Structure-activity relationship of anthelmintic cyclooctadepsipeptides

Article abstract of DOI:10.1271/bbb.110129

The relationship between cyclooctadepsipeptides and their anthelmintic efficacy was examined by converting the natural products, PF1022A, PF1022E and PF1022H. Some analoguessubstituted at the para position of the phenyllactate moiety showed higher or equivalent activity against the parasitic nematode, Ascaridia galli in chicken when compared with the parent compounds. It is suggested that lipophilicity and the polar surface area, in addition to structural requirements of the derivatives, influenced the anthelmintic efficacyin vivo.

Full text of DOI:10.1271/bbb.110129

Biosci. Biotechnol. Biochem., 75 (7), 1354–1363, 2011
Structure-Activity Relationship of Anthelmintic Cyclooctadepsipeptides
y
Makoto OHYAMA,1;2; Yumiko OKADA,3 Masaaki TAKAHASHI,3 Osamu SAKANAKA,3
1;2
Maki MATSUMOTO,3 and Kunio ATSUMI
1Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering,
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan
2Pharmaceutical Research Center, Meiji Seika Kaisha Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama,
Kanagawa 222-8567, Japan
3Chemistry, Manufacturing & Control Research Laboratories, Meiji Seika Kaisha Ltd.,
788 Kayama, Odawara, Kanagawa 250-0852, Japan
Received February 15, 2011; Accepted March 24, 2011; Online Publication, July 7, 2011
[doi:10.1271/bbb.110129]
The relationship between cyclooctadepsipeptides and
their anthelmintic efficacy was examined by converting
the natural products, PF1022A, PF1022E and PF1022H.
Some analogues substituted at the para position of the
phenyllactate moiety showed higher or equivalent
activity against the parasitic nematode, Ascaridia galli
in chicken when compared with the parent compounds.
It is suggested that lipophilicity and the polar surface
area, in addition to structural requirements of the
derivatives, influenced the anthelmintic efficacy in vivo.
al.12)). Although these synthetic methods are still highly
valued, the conversion of such natural products is of
practical use for them to be readily accessible. We report
here the conversion of PF1022s and the structure-
activity relationship of their analogues.
Results and Discussion
Conversion of PF1022A (1)
PF1022A (1) was converted as summarized in
Scheme 1. Catalytic oxidation of the benzene ring(s)
of 1 by using ruthenium(III) chloride afforded versatile
intermediates 11 and 12 possessing carboxyl group(s).
Benzothiazole and benzimidazole derivatives 13 and 14
were respectively prepared in accordance with the
method of Hendrickson and Hussoin13) from 11 and
12. Compound 12 was amidated to give compounds 15,
16, 17, 18, 19 and 20 by the usual coupling reagents with
amines. Nitration and successive hydrogenation of 1
yielded another key intermediate 228) which could be an
alternative source of PF1022H (8) and was converted to
the other benzothiazole derivatives 23, 24, and emodep-
side (25)14) which is one of the active ingredients of
ProfenderÒ launched in 2005 by Bayer HealthCare.
Key words: PF1022A; anthelmintic; structure-activity
relationship; depsipeptide
PF1022A (1) is a twenty-four-membered anthelmintic
cyclooctadepsipeptide that was isolated from the myce-
lial cake of Mycelia sterilia PF1022 by Sasaki et al.1) Its
analogues, PF1022B (2), C (3), D (4), E (5), F (6), G (7)
and H (8) were also found in the same culture.2–4) Before
these compounds were isolated, bassianolide (9)5) was
the only natural product consisting of four L-N-methyl-
amino acids and four D-2-hydroxy acids linked together
in a pattern giving the molecule a two-fold axis of
symmetry. Structurally analogous verticilide (FKI-1033,
10) has recently been reported as a ryanodine receptor
antagonist.6) The structures and biological activities of
these compounds are summarized in Fig. 1. These
compounds have specific biological activity against
their target organism without such other biological
effects as antibacterial activity and cytotoxicity.
PF1022A has been spotlighted in the animal health
field because of its high anthelmintic efficacy against
various parasites, even against benzimidazole-resistant
and ivermectin-resistant Haemonchus contortus,7) in
addition to its low toxicity.1) We therefore embarked
of this study on depsipeptide chemistry to clarify the
structure-activity relationship, with the objective of
developing a new anthelmintic drug. Total syntheses
of PF1022A and its related compounds were subse-
quently published within three years of its isolation
(Nishiyama et al.;8) Ohyama et al.;9) Dutton and
Nelson;10) Kobayashi et al.;11) and Scherkenbeck et
Conversion of PF1022E (5) and PF1022H (8)
Scheme 2 shows a summary of the method for
converting PF1022E (5). The nitrile group of compound
26 prepared from 5 was converted to tetrazole (27),
imidazole (30), thiazole (31), and the primary amine
(32) which was additionally modified to give the
dialkylaminoethoxy derivatives (33–38). Methylation
of 27 yielded two chromatographically separable iso-
mers (28 and 29). The Mitsunobu reaction was applied
to the phenolic hydroxyl group of 5 to afford the (S)-
prolinol derivative (39) and furfuryl derivative (40).
Derivatives 41–51 were prepared by typical O-alkyla-
tion of 5.
Compounds 52–60 were prepared from PF1022H (8)
in almost the same manner as that used for the
conversion of PF1022E. Scheme 3 summarizes the
reactions.
y
To whom correspondence should be addressed. Fax: +81-45-545-3120; E-mail: makoto.ohyama@meiji.com
Abbreviations: Tf2O, trifluoromethanesulfonic anhydride; DEAD, diethyl azodicarboxylate; TFA, trifluoroacetic acid; DCC, 1,3-dicyclohex-
ylcarbodiimide; HOBt, 1-hydroxybenzotriazole; PSA, polar surface area

Structure-Activity Relationship of Cyclodepsipeptides
1355
R1
R
N
O
R
O
O
O
O
O
R2
N
O
O
R4
N
O
O
O
R
O
N
R
R3
Anthelmintic effective Insecticidal
3)
R1
R2
R3
R4
R
IC50
dose [mg/kg] 1)
activity 2)
PF1022A (1)
PF1022B (2)
PF1022C (3)
PF1022D (4)
PF1022E (5)
PF1022F (6)
PF1022G (7)
PF1022H (8)
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
-
-
-
-
-
-
-
-
-
2
-
CH3
CH2
CH2
CH2
CH3
CH2
CH2
CH2
CH2
CH2
CH2
>20
>20
10
-
CH2
CH3
-
CH2
CH3
-
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
10-20
5-10
>20
>20
>10
-
-
CH2
OH
-
CH3
CH3
CH3
CH2
CH2
OH
OH
-
CH3
-
CH3
CH2
OH
Bassianolide (9) CH2CH(CH3)2
Verticilide (10) CH3
13 ppm
-
CH(CH3)2
(CH2)4CH3
CH(CH3)2
(CH2)4CH3
CH(CH3)2
(CH2)4CH3
CH(CH3)2
(CH2)4CH3
4.2 µM
1) >90% worm reduction against Ascaridia galli in chicken
2) lethal dosage against Bombyx mori
3) antagonistic activity against the ryanodine receptor from Periplaneta americana
Fig. 1. Structures and Biological Activities of PF1022s and Their Related Depsipeptides.
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R
R
a
N
N
N
O
O
O
O
O
+
O
CH CONH
2
N
O
15: R =
16: R =
17: R =
18: R =
N
N
N
R
O
O
O
O
O
O
O
O
O
CH CONHCH
2
N
O
O
O
2
N
N
N
CH CON
2
N
d
PF1022A (1)
11: R = CH2COOH
12: R = CH2COOH
N
N
CH CON
2
N
c
b
N
N
CH
14: R =
CH2
e
2
13: R =
N
H
19: R = CH2CONH
N CH2
S
20: R =
CH2CONH(CH2)2
N
O
N
O
O
O
O
O
R
N
O
O
N
R
O
N
S
O
O
R
O
O
O
O
N
O
O
N
O
N
O
O
N
O
O
N
O
O
S
h
21: R =NO2
22: R = NH2
O
N
R
f
23: R = NH2
24: R = H
g
j
i
25: R =
N
O
PF1022H (8)
Scheme 1. Conversion of PF1022A (1).
Reagents: a) RuCl3, H5IO6; b) 2-aminobenzenethiol, Tf2O, Ph3PO; c) 1,2-phenylenediamine, Tf2O, Ph3PO; d) DCC, HOBt, amine; e) fuming
HNO3; f) H2, Pd/C; g) (ClCH2CH2)2O, NaI, K2CO3; h) NH4SCN, Br2, CH3COOH; i) isoamyl nitrite; j) NaNO2, H2SO4 and then heating.

