Some bioactive natural products from chinese medicinal plants

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 21 © 2000 Elsevier Science B.V. All rights reserved

729

SOME BIOACTWE NATURAL PRODUCTS FROM CHINESE MEDICINAL PLANTS REN-SHENG XU Shanghai Institute of Materia Medica, Academia Sinica, Shanghai, 200031 China ABSTRACT: China is a country rich in medicinal plants because of its wide territory and variety of geography. Plentiful experience has been accumulated and recorded in the long history of traditional Chinese medicine, but the mechanisms of many Chinese medicines remain unclear. This fascinating research field has been attracting tremendous research efforts of scientists in chemistry, biology and medical sciences. Some successes have been achieved, from which the antimalarial artemisinin (qinghaosu) is an example. In this paper a few important recent researches on the bioactive components from the Chinese medicinal plants are discussed. These are the antitumor tubeimosides, isolated from bolbstemma panicnlatum\ cholinergic decumbenines from Corydalis decumbens\ antihipatitis sarmentosin from Sedum sarmentosium\ antiHIV active principle licochalcone A from Glycyrrhiza inflata\ cholinesterase inhibitor huperzine A from Huperzia serrata, which is already used for treatment of Alzheimer's disease in China; miotic agent baogongteng A from Erycibe obstusifolia; antifungal pseudolaric acid B from Pseudolarix kaempferi and insecticide stemona alkaloids from Stemona plants. Their structure elucidation, total syntheses, chemistry, bioactivities and some of the clinical trial results will be presented.

INTRODUCTION The recorded history of using medicinal herbs by Chinese people in treating diseases can be traced to thousands of years ago. Back in 1590, the prominent Chinese doctor Li Shi-zhen summarized a total of 1,892 Chinese medicines and a total of 11,096 formulae in his famous "Compendium of Materia Medica" (Ben-Cao-Gang-Mu). Today, a total of 12,807 Chinese medicines are officially documented, among which 11,146 are from plant source; 1,581 from animal source, and 80 from mineral source [1]. The number of the most commonly used Chinese medicines, however, is about 500. Most of them are included in the volume I of the Chinese Pharmacopoeia (1995 edition). As a result of its wide territory and variety of geography, China is rich in both medicinal plants and herb medicine culture. At the present time, Chinese medicine enjoys even more popularity compared to the modern western medicine in China. Both the traditional Chinese medicine (TCM) * Present address: 75 Los Altos Squar, Los Altos, CA 94022, USA

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REN-SHENG XII

and western medicine are practiced in hospitals all over China. There are large numbers of manufacturers of Chinese medicines and Chinese drug stores in China. Chinese medicine forms a unique school in the medical science. Some Chinese medicines have proven to be more effective in treatment of certain human diseases in comparison to western medicine. Rich experience has been accumulated and recorded in the long history of TCM, although the mechanisms of many Chinese medicines remain unclear. This fascinating research field has been attracting tremendous research efforts of scientists from China, Japan, and many other countries. While the major effort in the study of Chinese medicines has been focused on active principles, there are considerable efforts focused on the study of active fractions of herbs. Numerous evidences suggest that multiple components contained in an active fraction of an herb would play synchronism in treatment of disease, although a single component may play a major role and other components would enhance the bioactivity and reduce the toxicity of the fraction. In addition, multiple ingredients in formulae of TCM from different medicines may as well show similar synchronism. In research of the chemical components of Chinese medicines, the structures and bioactivities of more than a thousand new natural product compounds have been studied, published, and reviewed [2-5]. A notorious example is the study of antimalarial artemisinin (qinghaosu) and its derivatives [6,7]. In this paper a few important recent researches on the bioactive compounds from the Chinese medicinal plants are discussed. Most of them were developed within my research group, Department of Phytochemistry, Shanghai Institute of Materia Medica (SIMM), the Chinese Academy of Sciences. 1. ANTITUMOR TUBEIMOSIDES The bulb of Bolbostemma paniculatum [Maxim] Franquet (Family Cucurbitaceae) is used in China as a folk medicine for treating, curing tumors and warts [8]. The ground bulb was extracted with 75% ethanol, then the alcohol was removed by evaporation, the remaining water layer was defatted with chloroform and extracted with n-butanol, the butanol extract was then concentrated and chromatographed on normal and reverse silica-gel column, as well as preparative HPLC separation. Four saponins were separated, namely tubeimoside I, II, III and IV [9-11]. Structure studies showed tubeimoside I 1-1, II 1-2 and III 1-3 are rarely occurred macrocyclic saponin with an unusual dicrotalic acid bridge between two sugar chains in nature.

BIOACTIVE NATURAL PRODUCTS

" ^

731

t^ST

...

OH 1-5 1-7 1-8

R=H R = cx-L-Rha(l->2)-a-L-AraR = p-D-Xyl(l->3)-a-L-Rha(l->2)-a-L-Ara-

Scheme 1. Structures of tubeimosides.

Tubeimoside I 1-1, white needles, m.p. 250-252°C. [a] D 13.0° (c, 0.8, MeOH), according to its MS and 13C-NMR spectral data, has a molecular formula of C 63 H 98 0 2 9 , MW1318 (SIMS m/z 1319, [M+H]+, and m/z 1341, [M+Na]+). Mild acid hydrolysis of 1-1 afforded bayogenin, glucose, arabinose, rhamnose and xylose. 13C-NMR spectrum of 1-1 showed five anomeric carbon signals (8 94.1, 100.6, 103.5, 104.4, 106.4, 106.6) and three carbonyl signals (8 171.1, 171.2, 175.9). Signals at 8 94.1 and 175.9 indicated the presence of an ester glycosidic linkage at C-28 of bayogenin. Hydrolysis of 1-1 with dilute potassium hydroxide yielded prosapogenin 1-5 and trisaccharide, which was acid hydrolyzed to arabinose, rhamnose and xylose. The linkage position of arabinose and glucose in 1-5 was proposed by comparing its 13C-NMR spectrum with those of methyl ester of bayogenin, methyl-(5-D-gluco- and oc-L-arabino-pyranoside as 30-[a-L-arabinopyranosyl (!->2)-p-D-glucopyranosyl]-bayogenin. When

732

REN-SHENG Xtl

1-1 hydrolyzed with cellulase, terminal xylose was removed and prosapogenin 1-6 was obtained, which possessed three ester carbonyl groups (8 176.0, 171.2 and 171.1). When hydrolyzed 1-6 with 5% potassium carbonate the result obtained was a deacylated compound 1-7, containing only one ester carbonyl group (8 176.1, C-28 of 1-7). Hydrolysis of 1-1 with 0.5% potassium hydroxide gave deacylated compound 1-8 and dicrotalic acid (3-hydroxyl-3-methyl-pentanedioic acid) 1-9. A remarkable glycosidation shift 10.8 ppm (from 8 72.3 to 83.1) was found at C-3 of rhamnose in a comparative studies on 13C-NMR spectra of 1-6 and 1-8, indicating that the terminal xylose was attached to C-3 of that rhamnose. Comparison of 13C-NMR spectrum of 1-7 with that of the known [a-L-rhamnopyranosyl(l-2)-a-L-arabinopyranosyl]-3-0-acetyloleanolate, deduced the sequence of the sugar chain on the C-28 carbonyl group of 1-7 to be 3 - 0 - [ a - L - a r a b i n o p y r a n o s y l - ( l - > 2 ) - p - D glucopyranosyl-]-bayogenin-28-0-[a-L-rham-nopyranosyl-(l->2)-a-Larabinopyranosyl] ester. Finally the comparative studies on JH-NMR spectra of 1-6 and 1-7 using ^-JH-COSY (correlated spectroscopy) technique showed the signals of H-4 of rhamnose and terminal arabinose were significantly shifted to downfield by 1.57 ppm (8 4.32 to 5.89) and 1.23 ppm (8 4.33 to 5.56 ppm), respectively. These facts indicated the dicrotalic acid was bounded to C-4 of rhamnose and C-4 of terminal arabinose. Therefore the structure of 1-1 could be proposed as shown in the Scheme [9,10]. Tubeimoside II 1-2, colorless amorphous powder, C63H9803o, [oc]D 16.0° (c, 0.32, pyridine), SIMS showed the [M+H]+ion peak is at m/z 1335. It has one more hydroxy 1 group than that of 1-1. When comparing both 13C-NMR, can be deduced that its structure is 16-oc-hydroxytubeimoside I. Tubeimoside III 1-3, colorless amorphous powder, C^HiooC^i, [OC]D 7.9° (c, 0.52, pyridine), SIMS showed the [M+H]+ ion peak is at m/z 1365. It has P-D-glucopyranosyl(l->2)-p-D-glucopyranosyl linked with C-3 of bayogenin instead of oc-L -arabinopyranosyl-(l->2)-(}-Dglucopyranosyl in 1-2. The structure was determined in the same way as 1-3 [11]. Tubeimoside IV 1-4, white needles, m.p. 187-189°C, C41H70O14, [a] D 3.1°(c, 2.09, MeOH), FABMS showed the [M+Na]+ at m/z 809. Its *HNMR spectrum exhibited signals at 8 1.53, 1.02, 0.96, 0.88, 0.85 and 0.75 (all are singlet) for six methyl groups, 3.74 (2H, s)\ 3.89, 4.29 (each 1H, AB-system, J = 11.6Hz) for two hydroxymethene and 8 5.23 (1H, t, J = 6.8Hz) for a trisubstituted olefinic proton adjacent to methylene group (8 2.0, 2H, m). The "H^H COSY experiments of 1-4 showed the olefinic proton (8 5.23) is coupled with methyl (8 1.53) and hydroxymethene (8 3.74). The spectral data strongly suggested that 1-4 is a tetracyclic triterpenoid saponin. Comparing the 13C-NMR spectrum of 1-4 with that of the known actinostemmoside C indicated that both aglycone moieties

