Synthesis and Bioactivity of Analogs of Maculosin, a Host-Specific Phytotoxin Produced by Alternaria alternaria on Spotted Knapweed (Centaurea maculosa)

Article abstract of DOI:10.1021/jf960091c

Maculosin (cyclo-Pro-Tyr), a host-specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa), is an ideal prototype for creating a safe and an environmentally friendly antiknapweed herbicide. To evaluate this possibility

Full text of DOI:10.1021/jf960091c

3960
J. Agric. Food Chem. 1996, 44, 3960−3964
Syn th esis a n d Bioa ctivity of An a logs of Ma cu losin , a Host-Sp ecific
P h ytotoxin P r od u ced by Alter n a r ia a lter n a ta on Sp otted Kn a p w eed
(Cen ta u r ea m a cu losa )
Mikhail M. Bobylev,* Ludmila I. Bobyleva, and Gary A. Strobel
Department of Plant Pathology, Montana State University, Bozeman, Montana 59717
Maculosin (cyclo-Pro-Tyr), a host-specific phytotoxin produced by Alternaria alternata on spotted
knapweed (Centaurea maculosa), is an ideal prototype for creating a safe and an environmentally
friendly antiknapweed herbicide. To evaluate this possibility, a series of 18 maculosin analogs was
synthesized and tested in the greenhouse on whole knapweed plants by spray or brush application.
Interestingly, many of the maculosin analogs have significant potential as natural herbicides against
spotted knapweed. Even the simplest analog, cyclo-Pro-Phe, destroys two thirds of the spotted
knapweed foliage at a concentration of 6 × 10-2 mol/L within 15 days. Structure-activity
relationships are discussed.
Keyw or d s: Cyclodipeptides; herbicides; maculosin; spotted knapweed
INTRODUCTION
nothing is known about the influence of maculosin and
its analogs on whole knapweed plants because all
previous tests were performed on the detached and
punctured knapweed leaves according to the method
developed by Sugawara et al. (1985).
Spotted knapweed is one of North America’s most
noxious weeds (Harris and Cranston, 1979). Its inva-
sion of rangeland, roadsides, and pastures has caused
untold millions of dollars in loss in the northwestern
United States and southwestern Canada. Currently,
synthetic herbicides, especially Tordon, are successfully
used to combat spotted knapweed (Davis, 1990). How-
ever, it seems beneficial to look for safer and more
environmentally friendly herbicides among naturally
occurring phytotoxins. As we suggested earlier (Strobel
et al., 1987), weed pathogens are a very promising
source of bioactive natural products for weed control.
Maculosin [(I), (3S-cis)-hexahydro-3-[(4-hydroxyphenyl)-
methyl]pyrrolo[1,2-a]pyrazine-1,4-dione] is one of the
products found as a result of this novel approach.
For those reasons, we began a systematic investiga-
tion of the bioactivity of maculosin and its analogs
against spotted knapweed. In this report we examine
the influence of different substituents and certain
modifications of the structure in the part of the molecule
adjacent to the hydroxy group. This group is definitely
one of the most important structural features of macu-
losin, first because its presence results in a hundredfold
increase in activity from II to I, and second because it
is the primary site of the detoxification of maculosin by
the host plant (Park et al., 1994). Analogs studied in
the present work include compounds with various
substituents placed near the free hydroxy group (II-
Ia ,b,c); compounds with the protected hydroxy group
(IVa -f); compounds with the hydroxy group replaced
with other substituents, primarily halogens (Va -e); and
a series of compounds with no substituents at all, but
with increased size of aromatic rings (VI, VII) or with
more rigid plain structure (VIII). All synthesized
compounds, except I, II, and IIIc, are new. Both
maculosin and all its analogs were tested on whole
knapweed plants in the greenhouse.
