The Celastraceae from Latin America Chemistry and Biological Activity

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 18 © 1996 Elsevier Science B.V. All rights reserved.

739

The Celastraceae from Latin America Chemistry and Biological Activity

O. Muhoz, A. Penaloza, A.G. Gonzalez, A.G. Ravelo, LL. Bazzocchi and N X . Alvarenga 'Universidad de Chile, Facultad de Ciencias, Casilla 653-Santiago, Chile. C.P.N.O. Antonio Gonzalez, Universidad de La Laguna, Carretera La Esperanza 2, La LagunaTenerife. Espafia.

1. INTRODUCTION The CELASTRACEAE family was last reviewed in 1978 (Brtining & Wagner) [1] and since then a great deal of new chemical and phannacological information has accumulated. Celastraceae species have a long tradition of use in medicine and folk agriculture, especially in Asia and Latin America but also in other continents and undoubtedly took on a new lease of life in the seventies when the MAYTANSINOIDS, compounds with exceptional antitumoral properties, were discovered [I]. Nonetheless, the maytansinoids have not been made into a useful drug form as they cause serious gastro-intestinal damage when applied to rats [2]. For some time now several research laboratories have been intensively researching this fiamily, inspired by its broad and varied botanical distribution, the interesting chemical nature of its secondary metabolites, the complexity of the biogenetic processes which produce them, and most of all by the different types of pharmacological action displayed by preparations of its constituents. In Latin America (Mexico, Central America, the Caribbean and South America) this study is particularly momentous due in tfie main to a socio-economic and cultural climate which has not in the past lent itself either to sound development or the rational exploitation of the resources of the various countries involved. In the course of these research programmes, cytotoxic quinones, polyester sesquiterj)enes and pyridine-sesquiterpene alkaloids with antifeedant and/or insecticidal properties have been isolated from Latin American species, m particular those of the Maytenus genus which is extensively used by rural communities and tribes in the Andes and the Amazon basin. Recently some sesquiterpene alkaloids with immunosuppressive activity and sesquiterpenes with antitumoral activity have also been described [3].

740 As a general rule, the biosynthesis of skeletons belonging to the Celastraceae family is extremely specific, the triterpene-quinones and P-dihydro-agaroftiran type skeleton sesquiterpenes from these species having a notably high degree of oxidation. The presence of triterpene-quinones indicates the biosynthetic specificity' of the Celastraceae family since these compounds are synthesized in the roots and are virtually exclusive to the family. The next few pages are an update on the state of the Celastraceae family in Latin America, detailing the different chemical structures found and the results of the studies of biological activity carried out since the last publication on the subject [1].

2. THE SYSTEMATICS OF THE CELASTRACEAE FAMILY AND THE LATIN AMERICAN GENERA

THE CELASTRACEAE FAMILY

The Celastraceae family consists of about 55 genera and 850 species. According to Takhtajan [4], Hippocrateaceae would be subordinate to the Celastraceae. Systematically the Celastraceae family is arranged hierarchically as follows: Division: Spermatophyta; Subdivision: Magnoliophyta (Angiosperms); Class: Magnolitae (Dicotyledons); Order: Celastrales; Family: Celastraceae The Celastraceae family is probably related phylogenetically with the Aquifoliaceae; the presence of glandular discs around the ovary and the bright coloured aril in the Celastraceae are the principal differences between the two families. The Celastraceae family is pantropically distributed with radiation towards temperate or temperate-cold climates. In other words, the Celastraceae are principally concentrated in the tropical and subtropical regions and to a lesser extent in the temperate zones of the world (Figure la). The family is better represented in Central America and the West Indies than m South America except for the Maytenus genus [5] (15 species in Peru and 15 in Venezuela). They are found growing as upright trees, bushes and lianas and almost invariably have resin ducts or cells in the bast of the stems and leaves. The leaves are simple, usually alternate and opposite; stipules small and deciduous or missing; the flowers are small, fasciculate, actinomorphic, forming cymes, very occasionally racemes or inflorescence. The flowers are in general bisexual and rarely polygamodioecious plants. The petals are freestanding or coneshaped. The fruits are berries, capsules, drupes or samaras. The seeds generally have a coloured aril and contain embryos with large cotyledons and a relatively oleaginous endosperm. The chromosomic numbers described are X = 8,12,14.

741 The Celastraceae genera are very diversified; some of die taxa widi most species are: Maytenus (225; tropical), Salacia (200; tropical), Euonomynus (176; Himalayas, China and Japan), Hippocratea (120; tropical. South America, Mexico and the South of the USA), Cassine (40; South Africa, Madagascar, tropical Asia and the Pacific), Celastrus (30; principally Asia, some in Australia and in tropical and temperate zones of America), Elaodendrum (16; tropical and subtropical), Pachystima (5; North America) and Gyminda (3; Central America, Mexico and Florida). Gentry [5] compared the woody taxa found in Africa and America and commented that tiae genus Maytenus was in taxonomic terms closer to other genera of the same family found on the same continent than to other species of Maytenus from the other side of Ae Atlantic; this resemblance pattern is observed also at the level of secondary metabolites. Although there is no clear explanation for the foregoing, paleobotanic studies have shown that the paleoflora of South America and Africa were very similar in the late Paleozoic era (340-240 My) [6]. Bearing in mind that the early stages of Continental Drift between Africa and South America occurred about 135 My ago it can be argued diat many populations widely distributed in Gondwana were separated as the resuh of the Continental Drift (Figure lb) wliich meant that the split populations would develop independently Aereafter. Although few examples of Ae Celastraceae family have been found and only recently, fossilized remains of Celastrus show that during the Tertiary Age this genus was unrestrictedly distributed throughout America [7] and Europe [8]. It has even been suggested that the present distribution pattern of some species of American Celastrus corresponds better to a dispersion centre in Asia than to (Mie in Central America [7]. The phylogenetic history of the Celastraceae family, - and of other widely distributed families,appears to run parallel with that of the Continental Drift. Modem chemomolecular studies may help to determine the phylogenetic relationships of its different members, as well as with other families of the Celastrales order. If the theory of the Continental Drift is true, it is not surprising that the phylogenetic relationships of the Celastraceae with odier plant families should date back a long time. New chemotaxonomical studies are constantly appearing which relate the Celastraceae with tfie Lamiaceae, for instance. The presence in both femilies of sesquiterpenes, diterpcnes and triterpenes of high and specificfrmctionalityconfirms such a relationship [9].

742

Figure la.- Present-day distribution of the species of the Celastraceae family. As can be seen, this family has developed for preference in die tropics in the New and Old World, and is extending towards temperate or temperate/cold climates in the southern hemisphere.

Figure lb.- In overall terms, the Continental Drift is theorized as shown in this scheme. Some 200 My ago, all Ae continents formed a single mass, known as Pangea (a), which later split into two, Laurasia (NorA America, Europe and Asia) to Ae north and Gondwana (the Antarctic, South America, India, Australia and New Zealand) to the south (b). Still later Gondwana disintegrated, with India breaking off first and fmally colliding with Asia, then South America litting off from Africa, New Zealand from Antarctica and last of all, Australia from Antarctica (c &d).

743 3. SESQUITERPENES The salient feature of the family has been its wealth of sesquiterpenes with abnost a hundred of these compounds being isolated and characterized chemically. This group of metabolites very common in Latin American species have the eudesmane basic skeleton, and originate the wellknown p-dihydro-agarofuran skeleton polyester sesquiterpenes which are esterified by a series of common organic acids - acetic, benzoic, 3-furoic, trans-cinnamic acid, etc. (Table I).

A. POLYESTER SESQUITERPENES Sesquiterpene esters based on the dihydro-agarofuran moiety occur mainly within the Celastraceae family.

The basic polyhydroxy skeleton vary according to the position, number and

configuration of the esterresiduesin the dihydro-p-agarofuran sesquiterpene. The interest generated by polyester sesquiterpenes from the Celastraceae has increased in line with the complexity of the substances isolated and the possibility of their being applied to combat insect plagues instead of synthetic insecticides. The complexity and increasing numbers of these sesquiterpenes makes it difficult to arrange them systematically. They can however, be treated as derivatives of a basic polyhydroxy skeleton and thereafter organized in sunpler series. Accordingly, 37 series of P-dihydro-agarofuran type sesquiterpenes have been proposed ranging from a skeleton with two hydroxyls (boariol) to one with nine (euonyminol and isoueonyminol series) (Figure 2).

