Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor 2

Article abstract of DOI:10.1016/j.bmc.2014.12.007

N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists that were designed starting from our lead agonist (S)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Several of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds.

Full text of DOI:10.1016/j.bmc.2014.12.007

Accepted Manuscript
Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as se-
lective agonists of human formyl-peptide receptor 2
Enza Lacivita, Igor A. Schepetkin, Madia L. Stama, Liliya N. Kirpotina, Nicola
A. Colabufo, Roberto Perrone, Andrei I. Khlebnikov, Mark T. Quinn, Marcello
Leopoldo
PII:
S0968-0896(14)00845-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.007
BMC 11939
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 September 2014
28 November 2014
4 December 2014
Please cite this article as: Lacivita, E., Schepetkin, I.A., Stama, M.L., Kirpotina, L.N., Colabufo, N.A., Perrone, R.,
Khlebnikov,
A.I.,
Quinn,
M.T.,
Leopoldo,
M.,
Novel
3-(1H-indol-3-yl)-2-[3-(4-
methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor 2, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.007
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Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as Selective Agonists of
Human Formyl-Peptide Receptor 2
Enza Lacivita1, Igor A. Schepetkin2, Madia L. Stama1, Liliya N. Kirpotina2, Nicola A. Colabufo1,
Roberto Perrone1, Andrei I. Khlebnikov3,4, Mark T. Quinn2, and Marcello Leopoldo1*
Affiliation
1Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via
Orabona, 4, 70125, Bari, Italy
2Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717,
USA
3Department of Chemistry, Altai State Techical University, Barnaul, Russia
4Department of Biotechnology and Organic Chemistry, Tomsk Polytechinic University, Tomsk
634050, Russia
*Corresponding Authors: Marcello Leopoldo, Dipartimento di Farmacia – Scienze del Farmaco,
Università degli Studi di Bari “A. Moro”, via Orabona, 4, 70125, Bari, Italy. E-mail:
marcello.leopoldo@uniba.it; Phone: +39 080 5442798; Fax: +39 080 5442231
1

Abstract
N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles
in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the
resolution of pathological inflammatory reactions. We here report the synthesis and biological
evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl
peptide receptor-2 (FPR2) agonists, that were designed starting from our lead agonist (S)-3-(1H-
indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-
pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall
yields considerably higher than (S)-9a. Various of the new compounds showed agonist properties
comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used
to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also
assessed to obtain preliminary insight on drug-like properties of this class of compounds.
Keywords: Formyl peptide receptor, ureido propanamide, chiral agonist, Ca2+ mobilization,
neutrophil, metabolic stability
2

1. Introduction
Formyl peptide receptors (FPRs) comprise a functionally distinct G-protein coupled receptor
(GPCR) subfamily. In human and other primates, three subtypes of FPRs have been identified
(FPR1-3).1 FPR1 was initially reported as a high affinity receptor on the surface of neutrophils for
the prototypic N-formyl peptide formyl-methionine-leucine-phenylalanine (fMLF), and its gene was
cloned in 1990 from a differentiated HL-60 myeloid leukemia cell cDNA library.2,3 Two additional
human FPRs, designated as FPR2 and FPR3 were subsequently cloned using the FPR1 cDNA as a
probe.1 The cellular distribution of FPR1 and FPR2 in humans is largely overlapping: they have
been detected in monocytes/macrophages, neutrophils, immature dendritic cells, endothelial cells,
hepatocytes, astrocytes, microglial cells and fibroblasts. FPR3 is distributed in human
monocytes/macrophages and dendritic cells.4 Functionally, FPRs are considered to play relevant
roles in innate immunity and host defense mechanisms, including immune response to
microorganism infection, proinflammatory response in amyloidogenic diseases, host responses to
cell necrosis, and apoptosis.4-6 FPRs may also influence the expression and function of C-C
chemokine receptor type 5 and C-X-C chemokine receptor type 4 on human monocytes, two major
GPCR chemokine coreceptors of human immunodeficiency virus type 1 (HIV-1).7,8
Hence, there is evidence that bioactive ligands acting as FPR agonists or antagonists might
serve as useful therapeutics in host defense and as immunomodulatory activators to enhance
selective innate immune responses in order to reduce detrimental effects associated with
inflammation, infectious diseases, and cancer.9-13 Various endogenous and synthetic peptide FPR
agonists have been used to study potential therapeutic efficacy in models of pathological
processes.14 However, peptides are difficult to administer as therapeutic agents due to poor tissue
penetration, serum resistance, oral bioavailability and quick elimination, making small-molecule
compounds a better choice for future clinical development.15 Thus, the availability of non-peptide
small-molecule FPR ligands is clearly of substantial benefit in drug development and facilitating
structure-activity relationship analysis to model ligand binding features. To this end, a number of
3

synthetic nonpeptide FPR agonists and antagonists with a wide range of chemical diversity have
been identified (for a recent review see ref. 14). They include (Figure 1): benzimidazole derivatives
exemplified by compound 1,16 pyridazin-3(2H)-ones (compound 2),17 N-phenylurea derivatives
(compound 3),18 2-(N-piperazinyl)acetamide derivatives (compound 4),16 quinazolinones
derivatives such as the highly specific non-peptide FPR2 agonist 5 (designated in most publications
as Quin-C1),19 pyrazolone derivatives as the mixed FPR1/FPR2 agonist 6 (designated in most of
publications as “Compound 43”).20,21
Despite the increased number of FPR agonists and antagonists discovered in recent years,
pharmacokinetic analysis has been reported only for a limited number of compounds. Researchers
at Astra Zeneca have recently published three lead optimization studies on selective FPR1
antagonists.22-24 The authors were able to identify only three selective FPR1 antagonists endowed
with rat microsome intrinsic clearance (CLint) lower than 30 µL/min/mg, which was selected as lead
selection criteria for in vitro metabolic stability. These studies emphasized that it was challenging to
improve FPR1 potency while maintaining preferential pharmacokinetic properties.
As for FPR agonists, the only available data are related to the agonist 6, which showed
relatively low clearance (0.126 L/h/kg) and a half-life of 2.78 h after iv administration to rats (0.5
mg/kg).20 Therefore, considering that compound 6 is a mixed FPR1/FPR2 agonist, it is of interest to
identify selective FPR2 agonists endowed with pharmacokinetic properties suitable for studies in
vivo.
Recently, we have reported the identification of a group of 3-(1H-indol-3-yl)-2-[3-(4-
nitrophenyl)ureido]propanamide as agonists of human FPR1 and/or FPR2.25 These studies
originated from the finding that antagonists of gastrin-releasing peptide/neuromedin B receptors
(bombesin receptors), namely PD-176252 (7), PD168368 (8) (Figure 2), and related chiral
derivatives, were potent FPR1/FPR2 agonists.18 We evaluated 22 structural derivatives of
compound 7 for their ability to activate human neutrophils and HL-60 cells transfected with human
FPR1 or FPR2. While none of the compounds had affinity for bombesin receptors, 15 of the
4

compounds stimulated Ca2+ flux in FPR1/FPR2 transfected cells. Among these agonists, (S)-9a
(ST-12) (Table 1) emerged because it demonstrated potent agonist properties in both FPR2-HL60
cells and in human and murine neutrophils. However, the synthetic procedure for this compound
greatly limited its availability in amounts compatible with in vivo studies. To overcome this
limitation, and to increase our understanding of the use of such class of compounds as
pharmacological tool to study FPR2 in vitro and in vivo, we designed and synthesized a set of close
analogues of (S)-9 that could be obtained from easily accessible synthons. The new compounds
were tested for their ability to activate FPR2 in FPR2-HL60 cells and neutrophils from humans and
mice. Moreover, selected agonists were also assessed for their metabolic stability in vitro after
incubation with rat microsomes in comparison with compound 7, which is known to be active in
vivo.26
2. Chemistry
The target compounds were synthesized following the same synthetic route as our reference
compound (S)-9a. The synthesis of this compound starts from the key aryl-acetonitrile derivative
15a, which can be prepared following the six-step synthetic procedure depicted in Scheme 1. 2-
Methyl-5-hydroxypyridine is reacted with methyl iodide in the presence of NaH to give the
methoxy derivative 10, which was subjected to oxidation with 3-chloroperbenzoic acid to give N-
oxide 11. Reaction of 11 with acetic anhydride gave the ester 12, which was subsequently
hydrolized with KOH to give the alcohol 13. This intermediate was then transformed into the
corresponding chloroderivative 14 by means of thionyl chloride. Compound 14 underwent
nucleophilic substitution by cyanide ion to give the desired aryl-acetonitrile derivative 15a. The
aryl-acetonitriles 15c-f which were starting compounds for synthesis of the target compounds (S)-
9c-f and (R)-9c-f (Scheme 2) were readily available from commercial sources. The aryl-acetonitrile
15b was prepared in a one-step procedure from the commercially available 2-fluoro-4-
methylpyridine as previously described.27 The synthesis of the target compounds (S)-9b-f and (R)-
5

