Generation of a small library of cyclodepsipeptide PF1022A analogues using a cyclization-Cleavage method with oxime resin

Article abstract of DOI:10.1016/S0960-894X(01)00748-X

N-Methyloctadepsipeptides attached to an oxime resin were cyclized by heating them in refluxing ethyl acetate for 2 days to give cyclodepsipeptide PF1022A analogues. By using this method, we generated a small library of PF 1022A analogues (2), several of which possessed anthelmintic activity, based on an in vitro assay.

Full text of DOI:10.1016/S0960-894X(01)00748-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 353–356
Generation of a Small Library of Cyclodepsipeptide PF1022A
Analogues Using a Cyclization-Cleavage Method with Oxime Resin
Byung H. Lee,* Fred E. Dutton, David P. Thompson and Eileen M. Thomas
Discovery Research, Pharmacia Animal Health, Kalamazoo, MI 49001, USA
Received 16 October 2001; accepted 5 November 2001
Abstract—N-Methyloctadepsipeptides attached to an oxime resin were cyclized by heating them in refluxing ethyl acetate for 2 days
to give cyclodepsipeptide PF1022A analogues. By using this method, we generated a small library of PF 1022A analogues (2),
several of which possessed anthelmintic activity, based on an in vitro assay. # 2002 Elsevier Science Ltd. All rights reserved.
Helminths, especially parasitic nematodes, cause sub-
stantial health problems in humans and domestic ani-
mals. Currently, three distinct chemical classes are used
for broad spectrum control of gastrointestinal nema-
todes in veterinary medicine: benzimidazoles, imida-
zothiazoles, and macrocyclic lactones.1 None of these
drugs is ideally suited for all therapeutic situations, and
each class has been challenged by the development of
drug-resistant nematode strains.2 Expansion of the
anthelmintic arsenal is thus an urgent goal. The potent
antiparasitic activity of cyclodepsipeptide (CDP)
PF1022A 1 and its analogues was discovered by Japan-
ese scientists.3 Because PF1022A is unique both struc-
turally and in its mode of action, it represents a
promising new class of anthelmintics.
timely fashion. Recently, combinatorial technology has
emerged as a way to rapidly identify and optimize thera-
peutic agents.5 Herein we report a solid-phase method
for the rapid synthesis of PF1022A analogues and the
generation of a combinatorial library. The method uti-
lizes the oxime-funtionalized polystyrene resin devel-
oped by Kaiser,6 which has been used for making cyclic
peptides.7 Previously, we reported that cyclization of N-
methyl depsipeptides using a solid support.8
Using classical synthetic methods, resin-bound tetra-
depsipeptide 3 was prepared,8,9 and then linked at its
N-terminus with Boc-Leu-Lac-OH, a didepsipeptide, to
give hexadepsipeptide 48 (Scheme 1). A second didepsi-
peptide, Boc-Leu-Plac-OH, was then joined at the
N-terminus of 4 to give octadepsipeptide 5.8 Removal of
the Boc protecting group from peptide 5 gave 6, which
underwent macrolactamization in refluxing EtOAc (2
days) to give CDP 2b.8
Because the ring of the pipecolic acid residue (Pip) adds
macrocyclic ring strain, which tends to inhibit cycliza-
tion, we prepared in addition to 6 resin-bound octa-
depsipeptides 7 and 8 and cyclized them using Kaiser’s
method to determine the optimum position of Pip
within the octadepsipeptide chain (Scheme 2). The
position found in octadepsipeptide 6 proved optimal,
producing both the highest concentration of CDP per
milligram of resin and the greatest purity. On this basis
we utilized resin-bound tetradepsipeptide 3,10 which
contains the last four residues of 6, for the preparation
of a CDP library. To further understand the effect of
small rings on cyclization, we also prepared depsipep-
tides 9 and 10, which contain a proline residue. The
Three different syntheses of PF1022A have been repor-
ted.4 Each synthesis, however, requires multiple steps,
which severely limit the development of an SAR in a
*Corresponding author. Tel.:+1-616-833-6431; fax:+1-616-833-7721;
e-mail: bhlee@pharmacia.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00748-X

354
B. H. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 353–356
slightly smaller ring of proline introduced less macro-
cyclic strain (cf. cyclizations of octadepsipeptides 7 and
10). It is also less positionally sensitive (cf. cyclizations
of octadepsipeptides 9 and 10).