1356
M. OHYAMA et al.
44: R=Me
45: R=Et
46: R=nPr
47: R=iPr
48: R=CH2CH=CH2
49: R=nBu
Ar-OR
o
50: R=tBu
51: R=nOctyl
H
N
N
N
N
N
a
b
N
N
n
c
Ar-OCH2
Ar-OH
Z
Ar-OCH2
Ar-OCH2CN
Ar-OCH2
X
Y
N
28
PF1022E (5)
26
27
d, e, f
41: X=N, Y=Z=CH
42: Y=N, X=Z=CH
43: Z=N, X=Y=CH
+
H
N
N
N
k, l
h
N
N
m
Ar-OCH2
Ar-OCH2
d, g
N
29
30
Ar-OCH2CH2NH2
Ar-OCH2
Ar-OCH2
39
S
N
32
O
N
Ar-OCH2
H
40
i
j
31
Ar-OCH2CH2NR2
Ar-OCH2CH2N
n
33: R=Me
34: R=Et
37: n=1
38: n=2
35: R=nPr
36: R=nBu
Scheme 2. Conversion of PF1022E (5).
Ar is the benzene ring of the p-hydroxyphenyl-lactic acid moiety of PF1022E (5). Reagents: a) K2CO3, NaI, BrCH2CN; b) NaN3; c) K2CO3,
MeI; d) O,O0-diethyl dithiophosphate; e) MeI; f) aminoacetaldehyde dimethyl acetal, 6N-HCl; g) bromoacetaldehyde diethyl acetal, H2SO4;
h) H2 (3 atm), Pd/C; i) H2, Pd/C, aldehyde (for 33 and 34), or K2CO3, alkyl iodide (for 35 and 36); j) K2CO3, NaI, dibromoalkane; k) (S)-tert-
butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate, DEAD, Ph3P; l) TFA; m) 2-hydroxymethylfuran, DEAD, Ph3P; n) 2, 3, or 4-picolyl chloride,
Cs2CO3; o) NaH, alkylhalide (other than 50) or isobutene, H2SO4 (for 50).
c
Ar-OH
a, b
Ar-OCH2CH2NR2
Ar-OCH2CH2NH2
52
PF1022H (8)
53: R=Me
54: R=Et
d
f
g
Ar-OCH2CH2N
h
e
55
Ar-OR
Ar-OCH2
Ar-OCH2
57
Ar-OCH2CH2N
O
59: R=CH2Ph
60: R=tBu
O
N
58
56
Scheme 3. Conversion of PF1022H (8).
Ar is each benzene ring of the p-hydroxyphenyl-lactic acid moiety of PF1022H (8). Reagents: a) K2CO3, NaI, BrCH2CN; b) H2 (3 atm), Pd/C;
c) H2, Pd/C, formaldehyde (for 53) or acetoaldehyde (for 54); d) K2CO3, NaI, 1,4-dibromobutane; e) (BrCH2CH2)2O, NaI, K2CO3;
f) 2-chloromethylfuran, NaI, Cs2CO3; g) 2-picolyl chloride, NaI, Cs2CO3; h) K2CO3, benzylbromide (for 59) or isobutene, H2SO4 (for 60).
Structure-activity relationship for cyclooctadepsipep-
ability, and that all four leucines were necessary for high
biological activity.20) On the other hand, Jeschke et al.
have pointed out that there were highly efficacious
derivatives with more lipophilicity than PF1022A. The
latter case for the increased activity of the derivatives
was explained by the possible attribution of a more rigid
bioactive conformation.21,22) However, these present
studies were made with different species of parasites
and infected animals in our experiments. The effect of
lipophilicity may therefore vary according to such
conditions.
The cLogP and PSA values for the derivatives were
spotted on a chart to visualize the relationship between
the anthelmintic efficacy and physicochemical parame-
ters of the derivatives (Fig. 2). The cLogP and PSA
values for the highly efficacious compounds respectively
ranged from 4.3 to 7.6 and from 100 to 194. These
results indicate that both parameters had optimal ranges
for the bioavailability of the compounds. Some deriv-
atives showed, however, lower efficacy, although both
physicochemical parameters were in the optimal ranges.
tide
Although Sager,15) Harder16) and Weltz17) have
revealed the molecular target of PF1022A and emodep-
side to be
G protein-coupled receptor depsiphi-
lin[HC110-R], its structure remains unresolved, prob-
ably due to difficulty in crystallization. We therefore
considered how the structural difference in the deriva-
tives affected their efficacy in vivo.
Table 1 summarizes the structures of the derivatives
and their anthelmintic efficacy against Ascaridia galli,
as well as their calculated physicochemical parameters.
Calculated logarithm P(cLogP) is well known as one of
the indices of lipophilicity, and the polar surface area
(PSA) is used to explain intestinal absorption in
combination with cLogP.18,19) These parameters should
be taken into consideration for explaining the structure-
activity relationship in vivo. Scherkenbeck et al. have
recently reported in their studies on the conversion of
PF1022A that a significant increase in lipophilicity of
the derivatives resulted in a decrease of their bioavail-

Structure-Activity Relationship of Cyclodepsipeptides
1357
Table 1. Structure and Anthelmintic Efficacy of the PF1022A Derivatives
N
O
O
O
O
O
N
R2
O
O
R1
N
O
O
O
O
O
N
R1
CH2
CH3
R2
Anthelmintic efficacy1)
PSA
cLogP
6.06
4.37
5.33
2.68
3.63
4.59
1.43
3.74
6.81
5.52
4.21
3.28
4.96
4.40
5.01
2.50
5.52
3.50
5.41
5.65
4.34
5.67
4.55
4.60
4.67
5.09
6.56
Compound
PF1022A (1)
+++
100.84
CH2
CH2
PF1022D (4)
+
102.72
145.54
105.98
147.20
190.33
208.64
153.66
148.26
129.98
135.83
143.06
115.40
132.31
125.00
114.85
244.26
216.01
319.34
182.33
132.16
177.98
230.95
193.43
171.02
155.39
151.01
PF1022E (5)
-
CH2
CH2
OH
PF1022F (6)
CH3
CH3
CH3
++
PF1022G (7)
-
CH2
CH2
OH
OH
PF1022H (8)
-
CH2
OH
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
CH2COOH
= R1
CH2
= R1
-
CH2COOH
-
N
CH2
S
-
N
CH2
-
CH2
N
H
CH2CONH
CH2CONHCH2
CH2CON
N
O
-
CH2
CH2
N
-
N
N
-
CH2
CH2
N
CH2CON
-
N
+
-
CH2CONH
N
CH2
CH2
CH2
CH2CONH(CH2)2
N
O
= R1
-
CH2
CH2
NO2
NH2
= R1
= R1
= R1
= R1
-
N
-
CH2
NH2
S
N
-
CH2
S
+++
+++
-
CH2
CH2
N
O
CH2
OCH2CN
H
N
N
N
CH2
CH2
27
28
29
30
31
CH2
OCH2
N
N
N
N
N
+++
+++
+++
+++
CH2
CH2
OCH2
N
N
N
CH2
CH2
CH2
OCH2
N
H
N
CH2
OCH2
OCH2
N
S
CH2
N

1358
M. OHYAMA et al.
Table 1. Continued
R1
cLogP
5.83
6.90
7.96
9.02
6.42
6.99
5.85
6.79
6.14
6.14
6.14
5.98
6.51
7.04
6.86
6.78
7.57
7.21
9.70
5.61
7.73
6.78
4.60
7.53
6.22
9.20
8.35
PSA
143.78
106.28
104.01
104.01
105.22
104.60
103.98
127.46
121.37
126.47
125.48
111.58
104.26
103.75
102.41
105.65
103.39
110.56
103.33
113.61
109.43
106.82
134.36
155.60
140.51
107.20
120.26
Compound
R2
Anthelmintic efficacy1)
-
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
53
54
55
56
57
58
59
60
CH2
CH2
CH2
OCH2CH2NMe2
OCH2CH2NEt2
OCH2CH2NnPr2
CH2
-
CH2
CH2
CH2
++
CH2
CH2
CH2
OCH2CH2NnBu2
-
-
N
N
OCH2CH2
OCH2CH2
CH2
CH2
CH2
-
-
+++
+++
+++
-
CH2
OCH2
N
H
CH2
OCH2
CH2
CH2
O
N
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
OCH2
OCH2
OCH2
OMe
OEt
CH2
CH2
CH2
CH2
CH2
N
N
+++
+++
+++
+++
+++
+++
+++
-
OnPr
OiPr
CH2
CH2
OCH2CH=CH2
OnBu
CH2
CH2
CH2
CH2
CH2
OtBu
CH2
O(CH2)7CH3
OCH2CH2NMe2
= R1
= R1
= R1
= R1
= R1
= R1
= R1
= R1
-
-
CH2
CH2
CH2
OCH2CH2NEt2
-
N
N
OCH2CH2
OCH2CH2
-
O
-
CH2
CH2
CH2
CH2
OCH2
O
+
OCH2
N
-
OCH2Ph
OtBu
+
1) Score against Ascaridia galli in chicken at 5mg/kg dosage.
