Nada Ibrahim

Pascal Bonnet

Jean-Daniel Brion

Jean-François Peyrat

Jerome Bignon

Hélène Levaique

Béatrice Josselin

Thomas Robert

Pierre Colas

Stéphane Bach

Helene Levaique

Samir Messaoudi

Mouad Alami
email: mouad.alami@u-psud.fr

Abdallah Hamze
email: abdallah.hamze@u-psud.fr

Identification of a new series of flavopiridol-like structures as kinase inhibitors with high cytotoxic potency

Keywords: Favopiridol, cytotoxicity, Kinase inhibition, structure-activity relationship. Highlights

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Flavones-based scaffolds belong to privileged family of bioactive structures. [START_REF] Nicolaou | Natural Product-like Combinatorial Libraries Based on Privileged Structures. 1. General Principles and Solid-Phase Synthesis of Benzopyrans[END_REF][START_REF] Pojero | Targeting multiple myeloma with natural polyphenols[END_REF][START_REF] Jalili-Baleh | A review on flavonoid-based scaffolds as multi-target-directed ligands (MTDLs) for Alzheimer's disease[END_REF][START_REF] Kshatriya | In memory of Prof. Venkataraman: Recent advances in the synthetic methodologies of flavones[END_REF] This attribute is undoubtedly due to the enormous number of therapeutic benefits they exhibit. Particularly, in cancer diseases, flavonoids are known for their ability to inhibit important cell signaling proteins such as cyclin-dependent kinases (CDKs). [START_REF] Zi | Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins[END_REF][START_REF] Saisomboon | Antitumor effects of flavopiridol, a cyclin-dependent kinase inhibitor, on human cholangiocarcinoma in vitro and in an in vivo xenograft model[END_REF][START_REF] Wang | Design of wogonin-inspired selective cyclindependent kinase 9 (CDK9) inhibitors with potent in vitro and in vivo antitumor activity[END_REF] CDKs are wellknown to be the main key regulators of cell cycle progression, their hyperactivation is associated with several cancers. For this reason, CDK inhibitors are of great importance to explore as novel therapeutic agents for cancer treatment. [START_REF] Senderowicz | Development of cyclin-dependent kinase modulators as novel therapeutic approaches for hematological malignancies[END_REF] CDK4/6 inhibitors have already reach the drug market (Palbociclib approved by FDA in 2015, Abemaciclib and Ribociclib both approved by FDA in 2017) for the treatment of advanced breast cancer treatment. [START_REF] Roskoski | Properties of FDA-approved small molecule protein kinase inhibitors[END_REF] Earlier CDK inhibitors are described as purine derivatives and this is because they are ATP competitive ligands; for example dimethylaminopurine, [START_REF] Neant | 6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity[END_REF] olomoucine [START_REF] Veselý | Inhibition of Cyclin-Dependent Kinases by Purine Analogues[END_REF] roscovitine [START_REF] Meijer | Biochemical and Cellular Effects of Roscovitine, a Potent and Selective Inhibitor of the Cyclin-Dependent Kinases cdc2, cdk2 and cdk5[END_REF][START_REF] Teng | Recent advances in the development of cyclin-dependent kinase 7 inhibitors[END_REF][START_REF] Demange | Potent inhibitors of CDK5 derived from roscovitine: Synthesis, biological evaluation and molecular modelling[END_REF] and other potent purine based CDK inhibitors. [START_REF] Gray | Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors[END_REF] Like purines, flavones have also shown their potency in disrupting cell cycle and have been thus demonstrated as potent CDK inhibitors. [START_REF] Wang | The therapeutic potential of flavonoids[END_REF][START_REF] Zhang | Nitrogen-containing flavonoid analogues as CDK1/cyclin B inhibitors: Synthesis, SAR analysis, and biological activity[END_REF][START_REF] Vijayababu | Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression[END_REF]

Figure 1. Flavopiridol and mimics as potent CDKs inhibitors

The seminal discovery of flavopiridol (alvocidib) 1 (Figure . 1) whose structure was inspired by the natural antirheumatic flavonoid "Rohitukine" [START_REF] Harmon | The structure of rohitukine, the main alkaloid of Amoora rohituka (Syn. Aphanamixis polystachya) (meliaceae)[END_REF][START_REF] Naik | An antiinflammatory cum immunomodulatory piperidinylbenzopyranone from dysoxylum binectariferum : isolation, structure and total synthesis[END_REF][START_REF] Kumar | A chromatography-free isolation of rohitukine from leaves of Dysoxylum binectariferum: Evaluation for in vitro cytotoxicity, Cdk inhibition and physicochemical properties[END_REF][START_REF] Singh | Rohitukine inhibits NF-κB activation induced by LPS and other inflammatory agents[END_REF] led to the development of new synthetic flavonoids-based compounds as inhibitors of kinases. Alvocidib, the first CDKs (CDK1, [START_REF] Pojero | Targeting multiple myeloma with natural polyphenols[END_REF][START_REF] Kshatriya | In memory of Prof. Venkataraman: Recent advances in the synthetic methodologies of flavones[END_REF][START_REF] Saisomboon | Antitumor effects of flavopiridol, a cyclin-dependent kinase inhibitor, on human cholangiocarcinoma in vitro and in an in vivo xenograft model[END_REF][START_REF] Wang | Design of wogonin-inspired selective cyclindependent kinase 9 (CDK9) inhibitors with potent in vitro and in vivo antitumor activity[END_REF][START_REF] Roskoski | Properties of FDA-approved small molecule protein kinase inhibitors[END_REF] inhibitor to be tested in a clinical trial [START_REF] Lanasa | Final results of EFC6663: A multicenter, international, phase 2 study of alvocidib for patients with fludarabine-refractory chronic lymphocytic leukemia[END_REF][START_REF] Lin | Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease[END_REF][START_REF] Zeidner | Randomized multicenter phase II study of flavopiridol (alvocidib), cytarabine, and mitoxantrone (FLAM) versus cytarabine/daunorubicin (7+3) in newly diagnosed acute myeloid leukemia[END_REF] is the most active CDK9 inhibitor with IC 50 value of 20 nM. This drug is capable to induce regluated cell death with a block in the cell cycle at the G1/S and G2/M phases. [START_REF] Yeh | Up-regulation of CDK9 kinase activity and Mcl-1 stability contributes to the acquired resistance to cyclin-dependent kinase inhibitors in leukemia[END_REF][START_REF] Chen | Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia[END_REF][START_REF] Li | Cyclin-dependent kinase inhibitor, flavopiridol, induces apoptosis and inhibits tumor growth in drug-resistant osteosarcoma and Ewing's family tumor cells[END_REF] In 2004, it has received the Orphan Drug designation in chronic lymphocytic leukemia (CLL) from the FDA and the EMA respectively. [START_REF] Wiernik | Alvocidib (flavopiridol) for the treatment of chronic lymphocytic leukemia[END_REF][START_REF] Zeidner | Clinical activity of alvocidib (flavopiridol) in acute myeloid leukemia[END_REF] The extensive amount of medicinal chemistry efforts has been reported on the study of flavopiridol, aiming to understand the mechanism of action and the structural modifications in order to discover new CDKs inhibitors. [START_REF] Bharate | Discovery and Preclinical Development of IIIM-290, an Orally Active Potent Cyclin-Dependent Kinase Inhibitor[END_REF][START_REF] Jain | Cyclin-Dependent Kinase Inhibition by Flavoalkaloids[END_REF] As a result of this research, P-276-00 (Piramal) a flavopiridol analogue has reached advanced stages of clinical development for cancer treatment. [START_REF] Cassaday | A Phase II, Single-Arm, Open-Label, Multicenter Study to Evaluate the Efficacy and Safety of P276-00, a Cyclin-Dependent Kinase Inhibitor, in Patients With Relapsed or Refractory Mantle Cell Lymphoma[END_REF] Besides, previous SAR studies contributed to the identification of flavopiridol mimics (Figure 1) with selective inhibitory activity against CDK1 and 4, though with reduced potency. For example, the flavopiridol D-ring olefin analogue [START_REF] Pojero | Targeting multiple myeloma with natural polyphenols[END_REF] was reported as a CDK4 selective inhibitor [START_REF] Murthi | Structureactivity relationship studies of flavopiridol analogues[END_REF] thio-and oxaflavopiridol (3) were identified as CDK1 selective inhibitors. [START_REF] Kim | Cyclin-Dependent Kinase 1-Selective Inhibitors: Synthesis and Biological Effects[END_REF] In addition, flavopiridol structure-based design led to the discovery of 2-benzylidene-benzofuranone (4) as potent and selective inhibitor of CDK1 [START_REF] Schoepfer | Structure-Based Design and Synthesis of 2-Benzylidene-benzofuran-3-ones as Flavopiridol Mimics[END_REF] and recently P-276-00 [START_REF] Zi | Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins[END_REF] that was identified as a novel pan-selective CDK1 and -9 inhibitor. [START_REF] Joshi | In vitro antitumor properties of a novel cyclin-dependent kinase inhibitor, P276-00[END_REF] Flavopiridol inhibits several kinases with IC 50 values 30-300 nM. [START_REF] Czech | Antitumoral activity of flavone L-86-8275[END_REF] Actually, the advanced biology results created an increased demand for new small natural products derivatives [START_REF] Nicolaou | Natural Product-like Combinatorial Libraries Based on Privileged Structures. 1. General Principles and Solid-Phase Synthesis of Benzopyrans[END_REF] for drug discovery purposes. In order to emulate this approach, we found of great interest to explore new chemical space around the flavopiridol C ring with the purpose to identify unique structures with potential better cytotoxic activities. In this work, three series of flavopiridol analogues bearing various nucleus such as thiosugars (series 1), amino acids (series 2) as well as azoles moieties (series 3) tethered to the flavopiridol have been prepared (Figure 2). Their synthesis and in vitro cytotoxic antiproliferative activities as well as kinases inhibition are also presented.

Results and discussion

Chemistry

Although the chiral hydroxyl moiety on the D ring has been found to be essential for the anti CDK activity of flavopiridol, a recent study has demonstrated that replacing the chiral D ring with olefin analogue (tetrahydropyridyl) did not result in a major loss of CDK inhibitory activity. [START_REF] Murthi | Structureactivity relationship studies of flavopiridol analogues[END_REF] However, from a synthetic point of view, we thought it is practical to pursue our present work with olefin moiety on the D ring since its synthesis could be achieved with less number of steps. Therefore, we have decided to focus our effort on exploring further modifications around the C ring in order to elaborate unique new structures and to examine their impact on the biological activity.

We first became interested to introduce thiosugars, the stable glycomimics of O-glycosides, with the intention to increase the solubility of the compounds and to improve their pharmacological properties and activities through the Warburg effects (Calvaresi, E. C.; Hergenrother, P. J. Glucose conjugation for the specific targeting and treatment of cancer Chem Sci.

