First total synthesis of Mer-N5075A and a diastereomeric mixture of α and β-MAPI, new HIV-I protease inhibitors from a species of Streptomyces

Article abstract of DOI:10.1016/S0040-4020(01)00323-4

Mer-N5075A (1) and α-MAPI and β-MAPI (2 and 3) produced from a species of Streptomyces are new anomalous tetrapeptides having potential HIV-I protease inhibitory activity. The first total synthesis of 1 and a diastereomeric mixture of 2 and 3 was achieved

Full text of DOI:10.1016/S0040-4020(01)00323-4

TETRAHEDRON
Pergamon
Tetrahedron 57 +2001) 4311±4321
First total synthesis of Mer-N5075A and a diastereomeric
mixture of a and b-MAPI, new HIV-I protease inhibitors
from a species of Streptomyces
Yaeko Konda,a,p Yukihiro Takahashi,a Shiho Arima,a Noriko Sato,a Kazuyoshi Takeda,a
Kazuyuki Dobashi,b Masanori Babac and Yoshihiro Harigayaa
aSchool of Pharmaceutical Sciences, Kitasato University, 9-1 Shirokane 5 chome, Minato-ku, Tokyo 108-8641, Japan
bMercian Corporation, Central Research Laboratories, Bioresources Laboratory, Jounann 4-chome, Fjisawa-shi, Japan
cDivision of Human Retroviruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University,
Kagoshima 890-8520, Japan
Received 15 October 1999; accepted 12 March 2001
AbstractÐMer-N5075A +1) and a-MAPI and b-MAPI +2 and 3) produced from a species of Streptomyces are new anomalous tetrapeptides
having potential HIV-I protease inhibitory activity. The ®rst total synthesis of 1 and a diastereomeric mixture of 2 and 3 was achieved simply
by a route connecting two dipeptides. The synthetic method is applicable for synthesis of Mer-N5075A analogues, such as GE20372 A and B
+4 and 5) and other chemically modi®ed compounds. In addition, the inhibition of HIV-1Protease and anti HIV activity by the compounds 1,
a mixture of 2 and 3, and 24, 25, and 26 were described. q 2001Elsevier Science Ltd. All rights reserved.
1. Introduction
Stefanelli et al. isolated the novel HIV-I protease inhibitors,
GE20372 A +4) and B +5),5 which are structurally related to
1, 2, and 3. These tetrapeptides +1±5) show the structural
characteristics of a carboxyl group in one terminal, and an
alcohol or aldehyde group in the other terminal. They also
have a characteristic carbonyl linked between two amino
acid which form ureido. In spite of their interesting
Recently, development of therapeutic approaches for AIDs
has progressed rapidly. In particular, the combined use of
two or more drugs having different actions, such as
reverse transcriptase inhibitors1 and HIV +Human Immuno-
de®ciency Virus) protease inhibitors,2 has been shown to be
highly effective in the treatment of AIDs. The development
of HIV protease inhibitors is comparatively slow relative to
that of reverse transcriptase inhibitors, though several
peptide-based protease inhibitors have been synthesized
and shown to be highly effective in AIDS therapy,
which are exempli®ed by Indinavir2a and Saquinavir.2b
Hence, discovery of a new HIV protease inhibitor is
anticipated.
Mer-N5075A +1) is an anomalous tetrapeptide having potent
human immunode®ciency virus type I +HIV-I) protease
inhibition. It is isolated from Streptomyces chromofuscus
Mer-N5075, which was collected in Okinawa Prefecture,
Japan.3 Mer-N5075A belongs to the MAPI group4 of
compounds 2 and 3 having microbial alkaline protease
inhibition isolated from Streptomyces nigrescens. Recently,
Keywords: HIV-I protease inhibitor; syntheses; tetrapeptide; Streptomyces
species; ureido moiety.
p
Corresponding author. Tel.: 13-3444-4742; fax: 13-3444-4742;
e-mail: konday@platinum.pharm.kitasato-u.ac.jp
Figure 1. Mer-N5075A analogies having HIV-1protease inhibitory activity.
0040±4020/01/$ - see front matter q 2001Elsevier Science Ltd. All rights reserved.
PII: S0040-4020+01)00323-4

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Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
Scheme 1. Synthetic strategy for Mer-N5075A.
structural features and biological activity, their total
syntheses have not been reported. As a part of our synthetic
and biological studies on naturally occurring compounds
which inhibit HIV-protease, we focused our attention on 1
and its analogues. HIV-I protease inhibition of a-MAPI +2)
is reportedly ten times that of Mer-N5075A3 +1). The
terminal hydroxymethyl group of 1 is thought to be
converted to a compound having more potent activity,
such as a-MAPI and other analogous compounds, thus
leading to the development of effective therapeutic agents
for the treatment of AIDs. This background prompted us to
synthesize 1 and its analogues 2 and 3.
2. Results and discussion
Compound 1 consists of three ordinal amino acids, l-phenyl-
alanine +Phe), l-arginine +Arg), l-valine +Val) and +S)-
phenylalaninol +Pheol). Our synthetic strategy for 1 was to
connect two dipeptides 16 as a left-hand segment and 22 as a
right-hand segment composed of protected Arg and Phe, and
of Val and Pheol, respectively. This yields protected tetra-
peptide 24. We then remove the protective groups at the
same time to provide 1. The introduction of the ureido
group is an interesting key reaction for the synthesis of 1.
Several methods have been previously investigated and
reported7 concerning the introduction of a ureido group.
We effectively used N,N0-disuccinimidylcarbonate +DSC),8
which was developed by us as an activating reagent to intro-
duce the ureido group +Scheme 1).
Recently, we reported the total synthesis of 1 in a prelimi-
nary study.6 In this paper, we describe in detail the ®rst
synthesis of 1 and a and b-MAPI +2 and 3) +Fig. 1).
Scheme 2. Synthesis of active carbamate ester +9).

Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
4313
3. Synthesis of Mer-N5075A
Boc-ON in the presence of triethylamine afforded alcohol
18 in 67% yield. The alcohol 18 was etheri®ed with benzyl
bromide and sodium hydride in 90% yield. The Boc group
of 19 was removed by p-TsOH and then passed through
anion exchange resin to afford amine 20 in 90% yield.
The amine 20 was coupled with Boc-l-valine in the
presence of EDCI to give protected dipeptide 21 in 78%
yield. The amine 20 was alternatively synthesized from 17
in only one step as follows. The primary hydroxyl group of
17 was selectively etheri®ed by benzyl bromide and sodium
hydride to give the amine 20 quantitatively, which was
suf®ciently pure and thus used without further puri®cation.
Deprotection of the Boc group of 21 by treatment
with p-TsOH afforded the dipeptide salt 22 in 65% yield
+Scheme 4).
Dipeptide 16 was synthesized as follows. Compound 6 was
esteri®ed by treatment with di-tert-butyl dicarbonate9 in the
presence of DMAP +dimethylaminopyridine) in tert-BuOH,
thereby producing a tert-butyl ester 7 in 92% yield, followed
by the selective deprotection of the Boc group of 7 in the
presence of a tert-butyl ester according to the procedure
described by Goodacre et al.10 Thus, treatment of 7 with
p-toluenesulfonic acid +p-TsOH) gave the p-TsOH salt.
The salt was then passed through anion-exchange resin
+Amberlyst A-21) giving amine 8 in 92% yield from 7.
This was converted to the activated ester 9 by treatment
with DSC in 86% yield +Scheme 2).
The primary amino group of Boc-l-arginine +10) was
protected by the 4-methoxybenzenesulfonyl +Mbs) group
using Mbs chloride followed by puri®cation as a cyclo-
hexylamine salt; then, neutralization by citric acid afforded
the acid 11 in total yield of 89% from 10. Esteri®cation of 11
by benzyl alcohol in the presence of 1-ethyl-3-[3- +dimethyl-
amino)propyl] carbodiimide +EDCI) and DMAP gave the
ester 12 in 61% yield. Deprotection of the Boc group of
12 with p-TsOH gave the salt 13 in 99% yield. The salt
13 was then neutralized with N-methylmorpholine
+NMM)11 to give amine 14 and then connected with the
active ester 9 to afford the dipeptide 15 in total yield of
70% from 13. Debenzylation of 15 by catalytic hydrogena-
tion with Pd-C in ethanol gave the acid 16 in 99% yield
+Scheme 3).
