Prenylated hydroxystilbenes
11352310 · 2022-06-07
Assignee
Inventors
- Colin Charles Duke (Randwick NSW, AU)
- Van Hoan TRAN (Guildford NSW, AU)
- Rujee Kyokajee DUKE (Randwick NSW, AU)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C07C67/293
CHEMISTRY; METALLURGY
C07C37/055
CHEMISTRY; METALLURGY
C07C39/21
CHEMISTRY; METALLURGY
C07C67/297
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07C67/297
CHEMISTRY; METALLURGY
C07C39/21
CHEMISTRY; METALLURGY
C07C37/055
CHEMISTRY; METALLURGY
Abstract
A compound, composition, or method for treating cancer is disclosed. The compound (that can be used in the composition and/or method) can include ##STR00001##
or pharmaceutically acceptable salts or solvates thereof. R.sup.1a-R.sup.1d, R.sup.2-R.sup.3 (and R.sup.4 where applicable), and A---B can be as defined herein.
Claims
1. A compound according to formula (Ia): ##STR00049## or a pharmaceutically acceptable salt or solvate thereof, wherein: R.sup.1a is selected from CH.sub.2CH═C(CH.sub.3).sub.2, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHCH(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.1b is selected from, H, OH, OR.sup.4, CH.sub.2CH═C(CH.sub.3).sub.2, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHCH(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.1c is selected from H, OH, OR.sup.4a, CH.sub.2CH═C(CH.sub.3).sub.2, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHCH(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.1d is selected from H, OH, CH.sub.2CH═C(CH.sub.3).sub.2, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHCH(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; wherein at least one of R.sup.1b-1d is OH and at least one of R.sup.1a-1d is CH.sub.2CH═C(CH.sub.3).sub.2, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHCH(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.2 is selected from OH, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.3 is selected from OH, OR.sup.4, OCH.sub.2CH═C(CH.sub.3).sub.2, CH═CHC(CH.sub.3)═CH.sub.2, OCH═CHCH(CH.sub.3).sub.2, or OCH═CHC(CH.sub.3)═CH.sub.2; R.sup.4 is selected from, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl or benzyl; R.sup.4a is selected from, ethyl, propyl, isopropyl, butyl, isobutyl, butyl or benzyl; and A----B is selected from CH═CH, CH.sub.2CH.sub.2CH.sub.2, CH═CHX, or CH.sub.2-CH.sub.2X, where X═(CH.sub.2)pCH.sub.2 and p is an integer selected from the group consisting of 1 or 2, wherein the compound originates from propolis of the Lepidosperma genus.
2. A composition comprising a compound of claim 1 together with an acceptable excipient.
3. A method of treating inflammation, immunosuppression, skin aging, anti-bacterial infection or antifungal infection, comprising administering a compound according to claim 1 or a composition according to claim 2 to a subject in need thereof to treat the inflammation, immunosuppression, skin aging, anti-bacterial or antifungal infection.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
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(5)
(6)
(7)
(8)
(9)
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(11)
MODES FOR CARRYING OUT THE INVENTION
(12) In order to better understand the nature of the invention a number of examples will now be described as follows:
Materials
(13) Honey bees observed collecting from the sedges (Lepidosperma viscidum) were captured in plastic tubes, capped and frozen. Sections of the bee hind legs holding propolis were cut and pooled. Resin from plants were collected and stored at −20° C. until analysis. Plant samples were obtained and dried at 40° C. in a ventilated oven (Thermoline, NSW Australia) overnight to provide voucher specimens for identification by botanist, A/Prof Murray Henwood, John Ray Museum, University of Sydney, Sydney, Australia, and registered as Lepidosperma viscidum chemotype 1a, voucher number Duke 100222-42 and Lepidosperma viscidum chemotype 2a, voucher number Duke 100221-21.
(14) 76 Beehives made up of 3 10-frame boxes each fitted with a propolis mat under the hive cover lid were used to collect propolis samples. The individually numbered hives were used on location in the apiary sites situated in the central southern region of Kangaroo Island.
(15) Thin layer chromatography sheets precoated with silica gel 60 F254 and silica gel 60H for normal-phase short-column chromatography (NPSCC) were purchased from Merck. TLC plates were visualized with a UVGL-58 mineral-light lamp, Multiband UV-2544/366.
(16) All the chemicals used in the isolation and synthesis, including deuterated NMR solvents such as chloroform-d and methanol-d.sub.4, deuterated dimethyl sulfoxide (d.sub.6-dimethyl sulfoxide) were purchased from Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia). Solvents including hexane, dichloromethane, ethyl acetate, isopropanol, ethanol, methanol, and acetic acid were of analytical grade and purchased from Ajax Fine Chem, Taren Point, NSW, Australia or Asia Pacific Specialty Chemical Ltd (APS).
(17) Rotavapor model R-114 rotary evaporator with a water bath temperature ranging between 40-60° C. was used to evaporate the solvent fraction. Vacuum pump V-700 or Vacuubrand MD 4C NT diaphragm pump (Vacuubrand GMBH, Wertheim, Germany) with vacuum controller V-800 or V-850 is used. Final drying is carried out by a Napco 5831 vacuum oven (NAPCO, Salt Lake City, USA) using a DirectTorr vacuum pump (Sargent-Welch, Buffalo, USA).
(18) Preparative HPLC was performed on a Shimadzu preparative gradient LC-8A system on a reversed-phase column (Grace, Alltima C18 5 μM 22 mm ID×250 mm), injection volume 500 μL, eluted with methanol (75%) and water at 10 mL/min and detected at 280 nm with a UV-Vis detector (Shimadzu SPD-20A). Analytical HPLC was performed on Shimadzu UFLC, LC-20AD pump, SIL-20A HT autosampler, with a Hewlett-Packard Column, NUCLEOSIL 100C18, 5 μm, 4 mm×125 mm, injection volume 20 μL, eluted with methanol-water-acetic acid (70:29.8:0.2) at 1 mL/min and detected at 230 nm with a UV-Vis detector (Shimadzu SPD-20A).
(19) .sup.1H and .sup.13C Nuclear magnetic resonance (NMR) analyses were carried out on Varian 400 MHz System with a SMS autosampler (Palo Alto, Calif., USA). NMR spectra were referenced to tetramethylsilane (TMS). Mass spectra were obtained from a ThermoFinnigan TSQ 7000 (LC-MS/MS system) and a Finnigan Polaris Ion Trap MS/MS system (Finnigan, San Jose, USA) using an Xcalibur 1.2 data system.
Determination of the .SUP.1.H-NMR Chemical Profiles of the Plant and Propolis Specimens
(20) Resin samples from the base of stem (0.1 g), bee hind leg (0.01 g) and beehive propolis (1.0 g) were extracted with ethyl acetate at room temperature for 15 min. The extracts were filtered, dried under reduced pressure and analyzed by .sup.1H-NMR and HPLC. Samples from the sedge type-1 were found to contain prenylated hydroxystilbenes and cinnamates as major constituents, whereas those from sedge type-2 showed only prenylated stilbenes as major constituents as discussed above. Propolis samples were subsequently selected for isolation of the components.
Isolation and Identification of Prenylated Polydroxystilbenes from Propolis of Kangaroo Island
General Method
(21) Propolis (10 g) was extracted with dichloromethane at room temperature with stirring for 1 hr. The extract was subjected to purification using normal-phase short column chromatography (NPSCC). A step-wise gradient of mobile phase (2×100 mL) consisting of dichloromethane (DCM) and ethyl acetate (EtOAc) at 0, 1, 2, 4, 8, 10, 15, 20, 50 and 100% was employed to elute the components which were analysed by TLC and NMR. Further purification of the compounds, if required, was subsequently carried out on the same NPSCC with different mobile phases consisting of either hexane and EtOAc or hexane and isopropanol. Normal-phase preparative HPLC was also employed when required to further purify the compounds (Shimadzu LC-8A). The compounds were eluted through a silica column (Altima® silica 10 μm, 10 cm×250 cm) at a flow rate of 10 mL/min with a mobile phase of 2% isopropanol in hexane at ambient temperature. The elution of compounds was monitored with a UV detector (UV/Vis SPD-20A) at 280 nm. Structures and identity of these purified compounds were characterized by .sup.1H- and .sup.13C-NMR and mass spectrometry including high resolution mass spectrometry. Detailed structural analyses of the isolated compound were also carried out when needed by 2D-NMR using Gradient Heteronuclear Multiple Bond Coherence (GHMBC).
Isolated Prenylated Polyhydroxystilbene Derivatives
(E)-2-(3-methyl-2-buten-1-yl)-4′,5-dihydroxy-3,3′-dimethoxystilbene (USYDS1)
(22) ##STR00025##
(23) Light yellow liquid. C.sub.21H.sub.24O.sub.4 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 14.64 min. δ 7.16 (d, J=16 Hz, H), 7.07 (d, J=4 Hz, H), 6.94 (dd, J=8, 4 Hz, H), 6.86 (d, J=16 Hz, H), 6.78 (d, J=8 Hz, H), 6.63 (d, J=4 Hz, H), 6.34 (d, J=4 Hz, H), 5.06 (m, H), 3.89 (s, 3H), 3.77 (s, 3H), 3.39 (m, 2H), 1.80 (bs, 3H), 1.66 (bs, 3H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) δ 158.5, 154.4, 146.7, 145.6, 138.1, 130.6, 130.4, 130.3, 124.3, 123.7, 120.7, 120.6, 114.5, 108.2, 103.9, 98.2, 55.9, 55.7, 25.8, 24.5, 18.0; CI-MS: m/z 339 (M−1).sup.−, HRESIMS: m/z 341.1749 (M+H).sup.+, calcd 341.1747 for C.sub.21H.sub.25O.sub.4.
(E)-3-(3-methyl-2-butenyloxy)-4′,5-dihydroxy-3′-methoxystilbene (USYDS2)
(24) ##STR00026##
(25) Light yellow liquid. C.sub.20H.sub.22O.sub.4 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 15.46 min. δ 7.12 (d, J=4 Hz, 1H), 7.01 (d, J=16 Hz, 1H), 6.97 (dd, J=8, 4 Hz, 1H), 6.88 (d, J=16 Hz, 1H), 6.78 (d, J=8 Hz, 2H, 6.56 (m, 2H), 6.24 (m, 1H), 5.46 (m, 1H), 4.52 (d, J=4 Hz, 2H), 3.90 (s, 3H), 1.79 (bs, 3H), 1.76 (bs, 3H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) δ 160.2, 156.9, 146.7, 145.5, 139.9, 138.6, 129.9, 129.2, 126.2, 120.6, 119.4, 114.7, 108.4, 105.9, 105.4, 101.5, 65.0, 55.9, 25.8, 18.2; CI-MS m/z 325 (M−1).sup.−, HRESIMS m/z 327.15938 (M+H).sup.+, calcd 327.1591 for C.sub.20H.sub.23O.sub.4.
(E)-4-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxystilbene (USYDS13)
(26) ##STR00027##
(27) Light yellow liquid. C.sub.20H.sub.22O.sub.4 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 11.46 min. δ 7.08 (d, J=4 Hz, 1H), 6.93 (dd, J=8, 4 Hz, 1H), 6.91 (d, J=16 Hz, 1H), 6.78 (d, J=16 Hz, 1H), 6.77 (d, J=8 Hz, 1H), 6.46 (s, 2H), 5.23 (m, 1H), 3.89 (s, 3H), 3.28 (m, 2H), 1.76 (bs, 3H), 1.65 (bs, 3H); .sup.13C NMR-GHMBC (methanol-d.sub.4, 100 MHz): δ 157.4 (2C, C-3/5, H-2/6, H-1″), 149.3 (1C, C-3′, H-2′, H-5′, OCH.sub.3), 147.6 (1C, C-4′, H-2′, H-6′), 137.7 (1C, C-1′, H-beta), 131.4 (1C, C-1, H-alpha), 131.3 (1C, C-3″, H-1″, H-4″, H-5″), 128.7 (1C, C-beta, H-2′, H-alpha), 127.6 (1C, C-alpha, H-2/6, H-6′), 124.8 (1C, C-2″, H-1″, H-4″, H-5″), 121.2 (1C, C-6′, H-2′, H-beta), 116.5 (1C, C-5′),116.1 (1C, C-4, H-2/6, H-1″), 110.4 (1C, C-2′, H-beta), 105.9 (2C, C-2/6, H-2/6, H-alpha), 56.5 (1C, OCH.sub.3), 26.1 (1C, C-4″, H-5″), 23.5 (1C, C-1″), 18.1 (1C, C-5″, H-4″); CI-MS: m/z 325 (M−1).sup.−; HRMS: 325.14453 [M−1].sup.−, (calculated 325.14398 for C.sub.20H.sub.21O.sub.4).
(E)-3-(3-methyl-2-butenyloxy)-3′,4′,5-trihydroxystilbene (USYDS4)
(28) ##STR00028##
(29) Light yellow liquid. C.sub.19H.sub.20O.sub.4 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 18.62 min. δ 6.98 (d, J=4 Hz, 1H), 6.94 (d, J=16 Hz, 1H), 6.85 (dd, J=8, 4 Hz, 1H), 6.80 (d, J=16 Hz, 1H), 6.74 (d, J=8 Hz, 1H), 6.53 (m, 2H), 6.23 (m, 1H), 5.46 (m, 1H), 4.51 (d, J=4 Hz, 2H), 1.79 (bs, 3H), 1.76 (bs, 3H); .sup.13C NMR (methanol-d.sub.4, 100 MHz): δ 160.2, 158.2, 145.2, 145.1, 139.8, 136.9, 129.5, 128.5, 125.5, 119.9, 118.8, 114.9, 112.4, 105.2, 103.8, 100.7, 64.4, 24.4, 16.8; CI-MS: m/z 311 (M−1).sup.−; HREIMS: m/z 335.1252 (M+Na).sup.+, calcd 335.1259 for C.sub.19H.sub.20O.sub.4Na.
(E)-2,4-di(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxystilbene (USYDS6)
(30) ##STR00029##
(31) Off-white solid. C.sub.24H.sub.28O.sub.3 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 9.35 min. δ 7.30 (d, J=8 Hz, 2H), 7.11 (d, J=16 Hz, 1H), 6.80 (d, J=16 Hz, 1H), 6.76 (d, J=8 Hz, 2H), 6.63 (s, 1H), 5.21 (m, 1H), 5.10 (m, 1H), 3.41 (m, 2H), 3.35 (m, 2H), 1.81 (bs, 3H), 1.78 (bs, 3H), 1.68 (bs, 6H); CI-MS: m/z 363 (M−1).sup.−.
