Design and Synthesis of Novel N-Benzylidene Derivatives of 3-Amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine under Microwave, In Silico ADME Predictions, In Vitro Antitumoral Activities and In Vivo Toxicity

The synthesis of a series of new N-benzylidene derivatives of 3-amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine 10(a-l) bearing two points of molecular diversity is reported. These new compounds were synthesized in five steps including two steps under microwave dielectric heating. They were fully characterized using 1H and 13C NMR, FTIR and HRMS. The in silico physicochemical properties of compounds 10(a-l) were determined according to Lipinski’s rules of five (RO5) associated with the prediction of their bioavailability. These new compounds 10(a-l) were tested for their antiproliferative activities in fibroblasts and eight representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3, MCF7 and PANC1). Among them, the compounds 10h and 10i showed sub-micromolar cytotoxic activity on tumor cell lines (0.23 < IC50 < 0.3 μM) and no toxicity on fibroblasts (IC50 > 25 μM). A dose-dependent inhibition of Store-Operated Ca+2 Entry (SOCE) was observed in the HEK293 cell line with 10h. In vitro embryotoxicity and angiogenesis on the mCherry transgenic zebrafish line showed that 10h presented no toxic effect and no angiogenic effect on embryos with a dose of 5 μM at 72 hpf.

In this context and due to the synthetic potential around the 2-amino-4H-chromene platform and their chromeno [2,3-d]pyrimidine derivatives, the herein study was designed in order to identify novel potential anticancer compounds.In continuation of previous work [16], it seemed interesting to synthesize some new N-benzylidene derivatives of 3amino-4-imino-3,5-dihydro-4H-chromene [2,3-d]pyrimidines and to appraise their cytotoxic activities on a panel of tumoral cell lines.To better understand the pharmacological interest of these novel compounds, ADME predictions will be investigated.To complete the biological profile of the most active compounds on tumor cells, their effects will be evaluated on store-operated-calcium entry (SOCE) as this calcium entry is implicated in several human cancers [17].Owing to the fact that the zebrafish Danio rerio embryo model is accepted as a validated alternative assay to assess the acute toxicity of a molecule on this fish (OECD, N • 236) [18], the embryo toxicity evaluation will be examined with the selected relevant antitumor compounds and also associated with their possible effect on angiogenesis in the zebrafish embryo.

Synthesis Chemistry
In view of the relevance of N-arylidene derivatives of 3-amino-4-imino-3,5-dihydro-4H-chromene [2,3-d]pyrimidine as potential antitumor agents, the retrosynthesis analysis (Figure 2) showed that the desired starting compounds included three major buildingblocks.The first is the 2-amino-4H-chromene-3-carbonitrile platform, and the second is hydrazine, which provides the link between the platform and the N-arylidene moiety after condensation with an aromatic aldehyde.In this type of structure, the molecular diversity should come from the starting 2-amino-4H-chromene platform and aromatic aldehydes.
Pharmaceuticals 2024, 17, x FOR PEER REVIEW in order to identify novel potential anticancer compounds.In continuation of p work [16], it seemed interesting to synthesize some new N-benzylidene derivati amino-4-imino-3,5-dihydro-4H-chromene [2,3-d]pyrimidines and to appraise the toxic activities on a panel of tumoral cell lines.To better understand the pharmac interest of these novel compounds, ADME predictions will be investigated.To c the biological profile of the most active compounds on tumor cells, their effects evaluated on store-operated-calcium entry (SOCE) as this calcium entry is impli several human cancers [17].Owing to the fact that the zebrafish Danio rerio embry is accepted as a validated alternative assay to assess the acute toxicity of a mol this fish (OECD, N° 236) [18], the embryo toxicity evaluation will be examined selected relevant antitumor compounds and also associated with their possible e angiogenesis in the zebrafish embryo.