The salt 22 was neutralized with NMM to afford dipeptide
amine 23 quantitatively, which was condensed with the
dipeptide 16 in the presence of EDCI, hydroxybenzotriazole
+HOBt), and NMM to afford successfully the protected
tetrapeptide 24 in 90% yield from 22. The Mbs, tert-butyl,
and benzyl groups in 24 were simultaneously removed by
treatment with methanesulfonic acid12 and passed through
Amberlite IRA-410 to remove the acid, then puri®ed by
Sephadex LH-20 to afford Mer-N5075 A +1) in 60% yield.
1
The H and 13C NMR spectra of synthetic Mer-N5075A
were almost identical to those of the natural compound
1
described in the literature.3 Morever, the H and 13C NMR
spectra of synthetic Mer-N5075A perfectly coincided with
the spectra of the natural compound supplied by Mercian
Co., Ltd. Compound 1 showed the optical rotation value of
27
[a]D 224.48 +cˆ0.32, AcOH), which is close to the
Dipeptide 22 was synthesized as follows. Protection of
the amino group in phenylalaninol +17) by treatment with
reported value +[a]D28 227.68, cˆ0.11, AcOH) +Scheme 5).
Scheme 3. Synthesis of left-hand segment +16).

4314
Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
Scheme 4. Synthesis of right-hand segment +22).
4. Synthesis of the mixture of a and b-MAPI
the t-butyl and Mbs groups was performed by treatment with
methanesulfonic acid and passage through Amberlite IRA-
410 and Sephadex LH-20, which is the same method used
for 1. This gave the diastereomeric mixture of a and
b-MAPI +2 and 3) in 46% yield. These results show that
after the formation of a-MAPI +2), enolization of the
terminal aldehyde occurred under these acidic conditions
and 2 was epimerized to b-MAPI +3). We are planning to
analytically separate these diastereomers by HPLC or
preparative TLC to gain 2 and 3 as a single product. Further-
more, we will chemically synthesize 2 by converting MerN-
5075A +1) directly to 2 by Swern oxidation or Dessmartin
oxidation shown in Scheme 6.
In order to synthesize a-MAPI +2), debenzylation of the
tetrapeptide 24 was examined. Debenzylation did not
occur under either the conditions of catalytic hydrogenation
over Pd-C in EtOH, or those of hydrogen transfer using Pd-
black and Pd-C in EtOH. However, debenzylation of 24 by
catalytic hydrogenation with Pd-C in AcOH afforded the
alcohol 25 successfully in 92% yield. Oxidation of the
primary hydroxyl group of 25 by treatment with PDC in
the presence of molecular sieves and celite gave the alde-
hyde 26 in 60% yield as a single product which was
1
con®rmed from H and 13C-NMR spectra. Deprotection of
Scheme 5. Synthesis of 1.

Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
4315
Scheme 6. Synthesis of 2 and 3.
5. The inhibition of the HIV-1 protease and anti HIV
activity of 1, a mixture of 2 and 3 and related compounds
interesting to note that the inhibitory activity of compound
25, which has an alcohol group is superior to that of
compound 26, which has an aldehyde group at the terminal
position. The details of the experimental procedures will be
reported in Biol. Pharm. Bull in which the results of the
screening of HIV-1protease inhibition of nine synthetic
compounds related to Mer-N5075A, a and b-MAPI, and
GE20372-A will be shown. We will also discuss the struc-
tural biological activity relationship in future studies.
Measurement of the inhibitory activity of HIV-1protease
was carried out by our co-workers at Mercian Co., Ltd. in
the same manner as used in the case of Mer-N5075A.3 The
enzyme reaction was achieved using a substrate solution,
which was monitored by HPLC as described in the Experi-
mental Section. The IC50 values against the HIV-1protease
of compound 1, a mixture of 2 and 3, 24, 25, and 26 are
summarized in Table 1. Mer-N5075A and a mixture of
a-MAPI and b-MAPI +2 and 3) showed the HIV-1protease
inhibitory activity, although the values shown were rela-
tively low when compared with those in the literature
+lit.3 IC50 values +mM): Mer-N5075A 17.8; a-MAPI 1.3;
b-MAPI 18.3). One past study showed3 that the aldehyde
function at the terminal is more attributable to HIV-1
protease inhibitory action than the alcoholic function. It is
In 1993, Kaneto et al. reported that no anti HIV-1 activity
was detected when natural products 1 and 2 were subjected
to an in vitro system using MT-4 cell and HIV-1. We are
particulary interested in our anti HIV-1activity in vitro
system. Thus, we subjected synthetic compounds 1, a
mixture of 2 and 3, and 24 and 26 to an in vitro system
using MT-4 cell and HIV-1developed by our collaborator,
Dr. Baba. The system uses as control reagents nel®navir,
an HIV-1porotease inhibitor, and lamiudine, a reverse
transcriptase. The 50% effective concentration +EC50) and
50% cytotoxicity concentration +CC50) values are sum-
marized in Table 2.
Table 1. The HIV-1protease inhibitory activity of 1, a mixture of 2 and 3,
24, 25, 26
Compound
IC50 +mM)
Although compound 1, 2 and 3 showed HIV-1protease
inhibitory action, they did not show anti HIV-1activity,
as shown in Table 2. We believe the above result is due to
the fact that the samples are so hydrophilic that they are
miscible with the CD4 cell membrane, and thus they were
1
a mixture of 2 and 3
24
25
26
64.3
8.95
.100
68.2
.100

4316
Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
Table 2. The anti HIV activity of 1, a mixture of 2 and 3, 24, 26 in vitro
system using MT-4 cell and HIV-1
+n-hexane: AcOEtˆ2:1) to give 7 +1.11 g, 91.6%) as yellow
oil. Rfˆ0.53 +hexane:AcOEtˆ2:1). [a]D24132.048 +cˆ
1.08, CHCl3). IR +CHCl3) ymax cm21: 1490 +arom), 1700
+-NHCOO-), 1720 +COO-), 3430 +-NH-)HRFABMS m/z:
322.2017[M1H]1, Calcd for C18H28NO4: 322.2018. Anal.
Calcd for C18H28NO4: C, 67.26; H, 8.47; N,4.36. Found: C,
Compound
EC50 +mM)
CC50 +mM)
1
24
.100
.100
.4141
,1.7
,0.032
0.35
.100
.100
a mixture of 2 and 3
26
nel®navir
1
67.12; H, 8.39; N, 4.58. H NMR +300 MHz, CDCl3) d:
1.7
6.8
.20
1.39, 1.42 +each 9H, s, t-Bu, Boc), 3.05 +2H, d, Jˆ6.0,
Phe3-H2), 4.45 +1H, dt, Jˆ7.0, 6.0, Phe2-H), 4.95 +1H,
brd, Jˆ7.0, Phe2-NH), 7.12±7.35 +5H, m, Phe-arom.-H).
lamivudine
unable to invade the cell. Alternatively, their HIV-1
protease inhibitory activity is so weak that they do not effec-
tively inhibit anti HIV-1activity. It is noteworthy that a
mixture of 2 and 3, and 26 showed cytotoxicity. Details of
the experimental procedure for measuring the bioactivities
will be reported in Biol. Pharm. Bull in the near future
+Tables 1and 2).
7.1.2. l-Phenylalanine t-butyl ester 38). p-Toluenesulfonic
acid +2.06 g, 12.0 mmol) in EtOH +10 ml) was added to a
solution of 7 +3.85 g, 12.0 mmol) in ethyl ether +3 ml) and
stirred for 30 min at 08C. The reaction was continued for
further 24 h at room temperature. The mixture was concen-
trated in vacuo to give the p-toluenesulfonic acid salt
+4.59 g). mp 1348C +EtOH). Rfˆ0.50 +hexane/AcOEtˆ
2:1). [a]D26137.698 +cˆ1.04, CHCl3). IR +CHCl3)
y
max cm21: 1735 +-COO-), 2800±3300 +-SO3H), 3450
+-NH2). HRFABMS m/z: 222.1514 [M1H]1, Calcd for
6. Conclusion
1
C13H20NO2: 222.1494. H NMR +300 MHz, CDCl3) d: 1.42
+9H, s, t-Bu), 2.84 +1H, dd, Jˆ13.0, 8.0, Phe3-Ha), 3.04
+1H, dd, Jˆ13.0, 6.0, Phe3-Hb), 3.60 +1H, dd, Jˆ8.0, 6.0,
Phe2-H), 7.17±7.34 +5H, m, Phe-arom.-5H). Ethanolic solu-
tion +15 ml) of the salt +4.59 g, 11.7 mmol) was passed
through a column of anion exchange resin +Amberlist
A-21, 100 g, EtOH) to give 8 +2.43 g, 91.5% from 7).
