S1

Supporting Information

Mimicking the Intramolecular Hydrogen Bond: Synthesis, Biological Evaluation, and Molecular Modeling of Benzoxazines and

Quinazolines as Potential Antimalarial Agents

Sandra Gemma,†, √ Caterina Camodeca,†, √ Margherita Brindisi,†, √ Simone Brogi,†,√ Gagan Kukreja,†, √ Sanil Kunjir,†, √ Emanuele Gabellieri,†, √ Leonardo Lucantoni,×,√ Annette Habluetzel,×,√ Donatella Taramelli,†,#,√ Nicoletta Basilico,†,∀,√ Roberta Gualdani,∫ Francesco Tadini-Buoninsegni,∫ Gianluca Bartolommei,∫ Maria

Rosa Moncelli,∫ Rowena E. Martin,⊥ Robert L. Summers,⊥ Stefania Lamponi,†, √ Luisa Savini,†, √ Isabella Fiorini, †,√ Massimo Valoti,+ Ettore Novellino,†,∞ Giuseppe Campiani,†, √,* Stefania Butini†,√

†European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, Via Aldo Moro, 53100 Siena, Italy; √CIRM Centro Interuniversitario di Ricerche sulla Malaria, Università di Torino, Torino, Italy;

×Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino,

62032 Camerino (MC), Italy; #Dip. di Scienze Farmacologiche e Biomolecolari, and

∀Dip. di Scienze

Biomediche, Chirurgiche e Odontoiatriche, Università di Milano, Via Pascal 36, 20133 Milan; ∫Dept. of

Chemistry “Ugo Schiff,” University of Florence, 50019 Sesto Fiorentino, Italy; ⊥ Research School of

Biology, The Australian National University, Canberra ACT 0200, Australia; +

Dip. di Neuroscienze,

University of Siena, via A. Moro 2, Siena, Italy; ∞

Dip. di Chimica Farmaceutica e Tossicologica, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy

Corresponding Author *E-mail: campiani@unisi.it; Tel. +390577234172, Fax +390577234333

Table of Contents

Figure S1. Low energy conformers of isoquine (IQ), amodiaquine (AQ) and halofantrine ......... S2

Figure S2. Site of metabolism prediction for compound 6b,c ..................................................... S2

Figure S3. Ames test performed on S. typhimurium TA98 strains for compounds 5c,a and 6c,b S3

Figure S4. Ames test performed on S. typhimurium TA98 and TA100 strains in the presence of the liver fraction S9 for compounds 5a (panels A,B), 6b (panels C,D), and 6c (panels D,E) ............. S3

Figure S5 Superposition between docked poses of 6b and 6c and their low energy confomers ... S4

Figure S6 Docked poses of CQ, IQ, AQ and halofantrine .......................................................... S5

Figure S7 Superposition between initial docked structure of 6b and after 3ns of MD simulation S6

Figure S8 Superposition between initial docked structure of 6c and after 3ns of MD simulation S6

Figure S9 Plots derived from MD simulation............................................................................. S7

Table S1. Elemental analyses .................................................................................................... S8

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Figure S1. Low energy conformers of isoquine (IQ)(A), amodiaquine (AQ)(B) and halofantrine (C) in their monoprotonated forms. The H-bonds are represented by dotted black line. The picture was generated by Maestro (Schrödinger, LLC, New York, NY, 2011).

CYP 3A4 CYP 2C9 CYP 2D6

6b

6c

Figure S2. Site of metabolism prediction for compound 6b,c performed by P450 Site of Metabolism workflow distributed by Schrödinger (P450 Site of Metabolism Prediction, version 1.1, Schrödinger, LLC, New York, NY, 2011). The prediction for isoforms CYP2C9 and CYP2D6 combines induced-fit docking36 (which determines the accessibility of the compounds to the reactive centre of CYP) with a rule-based approach to intrinsic reactivity.37 In the case of CYP3A4, which possesses a highly flexibile binding site, only intrinsic reactivity was used to predict soft spots. The number of rays indicates the value of the Fe-accessibility, as determined by induced-fit docking. Each full ray represents a unit of accessibility, with the length of the final ray representing the decimal portion. The size of the circles corresponds to the overall ‘site of metabolism’ score; the color of the circle indicates the atomic intrinsic reactivity – with the highest level of intrinsic reactivity shaded dark red; a blue ring around the circle indicates that the atom passed through the CYP filtering stage. No ring indicates that the atom failed the CYP filtering stage.

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Figure S3. Ames test performed on S. typhimurium TA98 strains for compounds 5c (panel A), 5a (panel B), 6c (panel C), and 6b (panel D).

Figure S4. Ames test performed on S. typhimurium TA98 and TA100 strains in the presence of the liver fraction S9 for compounds 5a (panels A,B), 6b (panels C,D), and 6c (panels D,E).

