Synthetic Lethality in Pancreatic Cancer: Discovery of a New RAD51-BRCA2 Small Molecule Disruptor That Inhibits Homologous Recombination and Synergizes with Olaparib

Article abstract of DOI:10.1021/acs.jmedchem.9b01526

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

Full text of DOI:10.1021/acs.jmedchem.9b01526

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Article
Synthetic lethality in pancreatic cancer: discovery of a
new RAD51-BRCA2 small molecule disruptor that inhibits
homologous recombination and synergizes with olaparib
Greta Bagnolini, Domenico Milano, Marcella Manerba, Fabrizio Schipani, José-Antonio
Ortega, Dario Gioia, Federico Falchi, Andrea Balboni, Fulvia Farabegoli, Francesca De
Franco, Janet Robertson, Roberto Pellicciari, Isabella Pallavicini, Sebastiano Peri, Saverio
Minucci, Stefania Girotto, Giuseppina Di Stefano, Marinella Roberti, and Andrea Cavalli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01526 • Publication Date (Web): 10 Feb 2020
Downloaded from pubs.acs.org on February 10, 2020
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Synthetic lethality in pancreatic cancer: discovery of a new RAD51-BRCA2 small molecule
disruptor that inhibits homologous recombination and synergizes with olaparib
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Greta Bagnolini,§,†, Domenico Milano,§, Marcella Manerba,§, Fabrizio Schipani, Jose Antonio
§
§
§
§
§,†
†
Ortega, Dario Gioia, Federico Falchi, Andrea Balboni, Fulvia Farabegoli, Francesca De
¦
¦
¦
‡
‡
Franco, Janet Robertson, Roberto Pellicciari, Isabella Pallavicini, Sebastiano Peri, Saverio
‡,
§
,†
§,†
Minucci, Stefania Girotto, Giuseppina Di Stefano,† Marinella Roberti* and Andrea Cavalli*
§Computational & Chemical Biology, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa,
Italy.
†Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126
Bologna, Italy.
¦TES Pharma S.r.l., Via Palmiro Togliatti 22bis, I-06073, Corciano, Perugia, Italy.
Department of Biosciences, University of Milan, Via Celoria 26, 20100 Milan.
‡Department of Experimental Oncology at the IEO, European Institute of Oncology IRCCS, IFOM-
IEO Campus, Via Adamello 16, 20100 Milan.
†
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via S.
Giacomo 14, 40126 Bologna, Italy.
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ABSTRACT
Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates.
One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations.
Here, we exploit a new paradigm based on the possibility to trigger synthetic lethality using only
small organic molecules (dubbed “fully small-molecule-induced synthetic lethality”). We exploited
this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered
a dihydroquinolone pyrazoline-based molecule (35d), which disrupts the RAD51-BRCA2 protein-
protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous
recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with
olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in
BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic
lethality an innovative approach towards unmet oncological needs.
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Keywords: protein-protein interaction, protein-protein inhibitors, DNA repair, anticancer drugs,
PARP inhibitors.
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INTRODUCTION
Synthetic lethality is a new opportunity for discovering new anticancer molecules for
personalized targeted therapies. The concept derives from genetic studies in model organisms.1-5
Two genes are synthetically lethal if the perturbation of either gene alone has no effect on cell
viability, but the simultaneous impairment of both genes results in cell death. In principle, small
organic molecules can target the synthetically lethal partner of an altered gene in cancer cells, but
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not in normal cells. This creates opportunities to selectively kill cancer cells while sparing normal
cells.6-12
The DNA repair and DNA damage response (DDR) pathways are suitable for the application of
synthetic lethality as a novel anti-cancer therapeutic strategy.13-15 Genome instability is a hallmark
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of cancer. DNA damage occurs constantly in cells due to the continuous exposition to endogenous
and exogenous stressors. Consequently, cells have evolved a complex coordinated DDR, which
orchestrates a network of cellular processes to repair DNA damage and preserve genome integrity.
DDR thus prevents the transmission of altered genetic material to daughter cells and acts as a
tumor-suppressive barrier. Defects in DDR are associated with the accumulation of oncogenic
mutations and genome instability, and they contribute to cancer initiation and progression.
However, cancer cells with defects in one DDR pathway can become reliant on other pathways for
their survival. Targeting these other DDR pathways can potentially cause selective cancer cell death
through synthetic lethality. The classic example is the clinical application of poly (ADP-
ribose)polymerase (PARP) inhibitors in oncology patients with BRCA1/2 mutations. PARP is
crucial for repairing DNA single-strand breaks (SSBs), whereas BRCA 1/2 are important for
repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The
simultaneous impairment of both repair mechanisms results in cell-cycle arrest and apoptosis of
cancer cells through synthetic lethality. In 2014, olaparib was the first PARP inhibitor (PARPi)
approved to treat advanced ovarian cancer associated with defective BRCA genes.17 In 2018,
olaparib was approved to treat metastatic breast tumors associated with germline BRCA mutations.18
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In 2019, olaparib gained the FDA approval as first-line maintenance treatment of germline BRCA-
mutated metastatic pancreatic cancer. It appears to be a new treatment option for this disease,
which is one of the major unmet needs in oncology.19
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One of BRCA2’s key mechanisms in DDR is to recruit RAD51, an evolutionarily conserved
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recombinase, at the site of DSBs where it performs DSB repair through HR. Additionally, the
expression of RAD51 and the rate of RAD51-mediated HR are both elevated in a wide variety of
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cancers (e.g. breast, pancreatic). Moreover, the cellular amount of RAD51 is positively correlated
with resistance to radiotherapies or chemotherapies that induce DNA damage.22,23
The RAD51-BRCA2 interaction is mediated by eight well-conserved motifs, known as BRC
repeats.24-27 The X-ray crystallographic structure of the fourth BRC repeat (BRC4) is available in
complex with the catalytic domain of RAD51, making the RAD51-BRCA2 interaction suitable for
the structure-based design of small molecule inhibitors of protein-protein interactions (PPI). Indeed,
Lee et al. recently identified a small molecule inhibitor of RAD51-BRCA2 for potential cancer
treatment.28,29
In this context, we recently proposed a new anticancer drug discovery concept, dubbed “fully
small-molecules-induced synthetic lethality”.30,31 This concept combines RAD51-BRCA2
disruptors with olaparib to simultaneously impair two DNA repair pathways, thus mimicking the
synthetic lethality described above. We carried out a successful virtual screening campaign at the
FxxA pocket (i.e. zone I), one of the two RAD51 pockets responsible for BRC4 binding. This
allowed us to discover a series of triazole-based compounds. Compounds 1 and 2 were selected as
initial hit candidates (Figure 1). In line with our hypothesis, 2 increased the sensitivity to olaparib in
pancreatic cancer cells (BxPC-3) with fully functional BRCA2. Notably, this synergistic effect was
not observed in Capan-1, pancreas adenocarcinoma cells that lack BRCA2.30 Furthermore, to
discover more effective compounds, we conducted a chemical modification campaign around the
triazole moiety. We also improved the biological screening cascade with experiments to
characterize how the new compounds disrupt the RAD51-BRCA2 interaction and inhibit DSB
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repair. We obtained compound 3 (Figure 1) with an improved profile relative to the initial hits
according to a biochemical ELISA assay) and a clear mechanism of action, allowing synergy with
olaparib in cancer cells BxPC3, where olaparib is normally inactive. However, with 3, we could not
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fully reproduce the paradigm of synthetic lethality. This could be due to its low-level potency,
which did not cause HR inhibition greater than 40%. Additionally, the inherent resistance to
apoptosis of BxPC3 cells, which bear a mutant p53, could have further prevented apoptosis.