BIOACTIVE NATURAL PRODUCTS

733

were very similar, only the substituted position of a hydroxyl group in the side chain was different. The two carbon signals (8 14.0 and 68.2) of 1-4 suggested that the hydroxyl group is substituted on C-26. The glycosidic linkage positions could be determined by the glycosylation shifts 10.8 ppm for C-3 of aglycone and 8.8ppm for C-2 of glucose. Besides, the proton signals 8 3.75 for H-2 of glucose and 8 3.08 for H-3 aglycone in the •H-NMR spectrum of acetate of 1-4 were further confirmed the glycosidic linkage position. Finally, the coupling constants (6.7 and 7.4 Hz) of anomeric protons of 1-4 indicated that the anomeric configurations of glucose and arabinose were (3 and a. Thus the structure of tubeimoside IV 1-4 can be assigned as 3-0-[a-L-arabinopyranosyl(l->2)-p-Dglucopyranosyl]-3p, 7p, 18, 20, 26-pentahydroxy-(205)- dammar-24-ene (Scheme 1) [11]. Tanaka's group conducted the same research independently and reached the same results [12,13]. We have communicated and shared informations from time to time. Antitumor Activity of Tubeimoside I Group of BALB/c mice were used for in vivo tests by Yu et al [14]. When administered i.p. or i.m. 3 x 16 or 4 x 14 (mg/kg/day x days) tubeimoside I caused 71.5% and 64.0% inhibition of tumor growth within 20 days. (P< 0.001). In vitro test for 6 different human malignant cell lines IC50 (m mol/1) for line A-172 was 0.15; Goto-0.24; PANC-1--0.27; COLO320DM--0.43; Hela-0.79 and HGC-27--0.80. It was also noticed that tubeimoside I had no histologically detectable effects on mouse skin. These results showed tubeimoside I is a potent antitumor drug candidate for further investigation. 2. CHOLINERGIC DECUMBENINES The plant Corydalis decumbens (Thunb.) Pers. (Papaveraceae) and the crude alkaloid fraction separated from the plant have been used in folk medicine in China to treat hypertension, hemiplegia and pseudomyopia. Several isoquinoline alkaloids such as berberine, bicuculine, bulbocapnine, jatrihizine, palmatine, tetrahydropalmatine and minor new alkaloids decumbenine, decumbenine B and decumbenine C were separated from the alkaloid fraction of the plant by repeated chromatography on silica-gel column. The structure of decumbenine 2-1, decumbenine B 2-2 and decumbenine C 2-3 are determined by spectral analyses and comparison with known isoquinoline alkaloids (Scheme 2). Decumbenine 2-1, colorless cubic, m.p. 205-207°C, [ a ] D -45.6° (CHC13), C2oH17N06. Its UV spectrum absorptions are at A,max (loge) 222 (4.56), 297 (3.94), 326 (3.89) nm and IR spectrum absorption peaks are at

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REN-SHENG XH

vmax 930 and 1760 cm-'. 'H-NMR spectrum signals are at 5 2.46 (1H, s, NCH 3 ), 5.80, 6.06 (each 2H, s, 20CH 2 0), 3.98 (1H, d,J=5 Hz, H-l), 6.36 (1H, s, H-5), 6.64 (1H, s, H-8), 5.58 (1H, d, J= 5 Hz, H-9), 6.88 (1H, d, J= 8 Hz, H-4'), 7.13 (1H, d, J= 8 Hz, H-5') [15]. Decubenine B 2-2, colorless needle, m.p. 222-224°C. It has a molecular formula of C18H13NO5, according to its HRMS molecular ion M + at m/z 323.0776. Its UV spectrum absorption is at vmax 224, 280, 304, 340 ran. Its IR spectrum absorptions are at v max 3250, 1640, 1600, 1570, 1500, 1080 cm-1; its iH-NMR signals are at 8 9.14 (1H, s, H-l), 7.81 (1H, s, H4), 7.62 (1H, d, J= 8.6Hz, H-8), 7.30 (1H, d, J= 8.6Hz, H-7), 7.14 (1H, d,J= 8.0Hz, H-6'), 6.85 (1H, d, J= 8.0Hz, H-5'), 6.26 (2H, s, OCH 2 0), 6.10 (2H, s, OCH 2 0), 4.54 (2H, s, CH 2 0); ^C-NMR signals are at 5 152.3 (s, C-3), 151.3 (d, C-l), 123.6, 147.9, 147.5, 146.9, 140.4 (each s, C-Ar), 134.8 (s, C-4a), 124.0, 123.0, 111.9, 111.6, 107.6,(each
Scheme 2. Structures of decumbenines.

Decubenine C 2-3, brown needles, m.p. 211-213°C. It has molecular formula of Ci 9 H||N0 6 according to its HRMS molecular ion M+ at m/z 349.0548. Its UV spectrum absorptions at Xmax 237, 280, 318(sh.) ran are very similar to that of papaverine. Its IR spectrum absorptions are at vmax 1760, 1500, 1475, 1260, 1040, 940 cm-' and 'H-NMR signals are at 8 8.31 (1H, d, J = 6 Hz, H-3), 7.53 (1H, d, J = 6 Hz, H-4), 7.40 (1H, s, H-5), 7.12 (2H, s, H-8 and H-9), 6.21(2H, d,J= 7.5Hz, OCH 2 0), 7.06 (1H, d, J

BIOACTIVE NATURAL PRODUCTS

735

= 8 Hz, H-2'), 6.82 (1H,
* The details will be published in tetrahedron., by Xu, X.Y.; Qin, G.W.; Xu, R.S.

REN-SHENG XI!

736

Biological Activity of Decumbenine B* The synthetic decumbenine B 2-2 has been tested in the Department of Pharmacology of SIMM. The primary results showed decumbenine B has potent cholinergic activity. It inhibits the spontaneous contraction in the

0H

73%

NEto

2-6

NEt9 Reagents: a. Ag2O.NaOH. b. i) SOCl2,reflux: ii) HNEt2.C6H6.reflux. c. i) n-BuLi.THF, TMEDA,-78°C; ii) CH3I.

2

-7

/-? O.

2 - 10

°VJ^S

V

CH2OBn

JL

COOEt

2 - 11

O^

^X^

CH2OBn

k

ri

CH2OBzn

100% NBn 2 - 14 Reagents: e.H 2 NC 6 H,, ,C6H6,80PC. f. i)n-BuLi,THF,-78°C; ii) CICOOEt. g.HS(CH2)3SH,H0Ac,BF30Et2,CHCl3,RT h.LiAlH4,THF,reflux,8h. i. BnBr.NaH.THF.TBA. j . (CF3C00)2IPh, CH3CN-H20,RT. k.BnNH2,C6H6,reflux, lOh.

737

BIOACTIVE NATURAL PRODUCTS

(Scheme 3) contd..

2-7

+2-14

OBn 2 - 16

^ - Decumbenine B 2 - 2

Reagents: 1. LDA.dil.HCl . m. LiAlH4,THF,RT. n. 10%Pd/C,H 2 ,HOAc,RT. o. DDQ,C 6 H 6 , reflux. Scheme 3. Total synthesis of decubenine B.

intestine of mouse with maximum effect at 2 |Hg/ml. The parasympathomimetic activity of decumbenine B is similar to atropine, an antagonist of muscarinic agent, the inhibitory effect of atropine on the intestine is about 0.4 |ig/ml. The results also suggested that decumbenine B may block the spontaneous contraction by acting on muscarinic receptors. The research is in progress. 3. ANTIHEPATITIS SARMENTOSIN The new cyanogenic glycoside sarmentosin was separated from the whole plant Sedum sarmentosum Bunge (Crassulaceae). The plant is used in

738

REN-SHENG XH

Chinese folk medicine as an antibacterial agent and for treatment of chronic virus hepatitis. The antihepatitis principle was found to be a new ycyanogenic glycoside, namely sarmentosin, by coordinated separation with bioassay. The whole plant was ground and extracted with hot ethanol (95%), followed by evaporation of the alcohol from the extract, the remaining water layer was defatted with chloroform first and then passed through a granule carbon column, eluted with water and then 70% ethanol. The alcohol eluate was concentrated and chromatographed on silica-gel column, which was eluted with water saturated ethyl acetate and then ethyl acetate/methanol (8/1). The latter eluate was repeatedly chromatographed on silica-gel column with elution of ethyl acetate/acetone (3/2), detected by TLC, the water soluble gelatin substance, sarmentosin was obtained with 0.1% yield [17]. There are several other water soluble compounds, such as mannitol, leucine, valine, alanine, tyrosine, asparagine, and aspartic acid were also separated from the plant. Sarmentosin has a molecular formula of CnHi 7 N0 7 , [oc]D -17.4° (c, 0.62, H 2 0). Its structure was determined by spectral analyses and confirmed by X-ray crystallography as 2 - c y a n o - 4 - 0 - p - D glucopyranosyl-trans-buten-2-ol 3-1. Its UV absorption at ^ max 212 nm (log 8 4.02) and IR absorptions at vmax 3540-3240, 2235, 1640, 1110,1085 cm"1 indicated presence of unsaturated double bonds , nitrile and hydroxy 1 groups. 3-1 could be easily acetylated with acetyl anhydride and concentrated sulfuric acid to form colorless crystalic pentaacetyl sarmentosin with m. p. of 79-80°C. The acetyl compound has ] H-NMR signals at 5 1.91, 1.96, 2.01, 2.07 (15H, s, 5 acetyl group), 3.70 (1H, m, H5'), 4.13 (2H, d,J=5 Hz, H-6'), 4.48 (2H, d, J= 1 Hz , H-l), 4.60 (2H, s9 H-4), 4.57 (1H, overlapped with signal of H-4, H-l'), 4.96 (3H, m J= 8 Hz , H-2',3',4'). The 13C-NMR (D 2 0) signals of 3-1 are at 6 103.0 (
o

'CHpH

S~*< £ c ® x N = C CH 2 OH

0

3 1 Scheme 4. Isomerization of sarmentosin by Michael addition.

