Marion Duriez

Yassmina Mandouri

Bouchra Lekbaby

Hualin Wang

Aurélie Schnuriger

Francois Redelsperger

Ida Chiara

Marine Guerrera

Veronique Lefevre

James Fauveau

Ahodantin

Chiara Guerrera

Marine Lefevre

Veronique Fauveau

James Ahodantin

Ivan Quetier

Cerina Chhuon

Samir Gourari

Alexandre Boissonas

Upkar Gill

Patrick Kennedy

Nabil Debzi

Delphine Sitterlin

Mala K Maini

Dina Kremsdorf

Patrick Soussan
email: patrick.soussan@inserm.fr

Alternative splicing of Hepatitis B virus: a novel virus/host interaction altering liver immunity?

Keywords: HBV, liver fibrosis, HBSP, alternate splicing, macrophages, CCL2

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Constitutive splicing is an essential step for eukaryotic gene expression, generating mature mRNAs by removing introns and allowing exons to be joined together accurately. In contrast, alternate splicing (including exon skipping, intron retention, alternate 3' or 5' splice sites) contributes to the diversity of eukaryotic proteome [START_REF] Pohl | Alternative splicing of mutually exclusive exons--a review[END_REF] and involves in the control of expression of regulatory proteins in the viral context [START_REF] Hernandez-Lopez | Alternative splicing in human tumour viruses: a therapeutic target?[END_REF]. The splicing process is coordinated by the spliceosome, a large ribonucleoprotein complex, on specific RNA domains in association with ubiquitous transregulatory splicing factors including serinearginine-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNPs) families [START_REF] Han | Functional diversity of the hnRNPs: past, present and perspectives[END_REF].

The ability of Hepatitis B Virus (HBV) transcripts to undergo to alternate splicing (AS) has been well described in vitro and in the liver of patients with chronic infection (CHB) [START_REF] Susuki | Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome[END_REF][START_REF] Wu | Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected Hep G2 cells[END_REF]. The 3.5 kb pregenomic RNA (pgRNA) of HBV, which encodes for capsid and polymerase proteins, and constitutes the template for viral genome replication, can be alternatively spliced [START_REF] Seeger | Molecular biology of hepatitis B virus infection[END_REF]. The major pgRNA spliced variant, SP1RNA, has one third of the viral genome deleted ("intron 2447/489") and may account for up to 30% of total HBV pgRNA. SP1RNA can be packaged in core particles, reverse-transcribed and secreted. While pgRNA packaging leads to wild-type Dane particle secretion (wtHBV), the shorter SP1RNA constitutes a matrix for defective HBV circulating particles (dHBV), varying in proportion from 0% to more than 50% of HBV forms [START_REF] Terre | Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo[END_REF][START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF][START_REF] Soussan | Expression of defective hepatitis B virus particles derived from singly spliced RNA is related to liver disease[END_REF]. The regulation of SP1RNA during the course of liver disease remains poorly understood. However, recent reports in HBV infected patients have shown that the proportion of dHBV relates to viral replication, to interferon therapy failure [START_REF] Chen | Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy[END_REF] and increases with liver disease progression towards hepatocellular carcinoma (HCC) [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF]. SP1RNA allows the expression of HBV splicing-generated protein (HBSP). HBSP protein shares the N-terminal 46 amino-acids sequence of the viral polymerase fused to an original viral sequence constituting its C-terminal part (65 amino-acids). HBSP has been identified in liver tissues from patients with CHB [START_REF] Soussan | In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF], in whom it can induce an immune response [START_REF] Bayard | T-cell responses to hepatitis B splice-generated protein of hepatitis B virus and inflammatory cytokines/chemokines in chronic hepatitis B patients. ANRS study: HB EP 02 HBSP-FIBRO[END_REF]. The function of HBSP protein in liver pathogenesis remained uncertain, although in vitro studies had suggested an impact on cell viability, proliferation and migration [START_REF] Soussan | In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF][START_REF] Chen | Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity[END_REF] and more recently on hacking the TNF-α signaling pathway [START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-alpha-stimulated signaling pathways and limits the extent of liver inflammation[END_REF].

The previous observation that dHBV particles increase with liver disease progression prompted us to investigate whether and how regulation of HBV AS and liver pathogenesis are mechanistically linked. Because, the lack of robust animal models of liver pathogenesis induced by HBV infection, we decided to investigate the viral post-transcriptional regulation in HBV transgenic mice [START_REF] Cheng | Modeling hepatitis B virus infection, immunopathology and therapy in mice[END_REF]. Here we report that alteration of spliceosome machinery in HBV expressing cells is switched on by liver injury and enables a striking reduction in liver monocyte/macrophage recruitment through HBSP expression. Our findings reveal a novel paradigm whereby AS can generate a viral product able to inhibit immune-mediated inflammation and thereby down-modulate organ damage.

Materials and Methods

Mice and liver pathogenesis models

Inbred C57BL/6J HBV-transgenic mouse lineage 1.3.32 (TgHBV) has been previously described [START_REF] Guidotti | High-level hepatitis B virus replication in transgenic mice[END_REF]. TgHBV mice express and replicate HBV in the liver under the control of HBc promoter/enhancer-II. The two independent transgenic HBSP1 and HBSP2 mice strains carry HBSP gene (333 bp) encoding for the HBSP protein under the control of the HBx promoter/enhancer I (nt 832/1371) [START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-alpha-stimulated signaling pathways and limits the extent of liver inflammation[END_REF]. A rabbit β-globin intron was inserted between the HBx promoter/enhancer-I sequence and HBSP gene (genotype A). HBSP1 and HBSP2 transgenic mice were generated and expanded by back-crossing against the C57BL/6J strain (>12 back-crossings). C57BL/6J invalidated for CCR2 gene expression (CCR2KO) mice were also used [START_REF] Rodero | Immune surveillance of the lung by migrating tissue monocytes[END_REF]. All experiments were performed on 2-months old, heterozygous TgHBV, TgHBSP1, TgHBSP2 or homozygous CCR2 KO and corresponding littermate (WT) male mice. WT and TgHBV mice were treated for 7 weeks with carbon tetrachloride intraperitoneal injections (CCl 4 , 1ml/kg in oil, Sigma) twice a week to induce chronic liver fibrosis. Fulminant liver injury was generated by surgical bile duct ligation (BDL) using double sutures of bile duct near liver junction; WT and TgHBV mice were monitored for 2 days before sacrifice. Lipopolysaccharide intraperitoneal injections (LPS, 2.5mg/kg, Sigma) three times a week for 2 weeks were performed on WT and TgHBV mice to induce liver inflammation. TgHBSP, CCR2 KO and corresponding WT mice were treated twice a week with CCl 4 (1ml/kg in oil, Sigma) for 2 and/or 10 weeks. Acute liver injury was also performed in TgHBSP1 mice by a single intraperitoneal injection of CCl 4 (1ml/kg in oil). Thioacetamide intraperitoneal injections (TAA, 250mg/kg in PBS, Sigma) three times a week were performed on WT and TgHBSP1 mice for 2 weeks to induce liver fibrosis. TgHBV, TgHBSP and littermate WT were sacrificed 48 to 72 hours after the last injection. Mice livers were perfused with PBS-2% FCS and removed for analysis. Animals were maintained in pathogen-free conditions on 12-hour dark/light cycles and treated in accordance with EU regulations on animal care (Directive 86/609/EEC). All procedures were approved by the local animal care and use committee (Agreement A75-14-08; n° 02890 and 02891). Alanine aminotransferase (ALT) in serum was measured on a Cobas® 6000 analyzer (Roche), according to the manufacturer's instructions.

Human HBV infected liver samples

Patients were recruited from Mustapha Hospital (Alger, Algeria) and The Royal London Hospital (London, UK). Full ethical approval was obtained for the study; agreements by the National Health Ministry (according to legal article about clinical trials n°387 et 388 of 31/07/2006) and the Local Ethical Review Board at the Royal London Hospital. All patients recruited gave written informed consent. Liver samples, from biopsies surplus to diagnostic requirements, were obtained from 12 HBV-monoinfected patients. Patients were anti-HCV, anti-HDV and anti-HIV antibody negative and treatment naive. All patients had evidence of moderate liver damaged; scored by a pathologist according to the Metavir stage. ALT, HBeAg and HBsAg titres were determined using commercial assays (Roche, Biorad and Diasorin, respectively).

Additional methods are provided as supporting information.

Results

Increase in HBV alternate splicing upon liver injury

To assess whether liver injury impacts on HBV splicing regulation, TgHBV were treated with either CCl 4 (7 weeks/IP) or LPS (2 weeks/IP) to induce liver fibrosis or inflammation, respectively (FigS1 and Fig. S2). The amount of HBV pgRNA was reduced by a 5.3 fold and a 3.9 fold in LPS and CCl 4 treated TgHBV mice, respectively (Fig. 1A) as well as HBV viral load (FigS3). In contrast, HBV AS was significantly increased (Fig. 1A). Indeed, quantification of HBV pgRNA and SP1RNA-derived forms showed a significant increase of the SP1RNA proportion in both CCl 4 and LPS-treated TgHBV mice compared to control mice (oil-TgHBV vs CCl 4 -TgHBV: 32.5±9.6% vs 49.7±17.2%, p=0.04; PBS-TgHBV vs LPS-TgHBV: 27.6±8.7% vs 42.7±10.1%, p=0.01). Additionally, HBV AS upregulation was also observed in TgHBV mice with a BDL-induced fulminant hepatitis (control-TgHBV vs BDL-TgHBV: 30.6±4.9% vs 69.0±3.0%, p=0.001) (Fig. S4). These results may support an influence of liver damage on HBV post-transcriptional regulation.

Previous clinical studies suggested a correlation of HBV alternative splicing and the course of liver disease [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF] but the relevance of HBV pgRNA splicing to liver pathogenesis remains unknown. We therefore compared liver fibrosis or inflammation following CCl 4 or LPS treatment of TgHBV and their littermate wild-type (WT) control mice. Despite the well-known activities of HBV proteins on signaling pathways involved in inflammatory and fibrotic processes [START_REF] Zhao | Pathological functions of interleukin-22 in chronic liver inflammation and fibrosis with hepatitis B virus infection by promoting T helper 17 cell recruitment[END_REF], CCl 4 -treated TgHBV and WT mice displayed a comparable low stage of liver fibrosis (F2) on histological section (Fig. 1B). This was confirmed by quantification of collagen deposition and the expression of genes involved in liver fibrosis. Similarly, liver histology, staining for CD45+ cells and quantification of inflammation-inducible genes confirmed an equivalent degree of liver injury in LPS-treated WT and TgHBV mice (Fig. 1C and Fig. S5).

Regarding that several viral proteins may impact on liver disease, it was difficult to define the role of HBV AS and related HBSP expression in this experimental model. However, the lack of increase in CCl 4 -induced liver injury in TgHBV mice, expressing the complete HBV genome, contrasted with the exacerbations previously observed in transgenic mice expressing a subgenomic HBV sequence that is unable to express SP1RNA [START_REF] Chen | Increased susceptibility to liver injury in hepatitis B virus transgenic mice involves NKG2D-ligand interaction and natural killer cells[END_REF]. Considering the high level of SP1RNA in the liver of TgHBV mice, we therefore hypothesized that HBV AS, triggered by liver injury could limit the ensuing liver inflammation and fibrosis. To understand the role of SP1RNA during liver pathogenesis, we first investigated its regulation.

