7.99 (m, 1 H), 7.40e7.29 (m, 5 H), 6.54 (s, 1 H),
2.23e2.13 (m, 2 H), 1.91e1.82 (m, 2 H), 1.75e1.67(m, 4 H); 13C NMR
(126 MHz, DMSO‑d6) d ppm 173.40 (1 C, C]O), 154.20 (1 C, C]O),
143.99 (1 C, ArCH), 141.77 (1 C, ArCH), 139.52 (1 C, ArC), 136.00 (1 C,
ArC,131.61 (2 C, ArCH),126.99 (1 C, ArCH),123.51 (1 C, ArCH),119.54
(2 C, ArCH), 112.53 (1 C, ArC), 66.49 (1 C, Cgem), 36.38 (1 C, CH2),
36.29 (1 C, CH2), 23.69 (2 C, CH2); HRMS: Exact mass: 402.0691;
(MþH)þ: 403.0771; Observed mass: 403.0760; 2.83 ppm.
(19 ꢀ 100mm (5 m) C-18 Waters Xbridge, MeOH, pH ¼ 10); Yield:
m
10 mg, 20%; Appearance: white solid; LCMS: 2.00 min, m/z 423.1,
425.1 ((MþH)þ 1:1, 79/81Br), Purity>90%, Method D; 1H NMR
(500 MHz, DMSO‑d6) d 8.68 (s, 1H), 7.40e7.31 (m, 4H), 6.41 (s, 1 H),
3.59e3.48 (m, 1H), 2.75 (d, J ¼ 9.16 Hz, 2 H), 2.21 (s, 3 H), 2.14 (d,
J ¼ 12.03 Hz, 2 H), 2.07e1.97 (m, 2H), 1.83e1.74 (m, 2 H), 1.64 (m,
6 H), 1.40e1.51 (m, 2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm
1-[(4-bromophenyl)carbamoylamino]-N-pyrimidin-5-yl-
173.33 (1C, C]O) 154.20 (1 C, C]O) 139.78 (1 C, ArC) 131.40 (2 C,
ArCH) 119.37 (2 C, ArCH) 112.29 (1 C, ArC) 65.98 (1 C, Cgem) 53.81
(2 C, CH2) 48.61 (1 C, CH) 45.29 (1 C, CH3) 36.39 (2 C, CH2) 30.72 (2 C,
CH2) 23.75 (2 C, CH2); HRMS: Exact Mass: 422.1317; (MþH)þ
423.1397; Observed Mass: 423.1390; 1.66 ppm.
cyclopentanecarboxamide (23) Prepared according to procedure C
with intermediate 1-[(4-bromophenyl)carbamoylamino]cyclo-
pentanecarboxylic acid (47) (0.21 mmol) and 5-aminopyrimidine
(1.1 equiv., 0.23 mmol). Crude product was dry loaded on silica
and purified by flash column chromatography (silica gel, PE/EA 10–
>100%, EA/MeOH 2% gradient).; Yield: 37 mg, 43%; Appearance:
white solid; LCMS: 2.03 min, m/z 404.1, 406.1 ((MþH)þ 1:1, 79/81Br),
1-[(4-bromophenyl)carbamoylamino]-N-(1-
isopropylpyrazol-4-yl) cyclopentane carboxamide (27) Prepared
according to procedure C with intermediate 1-[(4-bromophenyl)
carbamoylamino] cyclopentanecarboxylic acid (47) (0.12 mmol)
and 1-isopropyl-1h-pyrazol-4-amine (1.1 equiv., 0.13 mmol). Crude
product was washed with diethyl ether to obtain desired product.;
Yield: 36 mg, 68%; Appearance: white solid; LCMS: 2.01 min, m/z
434.1, 436.1 (MþHþ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
Purity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 9.97 (s,
1 H), 9.03 (s, 2 H), 8.85 (s, 1 H), 8.70 (s, 1 H), 7.40e7.31 (m, 4 H), 6.60
(s, 1 H), 2.24e2.14 (m, 2 H), 1.90e1.80 (m, 2 H), 1.76e1.68 (m, 4 H);
13C NMR (126 MHz, DMSO‑d6)
d ppm 173.85 (1 C, C]O),154.20 (1 C,
C]O), 152.91 (1 C, ArCH), 147.83 (1 C, ArCH), 139.43 (1 C, ArC),
134.72 (1 C, ArC), 131.64 (2 C, ArCH), 119.58 (2 C, ArCH), 112.61 (1 C,
ArC), 66.39 (1 C, Cgem), 36.41 (2 C, CH2), 23.70 (2 C, CH2); HRMS:
Exact mass: 403.0644; (MþH)þ: 404.0724; Observed mass:
404.0723; 0.22 ppm.