Sesquiterpenes with two hydroxy groups

K

HO

Bovbt

Sesquiterpenes with three hydroxy groups OH

OH

OH

OH

OH

OH

HOi

^ - < Isocekxbicol

4{J-Hyd'«y-6-deo)y-oekxt3icol

744 Sesquiterpenes with four hydroxy groups CH2OH OH

Maikangunbi

OH

OH

OH 4 (J-Hyd-ocy-cebrbicol

OH

2p.4p-Dihydroy^
1 S-Hydrwy-celorbk^l

Sesquiterpenes with four hydroxy groups and one ketone group

2.3.13.1 STetra-deoxy-eMC3nlnol

Sesquiterpenes with five hydroxy groups CH2OH OH

OH

HO Pentahydroxy-agarofu ran

OH 15-Hydnay-celapanol

OH 4p- Hydros oeiapand

745 CH2OH

CHzOH

HO

OH

OH 2.3-DideGD^maytoi

3.4-Oidecsy-mayto(

OH

2.3.13.1 S-TetradeGKyisoeuoniminoi

OH

U-< OH 4-DeGD^magelland

Sesquiterpenes with five hydroxy and one ketone groups CH2OH

OH 3.4.13-TrideoDy«vonnd

Sesquiterpenes with six hydroxy groups CH2OH

HOSs^

746 CHaOH

OH

CH2OH

-

OH

ep-Hydroxy-pentahydroxy-agarofurano

2.3.13-Trideo)^oeuoniminol

CH2OH HO

OH

HO

2a .4p-DtTy(Jroxy.a^-celapanol

OH

2p .4p .Dihydroxy-S-epi-celapanol

Sesquiterpene with six hydroxy and one ketone groups CH2OH

r OH 3.13-Didecs^^-ey^3ninoi

747 Sesquiterpenes with seven hydroxy groups CH2OH

HO

CH2OH

^4-^

HO

0.

OH 4^-Hydroxy-aiatol

OH

May4oi

Sesquiterpene with eight hydroxy and one ketone groups CH2OH

CH2OH

Sesquiterpenes with nine hydroxy groups CH2OH

H2OH

isoeuonimnol Figure 2.- Polyhydroxy P-dihydro-agarofiiran skeleton sesquiterpenes

The structural elucidation of these sesquiterpenes, given the difficulty in determining the linking sites of the respective ester groups when more than three kinds of acid are involved as esters in the molecule, necessitates the use of nmr experiments (selective INEPT [10], Coloc [11] or HMBC 2D [12]), selective hydrolysis processes or X-ray crystallographic methods [13]. From comparison of the chemical shifts of similar compounds, it is not easy to establish the exact position of each ester.

748

Structures were deduced from spectroscopic studies, basically heteronuclear 'H-'^C correlations (HETCOR). The regiosubstitution characteristics were generally ascertained by using long-range correlation spectra with inverse detection, with a 2-D heteronuclear multiple bond connectivity techniques (HMBC) which located the substituents (benzoates, hydroxyls, acetates etc); ROESY and nOe experiments were used to complement information about conformational interaction and long-range relationships. The absolute configurations were determined in almost all cases by applying the exciton-chirality method to dibenzoyl derivatives. Total and/or partial hydrolysis of the esters and derivative preparations (acetates, benzoates, methoxyderivatives, etc.) provided further information. The 'H nmr spectra of these sesquiterp)enes show three well-defined absorption regions which are the main sources of analytical information: the high field region where signals for the methyl groups at C-12 and C-13 and, in the highest part of the spectrum, the characteristic methyl group at C-14 as doublet are all to be found. This latter is a useful diagnostic aid because this position tends to have a hydroxy substituent, and when this occurs, the doublet methyl signal disappears; secondly, the usual shift of the acetoxy groups is between 5 1.5 and 2.2 ppm; if the acetoxy shift is at 5 1.5, the relative position of the benzoyloxy group can to be determined as C-1 or C-9 on the decaline ring system since this group anisotropically shields the acyl groups in peri position to the ring. The H-7 equatorial proton appears as a doublet (J=3.0 Hz) between 6 2.5 and 3.0 depending on the relative position of the C-8 and C-6 protons with which it is normally coupled. The midspectrum between 5 3.0 and 6.5 is usually where the characteristic signals for the hydroxy acetate and/or benzoate geminal protons are to be seen together with those of the C-15 geminal protons which vary according to whether they are esterified or not. The shift and multiplicity of the signals usually provide information about Ae relative stereochemistry and regiochemistry of the substituents. The benzoate protons resonate in the low-field region between 7.0 and 8.0 ppm, making make it easy to distinguish them from the other ester groups of the p-dihydro-agarofuran system. Each of the signals exhibits the downfield chemical shift to be e3q>ected of the corresponding esterified OH group derivatives. Decoupling experiments and/or COSY, ROESY, etc. complete the information available. In general, the sesquiterpenes described to date have a stable configuration and conformation free from interconversions and normally determined by the configuration of the chiral centres C1, C-4, C-5, C-7 and C-10. This structural rigidity is also apparent in the stereochemistry and functionality of some stereocentres:

749 (a) C-1 and C-9 are usually esterified with C-1 regiosubstitution being a; (b) When there is substitution at C-6, it is always p; (c) If there are hydroxy group on C-4, it assumes a p equatorial position; (d) Stereochemistry and regiosubstimtion at C-2, C-3, C-8 and C-9 vary; (e) The oxo groups are usually at C-8 and less commonly at C-6. The following tables summarize the above data on the same lines as laid down by Wagner & BrUning[l].

TABLE 1. SESQUITERPENES ISOLATED FROM LATIN AMERICAN SPECIES 15

10

t L4^" V 13

1 Form

C-1

C-2

C-3

C-4

L 1

aOAc

aOAc

2H

H

pOAc

2

aOAc

aOAc

2H

H

3

aOAc

aOAc

2H

H

4

aOAc

aOAc

2H

5

aOAc

aOAc

2H

6

aOAc

aOAc

C.6

c-8

C-9

C-IS

Ref

2H

pOBz

OAc

14

pOAc

=o

pOBz

OAc

15

POAc

aOAc

POBz

OAc

15 1

H

POAc

aOAc

pOBz

OH

15

H

POAc

=o

pOBz

OH

15

2H

H

POAc

2H

pOBz

OH

14

7

aOAc

aOAc

2H

H

POAc

aOH

pOBz

OAc

15

1 8

aOAc

aOAc

2H

H

pOAc

aOAc

aOBz

OH

14

9

aOAc

aOAc

2H

H

POAc

aOH

pOBz

OH

15

10

aOAc

aOAc

2H

H

pOAc

aOH

aOBz

OH

14

11

aOAc

2H

2H

H

POAc

aOAc

pOBz

OAc

16

12

aOAc

2H

2H

H

POAc

2H

pOBz

OH

16

13

aOAc

2H

2H

H

pOAc

aOH

pOBz

OAc

16

14

aOBz

2H

2H

POH

POAc

pOAc

POBz

H

16

15

aOBz

POAc

POH

POH

pOAc

H

pOBz

H

17

16

aOBz

POAC

pOAc

pOH

pOAc

H

pOBz

H

17

17

aOBz

POH

POAc

pOH

POAc

H

pOBz

H

17

H

17

18

aOBz

pOAc

POH

pOH

H

H

pOBz

19

aOBz

pOAc

pOAc

pOH

H

H

pOBz

H

17

20

aOBz

pOAc

pOH

H

pOAc

H

pOBz

H

18,19

21

aOBz

POAc

POcA

pOH

pOBz

H

pOBz

H

17

22

aOBz

POAc

pOAc

pOH

H

pOAc

POBz

H

19

1

750

23

aOBz

pOAc

pOH

pOH

H

POAc

POBz

H

19

POH !

