Cytotoxic C-Benzylated Flavonoids from Uvaria chamae in methanol for 24 h. The filtrate and washes were evaporated to dryness: yield 77 mg, 36% by weight; 127 wmol Val, 7.7 pmol Gly (yield 32%).Total 14C counts recovered in the filtrate indicated a yield of 36%. The solid was recrystallized from ether-petroleum ether. Paper electrophoresis at pH 1.5 gave a single radioactive spot at Rval 0.48 (standards of Gly-Val,Asp, and cyclic Dca-Val appeared at 0.86,0.74, and 0.66, respectively); ‘H NMR (Varian HF, 220 MHz, MezSO-de) 6 0.91 (m, 6 H), 1.3-1.4 (broad m, cyclohexylaxial), 1.6-1.8 (broad m, cyclohexyl equatorial), 2.06 (m, 1 H,Val p-CH), 3.1 (m, tertiary CH), 4.1-4.2 (m,3 H, a-CH),3.8 (~,3H,OCH3),7.32 (d,2H, J = 8 H z , c y clohexyl NH), 7.68 (t, 1 H, J = 7 Hz, Gly NH), 8.64 (d, 1H, J =: 8 Hz, Val XH); I3C NMR (Bruker HX-90, 22.6 MHz, proton decoupled, Me2SO) (Me4Si 6 0) 6 19.1. 19.8 (Val,C.,), 25.5,33.2,51.6(cyclohexyl), 31.0 (Val Cp),44.8 (Gly C,,), 54.4 (OCHa),58.7 (Val CJ, 155 (Guan), 167.8 (Gly C=O), 169.7 (Val C=O). I3C assignments were based on the standards, cyclohexylamine, Gly-Val, and Arg.HC1.
Acknowledgment. We wish to thank Mr. Arun Dhundale for his assistance during the preliminary phase of this work.
J . Org. Chem., Vol. 42, No. 8,1977
1295
References and N o t e s Reported in part at the 170th National Meeting of the American Chemical Society, Chicago, Ill., Aug 1975;the work was supported by Grants AM 01260 and HL 12157 from the U S . Public Health Service and by a grant from the Hoffmann-La Roche Foundation. Abbreviations: [ “CIDCC, [ 1-”C dicyclohexylcarbodiimide; BOC,tertbutyloxycarbonyl; Lys(2,4-CI2Z), -2,4-dlchlorobenzyloxycarbonyllysine; Tos, ptoluenesulfonyl; DIEA, diisopropylethylamine; Res, 200-400 mesh resin beads of a 1 % cross-linkedcopolymer of styrene and divinylbenrene; Dca, N,N‘dicyclohexylamidino; TFA, trifluoroacetic acld TLC, thin layer chromatography. Other nomenclature and symbols follow the Tentatlve Rules of the IUPAC-IUB Commission on Biochemical Nomenclature, J. Blol. Chem., 241, 2491 (1966);242, 555 (1967);247, 977 (1972). R. B. Merrifleld, J. Am. Chem. SOC.,85, 2149 (1963). B. W.Erickson and R. 8. Merrifield in ”The Proteins”, Vol. II, 3rd ed, H. Neurath and R. H. Hili, Ed., Academic Press, New York, N.Y., 1976,pp
ryB
255-527. B. F. Gisin, Anal. Chim. Acta, 58, 248 (1972). R. S.Hcdges and R. B. Merrifield, Anal. Blochem., 65, 241 (1975). H. G. Khorana, Chem. Rev., 55, 145 (1953). I. Muramatsu, T. Hirabayashi, and A. Hagitani, Nlppon Kagaku Zasshl, 84, 855 (1963);Chem. Abstr., 80,12100c (1964). D. F. DeTar and R. Silverstein, J. Am. Chem. SOC., 88, 1013 (1966). D. F. DeTar and R. Silverstein, J. Am. Chem. Soc., 88, 1020 (1966). D. F. DeTar, R. Silverstein, and F. F. Rogers, Jr., J. Am. Chem. SOC., 88,
1024 (1966). F. Kurzer and K. Douraghi-Zadeh, Chem. Rev., 87, 107 (1967). H. Rink and B. Riniker, Helv. Chlm. Acta, 57, 831 (1974). Registry No.-IV, 5874-68-0;V, 61364-31-6;VI, 61348-54-7;VII, R. B. Merrifleldand M. A. Corigliano, unpublished results. G. Amiard and R. Hevmes. Bull. Soc.Chim. Fr.. 1360 (1956). 57944-26-0; VIII, 61364-32-7; DCC, 538-75-0; Gly-OEt HCl, 623-33-6; ---, (16jG. Amiard, R. HeymesandL. Velluz, U.S. Patent 2 797240 (June 25,1957); Gly HC1,6000-43-7;Gly, 56-40-6;Boc-Gly, 4530-20-5;Dca-Gly HCI, Chem. Abstr., 52, 4263 (1958). 61348-85-8;DIEA, 7087-68-5;[14C]urea,594-05-8;cyclohexylamine, (17)R. B. Merrifield, reported at the International Symposium on Peptide Syn108-91-8; l-cyclohexyl-2-cyclohexylamino-4-isopropyl-4,5-dithesis, Madison, Wis., May 30,1973. hydro-5-imidazolone, 61348-56-9; Val HCl, 17498-50-9; [14C]Dca(18)B. W. Erickson and R. B. Merrifield, J. Am. Chem. SOC., 95, 3757
.
