Synthesis of MEN11420, a glycosylated bicyclic peptide, by intramolecular double cyclization using a chloroimidazolinium coupling reagent

Article abstract of DOI:10.1016/S0040-4020(01)00011-4

The synthesis of MEN11420, a potent tachykinin receptor antagonist, has been achieved. The bicyclic glycosylated structure of MEN11420 was constructed via intramolecular double cyclization using CIP-mediated activation. The head to tail cyclization of the linear precursor, which contained an α-amino acid at its C-terminus, proceeded so rapidly that no serious racemization was apparent at the activated carboxyl function. The desired product was obtained without the need for purification of the intermediates throughout the synthesis.

Full text of DOI:10.1016/S0040-4020(01)00011-4

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 1749±1755
Synthesis of MEN11420, a glycosylated bicyclic peptide, by
intramolecular double cyclization using a chloroimidazolinium
coupling reagent
Kenichi Akajip and Saburou Aimoto
Institute for Protein Research, Osaka University, Suita, 3-2 Yamadaoka, Osaka 565-0871, Japan
Received 13 November 2000; accepted 15 December 2000
AbstractÐThe synthesis of MEN11420, a potent tachykinin receptor antagonist, has been achieved. The bicyclic glycosylated structure of
MEN11420 was constructed via intramolecular double cyclization using CIP-mediated activation. The head to tail cyclization of the linear
precursor, which contained an a-amino acid at its C-terminus, proceeded so rapidly that no serious racemization was apparent at the activated
carboxyl function. The desired product was obtained without the need for puri®cation of the intermediates throughout the synthesis. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
synthesis of bicyclic MEN10627 was reported by Pavone
et al.4 in 1995.
Tachykinins (TKs) comprise a family of peptides that are
widely distributed in the central and peripheral nervous
system and play a key role in neuronal stimulation.1 Of
the three mammalian's TKs (Substance P, Neurokinin A,
and Neurokinin B), Neurokinin A (NKA) is known to
play a role in bronchoconstriction, smooth muscle contrac-
tion and in¯ammation. Thus, antagonists of the Tachykinin
NK2 receptor which recognize Neurokinin A have a wide
range of potential therapeutic applications.2 MEN11420,
which is derived from the bicyclic Tachykinin analogue
MEN10627, is a potent and selective tachykinin NK2 recep-
tor antagonist as has been reported by Renzetti et al.3 in
1998. Structurally, MEN11420, cyclo{[Asn(b-d-GlcNAc)-
Asp-Trp-Phe-Dap-Leu]cyclo(2b±5b)} (Fig. 1), is a bicyclic
peptide with a (2-acetylamino-2-desoxy-b-d-glucopyrano-
syl)-l-asparaginyl residue in the place of the Met residue,
which is normally present in MEN10627. In spite of its
unique structure and speci®c activity for the NK2 receptor,
no detailed procedure for the synthesis of MEN11420 has,
to date, been reported, although, a Boc-based classical
Recently, we have developed a new coupling reagent,
2-chloro-1,3-dimethyl-2-imidazolinium
hexa¯uorophos-
phate (CIP),5 which in the presence of an additive (HOAt6
or DMAP7) permits the effective coupling of sterically
hindered a-dialkylamino acids or N-methylamino acids
(Fig. 2). Using this coupling reagent, we were able to
Figure 1. Structure of MEN11420.
Keywords: CIP; coupling reagent; intramolecular cyclization; MEN11420;
tachykinin antagonist.
Abbreviations: Bocˆtert-butoxycarbonyl; Butˆtert-butyl; CIPˆ2-chloro-
1,3-dimethyl-2-imidazolium hexa¯uorophosphate; Dapˆ2,3-diaminopro-
pionic acid; DIEAˆN,N-diisopropylethylamine; DIPCDIˆN,N0-diisopro-
pylcarbodiimide; DMAPˆ4-dimethylaminopyridine; Fmocˆ¯uoren-9-
ylmethyloxycarbonyl; HOAtˆ1-hydroxy-7-azabenzotriazole; HOBtˆN-
hydroxybenzotriazole; MALDI-TOFˆmatrix-assisted laser desorption
ionization time-of-¯ight; SPPSˆsolid phase peptide synthesis; TFAˆtri-
¯uoroacetic acid; TFEˆ2,2,2-tri¯uoroethanol.
p
Corresponding author. Tel.: 181-06-6879-8608; fax: 181-06-6879-
Figure 2. Structures of CIP, HOAt and DMAP.
