Synthesis, Fungicidal Activity, and Effects on Fungal Polyamine Metabolism of Novel Cyclic Diamines

Article abstract of DOI:10.1021/jf9608534

A number of novel, cyclic diamines were synthesized and examined for fungicidal activity as part of a continuing program of work on polyamine analogues. The novel synthetic cyclic diamines trans-1,2-bis(diethylaminomethyl)cyclopentane (compound 1) and trans-5,6-bis(aminomethyl)bicyclo[2.2.1]-hept-2-ene (compound 2) and the synthetic cyclic diamine 1,2-bis(dimethylaminomethyl)-4,5-dimethylcyclohexa-1,4-diene (compound 3) controlled the important crop pathogen Erysiphe graminis DC f.sp. hordei Marchai. Since E. graminis cannot be cultured in vitro, the effects of the three diamines on polyamine biosynthesis were studied using the fungal pathogen Pyrenophora avenae Ito & Kuribay. All three compounds were effective in reducing the growth of P. avenae in vitro and in altering polyamine levels. However, whereas compound 1 reduced concentrations of all three polyamines, compound 2 increased spermidine 2-fold and compound 3 had little effect on spermidine and spermine concentrations but reduced putrescine concentration by 69%. These changes in polyamine concentrations could not be correlated with changes in activities of biosynthetic enzymes. It seems therefore that although these novel cyclic diamines alter fungal polyamine metabolism, their effects on the growth of P. avenae may not be related to depletion of cellular polyamines.

Full text of DOI:10.1021/jf9608534

J. Agric. Food Chem. 1997, 45, 2341
−2344
2341
Syn th esis, F u n gicid a l Activity, a n d Effects on F u n ga l P olya m in e
Meta bolism of Novel Cyclic Dia m in es
Neil D. Havis and Dale R. Walters*
Department of Plant Science, SAC, Auchincruive, Nr Ayr KA6 5HW, United Kingdom
Fiona M. Cook and David J . Robins
Department of Chemistry, The University, Glasgow G12 8QQ, United Kingdom
A number of novel, cyclic diamines were synthesized and examined for fungicidal activity as part
of a continuing program of work on polyamine analogues. The novel synthetic cyclic diamines trans-
1,2-bis(diethylaminomethyl)cyclopentane (compound 1) and trans-5,6-bis(aminomethyl)bicyclo[2.2.1]-
hept-2-ene (compound 2) and the synthetic cyclic diamine 1,2-bis(dimethylaminomethyl)-4,5-
dimethylcyclohexa-1,4-diene (compound 3) controlled the important crop pathogen Erysiphe graminis
DC f.sp. hordei Marchal. Since E. graminis cannot be cultured in vitro, the effects of the three
diamines on polyamine biosynthesis were studied using the fungal pathogen Pyrenophora avenae
Ito & Kuribay. All three compounds were effective in reducing the growth of P. avenae in vitro and
in altering polyamine levels. However, whereas compound 1 reduced concentrations of all three
polyamines, compound 2 increased spermidine 2-fold and compound 3 had little effect on spermidine
and spermine concentrations but reduced putrescine concentration by 69%. These changes in
polyamine concentrations could not be correlated with changes in activities of biosynthetic enzymes.
It seems therefore that although these novel cyclic diamines alter fungal polyamine metabolism,
their effects on the growth of P. avenae may not be related to depletion of cellular polyamines.
Keyw or d s: Polyamines; cyclic diamines; fungicide; powdery mildew
INTRODUCTION
tion, and effects on polyamine metabolism in P. avenae
of a number of novel cyclic diamines.
