Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

HIDEYUKI ITO

1999 Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

HIDEYUKI ITO

1999 Studies on the Highly Oxidized Ellagitannins in Medicinal Plants

Contents

Introduction

Chapter I. Geraniin-Related Ellagitannins 5

1-1. Ellagitannins from Phyllanthus flexuosus 6 1- 1-1. Extraction and Isolation 1-1-2. Structures of Phyllanthusiins A - E

1-2. Ellagitannins from Geranium thunbergii 20 1-2-1. Extraction and Isolation 1-2-2. Structures of Geraniinic acids 8 and C, and Phyllanthusiin F

1-3. Ellagitannins from Acalypha hispida 28 1-2-1. Extraction and Isolation

1-2-2. Structures of Acalyphidins MI, M2 and DI

1-4. Geraniin-Related Ellagitannin monomers in Euphorbiaceae and Their Chemotaxonomical Significance 39

Chapter II. Ellagitannins Having a Gluconic Acid Core 44

II-I. Ellagitannins from Shepherdia argentea 44 11-1-1. Extraction and Isolation II -1-2. Structures of Shephagenins A and 8

11-2. Ellagitannins from Elaeagnus umbellata 49 11-2-1. Extraction and Isolation 11-2-2. Structures of Elaeagnatins A-G

-1- Introduction 11-3 . Taxonomical Significance of Ellagitannins Having A Gluconic Acid Core 61 A large group of polyphenolic compounds, "Tannins", are widely distributed in vegetable

11-4. Biogenesis of C-glucosidic Ellagitannin Dimers 62 kingdom, which are often encountered in our lives, being contained in tea, red wine, fruits, beverages and various medical plants. They are characterized as polyphenols possessing an Chapter III. Biological Activities of Highly Oxidized Ellagitannins astringent taste and ability to fonn colored solutions and precipitate with iron and other metals, and Related Polyphenols 66 in addition to binding properties with proteins such as albumin, gelatin and collagen as well as

III- I. Anti-Tumor Activity of Tannins and Related Polyphenols 66 alkaloids. The definition of tannins is proposed to be natural polyphenols with the aoove chemi­

III- I - I. In vitro assay; Inhibitory Effect on Epstein-Barr Virus 1 cal properties of molecular weights up to 500. ) Early Antigen Activation Early studies in tannin chemistry focused mostly on the characterization of components of 111 - 1-2. In vivo assay; Inhibitory Effect in Two-Stage Carcinogenesis Assay plant extracts used in the leather industry.2) On the other hand, numerous tannin-rich plants

have been used as folk medicines (hemostatics, antidiarrheic, diuretic, antiseptic, etc) and as Antibacterial Activity of Tannins and Related Polyphenols 111 -2. 2 consumed food and beverages in Asia, Europe, North America and Africa. , 3) However, the against Helicobacter pylori 71 chemistry of tannins in medical plants had been little investigated until 1970, because of diffi­

culty in the isolation of intractable, unstable, hardly separable tannins with closely related struc- Concluding Remarks 76 tures .•

Nevertheless, the number of reports dealing with the isolation and structural elucidation of Experimental 79 this class of natural polyphenols from medicinal plants and foods has increased markedly during Acknow ledgemen ts 94 the last two decades. The remarkable progress in the chemistry of tannins has been mostly

based on developments of modern analytical and isolation techniques and various spectroscopies References 95 including high-field NMR, CD, and FAB-MS.4)

List of Compounds 99 Generally, tannins are traditionally classified into two large groups, hydrolyzable tannins and

S condensed tannins. ) These names are based on their hydrolysis and condensation occurring in

the presence of acid or enzyme. The former with structural variation is further sub-divided into

gallotannins, ellagitannins and their oxidized metabolites (dehydroellagitannins), C-glucosidic

7 tannins,6) and complex tannins ), the last among which are composed of both hydrolyzable tan-

-1- -ii - nin and flavan units. Among those various types of hydrolyzable tannins characterized to date, On the other hand, although most of these ellagitannins have glucose core as a sugar unit in the ellagitannins have been attracted a considerable attentions because of their vast structural diver­ molecule, those having a gluconic acid residue as a polyalcohol have rarely been encountered in

8 sity and biological activities specific to structures ). Various biological activities including an­ nature. These ellagitannins have been hitherto found only in four plants IHippopha e rhamnoides l2 )

3 tioxidant, antiviral and antitumor activities were noticed and reported for different types of (Elaeagnaceae), Lagerstroemia speciosa, 13) L. subcostatal ) (Lythraceae) and Punica grana­

9 l4 ellagitanni ns. ) tum ) (Punicaceae)I, but their biological activity are not known at all. Among these plant fami ­

Among these ellagitannins having various biological acti vities, the oral administration of lies, the author has focused on the elaeagnaceous plants which have little been studied for tannin geraniin and the geraniin-containing extract from Geranium thunbergii to rats was found to constituents. Consequently, nine new ellagitannins with highly oxidized structures have been reduce the lipid peroxide concentration in serum and liver, which was raised by feeding the isolated from Shepherdia argentea and Elaeagnus umbel/ata, and their structures have been animals with peroxidized corn oil. 10) The levels of serum cholesterol, GOT, and GPT were also elucidated on the basis of spectral and chemical evidence. Five among them were ellagitannins

lowered. Ellagic acid, which exists in nature as a component unit of ellagitannins, was also based on the gluconic acid core, and the other four were C-glucosidic ellagitannin dimers which

found to show a potent anti-carcinogenic activity. I I) are regarded as the metabolites biogenetically produced by an intermolecular oxidative cou­

Geraniin is characterized by having a hydrated dehydrohexahydroxydiphenoyl (DHHDP) group pI i ng between monomers.

in the molecule, and is classified as dehydoellagitannins. The DHHDP group might originate This dissertation deals with the investigation on the isolation and structure elucidation of new

biogenetically from a hexahydroxydiphenoyl (HHDP) group that is a common constituent of 17 ellagitannins with highly oxidized structures from the five plant species in three families ellagitannins. The reactive DHHDP group might be further metabolized into various related (Euphorbiaceae, Geraniaceae and Elaeagnaceae). In addition, the biological activities such as acyl groups to lead a large number of mcxlified dehydroellagitannins that show various biologi­ in vitro and in vivo anti -tumor promoting effects and antibacterial activity against Helicobacter cal activities. Chebulagic acid is one of the typical example of modified dehydroellagitannin pylori have also been investigated for the tannins and related polyphenols obtained from the

probably derived from geraniin, and it was reported to exibit an anti-tumor activity and a potent above five plant species.

inhibitory effect against DNA-topoisomerase II. Geraniin and related compounds are consid­

ered to be metabolized in an animal, and then those metabolites may be activators or indirectly

act as mediators. In order to secure positive evidence of a contribution of these ellagitannins to

various biological activities, accumulation of much more new findings on structure-activity

relationships of this class of tannins would be needed. From this viewpoint, the author tried to

search for new tannins focusing on highly oxidized ellagitannins in nature and to estimate their

biological acti vity. The plants investigated in this study were (l) Phyllanthus flexuosus, (2)

Acalypha hispida belonging to Euphorbiaceae and (3) Geranium thunbergii (Geraniaceae).

-2- -3- Chapter I. Geraniin-Related Ellagitannins

Geraniin (1), a yellow crystalline dehydroellagitannin, was originally isolated from an official

anti-diarrheic in Japan, Geranium thunbergii (Geraniaceae) 15) and from Acer species

(Aceraceae).16) Its wide distribution in Euphorbiaceae as well as Geraniaceae was reported in 17 1982. ) Recently, its structure including absol ute configuration was unambiguously substanti­

ated by X-ray analysis, 18) which was the first example of chrystallographic analysis of free

ellagitannin. A large number of hydrolyzable tannins related with geraniin has revealed its

wider distribution in species of Cercidiphyllaceae, 16) Elaeocarpaceae, 19) Melastomataceae,20)

Rosaceae,21) Coriariaceae,22) Simaroubaceae, 16) and Betulaceae.23 )

The dehydroellagitannins and their metabolites have been found in the genera Euphorbia,

Mallotus, and Macaranga. Among them, chebulagic acid (2) is often found coexisting with a

major tannin, geraniin, in those genera, which could be producible by benzylic acid rearrange­

ment-like cleavage of cyclohexenetrione ring in the DHHDP group.8) The modified DHHDP

group and other analogues would biogenetically originate from chebuloyl group or its equiva-

lent. Geraniin is thus regarded as a key compound in the biogenesis of those modified

I dehydroellagitannins based on the C4-glucose core.

In order to survey new geraniin-related ellagitannins in nature, geraniin-rich plants I Phyllanthus

HO OHHO OH

H0-O----O-0H

co co OH H g / O-C-o-0H 731 0 -OH o 0 \ , co co co\ coI O~OH~ If O~QHO -- - ~OH HO~~~OHo OH 0 OH a b

-4- -5- flexuosus, Acalypha hispida (Euphorbiaceae) and Geranium thunbergii (Geraniaceae) I have been 1-1-1. Extraction and Isolation investigated and several new tannins with highly oxidized structures have been isolated.

The fresh leaves of P. flexuosus were extracted with ether and acetone to remove triterpene

1-1. Ellagitannins from Phyllanthusjlexuosus and other lipids. The residual materials were then extracted with aqueous acetone and filtered.

The filtrate was extracted successively with ether, CH2CI 2 and n-BuOH. The n-8uOH soluble

The plants belonging to the genus Phyllanthus (Euphorbiaceae) consist of approximately 600 portion was applied to repeated chromatography over Dia-ion HP-20, Toyopearl HW-40, MCI­

species, which are widely distributed throughout tropical and subtropical countries. This genus gel CHP-20P and Sephadex LH-20 to afford five new ellagitannins and a related polyphenol,

contains species that have useful medicinal applications?'+) A considerable number of these named phyllanthusiins A (3), B (4), C (5), D (6) and E (7), together with eight known polyphe-

species were previously examined and biological active constituents were reported. For ex­ ample, the antineoplastic bisabolene glycosides,25) phyllanthoside and phyllanthostatins 1,2 ~ and 3 2-.+,26) were isolated from P. accuminatus. Geraniin and related-polyphenols were isolated cti-fR

9 : R=H as inhibitors against angiotensin-converting enzyme (ACE) from "Paraparai mi", P. niruri27) 9a : R=Me which has traditionally been used for the treatment of urolitic disease and as a diuretic in Para- ~Rl-fR RO~OR guay. g% riR Phyllanthus flexuosus (Sieb. et Zucc.) Muell.-Arg. is a common shrub of the Central Japan- H2~c[nJ8-Q-OR R OR Himalayan geographic region.28) Bergenin and some terpenoids including several new triterpenes OR' OR 10 : R=R'=H and a diterpene were previously isolated from the bark of this plant. 13,29-34) Occurrence of 10a : R=R'=Me 10b : R=Me, R'=H bergenin, which is structurally related to hydrolyzable tannins, in this species, and the previous ~H1-fH finding of a wide distribution of geraniin in Euphorbiaceous plants, prompted to investigate f-O~OH g% OH other polyphenols in the leaves of this plant. The author has isolated thirteen polyphenols, Htc(nJ8-<>H including four new ellagitannins (phyllanthusiins A, 8, C and D) with highly oxidized structures R OH OR' OR and a new related polyphenol (phyllanthusiin E).

OH

2 12

-6- -7- nols, bergenin (8);~5) brevifolincarboxylic acid (9),36,37) corilagin (10), \5) chebulagic acid (2), 2/0-4 on the glucose core differs from that in 1.

38) repandusinic acid A (11),39) putranjivain A (12)40) and geraniinic acid B (13)41) (The struc- Besides the signals due to the corilagin moiety in the 'H-NMR spectrum of 3, mutually coupled ture of 13 which was first isolated as a new ellagitannin from Geranium thunbergii will be methine 103.46 (br d, 1=7.0 Hz)] and methylene 103.20 (dd, 1=7.0, 16.5 Hz); 0 3.20 (br d, described in section 1-2-2.). The known tannins were identified by direct comparison with au- 1= 16.5 Hz)1 protons, and an aromatic proton (07.17, 1H, s), which were attributable to those of thentic specimens or by comparison of their physicochemical data with those reported in the the 0-2/0-4 acyl group, were observed. The 13C_NMR spectrum of 3 showed fourteen carbon literature. resonances (four carbonyl carbons, eight Sp2 and two Sp3 carbons) due to the 0-2/0-4 acyl moi­

ety (Table 1-2). Among seven carbonyl carbons in total, two were assignable to a lactone (0

1-1-2. Structures of Phyllanthusiins A (3)-E (7) 162.2) and a carboxyl (0 169.3) carbon, based on the comparison with the B-ring carbons of

geraniinic acid B (13) (see 1-2-2). The chemical shifts of the A-ring carbon resonances were

The new compounds. except for phyllanthusiin E (7), were shown to be ellagitannins by the analogous to those of 13. These NMR features indicated that 3 was the regioisomer of 13

42 characteristic coloration with sodium nitrite and acetic acid reagent ) on thin-layer chroma- concerning the double bond in the 0-2/0-4 acyl group. tography (TLC). Methylation of 3 with diazomethane afforded a tridecamethylate (3a) that showed mlz 1134

(Mf ion peak in the positive electron impact mass (EI-MS) spectrum. Upon methanolysis with

Structure of Phyllanthusiin A (3) sodium methoxide in methanol, 3a yielded methyl tri-O-methylgallate (14) and dimethyl

hexamethoxydiphenate (IS). However, the product arisen from the 0-2/0-4 acyl moiety was

Phyllanthusiin A (3) was obtained as a pale yellow amorphous powder. The negative fast­ not detected as an outstanding spot on TLC. The linkage mode of the 0-2/0-4 acyl group with atom bombardment mass (FAB-MS) spectrum of 3 showed an (M-HY ion peak at mlz 951,

RO ORRO OR 7" indicating its molecular formula to be C4 ,H 280 2 The 'H-NMR spectrum of 3 showed a 2H OMe RO-Q---Q-OR singlet (07.14) and two I H singlets (07.05 and 6.66), ascribable to a galloyl and a HHDP group, Meooc-Q--OMe rgo cQ) OR OMe HC·/0 O-C~OR 14 I s , o~ respectively. The presence of a fully acylated C4 glucopyranose core was demonstrated by the -0 --./ ~ OR

• P1-J o 20~ coupling pattern and chemical shifts of the aliphatic proton signals, wHich were assigned by the I I CO CO ,. 7 A ~~ OR 'H_'H shift correlation spectroscopy (COSY) spectrum. These sugar proton signals were very MeOOC COOMe 15 similar to those of geraniin (1) (Table I-I). The 13 C_NMR spectrum of 3 was also closely similar 3 : R=H to those of 1, except for the signals due to the DHHDP group in 1, indicating that 3 has a 3a : R=Me -COLOC corilagin (10) moiety as a partial structure. In fact, partial hydrolysis of 3 in hot water yielded Fig. I-I. COLOC Correlations of 3 10. Phyllanthusiin A was thus assumed to be an ellagitannin in which only the acyl group at 0-

-8- -9- Tahlc I-I . IH-NMR Spectral Data of 1 and 3-61500 MHz. acetonc-d" + 0,0 (J in Hz)\

La"1 tbal 3 4 5 6

Glucose 6.SS (br s) H-I 6.60 (br 5) 6.60 (br 5) 6.30 (br s) 6.44 (br s) 6.37 (br s) 5.S4 (br s) H-2 5.60 (br 5) 5.60 (br 5) 5.42 (br s) 5.32 (br s) 5.54 (br s) 5.49 (br s) H-3 5.50 (br s) 5.60 (br 5) 5.64 (br s) 6.12 (br s) 5.57 (br s) 5.41 (br s) H-4 5.56 (br s) 5.46 (br s) 5.23 (br s) 5.15 (brs) 5.35 (br 5) 4.82 (br t, )=7.S) H-5 4.81 (m) 4.81 (m) 4.55 (br t, )=8.5) 4.76 (br t, )=8.5) 4.88 (br t, )=7.5) 4.75 (t, )= 10.5) H-6 4.93 (t,)=II) 4.78 (m) 4.79 (t, )= 11.5) 4.75 (t, )= 11.5) 4.83 (t, )= I 0) 4.33 (dd, )=8, II) 4.45 (dd, )=6,9) 4.31 (dd, )=8.5, 11.5) 4.32 (dd, J=8.5, 11.5) 4.32 (dd, )=7.5, 10) 4.38 (dd, J=7.5, 10.5)

Galloyl 7.15 (2H,s) H-2,6 7.22 (2H, 5) 7.15 (2H, s) 7.14 (2H, s) 7.14(2H, s) 7.13 (2H, s)

HHDP 7.05 (I H, s) H-3 7.13(IH,s) 7.08 (I H, 5) 7.05 (I H, 5) 7.05 (I H, s) 7.06 (l H, s) 6.64 (I H, s) 6.63(IH,s) 0 H-3' 6.71 (IH, s) 6.69 (l H, s) 6.66 (I H, 5) 6.58(IH,s) I A -ring 7.20 (I H, s) H-3' 7.25 (l H, 5) 7.28 (1 H, s) 7.17(IH,s) 7.12(lH,s) 7.06 (I H, s)

B-ring 4.89 (I H, d, J= 1.5) H-I" 5.16(IH,s) 4.72 (I H, d, J= 1.5) 4.S9 (I H, s) H-2" 5.52 (I H, d, )=2) H-3" 6.56 (I H, s) 6.26 (1 H, d, )=1.5) 4.43 (I H, d, )=2) 2.36 (I H, dd, J=6.5, 12) 6.28 (I H, d, J= I.S) 2.20 (l H, t, J= 12) H-4" 3.46 (1 H, br d, )=7) 4.S8 (I H, dd, )=6.5, 12) H-S" 3.20(lH,brdd,J=7,16.5) 3.26(IH,d,)=17) 3.09 (I H, br d,)= 16.S) 3.16 (I H, d,)= 17) 3.46 (I H, d, J=IS.5) H-7" 2.95 (I H, d, )=IS.5) 2.17 (3H, 5) H-9"

a) 400 MHz

~ '--"" to ~, :r: C1 C1 ;l n n n 'l n n n~· nnn'lnnr"J nrJnnn~ n'lnnnrJ~ 0- 00000000 00~' I I I I I I I ::3 0000~00a I I I I I 0 (b -\Ooo__JO\lfl~'-.NN-r:ro __J 0'1 'J) ~ '_.N N - 'ij Q.,V..l:.~N~O 8 o :: : :: :: :: :: :: :: :: ~ 0:: ~ +: ~ ~ -:00 ~~~~~~~ __J+-~...,.J~-~ v: , 3: lfl 0\ ~ N :r: .!" ------~ 0- o\\o\o\ONlfl~ 0'1 +:-'_.N+:---­ O\~'..;J~ON- O'I~'..;J~-N­ O\'-.N~-N O'I__J 0'10\0'1 \0 , '--"" lflNo\-oo+:-O\ lfl '_.N 00 lfl '_.N \0 lfl 00 lfl 0'1 lfl 00 lfl V"I O'Ilfl__J~O+:>'__J .p\Olfl-O '..;JNo\'-.N\OO z I V)VtN~o,o,N ~~OO~~O'N i.N6~~6~N :...-:...-~~O'OON ~~io6i.N ~~6~iooo ~::. 3: ~ - ;;0 ---- C/:J ~ c:: g ~ en o\\o\o\oN~Vt O'I~'-.N~--N O'I~(.;..)+:>'ON­ 0'1 +:>. '..;J +:>. - N - O\'_.N+:>'-N O'I__JO\O\__J\O lflNNlfllfl\oN ~:__l:__l:--l~:__l9 oolflO'llflooVllfl 0'1 v, __J ~ 0 +:>. 0\ ~\Ov'oo (.,)(.;..)O\NO- [ ::l ~~~66NO NN'-.NO\lfl-- NNVt~NVt6 :"'-6~NN~io Oo~iooow ~i.Nio~VtOO -0;: o g. ~ ~ en S ~ o :3 ------+, ~ O\O\'-.N+:-N+:-o\ O'I+:-'_.N+:--N­ O'I+:>''-.N+:>'ON­ 0'1 +:>. '..;J +:>. - N - O\'-.N+:--N O\__J 0\0\ 0'1 \0 ~ (") NN-o\ON\o lfllfloo~-O__J OOVlO'llflOOVllfl O'IVl__J~O~O'I +:>'\00\00 +:-'_.NO\ '_.N \0- ~ -::l ~0,600~ 6W~N~~ 2- wN:"'-io'gN~ N~~~N~~ ~N~~6~N i.N6~~~Ooio Q. c:: ~ !:: .!: ~ :3 ~ ::l I ~ .,~ ------~ __J__J~__Jlfloo-..l O\lfl~~--­ O'I~(.;..)+:>.ON- O'I~'-.N~-N­ 0\ '-.N ~ - N O\__J O'IO\__J \0 -N(.;..)__JO\-N lflOVl__JN__JO'I 00 lfl 0'1 lfl 00 Vl lfl O'I~__J~O~O\ +>.\OVlOO ~(.,)__J-O- .,. ::l Oo~:""~~~~ NOoVtwOoioOo ~N~~O~N i.N'!'~~:.,::..~~ ioo,OOo,N 00, 00 Vt 0, 00 ~ .,~ .s ~ (") =- -= 3: ::r :r: ~ N ::l r:ro ~ __J-__J~__JO\ ------O\'_.N~-N O'I__JO'IO\O'I\O l=S O'I~'-.N~--­ O'I~'-.N+:>.ON­ O\~(.;..)~-N­ ~ ~ '_.NO\lflO\oo~ oo\Olfl__J-__J\O O'IlflO'llfl__JlflV"l +:>'lfl__J~O~O'I ~\OlflOO NNO'INoo- VI 0- 5' (b Vtioo~:"::"~ :"::"~VtO:""i.NN NN~:"::"Ooo,N ~~~~~iio ~~ioOoN ~ioOVt:""Oo ::l .s ~ ('1) ~ ~ + N ------O\'-.NOlflOoo\oN~Vl O\~'-.N+:--NN O'I~'-.N~ON­ O'I~'..;J~-N­ O'I+:>'~-N O'I__JO'IO\__J\O o lflNO'IO\OO__JO'IlflN ~--J--J__J'-.NOO oolflO'lV"l__JVlV"l O'Ilfl__J+-O~__J v,OO\oo '_.N'_.NO\NO- ~ Il'g ~o~oOoioOoio~o 006~~~:"":"" ~i.N~v,io~i.N ~~00~;..;J~;-.N ~6:"'-~:"'- io:....~~~io .s ~ 13 glucose in 3 was substantiated by 'H_ C long-range COSY (COLOC) spectrum (JcH=7 Hz), in were assigned to the carboxyl carbons in the 0-2/0-4 acyl moiety as follows. In the COLOC which H-3' of the A-ring (6 7.17) was correlated with H-2 (6 5.42) of the glucose through three- spectrum of 4, one of the carboxyl carbons at 6 172.7 was correlated with both of methine bond couplings with a common ester carbonyl carbon (6 165.2). The other long-range correla­ protons (6 5.52 and 4.43) through two- and three-bond couplings. The methine proton at 6 4.43 tions are shown in Fig. 1-1 . Based on these data, the structure of phyllanthusiin A was elucidated (H-3") was also correlated with C-l' (6 116.8) and C-6' (6 150.8) of the A-ring. Similarly, the as 3. other methine proton at b 5.52 (H-2") exhibited cross peaks with the methine (b 56.7, C-3"),

quaternary (6 77.6, C-4") and A-ring C-6' carbon signals (b 150.8). Furthermore, methylene

Structure of Phyllanthusiin B (4) protons (C-5") were correlated with quaternary (C-4"), carboxyl (C-6") and ester carbonyl (B-

ring C-7") carbons (Fig. 1-2).