Identification of HBV alternate splicing factors

Characterization of pgRNA transregulatory splicing proteins remained a crucial prerequisite to investigate HBV AS regulation. Thus, HepG2 nuclear proteins interacting with HBV pgRNA were explored by RNA pull-down assay. Identification and relative quantification by Orbitrap mass-spectrometry showed in six independent experiments that 389 proteins were significantly associated with viral pgRNA (Fig. 2A and Table S1). Classification by gene ontology according to the biological functions allowed to distinct 5 groups representing 88% of associated-proteins (12% proteins remaining classified in group "other") (Fig. 2B). The most significant group was the "RNA processing" family, which was mainly composed of factors directly involved in RNA splicing (28/68 proteins; stained in Fig. 2C). Moreover, 30 additional proteins involved in RNA splicing and comprising hnRNPs proteins, were classified in further groups (Table S1). As illustrated, the proteins belonging to the "RNA processing" group have additional biological functions, which may contribute to the liver disease progression during infectious disease. Specific interaction of 9 transregulatory splicing factors, hnRNPAB, SF1, La, PSF and SRSF1,4,5,6,7 were confirmed in the pgRNA pull-down extract and not in controls (using antisense HBV pgRNA and empty beads) by western blot (Fig. 2D). The splicing factors SRSF2 and hnRNPH, absent or not significantly linked to pgRNA, were used as controls.

Splicing proteins are implicated in the regulation of HBV alternate splicing

The modulation of expression of HBV pgRNA interacting transregulatory splicing proteins (conserved inter-species) was investigated in liver extracts of TgHBV mice. Chemical induction of fibrosis led to an overexpression of PSF and SF1 proteins, whilst LPS-induced liver inflammation increased the expression of SRSF1 and La proteins (Fig. 3A). Despite the differential signature of splicing factors expression in both liver disease models, a comparable increase of SP1RNA proportion was observed. It was considered that AS regulation in eukaryotic cells has largely been driven by the alteration of cis-regulatory elements. These data provide additional evidence suggesting AS regulation and transregulatory proteins expression according to liver damage [START_REF] Fu | Context-dependent control of alternative splicing by RNA-binding proteins[END_REF].

Conservation of splicing factors in mammals led us to investigate their involvement on HBV AS in vitro. Once different human hepatoma cells were tested, impact of PSF, SRSF1 and La expression in viral AS regulation was investigated (Fig. 3 B-D). In Huh7 expressing HBV cells, La and PSF proteins increased SP1RNA proportion, whereas SRSF1 led to a decrease of viral splicing efficiency (Fig. 3E). This paradoxical decrease may be attributable to the fact that SRSF1 expression also considerably reduced HBV transcription, core and HBeAg expression (Fig. 3D), suggesting an impact on HBV RNA synthesis or metabolism. Taken together, these data emphasized for a possible influence of the microenvironment altered by liver injury on the expression of the HBV-regulatory splicing factors we had defined, driving regulation of the SP1RNA and consequently HBSP expression.

HBSP protein partially prevents CCl 4 -induced liver fibrosis

The impact of the HBV alternative splicing product HBSP on liver pathology was investigated in two transgenic mouse strains TgHBSP1&2 expressing the HBSP gene under the control of the weak hepato-specific HBx promoter (Fig. S6). The amount of HBSP RNA in TgHBSP mice was up to 10 fold lesser than SP1RNA expression in TgHBV mice (1.2x10 5 ±3.4x10 4 vs 1.2x10 6 ±9.9x10 5 copies/μg of liver RNAs, respectively) and remained stable with aging (FigS5) [START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-alpha-stimulated signaling pathways and limits the extent of liver inflammation[END_REF]. Since TgHBSP did not spontaneously develop liver pathogenesis, WT and TgHBSP mice were challenged for 2 weeks with CCl 4 or TAA to induce liver fibrosis (Fig. 4A, 4B, S6). Surprisingly, and despite a low fibrosis score (F0/F1) for all mice, TgHBSP mice treated with CCl 4 or TAA revealed a reduction in collagen deposition compared to their WT littermates.

To evaluate whether HBSP limited the extent of chemically-induced fibrogenesis and to investigate its biological impact on advanced stages of liver fibrosis, WT and TgHBSP1 mice were challenged with CCl 4 for 10 weeks (Fig. S6). As expected, all WT treated mice displayed severe fibrosis with periportal septa and developed cirrhosis (F4) (n=8/8) (Fig. 4C).

In contrast, TgHBSP1 treated-mice exhibited less marked fibrosis, with 75% animals having a F2 (n=2/8) and F3 (n=4/8) fibrosis score and only 25% reaching the F4 cirrhotic state (n=2/8). This observation was corroborated by a significant two-fold decrease in collagen deposition in TgHBSP1 liver tissue compared to WT mice upon CCl 4 treatment (Fig. 4C; 4.5±1.2% vs 8.4±1.2%, p=0.0002, respectively). The expression of pivotal fibrosis-related genes Col1a1, Desmin, Timp-1 and MMP-2 confirmed the lower sensitivity of HBSP mice to 10 weeks CCl 4 -induced fibrosis (Fig. 4D). In accordance with these results, α-SMA protein expressed by activated hepatic stellate cells (HSCs) was detected in a higher amount in the liver of WT compared to TgHBSP1 mice.

Recruitment of immune cells to the liver plays a central role in hepatic fibrogenesis [START_REF] Pellicoro | Liver fibrosis and repair: immune regulation of wound healing in a solid organ[END_REF]. To investigate whether this was altered by the expression of HBSP, intrahepatic leucocytes from 10 weeks CCl 4 -treated mice were isolated and characterized by flow cytometry. The CCl 4induced liver leucocyte infiltrate was significantly decreased in TgHBSP1 compared to WT mice (Fig. 5A). No significant difference in the intrahepatic upregulation of CD49b + NK, CD4 + and CD8 + T (±CD44 hi ) cells was observed between WT and TgHBSP1 mice treated with CCl 4 (Fig. 5B,5C). In contrast, the number and proportion of liver monocytes/ macrophages, defined as CD45 + F4/80 + CD11b + cells, were significantly reduced in TgHBSP1 compared to WT mice treated with CCl 4 (Fig. 5D; 2.6±0.5x10 5 vs 4.7±0.5x10 5 cells, p=0.02 and 14.5±2.1% vs 21.2±1.8% of CD45 + , p=0.02, respectively) (Fig. 5D). In addition, CCl 4 -challenged WT mice exhibited a higher periportal infiltrate of F4/80+ monocytes/macrophages than TgHBSP1 treated mice on stained liver sections (Fig. 5E).

Monocytes/macrophages contribute to liver fibrosis through the secretion of inflammatory cytokines, which directly activate HSCs [START_REF] Marra | Roles for chemokines in liver disease[END_REF]. In line with this, we confirmed that upon CCl 4induced chronic fibrosis, TgHBSP1 displayed a less pro-fibrotic inflammatory environment than WT mice, with a significant decrease in TGF-β, TNF-α and IL-1β genes expression (Fig. 5F) without differential distribution of monocyte/macrophage ontogeny markers CD163 and Marco (Fig. S8) in WT and TgHBSP mice [START_REF] Beattie | Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions[END_REF]. We next investigated the postulate that the decrease in monocyte/macrophage infiltrate in the HBSP-expressing liver could result from the impaired production of a chemokine involved in monocyte/macrophage survival and/or recruitment within the liver [START_REF] Karlmark | The fractalkine receptor CX(3)CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes[END_REF]. CCL1, CCL2, CCL3, CXCL10 and CX3CL1 transcripts were quantified and only CXCL10 and CCL2 were significantly reduced in the liver of TgHBSP1 compared to WT treated mice (Fig. 5F). Together, these results show that HBSP expression restricts partially chronic induced liver fibrosis.

HBSP impairs the monocyte/macrophage recruitment during liver injury

One explanation for the limited liver pathogenesis observed in TgHBSP1 mice could be a resistance of HBSP-expressing hepatocytes to CCl 4 -induced cytotoxicity. To investigate this possibility, WT and TgHBSP1 mice were treated with a single CCl 4 -injection to induce acute liver injury. In the context of acute hepatitis, TgHBSP1 and WT mice displayed comparable degrees of liver injury, with a similar increase in circulating ALT and equivalent central and periportal necrosis (Fig. 6A). The metabolism of CCl 4 in hepatocytes leads to reactive oxygen species (ROS) production and hepatocyte necrosis through lipid peroxidation accumulation. Consistent with this, ROS content assessment on liver extracts revealed a similar enhancement in TgHBSP1 and WT treated mice (Fig. 6B). In mouse strains, 48h after a single CCl 4 injection, liver apoptosis was rarely observed compared to necrosis and neither Desmin transcripts nor α-SMA protein expression were up-regulated, suggesting that HSCs were not yet activated (Fig. S9). Taken together, these results did not support a direct impact of HBSP expression on CCl 4 hepatocyte toxicity as an explanation for the reduced susceptibility of TgHBSP1 mice to liver damage in the chronic exposure model.

Instead we hypothesised that HBSP expression directly down-regulated hepatocyte chemokines content and thus monocyte/macrophage recruitment, accounting for the relative resistance to liver fibrosis in TgHBSP1 treated mice. Therefore, monocyte/macrophage recruitment was investigated by flow-cytometry in the acute CCl 4 hepatitis model. As previously observed during chronic exposure, the influx of intrahepatic leucocytes and monocytes/macrophages was significantly reduced in HBSP-expressing compared to WTmice with acute liver injury (Fig. 6C). The monocytes/macrophages decreased in TgHBSP1 mice included the inflammatory Ly6C hi CCR2 + subpopulation (Fig. 6C right panel: CCl 4 -WT vs CCl 4 -TgHBSP1: 8.8±0.1x10 5 vs 2.9±0.9x10 5 cells, p=0.003 and 13.8±2.3% vs 6.9±1.1% of CD45 + cells, p=0.01, respectively), recruited through a CCL2-dependent mechanism under CCl 4 treatment [START_REF] Baeck | Pharmacological inhibition of the chemokine C-C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly-6C(+) macrophage infiltration in mice[END_REF]. The diminished recruitment of monocytes/ macrophages in TgHBSP1 acute liver damage was only coupled to a decrease in CCL2 transcripts (Fig. 6D) and to a down-regulation of CCL2 protein in the liver (Fig. 6E). Reduced infiltration of monocytes/macrophages, known to be a major source of TNF-α [START_REF] Marra | Roles for chemokines in liver disease[END_REF], might also account for the decrease of TNF-α expression observed. The hepatic cells types producing CCL2 in the acute hepatitis model were further explored by immunofluorescence. CCL2 staining revealed a high signal around blood vessels and unexpectedly hepatocytes (counterstained by cytokeratin 18) were also clearly positive for CCL2 (Fig. 6F).

These data suggested that HBSP expression impacted on liver monocyte/macrophage recruitment through a down-regulation of hepatocyte CCL2 expression upon acute liver injury.