(500 MHz, DMSO‑d6)
d ppm 9.61 (s, 1 H) 8.62 (s, 1 H), 7.86 (s, 1 H),
7.42 (d, J ¼ 1.15 Hz, 1 H), 7.36e7.28 (m, 4 H), 6.43 (s, 1 H), 4.38 (quin,
J ¼ 6.73 Hz, 1 H), 2.13e2.04 (m, 2 H), 1.82e1.76 (m, 2 H), 1.69e1.62
(m, 4 H), 1.33 (d, J ¼ 6.87 Hz, 6 H); 13C NMR (126 MHz, DMSO‑d6)
1-[(4-bromophenyl)carbamoylamino]-N-(1,3-
d ppm 171.30 (1 C, C]O), 154.08 (1 C, C]O), 139.66 (1 C, ArC),
dimethylpyrazol-4-yl) cyclopentane carboxamide (24) Prepared
according to procedure C with intermediate 1-[(4-bromophenyl)
carbamoylamino] cyclopentane carboxylic acid (47) (0.14 mmol)
and 1,3-dimethyl-1H-pyrazol-4-amine (1.1 equiv., 0.16 mmol).
Crude product was purified by reverse phase column chromatog-
raphy (C18 12 g, Water pH-10/MeOH 90/10 / 100) to give final
product. Yield: 20 mg, 43%; Appearance: white solid; LCMS:
1.92 min, m/z 420.1, 422.1 (MþHþ 1:1, 79/81Br), Purity>95%, Method
131.45 (2 C, ArCH), 129.39 (1 C, ArCH), 121.71 (1 C, ArC), 119.29 (2 C,
ArCH), 117.90 (1 C, ArCH), 112.38 (1 C, ArC), 66.04 (1 C, Cgem), 52.84
(1 C, CH), 36.56 (2 C, CH2), 23.74 (2 C, CH2), 22.67 (2 C, CH3); HRMS:
Exact Mass: 433.1113; Observed Mass: 434.1187; 1.39 ppm.
1-[(4-bromophenyl)carbamoylamino]-N-(2-pyridyl)cyclo-
pentanecarboxamide (28) Prepared according to procedure C with
intermediate 1-[(4-bromophenyl)carbamoylamino] cyclopentane
carboxylic acid (47) (0.12 mmol) and 2-aminopyridine (1.1 equiv.,
0.13 mmol). The crude product was purified by preparative flash
D; 1H NMR (500 MHz, DMSO‑d6)
d ppm 8.96 (s, 1 H), 8.70 (s, 1 H),
7.41e7.33 (m, 4 H), 7.24 (s, 1 H), 6.52 (s, 1 H,), 3.66 (s, 3 H), 2.18e2.09
column chromatography (19 ꢀ 100mm (5
mm) C-18 Waters
Xbridge, MeOH, pH ¼ 10); Yield: 3.8 mg, 7.7%; Appearance: white
(m, 2 H), 2.06 (s, 3 H), 1.90e1.82 (m, 2 H), 1.71e1.65 (m, 4 H); 13C
NMR (126 MHz, DMSO‑d6) d ppm 172.91 (1 C, C]O),154.28 (1 C, C]
O), 139.73 (1 C, ArC), 133.32 (1 C, ArC), 131.89 (1 C, ArCH), 131.45
(2 C, ArCH), 119.35 (2 C, ArCH), 117.82 (1 C, ArC), 112.38 (1 C, ArC),
solid; LCMS: 2.08 min, m/z 403.1, 405.1 ((MþH)þ 1:1, 79/81Br), Pu-
rity>95%, Method D; 1H NMR (500 MHz, DMSO‑d6)
d 9.85 (s, 1H),
8.91 (s, 1H), 8.22 (d, J ¼ 4.81 Hz, 1H), 8.06 (d, J ¼ 8.17 Hz, 1H),
14

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
7.77e7.72 (m, 1H), 7.39e7.27 (m, 4H), 7.09e6.98 (m, 1H), 6.65 (s,
1H), 2.19e2.10 (m, 2H), 1.93e1.85 (m, 2H), 1.69e1.63 (m, 4H);
HRMS: Exact Mass: 402.0691; (MþH)þ 403.0771; Observed Mass:
403.0769; 0.5 ppm.