Synthesis Chemistry
In view of the relevance of N-arylidene derivatives of 3-amino-4-imino-3,5-d 4H-chromene [2,3-d]pyrimidine as potential antitumor agents, the retrosynthesis (Figure 2) showed that the desired starting compounds included three major b blocks.The first is the 2-amino-4H-chromene-3-carbonitrile platform, and the s hydrazine, which provides the link between the platform and the N-arylidene moi condensation with an aromatic aldehyde.In this type of structure, the molecular d should come from the starting 2-amino-4H-chromene platform and aromatic alde For the construction of desired 3-(N-phenylmethyliden)amino-4-imino-3,5-d 4H-chromeno [2,3-d]pyrimidine, the synthetic route of targeted compound 10 is d in Scheme 1. Concerning the 2-amino-4H-chromene-3-carbonitrile 4 as a key inter in this synthetic strategy, the preparation involved a two-step approach initiall oped in our laboratory [19] and well mastered practically in scale-up.In the first the synthesis of 2-imino coumarins 3(a-d), the first point of molecular diversity w duced by using four various aromatic aldehydes 1(a-d), which are, respectively, 1 ethylamino)salicylaldehyde; 1b, salicylaldehyde; 1c, 2-hydroxy-3-methoxybenza or ortho vanillin; and 1d, 2-hydroxynaphthaldehyde. Access to 2-imino couma very simple: reaction of commercial aldehyde 1(a-d) with propanedinitrile 2, in e solution at room temperature, in the presence of piperidine 0.5% mol.produced sired compounds 3 after work-up in good yields.In the second step, the prepar the 2-amino-3H-chromene-3-carbonitriles 4 resulted from the reduction of 2-im marins 3 with sodium borohydride at 0 °C in a cooled solution of MeOH during Due to the low solubility of these compounds 4 in MeOH, they were recovered us For the construction of desired 3-(N-phenylmethyliden)amino-4-imino-3,5-dihydro-4H-chromeno [2,3-d]pyrimidine, the synthetic route of targeted compound 10 is depicted in Scheme 1. Concerning the 2-amino-4H-chromene-3-carbonitrile 4 as a key intermediate in this synthetic strategy, the preparation involved a two-step approach initially developed in our laboratory [19] and well mastered practically in scale-up.In the first step for the synthesis of 2-imino coumarins 3(a-d), the first point of molecular diversity was introduced by using four various aromatic aldehydes 1(a-d), which are, respectively, 1a, 4-(diethylamino)salicylaldehyde; 1b, salicylaldehyde; 1c, 2-hydroxy-3-methoxybenzaldehyde or ortho vanillin; and 1d, 2-hydroxynaphthaldehyde. Access to 2-imino coumarins 3 is very simple: reaction of commercial aldehyde 1(a-d) with propanedinitrile 2, in ethanolic solution at room temperature, in the presence of piperidine 0.5% mol.produced the desired compounds 3 after work-up in good yields.In the second step, the preparation of the 2-amino-3H-chromene-3-carbonitriles 4 resulted from the reduction of 2-imino coumarins 3 with sodium borohydride at 0 With the underlying idea to introduce the pyrimidine fragment on 4H-chrome skeleton, it is necessary to add an electrophilic carbon to the 2-amino function of 4 chromene-3-carbonitrile 4 for hetero cyclization.For this, the transformation of the amino function of 4 into imidate 6 works well by the addition of 6 equivalents of eth orthoformate 5 at 110 °C under microwave dielectric heating [20] for 60 min catalyz with glacial acetic acid 0.5% mol., which afforded the desired functionalized imidat (Table 1) in moderate (6c: 52%) to good yields (6d: 90%).The interest of this method is th compound 6 is not soluble in the reaction mixture, which facilitates recovery using simp filtration.For the aza-annulation of imidate 6 into 4-imino-4,5-dihydro-3H-chromeno [2 d]pyrimidine 8, we applied a method described in the literature [21] employing the h drazine reagent 7. Applying this approach with slight experimental modifications, treated imidate 6 with 1.6 equivalents of aqueous solution of hydrazine (35% w/v) at roo temperature in EtOH for 2 h.As the reaction progressed, we noticed the low solubility 3-amino-4-imino-3,5-dihydro-4H-chromeno [2,3-d]pyrimidine 8 in the reaction med which, again, facilitates their recovery by simple filtration.These compounds 8(a-d) w prepared in yields ranging from 50 to 90%.If necessary, these intermediates 8 can be eas purified by recrystallization from EtOH.For the fifth and last step, the introduction of t second point of molecular diversity was realized by the condensation of 3-amino co pound 8 with aromatic aldehydes 9 or 1.After a set of experiments, optimal reaction co ditions for access of the desired 3-(N-phenylmethyliden)amino-4-imino-3,5-dihydro-4 chromeno[2,3-d]pyrimidine 10(a-i) and 10-(N-phenylmethyliden)amino-11-imino-11 naphto[1′,2′:5,6]pyrano [2,3-d]pyrimidine 10(j-l) were obtained from a mixture of aroma aldehyde 9 and compound 8 in absolute EtOH using a reaction temperature of 160 °C 60 min under microwave irradiation in the presence of piperidine 0.5% mol.After cooli down, filtration of the crude reaction mixture afforded compound 10, which was purifi using recrystallization from EtOH (10: 26-90% yield).The structure assignment of co pound 10 was evidenced by FTIR (C=N stretch, 1615 < ν < 1645 cm −1 ), by 1 H NMR for With the underlying idea to introduce the pyrimidine fragment on 4H-chromene skeleton, it is necessary to add an electrophilic carbon to the 2-amino function of 4Hchromene-3-carbonitrile 4 for hetero cyclization.For this, the transformation of the 2-amino function of 4 into imidate 6 works well by the addition of 6 equivalents of ethyl orthoformate 5 at 110 • C under microwave dielectric heating [20] for 60 min catalyzed with glacial acetic acid 0.5% mol., which afforded the desired functionalized imidate 6 (Table 1) in moderate (6c: 52%) to good yields (6d: 90%).The interest of this method is that compound 6 is not soluble in the reaction mixture, which facilitates recovery using simple filtration.For the aza-annulation of imidate 6 into 4-imino-4,5-dihydro-3H-chromeno [2,3-d]pyrimidine 8, we applied a method described in the literature [21] employing the hydrazine reagent 7. Applying this approach with slight experimental modifications, we treated imidate 6 with 1.6 equivalents of aqueous solution of hydrazine (35% w/v) at room temperature in EtOH for 2 h.As the reaction progressed, we noticed the low solubility of 3-amino-4imino-3,5-dihydro-4H-chromeno [2,3-d]pyrimidine 8 in the reaction media which, again, facilitates their recovery by simple filtration.These compounds 8(a-d) were prepared in yields ranging from 50 to 90%.If necessary, these intermediates 8 can be easily purified by recrystallization from EtOH.For the fifth and last step, the introduction of the second point of molecular diversity was realized by the condensation of 3-amino compound 8 with aromatic aldehydes 9 or 1.After a set of experiments, optimal reaction conditions for access of the desired 3-(N-phenylmethyliden)amino-4-imino-3,5-dihydro-4H-chromeno [2,3-d]pyrimidine 10(a-i) and 10-(N-phenylmethyliden)amino-11-imino-11Hnaphto[1 ′ ,2 ′ :5,6]pyrano [2,3-d]pyrimidine 10(j-l) were obtained from a mixture of aromatic aldehyde 9 and compound 8 in absolute EtOH using a reaction temperature of 160 • C for 60 min under microwave irradiation in the presence of piperidine 0.5% mol.After cooling down, filtration of the crude reaction mixture afforded compound 10, which was purified using recrystallization from EtOH (10: 26-90% yield).The structure assignment of compound 10 was evidenced by FTIR (C=N stretch, 1615 < ν < 1645 cm −1 ), by 1 H NMR for N-N=CH signal (8.24 < ν < 8.59 ppm) which is in agreement with an E-configuration [22].In mass spectrometry analysis (HRMS), the [M+H] + molecular ion signals for all compounds 10(a-l) were readily obtained as base signals.