Rfˆ0.13 +hexane:AcOEtˆ2:1). [a]D27110.228 +cˆ0.90,
CHCl3). IR +CHCl3) ymaxcm21: 1720 +-COO-), 3400
+-NH2). HRFABMS m/z: 222.1514 [M1H]1, Calcd for
The ®rst synthesis of a novel HIV-I protease inhibitor, Mer-
N5075 A and a mixture of a-MAPI and b-MAPI was
achieved. Each reaction involved in this procedure is
employable in a large scale preparation and applicable to
the synthesis of its analogues. Furthermore, Mer-N5075A,
its related compound 25, and a mixture of a and b-MAPI
showed the HIV-1protease inhibitory activity. We are
currently investigating synthesis of 4 and 5 and other
analogues to obtain more potent HIV-I protease inhibitors.
Notably, in the present study, a mixture of 2 and 3, 26 having
an aldehyde group at the terminal showed cytotoxicity.
1
C13H20NO2.: 222.1494. H NMR +300 MHz, CDCl3) dH:
1.42 +9H, s, t-butyl-H), 2.84 +1H, dd, Jˆ13.0, 8.0,
HRFABMS m/z: 222.1514 [M1H]1, Calcd for
C13H20NO2.: 222.1494. H NMR +300 MHz, CDCl3) d:
1
1.42 +9H, s, t-Bu), 2.84 +1H, dd, Jˆ13.0, 8.0, Phe3-Ha),
3.04 +1H, dd, Jˆ13.0, 6.0, Phe3-Hb), 3.60 +1H, dd, Jˆ8.0,
6.0, Phe2-H), 7.17±7.34 +5H, m, Phe-arom.-H).
7. Experimental
7.1. General
7.1.3. l-Phenylalanine t-butyl ester N-hydroxysuccin-
imide carboxamide 39). Compound
Melting points were taken on a Yanagimoto hot-stage and
1
are uncorrected. H and 13C NMR were recorded on a
Varian VXR-300, XL-400 spectrometers. The signals
8
+61.2 mg,
0.28 mmol) in acetonitrile +1ml) was added to a stirred
solution of N, N0-disuccinimidyl carbonate +70.0 mg,
0.27 mmol) in acetonitrile +4 ml) at room temperature
under argon. After 2.5 h, the mixture was concentrated in
vacuo. The residue was diluted with AcOEt +30 ml). The
organic layer was washed with 10% citric acid +7 ml£2),
saturated NaCl +5 ml£2), dried over Na2SO4, concentrated
in vacuo to give 9 as a crude product. +85.9 mg, 85.9%).
Rfˆ0.42 +hexane:AcOEtˆ1:3). mp 1268C +AcOEt).
Rfˆ0.42 +hexane/AcOEtˆ1:3). [a]D28188.798 +cˆ1.07,
CHCl3). IR +CHCl3) ymax cm21: 1730 +-COO-), 1780 +cyclic
imide), 3400 +-NH-). HRFABMS m/z: 385.1390 [M1Na]1,
Calcd for C18H22N2O6Na: 385.1376. Anal. Calcd for
C18H22N2O6: C, 59.66; H, 6.12; N,7.73. Found: C, 59.57;
H, 7.68; N, 7.68. 1H NMR +300 MHz, CDCl3) d: 1.41+9H, s,
t-Bu), 2.82 +4H, brs, NSu-CH2£2), 3.15 +2H, d, Jˆ5.5,
Phe3-H2), 4.46 +1H, dt, Jˆ7.0, 5.5, Phe2-H), 5.82 +1H, d,
Jˆ7.0, Phe2-NH), 7.15±7.35 +5H, m, Phe-arom.-5H).
1
were assigned by H-1H COSY, DEPT, HMQC, HMBC
experiments. Mass spectra were obtained on a JEOL-
JMX-DX 300 mass spectrometer +low resolution mass
spectrometry) and JEOL-JMS-AX505 HA mass spectro-
meter +high resolution mass spectrometry). Routine
monitoring of reactions was carried out using Merck 60
GF254 silica gel, glass-supported TLC plates. Flash column
chromatography was performed on silica gel 60 H +Merck).
Thin-layer chromatography +TLC) was done on silica gel 60
PF254 +Merck). Ethyl ether, tetrahydrofuran +THF) and
dichloromethane +CH2Cl2) was each distilled from sodium,
potassium hydride. Dimethylformaldehyde +DMF) was
dried by molecular sieves 3A.
7.1.1. N-t-Boc-l-phenylalanine t-butyl ester 37). Di-tert-
butyldicarbonate +1.65 g, 3.54 mmol), dimethylaminopyri-
dine +138 mg, 1.13 mmol) were added to a stirred solution
of N-t-boc-l-phenylalanine +6) +1.00 g, 3.77 mmol) in tert-
buthanol +8 ml) at room temperature under argon. After
30 min, the mixture was concentrated in vacuo and the
residue was puri®ed by ¯ash column chromatography
7.1.4. N-Boc-NG-methoxybenzenesulfonyl-l-arginine 311)
cyclohexylamine salt. 4-Methoxybenzenesulfonyl chloride
+1.7 g, 8.0 mmol) was added to a stirred solution of

Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
4317
N-Boc-l-arginine 10 +1.0 g, 3.2 mmol) in 80% aqueous ace-
tone +20 ml) at 08C maintained at pHˆ11.0±11.5 with 4N
NaOH through the addition. After 3 h, 4-methoxybenzene-
sulfonyl chloride +664.9 mg, 3.2 mmol) was again added.
After 18 h, the mixture was neutralized with 10% aqueous
citric acid and acetone was removed in vacuo. The aqueous
residue was washed with ether +6 ml£3), acidi®ed at
pHˆ3.0 with 10% aqueous citric acid, saturate with NaCl,
extracted with AcOEt +7 ml£3). The organic layer was
washed with 10% citric acid +7 ml), saturated NaCl
+6 ml£3), dried over Na2SO4, evaporated in vacuo to give
light yellow oil +1.7 g). To the oil in MeOH +3 ml), cyclo-
hexylamine +371mg, 3.7 mmol) was added, evaporated in
vacuo to give the 11 cyclohexylamine salt, quantitatively as
light yellow crystals. mp 508C +crude). HRFABMS m/z:
Arg2-NH), 6.27 +3H, brs, guanidine-NH£3), 6.88 +2H, d,
Jˆ9.0, Mbs-arom.-Ha), 7.28-7.40 +5H, m, benzyl), 7.76
+2H, d, Jˆ9.0, Mbs-arom.-Hb).
7.1.7. NG-methoxybenzenesulfonyl-l-arginine benzyl
ester p-toluenesulfonic acid salt 313). p-Toluenesulfonic
acid +1.0 g, 5.6 mmol) in THF +10 ml) was added to a
solution of 12 +1.0 g, 1.9 mmol)in THF +25 ml). After the
solution was stirred for 30 min at 08C, then stirred for
further 17 h at room temperature. The mixture was evapo-
rated in vacuo to give the salt 13 +1.82 g, 99%) as colorless
crystals. Rfˆ0.69 +hexane:AcOEtˆ1:3). [a]D2423.808
+cˆ1.00, CHCl3). IR +CHCl3) ymax cm21: 1500, 1570,
1590 +arom), 1680 +-CˆNH), 1740 +-COO-), 2800±3300
+-SO3H), 3400 +-NH2). HRFABMS m/z: 435.1735
[M1H]1, Calcd for C20H27N4O5S: 435.1702. 1H NMR
+300 MHz, CDCl3) d: 1.70 +2H, br, Arg4-H2), 1.95 +2H,
br, Arg3-H2), 2.32 +9H, s, p-TsOH-CH3£ 3), 3.18 +2H, br,
Arg5-H2), 3.77 +3H, s, Mbs-OCH3), 4.26 +1H, m, Arg2-H),
5.08, 5.10 +each 1H, d, Jˆ12.0, benzyl-CH2), 5.90 +6H, br,
NH31, guanidine-NH£3), 6.85 +2H, d, Jˆ9.0, Mbs-arom.-
H2), 7.16 +6H, d, Jˆ7.0, p-TsOH-arom.-Ha£3), 7.24 +5H,
m, benzyl-arom.-H), 7.68 +6H, d, Jˆ7.0, p-TsOH-arom-£3),
7.75 +2H, d, Jˆ9.0, Mbs-arom.-H).
