Discovery and Optimization of N-(4-(3-Aminophenyl)thiazol-2-yl)acetamide as a Novel Scaffold Active against Sensitive and Resistant Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b00547

Cancer is the second cause of deaths worldwide and is forecasted to affect more that 22 million people in 2020. Despite dramatic improvement in its care over the last two decades, the treatment of resistant forms of cancer is still an unmet challenge. Thus, innovative and efficient treatments are still needed. In this context, we report herein the synthesis and the evaluation of a new class of bioactive molecules belonging to the N-(4-(3-aminophenyl(thiazol-2-yl)acetamide family. Structure-activity relationships could be driven and resulted in the discovery of lead compound 6b. The latter display high in vitro potency against both sensitive and resistant cancer cell lines on three models: melanoma, pancreatic cancer, and chronic myeloid leukemia (CML). 6b leads to cell death by concomitant induction of apoptosis and autophagy, shows good pharmacokinetic properties, and demonstrates a significant reduction of tumor growth in vivo on A375 xenograft model in mice.

Full text of DOI:10.1021/acs.jmedchem.6b00547

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Discovery and optimization of N-(4-(3-aminophenyl)thiazol-2-yl)acetamide
as a novel scaffold active against sensitive and resistant cancer cells
Antoine Millet, Magali Plaisant, Cyril Ronco, Michaël Cerezo, Patricia
Abbe, Emilie Jaune, Elisa Cavazza, Stéphane Rocchi, and Rachid Benhida
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00547 • Publication Date (Web): 30 Aug 2016
Downloaded from http://pubs.acs.org on August 30, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery and optimization of Nꢀ(4ꢀ(3ꢀ
aminophenyl)thiazolꢀ2ꢀyl)acetamide as a novel
scaffold active against sensitive and resistant cancer
cells
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1#
2,3#
1#
2,3
2,3
Antoine Millet , Magali Plaisant , Cyril Ronco , Michaël Cerezo , Patricia Abbe , Emilie
2
,3
2,3
2,3,4*
1*
Jaune , Elisa Cavazza , Stéphane Rocchi
and Rachid Benhida .
1
2
Institut de Chimie de Nice UMR UNSꢀCNRS 7272, Nice, France ; INSERM, U1065, Centre
Méditerranéen de Médecine Moléculaire (C3M), Equipe Biologie et Pathologie des cellules
3
mélanocytaires : de la pigmentation cutanée au mélanome, Nice, France ; Université de Nice
4
Sophia Antipolis, UFR de Médecine, Nice, France ; Service de Dermatologie, Hôpital Archet
II, CHU Nice, France.
ABSTRACT
Cancer is the second cause of deaths worldwide and is forecasted to affect more that 22 million
people in 2020. Despite dramatic improvement in its care over the last two decades, the
treatment of resistant forms of cancer is still an unmet challenge. Thus, innovative and efficient
treatments are still needed. In this context, we report herein the synthesis and the evaluation of a
new class of bioactive molecules belonging to the Nꢀ(4ꢀ(3ꢀaminophenyl(thiazolꢀ2ꢀyl)acetamide
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
family. Structureꢀactivity relationships could be driven and resulted in the discovery of lead
compound 6b. The latter display high in vitro potency against both sensitive and resistant cancer
cell lines on 3 models: melanoma, pancreatic cancer and chronic myeloid leukemia (CML). 6b
leads to cell death by concomitant induction of apoptosis and autophagy, shows good
pharmacokinetic properties and demonstrates a significant reduction of tumor growth in vivo on
A375 xenograft model in mice.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Cancer is one of the major causes of death with approximately 8.2 million lethal cases in 2012.
According to the American Cancer Society, 14.1 million people affected by cancer have been
inventoried in 2012 and this number is expected to reach 22 million cases over the next two
1
decades, due in part to the increase in life expectancy. Cancer is characterized by the
uncontrolled growth of abnormal cells that have the potential to invade and to spread throughout
the body with the consequence of threatening the survival of the organism.
Different strategies can be considered to relieve patients diagnosed with cancer, all having
however their own limitations. When possible, surgery and radiation therapy are applied as first
treatment to most tumor masses, Surgery is the most effective intervention for localized tumors
but often damage large parts of healthy tissues or organs and the small tumors or the not visible
2
metastases cannot be removed. In all cases, a molecular treatment must be applied to kill the
3
remaining cells , and those treatments are characterized by their general lack of efficiency and a
significant to crippling toxicity for radioꢀ and chemotherapy agents. Ultimately, the overall
efficacy of the responses to cancer remains quite limited, especially in advanced stages of the
disease when metastases develop, for which treatments often only provide a mean to delay death
events for some months.
ACS Paragon Plus Environment
2

