Effect of prime-site sequence of retro-inverso-modified HTLV-1 protease inhibitor

Article abstract of DOI:10.1016/j.bmc.2014.02.050

The effects of additional substituents covering the prime-site of retro-inverso (RI)-modified HTLV-1 protease inhibitors containing a hydroxyethylamine isoster were clarified. Stereo-selective construction of the most potent isoster backbone was achieved by the Evans-aldol reaction. Addition of N-acetylated d-amino acid corresponding to the P₂′ site gave an RI-modified inhibitor showing superior inhibitory activity to the previous inhibitor. Inhibitory activities of the newly synthesized inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.

Full text of DOI:10.1016/j.bmc.2014.02.050

Bioorganic & Medicinal Chemistry 22 (2014) 2482–2488
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of prime-site sequence of retro-inverso-modified HTLV-1
protease inhibitor
Chiyuki Awahara a, Tadashi Tatsumi a, Saki Furuta b, Gen Shinjoh b, Hiroyuki Konno c, Kazuto Nosaka d,
Kazuya Kobayashi b, Yasunao Hattori b, Kenichi Akaji b,
⇑
a Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan
b Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
c Department of Biochemical Engineering, Graduate School of Science and Technology, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
d Department of Chemistry, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of additional substituents covering the prime-site of retro-inverso (RI)-modified HTLV-1 pro-
tease inhibitors containing a hydroxyethylamine isoster were clarified. Stereo-selective construction of
the most potent isoster backbone was achieved by the Evans-aldol reaction. Addition of N-acetylated
Received 4 February 2014
Revised 20 February 2014
Accepted 24 February 2014
Available online 4 March 2014
D
-amino acid corresponding to the P02 site gave an RI-modified inhibitor showing superior inhibitory
activity to the previous inhibitor. Inhibitory activities of the newly synthesized inhibitors suggest that
partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent
hydroxyethylamine inhibitor.
Keywords:
HTLV-1 protease
Inhibitor
Ó 2014 Elsevier Ltd. All rights reserved.
Retro-inverso
Hydroxyethylamine isoster
1. Introduction
topography of side-chain orientations is retained by RI modifica-
tion.6 Similar or improved interactions of RI-modified peptides with
Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus eti-
ologically associated with adult T-cell leukemia and a number of
other chronic diseases.1 HTLV-1 protease is an aspartic protease,
crucial for processing virus proteins. Thus, HTLV-1 protease is a suit-
able target for the development of inhibitors for therapeutic use. We
have previously reported the synthesis and structure–activity rela-
tionship of HTLV-1 protease inhibitors containing a transition-state
mimic, hydroxyethylamine dipeptide isoster.2 The results clearly
showed that the configurations at the hydroxyl- and side chain-
bearing asymmetric centers of the mimic have marked effects on
inhibitory activity. It was also shown that the P3–P01 sequence of
the substrate-based inhibitor is a core sequence for potent inhibi-
tory activity.3 Based on these studies, we found for the first time that
retro-inverso (RI) modification of the inhibitor containing the tran-
sition-state mimic can retain inhibitory activity.4
proteins or receptors have also been reported.7 However, few appli-
cations of the RI modification to protease inhibitors have been
reported. We found that RI modification of HTLV-1 protease inhib-
itors containing hydroxyethylamine dipeptide isoster 2 showed
inhibitory activity, suggesting that the RI-modified inhibitor re-
tained similar topography to the parent inhibitor 14 (Fig. 1b). The
relative relationship between the isoster hydroxyl and scissile site
substituent in 2 is the same as that in the parent inhibitor 1, in which
the side-chain substituent is supposed to interact with the protease
S1 pocket. The RI-modified inhibitor 2 contains D-amino acids cover-
ing the non-prime P2 to P5 sites, but it contains no additional prime
site amino acids except for the P01 site. In this paper, we examined
whether the additional D-amino acids or substituents covering the
prime site would have effects on inhibitory activity.
RI modifications involve a reversion of the peptide sequence
2. Results and discussion
accompanied by the replacement of each
L-amino acid with the cor-
responding
D
-amino acid5 (Fig. 1a). Fully or partially RI-modified
In the previous syntheses of RI-modified inhibitors, a stereo-
selective aldol reaction using a chiral auxiliary, 1-arylsulfonamido-
2-indanylrate, 8 was employed to evaluate the most potent configu-
ration at the hydroxyethylamine dipeptide isoster. Thus, all four dia-
stereomers containing different configurations at the isoster
hydroxyl site and scissile site substituent were stereo-selectively
biologically active peptides have been found to retain the properties
or biological activity of the parent peptides, since a similar
⇑
Corresponding author. Tel./fax: +81 75 595 4635.
E-mail address: akaji@mb.kyoto-phu.ac.jp (K. Akaji).
http://dx.doi.org/10.1016/j.bmc.2014.02.050
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

C. Awahara et al. / Bioorg. Med. Chem. 22 (2014) 2482–2488
2483
(a) Retro Inverso modification
reversion of peptide
sequence
replacement of L-amino
acid to D-amino acid
HOOC-ADBDCDDD-NH2
H2N-LALBLCLD-COOH
H2N-LDLCLBLA-COOH
(b) RI-modified HTLV-1 protease inhibitor
C-terminal
H2N
O
S2
S2
S1'
N-terminal
NH
S1'
OH
N
O
O
H
N
H
N
OH
O
O
H
N
N
I
N
H
N
N
N
H
O
O
N
O
O
N
H
O
H
S3
1 (substrate-based inhibitor)
Figure 1. Retro-inverso modification of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.