1
445.1757 [M1H]1, Calcd for C18H29N4O7S: 445.1754. H
NMR +300 MHz, CDCl3) d: 1.00±1.60 +11H, m, CHA-
11H), 1.41 +9H, s, t-Bu), 1.72 +2H, brd, Jˆ10.0, Arg4-H2),
1.99 +2H, brd, Jˆ10.0, Arg3-H2), 2.95 +1H, br, Arg5-Ha),
3.16 +1H, br, Arg5-Hb), 3.81 +3H, s, Mbs-OCH3), 3.89 +1H,
br, Arg2-H), 5.00±6.20 +3H, br, CHA-NH31), 5.74 +1H, d,
Jˆ6.0, Arg2-NH), 6.89 +2H, d, Jˆ9.0, Mbs-arom.-Ha), 7.76
+2H, d, Jˆ9.0, Mbs-arom.-Hb).
7.1.5. N-t-Boc-NG-methoxybenzenesulfonyl-l-arginine
311). After the solution of 11 cyclohexylamine salt
described above in AcOEt +40 ml) was acidi®ed with 10%
citric acid +10 ml), the aqueous layer was extracted with
AcOEt +10 ml£2). The organic layer was combined, washed
with saturated NaCl, dried over Na2SO4, evaporated in
vacuo to give 11 as light yellow crystals +1.8 g, 89% from
10). Rfˆ0.22 +CHCl3/MeOHˆ3:1). [a]D2311.928 +cˆ0.52,
MeOH). HRFABMS m/z: 445.1765 [M1H]1, Calcd
7.1.8. Dipeptide 15. A solution of the salt 13 +1.82 g,
1.87 mmol) in CH2Cl2 +25 ml) was neutralized with
N-methylmorpholine +568.3 mg, 5.6 mmol) and then,
carboxamide 9 +814.8 mg, 2.2 mmol) in CH2Cl2 +15 ml)
was added to the reaction mixture under argon and stirred
for 21h at room temperature. A solution of 9 +339.5 mg,
0.9 mmol)was added to the reaction mixture and stirred
further 3 h at room temperature. The mixture was diluted
in CHCl3 +500 ml). The organic layer was washed with
saturated NaHCO3 +20 ml£2), 10% citric acid +20 ml£2),
saturated NaCl +20 ml£2), dried over Na2SO4 and concen-
trated in vacuo. The residue was puri®ed by preparative
TLC +SiO2, CHCl3: MeOHˆ10:1) to give 15 +889.5 mg
70% from 13) as yellow crystals. mp 598C. Rfˆ0.59
+CHCl3: MeOHˆ10: 1). [a]D2819.728 +cˆ1.07, CHCl3).
IR +CHCl3) ymax cm21: 1500 +arom), 1680 +-NHCONH-),
1720 +-COO-), 3430 +-NH-).HRFABMS m/z: 682.2921-
[M1H]1, Calcd for C34H44O8N5S: 682.2911. Anal. Calcd
for C34H43N5O8S: C, 59.90; H, 6.36; N,10.27. Found: C,
1
for C18H29N4O7S: 445.1754. H NMR +300 MHz, CDCl3)
d: 1.41 +9H, s, t-Bu), 1.59 +2H, br, Arg4-H2), 1.69 +1H,
m, Arg3a-H), 1.81 +1H, m, Arg3b-H), 3.19 +2H, brs,
Arg5-H2), 3.81+3H, s, Mbs-OCH 3), 4.21H+1, br,
Arg1-H), 5.66 +1H, d, Jˆ8.0, Arg2-NH), 6.26 +1H, br,
guanidine-NH), 6.52 +2H, br, guanidine-NH£2), 6.90
+2H, d, Jˆ9.0, Mbs-arom.-Ha), 7.76 +2H, d, Jˆ9.0,
Mbs-arom.-Hb).
7.1.6. N-t-Boc-NG-methoxybenzenesulfonyl-l-arginine
benzyl ester 312). Benzyl alcohol +77.8 mg, 0.72 mmol),
EDCI +207 mg, 1.08 mmol), DMAP +7.3 mg, 0.06 mmol)
were added to a solution of 11 +1.33 mg, 0.30 mmol) in
THF +4 ml) and stirred for 24 h at room temperature under
argon. The mixture was diluted with AcOEt +25 ml). The
organic layer was washed with saturated NaCl +15 ml£2),
saturated NaHCO3 +15 ml£2), saturated aqueous NaCl
+15 ml£2) and then, dried over Na2SO4 and evaporated in
vacuo. The residue was puri®ed by preparative TLC +SiO2,
hexane: AcOEtˆ1:3) to give 12 as light yellow crystals
+98.1mg, 61%). mp 48±52 8C. Rfˆ0.28 +hexane:AcOEtˆ
1
59.80; H, 6.48; N, 9.98. H NMR +400 MHz, pyridine) d:
1.35 +9H, s, t-Bu), 1.54 +1H, m, Arg4-Ha), 1.70 +1H, m,
Arg4-Hb), 1.90 +2H, m, Arg3-H2), 2.88 +1H, dd, Jˆ13.0,
6.5, Phe3-Ha), 2.93 +1H, dd, Jˆ13.0, 6.5, Phe3-Hb), 3.13
+2H, br, Arg5-H2), 3.80 +3H, s, Mbs-OCH3), 4.31+1H, br,
Arg2-H), 4.51+H1 , dt,
Jˆ6.5, 6.0, Phe2-H), 5.05, 5.12
+each, 1H, d, Jˆ12.0, Bn-CH2), 5.86 +1H, br, Phe2-NH),
5.92 +1H, br, Arg2-NH), 6.86 +2H, d, Jˆ9.0, Mbs-arom.-
H2), 7.13±7.40 +10H, m, phenyl, benzyl arom.-H), 7.77 +2H,
d, Jˆ9.0, Mbs-arom.-H2).
1:3). [a]D2527.898 +cˆ0.71, CHCl3). IR +CHCl3) ymaxcm21
:
1500, 1560, 1590 +arom), 1620 +-NHCOO-), 1680
+-CˆNH), 1740 +-COO-), 3350 +-CˆNH), 3450 +-NH-).
HRFABMS m/z: 535.2260 [M1H]1, Calcd for
C25H35N4O7S: 535.2226. 1H NMR +300 MHz, CDCl3)d:
1.41 +9H, s, t-Bu), 1.57 +2H, m, Arg4-H2), 1.72 +2H, m,
Arg3-H2), 3.14 +1H, m, Arg5-Ha), 3.25 +1H, m, Arg5-Hb),
3.82 +3H, s, Mbs-OCH3), 4.26 +1H, m, Arg2-H), 5.12, 5.16
+each 1H, d, Jˆ12.0, benzyl-CH2), 5.31+1H, d, Jˆ8.0,
7.1.9. Debenzylated dipetide 16. 10% Palladium carbon
+1.09 g) was added to the solution of the dipeptide 15
+2.01g, 2.93 mmol) in acetic acid +30 ml), and mixture
was stirred under hydrogen gas for 2.5 h at room tempera-
ture and then, ®ltered. The ®ltrate was evaporated in vacuo
to give the acid 16 +1.74 g, 99%) as a light yellow oil.
Rfˆ0.07 +CHCl3: MeOHˆ5: 1). mp 79±888C +MeOH).
[a]D2819.368 +cˆ1.09, CHCl3). IR +CHCl3) ymax cm21
:

4318
Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
1500, 1570, 1600 +arom), 1630 +-NHCONH-), 1730
+-COO-), 3350 +-OH). HRFABMS m/z: 592.2436
[M1H]1, Calcd for C27H38N5O8S: 592.2441. 1H NMR
+300 MHz, CDCl3) d: 1.28 +9H, s, t-Bu), 1.51 +2H, m,
Arg4-H2), 1.58 +1H, m, Arg4-Hb), 1.90 +2H, m, Arg3-H2),
2.88 +1H, dd, Jˆ13.0, 6.5, Phe3-Ha), 2.93 +1H, dd, Jˆ13.0,
6.5, Phe3-Hb), 3.13 +2H, br, Arg5-H2), 3.80 +3H, s,
p-toluenesulfonic salt of 20 +118 mg, 95.3%) as colour-
less crystals.
The salt of 20 +118 mg, 0.30 mmol) in EtOH +5 ml) was
passed through a column of anion exchange resin
+Amberlist A-21) to give the amine 20 +65 mg, 90%
from 19) as colourless oil. Rfˆ0.50 +CHCl3: MeOHˆ
10:1). mp 110±1178C +EtOH). [a]D23129.078 +cˆ0.97,
CHCl3). IR +CHCl3) ymax cm21: 1600 +arom), 2800±3200
+-SO3H). HRFABMS m/z: 242.1566 [M1H]1, Calcd for
Mbs-OCH3), 4.31+1H, br, Arg2-H), 4.51+1H, dt,
Jˆ6.5,
6.0, Phe2-H), 5.05, 5.12 +each 1H, d, Jˆ12.0, Bn-CH2), 5.12
+1H, d, Jˆ12.0, Bn-CH2), 5.86 +1H, br, Phe2-NH), 5.92 +1H,
br, Arg2-NH), 6.86 +2H, d, Jˆ9.0, Mbs-arom.-H2), 7.13±
7.40 +10H, m, phenyl, benzyl arom.-H), 7.77 +2H, d, Jˆ9.0,
Mbs-arom.-H2).
