Exploring the size of the lipophilic unit of the soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115078

Soluble epoxide hydrolase (sEH) inhibitors are potential drugs for several diseases. Adamantyl ureas are excellent sEH inhibitors but have limited metabolic stability. Herein, we report the effect of replacing the adamantane group by alternative polycycli

Full text of DOI:10.1016/j.bmc.2019.115078

Bioorganic&MedicinalChemistry27(2019)115078
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploring the size of the lipophilic unit of the soluble epoxide hydrolase
inhibitors
Sandra Codonya, Elena Valverdea, Rosana Leivaa, José Breab, M. Isabel Lozab,
⁎
Christophe Morisseauc, Bruce D. Hammockc, Santiago Vázqueza,
a Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències de l’Alimentació, and Institute of Biomedicine (IBUB), Universitat de
Barcelona, Av. Joan XXIII 27-31, Barcelona E-08028, Spain
b Innopharma Screening Platform, Biofarma Research Group, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidad de Santiago
de Compostela, Spain
c Department of Entomology and Nematology, and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Soluble epoxide hydrolase (sEH) inhibitors are potential drugs for several diseases. Adamantyl ureas are ex-
cellent sEH inhibitors but have limited metabolic stability. Herein, we report the effect of replacing the ada-
mantane group by alternative polycyclic hydrocarbons on sEH inhibition, solubility, permeability and metabolic
stability. Compounds bearing smaller or larger polycyclic hydrocarbons than adamantane yielded all good in-
hibition potency of the human sEH (0.4 ≤ IC50 ≤ 21.7 nM), indicating that sEH is able to accommodate in-
hibitors of very different size. Human liver microsomal stability of diamantane containing inhibitors is lower
than that of their corresponding adamantane counterparts.
Adamantane
Inhibitor
Isocyanate
Soluble epoxide hydrolase
Urea
1. Introduction
hydrolysis of EETs to the corresponding dihydroxyeicosatrienoic acids
(DHETs), reducing the beneficial activities of EETs.12–15 The inhibition
Arachidonic acid (AA), a polyunsaturated fatty acid, plays im-
portant roles in cellular signaling as a second messenger and is also a
precursor for a wide variety of lipid mediators that are involved in
many physiological and pathophysiological processes. The first step in
the biosynthesis of these mediators, known as eicosanoids or oxylipins,
is an oxidation, which can be catalyzed by cyclooxygenases (COX), li-
poxygenases (LOX), and cytochrome P450 enzymes.1 Most research on
AA derivatives has focused on prostaglandins, processed by COX, and
leukotrienes, originated from LOX. Both types of metabolites are potent
inflammatory mediators and, consequently, several pharmaceuticals
have been produced to alleviate inflammatory conditions. These in-
cluded non-selective COX-1 and COX-2 inhibitors (e.g., ibuprofen, in-
domethacin), selective COX-2 inhibitors (e.g., celecoxib, etoricoxib),
and 5-LOX inhibitors (e.g., zileuton).2–4
of sEH in vivo by potent, selective inhibitors results in an increase of the
concentration of the EETs, reducing blood pressure and inflammatory
and pain states, thereby suggesting that sEH inhibitors may serve as
novel agents for treating hypertension, inflammatory diseases, pain
and, more recently, neurodegenerative diseases.16–21
X-ray crystallographic studies revealed that sEH has an active site
with a catalytic triad at the corner of an L-shaped hydrophobic pocket.
The triad includes a nucleophilic aspartic acid, which attacks the ep-
oxide carbon-highly polarized by hydrogen bonds with two tyrosine
residues, and a histidine-aspartic acid pair, which activates the hydro-
lysis of the acyl-enzyme intermediate.22 Therefore, lipophilic groups
such as cyclohexyl or adamantyl are commonly present in potent sEH
inhibitors in order to stablish hydrophobic interactions with the pocket.
In fact, the first sEH inhibitor to enter in clinical trials was AR9281, an
adamantyl urea (Fig. 1).23 Specifically, hundreds of sEH inhibitors
featuring a common structure of Ad-NH-C(O)-NH-R, where Ad is ada-
mantan-1-yl and R is alkyl, aryl or heterocyclyl groups, have been
synthesized and, subsequently, evaluated in several in vivo models
(Fig. 1).23–35 However, the poor metabolic stability of some ada-
mantane containing ureas could limit their usefulness to treat pa-
tients.36 Notwithstanding the high potency generally associated to
Comparatively, the third pathway remains relatively unexplored.
Cytochromes P450 enzymes transform AA to various biologically active
compounds, including epoxyeicosatrienoic acids (EETs).5,6 EETs are
reported to exhibit anti-inflammatory and anti-nociceptive properties
and are involved in the regulation of blood pressure and cellular
stress.7–11 Soluble epoxide hydrolase (sEH, EPHX2, E.C. 3.3.2.3), a
member of the α/β-hydrolase fold family of enzymes, catalyzes the
⁎ Corresponding author.
E-mail address: svazquez@ub.edu (S. Vázquez).
https://doi.org/10.1016/j.bmc.2019.115078
Received 3 May 2019; Received in revised form 31 July 2019; Accepted 25 August 2019
Availableonline26August2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
Fig. 1. Adamantyl-based sEH inhibitors.
Scheme 1. Synthesis of analogs containing a trifluorophenyl unit. Reagents and
conditions: (a) 2,3,4-trifluorophenylisocyanate, Et3N, anh. DCM, overnight.
Fig. 2. Polycyclic amines used in this study.
Scheme 2. Synthesis of diamantanamine 8.
adamantane-derived sEH inhibitors, alternative polycyclic hydro-
carbons have been scarcely evaluated.
In this work, a series of ring-contracted and ring-expanded analogs
of three potent adamantane sEH inhibitors, AR9281 (IC50 = 7.0 nM),23
t-AUCB (IC50 = 0.5 nM),37 and 1 (IC50 = 0.4 nM),38 were synthesized
and pharmacologically evaluated in order to test if alterations in the
size of the lipophilic unit attached to the urea significantly impact its
potency toward the human sEH (Figs. 1 and 2) as well as influencing
solubility, permeability and metabolic stability.
Scheme 3. Synthesis of t-AUCB analogs. Reagents and conditions: (a) tri-
phosgene, NaHCO3, DCM, 30 min; (b) t-4-[(4-aminocyclohexyl)oxy]benzoic
acid, Et3N, DCM, 30 °C, overnight; (c) t-4-[(4-aminocyclohexyl)oxy]benzoic
acid, Et3N, DMF, 50 °C, 3 days.
2. Results and discussion
2.1. Chemistry
Adamantyl ureas are typically synthesized by the reaction of ada-
mantyl isocyanates with a primary amine. Alternatively, the reaction of
amantadine (1-adamantylamine) with an isocyanate also furnishes
compounds. An alternative procedure was employed for the synthesis of
urea 24, involving the activation of 22 with 1,1′-carbonyldiimidazole
(Scheme 4).
adamantyl
ureas.
Taking
into
account
that
2,3,4-tri-
fluorophenylisocyanate is a commercially available compound, for the
preparation of the analogs of urea 1, we reacted this isocyanate with
four different amines, 4–7, featuring smaller polycyclic rings than
adamantane. Bisnoradamantane amines 4, 5 and 6 were synthesized
following reported procedures,39,40 while noramantadine 7 is com-
mercially available. For comparative purposes, we also synthesized,
using the same reaction, urea 1 and its isomer 10 (Scheme 1).
2.2. sEH inhibition and structure-activity relationships
The potency of the new compounds as human soluble epoxide hy-
drolase inhibitors was tested using a previously reported sensitive
fluorescent-based assay (Table 1).41
Within the series of the 2,3,4-trifluophenyl inhibitors, sequential
ring contraction from adamantanes 1 or 10 to noradamantane 14 and
to bisnoradamantane 13 resulted in a decrease of the inhibitory potency
(compare entries 1 and 2 vs 5 and 6, Table 1), likely because of the
reduction of hydrophobic interactions between the ring-contracted
moiety and the lipophilic pocket of the enzyme and the increase of
desolvation energy to transfer the molecule from the solution state to
the receptor cavity. This reduction in potency was also observed in the
other two series of sEH inhibitors (compare entry 11 vs 13, and entry 8
vs 9, Table 1).