H

pOBz i

POBz

H

20

POAc

POAc

pOBz

H

20

pOBz

H

21

21 1

24

aOBz

pOAc

POAc

1 25

aOBz

aOAc

H

pOH

26

aOBz

pOAc

H

pOH

pOBz

pOAc

27

aOBz

POAc

H

pOH

POAc

pOAc

POBz

H

pOBz

28

aOBz

POAc

H

pOH

H

POBz

H

20

29

aOBz

pOAc

H

POH

H

H

POBz

H

20 1

30

aOBz

H

H

pOH

H

H

pOBz

H

19

H

pOH

pOAc

H

POBz

H

19

POH

POH

POH

H

POBz

H

POAc

H

POCinn

H

21 1 22 1

H

H

H

18,23 I

H

24 1

: 31

aOBz

H

L32

aOBz

POAc

33

aOBz

^ A c L POH

POH

34

H

H

aOH

aOH

35

aOBz

2H

2H

pOH

POAc

2H

pOAc 1

36

aOBz

2H

2H

pOH

POAc

2H

POAc

OAc

24

1 37

aOBz

2H '

pOH

pOAc

pOH

aOAc

OAc

24 24

2H

H

38

aOCinn

pOAc

2H

POH

POAc

2H

pOAc

H

39

aOBz

2H

2H

POH

POAc

pOAc

aOAc

H

24

40

aOBz

2H

2H

POH

pOH

H

pOAc

H

24

41

aCinn

POH

H

POH

pOH

H

pOAc

H

25

42

oEp-

pOAc

2H

pOH

pOH

2H

pOAc

H

25 1

2H

2H

pOH

pOH

2H

pOAc

H

25 1

2H

2H

pOH

pOAc

2H

pOAc

H

25 1

Cinn

43

aCinn + Ep-Cinn

44

aCinn + Ep-Cinn

45

aOBz

2H

2H

pOH

pOAc

pOAc

aOAc

OAc

26

46

aOBz

2H

2H

POH

pOAc

==o

aOAc

OAc

26 1

! 47

aOBz

2H

2H

POH

pOH

pOH

aOAc

OAc

26

! 48

aOBz

2H

2H

POH

POH

pOBz

aOAc

OAc

26 1

49

aOBz

2H

2H

POH

pOAc

POH

aOAc

OAc

26

50

aOBz

2H

2H

pOH

pOAc

2H

pOAc

OAc

26

51

aOH

aOBz

2H

POH

POAc

2H

POBz

OAc

27 1

52

aOAc

aOBz

2H

POH

POAc

2H

53

aOAc

aOBz

2H

pOH

POAc [ aOBz

54

aOBz

pOAc

2H

POH

POAc

1 2H

55

aOBz

aOAc

2H

1 POH

POAc

2H

pOBz

56

aOAc

2H

2H

POH

POBz

2H

pOAc

2H

POH

pOAc

2H

1 pOBz

OBz

28

__iH

! POH

1. 2H

pOAc

OBz

28

1 57 _ aOAc L?8_._ ^ocOB^

2H pOAc

2H

pOBz

OAc

27

POBz

OAc

27

pOBz

H

28

H

i

28 1

OAc 1 28

751

L^i

aOAc

aOAc

2H

pOH

pOAc

=o

aOBz

OAc

i 60

aCinn

aOAc

2H

POH

POAc

=0

aOBz

H

29

61

aOAc

2H

2H

H

POAc

aOAc

pOFu

H

30

62

aOAc

2H

2H

pOH

POFu

2H

pOFu

H

30

2H

pOH

pOFu

aOAc

_pOFu

H

30

_63_

aOA£_ _ _ 2 H _

29

Ep-Cinn = Epoxycinnamate esters Cinn = Cinnamate ester 0-Fu = Furoate esters

The maytolins are an interesting instance of new sesquiterpenesfix>mthe Celastraceae characterized by the presence of a tetrahydro-oxepine nucleus. It would seem that these new types of skeleton are only biosynthesized by species of the MORTONIA genus, which consists of just four species, endemic to Mexico and the southern Unites States. The chemical study of three of these four species led to the isolation and characterization of eight new sesquiterpenes [3133](Figure3). OBz

OBz OBz

082 OBz

HO^i COOH 64 Mortonin A, R=H

66 Mortonin C

67 Mortonin D

68 Mortonol A, R=H 69 Mortonol B, R= OAc

65 Mortonin B, R=OAc

Figure 3. Sesquiterpenes isolated fix)m the genus Mortonia

The structures proposed

for MORTONINS A and B are the first recorded example of a

natural product in which ring B of the eudesmane skeleton undergoes oxidative cleavage to the the y-lactone. The subsequent isolation and characterization of the di-ester ketone MORTONOL (68) from M. greggi suggests that this sesquiterpene might be the biogenetic precursor of the whole MORTONIN series (Figure 4).

752

64.66

67

Figure 4. Possible formation of Mortonins sesquiterpenes

Boariol [18,23] is another new sesquiterpene isolated from the Chilean species M boaria Mol. which does not conform to the classic model of the sesquiterpenes previously described, and is in fact the simplest of all the compounds recorded from the Celastraceae. 'H and ^^C nmr studies showed the presence of a secondary and a tertiary OH, the latter at C-4 but with the opposite configuration to the customary p-hydroxyl at this position. The application of the Horeau method and an X-ray diffraction study confirmed the absolute configuration of the compound [18,23]. The absence of substituents at C-1, another notable feature of this structure, casts doubis on the biogenetic theory for the P-dihydro-agarofuran sesquiterpenes from this family which presumes that such substituents are present in nature. The possibility that boariol (34) might be an artifact was ruled out on the basis of two data: several sesquiterpenes with the classic C-4 d-OH configuration have been isolated from M. boaria Mol., even some with C-3 substitution; no products were obtained with carbonyl groups at C-3 and without hydroxy groups at C-4 which could have been hydrated non-stereospecifically via enol formation [18,23]. Fig. 5.

753 B. SESQUITERPENE ALKALOIDS Sesquiterpene alkaloids have similar structures to polyester sesquiterpenes except that the hydroxy groups of the eudesmane basic skeleton are esterified by nicotinic acid and/or its derivatives.

Little has been published about sesquiterpene alkaloids from American species

which tend to be found in the roots of the plants (Table II).

TABLE 11. N4AYT0LIN-TYPE SESQUITERPENE ALKALOIDS 15 10

f. M>" *13

Form

C-1

C-2

C-3

C-4

C-6

70

aOBz

2H

2H

pOH

pONic

71

aOCinn

POH

2H

pOH

pONic

72

aOBz

2H

2H

POH

pONic

73

aOBz

2H

2H

pOH

pONic

74

aOAc

aONic

2H

POH

I 75

aONic

aONic

2H

pOH

76

aOAc

aONic

2H

77

aOAc

aONic

78

aONic

79

aOAc

C-9

j C-8

C-15

Ref

pOAc

H

1 34

2H

POAc

H

34

POAc

aOAc

H

34

pOH

aOAc

H

34

pONic

aONic

pOBz

OAc

35

pOBz

aONic

POBz

OAc

35

pOH

pOBz

aONic

pOBz

OAc

35

2H

pOH

pOAc

aONic

POBz

OAc

35

aONic

2H

pOH

pOAc

aOBz

pOBz

OAc

35

aOBz

2H

H

pOAc

2H

PONic

OH

36

2H

'

80

aOAc

aOBz

2H

H

pOAc

2H

PONic

OAc

36

81

aOAc

aONic

2H

(30H

POH

aONic

pOBz

OAc

37

82

aONic

aONic

2H

pOH

pOH

aONic

pOBz

OAc

37

83

aONic

aONic

2H

pOH

1 pOAc

aONic

\ pOBz

OAc

37

1 ^"^ 1 85

aONic

aOAc

pOH

POH

aONic

POBz

OAc

: 37

aOAc

1 aONic

L ^QAc

1 aONic

2H

1 2H

L_H

1 j

1 pOBz 1 OAc 1 37 __..