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
One of the major causes of the failure of cancer chemotherapies is drug resistance, which
4
represents nowadays a critical clinical challenge in oncology. This phenomenon is encountered
either at the first attempt when tumors do not respond to treatment (intrinsic resistance), or over
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
the course of the treatment when tumors initially respond but then relapse (acquired resistance).
Overall, tumor cell resistance is a complex process that expresses itself simultaneously at
different levels: with first line nonꢀspecific barriers that diminish intracellular drug concentration
5a
like efflux pumps or metabolism, then with more specific and flexible defenses related to the
6
signaling pathways or to the molecular target. The development of the targeted therapies in the
past 15 years on which many hopes have relied is particularly prone to the apparition of acquired
7
resistances. Immunotherapies, although recent advances have demonstrated promising results in
8
terms of efficacy and benefit for patients, are also concerned by intrinsic and acquired
9
resistances. Thus, in parallel or in combination with current therapies, the research and
development of new drugs is of great importance to fight intrinsic resistance, to delay the
emergence of resistant cell populations or to restore their sensitivity to the level before the
apparition of resistance.
In this context, we describe herein the discovery and optimization of a new class of drugꢀlike
molecules with antiꢀproliferative activity against tumor cells. Based on initial findings showing
1
0
antiꢀproliferative activity of the wellꢀknown antiꢀdiabetes drug troglitazone, a new scaffold was
constructed by iterative structureꢀactivity relationships: Nꢀ(4ꢀ(3ꢀaminophenyl(thiazolꢀ2ꢀ
yl)acetamides. From this scaffold, a number of compounds was synthesized and were found to
exhibit significant activity against a panel of cancer cell lines, both sensitive and resistant to
respective standard treatment drugs. We focused on three important pathologies: (1) melanoma,
which is an aggressive form of skin cancer displaying one of the highest mortality rates and for
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7b
which resistance problems to recent Bꢀraf inhibitors is a major concern, (2) Chronic
Myelogenous Leukemia (CML), a largely studied and well characterized tumor cell model, as
demonstrated by the successful development of the kinase inhibitor imatinib, and (3) highly
deadly pancreatic cancer, for which the keystone treatment proposed relies on the old nucleoside
analog gemcitabine, which displays low response rates, high toxicity, severe adverse effects and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
very high resistance occurrence, leading to only few weeks enhancement in median survival.
From the initial hits structure, 29 active compounds divided into two series were synthesized
and evaluated for their cytotoxic potential on a melanoma A375 cell lines model. The 10 most
active compounds were screened on a panel of melanoma, CML and pancreatic cancer cells,
either sensitive or resistant to the standard drugs. The high cellular activities measured against
these three cancers, especially their capacity to affect resistant cells, together with the selectivity
observed over healthy cells, prompted us to study the pharmacological parameters of our lead
compound 6b in mice. No apparent toxicity was observable based on animal behavior, diet, and
organs weight, and the pharmacokinetic profile proved amply favorable to in vivo studies, which
were conducted on melanoma xenograft mice models and demonstrated the potent in vivo
1
1
activity of compound 6b.
RESULTS AND DISCUSSION
Chemistry. This work was initiated from the discovery in our laboratories that molecules
belonging to thiazolidinediones (TZD) class were displaying not negligible death induction of
1
0
melanoma cell lines by apoptosis. TZD, and particularly its most representative derivative
troglitazone (Figure 1), are known PPARγ nuclear receptor ligands, which are previously used
in the treatment of type 2 diabetes to increase insulin signaling and regulate glucose
ACS Paragon Plus Environment
4