Polyamine biosynthesis inhibitors can give effective
control of biotrophic fungal pathogens, such as rusts and
powdery mildews (Rajam et al., 1985; Walters, 1986;
West and Walters, 1988). Much of this early work used
enzyme-activated irreversible inhibitors, e.g. R-difluo-
romethylornithine (DFMO), an inhibitor of the polyamine
biosynthetic enzyme ornithine decarboxylase (ODC)
(Metcalf et al., 1978). Indeed, DFMO has been shown
to control a range of fungal pathogens in glasshouse
experiments and to control powdery mildew on barley
in field trials (Walters et al., 1992). Since powdery
mildew, for example, cannot be grown axenically, it has
been difficult to determine whether DFMO-induced
reductions in mildew infection are due to alterations in
polyamine biosynthesis. Nevertheless, DFMO was shown
to deplete intracellular concentrations of putrescine and
spermidine in the necrotrophic pathogen Pyrenophora
avenae (Foster and Walters, 1990). More recently, an
alternative method of polyamine perturbation has been
demonstrated using polyamine analogues (Porter and
Sufrin, 1986). As a result, a variety of polyamine
analogues have been shown to alter polyamine metabo-
lism in tumor cells leading to pronounced antiprolifera-
tive effects. In recent work, ketoputrescine, a commer-
cially available putrescine analogue, was shown to
possess fungicidal properties (Foster and Walters, 1993).
In previous papers, we have described the fungicidal
activity of a number of aliphatic putrescine analogues
(Havis et al., 1994a,b). This paper describes the syn-
thesis, as their salts and free bases, fungicidal evalua-
EXPERIMENTAL METHODS
Syn th esis of tr a n s-1,2-Bis(d ieth yla m in om eth yl)cyclo-
p en ta n e (1). trans-1,2-Cyclopentanedicarboxylic acid (0.5 g,
3.16 mmol) was heated under reflux with thionyl chloride (6
equiv) for 6 h. The excess thionyl chloride was then removed
under reduced pressure. The crude acid chloride was dissolved
in ether (5 mL) and was added dropwise to a stirred solution
of diethylamine (3.24 mL, 34 mmol, 10 equiv) in ether (10 mL)
cooled to 0 °C. The mixture was allowed to warm to room
temperature and stirred overnight. The precipitated diethyl-
amine hydrochloride was filtered off and extracted several
times with ether. The combined organic extracts were con-
centrated in vacuo to give trans-N,N,N′,N′-tetraethylcyclopen-
tane-1,2-dicarboxamide as a light yellow oil (0.73 g, 89%): 1H
NMR (90 MHz, CDCl3) 1.13 (12H, m), 1.75 (4H, m), 2.00 (2H,
m), and 3.20-3.55 ppm (8H and 2H, m).
To a solution of lithium aluminum hydride (0.45 g, 11.9
mmol) in dry ether (5 mL), under an atmosphere of nitrogen,
was added dropwise a solution of trans-N,N,N′,N′-tetraethyl-
cyclopentane-1,2-dicarboxamide (0.8 g, 2.98 mmol) in dry ether
(5 mL). When the addition was complete, the mixture was
heated at reflux for 1 h. The mixture was then cooled in an
ice bath, and excess hydride was decomposed by the dropwise
addition of water (1 mL), followed by a 15% solution of sodium
hydroxide (1 mL) and then water (2 mL) again. After 10 min
of vigorous stirring, the mixture was filtered with suction and
the granular precipitate was washed thoroughly with ether.
The filtrate was dried (MgSO4) and concentrated in vacuo to
give trans-1,2-bis(diethylaminomethyl)cyclopentane as a light
colored oil (0.71 g, 87%): IR (thin film) 2968, 2936, 2871, and
2796 cm-1; 1H NMR (200 MHz, CDCl3) 0.99 (12H, t), 1.31 (2H,
m) 1.46-1.82 (4H and 2H, m), 2.32 (4H, m), and 2.48 ppm
(8H, q); 13C NMR (50 MHz) 11.5 (CH3), 24.2 (CH2), 31.1 (CH2),
41.9 (CH), 47.0 (CH2), and 58.7 ppm (CH2); MS, m/ z 241 (M+
+ 1), 240 (M+, 7.7%), 211, 152, 138, 112, 99, 86 (100%), 72,
* Author to whom correspondence should be ad-
dressed (telephone 01292 525307; fax 01292 525314;
e-mail d.walters@au.sac.ac.uk).
S0021-8561(96)00853-9 CCC: $14.00
© 1997 American Chemical Society

2342 J. Agric. Food Chem., Vol. 45, No. 6, 1997
Havis et al.
and 58. (Found: C, 74.84; H, 13.10; N, 11.79; M+, 240.2564.
C15H32N2 requires C, 74.91; H, 13.42; N, 11.66; M+, 240.2565.)