1-[(4-bromophenyl)carbamoylamino]-N-(6-methyl-3-
pyridyl) cyclopentane carboxamide (29) Prepared according to
procedure C with intermediate 1-[(4-bromophenyl)carbamoyla-
mino] cyclopentane carboxylic acid (47) (0.12 mmol) and 5-amino-
2-methylpyridine (1.1 equiv., 0.13 mmol). The crude product was
purified by preparative flash column chromatography
dichloromethane (5 mL). The DCM solution was washed with ice-
cold aqueous KHSO4 solution 3 times. The organic layer filtered
through a phase separator. The solvent was evaporated under
reduced pressure to obtain the free acid. Yield: 50 mg, 80%;
Appearance: colourless liquid; LCMS: UV inactive; 1H NMR
(500 MHz, CHLOROFORM-d)
d ppm 11.10 (br s, 1 H), 5.18 (br d,
J ¼ 8.02 Hz, 1 H), 4.58e4.30 (m,1 H), 1.80e1.60 (m, 2 H), 1.44 (s, 9 H),
0.89e0.67 (m, 1 H), 0.55e0.42 (m, 2 H), 0.21e0.04 (m, 2 H).
Tert-butyl
N-[(1S)-1-(cyclopropylmethyl)-2-oxo-2-(3-
pyridylamino)ethyl]carbamate (51) Prepared according to pro-
cedure C with intermediate 49 (0.19 mmol) and 3-aminopyridine
(1.3 equiv.) The resulting product was purified by reverse phase
column chromatography (C-18, ACN/water pH-10 10:90 / 80:20)
to give desired product Yield: 30 mg, 35%; Appearance: white solid;
LCMS: 1.30 min, m/z 306.3 (MþH)þ Purity >90%, Method B; 1H NMR
(19 ꢀ 100mm (5 m) C-18 Waters Xbridge, MeOH, pH ¼ 10). Yield:
m
15 mg, 29%; Appearance: white solid; LCMS: 2.05 min, m/z 417.1,
419.1 ((MþH)þ 1:1, 79/81Br), Purity>95%, Method D; 1H NMR
(500 MHz, DMSO‑d6)
d
9.67 (s, 1H), 8.78 (s, 1H), 8.60 (d, J ¼ 2.86 Hz,
1H), 7.89 (dd, J ¼ 2.58, 8.31 Hz, 1H), 7.40e7.31 (m, 4H), 7.16 (d,
J ¼ 8.02 Hz, 1H), 6.59 (s, 1H), 2.39 (s, 3H), 2.20e2.12 (m, 2H),
1.89e1.82 (m, 2H), 1.70 (t, J ¼ 6.87 Hz, 4H); 13C NMR (126 MHz,
(500 MHz, CHLOROFORM-d)
d ppm 9.03e8.81 (m, 1 H), 8.59 (d,
J ¼ 2.29 Hz, 1 H), 8.31 (br. s., 1 H), 8.06 (br. s., 1 H), 7.21 (br. s., 1 H),
5.38 (d, J ¼ 7.45 Hz, 1 H), 4.39 (br. s., 1 H), 1.83e1.65 (m, 2 H),
1.47e1.45 (m, 9 H), 0.85e0.75 (m, 1 H), 0.56e0.46 (m, 2 H),
0.18e0.10 (m, 2 H).
DMSO‑d6) d ppm 173.24 (1 C, C]O), 154.22 (1 C, C]O), 152.09 (1 C,
ArC.), 140.94 (1 C, ArCH), 139.59 (1 C, ArC), 133.50 (1 C, ArC), 131.46
(2 C, ArCH), 127.66 (1 C, ArCH), 122.50 (1 C, ArCH), 119.39 (2 C,
ArCH), 112.46 (1 C, ArC), 66.47 (1 C, Cgem), 36.35 (2 C, CH2), 23.70
(2 C, CH2), 23.33 (1 C, CH3); HRMS: Exact Mass: 416.0848;
(MþH)þ:417.0928; Observed Mass: 417.0924; 0.96 ppm.
