Total synthesis and conformational studies of ceratospongamide, a bioactive cyclic heptapeptide from marine origin

Article abstract of DOI:10.1016/S0040-4020(02)00906-7

The first total synthesis of cis,cis-ceratospongamide (cyclo[L-Pro-L-Ile-Me-oxazoline-L-Phe-L-Pro-thiazole-L-Phe-]) was accomplished and confirmed by X-ray crystal analysis. The heating of cis,cis-ceratospongamide in DMSO converted it not to the trans,trans isomer but to the trans,trans-[D-allo-Ile]-ceratospongamide, which was confirmed by total synthesis. Its solution conformation was constructed by the dynamic simulated annealing method using ROE cross peaks, revealing a rounded and flat ring structure which is in contrast with the slim and tight structure of cis,cis isomer. The results shows that the trans,trans-[D-allo-Ile] isomer is the main thermal product of cis,cis-ceratospongamide.

Full text of DOI:10.1016/S0040-4020(02)00906-7

Tetrahedron 58 (2002) 8127–8143
Total synthesis and conformational studies of ceratospongamide,
a bioactive cyclic heptapeptide from marine origin
a
b
b
Fumiaki Yokokawa,a Hirofumi Sameshima, Yasuko In, Katsuhiko Minoura,
,
,
*
*
†
Toshimasa Ishidab and Takayuki Shioiria
,
aGraduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
bOsaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 18 April 2002; revised 2 July 2002; accepted 25 July 2002
Abstract—The first total synthesis of cis,cis-ceratospongamide (cyclo[L-Pro-L-Ile-Me-oxazoline-L-Phe-L-Pro-thiazole-L-Phe-]) was
accomplished and confirmed by X-ray crystal analysis. The heating of cis,cis-ceratospongamide in DMSO converted it not to the
trans,trans isomer but to the trans,trans-[D-allo-Ile]-ceratospongamide, which was confirmed by total synthesis. Its solution conformation
was constructed by the dynamic simulated annealing method using ROE cross peaks, revealing a rounded and flat ring structure which is in
contrast with the slim and tight structure of cis,cis isomer. The results shows that the trans,trans-[D-allo-Ile] isomer is the main thermal
product of cis,cis-ceratospongamide. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
symbiotica.7 This peptide consists of two L-phenylalanine
residues, one (L-isoleucine)-methyloxazoline residue, one
L-proline residue, and one (L-proline)-thiazole residue.
Interestingly, Gerwick and co-workers reported that cerato-
spongamide can be isolated as two stable isomers: the major
cis,cis isomer (1) and the minor trans,trans isomer (2)
around the two proline–phenylalanine amide bonds.
Although both isomers show moderate potency in the
brine shrimp toxicity assay (LD50¼13–19 mM), consider-
ably different activity is observed in terms of the inhibition
of secreted phospholipase A2 expression, a key enzyme in
the inflammatory cascade: ED50¼32 nM for 2 and is
inactive for 1. We were intrigued by the highly function-
alized macrocyclic ring structure as well as the difference in
biological activity exhibited by the conformational isomers
of ceratospongamide, and we therefore performed the total
synthesis and conformational analysis of ceratosponga-
mide.8 Herein, we report the total synthesis of cis,cis-
ceratospongamide (1) and its thermal product, trans,trans-
[D-aIle]-ceratospongamide (3), the X-ray single-crystal
analysis of 1, the solution conformation of 3 as determined
by 1H NMR spectroscopy and dynamic simulated annealing
(SA) methods, and the conformational stabilities of 1, 2
and 3 as determined by molecular mechanics/dynamics
calculations (Fig. 1).
Interest in marine organisms has been steadily increasing
primarily due to the presence of metabolites capable of
acting as potent bioactive agents for therapeutic use.1–4
Marine cyclic peptides have, in particular, shown a broad
range of bioactivities such as cytotoxic, tumor-promoting,
anticancer, antiviral, and anti-inflammatory activities. Many
of these cyclic peptides contain five-membered heterocycles
(oxazolines, oxazoles, thiazolines and thiazoles). Therefore,
the presence of heterocyclic building blocks as well as the
challenges in macrocyclization has stimulated the interest of
synthetic chemists in marine cyclic peptides.5 On the other
hand, upon the introduction of five-membered heterocycles
into the cyclic peptide sequence, the conformational
flexibility of the macrocycles is considerably reduced, and
usually a single secondary structure is preferred in the solid
state and in solution. Accordingly, a thorough understanding
of structural and conformational properties can facilitate the
studies concerning structure–activity relationships, which
are very helpful in the rational design of new potent drugs.6
Ceratospongamide is a bioactive oxazoline, thiazole-
containing cyclic heptapeptide isolated by Gerwick and
co-workers from the Indonesian red alga Ceratodictyon
spongiosum and the symbiotic sponge Sigmadocia
Keywords: cis,cis-ceratospongamide; trans,trans-[D-allo-Ile] cerato-
spongamide; thermodynamic isomerization; synthesis; conformation.
2. Results and discussion
2.1. Synthesis
*
Corresponding authors. Address: Novartis Pharma, Tsukuba Research
Institute, Tsukuba 300-2611, Japan. Tel.: þ81-298-65-2137; fax: þ81-
298-65-2308; e-mail: fumiaki.yokokawa@pharma.novartis.com; Tel.:
þ81-726-90-1069; fax: þ81-726-90-1068; e-mail: in@gly.oups.ac.jp
Present address: Graduate School of Environmental and Human Sciences,
Meijo University, Nagoya, 468-8502, Japan.
†
2.1.1. Retrosynthetic strategy. Our retrosynthetic analysis
of ceratospongamide is shown in Scheme 1. Since the
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00906-7

8128
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Figure 1. Structures of ceratospongamides.
Thz
S
L-Pro
L-Phe
macro-
lactamization
at Thz/Phe
S
O
N
cyclo-
dehydration
N
CO2H
H2N
O
O
N
N
H
N
N
O
O
ceratospongamide
H
N
H
H
N
NH
NH
N
N
H
N
O
O
L-Pro
L-Phe
O
O
HO
HO
L-aThr
O
O
L-Ile
4
5
macrolactamization
at Pro/Ile
O
S
N
Boc-L-Ile-L-aThr-L-Phe-L-Pro-Thz-OMe (7)
O
N
H
N
N
H
O
+
NH
N
Boc-L-Phe-L-Pro-OMe (8)
NH2
O
O
HO
HO2C
6
Scheme 1. Retrosynthetic analysis of ceratospongamide.
oxazoline ring is sensitive under acidic conditions and
readily epimerized at the chiral centers attached to the
heterocycle under mild acidic or basic conditions, the final
formation of the oxazoline ring by cyclodehydration of the
allo-L-threonine residue was employed to give the cyclic
peptide 4. Macrolactamization at the thiazole/L-phenyl-
alanine or L-proline/L-isoleucine amide bond of 4 was
chosen to avoid epimerization at the C-terminus to provide
HCl·HN(OMe)Me
DEPC, Et3N
Me
LiAlH4
N
CO2H
OMe
N
Boc
9
N
Boc
10
CHO
N
DMF
88 %
Et2O
O
Boc
11
HCl·H-L-Cys-OMe
Et3N
S
S
CMD
N
Boc
12
N
Boc
13
benzene
51 %
toluene
N
H
N
CO2Me
CO2Me
73 % (2 steps)
H
Scheme 2. Synthesis of Boc-L-Pro-Thz-OMe (13).

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
DEPC, Et3N, DMF
8129
Boc-L-Phe-OH (14)
HCl·H-L-Pro-OMe (15)
Boc-L-Pro-Thz-OMe (13)
Boc-L-Phe-L-Pro-OMe (8)
97 %
HCl-dioxane
DEPC, Et3N
DMF
HCl-dioxane
DEPC, Et3N
DMF
Boc-L-Phe-L-Pro-
Thz-OMe (16)
Boc-L-aThr-L-Phe-
L-Pro-Thz-OMe (18)
94 %
Boc-L-Phe-OH (14)
94 %
Boc-L-aThr-OH (17)
HCl-dioxane
DEPC, Et3N, DMF
75 %
Boc-L-Ile-L-aThr-L-Phe-L-Pro-Thz-OMe (7)
Boc-L-Ile-OH (19)
S
CO2Me
N
1. aq. LiOH, THF
2. HCl-dioxane
HCl-dioxane
DEPC, Et3N
O
Boc-L-Ile-L-aThr-L-Phe-
L-Pro-Thz-OMe (7)
N
H
HN
Boc
O
DMF
4
NH
3. a) DPPA, Et3N, DMF, 4 ˚C, 144 h
b) FDPP, i-Pr2NEt, DMF, 4 ˚C, 144 h
c) FDPP, i-Pr2NEt, DMF, rt, 50 h
d) HATU, i-Pr2NEt, DMF, 4 ˚C, 144 h
31 %
94 %
N
N
H
N
Boc-L-Phe-L-Pro-OMe (8)
47 %
63 %
18 %
aq. LiOH, THF
O
O
HO
O
20
O
S
N
aq. LiOH, THF
1. aq. LiOH, THF
2. HCl-dioxane
Boc-L-Ile-L-aThr-L-Phe-
L-Pro-Thz-OMe (7)
DEPC, Et3N
DMF
O
N
H
N
N
H
O
4
NH
3. a) DPPA, Et3N, DMF, rt, 74 h
b) FDPP, i-Pr2NEt, DMF, rt, 48 h
c) FDPP, i-Pr2NEt, DMF, rt, 74 h
d) HATU, i-Pr2NEt, DMF, rt, 137 h
23 %
99 %
N
Boc
Boc-L-Phe-L-Pro-OMe (8)
24 %
27 %
0 %
HCl-dioxane
NH
O
O
HO
21
CO2Me
O
S
F
F
F
F
N
N
O
O
-
N
Ph
Ph
PF6
NMe2
Deoxo-Fluor
4
+
N
N
(PhO)2P(O)N3
DPPA
P O
F
N
O
HATU
O
N
H
H
N
NH
O
CH2Cl2, -30 ~ -20 ˚C
N
N
NMe2
FDPP
54 %
O
O
(CH OCH CH ) NSF
3
Deoxo-Fluor
3
2
2 2
cis,cis-ceratospongamide (1)
Scheme 3. Total synthesis of cis,cis-ceratospongamide (1).
the linear peptide 5 or 6.9 For the synthesis of the linear
peptide 5 or 6, a [5þ2] convergent strategy was adopted to
give the pentapeptide 7 and dipeptide 8.
protected L-phenylalanine (14) with L-proline methyl ester
(15) using DEPC in 97% yield. Preparation of pentapeptide
segment 7 was initiated by removing the Boc protective
group of thiazole fragment 13 with hydrogen chloride in
dioxane followed by condensation with Boc-protected
L-phenylalanine (14) using DEPC in 94% yield. Iterative
removal of the Boc protective group followed by peptide
coupling with Boc-protected L-allo-threonine (17) and Boc-
protected L-isoleucine (19) gave pentapeptide segment 7.
After acidic deprotection of the Boc group of 7, segment
condensation with the acid derived from dipeptide 8
smoothly proceeded to give fully protected heptapeptide
20 in 94% yield. Similarly, deprotection of the Boc group of
8 and segment condensation with the acid derived from 7
also afforded linear peptide 21 in 99% yield. Then, the
comparison of the macrolactamization step at two sites
(Thz/Phe vs. Pro/Ile) was carried out. After saponification of
the methyl ester in 20 and 21 with aqueous lithium
hydroxide and acidic cleavage of the Boc group,
macrolactamizations of the resulting two free linear
peptides were performed in DMF solution (0.002 M) by
2.1.2. Total synthesis of cis,cis-ceratospongamide (1).
Since we have shown that optically pure thiazole amino
acids can be prepared by oxidation of thiazolidines with
chemical manganese dioxide (CMD) that is manufactured
industrially for batteries,10 we applied this methodology to
the synthesis of thiazole fragment 13. Coupling of Boc-
protected L-proline (9) with N,O-dimethylhydroxylamine
using diethyl phosphorocyanidate (DEPC, (EtO)2-
P(O)CN)11 afforded amide 1012 in 88% yield. After
reduction of amide 10 with lithium aluminum hydride,13
the resulting aldehyde 11 was condensed with L-cysteine
methyl ester to give thiazolidine 12 as a diastereomeric
mixture in 73% yield. Subsequent CMD oxidation of
thiazolidine 12 provided thiazole fragment 13 in 51%
yield (Scheme 2).
Dipeptide segment 8 was obtained by coupling of Boc-