Therefore, further biological experiments and new classes of RAD51-BRCA2 are needed to
confirm this paradigm and to assess its potential as an innovative anticancer strategy.
To this end, we report here on the identification of a new class of RAD51-BRCA2 disruptors
based on the dihydroquinolone pyrazoline moiety (4d-57d, Table 1, Scheme 2). We attempt to
depict general structure-activity relationships (SAR) of this new class of compounds as RAD51-
BRCA2 inhibitors, and outline the biological profile of the most promising derivative 35d (Table 1,
Scheme 2).
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ELISA assay. EC50 = 53±3M
ELISA assay. EC50 = 25±2M
ELISA assay. EC50 = 8±2 M

Figure 1. Structure of the previously identify triazoles 1-3.
RESULTS AND DISCUSSION
Hit identification and optimization
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Targeting protein-protein interactions (PPIs) is an attractive strategy for designing innovative
drugs for complex diseases such as cancer. Indeed, the first PPI inhibitors for cancer are now in
clinical development.32
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In this context, to identify RAD51-BRCA2 disruptors, we used the available X-ray crystallographic
structure of the fourth BRC repeat (BRC4) in complex with the catalytic domain of RAD51.24
BRC4 binds RAD51 in two different hydrophobic pockets (zone I and zone II, respectively). One
pocket (named zone I) can lodge BRC4’s FxxA motif (residues 1524-1527 of BRCA2) and is
critical for RAD51 multimerization. The other pocket (named zone II) can lodge the BRC4’s LFDE
motif (residues 1545-1548 of BRCA2) far from the oligomerization interface (Figure 2).24,33-35
Recently, we ran a successful virtual screening campaign based on high-throughput docking at the
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FxxA pocket to identify the first RAD51-BRCA2 disruptors. To increase the chemical diversity
and identify a novel class of RAD51-BRCA2 disruptors, we performed a second virtual screening
campaign targeting the LFDE binding pocket (see the Supporting Information). This binding pocket
is more evolutionarily conserved than the FxxA. Furthermore, mutation at the LFDE causes
cellular lethality and failure of RAD51 assembly in nuclear foci at the site of DNA breaks in vivo.
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This further suggests this pocket as a critical site for RAD51’s mechanism of action. To the best
of our knowledge, no inhibitor that binds the LFDE binding pocket has been reported so far in the
literature. This may open up new possibilities for combining molecules targeting zone I and zone II
towards a more in depth understanding of the mechanism of inhibition of RAD51-BRCA2
interaction.
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Figure 2. (A) RAD51−BRCA2 BRC repeat complex (PDB code: 1N0W). RAD51 is represented as
a surface, BRC4 as a cartoon. The two hot spots of the interaction between the proteins (Phe1524
and Phe1546) are highlighted in sticks; (B) ZONE II magnification showing the interacting residues
of BRC4 (yellow) and RAD51 (white).
Here, 42 small molecules were selected, purchased, and tested for their inhibitory activity using a
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competitive biochemical ELISA assay, as previously described by Rajendra et al. Among the
tested compounds, the commercially available dihydroquinolone pyrazoline derivative 4d
(
Figure 3 and 4) was the best candidate in terms of EC and chemical tractability. Its activity
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was confirmed by retesting the newly synthesized compound 4d (Scheme 2). Indeed, the
dihydroquinolone pyrazoline moiety is a core structure of compounds with different biological
targets.36,37 The binding mode to RAD51 of both enantiomers of 4d (Figure 3), as obtained by
induced-fit docking simulations, displays some points of interaction similar to those of the
crystallographic BRC4-RAD51 complex. Specifically, the docking model suggests that (i) the
fluorophenyl ring in position 5 of the pyrazoline lies (similarly to the Phe1549 of BRC4) in a
hydrophobic pocket outlined by the side chains of Leu204, Tyr205, Met251, Leu255, and Phe259
of RAD51; (ii) the carboxyl group of the pyrazoline side chain forms an ionic interaction with the
Arg250 (or Arg247) of RAD51, as does the side chain Glu1548 of BRC4. In addition, the model
suggests that the carbonyl and the nitrogen of the dihydroquinolone moiety, together with the
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carbonyl group of the pyrazoline side chain, establish hydrogen bonds with Arg254 and Glu258.
Notably, both enantiomers show the same global pattern of interactions.
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Figure 3. Both enantiomers of compound 4d docked into the LFDE binding site (site II) of
RAD51 (PDB code: 1N0W).
