Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl Nicotinamides

Article abstract of DOI:10.1002/cmdc.201700365

A number of trimethoxybenzoic acid anilides, previously studied as permeability glycoprotein (P-gp) modulators, were screened with the aim of identifying new anticancer agents. One of these compounds, which showed antiproliferative activity against resist

Full text of DOI:10.1002/cmdc.201700365

Accepted Article
Title: Investigating structural requirements for the antiproliferative
activity of biphenyl nicotinamides
Authors: Cosimo Damiano Altomare, Maria Majellaro, Angela
Stefanachi, Piero Tardia, Chiara Vicenti, Angelina Boccarelli,
Alessandra Pannunzio, Federica Campanella, Mauro
Coluccia, Nunzio Denora, Francesco Leonetti, Modesto de
Candia, and Saverio Cellamare
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ChemMedChem
10.1002/cmdc.201700365
FULL PAPER
Investigating structural requirements for the antiproliferative
activity of biphenyl nicotinamides
[
a]§
[a]§
[b]
[c]
[d]
Maria Majellaro, Angela Stefanachi, Piero Tardia, Chiara Vicenti, Angelina Boccarelli,
[
a]
[a]
[a]
[a]
Alessandra Pannunzio, Federica Campanella, Mauro Coluccia, Nunzio Denora, Francesco
[
a]
[a]
[a]
[a]
Leonetti, Modesto de Candia, Cosimo Damiano Altomare,* and Saverio Cellamare
[
a]
M. Majellaro, Dr. A.Stefanachi, Dr. A. Pannunzio, F. Campanella, Prof. M. Coluccia, Prof. N. De Nora, Dr. M.de Candia, Prof. S. Cellamare, Prof. C.
D. Altomare.
Department of Pharmacy-Drug Sciences, University of Bari, Via Orabona 4, 70125, Bari, Italy.
E-mail: cosimodamiano.altomare@uniba.it
[
[
b]
c]
Dr. P. Tardia.
D3-Drug Discovery and Development Department, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy
Dr. C. Vicenti.
Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari, Piazza Giulio Cesare 11, Bari, Italy.
70124.
[
d]
§]
Dr. A. Boccarelli.
Department of Biomedical Sciences and Human Oncology, University of Bari, Piazza Giulio Cesare 11, Bari, Italy. 70124.
These authors equally contributed to the work.
[
Abstract: A number of trimethoxybenzoic acid anilides, previously
studied as P-gp (permeability glycoprotein) modulators, were
screened with the aim of identifying new anticancer agents. One of
these compounds, which showed antiproliferative activity against
resistant MCF-7 cell line, was selected as the hit structure. The
replacement of the trimethoxybenzoyl moiety with the nicotinoyl one,
for overcoming solubility issues, led to synthesize a new series of N-
biphenyl nicotinoyl anilides, among which a nitro derivative (3)
displayed antiproliferative activity against MCF7 and MDA-MB231
submicromolar IC50 values against P-gp, but their poor aqueous
[
4]
solubility hampered a hit-to-lead development. Interestingly,
compound 1, which displayed a nanomolar affinity to P-gp, also
showed intrinsic cytotoxicity towards MCF7-adr cell line resistant
[
4]
to doxorubicin.
F
X
F
N
F
F
N
cells in the nanomolar range. Searching for a bioisoster of the NO
2
O
group led to a nitrile analogue (36) showing a strong increase in
activity against MCF-7 and MDA-MB231 cells. Compound 36
induced on MCF7 cells a dose-dependent accumulation of G2- and
F
HN
O
HN
O
F
O2N
O2N
HN
O
N
HN
O
N
HN
O
N
MeO
MeO
OMe
M-phase cell populations and
a decrease of S-phase cells.
OMe
Compared to vinblastine, a well known potent antimitotic agent,
compound 36 induced also a G1 arrest at low doses (20-40 nM), but
did not inhibit in vitro tubulin polymerization.
OMe
OMe
General structure of P-gp
modulating benzanilides
1
2
3
4
Figure 1. Structural modifications leading to hit identification.
Introduction
This evidence prompted us to screen the antiproliferative activity
of a series of trimethoxy and non-trimethoxy anilides (Table 1).
Due to their poor aqueous solubility, these compounds were
tested at a maximum concentration of 2.5 M, but none of them
showed effects against MCF7 and MDA-MB-231 cell lines. To
improve the solubility, the nicotinamide derivative 2 (Figure 1)
was synthesized as a strict analog of 1, but despite its higher
solubility it showed decreased P-gp affinity and loss of
The discovery of novel small molecule modulating processes
critically involved in the growth and survival of human tumors, is
one of the major challenges in oncological drug discovery
[
1]
research. Many organizations, both private and public, have
heavily invested in HTS (high throughput screening) campaigns
[
2]
of libraries of thousands-to-millions compounds.
Some years ago, our research group started a medium-
screening program of in-house focused chemical libraries aimed
at identifying new molecules with antiproliferative activity, first
[
4]
antiproliferative effects.
The nicotinamide moiety can be considered a privileged scaffold,
because of diverse biological activities shown by the large
amount of bioactive substances incorporating this fragment.
Representative examples of bioactive nicotinoyl derivatives are
shown in Figure 2, which includes the N-phenyl nicotinoyl
evaluating
glycoprotein) modulators.
a set of newly synthesized P-gp (permeability
[
3,4]
Multidrug resistance (MDR), which
is among the main causes of chemotherapy failure, is unrelated
to the pharmacological activity of drugs and could be triggered
by diverse mechanisms, including drug metabolism acceleration,
apoptotic pathway modulation, cellular damage repair and drug
[7]
derivative 16 as human sirtuin-2-selective inhibitor, 17 as
[
8]
[5]
sodium channel blocker, 18 as soluble epoxide hydrolase
efflux pump (e.g., P-gp) overexpression.
[
9]
[10]
inhibitor,
19 as HDAC1/HDAC2 inhibitor,
20 as anti-
Most of the investigated substances were biphenyl anilides of
trimethoxy benzoic acids, whose ability to modulate P-gp was
[
11]
[12]
leishmanial agent, and 21 as aquaporin 4 inhibitor. The N-
biphenyl-4-ylnicotinamide analog of 21, which may be
considered as a template of the compounds investigated herein,
proved to be active against a panel of bioassays, such as HIV-1
[4]
found to correlate with lipophilicity.
The most lipophilic
compounds, especially those able to form an intramolecular
hydrogen bond (IMHB), such as compound 1 (Figure 1), showed
1
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RNase H inhibition, MKP-3, HePTP, Hsp70, ER stress-induced
cell death and VHR1 in vitro high throughput screening.
Herein we report the synthesis of a new series of N-biphenyl
nicotinoyl anilides and evaluation of their antiproliferative activity
against MCF7 and MDA-MB231 cell lines. A starting point of this
study was that the ortho-nitro biphenyl derivative 3 showed
antiproliferative activity against MCF7 and MDA-MB231 cell
lines in the nanomolar range of concentrations. This result was
somehow supported by the activity reported by others for the
[
13]
F
O
H2N
HN
O
O
NC
O
N
N
H
H
N
N
H
N
F
ortho-nitrophenyl analog
apoptosis inducer identified by caspase-based HTS assay.
4 (Figure 1), which acts as an
N
[
6]
16
17
18
The search of the structural requirements for maintaining (or
increasing) the antiproliferative activity of the biphenyl
nicotinamide derivative 3 was carried out by synthesizing and
testing (i) the positional isomers (isonicotinamide and
picolinamide) and a few different congeners (substituents on the
distal phenyl of the biphenyl moiety), and (ii) the bioisosteric
O
O
O
HN
O
N
N
N
N
H
H
R2
R1
N
replacement of the ortho-NO
2
group.
19
20
21
Figure 2. Representative bioactive compounds bearing N-phenyl nicotinoyl
moiety.
[
a]
1
2
3
4
5
Cpd
X
R
R
R
R
R
5
C
3,5-diF
3,5-diF
3,5-diF
3,5-diF
NO
2
2
OCH
OCH
H
3
3
OCH
H
3
H
6
7
8
C
C
C
NO
OCH
H
3
NO2
H
NO
2
2
F
F
F
9
C
3,5-diF
NO
OCH
2
O
H
[
4]
1
0
N
C
C
C
C
N
-
3-F
F
H
H
H
11
NO
2
OCH
OCH
F
3
3
OCH
OCH
F
3
3
OCH
OCH
F
3
3
[
4]
1
2
3,5-diF
3,5-diF
3-COCH
-
NH
NH
2
2
13
4[3]
1
3
NO
2
2
OCH
OCH
3
3
OCH
OCH
3
3
OCH
OCH
3
3
[
3]
1
5
NO
Table 1. Previously and newly synthesized biphenyl benzamide derivatives lacking antiproliferative activity against MCF7 and MDA-MB-231 cell lines at 2.5 μM
[
a]
concentration. Synthesis details and analytical data in the references reported in squared brackets.
