Restricted conformation analogues of an anthelmintic cyclodepsipeptide

Article abstract of DOI:10.1021/jm020482k

Six analogues of the anthelmintic cyclodepsipeptide PF1022A were prepared, each containing a small ring fused to the macrocycle to restrict the number of conformations the larger ring can adopt. It was anticipated that such conformational changes could lead to enhanced biological activity and selectivity. The analogues form two series of three members each. In one series, a carbon-based molecular bridge joins the methyl of a leucine residue with the methyl of its closest lactic acid residue to form five-, six-, and seven-membered lactam rings. In the second series, a leucine residue is replaced with five-, six-, and seven-membered nitrogen heterocycles. Decreasing the size of the small ring in the lactam series increasingly distorts the macrocycle and consistently decreases activity relative to PF1022A. In the leucine series, a similar trend is observed. Molecular modeling of PF1022A along with the analogues described herein suggests that the ability to exist in a highly symmetrical conformational state is a necessary condition for biological activity.

Full text of DOI:10.1021/jm020482k

J . Med. Chem. 2003, 46, 2057-2073
2057
Restr icted Con for m a tion An a logu es of a n An th elm in tic Cyclod ep sip ep tid e
Fred E. Dutton,†,§ Byung H. Lee,*,† Sandra S. J ohnson,† Eileen M. Coscarelli,† and Pil H. Lee‡
Animal Health Discovery Research, Pharmacia Animal Health, Kalamazoo, Michigan 49001, and
Structural & Computational Chemistry, Pharmacia Corporation, Kalamazoo, Michigan 49001
Received October 25, 2002
Six analogues of the anthelmintic cyclodepsipeptide PF1022A were prepared, each containing
a small ring fused to the macrocycle to restrict the number of conformations the larger ring
can adopt. It was anticipated that such conformational changes could lead to enhanced biological
activity and selectivity. The analogues form two series of three members each. In one series,
a carbon-based molecular bridge joins the methyl of a leucine residue with the methyl of its
closest lactic acid residue to form five-, six-, and seven-membered lactam rings. In the second
series, a leucine residue is replaced with five-, six-, and seven-membered nitrogen heterocycles.
Decreasing the size of the small ring in the lactam series increasingly distorts the macrocycle
and consistently decreases activity relative to PF1022A. In the leucine series, a similar trend
is observed. Molecular modeling of PF1022A along with the analogues described herein suggests
that the ability to exist in a highly symmetrical conformational state is a necessary condition
for biological activity.
In tr od u ction
Ch em istr y
Compound 1 consists of eight residues in a floppy, 24-
membered ring: four N-methyl-L-leucines (MLeu), two
D-lactic acids (Lac), and two 3-phenyl-D-lactic acids
(PLac). Constructing a carbon-based bridge between the
N-methyl group of a leucine residue and the methyl
group of the adjacent lactic acid residue had the effect
of fusing a lactam ring to the macrocycle, thereby
reducing the number of conformations the macrocycle
can adopt. Five-, six-, and seven-membered lactams
(hereafter designated γ, δ, and ꢀ) were prepared and
elaborated as analogues of 1 (CDPs 2-4).
γ-La cta m CDP 2. A five-membered lactam (γ-lactam)
containing two chiral centers was prepared in four steps
from D-malic acid 8 and L-leucine benzyl ester 13
(Scheme 1). Stirring D-malic acid with 2,2-dimethoxy-
propane, which served as both reactant and solvent,
along with a catalytic amount of pyridinium p-toluene-
sufonate (PPTS) at room temperature for 2 days pro-
duced dioxolanone acid 10 in 97% yield. The initial two-
phase reaction mixture slowly gave way to a homoge-
neous solution and occasionally produced a small amount
of the related methyl ester 9, which could be removed
by recrystallization or chromatography. Often the crude
material was used and any ester present removed by
chromatography following subsequent reactions. Alde-
hyde 12, required for conversion to γ-lactam 15, was
most easily obtained by first reducing acid 10 to alcohol
11 with borane‚THF (∼100% yield) and then oxidizing
the alcohol to aldehyde 12 with PCC (73% yield).
Secondary amine 14 resulting from the reductive ami-
nation of aldehyde 12 with L-leucine benzyl ester 13 and
sodium cyanoborohydride in MeOH was not isolable but
immediately underwent an intramolecular displacement
of acetone from the dioxolanone ring to give γ-lactam
benzyl ester 15 in 53% yield.
Helminths, especially parasitic nematodes, cause
substantial health problems in humans and domestic
animals. Three broad classes of drugs are currently used
to treat gastrointestinal nematodes in veterinary medi-
cine: benzimidazoles, imidazothiazoles, and macrolac-
tones.1a None of these drugs is ideally suited for all
therapeutic situations, and certain nematode strains
have developed resistance to them. The potent anti-
parasitic activity of cyclodepsipeptide (CDP) PF1022A
1 (Figure 1) was discovered by J apanese scientists.1b
Because 1 is unique both structurally and in its mode
of action, it represents a promising new class of anthel-
mintics. Following our total synthesis of 1,2 we began
investigating the effects on biological activity resulting
from restricting the number of conformations that 1 can
adopt. It is well-established that geometry-constraining
effects, such as those produced by macrocyclic rings, are
nature’s way of rendering peptides biologically active.3
Additional constraints offer the possibility of increasing
both potency and selectivity. It was anticipated that
molecular modeling techniques applied to constrained
analogues of 1 might reveal information about the
conformational requirements of the receptor binding
site, which are presently unknown. With these objec-
tives in mind, we set about constructing a series of
conformationally restricted analogues of 1 at two dis-
tinct molecular sites: (1) between the N-methyl group
of a leucine residue and the methyl group of its adjacent
lactic acid residue where the construction of carbon-
based, molecular bridges led to CDPs 2-4; (2) at a
leucine residue where replacing it with nitrogen het-
erocycles produced CDPs 5-7.
* To whom correspondence should be addressed. Phone: 209-833-
6431. Fax: 269-833-7721. E-mail: byung.h.lee@pharmacia.com.
† Pharmacia Animal Health.
This was elaborated to γ-lactam CDP 2 using con-
ventional methods of peptide synthesis (Scheme 2).
N-Boc-N-methyl-L-leucine 16 was coupled with γ-lactam
§ Retired.
‡ Pharmacia Corporation.
10.1021/jm020482k CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/26/2003

2058 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
F igu r e 1.
Sch em e 1a
vinyl ether 27 as a 5:1 mixture of E and Z stereoisomers
in low yield (27-36%). Hydrolysis of the vinyl ether to
aldehyde 29 was problematic, but treatment with un-
dried acetone containing a trace of sulfuric acid at room
temperature produced sufficient quantities of the ho-
mologous aldehyde. The modest yield (44%) can be
partly accounted for by formation in 13% yield of the
corresponding dimethylacetal, 28. Aldehyde 29 was
subjected to reductive amination with L-leucine benzyl
ester 13 to give a 4:1 mixture of secondary amine 30
and δ-lactam benzyl ester 31 in approximately 33%
yield. The secondary amine was unstable with respect
to intramolecular cyclization and slowly underwent
lactamization over a period of 5 days at room temper-
ature to give a 6:1 mixture of δ-lactam benzyl ester 31
and amine 30. Heating amine 30 in toluene at 70 °C
for 90 min resulted in essentially complete conversion
to δ-lactam 31 having the R,S stereochemistry.
δ-Lactam 39, the S,S diastereomer of δ-lactam 31,
was prepared in better overall yield but required
adjusting a chiral center late in the synthesis (Scheme
4). Methyl 5-bromovalerate 32 and L-leucine benzyl
ester 13 were heated at 130 °C in DMF in the presence
of powdered sodium bicarbonate for 70-90 min to give
secondary amine 33 in 91% yield.
Unlike the γ-lactam synthesis where spontaneous
lactamization occurred between the secondary amine
moiety and its attached dioxolanone ring, here the much
less reactive ester moiety of secondary amine 33 re-
quired strong heating. During 18 h in refluxing xylene,
amine 33 underwent lactamization to produce δ-lactam
34 in 66% yield.
Hydroxylation of the R position of δ-lactam 34 was
accomplished in two steps. Bromination of 34 gave 35
in yields of 85-94%, which upon being heated at 150
°C for 65 min in formamide containing 1 equiv of water
produced the hydroxylated δ-lactam 36 in 87-92%
yields as a 1:1 mixture of R,S and S,S diastereomers.
Prolonged heating and higher temperatures led to a
reduction in yield due to O-formylation of the product
to give formate 37. In one such case, the hydroxylated
lactam was obtained in only 53% yield, while the
formylated lactam was formed in 21% yield.
Although either diastereomer was suitable for elabo-
ration, the mixture of both diastereomers was clearly
unacceptable. Therefore, δ-lactam 36 was subjected to
a Swern oxidation, which gave ketone 38 in 70-75%
yields. Reduction with D-glucose and Baker’s yeast
produced predominantly δ-lactam benzyl ester 39 hav-
ing the S,S stereochemistry but was not very consistent:
while g95% de (50% yield) was obtained on a 3 g scale.
a
(a) (CH3)2C(OCH3)2, PPTS, 25 °C, 53 h; (b) BH3‚THF, THF,
25 °C; (c) PCC, CH2Cl2, 25 °C; (d) HOAc, NaBH3CN, MeOH, 25
°C.
benzyl ester 15 (86%) and the benzyl group was removed
catalytically (98%) to give γ-lactam free acid 17. This
was coupled with benzyl 3-phenyl-D-lactate4 18 (87%)
followed by removal of the benzyl group (95%) to give
tetradepsipeptide free acid 19. Tetradepsipeptide free
amine 25 was prepared by coupling didepsipeptide free
acid 21 with didepsipeptide free amine 23 (80%) to give
tetradepsipeptide 24 followed by removal of the Boc
group with TFA (92%). Didepsipeptide free acid 21 was
prepared by removing the benzyl group (98%) from
didepsipeptide 20.2 Didepsipeptide free amine 23 was
prepared by removing the Boc group (90%) from didep-
sipeptide 22.2 Coupling acid 19 with amine 25 (55%)
followed first by removal of the Boc group (∼100%) and
then the benzyl group (97%) gave octadepsipeptide
amino acid 26, which underwent macrolactamization
with BOP reagent at high dilution (1 mM) to give
γ-lactam CDP 2 in 27% yield.
δ-La cta m CDP 3. The first attempt to prepare a six-
membered lactam (δ-lactam) by extending the synthetic
sequence used to prepare the γ-lactam gave the expected
product, 31, but proved impractical for elaboration to a
CDP because of poor yields in critical steps (Scheme 3).
However, because both chiral centers were present in
the starting materials, this synthetic sequence did allow
us to establish unambiguously the stereochemistry of
the δ-lactam resulting from a higher yielding, alterna-
tive synthesis described later. Aldehyde 12 was homolo-
gated by treating it with methoxymethyltriphenylphos-
phonium chloride and phenyllithium in ether to give

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2059
Sch em e 2a
a
(a) DCC, DMAP, CH2Cl2; (b) H2 (3 atm), 10% Pd/C, EtOH; (c) TFA, CH2Cl2, 25 °C; (d) BOP reagent, NMM, CH2Cl2, 1 mM, 0-25 °C,
3 days.
Sch em e 3a
Sch em e 4a
a
(a) Ph3P+CH2OCH3 Cl-, PhLi, 70:30 cyclohexane/Et2O, -78
a
(a) 13, NaHCO3 (powder), DMF, 130 °C, 90 min; (b) mixed
°C; (b) H2SO4 (trace), acetone, 25 °C; (c) 13, HOAc, NaBH3CN,
MeOH, 25 °C; (d) neat, 25 °C, 5 days or dry toluene, 70 °C, 90
min.
xylenes, reflux, 18 h; (c) (CH3)3SiCl, I2, Br2, Et3N, CH2Cl2, -20
°C; (d) H2O (1 equiv), HCONH2, 150 °C, 65 min; (e) 36, oxalyl
chloride, DMSO, Et3N, CH2Cl2, -78 °C; (f) Baker’s yeast, D-glucose,
H2O, 25 °C.
An earlier experiment on a 1 g scale gave only 70% de
although in 68% yield. (Reduction with K-selectride gave
inferior stereoselectivity: 58% de and 63% yield.) Ratios
of the two diastereomers were determined by averaging
sipeptide amino acid 43. Macrolactamization of 43 with
BOP-Cl at high dilution (1 mM) produced δ-lactam CDP
3 in 65% yield.
relative peak heights for several absorbances in the 13
C
ꢀ-La cta m CDP 4. The synthetic sequence used suc-
cessfully in the preparation of the δ-lactam failed when
applied to the synthesis of the ꢀ-lactam because the
corresponding homologous ester was too unreactive to
form a seven-membered lactam ring. The dioxolanone
sequence proved to be more successful (Scheme 6). The
required two-carbon homologation was achieved by
treating aldehyde 12 with (triphenylphosphoranyl-
idene)acetaldehyde in a Wittig reaction, which gave
unsaturated aldehyde 44 in 38-54% yields. The reac-
tion produced a ratio of E/Z isomers that varied con-
siderably between batches, ranging from 4:1 to 15:1. In
preliminary work, aldehyde 44 was subjected to reduc-
tive amination with L-leucine benzyl ester 13. Instead
of the expected secondary amine 47, this aldehyde gave
methoxylamine 45 in 39% yield as a 1:1 mixture of both
epimers, the methoxyl group having arisen from the
unexpected 1,4-addition of methanol to 44 prior to
NMR spectrum. Comparing the 1H and 13C NMR
spectra of this δ-lactam with those of δ-lactam 31 whose
stereochemistry (R,S) was unambiguously established
enabled us to assign the S,S stereochemistry to δ-lactam
39.
δ-Lactam benzyl ester 39 (g95% de) was coupled with
N-Boc-N-methyl-L-leucine 16 using Mitsunobu chemis-
try (84%) followed by removal of the benzyl group (99%)
to give δ-lactam free acid 40 with the required (inverted)
stereochemistry at the R position of the lactam ring
(Scheme 5). This was coupled with benzyl 3-phenyl-D-
lactate 18 (48%), and the Boc group was removed (94%)
to give tetradepsipeptide free amine 41. Removal of the
benzyl group from tetradepsipeptide 24 gave tetradep-
sipeptide free acid 42. Coupling amine 41 with acid 42
(58%) followed by first removing the Boc group (99%)
and then the benzyl group (93%) produced octadep-

2060 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
Sch em e 5a
removed by chromatography. Removal of the Boc group
(91%) gave tetradepsipeptide free amine 53. Amine 53
was coupled with tetradepsipeptide 42 (78%) and the
Boc group was removed (97%) followed by removal of
the benzyl group (94%) to give octadepsipeptide amino
acid 54. Macrolactamization of 54 with BOP-Cl (43%)
at high dilution (1 mM) gave ꢀ-lactam CDP 4.
To further investigate the effect on anthelmintic
activity of restricting the shape of the macrocycle, we
replaced one of the leucine residues of 1 with small,
nitrogen-containing heterocycles of five, six, and seven
members to give prolyl, pipecolyl, and azepanyl CDP
analogues (CDPs 5-7).
P r olyl CDP 5 (Sch em e 8). N-Boc-Proline 55 and
methyl D-lactate 56 were coupled (74%) and the methyl
ester was hydrolyzed (70%) to give didepsipeptide free
acid 57. Tetradepsipeptide free acid 58 was prepared
by coupling acid 57 and didepsipeptide free amine 23
(82%) followed by removal of the benzyl group (98%).
Coupling acid 58 with tetradepsipeptide free amine 25
(61%) followed by first removing the Boc group (90%)
and then the benzyl group (95%) gave octadepsipeptide
amino acid 59. Macrolactamization of 59 with BOP
reagent at high dilution (1 mM) gave prolyl CDP 5 in
12% yield.
a
(a) 16, DEAD, Ph3P, THF, 0-25 °C; (b) H2 (3 atm), 10% Pd/C,
EtOH; (c) 18, DCC, DMAP, CH2Cl2, 25 °C; (d) TFA, CH2Cl2, 25
°C; (e) DCC, DMAP, CH2Cl2; (f) BOP-Cl, DPEA, CH2Cl2, 1 mM,
0-25 °C, 48 h.
P ip ecolyl CDP 6 (Sch em e 9). Hexadepsipeptide
free acid 61 was prepared by coupling tetradepsipeptide
free acid 602 with didepsipeptide free amine 23 (80%)
followed by removal of the benzyl group (81%). N-Boc-
L-pipecolic acid 62 was coupled with benzyl L-lactate6
63 using Mitsunobu chemistry (24%) to invert the
stereochemistry of the lactic acid residue. Removal of
the Boc group (74%) gave didepsipeptide free amine 64.
Coupling acid 61 and amine 64 (24%) followed first by
removing the Boc group (84%) and then the benzyl
group (88%) gave octadepsipeptide amino acid 65.
Subsequent macrolactamization of 65 at moderate dilu-
tion (4 mM) using 1-methyl-2-chloropyridinium iodide
in CH2Cl2 gave pipecolyl CDP 6 in 50% yield.
reductive amination. Compound 45 provided an op-
portunity to test our hypothesis that the dioxolanone
ring is more reactive than an ordinary ester. Indeed,
when methoxylamine 45 was heated in refluxing xylene,
a slow conversion to ꢀ-lactam 46 resulted on the basis
of HPLC and mass spectral analyses. Heating for 90 h
was required to achieve a 90% conversion.
The problem caused by addition of methanol was
averted by reducing the double bond of aldehyde 44.
Hydrogenation at balloon pressure (72-90%) gave
saturated aldehyde 48 contaminated with acid 49. The
abundance of acid 49 varied from 6% to 26% and may
have been due to air oxidation of 44 during handling.5
Washing with aqueous NaHCO3 was not successful in
removing acid 49 probably because of its tendency to
chelate the sodium ion, which rendered the carboxylate
salt ether-soluble. Chromatography was used to remove
the acid from the more seriously contaminated batches.
Reductive aminations carried out on saturated aldehyde
48 using L-leucine benzyl ester 13 (65-74%) produced
secondary amine 50 having two chiral centers. When
heated in refluxing xylene, amine 50 underwent lac-
tamization to give ꢀ-lactam benzyl ester 51 in 54-79%
yield. The length of time (115-225 h) required to
achieve reasonable conversion varied considerably from
batch to batch, suggesting catalysis or inhibition by an
unknown agent whose abundance varied. The product
appears to have excellent thermal stability. NMR and
HPLC analyses showed ꢀ-lactam 51 to be optically pure;
all of the several batches of ꢀ-lactam gave a consistent
optical rotation of -36° to -37°.
Azep a n yl CDP 7. Preparation of azepanyl CDP 7
required the prior synthesis of 1-(tert-butoxycarbonyl)-
2-azepanecarboxylic acid 71 (Scheme 10). We prepared
oxazinone 66 following a literature procedure7 and
converted it in five steps to azepane 71. Generation of
the enolate of oxazinone 66 with sodium hexamethyl-
disilylamide and treatment with 1-iodo-5-chloropentane
furnished the homologated chloride, 67, in 73% yield.
Removal of the Boc group (87%) gave free amine 68,
which was cyclized to give bicyclic oxazine 69 in 45%
yield. Catalytic hydrogenation over PdCl2 (96%) gave
azepane 70, which upon treatment with di-tert-butyl
dicarbonate gave the Boc-protected azepane free acid
71 in 60% yield.
Acid 71 was coupled with benzyl D-lactate 72 (50%),
and the Boc group was removed (89%) to give didep-
sipeptide free amine 73 (Scheme 11). This was coupled
with hexadepsipeptide 61 (48%) followed first by re-
moval of the BOC group (90%) and then the benzyl
group (90%) to give octadepsipeptide amino acid 74. This
underwent macrolactamization when treated with
1-methyl-2-chloropyridinium iodide at moderate dilution
(7 mM) to give azepanyl CDP 7 in 34% yield.
ꢀ-Lactam 51 was further elaborated to ꢀ-lactam CDP
4 (Scheme 7). N-Boc-N-methyl-L-leucine 16 was coupled
with ꢀ-lactam benzyl ester 51 (71%), and the benzyl
group was removed (∼100%) to give ꢀ-lactam free acid
52. Coupling this product with benzyl 3-phenyl-D-
lactate4 18 gave the tetradepsipeptide (42%) along with
a lesser amount of diastereomer, which was easily