In order to obtain the ring-expanded analog 15, we started from
diamantanamine 8, which was synthesized in two steps from com-
mercially available diamantane, 16. Oxidation of 16 with sulfuric acid
followed by a reductive amination of ketone 17 by ammonium acetate
and NaCNBH3 led to amine 8 (Scheme 2). For the synthesis of the ring-
contracted and ring-expanded analogs of t-AUCB and AR9281, we first
prepared the required isocyanate by the reaction of the corresponding
polycyclic amine with triphosgene. The reaction of these isocyanates
with either t-4-[(4-aminocyclohexyl)oxy]benzoic acid (Scheme 3) or N-
acetyl-4-aminopiperidine, 22 (Scheme 4), furnished the desired
Nevertheless, the inhibitory potency was restored by the
2

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
and 28 showed IC50 values in the same range as that of AR9281
(compare entry 11 vs entries 14 and 15). Considering that 26 was
slightly more potent than its isomer 28, we synthesized two further
analogs derived from diamantane 8, i.e., the new ureas 15 and 21,
analogs of inhibitors 1 and t-AUCB, respectively. In line with the
aforementioned results, diamantane derivative 21 showed to be as
potent as t-AUCB (compare entry 8 vs 10, Table 1). However, within the
trifluorophenyl series, the diamantane derivative 15 was considerably
less potent than adamantane derivatives 1 or 10 (compare entries 1 and
2 vs 7, Table 1). The dissimilar behavior of 15 compared with 21 and 26
could be due to an opposite binding orientation of 15 compared to that
of 21 and 26, as observed previously for a different series of sEH in-
hibitors.43
Typically, steric parameters have stronger effects on the potency of
inhibitors against murine sEH rather than on the human sEH.32,41,43,44
For example, it has recently been reported that the progressive in-
troduction of one, two or three methyl groups in the bridgehead posi-
tions of the adamantane unit of t-AUCB did not lead to significant
changes in the IC50 values against the human enzyme, while leading to
a gradual increase in the IC50 values against the murine enzyme.32
However, when we tested the inhibition of the murine sEH by AR9281
and three analogs (24, 26 and 28), we did not find significant differ-
ences between their activities in human and murine species (Table 1).
Scheme 4. Synthesis of AR9281 analogs. Reagents and conditions: (a) 1,1′-
carbonyldiimidazole, 1,2-dichloroethane, 50 °C, 21 h; (b) Et3N, CHCl3, 50 °C,
24 h; (c) triphosgene, Et3N, DCM, 30 min; (d) DCM, Et3N, overnight; (e) DCM,
overnight.
2.3. Microsomal stability
Table 1
Inhibitory activities against the human and murine sEH.
It is known that the adamantane nucleus is prone to rapid meta-
bolism in vivo giving rise to a variety of inactive hydroxylated deriva-
tives. This results in low drug concentrations in blood and short in vivo
half-life. Metabolism studies have shown that the bridgehead hydro-
xylation (tertiary carbon) is favored over the secondary carbon posi-
tions, producing water-soluble hydroxyadamantane derivatives in the
liver, which are then easily excreted.45 Additionally, metabolic studies
showed that liver microsomes from phenobarbital-treated rats readily
metabolize diamantane to mono-, di- and possibly tri-hydroxy deriva-
tives.46,47 It is also known that several diamantanes are cytochromes
P450 inhibitors.48,49
Entry
Compound
IC50 (nM) human sEHa
IC50 (nM) murine sEHa
1
1
0.4
0.4
0.4
0.5
3.2
3.3
8.0
0.5
8.6
0.5
8.0
6.5
21.7
3.4
7.2
NDb
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.0
3.3
ND
5.0
1.8
2
10
3
11
4
12
5
13
6
14
7
15
8
t-AUCB
19
9
10
11
12
13
14
15
21
AR9281
24
Considering the aforementioned metabolism liability of the ada-
mantane and diamantane scaffolds, we assessed the in vitro stability of
some representative new ureas in human microsomes in order to ex-
amine the impact of the different hydrophobic units in their metabolic
stabilities.
25
26
28
a
Reported IC50 values are the average of three replicates. The fluorescent
assay as performed here has a standard error between 10 and 20% suggesting
that differences of two-fold or greater are significant. Because of the limitations
of the assay it is difficult to distinguish among potencies < 0.5 nM.41
As anticipated, diamantane derivatives were extremely labile com-
pounds, with their adamantane counterparts being considerably more
stable (compare entries 1 vs 6, 7 vs 9, and 10 vs 13 and 14, Table 2).
These results are in the line with what was expected, since diamantane
moiety features more tertiary carbon atoms than the adamantane ring,
which are prone to be hydroxylated.
b
ND: not determined.
introduction of two methyl groups in the bridgehead positions of the
bisnoradamantane moiety (compare entries 1 and 2 vs entries 3 and 4,
and entry 11 vs entry 12, Table 1), probably because the addition of the
methyl groups compensates the reduction in size from the adamantane
to the bisnoradamantane scaffold. Furthermore, the results showed that
the introduction of a methylene unit between the hydrophobic moiety
and the urea does not affect the potency of the compounds (compare
entry 3 vs 4, Table 1).
Finally, the bisnoradamantane and the noradamantane units seem
to have similar (compare entries 1 vs 3 and 4, and 1 vs 5 and 10 vs 12,
Table 2) or somehow reduced (compare entries 7 vs 8 and 10 vs 11,
Table 2) microsomal stability than adamantane.
2.4. Solubility and lipophilicity
In order to assess the impact of the polycyclic scaffold in the solu-
bility of the inhibitors, we experimentally determined their solubility in
a 1% DMSO: 99% PBS buffer solution.
Taking into account that the reduction of the polycyclic moiety from
adamantane to bisnoradamantane led, within the three series of in-
hibitors, to a reduction of the potency, we wondered if the opposite was
true. That is, whether an increase in the size of the lipophilic unit of the
inhibitor would lead to more potent compounds.
As expected, within the trifluorophenylurea series, the solubility
highly increases from diamantane 15 to adamantane 1 (18 and 57 μM,
respectively, Table 2) and then, slightly further increases to the nor-
adamantane 14 and to the bisnoradamantane 13 (65 and 82 μM, re-
spectively, Table 2). In fact, the diamantane derivatives were drama-
tically less soluble than their adamantane, noradamantane or
bisnoradamantane counterparts (compare entries 6 vs 1, 3, 4 and 5,
entries 9 vs 7 and 8 and entries 13 and 14 vs 10 and 11, Table 2).
With the aim of exploring the ring-expanded analogs, the ada-
mantane ring was replaced by the much larger diamantane moiety.
Somehow surprisingly, considering the substantial increase in size and
previous consideration of the adamantyl group as the marginal biggest
group as the N-substituent for sEH inhibitors,42 diamantane ureas 26
3

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
Table 2
displayed much lower permeability. As expected, benzoic acid deriva-
Solubility and microsomal stability of the new compounds.
tives t-AUCB and 21 were the less permeable compounds (Table 3).
Entry
Compound
Microsomal stabilitya
Solubility (μM)b
Lipophilicityc
3. Conclusions
1
1
34.3%
19.4%
30.0%
41.9%
30.2%
0.0%
57
4.1
3.9
4.1
3.5
3.8
4.5
3.7
3.4
3.9
2.1
2.2
1.8
2.5
2.6
2
10
NDd
66
Overall, it seems clear that the catalytic center of the sEH enzyme
can accommodate polycycles of different sizes, ranging from the small
bisnoradamantane moiety to the very large diamantane group.
Notwithstanding this, it appears, particularly within the t-AUCB and
AR9281 derivatives, that the replacement of the adamantane by larger
polycyclic rings, such as the diamantanes, is better than the replace-
ment by smaller ones.