1
C16H20NO: 242.1545. H NMR +300 MHz, CDCl3) d:
2.00 +2H, s, PheOH-NH2), 2.61+1H, dd, Jˆ13.5, 7.7,
PheOH 3-Ha), 2.82 +1H, dd, Jˆ13.5, 5.3, PheOH
3-Hb), 3.29 +1H, m, PheOH 2-H), 3.36 +1H, dd, Jˆ8.7,
7.0, PheOH 1-Ha), 3.50 +1H, dd, Jˆ8.7, 3.5, PheOH
1-Hb), 4.52 +1H, d, Jˆ12.0, benzyl-CH2), 4.55 +1H, d,
Jˆ12.0, phenyl-CH2), 7.15±7.40 +10H, m, phenyl,
benzyl-arom.-H).
7.1.10. N-3t-Butoxycarbonyl)-l-phenylalaninol 318). A
solution of Boc-ON +1.6 g, 6.6 mmol) in dioxane +3 ml)
was added to a mixture of l-phenylalaninol +17) +1 g,
6.6 mmol), triethylamine +1ml) and water +3 ml). The
mixture was stirred at room temperature for 3.5 h, then
concentrated in vacuo. The residue was puri®ed by prepara-
tive TLC +Silica gel, n-hexane: AcOEtˆ2:1) to give 18
+1.11 g, 66.9%) as colourless crystals. mp 93±958C.
Rfˆ0.47 +CHCl3: MeOHˆ10:1). [a]D26219.158C +cˆ1.07,
CHCl3). IR +CHCl3) ymax cm21: 1500 +arom), 1700
+-NHCOO-), 3450 +-NH-), 3650 +-OH)HRFABMS m/z:
2. NaH +1.59 g, 39.7 mmol) was added to a solution of
l-phenylalaninol +17) +3.00 g, 19.8 mmol) in DMF
+40 ml) at 08C and stirred for 2 h. After the evolution
of hydrogen gas deceased, benzyl bromide +2.60 ml,
21.8 mmol) was added at room temperature and stirred
1.5 h. DMF was removed in vacuo and the residue was
diluted with water +150 ml), extracted with CHCl3
+300 ml£3). The combined organic layer was washed
with saturated NaCl +150 ml), dried over Na2SO4,
concentrated in vacuo to give the crude product 20,
quantitatively +4.80 g) which was pure enough and used
in the next reaction without puri®cation. Physico-chemi-
1
252.1605 [M1H]1, Calcd for C14H22NO3: 252.1600. H
NMR +300 MHz, CDCl3) d: 1.42 +9H, s, t-Bu), 1.86 +1H,
brs, PheOH1-OH), 2.84 +2H, d, Jˆ8.0, PheOH3-H2), 3.58
+1H, dd, Jˆ10.0, 5.0, PheOH1-Ha), 3.70 +1H, brd, Jˆ10.0,
PheOH1-Hb), 3.90 +1H, br, PheOH2-H), 4.87 +1H, br,
PheOH 2-NH), 7.18±7.32 +5H, m, arom.-H).
1
cal data +Rf, HRFABMS) and the H NMR spectrum
agreed with those of 20 obtained from 19.
7.1.11. N-3t-Butoxycarbonyl)-l-phenylalaninol benzyl
ether 319). 60% NaH +320 mg, 8.0 mmol) was added to a
stirred solution of 18 in DMF +20 ml) at 08C under argon.
After 30 min, benzyl bromide +752 mg, 4.40 mmol) was
added to the reaction mixture. The mixture was stirred for
further 1.5 h at room temperature and evaporated in vacuo.
The residue was poured into ice water, extracted with CHCl3
+50 ml£4). The organic layer was washed with saturated
NaCl +25 ml£3), dried over Na2SO4 and concentrated in
vacuo. Residue was puri®ed by preparative TLC +Silica
gel, CHCl3: MeOHˆ10: 1) to give 19 +1.22 g, 90.0%) as
colourless oil. Rfˆ0.77 +CHCl3: MeOHˆ10: 1). [a]D282
4.898 +cˆ0.45, CHCl3)IR +CHCl3) ymaxcm21: 1500 +arom),
1690 +-NHCOO-), 3450 +-NH-). HRFABMS m/z: 364.1907
7.1.13. N-3tert-Butoxycarbonyl)-l-valyl-l-phenylalaninol
benzyl ether 321). N-+tert-Butoxycarbonyl)-l-valine
+110 mg, 0.51 mmol) and EDCI +98 mg, 0.51mol) were
added to a solution of compound 20 in CH2Cl2 +6 ml) at
08C under argon, and the mixture was stirried for 1h at
08C, and then for 24 h at room temperature. The mixture
was diluted with CHCl3 +68 ml), washed with saturated
NaHCO3 +13 ml£3), saturated NaCl +13£3), 1M HCl
+12.5 ml£3), saturated NaCl +13 ml£3), dried over
Na2SO4, evaporated in vacuo. The residue was puri®ed by
preparative TLC +Silica gel, n-hexane: AcOEtˆ2:1) to give
21 +0.18 g, 78.0%) as light yellow crystalls. mp 110±1138C
+AcOEt). Rfˆ0.66 +CHCl3/MeOHˆ10:1). [a]D24236.508
+cˆ0.40, CHCl3). IR +CHCl3) ymax cm21: 1450, 1490
+arom), 1660 +-NHCO-), 1700 +-NHCOO-). HRFABMS
m/z: 441.2735 [M1H]1, Calcd for C26H37N2O4: 441.2753
[M1H]. Anal. Calcd for C26H36N2O4: C, 70.88; H, 8.24;
N,6.36. Found: C, 70.64; H, 8.28; N, 6.33. 1H NMR
+400 MHz, CDCl3) d: 0.84 +3H, brd, Jˆ6.0, Val4-H3),
0.91+3H, d, Jˆ7.0, Val40-H3), 1.44 +9H, s, t-Bu), 2.09
+1H, m, Val3-H), 2.87 +1H, dd, Jˆ14.0, 8.0, PheOH3-Ha),
2.90 +1H, dd, Jˆ14.0, 7.5, PheOH3-Hb), 3.37 +1H, dd,
Jˆ10.0, 3.5, PheOH1-Ha), 3.39 +1H, dd, Jˆ10.0, 3.5,
PheOH1-Hb), 3.86 +1H, dd, Jˆ8.0, 6.0, Val2-H), 4.30
+1H, m, PheOH2-H), 4.46, 4.50 +each 1H, d, Jˆ12.0, ben-
zyl-CH2), 5.00 +1H, Jˆ8.0, Val2-NH), 6.24 +1H, Jˆ9.0,
PheOH-NH), 7.15±7.40 +10H, m, phenyl, benzyl-arom.-H).
1
[M1Na]1, Calcd for C21H27NO3Na: 364.1889. H NMR
+300 MHz, CDCl3) d: 1.44 +9H, s, t-Bu), 2.90 +2H, m,
PheOH 3-H2), 3.40 +1H, dd, Jˆ9.0, 3.5, PheOH 1-Ha),
3.41+H1 , dd,
Jˆ9.0, 4.0, PheOH 1-Hb), 3.88 +1H, br,
PheOH 2-H), 4.44, 4.52 +each, 1H, d, Jˆ12.0, benzyl-
CH2), 4.52, +each 1H, d, Jˆ12.0, benzyl-CH2), 4.88 +1H,
br, PheOH 2-NH), 7.10±7.50 +10H, m, PheOH, benzyl-
arom-H).
7.1.12. l-Phenylalaninol benzyl ether 320).