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
12
homeostasis. The independence between PPARγ activation and the cytotoxic effects of TZD
1
0a
observed on melanoma was subsequently demonstrated. From these promising preliminary
results, we started a medicinal chemistry study aimed at modifying the TZD structure in order to
overcome PPARγ activity and reach a full selectivity towards apoptosis of neoplastic cells. By
iterative structureꢀactivity relationships, we first showed that the thiazolidinedione part of
troglitazone was necessary for the antiꢀproliferative activity whereas the presence of the (1ꢀ
methylcyclohexyl)methoxy moiety rather provided affinity towards PPARγ receptors (data not
shown). It was later found that the thiazolidinedione moiety could be advantageously replaced by
a 2ꢀaminothiazole ring, and that the direct linkage, without the methylene group, to the phenyl
ring was also benefic. This leads to the identification of the first hits 4ꢀ(3ꢀaminophenyl)thiazolꢀ2ꢀ
amine and Nꢀ(4ꢀ(3ꢀaminophenyl)thiazolꢀ2ꢀyl)acetamide (Figure 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Structures of troglitazone and of the first hits identified
From these hit structures displaying moderate micromolar cellular activities (vide infra), we
explored modifications on the aniline nitrogen atom by attaching linear, cyclic or heteroaromatic
groups linked by an amide or sulfonamide function.
a
Scheme 1. Synthesis of analogues 4aꢀc and 5aꢀx
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagent and conditions : (a) Br , AlCl , diethyl ether, 0°C to r.t., 1h (97%); (b) Nꢀ
2
3
acetylthiourea, EtOH, 80°C, 30 min (92%); (c) NaBH , Pd/C (10% mol.), CH Cl /MeOH
4
2
2
(50:50), 0°C to r.t., 1h (83%); (d) acid chloride, pyridine, 0°C, 2h (61ꢀ82%); (e) sulfonyl
chloride, triethylamine, CH Cl /DMF (9:1), 25°C, (22ꢀ74%).
2
2
The synthesis of all analogs was performed as depicted in Scheme 1. The phenylꢀthiazole core
was prepared via a Hantzsch aminothiazole synthesis according to our previously reported
1
4
procedure. The first step consists in the αꢀbromination of metaꢀnitroacetophenone. The use of a
catalytic amount of aluminum trichloride under strict control of temperature and reaction time,
14
allowed obtaining only the desired monoꢀbrominated derivative in quantitative yields.
Then
bromoketone 1 is reacted with acetylthiourea under Hantzsch conditions to afford 2ꢀ
aminothiazole 2. Palladiumꢀhydride reduction of the nitro group gave the corresponding aniline
3
. The different derivatives were obtained by coupling aniline 3 with various acid chlorides (4aꢀ
c) or sulfonyl chlorides (5aꢀx). Alternatively, a Sonogashira coupling between 4ꢀiodoaryl 5x
derivative and the suitable terminal alkynes afforded compounds 6a and 6b (Scheme 2).
ACS Paragon Plus Environment
6

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 2. Synthesis of analogues 6aꢀb
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions : (a) 4ꢀiodosulfonyl chloride, triethylamine, CH Cl /DMF (9:1), 25°C,
2
2
20h (60%); (b) From 5x: Hexꢀ1ꢀyne or octꢀ1ꢀyne, DMF, triethylamine, Pd(OAc) , PPh , 50°C,
2h (72 to 89%).
2
3
The structures of all synthesized derivatives are summarized in
Table 1. The series A (2, 3, 4aꢀc, 5aꢀq) was first designed to explore the introduction of various
aromatic or heteroaromatic rings linked by an amide or sulfonamide function. Compounds of
series B (5rꢀx, 6aꢀb) were later synthesized to study the modulation in para position of
phenylsulfonyl group of 5f with various hydrophobic groups. In total 29 compounds were
synthesized and tested for their cytotoxic properties.
Table 1. Structure of all synthesized analogues.
Compound
R3
Compound
Series A
R3
4a
5h
5i
4b
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
c
5j
5k
5l
5a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5b
5c
5m
5d
5n
5o
5p
5e
5
f
5g
5q
Series B
5r
5w
5x
6a
5
s
5t
5u
6b
ACS Paragon Plus Environment
8

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5v
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Biological evaluation. Cytotoxic activity against melanoma sensitive cells. The cytotoxic
potency of the 22 compounds of series A was first evaluated using a cell viability assay on A375
human melanoma cells upon single dose treatment. All cell viability values are summarized in
Table 2.
Table 2. Evaluation of the activity of series A compounds
%
Cell
% Cell
viability
Compound
Compound
viability
47.3 ± 14.3
38.4 ± 10.1
40.0 ± 9.4
29.2 ± 3.6
18.3 ± 2.4
29.6 ± 11.0
45.7 ± 12.5
37.5 ± 5.9
36.8 ± 10.6
29.2 ± 3.6
72.2 ± 3.6
2
5g
5h
5i
51.2 ± 11.9
19.8 ± 3.5
26.3 ± 1.2
20.4 ± 6.2
3.3 ± 3.8
3
4a
4b
4c
5a
5j
5k
5l
46.5 ± 10.3
65.5 ± 11.1
60.7 ± 2.7
29.2 ± 5.9
59.5 ± 0.6
29.6 ± 14.6
5b
5m
5n
5o
5p
5q
5c
5d
5e
5f
a
Cell viability on A375 melanoma cells after a 48h treatment with 10µM of the indicated
compounds relative to a DMSO control ± SD from three independent experiments, as determined
by trypan blue exclusion assay.
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The structure of this first series of compounds and the measured activity allowed us to define
the first structureꢀactivity relationship. Our results showed that all compounds present an activity
in the medium to low micromolar range. 5ꢀmembered aromatic rings (4b, 5aꢀe) as well as
bicyclic heteroaromatics (5lꢀq) seem unfavorable, with viability values close or higher than the
parent aniline 3. Alkyl or aryl groups without Hꢀbond donating capability proved generally more
active than 3. Two interesting features are noteworthy. First, substitution with apolar groups on a
phenyl ring seems favorable (5gꢀk vs 5f) and para substitution preferred over meta position (5h
vs 5g, 5k vs 5j). Secondly, a sulfonamide is greatly preferred over an amide link (5k vs 4c).
These first findings prompted us to prepare a second series of 9 derivatives bearing apolar
groups in para position of a phenyl ring and linked on the aniline by a sulfonamide. The
activities of compounds of series B presenting different alkyl, halogen or alkyne groups are
summarized in Table 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Evaluation of the activity of series B compounds
%
Cell
% Cell
viability
Compound
a
Compound
a
viability
5
r
s
7.5 ± 1.2
2.2 ± 3.8
2.8 ± 4.8
1.5 ± 2.6
33.9 ± 14.0
5w
5x
6a
6b
26.3 ± 1.2
b
5
< 1.0
b
5
t
< 1.0
b
5
u
< 1.0
5
v
a
Cell viability on A375 melanoma cells after a 48 h treatment with 10µM of the indicated
compounds relative to a DMSO control ± SD from three independent experiments, as determined
b
by trypan blue exclusion assay. No viable cells.
With the exception of the bisꢀphenyl derivatives (5vꢀw), all compounds showed a strong
activity better than the parent 4ꢀtolyl group of 5h. Three compounds (5x, 6a and 6b)
ACS Paragon Plus Environment
10