(E)-2,4-di(3-methyl-2-buten-1-yl)-3,3′,4′,5-tetrahydroxystilbene (USYDS7)
(32) ##STR00030##
(33) Off-white solid. C.sub.24H.sub.28O.sub.4 .sup.1H NMR (methanol-d.sub.4, 400 MHz): R.sub.t 10.46 min. δ 7.07 (d, J=16 Hz, 1H), 6.94 (d, J=4 Hz, 1H), 6.79 (dd, J=8.4 Hz, 1H), 6.73 (d, J=16 Hz, 1H), 6.71 (d, J=8 Hz, 1H), 6.62 (s, 1H), 5.21 (m, 1H), 5.10 (m, 1H), 3.41 (m, 2H), 3.35 (m, 2H), 1.81 (bs, 3H), 1.78 (bs, 3H), 1.68 (bs, 6H); .sup.13C NMR (methanol-d.sub.4, 100 MHz): δ 153.23, 152.79, 145.04, 144.84, 134.98, 130.52, 130.26, 129.75, 128.67, 124.08, 123.94, 122.93, 118.56, 118.51, 115.32, 114.93, 112.30, 103.85, 24.54, 24.49, 24.46, 22.32, 16.74, 16.54; CI-MS: m/z 379 (M−1).sup.−1; HRMS: 379.19148 [M−1].sup.−, (calculated 379.19092 for C.sub.24H.sub.27O.sub.4).
(E)-2-(3-methyl-2-buten-1-yl)-3,3′,4′,5-tetrahydroxystilbene (USYDS8)
(34) ##STR00031##
(35) C.sub.19H.sub.20O.sub.4 .sup.1H NMR (acetone-d.sub.6, 400 MHz): δ 7.07 (d, J=2 Hz, 1H), 7.15 (d, J=16 Hz, 1H), 6.89 (dd, J=8, 2 Hz, 1H), 6.81 (d, J=8 Hz, 1H), 6.80 (d, J=16 Hz, 1H), 6.63 (d, J=2 Hz, 1H), 6.35 (d, J=2 Hz, 1H), 5.15 (t, J=7 Hz, 1H), 3.42 (d, J=7 Hz, 2H), 1.81 (s, 3H), 1.65 (s, 3H). .sup.13C NMR (acetone-d.sub.6, 100 MHz): δ 6 155.9, 155.8, 145.4, 145.3, 138.4, 130.1, 129.7, 129.4, 124.5, 123.9, 117.4, 119.0, 115.4, 112.9, 103.4, 101.6, 24.0, 25.0, 17.2. HRESIMS: (m/z) 313.1434 (M+H).sup.+, calcd 313.1440 for C.sub.19H.sub.21O.sub.4.
(E)-2-(3-methyl-2-buten-1-yl)-3′,4′,5-trihydroxy-3-methoxystilbene (USYDS9)
(36) ##STR00032##
(37) C.sub.20H.sub.22O.sub.4 .sup.1H NMR (acetone-d.sub.6, 400 MHz): δ 7.16 (d, J=16 Hz, 1H), 7.07 (d, J=2 Hz, 1H), 6.90 (dd, J=8, 2 Hz, 1H), 6.85 (d, J=16 Hz, 1H), 6.82 (d, J=8 Hz, 1H), 6.71 (d, J=2 Hz, 1H), 6.39 (d, J=2.3 Hz, 1H), 5.09 (1H, t, J=7 Hz, 1H), 3.78 (s, 3H), 3.40 (d, J=7 Hz, 2H), 1.80 (s, 3H), 1.64 (s, 3H). .sup.13C NMR (acetone-d.sub.6, 100 MHz): δ 158.5, 156.3, 145.3 (2C), 138.0, 130.1, 130.0, 129.6, 123.7, 124.2, 119.1, 118.9, 115.3, 112.9, 103.7, 98.1, 55.0, 25.0, 23.9, 17.2. HRESIMS: m/z 327.1592 (M+H).sup.+, calcd 327.1596 for C.sub.20H.sub.23O.sub.4.
(E)-2-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxystilbene (USYDS10)
(38) ##STR00033##
(39) C.sub.20H.sub.22O.sub.4 .sup.1H NMR: (acetone-d.sub.6, 400 MHz) δ 7.13 (d, J=16 Hz, 1H) 7.02 (dd, J=8, 2 Hz, 1H), 7.00 (s, 1H), 6.92 (d, J=8 Hz, 1H), 6.85 (d, J=16 Hz, 1H), 6.66 (d, J=2 Hz, 1H), 6.30 (d, J=2 Hz, 1H), 5.20 (t, J=7 Hz, 1H), 3.95 (s, 3H), 3.43 (d, J=7 Hz, 2H), 1.84 (s, 3H), 1.75 (s, 3H). .sup.13C NMR (acetone-d.sub.6, 100 MHz): δ 155.4, 154.4, 146.7, 145.7, 138.8, 133.6, 131.2, 130.1, 124.2, 122.5, 120.5, 117.7, 114.6, 108.4, 105.3, 102.5, 55.9, 25.8, 25.1, 18.0. HRESIMS: m/z 349.1411 (M+Na).sup.+, calcd 349.1416 for C.sub.20H.sub.22O.sub.4Na.
(E)-3-(3-methyl-2-butenyloxy)-4′,5-dihydroxystilbene (USYDS11)
(40) ##STR00034##
(41) C.sub.19H.sub.22O.sub.3 .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 7.38 (dd, J=7, 2 Hz, 2H), 7.00 (d, J=16 Hz, 1H), 6.84 (d, J=16 Hz, 1H), 6.82 (dd, J=7, 2 Hz, 2H), 6.64 (t, J=2 Hz, 1H), 6.56 (t, J=2 Hz, 1H), 6.33 (t, J=2 Hz, 1H), 5.50 (t, J=7 Hz, 1H), 4.51 (d, J=7 Hz, 2H), 1.81 (s, 3H), 1.76 (s, 3H). .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 160.4, 156.7, 155.4, 139.9, 138.4, 130.1, 128.8, 128.0 (2C), 126.3, 119.5, 115.6 (2C), 105.6, 105.5, 101.3, 64.9, 25.8, 18.2.
4-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxydihydrostilbene (USYDS12)
(42) ##STR00035##
(43) C.sub.19H.sub.22O.sub.4 .sup.1H NMR: (CD.sub.3OD, 400 MHz) δ 6.97 (d, J=8 Hz, 2H), 6.66 (d, J=8 Hz, 2H), 6.13 (s, 2H), 5.22 (t, J=7 Hz, 1H), 3.24 (d, J=7 Hz, 2H), 2.72 (m, 2H), 2.64 (m, 2H), 1.74 (s, 3H), 1.64 (s, 3H). .sup.13C NMR (CD.sub.3OD, 100 MHz): δ 155.5 (2C), 154.9, 140.3, 132.9, 129.4, 128.9 (2C), 123.6, 114.6 (2C), 112.3, 106.5 (2C), 38.0, 36.8, 24.5, 21.7, 16.5.
(E)-2-(3-methyl-2-buten-1-yl)-3-(3-methyl-2-butenyloxy)-3′,4′,5-trihydroxystilbene (USYDS14)
(44) ##STR00036##
(45) Colourless solid, yield 12 mg. ESI-MS: m/z 379 [M−1].sup.−, .sup.1H-NMR (methanol-d.sub.4 400 MHz): δ 7.07 (d, J=16 Hz, 1H), 6.96 (d, J=4 Hz, 1H), 6.80 (dd, J=8, 4 Hz, 1H), 6.79 (d, J=16 Hz, 1H), 6.74 (d, J=8 Hz, 1 H), 6.61 (d, J=4 Hz, 1H), 6.32 (d, J=4 Hz, 1H), 5.47 (m, 1H), 5.06 (m, 1H), 4.48 (m, 2H), 3.37 (m, 2H), 1.78 (m, 6H), 1.75 (m, 3H), 1.66 (m, 3H). .sup.13C-NMR (100 MHz, CD.sub.3OD): δ 16.75, 16.80, 23.84, 24.45, 24.54, 64.86, 99.04, 103.41, 112.39, 114.96, 118.71, 119.14, 120.14, 123.54, 123.97, 129.51, 129.70, 130.01, 136.78, 137.95, 145.08, 145.09, 155.59, 157.47. HRMS: 379.19148 [M−1].sup.−, (calculated 379.19092 for C.sub.24H.sub.27O.sub.4).
(E)-2,6-di(3-methyl-2-buten-1-yl)-3,3′,5,5′-tetrahydroxystilbene USYDS15
(46) ##STR00037##
(47) Colourless solid, yield 9 mg. ESI-MS: m/z 379 [M−1].sup.−, .sup.1H NMR (methanol-d.sub.4, 400 MHz): δ 6.91 (br t, 1H), 6.82 (d, J=16 Hz, 1H), 6.73 (d, J=1 Hz, 2H), 6.28 (s, 1H), 6.27 (d, J=16 Hz, 1H), 5.12 (m,2H), 3.26 (d, J=6 Hz 4H), 1.65 (m, 6H), 1.59 (m, 6H). .sup.13C-NMR (100 MHz, CD.sub.3OD): δ 16.75, 24.53, 25.60, 112.27, 114.86, 117.66, 118.24, 124.06, 124.63, 130.20, 133.33, 139.41, 144.73, 144.97, 153.04. HRMS: 379.19149 [M−1].sup.−, (calculated 379.19092 for C.sub.24H.sub.27O.sub.4).
(E)-2,6-di(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxystilbene USYDS18
(48) ##STR00038##
(49) Yield 5 mg. ESI-MS: m/z 393 [M−1].sup.−. .sup.1H NMR: (CD.sub.3OD, 400 MHz) δ 7.03 (d, J=2 Hz, 1H), 6.87 (d, J=17 Hz, 1H), 6.85 (dd, J=8, 2 Hz, 1H), 6.77 (d, J=8 Hz, 1H), 6.33 (d, J=17 Hz, 1H), 6.30 (s, 1H), 5.14 (br t, J=6 Hz, 2H), 3.88 (s, 3H), 3.26 (br d, J=6 Hz, 4H), 1.66 (br s, 6H), 1.60 (br s, 6H). .sup.13C NMR (CD.sub.3OD, 100 MHz): δ 18.37 (2C), 26.12 (2C), 27.20 (2C), 56.53, 102.58, 110.27, 116.42, 119.23 (2C), 121.03, 126.02 (2C), 126.31, 130.41 (2C), 131.7, 134.87, 140.95, 147.51, 149.26, 154.65 (2C). HRMS: 417.20363 [M+23].sup.+, (calculated 417.20418 for C.sub.25H.sub.30O.sub.4Na).
Chemical Modification and Synthesis of Prenylated Polyhydroxystilbenes
A. Rearrangement of O-prenyl to C-prenyl Polyhydroxystilbene
(50) A mixture of USYDS2 (5 mg) and Florisil (5 mg) in xylene (2 ml) was heated at 120° C. for 30 min in a microwave reactor (CEM Discover Microwave). The product was dried under reduced pressure and purified using NPSCC. Two rearrangement products were observed in a combined yield of approximately 30%.
(51) The major product observed was (E)-4-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxystilbene (USDYS13):
(52) ##STR00039##
(53) The minor product observed was (E)-2-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxystilbene (USYDS10).
B. Synthesis of Prenylated Polyhydroxystilbenes
1. Preparation of 3,5-dihydroxybenzoic Acid Methyl Ester (1)
(54) To a solution of 3,5-dihydroxybenzoic acid (10 g, 64.9 mmol) in anhydrous methanol (150 mL) acetyl chloride (2 mL, 28.1 mmol) was added dropwise. The mixture was heated at reflux under N.sub.2 for 17 hours. The solvent was evaporated and the residue was dissolved in ethyl acetate (100 mL), then washed with saturated sodium hydrogen carbonate (3×100 mL). The combined organic layers were washed with water (2×100 mL), dried over sodium sulfate, filtered and concentrated in vacuo to afford 2 as a colourless solid (9.80 g, 90%): mp 167-168° C. (lit..sup.1 mp 168-169° C.); .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.66 (s, 2H), 7.00 (d, J=2.4 Hz, 2H), 6.59 (t, J=2.4 Hz, 2H), 3.83 (s, 3H).
2. Preparation of 3-hydroxy-5-benzyloxybenzoic Acid Methyl Ester (2)
(55) To a suspension of NaH (60% dispersion in mineral oil, 3.3 g, 137.3 mmol) in anhydrous N,N-dimethylformamide (DMF) (100 mL) was added a solution of 2 (10 g, 59.5 mmol) in anhydrous DMF (30 mL) at 0° C. under N.sub.2, followed by the dropwise addition of benzyl bromide (6.4 mL, 53.8 mmol). The reaction mixture was stirred at room temperature for 2 hours, quenched with cold water (50 mL), acidified with cold 1 M HCl (20 mL) and extracted with diethyl ether (3×50 mL). The combined organic extracts were washed with water (2×50 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The product was purified using NPSCC using a step-wise gradient consisting of chloroform/ethanol to afford 2 as an off-white solid (4.5 g, 60%).sup.2: mp 97-98° C. (lit..sup.3 mp 98° C.); C.sub.15H.sub.14O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.01 (s, 1H), 7.43-7.33 (m, 5H), 7.24 (dd, J=2.0, 1.2 Hz, 1H), 7.20 (dd, J=2.0, 1.2 Hz, 1H), 6.70 (t, J=2.0 Hz, 1H), 5.07 (s, 2H), 3.90 (s, 3H).
3. Preparation of 3-hydroxy-5-benzyloxybenzoic Acid (3)
(56) To a solution of 2 (4.5 g, 16.7 mmol) in methanol (30 mL) was added 1 M NaOH (60 mL, 60 mmol). The resulting reaction mixture was stirred at 45° C. for 4 hours under N.sub.2, followed by the addition of 1 M HCl (50 mL) to acidify the solution before extraction with ethyl acetate (3×40 mL). The combined organic extracts were washed with water (2×30 mL) and dried over sodium sulfate. The solvent was evaporated in vacuo to afford 3 as an off-white solid (4.02 g, 94%): mp 196-198° C.; C.sub.14H.sub.12O.sub.4 .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.42-7.33 (m, 5H), 7.21 (dd, J=2.4, 1.2 Hz, 1H), 7.11 (dd, J=2.4, 1.2 Hz, 1H), 6.68 (t, J=2.4 Hz, 1H), 5.07 (s, 2H).