2013, 4, 2319-2333

). Then we envisioned the introduction of further groups like amino acids and azoles. These substituents will be introduced via a thioether (or amine) bonds on the para-iodo group of the 2-phenyl ring. (Figure 2). previously reported method for a large-scale synthesis of flavopiridol. [START_REF] Tabaka | Application of Modified Flavone Closure for the Preparation of Racemic L86-8275[END_REF] Condensation of 3,5-dimethoxy phenol with 1-methyl-4-piperidinone in acetic acid saturated with anhydrous hydrogen chloride gas provided compound 6 in 50% yield. Friedel-Craft acylation of the aromatic compound 6 was accomplished by reaction with acetic anhydride and BF 3 -OEt 2 followed by treatment of the resulting product under basic media in dichloromethane to afford 7 in 60 % yield.

Benzoylation of compound 7 using 2-chloro-4-iodobenzoyl chloride 4-iodobenzoyl chloride generated the benzoate 8a, and 8b in 82% and 93% yields respectively. The Baker Venkataraman step has been then accomplished with powdered KOH in hot pyridine which smoothly isomerized the benzoate 8 to 9 in quantitative yield, this step was followed by the dehydration reaction which under catalytic sulfuric acid in acetic acid at 100°C allowed the formation of flavone 10a and 10b in 68 and 56 % yields respectively. The final step of the demethylation of the methyl ether-protecting groups was carried out under solvent-free conditions by heating a mixture of 10 and pyridine.HCl at 210 °C for 4 hours. Under these conditions, the final compound could be then obtained in 56% yield. We recently reported an efficient method allowing the introduction of glycosyl thiols to various iodo(hetero)aryles, [START_REF] Bruneau | Stereoretentive Palladium-Catalyzed Arylation, Alkenylation, and Alkynylation of 1-Thiosugars and Thiols Using Aminobiphenyl Palladacycle Precatalyst at Room Temperature[END_REF] nucleic acids [START_REF] Probst | Efficient Buchwald-Hartwig-Migita Cross-Coupling for DNA Thioglycoconjugation[END_REF] and peptides , [START_REF] Montoir | Synthesis of aryl-thioglycopeptides through chemoselective Pd-mediated conjugation[END_REF] under simple and mild conditions using the palladium G3-Xanthphosbiphenyl precatalyst (Ref Chem Sci 2013 Buchwald et ACS Catal. 2015, 5, 1386-1396 ) . This method was adapted to our starting platform 11a-b, furnishing the first two series (a and b) in which thiosugars and sulfur containing amino acids groups were tethered to the flavones core via a thioether bond. Scheme 1. Synthetic route toward flavopiridol analogues. Substitution on the C ring with; Series A: thiosugars; Series B: aminoacids and alkyl chaines; Series C: heterocycles. General conditions for the palladium catalyzed coupling reaction for products 13 and 14 formation: Pd-G3-Xantphos 5-10 mol%, Et 3 N (3 equiv.), r.t. or 60°C, dioxane or dioxane/water. i: this compound was obtained from 13g after cleavage of THP under acidic media. ii: yield calculated over two steps. iii: Cs 2 CO 3 was used as base and the coupling was performed at 100 °C.

The thioglycoconjugation process could be performed rapidly and revealed to have a great tolerance towards functional groups (free OH and NH were well-tolerated). In order to develop a convergent synthetic strategy, the main challenge was to couple the unprotected iodo-flavopiridol 11 with the thiol-containing partners. Obviously, we were glad to observe again that precatalyst PdG3-Xantphos system was efficiently used for the first time with iodoflavopiridol substrates (11a and 11b) and protected thioglucose and galactose. Hence, with a catalytic amount of PdG3-Xantphos in a mixture of water and dioxane (see the experimental section for details) products (12a, 12c, 13a) were formed in good 50%, 75%, and 70 % yields respectively. The reaction was also successful with fully unprotectedthioglucose to generate compound 12b in a 64 % yield. The same conditions were applied with sulfur-containing amino acids N-acetylcysteine, N-Boc-cysteine, and a dipeptide serine-cysteine as well as alkyl chain bearing hydroxamic acid moieties to give compound (12d-h) in up to 92 % yield. Thus, a third series (series c) of flavopiridol analogues has been built successfully as well with thiolated heterocycles. Products 12j-r were obtained in up to 70 % yield and 13b-d in up to 60 %. However, introducing amino heterocycles was more challenging and needed more heating (100°C), and the replacement of Et 3 N with Cs 2 CO 3 as a base source. Product issued from this coupling of 11a with aniline was obtained in 35 % yield.

Biological results

In vitro antiproliferative activity

The antiproliferative effect of generated compounds has been assessed. The primary screening of series A, B and C was realized at one concentration of 10 µM on ovarian cancer cell line (SKOV3). Note here that 100% of cell viability was obtained for DMSO-treated cells. All compounds issued from series A and B did not show a growth inhibition superior to 50 %, while compounds from series C showed more interesting activities. Therefore, the IC 50 values were determined for series C compounds on the SKOV3 cell line (Table 1). In series A, compounds having a sugar unit were found ineffective (12a and 12b) or with moderate activity (compounds 12c and 13a). Derivatives containing amino acid moieties (Series B) also gave low to moderate cytotoxic activity (compounds 12d-h). Compounds issued from Series C having heterocyclic substituents, in particular, benzimidazole and imidazole ring were found to be the more active compounds, and they gave on IC 50 in the sub-micromolar range (compounds 12j, 12n, 12o, 13b, 13c). After this first screening of generated compounds on SKOV3, the five most active compounds were selected for evaluation against six additional human cancer cell lines and compared with the reference compound, the flavopiridol (Table 2).

Except compound 12n having imidazole group, all the other selected compounds were found active on the six tested cell lines with IC 50 values less than 1 μM. Overall, the presence of benzimidazole moieties seem to be important for the cytotoxic effect of these compounds. N-methyl benzimidazole derivatives showed more cytotoxic activity in comparison to their no methylated counterpart (compound 12j vs 12o). Finally, the presence of chlorine atom on the phenyl group tethered to chromenone ring display better activity, in comparison to the free-chlorine compounds (compound 13b vs 12j, and compound 12o vs 13c). This SAR led us to identify 13c as the most active compound, with an IC 50 < 100 nM on 6 cancer cell lines, and it was found more cytotoxic than the reference compound, the flavopiridol.

Table 2.

Antiproliferative activities of selected compounds against a panel of seven human cancer cell lines, IC 50 values are reported in nM. a SKOV3, ovarian cancer cells; b NCI-N87, gastric adenocarcinoma cells; c SKBR3, breast cancer cells; d PC3, prostate cancer cells; e MiaPaCa-2, pancreatic cancer cells; f HCT116, colon-carcinoma cells; g K562, immortalised myelogenous leukemia cell line.

Kinase enzymatic assays.

In order to study the mechanism of action of the more active compounds, we tested their effect on the inhibition of the enzymatic activities of protein kinases as putative targets. A panel of eight serine/threonine protein kinases was tested including cyclin-dependent kinases (HsCDK2/cyclin A, HsCDK5/p25, HsCDK9/cyclinT), glycogen synthase kinase-3 beta (HsGSK3β and SscGSK-3α/β), porcine casein kinase 1 (SscCK1δ/ε), Leishmania major casein kinase 1 (LmCK1), and Plasmodium falciparum glycogen synthase kinase-3 (PfGSK3 activity at 10 µM and 1 µM were used to characterize the bioactivity of the compounds during the primary screening campaign (Table 3). a Kinase activities in the presence of 10 or 1µM of tested compounds were measured by ADP-Glo luminescent assay (Promega, Madison, WI, USA), using 10 µM ATP. Data are mean (n = 2) expressed in % of maximal activity, compared with a DMSO control. The red color scale is used to highlight the values that are below 50% of residual kinase activity. (% of residual activity at 10 µM and 1 µM). ≥100 indicates that the compound cannot inhibit the enzymatic activity at the tested concentration. CC, concentration tested.

Overall, all tested compounds were found to inhibit slightly the activity of no human kinases, Lm-CK1, Pf-GSK3, and Ssc-CK1δ/ε kinases (only at high dose 10 µM). At low dose (1 µM), moderate to good activity was observed for Hs-CDK2/CyclinA, Hs-CDK5/p25, Hs-CDK9/CyclinT, Hs-GSK3β, and Ssc-GSK3α/β kinases.

Compounds displaying more than 70% inhibition at 10 μM were next tested over a wide range of concentrations (usually from 0.0003 to 10 μM) on human kinases and IC 50 values were determined from the dose response curves (Prism-GraphPad, GraphPad Software, San Diego, CA, USA). The results of the in vitro kinase assay were summarized in Table 4.

The cyclin-dependent kinases (CDKs), a subfamily of serine-threonine kinases, play an important role in the cell cycle regulation. Inhibition of cell cycle CDKs has been proven an efficient anticancer strategy. Concerning the inhibition of CDK2 and CDK5, the presence of a chlorine atom on the phenyl ring (compounds 13b and 13c) led to a decrease of the activity in comparison to the reference compound, the flavopiridol. However, no-chlorinated compounds (12j), (12o), and even imidazole derivative (12n) showed an interesting inhibition of these two kinases, similar to flavopiridol. Thus, compound 12j displayed the best activity with an IC 50 of 0.162 and 0.121 µM which is better than the reference compound. Cyclin-dependent protein kinase 9 (CDK9) has been shown to play an important role in the pathogenesis of malignant tumors. [START_REF] Ma | Cyclin-dependent kinase 9 (CDK9) is a novel prognostic marker and therapeutic target in osteosarcoma[END_REF] CDK9 inhibitors have demonstrated antitumoral activity in vitro. All the tested compounds in this study exhibited high inhibition toward this kinase with an IC 50 similar or better than the flavopiridol. Interestingly, compounds 12j and 12n displayed an IC 50 in the nM range. IC50 values are reported in μM. IC50 values. Activities were assessed in duplicate using the ADP-Glo luminescent assay (Promega), using 10 µM ATP.

b.

Kinases activities were assayed in duplicate.

GSK-3 is a ubiquitously expressed serine/threonine kinase, it is involved in many signaling pathways controlling many different key functions. Numerous studies show that GSK3 action supports cancer cells and suggest that its inhibition may have therapeutic benefits. The GSK-3 activity has been linked to many pathogeneses including colorectal cancer, diabetes, acute myeloid leukemia and Alzheimer's disease. [START_REF] Lo Monte | Identification of Glycogen Synthase Kinase-3 Inhibitors with a Selective Sting for Glycogen Synthase Kinase-3α[END_REF] The biological assay showed that all the tested compounds inhibit more efficiently human isoform  of GSK3 in comparison to the flavopiridol. Compound 13c displayed the best activity with an IC 50 of 59 nM, which is 20 folds more active than flavopiridol.

