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The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce)
The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce)
3739
established by both elemental analysis and high resolution mass measurement. The IR spectrum exhibited absorption maxima at 1650, 730 and 720 cm-’ typical of the presence of a cis disubstituted double bond. The mass spectrum showed a strong molecular ion peak at m/e 410 and satellite peaks at m/e 395 (M-15), 355 (M-55) and 328 (M-82); the latter fragments are probably formed by cleavage of ring A. The NMR spectrum of this compound was of interest and is briefly discussed. With benzene as solvent, multiplets centered at 7 4.57 and 4.60 were observed. These signals were attributed to the olefinic protons H, and H, in partial structure XX. In deuteriochloroform solution, the signal at 7 4.65 appeared to be a singlet while that at 7 4.62 was a very narrow multiplet about half the height of the other signal. Two other one-proton multiplets centered at 7 7.85 (triplet, J=3-0 c/s) and 8.04 (possible quartet; incompletely resolved) were observed in the spectrum of the benzene solution. These were assigned to the protons H, and H, in the position allylic to the double bond in XX. H. H.
xx An attempt was made to decouple the various signals. Thus irradiation of the sample at 7 4.58 caused a simplification of the signals at 7 7.90 and 8.04 due to the allylic protons. Conversely, irradiation at 7 8.04 caused the alteration of the low field proton region to two rather wide peaks of equal height. Because of the close similarity in chemical shift of the two pairs of protons concerned, the signals arising from the decoupling experiments still showed some degree of overlap and the results were inconclusive. The low coupling constants between the proton H, and the allylic protons H, and H, might at first sight appear rather puzzling. However, similar systems have been encountered by previous workers. Thus Meakins ef al. I9 have reported overlapping signals centered at 7 4.36 and 440 (w’/‘=4.5 c/s) for the olefinic protons in the compound, 5a-cholest-2-ene measured in Ccl, solution. The relative heights of the signals were 10 to 6. A similar situation was observed by these workers in the case of the triterpene lup-Zene (XXI) and by Djerassi et aI.” in the compound, cholesta2,8( 14)diendz-yl acetate (XXII). Hydrogenation of the olelln yielded a product, C,,H,,, m.p. 181-184O, which was identical in all respects with bserratane (XVIII). We, therefore, identify the olelin as A’+serratane (XIX) and to compound B we ascribe the structure, 3a-methoxy-2lahydroxydr4-serratenediol (IX).
__ XXI
6Ac
XXII
3740
J. P. KUINEY. 1. H. ROGERS and J. W. ROWE
The fifth component of the original extract was compound C. This substance was separated from B only with great difficulty by exhaustive chromatographic separations or by fractional recrystallization. This compound, m.p. 194-195”, [ai:* + 23O, had the empirical formula, C,,H,,02, and it was, therefore, a structural isomer of compounds A and B. In this case also the NMR and IR spectra showed the presence of OH and OMe groups, but no signals arising from an olefinic proton were seen. The NMR spectrum showed one proton triplets (J= 2.5 c/s) centered at T 6.54 and 7.22 indicating the configuration of both the OH and OMe functions to be axial. Acetylation of compound C gave the acetate derivative, C3JHs,03, m.p. 222-225 O, with the expected signals for a secondary acetate group in the NMR spectrum (Z 5.3 1, multiple& lH, and 7.95, singlet, 3H). The acetate group could be readily removed when the compound was treated with an excess of LAH and this procedure was utilized in the preparation of a pure specimen of compound C as the acetate could be more readily recrystallized. It has been well established by the work of Inubushi’s group that A”-serratene compounds are isomerized to the A’j anaiogues by moderate treatment with mineral acids.’ It was noted that this is an equilibrium reaction and that pure samples of the A” compounds were obtained only in the case where ketone functions were present in the original compounds. From the evidence so far described, we anticipated that compound C was a representative of the A’, series and it became necessary to carry out the acid isomerization of compound B to conclusively establish the structure. Following a procedure used by Inubushi, compound B acetate was isomerized by allowing it to stand in cold acetic acid-sulfuric acid for 24 hr. The reaction product was then deacetylated with LAH and the isomer&i compound B was separated from the unchanged A” material still present by preparative TLC on silica containing the dye stuff, Rhodamine B. L1The isomer&d material was a white crystalline solid, m,p. 190- 192’. The NMR spectrum of the rearranged material showed the absence of the olefinic proton signal at 7 4.65 and the presence of the other expected features. On the basis of the NMR and mass spectral data, and direct comparisons by TLC and quantitative IR techniques, compound C and isocompound B were identical. Compound C, therefore, has the structure, 3a-methoxy-2 1@-hydroxy-A”-serratene (XXIII). To the best of our knowledge, this is the first reported natural occurence of a A”-serratene derivative.
Finally, the presence of three other minor components in the original triterpene extract has been noted and structural work is now in progress. One can conclude that the Sitka spruce is indeed a rich source of triterpenes of the serratene family.
The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce)
3741
EXPERIMENTAL Mps were determined on a Kofler block and are uncorrected. Infrared (IR) spectra were taken as KBr pellets on a Perkin-Elmer Model 2 I spectrophotometer and the positions of absorption bands are quoted in wave numbers (cm-‘). NMR spectra were usually recorded at 60 MC/S on a Varian A60 instrument and at 100 MC/S, when indicated, on a Varian HAlOO instrument, using CDCI, as solvent; the line positions or centres of multiplets are given in the Tiers r scale with reference to TMS as the internal standard; the multiplicity, integrated areas and type of protons are indicated in parentheses. The mass spectra were measured on either an Atlas CH4 or an AEI MS9 mass spectrometer as specifically indicated. Samples were admitted to the ionization chamber using the direct insertion technique, the electron energy being maintained at 70 eV. The high resolution mass spectra for the determination of molecular formulae were obtained on the MS9 instrument. A detailed treatment of the mass spectral data is presented in the accompanying publication.” ORD curves were taken in MeGH sol on a JASCO UV/ORD/CD-5 spectropolarimeter. Plates for TLC were prepared using Woelm silica gel G as adsorbent. For the separation of mixed olefins, silica gel plates impregnated with AgNO, were prepared using 30 g of silica gel in 60 ml of 7% AgNO,aq, drying and activating in the usual way. As developing solvents, CHCI, and EtOAc-benzene (1 : 3) were used. In all cases, the spray reagent of choice was antimony pentachloride dissolved in Ccl, (1 :2). The spots became visible directly after spraying with this reagent. For column chromatography, neutral alumina (Woelm) was used throughout and deactivation was done by the addition of water. The approximate activity of the adsorbent utilized in specific experiments is indicated below. Isolation of Sitka spruce bark triterpenes. Bark was obtained from the butts of mature Sitka spruce trees (Picea sitchensis), Grays Harbor County, Washington State, and mechanically separated into a cork-rich fraction by hand sorting. The bark was air-dried and ground in a hammermill to pass a 20mesh sieve and was then extracted in a borosilicate glass Soxhlet extractor for I8 hr with petroleum ether (b.p. 40-600). The extract was taken to dryness to provide a crude mixture in a yield of 4.3%. The extract was dissolved in hot acetone (50 g extract in 500 ml acetone) and allowed to stand two days at ambient temp, whereupon white crystals were deposited. The crude crystalline deposit was filtered off and recrystallized once from hot acetone to give a crude white triterpene mixture, m.p. 250-255” in a yield of 15% of the starting extract. The filtrates were recovered as a yellow, tacky wax, m.p. 40”, from which further small quantities of crystalline triterpenes were obtained by chromatographic separation. Separation ojtriterpene extract. A sample of the crude extract (2.014 g) was dissolved in the minimum quantity of CHCI,. To the sol was added a few grams neutral alumina (Activity III) and the CHCI, was evaporated off from the slurry. The alumina coated with the extract was then transferred to the top of a column filled with deactivated alumina (100 g) in pet ether. Gradient elution with pet ether (b.p. 65I lOTbenzene followed by benzene-ether provided the various compounds in a fair state of purity. After further chromatography on individual fractions, where necessary, the composition of the original extract was estimated to be as shown below. Compound
C B E A F D G H
Solvent composition
pet. ether/ benzene 7:3 7:3 7:3 3:7 3:7 benzene/ether 9:l 8:2 8:2
Weight recovered (mg) from 2.014 g extract
% of extract
30 440 240 1100 not isolated
1.5 22.0 12.0 55.0
30 35 20
1.5 I.7 1.0
R/ (EtOAc/ benzene)
0.64 0.57 0.45 0.39 0.30 0.21
J. P. KUTNEY. I. H. ROGERSand J. W. ROWE
3142
Clearly the major components of the extract were the compounds designated by the letters A and B. Compound A. White crystals from CHCI,, m.p. 307.5-308”. 1x1:”+ 2.8”. ORD (c. 0.02; dioxan). 21”: I$,],,,,, o”, Ql,,, +46”, Ic#~l,, +9l”. 141, + 137’7 id],, +274”; v,,: 3550 (OH). 1100 (OMe) and 795 (trisubstituted double bond); UV. no absorption: NMR (65’ with external TMS): 4.6 I
I
(m, IH, oiefinic H), 6.46 (m. IH. H-C-OH).
6.58 (s. 3H, OMe). and 9.00-9.27
(23.8H. MC).
Mass spectrum (MS9): fragments at tn/e 441 (M-15). 438 (M-18) and 423 (M-33): 3 I6 and 269 (retro Diels-Alder); 221,220, 189 (allylic cleavage); other intense fragments at m/e 187, I47 and 135. (Found: C, 81.52; H, 11.48; 0. 7.01; Mol. wt. (MS9) 456. Calc. for C,,H,,O,: C. 81.08; H. 11.37;0,7.51%; Mol. wt. 456. High resolution mass measurement 456.398; Calc. for Cr,H,,O, 456.397. OMe (Zeisel) 7.01; Calc. for 10 Me 680%). Compound R White crystals from EtOH, m.p. 276277.5”. Ial:’ -554”; ORD (c, 002; MeOH), 21°: 141,, - 128’. I@I,,, -260’. 141,, -347”. \I$!,~ -433 , ty,,oo - 1290’; v,,,: 3650 (OH, weak). I103 (OMe), and 795 (trisubstituted double bond). UV. no absorption. NMR (100 MC/S): 4.71 (m. IH, olefinic H), 6-57 (t. J=2.5 I &-C-OMe).
c/s, IH,H-{OH),
6.72 (s, 3H, OMe), 7.25 (t. J=2.5
c/s. IH,
and 9.10-9.34 (21 .OH. Me). Mass spectrum (MS9): fragments at m/e 454 (M-2), 438
(M-18). 424 (M-32): 316, 284. 269 (retro Diels-Alder): 221. 220. 190, 189 (allylic cleavage); other intense fragments at m/e 187, 147 and 135. (Found: C, 8104; H, 11.37; 0, 764; Mol. wt (MS9) 456. Calc. for C,,H,,O,: C. 81.08; H. 11.37; 0, 7.51%; Mol. wt. 456. High resolution mass measurement 456.398; Calc. for C,,HIIO, 456.397. OMe (Zeisel) 6.97; Calc. for I OMe 6.80%). Compound B acerate. Compound B (520 mg) was treated with a mixture of dry, redistilled pyridine (IO ml) and Ac,O (IO ml) at room temp for 4 days. The reaction mixture was poured onto crushed ice and allowed to stand for 3 hr. The crude product was extracted into CHCI, (3 x 75 ml) and the combined CHCI., extracts washed with 5% HCI, 5% sodium bicarbonate and water. The recovered acetate (520 mg) was purified by column chromatography on deactivated alumina (Activity II, 50 g). Elution with pet ether-benzene (I7 : 3.500 ml) yielded the TLC pure compound (5 IO mg. 90% yield. RrCHCI, O-45). White crystals from EtOH, m.p. 205.5-207”. IaIi” -64.6”; v,,: 1733 and 1245 (OAc), 1103 0 (Me), and 792 (trisubstituted double bond); NMR (IO0 MC/S): 4.65 (m, IH, oletinic H). 5.28 (t, J=2.5 c/s. IH, H-- C!-0Ac).
6.70 (s. 3H. OMe). 7.23 (t, J=2.5
c/s. IH, H-
A -0Me).
7.94 (s, 3H. OAc).
I 9.07-9.30 (20.3H. Me). Mass spectrum (MS9): fragments at m/e 483 (M-15). 466 (M-32). 45 I (M-47) 438 (M-60) 423 (M-75); 3 16, 284. 269 (retro Diels-Alder); 262, 221, 203, 202, 190, 189 (allylic cleavage); other intense fragments at m/e 187 and 135. (Found: C, 79.55; H, 10.70; 0.9.80; Mol. wt. (MS9) 498. Calc. for C,,HIdO,: C, 79.46; H, 10-91; 0. 9.62%; Mol. wt. 498). Compound A ocefafe. Compound A (630 mg) was reacted as described above to yield the acetate (570 mg, yield 83%. R/CHCI, 0.21). White needles from EtOH. m.p. 199-201’. 1~1: +4.5’; v,,,: 1730 and 1250 (OAc), I 105 (OMe), and 795 (trisubstituted double bond). NMR (100 MC/S): 4.64 (m. lH. olefinic
I H), 5.28 (t, J = 2.5 c/s, IH, &C-OAc), 663 (s, 3H, OMe), 7.45 (q, J, = 119 c/s J2 = 40 c/s, I I lH.H-C-OMe). 7.93 (s. 3H, OAc). and 9.02-9.28 (23.OH. Me). Mass spectrum (MS9): fragments
I at m/e 483 (M-15). 438 (M-60). and 423 (M-75); 316, 284 and 269 (retro Diels-Alder); 262, 221, 204, 203, 190. 189 (allylic cleavage); other intense fragments at m/e 187 and 135. (Found: C, 79.43; H. 10.82; 0.9.55; Mol. wt. (MS9) 498. Calc. for C,,HsdO,: C, 79.46; H, 10.92; 0,9.63%; Mol. wt. 498). Compound B ketone. Compound B (I .O g) was dissolved in dry, redistilled pyridine (25 ml). This sol was added slowly to a slurry of chromic oxide (975 mg) in dry pyridine (10 ml) and the reaction mixture was stirred for 60 hr at room temp following the procedure of Sarrett.” The sol was poured into cold water (400 ml). allowed to stand for + hr. and filtered under suction. The dark brown ppt on the suction funnel was washed thoroughly with cold water and the crude ketone was then recovered by extraction with warm CHCI, (600 ml). The sol was washed with 5% aqueous HCI and water. The crude ketone (R,
The neutral triterpenes
CHCI,
0.45)
Elution
with benzene-pet
flakea from -228”.
EtOH,
I#],,,
1707 ()C=O),
(s, 3H,
OMe),
723
Mol.
cleavage);
Compound
-715”;
) ali0
-227’.
454.
ORD
-4
ld~l,,
795 ttisubstituted
Compound
C. 81.88:
H.
11.08:
0.
- 3 I .O. Addition
Further
elution
with benzene-ethyl has m.p.
effect alter 90 min; Y,,,:
22 I. 2 18.204,203,
7.04%:
(allylic Mol.
cleavage);
lxlC
-0.
