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

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.


■ 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 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 anticancer therapeutic strategy. 13−15 Genome instability is a hallmark of cancer. 16 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 (ADPribose)polymerase (PARP) inhibitors in oncology patients with BRCA1/2 mutations. PARP is crucial for repairing DNA single-strand breaks (SSBs), whereas BRCA1/2 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 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 One of BRCA2's key mechanisms in DDR is to recruit RAD51, an evolutionarily conserved recombinase, at the site of DSBs where it performs DSB repair through HR. 20 Additionally, the expression of RAD51 and the rate of RAD51-mediated HR are both elevated in a wide variety of cancers (e.g., breast, pancreatic). 21 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 wellconserved 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 (PPIs). 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 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 fully reproduce the paradigm of synthetic lethality. 31 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 (SARs) 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).

■ RESULTS AND DISCUSSION
Hit Identification and Optimization. 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 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 FxxA pocket to identify the first RAD51-BRCA2 disruptors. 30 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. This further suggests this pocket as a critical site for RAD51's mechanism of action. 33 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 toward a more in depth understanding of the mechanism of inhibition of RAD51-BRCA2 interaction.
Here, 42 small molecules were selected, purchased, and tested for their inhibitory activity using a competitive biochemical ELISA assay, as previously described by Rajendra et al. 33 Among the tested compounds, the commercially available dihydroquinolone pyrazoline derivative 4d (Figures 3  and 4) was the best candidate in terms of EC 50 and chemical tractability. Its activity 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 (similar to the Phe1546 of BRC4) in a hydrophobic pocket outlined by the side chains of Leu204, Tyr205, Met251, Leu255, and Phe259 of RAD51 and (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 carbonyl group of the pyrazoline side chain, establish hydrogen bonds with Arg254 and Glu258. Notably, both enantiomers show the same global pattern of interactions.
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 and methoxy, tert-butyl, and 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).
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 ELISA assay against the parent compound 4d (Table 1). This assay is effective in evaluating the ability of new molecules to compete with BRC4 to bind to RAD51. 30 Replacing the acyl chain of the pyrazoline nitrogen yielded compounds (5d−10d, Table 1) that had reduced inhibitory Scheme 1. Synthesis of Dihydroquinolone Pyrazoline Intermediates 86c−114c a a 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)indazolylpyrazoline 49d showed the best activity of the series with EC 50 = 0.95 ± 0.05 μM, while 33d−36d, 39d, 45d showed an activity very similar to that of the initial hit. A drop in potency was observed with compounds 37d, 42d−44d, 46d−47d, and 51d, while 38d, 40d−41d, 48d, and 50d were inactive. Finally, removing the 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 50 = 4 ± 0.5 μM; 4d-II, 10 ± 1 μM) to that of the parent 4d, suggesting 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 50 values ranging from 0.95 to 20 μM. Accordingly, 4d, 12d, 18d, 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 cellbased 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 fully functional BRCA2. 38 As shown in Table 2 and according to the rationale of our hypothesis, our preliminary screening consisted of 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 50 obtained with the ELISA test. 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 for 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.
To further assess the physical interaction between 35d and RAD51, a microscale thermophoresis (MST) assay was performed on the recombinant human RAD51 (see Experimental Section). The binding assay allowed us to determine the dissociation constant (K d ) for the RAD51-35d interaction and binding. The final binding curve ( Figure 5) shows that 35d binds to RAD51 with a K d value of 11 ± 6 μM. This is in agreement with the ELISA assay, supporting the initial hypothesis that 35d could act as a RAD51-BRCA2 disruptor.
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 μM (an upper limit in terms of solubility), and we estimated the dose causing a 50% inhibition of HR by applying the  μM.
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 μM 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 μM 35d. Furthermore, the percentage of RAD51-labeled nuclei measured in cultures exposed to 20 μM 35d was superimposable to that observed in cells exposed to the association of cisplatin with 20 μM 35d ( Figure 6C).
These results were in good agreement with the ELISA and MST outcomes, ultimately pointing to 35d as a novel 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 μM 35d, administered singularly or in association with 10 μM olaparib, was studied by evidencing nuclear γ-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 BxPC3cells) and are BRCA2-defective. 38 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.
The microscope pictures shown in Figure 7A showed increased evidence of γ-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 γ-H2AX labeling in nuclei appeared more evident than that observed in BxPC-3 cells.
Moreover, in agreement with data showing higher PARPi sensitivity for cells lacking functional HR, 17 these cultures showed increased nuclear labeling when exposed to olaparib. Notably, this labeling was not further enhanced by 35d coadministration. The percentage of γ-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 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 μM 35d, administered alone or in combination with 10 μM 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 35d/ 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 35d/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 μM olaparib ( Figure 8). The statistical analysis (see legend of Figure 8) compared the results for cultures treated with different doses of 35d or the 35d/ 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 μM 35d) to 0.0001 (15 and 20 μM 35d). When we applied the same analysis to the data of the cell death experiment, we found no statistically significant increase in cell death for olaparib when coadministered with 10−15 μM 35d. Notably, in cultures exposed to olaparib + 20 μM 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).
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 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 shows 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.
As expected, the sustained and increased DNA damage observed in BxPC3 cells treated with the 35d/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.
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 described 30,31 (Figure 9).
According to this method, CI < 0.8 indicates synergism while a result ranging from 0.8 to 1.2 indicates additive effects. This evaluation ( Figure 9) 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 20 μM concentration. The effects observed when 20 μM 35d is combined with 10 μM 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 μM 35d, a dose that did not relevantly affect (≈10%) HR ( Figure 6A). This result further supports the idea that the overadditive 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 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 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 μM cisplatin (Cpl) given separately or in combination with 20 μM 35d. Experimental details are reported in the Experimental Section. (B) Representative pictures showing DAPIstained 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 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. that this paradigm could be used to discover innovative anticancer therapies based on other lethal gene pairs using a similar medicinal chemistry strategy.