(2S)-2-amino-3-cyclopropyl-N-(3-pyridyl)propenamide HCl
(53) Prepared according to general procedure D with the inter-
mediate tert-butyl N-[(1S)-1-(cyclopropylmethyl)-2-oxo-2-(3-
pyridylamino)ethyl]carbamate (51) (0.13 mmol). The crude prod-
uct was used in the next step without further purification. Yield:
30 mg, 95%; Appearance: white solid; LCMS: 0.26 min, m/z 206.2,
(MþH)þ Purity >90%, Method B; 1H NMR (500 MHz, DMSO‑d6)
1-[(4-bromophenyl)carbamoylamino]-N-(5-methyl-3-
pyridyl) cyclopentane carboxamide (30): Prepared according to
procedure C with intermediate 1-[(4-bromophenyl)carbamoyla-
mino] cyclopentane carboxylic acid (47) (0.12 mmol) and 3-amino-
5-methylpyridine (1.1 equiv., 0.13 mmol). The crude product was
purified by preparative flash column chromatography
d
ppm 9.07 (d, J ¼ 2.29 Hz, 1 H), 8.53 (dd, J ¼ 5.16, 1.15 Hz, 1 H),
8.50e8.45 (m, 3 H), 8.45e8.42 (m, 1H), 7.79 (dd, J ¼ 8.59, 5.16 Hz,
1 H), 4.24e4.10 (m,1 H), 1.93e1.85 (m, 1 H), 1.71e1.61 (m, 1 H),
0.87e0.76 (m, 1 H), 0.47e0.34 (m, 2 H), 0.18e0.05 (m, 2 H).
2-amino-2-cyclopropyl-N-(3-pyridyl)acetamide HCl (54) Pre-
pared according to general procedure D with the intermediate (52)
(0.395 mmol). The crude product was used in the next step without
further purification. Yield: 85 mg, 95%; Appearance: off-white
solid; LCMS: 0.54 min, m/z 192.2 (MþH)þ Purity 90%, Method B;
(19 ꢀ 100mm (5 m) C-18 Waters Xbridge, MeOH, pH ¼ 10); Yield:
m
10 mg, 20%; Appearance: white solid; LCMS: 2.10 min, m/z 417.1,
419.1 ((MþH)þ 1:1, 79/81Br), Purity ¼ 88%, Method D; 1H NMR
(500 MHz, DMSO‑d6)
d ppm 9.66 (s, 1 H), 8.73 (s, 1 H), 8.55 (d,
J ¼ 2.29 Hz, 1 H), 8.05 (d, J ¼ 1.15 Hz, 1 H), 7.84 (t, J ¼ 2.00 Hz, 1 H),
7.39e7.26 (m, 4 H), 6.54 (s, 1 H), 2.23 (s, 3 H), 2.19e2.09 (m, 2 H),
1.87e1.78 (m, 2 H), 1.72e1.63 (m, 4 H); 13C NMR (126 MHz,
DMSO‑d6)
d
ppm 173.37 (1 C, C]O) 154.20 (1 C, C]O) 144.22 (1 C,
1H NMR (500 MHz, DMSO‑d6)
d ppm 12.09 (br s, 1 H), 9.16 (d,
ArCH) 139.56 (1 C, ArC) 138.75 (1C., ArCH) 135.65 (1 C, ArC) 132.65
(1 C, ArCH) 131.47 (2 C, ArCH) 127.34 (1 C, ArC) 119.37 (2 C, ArCH)
112.49 (1 C, ArC) 66.49 (1 C, Cgem.) 36.33 (2 C, CH2) 23.69 (2 C, CH2)
17.87 (1 C, CH3);; HRMS: Exact Mass: 416.0848; (MþH)þ: 417.0928;
Observed Mass: 417.0925; 0.72 ppm.
J ¼ 2.29 Hz, 1 H), 8.59e8.54 (m, 4 H), 7.86 (dd, J ¼ 8.31, 5.44 Hz, 1 H),
3.51 (dq, J ¼ 9.88, 5.11 Hz, 1H), 1.19e1.09 (m, 1H), 1.08e1.00 (m, 1H),
0.71e0.60 (m, 1H), 0.59e0.51 (m, 2H).