Maculosin is a host-specific fungal toxin produced by
Alternaria alternata on spotted knapweed (Centaurea
maculosa; Stierle et al., 1988). Its unique selectivity,
apparent safety, and simple structure, make maculosin
an ideal chemical lead for developing a safe and an
environmentally friendly antiknapweed herbicide. How-
ever, not much is known about structure-activity
relationships among maculosin analogs. Although mac-
ulosin was originally isolated along with a series of six
closely related cyclic dipeptides, only one (II) appeared
to be somewhat toxic to knapweed. Furthermore, there
does not seem to be literature examples of herbicide
activity among cyclic dipeptides as a whole (for the most
comprehensive and updated review on bioactive cyclic
dipeptides see Prasad, 1995). In addition, virtually
EXPERIMENTAL PROCEDURES
Melting points were determined on a Fisher-J ohns melting
point apparatus between two micro cover glasses and are
uncorrected. Elemental analysis was performed by Atlantic
Microlab Inc., Atlanta, GA. Infrared (IR) spectra on 3M
disposable polyethylene cards (#61) were recorded on a Bruker
IFS 25 spectrophotometer (ν, cm-1). Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on a Bruker AC300
spectrometer in CDCl3, with TMS as an internal reference;
chemical shifts are expressed as δ values in ppm. Optical
rotation was measured on a Perkin Elmer 241 MC polarimeter
in a 1-cm cell in a mixed solvent (CHCl3/MeOH, 1:1) at the
concentration of 1 g/dL at room temperature.
Most of the compounds I-VIII were obtained by the
standard coupling procedure from commercially available
BOC-protected amino acids and amino acids methyl esters,
followed by deprotection with formic acid and further cycliza-
* Author to whom correspondence should be ad-
dressed [telephone (406) 994-5147; fax (406) 994-1848;
e-mail uplmb@msu.oscs.montana.edu].
S0021-8561(96)00091-X CCC: $12.00
© 1996 American Chemical Society

Maculosin Analogs as Bioherbicides
J. Agric. Food Chem., Vol. 44, No. 12, 1996 3961
tion of the obtained formates in boiling 2-butanol (Nitecki et
al., 1968). O-Propyl- and O-butyl-L-tyrosine used in the
synthesis of compounds IVc and d were obtained by direct
alkylation of L-tyrosine with propyl and butyl bromides in
DMSO (Solar and Schumaker, 1966) and then transferred to
their BOC-derivatives by a standard reaction with BOC-ON
(Itoh et al., 1975). Compound IVf was obtained by alkylation
of IIIb with ethyl sulfate, as a further development of the
method originally designed for alkylation of BOC-protected
esters of tyrosine (Kolodziejczyk and Manning, 1980). Com-
pounds I and IIIa and b were obtained as molecular complexes
with benzene (1:1), as was first described for I (Tatsuno et al.,
1971). The intermediate linear dipeptide for the compound
IIIc appeared to be extremely sensitive to 1 N sodium bicar-
bonate solution and, for this reason, the reaction mixture after
coupling was washed only with water and 1 N citric acid
solution. This preventive measure might explain the fact that
the melting point of IIIc (194-196 °C) was significantly higher
than that reported in the literature (183-184 °C; Sviridov et
al., 1990; 159-164 °C; Bavykina et al., 1990).
compounds were dissolved in an auxiliary organic solution in
advance and diluted with water immediately before spraying.
For phytotoxicity evaluation at higher concentrations, only the
compounds with stable formulations and sufficient solubility
were used. All solutions were prepared in advance and used
within 2-3 h. Solutions were applied by wetting plants with
cotton swabs to provide complete and uniform coverage of both
leaf surfaces. This technique also allowed us to conserve
compounds. Five plants were used per each concentration.
Evaluation of Results. In the preliminary tests series, the
number and size of the necrotic lesions were not high enough
to allow for quantitative measurements. The results were
evaluated qualitatively in accordance with the following
scale: “+”, necrotic lesions present; “-”, no lesions.
In higher concentration tests, results were read three times,
on the 5th, 10th, and 15th days after treatment, and evaluated
visually based on the following scale: 10%, any damage area
<10% of the leaf surface; 30%, damage area within the range
of 10-50% of the leaf surface; 70%, damage area within 50-
90% of the leaf surface; 90%, any damage area >90% of the
leaf surface.
Yields, melting points, optical rotation, molecular formulas,
and elemental analysis of compounds I-VIII are recorded in
Table 1 and spectral data are in Table 2.
RESULTS AND DISCUSSION
Her bicid e Tests. Plants. The spotted knapweed plants
used in this study were individually grown in plastic pots in
pasteurized AquaGro soil mixture in a greenhouse (16 h day/8
h night). For preliminary tests, plants were used in the stage
of six to eight rosette leaves (∼8 weeks old). For phytotoxicity
evaluation at higher concentrations, plants were used in the
stage of 10 to 12 rosette leaves (∼12 weeks old).