I I05 (OMe). 8.91-9.25
407 (M-47);
Mol.
a fraction
I ‘. ORD
454.
Calc.
B ketone
for CI,H,,Or:
efh_vlene kerat.
(241
mg, Rf CHCI, Vacuum
MeOH).
21’:
C. 81.88;
Compound
H.
Diels-Alder);
221.
11.08;
0.
B ketone (250
derivative
(250
III.
7.04%:
Mol.
mg. yield
92%).
-56.5”.
Y,,,,,: I I IO (OMe).
(s. 4H.
-0-CH --
9.28 (21.3H, 79.26: Mol.
H.
CH
White
0.
crystals
and 795 (trisubstituted
-0-_).
6.79
Me). Mass spectrum 11.01;
rod-like
9.61;
(s.
(MS9):
Mol.
3H.
from LAH.
fragments
wt. (MS9)
498.
ether (I 9
: I,
7.23
4.62
fragments at m/e I90 and 189
H. I I .24; 0. 6.90: wt. 454).
The reaction was carried
while the water formed during trap.
The benzene sol was
(m.
on deacti-
200 ml) yielded the TLC
from benzene.
double bond); NMR: OMe).
of
of the Cotton
mg) was reacted with ethylene glycol
by means of a Dean-Stark
20 g). Elution with benzene-pet
+ 36”.
Addition
washed with water and the recovered crude ketal was purified by column chromatography vated alumina (Activity
0.23)
l&l,,,
218. 203.
C. 82.03;
acid hydrate (40 mg) as catalyst.
continuously
I.
sublimed
double bond): NMR:
H, Me). Mass spectrum (CH4):
out for 5 hr in refluxing benzene previously dried by distillation was removed
7.18:
on alumina (Activity
(300
(c. 0.02:
at m/e 147 and 135. (Found:
(I -5 ml) in the presence of p-toluenesulfonic the condensation
190. 189
wt. 454).
m.p. 262-263”.
and 795 (trisubstituted
284 and 269 (retro
other intense fragments
wt. (CH4)
Compound
ether (4: I) yielded
270-270.5.
1712 ()C==O),
422 (M-32).
frag-
pure material (740 mg. 65% yield. R,CHCI,
A. White flakes from EtOH,
olefmic H), 6.65 (s, 3H, OMe),
439 (M-15).
(CH4):
C. 8 I .58; H. I I .23; 0.
-929”; molecular amplitude -36.8. -2002O. I#],,, + 1680°. 141,,, sol of the ketone (002 and 0-06N) had virtually no effect on the amplitude
Ir#Jl,,, 0°, l&l,,, HCl to the MeOH
(m, lH,
ether (19: I) yielded TLC
largely unreacted compound
material (250-260’/0.07)
of HCI to
A (I. 133 g) was oxidized by the Sarrett procedure as described above
100 g). Elution with benzene-ethyl containing
White l$lssg
4.63 (tn, lH, olefinic H),
to yield the crude ketone (I. 174 g) which was purified by column chromatography 0.41).
50 g).
-137’.
Mass spectrum
1H. II
for C,,H,,O,:
III.
of the Cotton effect after 90
NMR:
at n;/e 147 and 135. (Found:
(Activity
21”: )41700
amplitude
double bond);
3743
(892 mg; yield 85%).
(c, O-02; MeOHk
I06 ‘; molecular
spruce)
alumina
-0CHJ. 891-9.17 (21.7H, Me). 4 and 407 (M-47); 284 and 269 (retro Diels-Alder);
(m
Calc.
A ketone.
on deactivated
had almost no effect on the amplitude
other intense fragments
wt. (CH4)
(Sitka
1000 ml) yielded the pure material
1100 (OMe,
ments at m/e 422 (M-32) (allylic
chromatography
: 17.
sol of the ketone (0.02N)
.
mm; v,.
by column ether (3
m.p. 240-242”.
lr#~l,,, -3332”.
the MeOH
671,
was purified
of the bark of Picea sitchensis
m.p. 4.65
317-318”
pure
(dec). lzlb
(m, IH. olefinic H). 6.04
IH,
and
9.01-
at m/e 399, 221 and 99 (very intense). (Found:
Calc.
for C,,H,,O,:
C, 79.52;
H. 10.84;
C,
0. 9.64%;
wt. 498).
Compound yield
A kefone ethvlene
the crude
(Activity
ketal (278
ketai.
mg).
Compound
This
by column
I, 30 g). Elution with benzene yielded the TLC
plates from pet ether. m.p. 317-319’(dec). bond);
NMR:
OMe).
9.00-9.27
4.63
intense). (Found: 10.84:
A ketone (245 mg) was reacted as described above to
was purified
0.
Desoxy Djerassi
(22.8
IH.
Mol.
compound
olefinic
H. Me).
C. 79.33;
9.64%; ef al.”
(m.
1~1: -4-4”; H).
6.02
vnuX: I I05 (OMe)
(s. 4H,
Mass spectrum (MS9):
H. 10.94; 0. 9.91;
Mol.
chromatography
on neutral
alumina
pure derivative (180 mg. 67% yield). Colourless and 795 (trisubstituted
-O-~-JJZ-O-).
fragments
wt. (MS9)
498.
6.61
at m/e 399, 221. Calc.
double (s.
3H.
189 and 99 (very
for C,,H,,O,:
C. 79.52;
H.
wt. 498).
B. Compound
The ketone (300
B ketone tosylhydrazone
was prepared by the method described by
mg) and ptoluenesulfonhydrazide
reagent (310
mg) were reacted
together in refluxing methanol (IO ml) in the presence of three drops of acetyl chloride as catalyst.
After
+ hr heating was terminated.
the reaction flask and contents allowed to cool to room temp. and the white
crystalline
by filtration.
(dec);
product
r’m..: 3180
removed (NH).
1635 (>C=N-_).
This material 1592
and
was shown to be TLC 1490 (aromatic),
pure: m.p.
1165 (-SO,-).
257-258” and 1095
J. P. KUTNEY, I. H. ROGERS and J. W. ROWE
3144
(OMe). (Found: C, 73.81; H. 10.00: N, 4.54; S. 4.96. Calc. for C,sH,,N?O,S: C. 73.26; H. 9.38; N. 4.50; s. 5.14%). The tosylhydrazone derivative (220 mg) was reduced by NaBH, (500 mg) in moist refluxing dioxan according to the method of Caglioti and Graaelli. ” After heating for 5 hr. the dioxan was evaporated and the product partitioned between benzene (I50 ml) and water. The benzene layer was washed with water. dried. and evaporated to yield the crude desoxy compound. This substance was purified by column chromatography on deactivated alumina (Activity III. 20 g). Elution with pet ether (200 ml) yielded the TLC pure desoxy compound (115 mg. 73% yield based on the ketone, RYCHCl, 0.7 1). White crystals from EtOH, m.p. 249.5-251”. 121: -49. I; v,,,: 1100 (OMe). and 737 (trisubstituted double bond); NMR: 4.70 (m. IH. olefinic H). 6.71 (s, 3H. OMe). 7.25 (m. IH.H--
A -0Me).
and 9.09-9.34 (20.2
I H. Me). Mass spectrum (CH4): fragments at m/e 425 (M-15). 408 (M-32). and 393 (M-47); 316.284 and 269 (retro Diels-Alder); 221. 204. 190, 189 (allylic cleavage); other intense fragments at m/e 187 and 135.(Found:C.84~82;H,11~94;0.3~71;Mol.wt.(CH4)440.Calc.forC,,H,,O:C.84.54;H.l1.82; 0. 3.65%; Mol. wt. 440). Desox?, compound A. Compound A ketone (330 mg) and p-toluenesulfonhydrazide reagent (330 mg) were reacted together in reiluxing benzene for 7 hr and most of the solvent removed by evaporation. The tosylhydrazone derivative which crystallized was filtered off and recrystallized from EtOH. m.p. 234237=’(dec); I’,“,=:3210 (NH). 1635 (>C=N-).
I165 (--SO?-_),
and 1107 (OMe); NMR: 2X10-2.72
(complex. 4H. aromatic). 6.63 (s, 3H. OMe). 7.55 (s, 3H. aromatic Me). and 8.96-9.25 (Me). (Found: C. 73.19; H. 9.54; N, 4.61: S. 5.20. Calc. for C,BH,,NrSO,: C. 73.26; H. 9.38; N, 4.50; S. 5.14%). The tosylhydrazone (390 mg) was reacted with NaBH, (500 mg) in refluxing dioxan as already described to yield the crude product (277 mg) which was purified by column chromatography on alumina (Activity 1.30 g). Elution with benzene yielded the pure desoxy compound (158 mg. 55% yield based on the ketone) White needles from Et&, m.p. 267-269”. IaI$ +338”; \P”: 1107 (OMc); NMR (100 MC/S): 4.60 (m. 1H. olefinic H), 6.60 (s. 3H. OMe), 7.36 (q, J,= Il.3 c/s.
J,=4.0
c/s,
lH,H-C-
I OMe). and 9.01-9.30 (21.5 H. Me). Mass spectrum (CH4): fragments at m/e 425 (M-15) 388 (M-52): 3 16, 284. 269 (retro Diels-Alder); 221, 204. 191 and 189 (allylic cleavage); other intense fragments at m/e 184 and 135. (Found: C. 84.68; H. 11.99; 0, 3.61; Mol. wt. (CH4) 440. Calc. for C,,H!,O: C. 84.54; H. 11.82; 0. 3.64%; Mol. wt. 440). Dihydro compound B. Compound B (10.25 mg, 22.5 micromoles) was hydrogenated in a microhydrogenation apparatus previously calibrated against pure maleic acid (7 determinations). Using a suspension of Adam’s catalyst (PtO,. 5.17 mg) in glacial AcOH. a smooth uptake of HZ (25.0 micromoles) was observed. On a larger scale. compound B (360 mg) was hydrogenated under atm press in glacial AcOH sol using Adam’s catalyst (100 mg). The uptake of H, over a period of 8 hr was 17.2 cc (talc. for one double bond 17.7 cc). The hydrogenation was repeated using fresh catalyst (50 mg) to complete the reaction and the crude product was dissolved in CHCI,. washed with 5% NaHCO,aq and water. The recovered product was TLC pure (366 mg. R,CHCI, 0.30). White needles from pet ether, m.p. 21 l-216’; v,,: 3500 (OH). 1088 (OMe) and no absorption at 795 (tri-substituted double bond); NMR: no olelinic H. 6.10 (m. IH. jj-
h -OH).
I
6.70 (s. 3H. OMe), 7.25 (m. 1H. H-C-OMe).