The score is expressed by +++ >90%, ++ 75-90%, + 50-75%, - <50% worm reduction.
This may have been due to a structural and/or charge
inadequacy in the binding affinity with the receptor as
will be discussed later.
Notwithstanding such difference, they had almost no
efficacy, possibly because of biodegradation and/or
mismatching with the putative receptor.
We next prepared diverse derivatives substituted at the
para position of a benzene ring by an alkyl chain, a
dialkylaminoethoxy group and a heterocycle, anticipating
other possible effects such as hydrophobic interaction,
electrostatic interaction and ꢀ-ꢀ stacking interaction.
O-alkyl derivatives 44–50 at the p-hydroxy group of 5
were comparable in efficacy to emodepside (25),
whether having a branched or straight chain. However,
i) Mono-substitutional effect on a phenyllactate
Carboxylic acid (12) and benzimidazole (14) both lost
anthelmintic activity. It would appear that a drastic
change to a benzene ring caused this adverse effect.
Amide derivatives 15–20 were mainly designed to
confirm the tolerance to a difference in the aromatic
ring and its distance from the depsipeptide backbone.

Structure-Activity Relationship of Cyclodepsipeptides
1359
Table 2. Anthelmintic Efficacy of the PF1022A Derivatives at Lower
Dosages.
350.00
300.00
250.00
200.00
150.00
100.00
50.00
Anthelmintic efficacy1Þ
2 mg/kg
1 mg/kg
PF1022A (1)
+++
+++
ꢂ
++
ꢂ
28
25
26
28
29
30
31
40
41
42
44
45
46
47
48
49
50
NT2Þ
+++
NT
NT
NT
+++
NT
ꢂ
+++
ꢂ
40
25
ꢂ
1
+
+++
+
0.00
2.00
4.00
6.00
8.00
10.00
12.00
cLogP
+++
+++
+++
+++
++
++
+
Fig. 2. Scatter Chart for the PF1022 Derivatives.
Each derivative was spotted according to its physicochemical
parameters. The anthelmintic score is expressed by , +++;
ꢀ, ++; , +; ꢁ, ꢂ. Representative highly efficacious compounds 1,
25, 28 and 40 are marked aside.
ꢂ
NT
+
+++
+++
+++
ꢂ
ꢂ
longer alkyl chains tended to decrease the efficacy (see
51, Table 2). These results indicate that, at least in
significant part, the side chain of the phenyllactate
moiety was bound to a hydrophobic spatially extended
pocket in the receptor. Derivatives possessing an
aliphatic amine such as the dialkylaminoethoxy groups
had almost no efficacy (33–39). A tertiary amine was
thought to be unfavorable for receptor-binding, com-
pound 35 being the only exception and an unknown
reason.
The heteroarylmethyl derivatives (28–31, and 40–42)
and the nitrile derivative (26) showed equivalent
efficacy. These derivatives shared the common property
of a hydrogen-bond-acceptor nitrogen or oxygen atom
which was exposed, unlike the tertiary amine deriva-
tives. Tetrazole is regarded as a carboxylic acid
equivalent, and the PSA value for the derivative (27)
was over-large for membrane permeability. Tetrazole
(27) therefore lost its efficacy, despite the methyltetra-
zoles (28 and 29) retaining theirs. In respect of the
pyridine derivatives, the 2-picolyl and 3-picolyl deriv-
atives (41 and 42) had higher efficacy than the 4-picolyl
derivative (43), probably because the position of the
putative hydrogen bond donor of the receptor was
rigidly regulated and the nitrogen atom of the 4-picolyl
group was incompatible with it.
1ÞScore against Ascaridia galli in chicken at the indicated dosage.
The score is expressed by +++ >90%, ++ 75–90%, + 50–75% and ꢂ
<50% worm reduction.
2ÞNT, not tested
Receptor-binding region
O
N
O
R1
O
O
O
N
O
R2
O
N
O
O
O
O
Hydrophobic
interaction
O
O
Region of
uncertain function
N
O
CH-π interaction
Hydrogen bond acceptor is favorable
Sterically hindered amine is unfavorable
Fig. 3. Putative Binding Mode of Derivative 40.
receptor-binding, and its duplication made the molecule
too large for bioavailability. The same would be true for
the decreased anthelmintic efficacy of the derivative
(59). In respect of derivatives 53–56, the most likely
cause for the lost activity was a charge inadequacy for
the binding affinity with the receptor, like the case of
derivatives 33–39 as already described.
The compounds showing an anthelmintic score of
+++ at a dosage of 5 mg/kg were next assayed at lower
doses, with the results shown in Table 2. Among these
compounds, the methyltetrazole (28) and furfuryl de-
rivative (40) showed higher efficacy than PF1022A (1)
and emodepside (25).
The putative binding mode is illustrated in Fig. 3
based on the substantially clarified structure-activity
relationship for the derivatives. The results obtained
here are exemplified by the fact that altering the
phenyllactate moiety significantly affected the anthel-
mintic efficacy. Unexplained parts of the structure may
have had such effects as making the conformation more
rigid, appropriately orienting the side chains, and
maintaining the bioavailability as a whole molecule by
controlling the physicochemical parameters represented
by the cLogP and PSA values.
ii) Bis-substitutional effect on phenyllactates
Carboxylic acid (11) and benzothiazoles (13, 23 and
24) prepared with the aim of altering the nature of the
benzene ring lead to a negative effect. It is suggested
that the benzene ring of PF1022A played an essential
role in binding to the receptor or stabilizing the
conformation via the intramolecular CH-ꢀ interaction
apparent in a crystalline structure.23) The derivatives
possessing nitro or amino groups (21 and 22) had no
efficacy, probably due to low affinity with the receptor,
based on the difference in electrostatic environment of
the benzene rings.
An attempt to introduce such functional groups as
furfuryl, 2-picolyl and t-butoxy to PF1022H (8), which
was effective for PF1022E (5), resulted in a worse effect
than that expected (57, 58 and 60). One of those
functional groups would presumably be required for
In summary, we provided versatile intermediates for
further conversion of PF1022s, and identified more

1360
M. OHYAMA et al.
chromatography (n-hexane/EtOAc ¼ 3=1 to 2/1) to give 4.07 g of the
bisbenzhydryl ester of 11 and 6.21 g of the monobenzhydryl ester of
12. To a solution of 0.95 g (0.78 mmol) of this bisbenzhydryl ester in
CH2Cl2 (3 mL) was added TFA (3 mL), and the mixture was stirred for
3.5 h at room temperature. Concentration of the reaction mixture and
purification by silica gel column chromatography (CHCl3=MeOH ¼
100=3 to 10/1) gave 0.62 g (0.70 mmol) of pure 11 as a white
amorphous powder. A mixture of 5.11 g (4.72 mmol) of the mono-
benzhydryl ester and 10%Pd/C (248 mg) in MeOH (100 mL) was
stirred overnight in a hydrogen atmosphere. The mixture was filtered
through CeliteÒ, and the resulting filtrate was concentrated under
reduced pressure. Purification by silica gel column chromatography
(CHCl3=MeOH ¼ 50=1 to 10/1) gave 4.08 g (4.45 mmol) of pure 12 as
a white amorphous powder.
potent derivatives than both the parent compound and
the launched product by considering the structure-
activity relationship. Even though these highly effica-
cious compounds have not yet been fully investigated,
continuing the search will lead us to find the best-in-
class depsipeptide anthelmintic agents with the help of
medicinal and computational chemistry, especially with
a breakthrough in elucidating the complex structure of
depsiphilin.
