Emile Roussel

Alexis Moréno

Nicolas Altounian

Christian Philouze

Basile Pérès

Aline Thomas

Olivier Renaudet

Pierre Falson

Ahcène Boumendjel

Chromones Bearing Amino Acid Residues: Easily Accessible and Potent Inhibitors of the Breast Cancer Resistance Protein ABCG2

Keywords: Chromones, BCRP, ABCG2, inhibitors, MDR, drug efflux

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Multidrug resistance (MDR) against anticancer drugs is one of the major causes of Three ABC exporters play an important role in the MDR phenotype, P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2), which are overexpressed in tumours and therefore are actively studied [START_REF] Borst | Mammalian ABC Transporters in Health and Disease[END_REF].

BCRP is the most recent discovered ABC human protein [START_REF] Allikmets | A Human Placenta-Specific ATP-Binding Cassette Gene (ABCP) on Chromosome 4q22 That Is Involved in Multidrug Resistance[END_REF][START_REF] Doyle | A Multidrug Resistance Transporter from Human MCF-7 Breast Cancer Cells[END_REF][START_REF] Miyake | Molecular Cloning of CDNAs Which Are Highly Overexpressed in Mitoxantrone-Resistant Cells[END_REF]. It is widely expressed in many organs like lungs, gut, liver, placenta and blood-brain barrier [START_REF] Zhou | The ABC Transporter Bcrp1/ABCG2 Is Expressed in a Wide Variety of Stem Cells and Is a Molecular Determinant of the Side-Population Phenotype[END_REF][START_REF] Leslie | Multidrug Resistance Proteins: Role of P-Glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in Tissue Defense[END_REF]. Moreover, it is naturally overexpressed in stem cells and present in organs responsible for absorption (intestine), elimination (liver and kidney) and distribution of drugs (blood-brain barrier and bloodplacental barriers) [START_REF] Tournier | Strategies to Inhibit ABCB1-and ABCG2-Mediated Efflux Transport of Erlotinib at the Blood-Brain Barrier: A PET Study on Nonhuman Primates[END_REF][START_REF] Kumar | Bioluminescent Imaging of ABCG2 Efflux Activity at the Blood-Placenta Barrier[END_REF]. BCRP is a monomer composed of 655 amino acids making it the smallest ABC protein so far reported [START_REF] Allikmets | A Human Placenta-Specific ATP-Binding Cassette Gene (ABCP) on Chromosome 4q22 That Is Involved in Multidrug Resistance[END_REF][START_REF] Doyle | A Multidrug Resistance Transporter from Human MCF-7 Breast Cancer Cells[END_REF][START_REF] Miyake | Molecular Cloning of CDNAs Which Are Highly Overexpressed in Mitoxantrone-Resistant Cells[END_REF]. BCRP is one of the ABC proteins that is functional once it is dimerized [START_REF] Allikmets | A Human Placenta-Specific ATP-Binding Cassette Gene (ABCP) on Chromosome 4q22 That Is Involved in Multidrug Resistance[END_REF][START_REF] Doyle | A Multidrug Resistance Transporter from Human MCF-7 Breast Cancer Cells[END_REF][START_REF] Miyake | Molecular Cloning of CDNAs Which Are Highly Overexpressed in Mitoxantrone-Resistant Cells[END_REF]. Recently, the 3D structure of human full-length BCRP by electron crystallography or cryo-electron microscopy was reported [14 -16].

Over the past three decades, the ABC transporters have been extensively investigated as targets for the development of inhibitors aiming to enhance and restore anticancer drugs potential [START_REF] Chen | Mammalian Drug Efflux Transporters of the ATP Binding Cassette (ABC) Family in Multidrug Resistance: A Review of the Past Decade[END_REF][START_REF] Iorio | Blood-Brain Barrier and Breast Cancer Resistance Protein: A Limit to the Therapy of CNS Tumors and Neurodegenerative Diseases[END_REF][START_REF] Hida | ATP-Binding Cassette Transporters in Tumor Endothelial Cells and Resistance to Metronomic Chemotherapy[END_REF]. In this regard, BCRP was the subject of extensive studies to conceive and develop in vivo effective inhibitors to be used as chemosensitizers that could work additively or synergistically with an anticancer drug [START_REF] Pires | Highly Potent and Non-Toxic, Chromone Inhibitors of the Human Breast Cancer Resistance Protein ABCG2[END_REF][START_REF] Honorat | MBL-II-141, a Chromone Derivative, Enhances Irinotecan (CPT-11) Anticancer Efficiency in ABCG2-Positive Xenografts[END_REF][START_REF] Hasanabady | ABCG2 Inhibition as a Therapeutic Approach for Overcoming Multidrug Resistance in Cancer[END_REF]. An important number of in cellulo effective inhibitors has been reported but only a few of them were subjected to the preclinical studies [START_REF] Wiese | BCRP/ABCG2 Inhibitors: A Patent Review (2009-Present)[END_REF]. Fumitremorgin (FTC) was the first selective inhibitor of ABCG2, which was considered clinically useless because of its high neurotoxicity [START_REF] Rabindran | Reversal of a Novel Multidrug Resistance Mechanism in Human Colon Carcinoma Cells by Fumitremorgin C[END_REF]. Nevertheless, it was used an inspiration tool for the development of analogs such as Ko143, less toxic and potent inhibitor [START_REF] Van Loevezijn | Inhibition of BCRP-Mediated Drug Efflux by Fumitremorgin-Type Indolyl Diketopiperazines[END_REF][START_REF] Allen | Potent and Specific Inhibition of the Breast Cancer Resistance Protein Multidrug Transporter in Vitro and in Mouse Intestine by a Novel Analogue of Fumitremorgin C[END_REF]. Both FTC and Ko143 are strong inhibitors of ABCG2 and composed of fragments found in some amino acids, such as tryptophane, leucine and proline. Otherwise, our previous work on the BCRP/ABCG2 protein led to the development of chromone derivatives as active, selective, nontoxic inhibitors. Among them, the MBL-II-141 (Figure 1 Based on the structure of MBL-II-141, the importance of hydrophobicity to the inhibition potential and inspired from FTC and Ko143, we focused our present study on chromones bearing one or two hydrophobic amino acids with the aim to develop new class of potent inhibitors that are biological active, non-cytotoxic and specific toward BCRP. The introduction of three amino acids was avoided due anticipated poor solubility that potentially hinder the biological evaluation. Hence, our study targeted 2 series of chromones-embedded amino acids analogues (Figure 1).

Results and Discussion

Chemistry

The 2 series of analogues shown in Figure 1 were synthesized according to the general synthetic pathway described in Scheme 1. The chromone moiety used as the building block for the synthesis of acids 6 and 7 was prepared by adapting our previous described method, using 2,6-dihydroxyacetophenone (1), bromobenzyl bromides and ethyl oxalate [START_REF] Pires | Highly Potent and Non-Toxic, Chromone Inhibitors of the Human Breast Cancer Resistance Protein ABCG2[END_REF][START_REF] Reis | Chromone as a Privileged Scaffold in Drug Discovery: Recent Advances: Miniperspective[END_REF][START_REF] Gaspar | A Valid Scaffold in Medicinal Chemistry[END_REF]]. An update of the general method is available in the experimental section allowing the synthesis of acids 6 and 7 in several gram scale. Structures of the new compounds were fully elucidated through NMR characterization. In addition, X-ray analysis of 9b and 9c confirmed that no racemization had occurred during the first coupling reactions (Figure 2). Supplementary data is available on request from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, quoting the deposition number CCDC-1874945 and -1874944 for 9b and 9c respectively.

As shown in

ABCG2 inhibition activity and cytotoxicity

The new compounds of the two series were evaluated for their ability to inhibit the efflux of mitoxantrone (MX), an anticancer drug substrate of BCRP that, being fluorescent, can be easily quantified by flow cytometry. The inhibition potency of the compounds was investigated in human embryotic kidney ABCG2-transfected cells, HEK293-ABCG2, and their negative control, HEK293 cells. Moreover, the intrinsic cytotoxicity of the compounds was evaluated using the MTT test on the same cell lines. Results are displayed in Table 1. a The percent inhibition of ABCG2 transport activity was studied by comparison with the reference inhibitor Ko143, which fully inhibited; the maximal inhibition for each derivative was measured at 1 mM. b Cytotoxicity was evaluated at 20 µM.

The values of inhibition %, EC 50 and cell viability are the mean of three independent experiments.

For the series I (compounds bearing one amino acid), the maximal percentage of inhibition at 1 µM of tested compound is close to the references, Ko143 and MBL-II-141. The inhibition activity is stronger with a bromine atom in position 2 (9a-e) than in position 4 (8a-d) of the benzyloxy group. Such positional effect could be observed by comparing 8a and 9a, displaying EC 50 of ~0.6 vs 0.10 µM, respectively. The importance of stereochemistry of the amino acid is marginal as can be observed with inhibitors 8c and 8d. Overall, for this series, the highest activity was reached with compounds 9a-e for which the hydrophobicity of the amino acid side chain and the position of the bromine atom are beneficial. Taken together, these results highlight the input of the amino acid hydrophobicity on the inhibition of BCRP. It should be highlighted that the introduction of less hydrophobic amino acid was deleterious for the inhibition activity (independent work, results not shown). The position of the bromine atom on the benzyloxy group influences moderately the inhibition activity. Finally, the introduction of a second hydrophobic amino acid is beneficial to reach a high level of ABCG2 inhibition at the expense of low solubility.

The cytotoxicity of chromone derivatives on ABCG2-transfected HEK293 cells showed a reliable cell viability for all compounds (Table 1). The absence of cytotoxicity prevails an excellent therapeutic index.

Selectivity toward ABCG2 versus ABCB1 and ABCC1

As introduced above, three exporters are mainly involved in the MDR phenotype, ABCB1, ABCC1 and ABCG2. Hence, we evaluated the selectivity of our compounds by measuring their ability to inhibit the drug efflux mediated by ABCB1 and ABCC1. As shown in Table 2, compounds tested at up to 10 µM did not prevent the efflux activity of rhodamine-123 for ABCB1 or calcein for ABCC1, indicating that are selective for ABCG2. More specifically, the five more potent compounds 9b-d and 13a-b displayed the highest selectivity at 1 µM and 10 µM. The values of inhibition % are the mean of three independent experiments.

Interactions of chromone derivatives with the active site of ABCG2

A docking study was successfully achieved using the cryo-EM structure of ABCG2-MZ29-Fab complex (PDB ID: 6HIJ) which contains two MZ29 molecules in the binding site [START_REF] Jackson | Structural Basis of Small-Molecule Inhibition of Human Multidrug Transporter ABCG2[END_REF] (see Supporting Info). For our study, our most active inhibitors (9c, 13a and 13b), MBL-II-141 and ko143 were docked into the active site of ABCG2 defined as comprising both MZ29 ligands.

Except for ko143, all the ligands best poses adopted a bent configuration that filled both MZ29 binding sites, thanks to the ligand internal flexibilities. The best pose of ligand ko143, a polycyclic and less flexible compound, binds between the two MZ29 binding sites (Figure 3). We observed a large variety of favorable interactions as ππ and hydrophobic interactions which are in agreement with the high docking scores and the inhibition activity determined in vitro (Figure 4).

The two dipeptides derivatives 13a and 13b have small structural difference where only the second amino acid is different (13a: (R,S)-Ile -(S)-Val; 13b: (R,S)-Ile -(S)-Leu).

Experimentally as well as by docking, no significant difference of inhibition activity and scores were observed. Note that the racemization that occurs on the first amino acid during the second peptide coupling reaction had no effect on the docking scores.