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
demonstrated activities below the threshold of the test with no cell survival at 10µM after 48h
treatment.
Over the two series, the general tendency proved that aliphatic substituents in para position are
the best tolerated on this scaffold. This first cell viability screening allowed us to select 10
compounds (3 of series A and 7 of series B) displaying activities below 20% at 10µM to study
their cytotoxic potential against different sensitive and resistant cell lines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
On the basis of the initial SAR deduced from tables 2 and 3, the 10 selected compounds (4c,
5h, 5k, 5rꢀu, 5x, 6aꢀb) were evaluated against resistant melanoma cell lines as well as against
one hematologic cancer (CML) and one solid cancer (pancreatic cancer), which are significantly
affected by resistance phenomena.
Evaluation against melanoma sensitive and resistant cell lines. The growth inhibition was tested
for melanoma against human A375 sensitive cells, and compared to the resistant cell lines A375
R5C3 (rendered resistant after in vitro excessive prolonged exposure to vemurafenib, the
standard BRaf inhibitor), and A375 RIV (rendered resistant after in vivo excessive prolonged
15
exposure to ). The cell viability values are depicted in Table 4 and Chart 1.
Table 4. Evaluation of the activity against sensitive and resistant melanoma cell lines
Cell viability at 10µM for 48hours
a
a
a
%
Cell viability
% Cell viability
% Cell viability
Compound
A375
sensitive)
A375 R5C3
(resistant)
A375
RIV(resistant)
(
4c
5h
5k
18.3 ± 2.4
19.8 ± 3.5
3.3 ± 3.8
63.8 ± 8.1
75.9 ± 4.3
49.6 ± 3.1
30.0 ± 5.8
53.3 ± 8.8
33.3 ± 6.7
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
r
s
7.5 ± 1.2
2.2 ± 3.8
2.8 ± 4.8
1.5 ± 2.6
80.1 ± 5.8
9.9 ± 6.2
15.6 ± 2.6
51.1 ± 6.8
8.4 ± 2.8
7.8 ± 4.7
26.7 ± 3.3
26.7 ± 3.2
5
b
c
c
5
t
< 1.0 (27.7 ± 0.9)
b
5u
< 1.0 (39.4 ± 1.9)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
5
6
x
< 1.0
10.0 ± 0.1
10.0 ± 5.8
b
a
< 1.0
b
b
c
b
c
6b
< 1.0
< 1.0 (83.0 ± 7.5)
< 1.0 (12.0 ± 1.2)
Vemurafenib
(5µM)
44 ± 3.6
101.4 ± 1.9
76.7 ± 3.3
a
Cell viability on A375 melanoma cells after a 48h treatment with 10 µM of the indicated
compounds relative to a DMSO control ± SD from three independent experiments, as determined
b
c
by trypan blue exclusion assay. No viable cells. Cell viability after a 48h treatment with 1µM
due to high potency at 10 µM.
Chart 1. Evaluation of the activity against sensitive and resistant melanoma cell lines
1
1
20.0
00.0
8
6
4
2
0.0
0.0
0.0
0.0
0.0
A375 (sensitive)
A375 R5C3 (resistant)
A375 RIV (resistant)
ACS Paragon Plus Environment
12

