Structure-based lead optimization and biological evaluation of BAX direct activators as novel potential anticancer agents

Article abstract of DOI:10.1021/jm501123r

The first direct activator of BAX, a pro-apoptotic member of the BCL-2 family, has been recently identified. Herein, a structure-based lead optimization turned out into a small series of analogues, where 8 is the most potent compound published so far. 8 was used as pharmacological tool to ascertain, for the first time, the anticancer potential of BAX direct activators and the obtained results would suggest that BAX direct activators are potential future anticancer drugs rather than venoms.

Full text of DOI:10.1021/jm501123r

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Article
Structure-based Lead Optimization and Biological Evaluation
of BAX Direct Activators as Novel Potential Anticancer Agents
mariano stornaiuolo, Giuseppe La Regina, Sara Passacantilli, Antonio Coluccia, Valeria La Pietra,
Mariateresa Giustiniano, Hilde Cassese, Salvatore Di Maro, Diego Brancaccio, Sabrina Taliani, armando
ialenti, Romano Silvestri, Claudia Martini, Ettore Novellino, Luciana Marinelli, and Gianluca Grassia
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501123r • Publication Date (Web): 10 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Structure-Based Lead Optimization and Biological
Evaluation of BAX Direct Activators as Novel
Potential Anticancer Agents
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Mariano Stornaiuolo, * Giuseppe La Regina, Sara Passacantilli, Gianluca Grassia, Antonio
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Coluccia, Valeria La Pietra, Mariateresa Giustiniano, Hilde Cassese, Salvatore Di Maro,
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Diego Brancaccio, Sabrina Taliani, Armando Ialenti, Romano Silvestri, Claudia Martini,
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Ettore Novellino, Luciana Marinelli *
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Dipartimento di Farmacia, Università di Napoli "Federico II", via D. Montesano 49, 80131
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Naples, Italy. Istituto Pasteur − Fondazione Cenci Bolognetti, Dipartimento di Chimica e
Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma,
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Italy. Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
KEYWORDS: Apoptosis, BCL-2, BAX, BAK, Rational Drug Design, Anticancer Drugs
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ABSTRACT
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The first direct activator of BAX, a pro-apoptotic member of the BCL-2 family, has been
recently identified. Herein, a structure-based lead optimization turned out into a small series of
analogs, where 8 is the most potent compound published so far. 8 was used as pharmacological
tool to ascertain, for the first time, the anticancer potential of BAX direct activators and the
obtained results would suggest that BAX direct activators are potential future anticancer drugs
rather than venoms.
INTRODUCTION
The lack of efficacy of marketed anticancer drugs is due, at least in part, to the tumor ability to
escape apoptosis. Thus, novel medicines, designed to activate the cell death machinery, have
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recently appeared as innovative anticancer drugs. In vertebrate, there are two major routes
leading to apoptosis, the activation of death receptors, or the mitochondrial outer membrane
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permeabilization (MOMP), which allows proteins such cytochrome C (cyt-C) to orchestrate the
activation of caspases and in turn the cell dismantling. MOMP is finely regulated by a complex
play among BCL-2 family members, which makes these proteins prime targets for therapeutic
3
intervention in cancer. Within this family, three main subgroups are known: a) the anti-
apoptotic members (e.g., BCL-2, BCL-X , MCL-1, A1) b) the pro-apoptotic members (e.g.,
L
4
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BAK and BAX), and c) the pro-apoptotic BH3-only proteins (e.g., BID, BIM, PUMA, BAD,
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NOXA). The evidence that, in cancer cells, the neutralization of antiapoptotic BCL-2 proteins
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unleashes mitochondrial apoptosis and that high levels of anti-apoptotic BCL-2 family members
are often associated with tumor resistance, led to a massive structure-aided medicinal chemistry
which culminated in the development of BH3 mimetics, such as the 4-[4-[[2-(4-
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chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-
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phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide (ABT737),
(4-[4-[[2-(4-
chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-
yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide
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(Navitoclax or ABT263), or the newly developed (4-[4-[[2-(4-chlorophenyl)-4,4-
dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4-
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ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide (ABT199)
However, despite their promises, some of these drugs, which are in diverse clinical phases for
human tumors treatment, were shown to cause side effects (thrombocytopenia, tumor lysis
1
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syndrome, etc.), or to be less efficient on specific kind of tumors, like MCL-1 overexpressing
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ones. Thus, in the search of novel small molecules as alternative modulators of cell death, the
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recent discovery of a new BAX activation site (also named trigger site or rear site) offered a
thrilling opportunity to develop a novel class of alternative apoptosis activators. As such, the
1
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identification of the first, and so far still unique, small molecule 1 (BAM-7) ( Figure 1) as BAX
direct activator, surely represents a milestone in the field. However, the results so far published
do not allow the understanding of BAX requirements for small ligands recognition and binding:
in fact, structure-activity relationship studies were just focused on derivatizations at the
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phenylhydrazono and phenylthiazole side groups of 1 with small groups with no improvement
1
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of activity. Moreover, neither the in vitro or in vivo anticancer property besides apoptotic
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effects on MEF cells, nor the capability of this lead to discriminate between cancer cells and
healthy cells was assessed.
Herein, we report the results of our lead optimization program, which culminated in the
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discovery
of
1-(4'-(dimethylamino)-5-hydroxy-[1,1'-biphenyl]-3-yl)-4-(2-(2-
ethoxyphenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (8 or BTC-8, Bax Targeting
Compound-8), which directly binds to BAX and, in tumor cell lines, induces MOMP with an
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EC one order of magnitude lower than that exhibited by 1 in cultured HuH7 cells. As such, it
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is the most active, low molecular weight, direct activator of BAX reported in the published
literature. To reveal the anticancer potential of 8, as illustrative example of BAX direct activator,
the latter was tested in a small panel of tumor cell lines and on an healthy cell line providing
encouraging results, which in turn prompted us to test 8 on a mouse model of lung carcinoma.
The in vivo preliminary study would suggest that BAX direct activators are potential future
anticancer drugs rather than venoms.
