Synthesis of anthelmintically active N-methylated amidoxime analogues of the cyclic octadepsipeptide PF1022A

Article abstract of DOI:10.1002/ps.590

The N-methylated amidoxime analogues of the cyclic octadepsipeptide PF1022A represent novel derivatives with activity against Trichinella spiralis Owen and Nippostrongylus brasiliensis Lane in vitro and against parasitic nematodes in mice and sheep. Some of them show better activity against Hymenolepis nana Siebold, Heterakis spumosa Schneider and Heligmosomoides polygyrus Dujardin in mice than the natural product PF1022A. In particular an improved efficacy against Haemonchus contortus Rudolphi and Trichostrongylus colubriformis Giles in sheep compared to the potent cyclic octadepsipeptide PF1022A and its mono-thionated derivative has been observed. Here we report on a specific modification at the N-methyl amide linkage by using the mono-thionated PF1022A, resulting in novel anthelmintically active backbone analogues of PF1022A.

Full text of DOI:10.1002/ps.590

Pest Management Science
Pest Manag Sci 58:1205–1215 (online: 2002)
DOI: 10.1002/ps.590
Synthesis of anthelmintically active
N-methylated amidoxime analogues of
the cyclic octadepsipeptide PF1022A
Peter Jeschke,1* Achim Harder,2 Georg von Samson-Himmelstjerna,3
Winfried Etzel,1 Wolfgang Gau,1 Gerhard Thielking1 and Gerhard Bonse2
1Bayer AG, Bayer CropScience, Research Global Chemistry Insecticides, Alfred-Nobel-Str 50, D-40789 Monheim, Germany
2Bayer AG, Animal Health Business Group, Agricultural Centre Monheim, D-51368 Leverkusen, Germany
3Institute of Parasitology, School of Veterinary Science, Buenteweg 17, D-30559 Hannover, Germany
Abstract: The N-methylated amidoxime analogues of the cyclic octadepsipeptide PF1022A represent
novel derivatives with activity against Trichinella spiralis Owen and Nippostrongylus brasiliensis
Lane in vitro and against parasitic nematodes in mice and sheep. Some of them show better activity
against Hymenolepis nana Siebold, Heterakis spumosa Schneider and Heligmosomoides polygyrus
Dujardin in mice than the natural product PF1022A. In particular an improved efficacy against
Haemonchus contortus Rudolphi and Trichostrongylus colubriformis Giles in sheep compared to the
potent cyclic octadepsipeptide PF1022A and its mono-thionated derivative has been observed. Here we
report on a specific modification at the N-methyl amide linkage by using the mono-thionated PF1022A,
resulting in novel anthelmintically active backbone analogues of PF 1022A.
# 2002 Society of Chemical Industry
Keywords: anthelmintic; N-methyl-amidoxime; cyclic octadepsipeptide; PF1022A; thionated depsipeptide;
Haemonchus contortus; Trichostrongylus colubriformis
1
INTRODUCTION
lated from Haemonchus contortus Rudolphi and thereby
induces influx of external calcium into cells.9 This
mode of action of an anti-nematodal drug is quite
different from the mechanism of action of the known
anthelmintics such as benzimidazoles, imidazothia-
zoles and macrocyclic lactones.10
PF1022A (Fig 1; 1), the active ingredient of the fungus
imperfectus Mycelia sterilia, is described as a powerful
broad-spectrum anthelmintic agent with low toxicity
in animals.1–4 This 24-membered cyclic octadepsi-
peptide was first isolated from natural sources by
Sasaki et al5 and prepared later through different total
syntheses.6–8 Recently it has been shown that
PF1022A binds to the amino terminus of a hepta-
helical, latrophilin-like transmembrane receptor iso-
The effects of structural modifications of PF1022A
on anthelmintic activity have been extensively studied
in order to increase the efficacy of the natural product
against parasites. However, the molecular design of
Figure 1. Structures of PF1022A (1)
and the mono-thionated PF1022A (2).
* Correspondence to: Peter Jeschke, Bayer AG, Bayer CropScience, Research Global Chemistry Insecticides, Alfred-Nobel-Str 50, D-40789
Monheim, Germany
E-mail: peter.jeschke@bayer-cropscience.com
(Received 11 February 2002; revised version received 9 May 2002; accepted 4 July 2002)
# 2002 Society of Chemical Industry. Pest Manag Sci 1526–498X/2002/$30.00
1205

P Jeschke et al
analogues has focused mainly on variations on the
a-amino acids or their N-alkyl substituents11 and on
the side chains of the a-hydroxy carboxylic acids.12,13
Conformationally restricted analogues of PF1022A
have been synthesized by fusing five-membered14 or
seven-membered lactam rings15 onto the macrocycle.
Beside aza-analogues of PF1022A in which the
a-carbons of the leucine residues are substituted by
nitrogen16 only limited attention has been paid to the
cyclic octadepsipeptide backbone. Recently, we have
described a useful method for the selective incorpora-
tion of one N-methylthioamide group into the back-
bone of PF1022A derivatives,17 resulting in eg the
mono-thionated PF1022A (Fig 1; 2). For each of the
resulting thionated analogues,18 the anthelmintic
activities against relevant intestinal nematodes in
livestock were found to be greater than those of the
parent cyclic octadepsipeptides. In the light of the
latter results, it has been suggested that the asym-
metric conformation influences the anthelmintic ac-
tivity of cyclic octadepsipeptides.14,18 Novel backbone
modifications with strong preference for an asym-
metric conformer of cyclic octadepsipeptides may give
valuable information about the conformational struc-
ture–activity relationships of PF1022A derivatives.