9a-f was accomplished as follows: aryl-acetonitriles 15a-f were cyclized with 1,5-dibromopentane
in the presence of NaH to give derivatives 16a-f that, after reduction with Raney nickel under
hydrogen pressure yielded the amines 17a-f (Scheme 2). (R)-Boc- or (S)-Boc-tryptophan were then
activated with carbonyldiimidazole and condensed with amines 17a-f to obtain the Boc-protected
derivatives (R)-18a-f and (S)-18b-f. Subsequently, these latter compounds were deprotected with
trifluoroacetic acid to obtain amines (R)-19-a-f and (S)-19-b-f. These intermediates were then
condensed with 4-methoxyphenylisocyanate to obtain the target compounds (R)-9-a-f and (S)-9-b-f.
3. Results and discussion
The aim of the present study was to identify FPR1/FPR2 agonists with a profile similar to
that of (S)-9a but more synthetically accessible, since its availability in a gram scale is limited.
Consequently, the availability of (S)-9a in a gram scale is also limited. This forced us to evaluate
possible substitutes of the 5-methoxy-2-pyridinyl ring in (S)-9a. Therefore, we searched for
commercially available or easily accessible analogs of the key intermediate 15a, and eventually
selected compounds 15b-f.
We systematically evaluated the enantiomeric pairs of target compounds (R)-9-b-f and (S)-
9-b-f (Table 1) because we found in our previous study that FPR1 and FPR2 were able to
discriminate between the enantiomers.25,28 For comparative purposes, we also synthesized (R)-9a
which was not studied previously. As can be seen from the data in Table 1, (S)-9a was 2.4-fold
more potent than (R)-9a for activating FPR2 in FPR2-HL60 cells, whereas a more pronounced
difference in potency was found between these enantiomers in activation of human neutrophils (>
14-fold). Removal of the methoxy group from the pyridyl ring of compounds (S)-9a and (R)-9a
gave compounds (S)-9e and (R)-9e, respectively. This modification had no significant impact on the
ability of the agonists to activate Ca2+ mobilization in FPR2-HL60 cells and in human neutrophils.
In contrast, in the case of 3-pyridyl positional isomers (S)-9d and (R)-9d, a marked
enantioselectivity was observed: while (S)-9d was able to activate FPR2 in FPR2-HL60 cells and in
6

human neutrophils, the (R)- enantiomer was not. A similar trend was observed for the enantiomer
pairs (S)-9b/(R)-9b and (S)-9f/(R)-9f where the (S)- isomer was active and the (R)- enantiomer was
inactive. Conversely, in the case of the enantiomer pairs of phenyl derivative 9c only the (R)-
enantiomer was active.
Collectively, these data confirm the ability of FPR2 to discriminate between the
enantiomers. To further investigate the enantiomer preference of our FPR2 ligands, all molecules
were overlaid on a previously refined three-subpocket pharmacophore model.18 The highest-score
superimpositions together with values of calculated similarity are shown in Table 2. The similarity
values depend equally on geometric and field similarity of a molecule and template. These field
descriptors (or field points) are extrema of electrostatic, steric and hydrophobic fields.29 Similar to
our previous finding,18,25 the active enantiomers of the ureidopropanamides reported here [(S)-9b,
(R)-9c, (S)-9d, and (S)-9f)] had better alignments than their corresponding inactive enantiomeric
conterparts. As examples, overlay of molecular conformations of the enantiomer pair (R)- and (S)-
9c is shown in Figure 3. Likewise, enantiomer (S)-9a with higher activity at FPR2 also had also
better aligment in comparison with its relatively low-active counterpart (R)-9a.
We previuosly reported that the substituted phenyl ring attached to the carbamide nitrogen
atom is always located in subpocked I.25 The present study is consistent with this observation: the 4-
methoxyphenyl group linked to the carbamide fragment occupies subpocked I of the
pharmacophore model for all compounds 9a-f. We also found that subpocket II is usually occupied
by a non-polar moiety of the active FPR2 enantiomer if such a moiety is present in a molecule.25 In
compounds 9a-f, possible moieties for location in subpockets II and III are represented by 3-indolyl
heterocycle and phenyl- or pyridyl-substituted cyclohexyl. Pyridyl and 3-indolyl fragments possess
polar nitrogen centers. The phenyl ring in most cases is also modified by polar methoxy group or
fluorine atom (Table 2). Thus, a non-polar moiety (1-phenylcyclohexyl) is present only in
enantiomers 9c. Among these enantiomers, the active agonist (R)-9c has the non-polar fragment in
subpocket II similarly to compounds investigated previously.25
7

According to chirality of the molecules investigated, the two fragments alternate between
subpockets II and III within enantiomeric pairs 9a, 9c, and 9f. Obviously, this is conditioned by the
chiral nature of the template itself. However, due to the high flexibility of molecular fragments,
both enantiomers in pairs 9b and 9d have similar locations of indolyl and aryl-cyclohexyl moieties
in subpockets II and III (Table 2), in spite of mutually inverse configurations of the chiral center.
However, R-enantiomers in these pairs were inactive. On the other hand, the same location of these
moieties in pair 9e is accompanied by relatively high agonist activity of both S- and R-enantiomers
(Table 2). A visual inspection of superimpositions of these molecules on the pharmacophore
template shows that active compounds have a specific orientation of the C=O group linked directly
to the chiral center. For active S-enantiomers, this carbonyl group is oriented towards 3-indolyl in
subpocket II, resulting in the appearance of a large blue field point between the C=O and 3-indolyl
moieties. For inactive R-enantiomers, the carbonyl group is oriented “outside” the template, and the
negative field point appears in another area. Nevertheless, for compound (R)-9e this carbonyl group
is also directed towards 3-indolyl (not shown). We suggest that such an orientation stipulates a high
activity of enantiomer (R)-9e, in spite of a lower total similarity value as compared with (S)-9e
(Table 2).
All FPR2 agonists listed in Table 1 were able to induce Ca2+ mobilization in human
neutrophils, except for (R)-9c and (S)-9f which were able to induce Ca2+ mobilization only in the
presence of probenecid. This particular feature had been already observed in the case of two
analogues of (S)-9a, which presented a nitro or a cyano group instead of the methoxy group on the
phenyl ring. Probenecid is an anion exchange protein inhibitor used for slowing dye leakage from
cells.30,31 The reason for the selective sensitivity of the Ca2+ flux response to probenecid for some of
these chiral ureidopropanamides is unclear. Literature data indicate that probenecid is a non-specific
inhibitor of multidrug resistance-associated proteins and can have different effects on several other
cellular targets, including transient receptor potential V2 (TRPV2) and various nonselective cation
channels.31 In addition, it has been proposed that primary myeloid cells maintain a subpopulation of
8

FPR in a low-affinity, possibly G protein-free state,32 which is not a feature of FPR-transfected HL-
60 cells. Because allosteric communication between the ligand-binding orthosteric site and the
cytoplasmic G-protein-binding surface is a fundamental feature of GPCRs,33 we suggest that some
FPR2 agonists with ureidopropanamide structure could stabilize this receptor in a G-protein-free
state, and additional agents (e.g., probenecid) may be required to reactivate G-protein coupling.
As far as the activity on FPR1-HL60 cells is concerned, both (S)-9a and (S)-9e stimulated
Ca2+ mobilization, whereas the corresponding enantiomers (R)-9a and (R)-9e were inactive. (S)-9b,
(R)-9c, (S)-9d, (R)-9e, and (S)-9f were also unable to stimulate Ca2+ mobilization in FPR1-HL60
cells, thus revealing an interesting preference for FPR2, at least in FPR-transfected HL60 cells.
Considering that (S)-9e was different from (S)-9d and (S)-9b only in the substitution pattern of the
pyridyl ring, it is apparent that minimal structural changes can deeply influence the activity of these
ureidopropanamides.
All compounds under investigation were active in mouse neutrophils, including compounds
(R)-9b, (S)-9c, (R)-9d, (R)-9f, which were not able to induce Ca2+ mobilization in human
neutrophils. The reason for this activity is currently not understood; however, we and others have
observed quite different response patterns to some agonists and/or antagonists in human and murine
neutrophils.14
Overall, the data presented here indicate that suitable structural modifications of reference
compound (S)-9a led to the identification of moderately potent FPR2 agonists that can be obtained
in gram scale quantity and could be possible candidates for studying in vivo effects of FPR2
activation.
Metabolic stability plays an important role in the success of drug candidates. First-pass
metabolism is one of the major causes of poor bioavailability and short half-life. Until recently,
metabolic stability was evaluated at a later stage of drug discovery and required laborious manual
manipulations. Instead, early screening of pharmacokinetic properties provides valuable and timely
information for medicinal chemists to optimize drug-like properties in parallel with activity
9