3
12
4
13
82
5
14
65
6
15
18
7
t-AUCB
19
93.5%
64.6%
14.6%
80.1%
59.3%
88.7%
0.0%
60
8
76
9
21
7
10
11
12
13
14
AR9281
24
> 100
> 100
ND
86
Of note, although the present results highlight the interest of dia-
mondoids as tools for investigating the size-limit of inhibitors,51 the low
solubility and the high metabolic lability of these derivatives severely
limits their potential use in medicinal chemistry.52
25
26
28
2.6%
85
a
Percentage of remaining compound after 60 min of incubation with human
microsomes obtained from Tebu-Xenotech in the presence of NADP at 37 °C.
Metabolism of testosterone was used as a positive control for metabolism
(22.4% remaining compound).
4. Experimental section
4.1. Chemistry
b
Solubility in a 1% DMSO: 99% PBS buffer solution, see experimental sec-
tion for details.
4.1.1. General
c
Lipophilicity refers to the consensus log Po/w value calculated using the
Commercially available reagents and solvents were used without
further purification unless stated otherwise. Preparative normal phase
chromatography was performed on a CombiFlash Rf 150 (Teledyne
Isco) with pre-packed RediSep Rf silica gel cartridges. Thin-layer
chromatography was performed with aluminum-backed sheets with
silica gel 60 F254 (Merck, ref. 1.05554), and spots were visualized with
UV light and 1% aqueous solution of KMnO4. Melting points were de-
termined in open capillary tubes with a MFB 595010M Gallenkamp.
400 MHz 1H and 100.6 MHz 13C NMR spectra were recorded on a
Varian Mercury 400 or on a Bruker 400 Avance III spectrometers.
500 MHz 1H NMR spectra were recorded on a Varian Inova 500 spec-
trometer. The chemical shifts are reported in ppm (δscale) relative to
internal tetramethylsilane, and coupling constants are reported in Hertz
(Hz). Assignments given for the NMR spectra of selected new com-
pounds have been carried out on the basis of DEPT, COSY 1H/1H
(standard procedures), and COSY 1H/13C (gHSQC and gHMBC se-
quences) experiments. IR spectra were run on Perkin-Elmer Spectrum
RX I, Perkin-Elmer Spectrum TWO or Nicolet Avatar 320 FT-IR spec-
trophotometers. Absorption values are expressed as wave-numbers
(cm−1); only significant absorption bands are given. High-resolution
mass spectrometry (HRMS) analyses were performed with an LC/MSD
TOF Agilent Technologies spectrometer. The elemental analyses were
carried out in a Flash 1112 series Thermofinnigan elemental micro-
analyzator (A5) to determine C, H, N and S. The structure of all new
compounds was confirmed by elemental analysis and/or accurate mass
measurement, IR, 1H NMR and 13C NMR. The analytical samples of all
the new compounds, which were subjected to pharmacological eva-
luation, possessed purity ≥95% as evidenced by their elemental ana-
lyses.
SwissADME program50 for five predictive log Po/w models (iLOGP, XLOGP3,
WLOGP, MLOGP and SILICOS-IT).
d
ND: not determined.
Finally, considering the right-hand side of the inhibitors, the acet-
ylpiperidine derivatives were the more soluble compounds, with the
two other series having similar solubility.
Of note, the experimental solubility values showed a good correla-
tion with the calculated lipophilicity values (see Table 2), the more
soluble AR9281 analogs being the compounds with the lowest lipo-
philicity. As expected, for any given right-hand side unit, the dia-
mantane derivatives showed always the higher lipophilicity.
2.5. Permeability
In order to evaluate the permeability of selected inhibitors, the
Caco-2 cell permeability model was used in this study. Apparent per-
meability values (Papp) were determined from the amount permeated
through the Caco-2 cell membranes at both apical-basolateral (A-B) and
basolateral-apical (B-A) direction (Table 3).
Of note, the size of the lipophilic unit of the sEH inhibitors seems to
be of little relevance regarding permeability, as evidenced through the
comparison within the three series of inhibitors: the trifluorophenyl
derivatives (compare entries 1 and 2 vs 3, Table 3), the benzoic acid
derivatives (compare entries 4 vs 5, Table 3) and the acetylpiperidine
derivatives (compare entries 6–8, Table 3). Regarding the right-hand
side of the ureas, acetylpiperidine derivatives were endowed with the
best permeability, while the trifluorophenyl compounds 1, 10 and 15
Table 3
4.1.2. Diamantan-3-one (17)
Permeability in the Caco-2 cell line of selected sEH inhibitors.
Diamantane, 16 (600 mg, 3.19 mmol), was suspended in conc.
H2SO4 96% (5 mL). The mixture was stirred at 75 °C for 10 h. The re-
action mixture was cooled to room temperature and poured on ice. This
aqueous solution was extracted with diethyl ether (3 × 50 mL). The
combined organic phases were dried over anh. Na2SO4 and filtered.
Evaporation of the organics gave a white solid (494 mg). This residue
was dissolved in DCM (20 mL) 25 g of neutrum alumina were added and
the solvent was evaporated obtaining a white solid. Then, hexane
(25 mL) was added, the suspension was stirred for 5 min and was fil-
trated (×2). Diethyl ether (50 mL) was added, the suspension was
stirred for 5 min and it was filtrated. Evaporation of the organics gave
17 as a white solid (440 mg, 69% yield). The spectroscopic data coin-
cide with those described in the bibliography.53,54
Entry
Compound
Papp A → B (nm/s)
Papp B → A (nm/s)
ERa
1
2
3
4
5
6
7
8
1
11.8
2.3
1.3
0.1
2.4
0.2
0.3
1.8
0
0.2
3.2
0.3
111
22.2
1.1
1.0
0.9
0
10
7.3
0.3
0.1
0.2
0.1
34.5
1.9
0.2
0.2
0.1
15
16.2
1.9
5.4
t-AUCB
21
210.3
67.5
180.2
146.9
191.6
53.7
3.1
2.4
24
159.2
156.2
208.3
2.8
31.6
19.6
38.5
26
13.6
20.2
28
a
The efflux ratio was calculated as ER = (Papp B → A)/(Papp A → B). See the
experimental section for further details. Permeability of estrone-3-sulfate and
colchicine were used as references.
4

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
2
1
2
4.1.3. Diamantane-3-amine (8)
F = 246 Hz, JC-F = 12 Hz, C4′), 147.5 (C, dd, JC-F = 243 Hz, JC-
Diamantan-3-one, 17 (583 mg, 2.88 mmol) was dissolved in IPA
(6 mL), followed by the addition of AcONH4 (3.33 g, 43.22 mmol). The
mixture was stirred at reflux for 1 h. Then, NaCNBH3 (1.26 g,
20.17 mmol) was added. The reaction mixture was stirred at reflux for
24 h. The dark solution was cooled down to room temperature and 10 N
NaOH was added until basic pH to quench the reaction. This mixture
was extracted with DCM (3 × 50 mL) and the combined organic phases
were dried over anh. Na2SO4 and filtered. Evaporation of the organics
gave a white solid (580 mg) which was dissolved in EtOAc and ex-
tracted with 2 N HCl. The aqueous layer was basified with 5 N NaOH
until basic pH and extracted with EtOAc. The combined organic phases
were dried over anh. Na2SO4 and filtered. Evaporation of the organics
in vacuo gave 8 as a white solid (390 mg, 66% yield). 1H NMR
(400 MHz, CDCl3) δ: 1.50 (dt, J = 12.8 Hz; J′ = 3.2 Hz, 1H), 1.61–1.83
(complex signal, 15H), 1.92–1.98 (complex signal, 2H), 2.91 (t,
J = 2.8 Hz, 1H, 3-H). 13C NMR (100.6 MHz, CDCl3) δ: 26.4 (CH), 31.1
(CH), 31.7 (CH2), 32.4 (CH), 36.6 (CH), 37.0 (CH), 37.6, 37.7, 37.8,
38.83 and 38.0 (1 CH and 4 CH2), 43.9 (CH), 56.4 (CH). HRMS-ESI+ m/
z [M+H]+ calcd for [C14H21N+H]+: 204.1747, found: 204.1753.
