Argifin; efficient solid phase total synthesis and evalution of analogues of acyclic peptide

Article abstract of DOI:10.1016/j.bmc.2009.02.047

An effective solid phase synthesis of Argifin, providing subsequent access to effective synthesis of analogues, was developed in 13% overall yield, as well as elucidating structure-activity relationships. The novel acyclic peptide 18b, prepared from a synthetic intermediate of Argifin, was found to be 70 times more potent as an inhibitor of Serratia marcescens chitinases B than Argifin itself.

Full text of DOI:10.1016/j.bmc.2009.02.047

Bioorganic & Medicinal Chemistry 17 (2009) 2751–2758
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Argifin; efficient solid phase total synthesis and evalution of analogues
of acyclic peptide
a
a
a
a
Toshiaki Sunazuka a, , Akihiro Sugawara , Kanami Iguchi , Tomoyasu Hirose , Kenichiro Nagai ,
*
Yoshihiko Noguchi a, Yoshifumi Saito a, Tsuyoshi Yamamoto a, Hideaki Ui a, Hiroaki Gouda b, Kazuro Shiomi a,
c
a,
*
¯
Takeshi Watanabe , Satoshi Omura
a Kitasato Institute for Life Sciences, Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
b School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan
c Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An effective solid phase synthesis of Argifin, providing subsequent access to effective synthesis of ana-
logues, was developed in 13% overall yield, as well as elucidating structure–activity relationships. The
novel acyclic peptide 18b, prepared from a synthetic intermediate of Argifin, was found to be 70 times
more potent as an inhibitor of Serratia marcescens chitinases B than Argifin itself
Ó 2009 Elsevier Ltd. All rights reserved.
Received 26 December 2008
Revised 17 February 2009
Accepted 19 February 2009
Available online 27 February 2009
Keywords:
Argifin
Chitinase
Solid phase
Serratia marcescens
1. Introduction
ylcarbamoyl-L-arginine moiety and the motif of the hydrolytic
pocket of the chitinases, critically contributing to expression of
Chitinases hydrolyze b-(1,4)-linked N-acetylglucosamine (Chi-
tin), which is one of the most abundant polysaccharides in nature.1
Since chitin is a major structural component of fungi and insects,2
with 18 chitinases playing important physiological roles in these
organisms, chitinase inhibitors are of considerable interest as po-
tential agents against fungi,3 insects,4 and malaria transmission.5
They also offer significant potential for treatment of asthma and
other diseases in humans.6 Despite their attractive potential for
medicinal usage, no practical use has been developed to date.
During screening for chitinase inhibitors, a new cyclic penta-
peptide Argifin (1) was isolated from the cultured broth of Gliocla-
dium sp. FTD-0668 by our group, and found to be a potent inhibitor
of Serratia marcescens chitinases (SmChi) with IC50 values of 0.025
nanomolar to micromolar range inhibitions.9
Design and development of practical and efficient strategies for
Argifin synthesis has been an important objective since the original
source, Gliocladium sp. FTD-0668, no longer produces this cyclic-
peptide. Therefore, a method for rapid and diverse synthetic route
of 1 is required the supply to the biological tests as well as the SAR
studies of Argifin. Herein, we report our efficient solid phase total
synthesis of 1, which also facilitates rapid synthesis of analogues,
using 2-chlorotrityl chloride resin along with HPLC purification.
Furthermore, during the total synthesis, we found that the acyclic
peptide, possessing a much-simplified structure, exhibited 70-fold
more potent inhibitory activity than that of 1 against SmChiB. Our
and 6.4 l
M against SmChiA and B, respectively (Fig. 1).7,8 The
structure of 1 was elucidated by amino acid analysis and detailed
1D and 2D-NMR experiments. Additionally, three-dimensional
structures of 1, in complex with SmChiB, were resolved by X-ray
crystallography9, resulting in detailed visualization of the interac-
tion of 1 with SmChiB. More importantly, there are at least four
H
N
O
O
NH
O
HO
O
NH
N
H
N
N
H
H
O
HN
N
O
x
concentrated hydrogen bond interactions between the N -meth-
IC50
=
0.025 0.0024 µM against SmChiA
6.4 1.0 µM against ChiB
>30
N
H
Sm
µM against SmChiC1
O
HO
O
Argifin (1)
* Corresponding authors.
E-mail addresses: sunazuka@lisci.kitasato-u.ac.jp (T. Sunazuka), omuras@insti.
kitasato-u.ac.jp (S. Omura).
Figure 1. Structure and IC50 values of Argifin (1).8
¯
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.047

2752
T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
PMB
N
work also facilitated elucidation of structure-activity relationships
(SAR) of the linear peptides.
Cl
Me
PMB
N
PMB
N
O
12
HN
N
NHBoc
N
NHBoc
R
NH2
N
NHBoc
R
Me
Me
2. Results and discussion
2.1. Chemistry
N
O
N
O
HN
N
Et3N, rt
NaH, THF
0 °C to 80 °C
1 h, 39%
13a; R = phenyl (58%)
3
11
13b; R = CF3CH2- (83%)
The first total synthesis of 1 was developed by I. M. Eggleston and
co-workers in 2005, utilizing solid phase peptide synthesis.10 Their
strategy, however, included liquid phase reaction sequences for
Scheme 2. Synthesis of 13a–b as a model study.
x
introduction of the N -methylcarbamoyl group during final stages
of total synthesis, with two-time HPLC separation due to its hydro-
philicity, indicating that an efficient strategy was still required,
especially to ensure rapid synthesis of analogues. Our retrosynthetic
analysis is outlined in Scheme 1. To avoid complications of the liquid
phase reactions, we envisaged the complete solid phase total syn-
thesis for all reaction sequences, except for final cleavage step from
the resin.
x
Initially, synthesis of the N -methylcarbamoyl group on resin is
required for solid phase total synthesis of 1.
x
To address this issue, we constructed the N -methylcarbamoyl
group in liquid phase as a model study (Scheme 2). Treatment of 1-
H-pyrazole-1-[N-(tert-butoxycarbonyl)]carboxamidine (11)12 with
N-methyl-N-(4-methoxybenzyl)carbamoyl chloride (12)13 in the
presence of NaH in THF afforded the corresponding urea com-
pound (3) in 39% yield. The guanidine formation involved N-meth-
ylcarbamoyl group (13a–b) being effectively introduced using
aniline and trifluoroethylamine at room temperature in 58% and
83% yields, respectively (Scheme 2), suggesting that this method
can not only be utilized for synthesis of 1, but also synthesis of sim-
Additionally,1-H-pyrazole-1-[N-(tert-butoxycarbonyl)-N’-(N-p-
methoxybenzylcarbamoyl-N-methyl)]carboxamidine (3) was de-
x
signed to simply introduce the N -methylcarbamoylguanidino
onto the NH2 group of Orn for solid phase synthesis.
From our synthetic perspective, 1 could be prepared from fully-
functional compound (2) with deprotection and cleavage from the
resin under acidic conditions. Resin-cyclic peptide (2) could be
synthesized using macrolactamization on resin and effective intro-
x
ilar natural products containing the N -methylcarbamoyl group,
such as Banyasin A.14
x
In possession of 3, the carboxylic acid at
a position in 6 was
duction of the N -methylcarbamoylguanidino group, together
loaded onto 2-chlorotrityl resin 5 in the presence of i-Pr2NEt in
DCM, resulting in 98% loading yield (1.4 mmol/g), calculated from
the weight of 6 after cleavage with 20% TFA in DCM. The Fmoc
group was removed with 20% piperidine in DMF, and subsequent
condensation with 7 using PyBop (benzotriazol-1-yloxytriopyrroli-
dinophosphonium hexa-fluorophosphite), i-Pr2NEt provided dipep-
tide (15). After subsequent removal of the Fmoc group (20%
piperidine in DMF), the use of HATU (N-[(dimethylamino)(3H-
1,2,3-triazolo(4,5-b)pyridin-3-yloxy)methylene]-N-methylmethana-
minium hexafluorophosphate) as the best coupling reagent with 8
afforded the tripeptide (16) with complete consumption of the
resulting 15. When PyBop or PyBrop (Bromo-tris-pyrrolidino-phos-
phonium hexafluorophosphate) as condensation reagents was
used, mixture of 15 and 16 were observed in LC–UV–MS, after
cleavage with 20% TFA in DCM. With 16 in hand, Fmoc deprotection
(20% piperidine in DMF) and coupling with 9 (PyBop, i-Pr2NEt)
afforded the tetrapeptide. The use of the same coupling procedure
with 10 and PyBop provided the linear pentapeptide, without
aspartimide formation, after cleavage with 20% TFA in DCM. After
deprotection of the Allyl (Pd(PPh3)4/dimedone in THF) and Fmoc
(20% piperidine in DMF) groups, macrolactamization of 4 with Py-
Bop in two cycles on resin furnished the corresponding cyclic com-
pound without oligomerization. To complete total synthesis, after
with 3 starting from the acyclic compound (4). Then, the linear
peptide composed blocks Fmoc-Asp(OAllyl)-OH (6), Fmoc-N-Me-
Phe-OH (7), Fmoc-Orn(Dde)-OH (8) [Dde; 1-(4,4-dimethyl-2,
6-dioxocyclohex-1-ylidene)ethyl]11
,
Fmoc-D-Ala-OH (9), and
Fmoc-Asp-Ot-Bu (10) can be assembled by solid phase synthesis,
using a 2-chlorotrityl link that is tolerant to reaction conditions
in Fmoc-based solid phase synthesis and stable under cleavage
conditions.