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Figure S5. (A) Superposition (RMSD 0.252) between docked pose of 6b (orange tube; potential energy = -187.192 Kcal/mol) and its lower energy conformer (grey tube; potential energy = -188.153 Kcal/mol). (B) Superposition (RMSD 0.207) between docked pose of 6c (purple tube; potential energy = -222.187 Kcal/mol) and its lower energy conformer (grey tube; potential energy = -223.276 Kcal/mol). The energy values were estimated by ECalc implemented in MacroModel (Schrödinger, LLC, New York, NY, 2011).

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Figure S6. (A) Docked pose of CQ (brown sticks) in the cavity of the hERG channel (cyan cartoon; α, β, γ, and δ refers to protein subunits). Despite the H-bonds formed by CQ with S649α and with the oxygen backbone of S649δ, only a weak π-π stacking between the quinoline moiety and the key residue Y652δ is observed. (B) Docked pose of IQ (green sticks) in the cavity of the hERG channel (cyan cartoon). Similarly to CQ, IQ interacts by polar contacts with S649α and with the oxygen backbone of S649δ, and by π-π stacking between the Y652δ and the quinoline moiety. (C) Docked pose of AQ (grey sticks) in the cavity of the hERG channel (cyan cartoon). AQ forms H-bond with residues S649δ and only a partial π-π stacking is observed between the quinoline moiety and the key residues Y652 and F656 from δ subunits. (D) Docked pose of halofantrine (yellow sticks) in the cavity of hERG channel (cyan cartoon). Halofantrine strongly interacts by π-π stacking with residue Y652δ (the distance between selected moiety is largely below 5 Å) and forms a series of H-bonds with S649α and T623α. These docking outputs (Goldscores are reported in the main text) and the comparison of interaction into hERG channel for reference compounds is in agreement with experimental results described in the Main Text. Similarly to 6b, only halofantrine is able to produce strong stacking with key residues into hERG channel and H-bonds with residues S649 and T623 (Figure 7A in the Main Text). These observations are in agreement with the nanomolar hERG channel inhibition potency experimentally observed for 6b and halofantrine. In addition, the pattern of interactions showed by CQ, IQ and AQ, although better than the pattern of interactions observed

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for 6c, in combination with docking score are again consistent with the observed micromolar affinity of these derivatives for hERG channel. Pictures were generated by PyMOL (Schrödinger, LLC, New York, NY, 2011).

Figure S7 Superposition between initial docked structure (orange) and final structure (light green) after 5 ns of MD of compound 6b bound to the hERG binding site.

Figure S8 Superposition between initial docked structure (purple) and final structure (sea green) after 5 ns of MD of compound 6c bound to the hERG binding site.

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6b 6c

Figure S9 Plots derived by Molecular Dynamics simulation. (A) Multivariate-plot for the number of H-bonds produced by 6b during 5 ns of MD. (B) Multivariate-plot for the number of H-bonds produced by 6c during 5 ns of MD. (C) Plot for the distance between quinazoline moiety of 6b and residues Y652 during 5 ns of MD (median value of distance: 3.92 Å). (D) Plot for the distance between quinazoline moiety of 6c and residues Y652 during 5 ns of MD (median value of distance: 6.34 Å). (E) Plot for the distance between quinoline moiety of 6b and residues Y652 during 5 ns of MD (median value of distance: 3.86 Å). (F) Plot for the distance between quinoline moiety of 6c

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and residues Y652 during 5 ns of MD (median value of distance: 5.83 Å). (G) Plot for the distance between phenyl ring of 6c and residues F656 during 5 ns of MD (median value of distance: 6.78 Å). The pictures were generated by Simulation Event Analysis implemented in Desmond.

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Table S1. Elemental analyses

Cpd Formula Calculated Found

C H N C H N

5a C19H18ClN3O 67.15 5.34 12.37 67.50 5.48 12.16

5b C20H20ClN3O 67.89 5.70 11.88 67.68 5.57 11.72

5c C24H20ClN3O 71.73 5.02 10.46 71.38 5.09 10.66

15a C19H15ClN4O 65.05 4.31 15.97 65.34 4.42 15.70

15b C20H17ClN4O 65.84 4.70 15.36 66.17 4.61 15.45

15c C24H17ClN4O 69.82 4.15 13.57 69.70 4.23 13.22

15d C28H21ClFeN4O 64.57 4.06 10.76 64.30 3.74 10.81

6a C19H19ClN4 67.35 5.65 16.54 67.49 5.28 16.78

6b C20H21ClN4 68.08 6.00 15.88 67.72 6.27 16.10

6c C24H21ClN4 71.90 5.28 13.98 72.28 5.02 13.60

16 C24H19ClN4O 69.48 4.62 13.50 69.67 4.57 13.63

21 C29H26ClN5O 70.22 5.28 14.12 70.12 5.64 14.00