Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster

Article abstract of DOI:10.1016/j.bmc.2010.02.019

Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new 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.2010.02.019

Bioorganic & Medicinal Chemistry 18 (2010) 2720–2727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors
containing a hydroxyethylamine isoster
Tadashi Tatsumi a, Chiyuki Awahara a, Hiromi Naka b, Saburo Aimoto b, Hiroyuki Konno a, Kazuto Nosaka a,
Kenichi Akaji a,
*
a Department of Medicinal Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan
b Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine
isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective
aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scis-
sile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially
modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxy-
ethylamine inhibitor.
Received 16 January 2010
Revised 10 February 2010
Accepted 11 February 2010
Available online 18 February 2010
Keywords:
HTLV-1 protease
Inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Retro inverso
Hydroxyethylamine isoster
1. Introduction
containing a hydroxyethylamine dipeptide isoster.7 We found the
configurations at the hydroxyl- and side chain-bearing asymmetric
Retro-inverso (RI) modifications involve a reversion of the pep-
tide sequence accompanied by the replacement of each -amino
acid with the corresponding
-amino acid1 (Fig. 1). Fully or par-
tially RI modified biologically active peptides have been found to
retain the properties or biological activity of the parent peptides,
since a similar topography of side chain orientations is retained
by the RI modification.2 The modification primarily reverses the
centers to have remarkable effects on the inhibitory activity. Eval-
uations of substrate specificity at the site of cleavage by this prote-
ase also revealed unique preferences for Pro at the P1’ position and
for Ile at the P2 position.8
L
D
2. Results and discussion
direction of amide bonds. The introduction of D-amino acids en-
Based on our previous studies on HTLV-1 protease inhibitors, ef-
fects of RI modifications of substrate-based inhibitors containing a
hydroxyethylamine isoster backbone were examined in the pres-
ent study (Fig. 2). Type-I RI inhibitor 2 based on the total RI mod-
ification of parent inhibitor 1 and type-II RI inhibitor 3 based on a
partial RI modification were designed and synthesized. In the pres-
ent RI modifications, Ile at the P2 position was replaced with (D)-Ile
to retain the side chain orientation after the modification, but the
chirality at the b-position of the side chain is not retained. The rel-
ative relationship between the isoster hydroxyl and scissile site
substituent in the type-II inhibitor 3 is the same as that in the par-
ent inhibitor 1, in which the side chain substituent is supposed to
interact with the protease S1 pocket.
hances the resistance of fully or partially RI modified peptides to
enzymatic degradation.3 Few applications of the RI modification
to protease inhibitors, however, have been reported, although sim-
ilar or improved interactions of RI modified peptides with proteins
or receptors have been reported.4 In this paper, we report the RI
modification of a HTLV-1 protease inhibitor containing the sub-
strate sequence which would improve its resistance to enzymatic
degradation without significantly affecting its inhibitory activity.
Human T-cell leukemia virus type 1 (HTLV-1) is a retro virus
etiologically associated with adult T-cell leukemia and a number
of other chronic diseases.5 HTLV-1 protease is an aspartic protease,
and crucial for processing of the virus’ proteins. Thus, HTLV-1 pro-
tease is a suitable target for the development of inhibitors for ther-
apeutic use. In previous papers,6 we reported the synthesis and
structure–activity relationship (SAR) of HTLV-1 protease inhibitors
Type-I RI inhibitor 2 was synthesized on a solid support using
an acid-stable succinate ester linker according to our previous re-
port6 (Scheme 1). (R,S)-Aminoalkylepoxide
4 derived from
(1R,2S)-1-arylsulfonamide-2-indanyl ester9 was reacted with H-
(D)-Ile-OAllyl and the resulting hydroxyethylamine product was
anchored to H-Gly-MBHA resin10 through a succinic acid linker
* Corresponding author. Tel./fax: +81 75 465 7659.
E-mail address: akaji@koto.kpu-m.ac.jp (K. Akaji).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.019

T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
2721
S4
P4
S2
P2
O
O
H
H
N
N
Substrate
(L-amino acids)
N
H
N
H
O
O
P3
S3
P1
S1
S4
P4
S2
P2
P2
P4
O
O
O
O
H
H
H
H
Retro-Inverso peptide
(D-amino acids)
N
N
N
N
N
N
N
N
H
H
H
H
O
O
O
O
P3
P1
P3
S3
P1
S1
Figure 1. Retro-inverso modification.
to give 6. After cleavage of the C-terminal allyl group of 6 with
[(C6H5)3P]4 Pd(0), H-(D)-Val-OAllyl was coupled using DIPCDI/
HOBt as coupling reagents. An aliquot of the product was treated
with HF to give a product having a major peak on analytical HPLC,
which suggests that no significant racemization occurred during
the coupling. Further elongation in the C-terminal direction with
H-(D)-Pro-NH2 was conducted in a similar manner to give resin
7, and an aliquot of 7 was similarly examined as above. The prod-
uct gave a single major peak when the coupling was conducted
with HATU/HOAt,11 whereas two major peaks were observed when
DIPCDI/HOBt were used as the coupling reagents.12 After cleavage
of the Boc group of the resin 7 with 50% TFA/CH2Cl2, Boc-(D)-Pro-
OH was coupled with DIPCDI/HOBt. The Boc group of the product
was similarly removed by TFA, and the resulting resin was treated
with acetic anhydride. The acetylated peptide was then cleaved
from the resin by treatment with HF at 4 °C. The succinic acid lin-
ker of the crude product was removed by treatment with AcONH4
at pH 9 for 20 h, and the crude product was purified by preparative
HPLC to give 2. The truncated analog 9 was similarly synthesized
from the intermediate 7.
struction of the isoster backbone containing a hydroxyl group
and scissile site substituent was achieved with a stereoselective al-
dol reaction,9 which made it possible to selectively prepare all four
diastereomers containing different configurations at the isoster hy-
droxyl site and scissile site substituent (Scheme 2). Boc-(D)-Pro-ol
10 was oxidized with SO3ꢀPy, and the resulting aldehyde was re-
acted with methoxymethyltriphenylphosphonium chloride at
50 °C for 3 h. The product 11 was treated with pyridinium p-tolu-
enesulfonate (PPTS) to afford the homologated aldehyde 12. A
known ester containing a chiral auxiliary, (1S,2R)-1-arylsulfonam-
ido-2-indanyl 4-methylvalerate 13,6 was then reacted with the
aldehyde 12. While the aldol condensation was conducted accord-
ing to our previous protocol,6 a mixture of diastereomers was ob-
tained in the present reaction. To confirm the configuration, each
diastereomer, separated by column chromatography, was hydro-
lyzed and the resulting b-hydoxycarboxlic acid was converted to
the oxazolidinone 22 or 23 through a Curtius rearrangement. From
the coupling constant of the vicinal protons obtained in a homonu-
clear decoupling experiment, it was supposed that the polar ester
14 has the syn configuration and the less polar ester 15 has the anti
configuration. NOE spectroscopy of oxazolidinones supported this
estimation.