Table 1.
Results  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.

10d
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 25 a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.

10e
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 25 a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.

10h
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 25 a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.  a Yield of isolated product.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log Po/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.

Physicochemical Properties of Compounds 10(a-l)
Lipinski's benchmark rule of five (RO5) defines desirable drug candidate physicochemical properties, such as the molecular weight MW < 500 Da, the octanol/water partition coefficient log P o/w < 5, the number of hydrogen bond donor HBD < 5, the number of hydrogen bond acceptor HBA < 10, the rotatable bonds RB < 10 [23] and completed now with the topological polar surface area tPSA < 140 Å 2 [24].The conventional RO5 has gained popularity as a "good rule of thumb" but is not specific for all drugs, especially for central nervous system (CNS) drugs [25].In the actual context, the physicochemical properties of compounds 10(a-l) are easily accessed on the SwissADME [26] web server [27], and the results are summarized in Table 2.For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotatable bonds RB (2 < RB10(a-l) < 6), we note that all of compound 10 is in agreement with  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotat-  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotat-  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotat-  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotat-  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA10(a-l) < 5), hydrogen bond donor HBD (HBD10(a-l) < 2), and rotat-  For compound 10, the log Po/w values are acceptable (2.86 < log Po/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen For compound 10, the log P o/w values are acceptable (2.86 < log P o/w < 4.07).The highest value (4.07) corresponds to compound 10a and the lowest values (2.66 and 2.86) correspond to compounds 10c and 10h bearing, not surprisingly, an ionizable phenolic function.For predicting drug solubility, the tPSA (topological polar surface area) is another interesting and effective descriptor for the transport properties of a molecule.The tPSA parameter describes the sum of the surface of its polar atoms (N, O) and the hydrogen atoms attached to them and, it is an interacting indicator of gastrointestinal absorption [28].For compounds 10(a-l), all of them have tPSA < 140 Å 2 (63.26 < tPSA 10(a-l) < 92.72 Å 2 ) and evidenced an acceptable permeability.To complete this, the calculated values of aqueous solubility (log S) showed that most of compound 10 is moderately soluble in water (−5.5 < log S 10(a-l) < −3.9).If we take into account the other parameters, such as hydrogen bond acceptor HBA (HBA 10(a-l) < 5), hydrogen bond donor HBD (HBD 10(a-l) < 2), and rotatable bonds RB (2 < RB 10(a-l) < 6), we note that all of compound 10 is in agreement with Lipinski's rule of five (RO5) (LV 10(a-l) = 0) [23].This means that these compounds should have potential efficient absorption and good oral bioavailability.