Conclusions

Years ago, we found that conveniently functionalized chromones offer interesting scaffolds for the design of ABCG2 inhibitors. As, the chemistry needed to access chromones scaffolds is now well mastered, it allows the building and refinement of new inhibitors with higher affinity and selectivity. The presence of a carboxylic acid function at the chromone scaffold allows the introduction of amino acid residues through peptidic coupling conditions. The amino acid residues chosen were those found in the FTC and its derivative Ko143, inhibitors.

As shown here, chromones bearing one bromine atom in position 2 of the bromobenzyloxy group and a hydrophobic mono-or dipeptide constitute promising leads as inhibitors of drug efflux mediated by ABCG2, being more active than the reference inhibitors Ko143 and MBL-II-141. Their easy chemical synthesis is a key advantage allowing the synthesis of multigram scale quantities of the three more potent compounds 9c, 13a and 13b. The three latter inhibitors are entering in preclinical trials (a patent was filed under the number FR1908485 on September 2019) dealing with the synthesis and inhibition activity of ABCG2 with chromones described in this manuscript. Note that attempts to introduce tripeptides on the chromone scaffold led to compounds with extreme low solubility that excluded any biological evaluation. Note: The purity of all products is above 95 % and was determined by HPLC or NMR.

Experimental Section

General procedures

General procedure A: To a solution of 2,6-dihydroxyacetophenone 1 (1,0 equiv.) in acetone (6 mL/mmol) were added simultaneously K 3 CO 3 (3,0 equiv.) and TBAB (0,4 equiv.) previously pounded together. The resulting solution was refluxed during 0.5 or 1 h and then the corresponding bromobenzyl bromide derivative (1,0 equiv.) in acetone (15 mL/mmol) was added drop by drop under inter atmosphere. The solution was refluxed during 4 or 5 h and monitored by TLC (Cyclohexane/Ethyl acetate 3:7). The solution was concentrated under vacuum and then poured into water before to be extracted with ethyl acetate. The combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum.

General procedure B: To a solution of 2 or 3 (1.0 equiv.) in dry THF (10 mL/mmol) wad added a solution of sodium ethoxide generated in-situ from sodium metal (6.0 equiv.) in dry ethanol (15 mL/mmol) at 0 °C and under inert atmosphere. Then, the resulting solution was stirred during 30 min at r.t. before to add the diethyl oxalate (4.0 equiv.) drop by drop.

The solution was warmed up at 50 °C until precipitation and then refluxed during 2 h and monitored by TLC (Cyclohexane/Ethyl acetate 1:1). Then, few drops of concentrated hydrochloric acid (37 %) were added to the suspension until a white colour change of the precipitate. The resulting suspension was refluxed during 1 h before to be cooled down at r.t.

Then, the suspension was concentrated under vacuum and poured into water before to be extracted with ethyl acetate. The combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum.

General procedure C: To a solution of 4 or 5 (1.0 equiv.) in a combination of THF (25 mL/mmol) and ethanol (8 mL/mmol) was added a solution of K 2 CO 3 (1.3 equiv.) in water (12 mL/mmol). Then the resulting solution was warmed up at 50 °C during 4 h and monitored by TLC (Cyclohexane/ethyl acetate 1:1). The solution was concentrated under vacuum and then poured into basified water (K 2 CO 3 20 %) and washed with ethyl acetate before to be acidified with HCl 37 %. The aqueous layer was extracted with ethyl acetate or a mixture of dichloromethane with a small amount of THF and methanol Then, the combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum. The combined organic layers were washed (3 times) with a solution of sodium bicarbonate (20 %). Then, the combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum.

General procedure E: To a solution of 8a or 9a (1.0 equiv.) in combination of THF (25 mL/mmol) and methanol (10 mL/mmol) was added a solution of LiOH (1.5 equiv.) in H 2 O (10 mL/mmol). The resulting solution was stirred 2 h at r.t. and monitored by TLC (Cyclohexane/Ethyl acetate 1:1). Then, the solution was concentrated under vacuum and poured into basified water (NaHCO 3 20 %) and washed (3 times) with ethyl acetate or a mixture of dichloromethane with a small amount of THF and methanol. The basic aqueous layer was acidified with a solution of hydrochloric acid 1 M, 6 M even concentrated hydrochloridric acid 37 % if needs and extracted with ethyl acetate. Then, the combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum.

1-(2-((4-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (2)

The crude was synthetized according to the general procedure A starting from

1-(2-((2-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (3)

The crude was synthetized according to the general procedure A starting from [M] + .

5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (7)

The crude was synthetized according to the general procedure C starting from 5 (

Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucinate (8a)

The crude was synthetized according to the general procedure D starting from 6 (0,300 g, 0.80 mmol) and L-isoleucine methyl ester hydrochloride (0,290 g, 1.60 mmol). It was purified by recrystallisation into methanol to afford 8a, crystals (0.318 g, 79 %).

C 24 H 24 BrNO 6 .

Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-tryptophanate (8c)

The crude was synthetized according to the general procedure D starting from 6 (0.100 g, 0.

Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-D-tryptophanate (8d)

The crude was synthetized according to the general procedure D starting from 6 (0,300 g, 0,80 mmol) and D-tryptophan methyl ester hydrochloride (0.408 g, 1.60 mmol

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucinate (9a)

The crude was synthetized according to the general procedure D starting from 7 (0,511 (M+H + ) 502.0865, found 502.0860.

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-leucinate (9b)

The crude was synthetized according to the general procedure D starting from 7 (0,500 g, 1,33 mmol) and L-leucine methyl ester hydrochloride (0.483 g, 2.66 mmol (M+H + ) 502.0865, found 502.0865.

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-valinate (9c)

The crude was synthetized according to the general procedure D starting from 7 (0,200 g, 0,53 mmol) and valine methyl ester hydrochloride (0.179 g, 1.07 mmol

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-phenylalaninate (9d)

The crude was synthetized according to the general procedure D starting from 7 (0,194 g, 0,52 mmol) and L-phenylalanine methyl ester hydrochloride (0.223 g, 1.03 mmol). It was purified by precipitation into ethyl acetate/cyclohexane (2:1) and then washed with diethyl ether to afford 9d, a pale yellow solid (0.187 g, 70 %). C 27 H 22 BrNO 6 .

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-tryptophanate (9e)

The crude was synthetized according to the general procedure D starting from 7 (0,100 g, 0,27 mmol) and L-tryptophan methyl ester hydrochloride (0.137 g, 0.54 mmol

(5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucine (10)

The crude was synthetized according to the general procedure E starting from 8a (0,671 g, 0.51 mmol). It was purified by recrystallisation in methanol and then washed with diethyl ether to afford 10, white crystals, (0.419 g, 51 %). C 23 H 22 BrNO 6 .

(5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucine (11)

The crude was synthetized according to the general procedure E starting from 9a (0,256 g, 0.51 mmol

Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucyl-Lleucinate (13b)

The crude was synthetized according to the general procedure D1 starting from 11 (0,082 g, 0.

Compounds

All chromone derivatives were dissolved in DMSO, and then diluted in DMEM high glucose medium. Stock solutions were stored at -20 °C and warmed to 25 °C just before to use. Flp-In293, NIH/3T3 and HEK293 cell lines were cultured and obtained in DMEM-High glucose with GlutaMAX TM supplemented with 10 % heat inactivated FBS and 1 % of penicillin/streptomycin in humidified atmosphere at 37 ºC with 5 % CO 2 . In addition, 200 µg/mL hygromycin B, 90 ng/mL colchicine or 750 µg/mL of G418 was added to the growth medium as selection agent for NIH/3T3, Flp-In293 and HEK293 transfected cells respectively. Specifically, for HEK293-MDR the monoclonal cell line was obtained by Fluorescence-Activated Cell Sorting (FACS) using the phycoerythrin-coupled 5D3 antibody (Santa Cruz Biotech) as a native expression reporter.

Cell lines and cultures

Cytotoxicity assays

The cytotoxicity of the compounds 8a-d, 9a-e, 12a-b and 13a-b was determined using a MTT colorimetric assay as reported in literature [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF][START_REF] Berridge | The Biochemical and Cellular Basis of Cell Proliferation Assays That Use Tetrazolium Salts[END_REF]. Briefly, cells were seeded into 96-well plates at a density of 1×10 5 cells/well for a total growth medium volume of 100 µL and incubated overnight. Following, 100 µL of fresh medium containing sequential concentrations of compounds to be tested (dissolved in DMSO in a 0, 1 and 10 μM concentration range)

were added to each well while DMSO control was fixed at 0.5% (v/v). After 72 h incubation, 22 μL of MTT dye in PBS (5 mg/mL) were added to each well and plates were incubated for an additional 4 h at 37 °C. After medium removal and drying the formazan dye crystals were solubilized with 200 μL of DMSO/ethanol (1:1, v/v). The absorbance was measured using spectrophotometry at 570 nm and 690 nm as reference wavelength. The effect of each compound on cell viability in all cell lines was calculated as the difference in absorbance between test wells and medium control wells.

Inhibition tests of MDR-related drug efflux

(a.u.) in the control cells (no substrate and no tested compound). All values are given as the geometric mean of fluorescence emission (a.u.) in a 655-730 nm filter (excitation 635 nm) measured in 5000 events. Assays were performed in triplicate. with a docking score of -10.23 kcal/mol, shows a slightly titled orientation, which avoids the binding of a second MZ29 ligand in cavity 1. Thus, as debated in the literature [START_REF] Jackson | Structural Basis of Small-Molecule Inhibition of Human Multidrug Transporter ABCG2[END_REF] and references therein), one cannot state that there is a single or two inhibitors that bind in the protein.

chemotherapy failure [ 1 - 3 ].

 13 Multidrug resistance (MDR) results in the loss of activity of anticancer agents against cells possessing the MDR phenotype. Multiple mechanisms could be involved in the emergence of MDR, including drug inactivation, target alteration, blockade of apoptosis, stimulation of DNA repair mechanisms and drug efflux [2,4,5]. The latter is caused by the overexpression of membrane proteins from the ABC superfamily that export anticancer drugs outside the cell, leading to the reduction of the drug concentration and ultimately decrease the therapeutic effect on cell proliferation.

  ) has shown promising in vivo activity [19,20,26-28].

Figure 1 .

 1 Figure 1. Structure of MBL-II-141 and the general structures of the new series

Scheme 1 .

 1 Scheme 1. (a) HCl•H 2 N-CH(R 1 )-COOCH 3 , TBTU, DIEA, DMF, RT, 12-24 h; (b) i-LiOH, THF/CH 3 OH/H 2 O,

Scheme 1 ,

 1 peptide coupling of chromone derivatives 6 or 7 with protected amino acids was realized in DMF with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) to afford compounds 8a-d and 9a-c in yields ranging from 28 to 90 %. Peptide coupling reactions could also be performed by using N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI,HCl) and 1-hydroxybenzotriazole (HOBt) in the presence of triethylamine or DIEA at room temperature. Overall, the best yields were obtained with TBTU/DIEA [31-33]. The saponification of esters 8a-d and 9a-e using lithium hydroxide in a mixture of THF/CH 3 OH/H 2 O allowed the formation of the corresponding carboxylic acid with 46 -83 % yields. The second peptide coupling reaction was performed in the same conditions as before to afford compounds 12a-b and 13a-b with 31 -41 % yields. Notably, during this second peptide-coupling reaction we observed a partial racemisation on the asymmetric carbon of the primarily introduced amino acid [32].

Figure 2 .

 2 Figure 2. ORTEP schemes for compounds 9b (left) and 9c (right) with ellipsoids drawn at the 50% probability level. Atoms were colored as bromide in dark red, oxygen in red, nitrogen in blue, carbon in grey and hydrogen in white.

