Synthesis and effects of oxadiazole derivatives on tyrosinase activity and SK-MEL-28 malignant melanoma cells

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1 Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 06 Synthesis and effects of oxadiazole derivatives on tyrosinase activity and human SK-MEL-8 malignant melanoma cells Mohd Fadhlizil Fasihi Mohd Aluwi a, Kamal Rullah a, b, Tan Huan Huan c, Chan Kok Meng c, Tan Si Jie a, Leong Sze Wei d, Ahmad Hasnan Mansor a, Bohari M Yamin e and Lam Kok Wai a, a Drugs and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 5000 Kuala Lumpur, Malaysia b Sekolah Tinggi Ilmu Farmasi Riau, Universitas Riau, Kampus Bina Widya Km.5, Simpang baru-pekanbaru, Indonesia c Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 5000 Kuala Lumpur. d Institute of Bioscience, Universiti Putra Malaysia, 00, Serdang, Selangor, Malaysia e School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 600 Bangi, Selangor, Malaysia Supplementary Data Synthesis and effects of oxadiazole derivatives on tyrosinase activity and SK-MEL-8 malignant melanoma cells Corresponding author. Tel.: ; david_lam@ukm.edu.my

2 . Chemistry.. General information All reagents were purchased from Aldrich and Merck and were used without further purification. All the solvents used in the syntheses were analysis and synthesis grade. The solvents used in spectroscopic measurements were spectroscopic grade. H and C MR spectra were recorded on a Bruker 500 MHz spectrometer. ESI-HRMS spectra were recorded on a Bruker microtof Mass Spectrometer. Single-crystal X-ray experiment was performed on a Bruker D-QUEST diffractometer (Bruker, AXS Inc., Madison, WI, USA). Melting points were determined on a STUART SMP0 melting point apparatus... H and C MR MR spectra of compound Compound. 5-(naphthalen--ylmethyl)-,,-oxadiazole--thiol. Brown solid. M.p 7-7 C. H MR (500 MHz, DMSO) δ 7.7 (d, J = 8. Hz, H), 7.6 (d, J = 8.7 Hz, H), 7.56 (d, J = 7.9 Hz, H), (m, H),.5 (s, H). C MR (6 MHz, DMSO) δ 77.87, 6.06,.5,.9, 9.56, 8.75, 8.6, 8.6, 6.75, 6.9, 5.77,.7, ESI-HRMS: (C H 0 OS) calc. [M+H].00, found O 6 5 SH D (m) 7.8 B (d) A (d) 7.7 E (s).5 C (d) 7.56 C (d) 7.56 A (d) 7.7 B (d) 7.6 D (m)

3 O 6 SH 7

4 MR spectra of compound Compound. 5-(naphthalen--ylmethyl)-,,-oxadiazole--thiol. Pale yellow solid. M.p 8-9 C. H MR (500 MHz, DMSO) δ 7.6, 7.60, 7.59, 7.59, 7.57, 7.55, 7., 7., 7., 7.0, 7.9, 7.5, 7.,.99. C MR (6 MHz, DMSO) δ 78.05, 6.,.09,.7,.8, 8.5, 7.85, 7.7, 7.69, 7.0, 6.6, 6.,.7. ESI-HRMS: (C H 0 OS) calc. [M+H].00, found O 6 5 SH B (s) 7.55 D (d) 7. A (m) 7.59 E (s).99 A (m) 7.59 B (s) 7.55 C (p) 7. D (d) 7. C (p)

5 O 6 SH 7

6 MR spectra of compound Compound. 5-(naphthalen--ylmethyl)--(piperidin--ylmethyl)-,,-oxadiazole-(H)-thione. colorless rod crystals. M.p C. H MR (500 MHz, dmso) δ 7.85 (dd, J =., 0.9 Hz, H), 7.80 (s, H), (m, H), 7.0 (d, J = 8.9 Hz, H),.8 (s, H),.5 (s, H),.57 (s, H),.58.8 (m, H). C MR (6 MHz, DMSO) δ 78.8, 6.9,.6,.5, 8.77, 8.00, 7.9, 7.0, 6.90, 6.55, 7.00, 5.,.6, 5.77,.68. ESI-HRMS: (C 9 H OS) calc. [M+H] 0.650, found O 6 S D (d) 7.0 B (s) 7.80 F (s).5 B (s) 7.80 A (dd) 7.85 E (s).8 G (s).57 H (m).0 A (dd) 7.85 C (m) 7.7 D (d) 7.0 C (m)