Phyllanthusiin B (4), a pale yellow amorphous powder, exhibited an (M-HY ion peak at mlz Methylation of 4 with dimethyl sulfate and potassium carbonate in dry acetone yielded a

I 969 in the negative FAB -MS spectrum, to indicate the molecular formula C4IH30028' The H- tridecamethyl derivative (4a) which showed the peaks at mlz 1153 (M+Hf and 1175 (M+Naf in

NMR spectrum of 4 showed close similarity to those of 4, except for the B-ring protons. Instead the FAB-MS spectrum. The 'H-NMR spectrum of 4a exhibited a 1H singlet (b 4.43) that disap- of the B-ring protons of 3, two mutually coupled methine protons 165.52,4.43 (each d, J=2 Hz)1 peared upon addition of 020. Thus, the presence of an aliphatic tertiary hydroxyl group in 4 was and isolated methylene protons Ib 3.26, 3.16 (each d, J= 17 Hz) I were observed in 4. Similarly, ascertained. the aromatic proton signals resembled those of 3 (Table I-I). The I3C-NMR spectrum of 4 in Methanolysis of 4a gave in addition to 14 and 15, a hexamethyl derivative (4b) which exhib- aliphatic carbon region showed resonances due to a methylene carbon (643.1), two methine C(A)-6' C(A)-4' C(A)-6' C(S) 4" C(A)-6\ C(S)-2"j C(S)-3" C(8)-5" carbons (6 81.6 and 56.7), and a quaternary carbon bearing an oxygen function (677.6) besides \ (ppm) II I I 'J glucose signals. Among the seven carbonyl carbon resonances, the signals at 6 172.7 and 171.8 7.0

H(GI c)- 1 >- H(Glc)-3 6.0

-t H(S)-2" HO RO HO OH 1--1 H(Glc )-2 :r 3 H(Glc)-4 TCO 7CO 30R 50 B~OB H(GI c) -5,6 H g6/ O -'C~OR H I 0 , 0 "=< .,... H(B)-3" , - 0 OR )-1° fO/'-tf ('.{:bc,o -,~o _ 08 ' H(GI c)-6 (J1 40 • 3 2 Me~OOCOH COOMe o b , -0 I W'>' ''H' '08 \ I MeOOC H"" OMe 8 rco OC T r; _ "\ ~ , ) ,. o J I \... OH1' MeOOC" " OMe , ,-- :~ ? I ,'-, H(8)-5" 6" ~ 0 OR 3.0 ,:,-,JIO ~CO ROOC t-+'" :r ' A H CO ~ '--'1/>- (I. 4b / , .:1 "\ : HOO ,.I' ,' , • 08 r 170 160 150 140 130 80 70 60 50 40 (ppm)

4 . R=H . - .... ~ COlOC 4a . R=Me

4 Fig. 1-2. COLOC Spectrum of (126 MHz, acetone-d6+020)

-12- - I3- ited an (M+Hf ion peak at mlz 457 in the FAB-MS spectrum. The IH-NMR spectrum of 4b showed an aromatic proton \67.08 (1 H, s) J, a methylene 162.89, 2.55 (each 1H, d, 1= 16.5 Hz) I and two methine 165.50,4.31 (each 1H, d, 1=1.5 Hz)1 signals. The a-configuration of benzylic

proton (H-3") of 4 was determined by nuclear Overhauser enhancement spectroscopy (NOESY)

which exhibited a remarkable NOE between the methine pro-

ton (H-3") and the anomeric proton of the glucose core (Fig. Sa 5 ~.-- 1-3) . . On the other hand, the small coupling constant (1=2 eOlOe ...... ~ NOE

Hz) between the methine protons (H-2" and 3") in the I H- Fig. 1-4. COLOC Correlations of 5 Fig. 1-5. Stereostructure and NOE Correlations of Sa NMR spectrum of 4 was indicative of their trans relation­

analysis. The IH_ and 13C-NMR spectra of 5 indicated the presence a corilagin unit, as shown in ship, ..B) establishing the f)-configuration at H-2". Based on Tables I-I and 1-2. The IH-NMR spectrum also exhibited methine proton signal [04.58 (dd, these data, the structure of phyllanthusiin B was concluded -- NOE

to be represented by the formula 4, although the absolute con- J=6.5, 12 Hz)] coupled with methylene proton signals [02.36 (dd,J=6.5, 12 Hz); 02.20 (t,J=12 Fig. 1-3. NOE Correlation of 4 figuration at C-4" remains undetermined. Hz)], and an isolated methine proton [04.59 (s)]. The 13 C-NMR spectrum showed five carbons

[0116.9 and 78.4 (quaternary carbons), 0 75.0 and 63.3 (methine carbons), and 046.6 (methyl-

Structure of Phyllanthusiin C (5) ene carbon)] in aliphatic region, which were analogous to those of the B-ring in 3 and 4. Among

these carbons, the signal at 0116.9 can be assigned to a geminal-diol or hemiacetal carbon based

The structure 5 of phyllanthusiin C was determined as follows. The molecular formula was on its chemical shift, similar to the corresponding signals of the B-ring in geraniin (la). In

determined to be C40H 30026 based on the negative FAB-MS Imlz 925 (M-HYI and the elemental addition, the resonances of C-4' and 6' of the A-ring in 5 were similar to those of phyllanthusiin

B (4), suggesting the presence of an ether bond at C-6'. The COLOC spectrum (J CH=8 Hz) of 5 RO OR RO OR0R R0-VV- revealed that one of the methylene proton signals (B-ring H-3") showed correlations with all CO CO OR Hg / O-C~OR '2J-?_o--i 0- \=( carbons of the B-ring by two- and three-bond couplings. The connectivities among the acyl r4 OR o 0 protons and glucose protons through three-bond couplings with the ester carbonyl carbon reso- \ I CO CO HO,,~t)~O R nances are illustrated in Fig. 1-4. ~rOR Hd RO Methylation of 5 with diazomethane afforded a dodecamethyl derivative (Sa), whose IH-NMR 5: R=H Sa : R=Me spectrum revealed a singlet (0 4.45) and a doublet (0 4.18) that disappeared upon addition of

-14- -15 - Structure of Phyllanthusiin D (6) HO MeO

co co OH co co OMe Hg / o- c nOMe H{i3- g-Q-OH 'J-~-0--1 0'={ ~ OH ~ OMe Phyllanthusiin 0 (6) was obtained as colorless fine needles. The IH-NMR spectrum of 6 was o 0 o 0 \ to \ to closely similar to that of geraniin (lb) having five-membered hemiacetal form at the OHHDP OH OH HO" : (I \\ HO" : fI \\ , ;; 1 \ OH , ;; I \ OMe group, except for extra signals due to an isolated methylene 16 3.46, 2.96 (each d, 1=IS.S Hz) I ~" ;; 0 OH ~", : 0 OMe Hc5' HO HeY Me6 and a methyl /6 2.17 (s) I groups in 6 (Table I-I). As mentioned above, crystalline geraniin 5 5a

readily forms an equilibrated mixture between six- and five-membered hemiacetal forms (la ~

MeO MeOOC Mea OMe Ib) of the OHHOP moiety in an aqueous acetone solution, to give a duplicated signal for each

co co OMe + o~M' proton in the 'H-NMR spectrum. However, such a phenomenon was not observed for 6. The H208 / -c~OMeO'={ -0 OMe o '3C_NMR spectrum of 6 also showed close similarity to that of except for extra three carbon pq 9a Ib OH OMe 10a resonances (0 206.4, SO.O and 32.0) attributable to a ketonic carbonyl , a methine and a methyl -

ene carbons, respectively, as shown in Table 1-2. The COLOC spectrum of 6 indicated that one HQ, CooMe CooMe COO Me COOMe ---', ,H r; '\ OMe -- ..... 6=.Q~ of the methylene protons (B-ring H-7") was correlated with a , (3-unsaturated ketone carbon [ HC{ 0 MeO OMe Ha~ through three-bond coupling, indicating that this methylene group located at S" position of the Chert 1- 1. Methylation of 5 B-ring. Methylation of 6 with dimethyl sulfate and potassium carbonate in dry acetone gave O indicating the presence of a tertiary and a secondary hydroxyl groups. On the other hand, 2°, deca-O-methylcorilagin (10a).44) Based on these data, phyllanthusiin 0 was characterized as methylation of 5 with dimethyl sulfate and potassium carbonate in dry acetone yielded deca-O­ the condensate of geraniin with acetone, as illustrated in the formula 6, which is consistent with methylcorilagin (10a)44) and methyl tri -O-methylbrevifolincarboxylate (9a). The formation of the negative FAB -MS data Imlz 991 (M-H)l

9a can be rationalized in terms of a series of reactions including a f3-elimination, keto-enol HO OHHO OH tautomerizaion and transesterification, as depicted in Chart I-I. H0VV-0H co co OH Stereochemistry of the B-ring in 5 was determined by NOESY measurement of 5a as follows. HOC: 6 / O -C ~OH 0 0 ~ 5 -0 1 OH The anomeric proton of the glucose core showed an NOE correlation with H- I" (64.74) of the 4 p,q 3 2 o 0 10'60 7'60 B-ring which also showed NOEs with a methoxy signal at 03.41 (C-S"-OMe) and a hydroxyl 3" 3' o B"" r; A '\ OH proton signal at 0 4. 18 (C-4"-OH), establishing the a-configurations for all of the functional HO ~. "OH r9H2 0 groups COMe and OH) on the B-ring, as shown in Fig. 1-4. Consequently, phyllanthusiin C was 8"C~H 3 6 assigned to the structure 5.

- 16- -17- The structure 6 deduced by the above data was verified by production of a condensate identi- Therefore, it is likely that phyllanthusiin D is an artefact formed during the extraction proce­ cal with phyllanthusiin D upon refluxing 1 in dry acetone containing trifluoroacetic acid for a dure.

week. The ROESY spectrum of 6 showed the ROE correlation between the anomeric proton of

the glucose and H- I" of the B-ring (04.89), which showed a remarkable correlation with one of Structure of Phyllanthusiin E (7)

the methylene protons (0 2.96, B-ring H-7") (Fig. 1-6). Consequently, the structure of

phyllanthusiin D was elucidated as the formula 6. Phyllanthusiin E (7), a brown amorphous powder, showed an (M-Hr ion peak at mlz 291 in the

Phyllanthusiin D may be an artifact, since it was isolated from the plant materials soaked in negative FAB-MS spectrum, corresponding to the formula C I3 HgOg. The IH-NMR spectrum of

acetone at the initial stage of extraction, and it was most likely to be produced by condensation 7 disclosed a singlet (0 7.38) and two 2H singlets (05.54 and 3.47). The presence of a carboxyl

of geraniin (1) with acetone in a weakly acidic condition. This compound was, however, not and two lactonic carbonyl (0 173.1, 163.8 and 163.3) carbons, and two methylene (0 68.8 and

produced when 1 was kept at room temperature for a few weeks in aqueous acetone that is 32.3) carbons was indicated by the 'H_13C heteronuclear shift correlation spectroscopy (HETCOR)

generally employed as the extraction solvent for tannins. Upon refluxing of 1 in an aqueous spectrum of 7. The remaining carbons were those due to eight Sp2 carbons in the '3C_NMR

acetone, compound 1 was slowly decomposed to corilagin (10) after 3 days, without formation spectrum. Based on these data and the COLOC spectrum of 7 as shown in Fig. 1-7, phyllanthusiin

of 6, by the normal phase HPLC analysis. On the other hand, 6 was reported to be isolated from E was determined as formula 7.

. I f G . h b .. 45) the aqueous acetone homogenate of cell suspension cu tures 0 eramum t un ergll.

H(8)-9"

H(B)-l" j)," J HO "hL-~l, L-t\..._

I~ F= H(Glc)-l

6 . 0 r- COLOC !! . !! F !! . o F H(B)-l" Fig. 1-7. COLOC Correlations of 7 4 . !!

• . 0 rI 3 .5 3 . 0 .. ~. ~ -- ROE 2.5 ~'~ L-~-r~,-.-~--~-r~~~~~ 7 . 0 6 . 5 6 . 0 5 . 5 5 . 0 4 . 5 4 .0 3 . 5 3 . 0 2 . 5 F2 (PPMI

Fig. 1-6. ROE Spectrum of 6 (500 MHz, acetone-d6+D20)

-18- -19- 1-2. Ellagitannins from Geranium thunbergii

HO OHHO OH HO~OH Geranium thunbergii Sieb et Zucco (Geraniaceae) which is rich in tannins has long been used co co 6 I OH OH as a remedy for intestinal disorders in Japan. In the early investigation of the leaf of this plant, H~~OO-C-o-~;,s'-o~ 0 - ["L-(' OH crystalline geraniin (1) as a major tannin constituent was isolated, and elucidated its unique OR' OR HO ~~-:> COOh o structure on the basis of spectroscopic and chemical evidence, 15) which was recently confirmed A-A' =; , 7 I I I I co co by X-ray crystallography. 18) Subsequent investigations of the leaf revealed the occurrence of COOH co HOOC H" " r; "\ OH HO·):tj-~OH three other dehydroellagitannins, furosinin (16), furosin (17) and didehydrogeraniin (18).46) These HOOC"'" - ~o OH ~XH HO HO tannins are all regarded as the metabolites of geraniin. In addition, a notable ellagitannin, 4 5 elaeocarpusin (19), which is a condensate of geraniin and ascorbic acid, was also obtained along

OH 47 ~H with geraniinic acid A (20) from water-soluble portion of the extract. ) Further investigation of _ H OH )...T0H r_ ~ ~ O~H CH~H OH co co co O-C~OH the polar fraction of the leaf has resulted in the isolation of additional new members of modified ICO OH 0 ~ °/- , I I OH 0 ~ dehydroellagitannins named geraniinic acids B (13), C (21) and phyllanthusiin F (22), along Hk!-0h ? q oc co coI coI ? q with some known tannins. oc co I I O~OH~ Ij '\ OH co co O~-;HO' - HO'~~~OHo OH 0 OH O~OH~ O~-Ij '\ OH HO' - 17 HO~~~OHo OH 0 OH 1-2-1. Extraction and Isolation 16

OH ~H A concentrated 70% aqueous acetone homogenate of the dried leaves of G. thunbergii was r_~ OH~;~HVOH co co co co o~ / OH I I extracted successively with Et 0, EtOAc and n-BuOH to give the respective extracts and the 2 Hi; /-0, ?-C-Q-OH ~ ~ 0 -OH water-soluble portion, The water-soluble extract was chromatographed over Dia-ion HP-20 and o 0 I \ oc co coI coI MCI-gel CHP-20P with aqueous MeOH to give three new ellagitannins, geraniinic acid B (13), O~OH~ Ij '\ OH O~QHO' - geraniinic acid C (21) and phyllanthusiin F (22), along with five known tannins, geraniin (1), HO'~~~OHo OH 0 OH 18 corilagin (10), phyllanthusiins B (4), C (5) and E (7).37) The known tannins were identified by direct comparison with authentic specimens.

-20- - 21- 13 HO OHHO OH unit as a partial structure of 13. These data suggested that 13 is an analog of 1. In the C_NMR HO OH HO OH 0H HO~OH H0-V-V spectrum of 13, the resonances due to two ester (or carboxyl) carbonyl carbons (6 171.1 and co co OH yO co OH ¢ / O-C~OH H1 / O-C-O-~ OH 161.2) and a methine carbon (6 80.l) signals were observed, instead of the signals attributable to H1-?_O-1 0 I-O. 0 - "==j o OH H OH OH a ketonic carbon (6 191.7), a hemiacetal carbon (696.1) and a geminal-diol carbon (692.4) in o 0 R o I co COOH HO~0~60.... 0 10 the B-ring of 1. From these NMR and MS data, 13 was presumed to have a lactone-carboxylic H b ...... ~H f ~ OH ItO _ - HO-Q--Q-OH o HO He) 0 OH HO OHHO OH acid structure in the B-ring of an acyl unit attached to 0-2/0-4. 19 20 If 13 has a dihydrocoumarin-type 6-lactone moiety as seen in chebulagic acid (2), 13 should I 1-2-2. Structures of Geraniinic acids B (13) and C (21), and Phyllanthusiin F (22) show an IR absorption band at around 1775 cm- and a significant upfield shift (ca. 6 ppm) of the

C-6' signal relative to C-4' in the 13 C_NMR, both of which are observed in 2.48) However, 13 did

Structures of Geraniinic acids B (13) and C (21) not exhibit such characterizations, to indicate that hydroxyl group at C-6' in geraniinic acid B

does not participate in lactone formation. The COLOC spectrum of 13 showed the methine

Geraniinic acid B (13) exhibited an (M+NH4f ion peak at m/z 970 in the electrospray ioniza- proton signal at c5 5.14 (B-ring H-2") correlated with two carbonyl carbon resonances at c5 171.1

1 tion mass spectrum (ESI-MS), corresponding to the molecular formula C41 H280 27' The H-NMR galloyl-H

spectrum of 13 showed a 2H singlet (6 7.19) and two I H singlets (67.06 and 6.64), due to a 1 HHDP-H

galloyl and an HHOP group, respectively. It also showed the signals of an aromatic proton at 6 / H(A)-3' '"

7.01 (s), a vinyl proton at 6 6.36 (d, J=1 Hz), and two methine protons at 65.33 (d, J=1 Hz) and H(Glc)-2 H(8)-S" 13 H(8)71311 ./H(8)-2" 65.14 (br s), besides the sugar protons characteristic of the 1C 4-glucopyranose (Table 1-8). The H(Glc)-6 H(Glc)-S H(Glc)-3 HO H(G IC)-4 " partial hydrolysis of 13 with hot water gave corilagin (10), indicating the presence of corilalgin ~ ~

:~~----~"~ , ------~~ I I ! I \ I I , ,I I I ,/ I " I I I I • ,I ,Ii i I J I " RO OR RO OR RO ORRO OR , ' , , / I I , , ' , " I /1 I, RO-Q---VOR RO-Q---VOR \ " I I I I 1 \ ,;" ,! I I \ , I , ' , I CO CO OR CO CO OR I , I I , o / O-C~ OR b / O-C~OR ,I H1-?_o--1 0- "'==j HJ-?_O~ o-"'==j H OR H OR o 0 oI 0, CO CO co CO H 3' 21 o B ~ " r; A ~ OR O ~ OR --...... -,,~~...---"--_~\--''''L~~ i I I t I J I I I o F HRO OR -:d"HAXR • t I ROOC,. 7.0 6.0 5.0 13 : R=H 21 : R=H (ppm) 13a : R=Me 21. : R=Me Fig. 1-8. IH-NMR Spectra of 13 and 21 (500 MHz, acetone-d +00) 6 2

-22- -23 - 0 (R)-hexamethoxydiphenate,15) [a]D +21 , upon methanolysis of tridecamethyl derivative (13a)

Table 1-3. 'H-NMR Spectral Data of 13 and 21 of 13. Based on these findings, the Structure of geraniinic acid B was determined as formula 13.

1500 MHz, acetone-dG + D~O (1 in Hz) I Geraniinic acid C (21) showed a pseudomolecular ion peak at m/z 970 (M+NH4) + in ESI-MS, 13 21

Glucose corresponding to the molecular formula C41~027 which is identical with that of geraniinic acid H- I 6.57 (br s) 6.67 (br s) H-2 5.32 (br s) 5.27 (br s) B (21). The spectral features (NMR, IR and CD spectra) of 21 showed close similarity to those H-3 5.36 (br s) 5.35 (br s) 5.44 (br s) 5.50 (br s) H-4 of 21. The only different point was a large coupling constant (J=6 Hz) between H-2" (6 5.63) H-5 4.61 (br dd, 1=8, 10) 4.73 (br dd, 1=8, 10) H-6 4.82 (br t, 1= II) 4.91 (brt,l=II) 4.27 (br dd, 1=8, II) 4.25 (br dd, J=8, 11) and H-3" (6 5.17) in the IH-NMR spectrum of 21. The (R)-configuration of C-3" in the B-ring

Galloyl was established by NOE experiment in a similar manner to In addition, the aUylic coupling H-2,6 7.19 (2H, s) 7.20 (2H, s) 21.

HHDP constant between H-3" and 5" was analogous with th.at of 13 «1 Hz). Thus the conformation of H-3 7.06 (I H, s) 7.10(IH,s) 6.65 (l H, s) H-3' 6.64 (1 H, s) the B-ring at 0-4 should be the same in these two compounds. A significant difference of the A-Ring H-3' 7.01 (tH, s) 6.93 (IH, s) chemical shift of H-2" signal between 13 and 21 was interpreted in term of an anisotropic effect

B-Ring of the A-ring. The proposed structure 21 for geraniinic acid C was substantiated by its methyla­ H-2" 5.14(lH. brs) 5.63 (I H. d. J=6) H-3" 5.33 (tH. d, J=l) 5.17 (I H, dd, J= 1,6) H-5" 6.36 (I H. d. J=l) 6.41 (IH,d,J=I) tion with dimethyl sulfate which afforded an expected tridecamethyl derivative (2la), together

with a byproduct, nona-O-methylcorilagin (lOb).

(B-ring C-1") and 161.2 (B-ring C-6") through three-bond couplings, indicating an n, f3-unsat­ urated &-lactone moiety in the B-ring of 13 (Fig. 1-9). The binding modes of the other acyl ...... " -- ... \ groups in 13 were also consistent with the long-range correlations in the COLOC spectrum. The \

NOESY spectrum of 13 showed an NOE between the anomeric proton of the glucose and H-3" ;..".Glc H-4 VVV' in the B-ring, establishing R-configuration at C-3", An allylic coupling (J=l Hz) was observed between H-3" and 5" in the 'H-NMR spectrum of 14 as well as Ib having a five-membered hemiacetal ring in the B-ring, to imply that the angle between C-3" - H-3" and C-5" - H-5" is ca. 90°. The trans-arrangement of H-2" and 3" in this conformation was consistent with their small coupling constants «I Hz).

Atropisomerism of the chiral HHDP was determined to be (R)-configuration by a large nega­ 13

5 44 tive Cotton effect at 225 nm (18\-1.2 x 10 ) in the circular dichroism (CD) spectrum. ) Fur- "---'~COLOC thermore, atropisomerism of the HHDP moiety in 13 was confirmed by formation of dimethyl Fig. 1-9. Stereostructure and COLOC Correlations of the 0-2/0-4 Acyl Moiety in 13

-24- -25 - Table 1-4. I H-NMR Spectral Data of 5 and 22 1500 MHz, acetone-df) + D 0 (J in Hz) 2 I Structure of Phyllanthusiin F (22) 5 22

Glucose H-I Phyllanthusiin F (22), obtained as an off-white amorphous powder, which was regarded as an 6.37 (br s) 6.21 (br s) H-2 5.54 (br s) 5.26 (br s) H-3 5.57 (br s) 4.52 (br s) analog of phyllanthusiin C (5) based on the following data. The 'H-NMR spectrum of 22~ re- H-4 5.35 (br s) 4.97 (br s) H-5 4.88 (br t, J=7.5) 4.28 (br dd, 1=6, 7) vealed a striking resemblance to those of 5, except for the lack of two I H singlet due to HHDP H-6 4.83 (t, J= 10) 4.21 (dd,1=7, II) 4.32 (dd, 1=7.5, 10) 3.96 (dd, 1=6, 11) protons and the upfield shifts of H-3 and H-6 (64.52 and 4.21 /3.96) of the glucose core relative Galloyl to the corresponding signals of 5. The ESI-MS of 22 showed an (M+NH4)+ ion peak at mlz 642, H-2,6 7.13 (2H, s) 7.IS(2H,s)

HHDP and its molecular formula was determined by high-resolution ESI-MS Imlz 642.1344 (M+NH4)+, H-3 7.06 (I H, s) H-3' 6.64(IH,s) calcd for C26H2401S+NH4 642.13061. Thus, 22 was assumed to have a structure lacking 0-3/0- A-Ring HHDP group in This assumption was substantiated by its hydrolysis with tannase yielding 6 5. H-3 ' 7.06(IH,s) 7.01 (I H, s)

22 and ellagic acid. The stereochemistry of the B-ring in 22 was established by NOE eXlPeri- B-Ring H- I" 4.59 (IH, s) 4.61 (IH, s) ment in an analogous way to that of 5. Consequently, the structure of phyllanthusiin F was H-3" 2.36 (IH, dd , 1=6.5,12) 2.34 (I H, dd, 1=7, 12) 2.20 (lH, t, J=12) 2.17 (IH, br t, 1=12) determined as 22. H-4" 4.58 (I H, dd, J=6.5, 12) 4.57 ( I H, dd, 1=7, 12)

HO OHHO OH

HO{'{--9-0H OH co co OH HOHC O-C~OH Hg / O-C~OH 'J-?-~--i 0- \=f A OH '2~?_o--i o--w.­ A OH o 0 \ I o 0 co co \ I co co Ha~OH Ho.J~)AoH ,. ~ 0 OH Hd HO . ~rOH Hd HO 22 5

-27- -26- 1-3. Ellagitannins from Acalypha hispida

Some Acalypha species of Euphorbiaceae have been used as folk medicines for treatment of HO - ", 'I ~ OH ~ /, - diarrhea and skin complaints in Southeast Asia. Acalypha hispida Burm. f. widely distributed in ~M/5 %~0 °c: ~~- HOH Asia is one of them, and its leaves have been used as a remedy for thrush and boils in China and --- ' H 4 3 2 , 0, Indonesia.49) Although the medicinal value of these plants is thought to be responsible for their R 1 : (1 'R)-DHHDP H tannin constituents, the poly phenolics in the plants have been little investigated. The author has 6 : (1'R)-Acetonyl-DHHDP isolated fifteen polyphenols including new ellagitannin monomers, named acalyphidins M, (~~) 26 and M2 (24), from the leaf extract of this plant. A geraniin dimer, designated as acalyphidin 0, (1'R)-DHHDP= (25), was also isolated as an acetonyl derivative (25a).

1-3-1. Extraction and Isolation

(1'R)-DHHDP (1'R)-Acetonyl-DHHDP= I 32 co The dried leaves of A. hispida were homogenized in 70% acetone. After concentration of the GG~H 2 0 OH homogenate, the precipitate deposited was collected by filtration and the concentrated solution G OG OG 31 was extracted with Et20, EtOAc and n-BuOH, successively. The precipitate was subjected to a

H CO-G combination of chromatography over Dia-ion HP-20, Toyopearl HW-40 and/or MCI -gel CHP- 2 O - G G-O 0 G-O~o O-G 20P, to furnish two new tannins, acalyphidins M, (23) and M2 (24), along with five known I o O-G G OOC HO HO OH OH a ~ ? AH tannins, geraniin (1), phyllanthusiin C (5), mallotusinin (26),50) euphorbins A (27) and B (28).51, H0-o-0**OH co nHOH HO HO CO co 52) The n-BuOH extract was similarly chromatographed to give a crude new ellagitannin dimer, H2 Cb/ 0 - G 'r-l:H ~_lH H0-VV0H acalyphidin D, (25) contaminated with excoecarianin (29),53) in addition to two flavonoids, tkI co co ? 9 H2C'O/O-G rutin and kaempferol 3-rutinoside,54) and four tannins, 9,37) 10,15) 29 and euphorbin 0 (30).55) (1'R)-DHHDP Penta-O-galloyl-~-D-glucose (31),56) furosin (32)15) and repandinin A (33)57) were isolated from 27 ft the EtOAc extract in the similar separation procedure. The known tannins were identified by (1'R)-DHHDP 28 direct comparison with authentic specimens or by comparison of their physicochemical data with those reported in the literature.