For

  the series II, comparison of compounds 12a-b and 13a-b highlighted again the slight contribution of the bromine atom in position 2 of the bromobenzyloxy moiety. Besides, the results obtained with that series also underlined the positive contribution of the second amino acid. The highest inhibitory activity of ABCG2 was obtained with compounds 13a-b (EC 50 = 0.07 ± 0.01 µM), regardless the nature of the side chain as long as it is hydrophobic. For comparison purposes, the concentration dependence of mitoxantrone efflux inhibition by Ko143, MBL-II-141 and the two most active compounds (9c and 13a) in HEK293-tranfected cells are provided in the supporting information section.

Figure 3 .

 3 Figure 3. Superposition of the best ranked poses of each ligand on the binding poses of MZ29 colored in cyan, 13a in purple (-12.5 kcal/mol); 13b in orange; MBL-II-141 in grey; 9c in dark blue (-10.94 kcal/mol) and ko143 in green (-10.1 kcal/mol).

Figure 4 .

 4 Figure 4. Illustrations of the poses in the binding pocket with respectively in green and cyan, hydrophobic and polar residues. (a) Docking of 9c in the chosen binding pocket of BCRP. (b) Docking of 13a in the chosen binding pocket of BCRP.

5. 1 .

 1 ChemistryNMR spectra were recorded on a 400 MHz Bruker Avance-400 instrument (400 MHz) or on a 500 MHz Bruker Avance-500 instrument (500 MHz). Chemical shifts (δ) are reported in ppm relatively to Me 4 Si used as an internal standard. Electrospray ionization (ESI) mass spectra were acquired by the Analytical Department of Grenoble University on an Waters Xevo G2-S Q TOF instrument with a nanospray inlet. Exact mass was given in (m/z). HPLC analyses were performed with an Agilent 1100 series using a diode array detector and a C18 reversedphase column (Nucleosil C18, Macherey-Nagel, 5 mm particle size, 125 mm x 3 mm) at 45 °C, with a mobile phase composed of A: water and TFA 0.1% and B: CH 3 OH and TFA 0.1% with a gradient 85:15 to 0:100 A:B over 14 min,1 mL/min, 10 µL injection, detection at 254 nm. Melting point (m.p.) expressed in degrees celsius (°C) were obtained on a Büchi melting point B540. Thin-layer chromatography (TLC) used Merck silica gel F-254 plates (thickness 0.25 mm). Unless otherwise stated, reagents were obtained from commercial sources (Alpha Aesar, Sigma-Aldrich and TCI) and were used without further purification.

  General procedure D: To a solution of 6 or 7 (1.0 equiv.) in dry DMF (20 mL/mmol) was added TBTU (2 equiv.) in the presence of DIEA (4 equiv.) under inert atmosphere. The solution was stirred during 30 min at r.t. Then, a solution of the amino acid methyl ester hydrochloride (2.0 equiv.) in DMF (10 mL/mmol) under inert atmosphere was added to the previous one. The resulting solution was stirred during 12 h to 24 h and monitored by TLC (Cyclohexane/Ethyl acetate 1:1). Then, the solution was concentrated under vacuum and poured into acidified water (HCl 1 M) and extracted with ethyl acetate. The combined organic layers were washed (3 times) with a solution of sodium bicarbonate (20 %). Then, the combined organic layers were washed (1 time) with water and brine before dried over magnesium sulfate, filtrated and evaporated under vacuum.General procedure D1: To a solution of 10 or 11 (1.0 equiv.) in dry DMF (20 mL/mmol) was added TBTU (2 equiv.) in the presence of DIEA (5 equiv.) under inert atmosphere. The solution was stirred during 30 min at r.t. Then, a solution of the amino acid methyl ester hydrochloride (2.0 equiv.) in DMF (10 mL/mmol) under inert atmosphere was added to the previous one. The resulting solution was stirred during 12 h to 24 h and monitored by TLC (Cyclohexane/Ethyl acetate 3:2). Then, the solution was concentrated under vacuum and poured into acidified water (HCl 1 M) and extracted with ethyl acetate.

  ABCC1-transfected, ABCB1-transfected and ABCG2-transfected, Flp-In-MDR, NIH/3T3-MDR [34] HEK293-MDR [35] were obtained as previously published elsewhere [36].

5. 2 . 6 .

 26 Selectivity assaysNIH/3T3 and Flp-In were seeded into a 96-well plates cells at a density of 5104 cells/well in 200 µL of medium and incubated overnight. Then, growth medium was changed with fresh medium containing the compounds 3a-h and 4a-c and in presence of 0.5 µM R123 (NIH/3T3) or 0.2 µM cAM (Flp-In) as fluorescence probe to a final concentration of DMSO 0,5% (v/v).Finally, medium was removed, washed with 100 µL of PBS and cells were trypsinized 5 min at 37°C thanks to 25 µL of trypsin. Then, each medium well was suspended with 175 µL of ice-cold PBS with 2% BSA.Intracellular fluorescence was measured with a MacsQuant VRB Analyzer flow cytometer (Miltenyi Biotec) with, at least, 5000 events recorded. While MX was excited at 635 nm and fluorescence emission recorded in a 655-730 nm window, R123 and cAM were excited at 488 nm and recorded in a 525/50 nm filter. The compound inhibition efficacy was estimated by using the previous equation. Assays were performed in triplicate.5.3. DockingThe structure of the ternary complex of human ABCG2 -MZ29-Fab (PDB ID: 6HIJ) was taken as our starting template[START_REF] Jackson | Structural Basis of Small-Molecule Inhibition of Human Multidrug Transporter ABCG2[END_REF]. It was solved by cryo-EM at a resolution of 3.56 Å. The protein exists as a homodimer, and two MZ29 molecules are bound at the central, inwardfacing cavity of ABCG2. We used the Protein Preparation Wizard tool to build an all-atom structure[START_REF] Sastry | Protein and Ligand Preparation: Parameters, Protocols, and Influence on Virtual Screening Enrichments[END_REF]. The missing loops of the protein lie beyond 30 Å of the ligands and were thus not considered in the calculations.The partial atomic charges for the protein and ligand atoms were derived from the OPLS3 force field[START_REF] Harder | OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins[END_REF]. All the water molecules were retrieved. The histidine residues were treated as neutral. The selection of the histidine enantiomers and the orientation of the asparagine and glutamine side-chains were chosen so as to maximize the hydrogen bond network. An allatom energy minimization with a 0.3 Å heavy-atom RMSD criteria for termination was performed using the Impref module of Impact and OPLS3 [41].Low-energy conformers of the chemical compounds, including MZ29, were generated using the LigPrep module. Relevant ionization and tautomeric states at pH comprised between 5 and 9 were generated using the Hammett and Taft methodology implemented in Epik. For compounds available as racemic mixtures, all stereoisomers were generated. The resulting conformations were energy-minimized using OPLS3 force field and a maximum of 32 conformations per ligand were generated and subjected to post-docking minimization[START_REF] Harder | OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins[END_REF].The obtained ligands were subjected to a rigid-receptor docking calculation using Glide in Extra Precision mode[START_REF] Friesner | Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy[END_REF]. The shape and physico-chemical properties of the binding site centered on one MZ29 molecule were mapped onto a cubic grid where the innerbox and outerbox have respective dimensions of 18*20*18 and 39*41*39 Å 3 , thus encompassing both MZ29 binding sites. The hydroxyls of Ser, Thr and Tyr residues were allowed to rotate.During the docking calculations, the parameters for van der Waals radii were scaled by 0.80 for receptor atoms with partial charges less than 0.15e. A maximum of 100 poses were retained and ranked according to the Glide XP docking scoring function. This docking protocol was able to recover the crystallographic binding pose of MZ29 ligand as the first-ranked pose with a Glide docking score of -10.26 kcal/mol (see Figures), and RMSD of thus allowing the binding of a second MZ29 ligand in cavity 1. The second-ranked pose of MZ29,

Table 1 .

 1 Inhibitory activity and cytotoxicity of chromones bearing amino acids residues.

	Entry	R 1	R 2	% Maximal inhibition at 1 µM a	EC 50 (µM)	% of Cell viability, at 10-20 µM b
	8a	(S)-CH(CH 3 )-CH 2 CH 3	/	115 ± 14	0.59 ± 0.08	/
	8b	(S)-CH 2 -Ph	/	92 ± 15	0.27 ± 0.11	84 ± 3
	8c	(S)-CH 2 -(3-indolyl)	/	96 ± 14	0.48 ± 0.07	77 ± 0
	8d	(R)-CH 2 -(3-indolyl)	/	85 ± 12	0.29 ± 0.05	102 ± 1
	9a	(S)-CH(CH 3 )-CH 2 CH 3	/	78 ± 9	0.10 ± 0.01	/
	9b	(S)-CH 2 -CH(CH 3 ) 2	/	115 ± 17	0.14 ± 0.04	87 ± 1
	9c	(S)-CH(CH 3 ) 2	/	87 ± 7	0.05 ± 0.03	76 ± 1
	9d	(S)-CH 2 -Ph	/	100 ± 14	0.10 ± 0.07	82 ± 6
	9e	(S)-CH 2 -(3-indolyl)	/	94 ± 17	0.6 -0.7	88 ± 1
	12a	(S,R)-CH(CH 3 )-CH 2 CH 3	(S)-CH(CH 3 ) 2	72 ± 7	0.25 ± 0.1	89 ± 22*
	12b	(S,R)-CH(CH 3 )-CH 2 CH 3	(S)-CH 2 -CH(CH 3 ) 2	52 ± 5	0.11 ± 0.03	101 ± 12*
	13a	(S,R)-CH(CH 3 )-CH 2 CH 3	(S)-CH(CH 3 ) 2	85 ± 7	0.07 ± 0.01	102 ± 11*
	13b	(S,R)-CH(CH 3 )-CH 2 CH 3	(S)-CH 2 -CH(CH 3 ) 2	83 ± 9	0.07 ± 0.01	104 ± 11*
	MBL-II-141	/	/	98 ± 5	0.13 ± 0.09	/
	Ko143	/	/	106 ± 1	0.09	/
	DMSO 0.5 %					104 ± 8

Table 2 .

 2 Selectivity of the compounds towards the ABC exporters mainly involved in the MDR phenotype.

	Entry	[Compound] (µM)	ABCB1	ABCC1	ABCG2
				% Maximal inhibition at 1 µM
		1	6 ± 2	23.0	92 ± 15
	8b
		10	7 ± 1	19.0	/
		1	7 ± 3	10.3	96 ± 14
	8c
		10	6 ± 1	17.4	/
		1	7 ± 1	11.9	85 ± 13
	8d
		10	6 ± 1	12.6	/
		1	/	5 ± 1	78 ± 9
	9a
		10	/	9 ± 5	/
		1	7 ± 1	20 ± 3	115 ± 17
	9b
		10	5 ± 1	19 ± 2	/
		1	8 ± 1	13 ± 1	87 ± 7
	9c
		10	7 ± 2	18 ± 1	/
		1	5 ± 1	26 ± 1	100 ± 14
	9d
		10	6 ± 1	29 ± 2	/
		1	7 ± 0	23 ± 3	94 ± 17
	9e
		10	5 ± 1	16 ± 3	/

  1 (1.500 g, 9.86 mmol). It was purified by a recrystallisation into ethyl acetate to afford 2, pale yellow crystals (2.321 g, 73 %). C 15 H 13 BrO 3 .