S1
C-terminal
N-terminal
S3
S1
2 (RI-modified inhibitor)
prepared by the combination of the indanylrate auxiliary and
L
- or
D
-
yield peptide resin 9. Completion of each coupling reaction was
confirmed by the ninhydrin test. The dipeptide isoster 8 (2.6 equiv)
was introduced by DIPCDI-HOAt12-mediated coupling (25 °C, 24 h),
and the N -Fmoc of the product was removed by treatment with
20% piperidine/DMF to give an intermediate 10. Acylation of the
amino acid. Based on these results, in this study, we focused on an
isoster having the most potent syn-configuration. The necessary
key intermediate 7 was stereo-selectively synthesized employing
a diastereo-selective aldol reaction using Evans oxazolidin-2-one
auxiliary9 (Scheme 1). Thus, 4-methylvaleric acid was condensed
with oxazolidine-2-one 3 according to the literature procedure to
yield desired product 4. The oxazolidinone 4 was treated with
slightly less equivalent of n-Bu2BOTf and then with a known alde-
hyde 54 to give the desired product 6 as a single diastereomer. With-
out further purification, the amide 6 was esterified with NaOMe
followed by hydrolysis with NaOH to yield N-Boc protected carbox-
ylic acid derivative 7 having the desired dipeptide isoster backbone
with 35% yield (in three steps). Use of an equivalent or slightly excess
equivalent of n-Bu2BOTf lowered the yield to 17–0%. Thiazolidine-2-
one auxiliary10 instead of the conventional Evans auxiliary gave no
desired product in this case.
a
terminal amino group on the resin was conducted using an excess
a
amount of carboxylic acid or chloride derivative to give the N -
modified peptide resin. Each peptide resin thus synthesized was
then treated with TFA (25 °C, 2 h) to cleave each inhibitor from
the resin. The crude product obtained by precipitation with ether
was purified by reverse-phase preparative HPLC to yield the de-
sired derivative 11–18 (overall yield, 7–29%). The homogeneity of
each synthesized compound was confirmed by analytical HPLC,
13
TOF-MS, and 1H NMR.
For further elongation to the P02 and P30 sites, each
D-amino acid
derivative was condensed to 10 using DIPCDI-HOBt as above with-
out difficulty (Scheme 3). Each peptide resin was then treated with
TFA (25 °C, 2 h) to yield the corresponding derivative 20–22. The
crude products isolated as above were purified by preparative
HPLC to give a single peak on analytical HPLC.13
The inhibitory activities of RI-modified inhibitors thus obtained
were examined using a synthetic mutant HTLV-1 protease and
substrate dodecapeptide according to our previous procedure.14
Cleavage of the substrate by a mutant protease in the presence of
the RI-modified inhibitor was monitored by analytical HPLC. The
rate of cleavage was estimated from the decrease in the substrate
during incubation. The inhibitory activity of each RI-modified
inhibitor was evaluated using the corresponding IC50 value ob-
tained from the sigmoidal dose–response curve (Table 1). The
experiment was repeated three times and obtained IC50 values be-
Condensation of the necessary D-amino acid derivatives for the
synthesis of RI-modified HTLV-1 protease inhibitors was carried
out by conventional Fmoc-based solid phase peptide synthesis
(SPPS) (Scheme 2). For the SPPS, Boc protecting group of the dipep-
tide isoster 7 was first converted to Fmoc derivative 8. Starting
from commercially available Rink amide resin (4-(20,40-dimethoxy-
phenyl-Fmoc-aminomethyl)-phenoxy resin)11, each
D-amino acid
derivative (2.6 equiv) corresponding to the P2 to P5 sites was suc-
cessively introduced by the combination of 20% piperidine/DMF
treatment and DIPCDI-HOBt-mediated coupling (25 °C, 2 h) to
O
1) 1.6M BuLi
2) 4-Methyl-
valeric acid
O
O
1M n-Bu2BOTf
then
HN
O
3
N
low 600
although the values over 1 mM gave rise to apparent deviation.
In contrast to our previous RI inhibitor 2 (IC50 = 160 M), com-
lM were in good agreement in three experiments,
O
76%
O
l
N
Boc
4
5
pound 11 having an N-terminal imino group without the acyl
group showed no inhibitory activity. N-benzoylated compound
12 showed weak inhibitory activity (IC50 = 1.1 mM), and insertion
of two atoms between the aromatic ring and carbonyl carbon
recovered inhibitory activities to nearly the same level as the pre-
vious compound 2. The effect was not correlated with the atom
OH
O
OH
O
1) NaOMe
2) NaOH
COOH
N
Boc
N
N
O
R
species (compounds 13 to 15, IC50 = 160 to 380 lM), which sug-
gests that the effect may be attributed to the planar aromatic ring
suitably situated away from the N-terminus. Substitution with
hydrophobic aliphatic groups instead of the aromatic ring de-
creased the inhibitory activities; substitution with a normal or sec-
ondary alkyl group maintained moderate inhibitory activity
7
8
(R=Boc); 35% in 3 steps
(R=Fmoc); 73% in 2 steps
6
1) TFA
2) Fmoc-OSu
Scheme 1.

2484
C. Awahara et al. / Bioorg. Med. Chem. 22 (2014) 2482–2488
1) piperidine
2) Fmoc-AA-OH/DIPCDI/HOBt
1) piperidine
8
H
N
2) /DIPCDI/HOAt
H
Fmoc
TFA
Fmoc N
DIle-DVal-DPro-DPro
9
3) piperidine
OH
OH
O
O
H
N
H
N
H
DIle-DVal-DPro-DPro
10
N
NH2
DIle-DVal-DPro-DPro
11
OH
1) BzCl
2) TFA
O
O
O
14
N
Bz
DIle-DVal-DPro-DPro NH2
12
O
15
O
1) ZCl
2) TFA
OH
OH
R
16
N
Z
DIle-DVal-DPro-DPro NH2
13
O
1) coupling
2) TFA
17
O
N
R
DIle-DVal-DPro-DPro NH2
18
O
14-18
Scheme 2.