Results and Discussion
Design Strategy
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The three-dimensional superimposition of BAX, alone (inactive state) and with BIM-BH3
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helix (active state) bound in the “rear site” clearly revealed that, upon BIM binding, significant
changes took place in the α1- α2 loop, which moves from a closed to an open conformation.
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Similar changes were observed upon 1 binding. As first step of our lead optimization protocol,
1
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docking experiments were performed, by means of Glide,
in order to reproduce the 1 binding
1
2
mode which was then overlapped onto that of BIM peptide. As shown in Figure 1a, the
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strongest interaction was observed between the carbonyl group of 1 pyrazolone core and the
BAX K21, a key residue which interacts with BIM as well (see Figure 1a, charge-charge
interaction with BIM E158). Notably, the ethoxyphenyl group of 1 lays adjacent to a presumed
hinge site for loop opening upon initiation of BAX activation and well overlaps onto BIM F159.
However, as shown in Figure 1a, BIM BH3 helix is much longer if compared to 1, holding five
further amino acids next to F159, (e.g., Y162 and Y163). On the opposite side, the
phenylthiazole portion of 1 well fitted on BIM L152 and I155, although lacks the terminal amino
group, that as in the case of BIM, engages charge reinforced hydrogen bonds with BAX D142
and E146, respectively. Based on these premises, we planned lead optimization studies of 1
aimed at understanding the BAX requirements and hopefully at improving the binding affinity.
Given the importance of the pyrazolone core, it was retained while a re-branching approach was
accomplished in the attempt to better mimic the essential features of the physiological activator
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(BIM BH3 only protein). Thus, as shown in Figure 1b, a number of different moieties were
considered. Basically, in the replacement of the 2-ethoxyphenyl-hydrazono group two
considerations were taken into account: i) substitution of hydrazone bridge with other spacer
groups, for synthetic reason ii) substitution of the ethoxyphenyl with bulkier aromatic moieties
(see compounds 2-7, and 15-16), in the attempt to approach the region occupied by BIM Tyr
residues (see Figure 1S in SI) which abut the hinge site for α1- α2 loop opening. Analogously,
for the replacement of the phenylthiazole group, the substitutions were planned based on the
following considerations: i) neither the N nor the S of the thiazole ring engaged interactions with
BAX, so that the thiazole ring could, in principle, be replaceable by other aromatic systems such
as phenyl ring, which can be easily substituted with the aim to establish further interactions with
other BAX residues (e.g. E17) ii) the para position of the biphenyl moiety is in proximity of
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D142 that together with E146 seizes the BIM amino terminal group (Figure 1a). Thus, we
inserted an exocyclic or endocyclic basic group at the terminal phenyl ring (8, 12 and 10
respectively). Compounds 9 and 13 were synthesized as negative control to test the importance
of the N,N-dimethyilamino group. Compound 14 was synthesized as negative control to test the
importance of the hydroxyl group on the biphenyl moiety. Finally, 11 was conceived as a
replacement of the 5-6 thiazole-phenyl system with a 6-5-phenyl-thiazole one.
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Figure 1. Design Strategy. a) Docked structure of 1 (pink) at the BAX rear site (green
surface). Interacting protein residues are highlighted as green sticks, the α1-α2 loop is depicted
as light green surface. BIM BH3 helix bound to the BAX rear site (PDB code: 2K7W), is colored
in transparent blue, with interacting residues depicted as blue sticks. b) Representation of the re-
branching approach. c) Binding mode of 8 within BAX rear site.
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CHEMISTRY
Synthesis of compounds 2-16 are depicted in Schemes 1-3. Compounds 2-5 and 16 were
prepared by reacting the 5-methyl-2-(4-phenylthiazol-2-yl)-2,4-dihydro-3H-pyrazol-3-one (17),
obtained by treatment of 2-hydrazinyl-4-phenylthiazole with ethyl acetoacetate in acetic acid at
reflux temperature for 3 h, with the appropriate benzaldehyde in the presence of piperidine in
boiling ethanol for 4 h (Scheme 1). Reaction of compound 17 with 2-(2-ethoxyphenyl)acetyl
chloride in the presence of potassium tert-butoxide in anhydrous tetrahydrofuran at 0 °C for 1 h
furnished the derivative 6, that was converted into the corresponding dione 7 by oxidation with
potassium permanganate in the presence of a catalytic amount of sulfuric acid in acetone at
reflux temperature for 12 h. Compounds 8, 12 and 15 were synthesized starting from the 2-(3-
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bromo-5-hydroxyphenyl)-5-methyl-2,4-dihydro-3 -pyrazol-3-one (19), that was treated with 2-
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ethoxybenzenediazonium chloride or naphthalene-2-diazonium chloride in the presence of
sodium acetate in ethanol at 0 °C for 2 h, and then coupled with the appropriate boronic acid in
the presence of sodium carbonate and Pd-catalyst in tetrahydrofuran at reflux temperature for 90
min under Ar stream (Scheme 2). Analogously, treatment of compound 19 with the appropriate
boronic acid and subsequent reaction with 2-ethoxybenzenediazonium chloride gave derivatives
9
, 11 and 13. Compounds 22-24 were prepared as above reported, starting from the 3-bromo-5-
hydrazinylphenol hydrochloride (18), obtained by reaction of 3-bromo-5-nitrophenol with tin(II)
chloride dihydrate in boiling ethyl acetate for 3 h, treatment with sodium nitrite and 37% HCl at
0
°C for 20 min and final reduction with tin(II) chloride dihydrate at 0 °C for 20 min. Derivatives
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0 and 14 were obtained similarly to 8, starting from pyrazolones 29 and 30, respectively
(
Scheme 3). Derivatives 29 and 30 were prepared as above reported, by treatment of 3-bromo-5-
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nitrophenol with the appropriate boronic acid, conversion into the corresponding
phenylhydrazine derivative and final cyclization with ethyl acetoacetate.