1 H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 7.62 -7.58 (m, 2H), 7.45 -7.41 (m, 2H), 7.30 (t, J = 8.3 Hz, 1H), 6.62 (dd, J = 8.4, 0.5 Hz, 1H), 6.52 (dd, J = 8.3, 0.7 Hz, 1H), 5.14 (s, 2H), 2.48 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 203.43, 159.52, 157.94, 135.96, 133.77, 131.41, 129.98, 121.15, 114.75, 109.63, 103.41, 69.30, 33.04. m.p. 114.8 -116.8 °C. MS (ESI) m/z 320 ( 79 Br), 322 ( 81 Br) [M] + .

  pale yellow crystals (2.583 g, 82 %). C 15 H 13 BrO 3 .The crude was synthetized according to the general procedure B starting from 2 (1.718 g, 5.35 mmol). It was purified by a recrystallisation into methanol or ethyl acetate to afford 4, Hz, 3H).13 C NMR (101 MHz, DMSO-d6) δ 176.39, 160.01, 157.70, 157.23, 150.19, 136.23, 135.29, 131.21, 128.90, 120.61, 115.43, 114.68, 110.54, 109.02, 69.22, 62.59, 13.87. m.p. 148.0 -149.4 °C. MS (ESI) m/z 402 ( 79 Br), 404 ( 81 Br) [M] + . H NMR (400 MHz, DMSO-d6) δ 7.76 (m, 1H), 7.66 -7.59 (m, 4H), 7.24 (dd, J = 8.4, 0.7 Hz, 1H), 7.14 -7.10 (m, 1H), 6.76 (s, 1H), 5.27 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 176.69, 161.44, 157.69, 157.35, 151.15, 136.27, 135.14, 131.20, 128.89, 120.58, 115.21,

	5.1.4. Ethyl 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (4)
	yellow crystals (1.516 g, 70 %). C 19 H 15 BrO 5 . 114.69, 110.58, 108.93, 69.23. m.p. 204.6 -205.3 °C. MS (ESI) m/z 374 ( 79 Br), 376 ( 81 Br)
	1 H NMR (400 MHz, DMSO-d6) δ 7.74 (m, 1H), 7.64 -7.56 (m, 4H), 7.25 -7.21 (m,
	1H), 7.13 -7.08 (m, 1H), 6.77 (s, 1H), 5.24 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1
	J = 8.0, 1.0 Hz, 1H), 7.51 (m, 1H), 7.33 (m, 1H), 7.30 -7.25 (m, 1H), 7.18 -7.13 (m, 1H),
	6.79 (s, 1H), 5.23 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.36 (m, 3H). 13 C NMR (101 MHz,
	DMSO-d6) δ 176.40, 160.01, 158.25, 157.43, 150.24, 135.70, 135.44, 132.17, 129.57, 129.12,
	127.85, 121.05, 115.43, 114.62, 110.78, 108.85, 69.72, 62.61, 13.87. m.p. 155.3 -157.1 °C.
	MS (ESI) m/z 403 ( 79 Br), 405 ( 81 Br) [M-H] + .
	1 H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 7.71 (dd, J = 8.0, 1.1 Hz, 1H), 7.61 (dd,
	J = 7.6, 1.6 Hz, 1H), 7.46 (m, 1H), 7.39 -7.29 (m, 2H), 6.67 (dd, J = 8.4, 0.5 Hz, 1H), 6.56 5.1.6. 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (6)
	(dd, J = 8.3, 0.7 Hz, 1H), 5.19 (s, 2H), 2.47 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ The crude was synthetized according to the general procedure C starting from 4 (1.586
	203.38, 159.60, 157.95, 135.31, 133.90, 132.72, 130.64, 130.41, 128.00, 123.04, 114.62, g, 3.93 mmol). It was purified by trituration into methanol and then washed with diethyl ether

1 (1.500 g, 9.86 mmol). It was purified by a recrystallisation into methanol or ethyl acetate to afford 3, 109.83, 103.19, 70.02, 32.86. m.p. 75.5 -77.4 °C. MS (ESI) m/z 321 ( 79 Br), 323 ( 81 Br)

[M+H] + , 319 ( 79 Br), 321 ( 81 Br) [M-H] + .

5.1.5. Ethyl 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (5)

The crude was synthetized according to the general procedure B starting from 3 (2.583 g, 8,04 mmol). It was purified by a recrystallisation into methanol or ethyl acetate to afford 5, yellow crystals (2.478 g, 76 %). C 19 H 15 BrO 5 . 1 H NMR (400 MHz, DMSO-d6) δ 8.11 (dd, J = 7.7, 1.4 Hz, 1H), 7.79 (m, 1H), 7.68 (dd, to afford 6, a white solid (1.259 g, 85 %). It's possible to recrystallize the desired product in methanol to afford white crystal. C 17 H 11 BrO 5 .

1

  .63, 157.04, 152.91, 136.29, 134.95, 131.21, 128.92, 120.59, 114.54, 112.73, 110.86, 108.99, 69.19, 57.24, 51.88, 35.48, 25.09, 15.37, 10.77. m.p. 117 °C. HRMS (ESI/QTOF) calcd for C 24 H 25 BrNO 6 (M+H + ) 502.0865, found 502.0853.

	5.1.9. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-phenylalaninate
	(8b)
	The crude was synthetized according to the general procedure D starting from 6 (0.472
	g, 1.26 mmol) and L-phenylalanine methyl ester hydrochloride (0.453 g, 2.52 mmol). It was
	purified by recrystallisation into methanol to afford 8b, crystals (0.551 g, 90 %).
	C 27 H 22 BrNO 6 .
	31, 171.22,
	159.22, 157.68, 156.94, 152.71, 137.20, 136.26, 135.08, 131.20, 129.05, 128.91, 128.31,
	126.62, 120.59, 114.50, 112.56, 110.60, 109.06, 69.21, 54.10, 52.20, 35.93. m.p.
	(Decomposition) 153.1 °C. HRMS (ESI/QTOF) calcd C 27 H 23 BrNO 6 (M+H + ) for 536.0709,
	found 536.0704.
	1 H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J = 7.9 Hz, 1H), 7.77 (t, J = 8.4 Hz, 1H), 7.64
	-7.56 (m, 4H), 7.34 (dd, J = 8.5, 0.7 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.70 (s, 1H), 5.25 (s,

2H), 4.37 (t, J = 7.7 Hz, 1H), 3.68 (s, 3H), 2.07 -1.96 (m, 1H), 1.58 -1.45 (m, 1H), 1.32 -1.19 (m, 1H), 0.95 -0.84 (m, 6H).

13

C NMR (101 MHz, DMSO-d6) δ 176.41, 171.40, 159.62,

1571 H NMR (400 MHz, DMSO-d6) δ 9.43 (d, J = 7.9 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.68 -7.54 (m, 4H), 7.30 (d, J = 12.6 Hz, 5H), 7.23 (d, J = 6.1 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.63 (s, 1H), 5.26 (s, 2H), 4.73 (dd, J = 13.5, 9.1 Hz, 1H), 3.68 (s, 3H), 3.26 (dd, J = 13.8, 5.2

Hz, 1H), 3.16 (dd, J = 13.6, 10.3 Hz, 1H).

13

C NMR (101 MHz, DMSO-d6) δ 176.

  g, 1.36 mmol) and L-isoleucine methyl ester hydrochloride (0.495 g, 2.72 mmol). It was purified by recrystallisation in methanol and then washed with diethyl ether to afford 9a,

	crystal, (0.352 g, 51 %). C 24 H 24 BrNO 6 .
	1 H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J = 7.7 Hz, 1H), 8.14 (d, J = 7.4 Hz, 1H), 7.82
	(m, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.52 (m, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (m, 1H), 7.17
	(d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 5.25 (s, 2H), 4.39 (t, J = 7.7 Hz, 1H), 3.70 (s, 3H), 2.10 -
	1.99 (m, 1H), 1.60 -1.48 (m, 1H), 1.33 -1.23 (m, 1H), 0.97 -0.86 (m, 6H). 13 C NMR (101
	MHz, DMSO-d6) δ 176.44, 171.41, 159.61, 157.38, 157.05, 152.97, 135.76, 135.11, 132.17,
	129.56, 129.14, 127.85, 121.04, 114.49, 112.75, 111.09, 108.81, 69.70, 57.26, 51.90, 35.48,
	25.09, 15.37, 10.77. m.p. 123.1 -125.7 °C. HRMS (ESI/QTOF) calcd for C 24 H 25 BrNO 6

  ). It was purified recrystallisation in methanol and then washed with diethyl ether to afford 9c, a pale yellow solid (0.126 g, 48 %). C 23 H 22 BrNO 6 .

	1 H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.82
	(m, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.51 (m, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33 (m, 1H), 7.16
	(d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 5.24 (s, 2H), 4.33 (m, 1H), 3.70 (s, 3H), 2.32 -2.19 (m, 1H),
	1.06 -0.92 (m, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 176.42, 171.34, 159.69, 157.41,
	157.07, 153.03, 135.76, 135.09, 132.17, 129.57, 129.18, 127.84, 121.07, 114.53, 112.74,
	111.10, 108.88, 69.76, 58.56, 51.90, 29.46, 19.04, 18.95. m.p. 127.0 -128.2 °C. HRMS
	(ESI/QTOF) calcd for C 23 H 23 BrNO 6 (M+H + ) 488.0709, found 488.0719.

  ). It was purified by precipitation into methanol and then washed with diethyl ether to afford 9e, a white solid (0.043 g, 28 %). C 29 H 23 BrN 2 O 6 .

	1 H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.36 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.7
	Hz, 1H), 7.83 (m, 1H), 7.69 (dd, J = 8.0, 0.9 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.51 (m, 1H),
	7.39 -7.28 (m, 3H), 7.25 (d, J = 2.2 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.07 (m, 1H), 6.98 (m,
	1H), 6.65 (s, 1H), 5.24 (s, 2H), 4.80 -4.70 (m, 1H), 3.69 (s, 3H). Two proton signals under
	water peak 13 C NMR (101 MHz, DMSO-d6) δ 176.36, 171.52, 159.18, 157.43, 156.95,
	152.81, 136.09, 135.74, 135.21, 132.17, 129.57, 129.15, 127.85, 127.03, 123.79, 121.05,
	121.02, 118.44, 118.04, 114.43, 112.55, 111.48, 110.83, 109.47, 108.89, 69.73, 53.80, 52.19,
	26.38. m.p. 250 -252.8 °C. HRMS (ESI/QTOF) calcd for C 29 H 24 BrN 2 O 6 (M+H + ) 575.0818,
	found 575.0821.

  .89, 108.99, 69.21, 57.19, 35.63, 25.04, 15.52, 10.96. m.p. 230.1 -230.4 °C. HRMS (ESI/QTOF) calcd for C 23 H 23 BrNO 6 (M+H + ) 488.0709, found 488.0715.

	1 H NMR (400 MHz, DMSO) δ 12.91 (s, 1H), 8.98 (d, J = 8.1 Hz, 1H), 7.78 (m, 1H), 7.67
	-7.57 (m, 4H), 7.37 (dd, J = 8.5, 0.6 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.72 (s, 1H), 5.26 (s,
	2H), 4.40 -4.29 (m, 1H), 2.07 -1.96 (m, 1H), 1.61 -1.45 (m, 1H), 1.38 -1.20 (m, 1H), 0.96

(d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).

13

C NMR (101 MHz, DMSO) δ 176.44, 172.23, 159.44, 157.63, 157.04, 153.12, 136.28, 134.91, 131.20, 128.93, 120.59, 114.54, 112.63, 110

  ). It was purified by recrystallisation into methanol and then washed with diethyl ether to afford 11, pale yellow crystals, (0.114 g, 46 %). C 23 H 22 BrNO 6 . J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).13 C NMR (101 MHz, DMSO-d6) δ 176.46, 172.23, 159.43, 157.39, 157.06, 153.18, 135.76, 135.07, 132.16, 129.57, 129.16, 127.84, 121.06, 114.49, 112.64, 111.12, 108.84, 69.73, 57.21, 35.62, 25.04, 15.52, 10.96. m.p.

