PARP-1 (pIC50 = 8.22); PARP-2 (pIC50 = 8.44); PI3Kα (pIC50 = 8.25); PI3Kδ (pIC50 = 8.13); PI3Kγ (pIC50 = 6.08); PI3Kβ (pIC50 = 6.54)

Significantly more apoptosis occurs when PARP/PI3K-IN-1 (Compound 15; 1 μM; 72 hours) is administered [1]. Following cells treatment with 1 μM of PARP/PI3K-IN-1 for 72 hours, the autophosphorylation levels of AKT and S6 were lowered, while the autophosphorylation level of ERK was increased. This suggests that the compound has the ability to both inhibit and activate the PI3K and ERK pathways [1]. BRCA1/2 expression in MDA-MB-468 cancer cells can be significantly downregulated at the mRNA level by PARP/PI3K-IN-1 (1 μM) [1]. In addition to demonstrating strong anti-proliferative action against BRCA-rich cells MDA-MB-231 and MDA-MB-468, PARP/PI3K-IN-1 also shown considerable inhibitory effect against BRCA-deficient cells HCC1937 and HCT116 [1].

Tumor growth is markedly inhibited by PARP/PI3K-IN-1 (intraperitoneal injection; 50 mg/kg; twice daily (BID) for 34 days) [1].

In Vivo Antitumor Effects Study[1]
On the basis of the excellent enzymatic and antiproliferative activities of compounds 14 and CAY10749 (PARP/PI3K-IN-1; compound 15) in vitro, we then evaluated their antitumor activities in vivo in MDA-MB-468 xenograft mouse model. Compounds 14 and 15, Olaparib, BKM120, and the combination of Olaparib and BKM120 were administered by intraperitoneal injection twice daily (BID) for 34 consecutive days. As shown in Figure 9, compounds 14 and CAY10749 (PARP/PI3K-IN-1; compound 15) significantly suppressed the tumor growth at a dose of 50 mg kg–1, and they were both well-tolerated with no mortality. The tumor suppression effects of 14 (TGI: 52.7%) and 15 (TGI: 73.4%) were both more effective than those of Olaparib (TGI: 28.5%) and BKM120 (TGI: 33.4%) and even the combination of Olaparib and BKM120 (TGI: 48.4%). It is also noteworthy that no significant weight fluctuations were observed during the whole process. The results suggest that dual PARP/PI3K inhibitors are superior to the single-target inhibitors in the antitumor efficacy.

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In Vivo Western Blot Analysis[1]
Because CAY10749 (PARP/PI3K-IN-1; compound 15) displayed more promising antitumor activity in vivo, it was submitted to mechanistic study to bolster our conclusion that the prominent antitumor effect of CAY10749 (PARP/PI3K-IN-1; compound 15) is indeed engendered by its PARP/PI3K dual-targeting capability. Western blot analysis of the excised tumor tissue from MDA-MB-468 tumor bearing mice was carried out. In good agreement with the in vitro Western blot analysis, compound 15 strongly inhibited the expression of pAKT and pS6, while it stimulated the expression of pERK and pETS1 (Figure 10). Moreover, 15 significantly downregulated the expression of BRCA1/2 and increased the level of cleaved PARP. In conclusion, our medicinal chemistry endeavor led to the discovery of compound 15, demonstrating that the dual-inhibition of PARP and PI3K with a single chemical entity is feasible.