Syn th esis of tr a n s-5,6-Bis(a m in om eth yl)bicyclo[2.2.1]-
h ep t-2-en e (2). To a cooled solution of fumaronitrile (2.73 g,
35 mmol) in ethanol (20 mL) was added dropwise, with
stirring, freshly distilled cyclopentadiene (2.55 g, 38 mmol).
When the addition was complete, the solution was concen-
trated in vacuo to half its original volume. The solution was
cooled to 0 °C, and an ice crystal was used to seed crystalliza-
tion. The product was recrystallized from ethanol to afford
trans-bicyclo[2.2.1]hept-2-ene-5,6-dinitrile (3.81 g, 75%): mp
92-94 °C [Blomquist and Winslow (1945) mp 95.5-96 °C]; IR
(CO); MS, m/ z 197 (M+ + 1), 196 (M+, 10.8%), 178, 151, 133,
107, 91 (100%), 77, and 51. (Found: M+, 196.0732. C10H12O4
requires M+, 196.0735 DMSO-d6.)
To a suspension of 4,5-dimethylcyclohexa-1,4-diene-1,2-
dicarboxylic acid (1 g, 5.1 mmol) in benzene (10 mL) was added
hexamethylphosphorous triamide (0.52 mL, 5.1 mmol) drop-
wise. The mixture was reheated at reflux for 20 min and then
allowed to cool to room temperature. Saturated sodium
bicarbonate solution (10 mL) was added, and the layers were
separated. The aqueous layer was extracted with dichlo-
romethane (3 × 20 mL), and the extracts were combined, dried
(MgSO4), and concentrated in vacuo to give a yellow oil (0.63
(KBr disc) 3448, 3072, 2998, and 2242 cm-1
;
1H NMR (200
g). The H NMR spectrum contained N,N,N′,N′-tetramethyl-
1
MHz, CDCl3) 1.64-1.81 (2H, m), 2.50-2.53 (1H, m), 3.16-
3.19 (1H, m), 3.40-3.45 (2H, m), and 6.37 ppm (2H, m); 13C
NMR (50 MHz) 34.5 (CH), 34.6 (CH), 46.2 (CH), 47.2 (CH),
48.2 (CH), 119.5 (CH), 119.9 (CH), 135.6 (CH), and 137.1 ppm
(CH); MS, m/ z 144 (M+, 0.3%), 117, 104, 90, 77, 66 (100%),
51, and 39. (Found: C, 75.10; H, 5.45; N, 19.44; M+, 144.0674.
C9H8N2 requires C, 75.00; H, 5.55; N, 19.44; M+, 144.0688.)
A three-neck flask was equipped with stopper, septum, and
condenser with nitrogen balloon. Lithium aluminum hydride
(1.06 g, 27.9 mmol) was put into the flask, and dry ether (15
mL) was added. trans-Bicyclo[2.2.1]hept-2-ene-5,6-dinitrile (1
g, 6.94 mmol) in dry ether (25 mL) was added dropwise over
20 min. Toward the end of the reaction the mixture became
thick and difficult to stir. After addition was complete, the
reaction was chilled to 0 °C and water (2 mL), 15% sodium
hydroxide solution (2 mL), and more water (5 mL) were added
sequentially and cautiously. The resulting white precipitate
was filtered off and extracted with dry ether (3 × 20 mL). The
ether extracts were dried (Na2SO4) and concentrated in vacuo
to give trans-5,6-bis(aminomethyl)bicyclo[2.2.1]hept-2-ene as
a colorless oil (0.98 g, 93%): IR (thin film) 3291, 2959, 2909,
2870, 2432, 1570, and 1458 cm-1; 1H NMR (200 MHz, CDCl3)
0.90 (1H, ddd), 1.36 (1H, d), 1.44 (1H, d), 1.60 (1H, ddd), 2.26
(1H, dd), 2.45 (1H, dd), 2.57 (1H, dd), 2.61 (1H, bs), 2.73 (1H,
dd), 2.84 (1H, bs), 6.06 (1H, dd), and 6.23 ppm (1H, dd); 13C
NMR (50 MHz) 44.6 (CH), 45.5 (CH), 46.0 (CH2), 46.9 (2 ×
CH2), 47.7 (CH), 48.3 (CH), 135.0 (CH), and 138.0 ppm (CH);
MS, m/ z 153 (M+ + 1), 152 (M+, 1.0%), 135, 122, 106, 91, 78,
69, 66 (100%), and 56. (Found: M+, 152.1314. C9H16N2
requires M+, 152.1314.)