1) Fmoc-DIle-OH
DIPCDI/HOBt
2) piperidine
OH
OH
O
O
TFA
H
N
H-DIle
N
H-DIle
DIle-DVal-DPro-DPro
19
N
NH2
DIle-DVal-DPro-DPro
20
10
1) Ac2O/Et3N/DMAP
2) TFA
1) Fmoc-DGln-OH/DIPCDI/HOBt
2) piperidine
3) TFA
OH
O
OH
O
N
N
NH2
DIle-DVal-DPro-DPro
21
DIle-DVal-DPro-DPro NH2
22
Ac-DIle
H-DGln-DIle
Scheme 3.
(compounds 16 and 17, IC50 = 520 and 420
group marked lowered the activity (compound 18, IC50 = 1 mM).
Further elongation of the corresponding -amino acids toward
l
M), but a tertiary alkyl
Thus, prime-site modification of the RI-modified HTLV-1 prote-
ase inhibitor based on hydrophobicity can improve inhibitory
activity, although only moderately. The results suggest that an
appropriately RI-modified inhibitor interacts with the protease in
a similar manner as the substrate-based inhibitor. Based on the
similar topography at side-chain orientations of the RI-modified
inhibitor, further optimization of the side-chain structures is now
D
the P02 and P03 sites marked lowered inhibitory activity when the
compounds had an N-terminal amino group (compounds 20 and
22, IC50 = 1.5 and 2.0 mM). Thus, the presence of a terminal ionic
group, regardless of the prime site position, showed strong nega-
tive effects on the interactions of the inhibitor and HTLV-1 prote-
underway by replacing the
D-Ile to D-allo-Ile. Replacements of D-
ase. Acylation of the P02 site
to yield compound 21 (IC50 = 110
D
-Ile increased the inhibitory activity
Pro to -Methyl Pro to suppress the main-chain cis/trans conver-
D
l
M) which was 1.5 times more
sion at the Pro residue are also underway.
potent than the previous RI inhibitor 2. These results suggest that
the small aliphatic or aromatic substitution situated three atom
distances from the P01 site imino group in the RI-modified inhibitor
can interact with the protease S02 pocket as with conventional sub-
strate-based statin-type inhibitor or hydroxymethylcarbonyl
3. Experimental
3.1. General
1H and 13C NMR spectra were measured with a Bruker AM-300
FT-NMR spectrometer in CDCl3 or CD3OD at 300 and 75 MHz,
respectively. Chemical shifts were relative to tetramethylsilane as
(HMC)-type inhibitors. In the crystal structures of HTLV-1 protease
15
complexed with those inhibitors,
the hydrophobic interactions
at these prime-sites were clearly demonstrated.

Table 1
compound
IC50 (µM)
compound
IC50 (µM)
OH
OH
OH
OH
O
O
O
O
N
H
N
DIle-DVal-DPro-DPro NH2
11
DIle-DVal-DPro-DPro NH2
16
no inhibition
520
O
O
OH
OH
OH
OH
OH
O
O
O
O
O
N
N
N
N
N
N
N
DIle-DVal-DPro-DPro NH2
17
DIle-DVal-DPro-DPro NH2
12
1100
160
380
290
160
420
1000
1500
110
O
H
N
NH2
NH2
DIle-DVal-DPro-DPro
18
DIle-DVal-DPro-DPro
2
O
O
OH
OH
OH
O
O
O
N
N
N
DIle-DVal-DPro-DPro NH2
13
NH2
O
DIle-DVal-DPro-DPro
20
H2N
HN
O
O
O
O
O
O
DIle-DVal-DPro-DPro NH2
21
DIle-DVal-DPro-DPro NH2
14
O
O
NH2
DIle-DVal-DPro-DPro NH2
22
DIle-DVal-DPro-DPro
15
2000
HN
H2N
H2NOC

2486
C. Awahara et al. / Bioorg. Med. Chem. 22 (2014) 2482–2488
an internal standard. The coupling constants were given in Hz.
Mass spectra were obtained on JMS-SX 102A and Bruker Daltonics
autoflex mass spectrometers. Optical rotations were determined
with a HORIBA SEPA-300 polarimeter. Preparative HPLC was per-
formed using a COSMOSIL 5C18-ARII column (10 ꢀ 250 mm) with
a linear gradient of CH3CN containing 0.1% TFA in 0.1% aqueous
TFA at a flow rate of 3.0 mL/min on a HITACHI ELITE LaChrom sys-
tem (OD, 220 nm). For analytical HPLC, a COSMOSIL 5C18-ARII col-
umn (4.6 ꢀ 150 mm) was employed with a linear gradient of
CH3CN containing 0.1% TFA in 0.1% aqueous TFA at a flow rate of
1.0 mL/min on a HITACHI ELITE LaChrom system (OD, 220 nm).