	4.9 Hz, 3H), 2.12 -1.90 (m, 2H), 1.59 -1.36 (m, 1H), 1.25 -1.11 (m, 1H), 0.99 -0.81 (m, 5.1.21. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucyl-L-
	12H). 13 C NMR (101 MHz, DMSO-d6) δ 176.43, 171.93, 171.64, 170.88, 170.67, 159.10, valinate (13a)
	158.94, 157.64, 157.01, 153.21, 136.29, 134.90, 131.19, 128.91, 120.58, 114.52, 112.61, The crude was synthetized according to the general procedure D1 starting from 11
	112.54, 110.83, 109.00, 69.21, 57.66, 57.42, 57.24, 56.72, 51.72, 51.53, 37.09, 35.99, 29.85, (0,091 g, 0.19 mmol) and L-valine methyl ester hydrochloride (0.064 g, 0.38 mmol). It was
	29.58, 25.65, 24.62, 19.02, 18.84, 18.35, 18.20, 14.95, 14.58, 11.37, 10.55. m.p. purified by precipitation into ethyl acetate and a minimum amount of cyclohexane and then
	(Decomposition) 268 °C. HRMS (ESI/QTOF) calcd for C 29 H 34 BrN 2 O 7 (M+H + ) 601.1549, washed with diethyl ether to afford 13a, pale yellow solid, (0.042 g, 37 %). C 29 H 33 BrN 2 O 7 .
	found 601.1545.
	1 H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.98 (d, J = 8.1 Hz, 1H), 7.78 (t, J = 8.4 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (d, J = 8.4 Hz, 0.5H), 8.60 -8.50 (m, 1H), 8.45
	Hz, 1H), 7.67 -7.57 (m, 4H), 7.37 (dd, J = 8.5, 0.6 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.72 (s, 5.1.20. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucyl-L-(d, J = 7.2 Hz, 0.5H), 8.13 (d, J = 7.5 Hz, 1H), 7.81 (m, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.52
	1H), 5.26 (s, 2H), 4.40 -4.29 (m, 1H), 2.07 -1.96 (m, 1H), 1.61 -1.45 (m, 1H), 1.38 -1.20 leucinate (12b) (m, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.33 (m, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 5.0 Hz,
	The crude was synthetized according to the general procedure D1 starting from 10 (0,123 g, 0.25 mmol) and L-leucine methyl ester hydrochloride (0.091 g, 0.50 mmol). It was purified by trituration into diethyl ether to afford 12b, pale yellow solid, (0.052 g, 34 %). 1H), 5.25 (s, 2H), 4.73 -4.65 (m, 0.5H), 4.54 -4.45 (m, 0.5H), 4.27 -4.21 (m, 0.5H), 4.21 -4.14 (m, 0.5H), 3.65 (d, J = 4.7 Hz, 3H), 2.13 -1.92 (m, 2H), 1.64 -1.47 (m, 1H), 1.28 -1.14 (m, 1H), 1.04 -0.70 (m, 12H). 13 C NMR (101 MHz, DMSO-d6) δ 172.31, 171.91, (m, 1H), 0.96 (d, 230.1 -230.4 °C. HRMS (ESI/QTOF) calcd for C 23 H 23 BrNO 6 (M+H + ) 488.0709, found C 30 H 35 BrN 2 O 7 . 171.88, 157.84, 157.83, 157.82, 157.51, 157.49, 157.46, 157.43, 157.42, 153.69, 153.67,
	488.0715. 1 H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 8.7 Hz, 0.5H), 8.62 (d, J = 7.8 Hz, 0.5H), 153.65, 135.92, 132.79, 130.34, 129.78, 128.33, 121.94, 114.74, 114.72, 114.70, 114.66,
	5.1.19. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucyl-L-8.54 (dd, J = 8.0, 4.5 Hz, 1H), 7.76 (m, 1H), 7.66 -7.56 (m, 4H), 7.35 (dd, J = 8.4, 4.2 Hz, 112.89, 112.87, 112.69, 111.52, 111.46, 109.48, 109.46, 109.37, 109.35, 70.33, 70.31, 58.31,
	valinate (12a) 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 5.25 (s, 2H), 4.58 (m, 0.5H), 4.39 (m, 58.30, 58.21, 58.05, 58.02, 57.96, 52.33, 52.26, 52.23, 52.21, 36.32, 30.00, 25.02, 19.31,
	0.5H), 4.36 -4.24 (m, 1H), 3.62 (d, J = 2.3 Hz, 3H), 2.03 -1.89 (m, 1H), 1.70 -1.55 (m, 19.17, 19.13, 18.66, 18.59, 15.29, 15.28, 10.85. m.p. (Decomposition) 277 °C. HRMS
	The crude was synthetized according to the general procedure D1 starting from 10 2H), 1.55 -1.45 (m, 2H), 1.24 -1.10 (m, 1H), 0.96 -0.79 (m, 12H). 13 C NMR (101 MHz, (ESI/QTOF) calcd for C 29 H 34 BrN 2 O 7 (M+H + ) 601.1549, found 601.1533.
	(0,123 g, 0.25 mmol) and L-valine methyl ester hydrochloride (0.084 g, 0.50 mmol). It was
	DMSO-d6) 176.43, 172.81, 172.60, 170.56, 170.42, 159.14, 158.95, 157.63, 157.01, 153.19,
	purified by trituration into diethyl ether to afford 12a, pale yellow solid, (0.061 g, 41 %).
	136.28, 134.90, 131.19, 128.91, 120.57, 114.51, 112.62, 112.54, 110.84, 109.00, 69.20, 57.29,
	C 29 H 33 BrN 2 O 7 .
	56.64, 51.86, 51.71, 50.39, 50.23, 36.98, 36.00, 25.59, 24.56, 24.23, 24.18, 22.78, 22.66,
	1 H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 8.6 Hz, 0.5H), 8.54 (d, J = 9.4 Hz, 1H), 21.28, 20.90, 14.99, 14.62, 10.54. m.p. (Decomposition) 273 °C. HRMS (ESI/QTOF) calcd
	8.43 (d, J = 7.5 Hz, 0.5H), 7.76 (t, J = 8.3 Hz, 1H), 7.60 (m, 4H), 7.35 (d, J = 8.2 Hz, 1H), for C 30 H 36 BrN 2 O 7 (M+H + ) 615.1690, found 615.1690.
	7.11 (d, J = 8.3 Hz, 1H), 6.69 (d, J = 5.4 Hz, 1H), 5.25 (s, 2H), 4.67 (dd, J = 8.8, 6.9 Hz,
	0.5H), 4.48 (t, J = 8.8 Hz, 0.5H), 4.22 (dd, J = 7.9, 6.9 Hz, 0.5H), 4.15 (m, 0.5H), 3.63 (d, J =

Acknowledgments

This work was supported by CBH-EUR-GS (ANR-17-EURE-0003) to AB and the Ligue Contre le Cancer for PF. protein 1, MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MX : Mitoxantrone; PAR : Parental; PBS : Phosphate buffered saline; R123 : Rhodamine 123; TBTU : 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; THF : Tetrahydrofuran

Cells were seeded into 96-well plates at a density of 510 4 cells/well in 200 µL of medium and incubated overnight. Then, growth medium was switched for fresh medium containing the compounds 8a-d, 9a-e, 12a-b or 13a-b and in the presence of 4 µM MX as fluorescent probe for BCRP-mediated efflux to a final concentration of 0.5 % DMSO (v/v). After 30 min incubation at 37°C, the medium was removed and cells were washed with 100 µL of DPBS followed by cells dissociation during 5 min at 37°C mediated by 25 µL of trypsin. Finally, trypsin was neutralized with 175 µL of ice-cold DPBS with 2% Bovine Serum Albumin (BSA) and cells were carefully resuspended. As selectivity assay, the same experiment was performed for P-gp-and MRP1-mediated efflux with 0.5 µM R123 or 0. Crystal structures determinations and refinements details of the compounds 9b and 9c are available in a PDF file free of charge.

Conflicts of interest

The authors declare no conflicts of interest regarding the publication of this manuscript.


Nicolas Dietrich

Mélanie Jimenez

Manuel Souto

Aaron W Harrison

Christophe Coudret

Eric Olmos

USING POP-CULTURE TO ENGAGE STUDENTS IN THE CLASSROOM

Keywords: General Public, Chemical Engineering, Collaborative / Communication / Writing, Humor / Puzzles / Games, Reactions /History, Philosophy/ Inquiry-Based/Discovery Learning, Physical Properties, Student-Centered Learning

Herein, we describe how video games, TV shows or movies have been used to provide an innovative framework for students to think about chemistry and chemical engineering.

The main objective of this paper is to show how science can be linked with pop culture, to provide educators with recent materials to use in classrooms, and to discuss the benefits and limitations of such tools. The videogames Fortnite, Spiderman and Angry Birds, the TV shows Game of Thrones and Breaking Bad, the Marvel movies, and the animated programs Raving Rabbids and Dragon Ball are used to illustrate different approaches to engage with students and encourage them to learn in a more recreational environment.

