Diphenyl ether derivatives occupy the expanded binding site of cyclohexanedione compounds at the colchicine site in tubulin by movement of the αT5 loop

Article abstract of DOI:10.1016/j.ejmech.2019.03.045

Microtubule targeting agents represent a very active arena in the development of anticancer agents. In particular, compounds binding at the colchicine site in tubulin are being deeply studied, and the structural information recently available on this binding site allows structure-directed design of new ligands. Structural comparison of our recently reported high resolution X-Ray structure of the cyclohexanedione derivative TUB075 bound to tubulin and the tubulin-DAMA-colchicine complex has revealed a conformational change in the αT5 loop. By a grid-based computational analysis of the tubulin-DAMA-colchicine binding site, we have identified a new favourable binding area in the colchicine-site that was unexplored by our lead TUB075. Thus, based on a structure-guided design, new cyclohexanedione derivatives have been synthesized and tested for tubulin binding and in cellular assays. As a result, we have identified diphenyl ether derivatives with IC₅₀ values around 10–40 nM against three different tumor cell lines and affinity constants for tubulin similar to that of colchicine around 10⁷ M^?1. As expected, they halted the cell cycle progression at G2/M phase at concentrations as low as 0.08 μM.

Full text of DOI:10.1016/j.ejmech.2019.03.045

European Journal of Medicinal Chemistry 171 (2019) 195e208
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Diphenyl ether derivatives occupy the expanded binding site of
cyclohexanedione compounds at the colchicine site in tubulin by
movement of the
aT5 loop
a
a
b
c
ꢀ
Oskia Bueno , Marta Gargantilla , Juan Estevez-Gallego , Solange Martins ,
b
a
c
a
ꢀ
ꢀ
ꢀ
J. Fernando Díaz , María-Jose Camarasa , Sandra Liekens , María-Jesús Perez-Perez ,
Eva-María Priego a
,
*
a
ꢀ
Instituto de Química Medica (IQM, CSIC), Juan de la Cierva 3, 28006, Madrid, Spain
b
ꢀ
Centro de Investigaciones Biologicas (CIB,CSIC), Ramiro de Maeztu 9, 28040, Madrid, Spain
c Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Microtubule targeting agents represent a very active arena in the development of anticancer agents. In
particular, compounds binding at the colchicine site in tubulin are being deeply studied, and the
structural information recently available on this binding site allows structure-directed design of new
ligands. Structural comparison of our recently reported high resolution X-Ray structure of the cyclo-
hexanedione derivative TUB075 bound to tubulin and the tubulin-DAMA-colchicine complex has
Received 19 December 2018
Received in revised form
28 February 2019
Accepted 18 March 2019
Available online 21 March 2019
revealed a conformational change in the aT5 loop. By a grid-based computational analysis of the tubulin-
DAMA-colchicine binding site, we have identified a new favourable binding area in the colchicine-site
that was unexplored by our lead TUB075. Thus, based on a structure-guided design, new cyclo-
hexanedione derivatives have been synthesized and tested for tubulin binding and in cellular assays. As a
result, we have identified diphenyl ether derivatives with IC50 values around 10e40 nM against three
Keywords:
Colchicine domain
Structure-based design
Affinity maps
different tumor cell lines and affinity constants for tubulin similar to that of colchicine around 107 Mꢀ1
.
Diphenyl ethers
As expected, they halted the cell cycle progression at G2/M phase at concentrations as low as 0.08 mM.
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
MTAs can be divided as microtubule-stabilizing agents [5], i.e.
placlitaxel (1) from the taxoid family, epothilone A (2) and lauli-
malilde (3) or as destabilizing agents like the vinca alkaloids such as
vinblastine (4), colchicine (5) or combretastatins (6, 7), among
others (Fig. 1) [3].
Microtubules are highly dynamic polymers composed of ab
-
tubulin heterodimers. These key components of the cytoskeleton of
eukaryotic cells are involved in pivotal biological functions such as
intracellular migration and transport, cell signalling, cell shape
maintenance, cell motility and mitosis [1]. Their crucial role in cell
division has made tubulin and microtubules important targets for
several conditions, especially for cancer, and therefore antitubulin
compounds represent the most successful class of antimitotic
agents in cancer chemotherapy [2]. To date, several chemical clas-
ses of microtubule targeting agents (MTAs) are known, both from
natural sources or synthetic derivatives [3,4].
At the molecular level, MTAs bind to ab-tubulin heterodimers at
six different binding sites that are referred to as the taxane,
colchicine, vinca, maytansine, laulimalide/peloruside and pironetin
sites, all of which have been structurally characterized by high
resolution X-ray crystallography and cryo-electron microscopy [6].
As recently reviewed [7], the mechanism of action of MTAs goes
far beyond than acting as antimitotic agents. Since microtubule
structure and function are crucial for cell motility and shape,
compounds affecting microtubules dynamics may lead to anti-
metastatic and antiangiogenic effects [8,9]. Tubulin destabilization
has also been linked to vascular disruption so that morphological
changes in the vascular endothelial cells increase vascular perme-
ability leading to blood flow occlusion. This is particularly relevant
At the cellular level, and according to their mechanism of action,
ꢀ
* Corresponding author. Instituto de Química Medica (CSIC), c/ Juan de la Cierva
3, E-28006, Madrid, Spain. Tel.: þ34915680040; fax: þ34915644853.
E-mail address: empriego@iqm.csic.es (E.-M. Priego).
https://doi.org/10.1016/j.ejmech.2019.03.045
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
Fig. 1. Chemical structures of selected microtubule-stabilizing (A) and microtubule-destabilizing (B) agents.
for tumor vessels because of their significant differences with
physiological vessels (i.e increased vascular permeability) [10].
Thus, treatment with vascular disrupting agents (VDAs) has been
proposed as an interesting antitumor approach leading to vascular
shutdown and tumor necrosis, although the outer rim of the tumor
remains unaffected, which support the use of VDAs combined with
other anticancer treatments [11]. Among VDAs, destabilizing
tubulin-colchicine-site binders have been extensively studied
[12,13].
accomplished.
2. Results and discussion
2.1. Affinity maps of the DAMA-colchicine-tubulin complex for the
design of new ligands
To identify new favourable binding areas unexplored by TUB075
at the colchicine domain, the corresponding affinity maps on the
tubulin-DAMA-colchicine complex (PDB ID: 1sa0) [14] were
generated by means of the computational tool cGRILL and the
lipophilic probe (CH3þ) [17,18]. As shown in Fig. 3A, three well-
defined affinity areas encircle DAMA-colchicine: area 1, located
The colchicine site is a hydrophobic pocket located near the
intra-dimer interface between the
a and b tubulin subunits [14].
Based on the structural information available, two distinct binding
modes of colchicine-site ligands have been characterized [15], with
compounds that bind deeper in the
compounds that bind closer to the interface between
b
subunit such as TN-16 (8), and
and
inside the b-subunit; area 2, covering the lower part of the trime-
a
b
thoxyphenyl ring of DAMA-colchicine (ring A); and area 3, where
the tropone ring of DAMA-colchicine (ring C) is located. When
TUB075 was superimposed onto this affinity maps, it became clear
that areas 1 and 2 were already occupied by rings A, B and D of
TUB75, whereas area 3 was scarcely explored by TUB075 although
the ethoxy group seemed to point towards it. Given the shape and
the lipophilic character of this unexplored area, we reasoned that,
keeping the oxygen atom as bridge, the incorporation of an addi-
tional aromatic ring (named ring E) linked to ring D, could fulfil this
affinity region, thus resulting in new diphenyl ether derivatives
(Fig. 3B).
subunits of a tubulin dimer, the prototype being DAMA-colchicine
(9) (Fig. 2A). We have recently reported the high resolution X-Ray
structure of the cyclohexanedione derivative TUB075 (10, Fig. 2A)
bound to ab-tubulin, compound that binds mostly at the b-subunit,
similar to TN-16 [16]. Superposition of the crystal structures of
tubulin-DAMA-colchicine and tubulin-TUB075 complexes revealed
a significant conformational change of the a-T5 loop (Fig. 2B). This
loop closed the TUB075 binding site in the tubulin-TUB075 com-
plex while it was flipped out in the DAMA-colchicine structure.
Based on this loop movement, it could be argued that DAMA-
colchicine makes uses of binding/affinity areas of the binding
domain that are not accessible for TUB075. Therefore, we have
dissected the binding mode of DAMA-colchicine with different
probes, using the computational tool cGRILL [17]. By unravelling
new potential affinity areas with cGRILL, structure-guided design of
TUB075 based derivatives has been carried out leading to the
synthesis of new cyclohexanedione derivatives with a distal
diphenyl ether moiety. In addition, the tubulin binding and the
antiproliferative evaluation of the new compounds have been
2.2. Chemistry
The synthesis of the required 2-phenoxyanilines (14a-j) was
carried out in a two-step process involving the nucleophilic aro-
matic substitution (SNAr) of 1-fluoro-2-nitrobenzene (11) with
differently functionalized phenols at positions 3 and/or 4, followed
by reduction of the nitro derivatives (Scheme 1). Thus, 2-
nitrodiphenyl ethers (12a-e) were prepared via
a modified

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
197
Fig. 2. (A) Chemical structures of TN-16, DAMA-colchicine and TUB075. (B) Movement of the
a-T5 loop. Superposition of tubulin-DAMA-colchicine complex (
a
-tubulin in magenta,
b-tubulin in blue marine and DAMA-colchicine in orange) with TUB075-tubulin complex (
a
-tubulin in light pink, b-tubulin in cyan and TUB075 in green). (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. (A) Affinity maps generated with the lipophilic probe CH3þ (green grid) onto tubulin-DAMA-colchicine (cyan sticks) complex and its superposition with TUB075 (magenta
sticks). (B) General structure of the new diphenyl ether derivatives. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article.)
described procedure [19]. Reaction of 1-fluoro-2-nitrobenzene (11)
with 3-fluoro (12a), 4-chloro (12b), 3-methoxy (12c), 4-methoxy
(12d) and 3,4-dimethoxy (12e) phenols in the presence of Cs2CO3
in acetonitrile at 80 ꢁC afforded the corresponding nitro derivatives
(13a-e) in excellent yields (88e99%). Under the same conditions,
the NH-Boc protected phenols (12f-h) [20e22] and 4-
hydroxyacetophenone (12i) reacted with 11 to yield the corre-
sponding nitro derivatives 13f-i. Finally, catalytic hydrogenation of
the halogenated compounds 13a-b in the presence of Pt/S or in the
presence of Pd/C for the rest of the compounds (13c-i), yielded the
corresponding 2-phenoxyanilines 14a-i. Aniline 14i was obtained
in 42% yield together with compound 14j by partial reduction of the
carbonyl group.
As shown in Scheme 2, reaction of 2-acetyl-5-
phenylcyclohexane-1,3-dione (15) [23] with the synthesized ani-
lines 14a-j in toluene at 110 ꢁC in the presence of 4 Å molecular
sieves [24] led to the final compounds 16a-j in moderate to good
yields. For comparative purposes, the unsubstituted derivative at

