Novel ureidopropanamide based N-formyl peptide receptor 2 (FPR2) agonists with potential application for central nervous system disorders characterized by neuroinflammation

Article abstract of DOI:10.1016/j.ejmech.2017.09.023

Formyl peptide receptor2 (FPR2) is a G-protein coupled receptor that plays critical roles in inflammatory reactions. FPR2-specific interaction can be possibly used to facilitate the resolution of pathological inflammatory responses by enhancing endogenous anti-inflammation systems. Starting from our lead agonist 5, we designed new ureidopropanamides derivatives able to activate FPR2 in transfected cells and human neutrophils. The new FPR2 agonists showed good stability towards oxidative metabolism in vitro. Moreover, selected compounds showed anti-inflammatory properties in LPS-stimulated rat primary microglial cells. (S)-3-(4-Cyanophenyl)-N-[[1-(3-chloro-4-fluorophenyl)cyclopropyl]methyl]-2-[3-(4-fluorophenyl)ureido]propanamide ((S)-17) emerged as prospective pharmacological tool to study the effects of FPR2 activation in the central nervous system (CNS) being able to reduce IL-1β and TNF-α levels in LPS-stimulated microglial cells and showing good permeation rate in hCMEC/D3 cells, an in vitro model of blood brain barrier. These results are very promising and can open new therapeutic perspectives in the treatment of CNS disorders characterized by neuroinflammation.

Full text of DOI:10.1016/j.ejmech.2017.09.023

Accepted Manuscript
Novel ureidopropanamide based N-formyl peptide receptor 2 (FPR2) agonists
with potential application for central nervous system disorders characterized by
neuroinflammation
Madia Letizia Stama, Joanna Ślusarczyk, Enza Lacivita, Liliya N. Kirpotina, Igor A.
Schepetkin, Katarzyna Chamera, Chiara Riganti, Roberto Perrone, Mark T. Quinn,
Agnieszka Basta-Kaim, Marcello Leopoldo
PII:
S0223-5234(17)30737-7
10.1016/j.ejmech.2017.09.023
EJMECH 9738
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 July 2017
Revised Date: 1 September 2017
Accepted Date: 14 September 2017
Please cite this article as: M.L. Stama, J. Ślusarczyk, E. Lacivita, L.N. Kirpotina, I.A. Schepetkin, K.
Chamera, C. Riganti, R. Perrone, M.T. Quinn, A. Basta-Kaim, M. Leopoldo, Novel ureidopropanamide
based N-formyl peptide receptor 2 (FPR2) agonists with potential application for central nervous system
disorders characterized by neuroinflammation, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.023.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
1

ACCEPTED MANUSCRIPT
Novel Ureidopropanamide Based N-Formyl Peptide Receptor 2 (FPR2) Agonists with
Potential Application for Central Nervous System Disorders Characterized by
Neuroinflammation
1
2
1
3
Madia Letizia Stama, Joanna Ślusarczyk, Enza Lacivita, * Liliya N. Kirpotina, Igor A.
3
2
4
1
3
Schepetkin, Katarzyna Chamera, Chiara Riganti, Roberto Perrone, Mark T. Quinn, Agnieszka
2
1
Basta-Kaim, Marcello Leopoldo
Affiliation
1
Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via
Orabona, 4, 70125 Bari, Italy
2
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of
Sciences, 12 Smetna St, 31-343 Krakow, Poland
3
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717,
USA
4
Dipartimento di Oncologia, Università di Torino, via Santena, 5/bis, 10126 Torino, Italy
*Corresponding Author: Enza Lacivita, Dipartimento di Farmacia – Scienze del Farmaco,
Università degli Studi di Bari “A. Moro”, via Orabona, 4, 70125, Bari, Italy. E-mail:
enza.lacivita@uniba.it; Phone: +39 080 5442750; Fax: +39 080 5442231

ACCEPTED MANUSCRIPT
KEYWORDS
Formyl peptide receptor 2, neuroinflammation, ureidopropanamides, microglia, metabolic stability,
blood-brain barrier permeation.
ABBREVIATIONS
FPR2, Formyl peptide receptor 2; NO, nitric oxide; IL-1β, interleukin 1β; LPS, lipopolysaccharide;
TNF-α, tumor necrosis factor α.

ACCEPTED MANUSCRIPT
Abstract
Formyl peptide receptor-2 (FPR2) is a G-protein coupled receptor that plays critical roles in
inflammatory reactions. FPR2-specific interaction can be possibly used to facilitate the resolution of
pathological inflammatory responses by enhancing endogenous anti-inflammation systems. Starting
from our lead agonist 5, we designed new ureidopropanamides derivatives able to activate FPR2 in
transfected cells and human neutrophils. The new FPR2 agonists showed good stability towards
oxidative metabolism in vitro. Moreover, selected compounds showed anti-inflammatory properties
in LPS-stimulated rat primary microglial cells. (S)-3-(4-Cyanophenyl)-N-[[1-(3-chloro-4-
fluorophenyl)cyclopropyl]methyl]-2-[3-(4-fluorophenyl)ureido]propanamide ((S)-17) emerged as
prospective pharmacological tool to study the effects of FPR2 activation in the central nervous
system (CNS) being able to reduce IL-1β and TNF-α levels in LPS-stimulated microglial cells and
showing good permeation rate in hCMEC/D3 cells, an in vitro model of blood brain barrier. These
results are very promising and can open new therapeutic perspectives in the treatment of CNS
disorders characterized by neuroinflammation.

ACCEPTED MANUSCRIPT
Introduction
Neuroinflammation is a complex multicellular process that plays a central role in a variety of
neurological diseases, including ischemia, neurodegenerative diseases, psychiatric and immune-
mediated disorders [1,2]. Increasing evidence suggests that microglia, the resident immune cells of
the central nervous system (CNS), play a pivotal role in inflammation-associated disorders. Under
normal conditions, activation of microglia cells exerts a protective role by regulating the response to
pathogens and promoting tissue repair through the release of anti-inflammatory and neurotrophic
factors [3]. On the other hand, chronic activation of immune responses can lead to the functional
switch of microglia from regulatory to neurotoxic leading to the excessive release of pro-
inflammatory cytokines, such as IL-1β, IL-18, and TNF-α, as well as of neurotoxic mediators like
nitric oxide (NO), prostaglandin E2, and superoxide anion [4]. Microglial activation is a
phenotypically and functionally dynamic process, which may progress differently depending on
aging, stage of disease or on the status of the brain environment [5,6].
In general, inflammation requires tight regulation and control: pro-inflammatory mediators operate
in a parallel and serial fashion to provoke the classical signs of inflammation, while anti-
inflammatory mediators ensure resolution of the inflammatory response. This latter aspect has
gained interest in recent years because several classes of peptidic or non-peptidic endogenous
factors have been identified as pro-resolving mediators [7]. Many pro- and anti-inflammatory
signals and pro-resolving circuits converge on a group of receptors that integrate contrasting cues to
determine the course of inflammation. Among these receptors is the formyl peptide receptor 2
(FPR2) [8,9], a G-protein coupled receptor that belongs to the formyl peptide receptor (FPR)
family, which includes also the subtypes FPR1 and FPR3. FPRs are expressed in several immune
cells, including neutrophils, monocytes/macrophages and microglia and are considered to play
relevant roles in innate immunity and host defense mechanisms and chemotaxis [9]. FPR2 is
functionally expressed in glial cells and astrocytes [10-12]. FPRs have homologs in fish and rodents

ACCEPTED MANUSCRIPT
[
13,14]. For example, Fpr2, a homolog of human FPR2, is localized in rat satellite glial cells and
neurons of dorsal root ganglions [15]. Expression of Fpr1 and Fpr2 was also reported in rat
neuronal stem cells [16]. Recently, expression of Fpr, which cross-reacts with anti-human FPR2
antibodies, was detected in rat brain cortex and hippocampus [17].
A prominent and unusual feature of FPR2 is that it can be activated by a variety of structurally
diverse agonists. Several endogenous peptides can activate FPR2 and are able to induce very
different biological effects. For example, FPR2 can mediate pro-inflammatory effects if activated
by N-formyl peptides, produced by bacteria and mitochondria to induce defense mechanisms, as
well as by prion protein PrP(106-126) and the amyloidogenic peptides serum amyloid A and β-
amyloid, which are associated with chronic inflammation and amyloidosis [9]. On the other hand,
FPR2 can mediate anti-inflammatory effects if activated by annexin A1, a glucocorticoid-regulated
protein that is involved in the adhesion mechanisms of leukocytes, as well as by lipoxin A4 (LXA4),
which is an anti-inflammatory lipid, the first endogenous FPR2 ligand to be identified [9,18]. The
ability of FPR2 to mediate contrasting effects is mechanistically related to the receptor dimerization
that can be induced in a ligand-specific manner [19].
Preclinical evidence generated with pharmacological tools or with knockout and transgenic mice
has suggested that LXA contributes to the resolution of inflammation through the activation of
4
FPR2, which in turn modulates chemokine and cytokine synthesis, inhibits neutrophils infiltration,
and promotion of phagocytosis [20-22]. Recently, stimulation of the resolution phase has been
proposed as a new therapeutic perspective for the treatment of chronic inflammatory diseases such
as chronic obstructive pulmonary disease, cystic fibrosis, rheumatoid arthritis, and also for CNS
diseases [23,24]. With this respect, recent data demonstrate that LXA inhibits microglial activation
4
and diminishes neuroinflammation after spinal cord hemisection [25]. Moreover, in rat hemorrhage,
the beneficial role of LXA in suppression of inflammation mediated by FPR2 and p38 MAPK
4
signaling pathways has been demonstrated [26]. It is worth emphasizing that the administration of
LXA in a transgenic mouse model of Alzheimer’s Disease is able to stimulate a pro-resolving
4