8130
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Figure 2. Stereoscopic molecular conformation of 1. The v torsion angles of two Pro amide bonds have cis orientation. No intramolecular hydrogen bonds are
formed.
using diphenyl phosphorazidate (DPPA),11,14 pentafluoro-
phenyl diphenylphosphinate (FDPP),15 or O-(7-azabenzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate (HATU).16 The cyclization at Thz/Phe site
smoothly proceeded and the yield by FDPP was higher
than those by DPPA and HATU. In contrast, the cyclization
at Pro/Ile site was sluggish, and no cyclized product was
obtained by using HATU. Accordingly, we carried out the
macrolactamization at Thz/Phe site with 1.5 equiv. of FDPP
and 4 equiv. of i-Pr2NEt at room temperature to give cyclic
peptide 4 in 63% yield from corresponding linear peptide
20. Finally, dehydrative cyclization of the allo-threonine
residue using bis(2-methoxyethyl)aminosulfur trifluoride
(Deoxo-fluor) to the oxazoline17 provided a single isomer of
ceratospongamide, which was completely identical with
natural cis,cis-ceratospongamide (1) as judged from 1H and
13C NMR spectra, HPLC, and TLC Rf values. Furthermore,
crystallization of synthetic cis,cis-ceratospongamide (1)
from chloroform–heptane mixture followed by X-ray
analysis allowed us to confirm that both Phe-Pro amide
bonds have cis orientation as described below. Accordingly,
our total synthesis and X-ray analysis have demonstrated
that the proposed structure of cis,cis-ceratospongamide (1)
by Gerwick and co-workers is correct (Scheme 3).
observed for the conformational torsion angles. The thiazole
and oxazoline rings are almost planar and are nearly
orthogonal to each other (dihedral angle¼80.28). The two
Pro cyclic rings exhibit typical Cb-exo-Cg-endo ring
puckering (C2-conformation).
The cyclic backbone chain of the molecule assumes a
distorted saddle-shaped rectangular structure. The backbone
structure of the thiazole-Phe-2-Pro-1 sequence forms a
warped plane; the distance between the N(Pro-1)-Ca(Pro-2)
˚
line and the C(4)–C(6) bond is about 2.5 A. This plane
forms one side of the rectangular long axes. The other side is
formed by the Ile-oxazoline-Phe-1 sequence, although the
backbone structure is considerably distorted from the
planarity because the oxazoline ring at the central position
forms a dihedral angle of 64.88 with respect to the amide
plane of Phe-1. Residues of Pro-1, Ile, Phe-1 and Pro-2 are
located at each corner of the ring structure, with the Ile side
chain and the oxazoline methyl group protruding above and
below the ring chain, and two Phe aromatic rings are
oriented antiparallel to each other along the ring structure.
One conformational feature of 1 is the absence of
intramolecular hydrogen bonds, which contribute to the
conformational stabilization of many cyclic peptides. All
peptide amide groups are approximately on the belt-plane of
cyclic backbone ring, thereby hindering the formation of the
intramolecular NH· · ·OvC hydrogen bonds. This indicates
that several restraints are imposed on the cyclic ring
structure, meaning the absence of conformational flexibility.
2.2. X-Ray crystal structure of 1
A stereoscopic view of cis,cis-isomer of the ceratosponga-
mide is shown in Fig. 2.18 Conformational torsion angles are
listed in Table 1. Because of the relatively high thermal
motion of each residue, the estimated standard deviation
No intermolecular hydrogen bonds or short contacts less
˚
than 3.5 A are observed between neighboring molecules,
˚
(esd) values for bond lengths (,0.1 A) and angles (0.5–18)
are somewhat larger than usual. However, these geometric
parameters are all within the range of those of similar-sized
depsipeptides, such as ascidiacyclamide19 and didemnin
B.20 Both Phe-Pro amide bonds take cis orientation of v
torsion angle (22.5 and 23.48), whereas the other peptide
bonds are all in the trans region. No notable anomaly was
although three water solvents molecules are included. These
solvents occupy the cavities created by the molecular
packing of 1, and hydrogen bonds are formed amidst them.
On the other hand, because of the tetragonal crystal
symmetry, the respective molecular packings along the a-
and b-axes are completely the same. Thus, such

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Table 1. Conformational torsion angles (degrees) of 1, 2, and 3
8131
spongamide (2). Gerwick and co-workers reported that
cis,cis-ceratospongamide (1) could be converted into the
trans,trans-isomer (2) by heating at 1758C in DMSO
according to HPLC monitoring.7 Therefore, we tried the
thermodynamic isomerization from our synthetic cis,cis-
ceratospongamide (1) to the trans,trans-isomer (2). As was
reported in their paper,7 thermal treatment of synthetic
cis,cis-ceratospongamide (1) at 1758C for 30 min in DMSO
gave a compound different from 1 in the isolated yield of 10–
30%(entry1). Thisthermalconversionproceededslowlyeven
below 1008C and was almost completed after 8 days (entry 2).
Interestingly, we also found that slightly acidic conditions (in
the presence of pyridinium p-toluenesulfonate) facilitated the
thermal conversion (1008C for 40 min, 75% isolated yield,
entry 3) as shown in Scheme 4.
1 (X-raya)
2 (MD)
3 (NMR/SA)
Pro-1
f
c
v
x1
x2
x3
x4
290.3 (8)
25.5 (7)
175 (1)
33.0 (7)
231.3 (7)
16.6 (7)
4.4 (6)
274.5
44.4
178.8
222.2
40.5
266.7
85.9
176.0
218.1
33.7
240.1
26.2
234.9
25.0
Ile/D-allo-Ile
f
c
v
x1,1
x1,2
x2
2100.3 (8)
217.5 (8)
2177.7 (8)
266.0 (8)
65.3 (8)
2103.5
30.1
2170.8
272.2
56.5
138.0
54.7
155.4
235.1
92.7
163 (1)
164.1
76.8
Oxazoline
Phe-1
fb
cc
v
124.0 (8)
27.6 (7)
177.1 (8)
138.7
268.7
2168.3
135.4
266.8
2173.7
Since MS measurements showed the same molecular weight
as 1, this thermal product could be a conformational and/or
structural isomer of 1. Therefore, we performed the total
synthesis of [D-allo-Ile]-ceratospongamide (3) using D-allo-
Ile instead of L-Ile. As shown in Scheme 5, the successive
attachment of Boc-D-aIle-OH (22) and Boc-L-Phe-L-Pro-
OMe (8) to Boc-L-aThr-L-Phe-L-Pro-Thz-OMe (18)
afforded the protected linear precursor 24, which, after
deprotection at C- and N-termini, underwent macro-
lactamization by using FDPP to give macrocycle 25. Final
construction of the oxazoline ring using Deoxo-fluor
afforded [D-allo-Ile]-ceratospongamide (3).
f
c
v
x1
x1,2
x2,1
2155.0 (8)
108.8 (8)
22.5 (6)
2175.8 (9)
70 (1)
2154.3
270.3
2171.7
241.9
106.0
298.6
136.1
2173.2
2176.0
85.6
2108 (1)
284.0
292.7
Pro-2
f
c
267.1 (7)
231.5 (7)
2177.5 (7)
33.1 (6)
240.3 (7)
30.8 (7)
29.2 (6)
263.2
252.8
2179.5
219.7
36.8
240.7
93.4
2158.8
237.6
41.3
vd
x1
x2
x3
x4
237.7
27.7
228.0
3.8
Surprisingly, synthetic peptide 3 was completely identical
with the thermal product of cis,cis-isomer (1) as judged
Thiazole
Phe-2
fe
cf
v
2176.5 (8)
22.0 (6)
174.6 (9)
2172.9
27.0
164.4
158.4
213.8
2169.4
1
from H and 13C NMR spectra and TLC Rf values. The
1
direct comparison of H NMR spectra for the synthetic 3
f
c
v
x1
x1,2
x2,1
2165.8 (8)
141.9 (8)
23.4 (6)
2170.9 (8)
85.1 (9)
294.1 (9)
2117.6
153.9
2156.0
176.4
102.5
2172.2
163.8
169.7
62.4
87.8
293.8
with natural trans,trans-ceratospongamide (2) showed a
good spectroscopic match, although there were subtle
differences around 5 ppm. Moreover, there were no
differences (Dd,0.4 ppm) in the reported data of 13C
NMR spectra between natural 2 and synthetic 3. In addition,
the optical rotation of synthetic 3 ([a]D¼238.8 (c 0.5,
CHCl3) was also the same to that of the natural 2
([a]D¼239.2 (c 0.5, CHCl3)). During the preparation of
this manuscript, Deng and Taunton published the total
synthesis of trans,trans-ceratospongamide (2) together with
cis,cis-ceratospongamide (1).21 Interestingly, their synthetic
trans,trans isomer (2) was completely identical with our
synthetic 3 in 1H and 13C NMR spectra. On the other hand,
Deng and Taunton reported21 that their synthetic trans,
trans-ceratospongamide was identical to naturally derived
one, and 1 was thermally transformed into an equilibrium
mixture of 1 and 2 in a ratio of 1:5.
287.7
The X-ray, MD and NMR/SA results are given for 1, 2, and 3, respectively.
The esd values of X-ray torsion angles in 1 are given in parentheses.
a
b
C2–N–C4–C6.
N–C4–C6–N(Phe-1).
c
d
Ca–C2–N(thiazole)–C4.
C2–N–C4–C6.
N–C4–C6–N(Phe-2).
e
f
crystallographic restraints may significantly affect the
molecular conformation of 1. The partial crystal packing
that shows the most crowded molecular association is
shown in Fig. 3. Two different interfaces could be observed:
one is between the right and central molecular layers, and
the other, between the central and left ones. The former
interface is formed by van der Waals forces such that the
uneven structures of the respective molecules are oriented in
such a way that they complement each other. In the latter
interface, the partial insertion of an L-Phe aromatic ring
into the concave ring structure of 1 is observed. However,
such interfaces are relatively weak, and each space created
by the molecular packing is occupied by three water
molecules.
To thoroughly understand the structure of our [D-allo-Ile]-
ceratospongamide (3), determination of the sequence and
the connection of amino acid residues and other units
(thiazole and methyloxazoline) was performed by a
1
combination of both one- and two-dimensional H NMR
spectral measurements (GCOSY, TOCSY and ROESY).
The results are given in Table 2, where the extensive
overlapping of the Phe-1 and Phe-2 aromatic protons made
complete peak assignment impossible.
2.3. Thermal product of 1 and structural analysis
The trans orientations of the two proline amide bonds were
shown by (i) the absence of ROEs of Ha(Pro-1)-Ha(Phe-2)/
We next investigated the synthesis of trans,trans-cerato-

8132
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Figure 3. Stereoscopic view of molecular interaction modes formed in the crystal structure. The left and central molecular layers running from the bottom to
the top are related to each other by a diad screw symmetry, and the central and right ones, by a diad symmetry. Open circles (red color) represent water
˚
˚
molecules (W). These water molecules are hydrogen bonded to one another: O(1)W· · ·O(2)W¼2.930 (6) A, O(1)W· · ·O(3)W¼2.63 (2) A,
˚
O(2)W· · ·O(3)W¼2.32 (2) A.
O
S
N
DMSO-d6
O
N
N
O
Thermal product
H
H
N
NH
1) no additive, 175 ˚C, 30 min; 10-30 % isolated yield
2) no additive, 100 ˚C, 8 days; 100 % conversion on 1H-NMR
3) PPTS, 100 ˚C, 40 min; 75 % isolated yield
N
N
O
O
4) PPTS, 60 ˚C, 39 h; 100 % conversion on 1H-NMR
O
cis,cis-Ceratospongamide (1)
H
N
NH
NH
NH
H
N
H
N
N
O
N
O
H
N
H+
O
O
O
O
O
O
O
Scheme 4. Thermodynamic isomerization of cis,cis-ceratospongamide (1).
Scheme 5. Total synthesis of trans,trans-[D-alle]-ceratospongamide (3).