■ INTRODUCTION

Attracting a general audience to chemistry and chemical engineering topics is a significant challenge [START_REF] Skluzacek | Summer Camp To Engage Students in Nutritional Chemistry Using Popular Culture and Hands-On Activities[END_REF][START_REF] Clapson | ChemEscape: Educational Battle Box Puzzle Activities for Engaging Outreach and Active Learning in General Chemistry[END_REF] and developing stimulating, alternative teaching methods is important for educators in all disciplines. In several articles, authors describe aspects of popular culture 3 to teach chemistry using resources that are part of everyday life to engage students more effectively. Chemistry classes have been supplemented with material from arts such as music [START_REF] Pye | Chemistry and Song: A Novel Way To Educate and Entertain[END_REF][START_REF] Last | Combining Chemistry and Music To Engage Students' Interest. Using Songs To Accompany Selected Chemical Topics[END_REF][6][START_REF] Ward | Songwriting to Learn: How High School Science Fair Participants Use Music to Communicate Personally Relevant Scientific Concepts[END_REF] (including jazz [START_REF] Crowther | Amino Acid Jazz: Amplifying Biochemistry Concepts with Content-Rich Music[END_REF] and opera [START_REF] André | Opera and Poison: A Secret and Enjoyable Approach To Teaching and Learning Chemistry[END_REF][START_REF] Cobb | Characters in Chemistry: A Celebration of the Humanity of Chemistry[END_REF] ) and paintings [START_REF] Uffelman | Teaching Science in Art: Technical Examination of 17th-Century Dutch Painting as Interdisciplinary Coursework for Science Majors and Nonmajors[END_REF][START_REF] Nivens | Meet Chemistry; Chemistry, Meet Art: Case Studies, Current Literature, and Instrumental Methods Combined To Create a Hands-On Experience for Nonmajors and Instrumental Analysis Students[END_REF][START_REF] Burke | Research with Undergraduates at the Intersection of Chemistry and Art: Surface-Enhanced Raman Scattering Studies of Oil Paintings[END_REF] (including fashion art [START_REF] Tallman | Introducing Students to Fundamental Chemistry Concepts and Basic Research through a Chemistry of Fashion Course for Nonscience Majors[END_REF] ), history [START_REF] Samet | Napoleon's Buttons: Teaching the Role of Chemistry in History[END_REF][START_REF] Bucholtz | Spicing Things Up by Adding Color and Relieving Pain: The Use of Napoleon's Buttons in Organic Chemistry[END_REF][START_REF] Bucholtz | Historical Examples Integrated into the Organic Chemistry Curriculum[END_REF][START_REF] Federico | ConfChem Conference on Case-Based Studies in Chemical Education: Chemistry of Pompeii and Herculaneum-A Case Study Course in Chemistry at the Interface of Ancient Technology and Archeological Conservation[END_REF] , archaeology [START_REF] Beilby | Archaeology, and Analytical Chemistry: A Synthesis of the Liberal Arts[END_REF][START_REF] Giménez | Finding Hidden Chemistry in Ancient Egyptian Artifacts: Pigment Degradation Taught in a Chemical Engineering Course[END_REF][START_REF] Harper | Lipid Residue Analysis of Archaeological Pottery: An Introductory Laboratory Experiment in Archaeological Chemistry[END_REF] , or literature [START_REF] Labianca | An Interdisciplinary Approach to Science and Literature[END_REF][START_REF] Liberko | Using Science Fiction To Teach Thermodynamics: Vonnegut, Ice-Nine, and Global Warming[END_REF][START_REF] Schwartz | Chemistry Education, Science Literacy, and the Liberal Arts[END_REF][START_REF] Spillane | What's Copenhagen Got To Do With Chemistry Class? Using a Play to Teach the History and Practice of Science[END_REF][START_REF] Herrick | Using a Poetry Reading on Hemoglobin To Enhance Subject Matter[END_REF][START_REF] Afonso | The Role of 'Popular' Books in Informal Chemical Education[END_REF] . As examples, educators illustrated chemistry with a Shakespeare's play [START_REF] Kloepper | Bringing in the Bard: Shakespearean Plays as Context for Instrumental Analysis Projects[END_REF] while others found inspiration in detective cases where chemistry was used by the perpetrator of a crime or in their identification [START_REF] Harper-Leatherman | True Apothecary: How Forensic Science Helps Solve a Classic Crime[END_REF][START_REF] Last | Chemistry in Victorian Detective Fiction: "A Race with the Sun[END_REF] . The chemical references from Ian Fleming's James Bond [START_REF] Last | Chemistry and Popular Culture: The 007 Bond[END_REF] series of novels were used to illustrate chemical reactions and substances (sedatives, rocket fuels, etc.). The Harry Potter novel series also offered an opportunity to reproduce wizardry experiments [START_REF] Copes | The Chemical Wizardry of J. K. Rowling[END_REF] in a chemistry lab (e.g. with invisible and color-changing inks, colored flame in a jam-jar). Famous characters from the Sherlock Holmes stories (from Conan Doyle's novels) have been used to create a fictional mystery based on chemistry [START_REF] Waddell | The Chemical Adventures of Sherlock Holmes: The Case of the Screaming Stepfather[END_REF][START_REF] Waddell | The Chemical Adventures of Sherlock Holmes: The Blackwater Escape[END_REF][START_REF] Shaw | The Chemical Adventures of Sherlock Holmes: The Serpentine Remains[END_REF] , as has a murder novel of Agatha Christie [START_REF] Southward | Precipitation of a Murder: A Creative Use of Strychnine Chemistry in Agatha Christie's The Mysterious Affair at Styles[END_REF] . Michael Crichton's novel Jurassic Park [START_REF] Hollis | Jurassic Park as a Teaching Tool in the Chemistry Classroom[END_REF] has been an inspiring source of discussions on the chemical defense of plants or chemicals used by animals for communication. Cartoons [START_REF] Kennepohl | Drawing Attention with Chemistry Cartoons[END_REF][START_REF] Giese | Connecting Current Literature, Cartoons, and Creativity: Incorporating Student-Created Cartoons in a Biochemistry Course to Enhance Learning[END_REF] and comic books [START_REF] Kakalios | The Materials Science of Marvel's The Avengers-Some Assembly Required[END_REF] can also illustrate chemical principles (e.g. microscale chemistry in Archie's comic book [START_REF] Szafran | Microscale Chemistry in the Comics[END_REF] or general chemistry in Dick Tracy [START_REF] Carter | Chemistry in the Comics: Part 1. A Survey of the Comic Book Literature[END_REF][START_REF] Carter | Chemistry in the Comics: Part 2. Classic Chemistry[END_REF][START_REF] Carter | Chemistry in the Comics: Part 3. The Acidity of Paper[END_REF][START_REF] Carter | Chemistry in the Comics: Part 4. The Preservation and Deacidification of Comic Books[END_REF] , DC Comics [START_REF] Ruekberg | A Chemistry Tidbit for Batman Fans[END_REF] or Marvel comics [START_REF] Kakalios | The Materials Science of Marvel's The Avengers-Some Assembly Required[END_REF] ).

Recently, lab safety rules have been presented to students with comics [START_REF] Di Raddo | Teaching Chemistry Lab Safety through Comics[END_REF] , graphic novels and mangas [START_REF] Kumasaki | Presenting Safety Topics Using a Graphic Novel, Manga, To Effectively Teach Chemical Safety to Students in Japan, Taiwan, and Thailand[END_REF] . Beyond novels and comics, movies are currently one of the biggest providers of pop-culture [START_REF] Frey | Put Some Movie Wow! In Your Chemistry Teaching[END_REF][START_REF] Baños I Díez | Using Feature Films as a Teaching Tool in Medical Schools[END_REF] . The list of movies used to illustrate chemistry is impressive, including for example Apollo 13 [START_REF] Goll | Teaching Chemistry Using the Movie Apollo 13[END_REF] , October Sky [START_REF] Goll | Teaching Chemistry Using October Sky[END_REF] , Star Trek [START_REF] Bormanis | Science Fictions and Fictional Science: A Brief Tour of Science in the Star Trek Universe[END_REF] and many others [START_REF] Wink | Almost Like Weighing Someone's Soul": Chemistry in Contemporary Film[END_REF][START_REF] Griep | Based on a True Story: Using Movies as Source Material for General Chemistry Reports[END_REF][START_REF] Taarea | The Elements Go to the Movies[END_REF][START_REF] Stengler | Beyond Teaching and Learning: Bringing Together Science and Society with and through Movies[END_REF][START_REF] Nelson | Hollywood Chemistry: When Science Met Entertainment[END_REF] . The omnipresent Marvel franchises often invoke various areas of chemistry and chemical engineering such as nanotechnology in the suits of Iron Man [START_REF] Kakalios | The Materials Science of Marvel's The Avengers-Some Assembly Required[END_REF] , properties of the fictional metal vibranium in Black Panther [START_REF] Collins | Vibranium, and the Periodic Table[END_REF] , the quantum realm in Ant-Man [START_REF] King | The Science (and the Scientists) Behind 'Ant-Man' -The New York Times[END_REF] , or material sciences in Spider-Man [START_REF] Allain | The Physics of Spider-Man's Webs[END_REF] . Television is also a good way to illustrate chemistry [START_REF] Slabaugh | Trends in Instruction of Chemistry by Films and Television[END_REF] and famous shows used for this purpose include The Price is Right [START_REF] Clark | The Price Is Right" for Your Classroom[END_REF] , The Big Bang Theory [START_REF] Li | Communicating the Nature of Science Through The Big Bang Theory: Evidence from a Focus Group Study[END_REF] , CSI [START_REF] Cass | Constructing Crimes: How the CSI Effect Is Created[END_REF] , The Simpsons 66 , Bones [START_REF] Milanick | Fact or Fiction? General Chemistry Helps Students Determine the Legitimacy of Television Program Situations[END_REF] , ER and House [START_REF] Millard | Television Medical Dramas as Case Studies in Biochemistry[END_REF] . Trending games are also an interesting pathway to involve students in general chemistry courses [START_REF] Dietrich | Using the "Red Bottle" Experiment for the Visualization and the Fast Characterization of Gas-Liquid Mass Transfer[END_REF][START_REF] Yang | Optical Methods to Investigate the Enhancement Factor of an Oxygen-Sensitive Colorimetric Reaction Using Microreactors[END_REF][START_REF] Yang | Local Investigations on the Gas-Liquid Mass Transfer around Taylor Bubbles Flowing in a Meandering Millimetric Square Channel[END_REF][START_REF] Dietrich | Bubble Formation at an Orifice: A Multiscale Investigation[END_REF][START_REF] Dietrich | Fast Measurements of the Gas-Liquid Diffusion Coefficient in the Gaussian Wake of a Spherical Bubble[END_REF][START_REF] Xu | Comparison of Three Different Techniques for Gas-Liquid Mass Transfer Visualization[END_REF][START_REF] Xu | Characterization of Bubble Shapes in Non-Newtonian Fluids by Parametric Equations[END_REF][START_REF] Xu | Mass Transfer and Diffusion of a Single Bubble Rising in Polymer Solutions[END_REF] . Educators have included pop-culture elements to solve educational escape games, e.g. to unveil the name of a super hero (Clark Kent from Superman) or famous gimmicks of a character [START_REF] Vergne | Escape the Lab: An Interactive Escape-Room Game as a Laboratory Experiment[END_REF][START_REF] Dietrich | Escape Classroom: The Leblanc Process-An Educational "Escape Game[END_REF] such as "Bazinga" from the TV series The Big Bang Theory. Moreover, while many educators have successfully used pop-culture themes to introduce their students to scientific concepts, educators have continually tried to use new techniques to engage their students, such as the creation of a Science Café on the pop-culture theme [START_REF] Burks | Where Science Intersects Pop Culture: An Informal Science Education Outreach Program[END_REF] . Video games 100 have become an increasingly important part of the entertainment industry, and they are also considered a form of art 101 ; surprisingly the use of videogames to illustrate chemistry or chemical engineering 102 is relatively unexplored in the literature even though pedagogical videogames exist [103][104][105] . Video games being used directly in education is an increasingly popular research topic and even just playing commercial video games has been shown to benefit important skills in adult learners like effective communication, executive function, and resourcefulness 106- 108 . Though these examples have been focused on skills-based learning, using video games for content-based learning in chemistry such as described below is beginning to be explored. The most notable example can be seen in the recent work by Smaldone, et al. where the authors presented a modified version of the popular video game Minecraft called PolyCraft World. In the game, the player collect resources and uses chemical refinement and synthesis techniques to craft equipment and materials in the game 109 . Initial results indicated that students who played the game learned advanced chemistry even without grading incentive or traditional classroom instruction. Given the difficulty of creating an engaging game content de novo, finding existing popular games to modify or for insight into how games can be used for educational purposes like PolyCraft World is an important resource. The main objective of this paper is to explore recent popculture references and the untapped potential of videogames for teaching purposes and more broadly propose new approaches to link chemistry/chemical engineering and pop culture. We present a range of activities inspired by videogames but also TV shows and recent movies, with their context and materials for implementation by the wider community. In a first section, three different activities that have been applied with students will be presented and the feedbacks from students' are discussed; in a second section, some additional activities used for outreach events are described.

■ ACTIVITIES

We report here activities related to the videogames Fortnite, the TV shows Games of Thrones and Breaking Bad and the movie Black Panther. All these activities were tested and evaluated with students' (see supplementary information for more details about the activities).

FORTNITE

Fortnite is an online video game developed in 2017 by Epic Games. The game mode includes a free-to-play battle royale game where up to 100 players fight in increasingly smaller spaces to be the last person standing. The game has cartoon graphics and does not present graphic violence such as bloodshed. Fortnite Battle Royale became a resounding success, drawing in more than 125 million players in less than a year and earning hundreds of millions of dollars per month. In early 2018, students of Tippecanoe High School in Ohio, USA, used a social media platform to challenge their professor to have a Fortnite-based final exam in chemistry. Although there is no report on how this story ended, it motivated the authors of this article to develop a new Fortnite-based protocol for chemistry classes that could be used by teachers facing a similar situation.

The videogame Fornite is more oriented toward physics than chemistry (e.g. bullet and rocket trajectories, amount of force per impact of projectiles, etc.). Nevertheless, in the game, once players have landed on the map, they must scavenge for weapons, resources and other items. The objective of this activity is to reproduce in the lab several items present in the Fortnite video game to illustrate simple chemical reactions.