Results and Discussion
by reacting 3',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-amine 1A with
nicotinoyl chloride, picolinoyl chloride and isonicotinoyl chloride,
respectively. Compound 36 and 37 were prepared by
condensation of 4-amino-4'-fluoro-[1,1'-biphenyl]-3-carbonitrile
11A with nicotinoyl chloride (36) or 3,4,5-trimethoxybenzoyl
chloride (37). Compound 32 was synthesized through hydrolysis
of 31 (Scheme 1).
The newly investigated biphenyl nicotinamide derivatives were
synthesized following the synthetic pathways shown in Schemes
1
and 2. Structural details along with the antiproliferative
activities, expressed as IC50 values, are reported in Tables 2 and
. A number of newly and previously synthesized derivatives
3
showing no activity at 2.5 M (solubility limit in the assay
conditions for most of them) are shown in Table 1.
A Suzuki reaction between the commercially available 5-chloro-
2-nitroaniline and 4-fluorobenzenboronic acid, carried out in the
above conditions, led to 12A, which yielded 28 after
condensation with nicotinoyl chloride. To prepare compound 35,
The first step of the synthesis consisted in a Suzuki reaction
between the suitable aniline and the aryl boronic acid to obtain
the biphenyl aniline derivatives 1A-11A (Scheme 1). Then, a
condensation between the biphenyl aniline and nicotinoyl
chloride or trimethoxybenzoylchloride, in dry dioxane in the
presence of DMAP and TEA, yielded compounds 24-27, 29-31,
the biphenylaniline 12A first underwent acetylation of the NH
group (13A) and next a reduction to NH . The biphenyl aniline
2
2
intermediate 14A was reacted with nicotinoyl chloride to yield
compound 35 (Scheme 2).
33-34, 36 and 37. Compounds 3, 22 and 23 were synthesized
2
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Scheme 1.
2 3 3 4 3 2
Reagents and conditions: a) aryl boronic acid, dioxane, 2M K CO , Pd(PPh ) , reflux; b) aryloyl chloride, dry dioxane, DMAP, Et N, reflux c) LiOH, THF/H O r.t.
F
F
F
Cl
a)
b)
c)
NH2
NO2
O
O
NH2
N
N
H
H
NO2
NO2
NH2
12A
13A
14A
d)
d)
F
F
O
N
H
N
O
NO2
N
H
O
NH
N
28
35
Scheme 2.
Reagents and conditions: a) aryl boronic acid, dioxane, Pd(PPh
DMAP, Et N, reflux.
)
3 4,
K
2
CO
3
3 2
2M, reflux; b) CH COCl, THF, r.t.; c) SnCl MeOH; d) nicotinoyl chloride, dioxane,
3
3
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General
structure
2
3
4
1
2
3
4
MCF7
(IC50 nM)
MDA-MB231
(IC50 nM)
[b]
cpd
3
X
X
X
R
R
R
R
[a]
[a]
log k’
C
C
N
C
C
C
C
C
C
-
N
N
C
C
N
N
N
N
N
-
C
C
C
N
C
C
C
C
C
-
3’,5’-diF
NO
NO
NO
NO
NO
NO
NO
NO
NO
-
2
2
2
2
2
2
2
2
2
-
-
H
250±70
100±70
229±95
0.89±0.01
-
[
6]
[c]
4
2
2
2
2
2
2
2
H
H
-
200±90
> 2500
> 2500
32±3
2
3
4
5
6
7
8
3’,5’-diF
3’,5’-diF
4’-F
H
> 2500
1.61±0.01
0.94±0.01
0.78±0.01
0.79±0.01
0.76±0.01
0.80±0.01
0.80±0.01
-
H
-
> 2500
H
H
H
H
H
-
58±10
3’-F
-
325±150
1400±500
30±10
68±50
> 2500
30±10
> 2500
4’-OCH
3’-OCH
3
3
-
-
[
d]
-
450±200
4.85±1.6
2730±1500
[
e]
CA4
-
-
-
3.3±0.47
Cisplatin
-
-
-
-
-
-
1000±150
-
1
Table 2. Antiproliferative activity data (IC50) against MCF7 and MDA-MB231 cell lines of R -substituted ortho-nitro-biphenyl nicotinamides and positional isomers.
[b]
[
a]
IC50 values (nM) coming from the mean of at least three independent experiments ± SEM, sampled in triplicate. Log of the capacity factor determined by
[
c]
[d]
[e]
4
RPLC in isocratic conditions (mobile phase: 65%MeOH/35% AcONH buffer 25 mM); structure in Fig. 1; structure in Scheme 2; Combretastatin A-4
1
2
MCF7
IC50 nM)
MDA-MB231
(IC50 nM)
[b]
General structure
cpd
X
R
R
[a]
[a]
log k’
(
2
3
3
3
3
3
3
3
9
0
1
2
3
4
5
6
C
N
C
C
C
C
C
C
C
3’,5’-diF
3’,5’-diF
4’-F
F
-
> 2500
> 2500
> 2500
> 2500
>2500
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
37±20
>2500
0.71±0.01
0.67±0.01
1.26±0.01
0.32±0.02
0.47±0.01
0.66±0.01
0.32±0.02
0.32±0.02
-
COOCH
COOH
3
4’-F
4’-F
SO
2
NH
2
4’-F
OCH
3
1200±400
> 2500
47±20
4’-F
NHCOCH3
CN
4’-F
[
b]
37
4’-F
CN
>2500
[
a]
Table 3. Effects on antiproliferative activity (IC50) of the replacements of ortho-nitro group in the biphenyl nicotinamide derivative 3. IC50 values (nM), coming
[
b]
from the mean of at least three independent experiments ± SEM, sampled in triplicate Log of the capacity factor determined by RPLC in isocratic conditions
[
c]
(
4
mobile phase: 65%MeOH/35% AcONH buffer 25 mM); 3,4,5-trimethoxybenzoyl analog (Scheme 1).
As shown by the IC50 values of the ortho-NO
2
derivatives in
A different spatial geometry of the biphenyl moiety in 24 caused
a drop of biological potency, as shown by the positional isomer
28 in which the distal 4’-F-phenyl is moved from para (in 24) to
meta position relative to NHCO.
The antiproliferative potency of the compounds in Table 2
appeared not dependent on the lipophilicity, as experimentally
assessed by reversed phase HPLC (log k’). Indeed, the most
active compounds 24 and 27 are almost isolipophilic (log k’ ca.
0.8) with the less active compounds, whereas the most lipophilic
derivative 22 (log k’ 1.6) did not show any activity up to 2.5 M
concentration.
Table 2, the 3’,5’-difluorinated biphenyl nicotinamide 3 displayed
a noteworthy antiproliferative activity against MCF7 and MDA-
MB231 cell lines (250 and 229 nM, respectively). Replacing the
nicotinamide moiety with the positional isomer moieties, i.e.
isonicotinamide (22) and picolinamide (23), resulted in a loss of
activity. Interestingly, N-(4-ethoxy-2-nitrophenyl)nicotinamide (4),
previously reported as apoptosis inducer in different cancer cell
lines (e.g., human breast cancer T47D, ZR75-1 and colorectal
[
6]
DLD-1) and re-synthesized in this study, proved to be almost
equipotent with 3 in inhibiting proliferation in both human breast
cancer cell lines MCF 7 and MDA-MB231.
We attempted to replace in the hit compounds 3 and 24 the
A further investigation of the structure activity relationships was
carried out by varying number and position of fluorine atoms (24
and 25) and replacing F with OCH (26 and 27) on the distal
3
ortho-NO
demonstrated in several cases,
and bioisosters (Table 3). Most of these derivatives are not able
to retain the activity expected for the OCH congener 34, and the
2
substituent, which may result a toxicoforic group as
[
14],[15]
with different substituents
phenyl ring. Within the limits of the chemical space examined, a
3
net gain in the cell growth inhibition potency over compound 3
was observed with 4’-F (24) and 3’-OCH (27) derivatives.