Table 1gives the analytical data for this library. The
average crude weight of the products is 13 mg and the
average concentration is 30%, based on HPLC analysis
of each sample. This means that our library produced
an average of 3.9 mg of actual product for each sample.
The structures of the products were confirmed by elec-
tro-spray, mass spectral analysis performed on the
appropriate fraction obtained by HPLC.
Having developed chemistry suitable to the application
of combinatorial methodology, we prepared eleven
didepsipeptides4c and used them along with resin-bound
tetradepsipeptide 3 to prepare a CDP library consisting
of 48 (8R1Â6R2) analogues. The structures of the
penultimate products (resin-bound before cyclization)
are shown in Scheme 3.
The highest concentrations of CDPs on resin were
obtained when two pipecolic acid residues were present
in the octadepsipeptide, namely peptides 24Â11–18.
Simple molecular modeling revealed that the single
pipecolic residue of 3 introduces a cisoid turn unfavor-
able to cyclization. This effect is reversed by the pre-
sence of a second pipecolic residue, which introduces a
similar, but offsetting, turn that enhances cyclization to
produce purities of 16–65%.
Biology
The in vitro activity of the CDP library was assessed
using the gastrointestinal nematode, Haemonchus con-
tortus.10 Each CDP was tested at a nominal concentra-
tion of 3 mM by adding 2.5 mL plate extract (adjusted in
volume with DMSO to give a stock solution of 2.5 mM)
to test tubes containing five parasites suspended in 2.5
mL culture medium. Motility levels were recorded at 1h
intervals, for a period of 24 h, using an automated
micromotility recording system.10 Negative controls
consisted of H. contortus incubates to which were added
0.1% DMSO. Positive controls contained 5 mM PF1 022A
or 0.1 mM Ivermectin (a widely-used anthelmintic agent),
both prepared in DMSO. Results from two separate
Scheme 1. Synthesis of the cyclodepsipeptides.
Scheme 3. Penultimate products, that is resin-bound octadepsipep-
tides before cyclization to CDPs.
Scheme 2. Determination of optimal pipecolic position.

B. H. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 353–356
Table 1. Analytical biological data for CDP library
355
Acknowledgements
R2
R1
19
20
21
22
23
24
A Genesis RSP 200 (Tecan) synthesizer was used to
make the CDP library. We gratefully acknowledge the
technical assistance of Gary J. Cleek in the use of this
instrument.
Leu-Lac Phe-Lac Ala-Lac Ileu-Lac Nleu-Plac Pip-Lac
11
Leu-Plac
19a,b
11.84c
27d
955e
93f
18
13.12
1
989
84
11
10.67
0
913
7189
13
13.24
11
15.56
11
11.71
25
1
7
0
38
65
955
1031
939
78
86
12
Phe-Plac
18
13.34
10
989
85
13
13.39
14
1023
4147
21
11.02
18
947
63
10
10
15.81
41
12
11.87
48
References and Notes
13.50
22
989
1. Lynn, R. C., Georgis’ Parasitology for Veterinarians; W. B.
Saunders Co: Philadelphia, 1995; p 247.
2. Prichard, R. Vet. Parasitol. 1994, 54, 259.
1065
973
0
1
13
Phe-Lac
1
2
1
2
1
2
1
9.18
65
4
1
2
1
8
3. Sasaki, T.; Takag, M.; Takasi, Y.; Miyadoh, S.; Okada, T.;
Koyama, M. J. Antibiot. 1992, 45, 692.
10.76
38
913
66
11.4
0.25
947
53
8.12
19
871
3160
10.88
45
913
13.54
54
989
4. (a) Ohyama, M. Biosci. Biotech. Biochem. 1994, 58, 1193.
(b) Scherkenbeck, J.; Plant, A.; Harder, A.; Mencke, N. Tetra-
hedron 1995, 31, 8459. (c) Dutton, F. E.; Nelson, S. J. J. Anti-
biot. 1994, 47, 1322.
5. (a) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor,
S. P.; Gordon, E. M. J. Med. Chem. 1994, 37, 1233. (b) Gor-
don, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P.; Gal-
lop, M. A. J. Med. Chem. 1994, 37, 1385.
6. (a) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45,
1295. (b) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982,
47, 3258. (c) Sasaki, T.; Kaiser, E. T. J. Org. Chem. 1991, 56,
3159.