H
N
O
O
Deprotection followed by
Boc
O
Cleavage from resin
N
t-BuO
NH
N
PMB
N
H
N
O
H
1
O
HN
O
N
N
H
O
O
PMB
N
O
N
N
NHBoc
2
Me
H
N
O
O
O
N
t-BuO
O
NH
NHDde
Dde=
3
O
H2N
HO2C
O
N
Macrolactamization
on resin
x
N
Dde deprotection (2% hydrazine in DMF), N -methylcarbamoylgua-
H
O
O
O
nidino formation with 3, followed by deprotection and cleavage
with 90% TFA in DCM from the resin, furnished 1 in overall 13% yield
after HPLC purification (Scheme 3). Spectral data and activities of
synthetic 1 were found to be identical with those of the natural 1.
Significantly, we found that an acyclic intermediate exhib-
ited better activity than the parent 1 against SmChiB, suggest-
ing a cyclic conformation might not be necessary to exhibit
inhibition of chitinase activity. The most important functional
O
Dde=1-(4,4-dimethyl-
2,6-dioxocyclohex-1-
ylidene)ethyl
4
O
FmocHN
OH
O
OH
t-BuO
9
FmocHN
Solid Phase
O
NHDde
10
FmocHN
O
Peptide Synthesis
HO
8
AllylO2C
Fmoc
N
x
NHFmoc
group to exhibit activity appears to be the N -methylcarba-
HO
moyl guanidine group, based on Argifin-SmChiB X-ray study.9
A similar result from the van Aalten group has been recently
reported using Aspergillus fumigatus chitinase B1 (AfChiB1) as
the enzyme for their assay.15 Most of their analogues, how-
ever, do not express inhibitory activity greater than that of
1 against AfChiB1.
HO
Cl
O
O
Cl
PS
7
6
PS=polystyrene
5
Scheme 1. Synthetic plan of 1.

T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
2753
OAllyl
activity on SmChiB except for Arg-series 20a–c (Table 1). In con-
trast, possessing the -Ala moiety increases activity in terms of
SmChiA. Furthermore, the addition of Asp moiety next to -Ala
1) 6, i-Pr2NEt,
1) 7, PyBop, i-Pr2NEt
DCM, 2 h
O
DCM/DMF, 2 h
D
5
NH2
D
2) 20% piperidine
in DMF, 1 h
O
2) 20% piperidine
in DMF , 1 h
enfeebles both activities. These correlations for all-series were
clearly demonstrated (sigmoidal graphs for Asp-series are provided
in Table 1). On the other hand, lack of the Asp unit next to N-Me-
Phe (in 19a–c and 20a–c) decreases activity of both SmChiA and B,
suggesting the Asp(OAllyl) plays a key role in the activity. Likewise,
the N-methyl-Phe moiety is also an important amino acid, indi-
cated by the finding that the Arg series display weak activity for
both SmChiA and B. Unfortunately, all of the acyclic compounds,
including 1, do not exhibit activity against SmChiC1.
O
14
H2N
NHDde
1) 8, HATU, i-Pr2NEt
OAllyl
OAllyl
O
DCM/DMF, 2 h
H
N
N
O
O
N
H
2) 20% piperidine
in DMF , 1 h
N
H
O
O
O
O
O
O
15
16
O
H2N
1) 10, PyBop, i-Pr2NEt
NH
DCM/DMF, 2 h
1) 9, PyBop, i-Pr2NEt
NHDde
DCM/DMF, 2 h
OAllyl
O
2) Pd(PPh3)4
N
O
dimedone, THF, 1 h
3) 20% piperidine
in DMF , 1 h
2) 20% piperidine
in DMF , 1 h
3. Conclusion
N
H
O
O
O
In summary, we have developed the efficient solid phase total
17
1) HATU, i-Pr2NEt,
DCM/DMF, 1 h
synthesis of 1, involving macrolactamization on resin as well as
construction of the N -methylcarbamoyl group, with single HPLC
90% TFA in DCM
x
(repeated 2 times)
1
4
2
purification, allowing us to speedily generate a variety of ana-
logues. It is notable that the novel acyclic peptide 18b exhibits
70-fold more potent activity against SmChiB than that of 1, indicat-
ing that the cyclic form is not necessary for anti-chitinase activity.
This means that we have identified not only a simplified structure
with potent inhibitory activity but also a new scaffold, which has
potent inhibitory activity, derived from the natural product. Fur-
ther studies for acyclic8 and cyclic analogues are continuing in
our laboratory.
overall yield 13%
in 15 steps
2) 2% hydrazine in DMF, 2 h
3) 3, i-Pr2NEt, DCM/DMF
Scheme 3. Solid phase total synthesis of 1.
We began our investigation of analogues by looking into nine
acyclic derivatives bearing the N -methylcarbamoylguanidino
x
group to elucidate the SAR against each SmChi isozyme. For syn-
thesis of acyclic peptides, we utilized the solid phase peptide syn-
thesis strategy using 2-chlorotrityl chloride resin. From the
synthetic perspective, elaboration of the acyclic analogues is out-
lined in Scheme 4. The carboxylic acids at each amino acid (6/7/
8) were loaded onto 5 in the presence of i-Pr2NEt in DCM, followed
by elaboration of appropriate amino acids (7/8/9/10) and
acetylation of terminal NH2 to furnish the acetylated products.
4. Experimental
4.1. General
Fmoc-Asp(OAllyl)-OH (6), Fmoc-N-Me-Phe-OH (7), Fmoc-D-Ala-
x
Deprotection of the Dde group and introduction of the N -meth-
OH (9), and Fmoc-Asp-Ot-Bu (10) were purchased from Watanabe
Chemical Industries, LTD. 2-Chlorotritylchlorie resin (5) was pur-
chased from NovaBiochem. Dry THF, toluene, and CH2Cl2 were pur-
chased from Kanto Chemical Co. Fmoc-Orn(Dde)-OH (8) was
prepared according to similar procedures. Precoated silica gel
plates with a fluorescent indicater (Merck 60 F254) were used for
analytical and preparative thin layer chromatography. Flash col-
umn chromatography was carried out with Kanto Chemical silica
gel (Kanto Chemical Co., Inc., Silica Gel 60 N, spherical neutral,
0.040–0.050 mm, Cat.-No. 37563-84). 1H NMR spectra were re-
corded at 270 MHz or 300 MHz or 400 MHz and 13C NMR spectra
were recorded at 67.5 MHz or 75 MHz or 100 MHz on JEOL JNM-
EX270 (270 MHz) or Varian VXR-300 (300 MHz) or Varian XL-
400 (400 MHz) or Varian UNITY-400 (400 MHz). The chemical
shifts are expressed in ppm downfield from internal solvent peaks
CH3OH (3.31, 4.84 ppm, 1H NMR), pyridine (8.71 (br), 7.55 (br),
7.19 (br) ppm, 1H NMR), CD3OD (49.0 ppm, 13C NMR), pyridine-
d5 (123.5, 135.5, 149.2 ppm, 13C NMR), D2O (the end of both fields;
0, 200 ppm, 13C NMR) and J values are given in hertz. The coupling
patterns are expressed by s (singlet), d (doublet), dd (double dou-
blet), t (triplet), m (multiplet) or br (broad). The All infrared spectra
were measured on a Horiba FT-210 spectrometer. High- and low-
resolution mass spectra were measured on a JEOL JMS-DX300
and JEOL JMS-AX505 HA spectrometer. Liquid chromatographic
analysis was conducted on a Hitachi ELITE LaChrom with Senshu
Pak-PEGASIL ODS-II (4.6u ꢀ 250 mm). Optical rotations were mea-
sured by JASCO DIP-370 polarimeter. Melting points were mea-
ylcarbamoylguanidino moiety afforded fully-functional compounds.