(2S)-2-[(4-bromophenyl) carbamoyl amino]-3-cyclopropyl-
N-(3 pyridyl) propenamide (11) Compound was prepared ac-
1-[(4-bromophenyl)carbamoylamino]-N-(6-chloro-3-
cording to procedure A with 1-bromo-4-isocyanato-benzene
pyridyl) cyclopentane carboxamide (31): Prepared according to
procedure C with intermediate 1-[(4-bromophenyl)carbamoyla-
mino] cyclopentane carboxylic acid (47) (0.12 mmol) and 5-amino-
2-chloropyridine (1.1 equiv., 0.13 mmol). Crude product was
washed with diethyl ether to obtain desired product. Yield: 40 mg,
75%; Appearance: white solid; LCMS: 2.15 min, m/z 437.0, 439.0,
441.0 ((MþH)þ 3:4:2, 79/81Br and 35/37Cl), Purity>95%, Method D; 1H
(0.1 mmol) and the intermediate (2S)-2-amino-3-cyclopropyl-N-
(3-pyridyl) propenamide HCl (53) (1 equiv., 0.1 mmol). Crude
product was purified by reverse phase column chromatography
(C18, acetonitrile/water pH ¼ 10 10/90 / 80/20). Yield: 27 mg,
66%; Appearance: white solid; LCMS: 1.45 min, m/z 403.1, 405.1
((MþH)þ 1:1, 79/81Br), Purity >95%, Method B 1H NMR (500 MHz,
DMSO‑d6)
d
ppm 10.40 (s, 1 H), 8.86 (s,1 H), 8.76 (d, J ¼ 1.72 Hz,1 H),
NMR (500 MHz, DMSO‑d6)
d
ppm 9.87 (s, 1 H), 8.69 (s, 1 H), 8.60 (d,
8.27 (dd, J ¼ 4.87,1.43 Hz, 1 H), 8.04 (ddd, J ¼ 8.31, 2.58,1.15 Hz,1 H),
7.43e7.31 (m, 5 H), 6.64 (d, J ¼ 8.02 Hz, 1 H), 4.46 (q, J ¼ 7.45 Hz,
1 H), 1.70 (dt, J ¼ 13.75, 6.87 Hz, 1 H), 1.52 (ddd, J ¼ 13.75, 7.45,
6.30 Hz, 1 H), 0.85e0.65 (m, 1 H), 0.46e0.36 (m, 2 H), 0.15e0.03 (m,
J ¼ 2.29 Hz, 1 H), 8.07 (dd, J ¼ 8.59, 2.86 Hz, 1 H), 7.41 (d, J ¼ 8.59 Hz,
1 H), 7.36e7.33 (m, 2 H), 7.31e7.28 (m, 2 H), 6.53 (s, 1 H), 2.19e2.10
(m, 2 H), 1.86e1.77 (m, 2 H), 1.71e1.64 (m, 4 H); 13C NMR (126 MHz,
DMSO‑d6)
d
ppm 174.05 (1 C, C]O) 154.70 (1 C, C]O) 143.91 (1 C,
2 H); 13C NMR (126 MHz, DMSO‑d6)
d ppm 171.85 (1 C, C]O),
ArC) 141.62 (1 C, ArCH) 139.98 (1 C, ArC) 136.20 (1 C, ArC) 131.94
(2 C, ArCH) 131.06 (1 C, ArCH) 124.50 (s, 1 C, ArCH) 120.02 (2 C,
ArCH) 113.09 (1 C, ArC) 66.99 (1 C, Cgem) 36.86 (2 C, CH2) 24.20
(2 C, CH2); HRMS: Exact Mass: 436.0302; (MþH)þ: 437.0382
Observed Mass: 437.0374; 1.83 ppm.
(2S)-2-(tert-butoxycarbonylamino)-3-cyclopropyl-propanoic
acid (49) The cyclohexylamine (CHA) salts need to be converted to
the free acid before they can be utilized in carbodiimide coupling.
Commercially available (2S)-2-(tert-butoxycarbonylamino)-3-
cyclopropyl-propanoic acid CHA salt was dissolved in
154.56 (1 C, C]O), 144.37 (1 C, ArCH), 140.93 (1 C, ArCH), 139.66
(1 C, ArC), 135.55 (1 C, ArC), 131.45 (2 C, ArCH), 126.25 (1 C, ArCH),
123.70 (1 C, ArCH), 119.47 (2 C, ArCH), 112.50 (1 C, ArC), 54.05 (1 C,
CH), 37.70 (1 C, CH2), 7.36 (1 C, CH), 4.41 (1 C, CH2), 4.13 (1 C, CH2);
HRMS: Exact Mass: 402.0691; (MþH)þ: 403.0770; Observed Mass:
403.0763; 1.65 ppm.