4. Preparation of 3-hydroxy-5-methoxybenzoic Acid (4)
(57) The title compound was prepared as described above by using methyl iodide instead of benzyl bromide to afford 4 as an off-white solid, mp 199-200° C. (lit..sup.4 mp 199-200° C.); C.sub.8H.sub.8O.sub.4 .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.01 (s, 2H), 7.16 (t, J=1.2 Hz, 1H), 7.13 (t, J=1.2 Hz, 1H), 6.15 (t, J=2.4 Hz, 1H), 3.82 (s, 3H).
5. Preparation of 3-acetoxy-5-benzyloxybenzoic Acid (5)
(58) To the solution of 3 (4.02 g, 17.6 mmol) in acetic anhydride (20 mL, 21.6 mmol) was added pyridine (10%, 0.15 mL, 1.76 mmol). The mixture was stirred at room temperature for 4 hours, quenched with water (30 mL), acidified by 0.1 M HCl (10 mL) and extracted with ethyl acetate (3×40 mL). The combined organic extracts were washed with water (2×30 mL), dried over sodium sulfate and filtered. The solvent was evaporated in vacuo and recrystallised from a mixture (1:1) of hexane/ethyl acetate to afford 5 as pinkish crystals (3.86 g, 96%).sup.5: mp 133-134° C.; C.sub.16H.sub.14O.sub.5 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.59 (dd, J=2.4, 1.2 Hz, 1H), 7.45 (dd, J=2.4, 1.2 Hz, 1H), 7.43-7.34 (m, 5H), 6.99 (t, J=2.4 Hz, 1H), 5.1 (s, 2H), 2.32 (s, 3H).
6. Preparation of 3-acetoxy-5-methoxybenzoic Acid (6)
(59) The title compound was prepared as described in 5 was used to give 6 as an off white solid: mp 151-153° C.; C.sub.10H.sub.10O.sub.5 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.50 (dd, J=2.0, 1.4 Hz, 1H), 7.43 (dd, J=2.0, 1.4 Hz, 1H), 6.90 (t, J=2.0 Hz, 1H), 3.86 (s, 3H), 2.32 (s, 3H).
7. Preparation of 3-methoxy-4-benzyloxybenzaldehyde (7)
(60) To a suspension of NaH (60% dispersion in mineral oil, 1.6 g, 66.6 mmol) in anhydrous DMF (50 mL) at 0° C. under N.sub.2 was added vanillin (4-hydroxy-3-methoxy benzaldehyde, 4 g, 26.3 mmol) in anhydrous DMF (20 mL) slowly via syringe, followed by the dropwise addition of benzyl bromide (3 mL, 25.2 mmol). The mixture was stirred at room temperature for 4 hours, quenched with cold water (30 mL), acidified with cold 1 M HCl (15 mL) and extracted with diethyl ether (3×40 mL). The combined organic extracts were washed with water (2×30 mL), dried over sodium sulfate and dried under reduced pressure to afford 7 as a colourless solid (6 g, 92%): mp 60-62° C. (lit..sup.6 mp 61-62° C.); C.sub.15H.sub.14O.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.84 (s, 1H)), 7.43-7.31 (m, 7H)), 7.00 (d, J=8.4 Hz, 1H)), 5.25 (s, 2H)), 3.95 (s, 3H)).
8. Preparation of 4-methoxy-3-benzyloxybenzaldehyde (8)
(61) The title compound was prepared as described in 7 to give 8 as a colourless solid: mp 61-63° C. (lit..sup.7 mp 61-63° C.); C.sub.15H.sub.14O.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.49-7.31 (m, 7H)), 7.00 (d, J=8.1 Hz, 1H)), 5.20 (s, 2H)), 3.97 (s, 3H)).
9. Preparation of 4-ethenyl-2-methoxy-1-benzyloxybenzene (9)
(62) To a suspension of methyltriphenylphosphonium bromide (6.5 g, 18.2 mmol) in anhydrous tetrahydrofuran (THF) (15 mL) under N.sub.2 at 0° C. was added potassium tert-butoxide (2.3 g, 20.5 mmol) and the reaction mixture was warmed to room temperature. A solution of benzyl vanillin (7) (4 g, 16.5 mmol) in anhydrous THF (15 mL) was added dropwise and stirred for 2 hours, then quenched with cold water (20 mL), acidified with 0.1 M HCl (20 mL), and extracted with ethyl acetate (3×40 mL). The combined organic extracts were washed with 20% aq. sodium chloride (20 mL) then dried under reduced pressure. The product was purified using NPSCC using a step-wise mobile phase consisting of hexane/ethyl acetate to afford 9 as a colourless solid (3.37 g, 85%); mp 53-54° C. (lit..sup.8 mp 50-51° C.); C.sub.16H.sub.16O.sub.2 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.45-7.28 (m, 5H), 6.99 (d, J=2 Hz, 1H), 6.90 (dd, J=4, 1 Hz, 1H), 6.83 (d, J=4 Hz, 1H), 6.68 (dd, J=17, 1 Hz, 1H), 5.64 (dd, J=17, 1 Hz, 1H), 5.17 (s, 2H), 5.14 (d, J=1 Hz, 1H), 3.92 (s, 3H).
10. Preparation of 5-ethenyl-2-methoxy-1-benzyloxybenzene (10)
(63) The title compound was prepared as described in 9 to give 10 as an off-white solid; mp 68-69° C. (lit..sup.9 mp 68-69° C.); C.sub.16H.sub.16O.sub.2 .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.48-7.31 (m, 5H), 7.01 (d, J=2 Hz, 1H), 6.97 (dd, J=8, 2 Hz, 1H), 6.85 (d, J=8 Hz, 1H), 6.64 (dd, J=17, 1 Hz, 1H), 5.56 (dd, J=17, 1 Hz, 1H), 5.17 (s, 2H), 5.12 (d, J=1 Hz, 1H), 3.92 (s, 3H).
11. Preparation of E-1-[3-acetoxy-5-benzyloxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (11)
(64) To a solution of N,N-bis(2,6-diisopropyl) dihydro imidazolium chloride (0.093 g, 10%, 0.217 mmol) and Pd(OAc).sub.2 (0.048 g, 10%, 0.217 mmol) in xylene (3 mL) under N.sub.2 at room temperature was added the acid chloride of 5 (0.7 g, 2.17 mmol) in xylene 2 mL, followed by the addition of 4-ethylmorpholine (0.04 mL, 0.316 mmol) and reagent 9 (0.627 g, 2.61 mmol) in xylene (3 mL). The mixture was heated at 130° C. for 18-22 hours. The solvent was evaporated in vacuo and the product was purified using NPSCC (hexane/ethyl acetate 3:1 as mobile phase) to give 11 as an yellow oil (0.33 g, 31.6%); C.sub.31H.sub.28O.sub.5 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.45-7.28 (m, 13H), 7.06 (d, J=2.0 Hz, 1H), 7.02 (d, J=17 Hz, 1H), 6.97 (dd, J=6, 2 Hz, 1H), 6.89 (m, 1H), 6.62 (t, J=2 Hz, 1H), 5.18 (s, 2H), 5.07 (s, 2H), 3.94 (s, 3H), 2.30 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 169.39, 159.78, 151.81, 149.79, 148.32, 139.82, 137.00, 136.57, 130.48, 129.78, 128.63 (2C), 128.58 (2C), 128.10, 127.88, 127.55 (2C), 127.24 (2C), 126.02, 119.98, 113.95, 112.02, 110.50, 109.48, 107.28, 71.0, 70.27, 56.02, 21.19; CI-MS m/z (%): 481 (M+1).sup.+, 503 (M+Na).sup.+; HRMS: m/z 503.1828 (M+Na).sup.+, calcd 503.1834 for C.sub.31H.sub.28O.sub.5Na.
12. Preparation of E-1-[3-acetoxy-5-benzyloxyphenyl]-2-[3-benzyloxy-4-methoxyphenyl]ethene (12)
(65) The title compound was prepared as described in 11 to condense the acid chloride of 5 with reagent 10. The product was recrystallised from a mixture (2:1) of hexane/toluene to afford yellowish needle crystals; C.sub.31H.sub.28O.sub.5 mp 133-134° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.49-7.29 (m, 10H), 7.07 (d, J=2 Hz, 1H), 7.05 (dd, J=8, 2 Hz, 1H), 6.97 (d, J=16 Hz, 1H), 6.95 (t, J=2 Hz, 1H), 6.89 (d, J=8 Hz, 1H), 6.84 (t, J=2 Hz, 1H), 6.82 (d, J=16 Hz, 1H), 6.62 (t, J=2 Hz, 1H), 5.18 (s, 2H), 5.07 (s, 2H), 3.94 (s, 3H), 2.30 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 169.39, 159.76, 151.80, 149.90, 148.34, 139.83, 137.05, 136.58, 129.94, 129.78, 128.64 (2C), 128.60 (2C), 128.11, 127.94, 127.58 (2C), 127.38 (2C), 125.88, 120.59, 112.00, 111.91, 111.81, 110.52, 107.22, 71.16, 70.27, 56.06, 21.18 (CH.sub.3CO); CI-MS m/z 481 (M+1).sup.+, 503 (M+Na).sup.+; FIRMS m/z 503.1832 (M+Na).sup.+, calcd 503.1834 for C.sub.31H.sub.28O.sub.5Na.
13. Preparation of E-1-[3-acetoxy-5-methoxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (13)
(66) The title compound was prepared as described in 11 to condense the acid chloride of 6 with reagent 9 to give the title compound as an off white solid; mp 104-106° C.; C.sub.25H.sub.24O.sub.5 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.45-7.30 (m, 8H), 7.07 (d, J=2 Hz, 1H), 7.03 (d, J=16 Hz, 1H), 6.98 (dd, J=8, 2 Hz, 1H), 6.90 (d, J=16 Hz, 1H), 6.88 (m, 1H), 5.18 (s, 2H), 3.95 (s, 3H), 3.83 (s, 3H), 2.31 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 169.41, 160.58, 151.83, 149.78, 148.31, 139.76, 137.00, 130.49, 129.72, 128.58 (2C), 127.88, 127.24 (2C), 126.07, 119.97, 113.94, 111.73, 109.67, 109.46, 106.54, 71.01, 56.02, 55.50, 21.18; CI-MS m/z 405 [M+1].sup.+; HRMS m/z 405.1695 (M+H).sup.+, calcd 405.1702 for C.sub.25H.sub.25O.sub.5.
14. Preparation of E-1-[3-hydroxy-5-benzyloxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (14)
(67) To a solution of stilbene 11 (0.04 g, 0.083 mmol) in mixed solvent (MeOH / THF/H.sub.2O, 3/3/3 mL) was added NaOH (0.02 g, 0.35 mmol) at 0° C. under N.sub.2. The reaction mixture was warmed up to room temperature and stirred for 3 hours. The solution was acidified with 0.1 M HCl (5 mL) and extracted with ethyl acetate (3×20 mL). The combined organic extracts were washed with 20% aq. sodium chloride (10 mL), dried over sodium sulphate and evaporated in vacuo. The product was purified using NPSCC (mobile phase of hexane/ethyl acetate) followed by recrystallisation to afford 14 as a colourless solid (0.016 g, 44.5%); mp 114-115° C.; C.sub.20H.sub.26O.sub.4 .sup.1H NMR NMR (400 MHz, Methanol-d.sub.4) δ 7.45-7.28 (m, 10H), 7.06 (d, J=2 Hz, 1H), 7.00 (d, J=16 Hz, 1H), 6.98 (dd, J=8, 2 Hz, 1H), 6.87 (d, J=8 Hz, 1H), 6.86 (d, J=16 Hz, 1H), 6.71 (t, J=2.0 Hz, 1H), 6.59 (t, J=2 Hz, 1H), 6.38 (t, J=2 Hz, 1H), 5.18 (s, 2H), 5.07 (s, 2H), 3.95 (s, 3H); .sup.13C NMR (100 MHz, Methanol-d.sub.4) δ 160.29, 156.78, 149.76, 148.19, 139.92, 136.98, 136.84, 130.65, 129.20, 128.61 (2C), 128.57 (2C), 128.02, 127.88, 127.49 (2C), 127.26 (2C), 126.54, 119.94, 113.96, 109.45, 105.97, 105.69, 101.54, 71.02, 70.01, 56.03; CI-MS: 461 (M+Na).sup.+, 439 (M+1).sup.+; HRMS m/z 439.1908 (M+H).sup.+, calcd 439.1909 for C.sub.29H.sub.27O.sub.4.
15. Preparation of E-1-[3-hydroxy-5-benzyloxyphenyl]-2-[3-benzyloxy-4-methoxyphenyl]ethene (15)
(68) Basic hydrolysis of 12 was carried out as described for compound 14 to give the title compound as a colourless solid; mp 117-119° C.; C.sub.29H.sub.26O.sub.4 .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.49-7.30 (m, 10H), 7.07 (d, J=2.0 Hz, 1H), 7.06 (dd, J=8.4, 2.0 Hz, 1H), 6.96 (d, J=16.0 Hz, 1H), 6.87 (d, J=8.0 Hz, 1H), 6.79 (d, J=16.4 Hz, 1H), 6.99 (t, J=1.6 Hz, 1H), 6.57 (t, J=2.0 Hz, 1H), 6.38 (t, J=2.4 Hz, 1H), 5.19 (s, 2H), 5.07 (s, 2H), 3.90 (s, 3H). .sup.13C NMR (100 MHz, Methanol-d.sub.4) δ 160.20, 158.30, 149.68, 148.25, 139.70, 137.41, 137.31, 130.62, 128.16, 128.07 (2C), 128.05 (2C), 127.52, 127.41 (3C), 127.15 (2C), 126.62, 120.35, 111.95 (2C), 105.67, 104.09, 101.11, 70.88, 69.56, 55.10; CI-MS: m/z 461 (M+Na).sup.+, 439 (M+1).sup.+; FIRMS m/z 439.1907 (M+H).sup.+, calcd 439.1909 for C.sub.29H.sub.27O.sub.4.