F = 9 Hz, C2′), 157.8 (C, CO). MS (DIP), m/z (%); significant ions: 338
(M+, 1), 149 [(C11H17
)
+, 43], 148 (86), 147 [(C6H4F3N)+, 42], 136
(19), 135 [(C10H15
)
+, 100], 119 (18), 107 (56), 106 (15), 105 (15), 93
(42), 91 (28), 79 (16), 77 (16). Elemental analysis: Calculated for
C18H21F3N2O: C 63.89, H 6.26, N 8.28. Found: C 63.83, H 6.52, N 8.26.
4.1.8. 1-(3,7-Dimethyl(tricyclo[3.3.0.03,7]oct-1-yl))-3-(2,3,4-
trifluorophenyl)urea (12)
From 3,7-dimethyltricyclo[3.3.0.03,7]octan-1-amine hydrocloride
(5·HCl) (61 mg) and following the general procedure a crude was ob-
tained. Column chromatography (SiO2, Hexane/Ethyl Acetate mixture)
followed by evaporation in vacuo of the appropriate fractions gave the
urea 12 (50 mg, 47% yield) as a white solid, mp 174–175 °C. IR (ATR) ν:
3335, 2957, 2930, 2882, 2158, 2005, 1686, 1656, 1637, 1621, 1565,
1509, 1471, 1308, 1289, 1242, 1204, 1165, 1154, 1118, 1081, 1064,
1020, 1009, 964, 946, 816, 796, 719, 694, 678, 657 cm−1
.
1H NMR
(400 MHz, CDCl3) δ: 1.15 [s, 6H, 3(7)-CH3], 1.39 [dd, J = 8.4 Hz,
J′ = 3.6 Hz, 2H, 4(6)-Ha], 1.64 [dd, J = 7.6 Hz, J′ = 3.6 Hz, 2H, 2(8)-
Ha], 1.76 [dd, J = 8.4 Hz, J′ = 3.0 Hz, 2H, 4(6)-Hb], 1.82 [d,
J = 7.6 Hz, 2H, 2(8)-Hb], 2.38 (t, J = 3.0 Hz, 1H, 5-H), 5.47 (broad s,
1H, 1-NH), 6.75 (broad s, 1H, 3-NH), 6.89 (m, 1H, 5′-H), 7.80 (m, 1H,
6′-H). 13C NMR (100.6 MHz, CDCl3) δ: 16.5 [CH3, C3(7)-CH3], 44.8
(CH, C5), 46.2 [C, C3(7)], 53.1 [CH2, C4(6)], 57.5 [CH2, C2(8)], 61.8
(C, C1), 111.4 (CH, dd, 2JC-F = 17.7 Hz, 3JC-F = 3.8 Hz, C5′), 115.0 (CH,
4.1.4. General procedure for the synthesis of the ureas 1 and 10–15
In a round-bottom flask equipped with a stir bar under nitrogen
atmosphere the appropriate amine hydrochloride (1.2 mmol) was
added to anh. DCM (~110 mM). To this suspension 2,3,4-tri-
fluorophenyl isocyanate (1 mmol) followed by triethylamine (7 mmol)
was added. The reaction mixture was stirred at room temperature
overnight. Then the solvent was removed in vacuo and the resulting
crude was purified by column chromatography.
3
2
3
t, JC-F = 5 Hz, C6′), 124.8 (C, dd, JC-F = 8 Hz, JC-F = 3.4 Hz, C1′),
1
1
2
2
139.7 (C, ddd, JC-F = 249 Hz, JC-F = 16.3 Hz, JC-F = 13.7 Hz, C3′),
2
3
142.1 (C, ddd, JC-F = 244 Hz, JC-F = 11.9 Hz, JC-F = 3.2 Hz, C4′),
141.4 (C, ddd, 1JC-F = 245 Hz, 2JC-F = 10 Hz, 3JC-F = 2.8 Hz, C2′), 154.6
(C, CO). MS (EI), m/z (%); significant ions: 324 (M+, 8), 268 (15), 148
(44), 147 [(C6H4F3N)+, 100], 146 (23), 136 (17), 134 (84), 122 (89),
121 (54), 120 (40), 119 (81), 110 (20), 109(51), 108 (50), 107 (28),
106 (16), 105 (21), 96 (17), 95 (70), 94 (54), 93 (52), 92 (18), 91 (44),
81 (17), 80 (18), 79 (37), 77 (33), 67 (24), 55 (16), 41 (23). Elemental
analysis: Calculated for C17H19F3N2O: C 62.95, H 5.90, N 8.64. Found:
C 63.12, H 6.17, N 8.48.
4.1.5. 1-(1-Adamantyl)-3-(2,3,4-trifluorophenyl)urea (1)
From adamantan-1-amine hydrochloride (2·HCl) (162 mg) and fol-
lowing the general procedure a crude was obtained. Column chroma-
tography (SiO2, Hexane/Ethyl Acetate mixture) followed by evapora-
tion in vacuo of the appropriate fractions gave the urea 1 (280 mg,
quantitative yield). The analytical sample was obtained by crystal-
lization from methanol. The spectroscopic data coincide with those
described in the bibliography.37
4.1.9. 1-(Tricyclo[3.3.0.03,7]oct-1-yl)-3-(2,3,4-trifluorophenyl)urea (13)
From tricyclo[3.3.0.03,7]octan-1-amine hydrochloride (4·HCl)
(19 mg) and following the general procedure a crude was obtained.
Column chromatography (SiO2, Hexane/Ethyl Acetate mixture) fol-
lowed by evaporation in vacuo of the appropriate fractions gave the
urea 13 (24 mg, 66% yield) as a white solid, mp 185–186 °C. IR (ATR) ν:
3331, 3105, 2970, 2943, 2894, 2159, 1656, 1640, 1620, 1563, 1510,
1467, 1318, 1288, 1244, 1204, 1171, 1107, 1076, 1065, 1017, 979,
4.1.6. 1-(2-Adamantyl)-3-(2,3,4-trifluorophenyl)urea (10)
From adamantan-2-amine hydrochloride (3·HCl) (166 mg) and fol-
lowing the general procedure a crude was obtained. Column chroma-
tography (SiO2, Hexane/Ethyl Acetate mixture) followed by evapora-
tion in vacuo of the appropriate fractions gave the urea 10 (270 mg,
94% yield). The analytical sample was obtained by crystallization from
EtOAc/pentane. The spectroscopic data were identical to those pre-
viously published.37
823, 800, 764, 723, 710, 690, 668, 645, 620 cm−1
.
1H NMR (400 MHz,
CDCl3) δ: 1.51 [d, J = 8.8 Hz, 2H, 4(6)-Ha], 1.74 [complex signal, 6H,
4(6)-Ha, 2(8)-H2], 2.34 [broad s, 2H, 3(7)-H], 2.42 (m, 1H, 5-H), 5.42
(broad s, 1H, 1-NH), 6.67 (broad s, 1H, 3-NH), 6.90 (m, 1H, 5′-H), 7.82
(m, 1H, 6′-H). 13C NMR (100.6 MHz, CDCl3) δ: 32.8 [CH, C3(7)], 43.0
(CH, C5), 46.6 [CH2, C4(6)], 51.1 [CH2, C2(8)], 61.6 (C, C1), 111.4
4.1.7. 1-[(3,7-Dimethyl(tricyclo[3.3.0.03,7]oct-1-yl)methyl]-3-(2,3,4-
trifluorophenyl)urea (11)
From (3,7-dimethyltricyclo[3.3.0.03,7]octan-1-yl)methanamine hy-
drochloride (6·HCl) (50 mg) and following the general procedure a
crude was obtained. Column chromatography (SiO2, Hexane/Ethyl
Acetate mixture) followed by concentration in vacuo of the appropriate
fractions gave the urea 11 (82 mg, 98% yield) as a white solid, mp
133–134 °C. IR (ATR) ν: 3323, 2952, 2881, 1637, 1621, 1570, 1510,
1474, 1292, 1244, 1177, 1045, 1001, 985, 809, 794, 755, 682,
2
3
3
(CH, dd, JC-F = 17.7 Hz, JC-F = 3.7 Hz, C5′), 115.1 (CH, t, JC-
2
3
F = 5.6 Hz, C6′), 124.8 (C, dd, JC-F = 8 Hz, JC-F = 3.5 Hz, C1′), 139.7
(C, dt, 1JC-F = 245 Hz, 2JC-F = 15 Hz, C3′), 142.2 (C, dd, 1JC-F = 225 Hz,
1
2
2JC-F = 12 Hz, C4′), 146.5 (C, dd, JC-F = 246 Hz, JC-F = 10 Hz, Ar-
C2′), 154.6 (C, CO). MS (DIP), m/z (%); significant ions: 296 (M+, 34),
268 (14), 267 (24), 254 (22), 147 [(C6H4F3N)+, 77], 146 (23), 119
(14), 95 (29), 94 (1 0 0), 82 (29), 81 (62), 80(20), 79(18). Elemental
analysis: Calculated for C15H15F3N2O: C 60.81, H 5.10, N 9.45. Found:
C 60.87, H 5.34, N 9.19.
