Running Title: Antimicrobial Activity of -Pinene Derivatives Synthesis, Antimicrobial Evaluation and Structure-Activity Relationship of -Pinene Derivatives Preeti Dhar ǂ, *, PuiYee Chan ǂ,, Daniel T. Cohen ǂ,, Fadi Khawam ǂ, Sarah Gibbons ǂ, Teresa Snyder-Leiby,σ, Ellen Dickstein, Prashant Kumar Rai δ,+, and Geeta Watal δ ǂ Department of Chemistry, State University of New York, 1 Hawk Drive, New Paltz, 12561 Department of Biology, State University of New York, 1 Hawk Drive, New Paltz, 12561 Department of Plant Pathology, University of Florida, Gainsville, Florida 32611, USA δ Drug Discovery & Development Division, Medicinal Research Lab, Department of Chemistry, University of Allahabad, Allahabad, 211002, India Corresponding Author Preeti Dhar dharp@newpaltz.edu Phone: 845-257-3797; Fax: 845 257-3791 Supporting Information Table of Contents Table of Contents... Error! Bookmark not defined. General Information... 2 Experimental Procedures... 2 References 8 Selected NMR Spectra... 9 Current affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Box 711, Rochester, NY 14642 Current affiliation: Department of Chemistry, Weinberg College of Arts and Sciences, 2145 Sheridan Road, Northwestern University, Evanston, IL 60208 + Current affiliation: Department of NMR, All India Institute of Medical Sciences, New Delhi 110029, India σ Current affiliation: Soft Genetics, State College 100 Oakwood place, PA-16803 1
S2 1 General Information 2 3 4 5 6 7 8 9 10 11 12 13 Silica gel 60 Å 70-230 or 230-400 mesh ASTM from Sigma-Aldrich was used for gravity column chromatography. TLC plates containing silica on aluminum/glass with a fluorescent indicator (254 nm), starting materials (+)- -pinene 98% and ( )- -pinene 98%, Lithium Aluminum Hydride (LAH) powder and all ACS grade solvents were purchased from Sigma-Aldrich and used as such. Chlorosulfonyl Isocyanate (CSI) 98%+ was purchased from Acros Organics. IR spectra were recorded on Perkin Elmer FTIR 1600 series in CH 2 Cl 2 solution and 1 H and proton decoupled 13 C NMR spectra were recorded on JEOL Eclipse 300 MHz NMR Spectrometer in CDCl 3. Chemical shifts are expressed in ppm relative to TMS as the internal standard. Data are reported as (ap = apparent, s = singlet, d = doublet, t = apparent triplet, q = quartet, m = multiplet, b = broad; coupling constant(s) in Hz; integration.) Synthesized compounds were > 95% pure as determined by 1 H-NMR. Experimental procedures that have been modified are provided below. 14 15 Experimental Procedures 16 17 18 19 20 21 22 9a (1S,2S,5R,7S)-2,8,8-trimethyl-4-oxo-3-azatricyclo[5.1.1.0 2,5 ]nonane-3-sulfonyl chloride (9a): To a N 2 purged flask with a magnetic stirring bar was placed (+)-α-pinene, 1a (5.46 g, 41.4 mmol) and anhydrous ether (45 ml). The solution was cooled to 73 C and chlorosulfonyl isocyanate (CSI) (3.48 ml, 41.4 mmol) was added drop wise by an addition funnel. The mixture was stirred for five hours and 2
S3 23 24 25 26 27 28 29 30 31 was put in refrigerator at 25 ºC for a week. The white crystalline solid was filtered off and recrystallized from ether/hexane at 73 ºC to afford 9a as colorless crystals (7.1 g, 64% yield). The R f value for this compound was 0.84 in 20% EtOAc/hexane. This compound gave a negative spot with 20% (w/v) ethanolic phosphomolybdic acid (PMA) solution and gave a positive spot with iodine vapor. Analytical data for 9a: 1 H NMR (300 MHz; CDCl 3 ): δ 4.26 4.18 (m, 1H), 2.54 2.40 (m, 1H), 2.42 2.19 (m, 2H), 2.08 1.96 (m, 1H), 1.82 1.57 (m, 2H), 1.15 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H); 13 C NMR (125 MHz; CDCl 3 ): δ 166.3, 75.0, 51.3, 48.5, 40.3, 39.4, 27.1, 26.9, 25.9, 23.5, 23.2; IR (film): 2982, 2876, 1805, 1404, 1265, 1167, 1130, 1099, 1052, 980, 894, 733 cm -1. IR and NMR were similar to what has been reported in the literature. 