Type-II RI inhibitors based on a partial RI modification were
synthesized by a conventional solution-phase procedure. Con-
S2
S1'
Hydroxyethylamine type inhibitor
(L-amino acids)
OH
NH
O
H
N
H
N
N
I
N
H
O
O
N
O
H
S3
1
S1
Total RI modification
S2
Partial RI modification
H2N
O
S2
S1'
S1'
NH2
N
O
O
N
H
OH
H
OH
O
O
N
O
H
NH
N
N
N
N
N
N
O
O
H
O
N
O
O
H
S3
2
S1
Type I RI inhibitor (D-amino acids)
Figure 2. Retro-inverso modification of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.
S3
3
S1
Type II RI inhibitor (D-amino acids)

2722
T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
O
O
H
N
OH
O
O
O
H
O
(a)
Boc-NH
N
(b), (c)
Boc-NH
O
O
O
H
Boc-NH
N
5
4
6
O
O
H
H
N
N
O
O
O
O
1) (d), (e)
2) (d), (f)
(g), (h)
(g), (i)
O
O
O
O
H
H
H
N
N
N
Boc-NH
N
N
N
N
N
H
H
O
O
O
NH2
O
NH2
O
O
8
7
(j), (k)
O
(g), (i)
(j), (k)
OH
OH
O
H
H
N
H
N
H
N
N
N
N
N
N
N
H
H
O
O
O
O
NH2
O
NH2
O
O
9
2
Scheme 1. Synthesis of type-I RI inhibitors. Reagents and conditions: (a) H-(D)-Ile-OAllyl in 2-propanol, 60 °C, 4 h and 25 °C, 18 h; (b) succinic anhydride/DMAP, 25 °C, 16 h;
(c) H-Gly-MBHA resin/DIPCDI/HOBt/DIEA, 25 °C, 18 h; (d) Pd[P(Ph)3]4, 25 °C, 2 h; (e) H-(D)-Val-OAllyl/DIPCDI/HOBt, 25 °C, 18 h; (f) H-(D)-Pro-NH2/coupling reagent, 25 °C, 2–
18 h; (g) TFA, 25 °C, 15 min; (h) Boc-(D)-Pro-OH/DIPCDI/HOBt, 25 °C, 1.5 h; (i) Ac2O/DIEA, 25 °C, 20 min; (j) HF, 4 °C, 1 h; (k) aq AcONH4, pH 8.5–9, 25 °C, 20 h.
Tos
NH
(a), (b)
(c)
OH
O
N
N
N
O
O
Boc
O
Boc
Boc
12
10
13
11
Tos
NH
Tos
NH
OH
OH
(d)
O
O
+
N
N
O
O
Boc
Boc
14
(e), (f), (g)
15
(e), (f), (g)
O
O
O
O
H
H
HN
HN
H
H
N
H
N
H
2.7 Hz
23
6.3 Hz
22
Scheme 2. Construction of the isoster backbone. Reagents and conditions: (a) SO3ꢀPy/DMSO, Et3N, 25 °C, 0.5 h; (b) ClꢁP+Ph3CH2OMe/tBuOK, 50 °C, 3 h; (c) PPTS, 25 °C, 4 h;
(d)1 M TiCl4/CH2Cl2, DIEA, ꢁ78 °C, 2 h; (e) 30%H2O2, LiOH, 25 °C, 24 h; (f) DPPA/Et3N, 80 °C, 0.5 h; (g) TFA, 25 °C, 15 min.
The isoster backbone thus prepared was then introduced into
the RI modified substrate sequence (Scheme 3). First, the b-
hydoxycarboxlic acid 18 was obtained by the hydrolysis of polar
ester 14 with LiOH. 18 was then coupled with a tetrapeptide syn-
thesized by a conventional solution-phase method using WSCDI/
HOBt as coupling reagents.13 The Boc group of the resulting prod-
uct 24 was removed with TFA and the product was reacted with
benzylisocyanate to afford the Diastereomer 3a of a Type-II RI
inhibitor. The diastereomer 3b was similarly synthesized from b-
hydoxycarboxlic acid 15 prepared from the chiral ester 13. Diaste-
reomers 3c and 3d of Type-II RI inhibitors were similarly synthe-
sized from (1R,2S)-1-arylsulfonamido-2-indanyl 4-methylval-
erates. Each diastereomer showed a single major peak at different
elution times on analytical HPLC.
The inhibitory activities of Type-I and -II RI inhibitors thus ob-
tained were examined using a synthetic dodecapeptide as a sub-
strate according to our previous procedure.6 Cleavage of the
substrate by a mutant HTLV-1 protease14 in the presence of the

T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
2723
H2N
O
14
OH
N
(e)
O
OH
O
O
O
H
N
HO
N
N
N
N
Boc
H
Boc
O
18
24
Boc-(D)-Ile-OH
(a), (f)
(a)
H2N
(b)
(c)
Boc-(D)-Val-OH
Boc-(D)-Pro-OMe
H-(D)-Pro-NH2
(a)
(b)
(d)
(a)
O
N
OH
O
O
O
H
N
N
N
N
H
O
N
O
H
3a
Scheme 3. Synthesis of type II RI inhibitor 3a. Reagents and conditions: (a) TFA, 25 °C, 0.5–2 h; (b) BOP/DIEA, 25 °C, 3 h-2 days; (c) 1 M NaOH, 25 °C, 2 days; (d) DPPA/DIEA,
25 °C, 2 days; (e) WSCDI/HOBt, 25 °C, 16 h; (f) benzylisocyanate/DIEA, 25 °C, 1.5 h.
RI inhibitor was monitored by analytical HPLC. The rate of cleavage
was estimated from the decrease in the substrate during the incu-
bation. The inhibitory activity of each RI inhibitor was evaluated
using the corresponding IC50 value obtained from the sigmoidal
dose–response curve15 (Table 1).