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Table 2. H- and 13C NMR spectral data (in ppm) for 3 with HMBC and ROESY correlations
8133
1
Position
1H
m
J (Hz)
13C
ROESYa
Pro-1
Ha
Hb
Hb
Hg
Hg
Hd
Hd
4.54
t
7.5
60.01
29.62
7.00–7.36 (w), 8.49 (s)
0.86 (w), 0.93 (w)
0.86 (w)
1.71
2.16
1.82
1.93
3.12
3.65
m
m
m
m
dt
m
25.07
46.69
9.4, 7.4
2.92 (m), 4.89 (m), 7.00–7.36 (m)
2.92 (m), 4.89 (s)
Ile
NH
Ha
Hb
Hg1
Hg1
(Hg2)3
(Hd1)3
8.49
4.97
1.83
1.21
1.30
0.86
0.93
d
10.1
10.1, 3.7
4.54 (s)
1.18 (m), 7.00–7.36 (m)
1.18 (m), 3.99 (w)
dd
m
m
m
d
48.31
36.85
25.71
1.18 (s)
1.18 (m), 1.71 (w), 2.16 (w), 3.68 (w), 3.99 (w)
1.18 (m), 1.71 (w), 3.99 (w)
7.0
7.4
14.06
11.57
t
Me-oxazoline
2
4
5
6
7
167.79
6.5
6.5
169.72
6.5
3.99
3.68
d
quint
73.18
79.35
0.86 (w), 0.93 (w), 1.83 (w), 7.06 (m), 7.00–7.36 (w)
0.86 (w), 7.00–7.36 (m)
1.18
d
21.53
0.86 (m), 0.93 (m), 1.30 (s), 1.83 (m), 4.97 (m)
Phe-1b
NH
Ha
Hb
Hb
7.06
4.87
3.22
3.38
d
8.5
3.99 (m)
3.87 (s)
3.87 (m)
3.87 (m)
ddd
dd
t
12.8, 8.5, 2.0
12.8, 2.0
12.8
52.81
37.14
Cg
137.45
Hd–Hz
Cd–Cz
CvO
7.00–7.36
125.85
168.48c
Pro-2
Ha
Hb
Hb
Hg
Hg
Hd
5.42
2.05
2.48
1.97
2.08
3.87
dd
m
m
m
m
m
8.2, 4.0
57.77
31.82
24.69
46.29
3.22 (m), 3.38 (w), 4.87 (s), 7.00–7.36 (m)
Thiazole
Phe-2b
2
4
5
6
169.89
147.69
123.66
159.13
8.15
s
NH
Ha
Hb
Hb
8.26
4.89
2.55
2.92
d
8.9
7.00–7.36 (w)
ddd
dd
dd
8.9, 7.8, 5.7
13.7, 7.8
13.7, 5.7
51.21
38.54
3.12 (m), 3.65 (s), 7.00–7.36 (m)
7.00–7.36 (w)
3.12 (m), 3.65 (w), 7.00–7.36 (w)
Cg
Hd–Hz
CvO
136.20
7.00–7.36
168.36c
ROESY correlation is listed only for the inter-residual ROE intensities observed in DMSO-d6 solution of 3. The letters s, m, and w in parentheses represent
strong, medium and weak ROE intensities, respectively.
a
Since the respective aromatic protons of Phe-1 and Phe-2 were not completely assigned on the NMR spectra, their ROE constraints were not included in the
model consideration.
The aromatic carbons (Cd–Cz) of Phe-1 and Phe-2 could not be separately assigned, although they correspond to one of 125.85, 126.21, 127.50, 127.96,
129.15, and 129.82 ppm.
These data are exchangeable.
b
c
Ha(Pro-2)-Ha(Phe-1) proton pairs and (ii) the strong ROEs
of Hd(Pro-1)-Ha(Phe-2)/Hd(Pro-2)-Ha(Phe-1) proton pairs
(Fig. 4). These trans assignments were also suggested by the
lack of any notable difference between the b and g carbon
chemical shifts (Ddbg) of Pro-1 and Pro-2 (4.6 and 7.1 ppm,
respectively), because the cis/trans conformational differ-
side of the oxazoline plane. These data suggest the
D-configuration of the Ile residue and the S-configuration
of the oxazoline C4 atom. Based on these NMR and
synthetic studies, it is concluded that the thermal product of
1 in DMSO solution results in the a-epimerization of the Ile
residue and the cis!trans conversion at the two Phe-Pro
amide bonds, consequently leading to the trans,trans-
[D-allo-Ile] isomer of 3. Furthermore, these results support
that the proposed structure of natural trans,trans-cerato-
spongamide (2) should be revised to trans,trans-[D-allo-Ile]
isomer (3).
22
ences correlate well with differential values of Ddbg and
the present values are significantly small as compared
with those of 1 (9.1 and 13.6 ppm, respectively).7 The ROEs
of (H7)3 (Me-oxazoline) with Hb/Hg/Hd (Ile) and H4
(Me-oxazoline) indicate their orientations on the same

8134
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Figure 4. Cross peaks of Hd(Pro-1)-Ha(Phe-2) and Hd(Pro-2)-Ha(Phe-1) proton pairs in partial ROESY spectrum of 3 in DMSO-d6 solution.
given in Table 3. Among them, 10 structures, which
˚
converged to less than 0.4 A for the averaged root mean
Table 3. Averaged RMS violation, RMS deviation and calculated energy
values of 50 converged structures
square (RMS) distance deviation of the backbone chains,
were selected to discuss the solution conformation of 3.
Their superposition is shown in Fig. 5. The most stable
conformer is shown in Fig. 6 and its conformational torsion
angles are given in Table 1.
˚
RMS violation (A)
0.041^0.009
0.710^0.298
˚
RMS deviation (backbone) (A)
Energies (kcal/mol)
Overall
Forcing potential
Bond
van der Waals
249.90^1.21
3.12^1.23
31.52^0.32
84.00^0.96
Based on these well-converged structures, it could be said
that 3 takes a solution conformation, where the amide bonds
of the two proline residues have the trans orientation. As a
whole, the ring structure of 3 may be very round and flat, as
compared with the slim and tight structure of 1. This is
obviously due to the difference in orientation between the
two Phe-Pro peptide bonds. Since the temperature depen-
dence of the NH protons of Ile, Phe-1 and Phe-2 is 25.45,
20.06 and 20.84 ppb/K, respectively, the amide protons of
Phe-1 and Phe-2 could form intramolecular hydrogen bonds
with the Phe-2 and Phe-1 carbonyl oxygens, respectively.
2.4. Solution conformation of 3
Three-dimensional (3D) models of 3 were constructed by
the dynamic SA method using the proton–proton distance
restraints derived from the ROE cross peaks. The ROE
constraints were not imposed on Phe-1 and -2 aromatic
protons, because of the lack of complete peak assignments.
Starting with 50 sets of conformations with random arrays
of atoms, energy-minimization trials were performed to
eliminate any possible source of initial bias in the folding
pathway, where the actual error function was minimized by
changing the respective f, C, v and x torsion angles. The
constructed NMR structures satisfied the distance con-
straints within the allowable range and one of four possible
f torsion angles within ^308. The structural statistics are
2.4.1. Molecular dynamics simulations of 1, 2, and 3. In
order to compare the relative stability among the confor-
mational energies of 1, 2, and 3, these isomers were
subjected to molecular dynamics (MD) calculations of
100 ps duration. The X-ray and NMR/SA structures listed in
Figure 5. Stereoscopic best-fit superposition of the 10 energetically stable converged structures. The aromatic rings of Phe-1 and Phe-2 were omitted for
clarity, because of the large variation of their orientations.

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
8135
Figure 6. Stereoscopic view of the most stable conformers of 3, derived from the NMR/SA method. Dotted lines represent possible intramolecular hydrogen
bonds.
Table 4. Averaged total energies, torsion angles, and distances between Ca
atoms of Phe-1 and Phe2 and between N atoms of thiazole and oxazoline
rings, together with their estimated standard deviations in parentheses
The time profiles of total energies, the v torsion angles of
the two Pro amide bonds, and the Ca(Phe-1)-Ca(Phe-2) and
N(thiazole)-N(Me-oxazoline) distances were monitored to
estimate their conformational fluctuations. The results are
summarized in Table 4. The respective systems were
observed to fluctuate within their stationary states through-
out the MD simulation for 100 ps, because the estimated
standard deviations of the total energies were all within
^5%. No conformational preference was observed among
the energies of 1, 2, and 3, and the conformational flexibility
was nearly the same, as determined from the respective
standard deviations of the torsion angles and the intra-
residual atomic distances. Also, the cis,trans conversions
of the v torsion angles were not observed for all conformers
during the MD simulation. Most conformers of 1 and 3 show
the same backbone conformations as the X-ray and NMR/
SA conformers, respectively. The most stable conformer of
2 in the MD simulation is given in Fig. 7, and the torsion
angles are given in Table 1. From these MD results, it could
be considered that each isomer assumes the conformation
with the energy minimum and there is no conformational
preference among them.
1
2
3
298 K
473 K
298 K
298 K
Total energy (kcal/mol)
v torsion angles (8)
Pro-1 amide bond
367 (9) 474 (13)
377 (9)
379 (8)
22 (16) 22 (15) 2171 (13) 179 (15)
21 (13) 26 (17) 2180 (11) 178 (12)
Pro-2 amide bond
˚
Distances (A)
Ca(Phe-1)–Ca(Phe-2) 7.5 (5)
N(thiazole)–N(Me-oxazoline) 4.5 (6)
7.5 (5)
4.4 (5)
7.9 (5)
6.6 (5)
7.8 (5)
6.6 (5)
Table 1 were used as starting conformers of 1 and 3, and that
of 2 was constructed by the dynamic SA method in
reference to the former two conformers. All MD calcu-
lations were performed at 298 K using Insight/Discover
software. No distance/torsional constraints were imposed to
enable the conformational space of the molecule to be fully
explored.
Figure 7. Stereoscopic view of the most stable conformer of 2, derived from MD simulations for 100 ps.

8136
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Scheme 6. Proposed mechanism of the thermal isomerization.
Since the conformational stability of 2 could be nearly the
same as that of 1, it is interesting to examine the possibility
of 1!2 conversion, because Gerwick et al.7 reported the
thermal conversion of 1 to 2, whereas the thermodynamic
isomerization of 1 by the same condition afforded 3. Thus,
the MD simulation of 1 was performed at a high temperature
(473 K). Consequently, no cis!trans conversion was
observed during the MD simulation for 100 ps (Table 4).
Although this simulation system as well as the simulation
time may be insufficient for deriving a conclusion, a high
energy barrier for their conformational conversion could be
considered, if this is even possible.
solved is the structural analysis of thermal product of 1 in
DMSO solution, because this is conflicting between
Gerwick–Taunton’s and ours. However, based on our
present studies, we were able to revise the originally
proposed structure of natural trans,trans-ceratospongamide
(2) to trans,trans-[D-allo-Ile]-isomer (3). In any event, the
present study indicated that a conformational similarity was
observed between 2 and 3. Because the conformation of 2 is
important for its highly potent inhibition of the expression
of secreted PLA2 in the inflammatory cascade,7 biological
activity for 3 would also be expected.
Based on this conformational analysis, we have proposed
that the thermodynamic isomerization of cis,cis-cerato-
spongamide (1) was initiated by the Ca epimerization of Ile
residue to provide the intermediate cis,cis-[D-allo-Ile]
isomer (27), which was immediately isomerized at the two
Phe-Pro amide bonds to produce the trans,trans-[D-allo-Ile]
isomer (3) as shown in Scheme 6. Since the acidic condition
facilitates the initial Ca epimerization of Ile residue via 26,
this thermodynamic isomerization in the presence of PPTs
smoothly proceeded at even 608C for 39 h (Scheme 4, entry
4). Deng and Taunton21 reported that trans,trans-cerato-
spongamide (2) could be obtained by macrolactamization
after forming the oxazoline. However, these studies
indicate that Ca epimerization of Ile residue should be
happened during the alkaline deprotection of both termini
or macrolactamization to produce not trans,trans isomer
(2), but trans,trans-[D-allo-Ile] isomer (3) as a minor
product.
3. Experimental
3.1. Synthesis
General. Melting points were measured with a YANACO
melting point apparatus and were uncorrected. Infrared
spectra were recorded on a SHIMADZU FT IR-8100
spectrometer. Optical rotations were measured on a DIP-
1000 digital polarimeter with a sodium lamp (l¼589 nm, D
line) and are expressed as follows: [a]TD (c g/100 mL,
solvent). 1H NMR spectra were recorded on a JEOL EX-270
(270 MHz), ALPHA 500 (500 MHz) or LAMBDA
(500 MHz) spectrometer. Chemical shifts are expressed in
ppm with tetramethylsilane or solvent as the internal
standard (CD3OD: d 4.78 ppm; DMSO-d6: d 2.49 ppm).
Data are reported in terms of: chemical shift, integration,
multiplicity (s¼singlet, d¼doublet, t¼triplet, q¼quartet,
br¼broad, m¼multiplet), coupling constants (Hz), and
assignment. 13C NMR spectra were recorded on a JEOL
EX-270 (67.8 MHz), ALPHA 500 (500 MHz) or LAMBDA
(500 MHz) spectrometer with complete proton decoupling.
Chemical shifts are reported in ppm with the solvent as the
internal standard (deuterochloroform: d 77.0 ppm; DMSO-
d6: d 39.5 ppm). Analytical thin layer chromatography was
performed on Merck Art. 5715, Kieselgel 60F254/0.25 mm
thickness plates. Visualization was accomplished with UV
The conformational analysis of ceratospongamide, a new
bioactive cyclic heptapeptide, is important for considering
its biologically active conformation. The present studies
established the total syntheses of 1 and its thermal product 3,
and made clear their molecular conformations and energy
stabilities, together with nearly the same conformational
stabilities of 2 and 3. The important issue that should be

