Chapter 9
Synthesis and Fungistatic Activity of Podocarpic Acid Derivatives 1
1
1
2
Edward J . Parish , Susan Bradford , Victoria J. Geisler , Patrick K. Hanners , Rick C. Heupel , Phu H. Le , and W. David Nes Downloaded by KTH ROYAL INST OF TECHNOLOGY on August 26, 2015 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch009
2
2
2
1
Department of Chemistry, Auburn University, Auburn, A L 36849 Plant and Fungal Lipid Group, Plant Development and Productivity Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710 2
As a class, octahydrophenanthrene lactones, podolactones, and related podocarpic acid derivatives have been reported to possess a wide variety of biological activities, including antileukemic activity, inhibition of plant cell growth, insect toxicity and antifungal properties. In the present study, a series of synthetic derivatives of podocarpic acid have been prepared by chemical synthesis and evaluated with respect to their ability to inhibit fungal growth. These compounds were evaluated against the Oomycetes- Phytophthora cactorum , Saprolesnia ferax, and Achlya bisexualis and the Ascomycetes-Gibberella fujikuroi. The results of these studies indicate that several of these new synthetic derivatives possess significant antifungal properties.
Podocarpic acid ( I ) was f i r s t isolated from the r e s i n of Podocarpus cupressins, an important timber tree which i s endemic to Java, and l a t e r from Podocarpus dacrydioides ("Kahikatea") and Dacrydium cupressinum ("Rimu"), trees which are found i n the timber regions of New Zealand (1). Since 1968, more than f o r t y oxygenated metabolites of podocarpic acid have been i s o l a t e d from various species of Podocarpus (2,3). Interest i n these n a t u r a l l y occurring and synthetic lactones, podolactones, and related podocarpic acid derivatives has been mainly due to the novel structures of these compounds and the various types of b i o l o g i c a l a c t i v i t y possessed by them. Octahydrophenanthrene lactones ( I I ) and related podocarpic acid derivatives ( I I I ) have been reported to possess hormonal and anti-inflammatory properties (4). Other s i m i l a r podolactones have been shown to i n h i b i t the expansion and d i v i s i o n of plant c e l l s (IV) (5-10), to have antileukemic a c t i v i t y (V) (11), to have a n t i b a c t e r i a l a c t i v i t y (12), to have insect t o x i c i t y properties (13-15), and to e x h i b i t antitumor a c t i v i t y (16-19). 0097-6156/87/0325-0140$06.00/0 © 1987 American Chemical Society
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. PARISH ET AL.
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141
OH
/
15
IS
C0 H
C0„R 23 I I I a, R =R=CH , R =H b, R7=R2=CH^, R =Br c, R^=CH ,R =R =H
o
o
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2
2
R=CH2CH
2
3
CH R 2
2
'"OCH
n
OH
IV
a, R =H, R =OH b, R7=CH , R =OH c, R^=H, R =SOCH
VI
HO VII
VIII
IX
a, R ^ P r , R =R =H 2
3
1
b,
R = Pr, R =R =0
c,
R - ^ P r , R =OH, R =H
d,
R =OH, R =R =H
1
2
3
2
1
2
Figure 1. Structures I-IX.
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
3
3
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ECOLOGY AND METABOLISM OF PLANT LIPIDS
Other reports have indicated that these types of compounds, as a c l a s s , possess s i g n i f i c a n t antifungal properties. The lactone (VI), f i r s t i s o l a t e d as a mold metabolite, was found to have s i g n i f i c a n t a c t i v i t y against a number of fungi (20). The momilactones A (VII) and Β (VIII) have been shown to be fungitoxic towards C. cucumerinum (21,22). In a recent report several oxidized r e s i n acid d e r i v a t i v e s of dehydroabietic acid (IX a-c) and 13-hydroxypodocarpic acid (IX d) were found to be highly f u n g i s t a t i c against P. p i n i . a c o n i f e r pathogenic fungi (23). I t was observed that mature trees were more r e s i s t a n t to fungal i n f e c t i o n and contained a greater quantity of oxidized r e s i n a c i d derivatives i n t h e i r r e s i n suggesting greater resistance. In view of t h e i r documented b i o l o g i c a l properties, i t appeared worthwhile to evaluate a series of synthetic intermediates derived from podocarpic a c i d f o r f u n g i s t a t i c a c t i v i t y against other plant pathogens. This report describes the preparation of these derivatives and the r e s u l t s obtained from incubations of each compound with cultures of selected species of oomycetous and ascomycetous fungi. Chemical Synthesis of Podocarpic Acid Derivatives Commercial podocarpic acid i s derived from natural sources. Several recent studies have been directed towards the t o t a l synthesis of t h i s r e s i n acid to assure adequate future supplies of t h i s material f o r use i n a g r i c u l t u r e and medicine (24,25). The goal of the present study was to prepare a s e r i e s of derivatives r e l a t e d to podocarpic acid f o r use i n s t r u c t u r e / a c t i v i t y studies designed to reveal functional groups responsible f o r the molecules f u n g i s t a t i c properties. Four s p e c i f i c modifications were planned: 1. 2. 3. 4.