and 9.10-9.24
(21.0
Me). Mass spectrum (MS9): fragments at m/e 456 (M-2), 440 (M-18), 426 (M-32), 411 (M-47), 393 (M-65). and 369 (M-89); no retro Diels-Alder; other fragments at m/e 221, 190, 189, 163. 150, 149 and 136. (Found:C,81.41;H, 11.48;0.7.15;Mol.wt.(MS9)458.Calc.forC,,H,,O,:C,81~l6;H. 11.86;0, 6.97%; Mol. wt. 458). Dihydro compound A. Compound A (10.54 mg, 23. I micromoles) was hydrogenates’ in a microhydrogenation apparatus at atm press using Adam’s catalyst (5.56 mg). No H, uptake was observed in EtOH sol, but use of glacial AcOH.instead resulted in the absorption of 22.3 micromoles. On a larger scale. compound A (200 mg) was hydrogenated as above using Adam’s catalyst (95 mg). After I5 hr the
The neutral triterpenes of the bark of Picen sirchensis (Sitka spruce)
3145
obaezvud H, uptake was 116 ml (CaIc 98 ml) and the reaction produd was isohtted as pkody described(R, CHC13 020). White uy&als from dioxan, mp. 288293”; Y,: 3558 (OH), 1082 0 (Me)
I and no absorption at 795 (trisubstituted double bond); NMR: no oleflnic proton, 6.59 (m, lH.H-COH), 664 (s. 3H, OMe), 9.02-9.26 (24.8 H, Me). Mass spectrum (MS9): fragments at m/e 440 (M-18). 426 (M-32), 385 (M-73). and 369 (M-89); no retro Diels-Alder; other fragments at m/e 245, 221. 189, 177, 176, 149 and 136. (Found: C, 81.29; H, 12.00; 0.6.66; Mol. wt. (MS9) 458. Calc. for C,,H,,O,: C. 81.16; H, 11.87; 0, 6.98%; Mol. wt. 458). Compound G. White crystals from EtOH. m.p. 289-291’. izlg - 12.3 ‘. Low solubility in common solventa; v, 9 354U (--OH), and 7.95 (trisuhatituted doubk bond); NMR: 4.65 (m, HI, oIcfinic H), 655 (t. lH, H-
I
-OH),
-OH),
6.80 (m. IH, jj-
and 9.04-9.32
(23-O H, Me). Mass spectrum
I (CH4): fragments at m/e 440 (M-2). 427 (M-IS), 424 (M- 18). and 409 (M-33); 302.284 and 269 (retro Diels-Alder); 221, 220. 207, 203, 190 and 189 (allylic cleavage); other intense fragments at m/e 234, 187, 149 and 135. (Found: C, 81.17; H, 11.26; 0, 7.35; Mol. wt. (CH4) 442. Calc. for &I&O,: C, 81.45; H, 11.31; 0, 7.24%; Mol. wt. 442). Compound G diacetute. A crude fraction of compound G obtained from the mother liquors during crystallization (75 mg) was acetylated as previously described using a mixture of equal volumes (10 ml) of Ac,O and dry pyridine at room temp. Isolation of the crude acetate in the usual way was followed by chromatographic separation on a column of deactivated alumina (Activity III, 10 g). Elution with benzene-pet ether (2 :3) yielded a white crystalline diacetate (45 mg) Examination on a silica chromatoplate (EtOAc-benzene, 1:4) showed the product to be pure diacetate (RfO.75. compound G R/0.41). White crystals from EtOH. m.p. 210-217’; NMR (100 MC/S): 4.65 (m. lH, olelinic H), 5.30 (t,J=2.5
I
c/s, IH, H-C-GAc),
I
5.47 (q, I, = 100, J, = 5.2 c/~ HI, H-C-GAc),
I
794 and 7098 (two
I
singlets, 6H, OAc). and 8.75-9.31 (19.6 H. Me). Mass spectrum (MS9): fragments at m/e 511 (M-15). 466 (M-60). 451 (M-75), 423 (M-103). 406 (M-120). 391 (M-135). and 363 (M-163); 203,202, 190 and 189 (allylic cleavage); and other fragments at m/e 187, 147 and 135. (Found: Mol. wt. (MS9) 526. Calc. for C,,H,,O,: Mol. wt. 526). Correlorion ofcompoundG with 2 I-episerratenediol. An authentic sample, isolated from pine bark by J. W. Rowe, was compared with compound G. White crystals, m.p. 284- 285’ (lit value 289-290“, 1~1: - 19’). Both compounds were identical on the basis of TLC, quantitative IR. NMR and mass spectral comparisons. Methyfation of compound A. A 3-necked LOOml flask was fitted with a high-speed stirrer, N, gas inlet tube, condenser with drying tube and a bopping funnel. Full precautions were taken to exclude moisture from the walls of the apparatus. Compound A (175 mg) was dissolved in warm, dry toluene (10 ml) and admitted to the reaction vessel via the dropping funnel. The apparatus was then thoroughly purged with purified grade N, gas and freshly cut K metal (300 mg) was added. The flask was warmed until the K was molten and stirring was commenced. After 2 hr. the reaction vessel was allowed to cool and an excess of pure dry Me1 (5 ml) was added from the dropping funnel. Stirring and heating were recommenced and the reaction was continued for a further 3 hr. AAer cooling, the excess K was destroyed by the addition of an excess of MeGH, the solvent was evaporated and the reaction products partitioned between CHCI, and water. The combined CHCI, extracts were washed with water, dried, and evaporated to yield the crude methyl ether. This product was purified by chromatography on a column of deactivated alumina (Activity III, 18 g). Elution with pet ether yielded first an oily liquid fraction (35 mg) and then the pure methyl ether (95 mg). Elution with benzene removed weacted compound A (50 mg). The methyl ether yielded white crystals from pet ether. m.p. 272-273’, Ial: - 16.4“; Y_: no absorption at 3500 (-OH), 1100 (OMe). and 793 (trisubstituted double bond); NMR: 4.70 (m. lH, oletinic II), 6.67 and 6.70 (two s, 6H, OMe), 7.20 (m. lH, H-
I
-0Me). 7.45 (m. lH, jj-0Me). 9.05-9.33 (21.2 H, Me). ‘i f Maas spectrum (CH4): fragments at m/e 455 (M-15). 438 (M-32) and 423 (M-47); 316, 284 and 269 (retro Diels-Alder); 221, 189 (allylic cleavage); other fragments at m/e 279, 187, 167 and 149. (Found:
J. P. KIJTNEY.I. H. ROGERS and J. W. ROWE
3146
C, 81.68; H, 11.45; 0.6.95; Mol. wt. (CH4) 470. Calc. for C,,H,,O,: C, 81.70; H, 11.49; 0.6.81%; Mol. wt. 470). Methylation of 2 1-episerratenediol and correlation with compound A. A sample of 2 1-episerratenediol (102 mg) was methylated according to the procedure outlined above using K metal (247 mg) and Me1 (5 ml). The crude product was separated on a column of deactivated alumina (Activity III, 20 g). Elution with pet ether yielded an unidentified oily liquid (45 mg). Further elution with benzene-pet ether (I : 9) removed the dimethyl ether (18 mg) and the use of benzene-pet ether (4 : 1) yielded first a fraction of a monomethyl ether (8 mg) and then a larger fraction of another monomethyl ether (43 mg). Finally elution with benzene removed some unreacted 2 I-episerratenediol (8 mg). The more plentiful monomethyl ether was purified by vacuum sublimation at 250-260”/0.05 mm to yield a white crystalline solid, m.p. 305-306’ (compound A. 304-305’). mixed m.p. 306-307’. These two compounds were identical on the basis of TLC, quantitative IR, NMR and mass spectral comparisons. The dimethyl ether derivative was also compared with compound A methyl ether. After vacuum sublimation, this compound had m.p. 277-278” (compound A methyl ether 272-273’). mixed m.p. 272-275”. These compounds also were shown to be identical by evidence similar to that used in the case of the monomethyl ether derivatives. Correlation of compound E with compound A ketone. The crude material was purified by three recrystalhxations from EtOH which gave white rod-like crystals, lap. 262-263”. 1a 1;’ - 16a)“.Vacuum sublimation raised the mp. to 270-271”. v,,. 1710 (OH), 1105 (OMc), and 795 (trisubstituted doubk bond); NMR (100 MC/S): 4.68 (m. IH, olefinic H), 6.73 (s, 3H, OMe), 7.34 (partly hidden multiplet,H-
I
-OMe), and F 8.94-9.28 (24.38. Me). (Found: C, 81.69; H, 11.78; 0.6.32; Mol. wt. (MS9) 454. Calc. for C,,H,,O,: C, 81.88; H. 11.08; 0, 7.04%; Mol. wt. 454. High resolution mass measurement 454.379. Calc. for C,,H,,02 454.381). Compound A ketone, m.p. 270-270.5”. mixed m.p. 270-271’. The compounds showed identical TLC behaviour and the quantitative IR spectra were superimposable. Dihydm compound B ketone. Dihydro compound B was oxidized with Jones’ reagent as described by Henbest” et al. The reagent was prepared by dissolving reagent grade chromic oxide (2.668 g) in cone H,SO, (2.13 ml) and diluting to 10 ml with distilled water. The dihydro compound (155 mg) was dissolved in warm acetone (30 ml) and placed in a 100 ml flask equipped with magnetic stirrer. Jones’ reagent (0.14 ml) was added and reaction allowed to proceed for 20 min. whereupon unreacted oxidising agent was destroyed by the addition of excess MeOH. The solvent was evaporated and the product partitioned between benzene and water. The benzene layer was washed with 5% Na,CO,aq, water, and dried over MgSO,. The benzene sol was run through a one inch bed of neutral silica to remove traces of chromous salt. Evaporation yielded the ketone (152 mg. R, CHCI, 0.62). White platelets from EtOH.
m.p. 167-169O; Y,, (CHCI,): 1705 ()C=O),
I
IH, H-C-OMe).
7.64 (m. -m--CO-),
and 1090 (OCH,). NMR: 6.66 (s, 3H, OMe), 7.20 (m, and 8.93-9.23
(22.4H.
Me). Mass spectrum
(MS9): fragments at m/e 441 (M-15). 424 (M-32). 409 (M-47), 385 (M-71), and 383 (M-73); prominent fragments at m/e 219, 189, 150, 149 and 136. (Found: C, 81.47; H, 11.23; 0, 7.25; Mol. wt. (MS9) 456. Calc. for C,,H,,O,: C, 81.52; H, 11.48; 0. 7.01%; Mol. wt. 456). Desoxy dihydro commnd B was prepared by three different methods: A. WoUfrKishner reduction. Dihydro B ketone (150 mg) was suspended in EtOH (15 ml) containing diethylene glycol(l.5 ml) and reacted in a 25 ml flask under reflux with hydrasine hydrate (O-75 ml) and KOH (0.14 g). After 30 min. the reagents had dissolved and the EtOH and excess hydrazine were then distilled off from an oil bath at 150”. Diethylene glycol(2 ml) was added and the oil bath temp raised to 190’. After 4 hr under reflux, the sol was cooled, poured into water (20 ml) and extracted with benzene (4 x 10 ml). The benzene extract was washed with water, dried and evaporated to yield a white crystalline solid (123 mg, 85% yield). This material was found to be TLC pure (Rr CHCI, 0.68). B. Borohydride reduction of dihydro B ketone tosylhydrazone. Dihydro B ketone (96 mg) was reacted with ptoluenesulfonhydrazide reagent (90 mg) in dry refluxing benzene and the water formed was removed by azeotropic distillation. After 3 hr the reaction was stopped and the product purified by chromatography on a neutral silica column (Activity 1. 15 g). Elution with benzene yielded a mixture of
The neutral triterpenes of the bark of Piceu sirchensis (Sitka spruce)
3747
unreacted ketone and tosylhydrazone (45 mg). Further elution with CHCI, yielded almost pure tosylhydrazone (113 mg) which was leached with a little cold MeOH and dried in racuo, m.p. 21 l-212” (dec); v,, (CHCI,): 3300 (NH), 1605 (aromatic), and 1085 (OMe); NMR: 2.10-2.75 (complex, 4H, aromatic), 670 (s, 3H, OMe), 722 (m, IH, jj
-Me),
758 (s, 3H, aromatic
Me), 92)8-9.28
--+
(overlapping signals, Me). In a second experiment, dihydro B ketone (600 mg) was reacted as above with ptoluenesulfonhydrazide reagent (600 mg) for 4 hr in benzene under reflux to yield a TLC pure sample of the tosylhydrazone. This product was reduced with NaBH, (I.70 g) in dioxan (55 ml). After IO hr under reflux. the solvent was removed in ~IXUOand the product recovered as previously described. Column chromatography on neutral silica (Activity I, 65 g) yielded the desoxy dihydro compound (380 mg, 66% yield). The desoxy dihydro compound B samples prepared in these two experiments, when recrystallized from EtOAc, could both be mechanically separated by hand sorting into platelet-type crystals, m.p. 188-194’. and needle-like crystals, m.p. 2 15-223’. The platelet crystals, which predominated in each of the two reduction experiments, gave superimposable quantitative IR spectra. Examination by VPC using a 20% silicone gum rubber (S.E. 30) column showed both samples to be mixtures of two components in the ratio 4: 1 with the corresponding pairs having identical retention times on the column. C. Hydrogenation of desoxy compound B. Attempts to hydrogenate desoxy compound B with Adam’s catalyst at atmos press in AcOH or AcOH-THF (I : I) sol were unsuccessful. The hydrogenation was successfully accomplished in a Parr bomb at 50 p.s.i. H, press using a sol of desoxy compound B (708 mg) in AcOH-CHCI, (I : I. 140 ml) with Adam’s catalyst (320 mg). After 3 hr. the catalyst was removed by filtration and the product recovered by evaporation of the solvent under reduced press. Examination by TLC (silica, CHCI,) showed that hydrogenation had occurred (yellow spot, starting material brown, similar R, value). The reaction product was purified by column chromatography on alumina (Activity I, 50 g). Elution with pet ether (b.p. 65-l IO’) removed dihydro desoxy compound B (687 mg, 97% yield). White prisms from pet ether, m.p. 195-198”. Examination by VPC under conditions above gave only one peak; v,,: I 100 (OMe), no absorption at 795 (trisubstituted double bond); NMR: no olegnic proton,
I
6.67 (s, 3H OMe), 7.20 (m, lH, H-C-OMe)
and 9.06-9.21 (Me) Mass spectrum (CH4): fragments
at m/e 440 (M-2). 427 (M-15). 410 (M-32). and 395 (M-47); other intense fragments at 272, 221, 190, 189, 177, 163, 150, 149 and 136. (Found: C, 83.96; H. 12.22; 0,3.68; Mol. wt. (CH4) 442. Calc. for C,,H,,O: C, 84.16; H, 12.22; 0, 3.62%; Mol. wt. 442). Ether clearage of desoxy dihydro compound B (44% HBr/AcOH). Desoxy dihydro compound B (370 mg) was reacted at room temp for 20 hr in a mixture of saturated HBr in AcOH (44%, 40 ml) and Ac,O (40 ml). All the material had dissolved before the reaction was terminated by pouring onto crushed ice. After standing for 2 hr. the product was extracted into CHCI,, washed with 5% KHCO,aq, water, and dried. The recovered product was a brown syrup containing some semi-crystalline material. This mixture was separated into two main fractions by column chromatography on deactivated alumina (Activity III, 36 g). Elution with pet ether yielded a colourless syrup (158 mg) which was apparently olefinic and showed two components when examined by TLC (AgNOJsilica, pet ether). Further elution with pet ether-CHCI, (9: I) yielded a white crystalline solid (137 mg); v,, (CHCI,): 1730 and 1230 (OAc), absence of Me (I 100): NMR: 5.36 (m. IH. H-
-0Ac). 7.92 (s, 3H. OAc). and 9.10-9.21 (Me). ! Further information was obtained on the olefin fraction as follows. NMR: 4.64 and 4.80 (weak m. olefinic H), 8.76 and 9.14-9.18 (Me). An examination by VPC using as liquid phase S.E. 30 gum rubber (20%) indicated the presence of at least 4 components which were not further examined. The acetate fraction was treated with LAH (500 mg) in dry refluxing THF for 4 hr and the excess reducing agent decomposed by the controlled addition of wet THF. Examination of the white crystalline product by TLC (silica, CHCI,) showed it to contain 3 components (R/0.55,0.48 and 0.28, yellow). The mixture was separated by column chromatography on deactivated alumina (Activity III, 20 g). Elution with pet ether-benzene (I 7 : 3) yielded first a mixture of the two less polar alcohols (50 mg) and then a pure fraction of the second alcohol (59 mg, alcohol B). Further elution with pet ether-benzene (I : I) yielded the third component (8 mg) which upon examination by TLC (silica, CHCI,) was seen to contain two compounds in the approximate ratio I :4. An attempt to separate the mixed fraction containing the
3148
J. P. KUTNEY.I. H. ROGERSand J. W. ROWE
of alcohols by further column chromatography on deactivated alumina (Activity III) was largely unsuccessful. The pure alcohol B, m.p. 219-221 O,was further examined as follows. Y,,,: 3560 (OH); NMR (100 lint pair
MC/S): 6.66 (triplet, J=3.0
c/s. 1H. H-
I
-OH) and 9.02-9.24 (23.7 H, Me). Mass spectrum F (MS9): fragments at m/e 413 (M-15), 410(M-18). and 395 (M-33); most intense fragments above m/e 100 occurred in the order 121, 136, 123, 109. 107, 137 and 149; other significant peaks were observed at 189, 190, 207, 231 and 259. (Found: Mol. wt. 428.400. Calc. for C&,0: Mol. wt. 428402). /ones oxidation 0/0koh0l B. Alcohol B (50 mg) was dissolved in warm spectral grade acetone (20 ml) in a flask equipped with a magnetic stirrer. Freshly prepared Jones reagent (0.3 ml) was added and the reaction was terminated after 20 min by the addition of reagent grade MeOH (5 ml). The crude ketone was recovered in the usual way and purified by column chromatography on deactivated alumina (Activity III, 10 g), yield, 44 mg. white crystals. m.p. 185-193” without recrystallization: ORD (c. 0.02, MeOH). 216: L@l,uoo”, tt$l,,, -40’. IRIS,, +2867“, h#~l,,, -2509O. 141, - 13%; molecular amplitude + 53.7. Addition of HCI to the MeOH sol of the ketone (0.05N) had almost no effect on the amplitude of the Cotton effect aRer 90 min; v,,:
no OH absorption,
1695 (X0);
NMR (100 MC/S): 7.52
(m. 2H, -m-CO-) and 8.89-9.13 (23.7 H, Me). Mass spectrum (CH4): fragment at m/e 411 (M-15); other important fragments at 274, 232, 205, 138 and 123. (Found: Mol. wt. 426.391. Calc. for C,H,,O: Mol. wt. 426.386). Borohydride reduction of ketone B rosylhydrazone. A sample of ketone B (30 mg) was reacted with tosylhydrazine reagent (3 I mg) in MeOH (3 ml) in a 25 ml flask quipped with reflux condenser. Acetyl chloride (3 drops) was used as catalyst and reaction was allowed to take place for 45 min under rAux. The white crystalline tosylhydrazone derivative was filtered off from the MeOH after cooling. The air 1590 (aromatic), and 1162 (dried material had m.p. 202-204’; v,,,: 3200 (NH), 1635 (C=N-), SO,-). The tosylhydrazone was transferred to a 50 ml flask equipped with reflux condenser. Sodium borohydride (55 mg) and spectral grade dioxan (15 ml) were added and reduction allowed to proceed for 10 hr under reflux. The solvent was evaporated and the reaction product taken up in CHCI,. The CHCI, extract was washed with water, dried and evaporated to yield a white crystalline solid (23 mg). Examination by TLC (silica, pet ether) showed mainly one component present. This was recovered by column chromatography from alumina (Activity I, 10 g). Elution with pet ether yielded a white crystalline solid (8 mg). This compound was recrystallized from spectral grade acetone. White leaf-like crystals, m.p. 184-186’. Authentic Bserratane (Inubushi), m.p. 184-187’. mixed m.p. 184-186“. The compounds showed identical TLC behaviour on both silica (R, 0.70, ginger) and alumina plates developed with pet ether. However, a-serratane could not be distinguished from p-serratane by TLC. Mass spectrum (CH4): fragment at m/e 397 (M-15); major fragments at 231, 192, 191. 163, 137 and 123. (Found: Mol. wt. 412408. Calc. for C,,,H,,: Mol. wt. 412.407). Ether clearage of desoxy dihydro compound B (36% HBr/ACOH~fsolution of Al-b-serratane. Dihydro desoxy compound B (590 mg) was suspended in a mixture of freshly prepared 36% HBr in glacial ACOH (50 ml) and Ac,O (50 ml). ARer stirring for 2.5 hr at 70-80’. the reaction mixture was poured onto crushed ice, allowed to stand for 2 hr and extracted into pet ether (b.p. 30-600). The sol was washed twice with water, dried over anhyd K&O,, filtered and evaporated to recover a semi-crystalline material with a foul odor. Examination by TLC (silica, CHCI,) showed the presence of two fractions (R, 0.80, brown; 0.49, brown). The mixture was separated by column chromatography on alumina (Activity I, 25 g). Elution with pet ether removed an olefinic fraction (457 mg). Further elution with benzene removed a mixed fraction (34 mg) containing olefinic and unreacted starting material. Finally, an acetate fraction (97 mg) was recovered by elution with benzene-ether (4 : 1); v,, (CHCI,): 17 IO and 1250 (OAc). no OMe (1100). This fraction was seen to contain polar impurities when examined by TLC and was not further studied. The olefin fraction was examined on a AgNO,/silica thin layer. Using pet ether as developing solvent, 6 spots (R/O-67, 0.61, 0.55. 0.47.0.42 and 0.33) were seen. The olefin mixture was separated on a column packed with AgNO, impregnated silica (20% AgNO,, 50 g). Elution with pet ether (b.p. 65-llO”, 25 ml fractions) removed all the material applied in 13 fractions according to the Table below. A white crystalline material was isolated in fractions 8 to 10.