8609; e-mail: akaji@protein.osaka-u.ac.jp
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00011-4

1750
K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
achieve the convergent syntheses of polythiazoline
alkaloids,8 peptaibols9 and cytostatic depsipeptide.10 These
synthetic studies suggest that the intramolecular coupling of
compounds, including sterically hindered glycosylated
amino acids, might be feasible using the CIP-additive as a
coupling reagent. Here, we describe the synthesis of
MEN11420 based on a general synthetic scheme via the
CIP-mediated intramolecular double cyclization of a
glycosylated linear precursor.
2. Results and discussion
The overall strategy for the synthesis is shown in Scheme 1.
A double intramolecular cyclization using the CIP reagent is
achieved by combining a head to tail cyclization and a
subsequent cyclization between the side chain functional
groups. In the ®rst cyclization of the precursor, which
contains an optically active a-amino acid at its C-terminus,
racemization at the activated carboxyl function presents a
Scheme 1.

K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
1751
Figure 3. HPLC and GC pro®les of the products obtained by pathway A. (i) HPLC pro®les of the crude glycosylated precursor 2 (a) and the monocylized crude
intermediate 5 (b). (ii) GC spectra of the monocyclized compound L and compound D.
serious problem. A low reaction rate for the condensation
would be expected to be one of the major reasons for
racemization.11 Thus, two different schemes for the ®rst
cyclization step were selected; one, which involved cycliza-
tion at the most hindered site between the glycosylated Asn
and Leu (Scheme 1, pathway A) and the other, which
involved the less hindered site between Dap(Boc) and Phe
(Scheme 1, pathway B). Each required linear precursor for
the above synthetic scheme was prepared by Fmoc-based
SPPS, since sugar residues, when covalently linked to the
peptide are not stable in anhydrous HF or the other strong
acids used for cleavage from the resin under classical Boc-
based procedures.12 As a solid support, an acid labile
2-chlorotritylchloride (Clt) resin13 was selected instead of
the conventional benzylalcohol-based Wang resin.
ninhydrin test was negative and the desired precursor resin
3 was obtained without dif®culty.
Each protected precursor peptide was then cleaved from the
corresponding resin by treatment with AcOH/TFE/CH2Cl2
(1:1:8) to yield the linear precursors 2 or 4 as powders. Each
Based on the above scheme, the protected linear precursor
resin 1, which was used in pathway A, was ®rst constructed
starting from the Fmoc-Leu-Clt resin prepared by published
procedures.13 Successive incorporation using a 2.5 equiv.
excess of Fmoc-Dap(Boc)-OH, Fmoc-Phe-OH, Fmoc-Trp-
OH and Fmoc-Asp(OBut)-OH were conducted by a
combination of piperidine-mediated deprotection and
DIPCDI-mediated coupling. The N-terminal glycosylated
residue, Fmoc-Asn(Ac3AcNH-b-Glc)-OH,14 was intro-
duced by the same procedure, except that a 1.3 equiv. excess
of the glycosylated amino acid and an extended coupling
reaction (6 h) were employed. At each condensation step,
the Kaiser ninhydrin test was negative. After the construc-
tion of the peptide chain, the N-terminal Fmoc group was
removed by treatment with piperidine to yield the side chain
protected precursor resin 1.
Another precursor resin 3, used in the pathway B, was then
constructed starting from the Fmoc-Phe-Clt resin. The
successive incorporation of Fmoc-amino acids, except for
Fmoc-Leu-OH, was conducted similarly as above. The
condensation of Fmoc-Leu-OH to the glycosylated Asn
was achieved by the CIP/HOAt method instead of the
conventional DIPCDI method because of the provable low
reactivity of sterically hindered glycosylated amino acid at
the N-terminus. At each condensation step, the Kaiser
Figure 4. HPLC pro®les of the products obtained by pathway B.
(i) Crude glycosylated precursor 4. (ii) Mono cyclized crude intermediate
5. p denotes the corresponding d-isomer. (iii) Double cyclized crude
intermediate 7.