N C

CH N / CH2OH 3-2

BIOACTIVE NATURAL

739

PRODUCTS

CH2OH) and 5 3.48 (1H, /n, -OCH-CH(CN)CH2OH) and the signal of olefinic proton disappeared. Its 13C-NMR signals are at 8 98.2 (d, C-lf), 80.0 (
-CH2CH2OH OAc

AcO' A £ 2 ° AcO,

.OCH2CH2CHCH2O OAc'

O

CH 3 CH 3

3-4 TsOH/MeOH OAc

f

AcO/V^-^°x A c O . \ _ ^ ^ \ ^ ^ ^

„OAc

Ph 3 CCI/ EtjN/DMAP

OCH 2 CH122CH< CHCH 2 OCPh 3 1 OH

ACOQ

3 - 6 PCC/NaOAc ^OAc AcO'^ V ^ AcO. OX ^

OCH 2 CH 2 CHCH 2 OH OAc 3- 5

(CH 3 ) 2 CO/ H C N HCN »

-*°\ * ^ \ OCH9CH9CCH9OCPhor~T~rT^ 2 2 3 ^ OAc - V II N a MeOH HC03/

OAc OH A c vO ' V ^ ^ Q v I AccA *^\ OCH2CH2CCH2OCP1^ ^ ^ V I OAc ICN 3-8

3-7

so 2 ci

JO Ac

^OAc

A c O " ^ — - Q

TMSI/CHCI3

^C?

H

OAc NC

OH

X

T

AcO^A——Q ^Q? A c O ^ ^ ^ S ^ O OAc

CH2OH

CH 2 OCPh 3

3 - 10 MeOH/Et3N/H20 3- 1

Scheme. 5. Total synthesis of sarmentosin.

H

3-9

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REN-SHENG XII

form cyanohydrin 3-8, which was subjected to dehydration with thionyl chloride in pyridine to convert to E-olefin 3-9 as the only isomer. Finally, selective cleavage of trityl ether of 3 - 9 with ( 1 trimethylsilyl)imidazole(TMSI) (1.2 equiv. TMSI in CHC13, N2) at room temperature yielded allyl alcohol 3-10 and subsequent deacetylation with MeOH-Et 3 N-H 2 0 (8:1:1) to form the sarmentosin. It was identified by spectral analyses and comparison with the authentic natural compound (Scheme 5) [18]. With a dosage of 100 mg per day, sarmentosin was proven to be active by clinical trial to lower the SGPT (serum glutamic pyruvic transaminase) level of patients suffering from chronic hepatitis. Isosarmentosin is not active against hepatitis [17]. 4. ANTIHIV LICOCHALCONE Licorice (Gan-Cao) is one of the famous Chinese medicines and is used in many formulae of TCM as antitoxin, antitussive, expectorant and as a sweetness ingredient. Among several species of Glycyrrhiza, Glycyrrhiza uralensis Fisch (leguminaceae) is the most popularly used. Glycyrrhiza inflata Bat., natively grown in Xinjian Region of China, is of the finest quality and is now also accepted in TCM. From the roots of Gl. inflata, a new chalcone, namely licochalcone, was separated by the auther [19]. The ground roots were extracted with chloroform and then chromatographed on polyamide column eluted with dilute alcohol to get the yellow solution. After concentration of the solution, it was purified by passing through an aluminum oxide column with elution of chloroform. The yellow powder was obtained upon evaporation of the chloroform and then was treated with dilute alcohol to form red needle crystal licochalcone. The yield is about 0.1%. Licochalcone, m.p. 136-138°C, C 2 iH 2 2 0 4 ) possessing chalcone character, was demonstrated by its UV absorption spectrum at A,max (loge): 256 (4.04), 315 (4.15), 380 (4.46) nm, IR absorption spectrum at vmax 1576, 1595, 1613, 1660 cnr1 and formation of violet precipitate with SbCl5. Its final structure 4-1 was determined by additional NMR spectrum analyses and chemical degradation. Its 1H-NMR signals showed the presence of AA'BB' aromatic protons at 8 6.94, 7.94 (each 2H, d,J = 8 Hz), two aromatic protons at 8 6.40, 7.40 (each 1H, s), two phenolic protons at 8 6.44 (2H, br), one aromatic methoxyl group at 8 3.78, two trans olefinic protons at 8 7.54, 8.00 (each 1H, d, J = 16Hz) and ArC(CH 3 ) 2 CH=CH 2 at 8 1.40 with ABC system at 8 6,14 (1H, q, J= 10, 16Hz), 5.26 (1H, d,J= 10Hz), 5.30 (1H, d,J= 16Hz). Alkali cleavage of licochalcone with 50% KOH gave acetophenone 4-2 and an aromatic aldehyde 4-3. When the latter was methylated with methyl iodide, hydrogenated with Adam's catalyst and oxidized with potassium

741

BIOACTIVE NATURAL PRODUCTS

OCH 3 H O N

HO OH

V

X <

^ S

OHC-/

\ - O H

KOH COCH, H3 4-2

A A.

4-3 l.Mel y / OCH3 V/2.H2/Pt02 I >A 3.KMn0 4

HOOC—/

\-OCH3

4-4

\ OH

J=\ HOOC-/

^

H3C ^ "

/J

O H

+

H 2 S0 4 /

H3C

g-CH3

Me 2 S0 4 OH

9,

"~M7

Scheme 6. Degradation of licochalcone.

permanganate to yield an aromatic acid 4-4. The structure of 4-4 was proven to be 3-a,a-dimethylpropyl-4,6-dimethoxy-benzoic acid by comparison with synthetic sample obtained through condensation of 2,4dihydroxybenzoic acid with isoprene followed by methylation with dimethyl sulfate. Thus the structure of 4-3 can be assigned as 3-a,adimethylallyl-4-hydroxyl-6-methoxy-benzaldehyde and licochalcone 4-1 is 4,4f-dihydroxy-2-methoxy-5-(a,a-dimethylallyl)-chalcone (Scheme 6) [19]. Synthesis of Licochalcone For further confirmation of the structure 4-1, the total synthesis was accomplished. When 2-methoxy-4-hydroxybenzaldehyde refluxed with bromoisoprene in acetone with presence of anhydride potassium carbonate an ether 4-5 was obtained. Compound 4-5 was put on Claisen rearrangement in butyric anhydride and N-dimethylaninine under nitrogen to yield butyrate 4-6, then hydrolyzed to 4-3. Finally, condensation of 4-3 with /7-hydroxyacetophenone was carried out in the presence of HC1 to produce licochalcone (Scheme 7). This was identified with authentic

742

REN-SHENG XU

natural one by spectral and TLC comparisons, and showing no depression of mixed melting point measurement [19]. OCHo OCHo

OHO-/

^V

O H

+BrCH2— g = C ^

K£O^OHC--^

^-OCH3-CH=c^ 4-5

(CH3CH2CH2C0)20

».

OH

OCOCH2CH2CH3

OCH3

NaOH

>

OHG-/

y/~OH

CH3CH2OH

Me2N-C 5 H 5

4-6 OCHo

"TX * «-Q^ ^* HOk

OH

COCH3

4-2

4-3

Scheme 7. Synthesis of licochalcone.

This research work was finished in 1971, but could only be published in 1979 due to the "cult-revolution" in China. In 1975, it was found the same structure, named as licochalcone A, which was isolated by Saitoh et al. from Xinjiang Glycyrrhiza (probably Gl globra) [20]. Their spectral data were the same as ours, but the reported melting point (101-102°C) was much lower than ours. In 1983 S. A. Khan and M. Krishnamurt synthesized licochalcone A starting from 2-hydroxyl-4-(0-prenyl)benzaldehyde 4-6, which was obtained by condensation of (3resorcylaldehyde with prenyl bromide. 4-6 was first methylated with dimethyl sulfate and then reacted withp-methoxymethoxyacetophenone in basic condition to get a chalcone 4-7. Upon Claisen rearrangement of 4-7 by using dimethylaniline and acetic anhydride at 190-195°C a chalcone 4-8 was obtained. Compound 4-8 was hydrolyzed in basic condition to 4-9 and then hydrolyzed in acid condition to form the final licochalcone A (Scheme 8) [21]. The conditions and procedures of the Claisen rearrangement in these two syntheses were different: in ours, the Claisen rearrangement was accomplished before condensation to obtain chalcone and latter performed the reaction after forming the chalcone. Besides, the condensation conditions for producing chalcone were different: ours was in acidic condition and Khan's method, it was in basic condition.

743

BIOACTIVE NATURAL PRODUCTS

OH

QH CH 3

OHO: - /

\-OH

+BrCHr-g=Cr^

K2CQ3^

Q H &

/

V.0CH2--CH==:G^ CH^ 4-6

OCH 3 Me2S04

•

/ OHO-Zv

/ .CH, '

^ A-OCH2

H3C02HCO^

\ - C O

CH 3

CH=cf KOH. EtOH

CH 3

OCH, CH3

H3COH2C

CR i

Me 2 N-C 5 H 5 A c 2 0

CH = Cf *CH,

190-195°C

OCH^ HoCOH 9 CO H3COH2O OCOCH3

OH

NaOH EtOH

]

4-9

4-8 OCHo HC1

HO

MeOH

Scheme 8. Khan's synthesis of licochalcone A.

There are hundreds of components that have been separated from licorice including the well known glycyrrhizine, a triterpenic acid glycoside, the main sweetness and antitoxin ingredient, many flavonoids and coumarins, among them licochalcone A is a specific chalcone in structure. Most chalcones have their functional groups in ring B and a hydroxy 1 group at C-2f of ring A which are easily transferred to flavone. In licochalcone A, the main functional groups are in ring B and no hydroxyl group at C-21 in ring A, and could not be converted to flavone.