One of these items is the "slurp juice", a consumable that adds shield and health points to the character. This item is represented by a two-colored viscous fluid with beads in a jar, which could be prepared in the chemistry lab with a teacher. The fluids can be made as slime paste using common material (hot water, a spoonful of borax, and glue) or chemical products (water, polyvinyl alcohol, and boric acid) in order to illustrate the mechanism of polymerization of polyvinyl alcohol 110 . This activity is recommended for middle school students', high school students' or even for beginners in chemistry at University level. Materials and methods for this activity are detailed in the supplementary information. Some glass beads and dyes (green for the bottom fluid and blue for the top fluid) can be added after the polymerization in order to improve the resemblance to the "real" slurp juice as depicted in Figure 1.a. The polymer unique properties (of both a solid and a liquid) can first be discussed in the classroom. Then experiments can be planned to answer the following questions: i)

How can you make the polymer stretch the farthest?

ii) Does the amount of borax added change the slime structure?

iii) What method of storage will make the polymer last the longest? A second famous item in the game is the "shield potion", a glowing blue liquid in a jar with gems floating inside. This item can be easily made using tonic water and a black light. The quinine (Figure 1.b) in tonic water will glow blue [111][112][113] , and the carbonic bubbles can perfectly mimic the gems (Figure 1.c). This experiment highlights the phosphorescence properties of quinine but fluorescein could also be used to show fluorescence effects 114 . Other products, such as energy drinks with B vitamins, milk, vanilla ice cream, caramel, and honey (to give a yellow color) could be used to produce a "stink bomb" (Figure 1.d) by adding few spoonfuls of table vinegar and hydrogen peroxide or directly with luminol to illustrate chemiluminescence [115][116][117] . The "stink bomb" can also illustrate chemical reaction and gas-liquid equilibrium as it is composed of ammonium hydrosulfide (NH4SH), an unstable compound that decomposes into ammonia and hydrogen sulfide. As soon as the container is broken (open), the dissolved ammonium sulfide rapidly decomposes and liberates copious amounts of the pungent gas. Landing. This episode is famous for its epic wildfire explosion during the Battle of Blackwater Bay. In the series, wildfire is a flammable liquid that is created and controlled by an Alchemist's Guild. When ignited, it can explode with tremendous force and the resulting fire cannot be extinguished with water. Wildfire is identifiable by the distinctive green hue of its flames and a bright green color in its liquid state.

GAME OF THRONES

Game of

The objective of this activity is to reproduce the flame. This activity is recommended as a demonstration only for high school or university students' but the wildfire must be made in the lab only by the educator with screen protection and a safety disclaimer [START_REF] Kumasaki | Presenting Safety Topics Using a Graphic Novel, Manga, To Effectively Teach Chemical Safety to Students in Japan, Taiwan, and Thailand[END_REF]120 (see hazard section).

When mixing boric acid with methanol; the reaction occurring is the synthesis of trimethyl borate, B(OCH3)3 depicted in Figure 2.a, and is as follows:

H3BO3 +3 CH3OH → B(OCH3)3 + 3 H2O (1)
Trimethyl borate burns distinctively green, as represented in Figure 3.a, due to the presence of boron:

4 B(OCH3)3 + 21 O2 → 4 BO3 + 12 CO2 + 18 H2O (2)
The experiment can be carried out with common products such as gas line antifreeze (methanol) and laundry booster/cleaning agent (borax -sodium borate) although this gives a mixture of orange and green flames due to the presence of sodium with the borate. This experimentation could be completed with the flame test to discuss the effect of ion on the flame color 121 , as done for the older pop culture reference Harry Potter [START_REF] Copes | The Chemical Wizardry of J. K. Rowling[END_REF] . More information about the experiment is given in the supplementary section.

BLACK PANTHER

Movies are the pop culture medium that is most widely used to illustrate science and chemical concepts, especially science fiction and superhero movies. Black Panther has been used recently to encourage students to think about an imaginary element, called Vibranium [START_REF] Collins | Vibranium, and the Periodic Table[END_REF] . In the movie, Wakanda's economy focuses on the production and use of this element, which has extraordinary chemical and physical properties. In this activity, the students were questioned on the possible place of Vibranium in the periodic table and its properties. The students' were separated in several groups and have to build a product with this element. A majority of the students developed a process to build the Vibranium steel, based on classical steel production (depicted in the supplementary section) whereas only very few groups worked on super-plastics or super-fertilizers based on vibranium. This overwhelming representation of steel production must be due to the influence of the movie, then an idea to avoid this behavior could be to impose a different product for each group, or to ask students for an alternative to the steel application. This activity is recommended as a project support or a discussion for university students' in chemistry or chemical engineering. The periodic table is a chemical concept that is easy to link with pop culture, and a large number of films include an element in their title [START_REF] Taarea | The Elements Go to the Movies[END_REF] . Many fictional elements are also present in the movies 128 (Table 1). As an activity for students, they could be asked to find an occurrence in a movie of a real or a fictional element and to discuss the properties of ■ HAZARDS Boric acid can be irritating for the eyes, skin, nose, throat and lungs, so it is recommended to wear rubber gloves when handling cleaning products, to wash away any cleaning product with water, and to avoid contact with nose, mouth, and eyes.

Boric acid is classified as toxic to reproduction and should not be handled by students.

The reaction involves fire therefore it should be conducted by a trained person in a safe area with a use of a protection shield. Prepare a lid to cover the container in order to quench the fire. Do not attempt to refill the container during or after the experiment.

Methanol can cause metabolic acidosis, neurologic sequelae, and even death, when ingested, so it is recommended to wear rubber gloves when handling cleaning products, to wash away any cleaning product with water, and to avoid contact with the nose, mouth, and eyes. Personal protective equipment such as dust mask, eyeshields, face shields and gloves should be used for the manipulation of the resazurin dyes.

■ STUDENT'S EVALUATION AND DISCUSSION

Students from three separate courses used these activities ("Fortnite", "Game of Throne", "Breaking Bad" and the "Black Panther") after attending a series of lectures ( 10 At the end of the activity, the teacher invited all students to evaluate the activities by completing a printed form containing ten questions with responses based on a Likert 134 scale (the response rate was 95%). Data are presented in Figure 3. In general, all statements showed high levels of agreement ("agree" and "strongly agree") on the benefits of pop culture, ranging from 60% to 92.8% of those surveyed. A majority of students (92.8%) enjoyed the use of pop culture in the courses and thought it was attractive and helped capture their attention (86.4%). A majority (81.6 %) also agreed that the use of pop culture elements helped them make connections between the theoretical aspects of the course and their application and helped improve their understanding (69.6%). Fewer (64.8 %) students agreed that the pop culture helped them to develop their critical thinking or made them think about aspects of the course (60%). It is worth noting that a majority thought that pop culture helped them to connect with other students (71.2%) and even more with educators (79.2%). Finally, a large majority (83.2%) think that the use of pop culture is an excellent educational tool that can complement the classical application of chemical engineering. In a free-response section of the questionnaire, students were asked to provide comments on the activities. One of them was "I will keep this exercise in mind all my life".

■ SUGGESTIONS FOR ADDITIONAL OUTREACH ACTIVITIES

In this section, five supplementary activities based on recent pop-culture references are proposed as a support for educative purpose ("Spiderman", "Angry birds", "Stranger Things & Chernobyl", "Raving rabbids" and "Dragon Ball"). All of them were performed with students' or visitors during open days or outreach forums. A specific evaluation is proposed at the end of this section to discuss the benefits of such activities. Completion of the collection and analysis of these samples grant the players research tokens that can be used to upgrade their suit and gadgets. Though a limited amount of the underlying scientific content is conveyed to the player in analyzing these spectra, it is very straightforward to create a puzzle game using a similar format that could be an effective way to teach concepts in atomic spectroscopy. An example of such a puzzle game is shown in Figure 4.a.

SPIDER-MAN

As pointed out in the movie

(a) In this puzzle, the player assigns the unknown absorption shown on the left as a simple sum of the individual atomic spectra using the emission spectra inventory on the right. Providing conceptual background about atomic absorption and emission spectroscopy and using known line positions of hydrogen atom (Balmer Series) or alkali atom spectra as shown in Figure 4.a conveys actual science to the player. In addition to assigning spectra using a spectral line inventory, an exercise could be envisaged using the Rydberg formula:

(b) (c)
! " = 𝑅𝑍 # $ ! $ ! " - ! $ # " & (3)
to predict the different electronic spectra by varying the nuclear charge and principal quantum numbers of hydrogenic atoms. 137 The puzzle game could also be expanded to other kinds of absorption spectroscopy such as infrared (IR) absorption.

This activity is recommended for middle school students' and high school students' as a game or a discussion in the classroom with a video of the game. This inventory-based video game puzzle may be well-suited for an electron impact mass spectrometry-based game as well. Instead, consider that your fragment inventory shown on the right is a molecular fragment inventory, and the player determines the molecular structure of the parent ion based on the fragmentation pattern. An interactive, video game puzzle could also include variables like varying the electron impact energies to show how the mass spectrum changes as a function of hard vs. soft ionization.

ANGRY BIRDS

Another famous video game is Angry Birds, a casual puzzle video game developed by Rovio Entertainment in 2009 138 . The gameplay revolves around players using a slingshot to launch birds at pigs stationed in or around various structures, with the goal of destroying all the pigs on the playing field. The Angry Birds series had a combined tally of over 2 billion downloads across all platforms and has been adapted in movies and television shows. In previous work, the franchise has been used as an introduction to the separation of waterborne pathogens using microfluidics 136 , channels in the micrometer range allowing for a precise control of fluid and particles at the micrometer scale 139 . In this activity, the analogy with pop culture icons was used to rapidly identify harmful pathogens in water samples. The wide range of particles that would normally be present in water but not visible to the naked eye due to their microscopic size are represented magnified using modelling clay. This activity is recommended for middle school students' and high school students' as hands-on activity. Some particles, representing pathogens that can cause a potential threat to human health, have facial expressions mimicking those from the video game Angry Birds for rapid identification (Figure 4.b). The overarching aim of the activity was then to engineer a suite of devices that replicate ongoing research in the field to isolate those "angry" pathogens and understand the chemistry associated with 1) the detection of those pathogens (fluorescence), the manufacturing process (e.g. bonding) of microfluidic devices (Figure 4.c) and how viscous liquids can be used to mimic at a macroscale a microfluidic environment 136,140 .

STRANGER THINGS & CHERNOBYL

Another TV shows that can be used for illustrating chemical reaction is Stranger Things, an American science fiction horror web television series created by the Duffer Brothers and released on Netflix in 2016. In the show a large tentacled monster named the Mind Flayer terrorizes the citizens of Hawkins, and in season 3, it expresses a huge desire to consume chemicals, most often poisonous (e.g. fertilizer and cleaning products). The reason is that the monster wants to create caustic reactions associated with this chemical consumption to cause violent explosive transformations into amorphous blobs of human biomass. This example is a very good tool to discuss acid-base reactions and pH. Chernobyl is a historical drama television miniseries created and written by Craig Mazin and directed by Johan Renck for HBO in 2019. The series centers around the Chernobyl nuclear disaster of April 1986 and the unprecedented cleanup efforts that followed. Chernobyl received widespread critical acclaim and became the highest rated TV show in history on some review platforms. The series is a very good example to discuss the operating principle of a nuclear power station, nuclear reactions and the principle of radioactivity 141 . Other major accidents can also be mentioned (Three Mile Island and Fukushima) in order to discuss the danger of this type of energy. Beyond the chemical aspect of the nuclear power plant, it is possible to encourage students to think about the series. For example, during episode 3, the basement of the plant is successfully drained, but a nuclear meltdown has begun, threatening to contaminate the groundwater. Authorities decide that a heat exchanger is needed under the plant to cool the reactor core and, according to the scientists, all the liquid nitrogen available in the Soviet Union will be required. This can be solved from a chemical engineering point of view, with a simple heat balance between the core of the plant and the nitrogen flowing below the power station as described Equation ( 4):

𝑄 = 𝑚 !"#$ . 𝐶 %,!"#$ . '( ') = 𝑈 *$+) $-!*+./$# . 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 0$+) 1-*+./$# . ∆𝑇 23 (4)
From this balance, the students can estimate, with some hypotheses on the parameters of the reactor core given in the supplementary material, the amount of nitrogen necessary to cool the power station down, from Equation 5:

𝑄 = 𝑊 4 ! . 𝐶 %,4 ! . 1𝑇 4 ! ,"5)3$) -𝑇 4 ! ,6.3$) 3 (5)
This show is thus a good example of the links between chemistry, chemical engineering, reactor design and a recent pop-culture hit that could be used as project or a discussion in the classroom for middle school students' and high school students'. to decipher the reaction of a sample rabbit. To make it more realistic, a library of images has been compiled in which the cartoonist has selected the lab's etiquette.