C. N4ACROCYCLIC SESQUITERPENE ALKALOIDS Celastraceae also elaborate other, more complex, alkaloids, also polyester sesquiterpenes, incorporating a macrocycle derived from an evonic, wilfordic, cassinic or other type p>Tidine dicarboxylic acid with an additional alkyl chain of the basic eudesmane cycle at C-3 and C-7 (Table III). Celastraceae alkaloids are well-documented for the European and Asian genera, particularly Catha, Celastrus, Euonymus and Trypterigium but are relatively rare among the Latin

754 American species. Except for a few from the Hippocratea, Peritassa and Orthosphenia genera, most new Celastraceae alkaloids have been obtained from species of Maytenus. As in the case of the polyester sesquiterpenes, structural elucidation has been based on 1H13C nmr correlations (HETCOR) and long range inverse detection (HMBC and HMQC). Relative configurations have been determined by the combined use of NOESY experiments. The absolute configuration of almost all the compounds was established by circular dichroism applications using the exciton chirality method in 1,2-dibenzoate systems. TABLE III. MACROCYCLIC ALKALOIDS FROM AMERICAN CELASTRACEAE (Wilfordate type)

!

Form

R'

R2

R3

COMPUESTO

Rcf|

35

86

OBz

OBz

OAc

EbcnifolincW-l

87

OBz

OBz

OH

Ebenifoline W.2

35

88

OBz

OAc

OAc

Euojaponine F

35

89

OAc

OAc

OAc

Euonine

35

1

90

OAc

OBz

Cangorinine W-I

36

1

91

OAc

OBz OBz

ONic

CingminiDe W-II

36

TABLE IV. MACROCYCLIC ALKALOIDS FROM AMERICAN CELASTRACEAE (Evoninoate type)

755

Rl

R^

R^

R4

R5

R7

R«

COMPUESTO

i ^^

OBz

OH

OH

OAc

OAc

OAc

H

OAc

EbcnifolincE-l

Rcf. 1 38 1

I 93

OB7

OAc

OH

OBz

OAc

OAc

H

OAc

Ebenifolinc E-2

38

R6

94

OBz

OAc

OH

OAc

OBz

OAc

H

OAc

Ebcnifolinc E-3

38

95

OBz

OAc

H

OAc

OAc

OAc

H

OAc

Ebenifolinc E-4

38

1^

OBz

OAc

OH

OH

OBz

OAc

H

OAc

Ebenifbline E-5

38

97

OBz

OH

OH

OBz

OAc

OAc

H

OAc

EuojaponineC

38

98

OBz

OAc

OH

OAc

OAc

OAc

H

OAc

Mayteine

38

99

OAc

OAc

OH

OAc

OAc

OAc

H

OAc

Euooymine

38

100

OAc OAc

OH

OBz

OAc

OAc

H

OAc

CangoriniE-I

39

101

OAc

OAc

OH

OAc

OBz

H

OAc OAc

Horridtne

40

I 102 OAc OAc

OH

OH

OAc

H

OH

OAc

Acanthotfaamine

41

103

OBz

CNMP

OH

OAc

OAc

OAc

H

OAc

Hippocrateine I

[AQ4_

OAc

CNMP

OH

OH

OAc

OAc

H

Mb

Hippocratcine n

42 J2

j

CNMP= 5 Carboxy-N-methylpiridonyl Mb= 2-Methylbutyroyl Orthosphenin (105) breaks the classical mould of the Celastraceae macrocyclic alkaloids described to date and is the only example of an evoninol nucleus with an oxo group at C-8 and residual cassinic acid. Its structure was ascertained by the spectroscopic methods mentioned above, hydrolysis and the preparation of derivatives [43]. Two new evoninate-type alkaloids have recently been described, peritassin A and B, obtained from species of the genus Peritassa. These structures are distinguished by the macrocyclic unit which consists of an evoninic acid isomer in which the pyridine ring of the dibasic acid is substituted at 4'-5' instead of the more usual substitution of evoninic acid at 2'-3' [44].

R«OAc PcritassineA R = OBz PeritassincB Figure 6. The Structures of Orthosphenin and Peritassin A and B

756 IV. DITERPENES In general, very little has been written about diterpenes from the Latin American Celastraceaeas these structures are not often found. Abietriene type diterpenes have been the general rule in the Celastraceae although the chemical study of the minor constituents of Orthosphenia mexicana and Rzedowskia tolantonguensis did enable pimarane type diterpenes to be isolated and chemically characterized [43,45] and the second of these species afforded a series of new diterpenes with an isopimarane skeleton, described for the first time in the Celastraceae. The structure of the diterpene 107 (C20H30O3) was established by spectroscopic methods and confirmed by x ray diffraction studies while, under nmr, the nor-diterpenes 109-113 proved to be structures with an exocyclic methylene and no carboxylic groups at C-4 and are assumed to be the result of an oxidative decarboxylation process as has occurred elsewhere. Orthosphenia mexicana yielded another new diterpene of the nor-isopimaradiene type (C19H28O3) related to the abovementioned products [43]; spectroscopic analysis and chemical trans-formations established its structure with a tertiary hydroxy 1, an a,p-unsaturated keto group and the presence of a typical vinyl system of the ABX type.

CH2OH

W^' Fig.

106 107 108

Rl (M COCHi CH2OH

R2

0 2H 2H

Ref 43 45 45

CHzOH

R2.„

^

Fig. 1 109

110 111 112 113

Rl 0 BOH aOH 0 POH

R2

H H H

(m OH

Ref

757 V. ALKALOIDS M. loesner Urb. and M buxifolia (A. Rich) Griseb collected on the island of Cuba have been extensively studied by H. Ripperger et al. [46-47] who isolated a series of new macrocyclic alkaloids of the spermidin type, commonly found in the Celastraceae family; the new alkaloids could be related to others akeady obtained by Kupchan's group.

feHs o H

Fig

R

COMPOUND

Ref

114

OH

Mayfoline

46

115

OAc

N( 1 )-acetyl-N{ 1 )-deoxymayfoline

46

"T^ ^

^3^^..^^' OAc

Fig.

R

COMPOUND

Ref

116

C^rl] jK^H^^H^H-

Loesenerine

47

Q')T{^-Qr\\^-Q\\-(Z')r{^-

17.18-Didehydroloesenerine

47

CnHs-CHOH-C4H4-

16,17-Didehydroloesenerin-18-ol

47

i 117

118

VI. TRITERPENES A. TRITERPENES FROM THE AMERICAN CELASTRACEAE The triterpenoids hitherto described for the Celastraceae almost invariably belonged to the FRIEDO-OLEANE series (including methylene quinones and phenolic compounds), LUPANE, OLEANE, GLUTINANE AND TARAXERANE series. Characteristic of the family are the triterpene methylene quinones synthesized in the roots of the plant and considered as taxonomic indicators and the same holds true for the American species. To date about 12 different endemic species belonging to eight different genera have been studied and 26 new triterpenes have been described as well as new triterpenoid dimeric structures. As is usual, all the species studied have a broad range of biological activity probably due to the presence in most, of triterpene methylene quinones of known biological effect such as pristimerin, celastrol, tingenone, iguesterin [48] etc.

758 Particularly interesting has been the case of Orthosphenia mexicam which yielded five new triterpene methylene quinones with a new carbon skeleton, a greater degree of conjugation than hitherto reported, an extra 14-15 double bond and a rearranged methyl at C-15. Its structure was elucidated by a succession of chemical transformations, spectroscopic methods and X ray diffraction which determined the absolute configuration of this compound [49,50]. TABLE V. METHYLENE QUINONE TRITERPENES

R4

R5

COMPOUND

Ref

0

Me

Netzahualcoyone

49

H? OH

Me

Netzahualcoyonol

50

Me

Netzahualcoyondiol

50

H? H-,

CO^Me

Netzahualcoyol

50

Me

Netzahualcoyene

50

R2

R3

COMPOUND Pristimerin

50

H

H? 0

H.

50

0

H7 OH

Tingenone

H

22-6-Hydroxy-tingenone

51

OH

0

H.

20-a-Hydroxy-tingenone

51

Celastrol

50

Rl

R2

CO^Me

OH

CO^Me

OH

H

C07Me

OH

H

CO^Me

OH

H

CO^Me

H

H

Rl CO^^CH:,

i COOH

R3

H

JH2_

ik^

Ref

!

759

R2

R»

COMPOUND

Rcf,

CO,CH,

Hz

Isopristtmerm in

52 1

H

0

Isotingcoooc m

. _52 . ]

TABLE VI. FRIEDELANE TYPE SKELETON TRITERPENES

[RJ CH, CH^ CH,

R3

R^

o o

H:

0

0

H:

R2

R6

COMPOUND

Rcf

H2 oOH H2 aOH 0

CH,

FricdcUmc-3,15-
53

1

CHi

15-a-Hydroxyfricdelin

53

1

CHi

15-a-Hydroxyfricdelffie-1 »3-iliooe

53

1

H2 0

CH,

Friedeliii

53

1

CH,

Fncdetane-1,3-
53

]

% H2

H2.

