Chimeric cyclodepsipeptides as mimetics for the anthelmintic PF1022A

Article abstract of DOI:10.1016/j.bmcl.2004.07.097

In the anthelmintic cyclooctadepsipeptide PF1022A didepsipeptide units have been exchanged for the β-turn mimics (d)-Pro-(l)-Pro and BTD in order to elucidate the functional role of the depsipeptide backbone. Analogues have been identified, which show an improved anthelmintical activity compared to the natural product. Preliminary structure-activity relationships suggest a symmetrical conformation to be the biological active one. In the anthelmintic cyclooctadepsipeptide PF1022A (1) didepsipeptide units have been exchanged for the β-turn mimetics (d)-Pro-(l)-Pro and BTD (7) in order to elucidate the functional role of the depsipeptide backbone. Compounds 12 and 14 are the first PF1022A analogues in which a substantial part of the PF1022A backbone has been replaced with an improvement of anthelmintical activity. Preliminary structure-activity relationships suggest a symmetric conformation to be the biological active one.

Full text of DOI:10.1016/j.bmcl.2004.07.097

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6129–6132
Chimeric cyclodepsipeptides as mimetics for
the anthelmintic PF1022A
Hubert Dyker,a Achim Harderb and Jurgen Scherkenbeckc,*
¨
aBayer Healthcare Pharma, 42096 Wuppertal, Germany
bBayer Healthcare Animal Health, 40789 Monheim, Germany
cBayer CropScience, 40789 Monheim, Germany
Received 10 May 2004; revised 21 July 2004; accepted 27 July 2004
Available online 18 October 2004
Abstract—In the anthelmintic cyclooctadepsipeptide PF1022A (1) didepsipeptide units have been exchanged for the b-turn mimetics
(D)-Pro-(L)-Pro and BTD (7) in order to elucidate the functional role of the depsipeptide backbone. Compounds 12 and 14 are the
first PF1022A analogues in which a substantial part of the PF1022A backbone has been replaced with an improvement of anthel-
mintical activity. Preliminary structure–activity relationships suggest a symmetric conformation to be the biological active one.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
leucine for proline or proline related cyclic amino acids
have been reported.11 A common strategy to restrict
the number of conformationsor to stabilize a particular
secondary structural element in a peptide is to replace
certain partsof the peptide for rigid, cyclic amino acids
or turn mimetics. In this paper we wish to outline the
Ôconformation-guidedÕ replacement of didepsipeptide
Nematode infectionsare a major cause of human mor-
bidity and mortality in the tropicsaswell asin temper-
ate climates.1 In animal health, nematode infectionsplay
a crucial role in cattle and sheep and are responsible for
enormous economic losses.2 Since the discovery of the
highly active anthelmintic macrolidesmilbemycin and
avermectin in the early seventies, reports on potent
new classes of anthelmintics have been scarce.3 One of
the most outstanding recently reported anthelmintic is
the cyclooctadepsipeptide PF1022A (1), the most active
member of a novel class of anthelminitic agents.4 During
the past years several total syntheses5 of PF1022A and
manifold structure–activity relationships have been
established.6 Additionally, the biosynthesis of
R3
O
O
O
N
N
O
O
R2
R4
O
O
O
7
PF1022A hasbeen elucidated and intensive investiga-
tionsinto the mode of action of thisnovel anthelmintic
are underway.8
O
N
N
O
O
R1
O
Most of the published PF1022A modifications refer to
side-chain alterations of the amino and hydroxycarb-
oxylic acids.9 Until now only a limited number of
R1 R2
PF1022A (1) Me Bzl
R3 R4
backbone modificationsusch asthe exchange of
N-
methylleucine for aza-leucine10 or the replacement of a
Me Bzl
Bzl Bzl
Me Bzl
Me Bzl
PF1022B
PF1022C
PF1022D
PF1022E
Bzl Bzl
Bzl Bzl
Me Me
Keywords: Anthelmintic; Cyclooctadepsipeptide; PF1022A; Turn
mimetic.
Me p-OH-Bzl Me Bzl
Bassianolide iPr iPr
iPr iPr
*
Corresponding author. Tel.: +49 2173 384688; fax: +49 2173
383342; e-mail: juergen.scherkenbeck@bayercropscience.com
Figure 1.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.097