S u b s t i t u t i o n of electron-withdrawing groups onto C (13) of the aromatic C r i n g (Scheme 1). V a r i a t i o n of the halogen at C (6) (Scheme 2). Formation of the lactones from each 6 α - bromo methyl ester d e r i v a t i v e (Scheme 3). S u b s t i t u t i o n of an acetate group f o r the methyl ester group at C (16) (Scheme 4).
The f i r s t modification, s u b s t i t u t i o n of the electron-with drawing halogen and n i t r o groups onto C (11) and/or C (13) of the aromatic r i n g , was based upon the well-known observation that the a n t i s e p t i c properties of phenols are enhanced by the introduction of these groups onto the phenolic r i n g (26). N i t r a t i o n was accomplished by reacting podocarpic acid ( I , Scheme 1) with n i t r i c acid i n acetic acid (27-30). The number of n i t r o groups introduced onto the aromatic r i n g was c o n t r o l l e d by the amount of n i t r i c acid used i n the reaction (one or two equivalents). The 13-nitro d e r i v a t i v e X was methylated with dimethyl s u l f a t e under basic condition to y i e l d XIV. A s i m i l a r methylation of I has been shown to produce methyl o-methyl podocarpate (XII) (4,16). Bromine was introduced at C (13) by the e l e c t r o p h i l i c s u b s t i t u t i o n of bromine into the aromatic ring of XII using bromine i n a c e t i c a c i d . The fact that t h i s reaction gives
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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only the monosubstituted 13-bromo derivative i s probably due to s t e r i c hindrance r e s u l t i n g from the angular methyl group i n the a x i a l o r i e n t a t i o n at p o s i t i o n 10 and the large s i z e of the bromine atom which would prevent s u b s t i t u t i o n at p o s i t i o n 11, the other ortho p o s i t i o n on the r i n g . In Scheme 2, benzylic oxidation of X I I , X I I I , and XIV using chromium t r i o x i d e produced the corresponding ketone derivatives I t has also recently been shown that X I I may be oxidized to the ketone XV under conditions of ozonolysis (31). Ketones XV and XVI were brominated using an adapted procedure derived from the work of B i b l e and Grove (4,16) to y i e l d the mono-and di-bromoketones XVIII and XIX. In order to e f f e c t the bromination of XVII, an alternate method was u t i l i z e d which gave ample quantities of bromoketone XX (32). The corresponding chloride derivatives (XXI and XXII) of XV and XVI were prepared by reaction with copper chloride and l i t h i u m chloride i n N,N-dimethylformamide (33). The assignments of the α - configuration to the halogen atoms at C (6) were v e r i f i e d using known coupling constants from the Si NMR spectra which are correlated to the x-ray structure determination of XVIII (16,34-37)· In Scheme 3, the α-bromoketones XVIII and XIX were converted to lactones XXIII and XXIV by refluxing i n c o l l i d i n e (4,16,38). By-products of t h i s reaction include the a, B-unsaturated ketone XXV which r e s u l t s from the dehydrobromination (38,39) of XVIII. Ketone XXVI r e s u l t s from a one-step dehydrobromination-decarbomethoxylation (40-43) of XIX. The acetate series of compounds (Scheme 4) was synthesized by hydride reduction of methyl 0-methyl podocarpate (XII) followed by a c e t y l a t i o n of the r e s u l t i n g alcohol XXVII (44). The acetate XXVIII was then oxidized at the benzylic p o s i t i o n to ketone XXIX. In contrast to the methyl ester d e r i v a t i v e s , halogenation at p o s i t i o n 6 (using methods described previously) of the corresponding keto acetates resulted i n two epimers, the 6 a- and 6 B- halogenated compounds, as w e l l as the dehydrohalogenation product XXXIV. The assignments of the a- and B-conf iguration to the halogen atoms at C (6) were determined from the Si NMR coupling constants at C (5) and C (16) as described previously. B i o l o g i c a l Evaluation of P o t e n t i a l Fungistatic Agents Podocarpic acid (I) and a number of i t s chemical derivatives (X-XXXIV) were evaluated f o r t h e i r p o t e n t i a l f u n g i s t a t i c a c t i v i t y as measured by t h e i r e f f e c t s on the growth of the fungi on s o l i d media (Table 1). A l l compounds evaluated were of 98% or greater p u r i t y ( t i c and g l c a n a l y s i s ) . Each structure was consistent with i t s ι 13 spectral analysis ( H NMR, C NMR, i r , and ms). Each compound was evaluated against Phytophthora cactorum (both with (B) and without (A) added c h o l e s t e r o l ) , Gibberella f u i i k u r o i (C), Saprolegnia ferax (D), and Achlya b i s e x u a l i s (both male (E) and female (F) s t r a i n s ) . The fungi were cultured as described i n references 45-47. P. cactorum. u n l i k e the other fungi, f a i l s to synthesize s t e r o l s (48) and requires s t e r o l to complete the reproductive phase of i t s l i f e cycle (49). Compounds (10 ug/ml) evaluated were dissolved i n a minimal amount of ethanol and introduced a s e p t i c a l l y into the s t e r i l i z e d X