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2061
Sch em e 6a
a
(a) Ph3PCHCHO, toluene, 80 °C, 90 min; (b) 13, HOAc, NaBH3CN, MeOH, 0-25 °C; (c) mixed xylenes, reflux, 115 h; (d) H2 (balloon
pressure), 5% Pd/C, EtOAc, 3 h; (e) 48, 13, HOAc, NaBH3CN, MeOH, 0-25 °C.
Sch em e 7a
Sch em e 8a
a
(a) DCC, DMAP, CH2Cl2; (b) H2 (3 atm), 10% Pd/C, EtOH; (c)
TFA, CH2Cl2, 25 °C; (d) BOP-Cl, DPEA, CH2Cl2, 1 mM, 0-25 °C,
22 h.
a
(a) DIC, DMAP, CH2Cl2; (b) 1 N NaOH, MeOH, 25 °C, 20 min;
(c) H2 (3 atm), 10% Pd/C, EtOH; (d) DCC, DMAP, CH2Cl2; (e) TFA,
CH2Cl2, 25 °C; (f) BOP reagent, NMM, CH2Cl2, 1 mM, 25 °C, 16
h.
Biology
The relative potency of 1 and the CDPs of this report
was determined in an anthelmintic assay utilizing
immunosuppressed jirds, Meriones unguiculatus, in-
fected with the ruminant parasite, Haemonchus con-
tortus, which has demonstrated utility as a model to
study anthelmintic efficacy8 and resistance.9 The con-
formational constraints introduced by the small rings
in both series of CDPs had a profound effect upon
biological activity and the shape of the macrocycle. The
five-membered rings in both series exhibited signifi-
cantly reduced biological activity relative to 1 (Table 1).
In the lactam series (CDPs 2-4), increasing the size of
the small ring tended to restore the macrocycle toward
its original shape and biological activity, suggesting that
the effects were indeed of a conformational nature and
not merely the result of a change in molecular composi-
tion. The situation is more complex in the case of the
three compounds (CDPs 5-7) in which a leucine residue
was replaced with a nitrogen heterocycle. Of these three
CDPs, CDP 6 containing the six-membered pipecolyl
ring had by far the best activity, suggesting that 6 is
capable of attaining a biologically active conformation
unavailable to 5. The activity of CDP 6 was comparable
to that of CDP 3. The seven-membered azepanyl ring
of CDP 7 seemingly constrained the macrocycle the
least. However, CDP 7 had essentially no biological
activity possibly because of the increased bulk of the
azepanyl ring relative to leucine. This is consistent with
earlier observations that replacing a leucine residue
often has a deleterious effect on anthelmintic activity.10
Molecular modeling of our CDPs has provided a further
explanation.
Molecu la r Mod elin g
To study the relationship between biological activity
and the structure of a compound, conformational analy-
sis and molecular matching were performed. Since no
structural information is available for these compounds,
except for NMR data on 1, and because of their extreme

2062 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
Ta ble 1. Percent Reduction of Haemonchus contortus in the
Sch em e 9a
J ird by CDPs When Dosed Orallya
reduction of Haemonchus contortus (%)
at the following CDP dosages
6.00
3.00
1.00
0.33
0.11
CDP
mg/jird
mg/jird
mg/jird
mg/jird
mg/jird
1
2
3
4
5
6
7
100
89a
69
69b
99
100
89
100
75
99
36
91
100
96
3
76
19
a
Result from dosing at 5.75 mg/jird. b Result from dosing at 2.75
mg/jird.
the NMR-based structures of 1 as references. NMR
studies11,12 show that 1 exists as a mixture of conformers
in solution: one is asymmetric, showing eight magneti-
cally nonequivalent residues; the other is symmetric,
showing only four magnetically nonequivalent residues.
From the rotating-frame Overhauser enhancement spec-
troscopy (ROSEY) and nuclear Overhauser effect spec-
troscopy (NOESY) data, it is clear that the asymmetric
conformer possesses a cis amide bond between the Lac
and MLeu residues. Two solution conformers of 1 were
obtained by using two sets of NMR NOESY data
obtained from methanol, one for the asymmetric con-
former (38 NOEs) and the other for the symmetric
conformer (34 NOEs). An ensemble of NMR structures
was generated using the method of dynamical simulated
annealing13 from a random array of atoms with the
upper bound distance restraints from the NOEs as
follows: very strong, <2.2 Å; strong, <2.7 Å; medium,
<3.3 Å; weak, <5.0 Å; very weak, <5.5 Å. The calcula-
tion produced a set of 25 structures with the lowest
restraint violations. For the analogues of 1, the Monte
Carlo multiple minimum (MCMM) technique14 was
applied to search conformational space by random
changes in torsion angles within the window of 10 kcal/
mol from the global minimum energy conformation.
Each structure was minimized with the Amber* poten-
tial energy force field,15 and only the unique structures
were kept. The number of conformations generated for
each compound with 5000 Monte Carlo steps ranged
from 419 for CDP 5 to 688 for CDP 4, reflecting the
flexibility of the compound. In a recent publication,11
Scherkenbeck, et al. obtained the symmetric conforma-
tion of 1 by MD simulations in CDCl3 using NMR-
derived NOEs as constraints. In this conformation, there
are two type II′ â-turns with Lac and MLeu as Ri and
a
(a) DIC, DMAP, CH2Cl2; (b) H2 (3 atm), 10% Pd/C, EtOH; (c)
DEAD, Ph3P, Et2O, 25 °C; (d) TFA, CH2Cl2, 25 °C; (e) 1-methyl-
2-chloropyridinium iodide, Et3N, CH2Cl2, 4 mM, 25 °C, 16 h.
Sch em e 10a
a
(a) 1-Iodo-5-chloropentane, [(CH3)3Si]2NNa, HMPA, THF, -78
°C; (b) TFA, CH2Cl, 25 °C, 4 h ; (c) K2CO3, CH3CN, reflux, 24 h;
(d) H2 (50 psi), PdCl2, 1:2 THF/EtOH, 20 h; (e) [(CH3)3CO]2O, Et3N,
7:3 THF/H2O, 25 °C, 16 h.
Sch em e 11a
R
i+3, respectively, causing hydrophobic collapse of the
two PLac-MLeu residues and the side chains of two
MLeu residues. At least one of their active analogues
also resembles the symmetric conformation of 1.
We identified this conformation among the 25 NMR
structures for the symmetric conformation of 1. This
conformation is shown in Figure 2. Assuming that this
might be the active conformation, we searched for a
similar conformation among the multiple conformations
of the analogues of 1. Two of the active analogues, CDP
3 and CDP 4, contain this conformation. The symmetric
conformation of each of these compounds is overlaid on
the symmetric conformation of 1 in Figures 3 and 4.
As are shown in the side views of these compounds,
there are hydrophobic collapses between the side chains
a
(a) DIC, DMAP, CH2Cl2; (b) TFA, CH2Cl2, 25 °C; (c) BOP-Cl,
Et3N, CH2Cl2, 0-25 °C, 16 h; (d) H2 (3 atm), 10% Pd/C, EtOH; (e)
1-methyl-2-chloropyridinium iodide, Et3N, CH2Cl2, 7 mM, 25 °C,
16 h.
flexibility, it is very difficult to identify the biologically
active conformation. Given these limitations, we at-
tempted to make some qualitative comparisons using

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2063
F igu r e 2. Symmetric conformation of 1 from NMR data.
F igu r e 4. (a) Overlay of CDP3 (pink) on 1 (green). (b) Side
view of CDP3.
analogues is their relative ability to achieve a sym-
metrical conformational state, as our modeling has
demonstrated.
Con clu sion
We prepared six conformationally constrained ana-
logues of the anthelmintic cyclodepsipeptide 1 by fusing
five-, six-, and seven-membered rings to its macrocycle.
The lactam CDPs 2-4 exhibit a steady progression in
relative biological activity as the size of the fused ring
increases, thereby reducing macrocyclic strain. Such a
progression in the leucine series is suggested by the
relative activities of CDPs 5 and 6. In the absence of
molecular modeling, the remaining analogue, 7, seems
to belie this progression. However, modeling indicates
that, despite the larger size of its fused ring, CDP 7 was
unable to achieve the symmetrical conformational state
necessary for biological activity.
Exp er im en ta l Section
Ch em istr y. An a lytica l Meth od s. Mass spectra from
electron impact (EI) ionization were determined with a Finni-
gan MAT 8230B double-focusing spectrometer. Samples were
introduced through the AUDEVAP probe inlet. A MicroMass
Platform II mass spectrometer was used to obtain electrospray
ionization (ES) data. FAB spectra were obtained via the liquid
secondary ion mass spectrometry (LSIMS) ionization technique
on a VG70SE double-focusing spectrometer utilizing a cesium
ion gun. These samples were analyzed in a matrix of 2-hy-
F igu r e 3. (a) Overlay of CDP4 (pink) on 1 (green). (b) Front
view of CDP4. (c) Side view of CDP4.
1
droxyethyl disulfide. H and 13C NMR spectra were obtained
using Bruker 300 and 400 MHz instruments. Proton spectra
of many of the intermediates showed the presence of more than
one conformer on the NMR time scale. The isopropylmethyl
groups of leucine often appeared as two, three, or more sets of
doublets because of conformers and are designated (2d, 6H),
(3d, 6H), etc. in the Experimental Section of this report.
Similar results were sometimes found with the tert-butyl
groups and N-methyl groups when a Boc group was present
and are designated (2s, 9H), (3s, 3H), etc. Other protons were
less likely to exhibit this effect. IR spectra were recorded using
a Digilab model FTS40 spectrometer. Samples were prepared
in a mineral oil mull, and the spectra were collected from 4000
to 500 cm-1 at a resolution of 2 cm-1 through the addition of
16 scans. A Perkin-Elmer Lambda 7 UV/vis spectrophotometer
was used to record UV spectra. Optical rotations were obtained
in MeOH using a Perkin-Elmer model 241 polarimeter.
Ma ter ia ls. D-Malic acid was purchased from Lancaster
Synthesis Inc., and N-Boc-N-methyl-L-leucine was purchased
from Bachem California. N-Boc-L-proline, methyl d-lactate,
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexaflu-
orophosphate (BOP Reagent), bis(2-oxo-3-oxazolidinyl)phos-
of MLeu and PLac as well as between the side chains
of the MLeu residues. Also seen from the front view,
these compounds with their fused rings cause the
overall conformation to fold more compared with 1. In
CDP6, the other active analogue, hydrophobic interac-
tions between different pairs of groups were observed,
for example, strong interactions between the phenyl ring
in the PLac and the six-membered heterocycle and also
between the side chains of the PLac and MLeu residues.
A similar symmetric conformation was not found in the
inactive analogues (CDPs 2, 5, and 7), although different
types of hydrophobic collapses were observed.
Given the limited experimental data, we cannot
conclusively identify the active conformations of 1 and
its analogues, but assuming that the symmetric confor-
mation of 1 is the biologically active one, then the most
salient difference between our active and inactive