To improve the RAD51-BRCA2 inhibitory activity of 4d, we conducted a chemical
modification campaign around the dihydroquinolone pyrazoline core. We synthesized a
chemical library that contained a variety of aromatic substitutions (red and blue regions) in
combination with modifications of the acyl chain moiety (green region) (Figure 4). All
compounds were synthesized and tested as racemic mixtures, after verifying that the
enantiomers of the hit compound 4d showed the same biochemical activity and the same
binding mode (Figure 3, details in the Biological Evaluation section). First, a series of different
acyl chains (namely acetyl, propionyl, 3-aminopropionyl, 4-amino-4-oxobutanoyl, 4-methoxy-
4
-oxobutanoyl, 3-(methylsulfonamido) propanoyl) was introduced on the pyrazoline nitrogen
(
5d-10d, Table 1). Next, the aromatic ring A was modified by replacing the fluorine atom with
different substituents, including chlorine and bromine atoms, methoxy, tert-butyl and
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trifluoromethyl groups, leaving the succinate acyl chain unchanged (11d-16d, Table 1). In
addition, the aromatic ring A was replaced by different substituted biphenyl or heterocycle
groups in order to probe its role (17d-32d, Table 1). The ring A was also modified in
combination with the propionyl (33d-35d, Table 1) or acetyl substitution on the pyrazoline
nitrogen (36d-51d, Table 1). Regarding the dihydroquinolone core, the chlorine atom in the C-
ring was removed, leaving the acyl chain unchanged and introducing some different
substituents in the phenyl ring A (52d-57d, Table 1).
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acetyl, propionyl, 3-aminopropionyl,
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-amino-4-oxobutanoyl, 4-methoxy-4-oxobutanoyl and
-(metylsulfonamido)propanoyl chains
OH
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unsubstituted ring
N
H
different substituted phenyl rings,
different substituted biphenyl rings,
different substituted heterocycles
4d
Figure 4. Overview of the optimization strategy of 4d for SAR exploration.
Chemistry
The desired dihydroquinolone pyrazoline derivatives 4d-6d and 11d-57d were achieved, taking
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advantage of a common synthetic strategy previously reported by Acker et al. The commercially
available 2-aminobenzophenones 58-59 treated with ethyl acetoacetate afforded the corresponding
methyl ketones 60a-61a, which underwent base-catalyzed condensation with the appropriate aryl
aldehydes 62-85 yielding the ,-unsaturated aryl ketones 86b-114b. In turn, 86b-114b were
treated with hydrazine monohydrate to yield the pyrazoline derivatives 86c-114c (Scheme 1). The
pyrazoline amines 86c-114c functionalized with succinic anhydride 115, acetic anhydride 116, or
propionic acid 117 afforded the corresponding desired dihydroquinolone pyrazoline derivatives
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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
d-6d and 11d-57d (Scheme 2). 7d (Scheme 3) was obtained by coupling 86c with the
commercially available 3-((tert-butoxycarbonyl)amino)propanoic acid 118 to achieve the Boc-
amino pyrazoline derivative 119. In turn, 119 was Boc-deprotected to afford the desired 7d. 8d
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
(Scheme 4) was obtained by HATU-mediated coupling of 4d with ammonium chloride. 9d (Scheme
5
) was afforded by coupling 86c with the commercially available 4-methoxy-4-oxobutanoic acid
20. The synthesis of 10d (Scheme 6) began with the commercially available methyl 3-amino-
1
propanoate 121, which was treated with methansulfonyl chloride 122 to afford the corresponding
methyl 3-(methylsulfonamido)propanoate 123, which underwent basic hydrolysis to give 3-
(methylsulfonamido)propanoic acid 124. The coupling reaction of 86c with 124 afforded the
desired 10d.
The aldehydes 67, 70, 74-84, not commercially available, were prepared following standard
procedures as reported in Supporting Information (Scheme S1- S9).
1
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Scheme 1. Synthesis of dihydroquinolone pyrazoline intermediates 86c-114c.
O
Ar
O
O
O
62-85
O
O
N NH
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
Ar
R1
R1
R1
R1
a
b
c
Ar
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
NH2
N
H
N
N
H
O
H
58-59
60a-61a
86b-114b
86c-114c
Cp
R
1
Ar
Cp
R
1
Ar
4-FPh
4-ClPh
4-BrPh
4-OMePh
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
9
0a
1a
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Cl
H
Cl
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
86b; 86c
87b; 87c
88b; 88c
89b; 89c
90b; 90c
91b; 91c
92b; 92c
93b; 93c
94b; 94c
95b; 95c
96b; 96c
97b; 97c
98b; 98c
99b; 99c
100b; 100c
101b; 101c
102b; 102c
103b; 103c
104b; 104c
105b; 105c
106b; 106c
107b; 107c
108b; 108c
109b; 109c
110b; 110c
111b; 111c
112b; 112c
113b; 113c
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
4-FPh
4-ClPh
4-BrPh
4-tert-BuPh
3-F-4-OMePh
4-(CF3)Ph
furan-2-yl
4'-F-[1,1'-biphenyl]-4-yl
4'-Cl-[1,1'-biphenyl]-4-yl
4'-Br-[1,1'-biphenyl]-4-y
4'-OMe-[1,1'-biphenyl]-4-y
1-methyl-1H-indol-5-yl
1-ethyl-1H-indol-5-yl
1-ethyl-1H-indazol-5-yl
2-ethyl-2H-indazol-5-yl
1-propyl-1H-indazol-5-yl
2-propyl-2H-indazol-5-yl
1-cyclohexyl-1H-indazol-5-yl
2-cyclohexyl-2H-indazol-5-yl
4-(1-ethyl-1H-indazol-5-yl)Ph
4-(2-ethyl-2H-indazol-5-yl)Ph
4-(1-propyl-1H-pyrazol-4-yl)Ph
4-FPh
4-OMePh
4-tert-BuPh
3-F-4-OMePh
4-(CF3)Ph
furan-2-yl
4'-F-[1,1'-biphenyl]-4-yl
4'-Cl-[1,1'-biphenyl]-4-yl
4'-Br-[1,1'-biphenyl]-4-y
4'-OMe-[1,1'-biphenyl]-4-y
1-methyl-1H-indol-5-yl
1-ethyl-1H-indol-5-yl
1-ethyl-1H-indazol-5-yl
2-ethyl-2H-indazol-5-yl
1-propyl-1H-indazol-5-yl
2-propyl-2H-indazol-5-yl
1-cyclohexyl-1H-indazol-5-yl
2-cyclohexyl-2H-indazol-5-yl
4-(1-ethyl-1H-indazol-5-yl)Ph
4-(2-ethyl-2H-indazol-5-yl)Ph
H
H
H
H
4-BrPh
3-BrPh
4-ClPh
4-OMePh
-
-
-
4-(1-propyl-1H-pyrazol-4-yl)Ph
3-BrPh
114b; 114c
H
4-(CF3)Ph
Reagents and conditions: (a) Ethyl acetoacetate, DMF, 120 °C µWave or reflux, 60a quantitative, 61a 53%; (b) KOH,
EtOH/H O 4:3 v/v (0.05 M), 0 °C to rt,53%-quantitative; (c) hydrazine monohydrate, EtOH, 110 °C µWave, 45 min., 64%-
2
quantitative
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8
9
1
1
1
1
1
1
1
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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
Scheme 2. Synthesis of final dihydroquinolone pyrazoline 4d- 57d.