Experimental
11. 1H-NMR (500 MHz, CD3OD) ꢁ: 0.83 (3H, d, J ¼ 6:5 Hz), 0.86
(3H, d, J ¼ 6:6 Hz), 0.89 (3H, d, J ¼ 6:5 Hz), 0.92–0.97 (12H, m),
0.99 (3H, d, J ¼ 6:7 Hz), 1.04 (3H, d, J ¼ 6:5 Hz), 1.25–1.97 (12H,
m), 1.42 (3H, d, J ¼ 6:9 Hz), 2.85 (2H, dd, J ¼ 7:1, 16.7 Hz), 2.87
(3H, s), 2.91 (2H, dd, J ¼ 6:9, 16.7 Hz), 2.98 (3H, s), 3.17 (3H, s), 3.28
(3H, s), 4.75 (1H, dd, J ¼ 4:0, 11.1 Hz), 5.18 (1H, q, J ¼ 6:8 Hz), 5.29
(1H, dd, J ¼ 4:5, 11.5 Hz), 5.44 (1H, dd, J ¼ 4:6, 11.5 Hz), 5.48 (1H,
dd, J ¼ 4:4, 11.9 Hz), 5.56 (1H, q, J ¼ 6:8 Hz), 5.83 (1H, t,
The anthelmintic efficacy was measured as described as follows in
the reference.1) Each sample was orally administered to chickens
infected with Ascaridia galli. The number of excreted worms was
counted every day for the next two weeks. The chickens were
sacrificed and the number of remaining worms was counted 2 weeks
after the administration. The efficacy rate was calculated in accordance
with the equation, efficacy rate[%] ¼ f(number of worms excreted)/
(total number of worms)g ꢁ 100: The anthelmintic efficacy is classi-
fied into four levels depending on the efficacy rate:
J ¼ 7:1 Hz), 5.90 (1H, t, J ¼ 7:1 Hz). ESI-TOFMS m=z: calcd. for
þ
C
42H68N4NaO16 ½M þ Naꢃþ, 907.4528; found, 907.4546.
þ þ þ > 90%; þ þ 75{90%; þ50{75%; ꢂ < 50%:
25
12. ½ꢂꢃD ꢂ70ꢀ (c 0.11, MeOH). 1H-NMR (500 MHz, CDCl3,
integral values are the sum of a 1:1 mixture of two conformers) ꢁ:
0.78–1.10 (54H, m), 1.12–1.92 (30H, m), 2.30 (1H, d, J ¼ 17:1 Hz),
2.60 (1H, dd, J ¼ 10:2, 17.9 Hz), 2.73 (1H, dd, J ¼ 5:9, 17.1 Hz),
2.79–3.28 (29H, m), 4.48 (1H, dd, J ¼ 4:5, 11.5 Hz), 4.58 (1H, dd,
J ¼ 4:7, 10.5 Hz), 5.07 (1H, q, J ¼ 6:7 Hz), 5.10 (1H, q, J ¼ 6:7 Hz),
5.21–5.69 (9H, m), 5.73 (1H, t, J ¼ 6:3 Hz), 5.79 (1H, dd, J ¼ 5:8,
cLogP values were calculated by LogP DB version 7.04 of ACD/
Labs, and PSA values were calculated by Sybyl-X version 1.2 of
Tripos after optimizing the structure by Discovery Studio version 3.0
of Accerlys. Optical rotation data were recorded with a Jasco DIP-370
polarimeter, and 1H-NMR data were recorded with a Jeol GSX-270
spectrometer or Jeol GSX-500 spectrometer. Chemical shift (ꢁ) data
are referenced to tetramethylsilane (0.0 ppm) or the residual solvent
peak as the internal standard (CDCl3, 7.26 ppm; CD3OD, 3.30 ppm).
PF1022A and its derivatives were present as a mixture of two
conformers in solution,1) therefore, NMR data of the major conformer
are listed unless otherwise noted. Some of the NMR data were
recorded with potassium thiocyanate in order to make it easier to
interpret them. Mass spectra were recorded with an Agilent 6530
Accurate-Mass Q-TOF LC/MS system. Column chromatography was
performed with Wakogel C-200, C-300 (Wako), or BW-820MH (Fuji
Silysia Chemicals). Methods of preparation and physicochemical data
for the compounds (15–20, 26, 34, 36–38, 40–49, 51–55, 57–60, and
PF1022H from 22) are available in Supplemental Materials (see Biosci.
Biotechnol. Biochem. Web site).
7.6 Hz), 5.84 (1H, t, J ¼ 6:7 Hz), 7.20–7.31 (10H, m). ESI-TOFMS
þ
m=z: calcd. for
939.4946.
C
47H72N4NaO14 ½M þ Naꢃþ, 939.4943; found,
(3S,6R,9S,12R,15S,18R,21S,24R)-6,18-bis(benzo[d]thiazol-2-yl-
methyl)-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-1,7,13,19-
tetraoxa-4,10,16,22-tetraazacyclo-tetracosan-2,5,8,11,14,17,20,23-oc-
taone (13). To
a solution of triphenylphosphine oxide (3.65 g,
13.12 mmol) in dichloroethane (20 mL) was added 9.5 mL of a 0.7 M
solution of Tf2O in CH2Cl2 in a nitrogen atmosphere at 0 ꢀC. After
stirring for 30 min at the same temperature, a mixture of 11 (1.16 g,
1.31 mmol) and 2-aminobenzenethiol (0.31 mL, 2.90 mmol) in di-
chloroethane (11 mL) was added dropwise, before the mixture was
heated to 80 ꢀC for 1 h. The reaction mixture was diluted with CHCl3
(50 mL) and washed with water (30 mL). The organic layer was dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. Purification by silica gel column chromatography
2,20-((2R,5S,8R,11S,14R,17S,20R,23S)-5,11,17,23-tetraisobutyl-
4,8,10,16,20,22-hexamethyl-3,6,9,12,15,18,21,24-octaoxo-1,7,13,19-
tetraoxa-4,10,16,22-tetra-azacyclo-tetracosane-2,14-diyl)diacetic acid
(11) and 2-((2R,5S,8R,11S,14R,17S,20R,23S)-14-benzyl-5,11,17,23-
tetraisobutyl-4,8,10,16,20,22-hexamethyl-3,6,9,12,15,18,21,24-octaoxo-
1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclo-tetracosan-2-yl)acetic acid
(12). A 4.0 g amount (4.21 mmol) of 1 and 9.15 g (40.14 mmol) of
orthoperiodic acid were dissolved in a mixture of CCl4 (8 mL), CH3CN
(8 mL) and water (12 mL). To this solution, 11.4 mg (approximately
0.043 mmol) of ruthenium (III) chloride n-hydrate was added at room
temperature. A 9.1 g amount (39.9 mmol) of orthoperiodic acid was
added twice at 2-h intervals, and the mixture was stirred overnight. The
mixture was further stirred on an ice bath for 50 min after adding 5 mL
of Et2O. The reaction mixture was diluted with water (30 mL) and
extracted twice with CHCl3 (30 mL, 30 mL). The organic layers were
combined, dried over anhydrous sodium sulfate, and evaporated to
dryness. The resulting residue was applied to silica gel column
chromatography (CHCl3=MeOH ¼ 20=1 to 8/1) to give 2.85 g of a
mixture of 11 and 12 with some impurities. This procedure was
repeated to obtain a sufficient mixture of 11 and 12. To a solution of
14.25 g of this mixture of 11 and 12 in EtOAc(120 mL) was added
dropwise a solution of 6.85 g (35.27 mmol) of diphenyl-diazomethane
in EtOAc (50 mL). The mixture was then stirred overnight at room
temperature. The reaction mixture was additionally stirred for 6 h after
adding acetic acid (1.0 mL), and successively washed with saturated
NaHCO3 (100 mL), 10% KHSO4 (100 mL), and brine (100 mL). The
organic layer was dried over anhydrous sodium sulfate and evaporated
to dryness. The resulting residue was applied to silica gel column
(CHCl3=EtOAc ¼ 8=1 to 10/3) gave 0.26 g of 13 as
a white
amorphous powder (19%). ½ꢂꢃD ꢂ65ꢀ (c 0.10, MeOH). 1H-NMR
(270 MHz, CDCl3) ꢁ: 0.55 (6H, d, J ¼ 6:6 Hz), 0.57 (3H, d,
J ¼ 6:6 Hz), 0.80 (3H, d, J ¼ 6:6 Hz), 0.85 (3H, d, J ¼ 6:6 Hz), 0.87
(3H, d, J ¼ 6:6 Hz), 0.92 (3H, d, J ¼ 6:2 Hz), 1.01 (3H, d, J ¼ 6:6 Hz),
1.04 (3H, d, J ¼ 7:5 Hz), 1.39 (3H, d, J ¼ 7:0 Hz), 1.10–1.81 (12H,
m), 2.84 (3H, s), 2.85 (3H, s), 3.13 (3H, s), 3.31 (3H, s), 3.42–3.74 (4H,
m), 4.48 (1H, dd, J ¼ 5:3, 9.7 Hz), 5.08 (1H, q, J ¼ 6:8 Hz), 5.25–5.57
(4H, m), 6.06 (1H, t, J ¼ 7:5 Hz), 6.15 (1H, t, J ¼ 7:3 Hz), 7.31–7.50
25
(4H, m), 7.80–7.95 (4H, m). ESI-TOFMS m=z: calcd. for
C
54H75N6O16S2 ½M þ Hꢃþ, 1063.4884; found, 1063.4885.