2-[(4-bromophenyl)carbamoylamino]-2-cyclopropyl-N-(3-
pyridyl)acetamide (12) Compound was prepared according to
procedure A with 1-bromo-4-isocyanato-benzene (0.32 mmol) and
the intermediate 2-amino-2-cyclopropyl-N-(3-pyridyl)acetamide
15

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
HCl (54) (1.1 equiv. 0.35 mmol). Crude product was purified by
reverse phase column chromatography (C18, acetonitrile/water
pH ¼ 10 10/90 / 80/20). Yield: 85 mg, 69%; Appearance: white
solid; LCMS: 1.37 min, m/z 389.1, 391.1 ((MþH)þ 1:1, 79/81Br), Purity
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;
>95%, Method B; 1H NMR (500 MHz, DMSO‑d6)
d ppm 10.35 (s, 1 H),
8.84 (s, 1 H), 8.78 (d, J ¼ 1.72 Hz,1 H), 8.28 (dd, J ¼ 4.58,1.15 Hz,1 H),
8.06 (ddd, J ¼ 8.31, 2.58, 1.15 Hz, 1 H), 7.40e7.33 (m, 5 H), 6.70 (d,
J ¼ 8.02 Hz, 1 H), 3.89 (t, J ¼ 7.73 Hz, 1 H), 1.16e1.09 (m, 1 H),
0.59e0.46 (m, 3 H), 0.38e0.34 (m, 1 H); 13C NMR (126 MHz,
Pathhunter detection kit (93-0001) 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.
Calcium mobilization assay protocol 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 concentration of 0.5x106/mL in F-12 nutrient
mixture (HAM) (21765-037, Gibco®) containing 10% Fetal Bovine
DMSO‑d6) d ppm 171.29 (1 C, C]O), 154.51 (1 C, C]O), 144.43 (1 C,
ArC), 140.80 (1 C, ArC), 139.57 (1 C, ArC), 135.57 (1 C, ArC.), 131.47
(2 C, ArCH), 126.14 (1 C, ArCH), 123.74 (1 C, ArCH), 119.47 (2 C,
ArCH), 112.56 (1 C, ArC), 56.59 (1 C, CH), 14.34 (1 C, CH), 2.98 (1 C,
CH2), 2.30 (1 C, CH2); HRMS: Exact Mass: 388.0535; (MþH)þ:
389.0613; Observed Mass: 389.0608; 1.32 ppm.
Methyl 2-(benzyloxycarbonylamino)-2-tetrahydropyran-4-
ylidene-acetate (55) To acetonitrile (10 mL) solution of methyl 2-
(benzyloxycarbonylamino)-2-dimethoxyphosphoryl-acetate (1 eq.,
2 g), 1,8-diazabicyclo[5.4.0]undec-7-ene (1 eq., 0.92 g) was added at
room temperature and stirred for 30 min. Acetonitrile (5 mL) so-
lution of tetrahydropyran-4-one (1 eq., 0.60 g) was added and
stirred overnight at room temperature. Water and ethyl acetate
were added to the reaction mixture, and the organic layer was
separated. The organic layer was washed with 2M HCl solution,
saturated brine, water. Then the organic phase was filtered through
the phase separator and concentrated under reduced pressure.
Crude product was purified via flash column silica (50 g silica,
PE:EtOAc 100:0, 70:30, 30:70) to obtain desired product as a white
solid. Yield: 800 mg, 40%; Appearance: white solid; LCMS: 1.42 min,
m/z 306.2 (MþH)þ, Purity >95%, Method B 1H NMR (500 MHz,
Serum (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 mL 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-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.
Isolation of human neutrophils from venous blood Blood was
collected from healthy donors in accordance with a protocol
approved by the Ludwig Maximilian University of Munich. Neu-
trophils were isolated from the blood using Polymorphprep (Axi-
Shield) following the manufacturer’s instructions. Isolated neu-
trophils were washed and resuspended in HBSS buffer containing
20 mM HEPES, 0.25% BSA, Ca2þ pH 7.4 (0.5x106 cells/mL) and used
on the day of the experiment.
CHLOROFORM-d) d ppm 7.45e7.29 (m, 5 H), 6.02 (br s, 1 H), 5.15 (s,
2 H), 3.97e3.51 (m, 7 H), 2.94 (br s, 2 H), 2.43 (t, J ¼ 5.44 Hz, 2 H);
Data corresponded to that reported in the literature [63,64].