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
8137
light, phosphomolybdic acid, or ninhydrin solution followed
by heating. Preparative thin layer chromatography was
performed on Merck Art. 5744, Kiselgel 60F254/0.5 mm
thickness plates. High resolution mass spectra (HRMS)
were measured at the Analytical Facility of Nagoya City
University. Solvents for extraction and column chromato-
graphy were of reagent grade. Liquid column chromato-
graphy was performed with forced flow (flash
chromatography of the indicated solvent mixture on silica
gel BW-820MH or BW-200 (Fuji Davison Co.)). Tetra-
hydrofuran (THF) was distilled from sodium metal/benzo-
phenone ketyl. Diethyl ether was distilled from lithium
aluminum hydride. Dichloromethane (CH2Cl2) was distilled
from calcium hydride. Toluene, dimethylsulfoxide (DMSO)
Pro-CbH2, CgH2), 3.15 (1H, br, thiazolidine-5), 3.23 (1H, m,
thiazolidine-4), 3.39 (2H, m, Pro-CdH2), 3.77 (3.79) (3H, s,
OCH3), 4.14 (1H, dd, J¼14.3, 7.1 Hz, NH), 4.47 (1H, br,
Pro-CaH), 4.88 (1H, d, J¼8.1 Hz, thiazolidine-2); 13C NMR
(67.8 MHz, CDCl3), minor rotamer in parentheses, d 22.7,
23.7 (23.6), 28.3 (28.5), 37.8 (38.7), 47.7 (47.2), 52.3, 58.1,
65.1, 68.0, 79.7. HRMS (EI) m/z calcd for C14H24N2O4S:
316.1457. Found: 316.1434.
3.1.3. Boc-L-Pro-Thz-OMe (13). A suspension of CMD
(7.56 g, 87.0 mmol) in benzene (9 mL) was refluxed with
stirring for 2 h using a Dean–stark apparatus (molecular
sieves type 4A). To the resulting suspension was added a
solution of the thiazolidine 12 (275 mg, 0.87 mmol) in
benzene (1 mL), and then the mixture was refluxed for 2 h.
The resulting mixture was filtered through a pad of celite
and the filtrate was concentrated. The residue was purified
by column chromatography (silica gel BW-820 MH,
hexane–EtOAc¼2:1) to afford thiazole 13 (139 mg,
0.44 mmol, 51%) as white crystals: mp 89–908C
(EtOAc–n-pentane); [a]2D4¼2107.8 (c 1.0, CHCl3); IR
nnmeaaxt (cm21) 3453, 1723, 1701, 1499, 1482, 1389, 1237; 1H
NMR (270 MHz, CDCl3), minor rotamer in parentheses, d
˚
and N,N-dimethylformamide (DMF) were dried over 4 A
molecular sieves. Triethylamine and N,N-diisopropylethyl-
amine were dried over potassium hydroxide. All other
commercially obtained reagents were used as received.
3.1.1. Total synthesis of cis,cis-ceratospongamide (1).
(2S)-2-(Methoxy-methylcarbamoyl)-pyrrolidine-1-car-
boxylic acid tert-butyl ester (10).12 To a solution of Boc-L-
Pro-OH (9) (10 g, 46.5 mmol) and N,O-dimethylhydroxyl-
amine hydrochloride (8.1 g, 83.0 mmol) in DMF (100 mL)
at 08C were successively added dropwise diethyl
phosphorocyanidate (13 mL, 86 mmol) and triethylamine
(17 mL, 122 mmol). The resulting solution was stirred at
08C for 2 h and at room temperature for 12 h. After dilution
with EtOAc, the mixture was washed with 1 M aq. KHSO4,
water, sat. aq. NaHCO3, water, and brine. The organic layer
was dried (Na2SO4), filtered, and concentrated. The residue
was purified by flash chromatography (BW-820MH,
hexane–EtOAc¼2:1–1:1–0:1) to afford the desired
t
1.33 (1.48) (9H, s, Bu), 1.90–1.98 (2H, m, Pro-CbH2),
2.05–2.35 (2H, br, CgH2), 3.61 (2H, br, Pro-CdH2), 3.95
(3.98) (3H, s, OCH3), 5.21 (1H, br, Pro-CaH), 8.01 (1H, s,
Thz-H); 13C NMR (67.8 MHz, CDCl3), minor rotamer in
parentheses, d 23.7 (24.4), 28.8, 34.8 (33.5), 47.2, 52.9, 60.1
(59.7), 80.8, 127.4 (127.7), 147.1, 153.8 (154.4), 162.2,
177.4. HRMS (EI) m/z calcd for C14H20N2O4S: 312.1144.
Found: 312.1109.
3.1.4. Boc-L-Phe-L-Pro-OMe (8). To a solution of Boc-L-
Phe-OH (14) (1.5 g, 5.65 mmol) and HCl–H-L-Pro-OMe
(15) (1 g, 6.04 mmol) in DMF (18 mL) at 08C were
successively added dropwise diethyl phosphorocyanidate
(0.94 mL, 6.20 mmol) and triethylamine (1.7 mL,
12.2 mmol). The resulting solution was stirred at 08C for
1 h and at room temperature for 11 h. After dilution with
EtOAc, the mixture was washed with 1 M aq. KHSO4,
water, sat. aq. NaHCO3, water, and brine. The organic layer
was dried (Na2SO4), filtered, and concentrated. The residue
was purified by flash chromatography (BW-820MH,
hexane–EtOAc¼1:1) to afford the desired product 8 as a
product 10 as a yellow oil (10.6 g, 41.0 mmol, 88%):
[a]2D6¼213.1 (c 1.1, CHCl3); IR n
(cm21) 1701, 1399,
neat
max
1165, 1123; 1H NMR (270 MHz, CDCl3), minor rotamer in
t
parentheses, d 1.42 (1.46) (9H, s, Bu), 1.86–2.23 (4H,
m, Pro-CbH2, CgH2), 3.20 (3H, s, OCH3), 3.40–3.58 (2H,
m, Pro-CdH2), 3.72 (3.78) (3H, s, N–CH3), 4.61 (4.67) (1H,
m, Pro-CaH); 13C NMR (67.8 MHz, CDCl3), minor rotamer
in parentheses, d 15.9 (16.0), 23.3 (23.9), 30.4 (29.5), 46.4
(46.7), 56.6 (56.3), 61.0 (61.1), 79.1 (79.3), 153.5 (154.1),
173.4 (172.9).
3.1.2. (2S)-2-(1-tert-Butoxycarbonyl-pyrrolidin-2-yl)-
thiazolidine-4-carboxylic acid methyl ester (12). To a
solution of amide 10 (852 mg, 3.3 mmol) in ether (10 mL) at
08C was added LiAlH4 (156 mg, 4.1 mmol). The resulting
solution was stirred at 08C for 15 min and quenched by
dropwise addition of 1 M aq. KHSO4. The mixture was
extracted with ether (£3). The combined organic extracts
were washed with brine, dried (Na2SO4), filtered, and
concentrated. The residue and HCl–H-L-Cys-OMe
(700 mg, 4.1 mmol) were dissolved in toluene (7.6 mL),
and to this suspension was added triethylamine (0.71 mL,
5.1 mmol) at 08C. The resulting suspension was stirred at
room temperature, filtered, and concentrated. The residue
was purified by flash chromatography (BW-820MH,
hexane/EtOAc¼2:1) to afford the desired product 12 as a
white solid (2.059 g, 5.47 mmol, 97%): mp 69–708C;
KBr
max
[a]2D6¼235.8 (c 1.0, CHCl3); IR n
(cm21) 3343, 1750,
1736, 1707, 1694, 1653, 1644, 1536, 1518, 1437, 1368; 1H
NMR (270 MHz, DMSO-d6, 1008C) d 1.32 (9H, s, Bu),
t
1.90 (3H, br, Pro-CgH2, CbH2), 2.17 (1H, br, Pro-CbH2),
2.79 (1H, dd, J¼14.0, 8.1 Hz, Phe-CbH2), 2.96 (1H, br, Phe-
CbH2), 3.42 (1H, m, Pro-CdH2), 3.63 (4H, s, Pro-CdH2,
OCH3), 4.37 (2H, br, Pro-CaH, Phe-CaH), 6.36 (1H, br,
NH), 7.25 (5H, m, PhH); 13C NMR (67.8 MHz, CDCl3),
minor rotamer in parentheses, d 24.9 (22.3), 28.4 (29.1),
39.2 (41.2), 46.8 (46.0), 52.2 (52.7), 53.2 (53.8), 58.9, 79.6
(77.2), 126.6 (126.8), 128.2 (128.4), 129.6 (129.2), 136.2,
155.0, 170.5, 172.1 (171.7). HRMS (EI) m/z calcd for
C20H28N2O5: 376.1998. Found: 376.1992.
yellow oil (758 mg, 2.4 mmol, 73%): [a]2D7¼286.3 (c 1.0,
3.1.5. Boc-L-Phe-L-Pro-Thz-OMe (16). Boc-L-Pro-Thz-
OMe (13) (1.806 g, 5.78 mmol) was treated with 4 M HCl–
dioxane (7 mL) at 08C. The mixture was stirred at room
temperature for 20 min. The mixture was concentrated and
CHCl3); IR n
(cm21) 3320, 1746, 1690, 1393, 1366,
neat
max
1165; 1H NMR (270 MHz, CDCl3), minor rotamer in
parentheses, d 1.47 (1.50) (9H, s, Bu), 1.71–1.99 (4H, m,
t