þ
(3S,6R,9S,12R,15S,18R,21S,24R)-6-((1H-benzo[d]imidazol-2-yl)
methyl)-18-benzyl-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexameth-
yl-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (14). The same procedure as that used for the
preparation of 13 afforded 248 mg (13%) of 14 as a white amorphous
25
powder from 1.68 g of 11 and 223 mg of 1,2-phenlenediamine. ½ꢂꢃD
ꢂ108ꢀ (c 0.11, MeOH). 1H-NMR (270 MHz, CD3OD, with KSCN)
ꢁ: 0.66 (3H, d, J ¼ 6:5 Hz), 0.77 (3H, d, J ¼ 6:5 Hz), 0.84 (6H, d,
J ¼ 6:5 Hz), 0.88 (3H, d, J ¼ 6:5 Hz), 0.90 (3H, d, J ¼ 6:2 Hz), 0.92
(3H, d, J ¼ 6:5 Hz), 0.95 (3H, d, J ¼ 6:7 Hz), 1.43 (3H, d, J ¼ 7:3 Hz),
1.46 (3H, d, J ¼ 7:0 Hz), 1.10–1.85 (12H, m), 2.90 (3H, s), 2.92 (3H,
s), 3.01 (3H, s), 3.15 (3H, s), 3.06–3.20 (2H, m), 3.33 (1H, dd,
J ¼ 10:0, 15.0 Hz), 3.48 (1H, dd, J ¼ 4:8, 15.0 Hz), 5.28–5.46 (6H,

Structure-Activity Relationship of Cyclodepsipeptides
1361
m), 5.54 (1H, dd, J ¼ 5:5, 9.0 Hz), 5.84 (1H, dd, J ¼ 4:9, 10.0 Hz),
7.21–7.35 (7H, m), 7.55 (2H, brs). ESI-TOFMS m=z: calcd. for
CDCl3) ꢁ: 0.67–0.90 (15H, m), 0.93 (3H, d, J ¼ 6:4 Hz), 1.01 (3H, d,
J ¼ 6:4 Hz), 1.03 (3H, d, J ¼ 5:7 Hz), 1.37 (3H, d, J ¼ 6:7 Hz), 1.38
(3H, d, J ¼ 6:7 Hz), 1.20–1.80 (12H, m), 2.79 (3H, s), 2.83 (6H, s),
3.06 (3H, s), 3.22 (1H, dd, J ¼ 7:3, 13.7 Hz), 3.33 (1H, dd, J ¼ 7:9,
13.7 Hz), 4.50 (1H, dd, J ¼ 5:6, 9.4 Hz), 5.09 (1H, q, J ¼ 6:7 Hz), 5.34
(1H, dd, J ¼ 4:8, 11.4 Hz), 5.36–5.54 (3H, m), 5.69 (1H, t,
J ¼ 7:6 Hz), 5.75 (1H, t, J ¼ 7:6 Hz), 7.42 (2H, dd, J ¼ 1:6, 8.6 Hz),
C53H77N6O12 ½M þ Hꢃþ, 989.5599; found, 989.5601.
þ
Compounds 15–20 were each prepared according to the same
procedure. A mixture of compound 12 (0.2 mmol), amine (0.3 mmol),
HOBt (0.4 mmol) and DCC (0.4 mmol) in THF (2 mL) was stirred at
room temperature for 5–20 h. The reaction mixture was then diluted
with EtOAc (50 mL) and successively washed with 5% KHSO4
(30 mL), saturated NaHCO3 (30 mL), and brine (30 mL). The organic
layer was dried over anhydrous sodium sulfate and evaporated to
dryness. The resulting residue was applied to silica gel column
chromatography (CHCl3=EtOAc ¼ 2=1 to 1/2) to give the desired
compound. The physicochemical data are available in Supplemental
Materials.
7.81–7.85 (2H, m), 8.08 (2H, d, J ¼ 8:6 Hz), 8.98 (1H, s), 8.99 (1H, s).
þ
ESI-TOFMS m=z: calcd. for
1085.4704; found, 1085.4706.
C
54H74N6NaO12S2
½M þ Naꢃþ,
Compounds 26, 41–49 and 51 were prepared according to the same
typical procedure (Method A or Method B).
Method A. A mixture of 5 (1.0 mmol), alkyl halide (2–10 mmol),
K2CO3 or Cs2CO3 (2–5 mmol) and NaI (0.1–1 mmol, if needed) in
acetone (20–30 mL) was stirred at room temperature for 3–24 h. The
reaction mixture was diluted with EtOAc (100 mL), and the organic
layer was successively washed with saturated NaHCO3 (70 mL), 10%
KHSO4 (70 mL), and brine (70 mL). The organic layer was dried over
anhydrous sodium sulfate and evaporated to dryness. The resulting
residue was applied to silica gel column chromatography to give the
desired compound.
Compounds 21, 22 and 25 were prepared according to the
reference.8)
(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,
18,22-hexamethyl-12,24-bis(4-nitrobenzyl)-1,7,13,19-tetraoxa-4,10,16,22-
tetraazacyclotetracosan-2,5,8,11,14,17,20,23-octaone (21) was pre-
pared as a white amorphous powder. ESI-TOFMS m=z: calcd. for
þ
52H78N7O16 ½M þ NH4ꢃþ, 1056.5505; found, 1056.5489. The other
Method B. A mixture of 5 (1.0 mmol), alkyl halide (3–10 mmol) and
NaH (1.9–2.4 mmol) in THF (20 mL) was stirred for 3–24 h at room
temperature in a nitrogen atmosphere. The reaction mixture was
poured into EtOAc (100 mL) and brine (60 mL) in separating funnel.
The organic layer was separated, dried over anhydrous sodium sulfate
and evaporated to dryness. The resulting residue was applied to silica
gel column chromatography to give the desired compound. The
physicochemical data are presented in Supplemental Materials.
C
physical properties were identical to those reported.8)
(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,
18,22-hexamethyl-12,24-bis(4-aminobenzyl)-1,7,13,19-tetraoxa-4,10,
16,22-tetraazacyclotetracosan-2,5,8,11,14,17,20,23-octaone (22) was
prepared as a white amorphous powder. ESI-TOFMS m=z: calcd. for
þ
C
52H78N6NaO12 ½M þ Naꢃþ, 1001.5575; found, 1001.5565. The
other physical properties were identical to those reported.8)
(3S,6R,9S,12R,15S,18R,21S,24R)-6-(4-((1H-tetrazol-5-yl)methoxy)
benzyl)-18-benzyl-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-
1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (27). A mixture of 502 mg (0.499 mmol) of 26, 103 mg
(1.423 mmol) of NaN3 and 208 mg (1.513 mmol) of triethylamine
hydrochloride in DMF (2.5 mL) was stirred at 80 ꢀC for 14 h. The
reaction mixture was diluted with EtOAc (50 mL), and successively
washed with 4% NaHCO3 (30 mL), 10% KHSO4 (30 mL) and brine
(30 mL). The organic layer was dried over anhydrous sodium sulfate
and evaporated. The resulting residue was applied to silica gel column
chromatography (CHCl3=MeOH ¼ 20=1 containing 0.05% acetic acid)
(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,
18,22-hexamethyl-12,24-bis(4-morpholinobenzyl)-1,7,13,19-tetraoxa-
4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,20,23-octaone (25)
was prepared as a white amorphous powder. ESI-TOFMS m=z: calcd.
for C60H91N6NaO14 ½M þ Hꢃþ, 1119.6593; found, 1119.6581. The
other physical properties were identical to those reported.8)
(3S,6R,9S,12R,15S,18R,21S,24R)-6,18-bis((2-aminobenzo[d]thiazol-
6-yl)methyl)-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-1,7,13,
19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,20,23-
octaone (23). To a solution of 1.009 g (1.030 mmol) of 22 and 482 mg
(6.33 mmol) of NH4SCN in acetic acid (9 mL) was added 1.3 mL
(2.0 mmol) of a 1.564 M solution of bromine in CHCl3 at 10–15 ꢀC.