Taken together, these results showed that compounds 12j and 12n which showed a moderate or low cytotoxic activity respectively, inhibit efficiently the activity of CDK9 kinase. Importantly, compound 13c which have the best cytotoxic activity displays a very good selectivity towards CDK9 and GSK3β kinases in comparison to CDK2 and 5, offering new potential targets for this compound.

Following the characterization of the kinases as putative targets driving the cytotoxicity phenotype, we then decided to explore the putative binding mode of the more active compound on the more affected target. We thus decided to select compound 13c and human GSK3 . The remaining % of maximal activity (compared with a DMSO control) were determined at ATP concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 µM. As shown on figure 3, the results obtained strongly suggest an ATP-competitive inhibition of GSK3 by 13c. The inhibition of the kinase activity is almost fully abrogated by the highest tested concentration of ATP (100 µM). Note here that this ATP competitive binding mode was also reported for the inhibition of CDKs by flavopiridol. [START_REF] Senderowicz | Development of cyclin-dependent kinase modulators as novel therapeutic approaches for hematological malignancies[END_REF] Fig. 3. Human GSK-3 inhibition by compound 13c at different ATP concentrations. GSK-3 kinase activities in the presence of 500 nM of 13c were measured by ADP-Glo luminescent assay using the mentioned ATP concentrations (from 3.125 to 100 μM). Data are mean (n = 3) ± SD expressed in % of residual activity, compared with a DMSO control.

Conclusion

We have shown successfully a convergent route to synthesize flavopiridol analogs modified at the C ring by means of PdG3-Xanthphos precatalyst. The cross-coupling was realized directly between no protected iodo-flavopiridol and thiol sugars, amino acids, and heterocycles under mild conditions. The synthesis allowed us to access three series of new and unique structures. Cytotoxic activities of benzimazoles containing flavopiridol presented in this work are comparable and even higher than flavopiridol. We showed also that the enzymatic activity of some of the tested kinase, including GSK3β, are potently affected by selected molecular hits. Among all tested compounds, compound 13c showed the best antiproliferative activity associated with an inhibition of CDK9, and GSK3β in the nM range. These results constitute a starting point enabling a better understanding of structure-activity relationships of this new series of compounds and should facilitate future elaboration to more efficient and selective inhibitors of CDK9 and GSK3β which may have applications in affecting the pathogenesis of cancer and Alzheimer's disease. Dual-or multi-target kinase inhibitors for developing new polypharmacological approaches can also be explored using these new flavopiridol derivatives.

Experimental

Materials

Solvents and reagents are obtained from commercial suppliers and were used without further purification. Analytical TLC was performed using Merck silica gel F254 (230-400 mesh) plates and analyzed by UV light or by staining upon heating with vanilin solution. For silica gel chromatography, the flash chromatography technique was used, with Merck silica gel 60 (230-400 mesh) and p.a. grade solvents unless otherwise noted. The 1 H NMR and 13 C NMR spectra were recorded in either CDCl 3 , MeOD, or DMSO-d6 on Bruker Avance 300 or 400 spectrometers. The chemical shifts of 1 H and 13 C are reported in ppm relative to the solvent residual peaks. IR spectra were measured on a Perkin Elmer spectrophotometer. High resolution mass spectra (HR-MS) were recorded on a MicroMass LCT Premier Spectrometer. Thiosugars were synthesized as according the following protocols. [START_REF] Floyd | Thiyl Glycosylation of Olefinic Proteins: S-Linked Glycoconjugate Synthesis[END_REF][START_REF] Gamblin | Glyco-SeS: Selenenylsulfide-Mediated Protein Glycoconjugation-A New Strategy in Post-Translational Modification[END_REF] The Xantphos palladium precatalyst third generation was synthesized according to literature protocol. [START_REF] Bruno | Design and preparation of new palladium precatalysts for C-C and C-N cross-coupling reactions[END_REF] 6 and Acetophenone 7 was prepared adopting reported methods spectral data are in agreement with the published ones. [START_REF] Siemann | A review and update of the current status of the vasculature-disabling agent combretastatin-A4 phosphate (CA4P)[END_REF] 4.2. Chemistry 4.2.1. Synthesis of the intermediates 4.2.1.1. 2-acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl 4-iodobenzoate (8a). A mixture of 7 (1.0 g, 0.0034 moles), 4-iododbenzoic acid chloride (1.8 g, 0.0068 moles) and DMAP (0.414 g, 0.0034 moles) was stirred overnight in DMF (40 mL) at r.t. After DMF was removed, the residue was dissolved in DCM and washed with saturated solution of bicarbonate, the organic layer was dried over MgSO 4 . The crude residue was purified by flash chromatography (DCM/MeOH 0  3 %). The product was obtained as orange solid in 82% yield. 1 H NMR (300 MHz, CDCl 3 ) δ 7.81 (q, J = 8.7 Hz, 4H), 6.42 (s, 1H), 5.55 (s, 1H), 3.92 (s, 3H), [START_REF] Jalili-Baleh | A review on flavonoid-based scaffolds as multi-target-directed ligands (MTDLs) for Alzheimer's disease[END_REF]

2-acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl 2-chloro-4-iodobenzoate (8b).

A mixture of 7 (0.800 g, 0.0027moles), 3-chloro,4-iododbenzoic acid chloride (1.400 g, 0.0046 moles) and DMAP (0.441 g, 0.0036 moles) was stirred overnight in DMF (30 mL) at r.t. After DMF was removed, the residue was dissolved in DCM and washed with saturated solution of bicarbonate, the organic layer was dried over MgSO 4 . The crude residue was purified by flash chromatography (DCM/MeOH 0  3%). The product was obtained as orange solid in 93% yield. 1 H NMR (300 MHz, CDCl 3 ) δ 7.88 (s, 1H), 7.72 (s, 2H), 6.43 (s, 1H), 5.64 -5.54 (m, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 2.96 (s, 2H), 2.53 (m, 5H), 2.34 (m, 5H). 13

2-(4-iodophenyl)-5,7-dimethoxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-chromen-4-one (10a).

According to the protocol of Tabaka et al. [START_REF] Tabaka | Application of Modified Flavone Closure for the Preparation of Racemic L86-8275[END_REF] A round-bottom flask equipped with an overhead stirrer was charged with 8a (1 g, 0.0019 moles) and pyridine (6 mL) and heated to 55 °C. Freshly pulverized KOH (158 mg, 2.82 mmol) was added with stirring and the heating continued for 3 hours. The resulting black mixture was poured into a mixture of H2O and HCl (concentrated) and allowed to stand for 30 min. While the mixture cooled, the pH was adjusted to 9 with Na 2 CO 3 (saturated), and the aqueous layer was extracted with 5% MeOH in CHCl 3 . The combined organic layers were washed with H 2 O and brine, dried (MgSO 4 ), filtered and concentrated under reduced pressure. The crude Compound 9 was used as it is in the next step of synthesis. A round-bottom flask was charged with 9 (1 g, 0.0019 moles), AcOH (20 mL), and H 2 SO 4 (concentrated, 2.2 mL) and heated to 100 °C. Heating was maintained for 2 h followed by cooling to rt overnight. The reaction was concentrated on a rotary evaporator, diluted with H 2 O, and neutralized by the addition of Na 2 CO 3 (saturated) to pH 9. The aqueous layer was extracted with 5% MeOH in CHCl 3 . The combined organics were dried (MgSO 4 ), filtered, and concentrated under reduced pressure. The product was purified by chromatography, DCM-MeOH (2 -> 4 %) to yield 10a as a yellow solid (68%). 1 H NMR (300 MHz, CDCl 3 ) δ 7.85 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 6.66 (s, 1H), 6.44 (s, 1H), 5.72 (s, 1H), 4.03 (s, 3H), 3.93 (s, 3H), 3.20 (m, 2H), 2.74 (m, 2H), 2.48 (m, 5H). 13 [START_REF] Tabaka | Application of Modified Flavone Closure for the Preparation of Racemic L86-8275[END_REF] A round-bottom flask equipped with an overhead stirrer was charged with 8a (1 g, 0.0019 moles) and pyridine (6 mL) and heated to 55 °C. Freshly pulverized KOH (158 mg, 2.82 mmol) was added with stirring and the heating continued for 3 hours. The resulting black mixture was poured into a mixture of H 2 O and HCl (concentrated) and allowed to stand for 30 min. While the mixture cooled, the pH was adjusted to 9 with Na 2 CO 3 (saturated), and the aqueous layer was extracted with 5% MeOH in CHCl 3 . The combined organic layers were washed with H 2 O and brine, dried (MgSO 4 ), filtered and concentrated under reduced pressure. The crude Compound 9b was used as it is in the next step of synthesis. A round-bottom flask was charged with 9 (280 mg, 0.5 mmol), AcOH (5.5 mL), and H 2 SO 4 (concentrated, 0.6 mL) and heated to 100 °C. Heating was maintained for 2 h followed by cooling to rt overnight. The reaction was concentrated on a rotary evaporator, diluted with H 2 O, and neutralized by the addition of Na 2 CO 3 (saturated) to pH 9. The aqueous layer was extracted with 5% MeOH in CHCl 3 . The combined organics were dried (MgSO 4 ), filtered, and concentrated under reduced pressure. The product was purified by chromatography, DCM-MeOH (2 -> 4 %) to yield 10b as a yellow solid (56%). 1 H NMR (300 MHz, CDCl 3 ) δ 7.89 (d, J = 1.5 Hz, 1H), 7.73 (dd, J = 8.2, 1.5 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 6.60 (s, 1H), 6.45 (s, 1H), 5.65 (s, 1H), 4.03 (s, 3H), 3.93 (s, 3H), 3.13 (d, J = 2.6 Hz, 2H), 2.67 (t, J = 5.5 Hz, 2H), 2.42 (m, 5H).

5,7-dihydroxy-2-(4-iodophenyl)-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-chromen-4-one (11a).

A round-bottom flask was charged with 10a (500 mg, 0.99 mmol) and pyridine hydrochloride (1.138 g, 9.9 mmol) and heated in an oil bath to 210-220 °C for 4 h. The mixture was neutralized and extracted with 5% MeOH in CHCl 3. The crude residue was purified by flash chromatography (DCM/MeOH 5  10 %). Yellow solid 56%. 1

2-(2-chloro-4-iodophenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-chromen-4-one (11b).

A round-bottom flask was charged with 10b (500 mg, 0.99 mmol) and pyridine hydrochloride (1.138 g, 9.9 mmol) and heated in an oil bath to 210-220 °C for 4 h. The mixture was neutralized and extracted with 5% MeOH in CHCl 3. The crude residue was purified by flash chromatography (DCM/MeOH 5  10%). Yellow solid 50%. 1 H NMR (300 MHz, MeOD) δ 8.05 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 6.53 (s, 1H), 6.36 (s, 1H), 5.81 (s, 1H), 2.98 (s, 2H), the other signals are overlapped with solvent.