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O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
Scheme 1. Reagents and conditions: (a) Cs2CO3, CH3CN, 80 ꢁC, 2e5 h, 88e99% yields; (b) for 13a,b: H2, Pt/S 5%, AcOEt, 30 psi, rt, 2 h, 74e84% yields; (c) for 13e-i: H2, Pd/C 10%,
AcOEt, 30 psi, rt, 1e4 h, 42e99% yields.
Scheme 2. Reagents and conditions: (a) Toluene, molecular sieves 4 Å, pressure tube, 110 ꢁC, 16 h, 28e99% yields; (b) TFA, CH2Cl2, rt, 20 min-1 h, 88e99% yields.
ring E (16k) was also synthesized from the commercially available
2-phenoxyaniline (14k). Finally, treatment of N-Boc derivatives 16f-
h with TFA yielded the free amino cyclohexanediones 16l-n.
potent antiproliferative compounds with IC50 in the sub-
micromolar range in all the cells lines tested. Thus, compound
16k with an unsubstituted phenyl ring provides IC50 values similar
or slightly better than those of the reference compound TUB075.
Incorporation of halogens at this phenyl ring, either a fluor at po-
sition 3 (16a) or a chlorine at position 4 (16b) did not improve the
antiproliferative activity. On the other hand, introduction of a
methoxy group at position 3, 4 or both (derivatives 16c, 16d and
16e, respectively) led to an improvement in the antiproliferative
2.3. Antiproliferative activity
The synthesized diphenyl ether derivatives were evaluated for
their anti-proliferative activity in three different cancer cell lines
[mouse leukemia (L1210), human lymphoblastic leukemia (CEM)
and human cervical carcinoma (HeLa) cells] and one endothelial
cell line [human microvascular endothelial cells (HMEC-1)]. Data
are collected in Table 1 and expressed as IC50 (50% inhibitory con-
centration) representing the concentration at which the com-
pounds reduce cell proliferation by 50%. Colchicine (5) and CA-4P
(7) are included as reference compounds while data from our
previous hit TUB075 (10) is included for comparative purposes [16].
The data collected in Table 1 revealed that the introduction of an
extra aromatic ring (ring E) in the structure of TUB075 led to highly
activities with IC50 values ranging from 0.04 to 0.16 mM, being the 3-
OMe derivative 16c the most potent among them. Interestingly,
compounds with amino groups in positions 3 or 4 of ring E, (16l and
16m, respectively) also provided very interesting antiproliferative
activities, with IC50 values ranging between 0.037 and 0.082 mM.
Finally, the double substituted compound 16n, with a methoxy
group at 3 and an amino group at 4, provided excellent anti-
proliferative activity against all the cell lines tested with IC50 values
between 0.012 and 0.033 mM.

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
199
Table 1
Anti-proliferative activity of the diphenylether derivatives (16a-n) in endothelial and tumor cell lines.
Tumor cells IC50
L1210
(m
M)a
Endothelial cells IC50
HMEC-1
(m
M)a
Comp.
CEM
HeLa
TUB075 (10)
0.19 0.05
0.19 0.01
0.18 0.0
0.10 0.02
16a
16b
16c
16d
16e
16i
0.33 0.10
0.25 0.01
0.058 0.014
0.14 0.08
0.065 0.003
0.19 0.02
0.17 0.06
0.19 0.02
0.041 0.007
0.12 0.02
0.16 0.00
0.55 0.30
0.50 0.40
0.093 0.022
0.16 0.03
0.054 0.014
0.13 0.01
0.36 0.036
0.26 0.030
0.059 0.001
0.083 0.004
0.14 0.03
0.22 0.01
0.18 0.01
16j
16k
16l
16m
16n
0.058 0.01
0.086 0,003
0.053 0.006
0.040 0.002
0.026 0.007
0.010 0.0006
0.013 0.0023
0.12 0.00
0.17 0.01
0.075 0.012
0.079 0.031
0.082 0.063
0.039 0.000
0.033 0.000
0.0087 0.0001
0.013 0.001
0.14 0.04
0.16 0.04
0.042 0.001
0.037 0.005
0.012 0.008
0.013 0.0004
0.011 0.001
0.041 0.008
0.047 0.019
0.029 0.003
0.0038 0.0011
0.0029 0.0001
Colchicine (5)
CA-4P (7)
a
IC50 (50% inhibitory concentration) is given as the mean SD of three independent experiments.
2.4. Tubulin binding
this series, were selected to further investigate their antimitotic
behaviour due to cell cycle arrest. Thus, we performed cell cycle
analysis on HMEC-1 cells at different concentrations (0.016, 0.08
In order to corroborate whether the antiproliferative activity of
these diarylethers was derived from their interaction with tubulin,
the effect on dimeric tubulin polymerization into microtubules was
tested in a turbidimetric assay including podophylotoxin as control.
As shown in Fig. 4, at the tested concentration all compounds
displayed a destabilizing effect of tubulin polymerization similar to
that of podophylotoxin.
In addition, binding affinities for all final compounds to tubulin
were determined by a competition experiment with R-PT as pre-
viously described [16,25]. The data obtained are collected in Table 2.
All the compounds showed a remarkable affinity for tubulin, with
Kb values ranging from 106 to 107 Mꢀ1. The best Kb values corre-
and 0.4
showed the typical distribution pattern of proliferating cells while
treatment with 0.08 or 0.4 M of 16m (Fig. 5A) or 16n (Fig. 6B)
mM) for 24 h. As shown in Fig. 5, control (untreated) cells
m
caused an accumulation in G2/M phase, indicating antimitotic ac-
tivity. Cell cycle analysis has also been performed with compounds
16m and 16n in HeLa cells, using colchicine as reference compound,
with similar results (Fig. S1).
2.6. Vascular disrupting activity
spond
to
compounds
16c
(Kb ¼ 15.65 ꢂ 106),
16m
(Kb ¼ 11.75 ꢂ 106 T) and 16n (Kb ¼ 18.05 x 106), and are similar to
that of colchicine (Kb ¼ 11.6 ꢂ 106 Mꢀ1). Interestingly, these com-
pounds were also among the ones that provided better anti-
proliferative activity in cell culture.
It is well established that compounds binding at the colchicine
site in tubulin are able to destroy the vasculature network formed
by endothelial cells. HMEC-1 cells were seeded on top of matrigel
and after 3 h a network of endothelial cells is visible (control). Then
derivatives 16m or 16n were added at different concentrations (2,
2.5. Cell cycle experiments
0.4 and 0.08
compounds led to vascular disrupting activity in a dose-dependent
manner, being visible still at 0.4 M.
mM). As can be seen in Fig. 6 the addition of the
Compounds 16m and 16n, two of the most active derivatives in
m
Fig. 4. Time course of tubulin polymerization at 37 ꢁC measured by 350 nm turbidimetry. Tubulin concentration: 25
mM, compounds 16c-n 30 mM.

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O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
Table 2
structure. Applying the computational tool cGRILL to the tubulin-
Binding constants for ab-tubulin.
DAMA-colchicine complex, and using CH3 probe, a new potential
binding area barely explored by TUB075 at the colchicine site was
identified. Thus, a series of diphenyl ether derivatives has been
synthesized and tested. The introduction of an extra aromatic ring
in the structure of TUB075 has led to highly potent antiproliferative
compounds, being compound 16n, with a methoxy group at 3 and
an amino group at 4, the most active among them, with IC50 values
Comp
Kb (Mꢀ1
)
Colchicine (5)
Podophyllotoxin
R-PT
TUB075 (10)
16c
16d
16e
16j
16k
11.6 ꢂ 106 (a) [26]
1.8 ꢂ 106 (a) [27]
3.2 ꢂ 106 [25]
(13 2) x 106 (a)
(15.65 5.55) x 106 (a)
(5.23 0.24) x 106 (a)
(2.24 0.33) x 106 (a)
(3.13 0.11) x 106 (a)
(6.46 0.91) x 106(a)
(5.56 1.25) x 106 (a)
(11.75 2.95) x 106 (a)
(18.05 8.05) x 106 (a)
between 0.012 and 0.033
m
M, and a tubulin binding constant value
of 18.05
x
106 Mꢀ1
, similar to that of colchicine
(Kb ¼ 11.6 ꢂ 106 Mꢀ1). Moreover, additional biological studies have
showed that compounds 16n and 16m held antimitotic activity by
an accumulation of cells in G2/M phase and a dose-dependent
vascular disrupting activity in the low micromolar range.
16l
16m
16n
bMean value of three experiments StdError
a
At 25 ꢁC.
4. Experimental
2.7. Docking studies
4.1. Chemistry procedures
To gain a better understanding on the potency and the binding
mode of the most potent compound 16n to tubulin, a docking study
was performed using the coordinates of the TUB075-tubulin com-
plex (pdb id: 6KFJ) as template. The best docking pose of 16n into
the colchicine domain in tubulin (Fig. 7A) showed that regions A, B,
C and D of 16n (showed in cyan sticks in Fig. 7A) occupies the same
pocket as TUB075 (represented in nude sticks) sharing also the
same orientation, which allows 16n to keep the crucial hydrogen-
bond interaction with Glu200, as reported for TUB075. Regarding
Melting points were obtained on a Reichert-Jung Kofler appa-
ratus and are uncorrected. The elemental analysis was performed
with a Heraeus CHN-O-RAPID instrument. The elemental compo-
sitions of the compounds agreed to within 0.4% of the calculated
values.
Electrospray mass spectra were measured on a quadrupole mass
spectrometer equipped with an electrospray source (Hewlett-
Packard, LC/MS HP 1100).
1H and 13C NMR spectra were recorded on a Varian INNOVA 300
operating at 299 MHz (1H) and 75 MHz (13C), respectively, a Varian
the new ring E, it is directed towards the interface between
subunits and the amino group present in this ring E is at hydrogen
bond distance (2.5 Å) to the backbone carbonyl of Thr 179, which is
located in the T5-loop. Moreover, superposition of this predicted
a and b
INNOVA-400 operating at 399 MHZ (1H) and 99 MHz
(
13C),
a
respectively, and a VARIAN SYSTEM-500 operating a 499 MHz (1H)
and 125 MHz (13C), respectively. Monodimensional 1H and 13C
spectra were obtained using standard conditions. 2D inverse proton
detected heteronuclear one-bond shift correlation spectra were
obtained using the Pulsed Field Gradient HSQC pulse sequence.
Data were collected in a 2048 ꢂ 512 matrix with a spectral width of
3460 Hz in the proton domain and 22,500 Hz in the carbon domain,
and processed in a 2048 ꢂ 1024 matrix. The experiment was opti-
mized for one bond heteronuclear coupling constant of 150 Hz. 2D
inverse proton detected heteronuclear long range shift correlation
spectra were obtained using the Pulsed Field Gradient HMBC pulse
sequence. The HMBC experiment was acquired in the same con-
ditions that HSQC experiment and optimized for long range
coupling constants of 7 Hz.
a
binding mode of 16n and the tubulin-DAMA-colchicine complex
(Fig. 7B) showed that ring E is overlapping with the tropone ring of
DAMA-colchicine, thus confirming our rational design of these new
derivatives.
3. Conclusions
Taken advantage of the high resolution X-ray structure of the
cyclohexanedione derivative TUB075 bound to ab-tubulin,
a
detailed structural comparison with the tubulin-DAMA-colchicine
complex has revealed a significant conformational change of the
a
T5 loop, that closed the TUB075 binding site in the tubulin-
TUB075 complex while it was flipped out in the DAMA-colchicine
Analytical TLC was performed on silica gel 60 F254 (Merck)
Fig. 5. Flow cytometric analysis of cell cycle distribution of HMEC-1 cells treated for 24 h with different concentrations of 16m (a), 16n (b).