8138
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
dried to afford the crude hydrochloride salt. To a solution of
Boc-L-Phe-OH (14) (1.53 g, 5.65 mmol) and the above
crude hydrochloride salt in DMF (19 mL) at 08C were
successively added dropwise diethyl phosphorocyanidate
(1 mL, 6.59 mmol) and triethylamine (1.8 mL, 13.0 mmol).
The resulting solution was stirred at 08C for 1 h and at room
temperature for 7 h. After dilution with EtOAc, the mixture
was washed with 1 M aq. KHSO4, water, sat. aq. NaHCO3,
water, and brine. The organic layer was dried (Na2SO4),
filtered, and concentrated. The residue was purified by flash
chromatography (BW-820MH, hexane–EtOAc¼2:1–1:1–
1:2) to afford the desired product 16 as an amorphous solid
(2.492 g, 5.42 mmol, 94%): [a]2D6¼276.7 (c 1.0, CHCl3);
IR nnmeaaxt (cm21) 3432, 3346, 1715, 1659, 1651, 1505, 1495,
1244; 1H NMR (270 MHz, CDCl3), minor rotamer in
was treated with MeOH (1 mL)–4N HCl–dioxane (8 mL)
at 08C. The mixture was stirred at room temperature for
20 min. The mixture was concentrated and dried to afford
the crude hydrochloride salt. To a solution of Boc-L-Ile-OH
(19) (637 mg, 2.75 mmol) and the above crude hydrochlo-
ride salt in DMF (10 mL) at 08C were successively added
dropwise
diethyl
phosphorocyanidate
(0.45 mL,
2.97 mmol) and triethylamine (0.84 mL, 6.05 mmol). The
resulting solution was stirred at 08C for 1 h and at room
temperature for 6 h. After dilution with EtOAc, the mixture
was washed with 1 M aq. KHSO4, water, sat. aq. NaHCO3,
water, and brine. The organic layer was dried (Na2SO4),
filtered, and concentrated. The residue was purified by flash
chromatography (BW-820MH, hexane–EtOAc¼1:5–0:1,
EtOAc–EtOH¼10:1) to afford the desired product 7 as a
t
parentheses, d 1.36 (1.40) (9H, s, Bu), 1.58 (2H, s,
white solid (1.399 g, 2.08 mmol, 75%): mp 111–1128C;
KBr
Pro-CgH2), 2.04 (2H, br, Pro-CbH2), 2.91–3.08 (2H, m,
Phe-CbH2), 3.35 (1H, m, Pro-CdH2), 3.71–3.74 (1H, m,
Pro-CdH2), 3.94 (3H, s, OCH3), 4.42 (1H, d, J¼7.9 Hz,
Phe-NH), 4.73 (1H, br, Phe-CaH), 5.36 (1H, m, Pro-CaH),
7.17–7.26 (5H, m, PhH), 8.03 (8.06) (1H, s, Thz-H); 13C
NMR (67.8 MHz, CDCl3) d 22.3, 27.6, 30.6, 36.9, 46.0,
51.1, 53.3, 57.8, 77.8, 125.6, 127.3, 127.7, 128.5, 136.7,
154.1, 160.5, 169.9, 171.2, 172.3. HRMS (EI) m/z calcd for
C23H29N3O5S: 459.1828. Found: 459.1840.
[a]2D6¼289.5 (c 1.0, CHCl3); IR n
(cm21) 3475, 3325,
max
1638, 1244; 1H NMR (270 MHz, DMSO-d6, 1008C) d
0.80–0.85 (6H, m, Ile-CgH3, Ile-CdH3), 1.05 (3H, d,
J¼6.1 Hz, aThr-CH3), 1.12–1.33 (2H, m, Ile-CgH2), 1.39
t
(9H, s, Bu), 1.74 (1H, br, –OH), 1.94–1.97 (2H, m,
Pro-CgH2), 2.17 (2H, br, Pro-CbH2), 2.83–2.97 (2H, m,
Phe-CbH2), 3.44 (2H, br, Pro-CdH2), 3.83 (3H, s, OCH3),
3.87–3.90 (2H, m, aThr-CbH, aThr-CaH), 4.41 (1H, br,
Ile-CaH), 4.79 (1H, br, Phe-CaH), 5.28 (1H, br, Pro-CaH),
6.31 (1H, d, J¼8.0 Hz, BocNH), 7.17 (5H, br, PhH), 7.48
(1H, d, J¼8.2 Hz, NH), 7.81 (1H, br, NH), 8.29 (1H, s,
Thz-H); 13C NMR (67.8 MHz, CDCl3), minor rotamer in
parentheses, d 11.4, 14.2, 15.6, 19.6, 24.75 (24.80), 28.3,
29.0, 37.8 (37.0), 39.1, 47.0 (47.1), 52.2 (51.0), 52.8, 57.7,
58.9 (59.1), 69.1, 80.1, 126.9 (127.2), 128.4 (128.6), 129.2
(129.3), 129.7 (129.4), 136.2 (135.8), 148.8, 155.8, 160.3
(160.2), 170.3 (170.6), 170.7 (171.8), 172.2 (172.0). HRMS
(EI) m/z calcd for C33H47N5O8S: 673.3145. Found:
673.3124.
3.1.6. Boc-L-aThr-L-Phe-L-Pro-Thz-OMe (18). Boc-L-
Phe-L-Pro-Thz-OMe (16) (1.378 g, 3.00 mmol) was treated
with 4 M HCl–dioxane (10 mL) at 08C. The mixture was
stirred at room temperature for 20 min. The mixture was
concentrated and dried to afford the crude hydrochloride
salt. To a solution of Boc-L-aThr-OH (17) (672 mg,
3.07 mmol) and the above crude hydrochloride salt in
DMF (10 mL) at 08C was successively added dropwise
diethyl phosphorocyanidate (0.5 mL, 3.30 mmol) and
triethylamine (0.92 mL, 6.63 mmol). The resulting solution
was stirred at 08C for 2 h and at room temperature for 6 h.
After dilution with EtOAc, the mixture was washed with
1 M aq. KHSO4, water, sat. aq. NaHCO3, water, and brine.
The organic layer was dried (Na2SO4), filtered, and
concentrated. The residue was purified by flash chromato-
graphy (BW-820MH, hexane–EtOAc¼1:2–0:1, EtOAc–
EtOH¼20:1–10:1) to afford the desired product 18 as a
3.1.8. Boc-L-Phe-L-Pro-L-Ile-L-aThr-L-Phe-L-Pro-Thz-
OMe (20). To a solution of Boc-L-Phe-L-Pro-OMe (8)
(222 mg, 0.59 mmol) in THF (2.4 mL) at 08C was added
0.5N aq. LiOH (2.4 mL, 1.2 mmol). The resulting solution
was stirred at 08C for 30 min and at room temperature for
30 min. After the reaction mixture was acidified by the
addition of 1 M aq. KHSO4, the resulting mixture was
extracted with CHCl3 (£3). The combined organic extracts
were dried (Na2SO4), filtered, and concentrated to afford the
crude Boc-L-Phe-L-Pro-OH. Boc-L-Ile-L-aThr-L-Phe-L-
Pro-Thz-OMe (7) (398 mg, 0.59 mmol) was treated with
4N HCl–dioxane (2 mL) at 08C. The mixture was stirred at
room temperature for 20 min. The mixture was concentrated
and dried to afford the crude hydrochloride salt. To a
solution of the above crude Boc-L-Phe-L-Pro-OH and the
above crude hydrochloride salt in DMF (2 mL) at 08C were
successively added dropwise diethyl phosphorocyanidate
(0.105 mL, 0.69 mmol) and triethylamine (0.18 mL,
1.30 mmol). The resulting solution was stirred at 08C for
1.5 h and at room temperature for 13 h. After dilution with
EtOAc, the mixture was washed with 1 M aq. KHSO4,
water, sat. aq. NaHCO3, water, and brine. The organic layer
was dried (Na2SO4), filtered, and concentrated. The residue
was purified by flash chromatography (BW-820MH,
hexane–EtOAc¼1:5–0:1, EtOAc–EtOH¼20:1–10:1) to
afford the desired product 20 as a white solid (508 mg,
0.55 mmol, 94%): mp 124–1258C; [a]2D5¼295.3 (c 1.0,
colorless viscous oil (1.581 g, 2.82 mmol, 94%):
[a]2D6¼290.7 (c 1.0, CHCl3); IR n
(cm21) 3325, 1723,
neat
max
1715, 1646, 1634, 1244, 1217; 1H NMR (270 MHz, DMSO-
d6) d 0.95 (3H, d, J¼6.1 Hz, aThr-CH3), 1.37 (9H, s, Bu),
t
1.95–1.97 (3H, m, Pro-CgH2, –OH), 2.13–2.18 (2H, m,
Pro-CbH2), 2.79–3.03 (2H, m, Phe-CbH2), 3.52 (2H, br,
Pro-CdH2), 3.71–3.74 (1H, m, aThr-CbH), 3.81 (3H, s,
OCH3), 3.88–3.91 (1H, m, aThr-CaH), 4.73–4.79 (1H, m,
Phe-CaH), 5.24–5.26 (1H, m, Pro-CaH), 6.61 (1H, d, J¼
8.7 Hz, BocNH), 7.19 (5H, br, PhH), 8.10 (1H, d, J¼7.5 Hz,
NH), 8.40 (1H, s, Thz-H); 13C NMR (67.8 MHz, CDCl3),
minor rotamer in parentheses, d 19.7 (19.6), 21.5, 28.3, 31.4
(34.3), 38.1 (40.3), 47.3 (46.3), 52.0 (52.3), 58.8 (59.0), 60.3
(64.0), 65.2, 69.1, 127.0 (126.8), 127.4 (127.2), 128.3
(128.5), 129.2 (129.4), 135.5 (135.4), 155.5, 161.6 (161.3),
170.4 (169.9), 171.0 (170.7), 171.9 (173.4). HRMS (EI) m/z
calcd for C27H36N4O7S: 560.2305. Found: 560.2311.
3.1.7. Boc-L-Ile-L-aThr-L-Phe-L-Pro-Thz-OMe (7). Boc-
L-aThr-L-Phe-L-Pro-Thz-OMe (18) (1.266 g, 2.75 mmol)