General procedure for the synthesis of compounds 12 and 13:

Coupling of 11a and 11b with thiol partners or with aniline:

A sealable tube purged with argon equipped with a cap was charged with 11a or 11b (1 equiv), thiosugars, thioheterocycles or aniline (1 to 1.5 equiv), PdG3-Xantphos (a mettre une ref pour dire que le PDG3 est synthétise selon le ChemSci 2013 de Buchwald car nous n'avons pas utilize du commercial) (5 to 10 mol%), the tube was flushed again with Argon, then degassed dioxane (and water when mentioned), and Et 3 N or Cs 2 CO 3 ( 1 to 6 equiv) were added. The mixture was stirred at r.t. or 60°C or at 100°C for the corresponding times (see details time below). Purification was performed with flash chromatography on silica gel using a gradient of methanol in dichloromethane or by preparative HPLC using a gradient of acetonitrile in water. -3,4,5-triyl triacetate (12a). 11a (20 mg, 0.04 mmol), -thiosugar (17 mg, 0.042 mmol), PdG3-Xantphos (5 mol%), Et 3 N (L) and dioxane (1 mL) were stirred at r.t. for 2 hours. The compound was purified over silica gel (DCM/MeOH, 5  7%) and obtained as yellow solid in 50% yield. 1 H NMR (300 MHz, DMSO) δ 12.88 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.02 (s, 1H), 6.33 (s, 1H), 5.73 (s, 1H), 5.55 (d, J = 10.1 Hz, 1H), 5.42 (t, J = 9.4 Hz, 1H), 4.95 (q, J = 9.5 Hz, 2H), 4.15 (dt, J = 23.9, 9.0 Hz, 3H), 3.19 (m br, 2H), 2.73 (m br, 2H), 2.43 (s, 6H), 1.96 (m, 12H). 13

(3R,4S,5S,6S)-2-(acetoxymethyl)-6-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4Hchromen-2-yl)phenyl)thio)tetrahydro-2H-pyran

5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-2-(4-(((2S,3S,4S,5S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)phenyl)-4H-chromen-4-one (12b)

. 11a (30 mg, 0.063 mmol), thiosugar (18 mg, 0.091 mmol), PdG3-Xantphos (8 mol%), Et 3 N (60 μL), water (0.2 mL) and dioxane (1 mL) were stirred at 60 °C for 2 hours. The compound was purified with over silica gel (DCM/MeOH, 5  15 %) and obtained as yellow solid in 64% yield. 1

(3S,4S,5S,6S)-2-(acetoxymethyl)-6-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4Hchromen-2-yl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate (12c)

. 11a (20 mg, 0.04 mmol), ß-thiosugar (17 mg, 0.042 mmol), PdG3-Xantphos (5 mol%), Et 3 N (50 μL), and dioxane (1 mL) were stirred at r.t. for 2 hours. The compound was purified with over silica gel (DCM/MeOH, 0  5%) and obtained as beige solid in 75 % yield. 1 H NMR (300 MHz, CDCl 3 ) δ 12.68 (s, 1H), 7.71 (d, J = 18.9 Hz, 4H), 6.61 (s, 1H), 5.90 (s, 1H), 5.49 (s, 1H), 5.31 (t, J = 10.0 Hz, 1H), 5.15 (d, J = 9.4 Hz, 1H), 4.98 (d, J = 9.5 Hz, 1H), 4.28-4.15 (m, 4H), 3.74 (s, 2H), 3.39 (s, 2H), 2.89 (s, 5H), 2.08 (m, 12 H). 13

(3R,4S,5S,6S)-2-(acetoxymethyl)-6-((3-chloro-4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4oxo-4H-chromen-2-yl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate (13a)

. 11b (50 mg, 0.098 mmol), ßthiosugar (40 mg, 0.11 mmol), PdG3-Xantphos (10 mol%), Et 3 N (100 μL), and dioxane (2 mL) were stirred at 60°C for 2 hours. The compound was purified with over silica gel (DCM/MeOH, 0  5 %) and obtained as yellow solid in 70% yield. 1 H NMR (300 MHz, MeOD) δ 7.73 (s, 1H), 7.62 (dd, J = 22.0, 8.0 Hz, 2H), 6.52 (s, 1H), 6.31 (s, 1H), 5.78 (s, 1H), 5.38 (t, J = 9.3 Hz, 1H), 5.22 (d, J = 10.0 Hz, 1H), 5.13 -4.93 (m, overlapped with solvent 5H), 4.36 -4.17 (m, 2H), 4.07 (s, 1H), 3.59 (s, 2H), 3.18 (s, 2H), 2.74 (s, 3H), 2.64 (s, 2H), 2.05 (dd, J = 22.9, 10.0 Hz, 12H). 13

(S)-methyl 2-((R)-2-(((benzyloxy)carbonyl)amino)-3-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4H-chromen-2-yl)phenyl)thio)propanamido)-3-hydroxypropanoate (12d

). 11a (30 mg, 0.063 mmol), thiodipeptide (27 mg, 0.075 mmol), PdG3-Xantphos (8 mol%), Et 3 N (60 μL), and dioxane (1.5 mL) were stirred at r.t. for overnight. The compound was purified with over silica gel (DCM/MeOH, 0  7 %) and obtained as orange solid in 90% yield. 1 H NMR (300 MHz, MeOD) δ 7.78 (d, J = 7.6 Hz, 2H), 7.51 (s, 2H), 7.40 -7.21 (m, 5H), 6.62 (s, 1H), 6.24 (s, 1H), 5.75 (s, 1H), 5.09 (s, 2H), 4.75 (s, 1H), 4.25 (s, 1H), 3.79 (d, J = 4.2 Hz, 2H), 3.71 (s, 3H), 3.58 (m, 4H), 3.14 (s, 2H), 2.73 (s, 3H), 2.62 (s, 2H). 13

N-acetyl-S-(4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4H-chromen-2-yl)phenyl)-Dcysteine (12e

). 11a (30 mg, 0.063 mmol), N-acetylcysteine (14 mg, 0.08 mmol), PdG3-Xantphos (8 mol%), Et 3 N (60 μL), water (0.2 mL) and dioxane (1 mL) were stirred at r.t. for overnight. The compound was purified with over silica gel (DCM/MeOH, 0  7 %) and obtained as yellow solid in 92% yield. 1 H NMR (300 MHz, DMSO) δ 7.88 (d, J = 7.7 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 6.89 (s, 1H), 6.36 (s, 1H), 5.74 (s, 1H), 4.36 (m,, 1H), 3.60 -3.39 (m, 3H), 3.27 (dd, J = 13.0, 7.7 Hz, 1H), 2.99 (s, 2H), 2.60 (s, 3H), 1.83 (s, 3H). 13

(S)-methyl 2-((tert-butoxycarbonyl)amino)-3-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4oxo-4H-chromen-2-yl)phenyl)thio)propanoate (12f)

. 11a (40 mg, 0.08 mmol), aminoacid (22 mg, 0.09 mmol), PdG3-Xantphos (6 mol%), Et 3 N (60 μL), and dioxane (1mL) were stirred at r.t. for overnight. The compound was purified with over silica gel (DCM/MeOH, 0  7%) and obtained as orange solid in 86% yield. 1 H NMR (300 MHz, CDCl 3 ) δ 12.73 (s, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 6.62 (s, 1H), 6.34 (s, 1H), 5.86 (s, 1H), 5.36 (s, 1H), 4.63 (s, 1H), 3.69 (s, 3H), 3.60 -3.35 (m, 4H), 3.10 (q, J = 7.3 Hz, 2H), 3.00 (s, 2H), 2.68 (s, 3H), 1.43 (s, 9H). 13

3-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4H-chromen-2-yl)phenyl)thio)-N-((tetrahydro-2H-pyran-2-yl)oxy)propanamide (12g)

. 11a (50 mg, 0.1 mmol), 3-mercapto-N-((tetrahydro-2H-pyran-2yl)oxy)propanamide (23 mg, 0.11 mmol), PdG3-Xantphos (6 mol%), Et 3 N (72 μL), and dioxane (1.5 mL) were stirred at r.t. for 2 h. The compound was purified on silica gel (DCM/MeOH, 0  7%) and obtained as orange solid in 76% yield. 1 H NMR (300 MHz, MeOD) δ 7.79 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 6.59 (s, 1H), 5.73 (s, 1H),4.78 (1H overlapped with solvent signal), 3.99 (t, J = 10.2 Hz, 1H), 3.60 (d, J = 10.1 Hz, 1H), 3.45 -3.22 (m, 6H), 2.93 (t, J = 5.3 Hz, 2H), 2.65 -2.44 (m, 6H), 1.91 -1.49 (m, 6H). 13 C NMR (75 MHz, MeOD) δ 182. 42, 168.61, 163.31, 162.98, 160.42, 154.23, 141.98, 128.26, 127.97, 127.40, 126.41, 125.20, 108.58, 103.59, 101.91, 61.64, 53.74, 51.65, 43.83, 31.96, 28.28, 27.56, 27.20, 24.80, 18. [d]imidazol-2-yl)thio)phenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4Hchromen-4-one (12j). 11a (30 mg, 0.063 mmol), 1H-benzo [d]imidazole-2-thiol (10 mg, 0.069 mmol), PdG3-Xantphos (8 mol%), Et 3 N (60 μL), water (200 μL) and dioxane (1 mL) were stirred overnight at 60°C. The compound was purified on silica gel (DCM/MeOH, 0  7%) and obtained as orange solid in 66% yield. 1

2-(4-((1H-benzo

4.2.2.11.