3
CN congener 36 proved to be one of the most active compounds
of the series, showing IC50 values comparable to those of 24 and
4
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2
7. HPLC monitoring showed that the ortho-CN biphenyl
sub-G1, G1, S, G2, M, and > G2/M cell populations (Figure 3I
and 3J).
nicotinamide derivative 36 is highly stable in PBS (100 mM, pH
7
.4), remaining intact after 24 h incubation, and stable enough in
Both 36 and vinblastine induced on MCF7 cells a dose-
dependent accumulation of G2- and M-phase cell populations
and a decrease of S-phase cells. However, unlike vinblastine,
compound 36 induced also a G1 arrest at low doses (20-40 nM).
Interestingly, both G1/G2 and M arrests were associated with
cell number reduction (Figure 3 H) along with an increase of
sub-G1 population, which appeared indicative of apoptosis.
These results suggest that compound 36, at low doses, behaves
differently from vinblastine, its action likely depending upon
activity on multiple pharmacological targets that seems engaged
with increasing concentrations. The effects of 36 and vinblastine
on MDA-MB231 cells were similar to those on MCF7 as far as
cell cycle modifications are concerned, i.e. dose-dependent
increase of G2 and M cells for both compounds, and additional
G1 increase for 36 at low doses (data not shown). On a molar
basis, however, vinblastine showed the same efficacy on the two
cell lines, whereas 36 was almost twice more effective on MDA-
MB231 cells. Overall, the divergent cell cycle status following
low- and high-dose, along with the higher inhibitory potency of
36 on the p53-defective MDA-MB231 cells, supports a multiple
target hypothesis for compound 36, stimulating investigation on
its mechanism of action.
100% human serum (half-live ca. 12 h).
The activity drop of 37 with respect to 36 ultimately proved the
superiority of the nicotinoyl over trimethoxybenzoyl moiety. Yet,
for the series of compounds in Table 2, no correlation appeared
to exist between the antiproliferative potency and lipophilicity.
A major outcome of this study was the evidence that the nitrile
group can be a successful replacement of the nitro group, as
demonstrated by the nanomolar antiproliferative potency of
compound 36. Actually, the number of nitrile-containing
pharmaceuticals has recently increased
medicinal chemistry of this functional group has been deeply
understood. The CN group should be biocompatible
stable to metabolic transformation.
[
16]
and the role in
[
17]
and
[
18]
This short polarized triple
[
19]
[20]
bond,
smaller than a methyl group,
is able to penetrate
narrow clefts, attaining polar interactions and HBs as acceptor
[
21]
[19],[22]
with amino acid side chains or water molecules.
It is
recognized that CN group/s may be introduced in bioactive
molecules for improving ADME properties, but also for
pharmacodynamic reasons (e.g., halogen bioisoster).
To investigate the possible targets of the biphenyl nicotinamide
derivatives, we started with examining the interference of
compound 36 in the cell cycle progression of two human breast
cancer cell lines, MCF-7 and MDA-MB-231, which differ for their
proliferative potential. Cell cycle progression is perturbed by a
variety of anticancer drugs, and investigating drug-dependent
modifications can contribute to the phenotypic characterization
[
23]
On the basis of a structural analogy between compound 36 and
the 6-chloro analog of 4, a previously reported potent inhibitor of
[
6]
tubulin polymerization (IC50 0.5 μM), we considered of a certain
interest to test a number of active biphenyl nicotinamides in a
cell-free tubulin polymerization assay. The progression of tubulin
polymerization was examined by monitoring the increase in
fluorescence emission at 420 nm (excitation wavelength is 360
nm) for 1 h at 37 °C. Vinblastine and paclitaxel, two known
microtubule-targeting agents acting as disassembly- and an
[
24]
of compounds under preclinical development.
Cell-cycle changes induced by compound 36 on MCF7 and
MDA-MB231 cells were investigated by the Operetta high-
content imaging system, using a multiparametric approach to
quantify DNA content (NuclearMask™ Blue Stain), DNA
synthesis (EdU-positive cells), and mitotic cells (pHH3-positive
cells). Nuclear DNA content evaluation is widely used for cell-
cycle phase identification in flow cytometry as well as in high
[27]
assembly-promoter, respectively, were used as controls. The
effects on tubulin polymerization of three substances are shown
in Figure 4 (i.e., 4 as reference inhibitor and its biphenyl analog
3, as well as the potent antiproliferative compound 36).
[25],[26]
content imaging systems.
MCF7 or MDA-MB231 cells
As expected, paclitaxel was a stabilizer of tubulin polymerization
growing in control (DMSO-treated) wells were stained with the
nuclear dye after formaldehyde fixation and imaged using a 20x
long working distance objective.
at 3 μM concentration, whereas vinblastine at the same
concentration acted as an effective inhibitor of tubulin
polymerization. The ortho-NO -phenyl nicotinamides 3 and 4
2
Harmony software was first used for image analysis (an example
is shown for MCF7 cells in Figure 3A), and PhenoLOGIC
algorithm was then applied to separate single cells from clumps.
From the single cell results, a histogram of nuclear dye intensity
sum was generated and the intensity corresponding to the
center of the G1 peak determined. Based on this value, a
histogram of normalized DNA (relative DNA content) was
obtained from DMSO-treated wells on a per-plate basis and
applied to all wells to identify sub-G1, G1, S, G2/M, and > G2/M
cell populations (Figure 3B). DNA histograms for MCF7 cells
treated with 36 or vinblastine at concentrations inducing similar
growth inhibitory effects (80 and 5 nM, respectively) are shown
in Figure 4 (inserts C and D). To provide a direct evaluation of
cells in S-phase and to separate G2 from M populations, EdU
incorporation and pHH3 expression were added to DNA content
measurements, as shown in Figure 4E and 4F, respectively. In
this way, the effect of 36 treatment on MCF7 cell cycle was
investigated and compared to that of vinblastine, by identifying
(Table 2) at 3 μM concentration displayed profiles similar to that
of vinblastine, whereas the ortho-CN-phenyl nicotinamide 36, a
more potent antiproliferative agent (Table 3), did not show any
appreciable effect on the progression of tubulin polymerization
(profile similar to control). Concentration-dependent effects of 3
on tubulin polymerization, indicating the inhibitory potency at
0.03-3 μM concentrations, are shown in SI (Fig. S1).
Apparently, the inhibitory effect of the tested nicotinoyl anilides
on tubulin polymerization in vitro do not relate with the
antiproliferative activity.
5
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(A)
(B)
(C) 36, 80 nM
(D) VB, 5 nM
(E)
(F)
(G)
(H)
(I)
(J)
Figure 3. High-content cell cycle analysis. (A) Example image of vehicle-treated MCF7 cells with nuclei stained with NuclearMask™ Blue Stain. DNA content
histograms of vehicle-treated MCF7 cells (B), and of cells treated with compound 36 (C) or vinblastine (VB) (D) at concentrations with similar cell number
reduction effects. By using the PhenoLOGIC algorithm, cell populations were separated based on normalized DNA content: <0.5, subG1; 0.5-1.35, G1; 1.35-1.70,
S; 1.70-2.5, G2/M; >2.5, >G2/M (red dotted lines). Representative images of vehicle-treated MCF7 cells stained with Alexa Fluor 488 (green, S-phase cells), and
anti-pHH3 (red, M-phase cells) antibodies are shown in (E) and (F), respectively, while a merging image from (A), (E) and (F) is shown in (G). (I) and (J),
vinblastine and 36 dose-response cell-cycle modifications determined using the multiparametric approach. (H) Effect of 36 or vinblastine treatment on cell number.
Values are the average of 5-8 technical replicates ± SD. * P < 0.05 versus vehicle-treated controls (Student’s t-test, two-tailed distribution, two sample equal
variance).
40,000
30,000
20,000
10,000
0
Figure 4. Effect of compounds 3,
(Cytoskeleton® kit). Tubulin polymerization was monitored by the increase
of fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37 °C.
4 and 36 on tubulin polymerization
[
28]
Control
Vinblastine
3
All the compounds were tested at
3 µM concentration. Paclitaxel and
vinblastine, at 3 µM concentration, were used as positive controls for tubulin
polymerization inhibition and stabilization, respectively. Data points are means
of triplicate experiments. The dose-response curve of compound 3 is reported
as an example in support information (Figure 5.SI).