The mixture was stirred at 40 ꢀC for 2 h and then cooled to 0 ꢀC. To this
solution was gradually added 15 mL of 28% NH4OH, the pH value
being 9.5 at that point. The mixture was extracted twice with EtOAc
(60 mL, 40 mL), and the organic layers were combined and succes-
sively washed with saturated NaHCO3 (50 mL) and brine (50 mL). The
organic layer was dried over anhydrous sodium sulfate and evaporated
to dryness. The resulting residue was applied to silica gel column
chromatography (CHCl3=MeOH ¼ 40=1 to 10/1) to give 748 mg of 23
25
to give 518 mg of 27 as a white amorphous powder (99%). ½ꢂꢃD ꢂ92ꢀ
(c 0.11, MeOH). 1H-NMR (270 MHz, CD3OD, with KSCN) ꢁ: 0.85–
0.96 (24H, m), 1.20–1.44 (4H, m), 1.46 (3H, d, J ¼ 6:7 Hz), 1.47 (3H,
d, J ¼ 7:0 Hz), 1.52–1.72 (4H, m), 1.75–1.85 (4H, m), 2.91 (3H, s),
2.92 (3H, s), 3.02 (6H, s), 3.02–3.21 (4H, m), 5.33–5.47 (6H, m), 5.45
(2H, s), 5.52 (1H, dd, J ¼ 5:6, 8.7 Hz), 5.55 (1H, dd, J ¼ 5:4, 8.7 Hz),
7.04 (2H, d, J ¼ 8:6 Hz), 7.24–7.38 (7H, m). ESI-TOFMS m=z: calcd.
for C54H82N9O13 ½M þ NH4ꢃþ, 1064.6032; found, 1064.6030.
þ
(3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-3,9,15,21-tetraisobutyl-
4,10,12,16,22,24-hexamethyl-18-(4-((1-methyl-1H-tetrazol-5-yl)methoxy)
benzyl)-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,
14,17,20,23-octaone (28) and (3S,6R,9S,12R,15S,18R,21S,24R)-6-
benzyl-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-18-(4-((2-
methyl-2H-tetrazol-5-yl)methoxy)benzyl)-1,7,13,19-tetraoxa-4,10,16,22-
tetraazacyclotetracosan-2,5,8,11,14,17,20,23-octaone (29). A mixture
of 188 mg (0.179 mmol) of 27, 74 mg (0.535 mmol) of K2CO3 and
0.11 mL (1.8 mmol) of MeI in acetone (3 mL) was stirred at room
temperature for 1 h. The insoluble materials were filtered off, and the
resulting filtrate was concentrated under reduced pressure. Purification
by preparative TLC (CHCl3=EtOAc ¼ 2=3) afforded 98 mg (51%) of
28 and 93 mg (49%) of 29 as white amorphous powders.
25
as a white amorphous powder (66%). ½ꢂꢃD ꢂ85ꢀ (c 0.16, MeOH).
1H-NMR (270 MHz, CD3OD, with KSCN) ꢁ: 0.82–1.00 (24H, m), 1.45
(6H, d, J ¼ 7:0 Hz), 1.10–1.88 (12H, m), 2.92 (6H, s), 3.03 (6H, s),
3.02–3.26 (4H, m), 5.30–5.51 (6H, m), 5.56 (2H, dd, J ¼ 6:4, 8.7 Hz),
7.26 (2H, dd, J ¼ 2:0, 8.8 Hz), 7.35 (2H, d, J ¼ 8:8 Hz), 7.59 (2H, d,
þ
þ
J ¼ 2:0 Hz). ESI-TOFMS m=z: calcd. for C54H77N8O12S2 ½M þ Hꢃ ,
1093.5102; found, 1093.5105.
(3S,6R,9S,12R,15S,18R,21S,24R)-6,18-bis(benzo[d]thiazol-6-yl-
methyl)-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-1,7,13,19-
tetraoxa-4,10,16,22-tetraazacyclo-tetracosan-2,5,8,11,14,17,20,23-oc-
taone (24). To a solution of 533 mg (0.488 mmol) of 23 in DMF (8 mL)
was added 0.8 mL (5.95 mmol) of isoamyl nitrite. The reaction mixture
was stirred at 65–70 ꢀC for 30 min. The resulting mixture was poured
into water (40 mL) and then extracted three times with CHCl3 (30 mL).
The organic layers were combined, dried over anhydrous sodium
sulfate and evaporated to dryness. The resulting residue was applied to
silica gel column chromatography (CHCl3=EtOAc ¼ 1=1 to
CHCl3=MeOH ¼ 30=1) to give 289 mg of 24 as a white amorphous
25
28. ½ꢂꢃD ꢂ92ꢀ (c 0.10, MeOH). 1H-NMR (270 MHz, CD3OD,
with KSCN) ꢁ: 0.85–0.97 (24H, m), 1.20–1.42 (4H, m), 1.47 (6H, d,
J ¼ 6:2 Hz), 1.53–1.71 (4H, m), 1.75–1.86 (4H, m), 2.92 (3H, s), 2.93
(3H, s), 3.01 (3H, s), 3.02 (3H, s), 3.02–3.21 (4H, m), 4.18 (3H, s),
5.31–5.59 (8H, m), 5.50 (2H, s), 7.06 (2H, d, J ¼ 8:9 Hz), 7.24–7.38
(7H, m). ESI-TOFMS m=z: calcd. for C55H80N8NaO13 ½M þ Naꢃþ,
þ
1083.5743; found, 1083.5738.
25
29. ½ꢂꢃD ꢂ91ꢀ (c 0.10, MeOH). 1H-NMR (270 MHz, CD3OD,
with KSCN) ꢁ: 0.79–1.00 (24H, m), 1.20–1.44 (4H, m), 1.47 (6H, d,
J ¼ 6:7 Hz), 1.52–1.72 (4H, m), 1.75–1.86 (4H, m), 2.92 (3H, s), 2.93
25
powder (56%). ½ꢂꢃD ꢂ74ꢀ (c 0.11, MeOH). 1H-NMR (270 MHz,

1362
M. OHYAMA et al.
(3H, s), 3.01 (3H, s), 3.02 (3H, s), 3.02–3.23 (4H, m), 4.38 (3H, s), 5.31
(2H, s), 5.32–5.52 (7H, m), 5.55 (1H, dd, J ¼ 5:5, 9.0 Hz), 7.02 (2H, d,
J ¼ 8:6 Hz), 7.29 (2H, d, J ¼ 8:6 Hz), 7.23–7.39 (5H, m). ESI-TOFMS
(1.80 mmol) of triethylamine and 240 mg (1.10 mmol) of di-tert-butyl
dicarbonate. The mixture was stirred at room temperature for 2.5 h and
then concentrated under reduced pressure to remove the dioxane. To
the resulting residue were added EtOAc (50 mL) and 5% citric acid
(30 mL). The organic layer was dried over anhydrous sodium sulfate
and evaporated. The resulting residue was applied to silica gel column
chromatography (CHCl3=EtOAc ¼ 2=1) to give 296 mg of the Boc
derivative of 32 as a white amorphous powder (58%). Compound 32
was quantitatively yielded by being treated with TFA and then
neutralized for use without further purification.
m=z: calcd. for C55H80N8NaO13 ½M þ Naꢃþ, 1083.5743; found,
þ
1083.5730.
(3S,6R,9S,12R,15S,18R,21S,24R)-6-(4-((1H-imidazol-2-yl)methoxy)
benzyl)-18-benzyl-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-
1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (30) and (3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-
3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-18-(4-(thiazol-2-
ylmethoxy) benzyl)-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetra-
(3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-18-(4-(2-(dimethylami-
no)ethoxy)benzyl)-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexameth-
yl-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (33). A mixture of 32 (283 mg, 0.282 mmol), 37%
formaldehyde (0.43 mL) and 10% Pd/C (65 mg) in EtOH (6 mL) was
stirred at room temperature for 8 h in a hydrogen atmosphere. After
removing the insoluble materials by filtration, the filtrate was
concentrated and subjected to silica gel column chromatography
(CHCl3=MeOH ¼ 20=1) to give 192 mg (69%) of 33 as a white
cosan-2,5,8,11,14,17,20,23-octaone (31).
A
2.07 g (2.06 mmol)
amount of 26 and 3.0 mL (16.1 mmol) of O,O0-diethyl dithiophosphate
were dissolved in a mixture of CHCl3 (2 mL), toluene (2 mL) and water
(0.35 mL), and the solution was stirred at 80 ꢀC for 30 min. The
reaction mixture was diluted with EtOAc (100 mL), and successively
washed with water (50 mL) and saturated NaHCO3 (50 mL). The
organic layer was dried over anhydrous sodium sulfate and evaporated.
The resulting residue was applied to silica gel column chromatography
(CHCl3=EtOAc ¼ 5=1 to 1/1) to give 1.67 g of the thioamide
intermediate as a white amorphous powder (78%). To a solution of
this thioamide (403 mg, 0.388 mmol) in acetone (40 mL) was added
MeI (0.25 mL, 4.02 mmol), and the mixture was stirred for 2 d at 30 ꢀC.