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We observed that at 10 µM, whatever the cell lines (sensitive or resistant), all the 10
compounds proved more active than the reference BꢀRaf inhibitor vemurafenib on A375
melanoma cells. An apparent correlation between the size of the apolar substituent in para
position and the activity could be drawn, the best compounds being those presenting a bulky
iodine atom or hydrophobic aliphatic chains. Five derivatives (5x, 6aꢀb) showed viabilities
below 10% for all cell lines, with the best product being the alkyne 6b displaying no cell survival
at 10 µM. The most interesting finding was their high activity on Bꢀraf inhibitor resistant cells,
compared to vemurafenib, which has no activity. This first finding suggests a new mechanism of
action of these molecules different from the inhibition of the Raf pathway, and therefore
prompted us to test these compounds on other cancer cell lines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Activity against chronic myeloid leukemia sensitive and resistant cell lines. After the promising
results obtained on melanoma cells, the 10 derivatives were tested against CML K562 cells,
either sensitive or resistant to the standard BCRꢀABL tyrosine kinase inhibitor imatinib. Indeed,
if imatinib has proven to be very effective in achieving high remission rates for CML patients, up
16
to 33% of them will develop acquired resistance, due in part to the wellꢀknown T315 mutation.
The viability values for these cell lines are resumed in Table 5 and Chart 2.
Table 5. Activity against sensitive and resistant K562 CML cell lines
Cell viability at 10µM for 48hours
a
a
Compound
% Cell viability
% Cell viability
K562 (sensitive)
K562 (resistant)
4
c
53.9 ± 6.0
15.3 ± 1.1
89.7 ± 3.7
38.5 ± 2.5
5h
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
k
31.0 ± 5.9
12.8 ± 3.3
51.6 ± 3.4
51.0 ± 11.3
19.4 ± 3.3
15.0 ± 2.5
26.9 ± 4.6
0.6 ± 0.1
25.6 ± 2.8
27.0 ± 3.6
60.9 ± 11.5
72.7 ± 8.1
35.1 ± 12.6
27.0 ± 3.6
35.1 ± 8.0
3.4 ± 3.4
5
r
s
5
5
t
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5u
5
6
x
a
6b
Imatinib (1µM)
9.7 ± 0.5
85.5 ± 17.7
a
Cell viability on K562 CML cells after a 48 h treatment with 10µM of the indicated
compounds relative to a DMSO control ± SD from three independent experiments, as determined
by trypan blue exclusion assay.
Chart 2. Activity against sensitive and resistant K562 CML cell lines
120.0
1
00.0
80.0
6
0.0
40.0
0.0
.0
K562 (sensitive)
K562 (resistant)
2
0
The 10 derivatives demonstrated also high activity on CML cell lines at 10 µM. The activity of
most derivatives proved in the same range for sensitive and resistant cells, implying a different
ACS Paragon Plus Environment
14

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mode of action than BCRꢀABL oncoprotein inhibition. The best activity values were obtained
again for 6b that leads to almost total mortality at the studied concentration.
Activity against pancreatic cancer sensitive and resistant cell lines. The choice to test our most
active compounds on pancreatic cell lines was driven by the poor diagnosis and overall survival
of patients affected by this cancer, and the few therapeutic options available in case of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
7
resistance. Indeed, the firstꢀline treatment relied almost exclusively on the nucleoside analog
gemcitabine, which displays a narrow therapeutic window and growing resistance phenomena
1
0
that dramatically reduce the global efficacy of the treatment. Therefore, the 10 most active
compounds of our series were evaluated against pancreatic cancer sensitive BxPCꢀ3 cells and
MIA PaCaꢀ2 cells resistant to gemcitabine. The activity of these compounds against these lines is
depicted in Table 6 and Chart 3.
Table 6. Activity against BxPCꢀ3 and MIA PaCaꢀ2 pancreatic cancer cell lines
Cellular viability at 10µM for
48hours
a
a
Compound
% Cell viability
% Cell viability
BxPCꢀ3
sensitive)
MIA PaCaꢀ2
(resistant)
(
4
c
1.5 ± 0.3
21.6 ± 1.8
0.6 ± 0.1
21.0 ± 5.3
4.3 ± 1.8
7.0 ± 1.4
49.1 ± 5.0
7.0 ± 0.7
40.7 ± 0.7
5
5
h
k
5
r
s
b
5
< 1.0
b
b
5t
< 1.0
< 1.0
b
b
5u
< 1.0
< 1.0
b
5x
< 1.0
15.8 ± 0.2
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b
b
b
6
a
< 1.0
< 1.0
< 1.0
b
6b
< 1.0
Gemcitabine (0.1 µM)
2.0 ± 0.4
86.5 ± 1.2
a
Cell viability on sensitive BxPCꢀ3 and resistant MIA PaCaꢀ2 pancreatic cells after a 48 h
treatment with 10 µM of the indicated compounds relative to a DMSO control ± SD from three
independent experiments, as determined by trypan blue exclusion assay. No viable cells
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
Chart 3. Activity against BxPCꢀ3 and MIA PaCaꢀ2 pancreatic cancer cell lines
1
20
00
80
1
6
4
2
0
0
0
0
BxPC3
MiaPACA
Our compounds proved particularly toxic to pancreatic cells and caused very high mortality at
0 µM for both sensitive and resistant cell lines.
1
Taking all these results together, we selected the most potent derivate 6b as lead compound for
further in vitro and in vivo studies of this new class of compounds.
ACS Paragon Plus Environment
16