In all tests, the only observed leaf damage consisted
of necrotic lesions of varying intensities. Symptoms
appeared within 2-5 days after treatment and the
lesions continued to develop for up to 15 days. In the
most successful cases, the lesions eventually covered the
whole leaf surface and killed the leaf completely. Some
leaves turned yellow, then dried, and died soon after
appearance of the first lesions, so symptom development
generally appeared as an acceleration of aging. No
other symptoms, such as retarded or accelerated growth,
wilting, or deformation were observed. The results of
the tests are recorded in Table 3.
To our great surprise, maculosin itself appeared to
be completely inactive on whole intact plants even at
the concentration of 4 × 10-2 mol/L. This result,
however, is quite consistent with the general picture of
natural product research on weeds: “a myriad of assays
have been utilized in the discovery of herbicidal natural
products”, and only “few of these compounds have
proven to have activity on intact weeds” (Stonard and
Miller-Wideman, 1994). All other compounds with the
free hydroxy group (IIIa -c) were inactive at the initial
concentration of 1 × 10-2 mol/L as well. Unfortunately,
Formulation. For preliminary tests, compounds were dis-
solved in a small amount of an auxiliary organic solution of
Tween 80 in organic solvent [ethanol or dimethylformamide
(DMF)], and then diluted with water to create 10-2
M
concentration. The resulting concentrations of the organic
solvent and the surfactant in the final solution were 5 vol %
and 5 mg/mL, respectively. For phytotoxicity evaluation at
higher concentrations, compounds were dissolved in an aux-
iliary solution of SilWet L-77 in DMF and diluted with water
to create 1 × 10-2 to 6 × 10-2 M concentrations. The resulting
concentrations of SilWet L-77 and DMF in the final solutions
are 2 mg/mL and 2 vol %, respectively. The same solvent/
surfactant solutions but without analogs were applied to
control plants.
Application. In preliminary tests, solutions were applied
to leaves by spraying. Two plants were taken per each
compound and sprayed simultaneously with 10 mL of the
solution. Some of the solutions were not stable enough and
precipitated in several minutes after preparation, so all

3962 J. Agric. Food Chem., Vol. 44, No. 12, 1996
Bobylev et al.
Ta ble 1. An a lytica l Da ta of New ly P r ep a r ed An a logs of Ma cu losin
found (calculated) (%)
compd
yield (%)
58
mp (°C)
[R]25 (c 1.0)
mol formula
C20H22N2O3
C
H
N
D
I
133-8
-132°
-199°
-103°
-79°
70.26
70.20
(70.98)
68.74
68.69
(68.83)
67.54
67.52
(67.78)
63.80
63.70
(64.43)
55.13
55.04
(55.08)
65.72
65.66
(65.68)
66.62
66.69
(66.65)
67.58
67.50