OH
O
O
NH
Succinic anhydride 115,
anhydrous THF,
µWave,120 °C, 45 min
N
N
O
N
O
Ar
Ar
R1
R1
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
7%-quantitative
N
N
H
H
86c-114c
4d, 11d-32d, 52d-57d
4
d: R = Cl; Ar = 4-FPh (62%)
25d: R = Cl; Ar = 2-ethyl-2H-indazol-5-yl (50%)
1
1
11d: R = Cl; Ar = 4-ClPh (74%)
26d: R = Cl; Ar = 1-propyl-1H-indazol-5-yl (86%)
1
1
1
1
1
1
1
1
2d: R = Cl; Ar = 4-BrPh (68%)
27d: R = Cl; Ar = 2-propyl-2H-indazol-5-yl (78%)
1
1
3d: R = Cl; Ar = 4-OMePh (86%)
28d: R = Cl; Ar = 1-cyclohexyl-1H-indazol-5-yl (87%)
1
1
4d: R = Cl; Ar = 4-tert-BuPh (81%)
29d: R = Cl; Ar = 2-cyclohexyl-2H-indazol-5-yl (95%)
1
1
5d: R = Cl; Ar = 3-F-4-OMe-Ph (51%)
30d: R = Cl; Ar = 4-(1-ethyl-1H-indazol-5-yl)Ph (81%)
1
1
6d: R = Cl; Ar = 4-(CF )Ph (71%)
31d: R = Cl; Ar = 4-(2-ethyl-2H-indazol-5-yl)Ph (49%)
1
3
1
7d: R = Cl; Ar = furan-2-yl (90%)
32d: R = Cl; Ar = 4-(1-propyl-1H-pyrazol-4-yl)Ph (quantitative)
1
1
18d: R = Cl; Ar = 4'-F-[1,1'-biphenyl]-4-yl (80%)
52d: R = H; Ar = 4-FPh (59%)
1
1
1
9d: R = Cl; Ar = 4'-Cl-[1,1'-biphenyl]-4-yl (77%)
53d: R = H; Ar = 4-BrPh (91%)
1
1
2
0d: R = Cl; Ar = 4'-Br-[1,1'-biphenyl]-4-yl (79%)
54d: R = H; Ar = 3-BrPh (7%)
1
1
21d: R = Cl; Ar = 4'-OMe-[1,1'-biphenyl]-4-yl (55%) 55d: R = H; Ar = 4-ClPh (67%)
1
1
2
2
2
2d: R = Cl; Ar = 1-methyl-1H-indol-5-yl (79%)
56d: R = H; Ar = 4-OMePh (67%)
1
1
3d: R = Cl; Ar = 1-ethyl-1H-indol-5-yl (49%)
57d: R = H; Ar = 4-(CF )Ph (71%)
1
1
3
4d: R = Cl; Ar = 1-ethyl-1H-indazol-5-yl (74%)
1
O
NH
N
N
N
O
Acetic anhydride 116,
anhydrous THF,
µWave,165 °C, 45 min
Ar
Ar
R1
R1
N
O
N
H
43%-quantitative
H
86c-90c, 92c-93c,
5d, 36d-51d
95c-103c, 108c
5
d: R = Cl; Ar = 4-FPh (47%)
44d: R = Cl; Ar = 4'-OMe-[1,1'-biphenyl]-4-yl (83%)
45d: R = Cl; Ar = 1-methyl-1H-indol-5-yl (75%)
46d: R = Cl; Ar = 1-ethyl-1H-indol-5-yl (70%)
47d: R = Cl; Ar = 1-ethyl-1H-indazol-5-yl (82%)
48d: R = Cl; Ar = 2-ethyl-2H-indazol-5-yl (77%)
49d: R = Cl; Ar = 1-propyl-1H-indazol-5-yl (90%)
50d: R = Cl; Ar = 2-propyl-2H-indazol-5-yl (85%)
1
1
36d: R = Cl; Ar = 4-ClPh (94%)
1
1
3
7d: R = Cl; Ar = 4-BrPh (73%)
1
1
3
8d: R = Cl; Ar = 4-OMePh (43%)
1
1
39d: R = Cl; Ar = 4-tert-BuPh (68%)
1
1
4
4
4
0d: R = Cl; Ar = 4-(CF )Ph (74%)
1
3
1
1d: R = Cl; Ar = furan-2-yl (73%)
1
1
2d: R = Cl; Ar = 4'-Cl-[1,1'-biphenyl]-4-yl (quantitative) 51d: R = Cl; Ar = 4-(1-propyl-1H-pyrazol-4-yl)Ph (81%)
1
1
43d: R = Cl; Ar = 4'-Br-[1,1'-biphenyl]-4-yl (80%)
1
O
N
NH
N
Propionic acid 117, HOBt,
Ar EDCI, DCM, 3h
N
O
Ar
R1
R1
21-59%
N
O
N
H
H
86c-89c
6d, 33d-35d
6
3
3
d: R = Cl; Ar = 4-FPh (21%)
1
3d: R = Cl; Ar = 4-ClPh (34%)
1
4d: R = Cl; Ar = 4-BrPh (36%)
1
35d: R = Cl; Ar = 4-OMePh (59%)
1
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Scheme 3. Synthesis of compounds 7d.