2064 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
phinic chloride (BOP-Cl), 1,3-dicyclohexylcarbodiimide (DCC),
1,3-diisopropylcarbodiimide (DIC), diethyl azodicarboxylate
(DEAD), N,N-diisopropylethylamine (DPEA), N-methylmor-
pholine (NMM), 2-chloro-1-methylpyridinium iodide, L-pipe-
colic acid, methyl 5-bromovalerate, and pyridinium p-tolu-
enesulfonate (PPTS) were purchased from Aldrich Chemical
Co., Inc. Methylene chloride, mixed xylenes, and DMF were
dried over 4 Å molecular sieves. Methanol was dried over 3 Å
molecular sieves. THF was dried and purified by distillation
from the sodium ketyl of benzophenone.
Gen er a l Ch em ica l P r oced u r es. 1. Gen er a l P r oced u r e
for Rem ovin g a Boc P r otectin g Gr ou p . The substrate was
dissolved in CH2Cl2 and sufficient TFA was added with stirring
to give a 10-20% TFA solution. The resulting reaction mixture
was stirred at 25 °C under an atmosphere of dry N2. Reaction
progress was monitored by TLC. The reaction was usually done
within 60 min. The reaction mixture was concentrated in
vacuo, and the residue was taken up in CH2Cl2 and washed
twice with saturated NaHCO3 and then with H2O followed by
saturated NaCl. The organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated. Final drying under high
vacuum gave the product.
N2. The malic acid and PPTS slowly formed a gum, which was
broken up with a spatula after 5 h. The gum slowly gave way
to a clear solution with stirring overnight. The reaction
mixture was stirred for 53 h and then concentrated. The
residue was taken up in EtOAc and filtered through silica gel
to remove the PPTS. The effluent was concentrated to give
dioxolanone acid 10 (97%) as an off-white solid, which some-
times contained a small amount of ester 9. The crude material
was often used without further purification. Recrystallization
from CH2Cl2/hexane gave pure dioxolanone acid 10 (13.80 g,
76%) as a white, crystalline solid. 1H NMR (400 MHz,
CDCl3): δ 1.56 (s, 3H), 1.61 (s, 3H), 2.85 (dd, J ) 6.5, 17.3 Hz,
1H), 2.99 (dd, J ) 3.9, 17.3 Hz, 1H), 4.71 (dd, J ) 3.9, 6.5 Hz,
1H), 10.64 (bs, 1H). MS (EI) m/z: 159 [M - CH3]. Anal.
(C7H10O5) C, H. Chromatography (20% EtOAc in hexane) of
the crude reaction product occasionally produced a small
1
amount (e3 wt %) of ester 9. H NMR (400 MHz, CDCl3): δ
1.56 (s, 3 H), 1.61 (s, 3 H), 2.80 (dd, J ) 6.6, 17.0 Hz, 1 H),
2.94 (dd, J ) 3.9, 17.0 Hz, 1 H), 3.74 (s, 3 H), 4.72 (dd, J )
3.9, 6.6 Hz, 1 H). 13C NMR (100 MHz, CDCl3): δ 26.20, 27.13,
36.46, 52.49, 71.02, 111.51, 169.95, 172.35. MS (ES) m/z: 211
[M + Na].
2. Gen er a l P r oced u r e for Rem ovin g a Ben zyl P r otect-
in g Gr ou p . The substrate was dissolved in absolute EtOH.
Palladium (10%) on activated carbon was added, and the
reaction mixture was hydrogenolyzed in a Parr shaker at 45-
50 psi of H2. The reaction was usually complete within 3 h.
The reaction mixture was flushed with N2 and filtered through
Celite. The Celite cake was washed thoroughly with absolute
EtOH, and the filtrates were combined and concentrated.
Residual EtOH, which could interfere with any subsequent
coupling reaction, was removed by twice dissolving the product
in EtOAc and concentrating. Drying under high vacuum at
25 °C gave the product. When both Boc and benzyl protecting
groups were to be removed, we found it generally advantageous
to remove the Boc group first followed by removal of the benzyl
group. This avoided the problem associated with retrieving the
resulting zwitterionic peptide from an acidic medium.
2-[(4R)-2,2-Dim et h yl-5-oxo-1,3-d ioxola n -4-yl]et h a n ol
(11). Dioxolanone acid 10 (13.6 g, 78.1 mmol) was dissolved
in THF (40 mL), and the mixture was cooled to 0 °C under an
atmosphere of N2. A solution of BH3 in THF (1 M, 100 mL,
100 mmol) was added dropwise over a period of 47 min. The
reaction mixture was stirred for 60 min at 0 °C and then for
2 h at 24 °C. The mixture was cooled to 0 °C, MeOH (50 mL)
was added dropwise, and the mixture was stirred for 5 min to
destroy the remaining borane. The mixture was concentrated
at or below 35 °C, MeOH (100 mL) was added, and the
resulting solution was concentrated. A final concentration from
EtOAc (100 mL) gave dioxolanone alcohol 11 (12.5 g, ∼100%)
as a clear, colorless oil. It was stored under N2 at 0 °C for
several days without deterioration. 1H NMR (400 MHz,
CDCl3): δ 1.55 (s, 3H), 1.61 (s, 3H), 1.97 (m, 1H), 2.14 (m,
1H), 3.82 (m, 2H), 4.56 (dd, J ) 4.9, 7.1 Hz, 1H).
3. Gen er a l P r oced u r e for Cou p lin g P ep t id es Usin g
DCC. The amine or alcohol was dissolved in CH2Cl2 along with
the carboxylic acid under an atmosphere of dry N2. The
solution was cooled to 0 °C, and DCC was added, usually in
the form of a 1 M solution of DCC in CH2Cl2. This was followed
immediately by the addition of solid DMAP. A precipitate of
dicyclohexylurea usually appeared within 2 min. The cooling
bath was removed, and the reaction mixture was stirred at
25 °C until the starting alcohol or amine had been mostly
consumed as indicated by TLC (usually within 3 h); reactions
seldom went to completion. The reaction mixture was filtered
to remove the urea and then concentrated. Any urea still
present was removed by dissolving the crude product in Et2O
and filtering a second time. The filtrate was concentrated and
dried under high vacuum to give the crude product.
4. Gen er a l P r oced u r e for Cou p lin g P ep t id es Usin g
DIC. The carboxylic acid and amine were dissolved in CH2-
Cl2, and the solution was cooled to 0 °C under an atmosphere
of dry N2. DIC (neat) was added followed immediately by
addition of DMAP, and the mixture was stirred at room
temperature. The reaction mixture was filtered to remove the
precipitated urea and the filtrate was concentrated to give
crude product.
5. Ch r om a togr a p h y. Preparative low-pressure chroma-
tography was performed using EM Science silica gel (230-
400 mesh) contained in a glass column or in a sintered glass
funnel. Products were eluted from the absorbent with varying
concentrations of EtOAc in hexane except where otherwise
noted. Small amounts of material, e.g., <1 g, were purified by
centrifugal elution from silica gel bonded to a glass rotor
contained in a Chromatotron.
Syn th esis of γ-La cta m Cyclod ep sip ep tid e (2). 2-[(4R)-
2,2-Dim eth yl-5-oxo-1,3-dioxolan -4-yl]acetic Acid (10). Pow-
dered D-malic acid 8 (13.94 g, 104 mmol) was combined with
2,2-dimethoxypropane (50 mL), PPTS (2.23 g, 8.91 mmol) was
added, and the two-phase mixture was stirred at 25 °C under
2-[(4R)-2,2-Dim eth yl-5-oxo-1,3-d ioxola n -4-yl]a ceta ld e-
h yd e (12). Dioxolanone alcohol 11 (17.8 g, 111 mmol) was
dissolved in CH2Cl2 (700 mL) and cooled to 0 °C. Solid
pyridinium chlorochromate (120 g, 555 mmol) was added all
at once, and the cooling bath was removed. The reaction
mixture was stirred at 25 °C for 4 h and then poured into Et2O
(1 L). The residual solid in the reaction flask was triturated
several times with Et2O, bringing the total volume of Et2O to
2 L. The combined Et2O solutions were filtered through Celite,
giving a clear, dark-orange filtrate. This was treated with
activated carbon (Darco G-60), and the mixture was stirred
intermittently for 20 min, after which it was filtered through
Celite giving a clear, pale-yellow filtrate. This was concen-
trated at less than 42 °C. The residue was dissolved in EtOAc
and similarly concentrated followed by drying under high
vacuum to give dioxolanone aldehyde 12 (12.9 g, 73%) as a
clear, brown-green oil. 1H NMR spectroscopy showed the
product to contain 90 mol % aldehyde and 10 mol % acid,
giving an adjusted yield of 65%. This material was stored
1
under N2 at 0 °C. H NMR (400 MHz, CDCl3): δ 1.57 (s, 3H),
1.62 (s, 3H), 2.91 (dd, J ) 7.2, 18.4 Hz, 1H), 3.09 (dd, J ) 3.5,
18.3 Hz, 1H), 4.78 (dd, J ) 3.5, 7.0 Hz, 1H), 9.74 (s, 1H).
L-Leu cin e Ben zyl Ester (13). L-Leucine (13.1 g, 100 mmol)
was combined with dioxane (60 mL) and H2O (40 mL), and
the resulting slurry was cooled to 0 °C. A solution of NaOH (1
N, 200 mL) was added followed by molten di-tert-butyl dicar-
bonate (49.3 g, 226 mmol). Additional dioxane (50 mL) was
used to rinse the di-tert-butyl dicarbonate into the reaction
flask. The cooling bath was removed, and the reaction mixture
was stirred at 25 °C for 23 h. The reaction mixture was diluted
with H2O (200 mL) and washed with pentane (4 × 300 mL).
The aqueous layer was cautiously acidified (foaming) with
citric acid to pH 1-3, and the mixture was then extracted with
EtOAc (2 × 350 mL). The extracts were combined, washed in
turn with H2O and saturated NaCl, and then dried over
anhydrous Na2SO4. Filtration and concentration of the filtrate

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2065
followed by drying under high vacuum gave N-Boc-L-leucine
(25.5 g, ∼100%) as a clear, colorless oil. H NMR (400 MHz,
give crude product, which upon chromatography (20% EtOAc
in hexane) produced the tetradepsipeptide intermediate (4.93
g, 87%) as a clear, pale-yellow oil. 1H NMR (400 MHz,
CDCl3): δ 1.87-1.99 (∼3d, 12H), 1.40 (m, 1H), 1.46 (s, 9H),
1.59 (m, 3H), 1.73 (m, 3H), 2.18 (m, 1H), 2.77 and 2.80 (2s,
3H), 3.10 (m, 2H), 3.15 (m, 2H), 4.73 (m 0.5H), 4.92 (m, 1.5H),
5.09 (d, J ) 12.1 Hz, 1H), 5.15 (d, J ) 12.0 Hz, 1H), 5.22 (m,
1H), 5.31 (m, 1H), 7.05-7.40 (m, 10H). MS (FAB) m/z: 681
[M + H]. This intermediate (2.48 g, 3.64 mmol), absolute EtOH
(100 mL), and 10% Pd on activated carbon (300 mg) were
combined and processed according to the general procedure
for removing a benzyl protecting group to give tetradepsipep-
1
CDCl3): δ 0.95 (d, J ) 6.5 Hz, 6H), 1.45 (s, 9H), 1.5-1.8 (m,
3H), 4.32 (bs, 1H), 4.93 (d, J ) 7.9 Hz, 1H), 6.13 (bs, 1H). The
bulk of this material (23.1 g, 100 mmol) was dissolved in DMF
(200 mL), and the solution was cooled to 0 °C. Cesium
carbonate (32.6 g, 100 mmol) was added, and the mixture was
stirred at 0 °C for 45-60 min. Benzyl bromide (11.9 mL, 17.1
g, 100 mmol) was added, and the reaction mixture was stirred
at 0 °C for 30 min and then at 25 °C overnight. The mixture
was poured into H2O (800 mL) and extracted with hexane (4
× 250 mL). The extracts were combined, washed in turn with
H2O (200 mL) and saturated NaCl, and then dried over Na2-
SO4. Filtration, concentration, and drying under high vacuum
gave benzyl N-Boc-L-leucinate (30.5 g, 95%) as clear, colorless
1
tide free acid 19 (2.05 g, 95%) as a glass. H NMR (400 MHz,
CDCl3): δ 0.85-1.00 (∼4d, 12H), 1.35-1.49 (m, 1H), 1.46 (s,
9H), 1.60 (m, 3H), 1.72 (m, 2H), 1.87 (m, 1H), 2.32 (m, 1H),
2.78 and 2.80 (2s, 3H), 3.14 (m, 1H), 3.19-3.38 (m, 3H), 4.78-
4.98 (m, 2H), 5.30 (m, 2H), 7.16-7.37 (m, 6H). MS (EI) m/z:
590 [M].
1
oil. [R]D -38° (c 0.99, MeOH). H NMR (400 MHz, CDCl3): δ
0.93 (2d, 6H), 1.44 (s, 9H), 1.46-1.54 (m, 1H), 1.60-1.73 (m,
2H), 4.36 (m, 1H), 4.90 (m, 1H), 5.14 (d, J ) 12.3 Hz, 1H),
5.20 (d, J ) 12.5 Hz, 1H), 7.37 (s, 5H). HRMS (FAB) m/z calcd
for C18H27NO4 + H1: 322.2018. Found: 322.2020. A portion
of this material (10.8 g, 33.6 mmol), CH2Cl2 (280 mL), and TFA
(70 mL) were combined and processed according to the general
procedure for removing a Boc protecting group, which gave
L-leucine benzyl ester 13 (7.58 g, ∼100%) as a clear, pale-yellow
oil. 1H NMR (400 MHz, CDCl3): δ 0.93 (2d, 6H), 1.47 (m, 1H),
1.60 (m, 1H), 1.79 (m, 1H), 2.15 (bs, 2H), 3.55 (m, 1H), 5.16 (s,
2H), 7.35 (bs, 5H).
N-Boc-N-Meth yl-L-leu cyl-D-la ctic Acid (21). Benzyl N-
Boc-N-methyl-L-leucyl-D-lactate2 20 (7.62 g, 18.7 mmol), ab-
solute EtOH (150 mL), and 10% Pd on activated carbon (1.50
g) were combined and processed according to the general
procedure for removing a benzyl protecting group to give
didepsipeptide free acid 21 (5.82 g, 98%) as a slightly turbid,
1
pale-yellow oil. H NMR (400 MHz, CDCl3): δ 0.89-1.00 (2d,
6H), 1.45 and 1.46 (2s, 9H), 1.50-1.80 (m, 6H), 2.81 (s, 3H),
4.74 and 4.84 (2m, 1H), 5.12 (m, 1H), 8.91 (bs, 1H).
Ben zyl (2S)-2-[(3R)-3-H yd r oxy-2-oxop yr r olid in yl]-4-
m eth ylp en ta n oa te (15) [γ-La cta m Ben zyl Ester 15]. A
solution of dioxolanone aldehyde 12 (6.40 g, 40.5 mmol) in
MeOH (25 mL) was added to a solution of L-leucine benzyl
ester 13 (8.96 g, 40.5 mmol) in MeOH (100 mL) at 0 °C under
an atmosphere of dry N2. Glacial HOAc (6.0 mL, 6.3 g, 105
mmol) was added, and the mixture was stirred for 30 min.
Powdered NaBH3CN (1.28 g, 20.3 mmol) was added, and the
mixture was stirred for 3 h at 25 °C. The reaction mixture
was poured into saturated NaHCO3 (200 mL), and the mixture
was swirled until CO2 evolution had abated. The mixture was
then extracted with Et2O (3×). The extracts were combined,
washed with saturated NaCl, dried (Na2SO4), and filtered. The
filtrate was concentrated to a turbid yellow oil. Chromatog-
raphy (30-70% EtOAc in hexane) gave γ-lactam benzyl ester
Ben zyl N-Meth yl-L-leu cyl-3-p h en yl-D-la cta te (23). Ben-
zyl N-Boc-N-methyl-L-leucyl-3-phenyl-D-lactate2 22 (8.57 g,
17.7 mmol), CH2Cl2 (50 mL), and TFA (6 mL) were combined
and processed according to the general procedure for removing
a Boc protecting group to give didepsipeptide free amine 23
1
(6.09 g, 90%) as a clear, pale-yellow oil. H NMR (400 MHz,
CDCl3): δ 0.75-0.82 (2d, 6H), 1.29 (t, J ) 7.2 Hz, 2H), 1.42
(bs, 1H), 1.49 (m, 1H), 2.20 (s, 3H), 3.08 (dd, J ) 9.8, 14.2 Hz,
1H), 3.18 (t, J ) 7.2 Hz, 1H), 3.28 (dd, J ) 4.0, 14.3 Hz, 1H),
5.15 (d, J ) 12.2 Hz, 1H), 5.20 (d, J ) 12.2 Hz, 1H), 5.32 (dd,
J ) 4.0, 9.9 Hz, 1H), 7.17-7.40 (m, 10H). HRMS (FAB) m/z
calcd for C23H29NO4 + H1: 384.2175. Found: 384.2183.
Ben zyl N-Boc-N-m eth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-
leu cyl-3-p h en yl-D-la cta te (24). Didepsipeptide free acid 21
(5.02 g, 15.8 mmol), didepsipeptide free amine 23 (6.06 g, 15.8
mmol), a solution of DCC in CH2Cl2 (1 M, 16 mL, 16 mmol),
DMAP (97 mg, 0.8 mmol), and CH2Cl2 (150 mL) were combined
and processed according to the general procedure for coupling
peptides using DCC. Chromatography (10-20% EtOAc in
hexane) of the crude reaction product gave tetradepsipeptide
1
15 (6.56 g, 53%) as a yellow oil. H NMR (400 MHz, CDCl3):
δ 0.92 (d, J ) 6.8 Hz, 3H), 0.94 (d, J ) 6.9 Hz, 3H), 1.47 (septet,
J ) 6.8 Hz, 1H), 1.73 (dd, J ) 7.4, 7.8 Hz, 2H), 1.92 (m, 1H),
2.42 (m, 1H), 3.33 (m, 2H), 4.15 (bs, 1H), 4.37 (dd, J ) 8.4, 8.7
Hz, 1H), 4.91 (dd, J ) 7.7, 8.4 Hz, 1H), 5.11 (d, J ) 12.3 Hz,
1H), 5.15 (d, J ) 12.3 Hz, 1H), 7.34 (m, 5H). HRMS (EI) m/z
calcd for C17H23NO4 305.1627. Found: 305.1615.
1
24 (8.68 g, 80%) as a clear, nearly colorless oil. H NMR (400
MHz, CDCl3): δ 0.80-1.00 (∼4d, 12H), 1.30-1.90 (m, 9H), 1.43
and1.46 (2s, 9H), 2.75 and 2.92 (2s, 1H), 2.84 (3s, 5H), 2.92 (s,
0.5H), 3.18 (bs, 2H), 4.68-5.42 (m, 4H), 5.09 (d, J ) 12.1 Hz,
1H), 5.16 (d, J ) 11.9 Hz, 1H), 7.10-7.40 (m, 10H). HRMS
(FAB) m/z calcd for C38H54N2O9 + H1: 683.3907. Found:
683.3911.
N-Boc-N-Meth yl-L-leu cyl-γ-la cta m F r ee Acid (17). N-
Boc-N-Methyl-L-leucine 16 (2.42 g, 9.86 mmol), γ-lactam benzyl
ester 15 (3.01 g, 9.86 mmol), a solution of DCC in CH2Cl2 (1
M, 9.9 mL, 9.9 mmol), DMAP (60 mg, 0.49 mmol), and CH2-
Cl2 (50 mL) were combined and processed according to the
general procedure for coupling peptides using DCC to give
crude product, which upon chromatography (20% EtOAc in
hexane) produced the elaborated γ-lactam benzyl ester (4.50
g, 86%) as a clear, colorless, viscous oil. MS (FAB) m/z: 533
[M + H]. This material (4.46 g, 8.37 mmol), absolute EtOH
(100 mL), and 10% Pd on activated carbon (700 mg) were
combined and processed according to the general procedure
for removing a benzyl protecting group to give γ-lactam free
acid 17 (3.63 g, 98%) as a white solid. 1H NMR (400 MHz,
CDCl3): δ 0.89-1.00 (∼3d, 12H), 1.43 (s, 9H), 1.52 (m, 2H),
1.75 (m, 4H), 1.96 (m, 1H), 2.53 (m, 1H), 2.76 and 2.78 (2s,
3H), 3.37 (m 1H), 3.50 (m, 1H), 4.70 (m, 0.5H), 4.86 (m, 1.5H),
5.38 (dd, J ) 8.1, 17.5 Hz, 1H), 8.0 (bs, 1H). MS (FAB) m/z:
443 [M + H], 465 [M + Na]. Anal. (C22H38N2O7) C, H, N.
Ben zyl N-Meth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-leu cyl-
3-p h en yl-D-la cta te (25). Tetradepsipeptide 24 (1.58 g, 2.31
mmol), CH2Cl2 (108 mL), and TFA (27 mL) were combined and
processed according to the general procedure for removing a
Boc protecting group to give tetradepsipeptide free amine 25
1
(1.24 g, 92%) as a clear, pale-yellow oil. H NMR (400 MHz,
CDCl3): δ 0.80-0.98 (∼5d, 12H), 1.28-1.90 (m, 7H), 1.46 (d,
J ) 6.9 Hz, 3H), 2.38 (s, 3H), 2.77 (s, 0.5H), 2.80 (s, 2H), 2.84
(s, 0.5H), 3.16 (m, 2H), 3.30 (t, J ) 7.3 Hz, 1H), 5.08 (d, J )
12.1 Hz, 1H), 5.14 (d, J ) 13.0 Hz, 1H), 5.25 (dd, J ) 5.2, 7.2
Hz, 1H), 5.34 (dd, J ) 5.0, 10.9 Hz, 1H), 5.48 (dd, J ) 6.8,
13.5 Hz, 1H), 7.10-7.41 (m, 10H). HRMS (FAB) m/z calcd for
C
33H46N2O7 + H1: 583.3383. Found: 583.3375.
N-Meth yl-L-leu cyl-γ-la cta m -3-p h en yl-D-la ctyl-N-m eth -
N-Boc-N-m et h yl-L-leu cyl-γ-la ct a m -3-p h en yl-D-la ct ic
Acid (19). γ-Lactam free acid 17 (3.50 g, 7.91 mmol), benzyl
3-phenyl-D-lactate4 18 (2.03 g, 7.91 mmol), a solution of DCC
in CH2Cl2 (1 M, 7.9 mL, 7.9 mmol), DMAP (48 mg, 0.40 mmol),
and CH2Cl2 (75 mL) were combined and processed according
to the general procedure for coupling peptides using DCC to
yl-L-leu cyl-D-la ct yl-N-m et h yl-L-leu cyl-3-p h en yl-D-la ct ic
Acid (26). Tetradepsipeptide free acid 19 (1.27 g, 2.15 mmol),
tetradepsipeptide free amine 25 (1.29 g, 2.21 mmol), a solution
of DCC (2.6 mL, 1 M, 2.6 mmol), DMAP (16 mg, 0.13 mmol),
and CH2Cl2 (75 mL) were combined and processed according
to the general procedure for coupling peptides using DCC.