HBSP modulates CCL2 expression in hepatocytes

Our results suggested that CCL2 production by hepatocytes contributes to the recruitment of monocytes/macrophages and consequently to liver damage. Next, we investigated the ability of HBSP to control the expression of CCL2. In line with our previous results [START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-alpha-stimulated signaling pathways and limits the extent of liver inflammation[END_REF] we showed that p65 (RelA) phosphorylation was impaired in the liver of TgHBSP1 compared to WT mice challenged for 10 weeks with CCl 4 (Fig. 7A). This result suggested an impact of NF-κB activation on CCL2 expression. Then, we observed in HeLa expressing HBSP cells a reduced level of IκBα phosphorylation at 5 minutes after TNF-α, treatment (Fig. 7B, left panel), together with a significant reduction of CCL2 transcription level compared to control cells 30 minutes post-treatment with TNF-α (Fig. 7B, right panel). A significant decrease of CCL2 secretion was also displayed in the supernatant of HepaRG expressing HBSP cells treated by TNF-α (Fig. 7C left panel; lanes 6 vs 5, respectively: 24.9±3.7 vs 33.7±2.3 ng/ml; p=0.008). In order to further explore CCL2 modulation by HBSP in a whole HBV genome context, a siRNA targeting the SP1RNA encoding HBSP was designed. In HBV-transfected HepaRG, siSP1RNA efficiently knocked down SP1RNA (6-fold decrease compared to scramble siRNA) without cross-reactivity on pgRNA expression (Fig. 7C middle panel). Upon TNF-α stimulation, CCL2 secretion was significantly higher in HepaRG cells in which SP1RNA had been knocked down than in those transfected with a scramble siRNA (Fig. 7D, lanes 10 vs 9: 46.3±4.0 vs 31.1±1.6 ng/ml, p=0.008).

To assess whether the reduction of CCL2 expression, through NF-κB, in HBSP expressing cells may account for the weaker liver fibrogenesis observed in TgHBSP, CCR2 invalidated (CCR2KO) (n=5) and control (n=5) mice were treated with CCl 4 . After 2 weeks of treatment, picro-sirius staining and western blot analysis of α-SMA expression revealed less marked liver fibrosis in CCR2KO mice, highlighting the main influence of this chemokine on fibrogenesis (Fig. 7E).

CCL2 expression relies on SP1RNA in liver of HBV chronic infected patients

Several lines of evidence indicate that subjects with CHB have alterations in chemokines that may drive pathogenesis [START_REF] Ren | Immune characteristics of different immune phases in natural course of chronic HBV infection[END_REF][START_REF] Tan | A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B[END_REF]. However the contribution of CCL2 recruitment of monocytes/macrophages has not been addressed in this context. Only few studies showed no correlation of circulating CCL2 levels with HBV disease stage but without information on CCL2 hepatic expression [START_REF] Tan | A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B[END_REF][START_REF] Lian | Expression profiles of circulating cytokines, chemokines and immune cells in patients with hepatitis B virus infection[END_REF]. Furthermore, although HBV splicing regulation was previously suggested during liver disease [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF][START_REF] Soussan | Expression of defective hepatitis B virus particles derived from singly spliced RNA is related to liver disease[END_REF][START_REF] Soussan | The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis[END_REF], the contribution of splicing factors expression was never considered. Therefore we investigated splicing factors and CCL2 expression in liver tissue surplus to diagnostic requirements available from 12 HBV monoinfected treatment-naive patients who were either undetectable or positive for HBV SP1RNA (Table S2). All had low-mild liver fibrosis (F0:F1:F2 fibrosis score in SP1RNA + n=1:4:1, in SP1RNA -n=0:6:0) with a high level of HBV RNA expression (SP1RNA + vs SP1RNA -groups: 1.3x10 7 ±5.3x10 6 vs 3.3x10 6 ±4.6x10 6 copies of HBV RNA/µg of liver RNA) (Fig. 8A). For 3/6 patients from each group, liver sample size allowed the additional study of the expression of transregulatory splicing proteins. Despite exhibiting a similar stage of liver disease, SP1RNA positive compared to negative liver samples displayed a higher expression of hnRNPAB together with La and SRSF1 splicing factors (Fig. 8B), which were also up-regulated in LPS treated HBV transgenic mice. In SP1RNA negative liver samples, La protein expression was mainly observed by immunohistochemistry in nucleus while it was increased and gathered in both nuclear and cytoplasmic compartments in SP1RNA positive liver samples (Fig. 8C), highlighting the shuttling of La protein [START_REF] Bayfield | Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)[END_REF]. Regarding CCL2 expression on patient liver sections, a differential staining was noticed with an increase in SP1RNA negative hepatocytes (Fig. 7D). The level of intrahepatic CCL2 transcripts was further quantified and SP1RNA positive liver samples displayed a 14 fold lower CCL2 expression compared to SP1RNA negative tissues (Fig. 8D; 0.13±0.04 vs 1.87±0.80, p=0.01, respectively).

In conclusion, enhancement of transregulatory splicing proteins expression and HBV pgRNA splicing in the liver of patients with CHB was associated with a reduction in CCL2 expression, as previously observed in transgenic mice and in vitro. Our findings support the critical role of infected hepatocyte through chemokine production in liver disease and suggest a novel feedback loop regulation displayed by HBV to counteract liver pathogenesis.

Discussion

Alternate splicing regulation of HBV transcripts was first described more than 20 years ago but its role in HBV disease has remained poorly defined [START_REF] Susuki | Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome[END_REF][START_REF] Wu | Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected Hep G2 cells[END_REF]. The proportion of dHBV has been shown to fluctuate considerably in chronic carriers and to be increased with the severity of liver pathogenesis [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF]. However the regulation of HBV AS and its impact on liver immunopathogenesis had not been investigated.

To decipher the mechanism of post-transcriptional regulation of HBV pgRNA, hepatic disease was induced in a HBV transgenic mouse model. In contrast to HBV infection models, transgenic mice present the benefice of a stable viral genome variability eliminating any potential interference of HBV cis-regulation on pgRNA/protein interactions. This model is sufficient to decipher the influence of liver damage on the viral post-transcriptional regulation even if the relevance of data obtained on liver pathogenesis is limited due to the transgene inducing HBV tolerance and to the constant amount of viral transcripts (including SP1RNA) and proteins. Interestingly, we showed that chemical liver damage led to a significant increase of HBV AS, suggesting an influence of the microenvironment on the splicing machinery. Indeed, the expression of transregulatory splicing proteins interacting with HBV pgRNA was affected by liver injuries in mice but also in liver of chronic HBV infected patients (Fig. 3/8). Transcriptomic analysis from liver diseases samples including hepatocarcinoma have previously suggested the role of such dysregulations of AS factors [START_REF] Chettouh | Mitogenic insulin receptor-A is overexpressed in human hepatocellular carcinoma due to EGFR-mediated dysregulation of RNA splicing factors[END_REF], here we reported an impact on HBV AS in vitro, in TgHBV mice and in CHB liver patients. Whilst the modulation of transregulatory splicing protein expression differed in liver inflammation and fibrosis in TgHBV treated mice, both liver damages triggered the same activation of viral AS. Characterization of molecular pathways [START_REF] Mallinjoud | Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin[END_REF] involved in the control of trans-regulatory splicing proteins and consequences on genome expression of hepatocyte during liver injury will be important to investigate in further clinical studies.

The main impact of AS regulation is an enhancement of SP1RNA, which encodes HBSP. Unexpectedly, HBSP expression in transgenic mice reduced the liver fibrosis induced by chemical treatment. Our results showed that HBSP expression in hepatocytes of treated mice downregulated their CCL2 expression and consequently reduced the recruitment of monocytes/macrophages. However, an effect on T cells could not be excluded considering trends observed in cytometry and cytokines expression data. These tendencies may result either from a domino effect on monocyte/macrophage recruitment or from an independent direct activity of HBSP on the adaptative immune system. Nevertheless, the direct impact of HBSP on hepatocyte CCL2 expression was confirmed in cells expressing HBSP, as well as in a whole HBV genome context after SP1RNA knockdown. As previously reported [START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-alpha-stimulated signaling pathways and limits the extent of liver inflammation[END_REF], once activated by inflammation, the NF-kB signaling pathway is partially hijacked by HBSP and as observed in vitro, may account for the downregulation of CCL2 expression. However, TgHBV displayed a similar fibrotic disease to WT mice, despite a non-significant 1.4 fold decrease of CCL2 expression (data not shown) and enhancement of viral AS. In parallel, CCL2-downregulation was found in SP1RNA positive liver patients in HBV chronic carriers with comparable liver injury. These data suggest the capacity of HBSP protein to antagonize liver fibrogenesis in a dynamic regulation of immune-mediated liver damage during the course of HBV infection.

Chemokines and their receptors orchestrate the chronology of immune responses during liver injury. Among these chemokines, CCL2 plays a central role in the hepatic recruitment of activated inflammatory monocytes/macrophages expressing Ly6C hi CCR2 + markers [START_REF] Marra | Roles for chemokines in liver disease[END_REF][START_REF] Baeck | Pharmacological inhibition of the chemokine C-C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly-6C(+) macrophage infiltration in mice[END_REF][START_REF] Dambach | Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse[END_REF]. Damaged hepatocytes have previously been suggested to be able to produce CCL2 [START_REF] Saiman | The role of chemokines in acute liver injury[END_REF][START_REF] Ziraldo | Central role for MCP-1/CCL2 in injury-induced inflammation revealed by in vitro, in silico, and clinical studies[END_REF] but were considered a minor source compared to activated HSCs and immune cells [START_REF] Saiman | The role of chemokines in acute liver injury[END_REF][START_REF] Ait-Goughoulte | Innate antiviral immune responses to hepatitis B virus[END_REF]. Our data highlight the significant contribution of CCL2 produced by hepatocytes in liver damage, since CCL2 downregulation in TgHBSP mice had a direct impact on liver pathogenesis. Therefore, reduced monocyte/macrophage infiltrates may account for the differential expression of cytokines and also for the recruitment of other immune cells in 10 weeks CCl 4 -treated mice, which seemed also partially affected, compared to control mice. Nevertheless, the main role of CCL2 downregulation was supported by the data obtained from the single CCl 4 -injection in TgHBSP mice as well as the similar impairment of liver fibrosis to those observed in CCR2KO treated mice, although altered immune composition at baseline may limit conclusion for this last model. Influence of monocytes/macrophages during HBV pathogenesis has been poorly explored even if the modulation of noncytolytic antiviral activity via their cytokine secretion was reported [START_REF] Boltjes | The role of Kupffer cells in hepatitis B and hepatitis C virus infections[END_REF][START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF][START_REF] Hosel | Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection[END_REF]. Additionally, monocytes/macrophages could participate in HBV specific adaptive immunity through the formation of intrahepatic myeloid-cell aggregates enabling local CD8 + T cells expansion (iMATEs) [START_REF] Huang | Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection[END_REF][START_REF] Nakamoto | Role of toll-like receptors in immune activation and tolerance in the liver[END_REF]. Thus, by countering monocyte/macrophage recruitment, HBSP might support the maintenance of a tolerogenic environment and favour immune escape [START_REF] Heymann | Liver inflammation abrogates immunological tolerance induced by Kupffer cells[END_REF]. Furthermore, the relationship between CCL2 and human HCC survival emphasized the clinical observation of HBV splicing enhancement prior to development of HCC and speculate about the role of HBSP in liver disease progression [START_REF] Li | Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy against hepatocellular carcinoma. Gut. 2015 Oct 9. Author names in bold designate shared co-first authorship CD11b + ) from WT and TgHBSP1 CCl 4 -treated mice (right). (E) Representative monocytes/ macrophages F4/80 staining on histological liver section (bar 250μm). (F) Cytokines (upper panel) and chemokines (lower panel) mRNA expression in the liver. Each dot represents a mouse; experiments performed on 10 weeks oil-WT (n=6), oil-TgHBSP1 (n=6)[END_REF].