653 cm−1
.
1H NMR (500 MHz, CD3OD) δ: 1.17 (s, 6H, C3(7)-CH3),
1.32–1.38 (complex signal, 4H, 4(6)-Ha and 2(8)-Ha), 1.42 (d,
J = 7.5 Hz, 2H, 2(8)-Hb), 1.59 (dd, J = 8 Hz, J′ = 3 Hz, 2H, 4(6)-Hb),
2.10 (t, J = 3 Hz, 1H, 5-H), 3.42 (s, 2H, CH2-N), 7.01 (m, 1H, 5′-H),
7.75 (m, 1H, 6′-H). 13C NMR (125.7 MHz, CD3OD) δ: 17.1 [CH3, C3(7)-
CH3], 42.7 (CH, C5), 43.8 [CH2, CH2-N], 48.5 [C, C3(7)], 52.3 (C,C1),
4.1.10. 1-(Tricyclo[3.3.1.03,7]non-3-yl)-3-(2,3,4-trifluorophenyl)urea
(14)
2
54.9 [CH2, C4(6)], 57.2 [CH2, C2(8)], 112.3 (CH, dd, JC-F = 17.7 Hz,
From tricyclo[3.3.1.03,7]nonyl-3-amine hydrochloride (7·HCl)
(100 mg) and following the general procedure, a yellow solid was ob-
tained (191 mg). Column chromatography (Hexane/Ethyl Acetate
3JC-F = 3.9 Hz, C5′), 116.7 (CH, C6′), 127.0 (C, d, JC-F = 6 Hz, C1′),
2
1
2
1
141.5 (C, dt, JC-F = 247 Hz, JC-F = 15 Hz, C3′), 143.6 (C, dd, JC-
5

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
mixture) gave urea 14 as a white solid (52 mg, 57% yield), mp
192–193 °C. IR (ATR) ν: 661, 757, 798, 956, 1005, 1021, 1052, 1083,
1101, 1155, 1176, 1248, 1287, 1325, 1382, 1429, 1470, 1509, 1563,
6H, 2′(6′)-Hax
,
9″-H2 and 6″(8″)-Hax], 1.86 [dd, J = 10.0 Hz,
J′ = 2.8 Hz, 2H, 2″(4″)-Hax], 1.98–2.05 [complex signal, 6H, 6″(8″)-Heq
and 3′(5′)-Heq and 2″(4″)-Heq], 2.13 [dd, J = 4 Hz, J′ = 12.8 Hz, 2H,
2′(6′)-Heq], 2.22 [broad s, 2H, 1″(5″)-H], 2.34 (t, J = 6.8 Hz, 1H, 7″-H),
4.39 (m, 1H, 1′-H), 6.96 [d, J = 9.2 Hz, 2H, 3(5)-H], 7.93 [d,
J = 8.8 Hz, 2H, 2(6)-H]. 13C NMR (100.6 MHz, CD3OD) δ: 29.1 [CH2,
C2′(6′)], 29.6 [CH2, C3′(5′)], 33.9 (CH2, C9″), 36.7 [CH, C1″(5″)], 42.3
[CH2, C6″(8″)], 42.9 (CH, C7″), 46.8 (CH, C4′), 48.2 [CH2, C2″(4″)],
63.3 (C, C3″), 74.0 (CH, C1′), 114.2 [CH, C3(5)], 121.2 (C, C1), 130.6
[CH, C2(6)], 157.9, (C, CO), 161.3 (C, C4), 166.5 (CO2H). HRMS-ESI-
1633, 1656, 2346, 2852, 2925, 3343 cm−1
.
1H NMR (400 MHz, CDCl3)
δ: 1.54–1.69 [complex signal, 4H, 9-H2 and 6(8)-Hax], 1.92 [dd,
J = 10.0 Hz, J′ = 2.8 Hz, 2H, 2(4)-Hax], 2.01–2.10 [complex signal, 4H,
6(8)-Heq and 2(4)-Heq], 2.25 [broad singlet, 2H, 1(5)-H], 2.40 [tt,
J = 6.8 Hz, J′ = 2.5 Hz, 1(7)-H], 7.00 (m, 1H, 5′-H), 7.69 (m, 1H, 6′-H).
13C NMR (100.6 MHz, CDCl3) δ: 35.9 (CH2, C9), 38.8 (CH, C1 and C5),
44.3 (CH2, C6 and C6), 44.8 (CH, C7), 49.9 (CH2, C2 and C4), 65.3 (C,
2
3
C3), 112.2 (CH, dd, JC-F = 18 Hz, JC-F = 4 Hz, C5′), 116.8 (CH, C6′),
126.9 (C, dd, J = 3 Hz, J′ = 8 Hz, C1′), 139.9 (C, dt, 1JC-F = 247.4, 2JC-
F = 14, C3′), 146–148 (complex signal, C4′ and C2′), 156.8 (C, CO).
Elemental analysis: Calcd for C16H17F3N2O·0.25 MeOH: C 61.31, H
5.70, N 8.80. Found C 61.51, H 5.94, N 8.55.
m/z [M−H]− calcd for [C23H30N2O4−H]−
: 397.2133, found:
397.2147.
4.1.14. Diamantane-3-isocyanate (20)
Triphosgene (110 mg, 0.368 mmol) was added in a single portion to
a
solution diamantane-3-amine (8) (150 mg, 0.73 mmol) in DCM
4.1.11. 1-(Diamant-3-yl)-3-(2,3,4-trifluorophenyl)urea (15)
(10.5 mL) and saturated NaHCO3 solution (4.5 mL). The resulting bi-
phasic mixture was stirred at room temperature for 30 min. Then, the
two phases were separated and the organic layer was washed with
brine, dried over anh. Na2SO4 and filtered. Evaporation in vacuo pro-
vided the isocyanate 20 as a white solid (152 mg, 90% yield), which
was used in the next step without further purification.
From diamantane-3-amine (8) (160 mg) and following the general
procedure, a solid was obtained (291 mg). Column chromatography
(Hexane/Dichloromethane mixture) gave the urea 15 (72 mg, 24%
yield) as a white solid, mp 199–200 °C. IR (ATR) ν: 678, 806, 1002,
1025, 1088, 1210, 1249, 1289, 1467, 1513, 1564, 1623, 1671, 1862,
1933, 1997, 2107, 2198, 2357, 2413, 2903 cm−1
.
1H NMR (400 MHz,
CDCl3) δ: 1.55–1.84 (complex signal, 17 diamantane-H), 3.87 (m, 1H, 3
diamantane-H), 5.57 (broad s, 1H, 1-NH), 6.90 (m, 1H, 5′-H), 7.09
(broad s, 1H, 3-NH), 7.74 (m, 1H, 6′-H). 13C NMR (100.6 MHz, CDCl3)
δ: 26.1 (CH), 30.1 (CH), 32.3 (CH), 32.7 (CH2), 36.3 (CH), 36.6 (CH),
37.2, 37.3, 37.35, 37.4 and 37.5 (1 CH and 4 CH2), 37.7 (CH2) 41.5
4.1.15. 4-((Trans-4-(3-(diamantan-3-yl)ureido)cyclohexyl)oxy)benzoic
acid (21)
4-[(Trans-4-aminocyclohexyl)oxy]benzoic acid hydrochloride55
(196 mg, 0.720 mmol) is dissolved in DMF (5 mL) and diamantane-3-
isocyanate (20) (150 mg, 0.65 mmol) was added followed by Et3N
(145 mg, 1.44 mmol). The reaction mixture was stirred at 50 °C for
3 days. The suspension was filtrated and the solvent was evaporated to
obtain a brown solid (315 mg) which was dissolved in DCM and washed
with 2 N HCl (2 × 20 mL). The organic layer was dried over anh.