-28 - -29- HO HO OH OH HO OH HO OH HO -Q----9 OH HO-t(--?-OH OMe CO co co co OH MeOOC-Q-OMe H2 o- G Cd/ H~ / O- C~OH OMe MeOOC Mea OMe )-~-O-j 0 "=< 14 H OH Methylation .. HO HO OH OH o OH Methanolysis G~C I PjJ-0M' '-co HO OH HO-Q-O-Q----9 OH ~hMe o 5 A ",-7 B ~ OH MeO~OMe HO HO CO CO II _ 9a MeOOC COOMe (I1-G 15 23

(1'R)-DHHDP .. ... ' R 30 Chart 1-2. Methylation of 23 Followed by Methanolysis 29 : R={1 'R)-DHHDP 298 : R=(1'R)-Acetonyl-DHHDP hexamethoxydiphenate (15). The partial hydrolysis of 23 with hot water gave corilagin (10) and

brevifolincarboxylic acid (9), thus establishing the locations of the galloyl and HHDP groups at 1-3-2. Structures of Acalyphidins M, (23), M2 (24) and D, (25) 0-1 and 0-3/0-6, respectively, on the glucose core. The position of the brevifolincarboxyl

group at 0-4 was deduced on the basis of a significant downfield shift of the H-4 signal (65.60), Structures of Acalyphidins M, (23) and M2 (24) compared with that of corilagin (10) (64.45). Based on these data, acaJyphidin M, was deter-

mined as 23, in which the configuration of methine carbon in the brevifolincarboxyl moiety Acalyphidin M, (23) was obtained as a light brown amorphous powder, which has the molecu­

remains to be established. Acalyprndin M J (23) was isolated at the first time from a natural

lar formula C40H280 25 as indicated by the positive FAB-MS (mlz 931 (M+Nafl and the elemen­ source, although 23 has been obtained upon treatment of geraniin (1) with triethylamine in tal analysis. The 'H-NMR spectrum of 23 showed a IH singlet at 6 7.16 and two I H singlets at aceto ni tri Ie. ~ 6.84 and 6.62, corresponding to a galloyl and an HHDP group, respectively (Table 1-5). The The structure 24 for acalyphidin M2 was determined as follows. The molecular formula sugar proton signals assigned by the 'H-'H COSY spectrum were characteristic of skew boat (C4IH28027) was deduced by the FAB-MS {mlz 975 (M+Nafl and the elemental analysis. The glucopyranose. In addition, the spectrum displayed mutually coupled methine proton 164.69 IH-NMR spectrum of 24 revealed the signals due to a gaJloyl16 7.14, (2H, s)l, an HHDP /6 7.05 (dd, 1=2,8 Hz)1 and methylene proton signals I~ 3.00 (dd, 1=8, 19 Hz) and 62.69 (dd, 1=2.,19 and 6.60 (each 1H, s)1 and 'C4-glucopyranose core (Table 1-5). Partial hydrolysis of 24 with hot Hz)1 in the aliphatic proton region, which were attributable to brevifolincarboxyl moiety in 23. water yielded corilagin (10), suggesting that the corilagin unit existed in 24. In addition, the The presence of brevifolincarboxyl group in 23 was also supported by the 13 C-NMR spectra I signals due to an aromatic IH singlet (67.46), two methine r~ 5.63 and 5.16 (each d, 1=6 Hz)/ data (6 193.6, 161.3, 150. 1, 147.8, 41.6 and 37.9). Furthermore, the acyl units of 21 were and a methylene 16 3.50 and 2.87 (each d, 1=18 Hz)1 group in its IH-NMR spectrum could be confirmed by the methylation of 23 followed by methanolysis yielding methyl tri-O­ assigned to those of 0-2/0-4 acyl unit. The '3C_NMR spectrum of 24 exhibited the signaJs due methylbrevifolincarboxylate (9a) besides methyl tri-O-methylgallate (14) and dimethyl

-30- -31- Table 1-5. 'H-NMR Spectral Data of 23 and 241500 MHz, acetone-d + O (J in Hz)1 6 2° tone carbon at & 165.8, which coupled with H-2" signal (Fig. 1-10). Based on the above data, the

23 24 acyl group attached to the glucose 0-2/0-4 was assumed to contain a dilactone unit. Glucose H-I 6.22 (br d, 1=5) 6.47 (br s) Methylation of 24 with dimethyl sulfate and potasium dicarbonate in dry acetone furnished H-2 4.18 (br d, 1=5) 5.56 (br s) H-3 4.83 (br d, 1=4) 5.70 (br s) deca-O-methyJcorilagin (lOa), and a tridecamethyl derivative (24a) whose structure was estab­ HA 5.60 (br d, 1=4) 5.32 (br d, 1=3) H-5 4.69 (t, 1=8) 4.74 (m) lished by its 'H-NMR and FAB-MS Imlz 1135 (M+Na)+1 spectra. H-6 4.45 (dd, 1=8, 11) 4.74 (m) 4.27 (dd, 1=8, II) 4.36 (m) The (R)-configuration at C-3" in the 0-2/0-4 acyl group was confirmed by the flame rotating

Galloyl nuclear Overhauser enhancement spectroscopy (ROESY) spectrum showing an ROE between H-2, 6 7.16 (2H, s) 7.14 (2H, s) H-3" and the anomeric proton signal of glucose core. In addition, the configuration at C-2" was HHDP H-3, 3' 6.84(IH,s) 7.05 (IH,s) 6.62 (I H, s) 6.60 (IH, s)

A -ring H-3' 7.06 (I H, s) 7.46 (IH, s) HA' 4.69 (1 H, dd, 1=2,8) H-5' 3.00 (I H, dd, 1=8, 19) 2.69 (I H, dd, J=2, 19) : :: :: ;:::;: I I 1&3 I I I I I III I I I I I B-ring 1114 I I, I I III I I I I I I ~ I! ill II I I I H-2" 5.63 (I H, d, 1=6) ------"'C'1e5 ~J=-= .=-=-~H- - ---fi : ': ;: H-3" 7.43 (IH,s) 5.16 (IH, d, 1=6) , 1118 ..... J- I I I H-5" 3.50 (lH, d, 1=18) 1e7:===~~~1--+------f- ~~' _L~-t : " I' 2.87 (IH, d, 1=18) lee I ______I I -,------1. ------i------~t-1 170 I I ': to four carboxyl carbons, six aromatic and four aliphatic carbons (& 171.5, 167.1, 165.8, 164.9, : ~ ------H------l--1

7.5 7.0 6.5 6.0 5.5 5.0 •. 5 • .0 146.9, 142.1, 139.7, 119.2, 116.6, 114.2, 74.3, 72.9,43. I and 40.9), ascribable to those of 2,4- 3.5 3.0 ppm acyl group, in addition to the resonances of corilagin moiety. The HMBC spectrum of 24 showed the three-bond correlation of the glucose H-2 signal (& 5.56) correlated with the ester carbonyl carbon (6 164.9), which in turn was associated with the A-ring H-3' (0 7.46) through three-bond coupling. Similarly, the methylene (H-5",o 3.50 and 2.87), and the methine signals (H-3", 0

5.16) were correlated with H-4 of glucose at 0 5.32 through a common ester carbonyl carbon

HMBC resonance at 0 171.5, respectively. The methylene H-5" also exhibited a cross peak with a 24: R=H lactone carbonyl carbon (0 167.1), which in turn showed a cross peak with H-2" methine (0 248: R=Me

5.63). On the other hand, the methine H-3" showed a three-bond correlation with another lac- Fig. 1-10. HMBC Spectrum of 24 lEster Carbonyl Regionl (500 MHz, acetone-d +0 0) 6 2

- 32 - - 33- determined on the basis of cis-arrangement between H-2" and H-3" as evidenced from their Table 1-6. IH NMR Data of 25a and 6 1500 MHz, acetone-d(, + D 0 (J in Hz) I coupling constant (1=6 Hz). Based on these data, the structure of acalyphidin M2 was concluded 2 to be represented by the formula 24, aJthough the configuration at C-4" remains unconfirmed. 25a 6 Glucose-I Glucose-II

Glucose (25) Structure of Acalyphidin D) H-I 6.35 (br s) 6.52 (br s) 6.55 (br s) H-2 5.4D (br s) 5.54 (br s) 5.54 (br s) H-3 5.46 (br s) 5.46 (br s) 5.49 (br s) Acalyphidin DI (25) was established as a dimer of geraniin (1), as follows. The dimeric nature H-4 5.37 (br s) 5.37 (br s) 5.41 (br s) H-5 4.73 (br t, J=8) of 25 was shown by the retention time similar to those of euphorbin A (27) and other dimers: on 4.76 (br t, J=8) 4.82 (t, J=7.5) H-6 4.66 (t, J=IO.5) 4.66 (t, J= 10.5) 4.75 (t, J=7.5) the normal phase HPLC. This compound was extremely difficult to be purified, since it was 4.30 (dd, J=8, 10.5) 4.36 (dd, J=8, 10.5) 4.38 (dd, J=7.5, 10.5) inevitably shown to be contaminated with a small amount of other dimers by the IH-NMR Galloyl H-2,6 7.12 (2H, s) 7.15 (2H, s) analysis, in spite of a single peak in both normal- and reversed-phase HPLC. The IH-NMR HHDP H-3 7.04 (each 1H, s) 7.05 (each I H, s) spectrum of the crude acalyphidin DI showed the signals due to the two set of methine and vinyl H-3' 6.62 (each 1H, s) 6.63 (each 1H, s) proton signals, characteristic of H-l" and H-3" of the DHHDP unit as those of geraniin, suggest- Valoneoyl H-A 7.02 (each IH, s) ing the presence of two DHHDP groups forming an equilibrium mixture of five- and six-mem- H-B 6.25 (each 1H, s) H-C 7.13 (each I H, s) bered hemiacetal structures. After various trials for its purification, acalyphidin DI was finaJly Acetonyl-DHHDP A-ring H-3', 3'" obtained in a pure state as its acetonyl derivative (25a), m/z 2000 (M+NH4)+ (ESI-MS), by 7.22, 7.16 (each 1H, s) 7.20(IH,s) B-ring H-I" 4.93 (each IH, d, J=I) 4.89 (I H, d, J=1.5) I column chromatography over MCI-gel CHP-20P of crude acetone adducts. The H-NMR spec- H-I "" 4.90 (each IH, d, J=l) H-3", ]"" 6.33,6.23 (each IH, d, J=J) 6.28 (IH, d,J=1.5) H-7",7"" 3.45,3.22 25a~ 3.46 Crude fraction 2.99, 2.92 (each IH,d,J=16) 2.95 (each IH,d,J=15.5) H-9",9'''' 2.14,2.06 (each 3H, s) 2.17 (3H, s) 29a I

trum of 25a exhibited a 2H singlet (6 7. 12) and five I H singlets (6 7.13, 7.04, 7.02, 6.62 and 50°C 2 hr 6.25), corresponding to the galloyl, HHDP and valoneoyI groups. These acyl groups were con­

firmed by production of 14, 15 and trimethyl octa-O-methylvaloneate (34) upon methanolysis

of the methylated compound 26a. The chemical shifts and coupling patterns of the sugar proton

Chart J-3. Reversed-Phase HPLC Profiles Before and After Acetonyl Preparation of the signals were closely similar to those of phyllanthusiin D (6) which corresponds to the acetonyl Fraction Containing 25

-34- - 35- derivative of geraniin (1). The presence of two acetonyl DHHDP groups in 25a was also de-

duced by the comparison of the signals due to the 0-2/0-4 acyl groups with those of 6 (Table 1-

Table 1-7. I3C NMR Data of 25a and 6 (126 MHz, acetone-d + O °) 6 2 6). These data implied that 25a was a dimer of 6 in which each monomer is linked with the

25a 6 25a 6 valoneoyl group. The I3C-NMR spectrum of 25a also supported this assumption (Table 1-7).

Glucose HHDP The position and orientation of the valoneoyl group were determined by the HMBC spectrum of C-I (1 ') 91.8,92.0 91.9 C-I 117.4 117.3 a C C-2 (2') 70.1,70.4 70.4 C-2 124.3 ) 124.3 ) 25a as follows (Fig. I-II). The glucose H-6' signal (04.66) showed a correlation with an aro- C-3 (3') 62.3,62.5 62.4 C-3 110.0 110.3 C-4 (4') 66.4,66.7 66.7 C-4 144.8b) 144.7 matic proton signal (66.25) through a common ester carbonyl carbon at 6 168.3. This aromatic C-5 (5') 72.8,73.3 73.1 C-5 137.4 137.8 proton signal was assigned to H-3' of the valoneoyl group, based on the two-bond correlation C-6 (6') 93.8,63.9 62.9 C-6 144.9b) I 45.()d) Galloyl C-7 166.1 166.4 C-I 119.9 120.1 C-I' 115.4 115.3 a C C-2,6 110.5 (2C) 110.6 (2C) C-2' 125.1 ) 125.4 ) C-3,5 145 .9 (2C) 146.1 (2C) C-3' 107.8 107.9 CA 139.8 14D.O CA' 145.6 145 .5 C-7 165.1 165.3 C-5' 136.4 136.5 Valoneoyl C-6' 145.3b) 145.3 <1) C-l 116.7 C-7' 168.7 168.7 C-2 123.7a) Acetonyl-DHHDP C-3 109.9 C-I, 1" 119.9 (2C) 120.1 C-4 144.6 C-2,2" 116.7,116.8 116.8 • acetone C-5 137.3 C-3, 3" 113.2,113.4 113.4 C-6 145.0b) CA,4" 147.6, 147.7 147.7 C-7 166.3 C-5,5" 137.7, 137.3 137.4 co co ,'H'I~ C-6,6" OH C-I' 117.1 146.7, 147.0 147.0 HO "_ - C-2' 125.la) C-7,7" 164.7, 164.8 164.8 HO~ HO 0 OH H C-3' 104.8 C-I', I'" 51.5,51.8 52.0 co . co C-2',2'" 145.4 (2C)b) Methylation CA' 146.9 145.5 ,'H '1~OH followed by HO _" - C-5' 137.0 C-3',3'" 126.5, 127.0 126.9 Methanolysis 25 HO~ He' 0 OH C-6' 14S . l b) C-4',4'" 197.S, 187.8 197.8 1% CF3COOH 10 C-7' 16S.3 C-S',S'" 80.6, SO.8 80.9 j C-I" 113.8 C-6',6'" 109.6, 109.S 109.8 C-2" 143.0 C-T 7'" 51.5, SI.8 SO.O OMe HOOC H HO H C-3" 136.9 C-8',8'" 206.3,206.4 206.4 MeOOC-Q-OMB HO:Q-O ~-!J '1_ '\ OH OMe CA" 140.2 C-9',9'" 31.7,31.8 32.0 HO OH CO ~ 14 C-5" 140.9 C-IO, 10'" 165.4, 165.5 165.5 15 H2~~~OC0-QOH C-6" 110.2 N OH C-7" 163.1 OH OH 34 a)-d) Values are interchangeable. 33

Chart 1-4. Acetonyl Preparation of 25 followed by reactions of 25a

-36- -37- 58 with an ethereal phenyl carbon at b 146.9. ) Further, partial hydrolysis of25a with 1% CF3COOH 1-4. Geraniin-Related Ellagitannin monomers in Euphorbiaceae and Their gave known isomallotinic acid (35), hence providing definite evidence for binding mode of the Chemotaxonomical Significance valoneoyl group as shown in the formula (Chart 1-4). The arrows in the formula 25a that indi­ cate the HMBC correlations clarified the binding mode of other acyl groups. Geraniin (1) and related dehydroellagitannins are characterized as the tannins having a hy­

The sterochemistry of acetonyl DHHOP units of 25a was confirmed by ROE correlations of drated OHHOP group in the molecule. The OHHOP group might originate biogenetically from

2 the H- J ' (H-I ''') of the B-ring with acetonyl methylene and anomeric protons (Fig. 1-11), as HHOP group that is a common constituent of ellagitannins. , 8) If it is true, the in vivo conver-

found in ROE of 6. Consequently, the structure of the acetonyl derivative of acalyphidin 0 1 was sion of HHOP to OHHOP unit seems to occur preferably at 0-2/0-4 or 0-3/0-6 positions on the

1 represented by the formula 25a. As upon derivatization of a OHHDP group in tannin into its C4 (or skew boat) glucopyranose core, as shown by fewer examples of dehydroellagitannins acetonyl derivative, the remainder of the molecule is proved to be unaffected as exemplified by possessing a OHHOP group at 0-4/0-6 or 0-2/0-3 on the 4C 1 glucose core. The reactive DHHDP

conversion of 1 into 6,37) the structure of acalyphidin 0 1 was concluded to be 25. group might be further metabolized into various related acyl groups to lead a large number of

Although many hydrolyzable tannin dimers composed of geraniin as a constituent monomer modified dehydroellagitannins. Geraniin is thus regarded as a key compound in the biogenesis

of modified dehydroellagitannins based on the IC glucose core.2,8) unit have been isolated from various Euphorbiaceous plants, acalyphidin 0 1 is the first example 4 of a dimer composed of two moles of geraniin. The dehydroellagitannins and their metabolites have been found in the genera Euphorbia,

Mallotus, Phyllanthus and Macaranga. Among them, chebulagic acid (11) was first isolated

from an Indian traditional medicine, myrobalans (Terminaria chebula), 38,59) and its constituent

unit, chebuloyl group, is considered to be biogenetically producible by benzylic acid rearrange­

ment-like cleavage of cyclohexenetrione ring in the DHHDP group (Chart 1_5).8) The modified

~ coI coI ~ ~ ~ HO ';~CO;o~ ~.}; ~COCo HO-Q--Q-OH ----... 0 ~ _ OH --.. H~~9~OH ~ HOOC - 'I_ ~ OH HO OH HO OH o 0 HO OH ) OHHO OH HOOC OH HO OH HHDP HO - ~ t ~ ~ ~ I I ~ I I co co coI co co co --.. .- °MO"HO OH - OH O~OHHO - HOOC~O"HO - OH 0 OH 0 OH o ) 0 HMBC ROE I DHHDP Chebuloyl 258 Chart 1-5 Fig. I-II. HMBC and ROE Correlations of 25a

- 38- -39- H0I=(0H H OH HO)=(OH ~OH COOH HHHOOH H ~-h H H O ~ OH HO ~O-b- OH H H ~h 7'l_ ~- h H ~ _H0r-.(H ~cr~ OH 0 0 C:{O HO HOH HO~H 0 H~H I OH I OH o ~H OH2C - 0 0 0 ~2 0-0 OCO ~F( OH O~H 14 OH I H OH 011:1 - OR"-0:"' OR OR OH r111 I 0-0:" Repanduslnlc acid A (11): R=H R '- Repandusinic acid B (53) : R=N Mallotinin (52) R=O, R '=H ' - N R R Repanduslnin (54) : R=P H Chebulagic acid (2) R=A Mallotusinic acid (39): R=(R)-DHHDP '------y----J?1 R Elaeocarpusin (19) R=B MalloJaponin (40) : R=B o Macarinin A (44): R=(R)-DHHDP Eumaculin E (56) Putranjivain A (12) : R=C Mallotunin (41) : R=C OH H HO Macarinin B (45): A=A H~O """"OHO Phyllathusiin A (3) : R=D o HO 7 OH H _ H ~ OH -_ H0~"" _ O 'I ~ OH Phyllathusiin B (4) : R=E H o CO H Phyllathusiin C (5) : R=F 0 H H~H90H ~Rl I 0 1\/1 ,H O -HO - : OH Geraniinic acid B (13) : R=K L 7 H' -- :-. " 0 o 0 H' - Geraniinic acid C (21) . R=Q ---! H l\ h 0rt:>-" OH C ! _,;=\OH 0 fO Mallotusinin (26) : R=I ~~~ R2 Euphormisin M2 (35) . R=L )1_O-..jOCO ~OH ~ Bixanin (36). R=J Mallonin (46) : ~'=G, R2=B, R3=R4=H ~ OH Amariinic acid (37) : R=M FuroSln (17) R =G, R2=(R)-DHHDP 3_ 4_ R OH P q ~ 3 ="'6 .." 1; Granatin B (38) : R=(S)-DHHDP Chebulanin (47) : A'=G, R2=A, R =A'4:H- _H :~ j~ CO CO ~Io O~O-O:" 3 4 2 o OH H Terchebln (48): R'=R =R =G, R =( - H~H O~H- 0 _.,.H'I Suplnanin (49)' R'-A3-R4 G A2 (SjR) DHHDP 1 • I. . - - =, = -DHHDP Furosinin (16) : R=OH HO'~~">=

HO**H Hff-0 Hp OHH HO HO l\ - CO CO H'" - O _HOl\/J {S)-DHHDP = H2t~6'R LO OOR ° AH 2 - 0 OCO ~ OH 0 HO OH R oH ? ? CO CO ~'oI ~ HO ~ Cg OC -etIJ OH o H .,H 'I ~ OH _ 0 - ",H 'I ~ HO OH o = ....--"----. HO'" H - H - OH Tanannln (57) ~o OH H 6H 0 OH HO~~O ;" "O~ Aleurinin A (58) R= -CH2-C-CN ~ II ~ CH2 1= Aleurinin C (59) R= -CH2-C-CH E= ....--"----. ....--"----. II 3 ~~: 1 1 1 1 0 0 COOHCO G= ? ? ~ ~-Q;H - 'I Galloylgeraniin (60) ° HOOC 1-1" 'I H ~ OH ~" _~ H _ °,H 0 HOOO'" - R= OH H _ 4HO 0 OH 0 AA_C~'H ~ O~ 'I ~ H OH o .' ~ H - OH A O- }- Co-QOH HO'" H - -CO ~ O - - H OH ° OH H~ dH ° OH K= OH Amarulone (61)

0&~HHO-OHH HO ~ CO CO N= p= HOVC~, \ I ~ ~ H H "0 ~

H~H••• H ~ H H o h 'I ~ H ? ? HOOO" - '--v---' ~o R o Carpinusin (62) R=(R)-DHHDP Hehoscopin A (63) R=(S)-DHHDP Euphorscopin (64)

Chart 1-6 Chart 1-7

-40- -41- DHHDP groups in phyllanthusiins A-D (3-6) and other analogues would biogenetically origi-

Tabl e I 8. Geraniin Related Ell agitannin Monomers from the Euphorbiaceae nate from chabuloyl group or its equivalent. High reactivity of the DHHDP group leading to

these metabolites from 1 or 2 was recently demonstrated by some reactions of 1 with thiol

compounds such as cysteine.60)

Although this class of hydrolyzable tannins may be frequently encountered in the plants pro-

Gc raniin (1) +++++++++ +++ ++ ++++++ + + + + ducing geraniin, the Euphorbiaceous plants produce, in particular, analogues with diverse struc- Ma ll otu si ni c acid (39) ++ + +++ + Mall ojaponin (40) ++ + + + tures including dimeric ellagitannins. Monomeric hydrolyzable tannins of this type hitherto Elaeocarpusin (19) ++ + + + Mallotunin (41 ) + + Mall onin (46) + + found in the Euphorbiaceae are shown in Charts 1-6 and 1-7. Mall otu sin in (26 ) + ++ + Terchehin (48) + + + + Mall otin in (52) + Among the polyphenolic compounds, hydrolyzable tannins have been suggested to be valu- Fu ros in (17) + + ++ ++ ++ + + f-u rosinin (16 ) able chemotaxonomic markers in elucidating the systematic and evolutional relationships be­ Dide hydroge ranii n ( 18) + Amariin (56 ) + Amarul one (61 ) + cause of their structural features characteristic of some families of the dicotyledon.80, 81 ) It Amariini c acid (37 ) + Rcpandu si nic ac id A ( II) + + + + + might be true for Euphorbiaceae, especially for the herbaceous Euphorbia species, for which Repandusini c acid B (53 ) + Rcpandu sinin (54) + Ge raniinic acid B ( 13) + + considerable confusion upon identification of species by only morphological features, is often Phyllanlhusiin A (3) + Phyllanlhusiin B (4) + + 80 Phyllanthusiin C (5) + encountered. ) The geraniin-related ellagitannins are distributed in the restricted families and Chebulagic ac id (2) + + + + + + + + C' hebulanin (47) + some are very characteristic of the Euphorbiaceae. The fingerprints of tannins of this class in Supinanin (49) + Tanarinin (57) + Macarinin A (44) + each plant might thus be useful to identify the species (Table 1-8). Macarinin B (45) + Macarinin C + Macaranin A (42) + Macaranin C (43) + PUlranji vain A ( 12) + + + Euphormi sin M2 (35) + Eumaculin E (55) + Aleurinin A (58) + Al eurinin C (59) + Carpinusin (62) + + + Heliosco pin A (63) + + Galloylgeraniin (60) + 3-Desgalloyltcrchcbi n (50) + Bixanin (36) + Euphorscopin (64) + Granatin B (38) + Virganin (51) +

- 42- - 43 - Chapter II. Ellagitannins Having a Gluconic Acid Core 11-1-2. Structures of Shephagenins A (65) and B (66)

Most of ellagitannins have glucose cores in the molecule. However, the ellagitannins possess­ 80th shephagenins A (65) and 8 (66) were characterized as ellagitannin having a gluconic

ing an oxidized glucose, gluconic acid core, have been found in four plant species belonging to acid core as follows. Shephagenin A (65), an off-white amorphous powder, exhibited the (M+H)+

12) 11) . 14) • b' I . I ... three families (Elaeagnaceae, Lythraceae ' and PUnIcaceae ). Their looglca activities and (M+Naf ion peaks at mlz 1121 and 1143, respectively, in the FA8-MS spectrum. The 'H_

have never been reported. This chapter deals with the structural diversity of ellagitannins hav- NMR spectrum of 65 showed a 2H singlet at 6 7.09 due to a galloyl group and five I H singlets

ing a gluconic acid core, which the author isolated, and their biological activities. (67. 10, 6.72, 6.65, 6.52 and 6.22) assignable to an HHDP group and a valoneoyl group. The

coupling pattern and chemical shifts of the sugar proton signals, which were assigned by 'H_'H

II-I. Ellagitannins from Shepherdia argentea COSY, were very similar to those of hippophaenin A (68). The I1C-NMR spectrum of 65 exhib­

ited five s/ carbon resonances (673.6,72.8,70.1, 39.9 and 64.7), and a carboxyl carbon signal

Some plants of Elaeagnus genus have been traditionally used as antidiarreic, and their medici­ at 6 168.8 besides those of the HHDP and valoneoyl groups (Table II-I), indicating the presence

nal usage would imply the presence of tannins and related polyphenols as the active constitu­ of a gluconic acid residue in the molecule. The acyl components were chemically substantiated

ents. Although the phytochemical study on the tannins and related polyphenols from Hippophae by acid hydrolysis of 65 affording gallic acid, ellagic acid and valoneic acid dilactone. Methy-

rhamnoides l2) (Elaeagnaceae) was reported previously, no report for other Elaeagnaceous plants lation of 65 with dimethyl sulfate and potassium carbonate in dry acetone yielded a

has been appeared. In a survey of tannins in Elaeagnaceous plants, the author examined the

OH tannin constituents of Shepherdia argentea Nutt. that is distributed in western North America. Co--Q-OH

HO I OH ~ OCH 2 HO . _~ C O-O~OH 11-1-1. Extraction and Isolation HO ~ C OOH ~o~ HO-Q-VOH OH OH OH OH

The 70% ethanol extract of the dried leaves of S. argentea was partitioned between water and 66

n-BuOH. The aqueous extract was subjected to chromatography aver Dia-ion and Mel-gel 65: R = H OH 65a: R = Me HO CO-OCH2 yO-Q-O H HO CHP-20P, to yield two new tannins named shephagenin A (65) and shephagenin B (66), and two o~6 OH HO HO cc/°\V'COOH 6) . 12 ) · . d . f' d b d' known tannins, strictinin (67) and hippophaenm A (68) ,which were I entl Ie y !fect HO C, OH HO Co - o~O\ yO -Q-OH HO OH HO~OH OH comparison with authentic specimens. HO H~6 HO OH OH OH OH OH 67 68

-44- -45 - nonadecamethyl derivative (65a), mlz 1387 (M+Hf , which upon methanolys is gave 14, ]5 and

33. In addition, the partial degradation of 65 in hot water yielded gallic acid and hippophaenin

~ :I: -0 A (68). The formation of 68 from 65 can be interpreted by the ether cleavage of the va loneoyl . ~ -0 :r: :r: ~:r: 'u C 0 .>-, ,.,., :c ~ -01U.o 0- 6' o 0 cu -t' c o..~ c C C C group followed by disproportionation reaction. 58) Based on these data, shephagenin A was ::J IU o C o c .2 8 o L- u-5 <;i "::: c L- ~ ~ ~~ 0 22 a 0'- I"_ assumed to be an ellagitannin having a gluconic acid core with a valoneoyl group instead of one 0 o ~ Cl...e ~ r' Ir, of the two HHDP groups in 68. ..0-

-0

;;., \() or, ~<. The (S)-configuration of the valoneoyl and HHDP group in 65 was established by a large Cotton ~ -N~~~\{)I"_ ~ ~~~~~~ :....~~,~ fr,~f._ r-i ~ -t I"_ tV I >.. - c § o G~GGG 0 GGUGUUG GUGUUUG UUGUGGU V "ti: 5 u ~ o effect at 229 nm (181 + 1.4 x 10 ) in the CD spectrum,82) Consequently, the structure of shephageni n co;) ..; :r: A was elucidated as formula 65 . ~ \() N- - ~ N ~ '"';". Shephagenin B (66) was obtained as an off-white amorphous powder. The FAB-MS of 66 II) :!: :r: :r: 'D N L- -0 -0 -0 ~ -o 'u 'u ·~ · u :r: .2 ('0 r", ('0 r", '"';", r", co;) ::. c ;:;;;- « 00 u :r: :r: :I: u :I: :r: :I: u showed the pseudo-molecular ion peaks at mlz 803 (M+H/ and 825 (M+Nat (ts 'H-NMR ~ "2il '2 '2 c "= Cil o..rU a Cl... Cl... Cl... 0 Cl... Cl... Cl... a ....,- a ::J <.) u 0 a a u a 0 0 u c o \.... ::J :r: :r: :r:"::: :r: :r: :r: ::J :2 spectrum exhibited two 2H singlets (67.15 and 7.14) and two IH singlets (6 6.73 and 6.65), 0 u -5 c 0 :r: :r: ~ 2 :r: :r: :co 0 c; Cil l"_ N N N l"_ N N N I"_ :i 0 r-r-, 11"', 11"', 11"', r", l"- I"- l"- II"', l"­ ~ e Ir , I,{j .,.;, II (; Cl... ~ I,{j I,{j I,{j I,{j I,{j .r. attributable to two galloyl groups and an HHDP group. On the other hand, the coupling pattern U ..0- Y -0 and the chemical shifts of the sugar proton signals were closely similar to those of shephagenin :I:

chemical shifts (65.02 and 4.07).58) However, those of 66 did not show such difference (6 4.44 '

- 46- -47 - and 4.23), indicating a lack of an HHDP group at 0-4/0-6 in 66. The location of the acyl groups 11-2. Ellagitannins from Elaeagnus umbellata in 66 was unequivocally established by the HMBC spectrum as follows. Two I H singlets (b

6.73 and 6.65) due to the HHDP group correlated with ester carbonyl carbons at b 169.2 (2C) The isolation and structural elucidation of shephagenins A (65) and B (66) having a gluconic through three-bond coupling, which in turn exhibited three-bond couplings with H-2 and H-3 acid core from 5hepherdia argentea and also their inhibitory effects on human immunodefi­ signals of the gluconic acid core. One of the galloyl protons at b 7.14 was correlated with H-6 of ciency virus (HIV)-J reverse transcriptase83) prompted to find further new ellagitannins from the sugar unit through three-bond coupling with the common ester carbonyl carbon at b 167.5. the elaeagnaceous plants. The author then examined the polyphenols of Elaeagnus umbellala

The remaining galloyl group should thus be at 0-4 of the gluconic acid core, although this Thunb. whose leaves and fruits have been traditionally used as a tonic and an astringent to treat connectivity was not observed in the HMBC. The CD spectrum of 66 showed a positive Cotton stomach and bowel disorders in Japan and China. Consequently, seven new ellagitannins,

4 effect at 234 nm (I HI +7.1 x 10 ) with ampl itude of a half of that of 65, confi rming the (5)­ elaeagnatins A (69), B (70), C (71), D (72), E (73), F (74) and G (75), along with fifteen known configuration of the HHDP group.82) Based on these findings, the structure of shephagenin B tannins and related polyphenols were isolated from the leaf extract. was determined as formula 66. 11-2-1. Extraction and Isolation

A concentrated sol ution of 70% aqueous acetone homogenate of the dried leaves of E. umbellala

was extracted with Et20, EtOAc and n-BuOH, successively, to afford respective extracts and

water-soluble portion. The n-BuOH extract was subjected to a combination of column chroma­

tography over Dia-ion HP-20, Toyopearl HW-40, MCI gel CHP-20P and Sephadex LH-20 to

give I, 6-di-O-galloyl-I3-D-glucose (76),84) pedunculagin (77),6) strictinin (67),6) punigluconin

(78),13) lagerstannin C (79),13) and brevifolincarboxylic acid (9).37) Similar chromatographic

separation of the water-sol uble portion led to the isolation of seven new tannins, elaeagnatins A

(69), B (70), C (71), D (72), E (73), F (74) and G (75), and 12 known polyphenols. The known

compounds were identified as 2, 3-(S)-HHDP-D-glucose (80),85) valoneic acid dilactone, 67,77,

hippophaenin A (68),12) 78, pterocarinin A (81),86) hippophaenin B (82),12) casuariin (83),6)

desgalJoylstachyurin (84),68) alienanin B (85)87,88) and casuglaunin A (86)89) by direct compari­

son with authentic specimens or by comparison of their physicochemical data with those re­

ported in the literature.