Cytotoxicity Assays of Compounds 10(a-l)
The potential antiproliferative activity of these novel 3-(N-phenylmethyliden)amino-4imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine 10(a-i) and 10-(N-phenylmethyliden) amino-11-imino-11H-naphto[1 ′ ,2 ′ :5,6]pyrano[2,3-d]pyrimidine 10(j-l) was evaluated in a panel of eight tumoral cell lines and in diploid skin fibroblasts used as normal cell lines for control.For these antitumor assays, chosen cell lines represent diverse types of human cancer, which are, respectively, human hepatocellular carcinoma (Huh7 D12), human colon adenocarcinoma (Caco2), human breast adenocarcinoma (MDA-MB231), MDA-MB468 for human breast carcinoma which correspond to the Triple Negative Breast Cancer (TNBC), human colon carcinoma (HCT116), PC3 as cell line initiated from bone metastasis of grade IV prostatic adenocarcinoma from a 62-year-old white man, MCF7 as breast cancer line isolated from a 69-year-old white woman and PANC1, as human pancreatic cancer cell line.The potential antiproliferative activity was preliminarily evaluated in vitro at a 25 µM single dose.The roscovitin and doxorubicin compounds were used in all experiments as reference standards because they are wide-spectrum anticancer agents.
For the compounds exhibiting strong growth inhibitory activity against a tumoral cell line (with a percentage of survival is <50%), they were used for a more detailed study to obtain full dose-response and survival curves using different concentrations (0.1-25 µM) for the determination of IC 50, which represented the concentration of the compound that kills 50% of the tumoral cells after 48 h incubation.The IC 50 was calculated from the full dose-response curve and used as a parameter for the antiproliferative activity of the new compounds.The results of these cytotoxicity assays for compounds 10(a-i) and compounds 10(j-l) are reported in Table 3.
The analysis of the results in Table 3 for in vitro antiproliferative activity assays highlights three categories of compounds.The first category concerns inactive products with IC 50 > 25 µM on tumor cancer cell lines and on fibroblasts.This concerns compounds 10(a-c), 10f and 10l.The second category is for compound 10 presenting moderate activity on tumor cell lines (1 < IC 50 < 25 µM) but is cytotoxic on fibroblasts (healthy skin cell); this is the case for compounds 10(d, e), 10g and 10(j, k).The last and the most interesting category concerns compounds with sub-micromolar cytotoxic activity on tumor cell lines (IC 50 < 1 µM) but without an effect on fibroblasts.For this most interesting category, compounds 10h and 10i (Figure 3) are in agreement with our selection criteria, i.e., a molecule active on tumor cells (IC 50 < 1 µM) and inactive on non-cancer cells (fibroblasts, IC 50 > 25 µM).tumor cell lines (1 < IC50 < 25 μM) but is cytotoxic on fibroblasts (healthy skin cell); this is the case for compounds 10(d, e), 10g and 10(j, k).The last and the most interesting category concerns compounds with sub-micromolar cytotoxic activity on tumor cell lines (IC50 < 1 μM) but without an effect on fibroblasts.For this most interesting category, compounds 10h and 10i (Figure 3) are in agreement with our selection criteria, i.e., a molecule active on tumor cells (IC50 < 1 μM) and inactive on non-cancer cells (fibroblasts, IC50 > 25 μM).tumor cell lines (1 < IC50 < 25 μM) but is cytotoxic on fibroblasts (healthy skin cell); this is the case for compounds 10(d, e), 10g and 10(j, k).The last and the most interesting category concerns compounds with sub-micromolar cytotoxic activity on tumor cell lines (IC50 < 1 μM) but without an effect on fibroblasts.For this most interesting category, compounds 10h and 10i (Figure 3) are in agreement with our selection criteria, i.e., a molecule active on tumor cells (IC50 < 1 μM) and inactive on non-cancer cells (fibroblasts, IC50 > 25 μM).tumor cell lines (1 < IC50 < 25 μM) but is cytotoxic on fibroblasts (healthy skin cell); this is the case for compounds 10(d, e), 10g and 10(j, k).The last and the most interesting category concerns compounds with sub-micromolar cytotoxic activity on tumor cell lines (IC50 < 1 μM) but without an effect on fibroblasts.For this most interesting category, compounds 10h and 10i (Figure 3) are in agreement with our selection criteria, i.e., a molecule active on tumor cells (IC50 < 1 μM) and inactive on non-cancer cells (fibroblasts, IC50 > 25 μM).

In Silico ADME Properties of Compounds 10h and 10i
To complete the physicochemical properties of compound 10 according to the Lipinski rule of five (RO5), we decided to predict for our compounds their gastrointestinal absorption (HIA-human gastrointestinal absorption) and BBB (Blood-Brain Barrier) penetration by using the W-Log P-versus-tPSA referential named "BOILED-Egg" model (Figure 4A) obtained from the SwissADME server [26].The "BOILED-Egg" model provides a quick, simple, easily repeatable, but statically unparalleled robust strategy for forecasting the passive gastrointestinal absorption and brain access of tiny compounds relevant for drug discovery and development.In this model, the "white region" is for the high

In Silico ADME Properties of Compounds 10h and 10i
To complete the physicochemical properties of compound 10 according to the Lipinski rule of five (RO5), we decided to predict for our compounds their gastrointestinal absorption (HIA-human gastrointestinal absorption) and BBB (Blood-Brain Barrier) penetration by using the W-Log P-versus-tPSA referential named "BOILED-Egg" model (Figure 4A) obtained from the SwissADME server [26].The "BOILED-Egg" model provides a quick, simple, easily repeatable, but statically unparalleled robust strategy for forecasting the passive gastrointestinal absorption and brain access of tiny compounds relevant for drug discovery and development.In this model, the "white region" is for the high probability of passive absorption by the gastrointestinal tract, and the "yellow region" (yolk) is for the high probability of brain penetration.Yolk and white areas are not mutually exclusive.The "gray region" concerns compounds that are predicted not to be absorbed in the gastrointestinal tract and not BBB permeant.The molecules are represented inside as either blue, which are substrates of P-glycoprotein, or red, which are not.In the BOILED-Egg model, we can see that compound 10h is located in the white region of high probability for passive gastrointestinal absorption and is not effluxed by P-glycoprotein (red circle).On the contrary, compound 10i is in the yellow area which means a high probability of passive BBB penetration and is also not effluxed by P-glycoprotein.This in silico result of the difference of BBB penetration between 10h (log P 10h 2.86) and 10i (log P 10i 3.17) is not surprising because 10i does not have a phenolic function (polar and ionizable group) [29].For the other compounds bearing a hydroxyl group in this series, which concerns 10b, 10e and 10k, the results are reported in Figure S1 of the Supplementary Information.probability of passive absorption by the gastrointestinal tract, and the "yellow region" (yolk) is for the high probability of brain penetration.Yolk and white areas are not mutually exclusive.The "gray region" concerns compounds that are predicted not to be absorbed in the gastrointestinal tract and not BBB permeant.The molecules are represented inside as either blue, which are substrates of P-glycoprotein, or red, which are not.In the BOILED-Egg model, we can see that compound 10h is located in the white region of high probability for passive gastrointestinal absorption and is not effluxed by P-glycoprotein (red circle).On the contrary, compound 10i is in the yellow area which means a high probability of passive BBB penetration and is also not effluxed by P-glycoprotein.This in silico result of the difference of BBB penetration between 10h (log P10h 2.86) and 10i (log P10i 3.17) is not surprising because 10i does not have a phenolic function (polar and ionizable group) [29].For the other compounds bearing a hydroxyl group in this series, which concerns 10b, 10e and 10k, the results are reported in Figure S1 of the Supplementary Information.For the bio-availability radar of compounds 10(h, i) in Figure 4B, which is the correlation of the six factors, LIPO (Lipophilicity): −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 (Unsaturation): 0.25 < Fraction Csp 3 < 1; FLEX (Flexibility): 0 < Number rotatable bonds < 9, we can see that molecules 10(h, i) violated just one criterion, i.e., unsaturation because both have fraction Csp 3 parameter < 0.25 (Table 2).The pink-colored zone is the suitable physicochemical space for oral bioavailability and results for the other compounds 10(a-l) are summarized in Table S1 of the Supplementary Information.For in silico prediction of drug-likeness model (Figure 4C), compound 10h presents, respectively, a score of −0.45 for 10h and −0.55 for 10i [30].In the analysis of all these in silico predictions and from a point of view of pharmacology, the most interesting compound 10 is molecule 10h because this one does not present a passive BBB penetration, this is the case of 10i, which is not effluxed by P-glycoprotein.