2- (4-(benzo[d]oxazol-2-ylthio)phenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4Hchromen-4-one (12k). 11a (30 mg, 0.063 mmol), benzo [d]oxazole-2-thiol (12 mg, 0.075 mmol), PdG3-Xantphos (10 mol%), Et 3 N (70 μL), and dioxane (1 mL) were stirred 2 hours at 60 °C. The compound was purified on silica gel (DCM/MeOH, 0  5%) and obtained as yellow solid in 60% yield. 1 H NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 8.12 (d, J = 7.7 Hz, 2H), 7.94 (s, 2H), 7.69 (s, 2H), 7.39 (d, J = 2.9 Hz, 3H), 7.11 (s, 1H), 6.37 (s, 1H), 5.86 (s, 1H), 2.88 (s, 4H), 2.65 (s, 3H), 2.55 (overlapped with solvent). 13 [d]imidazol-2-yl)thio)phenyl)-8-(1-methyl-1,2,3,6tetrahydropyridin-4-yl)-4H-chromen-4-one (12l). 11a (30 mg, 0.063 mmol), 6-methoxy-1H-benzo [d]imidazole-2-thiol (14 mg, 0.075 mmol), PdG3-Xantphos (10 mol%), Et 3 N (70 μL), and dioxane (1 mL) were stirred overnight at 60°C. The compound was purified on silica gel (DCM/MeOH, 0  7%) and obtained as yellow solid in 30% yield. 1 H NMR (300 MHz, MeOD) δ 7.93 (d, J = 7.9 Hz, 2H), 7.61 -7.39 (m, 3H), 7.06 (s, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.78 (s, 1H), 6.30 (s, 1H), 5.84 (s, 1H), 3.85 (s, 3H), 3.81 (s, 2H), 3.33 (overlapped with solvent), 2.89 (s, 3H), 2.74 (s, 2H). 13

5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-2-(4-(pyrimidin-2-ylthio)phenyl)-4H-chromen-4one (12m

). 11a (30 mg, 0.063 mmol), pyrimidine-2-thiol (10 mg, 0.075 mmol), PdG3-Xantphos (6 mol%), Et 3 N (100 μL), and dioxane (1 mL) were stirred overnight at 60°C. The compound was purified on silica gel (DCM/MeOH, 0  7%) and obtained as yellow solid in 69% yield. 1

2-(4-((1H-benzo[d]imidazol-2-yl)thio)-2-chlorophenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-chromen-4-one (13b

). 11b (30 mg, 0.059 mmol), 1H-benzo [d]imidazole-2-thiol (10 mg, 0.064 mmol), PdG3-Xantphos (10 mol%), Et 3 N (70 μL), and dioxane (1 mL) were stirred overnight at 60°C. The compound was purified on silica gel (DCM/MeOH, 0  5%) and obtained as yellow solid in 60% yield. 1 4-one (13d). 11b (20 mg, 0.04 mmol), pyrimidine-2-thiol (15 mg, 0.043 mmol), PdG3-Xantphos (10 mol%), Et 3 N (50 μL), and dioxane (2 mL) were stirred 2 hours at 60°C. The compound was purified on silica gel (DCM/MeOH, 0  5%) and obtained as yellow solid in 65% yield. 1

Biology

Cell culture and proliferation assay

Cancer cell lines were obtained from the American type Culture Collection (Rockville, USA) and were cultured according to the supplier's instructions. U87-MG cells were grown in Dulbecco minimal essential medium (DMEM) containing 4.5 g/L glucose supplemented with 10% FCS and 1% glutamine. All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Cell viability was assessed using Promega CellTiter-Blue reagent according to the manufacturer's instructions. Cells were seeded in 96-well plates (5 X 103 cells per well) containing 50 µL growth medium. After 24 h of culture, the cells were supplemented with 50 µL of the test compound dissolved in DMSO (less than 0.1% in each preparation). After 72 h of incubation, 20 µL of resazurin was added for 2 h before recording fluorescence (λex = 560 nm, λem = 590 nm) using a Victor microtiter plate fluorimeter (Perkin-Elmer, USA). The IC 50 value corresponds to the concentration of test compound that caused a decrease of 50% in fluorescence of drug-treated cells compared with untreated cells. Experiments were performed in triplicate.

Protein kinase assays

Kinase activities were measured according to the Promega's ADP-Glo assay described in Nguyen et al [START_REF] Nguyen | Kinase-Based Screening of Marine Natural Extracts Leads to the Identification of a Cytotoxic High Molecular Weight Metabolite from the Mediterranean Sponge Crambe tailliezi[END_REF] except for the following enzymes: (i) LmCK1 (from Leishmania major, recombinant, expressed in bacteria) was assayed in buffer A with 0.028 µg/µl of the following peptide: RRKHAAIGSpAYSITA as CK1-specific substrate; (ii) PfGSK3 (for Plasmodium falcicarum, recombinant, expressed in bacteria) was assayed in buffer A with 0.010 µg/µl of GS-1 peptide, a GSK3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE); (iii) SscCK1δ/ε (casein kinase 1δ/ε, porcine brain, native, affinity purified) was assayed in buffer A with 0.022 µg/µl of the following peptide: RRKHAAIGSpAYSITA ("Sp" stands for phosphorylated serine) as CK1-specific substrate; (iv) SscGSK3α/β (glycogen synthase kinase 3, porcine brain, native, affinity purified) isoforms were assayed in buffer A with 0.010 µg/µl of GS-1 peptide, a GSK3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE). The buffer solution A used in this study is composed of 10 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5 and 50 µg/ml heparin.

ATP competition assay

The activity of HsGSK3 was measured according to the Promega's ADP-Glo assay described in Nguyen et al (Ref).

Assays were performed in the absence or presence of (ni147)13c at 500 nM and increasing doses of ATP (from 3.125 to 100 μM). To plot and analyze the data, we used the software Prism-GraphPad (GraphPad Software, San Diego, CA, USA).

Figure 2 .

 2 Figure 2. Flavopiridol-based structural approach for the conception of a new series presented in this study Three different series of flavopiridol have been obtained in several step's synthetic pathways. First, the synthesis of flavopiridol analogues 11a-b (Scheme 1) has been achieved over seven steps following a reproducible and robust

Table 1 . Cell-based primary screening for selection of compounds with antiproliferative activity. compd 12a 12b 12c 12d 12e 12f 12g IC 50 (µM) a SKOV3

 1

		Na b	Na b	2.87 ± 1.9	4.3 ± 0.03	Na b	7.79 ± 1.6	6.85 ± 2.7
	compd	12h	12j	12k	12l	12m	12n	12o
	IC 50 (µM) a Na b	0.59 ± 0.07	2.0 ± 1.2	1.2 ± 0.5	Na b	0.29 ± 0.08	0.34 ± 0.013
	compd	12p	12q	12r	12t	13a	13b	13c
	IC 50 (µM) a 3.8 ± 1.2 Na b	Na b	1.2 ± 0.5	2.33 ± 1.6	0.33 ± 0.07	0.09 ± 0.045
	compd	13d	flavopiridol
	0.22 ± 0.06 a IC 50 values are reported in µM. The IC 50 is the concentration of compound required to decrease cell growth by 50%; IC 50 (µM) a Na b
	values represent the means ± SD of three experiments.
	b Na: not active, (IC 50 >10 µM).

13c 94 ± 45 49 ± 2 53.7 ± 4.1 85 ± 9.5 52.9 ± 6.8 81 ± 23 50.8 ± 2.8 flavopiridol 220 ± 60 111 ± 10.2 75 ± 3.2 110 ± 5 320 ± 21.5 20 ± 2.5 125 ± 24.2

  ). The percentage of residual

	Compound	SKOV3 a	NCI-N87 b	SKBR3 c	PC3 d	MiaPaCa-2 e	HCT116 f	K562 g
		595 ± 73	183 ± 81	253.8 ± 162	742 ± 21	852 ± 59	293 ± 7	194 ± 11
	12j
		295 ± 88	4796 ± 1271	352 ± 37	243 ± 88	448 ± 80	173 ± 18	300 ± 59
	12n
		344 ± 13	391 ± 86	249 ± 22.9	276 ± 64	361 ± 18	219 ± 18	345 ± 4.9
	12o
		333 ±72	12 ± 1.1	66.6 ± 2.5	326 ± 10	361 ± 18	170 ± 2	36 ± 4
	13b

Table 3 .

 3 Kinase-based primary screening. a

	compd	CC	Hs-CDK2/ CyclinA	Hs-CDK5/ p25	Hs-CDK9/ CyclinT	Hs-GSK3β Lm-CK1 Pf-GSK3	Ssc-CK1 δ/ε	Ssc-GSK3 α/β
	Flavopiridol	10 µM 1 µM	11 26	13 36	1 5	10 50	61 ≥100	70 ≥100	78 ≥100	16 70
	13b	10 µM 1 µM	22 46	14 42	3 2	4 14	35 81	64 ≥100	48 86	4 43
	12n	10 µM 1 µM	14 33	5 10	0 2	6 25	54 88	≥100 ≥100	43 78	11 53
	12o	10 µM 1 µM	25 51	26 41	-4 -2	-3 31	57 68	79 80	41 83	8 41
	13c	10 µM 1 µM	30 87	24 68	-2 1	2 12	38 62	46 80	62 90	10 31
	12j	10 µM 1 µM	5 35	4 14	10 8	-8 6	40 99	54 85	69 91	11 36

Table 4 .

 4 IC 50 values and structures of flavopiridol analogues (IC 50 in µM) a

	Compound	HsCDK2/CyclinA HsCDK5/p25 HsCDK9/CyclinT HsGSK3β
	Flavopiridol	0.379	0.272	0.011	1.184
	12j	0.162	0.121	0.008	0.117
	12o	0.349	0.284	0.014	0.160
	12n	0.469	0.195	0.009	0.356
	13b	1.556	0.550	0.015	0.216
	13c	1.725	1.093	0.064	0.059

a.

  .86 (s, 3H), 2.94 (s, 2H), 2.58 (s, 2H), 2.47 (s, 3H), 2.33 (s, 2H). HR-ESIMS: m/z 522.0797 [M + H] + (calcd for C 23 H 25 INO 5 , 522.0778).

  C NMR (75 MHz, CDCl 3 ) δ 159.94, 158.05, 139.44, 136.01, 134.89, 132.97, 128.66, 125.89, 116.71, 104.44, 99.01, 93.15, 55.89, 54.53, 52.18, 45.76, 32.04, 30.92, 29.31.

  C NMR (75 MHz, CDCl 3 ) δ 177.94, 161.02, 160.04, 155.30, 138.20, 131.27, 128.03, 127.37, 126.54, 108.70, 108.16, 97.84, 91.69, 56.45, 55.91, 54.96, 52.60, 46.02, 29.90, 1.02. HR-ESIMS: m/z 504.0670 [M + H] + (calcd for C 23 H 23 INO 4 , 504.0672). According to the protocol of Tabaka et al.

	4.2.1.4.	2-(2-chloro-4-iodophenyl)-5,7-dimethoxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-chromen-4-one
	(10b).

  5H).13 C NMR (75 MHz, DMSO) δ 159.97, 138.51, 128.38, 126.60, 109.97, 109.28, 105.96, 105.25, 98.73, 54.24, 51.78, 45.52, 34.20, 29.27. HR-ESIMS: m/z 476.0356 [M + H] + (calcd for C 21 H 19 INO 4 , 476.0359).

	H NMR (300 MHz,
	DMSO) δ 12.83 (s, 1H), 8.21 (s, 1H), 7.97 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.01 (s, 1H), 6.32 (s, 1H), 5.72
	(s, 1H), 3.17 (s, 2H), 2.71 (s, 2H), 2.41 (s,

  C NMR (75 MHz, DMSO) δ 182.68, 170.41, 169.96, 169.75, 169.54, 162.77, 162.22, 160.24, 154.28, 138.27, 130.24, 129.95, 127.64, 127.19, 126.49, 109.98, 109.34, 105.16, 104.26, 99.14, 82.69, 74.83, 73.31, 69.90, 68.51, 62.35, 54.53, 52.10, 45.48, 29.46, 29.21, 20.99, 20.84, 20.73;  max 2987, 2901, 1747, 1651, 1579, 1405, 1372, 1223, 1042, 909, 825, 563, 488 cm -1 ; HR-ESIMS: m/z 712.2058 [M + H] + (calcd for C 35 H 38 NO 13 S, 712.2064). HPLC purity, 98.5% (t R = 11.22 min).