Isosteric replacement of an amide bond in a peptide
backbone by an imino peptide bond or an amidine
group has been reported, but the concept of N-
methylated amidoxime or N-methylated N’-hydroxy-
amidine analogues of cyclic depsipeptides has not been
described in the literature so far. Several methods are
known for the synthesis of N-substituted amidines.19
Such a specific backbone modification of chemotactic
peptides by exchanging an amide bond for the corre-
sponding amidoxime appears to have a strong influ-
ence on the conformation.20
column filled with Kromasil C18 (Macherey-Nagel
GmbH & Co KG, Du¨ren, Germany). For preparative
column chromatography silica gel 60-Merck
(40–63mm) was used. Octanol–water partition coeffi-
cients (logP) were measured by a HPLC method using
reverse phase columns, the general principles of which
have been described elsewhere.21,22
2.2 Chemicals
The natural product PF1022A (1) used for thionation
reactions was kindly provided by Meiji Seika Kaisha,
Ltd (Tokyo). Mono-thionated PF1022A (2) was
synthesized from 1 by a route described in the
literature,
using
2,4-bis(4-phenoxyphenyl)-2,4-
dithioxo-1,3,2,4-dithiadiphosphetane (Belleau’s re-
agent).17,18 Hydroxylamine and acyl halides were
obtained commercially. O-Substituted hydroxyl-
amines were obtained commercially or prepared by
either of two general procedures. In one procedure
O-substituted hydroxylamines were prepared by
O-alkylation from N-hydroxyphthalimide and alkyl/
arylalkyl halides followed by hydrazinolysis, or by
Mitsunobu reaction23 of the appropriate alcohol with
N-hydroxyphthalimide in the presence of triphenyl-
phosphine and diethyl azodicarboxylate (DEAD)
followed by hydrazinolysis.
2.3 Synthesis
Figure 2 depicts the general synthetic pathway to
N-methylated amidoxime analogues (general structure
I), which were prepared by a method reported in the
literature.24,25
Representative preparations of the N-methylated
amidoxime analogues (I) and their respective trans-
formations into derivatives are given below.
In this paper we report an extension of our study on
the thionation of cyclic octadepsipeptides,17,18 where-
in the N-methyl amide group of PF1022A has been
formally replaced by a N-methylated amidoxime
fragment. The synthesis of these derivatives was
undertaken not only as a part of the investigations of
structure–activity relationships but also to test the
feasibility of using the N-methylated thioamide group
in cyclic octathiodepsipeptides, such as 2, to synthesize
novel backbone analogues.
2.3.1 Cyclo[-N-methyl-L-leucinyl-D-(methoxy-
imino)lactyl-N-methyl-L-leucinyl-D-phenyllactyl-N-
methyl-L-leucinyl-D-lactyl-N-methyl-L-leucinyl-D-
phenyllactyl-] (4, general procedure for 3 and 5–22)
To a solution of (2)17 (200.0mg; 0.20mmol) in aceto-
nitrile (5ml) were added O-methyl-hydroxylamine
hydrochloride
0.31mmol), Hg(OAc)2 (72.5mg; 0.22mmol) and
N,N-diisopropylethylamine (DIEA) (107.4ml;
(MeONH2 ꢀHCl)
(26.2mg;
1.25mmol) and the mixture was stirred at room
temperature. After 18h the reaction mixture was
treated again with MeONH2ꢀHCl, (26.2mg;
2
EXPERIMENTAL
0.31mmol),
mercury(II)
acetate
(72.5mg;
2.1 General
0.22mmol), and DIEA (107.4ml; 1.25mmol). After
6h stirring at room temperature, saturated aqueous
ammonium chloride solution (20ml) was added
followed by extraction with chloroform (4ꢁ15ml).
The dried organic phase was evaporated and the
residue was chromatographed on silica gel using
cyclohexaneþacetone (4þ1 by volume). Yield:
170mg (85%); m/z: 978 [M]þ; 1H NMR major
conformer: d 0.79 (d, 3H), 0.86 (d, 3H), 0.86 (d,
3H), 0.86 (d, 3H), 0.86 (d, 3H), 0.88 (d, 3H), 0.94 (d,
3H), 1.03 (d, 3H), 1.03 (d, 3H), 1.45 (d, 3H), 1.46–
1H and 13C NMR spectra were recorded in deutero-
chloroform (CDCl3), deuteroacetonitrile (CD3CN),
deuteroacetone (acetone-d6) or deuterodimethyl sulf-
oxide (DMSO-d6) with tetramethylsilane as the inter-
nal standard, using a Bruker Avance 600 instrument
(600MHz). Liquid chromatography mass spectra
(LC–MS) were obtained on a VG/Fisons Platform 1
with APCI ionization. Reverse phase (RP) high
pressure liquid chromatography (HPLC) was per-
formed using a Hewlett-Packard HP 1090 with a
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Pest Manag Sci 58:1205–1215 (online: 2002)

Anthelmintically active N-methylated amidone analogues of PF1022A
1.76 (m, 1H), 1.46–1.76 (m, 2H), 2.85 (s, 3H), 2.87
(s, 3H), 2.90 (s, 3H), 3.04 (s, 3H), 3.06 (d, 2H), 3.09
(d, 2H), 3.15 (d, 2H), 3.15 (d, 2H), 3.64 (s, 3H), 4.40
(dd, 1H), 4.64 (dd, 1H), 5.08 (q, 1H), 5.20 (q, 1H),
5.36 (dd, 1H), 5.36 (dd, 1H), 5.52 (dd, 1H), 5.70 (dd,
1H), 7.23–7.32 (m, 10H); 13C NMR major con-
former: d 29.3, 30.2, 30.9, 31.0 (NCH3), 53.9, 56.8,
56.9, 59.5 (NCH), 61.3 (NOCH3), 66.9, 68.1, 69.7,
71.1 (OCH), 152.9 (C=N), 170.2, 170.3, 170.3,
170.7, 171.1, 172.5, 172.5 (C=O).
Figure 2. Synthetic routes to the novel N-methylated amidoxime analogues of PF1022A (I).