CChCHx

3-Oxo-firiedooleaii-2S-oic metfayi ester

54

1

H7

H?

CH,

Maytenoic acid.

52-55

H2

CH,

Methyipopubiooate

55

H2

CH3

3^Hydroxy-2-oxofriedclan-20a-

55

H?

[CH,

0

H2

H?

CH^

0

H7

H7

|CH,

o o o

|ca>H

rco,cH^ [COjH

H7

H: fiOH 0

H2 H2

R5

cartrnxyik ac.

I CHt^OH 0

[CH^

0

1 CO^H __ _o

H: H2 H^ H: OH H^

H:

CH,

3-Oxofricdoolcan 20 a-hydroxy

52

H2

CH,OH

Canofilol

54

2a'Hydroxy populnonic ac.

51

i i 2 _ CH;

1

760 TABLE VII. FRIEDELANE TYPE SKELETON TRITERPENES

[RT" R2

R3

CX>MPOUND

Ref.

3-Hidroxy-2-oxofriedeliii-3-cne-20a-c«boxylic acid.

55 55

OH

CH,

H

OH

CH,

CH, 3-Hi(fax)xy-2-oxofiiedeian-3-aie-20a-indliyl caiboxyl

OH

CHO

H

Cangoronine

CH,

H

2-Oxofnedooiean-3-en-29-oic acid.

[H

52

J6

When the carboxy group at C-24 in cangorinin undergoes oxidative elimination, followed by oxidation of the A/B ring, pristimerin type triterpenes are obtained. The isolation (for the first time from a natural source) of both isopristimerin and isotingenone in the one plant bears out the biogenetic theory of the biomimetic conversion of pristimerin and tingenone, isopristimerin III and isotingenone III put forward some time ago by Monache et al [57].

TABLE VIII. FRIEDELANE TYPE SKELETON TRITERPENES

Rl CO:,H 1

H

R2

R3

H2 CH, 0 OH

COMPOUND

Ref

2,4(23)-Friedeladien-29-oic ac.

51

2,4(23>Friedeladicn'22B-hydroxy-21 -one

51

761 CHjOH

Figure 7. D:A-friedoolean-l-en-29-ol-3one (Ilicifolin) [52]

120

Figure 8. Transposed Friedo-olene Type Skeleton Triterpenes [58] 3-0X0-25(9-^8) abeo-fi4edoolean-(4)(23)-en-24->l-olida (119) y 3-oxo-25(9->8) abeo-J5nedoolean-(4)(23)-en-24-al (120) transposedfriedo-oleanetype skeletons isolated from Schaefferia cuneifolia

TABLE IX. OLEANE SKELETON TRITERPENES

762

R»

R2

=o

R3

R4

F5

R6

R7

R8

COMPOUND

ReJ

CH7OH

CH2OH

H

H

H

H

3-Oxo-28,29-
59

36,29-Dihydroxyolean-12-ene

59_

16,36,11 a-Trihydroxyolean-12-

60

12-ene OH

H

CH,

CH'^OH

H

H

H

H

OH

H

CH,

CH-,

OH

OH

H

H

cne OH

H

CH,

CH,

H

OH

H

H

36,11 a-EHhydroxyoIean-12-ene

60 J

OH

H

CH,

CHi

H

H

H

H

ft-Amyrm

60

OH

H

CH,

CH,

H

H

OH

H

36,15a-Dihydroxyolcan-12-eDC

61

OH

H

CH,

CO7H

H

H

H

H

Epikatonic ac.

55

CH,

CO-^H

H

H

H

H

Katononic ac.

55

=o OH

H

COOH

CH,

H

H

H

H

Oleanolic ac.

62

aOH

H

CH,

CO,H

H

H

H

aOH

Maytenfolic ac.

55

TABLE X. LUPANE SKELETON TRITERPENES

IRI i

R2 =0

R3

R4

R5

COMPOUND

Ref.

OH

CH,

H

3-Oxo-lup-20-en-30-ol

63,64

OH

H

OH

CH,

H

Lup-20-en-3B,30diol

63,64

OH

H

H

OH

H

Betulin

54,59,63

OH

H

H

CH,

H

Lupeol

54,60,62

OH

H

H

COOH

H

Betuiinic acid

62

H

CH,

aOH

3-Oxolup20-en-ll-ol

55

H

H

CH,

aOH

Li^)-cn-36,ll-diol

65

H

COOH

H

Betulonic acid

18

=0 OH =0

1

1

763 TABLE XI. OLEANANE A^« TYPE SKELETON TRITERPENES

Ri

R2

R3

R4

R5

COMPOUND

Ref.

OH

H

OH

H

H

Olean-18-ene-3B,166diol

66

H

OAc

H

H

3B, 16B-Diacctoxyolean-l 8-cnc

66

OH

H

H

16B-Hydroxyolean-18-en-3one

66

H

OH

H

16a-Hydroxyolean-18-en-3one

66

H

H

OH

3B, 11 a-Dihydroxyolean-18-ene

60

H

H

H

3-Oxo-olean-18-ene

51

OAc =0

=o H

OH =0

TABLE XIL GLEANAlsfE A^ TYPE SKELETON TRITERPENES

R2

COMPOUND

Ref. J

OH

CH:,OH

36,29-Dihydroxyglutm-5-ene

52,60

OH

CH,

3B,Hydroxyglutin-5-ene

60

Rl

TABLE XIII. OLEANANE A^ 1'12 xypE SKELETON TRITERPENES

764

^\ H

R? ^^ COMPOUND OH H 3 &-Hydroxyolean-9( 11), 12-diene =0

H

Ref.

60 60

3-Oxo-olean-9( 11). 12-diene

TABLE XIV. OLEANANE TYPE SKELETON TRITERPENES (with a hemiacetal 24-hydroxy-3-Keto) ~ »C02H

RJ

R2

R3

H

COMPOUND

Ref.

OAc

H

3-Acetoxy-salaspenmc ac.

63

OH

OH H

Orthosphenic ac.

63

OH

OH OH 66-Hydroxyorthosphenic ac.

50

OH

H

52,59,63

H

Salaspermico ac.

Friedo-oleane triterpenes with a hemiacetal 24-hydroxy-3-oxo grouping are exclusive to the Celastraceae and several have been isolated from South American species. It has been proposed that these compounds may be intermediates in the biogenetic pathway to the Celastraceae triterpene quinones.

B. DIMERIC TRITERPENES Triterpene dimers have recently been reported and are generally characterized by the presence of modified triterpene quinone monomer units. To date, ten dimers based on tingenone and/or pristimerin units have been described, most of them from the Maytenus genus [67]. A possible biogenetic route to triterpene quinones with an extra double bond (e.g., netzahualcoyone and derivatives) may involve dehydrogenation and subsequent pristimerin regrouping. Indeed, (Table V) two epimeric di-triterpene quinone ethers umbelatin a and p (118,119) have been isolated from the roots of Rzedowskia tolantonguensis [68]. The structures of pristimerin and netzahualcoyone were ascertained by the standard spectral methods. The ^H