Gly-Val, 61348-57-0; Gly-Val HCl, 61348-59-2; Dca-Gly-Val HC1, 61348-60-5; Boc-Ala, 15761-38-3; Ala-Gly-Val HCl, 61348-58-1; Boc-Leu, 13139-15-6; Boc-Ile, 13139-16-7; Boc-Val, 13734-41-3; Boc-Gly-Val,28334-73-8;Boc-Ile-Val,61348-61-6;Boc-Ala-Gly-Val, 56133-97-2;Boc-Leu-Ala-Gly-Val,61165-83-1.
(1973). (19)Recent Improvements in monitoring procedures have led to a marked reduction in the observed picrate titration of this peptide.
(20)R. H. Andreatta and H. Rink, Helv. Chlm. Acta, 58, 1205 (1973). (21)R. B. Merrifield, A. R. Mitchell, and J. E. Clarke, J. Org. Chem., 39, 660 (1974).
Cytotoxic C-Benzylated Flavonoids from Uvaria chamae William L. Lasswell, Jr., and Charles D. Hufford* Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, Mississippi 38677 Received September 13,1976 Ethanolic extracts of Uvaria chamae have demonstrated activity in vivo against P-388 lymphocytic leukemia in the mouse and in vitro against cells derived from human carcinoma of the nasopharynx (KB). Fractionation of these extracts yielded the known flavanones pinocembrin (1) and pinostrobin (2), the C-benzylated flavanones chamanetin (3), isochamanetin (4), and dichamanetin (5), and the C-benzylated dihydrochalcones uvaretin (6), isouvaretin (7). and diuvaretin (8).The structures were established by spectroscopic methods, chemical synthesis, and degradations. I n a previous communication2 t h e isolation and structure elucidation of t h e cytotoxic C-benzylated flavanones chamanetin3 (3) and isochamanetin (4) from t h e stem bark of Uvaria chamae were reported. We now wish to describe t h e total structure determination of these compounds. In addition, we wish t o describe the isolation and structure elucidation of t h e known flavanone pinocembrin (1) and t h e dibenzylated flavanone dichamanetin (5) isolated from stem bark extracts as well as t h e known flavanone pinostrobin (2).and C-benzylated dihydrochalcones uvaretin (6), isouvaretin (7), and diuvaretin (8), which were isolated from root bark extracts. Cytotoxicity5 residing in ethanolic extracts of the stem bark was concentrated in the ethyl acetate fraction of an ethyl acetate-water partition. Silicic acid chromatography of this fraction starting with initial eluent benzene followed by ether-benzene mixtures resulted in the isolation of four flavanones (1,3,4, and 5).
Scheme I
1
2
Compound 1 demonstrated UV, IR, and 1H NMR6 data consistent with a 5,7-dihydroxylated flavanone lacking B-ring’ substituents. Comparison of the isolated product with an authentic sample of pinocembrin verified structure 1. Based on spectroscopic evidence (1H NMR data in Table I) chamanetin (3) and isochamanetin (4) were designated as isomeric o -hydroxybenzyl derivatives of pinocembrin.2 Support for these assignments followed from their formation of
1296 J. Org. Chem., Vol. 42, No. 8,1977
Lasswell and Hufford
Table I.
lH NMR Spectra of Flavanonesa
Comwd
H-2b
480-39-7
1
480-37-5
2
5.53, dd (3.5,g.B) 5.50, dd (3.9,10.2) 5.60, dd (4.5,10.7) 5.68, dd (3.5,g.B) 5.65, dd (3.9,9.2)
Reeistrv no.