No inhibitory activity was detected for type-I RI inhibitors 2 and
9 even at 10 mM. In contrast, all diastereomers of type-II RI inhib-
itors showed inhibitory activity with IC50 values in the order of lM.
for the RI modification of a hydroxyethylamine backbone. Among
type-II RI inhibitors, 3a, with an R-configuration for the hydroxyl
group and S-configuration for the scissile site isobutyl substituent,
showed the most effective inhibitory activity (IC50 = 160 lM). The
diastereomers 3b and 3d having the reversed configuration of 3a
at the isoster hydroxyl group showed decreased inhibitory activi-
ties; one quarter or less the activity of 3a. Reversal of the isobutyl
substituent had a relatively small effect on the inhibitory activity.
These structure–activity relationships of type-II RI inhibitors are
consistent with those of parent hydroxylethylamine inhibitors,
These results indicate that the relative position of the isostere hy-
droxyl group for the isobutyl group directing the S1 site is critical
Table 1
IC50 values of RI inhibitors
Compound
IC50
OH
OH
H
N
H2N-Pro(D)-Val(D)-Ile(D)
Type I; 2
>10 mM
O
H
H2N-Pro(D)-Val(D)-Ile(D)
N
N
>10 mM
O
9
O
OH
OH
OH
OH
O
O
O
O
N
H2N-Pro(D)-Pro(D)-Val(D)-Ile(D)
H2N-Pro(D)-Pro(D)-Val(D)-Ile(D)
H2N-Pro(D)-Pro(D)-Val(D)-Ile(D)
H2N-Pro(D)-Pro(D)-Val(D)-Ile(D)
Type II; 3a
160
550
350
l
l
l
M
M
M
N
H
O
N
3b
3c
3d
N
H
O
N
N
H
O
N
1500 lM
N
H
O

2724
T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
which suggest that type-II RI inhibitors would interact with HTLV-
1 protease in the same manner as hydroxylethylamine inhibitors.
m), 2.87–2.96 (1H, m), 3.17 (1H, br d, J = 14.6 Hz), 3.51 (1H, br m),
3.65 (1H, br m), 4.65 (2H, m), 5.27 (1H, d, J = 10.6 Hz), 5.30–5.40
(1H, m), 5.85–5.98 (1H, m).
The parent inhibitor 1 (IC50 = 5.8 l
M),6b however, is still relatively
potent compared with a type-II RI modified inhibitor 3a. To im-
prove the inhibitory activities of the RI modified inhibitors, SAR
studies on side chain characteristics including (D)-alloisoleucine
are now underway.
To 150 mg of p-methylbenzhydrylamine (MBHA) resin
(0.66 mmol/g resin) swelled in DMF (1.5 mL) were added Fmoc-
Gly-OH (72 mg, 0.24 mmol), HOBt (37 mg, 0.24 mmol), DIPCDI
(39 lL, 0.24 mmol), and DIEA (42 lL, 0.24 mmol). The mixture was
In conclusion, retro-inverso (RI) modification of HTLV-1 prote-
ase inhibitors containing a hydroxyethylamine isoster backbone
was effective without significantly influencing the inhibitory activ-
ity. Structure–activity relationships regarding the isoster hydroxyl
group and scissile site substituent suggest that the RI modified
inhibitor would retain a similar topography to the parent inhibitor.
agitated for 90 min at 25 °C. After the resin was washed with DMF,
piperidine (20%) in DMF was added to the resin. After agitating for
a
20 min at 25 °C, the N -deprotected resin was washed with DMF.
To this resin were added the above carboxylic acid (78 mg,
0.16 mmol), HOBt (24 mg, 0.16 mmol), DIPCDI (25
and DIEA (27 L, 0.16 mmol). The mixture was agitated for 18 h at
25 °C, and washed with DMF. Acetic anhydride (46 L, 0.49 mmol)
and DIEA (84 L, 0.49 mmol) were added to the resin. After agitating
lL, 0.16 mmol),
l
l
l
3. Experimental
3.1. General
for 20 min at 25 °C, the product resin 6 was washed with DMF and
MeOH.
3.4. Ac-(D)-Pro-HEA(COCH2CH2CO-Gly-MBHA)-(D)-Ile-(D)-Val-
(D)-Pro-NH2 (8)
All solvents were of reagent grade. THF was distilled from so-
dium and benzophenone ketyl. CH2Cl2 was distilled from CaH2.
All commercial reagents were of the highest purity available. Ami-
no acid derivatives were purchased from Peptide Institute, Inc.
(Osaka, Minoh, Japan) or Watanabe Chemical, Inc. (Hiroshima, Ja-
pan). Analytical TLC was performed on silica gel (60 F-254, Plates
0.25 mm). Column chromatography was carried out on Wakogel
To the resin 6 (65 mg) in CHCl3/AcOH/NEM (37:2:1, 2 mL) was
added Pd[PPh3]4 (230 mg, 0.20 mmol), and the mixture was agi-
tated under darkness and an Ar atmosphere for 2 h. The resin
was washed with CHCl3, THF, and DMF, and then H-(D)-Val-OAllyl
(25 mg, 0.15 mmol), HOBt (23 mg, 0.15 mmol), DIPCDI (24
lL,
60 (particle size, 63–200
lm) or Wakogel FC-40 (particle size,
0.15 mmol), and DIEA (26 L, 0.15 mmol) were added. The mixture
l
20–40 m). Analytical and preparative HPLCs were performed
l
was agitated for 18 h at 25 °C, washed with DMF and MeOH, and
dried. The resin was similarly treated with Pd[PPh3]4 (230 mg,
0.20 mmol) as above, washed, and reacted with H-(D)-Pro-NH2
using a HITACHI ELITE LaChrom system (OD, 220 nm). 1H NMR
(300 MHz or 400 MHz) spectra were recorded on a Bruker AM-
300 or Bruker-DMX 400. Chemical shifts are expressed in ppm rel-
ative to TMS (0 ppm) or CHCl3 (7.28 ppm). High-resolution mass
spectra (HRMS) were obtained on a JMS-HX-110A (FAB) or a Bruker
Autoflex-II (MALDI-TOF). Optical rotations were recorded on a
HORIBA SEPA-300 polarimeter at the sodium D line.