3
4
6
Paclitaxel
10
20
30
40
50
60
Time / min
It is likely that the different behavior of the N-(2-
nitrophenyl)nicotinamides 3 and 4 (inhibitors) with respect to the
N-(2-cyanophenyl)nicotinamide derivative 36 (ineffective) may
6
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ChemMedChem
10.1002/cmdc.201700365
FULL PAPER
be related to the molecular flatness, which in turn should depend
upon the formation of intramolecular HB (IMHB) between the
amide NH (HB donor) and the HB acceptor group in ortho
(ESI) was performed with mass Q-TOF spectrometer (6530 mass Q-TOF
Agilent, Palo Alto, CA). In all cases, spectroscopic data are in agreement
with known compounds and assigned structures. Combustion analyses
were performed by Eurovector Euro EA 3000 analyzer (Milan, Italy) and
gave satisfactory results (C, H, N within 0.4% of calculated values).
[
28]
position (NO
2
or CN). In principle, IMHB formation should be
congeners (e.g., 3 and 4),
more favored in the case of 2-NO
2
rather than in the case of 2-CN congeners (e.g., 36). A broader
and more in-depth SAR study can help in establishing whether
and to what extent a more flat molecular geometry of the
biphenyl nicotinamides, resulting from IMHB formation, can be
causatively related to the inhibition of the tubulin polymerization.
Chromatographic settings. Purity determinations were carried out
using a Zorbax 300SB-C18 4.6 x 250mm, with 5 µm size particles, built
on a Waters double pump HPLC system in isocratic conditions. Injection
volumes were 5 μL, flow rate was 1 mL/min, detection was performed
with UV (λ=230 nm and 254 nm). Samples were prepared by dissolving
Conclusion
0
.1 mg/mL of the solute in 10% (v/v) DMSO and 90% (v/v) methanol.
Retention times (t were measured at least from three separate
injections, dead time (t ) was the retention time of deuterated water. The
mobile phase was filtered through a Nylon 66 membrane 0.45μm
Supelco, USA) before use.
r
)
0
A number of N-nicotinoyl biphenyl anilides were designed and
synthesized, and two new potent in vitro antiproliferative agents
(
(
24 and 27) were identified. The need to replace in these
compounds the potentially toxicoforic NO group, while
2
preserving their good antiproliferative activity, led to the
synthesis of a second series of biphenyl nicotinamides. Among
the investigated bioisosteric analogs, the nitrile-containing
analog 36 proved to be almost equipotent with the
corresponding nitro derivative 24, showing nanomolar IC50
values against two breast tumor cell lines, MCF7 and MBA-
General
procedure
for
the
Suzuki-Miyaura
cross-coupling
reaction:preparation of compounds 1A-12A (Scheme 1).
A suspension of the chosen aniline (5.0 mmol), the appropriated boronic
acid (7.5 mmol), and Pd(PPh (0.577 g, 0.50 mmol) in a aqueous
solution 2M of K CO (7.5 mL) and 1,4-dioxane (30 mL) was heated at
00 °C for 7 h under stirring. After cooling, the solvent was evaporated
3 4
)
2
3
MB231 (IC50 47 and 37 nM, respectively).
A cell-cycle
1
progression analysis on MCF7 cells, accomplished through
multiparametric automated microscopy, revealed a similar dose-
dependent accumulation of G2- and M-phase cell populations
and a decrease of S-phase for 36 and vinblastine, taken as
reference drug. On the other hand, compound 36, unlike
vinblastine, induced also a G1 arrest at low doses (20-40 nM).
Interestingly, G1/G2 and M arrests were associated with cell
number reduction, along with an increase of sub-G1 population
indicative of apoptosis. This finding suggests that 36, at low
concentrations, acts with a mechanism different from vinblastine.
Moreover, compound 36 did not show inhibitory effects on
tubulin polymerization. The possibility that the high
antiproliferative activity of compound 36 is associated to a multi-
target mechanism, which is appealing for a small molecule,
deserves further pharmacological investigation.
under vacuum to dryness and the obtained residue was treated with
dichloromethane. The suspension was filtered on celite, or silica gel and
the resulting residue was purified by chromatography.
3
',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-amine (1A):
1
Yield: 70%; HNMR (300 MHz, DMSO-d
6
) δ: 8.27 (d, J= 2.1 Hz, 1H), 7.82
(dd, J=2.2 Hz, J= 8.9 Hz, 1H), 7.61 (s br, 2H), 7.41 (d, J= 7.3 Hz, 2H),
7.16 (d, J=9.2 Hz, 1H), 7.09 (d, J=8.9 Hz, 1H); LRMS (ESI) m/z 249 [M-
-
H] .
4'-fluoro-3-nitro-[1,1'-biphenyl]-4-amine (2A):
1
Yield: 65%; H NMR (300 MHz, CDCl
3
) δ: 8.32 (d, J= 2.2 Hz, 1H), 7.59
(dd, J=2.1 Hz, J= 8.6 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.13 (t, J= 8.7 Hz, 2H),
-
6.89 (d, J= 8.7 Hz, 1H), 6.11 (s br, 2H); LRMS (ESI) m/z 231 [M-H] .
3'-fluoro-3-nitro-[1,1'-biphenyl]-4-amine (3A):
1
Yield: 70%; H NMR (300 MHz, CDCl ) δ: 8.37 (d, J= 2.2 Hz, 1H), 7.62
3
(
dd, J= 2.2 Hz, J= 8.6 Hz, 1H), 7.44 – 7.30 (m, 3H), 7.03 (t, J= 8.9 Hz,
-
1
H), 6.90 (d, J= 8.7 Hz, 1H), 6.15 (s br, 2H); LRMS (ESI) m/z 231 [M-H] .
Experimental Section
4'-methoxy-3-nitro-[1,1'-biphenyl]-4-amine (4A):
1
Yield: 29%; H NMR (300 MHz, CDCl
3
) δ: 8.32 (d, J= 2.2 Hz, 1H), 7.60
(dd, J=2.2 Hz, J=8.6 Hz, 1H), 7.55 – 7.41 (m, 2H), 7.02 – 6.93 (m, 2H),
6
.87 (d, J= 8.6 Hz, 1H), 6.07 (s br, 2H) 3.85 (s, 3H); LRMS (ESI) m/z 243
High analytical grade chemicals and solvents were purchased from
commercial suppliers. When necessary, solvents were dried by standard
techniques and distilled. After extraction from aqueous layers, the
organic solvents were dried over anhydrous sodium sulfate. Thin layer
chromatography (TLC) was performed on aluminum sheets pre-coated
with silica gel 60 F254 (0.2 mm) (E. Merck). Chromatographic spots were
visualized by UV light. Purification of crude compounds was carried out
by flash column chromatography on silica gel 60 (Kieselgel 0.040−0.063
-
[M-H] .
3'-methoxy-3-nitro-[1,1'-biphenyl]-4-amine (5A):
1
Yield: 60%; H NMR (300 MHz, CDCl
3
) δ: 8.38 (d, J= 2.2 Hz, 1H), 7.64
(dd, J= 2.2 Hz , J= 8.6 Hz, 1H), 7.37 (d, J= 7.9 Hz, 1H), 7.14 (d, J= 7.7
Hz, 1H), 7.09 – 7.06 (m, 1H), 6.89 (d, J= 8.6 Hz, 2H), 6.11 (s br, 2H),
-
3.87 (s, 3H); LRMS (ESI) m/z 243 [M-H] .
3,3',5'-trifluoro-[1,1'-biphenyl]-4-amine (6A):
1
Yield: 10% H NMR (300 MHz, CDCl
3
) δ: 7.24 – 7.13 (m, 2H), 7.06 – 6.97
mm, E. Merck) or by preparative TLC on silica gel 60 F254 plates or
1
(m, 2H), 6.83 (dd, J= 8.3 Hz, J= 8.9 Hz, 1H), 6.76-6.67 (m, 1H), 3.86 (s
crystallization. H NMR spectra were recorded in DMSO-d
6
or CDCl
3
at
-
br, 2H); LRMS (ESI) m/z 222 [M-H] .
300 MHz on a Varian Mercury 300 instrument. Chemical shifts (δ scale)
5
-(3,5-difluorophenyl)pyridin-2-amine (7A):
are reported in parts per million (ppm) relative to the central peak of the
solvent. Coupling constant (J values) are given in hertz (Hz). Spin
multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t
1
Yield: 70%; δ: H NMR (300 MHz, CDCl
3
) δ: 8.29 (d, J= 2.4 Hz, 1H), 7.62
(dd, J= 2.5 Hz, J= 8.5 Hz, 1H), 7.08 – 6.95 (m, 2H), 6.79-6.69 (m, 1H),
-
6.57 (d, J= 8.6 Hz, 1H), 4.58 (s br, 2H); LRMS (ESI) m/z 205 [M-H] .