(67.53)
67.63
67.57
(68.33)
72.22
72.19
(71.41)
59.60
59.51
(59.53)
63.08
62.98
(64.11)
60.41
60.39
(60.33)
52.14
52.05
(52.03)
45.51
45.45
(45.38)
58.18
58.12
(58.12)
73.49
6.56
6.59
(6.55)
6.58
6.62
(6.60)
6.25
6.27
(6.26)
5.75
5.80
(5.68)
4.94
4.96
(4.95)
6.58
6.63
(6.61)
7.03
7.04
(6.99)
7.33
7.36
(7.33)
7.58
7.61
(7.64)
6.44
6.46
(5.99)
5.96
5.98
(5.93)
5.83
5.86
(5.76)
5.45
5.46
(5.42)
4.69
4.71
(4.68)
4.06
4.09
(4.08)
5.26
5.28
(5.23)
6.18
8.37
8.34
(8.28)
11.49
11.40
(11.47)
7.98
7.92
(7.90)
7.61
II
82
54
37
44
51
61
69
59
54
133-5
131-6
123-133
194-6
162-7
136-8
143-6
125-8
143-5
135-40
154-6
172-4
184-5
197-200
212-23
208-12
191-4
151-3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
14H16N2O2
20H22N2O4
20H21ClN2O3
14H15N3O5
15H16N2O3
16H20N2O3
17H22N2O3
18H24N2O3
20H20N2O3
16H19ClN2O3
14H15FN2O2
14H15ClN2O2
14H15BrN2O2
14H15IN2O2
14H15N3O4
18H18N2O2
18H18N2O2
15H16N2O2
IIIa
IIIb
IIIc
IVa
IVb
IVc
IVd
IVe
IVf
Va
7.60
(7.51)
13.72
13.69
(13.76)
10.24
10.18
(10.21)
9.77
9.69
(9.72)
9.32
9.28
(9.26)
8.95
8.88
(8.85)
7.95
7.93
(8.33)
8.59
-162°
-111°
-101°
-96°
-79°
-72°
-74°
8.58
(8.68)
10.59
10.54
(10.68)
10.02
9.98
(10.05)
8.62
8.61
(8.67)
7.54
7.51
(7.56)
14.57
14.50
(14.52)
9.53
66
69
71
59
52
46
41
60
-112°
-98°
Vb
Vc
-82°
Vd
-79°
Ve
-96°
VI
-149°
-79°
73.40
(73.45)
73.61
73.57
(73.45)
70.17
6.23
(6.16)
6.21
6.26
(6.16)
6.31
9.45
(9.52)
9.40
VII
VIII
9.36
(9.53)
10.93
10.85
(10.93)
-126°
70.10
(70.29)
6.32
(6.29)
because of their insufficient solubility, III could not be
tested at higher concentrations.
as a whole: all other unsubstituted analogs VI-VIII
were also active in the preliminary test. However, only
II and VIII were soluble enough to allow for increased
concentrations. Compounds II appeared to be the most
potent among all tested compounds, and VIII was half
as active.
Among the compounds with protected hydroxy groups
(IV), only lower alkyl members (IVa -c, f; R2 ) Me, Et,
and Pr) were active in the preliminary test, and only
two (R2 ) Me, Et) were soluble enough to test them at
higher concentrations. Both IVa and IVb were less
active than either of unsubstituted analogs II and VIII.
Similar to IV, from all five compounds with the
replaced hydroxy group (V), only two with the smaller
substituents (Va and Vb, R3 ) F, Cl) were active and
only the smallest one (Va , R3 ) F) was soluble enough
In contrast to I, II retained its activity on whole
plants. This result is of special interest for several
important reasons. First, II has already been described
in the literature as phytotoxic to whole plants (Chen,
1960). Second, it is already the third Alternaria alter-
nata toxin [after AK toxins I and II (Nakashima et al.,