897
24
41
14
Ala-Plac
11
10.75
22
913
77
16
11.02
2120
947
10
8.24
27
871
56
10
11.01
34
913
79
10
13.57
54
989
15
15
9.17
897
77
53
15
Ileu-Plac
8
13.21
3
—
—
24
11.02
2
—
—
3
—
0
—
—
12
13.58
5
—
—
8
16.01
3
—
—
10
11.74
1
939
27
6
7. (a) Osapay, G.; Profit, A.; Taylor, J. W. Tetrahedron Lett.
1990, 31, 6121. (b) Xu, M.; Nishino, N.; Mihara, H.; Fuji-
moto, T.; Izumiya, N. Chemistry Lett. 1992, 191. (c) Kates,
S. A.; Sole, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.;
Albercio, F. Tetrahedron Lett. 1993, 34, 1549. (d) Jackson, S.;
DeGrado, W.; Dwivedi, A.; Parthasarathy, A.; Higley, A.;
Krywko, J.; Rockwell, A.; Markwaldo, J.; Wells, G.; Wexler,
R.; Mousa, S.; Harlow, R. J. Am. Chem. Soc. 1994, 116, 3220.
(e) Nishino, N.; Xu, M.; Mihara, H.; Fujimoto, T. Bull. Chem.
Soc. Jpn. 1992, 65, 991.
16
Ileu-Lac
14
10.66
11
897
52
9
10.94
6
—
—
1
—
0
—
—
12
10.86
13
879
66
8
13.50
11
955
48
8
8.88
43
863
0
17
Nleu-Lac
22
10.58
15
969
72
10
10.93
20
913
70
20
7.80
41
837
48
12
10.72
51
869
60
13
13.39
34
945
79
12
8.91
45
853
7
8. Lee, B. H. Tetrahedron Lett. 1997, 38, 757.
9. Synthesis of 3. Triphenylphosphine (TPP, 880 mg, 3.36
mmol), Boc-l-pipecolic acid (640 mg, 2.8 mmol), and benzyl
18 23
Nleu-Plac 13.24
12
13.33
7
999
6165
3
10.86
12
13.45
10
15.74
11
11.74
1
1
955
8
1
0
1
8
5
40
43
l-lactate (0.5 g, 2.8 mmol) were dissolved in Et2O (20 mL).
The resulting mixture was treated with diethyl azodicarboxy-
late (DEAD) (0.5 mL, 3.17 mmol in 5 mL of Et2O) at room
temperature over 20 min. The mixture was stirred for an
additional 1h and the precipitate removed by filtration. The
filtrate was concentrated and the residue purified by silica gel
chromatography (10% EtOAc in hexane) to give Boc-Pip-Lac-
923
955
1031
82
939
4
35
aCDP 2b.
bCrude weight (mg).
cHPLC Rt (min).
dPurity (%).
eMass spectra (M+Na).
1
OBn (0.88 g, 80%) as an oil. H NMR (400 MHz, CDCl3) d
fMotility (% reduction at 24 h).
1.1–2.3 (m, 18H), 2.8–3.0 (m, 1H), 3.8–4.1 (m, 1H), 4.7–5.2 (m,
4H), 7.2–7.4 (m, 5H). FABHRMS: m/e 392.2086
(C21H29NO6+H requires 392.2073). Boc-Pip-Lac-OBn (7.2 g,
18.39 mmol) was dissolved in CH2Cl2 (DCM) containing 10%
(v/v) TFA (200 mL). The reaction mixture was stirred for 1.5 h
and then slowly poured into saturated NaHCO3 aqueous
solution (200 mL) with rapid stirring. The mixture was trans-
ferred to a separatory funnel and shaken. The layers were
separated, and the aqueous layer extracted with DCM. The
organic layers were combined, washed with water, dried
(MgSO4), filtered, and concentrated to give Pip-Lac-OBn (5.25
g, 98% yield) as an oil. This was used without further pur-
ification. Boc-Leu-Plac-OH (5.0 g, 12.6 mmol) was dissolved
in DCM (10 mL) and treated with diisopropylcarbodiimide
(DIC) (2.2 mL, 12.6 mmol), dimethylaminopyridine (DMAP)
(244 mg, 2 mmol), and Pip-Lac-OBn (2.97 g, 12.4 mmol) at 0
ꢀC. The mixture was slowly warmed to room temperature and
stirred for 16 h. The precipitate was removed, and the filtrate
concentrated. The residue was purified by silica gel chromato-
experiments, four separate culture tubes per study,
are shown in Table 1. Results are expressed as %
reduction in motility, relative to 0.1% DMSO con-
trols. In these studies, 5 mM PF1022A reduced moti-
lity by 83%, and 0.1 mM Ivermectin by 85%. Several
products from the CDP library reduced H. contortus
motility by 80% or more following 24 h incubations.