ACCEPTED MANUSCRIPT
activation of microglia by reducing NF-κB activation and levels of pro-inflammatory cytokines and
chemokines and by increasing levels of anti-inflammatory IL-10. This alternative activation of
microglia translated into an improved phagocytic function with increased clearance of β-amyloid
plaques, reduced synaptotoxicity, and improvement of cognition [27].
A wide range of chemically diverse non-peptidic FPR agonists have been identified so far [28],
including benzimidazole derivatives exemplified by compound 1 [29], N-phenylurea derivatives
(
compound 2) [30], quinazolinones derivatives such as the highly specific non-peptide FPR2
agonist Quin-C1 (3) [31], and pyrazolone derivatives like the mixed FPR1/FPR2 agonist 4
designated in most of publications as “Compound 43”) [32] (see Chart 1). Recently, we reported
(
the identification of a group of 3-(1H-indol-3-yl)-2-[3-(4-substituted-phenyl)ureido]propanamide
derivatives as agonists of human FPR2, exemplified by compound 5 (Table 1, Chart 1) [33,34]. The
2
+
above agonists were characterized for their ability to induce intracellular Ca release. Whereas
agonists 3 and 4 exhibited anti-inflammatory properties in peripheral models of inflammation
[
35,36], there are no reports on the effects of FPR2 agonists in in vitro or in vivo models of
neuroinflammation.
Based on our previous studies, we aimed to identify new potent FPR2 agonists and to prove if the
new ureidopropanamide derivatives display anti-inflammatory and pro-resolving effects in an in
vitro model of neuroinflammation. Toward this aim, we have modified the structural framework of
5
taking into account that this agonist showed very low stability toward oxidative metabolism and
was rapidly degraded after incubation with rat liver microsomes, thus implying that 5 has a
pharmacokinetic profile not compatible with in vivo studies [34].
Results and Discussion
Study Design. As indicated above, 5 is a potent mixed FPR1/FPR2 agonist in FPR2-transfected
HL-60 cells and in human neutrophils [33] but displays very low stability toward oxidative

ACCEPTED MANUSCRIPT
metabolism (Table 1). Thus, in search of metabolically stable FPR2 agonists we selected from our
chemical library various 3-(1H-indol-3-yl)-2-[3-(4-substituted-phenyl)ureido]propanamide
derivatives and tested them for their susceptability to oxidative metabolism in vitro using rat liver
microsomes. We assessed microsomal stability as the percentage of recovery of parent compound
after a 30 minute incubation with liver microsomes preparation, as described previously [37].
Among the screened compounds, the enantiomeric pair (R)- and (S)-6, in which the cyclohexane
ring was replaced with the cyclopropane ring, had a percentage of recovery (15% and 11%,
respectively) higher than that of 5 (4%) (Table 1). In addition, the phenylcyclopropylmethyl moiety
is more synthetically accessible as compared to the (5-methoxypyridin-2-yl)cyclohexylmethyl
moiety of compound 5 [34]. Regarding interaction with FPR2, (R)-6 was previously characterized
2
+
as a weak FPR2 antagonist, whereas (S)-6 was able to induce Ca mobilization, albeit with low
potency (EC = 7.6 µM) [33]. Therefore, considering the slightly more favorable metabolic
5
0
stability profile and the synthetic accessibility, we selected (S)-6 as starting point for our study with
the aim of improving both potency in FPR2 activation and metabolic stability in vitro. We
systematically evaluated both enantiomers of the target compounds 7-17 (Table 1) because previous
studies have shown that the interaction of these chiral ureidopropanamides with FPRs can be highly
2
+
stereospecific [33]. We assessed the ability of the target compounds to induce Ca mobilization in
HL-60 cells transfected with h-FPR1 or h-FPR2 and in human neutrophils. Finally, considering that
2
+
the ability to induce Ca mobilization is a feature exhibited by both pro-inflammatory and pro-
resolving FPR2 agonists, the compounds showing the best combination of potency and metabolic
stability were further characterized in primary rat microglia cultures to assess their anti-
inflammatory properties.
Chemistry. Synthesis of the target compounds is depicted in Scheme 1. The key amines 22 and 23
were prepared from the corresponding nitriles 18 [38] and 19 [39], respectively, after hydrogenation
over Nickel-Raney. Amine 24 was prepared by reduction of nitrile 20 [40] with the borane-dimethyl

ACCEPTED MANUSCRIPT
sulfide complex in order to avoid reductive dehalogenation. The amines 21-24 were then condensed
with the commercially available (R)-Boc- or (S)-Boc-amino acids 25-27 after activation with N-N’-
carbonyldiimidazole to give the Boc-protected derivatives (R)- and (S)- 28-33. Subsequently, these
latter compounds were deprotected with trifluoroacetic acid to give amines (R)- and (S)- 34-39. The
target compounds were obtained by condensing amines (R)- and (S)- 34-39 with the appropriate
phenylisocyanate, except for compounds (R)-8, (S)-14, (S)-15, and (R)-17, which were prepared by
condensing the amines (R)-34, (S)-38, (R)-39 with the the appropriate aniline and N-N’-
carbonyldiimidazole.
Intrinsic Activity and Metabolic Stability of the Target Compounds. The first structural
modification on (R)- and (S)-6 was removal of the nitro substituent from the phenylureidic group or
its replacement with –OCH or –F substituents. This structural modification was inspired by
3
previous structure-activity relationship studies on ureidopropanamide derivatives, where the
introduction of a methoxy group [33] or an halogen [41] in the same position led to an improvement
in agonist potency. Indeed, (S)-7 and (S)-8 showed EC values lower than that of (S)-6. The effect
50
was more pronounced for the methoxy derivative (S)-7 (Table 1). In contrast, removal of the nitro
substituent of (S)-6 had little impact on its potency ((S)-9). Interestingly, replacement of the nitro
group with a methoxy group in (R)-6 shifted the intrinsic activity from antagonism to agonism ((R)-
7
)) [33]. This effect was not observed for derivatives (R)-8 and (R)-9, which were not able to induce
2
+
Ca mobilization. Note that the replacement of the nitro group with other substituents led to a
decrease in selectivity towards FPR1, as (S)-7, (S)-8, and (S)-9 were able to activate FPR1 as well.
In particular, (S)-9 showed a slight preference for FPR1 over FPR2. Finally, similarly to (S)-6,
compounds (S)-8 and (S)-9 behaved as partial agonists, whereas (S)-7 was characterized as a full
agonist at both FPR1 and FPR2.
Considering metabolic stability, it can be noted that this structural modification had a negative
influence, as the compounds (R)- and (S)-7-9, were less stable than (R)- and (S)-6 (Table 1). By

ACCEPTED MANUSCRIPT
comparing compounds 6 and 8, it can be speculated that the electronic properties of the substituent
on the phenyl ureidic moiety can have limited effects on metabolic stability of this group of
compounds.
We next evaluated the impact on both potency and metabolic stability of replacement of the
tryptophan core in (R)- and (S)-6 with that of unnatural amino acids. This structural modification
was inspired by the observation that different aminoacid cores were well tolerated in other FPR2
agonists with ureidopropanamide structure [28]. For this purpose, we selected the commercially
available 4-cyanophenylalanine and 3-pyridylalanine. In general, this structural modification was
beneficial. In fact, the introduction of 4-cyanophenylalanine led to a 10-fold increase in agonist
potency, as shown by (S)-10 compared to (S)-6, whereas (S)-11, which bears the 3-pyridylalanine
core, was only slighly more potent than (S)-6 (Table 1). Comparig the (R)- enantiomers with (R)-6,
2
+
a shift from antagonism to agonism was observed, as (R)-10 and (R)-11 were able to induce Ca
mobilization although with lower potency than the (S)-enantiomers. It can be also noted that (R)-
and (S)-10 behaved as full agonists, whereas (R)- and (S)-11 were characterized as partial agonists.
Finally, this structural modification led to a substantial increase in FPR1 agonist activity; in
particular, (S)-11 had an EC value for FPR1 in the low micromolar range. As for metabolic
50
stability, both enantiomeric pairs showed a percentage of recovery higher than 40%, with (S)-10
being the most stable compound of the set (56% recovery) (Table 1). These data suggest that
replacement of the tryptophan with an unnatural amino acid led to unfavorable interactions of the
molecule with the metabolic enzymes.
On the basis of these data, we selected the 4-cyanophenylalanine as the amino acid core for further
modifications to obtain new ureidopropanamide FPR2 agonists. First, we replaced the nitro
substituent of (R)- and (S)-10 with –OCH or –F with the aim of increasing agonist potency.
3
Additionally, we decorated the phenyl ring on the “right hand” of (R)- and (S)-10 with substituents
that could prevent interaction of the molecule with metabolic enzymes in an effort to further
increase metabolic stability (compounds 12-17). These modifications did not lead to improved