The reaction mixture was concentrated and dissolved in benzene
(4 mL). To this solution was added aminoacetaldehyde dimethylacetal
(0.04 mL, 0.37 mmol), and the mixture was refluxed for 90 min. A
4 mL amount of 6N HCl was added to this solution, and the mixture
was stirred at 100 ꢀC for 1 h. The reaction mixture was diluted with
EtOAc (50 mL), and successively washed with a mixture of 2N NaOH
(10 mL) and saturated NaHCO3 (30 mL), and brine (30 mL). The
organic layer was dried over anhydrous sodium sulfate and evaporated.
The resulting residue was applied to silica gel column chromatography
(CHCl3=MeOH ¼ 100=1 to 30/1) to give 231 mg of 30 as a white
amorphous powder (57%).
25
amorphous powder. ½ꢂꢃD ꢂ71ꢀ (c 0.13, MeOH). 1H-NMR (270 MHz,
CDCl3) ꢁ: 0.80–1.05 (27H, m), 1.25–1.80 (15H, m), 2.41 (6H, s), 2.73–
3.20 (20H, m), 4.08–5.75 (8H, m), 6.84 (2H, d, J ¼ 8:0 Hz), 7.14 (2H,
d, J ¼ 8:0 Hz), 7.20–7.35 (5H, m). ESI-TOFMS m=z: calcd. for
þ
C
56H86N5O13 ½M þ Hꢃþ, 1036.6222; found, 1036.6216.
(3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-18-(4-(2-(dipropylami-
no)ethoxy)benzyl)-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexameth-
yl-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (35). A mixture of 32 (500 mg, 0.498 mmol), K2CO3
(112 mg, 0.810 mmol) and 1-iodopropane (0.086 mL, 0.88 mmol) in
DMF (5 mL) was stirred overnight at room temperature. The reaction
mixture was diluted with EtOAc (50 mL) and washed with water
(50 mL). The organic layer was dried over anhydrous magnesium
sulfate and evaporated. The resulting residue was applied to silica gel
column chromatography (CHCl3=MeOH ¼ 20=1) to give 160 mg of 35
To a solution of 280 mg (0.270 mmol) of this thioamide just
obtained in toluene (1.5 mL) was added 0.5 mL (3.3 mmol) of
bromoacetaldehyde diethylacetal and conc. H2SO4 (2 drops). The
reaction mixture was stirred at 90 ꢀC for 30 min and then diluted with
EtOAc (30 mL). The organic layer was washed with saturated
NaHCO3 (20 mL), dried over anhydrous sodium sulfate and evapo-
rated. The resulting residue was applied to silica gel column
chromatography (CHCl3=EtOAc ¼ 4=1 to 2/1) to give 87 mg of 31
as a white amorphous powder (30%).
25
as a white amorphous powder (54%). ½ꢂꢃD ꢂ82ꢀ (c 0.21, MeOH).
1H-NMR (270 MHz, CDCl3) ꢁ: 0.80–1.05 (33H, m), 1.25–1.80 (19H,
m), 2.48 (4H, t, J ¼ 7:0 Hz), 2.75–3.15 (20H, m), 3.90–5.67 (8H, m),
6.79–6.82 (2H, m), 7.12–7.30 (7H, m). ESI-TOFMS m=z: calcd. for
þ
C
60H94N5O13 ½M þ Hꢃþ, 1092.6848; found, 1092.6848.
(3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-3,9,15,21-tetraisobutyl-
4,10,12,16,22,24-hexamethyl-18-(4-((S)-pyrrolidin-2-ylmethoxy) benzyl)-
1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,
20,23-octaone (39). To a solution of 522 mg (2.594 mmol) of (S)-tert-
butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate in THF (5 mL) were
added DEAD (0.16 mL, 1.02 mmol) and PPh3 (271 mg, 1.03 mmol).
After 30 min of stirring, 500 mg (0.518 mmol) of PF1022E (5) was
added, and the mixture was stirred overnight. DEAD (0.16 mL,
1.02 mmol) and PPh3 (271 mg, 1.03 mmol) were added again, and the
reaction mixture was stirred at room temperature for 10 d. After
concentration, the resulting residue was diluted with EtOAc (50 mL)
and washed with water (50 mL). The organic layer was dried over
anhydrous magnesium sulfate and evaporated. The resulting residue
was applied to silica gel column chromatography (CHCl3=MeOH ¼
20=1) to give 386 mg of the Boc derivative of 39 as a white amorphous
powder (65%). A 182 mg amount of 39 was obtained as a white
amorphous powder (51%) by treating with TFA, subsequent neutral-
ization and silica gel column chromatography (CHCl3=MeOH ¼ 20=1
20
30. ½ꢂꢃD ꢂ94ꢀ (c 0.12, MeOH). 1H-NMR (270 MHz, CDCl3)
ꢁ: 0.78–0.92 (18H, m), 0.95 (3H, d, J ¼ 6:0 Hz), 1.00 (3H, d,
J ¼ 6:8 Hz), 1.03 (3H, d, J ¼ 6:5 Hz), 1.20–1.78 (12H, m), 1.39 (3H,
d, J ¼ 7:3 Hz), 2.75 (3H, s), 2.81 (3H, s), 2.82 (3H, s), 3.00 (3H, s),
2.92–3.21 (4H, m), 4.42–4.52 (1H, m), 5.06–5.22 (1H, m), 5.13 (2H,
s), 5.34 (1H, dd, J ¼ 4:6, 11.5 Hz), 5.34–5.60 (3H, m), 5.61 (1H, t,
J ¼ 7:5 Hz), 5.67 (1H, t, J ¼ 7:5 Hz), 6.90 (2H, d, J ¼ 8:5 Hz), 7.07
(2H, s), 7.13 (2H, d, J ¼ 8:5 Hz), 7.19–7.34 (5H, m). ESI-TOFMS
m=z: calcd. for
C55H80N8O13
½M þ Hꢃþ, 1045.5862; found,
þ
1045.5860.
31. ½ꢂꢃD ꢂ102ꢀ (c 0.11, MeOH). 1H-NMR (270 MHz, CDCl3)
ꢁ: 0.78–0.92 (18H, m), 0.95 (3H, d, J ¼ 6:5 Hz), 1.01 (3H, d,
J ¼ 6:6 Hz), 1.03 (3H, d, J ¼ 6:6 Hz), 1.20–1.83 (12H, m), 1.40 (3H,
d, J ¼ 7:0 Hz), 2.73 (3H, s), 2.81 (3H, s), 2.83 (3H, s), 3.01 (3H, s),
2.92–3.21 (4H, m), 4.43–4.51 (1H, m), 5.08 (1H, q, J ¼ 6:7 Hz), 5.35
(2H, s), 5.30–5.72 (6H, m), 6.94 (2H, d, J ¼ 8:4 Hz), 7.17 (2H, d,
J ¼ 8:4 Hz), 7.20–7.35 (5H, m), 7.37 (1H, d, J ¼ 3:4 Hz), 7.80 (1H, d,
20
25
to 10/1). ½ꢂꢃD ꢂ80ꢀ (c 0.11, MeOH). 1H-NMR (270 MHz, CDCl3)
J ¼ 3:4 Hz). ESI-TOFMS m=z: calcd. for
C
56H80N5NaO13Sþ
ꢁ: 0.80–1.05 (27H, m), 1.36–2.01 (19H, m), 2.75–3.15 (19H, m),
3.65–3.71 (1H, m), 4.36–4.52 (1H, m), 4.97–5.21 (1H, m), 5.31–
5.67 (7H, m), 6.81–6.85 (2H, m)þ, 7.12–7.27 (7H, m). ESI-TOFMS
m=z: calcd. for C57H85N5NaO13 ½M þ Naꢃþ, 1070.6042; found,
1070.6034.
½M þ Hꢃþ, 1062.5473; found, 1062.5472.
(3S,6R,9S,12R,15S,18R,21S,24R)-6-(4-(2-aminoethoxy)benzyl)-18-
benzyl-3,9,15,21-tetraisobutyl-4,10,12,16,22,24-hexamethyl-1,7,13,19-
tetraoxa-4,10,16,22-tetraazacyclo-tetracosan-2,5,8,11,14,17,20,23-oc-
taone (32). A mixture of 26 (461 mg, 0.459 mmol), 10%Pd/C (50 mg)
and conc. HCl (0.2 mL) in EtOH (15 mL) was stirred at room
temperature in a hydrogen atmosphere at 3 atm for 16 h. The reaction
mixture was filtered, and the resulting filtrate was concentrated under
reduced pressure. The resulting residue was dissolved in dioxane
(10 mL) and water (10 mL). To this solution were added 0.25 mL
(3S,6R,9S,12R,15S,18R,21S,24R)-6-benzyl-3,9,15,21-tetraisobutyl-
18-(4-tert-butoxybenzyl)-4,10,12,16,22,24-hexamethyl-1,7,13,19-tetraoxa-
4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,20,23-octaone (50).