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
8139
CHCl3); IR nKmBaxr (cm21) 3496, 1645, 1559, 1455, 1246; 1H
NMR (270 MHz, DMSO-d6) d 0.78 (6H, br, Ile-CgH3, Ile-
CdH3), 1.00 (3H, d, J¼6.3 Hz, aThr-CH3), 1.28 (9H, s, tBu),
1.89 (7H, br, Ile-CgH2, Pro-CbH2, Pro-CdH2, –OH), 2.12
(2H, br, Pro-CgH2), 2.72–3.00 (4H, m, Phe-CbH2), 3.27–
3.72 (4H, m, Pro-CdH2), 3.81 (3H, s, OCH3), 4.16–4.27
(2H, m, aThr-CbH, Phe-CaH), 4.45 (1H, br, aThr-CaH),
4.73 (1H, br, Ile-CaH), 4.78 (1H, m, Phe-CaH), 5.24 (1H, m,
Pro-CaH), 7.01 (1H, d, J¼8.1 Hz, BocNH), 7.17–7.27
(10H, m, PhH), 7.74 (1H, d, J¼8.4 Hz, NH), 7.86 (1H, d,
J¼8.6 Hz, NH), 8.18 (1H, d, J¼7.4 Hz, NH), 8.29 (1H, s,
Thz-H); 13C NMR (67.8 MHz, CDCl3), minor rotamer in
parentheses, d 11.4, 15.7, 19.5 (19.2), 21.6, 24.6, 24.9, 25.2,
28.3, 31.5, 34.2, 36.7 (36.9), 38.2, 39.0, 40.5, 47.3 (47.5),
52.4 (52.5), 53.7 (53.5), 57.8 (57.4), 58.2 (58.1), 58.8, 59.4,
60.5 (60.3), 68.7, 80.8 (79.7), 126.8 (126.7), 126.9 (127.2),
127.5 (127.4), 128.4, 129.3 (128.8), 134.2, 135.5 (135.8),
146.1 (147.7), 155.1, 161.6, 168.8, 170.0, 170.4 (170.3),
171.2 (171.3), 171.6 (171.5), 172.2 (173.5). HRMS (FAB)
m/z calcd for C47H67N7O10S (MþH): 918.4435. Found:
918.4452.
J¼7.7 Hz, NH), 7.05 (2H, m, PhH), 7.19 (2H, m, PhH),
7.29–7.35 (6H, m, PhH), 7.91 (1H, d, J¼4.5 Hz, NH), 7.97
(8.01) (1H, s, Thz-H); 13C NMR (67.8 MHz, CDCl3), minor
rotamer in parentheses, d 11.4, 15.7, 16.2, 19.6, 24.8, 24.9,
28.4, 29.1, 30.9, 37.1 (37.4), 37.9, 39.2, 47.0 (47.2), 52.2
(52.0), 52.9 (52.8), 58.2 (57.8), 59.0 (59.1), 59.5, 69.1, 77.2
(80.0), 123.7 (123.6), 126.8, 127.1, 128.3 (128.4), 128.5
(128.8), 129.1 (129.3), 129.6, 135.4, 136.0, 148.7, 155.7,
160.2, 164.5, 169.8, 170.1, 170.5 (170.4), 171.9 (171.7),
172.0 (172.2). HRMS (FAB) m/z calcd for C47H67N7O10S
(MþH): 918.4435. Found: 918.4456.
3.1.10. Cyclo[L-Phe-L-Pro-L-Ile-L-aThr-L-Phe-L-Pro-
Thz] (4). To a solution of Boc-L-Phe-L-Pro-L-Ile-L-aThr-
L-Phe-L-Pro-Thz-OMe (18) (93 mg, 0.10 mmol) in THF
(0.4 mL) at 08C was added 0.5 M aq. LiOH (0.4 mL,
0.2 mmol). The resulting solution was stirred at 08C for
30 min and at room temperature for 30 min. After the
reaction mixture was acidified by the addition of 1 M aq.
KHSO4, the resulting mixture was extracted with CHCl3
(£3). The combined organic extracts were dried (Na2SO4),
filtered, and concentrated to afford the crude Boc-L-Phe-L-
Pro-L-Ile-L-aThr-L-Phe-L-Pro-Thz-OH. This crude hepta-
peptide was treated with 4 M HCl–dioxane (1 mL) at 08C.
The mixture was stirred at room temperature for 20 min.
The mixture was concentrated and dried to afford the crude
HCl-H-L-Phe-L-Pro-L-Ile-L-aThr-L-Phe-L-Pro-Thz-OH. To
a solution of the above crude HCl–H-L-Phe-L-Pro-L-Ile-L-
aThr-L-Phe-L-Pro-Thz-OH in DMF (51 mL) at 08C were
successively added dropwise pentafluorophenyl diphenyl-
phosphinate (59 mg, 0.15 mmol) and N,N-diisopropylethyl-
amine (0.071 mL, 0.41 mmol). The resulting solution was
stirred at room temperature for 50 h, and concentrated. The
residue was purified by flash chromatography (BW-820MH,
EtOAc–EtOH¼20:1–10:1) to afford the desired product 4
as a white solid (50 mg, 0.065 mmol, 63%): mp 168–
1698C; [a]2D7¼282.7 (c 1.0, CHCl3); IR nmKBaxr (cm21) 3453,
3.1.9. Boc-L-Ile-L-aThr-L-Phe-L-Pro-Thz-L-Phe-L-Pro-
OMe (21). To a solution of Boc-L-Ile-L-aThr-L-Phe-L-
Pro-Thz-OMe (7) (423 mg, 0.63 mmol) in THF (2.5 mL) at
08C was added 0.5N aq. LiOH (2.5 mL, 1.3 mmol). The
resulting solution was stirred at 08C for 30 min and at room
temperature for 30 min. After the reaction mixture was
acidified by the addition of 1 M aq. KHSO4, the resulting
mixture was extracted with CHCl3 (£3). The combined
organic extracts were dried (Na2SO4), filtered, and con-
centrated to afford the crude Boc-L-Ile-L-aThr-L-Phe-L-Pro-
Thz-OH. Boc-L-Phe-L-Pro-OMe (8) (299 mg, 0.79 mmol)
was treated with 4 M HCl–dioxane (2 mL) at 08C. The
mixture was stirred at room temperature for 20 min. The
mixture was concentrated and dried to afford the crude
hydrochloride salt. To a solution of the above crude Boc-L-
Ile-L-aThr-L-Phe-L-Pro-Thz-OH and the above crude
hydrochloride salt in DMF (2 mL) at 08C were successively
added dropwise diethyl phosphorocyanidate (0.11 mL,
0.72 mmol) and triethylamine (0.22 mL, 1.58 mmol). The
resulting solution was stirred at 08C for 1.5 h and at room
temperature for 18 h. After dilution with EtOAc, the
mixture was washed with 1 M aq. KHSO4, water, sat. aq.
NaHCO3, water, and brine. The organic layer was dried
(Na2SO4), filtered, and concentrated. The residue was
purified by flash chromatography (BW-820MH, hexane–
EtOAc¼1:5–0:1, EtOAc–EtOH¼20:1–10:1) to afford the
desired product 21 as a white solid (574 mg, 0.63 mmol,
99%): mp 106–1098C; [a]2D4¼287.2 (c 1.0, CHCl3); IR
nKmBaxr (cm21) 3410, 1752, 1638, 1541, 1453, 1171; 1H NMR
(270 MHz, CDCl3), minor rotamer in parentheses, d 0.90
(3H, d, J¼6.3 Hz, Ile-CgH3), 0.91 (3H, t, J¼7.3 Hz, Ile-
CdH3), 1.19 (1.12) (3H, d, J¼6.1 Hz, aThr-CH3), 1.32–1.39
1
1636, 1541, 1497, 1451; H NMR (270 MHz, CD3OD) d
0.89 (3H, d, J¼7.3 Hz, Ile-CgH3), 0.96 (3H, t, J¼6.6 Hz,
Ile-CdH3), 1.14–1.23 (2H, m, Ile-CgH2), 1.28 (3H, d,
J¼6.1 Hz, aThr-CH3), 1.46–1.70 (2H, m, Pro-CgH2), 1.89–
2.37 (7H, m, Ile-CbH2, Pro-CbH2, Pro-CgH2), 3.13 (2H, d,
J¼7.1 Hz, Phe-CbH2), 3.46 (2H, brd, J¼10.7 Hz, Phe-
CbH2), 3.56 (2H, m, Pro-CdH2), 3.85–4.05 (2H, m, Pro-
CdH2), 4.21–4.27 (1H, m, aThr-CbH), 4.38 (1H, d,
J¼5.1 Hz, aThr-CaH), 4.53 (1H, m, Pro-CaH), 4.75 (1H,
m, Ile-CaH), 4.96–5.14 (3H, m, Phe-CaH, Pro-CaH), 7.01
(1H, d, J¼8.1 Hz, BocNH), 7.20–7.29 (10H, m, PhH), 8.03
(1H, s, Thz-H); 13C NMR (67.8 MHz, CDCl3) d 11.8, 16.2,
20.1, 21.1, 24.3, 25.8, 29.7, 33.9, 36.8, 38.1, 38.3, 46.0,
47.4, 52.9, 53.8, 57.4, 58.1, 59.9, 62.8, 70.4, 123.9, 127.1,
127.7, 128.6, 129.1, 129.1, 131.1, 135.0, 136.5, 150.3,
160.9, 168.2, 169.7, 170.7, 170.9, 171.9. HRMS (FAB) m/z
calcd for C41H52N7O7S (MþH): 786.3649. Found:
786.3672.
t
(3H, m, Ile-CgH2, Ile-CbH), 1.43 (9H, s, Bu), 1.60–2.00
(5H, br, Pro-CbH2, OH), 2.12–2.26 (3H, m, Pro-CgH2), 2.38
(1H, m, Pro-CgH2), 2.93 (1H, d, J¼7.3 Hz, Phe-CbH2),
3.02–3.12 (3H, m, Phe-CbH2), 3.17–3.25 (3H, m, Pro-
CdH2), 3.39 (1H, m, Pro-CdH2), 3.75 (3.66) (3H, s, OCH3),
3.96 (1H, m, Phe-CaH), 4.12 (1H, q, J¼7.1 Hz, aThr-CbH),
4.23–4.42 (2H, m, aThr-CaH, Ile-CaH), 4.88 (1H, m, Phe-
CaH), 5.06–5.14 (2H, m, Pro-CaH), 5.41 (1H, d, J¼5.6 Hz,
BocNH), 6.81 (1H, d, J¼8.1 Hz, NH), 6.90 (6.95) (1H, d,
3.1.11. cis,cis-Ceratospongamide (1). To a solution of
cyclo[L-Phe-L-Pro-L-Ile-L-aThr-L-Phe-L-Pro-Thz] (4) (50 mg,
0.064 mmol) in CH2Cl2 (0.6 mL) at 2308C was added
Deoxo-fluor (0.024 mL, 0.13 mL). The reaction mixture
was stirred at 230 to 2208C for 20 min. The reaction
mixture was quenched by the addition of sat. aq. NaHCO3,
warmed to room temperature, and extracted with CHCl3.