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a
Scheme 1. Synthesis of Compounds 2-7 and 16
O
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N
N
S
R1
N
Me
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R = 1-benzhydryl
R = 1,1'-biphenyl-4-yl
R = naphthalen-2-yl
R = 1H-imidazol-2-yl
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6 R = phenyl
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O
O
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c
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NHNH2
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N
Me
Me
EtO
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O
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S
O
Me
EtO
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a
Reagents and reaction conditions: (a) ethyl acetoacetate, acetic acid, reflux temperature, 3 h,
62%; (b) appropriate benzaldehyde, piperidine, anhydrous ethanol, reflux temperature, 3 h, 28-
1%; (c) (i) potassium tert-butoxide, anhydrous tetrahydrofuran, -40 °C, 1 h; (ii) 2-(2-
ethoxyphenyl)acetyl chloride, 25 °C, 1 h, 74%; (d) potassium permanganate, catalytic sulfuric
acid, acetone, reflux temperature, 5%.
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a
Scheme 2. Synthesis of Compounds 8, 9, 11-13 and 15
OH
OH
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24 R = 4-isopropylphenyl
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1 R = thiophen-2-yl
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3 R = 4-isopropylphenyl
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OH
OH
OH
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OH
OH
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Me
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Me
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R = 2-ethoxyohenyl,
R = 4-(dimethylamino)phenyl
2 R = 2-ethoxyohenyl,
R = 3-aminophenyl
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1 R = naphthalen-2-yl
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5 R = naphthalen-2-yl,
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R = 4-(dimethylamino)phenyl
3
a
Reagents and reaction conditions: (a) tin(II) chloride dihydrate, ethyl acetate, reflux, 3 h; (b)
i) sodium nitrite, 37% hydrogen chloride, 0 °C, 20 min; (ii) tin(II) chloride dihydrate, 0 °C, 20
min, crude product; (c) ethyl acetoacetate, acetic acid, 80 °C, 3 h, 13%; (d) 2-
ethoxybenzenediazonium chloride or naphthalene-2-diazonium chloride, sodium acetate, 96°
ethanol, 0 °C, 2 h, 3-48%; (e) appropriate boronic acid, 2M sodium carbonate, dichloro[1,1'-
(
bis(diphenylphosphino)ferrocene]-palladium(II)
dichloromethane
tetrahydrofuran, reflux temperature, 90 min, Ar stream, 13-66%.
complex
(1:1),
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a
Scheme 3. Synthesis of Compounds 10 and 14
R1
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R1
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b,c
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NO2
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NHNH HCl
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2
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5 R = OH, R = pyridin-4-yl
27 R = OH, R = pyridin-4-yl
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6 R = H, R = 4-(dimethyl-
28 R = H, R = 4-(dimethyl-
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1 2
amino)phenyl
amino)phenyl
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EtO
Me
Me
0 R = OH, R = pyridin-4-yl
1
29 R = OH, R = pyridin-4-yl
30 R = H, R = 4-(dimethyl-
1
2
1
2
1
4 R = H, R = 4-(dimethyl-
1 2
1
2
amino)phenyl
amino)phenyl
a
Reagents and reaction conditions: (a) appropriate boronic acid, 2M sodium carbonate,
dichloro[1,1'-bis(diphenylphosphino)ferrocene]-palladium(II) dichloromethane complex (1:1),
reflux temperature, 90 min, Ar stream, 14-25%; (b) tin(II) chloride dihydrate, ethyl acetate,
reflux, 3 h; (c) (i) sodium nitrite, 37% hydrogen chloride, 0 °C, 20 min; (ii) tin(II) chloride
dihydrate, 0 °C, 20 min, crude product; (d) ethyl acetoacetate, acetic acid, 80 °C, 3 h, 12-13%;
(e) 2-ethoxybenzenediazonium chloride, sodium acetate, 96° ethanol, 0 °C, 2 h, 36-47%.
Biological Results
In vitro BAX Affinity, Selectivity Profile and SAR Rationalization. Affinities of candidate
compounds for recombinant BCl-2 family members were measured in vitro by Fluorescence
Polarization Assay (FPA). GST tagged full length BAX, untagged BCL-2 and BCL-X show
L
nanomolar affinity for FITC-BIM (Ki values of 200, 132 and 794 nM, respectively) (SI, Figure
1
1-12
2
S) in line with data previously reported.
The ability of 1-15 to displace bound FITC-BIM
was used to calculate their Ki for each of the three proteins (Figure 2 and Table 1). As previously
reported, 1 is selective on BAX over BCL-X (Figure 2 and Table 1). It displays however
L
affinity for BCl-2 despite this being somehow slightly lower than the one for BAX (Ki values of
3
.0 and 1.1 µM, respectively).
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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
Table 1. Ki (µM) of Compounds 1-15 for BAX, BCL-2 and BCL-X (FPA, FITC-BIM
displacement)
L
Compound BAX
BCL-2 BCL-XL
1
2
3
4
5
6
7
8
9
1.1
13.5
12.9
12.6
15.8
63.1
85.1
0.8
9.1
1.7
30.9
1.3
1.2
3.0
1.6
1.6
2.0
2.0
>100
>100
12.0
>100
>100
>100
>100
1.9
>100
>100
3.1
3.4
6.8
>100
>100
>100
>100
30.9
>100
>100
15.5
>100
3.4
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
0
1
2
1
1
13
14
1.3
1
2.5
4.2
33.9
1.9
1
1
5
6
2.4
In the range of concentration tested, the new compounds show either: higher affinity for BCl-2
over BAX and BCL-X (compounds 2 and 5), higher affinity for both BCl-2 and BCL-X over
L
L
BAX (compounds 3-5) and similar affinities for the three proteins (compounds 16) or for none of
them (compounds 6-7) (Table 1).
Compounds 8-10 show higher affinity for BAX over BCL-2 and BCL-X . Among them, 8
L
displays higher affinity for BAX (Ki = 0.8 µM) than 1 (Ki = 1.1 µM, Figure 2). 9 and 11 are
selective for BAX but they are somehow weaker binders than 8. 12 is specific for BAX, but its
BAX affinity is lower than 8, and comparable to that of 1. 13-15 display high affinity for BAX
but less specificity (Table 1) over the other two proteins.