NH
N
O
Cl
F
O
O
a
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
+
HO
N
O
N
H
H
118
86c
O
O
NH
NH2
O
O
N
N
N
O
N
O
Cl
F
Cl
F
b,c
N
N
H
H
7d
119
Reagents and conditions: (a) HOBt, EDCI, DCM, overnight, yield 25%; (b) HCl 4 M in dioxane, rt, 15 min; (c)
NaOH 0.5 M in EtOAc, rt, 15 min, yield 50%.
Scheme 4. Synthesis of compounds 8d.
OH
O
NH2
O
O
O
N
N
N
O
N
O
Cl
F
Cl
F
a
N
N
H
H
4d
8d
Reagents and conditions: (a) HATU (1.5 equiv), EDC (1.5 equiv), DCM, DMF, ammonium chloride (5.0
equiv), DIPEA (4.0 equiv) , rt, 26 h, yield 46%.
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9
1
1
1
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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
Scheme 5: Synthesis of compounds 9d.
O
O
O
NH
N
N
N
O
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
Cl
F
O
Cl
F
a
O
+
HO
N
O
N
O
H
H
86c
120
9d
Reagents and conditions: (a) HOBt (1.1 equiv), EDC (1.1 equiv), TEA (2.2. equiv) DCM, rt, 16 h, yield 42%.
Scheme 6. Synthesis of compounds 10d.
O
O
O
S
O
O
S
a
b
O
NH2
O
N
O
HO
N
O
H
H
121
123
124
O
O
S
NH
O
NH
N
N
N
O
Cl
F
Cl
F
c
124
+
N
H
O
N
H
86c
10d
Reagents and conditions: (a) TEA (5.0 equiv), anhydrous DCM, methansulfonyl chloride 122 (2.0 equiv), rt, 48
h, yield 82%; (b) MeOH/THF (1:1 v/v), 2M LiOH, rt, 16 h, yield 82%; (c) HOBt (1.1 equiv), EDC (1.1 equiv), TEA
(2.2. equiv) DCM, rt, 16 h, yield 15%.
Biological evaluation
To investigate the mechanism of action of the new dihydroquinolone pyrazoline derivatives,
different biological assays were performed. As a primary screening, the ability of compounds
5d-57d to inhibit RAD51-BRCA2 interaction was investigated with a competitive biochemical
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ELISA assay against the parent compound 4d (Table 1). This assay is effective in evaluating the
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
3
0
ability of new molecules to compete with BRC4 to bind to RAD51. Replacing the acyl chain of
the pyrazoline nitrogen yielded compounds (5d-10d, Table 1), which had reduced inhibitory
activity (5d, EC = 50 ± 10 M; 6d, EC50 = 34 ± 3 M) or were completely inactive (7d-10d).
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
5
0
These data indicate that no improvement in RAD51-BRCA2 inhibitory activity was achieved with
this subset of compounds relative to the parent 4d (EC = 16 ± 4 M). Replacing the fluorine atom
5
0
on ring A with bromine led to 12d (EC = 16 ± 2 M), which shows the same activity of the initial
5
0
hit 4d. The activity was affected when the fluorine was replaced with chlorine (11d, EC50 = 59 ± 8
M) and trifluoromethyl groups (16d, EC50 = 29 ± 2 M). The replacement of fluorine with

electron-donating groups yielded the inactive compounds (13d-15d). For the subset of
dihydroquinolone pyrazolines, in which the aromatic ring A was replaced by different
substituted biphenyl or heterocycle groups (17d-32d, Table 1), the N-methyl indole derivative
2
2d showed a fairly good potency with an EC = 2 ± 0.5 M, eight times higher than that of
5
0
the parent 4d (EC = 16 ± 4 M). 18d-21d, 23d, 25d, 30d, and 31d were active at micromolar
5
0
range, all very similar to the initial hit. A drop in activity was observed with compound 32d, while
derivatives 24d, 26d-29d were inactive. The replacement of the aromatic ring A in combination
with the substitution of the pyrazoline nitrogen with either propionyl or acetyl chain yielded
compounds 33d-51d. The 1-N-acetyl-5-(1-N-propyl)indazolyl-pyrazoline 49d showed the best
activity of the series with an EC = 0.95 ± 0.05 M, while 33d-36d, 39d, 45d showed an activity
5
0
very similar to that of the initial hit. A drop in potency was observed with compounds 37d, 42d-
4d, 46d-47d and 51d, while 38d, 40d-41d, 48d and 50d were inactive. Finally, removing the
4
chlorine atom on the dihydroquinolone core led to the completely inactive compounds 52d-57d,
suggesting an active role for the halogen. The enantiomers (4d-I and 4d-II) of the racemic hit
compound 4d, separated via reverse phase chiral chromatography, showed a very similar
inhibitory activity (4d-I, EC = 4 ± 0.5 M; 4d-II, 10 ± 1 M) to that of the parent 4d, suggesting
5
0
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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
no stereochemical preference of these compounds for the hypothesized molecular target RAD51
See Experimental Section). As expected for PPI disruptors, the SARs of the new series of
(
dihydroquinolone pyrazoline were rather complex to rationalize, with many cliffs and spikes that
were difficult to understand. Nonetheless, the SAR campaign allowed us to identify several
compounds with interesting EC values ranging from 0.95 to 20 M. Accordingly, 4d, 12d, 18d,
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
5
0
20d-23d, 30d-31d, 33d-36d, 39d, 45d, and 49d were submitted to cell-based study (Table 2).
Our working hypothesis is that compounds disrupting RAD51-BRCA2 interaction should affect
HR repair and increase the efficacy of PARPi in treating breast, ovarian, and pancreatic cancer. For
its clinical relevance, pancreatic adenocarcinoma was selected as our final model for cell-based
experiments, and BxPC3 cells were selected for a straightforward comparison with the previously
reported triazole derivatives.30,31 BxPC3 is derived from a human adenocarcinoma that expresses
3
8
fully functional BRCA2. As shown in Table 2 and according to the rationale of our hypothesis,
our preliminary screening consisted in verifying the efficacy of compounds in inhibiting cell HR
and/or in increasing the antiproliferative effect of olaparib. For each compound, both parameters
were verified using only one or two doses, in the range of the EC obtained with the ELISA test.