8140
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
The combined organic extracts were dried (Na2SO4),
filtered, and concentrated. The residue was purified by
flash chromatography (BW-820MH, EtOAc–EtOH¼1:0–
20:1) to afford the desired product 1 as colorless crystals
28.3, 31.3, 37.4 (37.3), 47.2 (46.3), 52.3 (52.4), 52.8 (53.0),
57.7 (57.5), 58.7 (58.1), 69.0, 77.2 (79.9), 126.9 (127.2),
127.5 (127.4), 128.4 (128.3), 128.7, 129.2 (129.3), 135.6
(135.5), 146.2 (147.6), 155.6, 161.5 (161.3), 170.3 (169.0),
170.5, 171.9 (172.4), 172.1 (173.5). HRMS (EI) m/z calcd
for C33H47N5O8S: 673.3145. Found: 673.3120.
(27 mg, 0.035 mmol, 55%): mp 236–2398C (benzene–n-
KBr
pentane); [a]2D6¼2136.5 (c 0.23, CHCl3); IR n
(cm21
)
max
3475, 1628, 1541, 1449; 1H NMR (500 MHz, CDCl3) d 0.83
(3H, d, J¼6.9 Hz, Ile-CgH3), 0.90 (3H, t, J¼7.5 Hz, Ile-
CdH3), 1.01–1.14 (2H, m, Ile-CgH2, Pro-CbH2), 1.31 (3H,
d, J¼6.0 Hz, Me-oxz-CH3), 1.34 (1H, m, Ile-CgH2), 1.58
(1H, m, Pro-CgH2), 1.73–1.79 (2H, m, Ile-CbH2, Pro-
CgH2), 1.88–1.95 (3H, m, Pro-CbH2, Pro-CgH2), 2.03–2.14
(1H, m, Pro-CbH2), 2.27–2.36 (1H, m, Pro-CbH2), 2.85
(1H, dd, J¼12.9, 6.6 Hz, Phe-CbH2), 2.89 (1H, m, Phe-
CbH2), 3.04 (1H, dd, J¼13.2, 8.1 Hz, Phe-CbH2), 3.19 (1H,
dd, J¼9.3, 1.5 Hz, Me-oxz-C4H), 3.42–3.59 (4H, m, Pro-
CaH, Pro-CdH2, Phe-CbH2), 3.69–3.76 (1H, m, Pro-CdH2),
3.93–4.01 (2H, m, Me-oxz-C5H, Pro-CdH2), 4.61–4.66
(1H, m, Ile-CaH), 4.71–4.80 (2H, m, Phe-CaH), 5.17 (1H,
m, Pro-CaH), 6.55 (1H, d, J¼9.6 Hz, Ile-NH), 6.61 (1H, d,
J¼8.7 Hz, Phe-NH), 7.16–7.36 (10H, m, PhH), 8.04 (1H, s,
Thz-H), 8.14 (1H, d, J¼6.3 Hz, Phe-NH); 13C NMR
(75.5 MHz, CDCl3) d 12.0, 16.0, 21.7, 21.9, 22.3, 25.1,
31.4, 35.5, 38.6, 39.5, 41.0, 46.9, 47.1, 51.8, 52.6, 54.2,
60.0, 61.6, 74.1, 81.9, 124.6, 127.0, 127.8, 128.6, 129.4,
129.9, 129.9, 136.5, 137.3, 149.1, 159.9, 169.2, 170.0,
170.0, 170.3, 170.9, 171.6. HRMS (FAB) m/z calcd for
C41H50N7O6S (MþH): 768.3543. Found: 768.3576.
3.1.13. Boc-L-Phe-L-Pro-D-aIle-L-aThr-L-Phe-L-Pro-
Thz-OMe (24). To a solution of Boc-L-Phe-L-Pro-OMe
(8) (182 mg, 0.48 mmol) in THF (1.2 mL) at 08C was added
1 M aq. NaOH (0.58 mL, 0.58 mmol). The resulting
solution was stirred at 08C for 1 h and at room temperature
for 1.5 h. After the reaction mixture was acidified by the
addition of 1 M aq. KHSO4, the resulting mixture was
extracted with CHCl3 (£3). The combined organic extracts
were dried (Na2SO4), filtered, and concentrated to afford the
crude Boc-L-Phe-L-Pro-OH. Boc-D-aIle-L-aThr-L-Phe-L-
Pro-Thz-OMe (23) (303 mg, 0.45 mmol) was treated with
4 M HCl–dioxane (2 mL) at 08C. The mixture was stirred at
room temperature for 20 min. The mixture was concentrated
and dried to afford the crude hydrochloride salt. To a
solution of the above crude Boc-L-Phe-L-Pro-OH and the
above crude hydrochloride salt in DMF (1.7 mL) at 08C
were successively added dropwise diethyl phosphoro-
cyanidate (0.083 mL, 0.55 mmol) and triethylamine
(0.15 mL, 1.04 mmol). The resulting solution was stirred
at 08C for 1 h and at room temperature for 12 h. After
dilution with EtOAc, the mixture was washed with 1 M aq.
KHSO4, water, sat. aq. NaHCO3, water, and brine. The
organic layer was dried (Na2SO4), filtered, and concen-
trated. The residue was purified by flash chromatography
(BW-820MH, hexane–EtOAc¼1:5–0:1, EtOAc–EtOH¼
20:1–10:1) to afford the desired product 24 as a white solid
(408 mg, 0.44 mmol, 99%): mp 66–678C; [a]2D6¼223.7 (c
1.0, CHCl3); IR nKmBaxr (cm21) 3346, 1717, 1638, 1522, 1453,
1246; 1H NMR (270 MHz, DMSO-d6) d 0.75 (3H, d,
J¼6.6 Hz, D-aIle-CgH3), 0.83 (3H, t, J¼7.4 Hz, D-aIle-
CdH3), 0.99 (3H, d, J¼6.1 Hz, aThr-CH3), 1.16–1.30 (3H,
3.1.12. Total synthesis of trans,trans-[D-aIle]-cerato-
spongamide (3). Boc-D-aIle-L-aThr-L-Phe-L-Pro-Thz-
OMe (23). Boc-L-aThr-L-Phe-L-Pro-Thz-OMe (18)
(343 mg, 0.746 mmol) was treated with MeOH (1 mL)–
4 M HCl–dioxane (8 mL) at 08C. The mixture was stirred at
room temperature for 20 min. The mixture was concentrated
and dried to afford the crude hydrochloride salt. To a
solution of Boc-D-aIle-OH (22) (228 mg, 0.762 mmol)
and the above crude hydrochloride salt in DMF (2.5 mL)
at 08C were successively added dropwise diethyl phosphoro-
cyanidate (0.14 mL, 0.922 mmol) and triethylamine
(0.23 mL, 1.66 mmol). The resulting solution was stirred
at 08C for 1 h and at room temperature for 14 h. After
dilution with EtOAc, the mixture was washed with 1 M aq.
KHSO4, water, sat. aq. NaHCO3, water, and brine. The
organic layer was dried (Na2SO4), filtered, and concen-
trated. The residue was purified by flash chromatography
(BW-820MH, hexane–EtOAc¼1:5–0:1, EtOAc–EtOH¼
10:1) to afford the desired product 23 as a white solid
(355 mg, 0.527 mmol, 71%): mp 87–898C; [a]2D6¼266.9 (c
1.0, CHCl3); IR nmneaaxt (cm21) 3282, 1738, 1726, 1636, 1538,
1505, 1455, 1244; 1H NMR (270 MHz, DMSO-d6, 1008C) d
0.81 (3H, d, J¼6.8 Hz, D-aIle-CgH3), 0.88 (3H, t, J¼7.3 Hz,
D-aIle-CdH3), 1.05 (3H, d, J¼6.1 Hz, aThr-CH3), 1.10–
t
m, D-aIle-CbH, D-aIle-CgH2), 1.27 (9H, s, Bu), 1.90 (7H,
br, Pro-CbH2, Pro-CdH2, –OH), 2.13 (2H, br, Pro-CgH2),
2.73–2.95 (4H, m, Phe-CbH2), 3.26–3.73 (4H, m, Pro-
CdH2), 3.81 (3H, s, OCH3), 3.97–4.03 (1H, m, Pro-CaH),
4.11–4.17 (1H, m, Phe-CaH), 4.37 (1H, br, aThr-CbH),
4.49 (1H, br, aThr-CaH), 4.74 (1H, m, D-aIle-CaH), 4.78
(1H, m, Phe-CaH), 5.24 (1H, m, Pro-CaH), 6.98 (1H, d,
J¼7.9 Hz, BocNH), 7.17–7.27 (10H, m, PhH), 7.79 (2H, t,
J¼8.1 Hz, NH), 8.18 (1H, d, J¼7.3 Hz, NH), 8.29 (1H, s,
Thz-H); 13C NMR (67.8 MHz, CDCl3), minor rotamer in
parentheses, d 11.7, 14.5 (14.6), 16.0 (15.95), 19.7 (19.8),
24.5, 25.5, 26.7, 28.2 (28.1), 31.6, 35.2, 38.6 (37.8), 47.7,
47.3, 52.4 (51.9), 55.8, 58.8 (59.4), 61.4 (61.1), 65.1, 67.3,
77.1 (79.9), 126.7, 126.8, 127.3 (127.1), 128.3 (127.9),
128.4 (128.6), 129.2 (129.4), 129.6, 135.3, 135.8 (135.7),
146.3 (147.5), 155.0 (155.7), 161.5 (161.4), 168.6 (168.9),
169.3, 170.7 (170.8), 171.3, 171.8, 172.2 (173.1). HRMS
(FAB) m/z calcd for C47H67N7O10S (MþH): 918.4435.
Found: 918.4464.
t
1.36 (4H, m, Ile-CgH2, Pro-CbH2), 1.41 (9H, s, Bu), 1.82
(1H, m, D-aIle-CbH2), 1.96 (2H, br, Pro-CgH2), 2.18 (1H,
br, –OH), 2.49 (2H, m, Phe-CbH2), 3.46 (1H, br, Pro-CdH2),
3.75 (1H, br, Pro-CdH2), 3.85 (3H, s, OCH3), 4.00–4.05
(1H, m, aThr-CbH), 4.26 (1H, m, aThr-CaH), 4.44 (1H, br,
D-aIle-CaH), 4.81 (1H, br, Phe-CaH), 5.21 (1H, br, Pro-
CaH), 6.21 (1H, d, J¼8.6 Hz, BocNH), 7.19 (5H, br, PhH),
7.52 (1H, d, J¼8.4 Hz, NH), 7.82 (1H, br, NH), 8.30 (1H, s,
Thz-H); 13C NMR (67.8 MHz, CDCl3), minor rotamer in
parentheses, d 11.7, 14.2 (14.3), 19.5 (19.3), 24.6, 26.3,
3.1.14. Cyclo[L-Phe-L-Pro-D-aIle-L-aThr-L-Phe-L-Pro-
Thz] (25). To a solution of Boc-L-Phe-L-Pro-L-Ile-L-aThr-
L-Phe-L-Pro-Thz-OMe (24) (189 mg, 0.21 mmol) in THF
(0.82 mL) at 08C was added 0.5 M aq. LiOH (0.82 mL,
0.41 mmol). The resulting solution was stirred at 08C for

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
8141
30 min and at room temperature for 1 h. After the reaction
mixture was acidified by the addition of 1 M aq. KHSO4, the
resulting mixture was extracted with CHCl3 (£3). The
combined organic extracts were dried (Na2SO4), filtered,
and concentrated to afford the crude Boc-L-Phe-L-Pro-D-
aIle-L-aThr-L-Phe-L-Pro-Thz-OH. This heptapeptide was
treated with 4 M HCl–dioxane (1 mL)–THF (1 mL) at 08C.
The mixture was stirred at room temperature for 20 min.
The mixture was concentrated and dried to afford the crude
HCl–H-L-Phe-L-Pro-D-aIle-L-aThr-L-Phe-L-Pro-Thz-OH.
To a solution of the above crude HCl–H-L-Phe-L-Pro-D-
aIle-L-aThr-L-Phe-L-Pro-Thz-OH in DMF (103 mL) at
08C were successively added dropwise pentafluorophenyl
diphenylphosphinate (119 mg, 0.31 mmol) and N,N-di-
isopropylethylamine (0.15 mL, 0.83 mmol). The resulting
solution was stirred at room temperature for 42.5 h, and
concentrated. The residue was purified by flash chroma-
tography (BW-820MH, EtOAc–EtOH¼1:0–20:1–10:1) to
afford the desired product 25 as a white powder (116 mg,
0.15 mmol, 72%): mp 188–1918C; [a]2D6¼258.3 (c 0.48,
CHCl3); IR nKmBaxr (cm21) 3475, 1630, 1541, 1453; 1H NMR
(270 MHz, CD3OD) d 0.91 (3H, d, J¼7.3 Hz, D-aIle-CgH3),
0.94 (3H, t, J¼6.6 Hz, D-aIle-CdH3), 1.25 (3H, d, J¼6.1 Hz,
aThr-CH3), 1.29–1.38 (2H, m, D-aIle-CgH2), 1.60 (2H, br,
Pro-CbH2), 1.88–2.00 (6H, m, D-aIle-CbH, Pro-CbH2,
Pro-CgH2), 2.28 (1H, m, Pro-CbH2), 2.81–2.94 (3H, m,
Pro-CdH2), 3.10 (1H, dd, J¼13.5, 7.3 Hz, Phe-CbH2),
3.24 (1H, dd, J¼12.9, 4.9 Hz, Phe-CbH2), 3.47 (2H, br,
Phe-CbH2), 3.69 (1H, br, Pro-CdH2), 4.10 (1H, m, aThr-
CbH), 4.35 (1H, d, J¼5.1 Hz, aThr-CaH), 4.46–4.57 (3H,
m, Pro-CaH, D-aIle-CaH, Phe-CaH), 4.97 (2H, m, Pro-CaH,
Phe-CaH), 7.28–7.33 (10H, m, PhH), 7.75 (1H, d, J¼
8.7 Hz, NH), 8.12 (1H, s, Thz-H), 8.25 (1H, d, J¼8.4 Hz,
NH), 8.69 (1H, d, J¼9.6 Hz), 9.41 (1H, d, J¼7.1 Hz, NH);
13C NMR (67.8 MHz, CDCl3) d 11.9, 14.6, 20.6, 21.4, 25.5,
26.8, 29.0, 33.2, 34.8, 39.7, 40.4, 45.3, 47.3, 52.4, 53.9,
56.4, 57.4, 58.1, 61.7, 70.4, 127.0, 127.1, 128.6, 128.7,
129.5, 129.5, 135.6, 136.3, 150.0, 160.1, 169.4, 170.3,
170.4, 170.5, 171.4, 171.7. HRMS (FAB) m/z calcd for
C41H52N7O7S (MþH): 786.3649. Found: 786.3661.
2.49 (1H, m, Pro-CbH2), 2.89 (1H, dd, J¼13.7, 6.4 Hz, Phe-
CbH2), 2.92 (1H, m, Pro-CdH2), 2.99 (1H, dd, J¼13.7,
7.3 Hz, Phe-CbH2), 3.20 (1H, dd, J¼13.4, 3.1 Hz, Phe-
CbH2), 3.48 (1H, t, J¼13.1 Hz, Phe-CbH2), 3.49 (1H, m,
Pro-CdH2), 3.92 (2H, m, Pro-CdH2), 3.94 (1H, d, J¼6.7 Hz,
oxz-C4H), 3.99 (1H, m, oxz-C5H), 4.31 (1H, t, J¼7.6 Hz,
Pro-CaH), 4.95–5.00 (2H, m, Phe-CaH, D-aIle-CaH), 5.09
(1H, dt, J¼10.4, 3.1 Hz, Phe-CaH), 5.55 (1H, dd, J¼7.9,
3.1 Hz, Pro-CaH), 6.80 (1H, d, J¼8.6 Hz, D-aIle-NH), 7.03
(1H, t, J¼7.3 Hz, Phe-H), 7.13–7.36 (10 H, m, Phe-H, Phe-
NH), 7.96 (1H, s, Thz-H), 8.48 (1H, d, J¼8.6 Hz, Phe-NH);
13C NMR (125 MHz, CDCl3) d 12.0, 13.8, 22.0, 25.0, 25.6,
26.3, 30.0, 32.0, 38.1, 38.3, 39.9, 46.5, 47.3, 50.2, 52.2,
52.9, 58.1, 61.6, 73.7, 80.8, 122.7, 126.5, 126.7, 128.1,
128.4, 129.7, 129.7, 135.8, 137.1, 149.1, 160.1, 168.4,
1
169.3, 170.1, 170.1, 170.4, 172.1. H NMR and 13C NMR
spectral data in DMSO-d6 are shown in Table 2. HRMS
(FAB) m/z calcd for C41H50N7O6S (MþH): 768.3543.
Found: 768.3550.
3.2. Thermal isomerization
3.2.1. Thermal conversion of 1 to 3. Synthetic cis,cis-
ceratospongamide (1, 3.2 mg, 0.0041 mmol) was dissolved
in DMSO (3 mL) and heated to 1758C. After 30 min,
the DMSO was removed under reduced pressure and the
residue was purified by flash chromatography (BW-820MH,
EtOAc–EtOH¼20:1–5:1) to afford trans,trans-[D-aIle]-
ceratospongamide (3, 1 mg, 0.0013 mmol, 31%).
1
3.2.2. H NMR monitoring of thermal conversion of 1
under neutral or acidic condition. A solution of cis,cis-
ceratospongamide (1, 2 mg, 0.0026 mmol) in DMSO-d6
(0.5 mL) was treated with PPTs (2 mg, 0.0080 mmol) and
warmed to 1008C. The thermal conversion was monitored
by 270 MHz 1H NMR.
3.3. X-Ray crystal analysis of 1
Single crystals of ceratospongamide (1) were grown from a
chloroform–heptane mixture as colorless plates by slow
evaporation at room temperature. For the subsequent X-ray
work, a single crystal of dimensions 0.2£0.3£0.5 mm3 was
used. A summary of crystallographic data is given in Table
5. Intensity data were collected in an v–2u scan mode by
3.1.15. trans,trans-[D-aIle]-Ceratospongamide (3). To a
solution of cyclo[L-Phe-L-Pro-D-aIle-L-aThr-L-Phe-L-Pro-
Thz] (25) (67 mg, 0.085 mmol) in CH2Cl2 (0.7 mL) at
2308C was added Deoxo-fluor (0.032 mL, 0.17 mmol). The
reaction mixture was stirred at 230 to 2208C for 20 min,
and then additional Deoxo-fluor (0.008 mL, 0.043 mmol)
was added. After 10 min, the reaction mixture was
quenched by the addition of sat. aq. NaHCO3, warmed to
room temperature, and extracted with CHCl3. The com-
bined organic extracts were dried (Na2SO4), filtered, and
concentrated. The residue was purified by flash chroma-
tography (BW-820MH, EtOAc–EtOH¼1:0–20:1–8:1) to
afford the desired product 3 as a colorless amorphous
˚
graphite-monochromated Cu Ka radiation (l¼1.5418 A)
on an automated AFC5R (Rigaku) diffractometer, where the
backgrounds were counted for 5 s at both extremes of each
reflection peak. The observed intensities were corrected for
the Lorentz and polarization effects, but not for the
absorption effect.
The crystal structures were solved by the direct method
using the SHELXS97 program.23 The reflections with
I.2s(I) were used for the structure refinement. The
refinements of non-H atoms were carried out by the full-
matrix least-squares calculations on F2o intensities with
anisotropic thermal parameters using the program
SHELXL97.24 H atomic positions of methyl groups were
found from the difference Fourier maps, and the others were
geometrically located. These were treated as riding with
fixed isotropic displacement parameters (Uiso¼1.2Ueq for
the associated C or N atoms, or Uiso¼1.5Ueq for methyl C
powder (37 mg, 0.047 mmol, 57%): mp 153–1558C; [a]2D5¼
KBr
max
238.8 (c 0.47, CHCl3); IR n
(cm21) 3475, 1638, 1541,
1449; 1H NMR (500 MHz, CDCl3) d 0.87 (3H, d, J¼6.7 Hz,
D-aIle-CgH3), 1.01 (3H, t, J¼7.3 Hz, D-aIle-CdH3), 1.16
(1H, m, D-aIle-CgH2), 1.27 (3H, d, J¼6.1 Hz, oxz-CH3),
1.50 (1H, m, D-aIle-CgH2), 1.79 (1H, m, Pro-CgH2), 1.92
(1H, m, Ile-CbH), 1.98 (1H, m, Pro-CbH2), 2.04 (1H, m,
Pro-CgH2), 2.10 (1H, m, Pro-CgH2), 2.19 (1H, m, Pro-
CbH2), 2.22 (1H, m, Pro-CgH2), 2.27 (1H, m, Pro-CbH2),