combined organic layer was dried over MgSO4, filtered, and con-
centrated. The residue was roughly purified by silica gel column
chromatography (hexane/EtOAc = 4:1). The crude product, without
further purification, was dissolved in MeOH/1 M NaOH (1:1,
10 mL) and stirred for 16 h. The reaction was quenched with 1 M
HCl. The mixture was extracted with EtOAc, dried over Na2SO4, fil-
tered, and concentrated. The residue was purified by silica gel col-
umn chromatography (CHCl3/MeOH/AcOH = 200:2.5:0.5) to yield 7
(210 mg, 35%, 3 steps) as a white powder. 1H NMR (300 MHz,
CDCl3): d 0.91 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H), 1.31–
1.53 (m, 2H), 1.47 (s, 9H), 1.56–1.75 (m, 3H), 1.83–2.00 (m, 4H),
2.58–2.64 (m, 1H), 3.27–3.36 (m, 2H), 3.73–3.75 (m, 1H), 4.15–
4.20 (m,1H). 13C NMR (75 MHz,CDCl3): d 21.7, 23.3, 26.2, 28.4,
31.2, 36.6, 39.5, 46.7, 48.5, 53.6, 68.3, 80.5, 157.1, 176.8. LRMALD-
3.2. (S)-4-Isopropyl-3-(4-methylpentanoyl)oxazolidin-2-one 4
A solution of n-BuLi (1.6 mL, 2.6 mmol, 1.6 M in hexane) was
added dropwise to a stirred solution of 3 (310 mg, 2.4 mmol) in
THF (5.0 mL) at ꢁ78 °C. The mixture was stirred at this tempera-
ture for 30 min. In a separate flask containing 4-methylvaleric acid
(0.30 mL, 2.4 mmol) and THF (5.0 mL) were added Et3N (0.40 mL,
2.9 mmol) and isobutylchloroformate (0.34 mL, 2.6 mmol) at 0 °C.
After stirring for 30 min, the lithio-(4S)-4-isopropyl-2-oxazolidi-
none was added at 78 °C and stirred for 1 h. The reaction was
quenched with saturated aqueous NaHCO3. The organic layer was
washed with water and brine, dried over Na2SO4, filtered, and con-
centrated. The residue was purified by silica gel column chroma-
tography (hexane/EtOAc = 6:1) to yield 4 (800 mg, 76%) as a
colorless oil. 1H NMR (300 MHz, CDCl3): d 0.88 (d, J = 6.9 Hz, 3H),
0.91–0.94 (m, 9H), 1.52–1.65 (m, 3H), 2.31–2.42 (m, 1H), 2.80–
2.91 (m, 1H), 2.94–3.05 (m, 1H), 4.20 (dd, J = 9.0, 3.3 Hz, 1H),
4.27 (t, J = 8.6 Hz, 1H), 4.41–4.46 (m, 1H). 13C NMR (75 MHz,
CDCl3): d 14.6, 17.9, 22.2, 22.3, 27.6, 28.3, 33.2, 33.5, 58.3, 63.2,
IMS, m/z 352.19 for [M+Na]+ (calcd for C17H31NNaO5 352.21). ½a 2D8
ꢂ
11 (CHCl3, c 1.5).
3.5. (S)-2-((R)-2-((R)-1-(((9H-Fluoren-9-
yl)methoxy)carbonyl)pyrrolidin-2-yl)-1-hydroxyethyl)-4-
methylpentanoic acid 8
To a solution of carboxylic acid 7 (210 mg, 0.64 mmol) in CH2Cl2
(5.0 mL) was added TFA (5.0 mL) and stirred for 1 h. The solvent
was removed under reduced pressure. The crude product was dis-
solved in DMF (5.0 mL) and then Et3N (0.27 mL, 1.9 mmol) and
Fmoc-OSu (320 mg, 0.95 mmol) were added to the solution. After
the mixture had been stirred for 3 h, the reaction was quenched
with saturated aqueous NH4Cl and AcOEt. The organic layer was
washed with water and brine, dried over MgSO4, filtered, and con-
centrated. The residue was purified by silica gel column chroma-
tography (CHCl3/MeOH/AcOH = 200:2.5:0.5) to yield 8 (210 mg,
73%) as a white powder. 1H NMR (300 MHz, CDCl3): d 0.91 (d,
J = 6.0 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H), 1.29–1.41 (m, 1H), 1.44–
1.55 (m, 2H), 1.59–1.75 (m, 3H), 1.90–2.00 (m, 3H), 2.58–2.64
(m, 1H), 3.37 (t, J = 6.5 Hz, 2H), 3.69–3.72 (m, 1H), 4.21–4.26 (m,
2H), 4.40 (dd, J = 10.2, 6.6 Hz, 1H), 4.49 (dd, J = 10.5, 6.9 Hz, 1H),
7.32 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.59 (d, J = 7.2 Hz,
2H), 7.76 (d, J = 7.5 Hz, 2H). 13C NMR (75 MHz,CDCl3): d 21.9,
23.6, 23.8, 26.4, 31.4, 36.9, 39.6, 46.7, 47.5, 48.7, 54.7, 67.8, 68.5,
120.2, 125.1, 125.2, 127.3, 128.0, 141.55, 141.57, 143.9, 144.0,
157.5, 177.3. HRFABMS, m/z 452.2435 for [M+H]+ (calcd for
154.0, 173.5. ½a D28
ꢂ
+81 (CHCl3, c 1.5).
3.3. (R)-tert-Butyl 2-((2R,3S)-2-hydroxy-3-((S)-4-isopropyl-2-
oxooxazolidine-3-carbonyl)-5-methylhexyl)pyrrolidine-1-
carboxylate 6
To a solution of 4 (800 mg, 3.5 mmol) in CH2Cl2 (5.0 mL) were
added n-Bu2BOTf (800 mg, 2.9 mmol) and DIEA (0.82 mL,
4.6 mmol) at 0 °C. After the mixture had been stirred for 30 min,
the reaction mixture was cooled to 78 °C, and
5 (390 mg,
C
27H34NO5 452.2437). ½a D22
ꢂ
2.8 (CHCl3, c 0.24).
