Synthesis and Fungicidal Activity of Alicyclic Diamines

Article abstract of DOI:10.1021/jf950772s

As part of an ongoing program of work on polyamine analogues, a number of alicyclic diamines were synthesized and examined for fungicidal activity. The alicyclic diamine 1,2-bis(aminomethyl)-4,5-dimethylcyclohexa-1,4-diene (BAD), synthesized as the dihydrochloride salt, controlled the important crop pathogen Erysiphe graminis f.sp hordei. Greatest control of E. graminis was achieved when BAD was applied 2 days postinoculation. The alicyclic diamines trans-4,5-bis(aminomethyl)-1,2-dimethylcyclohex-1-ene (trans-BAD) and the cis-isomer (cis-BAD), as well as 1,2-bis(amino-methyl)cyclopentene (BACP) and irons-1,2-bis(dimethylaminomethyl)cyclobutane (TCCBM), were also synthesized as their dihydrochloride salts. trans-BAD was found to possess greater fungicidal activity than the cis-isomer against E. graminis, while BACP and TCCBM both gave >80% control of powdery mildew infection. Since the powdery mildew fungus cannot be grown axenically, the effects of the alicyclic diamines on polyamine metabolism were examined using the oat leaf stripe pathogen, Pyrenophora avenae. BAD and derivatives had little effect on polyamine metabolism in the fungal pathogen P. avenae. It seems clear, therefore, that the antifungal activity of these derivatives may not be associated with altered polyamine metabolism.

Full text of DOI:10.1021/jf950772s

J. Agric. Food Chem. 1996, 44, 2835
−2838
2835
Syn th esis a n d F u n gicid a l Activity of Alicyclic Dia m in es
Neil D. Havis and Dale R. Walters*
Department of Plant Science, SAC, Auchincruive, Nr Ayr KA6 5HW, United Kingdom
William P. Martin, Fiona M. Cook, and David J . Robins
Department of Chemistry, The University, Glasgow G12 8QQ, United Kingdom
As part of an ongoing program of work on polyamine analogues, a number of alicyclic diamines
were synthesized and examined for fungicidal activity. The alicyclic diamine 1,2-bis(aminomethyl)-
4,5-dimethylcyclohexa-1,4-diene (BAD), synthesized as the dihydrochloride salt, controlled the
important crop pathogen Erysiphe graminis f.sp hordei. Greatest control of E. graminis was achieved
when BAD was applied 2 days postinoculation. The alicyclic diamines trans-4,5-bis(aminomethyl)-
1,2-dimethylcyclohex-1-ene (trans-BAD) and the cis-isomer (cis-BAD), as well as 1,2-bis(amino-
methyl)cyclopentene (BACP) and trans-1,2-bis(dimethylaminomethyl)cyclobutane (TCCBM), were
also synthesized as their dihydrochloride salts. trans-BAD was found to possess greater fungicidal
activity than the cis-isomer against E. graminis, while BACP and TCCBM both gave >80% control
of powdery mildew infection. Since the powdery mildew fungus cannot be grown axenically, the
effects of the alicyclic diamines on polyamine metabolism were examined using the oat leaf stripe
pathogen, Pyrenophora avenae. BAD and derivatives had little effect on polyamine metabolism in
the fungal pathogen P. avenae. It seems clear, therefore, that the antifungal activity of these
derivatives may not be associated with altered polyamine metabolism.
Keyw or d s: Polyamines; alicyclic diamines; fungicide; powdery mildew
INTRODUCTION
resultant solution was stirred for a further 60 min at this
temperature, after which time methanol (10 mL) was added.
The mixture was allowed to warm to room temperature and
filtered through a Celite pad, and the filtrate was concentrated
in vacuo to give an oil. Crystallization from ethyl acetate/
petroleum ether (40-60 °C) gave 1,2-bis(hydroxymethyl)-4,5-
dimethylcyclohexa-1,4-diene in 52% yield: mp 146-148 °C;
1H NMR (200 MHz, CDCl3) δ 4.16 (s, 4H), 2.76 (s, 4H), 1.65
(s, 6H); 13C NMR (50 MHz, CDCl3) δ 132.4 (C), 122.8 (CH3),
62.0 (CH2), 36.7 (CH2), 18.0 (CH3); IR (KBr disk) 3280, 2900,
2870, 2700, 1050, 1010, 990 cm-1; MS (m/ z) 168 (M+). Anal.