7 O 6 S

8 . X-ray Structural Characterization of Compound Table. Crystal data and structure refinement for compound. Identification code boly_0m Empirical formula C9 H O S Formula weight 9.5 Temperature 0() K Wavelength Å Crystal system Triclinic Space group P - Unit cell dimensions a = 7.09() Å α = 85.7 (). b = 8.095(5) Å β = (). c = 5.6(8) Å γ = 80.6 (). Volume 866.6(9) Å Z Density (calculated).0 Mg/m Absorption coefficient 0.97 mm - F(000) 60 Crystal size 0.0 x 0.0 x 0.0 mm Theta range for data collection. to 8.7. Index ranges -9<=h<=9, -0<=k<=0, -0<=l<=0 Reflections collected 59 Independent reflections [R(int) = ] Completeness to theta = % Refinement method Full-matrix least-squares on F Data / restraints / parameters / 0 / 7 Goodness-of-fit on F.09 Final R indices [I>sigma(I)] R = , wr = 0.09 R indices (all data) R = 0.090, wr = 0.0 Extinction coefficient n/a Largest diff. peak and hole 0.97 and e.å -

9 Table. Atomic coordinates (x 0 ) and equivalent isotropic displacement parameters (Å x 0 ) for compound. U(eq) is defined as one third of the trace of the orthogonalized U ij tensor. x y z U(eq) S() 79() 90() 0() 8() O() 5() 68() -66() 7() () 67() 68() 699() 5() () 7() 8056() 8() 8() () 77() 67() 89() 7() C() 78() 987() -69() 8() C() 6() 9795() -6() 50() C() 5866() 9() -08() () C() 76() 8996() -776() 60() C(5) 695() 88() -59() 70() C(6) 58() 77() -657() 7() C(7) 85() 789() -08() 6() C(8) 0() 859() -9() () C(9) 77() 860() -58() () C(0) 08() 988() -80() 0() C() 565() 98() -0() 9() C() 0() 7987() -() 6() C() 55() 59() 7() () C() 8() 590() 59() 0() C(5) 0() 57() 098() 5() C(6) -0() 586() 708() 55() C(7) -70() 566() 67() 65() C(8) 97() 665() 77() 7() C(9) 50() 68() 07() 5()

10 Table. Bond lengths [Å] and angles [ ] for compound. S()-C().69(8) O()-C().6() O()-C().68() ()-C().7() ()-().89() ()-C().5() ()-C().7() ()-C().() ()-C(9).6() ()-C(5).6() C()-C().5() C()-C(0).07() C()-H() C()-C().07() C()-H() C()-C(8).0() C()-C().9() C()-C(5).58() C()-H() C(5)-C(6).9() C(5)-H(5) C(6)-C(7).5() C(6)-H(6) C(7)-C(8).() C(7)-H(7) C(8)-C(9).() C(9)-C(0).6() C(9)-H(9) C(0)-C().5() C()-C().8() C()-H(A) C()-H(B) C()-H(A) C()-H(B) C(5)-C(6).506() C(5)-H(5A) C(5)-H(5B) C(6)-C(7).56() C(6)-H(6A) C(6)-H(6B) C(7)-C(8).5() C(7)-H(7A) C(7)-H(7B) C(8)-C(9).50() C(8)-H(8A) C(8)-H(8B) C(9)-H(9A) C(9)-H(9B) C()-O()-C() 06.5() C()-()-().95() C()-()-C() 7.7(5) ()-()-C() 0.0() C()-()-() 0.5() C()-()-C(9) 09.0() C()-()-C(5).()