To a solution of 701 mg (0.726 mmol) of 5 in CH2Cl2 (10 mL) were
added 1.4 mL of isobutene and 0.11 mL of H2SO4 at ꢂ40 ꢀC. After
stirring for 2 h at room temperature, 0.6 mL of triethylamine was added

Structure-Activity Relationship of Cyclodepsipeptides
at 0 ꢀC. The reaction mixture was concentrated under reduced pressure.
1363
References
The resulting residue was dissolved in 70 mL of EtOAc and
successively washed with 5% KHSO4 (70 mL) and brine (70 mL).
The organic layer was dried over anhydrous magnesium sulfate and
evaporated. The resulting residue was applied to silica gel column
chromatography (CHCl3=EtOAc ¼ 4=1) to give 578 mg of 50 as a
1) Sasaki T, Takagi M, Yaguchi T, Miyadoh S, Okada T, and
Koyama M, J. Antibiot., 45, 692–697 (1992).
2) Sasaki T, Kuwata M, Shimizu A, Takagi M, Kubota H, Okada
T, Uotani K, and Koyama M, Japan Kokai Tokkyo Koho, 1993-
170749 (July 9, 1993).
20
white amorphous powder (78%). ½ꢂꢃD ꢂ92ꢀ (c 0.1, MeOH). 1H-
NMR (270 MHz, CDCl3) ꢁ: 0.80–1.05 (27H, m), 1.32 (9H, s), 1.40
(3H, d, J ¼ 7:0 Hz), 1.10–1.80 (12H, m), 2.70–3.20 (16H, m), 5.30–
5.80 (8H, m), 7.00 (2H, d, J ¼ 8:5 Hz), 7.21–7.35 (7H, m). ESI-
3) Ohyama M, Okada Y, Nakagawa K, Takagi M, Okada T, Murai
Y, Yoneta T, and Iinuma K, Japan Kokai Tokkyo Koho, 1994-
184126 (July 5, 1994).
TOFMS m=z: calcd. for C56H88N5O13 ½M þ NH4ꢃþ, 1038.6379;
þ
4) Ohyama M, Takahashi M, Shigematsu Y, Sakanaka O, Murai Y,
and Iinuma K, WO1998/05655 (Dec. 2, 1998).
found, 1038.6382.
5) Suzuki A, Kanaoka M, Isogai A, Murakoshi S, Ichinoe M, and
Tamura S, Tetrahedron Lett., 2167–2170 (1977).
6) Omura S, Shiomi K, and Masuma R, WO2004/044214 (May
27, 2004).
(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,
18,22-hexamethyl-12,24-bis(4-(2-morpholinoethoxy)benzyl)-1,7,13,19-
tetraoxa-4,10,16,22-tetraazacyclotetracosan-2,5,8,11,14,17,20,23-oc-
taone (56). A mixture of 52 (232 mg, 0.217 mmol), NaI (40 mg,
0.267 mmol), K2CO3(180 mg, 1.305 mmol) and 2-bromoethylether
(0.07 mL, 0.50 mmol) in DMF (5 mL) was stirred at 80 ꢀC for 5 h.
The reaction mixture was poured into water (30 mL) and extracted
twice with CHCl3 (50 mL, 20 mL). The organic layers were combined,
dried over anhydrous sodium sulfate and evaporated. The resulting
residue was applied to silica gel column chromatography
(CHCl3=MeOH ¼ 20=1) to give 181 mg of 56 as a white amorphous
7) Samson-Himmelstjerna G, Harder A, Sangster NC, and Coles
GC, Parasitology, 130, 343–347 (2005).
8) Nishiyama H, Oogaki M, Yamanishi R, and Hara T, WO1993/
19053 (Sep. 30, 1993).
9) Ohyama M, Iinuma K, Isogai A, and Suzuki A, Biosci.
Biotechnol. Biochem., 58, 1193–1194 (1994).
10) Dutton FE and Nelson SJ, J. Antibiot., 47, 1322–1327 (1994).
11) Kobayashi M, Nanba T, Toyama T, and Saito A, Annual Report
of Sankyo Research Laboratories, 46, 67–75 (1994).
12) Scherkenbeck J, Plant A, Harder A, and Mencke N, Tetrahe-
dron, 51, 8459–8470 (1995).
20
powder (83%). ½ꢂꢃD ꢂ84ꢀ (c 0.19, MeOH). 1H-NMR (270 MHz,
CDCl3) ꢁ: 0.76–0.92 (18H, m), 1.00 (3H, d, J ¼ 7:0 Hz), 1.03 (3H, d,
J ¼ 6:7 Hz), 1.39 (3H, d, J ¼ 7:6 Hz), 1.21–1.83 (12H, m), 2.57 (8H, t,
J ¼ 4:5 Hz), 2.79 (4H, t, J ¼ 5:6 Hz), 2.83 (3H, s), 2.88 (3H, s), 2.96
(3H, s), 3.01 (3H, s), 2.90–3.14 (4H, m), 3.73 (8H, t, J ¼ 4:5 Hz), 4.07
(4H, t, J ¼ 5:6 Hz), 4.47 (1H, dd, J ¼ 5:6, 9.0 Hz), 5.07 (1H, q,
J ¼ 7:0 Hz), 5.18 (1H, dd, J ¼ 5:4, 10.0 Hz), 5.34 (1H, dd, J ¼ 4:6,
11.4 Hz), 5.41 (1H, q, J ¼ 7:6 Hz), 5.50 (1H, dd, J ¼ 3:9, 12.3 Hz),
5.57 (1H, t, J ¼ 7:9 Hz), 5.63 (1H, t, J ¼ 7:6 Hz), 6.82 (4H, d,
13) Hendrickson JB and Hussoin Md. S, J. Org. Chem., 52, 4137–
4139 (1987).
14) Harder A, Holden-Dye L, Walker R, and Wunderlich F,
Parasitol. Res., 97, S1–S10 (2005).
15) Saeger B, Schmitt-Wrede H-P, Dehnhardt M, Benten WP,
Krucken J, Harder A, Samson-Himmelstjerna G, Wiegand H,
¨
and Wunderlich F, FASEB J., 15, 1332–1334 (2001).
J ¼ 8:4 Hz), 7.14 (4H, d, J ¼ 8:4 Hz). ESI-TOFMS m=z: calcd. for
þ
16) Harder A, Schmitt-Wrede H-P, Krucken J, Marinovski P,
¨
C
64H99N6O16 ½M þ Hꢃþ, 1207.7118; found, 1207.7117.
Wunderlich F, Willson J, Amliwala K, Holden-Dye L, and
Walker R, Int. J. Antimicrob. Agents, 22, 318–331 (2003).
17) Welz C, Harder A, Schnieder T, Hoglund J, and
Samson-Himmelstjerna G, Parasitol. Res., 97, S22–S32 (2005).
18) Lipinski CA, Lombardo F, Dominy BW, and Feeney PJ, Adv.
Drug Del. Rev., 23, 3–25 (1997).
Acknowledgments
Part of this work was done at the Bioorganic
Chemistry Laboratory in the Faculty of Agriculture at
the University of Tokyo. The authors thank Professor
Akinori Suzuki, Professor Akira Isogai, Professor
Hiroshi Kataoka and the group members at that time.
The authors also thank the members of the Agricultural
and Veterinary Research Laboratories of Meiji Seika
Kaisha Ltd., for the measurements in the anthelmintic
assay.
19) Clark DE, J. Pharm. Sci., 88, 807–814 (1999).
20) Scherkenbeck J, Harder A, Plant A, and Dyker H, Bioorg. Med.
Chem. Lett., 8, 1035–1040 (1998).
21) Jeschke P, Harder A, Etzel W, Gau W, Thielking G, Bonse G,
and Iinuma K, Pest Manag. Sci., 57, 1000–1006 (2001).
22) Jeschke P, Harder A, Samson-Himmelstjerna G, Etzel W, Gau
W, Thielking G, and Bonse G, Pest Manag. Sci., 58, 1205–1215
(2002).
23) Kodama Y, Takeuchi Y, and Suzuki A, Sci. Reports of Meiji
Seika Kaisha, 31, 1–8 (1992).