Syn t h esis of 1,2-Bis(d im et h yla m in om et h yl)-4,5-d i-
m et h ylcycloh exa -1,4-d ien e Dih yd r och lor id e (3). 2,3-
Dimethyl-1,3-butadiene (1.64 g, 20 mmol), dimethyl acetyl-
enedicarboxylate (2.84 g, 20 mmol), and water (50 mL) were
heated at 60 °C for 24 h. The reaction mixture was then
cooled, and the precipitate was filtered off and washed with
water. The white solid was recrystallized from acetone to give
colorless crystals of dimethyl 4,5-dimethylcyclohexa-1,2-diene-
1,2-dicarboxylate (4.48 g, 73%): IR (KBr disc) 3446, 2956,
2859, 1740, 1724, and 1696 cm-1; 1H NMR (200 MHz, CDCl3)
1.66 (6H, s), 2.92 (4H, s), and 3.77 ppm (6H, s); 13C NMR (50
MHz) 17.8 (CH3), 33.9 (CH2), 52.0 (CH3), 121.4 (C), 132.6 (C),
and 168.2 ppm (CO); MS, m/ z 225 (M+ + 1), 244 (M+, 4.6%),
191, 177 (100%), 133, 105, 91, 77, and 59. (Found: C, 64.13;
H, 7.02; M+, 224.1055. C12H16O4 requires C, 64.25; H, 7.19%;
M+, 224.1049.)
4,5-dimethylcyclohexa-1,4-diene-1,2-dicarboxamide (with some
starting material): 1H NMR (90 MHz, CDCl3) 1.75 (6H, m),
2.30-2.80 (4H, m), 2.95 (6H, s), and 3.10 ppm (6H, s).
To a solution of lithium aluminum hydride (0.36 g, 9.47
mmol) in dry ether under an atmosphere of nitrogen was added
dropwise a solution of N,N,N′,N′-tetramethyl-4,5-dimethylcy-
clohexa-1,4-diene-1,2-dicarboximide (0.6 g, 2.4 mmol) in dry
ether (5 mL). When the addition was complete, the mixture
was heated at reflux for 1 h. The mixture was then cooled in
an ice bath, and excess hydride was decomposed by the
dropwise addition of water (0.5 mL), followed by 15% sodium
hydroxide solution (1 mL) and then water (ca. 2 mL) again.
After 10 min of vigorous stirring, the mixture was filtered with
suction. The granular precipitate was washed thoroughly with
ether (3 × 20 mL), and the filtrate was dried (MgSO4) and
concentrated in vacuo to give 1,2-bis(dimethylaminomethyl)-
4,5-dimethylcyclohexa-1,4-diene as an oil (0.12 g, 53%): IR
1
(thin film) 3802, 3422, 2918, 2866, 2523, and 1468 cm-1; H
NMR (200 MHz, D2O) 1.29 (3H, s), 1.32 (3H, s) 2.00-2.34 (4H,
m), 2.15 (6H, s), 2.16 (6H, s), and 2.71-3.02 ppm (4H, m); 13
C
NMR (50 MHz) 17.2 (CH3), 19.5 (CH3), 34.8 (CH2), 43.2 (CH3),
44.4 (CH3), 62.2 (CH2), 63.6 (CH2), 123.9 (C), and 129.1 (C);
MS, m/ z 223 (M+ + 1-2HCl), 222 (M+ - 2HCl, 3.8%), 164,
119, 105, 91, 77, 58 (100%), 42, and 30. (Found: M+ - 2HCl,
222.2108. C14H26N2 requires M+ -2HCl, 222.2096.)
Deter m in a tion of th e F u n gicid a l Activity of Cyclic
Com p ou n d s. The analogues were applied to plants, and
fungicidal activity was determined as described in detail
previously (Havis et al., 1994a). Briefly, the protectant and
curative activity of compounds 1-3 was studied using barley
infected with the powdery mildew fungus, Erysiphe graminis
f.sp. hordei, i.e. treatments were made 3 h preinoculation and
3 days postinoculation. In addition, the curative activity of
compounds 1-3 was examined using broad bean infected with
rust, Uromyces viciae-fabae (Pers.) Schroet, or chocolate spot,
Botrytis fabae Sardina. Inhibitors were applied to plants as
aqueous solutions in 0.01% Tween 20. Figures for percentage
leaf area infected on barley are the means of 20 replicates.