SAR towards BAX were rationalized on the basis of the complex between 1 and BAX, which
1
2
was previously obtained by a combined approach of docking and NMR experiments and on the
basis of docking of its analogue 8. Molecular docking of 8 within the BAX rear site resulted in a
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8
9
binding mode highly superimposable to that of 1 (Figure 1a and c). However, in line with the
lower Ki of 8, compared to that of 1, 8 reinforces its binding to BAX surface through an H-bond
with E17 by the ligand hydroxyl group and through a charge reinforced hydrogen bond with
D142 coupled with hydrophobic interactions between the methyl groups and D142 carbon chain.
According to the observed interactions, 9 (unsubstituted on the terminal phenyl ring) is less
active than 8 , 13 and 14. The importance of an amino group on the terminal phenyl ring is also
proved by the affinity of 12 compared to that of 9 and by the reduced specificity of 13 compared
to 8 and 12. All in all, SAR where the phenylthiazole group is replaced by other moieties clearly
demonstrate that such branch is replaceable by other aromatic systems, that if adequately
decorated bring to an improvement of BAX affinity. As regards the 2-ethoxyphenylhydrazono
group of 1, SAR were designed to explore: (i) bridges different from the hydrazono one and (ii)
possible, fruitful hydrophobic interactions established by different aromatic moieties. Among the
different bridges synthesized, just the ene one (16) seems to be well tolerated. The higher Ki of 6
and 7 seemed to be ascribable to the different geometries of ketomethylen- and diketo bridges
with respect to hydrazono group, which would bring the 2-ethoxyphenyl farther from L25, with
which an hydrophobic interaction was observed (Figure 1a). Although no H-bond between the
ethoxy group of 1 and K21 was observed with a semi-flexible docking approach, their proximity
would suggest a possible interaction, which can be also the reason for the slightly lower Ki of 1
with respect to that of 1. Analogs of 1, where the phenyl moiety were replaced by a benzhydryl
10
11
12
13
14
15
16
17
18
19
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(2), biphenyl (3) and naphthalene (4) group were all less active than the lead. Rationalization of
the lower Ki of 2-4 is somehow difficult as the benzhydryl, biphenyl and naphthalene group
would occupy the hinge site for loop opening, a region that undergoes to profound change upon
ligand binding and activation.
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1
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5
6
Cell Viability and Apoptosis Assays. Affinities and selectivity of 1-15 measured by FPA
well correlate with their potency on inducing apoptosis of MEF knock out cells. At the EC of 1,
50
all the compounds but 6, 7 and 11 induced apoptosis of MEF wt cells (Table 2 and 1S), while
0
1
2
3
4
5
6
7
8
9
0
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8
9
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1
2
3
4
5
6
7
8
9
0
-/-
-/-
they were ineffective on MEF BAX
BAK double knockout cells, indicating that they
ultimately lead to pro- apoptotic protein activation. 1, 8-10 and 12-15 are the most effective at
the concentration of 10 µM. 2-5 potencies are overall lower and partially affected by the absence
of BCL-2 as well as of Bid, Bad, Bak or Bax suggesting that, at least in MEF cells, BAX direct
activation is not their main mechanism of action (Table 1S). On the contrary, 1, 8-10 and 12-16
induce apoptosis independently by the absence of BCL-2, BID and BAD pointing to their target
being located downstream in the apoptotic pathway (Figure 3, Table 2 and Table 1S). 12, 13, 14
-/-
-/-
and 16 affected MEF BAX as well as in MEF BAK indicating that they can trigger apoptosis
-/-
either by BAK or by BAX. 8-10 and 15 similarly to 1 induced apoptosis in BAK but not in
-/-
BAX knockout cells and represent, among the tested ones, the candidate compounds with the
best features of selective BAX activators (Figure 3, Table 2 and Table 1S). MEF cells treated
with 8, the most potent compound of our series (EC of 8 on MEF-wt of 1.1 µM, EC of 1 on
50
50
MEF-wt of 7.7 µM) undergo apoptotic processes, as shown by the appearance of fragmented
nuclei and cell membrane blebbing. Moreover, its potency is reduced by the presence of the
1
6
caspase inhibitor Ac-DEVD-CHO (Figure 3), indicating that its mechanism of action involve
apoptotic effectors.
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Figure 2. 8 binds BAX at BIM binding site. Competition between 8 (upper panel) or 1 (lower
panel) and FITC-BIM for binding to BAX (green curve), BCL-2 (red curve) and BCL-XL (blue
curve). Mean of three experiments (s.d. are indicated).
Table 2. EC (µM) Values of Apoptosis Induction of Compound 8 or 1 Treatment on the
50
Indicated MEF Knockout Clones
-/-
-/-
-/-
-/-
-/-
-/-
Bax Bak
-/-
Compound
wt
Bcl2
Bad
Bid
Bak
Bax
1
8
7.7
1.1
3.2
0.6
3.5
0.8
4.0
0.7
6.0
1.3
>50
>50
>50
>50
Differently from MEF cells, tumor cells overcome pro-apoptotic stimuli by overexpressing
anti-apoptotic proteins or by downregulating pro-apoptotic ones. We thus decided to challenge
our compounds in inducing apoptosis in in vitro cultured tumor cells. Apoptosis was followed in
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5
6
Human Hepatoma HuH7 cells, that express wt BAX, high level of BCl-2 but low level of caspase
1
7
3
, the final effector of the apoptotic cascade. Under apoptotic stimuli they survive for longer
time than other tumor cells and, thus, they represent an ideal platform to look exclusively at the
early events of the apoptotic process.