-48 - -49- H OH OH H 11-2-2. Structure of Elaeagnatins A (69)-G (75) G= ~OH2C 3 -CO~H CCO~R R2 COOH 1 R3 OR Table II -2. Physicochemical data of elaeagnatin A-G (69- 75)

R1 R2 R3 R4 ESI-MS tR (min) tR (min) laJ A= Molecular Formula (M+NH )+ (Normal-phase) (Reversed-phase) (MeOlI) 68 (S)-HHDP G H ~ ~ H 4 ~COOH IsolventAI Isolvent DI 65 (S)-HHDP G A 65b (S)-HHDP H A R1 R2 R3 R4 69 C53 H4( P 35 1254 6.3 8 .3 + 62.3° 78 G H G H (S)-HHDP= 70 C1l2H540 52 1888 9.4 5.4 + 74.8° 81 H L G H 79 H H G H 71 C!l9H 511 ° 2056 9.9 6.3 82 OH H G A 88 (S)-HHDP H H 57 + 94.4° 72 H 2040 9.6 83 OH H H H HO '1_~ ~-~ OH C!!9 5f.P 5(i 10.2 +%.6° 73 C H(;2° (;1 2208 10.0 ]3.5 84 H OH H H H~ H H H 96 + 98.5° 89 OH H G H 74 C R2 H5(iOS3 ]906 9.5 ]2.0 + 50.8° 87 H OH G H 75 C 75 H 52 0~ 1738 9.4 11.5 + 89.5° 69 H L G A L= H~OH ~ Ho-r;t0H HOOH HO OH CO

"OH2C OH Structure of Elaeagnatin A (69) C~HO OCO -Q:~ /; H OH H

67 Elaeagnatin A (69) was obtained as a light-brown amorphous powder. Although the NMR

features ('H and I3 C) of 69 were complicated owing to multiple or broad signals arising from an

equilibration at sugar moiety, the sugar proton and carbon signals were found to be almost

superimposable on those of pterocarinin A (81). The difference in the 'H-NMR spectra between

HO~OH ~ 85

~ 69 HMBC

Fig. 11-1 . HMBC Correlations of 69 86

-50- -51- Table II-3. J H-NMR Spectral Data for the Sugar Moieties of Compounds 68, 86, 87 and 70-75 75 70 71 72 73 74 Proton 87 68 86 4.68 br s 4.75 br s 4.64 br s 4.92 br s 4.85 br s H-l 4.93 d 4.83 br s 4.74 br s (2) 4.92 br s 4.92 br s 4.93 br s 4.92 br s 5.17 br s H-2 4.86 t 4.93 br s 4.93 br s (2) 5.28 br s 5.20 br s 5.19 br s 5.26 br s 5.32 br s 5.28 br s H-3 4.98 t 5.21 br s (2) 5.76 br d 5.78 br d 5.74 br d 5.74 br d 5.73 dd 5.81 dd H-4 5.62 dd 5.80 dd (8.5) (7.5) (7) (7.5) (2, 7.5) (2, 10.5) (2, 9) (2,8) 5.35 dd 5.33 br d 5.33 dd 5.30 br d 5.27 br d 5.32 br d H-5 5.36 dd 5.36m (3.5, 7) (7.5) (7.5) (8) (3, 8.5) (3, 9) (8.5) 4.89 dd 4.93 dd 4.89 dd 4.87 dd 4.84 dd 4.90 dd H-6 4.84 dd 4.94 dd (3,13) (3.5, 13.5) (3.5, 12.5) (3, 13) (3, 13.5) (3, 12) (3, 13) (3.5, 13.5) 3.95 d 4.03 d 3.96 d 3.99 d 3.98 d 3.99 d 4.02d 4.07 d (13) I (13.5) (12.5) (13) (13.5) (12) VI (13) (13.5) N 6.05 d I 5.58 d 5.61 d 5.59 d H-I' (5) (4.5) (8) (5) 3.78 dd 5.38 d 5.38 d 4.72 dd 4.70 dd 5.43 d H-2' 5.36 d 4.70 dd (8,9.5) (9.5) (10) (2,5) (2.5,4.5) (4) (10) (2.5,5) 4.06 t 5.70 d 5.72 br d 5.44m 5.46m 4.57 dd H-3' 5.59 dd 5.46 t (1.5,4) (9.5) (I, 10) (5) (9.5) (10) 5.41 dd 5.51 dd 4.90 t 5.68 dd 5.44dd 5.81 d 5.76 br d 5.44m H-4' (3 .5,9.5) (1.5,9) (9.5) (1,9) (5, 11.5) (9) (9) 5.25 dd 5.84 dd 4.22 dd 5.60 dd 5.4lm 5.59 dd 5.55 dd 5.37 m H-5' (3 .5,9.5) (3,9) (5.5,9.5) (3.5,9) (3.,9) (3,9) 4.96 dd 4.97 dd 5.20 dd 5 .. 02 dd 4.99 dd 5.12 dd 5.13 dd 4.94 dd H-6' (3, (3) (3.5, 13.5) (3, 13) (5.5, (3) (3.5, 13.5) (2.5, 13.5) (3, 13 .5) (3, 13.5) 4.28 d 4.14d 3.79d 4.06d 4.29d 4.36 d 4.29d 4.25 d (13.5) (13) (13) (13.5) (13.5) ( 13.5) (13.5) (13)

500 MHz in acetone-d +0 0, coupling constants (J in Hz) are given in parentheses. 6 2

Table 11-4. J3C-N MR Spectral Data for the Sugar Moieties of Compounds 67, 68, 86, 88 and 70-75.

Carbon 87 68 67 86 70 71 72 73 74 75

C-I 65.5 41.2 40.7 40.6 41.5 41.6 41.6 40.4 C-2 81.0 81.7 80.9 80.9 81.7 81.7 82.0 81.9 C-3 70.9 73.5 74.1 74.5 73.8 73.7 75.6 74.7 C-4 73.3 74.0 74.6 75.0 73.9 74.1 73.7 72.8 C-5 72.0 71.2 72.2 72.4 71.4 71.6 71.7 70.1 C-6 64.5 63.8 63.4 63.6 64.1 64.1 64.2 63 .2 I VI w C-I' 168.6 95.5 67.0 168.7 169.4 67.2 67.4 171.1 94.7 I C-2 ' 72.6 72.7 76.7 72.4 72.7 76.6 76.6 73.6 72.0 C-3' 73.6 75.5 70.9 73.0 73.4 70.9 71.1 74.3 74.3 C-4' 70.1 74.5 75.1 70.7 70.4 75.5 75.7 71.3 73 .9 C-5' 70.1 73 .0 73 .0 69.3 69.4 72.9 72.8 69.8 72.5 C-6' 64.7 63 .6 64.1 63.4 63.6 64.1 64.5 65.3 63 .7

126 MHz in acetone-d(i+020 69 and 81 was indicated by the presence of an extra aromatic 1H singlet in the former. It showed the (M+NH4)+ ion peak at mlz 1254 in the ESI-MS, which is 150 mass units (corresponding to a galloyl group) larger than that of 81. Furthermore, methylation of 69 with dimethyl sulfate and (S)-HHDP= potassium dicarbonate in dry acetone followed by methanolysis furnished 14 and 33. Based on these data, it was suggested that 69 has a valoneoyl group instead of one of the two HHDP

1 2 groups in 81. The position and orientation of the valoneoyl group were determined by the 70: R -R =(S)-HHDP, R3=R4=H 1 2 3 4 ~O~ 71 : R -R =(S)-HHDP, R =A, R =H A= ~H 1 2 3 4 HMBC spectrum of 69 as follows. The singlet signal (b 6.20) at the highest field among the 74 : R =G, R =R =H , R =A COOH aromatic protons was assigned to HE-3, based on the two-bond correlation with the ethereal I 58 The H-NMR spectrum of elaeagnatin 8 (70) in the aromatic region exhibited a 2H singlet (b phenyl carbon at b 146.4. ) The HE-3 signal also displayed a correlation with H-6 of the glu- 7.13) and eight IH singlets(b7.12,6.92, 6.86 668 658 657 654and653)' th . cose core through three-bond couplings with a common ester carbonyl carbon (b 168.6), thus , . , . , . , . . In e aromatIc providing definite evidence for binding sites of the valoneoyl group as shown in the formula. region, implied that the monomeric constituent units of 70 are 68 and 87. It was further substan-

5 tiated by the close resemblance of the sugar carbon resonances in the '3 _ spectrum of 70 The CD spectrum of 69 showed an intensive positive Cotton effect at 236 nm (181 + 1.5 x 10 ), C NMR establishing (S)-configuration both of the chiral HHDP and valoneoyl groups.82) Thus, elaeagnatin to those of 68 and 87, except for a large upfield shift (ca. 25 ppm) of the C-I resonance relative to that of the latters (Table 11-4). This upfield shift indicated that a d' . . b A was assigned to the structure 69. ImenzatIOn etween 68 and 87 occurred through C-C coupling at this position (C-I), eliminating a water. Methylation of 70

with dimethyl sulfate and potassium carbonate in dry acetone followed by methanolysis af­ Structures of Elaeagnatins B (70), C (71) and F (74) 9 forded 14 and 15. Treatment of 70 with tannase 1) gave a monomeric hydrolysate besides gallic

and ellagic acids. The partial hydrolysate was identified with authentic lagerstannin A (88), EJaeagnatins 8 (70), C (71) and F (74) were established as C-glucosidic ellagitannin dimers, which prepared from 68. Atropisomerisms of the chiral HHDP groups were determined to be as follows. The dimeric nature of these tannins was shown by large retention times on the

5 90 (S)-series by a large positive Cotton effect at 235 nm (181 +3.3 x 10 ) in the CD spectrum.82) On normal-phase HPLC ) and the ESJ-MS data (Table Il-2). Based on the assignments of the 'H_ the basis of these data, the structure of elaeagnatin B was established as 70, which might be NMR signals by 'H_ 'H COSY spectra, 70,71 and 74 were deduced to be ellagitannin dimer, produced biogenetically by intermolecular C-C coupling accompanied by dehydration between composed of monomeric units having an open-chain glucose and a gluconic acid core. The the C-l glucose of 87 and the galloyl unit of 68. chemical shifts and coupling pattern of the open-chain glucose core in 70, 71 and 74 revealed The 'H-NMR spectrum of elaeagnatin C (71) showed a galloyl signal (b 7.11) and nine aro­ close similarity to those of stachyurin (87),6) except for an upfield shift of the H-J signal, as matic I H singlets (b 7.11, 7.04, 6.94, 6.89, 6.71,6.59,6.58,6.53 and 6.13), suggesting that 71 is shown in Table 11-3. The gluconic acid signals of 70 and 71 resembled those of hippophaenin A a C-glucosidic ellagitannin dimer having a valoneoyl group instead of an HHDP group in 70. (68),12) respectively, while those of 74 were closely comparable to those of punigluconin (78).13)

-55- -54- the valoneoyl group in 71 were at 0-4/0-6 of the gluconic acid core, as shown in formula 71. aromatic-H

IG CF J E The absolute configurations of the HHDP and the valoneoyl groups were established to be both H 1 o B (S) by the strong positive Cotton effects at 234 nm in the CD spectrum. 82 ) Consequently,

elaeagnatin C was assigned to the structure 71.

Fl (PPII) The 'H-NMR spectrum of elaeagnatin F (74) was analogous to those of 74, except for the

165 presence of an extra galloyl group and the lack of an HHDP group. A remarkable upfield shift of C 166 G H-3' signal (~ 1.15 ppm) in 74 was observed upon comparison of the sugar proton signals F 167 AH between 74 and 71 , indicating that the hydroxyl group at C-3' of the gluconic acid core is not 1 168 0 E J 169 acylated. The gluconic acid signals of 75 were closely comparable to those of punigluconin B 170 (78).13) In addition, the ESI-MS of 74 exhibited (M+NH,/ ion peak at mlz 1906 (C H 0 +NH ), 82 56 53 4 COOH 171 which is 150 mass units (C7 H20 4) lower than that of 71 (mlz 2056). Based on these data, 172 elaeagnatin F was assumed to be a C-glucosidic ellagitannin dimer in which an HHDP group at 7.0 8.5 8.0 5.5 5.0 (ppm) 0-2' 10-3' in 71 is replaced by a galloyl group at 0-2'. The location of acyl groups in 74 was

established by the HMBC measurement (Fig. 11-2). It showed a cross peak by three-bond cou-

pIing between the glucose H-6' signal (b 4.90) and the ester carbonyl carbon resonance (b 168.6),

which, in turn, correlated with the aromatic proton at b 6.18. This aromatic proton signal was

assigned to HE of the valoneoyl group based on its correlation through two-bond coupling with

the signal attributed to the ethereal carbon CE-4 (b 146.8) of the valoneoyl E-ring, and also f\ through three-bond coupling with CE-I (b 117.3).58) The location and orientation of the valoneoyl 74 HMBC group in 74 were thus established, and the other HMBC correlations substantiated the positions

Fig. 11 -2. HMBC Spectrum of 74 \ Ester Carbonyl Regionl (500 MHz, acetone-d6+D20) of the other acyl groups, other acyl, as shown in the formula 74. The (S)-configuration of the

valoneoyl and HHDP groups in 74 was determined by CD spectrum82 ) analogous to those of 70 Methylation of 71 and subsequent methanolysis yielded 33 in addition to 14 and 15, to confirm and 71. Based on these findings, the structure of elaeagnatin F was represented by the formula the presence of a valoneoyl group in the molecule. Enzymatic hydrolysis of 71 with tannase 74. gave a partial monomeric hydrolysate, which was identical with desgalloylshephagenin A (65b) obtained by the similar treatment of 70. These data indicated that the location and orientation of

-56- -57 - Structures of Elaeagnatins D (72) and E (73) and 336 mass units larger than that of 86, respectively. Based on these data, compounds 72 and

73 were presumed to be C-glucosidic ellagitannin dimers in which one HHOP group of 86 for 72 1 Elaeagnatins 0 (72) and E (73) were isolated as light-brown amorphous powders. These H- and two HHDPfor 73 are replaced by the valoneoyl groups, respectively. The linking mode and and 13 C_NMR spectra in the aliphatic region of 72 and 73 were closely comparable to those of orientation of the valoneoyl group in 72 and 73 were established by the HMBC spectra in the casuglaunin A (86), as shown in Tables 11-3 and 11-4, suggesting that these tannins are dirners similar manner to 74 (Fig. 11 -3). The CO spectra of 72 and 73 exhibited positive Cotton effects composed of stachyurin (87) and casuarinin (89) units. The distinction of the IH-NMR feature . h h 5 5 In t e sort-wavelength region (181234 +3.4 x J 0 and 181228 +2.8 x 10 ), confirming the (S)- of these tannins from that of 86 was revealed in the number of aromatic proton signals. Com- configurations for all of the HHOP and valoneoyl groups82) in each tannin. On the basis of these pound 72 showed a 2H singlet and eight I H singlets in the aromatic region, which include an data, the structures of elaeagnatins 0 and E were represented by 72 and 73, respectively. extra I H singlet relative to 86. On the other hand, 75 exhibited nine IH aromatic singlets. Upon methylation, followed by methanolysis, 72 yielded 14, 15 and 33, whereas 73 furnished 14 and Structure of Elaeagnatin G (75)

33 as main products. In the ESI-MS spectra, each pseudomolecular ion peak of 72 and 73 is 168

Elaeagnatin G (75) was obtained as a light-brown amorphous powder. The IH-NMR spectrum

of 75 exhibited the sugar proton signals characteristic of an open-chain glucose and a glucopy­

ranose with a 4C,-conformation. The chemical shifts of the former were closely similar to those

of 70-74, which were indicative of the existence of stachyurin (87) unit with C-C linkage but

not a hydroxyl group at the C-J position. Among the signals of 4CI-glucopyranose core, the

signals /03.78 (dd, 1=8,9.5 Hz) and 04.06 (t, 1=9.5 Hz) 1 of H-2' and H-3', assigned by the IH_ 72

, H: Hi n HMBC f\ 67 73 HMBC 75

Fig. 11-3. HMBC Correlations of 72 and 73 Fig. 11-4. HMBC Correlations of 75

-58 - -59- IH COSY, appeared at an upfield region, indicating the presence of free hydroxyl groups at the 11-3. Taxonomical Significance of Ellagitannins Having A Gluconic Acid Core concerned positions (C-2' and C-3'). Methylation of 75 followed by methanolysis gave 14, 15

and 33. A 2H singlet and seven 1H singlets in the aromatic proton signal region of 75 should be Ellagitannins belong to sub-group of hydrolyzable tannins, and have the HHDP group or a attributable to a valoneoyl, two galloyl and two HHDP groups, among which each one of the similar group derivable from the HHDP group in their molecules. Since most of the ellagitannins galloyl and HHDP groups participate in the C-C linkage to the glucose. The composiltion of have glucose core(s) as a sugar unit, ellagitannins having a gluconic acid, an oxidized glucose,

monomeric units was suggested by 13C_NMR spectrum of 75, which exhibited sugar carbon found in the limited plants constitute a unique group of natural polyphenols. Besides this type of resonances closely similar to those of strictinin (67) and the stachyurin moiety of 70-74 (Table ellagitannins isolated from Shepherdia argentea and Elaeagnus umbellata as described above, 11-4). The location and orientation of the valoneoyl group in 75 were determined by the HMBC analogous tannins were formerly found in four plants IHippophae rhamnoides l2 ) (Elaeagnaceae), spectrum in a similar manner to that of 74 (Fig. 11-4) . The binding modes of the other acyl Lagerstroemia speciosa 13) and L. subcostata 13) (Lythraceae), and Punica granatum 14) groups were further substantiated by connectivities among glucose proton-ester carbonyl car­ (Punicaceae)I. Distribution of this type of tannins in other Elaeagnaceous plants has been ex- bon-aromatic proton through three-bond coupling. The absolute configurations at the biphe­ amined by HPLC analysis (Table 11-5). As a result, hippophaenin A (68) and punigluconin (78) nyl moieties in 75 were confirmed to be all (S), based on the CD spectrum of 75, which showed were found to be widely distributed in Elaeagnaceous plants examined, suggesting that 82 a strong Cotton effect at 230 nm. ) On the basis of these data, the structure of elaeagnatin G ellagitannins having a gluconic acid core are characteristic of Elaeagnaceae and should be use- was represented by formula 75. ful as chemotaxonomical markers.

It is noteworthy that elaeagnatins B (70), C (71) and F (74) are the first examples of ellagitannin Table 1l-5. Distribution of Ellagitannin Having a Gluconic Acid Core in Elaeagnaceous Plants dimers composed of a C-glucosidic monomer and an ellagitannin monomer based on a gluconic ~ Ii) ~ Ci\ « ~ ~ ~ ~ ~ t::. 0- ~ t: t::. « co co t: E t: acid core. t: « u 'c t: c: t: t: t: co U LL QJ c: t: C 'c t: c: t: C

-60- - 61 - Dimers of this type could be regarded as being produced biogenetically by four types of routes 11-4. Biogenesis of C-glucosidic Ellagitannin Dimers as follows. Condensation of C-glucosidic ellagitannins should involve an intermolecular C-O

oxidative or C-C couplings between an anomeric carbon of open-chain glucose and an HHDP Several hundreds of hydrolyzable tannins have been isolated so far from a variety of the di- group on the other sugar unit. The C-O oxidative coupling between a galloyl group of one cotyledonous plants and their structures elucidated. They include more than 120 dimeric hydro- monomer and an HHDP group of the other monomer would result in a formation of the valoneoyl Iyzable tannins with diverse structures. Among the dimers, the number of C-glucosidic group (route A). Seven dimers in the Iythraceous plants were produced through this route. The ellagitannin dimers including elaeagnatins is now up to 26. intermolecular C-C coupling accompanied by dehydration between an anomeric carbon of one

Route A unit and an HHDP group of the other unit produces this dimer (route B). They include alienanin

H H Route C - ? [ Monomer] [Monomer] H Plant Family Tannin Plant Source

93 Reginins A (91 ), B (92 ) Lagerstroemia flos-regmae ) Lythraceae C (93) , 0 (94) [Monomer J Lagerstronin (95) Lagerstroemia flos-regina~3 ) Lythraceae 96 Lythraceae Lythcarins 0, (96) , O2 (97) Lytflrum salicaria ) 97 Heterophylliins B (98), C (99) Cory/us heterophylla ) Betulaceae Tannin Plant Source Plant Family

Elaeagnatins B (70)-G (75) E/aeagnus umbellata92 ) Elaeagnaceae Route B Casuglaunin A (86) E/aeagnus umbellata92 ) Elaeagnaceae Casuarina glaucc/'9) Casuarinaceae

OH I-~ [Monomer 1 - Route 0

Tanni n Plant Source Plant Family

8 Ali en anin A (100) Quercus a/iencf ) Fagaceae E/aeagnus umbellata92 ) Elaeagnaceae Alienanln B (85) - ~ H Quercus a/iencf8) Fagaceae Me/astoma ma/abathricun!7) Melastomataceae ""'~OH Rhoipte/ea chi/ianta9B) Rhoipteleaceae OH Robunn A (101 ) Quercus robu(!9) Fagaceae Quercus petraea' (0) Fagaceae Quercus crenata'O' ) Fagaceae Roburin B (102) Quercus robu(!9 ) Fagaceae Fagaceae Quercus petraea' OO) Tannin Plant Source Plant Family Roburin C (103) Quercus robu(!9 ) Fagaceae Quercus robu,w) Fagaceae Roburin 0 (104) Casuglaunin B (107) Casuanna glaucc/'9) Casuarinaceae Quercus petraea' (0) Fagaceae Anogeissinin (108) Anogelssus acummatalO2 ) Combretaceae Quercus crenata'O' ) Fagaceae Anogeissusins A (105), B (106) Anogeissus acuminata'02) Combretaceae

-63 - -62- 8 (85) in Elaeagnaceae, Fagaceae, Melastomataceae and Rhoiptelaceae. The six dimers of this plants including elaeagnatins B-G and casugJaunin A from E. umbel/ala were formed by route type were considerably distributed in Fagaceae. In the similar way to route B, this dimer was C. The coupling mode of the ellagitannin dimers of Elaeagnaceae thus seems to be characteris­ produced by the intermolecular C-C coupling between an anomeric position and a galloyl group tic of this plant family. (route C). Elaeagnatins 8-G (70-75) and casuglaunin A (86) are considered to be notably produced through this route from Elaeagnus umbellata (Elaeagnaceae). On the other hand, the complex tannin dimers having a f1avan-3-ol unit whose both 6- and 8-positions are attached through each anomeric positions of C-glucosidic ellagitannin monomers, were produced by the intermolecular C-C couplings accompanied by dehydration (route D). They include casuglaunin

OH 8 (107) and anogeissinin (108) from Casuarina glauca and Anogeissus acuminata, respectively.