Store-Operated Calcium Entry (SOCE) Assays for Compound 10h
Store-operated Ca +2 entry (SOCE) is a major pathway of Ca +2 entry in mammalian cells, which regulates a large variety of cellular functions including proliferation, apoptosis, motility and death.The SOCE is a ubiquitous receptor-activated Ca +2 entry route across the plasma membrane that is critical to many cellular functions in mammalian cells [31].Aberrant SOCE is directly related to carcinogenesis, cancer cell proliferation, chemoresistance, angiogenesis and metastasis [32,33].SOCE is supported mainly by the plasma membrane (PM) calcium selective channels of the Orai family and regulated by the Ca +2 sensing stromal interaction molecules (STIM1 and STIM2) located in the membrane of the endoplasmic reticulum (ER).
Today, it is well established that the depletion of ER Ca +2 induces STIM1 conformational changes and its translocation to ER-plasma membrane junctions where they activate Orai channels supporting SOCE [34,35].In many cancers, such as breast, liver, lung, gastric and colon, accumulating evidence exhibits augmented SOCE and modification of STIM1 and Orai protein expression.SOCE inhibition decreases the proliferation and metastasis of cancer cells, suggesting that SOCE may act as an oncogenic pathway [36].Also, the need and the challenge of discovering small molecules modulating SOCE [37] will be very important in cancer with the underlying idea to shoot out the uncontrolled problem of metastasis [38].
Regarding the fact that compound 10h or (3E) 3-N-(2-hydroxyphenylmethylidene) amino-4-imino-9-methoxy-3,5-dihydro-4H-chromeno [2,3-d]pyrimidine presents noticeable sub-micromolar cytotoxic activity on several tumor cell lines (10h: 0.23 < IC 50 < 0.3 µM on Huh7 D12, Caco2, MDA MB-231, MDA MB-468, HCT116, PC3 and MCF7) without toxicity on fibroblasts (IC 50 > 25 µM), we evaluated initially its inhibitory effect on SOCE in HEK293 cell lines (Human Embryonic Kidney).Thapsigargin (TG), an inhibitor of endoplasmic reticulum Ca +2 -ATPase, is used to deplete intracellular calcium stores in a Ca +2 -free solution and therefore activate SOCE.After the addition of 2 µM TG in a Ca +2 -free solution, HEK293 cells exhibited a rapid rise of intracellular Ca 2+ concentration ([Ca 2+ ] i ) (Figure 5A) corresponding to ER Ca 2+ stores depletion.Subsequent reintroduction of 1.8 mM CaCl 2 to the extracellular solution resulted in a sustained increase in [Ca 2+ ] i from baseline, which is consistent with a characteristic SOCE-mediated Ca +2 influx from the extracellular solution.Pre-treatment with compound 10h at a concentration ranging from 1.25 to 50 µM (addition after 5 min before recording iCa +2 level variation) partially inhibits SOCE in HEK293 cells in a dose-dependent manner (10-50 µM) (Figure 5B for % release and Figure 5C for % SOCE).The evaluation of the same compound 10h on a B cell line (PLP cell line) with the same protocol, presented no inhibitory effects on SOCE and Ca +2 release.In the context of a preliminary exploration of the functional effects of our molecules of interest and in the absence of a significant inhibition at a low concentration of compound 10h on the reference cell line we used (HEK293) no further exploration of the modulatory effect of this compound on cancer cell lines was initiated.release (.In the context of a preliminary exploration of the functional effects of our mole cules of interest and in the absence of a significant inhibition at a low concentration o compound 10h on the reference cell line we used (HEK293) no further exploration of the modulatory effect of this compound on cancer cell lines was initiated.(C).Tested compound 10h at a concentration ranging from 1.25 to 50 µM was added 5 min before recording intracellular Ca +2 level variation.Endoplasmic (ER) Ca +2 content is totally depleted in Ca +2 free buffer by injection of 2 µM Thapsigargin (TG) and SOCE is next measured by the addition of 1.8 mM Ca +2 in the extracellular medium.The magnitude of SOCE was estimated as the maximal values of the normalized F340/380 ratio following Ca +2 re-addition.Effects on cytosolic calcium variations of the same compound 10h were also evaluated on a B cell line (PLP cell line) without detecting any inhibitory effects on SOCE and Ca 2+ release.