Calcd for C10H16O2: C, 71.43; H, 9.52%. Found: C, 70.67; H,
9.43%.
Inhibitors of polyamine biosynthesis have been shown
previously to give effective control of biotrophic fungal
pathogens, such as rusts and powdery mildews (Rajam
et al., 1985; Walters, 1986; West et al., 1988). This early
work centered mostly on the use of enzyme-activated
irreversible inhibitors, e.g. R-difluoromethylornithine
(DFMO), an inhibitor of the polyamine biosynthetic
enzyme ornithine decarboxylase (ODC) (Metcalf et al.,
1978). In plant pathogenic fungi, DFMO produced a
depletion in the intracellular concentrations of putres-
cine and spermidine (Foster and Walters, 1990). How-
ever, 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
metabolism in tumor cells leading to pronounced anti-
proliferative effects. More recently, ketoputrescine, a
commercially 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 synthesis, as their salts, and fungicidal evaluation
of a number of alicyclic diamines.
To a solution of hydrazoic acid (1.0 M) in benzene (24 mL)
were added 1,2-bis(hydroxymethyl)-4,5-dimethylcyclohexa-1,4-
diene (1.68 g, 0.01 mol) in dry tetrahydrofuran (THF; 20 mL)
and diisopropyl azodicarboxylate (2.52 g, 0.025 mol) in dry
THF (10 mL). To this stirred solution was added triphenyl-
phosphine (10.48 g, 0.05 mol) in dry THF (35 mL) over 1 h.
The mixture was then stirred at 50 °C for a further 7 h, and
then water (5 mL) was added. The mixture was allowed to
cool and then partitioned between hydrochloric acid (1 M, 100
mL) and CH2Cl2 (100 mL). The aqueous layer was further
washed with CH2Cl2 (2 × 80 mL), and the water was removed
in vacuo to give a brown solid, which was dissolved in ethanol
and precipitated with ether to give 1,2-bis(aminomethyl)-4,5-
dimethylcyclohexa-1,4-diene dihydrochloride in 32% yield: 1H
NMR (200 MHz, D2O) δ 3.76 (s, 4H), 2.76 (s, 4H), 1.64 (s, 6H);
13C NMR (50 MHz, D2O) δ 140.2 (C), 123.2 (C), 40.2 (CH2),
36.0 (CH2), 19.6 (CH3); IR (KBr disk) 3420, 3010, 2920, 1660,
1640, 1507, 1480, 1450, 1380, 1110 cm-1; MS (m/ z) 166 (M+
- 2HCl). Anal. Calcd for C10H20N2Cl2: C, 50.21; H, 8.37; N,
11.72%. Found: C, 50.46; H, 8.17; N, 11.56%.
EXPERIMENTAL PROCEDURES
Syn th esis of 1,2-Bis(a m in om eth yl)-4,5-d im eth ylcyclo-
h exa -1,4-d ien e Dih yd r och lor id e (BAD) (1). To a solution
of diisobutyl aluminum hydride (DIBAL) in dichloromethane
(1.0 M, 45 mL, 0.045 mol) cooled to ice bath temperature under
nitrogen was added dimethyl 4,5-dimethylcyclohexa-1,4-diene-
1,2-dicarboxylate (2.24 g, 0.01 mol) (Kucherov and Grigoreva,
1961) in dry dichloromethane (30 mL) over 30 min. The
Syn th esis of tr a n s-4,5-Bis(a m in om eth yl)-1,2-d im eth -
ylcycloh exen e Dih yd r och lor id e (tr a n s-BAD) (2). To a
suspension of lithium aluminum hydride (1.52 g, 0.04 mol) in
dry THF (60 mL) at 0 °C was added dimethyl trans-1,2-
dimethylcyclohexene-4,5-dicarboxylate (Le Coq and Levas,
1966) (2.26 g, 0.01 mol) in dry THF (40 mL) over 30 min. The
resultant suspension was stirred for another hour at 0 °C, and
then saturated sodium sulfate (ca. 5 mL) was added dropwise.