1752
K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
crude product showed a single main peak on analytical
HPLC and was used for the next double cyclization without
further puri®cation (Figs. 3(i, part a) and 4(i)).
presence of DMAP under the same conditions as described
for the ®rst cyclization step. The reaction, which was moni-
tored by HPLC, also proceeded within 15 min in the
presence of an excess amount of CIP (Fig. 4(iii)). The
desired product was obtained as a major product on
HPLC, although several side reaction products were
observed in this second cyclization step. These side
products probably arose from the formation of oligomers
resulting from the intermolecular coupling reaction. The
O-acetyl groups of the crude double cyclized product
were ®nally removed by treatment with sodium methoxide
in methanol. The crude product, which was isolated by
adsorption to Diaion HP20, was puri®ed by preparative
reverse phase HPLC to yield the desired product in 4%
overall yield (calculated from the starting Clt resin, total
11 steps). The integrity of the puri®ed product was deter-
mined by analytical HPLC, amino acid analysis after acid
hydrolysis, MALDI-TOF MS and by 1H and 13C NMR. The
optical purity of the constituent amino acids in the synthetic
MEN11420 was con®rmed by gas chromatography using a
chiral column. The content of each d-amino acid was less
than 0.5%, a result of the hydrolysis conditions.
For the initial cyclization step, based on pathway A, the
head to tail cyclization of precursor 2 was conducted by
CIP-mediated activation in DMF in the presence of HOAt.
When the reaction was monitored by HPLC in the presence
of an excess amount of CIP (9 equiv.), the starting material
had disappeared within 15 min. Incomplete conversion to
the cyclized product was observed when a slight excess of
CIP was used, even when the reaction time was extended.
The use of HOBt as an additive also gave poor results and
gave a complex mixture.
The cyclized product, however, was found to be a mixture of
two compounds, as determined by HPLC analysis (Fig. 3(i,
part b)). Each compound had an amino acid composition
and mass value which was consistent with the theoretical
values, and there was no signi®cant difference between the
values of the two compounds. These results strongly suggest
that any difference in the two products would be re¯ected in
their optical purity. Thus, the optical purity of each
compound was examined by GC analysis using a chiral
column. The (penta¯uoropropionyl) amino acid n-butyl
esters, derived from a total hydrolysate of the each
compound, were analyzed in a Chirasil Val-L capillary
column. The analysis indicated that the compound L
contained all the expected l-amino acids, while the
compound D contained d-Leu instead of a l-Leu residue
(Fig. 3(ii)); this clearly shows that the racemization
occurred during the initial cyclization between the Leu
and glycosylated Asn residue. The relative content of the
racemized product to the desired product was estimated to
be 33% by HPLC. The content of the racemized product
decreased to 30% when the cyclization was conducted with
an additional additive, CuCl2,15 but no remarkable suppres-
sion of racemization was observed.
3. Conclusion
We report herein the synthesis of MEN11420, a potent
tachykinin NK2 receptor antagonist, based on a general
scheme for the synthesis of bicyclic glycopeptides. The
CIP-mediated intramolecular double cyclizations proceeded
within 15 min in the presence of HOAt or DMAP. The CIP-
mediated intramolecular cyclization between general
a-amino acids proceeded so rapidly that no serious race-
mization occurred at the activated carboxyl function.
Thus, the entire procedure, which includes the synthesis of
a glycosylated peptide on a solid support and the double
cyclization±deprotection steps in solution, could be
conducted in a reasonably short period of time (several
days) with no need for purifying the intermediates. A single
step HPLC puri®cation at the ®nal step of the synthesis was
suf®cient to obtain the optically pure MEN11420.
Initial cyclization between the less hindered Phe and
Dap(Boc) residues was then examined (Scheme 1, pathway
B). The coupling reaction was conducted with CIP/HOAt as
described for pathway A and the progress of the reaction
was monitored on HPLC. The linear precursor disappeared
within 15 min using a smaller amount of CIP (3 equiv.)
compared with those used in pathway A. The product
showed a single main peak, which contained a small
shoulder peak derived from the corresponding d-Phe isomer
(Fig. 4(ii)). The relative content of the racemized product
was estimated to be less than 6% by HPLC- and GC-
analysis. Thus, the CIP-mediated head to tail cyclization
between the general a-amino acid proceeded so rapidly
that no serious racemization had occurred, whereas the
cyclization of the precursor having a glycosylated amino
acid at its N-terminus proceeded more slowly, resulting in
a racemized product in a moderate yield. The crude cyclized
product was then treated with TFA/anisole to remove
the side chain protecting groups to yield monocyclized
hexapeptide derivative 6.