744

REN-SHENG XI)

Bioactivity of Licochalcone A It was reported licochalcone A inhibits the cytopathic activity of human immunodeficiency virus (HIV) by Okuda's group [22], A cell line named OKM-1, sensitive to the cytopathic activity of HIV, was established from the peripheral blood of a patient with adult T-cell leukemia. Giant cells due to the cytopathic activity, were formed within a day on co-culture with HIV-infected Molt-4 cell (OKM-1 : OKM-4 = 3:1). Licochalcone A inhibited the giant cell formation at concentration 20 |ig/ml without observable cytotoxicity. Besides, licochalcone A has a radical scavenging effect, inhibition of leukotriene synthesis in human polymorphonuclear neutrophils and xanthine oxidase. S. Shibata's group reported licochalcone A shows antiinflammatory effect and the anti-tumor promoting action [23]. A model of mouse-ear edema induced by A A (arachidonic acid) or TPA (12-0tetradecanoylpholbol 13-acetate) and papilloma on mouse back induced by TPA after initiation by a single application of DMBA (7,12-dimethylbenzaanthracene) was used for tests. The results showed 73.3% of tumorbearing mice were observed for control group without licochalcone A and only 26.7% of tumor-bearing mice for licochalcone A group after 20 weeks. P value is < 0.05. Recently M. Chen found licochalcone A possesses antileishmanial activity in mice infected with Leishmania major in hamsters infected with Leishmania donovani and inhibits in vitro growth of the human malaria parasite Plasmodium falciparum and protects mice from P. yoelii infection [24]. 5. CHOLINESETRASE INHIBITOR HUPERZINE A Huperzine A, a potent anticholinesterase alkaloid, and huperzine B were separated from a Chinese medicinal herb, Huperzia serrata (Thunb) Trev. {Lycopodium serratum Thunb.), (Lycopodiaceae), by Liu et al [25]. The plant was used in Zhejiang Province of China for treatment of some mental disorders. Huperzine A 5-1 has a molecular formula of Ci 5 H 18 N 2 0; m.p. 230°C; [oc]D-150.4°C (C, 0.5, MeOH). Its UV spectrum absorptions at A,max (log 8) 231 (4.0), 313 (3.89) nm, IR spectrum absorptions at v m a x 3180,1650,1550 cm-1 and ]H-NMR signals (Table 1) show the presence of a-pyridone ring in its molecule. The proton and carbon signals indicate existence of an endocyclic and an exocyclic double bond in the molecule. The final structure 5-1 was further determined by spectral analyses, including decoupling experiments, NOE measurements and comparison with the known structure of selagine 5-2. The difference between 5-1 and 5-2 is the situation of the olefinic proton at the endocyclic double bond: in

BIOACTIVE NATURAL PRODUCTS

745

5-2, its signal H-14 in 'H-NMR spectrum is singlet, but its signal H-8 in 5-1 is doublet, which indicated presence of a vicinal methine coupled with the olefmic proton in 5-1. Besides, irradiation of signal at H-7 in 5-1 led to decoupling of H-8 and H-6, and irradiation of H-14 produced NOE Table 1.

No

1H- and 13C- NMR Data of Huperzine A in CDCI3 in 5 ppm

,

H-N1MR(100 MHz)

13

C-NMR (22.63 MHz)

1 1

165.52 5

2

6.38 (1H, */, .72,3=9)

116.97*/

3

7.84 (1H, 4 .72,3=9)

140.25*/

4

122.95 5

5

142.59 5

6

2.76 (2H, AB part of ABX J6a,6P=16, J6a,7=3, J6p,7=0)

35.24 /

7

3.56 (lH,w)

8

3.58 (1H, 41/7,8=5)

10

1.62 (3H,*/, .710.11=7)

12.31*/

11

5.46 (lH,q, .710,11=7)

111.23*/

32.95 d 124.36*/

12

143.30 5

13

54.35 5

14

2.12 (2H,w)

15

49.25 / 134.09 5

16

1.46 (3H, s)

NH

13.20 (\Hybs)

Scheme 9. Structure of huperzine A 5-1, selagine 5-2 and huperzine B 5-3.

22.57*7

746

REN-SHENG XI!

enhancement at H-3, these facts are further proven the structure of 5-1. Recently Ayer fs group reinvestigated the structure of selagine due to the potent bioactivity of huperzine A. Selagine was isolated from the plant Lycopodium selago, collected near Grovels Jon in Sweden and compared with the original selagine, which was isolated by New Brunswick group, and huperzine A. They found all the three compounds are absolutely the same and the structure of selagine was determined incorrectly [26]. Total Synthesis of Huperzine A The structure of huperzine A was confirmed by total synthesis. There are several reports on total synthesis [27]. The following described was recently accomplished by Kozikowski's group [28,29]. The monoprotected diketone, 1,4-cyclohexanedione monoethylene ketal 5-4 was used as starting material. The pyrrolidine enamine of 5-5 was heated with acrylamide followed by hydrolysis to provide an 85:15 mixture of the lactam 5-6 and 5-7. After protection of the lactam nitrogen by benzylation, dehydrogenation of the latter mixtures afforded pyridone 5-8, then hydrogenolysis of its N-benzyl group, followed by O-methylation with methyl iodide and silver carbonate which yielded methoxypyridine 59, oc-carbomethoxylation of 5-9 gave the key intermediate (3-ketoester 510. The palladium-catalyzed bicycloannulation reaction of 5-10 using 1,1,3,3-tetramethylguanidine (TMG) as a base (to generate the 5,7dicarbanion equivalent) and 2-methylene-1,3-propanediol diacetate as the bis-electrophile in the presence of tetrakis(triphenylphosphine)-palladium (O) in refluxing dioxane resulted the methylene-bridge structure 5-11. The Wittig reaction of 5-11 with ethylidenetriphenylphosphorane in THF at 0°C to room temperature provided a 9:1 mixture of (Z)- and (£)-alkenes 512. The mixture was then subjected to an isomerization reaction with thiophenol and azobisisobutyronitrile (AIBN) in toluene at 85°C, leading to a 95:5 mixture of (£)- and (Z)-alkene 5-13 . Conversion of ester group of 5-13 to an amine was carried out by a two-step protocol: involving (1) alkaline hydrolysis of 5-13 in THF-methanol under reflux to afford 5-14 and (2) Curtius rearrangement of the acid 5-14 by diphenyl phosphorazidate/triethylamine treatment followed by methanolysis of the resulting isocyanate provided the carbamate 5-15. Trimethylsilyl iodide (TMSI) mediated deprotection of 5-15 in refluxing chloroform proceeded uneventfully, although partial isomerization of the exocyclic double bond to the endocyclic olefin, promoted presumably by adventitious hydroiodic acid, was observed. Treatment of the mixture with triflic acid in dioxane at 93°C yielded huperzine A 5-1 as the sole product in 84% yield. (Scheme There are several reports on synthetic studies on derivatives of huperzine A [30-32]. Most of them, however, did not show any

747

BIOACTIVE NATURAL PRODUCTS

enhancement in bioactivity except the shiff base product with isovanilline, called isovanihuperzine A, which has similar activity to huperzine A with less toxicity and is now under further studies [33].

OMe

N.

OMe

OMe 5-9

5-8

5-10

CH 0 ^

Ph 3 P^C 2 H 5 Br nBuLi OMe

5 - 11

N

1 \

PhSH, AIBN OMe

Me.

OMe

5 -12 5 - 13 ,CH2

20%NaOH THF, MeOH^ Me^

,CH2 N

U

^ COOH

(PhO)2P(0)N3/Et3N^

V - OOMe M P MeOH

Me,

5 - 14

5- 1

Scheme 10. Kozikowski's total synthesis of huperzine A.

OMe

5 - 15

REN-SHENG Xtl

748

Huperzine B has a molecular formula of Ci6H2oN20, m.p. 270-271°C, [oc]D-54.2° (C, 0.2, MeOH). It has UV spectrum absorptions at ?tmax (log £) 231 (3.95), 312 (3.85), and IR spectrum absorptions at vmax 3100, 1670, 1620,1610,1560 cm-1. The whole structure was proposed by spectral analyses as shown in 5-3. Its main difference from huperzine A is the absence of exocyclic double bond which form an additional piperidine ring in 5-3. Huperzine B does not possess anticholinesterase activity. Pharmacological Study Huperzine A (HupA) is a potent and selective cholinesterase (ChE) inhibitor. It enhances the learning and memory for patients with Alzheimer's disease remarkably. The anti-ChE activity studies were evaluated in vitro using acetylcholinesterase (AChE) (rat cortex homogenate) and butylcholinesterase (BuChE) (rat serum). HupA produces a marked concentration-dependent inhibition of AChE. Based on the concentration of inhibitor yield a 50% inhibition of enzyme activity (IC5o), the relative potencies of 5 inhibitors in the inhibition of AChE are E2020 > HupA > Tac (tacrine) > Phy (physostigmine) > Gal (galanthamine). HupA, however, inhibits BuChE at much higher concentration than needed for inhibition of AChE. On the basis of the ratios of BuChE : AChE, HupA is a much more selective inhibitor of AChE than E2020, Tac, Phy, or Gal. (Table 2). The AChE activity does not exhibit progressive decrease with the prolongation of incubation with HupA. The AChE activity recovered to 94% of the control after 5 times washing. These results indicate that the inhibitory manner of HupA is reversible and different from that of isoflurophate [34]. Table 2.

Anticholinesterase Activities of Cholinesterase Inhibitors in vitro

IC5o(nmolL_1) ChE-Inhibitor

Huperzine A Tacrine

BuChE 58894 74.6

Physostigmine

1259

E2020

5012.8

Galanthamine

12590

Ratio of IC50 AChE

BuChE/AChE

58.4

1008.5

93.0

0.8

251 13.6 1995

5.0 368.6 6.0

In a series of behavior studies, HupA improved cognitive performance in a broad range of animal models. Enhancement of learning and memory

BIOACTIVE NATURAL PRODUCTS

749

performance was documented in passive footshock avoidance, escape task of water maze and spatial discrimination of radial arm maze, as well as a delayed match-to-sample procedure in squirrel monkeys. The duration of improving effects with oral HupA on learning and memory retention processes are longer than Phy, Gal, or Tac. The magnitudes of improving effects produced by oral and intrapenitoneal administration are nearly equivalent, indicating that HupA has a higher efficacy with oral route than that of Phy. The improving effect of HupA is more potent on working memory than on reference memory compared with E2020 and Tac. This effect may be a benefit in Alzheimer's disease treatment. Treatment with HupA (0.25 mg/kg, p.o., once per day) for 8 days is as potent as acute administration in attenuating the scopolamine-induced amnesia, indicating that HupA induced no significant tolerance in the cognitive improvement. This finding is consistent with the inhibition of AChE. At improving dose, there is no evidence for nonspecific effects of HupA, such as alternating in running speed or locomotor activity. Compared to other cholinesterase inhibitor (ChEI), clearly HupA has a superior safety efficacy profile (Table 3) [35,36]. Table 3.

Efficacy (step-down passive avoidance performance) and Toxicity of Cholinesterase Inhibitors in Mice

Improving dose mg/kg, p.o.