1. p-Toluenesulfonic acid +50 mg, 0.30 mol) in EtOH
+0.3 ml) was dropped to a stirred solution of 19
+100 mg, 0.30 mmol) in ether +1.5 ml) during 1 h at
08C. After 30 min, the mixture was stirred for further
24 h at room temperature. The solvent was removed,
and the residue was crystallized with AcOEt to give
7.1.14. l-Valyl-l-phenylalaninol benzyl ether p-toluene-
surfonic acid salt 322). Anhydrous p-toluenesulfonic acid

Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
4319
+69 mg, 0.40 mmol) in EtOH +0.4 ml) was added to a stirred
solution of 21 +176 mg, 0.40 mmol) in ether +2.0 ml)
dropwise during 1h at 0 8C and stirred for 30 min. After
stirring for 24 h at room temperature, anhydrous p-toluene-
sulfonic acid +69 mg, 0.40 mmol) was again added to
the reaction mixture and stirred at 508C for 24 h. The
mixture was concentrated to give a crude product which
was recrystallized from AcOEt to give crude p-toluene-
sulfonic acid salt 22 +205 mg) which was recrystallized
from AcOEt to give colorless needles +133 mg, 65.0%).
mp. 177-1798C. Rfˆ0.47 +CHCl3: MeOHˆ10:1). [a]D252
20.68 +cˆ0.53, CHCl3). IR +CHCl3) ymax cm21: 1500, 1520,
1600 +arom), 1670 +-NHCO-), 2800±3200 +-SO3H), 3250
+-NH2). HRFABMS m/z: 341.2253[M1H]1, Calcd for
+t, Arg C-5), 51.24 +d, PheOH C-2), 53.86 +d, Arg C-2),
55.55 +q, Mbs-OCH3), 55.64 +d, Phe C-2), 59.40 +d, Val
C-2), 71.64 +t, PheOH C-1), 73.43 +t, benzyl-CH2), 81.45
+s, t-butyl-OC+CH3)3), 114.26 +d, Mbs-arom C-30, 50),
126.70, 127.06, 128.11 +each d, Phe-, PheOH-, benzyl-
arom. C-40), 128.43 +each d, benzyl-arom.- C-20, 60),
128.67, 128.85, 128.87, 129.48 +each d, Phe-, benzyl-,
PheOH-arom.- C30, 50), 128.72 +d, Mbs-arom.- C-20, 60),
129.91 +d, PheOH-arom.- C20, 60), 130.17 +d, Phe-arom.-
C20, 60), 137.74 +s, Phe-arom.- C-10), 138.30 +s, Mbs-arom
C-10), 139.02 +s, benzyl-arom C-10), 139.14 +s, PheOH-
arom C-10), 158.16 +s, Arg C-7), 158.67 +s, ureido-C),
162.15 +s, Mbs-arom.- C-40), 172.27 +s, Val C-1), 172.35
+s, Phe C-1), 173.46 +s, Arg C-1).
1
C21H29N2O2: 341.2229. H NMR +400 MHz, CDCl3) d:
0.81+3H, d, Jˆ7.0, Val4-H3), 0.89 +3H, d, Jˆ7.0, Val40-
H3), 2.08 +1H, sextet, Val3-H), 2.23 +3H, s, p-TsOH-CH3),
2.87 +1H, dd, Jˆ14.0, 8.0, PheOH3-Ha), 2.90 +1H, dd,
Jˆ14.0, 7.5, PheOH3-Hb), 3.37 +1H, dd, Jˆ10.0, 3.5,
PheOH1-Ha), 3.39 +1H, dd, Jˆ10.0, 3.5, PheOH1-Hb),
3.86 +1H, dd, Jˆ8.0, 6.0, Val2-H), 4.30 +1H, m, PheOH2-
H), 4.46 +1H, d, Jˆ12.0, Bn-CH2-H), 4.50 +1H, d, Jˆ12.0,
Bn-CH2-H), 5.00 +1H, Jˆ8.0, Val2-NH), 6.24 +1H, Jˆ9.0,
PheOH-NH), 7.15±7.40 +10H, m, phenyl, benzyl-arom.-H).
7.1.16. Mer-N5075A 31). Compound 24 +69.2 mg,
0.076 mmol) was dissolved in methanesulfonic acid
+0.38 ml, 5.83 mmol) and anisole +19 ml, 0.174 mmol) was
added and stirred for 3 h at room temperature. Ether
+6 ml£3) was added to the reaction mixture and decanted
to remove ether soluble portions to give the ether insoluble
portion as a light yellow oil +89.9 mg), which was puri®ed by
column chromatography +Amberlite IRA-410 acetate form,
H2O, Sephadex LH-20, MeOH) to provide 1 +27.0 mg, 60%)
as colourless granulars. Rfˆ0.15 +BuOH/AcOHˆ5/1, Silica
gel), 0.56 +MeOH/AcOHˆ100/1, Silica gel). mp 1828C.
HRFABMS: m/z: 598.3387 [M1H]1, Calcd for
7.1.15. Tetrapeptide 324). The solution of the salt 22
+685 mg, 1.31 mmol) in THF +12 ml) was neutralized with
N-methylmorphorine +278 mg, 2.75 mmol) at 08C under
argon to give a solution of amine 23; then a solution of 16
+774 mg, 1.31 mmol) in CH2Cl2 +10 ml), EDCI +276 mg,
1.44 mmol), HOBt +353 mg, 2.62 mmol) was added and
stirred for 1h under argon at 0 8C. After that the solution
was stirred further for 17 h at room temperature. The reac-
tion mixture was diluted with AcOEt +300 ml), washed with
saturated NaCl +15 ml£2), saturated NaHCO3 +15 ml), 10%
citric acid, dried over Na2SO4 and concentrated in vacuo to
give a light yellow oil +1.32 g), which was puri®ed by
preparative TLC +Silica gel, AcOEt) to give colourless
granules 24 +1.08 g, 90%) from 22.mp 878C +AcOEt).
Rfˆ0.46 +CHCl3/MeOHˆ10:1). [a]D21212.68 +cˆ1.03,
CHCl3). IR +CHCl3) ymax cm21: 1500, 1540, 1590 +arom),
1630 +-NHCO-), 1650 +-NHCONH-), 1710 +-COO-).
HRFABMS m/z: 914.4496 [M1H]1, Calcd for
C48H64N7O9S: 914.4486. Anal. Calcd for C48H63N7O9Sz1/
2H2O: C, 62.45; H, 6.99; N,10.62. Found: C, 62.62; H,
1
C30H44N7O6 598.3353. H NMR +400 MHz, DMSO-d6) d:
0.74 +3H, d, Jˆ7.0, Val3-CH3), 0.75 +3H, d, Jˆ6.5, Val3-
CH3), 1.42 +3H, m, Arg3-Ha, 4-H2), 1.65 +1H, br, Arg3-Hb),
1.88 +1H, m, Val3-H), 2.62 +1H, dd, Jˆ14.0, 8.0, PheOH3-
Ha). 2.62 +1H, dd, Jˆ14.0, 8.0, PheOH3-Ha), 2.82 +1H, 1H,
m, Phe3-Ha), 2.82 +1H, dd, Jˆ14.0, 5.5, PheOH3-Hb), 2.95
+2H, dd, Jˆ13.0, 5.5, Arg5-Ha, Phe3-Hb), 3.04 +1H, br,
Arg5-Hb), 3.24 +1H, dd, Jˆ10.0, 6.0, Phe-OH1-Ha), 3.31
+1H, dd, Jˆ10.0, 5.0, PheOH1-Hb), 3.88 +1H, m, PheOH2-
H), 3.97 +1H, m, Phe2-H), 4.04 +1H, dd, Jˆ9.0, 7.0, Val2-
H), 4.09 +1H, m, Arg2-H), 4.95 +1H, br, PheOH1-OH), 6.05
+1H, brd, Jˆ8.0, Phe2-NH), 6.68 +1H, brd, Jˆ8.0, Arg2-
NH), 7.05-7.28 +10H, m, Phe, PheOH-arom-H), 7.50-8.10
+3H, br, -C+ˆNH)-NH2), 7.73 +1H, br, Val2-NH), 7.82 +1H,
brd, Jˆ8.0, PheOH2-NH), 7.82 +1H, brd, Jˆ8.0, PheOH2-
NH), 8.90 +1H, br, Arg6-NH). 13C-NMR +100.6 MHz,
DMSO-d5) dc: 18.89, 19.89 +each q, Val3-CH3£2), 24.77
+t, Arg C-4), 29.17 +t, Arg C-3), 30.56 +d, Val C-3), 36.32
+t, PheOH C-3), 38.36 +t, Phe C-3), 40.20 +t, Arg C-5), 52.31
+d, PheOH C-2), 52.73 +d, Arg C-2), 56.60 +d, Phe C-2),
57.96 +d, Val C-2), 62.287 +t, PheOH C-1), 125.42, 125.82
+each d, Phe-, PheOH-arom C-40), 127.61, 128.02, 129.02,
129.48 +each dx2, Phe-, PheOH-arom C20, 30, 50, 60), 139.03
+s, Phe-arom C-10), 139.46 +s, PheOH-arom C-10), 157.45
+s, Arg C-7), 157.99 +s, ureido-C), 170.36 +s, Val C-1),
172.17 +s, Arg C-1). 175.44 +s, Phe C-1).