2066 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
Chromatography of the crude reaction product gave the
octadepsipeptide (1.48 g) as a clear, colorless oil. Proton NMR
showed 92 wt % product and 8 wt % EtOAc for a yield of 55%.
1H NMR (400 MHz, CDCl3): δ 0.79-1.00 (∼6d, 24H), 1.43 (s,
9H), 1.38-1.50 (m, 4H), 1.50-1.99 (m, 13H), 2.70-3.30 (m,
15H), 4.60-5.50 (m, 10H), 7.08-7.40 (m, 15H). HRMS (FAB)
m/z calcd for C64H90N4O15 + Na1: 1177.6300. Found: 1177.6275.
This material (1.45 g, 1.25 mmol), CH2Cl2 (100 mL), and TFA
(25 mL) were combined and processed according to the general
procedure for removing a Boc protecting group to give the
octadepsipeptide free amine (1.32 g, ∼100%) as a clear,
colorless, viscous oil. 1H NMR (400 MHz, CDCl3): δ 0.78-1.02
(∼6d, 24H), 1.15-1.96 (m, 18H), 2.40 (s, 3H), 2.43-3.33 (m,
12H), 3.48 (m, 1H), 4.91 (dd, J ) 5.2, 10.6 Hz, 1H), 5.00-5.39
(m, 7H), 5.49 (m, 1H), 7.10-7.40 (m, 15H). HRMS (FAB) m/z
calcd for C59H82N4O13 + H1: 1055.5956. Found: 1055.5920.
The octadepsipeptide free amine (1.29 g, 1.22 mmol), absolute
EtOH (150 mL), and 10% Pd on activated carbon (225 mg) were
combined and processed according to the general procedure
for removing a benzyl protecting group to give octadepsipeptide
amino acid 26 (1.14 g, 97%) as a tan powder. 1H NMR (400
MHz, CDCl3): δ 0.75-1.20 (m, 24H), 1.20-2.02 (m, 14H), 1.28
(d, J ) 7.1 Hz, ∼2.5H), 1.41 (d, J ) 6.8, ∼0.5H), 2.30-3.52
(m, ∼9H), 2.51 (s, ∼1H), 2.56 (s, ∼1H), 2.72 (s, ∼1H), 2.80 (s,
∼1H), 2.88 (s, ∼1H), 3.00 (s, ∼1H), 3.60 (m, 1H), 4.76-4.90
(m, 1H), 5.06-5.51 (m, 5H), 5.57 (dd, J ) 7.1, 13.8 Hz, 1H),
6.88 (bs, 2H), 7.11-7.32 (m, 10H). HRMS (FAB) m/z calcd for
again concentrated to a yellow solid (4.64 g). Chromatography
(10% EtOAc in hexane) gave a 5:1 E/Z mixture of vinyl ether
27 (620 mg, 27%) as a clear, pale-yellow oil. 1H NMR (400
MHz, CDCl3): δ 1.52 (s, 3H), 1.57 (s, 3H), 2.32-2.70 (m, 2H),
3.50 (s, 2.5 H), 3.60 (s, 0.5 H), 4.40 (m, 1H), 4.70 (m, 1H), 6.02
(d, J ) 6.2, 0.5 H), 6.40 (d, J ) 12.6, 2.5 H). 13C NMR (100
MHz, CDCl3): δ 26.3 (Z), 26.4 (E), 26.7 (Z), 27.4 (E), 30.2 (E),
56.2 (E), 60.0 (Z), 74.3 (Z), 75.0 (E), 95.7 (E), 99.4 (Z), 110.8
(E), 115.6 (Z), 149.3 (Z), 150.7 (E), 172.9 (E, Z). MS (EI) m/z:
186 [M].
3-[(4R )-2,2-Dim e t h yl-5-oxo-1,3-d ioxola n -4-yl]p r op a -
n a l (29) a n d (5R)-5-(3,3-Dim eth oxyp r op yl)-2,2-d im eth yl-
1,3-d ioxola n -4-on e (28). Vinyl ether 27 (490 mg, 2.63 mmol)
was dissolved in acetone (25 mL) and treated with sulfuric
acid (,1 drop) at 25 °C. After 70 min, TLC showed that all of
the aldehyde had been consumed. The reaction mixture was
treated with several drops of saturated NaHCO3 and concen-
trated to dryness. The residue was taken up in Et2O and
washed with H2O. The organic layer was dried (MgSO4),
filtered, and concentrated to give a yellow oil (275 mg)
consisting of a 78:22 molar ratio of aldehyde 29 (44%) to acetal
28 (13%). This material was used without further purification.
1H NMR (400 MHz, CDCl3): δ 1.54 (s, 3H), 1.58 (s, 3H), 1.77
(m, 0.6 H), 1.95 (m, 0.2 H), 2.09 (m, 0.8 H), 2.25 (m, 0.8 H),
2.62 (m, 1.6 H), 3.31 (s, 0.6 H), 3.33 (s, 0.6 H), 4.40 (m, 0.4 H),
4.47 (m, 0.8 H), 4.73 (s, 0.8).
Ben zyl (2S)-2-[(3R)-3-Hyd r oxy-2-oxop ip er id in yl]-4-m e-
th ylp en ta n oa te (31) [δ-La cta m Ben zyl Ester 31] a n d
Ben zyl (2S)-2-({3-[(4R)-2,2-Dim eth yl-5-oxo-1,3-d ioxola n -
4-yl]p r op yl}a m in o)-4-m eth ylp en ta n oa te (30). Aldehyde 29
(76 mg, 0.44 mmol) was dissolved in MeOH (1.5 mL), and the
mixture was cooled to 0 °C. L-Leucine benzyl ester 13 (98 mg,
0.44 mmol) was added followed by HOAc (0.06 mL), and the
reaction mixture was stirred for 30 min. Sodium cyanoboro-
hydride (14 mg, 0.22 mmol) was added, the mixture was stirred
for 5 min, and the cooling bath was removed. The progress of
the reaction was monitored by TLC, which showed many spots.
The reaction mixture was stirred for 4 h, more NaCNBH3 (20
mg) was added, and the mixture was stirred overnight, after
which TLC showed fewer spots. The mixture was poured into
saturated NaHCO3, mixed thoroughly, and twice extracted
with Et2O . The extracts were combined, washed with 10%
citric acid, dried (MgSO4), filtered, and concentrated. A second
concentration from EtOAc and drying under high vacuum gave
a colorless, slightly turbid oil. This was further purified by
chromatography (20% EtOAc in hexane) to give δ-lactam
benzyl ester 31 (46 mg, 33%), free of secondary amine 30, as
a clear, colorless oil. In the case where the reaction time was
only 2 h, a 4:1 mixture (18% combined yield) of secondary
amine 30 and δ-lactam 31 was obtained. After standing 5 days
at 25 °C, most of the amine had lactamized to give a 6:1
mixture of δ-lactam to amine. Complete conversion to δ-lactam
31 was achieved by heating the mixture at 70 °C in dry toluene
C
52H76N4O13 + H1: 965.5487. Found: 965.5464.
γ-La cta m Cyclod ep sip ep tid e (2). Octadepsipeptide amino
acid 26 (1.11 g, 1.15 mmol) was dissolved in CH2Cl2 (1150 mL)
to give a concentration of 1 mM, and the solution was cooled
to 0 °C. BOP reagent (534 mg, 1.21 mmol) was added, and the
mixture was stirred until it was completely dissolved. N-
Methylmorpholine (0.13 mL, 1.21 mmol) was added, and the
reaction mixture was stirred at 0 °C for 30 min and then for
3 days at 25 °C. The reaction mixture was concentrated to
about 100 mL and washed with saturated NH4Cl (250 mL).
The layers were separated, and the organic layer was dried
(MgSO4), filtered through Celite, and concentrated. The resi-
due was dissolved in EtOAc and again concentrated. Drying
under high vacuum gave a light, tan foam. This was further
purified by chromatography (30-60% EtOAc in hexane) to give
γ-lactam cyclodepsipeptide 2 (289 mg, 27%) as a white solid.
[R]25 -93° (c 0.62, MeOH). 1H NMR (400 MHz, CDCl3): δ
D
0.75-1.00 (m, 24H), 1.00-1.80 (m, 14H), 1.05 (d, J ) 6.3 Hz,
∼0.7H), 1.12 (d, J ) 6.9 Hz, ∼0.8H), 1.47 (d, J ) 6.8 Hz, 1.5H),
2.80 (s, 2.2H), 2.81 (s, 1.4H), 2.82 (s, 1.3H), 2.86 (s, 1.4H), 2.88
(s, 1.7H), 3.01 (s, 1H), 3.01-3.50 (m, 6H), 4.44 (m, 1H), 4.73-
5.71 (m, 7H), 7.17-7.34 (m, 10H). MS (FAB) m/z: 947 [M +
H], 1079 [M + Cs]. HRMS (FAB) m/z calcd for C52H74N4O12
+
Na1: 969.5201. Found: 969.5228. HPLC: tR ) 13.48 min (RP
8 column, 99.2% pure, gradient from 65% to 95% acetonitrile
+ 0.1% TFA/H2O + 0.1% TFA over 20 min); tR ) 13.41 min
(RP 4 column, 99.0% pure, gradient from 40% to 90% aceto-
nitrile + 0.1% TFA/H2O + 0.1% TFA over 20 min).
1
for 90 min. H NMR (400 MHz, CDCl3): δ 0.94 (s, 3H), 0.96
(s, 3H), 1.52 (m, 1H), 1.57-1.96 (m, 6H), 2.28 (m, 1H), 3.22
(m, 2H), 4.07 (dd, J ) 4.73, 6.46 Hz, 1H), 5.09 (d, J ) 12.3 Hz,
1H), 5.17 (m, 1H), 5.18 (d, J ) 12.0 Hz, 1H), 7.34 (m, 5H).
Syn th esis of δ-La cta m Cyclod ep sip ep tid e (3). Syn th e-
sis of δ-La cta m Ben zyl Ester (31). (5R)-5-[(E)-3-Meth oxy-
2-p r op en yl]-2,2-d im eth yl-1,3-d ioxola n -4-on e (27). A solu-
tion of phenyllithium in 70:30 cyclohexane/ether (1.8 M, 13.6
mL, 24.4 mmol) was added dropwise over a period of 10 min
to a slurry of (methoxymethyl)triphenylphosphonium chloride
(8.37 g, 24.4 mmol) in anhydrous ether (50 mL) at -20 to -5
°C under N2, which produced a red solution along with some
solid. The reaction mixture was stirred at 0 °C for 30 min and
then cooled to -78 °C. A solution of aldehyde 12 (1.93 g, 12.2
mmol) was added dropwise over a period of 15 min. The
reaction mixture was stirred at -78 °C for 45 min and then
at 25 °C for 2 h. Saturated NH4Cl (25 mL) was added dropwise.
Further dilution with H2O caused all solids to dissolve. The
mixture was shaken briskly, and the layers were separated.
The aqueous layer was extracted with ether, and the organic
layers were combined, washed in turn with H2O, saturated
NaHCO3, and saturated NaCl and then dried (Na2SO4). The
solution was filtered, concentrated, dissolved in EtOAc, and
HRMS (FAB) m/z calcd for
Found: 320.1862 (sic).
C18H25NO4 + H1: 320.1862.
Syn th esis of δ-La cta m Ben zyl Ester (39), a Dia ster eo-
m er of δ-La cta m Ben zyl Ester (31). Meth yl 5-[N-((1S)-1-
Ben zyloxycar bon yl-3-m eth ylbu tyl)am in o]pen tan oate (33).
Methyl 5-bromovalerate 32 (5.47 g, 28.1 mmol), L-leucine
benzyl ester 13 (6.21 g, 28.1 mmols), and powdered NaHCO3
(4.71 g, 56.1 mmol) were combined, and DMF (25 mL) was
added. The reaction mixture was heated at 130 °C for 90 min
and then cooled to room temperature. The mixture was diluted
with H2O and extracted with Et2O (3×). The extracts were
combined, washed in turn with H2O and saturated NaCl, and
then dried (Na2SO4). Filtration followed by concentration of
the filtrate gave secondary amine 33 (8.53 g, 91%) as a clear,
pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.88 (d, J ) 6.6
Hz, 3H), 0.91 (d, J ) 6.6 Hz, 3H), 1.49 (m, 3H), 1.67 (m, 4H),
1.83 (bs, 1H), 2.30 (m, 2H), 2.45 (m, 1H), 2.59 (m, 1H), 3.32