16. Preparation of E-1-[3-hydroxy-5-methoxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (16)
(69) Basic hydrolysis of 13 was carried out as described for compound 14 to give the title compound as a yellowish solid; mp 110-112° C. C.sub.23H.sub.22O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.45-7.28 (m, 5H), 7.07 (d, J=2 Hz, 1H), 7.00 (d, J=16 Hz, 1H), 6.98 (dd, J=8, 2 Hz, 1H), 6.87 (d, J=17 Hz, 1H), 6.85 (d, J=8 Hz, 1H), 6.62 (t, J=2 Hz, 1H), 6.57 (t, J=2 Hz, 1H), 6.31 (t, J=2 Hz, 1H), 5.17 (s, 2H), 3.94 (s, 3H), 3.81 (s, 3H); .sup.13C NMR (100MHz, CDCl.sub.3) δ 161.08, 156.86, 149.73, 148.16, 139.85, 136.96, 130.69, 129.11, 128.57 (2C), 127.90, 127.29 (2C), 126.61, 119.95, 113.97, 109.47, 105.72, 104.73, 100.72, 71.03, 56.03, 55.36; CI-MS, m/z 384 (M+Na).sup.+, 363 (M+1).sup.+; HRMS m/z 363.1592 (M+H).sup.+, calcd 363.1596 for C.sub.23H.sub.23O.sub.4.
17. Preparation of E-1-[3-(3-methyl-2-butenyloxy)-5-benzyloxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (17)
(70) ##STR00040##
(71) To the suspension of NaH (60% dispersion in mineral oil, 0.0051 g, 0.213 mmol) in anhydrous DMF (5 mL) under N.sub.2 at 0° C., stilbene 14 (0.04 g, 0.091 mmol) was added dropwise in DMF (3 mL), followed by the dropwise addition of 3,3-dimetylallyl bromide (0.011 mL, 0.091 mmol)..sup.2 The mixture was stirred at room temperature for 2-3 hours, then quenched with cold water (5 mL), acidified with cooled 0.1 M HCl (5 mL) and extracted with diethyl ether (3×10 mL). The combined organic extracts were washed with water (2×10 mL) and dried under reduced pressure. The product was purified using NPSCC (hexane/ethyl acetate as mobile phase) to give 17 as a yellowish oil (0.02 g, 43.3%); C.sub.34H.sub.34O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46-7.28 (m, 10H), 7.07 (d, J=2 Hz, 1H), 7.01 (d, J=16 Hz, 1H), 6.96 (d, J=2 Hz, 1H), 6.90 (d, J=16 Hz, 1H), 6.87 (d, J=8 Hz, 1H), 6.73 (t, J=2 Hz, 1H), 6.68 (t, J=2 Hz, 1H), 6.48 (t, J=2 Hz, 1H), 5.52 (m, 1H), 5.17 (s, 2H), 5.07 (s, 2H), 4.52 (d, J=7 Hz, 2H), 3.95 (s, 3H), 1.80 (d, J=1 Hz, 3H), 1.76 (d, J=1 Hz, 3H); .sup.13C NMR (100MHz, CDCl.sub.3) δ 160.21, 160.12, 149.78, 148.14, 139.49, 138.35, 137.03, 136.94, 130.76, 128.94, 128.59 (2C), 128.56 (2C), 127.94, 127.86, 127.54 (2C), 127.24 (2C), 126.97, 119.87, 119.53, 113.98, 109.43, 105.38, 105.32, 101.14, 71.01, 70.09, 64.84, 56.03, 25.85, 18.22; CI-MS: m/z 529 (M+Na).sup.+, 507 (M+1).sup.+; HRMS m/z 507.2528 (M+H).sup.+, calcd 507.2535 for C.sub.34H.sub.35O.sub.4.
18. Preparation of E-1-[3-(3-methyl-2-butenyloxy)-5-benzyloxyphenyl]-2-[3-benzyloxy-4-methoxyphenyl]ethene (18)
(72) ##STR00041##
(73) Prenylation of 15 was carried out as described for compound 17 to give the title compound as an off white solid; mp 88-90° C.; C.sub.34H.sub.34O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.49-7.32 (m, 10H), 7.08 (d, J=2 Hz, 1H), 7.06 (dd, J=8, 2 Hz, 1H), 6.97 (d, J=16 Hz, 1H), 6.89 (d, J=8 Hz, 1H), 6.82 (d, J=16 Hz, 1H), 6.71 (t, J=2 Hz, 1H), 6.66 (t, J=2 Hz, 1H), 6.47 (t, J=2 Hz, 1H), 5.53 (m, 1H), 5.20 (s, 2H), 5.07 (s, 2H), 4.52 (d, J=6 Hz, 2H), 3.91 (s, 3H), 1.80 (d, J=1 Hz, 3H), 1.75 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 161.04, 160.95, 150.51, 149.11, 140.24, 139.03, 137.81, 137.67, 130.91, 129.57, 129.27 (4C), 128.66, 128.58, 128.22 (2C), 128.05 (2C), 127.50, 121.10, 120.20, 112.48, 112.40, 105.95, 105.86, 101.60, 71.55, 70.47, 65.18, 56.36, 26.00, 18.33; CI-MS: m/z 529 (M+Na).sup.+, 507 (M+1).sup.+; HRMS m/z 507.2530 (M+H).sup.+, calcd 507.2535 for C.sub.34H.sub.35O.sub.4.
19. Preparation of E-1-[3-(3-methyl-2-butenyloxy)-5-methoxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (19)
(74) ##STR00042##
(75) Prenylation of 16 was carried out as described for compound 17 to give the title compound as off white crystal; mp 66-69° C.; C.sub.34H.sub.34O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.45-7.28 (m, 5H), 7.08 (d, J=2 Hz, 1H), 7.02 (d, J=16 Hz, 1H), 6.98 (d, J=2 Hz, 1H), 6.90 (d, J=16 Hz, 1H), 6.87 (d, J=8 Hz, 1H), 6.67 (t, J=2 Hz, 1H), 6.64 (t, J=2 Hz, 1H), 6.40 (t, J=2 Hz, 1H), 5.53 (m, 1H), 5.17 (s, 2H), 4.53 (d, J=7 Hz, 2H), 3.96 (s, 3H), 3.81 (s, 3H), 1.81 (d, J=1 Hz, 3H), 1.76 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 160.91, 160.23, 149.76, 148.14, 139.46, 138.35, 137.04, 130.78, 128.90, 128.56 (2C), 127.87, 127.25 (2C), 127.02, 119.86, 119.56, 113.98, 109.44, 104.96, 104.49, 100.37, 71.02, 64.82, 56.04, 55.36, 25.86, 18.22; CI-MS: m/z 453 (M+Na).sup.+; HRMS m/z 453.2036 (M+Na).sup.+, calcd 453.2042 for C.sub.28H.sub.30O.sub.4Na.
20. Preparation of (E)-3-(3-methyl-2-butenyloxy)-4′,5-dihydroxy-3′-methoxystilbene (20, Equivalent to USYDS2)
(76) To a solution of 17 (0.02 g, 0.035 mmol) in absolute ethanol (8 mL), was added 1,4-cyclohexadiene (3 mL, 0.030 mmol) and Pd-C (10%, 0.002 g). The mixture was stirred under N.sub.2 and refluxed at 80° C. for 4 hours. The solution was filtered and dried under reduced pressure to give an oil residue which was purified by high performance liquid chromatography (HPLC) to afford 20 as a yellowish oil; C.sub.20H.sub.22O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.02 (d, J=2 Hz, 1H), 7.00 (d, J=16 Hz, 1H), 6.99 (d, J=2 Hz, 1H), 6.91 (dd, J=8, 1 Hz, 1H), 6.85 (d, J=16 Hz, 1H), 6.64 (t, J=2 Hz, 1H), 6.56 (t, J=2 Hz, 1H), 6.33 (t, J=2 Hz, 1H), 5.53 (m, 1H), 4.52 (d, J=7 Hz, 2H), 3.95 (s, 3H), 1.82 (d, J=1 Hz, 3H), 1.76 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 160.38, 156.70, 146.67, 145.69, 139.87, 138.37, 129.73, 129.29, 126.15, 120.63, 119.49, 114.54, 108.24, 105.58, 105.38, 101.29, 64.85, 55.92, 25.85, 18.22; CI-MS: m/z 325 (M−1).sup.−; HRMS m/z 327.1588 (M+H).sup.+, calcd 327.1596 for C.sub.20H.sub.23O.sub.4.
21. Preparation of 3-(3-methyl-2-butenyloxy)-4′,5-dihydroxy-3′-methoxydihydrostilbene (21)
(77) ##STR00043##
(78) The title compound was obtained by hydrogenation of the double bond on the bridging C═C during removal of the benzyl group of compound 20. The title compound was obtained as a light yellow oil; C.sub.20H.sub.24O.sub.4 .sup.1H NMR 400 MHz, CDCl.sub.3) δ 6.84 (d, J=8 Hz, 1H), 6.69 (dd, J=8, 2 Hz, 1H), 6.62 (d, J=2 Hz, 1H), 6.34 (t, J=2 Hz, 1H), 6.26 (t, J=2.0 Hz, 1H), 6.24 (t, J=2 Hz, 1H),), 5.46 (m, 1H), 5.46 (s, 1H), 4.68 (s, 1H), 4.53 (d, J=7 Hz, 2H), 3.84 (s, 3H), 2.85 (m, 4H), 1.80 (d, J=0.4 Hz, 3H), 1.73 (d, J=0.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 160.11, 156.44, 146.22, 144.46, 143.73, 138.23, 133.61, 120.95, 119.55, 114.15, 111.10, 107.89, 107.58, 99.62, 64.72, 55.84, 38.29, 37.23, 25.84, 18.18; CI-MS: m/z 351 (M+Na).sup.+; HRMS m/z 351.1566 (M+Na).sup.+, calcd 351.1572 for C.sub.20H.sub.24O.sub.4Na.
22. Preparation of (E)-3-(3-methyl-2-butenyloxy)-3′,5-dihydroxy-4′-methoxystilbene (22)
(79) ##STR00044##
(80) Removal of benzyl group of 18 was carried out as described for compound 20 to give the title compound as a light yellow oil; C.sub.20H.sub.22O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.13 (d, J=2 Hz, 1H), 6.98 (d, J=16 Hz, 1H), 6.97 (dd, J=8, 2 Hz, 1H), 6.86 (d, J=8 Hz, 1H), 6.86 (d, J=16 Hz, 1H), 6.64 (t, J=2 Hz, 1H), 6.57 (t, J=2 Hz, 1H), 6.33 (t, J=2 Hz, 1H), 5.60 (s, 1H), 5.52 (m, 1H), 4.77 (s, 1H), 4.52 (d, J=9 Hz, 2H), 3.91 (s, 3H), 1.82 (d, J=1 Hz, 3H), 1.76 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 160.37, 156.69, 146.51, 145.75, 139.83, 138.36, 130.87, 128.92, 126.78, 119.51, 119.40, 111.84, 110.62, 105.66, 105.48, 101.33, 65.18, 56.36, 26.00, 18.33; CI-MS: m/z 349.1408 (M+Na).sup.+, calcd 349.1416 for C.sub.20H.sub.22O.sub.4Na.
23. Preparation of 3-(3-methyl-2-butenyloxy)-3′,5-dihydroxy-4′-methoxydihydro-stilbene (23)
(81) ##STR00045##
(82) The title compound was obtained by hydrogenation of double bond on the side chain during removal of the benzyl groups of compound 18. The title compound was obtained as a light yellow oil; C.sub.20H.sub.24O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.79 (d, J=2 Hz, 1H), 6.77 (d, J=4 Hz, 1H), 6.66 (dd, J=8, 2 Hz, 1H), 6.35 (t, J=2 Hz, 1H), 6.27 (m, 1H), 5.56 (s, 1H), 5.50 (m, 1H), 4.74 (s, 1H), 4.46 (d, J=7 Hz, 2H), 3.88 (s, 3H), 2.80 (m, 4H), 1.80 (d, J=1 Hz, 3H), 1.74 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 160.11, 156.42, 145.41, 144.80, 144.51, 138.18, 135.07, 119.73, 119.59, 114.59, 110.55, 107.81, 107.49, 99.64, 64.73, 55.00, 38.05, 36.89, 25.83, 18.18; CI-MS: m/z 351 (M+Na).sup.+; HRMS m/z 351.1566 (M+Na).sup.+, calcd 351.1572 for C.sub.20H.sub.24O.sub.3Na.
24. Preparation of E-1-[2-(3-methyl-2-butenyl)-5-hydroxy-3-benzyloxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (24)
(83) ##STR00046##
(84) To a solution of 17 (0.024 g, 0.061 mmol) in toluene (30 mL) was added 100-200 mesh Florisil (0.24 g, 10×) and heated at 110° C. under N.sub.2, for 4 hours. The reaction mixture was filtered, evaporated in vacuo and the red-brown residue was purified using NPSCC (hexane/ethyl acetate as mobile phase) to afford a brownish solid (0.014 g, 58.3%). The product was recrystallised from hexane/ethyl acetate (3:1) mixture to give 24 as an off white solid; mp 145-150° C.; C.sub.34H.sub.34O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46-7.28 (m, 10H), 7.20 (d, J=16 Hz, 1H), 7.06 (d, J=2 Hz, 1H), 6.97 (dd, J=8, 2 Hz, 1H), 6.88 (d, J=16 Hz, 1H), 6.87 (d, J=8 Hz, 1H), 6.68 (d, J=2 Hz, 1H), 6.40 (d, J=2 Hz, 1H), 5.18 (s, 2H), 5.17 (m, 1H), 5.05 (s, 2H), 4.69 (s, 1H), 3.94 (s, 3H), 3.48 (d, J=7 Hz, 2H), 1.73 (d, J=1 Hz, 3H), 1.67 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 157.60, 154.27, 149.76, 148.08, 138.31, 137.14, 137.04, 131.18, 130.59, 130.35, 128.56 (2C), 128.49 (2C), 127.86, 127.78, 127.23 (2C), 127.21 (2C), 124.74, 123.63, 121.13, 119.85,113.99, 109.47, 104.31, 99.53, 71.03, 70.29, 55.98, 25.77, 24.69, 18.01.