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
201
Fig. 6. (A) Vascular disrupting effects of 16m and 16n. (B) After 90 min the number of meshes, branching points and length of the tubes were quantified using Image J analysis.
Fig. 7. (A) Predicted binding mode of 16n (cyan) in the colchicine domain of ab-tubulin (a-subunit in blue and b subunit in magenta) and overlap with TUB075 (nude). Dashed lines
indicate hydrogen bonds and selected interaction residues are shown in sticks and labelled. (B) Superposition of 16n (cyan) and DAMA-colchicine (white). (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
precoated plates (0.2 mm). Spots were detected under UV light
(254 nm) and/or charring with ninhydrin or phosphomolibdic acid.
Separations on silica gel were performed by preparative cen-
trifugal circular thin-layer chromatography (CCTLC) on a Chroma-
totronR (Kiesegel 60 PF254 gipshaltig (Merck)), with layer thickness
of 1 and 2 mm and flow rate of 4 or 8 mL/min, respectively. Flash
column chromatography was performed in a Biotage Horizon
instrument.
Microwave reactions were performed using the Biotage Initiator
2.0 single-mode cavity instrument from Biotage (Uppsala). Exper-
iments were carried out in sealed microwave process vials utilizing
the standard absorbance level (400 W maximum power). The
temperature was measured with an IR sensor on the outside of the
reaction vessel.
4.1.1. General procedure [19] for the synthesis of 2-nitrodiphenyl
ethers (general procedure A)
To a mixture of Cs2CO3 (1.0 mmol) and the substituted phenol
(1.2 mmol) in anhydrous acetonitrile (2.5 mL/mmol), 1-fluoro-2-
nitrobenzene (11) (1.0 mmol) was added dropwise while stirring
under argon. After 2e5 h at 80 ꢁC, water (20 mL) and sat. aq.
NaHCO3 solution (20 mL) was added and the mixture was extracted
with ethyl acetate. The organic layer was dried over Na2SO4,
concentrated, and purified by flash chromatography (hexane/ethyl
acetate).

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O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
4.1.1.1. 1-(4-Fluorophenoxy)-2-nitrobenzene (13a). Following the
4.1.1.6. tert-Butyl
(3-(2-nitrophenoxy)phenyl)carbamate
(13f).
general procedure A, to a mixture of Cs2CO3 (482 mg, 1.48 mmol)
and 3-fluorophenol (12a) (162 mL, 1.70 mmol) in CH3CN (3.6 mL), 1-
Following the general procedure A, to a mixture of Cs2CO3 (694 mg,
2.13 mmol) and tert-butyl (3-hydroxyphenyl)carbamate (12f) [20]
(534 mg, 2.55 mmol) in CH3CN (5.3 mL), 1-fluoro-2-nitrobenzene
fluoro-2-nitrobenzene (11) (156 mL, 1.48 mmol) was added. After
4 h at 80 ꢁC, the residue was worked up and purified by flash
(11) (224 mL, 2.13 mmol) was added dropwise while stirring under
chromatography (hexane/ethyl acetate, 10:1) to yield 310 mg (90%)
argon. After 4 h at 80 ꢁC, the residue was worked up and purified by
flash chromatography (hexane/ethyl acetate, 4:1) to yield 650 mg
of 13a as a yellow oil. 1H NMR (DMSO‑d6, 300 MHz):
d 6.88 (dd,
J ¼ 8.3, 2.4 Hz, 1H, Ar), 7.00 (m, 1H, Ar), 7.06 (dd, J ¼ 8.5, 2.3 Hz, 1H,
Ar), 7.27 (dd, J ¼ 8.3, 1.2 Hz, 1H, Ar), 7.37e7.53 (m, 2H, Ar), 7.74 (m,
1H, Ar), 8.10 (dd, J ¼ 8.2, 1.7 Hz, 1H, Ar). MS (ES, positive mode): 234
(M þ H)þ. 1H NMR data are similar to those previously described
[28].
(92%) of 13f as a yellow oil. 1H NMR (DMSO‑d6, 400 MHz):
d 1.44 (s,
9H, (CH3)3), 6.64 (ddd, J ¼ 7.8, 2.3, 1.4 Hz, 1H, Ar), 7.15 (dd, J ¼ 8.4,
1.2 Hz, 1H, Ar), 7.22e7.31 (m, 3H, Ar), 7.36 (ddd, J ¼ 8.3, 7.5, 1.2 Hz,
1H, Ar), 7.69 (ddd, J ¼ 8.2, 7.4, 1.7 Hz, Ar), 8.06 (dd, J ¼ 8.2, 1.7 Hz, 1H,
Ar), 9.51 (br s, 1H, NH). MS (ES, positive mode): 331 (M þ H)þ, 661
(2 M þ H)þ.
4.1.1.2. 1-(4-Chlorophenoxy)-2-nitrobenzene (13b). Following the
general procedure A, to a mixture of Cs2CO3 (463 mg, 1.42 mmol)
and 4-chlorophenol (12b) (167 mL, 1.70 mmol) in CH3CN (3.6 mL), 1-
4.1.1.7. tert-Butyl
(4-(2-nitrophenoxy)phenyl)carbamate
(13g).
Following the general procedure A, to a mixture of Cs2CO3 (436 mg,
1.34 mmol) and tert-butyl(4-hydroxyphenyl)carbamate (12g) [21]
(337 mg, 1.61 mmol) in CH3CN (3.5 mL), 1-fluoro-2-nitrobenzene
fluoro-2-nitrobenzene (11) (149 mL, 1.42 mmol) was added. After
2 h at 80 ꢁC, the residue was worked up and purified by flash
(11) (141 mL, 1.34 mmol) was added dropwise while stirring under
chromatography (hexane/ethyl acetate 10:1) to yield 864 mg (99%)
argon. After 3 h at 80 ꢁC, the residue was worked up and purified by
flash chromatography (hexane/ethyl acetate, 4:1) to yield 512 mg
of 13b as a yellow oil. 1H NMR (DMSO‑d6, 300 MHz):
d 7.00e7.14 (m,
2H, Ar), 7.22 (dd, J ¼ 8.4, 1.2 Hz, 1H, Ar), 7.41 (m, 1H, Ar), 7.45e7.52
(m, 2H, Ar), 7.72 (m, 1H, Ar), 8.09 (dd, J ¼ 8.1, 1.7 Hz, 1H, Ar). 1H NMR
data are similar to those previously described [28].
(96%) of 13g as yellow oil. 1H NMR (DMSO‑d6, 400 MHz):
d 1.47 (s,
9H, (CH3)3), 6.99e7.05 (m, 3H, Ar), 7.29 (ddd, J ¼ 8.3, 7.4, 1.2 Hz, 1H,
Ar), 7.45e7.53 (m, 2H, Ar), 7.64 (ddd, J ¼ 8.5, 7.4, 1.7 Hz, 1H, Ar), 8.02
(dd, J ¼ 8.1, 1.7 Hz, 1H, Ar), 9.44 (br s, 1H, NH). MS (ES, positive
mode): 353 (M þ Na)þ, 661 (2 M þ H)þ.
4.1.1.3. 1-(3-Methoxyphenoxy)-2-nitrobenzene (13c). Following the
general procedure A, to a mixture of Cs2CO3 (925 mg, 2.84 mmol)
and 3-methoxyphenol (12c) (373 mL, 3.40 mmol) in CH3CN (7.1 mL),
4.1.1.8. tert-Butyl (2-methoxy-4-(2-nitrophenoxy)phenyl)carbamate
(13h). Following the general procedure A, to a mixture of Cs2CO3
(227 mg, 0.70 mmol) and tert-butyl(4-hydroxy-2-methoxyphenyl)
carbamate (12h) [22] (200 mg, 0.84 mmol) in CH3CN (1.8 mL), 1-
1-fluoro-2-nitrobenzene (11) (299 mL, 2.84 mmol) was added. After
3 h at 80 ꢁC, the residue was worked up and purified by flash
chromatography (hexane/ethyl acetate, 5:1) to yield 615 mg (88%)
of 13c as a yellow oil. 1H NMR (DMSO‑d6, 400 MHz):
d 3.75 (s, 3H,
fluoro-2-nitrobenzene (11) (74 mL, 0.70 mmol) was added drop-
CH3), 6.59 (ddd, J ¼ 8.2, 2.3, 0.8 Hz, 1H, Ar), 6.67 (t, J ¼ 2.4 Hz, 1H,
Ar), 6.79 (ddd, J ¼ 8.4, 2.4, 0.8 Hz, 1H, Ar), 7.16 (dd, J ¼ 8.4, 1.2 Hz, 1H,
Ar), 7.32 (t, J ¼ 8.2 Hz, 1H, Ar), 7.37 (ddd, J ¼ 8.4, 7.4, 1.2 Hz, 1H, Ar),
7.69 (ddd, J ¼ 8.3, 7.4, 1.7 Hz, 1H, Ar), 8.06 (dd, J ¼ 8.1, 1.7 Hz, 1H, Ar).
MS (ES, positive mode): 246 (M þ H)þ.1H NMR data are similar to
those previously described [28].
wise while stirring under argon. After 4 h at 80 ꢁC, the residue was
worked up and purified by flash chromatography (hexane/ethyl
acetate, 4:1) to yield 212 mg (96%) of 13h as a yellow oil. 1H NMR
(DMSO‑d6, 400 MHz):
d 1.45 (s, 9H, (CH3)3), 3.78 (s, 3H, OCH3), 6.58
(dd, J ¼ 8.7, 2.6 Hz, 1H, Ar), 6.87 (d, J ¼ 2.6 Hz, 1H, Ar), 7.08 (dd,
J ¼ 8.4, 1.2 Hz, 1H, Ar), 7.31 (ddd, J ¼ 8.4, 7.4, 1.2 Hz, 1H, Ar),
7.60e7.69 (m, 2H, Ar), 8.02 (br s, 1H, NH), 8.04 (dd, J ¼ 8.2, 1.7 Hz,
1H, Ar). MS (ES, positive mode): 361 (M þ H)þ, 383 (M þ Na)þ, 721
(2 M þ H)þ.
4.1.1.4. 1-(4-Methoxyphenoxy)-2-nitrobenzene (13d). Following the
general procedure A, to a mixture of Cs2CO3 (1.15 g, 3.54 mmol) and
4-methoxyphenol (12d) (528 mg, 4.25 mmol) in CH3CN (9 mL), 1-
fluoro-2-nitrobenzene (11) (374 mL, 3.54 mmol) was added drop-
4.1.1.9. 1-(4-(2-Nitrophenoxy)acetophenone (13i). Following the
general procedure A, to a mixture of Cs2CO3 (925 mg, 2.84 mmol)
and 4-hydroxyacetophenone (12i) (464 mg, 3.40 mmol) in CH3CN
wise while stirring under argon. After 3 h at 80 ꢁC, the residue was
worked up and purified by flash chromatography (hexane/ethyl
acetate 10:1) to yield 864 mg (99%) of 13d as a yellow solid. Mp
(7.1 mL), 1-fluoro-2-nitrobenzene (11) (299 mL, 2.84 mmol) was
76e78 ꢁC (lit [29] 75e76.5 ꢁC); 1H NMR (DMSO‑d6, 300 MHz):
d 3.76
added. After 5 h at 80 ꢁC, the residue was worked up and purified by
flash chromatography (dichloromethane/methanol, 20:1) to yield
613 mg (84%) of 13i as a white solid. Mp 104e106 ꢁC (lit [30]
(s, 3H, CH3), 6.96e7.03 (m, 3H, Ar), 7.04e7.12 (m, 2H, Ar), 7.28 (m,
1H, Ar), 7.63 (m, 1H, Ar), 8.02 (dd, J ¼ 8.1, 1.7 Hz, 1H, Ar). MS (ES,
positive mode): 246 (M þ H)þ. 1H NMR data are similar to those
previously described [19].
104.6 ꢁC). 1H NMR (DMSO‑d6, 400 MHz):
d 2.56 (s, 3H, CH3),
7.07e7.15 (m, 2H, Ar), 7.37 (dd, J ¼ 8.3, 1.2 Hz, 1H, Ar), 7.50 (m, 1H,
Ar), 7.79 (m, 1H, Ar), 7.98e8.03 (m, 2H, Ar), 8.14 (dd, J ¼ 8.2, 1.6 Hz,
1H, Ar). MS (ES, positive mode): 258 (M þ H)þ, 515 (2 M þ H)þ 1H
NMR data are similar to those previously described [30].
4.1.1.5. 1,2-Dimethoxy-4-(2-nitrophenoxy)benzene
(13e).
Following the general procedure A, to a mixture of Cs2CO3 (925 mg,
2.84 mmol) and 3,4-dimethoxyphenol (12e) (525 mg, 3.40 mmol)
in CH3CN (7.1 mL), 1-fluoro-2-nitrobenzene (11) (299
m
L,
4.1.2. General procedure for the synthesis of 2-aminodiphenyl
ethers (general procedure B)
2.84 mmol) was added. After 5 h at 80 ꢁC, the residue was worked
up and purified by flash chromatography (hexane/ethyl acetate,
10:1) to yield 767 mg (97%) of 13e as a yellow oil. 1H NMR
The corresponding 2-nitrodiphenyl ether was dissolved in ethyl
acetate (12 mL) in a pressure vessel and then 10% Pd/C or 5% Pt/S
(catalytic amount) was added. The vessel was hydrogenated at 30
psi for 1e5 h at room temperature. Then, the reaction mixture was
filtered and volatiles were removed under reduced pressure. In
most cases, the obtained 2-aminodiphenyl ethers were directly
used as such in the subsequent reaction without further
purification.
(DMSO‑d6, 400 MHz): d 3.74 (s, 3H, CH3), 3.76 (s, 3H, CH3), 6.61 (dd,
J ¼ 8.7, 2.8 Hz, 1H, Ar), 6.85 (d, J ¼ 2.8 Hz, 1H, Ar), 6.97e7.02 (m, 2H,
Ar), 7.27 (ddd, J ¼ 8.2, 7.4, 1.2 Hz, 1H, Ar), 7.63 (ddd, J ¼ 8.5, 7.4,
1.7 Hz, 1H, Ar), 8.02 (dd, J ¼ 8.1, 1.7 Hz, 1H, Ar). MS (ES, positive
mode): 276 (M þ H)þ. 1H NMR data are similar to those previously
described [19].