Methyl 2-amino-2-tetrahydropyran-4-yl-acetate (56) The
benzyl deprotection reaction was performed in the H-Cube reactor
(10% Pd/C CatCart®, full hydrogen mode, 50 ꢁC, 1.0 mL/min, one
cycle). The reaction mixture was concentrated under reduced
pressure. It was used in the next step without purification. Methyl
2-amino-2-tetrahydropyran-4-yl-acetate was obtained with high
yield as a colourless oil. Yield: 440 mg, 98%; Appearance: colourless
oil; LCMS: non-UV active; 1H NMR (500 MHz, CHLOROFORM-d)
d
ppm 4.03e3.96 (m, 2 H), 3.73 (s, 3 H), 3.37 (td, J ¼ 11.74, 1.72 Hz,
Human neutrophil static adhesion assay A static adhesion
assay was performed with the aim of examining the small molecule
agonists anti-inflammatory and proresolving properties. The day
before the assay, 96-well flat-bottomed plates were coated with
2 H), 3.29 (d, J ¼ 6.30 Hz, 1 H), 1.85 (tdt, J ¼ 2x11.89, 6.01,
2 ꢀ 3.72 Hz, 1 H), 1.63e1.40 (m, 6 H); Data corresponded to that
reported in the literature [63,64].
50 mL of cell adhesion molecules with the final concentration 1 mg/
4.3. General biological and ADMET procedures
mL: P-selectin (13025-H02H-100; SinoBiological) and ICAM- 1
(10346-H03H-100; SinoBiological). The coated plates were kept at
4 ꢁC overnight. The excess of solution was removed with a pipette
and the surface was blocked with BSA 2% for 1 h at room temper-
PathHunter® CHOeK1 FPRL1 cells (DISCOVERx) were grown in
the F-12 nutrient mixture (HAM) (21765-037, Gibco®) supple-
mented with 10% Fetal Bovine Serum (A3840401, Thermo Fisher
ature. Neutrophils, 120
m
L of neutrophils (0.5x106 cells/mL) per
Sci), Hygromycin B 800
Geneticin 300
m
g/mL (10687-010, Thermo Fisher Sci),
well, were pre-incubated with 30 mL of the agonists in non-coated
m
g/mL (10131-027, Thermo Fisher Sci). The cells were
plate (final dilution 1:5). Cells were incubated 30 min 37 ꢁC with
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.
gentle continuous rocking. The pre-incubated neutrophils were
added to the assay plate, 125 mL of pre-incubated neutrophils per
well. The reaction was incubated for 20 min at 37 ꢁC. Non-adherent
neutrophils were removed, and the fixation step was performed
with PFA 4%. Cells were stained with DAPI (Molecular Probes) for
b
-Arrestin Path Hunter® assay detection protocol To identify
novel FPR2 agonists PathHunter® CHOeK1 FPRL1 cells (DISCOV-
ERx) expressing complementing fragments of the -galactosidase
b
10 min and then washed gently with 200 mL PBS. Four pictures per
(
b
-gal) enzyme were used. One day before the assay, the cells were
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 processed using an ImageJ (National Institutes of
Health). Raw data were entered into GraphPad Prism 7.01 analysis
software (GraphPad Software, San Diego, CA, USA) for data pro-
cessing and visualisation. Each compound was tested in technical
diluted to a concentration of 0.5x106/mL in DISCOVERx plating
media (Assay CompleteTM Cell Plating 2 Reagent; 93e0563R2A).
The cells were plated out 10
mL per well of 384 well plate
(5,000 cells per well) and incubated 1 h at room temperature then
overnight at 37 ꢁC. 10 mM stock solutions of compounds were
16

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
triplicates.
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
Statistical data analysis GraphPad Prism 7.01 (GraphPad Soft-
ware, San Diego, CA, USA) was used for statistical analysis of the
data. For comparison of treatment groups with the control group
and P- value determination, 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.
PK studies. 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
Acquity UPLC BEH C8 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).
Microsomal stability assay The human and mouse liver mi-
crosomes (MLM) were obtained from BD Biosciences. The com-
pounds 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 Biosciences). The final compound con-
centration in the incubation is 1 mM, and the microsomal protein
(n
¼
12) were treated with either the vehicle (DMSO
concentration is 0.2 mg/mL. A sample was taken at t ¼ 0 and
quenched with two times volume of ice-cold methanol containing
an internal standard reference compound (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-MS/MS (Agilent Technologies G6410
series, triple quadrupole with MM-ESI ion source) using optimised
multiple reaction monitoring scans and a standard column gradient
(10%) þ PEG400 (35%) þ PG (25%) þ water (30%)) or the clear so-
lution of Compound 8 (10 mg/kg in vehicle).