■ CONCLUSIONS
Continuing our research line, we described a series of dihydroquinolone pyrazoline derivatives as a new class of RAD51-BRCA2 disruptors. Compound 4d was identified as a promising hit, and subsequent SAR efforts yielded 35d with the desired biological profile. As expected, 35d bound to its target (RAD51) and inhibited the protein−protein interaction between RAD51 and BRCA2. Importantly, it synergized and reproduced the paradigm of synthetic lethality in combination with olaparib in pancreatic cancer cells (BxPC3). These effects were strictly related to the extent of HR inhibition in a dosedependent trend. This is the most promising achievement of the current investigation and supports our working hypothesis that one can trigger synthetic lethality using only small organic molecules. Interestingly, the observed synthetic lethality was triggered by tackling two biochemically different mechanisms: enzyme inhibition (PARP) and protein−protein disruption (RAD51-BRCA2). This highlights how complex and diverse mechanisms of action can synergistically contribute to the same physiological and, in turn, pharmacological activity. We note, however, that 35d's low solubility may affect its metabolic and pharmacokinetic profile (DM/PK), preventing it from being studied further in in vivo cancer models. Structural tuning is therefore required (and currently ongoing) to discover more drug-like dihydroquinolone pyrazoline derivatives.
In conclusion, we have further shown that synthetic lethality may be a suitable framework for discovering innovative anticancer therapies. We are confident that this novel concept will open up several new avenues based on other lethal gene pairs to meet the medical needs in oncology.