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
203
4.1.2.1. 2-(3-Fluorophenoxy)aniline (14a). Following the general
procedure B, a solution of 13a (431 mg, 1.85 mmol) in ethyl acetate
(12 mL) in the presence of 5% Pt/S (catalytic amount) was subjected
to hydrogenation for 2 h at rt. Then, the reaction mixture was
filtered and the solvent was evaporated. The residue was purified
by flash chromatography (hexane/ethyl acetate, 5:1) to afford
316 mg (84%) of 14a as a colorless oil. 1H NMR (DMSO‑d6, 300 MHz):
evaporated to afford 490 mg (99%) of 14f as a pink oil. 1H NMR
(DMSO‑d6, 400 MHz): 1.44 (s, 9H, (CH3)3), 4.85 (br s, 2H, NH2), 6.47
(ddd, J ¼ 8.1, 2.4, 1.1 Hz, 1H, Ar), 6.54 (ddd, J ¼ 7.9, 7.2, 1.6 Hz, 1H, Ar),
6.75 (dd, J ¼ 8.0, 1.4 Hz, 1H, Ar), 6.79 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar), 6.90
(ddd, J ¼ 7.9, 7.2, 1.5 Hz, 1H, Ar), 7.08e7.19 (m, 3H, Ar), 9.37 (br s, 1H,
CONH). MS (ES, positive mode): 301 (M þ H)þ, 601 (2 M þ H)þ.
d
d
4.95 (br s, 2H, NH2), 6.52e6.60 (m, 1H, Ar), 6.62e6.72 (m, 2H, Ar),
4.1.2.7. tert-Butyl (4-(2-aminophenoxy)phenyl)carbamate (14g).
Following the general procedure B, a solution of 13g (275 mg,
0.83 mmol) in ethyl acetate (12 mL) in the presence of 10% Pd/C
(catalytic amount) was subjected to hydrogenation for 1 h at rt.
Then, the reaction mixture was filtered and the solvent was evap-
orated to afford 229 mg (92%) of 14f as a pink oil. 1H NMR
6.78e6.90 (m, 3H, Ar), 6.95 (m, 1H, Ar), 7.27e7.39 (m, 1H, Ar). MS
(ES, positive mode): 246 (M þ ACN þ H)þ. 1H NMR data are similar
to those previously described [28].
4.1.2.2. 2-(4-Chlorophenoxy)aniline (14b). Following the general
procedure B, a solution of 13b (63 mg, 0.252 mmol) in ethyl acetate
(12 mL) in the presence of 5% Pt/S (catalytic amount) was subjected
to hydrogenation for 2 h at rt. Then, the reaction mixture was
filtered and the solvent was evaporated. The residue was purified
by flash chromatography (hexane/ethyl acetate, 10:3) to afford
41 mg (74%) of 14b as a colorless oil. 1H NMR (DMSO‑d6, 300 MHz):
(DMSO‑d6, 400 MHz):
d 1.46 (s, 9H, (CH3)3), 4.86 (br s, 2H, NH2),
6.50 (m, 1H, Ar), 6.68 (dd, J ¼ 8.1, 1.4 Hz, 1H, Ar), 6.77 (dd, J ¼ 7.9,
1.7 Hz, 1H, Ar), 6.80e6.90 (m, 3H, Ar), 7.38 (d, J ¼ 8.5 Hz, 2H), 9.24
(br s, 1H, NH). MS (ES, positive mode): 301 (M þ H)þ, 601
(2 M þ H)þ.
d
5.76 (br s, 2H, NH2), 6.76e6.92 (m, 2H, Ar), 6.98e7.18 (m, 4H, Ar),
4.1.2.8. tert-Butyl
(4-(2-aminophenoxy)-2-methoxyphenyl)carba-
7.35e7.48 (m, 2H, Ar). MS (ES, positive mode): 220 (M þ H)þ with Cl
isotopic distribution. 1H NMR data are similar to those previously
described [19].
mate (14h). Following the general procedure B, a solution of 13h
(190 mg, 0.53 mmol) in ethyl acetate (12 mL) in the presence of 10%
Pd/C (catalytic amount) was subjected to hydrogenation for 4 h at
rt. Then, the reaction mixture was filtered and the solvent was
evaporated to afford 150 mg (86%) of 14h as a pink oil. 1H NMR
4.1.2.3. 2-(3-Methoxyphenoxy)aniline (14c). Following the general
procedure B, a solution of 13c (400 mg, 1.63 mmol) in ethyl acetate
(12 mL) in the presence of 10% Pd/C (catalytic amount) was sub-
jected to hydrogenation for 2 h at rt. Then, the reaction mixture was
filtered and the solvent was evaporated to afford 346 mg (98%) of
(DMSO‑d6, 400 MHz):
d 1.43 (s, 9H, (CH3)3), 3.75 (s, 3H, OCH3), 4.87
(br s, 2H, NH2), 6.33 (dd, J ¼ 8.7, 2.6 Hz,1H, Ar), 6.53 (m,1H, Ar), 6.69
(d, J ¼ 2.6 Hz, 1H, Ar), 6.75 (dd, J ¼ 8.0, 1.4 Hz, 1H, Ar), 6.79 (dd,
J ¼ 7.9, 1.6 Hz, 1H, Ar), 6.89 (m, 1H, Ar), 7.43 (d, J ¼ 8.7 Hz, 1H, Ar),
7.88 (br s, 1H, CONH). MS (ES, positive mode): 331 (M þ H)þ, 661
(2 M þ H)þ.
14c as an orange oil. 1H NMR (DMSO‑d6, 400 MHz):
d 3.71 (s, 3H,
CH3), 4.88 (br s, 2H, NH2), 6.42 (ddd, J ¼ 8.1, 2.3, 0.9 Hz, 1H, Ar), 6.47
(t, J ¼ 2.4 Hz, 1H, Ar), 6.55 (ddd, J ¼ 7.9, 7.2, 1.6 Hz, 1H, Ar), 6.62 (ddd,
J ¼ 8.3, 2.4, 0.9 Hz,1H, Ar), 6.80 (dd, J ¼ 7.9,1.6 Hz, 2H, Ar), 6.92 (ddd,
J ¼ 8.0, 7.3, 1.5 Hz, 1H, Ar), 7.20 (t, J ¼ 8.2 Hz, 1H, Ar). MS (ES, positive
mode): 216 (M þ H)þ, 257 (M þ ACN þ H)þ. 1H NMR data are similar
to those previously described [19].
4.1.2.9. 1-(4-(2-Aminophenoxy)phenyl)ethan-1-one (14i) and 1-(4-
(2-Aminophenoxy)phenyl)ethan-1-ol (14j). Following the general
procedure B, a solution of 13i (250 mg, 0.97 mmol) in ethyl acetate
(12 mL) in the presence of 10% Pd/C (catalytic amount) was sub-
jected to hydrogenation for 1 h at rt. Then, the reaction mixture was
filtered and the solvent was evaporated. The residue was purified
by flash chromatography (hexane/ethyl acetate 2:1). The fastest
moving fractions afforded 114 mg (52%) of 14i as a colorless oil. 1H
4.1.2.4. 2-(4-Methoxyphenoxy)aniline (14d). Following the general
procedure B, a solution of 13d (648 mg, 2.64 mmol) in ethyl acetate
(12 mL) in the presence of 10% Pd/C (catalytic amount) was sub-
jected to hydrogenation for 2 h at rt. Then, the reaction mixture was
filtered and the solvent was evaporated to afford 560 mg (98%) of
NMR (DMSO‑d6, 300 MHz):
d 2.50 (s, 1H, CH3), 4.96 (br s, 2H, NH2),
6.58 (m, 1H, Ar), 6.79e7.01 (m, 5H, Ar), 7.92 (d, J ¼ 8.4 Hz, 2H, Ar).
14d as a pink oil. 1H NMR (DMSO‑d6, 300 MHz):
d 3.71 (s, 1H, CH3),
MS (ES, positive mode): 228 (M þ H)þ.
4.88 (br s, 2H, NH2), 6.50 (m, 1H, Ar), 6.66 (dd, J ¼ 8.0,1.4 Hz,1H, Ar),
6.76 (dd, J ¼ 7.9, 1.7 Hz, 1H, Ar), 6.81e6.93 (m, 5H, Ar). MS (ES,
positive mode): 216 (M þ H)þ, 257 (M þ ACN þ H)þ. 1H NMR data
are similar to those previously described [31].
The slowest moving fractions afforded 93 mg (42%) of 14j as a
colorless oil. 1H NMR (DMSO‑d6, 300 MHz):
d
1.29 (d, J ¼ 6.4 Hz, 3H,
CH3), 4.67 (m, 1H, CH), 4.87 (br s, 1H, NH), 5.08 (d, J ¼ 4.1 Hz, 1H,
OH), 6.53 (m, 1H, Ar), 6.72e6.85 (m, 4H, Ar), 6.90 (m, 1H, Ar), 7.27
(d, J ¼ 7.7 Hz, 2H, Ar). MS (ES, positive mode): 230 (M þ H)þ.
4.1.2.5. 2-(3,4-Dimethoxyphenoxy)aniline (14e). Following the
general procedure B, a solution of 13e (400 mg, 1.45 mmol) in ethyl
acetate (12 mL) in the presence of 10% Pd/C (catalytic amount) was
subjected to hydrogenation for 2 h at rt. Then, the reaction mixture
was filtered and the solvent was evaporated to afford 343 mg (96%)
of 14e as a white solid. Mp 115e117 ꢁC. 1H NMR (DMSO‑d6,
4.1.3. General procedure for the reaction of 2-
acetylcylcohexanedione with anilines (general procedure C)
A solution of 2-acetylcylcohexanedione (15) (1.0 mmol), the
appropriate aniline (1.5 mmol) and 4 Å molecular sieves in toluene
(10 mL) was placed in an Ace pressure tube [23]. Then, the vessel
was sealed and heated in a microwave reactor at 110 ꢁC overnight.
After cooling, the solvent was evaporated and the residue was
purified by flash chromatography or by CCTLC in the Chromatotron.
400 MHz):
d 3.70 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 4.86 (br s, 2H,
NH2), 6.34 (m, 1H, Ar), 6.51 (m, 1H, Ar), 6.67e6.72 (m, 2H, Ar), 6.77
(m, 1H, Ar), 6.83e6.89 (m, 2H, Ar). MS (ES, positive mode): 246
(M þ H)þ, 287 (M þ ACN þ H)þ. 1H NMR data are similar to those
previously described [32].
4.1.3.1. 2-(1-((2-(3-Fluorophenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16a). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (189 mg,
0.82 mmol), 14a (250 mg, 1.23 mmol) and 4 Å molecular sieves in
toluene (5 mL). The resulting residue was purified by flash chro-
matography (hexane/ethyl acetate, 5:1) to afford 340 mg (99%) of
4.1.2.6. tert-Butyl (3-(2-aminophenoxy)phenyl)carbamate (14f).
Following the general procedure B, a solution of 13f (530 mg,
1.60 mmol) in ethyl acetate (12 mL) in the presence of 10% Pd/C
(catalytic amount) was subjected to hydrogenation for 2 h at rt.
Then, the reaction mixture was filtered and the solvent was
16a as a brown oil. 1H NMR (DMSO‑d6, 400 MHz):
d 2.44 (s, 3H,

