Novel D-erythro N-Octanoyl Sphingosine Analogs As Chemo- and Endocrine. Resistant Breast Cancer Therapeutics

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1 Page 11 of 32 Cancer Chemotherapy and Pharmacology Novel D-erythro N-Octanoyl Sphingosine Analogs As Chemo- and Endocrine Resistant Breast Cancer Therapeutics James W. Antoon, Jiawang Liu, Adharsh P. Ponnapakkam, Matthew M. Gestaut, Barbara S. Beckman, Maryam Foroozesh * Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA and Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA Corresponding Author: Barbara S. Beckman, PhD. bbeckman@tulane.edu, Phone: Keywords: Novel D-erythro N-Octanoyl Sphingosine, sphingolipids, breast cancer, chemoresistance, endocrine resistance, ceramide SUPPORTING INFORMATION ENCLOSED Page S2, S3: Experimental section containing synthesis of Analogs A-D. Page S4-S22: Identity and Degree of Purity of Compounds: Copies of 1 H NMR, 13 C NMR, HPLC spectra and elemental analytical data for analogs A-D S1

2 Cancer Chemotherapy and Pharmacology Page 12 of 32 SUPPORTING INFORMATION Experimental Section General All synthesis chemicals and solvents were purchased from Sigma-Aldrich Company except D- sphingosine/hcl purchased from CNH Technologies Inc. All coupling reactions were carried out under anhydrous conditions. The purities of the intermediates and the products were confirmed by HPLC (hp HEWLETT PACKARD SERIES 1050, phenomenex Gemini-NX 5u C18 110A) and thin layer chromatography (Whatman 250 µm LAYER, UV 254, Flexible plates for TLC and SILICA GEL IB2-F). Chromatography was performed on HPTLC Silica gel 60 F H NMR and 13 C NMR spectra were obtained on Varian 300 MHz NMR spectrometer. Mass spectral data was determined Agilent 6890N Network GC System with 5975C inert MSD. Elemental analysis was performed by Atlantic Microlab Inc. N-((2S,3R,E)-1,3-dihydroxyoctadec-4-en-2-yl)cyclohexanecarboxamide (analog A) At 0 C to the solution of 38 mg (0.29 mmol) of cyclohexanecarboxylic acid in 25 ml of anhydrous tetrahydrofuran (THF), 40 mg (0.29 mmol) of tert-butyl alcohol (HOBt) and 100 mg (0.29 mmol) of D-Sphingosine/HCl were added. After 5 min, 67 mg (0.33 mmol) of Dicyclohexylcarbodiimide (DCC) was added, and the ph of the solution was adjusted to 8 with 4-methylmorpholine. The reaction mixture was stirred at 0 C for 2 h and at room temperature overnight. On evaporation the residue was dissolved in 100 ml of ethyl acetate. The solution was washed successively with saturated sodium bicarbonate, 5% potassium bisulfate, and saturated sodium chloride, and the organic phase was separated and dried over anhydrous magnesium sulfate for 2 h. After filtration and evaporation under reduced pressure, the crude product was recrystallized in ethyl ether to obtain 60 mg (51%) of N-((2S,3R,E)-1,3- dihydroxyoctadec-4-en-2-yl)cyclohexanecarboxamide as colorless powder. GC/MS (m/e): 391 [M H 2 O] +. 1 H NMR (d 6 -DMSO, 300 MHz) /ppm = (d, J = 8.4 Hz, 1H), (dt, J = 15.3 Hz, J = 6.6 Hz, 1H), (dd, J = 15.3 Hz, J = 6.6 Hz, 1H), (d, J = 5.1 Hz, 1H), (t, J = 5.4 Hz, 1H), (q, J = 6.0 Hz, 1H), (m, 1H), (m, 2H), (m, 1H), (m, 2H), (m, 6H), 1.225~1.157 (m, 26H), (t, J = 6.6 Hz, 3H). 13 C NMR (d 6 -DMSO, 75 MHz) /ppm = , , , , , , , , , , , , , , , , Anal. Calcd for C 25 H 47 NO 3 : C, 73.30; H, 11.56; N, 3.42; O, Found: C, 72.99; H, 11.64; N, 3.74; O, N-((2S,3R,E)-1,3-dihydroxyoctadec-4-en-2-yl)pivalamide (analog D) At 0 C to the solution of 30 mg (0.29 mmol) of pivalic acid in 25 ml of anhydrous THF, 40 mg (0.29 mmol) of HOBt and 100 mg (0.29 mmol) of D-Sphingosine/HCl were added. After 5 min, 67 mg (0.33 mmol) of DCC was added, and the ph of the solution was adjusted to 8 with 4- methylmorpholine. The reaction mixture was stirred at 0 C for 2 h and at room temperature overnight. On evaporation the residue was dissolved in 100 ml of ethyl acetate. The solution was washed successively with saturated sodium bicarbonate, 5% potassium bisulfate, and saturated sodium chloride and the organic phase was separated and dried over anhydrous magnesium sulfate for 2 h. After filtration and evaporation under reduced pressure, the residue was purified by thin layer chromatography (Chloroform/Methanol 10/1) to obtain 0.50 g (45%) of N- S2

