Journal of Natural Products Vol. 47, No. 3 , pp. 445-452, May-Jun 1984
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CONSTITUENTS OF CANNABZS SATIVA, XXV. ISOLATION OF TWO NEW DIHYDROSTILBENES FROM A PANAMANIAN VARIANT HALAN. E&HLY,+ GUANG-ENMA,' CARLTONE. TURNER,^ and MAHMOUDA. E&HLY Research Institute of Pharmaceutical Sciences, Schwl of Pharmacy, University of Mississippi, University, MS 38677 ABSTRACT.-TWO new dihydrostilbene compounds (named cannabistilbenes I and 11) were isolated from a polar acidic fraction of a Panamanian variant of Cannabis sativa grown in Mississippi. The structure of cannabistilbene I was shown to be 3,4'-dihydroxy-5-methoxy-3'(3-rnethylbut-2-enyl)-dihydrostilbene(1)from spectral data which was confirmed by synthesis. There is spectral evidence to indicate that cannabistilbene I1 could be represented by either structure 3 or 4 .
A polar acidic fraction of a Panamanian variant of Cannabis sativa L. grown in Mississippi yielded the known spiro-indan compounds cannabispiran, iso-cannabispiran, dehydrocannabispiran, P-cannabispiranol, and two dihydrostilbene derivatives, canniprene and ~-{2(~-hydroxy-4-methoxyphenyl)-ethyl~-5-methoxyphenol ( 1, 2). Two polyhydroxylated cannabinoids, (+)-trans-cannabitriol(3) and cannabitetrol(4), vere also isolated. Further fractionation of an equivalent polar acidic fraction afforded two new dihydrostilbene derivatives with M+ 3 12 (compound A) and M+ 304 (compound B). The mass spectrum of compound A indicated that it was cleaved into two fragments: fragment A with mlz 137 (5%) and fragment B which appears at m/z 175 (100%). This pattern of fragmentation is very similar to that of canniprene (1, 5), which is a dihydrostilbene derivative. Further proof for a dihydrostilbene nucleus was found in the pmr spectrum. The bis-methylene bridge resonated at 6 2.80, and one aromatic methoxy appeared at 6 3.74. The presence of prenyl unit was indicated by a benzylic resonance at 6 3.33 (d, 2 H , J = 7 Hz), an olefinic proton at 6 5.25 (br, t , 1H) and two olefinic methyls at 6 1.78 (s, 6H). These data indicated that fragment B, with mlz 175, bears a hydroxy group and a prenyl unit. A comparison of the multiplicity of the aromatic protons (ring B) at 6 6.90-6.70 with that of 3-~2(3-hydroxy-4-methoxyphenyl)-enyl}-5-methoxyphenol suggested that the two substituents are ortho-disposed. Fragment A appearing at mlz 137 indicated that it carries one methoxy group and one hydroxy group. The multiplicity of the signals for the aromatic protons at 6 6.33-6.20 suggested that the hydroxy and methoxy groups are mta-disposed. Thus, compound A was proposed to be 3,4'-dihydroxy- 5- methoxy-3 '( 3 -methylbut-2-enyl)-dihydrostilbene(l),which we named cannabistilbene I. Final proof of the structure of cannabistilbene I was obtained through synthesis (Scheme 1). p-Hydroxybenzaldehyde (7)was converted into the dimethyl prop-2-ynyl ether (8). Semihydrogenation and Claisen rearrangement at 134" for 40 min gave the 3-prenylatedp-hydroxybenzaldehyde (10).This was benzylated, and the benzyl derivative (11) was used as the aldehyde component in a Wittig reaction with the phosphonium salt (15)to give the mono-benzylated stilbene derivative (16).Reduction with sodium and n-BuOH (6) resulted in selective reduction of the stilbene double bond and cleavage of the benzyl group without affecting the double bond of the prenyl unit to give a compound identical (gc, ir, pmr, ms) with the natural material (1). 'Currently a visiting professor from the Shangai Institute of Materia Medica, Chinese Academy of Sciences, People's Republic of China. 'Currently on leave of absence from the Research Institute of Pharmaceutical Sciences.