765 nmr spectrum of dimers showed two carbomethoxy groups, 12 angular methyls and six lowfield protons and correlation with those of pristimerin and related phenol, zeylasterone 2,3dimethyl ether (Tables XV and XVI) established the position of the C-23, C-25, C-26, C-28 and C-30 methyls. Treatment of pristimerin with 2,3-dichloro-5,6-dicyanoben2oquinone (DDQ) in dioxan afforded a mixture of four products separated by preparative tic. The least polar product was netzahualcoyone followed by the phenolic compound, 123; the other two products of medium polarity proved identical to the natural dimeric epimers 121 and 122 (Fig. 9). The behaviour of pristimerin in these conditions resembles the mechanistic theory of Barton about the formation of usnic acid [69]. These dimers, accordingly, could be synthesized by generating two radicals from a single precursor, in this case, pristimerin (Figure 10). These ideas have recently been bolstered by the discovery of another dimer, from M. umbelata, consisting of oxidated tingenone units. When tingenone was treated with DDQ in dioxan, various products were formed, two of which proved to be 124 and 125, confirming the proposed structures and the general way that methylene triterpene quinones act with DDQ [70]. Itokawa et al. [71] have recently isolated and characterized four new dimeric triterpenes from M. illicifolia (Paraguay), three of which have two units of pristimerin while the fourth (cangorosin B) has one pristimerin and one tingenone unit (Fig. 11). Magellanin [67], another example of a dimeric triterpene ether composed of modified pristimerin units (Fig. 12) was obtained from the roots of a Chilean species of the Maytenus genus. Its upper unit is modified pristimerin with an epoxide between C-3 and C-4; the structure of the lower is similar to that of cangorosin B. Sixty signals were observed in the ^^C nmr spectrum. The epoxide was confirmed by the chemical shift of C-3 and C-4 at 6 91.85 and 78.70 very like those described for Itokawa's dimer [71] and the presence of an aromatic A ring with a hydroxy 1 at C-3' and an ether bridge on C-2' could also be deduced from this sp)ectrum (Table XVII). Its ^H nmr spectrum unambiguously assigned the double bond between C-6' and C-T (5 6.63, dd, 1=2.66,10.20 Hz; 6 5.90, dd, J=2.48,10.20 Hz) and the H-l' proton (6 6.70, br s). The C-H couplings were determined by 2D heteronuclear inverse detection methods (HMQC and HMBC) (Tables XVIII and XIX) and its MS revealed fragments at m/z 464 (C30H40O4), 466 (C30H42O4), 480 and 450 corresponding to the two possible types of signal for cleavage around the ether group while lesserfragmentsreflected the typical rupture of pentacyclic triterpenes [67]. As with other dimers, magellanin may be the result of in vitro or in vivo auto-oxidation due to radical coupling [67].

766

+ 121 • 122

Netzahualcoyttno R«Ri«R2»H N«tz«hijatcoypn« R«OH; Ri •Ra «0

Fig. 9. Experimental Demostration oftfiePossible Biogenesis of Celastraceae Dimers

OOCH,

H-O

=o«' |DDQ

I>1 y;ir 121 •

0^

y ^

diketo radical

122

Fig. 10. Probable Formation of Dimers 121 and 122 by Radical Coi^ling

767

121 Ri=a-Me; R2=CCX)Me; R3=R4=H

Umbellatina

122 Ri=p-Me; R2=COOMe; R3=R4=H

Umbellatin p

124 Ri=a.Me; R2=H; R3=R4=0 125 Ri=a-Me; R2=H; R3=R4K) Fig. 11. Some Dimeric Triterpenesfromthe Celastraceae

C00CH3

CO2CH3

C00CH3

Cangorosin B

Cangorosin A Atropcangorosin A Dihydroatropocangorosin A CO2CH3

CO2CH3

Mageilanin Fig. 12. DimersfromM. ilicifolia and M. magellanica

768 TABLE XV. ^^c NMR ( 50 MHz ) Data ( 6, CDCb, Chemical Shifts in ppm Relative to Me4Si) of Pristimerin and Ethers 121 and 122 Pristimerin

122*

121'

C

jC

c

C

1

119.0(d)

! 110.8(d)

114.9(d)

11.3(d)

2

178.4(s)

179.2(8)

173.4(8)

188.0(8)

115.3(d) 174.0(8)

i3

146. l(s)

171.3(8)

145.3(8)

171.5(8)

144.7(8)

117.0(8)

j 91.2(8)

j 124.0(8)

92.1(8)

|5

127.5(s)

128.5(8)

132.0(8)

127.7(8)

6

133.9(d)

129.0(d)

189.6(8)

126.7(d)

7

118.1(d)

117.4(d)

126.3(d)

116.2(d)

u 8

169.9(8)

164.5(8)

151.3(8)

161.4(8)

9

42.9(8)

38.8(8)

44.0(8)

38.2(8)

124.0(8) 130.1(8) 189.0(8) 126.2(d) 150.5(8) 41.9(8)

lio

164.7(8)

137.7(8)

151.3(8)

137.7(8)

151.0(8)

11

33.6(t)

33.0{t)

34.1(t)

33.0(t)

34.2(t)

[l2

29.7(t)

29.5(t)

29.7(t)

29.7(t)

|l3

39.4(8)

39.1(8)

39.3(8)

39.0(8)

[l4 lis

45.0(8)

44.5(s)

44.5(s)

44.7(8)

28.7(t)

28.7(t)

29.4(t)

28.7(t)

|l6

36.4{t)

36.5(t)

36.5(t)

36.4(t)

29.8(t) 39.9(8) 44.2(8) 28.6(t) 36.5(t)

|l7

30.6(8)

30.7(s)

30.7(s)

30.6(s)

30.6(s)

|l8

44.4(d)

44.8(d)

44.8(d)

44.5(d)

44.7(d)

|l9

30.9(t)

30.9(t)

31.0(t)

30.9(t)

31.0(t)

bo

40.4(s)

40.6(8)

40.7(s)

40.5(8)

40.5(8)

21

29.9 (t)

29.8(t)

30.0(t)

29.9(t)

29.9(t)

|22

34.8(t)

34.9(t)

35.1(t)

34.9(t)

34.9(t)

[23

10.2(q)

24.7(q)

13.3(q)

22.5(q)

12.8(q)

[25

38.3(q)

37.8(q)

40.2(q)

37.6(q)

37.7(q)

|26

1 21.6(q)

21.0(q)

22.5(q)

1 20.9(q)

S 22.2(q)

|27

18.3(q)

18.3{q)

18.6(q)

18.3(q)

18-6(q)

|28

31.6(q)

31.7(q)

31.7(q)

31.6(q)

! 31.6(q)

179.0(s)

179.0(8)

|29

178.7(8)

179.0(8)

1179.0(s)

[30

32.7(q)

33.0(q)

j 33.0(q)

32.7(q)

32.8(q)

[31

1 31.6(q)

L5L6(q)

! 51.8(q)

1 51.5(q)

1 51.5(q)

^ The values of the pairs C and C may be interchanged.

1 j 1 1 1 j 1 1 1

1 1 1 1 1

769 TABLE XVI. ^H NMR ( 200 MHz ) Data (5, CDCI3, for The Methyls. Zeylasterone Pristimerin

121 H*

H

122

2,3-DmiethyI ether

H

H*

23.Me

2.21

1.37 2.72

1.41

2.73

25-Me

1.48

1.48

1.57

1.47

1.58

1.60

26-Me

1.26

1.26

1.26

1.27

1.25

1.32

2.66

27-Me

1.10

1.05

1.08

1.06

1.08

1.12

|28.Me

1.18

1.16

1.16

1.16

1.16

1.18

30-Me

0.53

0.52 0.54

0.53

0.54

0.60

1

TABLE XVU. C NMR. (100 MHz) (6, CDCI3) Pristimerin

Magellanin

Pristimerin

Magellanin

C

c 1

C.28

31.6

31.5

31.8

C-1

119.0

115.6

108.0 1

C-29

178.7

178.9

179.3

C-2

178.4

191.1

140.0 1

C-30

32.7

32.8

32.2

C-3

146.1

91.8

C.31

51.6

51.6

51.6

C-4

117.0

78.7

C.5

127,5

130.7

C.6

133.9

126.3

C-7

118.1

116.3

C-8

169.9

160.5

137.6 122.4 125.0 124.0 129.1 45.5

C-9

42.9

41.6

38.2

1 1 1 1 1 1

TABLE XVm. H NMR. (200 MHz) Magellanin H-1

CUCD

C.D.

6.06 d

6.07 d

J(Hz) (1.16)

(1.44) 6.42dd

C-10

164.7

173.7

143.7

H-6

C-11

33.6

32.9

31.2

J(Hz) (1.16,6.30)

C-12

29.7

29.7

29.5

C-13

39.4

39.0

38.9

C-14

45.0

44.5

44.3

H-y

6.70 brs

7.04 brs

C-15

28.7

28J

28^

H-6'

6,63 dd

6.60 dd

|c-16

36.4

36.4

36.3

J(Hz) (2.66,1020)

(2.85,9.88) 1

C-17

30.6

30.6

30.4

H-T

5.48 dd

C-18

44.4

44.4

44.2

\^&) (2.48,10.20)

IH-7

6.32 dd 5.92 d*

J(Hz) (6.30)

5.90 dd*

(1.44,6.46) 5.32 d (6.46)

(2.58,9.88)

[c-19

30.9

31.0

30.5

|c-20

40.4

40.4

40.5

|c-21

29.9

30.0

29.8 !