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
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0
1
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9
0
1
2
3
4
5
6
7
8
9
0
Apoptosis was followed in HuH7 by measuring MOMP, which inhibits mitocondrial
accumulation of the fluorescent probe Mitotracker Red. Among the tested compound, 8 was the
most potent in affecting probe accumulation (Figure 4 and Figure 3S). Other compounds of the
series (e.g., 2 and 3) affected mitochondrial potential only after 72 hours of treatment in line with
a reduced affinity for BAX but an increased affinity for BCL-2 or BCL-X , or both (Table 1 and
L
Figure 3S in SI). While 1 and 8 affected the cultures similarly at 10 µM, 8 induced a stronger
reduction in probe accumulation at 1 µM (Figure 4a). The higher potency of 8 over 1 was
quantified by a dose response curve revealing the EC of 1 and 8 in cultured HuH7 cells to be
5
0
8.2 µM and 700 nM, respectively (Figure 4b and Table 2S). Similarly to 1, 8 induces a
conformational change in BAX that exposes an epitope recognized by the anti-BAX 6A7
12
antibody, specific for the activated form of the protein (Figure 4S). As expected from a BAX
direct activator, 8 was able to induce the translocation of BAX to mitochondria, the cytochrome
C release from these organelles (Figure 5S and 6S), the activation of caspase 3 and the formation
of apoptotic nuclei (Figure 5S and 7S).
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Figure 3. 8 induces apoptosis in a BAX dependent way. Effect of 8 treatment on MEF cells.
Apoptotic rate of the indicated MEF clones upon 24 h of treatment with vehicle (0.1% DMSO,
gray bars), 1 (15 µM, white bars) or 8 (15 µM, white bars). The photographs represent
appearance of apoptotic cell membrane blebbing upon MEF treatment with 8 and its absence
when 8 treatment was done in the presence of the caspase inhibitor Ac-DEVD-CHO (10 µM).
-/-
Similarly to what we have already show in MEF BAX cells, 8 treatment had no effects on
the leukemic Jurkat (T- cell lymphoma) cell line. Jurkat cells express a mutated form of BAX
18
and rely only on BAK to induce MOMP. Jurkat cells resulted insensitive to 8 treatment,
confirming that our compound activates selectively BAX (Figure 6). Differently leukemic NB4
1
9
cells (promyelocytic), that express wt BAX , showed hallmarks of apoptosis already after 12
hours of treatment with 8.
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2
3
4
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6
7
8
9
0
Figure 4. 8 affects mitochondria in HuH7 cells. a) Mitochondrial activity of 8 and 1 treated
cells visualized by Mitotracker Red fluorescence uptake. Magnification bars are indicated. b)
After lysis of the cells absorbance of the dye was measured at 570 nm and plotted versus 8 or 1
concentration. EC were calculated by fitting values by non linear regression analysis of dose
5
0
response analysis and reported together with their 95% C.I. (error bars are indicated, n = 3).
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Figure 5. 8 induces growth arrest and cell death. a) The indicated cell lines cells were
incubated for 72 hours with the indicated concentration of 1 and 8 and then with Mitotracker.
Absorbance was measured after lysis of the cell. Values are the mean of at least three
experiment. Error bars (s.d.) are indicated. b) HuH7 cells were incubated for the indicated time
with 10µ 1 or 8 or vehicle alone. After the treatment cells were counted. Values are expressed as
percentage of control (cells at 0 minutes of treatment) and are representative of at least three
experiments.
Then, in order to have indication about the toxicity of 8 on healthy cells, we tested the effect of
the compound on primary healthy splenocytes. At concentration of 10 ꢀM, 8 did not alter the
apoptosis rate in primary cells, indicating that could selectively affect tumoral cells (Figure 6).
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6
This observation could be explained by the fact that, as seen for many other cancer drugs, 8
2
0
could affect predominantly highly proliferating cells rather than resting ones.
Compound 8 is able to affect mitochondrial membrane potential in all the tumor cell lines
tested (Figure 5). Like HuH7, Lewis lung carcinoma (LLC1) and human neuroblastoma (SHS5-
SY) cells were reported to be highly resistant to conventional pro-apoptotic drugs. Like for
HuH7, after treatment with 8, these cells did not show sign of death (visible in only the 20% of
cell (data not shown)), but they stop duplicating to undergo in a process of growth arrest. This
effect likely suggests that even in those tumor cell lines that have developed strategies to block
the initiation of the apoptotic process or its completion, BAX direct activation can hamper
mitochondrial activity, reduce intracellular energy content and ultimately affects cell growth.
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. 8 induces apoptosis in tumor cells but it does not alter apoptotic rate in healthy
splenocytes. The indicated tumor cell lines (a) or primary splenocytes (b) were incubated for 72
hours with 8 (10 µM). Apoptosis rates were detected by counting apopototic nuclei. Mean value
are reported together with their s.d. (n = 3 in a; n = 4 in b)
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9
In situ BAX Activation and In Vivo Tests. LLC1 cells resulted sensitive to 8 (Figure 5).
These cells well represent lung carcinoma, the first cause of adult mortality. To establish whether
8
would be efficacious in vivo, we used them in a murine Lewis lung carcinoma model, that
10
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reliably recapitulates human lung cancer in pathology, disease progression, clinical outcome, and
2
1
response to therapies. LLC1 cells have a high tropism for the lung and are able to form tumor
22
lesions that after 14 days are in exponential growth, making the drug treatment clinically
relevant. On day 0 tumor cells were injected into mice via the tail vein. From day 14 to day 17,
one group of mice were treated once a day with an intraperitoneal injection of 8 (1 mg/kg) or of
vehicle (1% DMSO). On day 17, animals were euthanized. The compound ability to induce an in
vivo activation of BAX was confirmed trough immunoprecipitation of lung lysates with 6A7
antibody (Figure 7). A visual inspection of the exposed lung of control mice showed very large
tumor lesions covering the lung surface, whereas the lungs of mice treated with 8 showed highly
size-reduced tumor lesions. Histological analysis using H&E staining of lung sections
demonstrated that after only 4 days of treatment, the ratio between tumor burden and lung area in
mice treated with 8 compared with control was significantly (P<0.01) decreased by 50% (Figure
8).