Alienanin A (100) HO OHOH OH Based on these data, most of these dimers that were produced by routes A and 8, were found Roburin A (101 ): R = OH. R' = H Roburin B (102) : R = X, R' =H in Lythraceae and Fagaceae, respecti vely. It is noteworthy that the dimers in elaeagnaceous Roburin C (103) : R = L. R' = H Roburin 0 (104) R = H, R' = OH OH HO OH OH

OH HO OH R-{j Hoho H HO OH HH~ H -_. OH HOHO ~H 0 I~ O _~H2 HO I '" y H H O~ O~ O H HO :;.- I 0" HO /. - o OH HO ~H ++ OH HO '" 0.0 co HO "I <;1 H~ I ~ co - o H2 ~ ~~\ H2 ff- HO OH co co I .....-0 H:;.- 0 R' 0 H co co I ,,- OH H ~ 0 P HO-X-~OH H 0 rf R 0 co Anogeissusin A (105) : R = H Ho ~H I HO - OHOH OH Anogeissusin B (106) : R = OH OH H OH 1 co * ~-/; H~OH HO ~ 0 - 0 H2 I HO OHOH OH HO OHOH OH ,-y- ,~ ___ 0 HO ~ I 0 - HO ~ -D " ,OH Reslnin A (91) : R =H , R' =OH H 0 co . Resinin B (92) : R = OH, R' = H Resinin C (93) : R = L, R' = H H

HO OHOH OH HO H Aesinin 0 (94) HO OH OH OH HO 1 '" Casuglaunin B (107) HO ..-; CO - OCH2 H HO 0 0 '" OH

R' ~ 0 H" OH OH R Ho Z: I,.: HO 1..-:: 0 --0 H2 0 R' HO :;.- 0-0 H

H 1 -' HO '" / ' HO OH OH OH OH 0 0 H 0 H L = H~O H____ H Ho--f.ft Lagerstronln (95) : R', A 4=OH, A2, R3=H Heterophylliin 8 (98) : R=A'=G H H 3 2 4 HO Ot--OH OH Lythcarin 0, (96) A' , A =H, A , A =OH Heterophyiliin C (99) : A=A' =HHOP 3 4 Lythcarin O2 (97) A \ A =OH. R2, A =H

-64- -65 - Chapter III. Biological Activities of Highly Oxidized Ellagitannins and Related using Raji cells (EBV genom-carring human lymphoblastoid cells) has recently been used as a

Polyphenols primary screening test for survey of anti tumor-promoting agents. 109) Potent inhibitors of the in vitro EBV-EA activation were thereafter investigated in the in vivo two-stage mouse skin car­

Recent investigations of tannins contained in medical plants, food, and beverages have re­ cinogenesis assay using 7, 12-dimethylbenzlalanthracene (DMBA) as an initiator and TPA as a vealed the structural diversity of ellagitannins. Inhibitory effects on topoisomerase-II 103) and tumor promoter. It has been reported that many natural products and synthetic compounds

l04 suppression of tumor virus gene expression ) by some ellagitannins have been found along which inhibited EBV-EA induction by tumor promoters have been found to act as inhibitors of with the anti-HIV effect lOS ) and scavenging effect on the superoxide radical. 106) As part of in vivo tumor promotion. biological studies of ellagitannins, anti-tumor activity 107) and antibacterial activity on Helicobacter The new modified dehydroellagitannins isolated in this thesis and their related polyphenols pylori have been revealed under collaborators with Drs. Tokuda and Hirai, for the newly found have been tested for the inhibitory effect on the in vitro EBV-EA activation, and on the in vivo highly oxidized ellagitannins and related polyphenols from the Euphorbiaceous, Geraniaceous two-stage carcinogenesis on mouse skin tumor promotion. and Elaeagnaceous plants in this work.

111-1-1. In vitro assay; Inhibitory Effect on Epstein-Barr Virus Early Antigen Activation

111-1. Anti-Tumor Activity of Tannins and Related Polyphenols

The EBV genome-carrying Iymphoblastoid cells (Raji cells derived from Burkitt's lymphoma)

A multi-stage theory as the mechanism of chemical carcinogenesis has been accepted, and were cultured in 10% FBS RPMI-1640 medium. The inhibition of EBV-EA activation was

. . • . 108) 6 cancer chemoprevention is regarded as one 0 f the e ffiIClent strategies lor cancer preventIon. assayed using Raji cells (virus non-producer type). 110) The indicator cells (Raji, I x 10 /mL)

Natural products and synthetic compounds to prevent the tumor-promoting step that is a long were incubated at 37°C for 48 hr in I mL of medium containing n-butyric acid (4 mmol) and

and reversible process in multi-stage carcinogenesis in humans have thus been searched for TPA 132 pmol=20 ng in dimethylsulfoxide (DMSO), 2 /-lLI as inducer, and various amounts of

extensively in recent years. Among various screening tests for the discovery of such active test compounds in 5 /-lL DMSO. Smears were made from the cell suspension, and the activated

substances is a convenient primary in vitro assay estimating inhibitory effect on Epstein-Barr cells that were stained by EBV-EA positive serum from nasopharyngeal carcinoma patients

virus early antigen (EBV-EA) acti vation induced by a well known tumor-promoter, 12-0- were detected by an indirect immunofluorescence technique. II I) In each assay, at least 500 cells

tetradecanoylphorbol-13-acetate (TPA). were counted, and the number of stained cells (positive cells) present was recorded. The aver­

Burkitt's lymphoma and nasopharyngeal carcinoma are found throughout the world, particu- age EBV-EA induction of the test compounds was expressed as a relative ratio to the control

larly the former in central Africa and the latter in southern China. These are regarded as the experiment (100%) which was carried out only with n-butyric acid (4 mmol) plus TPA (32

diseases induced by infection with EBV, an oncogenic herpes virus. Since the in vitro activator pmol).

of EBV are often correlated with in vivo tumor promoters, an assay for inhibitors of EBV-EA The ellagitannins and related polyphenols isolated from P.flexuosus, G. thunbergii, A. hispida,

-66- -67 - s. argentea and E. umbellata were evaluated for in vitro inhibition of EBV-EA acti vation in-

duced by TPA, and the res ults are shown in Table III- I . Among these tested compounds, bergenin Table III- I. Relative Ratio"i of EBV-EA Activation with Respect to Positive Control (100%) in the Presence of Tannins and Related Polyphenols. (8), brevifolincarboxylic acid (9), corilagin (10), alienanin B (85), cas uglaunin A (86) and

Concentration (mol ratio I TPA)bl stenophyllanin A (109) exhibited remarkable inhibitory effects on EBV-EA activati on. Their Test Compounds 1000 500 100 10 potencies were comparable to or much stronger (more than 65 % inhibition) than that of a posi ­

Phyllanthus flexuosus tive control , (-)-epigallocatechin gallate (110) (main polyphenol of green tea) 11 2) at a concentra- Geraniin (1) 14.6 (70yl 43.0 79.0 100.0 Phyllanthusiin A (3) 7.6 (70) 36.7 76.5 100.0 tion of 500 mol ration'PA. Phyllanthusiin B (4) 11.7 (70) 43 .3 78.2 100.0 Phyllanthusiin C (5) 15 .2 (70) 47.8 79.6 100.0 Among these inhibitors, compounds 85 and 109 belonging to C-glucosidic ellagitannins showed Phyllanthusiin D (6) 11.3 (70) 43.6 76.2 100.0 Bergenin (8) 0.0 (70) 26.5 44.3 92.3 the most potent activity with 81.5 and 79.1 % inhibition preserving the high viability of Raji Brevifolincarboxylic acid (9) 6.3 (70) 34.3 62.0 100.0 Corilagin (10) 2.6 (70) 25.3 75.4 95.7 cells, respectively, in comparison at a concentration of 500 mol ratior rPA. This implies that the Repandusinic acid A (11) 19.3 (60) 54.0 86.0 100.0 Putranjivain A (12) 17.6 (70) 54.0 86.0 100.0 activity might be specific to the structure and/or group. Although the structure-activity relation- Gcraniinic acid B (13) 9.3 (70) 38 .1 77.4 100.0 Shepherdia argentea ship among the C-glucosidic eJlagitannins is not clear, the presence of the HHDP group rather Shephagenin A (65) 17.9 (60) 55.3 87.2 100.0 Strictinin (67) 0.0 (70) 42.6 70.3 93 .8 than the valoneoyl group at 0 -4/0 -6 and of the C-C bond between C- I of gl ucose and the Hippophaenin A (68) 0.0 (70) 46.2 73 .5 94.6 Elaeagnus umbellata aromatic ring appears to be an important factor for exhibiting the inhibitory effect on EBV-EA Elaeagnatin A (69) 17.3(60) 60.2 84.1 100.0 Elaeagnatin 0 (72) 19.5 (60) 62.7 87.2 100.0 activation. Elaeagnatin E (73) 23.7 (60) 65.0 88.6 100.0 Elaeagnatin F (74) 17.4(60) 61.8 85.0 100.0 Compounds 9 and 10 are considered as compounds derived from cataboli sm of geraniin Elaeagnatin G (75) 13.6 (70) 58.7 83.7 100.0 Pedunculagin (77) 0.0 (70) 46.4 75 .7 95.8 (dehydroellagitannins). They showed much stronger inhibitory effect (9 : 74.7% and 10 : 65 .7% Punigluconin (78) 0.0 (70) 43.7 70.7 92.6 Lagerstannin C (79) 0.0 (70) 42.6 71.4 93 .0 inhibitions) than dehydroellagitannins (geraniin, phyllanthusiins A -D, geraniinic acid B, Pterocarinin A (81) 22.4 (70) 56.3 85.3 100.0 Hippophaenin B (82) 8.2 (70) 55.7 84.3 100.0 putranjivain A and repandusinic acid A; 46.0,,-,63.3% inhibitions). These results suggested that Casuariin (83) 0.0 (70) 48.9 73 .8 94.2 Alienanin B (85) 0.0 (70) 18.5 48 .0 70.5 9 and 10 might exhibit anti-tumor promoting effects as active metabolites upon oral administra- Casuglaunin A (86) 0.0 (70) 31.6 64.9 88.3 Stachyurin (87) 10.7 (60) 39.6 79.5 95 .2 tion of dehydroellagitannins. Casuarinin (89) 8.5 (70) 35 .3 62.0 90.0 Other tannins and related polyphenols Stenophyllanin A (1 09) 0.0 (70) 20.9 60.4 87.4 Ellagic acid 21.6 (70) 50.3 79.4 94.9 Epigallocatechin gallate (110) 6.4 (70) 34.9 68.1 87.7

a) Values represent relative percentages to the positive control value (100%). b) TPA concentration was 20 ng (32 pmol) ImL. .. . . c) Values in parentheses are viability percentage of Raji cells; unless otherwise stated, the viability percentages of Raji cells were more than 80%.

-69- - 68 - III-1-2. In vivo assay; Inhibitory Effect in Two-Stage Carcinogenesis Assay dence (%) of papillomas on mice treated with these tannins was 70% and 90% over the 20 week

period. In the experimental groups, the average number of papilloma per mouse was also re-

Animals were divided into three experimental groups, 15 mice each. The back of each mouse duced to about 67% relative to the control group in week 20 (Fig. 111-1 B). was shaved with surgical clippers, and the mice were topically treated with DMBA (1 ()() ~g, 390 Pentagalloyl-I3-D-gl ucose (31) classified as a gallotannin is known to be a potent anti -tumor 113 nmol) in acetone (0.1 mL) as an initiating treatment. One week after the initiation, papilloma promoter on mouse skin. ) The present study revealed that some C-glucosidic ellagitannins as formation was promoted twice a week by the application ofTPA (I ~g, J.7 nmol) in acetone (0.1 well as PGG also suppress and delay significantly tumor promotion of TPA on mouse skin. mL) to the skin. One hour before each treatment, the mice were treated with the samples (85 Alienanin B (85) was first isolated from Quercus aliena (Fagaceae)88) and later found also in nmol) in acetone (0.1 mL). The incidence of papillomas was examined weekly during 20 weeks. MeZastoma malabathricum (Melastomataceae).87) Stenophyllanin A (109) was similarly found

1 The in vivo anti -tumor promoting activity was then examined for the C-glucosidic tannins (85 in Quercus stenophylla (Fagaceae). 14) Melastoma malabathricum (Melstomataceae)87) and and 109) which showed potent activity in the short-term in vitro assay. Stachyurus praecox (Stachyuraceae).115) These plants as well as two plants in the present study

The in vivo activity was estimated by a two-stage carcinogenesis bioassay on mouse skin might thus warrant further study as sources for possible cancer chemopreventive agents. using DMBA as an initiator and TPA as a promoter. As seen in Fig. 111-1 A, the control animals showed a 100% incidence of papillomas at 8 weeks after promotion, whereas the treatment with 111-2. Antibacterial Activity of Tannins and Related Polyphenols against Helicobacter alienanin B (85) or stenophyllanin A (109) along with the initiator and promoter reduced the pylori percentage of tumor-bearing mice to 55% and 75%, respectively, even in week 15. The inci-

Helicobacter pylori was isolated from the gastric antrum of chronic gastritis in J983. 116) It is 100 10 A B a microaerobic Gram-negative bacterium living in the human stomach, and is associated with 80 8 various diseases such as gastritis, ulcerative of the duodenum and stomach, 117, 118) and gastric ~ ~ ~60 :::l 6 0 cancer. I 19) It is generally accepted that the majority of related diseases is curable once H. pylori ~ E ~ -0:: E E40o ~ 4 infection is successfully eradicated. In patients with H. pylori mucosal infections, eradication of 'a' '0.. 0: 0: 20 2 this microorganism seems to cure both infection and ulcer disease, and thus multi-drug therapy

by a combination of two or three among antibiotics, antacids and proton pump inhibitors has O ~HH"~KKr-.-----.----. 0 o 5 10 15 20 0 5 10 15 20 Weeks or promoti on Weeks of promoti on been applied. The anti-H. pylori activity of ellagitannins isolated in this work and their related Fig. III - I. Inhibition of TPA -induced tumor promotion by multiple application of alienanin B (85) polyphenols has then been examined. and stenophyllanin A (109). All mice were initiated with DMBA (390 nmol) and promoted with 1.7 nmol ofTPA given twice weekly starting I week after initiation. A: percentage of mice bearing pap­ H. pylori strains (NCTCI1638, ATCC43504 and A- J 3) were used, which were cultured on illomas. B: average numbers of papillomas per mouse . • . control TPA alone; 0 . TPA+85 nmol of 85; D, TPA+85 nmol of 109. 1.7% agar plates. The cells of each strain were inoculated on plates and incubated under mi-

-70- -71- croaerophilic culture conditions at 37°C for a few days, and then one loop of the colony was Table III-2. Antibacterial Activity of Tannins and Related Polyphenols against H. pylori suspended in serum-free PPLO agar medium, containing 0.2% dimethyl ~-cyclodextrin. The

MIC (f-lg/mL) suspensions were swabbed on agar plates and the plates were incubated at 37°C for 3 days. The Test Compounds mi ni mal i nhi bitory concentration (MIC) was defined as the lowest concentration yielding no NCrC ~J ATCCb) A-13b) visible growth, which was determined by the agar dilution method. Strictinin (67) 3.13 12.5 6.25 The anti-H. pylori activities of ellagitannins and related polyphenols are summarized in Table Tellimagrandin I (Ill) 3.13 12.5 12.5 Tellimagarndin II (1I2) 6.25 12.5 6.25 111-2. Among test compounds, tannins exhibited anti-H. pylori activities with MIC 3.13~50 !-tgl 6.25 12.5 12.5 Pentagallo)-Iglucosc (31) mL (NCTC strain). The hydrolyzable tannins almost showed potent activities (less than MIC Pedunculagin (77) 6.25 12.5 12.5 6.25 !-tg/mL; NCTC strain). Among them, corilagin (10), strictinin (67) and tellimagrandin I Casuarictin ( 113) 6.25 25.0 12.5

Corilagin (1 0) 3.13 12.5 6.25 (Ill) showed highest anti-H. pylori activity with MIC 3.13 !-tg/mL (NCTC strain). Corilagin Geraniin (1) 6.25 25.0 12.5 (10) was found in Phyllanthusflexuosus, Acalypha hispida, and Geranium thumbergii etc. and Casuarinin (89) 6.25 25.0 12.5 Hippophacnin A (68) 6.25 12.5 12.5 strictinin (67) was similarly found in Elaeagnus umbellata, Melastoma malabathricum, and Elacagnatin A (69) 12.5 25.0 50.0 Eucalyptus spp. etc. Tellimagrandin I is widely distributed in Eucalyptus, Rosa, Cornus spp. Agrimoniin (114) 6.25 25 .0 25.0 Nobotanin 8 (115) 6.25 12.5 12.5 and so on. It has been reported that green tea catechins had anti-H. pyLori activities, which Oenothein 8 (116) 6.25 12.5 12.5 gradually removed H. pylori from human patients infected with the microorganism. 120) In this Rugosin 0 (117) 6.25 50.0 25.0 Heterophylliin 8 (98) 6.25 25.0 12.5 HO Euphorbin C (lI8) 6.25 25.0 50.0 ~_OH ::~o: co -OCH, Alienanin 8 (85) 12.5 25.0 25.0 QJ HO ~ Co~o 4 ::,... lOR OH HO I Oenothein A (119) 6.25 50.0 25.0 OH ~ O~~H 25.0 12.5 Heterophylliin G (120) 6.25 HAJlOH Y OH HO OH Proc)anidin 8-1 (121) 50.0 100.0 >100.0 Tellimagrandin I (111) : A=OH Tellimagrandin II (112) : R=(l1)·OG ProC) anidin B-3 (122) 25.0 >100.0 50.0 Stenophyllanin A (109) Procyanidin B-4 (123) 25.0 >100.0 50.0 OH G= -CO-Q-OH Procyanidin 8-5 (124) 25.0 100.0 50.0 OH Procyanidin C-l (125) 100.0 100.0 >100.0 HOHOg~ h CO _ H2 H HO 0 0 _ L OH := I d'OH Epigallocatechin gallate (110) 12.5 50.0 25.0 co' «hoeo OH H0'(Y0,(V- HO \O~H OH OC OC Epicatechin gallate (126) 25.0 >100.0 50.0 ~'OR ' OH HO-Q--Q-OH Epicatechin (127) >100.0 >100.0 >100.0 HO HO OH OH Epigallocatechin gallate (110) : R=OH , R'=G Epicatechin gallate (126) : R=H, R'=G Casuarictin (113) a) Liquid medium, b) Solid medium Epicatechin (127) : R=A'=H

-72- -73 - assay, these ellagitannins showed more significant activities (MIC 3.13~ 12.5 ~g/mL; NCTC

strain) than green tea catechins, epigallocatechin gallate (110) (MIC 12.5 ~g/mL), epicatechin

gallate (126) (MIC 25 ~g/mL) and epicatechin (127) (MIC > 100 ~g/mL). The extracts of the H ,~ H /- co- 0 _ H2 H plants containing these ellagitannins or each ellagitannins are thus expected to be more effective H "', o-~o-c~ d loH ::*"! :o-~~'0 H*0-l ____ CH2OCO ~:~: H" \,0 ~ HO 0 J=\... ~ Q~~ ~ MH H H 0 oC o-oc~ OH \ 0 co 7, OH OH ~CO for the prevention or remedy of gastritis and gastric ulcers than green tea if taken as food. OHOCo

HOHO~"" -'" co--o - H2 HO '" HO ,~ CO-OCH2 OH HO :' OH caO°-;~40H"-?H o :r OH (JC0H HO :r -co' -'" OH HO". CO_O~~ 0--c0-9' 'O~ OH ,"~ I OH HO% ;b0 H ::;'OH 00:/ o HO "" OH ~H HO~"'" ~ , r HO /- CO--- OCH, o CH20, , HO OH , H HO"., co~ co* hOH HO HO ~ OH HO". CO _ O ~ r=fl R4 0 OH HO H"- ""-co ~ , OH HO HO ~'"o OCO ~OH ~ 9-~ , "... OH OH o0C OH HO"'" OH OH ,..., O~OHI H HO ~ OH ~ OH R1 R2 R3 R4 C4~C8 RugOSln D (117) HO H Oenotheln B (116) Procyanidin B-1 (121) OH H H OH Procyanidin B-3 (122) H OH H OH 111111111 Procyanidin B-4 (123) 1 C"'- HTH~ H~ n--1' CH2~:Z~H OH H OH OH H 111I1111t HOHO~~ -'" """--OCH, OH H '"" CO~ 0 C /- OH co OH HOr"-- , 0 oco :-. h H "'-c ". HO Co-OOC- ~ ~ ,,-loH H I *OH ~I DC ~OHOH HOO '" OH _ H~'" Hd¥0H OH 0H HO HO~O ~ h '1_~ OH H /- co--0- CH, 0 r-7Y HO H~ 10 co r=fH H". co-- OH ~OH , '\'~OH HfiicO/ -C0-Q0H H ~ OC"~OH~OHCl...... ' HO ~ c ~ OH OH -0 OH 'OH \"V

~ 0 o H~OHHO ~'" OH 'OH OH I 60 10 o CH2~ , H _.H H "', c~o..... C /- OH OH o He;: .' 'I 'l OH ~ 0 H • H ~ H ~ OC- 0 " C OH ,)·~)L..OH - - " OH ~ 7, HO~ OH 0 OH HO' 0 HO ?, "'- OH H ~"" OH H HO "'OH OH Euphorbln C (118) Oenothein A (119) ~ \"~OH

fIl "'OH HO~ OH OH Procyanidin C-1 (125) Procyanidin B-5 (124)

OH

Heterophylliin G (120) OH OH OH OH

-75 - -74- Concluding Remarks Consequently, nine new ellagitannins I shephagenins A (65) and B (66) from S. argentea, and

elaeagnatins A (69)-G (75) from E. umbellatal were isolated together with 23 known tannins

The plants that contain geraniin with the OHHOP group in the molecule have been used for and related polyphenols, and their structures elucidated. traditional medicines. On the other hand, geraniin and its related ellagitannins that have a highly Among them, shephagenins A (65), B (66), elaeagnatins B (70), C (71) and F (74) were addi­ metabolized OHHOP unit (acyl unit), have been revealed to be widely distributed in tional members of ellagitannins having a gluconic acid core in the molecule, suggesting that

Euphorbiaceous plants. In addition, ellagitannins having a gluconic acid as sugar unit have been this type of tannins might be characteristic constituents of the elaeagnaceous plants. In addi ­ rarely found in natural sources, but their biological activities have little been reported. This tion, elaeagnatins 8, C and 0 were the first examples of ellagitannin dimers composed of a C­ dissertation deals with the isolation and structural elucidation of such highly oxidized ellagitannins, glucosidic ellagitannin monomer and a monomer based on a gluconic acid core. and with some biological activities of their tannins. C-Glucosidic ellagitannin dimers, elaeagnatins B (70)-G (75), isolated from E. umbel/ata

seem to be biogenetically produced through intermolecular C-C coupling between an anomeric

1. Investigation of the geraniin-related ellagitannins of Phyllanthus flexuosus and Acalypha center of one monomer and a galloyl group of the other one. It is thus noticeable that these hispida (Euphorbiaceae), and Geranium thunbergii (Geraniaceae), has resulted in the isolation tannins are also specific constituents to Elaeagnaceae.

of I J new ellagitannins Iphyllanthusiins A (3)-F (22), acalyphidins MI (23), M 2 (24) (mono­ mers) and 0, (25) (dimers), and geraniinic acids B (13) and C (21) (monomers) I, along with 26 3. The ellagitannins and related polyphenols in this work were examined for anti-tumor promot­ known tannins and related polyphenols. The structures of these tannins were determined by ing activity and antibacterial activity against H. pylori. Bergenin (8), brevifolincarboxylic acid spectral analyses using various 20-NMR techniques and chemical method. (9), corilagin (10), alienanin 8 (85), casuglaunin A (86) and stenophyllanin A (109) were found

It is noteworthy that all of the new monomeric hydrolyzable tannins isolated in this work to exhibit potent inhibitory effects on EBV-EA activation induced by TPA. Among these in vitro could be regarded as metabolites produced through an oxidative modification at the highly reac­ inhi bitors, C-glucosidic ellagitannins, alienanin B (85) and stenophyllanin A (109) showed sig­ tive OHHOP moiety in geraniin. nificant anti-tumor promoting activity in the in vivo two-stage carcinogenesis assay using mouse.

Although many hydrolyzable tannin dimers possessing the geraniin part as a constituent mono­ As for antibacterial activity against H. pylori, corilagin (10), strictinin (67) and tellimagrandin I mer unit as exemplified by euphorbins (dimers composing of geraniin and pentagalloylglucose (111) exhibited strong antibacterial activity with 3.13 ~g/mL for H. pylori NCTC strain, respec­ or tellimagrandin II) have been hitherto isolated from various Euphorbiaceous plants, acalyphidin tively. Hippophaenin A (68) and pedunculagin (77), major tannins of E. umbellata, also showed

0 was the first example of a dimer composed of two moles of geraniin. potent activity with MIC 6.25 ~g/mL. The intake of the extracts of G. thunbergii and E. umbellata 1

containing these bioactive ellagitannins might thus be expected to be useful for prevention of

2. In order to search for a rare type of ellagitannins based on a gluconic acid core, polyphenol gastritis, peptic ulcer, and stomach and other cancers. Further investigation for significance of constituents of Shepherdia argentea and Elaeagnus umbellata (Elaeagnaceae) were investigated. these tannin-rich plants and foods are needed.

-76- -77- Experimental

General Method

Optical rotations were recorded on a JASCO DIP-IOOO or DIP-4 polarimeters. 'H and I1C- NMR spec­

tra were measured in acetone-dr,+D 20 and methanol-d~ on a Varian YXR-500 (500 MHz for 'H-NMR and 126 MHz for I.'C -NMR) instrument. Chemical shifts are given in D(ppm) values relative to that of the

, solvent lacetone-dr (DH 2.04; Dc 29.8), methanol-d~ (DH 3.35; Dc 49.8)] on a tetramethylsilane scale. ESI­ MS spectra were recorded on a Micromass Auto Spec OA-Tof mass spectrometer (solvent: 50%MeOH+0.I %AcONH~, flow rate: 20 I-tUmin). FAB-MS was taken on a YG 70-SE mass spectrom­ eter using 3-nitrobcnzyl alcohol as the matrix agent. CD spectra were measured on a JASCO J-720W spectrometer or J-500A spectrometer equipped with a DP-50 I N data processor. Normal-phase HPLC was conducted on a YMC-Pack SILA-003 (YMC Co., Ltd) (4.6 mm i.d. x 250 mm) or a Superspher Si 60 (Merck) (4.0 mm i.d. x 125 mm) column developed with n-hexane-MeOH-THF-formic acid (60: 45 : 15 : I) containing oxalic acid (500 mg/1.2 L) (solvent A), n-hexane-MeOH-THF-formic acid (55: 33 : II : I) containing oxalic acid (450 mg/L) (solvent B) or n-hexane-EtOAc (2 : I) (solvent C) (flow rate, 1.5 mLlmin; detection 280 nm) at room temperature. Reversed-phase HPLC was performed on a YMC­ Pack ODS-A A-302 (YMC Co., Ltd) or a LiChrospher RP-18 (Merck) column (4.6 i.d. x 150 mm) developed with 10mM H,PO~-lOmM KH1PO~-EtOH-EtOAc (45: 45 : 8: 2) (solvent D) or IOmM H3 PO~­ IOmM KH2PO~-C H,CN (9 : 9: 2) (solvent E) (flow rate, I mUmin) at 40°C. Detection was effected with a Shimadzu SPD-6A spectrophotometric detector at 280 nm. A YMC-PackA324 (YMC Co., Ltd) (10 i.d. x 300 mm) column was used for preparative HPLC. Analytical and preparative TLC were conducted with Kieselgel PF:!.">l (Merck) and spots were visualized by UV irradiation (254 nm) or by spraying NaN0 - 1 AcOH or FeCI.\ reagents. Solvents were evaporated under reduced pressure below 40°C.