  H NMR (300 MHz, MeOD) δ 8.45 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.68 (d, J = 7.7 Hz, 2H), 6.68 (s, 1H), 6.29 (s, 1H), 5.85 (s, 1H), 4.81 (signal overlapped with water), 3.97-3.93 (m, 3H), 3.73-3.68 (m, 1H), 3.52-3.48 (m, 4H), 3.38-3.31 (m, 5H overlapped with MeOD), 3.02 (s, 3H), 2.81 (s, 2H).13 C NMR (75 MHz, MeOD) δ 182.53, 163.66, 161.94, 161.11, 154.28, 140.72, 129.49, 128.85, 128.74, 126.36, 121.47, 109.99, 106.93, 104.23, 98.53, 86.76, 80.93, 78.35, 72.49, 70.04, 61.60, 51.97, 50.61, 41.68, 25.78;  max 2987, 2900, 1650, 1560, 1408, 1374, 1273, 1187, 1056, 825, 770, 745, 550 cm -1 ; HR-ESIMS: m/z 544.1649 [M + H] + (calcd for C 27 H 30 NO 9 S + , 544.1641). HPLC purity, >90% (t R = 8.24 min).

  C NMR (75 MHz, CDCl 3 ) δ 182.22, 170.47, 170.12, 169.98, 169.59, 162.91, 161.39, 160.98, 154.24, 138.33, 131.07, 129.09, 126.53, 124.07, 106.75, 105.61, 104.96, 99.72, 85.18, 77.46, 77.03, 76.61, 74.45, 71.82, 67.11, 61.28, 53.04, 51.59, 43.88, 27.49, 20.86, 20.70, 20.59;  max 2971, 2900, 1743, 1648, 1561, 1489, 1407, 1369, 1241, 1065, 825, 559 cm -1 ; HR-ESIMS: m/z 712.2076 [M + H] + (calcd for C 35 H 38 NO 13 S + , 712.2064); HPLC purity, 94.09 % (t R = 11.80 min).

  C NMR (75 MHz, MeOD) δ 182.11, 170.81, 170.03, 169.79, 169.54, 154.88, 138.05, 132.40, 132.14, 130.90, 130.39, 129.74, 128.20, 123.27, 110.29, 99.05, 83.51, 75.44, 73.69, 69.66, 68.07, 61.85, 52.76, 50.98, 42.53, 26.99, 19.40, 19.22, 19.13; HR-ESIMS: m/z 746.1682 [M + H] + (calcd for C 35 H 37 NO 13 SCl, 746.1674); HPLC purity, 94% (t R = 11.66 min).

  C NMR (75 MHz, MeOD) δ 182.39, 179.96, 178.43, 171.43, 170.46, 163.23, 160.71, 156.92, 154.24, 140.98, 128.47, 128.08, 127.63, 127.42, 126.51, 123.88, 108.09, 103.93, 103.70, 99.03, 66.43, 61.83, 56.83, 53.17, 52.11, 51.78, 51.32, 43.19, 33.51, 29.38, 27.42;  max 1648, 1561, 1405, 1370, 1241, 1065, 826, 741, 698, 560 cm -1 ; HR-ESIMS: m/z 704.2283 [M + H] + (calcd for C 36 H 38 N 3 O 10 S, 704.2278); HPLC purity, 92% (t R = 10.68 min).

  C NMR (75 MHz, DMSO) δ 182.50, 172.58, 169.56, 163.11, 162.16, 160.11, 154.22, 142.82, 127.84, 127.52, 127.11, 124.89, 108.65, 104.49, 104.14, 99.11, 53.33, 52.65, 51.27, 44.12, 34.41, 27.86, 22.93; HR-ESIMS: m/z 511.1543 [M + H] + (calcd for C 26 H 27 N 2 O 7 S, 511.1539).

  C NMR (75 MHz, CDCl 3 ) δ 182.41, 170.77, 162.66, 161.39, 161.18, 154.23, 140.80, 129.18, 128.91, 126.40, 125.89, 107.93, 105.11, 104.84, 99.63, 54.13, 53.27, 52.64, 52.22, 45.87, 44.86, 35.68, 28.91, 28.27, 8.74;  max 1648, 1576, 1417, 1363, 1273, 1160, 1098, 1012, 826, 697, 627, 561, 484 cm -1 ; HR-ESIMS: m/z 583.2110 [M + H] + (calcd for C 30 H 35 N 2 O 8 S, 583.2114); HPLC purity, > 90% (t R = 12.27 min).

  04;  max 1645, 1410, 1373, 1202, 1101, 1065, 1036, 896, 819, 749, 699, 621, 561 cm -1 ; HR-ESIMS: m/z 553.2015 [M + H] + (calcd for C 29 H 33 N 2 O 7 S, 553.2008); HPLC purity, >90 % (t R = 10.54 min). To a mixture of 12g in dioxane was added dropwise HCl in dioxane 4M (0.6 mL) and the mixture stirred for 30 minutes at r.t. the solvent was removed under vacuum. the product was purified by HPLC (water/ACN) to yield a yellow powder (81%). 1 H NMR (300 MHz, D 2 O) δ 8.37 (s, 1H), 7.04 (d, J = 5.6 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.00 (s, 1H), 5.89 (s, 1H), 5.39 (s, 1H), 3.00 (d, J = 23.6 Hz, 5H), 2.43 (s, 4H);  max 2971, 2900, 1648, 1561, 1409, 1366, 1273, 1187, 1066, 824, 770, 628, 558 cm -1 ; HR-ESIMS: m/z 469.1432 [M + H] + (calcd for C 24 H 25 N 2 O6S, 469.1433); HPLC purity, > 90% (t R = 9.34 min).

	4.2.2.9.	3-((4-(5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4H-chromen-2-yl)phenyl)thio)-N-
	hydroxypropanamide (12h).

  H NMR (400 MHz, DMSO) δ 12.66 (s, 1H), 7.80 (d, J = 7.5 Hz, 2H), 7.54 -7.27 (m, 4H), 7.04 (d, J = 3.0 Hz, 2H), 6.77 (s, 1H), 6.13 (s, 1H), 5.55 (m, 2H), 2.98 (d, J = 16.2 Hz, 2H), 2.33 (s, 4H), 2.19 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 182.13, 162.17, 159.78, 153.84, 144.98, 136.76, 130.41, 130.13, 127.13, 126.12, 122.40, 109.01, 104.86, 103.72, 98.78, 54.88, 54.12, 51.68, 45.09, 28.85;  max 1655, 1581, 1402, 1363, 1270, 829, 743, 560, 512, 495, 477 cm -1 ; HR-ESIMS: m/z 498.1494 [M + H] + (calcd for C 28 H 24 N 3 O 4 S, 498.1488); HPLC purity, 100% (t R = 10 min).

  C NMR (101 MHz, DMSO) δ 182.10, 161.99, 161.75, 160.30, 153.86, 151.37, 133.95, 131.91, 127.63, 125.13, 124.93, 122.23, 118.92, 110.53, 107.17, 105.67, 99.53, 98.69, 51.60, 50.05, 42.16, 25.74; HR-ESIMS: m/z 499.1333 [M + H] + (calcd for C 28 H 23 NO 5 S, 499.1328); HPLC purity, >90% (t R = 12.31 min).

	4.2.2.12.	5,7-dihydroxy-2-(4-((5-methoxy-1H-benzo

  C NMR (101 MHz, DMSO) δ 182.12, 162.49, 160.27, 153.83, 129.55, 127.37, 122.49, 112.27, 104.93, 103.87, 98.69, 55.51, 51.70, 50.11, 42.35, 25.97;  max 1650, 1403, 1100, 829, 698, 529, 511, 494, 487, 477, 460, 452 cm -1 ; HR-ESIMS: m/z 528.1594 [M + H] + (calcd for C 29 H 26 N 3 O 5 S, 528.1593); HPLC purity, 94.10% (t R = 10.08 min).

  2H).13 C NMR (75 MHz, MeOD) δ 157.73, 135.22, 126.68, 123.70, 117.73, 110.29, 105.00, 99.29, 53.15, 51.07, 43.00, 27.37;  max 2976, 1649, 1561, 1405, 1371, 1276, 1248, 1186, 1100,1014, 834, 768, 163.99, 161.28, 160.24, 153.67, 147.66, 141.32, 129.27, 129.02, 128.33, 127.59, 121.77, 121.63, 119.88, 119.30, 115.05, 114.66, 106.86, 103.48, 101.63, 98.55, 50.88, 49.44, 48.54, 41.59, 25.29 ; HR-ESIMS: m/z 441.1811 [M + H] + (calcd for C 27 H 25 N 2 O 4 , 441.1814).

	H NMR (300 MHz, MeOD) δ 8.56 (d, J = 5.0 Hz, 2H), 8.04 (d, J = 8.5
	Hz, 2H), 7.84 (d, J = 7.7 Hz, 2H), 7.21 (m, 1H), 6.87 (s, 1H), 6.33 (s, 1H), 5.91 (s, 1H), 3.85 (s, 2H), 3.34 (overlapped
	with solvent), 2.92 (s, 3H), 2.79 (s,

  H NMR (400 MHz, DMSO) δ 13.18 (s, 1H), 12.77 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.63 -7.50 (m, 3H), 7.25 (dd, J = 5.8, 2.9 Hz, 2H), 6.62 (s, 1H), 6.41 (s, 1H), 5.66 (s, 1H), 3.23 (s, 2H), 2.80 (s, 2H), 2.47 -2.36 (m, 5H).13 C NMR (101 MHz, DMSO) δ 181.67, 161.94, 159.94, 154.34, 143.89, 137.98, 132.21, 130.49, 129.63, 128.43, 126.78, 124.96, 108.69, 103.67, 52.85, 51.04, 43.70, 28.04;  max 1651, 1573, 1413, 1360, 1276, 1099, 743, 539, 530, 505, 495, 477, 460 cm -1 ; HR-ESIMS: m/z 532.1104 [M + H] + (calcd for C 28 H 23 N 3 O 4 SCl, 532.1098); HPLC purity, > 90% (t R =8.92 min). Xantphos (10 mol%), Et N (50 μL), and dioxane (1 mL) were stirred 2 hours at 60°C. The compound was purified on silica gel (DCM/MeOH, 3 5%) and obtained as yellow solid in 60% yield. 1 H NMR (300 MHz, MeOD) δ 7.81 -7.52 (m, 4H), 7.50 -7.28 (m, 2H), 6.55 (s, 1H), 6.37 (s, 1H), 5.79 (s, 1H), 4.02 -3.73 (m, 5H), 3.34-3.33 (m, 2H), 2.92 (m, 3H), 2.73 (s, 2H). 13 C NMR (75 MHz, MeOD) δ 162.64, 154.74, 138.17, 133.20, 130.05, 127.61, 124.02, 123.02, 121.31, 118.52, 110.53, 110.37, 98.63, 51.87, 50.49, 41.43, 30.13, 25.72;  max 1651, 1574, 1414, 1360, 1276, 1186, 1099, 820, 761, 743, 540, 530, 495, 477, 460 cm -1 ; HR-ESIMS: m/z 546.1255 [M + H] + (calcd for C 29 H 25 N 3 O 4 SCl + , 546.1254); HPLC purity, 91 % (t R = 11.02min).