* Author to whom correspondence should be ad-
dressed.
S0021-8561(95)00772-2 CCC: $12.00
© 1996 American Chemical Society

2836 J. Agric. Food Chem., Vol. 44, No. 9, 1996
Havis et al.
The solution was filtered, and the filtrate was concentrated
to give an oil, which on purification gave trans-4,5-bis-
(hydroxymethyl)-1,2-dimethylcyclohexene in 36% yield: mp
dicarboxamide (0.61 g, 89%): 1H NMR (90 MHz, CDCl3) δ 2.1
(4H, m), 2.85 (6H, s), 2.95 (6H, s), 3.7 (2H, m); IR (KBr disk)
3432, 2970, 2938, 1631 cm-1; MS (m/ z) 198 (M+), 154, 126,
100, 72 (100%). HRMS calcd for C10H18N2O2: M+ 198.1364
(23%). Found: 198.1369.
1
102-104 °C; H NMR (200 MHz, CDCl3) δ 3.61 (m, 4H), 3.48
(m, 2H), 1.76 (m, 4H), 1.53 (s, 6H); 13C NMR (50 MHz, CDCl3)
δ 124.6 (C), 66.2 (CH2), 40.7 (CH), 35.2 (CH2), 18.6 (CH3); IR
(thin film) 3300, 2890, 2880, 1410, 1380, 1080, 1030 cm-1; MS
(m/ z) 170 (M+). Anal. Calcd for C10H18O2: C, 70.59; H,
10.59%. Found: C, 70.65; H, 10.68%.
To a suspension of lithium aluminum hydride (0.25 g, 6.58
mmol) in anhydrous ether (10 mL) was added a solution of
trans-N,N,N′,N′-tetramethylcyclobutane-1,2 dicarboxamide (0.6
g, 3.03 mmol) in dry THF (5 mL) at a rate which maintained
gentle reflux. The mixture was then heated at reflux for 1 h
and hydrolyzed by the cautious addition of water (1 mL), 15%
sodium hydroxide solution (1 mL), and finally water (2 mL).
The fine white precipitate was filtered off, washed with ether
(3 × 10 mL), and discarded. The filtrate was concentrated to
give a colorless oil (0.4 g, 77%): 1H NMR (200 MHz, CDCl3) δ
1.57-2.46 (10H, m), 2.18 (12H, s); 13C NMR (50 MHz, CDCl3)
δ 25.2 (CH2), 38.5 (CH), 45.6 (CH3), 65.4 (CH2); IR (KBr disk)
3404, 2966, 2939, 2814, 2764, 1643, 1456 cm-1; MS (m/ z) 170
(M+), 125, 84, 58 (100%). HRMS calcd for C10H22N2: M+
170.1783. Found: 170.1784. The dihydrochloride was pre-
pared by precipitation from a solution of the base in dry ether
saturated with HCl gas.
Deter m in a tion of P r otecta n t a n d Cu r a tive Action of
BAD a ga in st P ow d er y Mild ew (Er ysiph e gr a m in is DC
f.sp h or d ei Ma r ch a l) on Ba r ley. Barley seedlings (Hordeum
vulgare L. cv. Golden Promise) were grown in Fison’s Leving-
ton compost in 36 cm seed trays. Plants were grown in a
glasshouse under natural daylight supplemented for 16 h daily
by 400 W mercury vapor lamps. The maximum temperature
was 24 °C during the day and 9 °C at night. Plants at Zadoks
growth stage 12 (second leaf unfolded) were sprayed before
and after inoculation with the powdery mildew fungus. BAD
and analogues were applied to plants in aqueous solution
containing 0.1 mL L-1 Tween 20. Barley seedlings were
sprayed to runoff with these solutions (usually 1 mM, unless
stated otherwise) using a Shandon spray unit, 3 h before or 3
days after inoculation with pathogen. To study the effects of
the time of application on infection, BAD was applied to barley
seedlings 1, 2, and 5 days pre- or postinoculation with mildew.