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2067
(m, 1H), 3.68 (s, 3H), 5.17 (s, 2H), 7.35 (s, 5H). MS (FAB) m/z:
336 [M + H].
Ben zyl (2S)-2-(2,3-Dioxo-1-p ip er id in yl)-4-m eth ylp en -
ta n oa te (38). A solution of DMSO (2.0 mL, 28.8 mmol) in CH2-
Cl2 (6 mL) was added in a slow stream to a solution of oxalyl
chloride (1.2 mL, 14.4 mmol) in CH2Cl2 (25 mL) at -78 °C,
and the mixture was stirred for 10 min. A solution of hydroxy-
δ-lactam 36 (4.18 g, 13.1 mmol) in CH2Cl2 (8 mL) was added
in a slow stream, and the reaction mixture was stirred at -78
°C for 60 min. Triethylamine (8.0 mL, 57.6 mmol) was added
in a slow stream at -78 °C. The cooling bath was removed,
and the mixture was stirred for 45 min at ambient tempera-
ture. The reaction mixture was poured into H2O and extracted
with CH2Cl2 (2×). The organic layers were combined and
washed in turn with 1 N H2SO4 and saturated NaHCO3 and
then dried (Na2SO4), filtered and concentrated under high
vacuum to a red oil, which solidified on standing overnight.
This was triturated with boiling hexane containing a small
amount of EtOAc and then chilled at 0 °C for 3 h. The solid
was removed by filtration and air-dried. Further drying to
constant weight under vacuum at 60 °C gave keto-δ-lactam
Ben zyl (2S)-4-Meth yl-2-(2-oxo-1-piper idin yl)pen tan oate
(34). Secondary amine 33 (8.53 g, 25.4 mmol) was combined
with mixed xylenes and heated under reflux for 42 h, during
which time the reaction mixture became orange but remained
clear. The solvent was removed under vacuum, and the residue
was purified by chromatography (20-30% EtOAc in hexane)
to give δ-lactam 34 (5.10 g, 66%) as a clear, light-yellow oil.
1H NMR (400 MHz, CDCl3): δ 0.94 (2d, 6H), 1.50 (m, 1H),
1.75 (m, 6H), 2.43 (m, 2H), 3.19 (m, 2H), 5.10 (d, J ) 12.4 Hz,
1H), 5.18 (d, J ) 12.3 Hz, 1H), 5.39 (dd, J ) 6.4, 9.5 Hz, 1H),
7.35 (m, 5H). MS (EI) m/z: 303 [M].
Ben zyl (2S)-2-(3-Br om o-2-oxo-1-p ip er id in yl)-4-m et h -
ylp en ta n oa te (35). δ-Lactam 34 (3.93 g, 13.0 mmol) was
dissolved in CH2Cl2 (100 mL), Et3N (9.0 mL, 65 mmol) was
added, and the reaction mixture was cooled to -20 °C.
Chlorotrimethylsilane (3.3 mL, 26 mmol) was added, and the
mixture was stirred for 7 min. Small crystals of iodine (4.9 g,
19 mmol) were added, and stirring was continued at -20 °C
for another 15 min, after which bromine (5.0 mL, 97 mmol)
was added and the reaction mixture was stirred at 0 °C for
2.3 h. The reaction was quenched by adding 10% Na2SO3 (50
mL). The mixture was transferred to a separatory funnel
where more Na2SO3 solution (100 mL) was added. The mixture
was shaken vigorously, causing it to become extremely dark.
After addition of another 100 mL of Na2SO3 solution and more
vigorous shaking, the mixture became light-yellow. The layers
were separated, and the aqueous layer was extracted with CH2-
Cl2 (2×). The extracts were combined and washed with 10%
Na2SO3 (150 mL), causing both layers to become pale-amber
in color. The layers were separated, and the organic material
was washed in turn with H2O and saturated NaCl and then
dried over Na2SO4. Filtration, concentration, and drying under
high vacuum gave a red-orange oil. This was purified by
chromatography (20% EtOAc in hexane) to give bromo-δ-
lactam 35 (4.20 g, 85%) as a mixture of diastereomers in the
form of a clear, very pale-yellow oil, which solidified on
standing for 30 min at room temperature. 1H NMR (400 MHz,
CDCl3): δ 0.94 (3d, 6H), 1.51 (m, 1H), 1.79 (m, 3H), 2.22 (m,
3H), 3.30 (m, 2H), 4.60 (m, 1H), 5.08-5.25 (m, 3H), 7.37 (m,
5H). HRMS (FAB) m/z calcd for C18H24BrNO3 + H1: 382.1018.
Found: 382.1021. Anal. (C18H24BrNO3) C, H, N, Br.
38 (3.12 g, 75%) as an off-white powder. [R]25 -47° (c, 0.97,
D
MeOH). 1H NMR (400 MHz, CDCl3): δ 0.93-0.97 (2d, J ≈ 6.6
Hz, 6H), 1.50 (m, 1H), 1.72-1.89 (m, 2H), 2.07-2.28 (m, 2H),
3.38-3.56 (m, 2H), 5.12 (d, J ) 12.2 Hz, 1H), 5.20 (d, J ) 12.2
Hz, 1H), 5.40 (dd, J ) 5.33, 12.2 Hz, 1H), 7.34 (m, 5H). 13C
NMR (100 MHz, CDCl3): δ 21.6, 22.1, 23.6, 25.4, 37.3, 38.9,
44.1, 55.5, 67.6, 128.6, 128.8, 129.0, 135.6, 158.5, 171.0, 191.5.
HRMS (EI) m/z calcd for
C18H23NO4: 317.1627. Found:
317.1621. Anal. (C18H23NO4) C, H, N.
Ben zyl (2S)-2-[(3S)-3-Hyd r oxy-2-oxop ip er id in yl]-4-m e-
th ylp en ta n oa te (39) [δ-La cta m Ben zyl Ester 39]. Baker’s
yeast (60 g) and D-glucose (6.0 g) were combined, H2O (160
mL) was added, and the mixture was swirled until the yeast
was completely wetted. Keto-δ-lactam 38 (3.03 g, 9.55 mmol)
was added as a powder. The clumps of solid, which formed
during the first hour, were broken up with a stirring rod. The
reaction mixture was stirred overnight at room temperature,
during which it increased severalfold in volume. The mixture
was filtered through Celite, an arduous process that required
repeated scraping of the Celite pad to facilitate filtration. When
the H2O had been mostly removed, the residue was mixed with
a large amount of sand, transforming the solids into a more
granular mixture, which facilitated stirring. The mixture was
stirred with EtOAc, and the solvent was removed by filtration.
This process was repeated four times. The filtrates were
combined and partially dried (Na2SO4), filtered, and concen-
trated to a brown oil. The oil was taken up in Et2O and filtered
to remove the insoluble material. Concentration of the filtrate
produced a clear, brown oil, which partially solidified on
standing at room temperature. Purification by chromatography
(20-40% EtOAc in hexane) gave δ-lactam benzyl ester 39 (1.81
g, 59%) as a clear, amber-colored oil. This material was
triturated with Et2O/hexane while warming, which caused it
to solidify. Filtration and air-drying gave δ-lactam benzyl ester
Ben zyl (2S)-2-(3-Hyd r oxy-2-oxo-1-p ip er id in yl)-4-m eth -
ylp en t a n oa t e (36) a n d Ben zyl (2S)-2-[3-(F or m yloxy)-2-
oxo-1-p ip er id in yl]-4-m et h ylp en t a n oa t e (37). Bromo-δ-
lactam 35 (3.90 g, 10.0 mmol) was dissolved in dry formamide
(75 mL) to which had been added H2O (0.18 mL, 10.0 mmol).
The reaction mixture was heated at 150 °C for 65 min, cooled
to room temperature, diluted with H2O, and extracted with
Et2O (3×). The extracts were combined, washed with H2O (2×)
and saturated NaCl, and then dried (Na2SO4), filtered, and
concentrated. The residue was purified by chromatography
(40% EtOAc in hexane) to give hydroxy-δ-lactam 36 (2.54 g,
80%) as a mixture of diastereomers in the form of a clear, light-
yellow oil, which solidified on standing to a waxy solid. 1H
NMR (400 MHz, CDCl3): δ 0.91-0.96 (3d, J ≈ 6.6 Hz, 6H),
1.50 (m, 1H), 1.60-1.98 (m, 5H), 2.28 (m, 1H), 3.14-3.20 (m,
2H), 3.48 (bs, 1H), 4.05 (m, 1H), 5.06-5.21 (m, 2H), 5.26 (dd,
J ) 5.4, 10.7 Hz, 1H), 7.35 (m, 5H). When this reaction was
performed on a larger scale (9.74 g of 35) and over a longer
reaction time (∼2 h), only a 53% yield (4.20 g) of hydroxy-δ-
lactam 36 was obtained along with a 21% yield (1.80 g) of
formate 37. These were separated by chromatography (15-
20% EtOAc in hexane) to give 37 as a clear, light-yellow oil.
1H NMR (400 MHz, CDCl3): δ 0.90-0.96 (3d, J ≈ 6.7 Hz, 6H),
1.50 (m, 1H), 1.68-2.03 (m, 5H), 2.20 (m, 1H), 3.27 (m, 2H),
5.08-5.42 (m, 4H), 7.36 (m, 5H), 8.15 (2s, 1H). 13C NMR (100
MHz, CDCl3): δ 20.3, 20.5, 21. 8, 21.9, 23.5, 23.6, 25.3, 25.4,
27.1, 27.2, 37.2, 37.3, 43.5, 44.3, 54.9, 55.1, 67.3, 67.3, 68.9,
69.4, 128.5, 128.6, 128.7, 128.8, 128.9, 129.0, 135.9, 160.4,
160.4, 167.3, 167.7, 171.5, 171.5. MS (FAB) m/z: 348 [M +
H], 695 [2M + H]. Anal. (C19H25NO5) C, H, N.
1
39 (1.52 g, 50%) as a nearly white powder. H and 13C NMR
showed the presence of only one diastereomer, suggesting an
optical purity of g95% de as determined by comparison with
δ-lactam benzyl ester 31 whose stereochemistry was unam-
biguously established by an alternative synthesis.16 1H NMR
(400 MHz, CDCl3): δ 0.90-0.96 (2d, J ≈ 6.6 Hz, 6H), 1.48 (m,
1H), 1.60-1.84 (m, 4H), 1.84-1.98 (m, 1H), 2.27 (m, 1H), 3.15-
3.30 (m, 2H), 3.63 (bs, 1H), 4.06 (dd, J ) 6.5, 10.9 Hz, 1H),
5.12 (d, J ) 12.3 Hz, 1H), 5.19 (d, J ) 12.3 Hz, 1H), 5.25 (dd,
J ) 10.7, 5.4 Hz, 1H), (7.34 (m, 5H). 13C NMR (100 MHz,
CDCl3): δ 20.2, 21.7, 23.5, 25.3, 28.3, 37.3, 43.1, 54.8, 67.3,
68.2, 128.5, 128.8, 129.0, 135.8, 171.7, 174.2. Anal. (C18H25
NO4) C, H, N.
-
N-Boc-N-Meth yl-L-leu cyl-δ-lactam Fr ee Acid (40). δ-Lac-
tam benzyl ester 39 (1.49 g, 4.66 mmol), N-Boc-N-methyl-L-
leucine 16 (1.14 g, 4.66 mmol), and triphenylphosphine (1.22
g, 4.66 mmol) were combined. THF (100 mL) was added, and
the mixture was stirred until all solids had dissolved. The
solution was cooled to 0 °C, and DEAD (0.88 mL, 5.60 mmol)
was added. The reaction mixture was stirred at room temper-
ature for 110 min and then concentrated. Chromatography

2068 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
(20% EtOAc in hexane) of the residue produced 2.30 g of clear,
colorless oil, which H NMR showed to contain 93 wt % product
MHz, CDCl3): δ 0.78-1.05 (5d, 24 H), 1.17-2.12 (m, 20 H),
2.40-3.30 (3s + m, 15 H), 3.47 (m, 1H), 5.09 (d, J ) 12.1 Hz,
1H), 5.14 (d, J ) 12.0 Hz, 1H), 5.19-5.57 (m, 7H), 7.03-7.41
(m, 15 H). The octadepsipeptide free amine (452 mg, 0.423
mmol), absolute EtOH (100 mL), and 10% Pd on activated
carbon (80 mg) were combined and processed according to the
general procedure for removing a benzyl protecting group. This
produced 403 mg of cream-colored solid, which 1H NMR
showed to consist of 96 wt % octadepsipeptide amino acid and
4 wt % EtOAc. This amounts to 387 mg (93%) of octadepsipep-
1
and 7 wt % EtOAc. This amounts to 2.14 g (84%) of the
elaborated δ-lactam benzyl ester. 1H NMR (400 MHz, CDCl3):
δ 0.93 (3d, 12 H), 1.45 (s, 9H), 1.48-2.19 (m, 10 H), 2.79 (m,
3H), 3.22 (m, 2H), 4.69-5.27 (m, 2H), 5.08 (d, J ) 12.3 Hz,
1H), 5.17 (d, J ) 12.3 Hz, 1H), 5.30 (m, 1H), 7.32 (m, 5H).
This material (2.11 g, 3.86 mmol), absolute EtOH (100 mL),
and 10% Pd on activated carbon (300 mg) were combined and
processed according to the general procedure for removing a
benzyl protecting group. This produced 1.83 g of white foam
and glass, which 1H NMR showed to contain 95 wt % product
and 5 wt % EtOAc. This amounts to 1.74 g (99%) of δ-lactam
free acid 40. 1H NMR (400 MHz, CDCl3): δ 0.93 (3d, 12 H),
1.38-2.24 (m, 10 H), 1.45 (s, 9H), 2.76 (2s, 3H), 3.30 (bs, 2H),
4.73 and 4.93 (2m, 1H), 5.22 (m, 2H), 9.40 (bs, 1H).
1
tide amino acid 43. H NMR (400 MHz, CDCl3): δ 0.78-1.08
(4d, 24 H), 1.28 (m, 4H), 1.40-2.02 (m, 16 H), 2.43-2.70 (s +
m, 3H), 2.70-3.38 (2s + m, 12 H), 3.74 (m, 1H), 5.00-5.60
(m, 7H), 6.69 (bs, 1H), 7.25 (m, 10 H).
δ-La cta m Cyclod ep sip ep tid e (3). Octadepsipeptide amino
acid 43 (388 mg, 0.396 mmol) was dissolved in CH2Cl2 (400
mL) to give a 1 mM solution, which was then cooled to 0 °C.
DPEA (0.17 mL, 1.0 mmol) was added followed by BOP-Cl (121
mg, 0.475 mmol), and the reaction mixture was stirred at 25
°C for 24 h. When TLC showed the reaction to be incomplete,
more DPEA (0.20 mL) and BOP-Cl (143 mg) were added. After
the mixture was stirred an additional 24 h, the reaction was
essentially complete. The reaction mixture was washed with
aqueous NaHCO3, filtered through Na2SO4, and dried (MgSO4).
Filtration, concentration, and drying under high vacuum
produced a solid, which was dissolved in EtOAc and filtered
through Celite to remove a small amount of insoluble material.
The filtrate was concentrated and then chromatographed (40-
50% EtOAc in hexane) to give an off-white solid. To remove
residual EtOAc, the solid was twice dissolved in 4:1 hexane/
CH2Cl2 and concentrated. Drying under high vacuum gave
δ-lactam cyclodepsipeptide 3 (249 mg, 65%) as a nearly
colorless, solid foam and glass. HPLC analysis showed this
material to be 99.8% pure and free of diastereomers (tR was
16.7 min on a 250 mm Vydac pH stable RP C8 column, with
product eluting with 40-90% CH3CN/H2O at 1 mL/min). 1H
NMR showed the product to consist of a mixture of conformers.
[R]25D -87° (c 1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ 0.70-
1.20 (m, 27 H), 1.20-2.30 (m, 19 H), 2.67-3.00 (5s, 9H), 3.00-
3.37 (m, 6H), 4.44 (m, 1H), 4.78 (m, 0.5 H), 5.05 (m, 0.5 H),
5.18-5.45 (m, 4H), 5.55-5.68 (m, 1H), 5.80-5.90 (m, 1H), 7.25
(m, 10 H). MS (FAB) m/z: 961 [M + H], 983 [M + Na], 1093
[M + Cs]. HRMS (FAB) m/z calcd for C53H76N4O12 + H1:
961.5538. Found: 961.5528. IR (KBr, cm-1): 1743 (ester
carbonyl), 1663 (amide carbonyl). UV (1%, 1 cm, MeOH) λmax
(ꢀ): 250 (5.0), 257 (4.6), 263 (3.4). Anal. (C53H76N4O12) C, H,
N.
Ben zyl N-Meth yl-L-leu cyl-δ-la cta m -3-p h en yl-D-la cta te
(41). δ-Lactam free acid 40 (1.71 g, 3.75 mmol), benzyl
3-phenyl-D-lactate4 18 (0.96 g, 3.75 mmol), a solution of DCC
in CH2Cl2 (1 M, 4.1 mL, 4.1 mmol), DMAP (23 mg, 0.19 mmol),
and CH2Cl2 (50 mL) were combined and processed according
to the general procedure for coupling peptides using DCC. The
crude reaction product was chromatographed (15-20% EtOAc
in hexane), which produced 1.28 g of clear, colorless oil. 1H
NMR showed this to contain 98 wt % product and 2 wt %
EtOAc. This amounts to 1.25 g (48%) of tetradepsipeptide.
HPLC analysis showed this material to consist of a 11:1
1
mixture of two diastereomers. H NMR (400 MHz, CDCl3): δ
0.93 (3d, 12 H), 1.46 (s, 9H), 1.35-2.03 (m, 10 H), 2.80 (2s,
3H), 3.01 (m, 2H), 3.16 (m, 2H), 4.70-5.55 (m, 6H), 7.12 (m,
2H), 7.27 (m, 5H), 7.36 (m, 3H). A second chromatography
fraction (0.82 g) contained a different mix of diastereomers.
The tetradepsipeptide (600 mg, 0.863 mmol), CH2Cl2 (16 mL),
and TFA (4 mL) were combined and processed according to
the general procedure for removing a Boc protecting group.
This produced tetradepsipeptide free amine 41 (483 mg, 94%),
which was used without further purification. 1H NMR (400
MHz, CDCl3): δ 0.93 (4d, 12 H), 1.37-2.37 (m, 11 H), 2.47 (s,
3H), 2.96-3.02 (m, 2H), 3.15 (m, 2H), 3.33 (t, J ) 7.25 Hz,
1H), 5.10 (d, J ) 12.1 Hz, 1H), 5.15 (d, J ) 12.0 Hz, 1H), 5.21-
5.33 (m, 2H), 5.43 (dd, J ) 5.20, 10.4 Hz, 1H), 7.11 (m, 2H),
7.27 (m, 5H), 7.37 (m, 3H).
N-Boc-N-m eth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-leu cyl-3-
p h en yl-D-la ctic Acid (42). Tetradepsipeptide 24 (11.8 g, 17.3
mmol), absolute EtOH (200 mL), and 10% Pd on activated
carbon (2.5 g) were combined and processed according to the
general procedure for removing a benzyl protecting group,
which gave tetradepsipeptide free acid 42 (10.3 g, ∼100%) as
Syn th esis of E-La cta m Cyclod ep sip ep tid e (4). (E)-4-
[(4R)-2,2-Dim eth yl-5-oxo-1,3-dioxolan -4-yl]-2-bu ten al (44).
Dioxolanone aldehyde 12 (9.65 g, 61.0 mmol) and (triphen-
ylphosphoranylidene)acetaldehyde (18.6 g, 61.1 mmol) were
combined with toluene (200 mL), and the mixture was heated
at 80 °C for 90 min. The reaction mixture was cooled to room
temperature and filtered through silica gel with Et2O. The
filtrate was concentrated to a mushy solid, which was tritu-
rated with 1:1 Et2O/hexane. Filtration followed by concentra-
tion of the filtrate and drying under high vacuum gave crude
product (9.09 g) as a clear, orange oil. Chromatography (20%
EtOAc in hexane) produced R,â-unsaturated aldehyde 44 (3.94
g, 46%) as an orange oil. [This material is sensitive to air
a white, solid foam. [R]25 -26° (c 1.13, MeOH). 1H NMR (400
D
MHz, CDCl3): δ 0.89 (m, 12 H), 1.23-1.86 (m, 18 H), 2.74-
3.05 (5s, 6H), 3.07-3.47 (m, 2H), 4.35-5.62 (8m, 4H), 7.02-
7.37 (m, 5H), 7.42 (bs, 1H). HRMS (FAB) m/z calcd for
C
31H48N2O9 + H1: 593.3438. Found: 593.3445. Anal. (C31H48
-
N2O9) C, H, N.
N-Meth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-
D-lactyl-N-m eth yl-L-leu cyl-δ-lactam -3-ph en yl-D-lactic Acid
(43). Tetradepsipeptide free acid 42 (461 mg, 0.778 mmol),
tetradepsipeptide free amine 41 (463 mg, 0.778 mmol), a
solution of DCC in CH2Cl2 (1 M, 0.8 mL, 0.8 mmol), DMAP
(4.8 mg, 0.039 mmol), and CH2Cl2 (20 mL) were combined and
processed according to the general procedure for coupling
peptides using DCC. This produced a solid foam and glass (940
mg), which was purified by chromatography (25-30% EtOAc
in hexane). From the appropriate fraction, 576 mg of clear,
yellow oil was obtained, which 1H NMR showed to contain 92
wt % product and 8 wt % EtOAc. This amounts to 530 mg
(58%) of octadepsipeptide. 1H NMR (400 MHz, CDCl3): δ 0.87
(4d, 24 H), 1.30-2.01 (m, 19 H), 1.45 (m, 9H), 2.75-3.28 (2s
+ m, 15 H), 4.41-5.50 (m, 8H), 5.09 (d, J ) 12.0 Hz, 1H), 5.14
(d, J ) 12.0 Hz, 1H), 7.05-7.44 (m, 15 H). The octadepsipep-
tide (512 mg, 0.437 mmol), CH2Cl2 (16 mL), and TFA (4 mL)
were combined and processed according to the general proce-
dure for removing a Boc protecting group. This gave the
octadepsipeptide free amine (462 mg, 99%). 1H NMR (400
1
oxidation.] H NMR (400 MHz, CDCl3): δ 1.56 (s, 3H), 1.61
(s, 3H), 2.75 (m, 1H), 2.91 (m, 1H), 4.57 (m, 1H), 6.25 (m, 1H),
6.80 (m, 1H), 9.54 (m, 1H). 13C NMR (100 MHz, CDCl3): δ
26.0, 27.4, 34.7, 72.8, 111.5, 136.3, 150.4, 172.0, 193.5. HRMS
(EI) m/ z calcd for C9H12O4: 184.0735. Found: 184.0743. [In
one case, high vacuum-drying of a faster eluting chromatog-
raphy fraction gave a small amount of methyl ester 9 as a
clear, yellow oil. This material sometimes formed during the
preparation of dioxolanone acid 10 and was harmlessly carried
forward in a synthesis until it was removed during a later
chromatography.]
Ben zyl (2S)-2-({3-Meth oxy-4-[(4R)-2,2-d im eth yl-5-oxo-
1,3-d ioxola n -4-yl]bu tyl}a m in o)-4-m eth ylp en ta n oa te (45).
L-Leucine benzyl ester 13 (401 mg, 1.81 mmol) was dissolved