All experiments were repeated twice.
Effects of Com p ou n d s 1-3 on Gr ow th , En zym e Activi-
ties, a n d P olya m in e Con cen tr a tion s in P . a ven a e. The
effects of compounds 1-3 on mycelial growth, polyamine
concentrations, and activities of ornithine decarboxylase (ODC;
EC 4.1.1.17) and S-adenosylmethionine decarboxylase (AdoMet-
DC; EC 4.1.1.50) were determined as described previously
(Foster and Walters, 1990; Havis et al., 1994a). Fungal tissue
was exposed to the diamines in liquid culture for 3 days.
Because of the very limited quantities of the compounds
available, it was not possible to expose the fungus to the
compounds for various periods of time.
Dimethyl 4,5-dimethylcyclohexa-1,4-diene-1,2-dicarboxylate
(2 g, 8.9 mmol) was dissolved in methanol (5 mL), and lithium
hydroxide monohydrate (2 g, 47.6 mmol) in methanol/water
(3:1) (40 mL) was added slowly. A yellow color appeared, and
stirring was continued for 24 h. After this time, the yellow
color disappeared and the mixture was washed with ether (3
× 20 mL) to remove any unreacted diester. Hydrochloric acid
(3 M, 10 mL) was added dropwise, and a cloudy suspension
appeared. Another portion of 3 M hydrochloric acid (5 mL)
was added dropwise, and the solution went clear (pH 2) and
was extracted with ethyl acetate (3 × 20 mL). The combined
ethyl acetate extracts were dried (Na2SO4) and concentrated
in vacuo to give a white solid of 4,5-dimethylcyclohexa-1,4-
diene-1,2-dicarboxylic acid (1.00 g, 57%): IR (KBr disc) 3421,
RESULTS AND DISCUSSION
Three novel cyclic diamines have been shown to
possess substantial fungicidal activity against E. grami-
nis on barley, when applied pre- or postinoculation. Best
control of E. graminis infection was achieved using
compound 1 (Figure 1), applied as a postinoculation
treatment (96%; Table 1). This compares favorably with
the level of disease control achieved with the commercial
fungicide propiconazole (98%; Table 1). The superior
control obtained with the commercial standard could be
2935, 2632, and 1694 cm-1 1H NMR (200 MHz, D2O and
;
DMSO) 1.59 (6H, s) and 2.84 ppm (4H, s); 13C NMR (50 MHz)
18.2 (CH3), 34.5 (CH2), 122.7 (C), 133.8 (C), and 172.8 ppm

Fungicidal Activity of Novel Cyclic Diamines
J. Agric. Food Chem., Vol. 45, No. 6, 1997 2343
Ta ble 2. Effect of Cyclic Com p ou n d s on th e Gr ow th of P .
a ven a e in Liqu id Cu ltu r e
treatmenta
mean wt (g)
treatmenta
mean wt (g)
control
compound 1
compound 2
1.20 ( 0.02
0.91 ( 0.03b
0.87 ( 0.04c
control
compound 3
3.07 ( 0.30
1.26 ( 0.22c
a
Compound 3 was used as the dihydrochloride salt and was
applied at 1.0 mM (i.e. 295 mg L-1); compounds 1 and 2 were used
as the free bases and applied at 1.0 mM (i.e. 240 and 152 mg L-1
,
respectively). b,c Significantly different from the control at P e
0.001 and P e 0.01, respectively.
F igu r e 1. Chemical structures of cyclic diamines.