optimization. Typically, liver microsomes are used to screen the primary form of metabolic
instability resulting from phase I oxidation. Among the studied compounds, we selected two pairs
of enantiomers for stability analysis, namely (R)- and (S)-9a and (R)- and (S)-9e because they were
the most promising agonists of the set. These compounds were evaluated for their in vitro metabolic
stability after incubation with rat microsomes, in the presence of an NADPH-generating system, in
comparison with compound 7 (Figure 1). Compound 7 was selected as a reference because it is a
close structural analog of (R)- 9a, (S)-9a, (R)-9e, and (S)-9e, and because it is known to be active in
vivo.26 The results (Table 3) indicated that compounds (R)- and (S)-9a and (R)- and (S)-9e were
rapidly metabolized by rat liver microsomes. Compounds (R)- and (S)-9e showed t1/2 = 0.6 min and
(R)- and (S)-9a showed t1/2 = 1.1 and 1.2 min, respectively. Compound 7 showed a slightly higher
stability in this assay with t1/2 = 4.4 min. The difference in metabolic stability between (S)-9a and 7
could be due to the presence of the nitro group in 4-position of the phenyl ring, in place of the
methoxy group, which deactivates the aromatic ring with respect to hydroxylation. In addition, the
presence of the α-methyl on the amide bond is likely to shield this bond from metabolic
degradation. In general, these results suggest a poor pharmacokinetic profile for compounds (R)-
and (S)-9a and (R)- and (S)-9e, as well as for reference compound 7. On the other hand, compound
7 is known to be active in vivo in various behavioral tests after i.p. administration in the rat and
guinea pig.26 One possible explanation is that in vivo binding of compound 7 to plasma protein or
tissues is high. High plasma protein binding remarkably decreases first pass metabolism of the
compound in the liver since it is only the free fraction that can be metabolized.34 Due to the close
similarity between the FPR1/FPR2 agonists (R)- and (S)-9a and (R)- and (S)-9b and compound 7 it
is likely that these compounds share a common metabolic fate in vivo. Nevertheless, further
pharmacokinetic studies are needed to fully elucidate the usability of these FPR2 agonists in vivo.
4. Conclusions
10

In summary, we have manipulated the structure of (S)-9a, a potent ureidopropanamide FPR
agonist previously studied in our laboratories, with the aim of identifying new potent FPR agonists
that can be synthesized starting from easily accessible synthons. We succeeded in our goal because
we identified various FPR2 agonists with good potencies and high selectivity over FPR1 receptor.
All studied compounds were overlaid to a previously refined pharmacophore model showing
interaction with the FPR2 receptor similar to that of the previously reported ureidopropanamide
derivatives. Finally, we have evaluated the in vitro metabolic stability of (R)- and (S)-9a and (R)-
and (S)-9e in order to gain preliminary information on the bioavailabity and drug-like properties of
these class of ligands. The tested compounds were rapidly metabolized by rat liver microsomes
showing an in vitro profile comparable to that of 7 (PD-176252), a ureidopropanamide derivative
that was selected as reference compound. While the metabolic stability data in vitro of (R)-and (S)-
9a and (R)-and (S)-9e were quite disappointing, the notion that compound 7 is in vivo active in
several animal models leaves room for further investigations of the pharmacokinetic properties of
our FPR2 agonists. In any case, further structural modifications of the ureidopropanamide
framework aimed to improve drug-like properties are warranted.
11

5. Experimental Section
5.1. Chemistry
Chemicals were purchased from Acros, Fluka, Sigma-Aldrich. 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 60A (63-200 µm) as the stationary phase. 1H NMR (300 MHz) and 13C NMR (75
MHz) spectra were recorded on a Varian Mercury-VX spectrometer. All spectra were recorded on
free bases. 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 one 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. All spectra were in accordance with the assigned structures.
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 ion mode. Elemental analyses (C,H,N)
of the target 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. The purity
of the target compounds listed in Table 1 was assessed by RP-HPLC and combustion analysis. All
compounds showed ≥ 95% purity. RP-HPLC analysis was performed on a Perkin-Elmer series 200
LC instrument using a Phenomenex Prodigy ODS-3 RP-18 column, (250 × 4.6 mm, 5 µm particle
size) and equipped with a Perkin-Elmer 785A UV/VIS detector setting λ= 254 nm. All target
compounds (Table 1) were eluted with CH3OH/H2O/Et3N, 8:2:0.01 at a flow rate of 1 mL/min.
Enantiomeric purity of the target compounds (R)- and (S)-9a-f was assessed by chiral HPLC
analysis on a Perkin-Elmer series 200 LC instrument using a Daicel ChiralCell 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.
12

All compounds showed enantiomeric excesses (e.e.) >95%. 4-Cyanomethyl-2-fluoropyridine (15b)
was prepared as described in the literature.27
5.1.1. 2-Methyl-5-methoxypyridine (10). To a stirred suspension of NaH (60% dispersed in oil)
(1.47 g, 36.8 mmol) in DMF (50 mL) at 0 °C under argon a solution of 5-hydroxy-2-methylpyridine
(4.00 g, 36.7 mmol) in DMF (20 mL) was addeed slowly, then the reaction mixture was allowed to
warm at room temperature. Methyl iodide (5.21 g, 36.7 mmol) was added to the reaction mixture
which was stirred for 1 h. The reaction mixture was quenched by addition of isopropanol (20 mL),
followed by H2O (20 mL) under argon. The reaction mixture was taken up in AcOEt and washed
with aqueous NaHCO3, dried (Na2SO4) and concentrated in vacuo to give compound 10 as a pure
liquid (2.40 g, 53% yield). 1H NMR (CDCl3): δ 2.49 (s, 3H), 3.83 (s, 3H), 7.05-7.16 (m, 2H), 8.19
(d, 1H, J= 2.8 Hz); GC/MS m/z 124 (M++1, 9) 123 (M+, 100), 108 (71), 80 (43).
5.1.2. 2-Methyl-5-methoxypyridine-N-oxide (11). To a solution of di 2-methyl-5-
methoxypyridine (10) (2.40 g, 19.5 mmol) in CH2Cl2 was added Na2SO4 (4.15 g, 29.25 mmol)
followed by 3-chloroperbenzoic acid (10.06 g, 58.3 mmol) and the mixture was stirred at room
temperature for about 48 h. The reaction mixture was filtered, and the white solid was washed with
CH2Cl2. The filtrate was concentrated and purified by colum chromatography (CHCl3/MeOH, 19:1,
as the eluent) to give compound 11 (0.92 g, 34% yield). 1H NMR: δ 2.45 (s, 3H), 3.81 (s, 3H), 6.84
(dd, 1H, J= 2.5, 8.8 Hz), 7.13 (d, 1H, J= 8.5 Hz), 8.10 (d, 1H, J= 2.5 Hz).
5.1.3. 2-Acetoxymethyl-5-methoxypyridine (12). A mixture of compound 11 (1.10 g, 7.9 mmol)
and acetic anhydride (5 mL) was warmed to reflux for 10 min. The reaction mixture was allowed to
cool to room temperature before being taken up in AcOEt, washed with aqueous NaHCO3. The
separated organic layer was dried (Na2SO4) and concentrated in vacuo to give a crude residue that
was chromatographed (CHCl3 as eluent). Compound 12 was obtained as a pale yellow oil (1.40 g,
13