Journal of Natural Products
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6
5
8
7
9
I
-1 SCHEME1.
Synthesis of 3,4’-dihydroxy-5-methoxy-3’-(3-methylbut-2-enyI)-dihydrostilbene.
The mass and pmr data of compound B indicated a new dihydrostilbene derivative which we named cannabistilbene 11. The mass spectrum showed two characteristic fragments at m/z 137 (fragment A) and at m/z 167 (base peak, fragment B). The pmr indicated that fragment A contains one hydroxy and one methoxy group meta-disposed (6 6.43-6.3 1, m , 3H), and fragment B contains one hydroxy and two methoxy groups.
1 2
3 4
R , = H , R,=OH, R,=isoprenyl, R4=H R,, R,=OCH,, R,=OH, R,=H R , = H , R,, R,=OCH,, R,=OH R,=H, R,, R,=OCH,, R,=OH
447
May-Jun 19841 ElSohly et a/. :Cannabis sativa New Dihydrostilbenes
The presence of a two protons singlet at 6 6.80 in the pmr spectrum suggested that fragment B is symmetric, and thus cannabistilbene I1 could be accommodated by structure 2. Synthesis of 2 was carried out following the procedure outlined in Scheme 2. Although the aromatic protons of fragment B of compound 2 appeared as a singlet in the prnr spectrum, their chemical shift was at a higher field (6 6.36) than those ofcannabistilbene 11. Study of the pmr spectra of related dihydrostilbenes, for example, canniprene ( 6 ) ,showed that the two ortho-aromaticprotons on the tetrasubstituted ring appear as a singlet around 6 6.80 as is the case with cannabistilbene 11. Thus, the relative distribution of substituents on ring B is proposed to be as shown in either structure 3 or 4 . The synthesis of both compounds is currently underway to prove the structure of cannabistilbene I1 unequivocally.
CH~OH
20
21
I
CHO
22
+
23 SCHEME2.
L2
15
Synthesis of 3,4’-dihydroxy-j’5,5’-trimethoxy dihydrostilbene
Several dihydrostilbene derivatives were previously isolated from Cannabis (5, 7) and synthesized ( 6 , 8). Batatasins 111-V, which are dihydrostilbenes isolated from Dimcoreu batatas, are known to have plant growth inhibitory properties ( 9 , 10). EXPERIMENTAL GENERAL EXPERIMENTAL PRoCrDURES.-Melting points were carried on a Thomas Hoover melting point apparatus and are uncorrected. The ir spectra were obtained on a Perkin-Elmer 281B recording spectrophotometer. Pmr spectra were recorded in the stated solvent on a Varian EM-390 90 MHz nrnr
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Journal of Natural Products
spectrometer using TMS as internal standard. Cmr spectra were taken on a JEOL FX 60 spectrometer operating at 15.03 MHz. Mass spectra were measured on a Finnigan 3200, MS/DS system. Gc analyses were performed using a 2% OV-17 column according to a previously published procedure (1 1). PLANTMATERIAL..-A Panamanian variant of C. sativa grown in Mississippi was used in this study. Herbarium specimens are deposited in the Herbarium, Research Institute of Pharmaceutical Sciences, University of Mississippi. EXTRACTIONAND FRACTIONATION.-Powdered 95% EtOH (see Figure 1).