Strengthened by previous clinical data reporting an increase of HBV defective circulating particles related to SP1RNA with liver disease severity [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF][START_REF] Redelsperger | Production of hepatitis B defective particles is dependent on liver status[END_REF][START_REF] Soussan | Expression of defective hepatitis B virus particles derived from singly spliced RNA is related to liver disease[END_REF], our present study suggests a new feedback loop involving HBV AS to counteract liver injury. As proposed in Figure 8E, the hepatic microenvironment altered by liver damage may contribute to enhance splicing factors involved in HBV SP1RNA production, and thus HBSP expression. Hepatocytes expressing HBSP exhibit reduced CCL2 secretion and consequently attenuated monocyte/ macrophage recruitment, thereby constraining inflammation. Although speculative at this stage, it is possible that hepatocytes expressing HBV SP1RNA are preferentially maintained by inhibition of local antiviral immunity, constituting a novel mechanism for viral persistence during chronic liver injury. To date, whether efficient viral suppression by antiviral therapy is accessible for the management of HBV infection, eradication of intracellular virus remains a challenge. CCL2 targeted immunotherapy in combination with nucleoside analog should contribute to facilitate an efficient HBV cure through a disruption of immune tolerance established during viral infection.

AS

  Fig figureROSreactive oxygen species

Fig. 1 .

 1 Fig.1. Liver injury increases HBV pgRNA alternate splicing in HBV transgenic mice (A) HBV pgRNA and SP1RNA expression (left) and SP1RNA proportion (right) quantified in the liver of TgHBV mice treated with oil/CCl 4 for 7 weeks or with PBS/LPS for 2 weeks. (B) Representative H&E and Picro-Sirius red stained histological liver section (left, bar 250μm) and quantification of collagen deposition (right) from 7 weeks oil, CCl 4 -WT and CCl 4 -TgHBV treated mice (upper panel). Fibrosis-related mRNA expression in the liver, each dot represents a mouse, experiments were performed on oil-WT (n=7), oil-TgHBV (n=7), CCl 4 -WT (n=9) and CCl 4 -TgHBV (n=8) mice (lower panel). C) Representative H&E

Fig. 2 .

 2 Fig. 2. Identification of cellular splicing factors interacting with HBV pgRNA(A) Volcano plot representation of HBV pgRNA-interacting proteins (700/1442) detected in 6 independent RNA pull-down assays (beads used as control) and identified by mass spectrometry (nanoRSLC-LTQ Orbitrap Velos). Proteins showing a significant interaction with HBV pgRNA (n=389/700) are depicted uppers the black line according to significance analysis. X-axis shows the difference of the average of the logarithm of Label Free Quantification (LQF) intensities between pgRNA and control; y-axis shows the negative logarithmic of t-test Welsh p-value. (B) Biological function groups of HBV pgRNA binding

Fig. 3 .

 3 Fig. 3. SP1RNA regulation is dependent on splicing-proteins expression (A) Splicing regulatory proteins expression in the liver of TgHBV mice treated with oil/CCl 4 for 7 weeks or with PBS/LPS for 2 weeks (SRSF2 and hnRNPH1 used as controls). (B) SP1RNA proportion (SP1RNA/(SP1RNA+pgRNA)x100) in Huh7, HepG2 and HepaRG cells expressing HBV after transient transfection of pHBV vector. (C) Huh7 co-tranfected cells with pHBV and PSF-GFP or SRSF1-GFP; PSF-GFP and SRSF1-GFP visualization and nucleus were counter stained with Hoescht (middle, bar 50μm); (D) pgRNA and SP1RNA quantification in HBV expressing Huh7 cells co-transfected with Neo (control), PSF, La or

Fig. 4 .

 4 Fig.4. HBSP expression protects against chemical-induced liver fibrosis Representative H&E and Picro-Sirius red stained histological liver section (bar 250μm) (left) and quantification of collagen deposition (right) from: (A) WT, TgHBSP1 and TgHBSP2 mice treated for 2 weeks with Oil (n=4), CCl 4 -WT (n=11), CCl 4 -TgHBSP1 (n=7) and CCl 4 -TgHBSP2 (n=7) mice. (B) WT and TgHBSP1 mice treated for 2 weeks with PBS (n=4 for both) or TAA-WT (n=7) and TAA-TgHBSP1 (n=8) mice. (C) WT and TgHBSP1 mice treated for 10 weeks with oil (n=6 for both WT and TgHBSP1) or CCl 4 (n=8 for both WT and TgHBSP1). (D) Liver fibrosis-related gene mRNA and α-SMA protein expression in the

Fig. 5 .Fig. 6 .

 56 Fig.5. HBSP expression reduces intrahepatic monocytes/macrophages infiltrate WT and TgHBSP1 mice were treated for 10 weeks with oil or CCl 4 . Intrahepatic leucocytes were isolated and analysed by flow cytometry. (A) Intrahepatic leucocyte cells number (left) and representative gating of CD4 + and CD8 + T cells (right). (B) CD4 + T (7-AAD -, CD3 + ,TCRβ + ,CD4 + ) and CD44 hi effector/memory CD4 + T cells number. CD8 + T (7-AAD -, CD3 + ,TCRβ + ,CD8 + ) and CD44 hi effector/memory CD8 + T cells number (left). (C) NK (7-AAD -,CD49b + ,CD3 -) cells number and representative gating. (D) Intrahepatic monocytes/ macrophage cells number, proportion and representative gating of (7-AAD, CD45 + , F4/80 + ,

Fig. 7 .

 7 Fig.7. Association between SP1RNA, HBSP and CCL2 expression in vitro (A, left panel) Western blot detection of p65 (RelA) total and its phosphorylated form (P-p65) on Ser536 in liver of TgHBSP and control mice treated by vehicle or CCl 4 during 10 weeks. (A, right panel) Quantification of P-p65 related expression in liver of TgHBSP and control mice treated by vehicle (n=4), by CCl 4 during 10 weeks in control (n=3) and TgHBSP (n=3) mice. (B, left panel) Total IκBα and phosphorylation level in HeLa cells transfected with pNeo or pHBSP plasmids and treated with TNF-α (25ng/ml) for 0, 5, 15 minutes. (B, right panel) Relative CCL2 mRNA quantification (normalized with GAPDH)

Fig. 8 .

 8 Fig.8. Association between SP1RNA, CCL2 and transregulatory splicing factors in HBV chronic carriers(A) HBV RNA and SP1RNA quantification in the liver of HBV chronic carriers (n=6 for undetectable (Und.) and n=6 for detectable SP1RNA groups). (B) Splicing factors expression in the liver of HBV chronic carriers with undetectable or detectable SP1RNA. (C) La and CCL2 immunochemistry on serial liver section counterstained with hematoxylin (upper and lower bar 50μm) from chronic HBV carrier with undetectable or detectable SP1RNA. (D) CCL2 mRNA quantification in the liver of HBV chronic carriers with undetectable (n=6) or detectable SP1RNA (n=6). (E) Schematic model for SP1RNA

J Hepatol. Author manuscript; available in PMC 2019March 25.

Acknowledgments

We thank N. Stadler, C. Cordier, B. Kane and F. Marliot for technical assistance; and Drs D. Auboeuf, C. Bourgeois, T. Heise, O. Ann for providing SRSF1 and PSF plasmids. We thank Drs ML. Michel, S. Briechler, H. Strick-Marchand, F. Pagès, D. Scott-Algara, A. Coulomb, J. Pol and T. Capiod for helpful scientific discussion. We thank animal facilities platform from Pitié and Broussais Universities. We thank FACS, proteomic and histological platforms of Broussais institute.

Financial support: This work was supported by grants from INSERM, UPMC, ANRS (n° N14015DR) and PHC-Tassili (11MDU826). MD was supported by ANRS (grant ASA14013DRA). YM was supported by French Ministry for Higher Education and Research and by the Ligue contre le Cancer (grant n° GB/MA/VSP-10504).

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Abbreviations

SR

serine-arginine rich protein hnRNP heterogeneous nuclear ribonucleoprotein protein

HBV

Hepatitis B Virus


Marwa Dhiabi

Ali Bouattour

Mehdi Fakhfakh

Souhir Abid

Ludovic Paquin

Thomas Robert

Stephane Bach

Jean-Pierre Bazureau
email: jean-pierre.bazureau@univ-rennes1.fr

Houcine Ammar

Practical approach to N-benzyl derivatives of 2-amino-8-methoxy-4H-chromene-3-carbonitrile by reductive amination: exploration of their effects against protein kinases and in silico ADME profiling

Keywords: 2-amino-4H-chromene, reductive amination, N-benzylation, Lipinski's descriptors, protein kinase, Pim1 inhibitor, Haspin inhibitor

The synthesis of 2-benzylamino-8-methoxy-4H-chromene-3-carbonitriles 7(a-e) has been realized in four steps, via reductive amination from the 2-amino-8-methoxy-4H-chromene-3-carbonitrile 4 as key intermediate platform with para-and meta-substituted benzaldehydes 5(a-e), in good overall yields. The physicochemical properties of 7(a-e) and also their aldimines 6(a-e) precursors have been determined using the Swiss ADME server platform according to the Lipinski's descriptors. Biological assays with a panel of six protein kinases such as HsCDK5-p25, HsCDK9/cyclin T, HsPim1, HsHaspin, SscGSK-3a/ and SscCK1/ showed that aldimines 6(a,b) and 6e are potentially interesting because they showed good percentage of residual activities against HsPim1 and HsHaspin.

Introduction

Chromenes and benzochromenes are notorious because many derivatives such as 2-amino-4Hchromene-3-carbonitriles (or their 3-carboesters analogues) exhibits a wide range of biological and pharmacological activities [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] . For examples in Figure 1, EPC 2407 (I) or crolibulin™ was identified in clinical phase I/II trials as drug-candidate 2 for the treatment of advanced solid tumors on anaplastic thyroid cancer (ATC). During the randomized phase II trial (NTC 01240590) [START_REF] Gulley | focus on anaplastic thyroid cancer (ATC)[END_REF] , the antitumoral activity of crolibulin™ plus cisplatin was compared to cisplatin alone on 40 enrolled patients for treatment of ATC. The 7dimethylamino derivative of crolibulin™ or MX5815 (II) [START_REF] Kasibhatla | Discovery and mechanism of action of a novel series of apoptosis inducers with potential vascular targeting activity[END_REF] was evaluated as apoptosis inducer (EC 50 50 nM) and inhibited cell growth of TD47D breast cancer cells [START_REF] Gourdeau | Anti-vascular and antitumor evaluation of 2-amino-4-(bromo-4,5-dimethoxyphenyl)-3-cyano-4H-chromenes, a novel series of anticancer agents[END_REF] with a GI of 37 nM. Among the 2-amino-4Hchromene derivatives, HA14-1 (III) was identified as a small non-peptidic ligand of Bcl-2 protein [START_REF] Manero | The small organic compound HA14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death[END_REF] using a de novo computer-aided design strategy based on the predicted structure of Bcl-2 protein [START_REF] Doshi | Structure activity relationship studies of ethyl-2-amino-6-bromo-4-(1cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA 14-1), an antagonist for antiapoptotic Bcl-2 proteins to overcome drug resistance in cancer[END_REF] . The diester derivative SV30 (IV) which is an analogue of pro-apoptotic molecule HA14-1 (III) was associated to lipid nanocapsules (LNCs) on F98 cells to improve its biological activities [START_REF] Weyland | Mitochondrial targeting by use of lipid nanocapsules loaded with SV30, an analogue of the small-molecule Bcl-2 inhibitor HA 14-1[END_REF] . For the naphtopyran derivative LY 290161 (V) [START_REF] Wood | Inhibition of mitosis and microtubule function through direct tubulin binding by a novel antiproliferative naphthopyran LY290181[END_REF] , it showed antiproliferative activity on microtubulin binding, particularly in the G2/M phase of the cell cycle [START_REF] Panda | Suppression of microtubule dynamics by LY290181[END_REF] .