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2069
in MeOH (5 mL), and the solution was cooled in an ice/water
bath under a nitrogen atmosphere. R,â-Unsaturated aldehyde
44 (334 mg, 1.81 mmol) was added followed by glacial HOAc
(0.2 mL, 210 mg, 3.49 mmol). The reaction mixture was stirred
at 0 °C for 30 min. Powdered NaBH3CN (56 mg, 0.89 mmol)
was added, and the mixture was stirred at room temperature
for 4 h. The reaction mixture was diluted with Et2O and poured
into a 1:1 solution of saturated NaHCO3 and H2O. The mixture
was shaken, the layers were separated, and the aqueous layer
was extracted with Et2O . The organic layers were combined,
washed in turn with Et2O and saturated NaCl, and then dried
(Na2SO4). Filtration and concentration of the filtrate led to a
slightly turbid, yellow oil (696 mg). This was purified by
chromatography (20% EtOAc in hexane) to give methoxyl-
amine 45 (296 mg, 39%) as a clear, light-yellow oil. NMR
spectroscopy showed this to be a 1:1 mixture of both epimers.
1H NMR (400 MHz, CDCl3): δ 0.89 (m, 6 H), 1.50 (m, 2 H),
1.55 (2s, 3 H), 1.60 (s, 3 H), 1.71 (m, 4 H), 1.90 (m, 1 H), 2.09
(m, 1 H), 2.56 (m, 1 H), 2.70 (m, 1 H), 3.30 (s, 1.5 H), 3.31 (m,
1 H), 3.34 (s, 1.5 H), 3.50 (m 0.5 H), 3.58 (m, 0.5 H), 4.47 (m,
0.5 H), 4.53 (m, 0.5 H), 5.18 (s, 2 H), 7.35 (s, 5 H). HRMS (FAB)
m/z calcd for C23H35NO6 + H1: 422.2542. Found: 422.2552.
The slow conversion of this material to ꢀ-lactam 46 by heating
it in refluxing xylene was monitored by mass spectrometry on
selected HPLC fractions during the 90 h required for 90%
completion.
light-yellow oil. [R]25D -7° (c 1.22, MeOH). 1H NMR (400 MHz,
CDCl3): δ 0.88 (d, J ) 6.6 Hz, 3H), 0.91 (d, J ) 6.6 Hz, 3H),
1.50 (m, 6H), 1.52 (s, 3H), 1.60 (s, 3H), 1.69 (m, 2H), 1.88 (m,
1H), ∼2.0 (bs, 1H), 2.48 (m, 1H), 2.60 (m, 1H), 3.32 (t, J ) 7.3
Hz, 1H), 4.35 (dd, J ) 4.3, 7.3 Hz, 1H), 5.18 (s, 2H), 7.36 (s,
5H). 13C NMR (100 MHz, CDCl3): δ 22.8, 22.9, 22.9, 25.2, 26.1,
27.5, 29.9, 31.7, 42.9, 48.0, 60.4, 66.7, 74.3, 110.7, 128.6, 128.7,
128.9, 136.1, 173.6, 176.0. HRMS (FAB) m/z calcd for C22H33
NO5 + H1: 392.2437. Found: 392.2437 (sic). Anal. (C22H33
NO5) C, H, N.
-
-
Ben zyl (2S)-2-[(3R)-3-Hyd r oxy-2-oxoa zep a n yl]-4-m eth -
ylp en ta n oa te (51) [E-La cta m Ben zyl Ester 51]. Secondary
amine 50 (2.55 g, 6.51 mmol) was dissolved in mixed xylenes
and heated at reflux under an atmosphere of dry N2 for 115
h. The reaction mixture was concentrated to a dark-brown oil
(2.56 g), which was dissolved in Et2O and treated with
activated charcoal for 20 min at room temperature. Filtration
through Celite and concentration gave crude product as a clear,
light-yellow oil. Chromatography (20% EtOAc in hexane)
followed by drying under high vacuum produced ꢀ-lactam
benzyl ester 51 (1.71 g, 79%) as a clear, pale-yellow oil. [R]25
D
1
-37° (c 1.12, MeOH). H NMR (400 MHz, CDCl3): δ 0.95 (d,
J ) 6.6 Hz, 3H), 0.98 (d, J ) 6.5 Hz, 3H), 1.30 (m, 1H), 1.42-
1.74 (m, 5H), 2.82 (m, 2H), 2.00 (m, 2H), 3.27 (m, 2H), 4.28
(dd, J ) 2.3, 11.4 Hz, 1H), 5.10 (d, J ) 12.3 Hz, 1H), 5.17 (d,
J ) 12.2 Hz, 1H), 5.31 (dd, J ) 4.4, 10.3 Hz, 1H), 7.36 (m,
5H). 13C NMR (100 MHz, CDCl3): δ 22.1, 23.5, 25.4, 27.5, 28.4,
33.9, 38.2, 46.2, 57.4, 67.4, 71.0, 128.5, 128.8, 129.0, 135.8,
171.5, 177.3. HRMS (EI) m/z calcd for C19H27NO4: 333.1940.
4-[(4R)-2,2-Dim eth yl-5-oxo-1,3-dioxolan -4-yl]bu tan al (48)
a n d 4-[(4R)-2,2-Dim eth yl-5-oxo-1,3-d ioxola n -4-yl]bu ta n o-
ic Acid (49). R,â-Unsaturated aldehyde 44 (3.19 g, 17.3 mmol)
was dissolved in EtOAc (200 mL). Palladium on activated
carbon (5 wt %, 180 mg) was added, and the mixture was
hydrogenated at balloon pressure with stirring at room tem-
Found: 333.1948. HRMS (FAB) m/z calcd for C19H27NO4
+
H1: 334.2018. Found: 334.2030. IR (mull, cm-1): 1739 (ester
carbonyl), 1642 (amide carbonyl). UV (1%, 1 cm, MeOH) λmax
(ꢀ): 257 (6.8), 262 (5.5), 266 (3.5). Anal. (C19H27NO4) C, H, N.
1
perature for 3 h. When H NMR indicated incomplete reaction,
the reaction mixture was flushed with N2, more catalyst was
(205 mg) added, and hydrogenation continued for another 3
h. After being flushed with N2, the reaction mixture was
vacuum-filtered through Celite and the filtrate was concen-
N-Boc-N-Meth yl-L-leu cyl-E-la cta m F r ee Acid (52). N-
Boc-N-methyl-L-leucine 16 (1.19 g, 4.86 mmol), ꢀ-lactam benzyl
ester 51 (1.62 g, 4.86 mmol), a solution of DCC in CH
2Cl2 (1
M, 4.9 mL, 4.9 mmol), DMAP (30 mg, 0.24 mmol), and CH2-
Cl2 (25 mL) were combined and processed according to the
general procedure for coupling peptides using DCC. Chroma-
tography (20% EtOAc in hexane) of the crude reaction product
and high vacuum-drying gave the elaborated ꢀ-lactam benzyl
1
trated to an oily residue (3.13 g). Since H NMR revealed an
unacceptably high level of acid 49 (26 mol % acid and 74 mol
% saturated aldehyde) apparently arising from air oxidation
of 44, the crude product was chromatographed (20-30% EtOAc
in hexane), which gave saturated aldehyde 48 (1.23 g) as a
clear, colorless oil. NMR indicated that this material was still
contaminated with acid 49 (9 wt %). Nonetheless, the material
was judged to be suitable for further synthesis.17 1H NMR (400
MHz, CDCl3): δ 1.53 (s, 3H), 1.61 (s, 3H), 1.79 (m, 3H), 1.95
(m, 1H), 2.50 (m, 2H), 4.40 (m, 1H), 9.78 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 17.9, 26.0, 27.5, 31.1, 43.5, 74.1, 110.9,
173.2, 201.8. MS (ES) m/z: 187 [M + H], 209 [M + Na]. Acid
49 (485 mg) was obtained free of aldehyde as a clear, pale-
ester (1.92 g, 71%) as a clear, colorless oil. [R]25 -33° (c 1.14,
D
1
MeOH). H NMR (400 MHz, CDCl3, rotamers): δ 0.97 (m, 12
H), 1.21-2.02 (m, 12 H), 1.46 (2s, 9H), 2.88 (2s, 3H), 3.30 (m,
2H), 4.81, (m, 0.5 H), 4.98 (m, 0.5 H), 5.06-5.26 (m, 3H), 5.31
(m, 1H), 7.34 (bs, 5H). 13C NMR (100 MHz, CDCl3): δ 14.4,
21.3, 21.6, 22.3, 23.0, 23.4, 23.6, 23.7, 24.9, 25.2, 25.4, 26.6,
26.7, 28.4, 28.7, 29.7, 30.4, 31.1, 31.9, 37.7, 38.0, 38.4, 45.7,
56.5, 56.8, 56.9, 57.3, 67.1, 74.0, 80.0, 80.4, 128.4, 128.5, 128.9,
136.0, 171.1, 171.6. HRMS (FAB) m/z calcd for C31H48N2O7 +
H1: 561.3539. Found: 561.3552. This material (1.89 g, 3.37
mmol), absolute EtOH (150 mL), and 10% Pd on activated
carbon (400 mg) were combined and processed according to
the general procedure for removing a benzyl protecting group,
which gave ꢀ-lactam free acid 52 (1.62 g, ∼100%) as a white,
1
yellow oil from a slower eluting chromatography fraction. H
NMR (400 MHz, CDCl3): δ 1.53 (s, 3H), 1.60 (s, 3H), 1.80 (m,
3H), 1.98 (m, 1H), 2.43 (m, 2H), 4.40 (m, 1H), 7.2-9.0 (bs, 1H).
13C NMR (100 MHz, CDCl3): δ 20.5, 26.0, 27.5, 31.0, 33.6, 74.1,
111.0, 173.3, 179.2. MS (ES) m/z: 201 [M - H], 203 [M + H],
225 [M + Na].
solid foam. [R]25 -27° (c 1.13, MeOH). 1H NMR (400 MHz,
D
CDCl3): δ 0.94 (m, 12 H), 1.45 (s, 9H), 1.50-2.05 (m, 12 H),
2.85 (s, 3H), 3.32 (m, 1H), 3.49 (m, 1H), 4.78 (m, 0.5 H), 4.95
(m, 0.5 H), 5.07 (m, 1H), 5.31 (m, 1H), 6.43-8.02 (bs, 1H).
HRMS (FAB) m/z calcd for C24H42N2O7 + H1: 471.3070.
Found: 471.3075.
Ben zyl (2S)-2-({4-[(4R)-2,2-Dim eth yl-5-oxo-1,3-dioxolan -
4-yl]bu tyl}a m in o)-4-m eth ylp en ta n oa te (50). A solution of
saturated aldehyde 48 (4.95 g, 26.6 mmol) in MeOH (50 mL)
was added to a solution of L-leucine benzyl ester 13 (5.88 g,
26.6 mmol) in MeOH (50 mL) at 0 °C under an atmosphere of
dry N2. Glacial HOAc (2.3 mL, 2.39 g, 39.9 mmol) was added,
and the mixture was stirred for 7 min (prolonged stirring is
deleterious) at 0 °C. A solution of NaBH3CN in THF (1 M, 17.8
mL, 17.8 mmol) was then added, and the reaction mixture was
stirred at room temperature for 30-45 min. The reaction
mixture was diluted with Et2O (200 mL) and poured into
aqueous NaHCO3 (200 mL). The mixture was shaken, and the
layers were separated. The aqueous layer was extracted with
Et2O (200 mL). The organic layers were combined and washed
with H2O (200 mL) and then with saturated NaCl (200 mL).
Filtration, concentration, and drying under high vacuum gave
crude product (10.8 g). Chromatography (20% EtOAc in
hexane) produced secondary amine 50 (6.89 g, 74%) as a clear,
Ben zyl N-Meth yl-L-leu cyl-E-la cta m -3-p h en yl-D-la cta te
(53). ꢀ-Lactam free acid 52 (2.96 g, 6.29 mmol), benzyl
3-phenyl-D-lactate4 18 (1.69 g, 6.46 mmol), a solution of DCC
in CH2Cl2 (1 M, 7.9 mL, 7.9 mmol), DMAP (40 mg, 0.33 mmol),
and CH2Cl2 (75 mL) were combined and processed according
to the general procedure for coupling peptides using DCC.
Chromatography (15% EtOAc in hexane) of the crude reaction
product followed by high vacuum-drying gave the tetradep-
sipeptide (1.88 g, 42%) as a clear, colorless oil. [R]25 -19° (c
D
1
1.14, MeOH). H NMR (400 MHz, CDCl3): δ 0.91 (m, 12 H),
1.10-2.00 (m, 12 H), 1.48 (s, 9H), 2.85-3.06 (m, 5H), 3.18 (bs,
2H), 4.78-5.44 (4 H), 5.06 (d, J ) 12.1 Hz, 1H), 5.13 (d, J )
12.0 Hz, 1H), 7.06-7.45 (m, 10 H). 13C NMR (100 MHz,