Plants were inoculated simply by dusting them with conidia
of E. graminis f.sp hordei. Infection intensity was assessed
6-10 days after inoculation by estimating the percentage leaf
area with pustules using a standard area diagram. Sporula-
tion usually occurred about 6-7 days after inoculation.
Effects of tr a n s-BAD a n d cis-BAD on Gr ow th , En zym e
Activities, a n d P olya m in e Con cen tr a tion s in P yr en o-
ph or a a ven a e. Because of limited supplies of experimental
test compounds, the effects of trans-BAD and cis-BAD on
mycelial growth, polyamine concentrations, and the activities
of ornithine decarboxylase (ODC; EC 4.1.1.17), S-adenosyl-
methionine decarboxylase (AdoMetDC; EC 4.1.1.50), and di-
amine oxidase (DAO; EC 1.4.3.6) were determined as described
previously (Foster and Walters, 1990; Havis et al., 1994b).
trans-4,5-Bis(Hydroxymethyl)-1,2-dimethylcyclohexene was
treated with hydrazoic acid as described above to give a brown
solid, which was recrystallized twice (ethanol/acetone) to give
trans-4,5-bis(aminomethyl)-1,2-dimethylcyclohexene dihydro-
chloride is 22% yield: 1H NMR (200 MHz, D2O) δ 2.85 (m,
4H), 1.96 (m, 4H), 1.60 (m, 2H), 1.47 (s, 6H); 13C NMR (50
MHz, D2O) δ 123.3 (C), 42.8 (CH2), 33.7 (CH), 30.8 (CH2), 19.0
(CH3); IR (KBr disk) 3450, 3030, 2900, 2850, 1610, 1500 cm-1
;
MS (m/ z) 168 (M+ - 2HCl). Anal. Calcd for C10H20N2Cl2: C,
49.79; H, 9.13; N, 11.62%. Found: C, 49.58; H, 9.15; N, 11.52.
Syn th esis of cis-4,5-Bis(a m in om eth yl)-1,2-d im eth yl-
cycloh exen e Dih yd r och lor id e (cis-BAD) (3). cis-1,2-Di-
methylcyclohexene-4,5-dicarboxylic anhydride (Grummitt and
Eudrey, 1960) was reduced with lithium aluminum hydride
as described above to give a yellow oil of cis-4,5-bis(hydroxy-
methyl)-1,2-dimethylcyclohexene in 36% yield: mp 72-74 °C;
1H NMR (200 MHz, CDCl3) δ 4.71 (s, 2H), 3.57 (m, 4H), 1.97
(m, 6H), 1.59 (s, 6H); 13C NMR (50 MHz, CDCl3) δ 123.9 (C),
63.5 (CH2), 38.4 (CH), 33.3 (CH2), 19.0 (CH3); IR (thin film)
3310, 2900, 1440, 1080, 1010, 690 cm-1; MS (m/ z) 152 (M+
H2O). Anal. Calcd for C10H18O2: C, 70.59; H, 10.59%.
Found: C, 70.76; H, 10.76%.
-
Treatment of this product with hydrazoic acid as described
above gave a brown solid, which was recrystallized (ethanol/
acetone) to give cis-4,5-bis(aminomethyl)-1,2-dimethylcyclo-
hexene dihydrochloride in 22% yield: 1H NMR (200 MHz, D2O)
δ 3.95 (m, 4H), 1.99 (m, 2H), 1.93 (m, 4H), 1.52 (s, 6H); 13C
NMR (50 MHz, D2O) δ 122.6 (C), 40.1 (CH2), 31.4 (CH), 28.9
(CH2), 19.4 (CH3); IR (KBr disk) 3450, 3030, 2900, 2850, 1610,
1500 cm-1; MS (m/ z) 168 (M+ - 2HCl). Anal. Calcd for
C10H20N2: C, 49.79; H, 9.13; N, 11.62%. Found: C, 49.85; H,
9.05; N, 11.25%.