Finally, cleavage from the resin, followed by deprotection of Boc and
PMB group under TFA conditions, readily furnished the nine acyclic
analogues (18–20a–c) in 21–78% yields (see Table 1).
2.2. In vitro evaluation
For determination of IC50 values against each SmChi isozyme,
nine acyclic compounds were subjected to a competition assay
with 4-methylumberiferyldiacetyl-chitobiose8,16 (Table 1). Inter-
estingly, Arg-(N-Me-Phe)-Asp (18a) and
Asp (18b) exhibited approximately 50–70-fold more potent activ-
ity against SmChiB (with 0.13 M and 0.091 M of IC50 values,
respectively) than that of parent 1, suggesting that the -Ala moi-
D-Ala-Arg-(N-Me-Phe)-
l
l
D
ety is not a crucial function to express competent inhibitory
1) Loading
AcHN
2) Peptide synthesis
3) Acetylation
NHDde
Cl
Cl
HO
O
Asp/Phe/Orn
O
O
5
1) Dde deprotection
2) guanidine formation
Boc
N
O
AcHN
NH
O
cleavage and
deprotection
AcHN
N
H
N
H
N
PMB
N
N
N
H
H
H
sured on
a Yanagimoto Micro Apparatus. Fluoroscence for
9 types of analogues
Asp series; 18a-c
Phe series; 19a-c
Arg series; 20a-c
measurments of chitinases inhibitory activities was measured by
fluorometer on a Labsystems Fluoroscan II. LC/UV-MS was per-
formed on a Waters 2795 Separation Module with AllianceÒ HT
and micromass ZQ (Column; Senshu Pak-PEGASIL ODS
O
HO
O
O
Scheme 4. General procedure of 9 types of linear analogues.

2754
T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
Table 1
IC50 results for 9 types of linear analogues
R
IC50
(lM)
Yieldsc (%)
SmChiAb
SmChiBb
SmChiCb
18a
18b
18c
19aa
19b
19c
20a
20b
20c
1
Ac
Ac-D-Ala
Ac-Asp-
Ac
Ac-D-Ala
Ac-Asp-
Ac
Ac-
Ac-Asp-
Argifin
0.14 0.0057
0.025 0.0015
1.1 0.061
0.75 0.072
0.090 0.014
8.4 1.0
31 1.2
6.6 0.64
32 7.6
0.025 0.0024
0.13 0.053
0.091 0.0038
1.0 0.19
3.7 0.67
2.7 0.63
30 6.8
260 55
64 12
110 17
6.4 1.0
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
78
44
20
22
40
30
58
35
21
13
D
D
D
-Ala
-Ala
-Ala
D
-Ala
a
Ref. 15.
b
c
The preparation of SmChiA, B, and C1; see Ref. 8.
Isolated yields after HPLC purification.
2u ꢀ 50 mm: Condition of HPLC; gradient 10% MeCN(0.05% TFA)/
H2O (0.1% TFA) to 100% MeCN(0.05% TFA) over 8 min, flow
0.3 mL/min, detect 200–400 nm, temp 20 °C). LC–UV was per-
formed on a ELITE LaChrom (Column; Senshu Pak PEGASIL ODS
20u ꢀ 250 mm with a flow rate of 8 mL/min. Mobile phase A was
0.05% TFA in MeCN , mobile phase B was 0.05% TFA in H2O).
Gradient 1 was T = 0 min, A = 15% ; T = 30 min, A = 98%. Gradient
2 was T = 0 min, A = 20%; T = 30 min, A = 98%. Gradient 3 was
T = 0 min, A = 25%; T = 30 min, A = 98%. Gradient 4 was T = 0 min,
A = 0%; T = 5 min, A = 5% ; T = 30 min, A = 98%.
4.3. N-(p-Methoxybenzyl)-N-methylamidoyl chloride 12
Although 12 has been reported by the Yoakim group (but there
is no spectra data for 12), we have synthesized 12 by using our
reaction sequence.
To a solution of p-methoxybenzaldehyde (30.00 mL, 248.0 mmol)
in MeOH (83.0 mL) was added 40% MeNH2 in water (17.30 mL) at
room temperature. After being stirred at room temperature for
1 h, the mixture was added NaBH4 (4.69 g, 124 mmol) at 0 °C and
then was stirred at 0 °C for 1 h. After the reaction was quenched
with 3 N HCl aq the solvent was removed in vacuo. The residue
was washed with CHCl3 (300 mL ꢀ 1) and the water layer was basi-
fied with 3 M NaOH aq (100 mL) at 0 °C to approximately pH 10.
The resulting water layer was extracted with CH2Cl2
(200 mL ꢀ 3), dried over Na2SO4, and concentrated in vacuo to pro-
duce the crude N-(p-methoxybenzyl)-N-methylamine, which is used
without further purification. To a solution of the crude N-(p-
methoxybenzyl)-N-methylamine in THF (330 mL) was carefully
added DIPEA (34.5 mL, 0.198 mmol), triphosgene (17.18 g,
0.058 mmol) at 0 °C. After being stirred for 1 h, the mixture was di-
luted with EtOAc (300 mL), quenched with satd NaHCO3 aq
4.2. Abbreviations
Ac = Acetyl, Boc = tert-butyloxycarbonyl, DCM = dichlorometh-
ane, Dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, DI-
PEA = N,N-diisopropylethylamine, DMF = N,N-dimethylformamide,
HATU; N-[(dimethylamino)(3H-1,2,3-triazolo(4,5-b)pyridin-3-yloxy)
methylene-]-N-methylmethanaminium hexafluorophosphate, Orn =
ornithin, PMB = p-methoxybenzyl, PyBop; benzotriazol-1-yloxytrio-
pyrrolidinophosphonium hexafluorophosphite. TFA = trifluoroacetic
acid, THF = tetrahydrofuran.

T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
2755
(100 mL) and separated. The mixture was washed with sat. NaH-
CO3 aq (100 mL ꢀ 1), satd NaCl aq (100 mL ꢀ 1), dried over Na2SO4,
and concentrated in vacuo. Flash column chromatography (hex-
ane/EtOAc = 10:1) afforded 12 (19.5 g, 0.032 mol) in 55% yield as
a colorless oil.
CH2 of Dde group), 2.29 (m, 1H, b position of Orn), 2.10 (m, 1H,
b position of Orn), 1.90 (m, 2H, d position of Orn), 0.95 (s, 6H, di-
methyl of Dde group), 13C NMR (67.5 MHz, pyridine-d5): 197.2
(x2), 175.2, 173.3, 157.4, 144.7, 144.4, 141.6 (ꢀ2), 128.0 (ꢀ2),
127.4 (ꢀ2), 125.6 (ꢀ2), 120.4 (ꢀ2), 108.0, 66.5, 54.7, 53.1 (ꢀ2),
47.8, 43.0, 30.0 (ꢀ2), 28.2 (ꢀ2), 26.1, 17.6, HR-MS (FAB, NBA):
calcd for C30H35O6N2: 519.2495 [M+H], found m/z: 519.2487
[M+H]+.
Rf = 0.70 (silica gel, hexane/EtOAc = 1:1), IR (KBr)
m
cmꢁ1: 2945
(m), 1736 (s), 1512 (m), 1248 (s), 1182 (m), 1070 (s), 1H NMR
(270 MHz, CDCl3) The mixture of rotamers was observed. d: 7.20
(m, 2H, Ph), 6.90 (m, 2H, Ph), [4.65 , 4.51 (s ꢀ 2, 2H, –PhCH2–), (rot-
amer)], 3.81 (s, 3H, O–CH3), [3.04, 2.97 (s ꢀ 2, 3H, –N–CH3), (rot-
amer)], 13C NMR (67.5 MHz, CDCl3) The mixture of rotamers was
observed. d: 159.4, [150.1, 149.1 (rotamer)], [129.5, 128.6, 127.3,
127.0 (rotamer)], [114.2, 114.1 (rotamer)], [55.3, 53.7 (rotamer)],
55.2, [37.5, 36.0 (rotamer)], HR-MS (FAB, PEG200 + 400): calcd
for C10H12O2NCl: 213.0557 [M+H], found m/z: 213.0556[M+H]+.