204
O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
CH3), 2.50e2.61 (m, 2H, H-4, H-6), 2.68e2.88 (m, 2H, H-4, H-6),
3.27 (tt, J ¼ 12.0, 4.1 Hz, 1H, H-5), 6.80 (dd, J ¼ 8.3, 2.3 Hz, 1H, Ar),
6.90 (m, 1H, Ar), 7.00 (td, J ¼ 8.4, 2.5 Hz, 1H, Ar), 7.16 (dd, J ¼ 8.2,
1.3 Hz, 1H, Ar), 7.22 (m, 1H, Ar), 7.28e7.35 (m, 5H, Ar), 7.37e7.47 (m,
2H, Ar), 7.52 (dd, J ¼ 7.9, 1.6 Hz,1H, Ar),14.84 (br s, 1H, NH). 13C NMR
855 (2 M þ H)þ. Anal. calc. for (C27H25NO4): C, 75.86; H, 5.89; N,
3.28. Found: C, 75.71; H, 5.98; N, 3.42.
4.1.3.5. 2-(1-((2-(3,4-Dimethoxyphenoxy)phenyl)amino)ethylidene)-
5-phenylcyclohexane-1,3-dione (16e). Following the general pro-
cedure C, an Ace pressure tube was charged with 15 (48 mg,
0.21 mmol), 14e (79 mg, 0.32 mmol) and 4 Å molecular sieves in
toluene (0.8 mL). The resulting residue was purified by CCTLC
(hexane/ethyl acetate, 2:1) to afford 73 mg (76%) of 16e as a white
(DMSO‑d6, 100 MHz):
d 19.8 (CH3), 36.0 (C-5), 45.2 (C-4, C-6), 105.8
(d, J ¼ 24.8 Hz, Ar), 108.6 (NHC]C), 110.6 (d, J ¼ 21.1 Hz, Ar), 113.9
(d, J ¼ 3.0 Hz, Ar), 120.3, 125.00, 126.5, 126.7, 127.7, 128.2, 128.5,
129.6 (Ar), 131.4 (d, J ¼ 9.9 Hz, Ar), 143.4, 149.9, 157.5 (d, J ¼ 11.0 Hz,
Ar), 162.8 (d, J ¼ 245.0 Hz, Ar), 172.5 (NHC]C). MS (ES, positive
mode): 416 (M þ H)þ. Anal. calc. for (C26H22FNO3): C, 75.17; H, 5.34;
N, 3.37. Found: C, 74.85; H, 5.46; N, 3.37.
solid. Mp 65e67 ꢁC 1H NMR (DMSO‑d6, 400 MHz):
d 2.48 (s, 3H,
CH3), 2.53e2.62 (m, 2H, H-4, H-6), 2.71e2.88 (m, 2H, H-4, H-6),
3.29 (m, 1H, H-5), 3.72 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 6.52 (dd,
J ¼ 8.7, 2.8 Hz, 1H, Ar), 6.76 (d, J ¼ 2.8 Hz, 1H, Ar), 6.90e6.96 (m, 2H,
Ar), 7.15e7.28 (m, 2H, Ar), 7.28e7.40 (m, 5H, Ar), 7.45 (dd, J ¼ 7.9,
1.6 Hz, 1H, Ar), 14.89 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
4.1.3.2. 2-(1-((2-(4-Chlorophenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16b). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (53 mg,
0.23 mmol), 14b (34 mg, 0.15 mmol) and 4 Å molecular sieves in
toluene (0.7 mL). The resulting residue was purified by CCTLC
(hexane/ethyl acetate,10:3) to afford 50 mg (75%) of 16b as a brown
d
20.0 (CH3), 36.1 (C-5), 45.2 (C-4, C-6), 55.7 (OCH3), 55.9 (OCH3),
104.7 (Ar), 108.5 (NHC]C), 110.4, 112.5, 117.8, 123.2, 126.3, 126.5,
126.7, 127.8, 128.5,129.3, 143.5, 145.7, 149.00, 149.8, 152.0 (Ar), 172.7
(NHC]C). MS (ES, positive mode): 458 (M þ H)þ, 915 (2 M þ H)þ.
Anal. calc. for (C28H27NO5): C, 73.51; H, 5.95; N, 3.06. Found: C,
73.32; H, 6.00; N, 3.24.
oil. 1H NMR (DMSO‑d6, 300 MHz):
d 2.45 (s, 3H, CH3), 2.54e2.62 (m,
2H, H-4, H-6), 2.68e2.88 (m, 2H, H-4, H-6), 3.27 (m, 1H, H-5),
6.98e7.04 (m, 2H, Ar), 7.11 (dd, J ¼ 8.2, 1.4 Hz, 1H, Ar), 7.22 (m, 1H,
Ar), 7.28e7.34 (m, 5H, Ar), 7.39e7.46 (m, 3H, Ar), 7.51 (dd, J ¼ 7.8,
1.7 Hz, 1H, Ar), 14.84 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
4.1.3.6. tert-Butyl
(3-(2-((1-(2,6-dioxo-4-phenylcyclohexylidene)
ethyl)amino)phenoxy)phenyl)carbamate (16f). Following the gen-
eral procedure C, an Ace pressure tube was charged with 15
(110 mg, 0.48 mmol), 14f (215 mg, 0.72 mmol) and 4 Å molecular
sieves in toluene (2 mL). The resulting residue was purified by
CCTLC (hexane/ethyl acetate, 2:1) to afford 153 mg (71%) of 16f as a
d
20.5 (CH3), 36.7 (C-5), 45.6 (C-4, C-6), 109.3 (NHC]C), 120.6,
120.60, 125.0, 127.2, 127.4, 128.2, 128.3, 128.8, 129.2, 130.2, 130.6,
144.1, 150.9,155.7 (Ar), 173.1 (NHC]C). MS (ES, positive mode): 432
(M þ H)þ with Cl isotopic distribution pattern. Anal. calc. for
(C26H22ClNO3): C, 72.30; H, 5.13; N, 3.24. Found: C, 71.95; H, 5.20; N,
3.38.
white solid. Mp 159e161 ꢁC. 1H NMR (DMSO‑d6, 400 MHz):
d 1.43 (s,
9H, (CH3)3), 2.44 (s, 3H, CH3), 2.52e2.64 (m, 2H, H-4, H-6),
2.68e2.84 (m, 2H, H-4, H-6), 3.27 (tt, J ¼ 12.2, 4.0 Hz, 1H, H-5), 6.54
(m, 1H, Ar), 7.08 (dd, J ¼ 8.2, 1.3 Hz, 1H, Ar), 7.15e7.25 (m, 4H, Ar),
7.28 (m, 1H, Ar), 7.28e7.37 (m, 4H, Ar), 7.41 (m, 1H, Ar), 7.49 (dd,
4.1.3.3. 2-(1-((2-(3-Methoxyphenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16c). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (65 mg,
0.28 mmol), 14c (92 mg, 0.42 mmol) and 4 Å molecular sieves in
toluene (1.1 mL). The resulting residue was purified by CCTLC
(hexane/ethyl acetate, 10:3) to afford 84 mg (70%) of 16c as an oil.
J ¼ 7.8, 1.6 Hz, 1H, Ar), 9.46 (br s, 1H, CONH), 14.85 (br s, 1H, NH). 13
C
NMR (DMSO‑d6, 100 MHz):
d 19.8 (CH3), 28.0 ((CH3)3), 36.0 (C-5),
46.3 (C-4, C-6), 79.3 (C(CH3)3), 108.5 (NHC]C), 107.7 111.5, 113.4,
119.8, 124.3, 126.5, 126.7, 127.5, 128.0, 128.5, 129.4, 130.0, 141.2,
143.5, 150.6, 152.6 (CONH), 156.4 (Ar), 172.5 (NHC]C). MS (ES,
positive mode): 513 (M þ H)þ. Anal. calc. for (C31H32N2O5): C, 72.64;
H, 6.29; N, 5.47. Found: C, 72.52; H, 6.34; N, 5.74.
1H NMR (DMSO‑d6, 400 MHz):
d 2.45 (s, 3H, CH3), 2.53e2.61 (m, 2H,
H-4, H-6), 2.68e2.87 (m, 2H, H-4, H-6), 3.28 (tt, J ¼ 12.1, 4.1 Hz, 1H,
H-5), 3.73 (s, 1H, OCH3), 6.51 (ddd, J ¼ 8.2, 2.4, 0.8 Hz, 1H, Ar), 6.59
(t, J ¼ 2.4 Hz, 1H, Ar), 6.74 (ddd, J ¼ 8.3, 2.5, 0.8 Hz, 1H, Ar), 7.08 (dd,
J ¼ 8.2, 1.3 Hz, 1H, Ar), 7.18e7.24 (m, 1H, Ar), 7.25e7.37 (m, 6H, Ar),
7.41 (td, J ¼ 7.8, 1.7 Hz, 1H, Ar), 7.49 (dd, J ¼ 7.8, 1.6 Hz, 1H, Ar), 14.87
4.1.3.7. tert-Butyl
(4-(2-((1-(2,6-dioxo-4-phenylcyclohexylidene)
ethyl)amino)phenoxy)phenyl)carbamate (16g). Following the gen-
eral procedure C, an Ace pressure tube was charged with 15
(100 mg, 0.43 mmol), 14g (195 mg, 0.65 mmol) and 4 Å molecular
sieves in toluene (1.9 mL). The resulting residue was purified by
CCTLC (hexane/ethyl acetate, 2:1) to afford 192 mg (87%) of 16g as a
(br s, 1H, NH). 13C NMR (DMSO‑d6, 125 MHz):
d 19.3 (CH3), 36.0 (C-
5), 45.1 (C-4, C-6), 55.3 (OCH3), 104.5 (Ar), 108.6 (NHC]C), 109.8,
110.2, 119.6, 124.3, 126.5, 126.7, 127.4, 128.0, 128.5, 129.4, 130.6,
143.4, 150.7, 157.1, 160.7 (Ar), 172.5 (NHC]C). MS (ES, positive
mode): 428 (M þ H)þ. Anal. calc. for (C27H25NO4): C, 75.86; H, 5.89;
N, 3.28. Found: C, 75.55; H, 6.01; N, 3.29.
white solid. Mp 108e110 ꢁC. 1H NMR (DMSO‑d6, 400 MHz):
d 1.46 (s,
9H, (CH3)3), 2.46 (s, 3H, CH3), 2.53e2.56 (m, 2H, H-4, H-6),
2.68e2.87 (m, 2H, H-4, H-6), 3.28 (m, 1H, H-5), 6.91e6.96 (m, 3H,
Ar), 7.18e7.25 (m, 2H, Ar), 7.30e7.34 (m, 4H, Ar), 7.36 (ddd, J ¼ 8.3,
7.5, 1.7 Hz, 1H, Ar), 7.44e7.48 (m, 3H, Ar), 9.38 (br s, 1H, CONH),
4.1.3.4. 2-(1-((2-(4-Methoxyphenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16d). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (100 mg,
0.43 mmol), 14d (139 mg, 0.65 mmol) and 4 Å molecular sieves in
toluene (1.8 mL). The resulting residue was purified by flash chro-
matography (dichloromethane/methanol, 10:0.1) to afford 156 mg
(85%) of 16d as a white solid. Mp 137e139 ꢁC. 1H NMR (DMSO‑d6,
14.88 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
d 19.9 (CH3),
28.1 ((CH3)3), 36.0 (C-5), 45.9 (C-4, C-6), 79.0 (C(CH3)3), 108.6
(NHC]C), 118.2, 119.4, 119.7, 123.4, 126.5, 126.6, 126.7, 127.9, 128.5,
129.3, 136.0, 143.5, 150.0, 151.6 (Ar), 152.8 (CONH), 172.5 (NHC]C).
MS (ES, positive mode): 513 (M þ H)þ. Anal. calc. for (C31H32N2O5):
C, 72.64; H, 6.29; N, 5.47. Found: C, 72.29; H, 6.39; N, 5.65.
400 MHz):
d 2.48 (s, 3H, CH3), 2.52e2.64 (m, 2H, H-4, H-6),
2.69e2.90 (m, 2H, H-4, H-6), 3.25 (m, 1H, H-5), 3.75 (s, 3H, OCH3),
6.88 (dd, J ¼ 8.3, 1.3 Hz, 1H, Ar), 6.94e7.03 (m, 4H, Ar), 7.16e7.27 (m,
2H, Ar), 7.29e7.38 (m, 5H, Ar), 7.45 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar), 14.90
4.1.3.8. tert-Butyl
ethyl)amino)phenoxy)methoxyphenyl)carbamate
Following the general procedure C, an Ace pressure tube was
charged with 15 (65 mg, 0.28 mmol), 14h (62 mg, 0.19 mmol) and
4 Å molecular sieves in toluene (0.8 mL). The resulting residue was
purified by CCTLC (hexane/ethyl acetate, 3:1) to afford 78 mg (76%)
(4-(2-((1-(2,6-dioxo-4-phenylcyclohexylidene)
(16h).
(br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
d
19.9 (CH3), 36.0 (C-
5), 45.6 (C-4, C-6), 55.5 (OCH3), 108.6 (NHC]C), 115.2, 117.6, 120.6,
123.2, 126.3, 126.5, 126.7, 127.8, 128.5, 129.3, 143.5, 148.7, 152.0,
156.0 (Ar), 172.6 (NHC]C). MS (ES, positive mode): 428 (M þ H)þ,