Figure 1: Bioactive 2-amino-4H-chromene derivatives reported in literature

Due to the potential pharmacological interest of the 2-amino-4H-chromene-3-carbonitrile platform (Figure 1), we were motived in our laboratory to explore the chemical reactivities of the carbonitrile function (electrophilic center) and the 2-amino group (nucleophilic center) for the construction of new heterocycle derivatives in combination to exploration of biological activities, such as their antiproliferative activities on tumor cell lines or inhibition activities of protein kinases (PKs) in a preliminary approach. In previous works, we showed that this platform (Figure 2) offers the possibilities to built tricyclic compounds (A) [START_REF] Bouattour | Microwave assisted a practical synthesis of 4-imino-3-phenyl-3,4-dihydro-1Hchromeno[2,3-d]pyrimidine-2(5H)-thione derivatives and exploration of their biological activities[END_REF] , amidines (B) [START_REF] Bouattour | An efficient and concise syntheses of N-3-substituted 9-methoxy-4H-[1]-benzopyrano[2,3-d]pyrimidine-4(5H)-imine and formamidine derivatives from methyl N-(3-cyano-8-methoxy-4H-[1]-benzopyran-2-yl)methanimidate[END_REF] and N-aryl derivatives (C) by trans-imination of 2-imino-precursors [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] . In continuation of these works, we decided to explore now the construction of N-benzyl derivatives (D) of 2amino-4H-chromene-3-carbonitrile, which are potentially the mono N-alkylated compounds of this platform using various benzyl halides. In silico analysis of the physicochemical properties (log P, partition coefficient; log S, water solubility; tPSA, topological polar surface aera; HBA and HBD, hydrogen bond acceptor and donor) will be also realized. Evaluation of their protein kinases (PKs) inhibition activities will be also evaluated in order to detect a potential PK inhibitor.

Material and methods

Chemistry

General information:

All the required chemical reagents and solvents for this work were purchased from Merck Sigma-Aldrich, Acros Organics Thermo Fisher Scientific France, VWR France and were used without further purification. Melting points were determined on a Kofler melting point apparatus and were uncorrected. Thin-layer chromatography (TLC) was accomplished on 0.2-mm precoated plates of silica gel 60 F-254 (Merck) and visualization was made with ultraviolet light (254 and 312 nm). Solvents were evaporated with a BUCHI rotary evaporator. Infrared (IR) spectra were recorded on a Jasco FT-IR 420 spectrophotometer apparatus using KBr pellets. [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR spectra were recorded on BRUKER AC 300 Avance III (300 MHz) spectrometer, [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR spectra on BRUKER AC 300 Avance III (75 MHz) spectrometer using respectively CDCl 3 or DMSO-d 6 with tetramethylsilane as internal reference. Chemical shifts are expressed in parts per million downfield (ppm). Data are given in the following order: δ value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), and number of protons, and coupling constants J is given in Hertz. The high-resolution mass spectra (HRMS) were enregistered in positive mode using a direct electrospray infusion (ESI), respectively on a Waters Q-Tof 2 and on a Thermo Fisher Scientific Q-Exactive spectrometers at the CRMPO platform of ScanMAT UAR 2025 CNRS (Bât. 11, Campus Beaulieu, Rennes, France). Elemental analyses (C, H and N) of 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e) were performed on a Flash Microanalyzer EA1112 CHNS/O Thermo Electron also in the CRMPO platform.

Copies of the spectra data including IR, 1 H-NMR, [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C-NMR, spectra of compounds 6(a-e) and 7(a-e) are provided in Supplementary Material.

2-Imino-8-methoxy-2H-[1]-benzopyran-3-carbonitrile (3)

To a stirred solution (550 rpm) at room temperature of 2-hydroxy-3-methoxybenzaldehyde 1 (1.52 g, 10 mmol) in absolute ethanol (20 mL) was added dropwise commercial propanedinitrile 2 (660.2 mg, 10 mmol) during 15 min in the presence of a catalytic amount of piperidine 0.5% mol. (425 mg, 5 mmol). Vigorous stirring was pursued during 4 hours at room temperature. Then, the reaction mixture was concentrated in a rotary evaporator under reduced pressure for the elimination of volatile, the desired product 3 was collected by filtration on a Büchner funnel (porosity N°4) and rinsed with absolute ethanol (3 x 10 ml). The 2-imino-8-methoxy-2H-[1]-benzopyran-3-carbonitrile 3 was dried under a high vacuum (10 -2 Torr) at 25°C for 1 hour to give 1.7 g of a yellowish powder (85% yield) and was further used without purification. Mp = 173-175°C. [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (DMSO-d 6 ) δ : 3.87 (s, 3H, OCH 3 ); 7.20-7.24 (m, 3H, H-5, H-6, H-7, Ar); 8.34 (s, 1H, H-4, Ar); 8.92 (br s, 1H, NH).

2-Amino-8-methoxy-4H-chromene-3-carbonitrile (4)

To a stirred and cooled suspension (550 rpm, 0°C) of 2-imino-8-methoxy-2H- [1]benzopyran-3carbonitrile 3 (2,4 g, 12 mmol) in 15 mL of methanol was added portionwise a commercial sodium borohydride 98% (232 mg, 6 mmol). Stirring at 0°C was pursued during 45 min. After stirring, the mixture was poured in 20 mL of deionized water at room temperature without stirring to improve decantation and precipitation. The resulting precipitate was collected by filtration on a Büchner funnel (porosity N°4) and washed with deionized water (3x 10 mL). The desired 2-amino-8-methoxy-4H-chromene-3-carbonitrile 4 was dried under high vacuum (10 -2 Torr) at 25°C for 1 h that gave a yellowish powder (2.303 g, 95% yield) and was further used without purification. Mp = 152-154 °C. IR (KBr, ν, cm -1 ): 1661 (C=C), 2186 (CN), 3332-3456 (NH 2 ). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (DMSO-d 6 ) δ: 3.43 (s, 2H, CH 2 ); 3.79 (s, 3H, MeO); 6.72 (d, 1H, J = 6.2 Hz, H-7, Ar); 6.92 (d, 1H, J 9.2 Hz, H-5, Ar); 7.03 (t, 1H, J = 9.2 Hz, H-6, Ar); 6.82 (br s, 2H, NH 2 ); [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

General procedure for the synthesis of 2-benzylidenamino-8-methoxy-4H-chromene-3-carbonitrile 6(a-e):

In a 50 mL two-necked round-bottomed flask, provided with magnetic stirrer and condenser, containing a solution of 2-amino-8-methoxy-4H-chromene-3-carbonitrile 4 (202 mg, 1 mmol) in 10 ml of absolute ethanol, was added dropwise a solution of various aromatic aldehydes (1 mol) in 5 ml of absolute ethanol in the presence of an equimolar quantity of piperidine (85 mg, 1 mmol) during 15 min. After complete addition, the reaction mixture was refluxed during 5 hours. After cooling down to room temperature, the precipitated material was collected by filtration on a Büchner funnel (porosity N°4), washed with cooled absolute ethanol (2 x 5 mL) and recrystallized from EtOH. Drying under high vacuum (10 -2 Torr) at 25°C for 1 h afforded the desired 2-benzylidenamino-8-methoxy-4H-chromene-3-carbonitrile 6 as a powder and was analyzed by 1 H, [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR and HRMS.

2-[(E)-(Phenylmethylene)amino]-8-methoxy-4H-chromene-3-carbonitrile (6a)

Compound 6a was prepared in 70% yield (203 mg) from benzaldehyde 5a (106 mg, 1 mmol) according to the standard procedure which gave 6a as yellowish powder, mp = 206-208 °C. IR (KBr, ν, cm -1 ): 1640 (C=N), 2200 (C≡N). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF]

2-[[(E)-(4-Dimethylaminophenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile (6b)

Compound 6b was prepared in 52% yield (189 mg) from 4-dimethylaminobenzaldehyde 5b (149 mg, 1 mmol) according to the standard procedure which gave 6b as yellowish powder, mp = 238-240 °C. IR (KBr, ν, cm -1 ): 1638 (C=N), 2195 (C≡N); 1 H-NMR (CDCl 3 ) δ : 3.11 (s, 6H, 2-CH 3 ); 3.79 (s, 2H, CH 2 ); 3.93 (s, 3H, MeO); 6.72 (d, 3H, J = 8.9 Hz, H-2', H-4',H-7, Ar); 6.84 (d, 1H, J = 7.6 Hz, H-5, Ar); 7.05 (t, 1H, J = 7.9 Hz, H-6, Ar); 7.91 (d, 2H, J = 8.7Hz, H-1', H-5', Ar); 8.81 (s, 1H, CH=N). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

2-[[(E)-(4-Methoxyphenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile (6c)

Compound 6c was prepared in 90% yield (288 mg) from 4-methoxybenzaldehyde 5c (136 mg, 1 mmol) according to the standard procedure which gave 6c as yellowish powder, mp = 204-206 °C. IR (KBr, ν, cm -1 ): 1645 (C=N), 2201 (C≡N). 1 H-NMR (CDCl 3 ) δ : 3.82 (s, 2H, CH 2 ); 3.91 (s, 3H, MeO); 3.94 (s, 3H, MeO); 6.73 (d, 1H, J = 7.7 Hz, H-7, Ar);6.85 (d, 1H, J = 8 Hz, H-5, Ar); 7.00 (d, 2H, J = 8.7 Hz, H-2', H-4', Ar); 7.07 (t, 1H, J = 8.0 Hz, H-6, Ar); 8.01 (d, 2H, J = 8.7 Hz, H-1', H-5', Ar); 8.88 (s, 1H, CH=N). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

2-[[(E)-(4-Chlorophenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile (6d)

Compound 6d was prepared in 80% yield (260 mg) from 4-chlorobenzaldehyde 5d (141 mg, 1 mmol) according to the standard procedure which gave 6d as yellowish powder, mp = 198-200 °C. IR (KBr, ν, cm -1 ): 1615(C=N), 2205 (C≡N). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H-NMR (CDCl 3 ) δ : 3.83 (s, 2H, CH 2 ); 3.93(s, 3H, MeO); 6.72 (d, 1H, J = 6.9 Hz, H-7, Ar); 6.85 (d, 1H, J = 7.5 Hz, H-5, Ar); 7.08 (t, 1H, J = 8.0 Hz, H-6, Ar); 7.47 (d, 2H, J = 8.5 Hz, H-2', H-4', Ar); 7.98 (d, 2H, J = 8.5 Hz , H-1', H-5', Ar); 8.89 (s, 1H, CH=N). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

2-[[(E)-(3-Methoxyphenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile (6e)