4.6. N-tert-Butoxycarbonyl-N’-(N-methyl-N-p-
methoxybenzyl)carbamoyl-N00-phenylguanidine 13a
To a solution of 3 (100 mg, 0.26 mmol) in acetonitrile (2.6 mL)
was added triethylamine (109 lL, 0.78 mmol), aniline (36 lL,
0.39 mmol) at room temperature. The mixture was stirred at room
temperature for 40 h. The solution was diluted with EtOAc (5 mL),
and was successively washed with sat. aq. NH4Cl (3 mL ꢀ 1), H2O
(3 mL ꢀ 1), and brine (5 mL ꢀ 1), dried over Na2SO4, and concen-
trated. The resulting colorless oil was purified by flash column
chromatography (hexane/EtOAc = 80:1–50:1) to afford 13a
(59.8 mg, 0.15 mmol) in 58% yield as a colorless oil.
4.4. 1-H-Pyrazole-1-(N-(tert-butoxycarbonyl)-N0-(N-methyl-N-p
-methoxybenzylcarbamoyl)-carboxamidine 3
Toasolutionof11(6.40 g, 30.4 mmol)inTHF(304 mL)wasadded
NaH (2.0 g, 45.6 mmol) at 0 °C. After being stirred at 0 °C for 10 min,
the reaction was allowed to warm up to room temperature and was
added a solution of 12 (19.5 g, 91.3 mmol) in THF (102 mL) before
heated up to reflux. The mixture was stirred at reflux for 1 h and
cooled to room temperature. After the mixture was diluted with
EtOAc (200 mL), quenched with satd aq NH4Cl (100 mL) and sepa-
rated, the organic layer was washed with H2O (100 mL ꢀ 1), dried
over Na2SO4 and concentrated to yield the crude product. Flash col-
umn chromatography (hexane/EtOAc = 10:1) afforded 3 (4.6 g,
11.9 mmol) in 39% yield as a colorless oil.
Rf = 0.39 (silica gel, hexane/EtOAc = 4:1), IR (KBr)
m
cmꢁ1: 3286
(w), 3151 (w), 2979 (w), 1712 (s), 1639 (s), 1512 (s), 1452 (s), 1412
(s), 1246 (s), 1153 (s), 1H NMR (270 MHz, CDCl3) The mixture of
rotamers was observed. d: 12.6 (s, 1H, –NH–, rotamer), [10.2, 10.1
(s, 1H, –NH–, rotamer)], 7.63 (d, J = 7.58 Hz, 1H, Ph), 7.53 (d,
J = 7.58 Hz, 1H, Ph), 7.43 (t, J = 7.26 Hz, 1H, Ph), 7.28-7.00 (complex
m, 4H, Ph), 6.86 (dd, J = 8.9, 2.3 Hz, 2H, Ph), [4.67, 4.53 (s ꢀ2, 2H, p-
OCH3PhCH2–, rotamer)], 3.79 (s, 3H, –OCH3), [3.04, 2.92 (s ꢀ2, 3H,
–N–CH3–, rotamer)], 1.53 (s, 9H, –COO(CH3)3), 13C NMR (67.5 MHz,
CDCl3) The mixture of rotamers was observed. d: 164.1, 158.6, 153.5,
151.6, [137.4, 137.3 (C ꢀ 1, Phe, rotamer)], [130.5, 130.1 (C ꢀ 1,
aromatic, rotamer)], [128.8, 128.5 (C ꢀ 2, aromatic),], 124.1, (Ph),
122.0 (C ꢀ 2, Ph), 113.8 (C ꢀ 2, aromatic), 82.8 (–OC(CH3)3), 55.2
(–OCH3), [52.3, 50.5, (–Ph–CH2–N(Me)–, rotamer)], [34.9, 33.2,
(–(O@C)N-CH3, rotamer)], 28.0 (C ꢀ 3, –OC(CH3)3), HR-MS (FAB,
PEG600+NaI): calcd for C22H29O4N4: 413.2189 [M+H], found m/z
413.2196 [M+H]+.
Rf = 0.41 (silica gel, hexane/EtOAc = 1:2), IR (KBr)
m
cmꢁ1: 2941
(w), 2359 (w), 1761 (s), 1641 (s), 1504 (s), 1242 (s), 1153 (s), 1H
NMR (270 MHz, CDCl3) The mixture of rotamers was observed. d:
8.24 (d, J = 18.2 Hz, 1H, pyrazole), 7.65 (d, J = 1.3 Hz, 1H, pyrazole),
7.32 (m, 2H, Ph), 6.87 (m, 2H, Ph), 6.44 (s, 1H, pyrazole), 4.56 (s, 2H,
p-OCH3PhCH2–), 3.83 (s, 3H, p-OCH3PhCH2–), 2.88 (s, 3H, N-CH3),
1.50 (s, 9H, O(CH3)3), 13C NMR (67.5 MHz, CDCl3) The mixture of
rotamers was observed. d: 160.5 (amidine), [158.8, 158.7 CH3O-
C6H4-, (rotamer)], 149.3 (–HN–C(@O)O-tBu), [142.5, 142.4 pyra-
zole, (rotamer)], [140.6, 139.7 –CH3N–C(@O)N–, (rotamer)],
[129.6, 128.8, 128.7 Ph, (rotamer)], 129.6 (pyrazole), [113.8,
113.6, Ph (rotamer)], 109.3 (pyrazole), [82.8, 82.7 –OC(CH3)3, (rot-
amer)], 55.1, (–OCH3), [53.0, 50.6 –Ph–CH2–N(Me)–, (rotamer)],
[34.5, 32.8 –N–CH3, (rotamer)], [27.9, 27.8 –OC(CH3)3, (rotamer)],
HR-MS (FAB, NBA): calcd for C19H26O4N5: 388.1985 [M+H], found
m/z: 388.1995 [M+H]+.
4.7. N-tert-Butoxycarbonyl-N0-(1,1,1-trisfluoroethyl)-N00-(N-
methyl-N-p-methoxybenzyl)-carbamoylguanidine 13b
To a solution of 3 (70 mg, 0.18 mmol) in THF/MeOH (1.8 mL/
1.0 mL) was added triethylamine (75
lL, 0.54 mmol), trifluoroeth-
ylamine (73 L, 0.27 mmol). The mixture was stirred at room
l
temperature for 25 h. The solution was diluted with EtOAc (2 mL),
and was successively washed with sat. aq. NH4Cl (2 mL ꢀ 1), H2O
(5 mL ꢀ 1), brine (5 mL ꢀ 1), dried over Na2SO4, and concen-
trated in vacuo. The resulting colorless oil was purified by flash
column chromatography (hexane/Et OAc = 80:1–50:1) to provide
13b (63.2 mg, 0.15 mmol) in 83% yield as a colorless oil.
4.5. Fmoc-Orn(Dde)-OH (8)
To a solution of Fmoc-Orn-OH hydrochloride (6.0 g, 0.015 mol)
in DCM–MeOH (7:3, 300 mL, 0.05 M) was added DIPEA (10.7 mL,
0.060 mol),
4,4-dimethyl-2,6-dioxocyclohexeylidene
(5.6 g,
Rf = 0.47 (silica gel, Hexane/EtOAc = 3:1), IR (KBr) m
cmꢁ1: 3330
0.030 mol) at room temperature. After being stirred at room tem-
perature for 48 h, the solvent was removed in vacuo. The residue
was diluted with EtOAc (300 mL), washed with 1 N aq HCl
(300 mL), satd aq NaCl (200 mL), dried over Na2SO4, and concen-
(w), 2983 (w), 1718 (s), 1637 (s), 1513 (s), 1457 (s), 1417 (s), 1246
(s), 1149 (s), 1H NMR (270 MHz, CDCl3) The mixture of rotamers was
observed. d: 12.4, 12.4 (s, 1H, –NH–, rotamer), 8.47, 8.46 (s, 1H,
–NH–, rotamer), 7.16 (t, J = 8.91, 2H, Ph), 6.84 (dd, J = 8.57,
2.31 Hz, 2H, Ph), [4.67, 4.49 (s ꢀ 2, 2H, p-OCH3PhCH2–, rotamer)],
4.02 (m, 2H, CH2CF3), 3.78 (s, 3H, OCH3), [3.00, 2.86 (s ꢀ 2, 3H, –
N–CH3–, rotamer)], 1.48 (s, 9H, –COO(CH3)3), 13C NMR (67.5 MHz,
CDCl3) The mixture of rotamers was observed. d: 163.6, 158.7,
154.4, 153.3, [130.4, 128.5 (rotamer, aromatic)], 128.8 (C ꢀ 2, aro-
matic), 124.0 (–CH2CF3, JCF = 276.5 Hz), 113.8 (C ꢀ 2, aromatic),
83.0 (–OC(CH3)3), 55.2 (–OCH3), [52.4, 50.4, (–Ph–CH2–N(Me)–,
rotamer)], 41.6 (–CH2CF3, JCF = 34.6 Hz), [34.7, 32.9, (–N–CH3, rot-
amer)], 28.0 (C ꢀ 3, –OC(CH3)3), HR-MS (FAB, PEG600+NaI): calcd
for C18H26O4N4F3: 419.1906 [M+H], found m/z 419.1905 [M+H]+.