1 32 33 34 35 36 37 38 39 40 41 42 43 44 10a (1S,2S,5R,7S)-2,8,8-trimethyl-3-azatricyclo[5.1.1.0 2,5 ]nonan-4-one (10a): β-lactam sulfonyl chloride 9a (0.50 g, 1.80 mmol) was dissolved in ether (2.5 ml) and added drop wise to the biphasic mixture of 20% aqueous Na 2 SO 3 (5 ml) and Et 2 O (2.5 ml) at 0 C. The aqueous phase was kept between ph 8-9 by the addition of 10% aqueous KOH solution. Following the addition the reaction mixture was stirred at 0 to 23 C for 48 hours. The organic layer was separated and the aqueous layer was extracted with Et 2 O (2 X 15 ml). The combined organic layers were dried over anhydrous MgSO 4, filtered, and concentrated to yield 10a as colorless crystals (0.27 g, 83%). The R f value was 0.04 in 20% EtOAc/Hex. This compound gave a positive spot with PMA solution and a negative spot with iodine vapor. Analytical data for 10a: 1 H NMR (300 MHz; CDCl 3 ): δ 5.89 (s, 1H), 2.85 (dd, J = 10.5, 1.8 Hz, 1H), 2.21 2.10 (m, 2H), 2.07 (dd, J = 6.5, 4.6 Hz, 1H), 2.00 1.79 (m, 2H), 1.52 (d, J = 11.0 Hz, 1H), 1.46 3
S4 45 46 47 48 (s, 3H), 1.30 (s, 3H), 0.89 (s, 3H); 13 C NMR (125 MHz; CDCl 3 ): δ 173.0, 59.0, 50.1, 49.8, 41.8, 40.0, 27.5, 25.6, 25.5, 24.8, 23.1; IR (film): 1745.7, 1264.1 cm -1. IR and NMR were similar to what has been reported in the literature. 1 The enantiomer of this compound 10 b was synthesized similarly starting from 9b. 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 11a 3a,4,4-trimethyl-2-oxohexahydro-3,5-methanocyclopenta[b]pyrrole-1(2H)-sulfonyl chloride (11a): In a flask with a magnetic stirring bar was placed β-lactam sulfonyl chloride 9a, (4.01 g, 14.4 mmol) and hexane (100 ml). The reaction mixture was refluxed for 24 hours. The resulting mixture was cooled to 23 C and evaporated to brown oil. The crude product was purified by flash column chromatography (150 g, Acros; 0.035-0.070 mm, pore diameter ca 6 nm). Using 20% EtOAc in hexanes as the eluent to yield 11a as colorless crystals (1.60 g, 40% yield). The R f value for this compound was 0.65 in 20% EtOAc/Hex. This compound gave a positive spot with 20% (w/v) ethanolic PMA solution and gave a negative spot with iodine vapor. Analytical data for 11a: 1 H NMR (CDCl 3 ): 4.22 (dd, J = 8.4, 1.9 Hz, 1H), 2.53 2.40 (m, 1H), 2.40 2.21 (m, 3H), 2.02 (ap t, J = 4.1 Hz, 1H), 1.81 1.60 (m, 2H), 1.15 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H); 13 C NMR (125 MHz; CDCl 3 ): δ 174.8, 68.8, 56.1, 49.5, 47.9, 46.2, 34.4, 33.2, 19.5, 18.8, 11.6; IR (film): 1778, 1406, 1264 cm -1. IR and NMR were similar to what has been reported in the literature. 1 65 4
S5 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 12a 3a,4,4-trimethylhexahydro-3,5-methanocyclopenta[b]pyrrol-2(1H)-one (12a): γ-lactam sulfonyl chloride 11a (0.44 g, 1.58 mmol) was dissolved in ether (2.5 ml) and added drop wise to the biphasic mixture of 20% aqueous Na 2 SO 3 (5 ml) and Et 2 O (2.5 ml) at 0 C. The aqueous phase was kept between ph 8-9 by the addition of 10% aqueous KOH solution. The mixture was stirred first at 0 C and slowly warmed up to room temperature. The reaction went on for approximately 48 hours. 1 Organic layer was separated and the aqueous layer was extracted with Et 2 O (2 X 15 ml). The combined organic layers were dried over anhydrous MgSO 4, filtered, and concentrated to yield 12a as colorless crystals (0.26 g, 72% yield). 