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Doseꢀresponse curves against various sensitive and resistant cell lines. First, the doseꢀactivity
response of 6b was evaluated at 10 different concentrations on the 7 studied cell lines. The
results are showed in Table 7 and Figure 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Table 7. Dose response values for 6b on the studied cancer cell lines
a
Cell line
Sensitivity
EC (µM, 48h)
50
A375
A375 RIV
A375 R5C3
K562
sensitive
resistant
resistant
sensitive
resistant
sensitive
resistant
0.56
0.48
1.32
1.42
1.62
0.38
0.64
K562
BxPCꢀ3
MIA PaCaꢀ2
a
Mean of 3 independent experiments; standard error was below 10%. Cell viability depending on
concentration plots measured at 10 different concentrations between 0.1 µM and 50 µM.
standard error was below 10%.
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Inhibitory curves of 6b against various cancer cell lines 6b displays a subꢀmicromolar
EC at 48h on melanoma and pancreatic cancer cells and reaches 1.5 µM against CML cells.
50
Toxicity and selectivity of 6b over nonꢀcancerous cells. We next investigated the toxicity of 6b
on two nonꢀcancerous cell lines: human keratinocytes and rapid dividing human fibroblasts. The
different cell lines were treated for 48h with 5 µM and 10 µM of 6b ꢀ respectively 10 and 20 fold
more than the EC on melanoma cells. The results are given in Table 8 and Chart 4.
50
Table 8. Toxicity of compound 6b on healthy and cancerous cell lines
Cell lines
5 µM
10 µM
a
KHN
91.2 ± 1.5
91.5 ± 0.9
c
93.4 ± 1.9
85.1 ± 0.3
b
FHN
A375
sensitive
c
<
1.0
< 1.0
ACS Paragon Plus Environment
18

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
c
A375 RIV
0.3 ± 0.1
< 1.0
a
Cell viability after a 48 h treatment with 10 µM of the indicated compounds relative to a
DMSO control ± SD from three independent experiments, as determined by trypan blue
exclusion assay. Human keratinocytes. Human fibroblasts. No viable cells.
a
b
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chart 4. Toxicity of compound 6b on healthy and cancerous cell lines
1
20
100
8
6
0
0
KHN
FHN
A375 sensitive
A375 RIV
40
2
0
0
DMSO
5 µM
10 µM
At 5 µM, no or marginal toxic effect of 6b was observed on normal cells whereas cancer cells
displayed almost no survival. At 10 µM, no significant toxicity was noted on keratinocytes and
fibroblasts. Total mortality of cancer cells was reached at this high dosage. These results show a
good selectivity of 6b towards cancer cells at its active concentration.
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3: compound 6b inhibits colony formation of A375 cells. Cells were plated at clonal
density and maintained in growth medium (CTR). One day after cell plating, 5 or 10 µM of 6b
was added to the culture medium. (A): Photographs of colony formation of A375 cells 10 days
after treatment. (B): Quantification of colony formation by measure of absorbance at 620 nm.
The results are expressed as percentages of the control. Data are mean ± SD of three independent
experiments performed in triplicate. ***, P < 0.001.
ACS Paragon Plus Environment
20