The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce) Fraction No.
Weight (mg)
R, Value of components
18 121 35 8 68 100 32 10
0.69 0.69 and 0.63 0.63 and 0.58 0.51 0.42 0.39 0.39 0.39
3149
Fractions 8-10 were bulked and rechromatographed on AgNO, impregnated silica (15 g). The main fraction from the second chromatography (67 mg) was recrystallized three times from spectral grade acetone. white needles, m.p. 188-191’; v,,: 1650,730 and 720 (cis disubstituted double bond), no OAc or OMe; NMR (100 MC/S) benzene: 4.57 and 460 (complex m, 2H. olefinic H), 7.85 (t, J= 2.0 c/s, IH, allylic H), 8.04 (m. IH, allylic H). and 9.01-9.13 (21.4 H. Me). Irradiation at r 8.04 caused the alteration of this region to two rather wide peaks of equal height. Alternately, irradiation at I 4.58 caused a simplification of the signals at 7.90 and 8.04 attributed to the allylic protons. The spectrum was also run in CDCI, sol and this permitted slightly better resolution of the oh&tic proton region which now showed a narrow triplet (J= 1.0 c/s) centred at 4.62 and an apparent singlet at 4.65. Mass spectrum (CH4): fragments observed at m/e 395 (M-15). 355 (M-55) and 328 (M-82); other prominent fragments at m/e 191. 189. 150, 137 and 123. (Found: C, 87.90; H. 12.43; Mol. wt. 410.392. Calc. for C&H,,: C. 87.80; H, 12.20%; Mol. wt. 410.391). Hydrogenation of A’-&serratune. A sample of AJ-/.l-serratane (38 mg) was hydrogenated in EtOAc sol (30 ml) in the presence of Adam’s catalyst (17 mg) for 2 hr. The recovered product was purified by chromatography on a short column of deactivated alumina (Activity III, 10 g). Elution with pet ether yielded a white solid (26 mg) which after recrystalliztion from acetone gave needlelike crystals, m.p. 175-180’. Vacuum sublimation raised the m.p. to 181-184’. The 8-serratane obtained from the previous ether cleavage reaction sequence (ex acetate) had m.p. 182-185”; mixed m.p. 181-184”. The two samples were identical on the basis of TLC. quantitative IR, NMR and mass spectral comparisons. (Found: C, 87.20; H. 12.65; Mol. wt. 412.407. Calc. for CrOHSz:C, 87.37; H. 12.62%; Mol. wt. 412.407). Isolation of compound C. Attempts to isolate pure compound C from mixtures with B by means of preparative scale thin layer or column chromatography were rather unrewarding because of the very similar R, values. A TLC pure sample of the compound (526 mg) was, however. obtained by column chromatography on deactivated alumina (Activity III. 1000 g) of a sample (20.638 g) of the total acetone soluble motha liquor after the removal of the main crystalline triterpeme fraction from the ortginal bark extra The eluting solvent used was pet ether-benzm (4:l). White needles from n-heptane, m.p. 194 195°~Izl’uD +23.l.v, _,: 3400 (OH. strong). 1IOU(OMe) and no trisubstituted double bond (795); NMR l
(100 MC/S): no olefinic proton, 6.54 (t. 5=2.5 5=2.5
c/s, IH, H-
I
c/s, IH, &-
-OH),
6.67 (s, 3H, OMe). 7.22 (1,
f
-OMe), and 9.04-9.24 (Me). Mass spectrum (CH4): fragments at m/e 454 ‘i (M-2). 441 (M-15). 438 (M-18). 424 (M-32), 409 (M-47) and 391 (M-65); no retro Diels-Alder; prominent fragments at m/e 236, 221. 203. 190, 189. 187, 147 and 135. (Found: Mol. wt. 456.395. Calc. for C,,H,,O>: Mol. wt. 456.398). Acefykxion of compound C. Compound C (108 mg) was acetylated in the usual way and the crude acetate (109 mg) was examined by TLC (silica. CHCI,, R, 0.53; compare compound C, 0.40). The product was chromatographed on deactivated alumina (Activity III, 20 g). Elution with pet etherbenzene (17:3) yielded the pure acetate (70 mg). Colourless rods from pet ether, m.p. 222-225’; v_: 1725 and 1250 (OAc). 1100 (OMe) and no trisubstituted double bond (795): NMR: 5.31 (m. IH.
jj-
I
-0Ac).
6.70 (s. 3H. OMe). 7.25 (m. IH. H-C-OMe).
7.95 (s. OAc). and 8.67-9.18
J. P. KUTNEY,I. H. ROGERSand J. W. ROWE
3750
Mass spectrum (MS9): fragments at m/e 483 (M-15), 466 (M-32), 452 (M-47) 438 (M-60), 432 (M-75). 406 (M-92) and 391 (M-107); no retro Diels-Alder; prominent fragments at m/e 255,221, 203, 190, 189, 187,147 and 135. (Found: Mol. wt. (MS9) 498. Calc for CssH,,Os: Mol. wt. 498). Acid isomerizatfon of compound E. This was performed according to a method described by Inubushi er ~1.~Compound B acetate (365 mg) was dissolved in AcOH (50 ml) and cooled to 0’. After the addition of a mixture of cone H,SO, (3 ml) in AcOH (4 ml), reaction was allowed to proceed 24 hr at room temp. The product was poured into a large excess of cold water, extracted into CHCI,, washed with 5% Na,CO,aq, water, and dried. Chromatography on a column of neutral alumina (Activity III) yielded a white solid (288 mg, pet ether eluant). This was deacetylated by treatment with excess powdered LAH in refluxing anhyd benzene for 3 hr. Workup of the reaction in the usual manner followed by passing the product through a column of neutral alumina yielded 220 mg of a mixture of iso-compound B and unreactcd compound B. The above mixture was separated by preparative TLC on three silica plates (20 x 20 cm) prepared in the usual fashion, except that a 0.1% aqueous sol of Rhodamine B was used to slurry the silica.” The plates were developed with CHCI, and the two bands could be clearly seen tier evaporation of the solvent. The less polar band was recovered from the plates, extracted with hot Ccl,, filtered through a bed of celite and evaporated to yield TLC pure iso-compound B (86 mg). White needles from pet ether, m.p. 190-192”; Y,,: 3400 (OH), 1095 (OMe), and no trisubstituted double bond (795); NMR; no
I
olefinic proton, 6.68 (m. lH, H-C-OH),
6-70 (s, 3H, OMe), 7.24 (m, lH, H-
I
c: -0Me)
and
I
9.05 to 9.26 (Me). Mass spectrum (MS9): fragments at m/e 441 (M-15) 438 (M-18). 424 (M-32), 409 (M-47) and 391 (M-65); no rktro Diels-Alder; prominent fragments at 236,221,204,203, 190,189,161, 149 and 135. (Found: Mol. wt. 456.394. Calc. for C,,H,,O,: 456.396). Correlation of compound C wifh iso-compund B. The compounds behaved identically when compared by TLC in two solvent systems. The quantitative IR spectra (KBr discs) were superimposable. Compound C, m.p. 194-195’; iso-compound B, m.p. 190-192’; mixture m.p. 190-194”. Properties of compound D. This product was isolated in a crude form in the chromatographic separation of the triterpene extract, but all attempts to purify the material by further column chromatography were unavailing. White crystals from EtOH, m.p. 245-252”; ORD (c. 0.02, MeOH), 21’: L$l,, -231’. ($~l,,, -231’. \@I,,, -2168’. 14),,, +461”, [b!,,, -2906’. Molecular amplitude -262;
v,:
3Mo (OH), 1707 (>c-o),
1387 and 1370 (geminal dimethyl) and 797 (trisubstituted double
I
--OH), 894-9.25 (22.6 H, Me). Mass Spec--? trum (CH4): fragments observed at m/e 425 (M-15). 422 (M-18). and 407 (M-33; no retro Diels-Alder; 218, 207, 203 and 189 (allylic cleavage); other prominent fragments at 177, 149, 147, 138 and 135. (Found: Mol. wt. 440.369. Caic. for C,,H,,O,: Mol. wt. 440.365). No further work has been performed due to the lack of sufficient material at this time. Properlies ofcompoundH. This product was also a minor component of the triterpene extract. The crude fraction (152 mg) as previously isolated was chromatographed a second time on a column of deactivated alumina (Activity III). The recovered material (59 mg) was recrystallized from EtOH, m.p. 256-270’; v,,,_: 3400-3300 (OH), 1387 and 1365 (geminal dimethyl) and 795 (trisubstituted double bond); NMR (100 MC/S): 4.72 (m, lH, olefinic H), 6.79 and 6.90 (overlapping t, J=S.O c/s, 2H,Hbond); NMR:465 (tn, HI, olefmic H), 684 (m, HI, B
A -OH),
8.78 and 9.06-9.35 (Me). Mass spectrum (MS9): fragments at m/e 427 (M-15). 424 (M-18).
I 409 (M-33). 406 (M-36) and 391 (M-5 I); 220,207 and 189 (allylic cleavage); other prominent fragments at 187, 147, 135, 127 and 121. (Found: C, 8066; I-I, 12-03; 0,750; Mol. wt. 442.380. Calc. for C,,Hs,Os: C, 81.45; H, 11.31; 0, 724%; MoL wt 442.381). Obviously this compound was impure and no further work was done owing to lack of material at this time. REFERENCES I Y. Inubushi, T. Sano and Y. Tsuda, Tetrahedron Leften 1303 (1964). ’ Y. Tsuda, T. Sane, K. Kawaguchi and Y. Inubushi, Ibid. 1279 (1964).
The neutral triter-penes of the bark of Piceu sifchensis
(Sitka spruce)
3751
’ Y. Inubushi, Y. Tsuda, T. Sane, T. Konita, S. Suzuki, H. Ageta and Y. Otake, Chem. Pharm. Bull. Japan IS, 1153 (1967). ’ J. W. Rowe, Tetrahedron Letters 2347 (1964). ’ J. W. Rowe and C. L. Bower, Ibid. 2745 (1965). 6 For leading refs, consult M. Martin-Smith and G. Subramanian, Phytochemistry 6,559 (1967); see also T. A. Bryce, M. Martin-Smith, G. Osske, K. Schreiber and G. Subramanian, Tetrahedron 23, 1283 (1967). ’ G. I. Poos. G. E. Arth. R. E. Beyler and L. H. Sarett. J. Am. Chem. SOC. 75,422 (1953). * C. Djerassi, 0. Halpem, V. Halpem and B. Riniker, Ibid. 80, 4001 (1958). ’ F. N. Lahey and M. V. Leeding, Proc. Chem. Sot. 342 (1958). ” C. Djerassi, L. A. Mitscher and B. J. Mitscher, J. Am. Chem. Sot. 81, 947 (1959). ” H. Audier, J. Bottin, A. Diara. M. Fetizon, P. Foy, M. Golfier and W. Vetter, Bull. Sot. Chim. Fr. 2292 (1964). ” Z. Pelah, D. H. Williams, H. Budzikiewicz and C. Djerassi, J. Am. Chem. Sot. 86,3722 (1964). ” L. Caglioti and P. Grasselli, Chem & Ind. 153 (1964). ” M. Fischer, Z. Pelah, D. H. Williams and C. Djerassi, Chem. Ber. 98, 3236 (1965). ” T. Obara and S. Abe. J. Chem. Sot. Jupn SO, 1487 (1959). I6 Y. Inubushi, Y. Tsuda, T. Sano and R. Nakagawa, Chem. Pharm. Bull. Japan 13, 104 (1965). ” P. Bladon, J. M. Fabian, H. B. Henbest, H. P. Koch and G. W. Wood, J. Chem. Sot. 2402 (195 1). ” S. Matsunaga, J. Okada and S. Uyeo, Chem. Comm. No. 21, 525 (1965); S. Uyco, J. Okada, S. Matsunaga and J. W. Rowe, Tetrahedron Zr, 2859 (1968). ” G. M. L. Cragg, C. W. Davey, D. N. Hall, G. D. Meakins, E. E. Richards and T. L. Whateley, J. Chem. Sot. (C), 1266 (1966). lo C. Djerassi, R. D. H. Murray and R. Villotti, J. Chem. Sot. 1160 (1965). ” J. Avigan, D. S. Goodman and D. Steinberg, J. Lfpid Res. 4, 100 (1963). ” J. P. Kutney, G. Eigendorf and I. H. Rogers, Tetrahedron 25, (1969).