LD50 (95% fiducial limits) mg/kg, p.o

Huperzine A

0.2

4.60(4.20-5.10)

Tacrine

16.0

53.10(46.90-60.30)

1

Physostigmine

0.3

1.96(1.69-2.28)

1

Galanthamine

2.0

27.10(25.30-29.00)

ChEI

Pharmacokinetic study showed HupA, absorbed rapidly, is distributed widely in the body and eliminated at a moderate rate. The blood level of HupA following i.v. or i.g. [3H]HupA in rats declined in phase two, the distribution phase and the elimination phase, with half-lives of 6.6 and 149 min (i.v.) and 10 and 203 min (i.g.) respectively. The oral bioavailability is 96.9%. In mice the radioactivities are highest in kidney, liver and present in all regions of the brain, particularly concentrated in frontoparietal cortex, striated cortex, hippocampus and in nucleus accumbens. The drugplasma binding rate was found to be 17%. HupA appeared to bind more tightly and specifically to acetylcholinesterase than the other AChE inhibitors. Compared to tacrine and donepezil, HupA has a longer half-life and the AChE-HupA complex has a lower dissociation rate, which may make it a more effective therapeutic agent. HupA appeared to be strongly specific for AChE, and such specificity suggested that it may be effective

750

REN-SHENG XU

with fewer adverse effects. HupA was also tested as a prophylactic drug against soman and other nerve poisons. Its long-lasting antidotal efficacy and low toxicity make this drug promising as a protective agent against chemical weapons [37]. Clinical Trials of HupA By using multicenter, prospective, double blind, parallel, placebo controlled and randomized method, 50 patients with average age 67 ± 1 1 (55-89) are administrated orally 0.2 mg (4 tablets) HupA and 53 patients with average age 66 + 11 (53-90) are given orally the same 4 tablets of placebo, bid for 8 weeks. The results indicated 58% (29/50) of patients treated with HupA showed improvement in their memory (P<0.01), cognitive (PO.01) and behavioral (P<0.01) functions. The efficacy of HupA is better than placebo (36% 19/53) (P<0.05). No severe side effect was found. As a result, HupA is a promising drug for symptomatic treatment of Alzheimer's disease [38]. HupA was used to treat 128 patients with myasthenia gravies. The clinical dose was commonly 0.4 mg/ day by i.m. in 10 day's duration. Its therapeutic effective rates were around 99% and duration action was 7 ± 6 hrs, while physostigmine was 4 + 5 hrs [39]. 6. MIOTIC AGENT BAOGONGTENG A Baogongteng A was isolated from Erycybe obtusifolia Benth (Convulaceae), a vine plant grown in Hainan island of China. The stem of the plant was used in folk medicine for treatment of rhaumatalgia. It was found to cause contraction of the pupils when administered its extract. The active principle, a new alkaloid, namely baogongteng A (BgtA), was isolated parallelly with pharmacological tests from the stems by Yao et al. [40,41]. It was the first cholinergic tropane alkaloid isolated from the natural source, since the common tropane alkaloids are known to possess the opposite anticholinergic property. The ground stem of the plant was percolated with 80% EtOH, after concentration of the extract the remaining water layer was acidified, defatted with chloroform, then basified with ammonium, and extracted with chloroform to get alkaloid extract, which was chromatographed on silica-gel column, eluted with EtOH/CHCl3 (1/1), guided by TLC and BtgA was obtained as a colorless viscous liquid. It is freely soluble in water and unstable in the air, but its benzoic acid salt (m.p.l61-162°C) is stable. The free base has a formula of C 9 H 15 N0 3 determined by elemental analyses and MS data m/z at 185 (M+), 142, 126, 108, 99, 80, 43, 31. It contains -NH, -OH and -OAc groups, which have been proven by IR spectrum absorptions vmax at 3400, 1740, 1260, 1035 cm"1 and l H-NMR

751

BIOACTIVE NATURAL PRODUCTS

signals at 8 1.98 (3H, s) and 2.6-2.8 (2H, br.). Its structure 6-1 was determined by spectral analyses and comparison with tropane alkaloids (Scheme 11) [40]. The nitrogen in its molecule is in -NH form. When it was methylated with formaldehyde and sodium borohydride its N-methyl compound 6-2 was obtained, the later is very similar to the known 6pacetoxy-pseudotropanol 6-3, obtained by reduction of 6(5-acetoxytropanone 6-4 with sodium borohydride. In compound 6-2, its ^ - N M R spectrum signal 8 5.15 (dd, J = 5.0,5.0 Hz) is similar to signal 8 5.05 {dd, J = 5.0,5.0 Hz) of that in 6-3. Therefore the acetoxyl group in BgtA is most possible at 6(3-position, just like in compound 6-3. The position of hydroxyl group in 6-1 was finally determined by a decoupling technique of its N-methyl acetyl compound 6-2. When irradiate on proton at C-l (8 3.45), the decoupling of protons are observed at C-7 (8 2.20) and the hydroxyl giminal proton at 8 4.5. Besides, when irradiate on proton signals at C-3 and C-4 (8 1.4-1.8) decouplings are observed the C-5 proton at 8 3.15 and the C-2 proton at 8 4.5. That means the hydroxyl group is at C2. Therefore the structure is determined as 2p-hydroxy-6(3-acetoxynortropane 6-1 (Scheme 11). Its absolute configuration was determined by reagent [Pr(dpm)3] induced CD split Cotton effect method, exciton chirality method and Horeau method as (2S, 6S)-2p-hydroxy-6p-acetoxynortropane. Except BgtA, Baogongteng C (BtgC), erycibelline, scopoline were also separated from the plant and from other two species: E. obtusifolia and E. elliptilimba. Structure of BgtC was determined as 6(3deacetyl-baogongteng A, but BgtC did not show miotic activity [42]. OH HCHO/NaBH 4

AcO.

ACO

V^\[^H

ft

6-2 NCH 3

NaBH 4

AcO, H

6-4

AcO.

O

OH 6-3

k

Scheme 11. Baogongteng A 6-1 and 6(3-acetoxy-pseudotropanol 6-3.

Total Synthesis of Baogongteng A The structure of baogongteng A was further confirmed by total synthesis by Xiang et al. [43]. 6(3-acetoxy-tropinone 6-4 was used as starting material for the synthesis. It was through bromination with liquid bromine in acetic acid, hydrolyzation with silver carbonate, and ketalization in thioglycol and adding boron fluoride dihydrate in ethyl ether solution to

752

REN-SHENG XI!

obtain the kitalized compound ,which was reduced with Reney Ni and oxidized with chromium oxide to get the C-2 ketone compound, it was now reduced with sodium borohydride and demethylated in toluene with 2, 2, 2-trichloroethyl chloroformate and potassium carbonate to get the final product, racemic baogongteng A (Scheme 12). It showed miotic activity with half potency of the natural one. NCH3 O H AcO

PH2SH

Ag 2 CQ 3

Br" 6-4 NCH3

NCH 3

„

AcO.

CrOo - AcO

AcO

NaBH A

OH

NH

AcO

1. ClCOOCH 2 CCl 3

iL? H

2. Z n / H O A c

6- 1 Scheme 12. Total synthesis of Baogongteng A.

In order to study the relationship between structure and miotic activity, several analogs were synthesized and the active derivatives are shown as below (Table 4). Rabbit pupils contraction study of above derivatives showed the basic skeleton of BtgA and its 6p-acetoxyl group are necessary in miotic action. Deacetylation of BgtA to 6(3-OH compound will reduce its activity. When the acetoxyl group in 6[i-position was replaced by benzoxyl or other groups, the compound lost its activity completely. All the active compounds had secondary amino-group on the bridge top of the compound. When it is derivatived to tertiary amino-compounds, the activity is reduced as a result. 2(3-hydroxyl group in BgtA is not necessary for activity. When the orientation of 2(3-hydroxyl group changed from (3 to a, its bioactivity was lost [44,45]. Pharmacology Studies of BgtA Since the free base of BgtA is not stable in the air and its benzoate (BABS) was used for all the pharmacological tests. The results was reviewed by Sen [46].

BIOACTIVE NATURAL PRODUCTS

753

Table 4. Bioactivity of Active Derivatives of BtgA

i

R2 No

R

Ri

*2

R3

Activity

11

H

Ac

H

OH

Cholinergic

2

H

Ac

H

H

Cholinergic

43

H

Ac

H

OAc

Cholinergic

H

Ac

CH 3 CH 2 COO

H

Cholinergic

5

H

Ac

(CH3)2CHCOO

H

Cholinergic

6

H

Ac

pCH3PhCOO

H

Cholinergic

H

Ac

OH

H

Cholinergic

H

Ac

PhCH=CHCOO

H

Cholinergic

1 9

CH 3

Ac

OAc

H

Cholinergic

1U 10

CH 3

Ac

pCH3PhCOO

H

Cholinergic

H

Ac

H

H

Cholinergic

CH 3

Ac

PhCOO

H

Anticholinergic

CH 3

Ac

/?N02PhC00

H

Anticholinergic

CH 3

Ac

pClPhCOO

H

Anticholinergic

CH 3

Ac

H

H

Anticholinergic & Cholinergic

r r

?