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
144
ECOLOGY AND METABOLISM OF PLANT LIPIDS Scheme 1 OH
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OH
XXI
R =H, R =C1
XXII
R-j=Br, R =C1
2
XX
2
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
9. PARISH ET AL.
Podocarpic Acid Derivatives
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Scheme 3
CH 0Ac o
XXXIV
XXX
R =H, R =Br
XXXI
R =H, R =C1
XXXII
R =Br, R =H
XXXIII
R ^ C l , R =H
1
1
1
2
XXIX
2
2
2
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
146
ECOLOGY AND METABOLISM OF PLANT LIPIDS
Table I .
Comparison of the f u n g i s t a t i c properties of podocarpic acid and i t s d e r i v a t i v e s
1
Fungal Species
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Compound
A
Β
C
D
Ε
F
I
66
82
94
62
68
74
X
75
97
94
96
96
76
XI
47
50
68
27
0
0
XIII
84
110
97
93
100
100
XIV
97
105
97
73
93
81
XV
97
97
95
79
76
66
XVI
92
104
90
96
88
88
XVII
91
101
100
93
99
—
XVIII
68
83
100
78
78
76
XIX
120
104
94
93
96
88
XX
58
86
102
100
78
80
XXI
72
86
96
91
88
76
93
91
96
88
XXII
107
110
XXIII
94
104
99
73
81
84
XXIV
68
85
94
100
54
74
XXV
115
98
96
8
84
75
XXVI
77
88
90
100
45
56
87
51
49
60
XXVII
125
84
XXVIII
122
101
93
84
71
87
XXIX
90
99
95
62
65
69
XXX
67
73
100
67
61
71
XXXI
143
99
98
91
74
77
XXXII
121
93
97
18
75
78
The values are expressed as a percentage of control's r a d i a l diameter obtained 3 to 12 days (depending on species) following inoculation with a 5mm plug.
A - P. cactorum
without added
c h o l e s t e r o l , Β - P. cactorum with added cholesterol, C Gibberella f u j i k u r o i . D - Saprolegnia ferax, Ε - Achlya b i s e x u a l i s , male s t r a i n , F - Achlya b i s e x u a l i s , female s t r a i n .
In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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agar-supplemented culture medium. The i n h i b i t i o n values obtained are expressed as percent of control by measurement of the r a d i a l diameter of fungal growth. Values greater than 100% represent an enhancement or stimulation of growth. Many of these compounds demonstrated varying degrees of s i g n i f i c a n t f u n g i s t a t i c (defined as i n h i b i t i o n of growth on s o l i d media) a c t i v i t y against one or more of the species in the study (Table I). In p a r t i c u l a r , d i n i t r o derivative XI demonstrated potent a c t i v i t y i n a l l assays. This may be due, i n part, to i t s resemblance to p i c r i c acid ( 2 , 4 , 6 - t r i n i t r o p h e n o l ) , a substance which i s known to complex with, and cause the i r r e v e r s i b l e precipation of protein (50,51). The lack of fungistatic properties of a s p e c i f i c compound to some but not a l l fungi tested may be due to the lack of mycelial uptake, a p o s s i b i l i t y which i s currently under study. Interestingly, cholesterol supplemented to P. cactorum was protective to the fungistatic properties resulting from the i n h i b i t i o n induced by some podocarpic acid derivatives. An increase, however, i n r a d i a l diameter induced by other derivatives does not necessarily imply a b e n e f i c i a l e f f e c t , since these mycelia appeared abnormal (cf. 49). In conclusion, these studies have indicated that chemical modification of the basic podocarpic acid structure can produce new compounds with antifungal a c t i v i t y . The results derived from t h i s study represent preliminary findings which are subject to further investigation. We anticipate that more detailed studies w i l l reveal information concerning the mechanism and mode of action of many of these compounds. In the meantime, we continue to develop new antifungal agents using selected natural products as model compounds.
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