Syn th esis of 1,2-Bis(a m in om eth yl)cyclop en ten e Dih y-
d r och lor id e (BACP ) (4). Dimethylcyclopentene-1,2-dicar-
boxylate (McDonald and Reitz, 1972) was reduced with lithium
aluminum hydride as described above to give an oil, which on
vacuum distillation (120-130 °C, 0.05 mmHg) gave 1,2-bis-
(hydroxymethyl)cyclopentene in 31% yield: mp 41-42 °C; 1H
NMR (200 MHz, CDCl3) δ 4.20 (s, H), 3.64 (bs, 2H), 2.47 (t,
4H), 1.85 (q, 2H); 13C NMR (50 MHz, CDCl3) δ 129.6 (C), 62.6
(CH2), 36.7 (CH2), 23.0 (CH2); IR (thin film) 3300, 2885, 2875,
1645, 1445, 1190 cm-1; MS (m/ z) 110 (M+ - H2O). Anal.
Calcd for C7H12O2: C, 65.63; H, 9.38%. Found: C, 65.60; H,
9.31%.
This diol was treated with hydrazoic acid (1.25 M) as
described above to give a brown solid, which was recrystallized
twice (ethanol/acetone) to give 1,2-bis(aminomethyl)cyclopen-
tene dihydrochloride in 21% yield: 1H NMR (200 MHz, D2O)
δ 3.63 (s, 4H), 2.35 (t, 4H), 1.79 (m, 2H); 13C NMR (50 MHz,
D2O) δ 136.0 (C), 37.3 (CH2), 34.8 (CH2), 22.3 (CH2); IR (KBr
disk) 3434, 2951, 2264, 2212, 1601, 1473, 1406, 1385, 1120,
937 cm-1; MS (m/ z) 109 (M+ - 2HCl - NH3). Anal. Calcd
for C7H16N2Cl2: C, 42.21; H, 8.04; N, 14.07%. Found: C, 42.13;
H, 8.17; N, 13.91%.
Syn th esis of tr a n s-1,2-Bis(d im eth yla m in om eth yl)cy-
clobu ta n e Dih yd r och lor id e (TCCBM) (5). The synthesis
of the free base was carried out by modifying the general
procedure of Quin et al. (1979). To a suspension of trans-1,2-
cyclobutanedicarboxylic acid (0.5 g, 3.47 mmol) in benzene (5
mL) was added hexamethylphosphorus triamide (0.63 mL, 3.47
mmol) dropwise. The mixture was heated to ca. 50 °C, stirred
for 30 min, and then cooled to room temperature; saturated
sodium bicarbonate solution (5 mL) was then added. The
layers were separated, and the aqueous layer was extracted
with dichloromethane (3 × 20 mL). The organic extracts were
combined, dried (MgSO4), concentrated, and washed with
hexane to yield trans-N,N,N′,N′-tetramethylcyclobutane-1,2-
RESULTS AND DISCUSSION
Syn th esis of Alicyclic Dia m in es. The synthesis of
compounds 1-3 was carried out by Diels-Alder cyclo-
addition reactions of 2,3-dimethylbutadiene with dif-
ferent alkenes to generate the six-membered rings,
followed by reduction to the diols and conversion into
the diamines (Figure 1). The five-membered diamine
was made by Dieckmann cyclization of R,R′-dibromo-
pimelate, followed by reduction and conversion into the
diamine (4). The four-membered diamine (5) was
prepared from trans-cyclobutane-1,1-dicarboxylic acid
via the bis(diethylamide).
F u n gicid a l Act ivit y of Alicyclic Com p ou n d s.