8

1 1*2 1 13

M

1 15

* double bond between C-2 and C-3

Ophthalmological study showed the miotic effect of 0.0125% BABS is equivalent to that of 2% pilocarpine eyedrop. The miotic effect of 0.1% BABS is a little weaker than that oyf 0.25% eserine eyedrop. 0.25% BABS, 2% pilocarpine and 0.05% eserine have no significant effect on normal intraocular pressure of rabbits. The prolonged use of 0.0125% of BABS eyedrop in rabbits' eyes was 3 times a day for 3 months. Slip-lamp, gonioscopic and ophthalmoscopic examinations, performed every month, showed there were no changes in cornea, iris, anterior chamber and fundus. According to the cumulative dose response curve obtained from isolated jejunum of rat, BABS is about 100 times more potent on intestinal

754

REN-SHENG XV

contraction than on pilocarpine. The KD for BABS and pilocarpine are 1,046 mM and 40.5 mM and the Emax for BABS and pilocarpine are 1.65 and 40.5 respectively. Therefore, the affinity of BABS to Mcholinoceptor is 39 times greater than that of pilocarpine, and the intrinsic activity of BABS-M-receptor complex is 1.65 times greater than that of pilocarpine-M-receptor complex. The acute LD50 of BABS was found to be 8.85 +1.2 mg/kg, when i.p. injected into mice. The toxic manifestations are mainly through cholinergic effects. The symptoms of intoxication are lacrimation, salivation, diarrhea, hypothermia, dyspnea, weakness and death. Atropine and scopolamine are specific antidotes against BABS intoxication, both could antagonize all these toxic symptoms and protect the animals from death. Pathological examinations of mice and monkeys, that had died from acute intoxication, revealed these are mainly bloodstasis in viscera. Clinical Trials of BtgA When different concentrations of BABS are locally applied into the eyes of normal volunteers, it showed the optimal concentration was 0.025%. With such concentration the miotic action began after 20 min and maintained for 8 hrs. When maximal miotic effect was attained, the diameter of the pupil was reduced to half-size. After applying eyedrop, the conjunction of volunteers turned reddish immediately and the congestion lasted for less than an hour, however, they did not have any discomfort. 178 eyes of patients with various types of primary glaucoma were observed. BABS lowered the intraocular pressure by 14 mmHg (PO.01) and pilocarpine by 13 mm Hg (P<0.01). There was no statistical significant difference between the two drugs in decreasing intraocular pressure. BABS increased the C-value by 0.09 (PO.01), while pilocarpine increased C-value by 0.15 (PO.01). There was no statistic significant difference between the two drugs as far as C-value is concerned. Their side effects are also similar [46]. 7. ANTIFUNGAL PSEUDOLARIC ACID B The root-bark of Pseudolarix kaempferi Gord. (Penaceae) was used in Chinese medicine as an antifungal agent to treat tinea pedis and other fungus diseases. The antifungal principle pseudolaric acid B 7-2, and pseudolaric acid A 7-1, C 7-3, C2 7-4, pseudolaric acid A-p-Dglucopyranosylester 7-5 as well as pseudolaric acid B - p - D glucopyranosylester 7-6 were separated by Zhou et al. [47] and Li et al. [48-50] independently from the root-bark of the plant. The benzene extract of the ground root-bark was treated with sodium bicarbonate and

755

BIOACTIVE NATURAL PRODUCTS

then the crude acid fraction was chromatographed on silica-gel column for further separation. The above mentioned have the same new carbon skeleton. Besides, two kaurane derivatives: pseudolaric acid D 7-7 and E 7-8 are also separated from the plant [51]. Table 5. 13C-NMR Data of Pseudolaric Acid B (7-2) and C (7-3) in 5 ppm

NO

Pseudolaric acid B

Pseudolaric acid C

C-l

33.2/

34.6 /

C-2

24.2/

24.1 /

C-3

49.2 d

53.5 c/

C-4

90.0 5

79.1 /

C-5

30.6/

33.3 /

C-6

20.0/

19.4/

C-7

134.4 5

134.0 5

C-8

141.6c/

142.9 c/

C-9

27.6/

26.8 /

CIO

55.2 5

54.6 5

C-l 1

83.6 5

83.8 s

C-12

28.4 c/

27.9 c/

C-13

144.4 c/

144.1 d

C-14

121.6c/

120.9 c/

C-15

138.6 c/

137.1c/

C-16

127.8 5

127.8 5

C-17

12.5 c/

12.1c/

C-l 8

173.2 5

175.3 5

C-19

I68.O5

I68.85

C-20

172.8 5

170.3 5

CH 3 0

52.0 q

51.5c/

CH3CO

21.7c/

-

CH3CO

164.4 5

-

REIN-SHENG XU

756

Pseudolaric acid B is the most antifungal active component. It is a colorless crystal, m.p. 165-167°C, [ot]D -37.3°C(c, 0.0233, MeOH). Its molecular formula was determined by mass spectrum as C ^ ^ O s - DI mass ion peaks are at m/z 414.1678 [M-H 2 0] + , 372.1586 [MCH3COOH]+ and 354.1488 [M - H 2 0 - CH3COOH]+. Its UV absorption at kmax 258 nm (log 8, 4.5), IR absorptions at vmax 2500-3000, 1740, 1719, 1709, 1688, 1640 cm->,and its 13C-NMR data (5 173.3, 172.8, 169.4 and 168.1) indicate existence of four carboxylic groups and one of them was in 8-lactone ring form. Presence of three double bonds (8 144.5, 141.7, 138.7, 134.5, 127.9, 121.8) predicted the compound involves three rings with a long side chain. The final structure was determined by spectral analyses including decoupling, NOE techniques and some chemical derivatizations (Table 5 and Scheme 13). Oxidation of Pseudolaric acid A 7-1 yielded pseudolaric acid B 7-2. Since the structure of pseudolaric acid A was determined by X-ray crystallography [52]. It was further confirmed structure of pseudolaric acid B. Structure of pseudolaric acid C 7-3 was determined as deacetyl pseudolaric acid B and structure of pseudolaric acid C2 7-4 was determined as demethyl pseudolaric acid B. The CD spectrum of pseudolaric acid B showed a negative Cotton effect at 252.5 nm (Ae -10.4) and positive Cotton effect at 218 nm (Ae +23.8) indicated the absolute structure of pseudolaric acid B is 3S, 45, \0R and UR [53]. 17

R

3

O O C ^ ^ ^ i 18 15

7-1

R, = CH 3 CO

7-2

Rj = CH3CO

7-3

R, = H

7-4

My I2CH3 OR, R2 = CH 3

R2 = COOCH 3 R2 = COOCH3

R, = CH3CO

R2 = COOH

19 -R2

R3 = H R3 = H R3=H R3 = H

7-5

R, = CH3CO

R 2 = CH 3

R 3 = |5-D-glc

7-6

R, = CH3CO

R 2 = COOCH3

R 3 = p-D-glc

COOH

Scheme 13. Structures of pseudolaric acids.

COOH

757

BIOACTIVE NATURAL PRODUCTS

Synthesis of Pseudolaric Acid B Synthesis of pseudolaric acid A was carried out by Pan et al. [54] (3-ketoester compound 7-10, on treatment with iodo-compound 7-11, potassium carbonate and 18-crown-6 in toluene gave 7-12, which was hydrolyzed to compound 7-13 in dilute acetic acid. When the compound 7-13 was treated with 0.5 equivalent of potassium t-butoxide in THF-t-BuOH (1 : 1) at room temperature for 1 hr, the key compound 7-14 was obtained. The tin compound 7-15, after treatment of butyl lithium, reacted with 7-14 to form the diol 7-16. Reaction of 7-16 with sodium hydride gave the 8 lactone 7-17 (Scheme 14). The remained steps are extension of the side chain and acetylation of the hydroxy 1 group at C-4 for accomplishment. EtOOC

EtOO<

7 - 13

7 - 14 COOR

TORT

Sn nBu3 7 - 15 THPO

~T&> 7- 17 Scheme 14. Pan's synthesis of pseudolaric acid B.

REN-SHENG XII

758

Antifungal Activity of Pseudolaric Acid B Antifungal activity of pseudolaric acid B 7-2 was tested in vitro and compared with amphotericin B by Li et al [55]. It showed pseudolaric acid B is against Candida species, Torulopsis petrophilum, Tribophyton mentagraphytes and Microsporum gypseum. It is not active against Cryptococcus neoformans, but amphotericin B is active against both C. albicans and C neoforrnans. This fact indicated that pseudolaric acid B may have different antifungal mechanism from amphotericin B. Neither the methylated derivative nor any of the hydrolyzed compounds of 7-2 are active against the tested microorganisms. Pseudolaric acid B-(3-Dpyranosylester 7-6 possesses only weak anticandidal activity and has no activity against T mentagrophytes or M. gypseum. Table 6 showed the minimum inhibitory concentration (MIC) of psudolaric acid B 7-2 and its potassium salt 7-9 determined by using the microbroth dilution assay. MIC value of 7-2 and 7-9 are within two dilutions of that of amphotericin B for most of the Candida species and T petrophilum. Initial evaluation of the minimum fungicidal concentration (MFC) was accomplished by subculturing from MIC tubes showing no growth onto drug-free agar plates. Using this technique, the MFC value of 7-2 and 7-9 for most of the Candida and Torulopsis species tested are within two dilution (twofold) of the MICs. It seems pseudolaric acid B has similar antifungal activity as amphotericin B, but has much less toxicity. Table 6.

Quantitative Activity of Pseudolaric Acid B (7-2) , its Potassium Salt (7-9) and Amphotericin B Against Candidas and Torulopsis Species.