Noteworthy, 8 seemed to be well tolerated since no mortalities, evidence of gross toxicity,
behavioral side-effects or body weight changes were observed in the four days of treatments.
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Figure 7. In vivo injected 8 induces in situ BAX activation. Homogenates of mouse lungs
treated or not with 8 were lysed and immunoprecipitated with the 6A7 monoclonal antibody.
Samples were boiled, run on a SDS-PAGE and decorated with a polyclonal antibody anti-BAX
(Only relevant part of the gel is shown).
A microscopic examination of heart, femoral muscle and healthy lung did not reveal any
pathologic change (data not shown). The in vivo efficiency of 8 in tumor mass reduction was of
relevance especially if a rough comparison with marketed anticancer drugs on similar tumor
models is done. In fact, cyclophosphamide (60 mg/kg), or adriamycin (1.25 mg/kg) gave a
2
3,24
reduction of 40% even in the case of prolonged treatments.
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Figure 8. In vivo activity of 8. a) Effect of the daily administration of 8 1mg/kg from day 14
to day 17 on tumor growth (dark violet staining). Microphotographies showing the entire lung
sections, the boxed areas in the upper-magnification H&E panels are shown at a higher
magnification in the lower panels. b) Graph showing the effect of 8 on tumor growth. The results
are expressed as tumor lesions/lung area ratio. Individual data points represent average value per
mouse; horizontal bars denote mean. P<0.01 vs control group.
CONCLUSION
11
Optimization of BAX direct activators has been already shown to be challenging . We here
show that 1 optimization can successfully lead to new hits with improved affinity for BAX.
However, minimal changes in the chemical structure of the hits can drastically change their
selectivity, affecting their affinity for other BCL-2 family members. In MEF cells whatever the
main target is, the event of binding leads always to apoptosis. However, in tumor cells a severe
unbalancing between pro-apopototic and anti-apopototic proteins exists, with the latter being
overexpressed in almost every tumor. Thus, ligands with low selectivity can be sequestered by
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5
5
5
5
6
anti-apoptotic proteins, that, ultimately, buffer them inhibiting the activation of pro-apoptotic
proteins.
As a result of our first round of 1 optimization, we here present 8. In vitro affinity of 8 for
BAX is only slightly higher than 1. On the contrary, it shows lower affinity for BCL-2 and BCL-
0
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0
-/-
X than 1. Its selectivity manifests in MEF BAX knockout cells, that, differently from many
L
other tested MEF knockout clones, are insensitive to 8 treatments. In cultured tumor cells it
induces MOMP with an EC of 700 nM, one order of magnitude lower than the one measured
50
for 1. As such, it is the most active, low molecular weight, direct activator of BAX reported in
the published literature. SAR data here developed clearly suggest that the phenyl-thiazol moiety
can be replaceable by other aromatic groups, and that the activity is increased by a terminal
positive charged group, that, according to a theoretical binding mode, would interact with
negatively charged BAX residues. 8 is able to induce BAX activation and translocation to
mitochondria, MOMP and consequent release of cyt-C in all the tumor cell line tested. Moreover
8
seems to be rather selective for cancer cells (HuH7, NB4, SHSY-5Y and LLC1) and
immortalized cells (MEF), leaving unperturbed apoptosis rate in healthy resting cells (healthy
splenocytes).
The in vivo efficiency of 8 in tumor mass reduction together with the absence of gross toxicity,
although just 4 days of treatment was applied, both would suggest that BAX direct activators
may really represent a novel promising class of anticancer agents rather than venoms.
EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were handled according to material safety data sheet of
the supplier and were used as purchased, without further purification. Organic solutions were
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dried over anhydrous Na SO . Evaporation of the solvents was carried out on Büchi Rotavapor
2
4
R-210 equipped with Büchi V-855 vacuum controller and Büchi V-700 (~ 5 mbar) and V-710 (~
mbar) vacuum pumps, and on Heidolph Hei-Vap G3B equipped with Vacuubrand PC 3001
2
10
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vacuum pump (~ 2 mbar). Column chromatography was performed on glass columns packed
with silica gel from Fluka and from Aldrich (70−230 mesh). Silica gel thin layer chromatography
(TLC) cards from Fluka and Macherey-Nagel (silica gel precoated aluminum cards with
fluorescent indicator visualizable at 254 nm) were used for TLC. Developed plates were
visualized by a Spectroline ENF 260C/FE UV apparatus. Melting points (mp) were determined
on Stuart Scientific SMP1 apparatus and on Stuart Scientific SMP30 apparatus and are
uncorrected. Infrared spectra (IR) were run on Perkin Elmer SpectrumOne FT-ATR
-
1
spectrophotometer. Band position and absorption ranges are given in cm . Mass spectral (MS)
and high resolution mass spectral (HRMS) analyses were recorded on AB Sciex API-2000 (MS)
and Thermo Fisher Scientific Inc. Orbitrap Exactive (HRMS) spectrometers equipped with an
1
13
ESI source. Proton ( H, 400.13 MHz) and carbon ( C, 100.6 MHz) nuclear magnetic resonance
spectra were recorded on Bruker Avance 400 and Varian Inova 400 spectrometers in the
indicated solvent and corresponding fid files processed by MestreLab Research S.L.
MestreReNova 6.2.1-769 and Bruker Topspin 3.2 software. Chemical shifts are expressed in δ
units (ppm) from tetramethylsilane. Elemental analyses of the compounds were found within
±0.4% of the theoretical values. The purity of tested compounds was found to be >95% by high
pressure liquid chromatography (HPLC) analysis. The HPLC system used (Thermo Fisher
Scientific Inc. Dionex UltiMate 3000) consisted of a SR-3000 solvent rack, a LPG-3400SD
quaternary analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array
detector, and an analytical manual injection valve with a 20 ꢀL loop. Samples were dissolved in
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acetonitrile at 10 mg/mL. HPLC analysis was performed by using a Thermo Fisher Scientific
Inc. Acclaim 120 C18 reversed-phase column (5 ꢀm, 4.6 mm ×250 mm,) at 30 ± 1 °C with an
isocratic gradient (acetonitrile:water = 90:10), flow rate of 1.0 mL/min and signal detector at 254
and 365 nm. Chromatographic data were acquired and processed by Thermo Fisher Scientific
Inc. Chromeleon 6.80 software.