2070 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
CDCl3): δ 14.5, 21.3, 21.6, 22.2, 23.0, 23.5, 23.8, 24.9, 25.1,
26.9, 28.4, 28.7, 30.0, 31.9, 35.0, 37.4, 37.7, 38.1. 5.5, 55.7, 55.8,
56.6, 57.3, 67.6, 73.9, 80.4, 127.4, 128.8, 128.9, 129.0, 130.3,
135.2, 135.8, 169.3, 171.4, 171.5. MS (ES) m/z: 731 [M + Na],
707 [M - H]. HRMS (FAB) m/z calcd for C40H56N2O9 + H1:
709.4064. Found: 709.4059. A slower eluting chromatography
fraction upon drying under high vacuum produced a diaste-
reomer of the tetradepsipeptide (1.04 g, 23%), which had 1H
NMR and 13C NMR spectra that were very similar to the
above. [R]25D +8° (c 0.94, MeOH). MS (ES) m/z: 731 [M + Na],
707 [M - H]. HRMS (FAB) m/z calcd for C40H56N2O9 + H1:
709.4064. Found: 709.4052. An intermediate fraction con-
tained a mixture of the two diastereomers (1.25 g, 28%) for a
total yield of coupled product amounting to 4.17 g (93%). The
major tetradepsipeptide diastereomer (1.72 g, 2.43 mmol), CH2-
Cl2 (20 mL), and TFA (5 mL) were combined and processed
according to the general procedure for removing a Boc protect-
ing group, which gave tetradepsipeptide free amine 53 (1.34
reaction came to a halt although TLC indicated the presence
of unreacted starting materials; addition of more DPEA and
BOP-Cl was without effect. The reaction mixture was concen-
trated to 800 mL and washed with aqueous NaHCO3. The
resulting emulsion was broken up by adding saturated NaCl.
The organic layer was filtered through anhydrous Na2SO4 and
dried (Na2SO4). Filtration, concentration, and drying under
high vacuum gave crude product. This was further purified
by chromatography (50% EtOAc in hexane) followed by high
vacuum-drying to give ꢀ-lactam cyclodepsipeptide 4 (628 mg,
43%) as a white, solid foam. [R]25 -97° (c 0.92, MeOH). 1H
D
NMR (400 MHz, CDCl3): δ 0.70-1.08 (m, 27H), 1.20-2.20 (m,
18 H), 2.65 (s, ∼0.2 H), 2.75 (s, ∼0.2 H), 2.78 (s, ∼2.8 H), 2.80
(s, ∼0.1 H), 2.82 (s, ∼2.8 H), 2.90 (s, ∼0.2 H), 2.97 (s, ∼2.7 H),
3.00-3.49 (m, 6H), 4.45 (t, J ) 7.5 Hz, 1H), 5.06 (dd, J ) 6.8,
13.8 Hz, 1H), 5.30 (dd, J ) 4.3, 11.8 Hz, 1H), 5.36 (dd, J )
2.7, 12.9 Hz, 1H), 5.44 (dd, J ) 4.2, 12.3 Hz, 1H), 5.53 (dd, J
≈ 3, 8.1 Hz, 1H), 5.65 (t, J ) 7.6 Hz, 1H), 5.71 (t, J ) 7.7 Hz,
1H), 7.23 (m, 10 H). 13C NMR (100 MHz, CDCl3): δ 14.4, 16.1,
21.3, 21.4, 21.5, 22.1, 23.6, 23.7, 24.4, 25.0, 25.5, 27.2, 28.4,
29.7, 30.8, 30.8, 31.5, 31.9, 36.7, 36.9, 38.0, 38.1, 38.4, 45.3,
54.5, 54.5, 55.9, 57.5, 69.0, 70.7, 71.6, 73.1, 127.4, 127.5, 128.8,
128.9, 128.9, 129.8, 135.4, 135.5, 169.9, 170.3, 170.5, 170.6,
171.1, 171.3, 171.4, 171.7. MS (ES) m/z: 997 [M + Na]. HRMS
(FAB) m/z calcd for C54H78N4O12 + H1: 975.5694. Found:
975.5711. Infrared (mull, cm-1): 1744 (ester carbonyl), 1666
(amide carbonyl). UV (1%, 1 cm, MeOH) λmax (ꢀ): 250 (4.7),
263 (3.7). Anal. (C54H78N4O12) C, H, N.
g, 91%) as a clear, colorless, viscous oil. [R]25 +6° (c 1.23,
D
MeOH). 1H NMR (400 MHz, CDCl3): δ 0.92 (m. 12 H), 1.19
(m, 1H), 1.40-2.03 (m, 11 H), 2.52 (s, 3H), 2.90-3.13 (m, 3H),
3.19 (m, 2H), 3.49 (t, J ) 7.3 Hz, 1H), 5.06 (d, J ) 12.0 Hz,
1H), 5.15 (d, J ) 12.0 Hz, 1H), 5.26 (t, J ) 5.7 Hz, 1H), 5.39
(m, 2H), 7.12-7.45 (m, 10 H). HRMS (FAB) m/z calcd for
C
35H48N2O7 + H1: 609.3539. Found: 609.3553.
N-Meth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-
D-lactyl-N-m eth yl-L-leu cyl-E-lactam -3-ph en yl-D-lactic Acid
(54). Tetradepsipeptide free acid 42 (1.26 g, 2.13 mmol),
tetradepsipeptide free amine 53 (1.30 g, 2.13 mmol), a solution
of DCC in CH2Cl2 (1 M, 2.3 mL, 2.3 mmol), DMAP (13 mg,
0.11 mmol), and CH2Cl2 (60 mL) were combined and processed
according to the general procedure for coupling peptides using
DCC. Chromatography of the crude reaction product followed
by drying under high vacuum gave the octadepsipeptide (1.99
Syn th esis of P r olin e Cyclod ep sip ep tid e (5). N-Boc-L-
P r olyl-D-la ctic Acid (57). N-Boc-L-proline 55 (2.15 g, 10
mmol), methyl D-lactate 56 (1.2 g, 12 mmol), DIC (1.6 mL, 10
mmol), DMAP (0.6 g, 5 mmol), and CH2Cl2 (25 mL) were
combined and processed according to the general procedure
for coupling peptides using DIC (reaction time of 1 h). In an
exception to the general procedure, the crude product was
partitioned between CH2Cl2 (150 mL) and 0.3 N HCl (100 mL).
The organic layer was washed with 5% K2CO3 (100 mL), dried
(MgSO4), and concentrated to give the didepsipeptide as an
g, 78%) as a white solid foam and clear glass. [R]25 -51° (c
D
1
1.02, MeOH). H NMR (400 MHz, CDCl3): δ 0.90 (m, 24 H),
1.32-2.08 (m, 21 H), 1.44 (bs, 9H), 2.74-3.29 (m, 12 H), 3.01
(s, 3H), 4.39-5.57 (6m, 8H), 5.05 (d, J ) 12.0 Hz, 1H), 5.13 (d,
J ) 12.0 Hz, 1H), 7.02-7.40 (m, 15 H). 13C NMR (100 MHz,
CDCl3): δ 14.5, 17.0, 21.6, 22.2, 23.5, 23.7, 25.0, 25.1, 25.2,
27.0, 28.7, 37.4, 37.9, 38.1, 55.7, 67.6, 68.2, 73.9, 74.2, 127.4,
128.7, 128.8, 128.9, 129.0, 129.9, 130.3, 135.8, 169.3, 171.4.
MS (ES) m/z: 1206 [M + Na], 1184 [M + H]. This material
(1.98 g, 1.67 mmol), CH2Cl2 (30 mL), and TFA (7.5 mL) were
combined and processed according to the general procedure
for removing a Boc protecting group, which gave the octadep-
oil (2.22 g, 74%), which slowly solidified. [R]25 -26.3° (c 0.89,
D
1
CHCl3). H NMR (400 MHz, CDCl3): δ 1.41 and 1.45 (s, 9H),
1.4-1.6 (m, 3H), 1.8-2.4 (m, 4H), 3.4-3.7 (m, 2H), 3.74 and
3.75 (2s, 3H), 4.3-4.4 (m, 1H), 5.13 and 5.18 (2q, 1H). HRMS
(FAB) m/z calcd for
C14H23NO6 + H1: 302.1603. Found:
302.1602. This material (2.1 g, 7.0 mmol) was dissolved in
MeOH (25 mL) and treated with 1 N NaOH (8 mL, 8 mmol)
at room temperature for 20 min. The mixture was poured into
H2O (20 mL) and extracted with Et2O (2 × 30 mL). The
aqueous layer was acidified with 1 N HCl (40 mL). The
resulting mixture was extracted with CH2Cl2 (3 × 30 mL). The
organic layer was dried (MgSO4) and concentrated to give
didepsipeptide free acid 57 as an oil (1.4 g, 70%), which slowly
sipeptide free amine (1.75 g, 97%) as a white solid foam. [R]25
D
1
-39° (c 1.35, MeOH). H NMR (400 MHz, CDCl3): δ 0.89 (m,
24 H), 1.06-2.05 (m, 22 H), 2.52 (m, 2H), 2.79-3.25 (m, 13
H), 3.58 (m, 1H), 5.04 (d, J ) 12.0 Hz, 1H), 5.12 (d, J ) 12.0
Hz, 1H), 5.17-7.59 (m, 7H), 7.00-7.40 (m, 15 H). 13C NMR
(100 MHz, CDCl3): δ 21.6, 22.2, 22.6, 22.8, 23.5, 23.6, 25.0,
25.1, 25.2, 32.1, 37.4, 38.1, 67.6, 73.9, 127.4, 128.7, 128.9, 128.9,
129.0, 129.8, 130.3, 135.8, 169.3, 171.4. HRMS (FAB) m/z calcd
for C61H86N4O13 + H1: 1083.6270. Found: 1083.6304. The
octadepsipeptide free amine (1.72 g, 1.59 mmol), absolute
EtOH (150 mL), and 10% Pd on activated carbon (360 mg) were
combined and processed according to the general procedure
for removing a benzyl protecting group, which gave octadep-
sipeptide amino acid 54 (1.48 g, 94%) as a tan, solid foam and
solidified. [R]25 -39.7° (c 0.90, CHCl3). 1H NMR (400 MHz,
D
CDCl3): δ 1.42 and 1.46 (s, 9H), 1.54 (d, 3H), 1.8-2.5 (m, 4H),
3.4-3.7 (m, 2H), 4.3-4.4 (m, 1H), 5.12 and 5.28 (q, 1H). HRMS
(FAB) m/z calcd for
288.1449.
C13H21NO6 + H1: 288.1447. Found:
N-Boc-L-p r olyl-D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-D-
la ctic Acid (58). Didepsipeptide free acid 57 (5.47 g, 19 mmol),
didepsipeptide free amine 23 (7.2 g, 19 mmol), DIC (3.27 mL,
20.9 mmol), DMAP (464 mg, 3.8 mmol), and CH2Cl2 (50 mL)
were combined and processed according to the general proce-
dure for coupling peptides using DIC (reaction time of 16 h).
The residue was chromatographed (10% EtOAc in hexane) to
give the tetradepsipeptide as an oil (10 g, 82%). 1H NMR (400
MHz, CDCl3): δ 0.9-2.3 (m, 23H), 2.7-2.9 (m, 3H), 3.0-3.6
(m, 4H), 4.3-5.4 (m, 6H), 7.1-7.4 (m, 10H). This material (5.0
g, 7.7 mmol), absolute EtOH (100 mL), and 10% Pd on
activated carbon (1.2 g) were combined and processed accord-
ing to the general procedure for removing a benzyl protecting
group to give tetradepsipeptide free acid 58 (4.18 g, 98%) as a
glass. [R]25 -23° (c 0.75, MeOH). 1H NMR (400 MHz,
D
CDCl3): δ 0.89 (m, 24 H), 1.12-2.08 (m, 21 H), 2.60 (m, 2H),
2.79 (m, 13 H), 3.89 (m, 1H), 5.00-5.61 (m, 7H), 7.28 (m, 10
H), 7.69 (bs, 2H). 13C NMR (100 MHz, CDCl3): δ 14.5, 16.6,
21.6, 21.9, 22.6, 23.2, 23.5, 23.6, 25.3, 25.3, 37.8, 60.7, 72.5,
126.8, 127.5, 128.5, 128.9, 129.8, 129.9, 170.7, 171.3. HRMS
(FAB) m/z calcd for C54H80N4O13 + H1: 993.5800. Found:
993.5825.
E-La cta m Cyclod ep sip ep tid e (4). DPEA (0.65 mL, 480
mg, 3.73 mmol) was added to a 1 mM solution of octadep-
sipeptide amino acid 54 (1.48 g, 1.49 mmol) in CH2Cl2 (1.5 L)
at 0 °C. After the mixture was stirred for 5 min, powdered
BOP-Cl (455 mg, 1.79 mmol) was added and the reaction
mixture was stirred at room temperature. After 22 h, the
solid. [R]25 -29.7° (c 0.80, CHCl3). 1H NMR (400 MHz,
D
CDCl3): δ 0.8-2.3 (m, 23H), 2.9-3.6 (m, 7H), 4.1-5.7 (m, 4H),
7.2-7.4 (m, 5H). HRMS (FAB) m/z calcd for C29H42N2O9
H1: 563.2968. Found: 563.2980.
+