In Vitro Embryotoxicity and Angiogenesis of Compound 10h on Zebrafish Danio rerio
Zebrafish embryos are increasingly used as in vivo models for drug screening, drug toxicity and safety assessments due to their original advantages.Zebrafish have orthologues for 70% of human proteins and comparable vertebrate anatomy, as well as paralogues of 84% of all known disease-related genes [39].The optical transparency of the zebrafish embryos makes it easy to visualize directly the changes in the developing vascular system [38].Since 2013, the zebrafish embryo has been accepted as a validated model for alternative assay to assess fish acute toxicity (OECD, N • 263) [18].Another interest in the zebrafish embryo model relies on the fact that the effects are evaluated on the entire vertebrate organism during the period of organogenesis.To evaluate in vivo embryotoxicity of compound 10h on zebrafish, we used the Tg(kdrl:Has.HRAS-mcherry) s896 zebrafish transgenic model and, sorafenib and erlotinib as reference drugs for comparison.Sorafenib is a multi-kinase inhibitor that reduces and decreases tumor angiogenesis and tumor cell proliferation in vivo inhibiting tumor growth of a broad spectrum of human tumor xenographs in athymic mice.Erlotinib is an anticancer drug and a potent inhibitor of intracellular EGRF phosphorylation.
The embryos were exposed to the test compounds (10h, sorafenib and erlotinb) according to standard protocols described in the literature [18,40].Fertilized eggs at 24-48 h post-fertilization (hpf) were selected and transferred to a 96-well plate (1 embryo/well).Concentrations used range from 0.021 to 5 µM and for compound 10h and sorafenib and 0.01 to 3 µM for erlotinib).Drugs were added to the respective well and the plate was then incubated at 28.5 • C. DMSO (0.6%) was used as vehicle control for the test compounds.The exposure duration continued up to 72 hpf because it is the endpoint of embryonic development [41].The dead embryos were eliminated, and the fertilized eggs were imaged at 0, 24, 48 and 72 hpf.The morphological deformities of zebrafish embryos were examined using an inverted Zeiss microscope, equipped with a monochromatic sCMOS camera (HAMAMATSU ORCA FLASH 4 LT) and a Colibri illumination system, controlled by Zen software 3.7 (Zeiss, Jena, Germany).
Representative images of the embryos (magnification: 2.5×) after exposure to compound 10h and sorafenib are presented in Figure 6, and in Figure S2 of Supplementary Information for erlotinib.For low doses of sorafenib, the normal development of the embryos is observed at 24 hpf whereas the morphological development is delayed at 48 hpf with an apparent toxicity at the 5 µM dose.Finally, the toxicity (4/5 of the embryos) is apparent at 72 hpf in embryos treated with 1.67 µM with an absence of angiogenesis of the vessels.Erlotinib treatment for 48 and 72 hpf, a complete hatching of the embryos is observed with a delay in vessel angiogenesis at the 0.38 µM dose (no toxicity at 1 µM dose for 72 hpf in agreement with a complete hatching of the embryos).Finally, 48 and 72 hpf treatment with our compound 10h, induces no toxic effect and results in complete hatching of the embryos without delay in vessel angiogenesis up to 5 µM.
Another interest of zebrafish Danio rerio is the possibility to deeply evaluate the effects of angiogenic compounds.In our model, the formation of most vessels is complete, the circulation is present, and the heart is beating after 28 hpf of development.Angiogenesis begins in the somites and in the anterior portion of the embryos.Many zebrafish blood vessels form by angiogenic sprouting and appear to require the same proteins required for blood vessel growth in mammals.By using the mCherry line transgenic zebrafish, it is possible to analyze the direct effect of a compound on the angiogenesis of dorsal vessels.Zebrafish embryos can survive up to a week without an intact circulatory system because defective vessel formation has no immediate lethal effect [42].
Images of dorsal vessels of mCherry Tg zebrafish exposed to compound 10h, sorafenib and erlotinib for 72 hpf are presented in Figure 7 (Zeiss microscope; magnification: 10×).Sorafenib at 1.67 µM induces an inhibition of the dorsal vessel vascularization confirming that this drug has an angiogenic effect.With erlotinib, the angiogenic effect appears at a 3.3 µM dose.For the compound 10h, we cannot observe an anti-angiogenic effect even with a 5 µM dose compared to untreated zebrafish embryos.
defective vessel formation has no immediate lethal effect [42].
Images of dorsal vessels of mCherry Tg zebrafish exposed to compound 10h, sorafenib and erlotinib for 72 hpf are presented in Figure 7 (Zeiss microscope; magnification: 10x).Sorafenib at 1.67 μM induces an inhibition of the dorsal vessel vascularization confirming that this drug has an angiogenic effect.With erlotinib, the angiogenic effect appears at a 3.3 μM dose.For the compound 10h, we cannot observe an anti-angiogenic effect even with a 5 μM dose compared to untreated zebrafish embryos.