1982, 1985)] containing phenylalanine, raising a ques-
tion about phytotoxicity of phenylalanine itself. Finally,
because II comprises a minor natural constituent of
different food products, for example, cheese (Roudot-
Algaron et al., 1993), cocoa (Pickenhagen et al., 1975),
or beer (Sakamura et al., 1978), that might tell about
its potential safety. It seems also that the absence of
substituents in the phenyl ring is beneficial for activity

Maculosin Analogs as Bioherbicides
J. Agric. Food Chem., Vol. 44, No. 12, 1996 3963
Ta ble 2. IR a n d 1H NMR Sp ectr a l Da ta of New ly P r ep a r ed An a logs of Ma cu losin
compd
IR (3M card 61), νmax (cm-1); 1H NMR (CDCl3), δ (J , Hz)
I
IR, 3255, 1667, 1614, 1593, 1516, 1473, 1463, 730, 719; 1H NMR, 7.05 (2Hd, 8.4), 6.78 (2Hd, 8.4), 5.94 (1Hs),
4.22 (1Hdd, 2.7, 9.9), 4.09 (1Ht, 7.25), 3.60 (2Hm), 3.47 (1Hdd, 4.0, 14.5), 2.78 (1Hdd, 9.9, 14.5), 2.34 (1Hm),
1.96 (3Hm)
II
IR, 3190, 1683, 1659, 1496, 1481, 1454, 753, 699; 1H NMR, 7.29 (5H), 5.61 (1Hs), 4.28 (1Hdd, 3.4, 10.6), 4.09
(1Ht, 7.6), 3.62 (3Hm), 2.78 (1Hdd, 10.8, 14.5), 2.33 (1Hm), 1.98 (3Hm)
IIIa
IR, 3268, 1654, 1518, 1473, 1463, 730, 719; 1H NMR, 7.36 (6Hs), 6.81 (1Hd, 8.0), 6.72 (1Hd, 1.8), 6.61 (1Hdd,
1.8, 8.0), 6.42 (1Hs), 6.22 (1Hs), 5.86 (1Hs), 4.20 (1Hdd, 3.4, 10.27), 4.09 (1Ht, 7.6), 3.61 (2Hm), 3.45 (1Hdd,
3.8, 14.5), 2.70 (1Hdd, 10.27, 14.5), 2.33 (1Hm), 1.97 (3Hm)
IIIb
IIIc
IVa
IVb
IVc
IVd
IVe
IVf
IR, 3231, 1664, 1512, 1473, 1463, 730, 719; 1H NMR, 7.36 (6Hs), 7.21 (1Hd, 1.9), 7.04 (1Hdd, 1.9, 8.3), 6.98
(1Hd, 8.3), 5.61 (1Hs), 4.23 (1Hdd, 3.3, 9.5), 4.09 (1Ht, 7.3), 3.58 (2Hm), 3.50 (1Hdd, 4.0, 14.7), 2.76 (1Hdd,
10.0, 14.7), 2.33 (1Hm), 1.99 (3Hm)
IR, 3231, 1664, 1512, 1473, 1463, 730, 719; 1H NMR, 10.49 (1Hs), 8.00 (1Hd, 2.1), 7.55 (1Hdd, 2.1, 8.6), 7.10
(1Hd, 8.6), 6.92 (1Hs), 4.33 (1Ht, 5.7), 4.10 (1Ht, 7.3), 3.58 (2Hm), 3.38 (1Hdd, 4.5, 14.7), 3.09 (1Hdd, 7.4,
14.7), 2.32 (1Hm), 1.99 (3Hm)
IR, 1671, 1513, 1473, 1463, 1249, 730, 719; 1H NMR, 7.14 (2Hd, 8.5), 6.90 (2 Hd, 8.6), 5.63 (1Hs), 4.22 (1Hdd,
3.2, 10.6), 4.08 (1Ht, 7.4), 3.80 (3Hs), 3.63 (2Hm), 3.55 (1Hdd, 3.6, 14.5), 2.74 (1Hdd, 10.6, 14.6), 2.32 (1Hm),
1.96 (3Hm)
IR, 1674, 1611, 1512, 1473, 1463, 1246, 730, 719; 1H NMR, 7.12 (2Hd, 8.6), 6.87 (2Hd, 8.6), 5.60 (1Hs), 4.22
(1Hdd, 3.0, 10.7), 4.08 (1Ht, 7.0), 4.02 (2Hq, 7.0), 3.62 (2Hm), 3.55 (1Hdd, 3.6, 14.7), 2.72 (1Hdd, 10.7, 14.6),
2.33 (1Hm), 1.97 (3Hm), 1.42 (3Ht, 7.0)
IR, 1674, 1611, 1512, 1473, 1463, 1247, 730, 719; 1H NMR, 7.12 (2Hd, 8.6), 6.87 (2Hd, 8.6), 5.60 (1Hs), 4.22
(1Hdd, 3.3, 10.7), 4.08 (1Ht, 7.6), 3.89 (2Ht, 6.6), 3.59 (2Hm), 3.55 (1Hdd, 3.5, 14.7), 2.72 (1Hdd, 10.7, 14.5),
2.33 (1Hm), 1.98 (3Hm), 1.83 (2Hm), 1.04 (3Ht, 7.4)