5
0
HR activity was assessed by evaluating the recombination rate between two transfected plasmids,
using a commercially available assay. This preliminary investigation allowed us to rapidly exclude
molecules showing i) low or no activity (4d, 12d, 18d, 20d, 22d, 23d, 33d, 45d, 49d), ii) poor
solubility in cell culture media (21d, 30d, 34d), iii) discrepancy between the data obtained in the
two different screening procedures such as HR inhibition not confirmed by increased olaparib
efficacy in the viability assay or, on the contrary, increased olaparib efficacy without HR inhibition
(31d, 36d), iv) a non-dose-dependent effect (39d). As for the emissucinic acid-containing
compounds (4d, 12d, 18d, 20d-23d, 30d-31d), one of the reasons of their lower potency in cells
might be the general poor permeability, likely related to the ionizable acid moiety. The data
reported in Table 2 show that 35d was the most promising compound in the HR activity test. We
then characterized 35d in additional biophysical and cell-based experiments.
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To further assess the physical interaction between 35d and RAD51, a microscale thermophoresis
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MST) assay was performed on the recombinant human RAD51 (see Experimental Section). The
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binding assay allowed us to determine the dissociation constant (K ) for the RAD51-35d interaction
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and binding. The final binding curve (Figure 5) shows that 35d binds to RAD51 with a K value of
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1 ± 6 M. This is in agreement with the ELISA assay, supporting the initial hypothesis that 35d
could act as a RAD51-BRCA2 disruptor.
Figure 5. MST analysis of His-hRAD51-35d binding. Titration curve of (RED-tris-NTA 2nd
Generation)-His-hRAD51 (80 nM) with increasing concentrations of 35d. Sigmoidal fitting curve
was obtained using the Affinity Analysis software of NanoTemper Technologies. MST data are the
average of three replicates.
To further characterize the effect of 35d on HR, the compound was administered at different
doses to BxPC3 cells for 5 h simultaneously with plasmid transfection; as shown in Figure 6A, it
produced a statistically significant dose-response effect in reducing cell HR. The compound was
tested up to 40 M (an upper limit in terms of solubility), and we estimated the dose causing a 50%
inhibition of HR by applying the polynomial regression to the collected data. The EC was 18.4
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M.
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An additional evidence of compromised HR was obtained by assessing the localization of
RAD51 in BxPC-3 nuclei after DNA damage. The results of this experiment are reported in Figure
6B,C. To obtain massive DNA damage, BxPC-3 cultures were exposed for 1 h to 50 M cisplatin.
The immunohistochemical staining of RAD51 shown in Figure 6B revealed evident nuclear foci in
cisplatin-treated cells, which appeared significantly reduced when the drug was administered in
association with 20 M 35d. Furthermore, the percentage of RAD51-labeled nuclei measured in
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cultures exposed to 20 M 35d was superimposable to that observed in cells exposed to the
association of cisplatin with 20 M 35d (Figure 6C).
These results were in good agreement with the ELISA and MST outcomes, ultimately pointing to
3
5d as a novel and RAD51-BRCA2 disruptor with a clear capability to interfere with HR.
The sustained inhibition of HR in cells should result in increased DNA damage, ultimately
leading to mutations and chromosome aberrations; these effects are expected to be further amplified
by PARP inhibition. The extent of DNA damage produced in cells treated for 48 h with 20 M 35d,
administered singularly or in association with 10 M olaparib, was studied by evidencing nuclear -
H2AX foci by immunofluorescence. The experiment was performed on both BxPC-3 and Capan-1
cultures. Capan-1 cells are derived from a human pancreatic adenocarcinoma (very similar to
3
8
BxPC3cells) and are BRCA2-defective. As a consequence, they do not operate RAD51-BRCA2-
dependent HR. The olaparib dose was selected on the basis of previously obtained results with the
same cell cultures.30,31 Results are reported in Figure 7A,B.
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Figure 6. (A): Effect on HR caused by 35d administered to BxPC3 cells during plasmid
transfection (5 h). HR was evaluated by real-time PCR, as described in the Experimental Section.
Data were statistically analyzed using the column statistics of Prism 5 software, which applies the
inferences analysis and the one-sample t-test. The observed inhibitory effect was significantly
different from 0 (the level of untreated cultures) for all tested doses, with p < 0.05. (B,C):
Immunofluorescence detection of RAD51 in BxPC-3 nuclei after a treatment with 50 M Cisplatin
(Cpl) given separately or in combination with 20 M 35d. Experimental details are reported in the
Experimental Sections. B: Representative pictures showing DAPI-stained cell nuclei and the
corresponding immune-labeling of RAD51 localization. In untreated cells (CTR), RAD51 labeling
is clearly evident in cytoplasm and does not appear in cell nuclei. In the pictures of Cpl-exposed
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cells, nuclear localization of the protein is clearly evident in 3 out of the 6 shown nuclei. A higher
magnification detail was included for this sample. C: The bar graph shows the percentage of
RAD51-labeled nuclei counted by two independent observers who analyzed the treated cultures.
Data were statistically evaluated by applying the one-way ANOVA, which indicated a significantly
increased nuclear RAD51 labeling caused by Cpl (p < 0.05) and no statistically significant
difference between cells treated with 35d and those exposed to 35d + Cpl.
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The microscope pictures shown in Figure 7A showed increased evidence of -H2AX labeling in
nuclei of BxPC-3 cells exposed to the compounds’ association, compared to the labeling observed
in cultures treated with olaparib. In Capan-1 cultures, the constitutive -H2AX labeling in nuclei
appeared more evident than that observed in BxPC-3 cells.