Pest Manag Sci 58:1205–1215 (online: 2002)
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P Jeschke et al
2.3.2 Cyclo[-N-methyl-L-leucinyl-D-(hydroxy-
imino)lactyl-N-methyl-L-leucinyl-D-phenyllactyl-N-
methyl-L-leucinyl-D-lactyl-N-methyl-L-leucinyl-D-
phenyllactyl-] (3)
0°C for 6h. The solvent was evaporated and the
residue was treated with chloroform (20ml). The
mixture was then extracted with hydrochloric acid
(1M) and twice with saturated sodium hydrogen
carbonate solution, and, after drying over anhydrous
magnesium sulfate, the solvent was removed under
vacuum. The residue was chromatographed on silica
gel using cyclohexaneþethyl acetate (1.5þ1 by
volume). Yield: 85mg (80%); m/z: 1036 [MþH]þ;
1H NMR (acetone-d6) major conformer: d 0.81 (d,
3H), 0.84 (d, 3H), 0.86 (d, 3H), 0.88 (d, 3H), 0.91 (d,
3H), 0.93 (d, 3H), 0.99 (d, 3H), 1.01 (d, 3H), 1.43 (d,
3H), 1.44 (d, 3H), 1.37–1.53 (m, 2H), 1.58–1.82 (m,
8H), 1.72–1.83 (m, 2H), 2.88 (s, 3H), 3.01 (s, 3H),
3.06 (m, 2H), 3.08 (s, 3H), 3.09 (s, 3H), 3.14 (m,
2H), 4.02 (dd, 1H), 5.14 (dd, 1H), 5.24 (dd, 1H),
5.29 (dd, 1H), 5.45 (q, 1H), 5.54 (dd, 1H), 5.56 (dd,
1H), 5.77 (q, 1H), 7.23–7.37 (m, 10H); 13C NMR
(acetone-d6) major conformer: d 14.4, 16.0, 16.9,
21.4, 21.5, 21.7, 22.5, 23.1, 23.2, 23.5, 23.6 (CH3),
25.1, 25.1, 25.3, 25.4 (CH), 31.3, 31.3, 31.7, 36.0
(NCH3), 37.8, 38.0 (CH2Ph), 37.4, 37.8, 37.9, 38.1
(CH2), 54.9, 55.0, 55.3, 57.4 (NCH), 64.6 (OCH2),
67.5, 68.9, 73.2, 74.8 (OCH), 127.6, 127.6, 129.2,
129.2, 130.4, 130.5 (Ph—C), 154.5 (C=N), 155.1,
168.7, 170.5, 171.0, 171.4, 171.5, 171.5, 171.7
(C=O).
Cyclo[-N-methyl-L-leucinyl-D-(benzyloxyimino)lac-
tyl-N-methyl-L-leucinyl-D-phenyllactyl-N-methyl-L-
leucinyl-D-lactyl-N-methyl-L-leucinyl-D-phenyllactyl-]
(12; 400.0mg; 0.37mmol) were hydrogenated in
methanol (40ml) in the presence of palladium-
charcoal [Pd content: 10% (200mg)] and concen-
trated hydrochloric acid (0.7ml) for approximately
20min. The reaction mixture was filtered and the
solvent was removed under vacuum. The residue was
chromatographed on RP-18 material using aceto-
nitrileþwater (8þ1 by volume). Yield: 110mg
(30%); m/z: 964 [M]þ; 13C NMR (DMSO-d6) major
conformer: d 153.7 (C=NOH).
2.3.3 Cyclo[-N-methyl-L-leucinyl-D-(acetoxyimino)-
lactyl-N-methyl-L-leucinyl-D-phenyllactyl-N-methyl-L-
leucinyl-D-lactyl-N-methyl-L-leucinyl-D-phenyllactyl-]
(23)
A mixture of 3 (200.0mg; 0.20mmol) in Ac2O
(1.0ml) was kept at 70°C for 30min. Following the
addition of saturated sodium hydrogen carbonate
solution (15ml) the mixture was extracted three times
with ethyl acetate (3ꢁ15ml). After drying over anhy-
drous magnesium sulfate the solvent was removed
under vacuum. The residue was first chromato-
graphed on silica gel using cyclohexaneþacetone
(10þ1 by volume) and then on RP-18 using
preparative HPLC. Yield: 80mg (35%); m/z: 1006
2.4 Biological assays
2.4.1 In vitro experiments
The larvae of the nematode Trichinella spiralis Owen
were isolated from skeletal muscles and diaphragms of
male mice of strain SPF/CFW1, 16–18g body weight
on receipt, and stored in sodium chloride solution
(9g litreꢂ1), supplemented with Canesten (20mg
mlꢂ1). Trichinella spiralis larvae were obtained from
pepsin-treated tissues from mice 50 days after injec-
tion. Twenty larvae per estimation were incubated in a
solution (2.0ml) containing Bacto Casitone, 20, yeast
extract 10, glucose 5, KH2PO4, 0.8, K2HPO4,
0.8g litreꢂ1, pH 7.2, supplemented with Sisomycin
(10mg mlꢂ1) and Canesten (1mg mlꢂ1). The test
compound (10mg) was dissolved in dimethylsulfoxide
(DMSO; 0.5ml) and added to the incubation medium
to give a final concentration of 100mg mlꢂ1. The
experiment was stopped after 5 days of incubation at
19°C. Activity was assessed on a scale of 0–3 where
3=full activity (all larvae dead); 2=good activity
(<100% but > 50% of the larvae dead); 1=weak
activity (>50% of larvae still alive) and 0=no activity
(number of living larvae equals that in the control).26
Adult nematodes (five males and females each) of
Nippostrongylus brasiliensis Lane were isolated from the
small intestine of female Wistar rats, stored in sodium
chloride solution (9g litreꢂ1), supplemented with
Sisomycin (20mg mlꢂ1) and Canesten (2mg mlꢂ1).
The incubation of each group of male or female worms
was performed in 1.0ml of medium, which was
collected for the estimation of the level of activity of
the acetylcholinesterase secreted by N brasiliensis.