4. Experimental
4.1. General
Solvents were reagent grade and were dried prior to use.
Fmoc amino acid derivatives, except for the diaminopro-
pionic acid (Dap) derivative, and the 2-chlorotrityl chloride
resin were obtained from Calbiochem Novabiochem and
were used without further puri®cation. Fmoc-Dap(Boc)-
OH was prepared according to the procedure for
Z-Dap(Boc)-OH16 with several modi®cations. For the
quanti®cation of Fmoc amino acids on the resin, the absorb-
ance at 301 nm after cleavage of the Fmoc group with
piperidine was measured according to the procedure
described by Meienhofer et al.17
Melting points are uncorrected. The 1H and 13C NMR
spectra were recorded on a 270 MHz (JEOL) or 400 MHz
(Bruker) spectrometer with TMS as an internal
standard. FAB-MS was obtained on a JEOL JMS-SX102A
The second intramolecular cyclization was conducted using
the crude deprotected product without further puri®cation:
the monocyclized product 6 was treated with CIP in the

K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
1753
spectrometer equipped with a JMA-DA7000 data system.
MALDI-TOF MS was obtained on a Voyager-DE Bio-
spectrometry Workstation of PerSeptive Biosystems. Gas
chromatography (GC) using Chirasil Val-L capillary
column was conducted according to published pro-
cedures.8,18 HPLC was carried out on a reversed phase
column which was eluted with CH3CN in 0.1% aq. TFA
and products were detected at OD 220 nm: Rt1, YMC
AM302 (4.6£150 mm), ¯ow rate 0.9 ml/min; Rt2, YMC-
Pack ProC18 AS 323 (10£250 mm), ¯ow rate 2.5 ml/min.
DMF. To this resin, Fmoc-Dap(Boc)-OH (2.5 equiv.) in
DMF, HOBt (2.5 equiv.), DIEA (2.5 equiv.) and DIPCDI
(2.5 equiv.) were added, and the mixture was agitated for
2 h at 258C. After washing the resin with DMF, the same
deprotection and condensation procedure was repeated for
the incorporation of Fmoc-Phe-OH, Fmoc-Trp-OH and
Fmoc-Asp(OBut)-OH. For the incorporation of Fmoc-
Asn(Ac3AcNH-b-Glc)-OH, a 1.3 equiv. excess of the
amino acid and other reagents were used. At each conden-
sation step, the results of a Kaiser ninhydrin test were nega-
tive after a single coupling reaction. The N-terminal Fmoc
group was removed by treatment with 20% piperidine/
DMF. The obtained resin was ®ltered, washed with DMF
and MeOH, and dried to yield 2.4 g of the desired hexa-
peptide resin 1.
4.1.1. Fmoc-Dap(Boc)-OH. To a stirred solution of bis[tri-
¯uoroacetoxy]-phenyliodine (6.5 g, 15 mmol) in DMF/H2O
(2:1, 60 ml), Fmoc-Asn-OH (3.37 g, 10 mmol) was added.
After 15 min, pyridine (1.6 ml, 20 mmol) was added and the
mixture was stirred for an additional 10 h. Solvent of the
mixture was removed by evaporation and the residue was
dissolved in H2O (100 ml). The solution was acidi®ed with
6N HCl and then washed with Et2O. The pH of the aqueous
phase was adjusted to 6 with 4N NaOH and the solution was
allowed to stand at 48C. The resulting precipitate was
®ltered, washed with Et2O and dried to yield 2.5 g (81%)
of Fmoc-Dap-OH as a solid.
(c) Cleavage. To 0.5 g (0.1 mmol) of the above peptide resin
1 (0.207 mmol/g), AcOH/TFE/CH2Cl2 (1:1:8, 20 ml) was
added and the mixture was agitated for 40 min at 258C.
The resin was ®ltered and washed with AcOH/TFE/
CH2Cl2 (1:1:8, 5 ml). The ®ltrate was evaporated to dryness
and the residue was dissolved in 2N AcOH. The solution
was lyophilized to yield 110 mg (84%) of 2 as a white
powder: Rt1 25.47 min [CH3CN (30±60%/30 min)],
MALDI-TOF MS, m/z 1266.47 for [M1H]1 (Calcd
1266.36 for C60H85N10O20). Amino acid analysis after 6N
HCl hydrolysis; Asp 1.45, Leu 1.00, Phe 1.06.