11 C(9)-()-C(5) 0.66() C()-C()-C(0).0(8) C()-C()-H() 9.5 C(0)-C()-H() 9.5 C()-C()-C().0(9) C()-C()-H() 9.5 C()-C()-H() 9.5 C()-C()-C(8) 8.97(8) C()-C()-C().85(9) C(8)-C()-C() 8.8(9) C(5)-C()-C().0() C(5)-C()-H() 9.5 C()-C()-H() 9.5 C()-C(5)-C(6) 0.() C()-C(5)-H(5) 9.9 C(6)-C(5)-H(5) 9.9 C(7)-C(6)-C(5) 0.7() C(7)-C(6)-H(6) 9.7 C(5)-C(6)-H(6) 9.7 C(6)-C(7)-C(8) 0.6() C(6)-C(7)-H(7) 9.7 C(8)-C(7)-H(7) 9.7 C()-C(8)-C(7) 9.(9) C()-C(8)-C(9) 8.5(7) C(7)-C(8)-C(9).(9) C(0)-C(9)-C(8).9(8) C(0)-C(9)-H(9) 9. C(8)-C(9)-H(9) 9. C(9)-C(0)-C() 9.5(8) C(9)-C(0)-C().7(8) C()-C(0)-C() 9.7(7) C()-C()-C(0).9(5) C()-C()-H(A) 09. C(0)-C()-H(A) 09. C()-C()-H(B) 09. C(0)-C()-H(B) 09. H(A)-C()-H(B) 07.9 ()-C()-O().7(5) ()-C()-C() 8.97(7) O()-C()-C() 7.76(5) ()-C()-O() 0.9() ()-C()-S().5() O()-C()-S().9() ()-C()-().7() ()-C()-H(A) 09. ()-C()-H(A) 09. ()-C()-H(B) 09. ()-C()-H(B) 09. H(A)-C()-H(B) 08.0 ()-C(5)-C(6).6(6) ()-C(5)-H(5A) 09. C(6)-C(5)-H(5A) 09. ()-C(5)-H(5B) 09. C(6)-C(5)-H(5B) 09. H(5A)-C(5)-H(5B) 08.0 C(5)-C(6)-C(7) 0.(8) C(5)-C(6)-H(6A) 09.6

12 C(7)-C(6)-H(6A) 09.6 C(5)-C(6)-H(6B) 09.6 C(7)-C(6)-H(6B) 09.6 H(6A)-C(6)-H(6B) 08. C(8)-C(7)-C(6) 09.60(8) C(8)-C(7)-H(7A) 09.7 C(6)-C(7)-H(7A) 09.7 C(8)-C(7)-H(7B) 09.7 C(6)-C(7)-H(7B) 09.7 H(7A)-C(7)-H(7B) 08. C(9)-C(8)-C(7).00(8) C(9)-C(8)-H(8A) 09. C(7)-C(8)-H(8A) 09. C(9)-C(8)-H(8B) 09. C(7)-C(8)-H(8B) 09. H(8A)-C(8)-H(8B) 08.0 ()-C(9)-C(8) 0.7(8) ()-C(9)-H(9A) 09.5 C(8)-C(9)-H(9A) 09.5 ()-C(9)-H(9B) 09.5 C(8)-C(9)-H(9B) 09.5 H(9A)-C(9)-H(9B) 08. Symmetry transformations used to generate equivalent atoms:

13 Table. Anisotropic displacement parameters (Å x 0 ) for compound. The anisotropic displacement factor exponent takes the form: -π [ h a* U h k a* b* U ] U U U U U U S() () 0() 6() -0() 6() -7() O() 5() () () -0() () -7() () () () () -7() () -7() () 6() 0() 9() -8() () -() () 6() 6() () -5() 0() -() C() 56() () 6() -() -9() -8() C() 7() 6() 60() -() -9() -() C() 8() 6() 7() () -() -6() C() 56() 5() 67() 6() 8() -9() C(5) 8() 68() 5() -() () -5() C(6) 9() 77() () -() 6() -() C(7) 7() 65() 7() -9() -() -9() C(8) 5() 8() () 0() -() -8() C(9) 5() () 5() -() -5() -0() C(0) 5() () 0() () -() 0() C() 9() 5() 7() -() 0() 8() C() 0() 9() 0() -9() 5() -() C() () () 7() -9() 6() -6() C() () 5() 6() -() -() -8() C(5) 0() 57() () -8() 0() -7() C(6) () 57() 68() -() () -() C(7) 7() 7() 5() -() 6() -7() C(8) 96() 95() () -5() 9() -6() C(9) 58() 66() 5() -() -() -7()