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
205
of 16h as a white solid. Mp 189e191 ꢁC. 1H NMR (DMSO‑d6,
400 MHz): 1.44 (s, 9H, (CH3)3), 2.47 (s, 3H, CH3), 2.53e2.66 (m, 2H,
(HNC]C), 118.4, 119.3, 124.0, 124.1, 126.5, 126.7, 127.3, 128.0, 128.5,
129.4, 130.2, 143.4, 150.8, 156.0 (Ar), 172.5 (HNC]C). MS (ES, posi-
tive mode): 398 (M þ H)þ.Anal. calc. for (C26H30N2O4): C, 78.59; H,
5.83; N, 3.52. Found: C, 78.30; H, 5.95; N, 3.58.
d
H-4, H-6), 2.69e2.91 (m, 2H, H-4, H-6), 3.28 (m, 1H, H-5), 3.76 (s,
3H, OCH3), 6.49 (dd, J ¼ 8.7, 2.6 Hz, 1H, Ar), 6.76 (d, J ¼ 2.6 Hz, 1H,
Ar), 7.00 (dd, J ¼ 8.2, 1.3 Hz, 1H, Ar), 7.20e7.26 (m, 2H, Ar), 7.32 (m,
4H, Ar), 7.38 (m, 1H, Ar), 7.47 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar), 7.57 (d,
J ¼ 8.7 Hz, 1H, Ar), 7.99 (br s, 1H, CONH), 14.89 (br s, 1H, NH). 13C
4.1.3.12. 2-(1-((2-(3-Aminophenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16l). To a solution of 16f (60 mg,
0.12 mmol) in dichloromethane (0.4 mL), TFA (0.4 mL, 4.68 mmol)
was added and the reaction mixture was stirred at room temper-
ature for 30 min. Then volatiles were removed and the residue was
purified by CCTLC (hexane/ethyl acetate, 1:1) to afford 47 mg (97%)
of 16l as a white solid. Mp 76e78 ꢁC. 1H NMR (DMSO‑d6, 400 MHz):
NMR (DMSO‑d6, 100 MHz):
d 19.9 (CH3), 28.1 ((CH3)3), 36.1 (C-5),
45.5 (C-4, C-6), 55.9 (OCH3), 79.1 (C(CH3)3), 103.3 (Ar),108.6 (NHC]
C), 109.9, 118.6, 122.6, 123.7, 126.5, 126.8, 128.0, 128.5, 129.4, 143.5,
151.3 (Ar), 151.9 (CONH), 153.0 (Ar), 172.6 (NHC]C). MS (ES, posi-
tive mode): 543 (M þ H)þ. Anal. calc. for (C32H34N2O6): C, 70.83; H,
6.32; N, 5.16. Found: C, 70.60; H, 6.33; N, 5.37.
d
2.46 (s, 3H, CH3), 2.53e2.63 (m, 2H, H-4, H-6), 2.68e2.88 (m, 2H,
H-4, H-6), 3.30 (m, 1H, H-5), 5.27 (br s, 2H, NH2), 6.08 (ddd, J ¼ 8.0,
2.4, 0.9 Hz, 1H, Ar), 6.16 (t, J ¼ 2.2 Hz, 1H, Ar), 6.34 (ddd, J ¼ 8.1, 2.2,
0.9 Hz, 1H, Ar), 6.98 (t, J ¼ 8.0 Hz, 1H, Ar), 7.04 (dd, J ¼ 8.3, 1.3 Hz, 1H,
Ar), 7.17e7.25 (m, 2H, Ar), 7.30e7.33 (m, 4H, Ar), 7.38 (td, J ¼ 7.8,
1.6 Hz, 1H, Ar), 7.45 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar), 14.87 (br s, 1H, NH).
4.1.3.9. 2-(1-((2-(4-Acetylphenoxy)phenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16i). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (26 mg,
0.11 mmol), 14i (39 mg, 0.17 mmol) and 4 Å molecular sieves in
toluene (0.5 mL). The resulting residue was purified by CCTLC
(hexane/ethyl acetate, 1:1) to afford 24 mg (48%) of 16i as a white
13C NMR (DMSO‑d6, 100 MHz):
d 19.8 (CH3), 36.0 (C-5), 45.3 (C-4, C-
6), 103.4, 105.4 (Ar), 108.5 (NHC]C), 109.7, 119.4, 123.7, 126.5, 126.7,
127.2, 127.8, 128.5, 129.3, 130.1, 143.5, 150.6, 151.0, 156.9 (Ar), 172.5
(NHC]C). MS (ES, positive mode): 413 (M þ H)þ. Anal. calc. for
(C26H24N2O3$H2O): C, 72.54; H, 6.09; N, 6.51. Found: C, 72.86; H,
5.84; N, 6.89.
solid. Mp 103e105 ꢁC. 1H NMR (DMSO‑d6, 400 MHz):
d 2.45 (s, 3H,
CH3), 2.54 (s, 3H, COCH3), 2.52e2.58 (m, 2H, H-4, H-6), 2.67e2.81
(m, 2H, H-4, H-6), 3.23 (tt, J ¼ 12.0, 4.0 Hz, 1H, H-5), 7.01e7.06 (m,
2H, Ar), 7.20e7.34 (m, 6H, Ar), 7.37 (td, J ¼ 7.7, 1.4 Hz, 1H, Ar), 7.48
(td, J ¼ 7.8, 1.7 Hz, 1H, Ar), 7.55 (dd, J ¼ 7.9, 1.7 Hz, 1H, Ar), 7.95e7.99
(m, 2H, Ar), 14.85 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
d
19.8 (CH3), 26.6 (COCH3), 36.0 (C-5), 45.0 (C-4, C-6), 108.6 (NHC]
4.1.3.13. 4-(2-((1-(2,6-Dioxo-4-phenylcyclohexylidene)ethyl)amino)
phenoxy)benzen-aminium 2,2,2-trifluoroacetate (16m). To a solu-
tion of 16g (66 mg, 0.13 mmol) in dichloromethane (0.4 mL), TFA
(0.4 mL, 5.07 mmol) was added and the reaction mixture was stir-
red at room temperature for 20 min. Then volatiles were removed
and the residue was triturated with dichloromethane and hexane
yielding 65 mg (99%) of 16m as a white solid. Mp 73e75 ꢁC. 1H NMR
C), 117.1, 121.2, 125.6, 126.5, 126.7, 128.3, 128.3, 128.5, 129.7, 130.8,
132.3, 143.4, 149.2, 160.4, 172.4 (NHC]C), 196.5 (COCH3). MS (ES,
positive mode): 440 (M þ H)þ. Anal. calc. for (C28H25NO4): C, 76.52;
H, 5.73; N, 3.19. Found: C, 76.12; H, 5.87; N, 3.24.
4.1.3.10. 2-(1-((2-(4-(1-Hydroxyethyl)phenoxy)phenyl)amino)ethyl-
idene)-5-phenylcyclohexane-1,3-dione (16j). Following the general
procedure C, an Ace pressure tube was charged with 15 (30 mg,
0.14 mmol), 14j (46 mg, 0.20 mmol) and 4 Å molecular sieves in
toluene (1 mL). The resulting residue was purified by CCTLC (hex-
ane/ethyl acetate, 1; 1) to afford 16 mg (28%) of 16j as a white solid.
(DMSO‑d6, 400 MHz): d 2.46 (s, 3H, CH3), 2.54e2.61 (m, 2H, H-4, H-
6), 2.69e2.86 (m, 2H, H-4, H-6), 3.29 (tt, J ¼ 12.1, 4.0 Hz, 1H, H-5),
7.00e7.07 (m, 3H, Ar), 7.15e7.20 (m, 2H, Ar), 7.20e7.36 (m, 6H, Ar),
7.41 (ddd, J ¼ 8.3, 7.3, 1.7 Hz, 1H, Ar), 7.50 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar),
14.89 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
d 19.9 (CH3),
Mp 60e62 ꢁC. 1H NMR (DMSO‑d6, 500 MHz):
d
1.31 (d, J ¼ 6.4 Hz,
36.0 (C-5), 45.9 (C-4, C-6), 108.6 (NHC]C), 119.1, 119.6, 122.3, 124.2,
126.6, 126.7, 127.2, 128.0, 128.5, 129.5, 132.2, 143.4, 150.8, 152.8 (Ar),
158.2 (q, J ¼ 34.2 Hz, CF3COOꢀ), 172.6 (NHC]C). MS (ES, positive
3H, (CH3)CHOH), 2.46 (s, 3H, CH3), 2.54e2.61 (m, 2H, H-4, H-6),
2.70e2.84 (m, 2H, H-4, H-6), 3.28 (m, 1H, H-5), 4.71 (qd, J ¼ 6.3,
4.1 Hz, 1H, CHOH), 5.16 (d, J ¼ 4.2 Hz, 1H, OH), 6.93e6.97 (m, 2H,
Ar), 7.00 (dd, J ¼ 8.2,1.3 Hz,1H, Ar), 7.20e7.27 (m, 2H, Ar), 7.28e7.33
(m, 4H, Ar), 7.34e7.37 (m, 2H, Ar), 7.39 (td, J ¼ 7.8, 1.7 Hz, 1H,
Ar),7.48 (dd, J ¼ 7.9, 1.6 Hz, 1H, Ar), 14.90 (br s, 1H, NH). 13C NMR
mode): 413 (M
(C28H25F3N2O5$H2O): C, 63.87; H, 4.79; N, 5.32. Found: C, 63.50; H,
þ
H)þ for C26H24N2O3. Anal. calc. for
4.74; N, 5.14.
(DMSO‑d6, 125 MHz):
d 19.9 (CH3), 26.0 ((CH3CHOH), 36.0 (C-5),
45.3 (C-4, C-6), 67.5 (CHOH), 108.6 (NHC]C), 118.2, 119.0, 123.9,
126.5, 126.3, 127.0, 127.1, 127.9, 128.5, 129.4, 143.2, 143.5, 151.1, 154.4
(Ar), 172.5 (NHC]C). MS (ES, positive mode): 442 (M þ H)þ.
Analytical HPLC (gradient 15e95% acetonitrile in 10 min): TR: 6.08;
area: 100%. (gradient 50e70% acetonitrile in 10 min): TR: 4.57; area:
100%.
4.1.3.14. 4-(2-((1-(2,6-Dioxo-4-phenylcyclohexylidene)ethyl)amino)
phenoxy)-2-methoxybenzenaminium 2,2,2-trifluoroacetate (16n).
To a solution of 16h (20 mg, 0.04 mmol) in dichloromethane
(0.5 mL), TFA (0.1 mL, 1.47 mmol) was added and the reaction
mixture was stirred at room temperature for 1 h. Then volatiles
were removed and the residue was triturated with dichloro-
methane and hexane yielding 18 mg (88%) of 16n as a white solid.
4.1.3.11. 2-(1-((2-Phenoxyphenyl)amino)ethylidene)-5-
phenylcyclohexane-1,3-dione (16k). Following the general proced-
ure C, an Ace pressure tube was charged with 15 (58 mg,
0.26 mmol), 2-phenoxyaniline (14k) (73 mg, 0.39 mmol) and 4 Å
molecular sieves in toluene (1.1 mL). The resulting residue was
purified by CCTLC (hexane/ethyl acetate, 2:1) to afford 70 mg (68%)
Mp 79e81 ꢁC. 1H NMR (DMSO‑d6, 400 MHz):
d 2.48 (s, 3H, CH3),
2.55e2.63 (m, 2H, H-4, H-6), 2.71e2.85 (m, 2H, H-4, H-6), 3.30 (tt,
J ¼ 12.1, 4.0 Hz, 1H, H-5), 3.82 (s, 3H, OCH3), 6.50 (dd, J ¼ 8.5, 2.5 Hz,
1H, Ar), 6.83 (d, J ¼ 2.5 Hz, 1H, Ar), 7.00 (dd, J ¼ 8.3, 1.3 Hz, 1H, Ar),
7.04 (d, J ¼ 8.5 Hz, 1H, Ar), 7.20e7.26 (m, 2H, Ar), 7.30e7.34 (m, 4H,
Ar), 7.39 (ddd, J ¼ 8.2, 7.4, 1.7 Hz, 1H, Ar), 7.48 (dd, J ¼ 7.9, 1.6 Hz, 1H,
of 16k as an oil. 1H NMR (DMSO‑d6, 400 MHz):
d
2.46 (s, 3H, CH3),
Ar), 14.88 (br s, 1H, NH). 13C NMR (DMSO‑d6, 100 MHz):
d 19.9 (CH3),
2.53e2.62 (m, 2H, H-4, H-6), 2.66e2.87 (m, 2H, H-4, H-6), 3.28 (m,
1H, H-5), 6.98e7.05 (m, 3H, Ar), 7.14e7.25 (m, 2H, Ar), 7.28 (dd,
J ¼ 7.6, 1.4 Hz, 1H, Ar), 7.30e7.38 (m, 5H, Ar), 7.40e7.44 (m, 2H, Ar),
7.49 (dd, J ¼ 7.9, 1.7 Hz, 1H, Ar), 14.89 (br s, 1H, NH). 13C NMR
36.1 (C-5), 45.4 (C-4, C-6), 56.1 (OCH3), 108.6 (NHC]C), 103.8, 110.6,
118.5, 120.3, 123.8, 126.6, 126.8, 128.0, 128.5, 129.4, 143.4, 150.9,
151.3 (Ar), 172.6 (NHC]C). MS (ES, positive mode): 443 (M þ H)þ
for C27H26N2O4. Anal. calc. for (C29H27F3N2O6): C, 62.59; H, 4.89; N,
5.03. Found: C, 62.22; H, 5.10; N, 5.21.
(DMSO‑d6, 100 MHz):
d 19.8 (CH3), 36.0 (C-5), 45.8 (C-4, C-6), 108.6