To a solution of Fmoc-Dap-OH (2.5 g, 8 mmol) in DMF
(10 ml), DIEA (2.8 ml, 16 mmol) and (Boc)2O (3.5 g,
16 mmol) were added and the mixture was stirred for 4 h
at 258C. The solvent was removed by evaporation and the
residue was extracted with AcOEt (50 ml). The organic
phase was washed with 5% aq. citric acid and then H2O,
dried over MgSO4, and evaporated. The residue was tri-
turated with hexane to yield Fmoc-Dap(Boc)-OH as an
amorphous powder. For storage, the product was converted
to its DCHA salt to yield 3.5 g (71%) of Fmoc-Dap(Boc)-
OH DCHA as a solid. For characterization, a part of the
product was treated with excess amount of TMSCHN2 in
hexane to yield Fmoc-Dap(Boc)-OMe as a powder: mp
4.1.3. H-Dap(Boc)-Leu-Asn(Ac3AcNH-b-Glc)-Asp(O-
But)-Trp-Phe-OH, 4. (a) Anchoring to Clt resin. To 2.0 g
(2.4 mmol) of 2-chlorotrityl chloride (Clt) resin, Fmoc-Phe-
OH (0.46 g, 1.2 mmol) in CH2Cl2/DMF (2:1, 10 ml) and
DIEA (1 ml, 5.7 mmol) were added. The mixture was
agitated for 1.5 h at 258C and the solvent was removed by
®ltration. MeOH/DIEA (9:1, 15 ml) was added to the resin
and the mixture was agitated for a further 30 min at 258C.
The resin was ®ltered, washed with MeOH and then dried to
yield 2.4 g of Fmoc-Phe-Clt resin, substitution at a level of
0.37 mmol/g.
23
150±1528C, [a]D ˆ116.8 (cˆ0.5, CHCl3), 1H NMR
(270 MHz, CDCl3) d 7.76 (d, Jˆ7.3 Hz, 2H), 7.61 (d,
Jˆ6.9 Hz, 2H), 7.40 (t, Jˆ6.9 Hz, 2H), 7.31 (t, Jˆ7.3 Hz,
2H), 5.93 (brd, Jˆ6.4 Hz, 1H), 4.89 (brt, Jˆ5.6 Hz, 1H),
4.50 (brs, 1H), 4.40 (d, Jˆ6.0 Hz, 2H), 4.22 (t, Jˆ6.6 Hz,
1H), 3.76 (s, 3H), 3.55 (brs, 2H), 1.44 (s, 9H). 13C NMR
(67.8 Hz, CDCl3) d 170.91, 156.40, 155.94, 143.63, 141.24,
127.66, 127.01, 125.07, 119.91, 80.00, 67.03, 54.90, 52.71,
47.06, 42.09, 28.20. FAB-MS, m/z 441.2031 for [M1H]1
(Calcd 441.2026 for C24H29N2O6).
(b) Condensation on Clt resin. Starting from the above
Fmoc-Phe-Clt resin, Fmoc-Trp-OH and Fmoc-Asp(OBut)-
OH were incorporated using DIPCDI by the same procedure
described for precursor 1. 1.51 g of the resulting Fmoc-
Asp(OBut)-Trp-Phe-Clt resin (3.03 g) was used for the
further chain elongation. For the incorporation of Fmoc-
Asn(Ac3AcNH-b-Glc)-OH, a 1.7 equiv. excess of the
amino acid and DIPCDI were used. For the coupling of
Fmoc-Leu-OH (2.5 equiv.) to the N-terminal glycosylated
amino acid, CIP/HOAt (2.5±2.0 equiv.) were employed.
Finally, Fmoc-Dap(Boc)-OH (2.5 equiv.) was incorporated
using DIPCDI (2.5 equiv.) and the N-terminal Fmoc group
was removed by treatment with 20% piperidine/DMF. At
each condensation step, the results of a Kaiser ninhydrin test
were negative after single coupling reaction. The obtained
resin was ®ltered, washed with DMF and MeOH, and dried
to yield 1.4 g of the desired hexapeptide resin 3.
4.1.2. H-Asn(Ac3AcNH-b-Glc)-Asp(OBut)-Trp-Phe-Dap-
(Boc)-Leu-OH, 2. (a) Anchoring to Clt resin. To 2.0 g
(2.1 mmol) of 2-chlorotrityl chloride (Clt) resin, Fmoc-
Leu-OH (0.19 g, 0.54 mmol) in CH2Cl2/DMF (2:1, 10 ml)
and DIEA (0.8 ml, 4.6 mmol) were added. The mixture was
agitated for 2 h at 258C and the solvent was removed by
®ltration. MeOH/DIEA (9:1, 8 ml) was added to the resin
and the mixture was agitated for a further 30 min at 258C.