25. Preparation of E-1-[2-(3-methyl-2-butenyl)-5-hydroxy-3-methoxyphenyl]-2-[3-methoxy-4-benzyloxyphenyl]ethene (25)
(85) ##STR00047##
(86) Rearrangment of 19 was carried out as described for compound 24 to give a pinkish solid: mp 161-162° C.; C.sub.28H.sub.30O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46-7.28 (m, 5H), 7.19 (d, J=16 Hz, 1H), 7.06 (d, J=2 Hz, 1H), 6.97 (dd, J=8, 2 Hz, 1H), 6.88 (d, J=16 Hz, 1H), 6.87 (d, J=8 Hz, 1H), 6.66 (t, J=2 Hz, 1H), 6.40 (t, J=2 Hz, 1H), 5.18 (s, 2H), 5.13 (m, 1H), 4.64 (s, 1H), 3.94 (s, 3H), 3.80 (s, 3H), 3.42 (d, J=6 Hz, 2H), 1.80 (d, J=1 Hz, 3H), 1.68 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 158.56, 154.35, 149.76, 148.07, 138.11, 137.04, 131.21, 130.63, 130.24, 128.56 (2C), 127.86, 127.22 (2C), 124.73, 123.61, 120.78, 119.84, 113.99, 109.42, 103.88, 98.21, 71.03, 55.97, 55.68, 25.77, 24.46, 17.98; CI-MS: m/z 453 (M+Na).sup.+, 431 (M+1).sup.+; HRMS m/z 431.2217 (M+1).sup.+, calcd 431.2222 for C.sub.28H.sub.31O.sub.4.
26. Preparation of (E)-2-(3-methyl-2-buten-1-yl)-3,4′,5-trihydroxy-3′-methoxy-stilbene (26, Equivalent to USYDS10)
(87) To a solution of 24 (0.02 g, 0.035 mmol) in absolute ethanol (6 mL) was added 1,4-cyclohexadiene (3 mL) and Pd—C (10%, 0.0035 mmol). The mixture was stirred under N.sub.2 and heated at 80° C. for 4 hours. The solution was filtered and evaporated in vacuo to give an oil residue which was purified using NPSCC (hexane/ethyl acetate as mobile phase) followed by normal phase HPLC (2:1 hexane/isopropanol as mobile phase) to afford the title compound as a light yellow oil: Data analogous to that of USYDS10.
27. Preparation of (E)-2-(3-methyl-2-buten-1-yl)-5,4′-dihydroxy-3′,3-dimethoxy-stilbene (27, Equivalent to USYDS1)
(88) The title compound was prepared using the procedure as described for compound 25 to give 27 as light yellow oil; C.sub.21H.sub.24O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.17 (d, J=16 Hz, 1H), 7.01 (dd, J=12, 2 Hz, 1H), 7.0 (d, J=2 Hz, 1H), 6.91 (d, J=8 Hz, 1H), 6.87 (d, J=16 Hz, 1H), 6.66 (t, J=2 Hz, 1H), 6.36 (t, J=2 Hz, 1H), 5.15 (m, 1H), 4.64 (s, 1H), 3.94 (s, 3H), 3.80 (s, 3H), 3.42 (d, J=7 Hz, 2H), 1.81 (d, J=1 Hz, 3H), 1.68 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 158.57, 154.37, 146.68, 145.60, 138.18, 130.61, 130.44, 130.27, 124.28, 123.67, 120.73, 120.58, 114.55, 108.26, 103.88, 98.18, 55.88, 55.71, 25.79, 24.48, 17.98; CI-MS: m/z 339 (M−1).sup.−; HRMS m/z 363.1566 (M+Na).sup.+, calcd 363.1572 for C.sub.21H.sub.24O.sub.4Na.
28. Preparation of 2-(3-methyl-2-buten-1-yl)-5,4′-dihydroxy-3′,3-dimethoxydihydro-stilbene (28)
(89) ##STR00048##
(90) The title compound was obtained by hydrogenation of double bond on the side chain during removal of the benzyl group of compound 27. The title compound was obtained as a light yellow oil; C.sub.21H.sub.26O.sub.4 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.86 (d, J=8 Hz, 1H), 6.70 (dd, J=8, 2 Hz, 1H), 6.64 (d, J=2 Hz, 1H), 6.30 (d, J=2 Hz, 1H), 6.24 (d, J=2 Hz, 1H), 5.48 (s, 1H), 5.07 (m, 1H), 4.67 (s, 1H), 3.85 (s, 3H), 3.78 (s, 3H), 3.28 (d, J=6 Hz, 2H), 2.82 (m, 4H), 1.74 (d, J=1 Hz, 3H), 1.66 (d, J=1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 158.63, 154.23, 146.23, 143.73, 142.01, 133.93, 130.59, 123.87, 120.92, 120.67, 114.21, 111.02, 107.96, 96.90, 55.84, 55.60, 37.19, 35.49, 25.76, 24.38, 17.95; CI-MS: m/z 365 (M+Na).sup.+; HRMS m/z 365.1723 (M+Na).sup.+, calcd 365.1729 for C.sub.21H.sub.26O.sub.4Na.
Biological Evaluations of Prenylated Polyhydroxystilbene Derivatives
1. Anticancer Activities of the Prenylated Polyhydroxystilbene Derivatives
(91) A) Seven prenylated polyhydroxystilbene derivatives, namely USYDS1, USYDS2, USYDS4, USYDS6, USYDS7, USYDS9 and USYDS13 were evaluated for inhibition of cell growth, as shown in table 1 below, against the 60 cell lines at a range of concentrations (1×10.sup.−8-1×10.sup.−4 M) at the National Cancer Institute (NCI), USA.
Methodology of the In Vitro Cancer Screen: General Method Adopted from NCI
(92) The human tumor cell lines were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical experiment, cells were inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C., 5% CO.sub.2, 95% air and 100% relative humidity for 24 h prior to addition of the drugs.
(93) After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition. Experimental drugs are solubilized in dimethyl sulfoxide (DMSO) at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/mL gentamicin. Aliquots of 100 μL of the drug were added to the appropriate microtiter wells already containing 100 μL of medium, resulting in the required final drug concentrations.
(94) Following drug addition, the plates were incubated for an additional 48 h at 37° C., 5% CO.sub.2, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4° C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μL of 80% TCA (final concentration, 16% TCA), and the absorbance was read on an automated plate reader at a wavelength of 515 nm. The GI.sub.50 value (concentration required for 50% inhibition of cell growth), TGI value (concentration required for total inhibition of cell growth) and LC.sub.50 value (concentration required for 50% cell lethality or death) were calculated for each of USYDS1, USYDS.sub.2, USYDS4, USYDS6, USYDS7, USYDS9 and USYDS13, and the results are presented in tables 1 to 3 below.
(95) TABLE-US-00001 TABLE 1 Effect of pPHOS USYDS1, USYDS2 and USYDS13 on human cancerous cells growth. Compounds USYDS1 (μM) USYDS2 (μM) USYDS13 (μM) GI.sub.50 TGI LC.sub.50 GI.sub.50 TGI LC.sub.50 GI.sub.50 TGI LC.sub.50 Leukemia CCRF-CEM 0.35 24.6 >100 5.06 >100 >100 6.56 >100 >100 HL-60 (TB) 0.02 0.06 68.9 1.34 9.77 >100 2.55 17.1 >100 K-562 0.04 11.7 >100 0.48 >100 >100 4.08 57.5 >100 MOLT-4 0.43 12.3 80.2 4.16 28.3 >100 2.95 13.7 >100 RPMI-8226 0.11 14.8 >100 4.32 52.8 >100 9.05 51.6 >100 SR 0.04 24.4 >100 0.54 >100 >100 2.80 >100 >100 Non-Small Cell Lung Cancer A549/ATCC 0.50 11.3 40.3 3.28 13.0 42.1 4.01 20.4 >100 EKVX 10.8 24.3 54.4 9.04 25.2 66.4 12.8 54.6 >100 HOP-62 0.44 16.0 42.2 4.44 18.5 43.4 5.66 20.4 53.7 HOP-92 0.06 12.3 39.6 3.98 21.1 60.1 5.96 30.5 >100 NCI-H226 3.13 13.3 45.6 5.43 19.1 46.4 10.9 50.1 >100 NCI-H23 0.28 15.1 62.5 2.89 23.6 >100 5.66 28.0 >100 NCI-H322M 11.8 25.7 55.9 9.64 22.7 52.3 5.90 28.5 >100 NCI-H460 0.23 1.38 42.0 2.47 10.9 58.7 3.09 15.6 >100 NCI-H522 0.02 — 44.8 1.14 3.57 14.7 2.13 59.8 25.6 Colon Cancer COLO 205 3.86 16.1 42.6 17.3 31.7 57.9 15.6 28.9 53.8 HCC-2998 1.07 11.5 35.8 4.61 17.3 46.4 11.0 30.6 85.3 HCT-116 0.30 11.1 40.4 2.09 11.9 40.0 4.33 15.6 45.6 HCT-15 0.05 16.1 >100 0.76 17.8 97.3 4.25 20.5 84.5 HT29 4.73 15.3 44.4 17.4 32.2 59.6 17.8 36.9 76.8 KM12 0.32 13.9 46.7 2.09 11.7 45.0 3.15 16.1 49.0 SW-620 0.04 15.1 44.3 0.53 14.0 40.7 3.98 15.9 42.8 CNS Cancer SF-268 0.25 14.1 50.2 4.50 19.2 51.6 6.17 32.9 >100 SF-295 0.65 16.2 55.7 3.94 14.7 49.2 5.22 23.5 77.5 SF-539 0.04 14.4 48.6 1.92 13.8 50.2 7.32 29.9 >100 SNB-19 0.17 19.3 51.5 5.02 20.4 53.6 8.02 24.2 64.5 SNB-75 0.03 20.2 72.0 2.14 10.3 55.8 5.47 36.6 >100 U251 0.37 10.9 36.0 2.96 14.7 41.0 5.47 20.7 57.6 Melanoma LOX IMVI 0.05 16.2 58.8 1.56 19.4 >100 4.48 20.2 80.0 MALME-3M 3.47 25.8 78.3 1.88 26.2 94.9 5.53 32.1 >100 M14 0.04 13.9 44.0 0.53 15.4 49.4 4.92 18.5 51.6 MDA-MB-435 0.03 13.8 43.0 0.34 11.0 41.3 3.08 15.9 >100 SK-MEL-2 0.32 10.6 38.0 1.25 9.59 41.6 3.09 9.37 40.2 SK-MEL-28 10.9 23.8 51.7 5.04 19.4 46.7 5.64 22.5 70.5 SK-MEL-5 0.05 0.75 5.99 1.58 11.5 34.3 2.50 8.54 29.2 UACC-257 2.53 17.7 45.4 7.89 20.6 46.0 5.35 18.1 44.0 UACC-62 1.62 18.3 46.6 3.73 15.9 44.6 5.30 19.7 58.8 Ovarian Cancer IGROV1 2.65 27.9 >100 5.84 46.7 >100 6.70 86.2 >100 OVCAR-3 0.26 — 37.3 3.02 10.7 36.1 7.98 39.8 >100 OVCAR-4 0.58 18.4 52.0 5.34 28.0 >100 9.68 74.8 >100 OVCAR-5 6.0 21.4 55.3 10.1 24.8 60.9 13.9 79.3 >100 OVCAR-8 0.08 11.7 50.0 2.80 14.8 44.6 5.48 59.8 >100 ADR-RES 0.03 — 70.7 0.40 4.95 >100 4.41 >100 >100 SK-OV-3 0.19 11.6 34.7 3.69 14.9 38.9 7.96 36.2 >100 Renal Cancer 786-0 0.48 14.0 >100 3.55 15.9 98.8 5.24 24.5 >100 A498 1.84 8.45 32.2 10.3 23.8 55.2 3.88 21.0 63.5 ACHN 0.07 14.0 44.6 3.76 17.5 52.1 5.11 >100 >100 CAKI-1 4.77 39.3 >100 6.13 >100 >100 5.90 >100 >100 RXF-393 0.03 — 39.1 1.35 5.46 23.6 5.48 18.9 46.5 SN12C 0.99 16.9 54.2 4.02 18.8 60.2 7.16 >100 >100 TK-10 4.10 15.7 40.5 4.69 14.3 40.1 6.98 31.4 >100 UO-31 0.25 16.0 51.3 2.60 16.9 48.9 4.16 38.1 >100 Prostate Cancer PC-3 0.42 16.1 47.9 3.84 18.7 65.8 6.36 >100 >100 DU-145 0.06 12.7 38.6 3.05 12.5 37.8 6.82 31.6 >100 Breast Cancer MCF7 0.23 13.0 52.9 1.24 15.0 41.0 0.68 19.9 82.7 MDA-MB-231 0.15 2.43 61.2 0.25 2.10 15.7 1.63 7.82 >100 BT-549 HS 578T 0.02 — >100 1.28 10.3 >100 2.40 15.0 >100 T-47D 0.72 19.8 67.4 5.66 33.6 >100 2.92 14.7 70.5 MDA-MB-468 0.36 8.37 33.2 2.72 9.41 36.8 1.68 7.63 >100