IR, 1676, 1512, 1473, 1463, 1247, 730, 719; 1H NMR, 7.12 (2Hd, 8.6), 6.87 (2Hd, 8.6), 5.59 (1Hbr s), 4.22 (1Hbd),
4.08 (1Ht, 7.6), 3.95 (2Ht, 6.5), 3.59 (3Hm), 2.72 (1Hdd, 10.7, 14.5), 2.34 (1Hm), 1.97 (3Hm), 1.77 (2Hm), 1.49
(2Hm), 0.98 (3Ht, 7.4)
IR, 1670, 1513, 1473, 1463, 1248, 730, 719; 1H NMR, 7.39 (5Hm), 7.14 (2Hd, 8.6), 6.96 (2Hd, 8.6), 5.65 (1Hs),
5.06 (2Hs), 4.22 (1Hdd, 3.1, 10.5), 4.07 (1Ht, 7.34), 3.64 (2Hm), 3.54 (1Hdd, 3.6, 14.6), 2.74 (1Hdd, 10.5, 14.6),
2.33 (1Hm), 1.94 (3Hm)
IR, 3,221 1673, 1502, 1475, 1426, 1284, 1257, 1111, 1063, 1040, 805, 754; 1H NMR, 7.38 (1Hdd, 2.2, 8.4), 7.25
(1Hd, 2.2), 6.88 (1Hd, 8.4), 5.78 (1Hbs), 4.23 (1Hdd, 3.4, 10.2), 4.08 (3Hq, 6.7), 3.61 (2Hm), 3.49 (1Hdd, 3.7,
14.7), 2.75 (1Hdd, 10.0, 14.6), 2.33 (1Hm), 1.97 (3Hm), 1.47 (3Ht, 6.7)
Va
Vb
IR, 3223, 1674, 1602, 1510, 1418, 1336, 1305, 1222, 1160, 1111, 825; 1H NMR, 7.21 (2Hdd, 5.4, 8.5), 7.03 (2Ht, 8.6),
5.89 (1Hs), 4.25 (1Hdd, 3.7, 9.5), 4.08 (1Ht, 7.5), 3.59 (3Hm), 2.85 (1Hdd, 9.7, 14.6), 2.34 (1Hm), 1.96 (3Hm)
IR, 3241, 1670, 1491, 1424, 1336, 1303, 1276, 1115, 1090, 1016, 813; 1H NMR, 7.32 (2Hd, 8.4), 7.18 (2Hd, 8.4),
5.64 (1Hs), 4.26 (1Hdd, 3.7, 10.0), 4.08 (1Ht, 7.5), 3.58 (2Hm), 3.54 (1Hdd, 3.7, 14.5), 2.82 (1Hdd, 10.0, 14.6),
2.34 (1Hm), 1.93 (3Hm)
Vc
Vd
Ve
VI
IR, 3223, 1684, 1670, 1488, 1420, 1336, 1303, 1115, 1071, 1011, 808; 1H NMR, 7.47 (2Hd, 8.3), 7.12 (2Hd, 8.3),
5.9 (1Hbs), 4.26 (1Hdd, 3.6, 9.7), 4.08 (1Hbs), 3.57 (3Hm), 2.83 (1Hdd, 9.5, 14.7), 2.33 (1Hm), 1.96 (3Hm)
IR, 3216, 1683, 1668, 1485, 1423, 1338, 1304, 1116, 1007, 804; 1H NMR, 7.66 (2Hbd), 6.99 (2Hd, 8.3), 5.9 (1Hbs),
4.25 (1Hdd, 3.1, 9.9), 4.07 (1Hbd, 5.98), 3.55 (3Hm), 2.80 (1Hdd, 9.5, 14.5), 2.33 (1Hbs), 1.97 (3Hm)
IR, 3220, 1684, 1604, 1514, 1437, 1347, 1314, 1109, 856, 737, 703; 1H NMR, 8.21 (2Hd, 8.6), 7.46 (2Hd, 8.6), 5.90
(1Hs), 4.37 (1Hdd, 4.1, 8.7), 4.10 (1Ht, 7.4), 3.61 (3Hm), 3.06 (1Hdd, 8.8, 14.6), 2.34 (1Hm), 1.97 (3Hm)
IR, 1667, 1664, 1634, 1473, 1462, 1428, 794, 768; 1H NMR, 8.03 (1Hm), 7.90 (1Hm), 7.83 (1Hd, 8.1), 7.55 (2Hm),
7.44 (1Ht, 8.0), 7.36 (1Hd, 6.9), 5.53 (1Hs), 4.42 (1Hd, 11.5), 4.33 (1Hdd, 3.2, 14.7), 4.05 (1Ht, 7.8), 3.66 (2Hm),
3.04 (1Hdd, 11.4, 14.6), 2.34 (1Hm), 2.08 (2Hm), 1.94 (1Hm)
VII
IR, 1668, 1473, 1462, 1418, 730, 719; 1H NMR, 7.81 (3Hm), 7.68 (1Hs), 7.50 (2Hm), 7.34 (1Hdd, 1.5, 8.4), 5.82
(1Hs), 4.37 (1Hdd, 2.8, 10.4), 4.08 (1Ht, 7.4), 3.77 (1Hdd, 3.6, 14.4), 3.61 (2Hm), 2.95 (1Hdd, 10.5, 14.4), 2.30
(1Hm), 1.95 (3Hm)
VIII
IR, 1667, 1473, 1462, 1417, 752, 730, 719; 1H NMR, 7.24 (4Hm), 4.93 (1Hd, 16.0), 4.45 (1Hd, 16.0), 4.08 (2Hm),
3.61 (2Hm), 3.41 (1Hdd, 4.3, 15.6), 3.11 (1Hdd, 11.1, 15.5), 2.41 (1Hm), 2.34 (1Hm), 1.98 (2Hm)
to evaluate its phytotoxicity at higher concentrations.