1 The R f value was 0.24 in 20% EtOAc:Hex. This compound gave a negative spot with 20% (w/v) ethanolic PMA solution and a positive spot with iodine vapor. Analytical data for 12a: 1 H NMR (300 MHz; CDCl 3 ): 6.24 (s, 1H), 3.34 (m, 1H), 2.26 2.11 (m, 1H), 2.13 1.97 (m, 1H), 1.89 (ap t, J = 4.1 Hz, 1H), 1.54 (dd, J = 12.9, 1.8 Hz, 1H), 1.25 (d, J = 13.1 Hz, 1H), 1.07 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ): δ 183.0, 60.6, 59.0, 48.5, 48.0, 47.5, 37.9, 33.2, 19.9, 19.2, 11.7; IR (film): 1693.8, 1421.8, 1264.1 cm -1. IR and NMR were similar to what has been reported in the literature. 3 82 83 84 85 13a 86 5
S6 87 88 89 90 91 92 (1S, 2S, 3R, 5S)-2-amino pinene-3-carboxylate (13a): Prepared according the reported literature. 2 (1R, 2R, 3S, 6R)-2-amino pinene-3-carboxylate 13b was synthesized starting from 10b. Analytical data for 13a: 1 H NMR (300 MHz; CDCl 3 ): δ 3.83 (q, 2H), 2.65 (dd, 1H), 2.00 (m, 1H), 1.87 (m, 1H), 1.59 (m, 2H), 1.43 (t, 3H), 1.01 (s, 3H), 0.97 (t, 1H), 0.93, (s, 3H), 0.90 (d, 1H), 0.69 (s, 3H); 13 C NMR (125 MHz; CDCl 3 ): δ 174.6, 59.9, 55.9, 54.8, 46.3, 39.7, 39.1, 31.1, 28.7, 28.1, 27.8, 23.5, 14.2; IR (film): 3300 (br), 2950, 2850, 1736 cm -1. IR and NMR were similar to what has been reported in the literature. 2 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 14a (1S, 2S, 3R, 5S)-2-Amino-3-hydroxymethylpinane (14a): To a slurry of LiAlH 4 (0.57 g, 15 mmol) and 110 ml of dry THF, amino ester 14a (10.1 g, 45 mmol) was added dropwise at 0 C. After refluxing for 4 hours (the reduction was monitored by TLC), the reaction mixture was cooled to 0 C and quenched by the addition of H 2 O (6.5 ml). The inorganic material was filtered off using a celite plug and washed with THF (10 ml), and Et 2 O (25 ml). The organic phase was washed with brine, dried over MgSO 4, filtered and concentrated. The pale-yellow oil was purified by flash column chromatography using ethyl acetate to remove impurities. The column was then flushed with ethanol to give the amino alcohol 14a. The enantiomer of this compound 14b was synthesized similarly starting from 13b. Analytical Data for 14a: 1 H NMR (300 MHz; CDCl 3 ): δ 3.62 (d, 1H), 3.47 (dd, 1H), 2.14 (m, 2H), 1.91 (m, 2H), 1.50 (t, 1H), 1.36 (m, 1H), 1.27 (s, 3H), 1.24 (s, 3H), 1.05, (d, 1H), 1.02 (s, 3H); 13 C NMR (125 MHz; CDCl 3 ): δ 66.0, 56.3, 55.9, 40.3, 39.2, 38.7, 32.2, 29.7, 28.1, 27.7, 23.5; IR (film): 3300(br), 2980, 2862 cm -1. IR and NMR were similar to what has been reported in the literature. 2 6
S7 109 Bioautographic assays 110 111 112 113 114 115 TLC-bioautographic assay was also run in 20% EtOAc:Hex on all the compounds and solvents used (hexanes, ethyl acetate, diethyl ether and methylene chloride) to further verify purity of compounds. In order to minimize the effects of diffusion that would arise due to different R f values of these compounds in a particular eluting system, we have used a modified bioautographic assay as described in the manuscript for reporting the zone of inhibitions for these compounds. 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 7
S8 132 133 134 135 References 1. Furst, G. T.; Wachsman, M. A.; Pieroni, J.; White, J. G.; Moriconi, E. J. Tetrahedron 1973, 29, 1675-1677 2. Szakonyi, Z.; Martinek, T.; Hetényi, A.; Fülöp, F. Tetrahedron: Asymmetry 2000, 11, 4571-4579. 3. Malpass, J. R. Tetrahedron Lett. 1972, 49, 4951-4954. 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 8
S9 156 Selected NMR Spectra 157 158 159 160 9
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