1.8 mmol) in CH2Cl2 (5.0 mL) was added. After the resulting mix-
ture had been stirred for 30 min, the temperature was raised up
to room temperature. The reaction mixture was cooled to 0 °C
and quenched with saturated aqueous NH4Cl and 30% H2O2. The
mixture was extracted with EtOAc and the whole was washed with
saturated aqueous NaHCO3, water, and brine. The combined organ-
ic layer was dried over MgSO4, filtered, and concentrated. The res-
idue was roughly purified by silica gel column chromatography
(hexane/EtOAc = 2:1) to yield 6. This compound was used next step
without further purification. 1H NMR (300 MHz, CDCl3): d 0.88 (d,
J = 6.6 Hz, 3H), 0.90–0.94 (m, 9H), 1.37–1.62 (m, 5H), 1.45 (s, 9H),
1.82–2.00 (m, 4H), 2.31–2.41 (m, 1H), 3.31 (brt, J = 6.2 Hz, 2H),
3.71–3.73 (m, 1H), 4.17–4.32 (m, 3H), 4.46–4.51 (m, 1H), 5.04
(m, 1H). 13C NMR (75 MHz, CDCl3): d 14.6, 17.9, 21.9, 23.4, 26.5,
28.3, 28.5, 31.1, 37.2, 39.6, 45.3, 46.4, 53.7, 58.9, 62.9, 69.2, 79.7,
154.0, 156.5, 175.1.
3.6. RI-peptide resin 10
The deprotected peptide resin 10 was constructed by Fmoc-
based solid phase peptide synthesis using Fmoc-NH-SAL-resin
(0.47 mmol/g, 66 mg, 0.037 mmol). Piperidine (20%) in DMF was
added to the resin. After agitating for 20 min, the resin was washed
with DMF. To this resin were added Fmoc-
HOBtꢃH2O (13 mg, 0.099 mmol), DIPCDI (15
DIEA (17 L, 0.099 mmol) in DMF. The mixture was agitated for
D
-AA-OH (0.099 mmol),
l
L, 0.099 mmol), and
l
2 h and was washed with DMF. Completion of the coupling reaction
was confirmed using Kaiser ninhydrin test. Following piperidine
treatments and couplings of Fmoc-AA-OH were repeated as above.
Finally, coupling of 8 (44 mg, 0.099 mmol) was carried out with
HOAt (13 mg, 0.099 mmol), DIPCDI (15
lL, 0.099 mmol), and DIEA
(17 L, 0.099 mmol). The mixture was agitated for 18 h, and the re-
sin was washed with DMF. After agitating with piperidine (20%) in
DMF for 20 min, the product resin 10 was washed with DMF.
l
3.4. (S)-2-((R)-2-((R)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)-1-
hydroxyethyl)-4-methylpentanoic acid 7
To a solution of the crude compound 6 in MeOH (5.0 mL) was
added NaOMe (300 mg, 5.5 mmol) at 15 °C and stirred for 16 h.
The reaction was quenched with saturated aqueous NH4Cl. The
mixture was extracted with EtOAc and the whole was washed with
1 M HCl, saturated aqueous NaHCO3, water, and brine. The
3.7. RI-peptide 11
TFA was added to the dried resin 10 and the mixture was agitated
for2 h. Themixturewasfiltered, andTFAwasremovedunderreduced

C. Awahara et al. / Bioorg. Med. Chem. 22 (2014) 2482–2488
2487
pressure. Ether was added and the precipitate was purified by pre-
parative HPLC to yield 11 (5.0 mg, 22% from Fmoc-NH-SAL-resin) as
a white powder. 1H NMR (300 MHz, CD3OD): d 0.90–1.06 (m, 18H),
1.17–1.27 (m, 1H), 1.50–1.59 (m, 3H), 1.63–1.79 (m, 3H), 1.87 (m,
3H), 1.95–2.16 (m, 8H), 2.18–2.38 (m, 3H), 2.47–2.53 (m, 1H),
3.46–3.89 (m, 4H), 3.94–4.00 (m, 1H), 4.17–4.30 (m, 1H), 4.38–
4.51 (m, 2H), 4.53 (dd, J = 8.1, 5.1 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H),
8.20 (d, J = 8.1 Hz, 1H). LRMALDIMS, m/z 635.26 for [M+H]+ (calcd
for C33H59N6O6 635.45).
was isolated and purified as above to yield 15 (6.0 mg, 21% from
Fmoc-NH-SAL-resin) as
white powder. 1H NMR (300 MHz,
a
CD3OD): d 0.86–0.91 (m, 12H), 0.94 (d, J = 6.6 Hz, 3H), 0.99 (d,
J = 6.9 Hz, 3H), 1.16–1.27 (m, 1H), 1.33–1.70 (m, 7H), 1.78–2.13
(m, 11H), 2.19–2.32 (m, 2H), 2.35–2.42 (m, 1H), 2.64 (t,
J = 7.2 Hz, 1H), 2.93 (t, J = 7.5 Hz, 1H), 3.37–3.49 (m, 2H), 3.59–
3.71 (m, 2H), 3. 83 (dd, J = 16.7, 7.1 Hz, 1H), 3.90–3.97 (m, 1H),
4.16–4.28 (m, 1H), 4.24 (d, J = 9.0 Hz, 1H), 4.30–4.39 (m, 1H),
4.41–4.45 (m, 2H), 4.65 (dd, J = 8.0, 5.0 Hz, 1H), 7.17–7.37 (m,
5H), 8.09 (br d, J = 8.4 Hz, 1H). LRMALDIMS, m/z 789.38 for
[M+Na]+ (calcd for C42H66N6NaO7 789.49).