3739
established by both elemental analysis and high resolution mass measurement. The IR spectrum exhibited absorption maxima at 1650, 730 and 720 cm-’ typical of the presence of a cis disubstituted double bond. The mass spectrum showed a strong molecular ion peak at m/e 410 and satellite peaks at m/e 395 (M-15), 355 (M-55) and 328 (M-82); the latter fragments are probably formed by cleavage of ring A. The NMR spectrum of this compound was of interest and is briefly discussed. With benzene as solvent, multiplets centered at 7 4.57 and 4.60 were observed. These signals were attributed to the olefinic protons H, and H, in partial structure XX. In deuteriochloroform solution, the signal at 7 4.65 appeared to be a singlet while that at 7 4.62 was a very narrow multiplet about half the height of the other signal. Two other one-proton multiplets centered at 7 7.85 (triplet, J=3-0 c/s) and 8.04 (possible quartet; incompletely resolved) were observed in the spectrum of the benzene solution. These were assigned to the protons H, and H, in the position allylic to the double bond in XX. H. H.
xx An attempt was made to decouple the various signals. Thus irradiation of the sample at 7 4.58 caused a simplification of the signals at 7 7.90 and 8.04 due to the allylic protons. Conversely, irradiation at 7 8.04 caused the alteration of the low field proton region to two rather wide peaks of equal height. Because of the close similarity in chemical shift of the two pairs of protons concerned, the signals arising from the decoupling experiments still showed some degree of overlap and the results were inconclusive. The low coupling constants between the proton H, and the allylic protons H, and H, might at first sight appear rather puzzling. However, similar systems have been encountered by previous workers. Thus Meakins ef al. I9 have reported overlapping signals centered at 7 4.36 and 440 (w’/‘=4.5 c/s) for the olefinic protons in the compound, 5a-cholest-2-ene measured in Ccl, solution. The relative heights of the signals were 10 to 6. A similar situation was observed by these workers in the case of the triterpene lup-Zene (XXI) and by Djerassi et aI.” in the compound, cholesta2,8( 14)diendz-yl acetate (XXII). Hydrogenation of the olelln yielded a product, C,,H,,, m.p. 181-184O, which was identical in all respects with bserratane (XVIII). We, therefore, identify the olelin as A’+serratane (XIX) and to compound B we ascribe the structure, 3a-methoxy-2lahydroxydr4-serratenediol (IX).
__ XXI
6Ac
XXII
3740
J. P. KUINEY. 1. H. ROGERS and J. W. ROWE
The fifth component of the original extract was compound C. This substance was separated from B only with great difficulty by exhaustive chromatographic separations or by fractional recrystallization. This compound, m.p. 194-195”, [ai:* + 23O, had the empirical formula, C,,H,,02, and it was, therefore, a structural isomer of compounds A and B. In this case also the NMR and IR spectra showed the presence of OH and OMe groups, but no signals arising from an olefinic proton were seen. The NMR spectrum showed one proton triplets (J= 2.5 c/s) centered at T 6.54 and 7.22 indicating the configuration of both the OH and OMe functions to be axial. Acetylation of compound C gave the acetate derivative, C3JHs,03, m.p. 222-225 O, with the expected signals for a secondary acetate group in the NMR spectrum (Z 5.3 1, multiple& lH, and 7.95, singlet, 3H). The acetate group could be readily removed when the compound was treated with an excess of LAH and this procedure was utilized in the preparation of a pure specimen of compound C as the acetate could be more readily recrystallized. It has been well established by the work of Inubushi’s group that A”-serratene compounds are isomerized to the A’j anaiogues by moderate treatment with mineral acids.’ It was noted that this is an equilibrium reaction and that pure samples of the A” compounds were obtained only in the case where ketone functions were present in the original compounds. From the evidence so far described, we anticipated that compound C was a representative of the A’, series and it became necessary to carry out the acid isomerization of compound B to conclusively establish the structure. Following a procedure used by Inubushi, compound B acetate was isomerized by allowing it to stand in cold acetic acid-sulfuric acid for 24 hr. The reaction product was then deacetylated with LAH and the isomer&i compound B was separated from the unchanged A” material still present by preparative TLC on silica containing the dye stuff, Rhodamine B. L1The isomer&d material was a white crystalline solid, m,p. 190- 192’. The NMR spectrum of the rearranged material showed the absence of the olefinic proton signal at 7 4.65 and the presence of the other expected features. On the basis of the NMR and mass spectral data, and direct comparisons by TLC and quantitative IR techniques, compound C and isocompound B were identical. Compound C, therefore, has the structure, 3a-methoxy-2 1@-hydroxy-A”-serratene (XXIII). To the best of our knowledge, this is the first reported natural occurence of a A”-serratene derivative.
Finally, the presence of three other minor components in the original triterpene extract has been noted and structural work is now in progress. One can conclude that the Sitka spruce is indeed a rich source of triterpenes of the serratene family.
The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce)
3741
EXPERIMENTAL Mps were determined on a Kofler block and are uncorrected. Infrared (IR) spectra were taken as KBr pellets on a Perkin-Elmer Model 2 I spectrophotometer and the positions of absorption bands are quoted in wave numbers (cm-‘). NMR spectra were usually recorded at 60 MC/S on a Varian A60 instrument and at 100 MC/S, when indicated, on a Varian HAlOO instrument, using CDCI, as solvent; the line positions or centres of multiplets are given in the Tiers r scale with reference to TMS as the internal standard; the multiplicity, integrated areas and type of protons are indicated in parentheses. The mass spectra were measured on either an Atlas CH4 or an AEI MS9 mass spectrometer as specifically indicated. Samples were admitted to the ionization chamber using the direct insertion technique, the electron energy being maintained at 70 eV. The high resolution mass spectra for the determination of molecular formulae were obtained on the MS9 instrument. A detailed treatment of the mass spectral data is presented in the accompanying publication.” ORD curves were taken in MeGH sol on a JASCO UV/ORD/CD-5 spectropolarimeter. Plates for TLC were prepared using Woelm silica gel G as adsorbent. For the separation of mixed olefins, silica gel plates impregnated with AgNO, were prepared using 30 g of silica gel in 60 ml of 7% AgNO,aq, drying and activating in the usual way. As developing solvents, CHCI, and EtOAc-benzene (1 : 3) were used. In all cases, the spray reagent of choice was antimony pentachloride dissolved in Ccl, (1 :2). The spots became visible directly after spraying with this reagent. For column chromatography, neutral alumina (Woelm) was used throughout and deactivation was done by the addition of water. The approximate activity of the adsorbent utilized in specific experiments is indicated below. Isolation of Sitka spruce bark triterpenes. Bark was obtained from the butts of mature Sitka spruce trees (Picea sitchensis), Grays Harbor County, Washington State, and mechanically separated into a cork-rich fraction by hand sorting. The bark was air-dried and ground in a hammermill to pass a 20mesh sieve and was then extracted in a borosilicate glass Soxhlet extractor for I8 hr with petroleum ether (b.p. 40-600). The extract was taken to dryness to provide a crude mixture in a yield of 4.3%. The extract was dissolved in hot acetone (50 g extract in 500 ml acetone) and allowed to stand two days at ambient temp, whereupon white crystals were deposited. The crude crystalline deposit was filtered off and recrystallized once from hot acetone to give a crude white triterpene mixture, m.p. 250-255” in a yield of 15% of the starting extract. The filtrates were recovered as a yellow, tacky wax, m.p. 40”, from which further small quantities of crystalline triterpenes were obtained by chromatographic separation. Separation ojtriterpene extract. A sample of the crude extract (2.014 g) was dissolved in the minimum quantity of CHCI,. To the sol was added a few grams neutral alumina (Activity III) and the CHCI, was evaporated off from the slurry. The alumina coated with the extract was then transferred to the top of a column filled with deactivated alumina (100 g) in pet ether. Gradient elution with pet ether (b.p. 65I lOTbenzene followed by benzene-ether provided the various compounds in a fair state of purity. After further chromatography on individual fractions, where necessary, the composition of the original extract was estimated to be as shown below. Compound
C B E A F D G H
Solvent composition
pet. ether/ benzene 7:3 7:3 7:3 3:7 3:7 benzene/ether 9:l 8:2 8:2
Weight recovered (mg) from 2.014 g extract
% of extract
30 440 240 1100 not isolated
1.5 22.0 12.0 55.0
30 35 20
1.5 I.7 1.0
R/ (EtOAc/ benzene)
0.64 0.57 0.45 0.39 0.30 0.21
J. P. KUTNEY. I. H. ROGERSand J. W. ROWE
3142
Clearly the major components of the extract were the compounds designated by the letters A and B. Compound A. White crystals from CHCI,, m.p. 307.5-308”. 1x1:”+ 2.8”. ORD (c. 0.02; dioxan). 21”: I$,],,,,, o”, Ql,,, +46”, Ic#~l,, +9l”. 141, + 137’7 id],, +274”; v,,: 3550 (OH). 1100 (OMe) and 795 (trisubstituted double bond); UV. no absorption: NMR (65’ with external TMS): 4.6 I
I
(m, IH, oiefinic H), 6.46 (m. IH. H-C-OH).
6.58 (s. 3H, OMe). and 9.00-9.27
(23.8H. MC).
Mass spectrum (MS9): fragments at tn/e 441 (M-15). 438 (M-18) and 423 (M-33): 3 I6 and 269 (retro Diels-Alder); 221,220, 189 (allylic cleavage); other intense fragments at m/e 187, I47 and 135. (Found: C, 81.52; H, 11.48; 0. 7.01; Mol. wt. (MS9) 456. Calc. for C,,H,,O,: C. 81.08; H. 11.37;0,7.51%; Mol. wt. 456. High resolution mass measurement 456.398; Calc. for Cr,H,,O, 456.397. OMe (Zeisel) 7.01; Calc. for 10 Me 680%). Compound R White crystals from EtOH, m.p. 276277.5”. Ial:’ -554”; ORD (c, 002; MeOH), 21°: 141,, - 128’. I@I,,, -260’. 141,, -347”. \I$!,~ -433 , ty,,oo - 1290’; v,,,: 3650 (OH, weak). I103 (OMe), and 795 (trisubstituted double bond). UV. no absorption. NMR (100 MC/S): 4.71 (m. IH, olefinic H), 6-57 (t. J=2.5 I &-C-OMe).
c/s, IH,H-{OH),
6.72 (s, 3H, OMe), 7.25 (t. J=2.5
c/s. IH,
and 9.10-9.34 (21 .OH. Me). Mass spectrum (MS9): fragments at m/e 454 (M-2), 438
(M-18). 424 (M-32): 316, 284. 269 (retro Diels-Alder): 221. 220. 190, 189 (allylic cleavage); other intense fragments at m/e 187, 147 and 135. (Found: C, 8104; H, 11.37; 0, 764; Mol. wt (MS9) 456. Calc. for C,,H,,O,: C. 81.08; H. 11.37; 0, 7.51%; Mol. wt. 456. High resolution mass measurement 456.398; Calc. for C,,HIIO, 456.397. OMe (Zeisel) 6.97; Calc. for I OMe 6.80%). Compound B acerate. Compound B (520 mg) was treated with a mixture of dry, redistilled pyridine (IO ml) and Ac,O (IO ml) at room temp for 4 days. The reaction mixture was poured onto crushed ice and allowed to stand for 3 hr. The crude product was extracted into CHCI, (3 x 75 ml) and the combined CHCI., extracts washed with 5% HCI, 5% sodium bicarbonate and water. The recovered acetate (520 mg) was purified by column chromatography on deactivated alumina (Activity II, 50 g). Elution with pet ether-benzene (I7 : 3.500 ml) yielded the TLC pure compound (5 IO mg. 90% yield. RrCHCI, O-45). White crystals from EtOH, m.p. 205.5-207”. IaIi” -64.6”; v,,: 1733 and 1245 (OAc), 1103 0 (Me), and 792 (trisubstituted double bond); NMR (IO0 MC/S): 4.65 (m, IH, oletinic H). 5.28 (t, J=2.5 c/s. IH, H-- C!-0Ac).
6.70 (s. 3H. OMe). 7.23 (t, J=2.5
c/s. IH, H-
A -0Me).
7.94 (s, 3H. OAc).
I 9.07-9.30 (20.3H. Me). Mass spectrum (MS9): fragments at m/e 483 (M-15). 466 (M-32). 45 I (M-47) 438 (M-60) 423 (M-75); 3 16, 284. 269 (retro Diels-Alder); 262, 221, 203, 202, 190, 189 (allylic cleavage); other intense fragments at m/e 187 and 135. (Found: C, 79.55; H, 10.70; 0.9.80; Mol. wt. (MS9) 498. Calc. for C,,HIdO,: C, 79.46; H, 10-91; 0. 9.62%; Mol. wt. 498). Compound A ocefafe. Compound A (630 mg) was reacted as described above to yield the acetate (570 mg, yield 83%. R/CHCI, 0.21). White needles from EtOH. m.p. 199-201’. 1~1: +4.5’; v,,,: 1730 and 1250 (OAc), I 105 (OMe), and 795 (trisubstituted double bond). NMR (100 MC/S): 4.64 (m. lH. olefinic
I H), 5.28 (t, J = 2.5 c/s, IH, &C-OAc), 663 (s, 3H, OMe), 7.45 (q, J, = 119 c/s J2 = 40 c/s, I I lH.H-C-OMe). 7.93 (s. 3H, OAc). and 9.02-9.28 (23.OH. Me). Mass spectrum (MS9): fragments
I at m/e 483 (M-15). 438 (M-60). and 423 (M-75); 316, 284 and 269 (retro Diels-Alder); 262, 221, 204, 203, 190. 189 (allylic cleavage); other intense fragments at m/e 187 and 135. (Found: C, 79.43; H. 10.82; 0.9.55; Mol. wt. (MS9) 498. Calc. for C,,HsdO,: C, 79.46; H, 10.92; 0,9.63%; Mol. wt. 498). Compound B ketone. Compound B (I .O g) was dissolved in dry, redistilled pyridine (25 ml). This sol was added slowly to a slurry of chromic oxide (975 mg) in dry pyridine (10 ml) and the reaction mixture was stirred for 60 hr at room temp following the procedure of Sarrett.” The sol was poured into cold water (400 ml). allowed to stand for + hr. and filtered under suction. The dark brown ppt on the suction funnel was washed thoroughly with cold water and the crude ketone was then recovered by extraction with warm CHCI, (600 ml). The sol was washed with 5% aqueous HCI and water. The crude ketone (R,
The neutral triterpenes
CHCI,
0.45)
Elution
with benzene-pet
flakea from -228”.
EtOH,
I#],,,
1707 ()C=O),
(s, 3H,
OMe),
723
Mol.
cleavage);
Compound
-715”;
) ali0
-227’.
454.
ORD
-4
ld~l,,
795 ttisubstituted
Compound
C. 81.88:
H.
11.08:
0.
- 3 I .O. Addition
Further
elution
with benzene-ethyl has m.p.
effect alter 90 min; Y,,,:
22 I. 2 18.204,203,
7.04%:
(allylic Mol.
cleavage);
lxlC
-0.