Na2SO4, filtered and evaporated. The resulting residue was crystallized
from hot DCM affording the urea 21 (111 mg, 37% yield) as a white
solid, mp 229–230 °C. IR (ATR) ν: 633, 695, 770, 845, 1031, 1052,
1088, 1163, 1243, 1312, 1504, 1568, 1599, 1710, 1956, 2020, 2237,
2346, 2496, 2868, 2904 cm−1. 1H NMR (400 MHz, CD3OD) δ: 1.36 (dq,
J = 3.2 Hz, J′ = 13.2 Hz, 2H, 3′(5′)-Hax], 1.52–1.63 [complex signal,
3H, 2′(6′)-Hax and 1 diamantane-H], 1.67–1.91 (complex signal, 17
diamantane-H), 2.02 [dd, J = 4.4 Hz, J′ = 13.2 Hz, 2H, 3′(5′)-Heq],
2.13 [dd, J = 3.6 Hz, J′ = 13.2 Hz, 2H, 2′(6′)-Heq], 3.58 (m, 1H, 4-H′),
3.73 (t, J = 2.8 Hz, 1H, 3″-H), 4.39 (m, 1H, 1′-H), 6.95 [d, J = 8.8 Hz,
2H, 3(5)-H], 7.94 [d, J = 8.8 Hz, 2H, 2(6)-H]. 13C NMR (100.6 MHz,
CD3OD) δ: 27.7 (CH), 31.1 [CH2, C2′(6′)], 31.7 [CH2, C3′(5′)], 31.8
(CH), 33.4 (CH), 33.6 (CH2), 38.0 (CH), 38.2, 38.6, 38.74, 38.77, 38.93,
38.95, 39.0, 43.3 (CH), 48.9 (CH, C4′), 55.2 (CH, C3″), 76.0 (CH, C1′),
111.4 (C, C1), 116.1 [CH, C3(5)], 132.8 [CH, C2(6)], 159.9, (C, CO),
163.0 (C, C4), 170.3 (CO2H). Elemental analysis: Calcd for
2
3
(CH, C4), 55.2 (CH, C3), 111.5 (CH, dd, JC-F = 18 Hz, JC-F = 4 Hz,
2
3
C5′), 115.1 (CH, C6′), 125.0 (C, dd, JC-F = 8 Hz, JC-F = 3 Hz, C1′),
1
2
1
141.1 (C, dt, JC-F = 247, JC-F = 16, C3′), 146.5 (C, dm, JC-
1
F = 246 Hz, C4′), 146.9 (C, dm, JC-F = 248 Hz, C2′), 154.6 (C, CO).
Elemental analysis: Calcd for C21H23F3N2O·0.25 CH2Cl2: C 64.18, H
5.96, N 7.04. Found: C 64.40, H 6.35, N 6.71.
4.1.12. Tricyclo[3.3.1.03,7]nonyl-3-isocyanate (18)
Tricyclo[3.3.1.03,7]nonyl-3-amine hydrochloride (7·HCl) (750 mg,
4.33 mmol) was suspended in DCM (52 mL) and aq. NaHCO3 (22 mL)
was added. Under argon atmosphere, the mixture was stirred and
cooled to 4 °C on an ice bath. Immediately, trisphosgene (642 mg,
2.16 mmol) was added. The mixture was stirred at 4 °C for 30 min. The
2 phases were separated and the organic layer was washed with brine
(2 × 30 mL). The organic phase was dried over anh. Na2SO4 and fil-
tered. Evaporation in vacuo of the organics gave 18 as a yellowish oil
(360 mg, 51% yield) which was used in the next step without further
purification.
4.1.13. 4-((Trans-4-(3-(tricyclo[3.3.1.03,7]non-3-yl)ureido)cyclohexyl)
oxy)benzoic acid (19)
C
28H36N2O4·0.1 CH2Cl2: C 71.34, H 7.71, N 5.92. Found: C 71.36, H
Under argon atmosphere, tricyclo[3.3.1.03,7]nonyl-3-isocyanate
(18) (250 mg, 1.53 mmol) was dissolved in anh. DCM (16 mL). 4-
[(trans-4-aminocyclohexyl)oxy]benzoic acid55 hydrochloride (497 mg,
1.83 mmol) and Et3N (619 mg, 6.12 mmol) were added. The reaction
mixture was stirred at 30 °C overnight. Water (60 mL) was added to the
resulting mixture and two phases were separated. The aqueous phase
was washed with DCM (2 × 50 mL) and acidified until pH = 2 with 5 N
HCl. This acid solution was extracted with DCM (5 × 30 mL) and the
organic layer was dried over anh. Na2SO4, filtered and evaporated. The
residue was dissolved in EtOAc, washed with 2 N HCl, dried over anh.
Na2SO4, filtered and evaporated. The organics were evaporated to af-
ford a yellowish oil (70 mg, 12% yield). Urea 19 was obtained by
crystallization from hot MeOH as white solid, mp 262–263 °C. IR (ATR)
ν: 3377, 3334, 2926, 2862, 1683, 1628, 1605, 1556, 1508, 1456, 1421,
1382, 1326, 1304, 1248, 1163, 1129, 1119, 1096, 1008, 953, 902, 847,
7.85, N 5.70.
4.1.16. N-(1-Acetylpiperidin-4-yl)-1H-imidazole-1-carboxamide (23)
N,N′-carbonyldiimidazole (400 mg, 2.46 mmol) was suspended in
anh. 1,2-dichloroethane (15 mL) under nitrogen. Then 1-acetyl-4-ami-
nopiperidine (22) (250 mg, 1.76 mmol) was added and the reaction
mixture was heated to 50 °C for 21 h. With an external ice bath, the
mixture was cooled down for 30 min. The resulting solid was collected
by filtration in vacuo and washed with 1,2-DCE (20 mL) affording 23
(312 mg, 75% yield) as a white solid, mp 191–193 °C. IR (ATR) ν: 3216,
3118, 3038, 2918, 2342, 2074, 1709, 1613, 1542, 1479, 1463, 1441,
1369, 1358, 1320, 1281, 1272, 1233, 1195, 1137, 1111, 1090, 1068,
1053, 1001, 984, 974, 916, 902, 859, 799, 748, 652 cm−1
.
1H NMR
(400 MHz, CDCl3) δ: 1.37 (complex signal, 2H, 3-Hax, 5-Hax), 1.99 (dm,
J = 12.8 Hz, J′ = 4 Hz, 1H) and 2.21 (dm, J = 12.8 Hz, J′ = 4 Hz, 1H)
(3′-Heq and 5′-Heq), 2.09 (s, 3H, COCH3), 2.69 (ddd, J = 13 Hz,
J′ = 2.6 Hz, 1H) and 3.21 (ddd, J = 13 Hz, J′ = 2.6 Hz, 1H) (2′-Hax and
775, 698, 636, 599 cm−1
.