ACCEPTED MANUSCRIPT
FPR2 agonist potency. In particular, the presence of a substituent in the 4-position of the phenyl
linked to the cyclopropyl ring negatively influenced the ability of these compounds to activate
FPR2 (Table 1). This effect was more evident for compounds (R)- and (S)-12 and (R)- and (S)-13,
which feature the bulky trifluoromethoxy group. Instead, (R)- and (S)-16 and (R)- and (S)-17,
bearing the small F– substituent in 4-position, showed EC values comparable to those of (R)- and
5
0
(S)-10 (Table 1). Moreover, decoration of the phenyl ring linked to the cyclopropyl moiety had
different impact on the FPR1 interaction, with (R)- and (S)-16 being potent FPR1 full agonists.
Introduction of bulky substituents on the phenyl linked to the cyclopropyl ring had a beneficial
effect on metabolic stability. In fact, derivates (R)- and (S)-13 showed very high percentage
recovery (> 80%) (Table 1). However, the methoxy-substituted derivatives showed lower stability
than that of the fluoro-substituted counterparts. On the other hand, the presence of the unnatural
amino acid core did not always translate into an improvement of metabolic stability. Derivatives
(R)- and (S)-14, and (R)- and (S)-16 showed metabolic stabilities comparable to those of
ureidopropanamides with the tryptophan core (Table 1).
Collectively, these data clearly indicate that the “right hand” of these ureidopropanamides plays an
important role in the interaction with FPR2 because structural modifications in this part of the
molecule greatly influence agonist potency. On the other hand, the steric hindrance of this part of
the molecule seems to be an important requisite for metabolically stable compounds. Therefore, it is
important to find the right balance in the dimension of the cyclopropyl phenyl moiety in order to
combine high potency with good metabolic stability.
We next evaluated the ability of the new compounds to activate FPR2 in human neutrophils. The
data show that neutrophils responded to all agonists that activated FPR-expressing HL-60 cells. The
2
+
only exceptions were (R)-8 and (R)-9, which were able to induce Ca mobilization in neutrophils
but not in FPR-expressing HL-60 cells. Among the compounds studied, (R)-11 and (S)-17 exhibited
EC values in the submicromolar range.
50

ACCEPTED MANUSCRIPT
Half-life and Intrinsic Clearance of Selected Compounds. According to our paradigm for
assessing metabolic stability of the new compounds, derivatives (R)- and (S)-10, (R)- and (S)-11,
(R)-15, (R)- and (S)-17, which all showed recovery higher than 20%, were further characterized by
evaluating half-life (t ) and intrinsic clearance (CL ) (Table 2). All selected compounds showed
1
/2
int
CL much lower than that of 5, confirming that the new derivatives were more stable than 5 with
int
respect to microsomal oxidative metabolism. Moreover, all compounds displayed t1/2 values
exceeding 15 min, which is reported as the lower limit for predicted low clearance compounds [42].
Among the new compounds, (S)-10, (R)-11, and (S)-17 showed a good balance between potency
and metabolic stability and were, thus, selected for further studies in order to assess their
pharmacological properties in an in vitro model of neuroinflammation.
Effect of Selected Compounds on Cell Viability and Metabolic Activity in Rat Primary
Microglial Cells. Initially, we evaluated the effect of (S)-10, (R)-11, and (S)-17 on cell viability and
metabolic activity in rat primary microglia cell cultures under basal conditions and after stimulation
with lipopolysaccharide (LPS). It is well known that LPS is a primary component of endotoxin
from Gram-negative bacteria cell walls [43,44], which binds mostly to Toll-like receptor 4 and
induces intracellular signaling resulting in the activation of mitogen activated protein kinases
(MAPKs) and NF-κB [45]. These proteins have been described as the key regulators of pro-
inflammatory factors production such as cytokines and NO, which may have cytotoxic effects and
may damage cells [46].
We evaluated the effect of (S)-10, (R)-11, and (S)-17 on cell viability by using the lactate
dehydrogenase (LDH) and MTT tests, two biochemical assays that evaluate different aspects of cell
death/viability processes. The LDH assay is a well-accepted paradigm to quantitatively assess cell
death after damage of the plasma membrane, which results in increased LDH efflux from injured
cells, and it is known that LPS induces impairements in cell membrane integrity. On the other hand,
MTT test quantifies mitochondrial activity by measuring the formation of a dark blue formazan

ACCEPTED MANUSCRIPT
product formed by reduction of the tetrazolium ring of MTT in living cells. Thus, these tests
provide an accurate and complete assessment of the impact of the selected compounds on the vital
status of microglial cells in culture.
We first tested the effect of (S)-10, (R)-11, and (S)-17 at different doses under basal conditions.
None of the compounds induced significant effects on LDH release or metabolic activity in the dose
range of 0.5-10 µM. On the other hand, exposure of the microglial cells to a 50 µM dose of all
compounds resulted in a significant increase in death processes. (S)-10 and (S)-17 also decreased
the conversion of MTT dye at a 50 µM dose (Figure 1).
Next, we evaluated the effect of the selected compounds after stimulation of the microglial cells
with LPS, which induces cell death processes by increasing LDH release as well as diminishing
metabolic activity. Interestingly, pre-treatment with (S)-10, (R)-11, and (S)-17 effectively blocked
LPS-induced cell death processes at the doses of 1 µM and 5 µM (Figure 1). On the other hand, no
effect was observed on metabolic activity in LPS-stimulated microglial cell cultures (Figure 1).
These data indicate that (S)-10, (R)-11, and (S)-17 did not induce either pro-inflammatory responses
or cytotoxicity in microglial cells. Moreover, our FPR2 agonists showed protective properties
against LPS treatment in the LDH assays.
Effect of Selected Compounds on Pro-Inflammatory Mediators Production in Rat Primary
Microglial Cells. To further characterize the anti-inflammatory properties of our FPR2 agonists, we
assessed their effect on the production of the pro-inflammatory mediators NO, IL-1β and TNF-α in
rat primary microglial cell cultures. Under normal conditions, NO is involved in many
physiological processes in the brain, such as regulation of proliferation, survival, differentiation of
the neurons, synaptic activity, and neural plasticity [47]. However, excessive NO synthesis leads to
the formation of reactive nitrogen species and neuronal cell death. Moreover, there is an intimate
relation between microglial activation, NO production, and neuroinflammation in the brain [48].
Also, inflammatory cytokines, such as IL-1β and TNF-α, have physiological functions in the brain

ACCEPTED MANUSCRIPT
which include effects on neurite outgrowth, neurogenesis, neuronal survival, and synaptic pruning
during brain development, synaptic transmission and synaptic plasticity [49]. However,
overproduction and exaggerated release of cytokines is associated with neuronal dysfunction.
We evaluated effects of the selected FPR2 agonists on the production of NO, IL-1β, and TNF-α in
rat primary microglial cultures under basal conditions and after stimulation with LPS. Furthermore,
to check if the observed effect is mediated through the interaction with FPR2, the microglia cells
were also pre-treated with the selective FPR2 antagonist WRW4.
(S)-10, (R)-11, and (S)-17 did not induce any change in NO level under basal conditions. The same
effect was observed when the cells were treated with WRW4 alone or in combination with the
selected agonists (Figure 2). Stimulation of the microglial cells with LPS increased the level of NO,
which was significantly attenuated by (S)-10 (5 µM and 10 µM), whereas no effect was observed
for (R)-11 and (S)-17. Moreover, pre-treatment of the microglial cells with WRW4 was not able to
reverse the anti-inflammatory effect on NO secretion evoked by (S)-10, suggesting that this effect
was mediated by the interaction with molecular targets different from FPR2 (Figure 2).
(S)-10, (R)-11, and (S)-17 (0.5 µM-10 µM) did not induce any change in the intracellular levels of
IL-1β and TNF-α production under basal conditions. As observed for NO production, the
antagonist WRW4, alone or in combination with the selected agonists did not affect IL-1β and
TNF-α production (Figures 3 and 4). Consistent with our previous studies, the stimulation with LPS
induced a significant up-regulation of both IL-1β and TNF-α production in the microglial cells [3].
Interestingly, (S)-10, (R)-11, and (S)-17 showed anti-inflammatory effects, being able to effectively
decrease LPS-evoked cytokine production (Figure 3 and 4). Pre-treatment with the antagonist
WRW4 was able to block the effect of (S)-17 on both IL-1β and TNF-α secretion, and of (R)-11
only on IL-1β production. In the case of (S)-10, WRW4 did not abolish the observed effect on both
pro-inflammatory cytokine release, suggesting that the anti-inflammatory effects of (S)-10 were
mediated by other molecular targets (Figure 3 and 4). Considering that the inhibition of IL-1β and