■ EXPERIMENTAL SECTION
Chemistry. General Chemical Methods. Solvents and reagents were obtained from commercial suppliers and used without further purification. If required, solvents were distilled prior to use. Automated column chromatography purifications were conducted using a Teledyne ISCO apparatus (CombiFlash Rf) with prepacked silica gel columns of different sizes (from 4 to 120 g). Mixtures of In BxPC-3 cells, coadministration of 35d and olaparib produced increased γ-H2AX labeling. A higher magnification detail was included for this sample. As expected, Capan-1 cells showed a constitutive γ-H2AX labeling that was highly increased by olaparib but was unaffected by 35d coadministration. (B) The bar graph shows the percentage of γ-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 olaparib and those exposed to olaparib + 35d. increasing polarity of cyclohexane and ethyl acetate or dichloromethane and methanol/ethanol were used as eluents. Preparative TLCs were performed using Macherey-Nagel precoated 0.05 mm TLC plates (SIL G-50 UV254). Microwave heating was performed using Explorer-48 positions instrument (CEM). NMR experiments were run on a Bruker Avance III 400 MHz spectrometer (400.13 MHz for 1 H and 100.62 MHz for 13 C), equipped with a BBI probe and Z-gradients, or on a Bruker FT NMR Avance III 600-MHz spectrometer (600.130 MHz for 1 H and 150.903 MHz for 13 C) equipped with a 5 mm CryoProbe QCI quadruple resonance, a shielded Z-gradient coil, and the automatic sample changer SampleJet NMR system. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide (DMSO-d 6 ) or deuterated chloroform (CDCl 3 ) as solvents. Chemical shifts for 1 H and 13 C spectra were recorded in parts per million using the residual nondeuterated solvent as the internal standard (for CDCl 3 , 1 H 7.26 ppm, 13 C 77.16 ppm; for DMSO-d 6 , 1 H 2.50 ppm, 13 C 39.52 ppm). UPLC−MS analyses were run on a Waters ACQUITY UPLC/MS system consisting of an SQD (single quadrupole detector) mass spectrometer equipped with an electrospray ionization interface and a photodiode array detector. The PDA range was 210−400 nm. The analyses were performed on either an ACQUITY UPLC HSS T3 C 18 column (50 × 2.1 mm i.d., particle size 1.8 μm) with a VanGuard HSS T3 C 18 precolumn (5 mm × 2.1 mm i.d., particle size 1.8 μm) (log D < 1) or an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm i.d., particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 mm × 2.1 mm i.d., particle size 1.7 μm) (log D > 1). The mobile phase was 10 mM NH 4  General Procedure A for the Synthesis of Quinolin-2(1H)one Acrolyl Intermediates. This procedure has been applied to the preparation of 86b−114b (Scheme 1). In a round-bottomed flask, the appropriate quinolin-2(1H)-one (60a−61a, 1.00 equiv) and potassium hydroxide (25.00 equiv) were stirred in EtOH/H 2 O (4:3 v/v, 0.05 M) at 0°C for 45 min prior to the addition of an appropriately substituted aryl aldehyde (62−85, 1.00 equiv). The reaction mixture was stirred overnight as it gradually reached room temperature. The reaction was quenched by slow addition of acetic acid (25.00 equiv). The crude was extracted with DCM/H 2 O (3 × 50 mL), the organic layer was then dried over Na 2 SO 4 , and the solvent was removed under reduced pressure. The desired compound was obtained after purification over silica gel unless otherwise noted.
General Procedures C 1 to Obtain the Final Desired Dihydroquinolone Pyrazoline Derivatives (Scheme 2). This procedure has been applied to synthesize 4d, 11d−32d, 52d−57d. In a microwaveable vessel, the appropriate pyrazol-3-ylquinolin-2(1H)- 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 coadministered with 10−15 μM 35d. In cultures exposed to olaparib + 20 μM 35d the evidence for cell death was markedly increased and statistically significant, with p < 0.0001. (B) After the 72 h treatment, BxPC3 cells were stained with vital dyes. As shown in the microscope pictures, the only culture displaying sharp evidence of cell death was that exposed to the combination of olaparib/20 μM 35d, as demonstrated by PI nuclear staining. one intermediate (86c−90c, 92c−93c, 95c−103c, 108c, 1.00 equiv) was dissolved in anhydrous THF (0.5 M). The succinic anhydride 115 (2.00 equiv) was added. The solution was microwaved (200 W) with stirring for 45 min at the appropriate temperature. The THF was removed under reduced pressure, and the organic layers were dissolved in DCM, washed with HCl aq pH 2 (3 × 30 mL), and dried over Na 2 SO 4 . The solvent was removed under reduced pressure, and the crude was purified over silica gel.
General Procedures C 2 to Obtain the Final Desired Dihydroquinolone Pyrazoline Derivatives (Scheme 2). This procedure has been applied to synthesize 5d, 36d−51d. In a microwaveable vessel, the appropriate pyrazol-3-ylquinolin-2(1H)-one intermediate (86c−90c, 92c−93c, 95c−103c, 108c, 1.00 equiv) was dissolved in anhydrous THF (0.5 M). The acetic anhydride 116 (2.00 equiv) was added. The solution was microwaved (200 W) with stirring for 45 min at the appropriate temperature. The THF was removed under reduced pressure, and the organic layers were dissolved in DCM, washed with HCl aq pH 2 (3 × 30 mL), and dried over Na 2 SO 4 . The solvent was removed under reduced pressure, and the crude was purified over silica gel.