Conclusions
Nine 3-(N-phenylmethylidene)amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d] pyrimidine 10(a-i) and three 10-N-(phenylmethylidene)amino-11-imino-11H-naphto[1 ′ ,2 ′ :5,6] pyrano [2,3-d]pyrimidine 10(j-l) were designed, synthesized in five steps including two steps under microwave irradiation.Compounds were evaluated in silico according to Lipinski's rules of five (RO5) and tested experimentally for their antiproliferative activities on a panel of eight tumoral cell lines associated with diploid skin fibroblasts as normal cell lines.The antitumoral assays revealed that compounds 10(h, i) presents submicrolar cytotoxic activity (0.23 < IC 50 < 0.8 µM) on tumor cell lines Huh7 D12, Caco2, MDA MB231, MDA MB468, HCT116, PC3 and MCF7.These two compounds have been assessed in silico for their ADME profile using the "BOILED-Egg" model and also for their bioavailability.Store-operated Calcium Entry (SOCE) assays of 10h in HEK293 cell lines revealed a dosedependent effect but with a minor inhibitory effect on SOCE at low concentrations inducing toxicity in cancer cell lines.In vitro embryotoxicity of compound 10h was performed on mCherry transgenic zebrafish line and compared to sorefenib and erlotinib.With a 5 µM dose of 10h at 72 hpf, complete hatching of the zebrafish embryos without delay in vessel angiogenesis is observed and without toxic effect.The molecule 10h can be considered as a potential bioactive "hit" compound for cancer and now additional work is in progress in our laboratory to complete its biological profile.

Chemistry Section-General Remarks
All reagents and solvents were purchased from Acros Fisher Scientific (Illkirch, France) and Merck Sigma-Aldrich (Saint-Quentin-Fallavier, France) were used without further purification.Thin-layer chromatography (TLC) was accomplished on 0.2-mm precoated plates of silica gel 60 F-254 (Merck, Rahway, NJ, USA) with appropriate eluent.Visualization was made with ultraviolet light (254 and 365 nm) or with a fluorescence indicator.Infrared (IR) spectra were recorded on a Jasco FT-IR 420 spectrophotometer apparatus (Jasco France, Lisses, France) using potassium bromide pellets. 1 H NMR spectra were recorded on BRUKER AC 300 Avance III (300 MHz) spectrometer, 13 C NMR spectra on BRUKER AC 300 Avance III (75 MHz) spectrometer using DMSO-d 6 with tetramethylsilane as an internal reference.Chemical shifts are expressed in parts per million downfield.Data are given in the following order: δ value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint: quintuplet, m, multiplet; br, broad), number of protons, coupling constants J is given in Hertz.The high-resolution mass spectra (HRMS) were recorded in positive mode using direct Electrospray infusion (ESI), respectively, on a Waters Q-Tof 2 or on a Thermo Fisher Scientific Q-Exactive spectrometers at the "Centre Régional de Mesures Physiques de l'Ouest" CRMPO ScanMAT (CRMPO platform, ScanMAT UAR 2025 CNRS, Rennes, France).Melting points were determined on a Kofler melting point apparatus and were uncorrected.Reactions under microwave irradiation (S2Wave platform ScanMAT UAR 2025 CNRS, Rennes) were realized in the Anton Paar Monowave 300 ® microwave reactor (Anton Paar France, Les Ulis, France) using borosilicate glass vials of 10 mL equipped with snap caps (at the end of the irradiation, cooling reaction was realized by compressed air).The microwave instrument consists of a continuous focused microwave power output from 0 to 800W.All the experiments were performed using the stirring option.The target temperature was reached with a ramp of 3 min and the chosen microwave power stayed constant to hold the mixture at this temperature.The reaction temperature is monitored using a calibrated infrared sensor and the reaction time includes the ramp period.The microwave irradiation parameters (power and temperature) were monitored by the Monowave software package for the Anton Paar Monowave 300 ® reactor.Solvents were evaporated with a BUCHI rotary evaporator (Villebon sur Yvette, France).

HEK293 Cell Culture
The HEK293 cell line was maintained in DMEM High Glucose with Sodium Pyruvate (110 mg/mL) and antibiotics (1% of penicillin/streptomycin) in a humidified incubator at 37 • C, supplemented with 5% of CO 2 .

Measurement of Intracellular Calcium Levels
Intracellular calcium levels were evaluated by recording changes in the fluorescence of the dual-wavelength fluorescent calcium-sensitive dye Fura-2AM using the plate reader FlexStation 3™ (Molecular Devices, Sunnyvale, CA, USA).Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak fluorescence emission at 510 nm after excitations at 340 and 380.Modifications in the 340/380 ratio reflect changes in intracellular-free Ca 2+ concentrations.The FlexStation 3™ temperature was set at 37 • C during data acquisition.Thapsigargin (2 µM) solution and Hepes-buffered solution with Ca 2+ containing (in mM): 135 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH were added from a 96-well reservoir plate during Calcium Mobilization Assay running at 100 and 750 s, respectively.Experimental setup parameters were optimized (pipette heights, volumes and rate of additions) to minimize disturbance of the cells while ensuring rapid mixing.The data were stored for later analysis by using SoftmaxPro (Molecular Devices), Excel v.16.66.1 and Graph Pad Prism v.8 software.
Measurement of Store-Operated Calcium (SOC) Entry ER Ca 2+ stores were depleted with 2 µM Thapsigargin an inhibitor of ER SERCA pumps under Ca 2+ -free conditions to determine the magnitude of intracellular Ca 2+ release from ER.The subsequent addition of extracellular Ca 2+ allowed the measurement of SOCE.The magnitude of SOCE was estimated as the maximal values of the normalized F340/F380 ratio following Ca 2+ re-addition.