3
4 58779-09-2
5
H-3' 2.67-3.60, (14.2) 2.70-3.50, (14.6) 2.50-3.50, (16.5) 2.67-3.60, (14.2) 2.60-3.60, (13.7)
Ca-OH
H-6
H-8
Misc
m
14.00
5.98
5.98
7.30-7.80,d m
m
13.90
6.13
6.13
m
12.60
6.10
m
13.33
m
13.13
7.50d 3.87e 6.50-7.33,d m 3.91f 6.67-7.50,d m 4.00f 6.50-7.80,d m 3.97f
6.26
a Values are in parts per million in acetoned6 solution. Multiplicities are indicated by the usual symbols: dd, double doublet; m, multiplet. Unmarked signals are singlets. Figures in parentheses are coupling constants in hertz. Center of the X portion of an ABX AB portion of an ABX system, JAB is in parentheses. The aromatic signals , system; parentheses include JAXand J B X respectively. in this category represent (1)the four B-ring aromatic protons for 1 and 2, (2) the nine B-ring and o-hydroxybenzyl aromatic protons for 3 and 4, and (3) the 13 B-ring and o-hydroxybenzyl aromatic protons for 5. e C7-OCH3. f ArCHzAr (two protons in 3 and 4;four protons in 5 ) .
Scheme I1
-
HO
KOH, EtOH
0
HO
OH 0
OH 0
OH O 4
9
3
0
Scheme I11
BF,.Et,O ____+
3+4+
dioxane,
"
O
R OH ? 0
+hydroxy-
HO
benzyl alcohol
5
1
t h e same dihydrochalcone (9) upon catalytic hydrogenation (Scheme 11) and their synthesis from pinocembrin and o hydroxybenzyl alcohol using boron trifluoride etherate in dioxane (Scheme 111). T h e dibenzylated product from this reaction was found t o correspond t o dichamanetin (51, a Cdibenzylated flavanone also isolated from stem bark extracts. T h e absolute stereochemistry in 3,4,and 5 follows from CD data which allows assignment of the 2 s configuration.2v8 Treatment of chamanetin (3)with ethereal diazomethane (Scheme IV) yielded four products: a monomethyl ether 3a, two dimethyl ethers 3b and 3c, and a trimethyl ether 3d. Treatment of isochamanetin (4)with ethereal diazomethane (Scheme V) yielded the dimethyl ethers 4a and 4b9 and the trimethyl ether 4c. Table I1 lists the spectroscopic data supporting the indicated methoxyl assignments in the methyl ethers of 3 and 4. The synthesis of chamanetin and isochamanetin established them as isomeric C-benzylated flavanones. However, placement of t h e o-hydroxybenzyl substituent a t t h e C-8 and C-6 positions of 3 and 4, respectively, depended upon t h e interrelation of certain methyl ethers of chamanetin and isochamanetin with methyl ether derivatives of the dihydrochalcones uvaretin (6) and isouvaretin (7) which were isolated from root bark extracts.
Scheme IV CHzNz
3-
CH30
0 OH 0
OH 0
3a
3b
OCH, 0 3c
OCH, 0 3d
J . Org.Chem., Vol. 42, No. 8,1977
Cytotoxic C-Benzylated Flavonoids from Uvaria chamae
1297
Scheme V
4a
4b
4c
Scheme VI
6
w
7a
7
Table 11. Spectral Data for 3,4, and Their Methyl Ethers ~
" NMR" Registry no.
Compd
Cs-OH
61462-95-1 61462-96-2 61462-97-3 61462-98-4 61462-99-5 61477-75-6 61463-00-1 61463-01-2 61463-02-3
3 3a 3b 3c 3d 4
12.60 13.50 12.70
4a 4b 4c
13.33 12.53
uv NaOAc shiftb
IR carbonyl band,' cm-1
+36 d d d d +28 d d d
1630 1630 1640 1662 1680 1630 1640 1678 1670
a All signals are singlets. The presence of a low-field, exchangeable signal indicates the presence of a hydroxyl group at C-5 which is hydrogen bonded to the carbonyl function.6 b Shift values refer to the 290-nm band (band 11) in the UV spectrum. A shift of -30 nm indicates a C-7 hydroxyl group.6 Carbonyl bands of unusually low value (