TABLE XIX. Three-bond^H-^^C

|c-22

34.8

34.8

36.0

coupling (HMBC) in Magellanin

|c-23

10.2

22.5

10.9

H-I

C-3,C-5.C-9

|c-25

38.3

34.9

22.2

H-6

C-4,C-8,C-10

|c-26

21.6

22.2

17.0

H-7

C-5,C-9,C-14

|c-27

18.3

18.3

17.5

H-r C-3',C-15'

•overlapping signals

1

770 VII. MISCELLANEOUS A number of heterogeneous natural products have been isolated from American species including aromatic and phenolic compounds, flavonoids, catechins etc. The following table indicates the main studies on the subject.

TABLE XX. SOME NON-CLASSIFIED PRODUCTS FROM THE CELASTRACEAE COMPOUND

COMPOUND

Ref.

2,6-Diacetoxy-7-hidroxy-8-metoxychromone

61

4,5-Dihydroblumenol A

72

1

Blumenol A

72

(-H'-O-methyl-epigallocatechin

65

Ouratea proanthocyanidin A

65

Dulcitol

65

j

Epicatechin

18

1

5'-0-Methylgallocatechin

18

1

4-Hydroxybenzalddiyde

18

Femiginol

9,73-75]

Sakuranctin

9, 73-75 1

Vni. TRANSFORMATIONS The Tenerife group which is responsible for about 70% of all the research published on the Latin American Celastraceae has concentrated on the isolation and structural characterization of secondary metabolites; ahnost incidentally they have also developed various transformations and partial syntheses to test biogenetic theories in vitro and prove structural correlations by means of chemical transformations [76]. Thus, a succession of transformations showed fiiedoleane triterpenes with hemiacetal 24hydroxy-3-keto grouping to be possible key intermediates in the biogaietic pathway of the Celastraceae triterpene quinones, and a triterpene with a hemiacetal group in the remote C-24 position was synthesized from fiiedelin, as shown in the scheme [76] (Fig. 13).

771

FrtocMki

(20% yield based on lactone) i) Na BH4 in ether ii) IBDA/12, py/CH2Cl2, 100 W tunsgten filament iii) t-butyl chromate/ ether iv) LiAlH4 V) K2O + n-bu4N^Cr/THF + (NH4)6Mo7024H2/K2C03, H2O2

Fig. 13. Synthesis of a Friedelane Triterpene with a 24-Hydroxy-3-oxo-hemiacetal Group

XI.

BIOLOGICAL ACTIVITY

A. ANTIFEEDANT ASSAYS It has been known for some time that some polyester sesquiterpenes of the p-dihydroagaroftiran type such as those IBrom the Celastraceae deter various msects from feeding. In China the powdered root bark of Celastrus angulatus [77] has been sprayed on crops to protect them against insect attack. Chemical and biological analysis has shown that the powder is active against various species of insects including the cucumber beetle (Aulacohora femoralis chinensis\ the crucifer beetle {Colaphellus lowringi), the cabbage work (Pieris rapae) and migrant locusts {Locusta migratoria migratorioides and Locusta migratoria manilensis). Wilfordin, tryptofordin and celangulin (Fig. 14) are antifeedant compounds obtained from extracts of the Celastraceae species Maytentis rigida [78], Trypterigium wilfordii [79] and Celastrus angulatus [77, 80], respectively, and as some products isolated from South American species have similar structural characteristics, they too have been assayed.

772

The insects used for assay were fifth-instar larvae of Spodoptera littoralis Boisduval {Lepidoptera, Noctuidae) and the methodology used to determined the FR50 was that described in references [18, 81]. Antifeedant activity has been discerned in 16 sesquiterpenes obtained from five endemic Latin American species. The results are set out in Table XXII. All compounds were active at a dosage of lO^g/cm^ with 72 the most active with FR5o<0.5 at a dose of 0.1 |ig/cm^. It is difficult to compare these resuhs with those given in the literature for other substances using different testing procedures [77]. However, the use of triphenyl tin acetate has previously been studied under similar conditions to those described herein with a FR5o=0.37 when the assay dose was 10 fag/cm^ [18, 81]. Therefore, more than eight of the new products in principle appear to be more active than triphenyl tin acetate, which is the standard for antifeedant assays. Compound 15 was compared using other standard methods [77], and the remaining sesquiterpenes showed moderate antifeedant activity against Spodoptera littoralis in the test applied.

PAC

Aco y

OAc

OBz

QOm

,.OAc

AcO*'.

OAc Wilfordin

Celangulin

Triptofordm

Fig. 14. Structure of Some Antifeedant Compounds from the Celastraceae

773 TABLE XXI. ANTIFEEDANT ACTIVITY OF SOME SESQUITERPENES ON SPODOPTERA LITTORALIS

OH 34 Boariol

Form

C-1

C-2

C-3

C-4

C'6

C-8

C-9

C-15

Source

1

aOAc

aOAc

H

H

pOAc

H

pOBz

CH3

M.ch.

3

aOAc

aOAc

H

H

pOAc

aOAc

POBZ

CH2OAC

M.ch.

4

aOAc

aOAc

H

H

pOAc

aOAc

pOBz

CH2OH

M.ch.

6

aOAc

aOAc

H

H

pOAc

H

pOBz

CH2OH

M.ch.

8

M.b.

M.b.

aOAc

aOAc

H

H

pOAc

aOAc

aOBz

CH2OH

M.ch.

15

OBz

POAC

POH

pOH

POAc

H

pOBz

CH3

M.m

16

OBz

POAc

pOAc

pOH

pOAc

H

POBz

CH3

M.b.

17

aOBz

POH

pOAc

pOH

pOAc

H

pOBz

CH3

M.b.

18

aOBz

POAc

pOH

pOH

H

H

pOBz

CH3

M.b.

20

aOBz

POAc

pOH

H

pOAc

H

pOBz

CH3

M.b.

22

aOBz

POAc

pOAc

pOH

H

pOAc

POBz

CH3

M.m.

34

H

H

OH

aOH

H

H

H

H

39

aOBz

H

H

pOH

pOAc

pOAc

aOAc

CH3

O.m. !

70

aOBz

H

H

pOH

PNic

H

POAc

CH3

Z.t.

72

OBz

H

H

pOH

pNic

pOAc

aOAc

CH3

Z.t.

1 73

aOBz

H

H

pOH

PNic

pOH

aOAc

CH3

O.m.

M.in.: Maytenus magellanica Lam. M.ch.: Maytenus chuhutensis Sjeg. O.m.: Orthosphenia mexicana Stand Leg. R.t.:

Rzedowskia tolantonguensis Med.

M.b.: Maytenus boaria Mo I.

M.b.

774 TABLE XXII. FEEDING RATIOS OF TEST COMPOUNDS

Compound

Dose (jig/'cm)

FR,,±S.M.D.

1

10

0.19 ±0.06

1

0.54 ± 0.09

3

10

0.13 ±0.05

1

0.71 ±0.09

4

10

0.07 ±0.16

1

0.53 ±0.32

6

10

0.36 ±0.09

8

10

0.16±0.15

1

0.65 ±0.25

10

0.24±0.19

15

1

0.44 ±0.12

16

10

0.56 ±0.11

17

10

0.13 ±0.05

18

10

0.63 ±0.11

20

10

0.24 ±0.19

22

10

0.38 ±0.05

34

10

0.52 ±0.10

39

10

0.12 ±0.07

1

0.40 ±0.02

70

10

0.68 ±0.14

72

10

0.04 ±0.03

1

0.15 ±0.15

0.1

0.45 ±0.28

10

0.74 ±0.06

73

j

i

FCj^ The ACDT/ACD proportion when 50% of CD has been consumed SNfi) Standard mean deviation.