1
7.02; N, 10.62. H NMR +400 MHz, pyridine-d5) d: 0.96,
0.99 +each 3H, d, Jˆ7.0, Val3-CH3£2), 1.38 +9H, s, t-Bu),
1.69 +2H, br, Arg4-H2), 1.82, 1.99 +each 1H, dt, Jˆ13.5, 6.5,
Arg3-H2), 2.27 +1H, octet, Jˆ7.0, Val3-H), 2.99, 3.07 +each
1H, dd, Jˆ13.5, 7.0, PheOH3-H2), 3.20, 3.25 +each 1H, dd,
Jˆ13.5, 6.0, Phe3-H2), 3.28 +2H, m, Arg5-H2), 3.58, 3.68
+each 1H, dd, Jˆ9.5, 4.5, PheOH1-H2), 3.64 +3H, s, Mbs-
OCH3), 4.49, 4.53 +each 1H, d, Jˆ12.0, benzyl-CH2), 4.75
+1H, m, PheOH2-H), 4.84 +1H, dd, Jˆ8.5, 7.0, Val2-H),
5.00 +hidden, Arg2-H), 5.10 +1H, dt, Jˆ8.0, 6.0, Phe2-H),
7.02 +2H, d, Jˆ9.0, Mbs-arom-H), 7.11 +1H, brd, Jˆ8.0,
Phe2-NH), 7.15±7.48 +15H, m, Phe-, PheOH-, benzyl-
arom.-H), 7.40 +1H, d, Jˆ8.0, Arg2-NH), 7.48 +hidden,
Val2-NH), 8.29 +2H, d, Jˆ9.0, Mbs-arom-H), 9.22 +1H,
brd, Jˆ8.0, PheOH2-NH). 13C-NMR +100.6 MHz, DMSO-
d6) dc: 18.96, 19.84 +each q, Val3-CH3£2), 25.97 +t, Arg
C-4), 28.04 +q, t-Bu-CH3), 31.04 +t, Arg C-3), 31.57 +d,
Val C-3), 37.82 +t, PheOH C-3), 39.13 +t, Phe C-3), 41.22
7.1.17. Debenzylation of 24. 10% palladium carbon
+384 mg) was added to a solution of 24 +767 mg,
0.84 mmol) in AcOH +15 ml) and stirred under a stream of
hydrogen at room temperature for 4.5 h. The reaction
mixture was ®ltered and the ®ltrate was evaporated in
vacuo to give 25 +635 mg, 92%) as colorless plates. mp
90±1108C +MeOH). Rfˆ0.30 +CHCl3/MeOHˆ3:1).
[a]D25211.078 +cˆ1.03, CHCl3). IR +CHCl3) ymax cm21
:
1590 +arom), 1650 +-NHCO-), 1670 +-NHCONH-), 1720

4320
Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
+-COO-), 3350 +-OH). HRFABMS m/z: 846.3832
[M1Na]1, Calcd for C41H57N7O9SNa: 846.3836.1H NMR
+400 MHz, pyridine-d5) d: 0.97, 1.00 +each 3H, d, Jˆ7.0,
Val3-CH3£2), 1.38 +9H, s, t-Bu-H), 1.71+2H, br, Arg4-H2),
1.82, 1.99 +each 1H, m, Arg3-H2), 2.32 +1H, m, Val3-H),
3.12, 3.23 +each 1H, dd, Jˆ13.0, 7.0, PheOH3-H2), 3.22
C-30, 50), 127.27, 127.35, 128.71 +each d, Phe-, PheCHO-,
Bn-arom C-40), 129.00 +d, Mbs-arom C-20, 60), 130.09
+d, PheCHO-arom C-20, 60), 130.36 +d, Phe-arom C-20,
60), 138.06 +s, Phe-arom C-10), 138.30 +s, Mbs-arom
C-10), 157.95, 157.99 +s, Arg C-7), 158.56, 158.59 +s,
ureido-C), 162.64 +s, Mbs-arom C-40), 172.17 +s, Val
C-1), 172.47 +s, Phe C-1), 173.54 +s, Arg C-1), 200.16 +d,
PheCHO C-1).
+2H, br, Phe3-H2), 3.31+2H, br, Arg5-H ), 3.64 +3H, s,
2
Mbs-OCH3), 3.97, 4.00 +each 1H, dd, Jˆ11.0, 5.0,
PheOH1-H2), 4.72 +1H, m, PheOH2-H), 4.85 +1H, t,
Jˆ8.0, Val2-H), 4.97 +1H, br, Arg2-H), 5.08 +1H, dt,
Jˆ8.0, 6.0, Phe2-H), 7.02 +2H, d, Jˆ8.5, Mbs-arom-H),
7.12±7.44 +10H, m, Phe, PheOH-arom-H), 7.16 +1H, d,
Jˆ8.0, Phe2-NH), 7.47 +1H, d, Jˆ8.0, Arg2-NH), 8.27
+2H, d, Jˆ8.5, Mbs-arom-H), 9.02 +1H, d,Jˆ8.0,
PheOH2-NH). 13C-NMR +100.6 MHz, pyridine-d5) dc:
18.89, 19.89 +each q, Val3-CH3£2), 26.00 +t, Arg C-4),
28.06 +q, t-Bu-CH3), 30.67 +t, Arg C-3), 31.42 +d, Val
C-3), 37.76 +t, PheOH C-3), 39.15 +t, Phe C-3), 41.18 +t,
Arg C-5), 53.83 +d, PheOH C-2), 54.04 +d, Arg C-2), 55.58
+q, Mbs-OCH3), 55.71+d, Phe C-2), 59.68 +d, Val C-2),
63.57 +t, PheOH C-1), 81.47 +s, t-Bu-OC+CH3)3), 114.29
+d, Mbs-arom C-30, 50), 126.57, 127.0 +each d, Phe-,
PheOH±arom C-40), 128.69 +d, Mbs-arom C-20, 60),
128.76, 128.81 +each d, Phe-, PheOH-arom C30, 50),
130.01, 130.18 +each d, Phe-, PheOH-arom C20, 60),
137.67 +s, Phe-arom C-10), 138.35 +s, Mbs-arom C-10),
139.68 +s, PheOH-arom C-10), 158.22 +s, Arg C-7), 158.81
+s, ureido-C), 162.19 +s, Mbs-arom C-40), 172.37 +s, Phe
C-1), 172.54 +s, Val C-1), 173.64 +s, Arg C-1).