In Vitro PARP Inhibition Assay[1]
PARP-1 and PARP-2 inhibition assays were carried out according to previously reported procedures.The inhibition of the tested compounds on PARP-1 and PARP-2 enzymatic activity in a cell-free system was determined by ELISA in 96-well plates. Each well was precoated with histone (20 μg/mL) diluted in 100 μL of PBS buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 150 mM NaCl, pH 7.4) and incubated at 37 °C overnight. After incubation, the plate was washed three times using 200 μL of PBST buffer (1x PBS containing 0.05% (v/v) Tween 20) and blocked with 200 μL of blocking buffer (1x PBST containing 5% (v/v) nonfat milk) at room temperature for 60 min. The plate was subsequently washed three times with 200 μL of PBST buffer as described above. Biotinylated-NAD+ (8 μM) and activator deoxyoligonucleotide (100 μg/mL) diluted in 70 μL of reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, pH 8.0) then were added into each well, followed by adding 10 μL of compound or solvent control at varying concentrations. Compounds to be tested were diluted in 10% (v/v) DMSO and tested in 10-dose with 3-fold serial dilution starting at a concentration of 1 μM. The reaction was initiated by the addition of 20 μL of PARP-1 or PARP-2 (10 ng/well) at 37 °C for 1 h. The reaction mixture was discarded, and the plate was washed three times with 200 μL of PBST buffer and tapped onto a clean paper towel as described above. Next, 50 μL of streptavidin-HRP was added to each well and incubated for 30 min at room temperature. The plate was washed three times with 200 μL of PBST buffer and tapped onto a clean paper towel as described above. Finally, 100 μL of ECL solution was added and incubated at room temperature for 15 min. Luminescent signal was measured using a multiwell spectrophotometer. The inhibition rate of PARP-1 or PARP-2 enzymatic activity was calculated as (Lu control – Lu treated/Lu control) × 100%. The concentration required for 50% inhibition of PARP-1 or PARP-2 enzymatic activity (IC50) was calculated using nonlinear regression with a normalized dose–response fit using Prism GraphPad software.

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In Vitro PI3K Inhibition Assay[1]
The assay was performed using an ADP-Glo Plus luminescence kinase assay kit. 50 μL of PI3K isoforms reaction mixture contains 10 mM Tris-HCl, pH 7.5, 25 μM ATP, 9.75 μM PIP2, 5% (v/v) glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 2% (v/v) DMSO at the following concentrations for each isoform: PI3Kα,β at 60 ng/mL; PI3Kγ at 8 ng/mL; and PI3Kδ at 45 ng/mL. Compounds to be tested were diluted in 10% (v/v) DMSO and tested in 10-dose with 3-fold serial dilution starting at a concentration of 1 μM. The assay plate was covered and incubated at room temperature (PI3Kα, PI3Kβ, and PI3Kγ for 1 h and PI3Kδ for 2 h). Next, 50 μL of ADP-Glo reagent was added and incubated for 40 min at room temperature followed by another 30 min incubation with 50 μL of kinase detection mixture. Luminescence signal was measured using a multiwell spectrophotometer. The IC50 values were calculated using nonlinear regression with normalized dose–response fit using Prism GraphPad software.

Apoptosis analysis[1]
Cell Types: MDA-MB-468 Cancer cell
Tested Concentrations: 1 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Caused a significant increase in apoptosis.

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Western Blot Analysis [1]
Cell Types: MDA-MB-468 cancer cells
Tested Concentrations: 1 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: After treating cells, the autophosphorylation levels of AKT and S6 diminished, and the autophosphorylation level of ERK increased.

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Cell Proliferation Inhibition Assay[1]
The human-cancer-cell lines MDA-MB-231, MDA-MB-468, HCT116, HCC1937, BxPC-3, A2780, Jurkat, DU145, A549, Caki-1, Ramos, and SW620 were maintained in RPMI1640 medium containing 10% (v/v) FBS at 37 °C in a 5% (v/v) CO2 humidified incubator. Cell proliferation assay was determined by the Cell Titer-Glo cell viability assay. Briefly, cells were passaged the day before dosing into a 96-well plate, allowed to grow for 12 h, and then treated with different concentrations (see Table S2 for details) of compound for 7 days at 5% CO2, 37 °C. After incubation, 100 μL of Cell Titer-Glo reagent was added to the assay plate, which was then incubated at room temperature for 10 min to stabilize luminescence signal and read by an Envision plate reader. The inhibition rate (%) = (1 – (RLU compound – RLU blank)/(RLU DMSO – RLU blank)) × 100%. The IC50 values were calculated using nonlinear regression with normalized dose–response fit using XLFit software.