1
[M]þ; H NMR (CD3CN) major conformer: d 0.78
(d, 3H), 0.82 (d, 3H), 0.82 (d, 3H), 0.84 (d, 3H), 0.89
(d, 3H), 0.90 (d, 3H), 0.94 (d, 3H), 0.96 (d, 3H), 1.35
(d, 3H), 1.41 (d, 3H), 1.49 (m, 1H), 1.51 (m, 1H),
1.53 (m, 1H), 1.55 (m, 1H), 1.55 (m, 1H), 1.57 (m,
1H), 1.58 (m, 1H), 1.59 (m, 1H), 1.60 (m, 1H), 1.69
(m, 1H), 1.69 (m, 1H), 1.70 (m, 1H), 1.96 (s, 3H),
2.81 (s, 3H), 2.88 (s, 3H), 2.94 (s, 3H), 2.97 (s, 3H),
3.00 (d, 1H), 3.05 (m, 2H), 3.07 (d, 1H), 3.92 (dd,
1H), 5.04 (dd, 1H), 5.17 (dd, 1H), 5.17 (dd, 1H),
5.34 (q, 1H), 5.46 (dd, 1H), 5.54 (dd, 1H), 5.67 (q,
1H), 7.21–7.32 (m, 10H); 13C NMR (CD3CN) major
conformer: d 16.6, 17.0 (CH3), 21.7, 22.6, 23.1, 23.5
(CH3), 25.5, 25.5, 25.7, 25.7 (CH), 31.7, 32.0, 32.0,
36.4 (NCH3), 38.0, 38.0, 38.0, 38.1 (CH2), 38.1,
38.1 (CH2Ph), 55.3, 55.3, 55.7, 57.6 (NCH), 68.0,
69.2, 72.3, 73.2 (OCH), 128.1, 129.4, 130.6
(Ph—C), 154.8 (C
171.7, 171.8, 171.9, 172.2 (C
=
N), 169.2, 171.1, 171.3, 171.3,
O).
=
2.3.4 Cyclo[-N-methyl-L-leucinyl-D-(ethoxycarbonyl-
oxyimino)lactyl-N-methyl-L-leucinyl-D-phenyllactyl-N-
methyl-L-leucinyl-D-lactyl-N-methyl-L-leucinyl-D-
phenyllactyl-] (24, general procedure for 25, 26, 27 and
28)
To a solution of 3 (105mg; 0.10mmol) in dry pyridine
(5ml) was added ethyl chloroformate (35.4mg;
0.32mmol) at 0°C and the mixture was stirred at
1208
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Anthelmintically active N-methylated amidone analogues of PF1022A
Details of the incubation and determination are
described by Rapson et al. 27 The level of activity of
the test compounds was assessed on a scale 0–3 where
3=full activity (95–100% enzyme inhibition); 2=
good activity (75–95% inhibition); 1=weak activity
(50–75% inhibition) and 0=negligible activity (<50%
inhibition).
press, were determined with the aid of a binocular
microscope (magnification ꢁ40). The activity against
the three nematodes was evaluated on a scale 0–3
where 3 represents cure (no parasites detectable), 2
effective (<20% of parasites remaining), 1 trace effect
(<50% of parasites remaining). Activity against the
tapeworm was also evaluated on a scale 0–3 where 3
represents cure (no parasite inclusive scolices detect-
able), 2 effective (some parasites expelled, tapeworm
strobila excreted), 1 trace effect (tapeworm strobila
excreted or macerated) and 0 ineffective (all parasites
remaining) (see Table 3).
2.4.2 Mixed-parasite infections in mice
For all experiments male mice of strain SPF/CFW1,
16–18g body weight on receipt, were used. Cages
constructed from Makrolon1 housed five animals
each. The mice were given water and ‘Sniff’ rat feed
13-mm pellets ad libitum. Mice received a mixed
inoculation with the tapeworms Hymenolepis nana
Siebold, and the nematodes Heligmosomoides polygyrus
(syn Nematospiroides dubius) Dujardin, Heterakis
spumosa Schneider and T spiralis.
2.4.3 Nematode infections in sheep
Sheep (Ovis aries L, Merino or Schwarzkopf breed,
25–35kg body weight) were infected experimentally
with 5000 Haemonchus contortus Rudolphi L3 and
treated with the test substance after the end of the pre-
potency period of the parasite. The test compounds
were administered orally in gelatine capsules. In the
case of Trichostrongylus colubriformis Giles sheep were
infected orally with 12000 L3, and similarly dosed
with test compound. Anthelmintic effects of the test
substances were measured as a function of the
reduction in the sheep faecal egg count. For the
purposes of counting eggs, freshly obtained faeces
from experimental animals were prepared using the
McMaster method as modified by Wetzel.30 The egg
counts were determined at regular intervals of 3 days
before and after treatment. The anthelmintic evalua-
tion was expressed as a function of the egg reduction
as follows: 3=>95%, 2=75–95%, 1=50–75% and
0= <50% egg reduction (see Table 4).
The infective material from H nana was collected
from mouse faeces 14–21 days post-infection (PI),
third-stage larvae (L3) of H polygyrus were collected
from mouse faeces 21 days PI, as described by Martin
et al,28 H spumosa eggs were obtained from female
worms isolated from the mouse colon 35–42 days PI
and were then incubated at 27°C for 3 weeks, and T
spiralis larvae were obtained from pepsin-treated
skeletal muscles and diaphragms of Wistar-W64 rats
20 days PI. The culturing methods are described in
Andrews et al29 and in the reference list therein. The
mixed-parasite infection was introduced into the mice
in a stepwise manner. They were first infected orally
with 90 embryonated H spumosa eggs, followed after 7
days with 100 T spiralis larvae; 27 days later, 60–70
larvae from H polygyrus were introduced and the final
infection, with 100 H nana eggs, followed after a
further 2 days. Test compounds (0.1g) were dissolved
or suspended in the emulsifier ‘Cremophor’ EI
(0.2ml). After addition of distilled water to give a
final volume of 5.0ml, a volume of between 0.1 and
0.5ml was orally applied per 20g mouse according to
the desired dose in mg kgꢂ1 body weight once daily for
4 consecutive days. One mouse received the highest
dose of 100mg kgꢂ1; if anthelmintic activity was
observed at this dose, lower dosages of 50 and
25mg kgꢂ1 were tested until anthelmintic activity
could no longer be detected. Treatment began on
day 46 PI and ended on day 49 PI. After a further 8
days the animals were killed with carbon dioxide and
then dissected. Hymenolepis nana were isolated from
the small intestine and the number of tapeworms was
estimated under a microscopically (magnification
ꢁ16). Heterakis spumosa were isolated from the
caecum and the colon, numbers also being determined
by microscopy. Removal of the duodenum and
dissection of this organ in a compressor, followed by
microscopic examination (magnification ꢁ40),
allowed the numbers of H polygyrus remaining to be
determined. The numbers of T spiralis present in about
1cm2 of abdominal muscle, removed by dissection and
compressed between two plastic sheets using a hand
3
RESULTS AND DISCUSSION
3.1 Chemistry
In this procedure, the high reactivity of the mono-
thionated PF 1022A (2) and of D-Lact1 thioamide was
used together with the high nucleophilicity of hy-
droxylamine derivatives. Thus, a desulfurization reac-
tion of 2 with O-substituted hydroxylamines in the
presence of mercury(II) acetate and/or mercury(II)
chloride and N, N-diisopropylethylamine (DIEA) in
acetonitrile afforded the N-methylated amidoxime
analogues (4–22).25 The N-methylated amidoxime
derivative 3 was prepared directly from the 2 and
hydroxylamine in the same manner as above or
alternatively was synthesized by simple removal of
the benzyl group (Bn) of the O-benzyl N-methyl-
amidoxime 12 by palladium-charcoal catalyzed hydro-
genation in the presence of hydrochloric acid. The
N-methylated amidoxime derivative 3 was allowed to
react with an excess of acetic acid anhydride at 70°C
forming the O-acetyl N-methylamidoxime 23 in 35%
yield. All further O-acylations of the hydroxyimino
group were carried out with three equivalents of the
corresponding alkyl or alkenyl chloroformates in dry
pyridine at low temperature to give the O-acylated
N-methylamidoxime derivatives (24–27).