206
O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
4.2. Biological methods
excitation and emission slits) were determined using a Jobin-Ybon
SPEX Fluoromax-2 (HORIBA, Ltd. Kyoto, Japan). Using these spectra
it is possible to calculate the free and the bound R-PT concentration
for each sample and thus to determine the binding constant of R-PT
for tubulin.
The human microvascular endothelial cell line HMEC-1 was
obtained from the Centers for Disease Control and Prevention
(Atlanta, GA, USA) and used from passage 17 till 27. Human cervical
carcinoma (HeLa) and human T-lymphoid (CEM) cells were ob-
tained from ATCC (Middlesex, UK). Cell lines were maintained in
culture for up to 3 months and grown in DMEM, supplemented
with 10% FBS, 0.01 M Hepes and 1 mM sodium pyruvate. All cells
were cultured in a humidified 5% CO2 incubator at 37 ꢁC.
Once Kb of R-PT is determined (5.1 ꢂ 106 M-1) this compound
could be used as a reference ligand as described in Ref. [36]. For that
purpose, the fluorescence emission of a previous mixed sample of
0.2
of increasing concentrations of the studied ligand in a black 96-well
plate (0; 0.05; 0.2; 0.5; 2; 5; 10; 30; 50; 70 M). The samples were
mM of R-PT and 0.2 mM of tubulin was evaluated in the presence
m
4.2.1. Cell proliferation
incubated 30 min at 25 ꢁC in a Varioskan plate reader (Thermo
Scientific Waltham, Massachusetts, USA) before the fluorescence
emission intensity at 456 nm (excitation 374 nm) was measured.
The data were analyzed and the binding constants determined
using Equigra V5.0 [37].
HMEC-1 cells were seeded in 48-well plates at 20,000 cells/well.
After 24 h, 5-fold dilutions of the compounds were added. The cells
were allowed to proliferate 4 days in the presence of the com-
pounds, trypsinized, and counted by means of a Coulter counter
(Analis, Belgium). Human cervical carcinoma (HeLa) cells were
seeded in 48-well plates at 10,000 cells/well. After 24 h, different
concentrations of the compounds were added. After 3 days of in-
cubation, the cells were trypsinized and counted in a Coulter
counter. Suspension cells (Mouse leukemia L1210 and human
lymphoid CEM cells) were seeded in 96-well plates at 60,000 cells/
well in the presence of different concentrations of the compounds,
allowed to proliferate for 96 h, and counted in a Coulter counter.
The 50% inhibitory concentration (IC50) was defined as the com-
pound concentration required to reduce cell proliferation by 50%.
4.2.5. Tubulin assembly time-course
Tubulin was diluted at a final concentration of 25 mM in a 10 mM
NaPi, 1 mM EGTA, 1 mM GTP (pH ¼ 7) buffer supplemented with
3.4 M glycerol. Each polymerization reaction was prepared in a 96-
well plate to a final volume of 100 mL with 30 mM of each of the
compounds tested (or the equivalent volume of DMSO for the
control). Polymerization was followed throughout time measuring
the scattering of light (
(Thermo Scientific Waltham, Massachusetts, USA).
l
¼ 350 nm) using a Varioskan plate reader
4.2.2. Cell cycle analysis
4.3. Computational methods
HMEC-1 cells were seeded in 6-well plates at 125,000 cells/well
in DMEM with 10% FBS. After 24 h, the cells were exposed to
different concentrations of the compounds. After 24 h, the DNA of
the cells was stained with propidium iodide using the CycleTEST
PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA). The DNA
content of the stained cells was assessed by flow cytometry on a
FACSCalibur flow cytometer and analyzed with CellQuest software
(BD Biosciences) within 3 h after staining. Cell debris and clumps
were excluded from the analysis by appropriate dot plot gating.
Percentages of sub-G1, G1, S, and G2/M cells were estimated using
appropriate region markers [33].
4.3.1. Affinity maps calculation
Binding pocket analysis of the tubulin-DAMA-colchicine was
carried out with cGRILL [17,18], a computational tools formally
equivalent to Goodford's program GRID [38]. Hydrogen atoms,
atom point charges and radii for all atoms in the complexes were
calculated by submission to Hþþ server (http://biophysics.cs.vt.
edu) in order to obtain their pqr format files. Grid center was
defined by selecting the corresponding ligand and a cubic box of
50 ꢂ 50 ꢂ 50 points and a 0.5 Å was set for the calculations. cGRILL
evaluates, at each grid point, the interaction energy between the
whole receptor and five different probes combining van der Waals
(Leonard-jones potential), electrostatic (Coulombic), and hydrogen
bonding [39] (geometry based) terms.
The five calculated affinity maps: lipophilic (CH3), hydrogen
bond acceptor (¼O), hydrogen bond donor (NH4þ), mixed
hydrogen bond donor-acceptor (OH) and hydrophobic-like (hy-
drophobic) were visualized and analyzed using the PyMOL plugin
provided with the software.
4.2.3. Tube formation
Wells of a 96-well plate were coated with 70 mL matrigel (10 mg/
mL, BD Biosciences, Heidelberg, Germany) at 4 ꢁC. After gelatini-
zation at 37 ꢁC during 30 min, HMEC-1 cells were seeded at
60,000 cells/well on top of the matrigel in 200 mL DMEM containing
10% FCS. After 3 h of incubation at 37 ꢁC, when the endothelial cells
had reorganized to form tube-like structures, the compounds were
added. Ninety minutes later, the cultures were photographed at
100ꢂ magnification and analyzed using digitalized analysis.
4.3.2. Docking of 16n
Compound 16n was used as ligand for the automated docking
experiments using TUB075 tubulin complex (PDB ID: 6KFJ) [16].
AMBER-compatible RESP point charges were used for 16n. The
Lamarckian genetic algorithm implemented in AutoDock 4.2 [40]
was used to generate the docked conformations within the putative
binding cavity by randomly changing the overall orientation of the
molecule as well as the torsion angles of all rotatable bonds. Default
settings were used except for the number of runs, population size,
and maximum number of energy evaluations, which were fixed at
250, 100 and 250.000, respectively. Rapid intra- and intermolecular
energy evaluations of each configuration was achieved by having
the receptor's atomic affinity potentials for aliphatic and aromatic
carbon, oxygen, nitrogen and hydrogen atoms precalculated in a
three-dimensional grid with a spacing of 0.375 Å. A distance-
dependent dielectric function was used in the computation of
electrostatic interactions.
4.2.4. Determination of binding constants
Proteins and ligands. Calf brain tubulin was purified as described
[34]. (R)-(þ)-ethyl 5-amino 2-methyl-1,2-dihydro-3-phenylpyrido
[3,4-b]pyrazin-7-yl carbamate (R-PT) [35] was a kind gift of Prof.
G.A. Rener, Organic Chemistry Research Department, Southern
Research Institute, Birminghm, Alabama. The compounds were
diluted in 99.8% D6-DMSO (Merck, Darmstadt, Germany) to a final
concentration of 20 mM and stored at ꢀ80 ꢁC.
Determination of binding constants. The binding constant of R-
PT [25] for dimeric tubulin was determined using the competition
method in 10 mM sodium phosphate, 0.1 mM GTP pH 7.0 at 25 ꢁC.
To do so 0.2
tubulin up to 10
with increasing amounts of R-PT up to 10
emission spectra (excitation 374 nm) of the samples (5 nm
m
M of R-PT was incubated with increasing amounts of
M and vice versa, 0.2 M of tubulin was incubated
M, the fluorescence
m
m
m