	4.2.2.20.	2-(2-chloro-4-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)phenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-
	tetrahydropyridin-4-yl)-4H-chromen-4-one (13c). 11b (20 mg, 0.04 mmol), 1-methyl-1H-benzo[d]imidazole-2-thiol (10
	mg, 0.043 mmol), PdG3-4.2.2.21. 2-(2-chloro-4-(pyrimidin-2-ylthio)phenyl)-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-4H-
	chromen-

  H NMR (300 MHz, MeOD) δ 8.60 (d, J = 4.7 Hz, 2H), 7.90 (s, 1H), 7.75 (s, 2H), 7.25 (d, J = 4.6 Hz, 1H), 6.55 (s, 1H), 6.31 (s, 1H), 5.78 (s, 1H), 3.52 (s, 2H), 3.11 (s, 2H), 2.65 (d, J = 16.7 Hz, 5H). 13 C NMR (75 MHz, MeOD) δ 172.91, 157.85, 136.00, 134.70, 133.38, 132.48, 131.15, 128.22, 123.68, 117.99, 110.42, 108.32, 99.16, 52.93, 51.05, 42.73, 27.30;  max 2987, 2900, 1651, 1557, 1409, 1376, 1259, 1183, 1100, 1057, 803, 769, 629, 549 cm -1 ; ESIMS: m/z 494.0943 [M + H] + (calcd for C 25 H 21 ClN 3 O 4 S, 494.0941); HPLC purity, 96% (t R = 10.49 min).

Acknowledgments

The authors acknowledge support of this project by the CNRS and University Paris-Sud and "La Ligue Contre le Cancer" throughout an "Equipe Labellisée 2014" grant. Our laboratory is a member of the Laboratory of Excellence LERMIT supported by a grant (ANR-10-LABX-33). The authors also thank the Cancéropôle Grand Ouest ("Marines molecules, metabolism and cancer" network), IBiSA (French Infrastructures en sciences du vivant: biologie, santé et agronomie) and Biogenouest (Western France life science and environment core facility network) for supporting the KISSf screening facility (FR2424, CNRS and Sorbonne Université), Roscoff, France.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/


Alexandre Eeckhoutte

Mathilde Saint-Ghislain

Manon Reverdy

Virginie Raynal

Sylvain Baulande

Guillaume Bataillon

Lisa Golmard

Dominique Stoppa-Lyonnet

Tatiana Popova

Claude Houdayer

Elodie Manié

Marc-Henri Stern
email: marc-henri.stern@curie.fr

Lack of evidence for CDK12 as an ovarian cancer predisposing gene

Keywords: CDK12, cancer susceptibility, ovarian carcinoma, pool sequencing, NGS

CDK12 variants were investigated as a genetic susceptibility to ovarian cancer in a series of 416 unrelated and consecutive patients with ovarian carcinoma and who carry neither germline BRCA1 nor BRCA2 pathogenic variant. The presence of CDK12 variants was searched in germline DNA by massive parallel sequencing on pooled DNAs. The lack of detection of deleterious variants and the observed proportion of missense variants in the series of ovarian carcinoma patients as compared with all human populations strongly suggests that CDK12 is not an ovarian cancer predisposing gene.

Acknowledgments

Introduction

Epithelial ovarian carcinoma (EOC) is a rare but dreadful disease and represents the 5th cause of death by cancer in women worldwide. The most frequent histology of this carcinoma is High Grade Serous Ovarian Carcinoma (HGSOC). The known risks factors of EOC are age, hormonal history and genetic factors [START_REF] Lheureux | Epithelial ovarian cancer[END_REF]. Genetic factors are involved in approximately 15% of HGSOC, often associated with the so-called hereditary breast and ovarian cancer syndrome (HBOC). BRCA1 and BRCA2, which code key actors of the homologous recombination (HR) DNA repair pathway, are the two major cancer predisposing genes. Heterozygote germline mutations of BRCA1 and BRCA2 lead to the most important increased risk to develop an HGSOC with a Relative Risk (RR) of 40 and 18, respectively [START_REF] Kuchenbaecker | Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers[END_REF]. They explain 65% to 75% of hereditary EOC. In addition, mutations of other genes belonging to the HR pathway, such as RAD51 paralogs, also participate to the HBOC syndrome [START_REF] Golmard | Contribution of germline deleterious variants in the RAD51 paralogs to breast and ovarian cancers[END_REF][START_REF] Loveday | Germline mutations in RAD51D confer susceptibility to ovarian cancer[END_REF][START_REF] Loveday | Germline RAD51C mutations confer susceptibility to ovarian cancer[END_REF]. A second group of predisposition genes belong to the MMR family (MLH1, MSH2, MSH6, PMS2), mutated in the Lynch syndrome. Lynch syndrome is associated with a RR of [3.6-13] for EOC, mainly of the endometrioid and clear cell subtypes, and explains ~10% of hereditary EOC [START_REF] Lheureux | Epithelial ovarian cancer[END_REF][START_REF] Bewtra | Hereditary ovarian cancer: a clinicopathological study[END_REF][START_REF] Helder-Woolderink | Ovarian cancer in Lynch syndrome; a systematic review[END_REF][START_REF] Bonadona | Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome[END_REF]. Strikingly, all these predisposition genes encode proteins involved in DNA maintenance.

Recently, CDK12 emerged as an important player in ovarian carcinoma. CDK12 (cyclindependent kinase 12) is one of the ten most frequently mutated genes in HGSOC (3% of the TCGA cohort) and is also mutated in 7% of metastatic castration-resistant prostate cancer [START_REF] Wu | Inactivation of CDK12 Delineates a Distinct Immunogenic Class of Advanced Prostate Cancer[END_REF].

CDK12 behaves as a classical tumor suppressor gene with bi-allelic somatic inactivation in tumors, with in most cases one deleterious mutation on one allele and one chromosomal partial deletion evidenced by loss of heterozygosity (LOH). We have previously shown that CDK12-inactivated tumors are associated with an unusual form of genomic instability named the TD-plus phenotype and characterized by hundreds of large tandem duplications of up to 10 megabases in size [START_REF] Popova | Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications[END_REF]. CDK12 is an essential gene during development as Cdk12 inactivation is embryonic lethal in mouse models [START_REF] Juan | Cdk12 is essential for embryonic development and the maintenance of genomic stability[END_REF]. CDK12 is a nuclear serine threonine kinase that dimerizes with Cyclin K (CCNK). Until recently, the only known targets of CCNK/CDK12 were serines 2 and 5 of the carboxy-terminal-domain (CTD) of the RNA polymerase II, required for elongation and end termination of transcription. In in-vitro studies, CDK12 is required for the expression of a subset of DNA Damage Response (DDR) genes, including BRCA1, FANCI, FANCD2 [START_REF] Blazek | The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes[END_REF], and conversely, CDK12-inactivated cell models are highly sensitive to PARP inhibitors [START_REF] Bajrami | Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity[END_REF]. Both in vitro studies and analyses of CDK12mutated tumors strongly suggest that CDK12 plays a role in genomic maintenance. Recent studies have showed that CDK12 acts by suppressing intronic polyadenylation events, including in DNA repair genes [START_REF] Tien | CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion[END_REF][START_REF] Krajewska | CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation[END_REF][START_REF] Dubbury | CDK12 regulates DNA repair genes by suppressing intronic polyadenylation[END_REF]. CDK12 phosphorylates 4E-BP1 to enable mTORC1dependent translation and maintains mitotic genome stability [START_REF] Choi | CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability[END_REF].

CDK12 as an important tumor suppressor gene in ovarian tumorigenesis pointed it as a potential predisposition gene for EOC. We explored this hypothesis by investigating the germline status of CDK12 in a series of 416 unselected consecutive and unrelated patients with EOC, negative for BRCA1 or BRCA2 mutations.

Material and methods

Patients

A series of blood DNA from 416 unselected consecutive and unrelated patients was assembled from the Genetic Department of Institut Curie, initially explored negative for BRCA1 or BRCA2 deleterious germline mutations, using the current techniques at time of diagnosis or reanalysis. Five of them were subsequently found to carry deleterious mutations of BRCA2 (1 case), RAD51C (1), RAD51D (1), PMS2 (1) and TP53 (1). All patients had personal history of EOC (mean age at diagnosis: 56 years-old) and benefited from genetic counseling. One hundred twenty-three of these patients had also developed one or more breast cancers. All patients have signed an informed consent for research of new cancer predisposing genes.

CDK12 sequencing in pooled DNA and positive control pools

Germline DNAs of the 416 patients were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The 416 DNAs were pooled into 52 equimolar pools of eight DNAs per pool, with an expected variant frequency of one mutated allele in 16 alleles (6.25%). Two additional pools were constituted as positive controls, containing seven patients' DNAs plus one tumor DNA with a known CDK12 mutation, c.137del/p.Lys46SerfsX11 and c.212dup/p.Glu72GlyfsX3 for pools #53 and #54, respectively. CDK12 coding sequence and flanking introns were sequenced using the TruSeq Custom Amplicon Low Input Kit (Illumina). Briefly, the design included 32 amplicons of 250 bp for a theoretical coverage of 100% on a cumulative target of 4.61 kb. The library was produced by PCR and ligation from 20ng of pooled genomic DNA, barcoded with 54 indexes, quantified (Bioanalyzer, Agilent) and pooled in an equimolar ratio. The library was then paired-end sequenced (PE250) with a MiSeq v2 Nano flow cell (Illumina).

Bioinformatics pipeline

Quality control was performed using FASTQC. Reads were aligned to the hg19 assembly with BWA MEM (v. 0.7.5a). Primers were soft-clipped with BAMclipper [START_REF] Au | BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing[END_REF]. BAM files were pre-processed with indels realignment and base quality score recalibration according to the GATK Best Practices (v. 3.5) [START_REF] Depristo | A framework for variation discovery and genotyping using next-generation DNA sequencing data[END_REF]. Variants were detected by Samtools mpileup (v. 1.7) [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF], HaplotypeCaller (v. 3.5) and Mutect2 (v. 3.5) . The union of all the variants called was annotated with ANNOVAR [START_REF] Wang | ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data[END_REF] according to different databases: EnsGene, COSMIC88, dbSNP151 and maximum allele frequency from 1000G, ExAC, ESP6500 and CG46. Variants were filtered out if: (i) synonymous, (ii) intronic and UTR located, (iii) biased for strand direction (outside of [0.4-0.6] ratio in a balanced site), (iv) frequency higher than 1% in any human reference population, (v) frequency higher than 1% in the patients' series, (vi) Variant Allele Frequency (VAF) < 1% and (vii) existing at a homozygous state in any individual. The remaining variants were then manually checked on IGV and five of them were discarded. A pileup approach was also implemented, with Samtools (v. 1.8) and a customized script to retain indels supported by more than five reads (Figure 1). An in silico prediction of splice defects was also performed using the MaxEntScan tool (MaxEnt; [START_REF] Yeo | Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals[END_REF]).