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P Jeschke et al
Table 1A. N-Methylated amidoxime analogues of PF1022A (I)
Structure
Molecular
No
R
Formula
Relative mass
Yield (%)
3
4
5
6
7
H
C52H77N5O12
C53H79N5O12
C54H81N5O12
C54H81N5O13
964.2
978.2
30
85
49
58
65
9
CH3
C2H5
992.2
CH2CH2OH
3-CF3-Phenoxyethyl
CH(CH3)2
1008.2
1152.3
1006.3
1004.2
1091.3
1075.3
1054.3
1088.7
1122.3
1072.3
1123.2
1106.7
1048.3
1044.3
1055.3
1089.7
1003.2
1006.2
1036.2
1034.2
1048.3
1064.3
1042.3
C61H84F3N5O13
C55H83N5O12
C55H81N5O12
C58H86N6O14
C58H86N6O13
C59H83N5O12
C59H82ClN5O12
C60H82F3N5O12
C59H82FN5O12
C59H81Cl2N5O12
8
9
CH2CH=CH2
CH2COMora
CH2COPyrb
Benzyl
36
28
76
15
72
32
66
37
15
81
20
17
77
24
35
80
59
17
52
38
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3-Cl-Benzyl
3-CF3-Benzyl
4-F-Benzyl
2,4-Cl2-Benzyl
2-Cl, 6-F-Benzyl
(S)-Tetrahydrofur-2-yl-methyl
Fur-2-yl-methyl
Pyrid-2-yl-methyl
2-Cl-Pyrid-5-yl-methyl
CH2CN
C59H81ClFN5O12
C57H85N5O13
C57H81N5O13
C58H82N6O12
C58H81ClN6O12
C54H78N6O12
C54H79N5O13
C55H81N5O14
C55H79N5O14
C56H81N5O14
C57H85N5O14
COCH3
COOC2H5
COOCH=CH2
COOCH2CH=CH2
COOCH(CH3)C2H5
SO2CH3
C53H79N5O14
S
a
N-Morpholino-carbonylmethyl=
.
b
N-Pyrrolidino-carbonylmethyl=
.
Methanesulfonylation of 3 to the corresponding O-
methylsulfonyl N-methylamidoxime 28 was easily
accomplished using three equivalents of methanesul-
fonyl chloride at 0°C. The oxime analogues (Fig 2; I)
were isolated in yields ranging from 9% to 85% after
purification by preparative column chromatography.
The N-methylated amidoxime analogues (I)
showed two to four sets of resonances (major and
minor sets) in their 1H NMR spectra. In compound 4,
for example, the ratio between these four sets of signals
was about 5:1.5:1.5:1 without a dynamic equilibration
at 298K in CDCl3 solution, thus showing that the
presence of (E/Z)-isomers and/or very stable confor-
mers without dynamic inter-conversion in solution.
Further spectroscopic analysis of the main conformer
using combination of two-dimensional NMR
a
(1H–1H-TOCSY, H–1H-ROESY, H–13C-HMQC,
1H–13C-HMBC) techniques in CDCl3 solution
showed only the asymmetric conformation, containing
a cis-amide bond between D-lactic acid (D-Lac5) and
MeLeu6. The cis-amide bond can be identified in the
ROESY NMR spectra by observing the ROE contacts
between Ca protons of neighbouring MeLeu/D-Lac
units. Compared with the Ca protons of MeLeu2,4,8 (d
4.64–5.36) the Ca protons of MeLeu6 resonates at a
higher field strength (d 4.40). However, analogues 23
and 24, carrying an O-acetyl group or an O-ethoxy-
carbonyl group instead of the O-methyl group,
1
1
1210
Pest Manag Sci 58:1205–1215 (online: 2002)

Anthelmintically active N-methylated amidone analogues of PF1022A
Table 1B. Analytical and spectroscopic data of the N-methylated amidoxime analogues of PF1022A (I)
HPLC b
Purity (ratio) c (%)
93.5 (20/80)
100
APCI-MS a
No [MþH]þ
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Rt (min)
[1H]NMR for =N—O—Z—CH—R’ d (ppm)
Z
e
964
978
17.54; 17.66d
18.34
20.05g
—
3.64 (3H, s)f
—
—
j
993
—
3.98 (1H, m), 3.88 (1H, m)f
3.94 (2H, t)h
—
1008
1152
1006
1005
1091
1076
1055
1088
1123
1072
1124
1106
1049
1044
1055
1089
1003
1006
1036
1037
1048
1064
1042
17.58
90.6
94.5
—
19.80
4.32 (2H, s)h
—
13.54
20.21, 20.31g
100
j
—
4.47 (1H, broad)i
—
4.37 (2H, dd)f,k
—
17.64
92.9
4.74 (1H, d), 4.40 (1H, d)f
4.56 (1H, d), 4.39 (1H, d)f
5.02 (1H, d), 4.94 (1H, d)f
—
17.95
21.23, 21.50g
19.61, 19.78d
97.8
j
—
—
—
99.1 (41.5/58.5) 4.93 (2H, m); 4.71 (1H, d), 5.06 (1H, d)h,l
19.50, 19.68d 100 (43.1/56.9)
—
5.03 (2H, m); 5.16 (1H, d), 4.80 (1H, d)h,l
4.98 (1H, d), 4.77 (1H, d)f
5.02 (2H, s), 4.99 (2H, dd)h,l
4.97 (2H, s)i
—
19.48
20.68, 21.16d
99.1
—
99.9 (56/44)
19.24, 19.44d 100 (70.8/29.2)
—
—
j
j
19.94g
—
—
4.76 (1H, d), 4.54 (1H, d)i
—
18.73
6.46, 6.58m
18.87
7.39, 7.49n
7.35n
100
—
98.8 (58.6/41.4) 5.08 (2H, broad)f
—
100
5.02 (1H, d), 4.78 (1H, d); 4.83 (1H, d), 4.72 (1H, d); 4.94 (2H, m)h,l
4.55 (1H, d), 4.15 (1H, d); 4.78 (1H, d), 4.32 (1H, d); 4.50 (2H, m)i,l
—
97.5 (6.5/93.5)
—
98.7
95.5
93.6
95.0
95.4
1.96 (3H, s)i
4.08 (2H, q)h
6.96 (1H, dd)i
4.53 (2H, ddd)i
4.58 (1H, m)h
2.84 (3H, s)i
CO
CO
CO
CO
CO
SO2
17.69
17.82
17.83
18.28
17.18
100
a Loop or LC-coupling, m/z for quasi-molecule ions related to the main isotopes of the elements.