(17 mg, 0.15 mmol), HOBt (23 mg, 0.15 mmol), DIPCDI (24
lL,
0.15 mmol), and DIEA (26 L, 0.15 mmol) for 18 h at 25 °C. The
l
product resin was treated with 50% TFA in CH2Cl2 for 15 min at
25 °C, and washed with CH2Cl2, DMF, and 5% DIEA in DMF. After
the resin was washed with DMF, Boc-(D)-Pro-OH (27 mg,
0.13 mmol), HOBt (19 mg, 0.13 mmol), DIPCDI (20
lL, 0.13 mmol),
3.2. Boc-NHCH(CH2CH(CH3)2)CH(OH)CH2-(D)-Ile-OAllyl (Boc-
HEA-(D)-Ile-OAllyl) (5)
and DIEA (22 L, 0.13 mmol) were added. The mixture was agi-
l
tated for 1.5 h at 25 °C, and washed with DMF. The resin was sim-
ilarly treated with 50% TFA in CH2Cl2 as above, washed, and reacted
with acetic anhydride (24 lL, 0.25 mmol), and DIEA (44 lL,
To a solution of Boc-aminoalkylepoxide (4)6a (40 mg, 170
lmol)
in 2-propanol (0.5 mL) was added H-(D)-Ile-OAllyl (320 mg,
1.7 mmol). The mixture was stirred at 60 °C for 4 h, and then at
25 °C for 18 h. The mixture was concentrated in vacuo and CHCl3
was added to the residue. The organic layer was washed succes-
sively with 5% aq citric acid and brine, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by flash chromatog-
raphy (hexane/AcOEt = 3:1) to give 5 (35 mg, 51%) as a colorless oil.
1H NMR (CDCl3) d: 0.88–0.94 (12H, m), 1.17–1.23 (1H, m), 1.25
(1H, s), 1.30–1.34 (1H, dd, J = 5.7, 4.5 Hz), 1.35–1.40 (1H, dd,
J = 10.0, 4.0 Hz), 1.43 (9H, s), 1.48–1.55 (1H, m), 1.67–1.77 (2H,
m), 2.38–2.43 (1H, br m), 2.82 (1H, br d, J = 10.8 Hz), 3.08 (1H, br
d, J = 5.9 Hz), 3.45 (1H, br m), 3.64 (1H, br m), 4.62 (2H, d,
J = 5.8 Hz), 4.76 (1H, br d, J = 9.4 Hz), 5.25 (1H, dd, J = 10.4,
1.4 Hz), 5.32–5.36 (1H, dd, J = 15.8, 1.4 Hz), 5.87–5.97 (1H, m).
0.25 mmol). After agitating for 20 min at 25 °C, the product resin
was washed with DMF and MeOH, and dried to yield 73 mg of 8.
3.5. Ac-(D)-Pro-HEA-(D)-Ile-(D)-Val-(D)-Pro-NH2 (2)
The resin 8 (58 mg) was treated with an anhydrous HF (5.0 mL) in
the presence of anisole (50 mL) at 4 °C for 60 min. After evaporation
of HF, ether (10 mL) was added to the reaction mixture. The resulting
precipitate was washed with ether (10 mL) and dissolved in 0.1%
aqueous TFA. The solution was freeze-dried to yield 12 mg of the
type I RI inhibitor precursor as a white amorphous powder. HRFAB
MS, m/z 751.4723 for [M+H]+ (calcd 751.4718 for C36H62N8O9).
The precursor product (11 mg) was dissolved in 0.1 aq TFA
(1.0 mL) and the pH of the solution was adjusted to 9.0. The mix-
ture was stirred at 25 °C for 20 h, and then the pH was adjusted
to 5.0 with 1 M AcOH. The solution was freeze-dried and the crude
product was purified by preparative HPLC [YMC Pro C18 column
(10 ꢂ 250 mm), CH3CN (15–45%/60 min) in 0.1% aq TFA, 2.5 ml/
min] to yield 2.1 mg (overall 7.3%) of 2 as a white powder. HRFAB
MS, m/z 595.4186 for [M+H]+ (calcd 595.4183 for C30H54N6O6).
3.3. Boc-HEA (COCH2CH2CO-Gly-MBHA)-(D)-Ile-OAllyl (6)
To a solution of 5 (59 mg, 150
succinic anhydride (19 mg, 190
l
mol) in CHCl3 (1 mL) were added
l
mol) and DMAP (5.3 mg, 44 mol),
l
and the mixture was stirred at 25 °C for 16 h. The mixture was
washed successively with 5% aq citric acid and brine, dried over
Na2SO4, and evaporated in vacuo. The residue was purified by flash
chromatography (hexane/AcOEt = 3:1) to give Boc-HEA (COCH2CH2-
COOH)-(D)-Ile-OAllyl (44 mg, 60%) as a colorless oil. 1H NMR (CDCl3)
d: 0.89–0.98 (12H, m), 1.24–1.29 (2H, m), 1.30–1.37 (1H, m), 1.44
(9H, s), 1.64–1.78 (2H, m), 2.03 (1H, br s), 2.37 (1H, br s), 2.67 (4H,
3.6. Ac-HEA-(D)-Ile-(D)-Val-(D)-Pro-NH2 (9); truncated analog
of type I RI inhibitor
To Boc-HEA(COCH2CH2CO-Gly-MBHA)-(D)-Ile-(D)-Val-OH, pre-
pared as above starting from 62 mg of MBHA resin (0.66 mmol/g re-

T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
2725
sin), in DMF (0.5 ml) were added H-(D)-Pro-NH2 (47 mg,
0.41 mmol), HOAt (8.4 mg, 62 mol), HATU (78 mg, 0.21 mmol),
and DIEA (18 L, 0.21 mmol). The mixture was agitated at 25 °C
zene sulfonamide (15); 1H NMR (CDCl3) d: 0.74 (3H, d, J = 6.6 Hz),
0.83 (3H, d, J = 6.6 Hz), 1.43 (9H, s), 1.20–2.12 (8H, m), 2.38 (1H,
m), 2.43 (3H, s), 2.85 (1H, d, J = 17.1 Hz), 3.06 (1H, dd, J = 17.1,
4.5 Hz), 3.28 (2H, m), 3.53 (1H, m), 4.26 (1H, m), 4.87 (1H, dd,
J = 9.6, 4.5 Hz), 5.48 (1H, dd, J = 4.5, 4.5 Hz), 5.77 (1H, d,
J = 3.6 Hz), 6.99 (1H, d, J = 9.6 Hz), 7.12–7.27 (4H, m), 7.30 (2H, d,
J = 8.4 Hz), 7.85 (2H, d, J = 8.4 Hz); HRFAB MS, m/z 615.3115 for
[M+H]+ (calcd 615.3104 for C33H47O7N2S).