BAD applied as a postinoculation spray provided very
substantial control of powdery mildew (E. graminis f.sp
hordei) on barley (93%; Table 1). Most effective control
of E. graminis was obtained when BAD was applied 2
days postinoculation, indicating curative properties for
this compound (Table 2). This effect was also observed

Fungicidal Activity of Alicyclic Diamines
J. Agric. Food Chem., Vol. 44, No. 9, 1996 2837
Ta ble 3. Effect of tr a n s-BADa a n d cis-BADb on th e
Gr ow th of P . a ven a e in Liqu id Cu ltu r e
treatment
mean wt (g)
lsd (P ) 0.05)
control
trans-BAD
cis-BAD
1.99
1.81
2.09
0.89
0.89
a
trans-BAD was used as the dihydrochloride at 211 mg L-1 (1
mM). cis-BAD was used as the dihydrochloride at 211 mg L-1 (1
b
mM).
Ta ble 4. Effect of tr a n s-BAD a n d cis-BAD on P olya m in e
Con cen tr a tion s in P . a ven a e
polyamine concn (µmol g-1 fresh wt)
treatmenta
control
putrescine
cadaverine spermidine
spermine
147.25 ( 20.99 54.02 ( 19.64 81.53 ( 8.12 86.87 ( 4.75
trans-BAD 126.01 ( 12.10 37.99 ( 7.34 72.79 ( 9.10 98.35 ( 5.69
cis-BAD
lsd (P )
0.05)
176.74 ( 46.30 34.03 ( 4.09 49.04 ( 6.15 118.48 ( 5.62
97.80 41.91 33.93 17.77
a
trans-BAD and cis-BAD were both used as their dihydrochlo-
rides at 211 mg L-1 (1 mM).
Ta ble 5. Effect of tr a n s-BAD a n d cis-BAD on En zym e
Activities in P . a ven a e
A. Enzyme Activity [pmol of CO2 (mg of protein)-1 h-1
]
lsd
(P ) 0.05)
lsd
(P ) 0.05)
treatmenta
control
trans-BAD 2.18 ( 0.55
cis-BAD
ODC
AdoMetDC
F igu r e 1. Chemical synthesis of alicyclic diamines.
1.26 ( 0.38
62.53 ( 23.90
22.12 ( 12.47
15.77 ( 2.76
Ta ble 1. Effects of BAD a n d Der iva tives on P ow d er y
Mild ew of Ba r ley
1.66
1.54
67.54
72.95
1.63 ( 0.40
% leaf area
infected
% disease
control
lsdc
(P ) 0.05)
B. Enzyme Activity [pmol of product (mg of protein-1) h-1
]
treatmenta
lsd
(P ) 0.05)
control
BADb
55.0
4.0
treatmenta
DAO
93
6.40
control
trans-BAD
cis-BAD
66.48 ( 0.95
50.50 ( 5.20
44.61 ( 3.15
control
trans-BAD
cis-BAD
37.33
8.70
14.20
20.11
21.73
77
62
5.53
6.01
a
trans-BAD and cis-BAD were both used as their dihydrochlo-
rides at 211 mg L-1
control
BACP
TCCBM
51.08
8.67
5.78
.
83
89
6.87
6.95
tion of rust uredospores on the leaf surface (Rajam et
al., 1989), and two putrescine analogues have been
shown to inhibit appressorium formation by uredospores
or Uromyces viciae-fabae (Reitz et al., 1995). Other
alicyclic compounds also controlled infection by E.
graminis. The most effective control was obtained with
postinoculation treatments of TCCBM (89%; Table 1).
Two isomers, trans-BAD and cis-BAD, exhibited dif-
fering fungicidal activities against E. graminis infection.
Thus, trans-BAD reduced mildew infection by 77%
(Table 1), while cis-BAD gave only 62% control of
mildew infection (Table 1). These differences, however,
were not significant. Similar effects were observed in
previous work in which the synthetic putrescine ana-
logue trans-1,4-diaminobut-2-ene (E-BED) was found to
possess greater fungicidal activity than the cis-isomer,
Z-BED (Havis et al., 1994a). The mechanism underly-
ing this difference in fungicidal activity is not known.
Since powdery mildew cannot be grown axenically,
the effects of these two isomers on polyamine biosyn-
thetic and catabolic enzymes and polyamine levels were
examined in the oat stripe pathogen, P. avenae. The
alicyclic compound produced little effect on growth or
polyamine metabolism in this fungus (Tables 3-5).