Compound 6e was prepared in 78% yield (250 mg) from 3-methoxybenzaldehyde 5e (136 mg, 1 mmol) according to the standard procedure which gave 6d as yellowish powder, mp = 212-214 °C. IR (KBr, ν, cm -1 ): 1640(C=N), 2205(C≡N). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (CDCl 3 ) δ : 3.84 (s, 2H, CH 2 ); 3.92 (s, 3H, MeO); 3.94 (s, 3H, MeO); 6.74 (d, 1H, J = 7.4 Hz, H-7, Ar); 6.86 (d, 1H, J = 7.9 Hz, H-5, Ar); 7.07 (d, 1H, J = 7.9 Hz, H-6, Ar); 7.12 (dd, 1H, J = 9.0, 3 Hz, H-3', Ar); 7.41 (t, 1H, J = 7.9 Hz, H-4', Ar); 7.58 (d, 1H, J = 7.5 Hz, H-5', Ar); 7.64 (s, 1H, H-1'); 8.92 (s, 1H, CH=N). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

General procedure for the synthesis of 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e):

In a 50 mL round-bottomed flask, provided with a magnetic stirrer and condenser, containing a suspension of 2-(benzylidene)amino-8-methoxy-4H-chromene-3-carbonitrile 6 (1.2 mmol) dissolved in 12 mL of cooled (0°C) tetrahydrofuran (THF) was added portionwise commercial sodium borohydride 98% (23 mg, 0.6 mmol). Vigorous stirring (500 rpm) at 0°C was pursued during 20 min. The reaction mixture was concentrated in a rotary evaporator under reduced pressure for the elimination of volatile and to the crude residue was added deionized water (15 mL) to ensure precipitation of compound 7. The insoluble material was collected by filtration on a Büchner funnel (porosity N°4) and washed with deionized water (3x10 mL). Compound 7 was dried under high vacuum (10 -2 Torr) at 25°C for 1 h, then recrystallized from absolute ethanol which, gave the desired 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7 as a powder analyzed by 1 H, [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR, HRMS and centesimal analysis.

2-(Phenylmethyl)amino-8-methoxy-4H-chromene-3-carbonitrile (7a)

Compound 7a was prepared in 96% yield (337 mg) from 2-[(E)-(phenylmethylene)amino]-8-methoxy-4Hchromene-3-carbonitrile 6a (348 mg, 1.2 mmol) according to the standard procedure which gave 7a as white powder, mp = 152-154 °C. IR (KBr, ν, cm -1 ): 2176 (C≡N), 3325 (NH). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (DMSO-d 6 ) δ : 3.42 (s, 2H, CH 2 ); 3.81 (s, 3H, MeO); 4.39 (d,2H, J = 6.4 Hz, CH 2 NH); 6.71 (dd, 1H, J = 7.6, 1.1 Hz, H-7, Ar); 6.93 (d, 1H, J = 8.1 Hz, H-5, Ar); 7.03 (t, 1H, J = 7.9 Hz, H-6, Ar); 7.23 (t, 1H, J = 7.8 Hz, H-3', Ar); 7.32 (t, 2H, J = 7.5 Hz, H-2', H-4', Ar); 7.38 (d, 2H, J =7.0 Hz, H-1', H-5', Ar); 7.73 (t, 1H, J = 6.4 Hz, NH). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] amino]-8-methoxy-4H-chromene-3-carbonitrile 6b (434 mg, 1.2 mmol) according to the standard procedure which gave 7b as yellowish powder, mp = 148-150 °C. IR (KBr, ν, cm -1 ): 2173 (C≡N), 3337 (NH). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (CDCl 3 ) δ : 3.54 (s, 2H, CH 2 ); 3.89 (s, 3H, MeO); 4.48 (d, 2H, J = 5.9 Hz, CH 2 NH); 4.96 (t, 1H, J = 5.3 Hz, NH); 6.77-6.63 (m, 3H, H-7, H-2', H-4', Ar); 6.81 (d, 1H, J = 8.1 Hz, H-5, Ar); 7.03 (t, 1H, J = 7.9 Hz, H-6, Ar); 7.26 (d, 2H, J = 8.5 Hz, H-1', H-5', Ar). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

2-(4-Methoxyphenylmethyl)amino-8-methoxy-4H-chromene-3-carbonitrile (7c)

Compound 7c was prepared in 97% yield (375 mg) from 2-[[(E)-(4-methoxyphenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile 6c (384 mg, 1.2 mmol) according to the standard procedure which gave 7c as white powder, mp = 156-158 °C. IR (KBr, ν, cm -1 ): 2172 (C≡N), 3332 (NH). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF]

2-(4-Chlorophenylmethyl)amino-8-methoxy-4H-chromene-3-carbonitrile (7d)

Compound 7d was prepared in 80% yield (314 mg) from 2-[[(E)-(4-chlorophenyl)methylene]amino]-8methoxy-4H-chromene-3-carbonitrile 6d (389.7 mg, 1.2 mmol) according to the standard procedure which gave 7d as yellowish powder, mp = 180-182 °C. IR (KBr, ν, cm -1 ): 12181 (C≡N), 3318(NH). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (CDCl 3 ) δ : 3.53 (s, 2H, CH 2 ); 3.86 (s, 3H, MeO); 4.52 (d, 2H, J = 6.3 Hz, CH 2 NH); 5.16 (t, 1H, J = 5.9Hz, NH); 6.69 (d, 1H, J = 7.4 Hz, H-7, Ar); 6.79 (d, 1H, J = 8.1 Hz, H-5, Ar); 7.03 (t, 1H, J = 8 Hz, H-6, Ar); 7.33 (s, 4H, H-1', H-5', H-2', H-4', Ar). [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF]

2-(3-Methoxyphenylmethyl)amino-8-methoxy-4H-chromene-3-carbonitrile (7e)

Compound 7e was prepared in 78% yield (302 mg) from 2-[[(E)-(3-methoxyphenyl)methylene]amino]-8-methoxy-4H-chromene-3-carbonitrile 6e (384 mg, 1.2 mmol) according to the standard procedure which gave 7e as withe powder, mp = 144-146 °C. IR (KBr, ν, cm -1 ): 2176 (C≡N), 3362 (NH). [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (CDCl 3 ) δ : 3.45 (s, 2H, CH 2 ); 3.72 (s, 3H, MeO); 3.77 (s, 3H, MeO); 4.45 (d, 2H, J = 6.2 Hz, CH 2 NH); 4.99 (t, 1H, J = 5.8 Hz, NH); 6.60 (d, 1H, J = 7.7 Hz, H-7, Ar); 6.70 (d, 1H, J = 8.2 Hz, H-5, Ar); 6.76 (dd, 1H, J = 8.3, 2.4 Hz, H-3', Ar); 6.87 (d, 2H, J = 8.1 Hz, H-1', H-5', Ar); 6.94 (t, 1H, J = 8 Hz, H-6, Ar); 7.19 (t, 1H, J = 7.8 Hz, H-4', Ar); [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR (CDCl 3 ) δ : 24.3 (CH 2 , C-4); 45.9 (C-11); 51.8 (C-3, C=); 55.2 (CH 3 , C-14'); 56.0 (CH 3 , C-8'); 110.4 (C-7, Ar); 113.2 (C-15, Ar); 113.4 (C-13, Ar); 119.9 (CN); 120.1 (C-5, Ar); 120.9 (C-17, Ar); 121.2 (C-10, Ar); 124.5 (C-6, Ar); 129.8 (C-16, Ar); 139.1 (C-9, Ar); 139.9 (C-12, Ar); 147.9 (C-2, Ar); 159.9 (C-8, Ar); 160.

Protein kinase assays

Kinase activities were assayed in appropriate kinase buffer, with either protein or peptide as substrate in the presence of 15µM [γ-33 P] ATP (3,000 Ci/mmol; 10 mCi/ml) in a final volume of 30 µl following the assay described in literature [START_REF] Bach | Roscovitine targets, protein kinases and pyridoxal kinase[END_REF] . Controls were performed with appropriate dilutions of dimethylsulfoxide. Full-length kinases are used unless specified. Peptide substrates were obtained from Proteogenix (Oberhausbergen, France).

 CDK5/p25 (human, recombinant, expressed in bacteria) was assayed in buffer A, with 0.8 µg/µl of histone H1 as substrate.

 CDK9/CyclinT (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed in buffer B (+ 0.15 mg/ml of BSA + 0.23 mg/ml of DTT) with 0.27 µg/µl of the following peptide: YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate.

 PIM1 (human proto-oncogene, recombinant, expressed in bacteria) was assayed in buffer A with 0.8 µg/µl of histone H1 (Sigma #H5505) as substrate.

 Haspin (human, kinase domain, amino acids 470 to 798, recombinant, expressed in bacteria) was assayed in buffer C with 0.007 µg/µl of Histone H3 (1-21) peptide (ARTKQTARKSTGGKAPRKQLA) as substrate;

 SscGSK-3/ (glycogen synthase kinase-3/, native, affinity purified from porcine brain extracts, Sus scrofra domesticus) was assayed in buffer B (+ 0.15 mg/ml of BSA + 0.23 mg/ml of DTT), with 0.010 µg/µl of GS-1 peptide, a GSK-3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE, "Sp" stands for phosphorylated serine).

 SscCK1/ (porcin casein kinase 1/e, native, affinity purified from porcine brain extracts, Sus scrofra domesticus) was assayed in buffer A, with 0.022 µg/µl of the following peptide: RRKHAAIGSpAYSITA as CK1-specific substrate.

Buffers:

(A) 60 mM -glycerophosphate, 30 mM p-nitrophenyl-phosphate, 25 mM MOPS (pH 7), 5 mM EGTA, 15 mM MgCl 2 , 1 mM DTT, 0.1 mM sodium orthovanadate;

(B) 10 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/ml heparin;

(C) MOPS 25 mM pH 7.5, 10 mM MgCl 2 .

Results and discussion

Chemistry and characterizations of synthesized compounds

Access to the planned 2-benzylamino-4H-chromene-3-carbonitrile 7 from the 2-amino-4H-chromene-3-carbonitrile platform 4 is outlined in Scheme 1.

Scheme 1: Synthetic route of the 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e). Reagent and conditions: i) piperidine 0.5% mol., EtOH, 25°C, 4 h. ii) NaBH 4 , MeOH, 25°C, 20 min. iii) piperidine, EtOH, 80°C, 5 h. iv) NaBH 4 , THF, 0°C, 20 min.