b
Conditions for analysis: column 125ꢁ4.0mm Kromasil 100 C18, 5mm; eluent, A=0.1% phosphoric acid, B=acetonitrile; gradient, 10% B (1min), 10–95% B
(17min), 95% B (6min); flow rate, 1.5ml minꢂ1; detection, UV 210nm.
c Ratio of geometrical (E/Z)-isomers around the C=N bond.
d Retention times of two conformers.
e 13C NMR (DMSO-d6): d 153.7 (C=N—OH)ppm.
f
CDCl3.
g
Conditions for analysis: column 125ꢁ2.1mm Kromasil 100 C18, 5mm; temperature 40°C; eluent, A=0.1% formic acid in water, B=0.08% formic acid in
acetonitrile; gradient, 10% B (1min), 10–95% B (17min), 95% B (6min); flow rate, 0.5ml minꢂ1; detection, mass spectrometer; micromass platform II, APCl ion
source.
h Acetone-d6.
i CD3CN.
j Not determined.
k Mixture of four conformers in a ratio of 2:2:1:1; 13C NMR (CDCl3): d 74.7 (=N—O—CH2—), 158.0 (C=N—O—)ppm.
l Signals of a mixture of conformers in solution.
m Conditions for analysis: column 75ꢁ4.6mm Kromasil 100 C18, 3.5mm; eluent, A=0.1% phosphoric acid, B=acetonitrile; gradient, 10% B (0.8min), 10–95% B
(6.73min), 95% B (1.57min); flow rate, 3.0ml minꢂ1; detection, UV 210nm.
n Conditions for analysis: column 75ꢁ4.6mm Kromasil 100 C18, 3.5mm; eluent, A=water, B=acetonitrile; gradient, 10% B (0.8min), 10–95% B (6.73min), 95% B
(1.57min); flow rate, 3.0ml minꢂ1; detection, UV 210nm.
exhibited two sets of signals in CD3CN with a ratio of
83:17 or in acetone-d6 solution with a ratio of 85:15.
Analogue 24, dissolved in acetone-d6, exists in two
conformations that are in dynamic equilibrium with
one another, as can be deduced from the correspond-
ing exchange cross peaks in its NOESY spectrum.
Further spectroscopic analysis showed in each case for
the minor conformer the asymmetric conformation,
containing two cis-amide bonds between the oxime of
D-lactic acid (D-Lac1) and MeLeu2 as well as between
D-Lac5 and MeLeu6. These observations suggest that
the number of conformers and the presence of cis-
amide bonds in solution depends strongly on the
nature of the substituent R in the N-methylated
amidoxime moiety of the analogues (I). When the
acyl substituent R, eg in the compounds 23 and 24, is
changed to an alkyl group, as in 4, a change in
flexibility of the PF1022A derivatives occurs. In other
words, the larger steric size of the substituted
O-carbonyl N-methylamidoxime moiety in 23 and 24
generally leads to an increased population of the
symmetric conformers compared to the asymmetric
conformers containing cis-amide bond(s).
In the same solvent, mono-thionated PF1022A (2)
showed a mixture of two asymmetric conformers, at a
ratio of 2:1,18 whereas PF1022A (1) exhibited a
mixture of the asymmetric and symmetric conformers,
at a ratio of 3:1.14
Pest Manag Sci 58:1205–1215 (online: 2002)
1211

P Jeschke et al
Table 2. Anthelmintic activities in vitro of the N-methylated amidoxime
analogues (I) in comparison with PF1022A (1), mebendazol and ivermectin
spectra, which resulted in the uncertainty of ꢃ0.3ppm
due to the lack of digital points in the f1-dimension.
The structural assigments of the macrocycles (I) were
based on the quasimolecule ion peaks [MþH]þ in the
LC mass spectra and characteristic resonances in the
Anthelmintic activity in vitro at 100mg mlꢂ1
Compound no
T spiralis
N brasiliensis
PF1022Aa
Mebendazold
Ivermectind
3b,c
2b
0b
0
3b,c
3b
3b
3
1
1H NMR spectra assignable of the H chemical shifts
of the incorporated [=N—O—R] fragment.
The relevant NMR (1H chemical shifts of
=
=
N—O—CH-R’,
=N—O—CO—CH-R’,
4
6
2
2
N—O—SO2—CH3 or 13C chemical shifts of
0
2
C
=
N—O—R) and physical characteristics of the
7
0
0
synthesized N-methylated amidoxime analogues (I)
are summarized in Table 1A and B. From the data
available, the question as to the presence of geome-
trical (E/Z)-isomers around the amidine C=N bond
for the N-methylated amidoxime analogues (I) cannot
be answered in detail.