1 Pseudolaric acid B MIC/MFC

Pseud, acid B (K + ) MIC/MFC

Amphotericin B MIC

C. albicans B311

1.56/3.12

3.12/6.25

1.56

C. albicans MCC 10231

1.56/3.12

1.56/6.25

3.12

C. parapsilosis ATCC 20224

3.12/12.5

3.12/25.0

1.56

C. stellatoidaes SA-1

1.56/6.25

3.12/6.25

3.12

C. tropicalis ATCC 20326

1.56/3.12

1.56/6.25

1.56

C.tropicalis ATCC 20021

1.56/3.12

3.12/6.25

3.12

C. Tripicalis LM 64

0.78/6.25

0.78/6.25

1.56

T. Petrophilum ATCC 20225

0.78/12.5

0.78/12.5

0.20

T. cremoris ^RKL1495

12.5/25.0

12.5/25.0

3.12

Fungi

BIOACTIVE NATURAL PRODUCTS

759

Antifertility Activity of Pseudolaric Acid B 7-2 The sodium bicarbonate solution of 7-2 was given s.c. , i.m., i.v. or p.o. induced the termination of early pregnancy either in rats or in rabbits. When it was given orally, 10 mg/kg daily, to 10 female rates on 10-12 days after mating, mid-term pregnancy of all rats was terminated . When 40 mg/kg of 7-2 was injected s.c or i.p. on 1-3 days after mating, it showed no estrogenic activity. It lowered the plasma progesterone levels, but progesterone did not antagonize the effects of 7-2 on early pregnancy in rats. The ED 50 of 7-1 and 7-2 in terminating the early pregnancy in rats given by gavage of single dose on 7 days after mating was determined to be 14.5 and 9.3 mg/kg respectively. The LD50 value of 7-1 and 7-2 given by gavage in rates were 220 and 130 mg/kg respectively. The therapeutic index (LD50/ED50) for 7-1 is 15 and for 7-2 is 14. Therefore pseudolaric acid A is less active but also less toxic than pseudolaric acid B [56]. 8. INSECTICIDE STEMONA ALKALOIDS The roots of Stemona plants (Stemonaceae) are one of the common Chinese medicines used as anticough agent and insecticides. The alcohol extract of the roots was shown active to kill insects such as: humanis captitis, pediculus humanis, bedbug, maggot, weevil, wiggler and other insects [57]. There are six species of stemona plants growing in China : Stemona tuberosa Lour. St. sessilifolia (Miq) French, et Savat, St. japonica (Blume) Miq., St. parviflora C.H. Wright, St, mairei Levi and Croomia japonica Miq. The first three plants are used in TCM and the latter three are used in folk medicine as insecticide, too. In the past, eleven stemona alkaloids have been separated from the roots of the first three Stemona plants and their structures were determined mostly by X-ray crystallography analyses due to the complexity of these structures. (Table 7 and Scheme 15) [58,59]. There are also several other stemona alkaloids, which have been reported, but their structures are unknown. In early 1949 Chinese chemist prof. Zhu Ren-hong started to study on stemona alkaloids from the roots of Stemona sessilifolia and separated an alkaloid, identified as stemonidine [60]. which was separated by Suzuki from St. japonica in 1929, its structure, however, was not clear at that time [61]. In 1981, this study was continued and the structure of stemonidine 8-34 was determined by using spectral analyses with the modern 2DNMR technique. Later it was found, that the structure of stemonidine is similar to stemospironine, isolated from St. sessifolia by Sakata in 1978 and determined its structure by X-ray crystallography [62,63]. At the same time, the structures of two new sterioisomer alkaloids, stemotinine 8-35 and isostemotinine 8-36, were studied. Both alkaloids were isolated

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from the roots of St. tuberosa, collected in Wenshan district, Yunnan province of China [62]. Table 7. Physical Data of the Known Stemona Alkaloids Formula

Structure

m.p.(°C)

MD

Plant Source

Tuberostemonine

C22H33NO4

8-1

86-88

-47° (CHCI3)

Stt

Bisdehydrotuberostemonine

C22H29NO4

8-2

176-178

106°(C 6 H 6 )

St. t.

I

Tuberostemon ine-A

C22H33NO4

8-3

120

-65° (EtOH)

St. t.

I

Oxotuberostemon ine

C22H31NO5

8-4

222

Stt

I

Stenine

C 1 7 H27N0 2

8-5

65-67

-30° (MeOH)

St. t

I

Stemonamine

C 1 8 H 2 3N04

8-6

172-174

0°(MeOH)

Stj.

I

Isostemonamine

C18H23N04

8-7

165-169

stj

1

Stemonine

C17H25NO4

8-8

151-152

-114° (EtOH)

St.j; St. s.

Protostemonine

C23H3lN06

8-9

172

147° (EtOH)

Stj.; St. s.

Stemofoline

C22H25NO5

8-10

87-89

273° (MeOH)

Stj.

Croomine

C18H27NO4

8 - 11

amorphous

9.6° (CHCI3)

Cr.j.

Alkaloid

St. I

Si. lubarosa; Si. j .

Si. japomca; Sl.s.

Si. scssil(folia; Cr. j .

Croomia japonica.

Stemotinine has a molecular formula of Ci8H25N05) determined by high resolution of mass spectrum with molecular ionM + at m/z 335.1751 (Calc. 335.1730). The presence of two singlets at 5 179.2 and 179.7 in the 13CNMR spectra and a strong IR absorption at 1764 cm-1 indicated containing two y-lactone rings in its molecule. This evidence together with the lack of olefmic carbons (NMR), lack of NH and OH groups (IR) and lack of UV absorbing chromophobes indicated that stemotinine consisted of a pentacyclic skeleton incorporating two y- lactones, one tertiary amine. Detailed JH-NMR (360 MHz) studies utilizing NOE, two dimensional J techniques led to full clarification of proton signals (Table 8 and Scheme 16); these data in conjunction with 13C-NMR results led to proposed structure of stemotinine as 8-35. The presence of y-lactone ring was supported by a mass peak at M+ -C5H7O2. Upon addition of DC1 to the CDCI3 solution of stemotinine, the signals of protons close to the nitrogen underwent downfield shifts and line broadening: H-3oc +0.09; H-5oc +0.12;

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761

Scheme 15. Structures of known stemona alkaloids.

H-5p + 0.18; H-6 +0.07; H-14 +0.32. It was also noted that in this case the signals of protons spatially close to the nitrogen lone pair, i.e. in this case H-14, underwent dramatic line broadening, whereas the shapes of

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other nearby protons, 3a, 5a, 5(5, and H-6 were affected much less. NOE study showed upon irradiation of H-3a of the molecule 3.6% NOE was observed at H-15a, i.e. the two protons are in proximity, and this shows that the most abundant population of the pendant y-lactone is as depicted in 8-35. Irradiation of H-5a 3.1% NOE was observed at H-3a; 2.0% at H5(3 and 6.4% at H-6. Irradiation of H-14(3 4.0% NOE was observed at H16(3 and irradiation of Me-11 and Me-16 3.4% and 4.0% NOE were observed at H-10p and H-15a respectively. Structure of stemotinine 8-35 was later proven by X-ray crystallography analysis [64]. Isostemotinine 8-36, Ci 8 H 2 5N0 5 , M+ at m/z 335.1742 (calc. 335.1730), closely resembles to stemotinine. The ^ - N M R spectrum signals of both alkaloids are also similar except for those due to H-10fs: in 8-35 they are at 8 2.61 (dd)/l.70 (dd), whereas in 8-36 they are at 2.10 (dd)l\J\ (dd), namely, the difference in 8 values are much smaller in 8-36 (Table 8; the H-7 and H-8's could not be fully analyzed). The closer chemical shifts of the two H-10's are attributable to lack of the ethereal oxygen anisotropic effect and thus lead to the 9-iso structure 8-36. Moreover, in 8-36 the perhydroazaazulene ring has an "8-CH2-down conformation" in view of the lack of W-type coupling between H-5a/H7 a and H-6/H-8a. This conformation is further supported by the dramatically broadening of the H-10a signal, as well as the H-14 signal, upon addition of trace amount of DC1, while the signal pattern of H-10(3 remains practically unaffected. As described below , this is accounted for by proximity of the H-10a to the nitrogen lone pair electrons; if the perhydroazaazulene ring adopted the "8-CH2-up conformation" (as in 835) the H-10a and H-10(3-protons would both be similarly situated with respect to the lone pair (see below), which is not the case. The Me-11 configuration rests on the fact that NOE's were observed between H10a/H-l 1 and between H-10(3/Me-11. The rest NOE are similar to 8-35.

Scheme 16. Structures of stemotinine 8-35 and isostemotinine 8-36.

The above studies on ]H-NMR spectra of these stemona alkaloids allowed discovery of a phenomenon and called " a line broadening effect of !H-NMR signal". Addition of a trace quantity of DCL resulted in extensive broadening of the signals due to protons spatially close to the nitrogen lone pair; later it was found that by adding an appropriate amount

BIOACTIVE NATURAL PRODUCTS

Table 8.

763

'H-NMR data of Stemotinine 8-35 and Isostemotinine 8-36 Stemotinnie

No

Isostemotinine

1.86

m

1.88

m

IP

1.91

m

1.92

m

2a

1.72

m

1.60

m

2P

1.98

m

2.15

m

3a

2.86

ddd

10.8 (2P), 8.8 (14p), 1.4 (8a)

2.93

ddd

10.8 (2P), 7.8 (14 p), 6.1 (2a)

5a

3.00

ddd

10.7 (5p), 6.3 (6), 1.4 (7a)

3.04

dd

10.4 (5p), 6.3 (6)

5P

3.22

d

10.7 (5a)

3.20

d

10.4 (5a)

6

4.59

m

6.3 (5a), 2.0 (7a), 2.0 (7p), 1.4 (8a)

4.68

ddd

6.3 (5a), 2.0 (7a), 2.0 (7p)

7a

1.81

m

13.5 (8P), 12.6 (7P), 5.9 (8a)

7P

1.62

bdd

12.6 (7a), 5.4(8p)i 1.8 (8a)

8a

1.55

ddt

13.5 (8P), 5.9 (7a), 1.8 (7P), 1.8(6)

8P

2.34

dt

13.5 (8a), 13.5 (7a), 5.4 (7P)

10a

2.61

dd

14.6(10P), 11.6(11)

2.10

dd

13.1 (10p), 10.0(11)

10 P

1.70

dd

14.6 (10a), 6.3(11)

1.71

dd

13.1 (10a), 12.6(11)

11

2.81

ddq

11.6 (10a), 7.7 (11-Me), 6.3 (10P)

2.80

ddq

12.6(1 Op), 10.0 (10a), 7.7 (11-Me)

11-Me

1.34

d

7.7(11)

1.28

d

7.7(11)

14 P

4.26

ddd

11.3 (15a), 8.8 (3a), 5.4 (15P)

4.14

ddd

11.3 (15a), 7.8 (3a), 5.4 (15P)

15a

1.48

ddd

12.6 (16P), 12,6 (15p), 11.3 (14P)

1.58

ddd

12.6 (16p), 12.6 (15P), 11.3 (14p)

15P

2.36

ddd

12.6 (15a), 9.0 (16P), 5.4 (14p)

2.36

ddd

12.6 (15a), 9.0 (16P), 5.4 (14P)

16P

2.67

ddq

12.6 (15a), 9.0 (15P), 7.5 (16-Me)

2.66

ddq

12.6 (15a), 9.0 (15P), 7.5 (16-Me)

16-Me

1.26

d

7.5 (16p)

1.28

d

M a

1

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REN-SHENG XI!