The resin was ®ltered, washed with MeOH and then dried in
vacuo to yield 2.1 g of Fmoc-Leu-Clt resin, substituted at a
level of 0.271 mmol/g.
(c) Cleavage. To 0.32 g (0.083 mmol) of the above peptide
resin 3 (0.26 mmol/g), AcOH/TFE/CH2Cl2 (1:1:8, 13 ml)
was added and the mixture was agitated for 40 min at
258C. The resin was ®ltered and washed with AcOH/TFE/
CH2Cl2 (1:1:8, 5 ml). The ®ltrate was evaporated to dryness
(b) Condensation on Clt resin. Piperidine (20%) in DMF
was added to the above resin and the mixture was agitated
for 20 min at 258C. The resin was ®ltered and washed with

1754
K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
and the residue was treated with H2O to afford 95 mg (90%)
of 4 as a powder: Rt1 21.19 min [CH3CN(30±60%/30 min)],
MALDI-TOF MS, m/z 1266.08 for [M1H]1 (Calcd
1266.36 for C60H85O20N10). Amino acid analysis after 6N
HCl hydrolysis; Asp 1.44, Leu 0.98, Phe 1.00.
1.1 mmol) and CIP (0.38 g, 1.3 mmol) were added, and
the mixture was stirred for 15 min at 258C. AcOH
(0.16 ml) was added to the reaction mixture. The solvent
was removed by evaporation and the residue was dissolved
in AcOEt (20 ml). The organic phase was washed with H2O,
dried over MgSO4, and then evaporated to dryness. The
crude product was used for the next deacetylation reaction
without any further puri®cation: Rt1 13.58 min [CH3CN
(30±60%/30 min)], MALDI-TOF MS, m/z 1188.83 for
[M1H]1 (Calcd 1188.10 for C51H65N10O16´CF3COOH).
Amino acid analysis after 6N HCl hydrolysis; Asp 1.71,
Leu 0.82, Phe 1.00.
4.1.4. Monocyclized intermediate, 5, by pathway A. To
the solution of linear precursor 2 (5 mg, 4 mmmol) in DMF
(1 ml), HOAt (5 mg, 40 mmol), CIP (13 mg, 47 mmol) and
DIEA (14 ml, 80 mmol) were added, and the mixture was
stirred for 15 min at 258C. HPLC analysis of the mixture
showed a major product (Compound L, Rt1 26.70 min)
accompanied with a side product (Compound D, Rt1
27.79 min) (Fig. 3(i)). Relative content of the compound
D was 33% on the HPLC. Both products were isolated on
4.1.6. MEN11420, 1. To a solution of the double cyclized
crude product 7 (55 mmol) in MeOH (5.0 ml), 28% NaOMe/
MeOH (0.32 ml, 0.17 mmol) was added and the mixture
was stirred for 20 min at 258C. The pH of the mixture was
adjusted to ca. 6 with AcOH and the solvent of the mixture
was evaporated in vacuo. The residue was dissolved in 0.1%
aq. TFA (10 ml). Diaion HP20 (ca. 5 g) was added to the
solution and the mixture was gently stirred for 30 min at
258C. The resin was ®ltered and washed with H2O. The
adsorbed product was then eluted with 80% CH3CN in
0.1% aq. TFA and the ®ltrate was lyophilized. The crude
product was puri®ed by preparative HPLC [YMC-Pack
ProC18AS323 (10£250 mm), CH3CN (25±32%/65 min)]
to yield 2.6 mg (overall 4.4%) of 1 as a white powder:
a
semipreparative column YMC-Pac ProC18AS323
(10£250 mm), which was eluted with CH3CN/0.1% TFA
[CH3CN 30±60% (60 min)]. Each product was then hydro-
lyzed for amino acid analysis and GC analysis. Amino acid
analysis after 6N HCl hydroloysis: Compound L; Asp 1.47,
Leu 1.03, Phe 1.00; Compound D; Asp 1.49, Leu 1.05, Phe
1.00. MALDI-TOF MS: Compound L; m/z 1362.68 for
[M1H]1 (Calcd 1362.34 for C60H38N10O19´CF3COOH);
Compound D; m/z 1362.45 for [M1H]1 (Calcd 1362.34
for C60H38N10O19´CF3COOH). GC-spectra of the acid
hydrolysate derived from Compound L and D [808C for
3 min, then 80 to 1908C (38C/min)] are shown in Fig. 3(ii).