I I05 (OMe). 8.91-9.25
407 (M-47);
Mol.
a fraction
I ‘. ORD
454.
Calc.
B ketone
for CI,H,,Or:
efh_vlene kerat.
(241
mg, Rf CHCI, Vacuum
MeOH).
21’:
C. 81.88;
Compound
H.
Diels-Alder);
221.
11.08;
0.
B ketone (250
derivative
(250
III.
7.04%:
Mol.
mg. yield
92%).
-56.5”.
Y,,,,,: I I IO (OMe).
(s. 4H.
-0-CH --
9.28 (21.3H, 79.26: Mol.
H.
CH
White
0.
crystals
and 795 (trisubstituted
-0-_).
6.79
Me). Mass spectrum 11.01;
rod-like
9.61;
(s.
(MS9):
Mol.
3H.
from LAH.
fragments
wt. (MS9)
498.
ether (I 9
: I,
7.23
4.62
fragments at m/e I90 and 189
H. I I .24; 0. 6.90: wt. 454).
The reaction was carried
while the water formed during trap.
The benzene sol was
(m.
on deacti-
200 ml) yielded the TLC
from benzene.
double bond); NMR: OMe).
of
of the Cotton
mg) was reacted with ethylene glycol
by means of a Dean-Stark
20 g). Elution with benzene-pet
+ 36”.
Addition
washed with water and the recovered crude ketal was purified by column chromatography vated alumina (Activity
0.23)
l&l,,,
218. 203.
C. 82.03;
acid hydrate (40 mg) as catalyst.
continuously
I.
sublimed
double bond): NMR:
H, Me). Mass spectrum (CH4):
out for 5 hr in refluxing benzene previously dried by distillation was removed
7.18:
on alumina (Activity
(300
(c. 0.02:
at m/e 147 and 135. (Found:
(I -5 ml) in the presence of p-toluenesulfonic the condensation
190. 189
wt. 454).
m.p. 262-263”.
and 795 (trisubstituted
284 and 269 (retro
other intense fragments
wt. (CH4)
Compound
ether (4: I) yielded
270-270.5.
1712 ()C==O),
422 (M-32).
frag-
pure material (740 mg. 65% yield. R,CHCI,
A. White flakes from EtOH,
olefmic H), 6.65 (s, 3H, OMe),
439 (M-15).
(CH4):
C. 8 I .58; H. I I .23; 0.
-929”; molecular amplitude -36.8. -2002O. I#],,, + 1680°. 141,,, sol of the ketone (002 and 0-06N) had virtually no effect on the amplitude
Ir#Jl,,, 0°, l&l,,, HCl to the MeOH
(m, lH,
ether (19: I) yielded TLC
largely unreacted compound
material (250-260’/0.07)
of HCI to
A (I. 133 g) was oxidized by the Sarrett procedure as described above
100 g). Elution with benzene-ethyl containing
White l$lssg
4.63 (tn, lH, olefinic H),
to yield the crude ketone (I. 174 g) which was purified by column chromatography 0.41).
50 g).
-137’.
Mass spectrum
1H. II
for C,,H,,O,:
III.
of the Cotton effect after 90
NMR:
at n;/e 147 and 135. (Found:
(Activity
21”: )41700
amplitude
double bond);
3743
(892 mg; yield 85%).
(c, O-02; MeOHk
I06 ‘; molecular
spruce)
alumina
-0CHJ. 891-9.17 (21.7H, Me). 4 and 407 (M-47); 284 and 269 (retro Diels-Alder);
(m
Calc.
A ketone.
on deactivated
had almost no effect on the amplitude
other intense fragments
wt. (CH4)
(Sitka
1000 ml) yielded the pure material
1100 (OMe,
ments at m/e 422 (M-32) (allylic
chromatography
: 17.
sol of the ketone (0.02N)
.
mm; v,.
by column ether (3
m.p. 240-242”.
lr#~l,,, -3332”.
the MeOH
671,
was purified
of the bark of Picea sitchensis
m.p. 4.65
317-318”
pure
(dec). lzlb
(m, IH. olefinic H). 6.04
IH,
and
9.01-
at m/e 399, 221 and 99 (very intense). (Found:
Calc.
for C,,H,,O,:
C, 79.52;
H. 10.84;
C,
0. 9.64%;
wt. 498).
Compound yield
A kefone ethvlene
the crude
(Activity
ketal (278
ketai.
mg).
Compound
This
by column
I, 30 g). Elution with benzene yielded the TLC
plates from pet ether. m.p. 317-319’(dec). bond);
NMR:
OMe).
9.00-9.27
4.63
intense). (Found: 10.84:
A ketone (245 mg) was reacted as described above to
was purified
0.
Desoxy Djerassi
(22.8
IH.
Mol.
compound
olefinic
H. Me).
C. 79.33;
9.64%; ef al.”
(m.
1~1: -4-4”; H).
6.02
vnuX: I I05 (OMe)
(s. 4H,
Mass spectrum (MS9):
H. 10.94; 0. 9.91;
Mol.
chromatography
on neutral
alumina
pure derivative (180 mg. 67% yield). Colourless and 795 (trisubstituted
-O-~-JJZ-O-).
fragments
wt. (MS9)
498.
6.61
at m/e 399, 221. Calc.
double (s.
3H.
189 and 99 (very
for C,,H,,O,:
C. 79.52;
H.
wt. 498).
B. Compound
The ketone (300
B ketone tosylhydrazone
was prepared by the method described by
mg) and ptoluenesulfonhydrazide
reagent (310
mg) were reacted
together in refluxing methanol (IO ml) in the presence of three drops of acetyl chloride as catalyst.
After
+ hr heating was terminated.
the reaction flask and contents allowed to cool to room temp. and the white
crystalline
by filtration.
(dec);
product
r’m..: 3180
removed (NH).
1635 (>C=N-_).
This material 1592
and
was shown to be TLC 1490 (aromatic),
pure: m.p.
1165 (-SO,-).
257-258” and 1095
J. P. KUTNEY, I. H. ROGERS and J. W. ROWE
3144
(OMe). (Found: C, 73.81; H. 10.00: N, 4.54; S. 4.96. Calc. for C,sH,,N?O,S: C. 73.26; H. 9.38; N. 4.50; s. 5.14%). The tosylhydrazone derivative (220 mg) was reduced by NaBH, (500 mg) in moist refluxing dioxan according to the method of Caglioti and Graaelli. ” After heating for 5 hr. the dioxan was evaporated and the product partitioned between benzene (I50 ml) and water. The benzene layer was washed with water. dried. and evaporated to yield the crude desoxy compound. This substance was purified by column chromatography on deactivated alumina (Activity III. 20 g). Elution with pet ether (200 ml) yielded the TLC pure desoxy compound (115 mg. 73% yield based on the ketone, RYCHCl, 0.7 1). White crystals from EtOH, m.p. 249.5-251”. 121: -49. I; v,,,: 1100 (OMe). and 737 (trisubstituted double bond); NMR: 4.70 (m. IH. olefinic H). 6.71 (s, 3H. OMe). 7.25 (m. IH.H--
A -0Me).
and 9.09-9.34 (20.2
I H. Me). Mass spectrum (CH4): fragments at m/e 425 (M-15). 408 (M-32). and 393 (M-47); 316.284 and 269 (retro Diels-Alder); 221. 204. 190, 189 (allylic cleavage); other intense fragments at m/e 187 and 135.(Found:C.84~82;H,11~94;0.3~71;Mol.wt.(CH4)440.Calc.forC,,H,,O:C.84.54;H.l1.82; 0. 3.65%; Mol. wt. 440). Desox?, compound A. Compound A ketone (330 mg) and p-toluenesulfonhydrazide reagent (330 mg) were reacted together in reiluxing benzene for 7 hr and most of the solvent removed by evaporation. The tosylhydrazone derivative which crystallized was filtered off and recrystallized from EtOH. m.p. 234237=’(dec); I’,“,=:3210 (NH). 1635 (>C=N-).
I165 (--SO?-_),
and 1107 (OMe); NMR: 2X10-2.72
(complex. 4H. aromatic). 6.63 (s, 3H. OMe). 7.55 (s, 3H. aromatic Me). and 8.96-9.25 (Me). (Found: C. 73.19; H. 9.54; N, 4.61: S. 5.20. Calc. for C,BH,,NrSO,: C. 73.26; H. 9.38; N, 4.50; S. 5.14%). The tosylhydrazone (390 mg) was reacted with NaBH, (500 mg) in refluxing dioxan as already described to yield the crude product (277 mg) which was purified by column chromatography on alumina (Activity 1.30 g). Elution with benzene yielded the pure desoxy compound (158 mg. 55% yield based on the ketone) White needles from Et&, m.p. 267-269”. IaI$ +338”; \P”: 1107 (OMc); NMR (100 MC/S): 4.60 (m. 1H. olefinic H), 6.60 (s. 3H. OMe), 7.36 (q, J,= Il.3 c/s.
J,=4.0
c/s,
lH,H-C-
I OMe). and 9.01-9.30 (21.5 H. Me). Mass spectrum (CH4): fragments at m/e 425 (M-15) 388 (M-52): 3 16, 284. 269 (retro Diels-Alder); 221, 204. 191 and 189 (allylic cleavage); other intense fragments at m/e 184 and 135. (Found: C. 84.68; H. 11.99; 0, 3.61; Mol. wt. (CH4) 440. Calc. for C,,H!,O: C. 84.54; H. 11.82; 0. 3.64%; Mol. wt. 440). Dihydro compound B. Compound B (10.25 mg, 22.5 micromoles) was hydrogenated in a microhydrogenation apparatus previously calibrated against pure maleic acid (7 determinations). Using a suspension of Adam’s catalyst (PtO,. 5.17 mg) in glacial AcOH. a smooth uptake of HZ (25.0 micromoles) was observed. On a larger scale. compound B (360 mg) was hydrogenated under atm press in glacial AcOH sol using Adam’s catalyst (100 mg). The uptake of H, over a period of 8 hr was 17.2 cc (talc. for one double bond 17.7 cc). The hydrogenation was repeated using fresh catalyst (50 mg) to complete the reaction and the crude product was dissolved in CHCI,. washed with 5% NaHCO,aq and water. The recovered product was TLC pure (366 mg. R,CHCI, 0.30). White needles from pet ether, m.p. 21 l-216’; v,,: 3500 (OH). 1088 (OMe) and no absorption at 795 (tri-substituted double bond); NMR: no olelinic H. 6.10 (m. IH. jj-
h -OH).
I
6.70 (s. 3H. OMe), 7.25 (m. 1H. H-C-OMe).
and 9.10-9.24
(21.0
Me). Mass spectrum (MS9): fragments at m/e 456 (M-2), 440 (M-18), 426 (M-32), 411 (M-47), 393 (M-65). and 369 (M-89); no retro Diels-Alder; other fragments at m/e 221, 190, 189, 163. 150, 149 and 136. (Found:C,81.41;H, 11.48;0.7.15;Mol.wt.(MS9)458.Calc.forC,,H,,O,:C,81~l6;H. 11.86;0, 6.97%; Mol. wt. 458). Dihydro compound A. Compound A (10.54 mg, 23. I micromoles) was hydrogenates’ in a microhydrogenation apparatus at atm press using Adam’s catalyst (5.56 mg). No H, uptake was observed in EtOH sol, but use of glacial AcOH.instead resulted in the absorption of 22.3 micromoles. On a larger scale. compound A (200 mg) was hydrogenated as above using Adam’s catalyst (95 mg). After I5 hr the
The neutral triterpenes of the bark of Picen sirchensis (Sitka spruce)
3145
obaezvud H, uptake was 116 ml (CaIc 98 ml) and the reaction produd was isohtted as pkody described(R, CHC13 020). White uy&als from dioxan, mp. 288293”; Y,: 3558 (OH), 1082 0 (Me)
I and no absorption at 795 (trisubstituted double bond); NMR: no oleflnic proton, 6.59 (m, lH.H-COH), 664 (s. 3H, OMe), 9.02-9.26 (24.8 H, Me). Mass spectrum (MS9): fragments at m/e 440 (M-18). 426 (M-32), 385 (M-73). and 369 (M-89); no retro Diels-Alder; other fragments at m/e 245, 221. 189, 177, 176, 149 and 136. (Found: C, 81.29; H, 12.00; 0.6.66; Mol. wt. (MS9) 458. Calc. for C,,H,,O,: C. 81.16; H, 11.87; 0, 6.98%; Mol. wt. 458). Compound G. White crystals from EtOH. m.p. 289-291’. izlg - 12.3 ‘. Low solubility in common solventa; v, 9 354U (--OH), and 7.95 (trisuhatituted doubk bond); NMR: 4.65 (m, HI, oIcfinic H), 655 (t. lH, H-
I
-OH),
-OH),
6.80 (m. IH, jj-
and 9.04-9.32
(23-O H, Me). Mass spectrum
I (CH4): fragments at m/e 440 (M-2). 427 (M-IS), 424 (M- 18). and 409 (M-33); 302.284 and 269 (retro Diels-Alder); 221, 220. 207, 203, 190 and 189 (allylic cleavage); other intense fragments at m/e 234, 187, 149 and 135. (Found: C, 81.17; H, 11.26; 0, 7.35; Mol. wt. (CH4) 442. Calc. for &I&O,: C, 81.45; H, 11.31; 0, 7.24%; Mol. wt. 442). Compound G diacetute. A crude fraction of compound G obtained from the mother liquors during crystallization (75 mg) was acetylated as previously described using a mixture of equal volumes (10 ml) of Ac,O and dry pyridine at room temp. Isolation of the crude acetate in the usual way was followed by chromatographic separation on a column of deactivated alumina (Activity III, 10 g). Elution with benzene-pet ether (2 :3) yielded a white crystalline diacetate (45 mg) Examination on a silica chromatoplate (EtOAc-benzene, 1:4) showed the product to be pure diacetate (RfO.75. compound G R/0.41). White crystals from EtOH. m.p. 210-217’; NMR (100 MC/S): 4.65 (m. lH, olelinic H), 5.30 (t,J=2.5
I
c/s, IH, H-C-GAc),
I
5.47 (q, I, = 100, J, = 5.2 c/~ HI, H-C-GAc),
I
794 and 7098 (two
I
singlets, 6H, OAc). and 8.75-9.31 (19.6 H. Me). Mass spectrum (MS9): fragments at m/e 511 (M-15). 466 (M-60). 451 (M-75), 423 (M-103). 406 (M-120). 391 (M-135). and 363 (M-163); 203,202, 190 and 189 (allylic cleavage); and other fragments at m/e 187, 147 and 135. (Found: Mol. wt. (MS9) 526. Calc. for C,,H,,O,: Mol. wt. 526). Correlorion ofcompoundG with 2 I-episerratenediol. An authentic sample, isolated from pine bark by J. W. Rowe, was compared with compound G. White crystals, m.p. 284- 285’ (lit value 289-290“, 1~1: - 19’). Both compounds were identical on the basis of TLC, quantitative IR. NMR and mass spectral comparisons. Methyfation of compound A. A 3-necked LOOml flask was fitted with a high-speed stirrer, N, gas inlet tube, condenser with drying tube and a bopping funnel. Full precautions were taken to exclude moisture from the walls of the apparatus. Compound A (175 mg) was dissolved in warm, dry toluene (10 ml) and admitted to the reaction vessel via the dropping funnel. The apparatus was then thoroughly purged with purified grade N, gas and freshly cut K metal (300 mg) was added. The flask was warmed until the K was molten and stirring was commenced. After 2 hr. the reaction vessel was allowed to cool and an excess of pure dry Me1 (5 ml) was added from the dropping funnel. Stirring and heating were recommenced and the reaction was continued for a further 3 hr. AAer cooling, the excess K was destroyed by the addition of an excess of MeGH, the solvent was evaporated and the reaction products partitioned between CHCI, and water. The combined CHCI, extracts were washed with water, dried, and evaporated to yield the crude methyl ether. This product was purified by chromatography on a column of deactivated alumina (Activity III, 18 g). Elution with pet ether yielded first an oily liquid fraction (35 mg) and then the pure methyl ether (95 mg). Elution with benzene removed weacted compound A (50 mg). The methyl ether yielded white crystals from pet ether. m.p. 272-273’, Ial: - 16.4“; Y_: no absorption at 3500 (-OH), 1100 (OMe). and 793 (trisubstituted double bond); NMR: 4.70 (m. lH, oletinic II), 6.67 and 6.70 (two s, 6H, OMe), 7.20 (m. lH, H-
I
-0Me). 7.45 (m. lH, jj-0Me). 9.05-9.33 (21.2 H, Me). ‘i f Maas spectrum (CH4): fragments at m/e 455 (M-15). 438 (M-32) and 423 (M-47); 316, 284 and 269 (retro Diels-Alder); 221, 189 (allylic cleavage); other fragments at m/e 279, 187, 167 and 149. (Found:
J. P. KIJTNEY.I. H. ROGERS and J. W. ROWE
3146