(96) TABLE-US-00002 TABLE 2 Effect of pPHOS USYDS4, USYDS9 and USYDS6 on human cancerous cells growth Compounds USYDS4 (μM) USYDS9 (μM) USYDS6 (μM) GI.sub.50 TGI LC.sub.50 GI.sub.50 TGI LC.sub.50 GI.sub.50 TGI LC.sub.50 Leukemia CCRF-CEM 3.50 >100 >100 2.14 >100 >100 2.96 9.17 82.1 HL-60 (TB) 2.59 6.04 >100 0.20 0.43 0.95 2.07 4.83 15.4 K-562 2.35 >100 >100 0.33 13.9 >100 1.46 4.13 22.6 MOLT-4 4.31 21.6 >100 1.27 6.77 >100 1.59 4.27 16.8 RPMI-8226 2.35 25.4 >100 0.45 >100 >100 2.34 5.61 62.0 SR 0.95 >100 >100 — — — — — — Non-Small Cell Lung Cancer A549/ATCC 4.74 25.6 >100 2.62 41.5 >100 2.69 8.42 33.7 EKVX 9.55 59.6 >100 26.4 >100 >100 7.19 21.4 54.4 HOP-62 6.49 26.3 83.4 2.87 >100 >100 5.11 17.5 44.0 HOP-92 1.92 13.6 62.3 0.62 >100 >100 1.86 4.67 14.5 NCI-H226 5.14 23.1 78.0 18.6 57.9 >100 1.47 30.1 61.5 NCI-H23 3.42 20.8 81.9 1.43 >100 >100 3.38 15.7 47.6 NCI-H322M 12.8 54.7 >100 0.76 >100 >100 1.16 25.5 56.2 NCI-H460 3.65 20.3 >100 0.47 10.3 75.7 1.89 3.90 8.06 NCI-H522 1.07 2.54 6.04 0.15 0.45 44.8 2.18 5.15 17.8 Colon Cancer COLO 205 9.72 23.0 53.5 21.7 83.6 >100 16.0 30.2 57.2 HCC-2998 5.16 19.0 53.4 4.18 21.2 >100 2.81 8.78 32.2 HCT-116 4.55 16.0 40.6 1.40 12.8 75.0 1.89 3.78 7.54 HCT-15 1.77 34.9 >100 0.43 >100 >100 2.13 6.26 48.2 HT29 16.3 32.5 64.7 18.6 46.6 >100 5.44 17.6 48.3 KM12 3.27 14.2 46.5 0.52 12.2 63.9 2.21 4.37 8.64 SW-620 2.97 16.6 46.5 0.31 15.3 75.0 1.84 3.54 6.82 CNS Cancer SF-268 3.89 23.2 >100 0.63 37.6 >100 2.78 8.97 44.3 SF-295 7.29 24.3 70.9 2.60 20.5 >100 6.78 20.8 53.2 SF-539 3.45 22.3 97.5 0.28 1.02 >100 4.48 17.6 57.1 SNB-19 10.5 25.8 63.8 0.55 >100 >100 6.38 20.6 52.7 SNB-75 1.77 9.11 48.3 0.24 — >100 6.67 21.4 54.0 U251 3.67 15.6 45.5 0.59 19.6 >100 2.33 5.31 16.7 Melanoma LOX IMVI 3.82 19.3 58.5 0.64 58.6 >100 1.76 3.42 6.68 MALME-3M 7.25 30.6 >100 0.62 >100 >100 2.80 8.56 37.1 M14 0.91 15.4 46.3 0.30 5.69 >100 2.82 8.46 33.4 MDA-MB-435 0.36 21.6 >100 0.13 0.42 >100 3.37 10.8 42.1 SK-MEL-2 2.18 6.57 31.0 2.17 12.1 >100 13.1 33.6 86.1 SK-MEL-28 9.18 27.6 77.9 8.14 >100 >100 11.2 24.0 51.3 SK-MEL-5 1.42 8.35 30.0 0.39 2.52 >100 2.29 6.17 22.1 UACC-257 5.44 19.4 48.6 6.15 59.2 >100 3.49 14.4 39.8 UACC-62 4.53 16.8 44.4 3.22 34.8 >100 10.3 23.5 53.8 Ovarian Cancer IGROV1 3.74 44.8 >100 4.57 >100 >100 5.23 17.7 54.1 OVCAR-3 3.98 15.0 44.9 0.90 3.84 24.8 2.10 4.31 8.85 OVCAR-4 3.98 50.4 >100 5.48 >100 >100 4.40 16.1 43.5 OVCAR-5 15.9 51.4 >100 30.7 >100 >100 5.86 20.0 53.3 OVCAR-8 4.94 31.1 >100 0.50 38.3 >100 2.95 9.51 42.9 ADR-RES 1.11 48.3 >100 0.29 3.69 >100 3.46 15.2 51.2 SK-OV-3 8.91 57.0 >100 0.50 5.12 >100 13.9 27.1 53.2 Renal Cancer 786-0 4.41 20.5 >100 2.99 18.7 >100 2.46 6.96 28.8 A498 9.07 23.5 55.2 10.3 53.5 >100 11.7 25.0 53.1 ACHN 4.57 >100 >100 0.66 >100 >100 3.94 14.6 50.4 CAKI-1 5.25 >100 >100 19.1 >100 >100 8.37 22.6 55.7 RXF-393 2.17 15.3 46.0 0.23 0.63 >100 2.15 5.39 18.7 SN12C 3.94 20.0 74.1 1.76 83.5 >100 2.85 7.98 30.8 TK-10 3.87 9.66 >100 5.34 41.2 >100 3.36 9.33 31.2 UO-31 3.31 20.7 66.4 0.46 >100 >100 2.51 7.50 33.5 Prostate Cancer PC-3 4.47 22.0 87.9 2.92 >100 >100 1.80 3.89 8.44 DU-145 6.37 44.6 >100 0.35 2.62 >100 3.47 12.9 40.1 Breast Cancer MCF7 2.57 18.2 51.1 2.81 >100 >100 4.02 18.0 60.5 MDA-MB-231 1.04 18.4 >100 0.38 2.61 >100 1.72 4.54 17.2 HS 578T 1.00 7.11 >100 0.20 0.73 >100 1.67 5.29 35.8 T-47D 2.13 6.93 >100 4.78 36.9 >100 5.03 20.3 57.9 MDA-MB-468 2.53 9.51 55.1 2.42 8.48 >100 2.30 6.39 26.0
(97) TABLE-US-00003 TABLE 3 Effect of pPHOS USYDS7 on human cancerous cells growth Compound USYDS7 (μM) GI.sub.50 TGI LC.sub.50 Leukemia CCRF-CEM 2.73 8.48 71.2 HL-60 (TB) 2.39 5.06 13.8 K-562 2.14 4.78 17.4 MOLT-4 2.01 5.19 26.5 RPMI-8226 2.31 6.01 61.1 Non-Small Cell Lung Cancer A549/ATCC 1.88 4.06 8.78 EKVX 4.25 14.7 44.9 HOP-62 3.67 15.0 51.6 HOP-92 1.52 4.21 14.2 NCI-H226 2.28 6.24 28.1 NCI-H23 1.81 5.22 29.5 NCI-H322M 5.29 18.1 44.7 NCI-H460 1.64 3.22 6.33 NCI-H522 1.61 3.57 7.92 Colon Cancer COLO 205 1.79 4.34 12.2 HCC-2998 1.95 4.26 9.34 HCT-116 1.57 3.02 5.82 HCT-15 1.29 3.78 22.0 HT29 3.64 10.8 45.8 KM12 1.93 3.88 7.79 SW-620 1.58 3.19 6.42 CNS Cancer SF-268 2.26 5.47 22.7 SF-295 3.37 11.4 44.6 SF-539 2.21 6.14 35.2 SNB-19 2.80 8.64 39.9 SNB-75 2.98 12.8 49.1 U251 1.49 2.93 5.74 Prostate Cancer PC-3 2.02 4.57 11.3 DU-145 2.65 7.62 28.3 Melanoma LOX IMVI 1.71 3.41 6.81 MALME-3M 1.97 4.46 10.4 M14 1.79 3.71 7.73 MDA-MB-435 2.29 6.47 31.7 SK-MEL-2 3.20 7.82 42.9 SK-MEL-28 3.65 11.8 36.7 SK-MEL-5 1.80 3.68 7.54 UACC-257 2.18 5.93 23.0 UACC-62 2.80 7.84 31.5 Ovarian Cancer IGROV1 3.12 8.39 36.7 OVCAR-3 1.82 3.36 6.19 OVCAR-4 2.85 11.1 66.8 OVCAR-5 3.04 11.6 39.0 OVCAR-8 1.68 3.65 7.95 ADR-RES 2.23 7.90 42.4 SK-OV-3 5.98 19.4 46.5 Renal Cancer 786-0 1.99 4.47 10.2 A498 11.1 23.8 50.8 ACHN 2.58 9.12 37.6 CAKI-1 4.24 16.2 49.0 RXF-393 1.53 3.23 6.80 SN12C 1.65 3.16 6.03 TK-10 3.62 11.2 34.3 UO-31 1.60 3.46 7.46 Breast Cancer MCF7 2.10 12.0 57.9 MDA-MB-231 1.49 3.89 10.7 HS 578T 1.84 8.09 50.9 T-47D 1.88 6.09 43.5 MDA-MB-468 2.12 4.84 13.9
(98) In summary, all the prenylated polyhydroxystilbene derivatives USYDS1 to USYDS9 and USYDS13 exhibited structure dependent inhibition of cancerous cell growth. In some cell lines, growth was inhibited at nano-molar concentrations. USYDS1 displayed the most potent activity followed by USYDS9 then USYDS2 in the inhibition of cancerous cell growth. The other pPHOS compounds tested were shown to be moderate inhibitors.
(99) Worthy of note is that these pPHOS required at least a 10 fold excess in concentration to cause cell death (LC.sub.50 values) or cause necrosis, over that required to inhibit cell growth (GI.sub.50 values). This indicates that the pPHOS were likely to cause the cancer cells to undergo programmed cell death (apoptosis) or cell cycle arrest.
(100) B) Two prenylated polyhydroxystilbene derivatives, namely USYDS10 and USYDS14 were evaluated for inhibition of cell growth, as shown in Table 4A and 4B below, against the cell lines indicated at a range of concentrations (1×10.sup.−8-1×10.sup.−4 M) at the National Cancer Institute (NCI), USA.
(101) TABLE-US-00004 TABLE 4A Inhibitory effect on cancer cells growth of USYDS10 GI50 TGI LC50 Leukemia HL-60(TB) 3.27E−7 1.88E−6 >1.00E−4 K-562 3.86E−7 3.17E−5 >1.00E−4 MOLT 5.96E−7 1.99E−5 >1.00E−4 RPMI 3.24E−7 1.63E−5 >1.00E−4 SR 1.88E−7 >1.00E−4 >1.00E−4 Non-Small Cell Lung Cancer A549/ATCC 6.60E−7 2.25E−5 >1.00E−4 EKVX 1.22E−5 7.34E−5 >1.00E−4 HOP 5.81E−7 1.77E−5 4.23E−5 HOP 3.98E−7 2.63E−5 9.32E−5 NCI-H226 5.35E−6 3.50E−5 >1.00E−4 NCI-H23 3.80E−7 1.52E−5 7.83E−5 NCI-H322M 1.18E−5 3.14E−5 8.37E−5 NCI-H460 3.81E−7 1.29E−5 6.61E−5 NCI-H522 3.49E−7 1.46E−5 4.22E−5 Colon Cancer COLO 1.53E−5 2.97E−5 5.77E−5 HCC-2998 1.81E−6 1.19E−5 3.98E−5 HCT-116 4.80E−7 1.30E−5 4.27E−5 HCT-15 4.98E−7 2.09E−5 >1.00E−4 HT29 5.72E−6 2.13E−5 6.90E−5 KM12 4.97E−7 1.53E−5 8.58E−5 SW-620 3.62E−7 2.15E−5 8.55E−5 CNS Cancer SF-268 1.40E−6 4.96E−5 >1.00E−4 SF-295 2.01E−6 1.91E−5 >1.00E−4 SF-539 3.76E−7 1.29E−5 4.17E−5 SNB-19 6.13E−7 2.28E−5 >1.00E−4 SNB-75 2.36E−7 1.22E−5 3.61E−5 U251 4.19E−7 1.62E−5 7.88E−5 Melanoma LOX IMVI 5.72E−7 1.93E−5 8.00E−5 MALME-3M 1.87E−5 4.17E−5 9.29E−5 M14 2.84E−7 1.42E−5 4.16E−5 MDA-MB-435 3.85E−8 2.56E−7 >1.00E−4 SK-MEL-2 5.76E−7 3.26E−5 >1.00E−4 SK-MEL-28 5.72E−7 1.69E−5 4.30E−5 SK-MEL-5 2.91E−7 1.27E−5 3.60E−5 UACC-257 1.17E−5 2.94E−5 7.37E−5 UACC-62 5.21E−7 1.55E−5 4.28E−5 Ovarian Cancer IGROV1 2.03E−6 >1.00E−4 >1.00E−4 OVCAR-3 3.72E−7 2.03E−5 >1.00E−4 OVCAR-4 1.03E−6 1.82E−5 5.77E−5 OVCAR-5 8.92E−6 2.80E−5 8.31E−5 OVCAR-8 4.67E−7 6.50E−5 >1.00E−4 NCI/ADR-RES 1.83E−7 8.04E−7 >1.00E−4 SK-OV-3 4.22E−7 1.14E−5 3.42E−5 Renal Cancer 786-0 6.81E−7 2.01E−5 7.45E−5 A498 2.17E−6 7.85E−6 3.48E−5 ACHN 9.03E−7 3.79E−5 >1.00E−4 CAKI-1 5.39E−7 6.68E−5 >1.00E−4 RXF 393 2.21E−7 3.76E−5 SN12C 6.71E−7 2.98E−5 >1.00E−4 TK-10 9.55E−6 5.15E−5 >1.00E−4 UO-31 8.78E−7 2.17E−5 9.52E−5 Prostate Cancer PC-3 2.27E−6 3.12E−5 >1.00E−4 DU-145 4.42E−7 2.05E−5 >1.00E−4 Breast Cancer MCF7 3.32E−7 1.30E−5 5.06E−5 MDA-MB-231/ATCC 6.42E−7 3.20E−5 >1.00E−4 HS 578T 2.92E−7 2.60E−5 >1.00E−4 BT-549 7.98E−7 1.90E−5 4.93E−5 T-47D 1.47E−6 2.47E−5 >1.00E−4 MDA-MB-468 3.73E−7 1.44E−5 4.49E−5
(102) TABLE-US-00005 TABLE 4B Inhibitory effect on cancer cells growth of USYDS14 GI50 TGI LC50 Leukemia HL-60(TB) 4.06E−6 1.63E−5 >1.00E−4 K-562 8.33E−7 1.42E−5 >1.00E−4 MOLT-4 4.03E−6 2.10E−5 >1.00E−4 RPMI-8226 4.07E−6 2.45E−5 >1.00E−4 SR 6.70E−7 2.46E−5 >1.00E−4 Non-Small Cell Lung Cancer A549/ATCC 4.33E−6 1.65E−5 4.55E−5 EKVX 6.45E−6 2.12E−5 5.17E−5 HOP-62 1.43E−6 3.77E−6 9.98E−6 HOP-92 1.49E−6 7.58E−6 3.29E−5 NCI-H226 1.72E−6 3.60E−6 7.52E−6 NCI-H23 2.92E−6 1.73E−5 5.76E−5 NCI-H322M 1.25E−5 2.57E−5 5.28E−5 NCI-H460 3.87E−6 1.49E−5 5.12E−5 NCI-H522 7.50E−7 2.26E−6 5.85E−6 Colon Cancer COLO 205 1.46E−5 2.85E−5 5.57E−5 HCC-2998 2.90E−6 1.06E−5 4.07E−5 HCT-116 4.20E−6 1.60E−5 4.66E−5 HCT-15 2.06E−6 1.04E−5 4.33E−5 HT29 1.40E−5 2.91E−5 6.09E−5 KM12 3.74E−6 1.44E−5 5.74E−5 SW-620 2.88E−6 1.67E−5 6.38E−5 CNS Cancer SF-268 4.65E−6 1.78E−5 4.85E−5 SF-295 3.91E−6 1.53E−5 4.31E−5 SF-539 2.96E−6 1.05E−5 3.78E−5 SNB-19 5.01E−6 2.01E−5 5.22E−5 SNB-75 1.79E−6 7.80E−6 2.93E−5 U251 3.09E−6 1.27E−5 3.94E−5 Melanoma LOX IMVI 1.36E−6 3.37E−6 8.35E−6 MALME-3M 3.14E−6 2.18E−5 5.48E−5 M14 2.95E−6 1.36E−5 5.00E−5 MDA-MB-435 3.73E−7 8.45E−6 4.00E−5 SK-MEL-2 3.72E−6 1.25E−5 4.12E−5 SK-MEL-28 3.05E−6 1.64E−5 4.16E−5 SK-MEL-5 5.54E−7 1.87E−6 4.46E−6 UACC-257 1.02E−5 2.35E−5 5.40E−5 UACC-62 2.61E−6 1.32E−5 3.91E−5 Ovarian Cancer IGROV1 3.65E−6 1.37E−5 6.54E−5 OVCAR-3 3.52E−6 1.35E−5 4.11E−5 OVCAR-4 2.98E−6 1.36E−5 3.86E−5 OVCAR-5 1.24E−5 2.58E−5 5.36E−5 OVCAR-8 3.59E−6 1.56E−5 6.49E−5 NCI/ADR-RES 7.80E−7 1.14E−5 4.93E−5 SK-OV-3 2.85E−6 1.37E−5 3.72E−5 Renal Cancer 786-0 3.44E−6 1.18E−5 4.00E−5 A498 1.30E−5 2.66E−5 5.46E−5 ACHN 2.98E−6 1.25E−5 3.65E−5 CAKI-1 3.59E−6 1.75E−5 4.54E−5 RXF 393 1.07E−6 3.58E−6 1.48E−5 SN12C 3.61E−6 1.51E−5 4.20E−5 TK-10 3.79E−6 1.21E−5 3.54E−5 UO-31 3.71E−6 1.79E−5 4.63E−5 Prostate Cancer PC-3 6.65E−6 2.16E−5 5.50E−5 DU-145 8.20E−6 2.11E−5 4.75E−5 Breast Cancer MCF7 2.74E−6 1.18E−5 4.19E−5 MDA-MB-231/ATCC 3.15E−6 1.43E−5 4.53E−5 HS 578T 2.71E−6 2.05E−5 >1.00E−4 BT-549 4.27E−6 1.63E−5 4.69E−5 T-47D 2.71E−6 7.84E−6 6.18E−5 MDA-MB-468 1.42E−6 4.12E−6 1.64E−5
(103) In summary, all the prenylated polyhydroxystilbene derivatives USYDS10 and USYDS14 exhibited structure dependent inhibition of cancerous cell growth.