(triplet), dd (double doublet), dt (double triplet), or m (multiplet). LRMS
7
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ChemMedChem
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FULL PAPER
1
Methyl 4-amino-4'-fluoro-[1,1'-biphenyl]-3-carboxylate (8A):
6
Yellow solid (yield: 27%); Mp>250 °C; H NMR (300 MHz, DMSO-d ) δ:
1
Yield: 17%; H NMR (300 MHz, CDCl
3
) δ: 8.06 (d, J= 2.3 Hz, 1H), 7.52-
12.27 (s br, 1H), 8.82 - 8.74 (m, 2H), 8.53 (d, J= 2.1 Hz, 1H), 8.27 – 8.19
(m, 2H), 8.16-8.07 (m, 1H), 7.79 – 7.69 (m, 1H), 7.66-7.56 (m, 2H), 7.35-
7
3
4
.44 (m, 3H), 7.16 – 7.04 (m, 2H), 6.74 (d, J= 8.5 Hz, 1H), 5.78 (s br, 2H),
-
-
.90 (s, 3H); LRMS (ESI) m/z 244 [M-H] .
7.25 (m, 1H); LRMS (ESI) m/z 354 [M-H] .
-amino-4'-fluoro-[1,1'-biphenyl]-3-sulfonamide (9A):
N-(3',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-yl)isonicotinamide. (23):
1
1
Yield: 35%; H NMR (300 MHz, CDCl
3
) δ: 7.97 (d, J= 2.2 Hz, 1H), 7.55
Yellow solid (yield: 17%); Mp:233-235 °C; H NMR (300 MHz, CDCl
3
) δ:
(
dd, J= 2.2 Hz, J= 8.4 Hz, 1H), 7.51-7.44(m, 2H), 7.11 (t, J= 8.7 Hz, 2H),
11.48 (s br, 1H), 9.11 (d, J= 8.9 Hz, 1H), 8.93 – 8.86 (m, 2H), 8.50 (d, J=
2.2 Hz, 1H), 7.94 (dd, J= 2.2 Hz, J= 8.9 Hz, 1H), 7.88 – 7.80 (m, 2H),
-
6
.88 (d, J= 8.4 Hz, 1H), 4.86 (s br, 4H). LRMS (ESI) m/z 265 [M-H] .
-
4
'-fluoro-3-methoxy-[1,1'-biphenyl]-4-amine (10A):
7.19 – 7.09 (m, 2H), 6.88 (t, J= 8.7 Hz, 1H); LRMS (ESI) m/z 354 [M-H] .
1
Yield: 66%. H NMR (300 MHz, CDCl
3
) δ: 7.51 – 7.43 (m, 2H), 7.12–7.03
N-(4'-fluoro-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (24):
1
(m, 2H), 7.00–6.94 (m, 2H), 6.76 (d, J= 7.6 Hz, 1H), 3.92 (s, 3H), 3.86 (s
Yellow solid (yield: 50%); Mp:177-178 °C; H NMR (300 MHz, CDCl
3
) δ:
-
br, 2H); LRMS (ESI) m/z 216 [M-H] .
11.39 (s br, 1H), 9.28 (d, J= 2.3 Hz, 1H), 9.05 (d, J= 8.8 Hz, 1H), 8.86 (dd,
J= 1.6 Hz, J= 4.8 Hz, 1H), 8.48 (d, J= 2.2 Hz, 1H), 8.35 – 8.25 (m, 1H),
7.93 (dd, J= 2.2 Hz, J= 8.8 Hz, 1H), 7.60 (dd, J= 5.2 Hz , J=8.8 Hz, 2H),
7.51 (dd, J= 4.8 Hz, J= 8.0 Hz, 1H), 7.19 (t, J= 8.6 Hz, 2H); LRMS (ESI)
4
-amino-4'-fluoro-[1,1'-biphenyl]-3-carbonitrile (11A):
1
Yield: 79%; H NMR (300 MHz, CDCl
3
) δ: 7.59 – 7.48 (m, 2H), 7.47–7.38
(
m, 2H), 7.16–7.05 (m, 2H), 6.81 (d, J= 8.5 Hz, 1H), 4.45 (s br, 2H);
-
-
LRMS (ESI) m/z 211 [M-H] .
m/z 336 [M-H] .
4
'-fluoro-4-nitro-[1,1'-biphenyl]-3-amine (12A):
N-(3'-fluoro-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide. (25):
1
1
Yield: 28%; H NMR (300 MHz, CDCl
3
) δ: 8.18 (d, J= 8.9 Hz, 1H), 7.60 –
Yellow solid (yield: 58%); Mp:185-186 °C; H NMR (300 MHz, DMSO-d
6
)
7
.52 (m, 2H), 7.15 (t, J= 8.7, 2H), 6.93 (d, J=1.8 Hz, 1H), 6.89 (dd, J= 1.9
δ: 10.98 (s br, 1H), 9.12 (d, J= 1.6 Hz, 1H), 8.81 (dd, J =1.6 Hz, J= 4.8
Hz, 1H), 8.33- 8.25 (m, 2H), 8.12 (dd, J=2.2 Hz, J=8.5 Hz, 1H), 7.81 (d,
J= 8.5 Hz, 1H), 7.71 – 7.50 (m, 4H), 7.32 – 7.20 (m, 1H); LRMS (ESI)
-
Hz, J= 8.9 Hz, 1H), 6.15 (s br, 2H). LRMS (ESI) m/z 231 [M-H] .
Synthesis of N-(4'-fluoro-4-nitro-[1,1'-biphenyl]-3-yl)acetamide (13 A):
-
0
.86 mmol (0,200 g) of 12A were dissolved in 4 ml of dry THF and 1.73
mmol (0.123 ml) of acetyl chloride were added. The mixture was stirred
for 72 h and then poured in a NH Cl saturated aqueous solution. The
m/z 336 [M-H] .
N-(4'-methoxy-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (26):
1
4
Yellow solid (yield: 42%) ; Mp:182-183 °C; H NMR (300 MHz, CDCl
3
) δ:
obtained precipitate was filtered and dried under vacuum.
11.37 (s br, 1H), 9.27 (d, J= 1.8 Hz, 1H), 9.01 (d, J= 8.8 Hz, 1H), 8.85 (dd,
J= 1.6 Hz, J= 4.8 Hz, 1H), 8.47 (d, J= 2.2 Hz, 1H), 8.29 (ddd J=1.7 Hz,
J=2.3 Hz, J=8.0 Hz, 1H), 7.93 (dd, J= 2.2 Hz, J=8.8 Hz,1H), 7.57 (d, J=
8.9 Hz, 2H), 7.51 (dd, J= 4.8 Hz, J= 8.0 Hz, 1H), 7.02 (d, J= 8.8 Hz, 2H),
1
Yield: 66%; H NMR (300 MHz, CDCl
3
) δ: 10.49 (s br, 1H), 9.04 (d, J=
2.0 Hz, 1H), 8.29 (d, J= 8.8 Hz, 1H), 7.68 – 7.60 (m, 2H), 7.35 (dd, J= 2.0
Hz, J= 8.8 Hz, 1H), 7.17 (t, J=8.7, 2H), 2.32 (s, 3H); LRMS (ESI) m/z 273
-
-
[
M-H] .
3.88 (s, 3H); LRMS (ESI) m/z 348 [M-H] .
Synthesis of N-(4-amino-4'-fluoro-[1,1'-biphenyl]-3-yl)acetamide (14A):
.36 mmol (0.100g) of 13 A were dissolve in 5 ml of MeOH and 1,8 mmol
0.406g) of SnCl were added and the mixture was stirred for 24h. The
N-(3'-methoxy-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (27):
1
0
Yellow solid (yield: 73%); Mp:154-155 °C; HNMR (300 MHz, CDCl
3
) δ:
(
2
11.40 (s br, 1H), 9.28 (d, J= 2.2 Hz, 1H), 9.04 (d, J= 8.8 Hz, 1H), 8.85 (d,
J= 3.3 Hz, 1H), 8.52 (d, J= 2.2 Hz, 1H), 8.30 (d, J= 7.7 Hz, 1H), 7.97 (dd,
J= 1.9 Hz, J= 8.8 Hz, 1H), 7.51 (dd, J=4.9 Hz, J= 7.9 Hz, 1H), 7.42 (t, J=
7.9 Hz, 1H), 7.23-7.19 (m, 1H), 7.14 (s, 1H), 6.97 (d, J= 8.5 Hz, 1H), 3.90
2 2
mixture was evaporated and purified by chromatography (CH Cl as
eluent) obtaining the desired product.