ACCEPTED MANUSCRIPT
TNF-α production by (S)-17 did not exhibit a clear dose response in the range from 0.5 to 10 µM,
we tested the effect of (S)-17 at lower doses. The data indicate that at 0.05 µM or 0.1 µM (S)-17
was not able to induce a significant effect on IL-1β and TNF-α production (Figure S4,
Supplementary Material).
Collectively, the data indicated that the selected FPR2 agonists did not induce pro-inflammatory
responses in resting microglial cells but exerted clear anti-inflammatory effects in the LPS-
stimulated cells. In the case of (S)-17 the anti-inflammatory effect was mediated by FPR2, whereas
(S)-10 and (R)-11 seem to exert these effects through other molecular targets.
Evaluation of Permeability in hCMEC/D3 Cells.
A critical characterization for prospective CNS-acting drugs is the ability to cross the blood brain
barrier (BBB), which acts as a highly lipophilic boundary. Compounds permeate BBB mainly by
passive diffusion mechanism, and several efflux systems prevent the entrance of xenobiotics into
CNS. To endorse the potential of compounds (S)-10, (R)-11, and (S)-17 as promising leads for the
development of neuroprotective agents, in vitro transport studies were undertaken. To this end, we
selected hCMEC/D3 cells, an immortalized human brain microvascular endothelial cell line, as an
in vitro model of BBB. hCMEC/D3 cells stably maintain most of the unique structural and
biochemical properties of brain endothelium in vivo, including tight junctions formation and the
expression of multiple efflux transporters of the ATP-binding cassette superfamily [50].
The permeation rate of compounds (S)-10, (R)-11, and (S)-17 across the cell monolayer in both
directions, i.e., apical-to-basolateral (AB) and basolateral-to-apical (BA), was assessed (Table 3).
Moreover, we evaluated the efflux ratio (ER) between BA and AB fluxes because ER greater than 3
can be taken as a figure of undesirable interaction with the efflux transporters (Table 3) [51].
Compound (S)-17 showed good permeation rates in both directions and ER value below 3, thus
envisaging good brain distribution and low interactions with the efflux transporters. On the other
hand, (S)-10 and (R)-11 showed lower permeation rates, especially in AB direction, which is more

ACCEPTED MANUSCRIPT
strongly influenced by the interaction with the efflux transporters, suggesting that (S)-10 and (R)-11
might have low brain distribution.
Conclusion
In summary, we have manipulated the structure of 5, a moderately potent ureidopropanamide FPRs
agonist previously studied in our laboratories, with the aim of identifying new potent FPR2 agonists
endowed with enhanced metabolic stability. Several of these new studied derivatives exhibited
agonist potency comparable to that of 5 and significantly higher t1/2 and CL , indicating that the
int
aim of improving stability towards oxidative metabolism was achieved. Analysis of (S)-10, (R)-11,
and (S)-17 in an in vitro model of neuroinflammation showed that they did not induce inflammatory
responses in resting rat primary microglial cell cultures but they were able to significantly reduce
the production of pro-inflammatory mediators when microglial cells were stimulated with LPS. In
particular, compound (S)-17 was able to reduce IL-1β and TNF-α levels in LPS-stimulated
microglial cells and this effect was mediated by FPR2 interaction because its effects were blocked
by pre-treatment of the cells with the FPR2 antagonist WRW4. Moreover, bidirectional transport
studies on hCMEC/D3 cells denoted good permeation rates of compound (S)-17 without suffering
from likely efflux transporters interactions, thus suggesting good brain permeation and CNS
distribution.
To the best of our knowledge, this is the first report on FPR2 agonists being studied in a model of
neuroinflammation in order to evaluate their ability to induce anti-inflammatory responses in
primary microglial cells. Among the studied agonists, (S)-17 emerges as a prospective
pharmacological tool to study the effects of FPR2 activation in the CNS because it elicits FPR2-
mediated anti-inflammatory effects in rat microglia and also displays suitable pharmacokinetic
characteristics. We believe that these data are promising considering that very recent studies have
indicated that stimulation of brain FPR2 with endogenous ligands such as LXA1 or annexin A1 [52]
is able to inhibit microglial activation and diminish neuroinflammation in several pathological

ACCEPTED MANUSCRIPT
conditions, including Alzheimer’s Disease and neuropathic pain. This can open new therapeutic
perspectives in the treatment of those CNS disorders characterized by neuroinflammation.
Experimental Section
1. Chemistry. Chemicals were purchased from Sigma-Aldrich, Alfa Aesar, and TCI Chemicals.
Unless otherwise stated, all chemicals were used without further purification. Thin layer
chromatography (TLC) was performed using plates from Merck (silica gel 60 F254). Column
chromatography was performed with 1:30 Merck silica gel 60 Å (63-200 µm) as the stationary
phase. Flash chromatographic separations were performed on a Biotage SP1 purification system
1
13
using flash cartridges pre-packed with KP-Sil 32−63 μm, 60 Å silica. H and C NMR spectra
were recorded on a Varian Mercury-VX spectrometer (300 MHz) or on a 500-vnmrs500 Agilent
spectrometer (500 MHz). All chemical shift values are reported in ppm (δ). For enantiomeric pairs,
NMR spectra for both enantiomers were recorded but the NMR spectrum of only the (R)-enatiomer
is reported in the experimental section. Recording of mass spectra was done on an HP6890-5973
MSD gas chromatograph/mass spectrometer; only significant m/z peaks, with their percentage of
relative intensity in parentheses, are reported. HRMS-ESI analyses were performed on a Bruker
Daltonics MicrOTOF-Q II mass spectrometer, mass range 50-800 m/z, electrospray ion source in
positive or negative ion mode. All spectra were in accordance with the assigned structures. The
purity of the target compounds listed in Table 1 was assessed by RP-HPLC and combustion
analysis. All compounds showed ≥ 98% purity. RP-HPLC analysis was performed on an Agilent
1260 Infinity Binary LC System equipped with a diode array detector using a Phenomenex Gemini
C-18 column (250 x 4.6 mm, 5 µm particle size). All target compounds (Table 1) were eluted with
CH OH/H O/Et N, 8:2:0.01 at a flow rate of 1 mL/min. Elemental analyses (C,H,N) of the target
3
2
3
compounds were performed on a Eurovector Euro EA 3000 analyzer. Analyses indicated by the
symbols of the elements were within ± 0.4 % of the theoretical values. Enantiomeric purity of the

ACCEPTED MANUSCRIPT
target compounds (R)- and (S)-7-17 was assessed by chiral HPLC analysis on a Perkin-Elmer series
200 LC instrument using a Daicel ChiralCel OD column (250 mm × 4.6 mm, 5 µm particle size)
and equipped with a Perkin-Elmer 785A UV/VIS detector setting λ= 230 nm. The compounds were
eluted with n-hexane/EtOH, 4:1, v/v at a flow rate of 0.8 mL/min. All compounds showed
enantiomeric excesses ≥ 98%.
The following compounds were prepared according to literature methods: 1-[(4-
trifluoromethoxy)phenyl]cyclopropanecarbonitrile (18) [38]; 1-(3-chloro-4-fluorophenyl)-
cyclopropanecarbonitrile (19) [39]; 1-(3-fluoro-4-methylphenyl)-cyclopropanecarbonitrile (20)
[
40]; 1-(phenylcyclopropyl)methylamine (21) [53]; (R)- and (S)-1-(1H-indol-3-ylmethyl)-2-[[(1-
phenylcyclopropyl)methyl]amino]-2-oxoethyl]carbamic acid, 1,1-Dimethylethyl Ester ((R)- and
S)- 28) [54]; (R)- and (S)-2-amino-3-(1H-indol-3-yl)-N-[(1-
(
phenylcyclopropyl)methyl]propanamide ((R)- and (S)-34) [54].
General Procedure for the Synthesis of the Amines 22 and 23.
Raney nickel was activated with 10 M KOH, then washed with H O, then with EtOH to remove
2
H O. The catalyst was then taken up in 2 N ethanolic ammonia and the appropriate nitrile (0.87
2
mmol) was added to the mixture which was then hydrogenated under 5 bar pressure of hydrogen at
50 °C for 15 h. The reaction mixture was then filtered through Celite and concentrated in vacuo to
give the desired amine as an oil.
[1-(4-Trifluoromethoxyphenyl)cyclopropyl]methanamine (22).
1
9
0% Yield. H NMR (CDCl ): δ 0.740.83 (m, 4H), 1.35 (br s, 2H), 2.76 (s, 2H), 7.13 (d, 2H, J= 8.3
3
+
Hz), 7.27 (d, 2H, J= 8.1 Hz). GC/MS: m/z 231 (M , 7), 203 (100), 134 (67), 115 (35).
[1-(3-Fluoro-4-methylphenyl)cyclopropyl]methanamine (23).
1
7
7% Yield. H NMR (CDCl ): δ 0.72–0.81 (m, 4H), 1.35 (br s, 2H), 2.23 (s, 3H), 2.74 (s, 2H),
3