Ta ble 3. Effect of Cyclic Com p ou n d s on P olya m in e
Con cen tr a tion s in P . a ven a e
Ta ble 1. Effect of Cyclic Com p ou n d s on In fection of
Ba r ley w ith th e P ow d er y Mild ew F u n gu s, E. gr a m in is
f.sp . h or d ei
polyamine concentration (mol g-1 fresh wt-1
)
treatmenta
putrescine
spermidine
spermine
postinoculation
treatment
preinoculation
treatment
control
136.3 ( 9.9
21.9 ( 4.0b
71.6 ( 9.7c
43.4 ( 18.6c
129.3 ( 16.0
57.9 ( 2.4c
260.9 ( 12.6
93.6 ( 8.4b
compound 1
compound 2
compound 3
262.8 ( 15.2b
129.9 ( 24.0
118.9 ( 12.1b
229.4 ( 41.5
leaf area
disease
leaf area
disease
treatmenta infected (%) control (%) infected (%) control (%)
a
Compound 3 was used as the dihydrochloride salt and applied
at 1.0 mM (i.e. 295 mg L-1); compounds 1 and 2 were used as the
control
34 ( 2.7
1 ( 0.3b
21 ( 2.3c
12 ( 2.3b
76 ( 2.1
67 ( 1.9c
37 ( 3.4b
43 ( 3.1b
compound 1
compound 2
compound 3
96
36
63
98
12
52
43
free bases and applied at 1.0 mM (i.e. 240 and 152 mg L-1
,
respectively). b,c Significantly different from the control at P e
0.001 and P e 0.01, respectively.
propiconazole 0.7 ( 0.2b
a
Compound 3 was used as the dihydrochloride salt and was
applied at 1.0 mM (i.e. 295 mg L-1), and compounds 1 and 2 were
used as free bases and applied at 1.0 mM (i.e. 240 and 152 mg
L-1, respectively). Compound 1, trans-1,2-bis(diethylaminometh-
yl)cyclopentane; compound 2, trans-5,6-bis(aminomethyl)bicyclo-
[2.2.1]hept-2-ene; compound 3, 1,2-bis(dimethylaminomethyl)-4,5-
dimethylcyclohexa-1,4-diene dihydrochloride. b,c Significantly dif-
ferent from the control at P e 0.001 and P e 0.005, respectively;
Ta ble 4. Effect of Cyclic Com p ou n d s on En zym e
Activities in P . a ven a e
enzyme activity [pmol of
CO2 (mg of protein)-1 h-1
]
treatmenta
ODC
AdoMetDC
control
259.5 ( 20.7
170.4 ( 9.0b
415.9 ( 28.5c
211.3 ( 10.4
53.7 ( 19.0
77.8 ( 21.4
49.9 ( 8.0
Propiconazole was used as a 0.1% solution (i.e. 250 mg L-1).
d
compound 1
compound 2
compound 3
140.4 ( 35.1
related to its formulation, since compound 1 was applied
as a simple aqueous solution with 0.01% Tween 20, and
formulation is known to improve the efficacy of fungi-
cidal compounds. For compounds 1 and 3, postinocu-
lation treatments were more effective than preinocula-
tion treatments (Table 1). This effect was also observed
with the synthetic putrescine analogues, E-BED and
E-TED (Havis et al., 1994a,b) and the novel, alicyclic
diamine BAD (Robins and Walters, 1993). This effect
could be related to perturbation of polyamine biosyn-
thesis in the germinating conidia on the leaf surface.
An inhibitor of ODC, DFMO, has previously been shown
to inhibit the germination of rust uredospores on the
leaf surface (Rajam et al., 1989). More recently, DFMO,
as well as the putrescine analogues E-BED and E-TED,
were shown to inhibit appressorium formation in the
rust fungus U. viciae-fabae (Reitz et al., 1995). Indeed,
it has been suggested that inhibitors of polyamine
biosynthesis exert their main effect against early fungal
development on the leaf surface (Walters, 1995).
Compounds 1-3 also exhibited limited activity against
low levels of B. fabae infection on broad bean when
applied as postinoculation treatments and gave poor
control of U. viciae-fabae on the same host (data not
shown). These results are markedly different from
those obtained with the aliphatic putrescine analogues,
E-BED and E-TED, both of which gave significant
control of U. viciae-fabae when applied as a postinocu-
lation treatment (Havis et al., 1994a,b). The reasons
for this difference are not known.
a
Compound 3 was used as the dihydrochloride salt and was
applied at 1.0 mM (i.e. 295 mg L-1); compounds 1 and 2 were used
as the free bases and applied at 1.0 mM (i.e. 240 and 152 mg L-1
,
respectively). b,c Significantly different from the control at P e
0.001 and P e 0.005, respectively.
growth of the fungus significantly (Table 2), each
produced different effects on polyamine levels and
enzyme activities.