trated. Recrystallization from EtOAc and hexane provided
(6.0 g, 0.0116 mol) in 75% yield as a colorless solid.
8
Rf = 0.35 (silica gel, CH3Cl–MeOH = 4:1), IR (KBr)
m
cmꢁ1: 3415,
ꢂ ¼ 4:32 (c
2958, 1574, 1464, 1450, 1267, 1149, 1034, 741, ½a D27
1.0, CH3Cl), mp = 150–152 °C, 1H NMR (270 MHz, pyridine-d5):
13.8 (br s, 1H, –COOH), 8.93 (d, J = 8.6 Hz, 1H, aromatic), 7.83 (d,
J = 7.6 Hz, 2H, aromatic), 7.70 (dd, J = 7.3, 3.3 Hz, 2H, aromatic),
7.39 (m, 2H, aromatic), 7.26 (m, 2H, aromatic), 4.88 (m, 1H,
–COOCH2CH–9-fluorenyl), 4.68 (m, 2H, –COOCH2-9-fluorenyl),
4.34 (t, J = 6.6 Hz, 2H), 2.59 (s, 3H, Me of Dde group), 2.47 (s, 4H,

2756
T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
4.8. Solid phase total synthesis of argifin (1)
4.8.6. General procedure for deprotection of Dde group
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in DMF (1.5 mL) for 1 h, and filtered. The MicroKan
microreactor was sequentially treated with a solution of 2% hydra-
zine monohydrate in DMF (1.5 mL). The mixture was vigorously
agitated at room temperature. After being agitated for 1 h, the
reaction was filtered, washed with DMF (5 mL ꢀ 4), DCM
(5 mL ꢀ 4), and dried in vacuo.
The solid phase total synthesis of Argifin (1) was carried out in a
MicroKan microreactor initially filled with ca. 30 mg of 2-chlorotri-
tyl resin 5 (1.4 mmol/g, purchased from NovaBioChem) and radio
frequency chip with a view to an application of a combinatorial
synthesis of an Argifin library in near future.
4.8.1. General procedure for loading of Fmoc-Asp(OAllyl)-OH 6
on to 2-chlorotrityl chloride resin 5
The MicroKan microreactor, containing 2-chlorotrityl resin 5,
was placed into a 20 mL screw vial, swollen in DCM (1.5 mL) for
1 h, and filtered. The MicroKan microreactor was treated with a
4.8.7. General procedure for guanidination
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in DCM (1.5 mL) for 1 h, and filtered. The MicroKan mic-
roreactor was sequentially treated with a 1.0 M solution of 3 in DCM
(126 lL) and N-diisopropylethylamine (43.9 lL, 252 lmol) in DCM/
cocktail of N-diisopropylethylamine (43.9 lL, 2.52 mmol), Fmoc-
DMF (4/1, 1.5 mL). The mixture was vigorously agitated at room
temperature. After being agitated for 2 h, the reaction was filtered,
washed with DMF (5 mL ꢀ 4), DCM (5 mL ꢀ 4), and dried in vacuo.
Asp(OAllyl)-OH (6) (49.8 mg, 0.126 mmol) in DCM (1.5 mL). The
mixture was vigorously agitated at room temperature. After being
agitated for 2 h, the reaction was quenched with MeOH (0.05 mL)
to cap remaining reactive sites and agitated for additional
15 min. the MicroKans were filtered, washed with DMF
(5 mL ꢀ 4), DCM (5 mL ꢀ 4), and dried in vacuo.
4.8.8. Cleavage from the resin
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in DCM (1.5 mL) for 1 h, and filtered. The MicroKan
microreactor was sequentially treated with a cocktail of 90% TFA/
DCM (1.5 mL). The mixture was vigorously agitated at room tem-
perature. After being agitated for 3 h, the reaction was filtered,
and dried in vacuo to provide the crude Argifin (24 mg, 84.5 %).
4.8.2. General procedure for deprotection of Fmoc group
The MicroKan microreactor was placed into a 20 mL screw
vial and swollen in DCM (1.5 mL) for 1 h, and filtered. The Micro-
Kan microreactor was sequentially treated with a solution of
20% piperidine in DMF (1.5 mL). The mixture was vigorously
agitated at room temperature. After being agitated for 1 h, the
reaction was filtered, washed with DMF (5 mL ꢀ 4), DCM
(5 mL ꢀ 4), and dried in vacuo.
The use of HPLC purification (15% MeCN/H2O) furnished
(3.8 mg, 13.4 %) as a colorless solid.
1
4.8.9. Synthetic argifin
IR (KBr) m
cmꢁ1: 3370, 3280, 3080, 2940, 1720, 1645, 1600, 1550,
1500, 1450, 1400, 1260, ½a D20
ꢂ
¼ ꢁ51:2 (c 1.0, H2O), 1H NMR
4.8.3. General procedure for peptide coupling
(400 MHz, D2O + 0.4% TFA) d: 7.36 (dd, J = 7.0, 7.0 Hz, 2H, e1, e2 posi-
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in DMF (1.5 mL) for 1 h, and filtered. The MicroKan
microreactor was treated with a cocktail of each amino acids
(3.0 equiv, Fmoc-N-Me-Phe-OH (7), Fmoc-Ala-OH (9), Fmoc-Asp-
Ot-Bu (10)), PyBop (3.0 equiv), N-diisopropylethylamine
(6.0 equiv) in DCM/DMF (4/1, 1.5 mL), except in the case of cou-
pling to dipeptide 15 when Fmoc-Orn(Dde)-OH 8 (3.0 equiv),
HATU (3.0 equiv), and N-diisopropylethylamine (6.0 equiv) in
DCM/DMF (4/1, 1.5 mL) were used. The mixture was vigorously
agitated at room temperature. After being agitated for 2 h, the
reaction was filtered, washed with DMF (5 mL ꢀ 4), DCM
(5 mL ꢀ 4), and dried in vacuo.
tion of MePhe), 7.29 (dd, J = 7.0, 7.0 Hz, 1H, f position of MePhe), 7.23
(d, J = 7.0 Hz, 2H, d1, d2 position of MePhe), 5.13 (dd, J = 11.0, 3.0 Hz,
1H,
J = 12.0, 2.5 Hz, 1H,
position of Arg), 4.18 (q, J = 7.0 Hz, 1H,
a
position of MePhe), 4.81 (m, 1H,
position of Asp1), 4.29 (dd, J = 11.5, 2.5 Hz, 1H,
position of Ala), 3.17 (dd,
a position of Asp2), 4.56 (dd,
a
a
a
J = 14.0, 3.0 Hz, 1H, b position of MePhe), 3.08 (dd, J = 16.5, 2.0 Hz,
1H, b position of Asp1), 3.05 (m, 1H, b position of MePhe), 3.01 (m,
2H, d position of Arg), 2.88 (s, 3H, NMe of MePhe), 2.88 (m, 2H, b posi-
tion of Asp1), 2.80 (m, 2H, b position of Asp2), 2.75 (s, 3H, NMe of
Arg), 2.52 (dd, J = 13.5, 13.5 Hz, 2H, b position of Asp2), 1.40 (m,
1H,
c position of Arg), 1.31 (d, J = 7.0 Hz, 3H, b position of Ala), 1.15
(m, 1H,
c
position of Arg), 1.13 (m, 1H, b position of Arg), ꢁ0.30 (m,
1H, b position of Arg), 13C NMR (100 MHz, D2O+0.4% TFA) d: 175.4
4.8.4. General procedure for deprotection of allyl group
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in THF (1.5 mL) for 1 h under N2 atmosphere, and
drained THF by a syringe. To the MicroKan microreactor was
sequentially added a solution of Pd(PPh3)4 (1.5 equiv), dimedone
(10 equiv) in THF (1.5 mL) through a syringe under N2 atmosphere
after this solution was stirred and activated at room temperature
for 1 h under N2 atmosphere. The mixture was vigorously agitated
at room temperature. After being agitated for 1 h, the reaction was
filtered, washed with THF (5 mL ꢀ 4), DMF (5 mL ꢀ 4), DCM
(5 mL ꢀ 4), and dried in vacuo.