2. Calculated Log Partition Coefficient (Log P) Values of Various Hydroxystilbenes
(104) The calculated Log P values for USYDS1 and USYDS2 and known hydroxystilbenes are presented in the table 5 below.
(105) TABLE-US-00006 TABLE 5 Calculated Log P values of various hydroxystilbenes Compound USYDS2 5.37 Rhapontigenin 2.82 Compound USYDS1 5.58 Compound USYDS1 without 3.49 a prenyl group. Resveratrol 3.14 Pinosylvin 3.68 Piceatannol 1.90
(106) The potent inhibition of pPHOS compounds, for example USYDS1 and USYDS.sub.2, may be explained in terms of their increased hydrophobicity, as demonstrated by their calculated Log partition coefficient (Log P) values. USYDS1 and USYDS2 have Log P values almost twice that of the hydroxystilbene resveratrol. The effects of Log P on therapeutic compounds relate primarily to tissue penetration and distribution. Higher Log P values will enable compounds to more easily cross cell membranes and enter cells.
3. Effect of Prenylated Polyhydroxystilbene Derivatives on UV-Irradiated Human Skin Cells
(107) Normal adult human keratinocytes (NHK) cells (Invitrogen, Vic, Australia) were cultured in keratinocyte growth medium Epilife supplemented with calcium and human keratinocyte growth supplement (HKGS, containing 0.2 ng EGF per mL, 5 mg insulin per mL, 5 mg transferrin per mL, 0.18 mg hydrocortisone per mL, and 0.2% bovine pituitary extract) (Invitrogen, Vic, Australia) in 12-well culture plates until the subconfluent state is reached. Cells were cultured to a density of 5×10.sup.3 cells per mL/well in a 24-well plate for 24 h at 37° C. in a humidified incubator with 5% carbon dioxide and tested according to the protocols described below:
Determination of Optimal Doses of UV Irradiation
(108) Cells, seeded at a density as described above, were washed twice with PBS (phosphate buffered saline) then irradiated with a range of UVA and UVB doses known as MED (minimal erythema dose, 1 MED=25.43/light intensity). Cells were replaced with growth medium and incubated for about 24 h at 37° C. in a humidified incubator with 5% carbon dioxide. Cell viability was measured using the MTS assay (CellTiter 96® AQ.sub.ueous One Solution Cell Proliferation Assay) (Promega, Vic Australia).
Rescue Assay
(109) Cells were washed twice with PBS, replaced with a thin layer of PBS, then irradiated with the optimal doses of UVA and UVB as determined above. Immediately after irradiation, cells were replaced with fresh culture medium containing the test samples at a range of concentrations, and further incubated in a humidified CO.sub.2 incubator at 37° C. for 24 hr. Supernatants were collected and kept at −80° C. until determination of PGE2 and cytokines (IL1, 6, 8, 10 & 12) concentration using ELISA kits.
Protective Assay
(110) Cells were washed twice with PBS, replaced with a thin layer of PBS containing different concentrations of the test compounds then irradiated with optimal doses of UVA and UVB as determined above. Immediately after irradiation, cells were replaced with fresh culture medium and further incubated in a humidified CO.sub.2 incubator at 37° C. for 24 h. Supernatants were be collected and kept at −80° C. until determination of PGE2 and cytokines (IL1, 6, 8, 10 & 12) concentration using ELISA kits.
(111) Sham-treated control cultures were handled identically but not exposed to UV irradiation. Stilbenes compounds including USYDS1, 2, 13, 5, and 7 and propolis extract were tested at 0.1, 1 and 10 μM or μg/mL.
(112) Results for the determination of optimal doses of UV irradiation revealed that at 1 MED of UVA and UVB irradiation, there is no significant effect on cell viability. Thus, this condition was chosen for investigation of the effects of stilbenes and propolis extract on levels of cytokines in the rescue assay.
(113) In preliminary investigation on modulation of cytokine productions in UV irradiated human epidermal keratinocytes (HEK) it was observed that a mixture of USYDS1 and USYDS2 moderately inhibit the production of IL-6, TGFα, G-CSF and GM-CSF (2-3 fold). However, it was found that the prenylated polyhydroxystilbene derivatives significantly increased IL-8 and IL-1rα production (4-5 fold) from UV irradiated cells. IL-8 is known to play a role in the onset of immunity response. IL-1rα (naturally occurring cytokine receptor antagonist) on the other hand plays an important role in inhibition of deleterious effect of IL-1 during inflammatory processes. Therefore, these preliminary results demonstrate that the prenylated polyhydroxystilbene derivates of the present invention may be good candidates for the treatment of conditions associated with immunity suppression and inflammation.
4. Antioxidant Activities of the Stilbenes and Propolis Extracts
1,1-Diphenyl-2-picrylhydrazyl (DPPH.SUP..circle-solid..) Scavenging Activity Assay
(114) The (1,1-diphenyl-2-picrylhydrazyl) DPPH assay is commonly used to test free radical scavenging ability of a compound or extracts of natural products by measurement of the reduction of DPPH.sup..circle-solid. radicals at 517 nm. In its radical form, DPPH.sup..circle-solid. shows a strong absorption at 517 nm due to its odd electron. Upon reduction by an antioxidant or radical scavenger, the absorption disappears and the resulting decolorization by scavenging the radical is stoichiometric with respect to the number of electrons taken up (DPPH.sup..circle-solid.+AH.fwdarw.DPPH:H+A.sup..circle-solid.). The DPPH assay was carried out in a stepwise procedure as described below.
(115) A methanolic solution of DPPH (0.1 mM) was stirred in a dark container at room temperature for 20 min. The solution was scanned between 400-750 nm to obtain a maximum wavelength (λmax, ˜510 nm). The concentration of the DPPH solution was adjusted with methanol to result in a maximum absorbance of approximately 1.0. Test samples at different concentrations and standard antioxidant solution (0.05 mL) were added to 0.95 mL of methanolic DPPH solution in a cuvette. Final concentrations of the test samples were 0.1, 1, 10, 50, 100 and 200 μM. The mixtures were shaken vigorously and allowed to stand in the dark for 30 min at room temperature. Absorbance of the resulting solution was measured at the maximum wavelength (˜510 nm). A decrease in absorbance indicated a free radical scavenging effect of the test samples. Dose response curves of the test samples were established to determine their IC.sub.50 values (concentrations that show 50% reduction in UV absorbance).
Results
(116) pPHOS compounds in this study exhibited a moderate to weak effect on free radical scavenging except for USYDS7 which exhibited a strong effect. These results are displayed in
5. Effect of Stilbene Derivatives on Nicotinamide Adenine Dinucleotide (NAD)-Dependent Deacetylase Sirtuin-2 (SIRT1)
(117) SIRT1 is a member of Sir2 family (class III) which is a NAD-dependent histone deacetylase. Deacetylation by SIRT1 enzyme can target many substrates including histone, tumor suppressor p53, forkhead transcription factor (FOXO), peroxisome proliferator-activated receptor-γ (PPARγ) and co-activator-1α (PGC-1α)..sup.10 SIRT1 has been shown to be involved in the regulation of many physiopathological processes like inflammation, cellular aging, apoptosis/proliferation, metabolism and cell cycle regulation (Chung, Yao et al. 2010). Accordingly, modulating SIRT1 activity could be a potential therapeutic target to control many diseases such as cancer, metabolic syndrome, obesity, neurodegenerative disorder, skeletal muscle dysfunction and aging-related diseases..sup.11
(118) SIRT1 assay kit (Cayman Chemical, Ann Arbor, Mich., USA) provides a fluorescence-based method for screening of SIRT1 inhibitors or activators. The assay was carried out according to the instructions from the manufacturer. In brief, the assay consists of two steps, both performed in the same plate. In the first step, the substrate, which comprises the p53 sequence Arg-His-Lys-Lys(ε-acetyl)-AMC (7-amino-4-methylcoumarin), is incubated with human recombinant SIRT1 along with its cosubstrate NAD.sup.+. Deacetylation sensitizes the substrate such that treatment with the developer in the second step releases a fluorescent product which was analysed using fluorometric plate reader at an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm. Stilbenes were assayed at three concentrations (1, 10 and 100 μM). Data represents two independent experiments each performed in triplicate.
Results
(119) All the stilbenes, except resveratrol, exhibited a concentration dependent inhibition of SIRT1 as shown in
6. Antibacterial Activities of USYDS1, USYDS2 and Ethanolic Extract of Sedge Type-1 Propolis
Summary
(120) The minimum inhibition concentration (MIC) screening was performed with 14 bacterial strains and 4 compounds. The MICs were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The MIC screening was performed on 96-well plates containing the compounds in serial 2-fold dilutions from 64 to 0.06 μg/ml. The bacterial inoculations were prepared in cation-adjusted Mueller Hinton medium broth from cultures grown on appropriate agar plates which are prepared freshly every week. The growth controls and sterile controls were included in each assay plates. The assay plates were incubated in an ambient-air incubator at 35±2° C. for 16-20 hr (24 hr for MRSA), and bacterial growth was observed and recorded. All MICs of reference compound levofloxacin in the MIC screening are within the standard range described in CLSI S100-A20. The potency of 3 test samples is the order of USYDS1>USYDS2>ethanolic propolis extract.
1. Materials
1.1. Strains
(121) TABLE-US-00007 Bacteria panel for MIC screening Cultivation MIC screening Microorganism Gram Strain .sup.1Resistance .sup.2Plasmid condition condition Escherichia coli G.sup.− ATCC TSA, ambient CAMHB, ambient (25922) air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Pseudomonas G.sup.− ATCC TSA, ambient CAMHB, ambient aeruginosa (27853) air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Klebsiella G.sup.− ATCC AMP, AZT, Yes TSA, ambient CAMHB, ambient pneumoniae (700603) CFX, CPD, air, 35 ± 2° C. air, 35 ± 2° C., 20 hr CAZ, CHL, PIP, TET Haemophilus G.sup.− ATCC Chocolate agar, HTM, ambient influenzae (49247) 5% CO.sub.2, air, 35 ± 2° C., 20 hr 35 ± 2° C. Acinetobacter G.sup.+ ATCC IMI TSA, ambient CAMHB, ambient calcoaceticus (51432) air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Enterococcus G.sup.+ ATCC VAN TSA + 5% sheep CAMHB, ambient faecium (700221) blood, ambient air, 35 ± 2° C., 20 hr air, 35 ± 2° C. Enterococcus G.sup.+ ATCC TSA + 5% sheep CAMHB, ambient faecalis (29212) blood, ambient air, 35 ± 2° C., 20 hr air, 35 ± 2° C. Streptococcus G.sup.+ ATCC TSA + 5% sheep CAMHB + 3% horse pyogenes (700492) blood, 5% CO.sub.2, blood, ambient air, 35 ± 2° C. 35 ± 2° C., 20 hr Streptococcus G.sup.+ ATCC PEN TSA + 5% sheep CAMHB + 3% horse pneumoniae (49619) blood, 5% CO.sub.2, blood, ambient air, 35 ± 2° C. 35 ± 2° C., 20 hr Streptococcus G.sup.+ Clinical ERY TSA + 5% sheep CAMHB + 3% horse pneumoniae isolate blood, 5% CO.sub.2, blood, ambient air, 35 ± 2° C. 35 ± 2° C., 20 hr Staphylococcus G.sup.+ ATCC TSA, ambient CAMHB, ambient aureus (29213) air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Staphylococcus G.sup.+ ATCC MET, OXA TSA, ambient CAMHB, ambient aureus (43300) air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Staphylococcus G.sup.+ Clinical LEV TSA, ambient CAMHB, ambient aureus isolate air, 35 ± 2° C. air, 35 ± 2° C., 20 hr Staphylococcus G.sup.+ Clinical MET, ERY, TSA, ambient CAMHB, ambient aureus isolate CLI air, 35 ± 2° C. air, 35 ± 2° C., 20 hr .sup.1Known resistance; .sup.2Known plasmid presence Abbreviation: TSA, trypticase soy agar; CAMHB, cation-adjusted Mueller Hinton broth; HTM, Haemophilus test medium; AMP, ampicillin; AZT, aztreonam; CFX, cefoxitin; CPD, cefpodoxime; CAZ, ceftazidime; CHL, chloramphenicol; PIP, piperacillin; TET, tetracycline; IMI, imipenem; VAN, vancomycin; PEN, penicillin; ERY, erythromycin; MET, methicillin; OXA, oxacillin; LEV, levofloxacin; CLI, clindamycin.