Compound 1 produced significant reductions in the
levels of putrescine, spermidine, and spermine (84%,
55%, and 64%, respectively; Table 3). The decrease in
putrescine concentration can be partly attributed to the
significant decrease in ODC activity observed in the
fungus, although, despite a small, insignificant increase
in AdoMetDC activity (Table 4), spermidine and sper-
mine levels were reduced. These reductions could be
related to the lower putrescine levels available for
conversion into higher polyamines.
Treatment with compound 2 led to significant reduc-
tions in the cellular concentrations of putrescine and
spermine (47% and 54%, respectively), while spermidine
levels were doubled in the fungal cells (Table 3). These
changes were accompanied by a significant increase in
ODC activity (60%; Table 4), which should have led to
an increase in putrescine levels in the cells. The
observed reduction in putrescine concentration may be
related to increased putrescine catabolism or increased
putrescine efflux from P. avenae grown in the presence
of compound 2, neither of which was examined in the
present work because of limited supplies of the test
compounds. The increased spermidine levels observed
are more difficult to explain, especially as AdoMetDC
activity was unaltered. It is known that spermidine can
be formed from acteylspermine in fungi (Large, 1992).
Since powdery mildew cannot be grown axenically,
the effects of the three novel cyclic diamines on polyamine
biosynthetic enzymes and polyamine levels were exam-
ined in the oat stripe pathogen, Pyrenophora avenae.
Interestingly, while all of the cyclic diamines reduced

2344 J. Agric. Food Chem., Vol. 45, No. 6, 1997
Havis et al.
Blomquist, A. T.; Winslow, E. C. Unsaturated nitriles as
dienophiles in the diene synthesis. J . Org. Chem. 1945, 10,
153-158.
Foster, S. A.; Walters, D. R. The effects of polyamine biosyn-
thesis inhibitors on mycelial growth, enzyme activity and
polyamine levels in the oat infecting fungus Pyrenophora
avenae. J . Gen. Microbiol. 1990, 136, 233-239.
Foster, S. A.; Walters, D. R. Fungicidal activity of the
polyamine analogue, keto-putrescine. Pestic. Sci. 1993, 37,
267-272.
Havis, N. D.; Walters, D. R.; Foster, S. A.; Martin, W. P.; Cook,
F. M.; Robins, D. J . Fungicidal activity of the synthetic
putrescine analogue (E)-1,4-diaminobut-2-ene. Pestic. Sci.
1994a , 41, 61-69.
Recent work has shown that changes in acetylpolyamines
occur in P. avenae exposed to inhibitors of spermidine
biosynthesis (Mackintosh and Walters, unpublished
results). It would be useful, therefore, to examine
changes in acetylspermine in P. avenae exposed to
compound 2, although this will require further synthesis
of the compound. Clearly, further work is needed to
elucidate the precise mechanism responsible for the
observed changes in polyamines in fungal tissue exposed
to compound 2 and to determine whether the observed
changes in polyamines are involved in the antifungal
activity of this compound.
Compound 3 reduced putrescine concentration in P.
avenae by 68% but had no effect on spermidine or
spermine concentrations (Table 3). The reduction in
putrescine was not accompanied by a significant de-
crease in ODC activity (Table 4). This would appear to
suggest that the putrescine reduction is due to either
increased putrescine catabolism or greater efflux from
the cells, although, again, further work is required if
the mechanisms responsible for these changes are to be
elucidated. Compound 3 also produced a large increase
in AdoMetDC activity (Table 4). This has been observed
previously with DFMO and the stereoisomer of E-BED,
(Z)-1,4-diaminobut-2-ene (Z-BED) (Foster and Walters,
1990; Havis et al., 1994a). In the case of DFMO, it was
suggested that the increase may be due to stabilization
of the active protein as found in animal systems (Foster
and Walters, 1990).
Havis, N. D.; Walters, D. R.; Martin, W. P.; Cook, F. M.;
Robins, D. J . Fungicidal activity of three putrescine ana-
logues. Pestic. Sci. 1994b, 41, 71-76.
Large, P. J . Enzymes and pathways of polyamine breakdown
in microorganisms. FEMS Microbiol. Rev. 1992, 88, 249-
262.