(–NHCO– of Ala), 175.2 (–NHCO– of Asp1), 174.4 (–NHCO– of Arg),
174.3(–NHCO–of Asp2), 171.5(
of Asp1), 170.3 (–NHCO– of MePhe), 155.3 (MeNHCONH–), 153.7
(MeNHCONHC(@NH)NH–), 137.5 (
position of MePhe), 129.8 (ꢀ2,
d1, d2 position of MePhe), 129.2 (ꢀ2, 1, 2 position of MePhe),
127.4 (f position of MePhe), 62.3 ( position of MePhe), 50.6 ( posi-
tion of Asp1), 50.0 ( position of Asp2), 49.7 ( position of Ala), 48.8
position of Arg), 40.8 (d position of Arg), 37.9 (b position of Asp2),
35.1 (b position of Asp1), 33.4 (b position of MePhe), 30.0 (NMe of
MePhe), 26.7 (b position of Arg), 26.1 (MeNHCO-), 24.1 ( position
cpositionof Asp2), 171.4(cposition
c
e
e
a
a
a
a
(a
c
of Arg), 16.9 (b position of Ala), HR-MS (FAB, thioglycerol + glycerol,
PEG600+NaI) calcd for C29H42O10N9: 676.3055 [M+H], found m/z:
676.3064 [M+H]+.
4.8.5. Cyclization
The MicroKan microreactor was placed into a 20 mL screw vial
and swollen in DCM (1.5 mL) for 1 h, and filtered. The MicroKan
microreactor was treated with a cocktail of HATU (2 equiv), DIPEA
(4 equiv), in DCM/DMF (4/1, 1.5 mL). The mixture was vigorously
agitated at room temperature. After being agitated for 2 h, the
reaction was filtered, washed with DMF (5 mL ꢀ 4), DCM
(5 mL ꢀ 4), and dried in vacuo. The same procedure was repeated
in this case.
x
4.9. N-Ac-Arg{N -(N-methylcarbanoyl)}-N-methyl-Phe-
Asp(OAllyl)-OH 18a
IR (KBr)
m
cmꢁ1: 3370, 3280, 3080, 3012, 1727, 1641, 1581,
1540, 1500, 1415, 1243, ½a D27
ꢂ
¼ ꢁ54:1 (c 1.0, MeOH), 1H NMR
(300 MHz, CD3OD) The mixture of rotamers was observed. d: 7.32
(dd, J = 7.0, 7.0 Hz, 2H, e1, e2 position of MePhe), 7.26–7.17 (com-

T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
2757
plex m, 3H, f position of MePhe, d1, d2 position of MePhe), 5.91
(complex m, 1H, b position of OAllyl), 5.31 (dd, J = 17.0, 5.0 Hz,
Ala), 1.29 (m, 1H,
c
position of Arg), 1.18 (m, 2H, b position of
Arg), 13C NMR (75 MHz, CD3OD) The mixture of rotamers was ob-
served. d: 175.2, 174.7, 174.6, 173.5, 172.1, 171.9, 171.7, 171.2,
156.2, 155.9, 139.4, 133.4, 130.8 (x2), 130.0 (x2), 129.6, 118.7,
66.7, 63.9, 50.8, 50.3, 48.9, 48.6, 41.8, 38.7, 36.7, 35.0, 30.3, 29.7,
28.5, 26.6, 22.5, 17.9, HR-MS (FAB, thioglycerol + glycerol,
PEG600+NaI) calcd for C34H50O12N9: 776.3579 [M+H], found m/z:
776.3579 [M+H]+.
1H,
of OAllyl) 5.06 (dd, J = 12.0, 3.0 Hz, 1H,
(m, 2H, of position of Asp2), 4.57 (dd, J = 5.0, 5.0 Hz, 1H,
tion of OAllyl), 4.30 (dd, J = 7.0, 2.0 Hz, 1H, position of Arg), 3.21
c
position of OAllyl), 5.21 (dd, J = 10.0, 1.0 Hz, 1H,
position of MePhe), 4.80
posi-
c position
a
a
a
a
(dd, J = 14.0, 2.0 Hz, 1H, b position of MePhe), 3.10–2.90 (complex
m, 3H, b position of MePhe, d position of Arg), 2.92 (m, 2H, b posi-
tion of Asp2), 2.86 (t, 3H, NMe of Arg), 2.78 (s, 1.5H, NMe of MePhe,
rotamer), 2.76 (s, 1.5H, NMe of MePhe, rotamer), 1.92 (s, 2H, Me of
x
4.12. N-Ac-Arg{N -(N-methylcarbanoyl)}-N-methyl-Phe-OH
Ac, rotamer), 1.89 (s, 1H, Me of Ac, rotamer), 1.61(m, 1H,
c
position
19a
of Arg), 1.28 (m, 1H, position of Arg), 1.16 (d, 2H, b position of
c
Arg), 13C NMR (75 MHz, CD3OD) The mixture of rotamers was ob-
served. d: 174.5, 174.2, 173.5, 171.7, 171.2, 155.9, 155.8, 139.4,
133.4, 130.8 (x2), 130.0 (x2), 129.6, 118.7, 66.6, 63.6, 50.4, 49.1,
42.0, 36.7, 35.1, 30.3, 30.0, 28.9, 26.6, 22.2, HR-MS (FAB, thioglyc-
erol + glycerol, PEG600+NaI) calcd for C27H40O8N7: 590.2957
[M+H], found m/z: 590.2938 [M+H]+.
IR (KBr) m
cmꢁ1: 3379, 3197, 3095, 3025, 1683, 1639, 1562,
1544, 1494, 1423, 1251, ½a D26
ꢂ
= –61.9 (c 1.0, MeOH), 1H NMR
(300 MHz, CD3OD) The mixture of rotamers was observed. d: 7.33
(dd, J = 7.0, 7.0 Hz, 2H,
plex m, 3H, f position of MePhe, d1, d2 position of MePhe), 5.02 (dd,
J = 12.0, 3.0 Hz, 1H, position of MePhe), 4.30 (dd, J = 7.0, 7.0 Hz,
1H, position of Arg), 3.24 (dd, J = 14.0, 7.0 Hz, 1H, b position of
e1, e2 position of MePhe), 7.28–7.18 (com-
a
a
x
4.10. N-Ac-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-N-methyl-Phe-
MePhe), 3.10–3.00 (complex m, 3H, b position of MePhe, d position
of Arg), 2.91 (s, 3H, NMe of MePhe), 2.78 (s, 1.5H, NMe of Arg, rot-
amer), 2.76 (s, 1.5H, NMe of Arg, rotamer), 1.90 (s, 1H, Me of Ac,
Asp(OAllyl)-OH 18b
IR (KBr)
m
cmꢁ1: 3415, 3300, 3080, 2940, 1725, 1675, 1600,
rotamer), 1.89 (s, 2H, Me of Ac, rotamer), 1.64 (m, 1H,
c
position
1538, 1486, 1450, 1400, 1200, ½a D27
ꢂ
¼ ꢁ59:1 (c 1.0, MeOH), 1H
of Arg), 1.28 (m, 1H,
c position of Arg), 1.18 (m, 2H, b position of
NMR (300 MHz, CD3OD) The mixture of rotamers was observed. d:
Arg), 13C NMR (75 MHz, CD3OD) The mixture of rotamers was ob-
served. d: 173.6, 173.4, 172.7, 156.0, 155.8, 139.1, 130.5 (ꢀ2),
130.0 (ꢀ2), 129.5, 62.8, 49.1, 41.9, 35.2, 30.8, 29.9, 29.7, 26.5,
22.3, HR-MS (FAB, thioglycerol + glycerol, PEG600 + NaI), calcd for
C20H31O5N6: 435.2356 [M+H], found m/z: 435.2346 [M+H]+.