of acid for the ^ - N M R line broadening effect of protons spatially close to the nitrogen lone pair became one sixth mole of the alkaloid. This effect was further used diagnostically to determine structures of other stemona alkaloids and nitrogen containing compounds, such as sinomenine, acetylsinoacutine and huperzine A [65,25]. On the basis of these studies, and by using the recently developed new NMR techniques including 1H" , H-COSY(correlated spectroscopy), HETCOR (heteronuclear correlation), ROESY(rotating frame Overhauser experiment spectroscopy), HMBC (*H detected heteronuclear multiple bond conectivity), HMQC (*H detected heteronuclear multiple-quantum coherence), as well as flash column chromatography, repeated preparative TLC and HPLC (normal or reverse phase) separation techniques, it was possible to separate dozens stemona alkaloids from the above mentioned China grown 6 species of Stemona plants and to determine structures of additional 24 new stemona alkaloids (Table 9 and Scheme 17) aside from the above mentioned known stemona alkaloids [66-74]. Among them St. parviflora and St. mairei were first studied. From them it was discovered that parvistemonine 8-38 has structure with new skeleton [71], the structure was then proven by X-ray crystallography and its absolute structure was determined to be 35, 9/?, 9aS, 105, llfl, 12/?, 135, 165, 185, and 205. In 1981, and later Go et al. determined the structure of stemoninine 8-27, separated from Stemona sessilifolia, by 2D NMR spectral analyses [75]. But they may misidentified that plant, it is most possibly isolated from 5/. tuberosa, as the ecology of stemona plants indicates, 5/. sessifolia is less likely to grow in Xichuan Province, China, where they are collected it, and our study indicated, the same alkaloid is separated from 5/. tuberosa, collected in Guantong province, China. Table 9.

Physical Data of New Discovered Stemona Alkaloids Formula

Structure

m.p. (°C)

la ID

Plant Sources

Tuberostemonol

C22H31NO5

8 - 12

amorphous

33.5° (MeOH)

St. t.

Neotuberostemonine

C22H33NO4

8-13

160-162

66.4° (EtOH)

St. t.

Bisdehydroneotube rostemonine

C22H29NO4

8 - 14

172-174

-32.0° (EtOH)

St. t.

Tuberostemonone

C22H3lN06

8 - 15

208-209

34.8° (MeOH)

St. t.

Tuberostemoninoamide

C22H3lN06

8-16

244-246

214.0° (EtOH)

St. t.

Stemonamide

C18H2iN05

8 - 17

182.5-184

-120.0° (Me 2 CO)

Stj.

Alkaloids

BIOACTIVE NATURAL PRODUCTS

765

(Table 9) contd.

Formula

Structure

m.p. (°C)

Isostemonamide

C18H2,N05

8 - 18

234-236

-177.0° (EtOH)

stl

Maistemonine

C 2 3 H29N0 6

8 - 19

205-207

-28.8° (MeOH)

St. m.; St. s.

Oxymaistemonine

C23H29NO7

8-20

217-219

44.6° (MeOH)

St. m.

Neostemonine

C 1 8 H 2 5 N04

8-21

198-200

245.0° (EtOH)

Stj.

B isdehy droneoste-mon i ne

C18H21NO4

8-22

218-221

187.0° (EtOH)

Stj.

Bisdehydroprotostemonine

C23H27NO6

8-23

192-194

169.0° (EtOH)

Stj.; St. s.

Tuberostemoamide

C12Hi7N03

8-24

amorphous

-28.1° (MeOH)

St. t.

C23H3lN0 6

8-25

165-167

-23.6° (EtOH)

St.j.;St.s.

Parvistemoamide

C12H19NO4

8-26

198 (HBr salt)

211.0° (MeOH)

St. p.

Stemoninine

C22H31NO5

8-27

113-115

-110.0° (EtOH)

St. t.

Bisdehydrostemo-ninine

C22H27NO5

8-28

176.5-177.5

-45.5° (Me 2 CO)

St. t.

Stemoenonine

C22H27NO7

8-29

192.5-195

10.5° (MeOH)

St. t.

Stemoninoamide

C17H23NO4

8-30

155-157

-94.0° (MeOH)

St. t.

L-stemofoline

C22H29NO5

8-31

76-78

-256.6° (MeOH)

St. p.

Oxystemofoline

C 2 2 H29N0 6

8-32

224-226

106.0° (MeOH)

St. p.

Methyloxystemofo-I ine

C23H3iN06

8-33

180-182

75.7° (MeOH)

St. p.

Stemonidine (Stemospiroline)

C 1 9 H29N0 5

8-34

119

5.4° (Me2CO)

St. s.;St.j.

Stemotinine

C18H25N05

8-35

207-208

91.7° (MeOH)

St. t.

Isostemotinine

C18H25N05

8-36

245-246

47.5° (MeOH)

St. t.

Tuberostemospiro-nine

C13H19NO4

8-37

245-246

-30.0° (MeOH)

St. t.

Parvistemonine

C22H33NO5

8-38

296 (HBr salt)

26.6° (MeOH)

St. p.

Didehydroparvistemonine

C 2 2 H29N0 5

8-39

220-222

-280.0° (MeOH)

St. p.

Parvistemoline

C17H25NO4

8-40

241-243

-24.7° (MeOH)

St.p

Alkaloids

Isoprotostemonine

la

St. /.; St. j . ; St.s are the same as indicated above. St. m. - St. mairei; St. p. = St. parviflora

ID

Plant Sources

1

REN-SHENG XI!

766

8 - 13

8 - 12

8 - 15

8 - 14

8 - 16

8 - 17

8 - 19 8 - 18

767

BIOACTIVE NATURAL PRODUCTS

(Scheme 17) contd.. Me Me

OM

8-21 8-20

8-23

OMe 8-24

8-25

Scheme 17. Structures of new stemona alkaloids-

2D-NMR techniques, especially HMBC and ROESY, are powerful tools for determining the steriostructure and proton and carbon signals assignment. The following are an example of these studies on the structure of tuberostemoninoamide. The relationship between long range of protonscarbons (HMBC) and proton-protons (ROESY) can be seen from the Scheme 19*. From the above structures we noticed all of stemona alkaloids contain a 4-azaazulene or perhydroazaazulene ring and most of them have ocmethyl-y-lactone ring linked at C-3 position, which can be detected by IR absorption at around 1760 cm-1, characteristic mass peak at M+ - 99 (M+ -C5H7O2) and doublet methyl signal at around 81.20. Some of these alkaloid structures contain another a-methyl-y-lactone ring in its left side. •The details will be published by Ye,Y.; Xu, R.S.

REN-SHENG XI!

768

8-29

Me

pMe

OH

8-32

Me

8-34

8-33

Me

OH

Me

-GfA 8-37

8 - 35

BIOACTIVE NATURAL PRODUCTS

769

(Sceheme 18) contd.. Me

8 - 39

8 - 38

Me

-

r s°\.

^Me

KTT>»

8-40 Sceheme 18. Structures of new stemona alkaloids-2.

The same species of the plant root contains different stemona alkaloids due to the growing area. For example, it was learned that stemotinine and isostemotinine are only contained in the roots of St. tuberosa growing in Wenshan district of Yunnan province, China. o

yH

Me>j Me O

Scheme 19. HMBC and ROESY of tuberostemonioamide 8-16.

All these structures can be categorized to six types: Alkaloids 8-12 to 8-16 belong to tuberostemonine 8-1 type; alkaloids 8-17 to 8-20 belong to maistemonine 8-19 type; alkaloids 8-21 to 8-26 belong to protostemonine 8-9 type; alkaloids 8-27 to 8-30 belong to stemoninine 8-27 type; alkaloids 8-31 to 8-33 belong to stemofoline 8-10 type; alkaloids 8-34 to 8-37 belong to croomine 8-11 type and alkaloids 8-38 to 8-40 belong to parvistemonine 8-38 type [74]. Separation and structure elucidation of

770

REN-SHENG XII

these stemona alkaloids may help to understand the biosyntheses of the alkaloids in Stemona plants and their taxonomy. Bioassay of the separated stemona alkaloids showed at 200 ppm isoprotostemonine 8-25 has antifeedent activity 33% to Spodoter litura. Bisdehydrotuberostemonine 8-14, bisdehydroneotuberostemonine 8-22 and bisdehydrostemoninine 8-28 possess some antileukemia P388 activities. The further bioassays are in progress . CONCLUSION The above presented materials cover only a small fraction of the TCM research field in China, and most of the researches are still in continuation. Today as Back-to-Nature has become a worldwide trend, TCM becomes an appealing and fascinating research field, attracting research efforts from all over the world. In recent years, TCM has been used in treatment of many difficult and complicated human diseases, such as AIDS, Cancers, Alzheimer's diseases, etc. Its efficacy has been supported by numerous clinical data. To further explore the mechanism of TCM and active principles become a challenge to all the natural product chemists, biological and medical researchers. Though TCM is originated from China, it belongs to the world. While more and more scientists are involved in the TCM research, we believe this research field will be fruitful and beneficial to the mankind in the 21 century. ACKNOWLEDGMENT The author is indebted to Prof. Ze-nei Chen, Department of Chemistry, Shanghai Second Medical University for his kind supply baogongteng A references and Prof. John E. Kuo, Department of Chemistry, Central Missouri State University, for his valuable suggestions and comments.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

Mei, Y.W. Chin. J. Information on TCM, 1996, 3, 5. Xu, R.S. Natural Products Chemistry, Academic Press, Beijing, 1993. Xu, R.S.; Xu, J.P.; Ye, Y. Intern. J. Orient. Med, 1992, 17, 90. Xu, R.S.; Zhu,Q.Z.; Xie, Y.Y. J. Ethnopharm.., 1985, 14, 223. Zhou, H.J.; Lu, Y.R. J. Chin. Pharm. Sci, 1992, /, 1. Luo, X.D.; Shen, C.C. Med Res. Rev. 1987, 7, 29. Zhou, W.S.; Xu, X.X. In Studies in Natural Products Chemistry, Atta- urRahman, Ed.; Elsevier Science B.V: Amsterdam, 1989 ; Vol. 3, pp 495-527. Fu, Z.C.; Li, Y.Y.; Zhou, L.Y., Zhong Cao Yao, 1983, 14, 15.

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