14 Table 5. Hydrogen coordinates ( x 0 ) and isotropic displacement parameters (Å x 0 ) for compound. x y z U(eq) H() H() H() H(5) H(6) H(7) H(9) H(A) H(B) H(A) H(B) H(5A) H(5B) H(6A) H(6B) H(7A) H(7B) H(8A) H(8B) H(9A) H(9B)

15 Table 6. Torsion angles [ ] for compound. C()-()-()-C() 0.7(8) C()-()-()-C() 7.6() C(0)-C()-C()-C() -0.7() C()-C()-C()-C(8) 0.6() C()-C()-C()-C() -79.(9) C()-C()-C()-C(5) 79.6() C(8)-C()-C()-C(5) -0.5() C()-C()-C(5)-C(6) 0.() C()-C(5)-C(6)-C(7) 0.() C(5)-C(6)-C(7)-C(8) -0.() C()-C()-C(8)-C(7) -79.(9) C()-C()-C(8)-C(7) 0.7() C()-C()-C(8)-C(9) -0.() C()-C()-C(8)-C(9) 79.88(7) C(6)-C(7)-C(8)-C() -0.() C(6)-C(7)-C(8)-C(9) -79.5() C()-C(8)-C(9)-C(0) -0.() C(7)-C(8)-C(9)-C(0) 79.05(9) C(8)-C(9)-C(0)-C() 0.() C(8)-C(9)-C(0)-C() -78.9(6) C()-C()-C(0)-C(9) 0.() C()-C()-C(0)-C() 79.9(7) C(9)-C(0)-C()-C() 98.9() C()-C(0)-C()-C() -80.0() ()-()-C()-O() -0.69(8) ()-()-C()-C() 78.9(7) C()-O()-C()-() 0.95(8) C()-O()-C()-C() (5) C(0)-C()-C()-() 0.() C(0)-C()-C()-O() -60.() ()-()-C()-O() 0.9(7) C()-()-C()-O() -7.5() ()-()-C()-S() -79.() C()-()-C()-S() 7.8() C()-O()-C()-() -0.76(6) C()-O()-C()-S() 79.00() C(9)-()-C()-() -70.5(5) C(5)-()-C()-() 67.() C()-()-C()-() -5.(8) ()-()-C()-() 7.9(9) C()-()-C(5)-C(6) (6) C(9)-()-C(5)-C(6) 59.6() ()-C(5)-C(6)-C(7) -57.6() C(5)-C(6)-C(7)-C(8) 5.7() C(6)-C(7)-C(8)-C(9) -5.9() C()-()-C(9)-C(8) 78.60(7) C(5)-()-C(9)-C(8) -58.8() C(7)-C(8)-C(9)-() 57.() Symmetry transformations used to generate equivalent atoms:

16 Table 7. Hydrogen bonds for compound [Å and ]. D-H...A d(d-h) d(h...a) d(d...a) <(DHA)

17 . Biology. Lineweaver-Burk plots for Kojic Acid and Rhodanine Figure Lineweaver-Burk plots for inhibition of kojic acid on the oxidation of L-DOPA by mushroom tyrosinase. Concentrations of kojic acid for curves 0- were 0, 5, 0, 5, and 0 μm, respectively. The inset represents the secondary plot of slope or Y-intercept versus the kojic acid concentration for determining the K I and K IS. The line was drawn using linear least square fit. 0.0 Slope K i =.98 M 0.5 Y-intercept K i ' = 0.97 M [I] ( M) [I] ( M) /v ( M/min) /[S] (mm) -

18 Figure Lineweaver-Burk plots for inhibition of rhodanine on the oxidation of L-DOPA by mushroom tyrosinase. Concentrations of rhodanine for curves 0- were 0, 0, 0, 0, and 80 μm, respectively. The inset represents the secondary plot of slope or Y-intercept versus the rhodanine concentration for determining the K I and K IS. The line was drawn using linear least square fit K i =.0 M Slope Y-intercept K i ' =.8 M [I] ( M) [I] ( M) /v ( M/min) /[S] (mm) -

19 . Computational studies. Molecular docking and dynamics simulation Fig.. RMSD plots of tyrosinase backbone in complex with compound (black line) and compound (red line) as a function of simulation time. Fig.. RMSF plot of selected tyrosinase residues in complex with compound during the final 5 ns of simulation.