3.8. RI-peptide 12
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added benzoyl
3.12. RI-peptide 16
chloride (10 lL, 0.075 mmol) and Et3N (15.6 lL, 0.112 mmol). The
mixture was agitated for 30 min, and the resin was washed with
DMF. After the resin was dried under reduced pressure, TFA was
added and the mixture was agitated for 2 h. The mixture was fil-
tered, and TFA was removed under reduced pressure. Ether was
added and the precipitate was purified by preparative HPLC to
yield 12 (2.8 mg, 10% from Fmoc-NH-SAL-resin) as a white powder.
1H NMR (300 MHz, CD3OD): d 0.86–0.92 (m, 12H), 0.95 (d,
J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H), 1.16–1.34 (m, 2H), 1.46–
1.65 (m, 5H), 1.67–1.89 (m, 4H), 1.95–2.14 (m, 8H), 2.18–2.29
(m, 2H), 2.41–2.48 (m, 1H), 3.06–3.09 (m, 1H), 3.46–3.58 (m,
3H), 3.62–3.75 (m, 3H), 3.78–3.87 (m, 1H), 3.89–4.01 (m, 1H),
4.20–4.28 (d, J = 9.3 Hz, 2H), 4.41–4.46 (m, 2H), 7.44–7.53 (m,
5H). LRMALDIMS, m/z 761.30 for [M+Na]+ (calcd for C40H62N6NaO7
761.46).
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added isovaleryl
chloride (20 drops from a syringe), Et3N (25 drops from a syringe),
and catalytic amount of DMAP. The mixture was agitated for
30 min, and the product was isolated and purified as above to yield
16 (2.4 mg, 9% from Fmoc-NH-SAL-resin) as a white powder. 1H
NMR (300 MHz, CD3OD): d 0.84–1.00 (m, 24H), 1.16–1.27 (m,
2H), 1.39–1.47 (m, 2H), 1.51–1.70 (m, 4H), 1.81–1.85 (m, 1H),
1.96–2.14 (m, 11H), 2.22–2.31 (m, 4H), 2.37–2.42 (m, 1H), 3.06–
3.09 (m, 1H), 3.46–3.55 (m, 3H), 3.62–3.72 (m, 2H), 3.79–3.87
(m, 1H), 3.91–3.98 (m, 1H), 4.24 (d, J = 9.0 Hz, 2H), 4.35–4.48 (m,
2H), 4.44 (d, J = 8.1 Hz, 1H). LRMALDIMS, m/z 741.36 for [M+Na]+
(calcd for C38H66N6NaO7 741.49).
3.13. RI-peptide 17
3.9. RI-peptide 13
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added 2-propyl-
pentanoic acid (14
0.085 mmol), DIPCDI (14
l
L, 0.088 mmol), HOBtꢃH2O (13 mg,
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added benzyl
chloroformate (10 drops from a syringe), Et3N (15 drops from a syr-
inge), and catalytic amount of DMAP. The mixture was agitated for
30 min, and the product was isolated and purified as above to yield
13 (3.8 mg, 13% from Fmoc-NH-SAL-resin) as a white powder. 1H
NMR (300 MHz, CD3OD): d 0.86–0.90 (m, 12H), 0.94 (d, J = 6.6 Hz,
3H), 0.99 (d, J = 6.6 Hz, 3H), 1.18–1.25 (m, 1H), 1.46–1.72 (m,
6H), 1.82–2.16 (m, 12H), 2.19–2.39 (m, 3H), 3.39–3.44 (m, 1H),
3.61–3.72 (m, 3H), 3.79–3.87 (m, 1H), 3.90–3.97 (m, 1H), 4.13
(brs, 1H), 4.22–4.25 (m, 1H), 4.41–4.47 (m, 2H), 4.65 (dd, J = 8.3,
5.3 Hz, 1H), 5.13 (s, 2H), 7.23–7.37 (m, 5H), 8.02–8.08 (m, 2H).
LRMALDIMS, m/z 791.77 for [M+Na]+ (calcd for C41H64N6NaO8
791.47).
l
L, 0.088 mmol), and DIEA (15 L,
l
0.088 mmol). The mixture was agitated for 2 h, and the product
was isolated and purified as above to yield 17 (2.0 mg, 7% from
Fmoc-NH-SAL-resin) as a white powder. 1H NMR (300 MHz, CD3-
OD): d 0.85–0.98 (m, 24H), 1.19–1.50 (m, 10H), 1.55–1.62 (m,
4H), 1.69–1.73 (m, 1H), 1.76–1.88 (m, 1H), 1.93–2.16 (m, 10H),
2.20–2.35 (m, 2H), 2.38–2.45 (m, 1H), 2.60–2.68 (m, 1H), 3.48–
3.61 (m, 3H), 3.63–3.72 (m, 2H), 3.67 (d, J = 8.7 Hz, 1H), 3.79–
3.87 (m,1H), 3.91–3.98 (m, 1H), 4.16–4.27 (m, 1H), 4.38–4.47 (m,
3H), 4.65 (dd, J = 8.1, 4.8 Hz, 1H), 7.99–8.10 (m, 2H). LRMALDIMS,
m/z 783.47 for [M+Na]+ (calcd for C41H72N6NaO7 783.54).