Experimental for Chapter I-I

Extraction and Isolation The fresh leaves (2.16 kg) of Pflexuosus, collected at Sekigahara, Gifu Prefecture, in summer, were first extracted with ether and acetone (each 10 Lx 6, 7 days) at room temperature to remove lipophylic compounds, and filtered . The residue was then extracted with 70% aqueous acetone and filtered. The filtrate was concentrated and further extracted with ether (700 mL x 6), CH1C1 1 (700 mL x 6) and n-BuOH (2 Lx 8). A part (200 g) of the n-BuOH extract (43 J g) was submitted to column chromatography over Dia-ion HP-20 (9 cm i. d. x 60 cm), developing with H 0 1 containing increasing amounts of MeOH in a stepwise gradient mode, to give the 10 % MeOH eluate fraction (I; 13.24 g), 20% MeOH eluate (fraction 2; 16.43 g), 40% MeOH eluate (fraction 3; 33.38 g), MeOH eluate (fraction 4 ; 24.76 g) and 70% aqueous acetone eluate (fraction 5; 0.13 g). Fraction I was further chromatographed over Toyopearl HW-40 (coarse) (5.0 cm i.d. x 50 cm) developing with 40% MeOH and 60% MeOH to yield bergenin (8) (2.4 g), brevifoJincarboxylic acid (9) (1.1 g), repandusinic acid A (11) (888 mg) and phyllanthusiin B (4) (87 mg). Fraction 2 was further purified by column chromatography over Toyopearl HW-40 (coarse) (20% MeOH - 30% - 40% - 60% -70%) to give 8 (3.7 g), 9 (678 mg), corilagin (10) (4.6 g), geraniinic acid B (13) (93 mg) and 11 (830 mg). The 70%

-78- -79- MeOH eluate (920 mg) was further purified by column chromatography over MCI gel CHP-20P to yield J= 17 Hz, B-ring H-5"), 3.29 (I H, d, J= 17 Hz, B-ring H-5"), 3.23, 3.6-+, 3.65, 3.70, 3.77, 3.85, 3.86, 3.87, 4 (50 mg). fraction 3 was similarly chromatographed over Toyopearl HW-40 (coarse) developing with 3.88,3.92,3.93 (each 3H, s, OMe), 3.66 (6H, s, OMe), 4.43 (I H, br s, B-ring 4"-OH), 4.48 (I H, d, J=2.0 50% MeOH -+ 60% -+ 70% to afford phyllanthusiin E (7) (255 mg), 10 (4.7 g), geraniin (1) (4.7 g), Hz, B-ring H-3"), 4.53 (I H, dd, J=8, 10.5 Hz, Glc H-6), 4.91 (I H, br t, J=8 Hz, GIc H-5), 5.06 (I H, t, phyllanthusiin C (5) (63 mg), putranjivain A (11) (116 mg) and phyllanthusiin A (3) (73 mg). J= I 0.5 Hz, Glc H-6), 5.25 (I H, br s, GIc H-4), 5.44 (I H, br s, Glc H-2), 5.66 (I H, d, J=2 Hz, B-ring H- Rechromatography of fraction 4 over Toyopearl HW-40 (fine) gave phyllanthusiin 0 (6) (476 mg) and 2"),6.21 (I H, br s, GIc H-3), 6.57 (I H, br s, GIc H-I), 6.88,7.05 (each IH, s, HHDP-H), 7.16 (I H, s, A­ chebulagic acid (2) (70 mg). ring H-3'), 7.24 (2H, s, galloyl-H). IJ C_NMR (acetone-dJ 6: 43.3 (B-ring C-5"), 55.7 (B-ring C-3"), 52. 1,53.9,56.0,56.4,56.6,56.8 (3C), 60.6, 60.7, 60.9, 61.0 (2C) (OMe x 13),61.3 (Glc C-3), 65.0 (GIc

, Phyllanthusiin A (3) A pale yellow amorphous powder, I a Ii) -94.5° (c= 1.0, acetone). UV Ama C-6), 67.4 (Glc C-4), 69.4 (GIc C-2), 73.1 (GIc C-5), 77.7 (B-ring C-4"), 82.0 (B-ring C-2"), 93.3 (Glc (MeOH) nm (log c): 222 (4.85),278 (4.47). CO (MeOH) 181 (nm): -8.5 x IO~ (241), -1.8 x 10.1 (265), -4.1 C-I), 105.5 (HHOP C-3'), 107.7 (2C) (galloyl C-2, 6), 109.0 (HHOP C-3), 109.21 (A-ring C-3'), 119. 16 x 10.1 (282). Negative rAB-MS mlz: 951 (M-H) . Anal. Calcd for C.lIH~RO~ 7· 6H~ 0: C, 46.22; H, 3.77. (A-ring C-I '), 121.4 (A-ring C-2'), 122. 1 (HHDP C-I), 124.4 (galloyl C-l), 124.8 (HHOP C -1), 127.0, Found: C, 46.33; H, 3.57. IH-NMR: see Table I- I. IIC_ NMR: see Table 1-2 . 129.8 (HHDP C-2, 2'), 139.0 (A-ring C-5 '), 144.1 (galloyl C-4), 144.6 (HHOP C-5'), 146.1 (HHOP ('_ 5), 153.37, 153.42 (HHOP C-6, 6'), 153.8 (HHOP C-4), 154.0 (A-ring C-4'), 154.7 (HHDP C-4'), 154.4 Partial Hydrolysis of Phyllanthusiin A (3) An aqueous solution (I mL) of phyllanthusiin A (I mg) (2C) (galloyl C-3, 5), 154.8 (A-ring C-6'), 164.6 (galloyl C-7), 165.1 (A-ring C-T), 166.0 (HHDP C-7), was heated in a boiling-water bath for 4 h, and the reaction mixture, after evaporation of the solvent, was 168.3 (HHDP C-T), 171.0 (B-ring C-6"), 171.7 (B-ring C-l "), 172.6 (B-ring C-7"). analYl.cd by HPLC (normal and reversed-phases) to detect corilagin (10). Methanolysis of the Tridecamethylate (4a) A mixture of 4a (10 mg) and 1% NaOMe (I mL) in Methylation of Phyllanthusiin A (3) A solution of 3 (10 mg) in EtOH (I mL) was treated with absolute MeOH (I mL) was kept standing overnight at room temperature and worked up as usual. Prepara­ ethereal CH ~ N , for 3 h at room temperature. After removal of the solvent, the residue was purified by tive TLC (light petroleum-CHCI,-acetone, 2: I : I) furnished methyl tri-O-methylgallate (14) II mg; El­ preparative TLC (benzene-acetone, 7: I ) to yield a tridecamethylate (3a) (3.2 mg) as a white amorphous MS mlz 226 (Mn, dimethyl hexamethoxydiphenate (15) 10.5mg; EI-MS mlz450 (Mf! and the hcxamethyl

0 ) (c=0.6, J= powder 10.1 1 - 118 acetone). EI-MS mlz: 1134 (Mf, 'H-NMR (acetone-dJ 6: 3.13 (IH, br d, derivative (4b) (1.5 mg), Ial l) _17° (c=0.5, acetone). FAB-MS mlz: 457 (M+H)+, 479 (M+Na)'. UV }.. 16.5 Hz, B-ring H-5"), 3.28 (lH, dd, J=7, 16.5 Hz, B-ring H-5"), 3.40 (lH, br d, J=7 Hz, B-ring B-4"), (MeOH) nm (log c): 221 (4.38),264 (3.84),300 (3.48). CD (MeOH) IOJ (nm): +1.1 x 10 1 (228), -1.9 x IO~ 3.39 3.65, 3.83, 3.87, 3.89, 3.90, 3.94 (each 3H, s, OMe), 3.66 (9H, s, OMe), 3.93 (6H, s, OMe), 4.43 (264), +0.7 x 10.1 (300). IH-NMR (acetone-d(.+D~O) 6: 2.55 (I H, d, 1=16.5 Hz, H-5 '), 2.89 (I H, d, J= 16.5 11 H, dd, 1=8, 11 Hz, Glucose (GIc) H-61, 4.63 (I H, br t, J=8 Hz, Gic H-5), 5.06 (I H, t, J=II Hz, Glc H- Hz, H-5'), 3.56, 3.64, 3.70, 3.85, 3.86, 3.93 (each 3H, s, OMe), 4.31 (J H, d, 1= 1.5 HI., H-3'), 5.50 (I H, d, 6),5.27 (I H, br s, Glc H-4), 5.48 (J H, br s, Glc H-2), 5.72 (J H, br s, Gic H-3), 6.46 (I H br s, GIc H-I), J= 1.5Hz, H-2'), 7.08 (J H, s, H-3). 6.95, 7.13 (I H, s, HHDP-H), 7.25 (2H, s, galloyl-H), 7.39 (I H, s, A-ring H-3 '). PhylJanthusiin C (5) A pale yellow amorphous powder, 10.1 1) -49° (c=1 .0, acetone). Anal. Calcd for Methanolysis of Tridecamethylate (3a) A mixture of 3a (0.5 mg) and J % NaOMe (0.05 mL) in C~ I HI() O~(: 5H " 0: C, 47.24; H, 3.94. Found: C, 47.38; H, 3.70. Negative FAB-MS mlz: 925 (M-H). UV MeOH (I mL) was left standing overnight at room temperature. The reaction mixture was acidified with An"" (MeOH) nm (log c): 223 (4.85),278 (4.49). CD 181 (nm): -3.3 x IO ~ (241), +0.4 x 10.1 (258), -4.1 x a few drops of AcOH and evaporated in vacuo. TLC analysis (benzene-acetone, 4: I) of the residue 10.1 (284). IH-NMR: see Table I-I. i.1C-NMR: see Table 1-2. showed the formation of methyl tri -O-methylgallate (14) (Rf 0.75) and dimethyl hexamethoxydiphenate (15) (Rf 0.36). Methylation of Phyllanthusiin C (5) a) A solution of 5 (50 mg) in EtOH (I mL) was treated with ethereal CH~N~ at room temperature for 5 h, and the reaction mixture, after removal of the solvent, was Phyllanthusiin B (4) A pale yellow amorphou powder, 10.1 1) -43° (c= 1.0, acetone). Anal. Calcd for purified by preparative TLC (light petroleum-CHCll-acetone, 2 : I : I) to give a dodecamethylate (Sa) (9 C H 0 AH 0: C, 47.22; H, 3.65. Found: C, 46.98; H, 3.71. Negative FAB-MS mlz: 969 (M-HY. UV .II I(\!!< ~ mg), as a white amorphous powder. FAB-MS mlz: 1095 (M+H)', I I 17 (M+Naf, IH-NMR (acetone-d) Am." (MeOH) nm (log c): 222 (4.89), 280 (4.50). CD (MeOH) 181 (nm): -2.8 X 10.1 (240), + 1.1 x 10.1 (256), 6: 2.29 (I H, br d, J= I 0.5 Hz, B-ring H-3"), 2.39 (I H, dd, J=6.5, 10.5 Hz, B-ring H-3"), 3.20, 3.41, 3.~ , -4.3 x 10.1 (283). IH-NMR: see Table 1-1. LlC-NMR: see Table 1-2. 3.70,3.85,3.86,3.88, 3.90,3.92,3.95 (each 3H. s, OMe), 3.67 (6H, s, OMe), 4.18 (I H, d, J=8.5 Hz, B­ ring 4"-OH), 4.45 (I H, s, B-ring 2"-OH), 4.55 (I H, dd, J=7.5, 10.5 Hz, Gic H-6), 4.65 (I H. ddd, J=6.5, Methylation of Phyllanthusiin B (4) A mixture of 4 (47 mg), (CHJ:S0.l (0.2 mL) and K ~ CO, (200 8.5, 10.5 Hz, B-ring H-4"), 4.74 (1 H, s, B-ring H-I "), 5.00 (I H, br t, J=7.5 Hz. GIc H-5), 5.12 (I H, t, mg) in dry acetone (5 mL) was stirred overnight at room temperature and then refluxed for 1 h. After J= I 0.5 Hz, Glc H-6), 5.44 (I H, br s, Gic H-4), 5.66 (I H, br s, GIc H-2), 5.69 (I H, br s, GIc H-3), 6.53 removal of the inorganic material by centrifugation, the reaction mixture was subjected to preparative (I H, br s, GIc H-I). 6.90, 7.06, 7.05 leach 1H, s, HHDP-H and A-ring H-3'), 7.25 (2H, s, galloyl-H). TLC (benzene-acetone, 7 : I), which gave a tridecamethylate (4a) (29 mg) as white solids, 10.11) -390 b) A mixture of 5 (30 mg), (CH)~S0.l (0.1 mL) and K2COI (100 mg) in dry acetone (5 mL) was stirred (c= 1.0, acetone). FAB-MS mlz: 1153 (M+H)+, 1175 (M+Na)+. 'H-NMR (acetone-d) 6: 3.20 (I H, d, overnight at room temperature and refluxed for 2 h. The reaction mixture was worked up as described

-80- -81- for the methylation of 3 to yield deca-O-methylcorilagin (lOa) (6 mg) and methyl tri-O­ achieved by monitoring HPLC (normal and reversed-phase). The 40% MeOH eluate (12 g) was further methylbrevifolincarboxylate (9a) (2.3 mg). purified by repeated column chromatography on MCI-gel CHP-20P with aqueous MeOH to give corilagin (10) (IA g), phyllanthusiins B (4) (60 mg), C (5) (103 mg), E (7) (142 mg), geraniin (1) (380 mg), Methanolysis of Dodecamethylphyllanthusiin C (Sa) A solution of Sa (2 mg) in absolute MeOH geraniinic acids B (13) (36 mg), C (21) (46 mg), and phyllanthusiin F (22) (17 mg). (I mL) containing a few drops of 1% NaOMe was left standing overnight at room temperature. After addition of AcOH followed by evaporation of the solvent, the residue was subjected to preparative TLC Geraniinic acid B (13) A pale yellow amorphous powder. lall) -770 (c= 1.7, acetone). ESI-MS ml (CHCI~-acetonc 20: I) to give 14, 15 and methyl tri-O-methylbrevifolincarboxylate (9a), which were z: 970 (M+NHy. HR ESI-MS mlz: 970.1190 (M+NHY, Calcd for C-l,H280~7+NH-I' 970.1162. UV AI1lIt\ J identified by co-chromatography on TLC with authentic samples. (MeOH) nm (log E): 219 (4.83), 275 (4.40). CD (MeOH) 181 (nm): -12 x IO (225), +0.2 x 10-1 (260), -3.4 x 10J (283). IR (KBr) em": 3380 (OH), 1704 (ester CO). 1599 (C=C). 'H-NMR: see Table 1-3. u C_ 0 Phyllanthusiin D (6) Colorless needles, mp 245-247°C (H20-MeOH). lall) _93 (c=1.0, acetone). NMR (acetone-dr.+D~O) D: 37.9 (B-ring C-3"), 63.9 (GIc C-6), 64.4 (GIc C-3), 65.9 (Glc C-4), 71.5 (GIc UV "-max (MeOH) nm (log E): 223 (4.82),280 (4.46). CD (MeOH) 181 (nm): -2.9 x 10-1 (240), + 4.9 x 10-1 C-2), 73.1 (Gic C-5), 80.1 (B-ring C-2"), 91.7 (Gic C-I), 148.6, (B-ring C-4"), 122.9 (B-ring C-5"), (260). -3.0 x 10-1 (287). Anal. Calcd for C-\-lHJ2027'5H20: C, 48.80; H, 3.88. Found: C, 48.86; H, 3.56. 107.9 (HHDP C-3'), /10.0, 111.9 (HHOP C-3, A-ring C-3'), 110.9 (2C) (galloyl C-2, 6), 115.0 (HHDP

Negative FAB-MS mlz: 991 (M-Hr. 'H-NMR: see Table 1-1. '3C-NMR: see Table 1-2. C-I '), 117.1 (HHDP C-I), 120.4 (galloyl C-1), 122.9 (B-ring C-5"), 123.3 (A-ring C-2'), 124.9 (HHDP C-2), 125.8 (HHDP C-2'), 136.4 (A-ring C-5'), 137.6, 138.3 (HHDP C-5, 5'), 139.5 (galloyl C-4), 144.2 Methylation of Phyllanthusiin D (6) A mixture of 6 (20 mg), (CH)~S0-l (0.1 mL) and K ~CO,~ (100 (2C), 144.4, 145.0 (2C), 145 .3 (HHDP C-4, 4',6,6', A-ring C-4', 6'), 145.9 (2C)(galloyl C-3, 5), 161.2 mg) in dry acetone (5 mL), was stirred overnight at room temperature and then refluxed for 17 h. Pre­ (B-ring C-6"), 164.5 (B-ring C-7"), 164.9 (galloyl C-7), 166.1 (HHDP C-7), 166.2 (A-ring C-T), 168.3 parative TLC (light petroleum-CHCI1-acetone, 6 : 3 : 2) after removal of the inorganic material furnished (HHDP C-T), 171.1 (B-ring C-I "). deca-O-methylcorilagin (lOa) (2.3 mg). Geraniinic acid C (2l) A pale yellow amorphous powder. laiD-85° (c=I.2, acetone). ESI-MS ml Preparation of Phyllanthusiin D (6) from Geraniin (I) A solution of 1 (50mg) in absolute acetone z: 970 (M+NHy. HR ESI-MS mlz: 970.1179 (M+NH/, Calcd for C-I,H"80~7+NH-I' 970. I 162. UV "-ma, (50 ml) containing CF~COOH (2 mL) was refluxed for J week. The concentrated reaction mixture was (MeOH) nm (log E): 219 (4.80),275 (4.38). CD (MeOH) 181: - 12.4 x 10-1 (225), +OA x 10-1 (260), -4.5 x submitted to column chromatography over MCI-gel CHP-20P, developing with H"O containing incrt::as­ 10J (283). IR (KBr) em": 3380 (OH), 1718 (ester CO), 1607 (C=C). 'H-NMR: see Table 1-3 . "C-NMR ing amounts of MeOH. The eluate with 40% MeOH gave phyllanthusiin 0 (6) (10 mg). (acetone-dr,+D~O) 6: 38.1 (B-ring C-3"), 63.8 (Glc C-6), 64.3 (Glc C-3), 65.8 (Glc C-4), 71.2 (GIc ('-2), 72.8 (Glc C-5), 78A (B-ring C-2"), 90.2 (Gic C-I), 107.9 (HHDP C-3 '), 110.5 (HHDP C-3), 11/.0 (2C) Phyllantbusiin E (7) A brown amorphous powder. UV Ama.\ (MeOH) nm (log E): 223 (4.36), 280 (galloyl C-2, 6), 111.8 (A-ring C-3 '), 113.1, 115. 1, 117.0 (HHDP C-I, I', A-ring C-I '), 120.3 (galloyl C­

1.1 (4.21),340 (3.97). Anal. Calcd for C HgOK' H20: C, 51.87; H, 3.57. Found: C, 50.32; H, 3.23. Negative I), 123A(B-ringC-5"), 124.9(A-ringC-2'), 125.1 (HHDPC-2), 125.8 (HHDPC-2'), 136.5 (A-ringC- FAB-MS mlz: 291 (M-H). 'H-NMR: see text. '~C- NMR (MeOH-d) &: 32.3 (C-13), 68.0 (C-II), 112.2 5'), 137.8 (2C)(HHDP C-5, 5'), 139.7 (galloyl C-4), 144.5, 145.0 (2C), 145.1, 145.5, 145.8 (HHDP C-4, (C-2), 112.7 (C-3), 114.2 (C-7), 114.8 (C-9), 139.5 (C-5), 140.9 (C-6), 143.6 (C-1 0), 150.9 (C-4), 162.4 4',6,6',A-ringC-4',6'), 146.0 (2C) (galloyl C-3,5), 149. 1 (B-ringC-4"), 162.0,164.5,164.8, /66.1, (C-8), 163 .8 (C-I), 173 . 1 (C-12). 166.3, 167.4, 168.3 (ester carbonyl).

Phyllanthusiin F (22) An off-white amorphous powder. lall) -16° (c=I.O, MeOH). ESI-MS mlz: Experimental for Chapter 1-2 642 (M+NHy. HR ESI-MS mlz: 642.1344 (M+NHY, Calcd for C2"H~0'H+NH-I' 642.1306. UV,,-max

(MeOH) nm (log E) : 230 (4.37), 280 (4.22). 'H-NMR: see Table \-4. '3C-NMR (acetone-dr,+D"O) 6: Plant Materials The leaves of G. thunbergii cultivated in the herbal garden of our Faculty were 46.6 (B-ring C-3"), 61.7 (Glc C-3), 62.76 (Glc C-6), 62.80 (B-ring C-I "),70.4 (GIc C-2), 71 .0 (Glc C-4), collected in August. A voucher specimen is deposited in the Herbarium, Faculty of Pharmaceutiical 74.7 (B-ring C-4"), 77.9 (Glc C-5), 78A (B-ring C-2"), 92.3 (Glc C-I), 110.1 (2C) (galloyl C-2, 6), 110.9 Sciences, Okayama University. (A-ring C-3 '), 116.8 (B-ring C-5"), 117.7 (A-ring C-2'), 118.8 (A-ring C-I '), 120.5 (galloYI C-I), 135.3 (A-ring C-5'), 139.4 (galloYI C-4), 146.0 (2C) (galloyl C-3, 5), 146.9 (A-ring C-4'), 149.6 (A-ring C-6'), Extraction and Isolation The dried leaves (997 g) of G. thunbergii were homogenized in acetone­ 165.31 (galloyl C-7), 165.28 (A-ring C-T), 174.2 (B-ring C-7"). H ~ O (7:3) (4 J.- x 3). The filtered homogenate was concentrated and extracted with Et~O, EtOAc and n­ BuOH, successively. The remaining aqueous solution was concentrated to give brownish residue (124 Partial Hydrolysis of 13 and 21 An aqueous solution (2 ml) of 13 (or 21; each I mg) was heated in g), and the aqueous extract was subjected to column chromatography over Oia-ion HP-20 with Hp and a boiling-water bath for 1.5 h. The reaction mixture was analyzed by HPLC IYMC Pack A~' 2 (O~S), 6 increasing amounts of MeOH in H ~ O (10%-20%-40%-60%) and MeOH. Fractionations \-vere mm i.d. x 150 mm; solvent E; 40°C; flow rate 1.3 mllmin; detection 280 nml to show the peaks due to

-82- - 83- corilagin (10) and ellagic acid. chromatographed over Toyopearl HW-40 (fine) (2.2 cm i.d. x 58 cm) with MeOH -H:!O (5:5-+6:4-+7:3)-+MeOH-H:!O-acetone (7:2: J-+6:2:2)-+acelone-H20 (7:3) in a stepwise gradient mode. Methylation of 13 and 21 A mixture of 13 (17 mg), (CH)2S0~ (0.12 ml) and K~CO, (100 mg) in The fractions showing similar HPLC patterns were combined and further purified by column chromatog­ acetone (5 ml) was stirred overnight at room temperature, and then refluxed for 8 h. After removal of the raphy over Scphadex LH-20 with EtOH andlor MCI-gel CHP-20P with aqueous MeOH to afford geraniin inorganic material by centrifugation, the supernatant was evaporated to dryness in vacuo. The product (1) (180 mg), mallotusinin (26) (4 mg), phyllanthusiin C (5) (20 mg), euphorbins A (27) (12 mg), B (28) was purified by preparative TLC (SiO" light petroleum-benzene-acetone 1:2: 1 vlv) to yield tridecamethyl (5 mg), and acalyphidin M, (23) (II mg). In order to obtain additional crops of the new tannins, the rest (c=0.8, derivative of 13, a pale yellowish am;rphous powder, lalll -1 28° acetone), 'H-NMR (acetone-d..) (32 g) of the precipitate was chromatographed over Dia-ion HP-20 (5.5 cm i.d. x 45 c m) with H:!O-+ 6: 7.30 (2H, s, gallo) I-H) , 7.22 ,6.91 (each I H, s, HHDP-H), 7.17 (I H, s, A-ring H-3 '),6.87 (I H, br s, MeOH-H:!O (2:8-+4:6-+6:4)-+MeOH in a stepwise gradient mode. The eluate with MeOH-H:!O (6:-+) Gle H- I), 6.42 (I H, d, J= I Hz, B-ring H-5"), 5.49 (I H, m, Glc H-4), 5.-+3 (1 H, m, Glc H-3), 5.40 (1 H, br was similarly fractionated by repeated column chromatography over Toyopearl HW40 (fine), Sephadex s, Glc H-2), 5.29 (I H, d, J= I Hz, B-ring H-2"), 5.29 (lH, t, J= I Hz, B-ring H-3"), 5.16 (I H, t, J= II Hz, LH-20 and MCI-gel CHP-20P to give fraction A/including acalyphidin 0 1 (25)1 (321 mg), acalyphidin Glc H-6), 4.81 (I H, br dd , J=8, II Hz, Gic H-5), 4.40 (I H, dd , J=8, II Hz, Gic H-6), 3.94, 3.90, 3.89, M , (23) (20 mg) and acalyphidin M:! (24) (16 mg). A part (29 g) of the n-BuOH e,tract (35 g) was 3.88,3.85,3.83,3.70,3.65, 3.50 (each 3H, s, OMe), 3.86, 3.68 (each 6H, s, OMe). Geraniinic acid C subjected to column chromatography over Dia-ion HP-20 (5.5 cm i.d. x 28 cm) and developed with (21) (16 mg) was methylated in a way similar to that described above to give nona-O-methylcorilagin H:!O-+MeOH-H:!O( 2:8-+4:6-+6:4) -+MeOH in stepwise gradient mode. The eluate with MeOH-H:!O (lOb) ( 1.0 mg) and the tridecamethyl derivative of 21 (4.7 mg), a pale yellowish amorphous powder, lali) (4:6) was further chromatographed over Toyopearl HW-40 (fine) and MCI-gel CHP-20P to give fraction - 1020 (c=D.7, acetone), 'H-NMR (acetone-d,) 6: 7.31 (2H, s, galloyl-H), 7.14,6.92 (each tH, s, HHDP­ B lincluding aealyphidin D, (25)1 (89 mg), rutin (34 mg), brevifolincarboxylie acid (9) (34 mg), H), 7.14 (J H, s, A-ring H-3 '),6.97 (I H, br s, Glc H-I), 6.48 (I H, d, J= I Hz, B-ring H-5"), 5.74 (I H, d, cxcoecarianin (29) (17 mg) and acalyphidin M :; (24) (8.7 mg). The eluate with MeOH-H 0 (6:4) was 1 J=6 Hz, B-ring H-2"), 5.57 (I H, m, GIc H-4), 5.36 (IH, m, GIc H-3), 5.31 (I H, br s, GIc H-2), 5.18 (lH, si milarly subjected to repeated column chromatographies to give kaempferol 3-rutinoside (120 mg), t, J=ll Hz, Ole H-6), 5.11 (I H, br d, J=6 Hz, B-ring H-3"), 4.82 (I H, br dd, 1=8, 11 Hz, Glc H-5), 4.40 corilagin (10) (19 mg) and euphorbin D (30) (52 mg). A part (6.0 g) of the EtOAc extract (20 g) was ( I H, dd, J=8, 11 Hz, GIc H-6), 3.94, 3.91,3.89,3.88,3.87,3.84,3.80,3.70,3.65,3.50,3.43 (each 3H, s, chromatographed over Toyopearl HW-40 (coarse) (2.2 em i.d . x 50 cm) with aqueous MeOH OMe), 3.68 (6H, S, OMe). (60~-+70%)-+MeOH-H:!O-aeetone (7:2: 1).60% MeOH eluate was rechromatographed over MCl-gel CHP-20P with aqueous MeOH to give furosin (17) (2.4 mg). The eluate of MeOH-H 0 -acetone (7:2: I) 1 Methanolysis of Tridecamethyl Derivatives of 13 and 21 The tridecamethyl derivatives of ]l3 and was also rechromatographed over MCI-gel CHP-20P with aqueous MeOH to give 1,2,3,4,6-penta-O­ 21 (each 8 mg) were separately methanolyzed with 1 % NaOMe (0.1 ml) in methanol (2 ml) at room galloyl-~-D-glucose (31) (12 mg) and repandinin A (32) (54 mg). temp. for 10 h. After acidification with a few drops of HOAc, the solvent was removed in vacuo. The residue was re-dissolved in acetone and purified by preparati ve TLC (Si02, benzene-acetone 15 : I) to Acalyphidin M, (23) A light brown amorphous powder. la/,) -43° (c=0.5, MeOH). UV A""" (MeOH) give methyl tri-O-methylgallate (1 mg) and dimethyl hexamethoxydiphenate (2 mg), lall) +21 ° (c= 1.1, nm (log E): 220 (4.98), 280 (4.46), 294 (4.38),360 (3.80). CD (MeOH) 181 (nm): -9.4 x 10J (240), +4.5 ~ ~ . 'J • acetone). x 10 (260), -8.8 x 10 (287), + 1.7 x 10 (323), -5.1 x 10 (348). FAB-MS mlz: 931 (M+Na) . Anal. Calcd for C.u ) H:;~ O :;:i· 5H:!O: C, 48.1 I; H, 3.50. Found: C, 48.04; H, 4.02. 'H-NMR: see Table 1-5. LlC- NMR Partial Hydrolysis of 5 An aqueous solution (I ml) of 5 (1 mg) was incubated at 37"C with 2 drops (acetone-d,,+O:; O) 6: 120.4 (C-I), J 10.6 (C-2), 146.0 (C-3), 138.2 (C-4), 146.0 (C-5), 110.6 (C-6) 166.0 of tannase prepared according to the literature. Reversed-phase HPLC (LiChrospher RP-18, 4 mm i.d. x (C-7)lgalloyl/, 116.4, IJ5.9(C-I, I'), 125.2, 125.4(C-2,2'), 109.5, 109.7(C-3,3'), 145.2(2C,C-4,4'), 250 mm; solvent E; flow rate 1.0 mIlmin; detection 280 nm) of the reaction mixture showed production 136.4, 137.0 (C-5, 5'), 144.8 (2C, C-6, 6'), 166.9, 168.4 (C-7, 7') / HHOPI, 193.6 (C-I), 1-+7.8 (C-2), of 22 (t 5.85 min) after 15 min. R 150.1 (C-3), 41.6 (C-4), 37.9 (C-5), 115.0 (C-I '), 115.4 (C-2'), 108.2 (C-3'), 139.6 (C-4'), 140.5 (C-5'), 143.6 (C-6'), 161.1, 172.0 (C-6, 7') I Brev /, 94.4 (C-l), 70.7 (C-2), 72.9 (C-3), 65.9 (C -4), 74.3 (C-5), 64.6 (C-6) /Glcl. Experimental for Chapter 1-3