7.33 (dd, J = 7.0, 7.0 Hz, 2H, e1, e2 position of MePhe), 7.26–7.17
(complex m, 3H, f position of MePhe, d1, d2 position of MePhe),
5.91 (complex m, 1H, b position of OAllyl), 5.30 (dd, J = 17.0,
11.0 Hz, 1H,
position of OAllyl), 5.11 (dd, J = 12.0, 4.0 Hz, 1H,
MePhe), 4.78 (m, 1H, position of Asp2), 4.57 (dd, J = 11.0 Hz,
6.0 Hz, 2H, of position of OAllyl), 4.28 (m, 2H, position of Ala,
position of Arg), 3.21 (dd, J = 14.0, 3.0 Hz, 1H, b position of
c
position of OAllyl), 5.22 (dd, J = 11.0, 7.0 Hz, 1H,
c
a
position of
x
a
4.13. N-Ac-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-N-methyl-Phe-
a
a
OH 19b
a
MePhe), 3.10–2.92 (complex m, 3H, b position of MePhe, d position
of Arg), 2.69 (s, 2H, NMe of MePhe), 2.67 (s, 1H, NMe of MePhe, rot-
amer), 2.88 (s, 3H, NMe of Arg), 2.83 (m, 2H, b position of Asp2),
1.89 (s, 1H, Me of Ac, rotamer), 1.87 (s, 2H, Me of Ac, rotamer),
IR (KBr) m
cmꢁ1: 3328, 3100, 3000, 2900, 1677, 1641, 1560,
1540, 1442, 1417, 1376, 1200,
NMR (300 MHz, CD3OD) The mixture of rotamers was observed. d:
½
a 2D6
ꢂ
¼ ꢁ31:4 (c 1.0, MeOH), 1H
7.34 (dd, J = 7.0, 7.0 Hz, 2H,
(complex m, 3H, f position of MePhe, d1, d2 position of MePhe),
5.15 (dd, J = 11.0, 5.0 Hz, 1H, position of MePhe), 4.24 (m, 2H,
position of Ala, position of Arg), 3.11 (dd, J = 12.0, 4.0 Hz, 1H, b
e1, e2 position of MePhe), 7.30–7.18
1.52 (m, 1H,
of Ala), 1.16 (m, 1H,
c
position of Arg), 1.20 (d, J = 6.7 Hz, 3H, b position
position of Arg), 1.03 (m, 2H, b position of
c
a
a
Arg), 13C NMR (75 MHz, CD3OD) The mixture of rotamers was ob-
served. d: 175.2, 174.2, 173.5, 172.1, 171.7, 171.1, 156.0, 155.8,
139.2, 133.4, 130.8 (ꢀ2), 129.6 (ꢀ2), 129.6, 118.8, 66.7, 63.5,
49.8, 49.1, 48.6, 42.0, 36.8, 35.2, 30.3, 29.8, 28.9, 26.6, 22.6, 18.0,
HR-MS (FAB, thioglycerol + glycerol, PEG600+NaI), calcd for
C30H45O9N8: 661.3345 [M+H], found m/z: 661.3310 [M+H]+.
a
position of MePhe), 3.17–3.00 (complex m, 3H, b position of
MePhe, d position of Arg), 2.96 (s, 3H, NMe of MePhe), 2.80 (s,
1H, NMe of Arg, rotamer), 2.77 (s, 2H, NMe of Arg, rotamer), 2.00
(s, 2H, Me of Ac, rotamer), 1.97 (s, 1H, Me of Ac, rotamer), 1.61
(m, 1H,
Ala), 1.23 (m, 1H,
c
position of Arg), 1.30 (d, J = 4.0 Hz, 3H, b position of
position of Arg), 1.09 (m, 2H, b position of
c
x
4.11. N-Ac-Asp-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-N-methyl-
Arg), 13C NMR (75 MHz, CD3OD) The mixture of rotamers was ob-
served. d: 174.8, 173.8, 173.3, 173.2, 156.0, 155.8, 138.9, 130.6
(ꢀ2), 130.0 (ꢀ2), 129.5, 64.5, 48.9, 48.6, 41.9, 35.3, 30.8, 29.8,
28.8, 26.6, 22.4, 18.0, HR-MS (FAB, thioglycerol + glycerol,
PEG600 + NaI), calcd for C23H36O6N7: 506.2727 [M+H], found m/z:
506.2716[M+H]+.
Phe-Asp(OAllyl)-OH 18c
IR (KBr)
m
cmꢁ1: 3423, 3300, 3000, 2900, 1725, 1670, 1600,
1536, 1500, 1434, 1396, 1200, ½a D27
ꢂ
¼ ꢁ28:1 (c 1.0, MeOH), 1H
NMR (300 MHz, CD3OD) The mixture of rotamers was observed. d:
7.32 (dd, J = 7.0, 7.0 Hz, 2H, e1, e2 position of MePhe), 7.23–7.17
(complex m, 3H, f position of MePhe, d1, d2 position of MePhe),
x
4.14. N-Ac-Asp-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-N-methyl-
5.91 (complex m, 1H, b position of OAllyl), 5.31 (dd, J = 17.0,
Phe-OH 19c
7.0 Hz, 1H,
sition of OAllyl), 5.08 (dd, J = 11.0, 3.0 Hz, 1H,
4.86 (m, 1H, position of Asp2), 4.73 (dd, J = 11.0, 5.0 Hz, 1H,
sition of Asp1), 4.58 (dd, J = 6.0 Hz, 6.0 Hz, 2H, of position of OAl-
lyl), 4.36 (m, 2H, position of Ala, position of Arg), 3.22 (dd,
J = 14.0, 3.0 Hz, 1H, b position of MePhe), 3.04 (dd, J = 13.0,
4.0 Hz, 2H, b position of Asp1), 3.01–2.97 (complex m, 3H, b posi-
tion of MePhe, d position of Arg), 2.92 (m, 2H, b position of Asp2),
2.85 (s, 3H, NMe of MePhe,), 2.77 (s, 1.5H, NMe of Arg, rotamer),
2.76 (s, 1.5H, NMe of Arg, rotamer), 1.99 (s, 3H, Me of Ac), 1.62
c
position of OAllyl), 5.21 (dd, J = 10.0, 2.0 Hz, 1H,
position of MePhe),
po-
c po-
a
IR (KBr) m
cmꢁ1: 3415, 3100, 3000, 2900, 1720, 1676, 1583,
a
a
1539, 1486, 1435, 1400, 1201, ½a D27
ꢂ
¼ ꢁ38:1 (c 1.0, MeOH), 1H
a
NMR (300 MHz, CD3OD) The mixture of rotamers was observed. d:
a
a
7.32 (dd, J = 7.0, 7.0 Hz, 2H,
(complex m, 3H, f position of MePhe, d1, d2 position of MePhe),
5.04 (dd, J = 11.0, 3.0 Hz, 1H, position of MePhe), 4.78 (dd,
J = 10.0, 6.0 Hz, 1H, position of Asp1), 4.25 (m, 2H, position of
Ala, position of Arg), 3.24 (dd, J = 12.0, 6.0 Hz, 1H, b position of
e1, e2 position of MePhe), 7.26–7.18
a
a
a
a
MePhe), 3.12 (dd, J = 12.0, 6.0 Hz, 2H, b position of Asp1), 3.08–
(m, 1H,
c position of Arg), 1.32 (d, J = 6.7 Hz, 3H, b position of
2.99 (complex m, 3H, b position of MePhe, d position of Arg),

2758
T. Sunazuka et al. / Bioorg. Med. Chem. 17 (2009) 2751–2758
2.96 (s, 3H, NMe of MePhe,), 2.79 (s, 1.5H, NMe of Arg, rotamer),
2.76 (s, 1.5H, NMe of Arg, rotamer), 1.98 (s, 1H, Me of Ac, rotamer),
(SmChiA-x10 dilution; SmChiB-x40 dilution; SmChiC1-x4 dilution
with 0.1 M phosphate buffer pH 7.0) (see the procedures for the
1.97 (s, 2H, Me of Ac, rotamer), 1.66 (m, 1H,
(d, J = 7.0 Hz, 3H, b position of Ala), 1.35 (m, 1H,
c
position of Arg), 1.29
position of Arg),
preparation of SmChiA, B, and C1), and 50 lL of 80 lM 4-meth-
ylumberiferyl-b-D-N,N’-diacethylchitobiose [4-MU-(GluNAc)2, Sig-
ma] in 0.1 M phosphate buffer (pH 7.0) were placed in each well of
a microplate, and incubated with 10lL of inhibitors in MeOH for
5 min. Fluorescence (excitation at 355 nm, emission at 460 nm)
was measured at intervals of 60 seconds by fluorometer (Fluoro-
scan II, Labsystems), and the rate of 4-methylumbelliferone pro-
duction was corrected by calibrating the quenching ratio of each
inhibitors using the mixture of the inhibitors and 4-methylumbel-
liferone (4MU).