ACCEPTED MANUSCRIPT
+
6
1
.74–6.82 (m, 2H), 7.07 (t, 1H, JH-H= 7.6 Hz, JH-F= 8.2 Hz), GC/MS: m/z 179 (M , 25), 151 (100),
47 (36), 133 (49), 109 (37).
[1-(3-Chloro-4-fluorophenyl)cyclopropyl]methanamine (24).
Borane-methyl sulfide complex as 10.0 M BH in excess methyl sulfide (4.6 mmol, 0.46 mL) was
3
dropped into an ice-cooled solution of 1-(3-chloro-4-fluorophenyl)cyclopropylcarbonitrile (0.30 g,
1.5 mmol) in anhydrous THF (10 mL), under stirring. After being refluxed for 3-4 h, the reaction
mixture was cooled at -10 °C and MeOH was added dropwise very carefully until gas evolution
ceased. The mixture was treated with 3 N HCl (20 mL) and was refluxed for 1h . After cooling, the
mixture was alkalized with 3 N NaOH and extracted with CH Cl (5 Õ 20 mL). The collected
2
2
organic layers were dried over Na SO and the solvent was evaporated under pressure to give the
2
4
1
pure amine as a colorless oil (0.25 g; 81% yield). H NMR (CDCl ): δ 0.73–0.82 (m, 4H), 1.37 (br
3
s, 2H), 2.76 (s, 2H), 7.07 (t, 1H, JH-H= 8.2 Hz, JH-F= 8.8 Hz), 7.17–7.22 (m, 1H), 7.36 (dd, 1H, J=
+
+
7
.0, 2.3 Hz). GC/MS: m/z 201 (M +2, 1), 199 (M , 4), 171 (100), 147 (27), 133 (60), 109 (29).
General Procedure for the Synthesis of Boc-protected Derivatives (R)- and (S)-29–33.
’
N,N -Carbonyldiimidazole (1.1 mmol) was added to a solution of (R)- or (S)-Boc-protected amino
acid (1.0 mmol), in anhydrous THF (10 mL) under N . The reaction mixture was stirred at room
2
temperature overnight, then a solution of the appropriate amine (1.0 mmol) in anhydrous THF was
added. The reaction mixture was stirred at room temperature for 6 h. Then, the solvent was removed
in vacuo and the residue was partitioned between EtOAc (20 mL) and H O (2 Õ 20 mL). The
2
aqueous layer was separated and extracted twice with EtOAc (20 mL). The collected organic layers
were dried (Na SO ) and evaporated in vacuo. The crude residue was purified through flash
2
4
chromatography (gradient eluition from 30% to 70% ethyl acetate in n-hexane) to give pure target
compound as a white solid.

ACCEPTED MANUSCRIPT
(R)-1-(4-Cyanophenylmethyl)-2-[[(1-phenylcyclopropyl)methyl]amino]-2-oxoethyl]carbamic
acid, 1,1-Dimethylethyl Ester ((R)-29).
1
7
3
4
8
5% Yield. H NMR (CDCl ): δ 0.76–0.89 (m, 4H), 1.38 (s, 9H), 2.96 (dd, 1H, J= 13.5, 6.4 Hz),
3
.07 (dd, 1H, J= 13.5, 7.6 Hz), 3.32 (dd, 1H, J= 14.1, 5.9 Hz), 3.4 (dd, 1H, J= 14.1, 5.9 Hz), 4.23–
.27 (m, 1H), 4.98 (br d, 1H), 5.8 (br t, 1H), 7.12–7.15 (m, 2H), 7.20–7.31 (m, 5H), 7.51 (d, 2H, J=
+
+
+
.8 Hz). ESI /MS m/z 442 (M+Na) , ESI /MS/MS m/z 342 (100).
S)–1-(4-Cyanophenylmethyl)-2-[[(1-phenylcyclopropyl)methyl]amino]-2-oxoethyl]carbamic
acid, 1,1-Dimethylethyl Ester ((S)-29).
(
+
+
+
6
4% yield. ESI /MS m/z 442 (M+Na) , ESI /MS/MS m/z 342 (100).
(R)-2-[[(1-Phenylcyclopropyl)methyl]amino]-2-oxoethyl]-1-(3-pyridinylmethyl)carbamic acid,
1
5
3
,1-Dimethylethyl Ester ((R)-30).
1
8% Yield. H NMR (CDCl ): δ 0.80–0.85 (m, 4H), 1.37 (s, 9H), 2.93 (dd, 1H, J= 13.5, 7.0 Hz),
3
.04 (dd, 1H, J= 14.1, 7.0 Hz), 3.38 (d, 2H, J= 5.3 Hz), 4.20–4.25 (m, 1H), 4.97 (br d, 1H), 5.91 (br
t, 1H), 7.13–7.20 (m, 5H), 7.23–7.26 (m, 1H), 7.46 (d, 1H, J= 7.6 Hz), 8.39 (d, 1H, J= 1.8 Hz), 8.47
+
+
+
(
dd, 1H, J= 4.7, 1.2 Hz). ESI /MS m/z 418 (M+Na) , ESI /MS/MS m/z 318 (100).
(S)- 2-[[(1-Phenylcyclopropyl)methyl]amino]-2-oxoethyl]-1-(3-pyridinylmethyl)carbamic acid,
1,1-Dimethylethyl Ester ((S)-30).
+
+
+
4
5% Yield. ESI /MS m/z 418 (M+Na) , ESI /MS/MS m/z 318 (100).
(R)-1-(4-Cyanophenylmethyl)-2-[[[(1-(4-trifluoromethoxyphenyl)cyclopropyl]methyl]amino]-
2
8
3
4
-oxoethyl]carbamic acid, 1,1-Dimethylethyl Ester ((R)-31).
1
4% Yield. H NMR (CDCl ): δ 0.79–0.89 (m, 4H), 1.38 (s, 9H), 2.99 (dd, 1H, J= 13.5, 6.5 Hz),
3
.13 (dd, 1H, J= 13.5, 7.0 Hz), 3.35 (dd, 1H, J= 13.5, 5.5 Hz), 3.42 (dd, 1H, J= 13.5, 5.5 Hz); 4.26–
.27 (m, 1H), 4.87 (br s, 1H), 5.92 (br t, 1H), 7.13 (d, 2H, J= 8.5 Hz), 7.19–7.21 (m, 2H), 7.26–7.28
+
+
+
(
m, 2H), 7.57 (d, 2H, J= 8.5 Hz). ESI /MS: m/z 526 (M+Na) , ESI -MS/MS: m/z 426 (100).

ACCEPTED MANUSCRIPT
(S)-1-(4-Cyanophenylmethyl)-2-[[[(1-(4-trifluoromethoxyphenyl)cyclopropyl]methyl]amino]-
2-oxoethyl]carbamic acid, 1,1-Dimethylethyl Ester ((S)-31).
+
+
+
8
7% Yield. ESI /MS: m/z 526 (M+Na) . ESI -MS/MS: m/z 426 (100).
(R)-1-(4-Cyanophenylmethyl)-2-[[[(1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-Dimethylethyl Ester ((R)-32).
1
8
1
1% Yield. H NMR (CDCl ): δ 0.75–0.84 (m, 4H), 1.38 (s, 9H), 2.23 (s, 3H), 2.98 (dd, 1H, J=
3
3.5, 6.6 Hz); 3.10 (dd, 1H, J= 13.5, 6.5 Hz); 3.34 (d, 2H, J= 5.4 Hz); 4.20–4.25 (m, 1H), 4.94 (br
d, 1H), 5.84 (br t, 1H), 6.74–6.82 (m, 2H), 7.07 (t, 1H, J= 8.0 Hz), 7.25 (d, 2H, J= 8.4 Hz), 7.55 (d,
+
+
+
2
H, J= 8.1 Hz). ESI /MS: m/z 474 (M+Na) ; ESI -MS/MS: m/z 374 (100).
S)-1-(4-Cyanophenylmethyl)-2-[[[(1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]amino]-2-
oxoethyl]carbamic acid,1,1-dimethylethyl ester ((S)-32).
(
+
+
+
7
1% Yield. ESI /MS: m/z 474 (M+Na) , ESI -MS/MS: m/z 374 (100).
(R)-2-[[[(1-(3-Chloro-4-fluorophenyl)cyclopropyl]methyl]amino]-2-oxoethyl]-1-(4-
cyanophenylmethyl)-carbamic acid, 1,1-Dimethylethyl Ester ((R)-33).
1
7
3
4
2
7% Yield. H NMR (CDCl ): δ 0.78–0.89 (m, 4H), 1.39 (s, 9H), 2.98 (dd, 1H, J= 13.5, 7.0 Hz),
3
.13 (dd, 1H, J= 13.5, 7.0 Hz), 3.32 (dd, 1H, J= 14.0, 5.5 Hz), 3.38 (dd, 1H, J= 14.0, 5.5 Hz), 4.24-
.28 (m, 1H), 4.94 (br s, 1H), 6.04 (br t, 1H), 7.02–7.06 (m, 2H), 7.20 (d, 1H, J=7.0 Hz), 7.28 (d,
+
+
+
H, J= 8.5 Hz), 7.58 (d, 2H, J= 8.0 Hz). ESI /MS: m/z 494 (M+Na) , ESI -MS/MS: m/z 394 (100).
S)- 2-[[[(1-(3-Chloro-4-fluorophenyl)cyclopropyl]methyl]amino]-2-oxoethyl]-1-(4-
cyanophenylmethyl)-carbamic acid, 1,1-Dimethylethyl Ester ((S)-33).
(
+
+
+
8
4% Yield. ESI /MS: m/z 494 (M+Na) , ESI -MS/MS: m/z 394 (100).
General Procedure for the Synthesis of Amines (R)- and (S)-35–39.
Trifluoroacetic acid (5 mL) was added to a solution of Boc-protected derivatives (R)- and (S)-29-33