1.2. Media and Reagents
(122) Trypticase soy agar (BD 211043), Cation-adjusted Mueller Hinton broth (BD 212322), Haemophilus test medium base (Fluka 51295), Hemin (Fluka 51280), β-NAD (Fluka 43410), Levofloxacin (Sigma 28266), Sheep blood (Quad Five 630-500), Lysed horse blood (Quad Five 205-500), 0.5 McFarland barium sulfate standard, Sterile 0.85% NaCl (w/v).
2. Methods
2.1. Prepare Bacterial Strains
(123) A. Revive bacterial strains from storage frozen (−80° C.) two days before the MIC screening. Streak onto surface of appropriate agar plates, and incubate the plates for 20-24 hr at 35±2° C. in an appropriate atmosphere.
(124) Streptococci: TSA II, 5% CO.sub.2
(125) Enterococci: TSA II, ambient air
(126) Haemophilus influenzae: chocolate agar, 5% CO.sub.2
(127) Other strains in the panel: TSA, ambient air
(128) B. Select 5-10 well-isolated colonies of similar morphology and restreak onto fresh agar plates using sterile loops. Incubate the plates for 20-24 hr at 35±2° C. in an appropriate atmosphere as above.
2.2. Prepare Compound Plates
(129) Compound stock solutions were prepared in 100% DMSO on the day of MIC screening and use immediately. Compound stock concentration=[(highest testing concentration) ×103 μl/3 μl] (e.g. if the required highest testing concentration is 64 μg/ml in assay plates, stock concentration=64×103/3=2.2 mg/ml). The potency of testing compound is assumed as 100% unless otherwise stated whereas the potency of reference compound is calculated according to manufacturer's analysis data.
(130) Eleven two-fold dilutions per compound were made then transferred 3 μl to each well of test plate. Final concentration of DMSO in the MIC screening is ˜3%.
2.3. Prepare Bacterial Inoculation
(131) A. Take out medium broth from 4° C. fridge and allow it to warm to room temperature.
(132) B. Transfer colonies from fresh culture plates into 5 ml of saline with sterile loops and mix well. Measure and adjust turbidity to 0.5 McFarland barium sulphate standard using a turbidity meter. Alternatively, transfer 1-2 colonies into 500 μl of saline and adjust OD625 to ˜0.1 using a plate reader.
(133) C. Dilute bacterial inoculum 1:280 for Gram-positive and fastidious strains and 1:400 for Gram-negative strains into corresponding medium broth (CAMHB, CAMHB+3% lysed horse blood, HTM) (e.g. 35.6 μl of inoculum into 10 ml of CAMHB or 25 μl of inoculum into 10 ml of CAMHB).
(134) H. influenzae: HTM
(135) Streptococci: CAMHB+3% lysed horse blood
(136) Other strains in the panel: CAMHB
2.4. Prepare Assay Plates
(137) A. Add 100 μl of the bacterial inoculum to each well of the compound plates except wells B12, D12, F12 and H12.
(138) B. Add 100 μl of medium broth to wells B12, D12, F12 and H12 of the compound plates.
(139) C. Stack four plates together and cover with a sterile plate lid. Incubate in an ambient-air incubator at 35±2° C. for 16-20 hours (24 hours for MRSA).
2.5.. Perform Colony Counts
(140) A. Dilute the bacterial inoculum (0.5 McFarland) to a serial of 10−1 to 10−7 in saline solution (e.g. 100 μl bacterial inoculum+900 μl of saline).
(141) B. Spread 100 μl of each dilution (10−4, 10−5, 10−6, and 10−7) onto CAMHA plates in triplication, let the liquid soak into the agar for 10 minutes, invert the agar plates and incubate for 24 hr at 35±2° C.
2.6. Record MICs and Calculate CFUs
(142) A. Open the compound plate layout in the compound management system, and check the assay plate barcode.
(143) B. Place the assay plate on the top of MIC reader, and adjust the magnification mirror to read each wells, record growth status as raw data. (Optional) Record photo image of each assay plates using high-speed high-resolution scanner.
(144) C. Determine MIC break points according to CLSI guideline.
(145) D. Count colonies and calculate CFU of bacterial inoculum.
3. Results
3.1. MIC Summary Table
(146) The MIC screening was performed with 14 bacterial strains (11 ATCC strains and 3 clinical isolates) and 4 compounds (USYDS1, USYDS2 and ethanolic extract of propolis and reference compound levofloxacin). The MICs are summarized in the below Table 6. The MIC values of reference compound Levofloxacin obtained in this study are within the standard range as described in S100-A20 [2]. The final concentration of DMSO in the MIC screening was ˜3%, and did not inhibit the growth of most microorganisms.
(147) TABLE-US-00008 TABLE 6 MICs (μg/ml) of USYDS1, USYDS2 and ethanolic extract of propolis against fourteen bacterial strains. Levofloxacin is reference compound. Ethanolic propolis Levofloxacin USYDS1 USYDS2 extract Compounds Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Acinetobacter calcoaceticus 4 4 >64 >64 >64 >64 >64 >64 Escherichia coli <0.0625 <0.0625 16 16 64 32 >64 >64 Enterococcus faecalis 1 1 16 16 32 32 64 64 (ATCC 29212) Enterococcus faecium >64 >64 8 16 32 32 64 32 (ATCC 700221) Haemophilus influenzae <0.0625 <0.0625 32 32 32 32 64 64 Klebsiella pneumoniae 1 1 >64 >64 >64 >64 >64 >64 Pseudomonas aeruginosa 1 1 >64 >64 >64 >64 >64 >64 Staphylococcus aureus 0.5 0.25 16 8 32 16 32 32 (ATCC 29213) Staphylococcus aureus 0.25 0.25 8 8 16 16 32 32 (ATCC 43300) Staphylococcus aureus 64 64 8 8 16 16 16 16 (Levofloxacin-resistant) Staphylococcus aureus 8 8 8 8 16 16 32 32 (MRSA, Erythromycin & clindamycin-resistant) Streptococcus pneumoniae 0.5 0.5 32 32 32 32 32 32 (ATCC 49619) Streptococcus pyogenes 0.5 0.5 64 64 64 64 >64 >64 (ATCC 700942) Streptococcus pneumoniae 1 1 32 32 32 64 64 >64 (Erythromycin-resistant)
4. Discussion
(148) The prenylated tetrahydroxystilbenes USYDS1 and USYDS2 showed a moderate anti-bacterial activities with the rank of potency of USYDS1>USYDS2>ethanolic extract of propolis.
7. The Effect of USYDS1, USYDS2 and USYDS10 Compounds of Kinases Activities
(149) The following is a list of kinases in the studied.
(150) TABLE-US-00009 No. Kinase 1 ABL2 2 AKT1 3 AKT2 4 AKT3 5 ALK1 6 AMPK(A1/B1/G1) 7 AMPK(A2/B1/G1) 8 Aurora A 9 Aurora B 10 Aurora C 11 AXL 12 BLK 13 BTK 14 CAMK1 15 CDK1/CyclinA2 16 CDK1/CyclinB 17 CDK2/CyclinA2 18 CDK3/CyclinE1 19 CDK4/CyclinD1 20 CDK4/CyclinD3 21 CDK5/CyclinP25 22 CDK6/CyclinD1 23 CDK6/CyclinD3 24 CDK7/CyclinH1/MNAT1 25 CHK1 26 c-KIT 27 c-KIT(V654A) 28 EGFR(T790M, L858R) 29 EphA1 30 EphA2 31 EphA3 32 EphA4 33 EphB1 34 EphB2 35 EphB3 36 ERK1 37 FGFR1(V561M) 38 FGFR2 39 FGFR3 40 FGR 41 FLT1(VEGFR1) 42 FLT3 43 GSK3α 44 HER2 45 IGF1R 46 InsR 47 KDR 48 LCK 49 NEK2 50 p38β 51 PDGFRβ 52 PI3Kα 53 PI3Kβ 54 PI3Kγ 55 PI3Kδ 56 PKAcα 57 PKAcβ 58 PKAcγ 59 PKCα 60 PKCβI 61 PKCγ 62 PKCε 63 PKCθ 64 PKCδ 65 PKCδ 66 PKC.Math. 67 PLK2 68 PLK3 69 RAF1 70 BRAF 71 BRAF(v599E) 72 RET 73 RON 74 SRC 75 TrkA 76 TrkB
Experiments
Materials
(151) Kinase-Glo(Plus)/ADP-Gloassay buffer
(152) 25 mMHEPES, 10 mMMgCl2, 0.01% Triton X-100, 100 μg/mLBSA, 2.5 mMDTT, pH 7.4.
(153) Caliper assay buffer
(154) 100 mMHEPES, 10 mMMgCl2, 100 μl/L Brij35 (30%), 1 mMDTT, pH 7.4.
Assay Substrates
(155) MBP protein, UnactiveMEK1, Rbprotein were purchased from SignalChem. Poly(glu:tyr)(4:1) was purchased from Sigma. PIP2 was purchased from Cayman. Peptide substrates were synthesized in HD Biosciences, China.
(156) ATP was purchased from Sigma. KinaseGloPlus reagent, KinaseGloreagent and ADP Gloreagent were purchased from Promega
Assay Procedure—Caliper Format
(157) Mix Kinases, substrate, ATP and compound in 96-well assay plate, total volume is 50 μL. Incubate assay plate at 30° C. for 1 hour. Stop reaction by adding 20 μL of 35 mM EDTA and transfer 26 μL stopped reaction to 384-well assay plate. Read the assay plate on the plate reader.
Assay Procedure—ADP-GloFormat
(158) Mix Kinases, substrate, ATP and compound in 384-well assay plate, total volume is 10 μl. Incubate assay plate at 30° C. for 1 hour. Add 10 μl/well of ADP GloReagent to the assay plate, incubate at 27° C. for 40 min.
(159) Add 20 μl/well Detection Reagent to the assay plate, incubate at 27° C. for 30 min. Read the assay plate on the plate reader
Assay Procedure—Kinase-Glo(Plus)Format
(160) Mix Kinase, substrate, ATP and compound in 384-well assay plate, total volume is 10 μl. Incubate assay plate at 30° C. for 1 hour. Add 10 μl/well KinaseGlo(Plus) reagent to the reaction mixture, and then incubate at 27° C. for 20 min. Read the assay plate on plate reader.
(161) Hundred percent effect was performed without compound and enzyme, but containing ATP and substrate.
(162) Zero percent effect was carried out without compound, but containing ATP, substrate and enzyme.
(163) SB202190 is reference compounds for kinase p38β; Staurosporine (STSP) is reference compound for the remaining kinases.
Results
(164) Kinases that inhibited more than 60% are summarised in the table below. It is interesting to note that all three compounds inhibited kinases TrKA and PI3Kδ and PI3Kγ. Both USYDS1 and USYDS10 appear to display similar inhibitory activities towards the kinases.
(165) TABLE-US-00010 Compound Kinases USYDS1 FLT3, TrkA, CDK2/CyclinA2, CDK4/CyclinD3, PI3Kα, PI3Kδ, PI3Kγ USYDS2 TrkA, PI3Kβ, PI3Kδ, PI3Kγ USYDS10 FLT3, TrkA, KDR, FLT1, CDK2/CyclinA2, PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ, CDK1/CyclinB
8. Acute Toxicity Study of USYDS1
Method
(166) A single mouse is given a single IP injection of 400 mg/kg; a second mouse receives a dose of 200 mg/kg IP and a third mouse receives a single dose of 100 mg/kg IP. The mice are observed for a period of 2 weeks. They are sacrificed if they lose more than 20% of their body weight or if there are other signs of significant toxicity. If all 3 mice must be sacrificed, then the next 3 dose levels (50, 25, 12.5 mg/kg) are tested in a similar way. This process is repeated until a tolerated dose is found. This dose is then designated the maximum tolerated dose (MTD) and is used to calculate the amount of material given to experimental mice during anti-tumor testing. The mice are allowed ad libitum feed and water. Drug was dissolved in 100% DMSO at concentration of 200 mg/mL.
Result
(167) TABLE-US-00011 Dose Death Survivor/ Group (mg/kg/dose) Route days Total day 15 Injection volume 1 100 IP None 1/1 0.5 μL/gm body wt 2 200 IP 1 0/1 1 μL/gm body wt 3 400 IP 1 0/1 2 μL/gm body wt
(168) The MTD of USYDS1 was determined as 100 mg/kg. This concentration indicates low mammalian toxicity and is being used for further anti-tumor testing. Hollow fiber assay (BEC/C) for USYDS1 is under progression. The hollow fibre assay is a preliminary rapid screen for assessing novel putative chemotherapeutic compounds against a range of cancer cell lines prior to their evaluation in the mouse xenograft model. The hollow fiber model has a shorter evaluation time and a reduced compound requirement compared to traditional xenograft models. The model allows for the effective selection of cancer cell types in the xenograft.
Chemotypes of Plants that Produce Resins as Sources of Prenylated Polyhydroxystilbene Derivatives
(169) Resins, gums or exudates obtained from plants of the Lepidosperma genus from different locations were analysed by quantitative .sup.1H-NMR (q-NMR) for prenylated polyhydroxystilbene content, which include C- and O-prenylated, O-methylated and non-O-methylated derivatives. Different proportions of these prenylated polyhydroxystilbene derivatives from the resins form a basis to classify the plants accordingly.
(170) There are at least 3 different chemotypes of Lepidosperma plants identified thus far and each of these plants display several sub-chemotypes. Type 1 is the most common plant which contains approximately equal proportion of both C- and O-prenylated derivatives. Type 2 plant contains only C-prenylated derivatives. Where as, type 3 plant contains no O-methylated prenylated polyhydroxystilbene derivatives.
(171) It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
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