Calculation and Data Analysis
Data analysis was performed using Softmax Pro and Graph Pad Prism.(Graph Pad): Ca 2+ concentration variations are estimated using the ratio of RFU at 340 and 380 nm (F340/F380) and for each measurement F340/F380 (∆F) ratio values were normalized to the initial basal ratio before TG addition (F0).F340/F380 (∆F/F0) normalization is the best estimation of calcium concentration changes.The inhibition results are expressed in terms of the percentage of response compared to the maximum response on the control condition.Inhibition is estimated as follows: % Inhibition = (100 − % response).4.2.3.In Vivo Zebrafish Danio rerio Assays Zebrafish Maintenance and Embryo/Larva Exposure Zebrafish (transgenic line Tg(kdrl:Hsa.HRAS-mCherry) s896 ) were housed in agreement with the European Union regulations concerning the protection of experimental animals (Directive 2010/63/EU) and approved by the ethics committee (CREEA: Comité Rennais d'Éthique en matière d'Expérimentation Animale) under permit number EEA B-35-040.Zebrafish were raised in our facilities (ImPACcell-Biosit, Rennes) using a ZebTEC Active Blue Stand Alone system (Tecniplast, Buguggiate, Italy) in recirculated water kept at 28.5 • C and spawned under standard conditions.Eggs were collected 2 h post-fertilization (hpf) and kept in glass Petri dishes containing water for subsequent examination under a binocular microscope to confirm fertilization.Developing embryos were distributed in 96-well plates (1 embryo per well) containing 200 µL of E3 embryonic medium (5 mM NaCl, 0.17mM KCl, 0.33 mM CaCl 2 , 0.3 mM MgSO 4 ) and kept in an incubator at 28.5 ± 0.5 • C (14/10 h light/dark photocycle).Chemical treatments were performed by adding DMSO alone (negative control), or test molecule.These experiments were performed in four technical replicates.

In Vivo Zebrafish Danio rerio Toxicity Assays
The experiments were performed using a Zeiss inverted fluorescent microscope (Observer Z1).The zebrafish toxicological test (Zebratox) was realized as follows: the 24 h post-fertilization embryos were arrayed by hand with one embryo per well in black-walled 96-well microplates for fluorescence assays (Perkin Elmer, Waltham, MA, USA).The embryos were treated before hatching from 0 to 72 h post-fertilization.The 20 mM DMSO stock chemicals were diluted in fish water and added to the wells at final concentrations from 0.0 2µM to 5 µM.The negative control (0.25% DMSO) was added to each replicate plate.The experiments were realized in triplicate in three independent plates.The embryos were incubated for 72 h at 28 • C and were imaged, after anesthesia by incubation at 4 • C for 30 min.The zebrafish fertilized eggs were provided by the ImPACcell-Biosit Zebrafish facilities (Rennes, France).

55a
Yield of isolated product.

Huh7 D12 :
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.

Huh7 D12 :
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.
Human hepatocellular carcinoma; Caco2: Caucasian colon adenocarcinoma cell; MDA MB231: Human breast adenocarcinoma; MDA MB468: Breast adenocarcinoma; human (Homo sapiens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-year-old white woman.PANC1: human pancreatic cancer cell line.a IC 50 values are expressed in µM and are the average of three assays, standard error ± 0.5 µM.b Selectivity Index in brackets, calculated according to SI = IC 50 fibroblasts/IC 50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.ens) cell lines, which correspond to the Triple Negative Breast Cancer (TNBC); HCT 116: human colon carcinoma; PC3: cell line initiated from bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old, white, male.MCF7: breast cancer cell line isolated in 1970 from a 69-yearold white woman.PANC1: human pancreatic cancer cell line.a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b Selectivity Index in brackets, calculated according to SI = IC50 fibroblasts/ IC50 on the corresponding tumoral cell line.c Used as positive control.NT: Not tested.

Figure 5 .
Figure 5. Effects of selected molecule 10h on ER Ca 2+ release and store-operated calcium entry (SOCE) measured in HEK293 cells.(A) Time-lapse recording of cytosolic calcium concentration variation (evaluated by changes in normalized fluorescence F340/F380 ratio).Inhibition of 3-(N-2hydroxyphenylmethylidene)amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine 10h on ER Ca +2 release (B) and store-operated Ca +2 entry (SOCE)(C).Tested compound 10h at a concentration ranging from 1.25 to 50 µM was added 5 min before recording intracellular Ca +2 level variation.Endoplasmic (ER) Ca +2 content is totally depleted in Ca +2 free buffer by injection of 2 µM Thapsigargin (TG) and SOCE is next measured by the addition of 1.8 mM Ca +2 in the extracellular medium.The magnitude of SOCE was estimated as the maximal values of the normalized F340/380 ratio following Ca +2 re-addition.Effects on cytosolic calcium variations of the same compound 10h were also evaluated on a B cell line (PLP cell line) without detecting any inhibitory effects on SOCE and Ca 2+ release.
2.5×.b results with erlotinib are reported in Figure S2 of Supplementary Information.
•C in a cooled solution of MeOH during 45 min.Due to the low solubility of these compounds 4 in MeOH, they were recovered using simple filtration on sintered glass (porosity N • 4).