1H NMR (400 MHz, CD3OD) δ: 1.36 (dq,
J = 3.2 Hz, J′ = 13.2 Hz, 2H, 3′(5′)-Hax], 1.51–1.66 [complex signal,
6

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
6′-Hax), 3.86 (dm, J = 13.6 Hz, 1H) and 4.67 (dm, J = 13.6 Hz, 1H) (2′-
Heq and 6′-Heq), 4.10 (m, 1H, 4′-H), 7.06 (dd, J = 1.6 Hz, J′ = 0.8 Hz,
1H, 4-H), 7.29 (broad d, J = 7.6 Hz, 1H, NH), 7.60 (dd, J = 1.6 Hz,
J′ = 1.2 Hz, 1H, 5-H), 8.29 (dd, J = 1.2 Hz, J′ = 0.8 Hz, 1H, 2-H). 13C
NMR (100.6 MHz, CDCl3) δ: 21.5 (CH3, COCH3), 31.4 and 33.1 (CH2,
C3′ and C5′), 40.9 and 45.6 (CH2, C2′ and C6′), 48.2 (CH, C4′), 116.2
(CH, C5), 130.3 (CH, C4), 136.2 (CH, C2), 148.5 (C, NHCNH), 169.2 (C,
COCH3). MS (DIP), m/z (%); significant ions: 169 (10), 168 (100), 153
(19), 126 (53), 125 (31), 85 (19), 84 (42), 83 (20), 82 (23), 81 (21), 68
(98), 57 (40), 56 (56), 55 (16). HRMS-ESI+ m/z [M+H]+ calcd for
[C11H16N4O2+H]+: 237.1346, found: 237.1345.
[broad singlet, 2H, 1′(5′)-H], 2.34 (t, J = 6.8 Hz, 1H, 7′-H), 2.74 (dt,
J = 11.2 Hz , J′ = 2.4 Hz, 1H, 2-Hax or 6-Hax), 3.13 (dt, J = 12 Hz,
J′ = 2.4 Hz, 1H, 6-Hax or 2-Hax), 3.71–3.85 (complex signal, 2H, 2-Heq
or 6-Heq and 4-H), 4.43 (d, J = 13.6 Hz, 1H, 6-Heq or 2-Heq), 4.89 (d,
J = 8.0 Hz, 1H, NH), 5.11 (s, 1H, NH). 13C NMR (100.6 MHz, CDCl3) δ:
21.4 (CH3, COCH3), 32.5 (CH2, C4 or C5), 33.7 (CH2, C5 or C4), 34.8
(CH2, C9′), 37.3 [CH, C1′(5′)], 40.7 (CH2, C2 or C6), 43.4 [CH2,
C6′(8′)], 43.7 (CH, C7′), 45.4 (CH2, C6 or C2), 46.7 (CH, C4), 49.30 and
49.32 (CH2, C2′ and C4′), 64.1 (C, C3′), 157.1 (CO, urea), 169.0 (CO,
COCH3). Elemental analysis: Calcd for C17H27N3O2·0.15C5H12: C 67.42,
H 9.18, N 13.29. Found: C 66.38, H 9.00, N 13.05.
4.1.17. 1-(1-Acetylpiperidin-4-yl)-3-(3,7-dimethyl(tricyclo[3.3.0.03,7
]
4.1.19. 1-(1-Acetylpiperidin-4-yl)-3-(diamant-3-yl)urea (26)
octa-1-yl)urea (24)
Diamantane-3-isocyanate (20) (155 mg, 0.67 mmol) was dissolved
in DCM (3 mL) and 1-acetyl-4-aminopiperidine (115 mg, 0.811 mmol)
dissolved in DCM (2 mL) was added. The mixture was stirred at room
temperature overnight. Evaporation of the solvent gave a white solid
(272 mg). Column chromatography (Dichloromethane/Methanol mix-
tures) gave the urea 26 (160 mg, 65% yield) as a white solid, mp
230–231 °C. IR (ATR) ν: 669, 727, 770, 808, 862, 917, 989, 1047, 1136,
1240, 1319, 1364, 1453, 1560, 1629, 1794, 1855, 1893, 1944, 1977,
2051, 2102, 2153, 2209, 2270, 2352, 2418, 2545, 2596, 2734, 2877,
In a round bottom flask equipped with a condenser apparatus and
magnetic stirrer a solution of 3,7-dimethyltricyclo[3.3.0.03,7]octan-1-
amine hydrochloride (5·HCl) (68 mg, 0.36 mmol) in chloroform (5 mL)
was prepared, to which was added N-(1-acetylpiperidin-4-yl)-1H-imi-
dazole-1-carboxamide (23) (172 mg, 0.73 mmol) followed by triethy-
lamine (0.06 mL, 0.40 mmol). The solution was heated to 50 °C for 25 h,
whereupon the reaction mixture was tempered to room temperature
and evaporated in vacuo to dryness (384 mg). Purification by column
chromatography (SiO2, Dichloromethane/Methanol mixture) afforded
24 (90 mg, 77% yield) as a white solid, mp 165–167 °C. IR (ATR) ν:
3359, 3244, 2947, 2878, 2170, 2034, 1960, 1613, 1556, 1477, 1443,
3020, 3071, 3275, 3316, 3494, 3566, 3688 cm−1
.
1H NMR (400 MHz,
CD3OD) δ: 1.32 [complex signal, 2H, 3(5)-Hax], 1.66–1.82 (complex
signal, 16H, diamantane-H), 1.85–1.98 (complex signal, 4H, 3-Heq, 5-
Heq, 2 diamantane-H), 2.10 (s, 3H, COCH3), 2.91 (dt, J = 11.2 Hz,
J′ = 2.8 Hz, 1H, 2-Hax or 6-Hax), 3.25 (dt, J = 11.2 Hz, J′ = 3.2 Hz, 2H,
6-Hax or 2-Hax), 3.71–3.78 (complex signal, 2H, 4-H and 1′-H), 3.85 (dt,
J = 14 Hz, J′ = 2.4 Hz, 2H, 6-Heq or 2-Heq), 4.29 (dt, J = 13 Hz,
J′ = 2.8 Hz, 2H, 2-Heq or 6-Heq). 13C NMR (100.6 MHz, CD3OD) δ: 21.2
(CH3, COCH3), 27.7 (CH), 31.8 (CH), 33.3 (CH2, C3 or C5), 33.4 (CH2),
33.6 (CH2), 34.1 (CH2, C5 or C3), 38.0, 38.1, 38.6, 38.7 (2 carbon),
38.91, 38.94 and 39.0 (3 CH2 and 5 CH, diamantane signals), 41.6
(CH2, C2 or C6), 43.3 (CH, C4′), 46.3 (CH2, C6 or C2), 47.7 (CH, C4),
55.9 (CH, C3′), 159.7 (C, CO urea), 171.5 (C, COCH3). HRMS-ESI+ m/z
[M+H]+ calcd for [C22H33N3O2+H]+: 372.2646, found: 372.2644.
1371, 1318, 1264, 1227, 1151, 1096, 1033, 978, 717, 639 cm−1
.
1H
NMR (400 MHz, CDCl3) δ: 1.11 [s, 6H, 3′(7′)-CH3], 1.22 [complex
signal, 2H, 2(6)-Ha], 1.34 [dd, J = 8.2 Hz, J′ = 3.4 Hz, 2H, 4′(6′)-Ha],
1.54 [dd, J = 7.4 Hz, J′ = 3.4 Hz, 2H, 2′(8′)-Ha], 1.70 [dd, J = 8.2 Hz,
J′ = 2.6 Hz, 2H, 4′(6′)-Hb], 1.75 [d, J = 7.6 Hz, 2H, 2′(8′)-Hb], 1.90 and
2.03 (complex signal, 2H, 3-Hax and 5-Hax), 2.07 (s, 3H, COCH3), 2.27
(t, J = 2.6 Hz, 1H, 5′-H), 2.75 (dt, J = 14.0 Hz, J′ = 2.8 Hz, 1H, 2-Hax
or 6-Hax), 3.14 (dt, J = 11.2 Hz, J′ = 2.8 Hz, 2H, 6-Hax or 2-Hax), 3.73
(broad d, J = 13 Hz, 2H, 6-Heq or 2-Heq), 3.83 (m, 1H, 4-H), 4.42 (broad
d, J = 13 Hz, 2H, 2-Heq or 6-Heq), 4.79 (d, J = 7.6 Hz, 1H, 1-NH), 5.18
(broad s, 1H, 3-NH). 13C NMR (100.6 MHz, CDCl3) δ: 16.5 [CH3,
C3′(7′)-CH3], 21.4 (CH3, COCH3), 33.6 [CH2, C3(5)], 40.6 (CH2, C2),
44.7 (CH, C5′), 45.3 (CH2, C6), 46.1 [C, C3′(7′)], 46.9 (CH, C4), 53.2
[CH2, C4′(6′)], 57.6 [CH2, C2′(8′)], 61.7 (C, C1′), 157.4 (C, CO urea),
169.0 (C, COCH3). MS (DIP), m/z (%); significant ions: 278 (10), 277
(58), 263 (20), 178 (10), 169 (25), 151 (18), 150 (22), 148 (25), 143
(100), 136 (26), 135 (43), 134 (31), 127 (16), 126 (23), 125 (17), 123
(11), 122 (86), 121 (29), 119 (25), 110 (22), 109 (86), 108 (48), 96
(18), 95 (62), 94 (29), 93 (16), 91 (15), 84 (31), 83 (16), 82 (33), 80
(11), 79 (12), 77 (11), 67 (11), 57 (13), 56 (25), 55 (17). Elemental
analysis: Calculated for C18H29N3O2: C 67.68, H 9.15, N 13.15.