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To demonstrate the effect of 6b on A375 melanoma cells viability, a clonogenic survival assay
was performed. Cells were plated at low density and treated with 5 or 10 µM of 6b. After 10
days, cells were fixed and stained with crystal violet in order to visualize the cell colonies
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
Figure 3A). Treatment with 6b at 5 µM and 10 µM induced a doseꢀdependent decrease in the
formation of colonies with respectively 77% and 94% of clonogenicity inhibition (Figure 3B).
Moreover, in the presence of 6b the colonies were less dense than the control colonies, which is
consistent with the effect of 6b on cell viability.
Preliminary results for the investigation of the mode of action of 6b. At this stage of the study,
we were interested in the mechanism of action of our new family of compounds. To this end, we
set up preliminary experiments aimed at investigating the mechanism of action of lead
compound 6b, and particularly the mechanism of cell death. Flow cytometry analysis on A375
melanoma cells was performed following the activation of caspase 3, a key marker of apoptosis
(Figure 4A). 6b induces a strong activation of apoptosis with 48% of cells positive for active
caspase 3. Western blot analyses on A375 melanoma cells were performed following the death
related markers Poly ADPꢀRibose Polymerase (PARP), a DNA repair enzyme substrate of
apoptosisꢀrelated caspase 3, and microtubuleꢀassociated protein 1A/1Bꢀlight chain 3 (LC3), a
protein involved in autophagy mechanisms (Figure 4B). Indeed, recent data clearly pointed out
the key role of autophagy in cancer initiation, progression, invasion, metastasis and, particularly,
1
8
in resistance events. Autophagy is a natural intracellular system that delivers cytoplasmic
constituents to the lysosome for their degradation. It plays a wide variety of complex
physiological roles including the regulation of cell homeostasis, metabolism and tumor immune
response. Although it is generally thought to be a protective mechanism for the cell, it may,
18c
under certain circumstances, also lead to cell death. Indeed, depending on the context and the
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
type of cancer, autophagy may suppress tumor growth or may allow cancer cells to overcome
1
9
metabolic stress.
We observed that 6bꢀstimulated cells displayed an increase of the cleavage of the PARP
enzyme, strongly arguing in favor of the induction of apoptosis by 6b (Figure 4). We also
analyzed whether 6b might induce autophagy. During autophagy, LC3ꢀI is converted to LC3ꢀII
through the ATG4ꢀdependent insertion of a phosphoethanolamine moiety before being recruited
into the membrane of the forming phagophore, a double membrane required for the recycling of
protein aggregates and organelles. Increased LC3ꢀII expression was detected after 6b treatment,
as shown in Figure 4. The significant conversion from LC3ꢀI to LC3ꢀII observed thus indicates a
maturation of autophagosomes and a contribution of autophagy to the global death event.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Compound 6b induces concomitant apoptotic and autophagic phenomena.
ACS Paragon Plus Environment
22

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A: Flow cytometry analysis of A375 melanoma cells treated with 10 µM of 6b for 48 hours and
labelled with FITCꢀconjugated antiꢀactive Caspase 3 antibody. The percentage of cells positively
labelled with antiꢀactive Caspase 3 antibody (apoptotic cells) is indicated for each condition.
Staurosporine was used as a positive control (1 µM for 12hours). B: Western blots showing the
cleavage of PARP enzyme and the conversion LC3ꢀI to LC3ꢀII. A375 melanoma cells were
treated for 24 hours with 10µM of 6b. Cell lysates were separated by SDS–PAGE and analyzed
by western blot using the indicated antibody. HSP60 were used as a loading control. One
representative experiment of three is shown.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To support these preliminary data, we studied LC3 expression by immunofluorescent staining
of melanoma cells treated with DMSO or 6b (Figure 5). Cells treated with 6b showed a dramatic
increase in LC3 staining compared with the control, characterized by the presence of LC3 puncta
(green staining) in the cytoplasm of treated cells, reminiscent of autophagosome formation.
These data clearly support that 6b also induces strong induction of autophagy.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hoechst
LC3B
Merge
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DMSO
6b
10µM
Figure 5. Immunofluorescence experiments on A375 melanoma cells showing the apparition of
autophagosomes. Immunofluorescence pictures of A375 melanoma cells treated with DSMO or
1
0µM of 6b for 12 hours. LC3B was labeled with antibody (green) and DNA was visualized
with Hoechst (blue).
We then evaluated the respective contribution of apoptosis and autophagy in the cell death
induced by 6b. We thus performed viability assays using specific LC3ꢀsiRNAs and the panꢀ
caspase inhibitor QVD to block apoptosis and/or autophagy processes, respectively (Figure 6).
ACS Paragon Plus Environment
24

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Analysis of the respective contribution of apoptosis and autophagy in the cell death.
A375 melanoma cells were transfected with a siRNA against LC3 (siLC3) or a siRNA control
(siCTR) and treated with a Pan Caspase Inhibitor (QVD) or DMSO. 24 hours after transfection,
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cells were treated with 10 µM of 6b or DMSO for 48 hours. (A) Viability of the cells was
estimated by trypan blue staining. The results are expressed as percentages of the control. Data
are mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 ; #, P <
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.05. (B) Cells were lysed and analyzed by western blotting using the indicated antibodies.
Hsp60 was used as a loading control. One representative experiment of three is shown.
As presented on western blot (Figure 6A), QVD and LC3ꢀsiRNAs inhibited PARP cleavage
and LC3BꢀII expression respectively in the presence or absence of 6b treatment. Independently,
inhibition of apoptosis or autophagy partially inhibited the cell death induced by 6b, whereas
concomitant inhibition of both mechanisms completely restored cell viability (Figure 6B). Taken
together, these data clearly indicate that apoptosis and autophagy act simultaneously and
contribute in similar proportions to 6bꢀinduced melanoma cell death. This result is particularly
interesting, as autophagy has been recently proposed as a promising target to overcome
2
0
resistance to classical apoptotic drugs. Special efforts are currently dedicated in our
laboratories to further elucidate the mode of action of these compounds and the signaling
pathways that might be involved.
Pharmacokinetic profile of 6b. In order to evaluate the potential of lead compound 6b for further
medicinal chemistry development, we investigated its in vitro and in vivo pharmacokinetic
properties.
As the chemical structure of the molecule presents no apparent weakness towards plasma
hydrolases, we evaluated directly its stability on mouse microsomes. Therefore, in vivo
pharmacokinetic experiments in mice were envisaged to study its distribution. 6b was formulated
in a diluted saline solution containing the emulsifier Kolliphor EL (see experimental section) and
ACS Paragon Plus Environment
26