In conclusion, we have developed a method for the
rapid preparation of N-methylcyclodepsipeptides using
a solid support and generated a small combinatorial
library of natural product analogues. Some of these
analogues were shown to possess anthelmintic activity,
based on results from an in vitro micromotility assay
against the gastrointestinal nematode, H. contortus.

356
B. H. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 353–356
graphy (20% acetone in hexane) to give Boc-Leu-Plac-Pip-Lac-
1
confirm structure and determine purity and resin concentra-
tion, a sample of 3 (30 mg) was suspended in DCM (1mL)
containing 10 mg of morpholine. The mixture was stirred for
16 h at room temperature to give the corresponding morpho-
linopeptide. A small aliquot (2 mL) was removed and analyzed
by HPLC (RP 8 column, gradient: 50–90% acetonitrile/
H2O+0.1% TFA over 20 min), which showed the mor-
pholinopeptide to be 98% pure (Rt=6.59 min). Insoluble
material was filtered off from the remaining morpholino-
peptide and the filtrate concentrated to give Boc-Leu-Plac-
Pip-Lac-morpholine as a thick oil (13 mg, 97% yield). 1H
NMR (400 MHz, CDCl3) d 0.8–2.3 (m, 27H), 2.5–3.9 (m,
15H), 4.5–5.6 (m, 4H) 7.1–7.4 (m, 5H). FABHRMS: m/e
646.3710 (C35H51N3O9+H requires 646.3703). Similarly
cleaved of resin to determine purity were: peptide 4 to give
OBn as an oil (4 g, 60% yield). H NMR (400 MHz, CDCl3) d
0.7–2.4 (m, 27H), 2.6–3.7 (m, 7H), 4.5–5.7 (m, 6H) 7.2–7.5 (m,
10H). FABHRMS: m/e 667.3608 (C37H50N2O9+H requires
667.3594). Boc-Leu-Plac-Pip-Lac-OBn (3.5 g, 5.3 mmol) was
dissolved in absolute EtOH (70 mL) and hydrogenolyzed for
17 h at 40 psi over 10% palladium on charcoal (1.0 g). The
reaction mixture was flushed with nitrogen, filtered, and con-
centrated to remove EtOH. The residue was dried under high
vacuum to give Boc-Leu-Plac-Pip-Lac-OH (2.9 g, 98%) as a
1
semi-solid. H NMR (400 MHz, CDCl3) d 0.8–2.3 (m, 27H),
2.7–3.9 (m, 7H), 4.5–5.8 (m, 4H) 7.2–7.4 (m, 5H).
FABHRMS: m/e 577.3132 (C30H44N2O9+H requires
577.3124). [a]D=À57.0ꢀ (c 0.97, DCM). Boc-Leu-Plac-Pip-
Lac-OH (2.15 g, 3.86 mmol), DMAP (610 mg, 5 mmol), and
Kaiser oxime (Novabiochem, 0.91mmol/g, 2 g, 1.82 mmol)
were suspended in DCM (80 mL). The mixture was treated
with DIC (0.94 mL, 5.4 mmol) and stirred for 16 h. The resin
was washed with DCM (2Â25 mL), MeOH (2Â25 mL), DMF
(2Â25 mL), and DCM (2Â25 mL). The washed resin was dried
in vacuo for 4 h to give 3 (2.67 g, 0.65 mmol/g of resin). To
Boc-Leu-Lac-Leu-Plac-Pip-Lac-morpholine,
99%
pure
(Rt=9.12 min), and peptide 6 to give Boc-Leu-Plac-Leu-Lac-
Leu-Plac-Pip-Lac-morpholine, 90% pure (Rt=13.34 min).
10. Geary, T. G.; Sims, S. M.; Thomas, E. M.; Vanover, L.;
Davis, J. P.; Winterrowd, C. A.; Klein, R. D.; Ho, N. F.;
Thompson, D. P. Expl. Parasitol. 1993, 77, 88.