98% yield). 1H NMR: δ 2.21 (s, 3H), 3.85 (s, 3 H), 5.28 (s, 2H), 7.19 (dd, 2H, J= 3.0, J= 8.5 Hz),
7.29 (d, 1H, J= 8.5 Hz), 8.28 (d, 1H, J= 2.7 Hz). GC/MS m/z 182 (M++1, 1) 181 (M+, 7), 138
(100).
5.1.4. 5-Methoxy-2-pyridinylmethanol (13). To a solution of compound 12 (1.40 g, 7.7 mmol) in
MeOH (30 mL) was added an excess of KOH and the mixture was refluxed 2 h. The solvent was
removed in vacuo, and the residue was partitioned between CH2Cl2 (20 mL) and H2O (20 mL). The
separated organic phase was dried (Na2SO4) and concentrated in vacuo to give compound 13 as a
pale yellow oil (1.00 g, 93% yield). 1H NMR (CDCl3): δ 1.82 (s, 1H), 3.87 (s, 3H), 4.70 (s, 2H),
7.18-7.24 (m, 2H), 8.25 (1H, J= 2.8 Hz); GC/MS m/z 140 (M++1, 3) 139 (M+, 44), 138 (100), 110
(71).
5.1.5. 2-Chloromethyl-5-methoxypyridine (14). To a solution of compound 13 (0.90 g, 6.5
mmoli) in CH2Cl2 was added dropwise excess SOCl2 (2 mL), and the mixture was refluxed for 2 h.
The solvent was removed in vacuo, and the residue was taken up in AcOEt and washed with
aqueous NaHCO3. The separated organic layer was dried (Na2SO4) and concentrated in vacuo.
Compound 14 was obtained as a pale yellow oil in quantitative yield. 1H NMR (CDCl3): δ 3.87 (s,
3H), 4.65 (s, 2H), 7.21 (dd, 1H, J= 2.8, 8.4 Hz), 7.39 (d, 1H, J= 8.4 Hz), 8.27 (1H, d, J= 2.8 Hz);
GC/MS m/z 159 (M++2, 9) 157 (M+, 28), 122 (100).
5.1.6. 5-Methoxy-2-pyridinylacetonitrile (15a). A mixture of derivative 14 (1.00 g, 6.3 mmol) and
KCN (0.41 g, 6.3 mmol) in EtOH (30 mL) and H2O (5 mL) was refluxed overnight. The mixture
was diluted with H2O (30 mL) and extracted with AcOEt (3 × 30 mL). The combined organic layers
were dried Na2SO4 and concentrated in vacuo. The crude residue was purified by colum
chromatography (CH2Cl2 as eluent) to give compound 15a as a yellow oil (0.37 g, 40% yield). 1H
NMR (CDCl3): δ 3.87 (s, 3H), 3.88 (s, 2H), 7.22 (dd, 1H, J= 3.2, 8.8 Hz), 7.34 (dd, 1H, J= 0.4, 8.4
14

Hz), 8.27 (1H, d, J= 2.8 Hz); GC/MS m/z 149 (M++1, 10) 148 (M+, 100), 133 (85), 105 (36), 78
(32).
5.2. General Procedure for the Synthesis of Compounds 16a-f
To a stirred suspension of NaH (60% dispersed in oil) (0.14 g, 3.4 mmol) in DMSO (20 mL) under
nitrogen at 15 °C, a solution of the appropriate acetonitrile (1.8 mmol) and 1,5-dibromopentane
(0.41 g, 1.8 mmol) in Et2O (4 mL) and DMSO (1 mL) was added dropwise over 1 h. The mixture
was allowed to warm to room temperature and stirred for an additional 24 h under nitrogen. The
reaction mixture was carefully quenched by the addition dropwise of isopropanol (5 mL), followed
by H2O 5 mL) 10 min later. The mixture was diluted with AcOEt and washed with H2O. The
aqueous layer was re-extracted with AcOEt, and the two organic layers were combined, dried
(Na2SO4), and concentrated in vacuo. The crude residue was chromatographed (n-hexane/AcOEt,
4:1, as eluent) to give the desired compound as a colorless oil.
5.2.1. 1-(5-Methoxy-2-pyridinyl)cyclohexanecarbonitrile (16a). 30% Yield. 1H NMR (CDCl3): δ
1.76-2.12 (m, 10H), 3.87 (s, 3H), 4.65 (s, 2H), 7.21 (dd, 1H, J= 2.8, 8.8 Hz), 7.51 (dd, 1H, J= 0.8,
9.6 Hz), 8.29 (1H, d, J= 3.2 Hz); GC/MS m/z 217 (M++1, 6), 216 (M+, 34), 176 (63), 161 (100).
5.2.2. 1-(2-Fluoro-4-pyridinyl)cyclohexanecarbonitrile (16b). 51% Yield. 1H NMR (CDCl3): δ
1.26-1.32 (m, 1H), 1.70-1.90 (m, 7H), 2.14 (app d, 2H), 7.05 (d, 1H, J= 1.4 Hz), 7.31 (dt, 1H, J=
2.3, 1.7 Hz), 8.25 (d, 1H, J= 5.5 Hz). GC/MS m/z 205 (M++1, 7), 204 (M+, 49), 149 (100).
5.2.3. 1-Phenylcyclohexanecarbonitrile (16c). 37% Yield. 1H NMR (CDCl3): δ 1.23-1.31 (m, 1H),
1.73-1.89 (m, 7H), 2.13-2.17 (m, 2H), 7.29-7.42 (m, 3H), 7.47-7.51 (m, 2H). GC/MS m/z 186
(M++1, 7), 185 (M+, 47), 130 (24), 129 (100).
15

5.2.4. 1-(3-Pyridinyl)cyclohexanecarbonitrile (16d). 81% Yield. 1H NMR (CDCl3): δ 1.28-1.40
(m, 1H), 1.77-1.90 (m, 7H), 1.97-2.16 (m, 2H), 7.30-7.35 (m, 1H), 7.61-7.64 (m, 1H), 8.45 (dd, 1H,
J= 4.7, 1.4 Hz), 8.53 (dd, 1H, J= 2.5, 0.8 Hz). GC/MS m/z 187 (M++1, 15), 186 (M+, 94), 171 (26),
131 (97), 130 (100).
5.2.5. 1-(2-Pyridinyl)cyclohexanecarbonitrile (16e). 74% Yield. 1H NMR (CDCl3): δ 1.28-1.40
(m, 1H), 1.77-1.90 (m, 7H), 1.97-2.16 (m, 2H), 7.21-7.25 (m, 1H), 7.59 (dt, 1H, J= 8.0, 1.0 Hz),
7.71 (td, 1H, J= 8.0, 1.9 Hz), 8.59-8.61 (m, 1H). GC/MS m/z 186 (M++1, 75), 185 (M+, 97), 157
(94), 146 (45), 131 (100).
5.2.6. 1-(4-Fluorophenyl)cyclohexanecarbonitrile (16f). 61% Yield. 1H NMR (CDCl3): δ 1.23-
1.31 (m, 1H), 1.73-1.89 (m, 7H), 2.12-2.16 (m, 2H), 7.04-7.11 (m, 2H), 7.42-7.49 (m, 2H). GC/MS
m/z 204 (M++1, 5), 204 (M+, 36), 147 (100).
5.3. General Procedure for the Synthesis of the Amines 17a-f
Raney nickel was activated with 10 M KOH, then washed with H2O, then with EtOH to remove
H2O. The Raney nickel was taken up in 2 N ethanolic ammonia and the appropriate nitrile 16a-f
(0.87 mmol) was added to the mixture which was then hydrogenated in autoclave under 5 bar
pressure of hydrogen at 50 °C for 15 h. The reaction mixture was filtered through Celite and
concentrated in vacuo to give the desired amine as a clear oil.
5.3.1. [1-(4-Methoxy-2-pyridyl)cyclohexyl]methanamine (17a). 90% Yield. 1H NMR (CDCl3): δ
1.14-1.55 (m, 10H, 2H D2O exchanged), 2.23-2.28 (m, 2H), 2.79 (s, 2H), 3.80 (s, 3H), 7.31 (d, 1H,
J= 8.0 Hz), 7.64 (td, 1H, J= 8.0, 1.9 Hz), 8.60-8.62 (m, 1H). GC/MS m/z 221 (M++1, 0.5), 191
(100), 162 (26), 123 (18).
16

5.3.2. [1-(2-Fluoro-4-pyridinyl)cyclohexyl]methanamine (17b). 91% Yield. 1H NMR (CDCl3): δ
1.18-1.37 (m, 4H), 1.52-1.65 (m, 6H), 2.10 (app d, 2H), 2.73 (s, 2H), 7.05 (d, 1H, J= 1.4 Hz), 7.31
(dt, 1H, J= 2.3, 1.7 Hz), 8.25 (d, 1H, J= 5.5 Hz). ESI+/MS m/z 209 (M+H)+, ESI-MS/MS m/z 192
(100).
5.3.3. (1-Phenylcyclohexyl)methanamine (17c). 73% Yield. 1H NMR (CDCl3): δ 1.14 (s, 2H, D2O
exchanged), 1.35-1.41 (m, 3H), 1.44-1.55 (m, 5H), 2.11-2.15 (m, 2H), 2.68 (s, 2H), 7.18-7.23 (m,
1H), 7.31-7.38 (m, 4H). GC/MS m/z 172 (0.2), 159 (52), 158 (100), 117 (26), 91 (91).
5.3.4. [1-(3-Pyridinyl)cyclohexyl]methanamine (17d). 86% Yield. 1H NMR (CDCl3): δ 1.14 (s,
2H, D2O exchanged), 1.35-1.41 (m, 3H), 1.44-1.55 (m, 5H), 2.11-2.15 (m, 2H), 2.68 (s, 2H), 7.30-
7.35 (m, 1H), 7.61-7.64 (m, 1H), 8.45 (dd, 1H, J= 4.7, 1.4 Hz), 8.53 (dd, 1H, J= 2.5, 0.8 Hz).
GC/MS m/z 186 (96), 171 (26), 130 (100).
5.3.5. [1-(2-Pyridinyl)cyclohexyl]methanamine (17e). 87% Yield. 1H NMR (CDCl3): δ 1.14-1.55
(m, 10H, 2H D2O exchanged), 2.23-2.28 (m, 2H), 2.79 (s, 2H), 7.08-7.12 (m, 1H), 7.31 (d, 1H, J=
8.0 Hz), 7.64 (td, 1H, J= 8.0, 1.9 Hz), 8.60-8.62 (m, 1H). GC/MS m/z 173 (11), 161 (100), 132
(50).
5.3.6. [1-(4-Fluorophenyl)cyclohexyl]methanamine (17f). 84% Yield. 1H NMR (CDCl3): δ 1.14-
1.52 (m, 8H, 2H D2O exchanged), 1.66-1.88 (m, 2H), 2.05-2.17 (m, 2H), 2.67 (s, 2H), 7.00-7.10
(m, 2H), 7.26-7.31 (m, 1H), 7.48-7.48 (m, 1H). GC/MS m/z 176 (40), 135 (15), 109 (100).
5.4. General Procedure for the Synthesis of BOC-Protected Derivatives (R)-18a-f and (S)-18b-
f.
17