Variant validation

The eight DNAs from each positive pool were analyzed independently by Sanger sequencing for the identified variant. Briefly, PCR was performed from 50 ng of DNA using specific primers and Taq Gold using standard protocols (Primers and conditions available on request) and sequenced using Big Dye Terminator kit V1 (3130XL, Applied Biosystems). Quality control of the electropherograms was performed using FinchTV (PerkinElmer) and sequences were analyzed using SeqScape (Applied Biosystems).

Prediction

In-silico predictions of deleterious consequences of the non-synonymous variants were performed using CADD (Combined Annotation Dependent Depletion) Phred score, SIFT (Sorting Intolerant from Tolerant), Polyphen-2 and VEP (Variant Effect Predictor).

CDK12 defect genomic signature

Tumor DNA extracted from frozen or FFPE blocs from CDK12-variant carriers were obtained from the institutional Biobank and the Department of Pathology, respectively. Genomic profiling was obtained by shallow Whole Genome Sequencing (sWGS, approximatively 1 read per base) of the tumor DNA on NovaSeq (Illumina). Adapters were trimmed with Cutadapt (v. 1.18). The number of reads in windows of 10 kb was extracted and normalized for GC content and mappability with ControlFREEC [START_REF] Boeva | Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data[END_REF]. The CDK12 TD plus pattern characteristic of CDK12 inactivation was visually checked [START_REF] Popova | Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications[END_REF].

Statistical power of the study

Assuming a distribution in CDK12 variants following a Poisson law, the theoretical frequency to detect at least one deleterious variant in a cohort of 927 cases with a power of 80 was calculated as the following: P(X≥1, λ) = 0.80 = 1-P(X=0, λ)

𝜆 = -𝑙𝑛(0.20) 927 = 1.7𝑒 -3

Results

The goal of this study was to evaluate CDK12 as a potential EOC predisposing gene. We thus defined the frequency of CDK12 germline variants in a series of 416 consecutive patients with ovarian carcinomas. The Region Of Interest (ROI) included 4525 bp corresponding to the coding sequence of CDK12, including 14 exons plus 2 base pairs of splicing sites. A pooled DNA sequencing approach was performed. The mean read depth on ROI was 920x and the majority of ROI (92%) displayed more than 320X coverage, corresponding to 20X per allele.

The lowest depth on ROI was ranging from 83X to 227X in the different pools (mean 143x).

The less covered regions were parts of exons 1 and 2, and the whole exon 10.

In addition to the two positive controls, a list of 151 variants was called by at least one of the variant calling methods. 21 different variants in 17 different pools were retained for validation after filtering. Ten predicted variants were not confirmed by Sanger sequencing, and thus were considered as false calls, whereas eleven of these variants were validated in 11 different patients, among which 10 were single nucleotide variants (SNVs) and one an in-frame 3-base deletion (Figure 1, Table 1). Out of these 11 variants, 9 were previously reported in the dbSNP database v151. All SNVs were missense variants with a predictive deleteriousness ranging from benign to moderate. One yet unreported SNP, c.A2712T, changes a glutamic acid codon conserved in all sequenced vertebrates up to lamprey and located within the kinase domain. However, the consequence of this change for aspartic acid was considered as mild.

No Loss of Function (LoF) variant, such as premature stop-gain, frameshift or splicing variant, was found in this series.

As compared with reported frequencies of these variants in the representative non-Finnish European population in the GnomAD database, none of these variants were significantly enriched in our series of EOC patients (Table 1). As CDK12 deleterious variants are embryonic-lethal at homozygous state, we considered only the missense SNPs reported in dbSNP151 and never found at homozygous state in any human population. The proportion of such missense SNPs was not significantly different in our series from that of the representative non-Finnish European population (10/832 alleles and 823/113650 alleles respectively; Fisher's exact test : p = 0.1453), which is in accordance with CDK12 not being an ovarian cancer predisposing gene.

Although no strong evidence supported the pathogenic effect of the identified variants, we further explored whenever possible the tumor of the corresponding variant carrier. We retrieved four EOC cases for whose tumor material was available. Given that CDK12 is a tumor suppressor gene, we first assessed the loss of the wild-type allele in tumors, according to the Knudson/two-hit hypothesis. Only one of the four EOC had a loss of the wild allele, and the three others retained the wild-type allele in the tumors. A key feature of CDK12inactivated tumors is a striking genomic profile enriched in numerous and very large tandem duplications, the TD-plus genomic signature [START_REF] Popova | Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications[END_REF], which was not found in the genomic profiles of the four tested EOCs with CDK12 variants (Table 1 and Figure 2).

Altogether, we found no evidence of deleterious CDK12 germline variants in our series of ovarian carcinoma, and so no evidence for its role as an ovarian susceptibility gene in the studied population.

Discussion

CDK12 recently emerged to play an important role in ovarian and prostatic carcinomas, as a tumor suppressor gene contributing to malignant transformation and genomic instability when inactivated. As such, CDK12 was a good candidate to also play a role in cancer predisposition. This study evaluated the incidence of CDK12 germline variants in a series of 416 unrelated and consecutive patients with ovarian carcinoma. A total of eleven CDK12 exonic variants were identified by massive parallel sequencing and validated by Sanger sequencing. None of the variants was a Loss of Function variant (LoF). However, one was in the kinase domain on a well conserved codon up to lamprey, but with a mild acid to acid change of coded amino-acid. Unfortunately, no tumor sample was available for further investigation of this case. We then compared the proportion of missense variants found in our series from that of the representative non-Finnish European population and found no statistically significant difference. We further mined four variants, for which tumor samples were retrieved and no evidence of CDK12 inactivation was found in these tumors.

Altogether, we found no evidence for a role of CDK12 as an ovarian cancer predisposition gene in an unbiased series of 416 BRCA1/2-wild-type patients with ovarian carcinoma.

Furthermore, the analysis of The Cancer Genome Atlas (TCGA) series of 511 ovarian serous cystadenocarcinomas identified 15 cases with the TD-plus phenotype and bi-allelic inactivation of CDK12, none of which carrying any deleterious germline variant [10 and Popova et al.,unpublished data]. These analyses of combined series (TCGA and in-house cases) had a power of 80% to detect at least one deleterious germline mutation in the hypothesis of a deleterious variant frequency of 1.7 10 -3 in EOC patients.

Interestingly, the number of LoF variants in Gnomad (n=6) is largely below the expected one, with a probability of being loss-of-function intolerant (pLI) score at 1 [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF], and an observed / expected (oe) score of 0.05 (gnomad.broadinstitute.org). This suggests that LoF variants are counter-selected in human populations, even at the heterozygous state. Interestingly, a report described the existence of a deleterious c.1047-2A>G germline CDK12 variant in 8 of the 106 HBOC cases tested (7.6%) in the Tatar population [START_REF] Brovkina | The Ethnic-Specific Spectrum of Germline Nucleotide Variants in DNA Damage Response and Repair Genes in Hereditary Breast and Ovarian Cancer Patients of Tatar Descent[END_REF], but the association with HBOC was not confirmed in a replication study [START_REF] Bogdanova | A Splice Site Variant of CDK12 and Breast Cancer in Three Eurasian Populations[END_REF]. If replicated, this would suggest that CDK12 variants could play a role in HBOC predisposition in some human populations, although strongly counter-selected in most human populations. The reason of this counter-selection is not clear. Cdk12 inactivation is embryonic lethal in mouse models, but heterozygous Cdk12 Δ/wt pups are viable and born with the expected frequency [START_REF] Juan | Cdk12 is essential for embryonic development and the maintenance of genomic stability[END_REF]. Clearly, long-term follow-up of these Cdk12 Δ/wt mice may be instrumental to unravel the mechanism of intolerance of LoF variants in Humans. In a more distant model in Drosophila, a decline of courtship learning was observed in CDK12 heterozygous flies [START_REF] Pan | Heterochromatin remodeling by CDK12 contributes to learning in Drosophila[END_REF]. This could be a plausible mechanism to explain the counter-selection of human heterozygous CDK12-mutant carriers, but caution should be taken given the evolutionary distance between flies and mammals.

In conclusion, our data evidenced the absence of deleterious CDK12 variants in patients with ovarian carcinoma, confirming the rarity of such variants in the general population, and making unlikely the existence of deleterious variants in more than 0.2% of EOC patients.

Thus our data do not support the role of CDK12 in ovarian carcinoma susceptibility. The origin of the intolerance of deleterious CDK12 variants in the population has yet to be explained.

Figure 1 .

 1 Figure 1. Variant calling workflow. Workflow of the CDK12 pool targeted-sequencing,

Figure 2 .

 2 Figure 2. Genome profiling of epithelial ovarian carcinoma carrying CDK12 variants.

  : • Synonymous, UTR & intronic variants • VAF < 1% • MAF in general/patient population > 1% • Strand biased in balanced site • Variants existing in a homologous state in individuals • Not validated on IGV

Figure 1

 1 Figure 1 Click here to access/download;Figure;Fig1_workflow_complet_ae.pptx

Figure 2

 2 Figure 2 Click here to access/download;Figure;Fig2_sWGS_CDK12_article.ppt

Table legends Table 1 . Description of CDK12 variants

 legends1

		Variant calling: union of
		HaplotypeCaller,
		MuTect2,
	Raw Reads	SAMTools mpileup
	FASTQC
		ANNOVAR
	QC files	ensGene, dbsnp151,
		cosmic88, popfreq_all
	Alignment on hg19
	BWA-MEM
	BAM files

. Ref: reference allele; Alt: alternative allele; Pop max freq: maximum allele frequency in any human population (Non-Finish European -NFEby default); AFR: AFRican population; EAS: East-Asian; EOC: Epithelial Ovarian Carcinoma; LOH: Loss Of Heterozygosity; ROH: Retention Of Heterozygosity; NA: Not Available; sWGS: shallow Whole Genome Sequencing; TD-Plus: tandem duplication-plus genome profile, characteristic of CDK12 inactivation.

Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Institut Curie. M. S-G and A. E. are supported by fellowships from the Université de Rouen and the Ligue Nationale Contre le Cancer, respectively. The Institut Curie ICGex NGS platform is funded by the EQUIPEX investissements d'avenir program (ANR-10-EQPX-03) and ANR10-INBS-09-08 from the Agence Nationale de la Recherche. We thank the patients.

We also acknowledge support from the Institut Curie for sample collection, banking and processing: the Biological Resource Center and its members (O. Mariani).