Metcalf, B. W.; Bey, P.; Danzin, C.; J ung, M. J .; Casara, P.;
Vevert, J . P. Catalytic irreversible inhibition of mammalian
ornithine decarboxylase (EC 4.1.1.17) by substrate and
product analogues. J . Am. Chem. Soc. 1978, 100, 2551-
2553.
Porter, C. W.; Sufrin, J . R. Interference with polyamine
biosynthesis and/or function by analogs of polyamines or
methionine as a potential anticancer chemotherapeutic
strategy. Anticancer Res. 1986, 6, 525-542.
Rajam, M. V.; Weinstein, L. H.; Galston, A. W. Prevention of
a plant disease by specific inhibition of fungal polyamine
biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6874-
8.
Various mechanisms for the inhibition of cell growth
by polyamine analogues have been proposed, including
inhibition of polyamine biosynthesis by direct enzyme
inhibition or regulation of polyamine biosynthetic en-
zymes (Porter and Sufrin, 1986). Our results would
appear to suggest that inhibition of the growth of P.
avenae was not achieved by direct inhibition of enzymes
of polyamine biosynthesis, although an effect via the
perturbation of polyamine catabolism or function, for
example, cannot be ruled out. A number of bis(benzyl)-
polyamine analogues have been shown to inhibit cell
growth by repression of polyamine biosynthesis and
direct binding to DNA, with subsequent disruption of
macromolecular function (Bitonti et al., 1989). It would
be useful to determine if this is a possible mode of action
for the novel cyclic diamines described in this paper.
The novel cyclic diamines described in this work, and
the aliphatic and alicyclic compounds described in
previous work (Havis et al., 1994a,b; Robins and Walters,
1993), exhibit effects on fungal growth that cannot be
correlated with depletion of intracellular polyamine
concentrations. Further work is necessary to elucidate
the mode of action of these compounds and, in particu-
lar, to determine whether the observed changes in
polyamine levels are related to the antifungal effects of
the compounds.
Rajam, M. V.; Weinstein, L. H.; Galston, A. W. Inhibition of
uredospore germination and germ tube growth by inhibitors
of polyamine metabolism in Uromyces phaseoli L. Plant Cell
Physiol. 1989, 30, 37-41.
Reitz, M.; Walters, D. R.; Moerschbacher, B.; Robins, D. J . The
effects of the synthetic putrescine analogues on germination
and appressorial formation in uredospores of Uromyces
viciae-fabae on artificial membranes. Lett. Appl. Microbiol.
1995, 21, 285-287.
Robins, D. J .; Walters, D. R. Alicyclic diamine plant fungicides.
U.K. Pat. Appl. GB 2 267 438 A, 1993.
Walters, D. R. The effects of a polyamine biosynthesis inhibitor
on infection of Vicia faba L. by the rust fungus, Uromyces
viciae-fabae (Pers.) Schroet. New Phytol. 1986, 104, 613-
619.
Walters, D. R. Inhibition of polyamine biosynthesis in fungi.
Mycol. Res. 1995, 99, 129-139.
Walters, D. R.; Havis, N. D.; Foster, S. A.; Robins, D. J . Control
of fungal diseases of arable crops using inhibitors of
polyamine biosynthesis. Proc. Br. Crop Prot. Conf. Pests Dis.
1992, 2, 645-651.
West, H. M.; Walters, D. R. The effects of polyamine biosyn-
thesis on infection of Hordeum vulgare L. by Erysiphe
graminis f.sp. hordei Marchal. New Phytol. 1988, 110, 193-
200.
Received for review November 5, 1996. Revised manuscript
received March 4, 1997. Accepted March 12, 1997.X
We are
grateful to the British Technology Group Ltd. for generous
financial support. SAC receives financial support from the
Scottish Office Agriculture, Environment and Fisheries
Department.
ACKNOWLEDGMENT
We are grateful to Dr. R. A’Court of BTG for advice
and guidance.
J F9608534
LITERATURE CITED
Bitonti, A. J .; Bush, T. L.; McCann, P. P. Regulation of
polyamine biosynthesis in rat hepatoma (HTC) cells by a
bisbenzyl polyamine analogue. Biochem. J . 1989, 257, 769-
774.
X Abstract published in Advance ACS Abstracts, May
1, 1997.