1
Yield: 87%; H NMR (300 MHz, DMSO-d
6
) δ: 9.46 (s br, 1H), 7.60–7.46
-
(
m, 2H), 7.33–7.16 (m, 4H), 7.09 (s, 1H), 6.92 (s br, 2H), 2.06 (s, 3H);
(s, 3H); LRMS (ESI) m/z 348 [M-H] .
+
LRMS (ESI) m/z 245 [M+H] .
N-(4'-fluoro-4-nitro-[1,1'-biphenyl]-3-yl)nicotinamide (28):
1
Synthesis of compounds 3, 22-31, 33-37.
Yellow solid (yield: 10%); Mp:190-191 °C; H NMR (300 MHz, DMSO-d
6
)
The intermediate anilines 1A-12A and 14A (1 mmol) were dissolved in
dry dioxane (0.1 M) and then nicotinoyl chloride hydrochloride (3 mmol),
DMAP (1 mmol ) and TEA (6 mmol) were added. The mixture was
refluxed for 18 hours. After cooling the obtained precipitate was removed
by filtration. The filtrate was evaporated and the residue was purified by
crystallization or silica column chromatography.
δ: 10.98 (s br, 1H), 9.12 (d, J= 1.8 Hz, 1H), 8.79 (dd, J=1.8 Hz, J= 4.7 Hz,
1H), 8.30 (dt, J= 1.8 Hz, J= 8.2 Hz, 1H), 8.09-8.04 (m, 2H), 7.82-7.78 (m,
2H), 7.71 (dd, J=1.8 Hz, J= 8.8 Hz, 1H), 7.60 (dd, J=5.2 Hz, J= 8.2 Hz,
-
1H) 7.39-7.33 (m, 2H). LRMS (ESI) m/z 336 [M-H] . .
N-(3,3',5'-trifluoro-[1,1'-biphenyl]-4-yl)nicotinamide (29):
1
White solid (yield: 30%); Mp:239-240 °C; H NMR (300 MHz, CDCl
3
) δ:
To synthesize compound 23 the commercially available isonicotinoyl
chloride was used for the reaction with compound 1A in the same
conditions.
9.15 (s, 1H), 8.83 (d, J= 4.8 Hz, 1H), 8.56 (t, J= 8.3 Hz, 1H), 8.24 (d, J=
8.2 Hz, 1H), 8.08 (s, 1H), 7.54 – 7.46 (m, 1H), 7.45 – 7.34 (m, 2H), 7.12-
-
7.05 (m, 2H), 6.86-6.76 (m, 1H); LRMS (ESI) m/z 327 [M-H] .
For compound 22 and 37 the starting aryloyl acid (1.5 mmol) was
suspended under argon in thionyl chloride (2.0 mL) and heated under
reflux for 2h. The unreacted excess of thionyl chloride was removed
under nitrogen flow to afford the corresponding aryloyl chloride. The solid
obtained was used without further purification for the reaction with the
aniline 1A and 11A, respectively, in the condition described above.
N-(5-(3,5-difluorophenyl)pyridin-2-yl)nicotinamide (30):
1
White solid (yield: 12%); Mp:181-182 °C; H NMR (300 MHz, DMSO-d
6
)
δ: 11.26 (s br, 1H), 9.14 (s, 1H), 8.82 (s, 1H), 8.75 (d, J= 3.3 Hz, 1H),
8.35 (d, J=8.0 Hz, 1 H), 8.27 (s, 2H), 7.61-7.50 (m, 3H), 7.31-7.18 (m,
-
1H); LRMS (ESI) m/z 310 [M-H] .
Methyl 4'-fluoro-4-(nicotinamido)-[1,1'-biphenyl]-3-carboxylate(31):
1
N-(3',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (3):
White solid (yield: 6%); Mp:189-190 °C; H NMR (300 MHz, CDCl
3
) δ:
1
Yellow solid (yield: 16%.); Mp:188-190 °C; H NMR (300 MHz, DMSO-d
6
)
12.19 (s br, 1H), 9.32 (d, J=2.0 Hz, 1H), 8.98 (d, J= 8.8 Hz, 1H), 8.81 (d,
J= 4.8 Hz, 1H), 8.38 – 8.31 (m, 1H), 8.29 (d, J= 2.3 Hz, 1H), 7.82 (dd,
J=2.3 Hz, J= 8.8 Hz, 1H), 7.62 – 7.54 (m, 2H), 7.48 (dd, J=4.8 Hz, J= 8.0
δ: 11.00 (s br, 1H), 9.12 (d, J= 1.7 Hz, 1H), 8.85 – 8.75 (m, 1H), 8.35 (d,
J= 2.0 Hz, 1H), 8.33 – 8.26 (m, 1H), 8.15 (dd, J= 2.0 Hz, J= 8.6 Hz, 1H),
-
7
.82 (d, J= 8.5 Hz, 1H), 7.65-7.56 (m, 3H), 7.35-7.26 (m,1H); LRMS (ESI)
Hz, 1H), 7.16 (t, J= 8.7 Hz, 2H), 4.01 (s,3H); LRMS (ESI) m/z 349 [M-H] .
-
m/z 354 [M-H] .
N-(4'-fluoro-3-sulfamoyl-[1,1'-biphenyl]-4-yl)nicotinamide (33):
N-(3',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-yl)picolinamide (22):
8
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ChemMedChem
10.1002/cmdc.201700365
FULL PAPER
1
White solid (yield: 15%); Mp>250 °C; H NMR (300 MHz, DMSO-d
6
) δ:
plates were further incubated at 37 °C for 72 h. Cells were fixed in situ by
the slow addition of cold TCA (50 μL, 50% w/v, final concentration 10%)
and incubated for 1 h at 4 °C. The supernatant was discarded, and the
plates were washed four times with tap water and air-dried.
Sulforhodamine- B solution (100 μL, 0.4% w/v, in 1% acetic acid) was
added to each well, and plates were incubated for 30 min at room
temperature. After staining, unbound dye was removed by washing five
times with acetic acid (1 %), and the plates were air-dried. Bound stain
was then solubilized in 10 mM 2-amino-2-(hydroxymethyl)-1,3-
propanediol (Trizma base), and the absorbance was read on an
automatic plate reader at 570 nm. The compound concentration able to
1
2
2.48 (s br, 1H), 9.21 (s, 1H), 8.82 (s, 1H), 8.45 – 8.37 (m, 1H), 8.03 (s,
H), 7.85 – 7.75 (m, 3H), 7.65 (s, 2H), 7.39 – 7.22 (m, 3H); LRMS (ESI)
-
m/z 370 [M-H]
N-(4'-fluoro-3-methoxy-[1,1'-biphenyl]-4-yl)nicotinamide (34):
1
White solid (yield: 33%); Mp:139-140 °C; H NMR (300 MHz, DMSO-d
6
)
δ: 9.14 (d, J= 1.7 Hz, 1H), 8.80 (dd, J=1.7 Hz, J=4.8 Hz, 1H), 8.58 (s ,
1
H), 8.55 (s, 1H), 8.29 – 8.21 (m, 1H), 7.58 – 7.52 (m, 2H), 7.47 (dd, J=
4
.9 Hz, J= 7.9 Hz, 1H), 7.24 – 7.19 (m, 1H), 7.17 – 7.08 (m, 3H), 4.01
-
(s,3H); LRMS (ESI) m/z 321 [M-H] .
N-(3-acetamido-4'-fluoro-[1,1'-biphenyl]-4-yl)nicotinamide (35):
1
White solid (yield: 25%); Mp:204-205 °C; HNMR (300 MHz, CDCl
3
) δ:
inhibit cell growth by 50% (IC50 ± SD) was then calculated from
9.74 (s br, 1H), 9.20 (s, 1H), 8.74 (s br, 1H), 8.46 (s, 1H), 8.18-8.14 (m,
semilogarithmic dose–response plots.
1
H), 7.70 (d, J= 8.2 Hz, 1H), 7.35-7.27 (m, 2H), 7.24-7.22 (m, 2H), 7.01
-
(t, J= 8.75 Hz, 2H), 2.13 (s, 3H); LRMS (ESI) m/z 348 [M-H] .