3 Page 13 of 32 Cancer Chemotherapy and Pharmacology ((2S,3R,E)-1,3-dihydroxyoctadec-4-en-2-yl)pivalamide as colorless powder. GC/MS (m/e): 382 [M H] +, 366 [M OH] +, 352 [M [ CH 2 =OH]] +. 1 H NMR (d 6 -DMSO, 300 MHz) /ppm = (d, J = 8.7 Hz, 1H), (dt, J = 15.3 Hz, J = 6.3 Hz, 1H), (dd, J = 15.3 Hz, J = 6.3 Hz, 1H), (d, J = 5.4 Hz, 1H), (t, J = 5.7 Hz, 1H), (q, J = 6.0 Hz, 1H), (m, 1H), (m, 2H), (m, 2H), (m, 22H), (s, 9H), (t, J = 6.6 Hz, 3H). 13 C NMR (d 6 -DMSO, 75 MHz) /ppm = , , , , , , , , , , , , Anal. Calcd for C 23 H 45 NO 3 : C, 72.01; H, 11.82; N, 3.65; O, Found: C, 72.12; H, 11.65; N, 3.66; O, (2S,3R,E)-2-((E)-benzylideneamino)octadec-4-ene-1,3-diol (analog C) A solution of 100 mg (0.29 mmol) of D-Sphingosine/HCl, 11.6 mg (0.29 mmol) of sodium hydroxide (NaOH), 0.05 ml of benzaldehyde in 10 ml of methanol was stirred at room temperature. After 1 hour, the solution was evaporated under reduced pressure, and the residue was washed with cooled methanol to yield 80 mg (71%) of (2S,3R,E)-2-((E)- benzylideneamino)octadec-4-ene-1,3-diol as colorless powder. GC/MS (m/e): 387 M +, 370 [M OH] +, 356 [M [ CH 2 =OH]] +. 1 H NMR (d 6 -DMSO, 300 MHz) /ppm = (s, 1H), (t, J = 3.6 Hz, 2H), (m, 3H), (m, 2H), (d, J = 4.2 Hz, 1H), (br, 1H), (m, 1H), (m, 1H), (m, 1H), (m, 1H), (m, 2H), 1.221~1.119 (m, 22H), (t, J = 6.6 Hz, 3H). 13 C NMR (d 6 -DMSO, 75 MHz) /ppm = , , , , , , , , , , , , , , , , , , , , Anal. Calcd for C 25 H 41 NO 2 : C, 77.47; H, 10.66; N, 3.61; O, Found: C 76.84; H, 10.82; N, 3.75; O, (2S,3R,E)-2-(benzylamino)octadec-4-ene-1,3-diol (analog B) To the solution of 80 mg (0.21 mmol) (2S,3R,E)-2-((E)-benzylideneamino)octadec-4-ene-1,3- diol in 10 ml of methanol, 20 mg (0.32 mmol) of sodium cyanoborohydride (NaBH3CN) was added. The reaction mixture was stirred at room temperature for 10 h, and evaporated under vacuum. The residue was purified by thin layer chromatography to give 30 mg (36%) of (2S,3R,E)-2-(benzylamino)octadec-4-ene-1,3-diol as colorless powder. GC/MS (m/e): 388 [M H] +, 372 [M OH] +, 358 [M [ CH 2 =OH]] +. 1 H NMR (d 6 -DMSO, 300 MHz) /ppm = 7.323~7.193 (m, 5H), (m, 2H), (d, J = 3.6 Hz, 1H), (br, 1H), (m, 1H), (s, 2H), (m, 1H), (m, 1H), (m, 1H), (m, 2H), (m, 22H), (t, J = 6.6 Hz, 3H). 13 C NMR (d 6 -DMSO, 75 MHz) /ppm = , , , , , , , , , , , , , , , Anal. Calcd for C 25 H 43 NO 2 : C, 77.07; H, 11.12; N, 3.60; O, Found: C, 75.79; H, 11.26; N, 3.55; O, S3

4 Cancer Chemotherapy and Pharmacology Page 14 of 32 Identity and Degree of Purity of Compounds 1 H NMR of Analog A S4

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6 Cancer Chemotherapy and Pharmacology Page 16 of C NMR of Analog A S6

7 Page 17 of 32 Cancer Chemotherapy and Pharmacology 1 H NMR of Analog B S7

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9 Page 19 of 32 Cancer Chemotherapy and Pharmacology 13 C NMR of Analog B S9

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11 Page 21 of 32 Cancer Chemotherapy and Pharmacology 1 H NMR of Analog C S11

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14 Cancer Chemotherapy and Pharmacology Page 24 of 32 1 H NMR of Analog D S14

15 Page 25 of 32 Cancer Chemotherapy and Pharmacology 13 C NMR of Analog D S15

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17 Page 27 of 32 Cancer Chemotherapy and Pharmacology HPLC spectra of Analogs A, B, C and D Column: Phenomenex Gemini-NX 5u C18 110A Flow:1 ml/min Sig = ; Ref = Mobile Phase: MeOH:H 2 O = 90:10 Retention Time = 7.52 Purity = 99.6% S17

18 Cancer Chemotherapy and Pharmacology Page 28 of 32 Column: Phenomenex Gemini-NX 5u C18 110A Flow:1 ml/min Sig = ; Ref = Mobile Phase: MeOH:H 2 O = 90:10 Retention Time = 7.98 Purity = 99.4% Column: Phenomenex Gemini-NX 5u C18 110A Flow:1 ml/min Sig = ; Ref = Mobile Phase: MeOH:H 2 O = 90:10 S18

19 Page 29 of 32 Cancer Chemotherapy and Pharmacology Retention Time = 7.16 Purity = 98.0% Column: Phenomenex Gemini-NX 5u C18 110A Flow:1 ml/min Sig = ; Ref = Mobile Phase: MeOH:H 2 O = 90:10 Retention Time = 6.57 Purity = 97.2% Elemental analytical data of Analogs A, B, C and D Analog Formula Calcd % Found % C H N C H N A C 25 H 47 NO B C 22 H 43 NO S19

20 Cancer Chemotherapy and Pharmacology Page 30 of 32 C C 25 H 41 NO D C 23 H 45 NO S20

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