Calculated for C18H29N3O2·1.0 H2O: C 64.07, H 9.26, N 12.45. Found: C
64.00, H 9.31, N 12.40.
4.1.20. Diamantane-4-isocyanate (27)
Diamantane-4-amine hydrochloride (9·HCl) (110 mg, 0.458 mmol)
was suspended in DCM (2 mL) and aq. NaHCO3 was added, followed by
triphosgene (68 mg, 0.23 mmol). The biphasic mixture was stirred at
room temperature for 30 min. The two phases were separated and the
organic layer was washed with brine. The organics were dried over anh.
Na2SO4, filtered and evaporated until 1 mL. The solution of isocyanate
(27) in DCM was used in the next step without further purification.
4.1.21. 1-(1-Acetylpiperidin-4-yl)-3-(diamant-4-yl)urea (28)
To the solution of diamantane-4-isocyanate (27) (105 mg,
0.46 mmol) in DCM (1 mL) from the previous step is added 1-acetyl-4-
aminopiperidine hydrochloride (22·HCl) (98 mg, 0.55 mmol) and DCM
(1 mL), followed by Et3N. The mixture was stirred at room temperature
overnight. DCM was added to the mixture and it was washed with 2 N
HCl (30 mL). The organics were dried over anh. Na2SO4, filtered and
evaporated to obtain a residue (48 mg). Column chromatography
(Dichloromethane/Methanol mixture) gave the desired urea (28)
(33 mg, 21% overall yield) as a beige solid, mp 195–196 °C. IR (ATR): ν:
3364, 2906, 2881, 2847, 1686, 1601, 1550, 1480, 1462, 1444, 1429,
1374, 1348, 1322, 1305, 1267, 1221, 1138, 1105, 1048, 1002, 986,
4.1.18. 1-(1-Acetylpiperidin-4-yl)-3-(tricyclo[3.3.1.03,7]non-3-yl)urea
(25)
Under argon atmosphere, tricyclo[3.3.1.03,7]nonane-3-isocyanate
(18) (360 mg, 2.20 mmol) was dissolved in anh. DCM (10 mL). 1-acetyl-
4-aminopiperidine (375 mg, 2.64 mmol) and Et3N (445 mg, 4.40 mmol)
were added. The reaction mixture was stirred at room temperature
overnight. The solvent was removed in vacuo and the residue was dis-
solved in EtOAc and washed with 2 N HCl. The organics were dried over
anh. Na2SO4, filtered and evaporated in vacuo affording a white yel-
lowish solid which was washed with acetone and EtOAc affording the
urea 25 as a yellowish solid (240 mg, 36% yield), mp 164–165 °C. IR
(ATR) ν: 638, 705, 783, 860, 904, 974, 992, 1059, 1139, 1230, 1269,
1318, 1361, 1429, 1555, 1620, 1659, 2351, 2919, 3328 cm−1. 1H NMR
(400 MHz, CDCl3) δ: 1.16–1.29 (complex signal, 2H, 4-Hax and 5-Hax),
1.47–1.61 [complex signal, 4H, 9′-H2 and 6′(8′)-Hax], 1.80 [dd,
J = 10.0 Hz, J′ = 2.8 Hz, 2H, 2′(4′)-Hax], 1.84–2.01 [complex signal,
6H, 6′(8′)-Heq, 2′(4′)-Heq, 4-Heq and 5-Heq], 2.07 (s, 3H, 8-H), 2.23
976, 918, 613, 597, 576 cm−1
.
1H NMR (400 MHz, CDCl3) δ: 1.19
(complex signal, 2H, 3-Hax and 5-Hax), 1.69–1.78 (complex signal, 10H,
diamantane-H), 1.83–1.94 (complex signal, 11H, 3-Heq, 5-Heq, 9 dia-
mantane-H), 2.08 (s, 3H, COCH3), 2.73 (dt, J = 11.6 Hz, J′ = 3.2 Hz, 1
H, 2-Hax or 6-Hax), 3.13 (dt, J = 11.6 Hz, J′ = 3.2 Hz, 2H, 6-Hax or 2-
H
ax), 3.71–3.85 (complex signal, 2H, 4-H and 6-Heq or 2-Heq), 4.36
(broad s, NH, urea), 4.42–4.50 (complex signal, 2H, 2-Heq or 6-Heq and
NH). 13C NMR (100.6 MHz, CDCl3) δ: 21.4 (CH3, COCH3), 25.6 (CH,
7

S. Codony, et al.
Bioorganic&MedicinalChemistry27(2019)115078
C9′), 32.4 (CH2, C3 or C5), 33.6 (CH2, C5 or C3), 36.6 (CH), 37.4 (CH2),
38.7 (CH), 40.7 (CH2, C2 or C6), 43.0 (CH2), 45.4 (CH2, C6 or C2), 46.9
(CH, C4), 49.8 (C, C4′), 156.6 (C, CO urea), 169.0 (C, COCH3). HRMS-
ESI+ m/z [M+H]+ calcd for [C22H33N3O2+H]+: 372.2646, found:
372.2657.
Acknowledgments
This work was funded by the Spanish Ministerio de Economía,
Industria y Competitividad (Grant SAF2017-82771-R to S.V.), the
European Regional Development Fund (ERDF), the Xunta de Galicia
(GRC2014/011 and ED431C2018-21) and the Generalitat de Catalunya
(2017 SGR 106). S.C., E.V. and R.L. acknowledge PhD fellowships from
the Universitat de Barcelona (APIF grant), the Institute of Biomedicine
of the University of Barcelona (IBUB), and the Spanish Ministerio de
Educacion, Cultura y Deporte (FPU grant), respectively. This work was
supported in part by the NIEHS Grant R01 ES002710 (to B.D.H.) and
NIEHS Superfund Research Program P42 ES004699.
4.2. Solubility
A 10 mM stock solution of the compound was serially diluted in
100% DMSO and 1 µL of this solution was added to a 384-well UV-
transparent plate (Greiner) containing 99 µL of PBS. The plate was in-
cubated at 37 °C for 2 h and the light scattering was measured in a
Nephelostar Plus reader (BMG LABTECH). The data was fitted to a
segmented linear regression for measuring the compound solubility.
Appendix A. Supplementary data
Supplementary data (1H and 13C NMR spectra of the compounds) to
this article can be found online at https://doi.org/10.1016/j.bmc.2019.
115078.
4.3. Microsomal stability
The human microsomes employed were purchased from Tebu-
Xenotech. The compound was incubated at 37 °C with the microsomes
in a 50 mM phosphate buffer (pH = 7.4) containing 3 mM MgCl2, 1 mM
NADP, 10 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate-
dehydrogenase. Samples (75 µL) were taken from each well at 0, 10, 20,
40 and 60 min and transferred to a plate containing 4 °C 75 µL acet-
onitrile and 30 µL of 0.5% formic acid in water were added for im-
proving the chromatographic conditions. The plate was centrifuged
(46,000g, 30 min) and supernatants were taken and analyzed in a
UPLC-MS/MS (Xevo-TQD, Waters) by employing a BEH C18 column
and an isocratic gradient of 0.1% formic acid in water: 0.1% formic acid
acetonitrile (60:40). The metabolic stability of the compounds was
calculated from the logarithm of the remaining compounds at each of
the time points studied.
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