High-content cell cycle analysis
N-(3-cyano-4'-fluoro-[1,1'-biphenyl]-4-yl)nicotinamide (36):
Treatment-induced modifications of cell cycle phases were investigated
using the high-content imaging system Operetta (Perkin Elmer Life
Sciences, Boston, MA), along with Harmony software and PhenoLOGIC
1
White solid (yield: 47%); Mp:223-224 °C; H NMR (300 MHz, DMSO-d
6
)
δ: 9.22 (d, J= 2.3 Hz, 1H), 8.86 (dd, J= 1.6 Hz, J= 4.8 Hz, 1H), 8.64 (d,
J= 8.7 Hz, 1H), 8.35 (s, 1H), 8.28 – 8.21 (m, 1H), 7.88 – 7.79 (m, 2H),
machine learning, according to a method recently described by Massey
-
[26]
7.57-7.47 (m, 3H), 7.22-7.14 (m, 1H); LRMS (ESI) m/z 316 [M-H] .
AJ.
Briefly, MCF7 or MDA-MB231 cells in complete culture medium
N-(3-cyano-4'-fluoro-[1,1'-biphenyl]-4-yl)-3,4,5-trimethoxybenzamide (37):
were seeded (4500 and 3000 cells/well, respectively) in a 96-well
collagen-coated plate (Perkin Elmer), allowed to attach for 24 h in normal
1
White solid (yield: 20%); Mp:220-221 °C; H NMR (500 MHz, DMSO-d
6
)
δ: 10.55 (s br, 1H), 8.19 (d, J= 2.2 Hz, 1H), 8.04 (dd, J= 2.3 Hz, J= 8.5
2
humified atmosphere supplemented with 5% CO , and then treated in the
Hz, 1H), 7.86 – 7.80 (m, 2H), 7.63 (d, J= 8.5 Hz, 1H), 7.34 (s, 3H), 7.33 –
same conditions for further 24 h with different concentrations of freshly
dissolved compounds. Cells were labelled with 10 μM (final
concentration/well) 5-ethynyl-2’-deoxyuridine (EdU) for 30 min. After EdU
incubation, media was removed immediately prior to fixation of adherent
cells with formaldehyde 3.7% (v/v) in PBS at room temperature for 15
min. Cells were washed twice with PBS and permeabilized with Triton X-
100 (Promega) for 15 min at room temperature. After two washes with
PBS, incorporated EdU was detected with Alexa Fluor 488 by a Click-iT®
EdU HCS Assay (C10350, Life Technologies) according to the
manufacturer’s recommended procedure. For phospho-histone H3 Ser28
detection, cells were incubated with 3% (w/v) bovine serum albumin
(BSA) overnight at 4 °C, and then incubated for 1 h at room temperature
in the dark with the anti-Phospho-Histone H3 primary antibody (Anti-
Phospho-Histone H3 pSer28, clone HTA28, SIGMA), 2 µg/ml final
concentration in BSA 3%. After three washes with 0.05% Tween® 20,
cells were incubated with the secondary antibody (Alexa Fluor® 647
Goat anti-Rat IgG, ThermoFisher Scientific), 10 µg/ml final concentration
in BSA 3%, for 1 h at room temperature in the dark. Following additional
three washes with 0.05% Tween® 20, nuclear staining was performed for
15 min at room temperature in the dark by NuclearMask™ Blue Stain
(Life Technologies), 100 µL/well. Finally, following two PBS washes, cells
were imaged with an Operetta high-content imaging system (Perkin-
Elmer) using a 20x long working distance objective. Typically, 10 fields
per well were imaged which equated to approximately 3000 cells/well.
Analysis sequence. Image acquisition and data analysis were performed
by using the Harmony ® Image Analysis Software with PhenoLOGIC
(Perkin-Elmer, Inc. MA, USA). Automated analysis was performed to
obtain as readout the total number of cells (for information and quality
control) and DNA content on a single-cell basis (nuclear mask intensity
sum). From the single cell results a histogram of nuclear mask sum was
generated (Excel software) and the intensity corresponding to the center
of the G1 peak determined. Based upon this value, a histogram of
normalized DNA (relative DNA content) was obtained from vehicle-
treated wells on a per-plate basis and applied to all wells to identify sub-
G1, G1, S, G2/M, and > G2/M cell populations. Multiparametric analysis
of cell-cycle was then performed by using relative DNA content values
along with mean marker intensity values of EdU-positive and PHH3-
-
7.30 (m, 1H), 3.86 (s, 6H), 3.74 (s, 3H); LRMS m/z 405 [M-H] .
Synthesis of 4'-fluoro-4-(nicotinamido)-[1,1'-biphenyl]-3-carboxylic acid
32) 0.1 mmol (0.035 g) of compound 11 were dissolved in 4 mL of a
mixture THF/water 3:1 and 0.1 mmol (0.003g) of LiOH were added. After
h the mixture was evaporated and the residue was treated with HCl 1N
(
3
(
5 mL) . The obtained precipitate was filtered and washed with water.
1
White solid (yield: 55%); Mp>250 °C; H NMR (300 MHz, DMSO-d
6
) δ:
1
8
2
3.91 (s br, 1H), 12.29 (s br, 1H), 9.14 (s, 1H), 8.81 (s, 1H), 8.70 (d, J=
.7 Hz, 1H), 8.36 – 8.23 (m, 2H), 7.97 (d, J= 8.0 Hz, 1H), 7.79 – 7.68 (m,
H), 7.68 – 7.56 (m, 1H), 7.30 (t, J= 8.7 Hz, 2H); LRMS (ESI) m/z 335.0
-
[M-H]
Pharmacological assays
Tumor Cell Lines and In Vitro Growth Inhibition Assay:
Two human tumor cell lines of breast MCF7 and MDA-MB 231 were
obtained from the National Cancer Institute, Biological Testing Branch
(Frederick, MD, USA). The cell lines MCF7 and MDA-MB231 were
maintained in the logarithmic phase at 37 °C in a 5% CO humidified air
2
in RPMI 1640 medium supplemented with 10% fetal calf serum and
respectively with, 1% glutamine (200 mM), 1% penicillin and
streptomycin (100 U/mL and 0.1 mg/mL) for MCF7 and 1,5% glutamine
(
200 mM), 2% penicillin and streptomycin (100 U/mL and 0.1 mg/mL) and
% Na Pyruvate (100 mM) for MDA-MB231. The growth-inhibitory effect
of the compounds under investigation was evaluated by using the
1
[
29]
Sulforhodamine-B (SRB) assay.
Briefly, cells were seeded into 96-well microtiter plates in culture medium
100 μL) at a plating density of 5000 and 10000 cells/well to MCF-7 and
(
MDA-MB-231 respectively. After seeding, microtiter plates were
incubated at 37 °C for 24 h prior to addition of the compounds. After 24 h,
samples of each cell line were fixed in situ with cold trichloroacetic acid
(TCA), to represent a measurement of the cell population at the time of
compound addition. The compounds to be tested (the stock solution was
prepared at a concentration of 1 mM in DMSO) were diluted freshly in
culture medium to the desired final concentrations (2.5–0.002 μM). After
the addition of different compound concentrations to triplicate wells, the
9
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ChemMedChem
10.1002/cmdc.201700365
FULL PAPER
[
[
6] S. X. Cai, B. Nguyen, S. Jia, J.Herich, J. Guastella, S. Reddy, B.
positive cells to specifically identify sub-G1, G1, S, G2, M, and > G2/M
cells.
Tseng, J. Drewe, S. Kasibhatla, J. Med. Chem. 2003, 46,
2474─2481.
7] T. Suzuki, M. N. Ahmed Khan, H. Sawada, E. Imai, Y .Itoh, K.
Yamatsuta, N. Tokuda, J. Takeuchi, T. Seko, H. Nakagawa, N.
Miyata, J. Med. Chem. 2012, 55, 5760─5773.
Statistical analysis
To determine concentration-dependent cell-cycle modifications,
comparisons were made between each dose and the respective vehicle-
only control. The cell fraction was calculated for each sample and paired
two-tailed Student’s t-test (two sample equal variance, P < 0.05) was
used to determine the significance of the response. Values represent the
means ± SD of 5-8 replicates.
[8] M. E. Kort, R. N. Atkinson, J. B. Thomas, I. Drizin, M. S. Johnson,
M. A. Secrest, R. J. Gregg, M. J. C. Scanio, L. Shi, A. H. Hakeem,
M. A. Matulenko, M. L. Chapman, M. J. Krambis, D. Liu, C.-C.
Shieh, X. Zhang, G. Simler, J. P. Mikusa, C. Zhong, S. Joshi, P.
Honore,; R. Roeloffs, S. Werness, B. Antonio, K. C. Marsh, C.R.
Faltynek, D. S. Krafte, M.F. Jarvis, B. E. Marron, Bioorg. Med.
Chem. Lett. 2010, 20, 6812─6815.
In vitro tubulin polymerization assay
[
9] A. B. Eldrup, F. Soleymanzadeh,; S. J. Taylor, I. Muegge, N. A.