775 B. ANTIINFLAMMATORY AND ANTIPYRETIC ACTIVITY Anti-inflammatory and antipyretic properties have recently been ascribed to global and purified methanol extracts obtained from the aerial part of Maytenus boaria (MoL), a quite widespread tree species in the rural areas of Chile and Argentina. The sesquiterpene extract showed significant antipyretic activity on fully-grown albino New Zealand rabbits following a modified pyrogenic test protocol [82, 83]. The study of antipyretic activity of the global methanolic extract was carried out at dosages ranging from 400 mg/kg to 1200 mg/kg, 800 mg/kg for the infusion and 30mg/kg for the enriched fraction. Antiinflammatory activity was studied using a dosage of 500 mg/kg of the global methanolic extract and 50 mg/kg of the enriched fraction. Assays showed that these substances exercised a pharmacologically significant anti-inflammatory effect [82, 83].

C.

CYTOSTATIC

ACTIVITY

OF

P-DIHYDRO-AGAROFURAN

SKELETON

SESQUITERPENES The products I, 3 and 6 [14] were assayed on cultures of P815 tumoral (mouse mast cell) and 3T3-LI non-tumoral (mousefibroblast)cells which were cultured in Dulbecco medium modified by Eagle, supplemented with 10% newborn calf serum, following the colorimetric method of Mosmann (1983) [84] which is based on the capacity of live cells to transform a colourless substrate into a coloured one. The cells (106 cl/ml) were incubated at 37**C in CO2 atmosphere on 96-hole cell culture plates together with the products to be studied predissolved in ethanol at concentrations of 40, 20, 10, 5, 2.5, 1.2, 0.6, 0.3, 0.16 and 0.08 ^ig/ml. After 22 h. incubation MTT (3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolio

bromide) pre-dissolved in saline

phosphate buffer, was added, left to incubate fw 2 h., after which HCl 0.04 N in isc^)ropanol was added. The optical density was measured on an ELISA spectrophotometer at 600 nm wavelength and the LD50 results were given in ^g/ml. These fmdings are set out in Table XXIII, and it can be seen that product 1 shows selective cytotoxicity to tumoral cells; its LD50 to P815 cells was much lower than that registered for 3T3-L1 (> 20 jig/ml) which means Aat further testing is in order.

TABLE XXIII: CYSTOSTATIC ACTIVITY P815

3T3-L1

1

6.2

>20

3

37

>20

6

36

>20

Compound

776 ANTIVIRAL ACTIVITY STUDY To study the antiviral activity. Type 1 simplex herpes virus, KOS stock (HSV-Hg) and Indiana vesicular stomatitis virus (VSV) were used and HeLa cells were cultured in Eagle-modified Dulbecco medium (EMDM), supplemented with 10% foetal calf serum, on plates with 24 holes. Monolayers of these cells were infected with HSV-1 or VSV at 0.5 ufp/cell or 0.01 ufp/cell, respectively, and later the product to be assayed, pre-dissolved in DMSO, was added in concentrations of 10, 20, 50, 100 and 200 ng/ml. After 48 h incubation for HSV-1 and 24 h for VSV, at 37°C in CO2 atmosphere, the cytopathic CPE effect was measured on a phase-contrasting microscope [85]. Protein synthesis was determined in the non-infected cells used as control and the HSV-1 and/or VSV infected cells, in order to obtain quantitative data on the toxicity and antivkal activity of the compounds assayed. Thus, 0.5 free medium methionine and 1 ^Ci/ml of methinione 32S were added to the cells and incubated at 37**C for 1 h. Thereafter the medium was siphoned off and the cells were washed with saline phosphate buffer and precipitated with 5% trichloroacetic acid (TCA). After 5 min, die acid was eliminated and the cells were washed twice with 96% ethanol,dried under an infrared light and dissolved in NaOH O.IN with DSS. A liquid scintillation counter was used for the recount. The results showed no antiviral activity for the dihydro-Pagarofuran sesquiterpenes.

ANTIMRICROBIAL AND CYTOSTATIC ACTIVITY OF TRITERPENES AND DIMER TRITERPENICS At the outset of this research programme into the American Celastraceae, it was already known that the meAylene quinone triterpenes pristimerin, tingenone, iguesterin, isoiguesterin and celastrol possessed a variety of biological properties. Campanelli et al [86] have studied the action mechanism of tingenone, previously described as antitumoral [87,89], and shown that the compound interacted with the bacterial DNA increasing its MT, probably by formation of extra hydrogen bonds between a hydroxy group in tingenone and the DNA phosphate groups. Moreover, tingenone exercises a marked inhibitory effect on DNA, RNA and protein synthesis in mouse fibroblast [90] and HeLa cells [91] as well as antibacterial activity, with a MIC of between 0.5 and 10 ^g/ml on Gram (+) bacteria [92] and none on Gram (-) bacteria [93]. Pristimerin, a compound structurally related to tingenone shows similar biological activity, being cytostatic and antibiotic [91,93]. Iguesterin is cytostatic to HeLa cells, isoiguesterin affects leukaemia and shows a moderate cytostatic activity on KB cells [94]. Celastrol, too, is known to be antibacterial to Gram positive bacteria [93]. Another series of methylene quinone triterpenes with a new type

777

of structure and extended conjugation consists of compounds isolated from various Latin American

species

such

as

netzahualcoyene,

netzahualcoyone,

netzahualcoyonol

and

netzahualcoyondiol [95-98]. With the exception of netzahualcoyene, these compounds show antibacterial activity' against Gram (+) bacteria but are inactive on Gram negative, the most active being netzahualcoyone which has an MIC of 1.5-1.6 fig/ml on Staphylococcus aureus. The presence of groups C=0 and OH in Ring E is indispensable and a ketone group at C-22 enhances the efficiency of this activity. Analysis of the minimum inhibitory concentrations of netzahualcoyone against Staphylococcus aureus shows it to be more active than some of the antibiotics used in clinical practice (Table XXIV).

TABLE XXIV. MIC against S. aureus of Netzacualcoyone and some antibiotic of clinical use. Antibiotic

MIC(^g/ml)

Netzahualcoyone

1.5-1.62

Oxolinic acid

1.0-4.0

Pipemidic acid

9.2

Cefradine

6.4

Cefradoxyle

1.6-6.4

Cefaclor

1.6-6.4

Cefotaxime

3.2

1

Netzahualcoyone inhibits various cellular processes: oxidative phosphorylation and the transport of glucose in sensitive bacteria [96,97] and also exerts a strong cytostatic activity on cultured HeLa cells; this activity disappears when the ftmctional groups in the A ring of the molecule are blocked (Table XXV).

TABLE XXV. Cytostatic Activity of Netzahualcoyone and Related Compounds Againts HeLa Cells. Compound Netzahualcoyone

ID50 (^g/ml) 0.1 1

Netzahualcoyondiol

1

Netzahualcoyonol Netzahualcoyene

<1 <1

Dimetox>'-dihydro-Netzahualcoyone

70.0

Mercaptopurine

0.1

778 Triterpene dimers with modified quinone or phenol skeletons also exhibit antimicrobial activity [68] and have also been synthesized [68,70]. Table XXVI shows the MIC (minimum inhibitory concentration) of the different products tested on Gram positive and Gram negative bacteria the former proving to be more susceptible to the activity of the compounds except for product 125 which had no effect whatoever on either. Bacillus species proved more sensitive than Staphylococcus aureus and this seems to be a general trait of quinone triterpenes such as netzahualcoyone, tingenol and pristimerin which are all more active on B. subtilis than S. aureus. The most active compound was 122, its MIC on B. subtilis (12 ^ig/ml) being particulary interesting. The dimers were less active than the monomers.

TABLE XXVI. MIC (mg/ml) of Dimer Compounds Against Gram Negative and Gram Positive Bacteria Bacteria

121

122

124

125

S. aureus

>100

70-80

>45

>100

B. subtilis

23-20

2-1

5-2.5 >100

B. cereus

23^0

10-8

30-25 >100

Salmonella sp

-

-

>45

>100

E. coli

>50

>50

>45

>100

-Not assayed All assays were carried out in triplicate

ACKNOWLEDGEMENTS

We are indebted to AIETI, CICYT Projects FAR 91-0472 and SAP 92-1028-CO2-01 and the Commission of European Communities, Project CI* 10505 ES(JR), for subsidies; we also are obliged to CONAF, for the plant material.

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