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General Procedure for the Preparation of Compounds 2-5 and 16. Example: 4-(2,2-
Diphenylethylidene)-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one (2). A mixture of
17 (0.21 g, 0.00084 mol), 2,2-diphenylacetaldehyde (0.16 g, 0.15 mL, 0.84 mmol) and piperidine
(0.071 g, 0.08 mL, 0.84 mmol) in ethanol (0.5 M) were heated to reflux for 3 h and evaporated.
The residue was purified by column chromatography (silica gel, dichloromethane:methanol =
1
99:1 as eluent) to furnish 2 (0.17 g, 47%), mp >220 °C with decomposition (from ethanol). H
NMR (DMSO-d ): δ 1.53 (s, 3H), 6.49 (s, 1H), 7.16 (d, J = 3.7 Hz, 2H), 7.22-7.38 (m, 10H),
6
-1
7.43 (t, J = 7.4 Hz, 2H), 7.72 (s, 1H), 7.96 ppm (d, J = 3.8 Hz, 2H). IR: ν 1616 cm . Anal.
(
C H N OS (435.54)) C, 74.46; H, 4.86; N, 9.65; S, 7.36; found C, 74.44; H, 4.83; N, 9.69; S,
2
7
21
3
+
7.34. MS (ESI) calcd for C H N OS: 435.1; found (M+H) : 436.1.
27
21
3
4-([1,1’-Biphenyl]-4-ylmethylene)-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one
(3). It was synthesized as 2 starting from 17 and [1,1'-biphenyl]-4-carbaldehyde. Yield 61%, mp
1
> 270 °C with decomposition (from ethanol). H NMR (DMSO-d ): δ 2.13 (s, 3H), 7.29-7.38 (m,
6
-
4H), 7.40-7.48 (m, 5H), 7.58-7.68 (m, 4H), 7.72 (s, 1H), 7.98 (d, J = 3.7 Hz, 2H). IR: ν 1625 cm
1
. Anal. (C H N OS (421.51)) C, 74.08; H, 4.54; N, 9.97; S, 7.61; found C, 74.04; H, 4.58; N,
26
19
3
+
9
.92; S, 7.59. MS (ESI) calcd for C H N OS: 421.1; found (M+H) : 422.3.
26 19 3
3-Methyl-4-(naphthalen-2-ylmethylene)-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one (4). It
was synthesized as 2 starting from 17 and 2-naphthaldehyde. Yield 37%, mp > 270 °C with
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decomposition (from ethanol). H NMR (DMSO-d ): δ 2.07 (s, 3H), 7.29-7.38 (m, 1H), 7.39-
6
7.50 (m, 5H), 7.70 (s, 1H), 7.75 (s, 1H), 7.80-7.90 (m, 3H), 7.93-8.03 ppm (m, 3H). IR: ν 1615
-1
cm . Anal. (C H N OS (395.48)) C, 72.89; H, 4.33; N, 10.63; S, 8.11; found C, 72.86; H, 4.31;
24
17
3
10
11
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+
N, 10.59; S, 8.09. MS (ESI) calcd for C H N OS: 395.1; found (M+H) : 396.1.
24 17
3
4
-((1H-Imidazol-2-yl)methylene)-3-methyl-1(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one (5).
It was synthesized as 2 starting from 17 and 1H-imidazole-2-carbaldehyde. Yield 28%, mp 189-
1
191 °C (from ethanol). H NMR (DMSO-d ): δ 1.98 (s, 3H), 7.18 (s, 1H), 7.26-7.48 (m, 5H),
6
-1
7
.62 (d, J = 3.6 Hz, 1H), 7.69 (s, 1H), 7.92 ppm (d, J = 3.8 Hz, 2H). IR: ν 1615 cm . Anal.
(C H N OS (335.38)) C, 60.88; H, 3.91; N, 20.88; S, 9.56; found C, 60.87; H, 3.90; N, 20.86;
1
7
13
5
+
S, 9.54. MS (ESI) calcd for C H N OS: 335.1; found (M+H) : 336.2.
1
7
13
5
4
-Benzylidene-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one
(16).
It
was
synthesized as 2 starting from 17 and benzaldehyde. Yield 30%, mp >270 °C with
1
decomposition (from ethanol). H NMR (DMSO-d ): δ 2.07 (s, 3H), 7.17-7.23 (m, 1H), 7.24-
6
7.35 (m, 5H), 7.39-7.46 (m, 3H), 7.70 (s, 1H), 7.97 ppm (d, J = 3.7 Hz, 2H). IR: ν 1360, 1495,
-1
1
622 cm . Anal. (C H N OS (345.42)) C, 69.54; H, 4.38; N, 12.17; S, 9.28; found C, 69.50; H,
20
15
3
+
4
.36; N, 12.17; S, 9.26. MS (ESI) calcd for C H N OS : 345.1; found (M+H) : 346.2.