3.14. RI-peptide 18
3.10. RI-peptide 14
To 10 (0.037 mmol) in CH2Cl2 were added trimethylacetyl chlo-
ride (10 drops from a syringe), Et3N (15 drops from a syringe), and
catalytic amount of DMAP. The mixture was agitated for 2 h, and
the product was isolated and purified as above to yield 18
(4.3 mg, 16% from Fmoc-NH-SAL-resin) as a white powder. 1H
NMR (300 MHz, CD3OD): d 0.85–0.92 (m, 12H), 0.94 (d, J = 6.6 Hz,
3H), 0.99 (d, J = 6.9 Hz, 3H), 1.15–1.33 (m, 1H), 1.26 (s, 9H), 1.37–
1.47 (m, 3H), 1.53–1.68 (m, 4H), 1.79–1.88 (m, 1H), 1.90–2.16
(m, 10H), 2.19–2.33 (m, 2H), 2.35–2.43 (m, 1H), 3.48–3.56 (m,
2H), 3.62–3.73 (m, 2H), 3.79–3.87 (m, 2H), 3.91–3.98 (m, 1H),
4.24 (d, J = 9.0 Hz, 1H), 4.36–4.47 (m, 3H), 4.65 (dd, J = 8.3, 5.0 Hz,
1H), 8.08 (br d, J = 8.4 Hz, 1H). LRMALDIMS, m/z 741.36 for
[M+Na]+ (calcd for C38H66N6NaO7 741.49).
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added trans-cin-
namic acid (15 mg, 0.093 mmol), HOBtꢃH2O (14 mg, 0.092 mmol),
DIPCDI (15 lL, 0.093 mmol), and DIEA (16 lL, 0.093 mmol). The
mixture was agitated for 2 h, and the product was isolated and
purified as above to yield 14 (8.2 mg, 29% from Fmoc-NH-SAL-re-
sin) as a white powder. 1H NMR (300 MHz, CD3OD): d 0.85–0.99
(m, 18H), 1.17–1.27 (m, 1H), 1.43–1.67 (m, 6H), 1.71–1.86 (m,
2H), 1.94–2.16 (m, 10H), 2.18–2.24 (m, 2H), 2.38–2.45 (m, 1H),
3.53–3.84 (m, 6H), 3.92–3.95 (m, 1H), 4.23–4.26 (m, 1H), 4.41–
4.49 (m, 3H), 4.63–4.67 (m, 1H), 6.95 (d, J = 15.6 Hz, 1H), 7.36–
7.42 (m, 3H), 7.57–7.67 (m, 3H), 8.07 (d, J = 8.4 Hz, 1H). LRMALD-
IMS, m/z 787.74 for [M+Na]+ (calcd for C42H64N6NaO7 787.47).
3.15. RI-peptide 20
3.11. RI-peptide 15
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added Fmoc-
Ile-OH (35 mg, 0.099 mmol), HOBtꢃH2O (15 mg, 0.099 mmol), DIP-
CDI (15 L, 0.099 mmol), and DIEA (17 L, 0.099 mmol). The mix-
D-
To 10 (0.037 mmol) in CH2Cl2/DMF (1:1) were added 3-phenyl-
propanoic acid (15 mg, 0.091 mmol), HOBtꢃH2O (14 mg,
l
l
0.092 mmol), DIPCDI (15
l
L, 0.093 mmol), and DIEA (16
lL,
ture was agitated for 2 h. After the resin was washed with DMF,
0.093 mmol). The mixture was agitated for 2 h, and the product
piperidine (20%) in DMF was added to the resin. After agitating

2488
C. Awahara et al. / Bioorg. Med. Chem. 22 (2014) 2482–2488
for 20 min, the deprotected resin was washed with DMF to yield
19. The resin was dried under reduced pressure, and then TFA
was added and the mixture was agitated for 2 h. The mixture
was filtered, and TFA was removed under reduced pressure. Ether
was added to the crude product to form the precipitate. The precip-
itate was washed with ether and purified by preparative HPLC to
yield 20 (7.5 mg, 27% from Fmoc-NH-SAL-resin) as a white powder.
1H NMR (300 MHz, CD3OD): d 0.85–1.02 (m, 21H), 1.06 (d,
J = 6.9 Hz, 3H), 1.15–1.30 (m, 2H), 1.44–1.69 (m, 7H), 1.74–1.77
(m, 1H), 1.81–1.90 (m, 1H), 1.93–2.16 (m, 11H), 2.19–2.25 (m,
2H), 2.38–2.42 (m, 1H), 3.52–3.61 (m, 3H), 3.64–3.73 (m, 2H),
3.78–3.87 (m, 1H), 3.90–3.97 (m, 1H), 4.02 (d, J = 6.3 Hz, 1H),
4.22–4.30 (m, 1H), 4.36–4.50 (m, 3H), 4.66 (dd, J = 8.0, 5.0 Hz,
1H), 8.07–8.10 (br d, J = 8.4 Hz, 1H). LRMALDIMS, m/z 748.27 for
[M+H]+ (calcd for C39H70N7O7 748.53).
4.8 Hz, 1H), 8.02 (br d, J = 8.4 Hz, 1H), 8.13 (br d, J = 8.7 Hz, 1H).
LRMALDIMS, m/z 876.29 for [M+H]+ (calcd for C44H78N9O9 876.59).
3.18. Measurement of inhibitory activity
Enzyme assays were carried out using the synthetic HTLV-1
protease mutant14 at an enzyme concentration of 5.6 nM. The reac-
tion mixture (0.5 M sodium acetate buffer, pH 5.6, containing 10%
glycerol, 10 mM DTT and 4 M NaCl) was analyzed on a Cosmosil
5C18-ARII column (4.6 ꢀ 150 mm), employing a linear gradient of
CH3CN (10–40%, 30 min) in aq 0.1% TFA. Each IC50 value was ob-
tained from a sigmoidal dose–response curve obtained from the
decrease of the substrate in the reaction mixture. Each experiment
was repeated 3 times.
Acknowledgment
3.16. RI-peptide 21
This work was supported in part by a Grant-in-aid for Scientific
research from the Japan Society for the Promotion of Science
(Grant 21590017 to K.A.).