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Western Blot Assay[1]
MDA-MB-468 cells were seeded into six-well plates and incubated overnight and then treated with or without different concentrations of compounds (BKM120 (1 μM), Olaparib (2 μM), BKM120 (1 μM) + Olaparib (2 μM), 14 (1 μM), and 15 (1 μM) for 6 h; medium with 1‰ (v/v) DMSO was used as the control. Cell samples were collected in ice-cold lysis buffer. Lysates were cleared by centrifugation at 14 000 rpm for 10 min at 4 °C, and supernatants were removed and assayed for protein concentration using the Pierce BCA Protein Assay Kit. Cell lysates were loaded to 8–12% SDS-PAGE and separated by electrophoresis. Separated proteins were then electrically transferred to polyvinylidene difluoride membranes, which were blocked with 5% bovine serum albumin/TBST for 1 h. Membranes were hybridized with the following primary antibodies: phospho-AKT (pAKT-Ser473), total AKT, phospho-S6 (pS6-Ser240), total S6, phospho-ERK (pERK-Thr202), total ERK, phospho-ETS1 (pETS1-Thr38), total ETS1, BRCA1, BRCA2, cleaved PARP, and β-actin in 1% nonfat dry milk. The bands were visualized using enhanced chemiluminescence after hybridization with a HRP-conjugated secondary antibody and then quantified by ImageJ software.

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Real-Time PCR[1]
The relative expression of BRCA1 or BRCA2 mRNA was detected using real-time PCR. Briefly, total RNA was extracted from cultured cells with TRIzol reagent according to the manufacturer’s instructions. Reverse transcription reactions were performed using PrimeScript RT reagent Kit with gDNA Eraser. For transcript quantification, SYBR Green-based qPCR was performed with PrimeScript RT Master Mix using a real-time PCR System. The human BRCA1 forward primer was 5′-GTCCCATCTGTCTGGAGTTGA-3′, and the reverse primer was 5′-AAAGGACACTGTGAAGGCCC-3′. The human BRCA2 forward primer was 5′-AAAGGACACTGTGAAGGCCC-3′, and the reverse primer was 5′-TTCTTCCTCTCTTTCATTGCG-3′. GAPDH was used as an internal control. The results were represented as fold changes relative to the internal control.

Animal/Disease Models: Sixweeks old male BALB/c nude mice, MDA-MB-468 cells [1]
Doses: 50 mg/kg
Route of Administration: ip; twice a day (BID) for 34 days
Experimental Results: Significant tumor inhibition grow.

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In Vivo Antitumor Activity Study[1]
Six-week-old male BALB/c nude mice were used. The animal permit number is SCXK (Peking) 2016-0011. Prior to implantation, MDA-MB-468 cells were harvested during exponential growth. The 2 × 10~6 cells were inoculated subcutaneously on the right flank of each BALB/c nude mouse. Mice were randomly divided into five treatment groups and a control group when the tumor size reached an approximate volume of 100 mm3. Olaparib (50 mg kg–1), BKM120 (27.5 mg kg–1), Olaparib + BKM120 (50 mg kg–1 + 27.5 mg kg–1), 14 (50 mg kg–1), and 15 (50 mg kg–1) were administered every 2 days for 34 days (6 mice per group) by intraperitoneal administration. The treatment with equal volume of PBS (5% DMSO, v/v) was used as the negative control. During treatment, tumor size and body weight were measured every 2 days. Tumor volume (V) was calculated using the equation V = ab2/2, where a and b stand for the longest and shortest diameters measured by vernier caliper, respectively.

[1]. Discovery of Novel Dual Poly(ADP-ribose)polymerase and Phosphoinositide 3-Kinase Inhibitors as a Promising Strategy for Cancer Therapy.J Med Chem. 2020 Jan 9;63(1):122-139.