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2071
L-P r olyl-D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-D-la ctyl-
N-m eth yl-L-leu cyl-D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-D-
la ctic Acid (59). Tetradepsipeptide free acid 58 (0.82 g, 1.46
mmol), tetradepsipeptide free amine 25 (0.895 g, 1.54 mmol),
a solution of DCC in CH2Cl2 (1 M, 1.65 mL, 1.65 mmol), DMAP
(40 mg, 0.33 mmol), and CH2Cl2 (20 mL) were combined and
processed according to the general procedure for coupling
peptides using DCC. The residue was chromatographed (10-
63 (0.5 g, 2.8 mmol) were added. The resulting mixture was
treated with a solution of DEAD (0.5 mL, 3.17 mmol) in Et2O
(5 mL) at room temperature over 20 min. The mixture was
stirred for an additional 1 h, and the precipitate was removed
by filtration. The filtrate was concentrated, and the residue
was purified by chromatography (10% EtOAc in hexane) to
1
give the didepsipeptide (0.37 g, 24%) as an oil. H NMR (400
MHz, CDCl3): δ 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). HRMS (FAB) m/z
calcd for C21H29NO6 + H1: 392.2073. Found: 392.2086. The
didepsipeptide (370 mg, 0.95 mmol), CH2Cl2 (13 mL), and TFA
(2 mL) were combined and processed according to the general
procedure for removing a Boc protecting group to give didep-
sipeptide free amine 64 (0.204 g, 74%) as a clear, pale-yellow
oil. It was used without further purification.
20% acetone in hexane) to give the octadepsipeptide as a solid
1
(1 g, 61%). [R]25 -51.8° (c 0.35, CHCl3). H NMR (400 MHz,
D
CDCl3): δ 0.8-2.3 (m, 42H), 2.7-3.6 (m, 15H), 4.3-5.6 (m,
10H), 7.1-7.5 (m, 15H). HRMS (FAB) m/z calcd for C62H86N4O15
+ Na1: 1149.5986. Found: 1149.5994. The octadepsipeptide
(1.0 g, 0.89 mmol), CH2Cl2 (27 mL), and TFA (3 mL) were
combined and processed according to the general procedure
for removing a Boc protecting group with the exception that
the reaction mixture was slowly poured into saturated K2CO3
(30 mL) with rapid stirring. The mixture was transferred to a
separatory funnel and shaken, and the layers were separated.
The aqueous layer was extracted with CH 2Cl2 and the organic
layers were combined, washed with H2O, dried (Na2SO4),
filtered, and concentrated to give octadepsipeptide free amine
as an oil (0.84 g, 90%). This material (0.80 g, 0.78 mmol),
absolute EtOH (100 mL), and 10% Pd on activated carbon (0.2
g) were combined and processed according to the general
procedure for removing a benzyl protecting group to give
octadepsipeptide amino acid 59 (0.7 g, 95%) as a solid. It was
used without further purification.
N-Meth yl-L-leu cyl-3-p h en yl-D-la ctyl-N-m eth yl-L-leu cyl-
D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-D-la ctyl-L-p ip ecolyl-
D-la ctic Acid (65). Hexadepsipeptide free acid 61 (417 mg,
0.48 mmol), didepsipeptide free amine 64 (0.141 g, 0.48 mmol),
DIC (0.09 mL, 0.51 mmol), DMAP (10 mg, 0.08 mmol), and
CH2Cl2 (20 mL) were combined and processed according to the
general procedure for coupling peptides using DIC (reaction
time of 16 h). Chromatography of the residue (20% acetone in
hexane) gave the octadepsipeptide as a semisolid (130 mg,
24%). HRMS (FAB) m/z calcd for C63H88N4O15 + H1: 1141.6324.
Found: 1141.6293. This material (130 mg, 0.11 mmol), CH2-
Cl2 (18 mL), and TFA (2 mL) were combined and processed
according to the general procedure for removing a Boc protect-
ing group to give the octadepsipeptide free amine as an oil
(0.1 g, 84%). This material (100 mg, 0.096 mmol), absolute
EtOH (50 mL), and 10% Pd on activated carbon (0.1 g) were
combined and processed according to the general procedure
for removing a benzyl protecting group to give octadepsipeptide
amino acid 65 (0.08 g, 88%) as a solid. It was used without
further purification.
P r olin e Cyclod ep sip ep tid e (5). Octadepsipeptide amino
acid 59 (550 mg, 0.59 mmol) was dissolved in CH2Cl2 (590 mL)
to give a 1 mM solution, which was treated with BOP reagent
(262 mg, 0.59 mmol) and N-methylmorpholine (0.065 mL, 0.60
mmol) at room temperature (reaction time of 16 h). The residue
was purified by chromatography (50% EtOAc in hexane) to
give proline cyclodepsipeptide 5 (60 mg, 12%) as a solid. [R]25
D
1
-65° (c 0.80, CHCl3). H NMR (400 MHz, CDCl3): δ 0.8-2.4
P ipecolyl Cyclodepsipeptide (6). Octadepsipeptide amino
acid 65 (80 mg, 0.084 mmol) was dissolved in CH2Cl2 (20 mL)
to give a 4 mM solution. Triethylamine (0.05 mL, 0.36 mmol)
was added followed by addition of 1-methyl-2-chloropyridinium
iodide (30 mg, 0.12 mmol, 1.4 equiv) at room temperature.
After being stirred for 16 h, the mixture was washed with 1 N
HCl (30 mL) and the organic layer was separated, dried
(MgSO4), and concentrated. The residue was purified by
chromatography (30% acetone in hexane) to give pipecolyl
(m, 37H), 2.7-3.3 (m, 15H), 3.5-6.0 (m, 8H), 7.2-7.4 (m, 10H).
HRMS (FAB) m/z calcd for C50H70N4O12 + H1: 941.4888.
Found: 941.4905. Anal. (C50H70N4O12‚H2O) C, H, N.
Syn th esis of P ip ecolyl Cyclod ep sip ep tid e (6). N-Boc-
N-m et h yl-L-leu cyl-3-p h en yl-D-la ct yl-N-m et h yl-L-leu cyl-
D-la ct yl-N-m et h yl-L-leu cyl-3-p h en yl-D-la ct ic Acid (61).
N-Boc-N-methyl-L-leucyl-3-phenyl-D-lactyl-N-methyl-L-leucyl-
D-lactic acid2 60 (4.0 g, 6.8 mmol), didepsipeptide free amine
23 (2.6 g, 6.8 mmol), CH2Cl2 (50 mL), DIC (1.17 mL, 7.5 mmol),
and DMAP (0.1 g, 0.8 mmol) were combined and processed
according to the general procedure for coupling peptides using
DIC. Chromatography of the crude reaction product (20%
acetone in hexane) gave the hexadepsipeptide as an oil (3.4 g,
cyclodepsipeptide 6 (40 mg, 50%) as a solid. [R]25 -74.1° (c
D
0.48, CHCl3). 1H NMR (400 MHz, CDCl3): δ 0.8-2.4 (m, 39H),
2.6-3.4 (m, 15H), 3.5-5.8 (m, 8H), 7.1-7.4 (m, 10H). HRMS
(FAB) m/z calcd for C51H72N4O12 + Na1: 955.5044. Found:
955.5039. Anal. (C51H72N4O12‚0.5H2O) C, H, N.
1
52%). H NMR (400 MHz, CDCl3): δ 0.8-1.9 (m, 45H), 2.6-
Syn th esis of Azep a n yl Cyclod ep sip ep tid e (7). ter t-
Bu tyl (3S,5S,6R)-3-(5-Ch lor op en tyl)-2-oxo-5,6-d ip h en yl-
4-m or p h olin eca r boxyla te (66). To a solution of tert-butyl
(2R,3S)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate7 66 (4 g,
11.3 mmol), 1-iodo-5-chloropentane (7.7 g, 33 mmol), THF (160
mL), and HMPA (18 mL) was added a solution of sodium bis-
(trimethylsilyl)amide in THF (1 M, 17 mL, 17 mmol) dropwise
at -78 °C. After 1.5 h, the dry ice bath was removed. After an
additional 1 h, the reaction mixture was poured into EtOAc,
washed with H2O and brine, dried (MgSO4), filtered, concen-
trated, and crystallized from hexanes to afford oxazinone 67
(3.8 g, 73%) as a yellow solid. [R]25D -62.8° (c 0.97, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 1.11 and 1.46 (2s, 9H), 1.3-2.4
(m, 8H), 3.57 (quintet, 2H), 4.8-5.3 (m, 2H), 5.94 (br s, 1H),
6.4-6.6 (m, 2H), 6.9-7.3 (m, 13H). HRMS (FAB) m/z calcd
for C26H32Cl1NO4 + H1: 458.2094. Found: 458.2098.
(3R,4S,10a S)-3,4-Dip h en ylocta h yd r o-1H-[1,4]oxa zin o-
[4,3-a ]a zep in -1-on e (69). Oxazinone 67 (2.55 g, 5.57 mmol)
was dissolved in a solution of TFA in CH2Cl2 (20%, 25 mL).
The reaction mixture was stirred for 3-4 h and then slowly
poured into saturated K2CO3 (30 mL) with rapid stirring. The
layers were separated, and the aqueous layer was extracted
with CH2Cl2. The organic phases were combined, washed with
H2O, dried (MgSO4), filtered, and concentrated to give the
deprotected oxazinone 68 (1.82 g, 87%) as an oil. This material
3.3 (m, 13H), 4.1-5.5 (m, 8H), 7.1-7.4 (m,15H). MS (FAB)
m/z: 958 [M + H]. This material (3.4 g, 3.55 mmol), absolute
EtOH (100 mL), and 10% Pd on activated carbon (1.1 g) were
combined and processed according to the general procedure
for removing a benzyl protecting group. The residue was dried
under high vacuum to give hexadepsipeptide free acid 61 (2.5
g, 81%) as a semisolid. It was used without further purifica-
1
tion. [R]25 -43.5° (c 1.0, CHCl3). H NMR (400 MHz, CDCl3):
D
δ 0.7-1.8 (m, 45H), 2.6-3.3 (m, 13H), 4.6-5.5 (m, 6H), 7.2-
7.4 (m, 10H). HRMS (FAB) m/z calcd for C21H29N1O6 + H1:
392.2073. Found: 392.2086.
N-Boc-L-p ip ecolic Acid (62). L-Pipecolic acid (517 mg, 4
mmol) was suspended in THF (20 mL) and treated with di-
tert-butyl dicarbonate (960 mg, 4.4 mmol) and Et3N (1.1 mL,
8 mmol). The mixture was refluxed for 2 h, cooled to room
temperature, and then stirred for 16 h. The precipitate was
removed by filtration, and the filtrate was concentrated. The
residue was dissolved in CH2Cl2 (30 mL) and washed with 0.5
N HCl (20 mL). The organic layer was separated, dried
(MgSO4), and concentrated to give N-Boc-L-pipecolic acid 62
(640 mg, 2.8 mmol, 70%).
Ben zyl L-P ip ecolyl-D-la cta te (64). N-Boc-L-pipecolic acid
62 (640 mg, 2.8 mmol) was dissolved in Et2O (20 mL), and
triphenylphosphine (880 mg, 3.36 mmol) and benzyl L-lactate6

2072 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Dutton et al.
was dissolved in acetonitrile (25 mL) and treated with K2CO3
(786 mg, 5.70 mmol) and KI (946 mg, 5.70 mmol). The mixture
was heated at reflux for 24 h, taken up in EtOAc (100 mL),
and washed with H2O. The organic layer was separated, dried
(MgSO4), and concentrated. The residue was purified by
chromatography (20% Et2O in hexane) to give bicyclic oxazine
under high vacuum to give octadepsipeptide amino acid 74
(135 mg, 90%) as a solid.
Azepan yl Cyclodepsipeptide (7). Octadepsipeptide amino
acid 74 (135 mg, 0.14 mmol) was dissolved in CH2Cl2 (20 mL)
to give a 7 mM solution, which was treated with Et3N (0.08
mL, 0.56 mmol) and 1-methyl-2-chloropyridinium iodide (50
mg, 0.20 mmol, 1.4 equiv). The reaction mixture was stirred
at room temperature for 16 h and washed with 1 N HCl (30
mL), and the organic layer was separated, dried (MgSO4), and
concentrated. The residue was purified by chromatography
(35% acetone in hexane) to give azepanyl cyclodepsipeptide 7
(45 mg, 34%) as a solid. [R]25D -76.0° (c 0.48, CHCl3). 1H NMR
(400 MHz, CDCl3): δ 0.8-2.0 (m, 41H), 2.1-3.1 (m, 13H), 3.2-
5.7 (m, 10H), 7.1-7.4 (m, 10H). HRMS (FAB) m/z calcd for
1
69 (800 mg, 45%) as an oil, which slowly solidified. H NMR
(400 MHz, CDCl3): δ 1.5-2.0 (m, 7H), 2.3-2.4 (m, 1H), 2.5-
2.6 (m, 1H), 2.7-2.9 (m, 1H), 3.97 (dd, J ) 3.5, 10.5 Hz, 1H),
4.24 (d, J ) 3.0 Hz, 1H), 5.94 (d, J ) 3.0 Hz, 1H), 6.7-7.3 (m,
10H). HRMS (FAB) m/z calcd for C21H23NO2 + H1: 322.1807.
Found: 322.1804.
(2S)-2-Azep a n eca r boxylic Acid (70). To a solution of
bicyclic oxazine 69 (100 mg, 0.31 mmol) in 1:2 THF/EtOH (21
mL) was added PdCl2 (30 mg). The reaction mixture was
hydrogenated at 50 psi for 20 h. The mixture was then purged
with N2, filtered through Celite to remove the catalyst, and
concentrated. The residue was crystallized from MeOH/acetone/
Et2O to give azepanecarboxylic acid 70 as a white solid (43
mg, 96%). [R]25D -23° (c 0.76, MeOH). Lit. value:18 [R]25D -21°
(c 1.02, H2O). 1H NMR (400 MHz, CD3OD): δ 1.6-2.1 (m, 7H),
2.3-2.5 (m, 1H), 3.2-3.4 (m, 2H), 4.1-4.2 (m, 1H).
C
52H74N4O12 + H1: 947.5381. Found: 947.5401. Anal. (C52H74
-
N4O12‚H2O) C, H, N.
Biology. The relative potency of PF1022 A and the CDPs
of this report was determined in an anthelmintic assay
utilizing immunosuppressed jirds, Meriones unguiculatus,
infected with the ruminant parasite Haemonchus contortus.
Outbred female jirds weighing 30-35 g (Charles River Labo-
ratories, Stone Ridge, NY) were used. Upon arrival, three jirds
each were randomly assigned to translucent polypropylene
cages (26.7 cm × 21.6 cm × 14.0 cm) containing wood shavings
and given commercial rodent chow (Purina 5002) and water
ad libitum. After a 2-day acclimation period, the jirds were
switched to a modification of the commercial chow containing
0.02% hydrocortisone (medicated) and remained on this chow
for the duration of the study. Five days after being placed on
medicated food, each jird was inoculated with ∼1000 ex-
sheathed infective stage larvae, which had been incubated for
1 h in Earle’s balanced salt solution as described by Conder
and J ohnson.8 Inoculations were administered per os using an
18 gauge dosing needle fitted to a 1 cm3 syringe. The lavae
were a strain of H. contortus originally obtained from Dr. K.
S. Todd, J r. (University of Illinois, Urbana, Illinois), which has
been maintained continuously in sheep at Pharmacia and
Upjohn. The jirds were allocated randomly by cage to treat-
ment groups (three animals/group) on day 10 postinoculation
(PI). The test compound was suspended in DMSO (17%)/
carboxymethylcellulose (83%) vehicle using a vortex mixer, and
the desired treatment dose was administered orally to each
jird in a 0.2 mL volume using an 18 gauge dosing needle fitted
to a 1 cm3 syringe. Control animals for each experiment were
dosed with vehicle alone. Treatment, necropsy, examination
of stomach contents, and determination of compound activity
were done as described by Conder.8 On day 3 posttreatment
(day 13 PI), all jirds were killed by CO2 inhalation, their
stomachs removed, longitudinally opened, and put in separate
scintillation vials containing 14 mL of distilled water. The vials
were vortexed and placed in a 37 °C water bath and incubated
for 5 h. Following incubation, 1 mL of formaldehyde solution
was added to each vial and the vials were stored for subse-
quent examination. Upon examination, the contents of each
vial were vortexed, the tissue was removed, and the entire vial
contents were examined for worms using a dissecting micro-
scope (15-45×). Percentage clearance for each compound was
determined using the following formula:
(2S)-1-(ter t-Bu toxycar bon yl)-2-azepan ecar boxylic Acid
(71). Azepanecarboxylic acid 70 (330 mg, 2.3 mmol) was
dissolved in 7:3 THF/H2O (20 mL) and treated with di-tert-
butyl dicarbonate (646 mg, 2.99 mmol) and Et3N (0.83 mL, 6
mmol). The mixture was stirred for 16 h at room temperature
and then partitioned between Et2O (30 mL) and H2O (20 mL).
The layers were separated, and the aqueous layer was acidified
to pH 2 with 1 N HCl. This was extracted with Et2O (2 × 25
mL) and all organic layers were combined, dried (MgSO4), and
concentrated to give N-Boc-azepanecarboxylic acid 71 (335 mg,
1
60%), which slowly solidified. H NMR (400 MHz, CDCl3): δ
1.1-2.0 (m, 7H), 1.43 and 1.48 (2s, 9H), 2.2-2.4 (m, 1H), 2.9-
3.1(m, 1H), 3.8-4.0 (m, 1H), 4.4-4.7 (2 m, 1H). HRMS (FAB)
m/z calcd for C12H21NO4 + H1: 244.1549. Found: 244.1549.
Ben zyl L-Azep a n yl-D-la cta te (73). N-Boc-azepanecarboxy-
lic acid 71 (200 mg, 0.82 mmol), benzyl D-lactate6 72 (148 mg,
0.86 mmol), a solution of DIC in CH2Cl2 (1 M, 0.86 mL, 0.86
mmol), DMAP (10 mg, 0.08 mmol), and CH2Cl2 (20 mL) were
combined and processed according to the general procedure
for coupling peptides using DIC (reaction time of 16 h). The
residue was chromatographed (10% EtOAc in hexane) to give
the didepsipeptide as an oil (166 mg, 50%). 1H NMR (400 MHz,
CDCl3): δ 1.1-2.0 (m, 11H), 1.42 and 1.49 (2s, 9H), 2.2-2.4
(m, 1H), 2.8-3.0 (m, 1H), 3.8-4.0 (m, 1H), 4.4-4.8 (m, 1H),
5.1-5.2 (m, 3H), 7.2-7.4 (m, 5H). MS (EI) m/z: 405 [M].
HRMS (FAB) m/z calcd for
C22H31NO6 + H1: 406.2229.
Found: 406.2236. This material (150 mg, 0.37 mmol), CH2Cl2
(9 mL), and TFA (1 mL) were combined and processed
according to the general procedure for removing a Boc protect-
ing group to give didepsipeptide free amine 73 (100 mg, 89%)
as a semisolid.
N-Meth yl-L-leu cyl-3-p h en yl-D-la ctyl-N-m eth yl-L-leu cyl-
D-la ctyl-N-m eth yl-L-leu cyl-3-p h en yl-D-la ctyl-L-a zep a n yl-
D-la ctic Acid (74). Hexadepsipeptide free acid 61 (347 mg,
0.40 mmol) and didepsipeptide free amine 73 (0.100 g, 0.333
mmol) were dissolved in CH2Cl2 (20 mL), and the solution was
cooled to 0 °C. BOP-Cl (204 mg, 0.80 mmol) and Et3N (0.14
mL, 1.0 mmol) were added, and the reaction mixture was
slowly warmed to room temperature where it was stirred for
16 h. The reaction mixture was concentrated, and the residue
was chromatographed (25% acetone in hexane) to give the
octadepsipeptide as a semisolid (184 mg, 48%). HRMS (FAB)
m/z calcd for C64H90N4O15 + Na1: 1177.6300. Found: 1177.6326.
This material (180 mg, 0.16 mmol), CH2Cl2 (18 mL), and TFA
(2 mL) were combined and processed according to the general
procedure for removing a Boc protecting group (reaction time
of 1.5 h) to give the octadepsipeptide free amine as a semisolid
(148 mg, 90%). The amine (148 mg, 0.155 mmol), absolute
EtOH (50 mL), and 10% Pd on activated carbon (50 mg) were
combined and processed according to the general procedure
for removing a benzyl protecting group. The residue was dried
percentage clearance )
{[(mean number of worms recovered from vehicle control
group) - (mean number of worms recovered from treated
group)]/(mean number of worms recovered from vehicle
control group)} × 100
Uniformity studies were run to refine the jird model and to
provide estimates of variance components to be used in
defining a mathematical model for the distribution of worm
burdens in the animals. Untreated vehicle control and stan-
dard anthelmintic treatments were evaluated in test groups
of three, six, and nine jirds per treatment group. Information
from these trials showed little effect in increasing the number
of jirds from three to nine in each treatment group/trial. A

Analogues of an Anthelmintic Cyclodepsipeptide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2073
(9) Conder, G. A.; J ohnson, S. S.; Guimond, P. M.; Geary, T. G.;
Lee, B. L.; Winterrowd, C. A.; Lee, B. H.; DiRoma, P. Utility of
a Haemonchus contortus/J ird (Meriones unguiculatus) Model for
Studying Resistance to Levamisole. J . Parasitol. 1991, 77, 83-
86.
cutoff was chosen that would result in no more than a 5% error
for a true 90% clearance compound when three jirds per trial
were used.
(10) Scherkenbeck, J .; Harder, A.; Plant, A.; Dyker, H. PF1022Asa
novel anthelmintic cyclooctadepsipeptide modification and ex-
change of the N-methyl leucine residues. Bioorg. Med. Chem.
Lett. 1998, 8, 1035-1040.
Ack n ow led gm en t. We are grateful to David Kloos-
terman for NMR data on PF1022A and J ames Blinn for
technical support in generating NMR-based structures.
(11) Schekenbeck, J .; J eschle, P.; Harder, A. PF1022A and Related
CyclodepsipeptidessA novel Class of Anthelmintics. Curr. Top.
Med. Chem. 2002, 2, 751-769.
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(16) In one case where this reaction was performed on a 1 g scale,
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de) of δ-lactam benzyl ester 39 and δ-lactam benzyl ester 31,
but the yield was 68%.
(17) This reaction gave variable results. Other batches required much
less reaction time, typically 1-3 h, and smaller amounts of
catalyst, and they contained fewer acid byproducts.
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