6130
H. Dyker et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6129–6132
Figure 2.
unitsfor the b-turn mimetics( D)-Pro-(L)-Pro and the
bicyclic ÔNagai–SatoÕ mimetic (BTD, 2) (Fig. 1).
The structure of PF1022A has recently been determined
by single-crystal X-ray analysis.12 Despite the occur-
rence of different space groups, structures determined
from crystals obtained from methanol, or acetone were
nearly identical. Both structures showed one cis amide
bond between (L)-MeLeu2 and (D)-Lac3, conferring
asymmetry on the molecule. However, in solution
PF1022A exists as a 3:1 mixture of two conformers,
which interconvert very slowly on the NMR time
scale.13 The main isomer corresponds to an asymmetric
conformer, while the minor isomer can be assigned to a
symmetric conformer with all four amide bonds in the
trans configuration. MD simulations in a H2O solvent
cage using NMR derived NOEÕsasconstraintsrevealed
two type II0 b-turnsin the ysmmetric conformation,
containing a (D)-PhLac- and a N-methyl-(L)-leucine resi-
due in the central i + 1 and i + 2 positions. The two
b-loopsinduce a hydrophobic collapes of the two
(D)-PhLac-(L)-MeLeu residues and the side chains of
MeLeu4 and MeLeu8, which significantly stabilizes the
symmetric conformation. The cis amide bondsbetween
Figure 3.
phenyllactic acid and N-methyl-(L)-leucine according to
standard procedures.17 Hexadepsipeptide 4 wascoupled
with 2 using BOP-Cl as the coupling reagent of choice for
N-methyl substituted amino acids.18 Removal of both
t
the Boc- and butyl-group with TFA provided the bicy-
clic linear PF1022 precursor 6 in 80% yield. Among sev-
eral coupling reagentstetsed PyBrop wasfound to
produce the highest yield (27%) of 9 in the entropically
critical macrocylization reaction.19 Formation of the ester
bond between 2 and 3 proved to be difficult. Best yields
of 5 were obtained with EDC/DMAP. Addition of HOBt
did not improve the situation and a two step active ester
coupling sequence using the trichlorophenyl ester failed
completely. Simultaneousdeprotection with TFA and
cyclization with BOP afforded the tricyclic PF1022 ana-
logue 7 in sufficient yield.
(D)-Lac3 and (L)-MeLeu4 in the asymmetric conforma-
2
tion forcesthe isde chainsof MeLeu
and MeLeu4
together, resulting in a NOE between MeLeu2Ha
and MeLeu4CH3Hd. An analogousNOE between
MeLeu8Ha and MeLeu6CH3d demonstrates that the
MeLeu6 side chain folds into the macrocyclic ring
system (Fig. 2).
Similarly to the rigid BTD-mimetic the linear (D)-Pro-
(L)-Pro also shows a strong b-turn stabilizing effect. 8
was synthesized analogously to 7 by coupling 3 with
the commercially available (D)-Pro-(L)-Pro, followed
by protecting group removal and macrocyclization
(Scheme 1).
2. Synthesis
The Nagai–Sato bicyclic dipeptide mimetic (BTD, 2)
was chosen because it shows an almost superimpossible
conformation on that of (D)-Phe-(L)-Pro residues in gra-
micidin S, which isknown to be a type II b-turn.14
0
Additionally, 2 has been used successfully by Alberg
and Schreiber15 asa rigidifying turn mimetic for
cyclosporin and by Bartlett and co-workers16 for a
structural mimetic of tendamistat, a 74-residue proteina-
ceousinhibitor of a-amylase (Fig. 3).
3. Discussion
The chimeric PF1022A analogues 7 and 8 have the turn
mimic in the favourable i + 1, i + 2 position while in
analogue 9 the BTD isshifted to the i, i + 1 position.20
NMR data together with MD simulations revealed that
all compoundsexist in only one conformation, and thus
confirm the conformation-stabilizing effect of the (D)-
Pro-(L)-Pro and the BTD turn mimetic. In particular
The synthesis of 2 followed the route developed by
Bartlett16 and Schreiber.15 The two linear hexadepsipep-
tides 3 and 4 were synthesized from (D)-lactic acid, (D)-