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
administered intravenously dosing at 1 mg/kg (n = 3 mice / group). As shown in Table 9, 6b
showed a good halfꢀlife time of over 45 min.
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
0
0
0
.8
.6
.4
.2
0
0
20
40
60
80
100
120
Time post-dose (min)
Figure 7. Concentrationꢀtime profile for compound 6b. Plasma samples were collected at regular
time after dosing, and quantification of 6b was performed using UHPLCꢀMS/MS.
6b could be measured readily after dosing and until the end of the experiment (2h postꢀdose),
showing a good plasma elimination halfꢀlife time T1/2 of 47 min. The compound penetrated
especially well into tissue components despite volume of distribution V values slightly lower
d
than expected considering the lipophilic features of the molecule (Table 9).
Table 9. Pharmacokinetic Parameters of 6b
Pharmacokinetic parameters
a
In vitro
Microsomal stability (min)
> 45
b
In vivo
T (min)
47 ± 4
43 ± 4
1
/2
Clearance (mL/min/kg)
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
V (L/kg)
2.91 ± 0.46
389 ± 35
d
AUC (ng.h/mL)
2
h
a
b
Mouse liver microsome stability T measured at 37°C. In vivo pharmacokinetic parameters
in mice at a dose of 1 mg/kg administered intravenously.
1
/2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The total clearance was moderate in plasma (43 mL/min/kg, liver blood flow is 90mL/min/kg,
so half of it is moderate at best) and thus the global exposure measured at the last time proved
particularly high with an AUC of 389 ng.h/mL for a 1 mg/kg dose. Furthermore, it is worth
2
h
noting that all mice behaved normally after intravenous injection and presented no apparent sign
of toxicity or behavior modification at this dose during the time of the study. Overall, the
pharmacokinetic parameters of 6b in mice proved very encouraging and fit the requirements for
the in vivo evaluation of the compound on tumor growth in xenografts models.
In vivo evaluation of 6b in mice xenogratfted with melanoma cells. Finally, to assess the
potential antineoplastic effect of our lead compound 6b in vivo, xenograft experiments were
6
performed. A375 melanoma cells (1.5x10 cells) were injected subcutaneously into 6 weeksꢀold
female athymic nude mice. After 5 days of tumor growth, the mice (n=6 in each group) were
treated by injection of either 6b with 0.7 mg/mouse/day, or the vehicle (Labrafil) for the control
group. The volume of the tumors was measured twice a week and the animals were euthanized
on day 31. Untreated control mice rapidly developed visible tumors, and a rapid tumor growth
was observed throughout the course of the study (Figure 8A). The treatment with 6b proved very
effective on tumor growth with mice showing a significantly attenuated development of tumors.
In addition, the tumors from the mice treated with 6b were markedly smaller compared to those
of the untreated control mice group (Figure 8B, 8C). These data clearly demonstrate an antiꢀ
neoplastic activity of our lead compound in vivo. Furthermore, no apparent toxicity on animals
was noticed over the course of the treatment based on their normal unmodified behavior
ACS Paragon Plus Environment
28

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compared to the controls, and a constant body mass suggesting an absence of diet disturbance
(Figure 8D).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. In vivo melanoma xenograft experiment of 6b. (A) Inhibition of tumor growth by 6b
6
using an A375 xenograft model. A375 melanoma cells (1.5.10 cells) were injected
subcutaneously in female immuneꢀdeficient BALB/c nu/nu (nude) mice. After 5 days, mice (n=6
in each group) were treated with 6b (0,7 mg/day) or Labrafil (the beginning of the treatment is
stamped by an arrow, figure 8A). The tumor growth curves were determined by measuring the
tumor volume. The bars indicate the mean ± SD. *, P < 0.05; **, P < 0.01 ***, P < 0.001. (B,C)
ACS Paragon Plus Environment
29