25
[a]D ˆ241.28 (cˆ0.1, H2O). Rt1 17.10 min [CH3CN
1
(25±35%/30 min)], H NMR (400 MHz, D2O at 458C) d
4.1.5. Double cyclization by pathway B. (a) Monocyclized
intermediate, 5. To the solution of linear precursor 4
(70 mg, 55 mmol) in DMF (9 ml), HOAt (19 mg,
0.14 mmol) and CIP (46 mg, 0.17 mmol) were added.
DIEA (43 ml, 0.25 mmol) was then added in three portions
to the mixture and the resulting solution was stirred for
15 min at 258C. HPLC analysis of the mixture showed a
single major peak with a small shoulder (relative content,
6%) (Fig. 4(ii)). The content of d-Phe isomer in the major
product was con®rmed by GC analysis using the same
procedure as described in pathway A synthesis. AcOH
(0.12 ml) was added to the reaction mixture. The solvent
of the reaction mixture was then removed by evaporation
and the resulting residue was dissolved in AcOEt (20 ml).
The organic phase was washed with H2O, dried over MgSO4
and evaporated to dryness. The crude product was used for
the next deprotection reaction without any further puri®ca-
tion: Rt1 24.30 min [CH3CN (30±60%/30 min)], MALDI-
TOF MS, m/z 1362.79 for [M1H]1 (Calcd 1362.34 for
C60H83N10O19´CF3COOH). Amino acid analysis after 6N
HCl hydrolysis; Asp 1.54, Leu 1.00, Phe 1.00.
7.68 (d, Jˆ7.5 Hz, 1H), 7.65 (d, Jˆ7.5 Hz, 1H), 7.57±
7.48 (m, 3H), 7.43±7.39 (m, 3H), 7.34 (t, Jˆ7.5 Hz, 1H),
7.00 (s, 1H), 5.02 (d, Jˆ9.8 Hz, 1H), 4.89 (m, 1H), 4.84 (m,
1H), 4.60 (m, 1H), 4.50 (m, 1H), 4.11 (dd, Jˆ14.3, 3.4 Hz,
1H), 3.97±3.95 (m, 1H), 3.89±3.86 (m, 2H), 3.84±3,68 (m,
1H), 3.59±3.47 (m, 2H), 3.53 (dd, Jˆ14.3, 3.4 Hz, 1H), 3.41
(dd, Jˆ14.2, 5.4 Hz, 1H), 3.21 (dd, Jˆ16.0, 3.9 Hz, 1H),
2.77 (dd, Jˆ16.0, 3.9 Hz, 1H), 2.11 (s, 3H), 1.80 (m, 2H),
1.07 (d, Jˆ5.6 Hz, 3H), 1.04 (d, Jˆ5.6 Hz, 3H). 13C NMR
(100 MHz, D2O at 258C) d 21.01, 22.43, 22.61, 24.78,
26.67, 34.19, 36.33, 36.61, 38.82, 39.72, 40.39, 49.22,
51.34, 54.37, 54.54, 55.17, 56.64, 60.72, 60.84, 69.62,
71.99, 74.29, 77.91, 78.89, 109.23, 112.26, 118.60,
119.93, 122.58, 123.82, 127.07, 127.55, 129.18, 129.65,
136.47, 137.40, 159.67, 163.56, 171.25, 171.60, 172.23,
173.17, 173.20, 174.58, 174.95, 175.04, 177.09. MALDI-
TOF MS, m/z 1062.32 for [M1H]1 (Calcd 1061.99 for
C45H59N10O13´CF3COOH). Amino acid analysis after 6N
HCl hydrolysis; Asp 1.95, Leu 1.07, Phe 1.00, Trp 0.56.
The GC chromatogram obtained after acid hydrolysis
showed that the content of each d-amino acid was less
than 0.5%.