ACCEPTED MANUSCRIPT
(
0.46 mmol) in CH Cl (20 mL). The reaction mixture was stirred at room temperature for 5 h and
2
2
basified with aqueous 1 M NaOH. The separated aqueous phase was extracted with CH Cl (2 × 20
2
2
mL). The combined organic layers were dried (Na SO ) and concentrated in vacuo to give the
2
4
desired compounds as pale yellow semisolids that were used without further purification.
(R)-2-Amino-3-(4-cyanophenyl)-N-[(1-phenylcyclopropyl)methyl]propanamide ((R)-35). 43%
1
Yield. H NMR (CDCl ): δ 0.87–0.97 (m, 4H), 1.72 (br s, 2H), 2.82 (dd, 1H, J= 14.0, 8.5 Hz), 3.21
3
(dd, 1H, J= 13.7, 4.4 Hz), 3.40 (dd, 1H, J= 13.9, 6.0 Hz), 3.45 (dd, 1H, J= 13.7, 6.0 Hz), 3.62 (dd,
+
1
3
H, J= 8.0, 4.5 Hz), 7.19–7.24 (m, 4H), 7.27–7.31 (m, 3H), 7.58 (d, 2H, J= 8.2 Hz). ESI /MS m/z
+
+
42 (M+Na) , ESI /MS/MS m/z 151(100).
(S)-2-Amino-3-(4-cyanophenyl)-N-[(1-phenylcyclopropyl)methyl]propanamide ((S)-35).
+
+
+
7
6% Yield. ESI /MS m/z 342 (M+Na) , ESI /MS/MS m/z 151(100).
(R)-2-Amino-N-[(1-phenylcyclopropyl)methyl]-3-(3-pyridinyl)propanamide ((R)-36).
1
9
3
7
3
0% Yield. H NMR (CDCl ): δ 0.81–0.87 (m, 4H), 1.53 (br s, 2H), 2.71 (dd, 1H, J = 13.5, 8.2 Hz),
3
.14 (dd, 1H, J= 14.1, 4.1 Hz), 3.36–3.49 (m, 2H), 3.55 (dd, 1H, J= 8.8, 4.1 Hz), 7.19–7.30 (m,
+
H), 7.52 (d, 1H, J= 7.6 Hz), 8.43 (d, 1H, J= 1.7 Hz), 8.5 (dd, 1H, J= 5.3, 1.7 Hz). ESI /MS m/z
+
+
18 (M+Na) , ESI /MS/MS m/z 226(100).
S)-2-Amino-N-[(1-phenylcyclopropyl)methyl]-3-(3-pyridinyl)propanamide ((S)-36).
(
+
+
+
Quantitative yield. ESI /MS m/z 318 (M+Na) , ESI /MS/MS m/z 226 (100).
(R)-2-Amino-3-(4-cyanophenyl)-N-[[1-(4-trifluoromethoxyphenyl)cyclopropyl]methyl]
propanamide ((R)-37).
1
9
3
3
2% Yield. H NMR (CDCl ): δ 0.82–0.93 (m, 4H), 1.47 (br s, 2H), 2.75 (dd, 1H, J= 13.5, 8.5 Hz),
3
.23 (dd, 1H, J= 13.5, 4.5 Hz), 3.41 (dd, 1H, J= 14.0, 6.00 Hz), 3.46 (dd, 1H, J= 14.0, 6.00 Hz),
.58 (dd, 1H, J= 8.8, 4.5 Hz), 7.12–7.15 (m, 2H), 7.25–7.28 (m, 3H), 7.29–7.31 (m, 2H), 7.59–7.62

ACCEPTED MANUSCRIPT
-
-
-
(
m, 2H). ESI /MS: m/z 402 (M-H) , ESI -MS/MS: m/z 285 (100), 116 (12).
(S)-2-Amino-3-(4-cyanophenyl)-N-[[1-(4-trifluoromethoxyphenyl)cyclopropyl]methyl]
propanamide ((S)-37).
-
-
-
9
0% Yield. ESI /MS: m/z 402 (M-H) , ESI -MS/MS: m/z 285 (100), 116 (13).
(R)-2-Amino-3-(4-cyanophenyl)-N-[[1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]
propanamide ((R)-38).
1
7
3
1
8
1% Yield. H NMR (CDCl ): δ 0.76–0.82 (m, 2H), 0.83–0.88 (m, 2H), 1.47 (br s, 2H), 2.23 (s,
3
H), 2.89–2.92 (m, 1H), 3.17–3.20 (m, 1H), 3.23–3.29 (m, 1H), 3.42–3.46 (m, 1H), 4.12–4.14 (m,
H), 6.83–6.86 (m, 2H), 7.06–7.09 (m, 1H), 7.13 (br t, 1H), 7.22 (d, 2H, J= 8.5 Hz), 7.46 (d, 2H, J=
-
-
-
.5 Hz). ESI /MS: m/z 350 (M-H) , ESI -MS/MS: m/z 233 (100), 116 (67).
S)-2-Amino-3-(4-cyanophenyl)-N-[[1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]
(
propanamide ((S)-38)
-
-
-
9
4% yield. ESI /MS: m/z 350 (M-H) , ESI -MS/MS: m/z 233 (100), 116 (61).
(R)-2-Amino-3-(4-cyanophenyl)-N-[[1-(3-chloro-4-fluorophenyl)cyclopropyl]methyl]
propanamide ((R)-39).
1
4
7% Yield. H NMR (DMSO-d ): δ 0.73–0.80 (m, 2H), 0.83–0.88 (m, 2H), 2.30 (br s, 2H), 2.64
6
(
(
(
dd, 1H, J= 13.0, 8.0 Hz), 2.88 (dd, 1H, J=13.0, 5.5 Hz), 3.20 (dd, 1H, J= 13.0, 5.5 Hz), 3.33–3.37
m, 1H), 3.40–3.42 (m, 1H), 7.18–7.21 (m, 1H), 7.28 (d, 1H, J= 9.0), 7.34 (d, 2H, J= 8.5 Hz), 7.38
-
-
-
dd, 1H, J= 6.5, 2.0 Hz), 7.70 (d, 2H, J= 8.4 Hz), 7.91 (br t, 1H). ESI /MS: m/z 370 (M-H) , ESI -
MS/MS: m/z 253 (100), 116 (25).
S)-2-Amino-3-(4-cyanophenyl)-N-[[1-(3-chloro-4-fluorophenyl)cyclopropyl]methyl]
(
propanamide ((S)-39).
-
-
-
9
5% Yield. ESI /MS: m/z 370 (M-H) , ESI -MS/MS: 253 (100), 116 (21).

ACCEPTED MANUSCRIPT
General Procedure for the Synthesis of the Final Compounds (Procedure A)
To a solution of the amine (R)- and (S)-34-39 (1.0 mmol) in anhydrous THF, a solution of the
appropriate 4-substitued phenylisocyanate (1.2 mmol) in the same solvent (10 mL) was added and
the reaction mixture was stirred at room temperature overnight. After removing the solvent in
vacuo, the residue was taken up in CHCl (20 mL) and washed with H O (2 Õ 20 mL). The
3
2
separated organic layers were dried over Na SO and concentrated under reduced pressure. The
2
4
crude residue was chromatographed (CHCl /AcOEt, 1:1 as the eluent). When necessary, the
3
obtained solid was further purified by crystallization from MeOH to give the final compounds.
(R)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[(1-phenylcyclopropyl)methyl]
propanamide ((R)-7).
1
1
9% Yield. H NMR (CDCl ): δ 0.56–0.74 (m, 4H), 3.06 (dd, 1H, J= 14.6, 8.2 Hz), 3.19–3.25 (m,
3
3H), 3.76 (s, 3H), 4.63–4.65 (m, 1H), 5.91 (br d, 1H), 6.00 (br t, 1H), 6.73 (d, 2H, J= 8.8 Hz), 6.83
(d, 1H, J= 1.8 Hz), 6.91–6.94 (m, 3H), 7.04–7.22 (m, 7H), 7.32 (d, 1H, J= 8.2 Hz), 7.62 (d, 1H, J=
1
3
8
1
1
.4 Hz), 7.97 (br s, 1H). C NMR (DMSO-d ): 171.9; 158.9; 154.8; 147.9; 136.5; 131.5; 128.3;
6
27.4; 125.6; 125.0; 123.0; 121.7; 119.8; 118.8; 114.5; 111.1; 109.7; 55.8; 53.6; 47.2; 36.4; 25.3;
-
-
-
2.7; 12.6. ESI /MS: m/z 481 (M-H) ; ESI -MS/MS: m/z 358 (100), 332 (44).
(S)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[(1-phenylcyclopropyl)methyl]
propanamide ((S)-7).
-
-
-
2
2% Yield. ESI /MS: m/z 481 (M-H) ; ESI -MS/MS: m/z 358 (100), 332 (43).
(S)-2-[3-(4-Fluorophenyl)ureido]-3-(1H-indol-3-yl)-N-[(1-phenylcyclopropyl)
methyl]propanamide ((S)-8).
1
4
1
1
3
1% Yield. H NMR (DMSO-d ): δ 0.64–0.81 (m, 4H), 2.84 (dd, 1H, J= 14.6, 7.0 Hz), 2.99 (dd,
6
H, J= 14.6, 5.3 Hz), 3.22 (dd, 1H, J= 13.5, 5.3 Hz), 3.38 (dd, 1H, J= 13.5, 5.8 Hz), 4.51–4.44 (m,
H), 6.22 (br d, 1H), 6.92 (t, 2H, J= 7.6 Hz), 6.98–7.00 (m, 2H), 7.10–7.21 (m, 6H), 7.28–7.34 (m,
-
H), 7.5 (d, 1H, J= 8.2 Hz), 8.02 (t, 1H, J= 5.3Hz), 8.66 (s,1H), 10.78 (s, 1H). ESI /MS: m/z 469