RABBIDS INVASION

Rabbids

Thus, along with the mandatory white lab coats, there is a board covered with scientific formulas, some from physics and some from chemistry. The surprise is that the chemistry ones are complex and related to a specific field of organic chemistry called "photochromism" (reproduced in Figure 5a) and a chemical reaction describing the light-induced coloration of a dye belonging to the spiropyran family is clearly visible 144 .

Photochromic dyes are commonly used in sunglasses, to adapt the optical density of the lenses to the surrounding luminosity. However, spiropyran dyes are rather unstable and fade away readily when used intensively. Thus, these dyes are now used for pedagogical or research purposes. A famous example is the commercially available "NitroBIPS", the photochemistry of which can be tested in the teaching lab 145,146 . The one on display in the Rabbid Invasion is the "1′,3′-dihydro-8-methoxy-1′,3′,3′-trimethyl- Genki dama (元気玉) is one of the most powerful attacks of Son Goku, the famous hero of the anime (illustrated in Figure 6). It consists of a giant sphere of vital energy provided by all the living cells surrounding Goku. Although similarities seem to exist with the well-known Kamé Hamé Ha (かめかめ波), no real description of its energy nature can be found. While this energy is indeed able to vaporize in some of the anime episodes, this energy also appears as mostly mechanical in others (buildings destruction, etc). One reasonable hypothesis is to assume that it behaves as a blackbody, whose spectral irradiance distribution is given by the Planck distribution.

This activity is recommended for middle high school or university students' (Chemical Engineering) as a project or a tutorial with professor.

Thus, let us consider that this sphere is a blackbody of temperature TGD, with a radius rGD = 10 m. Let also assume that the shortest distance between the sphere surface and Son Goku is p = 20 m (see Figure 6). It is also important to note that no value for the temperature TG seems available but, regarding the visible emission (bright blue) of the Genki dama, a reasonable temperature would be around 6000 K. The radiative emission of Son Goku should also be neglected and Son Goku is assumed to be a

With 𝐴 9: (m 2 ) the total area of the Genki dama, 𝐹 9:;<9 the view factor from the Genki-Dama sphere to Son Goku, assumed as the external surface of a coaxial cylinder (see Figure 6). 𝜎 = 5.67 × 10 ;= W/(m 2 K 4 ) is the Stefan-Boltzmann constant. The resolution of the problem is given in the supplementary section.

As some of these suggested additional activities were not tested in situ with students, they were presented to a panel of students' (N = 35 -Chemistry, Environment and Chemical Engineering -Academic year 2019/2020) during a scientific discussion (2 h) on the links between science and pop-culture in order to evaluate students'. The survey was conducted as an anonymous paper exercise, with students required to strongly agree, agree, neither/neutral, disagree or strongly disagree with a series of 10 statements. The majority of respondents were positive about educational benefits of pop culture with 82% of the respondents agreeing that the use of pop culture was a useful learning activity. More specifically, 75% of the students agreed (or strongly agreed) that pop culture had helped them apply chemistry/chemical engineering in a useful way.

Having just discussed about these pop-culture activities, the majority of respondents agreed (78%) that they would appreciate this approach in different subject areas of their cursus. These findings support the high level of student engagement and interaction observed by instructors when pop-culture is used. The data collected show that the majority of students enjoyed discussing science with a pop culture approach, in the open section of the survey some students' recommended to use it at a recreative moment between students' or with educators.

■ DISCUSSION

Pop-culture in classrooms can be beneficial as it creates engaging links between chemical concepts and their applications, and between educators' and students' interests. The objective is not to promote movies or video games but to connect and use the interest of students for these pop culture elements towards learning science.

Connection with recent pop culture elements such as those proposed in the present work could be used as support for demonstrating reactions, as side projects, analogies to communicate concepts and/or as a platform to start discussions. Educators need to be careful about inappropriate content depending on the student's age, to avoid spoiling anything for someone reading or watching a show, movie or book, to make sure the science involved is actually correct. It is also important to leave the students free to search chemistry during project in all types of media, recent or not, according to their interests to unleash their curiosity. Finally, pop-culture promotes critical thinking and cultural literacy, which are important skills for students to develop.

CONCLUSION

The present work provides creative and original activities based on pop culture (e.g.

Figure 1 .

 1 Figure 1. Example of Fortnite items that can be made in the chemistry lab: (a) "slurp juice" (b) the quinine molecule (c) "shield potion" (d) "stink bomb".

  Thrones is an American fantasy drama television series created by David Benioff and D. B. Weiss for HBO in 2011 118 . It is an adaptation of A Song of Ice and Fire, George R. R. Martin's series of fantasy novels, the first of which is A Game of Thrones, first published on August 1, 1996 119 . "Blackwater" is the ninth and penultimate episode of the second season of HBO's medieval fantasy television series. The entire episode is dedicated to the climactic Battle of the Blackwater, in which the Lannister army, commanded by acting Hand of the King, Tyrion Lannister, defends the city of King's

Figure 2 .

 2 Figure 2. (a) The triethyl borate molecule and an illustration of the "green fire" reaction from Game of Thrones (b) Synthesis of the dextroamphetamine Many other references from this series can be used for illustration, such as the reaction

  h) covering the topic of chemical engineering. The first two activities were used as a demonstration tool while the last two were done as a supplementary homework project. A total of 125 students participated to these activities and came from either a Chemical Engineering course (class 1, 53 students, in 2018; class 2, 51 students, in 2019) or a Chemical reaction master course (class 3, 21 students, 2019).

Figure 3 .

 3 Figure 3. Student responses relating to the use of pop-culture in the courses. Total number of respondents = 125 (academic year 2018/2019).

  Into the Spider-Verse, Peter Parker has a degree in chemical engineering and teaching materials can be developed from one of the most popular video games of 2018: Insomniac Games' Spider-Man. An important aspect of the game is the completion of missions that involve collecting PAH (PolyAromatic Hydrocarbons) samples, studying vehicle emissions, and determining the chemical composition of atmospheric particulate matter 135 . The video game directly simulates chemical analysis of these samples by having the player solve simplified versions of absorption spectra.

Figure 4 .

 4 Figure 4. (a) Puzzle game to assign an unknown absorption spectrum using an inventory of atomic emission spectra. (b) Particles made from modelling clay (FIMO®) are used to mimic heterogeneous particles present in drinking water samples. Those with "angry" faces model waterborne pathogens that can be harmful to humans and should be separated and detected to prevent outbreaks. (c) Macro-fluidic device made of modelling clay, a Plexiglass layer and silicon for bonding 136 .

  Invasions is an animated television series that premiered in 2013 142 . The show is based on the Raving Rabbids video game series produced by Ubisoft and created in 2006 143 . Among the hundreds of episodes of Rabbids invasions, developed by TeamTO for Ubisoft Motion Pictures, some, e.g. episode 17 of season 1 ("Rabbid Dreams" by Fabien Ouvrard & Mélanie Duval, 2014), involve scientific observations of those strange creatures. Part of the action takes place in a lab comprising an experiment room separated from a glass-walled observation office, where the scientists Gina and John try

6 -Figure 5 .

 65 Figure 5. (a) Reproduction of the molecule presented in the Raving Rabbids (b) experiment to illustrate the photochromism of the NitroBIPS molecule with a Raving Rabbid as a blank marker.

cylinder of height 𝑙 7 -

 7 𝑙 8 -𝑟 9: = 1.8 m and radius rSG = 0.3 m. In order to propose an original work to the student, we propose to calculate the net radiative flux from the Genki dama to Son Goku.

Figure 6 .

 6 Figure 6. Scheme of the problem

  video games, movies and TV series) to engage chemistry and chemical engineering students. The goal has been to show that chemistry and chemical engineering phenomena are widely present and play an essential role in recent pop culture as typified in the superhero movies, action video games or fantasy drama series.Instructors can stimulate students' interest in these domains by discussing the chemical content of such works during lectures, tutorials, by generating quizzes and assignment items based on occurrences in these videogames and movies, or by creating a stock of scientific trivia collected from popular culture sources. To conclude, popculture offers a wide range of possibilities for involving students in classroom, from hands-on activity to critical thinking, and from basic chemistry to chemical engineering.ASSOCIATED CONTENTSupporting InformationFortnite: Making "slurp juice"; Game of Thrones and Harry Potter: Making green fire; Breaking Bad: Synthesizing dexerine; The Black Panther movie: Proposing a process flow diagram for fabrication of "vibranium steel"; Raving Rabbids: Making color-changing paper; Dragon Ball: Calculating the net radiative flux from the Genki Dama to Son Goku (DOCX)

Table 1 .

 1 StarWars) or popular public figures (i.e. soccer players, rock stars) they like best in order to organize them in the periodic table according to their properties and/or names. Besides being a strategy to increase the attention of younger students for introducing the periodic table in classroom, relating the elements of the periodic table to pop culture characters is a very useful strategy to help memorize the groups and periods as well as to explain the properties of each element. List of fictional elements present in pop-culture media

	Name	Assumed	Reference
		Symbol

both, and to develop their creativity by linking these elements with the Mendeleev periodic table. Having a strong knowledge of the periodic table is also fundamental to understand the basic principles of chemistry and different strategies and games have been proposed to help students memorize the position of each element in the periodic table

129,130

.Recently, different periodic tables have been designed using fictional characters to be used as a mnemonic for high school students. For example, Disney characters have been organized in the periodic table relating each character to a property of the element (i.e. Boron (B) = Bambi, Bambi was Disney's fifth movie)

131

whereas Marvel, DC and Asterix characters have been periodically distributed in the periodic table by choosing characters whose names are reminiscent of the elements (i.e. Magnesium (Mg) = Magneto) 132,133 . This can be also an activity to be carried out in class where each student could choose other pop culture characters (i.e. The Simpsons,

AUTHOR INFORMATION

Nicolas DIETRICH E-mail: nicolas.dietrich@insa-toulouse.fr Christophe COUDRET E-mail: coudret@chimie.ups-tlse.fr ORCID number: 0000-0001-7334-5112 Note: The authors declare no competing financial interest.


Aurore Béral

Jacques Le Gouis

Vincent Allard

Is wheat



  Is wheat individual grain characteristics variance a source of genetic variability for water stress tolerance ? Aurore Beral 1 , Jacques Le Gouis 1 , Vincent Allard 1 , Christine Girousse 1 1 INRA, UCA, UMR 1095, Génétique, Diversité et Ecophysiologie des Céréales Contact: aurore.beral@inra.fr There is environmental and genetic variance for individual grain characteristics: Introduction: Grain yield is usually analyzed through its different average numeric components  However each component shows a large intragenotypic variance (eg grain size will vary depending on the tiller and the position on the spike)  Variance of these components has not be so far fully explored particularly in relation to stress tolerance (i) Understand precisely the structure of the variance associated to individual grain characteristics (ii) Stratify variance into inter-and intra-spike variances Issues:How to study these issues?

Fig. 1 :

 1 Fig. 1: Relationship between mean grain surface and grain surface variance for 228 genotypes Fig. 3: Grain surface distribution for 2 genotypes for 2 treatments

Fig. 2 :

 2 Fig. 2: Grain surface distribution for 2 genotypes Variability of distribution between genotypes showing different grain characteristics variance:  Genetic variance for grain size