10
11
13
14
16
17
20
21
22
23
24
25
27
28
2
2
2
1
0
0
0
0
0
2
0
0
1
0
1
0
3.2 Anthelmintic activity
Several in vitro studies revealed that PF1022A was very
0
1
0
1
active against T spiralis and N brasiliensis at 1mg
0
0
ml .
ꢂ1 28 In contrast, the new N-methylated amidoxime
3
0
derivatives (I) exerted full or only slight anthelmintic
activity against either nematode at the high concentra-
tion of 100mg mlꢂ1 (Table 2).
0
1
2
1
a Results taken from Reference 28.
b Concentration: 1mg mlꢂ1
c 0=no activity (number of living larvae equals that in the control)26 1=weak
activity (>50% of larvae still alive); 2=good activity (<100% but >50% of the
larvae dead); 3=full activity (all larvae dead).
In a further step we examined the anthelmintic
activities of the N-methylated amidoxime analogues
(I) in vivo using the mixed-parasite infection in mice.
PF1022A showed full anthelmintic efficacy at the oral
dose-rate of 50mg kgꢂ1 against H spumosa 28 and a
good activity against H polygyrus, but there was no
detectable effect against the tapeworm H nana 27
(Table 3).
.
d Commercial product; results taken from Reference 31.
The 13C chemical shifts were measured from
heteronuclear multiple quantum correlation (HMQC)
and heteronuclear multiple bond correlation (HMBC)
Two cyclic depsipeptides (14 and 23) displayed
Anthelmintic activity in mice
Compound no
H nana
100b/0c
H spumosa
H polygyrus
T spiralis larvae
PF1022Aa
50/3
10/3
25/2
100/0
0.5/2
25/3
100/0
100/3
2.5/0
25/0
Mebendazold
—
e
Ivermectind
—
0.25/2
25/2
e
3
4
25/0
50/3
50/0
50/0
50/0
5
7
9
100/3
100/2
50/3
100/1
100/1
50/0
100/0
100/1
50/1
100/0
100/0
50/0
11
13
14
17
18
21
23
24
26
100/3
100/3
50/0
100/0
100/2
50/3
100/0
100/0
50/0
100/0
100/0
50/0
50/3
50/1
50/0
50/0
100/3
100/3
50/0
100/0
100/1
50/3
100/1
100/0
50/0
100/0
100/0
50/0
100/0
100/3
100/1
100/0
100/0
100/0
100/0
100/0
a
Results taken from Reference 28.
b Dose in mg test substance kgꢂ1 body weight.
c
0=<50% egg reduction; 1=50–75% egg reduction; 2=75–95% egg reduction; 3=>95% egg
reduction.
d Commercial product; results taken from Reference 31.
Table 3. Anthelmintic activities of the N-methylated
amidoxime analogues (I) in comparison with
PF1022A (1), mebendazol and ivermectin
e
Not determined.
1212
Pest Manag Sci 58:1205–1215 (online: 2002)

Anthelmintically active N-methylated amidone analogues of PF1022A
anthelmintic activity against H spumosa (full activity at
50mg kgꢂ1) comparable to PF1022A (1), whereas the
N-methylated amidoxime 3 showed activity against H
polygyrus (full activity at 25mg kgꢂ1). The N-methyl-
amidoxime 3 was found to be more active against H
spumosa and H polygyrus at 25mg kgꢂ1 than the parent
nematode species. However, the introduction of an
allyloxycarbonyl group (25) as an alternative to the
ethoxycarbonyl group drastically reduced the anthel-
mintic efficacy. Nonetheless, the activity of the cyclic
octadepsipeptide 27, containing the branched, race-
mic (R,S)-sec-butyloxycarbonyl group at the N-
methylamidoxime fragment, was somewhat lower. A
comparison of the synthesized N-methylamidoximes
(I) containing different [=N-O-R]-fragments revealed
a tendency for the anthelmintic activity against the
abomasal nematode H contortus in sheep related to the
substituent R in the following order:
cyclooctadepsipeptide 1. Furthermore,
a limited
number of N-methylated amidoxime analogues
showed anthelmintic activity against the tapeworm H
nana in the gut (4, 9 and 17, full activity at 50mg
kgꢂ1). In sheep, N-methylated amidoxime analogues
(I) also displayed anthelmintic activity against the
sheep nematodes H contortus and but only compound
24 against T colubriformis (Table 4).
CO
CO
O
O
C2H5; CH3 > H > Fur-2-yl CH2 >
All the N-methylamidoxime derivatives (I) tested in
vivo in the mouse model were found to be fully active
against the abomasal sheep nematode H contortus at
HC¼CH2; ðR;SÞ-CO OCHðCH3ÞC2H5:
In summary, the N-methylated amidoxime 3, which
has a logP value comparable with that of the parent
compound PF1022A, as well as the more lipophilic
derivatives, 4 and 24, represent the most anthelminti-
cally active compounds in sheep. Finally, the marked
increase in anthelmintic activity against the nematode
H contortus in sheep of both derivatives 4 and 24 can be
explained on the basis of their bioactive asymmetric
conformation, containing a cis-amide bond. This
result is in agreement with previous findings.18
0.10mg kgꢂ1 and up to 0.01mg kgꢂ1
.