To the deprotected resin 19 prepared as above, acetic anhydride
(2.5 lL, 0.025 mmol), Et3N (5.1 lL, 0.037 mmol), and catalytic
amount of DMAP were added. The mixture was agitated for
10 min, and the product was isolated and purified as above to yield
21 (3.1 mg, 11% from Fmoc-NH-SAL-resin) as a white powder. 1H
NMR (300 MHz, CD3OD): d 0.85–0.96 (m, 21H), 0.99 (d, J = 6.9 Hz,
3H), 1.16–1.24 (m, 2H), 1.38–1.45 (m, 3H), 1.53–1.69 (m, 4H),
1.78–1.87 (m, 3H), 1.93–2.13 (m, 10H), 1.98 (s, 3H), 2.19–2.27
(m, 2H), 2.37–2.43 (m, 1H), 3.06–3.09 (m, 1H), 3.50–3.56 (m,
2H), 3.59–3.72 (m, 3H), 3.77–3.84 (m, 2H), 3.93–3.96 (m, 1H),
4.22 (br d, J = 9.0 Hz, 1H), 4.28–4.31 (m, 1H), 4.41–4.45 (m, 2H),
4.51 (br d, J = 9.6 Hz, 1H), 4.65 (dd, J = 8.1, 5.1 Hz, 1H). LRMALDIMS,
m/z 812.32 for [M+Na]+ (calcd for C41H71N7NaO8 812.53).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.050.
References and notes
1. Matsuoka, M. Oncogene 2003, 22, 5131.
2. Akaji, K.; Teruya, K.; Aimoto, S. J. Org. Chem. 2003, 68, 4755.
3. (a) Naka, H.; Teruya, K.; Bang, J. K.; Aimoto, S.; Tatsumi, T.; Konno, H.; Nosaka,
K.; Akaji, K. Bioorg. Med. Chem. Lett. 2006, 16, 3761; (b) Bang, J. K.; Teruya, K.;
Aimoto, S.; Konno, H.; Nosaka, K.; Tatsumi, T.; Akaji, K. Int. J. Peptide Res. Ther.
2007, 13, 173.
4. Tatsumi, T.; Awahara, C.; Naka, H.; Aimoto, S.; Konno, H.; Nosaka, K.; Akaji, K.
Bioorg. Med. Chem. 2010, 18, 2720.
3.17. RI-peptide 22
5. (a) Chorev, M.; Goodman, M. Acc. Chem. Rev. 1993, 26, 266; (b) Chorev, M.
Biopolymers (Peptide Science) 2005, 80, 67.
To the deprotected resin 19 prepared as above were added
6. (a) Verdini, A. S.; Viscomi, G. C. J. Chem. Soc., Perkin Trans. 1 1985, 697; (b) Fuller,
W. D.; Goodman, M.; Verlander, M. S. J. Am. Chem. Soc. 1987, 109, 4712; (c)
Verdini, A. S.; Dilvestri, S.; Becherucci, C.; Longobardi, M. G.; Parente, L.;
Peppoloni, S.; Perretti, M.; Pileri, P.; Pinori, M.; Viscomi, G. C.; Nencioni, L. J.
Med. Chem. 1991, 34, 3372.
7. (a) Lee, Y. S.; Agnes, R. S.; Davis, P.; Ma, S.; Badghisi, H.; Lai, J.; Porreca, F.;
Hruby, V. J. Med. Chem. 2007, 50, 165; (b) Sakurai, K.; Chung, H. S.; Kahne, D. J.
Am. Chem. Soc. 2004, 126, 16288.
8. (a) Ghosh, A. K.; Onishi, M. J. Am. Chem. Soc. 1996, 118, 2527; (b) Ghosh, A. K.;
Fidanze, S. J. Org. Chem. 1998, 63, 6146.
9. Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103, 2127.
10. Crimmins, M. T.; Chaudhary, K. Org. Lett. 2000, 2, 775.
11. Rink, H. Tetrahedron Lett. 1987, 28, 3787.
Fmoc-
D
-Gln-OH (36 mg, 0.098 mmol), HOBtꢃH2O (15 mg,
0.098 mmol), DIPCDI (15
l
L, 0.098 mmol), and DIEA (17 L,
l
0.098 mmol). The mixture was agitated for 2 h. After the resin
was washed with DMF, piperidine (20%) in DMF was added to
the resin. After agitating for 20 min, the deprotected resin was
washed with DMF. After the resin was dried under reduced pres-
sure, TFA was added and the mixture was agitated for 2 h. The
product was isolated and purified as above to yield 22 (5.8 mg,
16% from Fmoc-NH-SAL-resin) as
a
white powder. 1H NMR
(300 MHz, CD3OD): d 0.84–1.05 (m, 24H), 1.16–1.29 (m, 2H),
1.39–1.74 (m, 8H), 1.84–1.88 (m, 2H), 1.94–2.16 (m, 12H), 2.19–
2.33 (m, 2H), 2.36–2.54 (m, 3H), 3.38–3.44 (m, 1H), 3.60–3.72
(m, 3H), 3.79–3.86 (m, 2H), 3.89–3.94 (m, 1H), 3.97–4.01 (m,
1H), 4.21–4.28 (m, 1H), 4.35–4.49 (m, 4H), 4.66 (dd, J = 8.1,
12. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
13. The data are included in the Supplementary information.
14. Teruya, K.; Kawakami, T.; Akaji, K.; Aimoto, S. Tetrahedron Lett. 2002, 43, 1487.
15. Satoh, T.; Li, M.; Nguyen, J.-T.; Kiso, Y.; Gustchina, A.; Wlodawer, A. J. Mol. Biol.
2010, 401, 626.