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Figure 7. (A,B): Evaluation of DNA damage through immune detection of nuclear -H2AX foci
in BxPC-3 and Capan-1 cells exposed for 48 h to olaparib (10 µM) or 35d (20 µM), given alone or
in combination. (A): Representative pictures showing DAPI-stained cell nuclei and the
corresponding immune-labeling of -H2AX. In BxPC-3 cells, co-administration of 35d and
olaparib produced increased -H2AX labeling. A higher magnification detail was included for this
sample. As expected, Capan-1 cells showed a constitutive -H2AX labeling which was highly
increased by olaparib, but was unaffected by 35d co-administration. (B): The bar graph shows the
percentage of -H2AX -labeled nuclei counted by two independent observers who analyzed the
treated cultures. Data obtained in bxPC-3 cells were statistically evaluated by applying the one-way
ANOVA, which indicated a statistically significant difference between the cultures treated with
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olaparib and those exposed to olaparib + 35d. (C,D): Evaluation of micronuclei generation in
BxPC3 cells treated (72 h) with 35d and olaparib, given alone or in combination. C: Representative
pictures showing DAPI-stained cell nuclei. White asterisks indicate the presence of micronuclei. D:
The percentage of cells bearing micronuclei was estimated by two independent observers, by
analyzing 100-250 cells for each treatment sample. The obtained results were statistically analyzed
by applying the one-way ANOVA, which indicated a p value < 0.01.
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Moreover, in agreement with data showing higher PARPi sensitivity for cells lacking functional
17
HR, these cultures showed increased nuclear labeling when exposed to olaparib. Notably, this
labeling was not further enhanced by 35d co-administration. The percentage of -H2AX-labeled
nuclei measured in all treated cultures is shown in the bar graphs of Figure 7B.
The sustained and increased DNA damage produced in BxPC-3 nuclei generates chromosomal
aberrations which can be visualized through the presence of small DNA-staining bodies outside of
the main nucleus (micronuclei). The microscope pictures of Figure 7C show the appearance of this
feature in BxPC3 cells exposed for 72 h to 20 M 35d, administered alone or in combination with
1
0 M olaparib. The percentage of cells bearing micronuclei is reported in the graph of Figure 7D.
Notably, cells bearing micronuclei were markedly more frequent in cultures treated with the
3
5d/olaparib combination.
Taken together, the results reported in Figures 6 and 7 significantly support the requested
mechanism of action for 35d. Therefore, we conducted further experiments to test whether the
3
5d/olaparib combination would induce synthetic lethality.
We simultaneously evaluated cell viability and cell death, measured at 72 h in BxPC3 cells
exposed to 35d alone or in combination with 10 M olaparib (Figure 8). The statistical analysis (see
legend of Figure 8) compared the results for cultures treated with different doses of 35d or the
3
5d/olaparib combination (Figure 8A, upper panel). When applied to the data of the cell viability
experiment, this analysis indicated a statistically significant difference produced by 35d in
combination with olaparib in all the treated BxPC3 cultures, with p values ranging from 0.01 (10
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M 35d) to 0.0001 (15 and 20 M 35d). When we applied the same analysis to the data of the cell
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death experiment, we found no statistically significant increase in cell death for olaparib when co-
administered with 10-15 M 35d. Notably, in cultures exposed to olaparib + 20 M 35d (the
concentration producing the highest inhibition of HR), the evidence for cell death was markedly
increased and statistically significant, with p < 0.0001 (Figure 8A, lower panel).
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When cell death is a consequence of progressive DNA damage accumulation induced by
simultaneous PARP and HR inhibition, we would expect it to emerge gradually over the time. To
confirm and better characterize the lethality observed in cultures treated with the 35d/olaparib
combination, we therefore considered it inappropriate to conduct a simple evaluation of the
commonly used markers (e.g. caspase activation), since these can show very transient changes.
Instead, we observed cell morphology and reaction to vital dyes after the 72-h treatment, when the
experiment of Figure 8A indicated a significant level of cell death.
Figure 8B shows microscope pictures of BxPC3 cells stained with mixed DAPI and PI. The
simultaneous use of these two dyes can demonstrate cell death and indicate the death pattern.39
DAPI is cell-permeable and evidences nuclear morphology; healthy cells appear to display normal
nuclear morphology in the absence of PI staining, since this dye is not cell-permeable. Cells
undergoing apoptosis display nuclear condensation, which is indicated by increased DAPI staining.
PI staining indicates compromised membrane integrity, which characterizes necrotic cells and late-
apoptotic cells maintained in culture.
The microscope pictures show that untreated BxPC3 cells display only a moderate DAPI staining
of their nucleus. Nuclear DAPI staining is slightly increased in cells treated with 35d alone, but is
strikingly bright only in cells treated with the 35d/olaparib combination. Furthermore, PI staining
appeared only in these cultures, confirming the manifestation of synthetic lethality. The
simultaneous marked staining of these cells with both dyes could indicate an apoptotic phenomenon
followed by compromised membrane integrity because of cell persistence in culture.
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As expected, the sustained and increased DNA damage observed in BxPC3 cells treated with the
3
5d/olaparib combination (Figure 7) reproduced the desired mechanism of synthetic lethality
(Figure 8). These results are also relevant given the mutated p53 status of BxPC3 cells, which
should make them more resistant to mechanisms that induce cell death.
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Figure 8. (A): BxPC3 cell viability and death measured after 72 h exposure to 35d and 10 M
olaparib, given alone or in combination. Data were analyzed by two-way ANOVA using the two
treatments (35d and olaparib) as variables. In the cell viability experiment, Bonferroni post-test
indicated a statistically significant difference produced by olaparib co-administration in all BxPC3
cultures treated with the different 35d doses, with p values ranging from 0.01 (10 M 35d) to
0
.0001 (15 and 20 M 35d). The same analysis was applied to the data from the cell death
experiment and indicated that no statistically significant increase in cell death was produced by
olaparib when co-administered with 10-15 M 35d. In cultures exposed to olaparib + 20 M 35d
the evidence for cell death was markedly increased and statistically significant, with p < 0.0001.
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(B): After the 72-h treatment, BxPC3 cells were stained with vital dyes. As shown in the
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microscope pictures, the only culture displaying sharp evidence of cell death was that exposed to
the combination of olaparib/20 M 35d, as demonstrated by PI nuclear staining.
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Finally, the antiproliferative effect of the 35d/olaparib combination was also studied on the HR-
defective Capan-1 culture and on a non-neoplastic cell line derived from human kidney (HK-2).
Moreover, to evaluate the antineoplastic potency of the 35d/olaparib association, we also calculated
the combination index (CI) of the two compounds, using the procedure previously described30,31
(Figure 9).