This contrasts with the effects of aliphatic diamines,
which have been shown to perturb polyamine biosyn-
thesis (Havis et al., 1994a,b). Nevertheless, these
authors suggested that the aliphatic diamines did not
deplete intracellular polyamine concentrations suf-
a
Treatments were applied at 1 mM, i.e. 211, 211, 211, 199, and
243 mg L-1, respectively, and were applied as postinoculation
treatments. All compounds were used as salts (see Figure 2).
b
BAD, 1,2-bis(aminomethyl)-4,5-dimethylcyclohexa-1,4-diene di-
hydrochloride; trans-BAD, trans-4,5-bis(aminomethyl)-1,2-dimeth-
ylcyclohexene dihydrochloride; cis-BAD, cis-4,5-bis(aminomethyl)-
1,2-dimethylcyclohexene dihydrochloride; BACP, 1,2-bis(amino-
methyl)cyclopentene dihydrochloride; TCCBM, trans-1,2-bis(di-
methylaminomethyl)cyclobutane dihydrochloride. c lsd, least sig-
nificant difference.
Ta ble 2. Effects of Tim in g of BAD Ap p lica tion on
P ow d er y Mild ew of Ba r ley
% leaf area
infected
% disease
control
treatmenta
control
5 days preinoculation
2 days preinoculation
1 day preinoculation
1 day postinoculation
2 days postinoculation
5 days postinoculation
28.9 ( 2.5
24.0 ( 0.9
26.3 ( 4.3
22.8 ( 2.5
16.8 ( 2.0b
8.9 ( 1.2c
17.2 ( 1.9b
17
9
21
42
70
41
a
Treatments were applied at 1 mM, i.e. 211 mg L-1 b,cSignifi-
.
cantly different from the control at P e 0.005 and e 0.001,
respectively.
with the synthetic putrescine analogues E-BED and
E-TED (Havis et al., 1994a,b) and could be related to
perturbation of polyamine biosynthesis in the germinat-
ing conidia on the leaf surface. The ODC inhibitor,
DFMO, has already been shown to inhibit the germina-

2838 J. Agric. Food Chem., Vol. 44, No. 9, 1996
Havis et al.
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ficiently to account for the observed antifungal activity.
A similar situation exists in the present work. It is
important to remember, however, that effects of BAD
and derivatives on polyamine metabolism in developing
germlings of E. graminis cannot be ruled out. Since the
pathogen cannot be cultured axenically, the mode of
action of BAD against this fungus is not going to be
resolved easily.
It is interesting to note that a number of bis(benzyl)-
polyamine analogues have been shown to be potent
inhibitors of two strains of the human malaria parasite
Plasmodium falciparum in vitro (Bitonti et al., 1989).
These analogues were found to be 1000 times more
potent than the free amine analogues. It was suggested
that these compounds may not only repress polyamine
biosynthesis but exert cytotoxic effects by binding
directly to DNA and subsequently disrupt macro-
molecular synthesis. It would be useful to determine
whether BAD and its derivatives disrupt macromolecu-
lar synthesis in P. avenae.
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 two synthetic putrescine analogues on germination
and appressorium formation by uredospores on Uromyces
viciae-fabae on artificial membranes. Lett. Appl. Microbiol.
1996, 21, 285-287.
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-
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West, H. M.; Walters, D. R. The effects of polyamine biosyn-
thesis on infection of Hordeum vulgare L. by Erysiphe
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ACKNOWLEDGMENT
We are grateful to Dr. R. A’Court of BTG for advice
and guidance.
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Received for review November 22, 1995. Revised manuscript
received J uly 9, 1996. Accepted J uly 16, 1996.X
We are
grateful to the British Technology Group plc for generous
financial support. SAC receives financial support from the
Scottish Office Agriculture and Fisheries Department.
J F950772S
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.
X Abstract published in Advance ACS Abstracts, Sep-
tember 1, 1996.