c
1.00 (m, 2H, b position of Arg), 13C NMR (75 MHz, CD3OD) The mix-
ture of rotamers was observed. d: 174.4, 174.3, 173.8, 173.4, 172.5,
172.1, 156.0, 155.9, 138.8, 130.6 (ꢀ2), 130.1 (ꢀ2), 130.0, 63.1,
50.8, 49.7, 49.2, 41.9, 38.5, 35.1, 30.2, 29.9, 29.7, 26.6, 22.6, 18.1,
HR-MS (FAB, thioglycerol + glycerol, PEG600 + NaI), calcd for
C27H41O9N8: 621.2997 [M+H], found m/z: 621.3008 [M+H]+.
x
4.15. N-Ac-Arg{N -(N-methylcarbanoyl)}-OH 20a
IC50 values were determined from dose-response sigmoidal
curves, which were calculated by using KaleidaGraphÒ (Synergy
Software Inc., USA) with the experimental data.
IR (KBr)
m
cmꢁ1: 3316, 3116, 2921, 2850, 1685, 1640, 1560,
1545, 1508, 1425, 1253, 1205, ½a D24
ꢂ
¼ 2:06 (c 1.0, MeOH), 1H
NMR (300 MHz, CD3OD) d: 4.39 (s, 1H,
a position of Arg), 3.73–
3.00 (m, 2H, d position of Arg), 2.77 (s, 3H, NMe of Arg), 2.01 (s,
Acknowledgments
1H, Me of Ac), 1.73 (m, 1H,
c position of Arg), 1.33 (m, 1H, c posi-
tion of Arg), 1.14 (m, 2H, b position of Arg), 13C NMR (75 MHz,
CD3OD) d: 176.3, 173.4, 156.1, 155.8, 48.9, 41.8, 30.1, 26.5, 25.8,
22.4, HR-MS (FAB, thioglycerol + glycerol, PEG600 + NaI), calcd for
C10H20O4N5: 274.1545 [M+H], found m/z: 274.1515 [M+H]+.
This work was supported by the Grant of the 21st Century COE
Program, Ministry of Education Culture, Sports, Science and Tech-
nology. We thank the JSPS Research Fellowships for Young Scien-
tists to A.S. We also thank Ms. A. Nakagawa and Ms. N. Sato for
various instrumental analysis. K. Nagai acknowledges a Kitasato
University research grant for young researchers.
x
4.16. N-Ac-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-OH 20b
IR (KBr) m
cmꢁ1: 3369, 3083, 2948, 1675, 1650, 1560, 1546, 1461,
1425, 1379, 1203, ½a D23
ꢂ
¼ 11:2 (c 1.0, MeOH), 1H NMR (300 MHz,
References and notes
CD3OD) The mixture of rotamers was partly observed. d: 4.43 (dd,
J = 9.0, 5.0 Hz, 1H,
position of Ala), 3.16 (t, J = 7.0 Hz, 2H, d position of Arg), 2.76 (s,
3H, NMe of Arg), 1.98 (s, 3H, Me of Ac), 1.73 (m, 1H, position of
Arg), 1.35 (d, J = 7.0 Hz, 3H, b position of Ala), 1.31 (m, 1H, position
a position of Arg), 4.33 (dd, J = 14.0, 7.0 Hz, 1H,
1. Munro, C. A.; Gow, N. A. R. Med. Mycol. 2001, 39, 41.
2. Cabib, E.; Silverman, S. J.; Shaw, J. A. J. Gen. Microbiol. 1992, 138, 97.
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4. Sakuda, S.; Isogai, A.; Matsumoto, S.; Suzuki, A. J. Antibiot. 1987, 40, 296.
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Fidock, D. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14061; (b) Vinetz, J. M.;
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a
c
c
of Arg), 1.13 (m, 2H, b position of Arg), 13C NMR (75 MHz, CD3OD) The
mixture of rotamers was partly observed. d: 174.6, 174.2, 173.4, 156.0,
155.8, 48.9, 48.3, 41.8, 29.7, 29.3, 26.5, 22.4), 17.9. HR-MS (FAB, thi-
oglycerol + glycerol, PEG600 + NaI), calcd for C13H25O5N6: 345.1917
[M+H], found m/z: 345.1886[M+H]+.
6. Zhu, Z.; Zheng, T.; Homer, R. J.; Kim, Y. K.; Chen, N. Y.; Cohn, L.; Hamid, Q.; Elias,
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x
4.17. N-Ac-Asp-
D
-Ala-Arg{N -(N-methylcarbanoyl)}-OH 20c
¯
7. (a) Omura, S.; Arai, N.; Yamaguchi, Y.; Masuma, R.; Iwai, Y.; Namikoshi, M.;
Turberg, A.; Kölbl, H.; Shiomi, K. J. Antibiot. 2000, 53, 603; (b) Arai, N.; Shiomi,
¯
K.; Iwai, Y.; Omura, S. J. Antibiot. 2000, 53, 609; (c) Shiomi, K.; Arai, N.; Iwai, Y.;
IR (KBr)
m
cmꢁ1: 3359, 3087, 2958, 1716, 1652, 1558, 1550,
¯
Turberg, A.; Kölbl, H.; Omura, S. Tetrahedron Lett 2000, 41, 2141.
1455, 1434, 1392, 1201, ½a D24
ꢂ
¼ 5:66 (c 1.0, MeOH), 1H NMR
8. Hirose, T.; Sunazuka, T.; Sugawara, A.; Endo, A.; Iguchi, K.; Yamamoto, T.; Ui, H.;
¯
Shiomi, K.; Watanabe, T.; Sharpless, K. B.; Omura. S. J. Antibiot., submitted for
(300 MHz, CD3OD) The mixture of rotamers was observed. d: 4.79
publication.
(dd, J = 12.0, 7.0 Hz, 1H,
of Ala, position of Arg), 3.17 (dd, J = 7.0, 7.0 Hz, 2H, b position of
Asp1), 3.10–2.82 (m, 2H, d position of Arg), 2.77 (s, 3H, NMe of Arg),
1.97 (s, 3H, Me of Ac), 1.70 (m, 1H, position of Arg), 1.36 (d,
J = 6.0 Hz, 3H, b position of Ala), 1.14 (complex m, 3H, position
a position of Asp1), 4.38 (m, 2H, a position
¯
9. (a) Houston, D. R.; Shiomi, K.; Arai, N.; Omura, S.; Peter, M. G.; Turberg, A.;
a
Synstad, B.; Eijsink, V. G. H.; van Aalten, D. M. F. Proc. Natl. Acad. Sci. U.S.A. 2002,
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M.; Adams, D. J.; Shiomi, K.; Omura, S.; van Aalten, D. M. F. Chem. Biol. 2005, 12,
c
65.
c
10. Dixon, M. J.; Andersen, O. A.; van Aalten, D. M. F.; Eggleston, I. M. Bioorg. Med.
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of rotamers was observed. d: 174.9, 174.7, 173.8, 173.5, 172.1,
156.0, 155.9, 50.6, 49.7, 48.6, 41.8, 30.0, 29.6, 26.7, 22.5, 18.2,
HR-MS (FAB, thioglycerol + glycerol, PEG600 + NaI), calcd for
C17H30O8N7: 460.2140 [M+H], found m/z: 460.2156 [M+H]+.
11. (a) Bycroft, B. W.; Chan, W. C.; Chahabra, S. R.; Hone, N. D. J. Chem. Soc., Chem.
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4.18. Procedures for IC50 measurements against each SmChiA,
B, and C1
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Ten microliters of 0.1 M phosphate buffer (pH 7.0), 10
lL of
each inhibitors in MeOH, 30 L of diluted crude chitinase solution
l