(b) Double cyclized intermediate, 7. TFA-anisole (7.0±
0.24 ml) was added to the above mono cyclized product 5
and the mixture was stirred for 2.5 h at 258C. The TFA was
evaporated in vacuo and the residue was extracted with 2N
AcOH. The aqueous phase was washed with Et2O and
lyophilized to yield the side chain deprotected product 6
as a hygroscopic powder: MALDI-TOF MS, m/z 1205.94
for [M1H]1 (Calcd 1206.118 for C51H67N10O17´CF3-
COOH). Amino acid analysis after 6N HCl hydrolysis;
Asp 1.71, Leu 1.03, Phe 1.00.
Acknowledgements
The authors are grateful to Ms. Kayoko Oda (Kyoto
Pharmaceutical University) for collecting the FAB-MS data.
References
To the solution of the deprotected product 6 (55 mmol) in
DMF (9 ml), DIEA (48 ml, 0.27 mmol), DMAP (0.13 g,
1. Maggi, C. A.; Patacchini, R.; Rovero, P.; Giachetti, A.
J. Auton. Pharmacol. 1993, 13, 23±93.

K. Akaji, S. Aimoto / Tetrahedron 57 (2001) 1749±1755
1755
2. (a) Rovero, P.; Pestellini, V.; Maggi, C. A.; Patacchini, R.;
Regoli, D.; Giachetti, A. Eur. J. Pharmacol. 1990, 175, 113±
115. (b) McElroy, A. B.; Celgg, S. P.; Deal, M. J.; Ewan, G. B.;
Hagan, R. M.; Ireland, S. J.; Jordan, C. C.; Porter, B.; Ross,
B. C.; Ward, P.; Whittington, A. R. J. Med. Chem. 1992, 35,
2582±2591.
10. Akaji, K.; Hayashi, Y.; Kiso, Y.; Kuriyama, N. J. Org. Chem.
1999, 64, 405±411.
11. Carpino, L. A.; Ionescu, D.; El-Faham, A. J. Org. Chem. 1996,
2460±2465.
12. Pinzani, D.; Papini, A. M.; Vallecchi, M. E.; Chelli, M.;
Ginanneschi, M.; Rapi, G. Bioorg. Med. Chem. Lett. 1996,
6, 367±372.
3. Catalioto, R.-M.; Criscuoli, M.; Cucchi, P.; Giachetti, A.;
Giannotti, D.; Giuliani, S.; Lecci, A.; Lippi, A.; Patacchini,
R.; Quartara, L.; Renzetti, A. R.; Tramontana, M.; Arcamone,
F.; Maggi, C. A. Br. J. Pharmacol. 1998, 123, 81±91.
4. Pavone, V.; Lombardi, A.; Nasgtri, F.; Saviano, M.; Maglio,
O.; D'Auria, G.; Quartara, L.; Maggi, C. A.; Pedone, C.
J. Chem. Soc., Perkin II 1995, 987±993.
13. (a) Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu,
È
P.; Wenqing, Y.; Schafer, W. Tetrahedron Lett. 1989, 30,
3943±3946. (b) Barlos, K.; Gatos, D.; Kapolos, S.;
È
Papaphotiu, G.; Schafer, W.; Wenqing, Y. Tetrahedron Lett.
1989, 30, 3947±3950.
14. Inazu, T.; Kobayashi, K. Synlett 1993, 869±870.
15. (a) Nishiyama, Y.; Tanaka, M.; Saito, S.; Ishizuka, S.; Mori,
T.; Kurita, K. Chem. Pharm. Bull. 1999, 47, 576±578.
(b) Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.;
Kuwata, S. J. Chem. Soc., Chem. Commun. 1988, 419±420.
16. Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266±268.
17. Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Lhang, C.-D. Int. J. Peptide Protein Res.
1979, 13, 35±42.
5. Akaji, K.; Kuriyama, N.; Kiso, Y. Tetrahederon Lett. 1994,
35, 3315±3318.
6. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397±4398.
7. Hassner, A.; Krepski, L. R.; Alexanian, V. Tetrahedron 1978,
34, 2069±2076.
8. (a) Akaji, K.; Kiso, Y. Tetrahedron 1999, 53, 10685±10694.
(b) Akaji, K.; Kuriyama, N.; Kiso, Y. J. Org. Chem. 1996, 61,
3350±3357.
18. Frank, H.; Woiwode, W.; Nicholson, G.; Bayer, E. Liebigs
Ann. Chem. 1981, 354±365.
9. Akaji, K.; Tamai, Y.; Kiso, Y. Tetrahedron 1997, 53, 567±
584.