20 15
3
4
-(2-(2-Ethoxyphenyl)acetyl)-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one (6). A
suspension of potassium tert-butoxide (0.13 g, 1.2 mmol) in anhydrous tetrahydrofuran (15.0
mL), was cooled at -40 °C and a solution of 17 (0.25 g, 0.97 mmol) in the same solvent (10.0
mL) was added dropwise under Ar stream. The reaction mixture was stirred in the same
conditions for 1 h and a solution of 2-(2-ethoxyphenyl)acetyl chloride (0.31 g, 1.2 mmol) in the
same solvent (5.0 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 1 h,
diluted with water and extracted with ethyl acetate. Organic layer was washed with brine, dried
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and filtered. Evaporation of the solvent gave a residue that was purified by column
chromatography (silica gel, ethyl acetate:n-hexane = 1:1 as eluent) to furnish 6 (0.3 g, 74%), mp
1
1
05-110 °C (from ethanol). H NMR (DMSO-d ): δ 1.22 (t, J = 6.9 Hz, 3H), 2.25 (s, 3H), 3.97-
6
0
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4
5
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4.02 (m, 4H), 6.26 (s, 1H), 6.85 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 7.16 (d, J = 6.4 Hz,
1H), 7.24 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.90-7.94 ppm
-1
(
m, 3H). IR: ν 1780, 1784 cm . Anal. (C H N O S (419.50)) C, 65.85; H, 5.05; N, 10.02; S,
2
3
21
3
3
7
.64; found C, 65.80; H, 5.02; N, 10.06; S, 7.60. MS (ESI) calcd for C H N O S: 419.1; found
23 21 3 3
+
(
M+H) : 420.0. 2-(2-Ethoxyphenyl)acetyl chloride. A mixture of 2-(2-hydroxyphenyl)acetic acid
2.0 g, 13 mmol) and 37% hydrochloric acid (1.2 mL) in 96° ethanol (70.0 mL) was heated to
(
reflux for 3 h, cooled, diluted with water and extracted with ethyl acetate. Organic layer was
washed with brine, dried and filtered. Removal of the solvent gave ethyl 2-(2-
2
5
hydroxyphenyl)acetate (2.22 g, 94%), mp 65-67 °C (from petroleum ether), lit. 67-68 °C. A
mixture of ethyl 2-(2-hydroxyphenyl)acetate (2.22 g, 12 mmol), anhydrous potassium carbonate
(3.69 g, 26 mmol) and iodoethane (4.11 g, 2.1 mL, 26 mmol) in anhydrous N,N-
dimethylformamide (10.0 mL) was heated at 50 °C for 12 h. After cooling, the reaction mixture
was diluted with water and extracted with ethyl acetate. Organic layer was washed with brine,
dried and filtered. Removal of the solvent gave a residue that was purified by column
chromatography (silica gel, ethyl acetate:n-hexane = 3:7 as eluent) to furnish ethyl 2-(2-
1
ethoxyphenyl)acetate (2.22 g, 86%), oil. H NMR (DMSO-d ): δ 1.16 (t, J = 7.1 Hz, 3H), 1.27 (t,
6
J = 6.9 Hz, 3H), 3.55 (s, 2H), 3.96-4.07 (m, 4H), 6.86 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.1 Hz,
-1
1H), 7.15-7.23 ppm (m, 2H). IR: ν 1733 cm . A mixture of ethyl 2-(2-ethoxyphenyl)acetate
(4.86 g, 23 mmol) in 3N sodium hydroxide/96° ethanol (1:1, v/v) (180.0 mL) was heated to
reflux for 12 h. After cooling, the reaction mixture was made acidic with 6N hydrogen chloride
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and extracted with ethyl acetate. Organic layer was washed with brine, dried and filtered.
Removal of the solvent gave 2-(2-ethoxyphenyl)acetic acid (3.50 g, 83%), mp 99-102 °C (from
2
6 1
ethanol), lit 103 °C. H NMR (DMSO-d ): δ 1.28 (t, J = 6.7 Hz, 3H), 3.48 (s, 2H), 3.98 (q, J =
6
10
11
12
13
14
15
16
17
18
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.8 Hz, 2H), 6.85 (t, J = 6.9 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.14-7.31 (m, 2H), 12.13 ppm
-1
(broad s, disappeared on treatment with D O, 1H). IR: ν 1730, 2987 cm . To a mixture of 2-(2-
2
ethoxyphenyl)acetic acid (0.21 g, 1.2 mol) in anhydrous benzene (9.0 mL) was added thionyl
chloride (1.45 g, 0.9 mL, 12 mmol). The reaction mixture was stirred at 25 °C for 1 h and
evaporated to give 2-(2-ethoxyphenyl)acetyl chloride that was used without further purification.
1
-(2-Ethoxyphenyl)-2-(3-methyl-5-oxo-1-(4-phenylthiazol-2-yl)-4,5-dihydro-1H-pyrazol-4-
yl)ethane-1,2-dione (7). A mixture of 6 (0.25 g, 0.59 mmol), potassium permanganate (0.38 g,
.4 mmol) and a catalytic amount of 98% sulfuric acid in acetone (10.0 mL) was heated to reflux
2
for 12 h. Removal of the solvent gave a residue that was purified by column chromatography
(silica gel, ethyl acetate:n-hexane = 3:7 as eluent) to furnish 7 (0.013 g, 5%), mp 108-110 °C
1
(from ethanol). H NMR (DMSO-d ): δ 1.22 (t, J = 6.8 Hz, 3H), 2.29 (s, 3H), 4.06 (d, J = 6.7 Hz,
6
2H), 6.39 (s, 1H), 7.12-7.28 (m, 5H), 7.53 (d, J = 7.4 Hz, 2H), 7.74 (m, 1H), 7.86 (s, 1H), 8.19
-1
ppm (d, J = 6.6 Hz, 1H). IR: ν 1215, 2926 cm . Anal. (C H N O S (433.48)) C, 63.73; H,
2
3
19
3
4
4.42; N, 9.69; S, 7.40; found C, 63.70; H, 4.39; N, 9.72; S, 7.38. MS (ESI) calcd for
+
C H N O S: 433.1; found (M+H) : 434.4.
23
19
3
4
General Procedure for the Preparation of Compounds 8, 12, 15 and 22-26. Example: 1-
4'-(Dimethylamino)-5-hydroxy-[1,1'-biphenyl]-3-yl)-4-(2-(2-ethoxyphenyl)hydrazono)-3-methyl-
(
1H-pyrazol-5(4H)-one (8). A mixture of 20 (0.07 g, 0.17 mmol), 2M sodium carbonate (0.3 mL)
and (4-(dimethylamino)phenyl)boronic acid (0.034 g, 0.21 mmol) in tetrahydrofuran (0.6 mL)
was degassed for 10 min. Dichloro[1,1'-bis(diphenylphosphino)ferrocene]-palladium(II) (0.0082
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