O. Bueno et al. / European Journal of Medicinal Chemistry 171 (2019) 195e208
1348e1360. https://doi.org/10.2174/0929867322666150114163732.
207
Conflicts of interest
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M. Knossow, Insight into tubulin regulation from a complex with colchicine
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1038/nature02393.
The authors declare that they have no competing interests.
[15] A. Massarotti, A. Coluccia, R. Silvestri, G. Sorba, A. Brancale, The tubulin
Author contributions
colchicine domain:
a molecular modeling perspective, ChemMedChem 7
(2012) 33e42. https://doi.org/10.1002/cmdc.201100361.
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ꢀ
M-J.P.P. and E-M.P. designed the ligands. O.B. and M.G. per-
formed the chemical synthesis supervised by M-J.P.P. and M-J.C.
J.E.-G. performed the binding affinity assays supervised by J.F.D.
S.M. performed the biological assays supervised by S.L. M-J.P.P., S.L.
and E-M.P wrote the manuscript.
ꢀ
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M.-J. Camarasa, I. Barasoain, M.O. Steinmetz, J.F. Díaz, M.-J. Perez-Perez,
S. Liekens, E.-M. Priego, High-affinity ligands of the colchicine domain in
tubulin based on a structure-guided design, Sci. Rep. 8 (2018) 4242. https://
doi.org/10.1038/s41598-018-22382-x.
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novo design protocol guided by ligand efficiency indices (LEI), Methods in
molecular biology (Clifton, N. J.) 1289 (2015) 89e100.
[18] F. Gago, Avaliable at, http://farmamol.uah.es.
Acknowledgments
[19] J. Hwang, P. Li, W.R. Carroll, M.D. Smith, P.J. Pellechia, K.D. Shimizu, Additivity
of substituent effects in aromatic stacking interactions, J. Am. Chem. Soc. 136
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This project has been supported by the Spanish Ministerio de
Economía y Competitividad SAF2015-64629-C2-1-R (to EMP, MJPP.
and MJC) and BFU2016-75319-R (AEI/FEDER, UE) (JFD.) Financial
support from CSIC (Proyecto Intramural PIE-201668E079 to MJPP)
is also acknowledged. The authors acknowledge networking
contribution by COST action CM1407 “Challenging synthesis
inspired by nature e from natural products chemistry to drug
discovery” (to MJPP, SL, and JFD). JFD. is a member of the CIB
Intramural Program ‘‘Molecular Machines for Better Life’’ (MAC-
BET). We acknowledge Lizette Van Berckelaer, Eef Meyen and Sam
Noppen for excellent technical assistance. We thank ganadería
Fernando Díaz for calf brains for tubulin purification.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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