The preparation of key intermediate 4 involved a two-step approach developed in our laboratory [START_REF] Bouattour | Microwave assisted a practical synthesis of 4-imino-3-phenyl-3,4-dihydro-1Hchromeno[2,3-d]pyrimidine-2(5H)-thione derivatives and exploration of their biological activities[END_REF] . The first step is based on a facile hetero cyclization between 2-hydroxy-3-methoxybenzaldehyde or o-vanillin 1 and propanedinitrile 2 catalyzed with piperidine 0.5% mol. in ethanolic solution at room temperature, which afforded the 8-methoxy-2-imino-2H-[1]-benzopyran-3-carbonitrile 3 in 85% yield after work-up. In the second step, the imino-coumarin 3 was transformed into the 2-amino-8-methoxy-4H-chromene-3 carbonitrile 4 (in 75% yield) by reduction in methanolic solution of sodium borohydride. With the platform 4 in hand, initial attempts to obtain the desired N-benzylamino-4H-chromene-3-carbonitrile by direct Nalkylation of platform 4 with various benzyl halide were unsuccessful. After a set of experiments for the preparation of mono alkylated derivatives 7 in varying the following parameters as -reaction time range (6-24 hours), -the ratio of reagents (4 / benzyl halide: 1.05-1.1), -reaction temperature, -use of non-polar or polar solvents, we observed many difficulties to control mono alkylation or, to have a minimum of reactivity of the halide reagents. Therefore, we decided to abandon this N-alkylation approach and to explore the reductive amination. After optimization of the reaction conditions for transformation of platform 4 into the corresponding aldimine 6a with benzaldehyde 5a in the presence of 1 equivalent of piperidine in refluxed absolute ethanol (reaction time: 5 h, 6a: 70% yield), we extended this reaction with various aldehydes 5(b-c) in order to introduce molecular diversity. All the aldimines 6(a-e) were isolated from the reaction mixture by cooling, the insoluble compounds 6 were collected by a simple filtration and rinsed with cooled absolute ethanol. The desired compounds 6(a-e) were synthesized in yields ranging from 52 to 90% (Table 1). Structure of compounds 6(a-e) was substantiated by FTIR (CN stretch, 2196 <  (cm -1 ) < 2206), by [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR (N=CH signal, 8.81 <  < 8.92 ppm which is in agreement with a E-configuration [START_REF] Bjørgo | E-Z -Isomerism in aldimines[END_REF] and 13 C NMR (=CH,  160.50 ppm). In mass spectrometry analysis (HRMS) the [M+Na] + molecular ion signal for the five compounds 6 were readily obtained as base signal.

Table 1:

Results for the preparation of 2-[(E)-(benzylidene)amino]-8-methoxy-4H-chromene-3-carbonitrile 6(a-e) and 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e). Finally, treatment of aldimines 6(a-e) with sodium borohydride in cooled THF solution (0°C) during 20 min., afforded the reduced compounds 7(a-e). After elimination of the solvent in vacuo, the crude residue was triturated with deionized water and insoluble material was collected by filtration, and eventually recrystallized from absolute ethanol. Inspection of the data presented in Table 1 showed that reduction works well (7(a-e): 78-96% yield) and the targeted 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e) were prepared in good overall yields (32-56%). Before entering the biological tests, the five products 7 were characterized by 1 H, [START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR, FTIR, HRMS and centesimal analysis. Examination of their 1 H NMR spectra in DMSO-d 6 solution showed that the CH 2 Ar signal is located in the range 4.38 <  < 4.51 ppm and in the IR spectrum, important bands of absorption were observed between 3318 and 3361 cm -1 which can be attributed to the N-H stretching frequencies (Table 1). The physicochemical properties of compounds 6(a-e) and 7(a-e) were calculated with the Swiss ADME 16 server platform 17 . These physicochemical properties were evaluated based on Lipinski's rules [START_REF] Lipinski | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF] , considering log P (octanol/water partition coefficient) which indicates the concentration ratio of a neutral substance between two immiscible solvents in equilibrium and reveals the differential solubility of this substance in the two solvents. Log P is a very important parameter because it influences the bioavailability, permeability and toxicity of a drug candidate and a drug. Molecular weight (MW), aqueous solubility (log S), topological polar surface aera (tPSA), hydrogen bond donors (HBD), hydrogen bond acceptors (HBA) [START_REF] Lipinski | Lead-and drug-like compounds: the rule-of-five revolution[END_REF] , number of rotatable bonds (RB), the fraction of Csp 3 and drug-likeliness (violation of the 5 Lipinski rules) completed this in silico analysis (Table 2).

Structure of compound

Physicochemical properties and in silico ADME studies

The log P values of compounds 6(a-e) and 7(a-e) were acceptable, all under 5.0 and none of them negative. The highest values (3.75 and 3.80) correspond to 7b (R = p-NEt 2 ) and to 6d (R = p-Cl). According to the calculated values of aqueous solubility (log S), most of the aldimines 6(a-e) and the 2-benzylamino-8methoxy-4H-chromene-3-carbonitrile 7(a-e) are moderately soluble in water (-4 < log S 6(a-e) < -3.9 and -5 < log S 7(a-e) < -4.3). For predicting the drug solubility, there is another effective descriptor for transport properties of a molecule is the topological surface aera (tPSA) which described the sum of the surfaces of its polar atoms (N, O and Cl), and the hydrogen atoms attached to them. tPSA is an interacting indicator of gastrointestinal absorption and penetration of the brain-blood barrier (BBB). For compounds 6(a-e) and 7(a-e), tPSA values from 54 to 64 Å 2 evidenced an acceptable permeability. Complete analysis of the data in Table 2 shows that the 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e) and their corresponding precursors 6(a-e) are in agreement with Lipinski's rules of five (R05) [START_REF] Abad-Zapatero | A sorcerer's apprentice and the rule of five: from rule-of-thumb to commandment and beyond[END_REF] Using the SwissADME server 17 , the scale of similarity of synthetic compounds with the drug was used to evaluate criteria integrating lipophilicity (LIPO), size (MW), polarity (tPSA), solubility (log S), flexibility (FLEX, see also the number of rotatable bonds RB in Table 2), and saturation (INSATU). The ideal situation is that when all properties of these molecules are located in the pink region. That is to say when the basic constant properties are on the radar chart. The biocompatibility radar graphs and bioavailability of compounds 6(a-e) and 7(a-e) are reported in Table 3. Except for saturation, the radar plot shows whether all compounds 6 and 7 are in the pink area. It should be noted that the bioavailability score % (%Abs) of all compounds is 55%.

In vitro protein kinases assays

Now for the 2 nd part of this study related to the 2-benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e) and their aldimine precursors 6(a-e), we have been interested in exploring their biological properties, particularly on six different in vitro kinase assays. The protein kinases selected for these in vitro assays are the following: HsCDK5-p25 (Homo sapiens cyclin-dependant kinase 5-p25) [START_REF] Leclerc | Indirubins inhibit glycogen synthase kinase-3β and CDK5/P25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin dependent kinase inhibitors?[END_REF] ; HsCDK9/cyclin T (Homo sapiens cyclin-dependant kinase 9) [START_REF] Sonawane | Cyclin dependent kinase 9 inhibitors for cancer therapy[END_REF] ; HsPim1 (Homo sapiens Pim1, proto-oncogene, serine/threonine kinase) [START_REF] Blanco-Aparicio | Pim kinases in cancer: Diagnostic, prognostic and treatment opportunities[END_REF] ; HsHaspin (Homo sapiens Haspin, a mitotic serine/threonine protein kinase); SscGSK-3/ (Sus scofra domesticus glycogene synthase kinase-3/) [START_REF] Primot | Purification of GSK-3 by affinity chromatography on immobilized axin[END_REF] and SscCK1/ (Sus scofra domesticus casein kinase1/) [START_REF] Rena | D4476, a cell-permeant inhibitor of CK1, suppresses the sitespecific phosphorylation and nuclear exclusion of FOXO1a[END_REF] . All the selected enzymes presented interest for protein phosphorylation of serine, threonine and tyrosine residues in many regulatory mechanisms of cells. Deregulation of these protein kinases is mainly responsible of human diseases [START_REF] Buljan | Kinase interaction network expands functional and disease roles of human kinases[END_REF] .

In Table 4 are reported the results of in vitro kinase assays with the rate (%) of residual activities at 10 M and 1 M. Analysis of these results showed three categories of compounds. The first category is related to compounds which presented no significant protein kinase activity (rate > 60-70% at 10 M and 1 M).

The second category integrated compounds with moderate interest and the rate is less than or close to 50%. For the third category, the rate is less than 50% at 10 M and at 1 M, so the tested compounds have a marked effect on the catalytic activity. Results given in Table 4 showed that none compound 7 presents inhibition effects on this panel of these six protein kinases, but this does not mean that compounds 7 are inactive against protein kinases, it concerns only this panel. However, and among the 2-benzylidenamino-8-methoxy-4H-chromene-3-carbonitrile 6(ae), three compounds exhibited inhibitory activity on Pim1 and Haspin (Figure 3). For Pim1, this concerns the compound 6a (26% rate at 10 M) and 6b (55% rate at 1 mM); for Haspin, only the compound 6e is active (46% rate at 10 M).

Table 4: Effects of the 2-benzylidenamino-8-methoxy-4H-chromene-3-carbonitrile 6(a-e) and the 2benzylamino-8-methoxy-4H-chromene-3-carbonitrile 7(a-e) on the catalytic activity of six purified protein kinases. ATP concentration used in the kinase assays was 15 µmol/L. Results are expressed in % of maximal activity, i.e. measured in the absence of inhibitor but with an equivalent dose of DMSO (solvent of the tested compounds) (values are means, n = 2). Kinases are from human origin except when it is specified Ssc, Sus scrofa domesticus. NB: ≥100 indicates that the compound cannot inhibit the enzymatic activity at the tested concentration. * For Haspin, the kinase domain (amino acids 470 to 798) was expressed and analyzed.

Compoun

Conclusion

For this study, we have described a practical and new approach to 2-benzylamino-8-methoxy-4Hchromene-3-carbonitrile 7 with good overall yields ranging from 32 to 56%. The key step of this methodology is the reductive amination from the platform 4 which afforded indirectly an access to mono substitution of the amino function in position C-2 for this platform. The physicochemical properties (log P,

Figure 2 :

 2 Figure 2: Possibilities of molecular diversity offered by the 2-amino-4H-chromene-3-carbonitrile platform

  C NMR (100 MHz, CDCl 3 ) δ : 26.9 (CH 2 , C-4); 40.1 (2-CH 3 ); 56.0 (CH 3 O); 78.4 (C-3, C=); 110.7 (C-7, Ar); 111.4 (C-14, C-16, Ar); 119.1 (C-5, Ar); 119.3 (CN); 120.0 (C-6, Ar); 122.0 (C-10, Ar); 124.5 (C-13, C-17, Ar); 132.4 (C-12, Ar); 139.6 (C-9, Ar); 148.3 (C-8, Ar); 153.7 (C-15, Ar); 159.9 (C-2, Ar); 160.8 (C-11). HRMS (ES + , MeOH/CH 2 Cl 2 9:1), m/z: 356.1367 found (calculated for C 20 H 19 N 3 O 2 Na [M+Na] + requires 356.13695).

  C NMR (CDCl 3 ) δ : 27.0 (CH 2 , C-4); 55.5 (CH 3 , C-15'); 56.1 (CH 3 , C-8'); 81.1 (C-3, C=); 110.8 (C-7, Ar); 114.4 (C-14, C-16, Ar); 118.6 (C-10, Ar); 118.8 (CN); 120.0 (C-5, Ar); 124.7 (C-6, Ar); 128.0 (C-12, Ar); 132.3 (C-13, C-17, Ar); 139.3 (C-9, Ar); 148.3 (C-8, Ar); 159.2 (C-2, Ar); 160.9 (C-11, Ar); 163.7 (C-15). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 343.1057 found (calculated for C 19 H 16 N 2 O 3 Na [M+Na] + 343.10531).

  C NMR (CDCl 3 ) δ : 27.1 (CH 2 , C-4); 56.0 (CH 3 O); 83.4 (C-3, C=); 110.8(C-7, Ar); 118.1 (C-10, Ar); 118.5 (CN); 119.9 (C-5, Ar); 125.0 (C-6, Ar); 129.3(C-14, C-16, Ar); 131.3 (C-13, C-17, Ar); 133.5 (C-12, Ar); 139.2 (C-15, Ar); 139.3 (C-9, Ar); 148.3 (C-8, Ar); 158.6 (C-2, Ar); 160.2 (C-11). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 347.0556 found [M+Na] + (calculated for C 18 H 13 N 2 O 2 35 ClNa [M+Na] + requires 347.05578).