Concomitant inhibition of PARP and PI3K pathways has been recognized as a promising strategy for cancer therapy, which may expand the clinical utility of PARP inhibitors. Herein, we report the discovery of dual PARP/PI3K inhibitors that merge the pharmacophores of PARP and PI3K inhibitors. Among them, compound 15 stands out as the most promising candidate with potent inhibitory activities against both PARP-1/2 and PI3Kα/δ with pIC50 values greater than 8. Compound 15 displayed superior antiproliferative profiles against both BRCA-deficient and BRCA-proficient cancer cells in cellular assays. The prominent synergistic effects produced by the concomitant inhibition of the two targets were elucidated by comprehensive biochemical and cellular mechanistic studies. In vivo, 15 showed more efficacious antitumor activity than the corresponding drug combination (Olaparib + BKM120) in the MDA-MB-468 xenograft model with a tumor growth inhibitory rate of 73.4% without causing observable toxic effects. All of the results indicate that 15, a first potent dual PARP/PI3K inhibitor, is a highly effective anticancer compound.[1]

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PARP inhibitors have clinical effectiveness restricted to a small subgroup of patients with BRCA mutations. Recently, it was reported that PI3K inhibition could promote HR deficiency and sensitize BRCA-proficient tumors to PARP inhibition. Therefore, cotargeting of PARP and PI3K has been recognized as a promising chemotherapeutic strategy to expand the utility of PARP inhibitors. In our efforts to obtain dual PARP/PI3K inhibitors, lead compound 1 was designed by combining the pharmacophores of PARP and PI3K inhibitors. Subsequent structural optimization was conducted, focusing on increasing the inhibitory activities and improving inhibitory balance, and led to the candidate compounds 14 and 15. They both showed potent and well-balanced inhibitory activities against PARP-1 and PI3Kα with pIC50 values greater than 8.22. Compound 15 displayed more potent antiproliferative activity against a panel of BRCA-proficient cancer cells than Olaparib. Cellular mechanistic studies showed that compounds 14 and 15 strongly inhibited the growth of MDA-MB-468 cells through suppressing the PI3K signaling pathway, downregulating BRCA1/2 expression and inducing DNA damage and apoptosis. In the MDA-MB-468 cell-derived xenograft model, compounds 14 and 15 displayed excellent antitumor efficacy at a dose of 50 mg kg–1, which is considerably more efficacious than the single administration of Olaparib or BKM120 and even their combined administration. In view of the structure together with its encouraging in vitro and in vivo properties, compound 15 as a first dual PARP/PI3K inhibitor is worthy of further profiling. Our data demonstrate that dual PARP/PI3K inhibitors have a good synergistic effect and should be extensively evaluated as a new class of targeted therapy against a wide range of oncologic diseases.[1]

C33H28F4N8O3

660.6208

660.2220

C, 60.00; H, 4.27; F, 11.50; N, 16.96; O, 7.27

2337386-47-5

146014481

White to off-white solid powder

3.2

FC1C([H])=C([H])C(=C([H])C=1C(N1C([H])([H])C2=C(C(=NC(C3=C([H])N=C(C([H])=C3C(F)(F)F)N([H])[H])=N2)N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])C([H])([H])C1([H])[H])=O)C([H])([H])C1([H])C2=C([H])C([H])=C([H])C([H])=C2C(N=N1)=O

LIQDGVXNWJAUNO-UHFFFAOYSA-N

InChI=1S/C33H28F4N8O3/c34-25-6-5-18(14-26-19-3-1-2-4-20(19)31(46)43-42-26)13-22(25)32(47)45-8-7-21-27(17-45)40-29(41-30(21)44-9-11-48-12-10-44)23-16-39-28(38)15-24(23)33(35,36)37/h1-6,13,15-16H,7-12,14,17H2,(H2,38,39)(H,43,46)

4-[[3-[2-[6-amino-4-(trifluoromethyl)pyridin-3-yl]-4-morpholin-4-yl-6,8-dihydro-5H-pyrido[3,4-d]pyrimidine-7-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one

PARP/PI3KIN1; CAY10749; CAY-10749; PARP/PI3K-IN-1; CHEMBL4470724; SCHEMBL23493529; LIQDGVXNWJAUNO-UHFFFAOYSA-N; CAY 10749; BDBM50520037; PARP/PI3K IN 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~151.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (3.78 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)