8142
F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
Table 5. Summary of crystal data collections and structure refinement of 1
Molecular formula
Molecular weight
Crystal system
Space group
Unit cell dimensions
Volume
C41H49N7O6S·3H2O
821.98
Hexagonal
P41212
˚
˚
a¼b¼12.815 (2) A, c¼54.358 (9) A, a¼b¼g¼90.008
3
˚
8927 (2) A
Z, molecules/unit cell
F(000)
Density (calculated)
Absorption coefficient
Index ranges
8
3504
1.223 g cm23
1.133 mm21
0#h#15, 0#k#10, 0#l#65
No. of unique data measd
No. of reflections with I.2s(I)
Refinement method
Refinement weighting scheme
No. of variables refined
R(Rw)
4687
2394
Full least-squares on F 2
Calcd w¼1/[s 2(Fo2)þ(0.1000P)2] where P¼(Fo2þ2F2c)/3
516
0.0774 (0.2118)
0.1632 (0.2374)
1.313
0.653 and 20.239 e A
0.041/0.002
R(Rw) (all data)
Goodness-of-fit on F 2
Largest difference peak and hole
Max and averaged shift/esd
23
˚
atoms); their atomic positions were not included as variables
This SA method consists of the following three steps. In the
first step, an initial structure of the peptide is built with a
completely random array of atoms. Then, a target function is
determined in terms of the force constants of the covalent
bond (Fcovalent), repulsive van der Waals contact (Frepul),
and interproton distance (FROE). The distance-restraint
energy, FROE, is defined as the harmonic potential of
P
for the refinements. The function of w(F2o2Fc2)2 was
minimized by using the weighting scheme of w¼1/
[s 2(Fo2)þ(0.1000P)2], where P¼(Fo2þ2F2c)/3. Final R [¼
P
P
P
P
2 1/2
(lFol2lFcl)/ lFol]], Rw [¼( w(lFol2lFcl)2/ wlFol )
]
P
and S [¼( w(lFol2lFcl)2/(M2N))1/2, where M is the
number of reflections and N is the number of variables
used for the refinement] are also given in Table 5.
(
2
Kðri 2 riu=rilÞ if ri . riu or ri , ril
FROE
¼
Supplementary data sets (tables of final atomic coordinates,
anisotropic temperature factors, bond lengths, bond angles,
torsion angles of non-H atoms, and the atomic coordinates
of H atoms) have been deposited at the Cambridge
Crystallographic Data Centre (1: CCDC 183755) and are
available on request.
0
if ril # ri # riu
where ri is the calculated distance of an inter-proton pair i,
˚
˚
and ril and riu are the lower (1.6 A) and upper (5.0 A) limits
of its inter-proton distance, respectively. All proton–proton
distances showing ROE peaks, in spite of their ROE
intensities, were uniformly treated in the range of 1.6–
˚
3.4. Solution conformation of 3
5.0 A. The energy term for intermolecular hydrogen bonds
was not included in the model building. The force constant
(K) was finally chosen to be 25.0 kcal/mol. After minimiz-
ation by the steepest descents and successive conjugate
gradients method, the system was simulated for 50 ps at
1000 K. The global minimum of the target function was
initially investigated by substantially increasing the force
constants until they regained their full values. In the second
step, the temperature was decreased stepwise until
298 K. During this stage, the van der Waals repulsion
terms were set such that they be predominant. In the third
step, energy minimization was conducted again to refine the
obtained structure. For the distance constraints that involve
aromatic ring protons, that were not stereospecifically
assigned, the pseudo-atom treatment was used. Calculations
of other potential functions were performed according to the
protocol in the Insight II/Discover software. As input data
for constructing 3D structures, the restraints of f torsion
angles were not included in the calculations; instead, they
were used as indicators to estimate the reliability of the
constructed 3D structures.
3.4.1. NMR spectroscopy. All NMR measurements
(500 MHz for protons), except the temperature-variable
experiment, were performed at 292 K. A 9 mM degassed
solution of synthetic ceratospongamide (3) in DMSO-d6
was used for all NMR experiments. Chemical shifts were
measured relative to internal TMS at 0.0 ppm. Two-
dimensional GCOSY, TOCSY and ROESY spectra were
acquired in the phase-sensitive mode using standard pulse
schemes. Mixing times for ROESY and TOCSY spectra
were 400 and 80 ms, respectively. The time-domain matrix
consisted of 128£1024 complex data points and was zero-
filled to obtain a frequency domain matrix of 1024£1024
real data points. Thus, the spectra had digital resolutions of
0.4 Hz/point for the v1 and v2 directions. The 1H–13C shift
correlations were measured by HMQC and HMBC
spectroscopy using the standard pulse schemes. The
respective spectra were processed on a Sun SPARC
computer using Varian VNMRS software, applying normal
processing functions.
3.4.2. ROE restraint molecular modeling. 3D structures
of 3 that satisfy the ROE constraints of intramolecular
proton pairs were constructed by dynamic SA calcu-
lations25,26 using the Insight II/Discover 95 software.27
3.5. Molecular dynamics simulations of 1–3
Molecular dynamics calculations were performed using the
Insight II/Discover 95 program. The geometry optimization

F. Yokokawa et al. / Tetrahedron 58 (2002) 8127–8143
8143
was performed using the CVFF force field28 for peptides
and proteins. A target function was determined in terms of
the force constants for the covalent bond (Fcovalent) and
repulsive van der Waals contact (Frepul). Calculations of
these potential functions were performed according to the
protocol in the Insight II/Discover software. The confor-
mational energies in vacuo were minimized by the steepest
descents and conjugate gradients methods. The respective
systems were simulated for 100 ps by solving Newton’s
equation of motion. The simulation was conducted at 298 K,
a time step of 1.0 fs, a temperature relaxation time of
0.02 ps, and a period of update of non-bonded atom list of
25 fs.
J. Org. Chem. 1987, 52, 1252–1255. (b) Aoyama, T.; Sonoda,
N.; Yamauchi, M.; Toriyama, K.; Anzai, A.; Ando, A.; Shioiri,
T. Synlett 1998, 35–36. (c) Fujiwara, H.; Tojiki, K.;
Yokokawa, F.; Shioiri, T. Pept. Sci. 2000, 1999, 9–12.
(d) Sugiyama, H.; Yokokawa, F.; Shioiri, T. Org. Lett. 2000, 2,
2149–2152.
11. Takuma, S.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1982,
30, 3147–3153, and references therein.
12. The Weinreb amide 10 is currently commercially available
from Aldrich.
13. Fehrentz, J.-A.; Castro, B. Synthesis 1983, 676–678.
14. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972,
94, 6203–6205.
15. (a) Chen, S.; Xu, J. Tetrahedron Lett. 1991, 32, 6711–6714.
(b) Dudash, Jr. J.; Jiang, J.; Mayer, S. C.; Joullie, M. M. Synth.
Commun. 1993, 23, 349–356. (c) Deng, J.; Hamada, Y.;
Shioiri, T.; Matsunaga, S.; Fusetani, N. Angew. Chem. Int. Ed.
Engl. 1994, 33, 1729–1731. (d) Deng, J.; Hamada, Y.; Shioiri,
T. Synthesis 1998, 627–638.
Acknowledgements
We thank Professor W. H. Gerwick and Mr L. T. Tan for
comparison of our synthetic cis,cis-ceratospongamide and
its thermal product with the natural sample. The high-
resolution FAB mass spectra were measured by Dr Hideo
Naoki and Mr Tsuyoshi Fujita (Suntory Institute for
Bioorganic Research), to whom we are indebted. This
work was financially supported in part by a Grant-in-Aid for
Research in Nagoya City University (to F. Y.), the Uehara
Memorial Foundation (to F. Y.), the Fujisawa Foundation
(to F. Y.), and Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
16. Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber,
H.; Carpino, L. A.; Bienert, M. J. Org. Chem. 1996, 61,
8831–8838.
17. Phillips, A. D.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R.
Org. Lett. 2000, 2, 1165–1168.
18. (a) X-Ray and thermal isomerization works were presented at
36th Tennenbutsu Danwakai (Nagoya, Japan), July 15–17,
2001 and at 18th International Congress of Heterocyclic
Chemistry (Yokohama, Japan), July 29–August 3, 2001;
Abstracts; p 124. (b) Very recently, X-ray structure analysis of
1 was reported: Doi, M.; Yumiba, A.; Asano, A. Acta
Crystallogr. 2002, E58, o62–o64.
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