The diastereomers ((1R,2S)-N-[2,3-dihydro-2-((2R,3R)-2-isobu-
tyl-3-hydroxy-4-(N-Boc-(R)-pyrrolidin-2-yl)-1-oxobutoxy)indene-
1-yl]-4-methylbenzene sulfonamide 16 and (1R,2S)-N-[2,3-dihy-
dro-2-((2R,3S)-2-isobutyl-3-hydroxy-4-(N-Boc-(R)-pyrrolidin-2-
yl)-1-oxobutoxy)indene-1-yl]-4-methylbenzene sulfonamide 17)
were similarly prepared from (1R, 2S)-1-arylsulfonamido-2-inda-
nyl 4-methylvalerate (16); 1H NMR (CDCl3) d: 0.74 (d, J = 6.6 Hz,
2H), 0.83 (d, J = 6.6 Hz, 3H), 0.98–1.68 (m, 13H), 1.77–2.07 (m,
3H), 3.22–3.38 (m, 2H), 3.53 (m, 1H), 4.26 (m, 1H), 4.87 (dd,
J = 9.2, 4.8 Hz, 1H), 5.48 (dd, J = 4.5, 4.5 Hz, 1H), 5.76 (d,
J = 3.3 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 7.22–7.27 (m, 3H), 7.30 (d,
J = 8.1 Hz, 2H), 7.85 (d, J = 8.1 Hz, 2H). (17) 1H NMR (CDCl3) d:
0.79 (3H, d, J = 6.3 Hz), 0.85 (3H, d, J = 6.3 Hz), 1.08–1.56 (14H,
m), 1.82 (3H, m), 2.43 (3H, s), 2.62 (1H, m), 2.85 (1H, d,
J = 17.1 Hz), 3.05 (1H, dd, J = 17.1, 3.9 Hz), 3.20–3.36 (2H, m), 3.67
(1H, m), 4.02 (1H, m), 4.86 (1H, dd, J = 6.6, 4.5 Hz), 5.26 (1H, dd,
J = 4.5, 4.5 Hz), 5.63 (1H, d, J = 2.7 Hz), 6.36 (1H, d, J = 9.6 Hz),
7.21–7.44 (6H, m), 7.83 (1H, d, J = 8.4 Hz).
l
l
for 2 h. The Boc group of the product resin was removed and the
resulting amino group was acetylated as above to yield 76 mg of
dried Ac-HEA(COCH2CH2CO-Gly-MBHA)-(D)-Ile-(D)-Val-(D)-Pro-
NH2. HF treatment and removal of the linker at pH 8.5 were also
achieved as described for 2. The crude product was purified by
preparative HPLC to yield 1.7 mg (overall 15%) of 9 as a white pow-
der. HRFAB MS, m/z 498.637 for [M+H]+ (calcd 498.370 for
C25H47N5O5).
3.7. (1S,2R)-N-[2,3-dihydro-2-((2S,3R)-2-isobutyl-3-hydroxy-4-
(N-Boc-(R)-pyrrolidin-2-yl)-1-oxobutoxy)indene-1-yl]-4-
methylbenzene sulfonamide (14) and diasteromers (15, 16, and
17)
To a stirred solution of Boc-(D)-Pro-ol 10 (1.0 g, 5.0 mmol) in
DMSO/AcOEt (5 mL/10 mL) were added Et3N (2.1 mL, 15 mmol)
and SO3ꢀPy (2.4 g, 15 mmol). The mixture was stirred at 25 °C for
30 min and poured into ice-water. The aqueous phase was ex-
tracted with AcOEt. The organic phase was washed with H2O, dried
over Na2SO4, and rotary evaporated. The crude product was used
without further purification. To a suspension of methoxymethyltri-
phenylphosphine chloride (5.1 g, 15 mmol) in benzene was added
t-BuOK (1.9 g, 17 mmol). The mixture was stirred at 50 °C for
30 min, and then crude aldehyde obtained above was added to
the mixture. The whole solution was further stirred at 50 °C for
3 h and then quenched with saturated aqueous NH4Cl. The organic
phase was separated, and the remaining aqueous phase was ex-
tracted with AcOEt. The combined organic phase was washed with
H2O, dried over MgSO4, and rotary evaporated. The crude product
was partially purified by column chromatography (hexane/
AcOEt = 2:1) to yield 0.70 g (61%) of 11 (E/Z mixture) as an oil.
To a stirred solution of 11 (0.66 g, 2.9 mmol) in acetone
(30 mL) was added pyridinium p-toluenesulfonate (0.73 g,
2.9 mmol). The mixture was stirred at 25 °C for 4 h, and then
poured into saturated aqueous NaHCO3. The aqueous phase
was extracted with AcOEt. The organic phase was washed with
H2O, dried over MgSO4, and rotary evaporated. The crude prod-
uct of 12 was used in the next reaction without further purifica-
3.8. (2S,3R)-2-Isobutyl-3-hydroxy-4-(N-Boc-(R)-pyrrolidine-2-
yl)-butyric acid (18) and diasteromers (19, 20, and 21)
To a stirred solution of 14 (110 mg, 0.18 mmol) in THF/H2O (3:1,
8 mL) were added LiOHꢀH2O (30 mg, 0.72 mmol) and 30% aqueous
H2O2 (100 lL). The mixture was stirred at 25 °C for 24 h, and then
quenched with the addition of aqueous Na2SO3 and saturated
aqueous NaHCO3. The organic solvent was evaporated in vacuo.
The resulting aqueous solution was diluted with H2O, acidified
with 1 M HCl, and extracted with AcOEt. The organic layer was
washed with H2O, dried over MgSO4 and rotary evaporated to yield
32 mg (54%) of 18 as an amorphous powder. ½a D25
ꢁ16.1 (c 1.0,
ꢃ
CHCl3); 1H NMR (CDCl3) d: 0.91 (3H, d, J = 6.3 Hz), 0.93 (3H, d,
J = 6.6 Hz), 1.46 (9H, s), 1.02–2.07 (9H, m), 2.62 (1H, m), 3.32
(2H, m), 3.75 (1H, m), 4.16 (1H, m), 6.34 (1H, br s); HRFAB MS,
m/z 330.2284 for [M+H]+ (calcd 330.2280 for C17H32O5N).
tion. To
a solution of (1S,2R)-1-arylsulfonamido-2-indanyl 4-
methylvalerate 136a (0.60 g, 1.5 mmol) in CH2Cl2 (10 mL) was
added a 1 M solution of TiCl4 (1.8 mL) in CH2Cl2 at 4 °C. The mix-
ture was stirred at 25 °C for 15 min. To this solution was added
DIEA (1.0 mL, 6.0 mmol) and the mixture was stirred for 2 h at
25 °C. The resulting solution was added to a stirred solution of
aldehyde 12 (2.9 mmol) and a 1 M solution of TiCl4 (3.5 mL) in
CH2Cl2 (10 mL) at ꢁ78 °C. The mixture was stirred at ꢁ78 °C
for 2 h and then quenched by the addition of aqueous ammo-
nium chloride. The organic layer was washed with H2O, dried
over MgSO4, and rotary evaporated. The crude product was puri-
fied by silica gel column chromatography (hexane/AcOEt = 5:1).
Each diastereomixture was further purified by silica gel column
chromatography (hexane/AcOEt = 40:1 to 20:1) to yield 14 (polar
compound, 13%) and 15 (less polar compound, 20%) each as an
amorphous powder.
Hydrolysis of 15 (180 mg, 0.29 mmol) was similarly carried out
as above to yield 92 mg (95%) of (2S,3S)-2-isobutyl-3-hydroxy-4-
(N-Boc-(R)-pyrrolidine-2-yl)-butyric acid 19 as an amorphous
powder: ½a 2D5
ꢃ
ꢁ2.8 (c 1.0, CHCl3); 1H NMR (CDCl3) d: 0.92 (3H, d,
J = 6.6 Hz), 0.94 (3H, d, J = 6.6 Hz), 1.32–2.08 (18H, m), 2.42 (1H,
m), 3.33 (2H, dd, J = 7.5, 5.4 Hz), 3.62 (1H, d, J = 9.6 Hz), 4.15 (1H,
m), 6.59 (1H, br s); HRFAB MS, m/z 330.2284 for [M+H]+ (calcd
330.2280 for C17H32O5N).
Two additional diastereomers 20 and 21 were similarly pre-
pared from the ester 16 and 17. (2R,3R)-2-Isobutyl-3-hydroxy-4-
(N-Boc-(R)-pyrrolidine-2-yl)-butyric acid 20 from 16 (210 mg,
0.34 mmol): yield 116 mg (quantitative), ½a D25
ꢃ
12.38 (c 1.0, CHCl3);
1H NMR (CDCl3) d: 0.91 (3H, d, J = 6.6 Hz), 0.93 (3H, d, J = 6.6 Hz),
1.35–2.08 (18H, m), 2.47 (1H, m), 3.28–3.38 (2H, m), 3.63 (1H,
m), 4.17 (1H, m), 6.56 (1H, br s); MALDI TOF-MS, m/z 352.330 for
[M+Na]+ (calcd 352.210 for C17H31O5NNa). (2R,3S)-2-Isobutyl-3-
hydroxy-4-(N-Boc-(R)-pyrrolidine-2-yl)-butyric acid 21 from 17
Compound (14); 1H NMR (CDCl3) d: 0.79 (3H, d, J = 6.3 Hz), 0.85
(3H, d, J = 6.3 Hz), 1.43 (9H, s), 1.1–1.6 (6H, m), 1.79 (2H, m), 2.43
(3H, s), 2.62 (1H, m), 2.85 (1H, d, J = 17.1 Hz), 3.05 (1H, dd, J = 17.1,
4.5 Hz), 3.20–3.36 (2H, m), 3.66 (1H, m), 4.02 (1H, m), 4.86 (1H, dd,
J = 9.6, 4.5 Hz), 5.26 (1H, dd, J = 4.5, 4.5 Hz), 5.63 (1H, d, J = 2.7 Hz),
6.36 (1H, d, J = 9.6 Hz), 7.83 (2H, d, J = 8.4 Hz); HRFAB MS, m/z
615.3089 for [M+H]+ (calcd 615.3104 for C33H47O7N2S).
(280 mg, 0.46 mmol): yield 146 mg (97%), ½a D25
ꢃ
10.93 (c 1.0, CHCl3);
1H NMR (CDCl3) d: 0.91 (3H, d, J = 6.6 Hz), 0.93 (3H, d, J = 6.6 Hz),
1.13–2.10 (18H, m), 2.64 (1H, ddd, J = 8.4, 4.8, 4.5 Hz), 3.26–3.38
(2H, m), 3.77 (1H, m), 4.17 (1H, m), 6.62 (1H, br s); MALDI TOF-
MS, m/z 352.322 for [M+Na]+ (calcd 352.210 for C17H31O5NNa).
(1S,2R)-N-[2,3-dihydro-2-((2S,3S)-2-isobutyl-3-hydroxy-4-(N-
Boc-(R)-pyrrolidin-2-yl)-1-oxobutoxy)indene-1-yl]-4-methylben-

2726
T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
3.9. (4S,5R)-4-Isobutyl-5-(((R)-pyrrolidin-2-
luted with H2O, and washed with AcOEt. The aqueous solution
yl)methyl)oxazolidin-2-one (22)
was acidified with 1 N HCl and extracted with AcOEt. The organic
layer was washed with H2O, dried over MgSO4, and rotary evapo-
rated to yield 0.71 g (98%) of Boc-(D)-Ile-(D)-Val-(D)-Pro-OH. 1H
NMR (CDCl3) d: 0.83–1.60 (23H, m), 1.72–2.38 (6H, m), 3.67 (1H,
m), 3.85 (1H, m), 4.01 (1H, m), 4.53 (1H, dd, J = 7.5, 5.7 Hz), 4.60
(1H, dd, J = 8.7, 7.5 Hz), 5.13 (1H, d, J = 8.7 Hz), 6.98 (1H, d,
J = 8.7 Hz).
Boc-(D)-Pro-NH2 (0.21 g, 1.0 mmol) was treated with TFA as
above. The product was dissolved with CH2Cl2 (5 mL) and neutral-
ized with iPr2NEt. To the solution were added Boc-(D)-Ile-(D)-Val-
(D)-Pro-OH (0.39 g, 0.92 mmol), DPPA (0.22 mL, 1.2 mmol) and
iPr2NEt (0.19 mL, 1.1 mmol), and the mixture was stirred at 25 °C
for 2 days. The organic layer was washed with 10% citric acid, sat.
Na2CO3, and H2O, dried over MgSO4, and rotary evaporated. The
residue was purified by silica gel column chromatography
(CHCl3/MeOH = 50:1) to yield 0.40 g (56%) of Boc-(D)-Ile-(D)-Val-
(D)-Pro-(D)-Pro-NH2. 1H NMR (CDCl3) d: 0.80–1.60 (23H, m,
1.60–2.66 (8H, m), 3.38–4.67 (8H, m), 5.08–5.50 (1H, m), 6.60–
6.85 (1H, m), 7.62 (1H, br s), 8.13–8.78 (1H, m).
To a stirred solution of 18 (16 mg, 4.8
l
mol) in toluene (2.0 mL)
were added Et3N (10 L, 7.3 mol) and DPPA (10
l
l
l
L, 7.3 mol). The
l
mixture was stirred at 80 °C for 30 min. After cooling, the mixture
was rotary evaporated. The crude product was purified by silica gel
column chromatography (hexane/AcOEt = 1:1) to yield 14 mg
(88%) of (4S,5R)-4-isobutyl-5-((N-Boc-(R)-pyrrolidin-2-yl)methyl)-
oxazolidin-2-one. 1H NMR (CDCl3) d: 0.90 (3H, d, J = 6.6 Hz), 0.96
(3H, d, J = 6.6 Hz), 1.21–1.27 (1H, m), 1.49 (9H, s), 1.37–2.27 (8H,
m), 3.23–3.56 (2H, m), 3.83 (1H, ddd, J = 11.1, 7.5, 3.3 Hz), 3.93
(1H, br), 4.76 (1H, br), 6.50(1H, br). Nuclear overhauser and
exchange spectroscopy of the 1H NMR showed the NOE between
vicinal protons of the oxazolidin ring (Hs of 3.83 ppm (NH–CH)
and 4.76 ppm (O–CH)) clearly.
The product was dissolved in TFA (2 mL), and the mixture was
stirred at 25 °C for 15 min and rotary evaporated. The crude prod-
uct was purified by silica gel column chromatography (CHCl3/
MeOH = 50:1) to yield 22 (quantitative). 1H NMR (CDCl3) d: 0.88
(3H, d, J = 6.6 Hz), 0.94 (3H, d, J = 6.6 Hz), 1.18 (1H, m), 1.42 (1H,
m), 1.58 (1H, m), 1.81 (1H, m), 1.92–2.46 (5H, m), 3.35 (2H, br s),
3.79 (1H, m), 4.02 (1H, m), 4.88 (1H, dt, J = 6.3, 7.5 Hz), 6.75 (1H, s).
(4S,5S)-4-Isobutyl-5-(((R)-pyrrolidin-2-yl)methyl)oxazolidin-2-
one 23 was similary prepared from 19. 1H NMR (CDCl3) d: 0.90 (3H,
d, J = 6.6 Hz), 0.92 (2H, m), 1.27–1.53 (2H, m), 1.62 (1H, m), 1.81
(1H, m), 1.90–2.42 (5H, m), 3.37 (2H, br s), 3.65 (1H, m), 3.79
(1H, br s), 4.37 (1H, dt, J = 2.7, 7.5 Hz), 6.67 (1H, s). Nuclear overha-
user and exchange spectroscopy of 1H NMR of (4S,5S)-4-isobutyl-
5-((N-Boc-(R)-pyrrolidin-2-yl)methyl)oxazolidin-2-one was mea-
sured, but the NOE between vicinal protons of the oxazolidin ring
(Hs of 3.50 ppm (NH–CH) and 4.20 ppm (O–CH)) was very weak.
3.12. Type II RI inhibitor (3a) and diasteromers (3b, 3c, and 3d)
To a stirred solution of 18 (16 mg, 0.05 mmol) in THF (3 mL)
were added WSCDI HCl (12 mg, 0.06 mmol) and HOBt (11 mg,
0.06 mmol), and the mixture was stirred at 25 °C for 1 h. H-(D)-
Ile-(D)-Val-(D)-Pro-(D)-Pro-NH2, prepared from Boc-(D)-Ile-(D)-
Val-(D)-Pro-(D)-Pro-NH2 (150 mg, 0.30 mmol) and TFA (1 mL),
was dissolved in DMF, neutralized with iPr2NEt, and added to the
above solution of the activated ester of 18. The mixture was stirred
at 25 °C for 16 h, and diluted with AcOEt. The organic phase was
washed with 10% citric acid, sat.d Na2CO3, and H2O, dried over
MgSO4, and rotary evaporated. The residue was purified by silica
gel column chromatography (CHCl3/MeOH = 30:1) to yield 32 mg
(89%) of 24. 28 mg (0.04 mmol) of 24 was then treated with TFA
as before, and the residue was dissolved with CH2Cl2 (3 mL) and
neutralized with iPr2NEt. To the solution were added benzylisocy-
3.10. Boc-(D)-Ile-(D)-Val-(D)-Pro-OMe
Boc-(D)-Pro-OMe (2.3 g, 10 mmol) was treated with TFA at
25 °C for 30 min, and excess TFA was rotary evaporated. The resi-
due was dissolved in CH2Cl2 (30 mL) and neutralized with iPr2NEt.
To the solution were added Boc-(D)-Val-OH (2.6 g, 12 mmol) and
BOP (5.3 g, 12 mmol). The mixture was stirred at 25 °C for 3 h
and rotary evaporated. The residue was dissolved in AcOEt and
the organic phase was washed with 10% citric acid, sat.Na2CO3,
and H2O, dried over MgSO4, and rotary evaporated. The residue
was purified by silica gel column chromatography (CHCl3/
MeOH = 50:1 to 20:1) to yield 2.4 g (74%) of Boc-(D)-Val-(D)-Pro-
OMe as colorless oil. The product (0.54 g, 1.6 mmol) was then trea-
ted with TFA (5 mL) as above for 2 h. The product was dissolved in
CH2Cl2 (20 mL) and neutralized with iPr2NEt. To the solution were
added Boc-(D)-Ile-OH (0.38 g, 1.6 mmol) and BOP (0.79 g,
1.8 mmol). The mixture was stirred at 25 °C for 2 days and rotary
evaporated. The residue was dissolved in AcOEt and the organic
layer was washed with 10% citric acid, sat.Na2CO3, H2O, dried over
MgSO4, and rotary evaporated. The residue was purified by silica
gel column chromatography (CHCl3/MeOH = 50:1 to 30:1) to yield
0.60 g (84%) of Boc-(D)-Ile-(D)-Val-(D)-Pro-OMe as a white amor-
phous solid. 1H NMR (CDCl3) d: 0.80–1.68 (23H, m), 1.72–2.30
(6H, m), 3.72 (3H, s), 3.62–3.88 (2H, m), 3.96 (1H, m), 4.50 (1H,
dd, J = 8.7, 4.8 Hz), 4.61 (1H, dd, J = 8.7, 6.6 Hz), 4.97 (1H, d,
J = 8.7 Hz), 6.52 (1H, d, J = 8.7 Hz).
anate (7.0 lL, 0.06 mmol) and iPr2NEt (7.0 lL, 0.04 mmol), and the
mixture was stirred at 25 °C for 1.5 h. The solvent was evaporated
in vacuo and the residue was dissolved in AcOEt. The organic layer
was washed with 1 M HCl, sat.Na2CO3, and H2O, and dried over
MgSO4. The organic solvent was rotary evaporated, and the residue
was purified by silica gel column chromatography (CHCl3/
MeOH = 30:1) to yield 25 mg (86%) of partially purified 3a. The
product was further purified by preparative HPLC [YMC Pro C18
column (10 ꢂ 250 mm), CH3CN (40–80%/60 min) in 0.1% aq TFA,
2.5 mL/min] to yield homogeneous 3a as a white amorphous pow-
der (overall 43%): HPLC, 12.08 min [Cosmosil 5C18-ARII column
(4.6 ꢂ 150 mm), 1.0 mL/min, CH3CN (30–60%, 30 min)], 1H NMR
(CDCl3) d: 0.77–1.08 (18H, m), 1.08–2.65 (22H, m), 3.15–4.68
(14H, m), 6.32–7.00 (2H, m), 7.23–7.38 (5H, m). MALDI TOF MAS,
m/z 790.297 for [M+Na]+ (calcd 790.485 for C41H65O7N7Na).
Diasteromers 3b, 3c, and 3d were similarly prepared from 19,
20, and 21, respectively. Compound 3b; HPLC, 15.22 min [Cosmosil
5C18-ARII column (4.6 ꢂ 150 mm), 1.0 mL/min, CH3CN (30–60%,
30 min)], 1H NMR (CDCl3) d: 1H NMR (CDCl3) d: 0.82–1.17 (18H,
m), 1.34–2.63 (22H, m), 3.15–3.80 (7H, m), 3.98 (1H, m), 4.18–
4.65 (6H, m), 6.88–7.18 (2H, m), 7.23–7.38 (5H, m). MALDI TOF
MAS, m/z 790.893 for [M+Na]+ (calcd 790.485 for C41H65O7N7Na).
3c;
HPLC,
15.83 min
[Cosmosil
5C18-ARII
column
3.11. Boc-(D)-Ile-(D)-Val-(D)-Pro-(D)-Pro-NH2
(4.6 ꢂ 150 mm), 1.0 mL/min, CH3CN (30–60%, 30 min)], 1H NMR
(CDCl3) d: 0.75–1.14 (19H, m), 1.22–2.63 (21H, m), 3.14–3.33
(2H, m), 3.45–3.60 (2H, m), 3.62–3.80 (2H, m), 4.07 (1H, m),
4.18–4.65 (7H, m), 6.44 (1H, m), 7.23–7.39 (5H, m), 7.56 (1H, m).
MALDI TOF MAS, m/z 790.301 for [M+Na]+ (calcd 790.485 for
C41H65O7N7Na). Compound 3d; HPLC, 13.59 min [Cosmosil 5C18-
To
a solution of Boc-(D)-Ile-(D)-Val-(D)-Pro-OMe (0.75 g,
1.7 mmol) in MeOH (15 mL) was added 1 M NaOH (2.0 mL) and
the mixture was stirred at 25 °C for 2 days. The organic solvent
was rotary evaporated and the residual aqueous solution was di-

T. Tatsumi et al. / Bioorg. Med. Chem. 18 (2010) 2720–2727
2727
ARII column (4.6 ꢂ 150 mm), 1.0 mL/min, CH3CN (30–60%,
References and notes
30 min)], 1H NMR (CDCl3) d: 0.78–1.23 (19H, m), 1.23–2.68 (21H,
m), 3.15–4.92 (14H, m), 6.60–7.03 (2H, m), 7.18–7.38 (5H, m).
MALDI TOF MAS, m/z 790.316 for [M+Na]+ (calcd 790.485 for
C41H65O7N7Na).
1. (a) Chorev, M.; Goodman, M. Acc. Chem. Rev. 1993, 26, 266; (b) Chorev, M.
Biopolymers (Pept. Sci.) 2005, 80, 67.
2. (a) Verdini, A. S.; Viscomi, G. C. J. Chem. Soc., Perkin Trans. I 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.
3.13. Measurement of inhibitory activity
3. Gaston, F.; Granados, G. C.; Madurga, S.; Rabanal, F.; Lakhdar-Ghazal, F.; Giralt,
E.; Bahraoui, E. ChemMedChem 2009, 4, 570.
4. (a) Lee, Y. S.; Agnes, R. S.; Davis, P.; Ma, S.; Badghisi, H.; Lai, J.; Porreca, F.;
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Am. Chem. Soc. 2004, 126, 16288.
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 ꢂ 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 curve15 obtained from
the decrease of the substrate in the reaction mixture. Each exper-
iment was repeated three times.
5. Matsuoka, M. Oncogene 2003, 22, 5131.
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Acknowledgment
10. Matsueda, G. R.; Stewart, J. M. Peptides 1981, 2, 45.
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12. The product obtained by HF treatment of Boc-(D)-Pro-HEA-(D)-Ile-(D)-Val-(D)-
Pro-NH2 synthesized by DIPCDI coupling protocol also gave two major
products. The hydrolysate of each product was analyzed by gas
chromatography using a chiral capillary column.16 Each peak contains D- and
L-Val, respectively, which clearly show that remarkable racemization occurred
during the DIPCDI-mediated coupling of (D)-Pro to the C-terminal (D)-Val
residue17 (see the Supplementary data).
13. Kopple, K. D.; Nitecki, D. E. J. Am. Chem. Soc. 1962, 84, 4457.
14. Teruya, K.; Kawakami, T.; Akaji, K.; Aimoto, S. Tetrahedron Lett. 2002, 43, 1487.
15. A typical sigmoidal dose-response curve used for the estimation of IC50 values
is shown in the Supplementary data.
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 for K.A.).
Supplementary data
Supplementary data (DL analyses of type I RI inhibitors, a typi-
cal sigmoidal dose-response curve, and HPLC chromatogram for
compounds 3a–d) associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.019.
16. Abdalla, S.; Bayer, E.; Frank, H. Chromatographia 1987, 23, 83.
17. Frank, H.; Woiwode, W.; Nicholson, G.; Bayer, E. Liebigs Ann. Chem. 1981, 345.