leaves (7 kg) were extracted by percolation with
FRACTIONATION OF FRACTION F (EQUIVALENT).-This material (4.4g) was chromatographed on a silica gel 60 (E. Merck) column for flash chromatography (230-400 mesh) using 5% EtOAc in CH,CI, ( 1 .O liter) followed by 10% EtOAc in CH,CI, (500 ml), 15% EtOAc in CH,CI, (500 ml) and 20% EtOAc in CH,Cl, ( 2 . 5 liters). 207 fractions, each of 20 ml, were collected. ISOLATION OF CANNABISTILBENE 1.-Fractions 56-59 (29 mg), eluted with 5% EtOAc inCH,Cl,, showed two spots on precoated silica gel G plates with Rf values of 0.76 and 0.66 in 10% EtOAc in CH,CI, (system A). Further purification was achieved on precoated silica gel 60 F254plates (E. Merck) (20 Panamanian Variant of Cannabzr stivu Leaves (7 kg)
1
7
Percolates 1 & 2 (Extract A, 797 g)
Percolation with 95% EtOH (6 x 12 gal) Percolates 3-6
Extract B Partitioned as under extract A to yield a polar acidic fraction
Partitioned between CHCI, and H 2 0
CHCI, (fraction B)
H2O (fraction C)
4 Silica gel 60 column for flash chromatography starting with 5% EtOAc in CHKI,)
Partitioned between n-hexane and 3 N NaOH
+Hexane (fraction D)
3N NaOH (fraction E) Acidify with HC1 extract with Et,O Et,O (fraction F, 35.2 g)
207 fractions each of 20 ml collected
I
Fractions 56-59 Preparative chromatography
Cannabistilbene 1 FIGURE1.
Fractions 60-67 Preparative chromatography
Cannabistilbene I1
Flow chart for the fractionation of the ethanol extract of the leaves ofa Panamanian variant of Cannabis sativa grown in Mississippi.
May-Jun 19841 ElSohly et a / .:Cannabis sativa New Dihydrostilbenes
449
X 20, 0.25 mm) using system A. The band with Rf 0.76 was scraped and eluted with 5% MeOH in CHCI,. The residue obtained upon evaporation ofthe solvent (3 mg) was identical with canniprene (ir, ms, pmr). The band with Rf 0.66 was scraped and eluted with a MeOH-CHCI, mixture. Evaporation to dryness yielded a residue (5.7 mg) that was further purified on Whatman reversed phase plate KC18F using MeOH-H,O (8:2, system B). The major band (Rf0.39) was scraped, eluted with MeOH, and the solvent evaporated to yield 3 mg of a pure but oily residue (cannabistilbene I). Because of the scarcity of material, no attempts were carried out to crystallize it.
IDENTIFICATION OF CANNABISTILBENE I.-This cornpound was obtained as a light oil; gc RR, ir u max (film) 3340 (OH), 2845, 1610, 1590, 15 10, 1150 and (1.6) relative to 4-androstene-3,17-dione; 1060 cm-' ms m / z (rel. int., 9) 312 (M+, 4) obs. 312.17 19, calc. 312.1725 for CZOH24O3, 175 (loo), 157 (9). 145 (2.4), 137(5), 119(13.5), 105 (4), 91 (20.5);pmr(CDCl3)66.9O-6.70(m, 3H), 6.33-6.20 (m, 3H), 5.25 (t, br, lH), 3.74 (s, 3H), 3.33 (d, 2 H , J = 7 Hz), 2.80 (s, 4H), and 1.78 (s, 6H).
ISOLATIONOF CANNABISTILBENE II.-Fractions 60-67 (53 mg) eluted with 10% EtOAc in CH,CI, showed three spots with Rf values 0.76 (trace), 0.66, and 0.56 using system A. Gcims analysis revealed three compounds with molecular weight of 342, 3 12, and 304. Separation of the three components was achieved on silica gel 60 F,,, using system A, followed by reversed-phase chromatography on Whatman KC18F plates using system B. The band with Rf 0.59 in system B was scraped, eluted with MeOH, and the solvent was evaporated to yield 4 mg of a pure residue (tlc), which was labeled as cannabistilbene 11. IDENTIFICATION OF CANNABISTILBENE rr.-This compound was obtained as a colorless oil; gc RR, ms m/z (rel. int., %) 304 (M+ 3.5) obs. 304.1249, calcd (1.2) relative to 4-androstene-3,17-dione; 304.1310 for C,,H2,O5, 167 (100) and 137 (84); pmr (CDCI,) showed signals at 6 7.73 (s, l H , OH), 6.80 (5, 2H), 6.43-6.3 1 (m, 3H), 5.53 (br, s, l H , OH), 3.88 (s, 3H), 3.76 (s, 6H), and 2.68 (s, 4H). PREPARATIONOF CANNABISTILBENE I (l).