C, 81.68; H, 11.45; 0.6.95; Mol. wt. (CH4) 470. Calc. for C,,H,,O,: C, 81.70; H, 11.49; 0.6.81%; Mol. wt. 470). Methylation of 2 1-episerratenediol and correlation with compound A. A sample of 2 1-episerratenediol (102 mg) was methylated according to the procedure outlined above using K metal (247 mg) and Me1 (5 ml). The crude product was separated on a column of deactivated alumina (Activity III, 20 g). Elution with pet ether yielded an unidentified oily liquid (45 mg). Further elution with benzene-pet ether (I : 9) removed the dimethyl ether (18 mg) and the use of benzene-pet ether (4 : 1) yielded first a fraction of a monomethyl ether (8 mg) and then a larger fraction of another monomethyl ether (43 mg). Finally elution with benzene removed some unreacted 2 I-episerratenediol (8 mg). The more plentiful monomethyl ether was purified by vacuum sublimation at 250-260”/0.05 mm to yield a white crystalline solid, m.p. 305-306’ (compound A. 304-305’). mixed m.p. 306-307’. These two compounds were identical on the basis of TLC, quantitative IR, NMR and mass spectral comparisons. The dimethyl ether derivative was also compared with compound A methyl ether. After vacuum sublimation, this compound had m.p. 277-278” (compound A methyl ether 272-273’). mixed m.p. 272-275”. These compounds also were shown to be identical by evidence similar to that used in the case of the monomethyl ether derivatives. Correlation of compound E with compound A ketone. The crude material was purified by three recrystalhxations from EtOH which gave white rod-like crystals, lap. 262-263”. 1a 1;’ - 16a)“.Vacuum sublimation raised the mp. to 270-271”. v,,. 1710 (OH), 1105 (OMc), and 795 (trisubstituted doubk bond); NMR (100 MC/S): 4.68 (m. IH, olefinic H), 6.73 (s, 3H, OMe), 7.34 (partly hidden multiplet,H-
I
-OMe), and F 8.94-9.28 (24.38. Me). (Found: C, 81.69; H, 11.78; 0.6.32; Mol. wt. (MS9) 454. Calc. for C,,H,,O,: C, 81.88; H. 11.08; 0, 7.04%; Mol. wt. 454. High resolution mass measurement 454.379. Calc. for C,,H,,02 454.381). Compound A ketone, m.p. 270-270.5”. mixed m.p. 270-271’. The compounds showed identical TLC behaviour and the quantitative IR spectra were superimposable. Dihydm compound B ketone. Dihydro compound B was oxidized with Jones’ reagent as described by Henbest” et al. The reagent was prepared by dissolving reagent grade chromic oxide (2.668 g) in cone H,SO, (2.13 ml) and diluting to 10 ml with distilled water. The dihydro compound (155 mg) was dissolved in warm acetone (30 ml) and placed in a 100 ml flask equipped with magnetic stirrer. Jones’ reagent (0.14 ml) was added and reaction allowed to proceed for 20 min. whereupon unreacted oxidising agent was destroyed by the addition of excess MeOH. The solvent was evaporated and the product partitioned between benzene and water. The benzene layer was washed with 5% Na,CO,aq, water, and dried over MgSO,. The benzene sol was run through a one inch bed of neutral silica to remove traces of chromous salt. Evaporation yielded the ketone (152 mg. R, CHCI, 0.62). White platelets from EtOH.
m.p. 167-169O; Y,, (CHCI,): 1705 ()C=O),
I
IH, H-C-OMe).
7.64 (m. -m--CO-),
and 1090 (OCH,). NMR: 6.66 (s, 3H, OMe), 7.20 (m, and 8.93-9.23
(22.4H.
Me). Mass spectrum
(MS9): fragments at m/e 441 (M-15). 424 (M-32). 409 (M-47), 385 (M-71), and 383 (M-73); prominent fragments at m/e 219, 189, 150, 149 and 136. (Found: C, 81.47; H, 11.23; 0, 7.25; Mol. wt. (MS9) 456. Calc. for C,,H,,O,: C, 81.52; H, 11.48; 0. 7.01%; Mol. wt. 456). Desoxy dihydro commnd B was prepared by three different methods: A. WoUfrKishner reduction. Dihydro B ketone (150 mg) was suspended in EtOH (15 ml) containing diethylene glycol(l.5 ml) and reacted in a 25 ml flask under reflux with hydrasine hydrate (O-75 ml) and KOH (0.14 g). After 30 min. the reagents had dissolved and the EtOH and excess hydrazine were then distilled off from an oil bath at 150”. Diethylene glycol(2 ml) was added and the oil bath temp raised to 190’. After 4 hr under reflux, the sol was cooled, poured into water (20 ml) and extracted with benzene (4 x 10 ml). The benzene extract was washed with water, dried and evaporated to yield a white crystalline solid (123 mg, 85% yield). This material was found to be TLC pure (Rr CHCI, 0.68). B. Borohydride reduction of dihydro B ketone tosylhydrazone. Dihydro B ketone (96 mg) was reacted with ptoluenesulfonhydrazide reagent (90 mg) in dry refluxing benzene and the water formed was removed by azeotropic distillation. After 3 hr the reaction was stopped and the product purified by chromatography on a neutral silica column (Activity 1. 15 g). Elution with benzene yielded a mixture of
The neutral triterpenes of the bark of Piceu sirchensis (Sitka spruce)
3747
unreacted ketone and tosylhydrazone (45 mg). Further elution with CHCI, yielded almost pure tosylhydrazone (113 mg) which was leached with a little cold MeOH and dried in racuo, m.p. 21 l-212” (dec); v,, (CHCI,): 3300 (NH), 1605 (aromatic), and 1085 (OMe); NMR: 2.10-2.75 (complex, 4H, aromatic), 670 (s, 3H, OMe), 722 (m, IH, jj
-Me),
758 (s, 3H, aromatic
Me), 92)8-9.28
--+
(overlapping signals, Me). In a second experiment, dihydro B ketone (600 mg) was reacted as above with ptoluenesulfonhydrazide reagent (600 mg) for 4 hr in benzene under reflux to yield a TLC pure sample of the tosylhydrazone. This product was reduced with NaBH, (I.70 g) in dioxan (55 ml). After IO hr under reflux. the solvent was removed in ~IXUOand the product recovered as previously described. Column chromatography on neutral silica (Activity I, 65 g) yielded the desoxy dihydro compound (380 mg, 66% yield). The desoxy dihydro compound B samples prepared in these two experiments, when recrystallized from EtOAc, could both be mechanically separated by hand sorting into platelet-type crystals, m.p. 188-194’. and needle-like crystals, m.p. 2 15-223’. The platelet crystals, which predominated in each of the two reduction experiments, gave superimposable quantitative IR spectra. Examination by VPC using a 20% silicone gum rubber (S.E. 30) column showed both samples to be mixtures of two components in the ratio 4: 1 with the corresponding pairs having identical retention times on the column. C. Hydrogenation of desoxy compound B. Attempts to hydrogenate desoxy compound B with Adam’s catalyst at atmos press in AcOH or AcOH-THF (I : I) sol were unsuccessful. The hydrogenation was successfully accomplished in a Parr bomb at 50 p.s.i. H, press using a sol of desoxy compound B (708 mg) in AcOH-CHCI, (I : I. 140 ml) with Adam’s catalyst (320 mg). After 3 hr. the catalyst was removed by filtration and the product recovered by evaporation of the solvent under reduced press. Examination by TLC (silica, CHCI,) showed that hydrogenation had occurred (yellow spot, starting material brown, similar R, value). The reaction product was purified by column chromatography on alumina (Activity I, 50 g). Elution with pet ether (b.p. 65-l IO’) removed dihydro desoxy compound B (687 mg, 97% yield). White prisms from pet ether, m.p. 195-198”. Examination by VPC under conditions above gave only one peak; v,,: I 100 (OMe), no absorption at 795 (trisubstituted double bond); NMR: no olegnic proton,
I
6.67 (s, 3H OMe), 7.20 (m, lH, H-C-OMe)
and 9.06-9.21 (Me) Mass spectrum (CH4): fragments
at m/e 440 (M-2). 427 (M-15). 410 (M-32). and 395 (M-47); other intense fragments at 272, 221, 190, 189, 177, 163, 150, 149 and 136. (Found: C, 83.96; H. 12.22; 0,3.68; Mol. wt. (CH4) 442. Calc. for C,,H,,O: C, 84.16; H, 12.22; 0, 3.62%; Mol. wt. 442). Ether clearage of desoxy dihydro compound B (44% HBr/AcOH). Desoxy dihydro compound B (370 mg) was reacted at room temp for 20 hr in a mixture of saturated HBr in AcOH (44%, 40 ml) and Ac,O (40 ml). All the material had dissolved before the reaction was terminated by pouring onto crushed ice. After standing for 2 hr. the product was extracted into CHCI,, washed with 5% KHCO,aq, water, and dried. The recovered product was a brown syrup containing some semi-crystalline material. This mixture was separated into two main fractions by column chromatography on deactivated alumina (Activity III, 36 g). Elution with pet ether yielded a colourless syrup (158 mg) which was apparently olefinic and showed two components when examined by TLC (AgNOJsilica, pet ether). Further elution with pet ether-CHCI, (9: I) yielded a white crystalline solid (137 mg); v,, (CHCI,): 1730 and 1230 (OAc), absence of Me (I 100): NMR: 5.36 (m. IH. H-
-0Ac). 7.92 (s, 3H. OAc). and 9.10-9.21 (Me). ! Further information was obtained on the olefin fraction as follows. NMR: 4.64 and 4.80 (weak m. olefinic H), 8.76 and 9.14-9.18 (Me). An examination by VPC using as liquid phase S.E. 30 gum rubber (20%) indicated the presence of at least 4 components which were not further examined. The acetate fraction was treated with LAH (500 mg) in dry refluxing THF for 4 hr and the excess reducing agent decomposed by the controlled addition of wet THF. Examination of the white crystalline product by TLC (silica, CHCI,) showed it to contain 3 components (R/0.55,0.48 and 0.28, yellow). The mixture was separated by column chromatography on deactivated alumina (Activity III, 20 g). Elution with pet ether-benzene (I 7 : 3) yielded first a mixture of the two less polar alcohols (50 mg) and then a pure fraction of the second alcohol (59 mg, alcohol B). Further elution with pet ether-benzene (I : I) yielded the third component (8 mg) which upon examination by TLC (silica, CHCI,) was seen to contain two compounds in the approximate ratio I :4. An attempt to separate the mixed fraction containing the
3148
J. P. KUTNEY.I. H. ROGERSand J. W. ROWE
of alcohols by further column chromatography on deactivated alumina (Activity III) was largely unsuccessful. The pure alcohol B, m.p. 219-221 O,was further examined as follows. Y,,,: 3560 (OH); NMR (100 lint pair
MC/S): 6.66 (triplet, J=3.0
c/s. 1H. H-
I
-OH) and 9.02-9.24 (23.7 H, Me). Mass spectrum F (MS9): fragments at m/e 413 (M-15), 410(M-18). and 395 (M-33); most intense fragments above m/e 100 occurred in the order 121, 136, 123, 109. 107, 137 and 149; other significant peaks were observed at 189, 190, 207, 231 and 259. (Found: Mol. wt. 428.400. Calc. for C&,0: Mol. wt. 428402). /ones oxidation 0/0koh0l B. Alcohol B (50 mg) was dissolved in warm spectral grade acetone (20 ml) in a flask equipped with a magnetic stirrer. Freshly prepared Jones reagent (0.3 ml) was added and the reaction was terminated after 20 min by the addition of reagent grade MeOH (5 ml). The crude ketone was recovered in the usual way and purified by column chromatography on deactivated alumina (Activity III, 10 g), yield, 44 mg. white crystals. m.p. 185-193” without recrystallization: ORD (c. 0.02, MeOH). 216: L@l,uoo”, tt$l,,, -40’. IRIS,, +2867“, h#~l,,, -2509O. 141, - 13%; molecular amplitude + 53.7. Addition of HCI to the MeOH sol of the ketone (0.05N) had almost no effect on the amplitude of the Cotton effect aRer 90 min; v,,:
no OH absorption,
1695 (X0);
NMR (100 MC/S): 7.52
(m. 2H, -m-CO-) and 8.89-9.13 (23.7 H, Me). Mass spectrum (CH4): fragment at m/e 411 (M-15); other important fragments at 274, 232, 205, 138 and 123. (Found: Mol. wt. 426.391. Calc. for C,H,,O: Mol. wt. 426.386). Borohydride reduction of ketone B rosylhydrazone. A sample of ketone B (30 mg) was reacted with tosylhydrazine reagent (3 I mg) in MeOH (3 ml) in a 25 ml flask quipped with reflux condenser. Acetyl chloride (3 drops) was used as catalyst and reaction was allowed to take place for 45 min under rAux. The white crystalline tosylhydrazone derivative was filtered off from the MeOH after cooling. The air 1590 (aromatic), and 1162 (dried material had m.p. 202-204’; v,,,: 3200 (NH), 1635 (C=N-), SO,-). The tosylhydrazone was transferred to a 50 ml flask equipped with reflux condenser. Sodium borohydride (55 mg) and spectral grade dioxan (15 ml) were added and reduction allowed to proceed for 10 hr under reflux. The solvent was evaporated and the reaction product taken up in CHCI,. The CHCI, extract was washed with water, dried and evaporated to yield a white crystalline solid (23 mg). Examination by TLC (silica, pet ether) showed mainly one component present. This was recovered by column chromatography from alumina (Activity I, 10 g). Elution with pet ether yielded a white crystalline solid (8 mg). This compound was recrystallized from spectral grade acetone. White leaf-like crystals, m.p. 184-186’. Authentic Bserratane (Inubushi), m.p. 184-187’. mixed m.p. 184-186“. The compounds showed identical TLC behaviour on both silica (R, 0.70, ginger) and alumina plates developed with pet ether. However, a-serratane could not be distinguished from p-serratane by TLC. Mass spectrum (CH4): fragment at m/e 397 (M-15); major fragments at 231, 192, 191. 163, 137 and 123. (Found: Mol. wt. 412408. Calc. for C,,,H,,: Mol. wt. 412.407). Ether clearage of desoxy dihydro compound B (36% HBr/ACOH~fsolution of Al-b-serratane. Dihydro desoxy compound B (590 mg) was suspended in a mixture of freshly prepared 36% HBr in glacial ACOH (50 ml) and Ac,O (50 ml). ARer stirring for 2.5 hr at 70-80’. the reaction mixture was poured onto crushed ice, allowed to stand for 2 hr and extracted into pet ether (b.p. 30-600). The sol was washed twice with water, dried over anhyd K&O,, filtered and evaporated to recover a semi-crystalline material with a foul odor. Examination by TLC (silica, CHCI,) showed the presence of two fractions (R, 0.80, brown; 0.49, brown). The mixture was separated by column chromatography on alumina (Activity I, 25 g). Elution with pet ether removed an olefinic fraction (457 mg). Further elution with benzene removed a mixed fraction (34 mg) containing olefinic and unreacted starting material. Finally, an acetate fraction (97 mg) was recovered by elution with benzene-ether (4 : 1); v,, (CHCI,): 17 IO and 1250 (OAc). no OMe (1100). This fraction was seen to contain polar impurities when examined by TLC and was not further studied. The olefin fraction was examined on a AgNO,/silica thin layer. Using pet ether as developing solvent, 6 spots (R/O-67, 0.61, 0.55. 0.47.0.42 and 0.33) were seen. The olefin mixture was separated on a column packed with AgNO, impregnated silica (20% AgNO,, 50 g). Elution with pet ether (b.p. 65-llO”, 25 ml fractions) removed all the material applied in 13 fractions according to the Table below. A white crystalline material was isolated in fractions 8 to 10.