Partial Hydrolysis of 23 A solution of 23 (0.5 mg) in H!O (0.5 mL) was heated at 800 for 30 min. Plant material The leaves of Acalypha hispida were collected in Taiwan. A voucher specimen was The reaction mixture showed peaks identical with those of corilagin (1 0) and brevifolin carboxylic acid deposited at the Herbarium of the Faculty of Pharmaceutical Sciences, Okayama University. (9) on reversed-phase HPLC.

Extraction and Isolation The dried leaves (950 g) were homogenized (x 3) in acetone-H:;O (7:3) (12 Methylation of 23 followed by Methanolysis A mixture of 23 (2.5 mg), K CO (100 mg) and 1 l Lx 3 ) and the homogenate was filtered. The filtrate was concentrated and extracted with Et20 (I Lx 3), Me 2 S0~ (0.0 I mL) in dry acetone (2 mL) was stirred overnight at room temperature, and then refluxed EtOAc (I Lx 5) and n-BuOH satd with H:!O (I Lx 6). A part (8.5 g) of the preci pitate (I J 9 g) was for 2 hr. After removal of the inorganic material by centrifugation, the supernatant was evaporated to

- 84- - 85- dryness. The reaction mixture was directry methanolyzed with 1% NaOMe (0.1 mL) in MeOH (I mL) at Preparation of the Acetone Adduct of 25 Crude acalyphidin DI {fraction A (321 mg) land B (89 room temperature for 6 hr. After acidification with a few drops of AcOH, the solvent was removed in mg)1 described in the isolation procedure of tannins} was treated with ammonium formate (150 mg) 145 vacuo. The residue was subjected to preparative TLC to give methyl tri-O-methylgallate (14) (0.2 mg), mg in B I in acetone (10 mL) 15 mL in B I, and heated at 50° for 2 h. The residue after removal of the dimethyl hexamethoxydiphenate (15) (0.1 mg) and methyl tri-O-methylbrevifolincarboxylate (9a) (0.2 solvent was subjected to column chromatography over MCI-gel CHP-20P with H:O containing increas­ mg), which were identified by direct comparison with authentic samples (TLC, HPLC and MS). ing amounts of MeOH. The 40% MeOH eluate gave an acetyonyl derivative (25a) of acalyphidin 0 1(23 mg) 110 mg from B I, as a light brown amorphous powder,lall) -430 (c=O.4, MeOH). ESI-MS mlz: 2000 0 Acalyphidin M (24) A light brown amorphous powder. lall) _59 (c=0.5, MeOH). UV Ama, (MeOH) (M+NHy. UV An", (MeOH) nm (log E) : 224 (4.75),280 (4.46). CO (MeOH) 181 (nm): +3.2 X 10"' (208), 1 .."' nm (log E): 224 (4.75),280 (4.46). CD (MeOH) 181 (nm): +3.2 x 10 (208), -5.9 x 10 (236), +2.3 x 104 -5.9 X 10-1 (236), +2.3 X 10"' (263), -4.2 X 10-1 (288), +2.7 X 10"' (321). IH-NMR: see Table 1-6. 11C_NMR (263), -4.2 x 104 (288), +2.7 x 104 (321), -5 .1 x 103 (348). FAB-MS mlz: 975 (M+Na)+. Anal. Calcd for : see Table 1-7. C-lIH~NO~7·7H~O: C, 45.65; H, 3.92. Found: C, 45.98; H, 3.91. IH-NMR: see Table 1-5. LlC-NMR

(acetone-dr,+D~O) c): 120.4 (C-I), 110.6 (C-2), 146.0 (C-3), 138.2 (C-4), 146.0 (C-5), 110.6 (C-6), 166.0 Methylation of 25a followed by Methanolysis A mixture of 25a (2 mg), K~CO , (50 mg) and

(C-7) Igalloyll, 116.4. 115.9 (C-l, 1'), 12S.2, 125.4 (C-2, 2'), 109.S, 109.7 (C-3, 3'), 145.2 (2C, C-4, 4'), Me ~ S0-l (0.0 I ml) in dry acetone (2.5 ml) was stirred overnight at room temp, and then refluxed for 2 hr. 136.4,137.0 «('-5, 5'),144.8 (2C, C-6, 6'),166.9,168.4 (C-7, 7') [HHOP), 114.2 (C-I), 119.2 (C-2), A syrupy residue after removal of the inorganic material and solvent, was directly methanolyzed with 1% 116.6 «(,-3),146.9 «(,-4),139.7 (C-S), 142.1 (C-6), 164.9 (C-7) lA-ring), 16S.8 (C-l"), 72.9 (C-2"), 43.1 NaOMe (0.1 ml) in MeOH (I ml), and subjected to prep. TLC (Kieselgel PF~~, n-hexane-CHCI1-Me:CO, (C-r). 74.3 (C -4-"),40.9 «(,-5"), 167.1 (C-6"), 171.5 (C-T') IB-ring\, 91.3 (C-I), 70.4 (C-2), 62.4 (C-3), 4:6: I) to give 14, 15 and trimethyl octa-O-methylvaloneate (33), which were identified by comparison 67.3 «('-4-), 73.4 (C -5),63.9 (C-6) (Glc I· authentic samples (TLC and HPLC).

Partial Hydrolysis of 24 A solution of 24 (0.2 mg) in H~O (1.0 mL) was heated at 80° for 10 hr. Reversed-phase HPLC of the reaction mixture showed a peak identical with that of corilagin (10). Experimental for Chapter II-I

Methylation of 24 A mixture of 24 (10 mg) , Me2SO-l (0.02 mL) and K2CO, (200 mg) in dry acetone Plant Materials Leaves and stems of S. argentea were collected in Harney County, Oregon, U.S.A. (3 ml) was stirred overnight at room temperature and then refluxed for 2 hr. After removal of the ill1or­ A voucher specimen (Murata et aI., No.426) was deposited in the Herbarium, University of Tokyo (n). ganic material by centrifugation, the reaction mixture was subjected to preparative TLC (toluene-ac­ etone. 2: I), which gave deca-O-methylcorilagin (lOa) (1.3 mg) and tridecamethylate (24a) (1.8 mg). Extraction and Isolation The dried leaves (83 g) of S. argenlea were washed with acetone (I L), lOa: A \\ hite amorphous powder, IH-NMR (acetone-d,) c): 7.23 (2H, s, galloyl-H), 6.92, 6.81 (each 1H, and soaked twice in EtOH (each 1.2 L x I week), and then in 70% aqueous EtOH (1.2 L x I week) at s, HHDP-H). 3.94 (I H, d, 1=10 Hz, 4-0H), 3.90, 3.89, 3.88, 3.83, 3.68, 3.67 (6H), 3.66, 3.64, 3.16 (each room temperature to yield the EtOH extract (7 g) and 70% aqueous EtOH extract (19.5 g). The 70~ 3H, s, OMe), glucose protons see Table 1. 24a: A white amorphous powder, lall) _21° (c=0.5, MeOH). aqueous EtOH extract was partitioned between water and n-BuOH. A part (5 g) of the aqueous extract

FAB-MS ml;:. 1135 (M+H)'. 1157 (M+Na)". IH-NMR (acetone-dJ c): 7.25 (2H, s, galloyl-H), 7.10, 6.93 (16.2 g) was subjected to column chromatography over Oia-ion HP-20 with H ~O containing increasing (each I H, s, HHDP-H), 7.23 (I H, s, A-ring H-3 '),6.57 (1 H, br s, Glc H-I), 6.39 (I H, s, B-ring H-5"), amounts of MeOH in a stepwise gradient mode. The 10% MeOH eluate (439 mg) was rechromatographed

5.7-+ (I H, d, 1=8 Hz, B-ring H-2"), 5.67 (l H, br s, Glc H-3), 5.63 (IH, br s, Glc H-2), 5.32 (I H, m, Glc H­ over Toyopearl HW-40 (coarse grade) with H ~O- MeOH (8:2 -7:3 - 6:4 - 4:6) to give shephagenins -+),5.55 (I H, d, J=8 Hz, B-ring H-3"), 5.16 (I H, t, 1= II Hz, Glc H-S), 5.00 (1 H, dd, 1=8, 11 Hz, GIc H- A (65) (181 mg) and B (66) (8 mg) from the 30% MeOH eluate, and hippophaenin A (68) (44 mg) from 6),4.53 (IH, dd, J=8, 11 Hz, Glc H-6), 4.01,3.92,3.90,3.89,3.87,3.84,3.79,3.68,3.66 (9H), 3.60, 3.25 the 4-0% MeOH eluate. The 20% MeOH eluate (370 mg) from Dia-ion HP-20 column chromatography (each 3H, s. OMc). was purified by chromatography on Sephadex LH-20 (EtOH-MeOH) to yield trictinin (67) (17 mg).

Methylation of 24 followed by Methanolysis A mixture of 24 (0.5 mg), K ~COI (50 mg) and Me2S0 -l Shephagenin A (65) An off-white amorphous powder, laJ \)+116° (c=1.0, MeOH). UV A,,,,, (MeOH)

(0.0 I mL) in dr) acetone (2.0 mL) was stirred overnight at room temperature, and then refluxed for 2 hr. nm (log E): 229 (4.93), 277 (4.59). Anal. Calcd for C.;xH,~Olc·6H~O: C, 46.91 ; H, 3.58. Found: C, 46.83 ; Arter removal of the inorganic material by centrifugation, the supernatant was evaporated to dryness. A H, -+ .02. FAB-MS mlz: 1143 (M+Na( CD (MeOH) 181 (nm): + 1.4 x 10' (229), -2.3 x I O~ (278), +3.7 x solution of the mixture in MeOH (I mL) and 1% NaOMe (0.1 mL) was kept standing at room tempera­ 1O~ (280). IH-NMR (acetone-dr,+O:P) c) : 7.10,6.65, 6.22 (each tH, s, valoneoyl-H), 7.09 (2H, s, galJoyl­ ture for 6 hr. After acidification with AcOH, the solvent was removed in vacuo. The residue was re­ H), 6.72, 6.52 (each IH, s, HHDP-H), S.371IH, d, 1=9.5 Hz, Gluconic acid (GluA) H-2\, 5.57 (IH, d, dissolved in MeOH and analysed by normal-phase HPLC, which showed the production of methyl tri-O­ 1=9.5 Hz, GluA H-3), 5.72 (I H, d, 1=9.5 Hz, GluA H-4), 5.57 (I H, dd, 1=3.5,9.5 Hz, GluA H-5), 4.93

methylgallate (14) and dimethyl hexamethoxydiphenate (15). (IH, dd, 1=3.5,13 Hz, GluA H-6), 4.01 (IH, d, 1=13 Hz, GluA H-6). nC-NMR (acetone-dr,+ DcO): see Table II-I.

-86- - 87- (HHDP C-3 , 3'), 75 .8 (GluA C-2), 75.2 (GluA C-3), 71 .6 (GluA C-4), 68.4 (GluA C -5),66.1 (GluA C -6).

Acid Hydrolysis of 65 A solution of 65 (5 mg) in 3% H1S0~ was heated in a boiling water-bath for 8 h. After cooling, the reaction mixture was extracted with EtOAc. The EtOAc-soluble portion was Experimental for Chapter 11-2 analyzed by reversed-phase HPLC (solvent D) to detect gallic acid (tR 3.2 min), elJagic acid (tR 10.7 min) and valoneic acid dilactone (tR 8.4 min). The aqueous layer was neutralized with Amberlite IR-120 (OH form), and evaporated to dryness. The GC analysis of the syrupy residue after trimethylsilylation showed Extraction and Isolation The dried leaves (1 .7 kg) of E. umbellata, collected in July, were homog­ a peak identical with that of glucono-6-lactone (tR 7.1 min). enized in 70% acetone (10 L x 3), and the concentrated solution (1.6 L) was extracted with ether (1 Lx 6), ethyl acetate (1.5 Lx 6) and n-BuOH saturated with water (1.2 Lx 6), successively The H,O extract (190

Methylation of 65 A mixture of 65 (50 mg), anhydrous K1CO, (100 mg) and Me1S0-l (250 I-lL) in dry g) was chromatographed over Dia-ion HP-20 (6.7 cm i.d. x 65 cm) with H ~ O--aqueous MeOH (10'1(-- acetone (10 mL) was stirred overnight at room temperature, and then refluxed for 4 h. After removal of 20%--30% 40%--60% MeOH) MeOH--70% acetone-H~O. The eluate (8.4 g) from 10% MeOH the inorganic materials by filtration, the filtrate was concentrated in vacuo, and subjected to preparative was fractionated and purified by rechromatography over Toyopearl HW-4Q (coarse grade, 2.2 em i.d. x TLC (Kieselgel PF~ 'i-I ' light petroleum-CHC\-acetone, 2 : I : 2) to give the permethylated derivative 62 em) and/or MCI -gel CHP-20P (1.1 em i.d. x 32 cm) with aqueous MeOH followed by preparative

(65a) (20 mg) of shephagenin A as a white powder. Ia III + I 00.4° (c=0.5, MeOH). FAB-MS mlz 1387 HPLC IYMC A-312 (10 mm i.d. x 300 mm); solvent, 10 mM H 1 PO~-10 mM KH1PO.\-EtOH-EtOAc (47.5 (M+H)'. IH-NMR (acetone-d) 6: 7.37 (2H, s, galloyl-H), 7.23, 7.16,6.89,6.79,6.44 (each IH, s, HHDP : 47.5 : 4: I) I to gi\ c valoneie acid dilactone (5 mg), hippophaenins A (68) (1.2 g), B (82) (1.6 g), and valoneoyl-H), 5.60 (IH, dd, J=3, 9.5 Hz, GluA H-5/, 5.56 (lH, br d, J=lO Hz, GluA H-3), 5.54 (IH, puniglueonin (78) (15 mg), casuariin (83) (6 mg), elaeagnatin A (69) (1.4 g), alienanin B (85) (53 mg), d, J=8.5 Hz, GluA H-2), 5.52 (I H, br d, 1=9.5 Hz, GluA H-4), 4.89 (I H, dd, 1=3, 13.5 Hz, GluA H-6), casuglaunin A (86) (61 mg), elaeagnatins D (72) (20 mg), E (73) (10 mg), F (74) (20 mg) and G (75) (33 4.14 (I H, d, 1= 13.5, GluA H-6), 4.03,3.90,3.88,3.84,3.84,3.80,3.80,3.77,3.76, 3.74, 3.70, 3.64, 3.59 mg). The 20% MeOH (7.7 g) and 30% MeOH (6.8 g) eluates were separately subjected to column

(each 3H, s, OMe x 13),3.94,3.86,3.82 (each 6H, S, OMe x 6) chromatographies over Toyopearl HW-40 (coarse grade, 2.2 cm i.d. x 70 cm) with aqueous MeOH, MCI­ gel CHP-20P (1.1 cm i.d. x 33 cm) with H10 and aqueous MeOH, andlor Sephadex LH-20 (J.I cm i.d. x Methanolysis of65a An MeOH solution (I mL) of 65a (4 mg) containing I % NaOMe (50 I-lL) was 30 cm) with EtOH and finally purified by preparative HPLC IYMCA-312 (10 mm i.d. x 300 mm);

; left standing at room temperature for 10 h to give methyl tri-O-methylgallate (14) (tR 1.4 min), dimethyl solvent, 10 mM H,P0-l-IO mM KH ~ P0-l-EtOH - EtOAc (47.5: 47.5: 4: 1)lto yield 2, 3-(S)-HHDP-D­

; ; hexamethoxyldiphenate (15) (tR 2.6 min) and trimethyl octa-O-methylvaloneate (33) (tR 7.5 min), \vhich glucose (80) (46 mg), peduneulagin (77) (54 mg), 67 (20 mg), 68 (806 mg), pterocarinin A (81) (1 . 1 g), were shown to be identical with authentic samples by co-chromatography using HPLC and TLC (Kieselgel 82 (50 mg), desgalJoylstachyurin (84) (10 mg), 69 (39 mg), elaeagnatins B (70) (II mg) and C (71) (I I PF~~, light petroleum-CHCI,-acetone, 2 : 1 : I). HPLC conditions: Superspher Si60 (4 mm i.d. x 125 mg). The n-BuOH extract (65.8 g) was similarly fractionated and purified by a combination of column mm; Merck); solvent C, at room temperature. chromatographies over Diaion HP-20, Toyopearl HW-40 (coarse grade) and MCI-CHP-20P to give 68 (15 mg), lagerstannin C (79) (24 mg), 9 (3 mg), I, 6-di-O-galloyl-~-D-glucose (76) (14 mg), 77 (69 mg) Chemical Conversion of65 into 68 A solution of 65 (10 mg) in H~O (10 mL) was heated in a water and 67 (208 mg). The known compounds were identified by comparison of their physical data with the bath at 90°C, and the reaction process was monitored by HPLC, which showed the formation of gallic reported val ues.

; acid (lR; 1.8 min) and hippophaenin A (68) (t R 3.2 min). The residue obtained after removal of the solvent

) was subjected to chromatography over MCI-gel CHP-20P to give 68 (2 mg). The identity of 68 was Elaeagnatin A (69) A light-brown amorphous powder, lal l +62.3° (c=0.5, MeOH). Anal. Calcd for proved by co-chromatography with an authentic sample on HPLC. Compound 68 was further confirmed C, ., H.I(P ,, ·6H~O: C, 47.3; H, 3.9. Found: C, 47.3; H, 3.7. ESI-MS mlz: 1254 (M+NH)'. I-AB-MS mlz: by direct comparison of the IH-NMR spectrum with that of the authentic specimen. 1237 (M+Hf, UV A,,,,, (MeOH) nm (log E): 220 (4.79), 265 (sh 4.44). CD (McOH) 181 (nm): + 1.5 x I 0' (236), -2.S x (257), +4.1 x I (283). IH-NMR (aeetone-d,,+D:P) 6: (major tautomer) 7.07 (I H, S, H - 1O~ O~ I S, Shephagenin 8 (66) An off-white amorphous powder, lall) +142.5° (c=O.4, MeOH). UV Am,,, 6), 7.02 (2H, s, H(" -2, 6), 6.84 (I H, s, HI) -3), 6.45 (I H, HIl-3), 6.20 (I H, s, HI-3), 5.S5 II H, dd, J=2, 9 (MeOH) nm (log E): 218 (4.77), 268 (4.38). FAB-MS mlz: 803 (M+H)· , 825 (M+Na)'. CD (MeOH) 181 Hz, Gle H-41. 5.23 (I H, dd, 1=3, 9 Hz, GIc H-5), 5.12 (lH, brs, Glc H-2), 4.90 (lH, d, 1=2 Hz, GIc H-3), (nm): +7.1 x IO~ (234), -1.7 x 1O~ (260), +2.0 X 10~ (282). 'H-NMR (methanol-d) 6: 7.15,7.14 (each 4.81 (I H, dd, J=3, 13Hz, Gle H-6), 3.% (I H, d, 1= 13 Hz, Glc H-6), 3.9411 H, d, J=3.5 Hz, Iyxose (Lyx) 2H, s, galloyl-H), 6.73,6.65 (each I H, s, HHDP-H), 5.60 (I H, d, J=9.5 Hz, G1uA H-2), 5.90 (I H, br d, H-21, 3.93 (l H, dd, 1=6, 10 Hz, Lyx H-4), 3.85 (lH, dd, 1=3, 10Hz, Lyx H-3), 3.77 (I H, dd, 1=6, II Hz, J=9.5 Hz, GluA H-3), 5.82 (I H, br ct, 1=9.5 Hz, GluA H-4), 4.26 (I H, m, GluA H-5), 4.44 (I H, br d, Lyx H-5), 3.65 (I H, d, 1= II Hz, Lyx H-S), 3.55 (I H, brs, Glc H-I). I\C-NMR (MeOH-d) 6 : (major J=9.5 Hz, GluA H-6), 4.23 (IH, m, GluA H-6). I.\C-NMR 6: 171.3 (GluA C-I), 169.2 (2C) (HHDP C-7, tautomer) 45.9 (Glc C-I), 62.3 (Lyx C-S), 64.1 (Gle C-6), 66.0 (Lyx C-4), 69.8 (Gle C-5), 7 1.5 (2C) (Lyx C-2, 3), 72.8 (Glc C-4), 73.4 (Glc C-3), 74.6 (Glc C-2), 101.1 (Lyx C-I), 103.5 (CJj-3), 104.2 (C -3), 7'), 167.5 (galloyl C-7'), 166.6 (galloyl C-7), 145.5, 145.4 (galloyl C-3, 5,3',5'), 144.9 (2C) (HHDP C-4, I 4'), 143.6 (2C) (HHDP C-6, 6'), 139.3, 138.8 (galloyl C-4, 4'), 136.2, 136.1 (HHDP C-5, 5'), 126.2, 125.8 107.7 (CI)-3), 108.9 (CI-6), 108.9 (2C)(C, -2, 6), 113.9 (CI-I), 114.4 (CI,-I), 115.1 (CIl-I), 115.4 (CI-I),

(HHDP C-2, 2'), 120.4, 119.6 (galloyl C-I I'), 109.5 (2C), 109.4 (2C) (galloyl C-2, 6, 2', 6'), 107.0 (2C) II6.0(C 1 -I), 119.4(C(-I), 123.3 (C -2), /16.3, 123.5 (C -2,C -2), 125.5 (C,-2), 125.8 (C -3), 134.4 I II Il I

- 88- - 89- (C -5), 136.4 (CI-S), 136.7 (CD-S), 136.8 (C .-2), 138.0 (C~-S), 138.9 (C .-3), 139.3 (C -4), 139.5 (CrA), Il r , c Preparation of Lagerstannin A (88) from Hippophaenin A (68) An aq ueous sol ution of 68 (10 142.3 (C,-5), 142.7 (C\-4), 143.5 (Cn-6), 143.9 (C\-6), 144.1 (CE-6), 144.7 (C\)-6), 14S.1 (3C) (Cu-4, Cc- mg/3 mL) was incubated at 37°C for 72 h with tannase (10 drops). The reaction mixture after concentra­ 3,5), 145.6 (C[)-4), 14604 (CIA), 165.8 (Cc-7), 166.7 (CA-7), 167.7 (CI-7), 168.6 (C,-7), 168.7 (CJ)-7), tion was chromatographed over Dia-ion HP-20 with H20 and aqueous MeOH. The H,O eluate afforded 169.3 (CH-7). lagerstannin A (88) (3.2 mg). 88: A pale brown amorphous powder,lall) + 107.2° (c= I ~O, MeOH). FAB­