ACCEPTED MANUSCRIPT
-
-
13
(
M-H) , ESI -MS/MS: m/z 229 (6), 332 (14), 358 (100). C NMR (DMSO-d ): 170.7; 158.3; 154.6;
6
1
2
47.9; 136.5; 135.0; 128.3; 127.4; 125.6; 125.0; 123.1; 119.3; 118.8; 115.7; 109.6; 53.7; 47.0; 36.4;
-
-
-
5.3; 12.7. ESI /MS: m/z 469 (M-H) ; ESI -MS/MS: m/z 358 (100), 332 (12).
(R)-3-(1H-Indol-3-yl)-N-[(1-phenylcyclopropyl)methyl]-2-[3-(phenyl)ureido]propanamide
((R)-9).
1
3
2
6
7
1
1
5% Yield. H NMR (CDCl ): δ 0.49–0.74 (m, 4H), 3.09 (dd, 1H, J= 14.6, 8.8 Hz), 3.19–3.25 (m,
3
H), 3.72–3.76 (m, 1H), 4.65–4.73 (m, 1H), 6.09 (br s, 1H), 6.40 (br t, 1H), 6.83 (br s, 1H), 6.91–
.93 (m, 2H), 6.97–7.01 (m, 1H), 7.06–7.13 (m, 4H), 7.16–7.21 (m, 5H), 7.32 (d, 1H, J= 8.2 Hz),
1
3
.52 (br t, 1H), 7.65 (d, 1H, J= 8.2 Hz), 7.97 (s, 1H). C NMR (DMSO-d ): 171.7; 154.5; 147.9;
6
39.4; 136.5; 128.9; 128.3; 128.0; 127.4; 123.3; 121.6; 119.8; 118.8; 110.1; 109.5; 58.7; 46.9; 25.7;
-
-
-
2.7; 12.6. ESI /MS: m/z 451 (M-H) ; ESI -MS/MS: m/z 358 (100), 332 (16).
(S)-3-(1H-Indol-3-yl)-N-[(1-phenylcyclopropyl)methyl]-2-[3-(phenyl)ureido]propanamide
((S)-9).
-
-
-
1
5% Yield. ESI /MS: m/z 451 (M-H) , ESI -MS/MS: m/z 358 (100), 332 (10).
(R)-3-(4-Cyanophenyl)-2-[3-(4-nitrophenyl)ureido]-N-[(1-
phenylcyclopropyl)methyl]propanamide ((R)-10).
1
6
1
9% Yield. H NMR (DMSO-d ): δ 0.69–0.84 (m, 4H), 2.79 (dd, 1H, J= 13.5, 7.6 Hz), 2.96 (dd,
6
H, J= 13.5, 5.2 Hz), 3.18 (dd, 1H, J= 13.5, 5.3 Hz), 3.48 (dd, 1H, J= 14.1, 6.4 Hz), 4.51–4.58
(m,1H), 6.61 (br d, 1H), 7.11–7.17 (m, 2H), 7.19–7.27 (m, 5H), 7.53 (d, 2H, J= 9.1 Hz), 7.66 (d,
1
3
2
1
5
H, J= 8.2 Hz), 8.01 (d, 2H, J= 8.8 Hz), 8.22 (br t, 1H), 9.41 (br s, 1H). C NMR (DMSO-d ):
6
70.9; 154.1; 147.2; 143.7; 141.0; 132.3; 130.8; 128.5; 128.4; 126.5; 125.6; 119.3; 117.3; 109.7;
-
-
-
3.7; 47.0; 25.3; 12.7. ESI /MS: m/z 482 (M-H) , ESI -MS/MS: m/z 344 (100).
(S)-3-(4-Cyanophenyl)-2-[3-(4-nitrophenyl)ureido]-N-[(1-
phenylcyclopropyl)methyl]propanamide ((S)-10).

ACCEPTED MANUSCRIPT
-
-
-
3
9% Yield. ESI /MS: m/z 482 (M-H) ; ESI -MS/MS: m/z 344 (100).
(R)-2-[3-(4-Nitrophenyl)ureido]-N-[(1-phenylcyclopropyl)methyl]-3-(3-pyridinyl)-
propanamide ((R)-11).
1
3
1
1
5% Yield. H NMR (DMSO-d ): δ 0.73–0.85 (m, 4H), 2.73 (dd, 1H, J= 13.5, 7.6 Hz), 2.90 (dd,
6
H, J= 14.1, 4.7 Hz), 3.22 (dd, 1H, J= 14.1, 4.7 Hz), 3.45 (dd, 1H, J= 13.5, 5.9 Hz), 4,51–4.53 (m,
H), 6.65 (d, 1H, J= 7.6 Hz), 7.13–7.26 (m, 5H), 7.37 (m, 1H), 7.53 (d, 2H, J= 8.9 Hz), 8.1 (d, 2H,
1
3
J= 9.3 Hz), 8.22–8.30 (m, 3H), 8.36 (d, 1H, J= 4.7 Hz), 9.45 (s, 1H). C NMR (DMSO-d ): 171.1;
6
154.1; 150.7; 148.0; 147.2; 143.8; 141.0; 137.1; 133.2; 128.6; 128.5; 126.5; 125.6; 123.6; 117.2;
-
-
-
5
3.7; 47.1; 36.4; 25.3; 12.7; 12.6. ESI /MS: m/z 458 (M-H) , ESI -MS/MS: m/z 320 (100).
(S)- 2-[3-(4-Nitrophenyl)ureido]-N-[(1-phenylcyclopropyl)methyl]-3-(3-pyridinyl)-
propanamide ((S)-11).
-
-
-
2
8% Yield. ESI /MS: m/z 458 (M-H) ; ESI -MS/MS: m/z 320 (100).
(R)-3-(4-Cyanophenyl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(4-
trifluoromethoxyphenyl)cyclopropyl]methyl]propanamide ((R)-12).
1
7
1
4
2% Yield. H NMR (DMSO-d ): δ 0.70–0.87 (m, 4H), 2.75 (dd, 1H, J= 13.5, 8.1 Hz), 2.92 (dd,
6
H, J= 14.1, 5.1 Hz), 3.19 (dd, 1H, J= 14.1, 5.1 Hz), 3.42 (dd, 1H, J= 14.1, 6.0 Hz), 3.66 (s, 3H),
.46–4.53 (m, 1H), 6.20 (br d, 1H), 6.78 (d, 2H, J= 8.7 Hz), 7.17–7.21 (m, 4H), 7.26 (d, 2H, J= 8.4
1
3
Hz), 7.36 (d, 2H, J= 8.7 Hz), 7.68 (d, 2H, J= 8.1 Hz), 8.16 (br t, 1H), 8.39 (s, 1H). C NMR
(
DMSO-d ): 171.2; 158.9; 154.8; 144.8; 140.8; 140.2; 132.1; 131.7; 130.9; 126.0; 119.8; 118.6;
6
-
-
-
1
4
14.5; 113.9; 97.2; 55.8; 53.7; 47.0; 25.3; 12.7; 12.6. ESI -MS: m/z 551 (M-H) , ESI -MS/MS: m/z
28 (46), 402 (23), 385 (100).
(S)-3-(4-Cyanophenyl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(4-
trifluoromethoxyphenyl)cyclopropyl]methyl]propanamide ((S)-12).
-
-
-
7
6% Yield. ESI -MS: m/z 551 (M-H) , ESI -MS/MS: m/z 428 (50), 385 (100).