3.3 Structure–activity relationships
Replacement of one N-methylamide group in
PF1022A (1) with a N-methylamidoxime group, as
in 3 (logP =5.86), increased the anthelmintic activity
compared to both PF1022A (logP =5.84) and its
mono-thionated derivative (2) (logP =6.19).18 The
introduction of alkyl groups as substituents R in the
backbone N-methylamidoxime moiety in the com-
pounds (I), such as methyl (4), resulted in 25-fold
increase activity against H contortus in sheep compared
with the parent compound PF1022A. When the
substituent was heterocyclic and increased in size, as
in 19 (logP =7.05), the spectrum of anthelmintic
4
CONCLUSIONS
Several new N-methylated amidoxime analogues (I)
were synthesized using the mono-thionated PF1022A
(2) as a precursor. The N-methylated amidoxime
analogues (I) represent novel cyclic octadepsipeptide
derivatives of the anthelmintic agent PF1022A (1)
with improved activity in livestock animals. Although a
significant increase in lipophilicity should be critical
with respect to the bioavailability of cyclic octadepsi-
activity tended to be reduced compared to
4
(logP =6.73). In the series of the lipophilic O-acylated
N-methylamidoximes 24 (logP =6.23), 25 (logP =
6.32) and 27 (logP =6.67), the ethoxycarbonyl group
24 gave the highest ratings against both intestinal
Anthelmintic activity in sheep
Compound no
Log P a
H contortus
T colubriformis
0.25/0
PF1022A
5.84
0.25b/3c
0.01/0
0.10/3
20/3
d
—
mono-thionated PF1022Ae
6.19
d
—
—
d
Mebendazolf
Ivermectinf
3
20/3
d
—
0.20/3
0.10/3
0.01/2
0.01/3
0.10/3
0.01/3
0.10/2
0.10/2
0.20/3
d
—
5.86
d
—
d
4
19
24
25
27
6.73
7.05
6.23
6.32
6.67
—
d
—
0.10/3
d
—
d
—
a
LogP from HPLC (see Section 2.1).
b Dose in mg test substance kgꢂ1 body weight.
c
0=<50% egg reduction; 1=50–75% egg reduction; 2=75–95% egg reduction; 3=>95% egg
reduction.
d Not determined.
Table 4. Anthelmintic activities and lipophilicities of
the N-methylated amidoxime analogues (I) in
comparison with PF1022A (1), mono-thionated
PF1022A (2), mebendazol and ivermectin
e
Results taken from Reference 18.
f
Commercial product; results taken from Reference 32.
Pest Manag Sci 58:1205–1215 (online: 2002)
1213

P Jeschke et al
peptides,11 in the present study the more lipophilic
N-methylated amidoxime derivatives, 4 and 24,
proved more potent in their efficacy against the
10 Harder A and von Samson-Himmelstjerna G, Cyclooctadepsi-
peptides – a new class of anthelmintically active compounds,
Parasitol Res 88:481–488 (2002). DOI: 10.1007/s00436-002-
0619-2 (online: 2002).
abomasal nematode
H contortus in sheep than
11 Scherkenbeck J, Harder A, Plant A and Dyker H, PF1022A—a
novel anthelmintic cyclooctadepsipeptide. Modification and
exchange of the N-methyl leucine residue. Bioorg Med Chem
Lett 8:1035–1040 (1998).
PF1022A. Furthermore, depending on substitution
of the backbone N-methylamidoxime oxygen in (I),
anthelmintic activity can be increased. Some of the
anthelmintically active N-methylamidoxime analogues
(I) exist in CDCl3, CD3CN or acetone-d6 solution as a
mixture of symmetric and asymmetric conformers in
different ratios, containing a cis-amide bond. Forma-
tion of the bioactive asymmetric conformers and
increased activity of these N-methylamidoxime ana-
logues may be attributable to the substituents R (eg
steric factors) on the new N-methylamidoxime moiety
of the compound. In addition, the asymmetric con-
formation caused by isosteric replacement of a
N-methylamide bond with a N-methylamidoxime
enhances the cis-amide bond between D-Lac5 and
L-MeLeu,6 respectively.
12 Sakanaka O, Okada Y, Ohyama M, Matsumoto M, Takahashi
M, Murai Y, Iinuma K, Harder A and Mencke N, Derivatives
of cyclodepsipeptide, PF1022 substance (Bayer AG, Meiji
Seika Kaisha, Ltd), PCT Application 9 711 064 (1997).
13 Sakanaka O, Okada Y, Ohyama M, Takahashi M, Suzuki K,
Murai Y, Iinuma K, Harder A, Mencke N, Jeschke P and
Bonse G, Preparation of cyclodepsipeptide PF1022A deriva-
tives as anthelmintics (Meiji Seika Kaisha Ltd, Bayer AG),
PCT Application 9 837 088 (1998).
14 Scherkenbeck J, Dyker H, Gondol D, Harder A, Plant A and
Reichel F, Synthesis, conformational studies and anthelmintic
activity of a constrained PF1022A analogue. Pestic Sci 55:457–
461 (1999).
15 Dutton FE and Lee BH, Epsilon-lactam analogs of the
anthelmintic cyclodepsipeptide PF1022A. Tetrahedron Lett
39:5313–5316 (1998).
Mode of action and tolerance studies have not yet
been completed, but studies with PF1022A suggest
that the mechanism of action might rely on inter-
ference with a latrophilin-like receptor.9
16 Dyker H, Scherkenbeck J, Gondol D, Goehrt A and Harder A,
Azadepsipeptides: synthesis and evaluation of a novel class of
peptidomimetics. J Org Chem 66:3760–3766 (2001).
17 Jeschke P, Harder A, von Samson-Himmelstjerna G, Mencke N,
Bonse G, Iinuma K and Sakanaka O, Thiodepsipeptides for
combating endoparasites and a method for producing the same
(Bayer AG), PCT Application 9 843 965 (1998).
ACKNOWLEDGEMENTS
18 Jeschke P, Harder A, Etzel W, Gau W, Thielking G, Bonse G and
Iinuma K, Synthesis and anthelmintic activity of thioamide
analogues of cyclic octadepsipeptides such as PF1022A. Pest
Manag Sci 57:1000–1006 (2001).
The authors are very grateful to Rolf Steffes (chem-
istry), Rolf Suether and Brigitte Sto¨ppler (biology) for
skilful technical assistance.
19 Bushey DF and Hoover FC, Synthesis and stereochemistry of
amidoximes. J Org Chem 45:4198–4206 (1980).
20 Rao VS, Sauve G, Lajoie G and Belleau B, The solution
conformation of chemotactic peptides and their backbone-
modified analogs. Nuclear magnetic resonance and computer-
modeling studies with active amidoxime analogs of formyl-
methionyl-leucyl-phenylalanine. Spectroscopy (Ottawa) 4:153–
170 (1985).
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