Compounds Va showed virtually the same high activity
as II.
group by any means (protection as in III, replacement
as in IV, or total removal as in V) restored the activity.
Any further modification of the structure or any other
technique leading to improved penetration might also
result in increased activity. One such technique, using
a special penetrant SilWet L-77 instead of regular
surfactants, was applied to knapweed plants and re-
sulted in a threefold increase in activity.
Another limiting factor is poor translocation. Like
maculosin, its analogs do not move far from the place
of penetration. This lack of movement is evident by a
slow growth and restricted size of necrotic lesions.
These lesions do not spread to untreated parts of the
plant including newly grown parts.
Both penetration and translocation seem tightly con-
nected to solubility in aqueous systems. It is clear from
the pattern of the necrotic lesions that analogs of
maculosin do not penetrate inside the leaf through the
cuticle but only with the test solution through opened
stomata and hydathodes. It seems logical to assume
that increased solubility in aqueous systems will lead
to increased activity and vice versa. Solubility also
All these results show that some maculosin analogs
do have a significant potential as natural herbicides
against spotted knapweed. Even the simplest analog
II destroys two thirds of the spotted knapweed foliage
at the concentration of 6 × 10-2 mol/L. However, none
of the current analogs can control knapweed completely
because they do not affect younger leaves or buds and
thus allow the weed to regrow its foliage within 3-4
weeks after treatment. We do not have an explanation
for the resistance exhibited by younger leaves.
Penetration itself seems to be the most critical factor
affecting the activity of maculosin analogs. Maculosin,
which was two orders of magnitude more active than
II when applied to punctured leaves, was totally inactive
on whole intact plants. Evidently, the presence of free
hydroxy group is the major reason of the poor penetra-
tion and subsequent loss of activity. This conclusion is
supported both by inactivity of all other hydroxy analogs
III and by the fact that the elimination of the hydroxy

3964 J. Agric. Food Chem., Vol. 44, No. 12, 1996
Bobylev et al.
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phytotoxicity evaluation at
higher concentration
leaf damage in % at
pre-
liminary days after
concentration (c), c × 10-2
M
6
compd
test
treatment
0
1
2
4
I
II
-
5
10
15
0
2
4
18
33
44
30
48
50
57
64
66
+
IIIa
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IIIc
IVa
-
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5
10
15
5
10
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0
1
2
0
2
2
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7
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12
11
18
20
+
4
6
6
8
9
IVb
11
26
39
29
39
41
+
IVc
IVd
IVe
IVf
Va
+
-
-
+
5
10
15
0
2
4
20
40
49
42
50
55
+
Vb
Vc
Vd
Ve
VI
VII
VIII
+
-
-
-
+
+
5
10
15
4
9
10
8
15
19
15
22
26
24
36
40
+
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Further development of analogs of maculosin, espe-
cially targeting improvements in penetration and trans-
location, will certainly result in creating compounds of
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Received for review February 8, 1996. Revised manuscript
received August 16, 1996. Accepted August 30, 1996.X We
thank Montana Noxious Weed Trust Fund, the Montana
Agricultural Experimental Station, and Beim Foundation for
financial support.
J F960091C
X Abstract published in Advance ACS Abstracts, No-
vember 1, 1996.