H. Dyker et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6129–6132
6131
O
O
O
O
O
O
tBuO
N
N
N
N
N
N
O
O
O
H
tBuO
O
O
OH
O
O
O
O
O
O
4
3
1. 7, BOPCl, iPr2NEt, CH2Cl2
1. 7, EDC, DMAP, CH2Cl2,
rt, 12h, 37%
rt, 6h, 65%
2. TFA, CH2Cl2
rt, 2h, 85%
2. TFA, CH2Cl2
rt, 2h, 80%
NH2
O
O
O
O
O
O
O
O
O
N
HO
N
N
N
O
O
O
N
O
N
N
N
HO
O
O
O
O
O
O
NH2
H
S
O
O
O
S
5
6
H
BOP, iPr2NEt, CH2Cl2,
rt, 24h, 20%
PyBrop, iPr2NEt, CH2Cl2,
rt, 24h, 27%
O
O
O
O
O
O
O
O
O
NH
N
O
O
O
N
N
O
O
N
N
O
O
O
Ph
Ph
Ph
N
O
O
S
N
O
O
O
HN
S
O
O
O
O
O
O
O
O
N
N
N
O
N
N
N
O
O
7
8
9
Scheme 1.
the conformation of 8 resembles the symmetric conform-
ation of PF1022A indicating that (D)-Pro-(L)-Pro is
an adequate substitute for the (D)-PhLac-(L)-Leu unit
in PF1022A.
The synthetic compounds 7–9 were tested exclusively in
an animal test model, which provides a more realistic
picture of the anthelmintic potency compared to in vitro
screenings. In order to minimize animal usage in this
first screening stage anthelmintic activity was deter-
mined only against the sensitive helminth Haemonchus
contortus.21 Thus, sheep (Merino or Schwarzkopf breed,
25–35kg body weight) were infected experimentally with
5000 H. contortus L3 larvae and treated with the test
substance after the end of the pre-patency period of
the parasite.22 The test compounds were administered
orally in gelatine capsules or intravenously, as recently
described.23 Anthelmintic effects of the test substances
against H. contortus adultswere measured asa function
of the reduction in faecal egg count. For the purpose of
counting eggs, freshly obtained faeces from experimen-
tal animals were prepared using the McMaster method
asmodified by Wetzel and the egg count wascalculated
per gram of faeces.24 The egg countswere determined at
regular intervalsbefore and after treatment ( Fig. 4).
Figure 4.
Table 1.
Compound Dosage Eggsper gram of faeces
(mg/kg)
Reduction of
eggsper gram
of faecesafter
treatment (%)
Before
treatment
After
treatment
( SE)
Controla
0
0.1
6667
6142 ( 364)
0
8
100
43
95
98
59
9
1
1833 ( 71)
1000 ( 189)
1800 ( 235)
2500 ( 118)
3268 ( 1)
0.01
0.01
0.01
0.025
0.01
573 ( 182)
83 ( 21)
50 ( 8)
7
8
9
1345 ( 272)
1800 ( 141) 1638 ( 123)
Analogues 7 and 8 containing the mimeticsin the centre
of the b-turn gave full control of the nematode H. con-
tortus even at a dose of 0.01mgkgꢀ1. Thus, the two
a Controlswere not treated with any compound; egg counting was
undertaken at the corresponding times compared to treated animals.

6132
H. Dyker et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6129–6132
82, 97–101; (c) Saeger, H.-P.; Schmitt-Wrede, M.; Dehn-
hardt, W. P.; Benten, J.; Krucken, A.; Harder, A.;
¨
VonSamson-Himmelstjerna, G.; Wiegand, H.; Wunderl-
ich, F. FASEB J. 2001, 15, 1332–1334.
compoundsexert a 2 timeshigher anthelmintic activity
compared to PF1022A. Interestingly, compound 9, in
which the turn mimetic hasbeen placed in the unfavour-
able i, i + 1 position, was significantly less active against
H. contortus (Table 1).
9. Scherkenbeck, J.; Harder, A.; Plant, A.; Dyker, H. Bioorg.
Med. Chem. Lett. 1998, 8, 1035–1040.
10. Dyker, H.; Scherkenbeck, J.; Gondol, D.; Goehrt, A.;
Harder, A. J. Org. Chem. 2001, 66, 3760–3766.
11. (a) Dutton, F. E.; Lee, B. H.; Johnson, S. S.; Coscarelli, E.
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Scherkenbeck, J.; Dyker, H.; Gondol, D.; Harder, A.;
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H.; Dutton, F. E.; Thompson, D. P.; Thomas, E. M.
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Compounds 7 and 8 are the first analogues in which a
substantial part of the PF1022A backbone was replaced
for other residues with an improvement of anthelmintic
activity. The biological results indicate the symmetric
conformation might be the biological active one. Thus,
structures similar to 7 and 8 may serve as starting points
for second generation PF1022 analogues.
12. Kodama, Y.; Takeuchi, Y.; Suzuki, A. Sci. Rep. Meiji
Seika Kaisha 1992, 31, 1–8.
13. Unpublished internal results. See also Ref. 11a.
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17. See literature 6.
Acknowledgements
We thank D. Gondol, G. Muller and F. Reichel for their
¨
help in interpreting the NMR spectra and molecular
modeling support.
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687 (44). 9: 894 (39, [M + Na]+), 872 (56, [M + H]+), 783
(62), 733 (100).
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