MS mlz: 823 (M+Naf, 'H-NMR (acetone-d6+D10) 6: 6.64, 6.S7, 6.S2, 6.S1 (each 1H, s, HHDP-H), 5.78 Methylation of 69 followed by Metbanolysis A mixture of 69 (I mg), K1CO, (10 mg) and (CH)2S0.. (0.01 mL) in acetone (I mL) was stirred overnight at room temperature, then refluxed for 3 h. After (I H, dd, J=2, 10 Hz, GluA H-3), S.37 (lH, d, J=1O Hz, GluA H-2), S.26 (IH, dd, J=2, 9 Hz, GluA H-4), removal of the inorganic material by centrifugation, the supernatant was evaporated to dryness. The 4.81 (I H, dd, J=4, 12.5 Hz, GluA H-6), 4.31 (IH, dd, J=4, 9 Hz, GluA H-5), 3.90 (I H, d, J=12.5 Hz, residue was directly methanolyzed in 1% NaOMe in MeOH (l mL) at room temperature for 6 h. After GluA H-6). These physical data were consistent with the reported data. lJ) acidification with acetic acid and removal of the solvent, the residue was partitioned between EtOAc and H 0. The EtOAc soluble portion was treated with CH N (l mL) for 1 h and the solvent was eva(X)rated. Elaeagnatin C (71) A light-brown amorphous (X)wder, [aiD +94.4° (c=1.0, MeOH). UV An,.. (MeOH) 2 2 2 Normal phase HPLC (solvent C) of the reaction mixture showed peaks identical with those of the authen­ nm (log E): 221 (5.13),265 (4.79). Anal. Caled for C89H'>805]'22H20: C, 43.88; H, 4.22. Found: C, 43.84; tic methyl tri -O-methyl gallate (14) and trimethyl octa-O-methylvaloneate (33). H,4.03. ESI-MS mlz: 2056 (M+NHy. FAB-MS mlz: 2061 (M+Naf, CD (MeOH) 181 (nm): +3.0 x lOs ~ .. I ( 23- 4), -7.7 x 10 (261), +704 x 10 (284). H-NMR (acetone-d6+D~O) 6: 7.11 (2H, s, galloyl-H), 7.11,

Elaeagnatin B (70) A light-brown amorphous (X)wder, lall) +74.8° (c=1.0, MeOH). UV Am", (MeOH) 7.04, 6.94, 6.89,6.71,6.59,6.58, 6.S3, 6.13 (each IH, s, galloyl and HHDP-H), sugar protons, see Table II-3. "C-NMR(acetone-d~+D20)6: 103.6,105.1,106.9,107.1,107.7,107.8,108.0,109.3, 109.9(HHDP nm (log £): 222 (5.12), 261 (4.81). Anal. Calcd for CICH~0'i2'18H20: C, 44.85; H, 4.13. Found: C, 44.83; C-3,3', valoneoyl C-3, 3',6", galloyl C-6'), 109.S(2C)(galloyIC-2,6), 113.1, 11304, 114.2, 114.5, H, 4.14. ESI-MS mlz: 1888 (M+NH)'. FAB-MS mlz: 1871 (M+Hf, CD (MeOH) 181 (nm): +3.3 x lOs 114.6, IIS.2, lISA, 116.0, 116.2 (HHDPC-I, 1', valoneoyl C-I, 1'.1"), 119.6,119.8,121.7,123.0, (235), -7.9 x 10.1 (262), +6.0 x 10" (284). IH-NMR (acetone-dr,+D20) 6: 7. 13 (2H, s, galJoyl-H), 7.12, 6.92,6.86.6.68,6.58,6.57,6.54,6.53 (each 1H, s, galloyl and HHDP-H) , sugar protons, see Table 11-3. 123.8,124.6,124.7,125.3,125.4,125.7,126.9 (HHDP C-2, 2', valoneoyl C-2, 2', galloyl C-I), 134.5, 135.3,135.4,135.8,135.9,136.0,136.1,136.2,137.1, 137.3, 138.7, 139.1, 139.2 (HHDPC-S, 5', vaJoneoyl "C-NMR (acetone-d,,+D20) 6: 105.2, 106.7, 106.9, 107.3,10704,107.5, 108.1,109.9 (HHDP C-3, 3', galloyl C-6'), 109.4 (2C)(galloyl C-2, 6), 113 . 1, 113 A, 11404, 114.6, 114.8, lIS .3, 115 A, 116.1 (HHDP C-5, 5',2",3",4", galloyl CA, 3',4'), 142.0, 142.1 , 142.3, 14304, 143.6 (2C), 143.7, 143.8. 143.8, 143.9, C-I, I'), 120.0, 120.1,121.7,121.8,123.3,124.5,124.6,125.3,125.8, 125.9,126.0, 127.3 (HHDPC-2, 144.2, 144.3, 144.4, 144.S, 144.6, 144.7, 145.0, 145.2 (2C), 146.1, 146.4 (HHDP C-4, 4',6,6', valoneoyl (2C) 2', galJoyl C-l, 1',2').134.3,135.2,13504,135.5 (2C), 135.7, 136.1,136.9,137.0, 138.4(HHDPC-5,5', CA,6,5",galloyIC-3,S,3',5'), 165.5,166.8,167.7,167.8,168.1 (2C), 168.2,168.3,168.8, 168.9 (ester carbonyls, valoneoyl C-7), sugar carbons, see Table Il-4. galloyl C-4, 4'),142.0, 142.1,143.3, 143.4, 143048, 143.52, 143.S4, 143 .6, 143.8, 144.3 (5C), 144.4, 144.5, 144.8. 145.2 (2C), 146.0 (HHDP C-4, 6, 4',6', galloyl C-3, 5, 3',5'), 164.7, 165.1, 167.0, 167.1, 167.6,167.7,168.0,168.1,168.1,168.2 (ester carbonyls), sugar carbons, see Table IIA. Methylation of 71-76 followed by Methanolysis Methylation of individual tannins (each I mg) was performed in a way similar to that for 69 and 70 described above. Each reaction mi '(ture was Methylation of 70 followed by Methanolysis To a solution of 70 (I mg) in acetone (I mL) were directly methanolyzed in 1% NaOMe in MeOH (1 mL) at room temperature for 6 h. After a usual work­ up, the reaction mixtures obtained from the individual tannins were analyzed by normal phase HPLC added (CH)2S0.. (0.0 I mL) and K2CO, (10 mg), and the mixture was stirred overnight at room tempera­ ture and retluxed for 3 h. After centrifugation, the supernatant was evaporated off and the reaction (solvent C) to commonly detect the peaks identical with those of the authentic 14, 15 and 33. In the case mixture was directly methanolyzed in 1% NaOMe in MeOH (I mL) at room temperature for 6 h. After of 73, 15 was detected as a minor product that was produced by ether cleavage of the valoncoyl group on methylation. acidification with acetic acid and evaporation of the solvent, the residue was partitioned between EtOAc and H ~O. The ElOAc extract was further treated with CH2N2 (I mL) for 1 h and the solvent was evapo­ rated . The normal phase HPLC analysis (solvent C) of the residue revealed peaks identical with those of Partial Hydrolysis of 71 with Tannase A solution of 71 (0.2 mg) in H"O (0.5 mL) was treated with tannase at 37°C for 5 d. After the addition of EtOH, the reaction mixture was evaporated and analyzed by the authentic 14 and dimethyl hexamethoxydiphenate (IS). normal (solvent B) and reversed-phase HPLC (solvent D), which showed a peak due to a monomeric tannin identical with that of 65b obtained from shephagenin A (65). Partial Hydrolysis of 70 with Tannase A solution of 70 (0.2 mg) in H20 (0.5 mL) was treated with 9 1 tannase (3 drops) obtained from Aspergillus niger according to the literature. ) at 37°C for 5 d. After the addition of EtOH, the reaction mixture wa evaporated to dryness. The normal and reversed-phase Degalloylation of Shephagenin A (65) with Tannase A solution of 65 (20 mg) in H"O (20 mL) was HPLC (solvent Band C, respectively) showed, in addition to the peaks of gallic acid and ellagic acid, a incubated with tannase (4 mL) at 37°C for 6 d. After concentration of the reaction mixture, the product peak due to a monomeric partial hydrolyzate which was identical with that of lagerstannin A (88) pre­ was chromatographed over Dia-ion HP-20 with H"O-MeOH. The 10% MeOH eluate gave desgalloylshephagenin A (6Sb) (2.5 mg). 65b: A pale brown amorphous powder, FAA-MS mlz: 969 pared from 68.

-90- -91- (M+H)·. 'H-NMR (acetone-d(,+D 0) 6: 7.12,6.63, 6.S8, 6.53, 6.16 (each 1H, s, HHDP, valoneoyl-H), ,, 2 s, H -6), 7.12 (2H, s, H, -2,6), 7.10 (I H, s, H,-6), 7.02 (I H, s, H,-3), 6.93 (I H, s, HIJ-3), 6.54 (I H, s, HH- S.7S (I H, dd, 1=2, 10 Hz, GluA H-3), S.36 (IH, d, 1=10 Hz, GluA H-2), 5.30 (lH, dd, 1=1.5, 10 Hz, GluA 3),6.49 ( I H , S, HJ-3), 6.18 (I H, s, HI -3), sugar protons, see Table Il-3. "C-NMR (acetone-d( +D,O) 6 : H-4), 4.72 (IH, dd, 1=3.5, J 2 Hz, GluA H-6), 4.27 (I H, dd, 1=3.5, 8 Hz, GluA H-5), 3.82 (I H, d, 1= 12 I 04.9, IOS.9 ~ 107.1, 108.6, 110.0, 110.7 (CBIJ.',U-3, C,.-6), 109.2 (C,, -6), 110.2 (2C)«(', -2, 6), i10 .6 (2C) Hz, GJuA H-6). (C(;-2, 6), II).O(C,-I), IIS.I (CJ-I), IIS.2(CJ)-I), 116.0(C -J), 116.2«(,\-1), I 16.8 (CII-I), 117.3(C -I), , ,

120.7 (C(;- I ), 120.8 (Cc- I), 121.1 (CII-2), 121.7 (CII-I), 122.9 (C\-3), 123.7, 124.9, 12S.5, 126.5, 127.1, Elaeagnatin 0 (72) A light-brown amorphous powder, Ia II) +96.6° (c= 1.0, MeOH). UV An~1\ (MeOH) 128.2 (C \ II, D.I.U-2), 135.0 (Cu-S), 135.7 (Cj-S), 136.6 (CI-S), 136.6 (CI)-S), 136.9 (CI-S), 137.4 (C -2), , nm (log E): 222 (S.28), 268 (4.90). Anal. Calcd for CK9H'80",'21 H 0: C, 44.S0; H, 4.19. Found: C, 44.24; 2 137.7 (C,-5), 137.9 (C"A), 139.2 (2C) (Cc .o -4), 139.7 (CIA), 140.2 (C,-3), 142.7 (C\-4), 143.0 «('I-S), H,3.83. bSI-MS mlz: 2040 (M+NH)'. FAB-MS mlz: 2023 (M+Hf, CD (MeOH) 181 (nm): +3.4 x 10' 144. J (C,, -5) 142.8, 144.2, 144.3, 144.4, 144.6, 144.8 (C',B.IH.U-6), 144.9 (CIA), 145.1 (CjA), 14S.2 (C - (234), -8.6 x IO~ (261), +1.0 x lOs (284). 'H-NMR (acetone-de, + 0 20) &: 7.10 (2H, s, H(-2, 6), 7.09 ( IH, Il 4), 14S.6 (CIlA), 145.7 (2C) (Cc; -3, S), 14S.9 (2C) (C(, -3, S), 146.8 (CIA), 146.9 «('11- 3),166.0 (C( -7), s, H -6), 7.03 (I H, S, H -6), 6.92 (I H, S, Hj-3), 6.74 (I H, s, H -3), 6.56 (I H, s, H,,-3), 6.52 (1 H, s, H -3), , , Il IJ 166.1 (C(;-7), 166.9 (C,-7), 167.7 (C,,-7), 167.8 (C,-7), 167.9 (C,-7), 168.S(C -7), 168.6«('1- ) 169.2 (C - IJ 7 I 6.42 (I H, H -3), 6.19 (1 H, H -3), sugar protons, see Table II-3. '1C_NMR (acetone-d,+D 0) 6 : s, ,, s, , r 2 7), 169.S (CII -7), sugar carbons, see Table 11-4. 104.8, IOS.6, 105.8, 107.6,108.6 (2C), 109.9, 110.6 (CI3.,U. ,U,,-3, C,.,-6), 110.2 (2C) (C, -2, 6), IIS.O (CI)- 1),115.1 (Cf- - I), IIS.3 (CJ-I), 116.2 (C,-I), 116.2,116.3 (C" G-J), 116.7 (CII-I), 116.7 (C, ,-I), 117.2 (CI- Elaeagnatin G (75) A light-brown amorphous powder, lall) +89.SO (c= 1.0, MeOH). UV A""I\ (MeOH) 1), 117.6 (C(;-3), 120.2 (C(-I), 120.S(C,-l), 120.7 (C\-2), 122.5 (C,,-2), 122.S, 123.7, 124.7, 125.1, 126.3 , nm (log E): 217 (5.08), 264(4.71). Anal. Calcd for C7SHi20.j8·8H20: C, 48.29; H, 3.67. Found: C, 48.53;

126.8, 127.3, 127.9 (C,-3, Cn.ll.I,O.II.I,J-2), 135.0 (Cn-5), 13S.1 (CII-5), ].35.9 (C,,-5), 136.S (CJ-5), 136.8 H,3.90. ESI-MS mlz: 1738 (M+NH/. FAB-MS mlz: 1721 (M+Hf, CD (MeOH) 181 (nm): +2.2 x 105 (C,-5), 136.9 (C,-2), 137.1 (CIl-S), 137.6, 137.8 (C~ .,, -5), 139.0 (C,-3), 139.2 (C -4), 139.7 (C -4), 142.9 c I (230), -6.9 x IO~ (261), +6.5 x IO ~ (284). 'H-NMR (acetone-dr,+D20) 6: 7.41 (I H, s, H,-6), 7.IS (2H, s, (C,-5), 140.0,142.6,142.7, 143.6, 143.7, 144.1, 144.2, 144.4, 144.5, 14S.2, 14S.3, 14S.5 (C\Il.(..J. I-4, C,. H( .-2, 6), 7.12 (I H, s, H,-6), 6.90 (I H, s, HI;-3), 6.79 (I H, S, H (; -3), 6.60 (I H, s, HI)-3), 6.56 (I H, s, H -3), .-6), 144.9 (C .-4), 14S.0 (C -4), (C -5), 145.8 (2C) (C -3, 146.0 (C -4), 146.9 (2C) (C - Il 11.1>.1..(, ,11.1 ." " n 14S.6 , c S), II I 6.18 ( I H, s, H II -3), sugar protons, see Table II-3. I1C-NMR (acetone-d,,+020 ) 6 : 104.1, IOS.3, 106.9, 4,C -3), 165.0«('(, -7), 166. 1 (C -7), 167.2 (C -7), 168.1 (C -7), 168.2 (CJ)-7), 168.4 (C-7), 168.4 (C\-7) 3 , c , , , 111.4 (CII I(;.11- ), 107.S (C,)-3), 107.8 (CI.-3), 109.2 (C,-6), 109.S (2C) (C( -2, 6),114.3 (C,-I), 114.6 (C _ 168.7 (C -7), 169.2 «(',,-7), 169.S (C -7), 170.4 (C -7), sugar carbons, see Table 11-4. , n II 1), IIS.O(CIl-I), IIS.5(C\-I), 115.5(Co -I), 116.1 (CIl-I), 116.7(C,, -l), 1 18.4 (C\-3), 120.1 (C(. -I), 122.12 (C,-I), 122.S (C,-2), 122.6, 124.3, 125.6, 12S.8, 126.0, 127.S (C -2),134.3 (C -S) 135.3 (C -5) _ .\,B,D.I (.. II Il' IJ ' Elaeagnatin E (73) A light-brown amorphous powder, lall) +98.So (c=1.0, MeOH). UV Ama\ (MeOH) 13).7 (C,;-5), 136.1 (CI-S), 136.5 (CII-S), 136.9 (C,-2), 137.6 (CI-4), 138.S (C\-S), 139.2 (C(.-4), 139.4 nm (log E): 216 (S .27), 268 (4.94). Anal. Ca1cd for C96H62061'14H20: C, 47.18; H, 3.71. Found: C, 47.24; (2C) (C,-3, 4), 142.0 (2C) (C\A, 6), 142.4 (CI-S), 143.1 (CI-S), 143.3, 143.6, 143.6, 143.8 (C -6) s H,3.82. ESI-MS mlz: 2208 (M+NHy. FAB-MS mlz: 2191 (M+H( CD (MeOH) 181 (nm): +2.8 x10 144.1 (CI-4), 144.3 (C(;A), 144.5 (CI)-4) 144.8 (C -4), 145.2 (2C) (C('-3. 5),146.0 (C -3), II , 14~.~'(~~ ::-4): 5 (228), -7.3 x 10 ~ (260), +1.0 x 10 (284). 'H-NMR (acetone-dr,+D 0) 6: 7.11 (IH, s, H,-6), 7.09, 7.00 , 2 165.0 (Cc-7), 16S.4(C -7), 166.7 (C,-7), 166.9 (C\-7), 167.67 (C(;-7), 167.70 (C,-7), 167.9 (CI)-7), 168.0 ,, (each I H, S, H,.,-6), 7.03 (2H, s, Hc-2, 6), 6.93 (1 H, s, HI)-3), 6.72 ( I H, s, H J-3), 6.51,6.44 (each 1H, S, HH (C -7), 168.7 (CII-7), sugar carbons, see Table IIA. 11-3),6.32 (I H, s, H,-3), 6.22 (I H , S, H,,-3), sugar protons, see Table 1l-3. I1C-NMR (acetone - d(, +D ~ O) 6 : 105.0, 105 .S, 105.8, 106.0, 108.3, 108.7, 109.8, 111.2 (C~ . 'H.II.J.K-3, CI 1.-6), 109.9 (C,-6), 110.2 (2C) (C(, -2,6), IIS.0«(',)-I), 115.1 , IIS.6(C\.(;- I), 116.0(Cj- l), 116.3 (Cn-I), 116.4(C,, -I), 116.8 (2C) (Clo,"- 1),117.3, 117.4(C,, -I), 120.3 (Cc-I), 120.8 (C,-l), 122 .S (C,\-3), 122.7 (C,-2), 123.9 (C(;-3 ), 117.6,

120.S , 124.6, 124.8, 126.4, 126.7,127.4, 127.9(C\Il.D.E,Ci ,II,u-2), 134.9 (CB-5), 13S.1 (CII-S), 136.5 (CI)- 5), 136.9, 137.9 (C 1<;-5), 136.9 (C,,-S), 137.0 (C,,-5), 137.3 (CJ-5), 137.7 (2C)(C,.. , -2), 138.0, 138.9 (CI 1-3).139.2,139.8 (e, ,1--+) , 140.0, 140.1 (CI,I-4), 142.3,143.0 (C,.,-5), 142.8 (C\-4), 143.0,143.5,143.8,

144.0, 14-+.2, 144.S, 145.3 (C 'llIl, I (;,IIU-6), 144.8 (CIi-4), 144.9 (CJ-4), 14S.5 (CI-S), 145.6 (C,,-4), 145.7 (2C) (C, -3, S), 146.1 (ell--+). 146.8 (CI-4), 146.8 (C,,-4), 147.1 (C,-3), 164.9, 166.1 (C( .1-7), 167.2, 167.6

(C\ (,-7), 167.8, 168.2(C,, -7), 168.3 (C,)-7), 168.5 (C(-7), 168.6 (C,,-7), 169.1 (C,-7), 169.6 (CII -7), 170.S (C,,-7), sugar carbons, see Table 11-4.

Elaeagnatin F (74) A light-brown amorphous powder, lal,) +SO.8° (c=I.O, MeOH). UY An", (MeOH) nm (log E): 220 (S.14), 264 (4.78). Anal. Calcd for C~2Hy, O,.\·11 H20: C, 47.18; H, 3.77. Found: C, 47.42; H , 3.89. ESI-MS mlz: 1906 (M+NH)'. FAB-MS mlz: 1889 (M+Hf, CD (MeOH) 181 (nm): +2.S xlO' (232), -7.8 x IO~ (262), +8.8 x IO~ (284). 'H-NMR (acetone-d(,+D20) 6: 7.27 (2H, s, H(; -2, 6), 7.22 (I H ,

-92- -93 - References Acknowledgements

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-97- -96-

...... -~.., .. ~""'"" .. ~ ~ '- - .. ... ~ .~. ~----~ 100) Peng S., ScalbeltA., Monties B., Phytochemistry, 30, 775 (1991). List of Compounds 101) Tanaka T, Ueda N., Shinohara H., Nonaka G., Fujioka T, Mihashi K., Kouno I., Chem. Pharm. Bull., 44, 2236 (1996). 1 Geraniin 102) Lin T-l., Tanaka T, Nonaka G., Nishioka I., Young T-L., Chem. Pharm. Bull., 39, 1144 (1991). 2 Chebulagic acid 103) Kadota S., Takamori Y., Nyein K.N., Kikuchi T, Tanaka K., Ekimoto H., Chem. Pharm. Bull., 38, 3 Phyllanthusiin A 2687 (1990). 3a Tridecamethyl phyllanthusiin A 104) Tsai YJ., Aoki T., Murata H., Abe H., Sakagami H., Hatano T., Okuda T., Tanuma S., 1. BioI. 4 Phyllanthusiin 8 Chern., 267, 14436 (1992). 4a Tridecamethyl phyllanthusiin 8 105) Nakashima H. , Murakami T., Yamamoto N., Sakagami H., Tanuma S., Hatano T., Yoshida T., 4b Hexamethyl derivative from 4 Okuda T., Antiviral Res., 18,91 (1992). 5 Phyllamhusiin C 106) Hatano T., Edamatsu R., Hiramatsu M., Mori K., Fujita Y, Yasuhara T., Yoshida T., Okuda T, Sa Dodecamethyl phyllanthusiin C Chern. Pharm. Bull. , 37, 2016 ( 1989). 6 PhylJanthusiin D 107) Ito H., Miyake M., Nishitani E., Mori K., Hatano T., Okuda T., Konoshima T, Takasaki M., Kozuka 7 Phyllanthusiin E M., Mukaikawa T., Tokuda H., Nishino H., Yoshida T., Cancer Lett., 143,5 (1999). 8 Bergenin 108) Berenblum I., Cancer Res., 1,807 (1941). 9 Brevifolincarboxylic acid 109) Ito Y, Kawanishi M., Harayama T., Takabayashi S., Cancer Lett., 12,175 (1981). 9a Methyl tri-O-methylbrevifolincarboxylic acid 110) Konoshiama T., Takasaki M., Kozuka M. , Inada A., Nakanishi T., Tokuda H., Matsumoto T., 10 Corilagin Shoyakugaku Zasshi, 43, 135 ( 1989). lOa Oeca-O-methylcorilagin III) Henle G., Henle W., 1. Bacteriol, 91, 1248 (1966). lOb Nona-O-methylcorilagin 112) Yoshizawa S., Horiuchi H., Fujiki H., Yoshida T., Okuda T., Sugimura T., Phytotherapy Res., 1,44 11 Repandusinic acid A ( 1987). 12 Putranjivain A 113) Yoshizawa S., Horiuchi T., Suganuma M., Nishiwaki S., Yatsunami J., Okabe S., Okuda T, Muto 13 Geraniinic acid B Y., Frenkel K., Troll W., Fujiki H., Phenolic compounds in food and thier effects on health lI. 13a Tridecamethyl derivative from 13 Antioxidant & cancer prevention, ed. M.T. Huang, C.T Ho, and c.Y. Lee. 1992, Washington, DC.: 14 Methyl tri-O-methylgallate ACS s) mposium series 507. 316. IS Dimethy hexamethoxydiphenate 114) Nonaka G., Nishimura H., Nishioka 1.,1. Chem. Soc. Perkin Trans. 1,163 (1985). 16 Furosinin liS) Yasuhara T., Hatano T., Okuda T., Abstructs of papers, The II Oth Annual Meeting of Pharmaceuti- 17 Furosin cal Society of Japan, Sapporo, 1990, 224. 18 Didehydrogeraniin 116) Warren 1.R., Marshall B.1., Lancet, 1, 1273 (1983). 19 Elaeocarpusin 117) Lee A., Fox J., HaLell S., Infect. Immun., 61,1601 (1993). 20 Geraniinic acid A 118) Hopkins R.1 ., Morris J.1. , Am. 1. Med., 97, 265 (1994). 21 Geraniinic acid C 119) Parsonnet 1.. hicdman G.O., Vandersteen D.P., Chang Y, Vogelman J.H., Orentreich N., Sible) 21a TridecamethyJ derivative from 21 R.K., N. Engl. 1. Med .. 325,1127 (1991). 22 PhyIJanthusiin F 120) Unten R., Abstructs of papers, The Annual Meeting of the Japan Society for Bioscience, Biotech­ 23 Acalyphidin MI nolog) and Agrochemistry, Kyoto, 1996, 8. 24 Acalyphidin M2 24a Tridecamethyl derivative from 24 25 Acalyphidin 0 1 25a Acetonyl derivative from 25 26 Mallotusinin 27 Euphorbin A 28 Euphorbin B 29 Excoecarianin

-98- - 99- 29a Acetonyl derivative of 29 69 Elaeagnatin A 30 Euphorbin 0 70 Elaeagnatin B 31 1,2,3,4,6-Penta-O-galloyl-p-D-glucose 71 Elaeagnatin C 32 Repandinin A 72 Elaeagnati n 0 33 Trimcthyl octa-O-methylvaloneate 73 Elaeagnatin E 34 Isomallotinic acid 74 Elaeagnatin f 35 Euphormisin M2 75 Elaeagnatin G 36 Bixanin 76 1,6-0i-O-galloyl -p-D-glucose 37 Amariinic acid 77 Pedunculagin 38 Granatin B 78 Punigluconin 39 Mallotusinic acid 79 Lagerstannin C 40 Mallojaponin 80 2,3-(S)-HHOP-D-glucose 41 Mallotunin 81 Pterocarinin A 42 Macaranin A 82 Hippophaenin B 43 Macaranin C 83 Casuariin 44 Macarinin A 84 Oesgalloylstachyurin 45 Macarinin B 85 Alienanin B Mallonin 86 Cas ugl aunin A '"47 Chebulanin 87 Stachyurin 48 Terchebin 88 Lagerstannin A 49 Supinanin 89 Casuarinin 50 3-Desgalloylterchebin 90 Lagerstannin B 51 Virganin 91 Resinin A 52 Mallotinin 92 Resinin B 53 Repandusinic acid B 93 Resinin C 54 Repandusinin 94 Resinin 0 55 Eumaculin E 95 Lagerstonin 96 Lythcarin 0 56 Amariin 1

57 Tanarinin 97 Lythcarin O2 58 Aleurinin A 98 Heterophylliin B 59 Aleurinin C 99 Heterophylliin C 60 Galloylgeraniin 100 Alienanin A 61 Amarulone 101 Roburin A 62 Carpinusin 102 Roburin B 63 Helioscopin A 103 Roburin C 64 Euphorscopin 104 Roburin 0 65 Shephagenin A 105 Anogeissusin A 65a Nonadecamethyl derivative from 65 106 Anogeissusin B 6Sb Oesgalloylshephagenin A 107 Casuglaunin B 66 Shephagenin B 108 Anogeissinin 67 Strictinin 109 Stenophyllanin A 68 Hippophaenin A no Epigallocatechin gallate

- 100- - 101- 111 Tellimagrandin I 112 Tellimagrandin II 113 Casuarictin 114 Agrimoniin 115 Nobotanin 8 116 Oenothein 8 117 Rugosin D 118 Euphorbin C 119 Oenothein A 120 Heterophylliin G 121 Procyanidin 8-1 122 Procyanidin 8 -3 123 Procyanidin 8-4 124 Procyanidin 8-5 125 Procyanidin C-I 126 Epicatechin gallate 127 Epicatechin

- 102-