Farrow, D. Joseph, K. McKellop, C. C. Man, A. Kukulka, S. De
Lombaert, J. Med. Chem. 2009, 52, 5880–5895.
A number of selected compounds were tested at 3 µM concentration in a
tubulin polymerization assay kit (Cytoskeleton Inc., Denver, CO, USA).
Lyophilized tubulin (Cytoskeleton #T240) was used to evaluate the
tubulin polymerization inhibiting activity. Vinblastine and paclitaxel at 3.0
μM were used as positive controls for tubulin polymerization inhibition and
stabilization, respectively. All compounds were dissolved in DMSO and
[
10] J. L. Methot,; P. K. Chakravarty, M. Chenard, J. Close, J. C. Cruz,
W. K. Dahlberg, J. Fleming, C. L. Hamblett, J. E. Hamill, P.
Harrington, A. Harsch,; R. Heidebrecht, B. Hughes, J. Jung, C.M.
Kenific, A. M. Kral, P.T. Meinke, R. E. Middleton, N. Ozerova, D.L.
Sloman, M. G. Stanton, A. A. Szewczak, S. Tyagarajan, D. J.
Witter, J. P. Secrist,; T. A. Miller, Bioorg. Med. Chem. Lett. 2008,
18, 973─978.
further diluted with sterile water to obtain
a maximum DMSO
concentration of 0.1%, which was used as solvent control. Polymerization
was monitored by fluorescence enhancement due to the incorporation of
a fluorescent reporter into microtubules as polymerization occurs. In a 96
well plate, to a 0.5 μL of a 100x stock solution of each compound, 49.5
μL of supplemented tubulin supernatant were added. Incubation was
[
[
[
11] C. Reichwalda, O. Shimonyb, N. Sacerdoti-Sierrab, C. L. Jaffeb, C.
Kunicka, Bioorg. Med. Chem. Lett. 2008, 18, 1985─1989.
12] V. J. Huber, M. Tsujita, T. Nakada, Bioorg. Med. Chem. 2009, 17,
411–417.
13] National Center for Biotechnology Information. PubChem
done in
a temperature-controlled Multi-label Microplate Fluorimeter,
Compound
Database;
CID=279877,
(accessed
equipped with filters for excitation at 340-360 nm and emission at 420-
https://pubchem.ncbi.nlm.nih.gov/compound/279877
Dec 30, 2015).
460 nm (Victor 3 Model 1420-01296, Perkin-Elmer, Inc. MA, USA) at
[
14] G. F.Smith,. Prog Med Chem, 2011, 50,1─47.
3
7 °C, and fluorescence was measured at 460 nm every minute for 60
[
30]
[15] V. Purohit, A. K. Basu, Chem Res Toxicol, 2000, 13, 673-92.
min according the recommended procedures.
[
[
16] F. F. Fleming, L. Yao, P. C. Ravikumar, L. Funk, B. Shook, J. Med.
Chem. 2010, 53, 7902─7917
17] a) S. T. Murphy, H. L. Case, E. Ellsworth, S. Hagen, M. Huband, T.
Joannides, C. Limberakis, K. R. Marotti, A. M. Ottolini, M.
Rauckhorst, J. Starr, M. Stier, C. Taylor, T. Zhu, A. Blaser, W. A.
Denny, G. L. Lu, J. B. Smaill, F. Rivault, Bioorg. Med. Chem.Lett.
Acknowledgements
The authors would like to thank Giovanni Dipinto (Department of
Pharmacy-Drug Sciences, University of Bari) for the assistance
in Q-TOF Mass Spectrometry. The financial support by the
University of Bari Aldo Moro is gratefully acknowledged.
2
007, 17, 2150–2155. b) B. Markus, C.-H. Kwon, J. Pharm. Sci.
994, 83, 1729–1734.
1
[18] a) M. J. Boyd, S. N. Crane, J. Robichaud, J. Scheigetz, W. C.
Black,; N. Chauret, Q. Wang, F. Masse, R. M. Oballa, Bioorg. Med.
Chem. Lett. 2009, 19, 675─679. (b) A. W. Patterson, W. J. L.
Wood, M. Hornsby, S. Lesley, G. Spraggon, J. A.Ellman, J. Med.
Chem. 2006, 49, 6298─6307.
Keywords: Nicotinoyl anilides, Antiproliferative assay, High-
content cell cycle analysis, MCF-7 and MDA-MB231 tumor cell
lines.
[19] J.-Y. Le Questel, M. Berthelot, C. Laurence, J. Phys. Org. Chem.
2000, 13, 347─358.
[
20] E. L. Eliel, S. H. Wilen, L. N. Mander, Stereochemistry of Organic
Compounds, Wiley, 1994, p. 696─697.
[
21] N. Teno, T. Miyake, T. Ehara, O. Irie, J. Sakaki, O. Ohmori, H.
Gunji, N. Matsuura, K. Masuya, Y. Hitomi, K. Nonomuraa, M.
Horiuchia, K. Gohdaa, A. Iwasakia, I. Umemuraa, S. Tadaa, M.
Kometania, G. Iwasakia,; S. W. Cowan-Jacobb, M. Missbachb, R.
Lattmannb, C. Betscharta, Bioorg. Med. Chem. Lett. 2007, 17,
References:
[
[
[
1] D. W. Lee, Y-S. Choi, S. Y. Jee, M-Y. Lee, S. Y. Jeon, B. Ku, S.
Kim, S. H. Yi, D-H. Nam, Anal. Chem. 2014, 86, 535-542.
2] R. Zang, D. Li, I-C. Tang, J. Wang, S-T. Yang, Int. J. Biotech. Well.
Indus. 2012, 1, 31-51.
6
096–6100.
[
22] a) C. Laurence, K. A Brameld,. J. Graton, J.-Y. Le Questel, E.
Renault, J. Med. Chem. 2009, 52, 4073–4086. (b) A. Allerhand,
P.Schleyer, von Rague, J. Am. Chem. Soc. 1963, 85, 866–870.
23] T. Nogrady, D. F. Weaver, Medicinal Chemistry: A Molecular and
Biochemical Approach, Oxford University Press, 2005, p. 22.
3] R. Z. Pellicani, A. Stefanachi, M. Niso, A. Carotti, F. Leonetti, O.
Nicolotti, R. Perrone, F. Berardi, S. Cellamare, N. A. Colabufo, J.
Med. Chem. 2012, 55, 424-436.
[
[
[
24] F. Gasparri, A. Ciavolella, A. Galvani. Adv Exp Med Biol 2007, 604,
[
[
4] P. Tardia, A. Stefanachi, M. Niso, D.A. Stolfa,; G. F. Mangiatordi, D.
Alberga, O. Nicolotti, G. Lattanzi, A. Carotti, F. Leonetti, R.
Perrone, F. Berardi, A. Azzariti, N. A. Colabufo, S. Cellamare. J
Med Chem. 2014, 57, 6403─6418.
1
37-148.
25] G. K. Chan, T. L. Kleinheinz, D. Peterson, J. G. Moffat. PLoS One
013, 8(5), e63583.
2
[
[
26] A. J. Massey. PLoS One 2015, 10, e0134306.
27] B. T. Castle, S. McCubbin, L. S. Prahl, J. N. Bernens, D. Sept, D. J.
Odde. Mol Biol Cell 2017, doi: 10.1091/mbc.
5] a) P. D. W. Eckford; F. J. Sharom, Chem. Rev. 2009,109, 2989–
3011; b) B. C. Baguley, Methods Mol. Biol. 2010, 596, 1─14
1
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201700365
FULL PAPER
[
28] A. Stefanachi, G.F. Mangiatordi, P. Tardia, D. Alberga, F. Leonetti,
M. Niso, N.A. Colabufo, C. Adamo, O. Nicolotti, S. Cellamare.
Chem. Biol. Drug Des. 2016, 88, 820-831.
[
[
29] http://www.cytoskeleton.com/bk011p
30] V. Vichai, K. Kirtikara. Nature Protocols 2006, 1, 1112-1116.
1
1
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
R1
4-F
CN
X
R2
O
HN
N
Insert text for Table of Contents here.
A screening of a small library of trimethoxybenzoic and nicotinic acid anilides, previously studied as P-gp modulators, allowed us to
identify some N-biphenyl nicotinoyl anilides showing very interesting antiproliferative activities. The next hit refinement by a
biosisosteric approach, led us to observe potent antiproliferative activity of the cyano derivative 36 against MCF7 and MDA-MB231
cells lines.
1
2
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