N,N’-carbonyldiimidazole (1.1 mmol) was added to a solution of (1.0 mmol), in anhydrous THF (10
mL), under N2. The reaction mixture was stirred at room 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. The solvent was removed in vacuo and the residue was partitioned
between EtOAc (20 mL) and H2O (20 mL). The aqueous layer was extracted with EtOAc (2 × 20
mL) and the collected organic layers were dried (Na2SO4) and evaporated in vacuo. The crude
residue was chromatographed (CHCl3/EtOAc, 7:3, as eluent) to give pure target compound as a
white solid.
5.4.1. (R)-1-(1H-Indol-3-ylmethyl)-2-[[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((R)-18a). 35% Yield. 1H NMR (CDCl3): δ 1.20-
1.50 (m, 15H), 1.71-1.75 (m, 2H), 1.87-1.94 (m, 2H), 3.06-3.14 (m, 1H), 3.22-3.29 (m, 3H), 3.80
(s, 3H), 4.37-4.39 (m, 1H), 5.14-5.17 (m, 1H), 6.33 (br s, 1H), 6.84-6.87 (m, 1H), 6.91-6.99 (m,
2H), 7.11 (t, 1H, J= 7.4 Hz), 7.19 (t, 1H, J= 7.3 Hz), 7.33 (d, 1H, J= 8.0 Hz), 7.63 (d, 1H, J= 7.7
Hz), 8.01 (br s, 1H), 8.07 (br s, 1H). ESI/MS m/z 505 (M-H)-, ESI-MS/MS m/z 431 (100), 302 (61).
5.4.2. (R)-2-[[[1-(2-Fluoro-4-pyridinyl)cyclohexyl]methyl]amino]-2-oxoethyl]-1-(1H-Indol-3-
ylmethyl)-carbamic acid, 1,1-dimethylethyl ester ((R)-18b). 12% Yield. 1H NMR (CDCl3): δ
1.23-1.50 (m, 15H), 1.62-1.65 (m, 2H), 1.81-1.87 (m, 2H), 3.06-3.14 (m, 2H), 3.19-3.32 (m, 2H),
4.27-4.35 (m, 1H), 5.06 (br s, 1H), 5.35 (t, 1H, J= 2.9 Hz), 6.66 (dd, 1H, J= 8.5, 3.0 Hz), 6.96 (d,
1H, J= 1.9 Hz), 7.11-7.25 (m, 3H), 7.37 (d, 1H, J= 8.0 Hz), 7.61 (d, 1H, J= 7.5 Hz), 7.85 (s, 1H),
8.18 (s, 1H). ESI/MS m/z 493 (M-H)-, ESI-MS/MS m/z 419 (66), 289 (100).
5.4.3. (S)-2-[[[1-(2-Fluoro-4-pyridinyl)cyclohexyl]methyl]amino]-2-oxoethyl]-1-(1H-Indol-3-
ylmethyl)-carbamic acid, 1,1-dimethylethyl ester ((S)-18b). 43% Yield. ESI/MS m/z 493 (M-H)-,
ESI-MS/MS m/z 494 (13), 493 (100), 419 (9).
18

5.4.4. (R)-1-(1H-Indol-3-ylmethyl)-2-[[(1-phenylcyclohexyl)methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((R)-18c). 61% Yield. 1H NMR (CDCl3): δ 1.23-
1.50 (m, 15H), 1.52-1.60 (m, 2H), 1.84-1.90 (m, 2H), 3.01-3.11 (m, 2H), 3.02-3.64 (m, 2H), 4.29-
4.31 (m, 1H), 5.04 (br s, 1H), 5.18 (br s, 1H), 6.96 (d, 1H, J= 1.9 Hz), 7.12-7.14 (m, 5H), 7.36 (d,
1H, J= 8.2 Hz), 7.62 (d, 1H, J= 7.7 Hz), 8.05 (s, 1H). ESI/MS m/z 474 (M-H)-, ESI-MS/MS m/z
401 (26), 400 (100), 271 (87).
5.4.5. (S)-1-(1H-Indol-3-ylmethyl)-2-[[(1-phenylcyclohexyl)methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((S)-18c). 60% Yield. ESI/MS m/z 474 (M-H)-,
ESI-MS/MS m/z 401 (28), 400 (100), 271 (92).
5.4.6. (R)-1-(1H-Indol-3-ylmethyl)-2-[[[1-(3-pyridinyl)cyclohexyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((R)-18d). 83% Yield. ESI/MS m/z 475 (M-H)-,
ESI-MS/MS m/z 401 (87), 272 (100).
5.4.7. (S)-1-(1H-Indol-3-ylmethyl)-2-[[[1-(3-pyridinyl)cyclohexyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((S)-18d). 68% Yield. 1H NMR (CDCl3): δ 1.18-
1.49 (m, 15H), 1.58-1.61 (m, 2H), 1.85-1.90 (m, 2H), 3.04-3.14 (m, 1H), 3.04-3.44 (m, 1H), 3.20-
3.25 (m, 1H), 3.29-3.36 (m, 1H), 4.29-4.31 (m, 1H), 5.08 (br s, 1H), 5.32 (br s, 1H), 6.93 (d, 1H, J=
2.2 Hz), 7.03-7.24 (m, 4H), 7.36 (d, 1H, J= 8.0 Hz), 7.62 (d, 1H, J= 8.0 Hz), 8.25 (s, 1H), 8.31 (d,
1H, J= 1.7 Hz), 8.38 (dd, 1H, J= 4.7, 1.7 Hz). ESI/MS m/z 475 (M-H)-, ESI-MS/MS m/z 401 (82),
272 (100).
5.4.8. (R)-1-(1H-Indol-3-ylmethyl)-2-[[[1-(2-pyridinyl)cyclohexyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((R)-18e). 28% Yield. 1H NMR (CDCl3): δ 1.30-
19

1.50 (m, 17H), 1.72-1.85 (m, 1H), 1.89-2.00 (m, 1H), 3.11 (dd, 1H, J= 14.4, 7.6 Hz), 3.25 (dd, 1H,
J= 14.3, 5.2 Hz), 3.30 (d, 2H, J= 5.5 Hz), 4.39 (br s, 1H), 5.12 (br s, 1H), 6.44 (br s, 1H), 6.95-7.05
(m, 3H), 7.12 (dt, 1H, J= 7.0, 1.1 Hz), 7.19 (dt, 1H, J= 7.0, 1.1 Hz), 7.33 (d, 1H, J= 8.0 Hz), 7.49
(br t, 1H), 7.63 (d, 1H, J= 8.0 Hz), 8.01 (br s, 1H), 8.33 (br s, 1H). ESI-MS m/z 475 (M-H)-, ESI-
MS/MS m/z 401 (100), 272 (62).
5.4.9. (S)-1-(1H-Indol-3-ylmethyl)-2-[[[1-(2-pyridinyl)cyclohexyl]methyl]amino]-2-
oxoethyl]carbamic acid, 1,1-dimethylethyl ester ((S)-18e). 92% Yield. ESI/MS m/z 475 (M-H)-,
ESI-MS/MS m/z 401 (100), 272 (94).
5.4.10. (R)-2-[[[1-(4-Fluorophenyl)cyclohexyl]methyl]amino]-2-oxoethyl]-1-(1H-Indol-3-
ylmethyl)-carbamic acid, 1,1-dimethylethyl ester ((R)-18f). 78% Yield 1H NMR (CDCl3): δ
1.20-1.48 (m, 15H), 1.55-1.62 (m, 2H), 1.72-1.85 (m, 2H), 2.99-3.11 (m, 2H), 3.21-3.31 (m, 2H),
4.30-4.32 (m, 1H), 5.04 (br s, 1H), 5.16 (br s, 1H), 6.80 (app d, 3H), 6.97 (d, 1H, J= 2.2 Hz), 7.13-
7.24 (m, 3H), 7.37 (d, 1H, J= 7.9 Hz), 7.63 (d, 1H, J= 7.7 Hz), 8.07 (s, 1H). ESI/MS m/z 492 (M-
H)-, ESI-MS/MS m/z 418 (89), 289 (100).
5.4.11. (S)-2-[[[1-(4-Fluorophenyl)cyclohexyl]methyl]amino]-2-oxoethyl]-1-(1H-Indol-3-
ylmethyl)-carbamic acid, 1,1-dimethylethyl ester ((S)-18f). 63% Yield. ESI/MS m/z 492 (M-H)-,
ESI-MS/MS m/z 418 (100), 289 (49).
5.5. General Procedure for the Synthesis of Amines (R)-19a-f and (S)-19b-f.
Trifluoroacetic acid (5 mL) was added to a solution of (R)-18a-f, (S)-18b-f (0.46 mmol) in CH2Cl2
(20 mL). The reaction mixture was stirred at room temperature for 5 h and basified with aqueous
1M NaOH. The separated aquous phase was extracted with CHCl3 (2 × 20 mL). The combined
20

organic layers were dried (Na2SO4) and concentrated in vacuo to give the desired compounds as
pale yellow semisolids.
5.5.1. (R)-2-Amino-3-(1H-indol-3-yl)-N-[[1-(5-methoxy-2-
pyridinyl)cyclohexyl]methyl]propanamide ((R)-19a). 96% Yield. 1H NMR (CDCl3): δ 1.22-1.58
(m, 10H), 2.07-2.12 (m, 2H), 2.81 (dd, 1H, J= 14.0, 9.0 Hz), 3.29 (dd, 1H, J= 12.0, 3.8 Hz), 3.41
(d, 2H, J= 6.1 Hz), 3.62 (dd, 1H, J= 9.0, 4.2 Hz), 3.83 (s, 3H), 7.02 (d, 1H, J= 2.2 Hz), 7.11-7.22
(m, 4H), 7.29-7.37 (m, 2H), 7.65 (d, 1H, J= 7.7 Hz), 8.12 (br s, 1H), 8.24 (d, 1H, J= 2.5 Hz).
ESI/MS m/z 405 (M-H)-, ESI-MS/MS m/z 405 (14), 216 (100), 205 (85), 188 (51).
5.5.2. (R)-2-Amino-N-[[(1-(2-fluoro-4-pyridinyl)cyclohexyl]methyl]-3-(1H-indol-3-
yl)propanamide ((R)-19b). 90% Yield. ESI/MS m/z 392 (M-H)-, ESI-MS/MS m/z 263 (100), 177
(65).
5.5.3. (S)-2-Amino-N-[[(1-(2-fluoro-4-pyridinyl)cyclohexyl]methyl]-3-(1H-indol-3-
yl)propanamide ((S)-19b). Quantitative yield. 1H NMR (CDCl3): δ 1.23-1.50 (m, 10H), 1.62-1.65
(m, 2H), 1.81-1.87 (m, 2H), 3.06-3.14 (m, 2H), 3.19-3.32 (m, 2H), 4.27-4.35 (m, 1H), 6.66 (dd, 1H,
J= 8.5, 3.0 Hz), 6.96 (d, 1H, J= 1.9 Hz), 7.11-7.25 (m, 2H), 7.37 (d, 1H, J= 8.0 Hz), 7.61 (d, 1H,
J= 7.5 Hz), 7.85 (s, 1H), 8.18 (s, 1H). ESI/MS m/z 392 (M-H)-, ESI-MS/MS m/z 263 (100), 177
(54).
5.5.4. (R)-2-Amino-3-(1H-indol-3-yl)-N-[(1-phenylcyclohexyl)methyl]propanamide ((R)-19c).
93% Yield. 1H NMR (CDCl3): δ 1.23-1.70 (m, 10H), 1.98-2.05 (m, 2H), 2.82 (dd, 1H, J= 14.3, 8.5
Hz), 3.25-3.35 (m, 3H), 3.60 (dd, 1H, J= 8.3, 4.4 Hz), 6.84 (br t, 1H), 7.00-7.01 (m, 1H), 7.10-7.38
(m, 8H), 7.63-7.66 (m, 1H), 8.08 (br s, 1H). ESI/MS m/z 374 (M-H)-, ESI-MS/MS m/z 242 (100),
130 (62).
21

5.5.5. (S)-2-Amino-3-(1H-indol-3-yl)-N-[(1-phenylcyclohexyl)methyl]propanamide ((S)-19c).
98% Yield. ESI/MS m/z 374 (M-H)-, ESI-MS/MS m/z 242 (100), 130 (71).
5.5.6. (R)-2-Amino-3-(1H-indol-3-yl)-N-[[1-(3-pyridinyl)cyclohexyl]methyl]propanamide ((R)-
19d). 97% Yield. ESI/MS m/z 375 (M-H)-, ESI-MS/MS m/z 246 (100), 244 (57), 214 (50), 160
(65).
5.5.7. (S)-2-Amino-3-(1H-indol-3-yl)-N-[[1-(3-pyridinyl)cyclohexyl]methyl]propanamide ((S)-
19d). Quantitative yield. 1H NMR (CDCl3): δ 1.25-1.70 (m, 10H), 2.00-2.07 (m, 2H), 2.82 (dd, 1H,
J= 14.4, 8.7 Hz), 3.23-3.44 (m, 3H), 3.61 (dd, 1H, J= 8.7, 4.1 Hz), 6.97 (d, 1H, J= 1.9 Hz), 7.06-
7.30 (m, 3H), 7.36 (dd, 1H, J= 8.1, 0.7 Hz), 7.53-7.57 (m, 2H), 7.63 (d, 1H, J= 7.7 Hz), 8.24 (br s,
1H), 8.45 (dd, 1H, J= 4.7, 1.4 Hz), 8.53 (dd, 1H, J= 2.5, 0.8 Hz). ESI/MS m/z 375 (M-H)-, ESI-
MS/MS m/z 246 (100), 244 (58), 214 (52), 160 (70).
5.5.8. (R)-2-Amino-3-(1H-indol-3-yl)-N-[[1-(2-pyridinyl)cyclohexyl]methyl]propanamide ((R)-
19e). 98% Yield. ESI/MS m/z 375 (M-H)-, ESI-MS/MS m/z 244 (18), 214 (100), 160 (33).
5.5.9. (S)-2-Amino-3-(1H-indol-3-yl)-N-[[1-(2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-
19e). 97% Yield. 1H NMR (CDCl3): δ 1.25-1.54 (m, 10H), 2.04-2.10 (m, 2H), 2.86-2.95 (m, 1H),
3.21-3.45 (m, 3H), 3.78-3.82 (m, 1H), 7.02-7.34 (m, 6H), 7.52-7.60 (m, 3H), 8.41-8.50 (m, 1H),
8.73 (br s, 1H). ESI/MS m/z 375 (M-H)-, ESI-MS/MS m/z 244 (18), 214 (100), 160 (33).
5.5.10. (R)-2-Amino-N-[[1-(4-fluorophenyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)propanamide
((R)-19f). 94% Yield. 1H NMR (CDCl3): δ 1.25-1.76 (m, 10H), 1.82-1.96 (m, 2H), 2.84 (dd, 1H, J=
14.3, 8.5 Hz), 3.23-3.35 (m, 3H), 3.62 (dd, 1H, J= 8.3, 4.4 Hz), 6.93 (br s, 1H), 6.95-7.24 (m, 7H),
22

7.35-7.38 (m, 1H), 7.63 (d, 1H, J= 7.4 Hz), 8.18 (br s, 1H). ESI/MS m/z 392 (M-H)-, ESI-MS/MS
m/z 263 (100), 261 (42).
5.5.11. (S)-2-Amino-N-[[1-(4-fluorophenyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)propanamide
((S)-19f). 93% Yield. ESI/MS m/z 392 (M-H)-, ESI-MS/MS m/z 263 (100), 261 (41).
5.6. General Procedure for the Synthesis of the Final Compounds (R)-9a-f and (S)-9b-f.
To a solution of the amine (R)-19a-f, (S)-19b-f (1.0 mmol) in anhydrous THF, a solution of 4-
methoxyphenylisocyanate (1.2 mmol) in the same solvent (10 mL) was added and the reaction
mixture was stirred at room temperature for 5 h (TLC monitoring, CHCl3:AcOEt, 1:1 as the eluent).
After removing the solvent in vacuo, the residue was taken up in CHCl3 (20 mL) and washed with
H2O (2 × 20 mL). The separated organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The crude residue was chromatographed (CHCl3:AcOEt, 1:1 as the eluent). When
necessary, the obtained solid was further purified by crystallization from EtOAc/petroleum ether to
give the final compounds as a pale yellow powders.
5.6.1. (R)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]- N-[[1-(5-methoxy-2-
pyridinyl)cyclohexyl]methyl]propanamide ((R)-9a). 54% Yield. 1H NMR (DMSO-d6): δ 1.18-
1.46 (m, 9H), 1.64-1.75 (m, 1H), 1.92-1.94 (m, 1H), 3.10 (dd, 1H, J= 14.3, 8.2 Hz), 3.07-3.31 (m,
2H), 3.75 (s, 3H), 3.77 (s, 3H), 4.62-4.69 (m, 1H), 6.02 (d, 1H, J= 7.7 Hz), 6.47 (t, 1H, J= 5.1 Hz),
6.73-6.82 (m, 3H), 6.84-6.96 (m, 2H), 7.04-7.25 (m, 5H), 7.26-7.31 (m, 1H), 7.63 (d, 1H, J= 7.7
Hz), 8.00 (d, 1H, J= 3.0 Hz), 8.10 (s, 1H); 13C NMR (CDCl3): 22.3, 26.3, 29.4, 33.2, 33.8, 43.2,
49.1, 55.0, 55.2, 55.6, 55.8, 111.0, 114.5, 119.2, 119.3, 119.8, 121.7, 122.2, 122.7, 123.4, 123.5,
127.7, 132.1, 136.1, 136.4, 153.9, 155.9, 156.1, 156.2, 172.7, 111.0, 111.3, 114.5, 119.2, 119.3,
119.8, 121.7, 122.2, 122.7, 123.4, 123.5. ESI/MS m/z 554 (M-H)-, ESI-MS/MS m/z 431 (100), 504
23

(33), 302 (6). Compound melted at 119-121 °C (from n-hexane/CHCl3). Anal. (C32H37N5O4) C, H,
N.
5.6.2. (R)-N-[[(1-(2-Fluoro-4-pyridinyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((R)-9b). 23% Yield. ESI/MS m/z 542 (M-H)-, ESI-MS/MS
m/z 419 (91), 393 (100). Compound melted at 240-242 °C (from n-hexane/CHCl3). Anal.
(C31H34FN5O3) C, H, N.
5.6.3. (S)-N-[[(1-(2-Fluoro-4-pyridinyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((S)-9b). 48% Yield. 1H NMR (DMSO-d6): δ 1.07-1.56 (m,
9H), 1.84-1.88 (m, 1H), 1.99-2.04 (m, 1H), 2.76 (dd, 1H, J= 14.6, 8.0 Hz), 2.91 (dd, 1H, J= 14.7,
5.6 Hz), 3.00 (dd, 1H, 13.4, 5.7 Hz), 3.66 (s, 3H), 4.42 (q, 1H, J= 7.2 Hz), 6.11 (d, 1H, J= 8.0 Hz),
6.75-6.80 (m, 3H), 6.86 (dd, 1H, J= 8.5, 3.0 Hz), 6.95 (t, 1H, J= 7.0 Hz), 7.01-7.24 (m, 2H), 7.18-
7.24 (m, 2H), 7.30 (d, 1H, J= 8.0 Hz), 7.51 (d, 1H, J= 7.7 Hz), 7.60-7.68 (m, 2H), 8.08 (s, 1H),
8.37 (s, 1H); 13C NMR (DMSO-d6): 26.9, 31.2, 34.2, 38.2, 46.7, 54.8, 58.9, 60.4, 60.5, 60.6, 111.4,
119.3, 123.6, 124.4, 124.7, 126.3, 129.0, 132.8, 138.9, 141.5, 143.1, 159.3, 160.2, 165.2, 168.3,
177.7. ESI/MS m/z 542 (M-H)-, ESI-MS/MS m/z 419 (91), 393 (100). Compound melted at 230-
232 °C (from n-hexane/CHCl3). Anal. (C31H34FN5O3) C, H, N.
5.6.4. (R)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[(1-
phenylcyclohexyl)methyl]propanamide ((R)-9c). 40% Yield. 1H NMR (DMSO-d6): δ 1.11-1.46
(m, 9H), 1.87-2.01 (m, 2H), 2.79-3.00 (m, 3H), 3.67 (s, 3H), 4.44 (q, 1H, J= 4.4 Hz), 6.11 (d, 1H,
J= 8.0 Hz), 6.78 (d, 2H, J= 9.1 Hz), 6.95 (t, 1H, J= 7.3 Hz), 7.01-7.24 (m, 10H), 7.30 (d, 2H, J=
7.7 Hz), 7.51 (d, 1H, J= 7.7 Hz), 8.41 (s, 1H); 13C NMR (DMSO-d6): 22.4, 26.5, 29.3, 33.1, 33.5,
43.2, 50.7, 54.2, 55.7, 55.9, 110.6, 111.8, 112.0, 114.6, 118.8, 118.9, 119.2, 119.3, 119.7, 119.9,
121.5, 124.1, 124.3, 126.2, 127.4, 127.6, 128.1, 129.0. ESI/MS m/z 523 (M-H)-, ESI-MS/MS m/z
24

400 (100), 374 (56). Compound melted at 159-161 °C (from n-hexane/CHCl3). Anal. (C32H36N4O3)
C, H, N.
5.6.5. (S)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[(1-
phenylcyclohexyl)methyl]propanamide ((S)-9c). 44% Yield. ESI/MS m/z 523 (M-H)-, ESI-
MS/MS m/z 400 (100), 374 (52). Compound melted at 161-163 °C (from n-hexane/CHCl3). Anal.
(C32H36N4O3) C, H, N.
5.6.6. (R)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(3-
pyridinyl)cyclohexyl]methyl]propanamide ((R)-9d). 85% Yield. 13C NMR (DMSO-d6): 22.3,
26.4, 29.4, 33.1, 42.3, 50.2, 54.1, 55.7, 55.9, 110.6, 114.6, 118.8, 119.3, 119.7, 119.9, 124.0, 124.2,
128.1, 134.2, 135.5, 135.6, 136.7, 140.1, 147.4, 149.3, 149.5, 154.6, 155.5, 173.0. ESI/MS m/z 524
(M-H)-, ESI-MS/MS m/z 401 (100), 375 (96). Compound melted at 208-210 °C (from n-
hexane/CHCl3). Anal. (C31H35N5O3) C, H, N.
5.6.7. (S)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(3-
pyridinyl)cyclohexyl]methyl]propanamide ((S)-9d). 67% Yield. 1H NMR (CDCl3): δ 1.171.46
(m, 9H), 1.70-1.75 (m, 1H), 1.87-1.93 (m, 1H), 3.05-3.19 (m, 2H), 3.30 (dd, 1H, J= 13.5, 6.9 Hz),
3.75 (s, 3H), 4.60 (q, 1H, J= 7.2 Hz), 5.83 (d, 1H, J= 7.4 Hz), 6.06 (t, 1H, J= 6.2 Hz), 6.70-6.76
(m, 2H), 6.83 (d, 1H, J= 2.2 Hz), 6.98-6.11 (m, 3H), 7.16-7.35 (m, 5H), 7.58 (d, 1H, J= 3.0 Hz),
8.25-8.28 (m, 2H), 8.38 (s, 1H); ESI/MS m/z 524 (M-H)-, ESI-MS/MS m/z 401 (100), 375 (96).
Compound melted at 204-206 °C (from n-hexane/CHCl3). Anal. (C31H35N5O3) C, H, N.
5.6.8. (R)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(2-
pyridinyl)cyclohexyl]methyl]propanamide ((R)-9e). 46% Yield. 1H NMR (DMSO-d6): δ 1.08-
1.47 (m, 9H), 2.02-2.06 (m, 1H), 2.13-2.17 (m, 1H), 2.83 (dd, 1H, J= 14.8, 7.1 Hz), 2.96-3.09 (m,
25

2H), 3.66 (s, 3H), 4.43 (q, 1H, J= 7.1 Hz), 6.12 (d, 1H, J= 8.0 Hz), 6.74-6.80 (m, 3H), 6.97 (dt, 1H,
J= 7.7, 0.8 Hz), 7.00-7.11 (m, 3H), 7.17-7.23 (m, 3H), 7.29 (d, 1H, J= 8.0 Hz), 7.50-7.57 (m, 3H),
8.41 (s, 1H), 8.46 (dd, 1H, J= 4.7, 1.1 Hz); ESI/MS m/z 524 (M-H)-, ESI-MS/MS m/z 402 (30), 401
(100), 375 (44). Compound melted at 128-131 °C (from n-hexane/CHCl3). Anal. (C31H35N5O3) C,
H, N.
5.6.9. (S)-3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(2-
pyridinyl)cyclohexyl]methyl]propanamide ((S)-9e). 72% Yield. 13C NMR (DMSO-d6): 22.6,
26.4, 29.3, 32.9, 33.0, 45.7, 49.2, 54.1, 55.7, 55.8, 11.6, 111.9, 114.6, 118.9, 119.3, 119.8, 121.5,
122.2, 124.2, 128.1, 134.4, 136.8, 149.4, 154.6, 155.6, 155.5, 164.3, 172.8. ESI/MS m/z 524 (M-
H)-, ESI-MS/MS m/z 401 (100), 375 (49). Compound melted at 175-178 °C (from n-
hexane/CHCl3). Anal. (C31H35N5O3) C, H, N.
5.6.10. (R)-N-[[1-(4-Fluorophenyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((R)-9f). 53% Yield. 1H NMR (DMSO-d6): δ 1.16-1.56 (m,
10H), 1.84-1.88 (m, 1H), 2.92 (dd, 1H, J= 13.2, 5.0 Hz), 3.05-3.24 (m, 1H), 3.31 (dd, 1H, J=13.2,
7.4 Hz), 3.77 (s, 3H), 4.12 (q, 1H, J= 7.2 Hz), 6.71-6.79 (m, 7H), 6.90 (s, 1H), 7.08-7.14 (m, 3H),
7.19-7.25 (m, 3H), 7.35 (d, 1H, J= 8.0 Hz), 7.63 (d, 1H, J= 7.7 Hz), 8.11 (s, 1H); 13C NMR
(DMSO-d6): 22.3, 26.5, 29.3, 33.3, 33.7, 42.8, 54.2, 55.7, 55.9, 110.6, 114.6, 115.2, 115.5, 118.9,
119.7, 119.8, 128.1, 129.5, 134.2, 136.8, 140.9, 154.6, 155.5, 159.3, 162.6, 172.8. ESI/MS m/z 541
(M-H)-, ESI-MS/MS m/z 418 (100), 392 (70). Compound melted at 109-112 °C (from n-
hexane/CHCl3). Anal. (C32H35FN4O3) C, H, N.
5.6.11. (S)-N-[[1-(4-Fluorophenyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)-2-[3-(4-
methoxyphenyl)ureido]propanamide ((S)-9f). 40% Yield. ESI/MS m/z 541 (M-H)-, ESI-MS/MS
26

m/z 418 (100), 392 (63). Compound melted at 219-221 °C (from n-hexane/CHCl3). Anal.
(C32H35FN4O3) C, H, N.
5.7. Biological assays
5.7.1. Cell culture
Human promyelocytic leukemia HL-60 cells stably transfected with FPR1 (HL-60-FPR1) or
FPR2 (HL-60-FPR2) were cultured in RPMI 1640 medium supplemented with 10% heat-
inactivated fetal calf serum, 10 mM HEPES, 100 lg/mL streptomycin, 100 U/mL penicillin, and
G418 (1 mg/mL), as previously described.35 Wild type HL-60 cells were cultured under the same
conditions, but without G418.
5.7.2. Neutrophil isolation
Blood was collected from healthy donors in accordance with a protocol approved by the
Institutional Review Board at Montana State University. Neutrophils were purified from the blood
using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis
of red blood cells, as previously described.36 Isolated neutrophils were washed twice and
resuspended in HBSS without Ca2+ and Mg2+ (HBSS-). Neutrophil preparations were routinely
>95% pure, as determined by light microscopy, and >98% viable, as determined by trypan blue
exclusion.
For murine neutrophil isolation, bone marrow leukocytes were flushed from tibias and
femurs of BALB/c mice with HBSS, filtered through a 70-µm nylon cell strainer (BD Biosciences,
Franklin Lakes, NJ) to remove cell clumps and bone particles, and resuspended in HBSS at 106
cells/ml. Bone marrow neutrophils were isolated from bone marrow leukocyte preparations, as
described previously.37 In brief, bone marrow leukocytes were resuspended in 3 ml of 45% Percoll
solution and layered on top of a Percoll gradient consisting of 2 ml each of 50, 55, 62, and 81%
Percoll solutions in a conical 15-ml polypropylene tube. The gradient was centrifuged at 1600g for
27

30 min at 10°C, and the cell band located between the 61 and 81% Percoll layers was collected. The
cells were washed, layered on top of 3 ml of Histopaque 1119, and centrifuged at 1600g for 30 min
at 10°C to remove contaminating red blood cells. The purified neutrophils were collected, washed,
and resuspended in HBSS. All animal use was conducted in accordance with a protocol approved
by the Institutional Animal Care and Use Committee at Montana State University.
5.7.3. Ca2+ mobilization assay
Changes in intracellular Ca2+ were measured with a FlexStation II scanning fluorometer
using a FLIPR 3 calcium assay kit (Molecular Devices, Sunnyvale, CA) for human neutrophils and
HL-60 cells, as described previously.16 All active compounds were evaluated in parent (wild-type)
HL-60 cells for supporting that the agonists are inactive in non-transfected cells. Human
neutrophils or HL-60 cells, suspended in HBSS- containing 10 mM HEPES, were loaded with Fluo-
4 AM dye (Invitrogen) (1.25 µg/mL final concentration) and incubated for 30 min in the dark at 37
°C. After dye loading, the cells were washed with HBSS- containing 10 mM HEPES, resuspended
in HBSS containing 10 mM HEPES and Ca2+ and Mg2+ (HBSS+), and aliquotted into the wells of
flat-bottom, half-area-well black microtiter plates (2 x 105 cells/well). If indicated, 2 mM
probenecid was added 5 min before the assay. The compound of interest was added from a source
plate containing dilutions of test compounds in HBSS+, and changes in fluorescence were
monitored (λex = 485 nm, λem = 538 nm) every 5 s for 240 s at room temperature after automated
addition of compounds. Maximum change in fluorescence, expressed in arbitrary units over
baseline, was used to determine agonist response. Responses were normalized to the response
induced by 5 nM fMLF (Sigma Chemical Co., St. Louis, MO) for FPR1 HL-60 cells and
neutrophils, or 5 nM WKYMVm (Calbiochem, San Diego, CA) for FPR2 HL-60 cells, which were
assigned a value of 100%. Curve fitting (5-6 points) and calculation of median effective
concentration values (EC50) were performed by nonlinear regression analysis of the dose-response
curves generated using Prism 5 (GraphPad Software, Inc., San Diego, CA).
28

5.7.4. Stability Assays in Rat Liver Microsomes
Test compounds were pre-incubated at 37 °C with rat liver microsomes (0.5 mg/mL
microsomal protein) at 1 µM final concentration in 100 mM potassium phosphate buffer (pH 7.4)
for 10 min. Metabolic reactions were initiated by the addition of a NADPH regenerating system (β-
nicotinammide adenine dinucleotide phosphate, isocitric acid isocitric dehydrogenase, final isocitric
dehydrogenase concentration, 1unit/mL). Aliquots were removed (0, 5, 10, 20, 30, and 60 min) and
immediately mixed with an equal volume of cold acetonitrile containing internal standard (100
ng/mL tolbutamide and 100 ng/mL labetalol). Test compound incubated with microsomes without
NADPH regenerating system for 60 min was included. Quenched samples were centrifugated at
4000 rpm for 20 min, and the supernatants were collected for quantification analysis.
The relative loss of parent compound over the course of the incubation was monitored by
liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis was performed using a
Shimadzu LC 20-AD coupled with an API 4000 mass spectometer. LC separation was achieved
using a Phenomenex Synergi Hydro-RP 80A C18 (4 µm; 2.0 x 30 mm) with 0.1% formic acid in
water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) as eluents. Linear gradient
eluition started from 100% A to 100% B within 0.8 min. The flow rate was 800 µL/min and the
injection volume was 10 µL.
MS analysis was performed with an electrospray ionization (ESI) source in positive ion
mode. Instrument settings: nebulizer gas, nitrogen, 2.5 bar; dry gas, nitrogen, 10 L/min; dry heater
300 °C; collision gas: argon; capillary voltage: 5500 V. Concentrations were quantified by
measuring the area under the peak (M+H+) and converted to percentage of compound remaining,
using t = 0 peak area value as 100%. Natural logarithm of percentage remaining versus time data
for each compound was fitted to linear regression and the slope was used to calculate the
degradation half-life according to the equation:38
29