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Figure 9. (A): Antiproliferative effect caused by 35d and its association with olaparib, measured
in Capan-1 cells (which do not operate RAD51-BRCA2-dependent HR) and in normal
immortalized human renal cells (HK-2). The same procedures described for BxPC3 cells were used
here. (B): Combination indexes of the olaparib/35d association measured in the three used cell
lines, calculated according to the method previously described.30,31 For BxPC-3 cells, the data
reported in Figure 8A (Cell Proliferation) were used. Data were analyzed using the column statistics
and the one-sample t-test of Prism 5 software. For BxPC-3 cells, this test showed a statistically
significant difference from 0.8 for the combination of olaparib /20  35d. Values < 0.8 indicate
synergism between the two compounds.
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According to this method, a CI < 0.8 indicates synergism, while a result ranging from 0.8 and 1.2
indicates additive effects. This evaluation (Figure 9A,B) was performed for all the three studied cell
cultures; for BxPC-3 cells, the data reported in Figure 8A (cell viability experiment) were used for
the calculation of CI. Interestingly, in these cells the potency of the compound combination
increased in parallel with the 35d dose, reaching a statistically significant difference from 0.8 at the
2
0 M concentration. The effects observed when 20 M 35d is combined with 10 M olaparib may
thus indicate a synergism between the two compounds.
In HK-2 cultures, no dose of 35d appeared to significantly increase the antiproliferative power of
olaparib (two-way ANOVA). In Capan-1 cells, the same statistical evaluation showed significantly
increased antiproliferative effects for 35d/olaparib. However, in these cells, the CI did not show
increasing potency of the compound combination with dose escalation. Moreover, at all tested
doses, the value of CI was not significantly different from 1, which is the level measured in BxPC3
cells exposed to 10 M 35d, a dose which did not relevantly affect (≈ 10%) HR (Figure 6A). This
result further supports the idea that the over-additive effects in the 35d/olaparib combination arise
from and are strictly related to HR inhibition.
Overall, these data supported our working hypothesis that combining a RAD51-BRCA2 small
molecule disruptor with olaparib could be a valuable strategy for inducing synthetic lethality in
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cancer cell lines with fully functional BRCA genes and homologous recombination. This includes
pancreatic cancer, which is a major unmet need in oncology. We believe that this paradigm could be
used to discover innovative anticancer therapies based on other lethal gene pairs using a similar
medicinal chemistry strategy.
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1
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0
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Table 1. Structures and EC of compounds 4d-57d on ELISA Assay.
5
0
O
R2
Ar
N
N
O
R1
N
H
Compound
R1
R2
Ar
EC (μM)*
50
F
F
4
5
6
7
8
9
d
d
d
d
d
d
Cl
-CH
2
CH
2
COOH
16 ± 4
50 ± 10
34 ± 3
NA**
NA**
NA**
NA**
59 ± 8
16 ± 2
NA**
NA**
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-CH
3
-CH
2
CH
3
F
-CH
2
CH
2
NH
2
F
-CH
2
CH
2
C(O)NH
2
F
-CH
2
CH
2
C(O)
2
CH
3
F
F
1
1
1
1
1
0d
1d
2d
3d
4d
-CH
2
CH
2
NHS(O)
2
CH
3
-CH
2
2
2
2
CH
CH
CH
CH
2
2
2
2
COOH
COOH
COOH
COOH
Cl
Br
O
-CH
-CH
-CH
O
F
1
5d
Cl
-CH
2
CH
2
COOH
NA**
F
1
1
6d
7d
Cl
Cl
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
F
29 ± 2
F
O
NA**
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 102
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
Cl
Cl
Cl
Cl
-CH
-CH
-CH
-CH
2
2
2
2
CH
CH
CH
CH
2
2
2
2
COOH
COOH
COOH
COOH
F
Cl
Br
O
1
1
2
2
8d
9d
0d
1d
13 ± 1
25 ± 5
20 ± 2
20 ± 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
22d
Cl
Cl
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
2 ± 0.5
10 ± 2
N
N
2
3d
N
24d
25d
Cl
Cl
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
N
N
NA**
28 ± 4
N
N
N
2
6d
Cl
Cl
Cl
-CH
-CH
-CH
2
2
2
CH
CH
CH
2
2
2
COOH
COOH
COOH
NA**
NA**
NA**
N
27d
N
N
N
2
8d
Cl
Cl
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
N
29d
NA**
N
3
3
0d
1d
N
15 ± 3
N
N
Cl
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
17 ± 4
N
N
3
3
3
3
3
3
3
3
2d
3d
4d
5d
6d
7d
8d
9d
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
65 ± 5
8 ± 2
N
-CH
-CH
-CH
2
2
2
CH
CH
CH
3
3
3
Cl
Br
O
19 ± 1
19 ± 1
10 ± 0.7
38 ± 7
NA**
-CH
-CH
-CH
-CH
3
3
3
3
Cl
Br
O
15 ± 4
2
8
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Page 29 of 102
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
F
F
40d
1d
Cl
Cl
-CH
-CH
3
3
F
NA**
NA**
O
4
Cl
Cl
Cl
-CH
-CH
-CH
3
3
3
Cl
Br
O
42d
43d
44d
45 ± 8
70 ± 15
70 ± 6
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
4
4
5d
6d
Cl
Cl
-CH
-CH
3
3
18 ± 1
N
N
50 ± 15
N
4
4
7d
8d
Cl
Cl
-CH
-CH
3
3
N
N
40 ± 3
NA**
N
N
N
49d
Cl
-CH
3
0.95 ± 0.05
Cl
Cl
H
-CH
-CH
3
3
N
5
5
5
5
0d
1d
2d
3d
NA**
46 ± 9
NA**
NA**
N
N
N
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
F
H
Br
5
4d
H
-CH
2
CH
2
COOH
NA**
Br
5
5
5d
6d
H
H
-CH
-CH
2
2
CH
CH
2
2
COOH
COOH
Cl
NA**
NA**
O
F
5
7d
H
-CH
2
CH
2
COOH
F
NA**
F
*
*
: All points were tested in triplicate with error bars indicating the standard deviation.
*
NA: not active.
Table 2. Preliminary biological screening of compounds showing EC on ELISA assay ranging
5
0
from 0.95 to 20 μM.
Compound
EC ELISA (μM)
Preliminary Biological Screening
50
2
9
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