7.1.19. Mixture of a and b-MAPI 32 and 3). The
compound 26 +30.0 mg, 36.5 mmol) was dissolved in
methanesulfonic acid +184 ml, 2.80 mmol) and anisole
+9 ml, 84 mmol) was added. The mixture was stirred for
40 min. Ether +6 ml£3) was added to the reaction mixture
and decanted. Concentration of ether insoluble portion gave
light yellow oil +38.2 mg) which was puri®ed by column
chromatography +Amberlite IRA-410 acetone form, eluated
with water), followed by Sephadex LH-20 column chroma-
tography +eluated with MeOH) to afford colorless grannules
+2 and 3)+10.0 mg, 46%). 2 and 3: Rfˆ0.72 +MeOH/
AcOHˆ100: 1), mp 189±1988 +lit., 2: 204±2054c; 3: 221±
2124e); [a]D2526.38 +cˆ0.32, AcOH) +lit., 2: 218, cˆ1.0,
AcOH12c; 3: 0, cˆ1, AcOH12e). HRFABMS m/z: 596.3188
[M1H]1, Calcd for C30H42N7O6: 596.3197. 1H NMR
+400 MHz, DMSO-d6) d: 0.65 +3H, d, Jˆ7.0, Val3-CH3),
0.69 +3H, d, Jˆ6.8, Val3-CH3), 0.75 +3H, dd, Jˆ7.0, 1.3,
Val30*-CH3), 0.78 +3H, dd, Jˆ7.0, 2.0, Val30-CH3), 1.36
+2H, m, Arg3-Ha, Arg30-Ha), 1.42 +4H, m, Arg4-H2,
Arg40-H2), 1.66 +2H, m, Arg3-Hb, Arg30-Hb), 1.82 +1H,
quint, Val3-H), 1.92 +1H, quint, Val30-H), 2.72 +2H, dd,
Jˆ14.0, 10.0, PheCHO3-Ha, PheCHO30-Ha), 2.80 +2H,
dd, Jˆ10.5, 5.0, Phe3-Ha, Phe30-Ha), 2.94 +2H, m, Phe3-
Hb, Phe30-Hb), 2.97, 3.05 +each 2H, m, Arg5-H2, Arg50-H2),
3.14 +2H, dd, Jˆ14.0, 6.0, PheCHO3-Hb, PheCHO30-Hb),
3.96 +2H, m, Phe2-H, Phe20-H), 4.14 +2H, m, Val2-H,
Val20-H), 4.16 +2H, m, Arg2-H, Arg20-H), 4.32 +2H, m,
PheCHO2-H, PheCHO20-H), 6.05 +2H, d, Jˆ7.0, Phe2-
NH, Phe20-NH), 6.58, 6.60 +each 1H, d, Jˆ9.0, Arg2-NH,
Arg20-NH), 7.04-7.31+01H, m, Phe, PheCHO-arom-H),
7.28-7.86 +6H, br, -C+ˆNH)-NH2), 7.66, 7.71+each 1H, d,
Jˆ9.0, Val2-NH, Val20-NH), 8.50 +2H, d, Jˆ7.5,
PheCHO2-NH, PheCHO20-NH), 8.80 +2H, br, Arg6-NH,
Arg60-NH), 9.42, 9.47 +each 1H, brs, PheCHO1-H,
PheCHO10-H). 13C-NMR +100.6MHz, DMSO-d6) d:
17.37, 19.14 +each q, Val3-CH3£2), 17.52, 19.04 +each q,
Val30-CH3£2), 24.60, 24.64 +each t, Arg C-4, Arg C0-4),
29.15, 29.19 +each t, Arg C-3, Arg C0-3), 30.47 +d, Val
C-3, Val C0-3), 33.25, 33.33 +each t, PheCHO C-3, PheCHO
C0-3), 38.34, 38.36 +each t, Phe C-3, Phe C0-3), 40.20 +t, Arg
C-5, Arg C0-5), 52.46 +d, Arg C-2, Arg C0-2), 56.53, 56.58
+each d, Phe C-2, Phe C0-2), 57.21, 57.30 +each d, Val C-2,
Val C0-2), 59.46, 59.72 +each d, PheCHO C-2, PheCHO
C0-2), 125.43, 126.19, 127.59, 128.12, 128.15, 128.99,
129.11, 129.39 +each d, Phe, PheOH-arom C-20, 30, 40, 50,
60, C0-20, 30, 40, 50, 60), 137.44, 137.59 +each s, Phe-arom
C-10, C0-10), 139.36, 139.39 +each s, PheCHO-arom C-10,
C0-10), 157.27 +s, ureido-C, C0), 157.90, 157.95 +each s, Arg
C-7, Arg C0-7), 171.30, 171.36 +each s, Val C-1, Val C0-1),
172.19 +s, Arg C-1, Arg C0-1), 175.65 +s, Phe C-1, Phe
C0-1), 200.01, 200.07 +s, PheCHO C-1, PheCHO C0-1).
*The sign of H0 and C0 shows one of the diastereoisomer
in accordance with the other isomer which is described as H
and C.
7.1.18. Oxidation of tetrapeptide 25. PDC +51mg,
0.13 mmol), 4A-MS +300 mg), celite 545 +300 mg) were
added to the solution of 25 +100 mg, 0.12 mmol) in
CH2Cl2 +12 ml) and stirred for 3.5 h at room temperature
under argon. The reaction mixture was puri®ed by ¯ash
column chromatography +Silica gel, CHCl3/MeOHˆ20:1)
to afford colorless plates 26 +63 mg, 63%). mp 107±1148C
+MeOH). Rfˆ0.36 +CHCl3/MeOHˆ20:1). [a]D27222.138
+cˆ0.47, CHCl3)IR +CHCl3) ymax cm21: 1590 +arom),
1630 +-NHCONH-), 1650 +-NHCO-), 1710 +-CHO), 1730
+-COO-).HRFABMS m/z: 822.3828 [M1H]1, Calcd for
C41H56N7O9S: 822.3860 [M1H]. 1H NMR +400 MHz,
acetone-d6) d: 0.83, 0.86 +each 3H, d, Jˆ7.0, Val3-
CH3£2), 1.39 +9H, s, t-butyl-H), 1.57 +2H, br, Arg4-H2),
1.58 +1H, br, Arg3-Ha), 1.77 +1H, m, Arg3-Hb), 2.08 +1H,
m, Val3-H), 2.97 +1H, dd, Jˆ15.0, 9.0, Phe3-Ha), 2.99, 3.03
+each 1H, dd, Jˆ14.0, 6.0, PheCHO3-H2), 3.24 +1H, dd,
Jˆ15.0, 5.0, Phe3-Hb), 3.25 +2H, br, Arg5-H2), 3.86 +3H,
s, Mbs-OCH3), 4.29 +1H, dd, Jˆ9.0, 6.0, Val2-H), 4.33 +1H,
br, Arg2-H), 4.52 +1H, m, Phe2-H), 4.55 +1H, m, PheCHO2-
H), 6.19 +1H, brd, Jˆ8.0, Phe2-NH), 6.38 +1H, brd, Jˆ8.0,
Arg2-NH), 6.67 +2H, br, Arg-HN-C+ˆNH)-NHMbs), 7.00
+2H, d, Jˆ9.0, Mbs-arom-H), 7.18±7.31 +10H, m, Phe-
arom, PheCHO-arom-H), 7.43 +1H, brd, Jˆ9.0, Val2-NH),
7.82 +2H, d, Jˆ9.0, Mbs-arom-H), 7.80 +1H, br, Arg-HN-
C+ˆNH)-NHMbs), 7.86 +1H, br, PheCHO2-NH), 9.58 +1H,
br, PheCHO1-H). 13C-NMR +100.6MHz, DMSO-d6) d:
18.10, 19.70 +each q, Val3-CH3£2), 26.44 +t, Arg C-4),
28.13 +q, C+CH3)3), 29.80 +d, Val C-3), 30.20 +t, Arg
C-3), 34.89 +t, Phe C-3), 39.08 +t, PheCHO C-3), 41.01
+t, Arg C-5), 54.39 +d, Arg C-2), 55.81+d, Phe C-2),
55.87 +q, Mbs-OCH3), 59.23 +d, Val C-2), 60.80 +d,
PheCHO C-2), 81.73 +s, C+CH3)3), 114.50 +d, Mbs-arom

Y. Konda et al. / Tetrahedron 57 52001) 4311±4321
4321
3. Kaneto, R.; Chiba, H.; Dobashi, K.; Kojima, I.; Sasaki, K.;
Shibamoto, N.; Nishida, H.; Okamoto, R.; Akagawa, H.;
Mizuno, S. J. J. Antibiotics 1993, 46, 1622.
7.1.20. HIV-1 protease inhibition assay. Inhibition assay
of HIV-1protease was carried out as follows. A mixture of
4 mL of substrate solution +VSQNYPIV) +1mg/mL in
2MNaCl), 500 mM MES Buffer +pH 6.0), 3M NaCl,
10mM EDTA, 1% Triton X-100, 28 mL of enzyme solution,
8 mL of HIV protease +2 mg/ml), 4 mL of sample solution
was incubated for 2h at 378C. After that, 80 mL of D.W. was
added to the reaction mixture and heated for 2.5 h at 1008C
to stop the reaction. Then the mixture was centrifugated for
5 min at 12,000 rpm. The HPLC analysis was carried out
with 80 mL of the solution above under following condition.
Column: TSK gel ODS-120T; Eluent: 21% CH3CN/0.1%
TFA; Injection volume: 50 mL, Detection: 210 nm.
4. +a) Murano, S.; Watanabe, T. Agri. Biol. Chem. 1977, 41,
1313. +b) Murano, S.; Watanabe, T. Agri. Biol. Chem. 1978,
42, 2209. +c) Watanabe, T.; Fukuhara, K.; Murano, S.
Tetrahedron Lett. 1979, 46, 625. +d) Watanabe, T.; Murano,
S. Agri. Biol. Chem. 1979, 43, 243. +e) Shin-Watanabe, T.;
Fukuhara, K.; Murano, S. Tetrahedron 1982, 38, 1775.
5. Stefanelli, S.; Cavaletti, E.; Sarubbi, E.; Ragg, L.; Colombo,
L.; Selva, E. J. Antibiotics 1995, 48, 332.
6. Konda, Y.; Takahashi, Y.; Mita, H.; Takeda, K.; Harigaya, Y.
Chem. Lett. 1997, 345.
7. +a) Majer, P.; Randad, R. S. J. Org. Chem. 1994, 59, 1937.
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