ACCEPTED MANUSCRIPT
(R)-3-(4-Cyanophenyl)-2-[3-(4-fluorophenyl)ureido]-N-[[1-(4-
trifluoromethoxyphenyl)cyclopropyl]methyl]propanamide ((R)-13).
1
6
4% Yield. H NMR (DMSO-d ): δ 0.72–0.74 (m, 2H), 0.80–0.82 (m, 2H), 2.76 (dd, 1H J= 13.5,
6
8.5 Hz), 2.92 (dd, 1H, J= 13.5, 5.5 Hz), 3.20 (dd, 1H, J= 13.5, 5.5 Hz), 3.44 (dd, 1H, J= 14.0, 6.5
Hz), 4.49–4.53 (m, 1H), 6.32 (br d, 1H), 7.01–7.05 (m, 2H), 7.20 (d, 2H, J= 8.5 Hz), 7.25 (d, 2H,
J= 8.5 Hz) 7.29–7.34 (m, 2H), 7.35–7.38 (m, 2H), 7.69 (d, 2H, J= 8.5 Hz ), 8.24 (br t, 1H), 8.65 (s,
1
3
1
1
H). C NMR (DMSO-d ): 171.2; 162.9; 154.8; 144.8; 140.8; 140.2; 135.0; 132.1; 130.9; 129.7;
6
-
-
-
26.0; 119.3; 115.7; 113.9; 53.5; 47.1; 36.4; 25.3; 12.7; 12.6. ESI /MS: m/z 539 (M-H) , ESI -
MS/MS: m/z 428 (94), 385 (100).
S)-3-(4-Cyanophenyl)-2-[3-(4-fluorophenyl)ureido]-N-[[1-(4-trifluoromethoxyphenyl)
cyclopropyl]methyl]propanamide ((S)-13).
(
-
-
-
8
0% Yield. ESI /MS: m/z 539 (M-H) , ESI -MS/MS: m/z 428 (86), 385 (100).
(R)-3-(4-Cyanophenyl)-N-[[1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]-2-[3-(4-
methoxyphenyl)ureido]propanamide ((R)-14).
1
1
1
3% Yield. H NMR (DMSO-d ): δ 0.74–0.78 (m, 2H), 0.81–0.86 (m, 2H), 2.14 (s, 3H), 2.78 (dd,
6
H, J= 14.0, 8.0 Hz), 2.94 (dd, 1H, J= 14.0, 5.5 Hz), 3.18 (dd, 1H, J= 14.0, 5.5 Hz), 3.44 (dd, 1H,
J= 14.0, 6.5 Hz), 3.67 (s, 3H), 4.48–4.52 (m, 1H), 6.26 (br d, 1H), 6.78 (d, 2H, J= 8.8 Hz), 6.95–
.98 (m, 1H), 7.02 (d, 1H, J= 1.5 Hz), 7.12 (t, 1H, J= 8.0 Hz), 7.21 (d, 2H, J= 8.8 Hz), 7.26 (d, 2H,
6
1
3
J= 8.3 Hz), 7.67 (d, 2H, J= 8.5 Hz), 8.18 (br t, 1H), 8.46 (s, 1H). C NMR (DMSO-d ): 170.8;
6
1
2
61.1; 158.9; 154.8; 148.6; 140.8; 132.1; 130.9; 130.2; 120.5; 119.8; 114.5; 111.9; 55.8; 53.7; 47.0;
–
-
-
5.2; 14.5; 12.6. ESI /MS: m/z 499 (M-H) , ESI -MS/MS: m/z 376 (42), 333 (100).
(R)-3-(4-Cyanophenyl)-N-[[1-(3-fluoro-4-methylphenyl)cyclopropyl]methyl]-2-[3-(4-
fluorophenyl)ureido]propanamide ((R)-15).

ACCEPTED MANUSCRIPT
1
1
2
4
8
0% Yield. H NMR (DMSO-d ): δ 0.71–0.86 (m, 4H), 2.14 (s, 3H), 2.79 (dd, 1H, J= 13.7, 7.8 Hz),
6
.94 (dd, 1H, J= 13.7, 5.4 Hz), 3.19 (dd, 1H, J= 14.2, 5.0 Hz), 3.44 (dd, 1H, J= 14.2, 6.4 Hz), 4.51–
.52 (m, 1H), 6.36 (br d, 1H), 6.96 (td, 2H, J= J= 8.5, 1.5 Hz), 7.01–7.05 (m, 2H), 7.12 (t, 1H, J=
.0 Hz), 7.25 (d, 2H, J= 7.8 Hz), 7.30–7.35 (m, 2H), 7.67 (d, 2H, J= 8.5 Hz), 8.19 (br t, 1H), 8.70
1
3
(
s, 1H). C NMR (DMSO-d ): 171.7; 162.9; 154.8; 148.6; 140.9; 132.1; 130.9; 130.2; 120.8;
6
-
1
20.5; 119.3; 118.6; 115.7; 111.9; 109.8; 53.7; 47.0; 36.4; 25.2; 14.5; 12.6. ESI /MS: m/z 487 (M-
-
-
H) , ESI -MS/MS: m/z 376 (75), 333 (100).
(R)-N-[[1-(3-Chloro-4-fluorophenyl)cyclopropyl]methyl]-3-(4-cyanophenyl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((R)-16).
1
1
1
4
9% Yield. H NMR (DMSO-d ): δ 0.72–0.87 (m, 4H), 2.76 (dd, 1H, J= 13.5, 7.5 Hz), 2.92 (dd,
6
H, J= 13.5, 5.5 Hz), 3.14 (dd, 1H, J= 14.0, 5.0 Hz), 3.44 (dd, 1H, J= 14.0, 6.5 Hz), 3.67 (s, 3H),
.50 (td, 1H, J= 8.5, 6.0 Hz), 6.25 (br d, 1H), 6.78 (d, 2H, J= 9.0 Hz), 7.19–7.22 (m, 2H), 7.24–7.27
1
3
(
m, 4H), 7.45 (d, 1H, J= 7.5 Hz), 7.69 (d, 2H, J= 8.5 Hz), 8.22 (br t, 1H), 8.42 (s, 1H). C NMR
(
DMSO-d ): 170.8; 158.9; 155.7; 154.8; 147.0; 140.8; 132.1; 131.7; 132.1;130.9; 126.4; 120.3;
6
-
-
-
1
18.6; 119.8; 116.5; 114.5; 55.8; 53.7; 47.2; 36.4; 25.3; 12.7; 12.6. ESI -MS: m/z 519 (M-H) , ESI -
MS/MS: m/z 396 (50), 370 (30), 353 (100).
S)-N-[[1-(3-Chloro-4-fluorophenyl)cyclopropyl]methyl]-3-(4-cyanophenyl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((S)-16).
(
-
-
-
5
0% Yield. ESI -MS: m/z 519 (M-H) , ESI -MS/MS: m/z 396 (50), 370 (32), 353 (100).
(S)- 3-(4-Cyanophenyl)-N-[[1-(3-chloro-4-fluorophenyl)cyclopropyl]methyl]-2-[3-(4-
fluorophenyl)ureido]propanamide ((S)-17).
1
3
1
1
4% Yield. H NMR (DMSO-d ): δ 0.73–0.89 (m, 4H), 2.77 (dd, 1H, J= 13.7, 7.8 Hz), 2.93 (dd,
6
H, J= 13.7, 5.3 Hz), 3.15 (dd, 1H, J= 14.2, 5.4 Hz), 3.44 (dd, 1H, J= 14.2, 7 Hz), 4.48–4.53 (m,
H), 6.30 (br d, 1H), 7.01–7.06 (m, 2H), 7.25 (t, 2H, J= 7.8 Hz), 7.29–7.33 (m, 4H), 7.45 (dt, 1H,

ACCEPTED MANUSCRIPT
1
3
J= 6.0, 2.0 Hz), 7.69 (d, 2H, J= 6.4 Hz), 8.24 (br t, 1H), 8.63 (s, 1H). C NMR (DMSO-d ): 171.5;
6
158.3; 157,1; 156.4; 155.2; 154.9; 144.0; 141.6; 136.9; 132.3; 131.0; 130.7; 129.5; 119.5; 119.3;
-
-
-
1
16.9; 116.7; 115.7; 115.5; 109.6; 53.7; 46.9; 25.13; 12.7; 12.6. ESI /MS: m/z 507 (M-H) , ESI -
MS/MS: m/z 396 (100), 353 (89).
General Procedure for the Synthesis of the Final Compounds (Procedure B)
’
N,N -Carbonyldiimidazole (1.1 mmol) was added to a solution of aniline (1.0 mmol), in anhydrous
THF (10 mL), under N . The reaction mixture was stirred at room temperature overnight, then a
2
solution of the amine (R)-34, (S)-38, or (S)-39, (1.0 mmol) in anhydrous THF was added. The
reaction mixture was stirred until the reagents disappeared monitoring by TLC. Then, the solvent
was removed in vacuo and the residue was partitioned between EtOAc (20 mL) and H O (20 mL).
2
The separated aqueous layer was extracted twice with EtOAc (20 mL), then the collected organic
layers were dried (Na SO ) and evaporated in vacuo. The crude residue was chromatographed to
2
4
give pure target compound as a white solid. When necessary, the obtained solid was further purified
by crystallization from MeOH to give the final compound.
(R)-2-[3-(4-Fluorophenyl)ureido]-3-(1H-indol-3-yl)-N-[(1-phenylcyclopropyl)
methyl]propanamide ((R)-8).
1
1
1
1
3
9% Yield. H NMR (DMSO-d ): δ 0.65–0.80 (m, 4H), 2.84 (dd, 1H, J= 14.6, 7.0 Hz), 2.99 (dd,
6
H, J= 14.6, 5.3 Hz), 3.22 (dd, 1H, J= 13.5, 5.3 Hz), 3.38 (dd, 1H, J= 13.5, 5.8 Hz), 4.51–4.44 (m,
H), 6.22 (br d, 1H), 6.92 (t, 2H, J= 7.6 Hz), 6.98–7.00 (m, 2H), 7.10–7.21 (m, 6H), 7.28–7.34 (m,
1
3
H), 7.5 (d, 1H, J= 8.2 Hz), 8.02 (t, 1H, J= 5.3Hz), 8.66 (s,1H), 10.78 (s, 1H). C NMR (DMSO-
d6): 170.7; 158.3; 154.6; 147.9; 136.5; 135.0; 128.3; 127.4; 125.6; 125.0; 123.1; 119.3; 118.8;
-
-
-
1
15.7; 109.6; 53.7; 47.0; 36.4; 25.3; 12.7. ESI /MS: m/z 469 (M-H) , ESI -MS/MS: m/z 229 (6), 332
(14), 358 (100).