The neutral triterpenes of the bark of Picea sitchensis (Sitka spruce) Fraction No.
Weight (mg)
R, Value of components
18 121 35 8 68 100 32 10
0.69 0.69 and 0.63 0.63 and 0.58 0.51 0.42 0.39 0.39 0.39
3149
Fractions 8-10 were bulked and rechromatographed on AgNO, impregnated silica (15 g). The main fraction from the second chromatography (67 mg) was recrystallized three times from spectral grade acetone. white needles, m.p. 188-191’; v,,: 1650,730 and 720 (cis disubstituted double bond), no OAc or OMe; NMR (100 MC/S) benzene: 4.57 and 460 (complex m, 2H. olefinic H), 7.85 (t, J= 2.0 c/s, IH, allylic H), 8.04 (m. IH, allylic H). and 9.01-9.13 (21.4 H. Me). Irradiation at r 8.04 caused the alteration of this region to two rather wide peaks of equal height. Alternately, irradiation at I 4.58 caused a simplification of the signals at 7.90 and 8.04 attributed to the allylic protons. The spectrum was also run in CDCI, sol and this permitted slightly better resolution of the oh&tic proton region which now showed a narrow triplet (J= 1.0 c/s) centred at 4.62 and an apparent singlet at 4.65. Mass spectrum (CH4): fragments observed at m/e 395 (M-15). 355 (M-55) and 328 (M-82); other prominent fragments at m/e 191. 189. 150, 137 and 123. (Found: C, 87.90; H. 12.43; Mol. wt. 410.392. Calc. for C&H,,: C. 87.80; H, 12.20%; Mol. wt. 410.391). Hydrogenation of A’-&serratune. A sample of AJ-/.l-serratane (38 mg) was hydrogenated in EtOAc sol (30 ml) in the presence of Adam’s catalyst (17 mg) for 2 hr. The recovered product was purified by chromatography on a short column of deactivated alumina (Activity III, 10 g). Elution with pet ether yielded a white solid (26 mg) which after recrystalliztion from acetone gave needlelike crystals, m.p. 175-180’. Vacuum sublimation raised the m.p. to 181-184’. The 8-serratane obtained from the previous ether cleavage reaction sequence (ex acetate) had m.p. 182-185”; mixed m.p. 181-184”. The two samples were identical on the basis of TLC. quantitative IR, NMR and mass spectral comparisons. (Found: C, 87.20; H. 12.65; Mol. wt. 412.407. Calc. for CrOHSz:C, 87.37; H. 12.62%; Mol. wt. 412.407). Isolation of compound C. Attempts to isolate pure compound C from mixtures with B by means of preparative scale thin layer or column chromatography were rather unrewarding because of the very similar R, values. A TLC pure sample of the compound (526 mg) was, however. obtained by column chromatography on deactivated alumina (Activity III. 1000 g) of a sample (20.638 g) of the total acetone soluble motha liquor after the removal of the main crystalline triterpeme fraction from the ortginal bark extra The eluting solvent used was pet ether-benzm (4:l). White needles from n-heptane, m.p. 194 195°~Izl’uD +23.l.v, _,: 3400 (OH. strong). 1IOU(OMe) and no trisubstituted double bond (795); NMR l
(100 MC/S): no olefinic proton, 6.54 (t. 5=2.5 5=2.5
c/s, IH, H-
I
c/s, IH, &-
-OH),
6.67 (s, 3H, OMe). 7.22 (1,
f
-OMe), and 9.04-9.24 (Me). Mass spectrum (CH4): fragments at m/e 454 ‘i (M-2). 441 (M-15). 438 (M-18). 424 (M-32), 409 (M-47) and 391 (M-65); no retro Diels-Alder; prominent fragments at m/e 236, 221. 203. 190, 189. 187, 147 and 135. (Found: Mol. wt. 456.395. Calc. for C,,H,,O>: Mol. wt. 456.398). Acefykxion of compound C. Compound C (108 mg) was acetylated in the usual way and the crude acetate (109 mg) was examined by TLC (silica. CHCI,, R, 0.53; compare compound C, 0.40). The product was chromatographed on deactivated alumina (Activity III, 20 g). Elution with pet etherbenzene (17:3) yielded the pure acetate (70 mg). Colourless rods from pet ether, m.p. 222-225’; v_: 1725 and 1250 (OAc). 1100 (OMe) and no trisubstituted double bond (795): NMR: 5.31 (m. IH.
jj-
I
-0Ac).
6.70 (s. 3H. OMe). 7.25 (m. IH. H-C-OMe).
7.95 (s. OAc). and 8.67-9.18
J. P. KUTNEY,I. H. ROGERSand J. W. ROWE
3750
Mass spectrum (MS9): fragments at m/e 483 (M-15), 466 (M-32), 452 (M-47) 438 (M-60), 432 (M-75). 406 (M-92) and 391 (M-107); no retro Diels-Alder; prominent fragments at m/e 255,221, 203, 190, 189, 187,147 and 135. (Found: Mol. wt. (MS9) 498. Calc for CssH,,Os: Mol. wt. 498). Acid isomerizatfon of compound E. This was performed according to a method described by Inubushi er ~1.~Compound B acetate (365 mg) was dissolved in AcOH (50 ml) and cooled to 0’. After the addition of a mixture of cone H,SO, (3 ml) in AcOH (4 ml), reaction was allowed to proceed 24 hr at room temp. The product was poured into a large excess of cold water, extracted into CHCI,, washed with 5% Na,CO,aq, water, and dried. Chromatography on a column of neutral alumina (Activity III) yielded a white solid (288 mg, pet ether eluant). This was deacetylated by treatment with excess powdered LAH in refluxing anhyd benzene for 3 hr. Workup of the reaction in the usual manner followed by passing the product through a column of neutral alumina yielded 220 mg of a mixture of iso-compound B and unreactcd compound B. The above mixture was separated by preparative TLC on three silica plates (20 x 20 cm) prepared in the usual fashion, except that a 0.1% aqueous sol of Rhodamine B was used to slurry the silica.” The plates were developed with CHCI, and the two bands could be clearly seen tier evaporation of the solvent. The less polar band was recovered from the plates, extracted with hot Ccl,, filtered through a bed of celite and evaporated to yield TLC pure iso-compound B (86 mg). White needles from pet ether, m.p. 190-192”; Y,,: 3400 (OH), 1095 (OMe), and no trisubstituted double bond (795); NMR; no
I
olefinic proton, 6.68 (m. lH, H-C-OH),
6-70 (s, 3H, OMe), 7.24 (m, lH, H-
I
c: -0Me)
and
I
9.05 to 9.26 (Me). Mass spectrum (MS9): fragments at m/e 441 (M-15) 438 (M-18). 424 (M-32), 409 (M-47) and 391 (M-65); no rktro Diels-Alder; prominent fragments at 236,221,204,203, 190,189,161, 149 and 135. (Found: Mol. wt. 456.394. Calc. for C,,H,,O,: 456.396). Correlation of compound C wifh iso-compund B. The compounds behaved identically when compared by TLC in two solvent systems. The quantitative IR spectra (KBr discs) were superimposable. Compound C, m.p. 194-195’; iso-compound B, m.p. 190-192’; mixture m.p. 190-194”. Properties of compound D. This product was isolated in a crude form in the chromatographic separation of the triterpene extract, but all attempts to purify the material by further column chromatography were unavailing. White crystals from EtOH, m.p. 245-252”; ORD (c. 0.02, MeOH), 21’: L$l,, -231’. ($~l,,, -231’. \@I,,, -2168’. 14),,, +461”, [b!,,, -2906’. Molecular amplitude -262;
v,:
3Mo (OH), 1707 (>c-o),
1387 and 1370 (geminal dimethyl) and 797 (trisubstituted double
I
--OH), 894-9.25 (22.6 H, Me). Mass Spec--? trum (CH4): fragments observed at m/e 425 (M-15). 422 (M-18). and 407 (M-33; no retro Diels-Alder; 218, 207, 203 and 189 (allylic cleavage); other prominent fragments at 177, 149, 147, 138 and 135. (Found: Mol. wt. 440.369. Caic. for C,,H,,O,: Mol. wt. 440.365). No further work has been performed due to the lack of sufficient material at this time. Properlies ofcompoundH. This product was also a minor component of the triterpene extract. The crude fraction (152 mg) as previously isolated was chromatographed a second time on a column of deactivated alumina (Activity III). The recovered material (59 mg) was recrystallized from EtOH, m.p. 256-270’; v,,,_: 3400-3300 (OH), 1387 and 1365 (geminal dimethyl) and 795 (trisubstituted double bond); NMR (100 MC/S): 4.72 (m, lH, olefinic H), 6.79 and 6.90 (overlapping t, J=S.O c/s, 2H,Hbond); NMR:465 (tn, HI, olefmic H), 684 (m, HI, B
A -OH),
8.78 and 9.06-9.35 (Me). Mass spectrum (MS9): fragments at m/e 427 (M-15). 424 (M-18).
I 409 (M-33). 406 (M-36) and 391 (M-5 I); 220,207 and 189 (allylic cleavage); other prominent fragments at 187, 147, 135, 127 and 121. (Found: C, 8066; I-I, 12-03; 0,750; Mol. wt. 442.380. Calc. for C,,Hs,Os: C, 81.45; H, 11.31; 0, 724%; MoL wt 442.381). Obviously this compound was impure and no further work was done owing to lack of material at this time. REFERENCES I Y. Inubushi, T. Sano and Y. Tsuda, Tetrahedron Leften 1303 (1964). ’ Y. Tsuda, T. Sane, K. Kawaguchi and Y. Inubushi, Ibid. 1279 (1964).
The neutral triter-penes of the bark of Piceu sifchensis
(Sitka spruce)
3751
’ Y. Inubushi, Y. Tsuda, T. Sane, T. Konita, S. Suzuki, H. Ageta and Y. Otake, Chem. Pharm. Bull. Japan IS, 1153 (1967). ’ J. W. Rowe, Tetrahedron Letters 2347 (1964). ’ J. W. Rowe and C. L. Bower, Ibid. 2745 (1965). 6 For leading refs, consult M. Martin-Smith and G. Subramanian, Phytochemistry 6,559 (1967); see also T. A. Bryce, M. Martin-Smith, G. Osske, K. Schreiber and G. Subramanian, Tetrahedron 23, 1283 (1967). ’ G. I. Poos. G. E. Arth. R. E. Beyler and L. H. Sarett. J. Am. Chem. SOC. 75,422 (1953). * C. Djerassi, 0. Halpem, V. Halpem and B. Riniker, Ibid. 80, 4001 (1958). ’ F. N. Lahey and M. V. Leeding, Proc. Chem. Sot. 342 (1958). ” C. Djerassi, L. A. Mitscher and B. J. Mitscher, J. Am. Chem. Sot. 81, 947 (1959). ” H. Audier, J. Bottin, A. Diara. M. Fetizon, P. Foy, M. Golfier and W. Vetter, Bull. Sot. Chim. Fr. 2292 (1964). ” Z. Pelah, D. H. Williams, H. Budzikiewicz and C. Djerassi, J. Am. Chem. Sot. 86,3722 (1964). ” L. Caglioti and P. Grasselli, Chem & Ind. 153 (1964). ” M. Fischer, Z. Pelah, D. H. Williams and C. Djerassi, Chem. Ber. 98, 3236 (1965). ” T. Obara and S. Abe. J. Chem. Sot. Jupn SO, 1487 (1959). I6 Y. Inubushi, Y. Tsuda, T. Sano and R. Nakagawa, Chem. Pharm. Bull. Japan 13, 104 (1965). ” P. Bladon, J. M. Fabian, H. B. Henbest, H. P. Koch and G. W. Wood, J. Chem. Sot. 2402 (195 1). ” S. Matsunaga, J. Okada and S. Uyeo, Chem. Comm. No. 21, 525 (1965); S. Uyco, J. Okada, S. Matsunaga and J. W. Rowe, Tetrahedron Zr, 2859 (1968). ” G. M. L. Cragg, C. W. Davey, D. N. Hall, G. D. Meakins, E. E. Richards and T. L. Whateley, J. Chem. Sot. (C), 1266 (1966). lo C. Djerassi, R. D. H. Murray and R. Villotti, J. Chem. Sot. 1160 (1965). ” J. Avigan, D. S. Goodman and D. Steinberg, J. Lfpid Res. 4, 100 (1963). ” J. P. Kutney, G. Eigendorf and I. H. Rogers, Tetrahedron 25, (1969).