  C NMR (CDCl 3 ) δ : 27.1 (CH 2 , C-4); 55.5 (CH 3 , C-14'); 56.1 (CH 3 , C-8'); 82.8 (C-3, C=); 110.9 (C-13, Ar); 112.9 (C-7, Ar); 118.1 (C-10, Ar); 118.7 (CN); 120.0 (C-5, Ar); 120.0 (C-15, Ar); 123.9 (C-17, Ar); 124.9 (C-6, Ar); 129.8 (C-16, Ar); 136.4 (C-12, Ar); 139.5 (C-9, Ar); 148.3 (C-8, Ar); 158.9 (C-2, Ar); 160.0 (C-14, Ar); 161.7 (C-11). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 343.1056 found (calculated for C 19 H 16 N 2 O 3 Na [M+Na] + requires 343.10531).

  C NMR (DMSO-d 6 ) δ : 24.6 (CH 2 , C-4); 45.0 (CH 2 -NH, C-11); 49.9 (C-3, C=); 56.4 (C-8'); 111.4 (C-7, Ar); 120.1 (CN); 121.5 (C-10, Ar); 121.5 (C-5, Ar); 124.9(C-6, Ar); 127.4 (C-15); 128.0 (C-14, C-16, Ar); 128.3 (C-13, C-17, Ar); 138.9 (C-12, Ar); 134.3 (C-9, Ar); 147.9 (C-2, Ar); 160.4 (C-8, Ar

  C NMR (CDCl 3 ) δ : 24.4 (CH 2 , C-4); 40.6 (Me 2 N); 45.7 (C-11); 51.1 (C-3, C=); 56.1 (C-8'); 110.4 (C-7, Ar); 112.7 (C-14, C-16, Ar); 119.9 (CN); 121.0 (C-5, Ar); 121.5 (C-10, Ar); 124.4 (C-6, Ar); 125.9 (C-12, Ar); 129.1 (C-13, C-17, Ar); 139.1 (C-9, Ar); 148.0 (C-2, Ar); 150.2 (C-8, Ar); 160.4 (C-15, Ar

  H NMR (CDCl 3 ) δ : 3.54 (s, 2H, CH 2 ); 3.82 (s, 3H, MeO); 3.88 (s, 3H, MeO); 4.51 (d, 2H, J = 6.4 Hz, CH 2 NH); 5.04 (t, 1H, J = 5.6 Hz, NH); 6.69 (d, 1H, J = 7.2 Hz, H-7, Ar); 6.81 (d, 1H, J = 8.1 Hz, H-5, Ar); 6.89 (d, 2H, J = 8.6 Hz, H-2', H-4', Ar); 7.03 (t, 1H, J = 8.0 Hz, H-6, Ar); 7.31 (d, 2H, J = 8.5 Hz, H-1', H-5', Ar); 13 C NMR (CDCl 3 ) δ : 24.4 (CH 2 , C-4); 45.5 (C-11); 51.5 (C-3, C=); 55.3 (CH 3 , C-15'); 56.0 (CH 3 , C-8'); 110.4 (C-7, Ar); 114.1 (C-14, C-16, Ar); 119.9 (CN); 120.9 (C-5, Ar); 121.3 (C-10, Ar); 124.5 (C-6, Ar); 129.3 (C-13, C-17, Ar); 130.4 (C-12, Ar); 139.1 (C-9, Ar); 147.9 (C-2, Ar); 159.2 (C-8, Ar); 160.3 (C-15). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 345.1206 found (calculated for C 19 H 18 N 2 O 3 Na [M+Na] + requires 345.12051. Anal. Calcd for C 19 H 18 N 2 O 3 : C, 70.79; H, 5.63; N, 8.69. Found: C, 70.81; H, 5.66; N, 8.65.

  C NMR (CDCl 3 ) δ : 24.3 (CH 2 , C-4); 45.2 (C-11); 52.1(C-3, C=); 56.0 (CH 3 , C-8'); 110.4 (C-7, Ar); 119.9 (CN); 120.7 (C-5, Ar); 121.1 (C-10, Ar); 124.6 (C-6, Ar); 128.8 (C-14, C-16, Ar); 129.2 (C-13, C-17, Ar); 133.5 (C-15, Ar); 137.0 (C-12, Ar); 139.0 (C-9, Ar); 147.

Table 3 :

 3 for the following requirements: (a) a molecular weight < 500 g/mole; (b) hydrogen bond donors HBD < 5; (c) hydrogen bond acceptors HBA < 10 21 ; (d) Log P < 4.15 and (e) tPSA ≤ 140 Å 2 . This means that these compounds should have efficient absorption and good oral potential bioavailability 22 . Bioavailability radar related to the physicochemical properties of compounds 6(a-e) and 7(a-e). The pink colored zone is the suitable physicochemical space for oral bioavailability. LIPO (Lipophility): -0.7 < Log P < +5.0; SIZE: 150 g/mol < MW < 500 g/mol; POLAR (Polarity): 20 Å 2 < tPSA < 130 Å 2 ; INSOLU (Insolubility): -6 < Log S < 0; INSATU (Insaturation): 0.25 < Fraction Csp 3 < 1; FLEX (Flexibility): 0 < Number rotatable bonds < 9.

Figure 3 :

 3 Figure 3: 2-Benzylidenamino-8-methoxy-4H-chromene-3-carbonitrile 6(a,b) and 6e which are bioactive against protein kinases Pim1 and Haspin. Hs, Homo sapiens.

  ). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 315.1107 found (calculated for C 18 H 16 N 2 O 2 Na [M+Na] + requires 315.11095). Anal. Calcd for C 18 H 16 N 2 O 2 : C, 73.95; H, 5.52; N, 9.58. Found: C, 73.75; H, 5.48; N, 9.56.

	2.1.5.2 2-(4-Dimethylaminophenylmethyl)amino-8-methoxy-4H-chromene-3-carbonitrile (7b)
	Compound	7b	was	prepared	in	96%	yield	(419	mg)	from	2-[[(E)-(4-
	dimethylaminophenyl)methylene]

  ). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 358.1526 found (calculated for C 20 H 21 N 3 O 2 Na [M+Na] + requires 358.1526). Anal. Calcd for C 20 H 21 N 3 O 2 : C, 71.62; H, 6.31; N, 12.53. Found: C, 71.68; H, 6.36; N, 12.63.

  3 (C-14, Ar). HRMS (ES + , MeOH/CH 2 Cl 2 9:1) m/z: 345.1214 found (calculated for C 19 H 18 N 2 O 3 Na [M+Na] + requires 345.12151). Anal. Calcd for C 19 H 18 N 2 O 3 : C, 70.79; H, 5.63; N, 8.69. Found: C, 70.80; H, 5.62; N, 8.70.

  [START_REF] Patil | Chromenes: potential new chemotherapeutic agents for cancer[END_REF] H NMR and[START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR in CDCl 3 solution. d 1 H NMR and[START_REF] Bouattour | Synthesis of new 2-phenylamino-4H-chromene-3-carbonitrile derivatives and their effects on tumor cell lines and against protein kinases[END_REF] C NMR in DMSO-d 6 solution with TMS as internal reference. e 1 H NMR in CDCl 3 solution.

	6b		52	33	8.81	160.8	-	-
	6c		90	57	8.88	160.9	-	-
	6d		80	51	8.89	160.2	-	-
	6e		78	50	8.92	161.7	-	-
	7a		96	43	-		4.38 d	3325
	7b		96	32	-		4.48	3337
	7c		97	56	-		4.51	3332
	7d		80	41	-		4.51	3318
	7e		78	39	-		4.45	3362
	7	6 and	Yield (%) a	Overall yield (%) b	 CH= of 6 (ppm) c 	 CH2Ar of 7 (ppm) e	 NH of 7 (cm -1 )
	6a		70	45	8.88 d	162.4 d	-	-

a Yield of isolated product 6 and 7.

b

Overall yield of 6 and 7 calculated from compound 3. c

Table 2 :

 2 Results of physicochemical properties for compounds 6

(a-e) and 7(a-e).

	Prod.	Structure	MW (g/mol)	Log P	Log S	tPSA (Å 2 )	HBA HBD RB	Fract. Csp 3	LV
	6a		290.32	3.26	-3.89	54.61	4	0	3	0.11	0
	6b		361.44	3.95	-4.60	57.85	4	0	6	0.27	0
	6c		320.34	3.27	-3.95	63.84	5	0	4	0.16	0
	6d		324.76	3.80	-4.48	54.61	4	0	3	0.11	0
	6e		320.34	3.27	-3.95	63.84	5	0	4	0.16	0
	7a		293.33	3.13	-4.29	54.28	3	1	4	0.17	0
	7b		363.45	3.75	-5.00	57.62	3	1	7	0.32	0
	7c		322.36	3.10	-4.36	63.51	4	1	5	0.21	0
	7d		326.78	3.67	-4.89	54.28	3	1	4	0.17	0
	7e		322.36	3.13	-4.36	63.51	4	1	5	0.21	0
	MW, molecular weight; Log P, partition coefficient; Log S, water solubility; tPSA, topological surface aera;
	HBA, hydrogen bond acceptor; HBD, hydrogen bond donor: RB, rotatable bonds, LV, number of Lipinski's
	rule of 5 violations.

Acknowledgments

One of us (M.D.) wishes to thank the "Ministère de l'Enseignement Supérieur et de la Recherche de Tunisie" for the grant. Financial support of this program carried out under the French National Cancer Institute "Cancéropole Grand Ouest" in the "Molécules Marines, Métabolisme et Cancer" M3C network, is gratefully acknowledged. S. B. and T. R. thank IBiSA (French Infrastructures en Sciences du Vivant: Biologie, Santé et Agronomie) and Biogenouest (Western France life science and environment core facility network supported by the Conseil Régional de Bretagne) for supporting the KISSf screening facility (FR2424, CNRS and Sorbonne Université). The authors are grateful to the of the staff of the CRMPO analytical chemistry core facility for HRMS analysis (CRMPO platform, ScanMAT UAR 2025 CNRS at University of Rennes 1, Building 11A, Campus de Beaulieu, Rennes, France).

log S, tPSA, HBA, HBD, fraction Csp [START_REF] Gulley | focus on anaplastic thyroid cancer (ATC)[END_REF] , number of rotatable bond) have been established and results are in agreement with Lipinski's rules of five (R05). The ADME properties of the compounds 6(a-e) and 7(a-e) were also extensively studied in silico. Exploration of biological activities was realized against a panel of six protein kinases and among the 8-methoxy-4H-chromene-3-carbonitrile 6 and 7, only compounds 6(a,b) and 6e are potentially interesting because they showed good percentage of residual activities against Pim1 and Haspin. The current results are the beginning of a next relationship structure-activity (RSA) program for identification of a better promising protein kinase inhibitor derived from the 2-amino-4H-chromene-3carbonitrile platform. Works are in progress in our laboratory.

Declaration of Competing Interest

The authors declare that they have no known competing financial interest or personal relationships that have appeared to influence the work reported in this paper.

Supplementary material

Supplementary

data to this article can be found online at https://doi.org/10.1016/j.molstruc.2022.yyyyyy. Corresponding author : jean-pierre.bazureau@univ-rennes1.fr, phone: +33 223 236 603

Graphical abstract

Highlights

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: