Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]

Article abstract of DOI:10.1016/j.bmc.2016.10.031

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emiss

Full text of DOI:10.1016/j.bmc.2016.10.031

Bioorganic & Medicinal Chemistry 25 (2017) 277–292
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-activity relationship study towards non-peptidic positron
emission tomography (PET) radiotracer for gastrin releasing peptide
receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-
[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-
methyl-2-[3-(4-nitrophenyl)ureido]propionamide
Enza Lacivita a, Ermelinda Lucente a,1, Chantal Kwizera b, Ines F. Antunes b, Mauro Niso a, Paola De Giorgio a,
Roberto Perrone a,c, Nicola A. Colabufo a,c, Philip H. Elsinga b, Marcello Leopoldo a,c,
⇑
a Dipartimento di Farmacia – Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, Bari, Italy
b Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
c BIOFORDRUG s.r.l., Spin-off, Università degli Studi di Bari Aldo Moro, Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a
variety of human cancers, including prostate cancer, and therefore they represent a promising target for
in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-
peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-
(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)
ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This
antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m
was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [18F]
(S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 10 August 2016
Revised 14 October 2016
Accepted 27 October 2016
Available online 29 October 2016
Keywords:
Gastrin-releasing peptide
Positron emission tomography
Prostate cancer
Molecular imaging
G-protein coupled receptors
1. Introduction
Currently, two different classes of radiolabeled bombesin analogs
are under investigation. 1) Radiolabeled GRP-R agonists that inter-
The gastrin-releasing peptide receptor (GRP-R) (also known as
bombesin receptor 2, BB2) is a member of the mammalian bombe-
sin receptor family [1] and is receiving increasing attention
because of its high expression in several human tumors, including
prostate cancer, breast cancer, small cell lung cancer, ovarian can-
cer, endometrial cancer, and gastrointestinal stromal tumors [2–4].
Consequently, bombesin, a 14-amino acid peptide that binds
with high affinity to GRP-Rs, has been widely studied for the devel-
opment of positron emission tomography (PET) diagnostics [5,6].
nalize into tumor cells by endocytosis upon activation of the recep-
tor. These radiolabeled GRP-R agonists have been studied on the
assumption that radioactivity is accumulated with higher effi-
ciency in tumor cells, thus enhancing tumor-to-background ratio
[7]. 2) Radiolabeled GRP-R antagonists that do not internalize into
tumor cells but exhibit a persistent binding to the receptor result-
ing in an overall increased accumulation of radioactivity in tumor
tissue [8]. Even though the exact mode of action of the GRP-R
antagonists has not been fully elucidated yet, they represent an
interesting new class of peptidic vectors for the development of
radiotracers [9].
Abbreviations: GRP, gastrin-releasing peptide; CNS, central nervous system; PET,
positron emission tomography.
Corresponding author at: Dipartimento di Farmacia – Scienze del Farmaco,
Università degli Studi di Bari, Bari, Italy. Tel.: +39 080 5442798; fax: +39 080
5442231.
E-mail address: marcello.leopoldo@uniba.it (M. Leopoldo).
Present address: FARMALABOR SRL, Stabilimento via Pozzillo, zona ind., 76012
Canosa di Puglia (BT), Italy.
Apart from a strong and specific interaction with the target
receptor and reliable radionuclide introduction, an important req-
uisite of a radiotracer is high stability in vivo over the course of PET
imaging. However, radiolabeled peptides can be prone to consider-
able degradation after administration in vivo [10,11]. As a conse-
quence, an insufficient accumulation of the peptidic radiotracer
⇑
1
http://dx.doi.org/10.1016/j.bmc.2016.10.031
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

278
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
or accumulation of its fragments in target tissues combined with
high background radioactivity levels can occur. Thus, high stability
of the radiolabeled peptide is a crucial property for efficient in vivo
target visualization. A number of radiolabeled bombesin peptide
analogs have been developed for PET imaging of GRP-R positive
tumors (for recent reviews see Refs. [5,6]) but they showed quick
degradation in vivo [12–14]. Thus, the limitations imposed by pep-
tide pharmacokinetics with respect to binding and clearance
demonstrate the need of significant improvements of such radiola-
beled probes [15].
H
H
O
N
H
N
O
N
O
O2N
N
O
N
H
O
N
N
H
NH
NH
(S)-1a (PD-176252)
Fig. 1. Formula structures of reference compounds.
7
To overcome the above limitations, we have reasoned on the
possibility to develop small-molecule radiotracers targeting GRP-
Rs. Small molecules offer various advantages over peptides since
they can be suitably designed to modulate potency, selectivity,
lipophilicity, and cell permeability and therefore, to avoid poor tis-
sue penetration, poor serum stability, and quick elimination [16].
One additional advantage of targeting non-peptidic radiotracers
for GRP-Rs is the opportunity to access a radioligand labeled with
fluorine-18 which shows good nuclear characteristics for PET such
as low positron (b+) energy (0.64 MeV) and appropriate physical
half-life (109.7 min) [17]. In general, radiolabeling of biologically
active peptides with fluorine-18 might represent a considerable
challenge. Procedures for the direct radiolabeling with fluorine-
18 normally require harsh reaction conditions (strong bases, high
temperatures) not compatible with heat-sensitive biomolecules
[18]. Usually, site-specific radiolabeling of peptides with fluorine-
18 is achieved using suitable radiolabeled intermediates or pros-
thetic groups [19–22]. This approach includes multistep reaction
procedures and is, therefore, time-consuming. Thus, the aim of
the present study is to identify a non-peptidic fluorine-18 PET radi-
oligand, endowed with antagonist properties, for visualization of
GRP-R overexpression in tumor tissues. A search of the literature
revealed that the compound PD-176252 ((S)-1a, Fig. 1) is the only
non-peptidic GRP-R antagonist reported to date [23]. This antago-
nist was selected as starting point of the present study.
Subsequently, we analyzed the synthetic pathway to obtain (S)-
1a as originally disclosed in a patent application [26]. Apart from 4-
nitrophenylisocianate, the synthetic procedure required the amine
2a (Fig. 2) and (S)-a-methyltryptophan ((S)-12). The synthesis of
the amine 2a requires a time-consuming eight-step sequence,
characterized by very low overall yield (1.8%) [27]. As for (S)-12,
it is commercially available but the cost represents a limit to the
preparation of a large number of compounds. Actually, procedures
for the asymmetric synthesis of (S)-12 are reported in the literature
[28,29] but, at least in our hands, they did not yield the desired
amino acid in gram scale and/or with enantiomeric excess value
of over 98%. In the light of these considerations, the synthetic pro-
cedure of (S)-1a did not seem ideal to explore the structure-activity
relationships on a large number of (S)-1a analogs. Consequently,
we initially designed derivatives of (S)- and (R)-tryptophan instead
of (S)-12, because they were more affordable. Also, we evaluated
the bicyclic amines 2b–e (Fig. 2) as alternatives to 2a because they
are more synthetically accessible than 2a or commercially avail-
able. Thus, the first set of compounds of the present study included
derivatives (R)- and (S)-6b–d, (R,S)-6e, (S,S)-6e, and for compara-
tive purposes (S)-6a (Table 1).
The synthesis of the target compounds (S)-6a, (R)- and (S)-6b–d,
(R,S)-6e, (S,S)-6e is depicted in Scheme 1: Boc-protected R- or S-
tryptophan ((R)- or (S)-3) was first condensed with amine 2a–e,
to give derivatives (R)-4b–d, (S)-4a–d, (R,S)-4e, and (S,S)-4e. The
latter compounds were deprotected under acidic conditions to
afford amines (R)-5b–d, (S)-5a–d, (R,S)-5e, and (S,S)-5e. Condensa-
tion of these latter with 4-nitrophenylisocyanate gave the desired
target compounds.
2. Results and discussion
The GRP-R antagonist (S)-1a was identified by researchers at
Parke Davis in mid 1990’s via the application of a so-called ‘‘pep-
toid” drug design strategy [23]. The original paper reported that
(S)-1a displayed very high affinity for human cloned BB2 receptors
stably expressed in CHO cells (Ki = 0.17 nM). To further assess the
validity of (S)-1a as starting point for our study, we tested the
affinity of (S)-1a for GRP-Rs expressed in PC3 cells, a human pros-
tate cancer cell line with very high expression of GRP-Rs used in
xenograft models to test prospective PET radiotracers. Despite
(S)-1a is commercially available, its affinity for GRP-Rs in PC3 cells
has never been assessed until now.
The binding data were very disappointing because the new
compounds were devoid of GRP-R affinity (Table 1). Only com-
pounds (S)-6a, (R)-6b,d displayed measurable binding affinities
for GRP-Rs in PC3 cells (Ki = 4084–7039 nM), yet >80-fold lower
than (S)-1a. The difference of affinity between (S)-1a and (S)-6a
indicated that the a-methyltryptophan core is essential for affinity
at GRP-Rs of this class of compounds.
Taking a closer look at the original studies [23,30], we noted
that compound 7 (Fig. 1) was different from the GRP-R ligands ana-
logs of (S)-1a because of the presence of the N-methyltryptophan
Thus, the affinities of (S)-1a and of reference compounds hGRP
and bombesin were determined by measuring their ability to dis-
place [125I-Tyr4]bombesin from GRP-Rs in PC3 cells. The data
reported in Table 1 indicated that hGRP and bombesin displayed
Ki values comparable to those reported in the literature [24],
whereas (S)-1a showed a Ki value of 50 nM in PC3 cells quite differ-
ent from that reported (Ki = 0.17 nM) [23]. In addition, we evalu-
ated the affinity of (S)-1a also for human cloned BB2 receptors
and we found a Ki value of 61 nM which was again different from
that originally reported. Also another study has reported affinity
values of (S)-1a at GRP-Rs lower than Ki = 0.17 nM (IC50 = 230 nM
in Balb 3T3 cells; IC50 = 170 nM in HuTU-80 cells) [25]. These dif-
ferences might be due to the different cell lines used for the exper-
iments. In any case, our binding data on GRP-Rs in PC3 cells
supported the decision to select (S)-1a as the starting point for
our studies.
fragment in place of the
a-methyltryptophan one. Nonetheless,
compound 7 displayed low but sizeable binding affinity for GRP-
R (Ki = 1500 nM). Thus, we combined the structural features of 7
and (S)-1a by replacing the 2,6-diisopropylphenyl of 7 with the
4-nitrophenyl group (compounds (R)- and (S)-11f, Table 1). Then,
the cyclohexylmethyl group in (R)- and (S)-11f was replaced by a
bicyclic system as in compound (S)-1a (compounds (S)-11a,d,
Table 1).
The synthesis of final compounds (S)-11a,d, (R)- and (S)-11f is
depicted in Scheme 2. (R)- or (S)-N-benzyl-N-methyltryptophan
((R)- or (S)-8) [31] was condensed with amine 2a,d,f to give amides
(S)-9a,d, and (R)- and (S)-9f that were debenzylated by catalytic
hydrogenation to give N-methyl derivatives (S)-10a,d, (R)- and
(S)-10f. Condensation of these latter with 4-nitrophenylisocyanate
gave the desired target compounds.

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
279
Table 1
Binding affinities of tryptophan derivatives.
H
R1
*
O
N
N
O2N
R
O
N
H
NH
Compound
R1
R
–
GRP-Rs in PC3 cell
hBB2 receptors transfected in
CHO cells Ki S.E.M. [nM]a
membranes Ki S.E.M. [nM]a
(S)-1a
(S)-6a
–
H
50
3
61
5
7039 300
1724 149
O
O
N
(R)-6b
(S)-6b
H
H
4084 200
NTb
>5000 (30%)
>10,000 (26%)c
(R)-6c
(S)-6c
H
H
>10,000 (1%)
>10,000 (1%)
NT
NT
(R)-6d
(S)-6d
H
H
4133 40
>10,000 (18%)
NT
>5000 (46%)
N
(R,S)-6e
(S,S)-6e
H
H
>10,000 (6%)
>10,000 (12%)
NT
NT
S
(S)-11a
(S)-11d
CH3
CH3
>10,000 (6%)
>10,000 (15%)
>5000 (39%)
>5000 (18%)
O
N
N
(R)-11f
(S)-11f
CH3
CH3
>10,000 (30%)
>10,000 (11%)
>5000 (35%)
>5000 (39%)
hGRP
Bombesin
–
–
–
–
0.14 0.04
0.63 0.06
0.025 0.003
0.058 0.005
a
b
c
Data from three independent experiments.
Not tested.
Ki not determined. Percentage of inhibition measured at 10
l
M is given in parentheses.
R
O
O
N
N
a
c
d
b
O
OH
S
N
O
e
g
f
h
F
OH
O
N
F
N
F
k
l
j
i
Fig. 2. Formula structures of amines 2a–l (R-NH2).

280
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
Boc
HN
Boc
HN
O
O
O
O
O
H2N
H2N
1a,g
16a
A
B
H2N
R
R
N
OH
*
*
R
N
*
*
*
17a
OH
N
H
H
H
(S)-1j-l
Ar
Ar
(R)-6b-d
(S)-6a-d
(R,S)-6e
(S,S)-6e
C
A
B
12 (Ar = 3-indolyl)
(S)-12 (Ar = 3-indolyl)
14 (Ar = phenyl)
13a,g (Ar = 3-indolyl)
(S)-13j-l (Ar = 3-indolyl)
15a (Ar = phenyl)
NH
NH
NH
(R)-3
(S)-3
(R)-4b-d
(S)-4a-d
(R,S)-4e
(S,S)-4e
(R)-5b-d
(S)-5a-d
(R,S)-5e
(S,S)-5e
Scheme 3. Synthesis of
a-methyltryptophan-based target compounds 1a,g, 16a,
17a, and (S)-1j–l. Reagents and Conditions: (A) PyBOP, N-methylmorpholine, amine
2a,g,j–l, anhydrous THF, r.t., overnight; (B) 4-methoxy- or 4-nitrophenylisocianate,
anhydrous THF, 24 h, r.t.
Scheme 1. Synthesis of tryptophan-based target compounds (R)-6b–d, (S)-6a–d, (R,
S)-6e, and (S,S)-6e. Reagents and Conditions: (A) N,N0-carbonyldiimidazole, amine
2a–e, anhydrous THF, 24 h, r.t.; (B) trifluoroacetic acid, CH2Cl2, 5 h, r.t.; (C) 4-
nitrophenylisocianate, anhydrous THF, 15 h, r.t.
enantiomers with an analytical column proceeded with the desired
selectivity (a >0.8), the semi-preparative enantioseparation of 1a,g
and 17a was suboptimal (e.e. 90%). In the case of 16a the enan-
tioseparation was not even possible. Nonetheless, in order to have
indications of possible enantiopreference of the interaction of (S)-
1a analogs with GRP-R binding site, the separated enantiomers of
compounds 1a,g and 17a were submitted to radioligand binding
assays (Table 2). The data clearly indicated that the later eluting
enantiomer of all the three pairs possessed the highest binding
affinity. Since the later eluting enantiomer of 1a (compound
1aE2) displayed the same retention time of the enantiomerically
pure specimen of (S)-1a, the binding data suggested a very high
eudismic ratio of 1a enantiomers, showing an unprecedentedly
reported role of stereochemistry in the interaction of (S)-1a with
GRP-R.
Collectively, the binding data discussed until now indicated that
minor changes in the structure of (S)-1a were not tolerated. Thus,
the quest for more synthetically accessible analogs of (S)-1a
remained open. A study towards this aim is being published else-
where [32].
In order to achieve the aim of the study, we went back to the
original structure-activity relationship study on (S)-1a [23], where
it was reported that minor modifications on the aryl ring of the
‘‘right end” of the molecule might be tolerated. Therefore, we syn-
thesized 19F fluorinated analogs of (S)-1a that could be easily
obtained in the 18F radiolabeled form through conventional radio-
chemistry procedures. To this end, the most simple modification
was the replacement of (S)-1a methoxy group with the
2-fluoroethoxy ((S)-1m) (Table 2). In addition, we replaced the 5-
methoxy-2-pyridyl ring of (S)-1a with 4-(2-fluoroethoxy)phenyl,
2-fluoro-4-pyridyl, and 2-fluoro-5-pyridyl rings (compounds
(S)-1j–l) (Table 2).
The synthesis of target compounds (S)-1j–l required the key
amine intermediates 2j–l (Fig. 2). Amine 2j was synthesized
according to Scheme 4: 2b was first demethylated with 48% aque-
ous HBr to phenol 2i, then Boc protected to give derivative 18. This
phenolic derivative was alkylated with 2-fluoroethyltosylate to
give the intermediate 19, that, after deprotection, afforded amine
2j.
The synthesis of the key amines 2k,l (Scheme 4) paralleled the
published procedure for the synthesis of the amine 2a [27]. Nitriles
20k,l [33,34] were reacted with 1,5-dibromopentane in the pres-
ence of NaH to give the cyclohexane derivatives 21k,l. After hydro-
genation of the nitrile function of these latter compounds over
Nickel-Raney, the desired amines 2k,l were obtained. The final
compounds (S)-1j–l were prepared starting from amines 2j–l and
the amino acid (S)-12, as already illustrated in Scheme 3.
The synthesis of the final compound (S)-1m is depicted in
Scheme 5. Compound 2a was demethylated with 48% aqueous
HBr to give compound 2h. This latter amine was condensed with
(S)-12 to give the intermediate (S)-13h which, after reaction with
4-nitrophenylisocyanate, gave the phenolic derivative (S)-1h.
Then, this latter phenol was reacted with 2-fluoroethyl tosylate
to give the target compound (S)-1m.
CH3
N
CH3
N
CH3
O
O
O
Bn
Bn
HN
R
R
N
OH
N
*
*
*
(R)-11f
A
B
C
H
H
(S)-11f
(S)-11a,d
NH
NH
NH
(R)-8
(S)-8
(R)-9f
(S)-9f
(S)-9a,d
(R)-10f
(S)-10f
(S)-10a,d
Scheme 2. Synthesis of N-methyl-tryptophan-based target compounds (R)- and
(S)-11f, (S)-11a,d. Reagents and Conditions: (A) N,N0-carbonyldiimidazole, amine
2a,d,f, (B) H2 (5 atm), 10% Pd/C, ethanol; (C) N,N0-carbonyldiimidazole, 4-nitroani-
line, anhydrous THF, 24 h, r.t.
The binding affinity data listed in Table 1 indicated that com-
pounds (S)-11a,d, (R)- and (S)-11f were completely devoid of
GRP-R affinity (Ki >10,000 nM), indicating the failure of the hypoth-
esis that combining the structural features of 7 and (S)-1a would
give GRP-R ligands.
Consequently, we modified our strategy to bypass the use of (S)-
12 by synthesizing the new target compounds as racemic mixtures
starting from racemic 12, and then separating the desired optically
active target compounds by semi-preparative HPLC enantiosepara-
tion using a chiral stationary phase. Thus, keeping in mind the les-
son learned from the set of compounds listed in Table 1, the
backbone of the a-methyl amino acid was left unchanged and min-
imal structural variations with respect to (S)-1a were introduced.
In particular, the commercially available amine 2g was evaluated
as possible replacement of the amine 2a. Thus, we synthesized
the target racemic compound 1g which contains a tetrahydropyran
ring in place of the cyclohexane ring. Racemate 1a was prepared
for comparative purposes. Also,
1a was replaced by -methylphenylalanine leading to compound
16a, because (S)- -methylphenylalanine is more affordable than
(S)-12. The third modification was the replacement of the nitro
group of 1a with a methoxy group (compound 17a) in order to ver-
ify the introduction of a site for possible radiolabeling.
The synthesis of compounds 1a,g, 16a, and 17a is reported in
Scheme 3: the appropriate racemic aminoacid 12 or 14 was reacted
with the amine 2a,g to give the corresponding derivatives 13a,g
and 15a. Condensation of these latter with the appropriate
phenylisocyanate gave the desired target compounds.
Racemic mixtures 1a,g, 16a, and 17a were initially tested in
radioligand binding assays in order to obtain preliminary indica-
tions of the effect of these structural modifications (Table 2).
Among the four racemic mixtures, the highest affinity was shown
by 1a, again suggesting that even minor modifications on this
framework could negatively affect binding properties. Then, we
separated the enantiomers by semi-preparative HPLC using a cellu-
lose-based chiral stationary phase. Although the separation of the
a-methyltryptophan in compound
a
a

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
281
Table 2
Binding affinities of
a-methyl-tryptophan derivatives.
H
H
N
O
N
X
R
O
N
*
H
Ar
Compounds
X
Ar
R
GRP-Rs in PC3 cell membranes Ki S.E.M. [nM]a
129
7874 300
50
1a
NO2
NO2
NO2
3-Indolyl
3-Indolyl
3-Indolyl
5
O
1aE1b
(S)-1a
N
3
1g
NO2
NO2
NO2
3-Indolyl
3-Indolyl
3-Indolyl
1384 60
O
1gE1
>10,000 (8%)d
4621 110
1gE2c
O
16a
17a
17aE1
17aE2
NO2
Phenyl
1102 55
2466 80
>10,000 (7%)
837 50
O
O
OCH3
OCH3
OCH3
3-Indolyl
3-Indolyl
3-Indolyl
N
(S)-1j
(S)-1k
(S)-1l
(S)-1m
NO2
NO2
NO2
NO2
3-Indolyl
3-Indolyl
3-Indolyl
3-Indolyl
300 70
409 65
160 19
F
N
F
F
N
38
2
O
F
N
a
Data from three independent experiments.
E1: faster eluting enantiomer.
E2: later eluting enantiomer.
b
c
d
Ki not determined. Percentage of inhibition measured at 10
l
M is given in parentheses.
OH
NC
Ar
B
H2N
D
A
C
NC
R
NH2
Ar
20k: Ar = 2-F-4-Py
20l: Ar = 2-F-5-Py
21k: Ar = 2-F-4-Py
21l: Ar = 2-F-5-Py
2b
2i
R
2k,l
Boc
HN
Boc
OH
O
E
F
HN
NH2
2j
F
18
19
Scheme 4. Synthesis of intermediate amines 2j–l. Reagents and Conditions: (A) NaH (60% oil dispersion), 1,5-dibromopentane, Et2O/DMSO; r.t., 24 h; (B) H2 (5 atm), Nickel-
Raney, 2 M ethanolic NH3; (C) 48% aqueous HBr, reflux, 4–5 h; (D) (Boc)2O, Et3N, H2O/THF, r.t., overnight; (E) 2-fluoroethyl tosylate, Cs2CO3, DMF, reflux, 24 h; (F) 3 N HCl,
dioxane, r.t., overnight.
These modifications finally succeeded (Table 2): compounds
(S)-1l and (S)-1m retained high affinity for GRP-R, while (S)-1j
and (S)-1k were about 10-fold less potent than the parent (S)-1a.
On the basis of the binding affinities, compounds (S)-1l and (S)-
1m were subjected to further studies. Calcium flux studies showed
that compounds (S)-1a,l,m did not induce any calcium flux in PC3
cell culture. Instead, these compounds were able to reduce
dose-dependently the Ca2+ mobilization induced by bombesin,
indicating competitive antagonism at GRP-R (see Figs. S1–S3,
Supplementary Materials). The rank order of potency was (S)-
1m > (S)-1l > (S)-1a (Table 3).
Then, we evaluated if (S)-1a,l,m were stable in rat plasma for, at
least, the timeframe of a PET scan. All compounds showed the
desired stability having a degradation half-life (t1/2) longer than
2 h (Table 3). In vitro metabolism of compounds (S)-1a,l,m was
evaluated after incubation with rat hepatic microsomes in the
presence of an NADPH-generating system [35]. Compounds (S)-
1a,l underwent significant oxidative metabolism, whereas (S)-1m
was significantly more stable (Table 3).
Taken together, compound (S)-1m showed high stability in rat
plasma and towards oxidative metabolism in vitro in addition to
its high affinity and potency towards GRP-R, indicating its

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E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
O
H2N
OH
OH
O
N
H2N
H2N
A
B
C
H
N
N
N
NH
2a
2h
(S)-13h
H
H
N
O
N
OH
O
O2N
O
N
D
X
H
N
N
NH
(S)-1m (X = F)
18F](S)-1m (X = 18F)
[
(S)-1h
Scheme 5. Synthesis of the target compound (S)-1m and radiosynthesis of [18F](S)-1m. Reagents and Conditions: (A) 48% aqueous HBr, reflux, 4–5 h; (B) PyBOP, N-
methylmorpholine, (S)-12, anhydrous THF, r.t., overnight; (C) 4-nitrophenylisocianate, anhydrous THF, 24 h, r.t.; (D) 2-fluoroethyl tosylate, Cs2CO3, acetone, reflux, 8 h; or 2-
[
18F]fluoroethyl tosylate, NaH, DMF, 5 min, 125 °C.
Table 3
(7–14)]2 showed an IC50 value of 20 nM, which was comparable to
that of bombesin(1–14) (IC50 = 14 nM). In displacement binding
assays against [18F](S)-1m, Glu[Aca-BN(7–14)]2 showed the IC50
value of 0.10 nM. Thus, by comparing the IC50 values showed by
Glu[Aca-BN(7–14)]2 in the two experiments, we concluded that
Further characterization of compounds of (S)-1a,l,m.
Compound
IC50, nMa
Rat plasma
stability (t1/2, h)
Rat liver microsomal
stability (t1/2, min)
(S)-1a
(S)-1l
(S)-1m
290 77
201 30
121 13
>2
>2
>2
4.4b
2.5
20
[
18F](S)-1m binds at GRP-R with lower affinity than bombesin(1–
14), yet in the nanomolar range. Thus, [18F](S)-1m may display
favourable binding properties as a PET radiotracer for imaging
GRP-Rs.
a
EC50 of bombesin was 1.09 0.19 nM.
Data taken from Ref. [27].
b
Cell binding and dissociation kinetics of [18F](S)-1m were stud-
ied in PC3 cells. As depicted in Fig. 3B, the uptake of [18F](S)-1m in
PC3 cells gradually increased within 60 min of incubation, reaching
a plateau after this time point. This binding kinetics of [18F](S)-1m
is similar to the binding kinetics of other 18F-bombesin analogs
[36]. The maximum cellular associated radioactivity was found to
be 7% of the total added amount of radioactivity. The blocking
studies with Glu[Aca-BN(7–14)]2 revealed a decreased uptake to
4%. The dissociation kinetics of [18F](S)-1m was rapid, remaining
only 6% of the tracer within 120 min (Fig. 3C). This loosely bound
activity decreased exponentially with a half-life of 13 min. Thus,
considering the reversible high affinity binding and fast kinetics
shown by [18F](S)-1m, it is likely that the radioligand will show
high accumulation and fast washout of radioactivity during the
period of PET scan in vivo.
suitability as potential PET tracer. Of note, (S)-1m showed IC50
value (Table 3) in the same range as other 19F-bombesin analogs
such as Al19F-NOTA-4,7-lanthionine-BBN (114 3 nM) and Al19F-
NOTA-2,6-lanthionine-BBN (15 2 nM) which have shown good
properties as PET tracers in vitro as well as in vivo [36].
[
18F](S)-1m was synthesized by reaction of 2-[18F]fluoroethyl
tosylate with the corresponding phenolic precursor (S)-1h in pres-
ence of NaH followed by HPLC purification (Scheme 5). The total
synthesis time was 121 min. The obtained specific activity was
6.0 4.7 GBq/l
mol. In contrast to other [18F]fluoroethylation reac-
tions performed in our laboratories using the same purification
method for 2-[18F]fluoroethyl tosylate [37], the final radiochemical
yield was quite low (<5%) because of substantial defluorination. In
the HPLC-radiochromatogram, besides the peak of [18F](S)-1m, a
large peak was found at the void volume, most likely due to [18F]
fluoride. The formation of other byproducts can be ruled out as
the similar reaction under ‘cold’ conditions yielded only (S)-1m.
The radiochemical purity of the 2-[18F]fluoroethyl tosylate inter-
mediate was checked by TLC and UPLC and was >95%. However,
when reacting with the phenolic precursor (S)-1h in the presence
of NaH, the major part (>90%) of 2-[18F]fluoroethyl tosylate was
degraded to 18F-fluoride as it was concluded from HPLC analysis.
Nonetheless, [18F](S)-1m was obtained with a radiochemical yield
(RCY) of 3.4 0.3% (40–90 MBq) and a radiochemical purity >95%.
The stability of [18F](S)-1m was confirmed with UPLC within the
normal PET imaging time (2 h), revealing a radiochemical purity
>95% within 2 h (see Supplementary Materials, Fig. 4S), allowing
us to proceed with the in vitro experiments in PC3 cells.
3. Conclusions
During the years, GRP-R has emerged as a target for cancer
detection and treatment because of the different expression levels
in healthy and tumor tissues. To date, numerous radiolabeled bom-
besin analogs have been reported as imaging tools or tumor target-
ing vectors. However, the fast metabolic degradation of bombesin-
based radiotracers impacts their in vivo targeting capabilities and,
thus, represents a limitation for medical applications. Here we
have reported on the identification of the first non-peptidic PET
radiotracer for GRP-Rs [18F](S)-1m. Starting from the GRP-R antag-
onist (S)-1a, structure-activity relationships were explored in order
to find synthetically accessible non-peptidic GRP-R ligands. The
study showed that small changes in the backbone of the structure
of (S)-1a translated into a loss in affinity. Only small changes on the
pyridyl ring of (S)-1a were tolerated. In fact, 2-fluoroethoxy deriva-
tive (S)-1m was identified as a high affinity GRP-R antagonist,
endowed with stability in rat plasma and toward microsomal
oxidative metabolism in vitro. [18F](S)-1m was successfully synthe-
sized through conventional radiochemistry procedure, albeit in
low radiochemical yield. [18F](S)-1m confirmed high affinity for
The binding affinity of [18F](S)-1m for GRP-R was assessed in
whole PC-3 (GRPR +) cells (Fig. 3A). Results are plotted as sigmoid
curves for the displacement of [18F](S)-1m as a function of increas-
ing concentrations of the GRP-R probe Glu[Aca-BN(7–14)]2, previ-
ously characterized in our laboratories in competitive binding
studies in the same PC-3 (GRPR +) cells using the reference radioli-
gand 124I-[Tyr4]-BN(1–14) [38]. In those experiments, Glu[Aca-BN

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
283
Fig. 3. A) Inhibition of [18F](S)-1m binding to GRP-Rs on PC3 cells by Glu[Aca-BN(7–14)]2. B) Cellular uptake of [18F](S)-1m in PC3 prostate cancer cells. C) Dissociation
kinetics of [18F](S)-1m in PC3 cells.
GRP-Rs in whole PC3 cells and showed displaceable interaction
with GRP-R in vitro. Interestingly, [18F](S)-1m, compared to fluo-
rine-18-labeled bombesin agonists, showed similar cellular associ-
ated radioactive uptake and faster efflux kinetics. The promising
in vitro properties of [18F](S)-1m open new perspectives in the field
of GRP-R radiotracers suggesting that non-peptidic tracers might
have very different properties in terms of cell uptake and dissocia-
tion kinetics as compared to peptidic tracers. Future studies with
fluoropyridine (19k) [33]; 5-cyanomethyl-2-fluoropyridine (19l)
[34]; (S)-1,2,3,4-tetrahydro-1-naphthalenamine (2e) and cyclo-
hexylmethylamine (2f) were purchased from Sigma-Aldrich
(Milan, Italy), [4-(4-methoxyphenyl)tetrahydro-2H-pyran-4-yl]
methylamine (2g) was purchased from Life Chemicals Europe
GmbH (Munich, Germany), (S)-a-methyltryptophan ((S)-12) was
purchased from Acros Organics (Geel, Belgium).
[
18F](S)-1m or with optimized fluorinated analogs will allow to
4.1.1. General procedure for the preparation of compounds 2h and 2i
A mixture of the amine 2a (0.20 g, 0.91 mmol) or 2b (0.21 g,
0.96 mmol) and 48% aqueous HBr (5 mL) was refluxed for 4–5 h
(TLC monitoring). Then, the solvent was removed in vacuo and
the residue was washed several times with absolute ethanol, to
give the final hydrobromide salt as a white solid that was not fur-
ther purified (quantitative yield).
compare in vitro and in vivo properties of peptidic and non-peptidic
GRP-R PET radiotracers.
4. Experimental section
4.1. Chemistry
The purity of the target compounds (R)-6b–d, (S)-6a–d. (R,S)-6e,
(S,S)-6e, (R)-11f, (S)-11f, (S)-11a,d, 1a,g, 16a, 17a, (S)-1j–l, (S)-1m
was assessed by RP-HPLC and combustion analysis. All compounds
showed P95% purity. Column chromatography was performed
4.1.1.1. 1-Aminomethyl-1-(4-hydroxypyridin-2-yl)cyclohexane Hydro-
bromide (2h). 1H NMR (CD3OD): d 1.34–1.41 (m, 2H), 1.46–1.50 (m,
2H), 1.52–1.58 (m, 2H), 1.71–1.94 (m, 2H), 2.33 (app d, 2H), 3.29 (s,
2H), 3.30 (s, 3H, D2O exchanged), 8.02–8.03 (m, 2H), 8.32–8.33 (m,
1H), 10.07 (s, 1H); ESI-MS m/z 207 (M+H)+, ESI-MS/MS m/z: 190
(100).
with 1:30 Merck silica gel 60A (63–200 lm) as the stationary
phase. Melting points were determined in open capillaries on a
Gallenkamp electrothermal apparatus. Elemental analyses (C, H,
N) were performed on a Eurovector Euro EA 3000 analyzer. Analy-
ses indicated by the symbols of the elements were within 0.4% of
the theoretical values. 1H NMR spectra were recorded at 300 MHz
on a Varian Mercury-VX spectrometer. All spectra were recorded
on free bases. All chemical shift values are reported in ppm (d).
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. ESI-MS/MS analysis were performed with an Agilent
1100 Series LC-MSD trap System VL workstation. All spectra were
in accordance with the assigned structures. RP-HPLC analysis was
performed on a Perkin-Elmer series 200 LC instrument using a Phe-
4.1.1.2. 1-Aminomethyl-1-(4-hydroxyphenyl)cyclohexane Hydrobro-
mide (2i). 1H NMR (CD3OD): d 1.32–1.46 (m, 4H), 1.53–1.58 (m,
4H), 1.99–2.05 (m, 2H), 3.18 (app d, 2H), 3.30 (br s, 4H, D2O
exchanged), 6.82 (d, 2H, J = 8.5 Hz), 7.18 (d, 2H, J = 8.5 Hz); ESI+/
MS m/z: 206 (M+H)+, ESI-MS/MS m/z: 189 (100).
4.1.2. N-Boc-1-aminomethyl-1-(4-hydroxyphenyl)cyclohexane (18)
Amine 2i (0.14 g, 0.5 mmol) was dissolved in mixture of H2O/
THF (8 mL, 1:3, v/v,) then triethylamine (0.15 mL, 1.5 mmol) and
(Boc)2O (0.15 g, 0.5 mmol) were added. The reaction mixture was
stirred at room temperature overnight. Then, THF was removed
under reduced pressure and the aqueous residue was extracted
with AcOEt (2 ꢀ 20 mL). The combined organic layers were dried
over Na2SO4 and evaporated in vacuo. The crude residue was chro-
matographed (n-hexane/AcOEt, 4:1) to yield the title compound as
a semisolid (0.10 g, 70% yield). 1H NMR (CDCl3): d 1.32–1.46 (m + s,
13H), 1.53–1.58 (m, 4H), 1.99–2.05 (m, 2H), 3.10 (br s, 1H, D2O
exchanged) 3.18 (d, 2H, J = 6.3 Hz), 4.11–4.16 (m, 1H), 6.82 (d,
2H, J = 8.5 Hz), 7.18 (d, 2H, J = 8.5 Hz); ESI-MS m/z 304 (MꢁH)ꢁ,
ESI-MS/MS m/z: 204 (100).
nomenex Gemini C-18 column, (250 ꢀ 4.6 mm, 5
lm particle size)
and equipped with a Perkin-Elmer 785A UV/VIS detector setting
k = 254 nm. Compounds (R)-6b–d, (S)-6a–d. (R,S)-6e, (S,S)-6e, (R)-
11f, (S)-11f, (S)-11a,d, 1a,g, 16a, 17a, (S)-1j–l, (S)-1m were eluted
with CH3OH/H2O/Et3N, 7:3:0.01, v/v at a flow rate of 1.0 mL/min.
The following compounds were prepared as described in the lit-
erature: 1-aminomethyl-1-(4-methoxypyridin-2-yl)cyclohexane
(2a) [27]; 1-aminomethyl-1-(4-methoxyphenyl)cyclohexane (2b)
[39]; 1-(1-phenyl)cyclopropylmethylamine (2c) [40]; 1-amino-
methyl-1-(pyridin-2-yl)cyclohexane (2d) [41]; (S)- and (R)-N-ben-
zyl-N-methyl-tryptophan ((S)-8 and (R)-8) [31]; 4-cyanomethyl-2-

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E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
4.1.3. N-Boc-1-aminomethyl-1-(4-(2-fluoroethoxyphenyl)cyclohexane
(19)
4.1.6.1. 1-Aminomethyl-1-(2-fluoropyridin-4-yl)cycloexane (2k). 1H
NMR (CDCl3): d 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).
Freshly prepared 2-fluoroethyltosylate (0.23 g, 1.00 mmol) was
added to a suspension of 18 (0.13 g, 0.43 mmol) and Cs2CO3
(0.56 g, 1.72 mmol) in DMF. The reaction mixture was refluxed
for 24 h, then the solvent was removed in vacuo, the residue was
taken up with H2O (10 mL) and extracted with EtOAc
(2 ꢀ 10 mL). The combined organic phases were washed with
brine, dried over Na2SO4, and concentrated under reduced pres-
sure. The crude residue was chromatographed (n-hexane:EtOAc,
4:1 as eluent) to afford pure 19 (0.05 g, 35% yield). 1H NMR (CDCl3):
d 1.31–1.42 (m + s, 13H), 1.44–1.58 (m, 4H), 2.01–2.04 (m, 2H),
3.19 (d, 2H, J = 6.3 Hz), 4.10–4.18 (m, 2H), 4.24–4.27 (m, 1H),
4.66–4.69 (m, 1H), 4.82–4.85 (m, 1H), 6.91 (d, 2H, J = 8.8 Hz),
7.24 (d, 2H, J = 8.8 Hz); ESI-MS m/z 374 (M+Na)+, ESI-MS/MS m/z:
318 (100).
4.1.6.2. 1-Aminomethyl-1-(2-fluoropyridin-5-yl)cycloexane (2l). 1H
NMR (CDCl3): d 1.21–1.41 (m, 4H), 1.55–1.67 (m, 6H), 2.11 (app
d, 2H), 2.73 (s, 2H), 6.92 (dd, 1H, J = 8.5, 3.0 Hz), 7.75 (dt, 1H,
J = 8.5, 2.5 Hz), 8.17 (d, 1H, J = 2.2 Hz). ESI+/MS m/z 209 (M+H)+,
ESI-MS/MS m/z 192 (100), 136 (22).
4.1.7. General procedure for the preparation of compounds (R)-4b–d,
(S)-4a–d, (R,S)-4e, and (S,S)-4e
N,N0-Carbonyldiimidazole (1.1 mmol) was added to a solution of
(R)- or (S)-3 (1.0 mmol), in anhydrous THF (10 mL) under N2. The
reaction mixture was stirred at room temperature for 8 h, then a
solution of the appropriate amine (1.0 mmol) in anhydrous THF
was added. The reaction mixture was stirred at room temperature
for 24 h, then the solvent was removed in vacuo and the residue
was partitioned between in EtOAc (20 mL) and H2O (20 mL). The
aqueous phase was washed with EtOAc (2 ꢀ 20 mL) and the com-
bined organic layers were dried over Na2SO4 and evaporated in
vacuo. The crude was chromatographed on silica gel (CHCl3/EtOAc,
9:1, as eluent) to afford pure target compound.
4.1.4. 1-Aminomethyl-1-(4-(2-fluoroethoxyphenyl)cycloexane Hydro-
chloride (2j)
Compound 19 (0.44 g, 1.3 mmol) was solubilized in a mixture of
dioxane (5 mL) and 3 N HCl (5 mL) and stirred overnight. Then the
reaction mixture was evaporated under reduced pressure to give a
solid that was treated several times with absolute ethanol and
again concentrated by evaporation in order to remove residual
H2O to afford title compound (0.37 g, quantitative yield). 1H NMR
(CD3OD):
d 1.40–1.42 (m, 2H), 1.50–1.62 (m, 9H, 3H D2O
exchanged), 2.15–2.25 (m, 2H), 3.00 (s, 2H), 4.17–4.19 (m, 1H),
4.26–4.29 (m, 1H), 4.63–4.65 (m, 1H), 4.78–4.81 (m, 1H), 7.01–
7.05 (m, 2H), 7.35–7.39 (m, 2H); ESI-MS m/z 252 (M+H)+, ESI-MS/
MS m/z: 235 (100), 153 (30).
4.1.7.1. (R)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1(4-methoxyphenyl)-
cyclohexylmethyl]-2-oxoethyl]carbamate ((R)-4b). Colorless solid wax.
80% Yield. 1H NMR (CDCl3): d 1.23–1.61 (m + s, 17H), 1.78–1.85 (m,
1H), 2.98–3.12 (m, 2H), 3.20–3.31 (m, 2H), 3.75 (s, 3H), 4.27–4.32
(m, 1H), 5.10 (br d, 2H), 6.64 (br d, 2H), 6.76 (br d, 2H), 6.96 (d, 1H,
J = 2.5 Hz), 7.12–7.24 (m, 3H), 7.38 (d, 1H, J = 8.0 Hz), 7.64 (d, 1H,
J = 8.0 Hz), 8.03 (br s, 1H). ESI-MS m/z 504 (MꢁH)ꢁ, ESI-MS/MS m/z
430 (100), 301 (69).
4.1.5. General procedure for the synthesis of compounds 21k–m
To a stirred suspension of NaH (60% dispersion in mineral oil,
3.3 mmol) in DMSO (5 mL), under nitrogen at ꢁ15 °C, a solution
of the nitrile 19k–m (1.0 mmol) and 1.5-dibromopentane
(1.0 mmol) in ether and DMSO (4:1, v/v) was added dropwise over
1 h. The mixture was then allowed to warm at room temperature
and stirred for 24 h under nitrogen. Then, the reaction mixture
was carefully quenched by adding dropwise isopropanol (3 mL)
and water (5 mL). The reaction mixture was diluted with EtOAc
(30 mL) and washed first with H2O (20 mL), then with brine
(20 mL). The separated organic layer was dried (Na2SO4) and con-
centrated in vacuo. The crude residue was chromatographed (pet-
roleum ether/EtOAc, 7:3 as eluent) to give the target compound as
a colorless oil.
4.1.7.2. (R)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(1-phenyl)cy-
clopropylmethyl]-2-oxoethyl]carbamate ((R)-4c). Colorless solid
wax. 50% Yield. 1H NMR (CDCl3): d 0.62–0.74 (m, 4H), 1.40 (s,
9H), 1.61–1.69 (m, 2H), 3.10 (dd, 1H, J = 14.0, 6.0 Hz), 3.31 (dd,
1H, J = 14.0, 6.0 Hz), 4.32–4.38 (m, 1H), 5.13 (br s, 1H), 5.64 (br s,
1H), 6.93 (d, 2H, J = 2.5 Hz), 7.11–7.17 (m, 3H), 7.22 (dt, 1H,
J = 6.0, 2.1 Hz), 7.32–7.40 (m, 2H), 7.41–7.45 (m, 1H), 7.65 (d, 1H,
J = 8.0 Hz), 8.03 (br s, 1H). ESI-MS m/z 432 (MꢁH)ꢁ, ESI-MS/MS
m/z 358 (100), 229 (47).
4.1.7.3. (R)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(2-pyridyl)cy-
clohexylmethyl]-2-oxoethyl]carbamate ((R)-4d). Colorless solid
wax. 97% Yield. 1H NMR (CDCl3): d 1.30–1.50 (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 (MꢁH)ꢁ, ESI-MS/MS m/z 401
(100), 272 (62).
4.1.5.1. 1-Cyano-1-(2-fluoropyridin-4-yl)cyclohexane (21k). 51%
Yield. 1H NMR (CDCl3): d 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).
4.1.5.2. 1-Cyano-1-(2-fluoropyridin-5-yl)cyclohexane (21l). 70%
Yield. 1H NMR (CDCl3): d 1.25–1.33 (m, 1H), 1.70–1.90 (m, 7H),
2.15 (app d, 2H), 6.97 (dd, 1H, J = 8.5, 3.0 Hz), 7.92 (dt, 1H, J = 7.4,
2.5 Hz), 8.34 (d, 1H, J = 2.8 Hz). ESI+/MS m/z: 227 (M+Na)+, ESI-
MS/MS m/z: 227 (44), 145 (100).
4.1.7.4. t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[(1S)-1-(1,2,3,4-tetrahy-
dronaphthalen-1-yl)]-(2R)-2-oxoethyl]carbamate
((R,S)-4e). White
4.1.6. General procedure for the synthesis of amines 2k–m
To a solution of the nitrile 20k–m (1.0 mmol) in ethanolic NH3
(20 mL, 2 M), Nickel-Raney was added and the mixture was satu-
rated with H2 at 5 atm pressure. The mixture was stirred at 40 °C
for 22 h. Then, the reaction mixture was filtered through a Celite
pad and the filtrate was concentrated in vacuo to afford the target
amine (colorless oil) in quantitative yield.
powder. 65% Yield. 1H NMR (CDCl3): d 1.42 (s, 9H), 1.63 (br s, 3H),
1.82 (br s, 1H), 2.64 (t, 2H, J = 6.0 Hz), 3.16 (dd, 1H, J = 14.3,
7.7 Hz), 3.40 (dd, 1H, J = 14.4, 5.3 Hz), 4.42 (br s, 1H), 5.01–5.08 (m,
1H), 5.17 (br s, 1H), 5.87 (br d, 1H), 6.87 (br s, 1H), 7.07 (d, 2H,
J = 1.1 Hz), 7.10–7.23 (m, 4H), 7.37 (d, 1H, J = 9.6 Hz), 7.70 (d, 1H,
J = 8.0 Hz), 8.14 (br s, 1H). ESI–MS m/z 434 (M+H)+, ESI-MS/MS m/z
378 (100), 334 (25), 248 (14).

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
285
4.1.7.5. (S)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(5-methoxypyri-
din-2-yl)cyclohexylmethyl]-2-oxoethyl]carbamate ((S)-4a). Colorless
solid wax. 94% Yield. 1H NMR (CDCl3): d 1.20–1.52 (m, 17H), 1.71–
1.75 (m, 1H), 1.87–1.94 (m, 1H), 3.11–3.14 (m, 1H), 3.22–3.29 (m,
2H), 3.80 (s, 3H), 4.37–4.39 (m, 1H), 5.14–5.17 (m, 1H), 6.32 (br s,
1H), 6.85–6.99 (m, 4H), 7.11 (dt, 1H, J = 7.0, 1.1 Hz), 7.16 (dt, 1H,
J = 8.0, 1.1 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.0 Hz),
8.01 (br s, 1H), 8.07 (br s, 1H). ESI-MS m/z 505 (MꢁH)ꢁ, ESI-MS/
MS m/z 431 (100), 302 (65).
(d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.0 Hz), 8.09 (br s, 1H). ESI-MS
m/z 404 (MꢁH)ꢁ, ESI-MS/MS m/z 273 (39), 216 (100), 130 (18).
4.1.8.2.
(R)-2-Amino-3-(1H-indol-3-yl)-N-(1-phenylcyclopropyl-
methyl)propanamide ((R)-5c). Colorless solid wax. 72% Yield. 1H
NMR (CDCl3): d 0.81–0.89 (m, 4H), 1.50 (br s, 2H), 2.86 (dd, 1H,
J = 14.0, 8.0 Hz), 3.30 (dd, 1H, J = 14.0, 4.0 Hz), 3.32–3.51 (m, 2H),
3.65 (dd, 1H, J = 14.0, 4.0 Hz), 7.00 (d, 1H, J = 2.5 Hz), 7.11–7.44
(m, 9H), 7.64–7.67 (m, 1H), 8.10 (br s, 1H). ESI-MS m/z 334 (M
+H)+, ESI-MS/MS m/z 317 (100), 130 (30).
4.1.7.6. (S)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(4-methoxyphenyl)
cyclohexylmethyl]-2-oxoethyl]carbamate ((S)-4b). Colorless solid wax.
83% Yield. 1H NMR (CDCl3): d 1.39–1.60 (m, 18H), 1.79–1.86 (m,
1H), 2.99–3.12 (m, 2H), 3.20–3.26 (m, 2H), 3.75 (s, 3H), 4.25–4.35
(m, 1H), 5.06 (br s, 1H), 6.64 (d, 2H, J = 8.0 Hz), 6.75 (d, 2H,
J = 8.0 Hz), 6.96 (d, 1H, J = 2.2 Hz), 7.15 (dt, 1H, J = 7.0, 1.1 Hz), 7.23
(dt, 1H, J = 7.0, 1.1 Hz), 7.38 (d, 1H, J = 7.0 Hz), 7.63 (d, 1H,
J = 7.0 Hz), 8.10 (br s, 1H). ESI-MS m/z 504 (MꢁH)ꢁ, ESI-MS/MS m/z
430 (100), 301 (73).
4.1.8.3.
(R)-2-Amino-3-(1H-indol-3-yl)-N-[1-(pyridin-2-yl)cyclo-
hexylmethyl]propanamide ((R)-5d). Colorless solid wax. 90% Yield.
1H NMR (CDCl3): d 1.21–1.66 (m, 10H), 2.11–2.18 (m, 2H), 2.80
(dd, 1H, J = 14.6, 9.1 Hz), 3.29 (dd, 1H, J = 14.3, 4.2 Hz), 3.48 (d,
2H, J = 5.8 Hz), 3.62 (dd, 1H, J = 8.8, 4.1 Hz), 7.02 (d, 1H,
J = 2.5 Hz), 7.08–7.12 (m, 2H), 7.19 (dt, 1H, J = 8.1, 1.1 Hz), 7.24
(d, 1H, J = 8.0 Hz), 7.35 (d, 1H, J = 8.0 Hz), 7.43 (br t, 1H), 7.58–
7.65 (m, 2H), 8.17 (br s, 1H), 8.54–8.56 (m, 1H). ESI-MS m/z 377
(M+H)+, ESI-MS/MS m/z 360 (100), 332 (14), 191 (30).
4.1.7.7. (S)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(1-phenyl)cyclo-
propylmethyl]-2-oxoethyl]carbamate ((S)-4c). Colorless solid wax.
50% Yield. 1H NMR (CDCl3): d 0.62–0.74 (m, 4H), 1.40 (s, 9H),
1.61–1.69 (m, 2H), 3.10 (dd, 1H, J = 14.0, 6.0 Hz), 3.31 (dd, 1H,
J = 14.0, 6.0 Hz), 4.32–4.38 (m, 1H), 5.13 (br s, 1H), 5.64 (br s,
1H), 6.93 (d, 2H, J = 2.2 Hz), 7.10–7.17 (m, 3H), 7.22 (dt, 1H,
J = 6.0, 2.1 Hz), 7.32–7.40 (m, 2H), 7.41–7.45 (m, 1H), 7.65 (d, 1H,
J = 8.0 Hz), 8.10 (br s, 1H). ESI-MS m/z 432 (MꢁH)ꢁ, ESI-MS/MS
m/z 358 (100), 229 (47).
4.1.8.4. (2R)-2-Amino-3-(1H-indol-3-yl)-N-((1S)-1,2,3,4-tetrahydron-
aphtalen-1-yl)propanamide ((R,S)-5e). Colorless solid wax. 50%
Yield. 1H NMR (CDCl3): d 1.52 (br s, 2H), 1.66–1.82 (m, 3H),
1.98–2.06 (m, 1H), 2.68–2.81 (m, 2H), 3.06 (dd, 1H, J = 14.6,
8.5 Hz), 3.42 (dd, 1H, J = 13.5, 4.4 Hz), 3.78 (app q, 1H), 5.12–5.18
(m, 1H), 6.94–6.97 (m, 1H), 7.05–7.11 (m, 4H), 7.14 (dt, 1H,
J = 6.0, 1.0 Hz), 7.22 (dt, 1H, J = 6.0, 1.0 Hz), 7.37–7.41 (m, 2H),
7.73 (d, 1H, J = 7.0 Hz), 8.13 (br s, 1H). ESI-MS m/z 334 (M+H)+,
ESI-MS/MS m/z 317 (10), 204 (100), 187 (46).
4.1.7.8. (S)-t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-[1-(2-pyridyl)cy-
clohexylmethyl]-2-oxoethyl]carbamate
((S)-4d). Colorless solid
4.1.8.5.
(S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(5-methoxypyridin-2-
wax. 40% Yield. 1H NMR (CDCl3): d 1.30–1.50 (m, 17H), 1.72–1.85
(m, 1H), 1.89–2.00 (m, 1H), 3.09–3.30 (m, 2H), 3.33 (d, 2H,
J = 5.0 Hz), 4.39 (br s, 1H), 6.45 (br s, 1H), 6.95–7.05 (m, 3H), 7.11
(dt, 1H, J = 1.1, 7.0 Hz), 7.20 (dt, 1H, J = 7.0, 1.1 Hz), 7.33 (d, 1H,
J = 8.0 Hz), 7.49 (br t, 1H, J = 7.4 Hz), 7.63 (d, 1H, J = 8.0 Hz), 8.02
(br s, 1H), 8.32 (br s, 1H). ESI-MS m/z 475 (MꢁH)ꢁ, ESI-MS/MS
m/z 401 (100), 272 (59).
yl)cyclohexylmethyl]propanamide ((S)-5a). Colorless solid wax.
83% Yield. 1H NMR (CDCl3): d 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 = 14.0, 9.0 Hz),
3.41 (d, 2H, J = 6.0 Hz), 3.65 (dd, 1H, J = 14.0, 9.0 Hz), 3.83 (s, 3H),
7.02 (d, 1H, J = 2.5.0 Hz), 7.11–7.22 (m, 4H), 7.29–7.37 (m, 2H),
7.64 (d, 1H, J = 8.0 Hz), 8.12 (br s, 1H), 8.24 (dd, 1H, J = 1.2,
0.8 Hz). ESI-MS m/z 407 (M+H)+, ESI-MS/MS m/z 390 (100), 221
(34), 204 (26).
4.1.7.9. t-Butyl-N-[1-(1H-indol-3-ylmethyl)-2-((1S)-1,2,3,4-tetrahy-
dronaphthalen-1-yl)-(2S)-2-oxoethyl]carbamate
((S,S)-4e). White
powder. 70%. Yield 1H NMR (CDCl3): d 1.41 (s, 9H), 1.64 (br s,
3H), 1.91 (br s, 1H), 2.66 (app t, 2H, J = 6.2 Hz), 3.18 (dd, 1H,
J = 14.3, 8.0 Hz), 3.35 (dd, 1H, J = 14.3, 5.5 Hz), 4.42 (br s, 1H),
5.00 (br s, 1H), 5.17 (br s, 1H), 5.87 (br d, 1H), 6.68 (br s, 1H),
6.94–7.15 (m, 4H), 7.12 (dt, 1H, J = 7.2, 1.4 Hz), 7.34–7.37 (m,
1H), 7.69 (d, 1H, J = 7.7 Hz), 8.10 (br s, 1H). ESI-MS m/z 434 (M
+H)+, ESI-MS/MS m/z 378 (100), 334 (24), 248 (14), 204 (9).
4.1.8.6. (S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(4-methoxyphenyl)cy-
clohexylmethyl]propanamide ((S)-5b). Colorless solid wax. 85%
Yield. 1H NMR (CDCl3): d 1.36–1.55 (m, 10H), 1.92–1.96 (m, 2H),
2.84 (dd, 1H, J = 14.6, 8.5 Hz), 3.25–3.31 (m, 3H), 3.61 (dd, 1H,
J = 8.8, 4.7 Hz), 3.78 (s, 3H), 6.80–6.84 (m, 3H), 6.99 (d, 1H,
J = 2.2 Hz), 7.09–7.15 (m, 3H), 7.18–7.24 (dt, 1H, J = 8.0, 1.0 Hz),
7.37 (d, 1H, J = 8.2 Hz), 7.64 (d, 1H, J = 7.7 Hz), 8.07 (br s, 1H). ESI-
MS m/z 406 (M+H)+, ESI-MS/MS m/z 389 (100), 260 (23), 250 (23).
4.1.8. General procedure for the preparation of compounds (R)-5b–d,
(S)-5a–d, (R,S)-5e, and (S,S)-5e
4.1.8.7.
(S)-2-Amino-3-(1H-indol-3-yl)-N-(1-phenylcyclopropyl-
methyl)propanamide ((S)-5c). Colorless solid wax. 90% Yield. 1H
NMR (CDCl3): d 0.80–0.91 (m, 4H), 1.42 (br s, 2H), 2.85 (dd, 1H,
J = 14.0, 8.0 Hz), 3.30 (dd, 1H, J = 14.0, 4.0 Hz), 3.36–3.51 (m, 2H),
3.65 (dd, 1H, J = 14.0, 4.0 Hz), 7.00 (d, 1H, J = 2.5 Hz), 7.11–7.44
(m, 9H), 7.65 (br d, 1H, J = 3.9, 0.5 Hz), 8.11 (br s, 1H). ESI-MS m/
z 334 (M+H)+, ESI-MS/MS m/z 317 (100), 130 (24).
Trifluoroacetic acid (5 mL) was added to a solution of (R)-4b–d,
(S)-4a–d, (R,S)-4e, and (S,S)-4e in CH2Cl2 (20 mL). The reaction mix-
ture was stirred at room temperature for 5 h, basified with aqueous
1 M NaOH, and extracted with EtOAc (3 ꢀ 20 mL). The combined
organic layers were dried (Na2SO4) and concentrated in vacuo to
give a crude residue that was purified by column chromatography
(CHCl3/CH3OH, 19:1, as eluent) to afford the pure compound.
4.1.8.8.
(S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(pyridin-2-yl)cyclo-
4.1.8.1. (R)-2-Amino-3-(1H-indol-3-yl)-N-[1-(4-methoxyphenyl)cy-
clohexylmethyl]propanamide ((R)-5b). Colorless solid wax. 99%
Yield. 1H NMR (CDCl3): d 1.32–1.56 (m, 10H), 1.92–1.97 (m, 2H),
2.84 (dd, 1H, J = 14.3, 8.5 Hz), 3.25–3.31 (m, 3H), 3.62 (dd, 1H,
J = 8.5, 4.4 Hz), 3.80 (s, 3H), 6.80–6.84 (m, 3H), 6.99 (d, 1H,
J = 2.2 Hz), 7.11–7.18 (m, 3H), 7.21 (dt, 1H, J = 8.0, 1.0 Hz), 7.38
hexylmethyl]propanamide ((S)-5d). Colorless solid wax. 95% Yield.
1H NMR (CDCl3): d 1.22–1.68 (m, 10H), 2.10–2.19 (m, 2H), 2.80
(dd, 1H, J = 14.0, 8.8 Hz), 3.29 (dd, 1H, J = 14.0, 4.1 Hz), 3.48 (d,
2H, J = 9.0 Hz), 3.65 (dd, 1H, J = 9.1, 4.1 Hz), 7.02 (d, 1H,
J = 2.2 Hz), 7.08–7.17 (m, 2H), 7.19 (br t, 1H), 7.24 (d, 1H,
J = 8.0 Hz), 7.35 (d, 1H, J = 8.0 Hz), 7.43 (br t, 1H), 7.58–7.65 (m,

286
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
2H), 8.17 (br s, 1H), 8.53–8.56 (m, 1H). ESI-MS m/z 377 (M+H)+, ESI-
MS/MS m/z 360 (100), 246 (15), 191 (25).
MS m/z 496 (MꢁH)ꢁ, ESI-MS/MS m/z 358 (100), 229 (6). Anal.
(C28H27N5O4) C, H, N.
4.1.8.9. (2S)-2-Amino-3-(1H-indol-3-yl)-N-((1S)-1,2,3,4-tetrahydron-
aphtalen-1-yl)propanamide ((S,S)-5e). White powder. 97% Yield. 1H
NMR (CDCl3): d 1.50 (br s, 2H), 1.66–1.79 (m, 3H), 1.97–2.04 (m,
1H), 2.75 (app d, 2H), 2.98 (dd, 1H, J = 14.0, 6.0 Hz), 3.43 (dd, 1H,
J = 14.0, 6.0 Hz), 3.78 (q app, 1H), 5.13–5.18 (m, 1H), 7.06–7.15
(m, 6H), 7.21 (dt, 1H, J = 7.0, 1.0 Hz), 7.37–7.44 (m, 2H), 7.70 (d,
1H, J = 7.0 Hz), 8.16 (br s, 1H). ESI-MS m/z 332 (MꢁH)ꢁ, ESI-MS/
MS m/z 332 (100), 202 (40), 130 (31).
4.1.9.5.
(S)-3-(1H-Indol-3-yl)-N-[1-(5-methoxypyridin-2-yl)cyclo-
hexylmethyl]-2-[(4-nitrophenyl)-carbamoylamino]propanamide ((S)-
6a). 32% Yield. 1H NMR (DMSO-d6): d 1.05–1.42 (m, 7H), 1.84–2.14
(m, 2H), 2.84–2.91 (m, 1H), 2.99–3.06 (m, 2H), 3.25–3.27 (m, 2H),
3.69 (s, 3H), 4.47 (q, 1H, J = 6.9 Hz), 6.60 (d, 1H, J = 8.0 Hz), 6.91–
7.09 (m, 5H), 7.30 (d, 2H, J = 8.0 Hz), 7.50–7.61 (m, 4H), 8.10–
8.17 (m, 3H), 9.46 (s, 1H), 10.83 (s, 1H). 13C NMR (DMSO-d6):
171.8; 162.4; 158.9; 156.9; 152.1; 150.9; 146.7; 145.7; 141.3;
132.4; 130.4; 128.8; 126.1; 123.7; 123.4; 121.9; 116.4; 114.8;
58.7; 55.1; 53.8; 50.2; 45.3; 37.5; 37.4; 33.8; 30.8; 26.3; 22.9.
ESI-MS m/z 571 (M+H)+, ESI-MS/MS m/z 407 (100), 390 (23), 221
(14). Anal. (C31H34N6O5) C, H, N.
4.1.9. General procedure for the synthesis of the final compounds (R)-
6b–d, (S)-6a–d, (R,S)-6e, and (S,S)-6e
To a solution of the amine (R)-5b–d, (S)-5a–d, (R,S)-5e, and (S,S)-
5e (1.0 mmol) in anhydrous THF, a solution of 4-nitrophenyliso-
cyanate (1.2 mmol) in the same solvent (10 mL) was added and
the reaction mixture was stirred at room temperature for 15 h. After
removing the solvent in vacuo, the residue was taken up in EtOAc
(20 mL) and washed with H2O (2 ꢀ 20 mL). The separated organic
layer was dried over Na2SO4 and concentrated under reduced pres-
sure. The crude residue was chromatographed as detailed below.
The obtained solid was further purified by crystallization from
EtOAc/petroleum ether to give the final compound as a yellow
powder.
4.1.9.6.
(S)-3-(1H-Indol-3-yl)-N-[1-(4-methoxyphenyl)cyclohexyl-
((S)-
methyl]-2-[(4-nitrophenyl)-carbamoylamino]propanamide
6b). 10% Yield. 1H NMR (DMSO-d6): d 1.13–1.25 (m, 3H), 1.35–
1.44 (m, 4H), 1.80–1.97 (m, 2H), 2.71–2.90 (m, 2H), 2.97–3.04
(m, 1H), 3.21–3.28 (m, 2H), 3.62 (s, 3H), 4.48 (q, 1H, J = 6.9 Hz),
6.61 (d, 1H, J = 8.2 Hz), 6.72 (d, 2H, J = 8.8 Hz), 6.94 (t, 1H,
J = 7.0 Hz), 7.01–7.11 (m, 4H), 7.30 (d, 1H, J = 8.0 Hz), 7.39 (br t,
1H), 7.49–7.58 (d, 3H), 8.12 (d, 2H, J = 9.4 Hz), 9.46 (s, 1H), 10.82
(s, 1H). 13C NMR (DMSO-d6): 176.8; 158.9; 156.9; 152.1; 150.9;
146.7; 145.7; 141.3; 132.6; 130.4; 128.8; 126.1; 123.7; 123.4;
123.1; 121.9; 116.4; 114.8; 58.7; 53.8; 50.2; 45.3; 37.5; 37.4;
33.8; 30.8; 27.1. ESI-MS m/z 568 (MꢁH)ꢁ, ESI-MS/MS m/z 430
(100), 301 (5). Anal. (C32H35N5O5) C, H, N.
4.1.9.1.
(R)-3-(1H-indol-3-yl)-N-[1-(4-methoxyphenyl)cyclohexyl-
((R)-
methyl]-2-[(4-nitrophenyl)-carbamoylamino]propanamide
6b). 34% Yield. 1H NMR (DMSO-d6): d 1.12–1.18 (m, 3H), 1.29–
1.43 (m, 4H), 1.83–1.97 (m, 2H), 2.83–2.90 (m, 2H), 2.97–3.04
(m, 1H), 3.21–3.28 (m, 2H), 3.62 (s, 3H), 4.49 (q, 1H, J = 7.0 Hz),
6.52 (d, 1H, J = 8.0 Hz), 6.71 (d, 2H, J = 8.8 Hz), 6.94 (t, 1H,
J = 7.4 Hz), 7.01–7.11 (m, 4H), 7.30 (d, 1H, J = 8.0 Hz), 7.41 (t, 1H,
J = 5.9 Hz), 7.49–7.58 (m, 3H), 8.12 (d, 2H, J = 9.1 Hz), 9.39 (s, 1H),
10.82 (s, 1H). ESI-MS m/z 570 (M+H)+, ESI-MS/MS m/z 406 (100),
389 (14). Anal. (C32H35N5O5) C, H, N.
4.1.9.7. (S)-3-(1H-Indol-3-yl)-N-(1-phenylcyclopropylmethyl)-2-[(4-
nitrophenyl)-carbamoylamino]propanamide ((S)-6c). 30% Yield. 1H
NMR (DMSO-d6): d 0.66–0.75 (q, 2H, J = 9.0 Hz), 0.84 (br s, 2H),
2.84–2.94 (m, 1H), 2.99–3.06 (m, 1H), 3.20–3.26 (m, 1H), 3.36–
3.43 (m, 1H), 4.51 (q, 1H, J = 6.8 Hz), 6.51, (d, 1H, J = 8.0 Hz),
6.92–7.05 (m, 3H), 7.09–7.31 (m, 6H), 7.48–7.56 (m, 3H), 8.09–
8.12 (m, 3H), 9.40 (s, 1H), 10.79 (s, 1H). 1ESI-MS m/z 498 (M+H)+,
ESI-MS/MS m/z 360 (129), 334 (100), 317 (19). Anal.
(C28H27N5O4) C, H, N.
4.1.9.2. (R)-3-(1H-indol-3-yl)-N-(1-phenylcyclopropylmethyl)-2-[(4-
nitrophenyl)carbamoylamino]propanamide ((R)-6c). 37% Yield. 1H
NMR (DMSO-d6): d 0.69 (q, 2H, J = 9.7 Hz), 0.81–0.84 (m, 2H),
2.84–2.91 (m, 1H), 2.99–3.06 (m, 1H,) 2.20–2.27 (m, 1H), 3.36–
3.43 (m, 1H), 4.51 (q, 1H, J = 6.8 Hz), 6.51, (d, 1H, J = 7.9 Hz),
6.89–7.05 (m, 3H), 7.08–7.23 (m, 5H), 7.29 (d, 1H, J = 8.2 Hz),
7.48–7.56 (m, 3H), 8.11 (app d, 3H), 9.40 (s, 1H), 10.79 (s, 1H).
ESI-MS m/z 498 (M+H)+, ESI-MS/MS m/z 360 (12), 334 (100). Anal.
(C28H27N5O4) C, H, N.
4.1.9.8. (S)-3-(1H-Indol-3-yl)-N-[1-(pyridin-2-yl)cyclohexylmethyl]-
2-[(4-nitrophenyl)-carbamoylamino]propanamide
((S)-6d). 32%
Yield. 1H NMR (DMSO-d6): d 1.07–1.50 (m, 7H), 2.00–2.20 (m,
2H), 2.82–2.90 (m, 1H), 2.97–3.14 (m, 2H), 3.28–3.35 (m, 2H),
4.48 (q, 1H, J = 7.0 Hz), 6.53 (d, 1H, J = 8.0 Hz), 6.90–7.12 (m. 4H)
7.20 (d, 1H, J = 8.0 Hz), 7.30 (d, 1H, J = 8.0 Hz), 7.49–7.59 (m, 4H),
7.65 (br t, 1H), 8.10 (d, 2H, J = 9.4 Hz), 8.47 (q, 1H, J = 2.0 Hz), 9.40
(s, 1), 10.81 (s, 1H). 13C NMR (DMSO-d6): 171.9; 157.4; 154.2;
147.4; 140.9; 136.5; 136.3; 128.3; 127.8; 125.7; 123.9; 121.3;
118.9; 118.7; 117.1; 113.9; 111.7; 110.1; 55.1; 53.9; 49.0; 42.2;
33.5; 33.1; 29.0; 26.3; 22.1. ESI-MS m/z 541 (M+H)+, ESI-MS/MS
m/z 403 (13), 377 (100), 360 (30). Anal. (C30H32N6O4) C, H, N.
4.1.9.3. (R)-3-(1H-indol-3-yl)-N-[1-(pyridin-2-yl)cyclohexylmethyl]-
2-[(4-nitrophenyl)-carbamoylamino]propanamide
((R)-6d). 33%
Yield. 1H NMR (DMSO-d6): d 1.12–1.21 (m, 3H). 1.29–1.44 (m,
4H), 2.47–2.48 (m, 2H), 2.81–2.89 (m, 1H), 2.97–3.10 (m, 2H),
3.27–3.32 (m, 2H), 4.48 (q, 1H, J = 6.8 Hz), 6.51 (d, 1H, J = 8.0 Hz),
6.93 (t, 1H, J = 7.3 Hz), 7.00–7.12 (m, 3H), 7.19 (d, 1H, J = 8.0 Hz),
7.29 (d, 1H, J = 8.0 Hz), 7.49–7.57 (m, 4H), 7.66 (t, 1H, J = 5.6 Hz),
8.10 (d, 2H, J = 9.1 Hz), 8.47 (d, 1H, J = 3.6 Hz), 9.38 (s, 1H), 10.82
(s, 1H). ESI-MS m/z 541 (M+H)+, ESI-MS/MS m/z 403 (12), 377
(100). Anal. (C30H32N6O4) C, H, N.
4.1.9.9. 3-(1H-Indol-3-yl)-N-((1S)-1,2,3,4-tetrahydronaphthalen-1-
yl)-(2S)-[(4-nitrophenyl)-carbamoylamino]propanamide
((S,S)-
6e). 35% Yield. 1H NMR (DMSO-d6): d 1.59–1.66 (m, 2H), 1.80–
1.83 (m, 2H), 2.66 (br s, 2H), 3.02–3.18 (m, 2H), 4.56 (q, 1H,
J = 7.2 Hz), 4.88 (br s, 1H), 6.60 (d, 1H, J = 7.7 Hz), 6.67 (d, 1H,
J = 7.7 Hz), 6.90–7.12 (m, 5H), 7.16 (d, 1H, J = 1.7 Hz), 7.34 (d, 1H,
J = 8.0 Hz), 7.55–7.62 (d, 3H, J = 9.0 Hz), 8.12 (d, 2H, J = 9.4 Hz),
8.38 (d, 1H, J = 8.5 Hz), 9.45 (s, 1H), 10.88 (s, 1H). ESI-MS m/z 498
(M+H)+, ESI-MS/MS m/z 368 (100), 351 (27), 204 (21). Anal.
(C28H27N5O4) C, H, N.
4.1.9.4. 3-(1H-Indol-3-yl)-N-((1S)-1,2,3,4-tetrahydronaphthalen-1-
yl)-(2R)-2-[(4-nitrophenyl)carbamoylamino]propanamide
((R,S)-
6e). 12% Yield. 1H NMR (DMSO-d6): d 1.49–1.72 (m, 4H), 2.67–
2.68 (m, 2H), 3.02–3.09 (m, 2H), 4.55 (q, 1H, J = 6.6 Hz), 4.91 (br
s, 1H), 6.67 (d, 1H, J = 7.7 Hz), 6.93 (t, 2H, J = 7.0 Hz), 7.01–7.09
(m, 6H), 7.31 (d, 1H, J = 8.3 Hz), 7.55–7.59 (m, 3H), 8.13 (d, 2H,
J = 9.4 Hz), 8.47 (d, 1H, J = 8.5 Hz), 9.49 (s, 1H), 10.84 (s, 1H). ESI-

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
287
4.1.10. General procedure for the preparation of compounds (R)- or
(S)-9f, (S)-9a,d
J = 3.0 Hz). ESI-MS m/z 421 (M+H)+, ESI-MS/MS m/z 390 (51), 290
(100), 221 (23), 204 (14).
N,N0-Carbonyldiimidazole (1.1 mmol) was added to a solution of
(R)- or (S)-8 (1.0 mmol), in anhydrous THF (10 mL), under N2. The
reaction mixture was stirred at room temperature for 8 h, then
the solution of the appropriate amine (1.0 mmol) in anhydrous
THF was added. The reaction mixture was stirred at room temper-
ature for 24 h, then the solvent was removed in vacuo and the resi-
due was partitioned between EtOAc (20 mL) and H2O (20 mL). The
aqueous layer was extracted with EtOAc (2 ꢀ 20 mL) and the col-
lected organic layers were dried (Na2SO4) and evaporated in vacuo.
The crude residue was chromatographed (CHCl3/EtOAc, 9:1, as elu-
ent) to give pure target compound as a pale yellow solid.
4.1.11.2. (S)-2-[Methylamino-3-(1H-indol-3-yl)-N-[1-(pyridin-2-yl)-
cyclohexylmethyl]propanamide ((S)-10d). 60% Yield. 1H NMR
(CDCl3): d 1.25–1.49 (m, 5H), 1.62–1.64 (m, 4H), 2.04–2.16 (m,
2H), 2.10 (s, 3H), 2.73 (dt, 1H, J = 11.0, 4.0 Hz), 3.17–3.26 (m, 2H),
3.41–3.56 (m, 2H), 7.01 (d, 1H, J = 2.2 Hz), 7.07–7.14 (m, 2H),
7.19 (dt, 1H, J = 7.2, 1.1 Hz), 7.26–7.39 (m, 2H), 7.58–7.66 (m,
2H), 8.14 (br s, 1H), 8.55–8.58 (m, 1H). ESI-MS m/z 389 (MꢁH)ꢁ,
ESI-MS/MS m/z 358 (3), 260 (21), 228 (100), 157 (61), 130 (25).
4.1.11.3.
(R)-2-[Methylamino-3-(1H-indol-3-yl)-N-(cyclohexyl-
methyl)propanamide ((R)-10f). 70% Yield. 1H NMR (CDCl3): d 0.83–
0.97 (m, 2H), 1.14–1.28 (m, 3H), 1.36–1.48 (m, 1H), 1.63–1.68 (m,
6H), 2.27 (s, 3H), 2.85–2.93 (m, 1H), 3.02–3.18 (m, 2H), 3.28–3.49
(m, 2H), 7.06 (d, 1H, J = 2.2 Hz), 7.13 (dt, 1H, J = 7.0, 1.0 Hz) 7.20
(dt, 1H, J = 7.0, 1.0 Hz), 7.24 (br s, 1H), 7.37 (d, 1H, J = 8.0 Hz), 7.68
(d, 1H, J = 8.0 Hz), 8.20 (br s, 1H). ESI-MS m/z 312 (MꢁH)ꢁ, ESI-MS/
MS m/z 312 (69), 181 (51), 130 (100).
4.1.10.1.
(S)-2-[Benzyl(methyl)amino-3-(1H-indol-3-yl)-N-[1-(5-
methoxypyridin-2-yl)cyclohexylmethyl]propanamide ((S)-9a). 55%
Yield. 1H NMR (CDCl3): d 1.30–1.62 (m, 8H), 1.91–2.23 (m, 2H),
2.04 (s, 3H), 3.10 (dd, 1H, J = 14.5, 5.5 Hz), 3.37–3.62 (m, 6H), 3.81
(s, 3H), 7.03–7.10 (m, 7H), 7.14–7.21 (m, 4H), 7.34 (d, 1H,
J = 8.0 Hz), 7.55 (d, 1H, J = 8.0 Hz), 8.03 (br s, 1H), 8.20–8.21 (m,
1H). ESI-MS m/z 511 (M+H)+, ESI-MS/MS m/z 390 (100), 380 (73),
263 (50).
4.1.11.4.
(S)-2-[Methylamino-3-(1H-indol-3-yl)-N-(cyclohexyl-
methyl)]propanamide ((S)-10f). 90% Yield. 1H NMR (CDCl3):
d
4.1.10.2.
(S)-2-[Benzyl(methylamino-3-(1H-indol-3-yl)-N-[1-(pyri-
0.78–0.87 (m, 2H), 1.09–1.28 (m, 3H), 1.30–1.38 (m, 1H), 1.62–
1.66 (m, 6H), 2.38 (s, 3H), 2.94–3.10 (m, 3H), 3.18 (dd, 1H,
J = 14.5, 8.0 Hz), 3.39 (dd, 1H, J = 14.5, 6.0 Hz), 7.04–7.18 (m, 3H),
7.34–7.40 (m, 2H), 7.63 (d, 1H, J = 7.7 Hz), 8.57 (br s, 1H). ESI-MS
m/z 314 (M+H)+, ESI-MS/MS m/z 283 (69), 183 (100), 173 (22),
132 (15).
din-2-yl)cyclohexylmethyl]propanamide ((S)-9d). 75% Yield. 1H
NMR (CDCl3): d 1.28–1.60 (m, 8H), 1.95–2.09 (m, 2H), 2.23 (s,
3H), 3.07 (dd, 1H, J = 14.5, 5.5 Hz), 3.39–3.61 (m, 6H), 7.02–7.10
(m, 5H), 7.11–7.20 (m, 6H), 7.34 (d, 1H, J = 8.0 Hz), 7.53–7.60 (m,
2H), 8.02 (br s, 1H), 8.51–8.53 (m, 1H). ESI-MS m/z 481 (M+H)+,
ESI-MS/MS m/z 360 (100), 350 (92), 263 (61).
4.1.12. General procedure for the preparation of compounds (S)-11a,d,
(R)- and (S)-11f
To solution of 4-nitroaniline (1.0 mmol) in anhydrous THF
(10 mL) was added N,N0-carbonyldiimidazole (1.0 mmol), under
N2, and the reaction mixture was stirred at room temperature
4.1.10.3. (R)-2-[Benzylmethylamino-3-(1H-indol-3-yl)-N-(cyclohexyl-
methyl)]propanamide ((R)-9f). 70% Yield. 1H NMR (CDCl3): d 0.78–
0.89 (m, 2H), 1.07–1.18 (m, 3H), 1.29–1.38 (m, 1H), 1.60–1.67 (m,
5H), 2.39 (s, 3H), 3.05 (t, 3H, J = 6.3 Hz), 3.15 (dd, 1H, J = 14.5,
5.5 Hz), 3.46 (dd, 1H, J = 14.5, 7.0 Hz), 3.57–3.63 (m, 2H), 6.86 (br
t, 1H), 7.08–7.37 (m, 9H), 7.62 (d, 1H, J = 7.7 Hz), 8.04 (br s, 1H).
ESI-MS m/z 404 (M+H)+, ESI-MS/MS m/z 283 (100), 273 (87), 263
(63), 144 (23).
overnight.
A solution of amine (S)-10a,d, (R)- and (S)-10f
(1.0 mmol) in anhydrous THF was added and the resulting mixture
was stirred at room temperature for 24 h. Then the solvent was
removed in vacuo and methanol was added to the residue. The
solid was filtered off and the filtrate was evaporated under reduced
pressure. The crude residue was purified on a silica gel column by
eluting first with CHCl3, then with CHCl3/MeOH, 49:1. The obtained
solid was crystallized (EtOAc) to give a yellow solid.
4.1.10.4. (S)-2-[Benzylmethylamino-3-(1H-indol-3-yl)-N-(cyclohexyl-
methyl)]propanamide ((S)-9f). 30% Yield. 1H NMR (CDCl3): d 0.77–
0.90 (m, 2H), 1.06–1.19 (m, 3H), 1.23–1.39 (m, 1H), 1.57–1.68
(m, 5H), 2.39 (s, 3H), 3.05 (t, 2H, J = 6.5 Hz), 3.16 (dd, 1H, J = 14.5,
6.0 Hz), 3.38–3.67 (m, 4H), 6.86 (br t, 1H), 7.06–7.37 (m, 9H),
7.58–7.63 (m, 1H), 8.10 (br s, 1H). ESI-MS m/z 404 (M+H)+, ESI-
MS/MS m/z 283 (100), 273 (84), 263 (69), 144 (31).
4.1.12.1. (S)-N-[1-(5-Methoxypyridin-2-yl)-cyclohexylmethyl]-2-[3-(4-
nitrophenyl)-1-methyl-ureido]-3-(1H-indol-3-yl)propanamide
((S)-
11a). 12% Yield. 1H NMR (CDCl3): d 1.25–1.63 (m, 8H), 2.01–2.03
(m, 2H), 2.93 (s, 3H), 3.25 (dd, 1H, J = 15.0, 9.5 Hz), 3.42 (dt, 2H,
J = 12.6, 5.8 Hz), 3.75 (dd, 1H, J = 13.0, 6.0 Hz), 3.78 (s, 3H), 4.98
(br s, 1H), 6.87 (br s, 1H), 6.95 (br s, 2H), 6.99 (br s, 1H), 7.07–7.24
(m, 6H), 7.34–7.37 (m, 1H), 7.49 (br s, 2H), 8.11 (d, 1H, J = 3.0 Hz),
8.25 (br s, 1H). ESI-MS m/z 583 (MꢁH)ꢁ, ESI-MS/MS m/z 445
(100), 316 (92), 301 (14). Anal. (C32H36N6O5) C, H, N.
4.1.11. General procedure for the preparation of compounds (S)-10a,d,
(R)- and (S)-10f
A solution of N-benzyl derivatives (S)-9a,d, (R)- or (S)-9f in
ethanol (20 mL) was saturated with H2 (5 atm) in presence of a cat-
alytic amount of 10% Pd/C. The reaction mixture was stirred at
room temperature overnight. Then, the mixture was filtered
through a Celite pad and the filtrate was evaporated to dryness.
The crude residue was chromatographed (CHCl3/CH3OH, 19:1, as
eluent) to afford the pure compound as pale yellow oil.
4.1.12.2.
(S)-N-[1-(Pyridin-2-yl)-cyclohexylmethyl]-2-[3-(4-nitro-
((S)-
phenyl)-1-methylureido]-3-(1H-indol-3-yl)propanamide
11d). 6% Yield. 1H NMR (CDCl3): d 1.24–1.64 (m, 8H), 2.00–2.07
(m, 2H), 1.37–1.39 (m, 1H), 2.92 (s, 3H), 3.22–3.30 (m, 1H), 3.42–
3.49 (m, 2H), 3.64 (dd, 1H, J = 13.5, 6.0 Hz), 4.98 (br s, 1H), 6.84
(br s, 1H), 6.95 (br s, 1H), 7.00 (d, 1H, J = 1.2 Hz), 7.07–7.15 (m,
3H), 7.17–7.29 (m, 4H), 7.36 (d, 1H, J = 8.0 Hz), 7.48 (br s, 1H),
7.61 (dt, 1H, J = 7.7, 1.9 Hz), 8.08–8.11 (m, 1H), 8.15 (br s, 1H),
8.42–8.44 (m, 1H). ESI-MS m/z 553 (MꢁH)ꢁ, ESI-MS/MS m/z 415
(100), 286 (57). Anal. (C31H34N6O4) C, H, N.
4.1.11.1. (S)-2-[Methylamino-3-(1H-indol-3-yl)-N-[1-(5-methoxypyr-
idin-2-yl)cyclohexyl methyl]propanamide ((S)-10a). 46% Yield. 1H
NMR (CDCl3): d 1.25–1.44 (m, 4H), 1.53–1.61 (m, 4H), 1.91 (br s,
1H), 2.08–2.14 (m, 2H), 2.14 (s, 3H), 2.73–2.82 (m, 1H), 3.20–3.28
(m, 2H), 3.43 (dq, 2H, J = 13.2, 6.1 Hz), 3.84 (s, 3H), 7.01 (d, 1H,
J = 2.2 Hz), 7.09–7.22 (m, 4H), 7.31 (br s, 1H), 7.35 (d, 1H,
J = 8.0 Hz), 7.64 (d, 1H, J = 8.0 Hz), 8.10 (br s, 1H), 8.25 (d, 1H,

288
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
4.1.12.3.
(R)-N-Cyclohexylmethyl-2-[3-(4-nitrophenyl)-1-methy-
JH-F = 30.3 Hz, JH-H = 3.6 Hz), 4.70 (2H, JH-F = 48.0 Hz, JH-H = 3.6 Hz),
6.76 (d, 2H, J = 8.8 Hz), 6.92–7.06 (m, 4H), 7.26 (br t, 1H), 7.31 (d,
1H, J = 8.0 Hz), 7.53 (d, 1H, J = 7.7 Hz), 8.31 (s, 1H), 10.94 (s, 1H).
ESI-MS m/z 450 (MꢁH)ꢁ, ESI-MS/MS m/z 430 (100).
lureido]-3-(1H-indol-3-yl)propanamide ((R)-11f). 13% Yield. 1H
NMR (DMSO-d6): d 0.78–0.82 (m, 2H), 1.11–1.18 (m, 3H), 1.37–
1.39 (m, 1H) 1.60–1.76 (m, 5H), 2.92 (s, 3H), 2.94–3.15 (m, 4H),
5.02–5.07 (m, 1H), 6.92–7.07 (m, 3H), 7.27 (d, 1H, J = 8.0 Hz),
7.59–7.64 (m, 3H), 7.99 (br s, 1H), 8.09 (d, 2H, J = 9.4 Hz), 9.07
(br s, 1H), 10.78 (s, 1H). ESI-MS m/z 476 (MꢁH)ꢁ, ESI-MS/MS m/z
338 (100), 209 (24), 137 (17). Anal. (C26H31N5O4) C, H, N.
4.1.13.6. (S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(2-fluoropyridin-4-yl)-
cyclohexylmethyl]-2-methylpropionamide
((S)-13k). 1H
NMR
(CDCl3): d 1.10–1.32 (m + s, 7H), 1.37–1.41 (m, 4H), 1.64–1.69
(m, 1H), 1.72 (br s, 2H), 1.87–1.89 (m, 1H), 2.72 (d, 1H,
J = 14.3 Hz), 2.99–3.02 (m, 2H), 3.43 (d, 1H, J = 14.3 Hz), 6.65 (d,
1H, J = 0.8 Hz), 6.88–6.91 (m, 2H), 7.01–7.19 (m, 2H), 7.25–7.27
(m, 2H), 7.36–7.39 (m, 1H), 8.01 (d, 1H, J = 5.2 Hz), 8.30 (br s,
1H). ESI-MS m/z 407 (MꢁH)ꢁ, ESI-MS/MS m/z 228 (100).
4.1.12.4.
(S)-N-Cyclohexylmethyl-2-[3-(4-nitrophenyl)-1-methy-
lureido]-3-(1H-indol-3-yl)propanamide ((S)-11f). 16% Yield. 1H NMR
(DMSO-d6): d 0.75–0.86 (m, 2H), 1.04–1.21 (m, 3H), 1.32–1.38 (m,
1H) 1.57–1.60 (m, 5H), 2.92 (s, 3H), 2.94–3.15 (m, 4H), 4.91–5.02
(q, 1H, J = 7.8 Hz), 6.91–7.06 (m, 3H), 7.27 (d, 1H, J = 8.0 Hz), 7.59–
7.64 (m, 3H), 7.97 (br s, 1H), 8.10 (d, 2H, J = 9.3 Hz), 9.09 (br s,
1H), 10.77 (s, 1H). ESI-MS m/z 476 (MꢁH)ꢁ, ESI-MS/MS m/z 338
(100), 209 (24), 137 (17). Anal. (C26H31N5O4) C, H, N.
4.1.13.7. (S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(2-fluoropyridin-5-yl)-
cyclohexylmethyl]-2-methylpropionamide
((S)-13l). 1H
NMR
(CDCl3): d 1.23–1.60 (m + s, 13H), 1.76–1.88 (m, 2H), 2.72 (d, 1H,
J = 14.0 Hz), 3.21–3.27 (m, 2H), 3.41 (d, 1H, J = 14.0 Hz), 6.73 (dd,
1H, J = 8.8, 3.3 Hz), 6.90 (d, 1H, J = 2.2 Hz), 7.07–7.19 (m, 2H),
7.35–7.40 (m, 1H), 7.40 (br t, 1H), 7.48 (dt, 1H, J = 7.7, 2.8 Hz),
7.59 (d, 1H, J = 8.0 Hz), 7.96 (br d, 1H), 8.46 (br s, 1H). ESI-MS m/
z 407 (MꢁH)ꢁ, ESI-MS/MS m/z 279 (11), 278 (100), 276 (60).
4.1.13. General procedure for the preparation of compounds 13a,g,
15a, (S)-13h,J–l
To a solution of amino acid 12, 14, or (S)-12, (0.75 mmol) in THF
(50 mL) were added PyBOP (1.11 mmol), N-methylmorpholine
(3.00 mmol) and the amine 2a,g,h,j–l (0.90 mmol). The reaction
mixture was stirred at room temperature overnight. The mixture
was taken up with EtOAc (30 mL) and washed with H2O (30 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo.
The crude residue was chromatographed (CHCl3/MeOH, 19:1) to
afford the pure target compound as colorless oil in 80–90% yield.
4.1.14. General procedure for the preparation of compounds 1a,g, 16a,
17a, and (S)-1j–l
A mixture of the appropriate amine (1.0 mmol) and substituted
phenylisocyanate (1.2 mmol) in anhydrous THF (20 mL) was stir-
red at room temperature for 24 h. The solvent was evaporated
and the residue was treated with boiling MeOH. The mixture was
filtered and the filtrate was concentrated under reduced pressure.
The crude residue was chromatographed (CHCl3/EtOAc, 1:1, as elu-
ent) to afford the pure compound as a pale yellow solid.
4.1.13.1. 2-Amino-3-(1H-indol-3-yl)-N-[1-(5-methoxypyridin-2-yl)-
cyclohexylmethyl]-2-methylpropionamide (13a). 1H NMR (CDCl3): d
1.31 (s, 3H), 1.37–1.58 (m, 12H), 1.92–2.11 (m, 2H), 3.32–3.39
(m, 2H), 3.79 (s, 3H), 6.97 (d, 1H, J = 6.5 Hz), 7.01 (d, 1H,
J = 2.5 Hz), 7.05–7.19 (m, 3H), 7.32–7.36 (m, 1H), 7.56–7.61 (m,
2H), 8.19–8.20 (m, 1H), 8.33 (br s, 1H). ESI-MS m/z 421 (M+H)+,
ESI-MS/MS m/z 404 (88), 376 (20), 290 (95), 247 (100), 221 (39).
4.1.14.1. 3-(1H-Indol-3-yl)-N-[1-(5-methoxypyridin-2-yl)cyclohexyl-
methyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide (1a). 33%
Yield. 1H NMR (DMSO-d6): d 1.10–1.23 (m, 4H), 1.34–1.48 (m + s,
7H), 2.10 (br d, 2H), 2.99–3.07 (m, 2H), 3.23–3.28 (m, 2H), 3.66 (s,
3H), 6.55 (s, 1H), 6.80 (t, 1H, J = 7.2 Hz), 6.92–7.04 (m, 3H), 7.16
(d, 1H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.0 Hz), 7.40 (d, 2H, J = 7.7 Hz),
7.60 (d, 2H, J = 9.1 Hz), 8.13–8.17 (m, 2H), 9.50 (s, 1H), 10.80 (s,
1H). ESI-MS m/z 583 (MꢁH)ꢁ, ESI-MS/MS m/z 445 (100), 316 (8).
Anal. (C32H36N6O5) C, H, N.
4.1.13.2.
2-Amino-3-(1H-indol-3-yl)-N-[4-(4-methoxyphenyl)te-
trahydropyran-4-yl-methyl]-2-methylpropionamide (13g). 1H NMR
(CDCl3): d 1.33 (s, 3H), 1.56–1.98 (m, 8H), 3.40–3.65 (m, 4H),
3.69–3.79 (m, 2H), 3.75 (m, 3H), 6.70–6.75 (m, 2H), 6.90–6.93
(m, 2H), 6.96 (d, 1H, J = 2.2 Hz). 7.08–7.24 (m, 3H), 7.37 (d, 1H,
J = 6.0 Hz), 7.60 (d, 1H, J = 9.0 Hz), 8.53 (br s, 1H). ESI-MS m/z 422
(M+H)+, ESI-MS/MS m/z 405 (100), 291 (56), 248 (47), 158 (84).
4.1.14.2. 3-(1H-Indol-3-yl)-N-[4-(4-methoxyphenyl)tetrahydropyran-
4-ylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
(1g). 44% Yield. 1H NMR (CDCl3): d 1.22 (s, 3H), 1.61–1.92 (m, 4H),
2.96 (dd, 1H, J = 8.5, 13.8 Hz), 3.03 (d, 1H, J = 14.6 Hz), 3.29–3.48
(m + s, 7H), 3.56–3.63 (m, 2H), 5.89 (br s, 2H), 6.38 (d, 2H,
J = 9.0 Hz), 6.71–6.90 (m, 4H), 6.92 (d, 1H, J = 9.0 Hz), 7.13 (d, 1H,
J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.44 (d, 2H, J = 9.0 Hz), 7.99 (d,
2H, J = 9.0 Hz), 8.73 (s, 1H), 9.73 (s, 1H). ESI-MS m/z 584 (MꢁH)ꢁ,
ESI-MS/MS m/z 446 (100), 317 (8). Anal. (C32H35N5O6) C, H, N.
4.1.13.3. 2-Amino-N-[1-(5-methoxypyridin-2-yl)-cyclohexylmethyl]-
2-methyl-3-phenylpropionamide (15a). 1H NMR (CDCl3): d 1.27 (s,
3H), 1.29–1.58 (m, 12H), 1.92–2.11 (m, 2H), 3.27–3.36 (m, 2H),
3.82 (s, 3H), 7.09–7.17 (m, 4H), 7.19–7.24 (m, 3H), 7.59 (br s,
1H), 8.22 (t, 1H, J = 1.8 Hz). ESI-MS m/z 382 (M+H)+, ESI-MS/MS
m/z 337 (17), 247 (34), 221 (100), 204 (72).
4.1.13.4. (S)-2-Amino-3-(1H-indol-3-yl)-N-[1-(5-hydroxypyridin-2-
yl)cyclohexylmethyl]-2-methylpropionamide ((S)-13h). 1H NMR
(DMSO-d6): d 1.16–1.41 (m + s, 13H), 1.91–1.95 (m, 1H), 2.04–
2.07 (m, 1H), 3.03–3.25 (m, 4H), 6.90–7.04 (m, 5H), 7.29 (d, 1H,
J = 7.8 Hz), 7.51 (d, 1H, J = 7.8 Hz), 7.60 (br t, 1H, D2O exchanged),
8.04 (s, 1H), 9.55 (s, 1H, D2O exchanged), 10.88 (s, 1H, D2O
exchanged). ESI-MS m/z 405 (MꢁH)ꢁ, ESI-MS/MS m/z 405 (11),
274 (100), 216 (50), 205 (41), 188 (22).
4.1.14.3. N-[1-(5-Methoxypyridin-2-yl)cyclohexylmethyl]-2-methyl-
2-[3-(4-nitrophenyl)ureido]-3-phenylpropionamide (16a). 38% Yield.
1H NMR (CDCl3): d 1.15–1.20 (m, 2H), 1.25–1.37 (m, 2H), 1.41 (s,
3H), 1.51–1.51 (m, 4H), 1.96–2.11 (m, 2H), 3.18–3.30 (m, 3H),
3.44 (dd, 1H, J = 5.8, 12.9 Hz), 3.62 (s, 3H), 6.18 (br d, 1H), 6.92–
7.00 (m, 3H), 7.02–7.12 (m, 3H), 7.13–7.18 (m, 2H), 7.43–7.47
(m, 2H), 7.99–8.09 (m, 3H), 8.95 (s, 1H). ESI-MS m/z 544 (MꢁH)ꢁ,
ESI-MS/MS m/z 406 (100). Anal. (C30H35N5O5) C, H, N.
4.1.13.5.
(S)-2-Amino-3-(1H-indol-3-yl)-N-[4-(2-fluoroethoxy)
phenyl)cyclohexylmethyl]-2-methylpropionamide ((S)-13j). 1H NMR
(DMSO-d6): d 1.10–1.32 (m + s, 7H), 1.37–1.41 (m, 4H), 1.64–1.69
(m, 1H), 1.72 (br s, 2H), 1.87–1.89 (m, 1H), 2.73 (d, 1H,
J = 14.0 Hz), 2.99–3.02 (m, 2H), 3.10–3.17 (m, 1H), 4.14 (dt, 2H,
4.1.14.4. 3-(1H-Indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[1-
(5-methoxypyridin-2-yl)cycloexylmethyl]-2-methylpropionamide
(17a). 52% Yield. 1H NMR (DMSO-d6): d 1.13–1.23 (m, 4H), 1.31
(s, 3H), 1.37–1.49 (m, 4H), 2.13 (br d, 2H), 3.05 (dd, 1H, J = 12.9,

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
289
5.8 Hz), 3.18–3.26 (m, 1H), 3.34–3.38 (m, 2H), 3.65 (s, 3H), 3.69
(s, 3H), 6.04 (s, 1H), 6.81–6.86 (m, 3H), 6.94–7.00 (m, 3H), 7.18–
7.30 (m, 5H), 7.42 (d, 1H, J = 8.0 Hz), 8.17 (d, 1H, J = 3.0 Hz), 8.42
(s, 1H), 10.82 (s, 1H). ESI-MS m/z 568 (MꢁH)ꢁ, ESI-MS/MS m/z
445 (100), 419 (90), 316 (16). Anal. (C33H39N5O4) C, H, N.
chromatographed (CHCl3/EtOAc, 7:3 as eluent) to afford pure (S)-
1m (0.015 g, 28% yield). 1H NMR (DMSO-d6): d 1.10–1.32 (m, 4H),
1.43 (s, 3H), 1.47–1.56 (m, 4H), 2.00–2.13 (m, 2H), 3.03 (dd, 1H,
J = 12.9, 5.8 Hz), 3.10–3.17 (m, 3H), 4.14 (dt, 2H, JH-F = 30.3 Hz, JH-
H = 3.8 Hz), 4.53 (2H, JH-F = 40.4 Hz, JH-H = 3.8 Hz), 6.52 (s, 1H), 6.80
(t, 1H, J = 7.2 Hz), 6.92–7.04 (m, 3H), 7.16 (d, 1H, J = 8.8 Hz), 7.26
(d, 2H, J = 8.0 Hz), 7.40 (d, 2H, J = 7.7 Hz), 7.60 (d, 2H, J = 9.1 Hz),
8.13–8.17 (m, 2H), 9.50 (s, 1H), 10.80 (s, 1H). 13C NMR (DMSO-
d6): 171.9; 157.4; 154.2; 147.4; 140.9; 136.5; 136.3; 128.3; 127.8;
125.7; 123.9; 121.3; 118.9; 118.7; 117.1; 113.9; 111.7; 110.1;
84.4; 72.9; 55.1; 53.9; 49.0; 42.2; 33.5; 33.1; 29.0; 26.3; 22.9;
22.1. ESI-MS m/z 615 (MꢁH)ꢁ, ESI-MS/MS m/z 477 (100), 348 (5).
Anal. (C33H37FN6O5) C, H, N.
4.1.14.5. (S)-3-(1H-Indol-3-yl)-N-[1-(5-hydroxypyridin-2-yl)cyclo-
hexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
((S)-1h). 19% Yield: 1H NMR (DMSO-d6): d 1.10–1.23 (m, 4H), 1.34–
1.48 (m + s, 7H), 2.10 (br d, 2H), 2.99–3.07 (m, 2H), 3.23–3.28 (m,
2H), 6.55 (s, 1H), 6.80 (t, 1H, J = 7.2 Hz), 6.92–7.04 (m, 3H), 7.16
(d, 1H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.0 Hz), 7.40 (d, 2H, J = 7.7 Hz),
7.60 (d, 2H, J = 9.1 Hz), 8.13–8.17 (m, 2H), 9.50 (s, 1H), 10.80 (s,
1H), 10.99 (s, 1H). ESI-MS m/z 569 (MꢁH)ꢁ, ESI-MS/MS m/z 430
(100), 242 (8).
4.2. Semi-preparative HPLC enantioseparation on chiral stationary
phase
4.1.14.6. (S)-3-(1H-Indol-3-yl)-N-[1-[4-(2-fluoroethoxy)phenyl]cyclo-
hexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
((S)-1j). Yield: 33%. 1H NMR (DMSO-d6): d 1.13–1.18 (m, 4H), 1.36–
1.47 (m + s, 7H), 2.01 (br s, 2H), 2.92 (dd, 1H, J = 12.9 Hz) 3.21 (dd,
1H, J = 12.9 Hz), 3.38 (br s, 2H), 3.97–4.10 (m, 2H), 4.57–4.75 (m,
2H), 6.50 (s, 1H), 6.70 (d, 2H, J = 8.0 Hz), 6.77–6.82 (m, 1H), 6.92–
6.99 (m, 2H), 7.15–7.27 (m, 4H), 7.42 (d, 1H, J = 8.0 Hz), 7.63–
7.66 (m, 2H), 8.15–8.19 (m, 2H), 9.49 (br s, 1H), 10.82 (br s, 1H).
13C NMR (DMSO-d6): 176.8; 158.9; 156.9; 152.1; 150.9; 146.7;
145.7; 141.3; 132.6; 130.4; 128.8; 126.1; 123.7; 123.4; 123.1;
121.9; 116.4; 114.8; 84.4; 72.9; 58.7; 53.8; 50.2; 45.3; 37.5;
37.4; 33.8; 30.8; 27.1; 22.9. ESI-MS m/z 614 (MꢁH)ꢁ, ESI-MS/MS
m/z 476 (100), 347 (7). Anal. (C34H38FN5O5) C, H, N.
4.2.1. Instrumentation
The chromatographic apparatus consisted of a Shimadzu LC-
10AD Pump (Shimadzu Italia, Milan), a Rheodyne 7125 manual
injector equipped with a 200 ll sample loop (Jasco Europe, Italy,
Milan). A Merck Hitachi L-7400UV (Merck KGaA, Darmstadt, Ger-
many) was used as detector. Chromatograms were recorded with
a HSM Hitachi chromatography software. The column used was a
Chiralcel OD (cellulose tris(3,5-dimethylphenylcarbamate),
250 ꢀ 10 mm, 10
lm particle size)) purchased from Chiral Tech-
nologies Europe (Illkirch, France). HPLC-grade n-hexane and 2-pro-
panol (IPA) were obtained from Sigma-Aldrich (Milan, Italy);
triethylamine (TEA) was obtained from Fluka (Milan, Italy).
4.1.14.7. (S)-3-(1H-Indol-3-yl)-N-[1-(2-fluoropyridin-4-yl)cyclohexyl-
4.2.2. Chromatographic parameters
methyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
((S)-
The separation factor (a
) was calculated as k02/k01 and retention
1k). 1H NMR (DMSO-d6): d 1.06–1.09 (m, 2H), 1.21–1.26 (m, 2H),
1.39 (s, 3H), 1.51–1.54 (m, 4H), 2.03 (br d, 2H), 3.00 (dd, 1H,
J = 13.2, 5.2 Hz), 3.26–3.44 (m, 3H), 6.50 (br s, 1H), 6.77 (t, 1H,
J = 7.4 Hz), 6.94–7.02 (m, 3H), 7.15 (br d, 1H), 7.25 (d, 1H,
J = 8.3 Hz), 7.40 (d, 1H, J = 8.3 Hz), 7.53 (br t, 1H), 7.63 (d, 2H,
J = 9.1 Hz), 7.90 (d, 1H, J = 5.2 Hz), 8.15 (d, 2H, J = 9.1 Hz), 9.53 (br
s, 1H), 10.81 (br s, 1H). 13C NMR (DMSO-d6): 171.9; 166.0; 164.1;
157.4; 147.4; 143.5; 136.5; 136.3; 128.3; 127.8; 125.7; 123.9;
121.3; 118.9; 118.7; 117.1; 113.9; 111.7; 110.1; 55.1; 53.7; 49.0;
42.2; 33.5; 33.1; 29.0; 25.3; 22.9; 21.3. ESI-MS m/z 571 (MꢁH)ꢁ,
ESI-MS/MS m/z 433 (100), 304 (9). Anal. (C31H33FN6O4) C, H, N.
factors (k01 and k02) as k’1 = (t1 ꢁ t0)/t0 where t1 and t2 refer to
retention times of the first and second eluted enantiomers. The res-
olution factor (Rs) was calculated by the formula Rs = 2(t2 ꢁ t1)/
(w1 + w2) where w1 and w2 are the peak widths at base for the first
and second eluted enantiomers. The dead time of the column (t0)
was determined by injection of 1,3,5-tri-tert-butylbenzene.
4.2.3. Semipreparative enantioseparation of compounds 1a,g, 17a
Details on the separation of the enantiomerically enriched sam-
ples 1aE1, 1aE2, 1gE1, 1gE2, 17aE1, 17aE2 are given in Table 4.
4.3. Radiochemistry
4.1.14.8. (S)-3-(1H-Indol-3-yl)-N-[1-(2-fluoropyrid-5-yl)cyclohexyl-
methyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
((S)-
4.3.1. (S)-3-(1H-Indol-3-yl)-N-[1-[5-(2-[18F]fluoroethoxy)pyridin-2-yl]
cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
([18F](S)-1m)
1l). 10% Yield. 1H NMR (DMSO-d6): d 1.12–1.28 (m, 4H), 1.40 (s,
3H), 1.50–1.54 (m, 4H), 1.97–2.04 (m, 2H), 3.00 (dd, 1H, J = 13.2,
5.2 Hz), 3.26–3.44 (m, 3H), 6.48 (br s, 1H), 6.77–6.89 (m, 3H),
6.96 (t, 1H, J = 7.1 Hz), 7.25 (d, 1H, J = 8.0 Hz), 7.41 (d, 1H,
J = 8.0 Hz), 7.60–7.63 (m, 3H), 7.75 (dt, 1H, J = 8.0, 2.5 Hz), 8.12–
8.17 (m, 3H), 9.52 (br s, 1H), 10.81 (br s, 1H). 13C NMR (DMSO-
d6): 171.9; 163.1; 157.4; 154.2; 147.4; 140.9; 136.5; 136.3;
128.3; 127.8; 125.7; 123.9; 121.3; 118.9; 118.7; 117.1; 113.9;
111.7; 110.1; 55.1; 53.7; 49.0; 42.2; 33.5; 33.1; 29.0; 25.3; 22.9;
22.3. ESI-MS m/z 571 (MꢁH)ꢁ, ESI-MS/MS m/z 433 (100), 304 (6).
Anal. (C31H33FN6O54 C, H, N.
Aqueous [18F]fluoride (2–5 GBq) was produced by irradiation of
18O-water with an IBA 18/18 cyclotron via the 18O(p,n)18F nuclear
reaction. The [18F]-fluoride solution was passed through a SepPak
Light Accell plus QMA anion exchange cartridge (Waters, Milford,
MA, USA) to recover the 18O-enriched water. [18F]fluoride was
eluted with a solution of K2CO3 (1 mg/mL) into a V-vial containing
15 mg of Kryptofix 2.2.2. [18F]-Fluoride was dried by three consec-
utive azeptropical evaporations with dry acetonitrile. 2-[18F]-Fluo-
roethyl tosylate was prepared by fluorination at 125 °C for 5 min of
the ditosylate precursor (5 mg in 0.5 mL of dry acetonitrile) fol-
lowed by Sep-Pak Silica plus purification using 14 mL of hexane/
diethyl ether (3:1, v/v). After evaporation to dryness, 2-[18F]fluo-
roethyl tosylate was dissolved in DMF (0.8 mL) and added to a
solution of 1h (2 mg, 0.0035 mmol) and NaH (2 mg) in DMF
(0.2 mL). The reaction mixture was heated for 5 min at 125 °C.
Hereafter water (2 mL) was added to the cooled mixture. The reac-
tor was rinsed with an additional 1 mL of water. The reaction mix-
4.1.14.9. (S)-3-(1H-Indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]-
cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide
((S)-1m). 2-Fluoroethyltosylate (0.039 g, 0.18 mmol) was added to
a mixture of (S)-1h (0.050 g, 0.88 mmol) and Cs2CO3 (0.118 g,
0.36 mmol) in acetone. The reaction mixture was refluxed for 8 h
(TLC monitoring), then the solvent was removed in vacuo. The resi-
due was taken up with EtOAc, washed with H2O, dried over Na2SO4,
and concentrated under reduced pressure. The crude residue was
ture was purified by HPLC using
a microBondapak C18

290
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
Table 4
Experimental details for the separation of compounds 1aE1, 1aE2, 1gE1, 1gE2, 17aE1, 17aE2.
Compd
Mobile Phase
Flow rate
k0
a
ee%
1aE1a
1aE2b
n-Hexane/IPA (8:2, v/v)
4.5 mL/min
4.11
5.32
1.30
93
90
1gE1
1gE2
n-Hexane/IPA (8:2, v/v)
2.5 mL/min
2.5 mL/min
9.37
11.03
1.18
1.20
92
89
17aE1
17aE2
n-Hexane/IPA/TEA(8:2:0.01, v/v)
15.14
18.24
92
90
a
E1: faster eluting enantiomer.
E2: later eluting enantiomer.
b
(300 ꢀ 7.8 mm) with 10 mM sodium acetate/MeCN (4:6, v/v) as
mobile phase with a flow rate of 3 mL/min. The retention time
was 16 min. The collected HPLC-fraction was diluted with 20 mL
of water and passed through an C18-Sep-Pak. [18F](S)-1m was
eluted with ethanol (0.8 mL) and formulated with saline (5 mL).
Quality control was performed by UPLC, using a HSS T3 column
(20 mM Hepes pH 7.4, 3 mM MgCl2, 1 mM EDTA, 0.3% BSA) were
suspended 100 g of PC3 cells membranes or 0.32 g of human
l
l
cloned BB2 receptor, 0.02 nM [125I]-(Tyr4)-bombesin, test or refer-
ence compounds. The samples were incubated for 60 min at 27 °C.
The incubation was stopped by rapid filtration on Whatman GF/C
glass microfiber filters (presoaked in 0.5% polyethylenimine for
30 min). The filters were washed with 3 ꢀ 1 mL of ice-cold buffer
(50 mM Tris-HCl, pH 7.4). Nonspecific binding was determined in
(1.8
l
m, 3.0 ꢀ 50 mm) with the following gradient: t = 0–5 min:
90% 10 mM ammonium bicarbonate (pH 9.5) and 10% MeCN;
t = 5–6 min: 40% 10 mM ammonium bicarbonate (pH 9.5) and
60% MeCN; t = 6–7 min 70% 10 mM ammonium bicarbonate (pH
9.5) and 30% MeCN at a flow rate of 0.8 mL/min (retention time:
the presence of 1 lM hGRP. Approximately 90% of specific binding
was determined under these conditions. Concentration required to
inhibit 50% of radioligand specific binding (IC50) was determined
by using six to nine different concentrations of the drug studied
in two or three experiments with samples in duplicate. Apparent
inhibition constant (Ki) values were determined by nonlinear curve
fitting, using the Prism, version 3.0, GraphPad software [43].
[
18F]fluoride = 0.5 min, [18F](S)-1m = 4.75 min). Thus, [18F](S)-1m
was obtained within 120 min in 3.37 0.26% radiochemical yield
(decay-corrected). At the end of synthesis (EOS), the specific activ-
ity was 9.64 5.29 GBq/mmol and the radiochemical purity was
always higher than 95%.
4.5. Intracellular calcium mobilization
4.4. Receptor binding studies
Changes in intracellular Ca2+ was measured in human PC3 cells
with a InfiniteÒ Tecan multimode reader using the Fluo-4 DirectTM
Calcium Assay Kit (Molecular Probes, Inc.) as previously described
[9]. Cells (25,000 cells per well) were suspended in culture med-
ium, seeded in clear bottom black 96-well plates and cultured for
1 day at 37 °C and 5% CO2. After incubation time, the culture med-
4.4.1. General
PC3 human prostate cell line was obtained from Interlab Cell
Line Collection (ICLC, Genoa, Italy). Cell culture reagents were pur-
chased from EuroClone (Milan, Italy). Human cloned BB2 receptor
expressed in HEK-293 cells,
[
125I]-(Tyr4)-bombesin (2200 Ci/
mmol) were obtained from PerkinElmer Life and Analytical
Sciences (Boston, MA, USA). hGRP and bombesin were purchased
from Tocris Bioscience (Bristol, UK).
ium was removed and replaced with 50
lL/well of culture medium
and 50
l
L/well of Fluo-4 DirectTM dye dissolved in assay buffer con-
taining 5 mM probenecid. The plate was incubated for 90 min at
37 °C and 5% CO2. After addition of test compound, changes in flu-
orescence were recorded (kex = 494 nm, kem = 520 nm) every 5 s for
120 s. Maximum change in fluorescence, expressed in arbitrary
units over baseline, was used to determine agonist response. Max-
imum fluorescence was measured after addition of ionomycin and
baseline measurements were taken for untreated cells. The dose-
response curve for bombesin (Tocris Bioscience, UK) was obtained
4.4.2. Cells culture
PC3 cell lines was routinely cultured in RPMI 1640 supple-
mented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL
penicillin, 100 lg/mL streptomycin in a humidified incubator at
37 °C with a 5% CO2 atmosphere.
incubating cells with 5–6 concentrations of agonist (10ꢁ11
–
4.4.3. Saturation binding assay
10ꢁ6 M). The dose-response curves for the antagonists were
obtained incubating cells with 1 nM bombesin after 10 min pre-
treatment with different concentrations of test compounds
(10ꢁ10–10ꢁ5 M). All experiments were performed 3 times in tripli-
cate. Curve fitting (5–6 points) and calculation of median effective
concentration values (EC50) were performed by nonlinear regres-
sion analysis of the dose-response curves generated using Prism
3 [43].
The saturation experiments were carried out as described by
Benya et al. with minor modifications [24]. Membranes of human
PC3 prostate cells were prepared according to Colabufo et al. [42]
BB2 receptor were radiolabeled using [125I]-(Tyr4)-bombesin con-
centrations of 0.01–0.7 nM. Samples contained 100
branes, radioligand and hGRP (1 M) to determine non-specific
binding were incubated in a final volume of 500 L (Hepes 20 mM
lg of PC3 mem-
l
l
pH 7.4, 3 mM MgCl2, 1 mM EDTA, 0.3% BSA) for 60 min at 27 °C.
The suspension was filtered through Whatman GF/C glass microfi-
ber filters presoaked in 0.5% polyethylenimine for at least 30 min
prior to use. The filters were washed 3 ꢀ 1 mL ice-cold buffer
(50 mM Tris-HCl, pH 7.4). Scatchard parameters were determined
by nonlinear curve fitting, using the Prism, version 3.0, GraphPad
software [43] (Kd = 0.66 nM; Bmax = 358 fmol/mg of protein).
4.6. Serum stability
The rat serum was diluted in PBS (1:1, v/v) and pre-incubated at
37 °C for 5 min. The reaction was initiated by adding the appropri-
ate amount of the stock solution of the tested compounds in DMSO.
The final concentration of the tested compound was 10
amount of DMSO did not exceed 1.25%, v/v). Time samples aliquots
(100 l) were removed and immediately mixed with cold acetoni-
trile (400 l) containing the internal standard. Quenched samples
lM (the
4.4.4. Competition binding assays
BB2 receptor binding was carried out according to Benya et al.
with minor modifications [24]. In 0.5 mL of incubation buffer
l
l

E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
291
were centrifugated at 3500 rpm for 5 min and the supernatants
were directly injected for quantification analysis. Samples
(100 ll) were analyzed by using an Agilent 1260 Infinity Binary
LC System equipped with a diode array detector (Open Lab soft-
ware was used to analyze the chromatographic data) and a Phe-
and used for cell counting. Cell numbers were manually deter-
mined, using a phase-contrast microscope, a Bulker bright-line
chamber (depth, 0.1 mm:0.0025 mm2) and a hand-tally counter.
Cellular uptake of the radioactivity was corrected for the number
of viable cells. Results are expressed as percentage of incubated
radioactivity per million cells (mean SD).
nomenex Gemini C-18 column (250 ꢀ 4.6 mm,
5 lm particle
size). The samples were eluted using MeOH/water (70:30, v/v) as
eluent (1 mL/min). The in vitro serum half life (t1/2) was calculated
using the expression t1/2 = 0.693/b, where b is the slope found in
the linear fit of the natural logarithm of the fraction remaining of
the parent compound vs incubation time.
4.10. Dissociation kinetic study
For the dissociation study, 0.1 million PC-3 cells were plated per
well in 24-well plates one day before the experiments and kept at
37 °C in a humidified 5% CO2 atmosphere. On the day of the exper-
iments, the cells were washed twice with warm PBS and new RPMI
1640 was added to the wells. The cells were then incubated with
4.7. Stability assays in rat liver microsomes
50 l
l of [18F](S)-1m (ca 100 kBq/well) for 1 h at 37 °C to allow for
Test compounds were pre-incubated at 37 °C with rat liver
microsomes (Tebu-Bio, Milan, Italy) (1.0 mg/mL microsomal pro-
tein) at 10 lM final concentration in 100 mM potassium phosphate
maximum internalization. To remove unbound radioactivity, the
cells were washed twice afterwards with cold PBS and were then
incubated with warm RPMI 1640 medium at 37 °C for 0, 15, 30,
60 and 120 min in triplicate to allow for externalization. At the
end of each time point, the medium was discarded, the cells
buffer (pH 7.4) for 10 min. Metabolic reactions were initiated by
the addition of the NADPH regenerating system (containing
10 mM NADP, 50 mM glucose-6-phosphate, and 10 unit/mL glu-
cose-6-phosphate dehydrogenase, final glucose-6-phosphate
dehydrogenase concentration, 1 unit/mL). Aliquots were removed
at specific time endpoints and immediately mixed with an equal
volume of cold acetonitrile containing the internal standard. Test
compound incubated with microsomes without NADPH regenerat-
ing system was included. Quenched samples were centrifugated at
4500 rpm for 15 min and the supernatants were injected for quan-
washed with cold PBS, harvested with trypsin (100
resuspended in 900 L of RPMI 1640 (Vt = 1 mL). The cell suspen-
sions were collected for each well and the radioactivity was mea-
sured using gamma counter. 50 sample of each
suspension was mixed with 50 L of Trypan blue and used for cell
lL/well) and
l
a
A
lL
l
counting. Cell numbers were manually determined in the same
manner as mentioned above. Cellular uptake of the radioactivity
was corrected for the number of viable cells. Results are expressed
as the percentage of maximum intracellular radioactivity (remain-
ing activity at specific time-point/activity at time point 0,
mean SD).
tification analysis. Samples (100 lL) were analyzed by using an
Agilent 1260 Infinity Binary LC System equipped with a diode array
detector (Open Lab software was used to analyze the chromato-
graphic data) and
a
Phenomenex Gemini C-18 column
(250 ꢀ 4.6 mm, 5 m particle size). The samples were eluted using
l
CH3CN/20 mM ammonium formate pH 5.5 (70:30, v/v) as eluent
(1 mL/min). Concentrations were quantified by measuring the area
under the peak.
4.11. Competition study with [18F](S)-1m
PC-3 cells were plated in duplicate in a 24-well plate at a den-
sity of 0.1 million cells per well. After one day, the medium was
The in vitro half life (t1/2) was calculated using the expression
t1/2 = 0.693/b, where b is the slope found in the linear fit of the nat-
ural logarithm of the fraction remaining of the parent compound vs
incubation time [44]. In vitro half-life was then used to calculate
the intrinsic plasma clearance (CLint) according to the following
equation:
discarded and the cells were washed twice with PBS and 900
of warm RPMI 1640 medium was added per well. Next, 50 L of
18F](S)-1m (ca 100 kBq/well) solution was added in the absence
lL
l
[
or presence of different concentrations of unlabeled Glu[Aca-BN
(7–14)]2. After the cells were incubated for 60 min, the medium
was discarded. The cells were washed with cold PBS (3 ꢀ 0.5 mL/
0:693
1
CLint
:
ꢀ
In vitro t1=2 mg=ml microsomial protein
well), harvested with trypsin (100 lL) and resuspended in 900 lL
of RPMI. The cell suspensions were collected separately for each
well and the radioactivity was measured using a gamma counter.
A 50 lL sample of the suspension was mixed with 50 lL of trypan
4.8. [18F](S)-1m stability
blue and used for cell counting. Cell numbers were manually deter-
mined in the same manner as mentioned above. Cellular uptake of
the radioactivity was corrected for the number of viable cells.
Results are plotted as sigmoid curves for the displacement of
The formulated tracer (ethanol/water) was incubated at room
temperature and analyzed by UPLC at 1 and 2 h post incubation.
[
18F](S)-1m as a function of increasing concentrations of Glu[Aca-
4.9. Uptake study
BN(7–14)]2 (mean SD).
For uptake experiments, PC-3 cells were plated in 24-well
plates at a density of 0.1 million cells per well. After 24 h, the med-
ium was discarded, the cells were washed twice with warm and
new medium was added. Then, [18F](S)-1m (ca 100 kBq/well)
was incubated with cells at 37 °C for 0, 15, 30, 60 and 120 min in
Funding sources
The financial support by Progetto Idea Giovani Università degli
Studi di Bari (Prot. n. 26670 Tit.III/11, April 1, 2010) is gratefully
acknowledged.
triplicate to allow for cellular uptake. Glu[Aca-BN(7–14)]2 (20 lg)
was co-incubated with [18F](S)-1m in blocking groups at the same
time points. To remove unbound radioactivity, the cells were
washed twice with cold PBS and the cells were harvested with
Acknowledgment
trypsin (100
(Vt = 1 mL). The cell suspensions were collected for each well and
the radioactivity was measured using a gamma counter. A 50
sample of each suspension was mixed with 50 l of Trypan blue
lL/well) and resuspended in 900 lL of RPMI 1640
Dr. Marina Carrozzo (DiSTeBA, Università del Salento, Italy) is
gratefully acknowledged for enantiomeric separation of the race-
mic mixture of compounds 1a,g, 16a and 17a.
l
l
l

292
E. Lacivita et al. / Bioorg. Med. Chem. 25 (2017) 277–292
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