Cell Health Assay. In order to measure a variety of parameters
which leads to cell viability, cell death, cytotoxicity or cell prolif-
eration, 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 media. 2000 HepG2 cells per well were plated in
on an Acquity UPLC BEH C8 1.7 mm column (mobile phase: aceto-
nitrile and water with 0.05% acetic). The % turnover was obtained
by calculating the percentage difference of the peak areas, nor-
malised to the internal standard, at t ¼ 0 and t ¼ 30. Verapamil was
used as the standard compound.
25 m
L media of a 384 well plate and incubated at 37 ꢁC overnight.
The compounds were added from the serial dilution plates using
Biomek robot (automated 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 mea-
surements were imaged on the InCELL Analyzer 2000 using the
following four wavelength channels for each of the four dyes:
Hoechst (nuclei) - DAPI channel; Fluo-4 AM (cellular calcium) -
Fluorescein isothiocyanate (FITC) channel; Tetramethylrhodamine,
methyl ester (TMRM) (mitochondria) - Texas Red channel; TOTO-3
(dead nuclei) e Cy® 5 channel. Raw data were processed and
visualized in Genedata Screener analysis software. The cell mea-
surements were normalised 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.
Kinetic solubility measurements The kinetic solubility was
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. MM performed 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. MM and
TC prepared the first draft of the manuscript. All authors have given
approval to the final version of the manuscript.
measured by diluting a 4
compound into PBS buffer pH 7.4 in a filtration plate at a target
concentration of 200 M giving a final solution composition of 98:2
mL of 10 mM DMSO stock solution of the
m
PBS:DMSO. For each compound, measurements were performed in
triplicates with two standard compounds (Verapamil and Ketoco-
nazole) 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 compo-
sition of PBS:DMSO 80:20. A serial dilution for each compound was
then performed in flat bottomed plates in PBS:DMSO 80:20 with
Funding
This work was supported by the European Union’s Horizon 2020
research and innovation program under the Marie Sklodowska-
Curie grant agreement No. 675111.
Declaration of competing interest
concentrations 200
m
M, 100
m
M, 50
m
M, 25
m
M, 12.5
m
M and
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, AOG 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.
6.75 M. The UV absorbance was read across 230e400 nm at 1 nm
m
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 concentration in the filtrate for each
compound, and hence amount remaining in solution after 90 min
which is reported as the kinetic solubility.
LogD measurements LogD measurements were conducted us-
ing the shake flask method. Compound was diluted from 10 mM
DMSO stock solution into an Eppendorf containing equal amounts
of octanol and phosphate buffered saline (PBS) to give a final con-
Acknowledgment
The authors would like to acknowledge Dr David Tickle, Teresa
centration of 100
mM. The tubes were shaken for 12 h, centrifuged
Sementa and Sadhia Khan for performing the in vitro ADME testing.
17

M. Maciuszek, A. Ortega-Gomez, S.L. Maas et al.
European Journal of Medicinal Chemistry 214 (2021) 113194
(2016) 113e130.
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, and
Dr Bartolo Ferraro for in vivo studies.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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Abbreviations
ACN
Acetonitrile
ADME
BOC
Absorption, Distribution, Metabolism, Excretion
tert-Butyloxycarbonyl
BSA
CLint
Bovine Serum Albumin
Intrinsic Clearance
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DCC
DCM
DCE
Cardiovascular Diseases
40,6-Diamidino-2-phenylindole
N,N0-Dicyclohexylcarbodiimide
Dichloromethane
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Dichloroethane
DIPEA
DMAP
DMF
DMSO
EC50
N,N-Diisopropylethylamine
4-Dimethylaminopyridine
N,NeDimethylformamide
Dimethyl Sulfoxide
Half Maximal Effective Concentration
Efficacy
Emax
FPR2
Formyl Peptide Receptor 2
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
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
PK
Pharmacokinetics
RoI
Resolution of Inflammation
Receiver Operating Characteristic
Serum Amyloid A
Structure-Activity Relationship
Triethylamine
ROC
SAA
SAR
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TEA
TM
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T3P
THF
Propylphosphonic Anhydride
Tetrahydrofuran
VCAM
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